Design and synthesis of some new carboxamide and propanamide derivatives bearing phenylpyridazine as a core ring and the investigation of their inhibitory potential on in-vitro acetylcholinesterase and butyrylcholinesterase

Article abstract of DOI:10.1016/j.bioorg.2018.05.006

A series of new carboxamide and propanamide derivatives bearing phenylpyridazine as a core ring were designed, synthesized and evaluated for their ability to inhibit both cholinesterase enzymes. In addition, a series of carboxamide and propanamide derivat

Full text of DOI:10.1016/j.bioorg.2018.05.006

Accepted Manuscript
Design and Synthesis of some new carboxamide and propanamide derivatives
bearing phenylpyridazine as a core ring and the investigation of their inhibitory
potential on in-vitro acetylcholinesterase and butyrylcholinesterase
Burcu Kilic, Hayrettin O. Gulcan, Fatma Aksakal, Tugba Ercetin, Nihan Oruklu,
E. Umit Bagriacik, Deniz S. Dogruer
PII:
S0045-2068(18)30255-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.05.006
YBIOO 2359
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
14 March 2018
4 May 2018
7 May 2018
Please cite this article as: B. Kilic, H.O. Gulcan, F. Aksakal, T. Ercetin, N. Oruklu, E. Umit Bagriacik, D.S. Dogruer,
Design and Synthesis of some new carboxamide and propanamide derivatives bearing phenylpyridazine as a core
ring and the investigation of their inhibitory potential on in-vitro acetylcholinesterase and butyrylcholinesterase,
Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.05.006
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Design and Synthesis of some new carboxamide and propanamide
derivatives bearing phenylpyridazine as a core ring and the investigation of
their inhibitory potential on in-vitro acetylcholinesterase and
butyrylcholinesterase
Burcu Kilica, Hayrettin O. Gulcanb, Fatma Aksakalc, Tugba Ercetind, Nihan Oruklue, E. Umit
Bagriacike and Deniz S. Dogruera,*
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, Ankara, Turkey
bDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Eastern Mediterranean University, Gazimagosa, North Cyprus
cDepartment of Chemistry, Hacettepe University, Ankara, Turkey
dDepartment of Pharmacognosy, Faculty of Pharmacy, Eastern Mediterranean University, Gazimagosa, North Cyprus
eDepartment of Immunology, Faculty of Medicine, Gazi University, Ankara, Turkey
* Corresponding author E-mail address: denizdogruer2002@yahoo.com (Deniz S. Dogruer)
Keywords: Alzheimer’s disease; pyridazine; acetylcholinesterase inhibitory activity;
butyrylcholinesterase inhibitory activity; molecular docking.
Abstract
A series of new carboxamide and propanamide derivatives bearing phenylpyridazine as a core ring
were designed, synthesized and evaluated for their ability to inhibit both cholinesterase enzymes. In
addition, a series of carboxamide and propanamide derivatives bearing biphenyl instead of
phenylpyridazine were also synthesized to examine the inhibitory effect of pyridazine moiety on both
cholinesterase enzymes. The inhibitory activity results revealed that compounds 5b, 5f, 5h, 5j, 5l
pyridazine-3-carboxamide derivative, exhibited selective acetylcholinesterase (AChE) inhibition with
IC50 values ranging from 0.11 to 2.69 µM. Among them, compound 5h was the most active one (IC50
= 0.11 µM) without cytotoxic effect at its effective concentration against AChE. Additionally,
pyridazine-3-carboxamide derivative 5d (IC50 for AChE=0.16 µM and IC50 for BChE=9.80 µM) and
biphenyl-4-carboxamide derivative 6d (IC50 for AChE=0.59 µM and IC50 for BChE=1.48 µM)
displayed dual cholinesterase inhibitory activity. Besides, active compounds were also tested for their
ability to inhibit Aβ aggregation. Theoretical physicochemical properties of the compounds were
calculated by using Molinspiration Program as well. The Lineweaver-Burk plot and docking study
showed that compound 5h targeted both the catalytic active site (CAS) and the peripheral anionic site
(PAS) of AChE.
1.
Introduction
With respect to the increase in the average life expectancy, Alzheimer Disease (AD), the most
common form of age-related dementia, has become a major threat to the population over the age of 65
during the past several decades. According to the 2015 report of Alzheimer Disease International, the
number of patients with dementia is about 46.8 million, and it is expected to increase up to 131.5
million by 2050. Besides, it has been reported that AD is responsible for about more than 50 % of
dementia cases and is the third leading cause of death for the high-income countries following
ischemic heart disease and stroke [1, 2]. Hence, this situation indicates that AD is a serious health
problem.
Although the first AD case was described by Alois Alzheimer in 1906, the precise cause of AD is
still unclear due to its complex and multifactorial nature. However, several pathophysiological
elements such as amyloid-β (Aβ) aggregates, hyperphosphorylated tau-protein tangles, acetylcholine
(ACh) deficiency and oxidative stress are identified to be related to the pathophysiological processes
of the disease. Based on these, several hypotheses have been put forward to explain the cause of AD
[3-5]. Among them, amyloid β and tau hypotheses have been investigated extensively, however; the
drug candidates targeting them could not come into use due to their severe adverse effects or
inadequate clinic results [6, 7]. On the other hand, cholinergic hypothesis, the most studied one,
continues to be attractive for drug development studies [3-5]. Accordingly, clinical symptoms such as

difficulty in recalling recent events and other cognitive impairments are the consequence of reduced
cholinergic transmission, leading to the suggestion that all attempts to increase cholinergic
transmission might improve the cognitive functions. In this connection, the strategy for enhancing
cholinergic neurotransmission via the inhibition of cholinesterase enzymes, responsible for synaptic
cleavage of acetylcholine, has become an important means in the treatment of AD [8-11]. Indeed,
there are just three approved drugs called donepezil, galantamine, and rivastigmine, which are
cholinesterase inhibitors, and there is one drug called memantine, which is N-methyl-D-aspartic acid
(NMDA) receptor antagonist [3-5]. Although this validates the importance of cholinesterase inhibition
for the treatment of AD at one hand, it is obvious that the number of these drugs to be used in the
treatment of this illness is quite limited.
Acetylcholine is a neurotransmitter of the peripheral and central nervous system (CNS). It
provides neural conduction at the neuromuscular junctions in the peripheral nervous system and
mainly controls the memory and learning process in the CNS. Two enzymes that break down the
acetylcholine to terminate cholinergic transmission are the acetylcholinesterase (AChE) (EC 3.1.1.7)
and butyrylcholinesterase (BChE) (EC 3.1.1.8) enzymes. AChE is mainly synthesized in the
cholinergic nervous system and muscles, while BChE is primarily synthesized in the liver, and is the
dominant cholinesterase in the plasma. Although both can hydrolyze acetylcholine, their kinetics and
selectivity for substrate acetylcholine are different [12, 13].
Regarding the current drugs for the treatment of AD, although they differ each other with respect
to the organization of their active sites, both AChE and BChE are the potential targets to be focused
on. The active site of AChE consists of two primary binding sites, called the catalytic active site
(CAS) and the peripheral anionic site (PAS). CAS is located at the bottom of enzyme active-site gorge
and reveals the catalytic activity of the enzyme. On the other hand, PAS is located at the entrance of
the active site-gorge and enhances the catalytic activity of the enzyme by directing acetylcholine
towards the active site [14]. However, recent studies have reported that PAS also has a non-catalytic
function in such a way that it can interact with Aβ peptide and cause Aβ plaque formation by
triggering Aβ aggregation and these plaques mediate neurodegeneration observed in AD. Thus, the
design of the compounds having the ability to bind the both sites of the enzyme may provide both
cholinergic enhancement and Aβ aggregation inhibition [15]. Among the three AChEIs drugs, only
donepezil has dual binding sites inhibition for AChE. The general structure of BChE enzyme is highly
similar to the AChE. However, most of the aromatic residues in the AChE active site replaces with the
aliphatic amino acids in the BChE enzyme, which results in BChE active pocket being about 200 Å3
larger in volume. The increase in volume allows binding of the inhibitors to the BChE active pocket in
alternative conformations [16]. Moreover, in healthy individuals, the primary enzyme responsible for
the acetylcholine hydrolysis in CNS is the AChE. However, as the disease progresses, levels of AChE
decrease while the levels of BChE increase [17, 18]. Therefore, simultaneous inhibition of both
enzymes should provide additional benefits in the treatment of AD.
Taking into account the aspects stated above, within this research, it was aimed to design novel
pyridazine derivatives and corresponding biphenyl derivatives that have the potential to inhibit
cholinesterase enzymes.
For many years, our research group has focused on pyridazine derivatives associated with
different biological activities [19-22]. For the starting point of our studies, we have employed
minaprin, a known pyridazine derivative antidepressant drug. As classical to some of the CNS acting
agents, minaprine can interact with various neuroreceptors and it is a weak inhibitor of AChE (IC50
=
85 μM) [23]. Considering the structure of minaprine, it is apparent to observe how it also mimics the
structure of acetylcholine (Fig. 1). Therefore, a pyridazine derivative, minaprine, is a good tool to
design novel molecules that have higher potential to inhibit cholinesterase enzymes. Furthermore, we
preferred the bioisosteric replacement of the ester group of acetylcholine with an amide with the aim
of improving the hydrolytic stabilities of designed compounds. As a result, on the basis of these
findings and as a continuation of our research for new cholinesterase inhibitors [19, 24], we have
designed 6-(substitutedphenyl)pyridazine-3-carboxamide and 6-(substitutedphenyl)pyridazine-3-yl
propanamide derivatives along with the [1,1'-biphenyl]-4-carboxamide and ([1,1'-biphenyl]-4-
yl)propanamide derivatives employing the bioisosteric replacement of acetylcholine in minaprine
structure and remembering the Aryl-Spacer-Tertiary amine pharmacophore, which also exist in
minaprine, and within the current cholinesterase inhibitor drugs (Fig. 1). Subsequently, the synthesized

compounds were screened for their ability to inhibit both cholinesterase enzymes. Additionally, Aβ
aggregation inhibition, cytotoxicity and molecular docking studies were also performed for selected
compounds.
R
R’
R’’
Fig. 1. Design strategy and general structure of synthesized compounds.
2.
Result and discussion
2.1.
Chemistry
The synthetic scheme for the synthesis of the compounds are shown in Schemes 1, 2, 3, and 4.
Initially, 6-chloro-N-(2-substitutedethyl)pyridazine-3-carboxamide intermediates (1-4) were prepared
by commercially available 6-chloropyridazine-3-carboxylic acid and appropriate ethylamine
derivatives. It is important to note that the intermediates 1 to 4 have also been originally synthesized
compounds within this research. Next, the Suzuki cross-coupling reaction of the prepared intermediate
with suitable phenylboronic acid derivative afforded original corresponding N-(2-substitutedethyl)-6-
(phenyl/4-methoxyphenyl/4-methylsulfanylphenyl) pyridazine-3-carboxamide derivative (5a-l).
Compounds (6a-d), bearing biphenyl core, were synthesized by the reaction of commercially available
[1,1'-biphenyl]-4-carboxylic acid and appropriate ethylamine derivative in the presence of N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) and 4-(dimethylamino)pyridine
(DMAP). To obtain 3-substituted-N-[6-(phenyl/4-methoxyphenyl/4-methylsulfanylphenyl)pyridazin-
3-yl] propanamide derivatives (13a-i),
we synthesized both 6-(phenyl/4-methoxyphenyl/4-
methylsulfanylphenyl)pyridazin-3-amine intermediates (7-9) and 3-substitutedpropanoic acid
intermediates (10-12) as starting materials. Intermediates 7-9 were prepared by a previously reported
method via the Suzuki cross-coupling reaction using commercially available 6-phenylpyridazin-3-

amine and suitable phenylboronic acid derivative [25]. On the other hand, Michael addition reaction of
appropriate secondary amine derivative and methyl acrylate afforded esters which were further
hydrolyzed with NaOH to yield intermediates 10-12. Then, compound 13a-i were synthesized by the
reaction of obtained intermediates in the presence of EDC and DMAP. Finally, the treatment of
commercially available [1,1'-biphenyl]-4-amine and 3-chloropropionyl chloride gave the intermediate
14, which was treated with secondary amine derivatives to give corresponding N-([1,1'-biphenyl]-4-
yl)-3-substitutedpropanamide derivatives (15a-c).
Each of the title compounds synthesized are original except for compounds 6d and 15b. 6d has
CAS number 756780-04-8 but there are no registered reference and experimental data for this
compound on SciFinder. 15b is registered with CAS number 191168-70-4 and there is only one
reference about this compound [26]. The chemical structures of newly synthesized compounds were
verified by 1H NMR, 13C NMR, HRMS and elemental analysis. The 1H NMR, 13C NMR, mass spectra
and elemental analyses data of the compounds are consistent with the proposed structures. Chemical
shifts of protons as explained in details under the experimental section.
Scheme 1. Synthesis pathway of the compounds 5a-l. Conditions and reagents: (i) Oxalyl chloride, DCM, 2h, rt.
(ii) Ethylamine derivative, TEA, 1h, rt. (iii) Phenylboronic acid derivative, Pd(PPh3)4, 2M Na2CO3,
EtOH/toluene, reflux, under argon, 8h.
Scheme 2. Synthesis pathway of the compounds 6a-d. Conditions and reagents: (i) Ethylamine derivative, EDC,
DMAP, DCM, overnight, rt.

Scheme 3. Synthesis pathway of the compounds 13a-i. Conditions and reagents: (i) Pd(PPh3)4, 2M Na2CO3,
Toluene, reflux, under argon, 24h. (ii) DCM, overnight, rt. (iii) %5 NaOH, 30 min, 40 °C. (iv) EDC, DMAP,
DCM, overnight, rt.
Scheme 4. Synthesis pathway of the compounds 15a-c. Conditions and reagents: (i) 3-Chloropropionyl chloride,
DMAP, TEA, DMF, 25 min, rt. (ii) NaI, ACN, reflux, 2h. (iii) Amine derivative, K2CO3, 3h, rt.
2.2.
Cholinesterase inhibitory activities
The inhibitory activities of the synthesized compounds on AChE (from electric eel) and BChE
(from equine serum) were determined by the modified Ellman’s method using donepezil and
galantamine (10 μM) as the reference compounds. First, the percent inhibitions of the test compounds
were investigated. Then, the IC50 for those which have shown more than 60% inhibition at 10 µM was
measured and the results were reported in Table 1.
Regarding inhibitory activity results, the compounds displayed varying results at the percentage
inhibition tests. Especially, most of the carboxamide derivatives (5a-l and 6a-d) were found to
possess AChE inhibitory activity, but none of the propanamide derivatives (13a-i and 15a-c) displayed
remarkable activity on AChE. In addition, the carboxamide derivatives bearing biphenyl core (6a-d)
rather than phenylpyridazine (5a-l) exhibited lower potential to inhibit AChE.
The type of the tertiary amine groups also influenced the activity in varying ways. Compounds,
5h, 5l, 5d and 6d bearing 1-benzylpiperidine at the side chain were the most active inhibitors for
AChE (respectively, IC50 = 0.11, 0.12, 0.16 and 0.59 µM). On the other hand, compounds 5f, 5j, 5b
and 6b, bearing 1-benzylpiperazine showed lower AChE inhibition (respectively, IC50 = 1.63, 2.66,
2.69 µM and %39 inhibition). However, the introduction of phenylpiperazine (5a, 5e, 5i and 6a) or 4-
benzylpiperine (5c, 5g, 5k and 6c) at the side chain diminished or abolished the activity. These results

indicated and supported that 1-benzylpiperidine and 1-benzylpiperazine moieties are important for
optimal AChE inhibitory activity [27]. Furthermore, to improve the interaction with the PAS of AChE
[28], electron donating methoxy and methylsulfanyl substituents were introduced to the para position
of the phenylpyridazine ring, which resulted in similar AChE inhibitory activity with nonsubstituted
phenylpyridazine derivatives.
Among the tested compounds, compound 5h (IC50 = 0.11 µM) was found to be the most active
compound. The obtained activity was quite comparable to donepezil (IC50= 0.058 µM), however; it
was found to be much more potent in comparison to galantamine. Moreover, the compounds 5d (IC50=
0.16 µM) and 5l (IC50= 0.12 µM) exhibited similar potential. On the other hand, the title compounds
5b, 5f, 5j, and 6d (IC50= 2.69; 1.63; 2.66 and 0.59 µM) displayed a comparable activity when
considering the activity of galantamine, although they were not found as active as donepezil (IC50=
0.058 µM). Additionally, all of the active compounds (5h, 5l, 5d, 6d, 5f, 5j, 5b, 6b) showed much
more potent activity than minaprine (IC50 = 85 μM) [23].
With respect to BChE inhibitory activities, the tested compounds had no effect on BChE at 10
µM concentration, except for compounds 5d (IC50= 9.80 µM) and 6d (IC50= 1.48 µM). These
compounds containing 1-benzylpiperidine at the side chain are carboxamide derivatives. This
indicated that carboxamide structure and 1-benzylpiperidine moiety were also important for BChE
inhibition as well as AChE inhibition when compared with the propanamide structure and the other
tertiary amine derivatives used in this study. However, unlike AChE inhibition, the replacement of
phenylpyridazine (5d) with the biphenyl core led to an increase in BChE inhibitory activity (6d).
Compound 6d (IC50 = 1.48 µM) exhibited a higher BChE activity than donepezil (IC50 = 3.70 µM).
Moreover, another active compound 5d (IC50 = 9.80 µM) had a similar BChE inhibitory activity
compared to galantamine.

Table 1
Cholinesterase inhibitory activities of the synthesized compounds.
AChE
BChE
R
Comp
% inhibition
± SD (10 µM)
IC50 (µM)
± SEM
% inhibition
± SD(10 µM)
IC50 (µM)
± SEM
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
1-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
1-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
1-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
1-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
Phenylpiperazine
Benzylpiperazine
4-Benzylpiperidine
<%10
85 ± 0.7
30 ± 3.4
97 ± 0.2
<%10
n.d.
2.69 ± 0.001
n.d.
<%10
<%10
n.d.
n.d.
5a
5b
34 ± 4.6
65 ± 0.9
<%10
n.d.
5c
0.16 ± 0.001
n.d.
9.80 ± 0.001
n.d.
5d
5e
88 ± 0.7
<%10
1.63 ± 0.001
n.d.
<%10
n.d.
5f
23 ± 1.3
47 ± 1.9
<%10
n.d.
5g
96 ± 0.4
<%10
0.11 ± 0.001
n.d.
n.d.
5h
n.d.
5i
79 ± 1.0
12 ± 2.5
91 ± 0.2
<%10
2.66 ± 0.001
n.d.
<%10
n.d.
5j
14 ± 1.5
29 ± 2.9
<%10
n.d.
5k
0.12 ± 0.001
n.d.
n.d.
5l
n.d.
6a
39 ± 4.7
6 ± 2.5
95 ± 0.2
<%10
n.d.
27 ± 6.2
13 ± 3.0
86 ± 5.2
<%10
n.d.
6b
n.d.
n.d.
6c
0.59 ± 0.001
n.d.
1.48 ± 0.001
n.d.
6d
13a
13b
13c
13d
13e
13f
13g
13h
13i
15a
15b
15c
Donepezil
55 ± 3.9
<%10
n.d.
<%10
n.d.
n.d.
<%10
n.d.
<%10
n.d.
<%10
n.d.
25 ± 2.9
14 ± 4.9
<%10
n.d.
<%10
n.d.
n.d.
<%10
n.d.
n.d.
<%10
n.d.
26 ± 2.6
<%10
n.d.
<%10
n.d.
n.d.
<%10
n.d.
<%10
n.d.
<%10
n.d.
19 ± 3.7
7.5 ± 3.9
n.d.
21 ± 3.2
19 ± 3.6
n.d.
n.d.
n.d.
98 ± 1,0
89 ± 1,3
0.058 ± 0.001
n.d.
90 ± 0.5
20 ± 0.5
3.7 ± 0.001
n.d.
Galantamine
n.d. Not determined.
2.3.
Kinetic studies of enzyme inhibition
Kinetic studies were carried out to determine inhibition types of the most active compounds 5h
(for AChE) and 6d (for BChE). Lineweaver-Burk plots and replots of the slope versus concentration
were utilized to obtain the inhibition constants (Ki), as shown in Fig. 2. Accordingly, all compounds

were shown to have mixed-type inhibition. The obtained Ki values were consistent with the measured
IC50 values.
C o m p o u nd 5h
C o m p o u nd 5h
a
b
2 .0
1 .5
1 .0
0 .5
5 0
4 0
3 0
2 0
1 0
1 5 0 n M
1 0 0 n M
5 0 n M
K i
= 2 5 .3 5 n M
2 5 n M
0
n M
-5
5
1 0
1 5
2 0
-5 0 -2 5
0
2 5
5 0
7 5
1 0 0 1 2 5 1 5 0
-1 0
[S -1
]
m M -1
[5h ] nM
C o m p o u n d 6 d
C o m p o u n d 6 d
c
d
1 .0
0 .8
0 .6
0 .4
0 .2
5 0
8 0 0 n m
4 0
3 0
2 0
1 0
4 0 0 n M
2 0 0 n M
1 0 0 n M
K i
= 1 1 1 .7 n M
0
n M
-2 0
-1 0
1 0
2 0
3 0
4 0
-2 0 0
0
2 0 0
4 0 0
[I] nM
6 0 0
8 0 0
-1 0
[S -1 ] m M -1
Fig. 2. Lineweaver-Burk plot of 5h for AChE hydrolysis (a) and slope replot vs 5h concentration (b).
Lineweaver-Burk plot of 6d for BChE hydrolysis (c) and slope replot vs 6d concentration (d).
2.4.
Inhibition of self-induced and AChE-induced Aβ aggregation
According to the cholinesterase inhibitory activity results, the most potent six compounds (5b,
5d, 5f, 5h, 5j, and 6d) were selected for Aβ aggregation inhibition assay. To investigate the effect of
the selected compounds and references (Donepezil and phenol red) on the aggregation of Aβ1−42, the
reported thioflavin T-based fluorometric assay was performed at 100 µM concentration [29]. The
effects of each compound on the Aβ1–42 peptide self-aggregation and the AChE-induced aggregation
were summarized as the percent (%) inhibition data in Table 2.
According to the assay results, the test compounds were found to be more active for AChE-
induced aggregation than self-induced aggregation. Notably, 5b (%22.30) and 5j (%27.58) showed a
comparable potency on AChE-induced aggregation when considering the activity of donepezil
(%20.30). The results obtained for the reference molecules (donepezil, and phenol red) were consistent
with the literature data [30].
Table 2
Inhibition of Aβ aggregation of selected compounds.
Aβ aggregation
% inhibition ± SD
Self-induced
Aβ aggregation
% inhibition ± SD
AChE-induced
Compound (100 µM)
8.70 ± 0.04
n.i.
22.30 ± 0.39
10.49 ± 0.13
3.38 ± 0.058
n.i.
5b
5d
n.i.
5f
n.i.
5h
12.72 ± 0.17
n.i.
27.58 ± 0.10
n.i.
5j
6d
98.40 ± 0.12
12.9 ± 0.03
92.15 ± 0.05
20.3 ± 0.08
Phenol Red
Donepezil HBr
(n.i.) no inhibition

2.5.
Cytotoxicity test
The cytotoxic effect of compounds 5h and 6d was monitored in 3T3 cell lines to gain insight into
therapeutic potential of the compounds by using a commercially available 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT) assay. 5h was found to be nontoxic at its effective
concentration against AChE. On the other hand, 6d had low cytotoxicity for cells. According to this
results, pyridazine ring could be beneficial to improve the safety of compounds (Table 3).
Table 3
Cytotoxicity assay results of 5h and 6d.
Viability (%) of 3T3 cells
Compound
0.1 µM
96.7 ± 6.1
81.4 ± 3.9
1 µM
10 µM
97.9 ± 4.2
89.0 ± 3.3
80.6 ± 6.1
102.0 ± 3.9
5h
6d
2.6.
Assessment of physicochemical parameters
One of the biggest challenges in drug development is poor pharmacokinetic. Hence, the
calculation of molecular properties and BBB penetration scores of compounds were computed
employing the Molinspiration and admetSAR online services [31, 32]. Calculated descriptors such as
molecular weight, logP, topological polar surface area (tPSA), volume, number of hydrogen donors,
number of hydrogen acceptors and number of violations of Lipinski's rule and blood brain barrier
(BBB) permeability were presented in Table 4. Molecules violating more than one of these rules may
have problems with bioavailability. According to the calculated data, all compounds follow Lipinski's
rule. However, 6c, 6d, and 15c have one violation of Lipinski's rule. These three compounds which
have logP values over 5 are biphenyl derivatives. Thus it can be stated that phenylpyridazine system
enhances drug-likeness properties in addition to the inhibitory activity of the compounds. Based on the
predicted BBB values, all compounds are able to penetrate brain with a probability of more than %90,
therefore they are considered as CNS-active compounds.

Table 4
Calculated physicochemical parameters of the compounds.
Compound
MWa
logPa
tPSAa
Vola
nONa
nOHNHa
Vioa
BBBb
0.9736
0.9500
0.9500
0.9500
0.9813
0.9644
0.9644
0.9644
0.9714
0.9457
0.9457
0.9457
0.9906
0.9831
0.9835
0.9900
0.9861
0.9861
0.9861
0.9904
0.9904
0.9904
0.9831
0.9831
0.9831
0.9819
0.9819
0.9895
387.49
2.96
61.36
365.47
6
1
0
5a
401.51
400.53
400.53
417.51
431.54
430.55
430.55
433.58
447.61
446.62
446.62
385.51
399.54
398.55
398.55
387.49
401.51
400.53
417.51
431.54
430.55
433.58
447.61
446.62
385.51
399.54
398.55
2.66
3.95
4.00
3.02
2.72
4.00
4.05
3.39
3.10
4.38
4.43
4.54
4.25
5.53
5.58
3.49
3.19
4.47
3.54
3.25
4.53
3.92
3.62
4.91
4.80
4.50
5.79
61.36
58.12
58.12
70.59
70.59
67.35
67.35
61.36
61.36
58.12
58.12
35.57
35.57
32.34
32.34
61.36
61.36
58.12
70.59
70.59
67.35
61.36
61.36
58.12
35.57
35.57
32.34
382.27
386.32
386.32
391.02
407.82
411.86
411.86
400.16
416.96
421.01
421.01
373.78
390.58
394.63
394.63
365.47
382.27
386.32
391.02
407.82
411.86
400.16
416.96
421.01
373.78
390.58
394.63
6
5
5
7
7
6
6
6
6
5
5
4
4
3
3
6
6
5
7
7
6
6
6
5
4
4
3
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
0
0
0
0
0
0
0
0
0
0
0
1
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
13a
13b
13c
13d
13e
13f
13g
13h
13i
15a
15b
15c
MW: Molecular weight; logP: log octanol/water partition coefficient; tPSA: Total Polar Surface Area; nON:
number of Hydrogen acceptors; nOHNH: number of Hydrogen donors; Vol: Molecular volume; Vio: Violation
of Lipinski's rule. aCalculated with Molinspiration. bCalculated with admetSAR.
2.7.
Molecular docking
In order to predict the binding mode and affinities of newly synthesized compounds to target
enzymes AChE and BChE, docking was carried out. Herein, interaction with AChE and BChE was
discussed over the compounds 5h and 6d. Three-dimensional (3D) and two-dimensional (2D)
representation of the most energetically profitable poses of compounds 5h and 6d docked in the active
site of AChE was given in Fig. 3.

2D
3D
a)
b)
Fig. 3. 2D and 3D representation of docking poses for the compounds 5h (a) and 6d (b) in the active
site of AChE (PDB code: 1EVE)
AChE contains aromatic residues Trp84 and Phe330 at the catalytic active site and Trp279 at the
peripheral anionic site which are very important residues for ligand binding affinity and specificity
[27]. As seen from 3D views in Fig. 3, 5h and 6d were orientated along the active-site gorge of AChE.
5h and 6d docked to AChE with close affinity values of -10.0 and -9.9 kcal mol-1, correspondingly.
The interaction of 5h and 6d with the active-site gorge of AChE was dominated by hydrogen···π and
water-mediated contacts. Pyridazine ring of 5h interacted with Trp279 residue in the peripheral
anionic site of AChE and a water molecule through C–H···π and π···O–H interactions, respectively.
Also, π···O–H interaction between anisole ring of 5h and water molecule contributed to the
localization of the compound to the peripheral anionic site. Another C–H···π interaction was predicted
between piperazine ring of 5h and Tyr334 residue inside the PAS. Both of compounds 5h and 6d were
interacted with catalytic active site over the Phe330, Phe331 residues and water molecules. It was
predicted with the docking that compound 6d interacted only with Tyr334 in the peripheral anionic
site. Interaction with Trp279 observed in compound 5h was disappeared for compound 6d. Lower
AChE inhibitory activity of 6d contrary to 5h may be attributed to the absence of interaction with
Trp279 residue.
The most energetically profitable pose of compound 6d in the active site of BChE enzyme was
presented in Fig. 4 with 3D and 2D spaces.

2D
3D
Fig. 4. 2D and 3D representation of docking pose for the compound 6d in the active site of BChE
(PDB code: 1P0I)
The general structure of BChE enzyme is highly similar to the AChE. However, most of the
aromatic residues in the AChE active site replaces with the aliphatic amino acids in the BChE enzyme,
which results in BChE active pocket being about 200 Å3 larger in volume. This increase in volume
allows binding of the inhibitors to the BChE active pocket in alternative conformations [16]. Phe330
and Trp279 residues are conserved in AChE, but absent in BChE which can reflect affinity and
noncovalent interactions between BChE and compounds. For the best-scored pose of 6d to BChE
enzyme, binding affinity value of -8.6 kcal mol-1 was predicted. For compound 6d, piperidine group
interacted with Ser198 and His438 residues via hydrogen bonding, which is important for BChE
inhibition in the catalytic active site. In case of docking of 6d, π···O–H interaction between benzyl
group of compound and water molecule in the same region contributes to the stabilization of BChE. In
addition, the phenyl ring of 6d and Ser287 residue located near the acyl pocket interacted via π···C–H
interaction. Another π···C–H interaction was observed between the phenyl group of 6d and Gly283.
3.
Conclusion
The cholinesterase inhibitory activity results revealed that carboxamide series were suitable for
both cholinesterase inhibitions. Moreover, 6-phenylpyridazine and biphenyl moieties were found to be
optimal for AChE and BChE inhibition, respectively. Additionally, the physicochemical parameter
calculations of the synthesized compounds follow Lipinski's rules. Since biphenyl derivatives 6c, 6d,
and 15c have only one violation of Lipinski's rule, it can be considered that the pyridazine ring was
beneficial for AChE inhibitory activity as well as drug-likeness.
Enzyme kinetics and molecular docking studies indicated that the most potent AChE inhibitor 5h
(IC50 = 0.11 µM) could bind simultaneously to the CAS and PAS of AChE and this compound did not
show any cytotoxic effect. However, 5h presented no inhibitory activity for Aβ aggregation. On the
other hand, compounds 5b (%22.30) and 5j (%27.58) showed a comparable potency on AChE-
induced aggregation when considering the activity of donepezil (%20.30). Additionally, compound 6d
exhibited dual cholinesterase inhibitory activity (AChE IC50 = 0.59 µM; BChE IC50 = 1.48 µM).
As a result, 6-(substitutedphenyl)pyridazine-3-carboxamide and [1,1'-biphenyl]-4-carboxamide
can be considered as attractive core structures for the discovery of new multi target directed ligands
for AD therapy. Overall, our design studies employing minaprine, a very poor cholinesterase inhibitor,
have revealed very potent and original cholinesterase inhibitors derived from pyridazine.

4.
Experimental
4.1.
Chemistry
All the chemicals used for the synthesis of the compounds were purchased from commercial suppliers.
Melting points (mp) were recorded on an Schmelzpunkt SMP-II digital melting point apparatus and
1
values are uncorrected. Thin-layer chromatography (TLC) was performed on Merck 60F254 plates. H
(400 MHz) and 13C (100 MHz) NMR spectra were recorded with a Varian Mercury 400 MHz FT-
NMR spectrometer in DMSO-d6 and CDCl3 using tetramethylsilane as the internal standard (Faculty
of Pharmacy, Ankara University). All chemical shifts were recorded as δ (ppm) and coupling constants
(J) are reported as Hertz. The high resolution mass spectra (HRMS) were obtained on a Waters LCT
Premier XE Mass Spectrometer also coupled to an AQUITY Ultra Performance Liquid
Chromatography system at Faculty of Pharmacy, Gazi University, Ankara, Turkey. Elemental analysis
was performed on a Leco 932 CHNS instrument at Faculty of Pharmacy, Ankara University, Ankara,
Turkey, and the results were within ± 0.4% of the theoretical values.
4.1.1.
General procedure for the synthesis of 6-chloro-N-(2-substitutedethyl)pyridazine-3-
carboxamide intermediates (1-4)
6-Chloropyridazine-3-carboxylic acid (1 equivalent) was dissolved in DCM (10 ml). To this solution
catalytic amount of DMF and oxalyl choloride (1.1 equivalent) were added and the reaction mixture
was stirred at room temperature for 2h. After 2h, reaction mixture cooled at ice bath then, TEA (3
equivalent) and appropriate 2-substituted ethan-1-amine derivative (1.1 equivalent) were added and
stirred for additional 1h at room temperature. At the end of this period, reaction mixture was
evaporated to dryness then the precipitate boiled with water and petroleum ether respectively. The
resulting precipitate was filtered to yield intermediates (1-4), which were used for method A without
further purification.
4.1.1.1. 6-Chloro-N-(2-(4-phenylpiperazin-1-yl)ethyl)pyridazine-3-carboxamide (1)
Yield: 74%; mp: 143 °C. HRMS (ESI) [M+H]+ m/z for C17H21ClN5O calculated: 346.1435, found:
346.1438.
4.1.1.2. N-(2-(4-benzylpiperazin-1-yl)ethyl)-6-chloropyridazine-3-carboxamide (2)
Yield: 60%; mp: 145 °C. HRMS (ESI) [M+H]+ m/z for C18H23ClN5O calculated: 360.1591, found:
360.1612.
4.1.1.3. 3-(4-Benzylpiperidin-1-yl)-N-(6-chloropyridazin-3-yl)propanamide (3)
Yield: 40%; mp: 138 °C. HRMS (ESI) [M+H]+ m/z for C19H24ClN4O calculated: 359.1639, found:
359.1614.
4.1.1.4. N-(2-(1-benzylpiperidin-4-yl)ethyl)-6-chloropyridazine-3-carboxamide (4)
Yield: 56%; mp: 147 °C. HRMS (ESI) [M+H]+ m/z for C19H24ClN4O calculated: 359.1639, found:
359.1614.
4.1.2. General procedure for the synthesis of N-(2-substitutedethyl)-6-(phenyl/4-methoxyphenyl/4-
methylsulfanylphenyl)pyridazine-3-carboxamide derivatives (5a-l) (Method A)
Corresponding 6-chloro-N-(2-substitutedethyl)pyridazine-3-carboxamide intermediate (1 equivalent),
appropriate phenyl/4-methoxyphenyl/4-methylsulfanylphenylboronic acid (1.5 equivalent), 2M
Na2CO3 solution (0.55 ml for 1 mmol intermediate), EtOH (0.37 ml for 1 mmol intermediate) and
toluene (4.5 ml for 1 mmol intermediate) were mixed and stirred for a few minutes under inert

atmosphere. Then, tetrakis triphenylphosphine palladium (Pd(PPh3)4) (0.05 equivalent) were rapidly
added to this solution. Reaction mixture was refluxed under inert atmosphere for 8h. After completion
of the time, reaction mixture was evaporated to dryness and the obtained residue was purified by
column chromatography.
4.1.2.1. 6-Phenyl-N-[2-(4-phenylpiperazin-1-yl)ethyl]pyridazine-3-carboxamide (5a)
Following the method A, reaction of compound 1 (800 mg, 2.31 mmol) with phenylboronic acid (420
mg, 3.47 mmol) in the presence of Pd(PPh3)4 (130 mg, 0.11 mmol), 2M Na2CO3 solution (1.28 ml),
toluene (10.0 ml) and EtOH (0.85 ml) gave crude product then, it was purified by column
chromatography (EtOAc:MeOH, 95:5) and recrystallized from ACN. Yield: 110 mg, 12%; mp: 227
°C. 1H NMR (DMSO-d6) δ ppm: 9.14 (t, 1H, J = 5.6 Hz, amide H), 8.42 (d, 1H, J = 8.6 Hz, pyridazine
H), 8.25 (d, 1H, J = 8.6 Hz, pyridazine H), 8.23-8.21 (m, 2H, H2’, H6’), 7.63-7.59 (m, 3H, H3’, H4’,
H5’), 7.20 (t, 2H, J = 8.0 Hz, H3’’, H5’’), 6.93 (d, 2H, J = 8.0 Hz, H2’’, H6’’), 6.76 (t, 1H, J = 8.0 Hz,
H4’’), 3.55 (q, 2H, J = 6.4 Hz, -CONHCH2-), 3.15 (t, 4H, J = 4.8 Hz, piperazine H3, H5), 2.64-2.61 (m,
6H, piperazine H2, H6 and -CONHCH2CH2-). 13C NMR (CDCl3) δ ppm: 163.04, 160.70, 151.01,
135.47, 130.77, 129.21, 129.18, 127.39, 126.22, 124.82, 120.21, 116.39, 56.67, 52.93, 48.56, 36.02.
HRMS (ESI) [M+H]+ m/z for C23H26N5O calculated: 388.2137, found: 388.2126. Anal. Calcd. for
C23H25N5O: C, 71.29; H, 6.50; N, 18.07. Found: C, 70.99; H, 6.24; N, 18.29.
4.1.2.2. N-[2-(4-Benzylpiperazin-1-yl)ethyl]-6-phenylpyridazine-3-carboxamide (5b)
Following the method A, reaction of compound 2 (650 mg, 1.81 mmol) with phenylboronic acid (330
mg, 2.71 mmol) in the presence of Pd(PPh3)4 (105 mg, 0.09 mmol), 2M Na2CO3 solution (1.0 ml),
toluene (10.0 ml) and EtOH (0.80 ml) gave crude product then, it was purified by column
1
chromatography (EtOAc) and recrystallized from isopropanol. Yield: 135 mg, 19%; mp: 151 °C. H
NMR (DMSO-d6) δ ppm: 9.04 (t, 1H, J = 5.6 Hz, amide H), 8.39 (d, 1H, J = 8.8 Hz, pyridazine H),
8.23-8.18 (m, 3H, pyridazine H, H2’, H6’), 7.61-7.55 (m, 3H, H3’, H4’, H5’), 7.31-7.19 (m, 5H, H2’’, H3’’,
H4’’, H5’’, H6’’), 3.48-3.43 (m, 4H, -CONHCH2-, piperazine-CH2-phenyl), 2.54-2.37 (m, 10H,
13
piperazine 8H, -NHCH2CH2-). C NMR (DMSO-d6) δ ppm: 162.21, 159.63, 151.43, 138.17, 135.26,
130.53, 129.04, 128.67, 127.99, 127.11, 126.72, 126.00, 125.31, 61.99, 56.49, 52.59, 52.56, 36.34.
HRMS (ESI) [M+H]+ m/z for C24H28N5O calculated: 402.2294, found: 402.2299. Anal. Calcd. for
C24H27N5O: C, 71.79; H, 6.78; N, 17.44. Found: C, 71.61; H, 6.78; N, 17.37.
4.1.2.3. N-[2-(4-Benzylpiperidin-1-yl)ethyl]-6-phenylpyridazine-3-carboxamide (5c)
Following the method A, reaction of compound 3 (1000 mg, 2.79 mmol) with phenylboronic acid (510
mg, 4.18 mmol) in the presence of Pd(PPh3)4 (162 mg, 0.14 mmol), 2M Na2CO3 solution (1.5 ml),
toluene (12.5 ml) and EtOH (1.0 ml) gave crude product then, it was purified by column
chromatography (EtOAc:MeOH, 95:5) and recrystallized from MeOH. Yield: 590 mg, 53%; mp: 166
°C. 1H NMR (DMSO-d6) δ ppm: 9.03 (t, 1H, J =5.4 Hz, amide H), 8.41 (d, 1H, J = 8.8 Hz, pyridazine
H), 8.24 (d, 1H, J = 8.8 Hz, pyridazine H), 8.23-8.21 (m, 2H, H2’, H6’), 7.62-7.58 (m, 3H, H3’, H4’,
H5’), 7.27 (t, 2H, J = 7.6 Hz, H3’’, H5’’), 7.18-7.14 (m, 3H, H2’’, H4’’, H6’’), 3.48 (q, 2H, J = 6.4 Hz, -
CONHCH2-), 2.89 (d, 2H, J = 11.0 Hz, piperidine H2e, H6e), 2.53-2.49 (m, 4H, piperidine-CH2-phenyl
and -NHCH2CH2-), 1.92 (t, 2H, J = 11.0 Hz, piperidine H2a, H6a), 1.56-1.47 (m, 3H, piperidine H3e, H4,
H5e), 1.25-1.19 (m, 2H, piperidine H3a, H5a). 13C NMR (DMSO-d6) δ ppm: 162.19, 159.66, 151.47,
140.32, 135.29, 130.56, 129.08, 128.86, 128.00, 127.13, 126.02, 125.59, 125.34, 56.81, 53.18, 42.29,
37.30, 36.55, 31.73. HRMS (ESI) [M+H]+ m/z for C25H29N4O calculated: 401.2341, found: 401.2348.
Anal. Calcd. for C25H28N4O: C, 74.97; H, 7.05; N, 13.99. Found: C, 74.57; H, 6.92; N, 14.08.
4.1.2.4. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-phenylpyridazine-3-carboxamide (5d)
Following the method A, reaction of compound 4 (850 mg, 2.37 mmol) with phenylboronic acid (433
mg, 3.55 mmol) in the presence of Pd(PPh3)4 (137 mg, 0.12 mmol), 2M Na2CO3 solution (1.3 ml),
toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it was purified by column

1
chromatography (EtOAc) and recrystallized from ACN. Yield: 400 mg, 42%; mp: 170 °C. H NMR
(DMSO-d6) δ ppm: 9.20 (t, 1H, J = 5.8 Hz, amide H), 8.40 (d, 1H, J = 9.2 Hz, pyridazine H), 8.24-
8.20 (m, 3H, pyridazine H, H2’, H6’), 7.63-7.58 (m, 3H, H3’, H4’, H5’), 7.32-7.20 (m, 5H, H2’’, H3’’, H4’’,
H5’’, H6’’), 3.42-3.38 (m, 4H, piperidine-CH2-phenyl and -CONHCH2-), 2.77 (d, 2H, J = 11.5 Hz,
piperidine H2e, H6e), 1.89 (t, 2H, J = 11.5 Hz, piperidine H2a, H6a), 1.69 (d, 2H, J = 11.5 Hz, piperidine
H3e, H5e), 1.54 (q, 2H, J = 7.0 Hz, -NHCH2CH2-), 1.33-1.30 (m, 1H, piperidine H4), 1.22-1.12 (qd, 2H,
J = 11.5 Hz and 3.1 Hz piperidine H3a, H5a). 13C NMR (DMSO-d6) δ ppm: 162.32, 159.57, 151.70,
138.66, 135.32, 130.57, 129.10, 128.63, 128.00, 127.12, 126.66, 126.09, 125.32, 62.46, 53.20, 36.66,
35.77, 32.89, 31.81. HRMS (ESI) [M+H]+ m/z for C25H29N4O calculated: 401.2341, found: 401.2339.
Anal. Calcd. for C25H28N4O: C, 74.97; H, 7.05; N, 13.99. Found: C, 75.01; H, 6.85; N, 14.11.
4.1.2.5. 6-(4-Methoxyphenyl)-N-[2-(4-phenylpiperazin-1-yl)ethyl]pyridazine-3-carboxamide (5e)
Following the method A, reaction of compound 1 (770 mg, 2.23 mmol) with 4-methoxyphenylboronic
acid (507 mg, 3.34 mmol) in the presence of Pd(PPh3)4 (128 mg, 0.11 mmol), 2M Na2CO3 solution
(1.25 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it was purified by column
chromatography (EtOAc:MeOH, 96:4) and recrystallized from ACN. Yield: 285 mg, 31%; mp: 221
°C. 1H NMR (DMSO-d6) δ ppm: 9.07 (bt, 1H, amide H), 8.34 (d, 1H, J = 8.8 Hz, pyridazine H), 8.21-
8.18 (m, 3H, pyridazine H, H2’, H6’), 7.20 (t, 2H, J = 7.6 Hz, H3’’, H5’’), 7.14 (d, 2H, J = 9.2 Hz, H3’,
H5’), 6.92 (d, 2H, J = 7.6 Hz, H2’’, H6’’), 6.76 (t, 1H, J = 7.6 Hz, H4’’), 3.86 (s, 3H, -OCH3), 3.54 (q, 2H,
J = 6.4 Hz -CONH-CH2-), 3.14 (t, 4H, piperazine H3, H5), 2.63-2.60 (m, 6H, piperazine H2, H6 and -
13
CONHCH2CH2-). C NMR (CDCl3) δ ppm: 163.12, 161.95, 160.16, 150.89, 150.51, 129.13, 128.81,
127.85, 126.05, 123.87, 120.08, 116.34, 114.65, 56.74, 55.42, 52.95, 48.63, 36.12. HRMS (ESI)
[M+H]+ m/z for C24H28N5O2 calculated: 418.2243, found: 418.2241. Anal. Calcd. for C24H27N5O2: C,
69.04; H, 6.52; N, 16.77. Found: C, 69.02; H, 6.56; N, 16.84.
4.1.2.6. N-[2-(4-Benzylpiperazin-1-yl)ethyl]-6-(4-methoxyphenyl)pyridazine-3-carboxamide (5f)
Following the method A, reaction of compound 2 (550 mg, 1.52 mmol) with 4-methoxyphenylboronic
acid (348.5 mg, 2.29 mmol) in the presence of Pd(PPh3)4 (88 mg, 0.076 mmol), 2M Na2CO3 solution
(0.84 ml), toluene (9.0 ml) and EtOH (0.7 ml) gave crude product then, it was purified by column
1
chromatography (EtOAc) and recrystallized from EtOH-water. Yield: 330 mg, 50%; mp: 148 °C. H
NMR (DMSO-d6) δ: 9.06 (t, 1H, J = 5.6 Hz, amide H), 8.36 (d, 1H, J = 8.8 Hz, pyridazine H), 8.23-
8.18 (m, 3H, pyridazine H, H2’, H6’), 7.34-7.22 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 7.15 (d, 2H, J = 8.4
Hz, H3’, H5’), 3.86 (s, 3H, OCH3), 3.50-3.45 (m, 4H, -CONHCH2-, piperazine-CH2-phenyl), 2.55-2.39
(m, 10H, piperazine 8H, -NHCH2CH2-). 13C NMR (DMSO-d6) δ: 162.29, 161.34, 159.17, 150.83,
138.16, 135.26, 128.70, 128.63, 128.03, 127.46, 126.76, 125.85, 124.38, 114.54, 62.00, 56.53, 55.31,
52.59, 52.56, 36.27. HRMS (ESI) [M+H]+ m/z for C25H30N5O2 calculated: 432.2400, found: 432.2404.
Anal. Calcd. for C25H29N5O2: C, 69.58; H, 6.77; N, 16.23. Found: C, 69.34; H, 6.82; N, 16.15.
4.1.2.7. N-[2-(4-Benzylpiperidin-1-yl)ethyl]-6-(4-methoxyphenyl)pyridazine-3-carboxamide (5g)
Following the method A, reaction of compound 3 (750 mg, 2.09 mmol) with 4-methoxyphenylboronic
acid (476 mg, 3.13 mmol) in the presence of Pd(PPh3)4 (121 mg, 0.105 mmol), 2M Na2CO3 solution
(1.1 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it was purified by column
chromatography (EtOAc:MeOH, 96:4) and recrystallized from ACN. Yield: 438 mg, 49%; mp: 154
°C. 1H NMR (DMSO-d6) δ ppm: 8.97 (t, 1H, J = 5.4 Hz, amide H), 8.34 (d, 1H, J = 8.8 Hz, pyridazine
H), 8,21-8,17 (m, 3H, piridazin H, H2’, H6’), 7,26 (t, 2H, J = 7.6 Hz, H3’’, H5’’), 7.18-7.13 (m, 5H, H3’,
H5’, H2’’, H4’’, H6’’), 3.86 (s, 3H, -OCH3), 3.47 (q, 2H, J = 6.4 Hz, -CONHCH2-), 2.89 (d, 2H, J = 11.5
Hz, piperidine H2e, H6e), 2.52-2.49 (m, 4H, piperidine-CH2-phenyl and -CONHCH2CH2-), 1.91 (t, 2H,
J = 11,5 Hz, piperidine H2a, H6a), 1,54 (d, 2H, J = 11,5 Hz, piperidin H3e, H5e), 1,49-1,47 (m, 1H,
13
piperidine H4), 1.25-1.19 (m, 2H, piperidine H3a, H5a). C NMR (DMSO-d6) δ ppm: 162.24, 161.35,
159.17, 150.85, 140.28, 128.82, 128.59, 127.95, 127.49, 125.79, 125.55, 124.34, 114.53, 56.77, 55.30,
53.14, 42.25, 37.26, 36.47, 31.68. HRMS (ESI) [M+H]+ m/z for C26H31N4O2 calculated: 431.2447,

found: 431.2441. Anal. Calcd. for C26H30N4O2: C, 72.53; H, 7.02; N, 13.01. Found: C, 72.44; H, 6.76;
N, 13.15.
4.1.2.8. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-(4-methoxyphenyl)pyridazine-3-carboxamide (5h)
Following the method A, reaction of compound 4 (850 mg, 2.37 mmol) with 4-methoxyphenylboronic
acid (539 mg, 3.55 mmol) in the presence of Pd(PPh3)4 (137 mg, 0.12 mmol), 2M Na2CO3 solution
(1.3 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it was purified by column
chromatography (EtOAc:MeOH, 96:4) and recrystallized from ACN. Yield: 370 mg, 36%; mp: 171
°C. 1H NMR (DMSO-d6) δ ppm: 9.15 (t, 1H, J = 5.8 Hz, amide H), 8.33 (d, 1H, J = 8.4 Hz, pyridazine
H), 8.21-8.16 (m, 3H, pyridazine H, H2’, H6’), 7.32-7.21 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 7.14 (d, 2H,
J = 8.8 Hz, H3’, H5’), 3.86 (s, 3H, -OCH3), 3.42-3.37 (m, 4H, piperidine-CH2-phenyl and -CONHCH2-
), 2.77 (d, 2H, J = 11.5 Hz, piperidine H2e, H6e), 1.89 (t, 2H, J = 11.5 Hz, piperidine H2a, H6a), 1.69 (d,
2H, J = 11.5 Hz, piperidine H3e, H5e), 1.54 (q, 2H, J = 6.8 Hz, -CONHCH2CH2-), 1.32-1.29 (m, 1H,
piperidine H4), 1.22-1.12 (qd, 2H, J = 11.5 Hz and 3.4 Hz piperidine H3a, H5a). 13C NMR (DMSO-d6) δ
ppm: 162.44, 161.38, 159.14, 151.12, 138.68, 128.67, 128.04, 127.57, 126.70, 125.93, 124.39, 114.60,
62.49, 55.36, 53.24, 36.63, 35.81, 32.92, 31.83. HRMS (ESI) [M+H]+ m/z for C26H31N4O2 calculated:
431.2447, found: 431.2463. Anal. Calcd. for C26H30N4O2: C, 72.53; H, 7.02; N, 13.01. Found: C,
72.78; H, 6.92; N, 13.19.
4.1.2.9. 6-[4-(Methylsulfanyl)phenyl]-N-[2-(4-phenylpiperazin-1-yl)ethyl]pyridazine-3-carboxamide
(5i)
Following the method A, reaction of compound
1 (800 mg, 2.30 mmol) with 4-
(methylsulfanyl)phenylboronic acid (583 mg, 3.47 mmol) in the presence of Pd(PPh3)4 (133 mg, 0.12
mmol), 2M Na2CO3 solution (1.3 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it
was purified by column chromatography (DCM) and recrystallized from butanol. Yield: 210 mg, 21%;
mp: 233 °C. 1H NMR (CDCl3) δ ppm: 8.53 (bt, 1H, amide H), 8.32 (d, 1H, J = 9.0 Hz, pyridazine H),
8.06 (d, 2H, J = 8.4 Hz, H2’, H6’), 7.97 (d, 1H, J = 9.0 Hz, pyridazine H), 7.38 (d, 2H, J = 8.4 Hz, H3’,
H5’), 7.26 (t, 2H, J = 7.8 Hz, H3’’, H5’’), 6.93 (d, 2H, J = 7.8 Hz, H2’’, H6’’), 6.85 (t, 1H, J = 7.8 Hz,
H4’’), 3.72 (q, 2H, J = 6.0 Hz, -CONHCH2-), 3.26 (t, 4H, J = 4.8 Hz, piperazine H3, H5), 2.75-2.72 (m,
13
6H, piperazine H2, H6 and -CONHCH2CH2-), 2.54 (s, 3H, -SCH3). C NMR (CDCl3) δ ppm: 162.91,
160.06, 150.89, 142.80, 131.72, 129.12, 127.52, 126.28, 126.15, 124.17, 119.92, 116.26, 56.76, 53.00,
48.81, 36.30, 15.13. HRMS (ESI) [M+H]+ m/z for C24H28N5OS calculated: 434.2015, found: 434.2010.
Anal. Calcd. for C24H27N5OS: C, 66.48; H, 6.28; N, 16.15; S, 7.40. Found: C, 66.13; H, 6.24; N, 16.02;
S, 7.28.
4.1.2.10. N-[2-(4-Benzylpiperazin-1-yl)ethyl]-6-[4-(methylsulfanyl)phenyl]pyridazine-3-carboxamide
(5j)
Following the method A, reaction of compound
2 (550 mg, 1.52 mmol) with 4-
(methylsulfanyl)phenylboronic acid (385 mg, 2.29 mmol) in the presence of Pd(PPh3)4 (88 mg, 0.076
mmol), 2M Na2CO3 solution (0.84 ml), toluene (9.0 ml) and EtOH (0.7 ml) gave crude product then, it
was purified by column chromatography (EtOAc:MeOH, 97:3) and recrystallized from ACN. Yield:
1
300 mg, 46%; mp: 171 °C. H NMR (DMSO-d6) δ ppm: 9.09 (t, 1H, J = 5.6 Hz, amide H), 8.40 (d,
1H, J = 9.0 Hz, pyridazine H), 8.21 (d, 1H, J = 9.0 Hz, pyridazine H), 8.18 (d, 2H, J = 8.6 Hz, H2’,
H6’), 7.46 (d, 2H, J = 8.6 Hz, H3’, H5’), 7.34-7.22 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 3.51-3.45 (m, 4H, -
CONHCH2-, piperazine-CH2-phenyl), 2.56-2.39 (m, 13H, -SCH3, piperazine 8H, -NHCH2CH2-). 13C
NMR (DMSO-d6) δ ppm: 162.27, 159.15, 151.23, 142.08, 138.22, 131.37, 128.76, 128.09, 127.45,
126.82, 126.02, 125.84, 124.83, 62.06, 56.57, 52.66, 52.63, 36.35, 14.16.13C NMR (CDCl3) δ ppm:
162.91. 160.06. 150.89. 142.80. 131.72. 129.12. 127.52. 126.28. 126.15. 124.17. 119.92. 116.26.
56.76. 53.00. 48.81. 36.30. 15.13. HRMS (ESI) [M+H]+ m/z for C25H30N5OS calculated: 448.2171,
found: 448.2186. Anal. Calcd. for C25H29N5OS: C, 67.08; H, 6.53; N, 15.65; S, 7.16. Found: C, 66.74;
H, 6.42; N, 15.49; S, 7.14.

4.1.2.11. N-[2-(4-Benzylpiperidin-1-yl)ethyl]-6-[4-(methylsulfanyl)phenyl]pyridazine-3-carboxamide
(5k)
Following the method A, reaction of compound
3 (750 mg, 2.10 mmol) with 4-
(methylsulfanyl)phenylboronic acid (528 mg, 3.14 mmol) in the presence of Pd(PPh3)4 (121 mg, 0.105
mmol), 2M Na2CO3 solution (1.2 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it
was purified by column chromatography (EtOAc) and recrystallized from ACN. Yield: 520 mg, 56%;
1
mp: 183 °C. H NMR (DMSO-d6) δ ppm: 8.96 (t, 1H, J =5.6 Hz, amide H), 8.35 (d, 1H, J = 9.0 Hz,
pyridazine H), 8.19 (d, 1H, J = 9.0 Hz, pyridazin H), 8.15 (d, 2H, J = 8.6 Hz, H2’, H6’), 7.44 (d, 2H, J =
8.6 Hz, H3’, H5’), 7.24 (t, 2H, J = 7.4 Hz, H3’’, H5’’), 7.16-7.12 (m, 3H, H2’’, H4’’, H6’’), 3.45 (q, 2H, J =
6.2 Hz, -CONHCH2-), 2.86 (d, 2H, J = 11.3 Hz, piperidine H2e, H6e), 2.54 (s, 3H, SCH3), 2.50-2.47 (m,
4H, piperidine-CH2-phenyl and -NHCH2CH2-), 1.89 (t, 2H, J = 11.3 Hz, piperidine H2a, H6a), 1.54-1.45
(m, 3H, piperidine H3e, H4, H5e), 1.23-1.12 (m, 2H, piperidine H3a, H5a). 13C NMR (DMSO-d6) δ ppm:
162.23, 159.15, 151.26, 142.01, 140.32, 131.46, 128.86, 127.99, 127.42, 125.93, 125.59, 124.76,
56.80, 53.18, 42.29, 36.66, 37.29, 36.56, 31.73, 14.23. HRMS (ESI) [M+H]+ m/z for C26H31N4OS
calculated: 447.2219, found: 447.2212. Anal. Calcd. for C26H30N4OS: C, 69.92; H, 6.77; N, 12.54; S,
7.18. Found: C, 70.31; H, 6.67; N, 12.58; S, 7.22.
4.1.2.12. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-6-[4-(methylsulfanyl)phenyl]pyridazine-3-carboxamide
(5l)
Following the method A, reaction of compound
4 (850 mg, 2.37 mmol) with 4-
(methylsulfanyl)phenylboronic acid (598 mg, 3.56 mmol) in the presence of Pd(PPh3)4 (137 mg, 0.12
mmol), 2M Na2CO3 solution (1.3 ml), toluene (11.0 ml) and EtOH (0.9 ml) gave crude product then, it
was purified by column chromatography (EtOAc:MeOH, 97:3) and recrystallized from EtOAc. Yield:
1
610 mg, 58%; mp: 191 °C. H NMR (DMSO-d6) δ ppm: 9.15 (t, 1H, J = 6.2 Hz, amide H), 8.35 (d,
1H, J = 8.8 Hz, pyridazine H), 8.18 (d, 1H, J = 8.8 Hz, pyridazine H), 8.15 (d, 2H, J = 8.4 Hz H2’,
H6’), 7.44 (d, 2H, J = 8.4 Hz, H3’, H5’), 7.30-7.18 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 3.40-3.35 (m, 4H,
piperidine-CH2-phenyl, -CONHCH2-), 2.75 (d, 2H, J = 11.2 Hz, piperidine H2e, H6e), 2.54 (s, 3H,
SCH3) 1.87 (t, 2H, J = 11.5 , piperidine H2a, H6a), 1.66 (d, 2H, J = 11.2 Hz, piperidine H3e, H5e), 1.51
(q, 2H, J = 7.0 Hz, -NHCH2CH2-), 1.37-1.24 (m, 1H, piperidine H4), 1.20-1.13 (qd, 2H, J = 11.2 Hz
and 3. 3 Hz piperidine H3a, H5a). 13C NMR (DMSO-d6) δ ppm: 162.33, 159.05, 151.47, 141.96,
138.66, 131.48, 128.60, 127.96, 127.38, 126.62, 125.95, 125.92, 124.70, 62.44, 53.17, 36.66, 35.73,
32.91, 31.80, 14.21. HRMS (ESI) [M+H]+ m/z for C26H31N4OS calculated: 447.2219, found: 447.2223.
Anal. Calcd. for C26H30N4OS: C, 69.92; H, 6.77; N, 12.54; S, 7.18. Found: C, 69.68; H, 6.72; N, 12.44;
S, 7.17.
4.1.3.
General procedure for the synthesis of N-(2-substitutedethyl)-[1,1'-biphenyl]-4-carboxamide
derivatives (6a-d) (Method B)
A mixture of [1,1'-biphenyl]-4-carboxylic acid (1.1 equivalent) and EDC (1.2 equivalent) in DCM (10
ml) stirred at room temperature for 10 min then, DMAP (0.2 equivalent) was added to stirred mixture
and stirred for additional 5 min. To this mixture, appropriate 2-substituted ethan-1-amine derivative (1
equivalent) was added. After stirring overnight at room temperature, the reaction mixture was
evaporated to dryness then the precipitate boiled with water and petroleum ether respectively. The
resulting precipitate was filtered and crystallized from appropriate solvent.
4.1.3.1. N-[2-(4-Phenylpiperazin-1-yl)ethyl]-[1,1'-biphenyl]-4-carboxamide (6a)
Following the method B, reaction of 2-(4-phenylpiperazin-1-yl)ethan-1-amine (400 mg, 1.95 mmol)
with [1,1'-biphenyl]-4-carboxylic acid (425 mg, 2.14 mmol) in the presence of EDC (488 mg, 2.33
mmol) and DMAP (49 mg, 0.39 mmol) gave crude product then, it was recrystallized from butanol.
1
Yield: 300 mg, 40%; mp: 238 °C. H NMR (DMSO-d6) δ ppm: 8.45 (bt, 1H, amide H), 7.94 (d, 2H, J
= 8.0 Hz, H2, H6), 7.74 (d, 2H, J = 8.0 Hz, H3, H5), 7.72 (d, 2H, J = 7.6 Hz, H2’, H6’), 7.49 (t, 2H, J =
7.6 Hz, H3’, H5’), 7.40 (t, 1H, J = 7.6 Hz, H4’), 7.20 (t, 2H, J = 7.9 Hz, H3’’, H5’’), 6.92 (d, 2H, J = 7.9

Hz, H2’’, H6’’), 6.76 (t, 1H, J = 7.9 Hz, H4’’), 3.45 (q, 2H, J = 6.4 Hz, -CONHCH2-), 3.13 (t, 4H, J = 4,8
Hz, piperazine H3, H5), 2.61-2.54 (m, 6H, piperazine H2, H6 and -CONHCH2CH2-). 13C NMR (CDCl3)
δ ppm: 167.07, 151.16, 144.20, 140.04, 133.27, 129.14, 128.91, 127.95, 127.42, 127.25, 127.18,
119.87, 116.06, 56.37, 52.87, 49.23, 36.30. HRMS (ESI) [M+H]+ m/z for C25H28N3O calculated:
386.2232, found: 386.2227. Anal. Calcd. for C25H27N3O: C, 77.89; H, 7.06; N, 10.90. Found: C, 77.99;
H, 7.23; N, 10.81.
4.1.3.2. N-[2-(4-Benzylpiperazin-1-yl)ethyl]-[1,1'-biphenyl]-4-carboxamide (6b)
Following the method B, reaction of 2-(4-benzylpiperazin-1-yl)ethan-1-amine (300 mg, 1.37 mmol)
with [1,1'-biphenyl]-4-carboxylic acid (298 mg, 1.51 mmol) in the presence of EDC (315 mg, 1.64
mmol) and DMAP (34 mg, 0.28 mmol) gave crude product then, it was recrystallized from
1
isopropanol. Yield: 100 mg, 18%; mp: 141 °C. H NMR (DMSO-d6) δ ppm: 8.39 (t, 1H, J = 5.6 Hz,
amide H), 7.92 (d, 2H, J = 8.2 Hz, H2, H6), 7.76 (d, 2H, J = 8.2 Hz, H3, H5), 7.72 (d, 2H, J = 7.6 Hz,
H2’, H6’), 7.49 (t, 2H, J = 7.6 Hz, H3’, H5’), 7.41 (t, 1H, J = 7.6 Hz, H4’), 7.34-7.22 (m, 5H, H2’’, H3’’,
H4’’, H5’’, H6’’), 3.45 (s, 2H, piperazine-CH2-phenyl), 3.39 (q, 2H, J = 6.8 Hz, -CONHCH2-), 2.49-2.36
(m, 10H, piperazine and -CONHCH2CH2-). 13C NMR (DMSO-d6) δ ppm: 165.66, 142.50, 139.13,
138.18, 133.34, 128.89, 128.69, 128.01, 127.88, 127.68, 126.74, 126.37, 62.02, 56.86, 52.71, 52.58,
36.84. HRMS (ESI) [M+H]+ m/z for C26H30N3O calculated: 400.2389, found: 400.2387. Anal. Calcd.
for C26H29N3O: C, 78.16; H, 7.32; N, 10.52. Found: C, 77.95; H, 7.42; N, 10.62.
4.1.3.3. N-[2-(4-Benzylpiperidin-1-yl)ethyl]-[1,1'-biphenyl]-4-carboxamide (6c)
Following the method B, reaction of 2-(4-benzylpiperidin-1-yl)ethan-1-amine (350 mg, 1.60 mmol)
with [1,1'-biphenyl]-4-carboxylic acid (350 mg, 1.76 mmol) in the presence of EDC (369 mg, 1.92
mmol) and DMAP (39 mg, 0.32 mmol) gave crude product then, it was recrystallized from EtOH-
1
water. Yield: 300 mg, 47%; mp: 155 °C. H NMR (DMSO-d6) δ ppm: 8.42 (t, 1H, J = 5.4 Hz, amide
H), 7.92 (d, 2H, J = 8.6 Hz, H2, H6), 7.76 (d, 2H, J = 7.6 Hz, H3, H5), 7.72 (d, 2H, J = 7.6 Hz, H2’, H6’),
7.49 (t, 2H, J = 7.6 Hz, H3’, H5’), 7.40 (t, 1H, J = 7.6 Hz, H4’), 7.27 (t, 2H, J = 7.4 Hz, H3’’, H5’’), 7.19-
7.14 (m, 3H, H2’’, H4’’, H6’’), 3.40-3.34 (m, 2H, -CONHCH2-), 2.87 (d, 2H, J = 11.8 Hz, piperidine H2e,
H6e), 2.48-2.43 (m, 4H, piperidine-CH2-phenyl and -CONHCH2CH2-), 1.89 (t, 2H, J = 11,8 Hz,
piperidin H2a, H6a), 1,53 (d, 2H, J = 11,8 Hz, piperidin H3e, H5e), 1,47-1,42 (m, 1H, piperidine H4),
13
1.22-1.17 (m, 2H, piperidine H3a, H5a). C NMR (DMSO-d6) δ ppm: 165.60, 142.48, 140.29, 139.11,
133.31, 128.91, 128.86, 128.00, 127.90, 127.69, 126.75, 126.38, 125.59, 57.20, 53.31, 42.31, 37.29,
37.02, 31.69. HRMS (ESI) [M+H]+ m/z for C27H31N2O calculated: 399.2436, found: 399.2432 Anal.
Calcd. for C27H30N2O: C, 81.37; H, 7.59; N, 7.03. Found: C, 81.56; H, 7.66; N, 7.23.
4.1.3.4. N-[2-(1-Benzylpiperidin-4-yl)ethyl]-[1,1'-biphenyl]-4-carboxamide (6d)
Following the method B, reaction of 2-(1-benzylpiperidin-4-yl)ethan-1-amine (350 mg, 1.60 mmol)
with [1,1'-biphenyl]-4-carboxylic acid (350 mg, 1.76 mmol) in the presence of EDC (369 mg, 1.92
mmol) and DMAP (39 mg, 0.32 mmol) gave crude product then, it was recrystallized from EtOH-
1
water. Yield: 300 mg, 47%; mp: 137 °C. CAS Number: 756780-04-8. H NMR (DMSO-d6) δ ppm:
8.47 (t, 1H, J = 5.6 Hz, amide H), 7.93 (d, 2H, J = 8.4 Hz, H2, H6), 7.75 (d, 2H, J = 8.4 Hz, H3, H5),
7.72(d, 2H, J = 7.6 Hz, H2’, H6’), 7.49 (t, 2H, J = 7.6 Hz, H3’, H5’), 7.40 (t, 1H, J = 7.6 Hz, H4’), 7.33-
7.21 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 3.42 (s, 2H, piperidine-CH2-phenyl), 3.32-3.29 (m, 2H, -
CONHCH2-), 2.77 (d, 2H, J = 11.8 Hz, piperidine H2e, H6e), 1.89 (t, 2H, J = 11.8 Hz, piperidine H2a,
H6a), 1.67 (d, 2H, J = 11.8 Hz, piperidine H3e, H5e), 1.48 (q, 2H, J = 7.2 Hz, -CONHCH2CH2-), 1.32-
1.29 (m, 1H, piperidin H4), 1.21-1.14 (qd, 2H, J = 11.8 Hz and 3.2 Hz, piperidine H3a, H5a). 13C NMR
(DMSO-d6) δ ppm: 165.55, 142.43, 139.14, 138.62, 133.41, 128.91, 128.62, 127.97, 127.88, 127.70,
126.74, 126.64, 126.35, 62.44, 53.19, 36.80, 35.84, 32.93, 31.81. HRMS (ESI) [M+H]+ m/z for
C27H31N2O calculated: 399.2436, found: 399.2417 Anal. Calcd. for C27H30N2O: C, 81.37; H, 7.59; N,
7.03. Found: C, 81.50; H, 7.61; N, 7.25.

4.1.4. General
procedure
for
the
synthesis
of
6-(phenyl/4-methoxyphenyl/4-
methylsulfanylphenyl)pyridazin-3-amine intermediates (7-9)
6-Phenylpyridazin-3-amine
(7),
6-(4-methoxyphenyl)pyridazin-3-amine
(8)
and
6-(4-
(methylsulfanyl)phenyl)pyridazin-3-amine (9) were synthesized according previously reported method
[25]. Intermediates were recrystallized from toluene before using for method C. Yield for (7): 61%.;
mp: 153 °C. HRMS (ESI) [M+H]+ m/z for C10H9N3 calculated: 172.0875, found: 172.0869. Yield for
(8): 66%; mp: 165 °C. HRMS (ESI) [M+H]+ m/z for C11H11N3O calculated: 202.0980, found:
202.0975. Yield for (9): 68%; mp: 176 °C. HRMS (ESI) [M+H]+ m/z for C11H11N3S calculated:
218.0752, found: 218.0745.
4.1.5.
General procedure for the synthesis of 3-substitutedpropanoic acid intermediates (10-12)
To a mixture of 1-phenylpiperazine, 1-benzylpiperazine or 4-benzylpiperidine (2.0 g, 1 equivalent) in
DCM (15 ml), methyl acrylate (1.2 equivalent) was added. The mixture stirred at room temperature
overnight. After evaporation under reduced pressure, corresponding methyl ester was obtained and
hydrolyzed by heating at 40 °C for 30 min with 5% NaOH solution. End of this period, reaction
mixture was cooled to room temperature and neutralized with HCl. The resulting precipitate was
filtered, dried and used for the method C without further purification. Yield for 3-(4-phenylpiperazin-
1-yl)propanoic acid (10): 36%; mp: 196 °C. HRMS (ESI) [M+H]+ m/z for C13H18N2O2 calculated:
235.1447, found: 235.1452. Yield for 3-(4-benzylpiperazin-1-yl)propanoic acid (11): 31%; mp: 99 °C.
HRMS (ESI) [M+H]+ m/z for C14H20N2O2 calculated: 249.1603, found: 249.1595. Yield for 3-(4-
benzylpiperidin-1-yl)propanoic acid (12): 65%; mp: 150 °C. HRMS (ESI) [M+H]+ m/z for C15H21N2O
calculated: 248.1651, found: 248.1639.
4.1.6. General procedure for the synthesis of 3-substituted-N-(6-(phenyl/4-methoxyphenyl/4-
methylsulfanylphenyl)pyridazin-3-yl)propanamide derivatives (13a-i) (Method C)
A mixture of appropriate 3-substitutedpropanoic acid (10-12) (1.1 equivalent) and EDC (1.2
equivalent) in DCM (10 ml) stirred at room temperature for 10 min then, DMAP (0.2 equivalent) was
added to stirred mixture and stirred for additional 5 min. To this mixture, 3-substituted-N-(6-
(phenyl/4-methoxyphenyl/4-methylsulfanylphenyl)pyridazin-3-yl)propanamide intermediate (7-9) (1
equivalent) was added. After stirring overnight at room temperature, the reaction mixture was
evaporated to dryness then the precipitate boiled with water and petroleum ether respectively. The
resulting precipitate was filtered and crystallized from appropriate solvent.
4.1.6.1. 3-(4-Phenylpiperazine-1-yl)-N-(6-phenylpyridazin-3-yl)propanamide (13a)
Following the method C, reaction of 7 (300 mg, 1.75 mmol) with 10 (415 mg, 1.93 mmol) in the
presence of EDC (403 mg, 2.10 mmol) and DMAP (43 mg, 0.35 mmol) gave crude product then, it
1
was recrystallized from butanol. Yield: 210 mg, 31%; mp: 221 °C. H NMR (DMSO-d6) δ ppm: 11.46
(s, 1H, amide H), 8.41 (d, 1H, J = 9.4 Hz, pyridazine H), 8.22 (d, 1H, J = 9.4 Hz, pyridazine H), 8.09
(d, 2H, J = 6.8 Hz, H2’, H6’), 7.56-7.49 (m, 3H, H3’, H4’, H5’), 7.20 (t, 2H, J = 7.6 Hz, H3’’, H5’’), 6.94
(d, 2H, J = 7.6 Hz, H2’’, H6’’), 6.77 (t, 1H, J = 7.6 Hz, H4’’), 3.16 (t, 4H, J = 4.8 Hz, piperazine H3, H5),
2.74-2.69 (m, 4H, -COCH2CH2-), 2.61 (t, 4H, J = 4.8 Hz, piperazine H2, H6). 13C NMR (DMSO-d6) δ
ppm: 171.89, 155.08, 154.51, 150.89, 135.82, 129.42, 128.86, 128.80, 126.21, 125.83, 118.71, 118.44,
115.27, 53.26, 52.26, 48.11, 33.57. HRMS (ESI) [M+H]+ m/z for C23H26N5O calculated: 388.2137,
found: 388.2137. Anal. Calcd. for C23H25N5O: C, 71.29; H, 6.50; N, 18.07. Found: C, 70.91; H, 6.47;
N, 18.15.
4.1.6.2. 3-(4-Benzylpiperazine-1-yl)-N-(6-phenylpyridazin-3-yl)propanamide (13b)
Following the method C, reaction of 7 (250 mg, 1.46 mmol) with 11 (399 mg, 1.61 mmol) in the
presence of EDC (336 mg, 1.75 mmol) and DMAP (18 mg, 0.15 mmol) gave crude product then, it
1
was recrystallized from butanol. Yield: 150 mg, 31%; mp: 207 °C. H NMR (DMSO-d6) δ: 11.38 (s,

1H, amide H), 8.39 (d, 1H, J = 9.6 Hz, pyridazine H), 8.20 (d, 1H, J = 9.6 Hz, pyridazine H), 8.09 (d,
2H, J = 6.8 Hz, H2’, H6’), 7.56-7.49 (m, 3H, H3’, H4’, H5’), 7.31-7.23 (m, 4H, H2’’, H3’’, H4’’, H5’’, H6’’),
3.46 (s, 1H, piperazine-CH2-phenyl), 2.69-2.61 (m, 4H, -COCH2CH2-), 2.46-2.38 (m, 8H, piperazine).
13C NMR (DMSO-d6) δ ppm: 171.82, 155.06, 154.43, 138.15, 135.85, 129.31, 128.78, 128.61, 127.95,
126.68, 126.16, 125.68, 118.40, 61.90, 53.20, 52.48, 52.24, 33.53. HRMS (ESI) [M+H]+ m/z for
C24H28N5O calculated: 402.2294, found: 402.2287. Anal. Calcd. for C24H27N5O: C, 71.29; H, 6.78; N,
17.44. Found: C, 71.48; H, 6.39; N, 17.48.
4.1.6.3. 3-(4-Benzylpiperidin-1-yl)-N-(6-phenylpyridazin-3-yl)propanamide (13c)
Following the method C, reaction of 7 (400 mg, 2.33 mmol) with 12 (636 mg, 2.57 mmol) in the
presence of EDC (538 mg, 2.81 mmol) and DMAP (58 mg, 0.47 mmol) gave crude product then, it
1
was recrystallized from isobutanol. Yield: 185 mg, 20%; mp: 209 °C. H NMR (CDCl3) δ ppm: 12.33
(s, 1H, amide H), 8.53 (d, 1H, J = 9.4 Hz, pyridazine H), 8.04 (d, 2H, J = 6.8 Hz, H2’, H6’), 7.84 (d,
1H, J = 9.4 Hz, pyridazine H), 7.54-7.44 (m, 3H, H3’, H4’, H5’), 7.31-7.16 (m, 5H, H2’’, H3’’, H4’’, H5’’,
H6’’), 3.10 (d, 2H, J = 10.8 Hz, piperidin H2e, H6e), 2.73 (t, 2H, J = 6.0 Hz, -COCH2CH2-), 2.62-2.58
(m, 4H, -COCH2CH2- and piperidin-CH2-phenyl), 2.06 (t, 2H, J = 10.8 Hz, piperidin H2a, H6a), 1.74 (d,
2H, J = 10.8 Hz, piperidin H3e, H5e), 1.60-1.55 (m, 3H, piperidin H3a, H4, H5a). 13C NMR (CDCl3) δ
ppm: 172.29, 156.23, 154.71, 140.72, 136.36, 129.50, 129.10, 128.93, 128.21, 126.68, 125.82, 125.62,
119.20, 53.63, 53.26, 42.77, 38.27, 33.02, 32.08. HRMS (ESI) [M+H]+ m/z for C25H29N4O calculated:
401.2341, found: 401.2344. Anal. Calcd. for C25H28N4O: C, 74.97; H, 7.05; N, 13.99. Found: C, 74.66;
H, 7.12; N, 13.60.
4.1.6.4. N-[6-(4-Methoxyphenyl)pyridazin-3-yl]-3-(4-phenylpiperazin-1-yl)propanamide (13d)
Following the method C, reaction of 8 (350 mg, 1.74 mmol) with 10 (451 mg, 1.93 mmol) in the
presence of EDCI(403 mg, 2.10 mmol) and DMAP (43 mg, 0.35 mmol) gave crude product then, it
1
was recrystallized from butanol. Yield: 225 mg, 31%; mp: 251 °C. H NMR (DMSO-d6) δ ppm: 11.35
(s, 1H, amide H), 8.35 (d, 1H, J = 9.6 Hz, pyridazine H), 814 (d, 1H, J = 9.6 Hz, pyridazine H), 8.04
(d, 2H, J = 8.6 Hz, H2’, H6’), 7.20 (t, 2H, J = 7.6 Hz, H3’’, H5’’), 7.08 (d, 2H, J = 8.6 Hz, H3’, H5’), 6.94
(d, 2H, J = 7.6 Hz, H2’’, H6’’), 6.77 (t, 1H, J = 7.6 Hz, H4’’), 3.83 (s, 3H, -OCH3), 3.16 (bt, 4H,
piperazine H3, H5), 2.74-2.68 (m, 4H, -COCH2CH2-), 2.62 (bt, 4H, piperazine H2, H6). 13C NMR
(CDCl3) δ: 160.97, 156.00, 154.12, 152.48, 150.88, 129.10, 128.77, 127.97, 125.05, 120.07, 119.21,
116.37, 114.37, 55.34, 53.38, 52.57, 48.94, 32.98. HRMS (ESI) [M+H]+ m/z for C24H28N5O2
calculated: 418.2243, found: 418.2235. Anal. Calcd. for C24H27N5O2: C, 69.04; H, 6.52; N, 16.77.
Found: C, 69.20; H, 6.44; N, 16.83.
4.1.6.5. 3-(4-Benzylpiperazin-1-yl)-N-[6-(4-methoxyphenyl)pyridazin-3-yl]propanamide (13e)
Following the method C, reaction of 8 (390 mg, 1.94 mmol) with 11 (530 mg, 2.13 mmol) in the
presence of EDC (447 mg, 2.33 mmol) and DMAP (48 mg, 0.39 mmol) gave crude product then, it
1
was recrystallized from butanol. Yield: 295 mg, 35%; mp: 211 °C. H NMR (DMSO-d6) δ ppm: 11.31
(s, 1H, amide H), 8.34 (d, 1H, J = 9.2 Hz, pyridazine H), 8.13 (d, 1H, J = 9.2 Hz, pyridazine H), 8.05
(d, 2H, J = 8.8 Hz, H2’, H6’), 7.34-7.21 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 7.09 (d, 2H, J = 8.8 Hz, H3’,
H5’), 3.84 (s, 3H, -OCH3), 3.46 (s, 2H, piperazine-CH2-phenyl), 2.67-2.61 (m, 4H, -COCH2CH2-),
2.48-2.38 (t, 8H, piperazine). 13C NMR (DMSO-d6) δ ppm: 171.77, 160.42, 154.83, 154.02, 138.20,
128.67, 128.29, 128.02, 127.60, 126.75, 125.04, 118.49, 114.29, 61.95, 55.20, 53.28, 52.55, 52.29,
33.53. HRMS (ESI) [M+H]+ m/z for C25H30N5O2 calculated: 432.2400, found: 432.2386. Anal. Calcd.
for C25H29N5O2: C, 69.58; H, 6.77; N, 16.23. Found: C, 69.56; H, 6.60; N, 16.35.
4.1.6.6. 3-(4-Benzylpiperidin-1-yl)-N-[6-(4-methoxyphenyl)pyridazin-3-yl]propanamide (13f)
Following the method C, reaction of 8 (400 mg, 1.99 mmol) with 12 (541 mg, 2.19 mmol) in the
presence of EDC (458 mg, 2.39 mmol) and DMAP (49 mg, 0.40 mmol) gave crude product then, it
1
was recrystallized from isopropanol. Yield: 450 mg, 53%; mp: 200 °C. H NMR (CDCl3) δ ppm:

12.23 (s, 1H, amide H), 8.49 (d, 1H, J = 9.2 Hz, pyridazine H), 8.01 (d, 2H, J = 8.8 Hz, H2’, H6’), 7.79
(d, 1H, J = 9.2 Hz, pyridazine H), 7.30-7.16 (m, 5H, H2’’, H3’’, H4’’, H5’’, H6’’), 7.03 (d, 2H, J = 8.8 Hz,
H3’, H5’), 3.88 (s, 3H, -OCH3), 3.10 (d, 2H, J = 11.0 Hz, piperidin H2e, H6e), 272 (t, 2H, J = 5.8 Hz, -
COCH2CH2- ), 2.62-2.58 (m, 4H, -COCH2CH2- and piperidin-CH2-phenyl), 2.06 (t, 2H, J = 11.0 Hz,
piperidin H2a, H6a), 1.74 (d, 2H, J = 11.0 Hz, piperidin H3e, H5e), 1.60-1.58 (m, 3H, piperidin H3a, H4,
H5a). 13C NMR (CDCl3) δ ppm: 172.18, 160.88, 155.89, 154.26, 140.72, 129.09, 128.89, 128.20,
127.98, 125.81, 124.96, 119.26, 114.34, 55.37, 53.65, 53.26, 42.76, 38.25, 33.01, 32.06. HRMS (ESI)
[M+H]+ m/z for C26H31N4O2 calculated: 431.2447, found: 431.2448. Anal. Calcd. for C26H30N4O2: C,
72.53; H, 7.02; N, 13.01. Found: C, 72.52; H, 7.16; N, 12.86.
4.1.6.7. N-{6-[4-(Methylsulfanyl)phenyl]pyridazin-3-yl}-3-(4-phenylpiperazin-1-yl)propanamide (13g)
Following the method C, reaction of 9 (350 mg, 1.61 mmol) with 10 (415 mg, 1.77 mmol) in the
presence of EDC (370 mg, 1.93 mmol) and DMAP (39 mg, 0.32 mmol) gave crude product then, it
1
was recrystallized from butanol. Yield: 100 mg, 14%; mp: 243 °C. H NMR (CDCl3) δ ppm: 11.75 (s,
1H, amide H), 8.49 (d, 1H, J = 9.4 Hz, pyridazine H), 7.92 (d, 2H, J = 8.4 Hz, H2’, H6’), 7.80 (d, 1H, J
= 9.4 Hz, pyridazine H), 7.35 (d, 2H, J = 7.6 Hz, H3’, H5’), 7.26 (t, 2H, J = 8.0 Hz, H3’’, H5’’), 6.92-6.87
(m, 3H, H2’’, H4’’, H6’’), 3.41 (bt, 3H, 4H, piperazine H3, H5), 3.24-2.88 (m, 8H, piperazine H2, H6, -
COCH2CH2-), 2.50 (s, 3H, -SCH3). 13C NMR (CDCl3) δ ppm: 155.86, 154.39, 141.05, 137.74, 132.63,
129.19, 126.96, 126.41, 125.52, 120.51, 119.38, 116.61, 55.12, 52.47, 48.27, 32.62, 15.30. HRMS
(ESI) [M+H]+ m/z for C24H28N5OS calculated: 434.2015, found: 434.2025. Anal. Calcd. for
C24H27N5OS: C, 66.48; H, 6.28; N, 16.15; S, 7.40. Found: C, 66.35; H, 6.23; N, 15.98; S, 7.43.
4.1.6.8. 3-(4-Benzylpiperazin-1-yl)-N-{6-[4-(methylsulfanyl)phenyl]pyridazin-3-yl}propanamide (13h)
Following the method C, reaction of 9 (450 mg, 2.07 mmol) with 11 (566 mg, 2.28 mmol) in the
presence of EDC (476 mg, 2.48 mmol) and DMAP (51 mg, 0.41 mmol) gave crude product then, it
1
was recrystallized from EtOH. Yield: 150 mg, 16%; mp: 203 °C. H NMR (CDCl3) δ ppm: 11.94 (s,
1H, amide H), 8.51 (d, 1H, J = 9.2 Hz, pyridazine H), 7.96 (d, 2H, J = 88 Hz, H2’, H6’), 7.81 (d, 1H, J
= 9.2 Hz, pyridazine H), 7.40-726 (m, 7H, H3’, H5’, H2’’, H3’’, H4’’, H5’’, H6’’), 3.66 (s, 2H, piperazine-
CH2-phenyl), 2.88-2.62 (m, 12H, piperazine, -COCH2CH2-), 2.54 (s, 3H, -SCH3). 13C-NMR (CDCl3) δ
ppm: 155.77, 154.48, 140.92, 132.68, 129.53, 128.45, 127.53, 126.89, 126.36, 125.35, 119.30, 62.34,
53.16, 52.50, 51.93, 32.91, 15.36. HRMS (ESI) [M+H]+ m/z for C25H30N5OS calculated: 448.2171,
found: 448.2173. Anal. Calcd. for C25H29N5OS: C, 67.08; H, 6.53; N, 15.65; S, 7.16. Found: C, 67.04;
H, 6.47; N, 15.50; S, 7.20.
4.1.6.9. 3-(4-Benzylpiperidin-1-yl)-N-{6-[4-(methylsulfanyl)phenyl]pyridazin-3-yl}propanamide (13i)
Following the method C, reaction of 9 (450 mg, 2.07 mmol) with 12 (566 mg, 2.28 mmol) in the
presence of EDC (476 mg, 2.48 mmol) and DMAP (51 mg, 0.41 mmol) gave crude product then, it
was recrystallized from MeOH. Yield: 60 mg, 6%; mp: 198 °C. 1H NMR (DMSO-d6) δ ppm: 11.44 (s,
1H, amide H), 8.36 (d, 1H, J = 9.2 Hz, pyridazine), 8.18 (d, 1H, J = 9.2 Hz, pyridazine), 8.05 (d, 2H, J
= 8.6 Hz, H2’, H6’), 7.41 (d, 2H, J = 86 Hz, H3’, H5’), 7.27 (t, 2H, H3’’, H5’’), 718-7.15 (m, 3H, H2’’, H4’’,
H6’’), 2.91(d, 2H, J = 10,9 Hz, piperidin H2a, H6a), 2.65-2.58 (m, 4H, -COCH2CH2-), 254 (s, 3H, -
SCH3), 2,51 (d, .2H, piperidin-CH2-phenyl with DMSO), 1.92 (t, 2H, J = 10.9 Hz, piperidin H3e, H5e),
1.59-1.51 (m, 3H, piperidin H3e, H4, H5e), 1.24-1.21 (m, 2H, piperidin H3a, H5a). 13C NMR (DMSO-d6)
δ ppm: 171.96, 154.58, 154.35, 140.30, 140.22, 132.20, 128.83, 128.01, 126.55, 125.89, 125.60,
125.35, 118.44, 53.56, 52.78, 42.21, 37.29, 33.64, 31.67, 14.30. HRMS (ESI) [M+H]+ m/z for
C26H31N4OS calculated: 447.2219, found: 447.2203. Anal. Calcd. for C26H30N4OS: C, 69.92; H, 6.77;
N, 12.54; S, 7.18. Found: C, 69.53; H, 6.86; N, 12.55; S, 7.28.
4.1.7.
General procedure for the synthesis of N-([1,1'-biphenyl]-4-yl)-3-substitutedpropanamide
derivatives (15a-c) (Method D)

DMAP (72 mg, 0.59 mmol) and TEA (0.62 ml, 4.42) were added to [1,1'-biphenyl]-4-amine (1 g, 5.9
mmol) in dimethylformamide (8 ml) and the mixture cooled in the ice bath. To this cooled solution 3-
chloropropionyl chloride (0.85 ml, 8.87 mmol) was added dropwise and then reaction mixture was
stirred at room temperature for 25 min. At the end of this period, reaction mixture was poured into ice
water and the precipitate was filtered to yield N-([1,1'-biphenyl]-4-yl)-3-chloropropanamide (14),
which was used for the next step without further purification.
To solution of N-([1,1'-biphenyl]-4-yl)-3-chloropropanamide (1 equivalent) in 15 mL of ACN, NaI (3
equivalent) was added and the mixture was refluxed for 2 h and cooled to room temperature.
Subsequently, anhydrous potassium carbonate (2.5 equivalent) and appropriate amine derivative (2.5
equivalent) were added. Then the mixture was refluxed for 3 h. After evaporation under reduced
pressure, the crude product was purified by crystallization from an appropriate solvent.
4.1.7.1. N-([1,1'-Biphenyl]-4-yl)-3-(4-phenylpiperazin-1-yl)propanamide (15a)
Following the method D, reaction of 14 (400 mg, 1.54 mmol) with phenylpiperazine (0.58 ml, 3.84
mmol) in the presence of NaI (692 mg, 4.61 mmol) and anhydrous potassium carbonate (531 mg, 3.84
mmol) gave crude product then, it was recrystallized from butanol. Yield: 420 mg, 70%; mp: 215 °C.
1H NMR (CDCl3) δ ppm: 11.00 (s, 1H, amide H), 7.61-7.52 (m, 6H, H2, H3, H5, H6, H2’, H6’), 7.42 (t,
2H, J = 7.6 Hz, H3’, H5’), 7.33-7.29 (m, 3H, H4’, H3’’, H5’’), 6.98 (d, 2H, J = 7.4 Hz, H2’’, H6’’), 6.92 (t,
1H, J = 7.4 Hz, H4’’), 3.41 (t, 4H, J = 4.8 Hz, piperazine H3, H5), 2.84-2.80 (m, 6H, piperazine H2, H6
and -COCH2CH2-), 2.61 (t, 2H, J = 6.0 Hz, -COCH2CH2-). 13C NMR (DMSO-d6) δ ppm: 170.19,
150.97, 139.70, 138.69, 134.67, 128.86, 128.85, 126.93, 126.88, 126.17, 119.34, 118.75, 115.32,
53.72, 52.42, 48.19, 34.17. HRMS (ESI) [M+H]+ m/z for C25H28N3O calculated: 386.2232, found:
386.2233. Anal. Calcd. for C25H27N3O: C, 77.89; H, 7.06; N, 10.90. Found: C, 77.71; H, 7.07; N,
10.80.
4.1.7.2. N-([1,1'-Biphenyl]-4-yl)-3-(4-benzylpiperazin-1-yl)propanamide (15b)
Following the method D, reaction of 14 (250 mg, 0.96 mmol) with benzylpiperazine (0.42 ml, 2.40
mmol) in the presence of NaI (432 mg, 2.88 mmol) and anhydrous potassium carbonate (332 mg, 2.40
mmol) gave crude product then, it was recrystallized from butanol. Yield: 300 mg, 78%; mp: 146 °C.
1
CAS Number: 191168-70-4. H NMR (DMSO-d6) δ ppm: 10.18 (s, 1H, amide H), 7.68-7.60 (m, 6H,
H2, H3, H5, H6, H2’, H6’), 7.44 (t, 2H, J = 7.6 Hz, H3’, H5’), 7.34-7.24 (m, 6H, H4’, H2’’, H3’’, H4’’, H5’’,
H6’’), 3.45 (s, 2H, piperazine-CH2-phenyl), 2.64 (t, 2H, J = 6.8 Hz, -COCH2CH2-), 2.48-2.39 (m, 10H,
13
piperazine and -COCH2CH2-). C NMR (DMSO-d6) δ ppm: 170.11, 139.63, 138.61, 138.12, 134.58,
128.77, 128.70, 128.03, 126.85, 126.80, 126.77, 126.10, 119.24, 61.95, 53.64, 52.56, 52.36, 34.04.
HRMS (ESI) [M+H]+ m/z for C26H30N3O calculated: 400.2389, found: 400.2383. Anal. Calcd. for
C26H29N3O: C, 78.16; H, 7.32; N, 10.52. Found: C, 77.76; H, 7.20; N, 10.39.
4.1.7.3. N-([1,1'-Biphenyl]-4-yl)-3-(4-benzylpiperidin-1-yl)propanamide (15c)
Following the method D, reaction of 14 (250 mg, 0.96 mmol) with 4-benzylpiperidin (0.42 ml, 2.40
mmol) in the presence of NaI (432 mg, 2.88 mmol) and anhydrous potassium carbonate (332 mg, 2.40
mmol) gave crude product then, it was recrystallized from butanol. Yield: 300 mg, 78%; mp: 168 °C.
1H NMR (DMSO-d6) δ ppm: 10.18 (s, 1H, amide H), 7.68-7.60 (m, 6H, H2, H3, H5, H6, H2’, H6’), 7.44
(t, 2H, J = 7.6 Hz, H3’, H5’), 7.34-7.24 (m, 6H, H4’, H2’’, H3’’, H4’’, H5’’, H6’’), 3.45 (s, 2H, piperazine-
CH2-phenyl), 2.64 (t, 2H, J = 6.8 Hz, -COCH2CH2-), 2.48-2.39 (m, 10H, piperazine and -COCH2CH2-
). 13C NMR (DMSO-d6) δ ppm: 170.11, 139.63, 138.61, 138.12, 134.58, 128.77, 128.70, 128.03,
126.85, 126.80, 126.77, 126.10, 119.24, 61.95, 53.64, 52.56, 52.36, 34.04. HRMS (ESI) [M+H]+ m/z
for C27H31N2O calculated: 399.2436, found: 399.2433. Anal. Calcd. for C27H30N2O: C, 81.37; H, 7.59;
N, 7.03. Found: C, 81.75; H, 7.74; N, 7.07.
4.2.
Acetylcholinesterase and butyrylcholinesterase inhibition assays

AChE and BChE % inhibitory activities of the 28 test compounds at 10 μM concentration were
determined by the modified Ellman’s method [19, 33]. The formation of the yellow color was
measured at 412 nm using molecular devices, Versamax microplate reader. Electric eel AChE (type-
VI-S, EC 3.1.1.7) and equine serum BChE (EC 3.1.1.8), 5,5’-dithiobis-2-nitrobenzoic acid (DTNB),
acetylthiocholine iodide (ATI), butyrylthiocholine iodide (BTI) and Tris HCl were purchased from
Sigma Aldrich. Donepezil and Galantamine were used as the reference drug at 10 μM concentration.
Sample compounds were dissolved in DMSO, the concentration of DMSO in final reaction mixture
was 1%. At this concentration, DMSO has no inhibitory effect on both enzymes [34].
Briefly, AChE inhibitory activities of the compounds, each reaction mixture (final volume of 200
μL) contained 168 μL of 50 mM of Tris HCl buffer (pH 8.0), 10 μL of 6.8 mM DTNB solution, 10 μL
of AChE solution (0.4 U/mL), and 2 μL of each sample solution. After incubation for 10 min at 25 °C,
the reactions were initiated by the addition of 10 μL ATI solution (10 mM). The change of absorbance
was recorded for 10 min at 412 nm. As a control, an identical solution of the reaction mixture without
the sample solution was processed following the same protocol. The blank reading contained 178 μL
buffer, 10 μL DTNB, 2 μL DMSO and 10 μL ATI solution. BChE inhibitory activities of the test
compounds were measured through the same methodology described, except employing 0.8 U/mL
BChE solution as the enzyme source and the BTI solution (30 mM) as the substrate of the reaction.
Each experiment was performed in triplicate; absorbance values were corrected by subtracting the
absorbance of blank and the inhibition rate (%) was calculated.
Compounds displaying more than 60% inhibition were tested for IC50 determination. Assays were
performed with at least nine concentrations of selected compounds and IC50 values were determined
graphically from linear curves (log [inhibitor] vs. % inhibition) with the use of GraphPad Prism
software (Version 7.0). The results were displayed as mean ± SEM.
4.3.
Kinetic studies of enzyme inhibition
Compounds 5h (for AChE) and 6d (for BChE) were selected and kinetic studies performed
similar to enzyme inhibition assay. To obtain estimates of the inhibition type, reciprocal plots of 1/V
versus 1/[S] were constructed by using reported method with minor modifications [29]. The increase
of the absorbances was measured with different inhibitor concentrations (5h; 25, 50, 100, 150 nM and
6d; 100, 200, 400, 800 nM) and without inhibitor for proposed substrate concentrations (ATI; 0.05,
0.1, 0.2, 0.4 mM and BTI; 0.025, 0.05, 0.1, 0.2 mM). Slopes of the reciprocal plots were plotted
against the concentration of inhibitor, for estimation of Ki. All processes were assayed in triplicate and
data analysis was performed with GraphPad Prism software (Version 7.0). The results were displayed
as mean ± SEM.
4.4.
Aβ aggregation inhibition assay/Thioflavin T (ThT) assay
All the chemicals used for the assay purchased from Sigma-Aldrich and measurements carried out
at Thermo Biolite 24 well Multidish plate by using Thermo Varioskan Flash Microplate Reader. To
investigate the effect of the selected compounds and references (Donepezil and phenol red) on the
aggregation of Aβ1−42 at 100 mM concentration, a reported thioflavin T-based fluorometric assay was
performed [29]. Samples were dissolved in DMSO and analyzed in triplicate.
4.5.
Cytotoxicity test
4.5.1. Cell line and culture conditions
Cell line were provided by Immunology Department at Gazi University Medical School. Mouse
fibroblast NIH/3T3 (ATCC CRL1658) cell line were included in this study. Cell line was grown in
Dulbecco’s modified Eagle medium containing fetal bovine serum, 100 IU/mL penicillin, 100 mg/mL
streptomycin, 2 mM L-glutamine, and maintained in a humidified atmosphere of 5% CO2 at 37°C
(Sanyo, San Diego, California). Cells were subcultured when they were confluent.
4.5.2. MTT Assay

Cytotoxicity of compounds was assessed by using a commercially available 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Applied Science,
Indianapolis, Indiana). Briefly, target cells (5x103 in a volume of 0.1ml) were plated in 96-well flat
bottom culture plates (Greiner Bio-One, Germany) allowing cells to adhere to the bottom of the plate
at 37°C overnight. Cells were treated with various concentrations of 5h and 6d for 24 hours. MTT
reagent was added in the wells. Plates were incubated for 4 hours at 37°C. Cells were disrupted by
adding 0.1 ml of lysis buffer in the wells. After incubating them at 37°C for additional 24 hours, color
reaction was measured at 570 nm (with 670-nm reference) using a Synergy HT Multi-mode microplate
reader (Biotek, Winnoski, Vermont). All groups were performed in triplicates.
4.6.
Assessment of physicochemical parameters
Calculated descriptors (molecular weight, log P, topological polar surface area (tPSA), volume,
number of hydrogen donors, number of hydrogen acceptors and number of violations of Lipinski's
rule) of the compounds were computed employing the Molinspiration online service [31]. Blood brain
barrier (BBB) permeability values were calculated by admetSAR online service [32].
4.7.
Molecular docking
The crystal structures of the AChE and BuChE enzymes were retrieved from the RCSB Protein
Data Bank (http://www.rcsb.org/pdb/), under the accession codes 1EVE [27] and 1P0I [35],
respectively. Molecular Operating Environment (MOE) software [36] was used for molecular docking
of the ligands. The co-crystallized bound compounds and water molecules farther than 4.5 Å from
ligand or receptor were deleted from the crystal structure. Enzyme-ligand complexes were energy
minimized to a gradient of 0.01 kcal/(mol Å), and protonated by means of the force field AMBER99.
Charges on the enzyme and ligands were assigned using the force fields AMBER99 and MMF94X,
correspondingly. The active sites of enzymes were identified by the site finder application in MOE.
Triangle Matcher Algorithm and two rescoring functions, London dG and GBVI/WSA dG were used
to produce 30 poses of each ligand. All poses generated with docking were analyzed and the best-
scored pose with the lowest binding energy for each compound was selected for further investigation
of interactions with the corresponding enzyme.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Scientific and Technological Research Council of Turkey
(TUBITAK) [grant number 114S129].

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Graphical abstract

Highlights

A series of new carboxamide and propanamide derivatives bearing phenylpyridazine
or biphenyl as a core ring were synthesized.


Most of the carboxamide derivatives were selective for AChE.
The most active compounds inhibit human AChE at submicromolar/micromolar
concentration.

Kinetic and docking studies showed that most active compound was mixed type
inhibitor and binding both the catalytic active site (CAS) and the peripheral anionic
site (PAS) of AChE.