Endothelium dependent and independent mechanisms of vasorelaxant activity of synthesized 2,5-disubstituted-1,3,4-oxadiazole derivatives in rat thoracic aorta-ex vivo and molecular docking studies

Article abstract of DOI:10.2174/1570180812666150907203634

Background: Vasoconstriction is a major pathological feature of cardiovascular diseases involving endothelium dependent and independent mechanisms. Oxadiazole moiety appeared to be effective in various pathologies. Objective: The aim of the study was to s

Full text of DOI:10.2174/1570180812666150907203634

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441
Letters in Drug Design & Discovery, 2016, 13, 441-450
ISSN: 1570-1808
eISSN: 1875-628X
Endothelium Dependent and Independent Mechanisms of Vasorelaxant
Activity of Synthesized 2,5-disubstituted-1,3,4-oxadiazole Derivatives in
Rat Thoracic Aorta – Ex vivo and Molecular Docking Studies
Impact
Factor:
0.77
Zenab Attari1, Jayesh Mudgal1, Pawan G Nayak1, Nandakumar Krishnadas1, Revathi Rajappan2 and
N. Gopalan Kutty1,*
BENTHAM
S
CIENCE
1Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University,
Manipal, Karnataka, India
2Department of Pharmaceutical Chemistry, Manipal College of Pharmaceutical Sciences, Manipal
University, Manipal, Karnataka, India
Abstract: Background: Vasoconstriction is a major pathological feature of cardiovascular diseases
involving endothelium dependent and independent mechanisms. Oxadiazole moiety appeared to be ef-
fective in various pathologies.
Objective: The aim of the study was to synthesize and evaluate the mechanism of vasorelaxation ex-
hibited by synthesized oxadiazole derivatives.
Method: The 2,5-disubstituted-1,3,4-oxadiazole derivatives were synthesized by an efficient and simple method. The de-
rivatives were investigated for their ex-vivo vasorelaxant action on intact/denuded endothelium rat aortic rings precon-
tracted with norepinephrine/ phenylephrine/KCl.
Results: The contractions induced in the aortic rings by the addition of cumulative concentrations of norepinephrine,
phenylephrine, KCl and calcium were significantly antagonized by a derivative, OXD-Z2. In another experiment, verapa-
mil pretreatment inhibited phenylephrine and Ca2+-induced aortic contractions and OXD-Z2 did not alter verapamil-
induced inhibition. This indicated the role of L-type Ca2+-channels in the OXD-Z2-induced vasorelaxation via inhibition
of calcium influx. Further, atropine (muscarinic receptor antagonist), L-NAME (NO synthase inhibitor) and methylene
blue (non-selective cGMP inhibitor) inhibited OXD-Z2-induced relaxation in other sets of experiments. These results in-
dicate that OXD-Z2 also mediates vasorelaxation through NO release by muscarinic receptor activation. In addition, the
molecular docking studies showed that OXD-Z2 interacts with L-type Ca2+-channel, muscarinic (M2) receptor and eNOS.
Conclusion: Thus, it is deduced from the above findings that the vasorelaxant activity of OXD-Z2 involves muscarinic
receptor-mediated nitric oxide release in addition to direct inhibition of L-type Ca2+-channels.
Keywords: 2,5-disubstituted-1,3,4-oxadiazoles, aortic rings, endothelium, vasorelaxation, hypertension.
1. INTRODUCTION
laboratory demonstrated the vasorelaxant activity of 2, 5-
disubstituted-1, 3, 4-oxadiazole due to its ability to inhibit
calcium flux [5]. The present work was aimed to explore if
oxadiazole derivatives exert their vasorelaxant action
through mechanisms beyond calcium channel inhibition.
Thus, eight 2,5-disubstituted-1,3,4-oxadiazole derivatives
were synthesized by a simple method and evaluated for their
vasorelaxant actions in the rat thoracic aorta followed by
assessment of the involvement of endothelium-dependent or
–indepen-dent mechanism in their vasorelaxant action.
Endothelial dysfunction is the important underlying
pathological feature of cardiovascular diseases viz. hyperten-
sion, coronary artery disease, chronic heart failure, periph-
eral artery disease, diabetes, and chronic renal failure. Injury
to endothelia of blood vessels results in imbalance in cardio-
vascular homeostasis, eventually leading to endothelial dys-
function. This is characterized by pro-inflammatory states,
pro-thrombotic properties and reduced vasodilatation [1, 2].
Oxadiazole scaffold is a versatile pharmacophore, which
possesses diverse activities such as hypoglycaemic, vasore-
laxant and antihypertensive [3, 4]. A previous study in our
2. EXPERIMENTAL
2.1. Synthesis
*Address correspondence to this author at the Department of Pharmacology,
Manipal College of Pharmaceutical Sciences, Manipal University, Manipal -
576104, Karnataka, India; Tel: +91-820-2922482; Extn: 322;
The oxadiazole derivatives (coded as OXD-Z1 to
OXD-Z8) were synthesized by a well-established two-step
method with some modifications [5]. Briefly, isoniazid (b)
Fax: +91-820-2571998; E-mail: ng.kutty@manipal.edu
1875-628X/16 $58.00+.00
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442 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Attari et al.
Table 1. Characterization of 2,5-disubstituted-1,3,4-oxadiazole derivatives.
Benzaldehyde Derivatives
Compound Code
Molecular Formula
R1
R2
Mw
MP(ºC)
p-Anisaldehyde
p-Tolualdehyde
OXD-Z1
OXD-Z2
OXD-Z3
OXD-Z4
OXD-Z5
C16H15O3N3
C16H15O2N3
C15H12FO2N3
C15H12ClO2N3
C17H15O4N3
H
H
H
H
H
OCH3
CH3
297
281
285
301
325
98
102
104
114
210
p-Fluoro-benzaldehyde
p-Chloro-benzaldehyde
p-Hydroxy-benzaldehyde
F
Cl
OCOCH3
p-Dimethylamino-
benzaldehyde
OXD-Z6
C17H18O2N4
H
(CH3)2N
310
49
Vanillin
OXD-Z7
OXD-Z8
C18H17O5N3
C17H15O4N3
OCH3(m-)
OCOCH3
H
355
325
174
152
Salicylaldehyde
OCOCH3
was dissolved in methanol and ortho- or para- substituted
benzaldehyde (a) was added to the methanolic solution along
with a few drops of glacial acetic acid (Table 1). The mixture
was refluxed for 30 minutes. The intermediate product,
Schiff base (c) was filtered and recrystallized from alcohol.
Schiff base was heated for 5h with acetic anhydride on a
water bath.The reaction mixture (d) was then poured on to
crushed ice in a beaker. The solid that separated was recrys-
tallized from boiling water. The schematic representation of
synthesis is given in Fig. (1).
The characteristic properties of oxadiazole derivatives
were established through chemical, chromatographic and
spectroscopic methods. The strucures of the 1,3.4-oxadiazole
derivatives (OX-Z1 and OX-Z2) were confirmed through IR,
NMR and mass spectrometry. The remaining compounds
were characterized through their IR spectral data.
1. OXD-Z1 (1-[2-(4-methoxyphenyl)-5-(pyridine-4-yl)-
1,3,4-oxadiazol-3(2H)-yl] ethanone)
Mass - 297[M]+, 298[M+1]+, 255 m/z, 133 m/z.
1H NMR - 8.6 [d, 2H, -N(CH)2-)]; 6.8, 7.3, 7.6 [d, 6H,
Ar-H]; 6.9 [s, 1H, O-C(H)-N(COCH3)N]; 3.75 [s, 3H, -
OCH3]; 2.32 [s, 3H, -COCH3].
O
R2
R1
NH2
N
H
+
H
IR (KBr, cm-1) - 3047.63 (-Ar), 2933.83-2833.52 (-CH-
of Ar), 1670.41 (-COCH3), 1618.83-1410.10 (-C=N of Pyri-
dine), 1259.56 (C-O-C asymmetric), 1170.83 (-OCH3),
1068.60 (C-O-C symmetric).
N
O
Isoniazid (b)
Aldehyde (a)
CO
i) and refluxing for 30 min
2. OXD-Z2 (1-[2-(4-methylphenyl)-5-(pyridine-4-yl)-
1,3,4-oxadiazol-3(2H)-yl] ethanone)
CH
N
R2
N
H
Mass - 281 [M] +, 239 m/z, 78 m/z
R1
1H NMR - 8.8 [d, 2H, -N(CH2)-]; 7.2, 7.3, 7.7 [d, 6H,
Ar-H]; 7.07 [s, 1H, O-C(H)N(COCH3)N]; 2.36 [s, 3H, Ar-
CH3]; 2.33 [s, 3H, -COCH3]
Schiff base (Intermediate, c)
ii) and heating for 5 hours
N
R2
IR (KBr, cm-1) - 3032.20 (-Ar), 2920.32 (-CH- of Ar),
2845.10 (-CH3), 1670.41 (-COCH3), 1600.97-1410.01 (-C=N
of pyridine), 1257.63 (C-O-C asymmetric), 1066.67 (C-O-C
symmetric)
O
N
R1
N
N
COCH3
3. OXD-Z3 (1-[2-(4-fluorophenyl)-5-(pyridine-4-yl)-
1,3,4-oxadiazol-3(2H)-yl] ethanone)
IR (KBr, cm-1) - 3182.65 (-Ar), 2916.47 (-CH- of Ar),
16565.91 (-COCH3), 1406.15 (-C=N of Pyridine), 1298.14
(C-O-C asymmetric), 1149.61 (-F), 1064.74 (C-O-C sym-
metric).
2,5-disubstituted-1,3,4-oxadiazole (d)
i) 2-3 drops of glac. acetic acid
ii) acetic anhydride
1a. Schematic representation of synthesis of 2,5-disubstituted-1,3,4-
oxadiazole derivatives
CH3
4. OXD-Z4 (1-[2-(4-chlorophenyl)-5-(pyridine-4-yl)-
1,3,4-oxadiazol-3(2H)-yl] ethanone)
N
O
IR (KBr, cm-1) - 3032.20 (-Ar), 2850.88 (-CH- of Ar),
1668.48 (-COCH3), 1408.08 (-C=N of Pyridine), 1261.59
(C-O-C asymmetric), 1082.10 (C-O-C symmetric), 825.56 (-
Cl).
N
N
COCH3
Fig. (1). 1b. Structure of 2,5-disubstituted-1,3,4-oxadiazole deriva-
tive, OXD-Z2.

Vasorelaxation of Oxadiazole Derivatives in Rat Thoracic Aorta
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 443
5. OXD-Z5 (4-[3-acetyl-5-(pyridine-4-yl)-2,3-dihydro-
1,3,4-oxadiazol-2-yl] phenyl acetate)
IR (KBr, cm-1) - 3037.99 (-Ar), 1743.71 (-OCOCH3),
1662.69 (-COCH3), 1419.66 (-C=N of Pyridine), 1222.91
(C-O-C asymmetric), 1058.96 (C-O-C symmetric).
and result was analyzed using AD Instruments Power Lab
software. The aortic rings were equilibrated for 120 minutes
in Kreb’s solution, which was changed once every 20 min-
utes. The aortic rings were denuded by removing endothe-
lium using a 25-gauge needle tip. The absence of Ach-
induced relaxation was considered an indication of denuda-
tion.
6. OXD-Z6 (1-[2-(4-dimethylaminophenyl)-5-(pyridine-
4-yl)-1,3,4-oxadiazol-3(2H)-yl] ethanone)
IR (KBr, cm-1) - 2906.82 (-Ar), 2820.02 (-CH- of Ar),
1660.77 (-COCH3), 1437.02 (-C=N of Pyridine), 1232.55
(C-O-C asymmetric), 1163.11 (tertiary amine), 1064.74 (C-
O-C symmetric).
2.3.2. Vasorelaxant Effect of OXD-Z2 on Norepinephrine
(NE), Phenylephrine (PE) and KCl-induced Precontraction
in Rat Aortic Rings with and Without Endothelium
The contractions were induced by PE (1ꢀM), NE
(300nM) and KCl (60mM) in the aortic rings with or without
endothelium. The effect of OXD-Z2 was recorded on pre-
contracted aorta after cumulative addition of OXD-Z2
(10nM to 3mM) directly to the organ bath.
7. OXD-Z7 (4-[3-acetyl-5-(pyridine-4-yl)-2,3-dihydro-
1,3,4-oxadiazol-2-yl]-2-methoxyphenyl acetate)
IR (KBr, cm-1) - 3072.71 (-Ar), 2956.97 (-CH- of Ar),
1764.93 (-OCOCH3), 1656.91 (-COCH3), 1415.80 (-C=N of
Pyridine), 1213.27 (C-O-C asymmetric), 1161.19 (-OCH3),
1064.74 (C-O-C symmetric).
2.3.3. Effect of OXD-Z2 on Cumulative Concentration-
Response Curve of PE, NE and KCl
8. OXD-Z8 (2-[3-acetyl-5-(pyridine-4-yl)-2,3-dihydro-
1,3,4-oxadiazol-2-yl] phenyl acetate)
IR (KBr, cm-1) - 3037.99 (-Ar), 2976.26 (-CH- of Ar),
1759.14 (-OCOCH3), 1664.62 (-COCH3), 1415.80 (-C=N of
Pyridine), 1265.35 (C-O-C asymmetric), 1064.74 (C-O-C
symmetric).
Cumulative concentration-response curves (CRCs) for
PE (10pM to 300nM), NE (10pM to 300nM) and KCl
(10mM to 80mM) were obtained without and with incubat-
ing denuded aortic rings with OXD-Z2 (100nM and 300nM)
for 20 minutes. The effect of OXD-Z2 on the cumulative
CRCs of PE, NE and KCl was determined by comparing %
contraction in the presence and absence of OXD-Z2.
2.2. Drugs and Chemicals
2.3.4. Effect of OXD-Z2 on Cumulative Concentration-
Response of Ca2+-induced Contraction in Calcium Free
Depolarized Kreb’s Solution
As oxadiazole derivatives are water-insoluble at room
temperature, they were dissolved in dimethyl sulfoxide
(DMSO) and subsequent dilutions were made from stock
solution. The drugs and chemicals used in the study were
purchased from standard chemical suppliers.
The denuded aortic rings were incubated in calcium-free
Kreb’s solution for ten minutes, followed by replacement of
the solution with Ca2+-free isotonic depolarizing solution
containing a high concentration of K+ (100mM KCl). The
tissues were then incubated with OXD-Z2 (100nM and
300nM) for 15 minutes followed by recording of CRCs of
Ca2+ (1ꢀM to 30mM). The effect of OXD-Z2 on the cumula-
tive CRCs of Ca2+ was determined by comparing % contrac-
tion in the presence and absence of OXD-Z2.
2.3. Animals
Male Wistar rats, weighing 200-250 g were employed for
the study. The rats were housed 3 per cage and maintained in
the Central Animal Research Facility (CARF), Manipal Uni-
versity. The temperature of the room was maintained at
23 2°C with a 12 h light-dark cycle. The animal experiments
were carried out according to the guidelines of CPCSEA,
with the approval of the Institutional Animal Ethical Com-
mittee (IAEC) (vide letter # IAEC/KMC/54/2009-2010).
2.3.5. Effect of OXD-Z2 on Cumulative Concentration-
Response of PE and Ca2+ in the Aortic Rings Pretreated
with Verapamil
The CRC was recorded to assess the possible role of L-
type calcium channels (Ca2+-channels) in the OXD-Z2-
induced attenuation of contraction by PE and Ca2+. The aor-
tic rings were incubated with verapamil (10ꢀM) or OXD-Z2
(1ꢀM) with verapamil (10ꢀM) for 20 minutes before cumu-
lative CRCs of PE and Ca2+. The effect of OXD-Z2 on the
cumulative CRCs of PE and Ca2+in the presence of verapa-
mil was determined by comparing % contraction in the pres-
ence and absence of OXD-Z2.
2.3.1. Preparation of Aortic Rings for Tension Measure-
ment
The experiment was carried out by the method as previ-
ously established in our laboratory setup [5]. Rats were sacri-
ficed and the descending thoracic aorta was dissected after
removing the surrounding connective tissue and fat. The
bathing fluid for aorta was the Kreb’s solution of the compo-
sition: 118mM NaCl, 4.7mM KCl, 1.2mM MgSO4, 1.2mM
KH2PO4, 2.4mM CaCl2, 25mM NaHCO3 and 11mM glu-
cose. The aortic rings (2.5 mm) were mounted in an organ
bath filled with 20 ml Kreb’s solution at a resting tension of
2.0 g. The bath was maintained at 37ºC and aerated with a
mixture of 95% O2 and 5% CO2 gas. Isometric contractions
and relaxations were recorded using a force displacement
transducer (model MLT050/A, AD Instruments, Australia)
2.3.6. Investigation of Mechanisms Involving Endothelium
The CRCs were obtained after addition of different in-
hibitors to assess the involvement of mechanisms other than
Ca2+ in OXD-Z2-induced vasorelaxation. The aortic rings
were incubated with different inhibitors L-NAME (NG-nitro-

444 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Attari et al.
L-arginine-methyl ester) (10ꢁM, a non-selective NOS inhibi-
tor), indomethacin (10ꢁM, a COX inhibitor), glibenclamide
(10ꢁM, an ATP-sensitive K+-channel blocker), atropine
(10ꢁM, muscarinic receptor antagonist) or methylene blue
(10ꢁM, a guanylate cyclase inhibitor) and then the cumula-
tive concentrations of OXD-Z2 (10nM to 3mM) were added
to record relaxation of the precontracted aortic rings.
minimized up to the rms gradient of 0.01 using Merck
Molecular Force Field (MMFF). Conformers of compounds
were then generated by Monte Carlo method. In doing so, all
rotatable bonds of the ligand were selected and number of
seeds used for searching the conformational space was set 5.
All the conformers were then energetically minimized up to
the rms gradient of 0.01 and then saved in separate folder.
The active site selection was done by choosing the cavity
having maximum hydrophobic surface area. The docking
simulation was done using GRIP docking. The parameters
fixed for docking simulation was like this-number of place-
ment: 50, rotation angle of: 10°, exhaustive method, scoring
function: dock score. After docking simulation, the best
docked conformer of the ligand and receptor were merged
and their complex was then energetically optimized by defin-
ing radius of 10 Å measured from the docked ligand. Step-
wise energy optimization was done by first hydrogens; sec-
ond side chains and finally the backbone of receptor. The
optimized complexes were then checked for various interac-
tion of ligand with receptor like hydrogen bonding, hydro-
phobic bonding and Van der Waals’ interaction etc. The
binding affinity was evaluated by the binding free energy
(ꢂGb, kcal/mol) and binding interactions [10].
2.4. Data Analysis
All vasorelaxation responses recorded, were expressed as
percentage relaxation from the pre-constricted response of
PE, NE, KCl or calcium. Data are expressed as mean SEM
of 3 experiments. Contractile responses to NE, PE, KCl and
Ca2+ were expressed as the percentages of the maximum
contraction. Emax is the maximum % contraction induced by
PE, NE, KCl or Ca2+. The pD2 and EC50 values were calcu-
lated by non-linear regression analysis using Prism demo
version 6.0; Graph Pad Software, San Diego, CA, USA. The
results were analyzed by the “Student’s t-test” and ANOVA
followed by post hoc “Tukey’s test” at the significance level
p< 0.05, where applicable.
The pD2 values were calculated by the formula,
pD2 =pDx+Log[(EAm/EBm)-1]
3. RESULTS
3.1. Synthesis
where, pDx is negative log[OXD-Z2]M, EAm is % maximum
contraction induced by contractile agent/agonist, EBm is %
maximum contraction induced by contractile agent/agonist in
presence of OXD-Z2. pD2 value indicates a non-competitive
antagonism.
The list of eight oxadiazole derivatives synthesized and
their chemical identities are depicted in Table 1.
2.5. Molecular Docking
3.2. Vasorelaxant Effect of OXD-Z2 on Phenylephrine
(PE), Norepinephrine (NE) and KCl-induced Precontrac-
tion in Rat Aortic Rings with and Without Endothelium
Molecular docking studies were carried out by using
V.Life MDS 4.2 software. The PLP function was incorpo-
rated by the MDS V.Life Science software in the GRIP
docking method, which calculates the ligand-receptor bind-
ing affinity in terms of the PLP score. The PLP score was
designed to enable flexible docking of ligand to perform a
full conformational and positional search within a rigid bind-
ing site. All the optimized conformers were docked into ac-
tive binding site of L-type Ca2+-channel (PDB ID 1T0J) [6]
and eNOS (PDB ID 3HR4 and 3NOS) [7] and for muscarinic
receptor, the derivative was docked to M2 receptor (PDB ID
3UON). M2 receptor (PDB ID 3UON) was used as a tem-
plate for docking studies as the crystal structure of M1 recep-
tor was not available [8]. Water molecules and HET ATOM-
like bound ligand data were removed from the PDB file of
proteins during docking study. The crystal structure was re-
fined using V.Life Science’s MDS 4.2 software. The refine-
ment of the crude PDB structure of receptor was done by
completing the incomplete residues. The co-crystallized
ligand lying within the receptor was modified by assigning
missing bond order and hybridization states. The side chain
hydrogens were then added to the crystal structure and their
positions were optimized up to the rms gradient 1 by aggre-
gating the other part of the receptor. The optimized receptor
was then saved as mol file and used for docking simulation.
The 2D structure of the compound OXD-Z2 was built and
then converted into the 3D with the help of VLife MDS 4.2
software. The 3D structures were then energetically
NE (300nM), PE (1ꢁM) and KCl (60mM) produced a
sub-maximal contraction of aortic ring. All the oxadiazole
derivatives were analyzed for vasorelaxant activity in PE-
contracted aortic ring. OXD-Z1 and OXD-Z3 to OXD-Z8
failed to produce satisfactory vasorelaxation in thoracic aor-
tic rings (data not shown). However, cumulative addition of
OXD-Z2 (10nM to 3mM) produced a significant vasorelaxa-
tion in the pre-contracted aortic rings (Fig. 2). pEC50 and
EC50 values of OXD-Z2 are depicted in Table 2.
3.3. Effect of OXD-Z2 on Cumulative Concentration-
Response Curve of PE, NE and KCl
The concentration-dependent contraction of aortic ring
was obtained by the cumulative addition of PE (10pM to
300nM), NE (10pM to 300nM) and KCl (10mM to 80mM).
Incubation of the aortic ring with OXD-Z2 (100nM and
300nM) shifted the CRCs of PE, NE and KCl towards right
with change in Emax values, indicating antagonistic or vasodi-
latory activity of OXD-Z2 (Fig. 3). The shift in CRCs of PE
and KCl in the presence of OXD-Z2 (100nM) and OXD-Z2
(300nM) was significantly different in its absence (p<0.05)
(EC50 ratio are given in Table 3). The shift of CRC of NE was
significant in the presence of 300nM but not 100nM of
OXD-Z2. However, no difference was observed between
mean values of OXD-Z2 at the concentrations, 100 and 300nM

Vasorelaxation of Oxadiazole Derivatives in Rat Thoracic Aorta
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 445
Values are expressed as Mean SEM
Fig. (2). Vasorelaxant effect of OXD-Z2 in endothelium intact and denuded aortic rings pre-contracted with (a) PE (1ꢀM), (b) NE (300nM)
and (c) KCl (60mM).
Table 2. EC50and pEC50 values showing vasorelaxant effect of OXD-Z2 on aortic rings pre-contracted with PE, NE and KCl.
a
pEC50
EC50 (10-4 M) b
OXD-Z2 Against
With Endothelium
Without Endothelium
With Endothelium
Without Endothelium
PE
NE
3.49 0.06 R2=0.98
3.47 0.02 R2=0.99
3.61 0.06 R2=0.98
4.54 0.08 R2=0.98
4.33 0.04 R2=0.99
3.93 0.04 R2=0.99
3.2 (2.2-4.5)
3.3 (2.9-3.7)
2.4 (1.7-3.3)
0.28 (0.18-0.43)
0.45 (0.37-0.56)
1.1 (0.9-1.4)
KCl
a pEC50 values are mean SEM of 3 experiments
b EC50 values are expressed in 10-4 M concentration and 95% confidence interval in parentheses
*p<0.05 showed significant difference as compared to control group
Fig. (3). Effect of OXD-Z2 (100 & 300nM) on concentration-response curves of (a) PE, (b) NE and (c) KCl in rat aortic rings.
Table 3. EC50 ratio and Emax of cumulative concentration-response curve of PE, NE, KCl and Ca2+ in presence of OXD-Z2.
b
EC50 Ratio a
Emax%
OXD-Z2
(100nM)
Against
OXD-Z2
(300nM)
Against
OXD-Z2
(100nM)
Against
OXD-Z2
(300nM)
Against
CRC
PE
NE
72.21
41.46
3.6
200.1
6426
4.2
80.25
62.5
50.66
20.83
41.82
51.40
KCl
Ca2+
47.27
61.19
43.77
108.6
aEC50 ratio is the ratio of EC50 in presence of OXD-Z2 divided by EC50 of contractile agonist
bEmax is the maximum contraction by contractile agonist in presence of OXD-Z2

446 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Attari et al.
in PE, NE or KCl precontracted rings. The pD2 values of
OXD-Z2 (100nM) and OXD-Z2 (300nM) against PE-
induced contraction were found to be 7.24 and 7.27, respec-
tively. The pD2 values of OXD-Z2 (100nM) and OXD-Z2
(300nM) against NE-induced contractions were found to be
7.6 and 10.3, respectively. The pD2 values of OXD-Z2
(100nM) and OXD-Z2 (300nM) against KCl-induced con-
tractions were found to be 8.12 and 7.89, respectively.
3.5. Effect of OXD-Z2 on Cumulative Concentration-
Response Curves of PE and Ca2+ in the Aortic Rings Pre-
treated with Verapamil
Cumulative addition of PE (10pM to 3ꢃM) and Ca2+
(1ꢀM to 30mM) resulted in a concentration-dependent con-
traction of endothelium-denuded aortic ring. PE and Ca2+-
induced (Fig. 5) contractions in aortic ring were inhibited by
pretreatment with verapamil (10ꢃM) as well as OXD-Z2
(1ꢃM) with verapamil (10ꢃM). No statistical significant dif-
ference was observed between the two groups, verapamil and
OXD-Z2 with verapamil.
3.4. Effect of OXD-Z2 on Cumulative Concentration-
Response Curve of Ca2+-induced Contraction in Ca2+-free
Depolarized Kreb’s Solution
Cumulative addition of Ca2+ (1ꢀM to 30mM) produced a
concentration-dependent contraction of the aortic ring
suspended in the Ca2+-free depolarizing Kreb’s solution con-
taining 100mM KCl. Incubation of the aortic ring with
OXD-Z2 (100nM and 300nM) significantly antagonized
(Fig. 4) the Ca2+-induced contraction at p<0.05. The pD2
values of OXD-Z2 (100nM) and OXD-Z2 (300nM) against
Ca2+-induced contraction were found to be 7.63 and 7.44,
respectively. EC50 ratio indicated shift in the CRC of Ca2+ in
the presence of OXD-Z2 from CRC of Ca2+ without OXD-
Z2 with change in Emax value (Table 3).
3.6. Effect of OXD-Z2 on Vascular Tonus in Presence of
Inhibitors
Incubation of endothelium-intact aortic rings with inhibi-
tors, atropine, methylene blue and L-NAME was found to
significantly inhibit OXD-Z2-induced relaxation, whereas,
indomethacin and glibenclamide did not affect the OXD-Z2-
induced relaxation (Fig. 6). The shift in EC50 ratio indicates
the attenuation of OXD-Z2-induced vasorelaxation in pres-
ence of inhibitors. The EC50 ratio (EC50 shift) in the case of
L-NAME was less compared to other inhibitors, however,
significant difference was observed applying “Student’s t-
test” at p<0.05 (Table 4).
3.7. Molecular Docking
The docking studies of the compound OXD-Z2 were car-
ried out with L-type Ca2+-channel (1T0J.pdb) and eNOS
(3HR4.pdb and 3NOS.pdb) and for muscarinic receptor, the
derivative was docked with M2 receptor (PDB ID 3UON)
using V.Life MDS 4.2 software. The various binding interac-
tions of molecules with these receptors are shown in Table 5.
The docking result of the compound revealed that the com-
pound was energetically favourable in terms of dock score
especially with Ca2+-channel and muscarinic receptor which
was observed to be -49.5629 and -49.2814, kcal/mol respec-
tively than eNOS receptor (3NOS.pdb and 3HR4.pdb which
showed docking score of -46.1786 and –41.341 kcal/mol
respectively). The binding interaction of the compound was
found to be more favourable with M2 receptor (PDB ID
*p<0.05 showed significant difference as compared to control
group
Fig. (4). Effect of OXD-Z2 (100nM & 300nM) on concentration
response curves of Ca2+ in rat aortic rings.
*p<0.05 showed significant difference as compared to control group
Fig. (5). Effect of verapamil alone and in the presence of OXD-Z2 on cumulative concentration-response curves of (a) PE and (b) Ca2+-
induced contraction in rat aortic rings.

Vasorelaxation of Oxadiazole Derivatives in Rat Thoracic Aorta
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 447
Table 4. Effect of OXD-Z2 on vasomotor tonus in presence of various inhibitors.
Inhibitors
Atropine
Methylene Blue
L-NAME
Indomethacin
Glibenclamide
EC50 ratioa
6.89
8.37
1.04
0.52
0.32
aEC50 ratio is the ratio of EC50 in presence of OXD-Z2 plus inhibitors divided by EC50 in presence of OXD-Z2.
*p<0.05 showed significant difference as compared to control group
Fig. (6).Vasorelaxant effect of OXD-Z2 on NE-induced contraction in rat aortic rings pretreated with inhibitors; (a) Atropine, (b) Methylene
blue, (c) L-NAME, (d) Indomethacin and (e) Glibenclamide.
3UON) which showed the N- of the oxadiazole binds with
the amino acid residues, Arg 121A and Lys 383A through
hydrogen bonding and whereas C-of the pyridine ring binds
with the amino acid residue PHE1004A through ꢀ – ꢀ stack-
ing (Fig. 7). Hydrophobic and Van der Waal’s interactions
were found with various amino acids in the active site of the
receptor. These binding interactions reveal the importance of
N- of the oxadiazole and C- of the pyridine, so that the better
M2 receptor inhibitory activity was expected. Even though
the compound showed favorable binding interactions with all
other receptors, the H-bonding interaction was found only
with the M2 receptor. These studies gave us the insight for
the development of M2 receptor inhibitor in future.
4. DISCUSSION AND CONCLUSION
Eight oxadiazole derivatives with different substituents
were synthesized. An oxadiazole derivative coded as OXD-
Z2 among eight derivatives with a pyridine-3-yl ring at-
tached to carbon (C) in position 5, an acetyl group attached
to nitrogen (N) in position 3, and a toluyl group attached to
C in position 2 of the oxadiazole ring was chosen to evaluate

448 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5
Attari et al.
Table 5. Amino acids involved in the binding interaction between OXD-Z2-receptor complexes.
PDB ID
Hydrophobic Interaction
Van der Waal Interaction
1T0J
Lys 90A, Ala 335B, Pro 336B, Ile 338B, Glu 381B
Val 125A, Arg 121A, Lys 383A, Val 385A
Lys 90A, Asp 91A, Phe 92A, Arg 227A
3UON
Asn 58A, Arg 121A, Cys 124A, Pro 128A, Arg 135A
Asp 444A, Trp 445A, Asn 466A, Cys 99B, Gly 101B, Ser
102B, Asp 444B, Ala 446B, Asn 466B
Gly 101B, Ser 102B, Asp 444A, Trp 445B, Ala 446A,
Asn 466A
3NOS
3HR4
Glu 54F, Val 55F, Ala 57F, Asp 58F
Val 55F, Ala 57F, Glu 67F, Thr 70F, Met 71F, Ala 57H, Asp
58H, Glu 67H
Fig. (7). Binding interactions of OXD-Z2 with 3UON (M2-muscarinic receptor) in molecular docking studies.
mechanisms of vasorelaxation. OXD-Z2 was found to show
vasorelaxant action in phenylephrine (PE), norepinephrine
(NE) and KCl pre-contracted rat thoracic aorta with EC50
values given in Table 3. Incubation of the aortic ring with
OXD-Z2 shifted the concentration-response curve of PE, NE
and KCl towards the right showing inhibition of contraction
by OXD-Z2. The vasorelaxation produced by OXD-Z2 was
found to be slightly more in endothelium denuded aortic
rings than endothelium intact rings but not statistically sig-
nificant. Thus, it can be inferred from the results that OXD-
Z2 induced vasorelaxation may involve both endothelium-
dependent and -independent mechanisms. Therefore, a series
of ex vivo experiments were carried out to explore the
mechanism involved in OXD-Z2-induced vasorelaxation.

Vasorelaxation of Oxadiazole Derivatives in Rat Thoracic Aorta
Letters in Drug Design & Discovery, 2016, Vol. 13, No. 5 449
ing no role for K+-channel. However, it was seen earlier that
OXD-Z2 inhibited KCl-induced contraction. The above ob-
servation indicated that opening of K+-channel opening may
be involved in OXD-Z2-induced vasorelaxation at higher
concentrations [19]. Similarly, indomethacin also failed to
inhibit OXD-Z2-induced vasorelaxation, indicating that re-
laxing mediator such as prostanoids is not involved in the
OXD-Z2-induced vasorelaxation.
It is well known that PE and NE act on ꢀ-receptors and
generate inositol triphosphate (IP3) and diaceylglycerol
(DAG), releasing calcium from intracellular stores and acti-
vating protein kinase C, respectively. IP3 binds to its recep-
tors in the sarcoplasmic reticulum and releases Ca2+ to cause
contraction and opens receptor-dependent Ca2+-channel. The
contraction induced by PE and NE is due to calcium influx
either via opening of voltage-dependent Ca2+-channel or re-
ceptor-dependent Ca2+-channel [9, 11]. OXD-Z2 was found
The interaction between OXD-Z2 and various targets
were assessed by molecular docking studies. Three targets,
L-type Ca2+-channel, muscarinic receptor and eNOS were
chosen for docking studies on the basis of the above observa-
tions. eNOS was also chosen for molecular docking study, as
NO is a key player and OXD-Z2 activity was attenuated in
presence of L-NAME. The docking score ranged between -
41 to -49, which showed strong binding or interaction be-
tween OXD-Z2-receptor complexes. The compound, OXD-
Z2 showed hydrogen bonding and ꢄ – ꢄ stacking with mus-
carinic receptor.
to non-competitively antagonize PE (selective ꢀ -
1
adrenoceptor agonist), NE (non-selective ꢀ-adrenoceptor
agonist) and Ca2+-induced contraction suggesting that its
action may be through ꢀ- receptor and/or through inhibition
of Ca2+-channel. pD2 values (mentioned in the results) indi-
cated non-competitive antagonism. K+ also plays important
role in the regulation of smooth muscle membrane potential.
High K+ induces contraction of smooth muscle because of
depolarization of the cell membrane and increased calcium
influx through voltage-dependent Ca2+-channels [12]. OXD-
Z2 produced vasorelaxation in KCl-precontracted aortic ring,
which explained involvement of blockade of voltage-
dependent Ca2+-channel in the activity.
Both L-type and non L-type Ca2+-channels are involved
in the increase in calcium release during PE and NE-induced
contraction [13, 14] and verapamil blocks L-type (voltage-
dependent) Ca2+-channel in a concentration-dependent man-
ner [15]. The effect of OXD-Z2 on phenylephrine-induced
contraction in aortic rings pretreated with verapamil was
studied to investigate the role of L-type Ca2+-channel in
vasorelaxation. Pretreatment with verapamil inhibited both
PE and Ca2+-induced contractions in the aorta because of the
blockade of L-type Ca2+-channels. Incubation of OXD-Z2
along with verapamil further shifted the PE curve towards
right but failed to do so in calcium curve. The most probable
reason could be that calcium-induced contraction was com-
pletely abolished by verapamil and OXD-Z2 could not fur-
ther alter the contraction. Moreover, OXD-Z2 did not reverse
verapamil action as well and thus it can be inferred that
OXD-Z2-inhibited L-type Ca2+-channel, leading to aortic
vasorelaxation.
The role of Ca2+-channel in OXD-Z2-induced vasore-
laxation was observed in the above experiments. Another set
of experiments was performed to further explore the role of
NO in OXD-Z2 induced vasorelaxation in the endothelium
intact aorta. NO, known as endothelium derived relaxing
factor (EDRF), is associated with relaxation of blood vessels.
Various neuro-humoral and physical stimuli release NO
from endothelial cells. Various agents such as acetylcholine,
histamine, clonidine and adenosine stimulate specific endo-
thelial receptors and release NO which mediates relaxation.
NO activates soluble guanylyl cyclase and then increases the
production of cGMP, leading to protein kinase G activation.
This inhibits Ca2+ influx and produces vasorelaxation [16,
17]. The inhibitors, atropine (muscarinic receptor antago-
nist), L-NAME (NO synthase inhibitor) and methylene blue
(non-selective cGMP inhibitor) were found to inhibit the
OXD-Z2-induced relaxation suggesting that OXD-Z2 may
mediate relaxation through the release of NO-cGMP path-
way by acting on muscarinic receptor [18]. However, gliben-
clamide did not alter OXD-Z2-induced relaxation, suggest-
Thus, it can be deduced from the present ex vivo studies
on rat aortic rings and in silico molecular docking studies
that OXD-Z2 is a promising molecule which produces
vasorelaxation possibly by dual mechanisms: a) direct inhi-
bition of L-type calcium channel (endothelium-independent)
and b) agonistic action on muscarinic receptor through NO-
cGMP pathway (endothelium-dependent). However, further
studies need to be designed to assess in vivo safety and effi-
cacy in suitable animal models in order to prove its useful-
ness in cardiovascular diseases.
LIST OF ABBREVIATIONS
OXD-Z2 = methyl (toluyl) substituted-1,3,4-oxadiazole
NE
=
=
=
=
=
Norepinephrine
Phenylephrine
Calcium
PE
Ca2+
K+
Potassium
CRC
Concentration-response curve
CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flict of interest.
ACKNOWLEDGEMENTS
The authors are thankful to Manipal University for pro-
viding the facilities and instruments to perform the experi-
mental studies.
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Received: April 02, 2015
Revised: September 01, 2015
Accepted: September 11, 2015