5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible benzodiazepine analogues: Synthesis, receptor binding affinity and lipophilicity-dependent anti-seizure onset of action

Article abstract of DOI:10.1016/j.bioorg.2020.104504

A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles, possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor affinity and anti-seizure activit

Full text of DOI:10.1016/j.bioorg.2020.104504

Bioorganic Chemistry 106 (2021) 104504
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
5-[Aryloxypyridyl (or nitrophenyl)]-4H-1,2,4-triazoles as novel flexible
benzodiazepine analogues: Synthesis, receptor binding affinity and
lipophilicity-dependent anti-seizure onset of action
,
Latifeh Navidpoura *, Shabnam Shabania, Alireza Heidarib, Manouchehr Bashiric,
,
Azadeh Ebrahim-Habibid e, Soraya Shahhosseinic, Hamed Shafaroodif,
Sayyed Abbas Tabatabaic, Mahsa Toolabia
a Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 14176, Iran
b Department of Medicinal Chemistry, School of Pharmacy, Tehran Azad University of Medical Sciences, Tehran, Iran
c Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
d Biosensor Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
e Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
f Department of Pharmacology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran 14174, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
A new series of 5-(2-aryloxy-4-nitrophenyl)-4H-1,2,4-triazoles and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-triazoles,
possessing C-3 thio or alkylthio substituents, was synthesized and evaluated for their benzodiazepine receptor
affinity and anti-seizure activity. These analogues revealed similar to significantly superior affinity to GABAA/
benzodiazepine receptor complex (IC50 values of 0.04–4.1 nM), relative to diazepam as the reference drug (IC50
value of 2.4 nM). To determine the onset of anti-seizure activity, the time-dependent effectiveness of i.p.
administration of compounds on pentylenetetrazole induced seizure threshold was studied and a very good
1,2,4-triazole
Flexible benzodiazepines
GABAA/benzodiazepine receptor complex
Onset of action
PTZ induced seizure threshold
relationship was observed between the lipophilicity (cLogP) and onset of action of studied analogues (r2
=
0.964). The minimum effective dose of the compounds, determined at the time the analogues showed their
highest activity, was demonstrated to be 0.025–0.1 mg/kg, relative to diazepam (0.025 mg/kg).
1. Introduction
tolerance, withdrawal syndrome [4], and adverse events such as
cognitive impairments, sedation and drug interactions promotes the
Benzodiazepines (BZDs) are among the most important drugs pos-
sessing vast clinical use, ranging over different types of central nervous
system disorders, and act as anxiolytic, sedative/hypnotic, muscle
relaxant, and anticonvulsant agents [1]. BZDs operate through binding
to a specific domain of GABAA receptor, known as BZD receptor, and
allosterically modulate the action of GABA (γ-amino butyric acid). This
interaction results in increasing the opening frequency of chloride ion
channel [2] which appears to enhance the GABA inhibitory activity. Due
to their rapid onset of action, high efficacy rates and minimal toxicity,
BZDs remain important agents in the management of epilepsy and are
first choice for status epilepticus, and seizure associated with post-
anoxic insult [3]. They are also frequently indicated in the treatment
of febrile, acute repetitive and alcohol withdrawal seizures [2,3].
Despite their widespread clinical use, potential shortcomings including
researchers to seek for new BZDs with enhanced selectivity, safety and
efficacy. Recent studies have focused on identification of subtype se-
lective GABAA modulators [5,6].
Through past decades, several studies have examined the structur-
e–activity relationship of BZDs to describe the essential pharmacophore
elements, resulting into proposal of various models [7–11]. An aromatic
ring (A) and a coplanar proton-accepting group in appropriate distance
are two common features among these models. Furthermore, an out-of-
plane aromatic ring (C) could also enhance affinity of ligands to the
receptor [12–15].
Recently, the high-resolution cryo-electron microscopy structure of
subtype α1β3γ2L GABAA receptor bound to GABA and BZDs alprazolam
and diazepam (Fig. 1) has been reported [2,16]. The results showed that
these molecules occupy the canonical BZD-binding site at the 1/γ2
α
* Corresponding author.
E-mail address: navidpur@sina.tums.ac.ir (L. Navidpour).
https://doi.org/10.1016/j.bioorg.2020.104504
Received 28 March 2020; Received in revised form 19 November 2020; Accepted 19 November 2020
Available online 24 November 2020
0045-2068/© 2020 Elsevier Inc. All rights reserved.

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
interface, where they form extensive interactions. In case of diazepam,
the oxygen of carbonyl group and amidic nitrogen form hydrogen bonds
associated with redistribution of compounds from CNS to peripheral
tissues.
with
α1Ser205 and γ2Tyr58, respectively. Similar interactions were
Considering the aforementioned fact and to study the relationship
between the lipophilicity and onset of action of non-rigid BZDs, herein
we report the synthesis of a novel library of 5-(2-aryloxy-4-nitrophenyl)-
4H-1,2,4-triazole-3-thiones and 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-tri-
azole-3-thiones. The choice for bioisosteric replacement of A-ring with
nitrophenyl or pyridyl moieties had been based on their hydrophilic
nature, which, concomitant with different alkyl groups on thio moiety
would provide analogues with diverse range of lipophilicities. The
GABAA/benzodiazepine receptor complex binding affinity of the syn-
thesized compounds was evaluated, their onset of actin was determined
by a time-course study and the relationship between their cLogP and the
time the anti-seizure activity reaches the highest (Tmax) was figured out.
Finally, to study their in vivo potencies, the minimum effective dose of
each analogue was determined.
observed in alprazolam, between the triazole nitrogens and the afore-
mentioned amino acids. Phenyl substitutions in both diazepam (on C5),
and alprazolam (on C6) are involved in π-π interactions with α1Tyr210
and α1Phe100. The chlorine atoms at the C8 and C7 positions in al-
prazolam and diazepam interact with the
chains, respectively.
α1His102 and γ2Asn60 side
In previous works, diverse non-rigid structures, fulfilling all the
above requirements, were reported to have considerable anti-seizure
activity [17,18]. Furthermore, earlier studies have revealed that 3-
substituted-4H-1,2,4-triazoles (Fig. 1A) and 2-amino-1,3,4-oxadiazole
(Fig. 1B) could serve as comparable substitutions for triazole moiety
in alprazolam (Fig. 1) while maintaining its key features [17–21].
As a part of our ongoing program to develop new active non-rigid
benzodiazepines, we synthesized a novel class of non-rigid analogues
including a methylene bridge between the phenyl A-ring and 1,2,4-tria-
zole or amino-1,3,4-oxadiazole moieties (Fig. 1C and D), possessing
GABAA/benzodiazepine receptor complex binding affinity as low as
0.892–3.779 nM, in comparison with diazepam showing the affinity of
2.857 nM as the reference drug [21]. Beside retaining affinity for their
receptor, their anti-seizure activity evaluated 60 min post single i.p.
administration of the compounds was significantly lower than diaz-
epam. They increased the pentylenetetrazole induced seizure threshold
at 10–20 mg/kg dose, similar to diazepam at 0.05–0.1 mg/kg dose. This
observation could be attributed to the highly lipophilic character of the
synthesized compounds resulting in their early onset and duration of
anti-seizure actions.
2. Results and discussion
2.1. Chemistry
The synthetic reactions leading to 5-(2-aryloxy-3-pyridyl)-4H-1,2,4-
triazole-3-thiones,
5-(4-nitro-2-phenoxyphenyl)-4H-1,2,4-triazole-3-
thiones, and 2-amino-5-(4-nitro-2-phenoxyphenyl)-1,3,4-oxadiazole are
outlined in Schemes 1 and 2.
The starting material 2-phenoxy-4-nitrobenzoic acid (1) was pre-
pared by refluxing sodium salts of 2-chloro-4-nitrobenzoic acid and
phenol in dry DMF in the presence of catalytic amount of copper [21]. 2-
Aryloxynicotinic acids (9 and 10) were prepared by melting the sodium
salts of 2-chloronicotinic acid and appropriate phenol derivative [20].
Reaction of these acids with thionyl chloride followed by treatment of
resulting acid chlorides with thiosemicarbazide and subsequent cycli-
zation in basic media afforded the related 4H-1,2,4-triazole-3(2H)-thi-
ones (7, 11, and 12) [21]. Sonicating the compounds 7, 11 and 12 in the
presence of suitable alkyl halides in alkaline media (5–10 min) afforded
It is well understood that the onset and duration of action of single
bolus administration of a BZD depends on its lipid solubility [22]. BZDs
with higher lipid solubility have a more rapid onset of action and shorter
duration of action, if they do not produce active metabolites. Therefore,
it could be hypothesized that at 60 min post-administration of the syn-
thesized compound, absence of significant anti-seizure activity could be
Fig. 1. Diazepam, Alprazolam and representative examples of non-rigid benzodiazepines.
2

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
Scheme 1. Reagents and conditions: (a) H2SO4, Ethanol, reflux, 11 h; (b) N2H4⋅H2O, Ethanol, r.t., 24 h; (c) NaHCO3, Dioxane, r.t., 15 min, then CNBr, r.t., 2 h; (d)
KOH, Ethanol, reflux, 20 h; (e) SOCl2, reflux, 60 min; (f) H2NCSNHNH2, dry THF, ꢀ 5 ◦C to r.t.; 24 h; then NaOH 5%, reflux, 11 h; (g) RX, NaOH, Ethanol, Sonication,
5–10 min.
Scheme 2. Reagents and conditions: (a) SOCl2, reflux, 60 min; H2NCSNHNH2 in dry THF, ꢀ 5 ◦C to r.t.; 24 h; then NaOH 5%, reflux, 11 h; (b) RX, NaOH, Ethanol,
Sonication, 5–10 min.
3-alkylthio-4H-1,2,4-triazoles (8a-d, 13a-d, and 14a-d) [17,21]. Ester-
ification of acid 1 in dry ethanol in the presence of sulfuric acid resulted
in corresponding ester 2 [21]. Hydrazide 3 was prepared by treatment of
ester 2 with hydrazine hydrate in ethanol. The reaction of hydrazide 3
with cyanogen bromide afforded 2-amino-1,3,4-oxadiazole 4 [20,21].
Compound 4 rearranged to 3-ethoxy-4H-1,2,4-triazole analogue 5 by
being refluxed in ethanolic potassium hydroxide media [17,21].
2.2. Biological activity
2.2.1. Receptor binding affinity
The GABAA/Benzodiazepine Receptor complex binding affinity of
the title compounds was evaluated by their ability to displace [3H]-
flumazenil (Ro15-1788) from its specific binding site in rat cortical
membrane tissue [21,23]. The concentration of the tested compounds
(non-radioactive ligands) that inhibits the binding of [3H]-flumazenil by
3

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
50% is designated as IC50 values and is displayed in Table 1. In general,
most analogues revealed considerable in vitro activity, showing similar
or superior affinity to benzodiazepine receptors relative to diazepam as
the reference drug.
primary time-course studies were performed to determine the onset of
action for each analogue. The compounds with lower cLogP were chosen
for time-course studies. The effect of i.p. administration of 7, 8a-c, 11,
12, 13a-c, and 14b at 0.1 mg/kg dose at different time intervals before i.
v. administration of pentylenetetrazole (PTZ) on clonic induced seizure
threshold was investigated [24,25] and the results are displayed in
Figs. 2 and 3 and Table 1.
Within 5-(4-nitro-2-phenoxy)–2H-1,2,4-triazole-3(4H)-thione series
(7 and 8a-d), all compounds demonstrated higher affinity toward
benzodiazepine receptors with IC50 values of 0.13–0.54 nM compared
with diazepam with IC50 value of 2.4 nM as the reference drug. Upon
alkylation of the thio group, activity was retained, while substitution of
alkylthio with 3-ethoxy group resulted in compound 5 with less affinity
to benzodiazepine receptors (IC50: 7.06 nM). Bioisosteric replacement of
1,2,4-triazole-3-thione with 2-amino-1,3,4-oxadiazole provided com-
pound 4 with the highest affinity (IC50: 0.03 nM) to benzodiazepine
receptors, which is about 100 times more potent than diazepam as the
reference drug.
Next, the relationship between the time the anti-seizure activity
reaches the highest (Tmax) and calculated LogP was investigated. The
less lipophilic tautomers of 7, 11, 12 and compounds 8a, 13a, 13b, 13c,
14b were considered for correlation study. Resulting in the following
equation, a linear relationship with correlation coefficient of r2 = 0.964
was revealed.
Tm ax = (ꢀ 41.873)cLogP + 191.29
Bioisosteric replacement of 4-nitrophenyl A-ring with 3-pyridyl
moiety generated a new series of compounds (11, 12, 13a-d and 14a-
d) with high affinity, and IC50 values of 0.04–3.7 nM to BZD receptors.
Ortho-substitution of the phenoxy moiety (which is analogous to C-ring
of BZDs) with 2-chloro group and alkylation of the thio moiety on 1,2,4-
triazole ring retained the activity. In this subgroup, ethylthio (13b) and
propylthio (13c) analogues of 5-(2-phenoxy-3-pyridyl)-1,2,4-triazoles
(11 and 13a-d) and ethylthio derivative (14b) of 5-(2-(2-chlor-
ophenoxy)-3-pyridyl)-1,2,4-triazoles (12 and 14a-d) exhibited the
highest affinities with IC50 values of 0.05, 0.06 and 0.04 nM,
respectively.
Hence, a significant correlation was demonstrated between the lip-
ophilicity parameters and the onset of action of studied compounds.
Interestingly, the 1,2,4-triazole-3-thione analogues (7, 11 and 12)
having two tautomers in equilibrium with two different lipophilicity
parameters have revealed a short onset of action for the more lipophilic
tautomer and a long duration of action due to the activity of the less
lipophilic tautomer. These analogs can be considered as anti-seizure
analogs with rapid onset of action and long duration of action.
Finally, to assess the potency of each analogue, the minimum
effective dose of each compound at its Tmax was determined. For the rest
of the analogues (5, 8d, 13d, 14a, 14c, and 14d), their onset of action
was predicted using the equation which their minimum effective doses
at the predicted Tmax were assessed. According to the results, displayed
in Table 1 and Figs. 2 and 3, the compounds showed anti-seizure activity
at minimum effective doses of 0.025–0.1 mg/kg, while diazepam min-
imum effective dose is 0.025 mg/kg. Finally, among the investigated
2.2.2. The effect on clonic pentylenetetrazole-induced seizure threshold
Knowing the fact that the synthesized compounds have different
LogP and considering that the onset and duration of action of a single
bolus administration of a BZD depend on the lipid solubility of the drug,
Table 1
cLogP, receptor binding affinity (RBA), Tmax and minimum effective dose of 1,2,4-triazole-3-thiones and 2-amino-1,3,4-oxadiazole.
Compound
X
Y
RBAIC50 (nM)
Kd(nM)
cLogP
Tamax
Minimum Effective Dose (mg/kg)
(min)
7
=S/SH
SMe
SEt
–
0.24
N.D.d
0.13
0.54
0.13
0.03
7.06
0.74
N.D.
0.05
0.06
3.7
0.15
N.D.
0.07
0.35
0.08
0.02
4.26
0.45
N.D.
0.03
0.04
2.2
5.364/4.00b
4.11
1/25
10
0.1***,c
0.1**
8a
–
8b
–
4.623
1
0.05*
0.05*
0.1***
N.D.
8c
SPr
–
5.065
1
8d
SBn
–
5.898
N.D.
N.D.
N.D.
1/60
60
4
NH2
OEt
–
3.071
5
–
3.815
0.05**
0.1***
0.025*
0.025*
0.025*
0.1**
11
=S/SH
SMe
SEt
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
4.565/3.208
3.311
13a
13b
13c
13d
12
3.824
30
SPr
4.266
10
SBn
5.099
N.D.
1/35
N.D.
10
=S/SH
SMe
SEt
N.D.
0.15
0.04
0.5
N.D.
0.09
0.02
0.3
5.155/3.798
3.901
0.05**
0.05***
0.1***
0.1***
0.05*
0.025*
14a
14b
14c
14d
Diazepam
4.414
SPr
4.856
N.D.
N.D.
3.17/2.84
SBn
0.1
0.06
5.689
2.4
1.45
3.109
aTime when the highest activity is reached. The tested compounds were administered at 0.1 mg/kg, while Diazepam was administered at 0.025 mg/kg.
bLipophilicity parameter of two tautomers in equilibrium.
cStatistical analysis relative to CMC-treated mice group; *, p < 0.05; **, p < 0.001; ***, p < 0.0001.
dN.D. stands for not determined.
4

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
Fig. 2. Time Course (administered at 0.1 mg/kg) and Dose Response of anti-seizure activities of 7, 8a, 8b, 8c, 5, 11, 13a, 13b, 13c, 12 and 14b on PTZ-induced
seizure threshold. For Time-course studies of diazepam, it is administered at 0.025 mg/kg. Data are represented as mean ± SEM. Statistical analysis relative to CMC-
treated mice group; *, p < 0.05; **, p < 0.001; ***, p < 0.0001.
5

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
Fig. 3. Dose Response of anti-seizure activities of 5, 8b, 13d, 14a, 14c and 14d on PTZ-induced seizure threshold (administered at predicted Tmax using equation
[Tmax = (-41.873) cLogP + 191.29]). Data are represented as mean ± SEM. Statistical analysis relative to CMC-treated mice group; *, p < 0.05; **, p < 0.001; ***, p
< 0.0001.
analogues, 3-ethoxy-5-(4-nitro-2-phenoxyphenyl)-4H-1,2,4-triazole (5),
3-ethylthio-5-(2-phenoxy-3-pyridyl)-4H-1,2,4-triazole (13b) and 5-(2-
phenoxy-3-pyridyl)-3-propylthio-4H-1,2,4-triazole (13c) demonstrated
minimum effective dose of 0.025 mg/kg, similar to the reference drug
diazepam.
3. Molecular modeling (Docking) studies
The orientation of four potent BZD analogues, 3-ethylthio-1,2,4-tria-
zoles substituted with 4-nitro-2-phenoxyphenyl (8b), 2-phenoxypyridyl
(13b), and 2-(2-chlorophenoxy)pyridyl (14b); and 2-amino-1,3,4-oxa-
diazole compound (4) in the BZD-binding site of GABAA receptor
(6HUO) were examined using YASARA software package, version
15.7.12 [26,27]. To examine the method validity, the co-crystallized
ligands (alprazolam and diazepam in 6HUO and 6HUP, respectively)
were also docked.
For the alprazolam ligand, the conformation with the highest bind-
ing energy of + 11.43 kcal/mol matches exactly (RMSD: 0.14 Å) with
the position of the alprazolam in the co-crystallized complex (concern-
ing the sign of “binding energies”, please refer to the Experimental
section).
Fig. 4. The superimposition of Alprazolam (grey), 4 (cyan), 8b (magenta), 13b
(pink) and 14b (green). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
Similar pose of interaction was observed for diazepam ligand, with
binding energy of + 9.98 kcal/mol and RMSD of 0.31 Å, docked in co-
crystallized complex of diazepam and GABAA receptor (6HUP). In
docking of the synthesized BZD analogues, 4, 8b, 13b and 14b have
shown similar binding mode in alprazolam pocket of GABAA receptor
with binding energies of + 9.90, 9.71, 9.31 and 9.46 kcal/mol,
respectively.
4. Conclusion
A new series of non-rigid BZDs, 5-(2-aryloxyaryl)-4H-1,2,4-triazoles,
possessing C-3 thio or alkylthio substituents was synthesized and their
GABAA/benzodiazepine receptor complex binding affinity as well as the
lipophilicity dependency of their onset of anti-seizure action was stud-
ied. These compounds demonstrated similar to significantly superior
affinity towards the GABAA/benzodiazepine receptor complex, with a
significant correlation between their cLogP and onset of action. This led
us to conclude that in designing an anti-seizure candidate with appro-
priate onset and duration of action, the lipophilicity parameter is of
particular importance and should be carefully optimized. The highly
lipophilic analogues may not be suitable anti-seizure candidates, but
could be indicated for treatment of acute seizure attacks or in combi-
nation with other drugs to control the life threatening emergency states
such as status epilepticus. In addition, we suggest that the anti-seizure
assay performed at a fixed time may not be an acceptable experiment
for determination of GABAA/benzodiazepine receptor complex binding
affinity. To achieve the onset of action of a new investigational
analogue, a preliminary time-course study is necessary for determining
the relationship between the onset of action and lipophilicity of the
Fig. 4 shows the superimposed poses of structures 4, 8b, 13b, 14b,
and alprazolam, and Fig. 5 displays the important amino acids inter-
acting with the aforementioned compounds. Similar to alprazolam in co-
crystallized structure, the nitrogens in 1,2,4-triazoles and 1,3,4-oxadia-
zole interact with γ2Tyr58 and α1Ser205. 4-NH of 1,2,4-triazoles pro-
vides a hydrogen bond with γ2Phe77. In all studied compounds, the
phenoxy substituent which is a substitution for phenyl moieties in
diazepam (on C5), and alprazolam (on C6) are involved in
π-π in-
teractions with
interact with
α1Tyr210 and α1Phe100. Nitro substituents in 4 and 8b
α
1His102 and γ2Asn60 side chains, similar to chloro
substituent in alprazolam. 13b and 14b, possessing pyridine ring as a
bioisosteric replacement for nitrophenyl moiety display an interaction
between nitrogen of pyridine nucleus and α1His102 side chain.
6

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
Fig. 5. Docked poses of 4 (cyan), 8b (magenta), 13b (pink) and 14b (green) in the BZD-binding site of GABAA receptor (6HUO). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
compound.
filtered. The filtrate was acidified to obtain a yellow precipitate. The
resulting precipitate was recrystallized from ethanol/water to give 1.
Yield, 24.5%; m.p.162–164 ◦C; IR (KBr):
–
ν 2639–3381 (O H, COOH),
5. Experimental
5.1. Chemistry
1703 (C O), 1548, 1349 (NO ) cmꢀ 1; 1H NMR (CDCl3): δ 7.14 (d, J =
–
–
2
′
′
′
8.0 Hz, 2H, H2 ,6 ), 7.34 (t, J = 8.0 Hz, 1H, H4 ), 7.49 (t, J = 8.0 Hz, 2H,
′
′
H
3 ,5 ), 7.66 (d, J = 2.0 Hz, 1H, H3), 8.00 (dd, J = 2.0, 8.5 Hz, 1H, H5),
Melting points were determined using a Reichert-Jung hot-stage
microscope (Reichert-Jung, Germany) and are provided uncorrected.
Infrared spectra were recorded on a Nicolet Magna 550-FT spectrometer
(Nicolet, Madison, WI, USA). 1H NMR and 13C NMR spectra were
measured on a Bruker FT-500 MHz spectrometer (Bruker Bioscience,
USA) in CDCl3 or DMSO‑d6 and TMS as the internal standard, where J
(coupling constant) values were estimated in Hertz. Spin multiples are
given as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), b
(broad), dd (doublet of doublets), and td (triplet of doublets). Elemental
microanalyses were carried out with a Perkin-Elmer 240-C apparatus
(Perkin-Elmer, Beaconsfield, UK) and were within ± 0.4% of the theo-
retical values for C, H and N. All solvents and reagents were purchased
from Fluka, Acros, Sigma-Aldrich, and Merck Chemical Company.
8.31 (d, J = 8.5 Hz, 1H, H6), 10.73 (bs, 1H, H, COOH).
Anal. Calcd for C13H9NO5: C, 60.24; H, 3.05; N, 5.40. Found: C,
60.16; H, 3.28; N, 5.26.
5.1.1.1. Ethyl 4-nitro-2-phenoxybenzoate (2). To a stirred solution of 1
(1.03 g, 4 mmol) in dry ethanol (50 mL) was added sulfuric acid (0.5 mL)
and the reaction mixture was refluxed for 12 h. Then, the volatiles were
evaporated under reduced pressure, water (50 mL) was added to the
residue and the aqueous layer was extracted with ethyl acetate (3 × 50
mL). The organic layer was washed with saturated solution of sodium
bicarbonate and brine, then dried over anhydrous sodium sulfate. The
solvent was evaporated under reduced pressure to furnish 2 as a yellow
liquid. Yield, 87%; IR (KBr):
ν
1731 (C O), 1531, 1351 (NO ) cmꢀ 1; 1H
–
–
2
NMR (CDCl3): δ 1.30 (t, J = 7.0 Hz, 3H, CH3), 4.35 (q, J = 7.0 Hz, 2H,
5.1.1. 4-Nitro-2-phenoxybenzoic acid (1)
′
′
′
CH2), 7.03 (d, J = 8.0 Hz, 2H, H2 ,6 ), 7.20 (t, J = 8.0 Hz, 1H, H4 ), 7.40 (t,
To the solution of sodium (1.15 g, 50 mmol) dissolved in dry
methanol (50 mL) was added 2-chloro-4-nitrobenzoic acid (4.30 g, 20
mmol) and phenol (3.72 g, 40 mmol) and the mixture was stirred for 20
min at room temperature. Methanol was evaporated under reduced
pressure and dry THF was added to the residue twice and the solvent was
evaporated under reduced pressure. To the residue was added dry DMF
(50 mL) and copper powder and the mixture was refluxed for 16 h. Then,
1 L cold water was added to the reaction mixture, which was then
′
′
J = 8.0 Hz, 2H, H3 ,5 ), 7.74 (s, 1H, H3), 7.97 (d, J = 8.5 Hz, 1H, H5), 8.00
(d, J = 8.5 Hz, 1H, H6).
Anal. Calcd for C15H13NO5: C, 62.72; H, 4.56; N, 4.88. Found: C,
62.57; H, 4.43; N, 4.65.
5.1.1.2. 4-Nitro-2-phenoxybenzohydrazide (3). To a solution of 2 (0.862
g, 3 mmol) in ethanol (10 mL) was added hydrazine hydrate (0.75 mL,
15 mmol) and the reaction mixture was stirred for 24 h at room
7

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
temperature. The resulting yellow precipitate was filtered, washed with
5.1.1.5.2. 5-(2-Phenoxypyridin-3-yl)-2,4H-1,2,4-triazol-3-thione
◦
–
water, dried and recrystallized from ethanol/water to give 3. Yield,
(11). Yield, 46.5%; m.p. 233–236 C; IR (KBr):
ν
3419, 3088 (N H)
82%; m.p. 187–188 C; IR (KBr):
ν
cmꢀ 1; 1H NMR (DMSO‑d6): δ 7.20 (d, J = 7.5 Hz, 2H, H2 ,6 ), 7.25 (t, J =
◦
–
–
3350, 3061 (N H), 1691 (C O),
–
′
′
1543, 1344 (NO2) cmꢀ 1; H NMR (CDCl3): δ 7.14 (d, J = 7.5 Hz, 2H,
7.5 Hz, 1H, H4 ), 7.27 (dd, J = 5.0, 7.5 Hz, 1H, H5), 7.42 (t, J = 7.5 Hz,
1
′
′
′
′
′
′
′
′
H2 ,6 ), 7.35 (t, J = 7.5 Hz, 1H, H4 ), 7.50 (t, J = 7.5 Hz, 2H, H3 ,5 ), 7.65
(d, J = 2.0 Hz, 1H, H3), 8.03 (dd, J = 2.0, 9.0 Hz, 1H, H5), 8.59 (d, J =
9.0 Hz, 1H, H6), 10.44 (bs, 1H, NH).
2H, H3 ,5 ), 8.21 (dd, J = 2.0, 5.0 Hz, 1H, H6), 8.43 (dd, J = 2.0, 7.5 Hz,
1H, H4); 13C NMR (CDCl3): δ 114.81, 118.48, 121.91, 124.85, 127.32,
129.07, 138.54, 148.81, 152.92, 159.36, 166.71.
Anal. Calcd for C13H11N3O4: C, 57.14; H, 4.06; N, 15.38. Found: C,
56.96; H, 3.84; N, 15.04.
Anal. Calcd for C13H10N4OS: C, 57.76; H, 3.73; N, 20.73. Found: C,
57.60; H, 3.62; N, 20.54.
5.1.1.5.3. 5-(2-(2-Chlorophenoxy)pyridin-3-yl)-2,4H-1,2,4-triazole-3-
◦
thione (12). Yield, 49.26%; m.p. 247–249 C; IR (KBr):
ν 3427, 3088
5.1.1.3. 5-(4-Nitro-2-phenoxyphenyl)-1,3,4-oxadiazol-2-amine (4). To a
solution of 3 (0.683 g, 2.5 mmol) in 1,4-dioxane (5 mL) was added so-
dium bicarbonate (0.105 g, 1.25 mmol) in water (10 mL) and the
mixture was stirred for 5 min at room temperature. Subsequently,
cyanogen bromide (0.268 g, 2.5 mmol) was added and it was stirred for
an additional 2 h until a yellow precipitate was obtained. The precipitate
(N H) cmꢀ 1; 1H NMR (CDCl3): δ 7.18 (dd, J = 4.5, 7.5 Hz, 1H, H5), 7.27
–
′
′
(dd, J = 7.5, 2.0 Hz, 1H, H4 ), 7.31 (dd, J = 1.5, 7.5 Hz, 1H, H6 ), 7.38
′
′
(dt, J = 1.5, 7.5 Hz, 1H, H5 ), 7.51 (dd, J = 1.5, 7.5 Hz, 1H, H3 ), 8.19
(dd, J = 1.5, 4.5 Hz, 1H, H6), 8.26 (dd, J = 2.0, 7.5 Hz, 1H, H4); 13C NMR
(CDCl3): δ 116.52, 118.77, 124.54, 126.29, 126.92, 127.52, 129.89,
138.37, 148.65, 148.84, 159.66, 162.40, 166.91.
was filtered, washed with water and recrystallized from ethanol to give
◦
–
Anal. Calcd for C13H9ClN4OS: C, 51.23; H, 2.98; N, 18.38. Found: C,
51.02; H, 2.81; N, 18.25.
4. Yield, 95%, m.p. > 250 C; IR (KBr): 3446, 3320, 3083 (N H), 1547,
1343 (NO2) cmꢀ 1; 1H NMR (DMSO‑d6): 7.17 (d, J = 8.0 Hz, 2H, H2 ,6 ),
′
′
′
7.27 (t, J = 8.0 Hz, 1H, H4 ), 7.42 (bs, 2H, NH2), 7.48 (t, J = 8.0 Hz, 2H,
′
′
H
3 ,5 ), 7.64 (d, J = 2.0 Hz, 1H, H3), 8.10 (dd, J = 2.0, 8.5 Hz, 1H, H5),
5.1.1.6. General procedure for synthesis of 3-alkylthio-5-substituted-4H-
1,2,4-triazole. To a solution of triazole-3-thione (7, 11 and 12, 0.5
mmol) in ethanol (5 mL) and aqueous solution of sodium hydroxide
(10%, 1.2 mL, 0.75 mmol) was added alkyl halide (0.6 mmol). The
mixture was sonicated for 5–10 min. Then, to the reaction mixture was
added water, the aqueous layer was extracted with dichloromethane and
dried over sodium sulfate. The solvent was evaporated under reduced
pressure and the residue was purified by preparative TLC (hexane:
EtOAC, 4:1 for 7 and hexane:EtOAc, 9:1 for 11 and 12).
8.13 (d, J = 8.5 Hz, 1H, H6).
Anal. Calcd for C14H10N4O4: C, 56.38; H, 3.38; N, 18.78. Found: C,
56.22; H, 3.19; N, 18.65.
5.1.1.4. 3-Ethoxy-5-(4-nitro-2-phenoxyphenyl)-4H-1,2,4-triazole (5). To
a solution of 4 (0.507 g, 1.7 mmol) in ethanol (20 mL) was added po-
tassium hydroxide (0.392 g, 7 mmol) and the mixture was refluxed for
20 h. After cooling, the solvent was evaporated under reduced pressure.
Water was added to the residue and the mixture was neutralized with
hydrochloric acid until an orange precipitate was formed. The precipi-
tate was filtered, washed with water, dried and purified by column
chromatography (hexane/EtOAc, 9:1) to produce yellow crystals of 5.
5.1.1.6.1. 3-(4-Nitro-2-phenoxyphenyl)-5-methylthio-4H-1,2,4-tri-
azole (8a). Yield, 52%; m.p. 222–224 ◦C; IR (KBr):
ν 3259 (N H), 1523,
–
1346 (NO2) cmꢀ 1; 1H NMR (CDCl3): δ 2.68 (s, 3H, CH3), 7.17 (d, J = 7.5
′
′
′
Hz, 2H, H2 ,6 ), 7.37 (t, J = 7.5 Hz, 1H, H4 ), 7.52 (t, J = 7.5 Hz, 2H,
Yield, 17%; m.p. 198–200 ◦C; IR (KBr):
′
′
–
ν 3262 (N H), 1541, 1342
H3 ,5 ), 7.66 (d, J = 2.0 Hz, 1H, H3), 8.04 (dd, J = 2.0, 8.5 Hz, 1H, H5),
8.57 (d, J = 8.5 Hz, 1H, H6); 13C NMR (CDCl3): δ 14.29, 111.55, 117.39,
120.66, 121.98, 126.64, 129.98, 130.75, 148.46, 151.55, 153.50,
155.89.
(NO2) cmꢀ 1; 1H NMR (CDCl3): δ 1.47 (t, J = 7.0 Hz, 3H, CH3), 4.43 (q, J
′
′
= 7.0 Hz, 2H, CH2), 7.18 (d, J = 7.5 Hz, 2H, H2 ,6 ), 7.37 (t, J = 7.5 Hz,
′
′
′
1H, H4 ), 7.52 (t, J = 7.5 Hz, 2H, H3 ,5 ), 7.65 (d, J = 2.0 Hz, 1H, H3), 8.04
(dd, J = 2.0, 8.5 Hz, 1H, H5), 8.51 (d, J = 8.5 Hz, 1H, H6); 13C NMR
(CDCl3): δ 14.66, 65.83, 111.49, 118.11, 120.47, 120.63, 126.62,
130.57, 130.88, 149.06, 152.28, 153.50, 156.09.
Anal. Calcd for C15H12N4O3S: C, 54.87; H, 3.68; N, 17.06. Found: C,
54.69; H, 3.54; N, 16.93.
5.1.1.6.2. 3-Ethylthio-5-(4-nitro-2-phenoxyphenyl)-4H-1,2,4-triazole
(8b). Yield, 48%; m.p.184–185 ◦C; IR (KBr):
ν 3416 (N H), 1519, 1345
–
Anal. Calcd for C16H14N4O4: C, 58.89; H, 4.32; N, 17.17. Found: C,
58.76; H, 4.21; N, 17.05.
(NO2) cmꢀ 1; 1H NMR (CDCl3): δ 1.45 (t, J = 7.5 Hz, 3H, CH3), 3.21 (q, J
′
′
= 7.5 Hz, 2H, CH₂), 7.17 (d, J = 7.5 Hz, 2H, H2 ,6 ), 7.37 (t, J = 7.5 Hz,
′
′
′
1H, H4 ), 7.51 (t, J = 7.5 Hz, 2H, H3 ,5 ), 7.65 (s, 1H, H3), 8.03 (d, J = 8.5
Hz, 1H, H5), 8.57 (d, J = 8.5 Hz, 1H, H6); 13C NMR (CDCl3): δ 15.03,
26.45, 111.52, 118.08, 120.65, 121.95, 126.65, 130.72, 130.90, 149.23,
151.63, 153.44, 155.91.
5.1.1.5. General procedure for synthesis of 5-substituted-2,4H-1,2,4-tri-
azole-3-thiones. To the acid (1, 9 and 10, 5 mmol) was added thionyl
chloride (5 mL) and the reaction mixture was refluxed for 2 h. Then, the
volatile was evaporated under reduced pressure. The resulting acyl
chloride was dissolved in dry THF (5 mL) and add dropwise to the ice-
cold suspension of thiosemicarbazide (0.60 g, 8 mmol) in dry THF (5
mL). The mixture was stirred for 1 h at 0 ◦C and then overnight at room
temperature. The resulting precipitate was filtered, washed with water
and suspended in aqueous solution of sodium hydroxide (5%, 20 mL)
and refluxed for 6 h (for 9 and 10, 12 h). After cooling, the solution was
neutralized with acetic acid to reach a pH = 5–7 and the resulting
precipitate was filtered, washed with water, dried and purified by col-
umn chromatography (hexane:EtOAc, 1:1) to give 7, 11 and 12.
5.1.1.5.1. 5-(4-Nitro-2-phenoxyphenyl)-2,4H-1,2,4-triazole-3-thione
Anal. Calcd for C16H14N4O3S: C, 56.13; H, 4.12; N, 16.36. Found: C,
55.98; H, 3.94; N, 16.20.
5.1.1.6.3. 3-(4-Nitro-2-phenoxyphenyl)-5-propylthio-4H-1,2,4-triazole
(8c). Yield, 35%; m.p. 180–182 ◦C; IR (KBr):
ν 3264 (N H), 1525, 1341
–
(NO2) cmꢀ 1; 1H NMR (CDCl3): δ 1.06 (t, J = 7.0 Hz, 3H, CH3), 1.81 (m,
′
′
2H, CH2), 3.18 (t, J = 7.0 Hz, 2H, SCH2), 7.17 (d, J = 7.5 Hz, 2H, H2 ,6 ),
′
′
′
7.36 (t, J = 7.5 Hz, 1H, H4 ), 7.51 (t, J = 7.5 Hz, 2H, H3 ,5 ), 7.65 (d, J =
1.5 Hz, 1H, H3), 8.03 (dd, J = 1.5, 8.5 Hz, 1H, H5), 8.56 (d, J = 8.5 Hz,
1H, H6), 11.75 (bs, 1H, NH); 13C NMR (CDCl3): δ 13.34, 23.09, 34.09,
111.53, 118.08, 120.64, 121.97, 126.63, 130.72, 130.89, 149.22,
151.60, 153.45, 155.90.
(7). Yield, 64%; m.p. 243–245 ◦C; IR (KBr):
ν 3302, 3104 (N H), 1529,
–
1348 (NO2) cmꢀ 1; 1H NMR (CDCl3): δ 7.19 (d, J = 7.5 Hz, 2H, H2 ,6 ),
Anal. Calcd for C17H16N4O3S: C, 57.29; H, 4.52; N, 15.72. Found: C,
57.14; H, 4.37; N, 15.58.
′
′
′
′
′
7.38 (t, J = 7.5 Hz, 1H, H4 ), 7.53 (t, J = 7.5 Hz, 2H, H3 ,5 ), 7.68 (d, J =
2.0 Hz, 1H, H3), 8.07 (dd, J = 2.0, 8.5 Hz, 1H, H5), 8.12 (bs, 1H, NH),
8.60 (d, J = 8.5 Hz, 1H, H6).
5.1.1.6.4. 3-Benzylthio-5-(4-nitro-2-phenoxyphenyl)-4H-1,2,4-triazole
(8d). Yield, 14%; m.p. 182–184 ◦C; IR (KBr):
–
ν 3413 (N H), 1521,
1344 (NO2) cmꢀ 1; 1H NMR (CDCl3): δ 4.44 (s, 2H, CH2), 7.17 (d, J = 8.0
Anal. Calcd for C14H10N4O3S: C, 53.50; H, 3.21; N, 17.82. Found: C,
53.31; H, 3.14; N, 17.67.
′
′
′′
Hz, 2H, H2 ,6 ), 7.25 (t, J = 7.5 Hz, 1H, H4 ), 7.31 (t, J = 7.5 Hz, 2H,
′′ ′′
′
′′ ′′
H
3 ,5 ), 7.37 (t, J = 8.0 Hz, 1H, H4 ), 7.45 (d, J = 7.5 Hz, 2H, H2 ,6 ), 7.52
8

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
′
′
(t, J = 8.0 Hz, 2H, H3 ,5 ), 7.65 (d, J = 2.0 Hz, 1H, H3), 8.04 (dd, J = 2.0,
8.0 Hz, 1H, H5), 8.57 (d, J = 8.0 Hz, 1H, H6); 13C NMR (CDCl3): δ 36.38,
111.46, 118.08, 120.71, 121.83, 126.70, 127.40, 128.51, 128.96,
130.70, 130.91, 137.40, 149.27, 151.63, 153.37, 155.96.
Anal. Calcd for C21H16N4O3S: C, 62.36; H, 3.99; N, 13.85. Found: C,
62.20; H, 3.81; N, 13.74.
54.02; H, 3.76; N, 16.72.
5.1.1.6.11. 5-(2-(2-Chlorophenoxy)-3-pyridyl)-3-propylthio-4H-1,2,4-
◦
–
ν 3195 (N H)
triazole (14c). Yield, 46%; m.p. 181–184 C; IR (KBr):
cmꢀ 1; 1H NMR (CDCl3): δ 1.07 (t, J = 7.5 Hz, 3H, CH3), 1.83 (m, 2H,
CH2), 3.18 (t, J = 7.5 Hz, 2H, SCH2), 7.20 (dd, J = 5.0, 8.0 Hz, 1H, H5),
′
′
7.30 (dt, J = 2.0, 8.0 Hz, 1H, H4 ), 7.36 (dd, J = 2.0, 8.0 Hz, 1H, H6 ),
′
′
5.1.1.6.5. 3-Methylthio-5-(2-phenoxy-3-pyridyl)-4H-1,2,4-triazole
7.39 (dt, J = 2.0, 8.0 Hz, 1H, H5 ), 7.53 (dd, J = 2.0, 8.0 Hz, 1H, H3 ),
8.17 (dd, J = 2.0, 5.0 Hz, 1H, H6), 8.67 (dd, J = 2.0, 8.0 Hz, 1H, H4),
11.63 (bs, 1H, NH); 13C NMR (CDCl3): δ 13.37, 23.11, 34.12, 110.55,
119.68, 124.36, 127.07, 127.99, 130.61, 139.15, 148.36, 148.65,
151.40, 151.44, 158.75, 161.74.
(13a). Yield, 42% ; m.p.196–198 ◦C; IR (KBr):
ν
3200 (N H) cmꢀ 1; 1H
–
′
′
NMR (CDCl3): δ 2.66 (s, 3H, CH3), 7.18 (m, 3H, H4,2 ,6 ), 7.30 (t, J = 7.5
′
′
′
Hz, 1H, H4 ), 7.46 (t, J = 7.5 Hz, 2H, H3 ,5 ), 8.20 (dd, J = 2.0, 5.0 Hz, 1H,
H6), 8.67 (dd, J = 2.0, 7.5 Hz, 1H, H4), 11.83 (bs, 1H, NH); 13C NMR
(CDCl3): δ 14.58, 110.81, 119.37, 121.92, 125.96, 129.88, 138.92,
148.93, 151.73, 152.36, 159.71.
Anal. Calcd for C16H15ClN4OS: C, 55.41; H, 4.36; N, 16.15. Found: C,
55.27; H, 4.23; N, 16.02.
Anal. Calcd for C14H12N4OS: C, 59.14; H, 4.25; N, 19.70. Found: C,
59.02; H, 4.09; N, 19.55.
5.1.1.6.12. 3-Benzylthio-5-(2-(2-chlorophenoxy)-3-pyridyl)-4H-1,2,4-
triazole (14d). Yield, 27%; m.p. 146–148 ◦C; IR (KBr):
–
ν 3233 (N H)
5.1.1.6.6. 3-Ethylthio-5-(2-phenoxy-3-pyridyl)-4H-1,2,4-triazole
cmꢀ 1; 1H NMR (CDCl3): δ 4.44 (s, 2H, CH2), 7.19 (dd, J = 7.5, 8.0 Hz,
(13b). Yield, 45%; m.p. 164–166 ◦C; IR (KBr):
ν
3414 (N H) cmꢀ 1; 1H
1H, H5), 7.23–7.39 (m, 6H, H3 -5 ;4 -6 ), 7.45 (d, J = 7.0 Hz, 2H, H2 ,6 ),
–
′′ ′′
′
′
′′ ′′
′
NMR (CDCl3): δ 1.45 (t, J = 7.0 Hz, 3H, CH3), 3.20 (q, J = 7.0 Hz, 2H,
7.51 (dd, J = 1.5, 8.0 Hz, 1H, H3 ), 8.17 (dd, J = 2.0, 5.0 Hz, 1H, H6),
CH2), 7.16 (dd, J = 5.0, 7.5 Hz, 1H, H5), 7.19 (d, J = 8.0 Hz, 2H, H2 ,6 ),
8.69 (dd, J = 2.0, 8.0 Hz, 1H, H4), 11.85 (bs, 1H, NH); 13C NMR (CDCl3):
δ 36.45, 110.52, 119.71, 124.40, 127.11, 127.33, 128.01, 128.50,
129.01, 130.61, 137.54, 139.14, 148.36, 148.72, 151.47, 158.77,
161.19.
′
′
′
′
′
7.30 (t, J = 8.0 Hz, 1H, H4 ), 7.46 (t, J = 8.0 Hz, 2H, H3 ,5 ), 8.19 (dd, J =
1.5, 5.0 Hz, 1H, H6), 8.68 (dd, J = 1.5, 7.5 Hz, 1H, H4), 11.68 (bs, 1H,
NH); 13C NMR (CDCl3): δ 15.04, 26.46, 110.79, 119.34, 121.90, 125.94,
129.90, 138.90, 148.91, 151.76, 152.35, 159.73.
Anal. Calcd for C20H15ClN4OS: C, 60.83; H, 3.83; N, 14.19. Found: C,
60.83; H, 3.64; N, 14.02.
Anal. Calcd for C15H14N4OS: C, 60.38; H, 4.73; N, 18.78. Found: C,
60.21; H, 4.59; N, 18.61.
5.1.1.6.7. 5-(2-Phenoxy-3-pyridyl)-
3-propylthio-4H-1,2,4-triazole
5.2. Radioligand receptor binding assays
ꢀ 1
(13c). Yield, 37%; m.p. 138–141 ◦C; IR (KBr):
ν
3440 (N H) cm
;
–
1H NMR (CDCl3): δ 1.05 (t, J = 7.5 Hz, 3H, CH3), 1.82 (m, 2H, CH2), 3.17
5.2.1. Membrane preparation
(t, J = 7.5 Hz, 2H, SCH2), 7.17 (dd, J = 5.0, 8.0 Hz, 1H, H5), 7.19 (d, J =
The radioligand receptor binding studies were performed as previ-
ously described [21,23]. Briefly, Male Sprague–Dawley rats weighing
300–350 g (Pasteur Institute, Tehran, Iran) were anesthetized and
sacrificed. Their cortical membrane tissue was removed and homoge-
nized in 20 mL ice-cold Tris–HCl buffer (30 mM, pH 7.4) at medium
speed. The homogenates were centrifuged for 10 min at 600 g and the
resulting supernatant was centrifuged at 27,000g for 15 min. Ice-cold
buffer was used to wash the pellet 3 times by re-suspension and re-
centrifugation. After suspending the washed pellet in 20 mL buffer, in-
cubation was done at 37 ◦C for 30 min followed by centrifugation for 10
min at 27,000g. The pellet was washed once, and the final pellet was re-
suspended in 30 mL Tris–HCl buffer (50 mM, pH 7.4). All centrifugation
were performed at 4 ◦C. The concentration of protein was determined in
the membrane preparation by Bradford method using bovine serum al-
bumin (BSA) as a standard. The membrane preparation was stored at
ꢀ 20 ◦C until it was used 1–15 days later.
′
′
′
7.5 Hz, 2H, H2 ,6 ), 7.31 (t, J = 7.5 Hz, 1H, H4 ), 7.47 (t, J = 7.5 Hz, 2H,
′
′
H
3 ,5 ), 8.20 (dd, J = 2.0, 5.0 Hz, 1H, H6), 8.68 (dd, J = 2.0, 8.0 Hz, 1H,
H4); 13C NMR (CDCl3): δ 13.37, 23.12, 34.13, 110.79, 119.34, 121.92,
125.91, 129.84, 138.94, 148.86, 151.24, 151.62, 152.38, 159.70.
Anal. Calcd for C16H16N4OS: C, 61.52; H, 5.16; N, 17.93. Found: C,
61.38; H, 5.02; N, 17.78.
5.1.1.6.8. 3-Benzylthio-5-(2-phenoxy-3-pyridyl)-4H-1,2,4-triazole
(13d). Yield, 39%; m.p. 154–156 ◦C; IR (KBr):
ν
3193 (N H) cmꢀ 1; 1H
–
′
′
′′
NMR (DMSO‑d6): δ 4.42 (s, 2H, CH2), 7.16–7.18 (m, 4H, H5,2 ,6 , 4 ),
′′ ′′
′
′
′
′′ ′′
7.23–7.30 (m, 3H, H3 ,5 ,4 ), 7.42–7.45 (m, 4H, H3 ,5 ,2 ,6 ), 8.18 (dd, J =
1.5, 4.5 Hz, 1H, H6), 8.66 (dd, J = 1.5, 7.5 Hz, 1H, H4), 11.85 (bs, 1H,
NH); 13C NMR (CDCl3): δ 36.43, 110.70, 119.37, 121.96, 125.97,
127.32, 128.49, 128.98, 129.88, 137.57, 138.92, 148.95, 151.69,
152.35, 159.72.
Anal. Calcd for C20H16N4OS: C, 66.65; H, 4.47; N, 15.54. Found: C,
66.49; H, 4.35; N, 15.39.
5.1.1.6.9. 5-(2-(2-Chlorophenoxy)-3-pyridyl)-3-methylthio-4H-1,2,4-
5.2.2. Saturation studies
◦
To study the saturation binding of [3H]-flumazenil, seven different
concentrations of [3H]-flumazenil (ranging from 0.05 nM to 0.97 nM)
were used. The receptor binding affinity of [3H]-flumazenil (Kd) and the
BZD receptor density (Bmax) were determined by the amount of radio-
ligand required to saturate the receptors based on non-linear regression
analysis of the saturation curve data. The total binding (TB) (receptor +
radioligand) is the amount of binding of the radioligand in the absence
of non-radioactive ligand (diazepam). To determine the non-specific
–
ν 3190 (N H)
triazole (14a). Yield, 56%; m.p. 182–184 C; IR (KBr):
cmꢀ 1; 1H NMR (CDCl3): δ 2.68 (s, 3H, CH3), 7.20 (dd, J = 5.0, 7.5 Hz,
′
1H, H5), 7.28 (dt, J = 1.5, 7.5 Hz, 1H, H4 ), 7.36 (dd, J = 2.0, 7.5 Hz, 1H,
′
′
H6 ), 7.39 (dt, J = 2.0, 7.5 Hz, 1H, H5 ), 7.53 (dd, J = 2.5, 7.5 Hz, 1H,
′
H3 ), 8.18 (dd, J = 2.0, 5.0 Hz, 1H, H6), 8.70 (dd, J = 2.0, 7.5 Hz, 1H,
H4), 11.66 (bs, 1H, NH); 13C NMR (CDCl3): δ 14.58, 110.51, 119.69,
124.38, 127.08, 127.99, 130.60, 139.14, 148.36, 148.69, 151.51,
158.76, 162.33.
Anal. Calcd for C14H11ClN4OS: C, 52.75; H, 3.48; N, 17.58. Found: C,
52.63; H, 3.29; N, 17.44.
binding (NSB) of the radioligand, a large excess of diazepam (100 μM)
was used in parallel assays as the control experiment (receptor + radi-
oligand + excess of diazepam). Specific binding (SB) was measured at
various concentrations of radioligand by subtracting NSB from the TB
[21,23].
5.1.1.6.10. 5-(2-(2-Chlorophenoxy)-3-pyridyl)-3-ethylthio-4H-1,2,4-
◦
–
ν 3153 (N H)
triazole (14b). Yield, 52%; m.p. 198–199 C; IR (KBr):
cmꢀ 1; 1H NMR (CDCl3): δ 1.46 (t, J = 7.5 Hz, 3H, CH3), 3.21 (q, J = 7.5
Hz, 2H, CH2), 7.20 (dd, J = 5.0, 7.5 Hz, 1H, H5), 7.28 (dt, J = 1.5, 7.5 Hz,
′
′
1H, H4 ), 7.36 (dd, J = 1.5, 7.5 Hz, 1H, H6 ), 7.39 (dt, J = 1.5, 7.5 Hz, 1H,
5.2.3. Competition binding studies
′
′
H5 ), 7.53 (dd, J = 1.5, 7.5 Hz, 1H, H3 ), 8.17 (dd, J = 2.0, 5.0 Hz, 1H,
H6), 8.70 (dd, J = 2.0, 7.5 Hz, 1H, H4), 11.68 (bs, 1H, NH); 13C NMR
(CDCl3): δ 15.06, 26.47, 110.55, 119.68, 124.38, 127.08, 127.99,
130.60, 139.17, 148.36, 148.67, 151.45, 151.47, 158.76, 161.47.
Anal. Calcd for C15H13ClN4OS: C, 54.13; H, 3.94; N, 16.83. Found: C,
100 μg of the frozen membrane protein was thawed and resuspended
in Tris–HCl buffer (50 mM, pH 7.4) and incubated with 8.6 × 10-5 nmole
[3H]-flumazenil and increasing concentrations of newly synthesized li-
gands in a final volume 0.5 mL at 30 ◦C for 35 min. After incubation, the
assay was terminated by centrifugation at 1500 g for 5 min at 4 ◦C. The
9

L. Navidpour et al.
Bioorganic Chemistry 106 (2021) 104504
concentration of non-radioactive ligand that inhibits the specific binding
of [3H]-flumazenil (SB) by 50% is defined as IC50 value [21,23].
Acknowledgments
This work was supported by a grant from Research Council of Tehran
University of Medical Sciences (grant No: 94-04-33-31018).
5.2.4. Data analysis
All experiments were done in triplicates. The binding parameters of
[3H]-flumazenil were calculated from non-linear regression analysis of
the saturation curve data by using the activity base software package
(Program Prism, Graph Pad, San Diego, CA).
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Declaration of Competing Interest
The authors declare that they have no known competing financial
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11