Synthesis, α-glucosidase inhibition and in silico studies of some 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104002

In this study, a new series of 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives has been synthesized and screened for their α-glucosidase inhibitory potential. All molecules showed a considerable α-glucosidase inhibitory potential with IC₅₀ values ranging from 20.46 ± 0.21 to 0.18 ± 0.01 μg/mL when compared with the acarbose (IC₅₀ = 8.16 ± 0.12 μg/mL) as the standard. Compound 4 k having methoxy group on phenyl ring had the highest inhibitory effect with IC₅₀ = 0.18 ± 0.01 μg/mL value among the examined compounds. Electron-donating groups such as methyl and methoxy on the phenyl ring played an important role in the inhibition. Also, the Lineweaver-Burk plots analysis displayed that the inhibition type of 4k was the competitive mode like acarbose as standard. In silico studies were also performed to explore the binding interaction of the most active compound.

Full text of DOI:10.1016/j.bioorg.2020.104002

Journal Pre-proofs
Synthesis, α-glucosidase inhibition and in silico studies of some 4-(5-fluo-
ro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives
Emre Menteşe, Nimet Baltaş, Mustafa Emirik
PII:
S0045-2068(20)31299-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.104002
YBIOO 104002
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 April 2020
28 May 2020
7 June 2020
Please cite this article as: E. Menteşe, N. Baltaş, M. Emirik, Synthesis, α-glucosidase inhibition and in silico
studies of some 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104002
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2020 Elsevier Inc. All rights reserved.

Synthesis, α-glucosidase inhibition and in silico studies of some 4-(5-fluoro-2-
substituted-1H-benzimidazol-6-yl)morpholine derivatives
Emre Menteşe*, Nimet Baltaş, Mustafa Emirik
Department of Chemistry, Faculty of Arts and Sciences, Recep Tayyip Erdogan University, Rize, Turkey
E-mail: emre.mentese@erdogan.edu.tr
Fax: +904642234019
ABSTRACT
In this study, a new series of 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine
derivatives has been synthesized and screened for their α-glucosidase inhibitory potential. All
molecules showed a considerable α-glucosidase inhibitory potential with IC50 values ranging
from 20.46 ± 0.21 to 0.18 ± 0.01 µg/mL when compared with the acarbose (IC50= 8.16 ± 0.12
µg/mL) as the standard. Compound 4k having methoxy group on phenyl ring had the highest
inhibitory effect with IC50 = 0.18 ± 0.01 µg/mL value among the examined compounds.
Electron-donating groups such as methyl and methoxy on the phenyl ring played an important
role in the inhibition. Also, the Lineweaver-Burk plots analysis displayed that the inhibition
type of 4k was the competitive mode like acarbose as standard. In silico studies were also
performed to explore the binding interaction of the most active compound.
Keywords: Benzimidazole, morpholine, docking study, α-glucosidase inhibition
1. Introduction
In humans, glucosidase and amylase enzyme activities are involved in the breakdown of
dietary starches and sugars into glucose. The α-glucosidase activities are associated with two
small intestinal membrane-bound enzymes: maltase-glucoamylase and sucrase-isomaltase [1].
Alpha-glucosidase, a key carbohydrate digestion enzyme, leads to the breakdown of oligo-
and disaccharides from dietary complex carbohydrates [2]. The inhibition of α-glucosidase
plays an important role in treating type-2 diabetes reducing the absorption of
monosaccharides, especially glucose, and delaying the digestion of carbohydrates [2,3].
Nowadays, α-glucosidase inhibitors like miglitol, voglibose, and acarbose are approved for
the treatment of type-2 diabetes, but they show several adverse effects [3,4]. Additionally,
some published studies suggest that these inhibitors can be used for the treatment of cancer,
1

Pompe disease, and hepatitis [4-7]. Thus, a synthesis of potent new α-glucosidase inhibitors is
an attractive goal in the area of medicinal chemistry.
Benzimidazole is structurally similar to the purine and is found in the structure of
vitamin B12. Benzimidazole derivatives are decisive core structures utilized in drugs and
materials. From this fundamental structural similarity, it is not surprising that benzimidazole
containing molecules have been found to be biologically active small molecules [8,9].
Synthesis and evaluation of differently substituted benzimidazole analogs resulted in the
discovery of some important drugs like albendazole, mebendazole, omeprazole, and
thiabendazole [10-12]. Previous studies reveal that benzimidazole containing compounds are
found to possess antimicrobial, anticancer, antioxidant, antihelmintic, anti-inflammatory,
antiviral, antihypertensive, and anticoagulants activities [13-18]. Some of the benzimidazole
derivatives are reported to be potent inhibitors of urease and lipase [19-21]. Several 2-
substitutedbenzimidazole derivatives are observed to exhibit α-glucosidase inhibitory
properties (Scheme 1) [22-29]. It was demonstrated that α-glucosidase inhibitors mainly
depend upon the functional groups on 2-substitutedbenzimidazoles. The functional group on
the phenyl ring, such as the methoxy group, was the preferred substitution required for the
activity. Moreover, it became clear that insertion of halogen atoms in the benzimidazole
nucleus result in an increase of biological properties [23,30,31].
Cl
Br
Cl
O
N
N
N
N
N
H
OCH3
N N
N
H
O2N
N
N
NH
S
N
O
O
IC50= 26.931.43 M
SH
IC50= 32.630.50 M
IC50= 4.670.58 M
HO
HO
Br
N
N
N
N
N
OCH3
OCH3
OCH3
N
H
N
Cl
N
H
H
H
IC50= 43.500.90 M
IC50= 37.820.08 M
IC50= 21.150.08 M
IC50= 29.950.19 M
Scheme 1.Previously reported benzimidazoles as a potent α-glucosidase inhibitor with their
IC50 values.
Documented studies on the structure-activity relationship released that substitution at C-2
position of the benzimidazole system highly influences the biological activity. Fluorine and
morpholine are often encountered groups in potent bioactive compounds. Furthermore, the
fluorine atom enhances binding affinity to protein, changes physicochemical properties, and
2

increases metabolic stability [38-40]. The addition of these moieties on target molecules has
played a significant role in increasing biological activities [13,15,32]. Comparison of α-
glucosidase inhibition activity for synthesized compounds 4h, 4e, and previously reported
analogs are presented in Scheme 2, which shows that the addition of fluorine and morpholine
has a positive effect on inhibition properties [29].
Cl
Cl
F
N
N
N
H
N
H
N
O
IC50= 82.85 ± 3.76 µM
Br
IC50= 3.78 ± 0.05 µM
Br
F
N
N
N
H
N
H
N
O
IC50= 6.56 ± 0.11 µM
Not determined
a)
b)
Scheme 2. Comparison of α-glucosidase inhibition activity between compounds 4e, 4h (a),
and previously reported analogs (b) [26].
Therefore, promoted by these observations and in continuation to our interest in
benzimidazoles with their biological activities, herein some novel 5(6)-fluoro-2-
substitutedbenzimidazole derivatives containing morpholine skeleton at C-6(5) position were
synthesized and then investigated for their α-glucosidase inhibitory activity.
2. Results and Discussion
2.1. Chemistry
Synthetic approaches for the preparation of starting compounds (1, 2) and benzimidazole
derivatives (3a-j and 4a-k) are illustrated in Scheme 3. Compounds 1, 2, and Iminoester
hydrochlorides were synthesized according to the previously reported methods [33-35].The
target compounds (3a-j) were synthesized by the reaction of compound 1 and the
corresponding aldehydes in the presence of sodium dithionite (Na2S2O4) in an 87-94 % yield.
The reaction of compound 2 with iminoester hydrochlorides afforded compounds 4a-k. In
3

the1H NMR spectra of target molecules, NH signals were observed between 12.13-12.92 ppm.
The CH2 signals of compounds 4a-k were shown between 4.10-4.30 ppm. The signals for two
methylene protons of morpholine ring were observed around 2.92-3.01 ppm and 3.69-3.85
ppm.
F
NH2
NH2
Pd/C, EtOH
24 h. reflux
F
NO2
NH2
O
NH
F
NO2
NH2
N
DMSO
145-150 0C
N
Cl
O
O
2
1
NH.HCl
R2
MeOH, rt
R1 CHO
Na2S2O4
OC2H5
F
F
N
N
R2
R1
N
H
N
H
N
N
O
O
4a-k
3a-j
Scheme 3. Synthetic approaches for the preparation of target compounds
2.2. Inhibition of α-glucosidase
α-Glucosidase inhibition studies of the newly synthesized 4-(5-fluoro-2-substituted-1H-
benzimidazol-6-yl)morpholine derivatives (3a-j, 4a-k) were carried out according to our
previously reported protocol [32,36,37]. α-Glucosidase inhibitory activities of the target
molecules were shown in Table 1. Particularly, compound 4k having methoxy group on
phenyl ring had the highest inhibitory effect among the examined compounds. The second
most active compound is 4i having an electron-donating group on the phenyl ring at the
position-2 of benzimidazole. Along with this result, inhibitory activities of the compounds 3d,
3e, 3g, 3h, 3i, 4e-k, were significantly higher than those of acarbose. The structure-activity
relationship was mainly based upon bringing about the substitution of different groups on the
phenyl ring and position of the substituent. Compounds 3a and 4a having phenyl moiety with
no group had the lowest anti-α-glucosidase activity among all the compounds tested and
standards. Compounds 3g, 3h, and 3i bearing a 4-fluoro, 3-fluoro, and 2-fluoro groups on the
phenyl ring, respectively, were found to be more active inhibitors than acarbose. All three
compounds have the fluoro substituent on the phenyl ring, but the arrangement of the fluoro
group is different in them. This indicated that the difference in position of the substituents
4

greatly affects the α-glucosidase inhibitory potentials of the molecules (Scheme 4). Also,
compounds 4b, 4c, and 4d contain a 4-fluoro, 3-fluoro, and 2-fluoro substituents on phenyl
group, respectively. The inhibitory potential of compound 4d was found to be greater than
molecules 4b and 4c.
F
F
F
F
F
N
N
N
F
N
H
N
H
N
H
N
N
N
O
O
O
3g,
IC50= 5.46 ± 0.19 µg/mL
3h,
IC50= 1.96 ± 0.05 µg/mL
3i,
IC50= 0.34 ± 0.03 µg/mL
F
F
F
F
F
N
N
N
F
N
N
H
N
H
N
N
N
H
O
O
O
4b,
IC50= 16.34 ± 0.25 µg/mL
4c,
IC50= 12.28 ± 0.16 µg/mL
4d,
IC50= 9.42 ± 0.09 µg/mL
Scheme 4. The structure-inhibition relationship of compounds 3g, 3h, 3i, 4b, 4c, and 4d
Compound 4f (IC50 = 1.66 ± 0.06 µg/mL), which has 3-chloro substitution, was found to be
more active than compound 4e, which has 4-chloro substitution, and compound 4g, which has
2-chloro substitution. It was found that the difference of chloro substitution on the phenyl ring
greatly influences the inhibitory activity of these compounds (Scheme 5). A comparison
between the two series of halogenobenzyl analogues, namely, 3g-I vs 4e-g, reveals that the
chlorobenzyl derivatives (Scheme 5) are more active than the fluorobenzyl analogues
(Scheme 4).
Cl
Cl
F
F
F
N
N
N
Cl
N
H
N
H
N
H
N
N
N
O
O
O
4e,
IC50= 3.78 ± 0.05 µg/mL
4f,
IC50= 1.66 ± 0.06 µg/mL
4g,
IC50= 6.40 ± 0.33 µg/mL
Scheme 5.The structure-activity relationship of compounds 4e, 4f and 4g
Compound 4k having 4-OCH3 group on the phenyl part at position 2 of benzimidazole had
more potent α-glucosidase inhibitor with IC50 value = 0.18±0.01 as compared to compounds
4b (16.34 ± 0.25 µg/mL), 4e (3.78 ± 0.05 µg/mL), 4h (2.55 ± 0.11 µg/mL) and 4i (0.24 ± 0.02
5

µg/mL), having 4-F, 4-Cl, 4-Br, 4-CH3 substitution on the phenyl, respectively. It is clear that
electron-donating groups such as methyl and methoxy on the phenyl ring played an important
role in the inhibition (Scheme 6).
F
Br
Cl
F
F
F
N
N
N
N
H
N
H
N
H
N
N
N
O
O
O
4h,
IC50= 2.55 ± 0.11 µg/mL
4e,
IC50= 3.78 ± 0.05 µg/mL
4b,
IC50= 16.34 ± 0.25 µg/mL
OCH3
CH3
F
F
N
N
N
N
H
N
O
N
H
O
4i,
4k,
IC50= 0.18 ± 0.01 µg/mL
IC50= 0.24 ± 0.02 µg/mL
Scheme 6. The structure-activity relationship of compounds 4b, 4e, 4h, 4i, and 4k
Table 1. IC50 values of compounds 3a-i and 4a-k against α-glucosidase.
Compounds
3a
IC50 (µg/mL)
17.58 ± 0.23
R2
Compounds
4a
IC50 (µg/mL)
20.46 ± 0.21
R2*
R1
R2
H2C
H2C
0.9665
0.9475
3b
3c
3d
3e
13.16 ± 0.11
10.20 ± 0.13
5.65 ± 0.09
1.87 ± 0.07
0.9868
0.9689
0.9749
0.9651
4b
4c
4d
4e
16.34 ± 0.25
12.28 ± 0.16
9.42 ± 0.09
3.78 ± 0.05
0.8937
0.9579
0.8863
0.9547
F
Cl
H2C
H2C
NO2
F
HO
F
F3C
H2C
Cl
Br
H2C
3f
8.64 ± 0.10
5.46 ± 0.19
0.8946
0.8988
4f
1.66 ± 0.06
6.40 ± 0.33
0.9623
0.9712
F
Cl
H2C
3g
4g
F
Cl
6

3h
1.96 ± 0.05
0.97.76
4h
4i
H2C
H2C
Br
2.55 ± 0.11
0.24 ± 0.02
0.9629
0.9055
F
CH3
3i
0.34 ± 0.03
8.16 ± 0.12
0.9234
0.8755
F
H2C
H2C
Acarbose
4j
0.68 ± 0.04
0.18 ± 0.01
0.9246
0.9371
CH3
OCH3
4k
*R2 value indicates that the graph has linearity to calculate the IC50 value of the compounds and standard.
In the current study, the type of enzyme inhibition of 4k and acarbose was analyzed by
Lineweaver-Burk plots (Fig. 1a, b and Table 2). In the presence of 4k, Km value increased
(1.82 mg/mL, Fig. 1a) and Vmax value remained the same (153.84 mM/min.). From this point
of view, the Lineweaver-Burk plots analysis displayed that the inhibition type of 4k was the
competitive mode (Fig. 1).
Table 2. Type of α-glucosidase inhibition of molecule 4k and acarbose
Inhibitors p-NPG
Km (mg/mL)
0.50
Vmax (mM/min)
153.84
Type of Inhibition
-
IC50 (µg/mL)
-
R2
Control
Acarbose
4k
0.998
0.996
0.999
0.71
153.84
Competitive
Competitive
8.16 ± 0.12
0.18 ± 0.01
1.82
153.84
7

Fig. 1. Types of inhibition for compound 4k (Fig. 1a) and acarbose (Fig. 1b) as the standard.
2.3. In silico Docking Study
To screen for potential inhibition of the α-glucosidase, newly synthesized compounds were
docked against the crystal structure of yeast isomaltase from Saccharomyces cerevisiae with
Pdb id:3A4A and human lysosomal acid-α-glucosidase with Pdb id: 5NN8 [36,50]. A
summary of binding free energies and docking scores of synthesized compounds and
reference ligands are reported in Table 3. Similarly, Fig. 2 presents a graphical illustration and
interactions of compound 4k with amino acid residues at the active site of yeast isomaltase.
As the kinetic study revealed that compound 4k was a competitive inhibitor, active sites of
both enzymes were used in docking study. Furthermore, blind docking study was also
suggested that compound 4k were resulted better docking score in active site of target
structures rather than of allosteric sites.
Table 3. Docking scores and relative binding free energy of synthesized molecules (All values
were given in kcal/mol).
Yeast isomaltase
Human
Yeast isomaltase
Human
α-glucosidase
α-glucosidase
Comp. ΔG Bind
D. Score
ΔG Bind
D.Score Comp. ΔG Bind D. Score ΔG Bind D. Score
3a
3b
3c
-51.7
-39.1
-16.3
-7.7
-7.4
-6.6
-42.5
-53.4
-59.8
-7.4
-6.5
-6.5
4a
4b
4c
-43.1
-12.7
-41.1
-7.9
-8.2
-7.6
-58.8
-56.9
-57.7
-10.6
-8.9
-10.6
8

3d
3e
3f
3g
3h
3i
-49.5
-46.9
-45.3
-49.8
-44.7
-49.5
-28.6
-8.7
-8.2
-7.3
-6.9
-7.6
-8.0
-6.2
-56.9
-61.5
-44.2
-45.8
-43.3
-61.8
-56.8
-8.1
-6.9
-6.2
-8.5
-8.9
-9.5
-8.3
4d
4e
4f
4g
4h
4i
-30.3
-29.9
-31.1
-49.6
-54.5
-47.8
-41.8
-28.3
-8.1
-7.3
-7.6
-8.5
-7.7
-7.7
-7.5
-6.9
-58.9
-63.9
-58.5
-69.0
-60.1
-68.2
-65.1
-66.5
-9.4
-10.0
-8.1
-9.0
-10.8
-10.7
-10.0
-9.5
Acbs
4j
4k
Analysis of docking simulation to yeast isomaltase suggests that compound 4k having a 4-
methoxy group on the phenyl moiety was buried into the active site of enzyme doing
significant interactions with active site residues. Following the use of in silico methods, the
compound 4k showed that hydrogen bond interactions occurred between i) NH group of
diazole moiety of ligand and COO- group of glutamic acid (GLU277) ii) oxygen atom on
morpholine moiety of ligand and backbone of arginine (ARG315) iii) methoxy group of
ligand and NH group of arginine (ARG446) though the water molecules and water linked
hydrogen bonding with ASP312 and ARG315. Other prominent interactions are two π-π
stacking interactions that occurred via the benzene ring of the ligand and phenylalanine
(PHE303) and threonine (THR158) residues of the protein. The corresponding docking score
of 4k is −8.941 kcal/mol.
Methoxy group, which acts as hydrogen bond acceptor, allows more interaction to take place
and therefore increases α-glucosidase inhibition activity. It is clear that two rotatable bonds
placed on both sides of the benzimidazole skeleton increase the penetration ability of the
molecule 4k towards the active site of the enzyme.
Fig. 2. Ligand interaction diagram and binding pose of 4k at the active side of yeast
isomaltase
9

All the synthesized compounds (3a-i, 4a-k) were also docked to the binding site of human α-
glucosidase with scores in the range of -6.2 and -10.8 kcal/mol. The docking simulation of
4kagainst the crystal structure of human lysosomal acid-alpha-glucosidase showed similar
interactions with the yeast isomaltase binding site. The compound 4k which exhibits the
highest in vitro activity was bound into the receptor site making hydrogen bond with ALA97,
ILE98 and ARG275 residues. It was also observed that 4k exhibited hydrophobic interactions,
P-shaped π-π stacking interactions and halogen bonding. The interactions detail was given
Fig. 3 and Table 4 with distances.
Fig. 3. Ligand interaction diagram and binding pose of 4k at the active side of human α-
glucosidase
Table 4. The interactions detail of compound 4k at active site of human α-glucosidase
Residue
Distance
Ligand Atom
Protein Atom
Hydrogen Bonds
ALA97
ILE98
ILE98
ARG275
Halogen Bonds
GLN121
π- π Stacking
TRP126
2.73
CH of anisole
O of Backbone
O of Backbone
NH of Backbone
NH of guadinido
1.80
2.96
2.31
NH of imidazole
N of imidazole
O of morpholine
3.19
Fluorine
O of Backbone
3.86
3.90
TRP126
Hydrophobic Interactions
ALA93
ILE98
TRP126
3.94
3.70
3.43
10

As the MMGBSA binding energies are approximate free energies of binding, a more negative
value indicates stronger binding. The MMGBSA calculation of synthesized compounds for
both target protein was performed, and results were given in Table 3. According to binding
free energy calculations of the synthesized compounds for human alpha glucosidase enzyme,
the compounds 4a-k, 3i, 3e and 3c have more negative binding free energy than that of the
reference compound, Acarbose, meaning the formation of more stable enzyme-ligand
complexes. All contributions of ΔG Binding were illustrated in Fig. 4 graphically. The π-π
packing correlations and Generalized Born electrostatic solvation energy are the main
differences between Acarbose and synthesized compounds in general. It is also observed that
all studied compounds have better binding free energies for human α-glucosidase than that of
yeast isomaltase ones.
Fig. 4. MMGBSA Binding Free Energies (ΔG Bind) of all studied compounds and their contributions.
Colours show contributions and values show total ΔG Bind in kcal/mol. (Energy contributions ΔG
Bind Coulomb:Coulomb energy, ΔG Bind Covalent: Covalent binding energy, ΔG Bind vdW: Van der
Waals energy, Lipo: Lipophilic energy, Solv GB: Generalized Born electrostatic solvation energy,
Hbond: Hydrogen-bonding correction, Packing: π-π packing correction)
The trend in inhibitory effect of compounds 3i, 3h and 3g substituted with fluorine atom on
the ortho, meta and para positions of the phenyl substituent is similar to that of derivatives 4d,
4c and 4b bearing fluorine atom on equivalent positions of the benzyl substituent (Scheme 4).
However, activity between the two series reveals that compounds 3i, 3h and 3g with the
11

fluorophenyl group linked directly to the benzimidazole framework are more active than the
corresponding analogues 4d, 4c and 4b in which the aromatic ring is linked to the main
framework through the methylene group/bridge. The comparison of compounds 3i and 4d
interactions indicate that binding free energy of 3i shows more potential inhibition effect
against human α-glycosidase than that of compound 4d with -61.82 kcal/mol and -58.93
kcal/mol, respectively. Van der Waals energy contributions of binding free energies for 3i and
4d are -39.74 and -35.16 kcal/mol. Likewise, coulomb energy contribution is higher in
compound 3i. These differences are mostly related to that increased conjugative effect and
therefore π electron distribution of compounds 3i influences their non-covalent or
hydrophobic interactions with the receptors, in turn, increase the inhibitor potential of the
compounds including extended π system. In binding mode analyses, significant π-π stacking
interactions were found between the benzimidazole skeleton of compound 3i and the side
chain indole group of TRP126. In addition to this π-π stacking interactions, H-bonding
interaction was also found with GLN121. Similar interactions were found in binding mode
analyses of 4d. However, due to extended pi system, the π-π stacking and coulomb
interactions are stronger in 3i.
Fig. 5. Docked poses and interactions of 3i and 4d. (light blue dashed line: π−π stacking,
yellow dashed line: hydrogen bonding)
When the in vitro and in silico activities of compounds 4b, 4e and 4i are compared, the effect
of electron donating and electron withdrawing subsutients on the activity can be clarified. For
the mentioned three ligands, π conjugation flowed on the benzimidazole skeleton was
interrupted via CH2 group and F, Cl and CH3 groups were substituted to the para position of
the benzene ring, respectively. Thus, in vitro and in silico activities are listed as 4i> 4g> 4d.
12

As the electron withdrawal power increased on the aromatic benzene ring, the activity
decreased, while there was an obvious increase in activity in the case of an electron donating
group. This can be explained by increased cloumb and lipophilic interaction, which provides
the highest contribution to binding free energy by increasing charge density in the aromatic
ring as shown in Fig.4.
3. Conclusion
In conclusion, a new series of 4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine
derivatives was synthesized and then screened for their α-glucosidase inhibition properties.
Most of the target compounds (3a-j and 4a-k) showed remarkable α-glucosidase inhibitory
potentials. Compound 4k, which has methoxy group on phenyl ring, was found to be the most
active molecule of the series with an IC50 value of 0.18 ± 0.01 µg/mL. The Lineweaver-Burk
plots analysis displayed that the inhibition type of 4k was the competitive mode like acarbose
as standard. The corresponding docking score and binding free energy of 4k were estimated -
9.5 kcal/mol and -66.5 kcal/mol, respectively. It was observed from in vitro and in silico
study that electron-donating groups such as methyl, methoxy on phenyl ring and increased
conjugative effect played a significant role in the inhibition, could be a promising lead for
future investigation.
4. Experimental section
4.1. Chemistry
The chemicals were supplied from Merck, Aldrich, and Fluka. Melting points were
uncorrected and determined in open capillary tubes on a Büchioil-heated melting point
apparatus. Reactions were monitored by thin-layer chromatography (TLC) using precoated
aluminum sheets (silicagel60 F 2.54 0.2 mm thickness). The mobile phase was ethyl acetate
1
and hexane (2:1 or 3:1) and detection was made using UV light. H NMR and 13C NMR
spectra were recorded on a Varian-Mercury 400 (1H, 400 MHz; 13C, 100 MHz ) spectrometer
using DMSO-d6 as solvent and TMS as an internal standard. All chemical shifts were reported
in ppm. Elemental analyses were performed on a Carla Erba 1106 CHN analyzer (Heraeus,
Hanau, Germany); the experimental values were in agreement (± 0.4 %) with calculated ones.
4.2. Synthesis of compounds 3a-j
A solution of compound1 (10 mmol) and corresponding aldehyde (10 mmol) in EtOH(5 mL)
was prepared in a round bottom flask with a magnetic stir bar. Then, an aqueous solution of
13

Na2S2O4 (4 mmol) was added, and the resulting contents of reaction mixture in the round
bottom flask was stirred under refluxed for 4.5 h with stirring. After completion of the
reaction, the resulting solution was poured into the ice-water mixture. The precipitate was
collected by filtration and recrystallized from ethanol/water (4:1) to give pure product3a-j.
4.2.1. 4-(5-Fluoro-2-phenyl-1H-benzimidazol-6-yl)morpholine (3a)
Light yellow powder; Yield: 90 %, 142-143 °C. 1H-NMR: 3.01 (4H, bs, NCH2), 3.78 (4H, bs,
OCH2), 7.18 (1H, bs, ArH), 7.39-7.54 (4H, m, ArH), 8.13 (2H, d, J= 8 Hz, ArH), 12.87 (1H,
13
bs, NH). C-NMR: 51.93 (NCH2), 66.79 (OCH2), ArC:[126.61 (3C), 129.41 (3C), 130.12,
130.54, 137.13, 137.27] 152.03 (C=N), 153.33 (C-F, JCF= 240 Hz) Anal. calcd. (%) for
C17H16FN3O: C, 68.67; H, 5.42; N, 14.13. Found: C, 68.80; H, 5.50; N, 14.01.
4.2.2. 4-(2-(4-Chlorophenyl)-5-fluoro-1H-benzimidazol-6-yl)morpholine (3b)
Light yellow powder; Yield: 94 %, 239-240 °C. 1H-NMR: 2.96 (4H, t, J= 4 Hz, NCH2), 3.73
(4H, t, J= 4 Hz, OCH2), 7.13 (1H, d, J= 7.6 Hz, ArH), 7.36 (1H, d, J= 7.6 Hz, ArH), 7.53
(2H, d, J= 8.4 Hz, ArH), 8.01 (2H, d, J= 8.4 Hz, ArH), 12.87 (1H, bs, NH). 13C-NMR: 51.84,
52.87 (NCH2), 66.74 (OCH2), ArC:[102.97, 103.21, 104.22, 128.26, 129.26, 130.38, 134.73,
136.32, 137.36 (d, J= 12 Hz)], 150.85 (C=N), 153.42 (C-F, JCF= 235 Hz). Anal. calcd. (%)
for C17H15FClN3O: C, 61.54; H, 4.56; N, 12.67. Found: C, 61.67; H, 4.63; N, 12.75.
4.2.3. 4-(5-Fluoro-2-(4-nitrophenyl)-1H-benzimidazol-6-yl)morpholine (3c)
1
Orange powder; Yield: 94 %, 238 °C (dec.). H-NMR: 2.97 (4H, t, J= 4.4 Hz, NCH2), 3.74
(4H, t, J= 4.4 Hz, OCH2), 6.70 (1H, d, J= 8 Hz, ArH), 7.15 (2H, d, J= 8 Hz, ArH), 7.43 (1H,
d, J= 11.6 Hz, ArH), 7.80 (2H, d, J= 8Hz, ArH), 12.83 (1H, bs, NH). 13C-NMR: 51.60, 51.58
(NCH2), 66.64, 66.67 (OCH2), ArC:[101.66, 103.25, 111.78, 114.06, 116.19, 127.82, 129.10,
129.82, 131.10, 138.20 (d, J= 11 Hz)], 151.54 (C=N), 153.67 (C-F, JCF= 240 Hz). Anal.
calcd. (%) for C17H15FN4O3: C, 59.65; H, 4.42; N, 16.37. Found: C, 59.76; H, 4.51; N, 16.28
4.2.4. 2-(5-Fluoro-6-morpholino-1H-benzimidazol-2-yl)phenol (3d)
Light yellow powder; Yield: 92 %, 195-196 °C. 1H-NMR: 3.00 (4H, bs, NCH2), 3.77 (4H, bs,
OCH2), 6.97-7.02 (2H, m, ArH), 7.22 (1H, s, ArH), 7.34 (1H, t, J= 8 Hz, ArH), 7.49 (1H, d,
13
J= 12 Hz, ArH), 7.99 (1H, d, J= 8Hz, ArH), 10.24 (1H, s, OH), 12.99 (1H, bs, NH). C-
NMR: 51.80, 51.83 (NCH2), 66.64, 66.73 (OCH2), ArC:[110.11, 113.17, 115.52, 116.84,
117.55, 119.59, 119.91, 126.48, 127.54, 131.95, 136.84, 137.69 (d, J= 12 Hz)], 152.12 (C=N),
14

154.96, 156.56, 158.00. Anal. calcd. (%) for C17H16FN3O2: C, 65.17; H, 5.15; N, 13.41.
Found: C, 65.26; H, 5.23; N, 13.52
4.2.5. 4-(5-Fluoro-2-(2-(trifluoromethyl)phenyl)-1H-benzimidazol-6-yl)morpholine (3e)
1
Light brown powder; Yield: 88 %, 132-133 °C. H-NMR: 2.99 (4H, t, J= 4.4 Hz, NCH2),
3.76 (4H, t, J= 4.4 Hz, OCH2), 7.18 (1H, d, J= 6.4 Hz, ArH), 7.42 (1H, d, J= 12Hz, ArH),
7.71-7.75 (2H, m, ArH), 7.80 (1H, d, J= 6.8 Hz, ArH), 7.90 (1H, d, J= 8 Hz, ArH), 12.67
13
(1H, bs, NH). C-NMR: 51.91, 51.93 (NCH2), 66.75 (OCH2), ArC:[120. 07, 124.15 (CF3, d,
J= 272 Hz), 127.00 (q, JCF= 50 Hz), 127.72, 128.23, 128.33, 128.40 (d, JCF= 80 Hz), 130.49
(d, JCF= 20 Hz), 130.54, 132.57, 132.80, 137.33 (d, JCF= 11 Hz)], 150.04 (C=N), 153.32 (C-
F, d, JCF= 238 Hz). Anal. calcd. (%) for C18H15F4N3O: C, 59.18; H, 4.14; N, 11.50. Found:
59.10; H, 4.06; N, 11.62
4.2.6. 4-(2-(3-Bromo-4-fluorophenyl)-5-fluoro-1H-benzimidazol-6-yl)morpholine (3f)
White powder; Yield: 90 %, 197-198 °C. 1H-NMR: 2.98 (4H, t, J= 4 Hz, NCH2), 3.75 (4H, t,
J= 4.4 Hz, OCH2), 7.11 (1H, bs, ArH), 7.38 (1H, d, J= 12 Hz, ArH), 7.50 (1H, td, J= 8.0, 4.0
Hz, ArH), 8.09-8.13 (1H, m, ArH), 8.38 (1H, dd, J= 8.0, 2.0 Hz, ArH), 12.90 (1H, bs, NH).
13C-NMR: 51.84, 51.85 (NCH2), 66.74 (OCH2), ArC:[109.03 (d, JCF= 12 Hz), 117.80 (d,
JCF= 22 Hz), 127.93, 128.01, 128.68, 128.72, 131.33, 149.67], 151.38 (C=N), 159.34 (C-F, d,
JCF= 247 Hz). Anal. calcd. (%) for C17H14F2BrN3O: C, 51.80; H, 3.58; N, 10.66. Found: C,
51.91; H, 3.65; N, 10.58.
4.2.7. 4-(5-Fluoro-2-(4-fluorophenyl)-1H-benzimidazol-6-yl)morpholine (3g)
White powder; Yield: 93 %, 170-171 °C. 1H-NMR: 2.97 (4H, t, J= 4 Hz, NCH2), 3.75 (4H, t,
J= 4.4 Hz, OCH2), 7.14 (1H, d, J= 8 Hz, ArH), 7.33-7.39 (3H, m, ArH), 8.14 (2H, dd, J= 8.0,
4.0 Hz, ArH), 12.81 (1H, bs, NH). 13C-NMR: 51.88, 51.90 (NCH2), 66.75 (OCH2),
ArC:[116.01, 116.39 (d, JCF= 22 Hz), 127.15, 127.18, 128.80, 128.84, 137.07, 137.19, ],
151.17 (C=N), 153.30 (C-F, d, JCF= 237 Hz), 159.34 (C-F, d, JCF= 247 Hz). Anal. calcd. (%)
for C17H15F2N3O: C, 64.75; H, 4.80; N, 13.33. Found: C, 64.86; H, 4.89; N, 13.25
4.2.8. 4-(5-Fluoro-2-(3-fluorophenyl)-1H-benzimidazol-6-yl)morpholine (3h)
White powder; Yield: 90 %, 140-141 °C. 1H-NMR: 2.98 (4H, t, J= 4 Hz, NCH2), 3.75 (4H, t,
J= 4.4 Hz, OCH2), 7.08 (1H, bs, ArH), 7.28 (1H, t, J= 8 Hz, ArH), 7.40 (1H, bs, ArH), 7.56
(1H, q, J= 4.0 Hz, ArH), 7.88 (1H, d, J= 8 Hz, ArH), 7.95 (1H, d, J= 8 Hz, ArH), 12.92 (1H,
15

s, NH). 13C-NMR: 51.86 (NCH2), 66.74 (OCH2), ArC:[113.13 (d, JCF= 24 Hz), 116.68,
116.89, 122.63, 122.65, 131.51, 131.59, 132.77, 132.86], 150.68 (C=N), 162.88 (C-F, d, JCF=
242 Hz). Anal. calcd. (%) for C17H15F2N3O: C, 64.75; H, 4.80; N, 13.33. Found: C, 64.87; H,
4.88; N, 13.41
4.2.9. 4-(5-Fluoro-2-(2-fluorophenyl)-1H-benzimidazol-6-yl)morpholine (3i)
White powder; Yield: 87 %, 138-139 °C. 1H-NMR: 2.97 (4H, t, J= 4 Hz, NCH2), 3.76 (4H, t,
J= 4.4 Hz, OCH2), 7.22 (1H, bs, ArH), 7.34-7.43 (3H, m, ArH), 7.49-7.54 (1H, m, ArH), 8.18
13
(1H, t, J= 8Hz, ArH), 12.47 (1H, bs, NH). C-NMR: 51.88, 51.91 (NCH2), 66.75 (OCH2),
ArC:[116.81, 117.02, 118.33, 118.44, 125.20, 125.52, 130.29, 130.32, 132.01, 132.09,
137.37, 147.00], 151.78 (C=N), 152.25, 154.63, 158.46, 160.95. Anal. calcd. (%) for
C17H15F2N3O: C, 64.75; H, 4.80; N, 13.33. Found: C, 64.86; H, 4.87; N, 13.42
4.3. Synthesis of compounds 4a-k
A mixture of compound 2 (10 mmol) and corresponding iminoester hydrochlorides (12
mmol) in dry methanol (20 mL) was taken in a round bottom flask with a magnetic stir bar.
The reaction mixture was stirred for 10 h at room temperature. After the completion of the
reaction (monitored by TLC, ethyl acetate/hexane 2:1), the mixture was poured into water.
The precipitate was collected by filtration and recrystallized from ethanol/water (2:1) to give
pure product 4a-k.
4.3.1. 4-(2-Benzyl-5-fluoro-1H-benzimidazol-6-yl)morpholine (4a)
1
Light brown powder; Yield: 95 %, 82-83 °C. H-NMR: 2.92 (4H, t, J= 4 Hz, NCH2), 3.73
(4H, t, J= 4.4 Hz, OCH2), 4.12 (2H, s, CH2), 7.03 (1H, bs, ArH), 7.18-7.29 (6H, m, ArH),
12.21 (1H, s, NH). 13C-NMR: 35.34 (CH2), 52.00 (NCH2), 66.77 (OCH2), ArC:[126.85,
126.92, 128.88, 129.12, 136.30, 138.12], 151.62 (C=N), 154.33. Anal. calcd. (%) for
C18H18FN3O: C, 69.44; H, 5.83; N, 13.50. Found: C, 69.33; H, 5.76; N, 13.57.
4.3.2. 4-(5-Fluoro-2-(4-fluorobenzyl)-1H-benzimidazol-6-yl)morpholine (4b)
Light brown powder; Yield: 95 %, 132-133 °C. 1H-NMR: 2.95 (4H, t, J= 4 Hz, NCH2), 3.75
(4H, t, J= 4.4 Hz, OCH2), 4.14 (2H, s, CH2), 7.03 (1H, bs, ArH), 7.11-7.16 (2H, m, ArH),
13
7.32-7.36 (3H, m, ArH), 12.23 (1H, bs, NH). C-NMR: 34.43 (CH2), 52.00 (NCH2), 66.78
16

(OCH2), ArC:[98.99, 101.17, 105.29 (d, J= 24 Hz), 108.66, 115.46 (d, J=51Hz), 129.52,
131.01 (d, J= 8 Hz), 131.32, 134.23, 136.04, 136.60, 140.22], 151.58 (C=N), 154.26, 161.50
(CF, d, J= 241 Hz). Anal. calcd. (%) for C18H17F2N3O: C, 65.64; H, 5.20; N, 12.76. Found: C,
65.78; H, 5.29; N, 12.86
4.3.3. 4-(5-Fluoro-2-(3-fluorobenzyl)-1H-benzo[d]imidazol-6-yl)morpholine (4c)
Light brown powder; Yield: 90 %, 111-112 °C. 1H-NMR: 2.98 (4H, t, J= 4 Hz, NCH2), 3.85
(4H, t, J= 4.4 Hz, OCH2), 4.12 (2H, s, CH2), 6.84-6.88 (2H, m, ArH), 6.94 (1H, d, J= 8 Hz,
13
ArH), 7.01 (1H, d, J= 4 Hz, ArH), 7.13-7.19 (2H, m, ArH), 12.34 (1H, bs, NH). C-NMR:
35.24 (CH2), 51.77, 51.79 (NCH2), 67.01 (OCH2), ArC:[113.98, 114.19, 115.70 (d, J= 22
Hz), 124.39 (d, J= 4 Hz), 130.30, 130.39, 137.03 (d, J= 12 Hz), 138.77, 138.84, 138.84 ],
153.03 (C=N), 153.54 (C-F, d, J= 2540 Hz), 162.87 (C-F, d, J= 246 Hz). Anal. calcd. (%) for
C18H17F2N3O: C, 65.64; H, 5.20; N, 12.76. Found: C, 65.76; H, 5.28; N, 12.84.
4.3.4. 4-(5-Fluoro-2-(2-fluorobenzyl)-1H-benzo[d]imidazol-6-yl)morpholine (4d)
White powder; Yield: 91 %, 104-105 °C. 1H-NMR: 3.00 (4H, t, J= 4 Hz, NCH2), 3.86 (4H, t,
J= 4.0 Hz, OCH2), 4.21 (2H, s, CH2), 6.98-7.05 (3H, m, ArH), 7.17-7.27 (3H, m, ArH). 13C-
NMR: 29.11 (d, J= 4 Hz, CH2), 51.80, 51.83 (NCH2), 67.04 (OCH2), ArC:[115.50 (d, J= 16
Hz) 123.40, 123.50, 124.62, 124.65, 129.14, 129.22, 131.13 (d, J= 4 Hz), 136.87, 136.98],
152.50 (C=N), 153.48 (C-F, d, J= 239 Hz), 160.74 (C-F, d, J= 244 Hz). Anal. calcd. (%) for
C18H17F2N3O: C, 65.64; H, 5.20; N, 12.76. Found: C, 65.73; H, 5.28; N, 12.68
4.3.5. 4-(2-(4-Chlorobenzyl)-5-fluoro-1H-benzo[d]imidazol-6-yl)morpholine (4e)
Light brown powder; Yield: 95 %, 131-132 °C. 1H-NMR: 2.95 (4H, bs, NCH2), 3.75 (4H, bs,
OCH2), 4.14 (2H, s, CH2), 7.03 (1H, bs, ArH), 7.20 (1H, bs, ArH), 7.32 (2H, d, J= 8 Hz,
ArH), 7.37 (2H, d, J= 8Hz, ArH), 12.26 (1H, s, NH). 13C-NMR: 34.55 (CH2), 52.00 (NCH2),
66.78 (OCH2), ArC:[ 98.83 (d, J= 26 Hz), 101.19, 105.19 (d, J= 26 Hz), 108.69, 126.79,
127.60, 128.84, 131.06, 131.32, 131.70, 136.14, 136.56, 137.08, 138.42, 140.16, 151.59
(C=N), 153.93. Anal. calcd. (%) for C18H17FClN3O: C, 62.52; H, 4.96; N, 12.15. Found: C,
62.61; H, 5.04; N, 12.22.
4.3.6. 4-(2-(3-Chlorobenzyl)-5-fluoro-1H-benzo[d]imidazol-6-yl)morpholine (4f)
Light brown powder; Yield: 92 %, 105-106 °C. 1H-NMR: 2.93 (4H, t, J= 4 Hz, NCH2), 3.73
(4H, t, J= 4.4 Hz, OCH2), 4.15 (2H, s, CH2), 7.09 (1H, d, J= 8 Hz, ArH), 7.20-7.33 (4H, m,
17

13
ArH), 7.37 (1H, s, ArH), 12.13 (1H, bs, NH). C-NMR: 34.79 (CH2), 51.97, 51.99 (NCH2),
66.76 (OCH2), ArC:[126.95, 127.21, 127.93, 128.58, 129.03, 129.73, 130.50, 130.78, 133.28,
133.45, 136.41 (d, J= 11 Hz), 137.30, 140.51], 151.70, 153.65, 154.07. Anal. calcd. (%) for
C18H17FClN3O: C, 62.52; H, 4.96; N, 12.15. Found: C, 62.62; H, 5.04; N, 12.23
4.3.7. 4-(2-(2-Chlorobenzyl)-5-fluoro-1H-benzo[d]imidazol-6-yl)morpholine (4g)
Light brown powder; Yield: 90 %, 110-111 °C. 1H-NMR: 3.00 (4H, t, J= 4 Hz, NCH2), 3.86
(4H, t, J= 4.4 Hz, OCH2), 4.31 (2H, s, CH2), 7.02 (1H, d, J= 8 Hz, ArH), 7.14-7.16 (3H, m,
ArH), 7.26-7.28 (1H, m, ArH), 7.31-7.34 (1H, m, ArH). 13C-NMR: 33.45 (CH2), 51.80, 51.83
(NCH2), 67.04 (OCH2), ArC:[114.25, 127.37, 127.46, 128.80, 129.71, 131.12, 133.83,
134.31, 134.39, 136.90 (d, J= 11 Hz)], 152.46 (C=N), 153.47 (C-F, d, J= 238 Hz) Anal.
calcd. (%) for C18H17FClN3O: C, 62.52; H, 4.96; N, 12.15. Found: C, 62.62; H, 5.04; N,
12.23.
4.3.8. 4-(2-(4-Bromobenzyl)-5-fluoro-1H-benzo[d]imidazol-6-yl)morpholine (4h)
White powder; Yield: 93 %, 121-122 °C. 1H-NMR: 2.92 (4H, t, J= 4 Hz, NCH2), 3.73 (4H, t,
J= 4.4 Hz, OCH2), 4.10 (2H, s, CH2), 7.06 (1H, bs, ArH), 7.24 (2H, d, J= 8 Hz, ArH), 7.28
(1H, bs, ArH), 7.48 (2H, d, J= 8 Hz, ArH), 12.21 (1H, bs, NH). 13C-NMR: 34.61 (CH2),
51.98, 51.99 (NCH2), 66.76 (OCH2), ArC:[120.04, 120.12, 131.42, 131.70, 131.73, 136.29,
136.50, 137.48, 137.69], 151.65, 153.82, 154.02. Anal. calcd. (%) for C18H17FBrN3O: C,
55.40; H, 4.39; N, 10.77. Found: C, 55.50; H, 4.45; N, 10.70.
4.3.9. 4-(5-Fluoro-2-(4-methylbenzyl)-1H-benzo[d]imidazol-6-yl)morpholine (4i)
1
White powder; Yield: 90 %, 178-179 °C. H-NMR: 2.25 (3H, s, CH3), 2.95 (4H, bs, NCH2),
3.76 (4H, bs, OCH2), 4.07 (2H, s, CH2), 7.02 (1H, bs, ArH), 7.14 (2H, d, J= 8 Hz, ArH), 7.18
(2H, d, J= 8 Hz, ArH), 7.27 (1H, bs, ArH), 12.17 (1H, s, NH). 13C-NMR: 21.07 (CH3), 34.95
(CH2), 52.02 (NCH2), 66.79 (OCH2), ArC:[129.03, 129.39, 129.46, 135.05, 135.98], 152.28,
154.57. Anal. calcd. (%) for C19H20FN3O: C, 70.13; H, 6.20; N, 12.91. Found: C, 70.24; H,
6.29; N, 13.00
4.3.10. 4-(5-Fluoro-2-(3-methylbenzyl)-1H-benzo[d]imidazol-6-yl)morpholine (4j)
1
White powder; Yield: 89 %, 89-90 °C. H-NMR: 2.24 (3H, s, CH3), 2.92 (4H, bs, NCH2),
3.73 (4H, bs, OCH2), 4.07 (2H, s, CH2), 7.01 (1H, d, J= 8 Hz, ArH), 7.06-7.09 (3H, m, ArH),
13
7.15-7.18 (1H, m, ArH), 7.25 (1H, d, J= 12 Hz, ArH), 12.18 (1H, bs, NH). C-NMR: 21.41
(CH3), 35.31 (CH2), 51.99, 52.01 (NCH2), 66.77 (OCH2), ArC:[126.22, 127.50, 127.56,
18

128.79, 129.72, 136.21, 136.32, 137.97, 137.99], 151.63, 153.99, 154.40. Anal. calcd. (%) for
C19H20FN3O: C, 70.13; H, 6.20; N, 12.91. Found: C, 70.28; H, 6.30; N, 13.03
4.3.11. 4-(5-Fluoro-2-(4-methoxybenzyl)-1H-benzo[d]imidazol-6-yl)morpholine (4k)
White powder; Yield: 93 %, 186-187 °C. 1H-NMR: 2.92 (4H, t, J= 4 Hz, NCH2), 3.69 (3H, s,
OCH3), 3.73 (4H, t, J= 4.4 Hz, OCH2), 4.04 (2H, s, CH2), 6.85 (2H, d, J= 8 Hz, ArH), 7.05
(1H, bs, ArH), 7.19 (2H, d, J= 8Hz, ArH), 7.25 (1H, d, J= 12 Hz, ArH), 12.12 (1H, s, NH).
13C-NMR: 34.47 (CH2), 51.99, 52.02 (NCH2), 55.48 (OCH3), 66.77 (OCH2), ArC:[114.19,
114.31, 125.90, 126.75, 129.91, 129.98, 130.15, 131.74, 136.20 (d, J= 11 Hz)], 151.59,
153.95, 154.78, 158.40. Anal. calcd. (%) for C19H20FN3O2: C, 66.85; H, 5.91; N, 12.31.
Found: C, 66.97; H, 5.99; N, 12.40
4.4. α-Glucosidase inhibition assay and kinetic assay
α-Glucosidase inhibitory activity (from Saccharomyces cerevisiae, Sigma- Aldrich) of the
compounds (3a-i, 4a-k, and acarbose) was examined according to the published studies [36].
Acarbose (Sigma-Aldrich) was used as the standard inhibitor drug. The enzyme solution 20
U/mL was prepared in phosphate buffer (pH 6.8, 50 mM). All the compounds were prepared
as stock solutions in 70 % methanol, and then different concentrations were pre-pared in the
buffer (phosphate buffer, pH 6.8). 200 µL of the compound at different concentration, 5 µL of
the enzyme (20 U/mL) and 1245 µL of buffer were added and incubated for 15 min at 37 °C.
After the incubation period, 250 µL of p-nitrophenyl-α-dglucopyranoside (5 mM, Sigma-
Aldrich) was added in the mixture and incubated at 37 °C for another 10 minutes. Change in
absorbance was measured at 405 nm spectrophotometrically. The α-glucosidase inhibition
percentage was calculated as follows:
α-Glucosidase inhibition (%) = [(Acontrol-Asample) / Acontrol] x100
where A control is the activity of enzyme without compound/standardand Asample is the
activity of the enzyme with compound/standard at different concentrations. The concentration
of the inhibitor required for inhibiting 50 % of the enzyme activity under the assay conditions
was defined as the IC50. The enzyme activity was determined by using p-NPG as a substrate
in the buffer (pH 6.8). Increasing concentrations of p-NPG (0.20-2.4 mg/mL) and 5 µL of 20
U/mL α-glucosidase enzyme were added in a test tube and the mixture was incubated at 37 °C
for 14 min. The absorbance was measured at 405 nm. The obtained data were plotted as
19

1/activity (1/V) against 1/substrate concentration (1/[S]) according to the Lineweaver-
Burkegraph [36].
The best compound (4k) having with the lowest IC50 value in both synthesized compound
groups and acarbose were further studied to determine the type of α-glucosidase inhibition. In
the reaction media, the substrate concentrations were 0.20, 0.40, 0.80, 1.6 and 2.4 mg/mL in
the buffer (pH 6.8) containing enzyme solution (20 U/mL), with (4k, acarbose) or without
inhibitor solutions. The Lineweaver-Burkegraphs were obtained and inhibition patterns were
evaluated.
4.5. The protocol of docking simulation for α-Glucosidase inhibition
The 3D structure of α-glucosidase from Saccharomyces cerevisiae co-crystallized with
maltose (PDB ID: 3A4A) and crystal structure of human lysosomal acid-alpha-glucosidase,
GAA, in complex with acarbose (PDB ID: 5NN8) which has been crystalized with 2.45 Å
resolution, recently were used as the target α-glucosidase proteins [36,50]. Because X-ray
structure of S.cerevisiae α-glucosidase which is used in experimental part have not been
reported yet, we used the X-ray structure of S. cerevisiae isomaltase (PDB ID: 3A4A) with 71
% identity and 84 % similarity with the S. cerevisiae α-glucosidase for docking study [41]. To
elucidate the interaction between newly synthesized ligands and α-glucosidase, docking
simulations were performed using the Maestro Molecular Modeling platform (version 10.5)
by Schrödinger, LLC [42]. The X-ray crystal structure of the target protein was downloaded
from the Protein Data Bank (www.rcsb.org). The default Glide/XP [43,44] combined with
Induced Fit docking protocol (IFD) was applied for the prediction of binding poses and
interactions between the ligands and active site of the protein [45-48]. The rest of docking
procedures were performed as described in our previous articles [49].
The estimation of binding free energies of studied compounds was performed by using
MM/GBSA method which is based on the molecular dynamics simulations of the receptor-
ligand complexes [37]. The Protein-Ligand Interaction Profiler (PLIP) web service was used
to obtain the interactions table [51]. Blind Docking calculations were carried out with Blind
Docking Server, available at: http://bio-hpc.eu/software/blind-docking-server [52].
References
1. Sim L, Willemsma C, Mohan S, Naim H.Y, Pinto B.M, Rose D.R. J. Biological
Chem.2010;285:17763-17770.
20

2. Ghani U. Eur. J. Med. Chem.2015;103:133-162.
3. Hollander P. Drugs 1992;44:47-53.
4. Howe JD,Smith N, Lee MJR, Ardes-Guisot N, Vauzeilles B, Désiré J, Baron A, Blériot Y,
Sollogoub M, Alonzi DS, ButtersTD.Bioorg. Med. Chem.2013;21:4831-4838.
5. Park H, Hwang KY, Oh KH, Kim YH, Lee JY, Kim K. Bioorg Med Chem.2008;16:284-
292.
6. Storr SJ, Royle L, Chapman CJ, AbdHamid UM, Robertson JF, Murray A, Dwek RA,
RuddPM.Glycobiology2008;18:456-462.
7.Moorthy NSHN, Ramos MJ, Fernandes PA, Mini Rev. Med. Chem., 2012;12:713-720.
8. Rajasekhar S, Maiti B, Balamurali MM, Chanda K.Curr Org Synth. 2017;14:40-60.
9. Kumar BVS, Vaidya SD, Kumar RV, Bhirud SB, Mane RB. Eur J Med Chem.
2006;41:599-604.
10. Gaba M, Mohan C. Med Chem Res. 2016;25:173-210.
11. Velik J, Baliharova V, Fink-Gremmels J, Bull S, Lamka J, Skalova L. Res Vet Sci.
2004;76:95-98.
12. Kavitha CS, Hosamani AC, Seetharamareddy HR. Eur J MedChem. 2010;45:2048-2054.
13. Kahveci B, Menteşe E,Özil M, Ülker S, Ertürk M. MonatshChem.2013;144:993-1001.
14. Ng RA, Guan J, Lanter VC, Allan GF, Sbriscia T, Linton O, Lundeen SG, Sui Z. Bioorg
Med Chem Lett. 2007;17:784-788.
15. Pankaj K, Singh OS. Key Heterocycle Cores for Designing Multitargeting Molecules,
Elsevier, 2018.
16. Tuncbilek M, Kiper T, Altanlar N. Eur J Med Chem. 2009;44:1024-1033.
17. Bansal Y, Silakari O. Bioorg Med Chem. 2012;20:6208-6236.
18. Yılmaz F, Menteşe E, Baltaş N. Lett Drug Design Discov.2017;14:201-208.
19. Menteşe E, Yılmaz F, Emirik M, Ülker S, Kahveci B. Bioorg Chem. 2018;76:478-486.
20. Menteşe E, Bektaş H, Ülker S, Bekircan O, Kahveci B. Enzym Inh and Med Chem.
2014;29:64-68.
21. Zamana K, Rahima F, Taha M, Ullaha H, Wadood A, Nawaza M, Khana F, Wahabd Z,
Ali Shahe SA, Rehman AU, Kawdeg AN, Gollapalli M, Bioorg Chem. 2019;89:103024.
22. Özil M, Parlak C, Baltas N. BioorgChem. 2018;76:468-477.
23. Abdullah Zawawi NKN, Taha M, Ahmat N, Wadood A, Ismail NH, Rahim F, Azam SS,
Abdullah N. Bioorg Chem. 2016;64:29-36.
24. Dinparast L, Valizadeh H, Bahadori MB, Soltani S, Asghari B, Rashidi MR, J Mol Struct.
2016;1114:84-94.
25. Kumar JA, Tiwari AK, Ali AZ, Madhusudhana K, Reddy BS, Ramakrishna S, Raju BC. J
Enzym Inh Med Chem.2010;25:80-86.
26. Özil M, Emirik M, Yılmaz Etlik S, Ülker S, Kahveci B. Bioorg Chem. 2016;68:226-235.
27. Arshad T, Rasool N, Khan KM, Hussain S, Salar U, Ashraf M, Asghar H, Wadood A,
Riaz M, Taha M, Perveen S,Ismail NH. BioorgChem. 2017;72:21-31.
28. Taha M, Ismail NH, Imran S, Mohamad MH, Wadood A, Rahim F, Saad SM, Rehman A,
Khan KM. Bioorg Chem.2016;65:100-109.
29. Menteşe E, Ülker S, Kahveci B.Chem Heterocycl Comp. 2015;50:1671-1682.
30. Block JH, Beale JM, Wilson and Gilsvold’s text book of organic medicinal and
pharmaceutical chemistry, 11th Ed., Wolter Kluwer, London, 2006.
21

31. Lee LF, Schleppnik FM, Schneider RW, Campbell DH. J HeterocyclChem.1990;27:243-
245.
32. Matsumoto J, Miyamoto T, Minamida A, Nishimura Y, Egawa H, Nishimura H, J Med
Chem.1984;27:292-301.
33. Kus C, Altanlar N. Turkis J Chem. 2003;27:35-39.
34. Mentese E, Yilmaz F, Islamoglu F, Kahveci B, J Chem Res. 2015;39:206-208.
35. Pinner A. Die imidoether und ihre derivative, 1. Auflage, Oppenheim, Berlin,1982.
36. Tao Y, Zhang Y, Cheng Y, Wang Y, Zhang Y. Biomed Chromatogr.2013;27:148-155.
37. Lineweaver H, Burk D, J Am Chem Soc.1934;56:658-661.
38. Isanbor C, O’Hagan D. J Fluorine Chem.2006;127:303-319.
39. Jeschke P. Chem Bio Chem.2004;5:570-589.
40. Menteşe E, Yılmaz F, Ülker S, Kahveci B. Drug Res.2015;65:40-45.
41. Taha M, Shah SAA, Afifi M, Imran S, Sultan S, Rahim F, Khan KM. Bioorg Chem.
2018;77:586-592.
42. Schrödinger, 2015. Schrödinger Release (New York: LLC).
43.Halgren TA, Murphy RB, Friesner RA, Beard HS, Frye LL, Pollard WT, Banks JL, J Med
Chem. 2004;47:1750-1759.
44. Farid R, Day T, Friesner RA, Pearlstein RA. Bioorg Med Chem. 2006;14:3160-3173.
45. Friesner RA, Murphy RB, Repasky MP, Frye LL, Greenwood JR, Halgren TA,
Sanschagrin PC, Mainz DT. J Med Chem. 2006;49:6177-6196.
46. Sherman W, Beard HS, Farid R. Chem Biol Drug Des.2006;67:83-84.
47. ShermanW, Day T, Jacobson MP, Friesner RA, Farid R. J Med Chem. 2006;49:534-553.
48. Menteşe E, Akyüz G, Emirik M, Baltaş N. Bioorg Chem.2019;83:289-296.
49. Isanbor C, O’Hagan D. J Fluorine Chem.2006;127:303-319.
50. Roig-Zamboni V, Cobucci-Ponzano B, Iacono R, Ferrara MC, Germany S, Bourne Y,
Parenti G, Moracci M, Sulzenbacher G. Nat Commun.2017;8:1111-1111.
51. Salentin S, Schreiber S, Haupt VJ, Adasme MF, Schroeder M. Nucleic Acids Res.
2015;43(W1):W443‐W447.
52. Sánchez-Linares I, Pérez-Sánchez H, Cecilia JM, García JM. BMC Bioinformatics.
2012;13 Suppl 14(Suppl 14):S13.
22

Synthesis, α-glucosidase inhibition and in silico studies of some 4-(5-fluoro-2-
substituted-1H-benzimidazol-6-yl)morpholine derivatives
Emre Menteşe*, Nimet Baltaş, Mustafa Emirik
IC50 = 0.18 ± 0.01 µg/mL
- α-glucosidase inhibitory potential-
23

Declaration of interests
☐The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
24

Highlights
 4-(5-Fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives were synthesized.
 All the compounds were screened for their in vitro α-glucosidase inhibitory potential.
 All compounds showed outstanding α-glucosidase inhibitory potential
 In silico studies were carried out to explore the binding interaction of the most active
compound.
25