5.

215

Article

Biologically Potent Benzimidazole-

Based-Substituted Benzaldehyde
Derivatives as Potent Inhibitors for
Alzheimer’s Disease along with
Molecular Docking Study

Bushra Adalat, Fazal Rahim, Wajid Rehman, Zarshad Ali, Liaqat Rasheed, Yousaf Khan,
Thoraya A. Farghaly , Sulaiman Shams, Muhammad Taha, Abdul Wadood et al.

https://doi.org/10.3390/ph16020208
 pharmaceuticals

Article
Biologically Potent Benzimidazole-Based-Substituted
Benzaldehyde Derivatives as Potent Inhibitors for Alzheimer’s
Disease along with Molecular Docking Study
Bushra Adalat 1 , Fazal Rahim 1 , Wajid Rehman 1, *, Zarshad Ali 1 , Liaqat Rasheed 1 , Yousaf Khan 2 ,
Thoraya A. Farghaly 3 , Sulaiman Shams 4 , Muhammad Taha 5 , Abdul Wadood 4 , Syed A. A. Shah 6
and Magda H. Abdellatif 7

1 Department of Chemistry, Hazara University, Mansehra 21300, Pakistan

2 Department of Chemistry, COMSATS University, Islamabad 45550, Pakistan
3 Department of Chemistry, Faculty of Applied Science, Umm Al-Qura University,
P.O. Box 715, Makkah Almukkarramah 24382, Saudi Arabia
4 Department of Biochemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan
5 Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC),
Imam Abdul Rahman Bin Faisal University, P.O. Box 31441, Dammam 11099, Saudi Arabia
6 Faculty of Pharmacy, Universiti Teknologi MARA Cawangan Selangor Kampus Puncak Alam, Bandar,
Puncak Alam 42300, Malaysia
7 Department of Chemistry, College of Sciences, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
* Correspondence: [email protected]

Abstract: Twenty-one analogs were synthesized based on benzimidazole, incorporating a substi-

tuted benzaldehyde moiety (1–21). These were then screened for their acetylcholinesterase and
butyrylcholinesterase inhibition profiles. All the derivatives except 13, 14, and 20 showed various
inhibitory potentials, ranging from IC50 values of 0.050 ± 0.001 µM to 25.30 ± 0.40 µM against
Citation: Adalat, B.; Rahim, F.; acetylcholinesterase, and 0.080 ± 0.001 µM to 25.80 ± 0.40 µM against butyrylcholinesterase, when
Rehman, W.; Ali, Z.; Rasheed, L.; compared with the standard drug donepezil (0.016 ± 0.12 µM and 0.30 ± 0.010 µM, against acetyl-
Khan, Y.; Farghaly, T.A.; Shams, S.; cholinesterase and butyrylcholinesterase, respectively). Compound 3 in both cases was found to
Taha, M.; Wadood, A.; et al.
be the most potent compound due to the presence of chloro groups at the 3 and 4 positions of the
Biologically Potent
phenyl ring. A structure-activity relationship study was performed for all the analogs except 13, 14,
Benzimidazole-Based-Substituted
and 20, further, molecular dynamics simulations were performed for the top two compounds as well
Benzaldehyde Derivatives as Potent
as the reference compound in a complex with acetylcholinesterase and butyrylcholinesterase. The
Inhibitors for Alzheimer’s Disease
along with Molecular Docking Study. molecular dynamics simulation analysis revealed that compound 3 formed the most stable complex
Pharmaceuticals 2023, 16, 208. with both acetylcholinesterase and butyrylcholinesterase, followed by compound 10. As compared
https://doi.org/10.3390/ to the standard inhibitor donepezil both compounds revealed greater stabilities and higher binding
ph16020208 affinities for both acetylcholinesterase and butyrylcholinesterase.

Academic Editor: Thierry Besson

Keywords: benzimidazole; alzheimer disease; docking study; acetylcholinesterase and butyrylcholinesterase
Received: 7 December 2022
Revised: 27 January 2023
Accepted: 28 January 2023
Published: 30 January 2023 1. Introduction
Dementia is one of the major global challenges today. This dementia is mainly caused
by Alzheimer’s disease (AD) [1]. The number of Alzheimer’s patients increases day by day
Copyright: © 2023 by the authors.
as the population and age increase, by 2023 it will be up to 74.7 million people suffering
Licensee MDPI, Basel, Switzerland. from AD [2]. AD is a neurodegenerative disorder and reveals itself by the progressive loss
This article is an open access article of cognitive functions, speech impairment, and memory loss [3]. To understand the mecha-
distributed under the terms and nism of action of the disease, several pathways have been identified. Neurochemically, it
conditions of the Creative Commons is characterized by the constant shortage in cholinergic neurotransmission that affects the
Attribution (CC BY) license (https:// cholinergic neurons in the basal forebrain [4]. This loss of cholinergic neurons results in
creativecommons.org/licenses/by/ the decrease of AchE in the cognition related areas of the brain, such as the cerebral cortex
4.0/). and the hippocampus [5]. AchE catalyzes the hydrolysis of acetylcholine into choline and

Pharmaceuticals 2023, 16, 208. https://doi.org/10.3390/ph16020208 https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2023, 16, 208 2 of 17

acetic acid by two types of enzymes, acetylcholinesterase (AChE) and butyrylcholinesterase

(BuChE) [6]. The brains of AD patients are rich in the AchE enzyme, which is responsible
for the degradation of AchE [7]. Although at the cholinergic synapses the function of
AchE is well known, the physiological function of BuChE is not familiar. However, it was
observed that BuChE provides a backup to AChE when its activity decreases, and is active
at high concentrations of AChE [8]. Another non-cholinergic role of AchE is the formation
of senile plaque by the deposition of beta-amyloid (βA) [9]. In the beginning, therapeutic
strategies for treating diminished cholinergic neurotransmission were mainly focused on
AChE inhibitors, but studies have shown the significance of both AChE and BuChE [10].
Meanwhile, the inhibition of both these enzymes by a single molecule has been found to be
a more important therapeutic agent for the treatment of AD. Cholinesterase inhibitors are
classified into two classes: (i) specific, when they are used for the inhibition of AChE only;
and (ii) nonspecific when they inhibit both AChE and BuChE. Among the drugs approved
by the FDA, Tacrine and Rivastigmine are non-specific, while Donepezil and Galant-amine
are specific, inhibiting AchE only [11]. However, these drugs have limited efficiencies and
have various side effects, mostly at high doses.
Nitrogen containing heterocycles have received much attention in the field of drug
discovery to cure various diseases, also various inhibitors are present in the literature to
treat AD. Among these, benzimidazole is an important bioactive molecule, which is found
naturally in Vitamin B12, and is widely used for the synthesis of novel drugs [12,13]. The
heterocyclic ring of benzimidazole is also known as glyoxalin, 1,3-diazaole, imidazole,
and imidazole is a term frequently used to show the five membered heterocyclic system
containing tertiary N and imino functionalities in their structure. Imidazole rings are also
found in naturally occurring compounds such as alpha-amino acids, and many proteins,
such as purine, histamine, and biotin [14,15]. Benzimidazole scaffolds have been shown
to have a varied range of biological activities such as antifungal [16,17], anticancer [18],
antiulcer [19], antibacterial [20–22], anti-Alzheimer’s [23–25], and anti-inflammatory [26].
Various marketed drugs contain the benzimidazole moiety in their core structure; these
include: Nocodazole (anticancer), Tiabendazole (antifungal and ant parasitic), Flubendazole
(anthelmintic), and omeprazole (proton pump inhibitor).
Our research group is continuously trying to introduce new heterocyclic compounds
containing different moieties, for better therapeutic agency. Recently, we have reported
the benzimidazole bearing moiety in the skeleton for an α-glucosidase inhibitor [27,28], an
anti-Alzheimer’s inhibitor [29], and an alpha-glycosidase inhibitor [30–33], but still there
is a need to discover more compounds for future research. So, we identified interesting
profiles of substituted benzimidazole derivatives and screened them for anti-Alzheimer’s
activity as shown in Figure 1.
Pharmaceuticals 2023, 16, 208 3 of 17

Figure 1. Rationale of the current work [27–30].

2. Results and Discussion

2.1. Chemistry
Twenty-one scaffolds of 4-methoxybenzene-1,2-diamine and various substituted ben-
zaldehyde based benzimidazoles were synthesized. First of all, 4-methoxybenzene-1,2-
diamine (I, 0.5 mmol) with substituted benzaldehyde (0.5 mmol) in DMF (10 mL), in the
presence of the catalyst sodium meta-bisulfate (0.5 mmol), and the resulting mixture was
refluxed for 2–3 h to obtain the targeted derivatives (1–21) with the appropriate yield. Thin
Layer Chromatography (TLC) was employed for the monitoring of the reaction till the
conformation. Different spectroscopic techniques such as 13CNMR, 1HNMR, and HR-EIMS
were carried out to confirm the structure of the synthesized analogs (Scheme 1).

Scheme 1. Synthesis of benzimidazole analogs.

2.2. Molecular Docking Studies

We conducted a molecular docking analysis to understand the binding interactions
of compounds with active site residues of the selected enzyme. Based on the co-crystal of
the crystallographic structures, the synthesized compounds were docked into the active
sites of specific enzymes. The native inhibitor from the AChE was removed and then
Pharmaceuticals 2023, 16, 208 4 of 17

re-docked into the active site using MOE. This approach was used to validate the docking
procedure [34]. Utilizing PyMOL 2.3, the re-docked complex was then superimposed on the
reference co-crystallized ligand to calculate the root mean square deviation (RMSD) which
was predicted to be 0.56, revealing the validity of the docking protocol. The native inhibitor
was removed from the AChE and then re-docked according to the docking technique, every
compound was given a total of twenty conformations. The most active compounds’ top-
ranked conformations were chosen for future studies and visual inferences. The docking
results revealed that compound 3 showed excellent inhibitory potential against AChE
against both targets. In the case of the binding mode of this compound, we have found
that several key residues adopted key interactions with the essential compound moiety
as showed in Figure 2a. The detailed interaction profile revealed residues Tyr 334 (via
Pi-stacking) with the 5-ring. Also, we have found that this residue was further stabilized by
the residue Asp72, because of the proximity, and having Van der Walls interactions between
them. Several other Pi-stacking interactions were observed which might have a key role
in the stability of this compound in the active site of AChE, i.e., Ph. residues around this
moiety. The high potential might be due to the attached electron withdrawing groups at
the meta and para positions of the benzene. These groups withdraw some of the electrons
from the Pi-system, and subsequently, create a partial positive charge over the benzene ring,
and next this ring is compelled to adopt several Pi-stacking interactions with key residues.
Comparing this result with other similar compounds in the series also showed potential
against the target enzyme. Compounds 10 and 11 showed similar behaviours; these
compounds have similarly attached electron withdrawing groups at different positions.
The only differences are the attached hydroxyl group at the ortho position and the attached
Cl position. The lower potential compared to potent compounds might be due to the
attached OH group, which is categorized as an electron donating group. The potential of
this compound might be due to the Cl group withdrawing electrons from the Pi-system, and
ultimately the donating group donates electrons to stabilize the overall system, Figure 2b,c.
On the other hand, compound 3 showed high potential against BuChE. The mechanism
of inhibition might be due the reason discussed in the above section, Figure 2a. The
second potent compound in the series, 11, showed a good potential compared with the
least active compound. Though the differences found among the most active and second
ranked compounds is just the variation in the attached groups, which ultimately affects the
activity of the overall compound. The protein-ligand interaction profiles of other active
compounds are shown in Figure 2b,c. The computational and bioassay studies showed that
these compounds are the most active and significant compounds, which showed the best
potential against both the targets. The drug likenesses of the most active compounds are
presented in Table 1.

Table 1. Drug likeness of the most active compounds.

S.No Weight logP H-donor H-acceptor

Compound 3 293.15 4.11 1 2
Compound 10 309.15 3.65 2 3
Compound 11 293.15 4.14 1 2
Reference 380.51 1.04 1 3
Pharmaceuticals 2023, 16, 208 5 of 17

Figure 2. Docking interactions of the most active compound in the series, is for 3, 10 & 11 against the
AChE (a–c) and BuChE (a–c). Green dotted line represents bond.

2.3. Post Simulation Analysis

2.3.1. RMSD (Root Mean Square Deviation) Analysis
In order to assess the stability of the systems, the root mean square deviation (RMSD)
was calculated for each complex. Figure 3 displays the RMSD curve for the two best docking
score compounds 3 and 10, and a reference, in complex with AChE. All systems reported
an RMSD that ranged from 1 to 2 Å. The RMSD of compound 3 in complex with AChE
revealed a minor fluctuation between 5–10 ns and 20–25 ns, after that the system converged
and remained stable until the end of the 50 ns MD simulation. Compound 10 in complex
with AChE revealed fluctuations between 5–10 and 20–30 ns, but soon attained equilibrium
and remained stable during the whole 50 ns MD run. The reference compound donepezil
in complex with AChE was stable during the first 10 ns, after that the system showed major
fluctuations up to 30 ns. During this period, the RMSD increased to 2 Å. After that, the
system converged and attained stability and remained stable up to 50 ns MD. Further, the
RMSD analyses of compounds 3 and 10, and the reference, in complex with BuChE are
presented in Figure 4. The RMSD analyses demonstrated that the compounds were stable,
and fit into the binding pockets of AChE and BuChE. The compound 3 complex exhibits
the lowest RMSD among all the complexes, as shown in Figure 4. As compared to the
standard inhibitor, compounds 3 and 10 showed a greater stability for both of the targets.
Pharmaceuticals 2023, 16, 208 6 of 17

Figure 3. RMSD plot of compound 3 (blue color), compound 10 (magenta color), and donepezil (red
color) in complex with AChE.

Figure 4. RMSD plot of compound 3 (blue color), compound 10 (magenta color), and donepezil (red
color) in complex with BuChE.
Pharmaceuticals 2023, 16, 208 7 of 17

2.3.2. RMSF (Root Mean Square Fluctuation) Analysis

To take into account changes in the amino acid residue, the RMSF of the AChE and
BuChE backbone residues were calculated. To further understand how ligand binding
impacts the flexibility of each residue during the simulation, the RMSF was investigated.
The stability, stiffness, and compactness of the receptors were indicated by the amino acid
residues with the lowest RMSF values. Figure 5 shows the estimated RMSF values for
selected compounds and for the AChE complexes. Residues including Val 280, Leu 281,
Val 282, Asn 283, His284, Glu285, Trp286, His287, Val288, Leu289, Pro290, Ser 399, Trp 500,
Pro 501, and Pro 502 indicated a high degree of fluctuation, while the active site residues
such as Tyr 334, Asp72, Phe 288, Phe 331, Tyr 121 indicated great stability. Figure 5 displays
RMSF plots for compounds 3, 10, and the reference, in complex with AChE, while Figure 6
displays the RMSF plots for compounds 3, 10, and the reference, in complex with BuChE.

2.4. Binding Energy Calculation

Numerous techniques have been employed for virtual screening, docking, molecu-
lar dynamics, and MMPBSA free energy calculations of compounds. In this study, the
MMPBSA.py python script was used for calculating the binding energy [35]. The binding
energy of compound 3 in complex with AChE was found to be −56 kcal/mole, while
compound 3 in complex with BuChE was found to be −37 kcal/mole. The binding energies
of compound 10 in complex with AChE and BuChE were predicted to be −52 kcal/mole
and −31 kcal/mole, respectively. The delta G value of the best-docked complexes was
good as compared to the standard inhibitor donepezil. The results are shown in Table 2.

Figure 5. RMSF plot of compound 3 (blue color), compound 10 (magenta color), and donepezil (red
color) in complex with AChE.
Pharmaceuticals 2023, 16, 208 8 of 17

Figure 6. RMSF plot of compound 3 (blue color), compound 10 (magenta color), and donepezil (red
color) in complex with BuChE.

Table 2. MMGBSA analysis. All units are reported in kcal/mole.

Complex vdW EEL ESURF EGB ∆G TOTAL

Compound3_AChE −59.8288 −10.1721 −7.8161 5.5700 −56.4166
Compound10_AChE −57.0777 −2.0551 −
−6.1095 12.6979 −52.5444
−
Donepezil_ache −55.1897 −4.2318 −5.7198 14.9143 −50.6618
−
− Compound3_BuChE −41.5357 −2.6287 −4.2604 10.9467 −37.4781
Compound10_BuChE −35.5181 −5.0815 −3.4819 12.2147 −31.8668
Donepezil_BuChE −33.1783 −2.1543 −3.1661 11.2146 −30.1013

2.5. Pharmacokinetics (ADMET) vdW

Properties ofEEL
Finally Selected
ESURFCompounds
EGB ΔG TOTAL
Traditional drug design and
− discovery
− is a dangerous
− investment, which
− is commonly
exposed to unpredicted failures− in various
− stages of−the drug discovery and
− development.
One main reason for these failures is the efficiency and safety faults, which are related
− − − −
largely to absorption, distribution, metabolism, excretion (ADME) properties, and different
− − − −
toxicities (T). Therefore, rapid ADMET analysis is urgently needed to reduce the chance
− process.
of failure in the drug discovery − pkCSM −is an online server that − conveniently
performs six types of drug-likeness analysis (five rules of Lipinski and −one prediction
− − −
model), 31 ADMET endpoints prediction includes three basic properties, six absorptions,
three distributions, ten metabolisms, two excretions, and seven toxicities. pkCSM is a free
online server accessible at http://biosig.unimelb.edu.au/pkcsm/prediction.
ADMET properties have been studied for the three best compounds finally selected,
i.e., compounds 3, 10, and 11, along with the reference compound, having an effective IC50
value in vitro, with the best docking scores. All these compounds obeyed Lipinski’s rule of
five, according to which, “a drug like compound must not have more than 10 hydrogen
bond acceptors, not more than 5 hydrogen bond donors, a octanol-water coefficient not
Pharmaceuticals 2023, 16, 208 9 of 17

more than 5, and the molecular weight must be less than 500 Daltons”. They also had
ADMET properties in the required allotted range, which guarantees their drug likeness.
Assays were carried out for compounds 3, 10, and 11, and their ADMET properties are
shown in Tables 3 and 4.

Table 3. Pharmacokinetic (ADMET) properties.

Compound’s ID
Category Property with Unit
3 10 11 Donepezil
Water solubility (log mol/L) −4.959 −3.705 −8.852 −6.483
Absorption Intestinal absorption (%) 92.4 90.359 86.645 88.561
Skin permeability(log Kp) − −0.823 − −1.672− −2.178 − −2.728
− − − −
VDss (human) (log L/kg) − 0.487 − 0.196 − 0.803 − 0.943
Distribution BBB permeability − 0.458 − 0.21 − 0.453 − 0.514
− − − −
− − − −
CNS permeability (log PS) −1.394 −1.394 −0.914 −0.97
CYP2D6 substrate No No No No
− − − −
CYP3A4 substrate − No − −
No − Yes − Yes
− − −
Metabolism CYP1A2 inhibitor Yes Yes Yes No
CYP2C19 inhibitor No No Yes No
CYP2C9 inhibitor No No Yes No
Total Clearance(log ml/min/kg) 0.281 0.225 −0.294 0.685
Excretion
Renal OCT2 substrate No No −
− No No
−
AMES toxicity No No No No
Max. tolerated dose (human) (log mg/kg/day) 0.888 0.702 0.81 0.404
Toxicity Oral Rat Acute Toxicity (LD50) (mol/kg) 2.16 2.326 2.405 3.401
Hepatotoxicity No No No No
Skin Sensitisation Yes Yes No No

Table 4. Structure, acetylcholinesterase and butyrylcholinesterase inhibition activity of benzimidazole analogs.

AChE IC50 BuChE IC50

Code No. R
(µM ± SEM) (µM ± SEM)

1 2.10 ± 0.10 1.60 ± 0.10

2 5.10 ± 0.10 5.90 ± 0.10

3 0.050 ± 0.001 0.080 ± 0.001

4 3.40 ± 0.10 3.60 ± 0.10

Pharmaceuticals 2023, 16, 208 10 of 17

Table 4. Cont.

AChE IC50 BuChE IC50

Code No. R
(µM ± SEM) (µM ± SEM)

5 25.30 ± 0.40 25.80 ± 0.40

6 4.10 ± 0.10 5.10 ± 0.20

7 5.80 ± 0.10 5.90 ± 0.10

8 6.80 ± 0.20 4.70 ± 0.10

9 2.30 ± 0.10 2.10 ± 0.10

10 0.10 ± 0.001 0.40 ± 0.001

11 0.20 ± 0.001 0.30 ± 0.001

12 5.10 ± 0.10 5.80 ± 0.10

13 N. A. N. A.

14 N. A. N. A.
Pharmaceuticals 2023, 16, 208 11 of 17

Table 4. Cont.

AChE IC50 BuChE IC50

Code No. R
(µM ± SEM) (µM ± SEM)

15 0.80 ± 0.001 0.90 ± 0.001

16 1.30 ± 0.10 2.10 ± 0.10

17 6.10 ± 0.10 3.90 ± 0.10

18 11.30 ± 0.30 9.80 ± 0.30

19 9.20 ± 0.20 6.80 ± 0.20

20 N. A. 15.20 ± 0.30

21 2.10 ± 0.10 2.70 ± 0.10

Donepezil 0.016 ± 0.12 0.30 ± 0.010

2.6. Structure-Activity Relationship (SAR)

We have synthesized twenty-one scaffolds of substituted benzimidazole that exhibit vary-
ing degrees of cholinesterase inhibition potential when compared with the standard drug
Donepezil, having IC50 values of 0.016 ± 0.12 and 0.30 ± 0.010 for acetylcholinesterase and
butyrylcholinesterase, respectively. The potent compounds among the series were compounds
1–2, 4, 6–12, 15–16, and 9–21, having IC50 values ranging from 0.050 ± 0.001 to 5.80 ± 0.10 for
acetylcholinesterase, and 0.080 ± 0.001 to 5.90 ± 0.10 for butyrylcholinesterase. The most potent
compounds against AChE were, compound 3 (IC50 = 0.050 ± 0.001, 0.080 ± 0.001), which had
two chloro groups on the phenyl ring at positions 3 and 4, and compound 10 (IC50 = 0.10 ± 0.001),
which had a hydroxyl group at position 2 and chloro groups at positions 3 and 5. The most
potent compound against BuChE was compound 11 (IC50 = 0.30 ± 0.001), which had chloro
groups at positions 2 and 4, shown in Figure 7. This shows that the position of substituents will
affect the inhibition potential of analogs.
Pharmaceuticals 2023, 16, 208 12 of 17

Figure 7. SAR studies of compounds 3, 10, and 11.

Scaffolds 15 and 16 both have hydroxyl groups, but their positions are different.
In compound 15 the hydroxyl group is at the para position, and it has IC50 values of
0.80 ± 0.001 and 0.90 ± 0.001 for AChE and BuChE, respectively. While for analog 16,
the hydroxyl group is at the meta position, and the IC50 values of this compound are
1.30 ± 0.10 and 2.10 ± 0.10 for AChE and BuChE, respectively (Figure 8). This shows that
the ideal position for the hydroxyl group is para.

Figure 8. SAR studies of compounds 15 and 16.

Comparison of the nitro substituted compounds, 1 (IC50 = 2.10 ± 0.10, 1.60 ± 0.10),
2 (IC50 = 5.10 ± 0.10, 5.90 ± 0.10), and 4 (IC50 = 3.40 ± 0.10, 3.60 ± 0.10) shows that analog 1,
in which the nitro group is at the para position, is superior in activity to analogs 2 and 4,
which have the nitro group at the ortho and meta positions. Thus, positional changes of the
nitrogen atom causes a decline in the activity in the ortho and meta analogs (Figure 9).
Pharmaceuticals 2023, 16, 208 13 of 17

Figure 9. SAR studies of compounds 1, 2, and 4.

Similarly, if we compare analog 9 (2.30 ± 0.10, 2.10 ± 0.10), having one hydroxyl and
one methoxy group, with analog 21 (2.10 ± 0.10, 2.70 ± 0.10), having one hydroxyl and two
methoxy groups, analog 21 showed a better inhibition potential than analog 9. Analog 7,
having a benzoyloxy group on the phenyl ring also exhibited good inhibition, with an
IC50 value of 5.80 ± 0.10 for AChE, and 5.90 ± 0.10 for BuChE (Figure 10). While analog
6 (IC50 = 4.10 ± 0.10, 5.10 ± 0.20), having a methyl group, which is an electron donating
group, also showed potent inhibition, but less than analogs 3, 10, and 11. The SAR study
shows that the presence of electron withdrawing groups or electron donating groups on the
phenyl ring play a crucial role in the inhibition profile. The rest of the series of compounds
exhibited poor inhibition.

Figure 10. SAR studies of compounds 9 and 21.

3. Materials and Methods

3.1. Procedure for the Synthesis of Benzimidazole Analogs (1–21)
First of all, 4-methoxybenzene-1,2-diamine (I, 0.5 mmol), with variously substituted
benzaldehyde (0.5 mmol) in DMF (10 mL) in the presence of the catalyst sodium meta-
bisulfate (0.5 mmol), and the resulting mixture was refluxed for 2–3 h to obtain the targeted
derivatives (1–21) with appropriate yield. Thin Layer Chromatography (TLC) was em-
ployed for the monitoring of the reaction until the desired conformation. At the end, the
final product was separated, washed with distilled water, and then dried.

3.1.1. 5-Methoxy-2-(4-Nitrophenyl)-1H-Benzo[d]-Imidazole (1)

1HNMR (500 MHz, DMSO-d6): δ 11.31 (s, 1H, NH), 8.14 (d, J = 7.4 Hz, 2H, Ar-H),
8.10 (d, J = 7.9 Hz, 1H, Benzimidazole-H), 7.84 (s, 1H, Benzimidazole-H), 7.52 (d, J = 6.8 Hz,
1H, Benzimidazole-H), 7.40 (d, J = 7.3 Hz, 2H, Ar-H), 2.33 (s, 3H, -OCH3), 13CNMR (125 MHz,
DMSO-d6): δ 168.4, 163, 160.3, 149.7, 147.8, 143.3, 140.4, 126.1, 123.6, 121.3, 118.2, 117.7, 113.9, 45.6.
HR-EIMS: m/z Calcd. for C14H11N3O3 [M] + 269.0800; Found. 269.0796.
Pharmaceuticals 2023, 16, 208 14 of 17

3.1.2. 5-Methoxy-2-(3-Nitrophenyl)-1H-Benzo[d]-Imidazole (2)

1HNMR (500 MHz, DMSO-d6): δ 11.36 (s, 1H, NH), 8.18 (dd, J = 7.2, 2.0 Hz, 1H, Ar-H),
8.12 (d, J = 6.9 Hz, 1H, Benzimidazole-H), 7.87 (s, 1H, Benzimidazole-H), 7.58 (d, J = 6.4 Hz,
1H, Benzimidazole-H), 7.24 (s, 1H, Ar-H), 7.20 (t, J = 6.5 Hz, 1H, Ar-H), 7.06 (dd, J = 7.2,
1.9 Hz, 1H, Ar-H), 2.38 (s, 3H, -OCH3), 13CNMR (125 MHz, DMSO-d6): δ 168.5, 160.5,
149.6, 147.9, 143.1, 140.0, 134.7, 126.2, 123.7, 121.4, 118.4, 117.8, 113.0, 45.7. HR-EIMS: m/z
Calcd. for C14H11N3O3 [M] + 269.0800; Found. 269.0796.
Sample analysis can be found in Supplementary Materials.

3.2. Inhibition Assay Protocol of Acetylcholinesterase and Butyrylcholinesterase

The AChE/(BuChE) inhibitory test was measured using a spectrophotometric method
developed by Elman et al. [31,32]. A buffer solution containing phosphate (pH 8.0) of
volume 140 µL; 20 µL of each AChE/BuChE solution; and 20 µL of the test sample were
incubated at room temperature for 15 min. AChE/BuChE 10 µL was used to start the
reaction, followed by the addition of DTNB. ATCh or BTCh hydrolyzed the reaction of
DTNB with thiocholine for 15 min; unrestricted by AChE and BuChE enzymatic hydrolysis.
E − S/E × 100, where E&S represent enzyme activity with and without test samples, were
used to compute the percentage (percent) inhibition (30). Each sample’s inhibitory activity
was measured in terms of IC50 (g/mL) or µM. For all substances, the IC50 values were
derived using a generic graph. The graph was created in Excel and the IC50 values were
derived by taking Y = 50 and determining the x value as IC50 .

3.3. Molecular Docking

A molecular docking conformation study was carried out to understand the basics
of the binding modes of the synthesized compounds against the selected enzymes, acetyl-
cholinesterase (AChE) and butrylcholinesterase (BuChE), in order to corroborate the in vitro
and in silico results using the Molecular Operating Environment (MOE) software package.
Both targets’ crystal structures were extracted from the protein data bank (RCSB) by using
codes (PDB) 1ACL for AChE and 1P0P for BuChE. While the proteins and all synthesized
compounds were protonated and the energy was minimized by using the MOE Dock
modules default parameters, resulting in optimal structures for both the proteins and the
compounds. Docking investigations were conducted using these optimized structures of
the target proteins and compounds. Our earlier investigations have detailed descriptions
of the protocol [33,34].

3.4. Molecular Dynamics simulation

To confirm the stability of the docked complexes, a systematic molecular dynamics
simulation was run using the AMBER20 software. The TIP3P water model was used
to dissolve each system in a rectangular box, and the systems were then neutralized by
introducing counter ions [35]. Steepest descent minimization for 6000 cycles and conju-
gate gradient minimization for 3000 cycles were used to minimize the energy of all the
neutralized systems. The systems were quickly heated to 300 K after the completion of
the energy minimization. Then, each system underwent a two-step equilibration process
at a constant temperature of 300 K and 1 atm. The density was equilibrated for 2 ns in
the first stage using a weak constraint. The systems were then allowed to stabilize for
more than 2 ns without any constraints in the second step. The production phase was then
conducted for 50 ns. The particle mesh Ewald technique was employed for long-range
electrostatic interactions with a cutoff distance of 10.0 [36]. Covalent bonds were calculated
using the SHAKE algorithm [37,38]. The cpptraj package was used to evaluate trajectory
data while molecular dynamic simulations were carried out using the GPU-supported
pmemd.cuda [39,40].
Pharmaceuticals 2023, 16, 208 15 of 17

4. Conclusions
In conclusion, we have designed and synthesized a range of benzimidazole base
derivatives (1–21), and all of the synthesized derivatives were assessed for inhibition of
acetylcholinesterase and butyrylcholinesterase. All the synthetic analogs showed different
values of IC50 , ranging from 0.050 ± 0.001 to 25.30 ± 0.40 against acetylcholinesterase,
and from 0.080 ± 0.001 to 25.80 ± 0.40 against butyrylcholinesterase, as compared with
the standard drug donepezil (0.016 ± 0.12 µM against acetylcholinesterase, and
0.30 ± 0.010 µM against butyrylcholinesterase). Compounds 13, 14, and 20 did not show
any activity. Compound 3 in both cases showed excellent inhibitory potential due to the
presence of chloro groups at the 3 and 4 positions of the phenyl ring, and a structure-activity
relationship study was carried out for all the analogs except 13, 14, and 20. All the newly
synthetic compounds were characterized by various spectroscopic techniques to confirm
the structures, such as 1HNMR, 13CNMR, and HR-EIMS. A molecular docking study
was performed to understand the binding interactions between the analogs and proteins.
It is clear from the results that benzimidazole derivatives in the present work could be
considered as active neuroprotective therapeutics in the future.

Supplementary Materials: The following supporting information can be downloaded at:

https://www.mdpi.com/article/10.3390/ph16020208/s1, Spectral analysis.
Author Contributions: Methodology, B.A.; Software, Z.A., T.A.F., S.S. and A.W.; Validation, F.R. and
T.A.F.; Formal analysis, Z.A., L.R. and Y.K.; Investigation, S.A.A.S. and M.H.A.; Resources, M.H.A.;
Data curation, F.R. and M.T.; Writing—original draft, W.R. and Y.K.; Visualization, L.R.; Supervision,
W.R. All authors have read and agreed to the published version of the manuscript.
Funding: This work was financially supported by Higher Education Commission (HEC) Pakistan
through research grant under National Research Program for University Project No. 5721 & 5092.
One of us T.A.F. would like to thank the Deanship of Scientific Research at Umm Al-Qura University
for supporting this work by Grant Code: (22UQU4350477DSR13).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article and Supplementary Materials.
Conflicts of Interest: The authors declare that they have no known competing financial interests or
personal relationships that could have appeared to influence the work reported in this paper.

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