Design, synthesis and biological evaluation of 2,3-dihydro-5,6-dimethoxy-1H-inden-1-one and piperazinium salt hybrid derivatives as hAChE and hBuChE enzyme inhibitors

Article abstract of DOI:10.1016/j.ejmech.2020.112140

2,3-Dihydro-5,6-dimethoxy-2-[4-(4-alkyl-4-methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one halide salt derivatives as a novel donepezil hybrid analogs with the property of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) enzyme inhibition were designed and synthesized via N-alkylation reaction of 2,3-dihydro-5,6-dimethoxy-2-[4-(4-methylpiperazin-1-yl)benzylidene]-1H-inden-1-one with some alkyl halides. Biological tests demonstrated that most of the synthesized compounds have moderate to good inhibitory activities effect on cholinesterase enzymes. Among them, 10e showed the best profile as a selected compound for inhibition of hAChE (IC50 = 0.32) and hBuChE (IC50 = 0.43 μM) enzymes. Kinetic analysis and molecular docking led to a better understanding of this compound. Kinetic studies disclosed that 10e inhibited acetylcholinesterase in mixed-type and butyrylcholinesterase in non-competitive type. The toxicity results showed that 10e is less toxic than donepezil and has better inhibitory activity against hBuChE when compared to donepezil or Galantamine. Other performed experiments revealed that 10e has an anti-β amyloid effect which is capable of reducing ROS, LDH and MDA also possing positive effect on TAC. On the other hand, it has shown a good anti-inflammation effect.

Full text of DOI:10.1016/j.ejmech.2020.112140

European Journal of Medicinal Chemistry 191 (2020) 112140
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Design, synthesis and biological evaluation of 2,3-dihydro-5,6-
dimethoxy-1H-inden-1-one and piperazinium salt hybrid derivatives
as hAChE and hBuChE enzyme inhibitors
Sakineh Mozaffarnia b, Reza Teimuri-Mofrad a , Mohammad-Reza Rashidi b
a
,
, *
a
Department of Organic and Biochemistry, Faculty of Chemistry, University of Tabriz, Tabriz, Iran
Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 December 2019
Received in revised form
2,3-Dihydro-5,6-dimethoxy-2-[4-(4-alkyl-4-methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one
halide salt derivatives as a novel donepezil hybrid analogs with the property of acetylcholinesterase
(
AChE) and butyrylcholinesterase (BuChE) enzyme inhibition were designed and synthesized via N-
alkylation reaction of 2,3-dihydro-5,6-dimethoxy-2-[4-(4-methylpiperazin-1-yl)benzylidene]-1H-inden-
-one with some alkyl halides. Biological tests demonstrated that most of the synthesized compounds
have moderate to good inhibitory activities effect on cholinesterase enzymes. Among them, 10e showed
the best profile as selected compound for inhibition of hAChE (IC50 0.32) and hBuChE
IC50 ¼ 0.43 M) enzymes. Kinetic analysis and molecular docking led to a better understanding of this
9
February 2020
Accepted 9 February 2020
Available online 13 February 2020
1
a
¼
Keywords:
(
m
Alzheimer’s disease
Dual inhibition
AChE
compound. Kinetic studies disclosed that 10e inhibited acetylcholinesterase in mixed-type and butyr-
ylcholinesterase in non-competitive type. The toxicity results showed that 10e is less toxic than done-
pezil and has better inhibitory activity against hBuChE when compared to donepezil or Galantamine.
BuChE
Molecular modeling
Other performed experiments revealed that 10e has an anti-b amyloid effect which is capable of reducing
ROS, LDH and MDA also possing positive effect on TAC. On the other hand, it has shown a good anti-
inflammation effect.
©
2020 Elsevier Masson SAS. All rights reserved.
1
. Introduction
neuroinflammation, and oxidative stress [12e15]. The definitive
therapeutic of AD is unknown and finding a way to treat the disease
has become a challenge. One of the most popular treatments
existing is based on the cholinergic hypothesis by maintaining
acetylcholine (ACh) levels [16e19]. Also, other futuristic treatments
for AD include antioxidants, anti-inflammatory medicines, anti-
amyloid strategies, and hormones [20].
In the regulation of neurotransmitter ACh in the synapse of the
nervous system of the body, an enzyme interferes under the name
of acetylcholinesterase (AChE) playing an important role in the
hydrolysis of Ach [21e24].
The most important drugs used today to treat AD are AChE in-
hibitors, Donepezil, rivastigmine, galanthamine and tacrine [25,26].
Butyrylcholinesterase (BuChE) is another cholinesterase enzyme,
which plays a small role in the healthy brain for ACh hydrolysis, but
high levels of BuChE are found in AD patients acting as ACh
hydrolyzer in the absence of AChE. Inhibition of BuChE can increase
the cholinergic transmission in AD. Therefore, simultaneous inhi-
bition of AChE and BuChE enzymes can be a beneficial treatment
approach for AD [27e31].
Alzheimer’s disease (AD) is a multifactorial mental disorder and
one of the most common health issues which increasingly occurred
in developed countries with enhancing of the elderly population.
Indeed, AD is the most common type of dementia due to the
defective central nervous system (CNS). AD begins with short-term
memory loss and characterized by a progressive decrease or loss of
memory, cognitive activities and severe behavioral such as irrita-
bility, anxiety, and depression [1e9]. Unfortunately, with an in-
crease in life expectancy around the world, the number of people
with AD is growing and expected to be several multiplies until 2050
[
10,11]. Several factors can affect AD patients, including genetic,
pathological changes and dysfunction of neuro-cholinergic system,
the amplitude of unusual deposits of -amyloid (A ) peptide,
hyperphosphorylated tau protein, dyshomeostasis of biometals,
b
b
*
Corresponding author.
E-mail address: teymouri@tabrizu.ac.ir (R. Teimuri-Mofrad).
https://doi.org/10.1016/j.ejmech.2020.112140
223-5234/© 2020 Elsevier Masson SAS. All rights reserved.
0

2
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
Among existing medications, donepezil was introduced as non-
2. Results and discussion
toxicity, blood-brain barrier permeable, reversible and non-
competitive inhibitor of AChE in the USA for treatment of mild,
moderate, and severe AD [32e34]. Donepezil is one of the 1-
indanone analogus. 1-Indanone frameworks are available exten-
sively in natural and biologically synthetic compounds. Among its
biological activities are smooth muscle relaxant, anticancer, anti-
inflammatory activity, and acetylcholinesterase (AChE) inhibition.
Therefore, this combination has attracted the attention of chemists
and pharmacologists [35e38].
2.1. Chemistry
The synthetic route to the new 2,3-dihydro-5,6-dimethoxy-1H-
inden-1-one and piperazinium salt hybrid compounds is repre-
sented in schemes 1 and 2. Target compounds were prepared by
changing the functional group attached to the methylated nitrogen
of piperazine and act as the inhibitor of acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE) enzymes. Initially, 4-
Piperazine and its derivatives are important heterocyclic and
pharmacophores compounds. They exhibit many biological activ-
ities such as anti-cancer, antipsychotic, antioxidant, anti-angina,
antihistamine, anxiolytic, anti-microbial, anti-depressant, anti-
fungal and anti-HIV protease activity and found in the structure of
many medicines available in the market. They are also used in
making the central nervous system (CNS) active agents [39e43].
In this work, we tried to design and synthesize hybrid com-
pounds containing 2,3-dihydro-5,6-dimethoxy-1H-inden-1-one (a
key moiety of donepezil) and a piperazinium salt as new cholin-
esterase enzyme inhibitors with simultaneous AChE and BuChE
inhibitory activities (Fig. 1). Their effects on inhibition of AChE and
BuChE enzymes were studied. The most important goal in this
study was to synthesize compounds that could dual inhibition of
AChE and BuChE enzymes. The conversion of neutral nitrogen in
the parent compound 8 to the positive nitrogen in the ultimate
compounds resulted in an increase in the anti-AChE and anti-
BuChE activity. The structure of the synthesized compounds sug-
gests that they are capable of acting as a radical scavenger pos-
sessing the ability to trap radical oxygenated species (ROS). Among
the synthesized compounds, 2,3-dihydro-5,6-dimethoxy-2-[4-(4-
fluorobenzaldehyde (1) was reacted with 1-methylpiperazine (2)
ꢀ
in the presence of HDTMAB and K
2
CO
3
in DMF at 100 C resulting in
the formation of 4-(4-methylpiperazin-1-yl)benzaldehyde (3) in
the yield of 49% (Scheme 1) [44]. 4-(Bromoalkoxy)benzaldehydes
derivatives 6a-b were obtained from the reaction of 4-
hydroxybenzaldehyde (4) with appropriated dibromoalkane de-
ꢀ
rivatives 5a-b in the presence of K
2
CO
3
in acetone at 56 C, in 55
and 48% yields, respectively (Scheme 1) [45].
In the following, from the Aldol condensation reaction of 2,3-
dihydro-5,6-dimethoxy-1H-inden-1-one (7) with compounds 3
and 6a-b in the presence of NaOH 10% in EtOH at ambient tem-
perature, 2,3-dihydro-5,6-dimethoxy-2-[4-(4-methylpiperazin-1-
yl)benzylidene]-1H-inden-1-one
(8)
and
2,3-dihydro-5,6-
dimethoxy-2-[4-(bromoalkoxy)benzylidene]-1H-inden-1-ones
(9a-b) were obtained in 92, 86 and 82% yields, respectively (Scheme
2) [46,47].
The final products compounds 10a-m were synthesized via N-
alkylation reaction of compound 8 with various alkyl halides (R-X)
ꢀ
in the presence of KI in acetonitrile at 80 C, in good yields (Scheme
2) Compounds 10n and 10o with the simultaneous presence of two
1-indanone moieties in their structure were synthesized by the
reaction of compound 8 with 6a or 6b in the presence of KI in
(
3-chlorobenzyl)-4-methylpiperazinium-1-yl)benzylidine]-1H-
ꢀ
inden-1-one bromide (10e) was selected as the strongest inhibitor
of AChE and BuChE enzymes for further experiments that were
designed to analyze its various capabilities.
acetonitrile at 80 C with 55 and 50% yields, respectively (Scheme
2) [1,48].
2.2. In vitro inhibition studies of hAChE/hBuChE
The AChE and BuChE enzymes are known to be involved in AD
and compounds with inhibition of both AChE and BuChE are used
Scheme 1. Synthesis of 4-(4-methylpiperazin-1-yl)benzaldehyde (3) and 4-(bro-
moalkoxy)benzaldehydes (6a-b).
Fig. 1. Requirements in designing the structure of compounds 10a-o.

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
3
Scheme 2. Synthesis of compounds 8, 9a-b and 2,3-dihydro-5,6-dimethoxy-2-[4-(4-alkyl-4-methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one halide salt derivatives (10a-o)
as novel donepezil hybrid analogs.
for the treatment of AD. The value of inhibition of both AChE and
BuChE enzymes of newly synthesized compounds 10a-o were
investigated using Ellman’s spectrophotometric method [49] and
the enzymes obtained from human blood. Donepezil and gal-
antamin are used as a reference and the results are summarized in
Table 1. Most of the synthesized derivatives exhibit inhibitory ac-
tivity against both hAChE and hBuChE, IC50 values obtained for
hBuChE, compounds 10a and 10e showed simultaneous good
inhibitory effects on their activity. Compound 10a showed the
hAChE inhibitory activity (IC50 ¼ 0.37
donepezil while its inhibitory activity against hBuChE
M) was higher than donepezil. Both hAChE and
mM) lower than that of
(IC50 ¼ 0.43
m
hBuChE inhibitory activity in compound 10a are better than the
activity in Galantamin. Compound 10e showed hAChE inhibitory
’
hAChE and hBuChE are between 0.32 to 12.91
.43e25.2 M, respectively. Compound 8 which is considered as
the synthesized precursor has shown good inhibitory effects
mM
and
activity (IC50 ¼ 0.32
m
M) lower than of donepezil s and higher than
0
m
of Galantamin’s but in comparison to donepezil and Galantamin it
possesses
(IC50 ¼ 0.43
a
m
better inhibitory activity against hBuChE
M).
against hAChE (IC50 ¼ 0.32
mM), while it does not show any sig-
nificant effect on hBuChE.
The presence of a positive charge within the piperazinium
compounds 10 is expected not only increases interactions on the
active sites of the enzymes but also to entails a “locked-in” effect in
However, newly synthesized compounds 10a-o have shown
moderate effect on the inhibitory activity of enzymes hAChE and
Table 1
In vitro hAChE and hBuChE inhibitory activity of the synthesized compounds.
Compound
R-
X
IC50
(m
M) hAChE
IC50
(m
M) hBuChE
hAChE/hBuChE
a
8
e
e
0.32 ± 0.012
23.87%
e
a
a
9
9
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
a
b
e
e
6.162%
27.83%
5.16%
e
a
a
e
e
0%
e
0a
0b
0c
0d
0e
0f
0g
0h
0i
PhCH
2
-
Cl
Br
Br
Br
Br
Cl
Cl
Br
Br
Br
Br
Cl
Cl
Br
Br
e
0.37 ± 0.012
2.55 ± 0.062
1.99 ± 0.051
0.54 ± 0.025
0.32 ± 0.011
0.48 ± 0.021
0.38 ± 0.012
2.15 ± 0.061
0.54 ± 0.024
0.54 ± 0.03
4.25 ± 0.12
0.43 ± 0.025
1.1 ± 0.041
24 ± 0.02
0.43 ± 0.021
4.6 ± 0.13
0.65 ± 0.063
1.4 ± 0.071
0.43 ± 0.022
2.16 ± 0.081
3.24 ± 0.12
20.16 ± 2.1
21.6 ± 2.3
3.24 ± 0.1
3.6 ± 0.12
25.2 ± 2.3
6.48 ± 0.2
20 ± 0.01
0.86
0.55
3.06
0.38
0.74
0.22
0.12
0.11
0.02
0.17
1.18
0.02
0.17
1.2
4-BrPhCH
3-BrPhCH
2-BrPhCH
3-ClPhCH
2-ClPhCH
2
2
2
-
-
-
2
-
-
2
2
Py-2-yleCH e
CH
CH
CH
CH
CH
3
3
3
3
2
2 2
CH CH -
(CH
(CH
(CH
2
2
2
)
)
)
2
3
4
CH
CH
CH
2
2
2
-
-
-
0j
0k
0l
¼CHCH
2
-
0m
0n
0o
HOCH
2
CH -
2
b
c
(C19
(C21
e
H
H
17
O
O
4
)CH
)CH
2
-
-
21
4
2
0
0.83 ± 0.04
14.4 ± 15
31.6 ± 3
e
Donepezil
Galantamin
0.014 ± 0.0012
3 ± 0.18
0.001
0.095
e
e
a
Percent inhibition at a concentration of 100
Resulting from compound 9a.
Resulting from compound 9b.
mM.
b
c

4
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
the brain thus preventing peripheral cholinergic adverse effects
together with enabling prolonged duration of AChEIs action in
brain tissues [1,26].
Table 2
Kinetic of hAChE and hBuChE inhibition for the compounds 10a and 10e.
Compounds
hAChE
hBuChE
1
-Indanone moiety as a key part of the donepezil structure has
Ki (
mM)
KI (
m
M)
Ki (m
M)
KI (mM)
biological activity. To go on with this study compounds 10n and 10o
with the simultaneous presence of two 1-indanone moieties were
synthesized and were expected to have good inhibitory activity. But
results from biological testing as shown in Table 1 revealed that
mentioned compounds had only little effect on increasing inhibi-
tory activity than the most synthesized hybrid compounds and
were not suitable alternatives for this purpose.
1
1
0a
0e
0.5
1
0.5
0.5
0.4
0.5
0.4
0.5
Table 3
In vitro cytotoxicity of compounds 10a and 10e.
Compound
Cytotoxicity (IC50, mM)
2.3. Kinetic study of hAChE and hBuChE inhibitions of compounds
MCF-7
SH-SY5Y
HFF
1
0a and 10e
10a
0e
Donepezil
1.195
1.515
0.060
1.285
1.231
0.300
2.404
1.977
0.091
1
Toward a better understanding of the inhibition mechanism,
kinetic studies of compounds 10a and 10e as the strongest hAChE
and hBuChE inhibitors between other synthesized compounds
were performed (Fig. 2). In this study, a similar mode of action was
expected due to the structural similarity of these compounds. The
type of inhibition was determined from the analysis of the
LineweavereBurk plots. This pattern indicates non-competitive
cell lines were higher than that of donepezil which indicates that
these compounds are less toxic than donepezil.
2.5. Effect of 10e on the protection of PC12 against Ab (1-42)
inhibition for compound 10a with Ki values 0.5
for hAChE and hBuChE inhibitions, respectively. 10e has the non-
competitive inhibition for hBuChE with the Ki value of 0.5
while hAChE inhibition of this compound displayed mixed-type
inhibition with 1 M and 0.5 M Ki and KI values, respectively
Table 2). As previously reported, compounds with the non-
competitive type of inhibition, interact with enzyme PAS and
mixed-type inhibition is the result of interaction with both active
sites (PAS and CAS) of the enzyme [32,39].
m
M and 0.4
m
M
toxicity
mM
One of the key factors that play an important role in AD is the
formation of toxic aggregates of A . A peptides have two main
isoforms including A (1-40) and A (1-42). The advanced accu-
mulation of A causes oxidative stress and inflammation. Thus the
inhibitors of the A aggregation are known as the main therapeutic
strategy for AD [21,32]. Therefore, to complement the results and
explore the effect of the synthesized compounds on inhibition of A
1-42) aggregation, the most promising compound 10e was
examined by transmission electron microscopy (Fig. 3).
PC12 pheochromocytoma cells were selected because the cells
are vulnerable to A insult and have been used mostly to study
amyloid peptide neurotoxicity [50]. The A -induced PC12 cells
were incubated with different concentrations of 10e. According to
the MTT result, the PC12 cells were incubated with A (1-42) with
b
b
b
m
m
b
(
b
b
b
(
2.4. In vitro study of the toxicity profile of compound 10a and 10e
Cytotoxicity of compounds 10a and 10e were evaluated in hu-
b
b-
man foreskin fibroblast (HFF) cells, MCF-7 cells, and neuronal SH-
SY5Y cells. The MTT test was used to obtain the IC50 values (com-
pound concentration resulting in 50% inhibition of cell viability)
after 48 h treatment of HFF, MCF-7 and SH-SY5Y cells. The results
are summarized in Table 3. Donepezil has also been used as a
control of these cells. IC50 values of 10a and 10e in all three studied
b
b
or without concentration-dependent of compound 10e. Trans-
mission electron microscopy analysis was used for cell viability
comparison. As shown in Fig. 3, Ab-treated cells (Fig. 3b) experience
Fig. 2. LineweavereBurk plots of hAChE and hBuChE inhibition kinetics of the compounds 10a and 10e, (a) 10a hAChE inhibitory (b) 10a hBuchE inhibitory (c) 10e hAChE inhibitory
d) 10a hBuchE inhibitory.
(

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
5
markedly through direct interaction. It is demonstrated that the
pretreatment of cells with synthetic compound 10e (1 M and
M) can protect them against A induced cell death in a
concentration-dependent manner (Fig. 3c and d, respectively).
m
10
m
b
2.6. Effect of 10e on ROS levels
Previous studies suggested that oxidative injuries have an
important role in AD [32,51]. One of the symptoms of heightened
oxidative stress in various pathological conditions is increased
levels of reactive oxygen species (ROS). A
stimulate oxidative stress in the pathogenesis of AD. Thus, we can
predict that A (1-42) leads to ROS formation the oxidation of
b (1-42) is known to
b
neuronal proteins [21,52]. The anti-oxidant potential of the most
potent test compound 10e, was measured by appraising intracel-
0
lular levels of ROS in the cells affected by A
b
(1-42) using 4 ,6-
diamidino-2-phenylindole (DAPI), deoxynucleotidyltransferase
0 0
TUNEL) and 2 ,7 -dichlorofluorescine diacetate (DCFH-DA) assay.
(
Fig. 3. Transmission electron microscopy analysis of viability of PC12 cells were
ꢀ
DNA with DAPI and in the TUNEL method creates a highly fluo-
rescent complex in which the fluorescent intensity increased in the
presence of ROS. After passing the cell membrane, DCFH-DA un-
dergoes hydrolyzation by intracellular esterases to nonfluorescent
incubated with and without A
b
and 10e at 37 C for 24 h, (a) normal PC12 cells before
treatment, (b) PC12 cells incubated with A
PC12 cells were incubated with compound 10e (1
were incubated with compound 10e (10 M).
b
(1e42) (5 ìM), (c) the A
b-induced
m
M) (d) the A -induced PC12 cells
b
m
0
0
DCFH. DCFH is oxidized to the fluorescent 2 ,7 -dichlorofluorescine
DCF) in the presence of ROS (Fig. 4). As shown in Fig. 4, the
PC12 cells display low levels of ROS while significantly increases of
ROS levels were seen in the PC12 cells incubated with 5 M of A
(
reduced cell viability when compared to control cells (Fig. 3a). The
obtained TEM results offered that 10e inhibits A
b
aggregation
m
b
0
0
0
Fig. 4. Inhibition of ROS formation by 10e, detected by the conversion of 4 ,6-diamidino-2-phenylindole (DAPI), deoxynucleotidyltransferase (TUNEL) and 2 ,7 -dichlorofluorescine
diacetate (DCFH-DA) assay. Using a fluorescence confocal microscope helped to take color images, (a) control cells showing low levels of fluorescence (b) PC12 cells treated with
5
mM A
b (1-42) for 48 h showing increase in ROS (ced) reduction of ROS in Ab (1e42)-induced toxicity treated with 1 mM and 10 mM of 10e. (For interpretation of the references to
color in this figure legend, the reader is referred to the Web version of this article.)

6
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
Table 4
Action of 10e on the ROS formation in A
b
(1e42)-induced toxicity cells.
10e (
m
M)
ROS
Apoptosis
DCF
Control
20 ± 1
60 ± 10
50 ± 3
35 ± 5
18 ± 2
51 ± 11
51 ± 11
38 ± 4
Ab
5
mM
1
m
M
10 mM
Fig. 7. Effect of compound 10e on antioxidant capacity, in PC12 cells incubated with
, with and without 10e.
Ab
reaction of MDA and TBA under high temperature. Malondialde-
ꢀ
hyde is measured in acidic media and heat (90e100 C) colori-
metrically at 532 nm. A
in MDA levels [21,53]. A significant increase in MDA levels was seen
in the PC12 cells incubated with A (1-42) (5 M) for 8 h compared
to the control cell. As shown in Fig. 5, there are a sharp increase in
the MDA concentration in A (1e42)-induced toxicity cells (51.9%).
The addition of compound 10e (1 M and 10 M) to A (1e42)-
b-induced damage could cause an increase
b
m
b
m
m
b
induced toxicity cells provided a notable decrease in MDA levels in
a concentration-dependent manner (40.4e46.2%).
Fig. 5. MDA formation in A
b
(1-42) (5
mM)-induced toxicity in PC12 cells co-incubated
with or without 10e (1 M and 10
m
mM).
2.8. Effect of 10e on lactate dehydrogenase (LDH)
Lactate dehydrogenase (LDH) is a rather stable enzyme and an
oxidoreductase that catalyses the interconversion of pyruvate and
lactate and it is used to evaluate the occurrences of damage and
toxicity of tissue and cells. One of the symptoms of damage and
toxicity of tissue and cells is the increased levels of LDH. In AD, the
enzymatic activity increases compared to normal cells [53,54]. The
enzyme activity of lactate dehydrogenase is significantly increased
in A
normal cells (Fig. 6), suggesting that impaired function is a result of
its oxidative modification by A (1-42). As shown in Figure 6, 1
and 10 M concentration of 10e provided a decrease in LDH levels
16.6% and 27.5%), respectively. The concentration-dependent
manner of this test confirms the effect of the compound 10e on
(1e42)-induction of toxicity.
b (1e42)-induced toxicity cells (73.6%) when compared to
b
mM
m
(
Ab
Fig. 6. LDH release in A
b
(1e42)-induced toxicity cells with and without 10e.
2.9. Effect of 10e on TAC
The studies suggested that different antioxidants have the po-
tential in delaying the progression of AD. Compounds with inhi-
bition capability suitable for oxidation activities that occur under
the influence of atmospheric oxygen or reactive oxygen species are
called antioxidants. Antioxidants defend healthy brain against
oxidative damages that include antioxidant enzymes such as su-
peroxide dismutase (SOD), catalase (CAT) and free radical scaven-
gers such as ascorbate, vitamin E. In AD, there is a decrease in the
antioxidant defense system so oxidative stress can act as a risk
factor for AD [55e57]. The antioxidant activity of compound 10e
was determined by the oxygen radical absorbance capacity assay
using fluorescein. As can be seen in Fig. 7, there is a sharp decrease
(
1-42) for 48 h. On the other hand, ROS formation decreases in the
PC12 cells incubated with the same concentration of A (1-42) in
the presence of compound 10e as a free radical scavenger. As shown
in Table 4, the ROS formation in A (1e42)-induced toxicity cells is
three times as much as that of PC12 normal cells and the results
showed that compound 10e at 1 M and 10 M was able to reduce
this effect in a concentration-dependent manner.
b
b
m
m
2
.7. Lipid peroxidation inhibitory effect of 10e
Malonyldialdehyde (MDA) is one of the main intermediates
made during lipid peroxidation. MDA assay in biological sample
supplies a simple, reproducible, and standardized tool for evalua-
tion of lipid peroxidation. The MDA-TBA adduct formed by the
in the antioxidant system in A
in the presence of compound 10e (1
the antioxidant activity was observed.
b
(1e42)-induced toxicity cells, while
m
M and 10 M) an increase in
m

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
7
Fig. 8. Effect of 10e on LPS-induced production of TNF-a and IL-1b by human peripheral blood monocytes.
2
.10. mRNA expression levels of relevant genes
and 10e were performed, to obtain more information about binding
interplay with hAChEI and hBuChE in their hydrolytic active site.
The results showed that the derivatives could be inhibited by
binding to the active site of both cholinesterases. Docking results
showed that compounds studied in the crystal structure of hAChE
donepezil complex showed a similar situation to donepezil (Fig. 9)
and compound 8 (Fig. 10) having good performance with accept-
able binding energy. According to the results of molecular docking
of compounds 10a (Fig. 11) and 10e (Fig. 12) in hAchE, the com-
pound of 10a showed a binding energy of ꢁ13.14 kcal/mol and the
compound of 10e showed a binding energy of ꢁ13.85 kcal/mol,
while the binding energy relating to donepezil and compound 8
was equal to ꢁ11.71 and ꢁ11.3 kcal/mol, respectively. That low
energy reflects a better interaction between the ligand and the
enzyme. The methoxy group of 1-indanone moiety in the
Studies show that there is an impairment in microglial function
in AD in the presence of A . The microglial disorder might be
sustained by cytokines TNF- and IL-1 which play an important
role in the pathogenesis of inflammatory and this factor which can
accelerate AD progress. Therefore, anti-inflammatory compounds
of the ability to suppress TNF-
gested as effective agents in the treatment of AD [58,59].
As shown in Fig. 8, mRNA level of IL-1 and TNF- in A
cell line increased compared to that of the cell lines which were
treated with 1 mol and 10 mol of 10e peptide. Also when
compared with control a decrease in the levels of these genes was
observed in the cell line that treated with 10 mol of 10e. The exact p
values of gene expression results were shown in Fig. 8 and Table 5.
Gene expression results indicate a direct effect of 10e on the
reduction of inflammatory genes expression in dose dependent
manner.
b
a
b
a and IL-1b simultaneously are sug-
b
a
b treated
m
m
m
2
2
.11. Molecular modeling
.11.1. Molecular docking of compounds 10a and 10e with hAchE
and hBuChE
Molecular docking studies on the most active compounds 10a
Table 5
The exact p values of gene expression results.
Row
p TNF-
a
p IL-1b
Control with 10
mM
0.0330
0.0151
0.0001
0.0449
0.0107
0.0002
Fig. 10. Molecular docking of compound 8 in hAChE active sites.
A
A
b
b
with 1
with 10
m
M
m
M
Fig. 9. Molecular docking of donepezil in hAChE active sites.
Fig. 11. Molecular docking of compounds 10a in hAChE active sites.

8
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
Fig. 14. Molecular docking of compounds 8 in hBuChE active sites.
Fig. 12. Molecular docking of compounds 10e in hAChE active sites.
Fig. 15. Molecular docking of compounds 10a in hBuChE active sites.
Fig. 13. Molecular docking of donepezil in hBuChE active sites.
compounds 10a, 10e and 8 establishes H-bond with SER293 while
this group in donepezil forms H-bond with PHE295. Also, according
to the results obtained, compound 8 as the parent drug in this work
was well fit in to the hydrophobic pocket composed of amino acids
LEU289, SER293, PHE297, TRP286, TYR341, PHE338, and TYR337.
Both compounds 10a and 10e show the same results. They also
were interacted with the same amino acids as done for compound
8
, other amino acids that these compounds fit them as follows:
amino acids GLU292, ARG298, TYR124 and PHE295 placing around
-indanone and phenyl ring of benzylidine group and Amino acids
1
Fig. 16. Molecular docking of compounds 10e in hBuChE active sites.
TRP86, TYR341, TYR337, HIS447 and GLY448 in the catalytic anionic
site (CAS) placing around piperazinium salt moiety in compounds
1
0a and 10e. The quaternary nitrogen of this piperazinium ring
fit in to the hydrophobic pocket composed of amino acids PRO401,
CYS400, GLU404, ASP395, THR523, TYR396, PHE526, PRO527,
VAL529, and VAL361. Amino acids VAL233, SER235, PRO230,
MET302, and ASN228 fit with 1-indanone and phenyl ring of
benzylidine group and amino acids TYR396, TRP522 and CYS400
placed around the positive charge of piperazinium salt moiety in
the compound 10a. Docking result of compound 10e shows that
amino acids VAL233, SER235, LEU236, MET302, and ASN228 fit
with 1-indanone and phenyl ring of benzylidine group and amino
acids TYR396, PRO527, THR523, CYS400, PRO401, and TRP522 took
place around the positive charge of piperazinium salt moiety in
compound 10e. The quaternary nitrogen of the piperazinium ring in
compounds 10a and 10e facilitates ligand recognition through
binding to the TYR396 and the phenyl ring of the benzyl group in
facilitates ligand recognition through binding to the catalytic
anionic site (CAS) comprising TYR337. The phenyl ring of the benzyl
group in these compounds has p-p interaction with HIS447 of CAS.
Presence of more interactions with amino acids, except for the
common ones, between the parent drug and compounds 10a and
10e, may be a reason for better interactions of these compounds
with the hAchE enzyme.
Docking results showed that compounds studied in the crystal
structure of hBuChE showed a different situation to donepezil
(Fig. 13) and compounds 8 (Fig. 14) having good performance with
acceptable binding energy. According to the results of molecular
docking of compounds 10a (Fig. 15) and 10e (Fig. 16) in hBuChE, the
compound of 10a showed a binding energy of ꢁ10.5 kcal/mol and
the compound of 10e showed a binding energy of ꢁ10.6 kcal/mol,
while the binding energy relating to donepezil and compounds 8
was equal to ꢁ9.53 and ꢁ9.27 kcal/mol, respectively. That low
energy indicates the better interaction between the ligand and the
hBuChE enzyme. 1-Indanone methoxy group of compound 10e
establishes H-bond with Leu 236 while the compound of 10a,
donepezil and compounds 8 do not show any hydrogen bonding.
Additionally, according to the results, parent compound 8 was well
these compounds has p-p interaction with TRP522. The difference
in amino acids that parent compound 8 interacted with compared
to the ones which interacted with compounds 10a and 10e could be
a reason for better interactions observed in hBuChE enzyme.
2.11.2. Molecular docking of compound 10e with A
b
Molecular docking studies on the most active form of compound

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
9
appropriate dryer. Column chromatography was carried out by
using Silica gel 60 (Merck, Germany). TLC plates were visualized
using on UV light at 254 nm. Nuclear Magnetic Resonance spectra
1
13
for H NMR (400 MHz) and C NMR (100 MHz) were recorded by
using Bruker FT-400 spectrometer. CDCl or DMSO‑d were used as
solvent and chemical shifts were reported in values (ppm) relative
to internal Me Si and J values were reported in Hz. S, singlet; d,
3
6
d
4
doublet; t, triplet; q, quarted; dd, doublet of doublet; br, broad; m,
mutiplet were used for peak multiplicities as abbreviations. FT-IR
spectra were recorded by the Bruker Tensor 27 instrument in/on
KBr plate. The melting points were measured with an electro-
thermal apparatus MEL-TEMP model 1202 with open capillaries.
Elemental analysis was performed on an Elemen-tar Vario EL III
instrument.
4.2. Synthesis of 4-(4-methylpiperazin-1-yl)benzaldehyde (3) [44]
Fig. 17. Molecular docking of 10e with Ab peptide active sites.
The mixture of 4-fluorobenzaldehyde (2) (3 g, 24.19 mmol), 1-
methylpiperazine (1) (4.83 g, 48.38 mmol), HDTMAB (3.49 g,
1
0e was performed to understand the binding mode and the
9
.6 mmol) and K
2
CO
3
(6.67 g, 48.38 mmol) in DMF (12 ml) was
interaction of the compound with A
that this compound binds with A
transversely fitted along the major helix. The results of molecular
docking for compound 10e (Fig. 17) in A , showed binding energy
of ꢁ6.34 kcal/mol. Compound 10e did not show any hydrogen
bonding. 1-Indanone and phenyl ring of compound 10a, showed
hydrophobic interactions with residues HIS13, LEU17, LYS16, and
PHE20 and the positive charge of piperazinium salt moiety in
compound 10e have interaction with residues ALA21, VAL24, LYS28,
ILE31, LEU34, and MET31.
b
(1-42). The results showed
ꢀ
stirred for 5.5 h at 100 C. The reaction mixture was diluted with
water and was extracted with dichloromethane (3 ꢂ 10 ml) and the
organic layer was washed with water and brine, dried over sodium
sulfate and the solvent was evaporated using rotary evaporator. The
obtained yellow oil was purified on silica gel column chromatog-
raphy with hexane/ethyl acetate (4:6) as eluent. The yellow powder
b
(1-42). Compound 10e is
b
(2.4 g, 11.76 mmol) of compound 3 was obtained as the desired
product (49% yield).
4.3. General procedure for the synthesis of compounds 6a-b [45]
3
. Conclusion
Compounds 6a-b were synthesized as previously described in
the literature [45]. Potassium carbonate (3.89 g, 28.2 mmol) was
added to a solution of 4-hydroxybenzaldehyde (4) (3.52 g,
In summary, several novel 2,3-dihydro-5,6-dimethoxyinden 2-
4-(4-alkyl-4-methyl-piperazinium-1-yl)benzylidene)-1H-inden-
-one halides containing the 1-indanone moiety of donepezil and
[
1
2
2
8.9 mmol) in 20 ml dry acetone and the mixture was refluxed for
4 h. In a two-necked flask equipped with a condenser and addi-
piperazine ring were designed, synthesized and tested for their
biological activities on AD. Most of the compounds exhibited a good
inhibitory effect on AChE and BuChE, among them compounds 10a
and 10e displayed more inhibition activity. The preliminary
structure-activity relationships, molecular modeling, and Kinetic
study gave us valuable insight to understand the interactions be-
tween the enzyme and its ligand. Other biological tests of 10e such
as ROS, LDH, MDA, and TAC confirmed that this compound has the
potent antioxidant and anti-inflammatory activities suggesting
compound 10e to be beneficial for AD treatment. Alzheimer’s is a
multifactorial disease and could benefited from the development of
multifunctional compounds for curing AD which act on different
pathways simultaneously. Findings in the present study could be
taken in to consideration as a way for the progress of drug devel-
opment in AD treatment. By being capable to be carried out more
in vitro and in vivo tests include, inhibition investigation on
Monoamine oxidase (MAO), beta-amyloid peptide aggregation and
tau protein aggregation and measurement of the blood-brain-
barrier permeability and using liposomal carriers on the
mentioned compounds here. Also a notable result compound 8 can
be potentially used in the designing other hybrid drugs for Alz-
heimer’s disease.
tional funnel was added dibromoalkane (98.6 mmol) and 20 ml
acetone. Then the mixture of 4-hydroxybenzaldehyde (4) and po-
tassium carbonate in acetone mixture was added drop-wise with
stirring to the flask by additional funnel. The resulting solution was
heated to reflux under stirring for 72 h. Acetone was evaporated
using rotary evaporator apparatus. The mixture was extracted with
dichloromethane (3 ꢂ 20 ml) and the combined organic layer was
2 4
washed with water and brine, dried over anhydrous Na SO and
then dichloromethane was evaporated using rotary evaporator. The
residue was purified on silica gel column chromatography with
hexane/ethyl acetate (8:2) as eluent.
4.3.1. 4-(2-Bromoethoxy)benzaldehyde (6a)
From 8.5 ml (98.6 mmol) of 1,2-dibromoethane as reactant;
.58 g (15.6 mmol) of white solid was obtained (55% yield, mp.
0e45 C).
3
4
ꢀ
4
3
4
.3.2. 4-(4-Bromobutoxy)benzaldehyde (6b)
From 11.67 ml (98.6 mmol) of 1,4-dibromobutane as reactant;
.50 g (13.62 mmol) of white paste was obtained (48% yield).
.4. General procedure for the synthesis of compounds 8 and 9a-b
[45,47]
4
. Experimental section
2,3-Dihydro-5,6-dimethoxy-1H-inden-1-one (7) (1 g, 5.2 mmol)
4
.1. Materials and instruments
and benzaldehyde derivatives 3 or 6a-b (5.2 mmol) were dissolved
in ethanol (30 ml). Aqueous NaOH solution (10% W/W, 10 ml) was
added to this solution dropwise with stirring. The reaction mixture
was stirred for 48 h at room temperature. The precipitate was
All materials purchased from Merck and Sigma-Aldrich and
used without any further purification. Solvents were dried with an

10
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
filtered and washed with aqueous ethanol solution (50% V/V) and
then the solid product was dried under vacuum and used without
further purification.
NeCH
2
),7.10 (2H, d, J ¼ 8.7 Hz, AreH), 7.18 (2H, d, J ¼ 11.9 Hz, AreH),
7.34 (1H, s, ¼CH), 7.52e7.55 (3H, m, AreH), 7.63 (4H, m, AreH)
1
3
ppm; C NMR (100 MHz, DMSO):
7.2, 104.6, 108.3, 115.3, 126.2, 127.4, 129.2, 130.4, 130.6, 131.6, 132.2,
132.8, 133.4, 145.0, 149.4, 150.1, 155.2, 192.2 ppm. Anal. Calcd. For
33ClN (505.06): C 71.34, H 6.59, N 5.55; Found: C 71.41, H
6.63, N 5.48%.
d 31.9, 41.2, 44.9, 55.8, 56.2, 58.4,
6
4.4.1. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-methylpiperazin-1-yl)
benzylidene]-1H-inden-1-one (8)
C
30
H
2 3
O
From 1.06 g of compound 3 as reactant, 1.81 g (4.8 mmol) of
ꢀ
yellow solid was obtained (92% yield); mp. 220e222 C; FT-IR
(
1
(
KBr):
312 (CeN), 1127 (CeO) cm
3H, s, CH
), 2.63 (4H, t, J ¼ 8 Hz, NeCH), 3.36 (4H, t, J ¼ 8 Hz,
NeCH), 3.87 (2H, s, CH ), 3.94 (3H, s, OeCH ), 3.98 (3H, s, OeCH ),
.87 (2H, d, J ¼ 8.7 Hz, AreH), 6.92 (1H, s, AreH), 7.27 (1H, s, AreH),
n 2925 (CeH), 2862 (CeH), 1684 (C]O), 1610e1510 (C]C),
ꢁ1
1
4.5.2. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(4-bromobenzyl)-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
3
; H NMR (400 MHz, CDCl ): d 2.39
3
(10b)
2
3
3
From 0.197 g of 4-bromobenzyl bromide as reactant, 0.14 g of
6
ꢀ
13
yellow solid was obtained (87% yield), mp. 210e215 C; FT-IR (KBr):
2963 (CeH), 2837 (CeH), 1683 (C]O), 1597e1507 (C]C), 1306
7
.47 (1H, s, ¼CH), 7.52 (2H, d, J ¼ 8.7 Hz, AreH) ppm; C NMR
100 MHz, CDCl ): 31.0, 55.1, 55.2, 103.9, 106.0, 109.4, 119.7, 122.0,
22.8, 129.5, 129.8, 130.9, 132.2, 134.1, 135.3, 135.6, 140.9, 143.1,
43.6, 148.6, 154.5,191.7 ppm. Anal. Calcd. For C23 (378.47):
C 72.99, H 6.92, N 7.40; Found: C 72.85, H 6.84, N 7.47%.
n
(
1
1
3
d
ꢁ
1 1
(
CeN), 1250 (CeO) cm ; H NMR (400 MHz, DMSO‑d
s, CH ), 3.47e3.54 (5H, m, NeCH ), 3.79 (3H, s, OeCH
), 3.90 (3H, br, CH , Ne CH ), 4.76 (2H, s,
),7.10 (2H, d, J ¼ 8.6 Hz, AreH), 7.17 (2H, d, J ¼ 11.0 Hz, AreH),
6
):
d 3.08 (3H,
3
2
3
), 3.87 (4H, s,
26 2 3
H N O
OeCH
NeCH
7
3
, NeCH
2
2
2
2
.33 (1H, s, ¼CH), 7.57 (2H, d, J ¼ 8.2 Hz, AreH), 7.64 (2H, d,
4
.4.2. 2,3-Dihydro-5,6-dimethoxy-2-[4-(2-bromoethoxy)
13
J ¼ 8.6 Hz, AreH), 7.73 (2H, d, J ¼ 8.2 Hz, AreH) ppm; C NMR
100 MHz, DMSO‑d ): 31.8, 41.1, 44.8, 55.8, 56.1, 58.4e66.2, 104.6,
108.2,115.3,124.5,126.2,126.7,130.4,131.5,132.1,132.2,132.7,135.4,
45.0, 149.3, 150.0,155.1, 192.2. Anal. Calcd. For: C30
(628.41); C 57.34, H 5.13, N 4.46; Found: C 57.23, H 5.09, N 4.42%.
benzylidene]-1H-inden-1-one (9a)
(
6
d
From 1.19 g of Compound 6a as reactant, 1.81 g (4.5 mmol) of
ꢀ
white solid was obtained (86% yield), mp.170e172 C; FT-IR (KBr):
3
n
1
2 2 3
H32Br N O
028 (CeH), 2828 (CeH), 1683 (C]O), 1597e1504 (C]C), 1254
ꢁ
1 1
(
CeO), 540 (CeBr) cm ; H NMR (400 MHz, CDCl
J ¼ 6.2 Hz, CH eBr), 3.90e3.96 (5H, m, OeCH and CH
OeCH ), 6.96e6.98 (3H, m, AreH),
), 4.34 (2H, t, J ¼ 6.2 Hz, OeCH
.33 (1H, s, ¼CH), 7.54 (1H, s, AreH), 7.60 (2H, d, J ¼ 8.7 Hz, AreH)
3
):
d
3.66 (2H, t,
2
3
2
), 3.99 (3H, s,
3
2
4
.5.3. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(3-bromobenzyl)-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
10c)
7
13
ppm; C NMR (100 MHz, CDCl
03.9, 106.0, 106.4, 113.9, 127.9, 130.1, 130.8, 131.2, 132.4, 143.6,
48.4, 154.1, 157.9, 192.1 ppm. Anal. Calcd. For C20 19BrO (403.27):
C 59.57, H 4.75; Found: C 59.43, H 4.70%.
3
): d 27.7, 31.0, 55.1, 55.2, 66.7, 103.0,
(
1
1
From 0.197 g of 3-bromobenzyl bromide as reactant, 0.15 g of
H
4
ꢀ
yellow solid was obtained (93% yield); mp. 209e213 C; FT-IR (KBr):
n
2987 (CeH), 2838 (CeH), 1671 (C]O), 1582e1474 (C]C), 1303
ꢁ
1 1
(
CeN), 1247 (CeO) cm ; H NMR (400 MHz, DMSO‑d
s, CH ), 3.51e3.56 (4H, m, NeCH ), 3.63e3.66 (2H, m, NeCH
3H, s, OeCH ), 3.89 (3H, s, OeCH ), 3.93 (3H, br, CH , NeCH
1H, br, NeCH ), 4.83 (2H, s, NeCH
.19 (2H, d, J ¼ 6.4 Hz, AreH), 7.36 (1H, s, ¼CH), 7.51 (1H, t,
J ¼ 7.8 Hz, AreH), 7.65e7.67 (3H, m, AreH), 7.77 (1H, d, J ¼ 8.0 Hz,
6
): d 3.13 (3H,
4
.4.3. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-bromobutoxy)
3
2
2
), 3.82
), 3.96
benzylidene]-1H-inden-1-one (9b)
(
(
7
3
3
2
2
From 1.34 g of compound 6b as reactant, 1.85 g (4.3 mmol) of
2
2
),7.12 (2H, d, J ¼ 8.8 Hz, AreH),
ꢀ
white solid was obtained (82% yield), mp.172e176 C; FT-IR (KBr):
2
n
937 (CeH), 2878 (CeH), 1682 (C]O), 1600e1501 (C]C),
304e1244 (CeO), 540(CeBr) cm 1; 1H NMR (400 MHz, CDCl
1.93e1.99 (2H, m, CH ), 2.04e2.11 (2H, m, CH ), 3.49 (2H, t,
J ¼ 6.4 Hz,CH eBr), 3.90 (2H, s, CH ), 3.93 (3H, s, OeCH ), 3.98 (3H,
s, OeCH ), 6.92e6.96 (3H, m, AreH),
), 4.04 (2H, t, J ¼ 6.0 Hz, OeCH
.31 (1H, s,¼CH), 7.53 (1H, s, AreH), 7.58 (2H, d, J ¼ 8.7 Hz, AreH)
ꢁ
):
13
1
d
3
AreH), 7.88 (1H, br, AreH) ppm; C NMR (100 MHz, DMSO‑d
31.6, 40.9, 44.5, 55.6, 55.9, 58.3, 65.8, 104.4, 108.0, 115.0, 122.0,
26.0, 129.9, 130.2, 131.0, 131.2, 132.0, 132.3, 132.5, 133.2, 135.5,
44.6, 149.2, 149.8, 154.9, 191.7 ppm. Anal. Calcd. For:
6
):
2
2
d
2
2
3
1
3
2
1
C
5
7
30 2 2 3
H32Br N O (628.41): C 57.34, H 5.13, N 4.46; Found: C 57.40, H
.16, N 4.42%.
13
ppm; C NMR (100 MHz, CDCl
5.9, 104.0, 106.1, 113.8, 127.3, 130.2, 131.1, 131.2, 132.1, 143.5, 148.5,
54.1, 158.8, 192.1 ppm. Anal. Calcd. For C22 23BrO (431.33): C
3
): d 26.7, 28.3, 31.1, 32.2, 55.1, 55.2,
6
1
H
4
6
1.26, H 5.37; Found: C 61.16, H 5.40%.
4.5.4. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(2-bromobenzyl)-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
(10d)
4.5. General procedure for the synthesis of compounds 10a-m
[
1,48]
From 0.197 g of 2-bromobenzyl bromide as reactant, 0.13 g of
yellow solid was obtained (81% yield); mp. 181e185 C; FT-IR (KBr):
ꢀ
The mixture of compound 8 (0.098 g, 0.26 mmol) and appro-
n
2987 (CeH), 2841 (CeH), 1675 (C]O), 1592e1503 (C]C), 1302
ꢁ1 1
priated alkyl halides (R-X) (0.79 mmol) in acetonitrile (5 ml) was
refluxed for 24 h. The precipitate was filtered and washed with
acetonitrile, and then the solid product was dried under vacuum.
(CeN), 1255 (CeO) cm ; H NMR (400 MHz, DMSO‑d
s, CH ), 3.46e3.52 (2H, m, NeCH ), 3.63e3.67 (2H, m, NeCH
3.72e3.78 (2H, m, NeCH ), 3.83 (3H, s,OeCH ), 3.90 (3H, s,OeCH
, NeCH ), 3.99 (1H, br, NeCH
6
): d 3.17 (3H,
3
2
2
),
),
2
3
3
3
.95 (3H, br, CH
2
2
2
), 4.88 (2H, s,
4
.5.1. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-benzyl-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one chloride
10a)
From 0.099 g of benzyl chloride as reactant, 0.11 g of orange solid
NeCH
2
),7.12 (2H, d, J ¼ 8.9 Hz, AreH), 7.21 (2H, d, J ¼ 3.4 Hz, AreH),
7.38 (1H, s, ¼CH), 7.51 (1H, td, J ¼ 7.6, 1.6 Hz, AreH), 7.58 (1H, td,
(
J ¼ 7.5, 1.2 Hz, AreH), 7.67 (2H, d, J ¼ 8.8 Hz, AreH), 7.77 (1H, dd,
1
3
J ¼ 7.6, 1.5 Hz, AreH), 7.85 (1H, dd, J ¼ 7.9, 1.2 Hz, AreH) ppm;
C
ꢀ
was obtained (84% yield), mp. 188e195 C; FT-IR (KBr):
n
2963
6
NMR (100 MHz, DMSO‑d ): d 31.6, 40.9, 44.3, 55.6, 55.9, 58.5, 66.8,
(
1
3
CeH), 2837 (CeH), 1680 (C]O), 1596e1504 (C]C), 1306 (CeN),
104.5, 108.0, 115.0, 126.0, 127.0, 127.18, 128.2, 130.2, 131.2, 131.9,
132.5, 132.6, 132.8, 134.1, 136.1, 144.6, 149.2, 149.8, 154.9, 191.7 ppm.
ꢁ1 1
251 (CeO) cm ; H NMR (400 MHz, DMSO):
d
3.08 (3H, s, CH
), 3.64 (2H, s, NeCH ), 3.79 (3H, s,
), 3.91 (3H, s, CH , NeCH ), 4.77 (2H, s,
3
),
.50e3.54 (5H, m, NeCH
), 3.87 (3H, s, OeCH
2
2
Anal. Calcd. For: C30
2 2 3
H32Br N O (628.41): C 57.34, H 5.13, N 4.46;
OeCH
3
3
2
2
Found: C 57.28, H 5.11, N 4.49%.

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
11
4
.5.5. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(3-chlorobenzyl)-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
10e)
From 0.16 g of 3-chlorobenzyl bromide as reactant, 0.13 g of
DMSO‑d
108.0, 114.9, 125.9, 130.2, 131.2, 131.9, 132.5, 144.6, 149.2, 149.9,
154.9, 191.7 ppm; Anal. Calcd. For C26 33BrN (501.465): C 62.27,
H 5.59, N 5.58; Found: C 62.34, H 6.62, N 5.59%.
6
): d 10.4, 14.6, 31.6, 40.8, 45.9, 55.6, 55.9, 58.5, 63.9, 104.4,
(
H
2 3
O
ꢀ
orange solid was obtained (86% yield); mp. 219e224 C; FT-IR
KBr): 2988 (CeH), 2944 (CeH), 1672 (C]O), 1581e1471(C]C),
303 (CeN),1250 (CeO) cm ; H NMR (400 MHz, DMSO‑d
3H, s, CH ), 3.49e3.54 (4H, m, NeCH ), 3.63e3.65 (2H, m, NeCH
.82 (3H, s, OeCH ), 3.89 (3H, s, OeCH ), 3.92e3.95 (4H, m, CH
), 4.82 (2H, s, NeCH
),7.12 (2H, d, J ¼ 8.8 Hz, AreH), 7.20 (2H,
(
1
(
3
n
4.5.9. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-butyl-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
(10i)
ꢁ
1 1
6
):
d
3.13
),
3
2
2
3
3
2
,
From 0.108 g of 1-Bromobutane as reactant, 0.12 g of yellow
ꢀ
NeCH
2
2
solid was obtained (90% yield); mp. 247e250 C; FT-IR (KBr):
n
2959
d, J ¼ 7.17 Hz, AreH), 7.37 (1H, s, ¼CH), 7.57e7.68 (5H, m, AreH),
(CeH), 2865 (CeH), 1675 (C]O), 1593e1504 (C]C), 1301 (CeN),
13
ꢁ1
1
7
4
1
1
.74 (1H, s, AreH) ppm; C NMR (100 MHz, DMSO‑d
4.5, 55.6, 55.9, 58.3, 65.9, 104.4, 108.0, 115.0, 126.0, 129.7, 130.2,
30.4, 130.8, 131.2, 131.9, 132.0, 132.5, 132.8, 133.4, 144.6, 149.2,
49.9, 154.9, 191.8; Anal. Calcd. For C30 32BrClN (583.95): C
6
):
d
31.7, 40.9,
1252 (CeO) cm
J ¼ 7.4 Hz, CH ), 1.32e1.38 (2H, m, CH
(3H, s, CH ), 3.45e3.49 (2H, m, NeCH
3.74 (2H, br, NeCH ), 3.83 (3H, s, OeCH
(2H, s, CH
), 7.12 (2H, d, J ¼ 8.8 Hz, AreH), 7.21 (2H, d, J ¼ 6.8 Hz,
AreH), 7.38 (1H, s, ¼CH), 7.67 (2H, d, J ¼ 8.8 Hz, AreH) ppm;
NMR (100 MHz, DMSO‑d ): 13.5, 19.2, 23.0, 31.7, 40.9, 45.8, 55.7,
56.0, 58.6, 62.6, 104.5, 108.2, 115.0, 126.0, 130.3, 131.3, 132.07, 132.1,
132.5, 144.7, 150.0, 155.0, 191.9 ppm; Anal. Calcd. For C27 35BrN
;
H NMR (400 MHz, DMSO‑d
), 1.66e1.74 (2H, m, CH
), 3.54e3.61 (5H, m, NeCH
), 3.90 (3H, s, OeCH ), 3.95
6
):
d
0.95 (3H, t,
), 3.14
),
3
2
2
3
2
2
H
2
O
3
2
3
3
61.71, H 5.52, N 4.80; Found: C 61.59, H 5.48, N 4.76%.
2
13
C
4.5.6. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(2-chlorobenzyl)-4-
6
d
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one chloride
10f)
From 0.127 g of 2-chlorobenzyl chloride as reactant, 0.13 g of
(
H
2 3
O
(515.49): C 62.91, H 6.84, N 5.43; Found: C 62.56, H 6.83, N 5.42%.
ꢀ
dark yellow solid was obtained (93% yield); mp. 164e169 C; FT-IR
KBr): 2969 (CeH), 2836 (CeH), 1678 (C]O), 1595e1505 (C]C),
306 (CeN), 1251(CeO) cm ; H NMR (400 MHz, DMSO‑d
3H, s, CH ), 3.63e3.66 (3H, m, NeCH ), 3.71e3.73 (2H, m, NeCH
.82 (3H, s,OeCH ), 3.89 (3H, s,OeCH ), 3.94 (3H, br, CH , NeCH
.97 (1H, br, NeCH ), 4.90 (2H, s, NeCH
(
1
(
3
3
n
4.5.10. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-pentyl-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
(10j)
ꢁ
1 1
6
): d 3.16
3
2
2
),
),
3
3
2
2
From 0.119 g of 1-Bromopentane as reactant, 0.11 g of yellow
ꢀ
2
2
),7.12 (2H, d, J ¼ 8.8 Hz,
solid was obtained (80% yield); mp. > 250 C; FT-IR (KBr):
n
2943
AreH), 7.20 (2H, d, J ¼ 7.9 Hz, AreH), 7.36 (1H, s, ¼CH), 7.53 (1H, td,
J ¼ 7.5, 1.2 Hz, AreH), 7.60 (1H, td, J ¼ 8.0, 1.5 Hz, AreH), 7.65e7.69
(CeH), 2859 (CeH), 1675 (C]O), 1589e1497 (C]C), 1299 (CeN),
ꢁ
1
1
1251(CeO) cm
J ¼ 7.2 Hz, CH ), 1.26e1.39 (4H, m, CH
(3H, s, CH ), 3.44e3.48 (2H, m, NeCH
3.74e3.75 (2H, m, NeCH ), 3.82 (3H, s, OeCH
OeCH ), 3.95 (2H, s, CH
), 7.12 (2H, d, J ¼ 8.8 Hz, AreH), 7.21 (2H, d,
J ¼ 6.0 Hz, AreH), 7.37 (1H, s, ¼CH), 7.67 (2H, d, J ¼ 8.8 Hz, AreH)
;
H NMR (400 MHz, DMSO‑d
), 1.69e1.76 (2H, m, CH
), 3.54e3.60 (6H, m, NeCH
), 3.89 (3H, s,
6
):
d
0.90 (3H, t,
), 3.14
),
13
(
(
1
3H, m, AreH), 7.79 (1H, dd, J ¼ 7.5, 1.2 Hz, AreH); C NMR
3
2
2
100 MHz, DMSO‑d
08.2, 115.1, 125.5, 127.8, 130.7, 131.4, 132.1, 132.7, 136.1, 136.2, 144.8,
50.0, 155.0, 192.0; Anal. Calcd. For C30 (539.50): C
6
):
d
31.7, 41.0, 44.3, 55.7, 56.0, 58.5, 64.6, 104.5,
3
2
2
2
3
1
H32Cl
2
N
2
O
3
3
2
6
6.79, H 5.98, N 5.19; Found: C 66.86, H 5.92, N 5.22%.
13
6
ppm; C NMR (100 MHz, DMSO‑d ): d 13.7, 20.6, 21.7, 27.9, 31.7,
4
4
.5.7. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-((pyridine-2-yl)methyl)-
-methylpiperazinium)-1-yl)benzylidine]-1H-inden-1-one chloride
40.9, 45.8, 55.7, 56.0, 58.5, 62.8, 104.5, 108.1, 115.0, 126.0, 130.3,
131.3, 132.0, 132.6, 132.9, 144.7, 149.3, 150.0, 155.0, 191.9 ppm; Anal.
(10g)
Calcd. For C28
2 3
H37BrN O (529.52): C 63.51, H 7.04, N 5.29; Found: C
From 0.1 g of 2-(chloromethyl)pyridine as reactant, 0.1 g of dark
yellow solid was obtained (77% yield); mp. 210e214 C; FT-IR (KBr):
2959 (CeH), 2840 (CeH), 1674 (C]O), 1595 (C]C), 1302 (CeN),
63.55, H 7.01, N 5.23%.
ꢀ
n
4.5.11. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-hexyl-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
(10k)
From 0.13 g of 1-Bromohexane as reactant, 0.11 g of yellow solid
was obtained (78% yield); mp. > 250 C; FT-IR (KBr):
ꢁ
1 1
1
3
253(CeO) cm ; H NMR (400 MHz, DMSO‑d
6
):
), 376e378 (2H, m, NeCH
), 3.85e3.87 (1H, m, NeCH ), 3.88(3H, s, OeCH
), 4.89 (2H, s, NeCH
),7.12 (2H, d, J ¼ 8.7 Hz, AreH), 7.19 (2H, d,
J ¼ 9.2 Hz, AreH), 7.36 (1H, s, ¼CH), 7.54e7.57 (1H, m, AreH), 7.65
d
3.18 (3H, s, CH
), 3.81 (3H, s,
), 3.93 (2H, s,
3
),
.63e3.66 (4H, m, NeCH
2
2
OeCH
CH
3
2
3
ꢀ
2
2
n
2927 (CeH),
2858 (CeH), 1674 (C]O), 1591e1497 (C]C), 1300 (CeN), 1248
ꢁ1 1
(
7
d
2H, d, J ¼ 8.7 Hz, AreH), 7.76 (1H, d, J ¼ 7.6 Hz, AreH),
(CeO) cm ; H NMR (400 MHz, DMSO‑d
CH ), 1.30 (6H, m, CH ), 1.71 (2H, m, CH ), 3.14 (3H, s, CH
m, NeCH ), 3.55e3.57 (6H, m, NeCH ), 3.74e3.76 (2H, m, NeCH
3.81 (3H, s, OeCH ), 3.88 (3H, s, OeCH ), 3.93 (2H, s, CH ), 7.11 (2H,
d, J ¼ 8.8 Hz, AreH), 7.19 (2H, d, J ¼ 12.3 Hz, AreH), 7.35 (1H,
6
):
d
0.86 (3H, t, J ¼ 6.6 Hz,
), 3.50 (2H,
),
13
.96e8.01(1H, m, AreH) ppm; C NMR (100 MHz, DMSO‑d
6
):
31.8, 41.1, 46.8, 55.8, 56.1, 59.2,66.0, 104.6, 108.3, 115.2, 125.1, 126.1,
28.3, 130.4, 131.5, 132.2, 132.7, 137.9, 144.9, 148.8, 149.4, 150.1,
50.2, 155.1, 192.2 ppm; Anal. Calcd. For C29 32ClN (506.05): C
8.83, H 6.37, N 8.30; Found: C 68.75, H 6.29, N 8.41%.
3
2
2
3
2
2
2
1
1
6
3
3
2
H
3 3
O
13
s, ¼CH), 7.65 (2H, d, J ¼ 8.8 Hz, AreH) ppm; C NMR (100 MHz,
DMSO‑d ): 13.8, 20.9, 21.9, 25.4, 30.7, 31.7, 40.9, 45.9, 55.7, 56.0,
58.6, 62.8, 104.5, 108.2, 115.0, 126.0, 130.3, 131.3, 132.0, 132.6, 144.7,
149.2, 150.0, 155.0, 191.9 ppm; Anal. Calcd. For C29 39BrN
6
d
4
.5.8. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-propyl-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
10h)
From 0.097 g of 1-Bromopropane as reactant, 0.1 g of yellow
H
2 3
O
(
(543.55) C: 64.08, H: 7.23, N: 5.15 Found: C: 63.99, H: 7.20, N: 5.18%.
ꢀ
solid was obtained (77% yield); mp. 247e251 C; FT-IR (KBr):
n
2953
4.5.12. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(2-propen-1-yl)-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one chloride
(10 l)
From 0.06 g of 3-Chloro-1-propene as reactant, 0.09 g of dark
yellow solid was obtained (76% yield); mp. 219e223 C; FT-IR (KBr):
(
1
CeH), 2837 (CeH), 1672 (C]O), 1587e1500 (C]C), 1304 (CeN),
ꢁ
1
1
236(CeO) cm
;
H NMR (400 MHz, DMSO‑d
), 1.70e1.77 (2H, m, CH ), 3.15 (3H, s, CH
), 3.54e3.61 (6H, m, NeCH
), 3.90 (3H, s, OeCH
6
):
d
0.93 (3H, t,
), 3.42e3.46
J ¼ 7.3 Hz, CH
3
2
3
ꢀ
(
2H, m, NeCH
2
2
), 3.71e3.77 (2H, m,
), 3.95 (2H, s, CH ),
NeCH
2
), 3.83 (3H, s, OeCH
3
3
2
n 2966 (CeH), 2853 (CeH), 1677 (C]O), 1590e1505 (C]C), 1304
ꢁ
1 1
7
.12 (2H, d, J ¼ 8.8 Hz, AreH), 7.21 (2H, d, J ¼ 6.5 Hz, AreH), 7.37 (1H,
(CeN), 1249 (CeO) cm ; H NMR (400 MHz, DMSO‑d ):
6
d 3.17 (3H,
13
s, ¼CH), 7.67 (2H, d, J ¼ 8.9 Hz, AreH) ppm; C NMR (100 MHz,
s, CH ), 3.58 (5H, br, NeCH ), 3.61e3.62 (1H, m, NeCH ), 3.76e3.79
3
2
2

12
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
(
2H, m, NeCH
s, CH
), 4.25 (2H, d, J ¼ 7.1 Hz, NeCH
.07e6.17 (1H, m, ¼CH), 7.11 (2H, d, J ¼ 8.8 Hz, AreH), 7.20 (2H, d,
J ¼ 7.2 Hz, AreH), 7.36(1H, s, ¼CH), 7.66 (2H, d, J ¼ 8.8 Hz, AreH)
2
), 3.82 (3H, s, OeCH
3
), 3.89 (3H, s, OeCH
3
), 3.93 (2H,
m, AreH), 7.37 (1H, s, ¼CH), 7.40 (1H, s, ¼CH
2
d
), 7.65e7.71 (4H, m,
18.1, 25.4, 31.5, 31.7,
13
2
2
), 5.65e5.73 (2H, m, ¼CH
2
),
AreH) ppm; C NMR (100 MHz, DMSO‑d
6
):
6
40.8, 46.2, 55.6, 56.0, 58.6, 61.9, 67.0, 67.4, 104.5, 108.0, 115.0, 115.10,
126.1,127.8, 128.6,130.1, 130.3,131.0, 131.2,132.0,132.3, 132.6,133.4,
144.6, 144.8, 149.3, 149.9, 155.14, 159.5, 191.8 ppm. Anal. Calcd. For
13
ppm; C NMR (100 MHz, DMSO‑d
8.1, 64.4, 104.4, 108.1, 115.0, 125.3, 125.9, 127.9, 130.3, 131.2, 132.0,
32.5132.87, 144.6, 149.2, 149.9, 154.9, 191.8 ppm; Anal. Calcd. For
6
): d 31.6, 40.7, 46.1, 55.6, 55.9,
5
1
C
45
2 7
H49BrN O (809.80): C 66.74, H 6.10, N 3.46; Found: C 66.79, H
6.05, N 3.41%.
C
26
H31ClN
2 3
O (454.00): C 68.63, H 6.87, N 6.16; Found: C 68.56, H
6
.83, N 6.10%.
4.7. Biochemical studies. Cholinesterase inhibitory activities
4
.5.13. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-(2-hydroxyethyl)-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one chloride
10m)
From 0.063 g of 2-Chloroethan-1-ol as reactant, 0.1 g of yellow
Ellman’s method [49] was used to measure the inhibitory ac-
tivities of newly synthesized compounds and obtained the value of
the 50% inhibitory concentration (IC50) for in vitro hAChE and
hBuChE inhibitory activity of these compounds. In this method,
human erythrocytes AChE and human plasmatic BuChE were pro-
(
ꢀ
solid was obtained (84% yield); mp. > 250 C; FT-IR (KBr):
n 2997
0
(
CeH), 2887 (CeH), 1677 (C]O), 1591e1506 (C]C), 1304 (CeN),
duced from fresh blood [60]. 5, 5 -dithiobis (2-nitrobenzoic acid)
ꢁ
1 1
1
(
(
254 (CeO),1122 (CeO) cm ; H NMR (400 MHz, DMSO‑d
3H, s, CH ), 3.65e3.75 (10H, m, NeCH ), 3.82 (3H, s, OeCH
3H, s, OeCH ), 3.93 (4H, br, CH , OeCH
), 5.69 (1H, t, J ¼ 4.8 Hz,
6
):
d
3.26
(Ellman’s reagent, DTNB), phosphate buffer (PB, pH 7.4), acetylth-
iocholine (ATC) and butylthiocholine (BTC) were purchased from
Sigma-Aldrich. Glass cuvettes were used for measuring purposes.
3
2
3
), 3.89
3
2
2
OH), 7.11 (2H, d, J ¼ 8.5 Hz, AreH), 7.20 (2H, d, J ¼ 6.5 Hz, AreH),
All tests were carried out in sodium phosphate buffer (0.1 M of
13
0
7
(
6
1
.36 (1H, s, ¼CH), 7.65 (2H, d, J ¼ 8.5 Hz, AreH) ppm; C NMR
100 MHz, DMSO‑d ): 29.9, 31.6, 40.8, 47.0, 54.5, 55.6, 55.9, 59.3,
4.0, 104.4, 108.0, 114.5, 114.9, 125.9, 130.3, 131.2, 131.5, 131.9, 132.4,
44.6, 149.2, 149.9, 154.9, 191.7 ppm; Anal. Calcd. For C25 31ClN
458.99): C 65.42, H 6.81, N 6.10; Found: C 65.50, H 6.77, N 6.16%.
Na
2
HPO
4
with pH ¼ 8), 5, 5 -dithiobis (2-nitrobenzoic acid) (DTNB,
6
d
Ellman’s reagent) (3.5 mM, in Na
acetylthiocholine iodide (7 mM, in Na
the substrates of the enzymatic reaction. The inhibitory activities of
synthesized derivatives were obtained in five different concentra-
tions, and to get repeatability, each experiment was carried out
2
HPO
4
buffer with pH ¼ 7) and
buffer with pH ¼ 8) as
2
HPO
4
H
2
O
4
(
4
.6. General procedure for the synthesis of compounds 10n-o [1,48]
twice. The assay medium (1 ml) consisted of 550
buffer (pH ¼ 8), 150 L of DTNB, 150 L of substrate and 150
M) in test cuvettes, and 700 L of phosphate
m
L of phosphate
m
m
mL of
ꢁ
4
ꢁ10
The mixture of compound 8 (0.098 g, 0.26 mmol) and com-
inhibitor (10 - 10
buffer (pH ¼ 8), 150
m
ꢁ
4
pound 9a-b (0.26 mmol) in acetonitrile (5 ml) was refluxed for 24 h.
The precipitate was filtered and washed with acetonitrile, and then
the solid product was dried under vacuum.
m
L of DTNB and 150
m
L of inhibitor (10
-
ꢁ10
ꢀ
10
M) in control cuvettes were incubated for 5 min in 37 C. The
reaction was initiated by the immediate addition of 50
mL of the
enzyme. The activity was determined by measuring the increase in
absorbance at 412 nm in 5 min intervals using a spectrophotometer
Helios-Zeta (Thermospectronic, Cambridge, U.K.). hBuChE study
was conducted in a similar situation described above. Non-linear
and linear regressions were used to estimate the drug concentra-
tions inducing 50% inhibition of the hAChE or hBuChE activity [45].
4
.6.1. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-((2,3-dihydro-5,6-
dimethoxy-1H-inden-1-one-2-(4-ethoxybenzylidine-2-yl))-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one bromide
(10n)
From 0.1 g of 2,3-dihydro-5,6-dimethoxy-2-[4-(2-bromoethoxy)
benzylidine]-1H-inden-1-one (9a) as reactant, 0.11 g of dark yellow
ꢀ
solid was obtained (55% yield); mp. 230- 235 C; FT-IR (KBr):
n
2901
4.8. Kinetic evaluation of compounds 10a and 10e on hAChE and
(
CeH), 1680 (C]O), 1595e1505 (C]C), 1304 (CeN), 1171(CeO)
hBuChE inhibitions
ꢁ
1 1
cm ; H NMR (400 MHz, DMSO‑d
br, NeCH ), 3.830e3.835 (8H, m, OeCH
OeCH ), 3.908 (3H, s, OeCH ), 3.96e3.97 (4H, m, CH
2H, m, NeCH ), 4.11e4.14 (2H, m, NeCH ), 4.63(2H, m, OeCH
.13e7.16 (4H, m, AreH), 7.20 (1H, s, AreH), 7.21 (3H, s, AreH), 7.38
6
):
d
3.65 (3H, br, CH
, CH ), 3.902 (3H, s,
, NeCH ), 4.0
),
3
), 3.73(4H,
2
3
2
The kinetic studies were performed at five different concen-
trations of the acetylthiocholine iodide (0.1e1 mM) and butyrilth-
iocholine iodide (0.1e1 mM) substrates for hAChE and hBuChE
enzymes, respectively. The spectrometric Ellman’s method was
used for measuring the velocity of enzymatic reaction for com-
pounds 10a and 10e [49]. In each case, two different concentrations
of tested compound 10a were used to investigate the mechanism of
3
3
2
2
(
7
(
2
2
2
13
1H, s, ¼CH), 7.42 (1H, s, ¼CH), 7.66e7.71 (4H, m, AreH) ppm;
C
6
NMR (100 MHz, DMSO‑d ): d 29.9, 31.6, 40.8, 46.9, 55.6, 56.0, 58.6,
6
1
1
1.9, 67.0, 67.5, 104.6, 108.2, 115.1, 115.4, 126.1, 127.7, 128.6, 130.1,
30.3, 131.2, 132.0, 132.4, 132.6, 133.4, 144.6, 144.8, 149.2, 149.9,
55.1, 159.5, 192.2 ppm; Anal. Calcd. For C43 45BrN (781.74): C
6.07, H 5.80, N 3.58; Found: C 66.13, H 5.74, N 3.62%.
ꢁ
7
ꢁ6
ꢁ7
hAChE (5ꢂ10
and 5ꢂ10
mM) inhibitions. Another two different concentrations of
compound 10e were used for the kinetic evaluation of hAChE
mM) and hBuChE (5ꢂ10
and
ꢁ
10
6
H
2
O
7
6
ꢁ7
ꢁ6
ꢁ7
ꢁ7
(
5ꢂ10 and 10 mM) and hBuChE (510 and 710 mM) in-
4
.6.2. 2,3-Dihydro-5,6-dimethoxy-2-[4-(4-((2,3-dihydro-5,6-
hibitions. To obtain inhibition constants; i.e. Ki and KI values for
AChE and BuChE, reciprocal plots of 1/V vs. 1/[S] and 1/Vapp vs.
inhibitor concentration were drawn respectively (Line-
weavereBurk plot) [61]. Data analysis was performed with Micro-
soft Excel software.
dimethoxy-1H-inden-1-one-2-(4-butoxybenzylidine-4-yl))-4-
methylpiperazinium-1-yl)benzylidine]-1H-inden-1-one (10o)
From
0.11
g
of
2,3-dihydro-5,6-dimethoxy-2-[4-(4-
bromobutoxy)benzylidine]-1H-inden-1-one (9b) as reactant, 0.1 g
of yellow solid was obtained (48% yield); mp. 222e218 C; FT-IR
ꢀ
(
1
KBr):
303 (CeN), 1248 (CeO), 1125 (CeO) cm
DMSO‑d ): 1.81e1.82 (2H, m, CH ), 1.93 (2H, br, CH
CH ), 3.60 (8H, br, NeCH ), 3.71 (2H, br, NeCH ), 3.81 (3H, s,
OeCH ), 3.83 (3H, s, OeCH ), 3.89e3.93 (10H, m, OeCH , NeCH ),
.13e4.15 (2H, m, OeCH ), 7.06e7.12 (4H, m, AreH), 7.18e7.19 (4H,
n
2929 (CeH), 2830 (CeH), 1678 (C]O), 1592e1501 (C]C),
4.9. Evaluation of cytotoxicity in vitro
ꢁ
1
1
;
H NMR (400 MHz,
), 3.17 (3H, s,
6
d
2
2
The cytotoxicity of the test compounds was determined in human
foreskin fibroblast(HFF) cells, MCF-7cells andneuronalSH-SY5Ycells
using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) assay. MTT is reduced to formazan by the
3
2
2
3
3
3
2
4
2

S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
13
mitochondrial activity of viable cells. HFF, MCF-7 and SH-SY5Y cells
from the Pasteur Institute of Iran were purchased and cultured. The
cells were seeded at a concentration of 10 cells/well, into a 96-well
10e (1 mM and 10 mM) after 8 h of incubation. Using a thiobarbituric
acid reactive substance (TBARS) assay kit was measured intracel-
4
6
lular MDA production. 1- 10 cells per well were seeded in a six-
plate, in each well. After 24 h of incubation, in each well distributed
well plate, then collected in 200 ml of culture medium and soni-
cated for 3ꢂ5 s intervals at 40 V over ice. SDS Lysis solution (100 ml)
was added to the sample solution and mixed thoroughly. Then,
2
(
00
7.8e1000
the treatment, 10
incubated again for an additional 4 h period. Then, the medium was
carefullyremovedand200 LofDMSOwasadded forsolubilizationof
m
L
of test compounds at different final concentrations
ꢀ
m
M) and incubated at 37 C in 5% CO
2
for 48 h. At the end of
ꢀ
mL of MTT (2 mg/ml) was added and the cells were
250 ml of TBA reagent were added and incubated at 95 C for
45e60 min. After loading into a clear 96-well plate and the
absorbance at 535 nm was recorded using a microplate reader
(Thermoscintific- Finland Multiscan spectrophotometer (GO MUL-
TISCAN) [53,64].
m
formazan crystals in each well. The amount of formazan was
measured at 570 nm using an awareness technology (Florida, USA) at
570 nmwith a referencewavelength of 630 nm. In all determinations,
cytotoxicity is expressed as the concentration of the compound
resulting in 50% inhibition of cell viability.
4.13. Determination of lactate dehydrogenase (LDH)
LDH release is an indicator of neuronal injury and obtained ac-
4
.10. Ab toxicity
cording to the percentage leakage of LDH into the culture medium
to the total LDH activity per culture. Lactate dehydrogenase (LDH)
levels were measured in the culture medium after induction of
The experiment was done on 7-day-old cultures of PC12. PC12
cells were grown up in Dulbecco’s modified Eagle’s medium
DMEM) with 7% fetal bovine serum, 7% horse serum, 100 mg/ml
streptomycin, and 100 units/ml penicillin [62]. Then the cells were
incubated with 5 M A (1e42) ((rat/mouse) ab120959), in DMEM
cytoxicity with A
b with and without 10e. Lactate dehydrogenase
(
(
LDH) activity is according to the below reaction:
m
b
LDH
þ
Piruvate þ NADH ƒ! Lactate þ NAD
without serum. The plates were transferred to a 5% CO
and incubated at 37 C for 24 h. Then compound 10e with con-
centration of 1
incubated for 24 h [63].
2
incubator
ꢀ
that results from the oxidation of NADH and it is measured as the
rate of decrease in the absorbance at 340 nm, using a Thermo-
scintific- Finland Multiscan spectrophotometer (GO MULTISCAN)
mM and 10 mM was added to the incubation and
[
53,65].
4
4
.11. Measurement of the ROS production
.11.1. DCFH-DA assay
4
.14. TAC assay
0
0
The level of ROS was quantified using the 2 ,7 -
dichlorofluorescein-diacetate (DCFH-DA). In DCFH-DA assay, the
cells of each group were incubated with 10 M of DCFH-DA for
5 min at room temperature to allow the DCFH-DA to be incorpo-
From the solution consists of 10 mM phosphate-buffered saline
pH 7.2), 50
final concentration: 300
(
(
m
l of myoglobin solution, 20
ml of 3 mM ABTS solution
m
m
M), and 20 l of diluted plasma or Trolox
m
1
solution, 90
mL removed and added to 8 wells of 96-well microplate,
rated into any membrane-bound vesicles and also to let the diac-
etate group be cleaved by esterases. Then the nuclei of cells were
counterstained with DAPI and observed under a fluorescent mi-
croscope with excitation at 358 nm/emission at 461 nm and the
converted DCF product was measured using the multiple readers
with a fluorescence spectrophotometer, excitation at 485 nm and
emission at 530 nm. The fluorescence was measured using a fluo-
rescence spectrometer Thermoscintific- Finland Multiscan spec-
trophotometer (GO MULTISCAN).
ꢀ
mixed by vibration, and maintained at 25 C for 3 min. After the
addition of Ab, the reaction started at 450 nm with the microplate
reader (Thermoscintific- Finland Multiscan spectrophotometer (GO
MULTISCAN)) for 5 min (25 C) [66].
ꢀ
4
4
.15. Anti-inflammatory assay
.15.1. Gene expression analysis
mRNA expressions of the IL-1b and TNF-a genes were measured
by real-time PCR using the SYBR Green method. RNX-PLUS solution
was used to isolate total RNA (SinaClon, Tehran, Iran), and com-
plementary DNA (cDNA) was synthesized using a Revert Aid
Reverse Transcriptase Kit (Thermo Fisher, Waltham, MA, USA). Beta
actin was considered as an endogenous control for mRNA. The
4
.11.2. Tunnel assay
Several apoptotic cells were detected by Tunnel assay. In TUNEL
assay cells were fixed in 4% paraformaldehyde in PBS and pH 7.4 for
h at room temperature, slides were washed in PBS and incubated
with blocking solution (methanol 3% in H ) for 10 min at room
1
2 2
O
2 DDCT method was applied to calculate gene expressions relative
ꢁ
temperature. Then again washed with PBS and incubated with 0.1%
Triton X-100 in 0.1% sodium Citra for 2 min in ice. Slides were washed
to the housekeeping control of Beta actin.
twice with PBS. In a wet pan, 50
m
L of the reaction mixture was added
ꢀ
4.15.2. Statistical analysis
to each sample and then incubate at 37 C for 60 min. Slides were
washed three times with PBS. Then the nuclei of cells were stained
with DAPI and observed under a fluorescent microscope with exci-
tation at 500e550 nm/emission at 565-515 nm and excitation at 358
nm/emission at 461 nm for fluorcein fluorescent and DAPI, respec-
tively. Using a fluorescence spectrometer Thermoscintific- Finland
Multiscan spectrophotometer (GO MULTISCAN).
Statistical analysis was done by SPSS software (version 23.0;
SPSS Inc). Data analysis of four studied groups was carried out by
repeated measure One-Way ANOVA test. For graphs illustration,
version 7.00 of GraphPad Prism software (GraphPad Software, La
Jolla, CA, USA, www.graph pad.com) was used. p < 0.05 was
considered to be statistically significant.
4.12. Malonyl dialdehyde (MDA) analysis
4.16. Molecular docking studies
MDA is produced by a breakdown of the oxidative of cell
Human AchE (pdb 4Ey7), human BuchE (pdb 1p0p) and Ab (1-
membrane lipids and it is an index of lipid peroxidation. We
measured lipid peroxidation induced by A (5 M) with or without
42) (pdb 1iYT) were obtained from the Protein Data Bank
[22,67e71]. MGL tools 1.5.6 with AutoGrid4 and AutoDock4 were
b
m

14
S. Mozaffarnia et al. / European Journal of Medicinal Chemistry 191 (2020) 112140
used to set up and perform docking simulation [72,73]. Using
AutoDock Tools, target and ligand files were provided. Water
molecules were removed first of all. To perform docking calcula-
tions, Lamarckian genetic algorithms were employed as they are
implemented in AutoDock. The lowest energy docked conforma-
tion obtained from the AutoDock function was selected as the
binding mode. The relevant output resulting from AutoDock was
illustrated by Discovery studio 4.5.
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Declaration of competing interest
[
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
[
15] L. Jalili-Baleh, E. Babaei, S. Abdpour, S.N.A. Bukhari, A. Foroumadi, A. Ramazani,
M. Sharifzadeh, M. Abdollahi, M. Khoobi, A review on flavonoid-based scaf-
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Acknowledgements
Med.
Chem.
152
(2018)
570e589,
https://doi.org/10.1016/
j.ejmech.2018.05.004.
This project was financially supported by a grant from Iran Na-
tional Science Foundation (INSF) (ID No. 97015588). The authors
would like to acknowledge the financial support from University of
Tabriz, Tabriz University of medical sciences and the Deputy for
Research and Technology of Iranian Ministry of Health and Medical
Education.
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