Synthesis of benzimidazole–based analogs as anti alzheimer’s disease compounds and their molecular docking studies

Article abstract of DOI:10.3390/molecules25204828

We synthesized 10 analogs of benzimidazole-based thiosemicarbazide 1 (a–j) and 13 benzimidazole-based Schiff bases 2 (a–m), and characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase (AchE) and butyrylcholinester

Full text of DOI:10.3390/molecules25204828

molecules
Article
Synthesis of Benzimidazole–Based Analogs as Anti
Alzheimer’s Disease Compounds and Their
Molecular Docking Studies
Bushra Adalat 1, Fazal Rahim 1, Muhammad Taha 2,
*
, Foziah J. Alshamrani 3,
El Hassane Anouar 4 , Nizam Uddin 5, Syed Adnan Ali Shah 6,7 , Zarshad Ali 1 and
Zainul Amiruddin Zakaria 8,9,
*
1
Department of Chemistry, Hazara University, Mansehra 21300, Pakistan; busraadalat@yahoo.com (B.A.);
fazalstar@gmail.com (F.R.); Zarshad11@yahoo.com (Z.A.)
2
3
4
Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdulrahman Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia
Neurology Department, King Fahad Hospital of University, Imam Abdulrahman Bin Faisal University,
P.O. Box 1982, Dammam 34211, Saudi Arabia; Fshamrani@iau.edu.sa
Department of Chemistry, College of Science and Humanities in Al-Kharj, Prince Sattam Bin
Abdulaziz University, Al-Kharj 11942, Saudi Arabia; anouarelhassane@yahoo.fr
Department of Chemistry, University of Karachi, Karachi 75270, Pakistan; nizamuddin.abbasi@uok.edu.pk
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Universiti Teknologi MARA Cawangan
Selangor Kampus Puncak Alam, Bandar Puncak Alam, Selangor 42300, Malaysia; syedadnan@uitm.edu.my
Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, Bandar,
Puncak Alam, Selangor 42300, Malaysia
5
6
7
8
9
Department of Biomedical Science, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,
UPM Serdang, Selangor 43400, Malaysia
Integrative Pharmacogenomics Institute (iPromise), Universiti Teknologi MARA Cawangan Selangor
Kampus Puncak Alam, Bandar Puncak Alam, Selangor 42300, Malaysia
*
Correspondence: mtaha@iau.edu.sa (M.T.); zaz@upm.edu.my (Z.A.Z.); Tel.: +00966-5020-57370 (M.T.);
+603-8947-2111 (Z.A.Z.); Fax: +603-8943-6178 (Z.A.Z.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 August 2020; Accepted: 16 October 2020; Published: 20 October 2020
Abstract: We synthesized 10 analogs of benzimidazole-based thiosemicarbazide
1 (a–j) and 13
benzimidazole-based Schiff bases 2 (a–m), and characterized by various spectroscopic techniques and
evaluated in vitro for acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) inhibition activities.
All the synthesized analogs showed varying degrees of acetylcholinesterase and butyrylcholinesterase
inhibitory potentials in comparison to the standard drug (IC50 = 0.016 and 4.5
analogs ), compounds 1b, 1c, and 1g having IC50 values 1.30, 0.60, and 2.40
showed good acetylcholinesterase inhibition when compared with the standard. These compounds
also showed moderate butyrylcholinesterase inhibition having IC50 values of 2.40, 1.50, and 2.40 M,
respectively. The rest of the compounds of this series also showed moderate to weak inhibition.
While amongst the second series of analogs ), compounds 2c, 2e, and 2h having IC50 values of
1.50, 0.60, and 0.90 M, respectively, showed moderate acetylcholinesterase inhibition when compared
µM. Amongst these
1
(
a–
j
µM, respectively,
µ
2 (a–m
µ
to donepezil. Structure Aactivity Relation of both synthesized series has been carried out. The binding
interactions between the synthesized analogs and the enzymes were identified through molecular
docking simulations.
Keywords: synthesis; acetylcholinesterase; butyrylcholinesterase; molecular docking; Schiff base;
thiosemicarbazide; SAR1
Molecules 2020, 25, 4828; doi:10.3390/molecules25204828
www.mdpi.com/journal/molecules

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1. Introduction
Acetylcholinesterase (AchE) and butyrylcholinesterase (BchE) are enzymes that play an important
role in the hydrolysis of acetylcholine into choline and acetic acid [ ]. This results in the shortage
of acetylcholine in areas such as cortex and hippocampus of brain, which are associated with
high psychological functions [ ]. These enzymes are responsible for Alzheimer’s disease (AD)—a
1
2
progressive and an irreversible brain disorder that causes disturbance of the cholinergic system of the
brain. This disturbance may cause memory loss, disorientation, cognitive impairment, and difficulty
in thinking and problem solving [3–5]. In the aging society, AD is the main cause of dementia.
These enzymes help in the aggregation of neurotoxic beta amyloid, which causes neuronal cell
apoptosis. One of the approaches for treating AD is to target both acetylcholinesterase and
butyrylcholinesterase [6,7].
The AchE has two binding sites, a catalytic site that is responsible for acetylcholine hydrolysis
and the peripheral site for beta amyloid interaction. The AchE interactions with protein give rise to a
complex AchE–A
β, which causes neurotoxicity. AchE is present in the brain, muscles, and cholinergic
neurons while BchE is present in intestine, liver, kidneys, heart, lungs, and serum [8,9
]. It plays a
main role in the breakdown of ester containing compounds. Normally, AchE is dominant in the brain,
while BchE function rises when acetylcholine function gradually decreases in the brain of a patient
with Alzheimer’s disease. Therefore, there is an intense need for a drug that can inhibit the activity of
both AchE and BchE [10].
For the treatment of Alzheimer’s disease, several drugs have been approved by the FDA such as
donepezil, rivastigmine (Figure 1), tacrine, and galantamine [11]. However, hepatotoxicity, insufficient
activity, and gastrointestinal disturbance limit the use and applicability of these drugs [12–16].
To overcome the side effect of synthetic ChEs inhibitors, researchers have shown a great interest in the
isolation of these compounds as an alternative, and many non-toxic bioactive ChEs inhibitors have
been isolated from natural sources [17–19]. Amongst these, donepezil and galantamine are selective
to AchE, whereas tacrine and rivastigmine inhibit both AchE and BchE. The Benzimidazole nucleus
possesses a wide spectrum of activities that ranges from antimicrobial activities to being used against
the world most deadly diseases. It has gained importance in medicinal chemistry because of its affinity
for different enzymes and protein receptors.
Figure 1. Chemical structures of Alzheimer’s disease drugs: donepezil and rivastigmin.
Our research group has been continuously attempting to synthesize new heterocyclic moieties
in search of lead therapeutics. Recently, we reported benzimidazole-based oxadiazole as an
anti-alpha-glycosidase [20], benzimidazole-bearing bis-Schiff base as alpha glucosidase inhibitor [21–23],
and 2-mercaptabenzimidazole as alpha amylase inhibitor [24]. Benzimidazole is a potent urease
inhibitor [25]. Here, we report two new series of benzimidazole thiosemicarbazides and benzimidazole
Schiff bases as novel Alzheimer inhibitors.

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2. Results and Discussion
2.1. Synthesis of Benzimidazole-Based Thiosemicarbazide
We treated 1H-benzimidazole-2-thiol (1) (1-mmol) with methyl (4-bromomethyl) benzoate
(II) (1-mmol) in ethanol and refluxed it for 3–4 h to give methyl4-(((1H-benzimidazole-2yl)thio)
methyl) benzoate (III) as an intermediate product. This intermediate was then further treated
with hydrazine hydrate in ethanol and refluxed for 5–6 h to give 4-(((1H-benzimidazole-2-yl)thio)
methyl) benzohydrazides (IV) as a second intermediate product. This intermediate product is then
treated with various substituted isothiocyanates which resulted in the final product
1 (a–j) (Scheme 1,
Table 1). The reaction completion was monitored by TLC. The product was filtered and washed with
distilled water.
Scheme 1. Synthesis of benzimidazole thiosemicarbazide.
2.2. Synthesis of Benzimidazole Schiff Base
In first step, we synthesized benzimidazole analogs by treating 1-methyl-3,4-diaminobenzene
(V) with ethyl-4-formyl benzoate (VI) in dimethyl formide (DMF) along with sodium meta bi-sulfite
(Na2S2O5) as a base under reflux condition for 4 to 5 h to yield intermediate (VII) as product.
The intermediate (VII) was treated with benzene hydrate in ethanol and was refluxed for 4–5 h to
yield intermediate (VIII). In the third step, the intermediate (VIII) was treated with various aromatic
aldehydes in methanol in the presence of glacial acetic acid to synthesize our final desired products
2
(a–m) (Scheme 2, Table 2).
Scheme 2. Synthesis of benzimidazole Schiff bases.

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Table 1. Structure and acetylcholinesterase inhibition activities of benzimidazole thiosemicarbazide analogs.
S. No
R
AChEIC50 (µM)
BuChE IC50 (µM)
1a
11.30 ± 0.30
22.60 ± 0.50
1b
1.30 ± 0.10
2.40 ± 0.10
1c
0.60 ± 0.05
5.90 ± 0.10
1.50 ± 0.10
1d
29.10 ± 0.30
1e
1f
6.20 ± 0.10
8.50 ± 0.20
10.30 ± 0.20
12.90 ± 0.20
1g
1h
0.80 ± 0.05
2.40 ± 0.10
5.50 ± 0.10
7.30 ± 0.10
12.80 ± 0.20
13.40 ± 0.20
1i
1j
12.90 ± 0.20
0.016 ± 0.12
24.40 ± 0.30
4.5 ± 0.11
Standard drug
donepezil

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Table 2. Structures and acetylcholinesterase inhibition activities of benzimidazole Schiff bases.
S. No
R
AChEIC50 (µM)
BuChE IC50 (µM)
2a
3.40 ± 0.10
7.50 ± 0.30
2b
8.50 ± 0.20
12.10 ± 4.0
2c
1.50 ± 0.10
3.10 ± 0.10
4.10 ± 0.10
6.20 ± 0.10
2d
2e
0.60 ± 0.05
2.20 ± 0.10
2f
4.20 ± 0.10
7.60 ± 0.10
2g
10.70 ± 0.20
22.60 ± 0.10
2h
0.90 ± 0.05
2.20 ± 0.30
2i
2j
12.70 ± 0.20
16.60 ± 0.30
24.40 ± 0.30
26.36 ± 0.50
2k
2l
18.90 ± 0.40
2.30 ± 0.10
29.6 ± 0.50
5.15 ± 0.10
2m
21.10 ± 0.30
0.016 ± 0.12
32.50 ± 0.50
4.5 ± 0.11
Standard drug
donepezil

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2.3. Structure Activity Relationship
2.3.1. Acetylcholinesterase Activity of Benzimidazole-Based Thiosemicarbazide
We have synthesized ten analogs
them for ChE inhibition. All the analogs showed acetylcholinesterase inhibition of varying degrees
ranging from 0.60 0.05 M to 12.90 0.20 M. All the analogs showed good inhibitory activities.
1 (a–j) of benzimidazole-based thiosemicarbazide and evaluated
±
µ
±
µ
The analogs 1c and 1g were the most active in the series. These analogs have chloro groups on
the phenyl ring. Analog 1c having 2 chloro groups at position 2 and 3 of phenyl ring has an IC50
value of 0.60
value of 0.80
±
0.05
0.50
µ
M, while in analog 1g, the chloro groups are at position 3 and 4 with the IC50
M. These results showed that enzyme inhibition activity mainly depends on
±
µ
the substitution pattern of chloro group on the phenyl ring. Similarly, analog 1b having floro group
at position 4 on phenyl ring also showed a good inhibition with an IC50 value of 1.30
while analog 1a having bromo group at position 2 of phenyl ring has an IC50 value of 11.30
±
0.01
µ
M,
M.
µ
This shows that inhibition activity not only depends on the position of substituent but also on the
number of substituents attached to the phenyl ring. The analog 1h having one methyl group at position
4 has an IC50 value of 3.50
value of 7.30 0.10 M. These analogs also showed good inhibition. The difference in their inhibition
values may be due to the difference in the position of methyl group on the phenyl ring. The analog 1d
having 2 methyl groups—one at position 2 and the other at 6—has an IC50 value of 5.60 M. The analog
1f having nitro group at position 3 has an IC50 value of 6.20 M and analog 5 having nitro group at
position 4 has an IC50 value of 8.50 M. The analog 1j having phenyl ring has an IC50 value of 12.96 M.
The analogs ) showed varying degrees of butyrylcholinesterase inhibition ranging from
0.10 to 29.10 0.30 M when compared to the standard drug donepezil having an IC50 value
0.11 M. The most active analogs among the series were 1b
excellent butyrylcholinesterase inhibition with IC50 values of 2.40
12.90 0.20 M, and 2.40 0.10
±
0.01 µM, while analog 1i having methyl group at 3 position has an IC50
±
µ
µ
µ
µ
µ
1(a–j
1.50
of 4.5
±
±
µ
±
µ
,
±
1c
,
1e
,
1f, and 1g that showed
0.10, 10.30 0.20,
0.10, 1.50
±
±
±
µ
±
µ
M, respectively. The analogs 1b, 1c, and 1g were the most active.
These analogs have halogen atoms on different positions of the phenyl ring. This shows that the
presence and position of halogen atoms on phenyl ring plays a major role in inhibition. Analog 1a
showed moderate inhibition with the IC50 value of 22.60
position 2 on the phenyl ring. Analogs 1e and 1f have a nitro group on the phenyl ring. The slight
difference in the inhibition value may be due to their different positions on the phenyl ring. Similarly,
±
0.50 µM. This analog has a bromine atom at
analogs 1h and 1i having a methyl group on the phenyl ring have an IC50 value of 12.80
13.40 0.20 M, respectively. Both these analogs have electron donating groups at different positions
on the phenyl ring. The analog 1d showed a poor inhibition with an IC50 value of 29.10 0.30 M.
±
0.20 µM and
±
µ
±
µ
This analog has 2 methyl groups at position 2 and 6, respectively. Another least active analog was
analog 1j having a phenyl ring with an IC50 value of 24.40 ± 0.30 µM.
2.3.2. Acetylcholinesterase Inhibition Activity of Benzimidazole Schiff Bases
The synthesized benzimidazole Schiff base analogs were examined for AchE and BchE. All the
compounds exhibited good to moderate activity when compared with the standard drug donepezil
having an IC50 value of AchE 0.016
most active analog among the series, showing a good inhibition of both AchE and BchE with the IC50
values of 0.60 0.05 M and 2.20 0.10 M, respectively. This analog has two chlorine atomsone at
position 3 and the other at 4of the phenyl ring. Similarly, analog 2c also exhibited significant inhibition
of AchE and BchE with an IC50 value of 1.50 0.10 M and 4.10 0.10 M, respectively. This analog
±
0.12
µM and BchE 4.5
±
0.11 µM. Analog 2e was found to be the
±
µ
±
µ
±
µ
±
µ
has two chlorine atoms—one at position 3 and the other at 4—and a hydroxyl group at position 2.
The ChE inhibition data in Table 1 show that the substitution of chlorine atoms on the phenyl ring plays
a significant role in inhibition. The next active analog among the series is analog 2h having a dimethyl
amino group at position 4 on the phenyl ring. This analog also showed a significant inhibition of both
AchE IC50 = 0.90 ± 0.50 µM and BchE IC50 = 2.20 ± 0.10 µM.

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Similarly, the inhibitory potential of the compounds having hydroxyl group and methoxy group
on phenyl ring was also good, e.g., analog 2l has a hydroxyl group at position 4 on the phenyl ring and
a methoxy group at positions 3 and 5, and exhibited inhibition for AchE and BchE with IC50 values
of 2.30
3 and a methoxy group at position 4 showed a selective inhibition toward AchE with an IC50 value
of 4.20 0.1 M, and for BchE, the IC50 = 7.60 0.10 M. Analog 2d having a hydroxyl group on
the naphthalene ring also showed a good inhibition toward AchE, IC50 = 3.10 0.10 M, and BchE,
IC50 = 6.20 0.10 M. This shows that the hydroxyl group plays an important role in inhibition.
Similarly, analog 2a and 2b having a nitro group on the phenyl ring were more selective in inhibition
toward AchE with IC50 values 3.40 0.10 M and 8.50 0.20 M, while for BchE they showed a
weak inhibition with IC50 values of 7.50 0.30 M and12.10 4.0 M, respectively. The rest of the
±
0.10
µM and5.15
±
0.10 µM, respectively. Analog 2f having a hydroxyl group at position
±
µ
±
µ
±
µ
±
µ
±
µ
±
µ
±
µ
±
µ
compounds of the series showed weak inhibitory activities.
2.4. Molecular Docking Studies
The inhibition concentration values of benzimidazole thiosemicarbazide derivatives 1 (a
–j)
and benzimidazole Schiff bases ) as effective acetylcholinesterase inhibitors are displayed in
2 (a–m
Table 3. The acetylcholinesterase inhibition by the synthesized derivatives may be influenced by the
type, number, and positions of the substituted functional groups of their basic skeletons (Table 3).
To rationalize the observed acetylcholinesterase inhibition by the synthesized derivatives, molecular
docking has been carried out to determine the binding modes between the synthesized derivatives
1
(a–
j
) and ) from one side and the active residues of the acetylcholinesterase from another side.
2 (a–m
Table 3 gathers the calculated free binding energies of the stable complexes’ ligand–acetylcholinesterase,
the number of established intermolecular hydrogen bonding between the synthesized compounds and
active site residues of acetylcholinesterase, and the closest residues to the docked compounds and
their IC50 values.
All the complexes formed between the synthesized derivatives 1 (a
active residues of acetylcholinesterase displayed negative binding energies, which demonstrates that
acetylcholinesterase inhibition by synthesized derivatives 1 (a ) and ) is a thermodynamic
favorable process (Table 3). The docking of the best active benzimidazole thiosemicarbazides (1c 1b
and 1g), benzimidazole Schiff bases (2c 2h and 2e), and donepezil into the active binding site of
–j) and 2 (a–m) and the
–
j
2 (a–m
,
,
acetylcholinesterase are displayed in Figure 2.
Figure 2. Docked poses of the best active benzimidazole thiosemicarbazides (1c, 1b, and 1g blue color
representations), benzimidazole Schiff bases (2c, 2h, and 2e green color representations) and donepezil
(brown color representation) into the active binding site of acetylcholinesterase.

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Table 3. Free binding energies, hydrogen bonding, and number of closest residues to the docked
benzimidazole thiosemicarbazide derivatives 1 (a ) and benzimidazole Schiff bases ) into the
binding site of acetylcholinesterase, and their IC50 values.
–j
2 (a–m
Number of Closest
Residues to the Docked
Ligand in the Active Site
No. of
Compound
Free Binding
Energy (kcal/mol)
H–Bonds (HBs)
IC50 ± SEM
Benzimidazole Thiosemicarbazide Derivatives
1a
1b
1c
1d
1e
1f
1g
1h
1i
−12.01
−11.97
−12.94
−12.26
−11.38
−11.39
−12.58
−12.24
−12.38
−12.23
4
5
4
5
8
5
8
4
7
9
9
6
9
8
7
9
10
8
11.30 ± 0.30
1.30 ± 0.10
0.60 ± 0.05
5.90 ± 0.10
6.20 ± 0.10
8.50 ± 0.20
0.80 ± 0.05
5.50 ± 0.10
7.30 ± 0.10
12.90 ± 0.20
5
4
1j
Benzimidazole Schiff Bases
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
−6.11
−7.46
−6.10
−6.54
−6.80
−5.84
−8.10
−4.49
−0.73
−9.07
−5.96
−6.02
−5.92
−11.11
5
3
1
2
1
2
2
1
1
0
1
1
1
1
10
5
8
7
8
7
5
8
7
4
8
8
10
8
3.40 ± 0.10
8.50 ± 0.20
1.50 ± 0.10
3.10 ± 0.10
0.60 ± 0.05
4.20 ± 0.10
10.70 ± 0.20
0.90 ± 0.05
12.70 ± 0.20
16.60 ± 0.30
18.90 ± 0.40
2.30 ± 0.10
21.10 ± 0.30
0.016 ± 0.12
donepezil (Std)
For benzimidazole thiosemicarbazide derivatives, binding energies of the stable complexes
vary with a maximal variation of 1.56 kcal mol−1. In accordance with the MIC values, the best
active compound 1c with the IC50 value of 0.60
µ
M has the lowest binding energy of 13 kcal/mol.
−
These variations are low enough to be considered as a strong descriptor in rationalizing the observed
acetylcholinesterase inhibition. However, the number of hydrogen bonding, its distances, and the
intermolecular interactions between the substitute groups of 1 (a–j) and the active residues may help
in understanding the observed acetylcholinesterase inhibition. For instance, the compounds 1e and
1f differ by the position of the substituted nitro group at the aromatic ring, where in the former
the nitro group is substituted at para position while in the latter it is substituted at meta position
(Scheme 1). Experimentally, 1e shows a higher activity than 1f. Their corresponding complexes formed
with acetylcholinesterase display similar binding energies with a variation less than 0.01 kcal/mol−1
The higher acetylcholinesterase inhibition of 1e compared with 1f may refer to the number of hydrogen
bonding that was established by the former with the active residues of acetylcholinesterase compared
with the latter (Figure 3). As can be seen in Figure 3 and Table 3, in 1e, the nitro group at para
position forms three hydrogen bonds with amino acids GLY B:122, ALA B:204, and GLY B:121 of
acetylcholinesterase with distances 2.79, 3.00, and 3.23 Å, respectively. However, in 1f, the nitro group
at meta position forms one hydrogen bonding with TYR B:124 with a distance of 2.85 Å.
.

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1c
1e
1f
Figure 3. Three-dimensional (right) and two-dimensional (left) closest interactions between the active
site residues of acetylcholinesterase and benzimidazole thiosemicarbazide derivatives 1c, 1f, and 1f.
For benzimidazole Schiff bases, binding energies of the stable complexes vary with variations in the
range 0–8.34 kcal/mol−1 with respect to the stable one. Similar to the benzimidazole thiosemicarbazide
derivatives, the potency of benzimidazole Schiff bases to inhibit acetylcholinesterase is strongly
related to the substituted functional groups, which may increase the stability of benzimidazole Schiff
bases–acetylcholinesterase complexes. For instance, the compounds 2a and 2b differ by the position
of the substituted nitro group at the aromatic, where in the former the nitro group is substituted at
ortho position while in the latter it is substituted at meta position (Scheme 2). Experimentally, 2a shows
a higher activity than 2b. The higher acetylcholinesterase inhibition of 2a compared with 2b may
refer to the stability of the complex formed by the former with acetylcholinesterase, which displays a
binding energy variation of 1.35 kcal/mol−1 compared to the complex formed with 2b. The stability of
2a–acetylcholinesterase may refer to the number of hydrogen bonds 2a established with the active
residues of acetylcholinesterase compared with 2b (Figure 4). As can be seen in Figure 4 and Table 3,
in 2a–acetylcholinesterase, five hydrogen bonds are formed between 2a and amino acids ASP B:74,

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TYR B:72, SER B:203, GLY B:122 and SER B:125, while in 2b–acetylcholinesterase, three hydrogen bonds
are formed between 2b and amino acids ALA B:204, GLY B:121 and GLY B:122.
2a
2b
Figure 4. Three-dimensional (right) and two-dimensional (left) closest interactions between active site
residues of acetylcholinesterase and benzimidazole Schiff bases 2a and 2b.
3. Materials and Methods
3.1. General Procedure for the Synthesis of Benzimidazole-Based Thiosemicarbazide 1(a–j)
We treated 1H-benzimidazole-2-thiol (
1) (1-mmol) with methyl (4-bromomethyl) benzoate
(II) (1-mmol) in ethanol and refluxed it for 3–4 h to give methyl4-(((1H-benzimidazole-2yl)thio)
methyl) benzoate (III) as an intermediate product. This intermediate was then further treated with
hydrazine hydrate in ethanol and refluxed for 5–6 h to give 4-(((1H-benzimidazole-2-yl)thio) methyl)
benzohydrazides (IV) as a second intermediate product. This intermediate product is then treated
with various substituted isothiocyanate which gave the final product 1(a–j). The reaction completion
was monitored by TLC. The product was filtered and washed with distilled water.
2-(4-(((1H-benzo[d]imidazol-2◦-yl)thio)methyl)benzoyl)-N-(2-bromophenyl)hydrazine-1-carbothioamide (1a):
Yield (88%); m.p.: 314–315 C. IR
ν
cm−1 (KBr disk): 3460 cm−1 (NH stretch), 3163 cm−1 (Ar.C-H
stretch), 1641 cm−1 (C = O), 1582 cm−1 (C = N), 1552 cm−1 (C-N), 550 (C-Br); HNMR (500 MHz,
1
DMSO-d6):
Ar), 7.62 (d, J = 6.2 Hz, 1H, Ar), 7.57(m, 4H, Ar), 7.37 (t, J = 6.3 Hz, 2H, Ar), 7.19 (t, J = 6.2 Hz, 1H, Ar),
7.12(t, J = 6.3 Hz, 2H, Ar), 4.65 (S, 2H, S-CH2). 13CNMR (125 MHz, DMSO-d6):
182.3, 164.8, 150.2,
δ 12.58 (S,1H, NH), 10.5 (S,1H, NH), 9.8 (S,1H, NH), 9.5 (S,1H, NH), 7.89 (d, J = 5.8 Hz, 2H,
δ
144.1, 140.3, 139.2, 139.3, 137.3, 132.7, 132.0, 130.2, 128.6, 127.3 (2C), 127.6 (2C), 122.8 (2C), 121.9, 116.4,
116.2, 34.5.

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2-(4-(((1H-benzo[d]imidazol-2◦-yl)thio)methyl)benzoyl)-N-(4-fluorophenyl)hydrazine-1-carbothioamide (1b):
Yield (84%); m.p.: 319–320 C. IR
ν
cm−1 (KBr disk): 3490 cm−1 (NH stretch), 3185 cm−1 (Ar.C-H
stretch), 1645 cm−1 (C = O), 1582 cm−1 (C = N), 1552 cm−1 (C-N), 1050 (C-F); HNMR (500 MHz,
DMSO-d6): 12.58 (S,1H, NH), 10.49 (S,1H, NH), 9.73 (S,2H, NH), 7.88 (d, J = 6.4 Hz, 2H, Ar), 7.57 (m, 4H,
Ar), 7.36 (t, J = 5.9 Hz, 3H,Ar), 7.12 (t, J = 6.3 Hz, 3H,Ar), 4.65 (S, 2H,S-CH2). 13CNMR (125 MHz,
1
DMSO-d6):
δ 182.6, 165.1, 163.2, 150.1, 143.9, 140.8, 138.4, 138.6, 134.6, 130.4, 130.3, 127.4(2C), 127.1,
127.0, 123.3, 123.1, 116.3, 116.2, 115.9, 115.7, 34.6.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(2,3-dichlorophenyl) hydrazine-1-carbothioamide (1c):
◦
Yield (79%); m.p.: 321–322 C. IR
ν
cm−1 (KBr disk): 3396 cm−1 (NH stretch), 3159 cm−1 (Ar.C-H
stretch), 1649 cm−1 (C = O), 1571 cm−1 (C = N), 1550 cm−1 (C-N), 745 (C-Cl); HNMR (500 MHz,
DMSO-d6): 12.57(S, 1H, NH), 10.58 (S, 1H, NH), 9.92 (S, 1H, NH), 9.75 (S, 1H, NH) 7.88 (d, J = 6.2 Hz,
2H, Ar), 7.57 (t, J = 6.9 Hz, 4H, ArH), 7.35 (t, J = 5.95 Hz, 3H, Ar), 7.12 (t, J = 6.2 Hz, 2H, ArH), 4.61(S,
1
2H, S-CH2). 13CNMR (125 MHz, DMSO-d6):
δ 183.2, 165.2, 150.1, 145.1, 140.9, 139.2, 139.1, 138.2, 137.4,
132.9, 132.2, 129.8, 128.0, 127.5, 127.3, 127.3, 127.2, 123.4, 123.2, 116.1, 116.3, 34.5.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(2,6-dimethylphenyl) hydrazine-1-carbothioamide
(
1d): Yield (81%); m.p.: 311–312 ◦C. IR cm−1 (KBr disk): 3396 cm−1 (NH stretch), 3165 cm−1 (Ar.C-H
ν
stretch), 1650 cm−1 (C = O), 1576 cm−1 (C = N), 1548 cm−1 (C-N); 1HNMR (500 MHz, DMSO-d6): 12.58
(S, 1H, NH), 10.49 (S, 1H, NH), 9.54 (S, 1H, NH), 9.35 (S, 1H, NH), 7.8 (d, J = 6.6 Hz, 2H, Ar), 7.54 (m,
3H, Ar), 7.35 (d, J = 6.1 Hz, 2H, Ar), 7.23(m, 3H, Ar), 7.1 (m, 1H, Ar), 4.61(S, 2H, S-CH2). 13CNMR
(125 MHz, DMSO-d6):
δ 182.5, 165.2, 149.9, 143.5, 141.7, 138.8, 138.6, 135.8, 134.9, 134.7, 128.1, 128.3,
127.6(2C), 127.4, 127.3, 127.0, 122.8, 122.7, 115.6, 115.5, 34.7, 18.4(2C).
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(4-nitrophenyl)hydrazine-1-carbothioamide (1e):
◦
Yield (78%); m.p.: 319–320 C. IR
ν
cm−1 (KBr disk): 3426 cm−1 (NH stretch), 3195 cm−1 (Ar.C-H
stretch), 1652 cm−1 (C = O), 1579 cm−1 (C = N), 1546 cm−1 (C-N), 1530 (NO2), 1360 (NO2); 1HNMR
(500 MHz, DMSO-d6): 12.6 (S, 1H, NH), 10.58 (S, 1H, NH), 10.12 (S, 1H, NH), 10.07 (S, 1H, NH), 8.20
(d, J = 6.9 Hz, 2H, Ar), 7.8 (d, J = 5.61 Hz, 4H, Ar), 7.5 (m, 3H, Ar), 7.1 (m, 3H, Ar), 4.63(S, 2H, S-CH2).
13CNMR (125 MHz, DMSO-d6):
δ 182.2, 165.4, 149.5, 145.6, 144.9, 144.1, 140.6, 139.3, 139.2, 127.8, 127.7,
127.4, 127.3, 124.8, 124.7, 124.1, 124.0, 123.9, 123.7, 115.8, 115.6, 34.8.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(3-nitrophenyl)hydrazine-1-carbothioamide (1f):
Yield (83%); m.p.: 3316–317 ◦C. IR
ν
cm−1 (KBr disk): 3412 cm−1 (NH stretch), 3175 cm−1 (Ar.C-H
stretch), 1647 cm−1 (C = O), 1575 cm−1 (C = N), 1542 cm−1 (C-N), 1533 (NO2), 1355 (NO2); 1HNMR
(500 MHz, DMSO-d6): 10.60 (S, 1H, NH), 10.06 (S, 1H, NH), 8.4 (S, 1H, NH), 8.0 (S, 1H, NH), 7.9 (d, J =
6.3 Hz, 2H, Ar), 7.8 (d, J = 6.9 Hz, 3H, Ar), 7.68 (m, 4H, Ar), 7.13 (m, 3H, Ar), 4.64 (S, 2H, S-CH2).
13CNMR (125 MHz, DMSO-d6):
δ 182.7, 165.6, 149.2, 147.9, 142.1, 140.4, 139.1, 139.2, 138.3, 131.1, 129.1,
128.8, 128.7, 128.4, 128.0, 122.4, 122.2, 119.6, 119.4, 109.8, 109.8, 34.7.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(3,4-dichlorophenyl)hydrazine-1-carbothioamide (1g
)
Yield: (78%); m.p.: 312–313 ◦C. IR
ν
cm−1 (KBr disk): 3405 cm−1 (NH stretch), 3189 cm−1 (Ar.C-H
1
stretch), 1652 cm−1 (C = O), 1576 cm−1 (C = N), 1554 cm−1 (C-N), 780 (C-Cl); HNMR (500 MHz,
DMSO-d6): 12.59 (S, 1H, NH), 10.54 (S, 1H, NH), 9.97 (S, 1H, NH), 9.85 (S, 1H, NH), 7.8 (d, J = 6.6 Hz,
2H, Ar), 7.7 (d, J = 6.9 Hz, 1H, Ar), 7.6 (m, 4H, Ar), 7.5 (d, J = 6.5 Hz, 2H, Ar) 7.1 (m, 2H, Ar), 4.62
(S, 2H, S-CH2). 13CNMR (125 MHz, DMSO-d6):
δ 182.6, 165.5, 149.3, 142.1, 139.3, 137.8, 137.7, 135.6,
134.9, 131.2, 129.7, 129.6, 128.6, 128.6, 128.1, 128.0, 121.5, 121.6, 121.2, 116.3, 116.1, 34.6.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(p-tolyl)hydrazine-1-carbothioamide (1h): Yield
(80%); m.p.: 323–324 ◦C. IR
ν
cm−1 (KBr disk): 3360 cm−1 (NH stretch), 3180 cm−1 (Ar.C-H stretch),
2960 (C-CH3), 1645 cm−1 (C = O), 1574 cm−1 (C = N), 1555 cm−1 (C-N); 1HNMR (500 MHz, DMSO-d6):
12.59 (S, 1H, NH), 10.46 (S, 1H, NH), 9.68 (S, 1H, NH), 7.8 (d, J = 6.1 Hz, 2H, Ar), 7.5 (d, J = 6.3 Hz, 4H,
Ar), 7.27 (d, J = 6.2 Hz, 2H, Ar), 7.11(d, J = 5.9 Hz, 4H, Ar), 4.65 (S, 2H, S-CH2). 13CNMR (125 MHz,

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DMSO-d6):
δ 181.9, 165.6, 149.3, 141.9, 139.5, 137.2, 137.5, 136.6, 134.1, 129.7, 129.4, 128.5(2C), 128.3,
128.2, 125.9, 125.8, 121.2, 121.3, 117.3, 117.0, 34.7, 25.0.
2.-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-(m-tolyl)hydrazine-1-carbothioamide (1i) Yield:
(76%); m.p.: 313–314 ◦C. IR
ν
cm−1 (KBr disk): 3410 cm−1 (NH stretch), 3160 cm−1 (Ar.C-H stretch),
2980 (C-CH3), 1648 cm−1 (C = O), 1571 cm−1 (C = N), 1552 cm−1 (C-N); 1HNMR (500 MHz, DMSO-d6):
11.0 (S, 1H, NH), 10.47 (S, 1H, NH), 9.7 (S, 1H, NH), 9.6 (S, 1H, NH), 7.8 (d, J = 6.4 Hz, 2H, Ar), 7.5 (d, J =
6.6 Hz, 4H, Ar), 7.19 (m, 4H, Ar) 6.9 (d, J = 5.9 Hz, 2H, Ar), 4.6 (S, 2H, S-CH2). 13CNMR (125 MHz,
DMSO-d6):
δ 182.2, 165.5, 149.6, 141.7, 139.0, 137.9, 137.7, 137.5, 136.8, 129.6, 128.7, 128.6, 128.1, 128.0,
125.6, 125.5, 123.3, 123.0, 122.8, 117.8, 117.6, 34.7, 23.9.
2-(4-(((1H-benzo[d]imidazol-2-yl)thio)methyl)benzoyl)-N-phenylhydrazine-1-carbothioamide (1j) Yield: (77%);
m.p.: 309–310 ◦C. IR
ν
cm−1 (KBr disk): 3405 cm−1 (NH stretch), 3186 cm−1 (Ar.C-H stretch), 1651 cm−1
(C = O), 1572 cm−1 (C = N), 1550 cm−1 (C-N); 1HNMR (500 MHz, DMSO-d6): 12.59 (S, 1H, NH), 10.4
(S, 1H, NH), 9.75 (S, 1H, NH), 9.69 (S, 1H, NH), 7.8 (d, J = 6.5 Hz, 2H, Ar), 7.5 (d, J = 6.7 Hz, 4H, Ar), 7.3
(t, J = 6.1 Hz, 2H, Ar), 7.1(m, 5H, Ar), 4.6 (S, 2H, S-CH2). 13CNMR (125 MHz, DMSO-d6):
δ 10:182.7,
165.6, 149.2, 143.5, 140.2, 138.6, 138.3, 136.7, 128.8, 128.9, 128.6, 128.7, 128.1, 128.2, 127.3, 124.5(2C), 122.9,
122.8, 116.8, 116.5, 34.7.
3.2. General Procedure for the Synthesis of Benzimidazole-Based Schiff Bases 2(a–m)
In first step, we synthesized benzimidazole analogs by treating 1-methyl-3,4-diaminobenzene
(V) with ethyl-4-formyl benzoate (VI) in dimethyl formide (DMF) along with sodium meta bi-sulfite
(Na2S2O5) as a base under reflux condition for 4 to 5 h to yield intermediate (VII) as product.
The intermediate (VII) was treated with benzene hydrate in ethanol and was refluxed for 4–5 h to
yield intermediate (VIII). In the third step, the intermediate (VIII) was treated with various aromatic
aldehydes in methanol in the presence of glacial acetic acid to synthesize our final desired products
2 (a–m).
4-(5-methoxy-1H-benzo[d]imidazol-2-yl)-N0-(2-nitrobenzylidene)benzohydrazide (2a): Yield (81%); m.p.: IR
ν
cm−1 (KBr disk): 3365 cm−1 (NH stretch), 3174 cm−1 (Ar.C-H stretch), 1653 cm−1 (C = O), 1572 cm−1
(C = N), 1551cm−1 (C-N), 1525 (NO2), 1345 (NO2) 1160 (C-O-C); 1HNMR (500 MHz, DMSO-d6): 12.3
(S,1H, NH), 9.6 (S,1H, NH), 8.3 (d, J = 6 Hz, 2H, Aromatic H), 8.1 (d, J = 6.6 Hz, 2H,Aromatic H), 8.0
(m, 3H, Aromatic H), 7.7 (d, J = 6.5 Hz, 2H, Aromatic H), 7.6 (S,1H, Aromatic H), 7.4 (d, J = 6.7 Hz,1H,
Aromatic H), 3.5 (S, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.4, 157.6, 153.3, 147.7, 142.5, 140.2,
138.6, 135.1, 134.5, 133.7, 132.4, 130.8, 130.7, 130.2, 129.5, 128.1, 128.0, 124.8, 117.8, 112.4, 10I.3, 57.3.
4-(5-methoxy-1H-benzo[d]imidazol-2-yl)-N0-(3-nitrobenzylidene)benzohydrazide (2b): Yield (84%); m.p.:
311–312 ◦C. IR
ν
cm−1 (KBr disk): 3360 cm−1 (NH stretch), 3181 cm−1 (Ar.C-H stretch), 1654 cm−1
(C = O), 1578 cm−1 (C = N), 1557 cm−1 (C-N), 1528 (NO2), 1348 (NO2) 1168 (C-O-C); 1HNMR (500 MHz,
DMSO-d6): 11.6 (S,1H, NH), 9.9 (S,1H, N = CH), 8.8(S,1H, Aromatic H), 8.5(S,1H, Aromatic H), 8.3(d, J =
6.8 Hz,1H, Aromatic H), 8.1(m, 3H, Aromatic H), 7.7 (d, J = 6 Hz, 2H Aromatic H), 7.6 (m, 2H Aromatic),
7.5(d, J = 6.3 Hz,1H Aromatic H), 3.5(S, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.8, 157.9,
154.1, 147.5, 145.8, 140.1, 138.9, 135.0, 134.8, 132.9, 131.9, 130.6, 130.5, 129.8, 128.3, 128.3, 126.0, 120.8,
116.9, 110.8, 102.5, 56.7.
N0-(3,4-dichloro-2-hydroxybenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl) benzohydrazide (2c): Yield
(85%); m.p.: 319–320 ◦C. IR
ν
cm−1 (KBr disk): 3570 cm−1 (OH stretch), 3355 cm−1 (NH stretch), 3160
cm−1 (Ar.C-H stretch), 1643 cm−1 (C = O), 1576 cm−1 (C = N), 1554 cm−1 (C-N), 1180 (C-O), 768 (C-Cl);
1HNMR (500 MHz, DMSO-d6): 11.7 (S, 1H, NH), 8.7(S,1H, NH), 8.6 (S, 1H, OH), 8.3(S,1H,N = CH),
8.2(d, J = 6 Hz, 2H, Aromatic H), 8.1(d, J = 6.7 Hz, 2H, Aromatic H), 8.0(d, J = 6.9 Hz, 2H, Aromatic)
7.3(m,1H, Aromatic H), 3.8 (S, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.5, 162.3, 158.1, 153.2,
147.8, 140.3, 139.1, 138.6, 134.9, 133.3, 130.6, 130.6, 129.9, 127.9, 127.8, 125.2, 123.6, 119.2, 117.4, 111.8,
101.4, 56.8.

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N0-((3-hydroxynaphthalen-2-yl)methylene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2d) Yield:
(77%); m.p.: 304–305◦C. IR cm−1 (KBr disk): 3540 cm−1 (OH stretch), 3320 cm−1 (NH stretch), 3174
ν
cm−1 (Ar.C-H stretch), 1649 cm−1 (C = O), 1579 cm−1 (C = N), 1558 cm−1 (C-N), 1140 (C-O); 1HNMR
(500 MHz, DMSO-d6): 12.7(S, 1H, NH), 9.9(br s, 1H, 0H), 9.8(S, 1H, NH), 9.5(S, 1H, N = CH), 8.4(d, J =
6.4 Hz, 2H, Aromatic H) 8.3(d, J = 5.8 Hz, 1H, Aromatic H), 7.9(m, 4H, Aromatic H), 7.8(d, J = 6.6 Hz,
2H, Aromatic H), 7.5(m, 2H, Aromatic H), 7.3(d, J = 6.4 Hz, 1H), 7.1(d, J = 7.2 Hz, 1H, Aromatic H),
3.9(S, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.4, 158.2, 157.5, 153.1, 146.8, 140.3, 139.8, 138.6,
134.1, 132.9, 129.8, 129.7, 128.2, 127.4, 127.5, 126.3, 125.8, 124.9, 123.5, 117.3, 116.2, 111.8, 111.3, 105.9,
100.8, 56.3.
N’-(3,4-dichlorobenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2e): Yield (72%); m.p.:
306–307 ◦C. IR
ν
cm−1 (KBr disk): 3415 cm−1 (NH stretch), 3176 cm−1 (Ar.C-H stretch), 1646 cm−1 (C =
O), 1579 cm−1 (C = N), 1558 cm−1 (C-N), 1143 (C-O), 790 (C-Cl); 1HNMR (500 MHz, DMSO-d6): 12.1(S,
1H, NH), 9.5(S, 1H, NH), 8.6(S, 1H, N = CH), 8.4(br s, 1H Aromatic H), 8.3 (d, J = 6 Hz, 2H, Aromatic
H), 8.1(m, 3H, Aromatic H), 7.7(d, J = 6.6 Hz, 2H, Aromatic H), 7.5(S, 1H, Aromatic H), 7.4(d, J = 6.4
Hz,1H, Aromatic H), 3.7(S, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 163.9, 156.8, 153.1, 140.4,
139.6, 137.9, 135.2, 134.2, 133.8, 133.4, 132.9, 130.6, 130.1, 129.8, 129.9, 128.1, 127.6, 127.6, 115.8, 110.5,
100.6, 56.9.
N0-(3-hydroxy-4-methoxybenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2f); Yield
(87%); m.p.: 312–313◦C. IR
ν
cm−1 (KBr disk): 3563 cm−1 (OH stretch), 3326 cm−1 (NH stretch), 3171
cm−1 (Ar.C-H stretch), 1653 cm−1 (C = O), 1567 cm−1 (C = N), 1551 cm−1 (C-N), 1172 (C-O); 1HNMR
(500 MHz, DMSO-d6): 11.9(S, 1H, NH), 9.9(S, 1H, NH), 8.3(br s, 1H, OH), 8.1(S, 1H, N = CH), 7.7(d, J =
6.7 Hz, 2H, Aromatic H), 7.6(m, 2H, Aromatic H), 7.4(S,1H, Aromatic H), 7.2(m, 2H, Aromatic H), 6.8(d,
J = 6.8 Hz, 1H, Aromatic H), 6.6(m, 2H, Aromatic H), 3.8(S,6H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.7, 156.2, 153.0, 152.6, 148.2, 146.9, 140.5, 138.6, 134.2, 133.0, 130.9, 130.1, 130.0, 127.7, 127.6, 123.0,
116.2, 115.3, 112.8, 111.2, 101.4, 57.4, 56.2.
N0-(anthracen-9-ylmethylene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2g): Yield (83%);
m.p.: 307–308 ◦C. 1HNMR (500 MHz, DMSO-d6): 11.7(s,1H, NH), 10.7(s,1H,NH), 8.2(s, 1H, N = CH),
8.1(d, J = 7.3 Hz, 2H, Aromatic H), 7.8(m, 2H, Aromatic H), 7.6(d, J = 7 Hz, 2H, Aromatic H), 7.4(d, J
= 6.8 Hz, 1H, Aromatic H), 7.3(m, 4H, Aromatic H), 7.0(m, 3H, Aromatic H), 6.9(s,1H, Aromatic H),
6.8(d, J = 6 Hz, 1H, Aromatic H), 3.7(s, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.2, 157.1,
152.9, 143.0, 140.2, 137.9, 134.2, 132.8, 132.1, 132.0, 130.1, 130.0, 128.9, 128.9, 128.8, 128.2, 128.2, 128.1,
128.1, 127.6, 127.5, 125.8, 125.8, 125.5, 125.5, 124.1, 116.8, 112.1, 100.9, 56.3.
N0-(4-(dimethylamino)benzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2h): Yield (77%);
m.p.: 317–318 ◦C. IR
ν
cm−1 (KBr disk): 3410 cm−1 (NH stretch), 3171 cm−1 (Ar.C-H stretch), 1658
cm−1 (C = O), 1572 cm−1 (C = N), 1554 cm−1 (C-N), 1147 (C-O); 1HNMR (500 MHz, DMSO-d6): 12.8(s,
1H, NH), 11.6(s, 1H, NH), 8.5(s, 1H, N = CH), 8.0(d, J = 6 Hz, 2H, Aromatic H), 7.5(d, J = 6.5 Hz, 1H,
Aromatic H), 7.4(m, 4H, Aromatic H), 7.3(s, 1H, Aromatic H), 6.8(d, J = 6.5 Hz, 2H, Aromatic H), 6.7(d,
J = 6.6 Hz, 1H, Aromatic), 3.0(s, 3H, OCH3), 2.9(6H, N-(CH3)2). 13CNMR (125 MHz, DMSO-d6):
δ
164.3, 157.0, 153.7, 152.8, 147.1, 140.1, 137.8, 134.6, 132.6, 130.2, 130.2, 128.4, 128.5, 127.5, 127.4, 123.0,
116.9, 112.4, 112.3, 111.7, 100.8, 56.3, 42.5, 42.5.
N0-(4-(benzyloxy)benzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2i): Yield: (85%);
m.p.: 310–311 ◦C. IR
ν
cm−1 (KBr disk): 3440 cm−1 (NH stretch), 3164 cm−1 (Ar.C-H stretch), 1659
cm−1 (C = O), 1582 cm−1 (C = N), 1552 cm−1 (C-N), 1191 (C-O); 1HNMR (500 MHz, DMSO-d6): 11.8(s,
1H, NH), 8.7(s, 1H, NH), 8.3(s, 1H, N = CH), 8.2(d, J = 7.0 Hz, 2H, Aromatic H), 7.8(d, J = 6.4 Hz, 2H,
Aromatic H), 7.7(m, 2H, Aromatic H), 7.2(d, J = 5.9 Hz, 3H, Aromatic H), 6.9(d, J = 6.6 Hz, 1H, Aromatic
H), 6.8(s, 2H, Aromatic), 3.7(s, 3H, OCH3), 3.0(m, 6H, NCH3),. 13CNMR (125 MHz, DMSO-d6):
δ 164.1,
161 8, 156.8, 153.2, 147.4, 140.2, 138.4, 137.1, 134.4, 132.9, 130.1, 130.1, 130.2, 130.2, 129.1, 129.1, 127.9,
127.8, 127.7, 127.4, 127.1, 127.0, 116.9, 113.9, 113.8, 109.8, 100.5, 72.1, 56.6.

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4-(5-methoxy-1H-benzo[d]imidazol-2-yl)-N0-(naphthalen-1-ylmethylene)benzohydrazide (2j): Yield (80%);
m.p.: 303–304 ◦C. IR
ν
cm−1 (KBr disk): 3392 cm−1 (NH stretch), 3179 cm−1 (Ar.C-H stretch), 1645 cm−1
(C = O), 1576 cm−1 (C = N), 1552 cm−1 (C-N), 1173 (C-O); 1HNMR (500 MHz, DMSO-d6): 12.1(s, 1H,
NH), 9.0(s, 1H, NH), 8.8(s, 1H, N = CH), 8.2(s, 1H, Aromatic H), 8.0(m, 6H, Aromatic H), 7.8(d, J
= 7.2 Hz, 2H, Aromatic H), 7.6(m, 3H, Aromatic H), 7.5(d, J = 6.8 Hz, 1H, Aromatic H), 7.3(s, 1H,
Aromatic H), 2.9(s, 3H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.5, 156.8, 152.9, 146.9, 140.0, 138.6,
136.5, 134.3, 134.0, 133.1, 129.9, 129.8, 129.8, 128.4, 128.1, 128.1, 127.8, 127.7, 127.1, 127.0, 126.4, 126.3,
117.3, 111.8, 100.6, 56.8.
N0-(3-bromo-2,4-dimethoxybenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2k): Yield
(75%); m.p.: 310–311 ◦C. IR
ν
cm−1 (KBr disk): 3405 cm−1 (NH stretch), 3160 cm−1 (Ar.C-H stretch),
1655 cm−1 (C = O), 1573 cm−1 (C = N), 1552 cm−1 (C-N), 1182 (C-O), 742 (C-Br); 1HNMR (500 MHz,
DMSO-d6): 12.0(s, 1H, NH), 8.9(s, 1H, NH), 8.7(s, 1H, N = CH), 8.2(d, J = 6.7 Hz, 2H, Aromatic H),
8.1(d, J = 6.2 Hz, 2H, Aromatic H), 7.6(s, 1H, Aromatic H), 7.5(d, J = 6.2 Hz, 1H, Aromatic H), 7.4(s, 2H,
Aromatic H), 7.3(d, J = 6.1 Hz, 1H, Aromatic H), 3.4(s, 9H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ
164.8, 157.1, 155.0, 153.2, 149.6, 146.5, 139.8, 138.6, 134.4, 133.0, 130.1, 130.0, 127.9, 127.8, 119.3, 117.3,
116.7, 116.0, 114.9, 110.9, 101.5, 56.8, 55.7, 55.2.
N0-(3-hydroxy-2,4-dimethoxybenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl) benzohydrazide (2l): Yield
(78%); m.p.: 317–318 ◦C. IR
ν
cm−1 (KBr disk): 3512 cm−1 (OH stretch), 3365 cm−1 (NH stretch), 3170
cm−1 (Ar.C-H stretch), 1658 cm−1 (C = O), 1574 cm−1 (C = N), 1552 cm−1 (C-N), 1168 (C-O); 1HNMR
(500 MHz, DMSO-d6): 12.2(s, 1H, NH), 8.4(s, 1H, N = CH), 8.2(d, 6.2 Hz, 2H, Aromatic H), 7.7(m, 2H,
Aromatic H), 7.6(d, J = 6 Hz, 2H, Aromatic H), 7.2(d, J = 6.1 Hz, 1H, Aromatic H), 6.7(d, J = 6.1 Hz, 1H,
Aromatic H), 6.6(s, 1H, Aromatic H), 3.0(s, 9H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.6, 156.5,
152.9, 149.5, 149.3, 147.0, 140.1, 139.9, 138.3, 134.6, 132.7, 129.9, 129.9, 128.5, 127.5, 127.4, 116.8, 110.9,
104.8, 104.7, 101.5, 56.1, 56.1, 55.9.
N0-(2,4-dimethoxybenzylidene)-4-(5-methoxy-1H-benzo[d]imidazol-2-yl)benzohydrazide (2m): Yield (81%);
m.p.: 307–308 ◦C. IR
ν
cm−1 (KBr disk): 3438 cm−1 (NH stretch), 3164 cm−1 (Ar.C-H stretch), 1656 cm−1
(C = O), 1576 cm−1 (C = N), 1554 cm−1 (C-N), 1170 (C-O); 1HNMR (500 MHz, DMSO-d6): 12.0(s,1H,
NH), 11.6(s, 1H, NH), 8.5(s, 1H, N = CH), 8.3(d, J = 6.3 Hz, 2H, Aromatic H), 8.1(m, 3H, Aromatic H),
7.7(d, J = 6 Hz, 1H, Aromatic H), 7.4(d, J = 6.8 Hz, 1H, Aromatic H), 6.77(d, J = 6.6 Hz, 2H, Aromatic
H), 6.70(d, J = 6.3 Hz, 1H, Aromatic H), 2.98(s, 9H, OCH3). 13CNMR (125 MHz, DMSO-d6):
δ 164.1,
163.5, 159.9, 156.8, 153.0, 146.5, 139.9, 138.2, 134.7, 133.4, 132.9, 130.1, 130.1, 128.1, 128.0, 116.8, 112.0,
109.6, 105.9, 102.3, 100.7, 56.1, 55.9, 55.9.
3.3. Docking Studies
The intermolecular binding modes between benzimidazole thiosemicarbazide derivatives 1 (
a–j)
and benzimidazole Schiff bases 2 ( ), and the active residues of acetylcholinesterase have been
a–m
examined using Autodock package [26]. The acetylcholinesterase and its original docked donepezil
geometries were download from the RCSB data bank web site (PDB code 4EY7) [27]. The active
site is identified based on co-crystallized receptor–ligand complex structure of acetylcholinesterase.
The re-docking of the original ligand donepezil into the active site is well reproduced with a RMSD
value of 0.63 Å. Molecular geometries of benzimidazole-based analog derivatives were minimized
at Merck molecular force field 94 (MMFF94) level44, and saved as PDB files. A docking study was
performed using Lamarckian genetic algorithm, with 500 as the total number of run for the binding
sites for the original ligands of the synthesized derivatives. In each respective run, a population of 150
individuals with 27,000 generations and 250,000 energy evaluations were employed. The operator
weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. The docking
calculations have been carried out using an Intel (R) Core (TM) i5-3770 CPU @ 3.40 G Hz workstation.

Molecules 2020, 25, 4828
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4. Conclusions
In this study, ten analogs of benzimidazole thiosemicarbazide
benzimidazole Schiff bases
1
(
a
–
j
) and 13 analogs of
1
2
(
a
–
m
) were synthesized and characterized through 13C NMR, H,
and HR-EIMS. All the synthesized analogs of the two series were examined for their acetylcholinesterase
and butycholinesterase inhibitory potentials which exhibited varying degrees of biological activities.
Amongst the synthesized analogs of series 1, a
0.60 0.50, and 2.40 0.10 M, respectively, exhibited excellent acetylcholinesterase inhibition
potential as compared to their inhibition potential to donepezil, and these analogs also showed excellent
butycholinesterase inhibition with IC50 values of 2.40 0.10, 1.50 0.10 M, and 2.40 0.10 M as
–
j
), 1b
,
1c, and 1g having IC50 values 1.30
±
0.10,
±
±
µ
±
±
µ
±
µ
compared to their inhibition potential to donepezil. While other analogs of the series showed good to
moderate activities (Table 1), the rest of the compounds of this series also showed moderate to weak
inhibition. The analogs of the second series
of 1.50 0.10, 0.60 0.05, and 0.90 0.05
inhibitory potentials and also the same analogs 2c
and 2.20 0.30 M showed that this series displayed excellent butycholinesterase inhibition when
2
(
a
–
m
) compounds 2c
M, respectively, showed excellent acetylcholinesterase
2e, and 2h with IC50 values of 4.10 0.10, 2.20 0.10,
, 2e, and 2h having IC50 values
±
±
±
µ
,
±
±
±
µ
compared to the standard drug donepezil (Table 2). The other analogs of this series showed good to
moderate activity. SAR studies of these analogs was established through molecular simulation studies.
Author Contributions: Conceptualization, M.T. and F.J.A.; methodology, F.R. and B.A.; software, E.H.A.;
validation, E.H.A. and B.A.; formal analysis, N.U.; investigation, F.J.A.; resources, N.U.; data curation, M.T.
and Z.A.Z.; writing—original draft preparation, F.R.; writing—review and editing, S.A.A.S. and Z.A.Z.;
visualization, Z.A.; supervision, Z.A.Z.; project administration, F.J.A. and M.T.; funding acquisition, F.J.A.
and Z.A. All authors have read and agreed to the published version of the manuscript.
Funding: The authors would like to acknowledge the Higher Education Commission of Pakistan for providing a
research grant under the National Research Program for Universities (Project Nos. 5721 & 5092).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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