Novel pyridazinone derivatives as butyrylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2019.103304

In the current study, forty-four new [3-(2/3/4-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl carbamate derivatives were synthesized and evaluated for their ability to inhibit electric eel acetylcholinesterase (EeAChE) and equine butyrylcholinesterase (eqB

Full text of DOI:10.1016/j.bioorg.2019.103304

BioorganicChemistry92(2019)103304
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel pyridazinone derivatives as butyrylcholinesterase inhibitors
⁎
Yasemin Dundara, , Ozge Kuyrukcua, Gokcen Erena, F. Sezer Senol Denizb, Tijen Onkola,
Ilkay Erdogan Orhanb
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
b Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, 06330 Ankara, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the current study, forty-four new [3-(2/3/4-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl carbamate
derivatives were synthesized and evaluated for their ability to inhibit electric eel acetylcholinesterase (EeAChE)
and equine butyrylcholinesterase (eqBuChE) enzymes. According to the inhibitory activity results, [3-(2-meth-
oxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl heptylcarbamate (16c, eqBuChE, IC50 = 12.8 μM; EeAChE, no in-
hibition at 100 μM) was the most potent eqBuChE inhibitor among the synthesized compounds and was found to
be a moderate inhibitor compared to donepezil (eqBuChE, IC50 = 3.25 μM; EeAChE, IC50 = 0.11 μM). Kinetic
and molecular docking studies indicated that compounds 16c and 14c (hexylcarbamate derivative, eqBuChE,
IC50 = 35 μM; EeAChE, no inhibition at 100 μM) were mixed-type inhibitors which accommodated within the
catalytic active site (CAS) and peripheral anionic site (PAS) of hBuChE through stable hydrogen bonding and π-π
stacking. Furthermore, it was determined that [3-(2-methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl (4-me-
thylphenyl)carbamate 7c (eqBuChE, IC50 = 34.5 μM; EeAChE, 38.9% inhibition at 100 μM) was the most active
derivative against EeAChE and a competitive inhibitor binding to the CAS of hBuChE. As a result, 6-(2-meth-
oxyphenyl)pyridazin-3(2H)-one scaffold is important for the inhibitory activity and compounds 7c, 14c and 16c
might be considered as promising lead candidates for the design and development of selective BuChE inhibitors
for Alzheimer’s disease treatment.
Alzheimer’s disease
Pyridazinone derivatives
Acetylcholinesterase inhibitors
Butyrylcholinesterase inhibitors
Docking
1. Introduction
neurotransmission through inhibition of acetylcholinesterase (AChE)
enzyme that hydrolyses ACh. The AChE inhibitors donepezil, rivas-
Dementia is
a
cognitive impairment syndrome which affects
tigmine, and galantamine are used for symptomatic treatment for mild-
to-moderate AD [6–8]. In the later stages of AD, the level of AChE
enzyme progressively decreases due to dramatic cholinergic synapse
and neuronal loss. However, the butyrylcholinesterase (BuChE) levels
increase significantly and BuChE plays a critical role for ACh hydrolysis
in the late stage of AD [9–11]. In addition, both enzymes, in particular,
BuChE, are also found to associate with neurotoxic amyloid β plaques
and neurofibrillary tangles in human AD brain tissue [12,13]. Thus, in
recent years, studies on selective BuChE inhibitors have been increased
and are considered as an alternative for the treatment of patients with
mild-to-late stages of AD [9–11,14,15].
memory, cognitive abilities, and behavior. According to the World
Health Organization, there are approximately 50 million dementia
patients globally and this number is estimated to grow to 152 million by
2050. The most common form of dementia is Alzheimer's disease (AD),
accounting for about 60–70% of cases [1,2]. AD, an irreversible neu-
rodegenerative disorder, leads to the progressive loss of cognitive and
intellectual function, reduced ability to perform the activities of daily
living as well as the emergence of behavioral and psychological
symptoms such as depression, apathy, anxiety, agitation, irritability,
delusion, and hallucination [3,4]. There is no cure for AD at the mo-
ment, where people aged 65 and older survive an average of 4–8 years
after diagnosis of Alzheimer’s type of dementia [5].
In our previous work, [3-(4-substitutedphenyl)-6-oxopyridazin-
1(6H)-yl)methyl phenylcarbamate derivatives were synthesized and
screened for their inhibitory activity on cholinesterase enzymes. Among
the compounds, [3-(4-methoxyphenyl)-6-oxopyridazin-1(6H)-yl)me-
thyl phenylcarbamate derivative showed inhibitory activity against
both AChE and BuChE [16]. Previous studies by different research
Neuropathological features of the AD are characterized by amy-
loidal plaques, neurofibrillary tangles, cholinergic neurons damage, and
abnormally decreased levels of the acetylcholine (ACh). Most of the
current drugs for the AD are mainly based on enhancing cholinergic
⁎ Corresponding author.
E-mail address: yasemina@gazi.edu.tr (Y. Dundar).
https://doi.org/10.1016/j.bioorg.2019.103304
Received 30 May 2019; Received in revised form 16 September 2019; Accepted 17 September 2019
Availableonline18September2019
0045-2068/©2019PublishedbyElsevierInc.

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Scheme 1. Reagents and conditions: i) 105–108 °C,
3 h; then NH2NH2·H2O, reflux, 2 h. ii) 37% for-
malin, reflux, 2 h. R1: 4-methoxyphenyl (a, 1st
serie); 3-methoxyphenyl (b, 2nd serie); 2-methox-
yphenyl (c, 3rd serie).
groups have shown that the arylcarbamate or long chain alkylcarba-
mate group is a suitable fragment to achieve selective BuChE inhibition
[15,17–20]. Based on these findings, in order to obtain more potent and
selective BuChE inhibitors, [3-(2/3/4-methoxyphenyl)-6-oxopyridazin-
1(6H)-yl)methyl substitutedphenyl/ alkylcarbamate derivatives were
synthesized and evaluated for their inhibitory effects on EeAChE and
eqBuChE enzymes. Additionally, kinetic and molecular docking studies
were performed for the selected compounds.
According to the activity results, in the first serie (3a-17a) bearing
6-(4-methoxyphenyl)pyridazin-3(2H)-one moiety; all compounds ex-
cept 6a (4.6%) and 14a (20.1%) had no inhibitory effect on EeAChE at
100 μM. Concerning the activity of this serie against eqBuChE, the in-
hibitory activity remained below 15% in the aromatic, short alkyl
chain, and dimethyl carbamate derivatives (3a-11a, 17a). The sig-
nificant increase in activity was observed with butyl derivative (12a,
27.5%). The pentyl (13a), hexyl (14a), cyclohexyl (15a) derivatives
showed inhibitory activity of 36.4%, 48.9%, 51.5% (IC50 = 97.2 μM),
respectively. However, the heptyl derivative (16a) exerted an in-
hibitory activity of 33.2% (Fig. 1).
2. Results and discussion
On the other hand, the activity results of the second serie bearing 6-
(3-methoxyphenyl)pyridazin-3(2H)-one moiety were evaluated; only 4-
fluorophenylcarbamate (4b, 11.1%), 4-methoxyphenylcarbamate (6b,
5.9%) and hexylcarbamate (14b, 16.6%) derivatives had inhibitory
effect on EeAChE. Regarding the activity of this serie against eqBuChE,
similar to the first serie, the aromatic, short alkyl chain, and dimethyl
carbamate derivatives (3b-11b, 17b) showed no inhibitory activity or
less than 15%. An increase in activity was observed with butyl deri-
vative (12b) which showed 20.9% inhibition. Pentyl (13b), hexyl
(14b), and cyclohexyl (15b) derivatives exhibited 49.4%, 55.0%
(IC50 = 76.1 μM), and 43.0% inhibitory activity, respectively. The
heptyl derivative (16b) was tested at 50 μM due to the solubility pro-
blem and showed the inhibitory activity of 31.9% at this concentration
(Fig. 1).
2.1. Chemistry
The synthetic routes for the target compounds are outlined in
Schemes 1 and 2. The starting compounds, 6-(substitutedphenyl)pyr-
idazin-3(2H)-one derivatives (1a, 1b, 1c) was readily prepared by the
reaction of glyoxylic acid monohydrate, appropriate acetophenone, and
hydrazine hydrate. These compounds were then reacted with for-
maldehyde to obtain hydroxymethyl derivatives (2a, 2b, 2c) as shown
in Scheme 1. The compounds except N-methyl and N,N-dimethyl car-
bamate derivatives were afforded by the reaction of hydroxymethyl
derivatives with appropriate isocyanate in the presence of N-methyli-
midazole (Scheme 2). Methylamine and 1,1-carbonyldiimidazole were
used instead of toxic methyl isocyanate in the synthesis of N-methyl
carbamate derivatives (8a, 8b, 8c) (Scheme 2). N,N-dimethylcarbamate
derivatives (17a, 17b, 17c) were prepared by the reaction of N,N-di-
methylcarbamoyl chloride, hydroxymethyl derivatives, and N-methy-
limidazole (Scheme 2).
The third serie carrying 6-(2-methoxyphenyl)pyridazin-3(2H)-one
moiety is the group with the most active compounds. According to the
results of EeAChE inhibition assay; only phenyl (3c), 4-chlorophenyl
(5c), 4-methoxyphenyl (6c), and 4-methylphenyl (7c) derivatives
showed 24.7%, 15.4%, 38.2% and 38.9% inhibitory activity, respec-
tively. As far as the activity against eqBuChE is concerned, methyl to
butyl chain and dimethyl carbamate derivatives (8c-12c) showed less
than 15% inhibitory activity. The activity increased as the chain ex-
tended, that pentyl (13c), hexyl (14c), cyclohexyl (15c), heptyl (16c)
2.2. AChE and BuChE inhibitory activity
Inhibitory activities of the synthesized compounds on EeAChE and
eqBuChE were evaluated by Ellman’s method [21–23]. The results are
given as the percentage of enzyme inhibition at 100 μM (Fig. 1). IC50
values were calculated for compounds which exhibited inhibition of
more than 50% (Table 1). Donepezil and galantamine were used as the
reference compounds tested at 10 μM concentration.
derivatives
showed
with
49.5%
(IC50 = 99.4 μM),
68.4%
(IC50 = 35.0 μM), 38.2%, 76.9% (IC50 = 12.8 μM) inhibitory activity,
respectively. Different from the other series, the aromatic carbamate
Scheme 2. Reagents and conditions: i) CDI, AcCN, rt, 3 h; then CH3NH2, K2CO3, rt, 3 h. ii) appropriate isocyanate, NMI, AcCN, rt, 24 h. iii) DMCC, NMI, K2CO3,
AcCN, rt, 24 h.
2

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 1. EeAChE and eqBuChE inhibitory activity of the synthesized compounds (3a-17a, 3b-17b, 3c-17c) and the references. Data are indicated as percentage of
inhibition at 100 µM SD (n = 4). Donepezil and galantamine were used as the reference drugs at 10 μM concentration.
derivatives were also found to display inhibitory activity on eqBuChE.
The phenyl (3c), 4-fluorophenyl (4c), 4-chlorophenyl (5c), 4-methox-
yphenyl (6c), and 4-methylphenyl (7c) carbamate derivatives showed
65.4% (IC50 = 50.3 μM), 30.4%, 25.0%, 53.4% (IC50 = 92.6 μM), and
66.6% (IC50 = 34.5 μM), respectively (Fig. 1).
while improvement in activity was observed in the following order:
methyl (7c) > H (3c) > methoxy (6c) group. Compound 7c was
found to be 2.6 fold more active than 6c against eqBuChE. Besides,
compounds 6c (38.2%) and 7c (38.9%) derivatives showed similar
activity pattern in EeAChE.
In generally, the synthesized compounds exhibited a better in-
hibitory activity against eqBuChE compared with EeAChE. However,
none of the compounds was found to be more active than references
donepezil and galantamine. When the structure-activity relationships
were evaluated for eqBuChE, the position of the methoxy substituent in
the phenylpyridazinone ring was found to be important, and it was
suggested that the presence of ortho methoxy group increased the in-
hibitory activity. Regarding the importance of the substituent in car-
bamate group, especially, long-chain carbamate groups such as hexyl or
heptyl was enhanced the eqBuChE inhibitory activity. Although the
heptyl carbamate derivative 16c was the most active eqBuChE inhibitor
among the synthesized compounds, it was four-fold less active than
donepezil. Butyl chain was the critical length at which inhibitory ac-
tivity begins increasing significantly. Regarding the third serie, among
the substituted aromatic carbamate derivatives, electron withdrawing
groups like fluoro (4c), chloro (5c) groups decreased inhibitory activity
For the eqBuChE inhibition mechanism of the selected compounds
7c, 14c, and 16c was determined by enzyme kinetic study. The
Lineweaver-Burk plots and replots of the slope versus concentration
were utilized to obtain the inhibition constant (Ki) (Table 1 and Fig. 2).
Compound 7c, 4-methylphenylcarbamate derivative, displayed a com-
petitive inhibition while hexylcarbamate derivative 14c and heptyl-
carbamate derivative 16c showed a mixed-type inhibition. The ob-
tained Ki values were consistent with the measured IC50 values.
Obtained plots of the inhibitors are shown in Fig. 2.
To elucidate the time dependent inhibition, the IC50 shift assay was
carried out by pre-incubating the eqBuChE with (i) no compound, (ii)
selected compounds, and (iii) rivastigmine as the reference compound
for different time periods (40, 20, 10 min). Rivastigmine, the pseudo-
irreversible inhibitor, showed time-dependent inhibition with 3.1 fold
shift in IC50. In case of tested compounds, compound 7c, 14c and 16c
exhibited similar inhibition profile with 1.47, 1.8, and 2.8 fold shift IC50
Table 1
EeAChE and eqBuChE inhibitory activity of the selected compounds and references.
O
N
N
O
O
R1
NH
R2
Comp.
R1
R2
EeAChE
eqBuChE
Inhibition (%)a
nib
IC50 (µM)
Inhibition (%)a
IC50 (µM)
Ki (µM)
Inhibition mechanism
Competitive
15a
4-OCH3
3-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
2-OCH3
cyclohexyl
hexyl
ndc
nd
nd
nd
nd
nd
nd
nd
51.5
55.0
65.4
53.4
66.6
49.5
68.4
76.9
65.5
37.9
5.6
1.2
2.7
0.9
1.0
1.0
0.4
1.2
1.8
1.2
97.2
76.1
50.3
92.6
34.5
99.4
35.0
12.8
3.25
nd
0.6
14b
16.6
24.7
38.2
38.9
ni
5.8
2.2
1.0
2.1
2.5
3.4
4.0
3.2
0.3
2.6
0.8
0.16
3c
phenyl
6c
4-methoxyphenyl
4-methylphenyl
pentyl
7c
33.3
13c
14c
hexyl
ni
30.2
12.0
Mixed-type
Mixed-type
16c
heptyl
ni
Donepezild
Galantamined
89.1
86.0
0.1
2.1
0.11
3.7
0.03
0.5
a
b
c
Data are indicated as percentage of inhibition at 100 µM
ni: no inhibition.
SD (n = 4).
nd: not determined.
d
Donepezil and galantamine were used as the reference drugs at 10 μM concentration.
3

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 2. Kinetic study on the mechanism of eqBuChE inhibition by 7c, 14c, and 16c. Lineweaver-Burk plots: 7c (A), 14c (C), 16c (E). Slope replot vs inhibitor
concentration: 7c (B), 14c (D), 16c (F).
values, respectively. The time-dependent decrease in IC50 values of the
compounds suggested that our compounds may be a reversible inhibitor
or a pseudo-irreversible inhibitor or an irreversible inhibitor. Although
carbamate derivatives are generally known to inhibit ChEs by a me-
chanism involving covalent interaction between their carbamate group
and the catalytic serine, our compounds do not participate this type of
interaction with active site serine. However, being an irreversible or a
pseudo-irreversible inhibitor could not be suggested for the compounds
7c, 14c and 16c. Therefore, our compounds were proposed as re-
versible with tight-binding inhibition [24,25] which is known to show
kinetics similar to covalent inhibition may the explanation for the
binding mechanism for the compounds tested.
2.3. Molecular docking studies
To explore possible binding interactions of potential inhibitors,
compounds 14b, 3c, 6c, 7c, 14c, and 16c were selected to dock into
EeAChE (PDB: 1C2O), hAChE (PDB: 4EY7), and hBuChE (PDB: 4TPK)
using Glide module implemented in Schrödinger Small-Molecule Drug
Discovery Suite.
The compounds showing dual inhibition of AChE and BuChE fit into
the binding site of EeAChE interacting with the CAS and PAS regions. In
the case of compounds 3c, 6c, and 7c, the 2-methoxyphenyl ring was
bound to the PAS, forming π-π stacking interactions with TRP286. The
pyridazinone and phenyl rings adjacent to the carbamate moiety ex-
tended beyond the CAS and stabilized by a hydrogen bond between
4

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 3. Proposed binding modes for compounds 14b (A), 3c (B), 6c (C), and 7c (D) in EeAChE active site (PDB: 1C2O). The compounds are presented as green ball
and sticks with nitrogen and oxygen atoms in blue and red, respectively. The involved residues are named and showed as white sticks. Hydrogen bonds and π-π
stacking contacts are represented with yellow and cyan dashed lines, respectively.
carbonyl oxygen and PHE295 backbone and π-π stacking contacts with
TYR337, respectively. For compound 14b, the interactions obtained
were a π-π stacking contact between 3-methoxyphenyl ring and TYR72
from the PAS and a hydrogen bond between carbonyl oxygen and
PHE295 backbone. Unlike the compounds 3c, 6c, and 7c, compound
14b showed no contact with TYR337 from the CAS due to replacement
of the ring with alkyl chain (Fig. 3).
orientation of the molecule differed from the proposed binding mode in
EeAChE active site. 4-Methoxyphenyl moiety positioned in the mouth of
the gorge by a π-π stacking contact with TRP286 and a water mediated
hydrogen bond with SER293 whereas the pyridazinone ring formed a π-
π stacking contact with TYR337 from oxyanion hole. Besides, addi-
tional hydrogen bonds were observed between the PAS residue TYR124
and the carbonyl oxygen of the carbamate group and oxygen atom of 2-
methoxyphenyl ring through water network. Regarding compound 7c,
the presence of water molecules provided a hydrogen bond with
TYR124. Moreover, additional π-π stacking contacts which enhanced
the stability of inhibitor:enzyme complex compared to EeAChE were
observed in the PAS and the oxyanion hole with TYR341 and TRP86,
respectively.
Since the X-ray crystal structures of EeAChE do not contain any
water molecules, the selected compounds were also docked into hAChE
in order to identify the impact of water molecules on binding pattern as
shown in Fig. 4. Considering large mobility of some residues to ac-
commodate ligands [26] in AChE structures, the flexibility of some side
chains such as TRP286, PHE338 have been taken into account in
docking studies by Schrödinger’s Induced Fit Docking (IFD) protocol
[27]. The results demonstrated that keeping the structural water mo-
lecules in active site provided favorable hydrogen bonds with TYR124
for all compounds and SER293 for compound 6c. For compound 14b
anchored the PAS via TRP286 instead of TYR72 in EeAChE. The car-
bonyl oxygen of carbamate moiety formed a hydrogen bond with
TYR124 from the PAS through HOH954. The PAS interaction of the
compound 3c with TRP286 was replaced with TYR124 via HOH737
mediated hydrogen bond in EeAChE. In the case of compound 6c, the
Although enzyme inhibition studies were performed by eqBuChE,
molecular docking studies were carried out using hBuChE since there is
no available x-ray structure of eqBuChE. According to molecular
docking results performed with hBuChE, orientation of the compounds
was mainly driven by the interactions with the CAS comprising re-
sidues. Unlike the obtained binding modes in AChE, none of the com-
pounds interacted with the PAS residues except compound 3c.
As depicted in Fig. 5, the binding mode of compound 14b suggested
that the pyridazinone moiety was bound to choline binding site by a π-π
5

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 4. Proposed binding modes for compounds 14b (A), 3c (B), 6c (C), and 7c (D) in hAChE active site (PDB: 4EY7). The compounds are presented as green ball and
sticks with nitrogen and oxygen atoms in blue and red, respectively. The involved residues and structural water molecules are named and showed as white sticks.
Hydrogen bonds and π-π stacking contacts are represented with yellow and cyan dashed lines, respectively.
stacking contact with TRP82. 3-Methoxyphenyl ring was oriented to-
ward THR120 which was located between choline binding site and the
PAS forming a hydrogen bond, whereas another hydrogen bond was
observed between nitrogen atom of carbamate group and ALA328.
Compound 3c was found to be the one among the all compounds tested
that bound to the PAS by interacting ASP70 via water network. In ad-
dition to this, water mediated hydrogen bonds with SER79, the catalytic
triad member SER198, GLY117 from oxyanion hole, and THR120 and a
π-π stacking contact with PHE329 were found to be important for the
stabilization of inhibitor:enzyme complex. In the case of compound 6c,
2-methoxyphenyl ring was located in the pocket forming acyl binding
site and oxyanion hole π-π stacking contacts with TRP231 and PHE329,
respectively. A hydrogen bond between carbonyl oxygen of carbamate
group and THR120 was also observed. The compound 7c was found to
be interacted with only oxyanion hole by forming π-π stacking contacts
with TRP82 and PHE329.
measured for the compounds, but none of them showed proximity to
SER203 (Tables 2 and 3).
The compounds 14c and 16c exhibited inhibitory potency for only
BuChE occupied the region between anionic site and acyl pocket
(Fig. 6). Both compounds bound to oxyanion hole via π-π stacking
contact between 2-methoxyphenyl ring and PHE329 and water medi-
ated hydrogen bond with GLY117, while forming a hydrogen bond
between oxygen atom of pyridazinone moiety and THR120. Both
compounds interacted the catalytic triad member SER198 through
water mediated hydrogen bond. However, the orientation of compound
14c allowed to form hydrogen bonds with SER79 and ASP70 from the
PAS by water network which was not observed for compound 16c and
the π-π stacking contact with TRP231 was observed for only compound
16c.
Molecular docking results obtained showed that there is a good
correlation between predicted binding modes and BuChE inhibition of
the compounds tested regarding glide scores and the residues interacted
and the carbamate group of the compounds was not sufficiently close to
react with catalytic triad member SER198 (Table 4).
Molecular docking results considering glide scores and the residues
interacted were found to be in accordance with in vitro data obtained.
The distances between carbamate moiety of the compounds and cata-
lytic triad member SER203 that ranges from 6.22 Å to 6.40 Å were
As depicted in Fig. 7A, the compounds 7c, 14c, and 16c, which
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Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 5. Proposed binding modes for compounds 14b (A), 3c (B), 6c (C), and 7c (D) in hBuChE active site (PDB: 4TPK). The compounds are presented as green ball and
sticks with nitrogen and oxygen atoms in blue and red, respectively. The involved residues and structural water molecules are named and showed as white sticks.
Hydrogen bonds and π-π stacking contacts are represented with yellow and cyan dashed lines, respectively.
carbamate moiety which interacted the PAS residue was found to be
very similar to that of compound 14c.
Table 2
Molecular interaction of EeAChE active site with the compounds docked.
Although similarities between AChE and BuChE active sites re-
garding the shape and the residue arrangement were available, BuChE
has a larger catalytic site than AChE [28,29] (Fig. 7B). Therefore, this
structural difference was suggested to rationalize the observed lacking
in vitro AChE inhibition over BuChE, for instance compounds 14c and
16c. Compound 14c selectively inhibiting BuChE with respect to AChE,
bound to hBuChE in U-shaped conformation while having less con-
tracted pose in AChE active site. If compound 14c binds to AChE in the
same conformation as in BuChE, it would lead to clashes between the
alkyl chain and residues PHE295 and PHE297. Due to steric hindrance,
compound 14c preferred a linear conformation in AChE active site
through only π-π contacts between terminal phenyl ring and TRP286,
the PAS region residue. Due to having alkyl chain instead of aromatic
rings as in compounds 14b, 3c, 6c, 7c, there is lack of π-π interaction
which leads less stable enzyme:inhibitor complex for BuChE inhibitors
compared to that of the compounds exhibited dual inhibition. It is also
observed that there is an agreement with the chain length and inhibi-
tion% values for BuChE. Increased chain length might help compound
to transfer into a flexible conformation in order to occupy the larger
active site of BuChE. Finally, bearing alkyl chain that enables the U-
shaped conformation may enhance the BuChE selectivity over AChE.
Comp.
Glide score (kcal/
Interacting residues
Distance carbamate-
mol)
SER203 (Å)
14b
3c
−6.615
−7.586
TYR72 (π-π stack);
PHE295 (H-bond)
TRP286, TYR337 (π-π
stack);
6.40
6.22
PHE295 (H-bond)
TRP286, TYR337 (π-π
stack);
6c
7c
−7.450
−7.791
6.32
6.39
PHE295 (H-bond)
TRP286, TYR337 (π-π
stack);
PHE295 (H-bond)
exhibited the highest inhibitory effect against hBuChE among the
compounds tested, preferred to occupy the CAS region between oxya-
nion hole and acyl binding site. Kinetic studies revealed that compound
7c was a competitive inhibitor, while compounds 14c and 16c were
mixed-type inhibitors of hBuChE. Molecular docking studies were in
good agreement with kinetic studies since the competitive inhibitor 7c
interacted with CAS and the mixed-type inhibitor 14c exerted a dual
CAS-PAS binding. Although the interaction with the PAS was not ob-
served for mixed-type inhibitor 16c, its orientation of nitrogen atom of
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Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Table 3
Molecular interaction of hAChE active site with the compounds docked.
Comp.
Glide score (kcal/mol)
Interacting residues
Distance carbamate-SER203 (Å)
9.38
14b
−8.815
TRP286 (π-π stack);
PHE295 (H-bond);
TYR124 (HOH954 mediated H-bond)
TYR337 (π-π stack);
3c
6c
−9.576
9.43
PHE295 (H-bond);
TYR124 (HOH737 mediated H-bond)
TRP286, TYR337 (π-π stack);
−10.290
10.97
TYR124 (HOH954, HOH955 mediated H-bond);
TRP286 (HOH953, HOH793 mediated H-bond);
SER293 (HOH953, HOH793, HOH805 mediated H-bond)
TRP86, TRP286, TYR337, TYR341 (π-π stack);
PHE295 (H-bond);
7c
−10.025
8.62
TYR124 (HOH954 mediated H-bond)
Fig. 6. Proposed binding modes for compounds 14c (A), 16c (B) in hBuChE active site (PDB: 4TPK). The compounds are presented as green ball and sticks with
nitrogen and oxygen atoms in blue and red, respectively. The involved residues and structural water molecules are named and showed as white sticks. Hydrogen
bonds and π-π stacking contacts are represented with yellow and cyan dashed lines, respectively.
3. Conclusion
Table 4
Molecular interaction of hBuChE active site with the compounds docked.
In this paper, three series of (6-oxo-3-substitutedpyridazin-1(6H)-yl)
methyl carbamate derivatives were synthesized and evaluated for their
ability to inhibit EeAChE and eqBuChE. All compounds except 6a, 4b,
and 6b, displayed inhibitory activity against eqBuChE. It is observed
that the compounds with short alkyl chain were not active in all series,
Comp.
Glide score
Interacting residues
Distance carbamate-
(kcal/mol)
SER198 (Å)
14b
3c
−6.879
−7.704
TRP82 (π-π stack);
9.67
8.86
THR120, ALA328 (H-bond)
PHE329 (π-π stack);
while the long-chain carbamate derivatives showed
a significant
THR120 (H-bond);
GLY117, SER198 (HOH781,
HOH767 mediated H-bond);
ASP70, SER79 (HOH736
mediated H-bond)
eqBuChE inhibitory activity. Only in the third serie, aromatic carba-
mate derivatives 3c, 6c, 7c exhibited inhibitory activity against both
EeAChE and eqBuChE. Besides, it is determined that the presence of the
6-(2-methoxyphenyl)pyridazin-3(2H)-one moiety increased the in-
hibitory activity. In total, eight compounds showed 50% or more in-
hibitory activity for eqBuChE and IC50 values were calculated. Among
these compounds, 15a, 13c, 14c, and 16c were inhibited solely
eqBuChE at tested concentration. The heptylcarbamate derivative 16c
(IC50 = 12.8 µM) was found to be the most active compound. For the
determination of the inhibitory mechanism, 7c, 14c, and 16c were
selected for enzyme kinetic study. Obtained results indicated that
compound 7c was a competitive inhibitor, while compounds 14c and
16c were mixed-type inhibitors of eqBuChE. Additionally, Ki values of
the selected compounds were consistent with the IC50 values. Molecular
docking results also supported the findings from kinetic study in which
the competitive inhibitor 7c interacted with CAS and the mixed-type
inhibitor 14c exerted a dual CAS-PAS binding. Although the interaction
with the PAS was not observed for mixed-type inhibitor 16c, its
6c
−7.815
TRP231, PHE329 (π-π stack);
THR120 (H-bond)
8.61
7c
−7.921
−7.850
TRP82, PHE329 (π-π stack)
PHE329 (π-π stack);
6.52
8.87
14c
THR120 (H-bond);
ASP70, SER79 (HOH736
mediated H-bond);
GLY117, SER198 (HOH781,
HOH767 mediated H-bond)
TRP231, PHE329 (π-π stack);
THR120 (H-bond);
16c
−8.664
7.98
GLU197 (HOH781 mediated
H-bond);
GLY117, SER198 (HOH781,
HOH767 mediated H-bond)
8

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
Fig. 7. (A) The superimposition of docked compounds 7c (yellow), 14c (grey), and 16c (grey) inside hBuChE active site (PDB: 4TPK). All compounds are presented as
ball and stick with nitrogen and oxygen atoms in blue and red, respectively. Residues of acyl binding pocket, oxyanion hole, PAS and catalytic triad are shown in
blue, orange, cyan and magenta sticks, respectively. (B) Superimposed binding modes of compound 14c inside hAChE (green) and hBuChE (yellow) active sites.
External surfaces of hAChE and hBuChE gorge cavities are presented as pink solid and blue mesh, respectively. The key residues of hAChE are colored pink, while the
corresponding residues are colored blue.
orientation of nitrogen atom of carbamate moiety which interacted the
PAS residue was found to be very similar to that of compound 14c.
According to the IC50 shift assay, compounds 7c, 14c, and 16c ex-
hibited a time-dependent inhibition against eqBuChE. Although the
time-dependent inhibition of compounds showed similarity with that of
rivastigmine, molecular docking studies indicated that compounds do
not inhibit eqBuChE by a mechanism involving covalent bond forma-
tion at the active site serine. A reversible with tight-binding inhibition
of eqBuChE which is known to show kinetics similar to covalent irre-
versible inhibitors was proposed as a binding mechanism for the com-
pounds tested because of the π-π stacking interactions with especially
PHE329. In conclusion, these compounds could be considered as pro-
mising lead candidates for the design and development of drugs against
AD.
under reflux for 2 h. At the end of the period, the reaction medium was
cooled, the precipitate was filtered and crystallized from the appro-
priate solvent.
4.1.2. General procedure for the synthesis of 2-(hydroxymethyl)-6-
substitutedpyridazin-3(2H)-one derivatives (2a, 2b, 2c)
A mixture of 5.05 g (0.025 mol) of 6-substitutedpyridazin-3(2H)-one
(1a, 1b, 1c) and 30 mL of 37% formalin was heated under reflux for 2 h.
At the end of the period, the reaction medium was cooled to room
temperature, 30 mL water was added, and the precipitated product was
washed with water and filtered.
4.1.3. General procedure for the synthesis of carbamate derivatives (3a, 3b,
3c-7a, 7b, 7c and 9a, 9b, 9c-16a, 16b, 16c)
Appropriate isocyanate derivative (0.001 mol) and N-methylimida-
zole (0.016 mL, 0.016 g, 0.0002 mol) were added to mixture of 2-(hy-
droxymethyl)-6-substitutedpyridazin-3(2H)-one derivative (2a, 2b, 2c)
(0.23 g, 0.001 mol) in 10 mL acetonitrile. The mixture was stirred at
room temperature for 24 h. Then 50 mL ice-water was added, pre-
cipitated product was filtered, and crystallized from the appropriate
solvent.
4. Experimental part
4.1. Chemistry
All reagents and solvents were purchased from Sigma-Aldrich and
used without further purification. Melting points were determined with
an Electrothermal-9200 Digital Melting Point Apparatus and were un-
corrected. 1H NMR and 13C NMR spectra were recorded in DMSO-d6 on
a Varian Mercury 400, 400 MHz High Performance Digital FT-NMR
spectrometer at the NMR facility of Faculty of Pharmacy, Ankara
University. All chemical shifts were recorded as δ (ppm). Microanalyses
for C, H, and N were performed on a Leco-932 at Faculty of Pharmacy,
Ankara University, and they were within the range of 0.4% of the
theoretical value. HRMS spectra were taken on a Waters LCT Premier
XE orthogonal acceleration time-of-flight (oa-TOF) mass spectrometer
using ESI (+) or ESI (−) methods. The synthesis of the compounds 1a
[30], 2a [31], 3a [16], 1b [30], 1c [30] were previously reported.
4.1.4. General procedure for the synthesis of N-methylcarbamate
derivatives (8a, 8b, 8c)
The mixture of 2-(hydroxymethyl)-6-substitutedpyridazin-3(2H)-
one derivative (2a, 2b, 2c) (0.269 g, 0.00116 mol) and 1,1-carbo-
nyldiimidazole (0.379 g, 0.00234 mol) in 10 mL acetonitrile was stirred
at room temperature for 3 h. Then potassium carbonate (0.160 g,
0.00116 mol) and methylamine (0.066 mL, 30% aqueous solution,
0.059 g, 0.00116 mol) was added to reaction mixture and stirred at
room temperature for 3 h. At the end of the time, reaction mixture was
poured into 50 mL of ice-water; the precipitate formed was filtered and
crystallized from the appropriate solvent.
4.1.1. General procedure for the synthesis of 6-substitutedpyridazin-3(2H)-
one derivatives (1a, 1b, 1c)
4.1.5. General procedure for the synthesis of N,N-dimethylcarbamate
derivatives (17a, 17b, 17c)
The mixture of glyoxylic acid monohydrate (5.52 g, 0.06 mol) and
appropriate acetophenone derivative (27.04 g, 0.18 mol) was heated at
105–108 °C for 3 h. At the end of the time, the reaction medium was
cooled to room temperature and to added 24 mL of water and 4.8 mL of
conc. ammonium hydroxide. Then, the mixture was extracted with di-
chloromethane (2 × 50 mL), and 2.9 mL (0.06 mol) hydrazine hydrate
was added onto the separated aqueous phase. The mixture was heated
To mixture of 2-(hydroxymethyl)-6-substitutedpyridazin-3(2H)-one
derivative (2a, 2b, 2c) (0.23 g, 0.001 mol), N-methylimidazole
(0.016 mL, 0.016 g, 0.0002 mol) and potassium carbonate (0.138 g,
0.001 mol) in 10 mL acetonitrile was added dimethylcarbamoyl
chloride (0.09 mL, 0.107 g, 0.001 mol) and stirred at room temperature
for 24 h. At the end of the time, reaction mixture poured into 50 mL ice/
9

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
water, obtained product was filtered and purified by preparative TLC in
dichloromethane:methanol (100:3) solvent system.
methylphenyl)carbamate, 7a. Crystallized from ethanol/water to yield
44%; m.p. 213 °C; 1H NMR (DMSO-d6) δ: 9.82 (1H, s, NH), 8.09 (1H, d,
J: 9.6 Hz, pyridazine H5), 7.86 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.35
(2H, d, J: 8 Hz, phenyl H2′,6′), 7.12–7.04 (5H, m, pyridazine H4,
phenyl H3,5, phenyl H3′,5′), 6.08 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.23
(3H, s, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.53, 158.84, 152.00,
144.11, 136.05, 131.83, 131.65, 130.40, 129.18, 127.43, 126.22,
118.32, 114.35, 72.91, 55.30, 20.30 ppm; Anal. calc. for C20H19N3O4:
C, 65.74; H, 5.24; N, 11.50; found: C, 66.10; H, 5.426; N, 11.52%;
HRMS calc. for C20H20N3O4 [M + H]: 366.1454, found: 366.1457.
4.1.5.1. 6-(4-Methoxyphenyl)pyridazin-3(2H)-one, 1a. Crystallized from
water to yield 58.2%; m.p. 194 °C; HRMS calc. for
[M + H]: 203.0821, found: 203.0828 [30].
C11H11N2O2
4.1.5.2. 2-(Hydroxymethyl)-6-(4-methoxyphenyl)pyridazin-3(2H)-one,
2a. The crude product was used without further purification due to the
stability problem. Yield 86.2%; m.p. 145 °C; HRMS calc. for
C
12H13N2O3 [M + H]: 233.0926, found: 233.0932 [31].
4.1.5.8. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
methylcarbamate, 8a. Crystallized from acetone/water to yield 54.8%;
m.p. 155 °C; 1H NMR (DMSO-d6) δ: 8.04 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.81 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.27 (1H, q, NH), 7.06–7.01
(3H, m, pyridazine H4, phenyl H3,5), 5.92 (2H, s, CH2), 3.79 (3H, s,
OCH3), 2.57 (3H, d, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.50, 158.76,
155.22, 143.91, 131.68, 130.34, 127.39, 126.28, 114.33, 72.76, 55.30,
26.89 ppm; Anal. calc. for C14H15N3O4: C, 58.13; H, 5.23; N, 14.53;
found: C, 58.29; H, 5.382; N, 14.49%; HRMS calc. for C14H16N3O4
[M + H]: 290.1141, found: 290.1131.
4.1.5.3. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
phenylcarbamate, 3a [16]. Crystallized from acetonitrile to yield 82.6%;
m.p. 198 °C; 1H NMR (DMSO-d6) δ: 9.91 (1H, s, NH), 8.05 (1H, d, J:
9.6 Hz, pyridazine H5), 7.83 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.45 (2H,
d, J: 8 Hz, phenyl H2′,6′), 7.26 (2H, t, phenyl H3′,5′), 7.09 (1H, d, J:
10 Hz, pyridazine H4), 7.02 (2H, d, J: 8.8 Hz, phenyl H3,5), 6.98 (1H, t,
phenyl H4′), 6.07 (2H, s, CH2), 3.78 (3H, s, OCH3) ppm; 13C NMR
(DMSO‑d6) δ: 160.48, 158.78, 151.96, 144.07, 138.56, 131.76, 130.33,
128.73, 127.37, 126.15, 122.70, 118.24, 114.28, 72.92, 55.23 ppm;
Anal. calc. for C19H17N3O4.3/4H2O: C, 62.54; H, 5.11; N, 11.52, found:
C, 62.32; H, 4.89; N, 11.39%; HRMS calc. for C19H18N3O4 [M + H]:
352.1297, found: 352.1296.
4.1.5.9. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
ethylcarbamate, 9a. Crystallized from ethanol/water to yield 73.85%;
m.p. 80 °C; 1H NMR (DMSO-d6) δ: 8.04 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.81 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.39 (1H, t, NH), 7.06–7.01
(3H, m, pyridazine H4, phenyl H3,5), 5.91 (2H, s, CH2), 3.79 (3H, s,
OCH3), 3.01–2.98 (2H, m, CH2), 0.98 (3H, t, CH3) ppm; 13C NMR
(DMSO-d6) δ: 160.50, 158.76, 154.51, 143.91, 131.68, 130.35, 127.40,
126.28, 114.33, 72.62, 55.30, 35.06, 14.84 ppm; Anal. calc. for
4.1.5.4. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
fluorophenyl)carbamate, 4a. Crystallized from acetonitrile to yield
70.4%; m.p. 212 °C; 1H NMR (DMSO-d6) δ: 9.95 (1H, s, NH), 8.06
(1H, d, J: 9.6 Hz, pyridazine H5), 7.82 (2H, d, J: 9.2 Hz, phenyl H2,6),
7.44 (2H, dd, J1: 8.8 Hz, J2: 5.2 Hz, phenyl H2′,6′), 7.13–7.07 (3H, m,
pyridazine H4, phenyl H3′,5′), 7.02 (2H, d, J: 8.8 Hz, phenyl H3,5),
6.06 (2H, s, CH2), 3.78 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ:
160.54, 158.83, 157.81 (d, J: 238 Hz, F-phenyl H4′), 152.11, 144.14,
134.96, 131.86, 130.40, 127.42, 126.20, 119.98, 115.39 (d, J: 22.5 Hz,
F-phenyl H3′,5′), 114.35, 73.02, 55.29 ppm; Anal. calc. for
C
15H17N3O4.1/2H2O: C, 57.68; H, 5.81; N, 13.45; found: C, 57.36; H,
6.123; N, 13.42%; HRMS calc. for C15H18N3O4 [M + H]: 304.1297,
found: 304.1288.
4.1.5.10. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
C
19H16FN3O4: C, 61.79; H, 4.37; N, 11.38, found: C, 61.81; H, 4.523;
propylcarbamate, 10a. Crystallized from acetone/water to yield 84.6%;
m.p. 101 °C; 1H NMR (DMSO-d6) δ: 8.04 (1H, d, J: 10 Hz, pyridazine
H5), 7.81 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.41 (1H, t, NH), 7.06–7.01
(3H, m, pyridazine H4, phenyl H3,5), 5.91 (2H, s, CH2), 3.79 (3H, s,
OCH3), 2.92 (2H, q, CH2), 1.40–1.35 (2H, m, CH2), 0.79 (3H, t, CH3)
ppm; 13C NMR (DMSO-d6) δ: 160.49, 158.76, 154.72, 143.91, 131.67,
130.34, 127.39, 126.27, 114.32, 72.62, 55.29, 42.02, 22.47,
11.15 ppm; Anal. calc. for C16H19N3O4.1/2H2O: C, 58.89; H, 6.18; N,
12.88; found: C, 59.04; H, 6.384; N, 12.99%; HRMS calc. for
N, 11.46%; HRMS calc. for C19H17FN3O4 [M + H]: 370.1203, found:
370.1204.
4.1.5.5. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
chlorophenyl)carbamate, 5a. Crystallized from acetonitrile to yield
77.6%; m.p. 217 °C; 1H NMR (DMSO-d6) δ: 10.06 (1H, s, NH), 8.07
(1H, d, J: 10 Hz, pyridazine H5), 7.82 (2H, d, J: 8.8 Hz, phenyl H2,6),
7.46 (2H, d, J: 8.8 Hz, phenyl H2′,6′), 7.32 (2H, d, J: 9.2 Hz, phenyl
H3′,5′), 7.09 (1H, d, J: 9.6 Hz, pyridazine H4), 7.02 (2H, d, J: 8.8 Hz,
phenyl H3,5), 6.06 (2H, s, CH2), 3.78 (3H, s, OCH3) ppm; 13C NMR
(DMSO-d6) δ: 160.55, 158.84, 151.96, 144.16, 137.64, 131.88, 130.40,
128.72, 127.43, 126.43, 126.18, 119.76, 114.35, 73.10, 55.30 ppm;
Anal. calc. for C19H16ClN3O4: C, 59.15; H, 4.18; N, 10.89, found: C,
58.86; H, 4.290; N, 10.97%; HRMS calc. for C19H17ClN3O4 [M + H]:
386.0908, found: 386.0909.
C16H20N3O4 [M + H]: 318.1454, found: 318.1451.
4.1.5.11. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl propan-
2-ylcarbamate, 11a. The crude product was purified by preparative TLC
using dichloromethane-methanol (100:3) solvent system. Yield 49.9%;
m.p. 77 °C; 1H NMR (DMSO-d6) δ: 8.05 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.82 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.35 (1H, d, NH), 7.07–7.02
(3H, m, pyridazine H4, phenyl H3,5), 5.91 (2H, s, CH2), 3.79 (3H, s,
OCH3), 3.62–3.57 (1H, m, CH), 1.01 (6H, d, CH3) ppm; 13C NMR
(DMSO-d6) δ: 160.49, 158.76, 153.81, 143.90, 131.65, 130.33, 127.38,
126.27, 114.32, 72.52, 55.30, 42.39, 22.38 ppm; Anal. calc. for
4.1.5.6. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
methoxyphenyl)carbamate, 6a. Crystallized from acetonitrile to yield
74%; m.p. 210 °C; 1H NMR (DMSO-d6) δ: 9.71 (1H, s, NH), 8.06 (1H, d,
J: 10 Hz, pyridazine H5), 7.82 (2H, d, J: 8.8 Hz, phenyl H2,6), 7.34 (2H,
d, J: 8 Hz, phenyl H2′,6′), 7.07 (1H, d, J: 10.4 Hz, pyridazine H4), 7.02
(2H, d, J: 8.4 Hz, phenyl H3,5), 6.84 (2H, d, J: 8.8 Hz, phenyl H3′,5′),
6.04 (2H, s, CH2), 3.78 (3H, s, OCH3), 3.67 (3H, s, OCH3) ppm; 13C
NMR (DMSO-d6) δ: 160.53, 158.83, 154.99, 152.13, 144.09, 131.81,
131.60, 130.39, 127.42, 126.22, 119.86, 114.35, 114.00, 72.90, 55.29,
55.14 ppm; Anal. calc. for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02,
found: C, 62.86; H, 5.154; N, 10.96%; HRMS calc. for C20H20N3O5
[M + H]: 382.1403, found: 382.1404.
C
16H19N3O4.3/4H2O: C, 58.08; H, 6.25; N, 12.70, found: C, 58.14; H,
6.076; N, 12.32%; HRMS calc. for C16H20N3O4 [M + H]: 318.1454,
found: 318.1443.
4.1.5.12. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
butylcarbamate, 12a. Crystallized from ethanol/water to yield 60.6%;
m.p. 72 °C; 1H NMR (DMSO-d6) δ: 8.05 (1H, d, J: 10 Hz, pyridazine H5),
7.82 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.40 (1H, t, NH), 7.07–7.01 (3H,
m, pyridazine H4, phenyl H3,5), 5.92 (2H, s, CH2), 3.79 (3H, s, OCH3),
2.97 (2H, q, CH2), 1.37–1.33 (2H, m, CH2), 1.26–1.22 (2H, m, CH2),
0.82 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.49, 158.76, 154.70,
4.1.5.7. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
10

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
143.91, 131.65, 130.33, 127.39, 126.28, 114.31, 72.57, 55.29, 38.87,
31.33, 19.33, 13.59 ppm; Anal. calc. for C17H21N3O4.1/3H2O: C, 60.52;
H, 6.47; N, 12.46; found: C, 60.59; H, 6.822; N, 12.51%; HRMS calc. for
5.530; N, 13.15%; HRMS calc. for C15H18N3O4 [M + H]: 304.1297,
found: 304.1296.
C
17H22N3O4 [M + H]: 332.1610, found: 332.1609.
4.1.5.18. 6-(3-Methoxyphenyl)pyridazin-3(2H)-one,
1b. Crystallized
from water to yield 60%; m.p. 185 °C; HRMS calc. for C11H11N2O2
[M + H]: 203.0821, found: 203.0830 [30].
4.1.5.13. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
pentylcarbamate, 13a. Crystallized from ethanol/water to yield 57.2%;
m.p. 67 °C; 1H NMR (DMSO-d6) δ: 8.07 (1H, d, J: 10 Hz, pyridazine H5),
7.84 (2H, d, J: 8.8 Hz, phenyl H2,6), 7.42 (1H, t, NH), 7.09–7.03 (3H,
m, pyridazine H4, phenyl H3,5), 5.94 (2H, s, CH2), 3.81 (3H, s, OCH3),
2.98 (2H, q, CH2), 1.41–1.37 (2H, m, CH2), 1.26–1.21 (4H, m, CH2),
0.83 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.49, 158.76, 154.69,
143.90, 131.64, 130.33, 127.38, 126.28, 114.30, 72.57, 55.29, 40.19,
28.88, 28.35, 21.75, 13.84 ppm; Anal. calc. for C18H23N3O4.1/3H2O: C,
61.52; H, 6.79; N, 11.96; found: C, 61.59; H, 6.909; N, 11.91%; HRMS
calc. for C18H24N3O4 [M + H]: 346.1767, found: 346.1774.
4.1.5.19. 2-(Hydroxymethyl)-6-(3-methoxyphenyl)pyridazin-3(2H)-one,
2b. The crude product was used without further purification due to the
stability problem. Yield 78.7%; m.p. 133 °C; HRMS calc. for
C12H13N2O3 [M + H]: 233.0926, found: 233.0935.
4.1.5.20. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]
methylphenylcarbamate, 3b. Crystallized from acetonitrile to yield
68.6%; m.p. 198–201 °C; 1H NMR (DMSO-d6) δ: 9.92 (1H, s, NH),
8.10 (1H, d, J: 10 Hz, pyridazine H5), 7.46–7.36 (5H, m, phenyl H2,5,6,
phenyl H2′,6′), 7.26 (2H, t, phenyl H3′,5′), 7.12 (1H, d, J: 10 Hz,
pyridazine H4), 7.03–6.97 (2H, m, phenyl H4, phenyl H4′), 6.09 (2H, s,
CH2), 3.77 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 159.69, 158.93,
151.98, 144.09, 138.58, 135.19, 132.10, 130.40, 130.11, 128.79,
122.78, 118.27, 115.40, 111.29, 72.91, 55.20 ppm; Anal. calc. for
4.1.5.14. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
hexylcarbamate, 14a. Crystallized from ethanol/water to yield 75.12%;
m.p. 71 °C; 1H NMR (DMSO-d6) δ: 8.05 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.82 (2H, d, J: 8.8 Hz, phenyl H2,6), 7.40 (1H, t, NH), 7.07–7.02
(3H, m, pyridazine H4, phenyl H3,5), 5.92 (2H, s, CH2), 3.79 (3H, s,
OCH3), 2.96 (2H, q, CH2), 1.36–1.34 (2H, m, CH2), 1.22–1.20 (6H, m,
CH2), 0.81 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.49, 158.77,
154.69, 143.89, 131.62, 130.32, 127.37, 126.28, 114.29, 72.54, 55.28,
40.23, 30.91, 29.17, 25.82, 21.99, 13.84 ppm; Anal. calc. for
C19H25N3O4.1/4H2O: C, 62.71; H, 7.06; N, 11.55; found: C, 62.88; H,
7.347; N, 11.61%; HRMS calc. for C19H26N3O4 [M + H]: 360.1923,
found: 360.1921.
C
19H17N3O4: C, 64.95; H, 4.88; N, 11.96, found: C, 64.74; H, 5.079; N,
12.06%; HRMS calc. for
352.1295.
C19H18N3O4 [M + H]: 352.1297, found:
4.1.5.21. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
fluorophenyl)carbamate, 4b. Crystallized from acetonitrile to yield
80.2%; m.p. 192–193 °C; 1H NMR (DMSO-d6) δ: 9.97 (1H, s, NH),
8.11 (1H, d, J: 10 Hz, pyridazine H5), 7.46–7.37 (5H, m, phenyl H2,5,6,
phenyl H2′,6′), 7.13–7.09 (3H, m, phenyl H3′,5′, pyridazine H4), 7.03
(1H, dd, J1: 7.8 Hz, J2: 2.4 Hz, phenyl H4), 6.08 (2H, s, CH2), 3.77 (3H,
s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 159.70, 158.94, 157.83 (d, J:
239.38 Hz, F-phenyl H4′), 152.10, 144.11, 135.19, 134.95, 132.12,
130.41, 130.11, 119.99, 118.28, 115.40 (d, J: 22.64 Hz, F-phenyl H3′,
5′), 115.38, 111.32, 72.98, 55.21 ppm; Anal. calc. for C19H16FN3O4: C,
61.79; H, 4.37; N, 11.38, found: C, 61.43; H, 4.494; N, 11.40%; HRMS
calc. for C19H17FN3O4 [M + H]: 370.1203, found: 370.1194.
4.1.5.15. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
cyclohexylcarbamate, 15a. Crystallized from ethanol/water to yield
50.9%; m.p. 144 °C; 1H NMR (DMSO-d6) δ: 8.04 (1H, d, J: 9.6 Hz,
pyridazine H5), 7.81 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.36 (1H, d, NH),
7.06–7.01 (3H, m, pyridazine H4, phenyl H3,5), 5.90 (2H, s, CH2), 3.79
(3H, s, OCH3), 3.26–3.24 (1H, m, cyclohexyl H1), 1.73–1.48 (5H, m,
cyclohexyl equatorial), 1.22–1.05 (5H, m, cyclohexyl axial) ppm; 13C
NMR (DMSO-d6) δ: 160.49, 158.77, 153.84, 143.90, 131.65, 130.32,
127.38, 126.27, 114.31, 72.57, 55.29, 49.48, 32.48, 25.07, 24.49 ppm;
Anal. calc. for C19H23N3O4: C, 63.85; H, 6.49; N, 11.76; found: C, 63.85;
H, 6.803; N, 11.67%; HRMS calc. for C19H24N3O4 [M + H]: 358.1767,
found: 358.1763.
4.1.5.22. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
chlorophenyl)carbamate, 5b. Crystallized from acetonitrile to yield
76.6%; m.p. 194–195 °C; 1H NMR (DMSO-d6) δ: 10.08 (1H, s, NH),
8.11 (1H, d, J: 10 Hz, pyridazine H5), 7.47–7.37 (5H, m, phenyl H2,5,6,
phenyl H2′,6′), 7.32 (2H, d, J: 9.2 Hz, phenyl H3′,5′), 7.11 (1H, d, J:
10 Hz, pyridazine H4), 7.03 (1H, dd, J1: 8.2 Hz, J2: 2.4 Hz, phenyl H4),
6.09 (2H, s, CH2), 3.77 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ:
159.69, 158.93, 151.94, 144.12, 137.62, 135.17, 132.13, 130.41,
130.11, 128.72, 126.45, 119.75, 118.27, 115.38, 111.32, 73.06,
55.21 ppm; Anal. calc. for C19H16ClN3O4: C, 59.15; H, 4.18; N, 10.89,
found: C, 58.88; H, 4.286; N, 10.95%; HRMS calc. for C19H17ClN3O4
[M + H]: 386.0908, found: 386.0891.
4.1.5.16. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
heptylcarbamate, 16a. Crystallized from ethanol/water to yield 57.6%;
m.p. 76 °C; 1H NMR (DMSO-d6) δ: 8.07 (1H, d, J: 10 Hz, pyridazine H5),
7.84 (2H, d, J: 8.8 Hz, phenyl H2,6), 7.41 (1H, t, NH), 7.08–7.03 (3H,
m, pyridazine H4, phenyl H3,5), 5.94 (2H, s, CH2), 3.81 (3H, s, OCH3),
2.98 (2H, q, CH2), 1.39–1.36 (2H, m, CH2), 1.23–1.21 (8H, m, CH2),
0.83 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 160.49, 158.76, 154.69,
143.89, 131.63, 130.33, 127.38, 126.29, 114.30, 72.52, 55.29, 40.22,
31.17, 29.20, 28.34, 26.11, 21.99, 3.89 ppm; Anal. calc. for
4.1.5.23. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
C
20H27N3O4: C, 64.32; H, 7.29; N, 11.25; found: C, 64.00; H, 7.673;
methoxyphenyl)carbamate, 6b. Crystallized from acetonitrile to yield
56.1%; m.p. 183 °C; 1H NMR (DMSO-d6) δ: 9.73 (1H, s, NH), 8.10 (1H,
d, J: 10 Hz, pyridazine H5), 7.46–7.33 (5H, m, phenyl H2,5,6, phenyl
H2′,6′), 7.11 (1H, d, J: 10 Hz, pyridazine H4), 7.04–7.01 (1H, m, phenyl
H4), 6.84 (2H, d, J: 8.8 Hz, phenyl H3′,5′), 6.06 (2H, s, CH2), 3.77 (3H,
s, OCH3), 3.68 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 159.69,
158.93, 155.01, 152.11, 144.05, 135.21, 132.08, 131.58, 130.40,
130.11, 119.86, 118.27, 115.38, 113.99, 111.30, 72.84, 55.21,
55.14 ppm; Anal. calc. for C20H19N3O5: C, 62.99; H, 5.02; N, 11.02,
found: C, 62.96; H, 5.232; N, 10.99%; HRMS calc. for C20H20N3O5
[M + H]: 382.1403, found: 382.1400.
N, 10.97%; HRMS calc. for C20H28N3O4 [M + H]: 374.2080, found:
374.2086.
4.1.5.17. [3-(4-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
dimethylcarbamate, 17a. The crude product was purified by preparative
TLC using dichloromethane-methanol (100:3) solvent system. Yield
13%; m.p. 120 °C; 1H NMR (DMSO-d6) δ: 8.05 (1H, d, J: 9.6 Hz,
pyridazine H5), 7.82 (2H, d, J: 9.2 Hz, phenyl H2,6), 7.07–7.02 (3H,
m, pyridazine H4, phenyl H3,5), 5.94 (2H, s, CH2), 3.79 (3H, s, OCH3),
2.82 (3H, s, CH3), 2.78 (3H, s, CH3) ppm; 13C NMR (DMSO-d6) δ:
160.49, 158.72, 154.24, 143.96, 131.73, 130.35, 127.42, 126.31,
114.33, 73.72, 55.29, 36.03, 35.53 ppm; Anal. calc. for
4.1.5.24. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
C
15H17N3O4.1/2H2O: C, 57.68; H, 5.81; N, 13.45; found: C, 57.59; H,
methylphenyl)carbamate, 7b. Crystallized from acetonitrile to yield
11

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
67.2%; m.p. 191 °C; 1H NMR (DMSO-d6) δ: 9.83 (1H, s, NH), 8.14 (1H,
d, J: 10 Hz, pyridazine H5), 7.46–7.39 (3H, m, phenyl H2,5,6), 7.34
(2H, d, J: 8 Hz, phenyl H2′,6′), 7.14 (1H, d, J: 9.6 Hz, pyridazine H4),
7.09 (2H, d, J: 8 Hz, phenyl H3′,5′), 7.06–7.04 (1H, dd, J1: 7.8 Hz, J2:
2.8 Hz, phenyl H4), 6.09 (2H, s, CH2), 3.79 (3H, s, OCH3), 2.23 (3H, s,
CH3) ppm; 13C NMR (DMSO-d6) δ: 159.68, 158.92, 151.98, 144.06,
136.02, 135.20, 132.07, 131.65, 130.39, 130.10, 129.16, 118.26,
115.38, 111.29, 72.86, 55.20, 20.30 ppm; Anal. calc. for C20H19N3O4:
C, 65.74; H, 5.24; N, 11.50, found: C, 65.60; H, 5.406; N, 11.57%;
HRMS calc. for C20H20N3O4 [M + H]: 366.1454, found: 366.1452.
158.85, 154.66, 143.86, 135.25, 131.91, 130.33, 130.07, 118.22,
115.23, 111.36, 72.56, 55.21, 39.90, 31.31, 19.32, 13.56 ppm; Anal.
calc. for C17H21N3O4: C, 61.62; H, 6.39; N, 12.68, found: C, 61.55; H,
6.626; N, 12.68%; HRMS calc. for C17H22N3O4 [M + H]: 332.1610,
found: 332.1601.
4.1.5.30. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
pentylcarbamate, 13b. Crystallized from ethanol/water to yield 81.9%;
m.p. 99 °C; 1H NMR (DMSO-d6) δ: 8.11 (1H, d, J: 10.4 Hz, pyridazine
H5), 7.47–7.39 (4H, m, phenyl H2,5,6, NH), 7.10 (1H, d, J: 9.6 Hz,
pyridazine H4), 7.07–7.04 (1H, m, phenyl H4), 5.96 (2H, s, CH2), 3.82
(3H, s, OCH3), 2.98 (2H, q, CH2), 1.40–1.35 (2H, m, CH2), 1.27–1.19
(4H, m, CH2), 0.83 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 159.68,
158.86, 154.65, 143.86, 135.25, 131.91, 130.33, 130.06, 118.22,
115.22, 111.37, 72.55, 55.22, 40.20, 28.86, 28.34, 21.73, 13.82 ppm;
Anal. calc. for C18H23N3O4: C, 62.59; H, 6.71; N, 12.17, found: C, 62.31;
H, 6.960; N, 12.08%; HRMS calc. for C18H24N3O4 [M + H]: 346.1767,
found: 346.1767.
4.1.5.25. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
methylcarbamate, 8b. Crystallized from acetone/water to yield 38.8%;
m.p. 114 °C; 1H NMR (DMSO-d6) δ: 8.09 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.46–7.38 (3H, m, phenyl H2,5,6), 7.29 (1H, q, NH), 7.09 (1H, d,
J: 10 Hz, pyridazine H4), 7.05–7.02 (1H, m, phenyl H4), 5.94 (2H, s,
CH2), 3.80 (3H, s, OCH3), 2.57 (3H, d, N-CH3) ppm; 13C NMR (DMSO-
d6) δ: 159.69, 158.86, 155.19, 143.88, 135.27, 131.95, 130.35, 130.10,
118.25, 115.27, 111.36, 72.76, 55.24, 26.89 ppm; Anal. calc. for
C
14H15N3O4: C, 58.13; H, 5.23; N, 14.53, found: C, 58.18; H, 5.370;
4.1.5.31. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
hexylcarbamate, 14b. Crystallized from ethanol/water to yield 73.7%;
m.p. 85 °C; 1H NMR (DMSO-d6) δ: 8.08 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.45–7.37 (4H, m, phenyl H2,5,6, NH), 7.08 (1H, d, J: 10 Hz,
pyridazine H4), 7.04–7.01 (1H, m, phenyl H4), 5.93 (2H, s, CH2), 3.79
(3H, s, OCH3), 2.95 (2H, q, CH2), 1.37–1.32 (2H, m, CH2), 1.23–1.19
(6H, m, CH2), 0.80 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 159.68,
158.85, 154.65, 143.85, 135.25, 131.90, 130.33, 130.06, 118.22,
115.22, 111.37, 72.54, 55.22, 40.23, 30.88, 29.15, 25.81, 21.97,
13.83 ppm; Anal. calc. for C19H25N3O4: C, 63.49; H, 7.01; N, 11.69,
found: C, 63.69; H, 7.076; N, 11.62%; HRMS calc. for C19H26N3O4
[M + H]: 360.1923, found: 360.1919.
N, 14.50%; HRMS calc. for C14H16N3O4 [M + H]: 290.1141, found:
290.1154.
4.1.5.26. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
ethylcarbamate, 9b. Crystallized from ethanol/water to yield 72.5%;
m.p. 112 °C; 1H NMR (DMSO-d6) δ: 8.11 (1H, d, J: 10.4 Hz, pyridazine
H5), 7.47–7.39 (4H, m, phenyl H2,5,6, NH), 7.10 (1H, d, J: 9.6 Hz,
pyridazine H4), 7.06–7.04 (1H, m, phenyl H4), 5.97 (2H, s, CH2), 3.82
(3H, s, OCH3), 3.07–3.00 (2H, m, CH2), 1.01 (3H, t, CH3) ppm; 13C
NMR (DMSO-d6) δ: 159.69, 158.87, 154.49, 143.89, 135.27, 131.94,
130.35, 130.10, 118.25, 115.27, 111.37, 72.63, 55.24, 35.08,
14.82 ppm; Anal. calc. for C15H17N3O4: C, 59.40; H, 5.65; N, 13.85,
found: C, 59.49; H, 5.446; N, 13.83%; HRMS calc. for C20H20N3O5
[M + H]: 382.1403, found: 382.1400.
4.1.5.32. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
cyclohexylcarbamate, 15b. Crystallized from acetonitrile/water to yield
67.5%; m.p. 148 °C; 1H NMR (DMSO-d6) δ: 8.08 (1H, d, J: 9.6 Hz,
pyridazine H5), 7.45–7.37 (4H, m, phenyl H2,5,6, NH), 7.08 (1H, d, J:
10 Hz, pyridazine H4), 7.04–7.01 (1H, m, phenyl H4), 5.93 (2H, s,
CH2), 3.79 (3H, s, OCH3), 3.26–3.24 (1H, m, cyclohexyl H1), 1.73–1.48
(5H, m, cyclohexyl equatorial), 1.22–1.01 (5H, m, cyclohexyl axial)
ppm; 13C NMR (DMSO-d6) δ: 159.69, 158.87, 153.82, 143.86, 135.25,
131.92, 130.33, 130.08, 118.23, 115.23, 111.39, 72.52, 55.23, 49.51,
32.48, 25.07, 24.49 ppm; Anal. calc. for C19H23N3O4: C, 63.85; H, 6.49;
N, 11.76, found: C, 63.91; H, 6.834; N, 11.71%; HRMS calc. for
4.1.5.27. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
propylcarbamate, 10b. Crystallized from ethanol/water to yield 80.9%;
m.p. 101 °C; 1H NMR (DMSO-d6) δ: 8.11 (1H, d, J: 10.4 Hz, pyridazine
H5), 7.48–7.40 (4H, m, phenyl H2,5,6, NH), 7.11 (1H, d, J: 9.6 Hz,
pyridazine H4), 7.07–7.04 (1H, m, phenyl H4), 5.96 (2H, s, CH2), 3.82
(3H, s, OCH3), 2.96 (2H, q, CH2), 1.43–1.38 (2H, m, CH2), 0.82 (3H, t,
CH3) ppm; 13C NMR (DMSO-d6) δ: 159.69, 158.87, 154.70, 143.88,
135.26, 131.94, 130.35, 130.09, 118.25, 115.26, 111.37, 72.62, 55.24,
42.03, 22.47, 11.15 ppm; Anal. calc. for C16H19N3O4: C, 60.56; H, 6.03;
N, 13.24, found: C, 60.68; H, 6.242; N, 13.14%; HRMS calc. for
C19H24N3O4 [M + H]: 358.1767, found: 358.1765.
C
16H20N3O4 [M + H]: 318.1454, found: 318.1458.
4.1.5.33. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
heptylcarbamate, 16b. Crystallized from ethanol/water to yield 64.3%;
m.p. 96 °C; 1H NMR (DMSO-d6) δ: 8.09 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.45–7.37 (4H, m, phenyl H2,5,6, NH), 7.08 (1H, d, J: 10 Hz,
pyridazine H4), 7.04–7.02 (1H, m, phenyl H4), 5.93 (2H, s, CH2), 3.79
(3H, s, OCH3), 2.95 (2H, q, CH2), 1.37–1.33 (2H, m, CH2), 1.24–1.18
(8H, m, CH2), 0.81 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 159.69,
158.86, 154.65, 143.85, 135.26, 131.91, 130.33, 130.06, 118.22,
115.23, 111.37, 72.53, 55.22, 40.23, 31.15, 29.19, 28.31, 26.10,
21.98, 13.88 ppm; Anal. calc. for C20H27N3O4: C, 64.32; H, 7.29; N,
11.25, found: C, 64.27; H, 7.547; N, 11.12%; HRMS calc. for
4.1.5.28. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl propan-
2-ylcarbamate, 11b. Crystallized from ethanol/water to yield 74.5%;
m.p. 126 °C; 1H NMR (DMSO-d6) δ: 8.11 (1H, d, J: 10 Hz, pyridazine
H5), 7.48–7.37 (4H, m, phenyl H2,5,6, NH), 7.11 (1H, d, J: 9.6 Hz,
pyridazine H4), 7.07–7.04 (1H, m, phenyl H4), 5.95 (2H, s, CH2), 3.82
(3H, s, OCH3), 3.62–3.59 (1H, m, CH), 1.05 (6H, d, CH3) ppm; 13C NMR
(DMSO-d6) δ: 159.69, 158.86, 153.78, 143.87, 135.26, 131.92, 130.33,
130.08, 118.24, 115.25, 111.37, 72.53, 55.24, 42.41, 22.37 ppm; Anal.
calc. for C16H19N3O4: C, 60.56; H, 6.03; N, 13.24, found: C, 60.52; H,
6.271; N, 13.16%; HRMS calc. for C16H20N3O4 [M + H]: 318.1454,
found: 318.1456.
C20H28N3O4 [M + H]: 374.2080, found: 374.2067.
4.1.5.34. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
4.1.5.29. [3-(3-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
dimethylcarbamate, 17b. Crystallized from water to yield 28.2%; m.p.
95 °C; 1H NMR (DMSO-d6) δ: 8.10 (1H, d, J: 9.6 Hz, pyridazine H5),
7.46–7.38 (3H, m, phenyl H2,5,6), 7.09 (1H, d, J: 9.6 Hz, pyridazine
H4), 7.05–7.03 (1H, m, phenyl H4), 5.96 (2H, s, CH2), 3.80 (3H, s,
OCH3), 2.82 (3H, s, CH3), 2.79 (3H, s, CH3) ppm; 13C NMR (DMSO-d6)
δ: 159.68, 158.81, 154.22, 143.92, 135.30, 132.00, 130.36, 130.10,
118.28, 115.27, 111.39, 73.70, 55.24, 36.04, 35.53, ppm; Anal. calc.
butylcarbamate, 12b. Crystallized from ethanol/water to yield 59.9%;
m.p. 84 °C; 1H NMR (DMSO-d6) δ: 8.09 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.45–7.38 (4H, m, phenyl H2,5,6, NH), 7.08 (1H, d, J: 9.6 Hz,
pyridazine H4), 7.05–7.02 (1H, m, phenyl H4), 5.94 (2H, s, CH2), 3.80
(3H, s, OCH3), 2.97 (2H, q, CH2), 1.37–1.31 (2H, m, CH2), 1.26–1.20
(2H, m, CH2), 0.82 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 159.67,
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BioorganicChemistry92(2019)103304
for C15H17N3O4: C, 59.40; H, 5.65; N, 13.85, found: C, 59.14; H, 6.001;
N, 13.97%; HRMS calc. for C15H18N3O4 [M + H]: 304.1297, found:
304.1306.
4.1.5.41. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
methylphenyl)carbamate, 7c. Crystallized from ethanol/water to yield
60%; m.p. 144 °C; 1H NMR (DMSO-d6) δ: 9.79 (1H, s, NH), 7.76 (1H, d,
J: 9.6 Hz, pyridazine H5), 7.47–7.43 (2H, m, phenyl H4,6), 7.31 (2H, d,
J: 8.8 Hz, phenyl H2′,6′), 7.15 (1H, d, J: 8.8 Hz, phenyl H3), 7.07–7.00
(4H, m, phenyl H3′,5′,5, pyridazine H4), 6.03 (2H, s, CH2), 3.81 (3H, s,
OCH3), 2.21 (3H, s, CH3) ppm; 13C NMR (DMSO-d6) δ: 158.96, 156.81,
151.97, 144.74, 136.03, 135.75, 131.63, 131.30, 129.67, 129.17,
128.66, 123.62, 120.77, 118.28, 111.96, 72.89, 55.72, 20.30 ppm;
Anal. calc. for C20H19N3O4: C, 65.74; H, 5.24; N, 11.50, found: C, 65.97;
H, 5.185; N, 11.49%; HRMS calc. for C20H20N3O4 [M + H]: 366.1454,
found: 366.1452.
4.1.5.35. 6-(2-Methoxyphenyl)pyridazin-3(2H)-one,
from ethanol/water to yield 72.4%; m.p. 170 °C; HRMS calc. for
11H11N2O2 [M + H]: 203.0821, found: 203.0821 [30].
1c. Crystallized
C
4.1.5.36. 2-(Hydroxymethyl)-6-(2-methoxyphenyl)pyridazin-3(2H)-one,
2c. The crude product was used without further purification due to the
stability problem. Yield 90%; m.p. 143–144 °C; HRMS calc. for
C
12H13N2O3 [M + H]: 233.0926, found: 233.0935.
4.1.5.42. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
methylcarbamate, 8c. Crystallized from acetone/water to yield 37.8%;
m.p. 149 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine
H5), 7.47–7.42 (2H, m, phenyl H4,6), 7.27 (1H, q, NH), 7.15 (1H, d, J:
8.8 Hz, phenyl H3), 7.04 (1H, t, J: 7.2 Hz, phenyl H5), 6.98 (1H, d, J:
9,6 Hz, pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.55 (3H,
d, CH3) ppm; 13C NMR (DMSO-d6) δ: 158.89, 156.80, 155.18, 144.52
135.60, 131.25, 129.68, 128.61, 123.66, 120.76, 111.94, 72.73, 55.71,
26.87 ppm; Anal. calc. for C14H15N3O4: C, 58.13; H, 5.23; N, 14.53,
found: C, 57.90; H, 5.283; N, 14.46%; HRMS calc. for C14H16N3O4
[M + H]: 290.1141, found: 290.1142.
4.1.5.37. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
phenylcarbamate, 3c. Crystallized from ethanol/water to yield 70.2%;
m.p. 155 °C; 1H NMR (DMSO-d6) δ: 9.90 (1H, s, NH), 7.75 (1H, d, J:
9.6 Hz, pyridazine H5), 7.46–7.43 (4H, m, phenyl H4,6, phenyl H2′,6′),
7.26 (2H, t, J: 8 Hz, phenyl H3′,5′), 7.14 (1H, d, J: 8.8 Hz, phenyl H3),
7.05–6.97 (3H, m, pyridazine H4, phenyl H5, phenyl H4′), 6.05 (2H, s,
CH2), 3.80 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 158.97, 156.82,
151.97, 144.77, 138.60, 135.78, 131.31, 129.68, 128.79, 128.67,
123.62, 122.76, 120.77, 118.26, 111.96, 72.95, 55.73 ppm; Anal.
calc. for C19H17N3O4: C, 64.95; H, 4.88; N, 11.96, found: C, 65.03; H,
5.130; N, 11.89%; HRMS calc. for C19H18N3O4 [M + H]: 352.1297,
found: 352.1297.
4.1.5.43. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
ethylcarbamate, 9c. Crystallized from ethanol/water to yield 48%; m.p.
116 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine H5),
7.47–7.39 (3H, m, NH, phenyl H4,6), 7.15 (1H, d, J: 8 Hz, phenyl H3),
7.04 (1H, t, J: 7.6 Hz, phenyl H5), 6.98 (1H, d, J: 9.6 Hz, pyridazine
H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 3.03–2.96 (2H, m, CH2),
0.98 (3H, t, CH3) ppm; 13C NMR (DMSO-d6) δ: 158.91, 156.81, 154.49,
144.53, 135.60, 131.26, 129.68, 128.61, 123.67, 120.75, 111.95,
72.62, 55.72, 35.06, 14.82 ppm; Anal. calc. for C15H17N3O4: C, 59.40;
H, 5.65; N, 13.85, found: C, 59.48; H, 5.724; N, 13.81%; HRMS calc. for
C15H18N3O4 [M + H]: 304.1297, found: 304.1296.
4.1.5.38. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
fluorophenyl)carbamate, 4c. Crystallized from ethanol/water to yield
45.6%; m.p. 158 °C; 1H NMR (DMSO-d6) δ: 9.95 (1H, s, NH), 7.75 (1H,
d, J: 9.6 Hz, pyridazine H5), 7.47–7.42 (4H, m, phenyl H4,6, phenyl
H2′,6′), 7.15–7.00 (5H, m, pyridazine H4, phenyl H3,5, phenyl H3′,5′),
6.04 (2H, s, CH2), 3.80 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ:
158.97, 157.8 (d, J: 234.7 Hz, F-phenyl H4′), 156.82, 152.08, 144.78,
135.78, 134.94, 131.31, 129.67, 128.67, 123.61, 120.77, 119.91,
115.39 (d, J: 22.4 Hz, F-phenyl H3′,5′), 111.96, 73.00, 55.72 ppm;
Anal. calc. for C19H16FN3O4: C, 61.79; H, 4.37; N, 11.38, found: C,
61.65; H, 4.475; N, 11.47%; HRMS calc. for C19H17FN3O4 [M + H]:
370.1203, found: 370.1188.
4.1.5.44. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
propylcarbamate, 10c. Crystallized from acetone/water to yield 31.8%;
m.p. 146 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.47–7.40 (3H, m, NH, phenyl H4,6), 7.15 (1H, d, J: 8.4 Hz, phenyl
H3), 7.04 (1H, td, J1: 7.6 Hz, J2: 1.2 Hz, phenyl H5), 6.98 (1H, d, J:
9.6 Hz, pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.92 (2H,
q, CH2), 1.40–1.35 (2H, m, CH2), 0.79 (3H, t, CH3) ppm; 13C NMR
(DMSO-d6) δ: 158.89, 156.80, 154.68, 144.51, 135.58, 131.25, 129.66,
128.60, 123.66, 120.73, 111.94, 72.60, 55.71, 42.01, 22.45,
11.13 ppm; Anal. calc. for C16H19N3O4: C, 60.56; H, 6.03; N, 13.24,
found: C, 60.74; H, 6.180; N, 13.26%; HRMS calc. for C16H20N3O4
[M + H]: 318.1454, found: 318.1441.
4.1.5.39. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
chlorophenyl)carbamate, 5c. Crystallized from ethanol/water to yield
41.5%; m.p. 162 °C; 1H NMR (DMSO-d6) δ: 10.06 (1H, s, NH), 7.75 (1H,
d, J: 9.6 Hz, pyridazine H5), 7.47–7.43 (4H, m, phenyl H4,6, phenyl
H2′,6′), 7.34–7.31 (2H, m, phenyl H3′,5′), 7.15 (1H, d, J: 8.4 Hz, phenyl
H3), 7.05–7.00 (2H, m, pyridazine H4, phenyl H5), 6.05 (2H, s, CH2),
3.80 (3H, s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 158.89, 156.74,
151.85, 144.72, 137.54, 135.73, 131.25, 129.59, 128.64, 128.60,
126.35, 123.52, 120.69, 119.66, 111.89, 72.99, 55.66 ppm; Anal.
calc. for C19H16ClN3O4: C, 59.15; H, 4.18; N, 10.89, found: C, 59.21;
H, 4.125; N, 10.82%; HRMS calc. for
386.0908, found: 386.0896.
C19H17ClN3O4 [M + H]:
4.1.5.45. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl propan-
2-ylcarbamate, 11c. Crystallized from ethanol/water to yield 64.1%;
m.p. 115 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine
H5), 7.47–7.42 (2H, m, phenyl H4,6), 7.34 (1H, d, NH), 7.15 (1H, d, J:
8.4 Hz, phenyl H3), 7.04 (1H, td, J1: 7.6 Hz, J2: 1.2 Hz, phenyl H5), 6.98
(1H, d, J: 9.6 Hz, pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3),
3.60–3.55 (1H, m, CH), 1.02 (6H, d, CH3) ppm; 13C NMR (DMSO-d6) δ:
158.89, 156.79, 153.76, 144.50, 135.56, 131.23, 129.64, 128.58,
123.64, 120.72, 111.93, 72.50, 55.70, 42.36, 22.35 ppm. Anal. calc.
for C16H19N3O4: C, 60.56; H, 6.03; N, 13.24, found: C, 60.51; H, 6.115;
N, 13.18%; HRMS calc. for C16H20N3O4 [M + H]: 318.1454, found:
318.1441.
4.1.5.40. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
(4-
methoxyphenyl)carbamate, 6c. Crystallized from ethanol/water to
yield 16.3%; m.p. 157 °C; 1H NMR (DMSO-d6) δ: 9.71 (1H, s, NH),
7.75 (1H, d, J: 9.6 Hz, pyridazine H5), 7.47–7.42 (2H, m, phenyl H4,6),
7.33 (2H, d, J: 8.8 Hz, phenyl H2′,6′), 7.14 (1H, d, J: 8.8 Hz, phenyl
H3), 7.05–6.99 (2H, m, phenyl H5, pyridazine H4), 6.84 (2H, d, J:
8.8 Hz, phenyl H3′,5′), 6.02 (2H, s, CH2), 3.80 (3H, s, OCH3), 3.67 (3H,
s, OCH3) ppm; 13C NMR (DMSO-d6) δ: 158.97, 156.82, 154.99, 152.10,
144.72, 135.74, 131.59, 131.30, 129.68, 128.67, 123.63, 120.77,
119.84, 113.99, 111.96, 72.87, 55.73, 55.14 ppm; Anal. calc. for
C
20H19N3O5: C, 62.99; H, 5.02; N, 11.02, found: C, 62.82; H, 5.116;
4.1.5.46. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
butylcarbamate, 12c. Crystallized from ethanol/water to yield 58%;
m.p. 95 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine H5),
N, 11.02%; HRMS calc. for C20H20N3O5 [M + H]: 382.1403, found:
382.1404.
13

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
7.47–7.38 (3H, m, NH, phenyl H4,6), 7.15 (1H, d, J: 8 Hz, phenyl H3),
7.04 (1H, td, J1: 7.2 Hz, J2: 0.8 Hz, phenyl H5), 6.98 (1H, d, J: 9.6 Hz,
pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.96 (2H, q,
CH2), 1.36–1.31 (2H, m, CH2), 1.26–1.20 (2H, m, CH2), 0.82 (3H, t,
CH3) ppm; 13C NMR (DMSO-d6) δ: 158.90, 156.81, 154.67, 144.51,
135.57, 131.24, 129.66, 128.59, 123.67, 120.73, 111.94, 72.57, 55.70,
39.91, 31.30, 19.32, 13.56 ppm. Anal. calc. for C17H21N3O4: C, 61.62;
H, 6.39; N, 12.68, found: C, 61.70; H, 6.630; N, 12.75%; HRMS calc. for
dimethylcarbamate, 17c. The crude product was purified by preparative
TLC using dichloromethane-methanol (100:3) solvent system. Yield
33.9%; m.p. 100 °C; 1H NMR (DMSO-d6) δ: 7.75 (1H, d, J: 9.6 Hz,
pyridazine H5), 7.44 (2H, d, J: 7.6 Hz, phenyl H4,6), 7.15 (1H, d, J:
8 Hz, phenyl H3), 7.05 (1H, td, J1: 7.6 Hz, J2: 0.8 Hz, phenyl H5), 6.98
(1H, d, J: 10 Hz, pyridazine H4), 5.93 (2H, s, CH2), 3.82 (3H, s, OCH3),
2.81 (3H, s, CH3), 2.79 (3H, s, CH3) ppm; 13C NMR (DMSO-d6) δ:
158.84, 156.79, 154.20, 144.50, 135.59, 131.26, 129.74, 128.65,
123.62, 120.77, 111.94, 73.70, 55.70, 36.01, 35.53 ppm; Anal. calc.
for C15H17N3O4·H2O: C, 56.07; H, 5.96; N, 13.08, found: C, 56.13; H,
5.525; N, 12.80%; HRMS calc. for C15H18N3O4 [M + H]: 304.1297,
found: 304.1293.
C
17H22N3O4 [M + H]: 332.1610, found: 332.1610.
4.1.5.47. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
pentylcarbamate, 13c. Crystallized from ethanol/water to yield 88.3%;
m.p. 100 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine
H5), 7.47–7.38 (3H, m, NH, phenyl H4,6), 7.15 (1H, d, J: 8.4 Hz, phenyl
H3), 7.03 (1H, td, J1: 7.2, J2: 0.8 Hz, phenyl H5), 6.98 (1H, d, J: 9.6 Hz,
pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.95 (2H, q,
CH2), 1.38–1.33 (2H, m, CH2), 1.24–1.17 (4H, m, CH2), 0.81 (3H, t,
CH3) ppm; 13C NMR (DMSO-d6) δ: 158.90, 156.81, 154.66, 144.51,
135.57, 131.24, 129.66, 128.60, 123.66, 120.72, 111.94, 72.57, 55.70,
40.18, 28.86, 28.35, 21.75, 13.84 ppm; Anal. calc. for C18H23N3O4: C,
62.59; H, 6.71; N, 12.17, found: C, 62.88; H, 6.795; N, 12.21%; HRMS
calc. for C18H24N3O4 [M + H]: 346.1767, found: 346.1756.
4.2. Cholinesterase inhibition assay
AChE and BuChE (inhibitory%) activities of the synthesized com-
pounds were determined by the modified Ellman’s method [21–23].
Electric eel AChE (type-VI-S, EC 3.1.1.7), equine BuChE (EC 3.1.1.8),
acetylthiocholine iodide, butyrylthiocholine chloride, 5,5′-dithio-bis-(2-
nitrobenzoic acid) (DTNB), donepezil hydrochloride, and galantamine
hydrobromide were purchased from Sigma-Aldrich. The assay was
carried out by VersaMax Molecular Devices microplate reader.
For AChE inhibitory assay; 140 μL of 0.1 mM sodium phosphate
buffer (pH = 8), 20 μL of test solution and 20 μL of AChE (0.2 unit/mL)
solution were added in a 96-well microplate. After 10 min preincuba-
tion at room temperature, 10 μL of DTNB (10 mM) was added into each
well followed by 10 μL of acetylthiocholine iodide (0.4 mM).
Absorbance of the yellow end product was measured at 412 nm uti-
lizing a 96-well microplate reader. Percentage of inhibition of AChE
was determined by comparison of rates of reaction of samples relative
to blank sample (ethanol in phosphate buffer pH = 8) using the formula
(E-S)/E × 100, where E is the activity of enzyme without test sample
and S is the activity of enzyme with test sample. The experiments were
done in quadruplicate and the results were expressed as average values
with SD (Standard Deviation). For BuChE inhibitory assay, the same
procedure as described above was followed except for the use of en-
zyme and substrate, which were BuChE (0.4 unit/mL) and butyr-
ylthiocholine chloride (0.2 mM), respectively. Compounds displayed
more than 50% inhibition were tested for IC50 determination. Each IC50
value was calculated by non-linear regression analysis using the
GraphPad Prism program and determined from at least two in-
dependent experiments.
4.1.5.48. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
hexylcarbamate, 14c. Crystallized from ethanol/water to yield 80.6%;
m.p. 89 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 9.6 Hz, pyridazine
H5), 7.47–7.38 (3H, m, NH, phenyl H4,6), 7.14 (1H, d, J: 8 Hz, phenyl
H3), 7.03 (1H, t, J: 7.2 Hz, phenyl H5), 6.97 (1H, d, J: 9.6 Hz,
pyridazine H4), 5.90 (2H, s, CH2), 3.80 (3H, s, OCH3), 2.94 (2H, q,
CH2), 1.36–1.31 (2H, m, CH2), 1.22–1.19 (6H, m, CH2), 0.81 (3H, t,
CH3) ppm; 13C NMR (DMSO-d6) δ: 158.83, 156.74, 154.59, 144.44,
135.50, 131.18, 129.59, 128.53, 123.60, 120.65, 111.88, 72.49, 55.64,
40.14, 30.82, 29.07, 25.74, 21.91, 13.77 ppm; Anal. calc. for
C
19H25N3O4: C, 63.49; H, 7.01; N, 11.69, found: C, 63.93; H, 6.938;
N, 11.64%; HRMS calc. for C19H26N3O4 [M + H]: 360.1923, found:
360.1915.
4.1.5.49. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
cyclohexylcarbamate, 15c. Crystallized from acetone/water to yield
47.7%; m.p. 168 °C; 1H NMR (DMSO-d6) δ: 7.75 (1H, d, J: 9.6 Hz,
pyridazine H5), 7.49–7.45 (2H, m, phenyl H4,6), 7.39 (1H, d, NH), 7.17
(1H, d, J: 8 Hz, phenyl H3), 7.06 (1H, t, J: 7.2 Hz, phenyl H5), 7.00 (1H,
d, J: 9.6 Hz, pyridazine H4), 5.92 (2H, s, CH2), 3.83 (3H, s, OCH3),
3.27–3.25 (1H, m, cyclohexyl H1), 1.75–1.50 (5H, m, cyclohexyl
equatorial), 1.24–1.04 (5H, m, cyclohexyl axial) ppm. 13C NMR
(DMSO-d6) δ: 158.91, 156.81, 153.82, 144.52, 135.57, 131.25,
129.67, 128.59, 123.66, 120.73, 111.94, 72.56, 55.71, 49.46, 32.47,
25.08, 24.48 ppm. Anal. calc. for C19H23N3O4: C, 63.85; H, 6.49; N,
11.76, found: C, 64.09; H, 6.672; N, 11.68%; HRMS calc. for
4.3. Time-dependent inhibition assay
Time-dependent inhibition of eqBuChE was studied using IC50 shift
approach [32–34]. In this assay, the IC50 values of the selected com-
pounds (7c, 14c, and 16c) and reference (rivastigmine, pseudo-irre-
versible inhibitor) were determined by three different pre-incubation
times (10, 20, 40 min). Briefly; test compounds (20 μL) were allowed to
pre-incubate with eqBuChE (20 μL, 0.4 unit/mL) for various periods of
time in 140 μL of 0.1 mM sodium phosphate buffer (pH = 8) at room
temperature. Following the pre-incubation step, the substrate butyr-
ylthiocholine chloride (10 μL, 0.2 mM) and DTNB (10 μL, 10 mM) were
added to the incubation mixture and the change in the absorbance was
recorded. All measurements were carried out in triplicate and were
C
19H24N3O4 [M + H]: 358.1767, found: 358.1768.
4.1.5.50. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
heptylcarbamate, 16c. Crystallized from ethanol/water to yield 34.9%;
m.p. 89 °C; 1H NMR (DMSO-d6) δ: 7.73 (1H, d, J: 10 Hz, pyridazine H5),
7.47–7.38 (3H, m, NH, phenyl H4,6), 7.15 (1H, d, J: 8 Hz, phenyl H3),
7.04 (1H, td, J1: 7.6 Hz, J2: 0.8 Hz, phenyl H5), 6.98 (1H, d, J: 9.6 Hz,
pyridazine H4), 5.90 (2H, s, CH2), 3.81 (3H, s, OCH3), 2.94 (2H, q,
CH2), 1.37–1.34 (2H, m, CH2), 1.23–1.20 (8H, m, CH2), 0.82 (3H, t,
CH3) ppm; 13C NMR (DMSO-d6) δ: 158.89, 156.80, 154.65, 144.50,
135.56, 131.24, 129.65, 128.59, 123.66, 120.71, 111.94, 72.56, 55.70,
40.20, 31.16, 29.18, 28.32, 26.10, 21.98, 13.88 ppm; Anal. calc. for
expressed as mean
SD. The IC50 values were calculated using
GraphPad Prism software.
4.4. Kinetic studies of cholinesterase inhibition
C
20H27N3O4: C, 64.32; H, 7.29; N, 11.25, found: C, 64.55; H, 7.352; N,
To investigate the mechanism of action of the selected compounds
(7c, 14c, 16c) on BuChE, a kinetic analysis was performed by using
Ellman’s method [35–37]. The experiments were carried out by varying
the concentration of the substrate (butyrylthiocholine chloride, from
0.013 mM to 0.054 mM) in the presence of different concentration of
11.27%; HRMS calc. for
374.2066.
C20H28N3O4 [M + H]: 374.2080, found:
4.1.5.51. [3-(2-Methoxyphenyl)-6-oxopyridazin-1(6H)-yl]methyl
14

Y. Dundar, et al.
BioorganicChemistry92(2019)103304
the inhibitors (7c and 14c: 100 µM, 50 µM, 10 µM, 5 µM; 16c: 50 µM,
10 µM, 5 µM, 1 µM). Mode of inhibition of the enzyme was assayed by
Lineweaver-Burk plot of the inverse of the velocities (1/V) versus the
inverse of the substrate concentration 1/[S] (mM−1). Slopes of the re-
ciprocal plots were then plotted against the concentration of inhibitor,
for estimation of Ki. All processes were assayed in triplicate.
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cholinesterase inhibitors for treatment of Alzheimer’s disease, Eur. J. Med. Chem.
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M. Zivin, A. Podkowa, F. Nachon, X. Brazzolotto, J. Stojan, J. Kos, N. Coquelle,
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4.5. Molecular docking
The molecular docking studies were carried out using Glide and
Induced fit docking protocols implemented in the Schrödinger Small-
Molecule Drug Discovery Suite (Small-Molecule Drug Discovery Suite
2018-1, Schrödinger, LLC, New York, NY, 2018) with an iMac 3.2 GHz
quad-core Intel Core i5 processor with 8 GB of memory on one CPU. The
compounds which were built via builder panel in Maestro were sub-
jected to ligand preparation by LigPrep (Schrödinger Release 2018-1:
LigPrep, Schrödinger, LLC, New York, NY, 2018) using default condi-
tions. The crystal structures of the EeAChE (PDB:1C2O) [38], hAChE
(PDB: 4EY7) [39] and the hBuChE (PDB: 4TPK) [40] were retrieved
from the Protein Data Bank. The proteins were prepared using the
Protein Preparation Wizard tool. Hydrogen atoms were added followed
by assignment of all atom charges and atom types. Finally, energy
minimization and refinement of the structures were done up to 0.3 Å
RMSD by applying OPLS-2005 force field. Centroid of the active site
residues was defined as the grid box. Van der Waals (vdW) radius
scaling factor 1.00, partial charge cutoff 0.25, and OPLS-2005 force
filed were used for receptor grid generation. The compounds prepared
by LigPrep were docked into hBuChE using the extra-precision (XP)
docking mode of the Glide without using any constraints and a 0.80
vdW radius scaling factor and 0.15 partial charge cutoff [41]. The
protocol facilitates docking by ligand flexibility and generation of
multiple conformers within the rigid receptor. The compounds pre-
pared by LigPrep were docked into AChE using the IFD protocol [27]
which considers flexibility to both compounds and receptor. The fol-
lowing residues ASP74, TRP86, TYR124, TYR133, SER203, TRP286,
PHE295, PHE297, TRY337, PHE338 and HIS447 lining the binding site
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Acknowledgments
This work was supported by The Scientific and Technological
Research Council of Turkey (TUBITAK) (grant number 117S872).
Declaration of Competing Interest
The authors declare no conflict of interest.
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