Biological evaluation and molecular docking studies of 4-aminobenzohydrazide derivatives as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.molstruc.2021.130918

Nowadays, inhibition of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes have emerged as an encouraging approach in the treatment of dementia and remission of symptoms of Alzheimer's disease. Therefore, inhibition of cholinesterase

Full text of DOI:10.1016/j.molstruc.2021.130918

Journal of Molecular Structure 1244 (2021) 130918
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Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Biological evaluation and molecular docking studies of
4-aminobenzohydrazide derivatives as cholinesterase inhibitors
Zuleyha Almaza,∗, Aykut Oztekinb, Ayse Tan c, Hasan Ozdemird
a Department of Molecular Biology and Genetics, Faculty of Arts and Sciences, Mus Alparslan University, Mus, 49250, Turkey
b Agri Ibrahim Cecen University, Vocational School of Health Services, Department of Medical Services and Techniques, Agri, 04100, Turkey
c Vocational School of Technical Sciences, Mus Alparslan University, Mus, 49250, Turkey
d Atatürk University, Science Faculty, Chemistry Department, Erzurum, 25240, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
Nowadays, inhibition of the acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes
have emerged as an encouraging approach in the treatment of dementia and remission of symp-
toms of Alzheimer’s disease. Therefore, inhibition of cholinesterases is one of the main targets by
researchers. Benzohydrazides are biologically active compounds and have various pharmacological ef-
fects, bearing these in mind, we investigated the inhibitory effects of some mono or di-substituted 4-
aminobenzohydrazide derivatives (1a-11a) against AChE and BChE. For this purpose, we studied the inhi-
bition effects (IC50, Ki values, and inhibition types) of these molecules on AChE and BChE enzymes. Based
on the results, compound 3a showed potent AChE and BChE inhibition (IC50 = 0.59 and 0.15 μM). The Ki
values of the compounds (3a, 4a, and 8a) showing the best inhibition effect against AChE and BChE were
calculated and these values ranged from 0.10 0.04 to 5.10 2.14 μM. To determine the possible binding
mechanisms with the active sites of both enzymes of compounds 3a, 4a, and 8a having strong inhibitory
effects, docking analyses were performed. According to the docking results, compound 3a showed the
best binding affinity (-7.3 kcal/mol for AChE and -6.8 kcal/mol for BChE) against both enzymes.
Received 23 February 2021
Revised 3 June 2021
Accepted 14 June 2021
Available online 18 June 2021
Keywords:
Benzohydrazides
AChE
BChE
Enzyme inhibition
Molecular docking
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
tomatic treatment of moderate AD [5]. AChE is a membrane-bound
enzyme and is found in brain, muscles and cholinergic neurons [6].
Alzheimer’s disease (AD) is considered a neurodegenerative dis-
ease that progresses over time and is very difficult to diagnose at
an early stage [1]. Dementia is the most common type of men-
tal pathology seen in middle-aged and elderly individuals. The
most important changes detected in the brains of individuals with
AD are synapse loss with neuron death, amyloid plaque forma-
tion, and decreased neurotransmitter content. Since the most dra-
matic abnormalities occur in the cholinergic system, this situa-
tion is referred to as the cholinergic hypothesis of AD. There-
fore, AD is mainly associated with change in cholinesterase (ChE)
metabolism and degeneration [2]. ChE’s are hydrolyzed acetyl-
choline and two types, namely acetylcholinesterase (AChE) and
butyrylcholinesterase (BChE). About 80% of acetylcholine is hy-
drolyzed by AChE, while BChE plays a complementary role[3]. ChE
inhibitors have been supported by the cholinergic hypothesis with
the finding that they have positive effects on cognition in AD [4].
These inhibitory compounds have been suggested for the symp-
AChE lowers neurotransmitter levels by hydrolyzing ACh to acetate
and choline. It terminates the communication between nerve cells,
thus playing an important role in cholinergic transition [5]. BChE is
found in the liver, pancreas, blood serum and central nervous sys-
tem (CNS) [7]. In AD, AChE decreases by up to 45% while BChE
increases by up to 90%. Thus BChE compensates for the role of
AChE. The development of ChE inhibitors that act as dual-target
inhibitors of AChE and BChE is therefore very important [3]. There
are a number of drugs for treating the most common symptoms of
dementia [8]. These drugs, which partially inhibit AChE, can cross
the blood–brain barrier. They can increase ACh levels and poten-
tiate their physiological effects. In this way, they can also pro-
vide symptomatic relief [9]. Commonly prescribed anti-AD drugs,
which are cholinesterase inhibitors, act by increasing ACh levels
through inhibition of this enzyme [10]. Unfortunately, the medi-
cations developed have only been able to relieve AD symptoms.
But, if brain cell damage is severe, the ability of cholinesterase in-
hibitors for curing the symptoms becomes less and less. It was de-
termined that the effects of cholinesterase inhibitors reported in
studies differ in for each individual [11]. The first drug approved
for the treatment of AD (in 1993) is tacrine, an AChE and BChE
∗
Corresponding author.
E-mail address: z.turkoglu@alparslan.edu.tr (Z. Almaz).
https://doi.org/10.1016/j.molstruc.2021.130918
0022-2860/© 2021 Elsevier B.V. All rights reserved.

Z. Almaz, A. Oztekin, A. Tan et al.
Journal of Molecular Structure 1244 (2021) 130918
inhibitor. However, the use of tacrine has been limited because it
causes side effects such as nausea, diarrhea, dizziness, and vomit-
ing. More tolerated and less toxic drugs were approved instead of
tacrine. To date, four cholinesterase inhibitors with different phar-
macological and pharmacokinetic profiles have been licensed for
the symptomatic treatment of AD, such as rivastigmine, donepezil
and galantamine, along with tacrine [12]. These drugs will con-
tinue to be developed as they are a proven symptomatic therapy
with a known goal. In the light of all this information, the syn-
thesis of new molecules has increased as cholinesterase inhibitors,
which are an important treatment option for AD. In order to con-
tribute to these pharmacological studies, we focused on the effect
of benzohydrazide derivatives against AChE and BChE. Benzohy-
drazides, which are aromatic derivatives of hydrazine and benzoic
acid derivatives, have become important due to their various phar-
macological effects. It has also been found that benzohydrazide
derivatives have inhibitory activities against AChE and BChE. In
the recent studies, amide benzohydrazides have emerged as poten-
tial candidates as anticholinesterases and have remarkable inhibi-
tion potential against AChE and BChE [13]. It has also been found
that the 6-substituted-3 (2H) –pyridazinone 2-yl-propionhydrazide
derivatives (expecially 4 -Fluorophenyl) exhibits significant AChE
inhibitory activities [14]. Furthermore, they have been found to act
as analgesic and anti-inflammatory agents [15,16]. In addition, ben-
zohydrazide derivatives have significant antitubercular and anti-
HIV activities [17,18]. In the light of above, in this study, mono
or di-substituted 4-aminobenzohydrazide derivatives were synthe-
sized and their inhibition potential on AChE and BChE enzymes
was examined in vitro by comparing with a reference drug tacrine.
formation, amyloid plaques and neuron loss [22]. We evalu-
ated the inhibitory effects of 11 different 4-aminobenzohydrazide
derivatives and tacrine on AChE and BChE under in vitro
conditions in the present study. The concentration of the 4-
aminobenzohydrazide derivatives (1a-11a) required to inhibit 50%
of the enzyme activities were calculated from different inhibitor
concentrations and are summarized in Table 1. Compound 3a
showed a better AChE inhibitory effect than tacrine, an AChE in-
hibitor used therapeutic agent in AD treatment. Three compounds
(4a, 8a, and 10a) showed notable inhibitory effects against AChE
and BChE with IC50 values. The IC50 values for compound 3a,
which showed the best inhibition activity from these derivative in-
hibitors, were 0.59 μM for AChE and 0.15 μM for BChE. A selectiv-
ity index (SI) was calculated to understand whether or not a given
compound was selective for enzymes. Thereafter, Lineweaver-Burk
plots were drawn to determine the Ki values and inhibition types
of the compounds (3a, 4a, 8a) showing the best inhibitory effect
against both AChE and BChE enzymes. As seen in Table 2, Ki val-
ues range from 0.28 0.02 to 5.10 2.14 for AChE and 0.10 0.04
to 3.84 1.76 for BChE. Compound 3a had the best Ki value, which
also had the best docking score with AChE and BChE. These three
compounds for AChE, compound 4a for BChE showed a noncom-
petitive inhibition type. Compounds 3a and 8a showed competitive
inhibition type for BChE. Activity (%)-[3a] and Lineweaver–Burk
graphs of compound 3a showing the best activity against AChE and
BChE are given in Figs. 2 and 3 respectively.
When the structure-activity relationships of the compounds are
examined, compound 3a bearing the Br and F substituents at the
C3 and C5 positions of the benzene ring showed the best inhibitory
activity against AChE and BChE. In contrast to 3a, it was seen to
have weaker the inhibition potential of compound 1a, which con-
tains the Cl substituent at the C5 position and Br substituent at
the C3 position of the benzene ring. Compounds 4a, 8a, and 10a,
which contain the methoxy group, Br or Cl were attached to the
C2 position of the benzene ring respectively, showed significant in-
hibitory activities against both AChE and BChE. The Br substituent
at the C2 position of the benzene ring of compound 8a, compared
to the C3 position, decreased significantly the inhibition potential
for both enzymes. The methoxy, Br, and Cl groups at the C2 and
C3 positions of the benzene ring (for compounds 5a, 9a, and 11a,
respectively), were significantly decreased the inhibitory activities
against both enzymes. While the methyl group at the C2 of the
benzene ring of compound 6a showed an approximately two-fold
reduction in inhibition of AChE compared with the methyl group
at the C3 of compound 7a. On the other hand in inhibition with
BChE of 6a, a slightly increase was observed.
2. Results and discussion
2.1. Chemistry
Methyl aminobenzoate derivatives (1–11) were pur-
chased from fluorochem ltd U.K. In our previous studies, 4-
aminobenzohydrazide derivatives (1a-11a) were synthesized from
methyl 4-aminobenzoate derivatives according to previous de-
scribed procedure [19-21] (Fig. 1). For this purpose, compounds
1–11 were reacted with hydrazine to obtain the target compounds
(1a-11a) in good yields.
2.2. Anti-cholinesterase activity and enzyme kinetics studies
ChE inhibitors are commonly used in the treatment of AD,
which is known for decreased cholinergic transmission, knot
Fig. 1. The general synthesis pathway of mono or disubstituted 4-aminobenzohydrazides (1a-11a).
2

Z. Almaz, A. Oztekin, A. Tan et al.
Journal of Molecular Structure 1244 (2021) 130918
Table 1
IC50 values (μM) and SI indexes of the 4-aminobenzohydrazide derivatives (1a-11a).
Inhibitors
AChE
R2
BChE
R2
SI indexa
1a
346.50
0.957
441.43
0.985
0.981
0.961
1.27
0.50
0.25
2a
3a
173.28
0.59
0.986
0.989
86.63
0.15
4a
2.82
0.996
3.63
0.993
1.29
5a
693.14
346.57
693.14
4.62
0.977
0.935
0.953
0.925
0.956
0.925
>1000
573.92
428.12
3.81
-
–
6a
0.979
0.967
0.961
0.958
0.974
1.65
0.62
0.82
0.69
7.45
7a
8a
9a
693.14
475.98
10a
11a
8.45
63
693.14
0.71
0.964
0.982
>1000
-
–
∗
Tacrine
0.11
0.987
0.15
aCalculated by the formula IC50 BChE/IC50 AChE for each compound to show selectivity toward either enzyme.
∗Used as a positive control for AChE and BChE enzymes.
Comparing our IC50 values with a study examining the bi-
Table 2
ological activities of sulfonyl hydrazones, benzoyl hydrazones,
and chalcone derivatives, the authors found that the IC50 values
showing the best AChE inhibitory activity were 10.16 0.80 μM
for [N’-(1-(4-morpholinophenyl)ethylidene)−2-(trifluoromethoxy)
benzenesulfonohydrazide], and 17.20 1.86 μM for [4-Chloro-N’-
(1-(4-morpholinophenyl)ethylidene)benzohydrazide] while our
5-Fluoro-substituted compound 3a was in the 0.59 μM. In the
Ki values (μM) and inhibition types of compounds 3a, 4a and 8a having strong
inhibitory effects against AChE and BChE.
Inhibitors
AChE (μM)
Inhibition type
BChE (μM)
Inhibition type
3a
4a
8a
0.28 0.02
2.95 0.55
5.10 2.14
Noncompetitive
Noncompetitive
Noncompetitive
0.10 0.04
1.37 0.60
3.84 1.76
Competitive
Noncompetitive
Competitive
3

Z. Almaz, A. Oztekin, A. Tan et al.
Journal of Molecular Structure 1244 (2021) 130918
Fig. 2. IC50 graph and Lineweaver-Burk graph of compound 3a for AChE.
Fig. 3. IC50 graph and Lineweaver-Burk graph of compound 3a for BChE.
same study BChE assay, the authors found that the IC50 values
indicated the best BChE inhibitory activity were 16.52 0.80 μM
for [[N’-(1-(4-morpholinophenyl) ethylidene)−2-(trifluoromethoxy)
benzenesulfonohydrazide] and 19.21 0.75 for μM [4-(methylthio)-
bridge, and attractive charge interactions with Glu198 residue. The
Br substituent on the benzene ring of 3a formed π–alkyl inter-
actions with Phe293, His443, and Phe334 residues. The benzene
ring of the compound formed π–π T-shaped interactions with
Trp82, Tyr333 residues. In the interaction with BChE, compound
3a formed van der Waals interactions with Gly437, Ser196, Tyr126,
Gly114, Asn81, Thr118, and Asp68 residues. The amine group on
the benzene ring and the hydrazide moiety of 3a formed conven-
tional hydrogen bonds with Glu195 and Trp80 residues, respec-
tively. While the Br substituent on the benzene ring of 3a formed
π–alkyl interactions Trp80 and His436 residues, the F substituent
formed halogen (fluorine) interactions Glu195 and Gly113 residues.
Finally, the carbonyl group formed a carbon-hydrogen bond with
Gly119 residue.
N’-(1-(4-morpholinophenyl)
ethylidene)benzohydrazide]
[23].
Concordantly, we observed that the IC50 value for compound 3a
showing best BChE inhibition was in the 0.15 μM.
2.3. Molecular docking
Compounds 3a, 4a, and 8a showed significant inhibition ef-
fects against AChE and BChE. Therefore, their molecular docking
studies were carried out on the compounds. The compounds were
similarly docked into the active sites of both receptors. Accord-
ing to the docking results, the compounds could easily bound
with the active sites of AChE and BChE. 2D ligand-receptor in-
teraction diagrams of the compounds with AChE and BChE are
presented in Fig. 5. While the binding energy values of com-
pounds 3a, 4a, and 8a in the interaction with AChE, were calcu-
lated as −7.3 kcal/mol, −7.0 kcal/mol, and −6.9 kcal/mol, those
of 3a, 4a, and 8a in the interaction with BChE were calculated as
−6.8 kcal/mol, −6.1 kcal/mol, and −6.4 kcal/mol, respectively. The
best binding poses and 3D ligand-receptor interaction diagrams of
compound 3a, which is the best inhibitor against both receptors,
are presented in Fig. 4 for AChE and Fig. 6 for BChE. In the in-
teraction with AChE, compound 3a formed van der Waals interac-
tions with Tyr115, Trp113, Ile447, Gly444, Tyr129, Ser 199, Gly117,
Gly118, and Tyr120 residues. The hydrazide moiety of compound
3a formed a conventional hydrogen bond with Gly116, a π–cation
interaction with Trp82, a carbon-hydrogen bond with Gly444, salt
3. Experimental
3.1. Chemicals
Purified AChE from electric eel (Electrophorus electricus) and
butyrylcholinesterase from equine serum, acetylthiocholine iodide
ꢀ
(AChI), butyrylcholine iodide (BChI), 5,5 dithiobis-2-nitrobenzoic
acid (DTNB,), dimethylsulfoxide (DMSO) and other chemicals were
obtained from (Sigma-Aldrich, St. Louis, MO).
3.2. Synthesis of 4-aminobenzohydrazide derivatives (1a-11a)
4-aminobenzohydrazide derivative compounds (1a-11a) were
synthesized from commercially available 4-aminobenzoate forms
according to the protocol previously described [21]. Experimental
details had been presented in our previous studies [19,20].
4

Z. Almaz, A. Oztekin, A. Tan et al.
Journal of Molecular Structure 1244 (2021) 130918
Fig. 4. The best binding pose and 3D interaction diagram of compound 3a with AChE,.
Fig. 5. 2D ligand-receptor interaction diagrams of compounds: A) 3a-AChE, B) 4a-AChE, C) 8a- AChE, D) 3a-BChE, E) 4a-BChE, F) 8a-BChE.
Fig. 6. The best binding pose and 3D interaction diagram of compound 3a with BChE.
3.3. In vitro inhibition assay of cholinesterases
as a substrate. DTNB was used for determination of AChE and BChE
˙
enzyme activitiy. Initially, 2 mg of each inhibitor synthesized was
The inhibitory effects of synthesized (1a-11a) inhibitory
molecules on AChE and BChE activity were measured using a
modification of Ellman’s spectrophotometric method [24] as de-
scribed previously [25,26]. The essence of this method is to form
taken, dissolved in 1 mL of DMSO and diluted in various concentra-
tions with deionized water. The reaction system was composed of
2-200 μL inhibitor sample, 50 μL (AChI)/(BChI), 100 μL of buffer
(1 M, pH 8.0; Tris-HCl buffer for the AChE assay and phosphate
buffer for the BChE assay), 50 μL DTNB, and 20μL enzyme (0.2
units/mL for the AChE assay and 0.3 units/mL for the BChE assay).
ꢀ
5-mercapto-2-nitrobenzoic acid by reacting thiocholine with 5,5 -
Dithio-bis (2-nitro-benzoic acid)(DTNB). AChI and BChI were used
5

Z. Almaz, A. Oztekin, A. Tan et al.
Journal of Molecular Structure 1244 (2021) 130918
The absorbance of the reaction mixture starting with the enzyme
addition was measured at 412 nm for 5 min on a Shimadzu UV-
1800 Spectrophotometer. Activity (%) - [1a-11a] (inhibitory concen-
tration) was plotted to determine the inhibition effect of derivative
molecules on AChE and BChE. IC50 values were obtained by activ-
ity (%) versus compound plots. Three different inhibitory concen-
trations were used to calculate Ki values. Lineweaver - Burk curves
were plotted and calculations were performed [27].
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ment of AD. In view of these studies, we synthesized some 4-
aminobenzohydrazide derivatives (1a-11a) and investigated their
inhibitory properties against AChE and BChE. The results showed
that some of these compounds have moderate to high inhibitory
activity. Compound 3a is a good inhibitor for both AChE and BChE.
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Declaration of Competing Interest
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The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
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CRediT authorship contribution statement
Zuleyha Almaz: Data curation, Investigation, Methodology,
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Writing
– original draft, Writing – review & editing. Aykut
Oztekin: Visualization, Writing – review & editing. Ayse Tan: Vi-
sualization, Writing – review & editing. Hasan Ozdemir: Method-
ology.
˙
[25] Z. Köksal, Z. Alım, S. Bayrak, I. Gülçin, H. Özdemir, Investigation of the ef-
fects of some sulfonamides on acetylcholinesterase and carbonic anhydrase en-
zymes, J. Biochem. Mol. Toxicol. 33 (5) (2019) e22300.
˙
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