Organocatalyzed solvent free an efficient novel synthesis of 2,4,5-trisubstituted imidazoles for α-glucosidase inhibition to treat diabetes

Article abstract of DOI:10.1016/j.bioorg.2014.11.006

A new and efficient solvent free synthesis of 2,4,5-trisubstituted imidazoles (3a-3j) was achieved by N-acetyl glycine (NAG) catalyzed three components condensation of aldehydes, benzil and ammonium acetate. Our synthetic methodology accommodated a range of various substituted alkyl and aryl aldehydes. Evaluation of α-glucosidase inhibitory activity of these imidazole derivatives revealed that most of them presented good α-glucosidase inhibition at low micro-molar concentrations. Among the synthesized compounds, compound 3c, bearing the ortho-hydroxy phenyl substituent at position 2 displayed the highest inhibitory activity with an IC₅₀ value 74.32 ± 0.59 μM. In silico molecular docking for all compounds and computational studies of the most active compound 3c were also performed.

Full text of DOI:10.1016/j.bioorg.2014.11.006

Accepted Manuscript
Organocatalyzed solvent free an efficient novel synthesis of 2,4,5-trisubstituted
imidazoles for α-glucosidase inhibition to treat diabetes
Muhammad Yar, Marek Bajda, Sohail Shahzad, Nisar Ullah, Mazhar Amjad
Gilani, Muhammad Ashraf, Abdul Rauf, Ayesha Shaukat
PII:
S0045-2068(14)00113-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.11.006
YBIOO 1774
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
9 August 2014
Please cite this article as: M. Yar, M. Bajda, S. Shahzad, N. Ullah, M.A. Gilani, M. Ashraf, A. Rauf, A. Shaukat,
Organocatalyzed solvent free an efficient novel synthesis of 2,4,5-trisubstituted imidazoles for α-glucosidase
inhibition to treat diabetes, Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.11.006
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Organocatalyzed solvent free an efficient novel synthesis of 2,4,5-
trisubstituted imidazoles for α-glucosidase inhibition to treat diabetes
∗
Muhammad Yar,a, Marek Bajda,b,c Sohail Shahzad,a,d, Nisar Ullah,e Mazhar Amjad Gilani,f
Muhammad Ashraf,g Abdul Rauf,d Ayesha Shaukatg
aInterdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore,54000,
Pakistan
bFaculty of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland
cDepartment of Physicochemical Drug Analysis, Faculty of Pharmacy, Jagiellonian University Medical College, Medyczna 9,
30-688 Cracow, Poland
dDepartment of Chemistry, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
eDepartment of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran, 31261, Saudi Arabia
fDepartment of Chemical Engineering, COMSATS Institute of Information Technology, Lahore, 54000, Pakistan
gDepartment of Biochemistry & Biotechnology, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan
Abstract:
A new and efficient solvent free synthesis of 2,4,5-trisubstituted imidazoles (3a–3j) was
achieved by N-acetyl glycine (NAG) catalyzed three components condensation of
aldehydes, benzil and ammonium acetate. Our synthetic methodology accommodated a
range of various substituted alkyl and aryl aldehydes. Evaluation of α-glucosidase
inhibitory activity of these imidazole derivatives revealed that most of them presented
good α-glucosidase inhibition at low micro-molar concentrations. Among the synthesized
compounds, compound 3c, bearing the ortho-hydroxy phenyl substituent at position 2
*Corresponding author. Tel.: +92-42-111001007; fax: 0092-42-35321090
(M.Y.); E-mail addresses: drmyar@ciitlahore.edu.pk (M. Yar),
1

displayed the highest inhibitory activity with an IC50 value 74.32 0.59 μM. In silico
molecular docking for all compounds and computational studies of the most active
compound 3c were also performed.
Keywords: Imidazoles, α-glucosidase inhibition, diabetes, organocatalysis, molecular
modelling, baker's yeast
1. Introduction:
Diabetes mellitus is a group of metabolic disorders which result from defects in both
regulations of insulin secretion and/or insulin action. As a result, the glucose concentration
in the blood increases (hyperglycemia) and over time causes serious damage to many of
the body's organs, especially the nerves and blood vessels [1]. Since 1980, the number of
people in the world with diabetes has increased from 153 million to 347 million in 2008
[2]. Based on the WHO projection, diabetes will become seventh leading cause of death
globally by 2030. One therapeutic approach for diabetes is to control the postprandial
glucose levels and suppress postprandial hyperglycemia by inhibition of carbohydrate-
hydrolyzing enzymes, such as α-glucosidase [3, 4]. α-Glucosidase inhibitors reversibly
inhibit digestive α-glucosidase, which retard the liberation of glucose from dietary
complex carbohydrate and starch, delaying absorption of glucose into the bloodstream and,
thus reducing plasma glucose levels. Inhibition of α-glucosidase has drawn a special
interest by the pharmaceutical research community as a remedy for the treatment of
carbohydrate mediated diseases such as cancer, viral infections, diabetics and hepatitis [5-
7]. The inhibitors of α-glucosidase are known to possess antitumor, antidiabetics, antiviral,
and immunoregulatory activities [8-10]. In addition, α-glucosidase inhibitors such as
deoxynojirimycin (DNJ), N-butyl-deoxynojirimycin (NB-DNJ) and castanospermine are
2

potent inhibitors of the HIV replication and HIV mediated syncytium formation in vitro
[10].
Currently, glucosidic based α-glucosidase inhibitors such as acrabose [11], miglitol [12],
voglibose [13] and nojirimycin [14] are clinically used to control blood glucose levels of
patients (Figure 1). Although effective, these inhibitors suffer from liabilities such as
abdominal distension, flatulence, meteorism and possibly diarrhea [15]. In order to search
for more effective and safer α-glucosidase inhibitors, efforts have been devoted in the
development of non-glucosidic based inhibitors [16, 17].
OH 8
7
6
9 HO
5
OH
N
1
4
2
HO
OH
3
H3C
HO
11
N
H
O
OH
HO
10
OH
O
OH O
OH
HO
OH
OH
O
O
OH
Miglitol
OH
Acarbose
O
HO
OH
OH
H
N
HO
HO
OH
OH
HO
HO
OH
HO
N
H
OH
OH
Deoxynojirimycin
Voglibose
Figure 1: The α-glucosidase inhibitors: acarbose, voglibose, deoxynojirimycin and miglitol.
Substituted imidazoles are known to possess a range of therapeutic effects such as potent
and selective inhibitors of p38 MAP kinase [18], B-Raf kinase [19], transforming growth
factor b1 (TGF-b1) type 1 activating receptor-like kinase (ALK5) [20], cyclooxygenase-2
(COX-2) [21], and biosynthesis of interleukin-1 (IL-1) [22]. In addition, SB-220109
(Figure 2) is reported as potent p38α recaptulates protein kinase inhibitor [23].
3

F
N
OH
N
H
N
Figure 2: The molecular structure of SB-220109.
Generally 2,4,5-trisubstituted imidazoles are synthesized by three component
cyclocondensation of a 1,2-diketone, α-hydroxyketone or α-ketomonoxime with an
aldehyde and ammonium acetate using a variety of different catalysts including L-proline
[24], montmorillonite K10 [25], zeolite [25], nano sulfated zirconia [25], 1-ethyl-3-
methylimidazole acetate [EMIM]OAc [26], zirconium (IV) acetylacetonate (Zr(acac)4)
[27], 1-(2-hydroxyethyl)-3-methyl imidazoliumtetrafluoroborate ([HeMIM]BF4) [28],
acetic acid (HOAc) [29], tetrabutylammonium bromide (TBAB) [30], polymer supported
zinc chloride [31], supported ionic liquid like phase (SILLP) [32], Nano-SnCl4 . SiO2 [33],
L-cysteine [34], 1-methylimidazolium trifluoroacetate ([Hmim]TFA) [35] and potassium
dihydrogen phosphate [36].
Most of these synthetic methods suffer from one or more serious limitations including
laborious and complex work-up and purification, significant amounts of waste materials,
strongly acidic conditions, occurrence of side reactions, low yields, use of expensive
reagents and require elevated temperatures. To overcome these shortcomings, the
development of a new organocatalytic system to meet the criteria of a mild, efficient, and
environmentally benign protocol for the synthesis of substituted imidazoles is highly
desirable. Recently, we have found and reported the use of N-acetyl glycine (NAG) as an
4

efficient organocatalyst in the synthesis of pyrimidines, where the required pyrimidines
were achieved in significant to good yields. An easy method was established for the
removal of NAG from the reaction mixture after the reaction and the subsequent
purification of the targeted pyridmides was straight forward as well [37]. In continuation of
our ongoing medicinal chemistry research endeavours [38-40] herein, we disclose a novel
and facile NAG organocatalyzed synthesis of 2,4,5-trisubstituted imidazoles under mild
and solvent free reaction conditions. The advantages of using N-acetyl glycine (NAG) as
an efficient organocatalyst in the synthesis of 2,4,5-trisubstituted imidazoles were included
the easy-work up and purification, one pot and one step, avoidance from any acidic
conditions, better yields, solvent-free and thus environmentally friendly. In vitro α-
glucosidase inhibitory activity and in silico molecular docking studies of all compounds
are also included to demonstrate the binding pattern with the active site of the targeted
protein.
2. Results and discussion
2.1. Chemistry
The desired 2,4,5-trisubstituted imidazoles 3a–3j [25, 32-35, 41, 42] bearing different
substituents were prepared in good to excellent yields (67–85%) as shown in Scheme 1.
The preparation of 3a–3j was ensued from condensation of benzil, ammonium acetate and
the corresponding aldehydes 2a–2j, catalyzed by NAG. Notably, a variety of functional
groups such as hydroxyl, methoxy and chloro were tolerated under our new reaction
protocol. The chemical structures of all the synthesized compounds were established with
the aid of spectroscopic and physical methods.
5

O
O
N
H
O
H
O
H3C
Ph
Ph
N
O
NAG
R
Ph
2 NH4OAc
R-CHO
N
H
Ph
150 οC, 2 h
3a-3j
67-85%
R = Ar or alkyl
Scheme 1: Synthetic protocol for the NAG catalyzed synthesis of 2,4,5-trisubstituted
imidazoles.
2.2. α-Glucosidase inhibition assay
A recurring aspect in enzyme catalyzed reaction mechanisms is the use of proton-transfer
processes to affect catalysis. The classic example of this phenomenon is the serine
proteases [44], for instance chymotrypsin, where the nucleophilicity of the active-site
serine hydroxyl group is enhanced by a histidine-side-chain imidazole nucleus mediated
indirect co-ordination to an anionic aspartate residue, in the so-called 'catalytic triad'. The
well-characterized general acid-base catalysis employed by glucosidases to ensue cleavage
of the glycosidic linkage (for reviews of this topic, see [45] and [46] and references cited
therein) led us to speculate that placement of an imidazole unit between the 'acid' and 'base'
residues would result in shuttling of a proton between the two groups 'through' the
imidazole nucleus. The proposed mechanism of the interaction of the imidazole with the
active site of the β-glucosidase enzyme has been elaborated in Scheme 2 [47]. Similar
interactions could also be expected among other glucosidases and potential acid catalysts
such as histidine, tyrosine or Mg+2 [45]. Compounds that would bind with glucosidases in
the aforementioned manner is expected to be potential inhibitors of such enzymes [48].
6

Balba et al., determined the in vivo and in vitro α-glucosidase inhibition activities of 4,5-
diphenylimidazole-2-thione. It was described that 4,5-diphenylimidazole-2-thione
demonstrated in vitro α-glucosidase inhibition by non-competitive reversible mechanism
[43].
O
H
O
O
O
H
N
N
N
H
N
H
O
O
O
O
Scheme 2: Mode of interaction of imidazole with active-site catalytic residues of
glucosidase enzyme
In the current studies, all the synthesized imidazole derivatives (3a–3j) were evaluated for
α-glucosidase inhibitory activity which revealed that compounds 3c, 3f and 3g were found
to be active, showing α-glucosidase inhibition from 96.63 1.11% to 97.63 1.29% at 0.5
mM with IC50 values ranged between106.57 0.86 to 74.32 0.52 μM (Table 1). Acarbose
was used as a standard inhibitor.
2.3. Structure activity relationship
In order to delineate the structure–activity relationship and to get an optimized α-
glucosidase inhibitor, both benzaldehyde and substituted benzaldehydes as well as alkyl
aldehydes were used in the synthesis of these substituted imidazole derivatives (3a–3j).
7

These substituents include electron donating groups such as hydroxly, methoxy and
electron withdrawing group such as chloro substituent. Compound 3c, bearing 2-
hydroxyphenyl moiety at the 2-position of the imidazole ring, was proved to be the most
active one with an IC50 value of 74.32 0.52μM. Changing the position of the hydroxyl
group in the phenyl ring from C-2 to C-4, producing 3f (97.12 0.71 μM) slightly
diminished the activity. From the activity pattern it can be ascertain that the presence of the
hydroxy group in the phenyl ring plays a significant role in the higher activitiy of these
compounds and the activity is higher when hydroxyl group is present at C-2 of the phenyl
ring (compounds 3c vs 3f). Likewise compound 3g (106.57 0.86 μM) possessing hydroxyl
group in the phenyl ring at C-2 was also reasonably active compared to the rest of the
compounds in the series that do not contain hydroxyl group in the phenyl ring (Table 1).
Compound 3a, bearing unsubstituted phenyl ring was turned out to be inactive and when a
chlorine atom was introduced at the C-4 position of the 2-phenyl ring, the resulting
derivative 3b (458.32 1.14 μM) showed modest activity. Similarly, compound 3d
(359.42 1.12 μM), possessing a methoxy group at C-3 turned out to be modestly active
compared to its parent compound 3a (Table 1).
8

Table 1: Percent yield, α-glucosidase inhibition activity and IC50 values (mean SEM, n=3) of
2,4,5-trisubstituted imidazoles (ND, not determined).
Ph
N
R
N
Ph
H
(3a-3j)
Entry
Products
R
Yield (%)
% Inhibition
(0.5 mM)
IC50 (μM)
1.
2.
3a
3b
78
76
28.75 2.18
51.53 2.37
ND
Cl
458.32 1.14
HO
3.
4.
3c
70
62
97.63 1.29
89.23 2.45
74.32 0.52
359.42 1.12
ND
OMe
3d
MeO
5.
6.
3e
76
85
11.09 1.41
97.18 1.45
OH
3f
97.12 0.71
9

HO
OMe
7.
3g
71
96.63 1.11
106.57 0.86
OMe
OMe
8.
9.
3h
3i
67
20.76 1.16
8.76 1.76
ND
ND
CH3
67
CH3
CH3
10.
3j
73
17.56 1.85
92.23 0.14
ND
11.
Acarbose
-
-
38.25 0.12
2.3. Molecular Modelling
The novel compounds 3a-3j were tested in α-glucosidase assay and displayed different
activities. The most active inhibitor 3c ortho-hydroxy analogue of 3a was 2 fold less potent
than reference compound - acarbose while the parent compound 3a was almost inactive.
Therefore, we decided to perform docking studies to find the possible binding mode for all
derivatives and to explain the reasons of the varied potency.
As α-glucosidase from baker’s yeast was utilized in biological experiments, we needed to
use the enzyme from the same source for in silico modelling. The crystal structure of
Saccharomyces cerevisiae α-glucosidase has not been available so far and therefore, we
performed docking studies with the homology model, built and described previously [49].
It was characterized by high quality. The active site contained important, from the
10

viewpoint of hydrolysis, amino acids: two histidines (His111, His348), two aspartates
(Asp214, Asp349) and one glutamate (Glu276).
It was noted that all compounds presented one main, shared binding mode but the docking
runs were really converged only for the active derivatives (with determined IC50 values).
Moreover, the hydrophilic substituents such as hydroxyl groups were responsible for the
interactions, essential for the activity. The core of molecules was engaged in some
interactions but they were not enough to provide the high inhibition of enzyme.
The imidazole ring created π - cation interaction with Arg439, and its NH fragment was
involved in hydrogen bond with Asp349. The phenyl group in position 5 of imidazole
formed other π - cation interaction with Arg312, and phenyl in position 4 weak CH-π
interaction with Phe158. The third phenyl moiety in position 2 (derivatives 3a-3h) created
π-π stacking interaction with Phe177. In case of compounds 3i and 3j, replacement of that
phenyl group by alkyl chain was responsible for very low activity due to lack of π-π
interaction. Introduction of hydrophilic substituents with H-bond donating properties in the
third phenyl ring led to extra interactions and increase of the activity. The hydroxyl group
-
in ortho position (compound 3c, Figure 3) was both donor (OH ꢀ OOC of Asp68) and
+
acceptor (HO ꢁ H2N-C(=NH2 )-NH2 of Arg439) of hydrogen bonds. Movement of
hydroxyl substituent into para position (compound 3f) caused small decrease of potency in
comparison with ortho substituted inhibitor 3c because OH group formed only one H-bond
-
with Asp214 (OH ꢀ OOC of Asp214). Further modifications, including introduction of
methoxy substituents decreased the activity in comparison with hydroxy derivatives (3c,
3f) because methoxy groups were only H-bond acceptors (compounds 3e, 3d, 3h), and in
case of derivative 3e (o-methoxy analogue of 3a) additionally led to steric clash. Only
11

compound 3g, which contained both hydroxyl and methoxyl substituents, displayed similar
activity to inhibitor 3f due to hydrogen bond between hydroxyl group in ortho position and
Arg439, which decreased undesirable effect of phenyl rotation, caused by presence of -
OCH3. Compound 3b with chlorine atom was weak inhibitor. Halogen bond between Cl
and Asp68 wasn't enough to increase the activity in a large extent.
It seems that introduction of some extra hydroxyl substituents in phenyl rings could
improve the inhibitory potency of novel derivatives.
Comparing scoring function for all derivatives (3a-3j) with score for acarbose, it was noted
that they had been worst assessed than reference inhibitor. It remained in accordance with
experimental results and suggested that some further derivatives should be developed.
Figure 3: General view of baker's yeast α-glucosidase with docked compound 3c (left). Detailed
binding mode of inhibitor 3c (right; black lines represent hydrogen bonds).
12

3. Computational Studies
Molecular electrostatic potential (MEP) is associated with the electronic density and it
describes the sites for electrophilic and nucleophilic reactions as well as hydrogen bonding
interactions [50, 51]. Usually, the preferred nucleophilic site is represented by red color and
the preferred electrophilic site is represented by blue color. The different values of the
electrostatic potential at the surface are represented by different colors. Potential increases in
the order red < orange < yellow < green < blue. The color code of the map is in the range
between −0.0693 a.u. (deepest red) and 0.0693 a.u. (deepest blue) in the compound, where
blue shows the strongest attraction and red shows the strongest repulsion. Regions of negative
V(r) is usually associated with the lone pair of electronegative atoms.
From the docking studies, it was revealed that NH fragment of the imidazole and the hydroxy
group attached to aromatic ring was responsible for making hydrogen bonds making the
molecule more potent. It is further supported by generating molecular electrostatic potential
map of the most active molecule 3c (Figure 4). The blue regions (NH and OH groups)
indicate the most electrophilic sites of the molecule and are involved in hydrogen bonding.
13

Figure 4: Molecular electrostatic potential map of 3c calculated at B3LYP/6-31G(d,p).
4. Conclusion
In conclusion, 2,4,5-trisubstituted imidazoles were synthesized by employing a new
catalyst (NAG) under solvent free reaction conditions. The in-vitro α-glucosidase
inhibitory activity of these compounds revealed that compounds 3c, 3f and 3g, all bearing
a hydroxyl group in the 2-phenyl ring, were found to be significantly active. Compound 3c,
being the most active, was subjected to in silico molecular docking and computational
studies. Finding of these inhibitors may lead to future non-glucosidic based inhibitors.
4. Experimental
4.1. General methods
Infrared spectra were recorded on a photo acoustic mode at the frequency range of 4000–
400 cm-1 with 256 consecutive scans at a 8 cm-1 resolution on a Thermo-Nicolet 6700 P
FTIR Spectrometer (USA).1H NMR spectra were recorded on a AC-Bruker 300 MHz
spectrometer in CDCl3 and DMSO-d6 containing TMS as internal standard. Splitting
14

patterns were as follows s (singlet), d (doublet), dt (doublet of triplet), dd (double
doublets), t (triplet), and m (multiplet). All J values are given in Hz and chemical shifts in
δ-units. The progress of all reactions was monitored by TLC, which was performed on 2.0
× 5.0 cm aluminum sheets precoated with silica gel 60F254 to a thickness of 0.25 mm
(Merck).The chromatograms were visualized under ultraviolet light (254–366 nm).The
melting points were determined on a capillary melting point apparatus and are uncorrected.
Mass spectra were recorded on a Varian MAT 312 double focusing spectrometer,
connected to an IBM-AT compatible PC computer system. Chemical reagents were
purchased from the Merck Chemical Company in high purity. All the materials were of
commercial grade reagent.
4.2. General procedure for the synthesis of 2,4,5-trisubstituted imidazoles (3a-3j)
A mixture of 1 g benzil (1 mmol), 0.733 g ammonium acetate (2mmol), 0.557 g N-acetyl
glycine (1mmol) and the corresponding aldehyde (1mmol) was ground in a mortar and
pestle while mixed thoroughly. The mixture was then transferred to a 50 mL round bottom
flask, equipped with an efficient guard tube and was heated under stirring 150 °C for 2
hours. Progress of the reaction was monitored by a TLC. After completion of the reaction,
the reaction mixture was diluted with 5 % NaHCO3 and extracted with ethyl acetate. The
organic layer was separated, dried over Na2SO4 and evaporated. The resulting residues
were kept overnight at -18 °C to afford solid, which was recrystallized from methanol to
obtain pure products 3a-3j. The physical and spectral data of compounds 3a–3j were in
complete agreement with previous reports [25, 32-35, 41, 42].
4.2.1. 2,4,5-Triphenyl-1H-imidazole (3a)
15

White solid, yield: 67%; m.p: 270-272 °C; FT-IR (υmax, cm-1): 3230 (N-H), 3058 (Ar C-
H), 2851 (Alkyl C-H), 1678 (C=N), 1600 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ
12.61 (s, 1H, NH), 8.16 (d, 2H, Ar-H), 7.69-7.09 (m, 13H, Ar-H); MS (EI) m/z 296 (M+).
4.2.2. 2-(4-Chlorophenyl)-4,5-diphenyl-1H-imidazole (3b)
Pale yellow solid, yield: 67%; m.p: 260-265 °C; FT-IR (υmax, cm-1): 3227 (N-H), 3059 (Ar
1
C-H), 2839 (Alkyl C-H), 1678 (C=N), 1599 (C=C); H-NMR (400 MHz, DMSO-d6): δ
12·75 (s, 1H, NH), 7.96 (d, 2H, Ar-H), 7.43-7.22 (m, 12H, Ar-H); MS (EI) m/z 330 (M+).
4.2.3. 2-(4,5-Diphenyl-1H-imidazol-2-yl)phenol (3c)
Pale yellow solid, yield: 67%; m.p: 200-202 °C; FT-IR (υmax, cm-1): 3228 (N-H), 3061 (Ar
1
C-H), 2841 (Alkyl C-H), 1677 (C=N), 1594 (C=C); H-NMR (400 MHz, DMSO-d6): δ
13.01 (s, 1H, NH), 9.61 (s, 1H, O-H), 8.32 (d, 1H, Ar-H), 7.60-6.73 (m, 13H, Ar-H); MS
(EI) m/z 312 (M+).
4.2.4. 2-(3-Methoxyphenyl)-4,5-diphenyl-1H-imidazole (3d)
White solid, yield: 67%; m.p: 260-262 °C; FT-IR (υmax, cm-1): 3058 (N-H), 3055 (Ar C-
H), 2845 (Alkyl C-H), 1676 (C=N), 1589 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ 12.97
(s, 1H, NH), 7.66-6.90 (m, 14H, Ar-H), 3.88 (s, 3H, OCH3); MS (EI) m/z 326 (M+).
4.2.5. 2-(2-Methoxyphenyl)-4,5-diphenyl-1H-imidazole (3e)
Pale yellow solid, yield: 67%; m.p: 210-212 °C; FT-IR (υmax, cm-1): 3232 (N-H), 3060 (Ar
1
C-H), 2839 (Alkyl C-H), 1677 (C=N), 1585 (C=C); H-NMR (400 MHz, DMSO-d6): δ
12.91 (s, 1H, NH), 8.53 (d, 1H, Ar-H), 7.59-7.02 (m, 13H, Ar-H), 4.04 (s, 3H, OCH3); MS
(EI) m/z 326 (M+).
4.2.6. 4-(4,5-Diphenyl-1H-imidazol-2-yl)phenol (3f)
16

Yellow solid yield: 67%; m.p: 235-237 °C; FT-IR (υmax, cm-1): 3227 (N-H), 3055 (Ar C-
H), 2844 (Alkyl C-H), 1679 (C=N), 1594 (C=C); 1H-NMR (400 MHz, DMSO-d6): δ 12.35
(s, 1H, NH), 9.54 (s, 1H, O-H), 7.89 (d, 2H, Ar-H), 7.53-7.21 (m, 10H, Ar-H), 6.87 (d, 2H,
Ar-H); MS (EI) m/z 312 (M+).
4.2.7. 2-(4,5-Diphenyl-1H-imidazol-2-yl)-6-methoxyphenol (3g)
Pale yellow solid, yield: 67%; m.p: 170-172 °C; FT-IR (υmax, cm-1): 3236 (N-H), 3063 (Ar
1
C-H), 2842 (Alkyl C-H), 1690 (C=N), 1589 (C=C); H-NMR (400 MHz, DMSO-d6): δ
12.95 (s, 1H, NH), 9.61 (s, 1H, O-H), 8.32 (d, 1H, Ar-H), 7.60-6.73 (m, 13H, Ar-H), 3.89
(s, 3H, OCH3); MS (EI) m/z 342 (M+).
4.2.10. 2-(3,4-Dimethoxyphenyl)-4,5-diphenyl-1H-imidazole (3h)
Pale yellow solid, yield: 67%; m.p: 213-215 °C; FT-IR (υmax, cm-1): 3231 (N-H), 3066 (Ar
1
C-H), 2838 (Alkyl C-H), 1680 (C=N), 1602 (C=C); H NMR (400 MHz, DMSO-d6): δ
12.93 (s, 1H, NH), 7.73 (d, 1H, Ar-H), 7.26-6.86 (m, 12H, Ar-H), 3.94 (s, 3H, OCH3); MS
(EI) m/z 356 (M+).
4.2.8. 2-Methyl-4,5-diphenyl-1H-imidazole (3i)
Pale yellow solid, yield: 67%; m.p: 240-242 °C; FT-IR (υmax, cm-1): 3215 (N-H), 3067 (Ar
1
C-H), 2848 (Alkyl C-H), 1681 (C=N), 1561 (C=C); H NMR (400 MHz, DMSO-d6): δ
13.11 (s, 1H, NH), 7.55–7.34 (m, 10H, Ar-H), 2.56 (s, 3H, CH3); MS (EI) m/z 234 (M+).
4.2.9. 4,5-Diphenyl-2-(propan-2-yl)-1H-imidazole (3j)
17

Pale yellow solid, yield: 67%; m.p: 230-232 °C; FT-IR (υmax, cm-1): 3230 (N-H), 3063 (Ar
1
C-H), 2839 (Alkyl C-H), 1686 (C=N), 1584 (C=C); H NMR (400 MHz, DMSO-d6): δ
12.93 (s, 1H, NH), 7.66-7.26 (m, 10H, Ar-H), 3.18 (d, 1H, C-H), 1.25 (d, 6H, CH3); MS
(EI) m/z 262 (M+).
4.3. α-Glucosidase inhibition assay
A slightly modified method of Pierre et al. (1978) was adopted for α-glucosidase inhibition
assay. A 100 μL assay mixture containing 70 μL phosphate buffer saline (50 mM at pH
6.8), 10 μL corresponding test compounds dissolved in methanol/DMSO (0.5 mM) and 10
μL (0.057 units) α-glucosidase enzyme were mixed, pre incubated for 10 min at 37 °C and
the absorbance was recorded at 400 nm using Synergy HT BioTek (USA) 96-well plate
reader. The reaction was initiated by the addition of 10 μL p-nitrophenyl-α-D-
glucopyranoside (substrate, 0.5 mM/well, code No. N1377 from Sigma Inc).
The change in absorbance of p-nitrophenol formed was recorded at 400 nm after 30 min of
incubation at 37 °C. Both positive and negative controls were run. Acarbose was used as
positive control at 0.5 mM concentration. All experiments were carried out in triplicate and
results are mean SEM. For the determination of IC50 values, suitable dilutions of active
compounds were carried out for the assay. Data was computed for the determination of
IC50 values using EZ-Fit Enzyme kinetics software (Perrella Scientific Inc. Amherst,
USA).
The percent inhibition was calculated by the following equation;
Inhibition(%)= 100-(Abs. of test/Abs. of control) ×100
18

α-Glucosidase (Cat No. 5003-1KU Type I) from Saccharomyces cereviciae has been used
in the assay because of the structural and functional similarities between the yeast
(eukaryote) and mammalian enzyme (eukaryote).
4.4 Molecular modelling studies
The three-dimensional structures of the compounds were built with Corina on-line tool
[53] and subsequently prepared in Sybyl X-1.1 [54]. All atom and bond types, protonation
states were checked and later Gasteiger-Marsili charges were added. The homology model
of the baker’s yeast α-glucosidase, developed in the previously described way [49], was
used for dockings with Gold 5.1 software [55]. All histidine residues were set as Nε (HSE)
tautomers and ligand (glucose which was taken into account during model building) was
removed. The active site was defined as all amino acid residues within 15 Å from that
glucose molecule. Dockings were performed using genetic algorithm with default settings.
GoldScore function and analysis of binding mode were applied to find final ligand poses.
The docking method was previously validated, based on reference inhibitor - acarbose
[49]. Results were viewed in PyMOL 0.99rc2 [56].
4.5 Computational studies:
The processes based on the ‘‘recognition’’ of one molecule by another, as in drug-receptor,
and enzyme substrate interactions are related to electrostatic potential V(r), because the two
species come close to each other through their potentials. For the systems studied the MEP
values were calculated as described previously, using the following equation [52].
19

Where the summation runs over all the nuclei A in the molecule and polarization and
reorganization effects are ignored. ZA is the charge of the nucleus A, located at RA and ρ(r') is
the electron density function of the molecule. All calculations were performed with Gaussian
09 [57]. The geometries of the structures were optimized at hybrid B3LYP [58, 59] method
with 6-31G (d,p) basis set.
Acknowledgments
Molecular modelling studies were financially supported by Polish National Center of
Science, Postdoctoral Research Grant No. DEC-2012/04/S/NZ2/00116.
Conflict of interest
Authors declare no conflict of interest
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23

Graphical Abstract
Molecular electrostatic potential map of 3c
Binding mode of α-glucosidase inhibitor 3c
(IC50 74.32 0.59 μM)
24

High lights
•
•
•
•
•
A novel method has been developed for the facile and efficient synthesis of imidazole derivatives
This simple and versatile protocol yielded the targeted compounds in good to excellent yields
Most of the synthesized compounds have exhibited potent α-glucosidase activity
Generally ortho-hydroxy phenyl substituted compounds showed more promising inhibition
Compound 3c presented the highest inhibitory activity with IC50 value 74.32 0.59 μM
25