New 4,5-diphenylimidazole-acetamide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors: synthesis, in vitro and in silico enzymatic and toxicity evaluations

Article abstract of DOI:10.1007/s00706-021-02779-7

Herein, a new series of 4,5-diphenylimidazole-acetamide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors was designed and synthesized. All the synthesized compounds exhibited excellent inhibition potencies (IC₅₀ values = 55.6–149.2 μM)

Full text of DOI:10.1007/s00706-021-02779-7

Monatshefte für Chemie - Chemical Monthly
https://doi.org/10.1007/s00706-021-02779-7
ORIGINAL PAPER
New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids
as potent α‑glucosidase inhibitors: synthesis, in vitro and in silico
enzymatic and toxicity evaluations
Nima Sepehri1 · Homa Azizian2 · Reza Ghadimi3 · Fahimeh Abedinifar4 · Somayeh Mojtabavi5 ·
Mohammad Ali Faramarzi5 · Ali Akbar Moghadamnia6 · Ebrahim Zabihi6 · Gholamhossein Mohebbi7 ·
Bagher Larijani4 · Haleh Hamedifar8 · Maryam Mohammadi‑Khanaposhtani6 · Mohammad Mahdavi4
Received: 9 January 2021 / Accepted: 29 April 2021
© Springer-Verlag GmbH Austria, part of Springer Nature 2021
Abstract
Herein, a new series of 4,5-diphenylimidazole-acetamide-1,2,3-triazole hybrids as potent α-glucosidase inhibitors was
designed and synthesized. All the synthesized compounds exhibited excellent inhibition potencies (IC50 values=55.6–149.2
μM) against α-glucosidase when compared with the standard inhibitor acarbose (IC50 =750.0 μM). Among the newly synthe-
sized compounds, 4-methyl, 4-methoxy, and 2,3-dichloro derivatives exhibited the highest anti-α-glucosidase activities and
were also non-cytotoxic against human normal dermal fbroblast cells. In silico druglikeness, ADME, and toxicity studies of
these compounds were performed and obtained results were compared with acarbose. All the synthesized compounds were
also inactive against α-amylase in comparison to acarbose. Kinetic study of the most potent compound, 4-methyl derivative,
against α-glucosidase demonstrated that this compound is a competitive inhibitor. Furthermore, in silicoinduced ft docking
and molecular dynamic studies were performed to further investigate the interaction, orientation, and conformation of these
compounds over the active site of α-glycosidase.
Graphic abstract
Keywords α-Glucosidase · Kinetic study · Docking study · 4,5-Diphenylimidazole · Acetamide · 1,2,3-Triazole
Introduction
and/or insulin action. High glucose levels over a long time
causes serious problems in the vital organs of the human
body such as eyes, nerves, and kidneys. The number of
diabetic patients is increasing rapidly, and according to the
World Health Organization’s prediction, by 2030 diabetes
will become the seventh leading cause of death. In patients
with impaired insulin production, the main treatment is
insulin injection, but in patients with insulin dysfunction,
the main treatment is suppressing postprandial hyperglyce-
mia by drugs such acarbose and metformin [1, 2]. Acarbose
Diabetes mellitus is one of the most common metabolic
disorders which lead to increase glucose concentration
in the blood because of the defects in insulin production
* Maryam Mohammadi-Khanaposhtani
maryammoha@gmail.com
* Mohammad Mahdavi
momahdavi@tums.ac.ir
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

N. Sepehri et al.
inhibited the intestinal α-glucosidase, which is responsible
for the release of glucose from carbohydrates (oligosac-
charides and disaccharides) into the bloodstream [3]. In
addition to α-glucosidase, acarbose also inhibits pancreatic
α-amylase that is other important carbohydrate-hydrolyzing
enzyme [4, 5]. This enzyme is responsible for convert poly-
saccharides to oligosaccharides and disaccharides. It is well
documented that inhibition of α-amylase increased secre-
tion of undigested polysaccharides to long intestine and led
to gastrointestinal side efects such as fatulence, diarrhea,
abdominal discomfort, pain, and bloating [6]. On the other
hand, in addition to diabetes, inhibition of α-glucosidase can
be useful in the other carbohydrate mediated diseases such
as cancer and viral infections [7, 8].
compounds against the normal human cell line was deter-
mined. Furthermore, druglikeness, ADME, and toxicity
of these new compounds were also predicted by in silico
studies.
Results and discussion
Chemistry
The synthetic route for 4,5-diphenylimidazole-acetamide-1,2,3-
triazole hybrids 9a–9m has been depicted in Scheme 1.
It was started from the reaction of 4-(prop-2-ynyloxy)
benzaldehyde (1), benzil (2), and ammonium acetate (3)
in DMF at refux for 1 h to give 4,5-diphenyl-2-[4-(prop-
2-ynyloxy)phenyl]-1H-imidazole (4). On the other hand,
reaction between aniline derivatives 5a–5m and chloroacetyl
chloride (6) in DMF at room temperature for 30 min let to
produce 2-chloro-N-phenylacetamide derivatives 7a–7m.
Then, the latter compounds and sodium azide reacted in the
presence of Et3N in the mixture of H2O/t-BuOH at room
temperature for 1 h to give in situ azide derivatives 8a–8m.
Finally, mixture of 4,5-diphenyl-2-[4-(prop-2-ynyloxy)
phenyl]-1H-imidazole, CuSO4, and sodium ascorbate was
added to the freshly prepared azide derivatives 8a–8m and
the reaction was continued at room temperature for 18–22 h
to give the corresponding products 9a–9m.
4,5-Diarylimidazoles are found in many biological
active compounds with anticancer, anti-cyclooxygenase-2,
anti-bacterial, and anti-inflammatory activities [9–12].
Furthermore, several 4,5-diarylimidazole derivatives with
anti-α-glucosidase activity have been reported (Fig. 1)
[13–15]. For example, 4,5-diphenylimidazole-1,2,3-triazole
derivatives A exhibited high inhibitory activities against
α-glucosidase (IC50 values = 90.4–246.7 μM comparing
with acarbose IC50 = 750.0 μM) (Fig. 1) [15]. One of the
other important scafolds for design of new hybrid molecules
with various biological activities is 1,2,3-triazole ring [16].
This ring observed in the potent α-glucosidase inhibitors
such as benzimidazole-acetamid-1,2,3-triazole derivatives
B (IC50 values=25.2–176.5 μM comparing with acarbose
IC50 =750.0 μM) (Fig. 2) [17, 18]. Because of the presence
of 4,5-diphenylimidazole, acetamid-1,2,3-triazole moieties
in the potent α-glucosidase inhibitors A and B, herein, we
decided to design and synthesize of 4,5-diphenylimidazole-
acetamide-1,2,3-triazole derivatives C as new α-glucosidase
inhibitors (Fig. 2). To evaluate of interactions of these com-
pounds with α-glucosidase, kinetic, docking, and molecular
dynamic studies of these compounds were also performed.
Moreover, in vitro cytotoxic effects of the synthesized
In vitro α‑glucosidase inhibitory activity
Anti-α-glucosidase activity of the synthesized 4,5-diphe-
nylimidazole-acetamide-1,2,3-triazole hybrids 9a-9m was
determined in vitro against yeast α-glucosidase (Saccharo-
myces cerevisiae, EC 3.2.1.20) and compared with acarbose
as standard inhibitor [19]. The obtained result demonstrated
that all the compounds 9a–9m are more potent than acar-
bose (IC50 = 750.0 12.5 μM) and among them, the most
Fig. 1 Design strategy for new 4,5-diphenylimidazole-acetamide-1,2,3-triazole derivatives 9
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
Fig. 2 Kinetics of α-gluosidase inhibition by acarbose and compound
9b. a and c Lineweaver–Burk plot in the absence and presence of dif-
ferent concentrations of acarbose and compound 9b, respectively; b
and d the secondary plot between Km and various concentrations of
acarbose and compound 9b, respectively
potent compounds were compounds 9b, 9d, and 9h with
IC50 values 55.6 0.4, 66.7 0.6, and 78.7 0.7 μM, respec-
tively. Moreover, the remaining compounds with the IC50
values ranging from 87.7 0.8 to 149.2 1.5 μM exhibited
α-glucosidase inhibitory activities around 8.6-5 folds more
than acarbose.
To optimize the anti-α-glucosidase activity, the substitu-
ent on the phenyl ring of N-phenylacetamide moiety was
altered. Among the N-phenylacetamide derivatives 9a–9m,
the most potent compound was 4-methyl derivative 9b.
Removing the methyl substituent or replace it with meth-
oxy or phenoxy substituents, led to a slightly decrease in the
Scheme 1
1 3

N. Sepehri et al.
inhibitory activity as observed in the compounds 9a, 9d, and
9m, respectively. As can be seen in Table 1, adding a bro-
mine atom to 4-methylphenyl group dramatically decreased
inhibitory activity (compound 9b vs. compound 9c).
compound 9i, led to a signifcant decrease in the inhibi-
tory activity. In addition, inhibitory activity of the com-
pounds 9e–9g with a chloro substituent, demonstrated
that translocation of chlorine atom on the phenyl group of
N-phenylacetamide moiety had significant effect on
α-glucosidase inhibitory activity, and 2-chloro was preferred
[order of inhibitory activity: 2-Cl (compound 9e)>3-Cl (com-
pound 9f)>4-Cl (compound 9g)]. Replacement of chlorine
atom at 3-position with bromine atom (compound 9k) or 2-posi-
tion with nitro group (compound 9l) increased α-glucosidase
inhibition. Moreover, adding a fuorine atom to 4-position of
3-chlorophenyl derivative 9f led to a signifcant decrease in the
inhibitory activity as observed in the compound 9j.
Among the chloro-substituted derivatives 9e–9j, com-
pound 9h with 2,3-dichlorophenyl moiety was the most
potent compound. Removing the 3-chloro substituent, as
in compound 9e, led to slightly decrease in the inhibitory
activity while movement of 2-chloro to 5-position, as in
Table 1 α-Glucosidase and α-amylase inhibitory activities of imida-
zole-acetamide-1,2,3-triazoles derivatives 9a–9m
The comparison of IC50 values of the new 4,5-diphe-
nylimidazole-acetamide-1,2,3-triazole derivatives 9 with
their corresponding analogs of 4,5-diphenylimidazole-1,2,3-
triazole series A revealed that presence of acetamid unit
increased α-glucosidase inhibitory activity (Scheme 2)
[15]. Therefore, it seems that our strategy to replacement
of benzyl moieties of compounds A with N-phenylaceta-
mide moieties led to increase in inhibitory activity against
α-glucosidase.
Compound
R
IC50/μM
α-glucosidase
α-amylase
In contrast, this comparison between new compounds 9
and reported benzimidazole-acetamid-1,2,3-triazole deriv-
atives B revealed that, with the exception of 4-methoxy
derivative of 4,5-diphenylimidazole series, benzimidazole
derivatives were more potent than their analogs of 4,5-diphe-
nylimidazole series (Scheme 3) [17].
9a
H
87.7 0.8
55.6 0.4
129.3 1.3
66.7 0.6
107.6 1.1
116.6 1.2
125.2 1.3
78.7 0.7
134.5 1.4
149.2 1.5
98.2 1.0
94.7 0.9
83.7 0.8
750.0 12.5
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
108 0.71
9b
4-CH3
2-Br-4-CH3
4-OCH3
2-Cl
3-Cl
4-Cl
2,3-dichloro
3,5-dichloro
3-Cl-4-F
3-Br
2-Nitro
4-O-Ph
–
9c
9d
9e
9f
α-Amylase inhibition assay of the synthesized compounds
was also performed and obtained results demonstrated that
all new compounds 9a–9m were inactive against α-amylase
(IC50 > 200 μM) when compared with standard inhibitor
acarbose (IC50 =108 0.71 μM) [19].
9g
9h
9i
9j
9k
9l
Kinetic study
9m
Acarbose
To determine the interaction type of the synthesized com-
pounds with α-glucosidase and to compare them with
Scheme 2
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
Scheme 3
standard drug, kinetic studies was performed on the acarbose
and the most active compound 9b [20]. The type of inhi-
bition these compounds and their inhibitory constant were
indicated by Lineweaver–Burk plots and the secondary re-
plot of these plots. As shown in Fig. 2a, c, with increasing
concentration of acarbose and compound 9b, Vmax values
were not afected, while the Km values increased. There-
fore, the latter compounds are competitive inhibitors against
α-glucosidase. Ki values of acarbose and compound 9b were
737 and 50 µM, respectively (Fig. 2b, d).
Molecular docking study
Molecular docking study was carried out to evaluate the
interaction modes of the newly synthesized compounds
9a–9m in the modeled α-glycosidase [21–26]. The location
and residue specifcation of the modeled α-glycosidase active
site is shown in Fig. 3a. According to Yamamoto et al. inves-
tigation, the α-glycosidase pocket shape active site located
at the interface of domain A and B, which contain conserve
residues around the substrate-binding site [27]. There are
three conserve acidic residues including Asp214 (Asp215),
Asp349 (Asp352), and Glu277 (Glu276), which have the
Fig. 3 Representation of α-glycosidase active site. a The environment
of α-glycosidase active site located at the interface of domain A and
B. Domain A, B, and C are colored in yellow, blue, and red, respec-
tively. b Close-up representation of active site, the modeled α-D-
glucose and the corresponding re-docked form represented in green
and cyan color, respectively
1 3

N. Sepehri et al.
catalytic role [28, 29]. His279 (His280), Phe157 (Tyr158),
and the loop consisting of residues 310–315 located at the
entrance of the active site, while Tyr72 situated at the bottom
of the active site. In addition, Asp68 (Asp69) and Arg443
(Arg442) placed at the wall of the active site which have
the role in recognition of non-reducing end of the glucose
(numbering in the parenthesis indicate the residue number-
ing according to the isomaltase structure). Finally, residues
including Arg439, His111, His348, Arg212, and Gln181 are
the residues around the substrate-binding pocket.
4,5-diphenyl-1H-imidazol-phenoxy moiety pointed to the
mouth of the active site in the manner that 4,5-diphenyl
rings stacked between the two loops at the large hydrophobic
entrance of the active site; one from domain A (in yellow)
and the other one from domain B (in blue). In addition, the
1,2,3-triazol ring and the N-phenylacetamide moiety posi-
tioned at the bottom of the U conformer and along domains I
and II, respectively (Fig. 4a). Figure 4b shows the horizontal
U-shape orientation of compounds 9c, 9f, 9i, 9e, 9g, 9l in
to the active site in which the 4,5-diphenyl-1H-imidazol-
phenoxy moiety and N-phenylacetamide moiety at the mouth
of U structure pointed toward the conserved region I and II
and the 1,2,3-triazol ring which is at the bottom of the U
structure directed toward the active site mouth.
The reliability of the applied docking protocol was
assessed by re-docking of α-D-glucose into the active site of
the α-glucosidase. The key characteristic of a good docking
program is its ability to reproduce the actual binding modes
of ligands. To test this, α-D-glucose taken out of the mod-
eled protein–ligand complex and re-docked into its binding
site. The docked binding mode is then compared with the
modeled binding mode, and the RMSD is calculated; suc-
cessful binding mode prediction is considered if the RMSD
is below a certain value (usually<2.0 Å). Figure 3b shows
the superimposed structures between the docked and the
modeled α-D-glucose over α-glucosidase, which has RMSD
acceptable (RMSD value = 0.3 Å). According to Fig. 3b,
the docked glucose molecule is bound at the bottom of the
active site pocket by ten hydrogen bonds and it stacks against
Tyr72.
The molecular interactions of the best conformational
pose and energy valued docked complex of compounds 9b,
9d, and 9h with higher inhibition activity and compounds
9e, 9g, and 9i with the relative lower inhibition activity illus-
trated in Fig. 5.
Compounds 9b, 9d, and 9h shows similar orientation and
interaction which is matched to the conformation depicted
in Fig. 4a. In all the mentioned compounds the rings related
to 1H-imidazol, 1,2,3-triazol, and the phenoxy moiety sta-
bilized through π-π stacking and T-shape π-π hydrophobic
interaction with the side-chain group of Phe157. In addi-
tion, the 1H-imidazol ring and the phenoxy oxygen atom (O)
formed H-bond interaction with the backbone (C=O) of the
Phe157 and side chain of Arg439, respectively (Fig. 5a–c).
Figure 5d–f provides the interaction details of compounds
9e, 9g, and 9i in which the 1H-imidazol moiety stabilized
through H-bond and π-π stacking interaction with Tyr313
and Phe311, respectively. Furthermore, the acetamide linker
After confrmation of modeled α-glycosidase, the top
scoring pose of all the title compounds inside the active
site was analyzed. Figure 4 represents that the synthesized
compounds have two diferent ftting-in conformers. Both
of the conformers are in U-shape structure but have dif-
ferent orientations inside the active site. The frst one is
related to the compounds 9b, 9d, 9k, and 9h in which the
Fig. 4 a Docked representation of the compounds 9b, 9d, 9k, and
9h over the α-glycosidase active site. b Docked representation of the
compounds 9c, 9f, 9i, 9e, 9g, and 9l over the α-glycosidase active site
(domain A and B are colored in yellow and blue, respectively. The
docked compounds colored in green. I, II, III, and IV are the four
α-glycosidase active site conserved regions and the dash lines depicts
the mouth of the active site)
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
Fig. 5 3D representation of ligand-residue interactions of compounds 9b (a), 9d (b), 9h (c), 9e (d), 9g (e), and 9i (f) over α-glycosidase active
site. (Domain A and B are colored in yellow and blue, respectively)
provide H-bond interaction through with the backbone car-
bonyl group of Ser156 and Phe157, respectively.
inhibition activity. In addition, the presence and the essen-
tial interaction role of Phe157 from the B domain side of
the active site mouth besides Arg312 from the opposite site
over the A domain side of the active site mouth have been
identifed for compounds stabilization over the α-glycosidase
active site.
Based on the observed IFD result, it can be proposed
that our synthetic compounds reside in to the α-glycosidase
active site through two diferent orientations with one of
them more corresponding to the higher α-glycosidase
1 3

N. Sepehri et al.
around 2 Å and used to investigate the structural specifcity
of α-glycosidase-ligand complexes.
Molecular dynamic investigation
The fexibility of protein structure can be evaluated
through investigating the RMSF value in which evaluate
the fuctuation of the protein backbone structure from its
average position throughout the whole simulation time.
The residue RMSF values of α-glycosidase apo enzyme
and the enzyme in bounded state showed that, the apo
enzyme (yellow-colored dash box) had higher RMSF fuc-
tuations compared to the glycosidase bound-states (green-
and red-colored dash boxes) (Fig. 7a).
Additionally, MD simulation was performed to understand
the efect of the compound over the enzyme active site [30].
For this purpose, the structural perturbations incurred by
the most potent compound 9b has been investigated through
study the RMSD, RMSF, and its efect on the active site
environment in comparison to acarbose as α-glycosidase
standard inhibitor and the apo enzyme (Fig. 6).
Root mean square deviation (RMSD) of the enzymes’
backbone was analyzed over 20 ns MD simulation to study
the stability of the protein–ligand complex trajectories.
The RMSD value of the apo α-glycosidase depicts broad
fuctuations throughout simulation time which is higher
than the two enzyme complexes. The RMSD value sharply
increased in the frst 4 ns and steadily increased up to 16 ns
and become more stable for the last 4 ns of simulation time
with the value of 2.5 Å. The RMSD value of α-glycosidase
complexed with acarbose was stable until 12 ns and slightly
increased through the next 4 ns and become steady for the
rest of the simulation time with the lower RMSD value of
2 Å.
According to the result, the mentioned lower RMSF
value occurs upon ligand binding to the enzyme, in which
residues movement decrease as a result of non-bonding
interaction between the ligand and the enzyme active site.
Moreover, Fig. 7a depicts that there are four structural
segments which revealed different RMSF pattern upon
ligand binding including; B domain loop (pink dash box), the
active site lid (pin dash box), A domain and B domain sides
of the active site entrance (orange and blue dash boxes).
Based on results shown on Fig. 5a, b, the RMSF value
of the B domain loop residues would have decreased in
glycosidase/acarbose and compound 9b bound-state rather
than apo enzyme which is due to the presence of non-
bonding interaction related to Phe157.
Comparing the apo enzyme, the RMSD value of
α-glycosidase complexed with compound 9b is highly simi-
lar as acarbose bounded state especially for the frst 6 ns and
slightly increased due to the higher number of ring and fex-
ibility which makes it slightly deviate from the initial struc-
ture for the rest of simulation time with the same RMSD as
acarbose bounded state (2 Å).
In contrast, although the fexibility of the active site
lid was the highest in enzyme acarbose bound state, the
mentioned segment fexibility revealed the lowest in gly-
cosidase complexed with compound 9b. To investigate the
proposed reason of the mentioned observation, acarbose
and compound 9b interacted with several residues located
both on A and B domain side of the active site mouth
(Fig. 7b). Although acarbose showed more interaction with
the A side domain of active site entrance (orange dash line
To be brief, the RMSD value of the bounded state
enzymes obviously deviated from the apo enzyme at the frst
time of simulation and obviously decreased as a result of
α-glycosidase structural rigidity due to active site bounding
state. The RMSD value for the last 5 ns steadily equilibrated
Fig. 6 RMSD of the
α-glycosidase backbone in com-
plexed with acarbose (in green),
compound 9b (in red) and the
apo enzyme (in yellow) for over
20-ns MD simulation time
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
Fig. 7 RMSF plot of the α-glycosidase backbone in complexed with
compound 9b (in red) and acarbose (in green) and the apo enzyme
(in yellow) for over 20-ns MD simulation time (a). Ligand binding
location of glycosidase in bound and unbound-state for over 20 ns
MD simulation time (α-helical and ß-strand regions are highlighted
in red and blue backgrounds, respectively) (b). 3D representation of
α-glycosidase structure. Enzyme domain of A, B, and C are colored
in yellow, blue, and orange, respectively. The fexible regions corre-
spond to B domain loop and active site lid are colored in pink (c).
Close-up representation of α-glycosidase active site (d)
in glycosidase/acarbose), compound 9b provided higher
number of interactions with the residues of B domain side
(blue dash line in glycosidase/compound 9b). Therefore, it
can be proposed that the more ligand interaction with the
B domain sides of the active site mouth, the lower RMSF
of the active site lid.
Moreover, Fig. 7c, d represents the organization of the
α-glycosidase three main domains; A, B, and C along with
1 3

N. Sepehri et al.
the close-up representation of the active site mouth with
the corresponding residues of A and B domains at the both
sides of active site entrances.
9b than acarbose which also supported by experimental
assay.
In summary, based on the RMSF plot, acarbose and com-
pound 9b have almost the same interaction pattern through
the whole α-glycosidase structure. The only dissimilarity
comes from active site lid fexibility in which we can pro-
posed that compound 9b along with more interaction over
the B domain side of the active site entrance has domi-
nant efect in rigidity of active site lid rather than acarbose
which has more interaction with the A domain side of the
α-glycosidase active site entrance.
Cytotoxicity against normal human cells
Cytotoxicity of the most active compounds 9b, 9d, and
9h was evaluated against human dermal fbroblasts (HDF)
cells using by MTT assay [19]. Obtained results revealed
that at 80 µM, these compounds were non-cytotoxic
against studied cell line.
Figure 8 represents the detailed orientation and interac-
tions that occurred more than 30% of the simulation time
during the equilibrated phase over α-glycosidase complexed
with compound 9b.
In silico druglikeness, ADME, and toxicity studies
Druglikeness/ADME/T properties of the compounds 9b,
9d, 9h, and acarbose were evaluated in silico using by
online software PreADMET and the predicted results were
shown in Table 2 [31]. As can be seen in Table 2, our syn-
thesized compounds and acarbose violated rule of fve and
are drug like according to MDDR like rule. Acarbose and
compounds 9b, 9d, and 9h have poor to medium Caco-2
cell permeability while all studied compounds have low
blood brain barrier (BBB) nd skin permeability. On the
other hand, compounds 9b, 9d, and 9h have high human
oral absorption (HIA) while acarbose does not show HIA.
Predicting the toxicity of selected compounds by Pre-
ADMET server demonstrated that our synthesized com-
pounds, unlike acarbose, were not mutagenic (Ames test).
Furthermore, compounds 9b, 9d, and 9h are medium risk
in term of cardiotoxicity while cardiotoxicity of acarbose
is ambiguous. Synthesized compounds 9b, 9d, and 9h,
unlike acarbose, were not carcinogen on mouse. On the
other hand, in term of carcinogenicity on rat, compound
9h and acarbose were negative.
It represents two important structural moieties in stabi-
lizing compound 9b at the mouth of the active site. The
frst one is the imidazole ring which interacts with Phe157
located on B domain side of active site entrance through
H-bond interactions for the whole simulation time (99%) and
the next one, is the central part of the phenoxy ring which
interacts with Arg312 located on A domain side through
π-cation interaction for 78% of the simulation time. Along
to the interactions which stabilized compound 9b in front of
the active site entrance, the acetamide moiety can provide
H-bond interaction with Thr215 at the bottom of the active
site for about 55% of simulation time.
In addition to the interaction analysis, the Prime/MM-
GBSA module was used to estimate the strengths of inter-
actions between the ligand–protein complexes which gen-
erated by the clustering method. ΔGbind of α-glycosidase/
compound 9b complex and α-glycosidase/acarbose complex
were estimated to be−349.26 and−261.63 kJ/mol, respec-
tively, revealing stronger binding interaction of compound
Fig. 8 The detailed orientation and ligand atom interactions that occurred more than 30.0% of the simulation time during the equilibrated phase
over α-glycosidase complexed with compound 9b
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
Table 2 Druglikeness/ADME/T
Profle of the most potent
Druglikeness/ADME/Ta
Compound
compounds 9b, 9d, and 9 h
9b
9d
9h
Acarbose
Rule of fve
MDDR like rule
Caco2
HIA
BBB
Skin permeability
Ames test
hERG inhibition
Carcino mouse
Carcino rat
Violated
Drug-like
26.095
95.076467
2.93397
−2.61499
Non-mutagen
Medium risk
Negative
Positive
Violated
Drug-like
34.0086
95.295168
1.31653
−2.72782
Non-mutagen
Medium risk
Negative
Violated
Drug-like
39.3674
95.513821
4.71572
−2.62073
Non-mutagen
Medium risk
Negative
Violated
Drug-like
9.44448
0.000000
0.0271005
−5.17615
Mutagen
Ambiguous
Positive
Positive
Negative
Negative
aThe recommended ranges for Caco2:<25 poor,>500 great, HIA:>80% is high<25% is poor,
BBB= −3.0–1.2, and Skin_Permeability= −8.0 to −1.0
disks). Mass spectra for several synthesized compounds
were obtained with an Agilent Technology (HP) mass
Conclusion
spectrometer operating at an ionization potential of 70 eV.
Finally, elemental analysis was performed by an Elementar
Analysen system GmbH VarioEL CHN mode. Compounds
8a–8m were obtained in situ according to described pathway
in our previous work [17].
In conclusion, we presented hybrid structures of 4,5-diphe-
nylimidazole-acetamide-1,2,3-triazole, and acetamide moie-
ties as new α-glucosidase inhibitors. The results of anti-α-
glucosidase activity of the synthesized compounds 9a–9m
revealed that all these compounds were more potent than
standard inhibitor acarbose. Among them, compounds 9b,
9d, and 9h exhibited the highest α-glucosidase inhibitory
activities and were also non-cytotoxic against HDF cells.
Moreover, the most potent compound 9b was a competitive
inhibitor against α-glucosidase; thus, the molecular mode-
ling study was performed in the active site of α-glucosidase.
This study permitted us to evaluation of interaction modes of
the synthesized compounds in the α-glucosidase active site.
Furthermore, molecular dynamic simulations showed that
compound 9b oriented by vertical U-shape structure into
the active site from mouth to the bottom and stabilized the
enzyme domains by interacting with the interface of domain
A and domain B through long lasting non-binding interac-
tions with Phe157, Thr215, His239, and Arg312 which have
the signifcant role for inhibition activity of the mentioned
compound. In silico druglikeness/ADME/T studies revealed
that the most potent compounds can be druglikeness agents
with appropriate ADME properties and toxicity profle when
compared with acarbose.
4,5‑Diphenyl‑2‑[4‑(prop‑2‑ynyloxy)phenyl]‑1H‑imida‑
zole (4, C24H18N2O) A mixture of 4-(prop-2-yn-1-yloxy)
benzaldehyde (1, 1 mmol), benzil (2, 1 mmol), and ammo-
nium acetate (3, 10 mmol) in 10 cm3 acetic acid was heated
at refux for 1 h. Upon completion of the reaction (checked
by TLC), the obtained mixture was poured into cold water,
the precipitate was fltered of, and washed with cold water
to aford desired product 4. White powder; yield: 85%;
m.p.: 203–205 °C; IR (KBr): v = 3417, 3329, 3068 cm−1;
1H NMR (300 MHz, DMSO-d6): δ=3.61 (s, 1H, CH), 4.87
(s, 2H, CH2), 7.11 (d, J=7.56 Hz, 2H, H2, H6), 7.26–7.37
(m, 6H, Ph), 7.52–7.54 (m, 4H, Ph), 8.02 (d, J= 8.13 Hz,
2H, H3, H5), 11.99 (s, 1H, NH) ppm; 13C NMR (75 MHz,
DMSO-d6): δ = 55.97, 78.80, 115.47, 115.76, 124.29,
127.12, 128.85, 145.96, 157.82, 172.51 ppm; MS (70 eV):
m/z=350 (M+).
General procedure for the synthesis
of 4,5‑diphenylimidazole‑1,2,3‑triazole‑acetamide
hybrids 9a–9m
Experimental
A mixture of 4,5-diphenyl-2-[4-(prop-2-ynyloxy)phenyl]-
1H-imidazole (4, 1 mmol), CuSO4·5H2O (7 mol%), and
catalytic amounts of sodium ascorbate was added to the
freshly prepared azide derivatives 8a–8m and stirred at room
temperature for 18–22 h. Upon completion of the reaction
(monitored by TLC), the obtained mixture was poured into
crushed ice. After that, the precipitates were fltered of,
Melting points of 4,5-diphenylimidazole-acetamide-1,2,3-
triazole 9a–9m were determined with a Kofer hot stage
1
apparatus. H and 13C NMR spectra of these derivatives
were obtained with a Bruker FT-500, using TMS as an
internal standard. IR spectra of the title compounds recoded
with a Nicolet Magna FTIR 550 spectrophotometer (KBr
1 3

N. Sepehri et al.
washed with water, and purifed by recrystallization from
ethanol to give the target derivatives 9a–9m.
13C NMR (500 MHz, DMSO-d6): δ=52.6, 55.6, 61.6, 114.5,
115.3, 121.3, 123.9, 126.8, 127.2, 131.9, 142.8, 146.0,
156.0, 158.7, 164.1 ppm; MS (70 eV): m/z=556 (M+).
2‑[4‑[[4‑(4,5‑Diphenyl‑1H‑imidazol‑2‑yl)phenoxy]‑
methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑N‑phenylacetamide (9a,
C32H26N6O2) White powder; yield: 75%; m.p.: 192–194 °C;
IR (KBr): v=3419, 3062, 1692 cm−1; 1H NMR (500 MHz,
DMSO-d6): δ=5.24 (s, 2H, CH2), 5.36 (s, 2H, CH2), 7.07
(t, J = 7.5 Hz, 1H, H4ʹ), 7.17 (d, J = 8.9 Hz, 2H, H2, H6),
7.28–7.36 (m, 8H, Ph, H3ʹ, H5ʹ), 7.50–7.51 (m, 4H, Ph),
7.57 (d, 2H, J = 8.5 Hz, H2ʹ, H6ʹ), 8.02 (d, J = 9 Hz, 2H,
H3, H5), 8.25 (s, 1H, triazole), 10.47 (s, 1H, NH), 12.65 (s,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): δ=52.4,
61.6, 115.3, 123.9, 126.7, 127.2, 127.5, 127.9, 128.8, 139.2,
142.8, 146.0, 158.7, 165.7 ppm; MS (70 eV): m/z = 526
(M+).
N‑(2‑Chlorophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑imida‑
zol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]aceta‑
mide (9e, C32H25ClN6O2) White powder; yield: 72%; m.p.:
184–186 °C; IR (KBr): v=3421, 3070, 1684 cm−1; 1H NMR
(500 MHz, DMSO-d6): δ=5.20 (s, 2H, CH2), 5.50 (s, 2H,
CH2), 7.18 (d, J = 9 Hz, 2H, H2, H6), 7.2–7.60 (m, 12H,
Ph), 7.75 (d, J = 8 Hz, 1H, H3ʹ), 8.05 (d, J = 8.5 Hz, 2H,
H3, H5), 8.3 (s, 1H, triazole), 10.27 (s, 1H, NH), 12.5 (bs,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): δ=52.4,
61.6, 115.3, 123.9, 126.9, 126.4, 127.2, 127.5, 128.0, 128.6,
128.8, 129.1, 130.1, 131.7, 134.6, 142.9, 146.0, 158.7, 166.4
ppm.
2‑[4‑[[4‑(4,5‑Diphenyl‑1H‑imidazol‑2‑yl)phenoxy]‑
methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑N‑(p‑tolyl)acetamide
(9b, C33H28N6O2) White powder; yield: 77%; m.p.: 199–
N‑(3‑Chlorophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑imida‑
zol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]aceta‑
mide (9f, C32H25ClN6O2) White powder; yield: 72%; m.p.:
206–208 °C; IR (KBr): v=3419, 3069, 1684 cm−1; 1H NMR
(500 MHz, DMSO-d6): δ=5.25 (s, 2H, CH2), 5.40 (s, 2H,
CH2), 7.1–7.20 (m, 3H, H4ʹ, H2, H6), 7.3–7.4 (m, 7H, Ph,
H3ʹ), 7.50 (d, J=7.5 Hz, 1H, H6ʹ), 7.55 (m, 4H, Ph), 7.8 (s,
1H, H2ʹ), 8.05 (d, 2H, J=8.5 Hz, H3, H5), 8.3 (s, 1H, tria-
zole), 10.7 (s, 1H, NH), 12.5 (bs, 1H, NH) ppm; 13C NMR
(500 MHz, DMSO-d6): δ=52.7, 61.6, 115.3, 118.1, 119.2,
123.9, 124.0, 126.8, 127.2, 128.9, 131.1, 133.7, 140.3,
142.9, 146.0, 158.7, 165.1 ppm.
1
201 °C; IR (KBr): v = 3417, 3065, 1689 cm−1; H NMR
(500 MHz, DMSO-d6): δ = 2.1 (s, 3H, CH3), 5.20 (s, 2H,
CH2), 5.35 (s, 2H, CH2), 7.12 (d, J = 8 Hz, 2H, H2, H6),
7.15 (d, J=8.5 Hz, 2H, H3ʹ, H5ʹ), 7.25–7.45 (m, 7H, Ph),
7.5 (d, J=7.5 Hz, 2H, H2ʹ, H6ʹ), 7.55 (m, 3H, Ph), 8.05 (d,
J=8.5 Hz, 2H, H3, H5), 8.3 (1H, s, triazole), 10.5 (1H, s,
NH), 12.5 (1H, bs, NH) ppm; 13C NMR (500 MHz, DMSO-
d6): δ=21.0, 52.7, 61.6, 115.3, 119.7, 123.9, 126.8, 127.2,
129.7, 133.2, 136.4, 142.9, 158.7, 164.4 ppm; MS (70 eV):
m/z=540 (M+).
N‑(4‑Chlorophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑imida‑
zol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]aceta‑
mide (9g, C32H25ClN6O2) White powder; yield: 68%; m.p.:
185–187 °C; IR (KBr): v=3418, 3068, 1687 cm−1; 1H NMR
(500 MHz, DMSO-d6): δ=5.25 (s, 2H, CH2), 5.40 (s, 2H,
CH2), 7.15 (d, J=9 Hz, 2H, H2,6), 7.2–7.4 (m, 5H, Ph), 7.41
(d, J=9 Hz, 2H, H3ʹ, H 5ʹ), 7.5–7.6 (m, 5H, Ph), 7.61 (d,
J=9 Hz, 2H, H2ʹ,H6ʹ), 8.10 (d, J=9 Hz, 2H, H3,H5), 8.3 (s,
N‑(2‑Bromo‑4‑methylphenyl)‑2‑[4‑[[4‑(4,5‑diphe‑
nyl‑1H‑imidazol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑tria‑
zol‑1‑yl]acetamide (9c, C33H27BrN6O2) White powder;
yield: 79%; m.p.: 209–211 °C; IR (KBr): v = 3419, 3068,
1
1685 cm−1; H NMR (500 MHz, DMSO-d6): δ = 2.29 (s,
3H, CH3), 5.26 (s, 2H, CH2), 5.45 (s, 2H, CH2), 7.1–7.20
(m, 3H, H2, H6, H5ʹ), 7.25–7.40 (m, 6H, H3ʹ, Ph), 7.5–7.6
(m, 6H, H6ʹ, Ph) 8.05 (d, J = 8.5 Hz, 2H, H3, H5), 8.3 (s,
1H, triazole), 10.01 (s, 1H, NH), 12.5 (bs, 1H, NH) ppm;
13C NMR (500 MHz, DMSO-d6): δ=20.5, 52.4, 61.6, 115.3,
118.1, 123.9, 126.8,127.2, 129.3, 133.2, 133.3, 137.8, 142.8,
146.0, 158.7, 165.2 ppm.
1H, triazole), 10.5 (s, 1H, NH), 12.5 (s, 1H, NH) ppm; 13
C
NMR (500 MHz, DMSO-d6): δ=52.7, 61.6, 115.3, 121.3,
123.4, 126.8, 127.2, 127.8, 128.8, 129.3, 137.3, 142.9,
146.0, 158.1, 164.9 ppm.
N‑(2,3‑Dichlorophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑im‑
idazol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑
acetamide (9h, C32H24Cl2N6O2) White powder; yield:
66%; m.p.: 210–212 °C; IR (KBr): v = 3426, 3071, 1681
2‑[4‑[[4‑(4,5‑Diphenyl‑1H‑imidazol‑2‑yl)phenoxy]‑
methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑N‑(4‑methoxyphenyl)‑
acetamide (9d, C33H28N6O3) White powder; yield: 75%;
m.p.: 202–204 °C; IR (KBr): v = 3421, 3063, 1681 cm−1;
1H NMR (500 MHz, DMSO-d6): δ = 3.73 (s, 3H, OCH3),
5.24 (s, 2H, CH2), 5.36 (s, 2H, CH2), 6.92 (d, J = 8.5 Hz,
2H, H3ʹ, H5ʹ), 7.20 (d, J=8.5 Hz, 2H, H2, H6), 7.25–7.60
(m, 12H, Ph, H2ʹ, H6ʹ), 8.05 (d, J=8.5 Hz, 2H, H3, H5), 8.3
(1H, s, triazole), 10.5 (s, 1H, NH), 12.5 (bs, 1H, NH) ppm;
1
cm−1; H NMR (500 MHz, DMSO-d6): δ = 5.26 (s, 2H,
CH2), 5.50 (s, 2H, CH2), 7.15 (d, J=8.5 Hz, 2H, H2, H6),
7.25 (m, 1H, Ph), 7.30 (t, J = 7.5 Hz, 2H, Ph), 7.36–7.40
(m, 2H, Ph), 7.44 (t, J=7.5 Hz, 2H, Ph), 7.55 (m, 3H, Ph),
7.75 (d, J = 8 Hz, 1H, H6ʹ), 8.05 (d, J = 8.5 Hz, 2H, H3,
H5), 8.3 (s, 1H, triazole), 10.27 (s, 1H, NH), 12.5 (bs, 1H,
1 3

New 4,5‑diphenylimidazole‑acetamide‑1,2,3‑triazole hybrids as potent α‑glucosidase…
NH) ppm; 13C NMR (500 MHz, DMSO-d6): δ=52.4, 61.6,
115.3, 123.9, 126.8, 127.2, 127.5, 128.1, 128.1, 128.6,
128.8, 129.1, 131.7, 132.5, 135.7, 136.6, 137.3, 142.9,
146.0, 158.7, 165.5 ppm.
61.5, 115.3, 125.5, 126.0, 126.4, 126.8, 127.2, 130. 8, 134.6,
142.9, 158.7, 165.4 ppm; MS (70 eV): m/z=571 (M+).
2‑[4‑[[4‑(4,5‑Diphenyl‑1H‑imidazol‑2‑yl)phenoxy]‑
methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑N‑(3‑phenoxyphenyl)‑
acetamide (9 m, C38H30N6O3) White powder; yield: 65%;
m.p.: 193–195 °C; IR (KBr): v = 3423, 3065, 1682 cm−1;
1H NMR (500 MHz, DMSO-d6): δ=5.25 (s, 2H, CH2), 5.36
(s, 2H, CH2), 6.76 (d, J=6.5 Hz, 1H, H3ʹ), 7.05 (d, J=8.5
Hz, 2H, H2, H6), 7.15–7.60 (m, 18H, Ph, H2ʹ, H4ʹ, H5ʹ),
8.05 (d, J=8.5 Hz, 2H, H3, H5), 8.3 (s, 1H, triazole), 10.50
(s, 1H, NH), 12.5 (bs, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): δ=52.7, 61.6, 109.3, 114.2, 114.4, 115.3, 119.
5, 123.9, 124.2, 126.8, 127.2, 127.5, 128.6, 128.7, 128.8,
128.9, 129.1, 130. 6, 130.8, 140.4, 142.7, 146.0, 156. 7,
157.8, 158.7, 164.8 ppm.
N‑(3,5‑Dichlorophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑im‑
idazol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑
acetamide (9i, C32H24Cl2N6O2) White powder; yield: 71%;
m.p.: 193–195 °C; IR (KBr): v=3424, 3069, 1684 cm−1; 1H
NMR (500 MHz, DMSO-d6): δ = 5.25 (s, 2H, CH2), 5.40
(s, 2H, CH2), 7.15 (d, J = 9 Hz, 2H, H2, H6), 7.30–7.55
(m, 11H, Ph, H4ʹ), 7.65 (d, J=2 Hz, 2H, H2ʹ,6ʹ), 8.05 (d,
J = 9 Hz, 2H, H3, H5), 8.3 (s, 1H, triazole), 10.87 (s, 1H,
NH), 12.5 (bs, 1H, NH) ppm; 13C NMR (500 MHz, DMSO-
d6): δ=53.7, 61.6, 115.3, 117.9, 123.6, 123.9, 126.8, 127.2,
134.7, 141.1, 142.9, 146.0, 158.7, 166.5 ppm.
N‑(3‑Chloro‑4‑fluorophenyl)‑2‑[4‑[[4‑(4,5‑diphe‑
nyl‑1H‑imidazol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑tria‑
zol‑1‑yl]acetamide (9j, C32H24ClFN6O2) White powder;
yield: 67%; m.p.: 196–198 °C; IR (KBr): v = 3426, 3066,
α‑Glucosidase and α‑amylase inhibition assays
In vitro α-glucosidase and α-amylase inhibition assays of
compounds 9a–9m were performed according to our previ-
ous work [19].
1
1683 cm−1; H NMR (500 MHz, DMSO-d6): δ = 5.26 (s,
2H, CH2), 5.39 (s, 2H, CH2), 7.15 (d, J=8.5 Hz, 2H, H2,
H6), 7.2–7.6 (m, 10H, Ph), 7.90 (d, J= 7.5 Hz, 1H, H2ʹ),
8.05 (2H, d, J= 9 Hz, H3, H5), 8.3 (s, 1H, triazole), 10.7
(s,1H, NH), 12.5 (s, 1H, NH) ppm; 13C NMR (500 MHz,
DMSO-d6): δ=52.6, 61.6, 115.3 (d, J=87.5 Hz), 120.1 (d,
J=28 Hz), 121.2, 123.9, 126.8, 127.2, 142.9, 146.0, 158.7,
165.0 ppm.
Kinetic studies
Kinetic studies were carried out to determine inhibition
mode of standard drug acarbose and the most potent com-
pound 9b. The 20 mm3 of enzyme solution (1 U/cm3) was
incubated with different concentrations of acarbose (0,
400, 600, and 800 μM) or compound 9d (0, 15, 35, and 55
μM) for 15 min at 30 °C. The enzymatic reaction was then
started by adding diferent concentrations of substrate (1–4
mM), and change in absorbance was measured at 405 nm
for 20 min using spectrophotometer (Gen5, Power wave xs2,
BioTek, America) [20].
N‑(3‑Bromophenyl)‑2‑[4‑[[4‑(4,5‑diphenyl‑1H‑imida‑
zol‑2‑yl)phenoxy]methyl]‑1H‑1,2,3‑triazol‑1‑yl]aceta‑
mide (9 k, C32H25BrN6O2) White powder; yield: 65%; m.p.:
181–183 °C; IR (KBr): v=3423, 3066, 1681 cm−1; 1H NMR
(500 MHz, DMSO-d6): δ=5.30 (s, 2H, CH2), 5.50 (s, 2H,
CH2), 7.15 (d, J = 9 Hz, 2H, H2, H6), 7.3–7.55 (m, 13H,
Ph, H4ʹ, H5ʹ, H6ʹ), 7.95 (1H, s, H2ʹ), 8.05 (2H, d, J=9 Hz,
H3, H5), 8.3 (s, 1H, triazole), 10.6 (s, 1H, NH), 12.5 (bs,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): δ=52.7,
61.6, 115.3, 118.5, 122.1, 122.1, 123.9, 126.8, 126.9, 127.2,
131.4, 140.2, 142.9, 146.0, 158.7, 166.1 ppm.
Molecular docking study
To fnd out the interaction modes of the synthesized com-
pounds over the α-glycosidase, Maestro Molecular Modeling
platform (version 10.5) by Schrödinger, LLC was performed
[21]. There was not any crystallographic structure of Sac-
charomyces cerevisiae α-glucosidase, so the homology mod-
eled structure was constructed using the crystal structure
of S. cerevisiae isomaltase in complex with α-D-glucose
(PDB id: 3A4A) as target structure [22]. The latter enzyme
has 72% identical and 85% shares sequence similarity with
S. cerevisiae α-glucosidase. Despite a small diference in
the structures of their active site pockets, such as steric hin-
drance by two residues, the structure of the active site of
α-glucosidase is highly similar to that of isomaltase. There-
fore, the active site of α-glucosidase was defned around the
location of α-D-glucose after structural superimposing of
2‑[4‑[[4‑(4,5‑Diphenyl‑1H‑imidazol‑2‑yl)phenoxy]‑
methyl]‑1H‑1,2,3‑triazol‑1‑yl]‑N‑(2‑nitrophenyl)aceta‑
mide (9l, C32H25N7O4) White powder; yield: 69%; m.p.:
1
218–220 °C; IR (KBr): v = 3423, 3064, 16,852 cm−1; H
NMR (500 MHz, DMSO-d6): δ = 5.26 (s, 2H, CH2), 5.47
(s, 2H, CH2), 7.18 (d, J=8.5 Hz, 2H, H2, H6), 7.2–7.45 (m,
7H, Ph), 7.5–7.55 (m, 4H, Ph, H4ʹ), 7.7–7.9 (2H, m, H3ʹ,
H5ʹ), 8.0 (d, J=8.5 Hz, 1H, H6ʹ), 8.03 (d, J=8.5 Hz, 2H,
H3, H5), 8.3 (s, 1H, triazole), 10.8 (s, 1H, NH), 12.5 (bs,
1H, NH) ppm; 13C NMR (500 MHz, DMSO-d6): δ=52.4,
1 3

N. Sepehri et al.
α-glucosidase over isomaltase followed by energy minimiza-
tion and short molecular dynamic to release any undesired
strain.
protein–ligand complex. During the simulation, every 1000
ps of the actual frame was stored. The dynamic behavior
and structural changes of the systems were analyzed by the
calculation of the root mean square deviation (RMSD) and
RMSF. Subsequently, the energy-minimized structure calcu-
lated from the equilibrated trajectory system was evaluated
for investigation of each ligand–protein complex interaction.
The 2D structures of all synthesized compounds were
drawn in Marvin 15.10.12.0 program (http://www.chema
xon.com) and converted into pdb fle. The Protein Prepara-
tion Wizard and the LigPrep module were used to prepare
protein and ligand structures properly [23–25]. The missing
side chains of the proteins were flled using the Prime tool
and missing residues were updated.
Prime MM‑GBSA
The accurate side-chain and backbone fexibility during
ligand binding at the active site of α-glycosidase were pre-
dicted by IFD method using Glide software (Schrödinger
LLC 2018, USA) [26]. Because the kinetic study of the most
potent compound revealed competitive type inhibition mode
for this compound, the α-glucosidase active site was used
to generate the grid for IFD calculation. The maximum 20
poses with receptor and ligand van der waals radii of 0.7
and 0.5, respectively considered. Residues within 5 Å of
the α-D-glucose at the active site were refned followed by
side-chain optimization. Structures whose prime energy is
more than 30 kcal/mol are eliminated based on extra pre-
cious Glide docking.
The ligand-binding energies (ΔGbind) were calculated for
compound 9b and acarbose using Molecular mechan-
ics/generalized born surface area (MM-GBSA) modules
(Schrödinger LLC 2018) (75) based on the following
equation:
[
]
ΔGbind = Ecomplex − Ereceptor + Eligand
,
where ΔGbind is the calculated relative free energy which
includes both ligand and receptor strain energy. Ecomplex
is the MM-GBSA energy of the minimized complex, and
Eligand is the MM-GBSA energy of the ligand after remov-
ing it from the complex and allowing it to relax. Ereceptor is
the MM-GBSA energy of relaxed protein after separating it
from the ligand. The MM-GBSA calculation was performed
based on the clustering method for energy calculation.
Molecular dynamic simulation
Molecular dynamic (MD) simulation of this study was per-
formed using the Desmond v5.3 module implemented in
Maestro interface (from Schrödinger 2018–4 suite) [30].
The appropriate pose for MD simulation procedure of the
compounds was achieved by docking method.
In vitro cytotoxicity assay
Cytotoxicity evaluation on compounds 9b, 9d, and 9h was
performed according to our previous works [19].
To build the system for MD simulation, the protein–ligand
complexes were solvated with SPC explicit water molecules
and placed in the center of an orthorhombic box of appropri-
ate size in the Periodic Boundary Condition. Sufcient coun-
ter-ions and a 0.15 M solution of NaCl were also utilized to
neutralize the system and to simulate the real cellular ionic
concentrations, respectively. The MD protocol involved min-
imization, pre-production, and fnally production MD simu-
lation steps. In the minimization procedure, the entire system
was allowed to relax for 2500 steps by the steepest descent
approach. Then, the temperature of the system was raised
from 0 to 300 K with a small force constant on the enzyme to
restrict any drastic changes. MD simulations were performed
via NPT (constant number of atoms, constant pressure i.e.
1.01325 bar and constant temperature, i.e. 300 K) ensem-
ble. The Nose‐Hoover chain method was used as the default
thermostat with 1.0-ps interval and Martyna-Tobias-Klein as
the default barostat with 2.0-ps interval by applying isotropic
coupling style. Long‐range electrostatic forces were calcu-
lated based on Particle-mesh-based Ewald approach with the
he cut‐of radius for columbic forces set to 9.0 Å. Finally, the
system subjected to produce MD simulations for 20 ns for
In silico druglikeness/ADME/T study
In silico druglikeness/ADME/T studies on compounds 9b,
9d, 9h, and acarbose (standard drug) were performed using
the preADMET online server (http://preadmet.bmdrc.org/)
[31].
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s00706-021-02779-7.
Acknowledgements This work was financially supported by the
National Institute for Medical Research Development (NIMAD) (the
grant number: 972514). The ethics code for this study is IR.NIMAD.
REC.1398.036 (http://ethics.research.ac.ir/IR.NIMAD.REC.1398.036).
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Publisher’s Note Springer Nature remains neutral with regard to
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Authors and Afliations
Nima Sepehri1 · Homa Azizian2 · Reza Ghadimi3 · Fahimeh Abedinifar4 · Somayeh Mojtabavi5 ·
Mohammad Ali Faramarzi5 · Ali Akbar Moghadamnia6 · Ebrahim Zabihi6 · Gholamhossein Mohebbi7 ·
Bagher Larijani4 · Haleh Hamedifar8 · Maryam Mohammadi‑Khanaposhtani6 · Mohammad Mahdavi4
1
2
3
6
7
8
Nano Alvand Company, Avicenna Tech Park, Tehran
University of Medical Sciences, Tehran, Iran
Cellular and Molecular Biology Research Center, Health
Research Institute, Babol University of Medical Sciences,
Babol, Iran
Department of Medicinal Chemistry, School of Pharmacy,
Iran University of Medical Sciences, Tehran, Iran
The Persian Gulf Marine Biotechnology Research Center,
The Persian Gulf Biomedical Sciences Research Institute,
Bushehr University of Medical Sciences, Bushehr, Iran
Social Determinants of Health Research Center, Health
Research Institute, Babol University of Medical Sciences,
Babol, Iran
CinnaGen Medical Biotechnology Research Center, Alborz
University of Medical Sciences, Karaj, Iran
4
5
Endocrinology and Metabolism Research Center,
Endocrinology and Metabolism Clinical Sciences Institute,
Tehran University of Medical Sciences, Tehran, Iran
Department of Pharmaceutical Biotechnology, Faculty
of Pharmacy, Tehran University of Medical Sciences, Tehran,
Iran
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