Synthesis, in vitro and in silico enzymatic inhibition assays, and toxicity evaluations of new 4,5-diphenylimidazole-N-phenylacetamide derivatives as potent α-glucosidase inhibitors

Article abstract of DOI:10.1007/s00044-021-02734-5

α-Glucosidase is responsible for glucose release of oligosaccharides and disaccharides in the intestine and increase postprandial hyperglycemia. Inhibition of this enzyme is a beneficial therapeutic method for glycemic control in diabetes. This study deals with the design and synthesis of 4,5-diphenylimidazole-N-phenylacetamide derivatives 7a–l and the screen of these compounds for their potential for α-glucosidase inhibition. All the synthesized compounds exhibited superior α-glucosidase inhibition (IC₅₀ = 90.0–598.5 μM) as compared to standard inhibitor acarbose (IC₅₀ = 750.0 μM). In contrast, these compounds were inactive against α-amylase. Among the synthesized compounds, compound 7h was the most potent inhibitor of this library and was a competitive inhibitor into α-glucosidase with K_i value = 86.3 μM. Docking study of the most potent compounds was performed to evaluate the binding interactions of these compounds with the active site of enzyme and to determine of binding energies of ligand–enzyme complexes. The results of this in silico study are in complete agreement with the results obtained from in vitro α-glucosidase inhibition assay. Docking study of the most potent compound demonstrated that it interacted with important residues in the active site of α-glucosidase. In vitro cytotoxic activity of the most potent compounds and in silico druglikeness/ADME/toxicity study of these compounds were evaluated.

Full text of DOI:10.1007/s00044-021-02734-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:1273–1283
https://doi.org/10.1007/s00044-021-02734-5
ORIGINAL RESEARCH
Synthesis, in vitro and in silico enzymatic inhibition assays, and
toxicity evaluations of new 4,5-diphenylimidazole-N-
phenylacetamide derivatives as potent α-glucosidase inhibitors
1
Atefeh Nikraftar2 Mohammad Sadegh Asgari3 Mehdi Emadi4
● ● ●
●
●
●
●
Maryam Mohammadi-Khanaposhtani
Somayeh Mojtabavi5 Mohammad Ali Faramarzi5 Hossein Rastegar6 Bagher Larijani7 Mohammad Mahdavi7
●
Received: 16 December 2020 / Accepted: 28 April 2021 / Published online: 15 May 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
α-Glucosidase is responsible for glucose release of oligosaccharides and disaccharides in the intestine and increase
postprandial hyperglycemia. Inhibition of this enzyme is a beneficial therapeutic method for glycemic control in diabetes.
This study deals with the design and synthesis of 4,5-diphenylimidazole-N-phenylacetamide derivatives 7a–l and the screen
of these compounds for their potential for α-glucosidase inhibition. All the synthesized compounds exhibited superior α-
glucosidase inhibition (IC50 = 90.0–598.5 µM) as compared to standard inhibitor acarbose (IC50 = 750.0 µM). In contrast,
these compounds were inactive against α-amylase. Among the synthesized compounds, compound 7h was the most potent
inhibitor of this library and was a competitive inhibitor into α-glucosidase with Ki value = 86.3 μM. Docking study of the
most potent compounds was performed to evaluate the binding interactions of these compounds with the active site of
enzyme and to determine of binding energies of ligand–enzyme complexes. The results of this in silico study are in complete
agreement with the results obtained from in vitro α-glucosidase inhibition assay. Docking study of the most potent
compound demonstrated that it interacted with important residues in the active site of α-glucosidase. In vitro cytotoxic
activity of the most potent compounds and in silico druglikeness/ADME/toxicity study of these compounds were evaluated.
●
●
●
Keywords α-Glucosidase 4,5-Diphenylimidazole N-Phenylacetamide Molecular modeling
Introduction
classified to two main types: type 1, that is, insulin dependent
and due to defects in pancreatic β-cells in insulin secretion
and type 2 diabetes, that is, non-insulin dependent and insulin
resistance in body cells is usually observed in it [2, 3]. The
high prevalence and increasing number of diabetic patients
has led to the use of various treatment methods to control
blood sugar in these patients. The main treatment for type 1
diabetes is insulin, and the main treatment for type 2 diabetes
Diabetes mellitus is a metabolic disorder that affect people of
different ages, from children to the elderly [1]. This disease is
Supplementary information The online version contains
supplementary material available at https://doi.org/10.1007/s00044-
021-02734-5.
* Maryam Mohammadi-Khanaposhtani
4
Electrical and Computer Engineering Department, Babol
Noshirvani University of Technology, Babol, Iran
maryammoha@gmail.com
* Mohammad Mahdavi
5
Department of Pharmaceutical Biotechnology, Faculty of
Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
momahdavi@tums.ac.ir
6
Cosmetic Products Research Center, Iranian Food and Drug
Administration, MOHE, Tehran, Iran
1
Cellular and Molecular Biology Research Center, Health Research
Institute, Babol University of Medical Sciences, Babol, Iran
7
Endocrinology and Metabolism Research Center, Endocrinology
and Metabolism Clinical Sciences Institute, Tehran University of
Medical Sciences, Tehran, Iran
2
Department of Medicinal Chemistry, Faculty of Pharmacy, Islamic
Azad University, Ayatollah Amoli Branch, Amol, Iran
3
School of Chemistry, College of Science, University of Tehran,
Tehran, Iran

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Fig. 1 Hydrolysis of
carbohydrates by α-amylase and
α-glucosidase
is oral medications that lower postprandial hyperglycemia
[4, 5]. One of the important classes of the latter medications
are α-glucosidase inhibitors, such as acarbose, voglibose, and
miglitol [6]. These drugs effectively inhibited α-glucosidase
that is responsible for breaking down oligosaccharides and
disaccharides to monosaccharides and decreased glucose level
in blood [7]. Acarbose showed intestinal complications such
as flatulence, bloating, cramping, and abdominal pain that
several enzymatic studies have suggested that α-amylase
inhibition is the cause of these side effects [8]. α-Amylase is
an important carbohydrate hydrolyzing enzyme that converts
polysaccharides to oligosaccharides and disaccharides. α-
Amylase inhibition in addition to helping to lower blood
glucose leads to the secretion of undecomposed poly-
saccharides to long intestine and causes intestinal complica-
tions. Mechanism of actions of α-glucosidase and α-amylase
is schematically shown in Fig. 1.
derivative also exhibited various biological activities [11].
This moiety is found in the several potent α-glucosidase
inhibitors, such as compounds A and B (Fig. 2) [15, 16].
Furthermore, 4,5-diphenylimidazole-2-phenoxy derivatives
C also exhibited high inhibitory activity against α-glucosi-
dase [17]. On the other hand, N-phenylacetamid moiety is
found in the several series of new potent α-glucosidase
inhibitors such as compounds D–F (Fig. 2) [18–20]. There-
fore, herein, novel structures 7 by connecting 4,5-dipheny-
limidazole-2-phenoxy to N-phenylacetamid were designed,
synthesized, and evaluated as new α-glucosidase inhibitors.
Material and methods
General chemistry
Nitrogen-containing heterocycles have broad range of
biological activities and are important blokes for drug design
[9]. One of the most important of these heterocycles is imi-
dazole, which is found broadly in the natural and synthetic
molecules with biological properties, such as antifungal, anti-
inflammatory, antitubercular, anticancer, and antiviral activ-
ities [10–14]. 4,5-Diphenylimidazole as a valuable imidazole
Melting points of 4,5-diphenylimidazole-N-phenylacetamide
derivatives 7a–l were carried out on a Kofler hot stage
apparatus and are uncorrected. Infrared spectra were recorded
using KBr disks for solid materials on a Nicolet Magna FTIR
1
550 instrument. The H nuclear magnetic resonance (NMR)
and 13C NMR spectra were run on a Bruker FT-500. Com-
pounds were dissolved in dimethyl sulfoxide (DMSO)-d6, and

Medicinal Chemistry Research (2021) 30:1273–1283
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Fig. 2 Design strategy for 4,5-
diphenylimidazole-N-
phenylacetamide derivatives 7
as new α-glucosidase inhibitors
chemical shifts were referenced to tetramethylsilane. Mass
spectra were obtained on an Agilent Technology (HP; MS
Model: 5973 Network Mass Selective Detector) analysis
system. Elemental analyses were performed on an Elementar
Analysensysteme GmbH Vario EL CHNS mode instrument.
dimethylformamide (DMF) were stirred at 100 °C for 8 h.
Then the reaction mixture was poured into cold water, the
precipitate was filtered off, and washed with water to afford
4,5-diphenylimidazole-N-phenylacetamide derivatives 7a–l.
2-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)-N-
General procedure for the synthesis of 2-chloro-N-
phenylacetamide derivatives (3a–l)
phenylacetamide (7a)
White solid; isolated yield: 82%, mp 179–181 °C. Infrared
1
A mixture of aniline derivatives 1a–l (1 mmol) and chlor-
oacetyl chloride 2 (1.2 mmol) in acetone (15 mL) was stir-
red at room temperature for 30 min. Then the reaction
mixture was poured in water and the obtained precipitate
was filtered and washed with water to obtain pure 2-chloro-
N-phenylacetamide derivatives 3a–l.
(IR) (KBr, υ): 3289, 3052, 2935, 1637 cm−1. H NMR
(500 MHz, DMSO-d6) δ 12.54 (s, 1H), 8.03 (d, J = 8.5 Hz,
2H), 7.73 (d, J = 7.6 Hz, 2H), 7.59–7.56 (m, 3H),
7.54–7.52 (m, 4H), 7.45–7.42 (m, 2H), 7.41–7.32 (m, 5H),
7.08 (d, J = 8.6 Hz, 2H), 4.51 (s, 2H, O-CH2). 13C NMR
(126 MHz, DMSO-d6) δ 170.27, 164.37, 158.38, 145.97,
139.69, 132.89, 130.98, 130.84, 130.58, 129.53, 129.40,
129.25, 129.14, 129.00, 128.95, 128.73, 127.10, 125.66,
124.15, 115.36, 67.25. MS (70 eV, m/z [M+]) = 445. Anal
Calcd for C29H23N3O2, C, 78.18, H, 5.20, N, 9.43. Found C,
78.16, H, 5.27, N, 9.41.
General procedure for the synthesis of 2-(4-
formylphenoxy)-N-phenylacetamides (5a–l)
A suspension of 2-chloro-N-phenylacetamide derivatives 3a–l
(1 mmol), 4-hydroxybenzaldehyde 4 (1 mmol), KI (1 mmol),
and K2CO3 (2 mmol) in acetone (15 mL) was stirred at 80 °C
for 4 h. Similar to previous step, the reaction mixture was
poured in water and residue was filtered and washed with water
to obtain pure 2-(4-formylphenoxy)-N-phenylacetamides 5a–l.
2-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)-N-(p-tolyl)
acetamide (7b)
White solid; isolated yield: 84%, mp 204–206 °C. IR (KBr,
υ): 3277, 3069, 2921, 1655 cm−1 1H NMR (500 MHz,
.
General procedure for the synthesis of 4,5-
diphenylimidazole-N-phenylacetamide derivatives
(7a–l)
Chloroform-d) δ 12.84 (s, 1H), 7.99 (d, J = 8.7 Hz, 2H),
7.88 (d, J = 7.4 Hz, 4H), 7.78–7.68 (m, 1H), 7.58–7.55 (m,
1H), 7.49–7.44 (m, 4H), 7.44–7.38 (m, 3H), 7.35 (d, J =
8.8 Hz, 2H), 7.30–7.27 (m, 1H), 7.16–7.08 (m, 1H), 5.25 (s,
2H, O-CH2), 2.17 (s, 3H, CH3). 13C NMR (126 MHz,
DMSO-d6) δ 170.58, 158.40, 145.97, 138.41, 136.47,
2-(4-Formylphenoxy)-N-phenylacetamides 5a–l (1 mmol),
benzil 6 (1 mmol), ammonium acetate (10 mmol), and

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134.01, 132.59, 131.43, 128.86, 128.19, 127.51, 127.14,
125.34, 124.03, 122.20, 120.94, 119.65, 118.15, 115.36,
115.12, 115.12, 65.02, 19.10. MS (70 eV, m/z [M+]) = 459.
Anal Calcd for C30H25N3O2, C, 78.41, H, 5.48, N, 9.14.
Found C, 78.44, H, 5.41, N, 9.13.
N-(2,6-dichlorophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-2-yl)
phenoxy)acetamide (7f)
White solid; isolated yield: 84%, mp 199–201 °C. IR (KBr,
υ): 3336, 2953, 1649 cm−1. 1H NMR (500 MHz, DMSO-d6)
δ 12.58 (s, 1H), 8.06 (d, J = 8.4 Hz, 2H), 7.64–7.47 (m,
8H), 7.46–7.32 (m, 6H), 7.17 (d, J = 8.9 Hz, 2H), 4.85 (s,
2H, O-CH2). 13C NMR (126 MHz, DMSO-d6) δ 167.62,
157.75, 147.99, 145.83, 140.30, 138.80, 136.48, 135.52,
133.87, 132.96, 132.31, 130.44, 128.85, 128.24, 127.24,
125.91, 125.44, 124.72, 124.22, 115.50, 67.61. MS (70 eV,
m/z [M+]) = 514. Anal Calcd for C29H21Cl2N3O2, C, 67.71,
H, 4.11, N, 8.17. Found C, 67.76, H, 4.12, N, 8.15.
2-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)-N-(4-
ethylphenyl)acetamide (7c)
White solid; isolated yield: 83%, mp 167–169 °C. IR
(KBr, υ): 3271, 3073, 2938, 1631 cm−1 1H NMR
.
(500 MHz, DMSO-d6) δ 12.67 (s, 1H), 8.10–7.95 (m,
3H), 7.58–7.50 (m, 5H), 7.43–7.35 (m, 5H), 7.33–7.24
(m, 3H), 7.11–6.95 (m, 3H), 4.77 (s, 2H, O-CH2), 2.69
(q, J = 7.0 Hz, 2H, CH2), 1.26 (t, J = 7.2 Hz, 3H, CH3).
13C NMR (126 MHz, DMSO-d6) δ 170.59, 158.42,
145.98, 138.95, 136.90, 134.85, 133.82, 132.77, 131.52,
129.98, 128.86, 128.19, 127.52, 127.16, 125.62, 124.00,
121.56, 119.57, 117.80, 115.12, 65.02, 56.52, 19.03. MS
(70 eV, m/z [M+]) = 473. Anal Calcd for C31H27N3O2, C,
78.62, H, 5.75, N, 8.87. Found C, 78.58, H, 5.67,
N, 8.82.
N-(2-bromophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-2-yl)
phenoxy)acetamide (7g)
White solid; isolated yield: 85%, mp 236–238 °C. IR (KBr, υ):
1
3326, 2987, 1648 cm−1. H NMR (500 MHz, DMSO-d6) δ
12.59 (s, 1H), 8.10 (d, J = 7.8 Hz, 2H), 7.89 (d, J = 7.5 Hz,
1H), 7.71 (d, J = 7.6 Hz, 1H), 7.60–7.50 (m, 4H), 7.49–7.27
(m, 7H), 7.25–7.03 (m, 4H), 4.87 (s, 2H, O-CH2). 13C NMR
(126 MHz, DMSO-d6) δ 167.12, 157.99, 145.91, 140.26,
137.61, 135.86, 134.33, 133.14, 131.59, 130.38, 128.77,
128.43, 127.79, 127.47, 127.23, 125.87, 124.60, 123.43,
121.85, 120.32, 117.44, 115.52, 115.52, 67.56. MS (70 eV, m/
z [M+]) = 523. Anal Calcd for C29H22BrN3O2, C, 66.42, H,
4.23, N, 8.01. Found C, 66.44, H, 4.21, N, 8.08.
2-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)-N-(4-
methoxyphenyl)acetamide (7d)
White solid; isolated yield: 82%, mp 193–195 °C. IR (KBr,
υ): 3279, 3071, 2920, 1644 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 12.56 (s, 1H), 8.02 (d, J = 8.4 Hz, 2H),
7.62–7.17 (m, 15H), 7.04 (d, J = 8.4 Hz, 2H), 4.76 (s, 2H,
O-CH2), 3.74 (s, 3H, O-CH3). 13C NMR (126 MHz,
DMSO-d6) δ 170.58, 158.39, 145.97, 140.02, 138.08,
136.53, 135.14, 133.49, 132.05, 130.71, 128.86, 128.04,
127.12, 125.64, 124.05, 122.18, 120.99, 119.31, 117.93,
115.11, 65.03, 55.67. MS (70 eV, m/z [M+]) = 475. Anal
Calcd for C30H25N3O3, C, 75.77, H, 5.30, N, 8.84. Found C,
75.83, H, 5.28, N, 8.83.
N-(3-bromophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-2-yl)
phenoxy)acetamide (7h)
White solid; isolated yield: 86%, mp 214–216 °C. IR (KBr,
υ): 3273, 3072, 2937, 1643 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 12.53 (s, 1H), 8.02 (d, J = 7.8 Hz, 2H), 7.62 –
7.48 (m, 6H), 7.47 – 7.17 (m, 9H), 7.04 (d, J = 7.8 Hz, 2H),
4.75 (s, 2H, O-CH2). 13C NMR (126 MHz, DMSO-d6) δ
170.57, 158.39, 145.97, 138.95, 136.93, 135.92, 134.52,
133.23, 131.34, 128.81, 127.11, 124.05, 122.73, 121.52,
120.80, 119.71, 118.63, 117.69, 116.60, 115.11, 115.11,
113.36, 111.63, 65.03. MS (70 eV, m/z [M+]) = 523. Anal
Calcd for C29H22BrN3O2, C, 66.42, H, 4.23, N, 8.01. Found
C, 66.49, H, 4.22, N, 8.06.
N-(4-chlorophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-2-yl)
phenoxy)acetamide (7e)
White solid; isolated yield: 86%, mp 239–241 °C. IR (KBr,
υ): 3330, 2905, 1742, 1647 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 13.58 – 12.21 (m, 1H), 8.04 (d, J = 8.9 Hz,
2H), 7.65–7.35 (m, 13H), 7.31 (t, J = 7.3 Hz, 2H), 7.06 (d,
J = 8.9 Hz, 2H), 4.77 (s, 2H, O-CH2). 13C NMR (126 MHz,
DMSO-d6) δ 170.54, 158.57, 150.64, 145.87, 138.73,
133.33, 132.69, 130.80, 128.90, 128.25, 127.67, 127.32,
125.72, 124.60, 123.52, 122.03, 120.97, 118.84, 115.39,
115.16, 115.16, 65.01. MS (70 eV, m/z [M+]) = 479. Anal
Calcd for C29H22ClN3O2, C, 72.57, H, 4.62, N, 8.75. Found
C, 72.52, H, 4.61, N, 8.69.
N-(4-bromophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-2-yl)
phenoxy)acetamide (7i)
White solid; isolated yield: 86%, mp 224–226 °C. IR (KBr,
υ): 3325, 2954, 1649 cm−1. 1H NMR (500 MHz, DMSO-d6)
δ 13.01 – 11.95 (m, 1H), 8.03 (d, J = 8.5 Hz, 2H), 7.60 –
7.23 (m, 15H), 7.05 (d, J = 8.5 Hz, 2H), 4.76 (s, 2H, O-
CH2). 13C NMR (126 MHz, DMSO-d6) δ 170.58, 158.41,

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145.98, 136.09, 134.81, 133.08, 132.27, 131.13, 130.27,
128.86, 128.18, 127.51, 127.16, 125.77, 125.09, 124.00,
122.28, 121.28, 119.55, 115.12, 65.02. MS (70 eV, m/z
[M+]) = 523. Anal Calcd for C29H22BrN3O2, C, 66.42, H,
4.23, N, 8.01. Found C, 66.40, H, 4.25, N, 8.01.
Pharmacology
In vitro α-glucosidase inhibition assay
α-Glucosidase inhibition assay was evaluated on yeast α-
glucosidase (Saccharomyces cerevisiae, EC3.2.1.20, 20 U/
mg) according to the previous reports [21–24]. First, the
various concentrations of compounds 7a–l (20 μl), α-glu-
cosidase solution (20 μl), and potassium phosphate buffer
(135 μl) were added to a 96-well plate and incubated at
37 °C for 10 min. Then p-nitrophenyl glucopyranoside
(substrate, 25 μl, 4 mM) was added to the latter plate and
allowed to incubate at 37 °C for 20 min. Finally, the
absorbance changes were measured at 405 nm by using
spectrophotometer (Gen5, Power wave xs2, BioTek,
America). Solvent (DMSO with final concentration of 10%)
was used as negative control and acarbose was used as
positive control. IC50 values of title compounds 7a–l were
calculated from nonlinear regression curve using the Logit
method.
2-(4-(4,5-diphenyl-1H-imidazol-2-yl)phenoxy)-N-(2-methyl-
3-nitrophenyl)acetamide (7j)
White solid; isolated yield: 81%, mp 195–197 °C. IR (KBr,
υ): 3288, 3098, 2963, 1655 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 12.58 (s, 1H), 8.03 (d, J = 8.3 Hz, 2H),
7.61–7.45 (m, 6H), 7.45–7.21 (m, 9H), 7.05 (d, J = 8.3 Hz,
2H), 4.76 (s, 2H, O-CH2), 2.07 (s, 3H, CH3). 13C NMR
(126 MHz, DMSO-d6) δ 170.58, 158.40, 145.98, 136.68,
134.99, 133.13, 131.55, 130.59, 129.76, 128.86, 128.16,
127.51, 127.14, 126.12, 125.08, 124.03, 122.45, 120.83,
118.76, 117.90, 115.12, 65.02, 19.03. MS (70 eV, m/z
[M+]) = 504. Anal Calcd for C30H24N4O4, C, 71.42, H,
4.79, N, 11.10. Found C, 71.44, H, 4.76, N, 11.16.
N-(3-chloro-2-methylphenyl)-2-(4-(4,5-diphenyl-1H-
In vitro α-amylase inhibition assay
imidazol-2-yl)phenoxy)acetamide (7k)
The inhibitory activity of the newly synthesized compounds
7a–l against α-amylase was obtained according to used
method in our previous work [23].
White solid; isolated yield: 83%, mp 166–168 °C. IR (KBr,
υ): 3283, 3086, 2963, 1650 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 12.43 (s, 1H), 8.23 (d, J = 8.3 Hz, 1H), 8.14
(d, J = 8.3 Hz, 1H), 8.09 (d, J = 8.9 Hz, 2H), 7.80 (t, J =
7.8 Hz, 1H), 7.57–7.52 (m, 4H), 7.42–7.29 (m, 7H), 7.19 (d,
J = 8.9 Hz, 2H), 4.86 (s, 2H, O-CH2), 2.05 (s, 3H, CH3).
13C NMR (126 MHz, DMSO-d6) δ 195.29, 180.73, 167.92,
156.48, 144.75, 143.98, 142.84, 141.38, 140.41, 139.08,
138.34, 136.95, 136.02, 134.06, 132.71, 131.35, 130.07,
129.99, 127.18, 125.37, 123.63, 122.65, 120.98, 64.04,
18.85. MS (70 eV, m/z [M+]) = 493. Anal Calcd for
C30H24ClN3O2, C, 72.94, H, 4.90, N, 8.51. Found C, 72.91,
H, 4.88, N, 8.50.
Kinetic study
For kinetic study, the most potent compound 7h was
selected and 20 μl of it in four different concentrations 0, 30,
60, and 90 μM was incubated with 20 μl of α-glucosidase
solution (1 U/ml) for 15 min at 30 °C. After that, the
enzymatic reaction was started by adding 20 μl of p-nitro-
phenyl glucopyranoside as substrate in concentrations
1–4 mM and the absorbance changes was determined for
20 min at 405 nm by using spectrophotometer (Gen5, Power
wave xs2, BioTek, America).
N-(2-chloro-3-fluorophenyl)-2-(4-(4,5-diphenyl-1H-imidazol-
2-yl)phenoxy)acetamide (7l)
Molecular modeling study
White solid; isolated yield: 87%, mp 177–19 °C. IR (KBr,
Modeled α-glucosidase was constructed according to the
described method by Imran et al. [25, 26]. At first, a
search was performed by using SWISS-MODEL to find
an appropriate protein in the protein data bank (PDB) with
a high sequence similarity with S. cerevisiae α-glucosi-
dase (used form of enzyme in enzymatic assay). As a
result, isomaltase from S. cerevisiae (PDB code 3A4A)
was selected with 72% identical and shares 85% similarity
with the S. cerevisiae α-glucosidase. Then this enzyme
was subjected through sequence alignment and homology
model using automated homology modeling pipeline
SWISS-MODEL (managed by Swiss Institute of
υ): 3287, 3080, 2966, 1651 cm−1 1H NMR (500 MHz,
.
DMSO-d6) δ 12.56 (s, 1H), 8.07 (d, J = 8.5 Hz, 2H), 7.80 (t,
J = 7.5 Hz, 1H), 7.55 (s, 5H), 7.47–7.39 (m, 3H), 7.38–7.30
(m, 3H), 7.27 – 7.20 (m, 2H), 7.14 (d, J = 8.5 Hz, 2H), 4.89
(s, 2H, O-CH2). 13C NMR (126 MHz, DMSO-d6) δ 167.52,
164.28, 158.32, 149.55, 145.94, 137.31, 135.81, 130.39,
129.41, 129.32, 128.97, 128.81, 128.70, 128.23, 128.13,
127.81, 127.55, 127.41, 127.32, 127.17, 126.86, 126.01,
125.46, 124.36, 120.87, 120.50, 120.37, 115.36, 67.39. MS
(70 eV, m/z [M+]) = 497. Anal Calcd for C29H21ClFN3O2,
C, 69.95, H, 4.25, N, 8.44. Found C, 69.91, H, 4.24, N, 8.49.

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Medicinal Chemistry Research (2021) 30:1273–1283
Bioinformatics) and the quality of the obtained modeled
enzyme (in pdb format) was verified using PROCHECK
[27]. Pdb format of the modeled enzyme was converted to
pdbqt coordinate by Auto dock Tools. Three-dimensional
structures of the most potent compounds and standard
inhibitor acarbose were built by MarvineSketch 5.8.3,
2012, ChemAxon (http://www.chemaxon.com) and were
saved as pdb format and converted to pdbqt coordinate
using Auto dock Tools. These pdbqts were used as an
input file for the AUTOGRID program. In the latter pro-
gram, for each atom type in the ligand, maps were cal-
culated with 0.375 Å spacing between grid points and
center of grid box was placed at x = 12.5825, y = 7.8955,
and z = 12.519 Å with dimensions 40 × 40 × 40 Å. Flex-
ible ligand dockings were applied for the selected ligands.
Each docked system was carried out by 50 runs of
AUTODOCK search by the Lamarckian genetic algo-
rithm. The best docking pose in term of binding energy
for each ligand was selected for study of the interaction
mode between α-glucosidase and ligand.
Results and discussion
Chemistry
The synthetic routes for the synthesis of 4,5-diphenylimi-
dazole-N-phenylacetamide derivatives 7a–l has been depic-
ted in Scheme 1. This synthesis process was started from the
reaction between aniline derivatives 1a–l and chloroacetyl
chloride 2 in acetone at room temperature for 30 min to give
2-chloro-N-phenylacetamide derivatives 3a–l. The latter
compounds reacted with 4-hydroxybenzaldehyde 4 in the
presence of KI and K2CO3 in acetone at 80 °C for produce 2-
(4-formylphenoxy)-N-phenylacetamides 5a–l. In the final
step, 2-(4-formylphenoxy)-N-phenylacetamides 5a–l and
benzil 6 in the presence of ammonium acetate in DMF at
100 °C converted to desired 4,5-diphenylimidazole-N-phe-
nylacetamide derivatives 7a–l.
α-Glucosidase and α-amylase inhibitory activities
In vitro anti-α-glucosidase activity of the synthesized 4,5-
diphenylimidazole-N-phenylacetamide derivatives 7a–l was
evaluated and the obtained results were compared with
In vitro cytotoxicity assay
In vitro cytotoxic activity of the most potent compounds 7h,
7a, and 7d was determined by 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazolium bromide (MTT) assay according to
literature [28]. For this propose, normal and cancer human
cell lines were used.
acarbose as a standard α-glucosidase inhibitor (IC50 =
750.0 9.6 µM). As can be seen in Table 1, the IC50 values
of the studied compounds demonstrated that all the syn-
thesized compounds 7a–l were more potent than acarbose
(IC50s ≤ 598.5 8.0 µM). In the synthesis of these com-
pounds, aniline and various aniline derivatives with
electron-donating groups such as methyl, ethyl, and meth-
oxy and electron-withdrawing groups such as chloro,
bromo, nitro, and fluoro were used in order to optimize α-
glucosidase inhibition. The most potent compounds were 3-
bromo and un-substituted compounds 7h and 7a with IC50
values = 90.0 2.5 and 103.6 4.0 μM, respectively.
In silico druglikeness/ADME/toxicity study
In silico druglikeness/ADME/toxicity properties of standard
drug acarbose and the most potent compounds 7h, 7a, and
7d were predicted by using the preADMET online server
(http://preadmet.bmdrc.org/) [29].
Scheme 1 Synthetic routes to the target compounds 7a–l: (a) Acetone, r.t .30 min; (b) KI, K2CO3, Acetone, 80 °C, 4 h; (c) NH4OAC, DMF,
100 °C, 8 h

Medicinal Chemistry Research (2021) 30:1273–1283
1279
Table 1 In vitro α-glucosidase and α-amylase inhibitory activities of
the synthesized compounds 7a–l
Kinetic study against α-glucosidase
Kinetic study of the most potent compound 7h was carried out
in order to evaluate mechanism of α-glucosidase inhibition of
the new compounds. As was shown in Fig. 3a, with increase
of concentration of the compound 7h, Vmax remained
unchanged while Km increased. Therefore, compound 7h was
a competitive inhibitor against α-glucosidase. The value of Ki
for compound 7h was 86.3 0.11 μM (Fig. 3b).
Compound
R
IC50 (μM)a
α-Glucosidase molecular modeling study
α-glucosidase
α-amylase
7a
H
103.6 4.0
411.5 7.3
402.0 7.0
154.1 4.7
314.5 6.5
161.5 6.6
179.8 4.4
90.0 2.5
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
>500
108 0.71
In order to clarify interaction modes of the newly synthe-
sized compounds in the active site of α-glucosidase, mole-
cular modeling was conducted on the modeled form of this
enzyme using Auto Dock Tools (version 1.5.6) [21]. For
docking purposes, acarbose as standard inhibitor and most
potent compounds 7h and 7a were selected and docked in
the α-glucosidase active site. The superimposed structure of
acarbose and the most potent compound 7h in the active site
of target enzyme is shown in Fig. 4a. Analysis of the best
pose of acarbose revealed that this inhibitor with binding
energy of −4.04 kcal/mol, interacted with the α-glucosidase
active site residues Gln322, Thr301, Glu304, Ser308,
Arg312, Thr307, and Asn241 via hydrogen bonds (Fig. 4b).
Furthermore, acarbose formed a hydrophobic interaction
with His279, several non-classical hydrogen bonds with
Arg312, Glu304, Val305, and His239, and two unfavorable
interactions with Arg312 and Thr307.
The most potent compound 7h established a hydrogen bond
with Thr307 via NH unit of imidazole ring and a π-anion
interaction with Glu304 via phenoxy group (Fig. 5a). Imida-
zole ring and phenoxy group also interacted with residue
Pro309 through hydrophobic interactions. This amino acid also
established a non-classical hydrogen bond with compound 7h.
Phenyl rings attached to imidazole ring interacted with Ala326
and Val316 via hydrophobic interactions. Furthermore, N-3-
bromophenylacetamid moiety of compound 7h established the
following interactions: two hydrophobic interactions with
residues Phe158 and Arg312 through 3-bromo substituent and
phenyl ring, respectively, and a non-classical hydrogen bond
with residue Phe311 through carbonyl unit.
7b
4-CH3
4-Ethyl
4-OCH3
4-Cl
7c
7d
7e
7f
2,6-Dichloro
2-Br
7g
7h
3-Br
7i
4-Br
598.5 8.0
396.0 6.0
303.1 6.0
215.3 5.0
750.0 9.6
7j
2-CH3-3-NO2
2-CH3-3-Cl
2-Cl-3-F
—
7k
7l
Acarbose
Furthermore, 4-methoxy derivative 7d, 2,6-dichloro deri-
vative 7f and 2-bromo derivative 7g exhibited good anti-α-
glucosidase activity (IC50 values ≤179.8 4.4 µM).
The α-glucosidase inhibitory activity of 4,5-diphenyli-
midazole-N-phenylacetamide derivatives 7a–l demonstrated
that 3-bromo derivative 7h showed the most potent activity,
while 4-bromo derivative 7i was less active compound
among the synthesized compounds. Moreover, movement
of bromo substituent of 3-position to 2-position, as in
compound 7g, led to a moderate decrease in the inhibitory
activity.
The second potent compound among the synthesized
compounds was un-substituted compound 7a. Introduction
of 4-methyl, 4-ethyl, or 4-chloro on N-phenylacetamide ring
dramatically deteriorated anti-α-glucosidase as observed in
compounds 7b, 7c, and 7e while introduction of 4-methoxy
substituent on the latter ring, as in compound 7d, slightly
decreased inhibitory activity in comparison to un-
substituted compound 7a.
The comparison of interaction modes of the most potent
compound 7h containing N-3-bromophenylacetamid moiety
with the second potent compound 7a with N-phenylacetamid
moiety demonstrated that compound 7h established an
additional hydrophobic interaction with active site residue
Phe158 via 3-bromo substituent (Fig. 5). It should also be
noted that compound 7a created an additional hydrophobic
interaction with Pro309 via 5-phenyl ring in comparison to
compound 7h (Fig. 5b). Other established interactions are the
same in the both compounds 7h and 7a. Details of interaction
modes of compounds 7h and 7a are listed in Table 2.
Among the two-substituted compounds 7f and 7j–l, 2,6-
dichloro derivative 7f exhibited the best inhibitory activity.
α-Amylase inhibition assay of the synthesized com-
pounds 7a–l was also carried out and obtained data revealed
that all the title compounds (IC50 values >500 μM) in
comparison to acarbose (IC50 = 108 0.71 μM) were
inactive.

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Medicinal Chemistry Research (2021) 30:1273–1283
Fig. 3 a The inhibition type and b Ki value determination of the most potent compound 7h
Fig. 4 (a) Superimposition structure of acarbose (cyan) and the most potent compound 7h (blue); (b) interaction mode of acarbose in the active site
of α-glucosidase
Fig. 5 (a) The interaction modes of the most potent compounds 7h and (b) 7a in the active site of α-glucosidase

Medicinal Chemistry Research (2021) 30:1273–1283
Table 2 Interaction mode details
1281
Compound Interaction
Interacting unit of the ligand
Amino acid Distance (Å)
of the compounds 7h and 7a
7h
Hydrophobic
Hydrophobic
A phenyl ring of 4,5-
diphenylimidazole moiety
Ala326
Val316
4.55
5.38
A phenyl ring of 4,5-
diphenylimidazole moiety
H-bond
NH unit of imidazole ring
Imidazole ring
Thr307
Pro309
Pro309
Glu304
Phe311
2.09
4.42
4.86
3.41
3.07
Hydrophobic
Hydrophobic
π-anion
Phenoxy group
Phenoxy group
-CH2-C=O
Non-classical
hydrogen bond
Non-classical
-CH2-C=O
Pro309
3.34
hydrogen bond
Hydrophobic
Hydrophobic
Hydrophobic
N-3-Bromophenyl moiety
N-3-Bromophenyl moiety
Arg312
Arg312
Ala326
4.04
5.28
5.17
7a
A phenyl ring of 4,5-
diphenylimidazole moiety
Hydrophobic
Hydrophobic
A phenyl ring of 4,5-
diphenylimidazole moiety
Val316
Pro309
5.01
5.31
A phenyl ring of 4,5-
diphenylimidazole moiety
H-bond
NH unit of imidazole ring
Imidazole ring
Thr307
Pro309
Pro309
Glu304
Phe311
2.01
4.23
5.14
3.41
2.99
Hydrophobic
Hydrophobic
π-anion
Phenoxy group
Phenoxy group
-CH2-C=O
Non-classical
hydrogen bond
Non-classical
hydrogen bond
-CH2-C=O
Pro309
Arg312
3.37
4.7
Hydrophobic
N-phenyl moiety
Table 3 Cytotoxic activity of compounds 7j, 7k, and 7a
The calculation of binding energies of acarbose
(−4.04 kcal/mol) and compounds 7h (−11.32 kcal/mol) and
7a (−10.65 kcal/mol) demonstrated that our newly synthe-
sized compounds bind to α-glucosidase more easily than
acarbose. These findings are in agreement with the obtained
date of in vitro enzyme inhibition assay (Table 1, com-
pounds 7h and 7a vs. acarbose).
Compound
Cytotoxicity (IC50 μM)a
MCF-7
HDF
7j
>200
>200
7k
>200
>200
7a
>200
>200
Etoposide
22.08 0.39
92.7 1.2
In vitro cytotoxicity assay
aValues are the mean SD
In vitro cytotoxicity of compounds 7h, 7a, and 7d was
evaluated against normal cells HDF and cancer cells MCF-7
by MTT assay [28]. As can be seen in Table 3, the selected
compounds were non-cytotoxic at 200 µM against studied
cells in comparison to standard cytotoxic agent etoposide.
PreADMET [29]. The obtained results are listed in Table 4.
As can be seen in this table, compounds 7a and 7d are drug-
like in terms of Rule of Five and MDDR-like rule while
compound 7h and acarbose only follow MDDR-like rule.
Our new compounds 7h, 7a, and 7d have Caco2 cell per-
meability and human oral absorption higher of acarbose.
The predicted values of blood–brain barrier permeability for
any of the studied compounds are not in the acceptable
range. Predicting the toxicity of the all selected compounds
showed that all the studied compounds were mutagenic
In silico druglikeness, ADME, and toxicity studies
Druglikeness/ADME/T properties of acarbose as standard
drug and new compounds 7h, 7a, and 7d as the most potent
inhibitors were predicted by the online software

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Medicinal Chemistry Research (2021) 30:1273–1283
Table 4 Druglikeness/ADME/T profile of the most potent compounds
7h, 7a, and 7d
Compliance with ethical standards
Conflict of interest The authors declare no competing interests.
Druglikeness/
ADME/Ta
Compound
7h
7a
7d
Acarbose
Violated
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
Rule of Five
Violated
Suitable
Suitable
MDDR-like rule Drug-like Drug-like Drug-like Drug-like
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42.8665
53.6636
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