A simple and efficient synthesis of novel inhibitors of alpha-glucosidase based on benzimidazole skeleton and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2016.08.011

A novel series of benzimidazole derivatives were prepared starting from o-phenylenediamine and 4-nitro-o-phenylenediamine with iminoester hydrochlorides. Acidic proton in benzimidazole was exchanged with ethyl bromoacetate, then ethyl ester group was transformed into hydrazide group. Cyclization using CS₂/KOH leads to the corresponding 1,3,4-oxadiazole derivative, which was treated with phenyl isothiocyanate resulted in carbothioamide group, respectively. As the target compounds, triazole derivative was obtained under basic condition and thiadiazole derivative was obtained under acidic condition from cyclization of carbothioamide group. Most reactions were conducted using both the microwave and conventional methods to compare yields and reaction times. All compounds obtained in this study were investigated for α-glucosidase inhibitor activity. Compounds 6a, 8a, 4b, 5b, 6b and 7b were potent inhibitors with IC₅₀ values ranging from 10.49 to 158.2?μM. This has described a new class of α-glucosidase inhibitors. Molecular docking studies were done for all compounds to identify important binding modes responsible for inhibition activity of α-glucosidase.

Full text of DOI:10.1016/j.bioorg.2016.08.011

Bioorganic Chemistry 68 (2016) 226–235
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
A simple and efficient synthesis of novel inhibitors of alpha-glucosidase
based on benzimidazole skeleton and molecular docking studies
a
a
b
c
Musa Özil a, , Mustafa Emirik , Semiha Yılmaz Etlik , Serdar Ülker , Bahittin Kahveci
⇑
a Department of Chemistry, Recep Tayyip Erdogan University, 53100 Rize, Turkey
b Department of Biology, Recep Tayyip Erdogan University, 53100 Rize, Turkey
c Department of Nutrition and Dietetics, Faculty of Health Sciences, Karadeniz Technical University, Trabzon, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of benzimidazole derivatives were prepared starting from o-phenylenediamine and
4-nitro-o-phenylenediamine with iminoester hydrochlorides. Acidic proton in benzimidazole was
exchanged with ethyl bromoacetate, then ethyl ester group was transformed into hydrazide group.
Cyclization using CS2/KOH leads to the corresponding 1,3,4-oxadiazole derivative, which was treated
with phenyl isothiocyanate resulted in carbothioamide group, respectively. As the target compounds, tri-
azole derivative was obtained under basic condition and thiadiazole derivative was obtained under acidic
condition from cyclization of carbothioamide group. Most reactions were conducted using both the
microwave and conventional methods to compare yields and reaction times. All compounds obtained
Received 5 August 2016
Revised 22 August 2016
Accepted 23 August 2016
Available online 24 August 2016
Keywords:
Benzimidazole
Microwave
in this study were investigated for
7b were potent inhibitors with IC50 values ranging from 10.49 to 158.2
class of -glucosidase inhibitors. Molecular docking studies were done for all compounds to identify
important binding modes responsible for inhibition activity of -glucosidase.
Ó 2016 Elsevier Inc. All rights reserved.
a
-glucosidase inhibitor activity. Compounds 6a, 8a, 4b, 5b, 6b and
Molecular docking
lM. This has described a new
a-Glucosidase
a
a
1. Introduction
with a number of advantages of the eco-friendly approach in the
framework of green chemistry [18–21].
The wide range of biological activities of benzimidazole has
made it a preferred structure for the modern drug discovery
[1,2]. Benzimidazole structures are classified under several ATC
groups such as antineoplastic-abemaciclib, antibiotic-ridinilazole,
histamine H4 receptor antagonist-toreforant and the proton pump
inhibitor-tegoprazan, which represents substances used in both
human and veterinary medicine [3,4] (Fig. 1). Thus, benzimidazoles
of both synthetic and natural sources are the key components of
many bioactive compounds [5–10].
In the literature, a number of synthetic routes have been devel-
oped in recent years to bring out new reagents for the synthesis of
benzimidazoles. However, the major drawbacks of these methods
are the harsh reaction conditions, unsatisfactory yields, use of
expensive reagents and long reaction time [11–17]. Therefore,
there is a need for an efficient synthetic route for benzimidazoles,
which is short and economical. Also, the use of microwave irradia-
tion in chemical reactions leads to easier work-up, higher yields,
enhanced reaction rates, and sometimes, to selective conversions
The inhibitors of glucosidase enzyme are highly useful for med-
ical therapies, such as diabetes, cancer, hyperlipoproteinemia, and
obesity HIV [22–25].
a-Glucosidase inhibitors such as acarbose,
miglitol, and voglibose are clinically used in the effective treatment
of type-2 diabetes mellitus [26]. These inhibitors effectively
decrease the postprandial glucose levels in type-2 diabetic patients
[27]. However, they cause various side effects, such as diarrhea,
abdominal and flatulence discomfort [28]. Therefore, designing of
new
reasonable claim [29]. The benzimidazole derivatives exhibited
-glucosidase [30–32] as well as antidiabetic [33] activity; hence
a-glucosidase inhibitors with minimum side effects is still a
a
it is important to obtain novel benzimidazole derivatives as
antidiabetic compounds.
It would be worthwhile to design and synthesize some new
benzimidazole derivatives bearing oxadiazole, triazole and thiadi-
azole moiety and to screen them for potential biological activities.
In this study we obtained benzimidazoles from iminoester
hydrochlorides by both microwave irradiation and conventional
methods. In the next step, we synthesized benzimidazole deriva-
tives containing ester, hydrazide, carbothioamide group, and
oxadiazole, triazole, and thiadiazole ring. The biological properties
of the synthesized compounds were evaluated in terms of
⇑
Corresponding author.
E-mail address: musa.ozil@erdogan.edu.tr (M. Özil).
http://dx.doi.org/10.1016/j.bioorg.2016.08.011
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
227
F
N
N
H
N
F
N
CH3
N
N
N
H
N
N
N
N
N
N
H
N
CH3
H
3C
H
3C
ridinilazole
abemaciclib
H
3C
CH3
N
N
H
N
N
N
HN
H
CH3
N
H
N
O
F
O
F
CH3
N
CH3
H
3C
O
CH3
toreforant
tegoprazan
Fig. 1. Some drugs containing benzimidazole structure.
a
-glucosidase inhibitory activities. In addition, docking studies
diazol-2-amine (8a,b) were realized through intramolecular
cyclization of thiosemicarbazides 6a,b in the presence of cold con-
centrated sulfuric acid (Scheme 1). As is known that compounds
with the structures like 7a,b can exist both in thiol and thione tau-
tomericforms, and the NH signal due to thioneform is a moreupfield
singlet than the SH signal derived from thiol form [19]. According to
the IR spectroscopic data of compounds 7a,b the presence of SAH
stretching band at 2663 and 2720 cmÀ1, respectively, and the
absence of an absorption at about 1260–1120 cmÀ1 region charac-
teristic for the C@S stretching vibrations showed that these com-
pounds were in the thiol form.
were presented to explore their complementarity with the speci-
fied binding pockets as well.
2. Results and discussion
2.1. Chemistry
The synthetic process was started from the reaction of 2-(2,4-
dichlorophenyl)acetonitrile with ethanol under HCl(g) producing
ethyl 2-(2,4-dichlorophenyl)acetimidate hydrochloride (1) accord-
ing to the literature [34]. Synthesis of benzimidazoles (2a,b) from
ethyl 2-(2,4-dichlorophenyl) acetimidate hydrochloride (1) and
o-phenylenediamine or 4-nitro-o-phenylenediamine in both con-
ventional and microwave method, make the procedure a clean,
efficient, and cheap method to afford a variety of useful hetero-
cyclic compounds.
The structures of new compounds were confirmed by FT-IR, 1H
NMR, 13C NMR spectroscopy and mass spectrometry. All synthe-
sized compounds were screened for their biological activities.
2.2.
a-Glucosidase inhibitory assay
All compounds were evaluated with regard to
a-glucosidase
Compound 2a,b reacted with ethyl bromoacetate under both
microwave and conventional conditions to produce benzimidazole
esters 3a,b. Compound 3a,b was further treated with an excess of
hydrazine hydrate to produce hydrazide 4a,b in moderate yield
(68%, 81%) under conventional heating and in high yield (82%,
96%) under microwave heating. The reaction of compounds 4a,b
with carbon disulfide in an alkaline medium under conventional
and microwave methods followed by acidification resulted in
1,3,4-oxadiazole ring closure producing benzimidazole thiols 5a,b.
Thiosemicarbazides are known as miscellaneous compounds for
the synthesis of different heterocyclic ring systems containing
sulfur and nitrogen [35]. Compounds 6a,b which were thiosemi-
carbazides derivatives prepared by the reaction of 4a,b and phenyl
isothiocyanate in ethanol, are useful intermediate for the synthesis
of 1,3,4-thiadizaole and 1,2,4-triazole derivatives (Scheme 1).
The cyclization of compounds 6a,b a with 2 N aqueous NaOH
solution resulted in the formation of 5-{[2-(2,4-dichlorobenzyl)-(7
-nitro)-1H-benzimidazol-1-yl]methyl}-4-phenyl-4H-1,2,4-triazole-
3-thiol (7a,b) under both microwave irradiation and conventional
method. On the other hand, the synthesis of 5-{[2-(2,4-dichloroben
zyl)-(6-nitro)-1H-benzimidazol-1-yl]methyl}-N-phenyl-1,3,4-thia
inhibition and compounds 6a, 8a, 4b, 5b, 6b and 7b showed inhi-
bition at various concentrations. No significant inhibitory effect
was detected for other compounds. Among the tested compounds,
4b, 5b and 7b showed the most significant
tion. These compounds inhibited -glucosidase activity by
99 1% at a concentration of 10 M (Table 1). Acarbose, known
as -glucosidase inhibitor used as anti-diabetic drug, showed an
a-glucosidase inhibi-
a
l
a
inhibitory effect by 83.3 1.5% at the same concentration. IC50 val-
ues of compounds 4b, 5b and 7b were determined as 10.49 0.91,
26.93 1.43, and 40.45 1.55 respectively (Table 1). These com-
pounds have a considerable inhibition potential. It was concluded
from the biological results for compounds 4b (IC50 10.49 0.91), 5b
(IC50 26.93 1.43), and 7b (IC50 40.45 1.55) that both electron
donating groups i.e. ANHA, ANH2, and ASH at hydrazide, oxadia-
zole, and triazole cyclics which are substituted at N À 1 position
of benzimidazole, as well as electron withdrawing group i.e.
ANO2 on benzimidazole ring play an effective role in this assay.
The binding interactions of promising compounds (4b, 5b, 7b)
were proved by using molecular docking.
Additionally, we have calculated the logP values of these
compounds using the ClogP software to gain some insight on the

228
M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
NH2
N
Cl
Cl
Cl
Cl
EtOH
HCl(g)
N
H
R
Cl
R
NH2
N
NH
C
.HCl
Cl
EtO
a
R:
series
-NO2 b series
H,
2a,b
,
1
BrCH2COOEt
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
NH2NH2
CS2 / KOH
R
R
R
O
O
N
N
OEt
O
NHNH2
Cl
HS
4a,b
3a,b
5a,b
PhNCS
Cl
N
N
R
Cl
O
NH
Ph
HN
HN
S
H
2SO4
NaOH
6a,b
N
N
N
N
Cl
Cl
Cl
Cl
R
R
N
N
N
N
S
N
Ph
HN
HS
Ph
7a,b
8a,b
Scheme 1. Synthetic pathway for the synthesis of benzimidazole derivatives.
influence of this important physico-chemical property on
glucosidase inhibition (Table 1).
a
-
2.3. In silico docking studies
The high values of logP, show parallelism with hydrophobic of
the molecular, and affinity of a compound for the hydrophobic part
of cell membrane. A ‘‘Rule of 5” property is a set of simple molec-
ular clarifying techniques used by Lipinski in formulating his ‘‘Rule
of 5” [36]. The rule cases, that, most ‘‘drug-like” molecules have
logP 6 5.
The docking scores of the studied compounds with 3TOP were
given in Table 1. The docking poses of the most active ligands 4b,
5b and 7b were illustrated in Figs. 2, 3 and 4 respectively. The
docking scores have a positive correlation with the experimental
results, in general. The crystal ligand acarbose binds with forming
H-bonding connections between OH groups of the acarbose and

M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
229
Table 1
-Glucosidase inhibitory effects and IC50 values of selected synthesized compounds
(at final concentration of 10 M). Acarbose was used as positive control.
4. Experimental
a
l
4.1. General information
Compound
a-glucosidase
Melting points were determined on capillary tubes on a Büchi
oil heated melting point apparatus and uncorrected. The IR spectra
were recorded on a Perkin Elmer Spectrum 100 FTIR Spectrometer
using KBr pellets. The 1H and 13C NMR spectra were recorded on an
Agilent Premium spectrometer (400 and 100 MHz, respectively) in
DMSO-d6 with TMS as internal standard. Heated electrospray
ionization mass spectroscopy (MS-MS) (H-ESI) was investigated
using Thermo Scientific TSQ Quantum Access MAX Triple Stage
Quadrupole Mass Spectrometer. Elemental analyses were per-
formed on a Carlo Erba 1106 CHN analyzer. A monomode CEM-
Discover microwave apparatus was used at 300 W power in the
standard configuration as delivered, including its software. All
experiments were carried out in microwave process vials (35 mL)
with control of the temperature by infrared detection temperature
sensor. The temperature was computer-monitored and maintained
constant by a discrete modulation of delivered microwave power.
The pulses of microwave are continuous for the time interval. After
completion of the reaction, the vial was cooled to 60 °C via air-jet
cooling. The ClogP values were obtained using ChemDraw Profes-
sional Version 15.0 software. All chemicals, solvents, and reagents
were of reagent grade quality and were used as purchased from
commercial sources. Ethyl (2,4-dichlorophenyl)acetimidate
hydrochloride (1) was prepared according to the reported proce-
dure [34].
% Inhibition
IC50
(
l
M)
ClogP
Docking score
2a
3a
4a
5a
6a
7a
8a
2b
3b
4b
5b
6b
7b
8b
Acarbose
–
–
–
–
–
–
5.06
5.15
3.15
3.88
5.20
6.20
6.66
4.74
4.97
2.98
3.71
4.80
6.03
6.49
À6.83
À5.15
À5.03
À7.21
À6.99
À6.53
À7.05
À5.88
À4.53
À8.23
À8.82
À6.23
À8.05
À4.76
À8.27
76
96
–
3
5
253.84 7.02
141.17 4.02
–
158.24 3.41
–
99
–
2
–
–
99
99
95
99
–
1
1
1
1
10.49 0.91
26.93 1.43
93.03 2.20
40.45 1.55
–
83.3 1.5
13.34 1.26
–: not determined.
the oxygen atom of ASP1370, ASP1279, ASP1157 and ASP1526
amino acid residues.
The docking pose of compound 4b was shown in Fig. 2. The
docking study predicted that compound 4b binds to enzyme form-
ing a hydrogen bond with TRP1369 residue through the C@O
groups of the ligand and the salt bridge through the NOO moiety
at the binding cavity. The other interaction involves
interaction with PHE1560.
Fig. 3 demonstrates the docking pose of compound 5b. The
prominent interaction of compound 5b with enzyme is the
hydrogen bond interaction formed between the nitrogen atom
p–p stacking
4.2. General procedure for the synthesis of benzimidazoles
Conventional method.
A
mixture of o-phenylenediamine
(1.08 g, 0.01 mol) or 4-nitro-o-phenylenediamine (1.53 g,
0.01 mol) and iminoester hydrochloride (2.69 g, 0.01 mol) in
50 mL dry methanol was taken in a round bottom flask. The
solution was stirred for 12 h at room temperature. After the
completion of the reaction (monitored by TLC, ethyl acetate/hex-
ane 3:1), the mixture was poured into water. The precipitate was
collected by filtration, washed with hot-water to suspend from
unreacted compounds and recrystallized from ethanol/water
(1:3) to give pure 2a–2b.
Microwave method. A mixture of o-phenylenediamine (0.54 g,
0.005 mol) or 4-nitro-o-phenylenediamine (0.77 g, 0.005 mol)
and iminoester hydrochloride (1.35 g, 0.005 mol) in 15 mL dry
methanol was irradiated in closed vessels with pressure control
at 65 °C for 10 min (hold time) at 300 W maximum power. Work
up and purification procedures were carried out as described
above.
of ligand and THR1586 residue of
other arresting interactions are the salt bridge occurring between
the NOO moiety of ligand and TRP1369 residue of -glucosidase
stacking interaction with TYR1258 amino acid
a-glucosidase protein. The
a
protein and
residue.
p–p
The docking pose of compound 7b was demonstrated in Fig. 4.
At the binding site of -glucosidase, PHE1560, PHE1559 and
TRP1355 residues construct the stacking interaction with ben-
zyl ring of the ligand 7b. The hydrophobic interactions are mainly
formed by ILE1587, TRP1369, TRY1251 and MET1421 amino acid
residues. GLN1561, HIE1584 and THR1586 residues form polar
interactions with the ligand 7b.
a
p–p
3. Conclusion
The aim of this work was to design, synthesize and evaluate for
-glucosidase inhibition of novel benzimidazole derivatives con-
4.2.1. 2-(2,4-Dichlorobenzyl)-1H-benzimidazole (2a)
a
Yield: 2.52 g, (91%) (for conv. method), 1.32 g, (95%), (for mw
method), mp 196–198 °C; IR (KBr): 3086 (NH), 2999 (ArACH),
2950 (Alip-CH), 1589 (C@N), 740, 721 (CACl). 1H NMR (400 MHz,
DMSO-d6) d: 4.28 (s, 2H, CH2), 7.09–7.12 (m, 2H, Ar), 7.38 (d,
J = 7.8 Hz, 1H, Ar), 7.42 (d, J = 7.8 Hz, 1H, Ar), 7.47 (d, J = 8.0 Hz,
2H, Ar), 7.61 (s, 1H, Ar), 12.26 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) d: 32.6 (CH2), 121.5, 121.8, 121.8, 122.0, 127.9, 129.2,
132.7, 133.3, 134.7 (ArÁC), 152.2 (C@N). Anal. Calcd for C14H10N2-
Cl2%: C 60.67, H 3.64, N 10.11; found %: C 60.65, H 3.62, N 10.09;
Mass spectrum, m/z (Irel, %): 277.26 [M+H]+ (100) [37].
taining ester, hydrazide, carbothioamide groups, oxadiazole, tria-
zole and thiadiazole rings. Most of the compounds were
synthesized by using both microwave-assisted and conventional
methods. The synthetic approach provides rapid, inexpensive,
and efficient routes for the access to new benzimidazole deriva-
tives in good yields by microwave technique. Synthesized benzim-
idazole derivatives were evaluated for their
inhibitory potential and identified three compounds as potent
glucosidase inhibitor. These compounds showed IC50 values rang-
ing from 10.49 0.91 (4b), 26.93 1.43 (5b), and 40.45 1.55
a-glucosidase
a-
(7b)
lM. Through molecular docking study, we have concluded
that the presence of nitro group on benzimidazole ring is highly
important. If nitro group is replaced with hydrogen, the exhibited
potential either reduces many times or brings about complete
inactivity.
4.2.2. 2-(2,4-Dichlorobenzyl)-6-nitro-1H-benzimidazole (2b)
Yield: 2.87 g, (89%) (for conv. method), 1.50 g, (93%), (for mw
method), mp 211–213 °C; IR (KBr): 3281 (NH), 3063 (ArACH),
2982 (Alip-CH), 1590 (C@N), 1516, 1340 (ANO2), 736, 688 (CACl).

230
M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
Fig. 2. The 2D ligand interaction diagram and predicted binding mode of compound 4b.
1H NMR (400 MHz, DMSO-d6) d: 4.38 (s, 2H, CH2), 7.43
(d, J = 8.0 Hz, 1H, Ar), 7.48 (d, J = 8.0 Hz, 1H, Ar), 7.61 (d,
J = 8.4 Hz, 1H, Ar), 7.65 (s, 1H, Ar), 8.06 (d, J = 8.4 Hz, 1H, Ar), 8.37
(s, 1H, Ar), 12.97 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d:
32.7 (CH2), 108.5, 111.9, 116.0, 128.0, 129.3, 133.1, 133.5, 133.9,
4.3. General procedure for the synthesis of benzimidazole esters
Conventional method. Dry K2CO3 (3.45 g, 0.025 mol) was added
to a solution of compound 2a (2.77 g, 0.01 mol) or 2b (3.23 g,
0.01 mol) in absolute acetone (50 mL), and the mixture was stirred
for 30 min at room temperature. Then, ethyl bromoacetate 1.5 mL,
(0.012 mol) was added to the solution and stirring was continued
overnight. After the completion of the reaction (monitored by
134.4, 134.9, 137.3, 142.8 (ArÁC), 143.8 (C@N); Anal. Calcd for C14
-
H9N3O2Cl2%: C 50.67, H 2.29, N 13.64; found %: C 50.64, H 2.28, N
13.66; Mass spectrum, m/z (Irel, %): 322.12 [M+H]+ (100).

M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
231
Fig. 3. Predicted binding mode and 2D ligand interaction diagram of compound 5b.

232
M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
Fig. 4. Predicted binding mode and 2D ligand interaction diagram of compound 7b.
TLC, eluent AcOEt–hexane, 3:1), the mixture was added to ice
water and filtered, afterwards the solid residue was recrystallized
from ethanol to give pure 3a–3b.
Microwave method. The mixture of compound 2a (1.39 g,
0.005 mol) or 2b (1.61 g, 0.005 mol) and dry K2CO3 (3.5 g,
0.026 mol) in absolute acetone (15 mL) was irradiated with

M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
233
microwaves at 100 °C for 5 min (hold time) at 300 W maximum
power. Then ethyl bromoacetate (1.4 mL, 0.012 mol) was added
to the reaction mixture and the irradiation was continued for addi-
tional 15 min at the same conditions. Work up and purification
procedures were carried out as described above.
C16H14N4OCl2%: C 55.03, H 4.04, N 16.04; found %: C 55.01, H
4.06, N 16.01; Mass spectrum, m/z (Irel, %): 349.28 [M+H]+ (100).
4.4.2. 2-[2-(2,4-Dichlorobenzyl)-6-nitro-1H-benzimidazol-1-yl]aceto
hydrazide (4b)
Yield: 3.20 g, (81%) (for conv. method), 1.90 g, (96%), (for mw
method), mp 256–258 °C; IR (KBr): 3313, 3293 (NH2, NH), 3100
(ArACH), 3057 (Alip-CH), 1651 (C@O), 1620 (C@N), 1517, 1338
(ANO2), 738, 685 (CACl). 1H NMR (400 MHz, DMSO-d6) d: 4.39 (s,
2H, NH2), 5.03 (s, 2H, CH2), 5.09, (s, 2H, N-CH2), 7.64 (d,
J = 8.2 Hz, 1H, Ar), 7.70 (d, J = 8.2 Hz, 1H, Ar), 7.78 (s, 1H, Ar), 8.06
(d, J = 7.8 Hz, 1H, Ar), 8.16 (d, J = 7.8 Hz, 1H, Ar), 8.42 (s, 1H, Ar),
9.60 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d: 31.2 (CH2),
45.3 (N-CH2), 107.7, 111.1, 114.8, 117.8, 119.3, 127.9, 129.2,
132.9, 133.5, 133.8, 134.9, 135.5, 140.6, 141.7, 142.9, 143.2,
147.1, 157.6 (ArÁC), 159.0 (C@N), 165.8 (C@O). Calcd for C16H13N5
O3Cl2%: C 48.75, H 3.32, N 17.77; found %: C 48.78, H 3.33, N
17.76; Mass spectrum, m/z (Irel, %): 394.22 [M+H]+ (100).
4.3.1. Ethyl [2-(2,4-dichlorophenyl)-1H-benzimidazol-1-yl]acetate
(3a)
Yield: 2.55 g, (70%) (for conv. method), 1.6 g, (88%), (for mw
method), mp 164–166 °C; IR (KBr): 2918, 2849 (Alip-CH), 1740
(C@O), 1589 (C@N), 1198 (CAO), 742, 721 (CACl). 1H NMR
(400 MHz, DMSO-d6) d: 1.16 (t, J = 8.0 Hz, 3H, CH3), 4.10 (q,
J = 8.0 Hz, 2H, CH2), 4.29 (s, 2H, CH2), 5.23 (s, 2H, N-CH2), 7.12–
7.20 (m, 2H, Ar), 7.34 (d, J = 8.0 Hz, 1H, Ar), 7.39 (d, J = 8.0 Hz, 1H,
Ar), 7.48 (d, J = 8.0 Hz, 1H, Ar), 7.52 (d, J = 8.0 Hz, 1H, Ar), 7.62 (s,
1H, Ar). 13C NMR (100 MHz, DMSO-d6) d: 14.4 (CH3), 30.8 (CH2),
44.9 (N-CH2), 61.8 (O-CH2), 110.5, 119.1, 122.1, 122.5, 127.8,
129.1, 132.7, 133.4, 134.2, 134.8, 136.0, 142.5 (Ar C), 152.9
(C@N), 168.4 (C@O). Anal. Calcd for C18H16N2O2Cl2%: C 58.47, H
4.04, N 8.02; found %: C 58.44, H 4.04, N 8.00; Mass spectrum,
m/z (Irel, %): 363.35 [M+H]+ (100).
4.5. General procedure for the synthesis of benzimidazole oxadiazoles
Conventional method. A solution of KOH (0.28 g, 0.005 mol) in
water (5 mL) was added dropwise to a solution of compound 4a
(1.75 g, 0.005 mol) or 4b (1.97 g, 0.005 mol) and CS2 (0.37 mL,
0.005 mol) in ethanol (30 mL). The reaction mixture was refluxed
24 h until the evolution of hydrogen sulfide ceased. After the com-
pletion of the reaction (TLC, eluent AcOEt), the solvent was evapo-
rated under reduced pressure, and a viscous solid appeared. It was
dissolved in water (50 mL) and acidified with 4 N hydrochloric acid
to pH 4. The precipitated solid was filtered and recrystallized twice
from ethanol to give pure 5a–5b.
Microwave method. A solution of KOH (0.28 g, 0.005 mol) in
water (5 mL) was added dropwise to a solution of compound 4a
(1.75 g, 0.005 mol) or 4b (1.97 g, 0.005 mol) and CS2 (0.37 mL,
0.005 mol) in ethanol (15 mL) in a microwave vial. The mixture
was irradiated with microwaves at 130 °C for 15 min (hold time)
at 300 W maximum power. Work up and purification procedures
were carried out as described above.
4.3.2. Ethyl [2-(2,4-dichlorophenyl)-6-nitro-1H-benzimidazol-1-yl]
acetate (3b)
Yield: 3.39 g, (83%) (for conv. method), 1.74 g, (85%), (for mw
method), mp 140–142 °C; IR (KBr): 3095 (ArACH), 2979 (Alip-
CH), 1735 (C@O), 1519, 1335 (ANO2), 1228 (CAO), 735, 685 (CACl).
1H NMR (400 MHz, DMSO-d6) d: 1.17 (t, J = 8.0 Hz, 3H, CH3), 4.11
(q, J = 8.0 Hz, 2H, CH2), 4.38 (s, 2H, CH2), 5.39 (s, 2H, N-CH2), 7.72
(d, J = 7.8 Hz, 1H, Ar), 7.77 (d, J = 7.8 Hz, 1H, Ar), 7.82 (s, 1H, Ar),
8.07 (d, J = 8.0 Hz, 1H, Ar), 8.15 (d, J = 8.0 Hz, 1H, Ar), 8.44 (s, 1H,
Ar). 13C NMR (100 MHz, DMSO-d6) d: 14.4 (CH3), 31.0 (CH2), 45.4
(N-CH2), 61.9 (O-CH2), 108.0, 111.3, 115.3, 118.0, 118.4, 119.4,
127.9, 129.2, 133.0, 133.5, 133.6, 134.9, 140.5, 141.6, 143.2,
146.9, 157.3, 158.7 (C@N), 168.0 (C@O). Anal. Calcd for C18H15N3
O4Cl2%: C 51.80, H 3.32, N 10.66; found %: C 51.78, H 3.34, N
10.62; Mass spectrum, m/z (Irel, %): 408.64 [M+H]+ (100).
4.4. General procedure for the synthesis of bibenzimidazole hydrazides
4.5.1. 5-{[2-(2,4-Dichlorobenzyl)-1H-benzimidazol-1-yl]methyl}-
1,3,4-oxadiazole-2-thiol (5a)
Conventional method. Compound 3a (3.64 g, 0.01 mol) or 3b
(4.08 g, 0.01 mol) and hydrazine hydrate (5 mL, 0.10 mol) were
dissolved in dry ethanol (30 mL) and stirring was continued over-
night. After the completion of the reaction (monitored by TLC, elu-
ent AcOEt–hexane, 3:1), the residue was filtered off. The product
was subsequently recrystallized from ethanol to give pure 4a–4b.
Microwave method. Compound 3a (1.82 g, 0.005 mol) or 3b
(2.05 g, 5 mmol) and hydrazine hydrate (2.5 mL, 0.05 mol) were
dissolved in dry ethanol (15 mL) and irradiated with microwaves
at 90 °C for 10 min (hold time) at 300 W maximum power. Work
up and purification procedures were carried out as described
above.
Yield: 1.27 g, (65%) (for conv. method), 1.41 g, (72%), (for mw
method), mp 248–250 °C; IR (KBr): 3053 (ArACH), 2409 (SH),
1622, 1585, 1560 (C@N), 745, 718 (CACl). 1H NMR (400 MHz,
DMSO-d6) d: 4.40 (s, 2H, CH2), 5.78 (s, 2H, N-CH2), 7.17–7.27 (m,
2H, Ar), 7.31 (d, J = 8.0 Hz, 1H, Ar), 7.37 (d, J = 8.0 Hz, 1H, Ar),
7.55 (d, J = 8.0 Hz, 1H, Ar), 7.60 (d, J = 8.0 Hz, 1H, Ar), 7.63 (s, 1H,
Ar), 10.75 (s, H, NH). 13C NMR (100 MHz, DMSO-d6) d: 30.8 (CH2),
38.5 (N-CH2), 110.6, 119.2, 122.6, 132.0, 127.8, 129.1, 133.2,
133.9, 134.3, 134.8, 135.5, 142.7 (ArAC), 152.7 (C@N), 159.5
(C@N), 178.4 (C@S). Calcd for C17H12N4Cl2OS%: C 52.18, H 3.09, N
14.32; found %: C 52.20, H 3.08, N 14.31; Mass spectrum, m/z (Irel
,
%): 391.28 [M+H]+ (100).
4.4.1. 2-[2-(2,4-Dichlorobenzyl)-1H-benzimidazol-1-yl]aceto
hydrazide (4a)
4.5.2. 5-{[2-(2,4-Dichlorobenzyl)-6-nitro-1H-benzimidazol-1-yl]
methyl}-1,3,4-oxadiazole-2-thiol (5b)
Yield: 2.38 g, (68%) (for conv. method), 1.44 g, (82%), (for mw
method), mp 235–237 °C; IR (KBr): 3273, 3138 (NH2, NH), 3099
(ArACH), 2960 (Alip-CH), 1662 (C@O), 1564 (C@N), 741, 716
(CACl). 1H NMR (400 MHz, DMSO-d6) d: 4.34 (s, 2H, NH2), 4.87
(s, 2H, CH2), 5.23 (2H, s, N-CH2), 7.11–7.20 (m, 2H, Ar), 7.34 (d,
J = 8.2 Hz, 1H, Ar), 7.38 (d, J = 8.2 Hz, 1H, Ar), 7.44 (d, J = 8.0 Hz,
1H, Ar), 7.49 (d, J = 8.0 Hz, 1H, Ar), 7.63 (s, 1H, Ar), 9.54 (1H, s,
NH). 13C NMR (100 MHz, DMSO-d6) d: 30.9 (CH2), 44.9 (N-CH2),
110.3, 119.01, 121.9, 122.3, 127.8, 129.1, 132.6, 133.3, 134.5,
134.8, 135.9, 142.5 (ArÁC), 153.2 (C@N), 166.3 (C@O). Calcd for
Yield: 1.46 g, (67%) (for conv. method), 1.64 g, (75%), (for mw
method), mp 148–150 °C; IR (KBr): 3070 (ArACH), 2919 (Alip-
CH), 2728 (SAH), 1519, 1338 (ANO2), 734, 686 (CACl). 1H NMR
(400 MHz, DMSO-d6) d: 4.47 (s, 2H, CH2), 5.89, 5,95 (s, 2H,
N-CH2), 7.74 (d, J = 7.6 Hz, 1H, Ar), 7.87 (d, J = 7.6 Hz, 1H, Ar),
7.91 (s, 1H, Ar), 8.08 (d, J = 8.0 Hz, 1H, Ar), 8.20 (d, J = 8.0 Hz, 1H,
Ar), 8.45 (s, 1H, Ar), 10.71 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) d: 31.1 (CH2), 39.1 (N-CH2), 108.1, 111.4, 115.4, 118.3,
118.8, 119.6, 127.9, 129.2, 133.1, 133.5, 134.9, 140.1, 141.7,
143.6, 147.0 (Ar C), 157.2, 159.0 (C@N), 178.4 (C@S). Calcd for

234
M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
C17H11N5Cl2O3S%: C 46.80, H 2.54, N 16.05; found %: C 46.81, H
up and purification procedures were carried out as described
above.
2.55, N 16.02; Mass spectrum, m/z (Irel, %): 436.29 [M+H]+ (100).
4.6. General procedure for the synthesis of benzimidazole
carbothioamides
4.7.1. 5-{[2-(2,4-Dichlorobenzyl)-1H-benzimidazol-1-yl]methyl}-4-
phenyl-4H-1,2,4-triazole-3-thiol (7a)
Yield: 0.70 g, (30%) (for conv. method), 0.56 g, (48%), (for mw
method), mp 165–167 °C; IR (KBr): 3060, 3024 (ArACH), 2966
(Alip-CH), 2663 (SH), 1589, 1570 (C@N), 742, 687 (CACl). 1H
NMR (400 MHz, DMSO-d6) d: 4.04 (s, 2H, CH2), 5.42 (s, 2H, N-
CH2), 7.10–7.36 (m, 9H, ArAH), 7.50 (d, J = 8.0 Hz, 2H, Ar), 7.60 (s,
1H, Ar), 13.89 (s, 1H, SH). 13C NMR (100 MHz, DMSO-d6) d: 30.5
(ACH2), 43.0 (N-CH2), 110.6, 119.0, 122.1, 122.5, 127.7, 128.5,
130.0, 132.7, 133.1, 133.3, 134.1, 134.7, 135.6, 142.4 (ArAC),
147.9, 152.5 (C@N), 169.2 (CASH). Calcd for C23H17N5Cl2S%: C
59.23, H 3.67, N 15.02; found %: C 59.21, H 3.66, N 15.05; Mass
spectrum, m/z (Irel, %): 466.39 [M+H]+ (100).
Conventional method.
A solution of compound 4a (3.50 g,
0.01 mol) or 4b (3.94 g, 0.01 mol) and phenyl isothiocyanate
(1.11 mL, 0.01 mol) in ethanol (30 mL) was refluxed with stirring
for 4 h. The progress of the reaction was checked with TLC (eluent
AcOEt–hexane, 3:1). The mixture was cooled to room temperature
and the residue was filtered off. The product was subsequently
recrystallized from ethanol to give pure 6a–6b.
Microwave method.
A solution of compound 4a (1.75 g,
0.005 mol) or 4b (1.97 g, 0.005 mol) and phenyl isothiocyanate
(0.56 mL, 0.005 mol) in ethanol (20 mL) was irradiated with micro-
waves at 120 °C for 12 min (hold time) at 300 W maximum power.
Work up and purification procedures were carried out as described
above.
4.7.2. 5-{[2-(2,4-Dichlorobenzyl)-7-nitro-1H-benzimidazol-1-yl]
methyl}-4-phenyl-4H-1,2,4-triazole-3-thiol (7b)
Yield: 1.72 g, (67%) (for conv. method), 1.01 g, (79%), (for mw
method), mp 138–140 °C; IR (KBr): 3051 (ArACH), 2920 (Alif-
CH), 2720 (SH), 1620, 1588 (C@N), 1519, 1336 (ANO2), 737, 694
(CACl). 1H NMR (400 MHz, DMSO-d6) d: 4.22 (s, 2H, CH2), 5.55 (s,
2H, N-CH2), 7.27–7.62 (m, 5H, ArAH), 7.77 (d, J = 8.0 Hz, 2H, Ar),
7.83 (s, 1H, Ar), 8.04 (d, J = 7.8 Hz, 1H, Ar), 8.14 (d, J = 7.8 Hz, 1H,
Ar), 8.38 (s, 1H, Ar), 13.89 (s, 1H, SH). 13C NMR (100 MHz, DMSO-
d6) d: 30.9 (ACH2), 45.5 (N-CH2), 110.6, 111.4, 115.2, 118.3,
119.2, 127.9, 128.5, 129.2, 130.0, 130.3, 132.9, 133.2, 133.4,
134.8, 140.2, 140.6, 141.5, 143.3 (ArAC), 147.4, 157.0 (C@N),
169.3 (CASH). Calcd for C23H16N6Cl2O2S%: C 54.02, H 3.15, N
16.43; found %: C 54.04, H 3.16, N 16.40; Mass spectrum, m/z
(Irel, %): 511.47 [M+H]+ (100).
4.6.1. 2-{[2-(2,4-Dichlorobenzyl)-1H-benzimidazol-1-yl]acetyl}-N-
phenylhydrazine carbothioamide (6a)
Yield: 4.12 g, (85%) (for conv. method), 2.21 g, (91%), (for mw
method), mp 148–150 °C; IR (KBr): 3436, 3333 (NH), 3162
(ArACH), 2966 (Alif-CH), 1708 (C@O), 1594 (C@N), 1184 (C@S),
742, 692 (CACl). 1H NMR (400 MHz, DMSO-d6) d:4.34 (s, 2H,
CH2), 5.07 (s, 2H, N-CH2), 7.15–7.40 (m, 9H, ArAH), 7.51 (d,
J = 8.0 Hz, 2H, Ar), 7.63 (s, 1H, Ar), 9.72 (s, 1H, NH), 10.34 (s, 1H,
NH), 10.53 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d: 30.9
(CH2), 44.9 (N-CH2), 110.5, 119.0, 122.0, 122.4, 127.8, 129.08,
132.6, 132.7, 133.3, 134.5, 134.9, 136.0, 139.4 (ArÁC), 142.5
(C@N), 153.2 (C@O), 153.3 (C@S). Calcd for C23H19N5Cl2OS%: C
57.03, H 3.95, N 14.46; found %: C 57.00, H 3.94, N 14.47; Mass
spectrum, m/z (Irel, %): 484.45 [M+H]+ (100).
4.8. General procedure for the synthesis of benzimidazole thiadiazoles
4.6.2. 2-{[2-(2,4-Dichlorobenzyl)-6-nitro-1H-benzimidazol-1-yl]
acetyl}-N-phenyl hydrazinecarbothioamide (6b)
A solution of compound 6a (2.42 g, 0.005 mol) or 6b (2.65 g,
0.005 mol) was dissolved in ethanol (20 mL). Then, concentrated
sulfuric acid (5 mL) was slowly added to an ice cooled and stirred
solution over a period of 15 min. The reaction mixture was stirred
at room temperature for 24 h. Then, the mixture was poured into
equal volume of ice-cold water, neutralized to pH 8 with ammonia,
and the obtained residue was filtered and recrystallized from ethyl
acetate-petroleum benzene (3:1) to give pure 8a–8b.
Yield: 3.92 g, (74%) (for conv. method), 2.25 g, (85%), (for mw
method), mp 231–233 °C; IR (KBr): 3332, 3216 (NH), 2969 (Alif-
CH), 1696 (C@O), 1594 (C@N), 1508, 1337 (ANO2), 1200 (C@S),
735, 691 (CACl). 1H NMR (400 MHz, DMSO-d6) d: 4.42 (s, 2H,
CH2), 5.22 (s, 2H, N-CH2), 7.17–7.65 (m, 5H, ArAH), 7.77 (d,
J = 7.8 Hz, 2H, Ar), 7.88 (s, 1H, Ar), 8.07 (d, J = 8.2 Hz, 1H, Ar), 8.17
(d, J = 8.2 Hz, 1H, Ar), 8.43 (s, 1H, Ar), 9.73 (s, 1H, NH), 10.59
(s, 1H, NH), 11.03 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) d:
31.2 (CH2), 45.4 (N-CH2), 111.3, 115.3, 115.9, 117.9, 118.3, 119.3,
127.8, 127.9, 129.2, 132.9, 133.5, 133.8, 133.9, 135.0, 139.3,
140.7, 141.7, 143.0 (ArÁC), 147.1 (C@N), 157.7 (C@O), 159.1
(C@S). Calcd for C23H18N6Cl2O3S%: C 52.18, H 3.43, N 15.87; found
%: C 52.20, H 3.42, N 15.88; Mass spectrum, m/z (Irel, %): 529.31 [M
+H]+ (100).
4.8.1. 5-{[2-(2,4-Dichlorobenzyl)-1H-benzimidazol-1-yl]methyl}-N-
phenyl-1,3,4-thiadiazol-2-amine (8a)
Yield: 1.87 g, (80%), mp 133–135 °C; IR (KBr): 3227 (NH), 3069
(ArACH), 2984 (Alifatik CH), 1597 (C@N), 774, 693 (CACl). 1H NMR
(400 MHz, DMSO-d6) d: 4.64 (s, 2H, CH2), 6.06 (s, 2H, N-CH2), 7.14–
7.57 (m, 7H, ArAH), 7.72 (d, J = 8.4 Hz, 2H, Ar), 7.79 (s, 1H, Ar), 7.84
(d, J = 8.0 Hz, 2H, Ar), 10.50 (s, 1H, NH). 13C NMR (100 MHz, DMSO-
d6) d: 31.4 (ACH2), 45.8 (N-CH2), 111.5, 118.6, 123.4, 128.5, 129.3,
129.8, 130.2, 133.6, 134.0, 134.6, 135.5, 135.8, 140.0, 141.5 (ArAC),
153.2, 155.4 (C@N), 166.3 (CANH). Calcd for C23H17N5Cl2S%: C
59.23, H 3.67, N 15.02; found %: C 59.25, H 3.67, N 15.00; Mass
spectrum, m/z (Irel, %): 466.46 [M+H]+ (100).
4.7. General procedure for the synthesis of benzimidazole triazoles
Conventional method.
A solution of compound 6a (2.42 g,
0.005 mol) or 6b (2.65 g, 0.005 mol) was dissolved in ethanol
(30 mL). Then, 4 N aqueous NaOH (1 mL) was added to the solution
and refluxed with stirring for 24 h. After the completion of the
reaction (monitored by TLC, eluent AcOEt), the mixture was added
to ice water (100 mL) and acidified to pH 6–7 with dil. HCl. The
resulting precipitate was filtered, washed with water, and recrys-
tallized from ethyl acetate to give pure 7a–7b.
4.8.2. 5-{[2-(2,4-Dichlorobenzyl)-6-nitro-1H-benzimidazol-1-yl]
methyl}-N-phenyl-1,3,4-thiadiazol-2-amine (8b)
Yield: 2.20 g, (86%) mp 188–190 °C; IR (KBr): 3255 (NH), 2921
(Alifatik CH), 1602 (C@N), 1510, 1338 (ANO2), 748, 689 (CACl).
1H NMR (400 MHz, DMSO-d6) d: 4.53 (s,2H, CH2), 6.00 (s, 2H,
CH2), 6.95–7.39 (m, 5H, ArAH), 7.52 (d, J = 8.0 Hz, 2H, Ar), 7.61 (s,
1H, Ar), 8.09 (d, J = 8.0 Hz, 1H, Ar), 8.20 (d, J = 8.2 Hz, 1H, Ar),
8.45 (s, 1H, Ar), 10.34 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6)
d: 31.5 (CH2), 42.4 (N-CH2), 107.9, 111.4, 117.9, 118.2, 122.5,
Microwave method.
A mixture of compound 6a (1.21 g,
0.0025 mol) or 6b (1.33 g, 0.0025 mol) and 2 N aqueous NaOH
(1 mL) in ethanol (10 mL) was irradiated with microwaves at
110 °C for 20 min (hold time) at 300 W maximum power. Work

M. Özil et al. / Bioorganic Chemistry 68 (2016) 226–235
235
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127.9, 129.2, 129.5, 133.5, 133.0, 134.9, 139.9, 140.8, 143.2, 143.5
(ArAC), 147.1, 154.0 (C@N), 165.7 (CANH). Calcd for C23H16N6Cl2-
O2S%: C 54.02, H 3.15, N 16.43; found %: C 53.99, H 3.14, N 16.45;
Mass spectrum, m/z (Irel, %): 511.39 [M+H]+ (100).
4.9. a-Glucosidase inhibition assay
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compounds were dissolved in DMSO. In 96-well microtiter plates,
a
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20
buffer were added and incubated for 15 min at 37 °C. After incuba-
tion, 25 L of p-nitrophenyl- -glucopyranoside (2 mM, Sigma
lL of test sample, 20 lL of enzyme (20 mU/mL) and 135 lL of
l
a-D
Aldrich) was added and change in absorbance was monitored for
20 min at 400 nm. Test compound was replaced by DMSO (10%
final) as control. Acarbose (Sigma-Aldrich) was used as a standard
inhibitor. The assays were done in triplicate. The IC50 value was
determined as the concentration of compound that give 50% inhi-
bition of maximal activity.
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4.10. Docking studies
The molecular docking study which has a significant impor-
tance to discover the ligand-protein interactions was performed
to observe the binding strength and pattern. The 3D protein struc-
tures for C-terminal domains of human intestinal a-glucosidase
(PDB ID: 3TOP) was used as molecular target. The crystal structure
complexed with an original ligand (acarbose) was retrieved from
RCSB Protein Data Bank and then submitted to protein preparation
module of Schrodinger’s Maestro molecular modeling package.
3D structure of each ligand was submitted to geometry opti-
mization in g09 program at the level of DFT/B3LYP/6-311G+(d,p)
[38]. All ligands were then set to the physiological pH (pH = 7.4)
at the protonation step of Maestro molecular modeling suite and
geometry optimizations were repeated. All molecules are docked
at the binding sites of a-glucosidase enzyme. The default Glide/
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site residues were considered by Induced Fit Docking (IFD)
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Acknowledgements
The authors gratefully acknowledge financial support from
Recep Tayyip Erdog˘an University, BAP, Rize, Turkey, through Pro-
ject number 2013.102.02.2.
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