A new series of pyridazinone derivatives as cholinesterases inhibitors: Synthesis, in vitro activity and molecular modeling studies

Article abstract of DOI:10.1016/j.pharep.2019.07.006

Background: The pyridazinone nucleus has been incorporated into a wide variety of therapeutically interesting molecules to transform them into better drugs. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are known to be serine hydrolase enzymes responsible for the hydrolysis of acetylcholine (ACh). Inhibition of cholinesterases is an effective method to curb Alzheimer's disease. Here, we prepared 12 new 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(nonsubstituted/4-substituted benzenesulfonohydrazide) derivatives and evaluated their inhibitory effects on AChE/BChE in pursuit of potent dual inhibitors for Alzheirmer's Disease. We also tried to get insights into binding interactions of the synthesized compounds in the active site of both enzymes by using molecular docking approach. Method: We obtained our compounds by the reaction of various substituted/nonsubstituted benzenesulfonic acid derivatives with 6-substitutedphenyl-3(2H)-pyridazinone-2-yl acetohydrazide and determined their anticholinesterase activities according to the Ellman's method. Molecular docking studies were done using Glide and the results were evaluated on Maestro (Schr?dinger, LLC, New York, NY, 2019). Results: The title compounds showed moderate inhibition at 100 μg/ml against both enzymes, yet with better activity against BChE. Compound VI_2a emerged as a dual inhibitor with 25.02% and 51.70% inhibition against AChE and BChE, respectively. Conclusion: This study supports that novel pyridazinone derivates may be used for the development of new BChE inhibitory agents. It was less potent than the reference drugs, yet promising for further modifications as a lead. The ability of the compounds to adopt energetically more favourable conformations and to engage in more key interactions in the ECBChE active gorge explains their better activity profile against ECBChE.

Full text of DOI:10.1016/j.pharep.2019.07.006

Accepted Manuscript
Title: A New Series of Pyridazinone Derivatives as
Cholinesterases Inhibitors: Synthesis, In Vitro Activity and
Molecular Modeling Studies
¨
¨
Authors: Azime Berna Oz c¸ elik, Zeynep Ozdemir, Suat Sari,
Semra Utku, Mehtap Uysal
PII:
S1734-1140(19)30266-X
DOI:
Reference:
https://doi.org/10.1016/j.pharep.2019.07.006
PHAREP 1084
To appear in:
Received date:
Revised date:
Accepted date:
17 April 2019
8 July 2019
19 July 2019
¨
¨
Please cite this article as: Oz c¸ elik AB, Ozdemir Z, Sari S, Utku S, Uysal M, A
New Series of Pyridazinone Derivatives as Cholinesterases Inhibitors: Synthesis, In
Vitro Activity and Molecular Modeling Studies, Pharmacological Reports (2019),
https://doi.org/10.1016/j.pharep.2019.07.006
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A New Series of Pyridazinone Derivatives as Cholinesterases Inhibitors:
Synthesis, In Vitro Activity and Molecular Modeling Studies
Running title: Anticholinesterase, pyridazinone derivatives
1
*
2
3
4
1
Azime Berna Özçelik , Zeynep Özdemir , Suat Sari , Semra Utku , Mehtap Uysal
1
Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
2
İnönü University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Malatya, Turkey
Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Ankara, Turkey
Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Mersin, Turkey
3
4
*
Correspondence: Assoc. Prof. Azime Berna ÖZÇELİK, Gazi University, Faculty of Pharmacy,
Department of Pharmaceutical Chemistry, 06100, Ankara, Turkey.
Phone: +90 312 202 32 22
Fax:
+90 312 223 50 18
e-mail: brndemirci@yahoo.com

GRAPHİCAL ABSTRACT
A series of 3(2H)-pyridazinone derivatives were synthesis and evaluated their
acetylcholinesterase (AChE) and buthyrylcholinesterase (BChE) inhibitory activity.
Compound VI2a emerged as a dual inhibitor with 25.02% and 51.70% inhibition against AChE
and BChE, respectively. Through molecular docking studies we tried to provide insights into
the inhibition mechanism of our compounds against both enzymes at molecular level.
Research Highlights




12 new 3(2H)-pyridazinone derivative compounds have been synthesized.
The anticholinesterase activity has been examined according to the Ellman Method.
The structure-activity relationship of the synthesized compounds have been studied.
Molecular docking studies have been performed.
Abstract
Background: The pyridazinone nucleus has been incorporated into a wide variety of
therapeutically interesting molecules to transform them into better drugs. Acetylcholinesterase
(AChE) and butyrylcholinesterase (BChE) are known to be serine hydrolase enzymes
responsible for the hydrolysis of acetylcholine (ACh). Inhibition of cholinesterases is an
effective method to curb Alzheimer’s disease. Here, we prepared 12 new 6-substituted-3(2H)-
pyridazinone-2-acetyl-2-(nonsubstituted/4-substituted benzenesulfonohydrazide) derivatives
and evaluated their inhibitory effects on AChE/BChE in pursuit of potent dual inhibitors for
Alzheirmer’s Disease. We also tried to get insights into binding interactions of the synthesized
compounds in the active site of both enzymes by using molecular docking approach.

Method: We obtained our compounds by the reaction of various substituted/nonsubstituted
benzenesulfonic acid derivatives with 6-substitutedphenyl-3(2H)-pyridazinone-2-yl
acetohydrazide and determined their anticholinesterase activities according to the Ellman’s
method. Molecular docking studies were done using Glide and the results were evaluated on
Maestro (Schrödinger, LLC, New York, NY, 2019).
Results: The title compounds showed moderate inhibition at 100 µg/ml against both enzymes,
yet with better activity against BChE. Compound VI2a emerged as a dual inhibitor with 25.02%
and 51.70% inhibition against AChE and BChE, respectively.
Conclusion: This study supports that novel pyridazinone derivates may be used for the
development of new BChE inhibitory agents. It was less potent than the reference drugs, yet
promising for further modifications as a lead. The ability of the compounds to adopt
energetically more favourable conformations and to engage in more key interactions in the
ECBChE active gorge explains their better activity profile against ECBChE.
Abbreviations: AChE, acethylcholine esterase; BChE, Buthyrylcholine esterase; AD,
Alzheimer’s disease; PAS, peripheral anionic site; CBS, cation-binding pocket; SEM, standard
error mean
Keywords: AChE inhibitor / BChE inhibitor / 3(2H)-Pyridazinone / Hydrazone / Molecular
modelling.

Introduction
Pyridazines are six-membered dinitrogen heterocycles often found in three different
isomers, namely pyridazine (1,2-diazine), pyrimidine (1,3-diazine) and pyrazine (1,4-diazine)
1]. Nitrogen-containing heteroaromatic rings make up part of the bioactive compound space
[
due to the coordination capability of nitrogen [2]. Recent studies have found that pyridazine is
one of the most developable heteroaromatic rings for new drugs with eligibility to form ligands
towards different targets, it is also found in the structure of natural compounds displaying
various biological activities. Applications of substituted pyridazines as antidepressant,
antihypertensive, anticonvulsant, antibacterial, diuretic, anti-HIV, anticancer, anti-
inflammatory, analgesic, cardiovascular, and neuroprotective agents are available in the
literature [3-13]. Some examples for medical use of pyridazine derivatives are zardaverine and
imazodane as cardiotonic PDE III inhibitors and emorfazone as an analgesic agent (Figure 1)
[
6-8]. Xing et al. synthesized 2,6-disubstituted pyridazinone analogs and investigated their
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitor activities. Among
them, compound 4 was reported to be highly potent and selective against the cited enzymes
(Figure 1) [14].
FIGURE 1
Figure 1. Structure of the drugs bearing pyridazinone skeleton and compound 4 [14].
Alzheimer disease (AD) is well-known for over a century, however, there is no concrete
therapeutic treatment prevailed [15-17]. There is relationship between AD and changes in many
neurotransmitters, including cholinergic, noncholinergic, serotonergic, dopaminergic,
aminoacidergic, and neuropeptidergic; but the relationship between changes in the cholinergic
system and AD is the most well-known. According to this relationship described as cholinergic
hypothesis, decrease in acetylcholine and choline acetyl transferase levels is associated with
AD [18]. Post-mortem brain studies and altered cholinergic symptoms such as degeneration of
neurons controlling neocortical regions, loss of cholinergic terminals in the cerebral cortex, loss
of choline acetyl transferase activity and acetyl choline synthesis, decreased cholinergic
receptivity and increased AChE activity, which can be seen in many Alzheimer's patients
[
19,20]. For this reason, in the treatment of AD increasing acetylcholine level by suppressing
AChE enzyme is one of the frequently used treatment modalities. On this basis, AChE inhibitors
AChEIs) have become the leading strategy for the development of anti-AD agents. The current
(

interest in these drugs has received considerable attention, too [21-23]. Some AChEIs, such as
tacrine, donepezil, rivastigmine, and ensaculin (Figure 2), show modest improvement in
memory and cognitive functions, and have been used to treat AD clinically for a long time. But
only ensaculin, a coumarin derivative composed of benzopyran and a piperazine substituted
moiety, has appeared to prevent or slow down the progressive neurodegeneration [22-25].
Ensaculin has been used clinically for the treatment of AD as an AChEI for a long time.
Although BChE is sparsely expressed in humans, recent studies proved that AChE
inhibition alone may not be sufficient to reduce cholinesterase activity and choline levels may
elevate since BChE makes up for the lost AChE activity. Furthermore, it is known that BChE
expression is increased during AD [26,27]. Several studies, so far, have emphasized the
importance of dual ChE inhibition in AD and other neurological disorders with cognitive
decline. Drugs such as rivastigmin are efficacious in AD thanks to their dual-inhibitory
character [28].
FIGURE 2
Figure 2. Structure of some FDA-approved AChEI in AD.
Zhou et al. designed and synthesized three different groups of coumarin-like compounds
(A, B, C) bearing phenylpiperazine rings as substitution to evaluate their potential for treating
AD (Figure 3) [25]. Zhou et al. also reported three hypothesis for the AChEI activity of their
derivatives: (1) the coumarin ring, 2-(H)-chromen-2-one, a heterocyclic moiety involved in
ensaculin’s structure are responsible for cognitive functions and compatible with high anti-
AChE potency, interacting with the peripheral anionic site (PAS); (2) the nitrogen atom from
the phenylpiperazine group acting as the positive charge center presented in many potent AChE
inhibitors, which can interact with the catalytic center of AChE demonstrated by the X-ray
crystallographic studies of the AChE-donepezil and AChE-galantamine complexes, and (3) the
phenyl ring is connected with the piperazine ring interacting with the cation-binding pocket
(CBS) as shown in Figure 3 [25]. In addition, a linker chain bearing different numbers of carbon
atom at the second position of the pyridazinone ringmight interact with the residues lining the
wall of the AChE gorge.
FIGURE 3
Figure 3. Structural hypothesis for AChEIs and some designed compounds from the
literature [25]

In view of the above mentioned findings, we designed and synthesized the title
compounds (VI1a-1d, VI2a-2d, VI3a-3d) to provide the structural requirements for AChEI and
BChEI activities (Figure 4). We in vitro evaluated their AChE and BChE inhibitory effects
using Ellman’s method, the most commonly used method to measure cholinesterase activity
[
29].
FIGURE 4
Figure 4. Structural hypothesis for the title compounds [25].
The CBS moiety was designed as substituted phenyl ring and the structural modifications
were focused on substitutions on the phenyl rings expected to interact with the CBS and PBS.
Methoxy, chloro, bromo, and fluoro groups were included as substitutions on these rings at
varying positions and combinations. Using molecular docking approach, we tried to predict
binding interactions of our derivatives with AChE and BChE active sites and get insights into
the structural attributes that affect their activities against these enzymes.
Materials and Methods
Chemistry
All chemicals used in this study were purchased from Aldrich, Fluka AG and E. Merck. 4-
oxo-4-phenylbutanoic acid derivatives I1-3, 6-substitutedphenyl-4,5-dihydropyridazin-3(2H)-
one derivatives II1-3, 6-substitutedphenylpyridazin-3(2H)-one derivatives III1-3, 6-
(
substitutedphenyl) piperazine-1-yl)-3(2H)-on-2-yl acetate derivatives IV1-3 and 6-
substitutedphenyl)piperazine-1-yl)-3(2H)-on-2-yl acetohydrazide derivatives V1-3 were
(
synthesized according to literature methods [30-33]. All title compounds VI1a-1d, VI2a-2d, VI3a-
3
d
were synthesized first time in this study according to the literature methods [34-36].
The purity of the compounds was checked by TLC with Merck Kieselgel F254 plates.
Melting points were determined on Electrothermal 9200 melting points apparatus and the values
were uncorrected. IR spectra were recorded on a Perkin Elmer Spectrometer by ATR technique.
1
13
H-NMR and C-NMR spectra were recorded on a Brucker Avonce Ultrashield FT-NMR
spectrometer in DMSO and CDCl at 300 MHz in Inonu University, Malatya. The mass spectra
HRMS) of the compounds were recorded on Waters Acquity Ultra Performance Liquid
3
(
Chromatograpy Micromass which combined LCT PremierTM XE UPLC/MS TOFF
spectrophotometer (Waters Corp, Milford, USA) by ESI+ and ESI- techniques.
Synthesis of 4-oxo-4-phenylbutanoic acid derivatives (I1-3
)

A mixture of 0.275 mol aluminum chloride, 20 ml carbon disulfide, and 0.25 mol
succinicanhydride was added portion wise in standard conditions to a mixture of 0.25 mol 2-
fluoroanisole and 50 ml carbon disulfide. Then the mixture was refluxed for 4 h at 40-50 °C.
After cooling, the mixture was poured onto ice water and the precipitate was collected, dried
and recrystallized from water [37].
Synthesis of 6-substitutedphenyl-4,5-dihydropyridazin-3(2H)-one derivatives (II1-3
)
0
.01 Mol 4-(2-fluoro-4-methoxyphenyl)-4-oxobutanoic acid and 0.015 mol hydrazine
hydrate (0.85 ml, 55%) in 30 ml ethanol were refluxed for 4 h. The reaction mixture was cooled
and precipitate thus formed was collected by filtration, dried, and crystallized from ethanol [30].
Synthesis of 6-substitutedphenylpyridazin-3(2H)-one derivatives (III1-3
Method 1:
)

A solution of 0.043 mol bromine in 25 ml glacial acetic acid was added drop wise to a
solution of 0.039 mol 6-(2-fluoro-4-methoxyphenyl)-4,5-dihydro-3(2H)-pyridazinone in 100
ml glacial acetic acid at 60-70 °C, then the reaction mixture was refluxed for 3 h. After cooling
to 5°C, it was poured into ice water and converted to the free base form with ammonium
hydroxide. The precipitate was collected by filtration, washed with water until neutral, dried,
and crystallized from ethanol-water [38].

Method 2:
Glyoxylic acid (0.05 mol) and 4-chloro/bromo/methylacetophenone (0.15 mol) were heated
o
o
at 100–105 C for 2 h. At the end of this period, the reaction mixture was cooled to 40 C and
5% ammonium hydroxide solution was added to the reaction mixture until the pH of the
2
medium reached 8. Then, the reaction mixture was extracted with dichloromethane (20 ml), the
aqueous layer was separated and refluxed in the presence of hydrazine hydrate (0.05 mol) for
2
h. After the completion of the reaction, the mixture was cooled to room temperature. The
resulting precipitate was filtered to give compounds C1-3 [39-41].
Synthesis of ethyl 6-(substitutedphenyl)piperazine-1-yl)-3(2H)-on-2-yl acetate derivatives
(IV1-3)
A mixture of 0.01 mol proper 6-substituted phenylpyridazin-3(2H)-one derivative (III1-
), 0.02 mol (2.2252 ml) ethyl bromoacetate, and 0.02 mol (2.7636 g) potassium carbonate in
3
acetone (40 ml) was refluxed overnight. After the mixture was cooled, the organic salts were

filtered off, the solvent was evaporated, and the residue was purified by crystallization from
methanol to give the esters [36].
Synthesis of 6-(substitutedphenyl)piperazine-1-yl)-3(2H)-on-2-yl acetohydrazide derivatives
(
1-3
V )
Hydrazine hydrate (99%, 3ml) was added to a solution of 0.01 mol proper ethyl 6-
substituted phenyl)piperazine-1-yl)-3(2H)-on-2-yl acetate derivative (IV1-3) in 25 ml methanol
(
and stirred for 3h at room temperature. The precipitate obtained was filtered off, washed with
water, dried, and crystallized from ethanol [34].
General
procedure
for
preparation
of
N’-[(substitutedphenyl)sulfonyl]-2-(6-
(substitutedphenyl)-3(2H)-pyridazinone-2-yl)acetohydrazide (VI1a-1d, VI2a-2d, VI3a-3d
)
Substituted benzenesulfonyl chloride (1 mmol) was added to a solution of proper 6-
(substituted phenyl)piperazine-1-yl)-3(2H)-on-2-yl acetohydrazide derivative (V1-3) (1 mmol)
in pyridine (10 ml) at 0 °C. The resulting mixture was stirred at room temperature for 5 h. At
the end of this period, the reaction mixture was poured into ice water. The precipitate was
filtered, dried, and crystallized from an appropriate solvent. The compounds were identified by
1
13
IR, H-NMR, C-NMR and mass spectra.
All spectral data of the compounds were in accordance with the assigned structures as
shown below.
2
-(3-(2-Fluoro-3-methoxyphenyl)-6-oxopyridazin-1(6H)-yl)-N'-
(phenylsulfonyl)acetohydrazide (VI1a
)
o
1
Yield: 59%; mp: 208 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 3.91 (3H; s; -OCH
3
), 4.69
4
,5
(
2H; t; -N-CH
2
-C=O), 7.05-8.09 (10H; m; phenyl protons and pyridazinone H ), 10.02 (1H; s;
1
3
-
NH-C=O) and 10.42 (1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.43 (1C; -
2
3
OCH
3
), 56.12 (1C; -N-CH
2
-C=O), 112.99 (1C; phenyl C ), 113.36 (1C; phenyl C ), 113.95 (1C;
4
5
6
phenyl C ), 122.46 (1C; phenyl C ), 126.97 (1C; phenyl C ), 127.51 (1C; 2-fluoro-3-
5
6
methoxyphenyl C ), 128.81 (1C; 2-fluoro-3-methoxyhenyl C ), 129.51 (1C; 2-fluoro-3-
4
5
1
methoxyhenyl C ), 130.87 (1C; pyridazinone C ), 132.91 (1C; 2-fluoro-3-methoxyhenyl C ),
6
4
1
38.93 (1C; pyridazinone C ), 148.24 (1C; pyridazinone C ), 149.96 (1C; 2-fluoro-3-
2
3
1
methoxyphenyl C ), 153.19 (1C; 2-fluoro-3-methoxyphenyl C ), 158.65 (1C; phenyl C ),

3
1
65.51 (1C; CH
2
-N-C=O), 170.67 (1C; pyridazinone C ). C19
H17FN
4
O S. MS (ESI+)
5
calculated: 433.0982, Found: m/e 433.0984 (M+H; % 100.0).
2
-(3-(2-Fluoro-3-methoxyphenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-
fluorophenylsulfonyl)acetohydrazide (VI1b
)
o
1
Yield: 94%; mp: 233 C; H-NMR (DMSO-d
2H; t; -N-CH -C=O), 7.23-8.09 (9H; m; phenyl protons and pyridazinone H ), 9.80 (1H; s; -
NH-C=O) and 10.49 (1H; s; -NH-SO
6
, 300 MHz): δ (ppm) 3.90 (3H; s; -OCH
3
), 4.70
4
,5
(
2
1
3
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.55 (1C; -OCH
3
),
2
3
5
1
6.08 (1C; -N-CH
2
-C=O), 112.95 (1C; 4-fluorophenyl C ), 113.89 (1C; 4-fluorophenyl C ),
6
5
15.81 (1C; 4-fluorophenyl C ), 116.11 (1C; 4-fluorophenyl C ), 122.41 (1C; 2-fluoro-3-
5
6
methoxyphenyl C ), 127.02 (1C; 2-fluoro-3-methoxyhenyl C ), 129.47 (1C; 2-fluoro-3-
4
5
1
methoxyhenyl C ), 130.87 (1C; pyridazinone C ), 135.12 (1C; 2-fluoro-3-methoxyhenyl C ),
6
4
4
1
1
1
42.38 (1C; pyridazinone C ), 148.09 (1C; pyridazinone C ), 149.96 (1C; 4-fluorophenyl C ),
2
3
53.19 (1C; 2-fluoro-3-methoxyphenyl C ), 162.87 (1C; 2-fluoro-3-methoxyphenyl C ),
1
-N-C=O), 170.66 (1C; pyridazinone C3).
65.56 (1C; 4-fluorophenyl C ), 166.20 (1C; CH
2
C
19
H
16
F
2
N
4
5
O S. MS (ESI+) calculated: 451.0888, Found: m/e 451.0879 (M+H; % 100.0).
2
-(3-(2-Fluoro-3-methoxyphenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-
chlorophenylsulfonyl)acetohydrazide (VI1c
)
o
1
Yield: 89%; mp: 228 C; H-NMR (DMSO-d , 300 MHz): δ (ppm) 3.90 (3H; s; -OCH3), 4.70
6
4
,5
(
2H; t; -N-CH2-C=O), 7.03-8.04 (9H; m; phenyl protons and pyridazinone H ), 10.17 (1H; s;
1
3
-
NH-C=O) and 10.50 (1H; s; -NH-SO
2
). C-NMR (DMSO-d , 300 MHz), δ 52.56 (1C; -
6
2
OCH
3
), 56.12 (1C; -N-CH -C=O), 112.99 (1C; 4-chlorophenyl C ), 113.26 (1C; 4-chlorophenyl
2
3
6
5
C ), 113.96 (1C; 4-chlorophenyl C ), 122.41 (1C; 4-chlorophenyl C ), 123.87 (1C; 2-fluoro-3-
5
6
methoxyphenyl C ), 126.96 (1C; 2-fluoro-3-methoxyhenyl C ), 129.51 (1C; 2-fluoro-3-
4
5
1
methoxyhenyl C ), 130.93 (1C; pyridazinone C ), 136.09 (1C; 2-fluoro-3-methoxyhenyl C ),
6
4
4
1
1
1
37.76 (1C; pyridazinone C ), 142.41 (1C; pyridazinone C ), 148.11 (1C; 4-chlorophenyl C ),
2
3
49.58 (1C; 2-fluoro-3-methoxyphenyl C ), 153.21 (1C; 2-fluoro-3-methoxyphenyl C ),
1
3
58.65 (1C; 4-chlorophenyl C ), 165.59 (1C; CH2-N-C=O), 170.62 (1C; pyridazinone C ).
MS (ESI+) calculated: 467.0592, Found: m/e 467.0590 (M+H; % 100.0).
C
19
H
16ClFN
4
O S
5 .
2
-(3-(2-Fluoro-3-methoxyphenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-trifluoromethyl
phenylsulfonyl)acetohydrazide (VI1d
)

o
1
Yield: 87%; mp: 240 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 3.90 (3H; s; -OCH
3
), 4.70
4
,5
(
2H; t; -N-CH
2
-C=O), 7.01-8.10 (9H; m; phenyl protons and pyridazinone H ), 10.35 (1H; s;
1
3
-
NH-C=O) and 10.56 (1H; s; -NH-SO
2
). C-NMR (DMSO-d , 300 MHz), δ 52.59 (1C; -
6
OCH
3
), 56.11 (1C; -N-CH
2
-C=O), 112.94 (1C; -CF
3
), 113.20 (1C; 4-trifluoromethylphenyl C2),
3
6
1
4
3
13.92 (1C; 4-trifluoromethylphenyl C ), 122.41 (1C; 4-trifluoromethylphenyl C ), 125.22 (1C;
5
5
-trifluoromethylphenyl C ), 125.98 (1C; 2-fluoro-3-methoxyphenyl C ), 127.02 (1C; 2-fluoro-
6
4
-methoxyhenyl C ), 128.55 (1C; 2-fluoro-3-methoxyhenyl C ), 130.90 (1C; 4-
4
5
trifluoromethylphenyl C ), 132.28 (1C; pyridazinone C ), 141.13 (1C; 2-fluoro-3-
1
6
4
methoxyhenyl C ), 142.88 (1C; pyridazinone C ), 148.11 (1C; pyridazinone C ), 148.25 (1C;
2
3
2
-fluoro-3-methoxyphenyl C ), 149.97 (1C; 2-fluoro-3-methoxyphenyl C ), 153.21 (1C; 4-
1
3
trifluoromethylphenyl C ), 158.64 (1C; CH2-N-C=O), 165.71 (1C; pyridazinone C ).
MS (ESI+) calculated: 501.0856, Found: m/e 501.0940 (M+H; % 100.0).
C
20
H
16
F
4
N
4
O S
5 .
2
-(3-(4-Chlorophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(phenylsulfonyl)acetohydrazide (VI2a
)
o
1
Yield: 86%; mp: 198 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.71 (2H; t; -N-CH
2
-C=O),
4
,5
7
.10-8.10 (11H; m; phenyl protons and pyridazinone H ), 10.03 (1H; s; -NH-C=O) and 10.44
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.77 (1C; -N-CH
2
-C=O), 127.51 (1C;
2
3
4
5
phenyl C ), 127.56 (1C; phenyl C ), 127.77 (1C; phenyl C ), 128.83 (1C; phenyl C ), 128.91
6
2
3
(
1C; phenyl C ), 129.42 (1C; 4-chlorophenyl C ), 129.61 (1C; 4-chlorophenyl C ), 131.02 (1C;
5
5
6
pyridazinone C ), 132.95 (1C; 4-chlorophenyl C ), 133.62 (1C; 4-chlorophenyl C ), 134.25
6
4
1
(
1C; pyridazinone C ), 137.30 (1C; pyridazinone C ), 138.90 (1C; 4-chlorophenyl C ), 142.52
4
1
(
1C; 4-chlorophenyl C ), 158.71 (1C; phenyl C ), 165.45 (1C; CH -N-C=O), 170.62 (1C;
2
3
pyridazinone C ). C18
H15ClN
4
4
O S. MS (ESI+) calculated: 419.0581, Found: m/e 419.0591
(M+H; % 100.0).
2
-(3-(4-Chlorophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-
fluorophenylsulfonyl)acetohydrazide (VI2b
)
o
1
Yield: 79%; mp: 212 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.71 (2H; t; -N-CH -C=O),
2
4
,5
7
.06-8.11 (10H; m; phenyl protons and pyridazinone H ), 10.09 (1H; s; -NH-C=O) and 10.48
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.59 (1C; -N-CH
2
-C=O), 115.84 (1C;
2
3
5
4
-chlorophenyl C ), 116.14 (1C; 4-chlorophenyl C ), 116.79 (1C; 4-chlorophenyl C ), 127.55
6
2
3
(
1C; 4-chlorophenyl C ), 128.91 (1C; 4-fluorophenyl C ), 129.61 (1C; 4-fluorophenyl C ),
5
5
6
1
1
30.63 (1C; pyridazinone C ), 131.06 (1C; 4-fluorophenyl C ), 132.95 (1C; 4-fluorophenyl C ),
6
4
1
34.25 (1C; pyridazinone C ), 135.14 (1C; pyridazinone C ), 142.54 (1C; 4-chlorophenyl C ),

1
4
1
58.71 (1C; 4-fluorophenyl C ), 162.87 (1C; 4-chlorophenyl C ), 165.47 (1C; 4-fluorophenyl
4
3
C ), 166.20 (1C; CH
2
-N-C=O), 170.58 (1C; pyridazinone C ). C18
H
14ClFN
4
4
O S. MS (ESI+)
calculated: 437.0487, Found: m/e 437.0493 (M+H; % 100.0).
2
-(3-(4-Chlorophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-
chlorophenylsulfonyl)acetohydrazide (VI2c
)
o
1
Yield: 86%; mp: 244 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.71 (2H; t; -N-CH -C=O),
2
4
,5
7
.06-8.11 (10H; m; phenyl protons and pyridazinone H ), 10.19 (1H; s; -NH-C=O) and 10.51
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.61 (1C; -N-CH -C=O), 127.56 (2C;
2
1
2,6
1
3
1
5
4
1
-chlorophenyl C ), 128.93 (1C; 4-chlorophenyl C ), 129.52 (1C; 4-chlorophenyl C ),
5
2
3,5
2
29.81 (1C; pyridazinone C ), 131.08 (2C; 4-chlorophenyl C ), 132.95 (2C; 4-chlorophenyl
2
,6
6
4
1
C ), 134.26 (1C; pyridazinone C ), 136.07 (1C; pyridazinone C ), 137.73 (1C; 4-chlorophenyl
1
2
1
1
4
C ), 137.85 (1C; 4-chlorophenyl C ), 142.57 (1C; 4-chlorophenyl C ), 158.87 (1C; 4-
2
4
3
chlorophenyl C ), 165.52 (1C; CH
2
-N-C=O), 170.56 (1C; pyridazinone C ). C18
H14Cl
2
N
4
4
O S.
MS (ESI+) calculated: 453.0191, Found: m/e 453.0186 (M+H; % 100.0).
2
-(3-(4-Chlorophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-trifluoromethyl
phenylsulfonyl)acetohydrazide (VI2d
)
o
1
Yield: 82%; mp: 229 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.71 (2H; t; -N-CH -C=O),
2
4
,5
7
.05-8.12 (10H; m; phenyl protons and pyridazinone H ), 10.34 (1H; s; -NH-C=O) and 10.55
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.64 (1C; -N-CH
2
-C=O), 125.96 (1C;
1
2,6
-CF
3
), 126.01 (1C; 2-chlorophenyl C ), 127.52 (2C; 4-trifluoromethylphenyl C ), 128.56 (2C;
-trifluoromethylphenyl C ), 128.89 (4C; 2-chlorophenyl C2,3,5,6), 129.58 (1C; 4-
3
,5
4
1
5
trifluoromethylphenyl C ), 131.05 (1C; pyridazinone C ), 132.92 (1C; 4-trifluoromethylphenyl
4
6
4
C ), 134.26(1C; pyridazinone C ), 142.54(1C; pyridazinone C ), 142.87 (1C; 4-chlorophenyl
4
3
C ), 158.70 (1C; CH
2
-N-C=O), 165.64 (1C; pyridazinone C ). C19
H14ClF N
3 4
4
O S. MS (ESI+)
calculated: 487.0455, Found: m/e 487.0462 (M+H; % 100.0).
2
-(3-(4-Bromophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(phenylsulfonyl)acetohydrazide (VI3a
)
o
1
Yield: 78%; mp: 215 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.76 (2H; t; -N-CH
2
-C=O),
4
,5
7
.15-8.15 (11H; m; phenyl protons and pyridazinone H ), 10.08 (1H; s; -NH-C=O) and 10.49
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.56 (1C; -N-CH -C=O), 122.94 (1C;
2
4
3
2
phenyl C ), 123.00 (1C; phenyl C ), 127.51 (1C; 4-bromophenyl C ), 127.81 (1C; 4-
6
5
2
6
bromophenyl C ), 128.83 (1C; phenyl C ), 129,42 (1C; phenyl C ), 129,62 (1C; phenyl C ),

3
5
1
30.98 (1C; 4-bromophenyl C ), 132.92 (1C; 4-bromophenyl C ), 133.62 (1C; 4-bromophenyl
4
5
6
C ), 137.30 (1C; pyridazinone C ), 138.90 (1C; pyridazinone C ), 142.60 (1C; pyridazinone
4
1
4
C ), 158.71 (1C; phenyl C ), 158.88 (1C; 4-bromophenyl C ), 165.43 (1C; CH -N-C=O),
2
3
1
4
70.61 (1C; pyridazinone C ). C18
63.0102 (M+H; % 100.0).
H15BrN
4
4
O S. MS (ESI+) calculated: 463.0076, Found: m/e
2
-(3-(4-Bromophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-fluorophenylsulfonyl)acetohydrazide
(
VI3b)
o
1
Yield: 87%; mp: 254 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.76 (2H; t; -N-CH -C=O),
2
4
,5
7
.05-8.09 (10H; m; phenyl protons and pyridazinone H ), 10.23 (1H; s; -NH-C=O) and 10.56
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.62 (1C; -N-CH
2
-C=O), 123.00 (1C;
6
2
5
4
-bromophenyl C ), 127.81 (1C; 4-bromophenyl C ), 128.97 (1C; 4-bromophenyl C ), 129.51
5
3,5
2,6
(
1C; 4-bromophenyl C ), 129.61 (2C; 4-fluorophenyl C ), 129.82 (2C; 4-fluorophenyl C ),
5
1
6
1
1
31.04 (1C; pyridazinone C ), 131.85 (1C; 4-bromophenyl C ), 133.32 (1C; pyridazinone C ),
4
1
37.74 (1C; pyridazinone C ), 137.84 (1C; 4-fluorophenyl C ), 142.65 (1C; 4-bromophenyl
4
4
-N-C=O), 170.56 (1C; pyridazinone C3).
C ), 158.72 (1C; 4-fluorophenyl C ), 165.51 (1C; CH
2
C
18
H
14BrFN
4
O
4
S MS (ESI+) calculated: 481.2964, Found: m/e 480.9975 (M+H; % 100.0).
2
-(3-(4-Bromophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-
chlorophenylsulfonyl)acetohydrazide (VI3c
)
o
1
Yield: 73%; mp: 225 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.70 (2H; t; -N-CH -C=O),
2
4
,5
7
.06-8.11 (10H; m; phenyl protons and pyridazinone H ), 10.09 (1H; s; -NH-C=O) and 10.48
1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.60 (1C; -N-CH
2
-C=O), 115.84 (2C;
2
,6
5
5
4
-bromophenyl C ), 116.14 (1C; 4-bromophenyl C ), 122.99 (1C; 4-bromophenyl C ), 127.80
3
,5
2,6
5
(
2C; 4-chlorophenyl C ), 129.61 (2C; 4-chlorophenyl C ), 130.63 (1C; pyridazinone C ),
1
6
4
1
1
30.76 (1C; 4-bromophenyl C ), 131.02 (1C; pyridazinone C ), 131.83 (1C; pyridazinone C ),
1
4
33.31 (1C; 4-chlorophenyl C ), 135.15 (1C; 4-bromophenyl C ), 142.61 (1C; 4-chlorophenyl
4
3
C ), 158.71 (1C; CH
2
-N-C=O), 165.416 (1C; pyridazinone C ). C18
H
14BrClN
4
4
O S. MS (ESI+)
calculated: 497.7480, Found: m/e 498.9678 (M+H; % 100.0).
2
-(3-(4-Bromophenyl)-6-oxopyridazin-1(6H)-yl)-N'-(4-trifluoromethyl
phenylsulfonyl)acetohydrazide (VI3d
)
o
1
Yield: 70%; mp: 255 C; H-NMR (DMSO-d
6
, 300 MHz): δ (ppm) 4.72 (2H; t; -N-CH -C=O),
2
4
,5
7
.04-8.11 (10H; m; phenyl protons and pyridazinone H ), 10.35 (1H; s; -NH-C=O) and 10.56

1
3
(
1H; s; -NH-SO
2
). C-NMR (DMSO-d
6
, 300 MHz), δ 52.66 (1C; -N-CH
2
-C=O), 121.61 (1C;
1
2
-CF
3
), 123.001 (1C; 2-bromophenyl C ), 125.23 (1C; 4-trifluoromethylphenyl C ), 125.95 (1C;
6
3
4
-trifluoromethylphenyl C ), 126.00 (1C; 4-trifluoromethylphenyl C ), 126.59 (1C; 4-
5
2
3
trifluoromethylphenyl C ), 127.75 (1C; 4-bromophenyl C ), 127.82 (1C; 4-bromophenyl C ),
5
6
1
28.55 (1C; 4-bromophenyl C ), 129.58 (1C; 4-bromophenyl C ), 130.99 (1C; 4-
1
5
trifluoromethylphenyl C ), 132.27 (1C; pyridazinone C ), 133.36 (1C; 4-trifluoromethylphenyl
4
6
4
C ), 141.10 (1C; pyridazinone C ), 142.87 (1C; pyridazinone C ), 158.87 (1C; 4-bromophenyl
4
3
C ), 165.64 (1C; CH
2
-N-C=O), 170.62 (1C; pyridazinone C ). C19
H14BrF
3
N
4
4
O S. MS (ESI+)
calculated: 531.3042, Found: m/e 530.9960 (M+H; % 100.0).
Pharmacology
Anticholinesterase Activity
The in vitro inhibition of AChE and BChE by the newly synthesized title compounds was
determined by the method of Ellman et al. [29] using galantamine (CAS 357-70-0) as reference.
Electric eel AChE (Type-VI-S, EC 3.1.1.7, Sigma, St. Louis, MO, USA) and horse serum BChE
(EC 3.1.1.8, Sigma) as the enzyme sources, acetylthiocholine iodide and and butyrylthiocholine
chloride (Sigma) as substrates, and 5,5’-dithio-bis(2-nitrobenzoic)acid (DTNB) were also used
in the anticholinesterase activity determination. All reagents and conditions were the same as
described recently [42]. Briefly, in this method, 140 µl 0.1 mM sodium phosphate buffer
(pH8.0), 20 µl DTNB, 20 µl test solution, and 20 µl AChE/BChE solution were added by
multichannel automatic pipette (Gilson pipetman, Middleton, USA) in a 96-well microplate and
o
incubated for 15 min at 25 C. The reaction was then initiated with the addition of 10 µl
acetylthiocholine iodide/butyryl-thiocholine chloride. The hydrolysis of acetylthiocholine
iodide/butyrylthiocholine chloride was monitored by the formation of the yellow 5-thio-2-
nitrobenzoate anion as a result of the reaction of DTNB with thiocholines catalyzed by the
enzymes at a wavelength of 412 nm utilizing a 96-well microplate reader (VersaMax Molecular
Devices, North Carolina, USA). The measurements and calculations were evaluated using
Softmax PRO 4.3.2.LS software (Softmax Moleculer Devices, Downingtown, USA). The
percentage of inhibition of AChE/BChE was determined by comparing the rates of the
reactions of the samples relative to blank samples (ethanol in phosphate buffer pH= 8) using
the formula (E-S)/E × 100, where E is the activity of the enzyme without test sample and S is
the activity of the enzyme with the test sample. The experiments were done in triplicate and the
results were expressed as average values with S.E.M. (standard error mean).

Molecular Modeling Methodology
Sequence alignment for EeAChE (O42275) and ECBChE (Q9N1N9) was performed using
BLAST and Clustal Omega. The N terminal insertion residues of ECBChE were removed. The
template structures, TcAChE (PDB ID: 6EUC [43]) and hBChE (PDB ID: 4TPK [44]), were
downloaded from the RCSB protein data bank (www.rcsb.org) [45,46]. An initial 100 model
structures were built by MODELLER (v9.19) for each enzyme using the sequence alignments
and the template structures. The best model for each enzyme was selected according to the
DOPE scores. Loop refinement was performed for the EeAChE model, which gave another 100
candidate models, again, the one with best DOPE score was selected. The final EeAChE and
ECBChE models were submitted to PROCHECK server for analyses.
The model structures were prepared for docking using the Protein Preparation Wizard of
Maestro (Schrödinger, LLC, New York, NY, 2019) [46]. In this process, the ionization and
tautomeric states were generated by Epik (Schrödinger, LLC, New York, NY, 2019) and the
proton orientations were set by PROPKA [47]. Restrained minimization was run using Impact
(Schrödinger, LLC, New York, NY, 2019) with convergence of heavy atoms to an RMSD of 3
Å. The 3-D models of the ligands were created using MacroModel (Schrödinger, LLC, New
York, NY, 2019), prepared for docking using LigPrep (Schrödinger, LLC, New York, NY,
2
019) to include possible ionization and tautomeric states, and optimized using OPLS_2005
force field and conjugate gradient method [48]. The ligands were docked to the grids prepared
by Grid generation module of Maestro using the central coordinates 56.74 164.35 183.69 for
EeAChE and 4.33 10.44 14.00 for ECBChE. Docking procedure was performed 50 times for
each ligand by Glide (Schrödinger, LLC, New York, NY, 2019) at extra precision with post-
docking minimization enabled [49-51]. The docking score of each ligand was determined from
the best pose upon visual evaluation on Maestro. RM0 and 3F9 were re-docked to their
respective binding sites to test the performance of the docking methodology; the RMSD values
of the resulting docking poses were 1.60 and 0.79, respectively.
Results
Chemistry
The title compounds (VI1a-1d, VI2a-2d, VI3a-3d) were synthesized according to the literature
methods as outlined in Scheme 1.
SCHEME 1
Scheme 1. Synthesis of the VI1a-1d, VI2a-2d, VI3a-3d
.

Synthesis of the compounds was initiated by obtaining benzoyl propanoic acid
derivatives (I1-3) in the presence of succinic anhydride and substituted benzene by anhydrous
aluminum chloride catalysis. Subsequently, the reaction of these compounds with hydrazine
hydrate led to the formation of 4,5-dihydro-3(2H) -pyridazinone (II1-3) [52,53]. 6-Substituted-
3
(2H)-pyridazinone derivatives (III1-3) were obtained by hydrolysis of II1-3 heated in glacial
acetic acid [34]. A very useful and easy-to-use method was also tested for synthesis of
compound III1-3, which Schmidt and Druey developed in 1954 for the synthesis of pyridazinone
compounds [54]. The reaction is based on the formation of 3(2H)-pyridazinone derivatives by
condensation of 1,2-dicarbonyl compound with monosubstituted or unsubstituted hydrazine
and the carboxyl derivative containing active methylene group. Dicarbonyl compounds
including 1,2-diketones, α-ketoacids or glyoxal and esters, malonic acid, acetoacetic acid,
cyanoacetic acid, benzoylacetic acid or hippuric acid esters are used as α-methylene reactive
group [55]. In this study, glyoxalic acide was used as 1,2-dicarbonyl compound. Glyoxalic acid
and acetophenone derivatives were heated with hydrazine hydrate in acidic medium to give
3
(2H)-pyridazinone derivatives (III1-3). Ethyl 6-substituted-3(2H)-pyridazinone-2-ylacetate
derivatives (IV1-3) were obtained by the reaction of III1-3 with ethyl bromoacetate in the
presence of CO in acetone [34]. Then, 6-substituted-3(2H)-pyridazinone-2-
K
2
3
ylacetohydrazide derivatives (V1-3) were synthesized by the condensation reaction of IV1-3 with
hydrazine hydrate [34]. Ultimately, the title compounds bearing benzenesulfonohydrazide
structure were obtained by the reaction of V1-3 with substituted/nonsubstituted
benzenesulfonylchlorides. All of the title compounds were reported for the first time in this
study. The reaction yields ranged approximately from 60% to 90%. Compound VI1b was
synthesized with the highest yield (94%) while compound VI1a with the lowest yield (59%).
The physical and spectral properties of the starting compounds were in accordance with the
1
literature [35,56-60]. Molecular structures of title compounds were confirmed by IR, H-NMR,
13C-NMR, and mass spectral data. Their molecular structures, yields, and melting points are
given in Table 1.
TABLE 1
Table 1. Molecular structures, yields and melting points of VI1a-1d, VI2a-2d, VI3a-3d
Pharmacology

In vitro AChE and BChE inhibition of VI1a-1d, VI2a-2d, VI3a-3d was determined by
the method of Ellman et al. [29] using galantamine as reference. All compounds showed
higher inhibitory activity against BChE than AChE. The activity data of the compounds
were summarized as percentage inhibition ± standard deviation (SD) values at 50 and 100
µg/ml concentration in Table 2.
TABLE 2
Table 2. AChE and BChE inhibition percentages of the title compounds with ± standard
error mean (SEM)
The compounds did not show AChE inhibition at 50 μg/ml. Compound VI1c and VI2c did
not show AChE inhibition at 100 μg/ml, either. The inhibition % of VI3b and VI3d could not
be measured because of precipitation at 100 µg/ml. The compounds were less potent than
reference drugs, such as galantamine, donepezil, rivastigmine, physostigmine and tacrine but
almost twice more potent against BChE than AChE. VI2a, bearing 4-chlorophenyl ring,
showed the best AChE inhibition with 25.02% and compound VI2a showed the highest BChE
inhibitory activity with a value of 51.70%. While compound VI2c did not show AChE
inhibitory activity, it was found to be the second most potent against BChE with a value of
5
1.36%. Compounds VI2c and VI2d showed high BChE inhibitor activity at low concentration
compared to other compounds.
Molecular modelling studies
Homology modelling and structural evaluation of the enzyme structures
We built homology model of Electrophorus electricus (electric eel) AChE (EeAChE) and
Equus caballus (horse) BChE (ECBChE) using comparative modelling techniques. The
available crystal structures of EeAChE lack structural quality, resolution, and co-crystallized
ligand to be useful in modelling studies and there is no crystal structure available for ECBChE.
Homology modelling was performed by simply aligning the sequences of these enzymes with
the most homologous sequences of the proteins with available 3D structure, Tetronarce
californica AChE (TcAChE) and Homo sapiens BChE (hBChE), and threading the target model
structures. (For the alignments, conserved and key residues see Figure S1 and S2 of Supporting
Information). Sequence identitiy between EeAChE and TcAChE The co-crystallized inhibitors,

(E)-3-hydroxy-6-(5-morpholinopentyl)picolinaldehyde oxime (RM0) in TcAChE and N-
{
[(3R)-1-(2,3-dihydro-1H-inden-2-yl)piperidin-3-yl]methyl}-N-(2-
methoxyethyl)naphthalene-2-carboxamide (3F9) in hBChE active gorge, as well as the solvent
molecules, were transferred to the respective model structures using a specific MODELLER
script [61]. A long insertion loop between residues 439-470 of EeAChE was optimized although
these residues are away from the active gorge. In the case of ECBChE an insertion loop at the
N terminal, which is far from the catalytic site, was removed. The PROCHECK analyses of the
models confirmed their structural quality. The Ramachandran plot showed only 0.4% of the
residues in the disallowed region for both models (See Figure S3 and S4 of Supporting
Information), 99.6% of the planar groups of EeAChE and were within limits and this value was
1
2
00% for ECBChE. Regarding bad contacts, bond lengths, and angles both models were "1 1
" in Morris et. al classification [62].
Molecular docking and binding interactions
The catalytic gorge of EeAChE accommodates several water molecules which take part in the
water-mediated H bond network between RM0 and the receptor just like TcAChE, as well as
π- π and π-cation interactions (Figure 5) [43]. In these interactions residues such as Tyr146,
Ala226, Glu224, Trp304, Tyr355, Phe356, and His494 stood forward with direct or water-
mediated contacts. The same interactions were observable with the corresponding residues in
the template crystal structure. The counterparts of these residues in the Homo sapiens isozyme
(HSAChE) reportedly play key role in accommodation of potent AChE ligands (see Table S1
in Supporting Information). For example, according to the literature studies, Tyr72 and Trp286,
so-called PAS residues, stack with aromatic moieties of inhibitors close to the gorge entry.
Asp74 (a PAS residue), Gly122, Glu202 (a choline binding pocket), Ala204, along with Tyr72
are known to participate in H bonds, some of which are water-mediated. Both Tyr337, a
choline-binding pocket residue, and Phe338, which is an acyl-binding residue, are important
for packing ligands in the cavity tightly. His447 is a member of the catalytic triad responsible
for the esterase activity [60,63,64]. We observed in docking studies that galantamine also made
water-mediated H bonds with Gu224 and His494 and π-π interaction with Tyr146 (Figure 5).
FIGURE 5
Figure 5. Binding interaction of RM0 (green), galantmine (gray), 1a (teal) and 2a (orange) (A-
D, respectively) in EEChE active gorge. Ligands and water molecules are showed as color
sticks, the active gorge residues in sticks and the protein backbone in tubes.

Our compounds bound the active gorge of EeAChE with high affinity (Table 3). Especially 1a
and 2a, which were the most potent AChE inhibitors in the series, were among the derivatives
with good scores. They bound to the EeAChE active site with the phenylpyridazinone moiety
stretching to the bottom of the gorge and the phenylsulfonyl moiety occupying the PAS as
demonstrated in Figure 5. Phenylpyridazinone moiety was mostly in hydrophobic contacts with
the acyl- and choline-binding pocket residues of the CAS, defying the hypothesis of Zhou et al.
[
25]. Trp108, Trp304, and Tyr355 were among key residues in these interactions (see Figure
S5 of Supporting Information for more details). Our compounds, however, failed in other key
interactions listed above, especially H bonds and interactions with the catalytic triad, which
could account for their limited potency against EeAChE compared to galantamine. Especially,
lack of a positive charge group, a common pharmacophore among known AChE inhibitors, is
one of the reasons for this situtation.
TABLE 3
Table 3. Docking scores
The complex of ECBChE and 3F9, a potent hBChE inhibitor [44], features several key
interactions with the catalytic residues of the enzyme directly or mediated with water. Glu197
(choline-binding pocket), Trp231 and Phe329 (acyl-binding pocket), Asp70 and Tyr332 (PAS)
are among the important residues to engage in these interactions (Figure 6). The exact same
interactions with the exact same residues are observable between 3F9 and hBChE crystal
structure as expected given the high level of homology between the two structures [44].
Docking pose of galantamine features interactions with Trp82 and Glu197 (choline-binding
pocket) and His438 (catalytic triad) (Figure 6).
The title compounds fit in ECBChE active site with high affinity (Table 4). Their scores were
as good as that of galantamin and potent derivatives ,for example, 2a and 2c were among the
top scoring compounds. The compounds were observed to bind to the active gorge of ECBChE
assuming a “V” shape taking a twist around the center of mass, unlike their straight binding
orientations in EeAChE (Figure 6). The reason for this is the topology of the active gorge of
BChE enzymes, which allows more conformational freedom in order to accommodate BCh,
which has a bulkier acyl group than ACh [65]. In Figure 6 2a and 2c are depicted docked to the
enzyme and the key residues in their interactions include Trp82, Trp231, Leu285, and Phe329.

Along with conformational freedom, the title compounds engaged more in key interactions with
ECBChE than with EeAChE (see Figure S6 of Supporting Information for more details), which
probably made them more potent against the former and resulted in better docking scores (Table
4
). Most of these residues were showed to be important for competitive inhibition through
experimental and theoretical studies [44,60,66,67]. The binding mode of the title compounds in
ECBChE active site were in accordance with the hypothesis presented by Zhou et al. [25] as
summarized in Figure 4, which was not the case with EeAChE.
FIGURE 6
Figure 6. Binding interaction of 3F9 (green), galantmine (gray), 2a (orange) and 2c (magenta)
(A-D, respectively) in ECBhE active gorge. Ligands and water molecules are showed as color
sticks, the active gorge residues in sticks and the protein backbone in tubes.
Discussion
As a continuation of our research interest in developing novel 3(2H)-pyridazinone
derivatives having AChEI and BChEI activities and establishing new relationships between
their structure and activity, we designed and synthesized a series of 6-substituted-3(2H)-
pyridazinone-2-acetyl-2-(nonsubstituted/4-substituted
benzenesulfonohydrazide)
and
evaluated their AChE and BChE inhibitory activities according to the Ellman’s method. The
title compounds showed moderate inhibition at 100 µg/ml and they were more active against
BChE. Compound VI2a emerged as a dual inhibitor with 51.70% and 25.02% inhibition against
AChE and BChE, respectively. In general, halogen substitution on benzalhydrazone phenyl ring
leads to an increase in AChE and BChE inhibition. Cl and CF substitutions at the para position
3
of the benzalhydrazone phenyl ring also improved the anti-BChE activity. Adversely, m-
substitutions on the benzalhydrazone phenyl ring with strong electron-donating groups such as
OCH reduced the AChE and BChE inhibition. It was less potent than the reference drugs, yet
3
promising for further modifications as a leader.
With homology modelling we obtained reliabale and validated structure models of
EeAChEand ECBChE. Molecular docking studies were very insightful for the structural
features of the title compounds to engage in key interactons, as well as their shortcomings for
ideal fit. Although the compounds showed high affinity to the enzymes, the lack of a positive
charge group, for example, was apparent in docking studies, which could be a key aspect for
future optimization of these derivatives into better inhibitors. The ability of the compounds to

adopt energetically more favourable conformations and to engage in more key interactions in
the ECBChE active gorge explains their better activity profile against ECBChE.
Conflict of interest
All authors declare no conflict of interest connected with this paper.

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Figure captions:
Figure 1. Structure of the drugs bearing pyridazinone skeleton and compound 4 [14].
Figure 2. Structure of some FDA-approved AChEI in AD.
Figure 3. Structural hypothesis for AChEIs and some designed compounds from the literature
[
25].
Figure 4. Structural hypothesis for the title compounds [25].
Figure 5. Binding interaction of RM0 (green), galantmine (gray), 1a (teal) and 2a (orange) (A-
D, respectively) in EeAChE active gorge. Ligands and water molecules are showed as color
sticks, the active gorge residues in sticks and the protein backbone in tubes.
Figure 6. Binding interaction of 3F9 (green), galantmine (gray), 2a (orange) and 2c (magenta)
(A-D, respectively) in ECBhE active gorge. Ligands and water molecules are showed as color
sticks, the active gorge residues in sticks and the protein backbone in tubes.
Scheme 1. Synthesis of the VI1a-1d, VI2a-2d, VI3a-3d
.