Study on the interaction of triaryl-dihydro-1,2,4-oxadiazoles with α-glucosidase

Article abstract of DOI:10.1007/s40199-019-00322-y

Purpose: One of the therapeutic approaches in the management of Type 2 diabetes is delaying the absorption of glucose through α-glucosidase enzymes inhibition, which can reduce the incidence of postprandial hyperglycemia. The existence of chronic postpran

Full text of DOI:10.1007/s40199-019-00322-y

DARU Journal of Pharmaceutical Sciences
https://doi.org/10.1007/s40199-019-00322-y
RESEARCH ARTICLE
Study on the interaction
of triaryl-dihydro-1,2,4-oxadiazoles with α-glucosidase
Arefeh Khosravi1 & Gholamhassan Vaezi1,2 & Vida Hojati1 & Khosrou Abdi3
Received: 3 June 2019 /Accepted: 17 December 2019
#
Springer Nature Switzerland AG 2020
Abstract
Purpose One of the therapeutic approaches in the management of Type 2 diabetes is delaying the absorption of glucose through
α-glucosidase enzymes inhibition, which can reduce the incidence of postprandial hyperglycemia. The existence of chronic
postprandial hyperglycemia impaired the endogenous antioxidant defense due to inducing oxidative stress induced pancreatic β-
cell destruction through uncontrolled free radicals generation such as ROS, which in turn, leads to various macrovascular and
microvascular complications. This study aimed to synthesize 2-aryl-4,6-diarylpyrimidine derivatives, screen their α-glucosidase
inhibitory activity, perform kinetic and molecular docking studies.
Methods A series of 3,4,5-triphenyl-4,5-dihydro-1,2,4-oxadiazole derivatives were synthesized and their α-glucosidase inhib-
itory activity was screened in vitro. Compounds 6a-k were synthesized via a two-step reaction with a yield between 65 and 88%.
The structural elucidation of the synthesized derivatives was performed by different spectroscopic techniques. α-Glucosidase
inhibitory activity of the oxadiazole derivatives 6a-k was evaluated against Saccharomyces cerevisiae α-glucosidase.
Results Most of the synthesized compounds demonstrated α-glucosidase inhibitory action. Particularly compounds 6c, 6d and
6 k were the most active compounds with IC50 values 215 3, 256 3, and 295 4 μM respectively. A kinetic study performed
for compound 6c revealed that the compound is a competitive inhibitor of Saccharomyces cerevisiae α-glucosidase with Ki of
122 μM. The docking study also revealed that the two compounds, 6c and 6 k, have important binding interactions with the
enzyme active site.
Conclusion The overall results of our study reveal that the synthesized compounds could be a potential candidate in the search for
novel α-glucosidase inhibitors to manage the postprandial hyperglycemia incidence.
.
.
.
Keywords 1,2,4-oxadiazoles α-Glucosidase activity Docking Kinetic study
Background
A serious public health issue globally with multiple etiologies
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s40199-019-00322-y) contains supplementary
material, which is available to authorized users.
is Diabetes mellitus (DM) which is characterized by a failure
of glucose homeostasis. The important result of the metabolic
disturbances of carbohydrate, fat, and protein due to absolute
or relative defects in insulin secretion and/or insulin action has
been recognized as DM [1]. Type 2 diabetes mellitus (T2DM)
accounts approximately 90% of all diabetic cases, which in-
dicates a strict increase in its prevalence partly due to ageing
population, overweight, and obesity [2]. Even though T2DM
can be managed with physical exercise, diet, and modern
drugs, there are several cases need to drug therapy. The cur-
rently available oral hypoglycemic drugs exhibit adverse side
effects on an extended use including edema
(Thiazolidinediones), hypoglycemia (Sulphonylureas),
* Gholamhassan Vaezi
gh.vaezi@yahoo.com
* Khosrou Abdi
khmabdi@tums.ac.ir
1
Department of Biology, Damghan branch, Islamic Azad University,
Damghan, Iran
2
Department of Chemistry, Islamic Azad University, North Tehran
Branch, Tehran, Iran
3
Department of Medicinal Chemistry and Radiopharmacy, Tehran
University of Medical Sciences , Tehran, Iran

DARU J Pharm Sci
weight gain (Sulphonylureas and Thiazolidinediones), folate
and B12 malabsorption and lactic acidosis (Metformin), and
gastrointestinal symptom (Acarbose) [3]. The currently avail-
able α-glucosidase inhibitors, such as acarbose has showed
several side effects including hypoglycemia at higher doses,
liver problems, diarrhea, and lactic acidosis. Therefore, there
is an urgent need to discover and develop potential α-
glucosidase inhibitors in recent years.
Materials and methods
Materials
All chemicals compounds and reagents were purchased from
Merck (Darmstadt, Germany). Melting points were determined
with a Kofler hot stage apparatus (Reichert, Vienna, Austria)
1
and are uncorrected. H NMR and 13C NMR spectra were
In the management of DM, the inhibition of
carbohydrate-hydrolyzing enzymes and regulating those
constituents that cause the glucose uptake from the blood-
stream is important [4]. In T2DM when the insulin secre-
tion becomes insufficient or the cells fail to recognize the
secreted insulin, cells glucose absorption increases which
results in elevated glucose level that leads to postprandial
hyperglycemia [5]. On the other hand, long term high plas-
ma level of glucose causes to impair the endogenous anti-
oxidant agents such as glutathione, coenzyme-Q and α-
lipoic acid. Decreasing the level of this antioxidant in-
creases the level of reactive oxygen species (ROS), hy-
droxyl and nitric oxide (NO) radicals that induces pancre-
atic β-cell destruction. Furthermore, uncontrolled free rad-
icals generation leads to various microvascular,
macrovascular complications, retinopathy, cardiovascular
diseases, and nephropathy [6–9]. Thus in T2DM patients,
delaying the absorption of digestible dietary carbohydrates
is an important therapeutic approach to control the post-
prandial hyperglycemia to reduce the impact of T2DM
through α-glucosidase enzyme inhibition [10, 11]. α-
Glucosidase is the important catalyzing enzyme that in-
volves in the process of the hydrolysis of 1,4-α-glycosidic
linkages of carbohydrates and converts into monosaccha-
rides (α-glucose), which can be absorbed into the blood
[12]. The inhibition of this enzyme is the most valuable
therapeutic approaches to delay the absorption of glucose
and dropping the postprandial blood glucose level, which
can potentially reduce the impact of T2DM [11]. On these
bases, various small molecules both synthetic and isolates
of natural product extracts have been reported with poten-
tial α-glucosidase inhibitory activities [13, 14].
On the other side, there are some reports on α-glucosidase
inhibitory activity of 1,2-diaryl heterocycles. For example a
series of 5,6-diphenyl-1,2,4-triazine derivatives (Fig. 1a)
showed good activity for inhibition of α-glucosidase enzyme
[15]. Other heterocyclic rings such as imidazole and pyrrole as
central core contains 1,2-diaryl substitution (Fig. 1b, c) have
been reported as anti-hyperglycemic agents [16, 17].
With respect to the above background, in the current
research of new, simple and easily obtained compounds
that could be active against α-glucosidase, herein, we
have reported synthesis and evaluation of glucosidase
inhibitory activity of new 4,5-dihydro-3,4,5-triary-1,2,4-
oxadiazole derivatives.
recorded with Bruker FT-500 and Bruker FT-125 spectrometer
(Bruker, Darmstadt, Germany), respectively, using TMS as the
internal standard and CdCl3 as a solvent. The chemical shifts
(δ) values were expressed in parts per million (ppm), the cou-
pling constants (J) in hertz and spin multiplicities are given as s
(singlet), d (doublet), t (triplet), and m (multiplet).
Chemistry
General pSrocedure for synthesis
of 3,4,5-triaryl-4,5-dihydro-1,2,4-oxadiazole
To a solution of N-benzylideneaniline derivative (1 eq) in ether
was added N-hydroxybenzimidoyl chloride (1 eq) and
triethylamine (1.2 eq) successively. The reaction mixture was
stirred at room temperature overnight. The solvent was evaporat-
ed under reduced pressure. The residue was crystallized in acetic
acid-water to yield 3,4,5-triaryl-4,5-dihydro-1,2,4-oxadiazoles.
Compound 6a was prepared as the published procedures [18]
and the structure was confirmed with reported spectroscopic data.
3-(4-chlorophenyl)-4,5-diphenyl-4,5-dihydro-1,2,4-oxadiazole
(6b)
Yield: 73%. Mp: 133-135 °C. 1HNMR (CdCl3) δ; 6.53(s, 1H,
H5-oxadiazole), 6.81(d, J = 7.5 Hz, 2H), 7.10–7.14(m, 1H),
7.19–7.22(m, 2H), 7.32(d, J = 8 Hz, 2H), 7.45(bs, 3H), 7.55(d,
J = 8 Hz, 2H), 7.59(bs, 2H). 13CNMR (CdCl3) δ; 154.4, 141.0,
138.8, 136.6, 129.9, 129.7, 129.3, 129.0, 128.9, 127.2, 125.9,
124.3, 123.9, 100.6. Mass (M/z, (%)); 334 (45), 257(5), 228
(100), 180 (47), 105 (21), 77 (49), 57 (38). Anal. Calcd. For
C20H15ClN2O: C, 71.75; H, 4.52; N, 10.59. Found: C, 71.54;
H, 4.63; N, 10.47.
3,4-bis(4-chlorophenyl)
-5-phenyl-4,5-dihydro-1,2,4-oxadiazole (6c)
Yield: 88%. Mp: 143-144 °C. 1HNMR (CdCl3) δ; 6.49(s, 1H,
H5-oxadiazole), 6.73(d, J = 7.4 Hz, 2H), 7.16(d, J = 7.5 Hz,
2H), 7.34(d, J = 7.4 Hz, 2H), 7.46(m, 3H), 7.53(d, J = 7.5 Hz,
2H), 7.57(m, 2H). 13CNMR (CdCl3) δ; 154.1, 139.4, 138.4,
136.8, 131.5, 130.1, 129.5, 129.2, 129.2, 129.0, 127.2, 125.5,
123.5, 100.5. Mass (M/z, (%)); 368 (37), 352 (10), 246(50),
213 (11), 185 (36), 129 (44), 111 (40), 83 (77), 77 (40), 55

DARU J Pharm Sci
Fig. 1 Chemical structures of
some compounds with α-
glucosidase inhibitory and anti-
hyperglycemia activity
(100). Anal. Calcd. For C20H14Cl2N2O: C, 65.06; H, 3.82; N,
7.59. Found: C, 65.26; H, 3.61; N, 7.44.
7.05(t, 2H, J = 8.0 Hz), 7.15(d, J = 8.5 Hz, 2H), 7.27(d, J =
8.5 Hz, 2H), 7.46(d, J = 8.0 Hz, 2H), 7.56–7.60(m, 2H).
13CNMR (CdCl3) δ: 165.0, 163.0, 154.1, 139.5, 138.3,
131.4, 130.1, 130.0, 130.0, 129.4, 128.9, 127.1, 125.5,
121.2, 116.2, 116.0, 100.4, 21.4. Mass (M/z, (%)); 366 (25),
246(100, 212 (29), 95 (18), 69 (35). Anal. Calcd. For
C21H16ClFN2O: C, 68.76; H, 4.40; N, 7.64. Found: C,
68.39; H, 3.99; N, 7.78.
3-(4-chlorophenyl)-4-(4-fluorophenyl)
-5-phenyl-4,5-dihydro-1,2,4-oxadiazole (6d)
Yield: 78%. Mp: 143-145 °C. 1HNMR (CdCl3) δ; 6.45(s, 1H,
H5-oxadiazole), 6.78–6.82(m, 2H), 6.88–6.92(t, 2H, J = 9.0),
7.32(d, J = 8.5 Hz, 2H), 7.45–7.48(m, 3H), 7.52(d, J = 8.5 Hz,
2H), 7.55–7.60(m, 2H). 13CNMR (CdCl3) δ; 161.7, 154.6,
138.3, 137.0, 136.7, 130.00, 129.6, 129.2, 129.0, 128.9,
127.3, 126.8, 126.8, 123.6, 116.4, 116.3, 101.0. Mass (M/z,
(%)); 352 (25), 289 (19), 246 (100), 198 (27), 153 (21). Anal.
Calcd. For C20H14ClFN2O: C, 68.09; H, 4.00; N, 7.94. Found:
C, 68.31; H, 3.91; N, 7.67.
3-(4-chlorophenyl)-4-(4-fluorophenyl)-5-(p-tolyl)
-4,5-dihydro-1,2,4-oxadiazole (6 g)
Yield: 65%. Mp: 125-128 °C. 1HNMR (CdCl3) δ; 2.38(s, 3H,
CH3), 6.42(s, 1H, H5-oxadiazole), 6.72–6.808(m, 2H), 6.89(t,
J = 7.1 Hz), 2H), 7.26(d, J = 8.0 Hz, 2H), 7.33(d, J = 8.5 Hz,
2H), 7.45(d, J = 8.0 Hz, 2H), 7.51(d, J = 8.5 Hz, 2H).13CNMR
(CdCl3) δ; 161.6, 159.7, 154.6, 140.1, 136.9, 136.6, 135.3,
129.6, 129.2, 129.0, 127.3, 126.8, 126.7, 123.7, 116.4,
116.2, 101.0, 21.4. Mass (M/z, (%)); 366 (18), 246(100),
213 (32), 95 (27), 69 (34). Anal. Calcd. For C21H16ClFN2O:
C, 68.76; H, 4.40; N, 7.64. Found: C, 68.46; H, 4.19; N, 7.39.
3,4-bis(4-chlorophenyl)-5-(p-tolyl)
-4,5-dihydro-1,2,4-oxadiazole (6e)
Yield: 86%. Mp: 120-122 °C. 1HNMR (CdCl3) δ; 2.41(s, 3H,
CH3), 6.46(s, 1H, H5-oxadiazole), 6.72(d, J = 8.5 Hz, 2H),
7.16(d, J = 8.5 Hz, 2H), 7.26(d, J = 8.0 Hz, 2H), 7.34(d, J =
8.0 Hz, 2H), 7.45(d, J = 8.0 Hz, 2H), 7.52(d, J = 8.0 Hz, 2H).
13CNMR (CdCl3) δ; 154.1, 140.1, 139.4, 136.8, 135.3, 131.4,
129.6, 129.4, 129.2, 129.1, 127.1, 125.5, 123.6, 100.5, 21.4.
Mass (M/z, (%)); 382 (33), 262(100), 229 (34), 192 (11), 125
(17), 91 (18). Anal. Calcd. For C21H16Cl2N2O: C, 65.81; H,
4.21; N, 7.31. Found: C, 65.66; H, 4.09; N, 7.25.
3-(4-chlorophenyl)-5-(4-methoxyphenyl)
-4-phenyl-4,5-dihydro-1,2,4-oxadiazole (6 h)
Yield: 72%. Mp: 119-122 °C. 1HNMR (CdCl3) δ; 3.85(s, 3H,
OCH3), 6.49(s, 1H, H5-oxadiazole), 6.79(d, J = 7.5 Hz, 2H),
6.96 (d, J = 8.0 Hz, 2H), 7.13(m, 1H), 7.19(t, J = 7.5 Hz, 2H),
7.31(d, J = 8.0 Hz, 2H), 7.50–7.56(m, 4H). 13CNMR (CdCl3)
δ; 160.83, 154.36, 140.86, 136.49, 130.80, 129.30, 129.23,
128.97, 128.70, 125.86, 124.44, 124.06, 114.21, 100.52,
55.35. Mass (M/z, (%)); 364 (36), 228 (100), 216 (41), 1135
(29), 77 (39). Anal. Calcd. For C21H17ClN2O2: C, 69.14; H,
4.70; N, 7.68. Found: C, 69.26; H, 4.55; N, 7.54.
4-(4-chlorophenyl)-3-(4-fluorophenyl)-5-(p-tolyl)
-4,5-dihydro-1,2,4-oxadiazole (6f)
1
Yield: 86%. Mp: 80-82 °C. HNMR (CdCl3) δ; 2.43(s, 3H,
CH3), 6.46(s, 1H, H5-oxadiazole), 6.72(d, J = 8.5 Hz, 2H),

DARU J Pharm Sci
3-(4-chlorophenyl)-4-(4-fluorophenyl)-5-(4-methoxyphenyl)
-4,5-dihydro-1,2,4-oxadiazole (6i)
concentrations of the target compounds 6a-k (20 μL), and
135 μL of potassium phosphate buffer were added to the 96-
well plate and incubated at 37 °C for 10 min. Then, 25 μL of
4 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.05 M
phosphate buffer (pH = 6.8) was added to each well and the
reaction mixture was allowed to incubate for 20 min at 37 °C.
Finally, the reaction was terminated by adding 50 μL of 0.2 M
sodium carbonate solution and then, the change in absorbance
was measured at 405 nm with a spectrophotometer (Gen5,
Power wave xs2, BioTek, America). DMSO and acarbose
were used as the control and standard inhibitor, respectively.
The α-glucosidase inhibitory activity was expressed as the
percentage of enzyme inhibition for each synthesized com-
pound and calculated by the following formula [19, 20]:
% Inhibition = (Control Absorption-Sample Absorption)/
(Control Absorption) × 100.
Yield: 76%. Mp: 98-102 °C. 1HNMR (CdCl3) δ; 3.88(s, 3H,
OCH3), 6.40(s, 1H, H5-oxadiazole), 6.76–6.79(m, 2H), 6.89
(t, J = 9.0 Hz, 2H), 6.97(d, J = 8.5 Hz, 2H), 7.32(d, J = 8.5 Hz,
2H), 7.49(d, J = 8.0 Hz, 4H). 13CNMR (CdCl3) δ; 161.7,
160.9, 159.7, 154.6, 136.8, 136.4, 130.2, 129.2, 129.0,
128.9, 126.9, 126.9, 123.7, 116.4, 116.2, 114.2, 100.9, 55.4.
Mass (M/z, (%)); 382 (13), 258(50), 246 (88), 138 (54), 123
(100), 95 (52), 69 (41). Anal. Calcd. For C21H16ClFN2O2: C,
65.89; H, 4.21; N, 7.32. Found: C, 65.53; H, 4.11; N, 7.46.
4-(4-chlorophenyl)-3-(4-fluorophenyl)-5-(4-methoxyphenyl)
-4,5-dihydro-1,2,4-oxadiazole (6j)
1
Yield: 71%. Mp: 80-83 °C. HNMR (CdCl3) δ; 3.88(s, 3H,
The concentration of the target compounds required to in-
hibit 50% of the enzyme activity (IC50) values was obtained
from non-linear regression curve using the Logit method.
OCH3), 6.45(s, 1H, H5-oxadiazole), 6.71(d, J = 8.5 Hz, 2H),
6.97 (d, J = 8.5 Hz, 2H), 7.05(t, J = 9.0 Hz, 2H), 7.17(d, J =
8.5 Hz, 2H), 7.49(d, J = 9.0 Hz, 2H), 7.55(m, 2H). 13CNMR
(CdCl3) δ; 165.0, 163.0, 154.0, 140.1, 139.5, 135.4, 131.3,
130.1, 130.0, 129.6, 129.4, 127.2, 125.5, 121.4, 116.1,
116.0, 100.4, 54.9. Mass (M/z, (%)); 382 (21), 246 (100),
233 (19), 135 (13). Anal. Calcd. For C21H16ClFN2O2: C,
65.89; H, 4.21; N, 7.32. Found: C, 65.73; H, 3.98; N, 7.40.
Enzyme kinetic studies
The mode of inhibition of the most active compound (6c),
identified with the lowest IC50, was investigated against
alpha-glucosidase activity with different concentrations of p-
nitrophenyl α -D-glucopyranoside (2–10 mM) as substrate in
the absence and presence of 6c at different concentrations (0,
105, 160 and 215 μM). A Lineweaver–Burk plot was generated
to identify the type of inhibition and the Michaelis–Menten
constant (Km) value was determined from plot between recip-
rocal of the substrate concentration (1/[S]) and reciprocal of
enzyme rate (1/V) over various inhibitor concentrations.
Experimental inhibitor constant (Ki) value was constructed by
secondary plots of the inhibitor concentration [I] versus Km.
3,4-bis(4-chlorophenyl)-5-(4-methoxyphenyl)
-4,5-dihydro-1,2,4-oxadiazole (6 k)
1
Yield: 66%. Mp: 79-81 °C. HNMR (CdCl3) δ; 3.85(s, 3H,
OCH3), 6.45(s, 1H, H5-oxadiazole), 6.71(d, J = 9.0 Hz, 2H),
6.96(d, J = 9.0 Hz, 2H), 7.15(d, J = 8.5 Hz, 2H), 7.34(d, J =
8.5 Hz, 2H), 7.48–7.51(m, 4H). 13CNMR (CdCl3) δ; 160.4,
159.1, 139.3, 136.7, 131.5, 130.2, 129.4, 129.2, 129.1, 128.7,
125.7, 123.7, 114.3, 100.5, 55.4. Mass (M/z, (%)); 398 (20),
262(100), 246 (230), 135 (19), 111 (17). Anal. Calcd. For
C21H16Cl2N2O2: C, 63.17; H, 4.04; N, 7.02. Found: C,
63.36; H, 3.91; N, 7.29.
Molecular docking
To develop more understanding in the mode of binding of the
synthesized compounds, molecular docking studies were car-
ried out. AutoDock version 4.2.6, AutoDock Tools (ADT)
version 1.5.6 and Discovery studio v16.1.0.15350 were used
to conduct the docking studies. The most potent compounds
from the series was generated as ligand with their 3D struc-
ture, 6c and 6d using chemDraw Ultra 12.0.2 version of
Cambridge University. Since there is no report yet about the
crystal structure of α-glucosidase from Saccharomyces
cerevisiae for preparing the protein for docking, we retrieved
the crystal structure of isomaltase from Saccharomyces
cerevisiae (PDB ID: 3A4A; Resolution 1.6 Å) from protein
Data Bank (www.rcsb.org/pdb/). Then, the protein structure
was prepared by removing the water molecules and original
inhibitors. The ligand and protein pdbqt files were prepared
and grid box formation was accomplished using AutoDock
Biological evaluation
In vitro α-glucosidase inhibition activity
The α-glucosidase inhibitory activity of the synthesized com-
pounds was evaluated using p-nitrophenyl-α-D-
glucopyranoside (pNPG) as a substrate based on previously
reported method (Nikookar et al., 2018). The α-glucosidase
enzyme (EC3.2.1.20, Saccharomyces cerevisiae, 20 U/mg)
and substrate (pNPG) were purchased from Sigma-Aldrich.
The desired concentrations of enzyme were prepared in potas-
sium phosphate buffer (pH 6.8, 50 mM), and the target com-
pounds 6a-k were dissolved in DMSO (10% final concentra-
tion). A volume of 20 μL of α-glucosidase solution, different

DARU J Pharm Sci
Tools. AutoGrid was used in order to prepare the grid map
using a grid box. The dimension of the grid size was set to
60 × 60 × 60 with grid spacing of 0.375 Å. The center of grid
box was designated in coordinates x = 21.277, y = −0.773,
and z = 18.648. Fifty runs of the AUTODOCK search by the
Lamarckian genetic algorithm were performed for each
docked system. So as to analyze the interactions between the
target enzyme and the inhibitor, the best pose of each ligand
was designated and the result was visualized with Discovery
Studio Visualizer v16.1.0.15350 (Accelrys, San Diego, USA).
preparation of 1,2,4-oxadiazoles is involved of the 1,3-dipolar
cycloaddition of benzonitrile oxide and imines [18]. There are
some reports that describe the dimerization of benzonitrile ox-
ide within a few minutes to several days related to nature of
substituted group in aryl ring [21]. As illustrated in scheme 1,
an easy method for preparation of the final compounds (6)
from condensation of 4-substituted benzohydroxyiminoyl
chlorides (5) and imines (3) in diethyl ether was used. 4-
Substituted benzohydroxyiminoyl chlorides were prepared
from arylaldehyde oximes and N-chlorosuccinimide in DMF
as reported procedure [22]. The compounds were characterized
by 1H and 13C nuclear magnetic resonance, Mass spectroscopy
and CHN analysis. In 1HNMR spectra of the derivatives (6) a
typical singlet signal at 6.45–6.52 ppm range is attributed to
the H5-oxadiazole ring. Other marker peaks in 13CNMR spec-
tra of compound 6 are the signals at 123 and 100 ppm that has
been referred to C3 and C5 of oxadiazole ring, respectively.
General procedure for preparation of 6a-k was reported.
Results and discussion
Chemistry
The procedure for preparation of compounds 6 has been illus-
trated In Scheme 1. One of the reported synthesis route for
Scheme 1 Synthesis pathway for preparation of 6a-k

DARU J Pharm Sci
In vitro α-glucosidase inhibition activity
for 6a-k than the standard drug, acarbose. Optimization of the
α-glucosidase inhibitory activity of 3,4,5-triaryl 1,2,4-
oxadiazoles derivatives was performed using different substi-
tution on 4 position of the phenyl rings. The differences in the
pattern of inhibition of the synthesized compounds are mainly
due to the nature of the substituents on the phenyl moiety.
The most potent compounds were 6c, 6d, and 6 k with IC50
values 215 3, 256 3 and, 295 4 μM, respectively while
the IC50 value of acarbose was 750 8.7 μM (Table 1). The
other target compounds showed a moderate inhibitory activity
around 1.64–2.45 folds more than acarbose with the IC50
values in the range of 305 4 to 455 6 μM. The comparison
between activities of synthesized compounds shows that the
substitution on the aryl ring is critical for inhibition of α-
glucosidase as compound 6a was completely inactive in
2 mM. Substitution on R3 especially chlorine atom increases
the activity. The best group in R1 is hydrogen if chlorobenzene
has been substituted as the aryl number 3. In this case, both of
The activity of the target compounds 6a-k was assessed in vitro
against yeast α-glucosidase (EC 3.2.1.20) inhibition. The usage
of this source of enzyme has been reported for inhibitory prop-
erties of α-glucosidase in numerous papers [15, 16, 19, 23–25,
and]. A slightly different classification of α-glucosidases that is
based on recognition of homologous sites of the enzymes’ amino
acid sequences has been reported [26]. 3D structures have also
been obtained for the enzymes from A. oryzae, pig pancreas,
A. niger, barley, and Pseudomonas stutzeri. All these structures
have A similar topological features has been found in all these
structres. In all these enzymes, the active site is a slit formed at
the C-terminus of the β-sheet of the (β/α) 8 barrel [27].
Furthermore, a closeness results has been reported for assessment
of α-glucosidase inhibitory activity using two different kind of
enzyme (α-glucosidase EC 3.2.1.20; Sigma and rat intestinal α-
glucosidase source). For kaempferol, a natural product separated
from herb, IC50 were 61 μM and 59 μM respectively. The per-
centage of enzyme inhibition at concentration of 0.83 μM was
reported as 68% and 55% respectively [28]. In this study, yeast
α-glucosidase was used as a eukaryotic enzyme. Compound 6c
was bound to active site on the enzyme and competed with the
substrate for binding to the active site, so the type of inhibition
was competitive inhibition. The results were compared with
acarbose, the marketed α-glucosidase inhibitor, as a standard
drug. The activity of acarbose has been reported in a range of
600–800 μM related to source of the enzyme. In our experiments
a 750 12 μM was found for IC50 of acarbose in triplicated tests.
The in vitro α-glucosidase inhibitory activity was carried out
based on the previously reported method [23]. The results were
described as the concentration of the target compounds required
to inhibit 50% of the α-glucosidase activity (IC50 values) [29]
and are summarized in Table 1.
The biological activity of the target compounds 6a-k was
illustrated in Table 1 that shows the better inhibitory activity
Table 1 α-Glucosidase inhibition assay of 6a-k
Compounds
R1
R2
R3
IC50(μM)
Acarbose
6a
750 12
inactive
H
H
H
Cl
F
H
6b
H
Cl
Cl
Cl
Cl
F
346
215
256
455
318
362
305
444
314
295
5
3
3
6
4
5
4
6
4
4
6c
H
6d
H
6e
Me
Cl
Cl
F
6f
Me
6 g
6 h
6i
Me
Cl
Cl
Cl
F
OCH3
OCH3
OCH3
OCH3
H
F
Fig. 2 Kinetics of alpha-glucosidase inhibition by 6c. (a) The
Lineweaver-Burk plot in the absence and presence of different concen-
trations of 6c; (b) The secondary plot between Km and various concen-
trations of 6c
6j
Cl
Cl
6 k
Cl

DARU J Pharm Sci
Fig. 3 Possible mode of interaction between the most potent compounds, 6c and 6d, and the enzyme active site
chlorine and fluorine atom has been tolerated as R2 position
and show better activity in comparison of hydrogen.
molecular docking techniques are used to assess the binding
affinity and orientation of drug molecules in the possible tar-
get’s site. Basically, the goal of molecular docking is accurate
structural modeling and correct prediction of activity.
Therefore, molecular docking studies have shaped the ap-
proach of drug design to a situation where the structure of
the drug molecule will be designed based on the information
obtained from the 3D binding fit on the receptor site [30].
After biological investigations, the possible mode of inter-
action between the most potent compounds, 6c and 6d with the
enzyme active site were check by docking study. In the case of
compound 6c, PHE303, PRO312 and ARG315 residues of the
active site had П-alkyl interaction with the 6c. ASP307 residue
has also showed the П-anion interaction with the 6c. There are
also some other van der waals interaction between 6c and other
residues in the active site. For compound 6d, PRO312 and
ARG315 residues showed П-alkyl interaction with the 6d.
HIS280 and ASP307 residues had also showed the П-anion
Enzyme kinetic studies
AccordingtoFig.2a,theLineweaver-BurkplotshowedthattheKm
gradually increased and Vmax remained unchanged with increasing
inhibitor concentration indicating a competitive inhibition. The
results show 6c bind to active site on the enzyme and compete with
the substrate for binding to the active site. Furthermore, plot of the
Km versus different concentration of inhibitor gave an estimate of
the inhibition constant, Ki of 213 μM (Fig. 2b).
Docking study
In the modern area of drug discovery, molecular docking tech-
niques have been widely applied in order to understand the
interaction of the drugs with the receptors. In particular

DARU J Pharm Sci
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Conclusion
In summary, a series of triaryl-dihydro-1,2,4-oxadiazoles de-
rivatives were synthesized via condensation of N-
benzylideneaniline derivative and N-hydroxybenzimidoyl
chloride in diethyl ether in triethylamine. The reported deriv-
atives exhibited α-glucosidase inhibition activity, among of
them were better than reference compound (acarbose).
Compounds 6c, 6d, and 6 k showed potent activity among
the series. Compound 6c bearing chlorine substituent on R2
and R3 exhibited the most potent α-glucosidase inhibitory
activity with an IC50 value of 215 3 μM. The kinetic study
showed that 6c bind to active site on the enzyme and compet-
ed with the substrate for binding to the active site.
Furthermore, plot of the Km versus different concentration of
inhibitor gave an estimate of the inhibition constant, Ki of
213 μM. Similarly, the molecular docking study also revealed
that the two compounds have important binding interactions
with the enzyme active site and can bind to the enzyme freely
than the reference standard acarbose. Possible mode of inter-
action between the most potent compounds, 6c and 6d, with
the active site of enzyme was studied. The П-anion and П-
alkyl interaction were distinguished between 6c and 6d with
the residue in the active site. The outcomes from the molecular
docking studies of compounds 6c and 6d supported the results
obtained from the in vitro assay. In conclusion, the synthe-
sized compounds of this class of heterocyclic motifs have
shown promising results for further development of potent,
selective and efficacious inhibitors against the α-glucosidase
enzyme that could be a potential candidate for design anti-
diabetes new agents.
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Acknowledgements The authors would like to express their thankfulness
to Tehran University of Medical Sciences for providing the research data
facilities.
17. Goel A, Agarwal N, Singh FV, Sharon A, Tiwari P, Dixit M, et al.
Antihyperglycemic activity of 2-methyl-3,4,5-triaryl-1H-pyrroles
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Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
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