Novel agonists of benzodiazepine 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

Article abstract of DOI:10.1016/j.bmc.2014.12.016

Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical practice. In spite of their benefits they have several side effects, so synthesis of new agonists of these receptors to get more specific effect and better profile

Full text of DOI:10.1016/j.bmc.2014.12.016

Accepted Manuscript
Novel agonists of benzodiazepine 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
Mehrdad Faizi, Sara Dabirian, Hamed Tajali, Fatemeh Ahmadi, Elham Rezaee
Zavareh, Soraya Shahhosseini, Sayyed Abbas Tabatabai
PII:
S0968-0896(14)00864-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.016
BMC 11948
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 October 2014
10 December 2014
11 December 2014
Please cite this article as: Faizi, M., Dabirian, S., Tajali, H., Ahmadi, F., Zavareh, E.R., Shahhosseini, S., Tabatabai,
S.A., Novel agonists of benzodiazepine 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, Bioorganic & Medicinal Chemistry
(2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.12.016
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Novel agonists of benzodiazepine 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
Mehrdad Faizia, Sara Dabirianb, Hamed Tajalia, Fatemeh Ahmadib, Elham Rezaee Zavarehb
Soraya Shahhosseinib, Sayyed Abbas Tabatabaib,*
a)
Department of Pharmacology and Toxicology, School of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
b) Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University
of Medical Sciences, Tehran, Iran
Corresponding author: Sayyed Abbas Tabatabai*
Tel: 00982188200093
Fax: 00982188665341
Email: sa_tabatabai@sbmu.ac.ir
Keywords: Synthesis, Benzodiazepine agonists, Binding assay, Hypnotic activity.
Abstract
Agonists of benzodiazepine (BZD) binding site in GABA receptors are widely used in clinical
practice. In spite of their benefits they have several side effects, so synthesis of new agonists
of these receptors to get more specific effect and better profile of adverse drug reactions is
still continued. Novel BZD agonists were designed based on the pharmacophore/receptor
model of BZD binding site of GABAA receptor. Energy minima conformers of the designed
compounds and estazolam, a known BZD receptor agonist, were well superimposed in
conformational analysis. Docking studies revealed that the carbonyl group of the compound
4c ,3-(2-chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one, was
near the nitrogen moiety of triazole ring of estazolam providing the hydrogen bond acceptor
in proper direction in the BDZ-binding site of GABAA receptor model (α1β2ϒ2). The
designed compounds were synthesized and their in vitro affinity for the central BZD receptor
was determined. Most of the novel compounds had better affinity for the BZD site of action
1

on GABAA receptor complex than diazepam. Finally, the novel compound 4c with the best
affinity in radioligand receptor binding assay (Ki=0.42 nM and IC50= 0.68 nM) was selected
as candidate for in vivo evaluation. This compound showed significant hypnotic activity and
weak anticonvulsant effect with no impairment on learning and memory performance in
mouse. The pharmacological effects of the compound 4c were antagonized by flumazenil, a
BZD antagonist, which confirms the involvement of BZD receptors in the biological effects
of the novel ligand.
1. Introduction
Agonists of benzodiazepine (BZD) receptor, are extensively used in the treatment of epilepsy,
anxiety, muscle cramps and sleep disorders.1 The pharmacological effects of BZDs result
from their affinity for a specific binding site on the GABAA receptors, known as the BZD
receptor.2-4 BZD receptor agonists modulate the allosteric GABA binding site and increase
the opening of a selective chloride ion channel that causes hyperpolarization of the post-
synaptic membrane.1 Since BZDs have some adverse effects, 5 development of the novel BZD
receptor agonists is still continued and a great deal of attention. Among all pharmacophores
proposed for binding to the BZD receptors, an aromatic ring and a coplanar proton-accepting
group in suitable distance are essential for interaction with the receptor. In addition, binding
to the receptor could be potentiated by second out-of-plane aromatic ring.6, 7 According to this
structure-activity relationship (SAR) and in continuance of our previous studies on some
simple non-rigid derivatives with five member heterocycle rings such as triazoles,
6, 8-16
oxadiazoles, and thiadiazoles,
We designed some structures with all suggested
requirements (Fig. 1) and performed conformational analysis followed by superimposition of
energy minima conformers of the designed compounds on estazolam, a known BZD agonist
to confirm whether they could mimic the structure of a BZD agonist. The synthesized
compounds were evaluated for their in vitro affinity to BZD receptor by radioligand receptor
binding assay. The compound, which had the best affinity to BZD receptor, was studied in
vivo for its pharmacological effects. The anticonvulsant action was determined using
pentylenetetrazole (PTZ)-induced lethal convulsion and maximal electroshock (MES) tests.
Mouse learning and memory impairment was evaluated by passive avoidance test and the
hypnotic effect was measured through potentiating of pentobarbital sleeping time model.
Finally, to confirm the mode of action of the synthesized compound, the effect of flumazenil,
2

a BZD receptor antagonist, on the pharmacological activity of the compounds was
determined.
2. Results and discussion
2.1. Conformational analysis
Conformational analysis of the designed compounds and estazolam were performed through
MMX force field method followed by AM1 calculation. Figure 2 represents the
superimposition of the energy minima conformer of the compound 4c on estazolam as a
known BZD agonist. Obviously, the main BZD pharmacophores, aromatic rings and proton
accepting groups are in the same orientation which means the designed compound could well
mimic the agonistic shape of BZD ligands.
2.2. Chemistry
The designed compounds were synthesized according to scheme l. Methyl benzoate was
reacted with hydrazine hydrate in methanol at room temperature to give corresponding
hydrazide 16 which was treated with cyanogen bromide to close the 1,3,4-oxadiazole ring
,compound 2, with 62% yield.6 Compounds 4a-4c were prepared in acceptable yield by the
reaction of 2 with ethyl acetoacetate to afford the intermediate 3 followed by treatment with
proper substituted amine.17 Finally, compounds 5a-5c were synthesized through the reaction
of the 4a-4c with hydrazine hydrate in good yields.18
2.3. Affinity for the central BZD receptors
According to the Table 1, all of the synthesized compounds except 5b showed more affinity
for BZD binding site in rat brain than diazepam, a classic central BZD receptor (CBR)
agonist. In triazolopyrimidinone series, the most potent analogue is the compound 4c having
chloro substituent on 2 position of the benzyl group with Ki and IC50 values of 0.42 nM and
0.68 nM respectively. This result is in agreement with SAR of BZD receptor ligands.19
However in 1,2,4-triazole series, unsubstituted analogue 5a showed the best affinity for the
CBR with Ki value of 0.53 nM and IC50 value of 0.87. This difference between two series may
be due to small dissimilarity of the benzyl group orientation in receptor binding site.
2.4. Molecular modeling (docking) studies
Interaction of the most potent BZD ligand 4c, 3-(2-chlorobenzyl)-5-methyl-2-phenyl-
[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one,with amino acids of diazepam-binding pocket of
3

GABAA receptor (α1β2ϒ2) and the orientation of this ligand along with estazolam in the
BZD-binding site that examined by a flexible docking experiment using AutoDock 4.2.3
software were observed in Figure 3(a, b). It is obvious that the carbonyl group of the 4c was
in the same direction of the nitrogen moiety of estazolam for hydrogen binding with α1
Thr206 and α1 Tyr209 and also the phenyl ring of the 4c may bind in a lipophilic pocket
formed byϒ2 Phe77 and ϒ2 Val190 of the receptor. In addition, ligand 5a, 3-amino-4-benzyl-
5-phenyl-4H-1,2,4-triazole, was docked in the BZD-binding site of GABAA receptor and
shown in Figure 4 (a, b). This figure indicated that the benzyl group of this ligand has
different orientation relative to same group of 4c in the active site.
2.5. Pharmacological evaluations
2.5.1. PTZ-induced lethal convulsion test and MES tests
Compound 4c showed the highest affinity for the BZD receptors among the novel compounds
and was selected for pharmacological tests to evaluate the efficacy of the compound. As
shown in Table 2, compound 4c showed weak anticonvulsant effect in both MES and PTZ
models compared to diazepam. The anticonvulsant potency of the novel compound was
significantly lower than diazepam in both tests (P<0.05). In both PTZ and MES tests,
flumazenil (10 mg/kg) was able to prevent the anticonvulsant activity of the compound. The
antagonistic effect of flumazenil indicates that BZD receptors are highly involved in the
anticonvulsant effects of the novel compound.
2.5.2. Passive avoidance test
The latency to enter the dark compartment in passive avoidance test in testing day is shown in
Figure 5. Midazolam as positive control showed significant decrease in latency compared to
control (P<0.001) indicating anterograde amnesia. However, compound 4c in doses of 0.325,
0.65, 1.25, and 2.5 mg/kg did not show any significant effect on passive avoidance test. These
results indicate that compound 4c has minimum effect on anterograde memory.
2.5.3. Potentiation of pentobarbital induced sleeping time
The duration of losing of righting reflex (considered as sleeping time) following injection of
pentobarbital and the novel compound is shown in Figure 6. Diazepam (1 mg/kg) as positive
control increased the sleeping time (P<0.001). The novel compound in dose of 0.625 mg/kg
did not show any effect on sleeping time induced by pentobarbital. However, in doses of 1.25,
4

2.5, and 5 mg/kg, it was able to increase the sleeping time (P<0.001 for all three doses) and
the effect was dose-dependent. Flumazenil (10 mg/kg) prevented the effect of the compound
4c (P<0.001). This test revealed that compound 4c has hypnotic effect by acting on BZD
receptor.
2.5.4. Preliminary evaluation of toxicity
Injection of 50 and 150 mg/kg, which are 10 and 30 times more than the hypnotic dose of the
compound 4c respectively (ED50 = 5mg/kg), did not show any significant toxicity reactions
except sedation and hypnosis. It seems that this compound is probably safe enough to precede
future studies. Obviously, complete toxicity evaluation tests are necessary in future studies.
3. Conclusions
In summary, the derivatives of 1,2,4-triazolo[1,5-a]pyrimidinone and 1,2,4-triazole-3-amine
as novel BZD agonists were investigated. Most of the novel synthesized compounds showed
better affinity for CBR than diazepam in radioligand binding assay. Compound 4c with the
highest affinity for CBR was evaluated in biological assay. This compound revealed
considerable hypnotic and weak anticonvulsant activity beside no impairment on learning and
memory. Since most of the potent BZD agonists affect learning and memory, the developed
scaffold could be a valuable lead compound in designing the new class of BZD receptor
ligands with no memory defect. It seems that the synthesized compounds have binding
affinity for the GABAA receptors with α1 subunit due to the fact that the GABAA receptor
with this subunit is responsible for the hypnotic effect and part of seizure protection of BZD
20
agonists. Since the effect on memory is mediated through the GABAA receptors with α5
subunit, 20 the designed compounds may have no affinity for this subunit. In this study, we did
not test the binding of the compound to the specific subunits of GABAA receptor and proving
this hypothesis needs further studies.
4. Experimental
4.1. Calculations
Conformational analysis of the synthesized compounds and estazolam were performed
preliminarily through MMX force field method followed by AM1 calculation implemented in
HyperChem8 software (Hypercube, Inc.). The global energy minima conformers of the
designed compounds on corresponding conformer of estazolam molecule, a known BZD
agonist, were superimposed.
5

4.2. Materials and instrumentation
Melting points (mp) were determined using the Electrothermal 9100 apparatus and are
uncorrected. Infrared spectra were acquired on a Perkin-Elmer 1420 ratio recording
spectrometer. A Bruker FT-500 MHz instrument (Bruker Biosciences, USA) was used to
1
obtain H-NMR spectra; chloroform-d and DMSO-d6 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. HPLC Agilent system and elemental analyser were
used to obtain LC Mass spectra and elemental analysis respectively. All chemicals and
reagents were obtained commercially from Aldrich or Merck Company and were used
without further purification.
4.3. Benzoic acid hydrazide (1)
28ml (222 mmol) of methyl benzoate and 54 ml (1.11 mol) hydrazine hydrate were stirred for
12 h at room temperature in methanol (10 ml).After this time, the solvent was evaporated and
white precipitates washed with diethyl ether, to give 25 g (83%) of 1. mp: 103-106°C; IR:
(KBr) ν (cm-1) 3316, 3235, 3033, 1667; Mass m/z (%):136 (M+, 59), 122 (40), 106 (64), 105
(100), 77(100). Anal. Calcd for C7H8N2O: C, 61.75; H, 5.92; N, 20.58. Found: C, 61.55; H,
5.98; N, 20.67.
4.4. 2-Amino-5-phenyl-1,3,4-oxadiazole (2)
15 g (110 mmol) of 1 and 13 g (122 mmol) cyanogens bromide were dissolved in methanol
(100 mL) and were refluxed for 4h. The solvent was evaporated and the residue recrystallized
from isopropyl alcohol and water to give 11 g (62%) of 2. mp: 246-248°C; IR: (KBr) ν (cm-1)
3305, 3123, 1663, 1651; Mass m/z (%):161 (M+, 100), 118 (98), 105 (74), 103 (70), 91 (61),
77 (87). Anal. Calcd for C8H7N3O: C, 59.62; H, 4.38; N, 26.07. Found: C, 59.85; H,
4.40; N, 25.87.
4.5. 7-Methyl-2-phenyl-5H-[1,3,4]oxadiazolo-[3,2-a]pyrimidine-5-one (3)
10g (62 mmol) of 2 and 40ml (317 mmol) ethyl acetoacetate were refluxed for 48 h. The
excess ethyl acetoacetate was evaporated and the crude product was purified by
chromatography on silica gel column. Elution with ethyl acetate/hexane (70:30) gave 4g
(30%) of 3. Mp: 179-180°C; IR: (KBr) ν (cm-1)1698, 1632, 1607 ; Mass m/z (%):227 (M+, 9),
6

143 (10), 110 (29), 105 (100), 103 (37), 77 (100); 500 MHz 1H-NMR (CDCl3):δ ppm: 2.46
(3H, s, CH3), 6.32 (1H, s, H-C6 of oxadiazolopyrimidinone ring), 7.61 (2H, t, J = 7.5 Hz, H3
& H5 of phenyl ring), 7.70 (1H, t, J = 7.5 Hz, H4 of phenyl ring), 8.18 (2H, d, J = 7.5 Hz, H2
& H6 of phenyl ring). Anal. Calcd for C12H9N3O2: C, 63.43; H, 3.99; N, 18.49. Found: C,
63.30; H, 4.03; N, 18.56.
4.6. General procedure for the synthesis of compounds 4a-4c
500mg (2.2 mmol) of 7-methyl-2-phenyl-5H-[1,3,4]oxadiazolo-[3,2-a]pyrimidine-5-one and
4ml (35mmol) substituted amine were refluxed for 5 h. The unreacted amine was evaporated
and the remnant was washed with diethyl ether. Finally, the precipitate was filtered and
recrystallized from methanol.
4.6.1. 3-Benzyl-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one (4a)
Yield: 68%; mp: 150-152°C; IR: (KBr) ν (cm-1) 1680; Mass m/z (%):316 (M+, 62), 315 (M+-
1
1, 21), 212 (32), 110 (67), 91 (100), 77 (14) ;500 MHz H-NMR (CDCl3):δ ppm:2.31 (3H, s,
CH3), 5.38 (2H, s, CH2), 6.07 (1H, s, H-C6 of triazolopyrimidinone ring), 7.14 (2H, d, J = 6.8
Hz, H2' & H6' of benzyl ring), 7.25-7.30 (3H, m, H3', H4' & H5' of benzyl ring), 7.55 (2H, t, J =
7.5 Hz, H3 & H5 of phenyl ring), 7.63 (1H, t, J = 7.5 Hz, H4 of phenyl ring), 7.69 (2H, d, J =
7.5 Hz, H2 & H6 of phenyl ring). Anal. Calcd for C19H16N4O: C, 72.13; H, 5.10; N, 17.71.
Found: C, 72.32; H, 5.04; N, 17.64.
4.6.2. 3-(4-Fluorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one
(4b)
Yield: 68%; mp: 185-187°C; IR: (KBr) ν (cm-1) 1687; Mass m/z (%):(M+, 21), 110 (50), 109
1
(100) ;500 MHz H-NMR (DMSO):δ ppm: 2.31 (3H, s, CH3), 5.35 (2H, s, CH2),6.06 (1H, s,
H-C6 of triazolopyrimidinone ring), 7.11 (2H, t, J = 8.4 Hz, H3' & H5' of benzyl ring), 7.19-
7.21 (2H, m, H2' & H6' of benzyl ring), 7.56 (2H, t, J = 7 Hz, H3 & H5 of phenyl ring), 7.61-
7.69 (3H, m, H2, H4 & H6 of phenyl ring). Anal. Calcd for C19H15FN4O: C, 68.25; H, 4.52; N,
16.75. Found: C, 68.05; H, 4.56; N, 16.87.
4.6.3. 3-(2-Chlorobenzyl)-5-methyl-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(3H)-one
(4c)
Yield: 69%; mp: 220-222°C; IR: (KBr) ν (cm-1) 1691; Mass m/z (%):350 (M+, 11), 315(100),
125 (100), 103 (34), 94 (67), 89 (60) ;500 MHz 1H-NMR (CDCl3):δ ppm: 2.40 (3H, s, CH3),
7

5.50 (2H, s, CH2), 6.22 (1H, s, H-C6 of triazolopyrimidinone ring), 6.98 (1H, d, J = 7.6 Hz,
H6' of benzyl ring), 7.27 (1H, t, J = 7.6 Hz, H5' of benzyl ring), 7.33 (1H, t, J = 7.6 Hz, H4' of
benzyl ring), 7.46-7.51 (3H, m, H3' of benzyl ring, H3 & H5 of phenyl ring), 7.56-7.64 (3H, m,
H2, H4 & H6 of phenyl ring). Anal. Calcd for C19H15ClN4O: C, 65.05; H, 4.31; N, 15.97.
Found: C, 64.79; H, 4.37; N, 16.08.
4.7. General procedure for the synthesis of compounds 5a-5c
500mg (1.6 mmol) of compounds 4a-4c was added in the mixture of 8 ml hydrazine hydrate
and 2 ml water and stirred for 3 h at room temperature. The reaction mixture was cooled at
4°C overnight and recrystallized from ethanol plus a few drops of water to give corresponding
final products.
4.7.1. 3-Amino-4-benzyl-5-phenyl-4H-1,2,4-triazole (5a)
Yield: 83%; mp: 185-187°C; IR: (KBr) ν (cm-1) 3309, 3126, 1659; Mass m/z (%):273 (M+ +
23), 251 (M+ + 1) ;500 MHz 1H-NMR (DMSO):δ ppm: 5.16 (2H, s, CH2), 6.03 (2H, s, NH2),
6.96 (2H, d, J = 7.6 Hz, H2' & H6' of benzyl ring), 7.24 (1H, t, J = 7.6 Hz, H4' of benzyl ring),
7.30 (2H, t, J = 7.6 Hz, H3' & H5' of benzyl ring), 7.40-7.42 (3H, m, H3, H4 & H5 of phenyl
ring), 7.47-7.49 (2H, m, H2 & H6 of phenyl ring). Anal. Calcd for C15H14N4: C, 71.98; H,
5.64; N, 22.38. Found: C, 71.81; H, 5.70; N, 22.49.
4.7.2. 3-Amino-4-(4-fluorobenzyl)-5-phenyl-4H-1,2,4-triazole (5b)
Yield: 83%; mp: 177.5-179.5°C; IR: (KBr) ν (cm-1) 3327, 3074, 1644; Mass m/z (%):291(M+
+ 23), 269 (M+ + 1) ;500 MHz 1H-NMR (DMSO):δ ppm: 5.14 (2H, s, CH2), 6.06(2H,s, NH2),
6.99 (2H, dd, J = 8.4, 5.5 Hz, H2' & H6' of benzyl ring), 7.14 (2H, t, J =8.4 Hz, H3' & H5' of
benzyl ring), 7.40-7.50 (5H, m, of phenyl ring). Anal. Calcd for C15H13FN4: C, 67.15; H, 4.88;
N, 20.88. Found: C, 67.30; H, 4.85; N, 20.83.
4.7.3. 3-Amino-4-(2-chlorobenzyl)-5-phenyl-4H-1,2,4-triazole (5c)
Yield: 84%; mp: 180.5-182°C; IR: (KBr) ν (cm-1) 3465, 3389, 3318, 1626; Mass m/z
(%):307 (M+ + 23), 285 (M+ + 1); 500 MHz 1H-NMR (CDCl3):δ ppm: 4.65 (2H, s, CH2),
5.16 (2H, s, NH2),6.99 (1H, dd, J = 8.0, 1.7 Hz, H6' of benzyl ring), 7.31-7.39 (2H, m, H4' &
H5' of benzyl ring), 7.41-7.47 (3H, m, H3' of benzyl ring, H3 & H5 of phenyl ring), 7.49-7.54
(3H, m, H2, H4 & H6 of phenyl ring). Anal. Calcd for C15H13ClN4: C, 63.27; H, 4.60; N,
19.68. Found: C, 63.46; H, 4.56; N, 19.57.
8

4.8. Radioligand receptor binding assay
All of the binding assays were done in triplicates. The radioligand receptor binding studies
were done as previously described.21-22 Briefly, the cortical membrane tissue of Male
Sprague-Dawley rats weighing 300-350g (Pasteur Institute, Tehran, Iran) was homogenized in
20 mL ice-cold Tris-HCl buffer (30 mM, pH 7.4) using a Silent S homogenizer (Heidolph,
Germany) at medium speed. The homogenate was centrifuged at 600 g for 10 min using a
Beckman Coulter L90K centrifuge. The resulting supernatant was centrifuged at 27000 g for
15 min followed by three centrifugations and resuspensions cycles. The washed pellet was
suspended in 20 mL buffer, incubated at 37°C for 30 min and then centrifuged for 10 min at
27000 g. The pellet was washed once, and the final pellet was re-suspended in 30 mL Tris-
HCl buffer (50 mM, pH 7.4). All of the centrifugation was performed at 4°C. 23-25 The amount
of protein was estimated in the membrane preparation by the Bradford method using bovine
serum albumin (BSA) as a standard. 26 The membrane preparation was stored at -20°C until it
was used 1-15 days later. For binding studies, 100 µg of frozen membrane was thawed,
resuspended into Tris-HCl buffer (50 mM, pH 7.4), and incubated with 7 different
concentrations of [3H]-flumazenil at 30°C for 35 min. The incubation was terminated by the
centrifugation of reaction mixture at 1500 g for 4 min at 4°C. The total binding (TB) (receptor
+ radioligand), non-specific binding (NSB) (receptor + radioligand + excess diazepam), and
specific binding (SB) (TB-NSB) were measured at various radioligand concentrations. The
amount of radioligand required to saturate the receptors was used to determine the receptor
binding affinity of [3H]-flumazenil (Kd) and the BZD receptor density (Bmax) based on non-
linear regression analysis of the saturation curve data.27 The Kd and Bmax for [3H]-flumazenil
are 1.35 o.316nM and 0.638 0099 Pmol/mg respectively. In competition studies, 100 µg
of membrane protein in Tris-HCl buffer (50 mM, pH 7.4) was incubated with 8.6×10-5nmol
[3H]-flumazenil and increasing amount of newly synthesized ligands in a final volume 0.5mL
at 30°C for 35 min.24 After incubation, the assay was terminated by centrifugation (1500 g,
4C, 5 min). Binding (receptor + radioligand) and NSB were measured at various
concentrations of unlabeled ligand. TB is determined in the absence of any added competitor
(non-radioactive ligand). The concentration of non-radioactive ligand that inhibits the binding
of [3H]-flumazenil by 50% is IC50 value.27-29 All of experiments were done in triplicates.
Nonspecific binding (NSB) was determined in parallel assays performed in the presence of
100 µM diazepam.
4.9. Docking studies
9

The homology model of the Diazepam bound GABAA receptor (α1β2ϒ2) developed by Ernst
et al. was retrieved the supplementary material of their published paper.30 The structures of
compounds were investigated using the Lamarckian genetic algorithm search method
implemented in AutoDock 4.2.3 software (http://autodock.scripps.edu). The receptors were
kept rigid, and ligands were allowed to be flexible. Polar hydrogens and Kollman united atom
partial charges were added to the individual protein atoms. Each structure was energy
minimized under MM+ method in HyperChem8 software and converted to pdbqt format file
using AutoDockTools version1.5.6rc3 (http://mgltools.scripps.edu). A docking grid box was
built with 40, 40 and 40 points in 42.820, 44.4360 and 6.8690 directions and the number of
generations and maximum number of energy evaluations was set to 100 and 2,700,000,
respectively. Docking results were clustered with a root mean square deviation (RMSD) of
0.5 Å and evaluated by Pymol software version 1.5.0.1 (http://pymol.findmysoft.com).
4.10. Pharmacological evaluations
4.10.1. Animals and drugs
Male NMRI albino mice (weighing 18-22 g; 12 weeks old) were used for pharmacological
evaluations of the novel compound (4c). Animals were purchased from Pasteur Institute, Iran.
They were transferred to the animal house of School of Pharmacy 7 days before the
experiments and housed in a 12 hours light/dark condition with a controlled temperature and
humidity. During the housing, animals had free access to standard food and water. For all the
experiments, the mice were transferred to the lab 60 minutes before starting the tests to get
habituated to the lab environment. Animals were divided to experimental groups randomly
and each mouse was used only one time for the experiments. Before starting the experiments,
proposal of the experiments were sent to institutional animal care and use committee of
Shahid Beheshti University of Medical Sciences and approval of the committee was acquired.
All the experiments were performed in accordance with National Institute of Health (NIH)
Principles of Laboratory Animal Care (NIH publication #85-23). In all experiments the
minimum numbers of mice were used and sufficient attentions were considered to reduce pain
or discomfort of animals. The novel compound 4c, diazepam, as reference BZD agonist, and
flumazenil, as antagonist of BZD receptors, were dissolved in mixture of DMSO and water
(1:10) and administered 5 ml/kg; i.p.; 30 minutes before the experiments. PTZ, pentobarbital,
10

and midazolam were dissolved in distilled water and injected i.p. (volume of injection was 5
ml/kg).
4.10.2. PTZ-induced lethal convulsion test and MES tests
The anticonvulsant activity of the novel compound was determined in two models of seizure,
PTZ and MES induced seizure.23, 31 In PTZ model, the ability of the novel compound to
protect mice against lethal dose of PTZ (100 mg/kg) was evaluated. Animals were closely
observed for 30 minutes after injection of PTZ and the number of dead mice was reported. In
MES test, the ability of the novel compound to prevent MES induced seizure in mice was
evaluated. In this test, occurrence of hind limb tonic extension (HLTE) in mice following
applying MES (60 Hz, 37.2 mA and 0.25 s) was assessed. The electrical current was applied
through ear electrodes and mice were observed for 30 seconds for incidence of HLTE.
4.10.3. Passive avoidance test
Step throw passive avoidance test was used for evaluation of anterograde amnesia induced by
the novel compound 4c and midazolam (1mg/kg, i.p.), a BZD with amnestic effects.1 The
passive avoidance cage had two different compartments separated by a sliding door. One
compartment was black and dark and another one was white and lighted. On training day,
mouse was placed in the white compartment, facing away from sliding door for 30 seconds.
Then, the sliding door was opened and mouse was able to enter the dark compartment. When
the mouse entered the dark compartment with four paws, mouse got a foot shock (0.5 mA, 2
seconds). On the testing day (after 24 hours), mouse was placed in lighted compartment and
the latency to enter the dark compartment was reported.
4.10.4. Potentiation of pentobarbital sleeping time
Pentobarbital (65 mg/ kg i.p.) was injected 30 min after administration of the novel compound
4c and diazepam (1 mg/kg). The duration of a loss of the righting reflex was considered as the
sleep time. Mice were placed on an electric blanket during the experiment to prevent
hypothermia.32, 33
4.10.5. Preliminary evaluation of toxicity
For evaluation of toxicity of the novel compound 4c, 10 mice were selected randomly. Doses
of 50 and 150 mg/kg, which are 10 and 30 times more than the ED50 of hypnotic dose, were
used in this study and animals were observed for 24 hours after injection of the compound 4c.
11

4.11. Data Analysis
The binding parameters of [3H]-flumazenil (Kd and Bmax) and newly synthesized ligands were
calculated from non-linear regression analysis of the saturation and competitive curve data by
using the activity base software package (Program Prism, Graph Pad, San Diego, CA). The
IC50s of the novel compounds in binding studies were analyzed using one way ANOVA with
Tukey’s HSD post-hoc test. In PTZ and MES tests, probit-regression method and SPSS
software (Chicago, IL; version 13) was used to determine ED50 of the novel compound 4c and
diazepam. Fisher’s exact probability test was used to analyze the difference between the ED50
of the novel compound and diazepam. In PTZ and MES tests, the data were presented as
mean with 95% confidence intervals and in the rest of the pharmacological evaluations, one-
way ANOVA with Tukey’s HSD post-hoc test was used and all the data were presented as
mean + SEM. P<0.05 considered statistically significant.
5. Acknowledgment
This work was supported by grant from the Research Council of Shahid Beheshti University
of Medical Sciences and Iran National Science Foundation (INSF). We would like to
acknowledge the Radiopharmacy Laboratory, School of Pharmacy, Shahid Beheshti
University of Medical Sciences for radioligand receptor binding studies.
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14

Scheme 1. Reagents and conditions: (a): NH2NH2.H2O, Methanol, rt, 12 h; (b): BrCN, Methanol, reflux, 4 h; (c):
CH3COCH2COOEt, reflux, 48 h; (d): substituted amine, reflux, 4 h; (e): NH2NH2.H2O, H2O/Dioxane, reflux, 6 h.
15

Figure 1. The structure of the designed compounds and estazolam. The main pharmacophores have been
conserved.
Figure 2. Stereo view of the superimposition of the energy minima conformers of estazolam (top left, violet) and
compound 4c (top right).

Figure 3. (a) Interaction of 4c with amino acids of diazepam-binding pocket of GABAA receptor (α1β2ϒ2). (b)
Overlay of 4c (blue) and estazolam (magenta) in pocket of GABAA receptor (α1β2ϒ2).
Figure 4. (a) Interaction of 5a with amino acids of diazepam-binding pocket of GABAA receptor (α1β2ϒ2). (b)
Overlay of 5a (magenta) and 4c (blue) in pocket of GABAA receptor (α1β2ϒ2).

Passive Avoidance
400
300
200
100
0
***
Figure 5. Effect of the novel compound on anterograde memory in passive avoidance test: The delay time to
entrance to the dark compartment in testing day is shown. Data are presented as mean + SEM. In all groups n=8.
*** represents P<0.001 compared to control. 1 mg/kg of midazolam was used in this experiment.

Pentobarbital test
120
100
80
60
40
20
0
***
***
***
***
Figure 6. Hypnotic effect of the novel compound in pentobarbital test: The sleeping time (duration of loss of
righting reflex) is shown. Data are presented as mean + SEM. In all groups n=8. *** represents P<0.001
compared to control. 10 mg/kg of flumazenil was used in this experiment.

Table 1. In vitro binding affinities of the novel compounds tested on 3H-flumazenil binding to
benzodiazepine receptor.
Compound
Structure
Ki (nM)
1.46
IC50 SEM
(nM)
4a
2.39 0.99
4b
0.97
0.42
1.59 0.103
O
CH3
N
N
N
N
4c
5a
0.68 0.14
Cl
0.53
2.73
0.87 0.11
5b
4.45 0.034
5c
1.37
1.53
2.24 0.108
2.5 0.066
Diazepam
16

Table 2. PTZ-induced lethal convulsion test and MES tests of the compound 4c
ED50 mg/kg a
Compound
PTZ
MES
4c
Diazepam
56.57 (32.23 - 99.28)
0.75 (0.56 - 0.96)
15.01 (8.21 - 30.02)
0.74 (0.45 - 1.07)
a n = 10, 95% Confidence limits in parentheses
17

18