Discovery of amide-functionalized benzimidazolium salts as potent α-glucosidase inhibitors

Article abstract of DOI:10.3390/molecules26164760

α-Glucosidase inhibitors (AGIs) are used as medicines for the treatment of diabetes mellitus. The α-Glucosidase enzyme is present in the small intestine and is responsible for the breakdown of carbohydrates into sugars. The process results in an increase in blood sugar levels. AGIs slow down the digestion of carbohydrates that is helpful in controlling the sugar levels in the blood after meals. Among heterocyclic compounds, benzimidazole moiety is recognized as a potent bioactive scaffold for its wide range of biologically active derivatives. The aim of this study is to explore the α-glucosidase inhibition ability of benzimidazolium salts. In this study, two novel series of benzimidazolium salts, i.e., 1-benzyl-3-{2-(substituted) amino-2-oxoethyl}-1H-benzo[d]imidazol-3-ium bromide 9a–m and 1-benzyl-3-{2-substituted) amino-2-oxoethyl}-2-methyl-1H-benzo[d] imidazol-3-ium bromide 10a–m were screened for their in vitro α-glucosidase inhibitory potential. These compounds were synthesized through a multistep procedure and were characterized by¹H-NMR,¹³C-NMR, and EI-MS techniques. Compound 10d was identified as the potent α-glucosidase inhibitor among the series with an IC₅₀ value of 14 ± 0.013 μM, which is 4-fold higher than the standard drug, acarbose. In addition, compounds 10a, 10e, 10h, 10g, 10k, 10l, and 10m also exhibited pronounced potential for α-glucosidase inhibition with IC₅₀ value ranging from 15 ± 0.037 to 32.27 ± 0.050 μM when compared with the reference drug acarbose (IC₅₀ = 58.8 ± 0.12 μM). A molecular docking study was performed to rationalize the binding interactions of potent inhibitors with the active site of the α-glucosidase enzyme.

Full text of DOI:10.3390/molecules26164760

molecules
Article
Discovery of Amide-Functionalized Benzimidazolium Salts as
Potent α-Glucosidase Inhibitors
Imran Ahmad Khan 1, Matloob Ahmad 1,
* *
, Usman Ali Ashfaq 2, Sadia Sultan 3,4,* and Magdi E.A. Zaki 5,
1
Department of Chemistry, Government College University, Faisalabad 38000, Pakistan;
imrankhan707470@gmail.com
2
3
4
5
Department of Bioinformatics and Biotechnology, Government College University,
Faisalabad 38000, Pakistan; ashfaqua@gcuf.edu.pk
Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus,
Bandar Puncak Alam 42300, Selangor Darul Ehsan, Malaysia
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA,
Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor Darul Ehsan, Malaysia
Department of Chemistry, Faculty of Science, Imam Mohammad Ibn Saud Islamic University (IMSIU),
Riyadh 11623, Saudi Arabia
*
Correspondence: Matloob.ahmad@gcuf.edu.pk (M.A.); drsadia@uitm.edu.my (S.S.);
mezaki@imamu.edu.sa (M.E.A.Z.)
Abstract: α-Glucosidase inhibitors (AGIs) are used as medicines for the treatment of diabetes mellitus.
The -Glucosidase enzyme is present in the small intestine and is responsible for the breakdown of
α
carbohydrates into sugars. The process results in an increase in blood sugar levels. AGIs slow down
the digestion of carbohydrates that is helpful in controlling the sugar levels in the blood after meals.
Among heterocyclic compounds, benzimidazole moiety is recognized as a potent bioactive scaffold for
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
its wide range of biologically active derivatives. The aim of this study is to explore the
inhibition ability of benzimidazolium salts. In this study, two novel series of benzimidazolium
salts, i.e., 1-benzyl-3-{2-(substituted) amino-2-oxoethyl}-1H-benzo[d]imidazol-3-ium bromide 9a
and 1-benzyl-3-{2-substituted) amino-2-oxoethyl}-2-methyl-1H-benzo[d] imidazol-3-ium bromide
α-glucosidase
ꢀꢁꢂꢃꢄꢅꢆ
Citation: Khan, I.A.; Ahmad, M.;
Ashfaq, U.A.; Sultan, S.; Zaki, M.E.A.
Discovery of Amide-Functionalized
Benzimidazolium Salts as Potent
α-Glucosidase Inhibitors. Molecules
2021, 26, 4760. https://doi.org/
10.3390/molecules26164760
–m
10a
–
m
were screened for their in vitro α-glucosidase inhibitory potential. These compounds were
1
synthesized through a multistep procedure and were characterized by H-NMR, 13C-NMR, and
EI-MS techniques. Compound 10d was identified as the potent
series with an IC50 value of 14 0.013 M, which is 4-fold higher than the standard drug, acarbose.
In addition, compounds 10a 10e 10h 10g 10k 10l, and 10m also exhibited pronounced potential
for -glucosidase inhibition with IC50 value ranging from 15 0.037 to 32.27 0.050 M when
compared with the reference drug acarbose (IC50 = 58.8 0.12 M). A molecular docking study
α-glucosidase inhibitor among the
±
µ
,
,
,
,
,
Academic Editor: René Csuk
α
±
±
µ
±
µ
Received: 30 June 2021
Accepted: 1 August 2021
Published: 6 August 2021
was performed to rationalize the binding interactions of potent inhibitors with the active site of the
α-glucosidase enzyme.
Keywords: benzimidazole derivatives; benzimidazolium salts; molecular docking; anti-diabetic
studies; α-glucosidase inhibition
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
The benzimidazole ring system has established its reputation amongst heterocyclic
templates for its biologically active derivatives. Benzimidazole derivatives have displayed
Copyright:
© 2021 by the authors.
exceptional clinical and biological applications, such as anticancer [
antimalarial [ ], antibacterial [ ], anti-HIV [ ], anti-inflammatory [ ], antioxidant [
tiprotozoal [ ], and enzyme inhibitory activities [ ]. In addition, it serves as an essential
1
], antifungal [
2],
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
3
8
4
5
6
7], an-
9
component of various commercially available biocides [10,11].
Benzimidazolium salts (derived from benzimidazole) have been extensively explored
the for design and discovery of new drugs [12
–14]. Particularly, in organometallic chemistry,
Molecules 2021, 26, 4760. https://doi.org/10.3390/molecules26164760
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4760
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benzimidazolium salts have emerged as an influential class of ligands due to strong
σ
-
donor and weak
π
-accepting ability. Besides multiple synthetic and catalytic applications,
benzimidazolium salts are also proven as effective antibacterial
1 [15], anticancer 2 [16],
antimalarial, and anti-leishmanial agents 3 [17,18] (Figure 1).
Figure 1. The chemical structures of biologically active benzimidazolium salts.
Diabetes mellitus is a prevailing disorder, arisen due to a high level of blood glucose.
-Glucosidase enzyme is responsible for the lysis of -glucopyranoside bonds in dis-
The
α
α
accharides and oligosaccharides to form monosaccharides, ultimately regulating the blood
glucose availability and postprandial hyperglycemia in type-2 diabetic patients [19]. The
role of
sugar-lowering agents [20]. At present, a number of
bose, voglibose, and miglitol have been discovered [21]. These
capable to interrupt the hydrolysis of carbohydrates and thus, reduce postprandial blood
glucose levels [22 23]. However, continuous intake of these drugs may cause severe side
effects such as diarrhea, body pain, and gastrointestinal disorders [24 25]. Subsequently,
-glucosidase inhibitors associated with minimum side effects is
α
-glucosidase inhibitors is to reduce hyperglycemia, thus considered as first-line
-glucosidase inhibitors such as acar-
-glucosidase inhibitors are
α
α
,
,
the development of new
the need of the time.
α
Over the recent years, heterocyclic compounds have been extensively studied as
-glucosidase inhibitors [26 28]. More specifically, the benzimidazole ring system has
emerged as an efficient template for new and potential anti-diabetic agents [29 31]. Benzim-
idazolium salts having -glucosidase inhibitory potential have appeared in a few literature
reports [32]. To the best of our knowledge, this research work is among the few successful
efforts to explore benzimidazolium salts as -glucosidase inhibitors. Keeping in view with
α
–
–
α
α
evidence from the literature for the biological activities of benzimidazolium salts, we have
synthesized a library of novel 1,3-disubstituted benzimidazolium salts and have screened
them for α-glucosidase inhibition studies. Furthermore, the structure-activity relationship
has been established with the help of molecular docking studies.
2. Results and Discussion
2.1. Chemistry
Twenty-six benzimidazolium salts 9(a–m) and 10(a–m) bearing amide functionality
have been synthesized by following the methodology as illustrated in Scheme 1. It in-
volves the reaction of o-phenylenediamine and respective carboxylic acid for 3 to 5 h to
give 1H-benzimidazole and 2-methyl-1H-benzimidazole. These precursor benzimidazoles
were treated with benzyl chloride in the presence of a base and using acetonitrile as a
solvent to form corresponding N-benzylatedbenzimidazoles in 65–85% yields. Quater-
nization of the synthesized N-benzylbenzimidazole precursors with a range of substituted
α
-bromoacetamides resulted in the formation of targeted benzimidazolium salts 9(a–m)

Molecules 2021, 26, 4760
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and 10
(
a–
m
). The chemical reaction was monitored by TLC using chloroform: methanol
(4:1) mixture as a mobile phase. After completion of the reaction, products were filtered,
washed, and purified using column chromatography.
Scheme 1. The lay-out for the synthesis of targeted benzimidazolium salts.
2.2. Characterization
The formation of benzimidazolium salts was confirmed by 1H-NMR, 13C-NMR, and
mass spectrometry (Supplementary Materials File). A characteristic sharp singlet between
9.91 and 10.3 ppm is observed for NCHN protons (imidazole ring) of compounds 9a–m.
For all benzimidazolium salts, protons of the methylene group attached to phenyl (–CH2–
Ph) resonated at 5.51–5.86 ppm as a sharp singlet whereas the occurrence of another
slightly downfield singlet in the range of 5.66–6.00 ppm is integrated for two protons of
methylene linker of acetamide moiety. The acetanilide NH in benzimidazolium bromides
appeared as an isolated singlet at 10.0–11.78 ppm. In 1H-NMR spectra of 9i
, 9j, 9k, 10i, 10j,
and 10k, a singlet peak in the range of 2.25–2.30 ppm confirmed the presence of methyl
substituent. Similarly, the appearance of a singlet at 3.71–3.91ppm corresponded to the
methoxy group of compounds 9l
, 9m, 10l, and 10m. Aromatic protons of benzimidazolium
salts 9(a–m) and 10
(a–m
) were detected in the range of 6.90–8.61ppm. In the case of 13C-
NMR spectra, signals of methylene carbons and carbonyl carbons were observed in the
range of 49–52 ppm and 162.6–166 ppm respectively. Furthermore, mass spectrometry data
also confirmed the formation of title compounds. The NMR data of similar functionalities
in the literature [33,34] was found supportive of our findings.
2.3. In Silico Studies (Molecular Docking)
The in silico techniques have established their reputation in the field of drug discovery.
The study is helpful in identifying the potential biologically active compounds as the targets
for synthesis, thus minimizing the hardships of laborious synthetic work. Considering
the effectiveness of this tool, we executed the molecular docking studies of a library of

Molecules 2021, 26, 4760
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benzimidazolium salts. Molecular docking was performed with the help of the MOE
software to screen out the compounds having the best residual interactions with the
receptor protein. Compounds were ranked on the basis of docking scores, rmsd values,
and interactions with the selected pocket of the receptor enzyme. Results of molecular
docking are presented in Figures 2 and 3 and Table 1.
Thr
204
Trp
406
Arg
202
Arg
526
Asp
542
Asp
203
Asp
443
Met
444
Ser
448
Phe
575
Asp
327
+
N
H
N
N
Cl
O
Ile
364
10d
10a
Trp
539
Asp
571
Trp
441
His
600
-
O
+
N
O
N
Asp
327
O
+
N
N
H
Phe
575
Ile
364
Asp
203
Thr
204
Lys
480
Trp
406
Ser
448
Asp
542
Asp
443
Thr
205
Ile
328
Arg
526
Tyr
299
Met
444
10h
Asn
449
10g
Figure 2. Cont.

Molecules 2021, 26, 4760
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10l
10m
Figure 2. 2D interaction modes of active compounds (10a
, 10d, 10g, 10h, 10l, and 10m). The blue color indicates ligand
exposure and the green color dotted lines indicates the interactions of receptor enzyme with ligands.
Figure 3. 3D interaction modes of active compounds (10a
, 10d, 10g, 10h, 10l, and 10m). Color portion shows the pocket.
Selected and red portions indicate the ligand exposure points.

Molecules 2021, 26, 4760
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Table 1. In silico screening results of benzimidazolium salts 9a–m and 10a–m.
Compound
10a
−10.87
1.75
10d
−10.85
1.49
10g
−11.83
1.59
10h
−10.94
1.52
10l
−13.5
1.08
10m
−13.43
1.74
Acarbose
Docking Score
(Kcal/mol)
−16.18
2
Rmsd Value (Å)
His600,
Asp542,
Arg526,
Asp327,
Met444,
Lys480
Interacting
Residues
Asp203,
Asp327
Asp327,
Asp203
Asp203,
Asp542
Asp542,
Asp443,
Asp203,
Asp542
Asp203
Among the screened derivatives, the benzimidazolium salts 10a
–
m
showed good
docking scores and significant interactions with the selected pocket of the receptor protein
in comparison to the benzimidazolium salts 9a (Table 1, Figure 2). The binding energy
values of most of these derivatives were found close to the standard drug, acarbose
16.18 Kcal/mol). These ligands also occupied the selected pocket of the receptor enzyme
–m
(
−
effectively, as shown in 3D maps (Figure 3).
Most of the ligands interacted with Asp203, Asp327, and Asp542 residues among the
reference/selected residues (Asp203, Asp542, Asp327, His600, Arg526) as revealed by the
2D maps and were considered responsible for the inhibition of the α-glucosidase enzyme
(Figure 2). Nakamura and group had also observed the interactions of Asp203, Asp327,
and Asp542 residues with the salacinol and its derivatives during in silico screening against
α
-glucosidase enzyme [35]. Similarly, the aglycone of curculigoside A, and derivatives,
blocked the activity of -glucosidase enzyme via bindings with Asp203, Asp327, and
α
Asp542 residues [36]. In silico analysis of anthocyanidins and anthocyanins proved them
good inhibitors of the α-glucosidase enzyme. These compounds also exhibited inhibitory
action by interacting with the selected residues, Asp203, Asp327, and Asp542 [37].
Prominently, the derivatives 10a
, 10d, 10g, 10h, 10l, and 10m exhibited best docking
scores ( 10.06 to 11.15 Kcal/mol) and rmsd values less than 2Å (Table 1). Regarding
−
−
the interaction modes, the compounds 10a and 10d showed interactions with the common
residues, Asp203 and Asp327 (Figure. 2). The docking scores for these ligands were found
to be
−
10.87 and
−
10.85 Kcal/mol, respectively. Similarly, the ligands 10h and 10m also
10.94 and 13.43 Kcal/mol) and bindings with common
exhibited good docking scores (
−
−
residues, Asp203 and Asp542 (Figure 2). The ligands 10g and 10m showed interactions with
Asp203 and Asp542, respectively. In order to check the validity of docking experiments,
acarbose was also docked into the selected pocket of the enzyme and it showed binding
interactions with selected residues, His600, Asp542, Arg526, and Asp327 (Figure 4). It
is important to note that the presence of methyl, bromo, chloro, methylene, acetamide
groups, and imidazole ring were found indispensable for the in silico inhibition of the
α-glucosidase enzyme.

Molecules 2021, 26, 4760
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Figure 4. 2D (Left) and 3D (Right) interaction modes of the standard drug, acarbose.
2.4. In Vitro α-Glucosidase Inhibitory Activity
Benzimidazole derivatives have been reported as excellent inhibitors for
α
-glucosidase,
dipeptidyl Peptidase IV, aldose reductase, and diacylglycerol acyltransferase [38
this study, two series of benzimidazolium salts that are quaternary derivatives of benzimi-
dazole have been synthesized in search of potent -glucosidase inhibitors. The compounds
) and 10 ) were screened for in vitro α-glucosidase inhibitory activity according
to reported protocol by using acrobose as a reference drug. The synthesized benzim-
idazolium salts exhibited a varying degree of -glucosidase inhibitory potential with
IC50 values ranging from 14 to 279.28 M presented in Table 2. The structure–activity
relationship established the role of various substituents on phenyl rings and their posi-
tions with biological activities. First of all, compounds 9a were found to be inactive
or weakly active against the enzyme, while compounds having C2-substituted benzimi-
dazolium salts 10a demonstrated excellent -glucosidase inhibitory profile (Table 2).
These results strengthen the fact that C2-substituted benzimidazole variants are more
susceptible to -glucosidase inhibition. Recently, Mentes and his coworkers synthesized
4-(5-fluoro-2-substituted-1H-benzimidazol-6-yl)morpholine derivatives and indicated that
,39]. In
α
9(a–
m
(a–m
α
µ
–
m
–m
α
α
C2 substitution of benzimidazole has a significant effect on
α-glucosidase inhibitory activi-
ties [40]. The strong impact of C2 substituted benzimidazole on α-glucosidase and urease
inhibitory activities is observed by other researchers too [41]. Predominately, compound
10d (1-benzyl-3-(2-((3-bromophenyl)amino)-2-oxoethyl)-2-methyl-1H-benzo[d]imidazole-
3-ium bromide) exhibited the highest
14 + 0.013 µM, which is 4-fold higher than reference drug. Compounds 10m
10l
10e, and 10k exhibited better activity than acarbose with an IC50 value of 16.16 ± 0.026,
17 ± 0.067, 19 0.086, 22 0.040, 25 0.011, 30 0.049 and 32.27 0.050 M, respectively.
Compounds 10c 10d 10e having ortho, meta, and para-substituted bromo group
α
-glucosidase inhibitory potential with IC50 value
,
10h 10g 10a
,
,
,
,
±
±
±
±
±
µ
,
,
on phenyl rings, showed higher activity than acarbose (standard drug). The shifting of
bromo from position 2 to position 3 led to a dramatic increase in activity. Among all of
the halo substituted derivatives, 10d was the most active with the highest
inhibitory potential. Replacing of the bromo substituent with the chloro substituent caused
a slight decline in the -glucosidase inhibitory potential for 10a and 10b, as the compound
α-glucosidase
α
appeared as a moderate to poor inhibitor. It is reflected that nature and change in position
of the halo-group have a notable effect on the α-glucosidase inhibitory activity.

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Table 2. In vitro α-glucosidase inhibitory (IC50) activities of synthesized benzimidazolium salts.
Sr No
1
Code
9a
R
H
R1
3-Cl
IC50 (µM)
150.7 ± 0.045
169.16 ± 0.023
70.01 ± 0.037
171.11± 0.089
198 ± 0.103
241 ± 0.027
215.73 ± 0.112
370 ± 0.032
259.86 ± 0.043
200.14 ± 0.039
201.13 ± 0.105
74.94 ± 0.028
279.28 ± 0.030
22 ± 0.040
60 ± 0.041
46 ± 0.106
14 ± 0.013
30 ± 0.049
60 ± 0.025
19 ± 0.086
17 ± 0.067
100 ± 0.048
60 ± 0.041
2
9b
H
4-Cl
3
9c
H
2-Br
4
9d
H
3-Br
5
9e
H
4-Br
6
9f
H
2-NO2
3-NO2
4-NO2
2-CH3
3-CH3
4-CH3
2-OCH3
4-OCH3
3-Cl
7
9g
H
8
9h
H
9
9i
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
9j
H
9k
H
9l
H
9m
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
Acarbose
H
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
-
4-Cl
2-Br
3-Br
4-Br
2-NO2
3-NO2
4-NO2
2-CH3
3-CH3
4-CH3
2-OCH3
4-OCH3
-
32.27 ± 0.050
25± 0.011
16.16 ± 0.026
58.8 ± 0.015
Among nitro-substituted compounds, 10g and 10h, having a substitution at the meta
and para positions, demonstrated promising -glucosidase inhibitory results due to a
α
strong electron-withdrawing effect. However, 10f, having nitro-substitution at the ortho
position, showed considerable decline in activity.
The bioactivity results of 10l and 10m benzimidazolium salts with methoxy substi-
tution at the ortho and para positions, also showed their effectiveness for α-glucosidase
inhibition. Higher activities of these compounds containing an electron-donating methoxy
group are due to resonance stabilization of the ortho and para positions. Contrary to these
results, methyl-substituted derivatives 10i–k showed varying degrees of α-glucosidase
inhibitory potential. Compounds 10i and 10j containing methyl groups at the ortho and
meta positions appeared as poor inhibitors, whereas, 10k having methoxy at the para
position was found to be moderately active (Figure 5).
It is clear from direct comparison of halo and methoxy/methyl-substituted compounds
that there is a slight decrease in the
substituted benzimidazolium salts. The decreasing order of
potential of the active compounds can be listed as 10d 10m
10e 10k 10c > acarbose. Compounds 9m in the first series, and 10i in the second
α
-glucosidase inhibitory profile for methoxy/methyl-
-glucosidase inhibitory
10h 10g 10a 10l
α
>
>
>
>
>
>
>
>

Molecules 2021, 26, 4760
9 of 18
series, having electron-donating substitution (methoxy and methyl), exhibited the lowest
α
-glucosidase inhibitory results in their respective series. It can be concluded from the
above IC50 results that the position of both electron-donating and withdrawing groups on
the phenyl ring have a vital role in determining the efficacy of α-glucosidase inhibition.
Figure 5. The chemical structures of potent α-glucosidase inhibitors.

Molecules 2021, 26, 4760
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3. Materials and Methods
3.1. General
All chemicals and solvents used in this synthesis were purchased from Sigma-Aldrich
(Munich, Germany) and Daejung Chemicals (Siheung-si, South Korea). Solvents such
methanol, acetonitrile, and chloroform used in this synthesis were purified and dried by
a solvents purification system. Melting points were recorded by using the Stuart SMP
40 melting point apparatus. Spectroscopic studies (1H and 13C-NMR) were carried out by
using the Bruker 600 MHz spectrometer in DMSO-d6 solvent.
3.2. General Procedure for the Preparation of Benzimidazole Precursors 6(a–b)
o-Phenylenediamine
4 (5.4 g, 0.05 mol) and formic acid or acetic acid (0.075 mol) were
mixed in a round bottom flask. The mixture was heated during stirring at 100 ◦C for 3 h.
Afterwards, the contents of the flask were cooled to room temperature and an aqueous
solution of sodium hydroxide (10% w/v) was added dropwise to attain a pH~8. The
ppts. of the corresponding benzimidazole were formed, which were filtered, washed with
ice-cold water, and dried. The products were purified by recrystallization from methanol.
1H-Benzimidazole (6a): Yield: 85%; m.p. 172 ◦C (Literature m.p. 171–173 ◦C [42]).
2-Methyl-1H-Benzimidazole (6b): Yield: 81%; m.p. 176 ◦C (Literature m.p. 176–178 ◦C [42]).
3.3. General Procedure for Preparation of N-Benzylatedbenzimidazoles 7(a–b)
A mixture of benzimidazole 6a or 6b (0.05 mol), CTAB (0.05 mol), acetonitrile (30 mL),
and K2CO3 (0.1 mol) was stirred at room temperature. Benzyl chloride (0.075 mol) was
added drop-wise to the reaction mixture over a period of 5 min. Afterwards, after stirring,
and the reaction mixture was subjected to reflux for 8 h. The progress of the reaction was
monitored by using TLC in a mixture of chloroform and methanol in a 4:1 ratio as a mobile
phase. After the completion of the reaction, the contents of the flask were cooled to room
temperature and poured into crushed ice. The crude product thus obtained was purified
by column chromatography.
1-Benzyl-1H-benzimidazole (7a): White crystalline solid; Yield: 85%; m.p. 118 ◦C (Literature
m.p. 115–118 ◦C [43
,
44]); 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 5.60 (s, 2H, CH2-Ph),
7.10–7.22 (m, 3H, Ar-H), 7.30–7.35 (m, 5H, Ar-H), 7.75 (d, J = 7.8 Hz, 1H, Ar-H), 7.86 (s, 1H,
CH=N). 13C-NMR (DMSO-d6, 150 MHz)
(ppm): 48.8, 111.0, 121.5 (2C), 122.5, 124, 128
(2C), 128.5, 130, 134, 135.7, 144.1, 144.8 (C = N).
δ
1
1-Benzyl-2-methyl-1H-benzimidazole (7b): Waxy solid; Yield: 65%; H-NMR (DMSO-d6,
600 MHz (ppm): 2.45 (s, 3H, CH3), 5.30 (s, 2H, CH2-Ph), 7.10 (t, 3H, Ar-H), 7.35–7.42 (m,
5H, Ar-H), 7.70 (d, J = 7.2 Hz, 1H, Ar-H). 13C-NMR (DMSO-d6, 150 MHz)
(ppm): 14.3,
)
δ
δ
46.4, 110.0, 121.5, 122.5 (2C), 124, 126 (2C), 127.5, 129, 133, 135.7, 141.2, 143.0 (C = N).
3.4. General Procedure for the Preparation of Benzimidazolium Salts 9(a–m)
1-Benzyl-1H-benzimidazole 7a (0.22 g, 1 mmol) was dissolved in acetonitrile (30 mL)
in a round bottom flask. The solution of 2-bromo-N-arylacetamides 8(a–m) (1 mmol)
in acetonitrile (10 mL) was added dropwise in the round bottom flask and the reaction
mixture was refluxed for 12–14 h. The completion of the reaction was observed by thin-
layer chromatography. Finally, the contents were cooled in an ice bath to obtain the crude
solid salts which were purified by column chromatography.
1-Benzyl-3-[2-{(3-chlorophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9a):
◦
1
White crystalline solid, Yield: 80%; m.p. 205–207 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 5.63 (s, 2H, CH2-Ph), 5.89 (s, 2H, CH2-CO), 7.19 (d, 1H, J = 7.98 Hz, Ar-H), 7.41
δ
(t, 2H, J = 8.07 Hz, Ar-H), 7.45 (t, 2H, J = 7.38 Hz, Ar-H), 7.51 (d, 1H, J = 8.28 Hz, Ar-H),
7.54 (d, 2H, J = 7.32 Hz, Ar-H), 7.67–7.72 (m, 2H, Ar-H), 7.81 (s, 1H, Ar-H), 8.01 (d, 1H,
J = 7.68 Hz, Ar-H), 8.07 (d, 1H, J = 7.86 Hz, Ar-H), 9.97 (s, 1H, NCHN), 11.02 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 49.2, 50.0, 113.8, 114.0, 117.7, 118.8, 123.8, 126.8,

Molecules 2021, 26, 4760
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127.0, 128.2 (2C), 128.4, 129.1(3C), 130.4, 130.7, 133.2, 133.7, 139.5, 143.5, 164.0 (C=O). MS
(ESI+): m/z calcd for C22H19ClN3O+ [M − Br]+ 376; found 377 [M − Br + H]+.
1-Benzyl-3-[2-{(4-chlorophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9b):
◦
1
White crystalline solid; Yield: 78%; m.p. 197–198 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 5.62 (s, 2H, CH2-Ph), 5.89 (s, 2H, CH2-CO), 7.39- 7.42 (m, 2H, Ar-H), 7.43–7.46 (m,
δ
3H, Ar-H), 7.54 (d, 2H, J = 7.2 Hz, Ar-H), 7.65–7.67 (m, 3H, Ar-H), 7.68–7.71 (m, 1H, Ar-H),
8.01 (d, 1H, J = 6.72 Hz, Ar-H), 8.07 (d, 1H, J = 6.96 Hz, Ar-H), 9.98 (s, 1H, NCHN), 10.96 (s,
1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 49.2, 50.0, 113.8, 114.0, 121.0 (2C), 126.8,
127.0, 127.6, 128.2 (2C), 128.8 (2C), 129.0 (2C), 130.4, 132.0, 132.0, 133.7, 137.1, 143.6, 163.6
(C=O): MS (ESI+): m/z calcd for C22H19ClN3O+ [M − Br]+ 376; found 376 [M − Br]+.
1-Benzyl-3-[2-{(2-bromophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9c):
◦
1
White crystalline solid; Yield: 78%; m.p. 192–194 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 5.68 (s, 2H, CH2-Ph), 5.87 (s, 2H, CH2-CO), 7.19 (t, 1H, J = 7.2 Hz, Ar-H), 7.39 (t,
δ
2H, J = 7.2 Hz, Ar-H), 7.42 (q, 2H, J = 12.6 Hz, J = 14.4 Hz, Ar-H), 7.2 (t, 2H, J = 8.75 Hz,
Ar-H) 7.60 (d, 1H, J = 7.26 Hz, Ar-H), 7.66–7.72 (m, 3H, Ar-H), 8.00 (d, 1H, J = 8.00 Hz,
Ar-H), 8.06 (d, 1H, J = 8.06 Hz, Ar-H), 10.04 (s, 1H, NCHN), 10.50 (s, 1H, NH). 13C-NMR
(DMSO-d6, 150 MHz) δ (ppm): 49.0, 50.0, 113.9 (2C), 126.8, 127.0, 127.5, 128.2 (5C), 128.8
(4C), 129.0, 131.0, 132.9, 133.8, 143.7, 164.1 (C=O). MS (ESI+): m/z calcd for C22H19BrN3O+
[M − Br]+ 420.0706; found 420.0714 [M − Br]+.
1-Benzyl-3-[2-{(3-bromophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9d):
◦
1
White crystalline solid; Yield: 85%; m.p. 176–178 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 5.62 (s, 2H, CH2-Ph), 5.88 (s, 2H, CH2-CO), 7.31–7.36 (m, 2H, Ar-H), 7.42 (t, 1H,
δ
J = 8.46 Hz, Ar-H), 7.41–7.46 (m, 2H, Ar-H), 7.53–7.55 (m, 3H, Ar-H), 7.67–7.72 (m, 2H,
Ar-H), 7.95 (t, 1H, J = 3.6 Hz, Ar-H), 8.01 (d, 1H, J = 6.93 Hz, Ar-H), 8.07 (d, 1H, J = 6.93 Hz,
Ar-H), 9.96 (s, 1H, NCHN), 10.99 (s, 1H, NH).13C-NMR (DMSO-d6, 150 MHz)
δ (ppm):
49.2, 50.0, 113.8, 114.0, 118.1, 121.6, 121.7, 126.7, 126.8, 127.0, 128.8, 128.2 (2C), 129.1 (2C),
130.4, 131.0, 132.0, 133.7, 139.7, 143.6, 163.9 (C=O). MS (ESI+): m/z calcd for C22H19BrN3O+
[M − Br]+ 420; found 420 [M − Br]+.
1-Benzyl-3-[2-{(4-bromophenyl)amino)}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9e):
◦
1
White crystalline solid; Yield: 75%; m.p. 150–152 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 5.60 (s, 2H, CH2-Ph), 5.88 (s, 2H, CH2-CO), 7.40 (t, 1H, J = 7.26 Hz, Ar-H), 7.45
δ
(t, 2H, J = 7.35 Hz, Ar-H), 7.52–7.55 (m, 3H, Ar-H), 7.55 (t, 1H, J = 3.4 Hz, Ar-H), 7.6 (d,
2H, J = 8.8 Hz, Ar-H), 7.76–7.71 (m, 2H, Ar-H), 8.0 (d, 1H, J =8.3 Hz, Ar-H), 8.06 (d, 1H,
J = 7.9 Hz, Ar-H), 9.95 (s, 1H, NCHN), 10.92 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
(ppm): 49.2, 50.0, 113.8, 114.0, 115.7, 121.3, 126.8, 127.0, 128.2 (3C), 128.8, 129.1 (3C), 130.4,
131.8 (2C), 132.0, 133.7, 143.6, 144.20, 163.6 (C=O). MS (ESI+): m/z calcd for C22H19BrN3O+
[M − Br]+ 420; found 420 [M − Br]+.
1-Benzyl-3-[2-{(2-nitrophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9f): White
crystalline solid; Yield: 74%; m.p. 142–143 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ (ppm):
5.69 (s, 2H, CH2-Ph), 5.8 (s, 2H, CH2-CO), 7.38–7.41 (m, 1H, Ar-H), 7.44 (q, 3H, J = 7.4 Hz,
Ar-H), 7.52 (d, 2H, J = 7.2 Hz, Ar-H), 7.67–7.73 (m, 3H, Ar-H), 7.77 (t, 1H, J = 7.8 Hz,
Ar-H), 8.0 (t, 3H, J = 7.35 Hz, Ar-H), 9.99 (s, 1H, NCHN), 11.2 (s, 1H, NH). 13C-NMR
(DMSO-d6, 150 MHz) δ (ppm): 49.0, 50.0, 113.8, 114.0, 121.0, 125.0 (2C), 126.8, 127.0, 128.2
(3C), 128.8, 129.0 (3C), 130.0, 132.0, 134.1, 142.8, 143.5, 164.2 (C=O). MS (ESI+): m/z calcd
for C22H19N4O3+ [M − Br]+ 387; found 388 [M − Br + H]+.
1-Benzyl-3-[2-{(3-nitrophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9g): White
crystalline solid; Yield: 75%; m.p. 162–163 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ (ppm):
5.63 (s, 2H, CH2-Ph), 5.86 (s, 2H, CH2-CO), 7.38–7.41 (m, 1H, Ar-H), 7.44 (t, 2H, J = 14.7 Hz,
Ar-H), 7.52 (d, 2H, J = 7.86 Hz, Ar- H), 7.66–7.71 (m, 3H, Ar-H), 7.90 (d, 1H, J = 7.68 Hz,
Ar-H), 7.97–8.00 (m, 2H, Ar-H), 8.05 (d, 1H, J = 7.68 Hz, Ar-H), 8.61 (s, 1H, Ar-H), 9.93 (s,
1H, NCHN), 11.27 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 49.2, 50.0, 113.4,

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113.8, 114.0, 118.6, 125.3, 126.8, 127.0, 128.2 (2C), 128.87, 129.1 (2C), 130.4 (2C), 130.5, 132.0,
+
139.2, 143.5, 148.0, 164.3 (C=O). MS (ESI+): m/z calcd for C22H19N4O3 [M
−
Br]+ 387;
found 387 [M − Br]+.
1-Benzyl-3-[2{(4-nitrophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9h): White
◦
crystalline solid; Yield: 70%; m.p. 152–153 C. 1H-NMR (DMSO-d6, 600 MHz)
δ (ppm): 5.64
(s, 2H, CH2-Ph), 5.87 (s, 2H, CH2-CO), 7.41 (t, 1H, J = 7.2 Hz, Ar-H), 7.45 (t, 2H, J = 7.35 Hz,
Ar-H), 7.52 (d, 2H, J = 7.26 Hz, Ar-H), 7.68–7.71 (m, 2H, Ar-H), 7.85 (d, 2H, J = 9.1 Hz,
Ar-H), 8.01 (t, 1H, J = 9.2 Hz, Ar-H), 8.07 (d, 1H, J = 7.8 Hz, Ar-H), 8.28 (d, 2H, J = 9.18 Hz,
Ar-H), 9.91 (s, 1H, NCHN), 11.34 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm):
49.3, 50.0, 113.8, 114.0, 119.15 (2C), 125.1 (2C), 126.8, 127, 128.2 (2C), 128.9, 129.1 (2C), 130.4,
+
132.0, 133.7, 142.7, 143.6, 144.2, 164.6 (C=O). MS (ESI+): m/z calcd for C22H19N4O3 [M
−
Br]+ 387; found 387 [M − Br]+.
1-Benzyl-3-{2-oxo-2-(o-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (9i): White crys-
talline solid; Yield: 90%; m.p. 162–163 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
(ppm): 2.3 (s,
δ
3H, CH3), 5.67 (s, 2H, CH2-Ph), 5.9 (s, 2H, CH2-CO), 7.13 (t, 1H, J = 7.14 Hz, Ar-H), 7.19 (t,
1H, J = 7.11 Hz, Ar-H), 7.2 (d, 1H, J = 7.32 Hz, Ar-H), 7.39 (t, 1H, J = 7.2 Hz, Ar-H), 7.44 (t,
3H, J = 7.26 Hz, Ar-H), 7.53 (d, 2H, J = 7.26 Hz, Ar-H), 7.66–7.73 (m, 2H, Ar-H), 8.0 (d, 1H,
J =8.2 Hz, Ar-H), 8.07 (d, 1H, J = 8.28 Hz, Ar-H), 10.0 (s, 1H, NCHN), 10.14 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 17.9, 49.0, 50.0, 113.7, 113.9, 124.9, 126.1, 128.2
(4C), 128.8 (4C), 129.0 (3C), 130.5 (2C), 131.8, 143.6, 163.6 (C=O). MS (ESI+): m/z calcd for
C23H22N3O+ [M − Br]+ 356; found 357 [M − Br + H]+.
1-Benzyl-3-{2-oxo-2-(m-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (9j): White crys-
talline solid; Yield: 88%; m.p. 132–134 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ (ppm): 2.3
(s, 3H, CH3), 5.60 (s, 2H, CH2-Ph), 5.88 (s, 2H, CH2-CO), 6.92 (d, 1H, J = 7.6 Hz, Ar-H),
7.22 (t, 1H, J = 7.8 Hz, Ar-H), 7.39–7.40 (m, 2H, Ar-H), 7.42–7.46 (m, 3H, Ar-H), 7.53 (d,
2H, J = 7.3 Hz, Ar-H), 7.65–7.71 (m, 2H, Ar-H), 7.99 (d, 1H, J = 7.98 Hz, Ar-H), 8.04 (d, 1H,
J = 8.04 Hz, Ar-H), 9.99 (s, 1H, NCHN), 10.74 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
(ppm): 21.1, 49.2, 50.0, 113.8, 116.5, 119.8, 124.7, 126.8, 127.0, 128.2 (3C), 129.0 (3C), 130.4,
132.0, 133.7, 137.1, 138.0, 138.2, 143.5, 163.3 (C=O). MS (ESI+): m/z calcd for C23H22N3O+
[M − Br]+ 356; found 356 [M − Br]+.
1-Benzyl-3-{2-oxo-2-(p-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (9k): White crys-
talline solid; Yield: 90%; m.p. 148–149 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ (ppm): 2.26
(3H, s, CH3), 5.59 (s, 2H, CH2-Ph), 5.88 (s, 2H, CH2-CO), 7.16 (d, 2H, J = 8.34 Hz, Ar-H),
7.39–7.40 (m, 1H, Ar-H), 7.45 (t, 2H, J = 6 Hz, Ar-H), 7.52 (dd, 4H, J = 7.8, 16.1 Hz, Ar-H),
7.66–7.71 (m, 2H, Ar-H), 8.03 (d, 1H, J = 7.32 Hz, Ar-H), 8.06 (d, 1H, J = 7.38 Hz, Ar-H), 9.99
(s, 1H, NCHN), 10.72 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 20.4, 49.1, 49.9,
113.8 (2C), 119.3 (3C); 126.9, 128.2 (4C); 128.8 (5C), 129.0, 129.3, 133.7, 143.5, 163.1 (C=O).
MS (ESI+): m/z calcd for C23H22N3O+ [M − Br]+ 356; found 357 [M − Br + H]+.
1-Benzyl-3-[2-{(2-methoxyphenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9l):
◦
1
White crystalline solid; Yield: 92%; m.p. 188–189 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 3.88 (s, 3H, OCH3), 5.67 (s, 2H, CH2-Ph), 5.87 (s, 2H, CH2-CO), 6.90 (t, 1H,
δ
J = 7.20 Hz, Ar-H), 7.09 (d, 1H, J = 8.04 Hz, Ar-H), 7.13 (t, 1H, J = 7.68 Hz, Ar-H), 7.38 (t, 1H,
J = 7.2 Hz, Ar-H), 7.48 (t, 2H, J = 7.2 Hz, Ar-H), 7.52 (d, 2H, J = 7.38 Hz, Ar-H), 7.64–7.70
(m, 2H, Ar-H), 7.88 (d, 1H, J = 7.90 Hz, Ar-H), 8.00 (dd, 2H, J = 8.2, 15.99 Hz, Ar-H), 10.03
(s, 1H, NCHN), 10.07 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 49.2, 49.9, 55.8,
111.4, 113.7, 113.8, 120.3, 121.9, 125.4, 126.2, 126.8, 127.0, 128.2 (2C), 128.8, 129.0 (2C), 130.4,
131.8, 133.7, 143.5, 149.8, 163.6 (C=O). HRMS (ESI+): m/z calcd for C23H22N3O+ [M
−
Br]+
372.1707; found 372.1714 [M − Br]+.
1-Benzyl-3-[2-{(4-methoxyphenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide (9m):
White crystalline solid; Yield: 90%; m.p. 171–173 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
(ppm): 3.71 (s, 3H, OCH3), 5.58 (s, 2H, CH2-Ph), 5.87 (s, 2H, CH2-CO), 6.90–6.93 (m, 2H,
δ

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Ar-H), 7.37–7.39 (m, 1H, Ar-H), 7.43 (t, 2H, J = 14.7 Hz, Ar-H), 7.53 (dd, 4H, J = 3.4, 8.3 Hz,
Ar-H), 7.64–7.69 (m, 2H, Ar-H), 7.99 (d, 1H, J = 7.92 Hz, Ar-H), 8.08 (d, 1H, J = 7.98 Hz,
Ar-H), 10.00 (s, 1H, NCHN), 10.69 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm):
49.1, 50.0, 55.2, 113.8, 114.0, 121.0 (2C), 126.8, 127, 128.2 (3C), 128.8, 129.0 (3C), 130.4, 131.2,
132.0, 133.7, 143.5, 155.70, 162.8 (C=O). MS (ESI+): m/z calcd for C23H22N3O+ [M
372; found 373 [M − Br + H]+.
−
Br]+
3.5. General Procedure for the Preparation of Benzimidazolium Salts 10(a–m)
1-Benzyl-2-methyl-1H-benzimidazole 7b (0.23 g, 1 mmol) was dissolved in acetoni-
trile (30 mL) in a round bottom flask. The solution of 2-bromo-N-arylacetamides
8(a–m)
(1 mmol) in acetonitrile (10 mL) was added dropwise in the round bottom flask and the
reaction mixture was refluxed for 12–14 h. The completion of the reaction was observed by
thin-layer chromatography. Finally, the contents were cooled in an ice bath to obtain the
crude solid salt which was purified by column chromatography.
1-Benzyl-3-[2-{(3-chlorophenyl)amino}-2-oxoethyl]-2-methy◦l-1H-benzo[d]imidazol-3-ium bromide
(
10a): White crystalline solid; Yield: 74%; m.p. 210–213 C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 2.94 (s, 3H, Benzimidazole-CH3), 5.52 (s, 2H, CH2-Ph), 5.83 (s, 2H, CH2-CO), 7.17
(d, 1H, J = 9.52 Hz, Ar-H), 7.31 (d, 2H, J = 9.2 Hz, Ar-H), 7.35–7.41 (m, 4H, Ar-H), 7.44 (t,
1H, J = 9.6 Hz, Ar-H), 7.61–7.67 (m, 2H, Ar-H), 7.74 (s, 1H, Ar-H), 7.94 (d, 1H, J = 9.4 Hz,
Ar-H), 7.89 (d, 1H, J = 8.84 Hz, Ar-H), 10.94 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ
(ppm): 11.3, 48.2, 48.7, 113.3, 113.7, 118.5, 119.6, 124.5, 127.1, 127.2, 127.6 (2C), 129.0, 129.6
(2C), 131.1, 131.2, 131.8, 133.7, 134.3, 139.8, 153.6, 164.0 (C=O). MS (ESI+): m/z calcd for
C23H21ClN3O+ [M − Br]+ 390; found 391 [M − Br + H]+.
1-Benzyl-3-[2-{(4-chlorophenyl)amino}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazol-3-ium bromide
◦
10b): White crystalline solid; Yield: 74%; m.p. 178–180 C. 1H-NMR: (DMSO-d6, 600 MHz)
(
δ
(ppm): 2.95 (s, 3H, Benzimidazole-CH3), 5.52 (s, 2H, CH2-Ph), 5.66 (s, 2H, CH2-CO),
7.33(d, 2H, J =17.4 Hz, Ar-H), 7.36 (t, 2H, J = 4.9 Hz, Ar-H), 7.39–7.42 (m, 3H, Ar-H), 7.60 (d,
2H, J = 8.94 Hz, Ar-H), 7.65 (q, 2H, J1 = J2 = 18 Hz, Ar-H), 7.95 (d, 1H, J =7.85 Hz, Ar-H),
7.99 (d, 1H, J =7.74 Hz, Ar-H), 10.88 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm):
10.8, 47.7, 48.2, 112.8, 113.2, 121.1 (2C), 126.5, 127.1 (2C), 127.8, 128.5, 128.8 (2C), 129.1 (3C),
130.5, 131.3, 133.8, 136.8, 153.2, 163.3 (C=O). HRMS (ESI+): m/z calcd for C23H21ClN3O+
[M − Br]+ 390.1368; found 390.1374 [M − Br]+.
1-Benzyl-3-[2-{(2-bromophenyl)amino)}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazol-3-ium bromide
(
10c): White crystalline solid; Yield: 76%; m.p. 252 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 3.0 (s, 3H, Benzimidazole-CH3), 5.64 (s, 2H, CH2-Ph), 6.0 (s, 2H, CH2-CO), 7.20 (t,
1H, J = 7.5 Hz, Ar-H), 7.32–7.42 (m, 6H, Ar-H), 7.63 (q, 2H, J1 = J2 = 7.5 Hz, Ar-H), 7.7 (q, 2H,
J1 = J 2= 8.2 Hz, Ar-H), 7.98 (d, 1H, J = 8.2 Hz, Ar-H), 8.03 (d, 1H, J = 8.2 Hz, Ar-H), 10.3 (s,
1H, NH), 13C-NMR (DMSO-d6, 150 MHz)
δ
(ppm): 11.0, 47.7, 48.2, 113.0, 113.2, 113.6, 118.7,
125.5, 126.5, 126.6, 127.1 (2C), 128.5, 129.1 (2C), 130.5, 130.6, 131.4, 133.8, 139.1, 148.0, 153.2,
164.0 (C=O). MS (ESI+): m/z calcd for C23H21BrN3O+ [M Br]+ 434; found 434 [M Br]+.
−
−
1-Benzyl-3-[2-{(3-bromophenyl)amino}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazol-3-ium bromide
10d): White crystalline solid; Yield: 80%; m.p. 242 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
(ppm): 3.0 (s, 3H, Benzimidazole-CH3), 5.60 (s, 2H, CH2-Ph), 5.90 (s, 2H, CH2-CO),
(
δ
7.31–7.35 (m, 4H, Ar-H), 7.3 (t, 1H, J = 7.3 Hz, Ar-H), 7.4 (t, 2H, J = 7.3 Hz, Ar-H), 7.5 (d, 1H,
J = 7.3 Hz, Ar-H), 7.6–7.7 (m, 2H, Ar-H), 8.0 (s, 1H, Ar-H), 8.1 (d, 1H, J = 8.05 Hz, Ar-H), 8.04
(d, 1H, J = 7.75 Hz, Ar-H), 11.0 (s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 11.0,
47.8, 48.2, 113.0, 113.3, 118.2, 121.6, 121.8, 126.5, 126.6, 126.7, 127.1 (2C), 128.4, 129.1 (2C),
130.6, 131.0, 131.4, 134.0, 139.6, 153.2, 163.6 (C=O). MS (ESI+): m/z calcd for C23H21BrN3O+
[M − Br]+ 434; found 434 [M − Br + H]+.
1-Benzyl-3-[2-{(4-bromophenyl)amino}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazol-3-ium bromide
◦
10e): White crystalline solid; Yield: 82%; m.p. 212 C. 1H-NMR (DMSO-d6, 600 MHz)
(
δ
(ppm): 2.99 (s, 3H, Benzimidazole-CH3), 5.66 (s, 2H, CH2-Ph), 5.91 (s, 2H, CH2-CO),

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7.33–7.43 (m, 5H, Ar-H), 7.52–7.54 (m, 2H, Ar-H), 7.68–7.61 (m, 4H, Ar-H), 7.98 (d, 1H,
J = 7.4 Hz, Ar-H), 8.11 (d, 1H, J = 7.3 Hz, Ar-H), 11.54 (s, 1H, NH). MS (ESI+): m/z calcd for
C23H21BrN3O+ [M − Br]+ 434; found 435 [M − Br + H]+.
1-Benzyl-2-methyl-3-[2-{(2-nitrophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide
10f): White crystalline solid; Yield: 72%; m.p. 160–161 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): (500 MHz, DMSO-d6) δ (ppm): 2.98 (s, 3H, Benzimidazole-CH3), 5.73 (s, 2H,
CH2-Ph), 5.91 (s, 2H, CH2-CO), 7.31–7.34 (m, 2H, Ar-H), 7.37–7.43 (m, 3H, Ar-H), 7.62–7.66
(m, 4H, Ar-H), 7.74–7.77 (m, 1H, Ar-H), 7.97 (t, J = 6.7 Hz, 2H, Ar-H), 8.09 (d, J = 8.0 Hz,
+
1H, Ar-H), 11.78 (s, 1H, NH). MS (ESI+): m/z calcd for C23H21N4O3 [M
−
Br]+ 401; found
402 [M − Br + H]+.
1-Benzyl-2-methyl-3-[2-{(3-nitrophenyl)amino}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide
◦
(
10g): White crystalline solid; Yield: 73%; m.p. 180–182 C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 2.98 (s, 3H, Benzimidazole-CH3), 5.61 (s, 2H, CH2-Ph), 5.90 (s, 2H, CH2-CO), 7.34
(d, 2H, J = 7.26 Hz, Ar-H), 7.37 (t, 1H, J = 7.2 Hz, Ar-H), 7.42 (t, 2H, J = 7.5 Hz, Ar-H),
7.63 (dd, 1H, J = 1.1, 7.32 Hz, Ar-H), 7.65–7.68 (m, 3H, Ar-H), 7.91 (d, 1H, J = 8.16 Hz,
Ar-H), 7.95–8.0 (m, 2H, Ar-H), 8.04 (d, 1H, J = 7.78 Hz, Ar-H), 11.29 (s, 1H, NH). 13C-NMR:
(DMSO-d6, 150 MHz)
δ (ppm): 11.0, 47.8, 48.2, 113.0, 113.2, 113.6, 118.7, 125.5, 126.5, 126.6,
127.1 (2C), 128.5, 129.1 (2C), 130.5, 130.6, 131.4, 133.8, 139.1, 148.0, 153.2, 164.0 (C=O). MS
(ESI+): m/z calcd for C23H21N4O3+ [M − Br]+ 401; found 401 [M − Br]+.
1-Benzyl-2-methyl-3-[2-{(4-nitrophenyl)amino)}-2-oxoethyl]-1H-benzo[d]imidazol-3-ium bromide
◦
10h): White crystalline solid; Yield: 76%; m.p. 222–224 C. 1H-NMR (DMSO-d6, 600 MHz)
(
δ
(ppm): 2.96 (s, 3H, Benzimidazole-CH3), 5.60 (s, 2H, CH2-Ph), 5.87 (s, 2H, CH2-CO),
7.32 (d, 2H, J = 7.4 Hz, Ar-H), 7.37 (t, 1H, J = 7.2 Hz, Ar-H), 7.41 (t, 2H, J = 7.2 Hz, Ar-H),
7.62–7.67 (m, 2H, Ar-H), 7.84 (d, 2H, J = 8.8 Hz, Ar-H), 7.96 (d, 1H, J = 7.9 Hz, Ar-H), 8.02
(d, 1H, J = 7.53 Hz, Ar-H), 8.25 (d, 2H, J = 9.2 Hz, Ar-H), 10.88 (s, 1H, NH). 13C-NMR
(DMSO-d6, 150 MHz)
δ (ppm):10.8, 47.9, 48.2, 113.0, 113.2, 119.4 (2C), 125.0 (2C), 126.6,
126.7, 127.1 (2C), 128.5, 129.1 (2C), 130.5, 131.3, 133.8, 143.0, 144.0, 153.2, 164.2 (C=O). MS
(ESI+): m/z calcd for C23H21N4O3+ [M − Br]+ 401; found 402 [M − Br + H]+.
1-Benzyl-2-methyl-3-{2-oxo-2-(o-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (10i):
◦
1
White crystalline solid; Yield: 84%; m.p. 168–170 C. H-NMR (DMSO-d6, 600 MHz)
(ppm): 2.25 (s, 3H, CH3), 2.51 (s, 3H, Benzimidazole-CH3), 5.59 (s, 2H, CH2-Ph), 5.87
δ
(s, 2H, CH2-CO), 7.11–7.19 (m, 2H, Ar-H), 7.24 (d, 1H, J = 8.60 Hz, Ar-H), 7.30–7.41 (m,
6H, Ar-H), 7.63.7–68 (m, 2H, Ar-H), 7.99 (q, 2H, J1 = J2 = 9.6 Hz, Ar-H),10.08 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 11.3, 18.2, 48.0, 48.7, 113.2, 113.8, 125.5, 126.5,
126.6, 127.0, 127.1, 127.6 (2C), 129.0, 129.6 (2C), 131.0, 131.1, 132.0, 132.4, 134.4, 135.6, 153.6,
164.0 (C=O). MS (ESI+): HR m/z calcd for C24H24N3O+ [M
−
Br]+ 370.1914; found 370.1917
[M − Br]+.
1-Benzyl-2-methyl-3-{2-oxo-2-(m-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (10j):
White crystalline solid; Yield: 82%; m.p. 192–194 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 2.26 (s, 3H, CH3), 2.95 (s, 3H, Benzimidazole-CH3), 5.51 (s, 2H, CH2-Ph), 5.87 (s, 2H,
CH2-CO), 6.94 (d, 1H, J = 7.5 Hz, Ar-H), 7.22 (t, 1H, J = 7.8 Hz, Ar-H), 7.32 (d, 2H, J = 7.2 Hz,
Ar-H), 7.36 (q, 2H, J1 = J2 = 7.2 Hz, Ar-H), 7.41 (t, 3H, J = 7.39 Hz, Ar-H), 7.62–7.67 (m, 2H,
Ar-H), 7.95 (d, 1H, J = 8.01 Hz, Ar-H), 7.98 (d, 1H, J =8.01 Hz, Ar-H), 10.68 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 10.8, 21.0, 47.7, 48.1, 112.8, 113.2, 116.6, 120.0,
125.0, 126.5, 126.6, 127.1(2C), 128.5, 128.8, 129.1 (2C), 130.6, 131.4, 133.8, 137.8, 138.3, 153.1,
163.0 (C=O). MS (ESI+): m/z calcd for C24H24N3O+ [M − Br]+ 370; found 370 [M − Br]+.
1-Benzyl-2-methyl-3-{2-oxo-2-(p-tolylamino)ethyl}-1H-benzo[d]imidazol-3-ium bromide (10k):
White crystalline solid; Yield: 82%; m.p. 220–221 ◦C. 1H-NMR (DMSO-d6, 600 MHz)
δ
(ppm): 2.26 (s, 3H, CH3), 2.96 (s, 3H, Benzimidazole-CH3), 5.51 (s, 2H, CH2-Ph), 5.90 (s,
2H, CH2-CO), 7.15 (d, 2H, J = 8.3 Hz, Ar-H), 7.33 (d, 2H, J = 7.17 Hz, Ar-H), 7.36 (t, 1H,
J = 7.4 Hz, Ar-H), 7.41 (t, 2H, J = 7.3 Hz, Ar-H), 7.48 (d, 2H, J = 8.41 Hz, Ar-H), 7.62–7.67 (m,

Molecules 2021, 26, 4760
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2H, Ar-H), 7.98 (d, 1H, J = 7.5 Hz, Ar-H), 8.03 (d, 1H, J = 7.5 Hz, Ar-H), 10.64 (s, 1H, NH).
13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 10.8, 20.4, 47.8, 48.1, 112.8, 113.0, 113.3, 119.4 (2C),
127.1(2C), 128.4, 129.0 (3C), 129.3 (2C), 130.6, 131.5, 133.1, 134.0, 135.6, 153.1, 163.0 (C=O).
MS (ESI+): m/z calcd for C24H24N3O+ [M − Br]+ 370; found 370 [M − Br]+.
1-Benzyl-3-[2-{(2-methoxyphenyl)amino}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazol-3-ium bro-
◦
1
mide (10l): White crystalline solid; Yield: 84%; m.p. 244–246 C. H-NMR (DMSO-d6,
600 MHz (ppm): 2.97 (s, 3H, Benzimidazole-CH3), 3.91 (s, 3H, OCH3), 5.67 (s, 2H, CH2-
)
δ
Ph), 5.91 (s, 2H, CH2-CO), 6.90–7.14 (m, 3H, Ar-H), 7.35–7.42 (m, 5H, Ar-H), 7.64–7.67 (m,
2H, Ar-H), 7.92 (d, 1H, J = 9.05 Hz, Ar-H), 8.0 (dd, 2H, J = 9.27, 9.61 Hz, Ar-H) 10.0 (s, 1H,
NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 11.4, 48.4, 48.7, 56.3, 112.0, 113.3, 113.8,
120.8, 122.3, 125.8, 127.0, 127.1, 127.6 (3C), 129.0, 131.1 (3C), 132.0, 134.5, 150.2, 153.8, 164.0
(C=O). MS (ESI+): m/z calcd for C24H24N3O2+ [M − Br]+ 386; found 386 [M − Br]+.
1-Benzyl-3-[2-{(4-methoxyphenyl)amino}-2-oxoethyl]-2-methyl-1H-benzo[d]imidazole-3-ium bro-
◦
mide (10m): White crystalline solid; Yield: 82%; m.p. 235–238 C. 1H-NMR (DMSO-d6,
600 MHz) δ (ppm): 2.96 (s, 3H, Benzimidazole-CH3), 3.72 (s, 3H, OCH3), 5.51 (s, 2H, CH2-
Ph), 5.88 (s, 2H, CH2-CO), 6.92 (d, 2H, J = 9.06 Hz, Ar-H), 7.33 (d, 2H, J = 7.2 Hz, Ar-H),
7.37 (t,1H, J = 7.2 Hz, Ar-H), 7.41 (t, 2H, J = 7.3 Hz, Ar-H), 7.50 (d, 2H, J = 9.03 Hz, Ar-H),
7.62–7.67 (m, 2H, Ar-H), 7.96 (d, 1H, J = 7.93 Hz, Ar-H), 8.01 (d, 1H, J = 7.93 Hz, Ar-H) 10.63
(s, 1H, NH). 13C-NMR (DMSO-d6, 150 MHz)
δ (ppm): 10.8, 47.7, 48.2, 55.2, 112.8, 113.2,
114.0 (2C), 121.1 (2C), 126.5, 126.6, 127.1 (2C), 128.5, 129.1 (2C), 130.6, 131.0, 131.4, 134.0,
+
153.2, 155.8, 162.6 (C=O). MS (ESI+): m/z calcd for C24H24N3O2 [M
−
Br]+ 386; found 387
[M − Br + H]+.
3.6. In Silico α-Glucosidase Inhibition Studies
The in silico analysis of all the benzimidazolium salts along with the reference acarbose
was carried out by using the MOE Ver.2014.0901 (Molecular Operating Environment,
Chemical Computing Group ULC., Montreal, QC, Canada).
3.6.1. Ligand Preparation
The software (ChemDraw Ultra 12 + Serial, Perkin Elmer, MA, USA) was utilized
to draw the structures of all these compounds. In order to display these files in MOE,
structures of these compounds were saved in the MDL file (“.sdf”). The 2D structure of
reference acarbose in the SDF file was retrieved from NCBI Pubchem. Afterwards, 3D
protonation and energy minimizations of compounds as well as acarbose were executed
under default parameters in MOE.
3.6.2. Protein Preparation
The 3D structure of Saccharomyces cerevisiae α-glucosidase (PDB ID: 2QMJ) was down-
loaded from the Protein Data Bank (http://www.rcsb.org/pdb, accessed on 10 June 2021).
The binding pocket with the protein residues (Asp203, Asp542, Asp327, His600, Arg526)
was selected and the interactions of the ligand (acarbose) were checked. Afterwards ligand,
solvent molecules, and unknown atoms from the receptor protein were removed. The
active sites from the 3D structure of the protein were determined using MOE-Site Finder
Module. The active sites of the ligand consist of Asp203, Tyr299, Asp327, Ile328, Asp366,
Trp406, Trp441, Asp443, Met444, Arg526, Trp539, Gly541, Asp542, Asp571, Phe575, Arg598,
and His600 residues. These sites were occupied by dummy atoms and the protein was
3D-protonated and energy-minimized using default parameters in the MOE software.
3.6.3. Molecular Docking
Ten conformations of all the ligands were docked into the selected site of the en-
zyme. The MOE-Dock module was used to access binding modes of ligands with the
α
-glucosidase enzyme. The parameters, placement: triangle matcher, rescoring: London
dG, refinement: forcefield, retain: 10 were used. The root mean square deviation rmsd val-

Molecules 2021, 26, 4760
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ues and docking scores of their top-ranked conformations were used to study the binding
modes of ligands. Results of the molecular docking are depicted in Table 1.
3.7. In Vitro α-Glucosidase Inhibition
The literature protocol was used to assess
α-glucosidase inhibition of synthesized
compounds [45]. The solution of the -glucosidase enzyme (obtained from Saccharomyces
α
cerevisiae (Sigma-Aldrich, Munich, Germany)) was prepared in phosphate buffer (pH 6.8,
100 mM). The synthesized compounds were dissolved in dimethyl sulfoxide (DMSO). A
mixture of 40
12.5 L of each sample was prepared in 96-well microtiter plates and incubated for 5 min
at 37 ◦C. Afterwards, 40
L of 5 mM p-nitrophenyl- -D-glucopyranoside (Sigma-Aldrich,
µL of the α-glucosidase enzyme (0.5 U/mL), 120 µL of phosphate buffer, and
µ
µ
α
Munich, Germany) was poured into each well and further incubated for an additional
◦
half-hour. The absorbance at 405 nm and 37 C was noted using a microplate reader.
Acarbose (Sigma-Aldrich, Munich, Germany) and DMSO were used as reference inhibitors
and negative control, respectively. The tests were conducted in triplicate and percent
inhibitions were calculated using the following formula:
% Inhibition = [(A control − A sample)/A control] × 100
where ‘A’ stands for absorbance.
The IC50 values of the synthesized compounds and acarbose were also determined.
4. Conclusions
In conclusion, two novel series of benzimidazolium salts bearing amide function-
ality, -glucosidase inhibitory
9
(
a–
m
) and 10
(
a
–
m
), were synthesized and screened for
α
potential. The C2-substituted benzimidazolium derivatives 10
(a–m) emerged as excellent
α
-glucosidase inhibitors. The bromo-substituted compound 10d exhibited the best activity
among the series, with an IC50 value of 14 + 0.013 M which is 4-fold higher than the
µ
standard drug. Moreover, molecular docking studies of the most active compounds were
carried out to find the interaction mode and structure-activity relationship. This study,
being pioneering in its nature, will possibly be a landmark for further research for the
discovery of safer drugs for the treatment of diabetic mellitus.
1
Supplementary Materials: The following are available online: H-NMR, 13C-NMR, and MS spectra
of the synthesized compounds having potent enzyme inhibition activities.
Author Contributions: Conceptualization, M.A. and U.A.A.; methodology, I.A.K., M.A. and U.A.A.;
synthesis, I.A.K. and M.A.; characterization, I.A.K., M.A., S.S. and M.E.A.Z.; enzyme inhibition
studies, I.A.K., M.A. and U.A.A.; writing—original draft preparation, I.A.K., M.A., S.S., U.A.A. and
M.E.A.Z.; writing—review and editing, I.A.K., M.A., S.S., U.A.A. and M.E.A.Z.; supervision, M.A.
All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Higher Education Commission of Pakistan, grant number
NRPU-5614.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available in the Supplementary
Materials.
Acknowledgments: The authors (I.A.K., M.A. and U.A.A.) greatly acknowledge the Government
College University, Faisalabad for support.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.

Molecules 2021, 26, 4760
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