Synthesis, α-glucosidase inhibitory, cytotoxicity and docking studies of 2-aryl-7-methylbenzimidazoles

Article abstract of DOI:10.1016/j.bioorg.2016.02.004

Benzimidazole analogs 1-27 were synthesized, characterized by EI-MS and ¹H NMR and their α-glucosidase inhibitory activities were found out experimentally. Compound 25, 19, 10 and 20 have best inhibitory activities with IC₅₀ values 5

Full text of DOI:10.1016/j.bioorg.2016.02.004

Accepted Manuscript
Synthesis, α-Glucosidase inhibitory, Cytotoxicity and Docking Studies of 2-
Aryl-7-methylbenzimidazoles
Muhammad Taha, Nor Hadiani Ismail, Syahrul Imran, Muhammad Helmi
Mohamad, Abdul Wadood, Fazal Rahim, Syed Muhammad Saad, Ashfaq ur
Rehman, Khalid Mohammed Khan
PII:
S0045-2068(16)30012-8
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.02.004
YBIOO 1880
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 December 2015
12 February 2016
12 February 2016
Please cite this article as: M. Taha, N.H. Ismail, S. Imran, M.H. Mohamad, A. Wadood, F. Rahim, S.M. Saad, A.u.
Rehman, K.M. Khan, Synthesis, α-Glucosidase inhibitory, Cytotoxicity and Docking Studies of 2-Aryl-7-
methylbenzimidazoles, Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.02.004
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Synthesis, α-Glucosidase inhibitory, Cytotoxicity and Docking Studies of 2-
Aryl-7-methylbenzimidazoles
Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Syahrul Imrana,b, Muhammad Helmi Mohamada,b,
Abdul Wadoodc, Fazal Rahimc, Syed Muhammad Saadd, Ashfaq ur Rehmanc, Khalid
Mohammed Khand
aAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA
(UiTM), Puncak Alam Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia.
bFaculty of Applied Science Universiti Teknologi MARA (UiTM), 40450, Shah Alam,
Selangor, Malaysia.
cDepartment of Chemistry, Hazara University, Mansehra-21120, Khyber Pukhtunkhwa,
Pakistan
dH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan.
Abstract
Benzimidazole analogs 1-27 were synthesized, characterized by EI-MS and 1HNMR and their
α-glucosidase inhibitory activities were found out experimentally. Compound 25, 19, 10 and
20 have best inhibitory activities with IC50 values 5.30 ± 0.10, 16.10 ± 0.10, 25.36 ± 0.14 and
29.75 ± 0.19 respectively against α-glucosidase. Compound 6 and 12 has no inhibitory
activity against α-glucosidase enzyme among the series. Further studies showed that the
compounds are not showing any cytotoxicity effect. The docking studies of the compounds as
well as the experimental activities of the compounds correlated well. From the molecular
docking studies, it was observed that the top ranked conformation of all the compounds fit
well in the active site of the homology model of α-glucosidase.
Keywords: 4-Methylbenzimidazol; α-glucosidase inhibitors; antihyperglycemia; structure-
activity relationship, cytotoxicity Docking studies
Corresponding authors:
E-mail: taha_hej@yahoo.com and muhamm9000@puncakalam.uitm.edu.my, Tel: 0060193098141
1

1.
Introduction
Benzimidazole is a fused heterocyclic compound formed by the merger of benzene and
imidazole. This nuclues has a variety of therpeutical potentials which have greatly appealed
the medicinal chemists. Anticancer [1], antiinflammatory [2], antimicrobial [3], antioxidant
and antiglycation [4], α-Glucosidase [5], β-glucuronidase [6], Carbonic anhydrase [7],
antiulcer [8], and antiviral [9] are some of the properties associated with this medicinally
important skeleton. Some anticancer drugs based on benzimidazole, such as Bendamustin
[10], Hoechst 33258 [11], and nocodazole [12] (Figure-1) are in preclinical or clinical trials.
Figure-1. Benzimidazole that had been used in clinical and preclinical stages
α-Glucosidase (EC 3.2.1.20) is a hydrolase enzyme that is found in the the intestinal cells at
the brush border surface. The breakdown of carbohydrates into glucose is aided by this
enzyme by the hydrolysis mechanism, as the human intestine is only capable of absorbing
glucose monomers into the blood stream [13]. Digestion of carbohydrates and processing of
glycoprotiens have essentially dependence upon the enzymatic action of α-glucosidase.
Therefore diseases like diabetes, cancer, and HIV are susceptable to be cured by inhibitors of
α-glucosidase [14-16]. Some of the synthetic α-glucosidase inhibitors (AGI’s), such as
acarbose, miglitol, and voglibose have gained much attention during the last two decades, in
the field of medicinal chemistry [17,18]. These inhibitors deccelerate the digestion process of
oligosaccharides and polysaccharides into coressponding monomers and consequently lower
the glucose level in blood [19]. These inhibitors, in contrast to the other antihyperglycemic
drugs which modulate certain biochemical processes, act locally in the human intestine
[20,21]. However, the clinical use of these is associated with side effects such as abdominal
discomfort, diarrhea, and flatulence [22,23]. Hence, designing of new α-glucosidase inhibitors
with no or minimum side effects is still a reasonable demand. The benzimidazole derivatives
showed α-glucosidase [24] as well as antidiabetic [25] activity, therefore we design this
project to further investigate the potential of benzimidazole as antidiabetic compounds.
2

2.
Results and Discussion
2.1. Chemistry
In continuous effort to discover potent enzyme inhibitors [26], twenty six benzimidazole
derivatives were successfully synthesized and purified. The 3-methyl-o-phenylenediamine is
reacted with various benzaldehydes substituted with hydroxyl group, methyl group, methoxy
group, nitro group, chlorine, fluorine on different carbon. The synthesis of benzimidazoles
occurred via condensation of arylaldehye and 3methyl-phenylenediamine in the presence of
Na2S2O5 as oxidizing reagents in DMF (Scheme-1).
Scheme-1. Synthesis of benzimidazole derivatives (1-27)
The crude products were further recrystallized from methanol to afford pure compounds. The
structures of the benzimidazoles 1-27 were determined using spectroscopic techniques such as
NMR, MS and were further confirmed using CHN analysis.
2.2. Enzyme Inhibitory Studies
Survey on structure-activity relationship (SAR) suggests that the variation of different
substituents on the aryl part at C-2 is responsible for varied level of α-glucosidase inhibitory
potential of 2-aryl-7-methylbenzimidazole 1-27.
Compound 25 (IC50 = 5.30 ± 0.10 µM) showed the highest activity among the series of
twenty-seven analogs of 2-aryl-7-methylbenzimidazoles (1-27). It was found to be one
hundred and sixty two (162) folds more active than the standard drug, acarbose (IC50 = 856.45
± 5.60 µM). The presence of chloro group at C-2′ must have been in best compatibility mode
to interact with the enzyme, α-glucosidase. This inhibitory potential of chloro group at C-2′,
drastically reduced to twenty-eight (28) folds, when the chloro group moved to C-4′, as in
compound 27 (IC50 = 145.28 ± 1.82 µM). Activity was further halved when chloro group
changed its position from C-4′ to C-3′, as in compound 26 (IC50 = 325.02 ± 2.86 µM). This
3

chlorine substitution trend of ortho (C-2′) > para (C-4′) > meta (C-3′), for the activity was
durther supported by the three regio-isomers of fluorinated anolgs, as well. Fluorine at C-2′, as
in compound 8 (IC50 = 34.42 ± 0.35 µM), was found to have seven-folds lesser activity than
its corresponding chlorinated derivative (compound 25). The para (C-4′) fluorinated analog,
as in compound 16 (IC50 = 135.70 ± 1.20 µM), had a drop in activity upto four-folds.
Switching of fluoro group from C-4′ to C-3′, as in compound 9 (IC50 = 165.01 ± 1.28 µM),
resulted with further decline in activity.
Among the monohydroxylated anaologs, same o > p > m activity trend was followed.
Hydroxy at C-2′ as in compound 19 had an IC50 value of 16.10 ± 0.10 µM, which lowered to
six folds upon movement of hydroxyl from C-2′ to C-4′, as in compound 17 (IC50 = 95.60 ±
0.92 µM). Placement of hydroxy group at meta (C-3′), as in compound 18 (IC50 = 325.69 ±
2.68 µM), resulted in decrement of twenty folds activity with respect to its ortho analog
(compound 19).
Dihydroxylation at ortho (C-2′) and para (C-4′), as in compound 2 (IC50 = 60.90 ± 0.55 µM),
showed the activity intermediate to individual ortho (compound 19) and para (compound 17)
anologs of monohydroxy substitutions. Movement of hydroxy from C-4′ to C-3′, as in
compound 15 (IC50 = 85.60 ± 0.75 µM) slightly lowered the activity. Hydroxy at C-5′ from C-
4′, as in compound 13 (IC50 = 125.34 ± 1.06 µM) halved the activity. The presence of two
hydroxyl groups at C-3′ and C-4′, as in compound 10 (IC50 = 25.36 ± 0.14 µM), doubled the
activity with respect to compound 2.
Although merely methoxylation at the aryl part did not show any activity (compounds 1, 22,
and 23) but methoxylation along with hydroxylation showed some inhibitory potentials. The
meta linkage of hydroxy and methoxy, as in compound 20, showed a good inhibitory potential
with IC50 value 29.75 ± 0.19 µM. The activity was dropped to 45.20 ± 0.19 µM for the para
linkage of hydroxy and methoxy, as in compound 14. A further decline in activity was
observed for compound 21 (IC50 = 225.80 ± 2.05 µM), which had hydroxy and methoxy
substituted ortho to each other.
Nitro substitutions at C-3′ (compound 5) or C-4′ (compound 6) did not show any activity,
however the ortho (C-2′) substitution, as in compound 7 (IC50 = 44.62 ± 0.42 µM) showed
nineteen folds better activity than the standard drug, acarbose. Methylation at the aryl part
(compounds 3 and 4) was found to be useless to inhibit the enzymatic action of α-glucosidase.
Among the heterocycles, replacement of pyidines with phenyl (compound 12 and 24) did not
show any activity but thiophene redeemed the activity with IC50 value of 225.05 ± 2.18 µM.
The compounds were not showing any inhibition against cytotoxicity activity.
4

Table-1. α-Glucosidase inhibitory activity of 2-aryl-7-methylbenzimidazoles (1-27)
Compounds
Structures
IC50 ± SEMa [M]
N. A.b,c
1
60.90 ± 0.55
N. A.b,c
2
3
4
5
6
7
8
N. A.b,c
N. A.b,c
N. A.b,c
44.62 ± 0.42
34.42 ± 0.35
5

165.01 ± 1.28
25.36 ± 0.14
9
10
225.05 ± 2.18
N. A.b,c
11
12
125.34 ± 1.06
45.20 ± 0.39
85.60 ± 0.75
13
14
15
135.70 ± 1.20
95.60 ± 0.92
16
17
325.69 ± 2.68
18
6

16.10 ± 0.10
29.75 ± 0.19
225.80 ± 2.05
N. A.b,c
19
20
21
22
N. A.b,c
23
24
25
725.50 ± 3.78
5.30 ± 0.10
325.02 ± 2.86
26
27
145.28 ± 1.82
856.45 ± 5.60
Acarbose
(Standard Drug)
7

SEMa is the Standard Error of the Mean; N. A.b means Not Active, C # the maximum concentration of all
inactive compound was tested upto 1 mM.
2.3. Homology modeling and docking simulation
The crystallographic structure for α-glucosidase enzyme has not been solved up-to yet.
However, only few homology models have been reported [27-30] so, we developed the 3D
model for α-glucosidase by comparative homology modeling technique using the same
etiquette as described by Fazal et al. [31]. The primary sequence of α -glucosidase was
retrieved from UniProt protein resource data bank (http://www.uniprot.org/) under the access
code P53341. Template search was performed by means of MOE Search tools against the
PDB data bank implemented in MOE 2010-11. The 1.30 Å resolving crystallographic
structure of Saccharomyces cerevisiae isomaltase (PDB code 3AJ7) [32] with 72.4% of
sequence identity with the target were selected as the template for modeling. The 3D structure
of α-glucosidase for Saccharomyces cerevisiae was built by means of MOE homology
modeling tools. The developed 3D model was subjected to energy minimization up to 0.05
gradients. Before docking, ligands and protein were prepared using MOE 2010-11. 3D
structure of all synthesized compounds was built using the Molecular Builder program
implemented in MOE. Finally, a database was created in which all the ligands were converted
into their respective 3D structures and this database was used as input file MOE-docking.
Subsequently, the energy of compounds present in the database was minimized up to 0.05
Gradient using MMFF94x force field. Energy minimization of the database was followed by
the preparation of protein for docking purposes. Most macromolecular crystal structures
contain little or no hydrogen coordinate data due to limited resolution and thus protonation
was accomplish prior to docking using Protonate 3D tools implemented in MOE. Protonation
was followed by energy minimization up to 0.05 Gradient using Amber99 force field. The
database was docked into the active site of protein using the Triangular Matching docking
method and 30 conformations of each Ligand protein complex were generated with docking
score (S). Each complex was analyzed for interactions and their 3D images were taken.
2.4. Interaction analysis
Benzimidazole analogs were synthesized and their inhibitory activities were found out
experimentally. The highly potent compounds from the series were selected on the basis of
IC50, which was determined experimentally. From the molecular docking studies, it was
observed that the top ranked conformation of all the compounds fit well in the active site of
8

the homology model of a-glucosidase. Compounds 25, 19, 10, and 20 have best inhibitory
activity with IC50 values 5.30 ± 0.10, 16.10 ± 0.10, 25.36 ± 0.14 and 29.75 ± 0.19 respectively
against α-glucosidase and compound 6 and 12 has no inhibitory activity against α-glucosidase
enzyme. The docking studies of the compounds as well as the experimental activities of the
compounds correlated well.
Compound 25 was found with a best fit in the binding pocket of the α-glucosidase
enzyme with the docking score -8.7211 and have shown good interactions with the active site
residues (Arg 439) of the receptor protein. Arg 439 was observed making two arene-cation
interaction with the π system of the benziimidazole moiety of the compound. The 3D
interactions of the compound with the receptor α-glucosidase protein was shown in the
Figure 2a. The substituted chlorobenzene group showed inertness regarding interaction in
this compound and this might be due to the electron withdrawing inductive effect of chlorine
which deactivte the aromatic benzene ring. In compound 19 the chlorine is replaced by OH
group which is active in bonding formation and thus here this group interacts with the target
protein.
Compound 19 showed good docking score of -8.9503 and Glu 276 formed hydrogen
bonds with the hydrogen atoms of the –OH moiety of the compound. Arg 439 made an arene-
cation interaction with one of the imidazole moiety of the compound. In compound 10 there
are two OH group with the substituted moiety and the thus the number of interactions
increases as shown in Figure 2b.
Figure-2. Docking pose of the compound 25 and 19 in the active site of α-glucosidase
enzyme.
Compound 10 have good IC50 (25.36 ± 0.14) as well as good docking score i-e -11.3224.
This compound had shown four interactions with the Arg 439, Asp 408, Arg 312 and Tyr
9

313 as shown in the figure 3. Arg 312 and Tyr 313 interact with the hydroxyl group of
catechol moiety of the ligand at one end and Asp 408 with the OH of the nearby atom of
catechol group at the other end. Arg 439, additionally, made an arene-cation linkage with the
the π electronic cloud of the same catechol moiety of the compound (Figure 3a). In
compound 20 one of the OH group is replaced by methoxy group which resulted in the non
involvement of the remaining OH group in bonding directly. Compound 20 have good IC50
(29.75 ± 0.19) as well as good docking score i-e -10.1882. This compound had shown three
interactions with the Arg 439 and Phe 177 as shown in the Figure 3b. Arg 439 interacts with
the imidazol and methoxyphenol rings of the ligand via arene-cation bonds. Phe 177 made an
arene- arene linkage with the the π electronic cloud of the benzimidazole moiety of the
compound. From the above theoritical approach we have observed that the interaction
between ligand and protein increases as the polar groups (OH) increase. However their
attachement to the compounds may alter the various other drug like properties i.e. TPSA,
LogS, LogP, Molecular weight and Toxicity as reflected from their grading by experimental
values i.e. IC50. The inactive compounds also showed here a poor behavior in the binding
mode. The poor or no binding interaction of these compounds might be attributed to the less
active substituted groups like NO2 and OCH3. The placement of inactive compound 6 in the
binding pocket of protein is shown in Figure 3c having no interaction.
10

Figure-3. Docking pose of the compound 10, 20 and 06 in the active site of α-glucosidase
enzyme.
3. Conclusion
We had synthesized benzimidazole derivatives in this study and evaluated for α-glucosidase
inhibition. Substituent effect showed that compounds with hydroxyl and halides are capable
of inhibiting the activity at a concentration as low as 16.10 and 5.30 µM, respectively.
Molecular docking studies have correlated well with biological inhibitory assay. Analysis of
the binding mode clearly shows the increase in hydrophilicity of substituents played the most
important role in the activity profile. Further studies on cytotoxicity showed that the
compounds do not exhibit cytotoxicity activity.
4. Experimental
4.1. General
NMR experiments had been carried out using UltraShield Bruker FT NMR 500 MHz.
Electron impact mass spectra (EI-MS) were recorded on a Finnegan MAT-311A, Germany.
CHN analysis was performed on a Carlo Erba Strumentazion-Mod-1106, Italy. Reaction
progress was monitored using thin layer chromatography (TLC) which was performed on pre-
11

coated silica gel aluminum plates (Kieselgel 60, 254, E. Merck, Germany). Chromatograms
were visualized by UV at 254 and 365 nm.
4.2. Assay for α-Glucosidase Inhibitory Activity
The 135 µL of 50 mM phosphate saline buffer pH (6.8), solution of enzyme (20 µL), and 20
µl of test sample with 70% DMSO was added to 96-well plate. The samples were incubated
for 15 minutes. pre- read of the plate was taken after incubation using SpectraMax. substrate
(pNPG) (25 µL) was added before final reading was taken at 400 nm on Spectra Max. The
percent inhibition was calculated using the following formula:
[(absorbance for control - absorbance for sample)/ absorbance for control] x 100%
4.3. Cytotoxicity activity
The Neutral Red cytotoxicity assay is based on the initial protocol described [33] with some
modifications. Briefly, the cells (1x104/well) were seeded in 96-well microtiter plates (Nunc)
and allowed to grow for 24 hours before treatment. After 24 hours of incubation, the cells
were treated with six different concentrations (0.1-100 µg/mL) of test compounds, in three
replicates. The plates were incubated for 72 h at 37°C in a 5% CO2 incubator. A stock
solution was obtained by dissolving the test compounds in DMSO. Further dilution to
different tested concentrations were then carried out ensuring that the final concentration of
DMSO in the test and control wells was not in excess of 1% (v/v). No effect due to the
DMSO was observed. Doxorubicin was used as the positive control. The well containing
untreated cells was the negative control. At the end of the incubation period, the media were
replaced with medium containing 50 µg/mL of Neutral Red. The plates were incubated for
another 3 hours to allow for uptake of the vital dye into the lysosomes of viable and injured
cells. After the incubation period, the media were removed and cells were washed with the
neutral red washing solution. The dye was eluted from the cells by adding 200 µL of Neutral
Red resorb solution and incubated for 30 minutes at room temperature with rapid agitation on
a microtiter plate shaker. Dye absorbance was measured at 540 nm using a spectrophotometer
ELISA plate reader.
4.4. General procedure for the synthesis of compounds 1-27
In a typical reaction, sodium metabisulfite (Na2S2O5) (20 mL) was added to a stirred mixture
of 3-methyl-o-phenylenediamine (2.12 mmol) and substituted aromatic aldehydes (2.16
12

mmol) in N,N-dimethylformamide (DMF) in 100 mL round bottom flask. The reaction
mixture was refluxed for 4 hours at 130°C at atmospheric pressure, the progress of reaction
was monitored by TLC. After completion of reaction, reaction mixture was allowed to cool to
room temperature. Addition of 30 mL of water resulted in precipitation of a solid material.
The solid benzimidazole was filtered and dried overnight. The solid benzimidazole
derivatives obtained inhigh yields. Recrystallization from methanol afforded pure products.
4.4.1. 2-(3′,4′-Dimethoxyphenyl)-7-methyl-1H-benzo[d]imidazole (1)
m.p. 256-257 °C; IR ν cm-1 (KBr disk): 2951 (NH stretch), 1607 (C=N), 1433 (C=C), 1380
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 12.12 (s, 1H, NH), 7.82 (s, 1H), 7.76 (s, 1H), 7.43
(s, 1H), 7.32 (d, 1H, J = 8.0 Hz), 7.14 (d, 1H, J = 8.5 Hz), 7.09 (t, 1H, J = 7.5 Hz), 6.98 (d,
1H, J = 7.5 Hz), 3.90 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 2.58 (s, 3H, CH3), 13C NMR (150
MHz, DMSO-d6): δ 153.88, 151.91, 149.19, 138.45, 137.42, 125.16, 124.84, 124.60,
124.32(s), 120.84, 114.27, 113.23, 109.56, 56.82, 18.13; HREI-MS: m/z calcd for
C16H16N2O2 [M]+ 268.1212; Found 268.1216; Anal. Calcd for C16H16N2O2, C, 71.62; H, 6.01;
N, 10.44; Found C, 71.63; H, 5.99; N, 10.46
4.4.2. 2-(2′,4′-Dihydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (2)
m.p. 248-249 °C; IR ν cm-1 (KBr disk): 3234 (NH stretch), 1603 (C=N), 1483 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 13.39 (s, 1H, NH), 12.90 (s, 1H, OH), 10.03 (s, 1H, OH), 7.95
(d, 1H, J = 11.0 Hz), 7.43 (s, 1H), 7.16 (s, 1H), 7.07 (s, 1H), 6.54 (d, 1H, J = 13.0 Hz), 2.57
13
(s, 3H, CH3), C NMR (150 MHz, DMSO-d6): δ160.52, 158.62, 151.97, 138.25, 137.34 (s),
129.73 (s), 125.09, 124.52, 124.34, 114.38, 109.59, 103.94, 103.03, 19.07 ; HREI-MS: m/z
calcd for C14H12N2O2 [M]+ 240.0899; Found 240.0903; Anal. Calcd for C14H12N2O2, C,
69.99; H, 5.03; N, 11.66; Found C, 70.01; H, 5.04; N, 11.64
4.4.3. 2-(3′-Methylphenyl)-7-methyl-1H-benzo[d]imidazole (3)
m.p. 218-219 °C; IR ν cm-1 (KBr disk): 3168 (NH stretch), 1627 (C=N), 1432 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 12.76 (s, 1H, NH), 8.05 (s, 1H), 8.00 (s, 1H), 7.47 (t, 2H, J =
9.0 Hz), 7.31 (d, 1H, J = 7.5Hz), 7.12 (t, 1H, J = 8.0 Hz), 7.00 (d, 1H, J = 7.5 Hz), 2.58 (s,
13
3H, CH3), 2.51 (s, 3H, CH3), C NMR (150 MHz, DMSO-d6): δ 149.19, 138.45, 137.42,
137.52, 133.48, 131.29, 127.95, 126.50, 125.12, 124.60, 124.28, 124.16, 114.38, 21.25, 18.17;
HREI-MS: m/z calcd for C15H14N2 [M]+ 222.1157; Found 222.1163; Anal. Calcd for
C15H14N2, C, 81.05; H, 6.35; N, 12.60; Found C, 81.04; H, 6.37; N, 12.58
13

4.4.4. 2-(4′-Methylphenyl)-7-methyl-1H-benzo[d]imidazole (4)
m.p. 184-185 °C; IR ν cm-1 (KBr disk): 3623 (NH stretch), 1620 (C=N), 1437 (C=C), 1311
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 12.12 (s, 1H, NH), 8.11 (d, 2H, J = 8.0 Hz), 7.40
(s, 1H), 7.37 (d, 2H, J = 8.0 Hz), 7.10 (t, 1H, J = 7.5 Hz), 7.00 (d, 1H, J = 7.0 Hz), 2.57 (s,
13
3H, CH3), 2.39 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 149.67, 139.81, 138.25,
137.64, 131.17, 129.74, 129.74, 126.89, 126.89, 125.06, 124.60, 124.21, 114.51, 21.02, 18.25;
HREI-MS: m/z calcd for C15H14N2 [M]+ 222.1157; Found 222.1164; Anal. Calcd for
C15H14N2, C, 81.05; H, 6.35; N, 12.60; Found C, 81.04; H, 6.33; N, 12.61
4.4.5. 2-(3′-Nitrophenyl)-7-methyl-1H-benzo[d]imidazole (5)
m.p. 191-192 °C; IR ν cm-1 (KBr disk): 3219 (NH stretch), 1620 (C=N), 1520 (C=C), 1348
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 12.95 (s, 1H, NH), 9.12 (s, 1H), 8.69 (s, 1H), 8.34
(d, 1H, J = 8.0 Hz), 7.88 (t, 1H, J = 8.5Hz), 7.53 (s, 1H), 2.51 (s, 3H, CH3); 13C NMR (150
MHz, DMSO-d6): δ 149.12, 149.05, 138.45, 137.54, 134.63, 133.12, 132.08, 125.06, 124.52,
124.14, 124.39, 121.32, 114.37, 18.21; HREI-MS: m/z calcd for C14H11N3O2 [M]+ 253.0851;
Found 253.0847; Anal. Calcd for C14H11N3O2, C, 66.40; H, 4.38; N, 16.59; Found C, 66.41;
H, 4.37; N, 16.57;
4.4.6. 2-(4′-Nitrophenyl)-7-methyl-1H-benzo[d]imidazole (6)
m.p. 274-275 °C; IR ν cm-1 (KBr disk): 3192 (NH stretch), 1624 (C=N), 1438 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 12.95 (s, 1H, NH), 8.48 (d, 2H, J = 8.5 Hz), 8.44 (d, 2H, J =
9.0 Hz), 7.47 (s, 1H), 7.19 (t, 1H, J = 7.5 Hz), 7.08 (d, 1H, J = 7.5 Hz), 2.61 (s, 3H, CH3); 13C
NMR (150 MHz, DMSO-d6): δ 149.68, 149.13, 138.25, 137.47, 134.86, 127.21, 127.21,
125.72, 125.72, 125.10, 124.68, 124.21, 114.37, 18.25; HREI-MS: m/z calcd for C14H11N3O2
[M]+ 253.0851; Found 253.0859; Anal. Calcd for C14H11N3O2, C, 66.40; H, 4.38; N, 16.59;
Found C, 66.38; H, 4.39; N, 16.61
4.4.7. 2-(2′-Nitrophenyl)-7-methyl-1H-benzo[d]imidazole (7)
m.p. 259-261 °C; IR ν cm-1 (KBr disk): 3297 (NH stretch), 1617 (C=N), 1439 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 11.76 (s, 1H, NH), 8.20 (t, 1H, J = 1.5 Hz), 7.63 – 7.58 (m,
1H), 7.48 – 7.36 (m, 3H), 7.19 (t, 1H, J = 7.5 Hz), 7.06 (d, 1H, J = 11.5 Hz), 2.60 (s, 3H,
13
CH3); C NMR (150 MHz, DMSO-d6): δ 150.18, 145.62, 138.31, 137.48, 135.29, 132.21,
129.46, 128.05, 125.10, 124.72, 124.47, 124.36, 114.28, 18.27; HREI-MS: m/z calcd for
14

C14H11N3O2 [M]+ 253.0851; Found 253.0856; Anal. Calcd for C14H11N3O2, C, 66.40; H, 4.38;
N, 16.59; Found C, 66.38; H, 4.37; N, 16.58
4.4.8. 2-(2′-Fluorophenyl)-7-methyl-1H-benzo[d]imidazole (8)
m.p. 144-145 °C; IR ν cm-1 (KBr disk): 3097 (NH stretch), 1603 (C=N), 1439 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 12.12 (s, 1H, NH), 8.05 (d, 1H, J = 7.0 Hz), 8.00 (d, 1H, J =
7.5 Hz), 7.62 (m, 1H), 7.42 (s, 1H), 7.35 (dt, 1H, J = 2.5 Hz, 6.0 Hz), 7.14 (t, 1H, J = 7.5 Hz),
7.03 (d, 1H, J = 7.5 Hz), 2.57 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 161.49 (d, J
= 262.3 Hz), 147.55, 147.47, 138.36, 137.42, 130.60, 128.82, 126.19, 124.87, 124.65, 124.41,
120.18, 117.68, 114.34, 18.19; HREI-MS: m/z calcd for C14H11FN2 [M]+ 226.0906; Found
226.0912; Anal. Calcd for C14H11FN2, C, 74.32; H, 4.90; N, 12.38; Found C, 74.33; H, 4.91;
N, 12.37
4.4.9. 2-(3′-Fluorophenyl)-7-methyl-1H-benzo[d]imidazole (9)
m.p. 174-175 °C; IR ν cm-1 (KBr disk): 3097 (NH stretch), 1612 (C=N), 1426 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 9.81 (s, 1H, NH), 8.39 (s, 1H, OH), 7.64 (d, 1H, J = 2.5 Hz),
7.54 (dd, 1H, J = 1.5 Hz, 1.5 Hz), 7.44 (d, 1H, J = 8.0 Hz), 7.21 (s, 1H), 7.12 (s, 1H), 6.96 (d,
3H, J = 8 Hz), 2.58 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 164.26 (d, J = 261.3
Hz), 149.17, 138.34, 137.47, 131.68, 130.54, 125.09, 124.52, 124.32, 120.18, 114.86, 114.32,
112.49, 18.14; HREI-MS: m/z calcd for C14H11FN2 [M]+ 226.0906; Found 226.0899; Anal.
Calcd for C14H11FN2, C, 74.32; H, 4.90; N, 12.38; Found C, 74.34; H, 4.89; N, 12.36
4.4.10. 2-(3′,4′-dihydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (10)
m.p. 251-253 °C; IR ν cm-1 (KBr disk): 3098 (NH stretch), 1614 (C=N), 1445 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 9.57 (s, 1H, NH), 8.07 (dd, 1H, J = 1.0 Hz, 1.0 Hz), 8.00 (d,
2H, J = 7.5 Hz), 7.90 (m, 1H), 7.79 (m, 1H), 7.43 (m, 1H), 7.16 (t, 1H, J = 7.0 Hz), 7.06 (d,
13
1H, J = 7.5 Hz), 2.73 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 148.96, 147.61,
138.36, 137.47, 125.09, 124.52, 124.21, 120.51, 119.18, 115.29, 114.34, 113.18, 18.29;
HREI-MS: m/z calcd for C14H12N2O2 [M]+ 240.0899; Found 240.0904; Anal. Calcd for
C14H12N2O2, C, 69.99; H, 5.03; N, 11.66; Found C, 70.01; H, 5.01; N, 11.64
4.4.11. 7-methyl-2-(thiophen-2′-yl)-1H-benzo[d]imidazole (11)
m.p. 278-279 °C; IR ʋ cm-1 (KBr disk): 3065 (NH stretch), 1567 (C=N), 1423 (C=C), 1311
(C-N), 718 (C-S); 1H NMR (500 MHz, DMSO-d6): δ 12.83 (s, 1H, NH), 7.94 (s, 1H), 7.72 (d,
15

1H, J = 4.0 Hz), 7.42 (s, 1H), 7.23 (s, 1H), 7.09 (d, 1H, J = 8.0 Hz), 6.99 (s, 1H), 2.55 (s, 3H,
CH3); 13C NMR (150 MHz, DMSO-d6): δ 143.95, 139.82, 138.34, 137.42, 130.14, 127.69,
127.39, 124.87, 124.67, 124.21, 114.33, 18.27; HREI-MS: m/z calcd for C12H10N2S [M]+
214.0565; Found 214.0565; Anal. Calcd for C12H10N2S, C, 67.26; H, 4.70; N, 13.07; Found
C, 67.25; H, 4.71; N, 13.08
4.4.12. 7-methyl-2-(pyridin-3′-yl)-1H-benzo[d]imidazole (12)
m.p. 234-235 °C; IR ν cm-1 (KBr disk): 3092 (NH stretch), 1618 (C=N), 1437 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 12.99 (s, 1H, NH), 9.37 (s, 1H), 8.69 (d, 1H, J = 3.5 Hz), 8.54
(d, 1H, J = 7.0 Hz), 7.61 (t, 1H, J = 4.5 Hz), 7.48 (s, 1H), 7.15 (t, 1H, J = 7.5 Hz), 7.05 (d,
1H, J = 7.0 Hz), 2.60 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 150.02 (s), 149.83
(s), 149.52 (s), 138.45 (s), 137.34 (s), 135.64 (s), 132.13 (s), 128.09 (s), 124.92 (s), 124.42 (s),
124.11 (s), 114.30 (s), 18.27; HREI-MS: m/z calcd for C13H11N3 [M]+ 209.0953; Found
209.0948; Anal. Calcd for C13H11N3, C, 74.62; H, 5.30; N, 20.08; Found C, 74.60; H, 5.31; N,
20.10
4.4.13. 2-(2′,5′-dihydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (13)
m.p. 236-238 °C; IR ν cm-1 (KBr disk): 3082 (NH stretch), 1623 (C=N), 1449 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 9.19 (s, 1H, NH), 7.51 (s, 1H), 7.26 (s, 1H), 7.17 (s, 1H), 6.92
13
(s, 2H), 2.60 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 152.68, 152.24, 151.41,
138.28, 137.53, 125.12, 124.71, 124.20, 118.97, 116.73, 116.08, 114.39, 114.28, 18.32;
HREI-MS: m/z calcd for C14H12N2O2 [M]+ 240.0899; Found 240.0910; Anal. Calcd for
C14H12N2O2, C, 69.99; H, 5.03; N, 11.66; Found C, 69.97; H, 5.05; N, 11.64
4.4.14. 2-(2′-hydroxy-5′-methoxyphenyl)-7-methyl-1H-benzo[d]imidazole (14)
m.p. 264-266 °C; IR ν cm-1 (KBr disk): 3085 (NH stretch), 1601 (C=N), 1442 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 11.89 (s, 1H, NH), 7.68 (s, 1H), 7.48 (d, 1H, J = 8.0 Hz), 7.21
(t, 1H, J = 7.5 Hz), 7.10 (d, 1H, J = 7.5 Hz), 7.01 (d, 1H, J = 6.0 Hz), 6.98 (d, 1H, J = 9.0 Hz),
3.80 (s, 3H, OCH3), 2.58 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 155.68, 153.20,
151.47, 138.25, 137.54, 125.08, 124.63, 124.41, 117.13, 115.92, 114.79, 114.21, 113.15,
56.03, 18.25; HREI-MS: m/z calcd for C15H14N2O2 [M]+ 254.1055; Found 254.1048; Anal.
Calcd for C15H14N2O2, C, 70.85; H, 5.55; N, 11.02; Found C, 70.83; H, 5.57; N, 11.03
16

4.4.15. 2-(2′,3′-dihydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (15)
m.p. 258-259 °C; IR ν cm-1 (KBr disk): 3097 (NH stretch), 1613 (C=N), 1435 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 13.30 (s, 1H, OH), 12.12 (s, 1H, NH), 9.30 (s, 1H, OH), 7.55
(d, 2H, J = 7.5 Hz), 7.50 (d, 1H, J = 7.5 Hz), 7.25 (t, 1H, J = 7.5 Hz), 7.15 (d, 1H, J = 7.0 Hz),
6.96 (d, 1H, J = 7.5 Hz), 6.87 (t, 1H, J = 8.0 Hz), 2.60 (s, 3H, CH3); 13C NMR (150 MHz,
DMSO-d6): δ 151.92, 148.09, 147.56, 138.35, 137.44, 125.00, 124.62, 124.31, 122.19,
120.95, 117.01, 114.31, 112.39, 18.23; HREI-MS: m/z calcd for C14H12N2O2 [M]+ 240.0899;
Found 240.0892; Anal. Calcd for C14H12N2O2, C, 69.99; H, 5.03; N, 11.66; Found C, 70.01;
H, 5.02; N, 11.65
4.4.16. 2-(4′-Fluorophenyl)-7-methyl-1H-benzo[d]imidazole (16)
m.p. 142-143 °C; IR ν cm-1 (KBr disk): 3119 (NH stretch), 1608 (C=N), 1443 (C=C), 1316
(C-N), 1234 (C-F); 1H NMR (500 MHz, DMSO-d6): δ 12.75 (s, 1H, NH), 8.27 (d, 1H, J = 6.0
Hz), 8.25 (d, 1H, J = 5.5 Hz), 7.42 (t, 3H, J = 9.0 Hz), 7.11 (t, 1H, J = 7.5 Hz), 7.01 (d, 1H, J
= 7.0 Hz), 2.57 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 165.89 (d, J = 262.3 Hz),
149.76, 138.25, 137.47, 129.25, 129.25, 128.69, 125.13, 124.67, 124.21, 116.14, 116.14,
114.51, 18.21; HREI-MS: m/z calcd for C14H11FN2 [M]+ 226.0906; Found 226.0913; Anal.
Calcd for C14H11FN2, C, 74.32; H, 4.90; N, 12.38; Found C, 74.34; H, 4.91; N, 12.36
4.4.17. 2-(4′-Hydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (17)
m.p. 246-248 °C; IR ν cm-1 (KBr disk): 3271 (NH stretch), 1597 (C=N), 1492 (C=C), 1375
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 13.07 (s, 1H, OH), 11.63 (s, 1H, NH), 8.16 (d, 2H,
J = 9.0 Hz), 7.42 (d, 1H, J = 8.0 Hz), 7.18 (s, 1H), 7.15 (d, 1H, J = 8.5 Hz), 7.09 (d, 1H, J =
13
7.5 Hz), 2.47 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 160.71, 149.76, 138.32,
137.48, 126.98, 126.98, 124.79, 124.42, 124.28, 120.92, 116.43, 116.43, 114.28, 18.28;
HREI-MS: m/z calcd for C14H12N2O [M]+ 224.0950; Found 224.0946; Anal. Calcd for
C14H12N2O, C, 74.98; H, 5.39; N, 12.49; Found C, 74.97; H, 5.38; N, 12.47
4.4.18. 2-(3′-Hydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (18)
m.p. 239-241 °C; IR ν cm-1 (KBr disk): 3274 (NH stretch), 1593 (C=N), 1494 (C=C), 1382
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 12.92 (s, 1H, OH), 11.91 (s, 1H, NH), 7.86 – 7.84
(m, 2H), 7.53 (t, 1H, J = 8.0 Hz), 7.47 (d, 1H, J = 8.0 Hz), 7.22 (t, 1H, J = 7.5 Hz), 7.17 (s,
1H), 2.62 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 157.78 (s), 149.19 (s), 138.36 (s),
137.48 (s), 132.26 (s), 130.84 (s), 125.09 (s), 124.72 (s), 124.41 (s), 119.25 (s), 117.09 (s),
17

114.41 (s), 114.34 (s), 18.29; HREI-MS: m/z calcd for C14H12N2O [M]+ 224.0950; Found
224.0942; Anal. Calcd for C14H12N2O, C, 74.98; H, 5.39; N, 12.49; Found C, 74.99; H, 5.37;
N, 12.51
4.4.19. 2-(2′-Hydroxyphenyl)-7-methyl-1H-benzo[d]imidazole (19)
m.p. 224-225 °C; IR ν cm-1 (KBr disk): 3272 (NH stretch), 1596 (C=N), 1490 (C=C),
1388.83 (C-N); 1H NMR (500 MHz, DMSO-d6): δ 13.16 (s, 1H, NH), 8.05 (s, 1H), 7.44 (s,
1H), 7.40 (t, 1H, J = 7.0 Hz), 7.20 (t, 1H, J = 7.5 Hz), 7.10 (d, 1H, J = 7.5 Hz), 7.05 (t, 2H, J
= 8.5 Hz), 2.59 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 159.62, 152.13, 138.38,
137.24, 131.09, 129.75, 125.13, 124.52, 124.21, 121.01, 118.52, 114.30, 111.36, 18.21;
HREI-MS: m/z calcd for C14H12N2O [M]+ 224.0950; Found 224.0958; Anal. Calcd for
C14H12N2O, C, 74.98; H, 5.39; N, 12.49; Found C, 75.02; H, 5.41; N, 12.52
4.4.20. 2-(2′-Hydroxy-4′-methoxyphenyl)-7-methyl-1H-benzo[d]imidazole (20)
m.p. 208-209 °C; IR ν cm-1 (KBr disk): 3302 (NH stretch), 1637 (C=N), 1407 (C=C), 1288
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 13.20 (s, 1H, NH), 8.04 (d, 1H, J = 8.0 Hz), 7.46
(d, 1H, J = 7.5 Hz), 7.20 (t, 1H, J = 7.5 Hz), 7.10 (d, 1H, J = 7.0 Hz), 6.67 (d, 1H, J = 7.2 Hz),
13
6.61 (s, 1H), 3.82 (s, 3H, OCH3), 2.58 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ
161.56, 160.33, 152.13, 138.25, 137.47, 130.08, 125.10, 124.69, 124.21, 114.33, 108.05,
107.58, 102.81, 56.13, 18.33; HREI-MS: m/z calcd for C15H14N2O2 [M]+ 254.1055; Found
254.1063; Anal. Calcd for C15H14N2O2, C, 70.85; H, 5.55; N, 11.02; Found C, 70.82; H, 5.57;
N, 11.03
4.4.21. 2-(3′-Hydroxy-4′-methoxyphenyl)-7-methyl-1H-benzo[d]imidazole (21)
m.p. 261-263 °C; IR ν cm-1 (KBr disk): 3092 (NH stretch), 1602 (C=N), 1437 (C=C);7 1H
NMR (500 MHz, DMSO-d6): δ 9.39 (s, 1H, NH), 7.67 (s, 1H), 7.66 (d, 1H, J = 8.5 Hz), 7.43
(d, 1H, J = 8.0 Hz), 7.19 (d, 1H, J = 7.5 Hz), 7.16 (d, 1H, J = 8.5 Hz), 7.09 (d, 1H, J = 7.0
13
Hz), 3.88 (s, 3H, OCH3), 2.58 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 149.58,
149.08, 148.11, 138.25, 137.46, 125.01, 124.84, 124.52, 124.21, 119.09, 115.16, 114.30,
110.32, 56.71, 18.14; HREI-MS: m/z calcd for C15H14N2O2 [M]+ 254.1055; Found 254.1061;
Anal. Calcd for C15H14N2O2, C, 70.85; H, 5.55; N, 11.02; Found C, 70.86; H, 5.54; N, 11.01
4.4.22. 2-(3′-Methoxyphenyl)-7-methyl-1H-benzo[d]imidazole (22)
18

m.p. 242-243 °C; IR ν cm-1 (KBr disk): 3082 (NH stretch), 1607 (C=N), 1497 (C=C), 1251
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 11.89 (s, 1H, NH), 7.82 – 7.80 (m, 2H), 7.56 (t,
1H, J = 8.0 Hz), 7.51 (d, 1H, J = 8.0 Hz), 7.26 (t, 1H, J = 7.5 Hz), 7.19 – 7.15 (m, 1H), 3.90
13
(s, 3H, OCH3), 2.62 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 162.59, 149.19,
138.37, 137.46, 131.67, 129.02, 125.07, 124.52, 124.30, 119.41, 114.51, 114.27, 114.01,
56.13, 18.25; HREI-MS: m/z calcd for C15H14N2O [M]+ 238.1106; Found 238.1109; Anal.
Calcd for C15H14N2O, C, 75.61; H, 5.92; N, 11.76; Found C, 75.59; H, 5.90; N, 11.78
4.4.23. 2-(4′-Methoxyphenyl)-7-methyl-1H-benzo[d]imidazole (23)
m.p. 172-174 °C; IR ν cm-1 (KBr disk): 3197 (NH stretch), 1627 (C=N), 1419 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 12.53 (s, 1H, NH), 8.18 (d, 2H, J = 9.0 Hz), 7.46 (d, 1H, J =
8.0 Hz), 7.21 (s, 1H), 7.19 (d, 1H, J = 8.5 Hz), 7.11 (d, 1H, J = 7.5 Hz), 3.87 (s, 3H, OCH3),
13
2.59 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 161.69, 149.46, 138.36, 137.49,
126.79, 126.79, 125.43, 124.97, 124.42, 123.31, 114.85, 114.11, 114.11, 56.02, 18.13; HREI-
MS: m/z calcd for C15H14N2O [M]+ 238.1106; Found 238.1158; Anal. Calcd for C15H14N2O,
C, 75.61; H, 5.92; N, 11.76; Found C, 75.63; H, 5.91; N, 11.77
4.4.24. 7-Methyl-2-(pyridin-4′-yl)-1H-benzo[d]imidazole (24)
m.p. 164-165 °C; IR ν cm-1 (KBr disk): 3107 (NH stretch), 1608 (C=N), 1443 (C=C), 1315
(C-N); 1H NMR (500 MHz, DMSO-d6): δ 13.16 (s, 1H, NH), 8.76 (d, 2H, J = 5.5 Hz), 8.17
(s, 1H), 8.10 (s, 1H), 7.54 (s, 1H), 7.17 (s, 1H), 7.06 (s, 1H), 2.60 (s, 3H, CH3); 13C NMR
(150 MHz, DMSO-d6): δ 150.68, 150.37, 149.52, 140.25, 138.31, 137.27, 125.17, 124.67,
124.19, 123.43, 123.81, 114.40, 18.17; HREI-MS: m/z calcd for C13H11N3 [M]+ 209.0953;
Found 209.0957; Anal. Calcd for C13H11N3, C, 74.62; H, 5.30; N, 20.08; Found C, 74.61; H,
5.32; N, 20.10
4.4.25. 2-(2′-Chlorophenyl)-7-methyl-1H-benzo[d]imidazole (25)
m.p. 227-229 °C; IR ν cm-1 (KBr disk): 3092 (NH stretch), 1606 (C=N), 1433 (C=C); 1H
NMR (500 MHz, DMSO-d6): δ 11.58 (s, 1H, NH), 7.86 (d, 1H, J = 7.0 Hz), 7.67 (d, 1H, J =
8.0 Hz), 7.58 – 7.52 (m, 1H), 7.45 (d, 1H, J = 8.0 Hz), 7.16 (t, 1H, J = 8.0 Hz), 7.05 (d, 1H, J
= 7.0 Hz), 2.56 (s, 3H, CH3); 13C NMR (150 MHz, DMSO-d6): δ 147.18, 138.39, 137.47,
132.10, 131.09, 130.73, 130.58, 128.09, 127.66, 124.90, 124.67, 124.32, 114.20, 18.31;
HREI-MS: m/z calcd for C14H11ClN2 [M]+ 242.0611; Found 242.0617; Anal. Calcd for
C14H11ClN2, C, 69.28; H, 4.57; N, 11.54; Found C, 69.30; H, 4.59; N, 11.51
19

4.4.26. 2-(3′-Chlorophenyl)-7-methyl-1H-benzo[d]imidazole (26)
m.p. 214-216 °C; IR ν cm-1 (KBr disk): 3092 (NH stretch), 1612 (C=N), 1441 (C=C); 1H
NMR (5MHz, DMSO-d6): δ 11.84 (s, 1H, NH), 8.29 (s, 1H), 8.19 (d, 1H, J = 6.0 Hz), 7.64 (d,
2H, J = 6.5 Hz), 7.49 (d, 1H, J = 8.0 Hz), 7.22 (t, 1H, J = 7.5 Hz), 7.12 (d, 1H, J = 7.5 Hz),
13
2.60 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 149.07, 138.25, 137.47, 133.78,
132.17, 130.56, 128.41, 128.10, 125.79, 125.03, 124.72, 124.30, 114.51, 18.14; HREI-MS:
m/z calcd for C14H11ClN2 [M]+ 242.0611; Found 242.0614; Anal. Calcd for C14H11ClN2, C,
69.28; H, 4.57; N, 11.54; Found C, 69.27; H, 4.56; N, 11.55
4.4.27. 2-(4′-Chlorophenyl)-7-methyl-1H-benzo[d]imidazole (27)
m.p. 144-145 °C; IR ν cm-1 (KBr disk): 3097 (NH stretch), 1603 (C=N), 1439 (C=C),
1095.34 (C-N); 1H NMR (500 MHz, DMSO-d6): δ 11.35 (s, 1H, NH), 8.24 (d, 2H, J = 7.0
Hz), 7.69 (d, 2H, J = 8.5 Hz), 7.47 (d, 1H, J = 8.0 Hz), 7.20 (t, 1H, J = 7.5 Hz), 7.10 (d, 1H, J
13
= 7.5 Hz), 2.59 (s, 3H, CH3); C NMR (150 MHz, DMSO-d6): δ 149.67, 138.45, 137.34,
134.57, 134.29, 129.35, 129.35, 128.54, 128.54, 125.80, 124.65, 124.34, 114.41, 18.21;
HREI-MS: m/z calcd for C14H11ClN2 [M]+ 242.0611; Found 242.0607; Anal. Calcd for
C14H11ClN2, C, 69.28; H, 4.57; N, 11.54; Found C, 69.29; H, 4.55; N, 11.56
Acknowledgements
Authors would like to acknowledge The Ministry of Agriculture (MOA) Malaysia and
Universiti Teknologi MARA for the financial support under MOA grant file No. 100-RMI/
MOA 16/6/2 (1/2013) and Atta-ur-Rahman Institute for Natural product Discovery (RiND) to
provide excellent designed lab and facility for the research and all technical and non-technical
staff for a lot of support for this work.
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Graphical Abstract
Synthesis, α-Glucosidase inhibitory, Cytotoxicity and
Docking Studies of 2-aryl-7-methylbenzimidazoles
Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Syahrul Imrana,b, Muhammd Helmi Bin Mohamada,b,
Abdul wadoodc, Fazal Rahimc, Syed Muhammad Saadd, Ashfaq ur Rehmanc, Khalid Mohammed Khand
aAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam
Campus, 42300, Bandar Puncak Alam, Selangor, Malaysia.
bFaculty of Applied Science Universiti Teknologi MARA (UiTM), 40450, Shah Alam, Selangor, Malaysia.
cDepartment of Chemistry, Hazara University, Mansehra-21120, Khyber Pukhtunkhwa, Pakistan
dH. E. J. Research Institute of Chemistry, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi-75270, Pakistan.
Synthesis of benzimidazole derivatives (1-27) as α-glucosidase inhibitor

Research Highlights
 Synthesis of 27 benzimidazole derivatives
 In-vitro α-glucosidase inhibitory activity
 Cytotoxicity
 Docking studies were carried out to confirm binding of active compounds with enzyme
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