Design, synthesis and biological activity of novel tacrine-isatin Schiff base hybrid derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103006

A series of novel tacrine-isatin Schiff base hybrid derivatives (7a-p)were designed, synthesized and evaluated as multi-target candidates against Alzheimer's disease (AD). The biological assays indicated that most of these compounds displayed potent inhibitory activity toward acetylcholinesterase (AChE)and butyrylcholinesterase (BuChE)and specific selectivity for AChE over BuChE. It was also found that they act as excellent metal chelators. The compounds 7k and 7m were found to be good inhibitors of AChE-induced amyloid-beta (Aβ)aggregation. Most of the compounds inhibited AChE with the IC₅₀ values, ranging from 0.42 nM to 79.66 nM. Amongst them, 7k, 7m and 7p, all with a 6 carbon linker between tacrine and isatin Schiff base exhibited the strongest inhibitory activity against AChE with IC₅₀ values of 0.42 nM, 0.62 nM and 0.95 nM, respectively. They were 92-, 62- and 41-fold more active than tacrine (IC₅₀ = 38.72 nM)toward AChE. Most of the compounds also showed a potent BuChE inhibition among which 7d with an IC₅₀ value of 0.11 nM for BuChE is the most potent one (56-fold more potent than that of tacrine (IC₅₀ = 6.21 nM)). In addition, most compounds exhibited the highest metal chelating property. Kinetic and molecular modeling studies revealed that 7k is a mixed-type inhibitor, capable of binding to catalytic and peripheral site of AChE. Our findings make this hybrid scaffold an excellent candidate to modify current drugs in treating Alzheimer's disease (AD).

Full text of DOI:10.1016/j.bioorg.2019.103006

Accepted Manuscript
Design, synthesis and biological activity of novel tacrine-isatin Schiff base hy-
brid derivatives
E. Riazimontazer, H. Sadeghpour, H. Nadri, A.H. Sakhteman, T. Tüylü Kü ç
ükkılınç, R. Miri, N. Edraki
PII:
S0045-2068(18)31416-0
https://doi.org/10.1016/j.bioorg.2019.103006
103006
DOI:
Article Number:
Reference:
YBIOO 103006
Bioorganic Chemistry
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Received Date:
Revised Date:
Accepted Date:
8 December 2018
10 March 2019
19 May 2019
Please cite this article as: E. Riazimontazer, H. Sadeghpour, H. Nadri, A.H. Sakhteman, T. Tüylü Kü ç ükkılınç, R.
Miri, N. Edraki, Design, synthesis and biological activity of novel tacrine-isatin Schiff base hybrid derivatives,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103006
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Design, synthesis and biological activity of novel tacrine-isatin Schiff base
hybrid derivatives
E. Riazimontazera, H. Sadeghpoura,*, H. Nadrib, A.H. Sakhtemana,**, T. Tüylü Küçükkılınçc, R.
Miria,d, N. Edrakid
a Department of Medicinal Chemistry, Faculty of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran.
b
Department of Medicinal Chemistry, Faculty of Pharmacy and pharmaceutical sciences research center, Shahid Sadoughi
University of Medical Sciences, Yazd, Iran.
c Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, Sihhiye-Ankara, Turkey
d Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.
*Corresponding author. Tel.: +98 713 2424127; Fax: +98 713 2424126
E-mail: sadeghpurh@sums.ac.ir
**Co-corresponding author. Tel.: +98 713 2424127; Fax: +98 713 2424126
E-mail: asakhteman@sums.ac.ir
Abstract
A series of novel tacrine-isatin Schiff base hybrid derivatives (7a-p) were designed,
synthesized and evaluated as multi-target candidates against Alzheimer’s disease (AD). The
biological assays indicated that most of these compounds displayed potent inhibitory activity
toward acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and specific selectivity
for AChE over BuChE. It was also found that they act as excellent metal chelators. The
compounds 7k and 7m were found to be good inhibitors of AChE-induced amyloid-beta (Aβ)
aggregation. Most of the compounds inhibited AChE with the IC50 values, ranging from 0.42 nM
to 79.66 nM. Amongst them, 7k, 7m and 7p, all with a 6 carbon linker between tacrine and isatin
Schiff base exhibited the strongest inhibitory activity against AChE with IC50 values of 0.42 nM,
0.62 nM and 0.95 nM, respectively. They were 92-, 62- and 41-fold more active than tacrine
(IC50 = 38.72 nM) toward AChE. Most of the compounds also showed a potent BuChE inhibition
among which 7d with an IC50 value of 0.11 nM for BuChE is the most potent one (56-fold more
potent than that of tacrine (IC50 = 6.21 nM)). In addition, most compounds exhibited the highest
metal chelating property. Kinetic and molecular modeling studies revealed that 7k is a mixed-
type inhibitor, capable of binding to catalytic and peripheral site of AChE. Our findings make
this hybrid scaffold an excellent candidate to modify current drugs in treating Alzheimer’s
disease (AD).
Keywords: Alzheimer’s disease, Acetylcholinesterase, Butyrylcholinesterase, Cholinesterase
inhibitors, Tacrine, Isatin Schiff base, Amyloid-beta aggregation, Metal chelation.
1

1. Introduction
Alzheimer’s disease (AD) is a progressive and fatal degenerative disorder in the central
nervous system leading to the most common form of dementia in aged population [1]. Patients
suffer from AD have symptoms such as memory loss and other cognitive disabilities. The slowly
developing symptoms are serious enough to interfere with daily tasks that eventually lead to
mortality. At the moment, about 40 million people worldwide are affected by AD and by 2050 it
will affect 150 million people [2]. Constant care and related services cost billions of dollars
annually, including medical care, and human resources. Today, AD is considered as one of the
most expensive diseases and the expenses is expected to increase by fivefold in the year 2050 [3,
4]. Although some cases of AD were observed before the age of 65, old age is one major known
risk factor for this disease [5]. Since the current AD treatments are not curable, finding better
treatment strategies has become a worldwide endeavor. Most studies focused on finding
remedies, to retard its onset and stop it from progression [6, 7].
Autopsy studies and clinical diagnoses have confirmed major pathological hallmarks for
AD in the basal forebrain, cortex and amygdale [8]. Intracellular formation of neurofibrillary
tangles (NFTs) and neuropil threads (NTs) resulting from abnormal hyperphosphorylation of tau
protein is of a great importance [9]. In addition, the aggregation and extracellular deposition of
amyloid beta (Aβ) protein is the cause of plaque formation [10]. Recently, these small oligomers
of β-amyloid and tau protein have gained considerable attention. It is associated with the
hydrolysis of the amyloid precursor protein (APP) by β-secretase 1 (BACE-1) [11]. They were
found to be effective in activating apoptotic cascade leading to cell death [12]. Other factor
involved in AD is reduced cholinergic transmission and acetylcholine (ACh) levels which makes
it a prominent strategy for developing cholinesterase inhibitors (ChEIs). The aim of
cholinesterase inhibitors is to increase the levels of acetylcholine through inhibition of
cholinesterases (ChEs) [13]. Acetylcholinesterase (AChE) is also accountable for several non-
catalytic pro-aggregation activity [14]. Peripheral anionic site (PAS) amino acid residues in
AChE can interact with Aβ protein to produce AChE-Aβ complex, found to be entangled in Aβ
aggregates [15]. Aβ as a metalloprotein has selective high- and low-affinity metal binding sites
[16]. High level of iron, copper and zinc in cerebral β-amyloid deposits can also speed up Aβ
aggregation [17]. Metal chelators and ROS scavengers have potential therapeutic capacity in
treating AD due to increased level of metal ions in plaques [18]. Based on distinctive structural
and biochemical properties of AChE, molecules with specific interaction with catalytic active
site (CAS) and peripheral anionic site (PAS) residues can be important in AChE inhibition.
These agents are also helpful in preventing Aβ aggregation facilitated by AChE [19, 20]. The
hydrolyzing effect and therefore termination of ACh activity in case of both cholinesterase
enzymes (AChE and BuChE) plays an important role in different regions of the brain. In AD, a
decrease in AChE turnover by up to 85% is observed in particular parts of the brain. In contrast,
Butyrylcholinesterase (BuChE) levels are amplified during the development of AD [21]. Indeed,
the increase ratio of BuChE to AChE in AD brain can improve the supportive role of BuChE in
2

hydrolyzing excess of ACh. In addition, formation of hallmarks such as amyloid rich neuritic
plaques and neurofibrillary tangles were discovered by increasing levels of BuChE [22].
The first and the only class of drugs in the market, for treating AD are cholinesterase
inhibitors, such as tacrine, donepezil, rivastigmine, galantamine and memantine [23-25]. Thus, as
an efficient therapy, incorporation of a known AChE inhibitor, which is linked with another
moiety to create bi- or multifunctional hybrid exerts beneficial properties in the treatment of AD
[26]. The AChE and BuChE moieties of the hybrid structures are constructive in the interaction
of molecules with PAS as well as CAS [27]. Tacrine (1,2,3,4-tetrahydroacridin-9-amine) is a
reversible inhibitor of cholinesterase and the first FDA approved drug for AD. Although, its side
effects such as serious hepatotoxicity has limited its usage in therapeutic application; hence,
modifying this compound is still of great interest [28]. With respect to designing hybrid
structures, some multi-target compounds were designed by connecting tacrine with another
fragment through a proper linker in order to develop potent ChEIs with additional activities [29].
For example, tacrine-rhein hybrids [30], tacrine-coumarin hybrids [31], donepezil-tacrine hybrids
[32] and tacrine-melatonin hybrids [33] are reported as multifunctional agents.
In this work, isatin (1H-indole-2,3-dione) was selected as a molecular fragment to design
a novel tacrine-isatin Schiff base hybrid with multi-target characteristic. It is an endogenous low
molecular weight non-peptide compound which is used for the synthesis of large variety of
heterocyclic compounds and has many interesting biological activities [34, 35]. It is found in
mammalian tissues and body fluids and its modulator function has been the subject of several
studies [36, 37]. The studies suggested that isatin increased ACh levels by inhibiting AChE
activity. Some derivatives such as isatin Mannich bases were considered to develop new
cholinesterase and carbonic anhydrase (CA) inhibitors [38]. Isatin as an endogenous inhibitor of
monoamine oxidase (MAO), especially MAO-B, may play a role in the regulation of the brain
levels of acetylcholine and dopamine, which are reduced in age dependent neurodegenerative
diseases such as AD and parkinson [39-41]. The neuroprotective property of isatin is also mainly
related to the inhibition of monoamine oxidase. Isatin as a bioregulator has a selective effect on
certain protein-protein interactions (PPI) [42, 43]. It may influence interaction between amyloid-
beta and its crucial intracellular targets and this opens new possibilities in pharmacological
prevention of amyloid-beta toxicity [44, 45]. The scavenging and antioxidant activity of isatin
derivatives were also reported [46, 47]. Amongst all activity profiles, the inhibition of enzymes
(cholinesterase, carbonic anhydrase, MAO-B), the effect on protein aggregations,
neuroprotective properties and antioxidant activity can make isatin a good candidate to design
multifunctional molecules against neurodegenerative diseases.
The other approach to design tacrine-isatin Schiff base hybrids was based on structural
modification of donepezil by using indole moiety as a bioisosteric substitute of the indanone
moiety of donepezil which is available in isatin structure [48-50]. Some isatin bioisosteric
structures have been reported as dual inhibitors of AChE and BACE-1 as well [51, 52].
Structures of several previously reported compounds related to donepezil and isatin structure
with their activity profiles have been shown in scheme 1.
3

Scheme 1. Structures of some anti-Alzheimer compounds related to donepezil and isatin structures with their
biological properties.
As the main purpose of the present study, a selection of novel tacrine-isatin Schiff base
hybrid derivatives was consequently synthesized and their structures were clarified. The
synthesized compounds were evaluated for their in vitro activity as inhibitors of AChE, BuChE,
and amyloid-beta anti-aggregation activity, with metal chelation properties.
4

2. Result and discussion
2.1. Chemistry
The tacrine-Schiff base hybrids 7a-p were synthesized by the following route depicted in
(scheme 2). At the beginning of this synthetic strategy, tacrine 2 was prepared according to the
well-known Friedländer condensation reaction between anthranilonitrile and cyclohexanone. The
reaction was promoted by a lewis acid, aluminium chloride, as a catalyst [53]. The subsequent
step was done by the reaction of tacrine with dibromoalkanes in the presence of sodium hydride
in a dry condition to provide the intermediates 3a and 3b. The optimal linker length in terms of
the possibility of reaction and eligible yield was n = 5, 6. Afterwards, isatin reacted with
different aniline derivatives, substituted by electron withdrawing and electron donating groups in
para, metha and ortho positions, to produce isatin-Schiff base derivatives as another key
intermediates 6a-h. Isatin-Schiff base compounds were obtained by condensation reaction under
reflux in ethanolic solution in the presence of catalytic amount of acetic acid for 5-6 h in good
yields [54]. Finally, 3a or 3b was reacted with the mentioned isatin-Schiff base derivatives 6a-h
to form a desired compounds 7a-p in good yields. Nucleophilic reactions in the presence of
potassium carbonate at room temperature and dry DMF afforded desired target compounds 7a-p
in good yields.
5

NH2
HN
N
Br
n
3a n=5
3b n=6
CN
a
b
NH2
N
1
2
R2
R1
R3
R2
N
O
R1
H2N
R3
c
O
+
O
N
H
N
H
6a R1=H R2=H R3=H
6f R1=H R2=H R3=Cl
6g R1=H R2=H R3=Me
6h R1=H R2=H R3=OMe
6b R1=Cl R2=H R3=H
6c R1=H R2=Cl R3=H
6d R1=H R2=Me R3=H
6e R1=H R2=NO2 R3=H
4
5
R2
R1
R2
R3
R1
R3
N
HN
Br
N
n
O
N
d
+
O
N
N
HN
H
n
3a, 3b
6a-i
N
7a n=5 R1=H R2=H R3=H
7b n=5 R1=Cl R2=H R3=H
7c n=5 R1=H R2=Cl R3=H
7d n=5 R1=H R2=Me R3=H
7e n=5 R1=H R2=NO2 R3=H
7f n=5 R1=H R2=H R3=Cl
7g n=5 R1=H R2=H R3=Me
7h n=5 R1=H R2=H R3=OMe
7i n=6 R1=H R2=H R3=H
7j n=6 R1=Cl R2=H R3=H
7k n=6 R1=H R2=Cl R3=H
7l n=6 R1=H R2=Me R3=H
7m n=6 R1=H R2=NO2 R3=H
7n n=6 R1=H R2=H R3=Cl
7o n=6 R1=H R2=H R3=Me
7p n=6 R1=H R2=H R3=OMe
Scheme 2. Synthesis of tacrine-isatin Schiff base hybrids 7a-p. Reagents and conditions: (a) i) cyclohexanone,
toluene, AlCl3, reflux, 24h; ii) NaOH, reflux, 24h; (b) Br(CH2)nBr (n=5,6), NaH, dry DMF, r.t., 5h (c) acetic acid,
ethanol, reflux (d) anhydrous K2CO3, dry DMF, r.t., 24h.
2.2. Structure-Activity relationship of the compounds 7a-p
Based on the distance between CAS and PAS of the AChE, different structures of
tacrine-isatin Schiff base with 5 and 6 carbon linkers were synthesized. The aim was to achieve
the optimal linker length for the activity of this series of compounds. Another perspective of this
study was to investigate the role of electron donating and electron withdrawing groups at
different positions of isatin. For this purpose, different groups including -Me, -OMe, -NO2, -Cl,
6

were introduced to 2-, 3- or 4-position of isatin Schiff base moiety (phenyl ring), to afford the
compounds 7b-h and 7j-p. Synthesis of the compounds 7a-p is depicted in Scheme 2.
After obtaining all the target compounds, their inhibitory activities against
acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BuChE, from equine
serum) were determined according to Ellman’s method, using tacrine as a reference compound
[55]. The IC50 values of all tested compounds and their selectivity index for eelAChE over
eqBuChE are summarized in Table 1. Hence, it can be concluded that most of the tested
compounds (7a-7p) are potent ChE inhibitors with IC50 values ranging from 0.42 nM to 79.66
nM. Compounds (7i-p) with six-carbon linker between tacrine moiety and isatin Schiff base
showed inhibitory activity toward AChE better than compounds (7a-h) with five-carbon linker.
Furthermore, the optimal linker length, seen in the 6-carbon compounds afforded better
selectivity towards AChE (Table1). Amongst the compounds with 6 carbon linker, compounds
7k, 7m and 7p substituted with 3-chloro group (IC50 = 0.42 nM), 3-nitro group (IC50 = 0.62 nM)
and 4-methoxy group (IC50 = 0.95 nM) had the highest AChE inhibitory activity. The activity of
these compounds is 92-, 62- and 41-fold more potent than that of tacrine (IC50 = 38.72 nM for
AChE). In 6 carbon linker series, 7j with IC50 = 2.79 nM exerted the minimum inhibitory activity
against AChE, which is 14-fold more potent than tacrine. Both potential ligands had chloro and
nitro groups as electron-withdrawing substituent at meta position (7k, 7m). Moving the chloro
group at 3-position of isatin Schiff base ring in compound 7k (IC50 = 0.42 nM) to 2 and 4-
position in compound 7j (IC50 = 2.79 nM) and 7n (IC50 = 1.63 nM) resulted in a significant
reduction in AChE inhibition. The results revealed that the inhibitory activity for AChE could be
increased with an electron-deficient substituent at meta position. Amongst 5 carbon linker series
for AChE, compounds 7c (IC50 = 15.37 nM) and 7e (IC50 = 7.30 nM) showed to have higher
potency than others, which is 2.5- and 5-fold more potent than tacrine. In this case, the same
pattern was perceived as well as the compounds with 6 carbon linker. Both 7c and 7e bearing
chloro and nitro withdrawing groups in meta position. These results implied that n = 6 might be a
suitable length for linker to bind simultaneously to PAS and CAS of AChE.
The activity of the compounds against BuChE is shown in Table 1. Compound 7d with a
five carbon linker exhibited the strongest inhibition against BuChE with IC50 value of 0.11 nM,
which was 56-fold more potent than that of tacrine (IC50 = 6.21 nM). However, for other
compounds, a comparative trend was observed with lengthening the linker and changing the
substituted groups. As a result, in compounds bearing nitro at meta position and methoxy at para
position, changing linker length from 5 to 6 decreased BuChE inhibition ((7e, 7m) and (7h, 7p)).
In contrast, in case of chloro analogues, an increase in BuChE inhibitory activity was observed
by lengthening alkyl chain spacer from 5 to 6 carbon atoms ((7b, 7j), (7c, 7k) and (7f, 7n)). In
methyl substituted compounds 7d, 7l, 7g and 7o a different pattern was figured out. 3-methyl in 5
carbon linker compound (7d) showed a higher potency than its congener with 6 carbon linker
(7l). On the contrary, in compounds with methyl at position 4 (7g, 7o), lengthening the linker
from 5 to 6 gave higher BuChE inhibitory activity.
7

On the other hand, by replacing chloro group at 3-position of isatin Schiff base moiety
(compound 7k, IC50 = 0.42 nM) with nitro group in compound 7m (IC50 = 0.62 nM), the AChE
inhibitory activity dropped slightly, while the BuChE inhibitory activity decreased significantly
(compound 7k with IC50 = 0.57 nM and compound 7m with IC50 = 3.52 nM). It was concluded
that nitro group was unfavorable for BuChE inhibition, and such an unbalanced decline in AChE
and BuChE inhibition led to the high selectivity index (SI = 5.677) for 7m. The same pattern was
observed for 7p with methoxy group at para position (IC50 = 0.95 nM for AChE and IC50 = 6.05
nM for BuChE), which resulted in the highest selectivity index (SI = 6.368). It could be inferred
that 6 carbon linker with substituent at para position causes an increase in the selectivity index of
compounds.
Table 1. Inhibition of eelAChE and eqBuChE by compounds 7a-p.
Compound
n
R1
R2
R3
IC50 (nM)
eelAChEa
Selectivity
indexc
0.07
eqBuChEb
2.67 ± 0.14
2.49 ± 0.44
1.34 ± 0.02
0.11 ± 0.02
3.14 ± 0.11
2.96 ± 0.31
2.80 ± 0.67
7a
7b
7c
7d
7e
7f
5
5
5
5
5
5
5
H
Cl
H
H
H
H
H
H
H
H
H
39.98 ± 0.23
35.94 ± 1.02
15.37 ± 0.58
57.85 ± 3.36
7.30 ± 0.02
16.32 ± 0.37
79.66 10.15
0.07
Cl
H
0.09
Me
NO2
H
H
0.00
H
0.43
Cl
Me
0.18
7g
H
0.03
± 0.98
7h
7i
5
6
6
6
6
6
6
6
6
H
H
Cl
H
H
H
H
H
H
H
H
OMe
H
35.17
0.59 ± 0.05
4.19 ± 0.13
2.14 ± 0.18
0.57 ± 0.10
0.46 ± 0.02
3.52 ± 0.44
2.86 ± 0.02
0.94 ± 0.09
6.05 ± 0.38
6.21 ± 0.58
0.02
1.66
0.77
1.36
0.19
5.68
1.75
0.35
6.37
0.16
2.52 ± 0.26
2.79 ± 0.14
0.42 ± 0.01
2.41 ± 0.08
0.62 ± 0.06
1.63 ± 0.13
2.69 ± 0.17
0.95 ± 0.21
38.72 ± 3.16
7j
H
H
7k
Cl
Me
NO2
H
H
7l
H
7m
7n
H
Cl
7o
H
Me
OMe
7p
H
Tacrine
a The 50% inhibitory concentration (means ± SD of three experiments) of AChE
from electric eel.
b The 50% inhibitory concentration (means ± SD of three experiments) of BuChE
from equine serum.
c AChE selectivity index = IC50 (eqBuChE)/ IC50 (eelAChE).
2.3. Kinetic study of AChE inhibition
To survey the inhibition mechanism of AChE, compound 7k as the most potent inhibitor
was selected for kinetic studies by analyzing Lineweaver–Burk plots. This method provides
information about the mode of inhibition and binding of these inhibitors. This graphical plot
represents reciprocal velocity versus reciprocal substrate ATChI (acetylthiocholine iodide) on
three increasing concentrations for the different inhibitor concentrations. As shown in Fig. 1,
8

increased slopes (from 0.03 to 0.195) and intercepts (from 0.35 to 0.85) at following increasing
concentration of the inhibitor 0, 1.13, 2.22 and 4.32 nM, indicated a mixed-type inhibition. This
pattern indicates that compound 7k might have affinity to peripheral anionic site (PAS) as well
as catalytic anionic site (CAS) of AChE. Previous studies showed that some amino acids in PAS
of AChE are responsible for interaction with Aβ peptide, which might induce the deposition of
neurotoxic Aβ fibrils. As a result, inhibitors with the ability to bind to both CAS and PAS of
AChE and mixed-type inhibition mechanism might be able to reduce amyloid plaque formation
as well as AChE inhibitory activity. Compound 7k, with a six carbon linker length between
tacrine and isatin Schiff base, can concurrently interact with the CAS and PAS of AChE that has
potential inhibitory activity to AChE- induced Aβ aggregation. As a secondary plot, by applying
the slope values of the Lineweaver-Burk plots versus varying concentration of 7k, the inhibition
constant value, Ki, equal to 0.41 nM was found for compound 7k (Fig. 1).
Fig. 1. Kinetic of AChE inhibition by compound 7k: Left) Lineweaver-Burk plot, Right) Lineweaver-Burk
secondary plot.
2.4. Molecular docking
2.4.1. Docking study with AChE
As an attempt to find the binding pose of the most potent compounds 7k, 7m and 7p in
the active site of AChE, the ligand-protein docking was carried out by means of Auto Dock Vina
(1.1.2). The Protein Data Bank (PDB) contains variety of crystal structures of
acetylcholinesterase complexed with different ligands. For this aim, five 3D X-ray crystal
structures of AChE were retrieved from Protein Data Bank (PDB). Amongst them, 4EY7, 1E66
and 1EVE were selected based on optimal self-docking criteria of RMSD < 2. The best docking
validation score belonged to 1E66 (RMSD=0.60). The obtained parameters from validation step
9

were used for cross-docking of the synthesized compounds. After termination of cross-docking
simulation, the best pose for each ligand was selected for visualization and analysis. The results
of cross-docking studies in terms of binding energies together with the residues involved in the
interaction are shown in Table 2 for all three PDB codes. As it can be seen from the results of
interaction for three PDB codes, all are in good agreement. The molecular docking results for
representative compounds 7k, 7m and 7p showed that the ligands were well oriented towards the
bottom of the active site gorge.
As shown in Fig. 2, the tacrine moiety of 7k, 7m and 7p were situated at the CAS of
AChE (1E66). Tacrine ring and protonated nitrogen created π-π stacking interaction and cation-π
interaction with the indole ring of Trp84 and phenyl ring of Phe330. The positively charged
nitrogen of tacrine made a hydrogen bond with His440. In addition, the positively charged
nitrogen of Schiff base could interact with Trp279 via a cation-π interaction and also showed π-π
stacking interaction with the Schiff base ring in the PAS of AChE (1E66).
The nitrogen attached to the linker by tacrine in 7p, interacted with Trp84 by cation-π
interaction in the CAS and the protonated nitrogen of isatin created cation-π interaction with
Tyr334 in the PAS of AChE (1EVE). In contrast, the tacrine ring in 7k and 7m adopted an
inverse orientation through π-π stacking against the indole ring of Trp 279 in PAS, while Schiff
base ring formed π-π interaction with Trp84 in the CAS. Also, Tyr334 and Phe330 adopted
cation-π interactions through protonated nitrogen of isatin and nitrogen of Schiff base in the CAS
of AChE (1EVE). Thy121 and Tyr334 are the two residues responsible for interactions with the
mid-gorge of AChE (1EVE) (Fig. 2).
The tacrine ring in 7k, 7m and 7p adopted π-π stacking orientation by Trp86 and cation-π
interaction by Trp86 and Tyr337. Nitrogen attached to the linker by tacrine, at the CAS was
responsible for the two interactions. In 7m, nitrogen of Schiff base interacted with Trp286 ring
through cation-π interaction with the PAS of AChE (4EY7). Formation of hydrogen bonds was
observed between hydroxyl groups of Tyr124 and Phe295 and carbonyl group in isatin at the
PAS of AChE (4EY7). This led to more stabilized interaction. Isatin could bind to the mid-gorge
site of AChE (4EY7) through a cation-π interaction between its protonated nitrogen atom and
Tyr341.
The above kinetic study implied that 7k, 7m and 7p were a dual site binding inhibitor for
AChE. In fact, the present molecular docking results confirmed that these compounds bind to
CAS, PAS and mid-gorge site of AChE simultaneously. These results were consistent with our
molecular design for AChE inhibition.
10

7k (1E66)
7m (1E66)
7p (1E66)
7k (1EVE)
7m (1EVE)
7p (1EVE)
7k (4EY7)
7m (4EY7)
7p (4EY7)
Fig. 2. The best pose of the most active compounds 7k, 7m and 7p with different x-ray crystal extructures (PDB
code: 1E66, 1EVE, 4EY7) in the active site of AChE; and the residues of the active site involved in ligand binding.
11

Table 2. RMSD values and binding energies of compounds 7k, 7m and 7p with the residues involved in the
intractions.
PDB Code
1E66
RMSD
of self-docking
0.60
Binding Energy (Kcal.mol-1)
7k
7m
7p
Tacrine
CAS
7k
7m
7p
Tacrine
CAS
Trp84
Phe330
His440
PAS
CAS
Trp84
PAS
CAS
Trp84
Phe330
His440
PAS
-13.4 -13.7 -12.8
-10.0
Trp279
Trp279
Trp279
Phe330
Trp84
His440
Phe330
His440
1EVE
4EY7
0.86
0.98
-12.6 -12.3 -12.0
-13.2 -13.8 -13.0
-8.4
-8.8
Trp84
Tyr334
Phe330
Trp86
Trp279 Tyr334 Trp279
Phe330
Trp84
Tyr121
Tyr334
Tyr341
Phe331
Phe330
Asp72
Tyr341
Trp86
Tyr341
Trp86
Phe338
Tyr337
His447
Tyr124
Tyr337 Phe295 Tyr337 Trp286 Tyr337 Phe295
Tyr124
2.4.2. Docking study with BuChE
Due to the significant potency of target compounds on BuChE, ligand-protein docking
was also conducted to predict the binding poses of the representative compounds 7d, 7h and 7l in
the active site of BuChE. The 3D coordinate of the human BuChE (PDB ID: 4XII) was retrieved
and prepared as described for AChE. Docking of 7d, 7h and 7l was performed after a self-
docking validation procedure (RMSD=1.59 Aº). As depicted in Fig. 3, compounds 7d, 7h and 7l
were well fitted in the active site cavity of BuChE. The fused tacrine ring was oriented to
hydrophobic pocket composed of Trp82 with cation- interaction and
stacking interaction.
휋
휋 ‒ 휋
In 7d, the oxygen atom of the isatin ring contributed to hydrogen-binding with Thr120. In 7h, the
-cation interaction and stacking interaction formed by Tyr332 with nitrogen and phenyl
휋
휋 ‒ 휋
ring of Schiff base. In 7l, -cation interaction with Tyr332 and protonated nitrogen of isatin ring
휋
were seen. The results showed that all the three compounds could bind to CAS and PAS of the
BuChE (table 3).
12

7d (4XII)
7h (4XII)
7l (4XII)
Fig.3. The best pose of the most active compounds 7d, 7h and 7l with x-ray crystal extructure (PDB code: 4XII) in
the active site of BuChE; and the residues of the active site involved in ligand binding.
Table 3. RMSD values and binding energies of compounds 7k, 7m and 7p with the residues involved in the
interactions.
PDB Code
RMSD
of self-docking
1.59
Binding Energy (Kcal.mol-1)
7d
7h
7l
Tacrine
CAS
7d
7h
7l
Tacrine
CAS
PAS
CAS
PAS
CAS
PAS
4XII
-11.8 -11.4 -11.5
-8.2
Trp82 Thr120 Trp82 Tyr332 Trp82 Tyr332
Trp82
2.5. Ferrous (Fe2+) Ions chelating activity
Fe2+ as a transition metal ion has the ability to form free radicals by gaining or losing
electrons. Therefore, reactive oxygen species formation can be prevented by the chelation of
metal ions with chelating agents. In the present study, chelation power assay was done on the
new tacrine isatin Schiff base derivatives by assessing their ability to compete with ferrozine for
ferrous ion [56]. In the presence of chelating agents, the formation of ferrozine-Fe2+ complex
with a magenta color is not completed, leading to reduced absorbance. Our data is shown in table
4. Most of the compounds have significant chelation power (33%- 81%) in comparison with
tacrine (83%) and EDTA (84%) as a standard iron chelator (Fig. 4). It was observed that 7k and
7p exhibited significant capacity to chelate ferrous ion with the value of 81% and 80%. It can be
inferred that amino group conformation linked by tacrine to the spacer is the most efficient iron
chelating site. 2-keto and 3-imine groups on isatin part of structures are also responsible for iron
chelation. The high affinity of tacrine isatin Schiff base derivatives to ferrous ion is of great
significance, because it was proposed that the transition metal ions contribute to the oxidative
damage in neurodegenerative disorders such as Alzheimer’s [57].
13

Table 4. Metal chelating percentage of synthesized compounds determined by a colorimetric method.
Compound
% of Fe2+ chelating at 400 μM
± 4.4
± 3.7
± 0.4
± 1.4
7a
7b
64.95
74.47
78.93
77.63
46.37
73.68
74.94
46.33
74.45
43.15
81.52
73.38
33.51
72.42
64.06
80.24
83.56
84.71
7c
7d
± 5
.0
7e
± 3.2
± 3.5
± 5.8
± 2.9
± 3.3
± 1.9
± 6.0
± 5.5
± 4.5
± 6.7
± 2.3
± 1.0
± 0.9
7f
7g
7h
7i
7j
7k
7l
7m
7n
7o
7p
Tacrine
EDTA
Fig. 4. Metal chelating activity of compounds 7a-p against EDTA as a standard iron chelator.
2.6. Inhibition of AChE-induced and self-induced Aβ aggregation
As several studies have found, AChE directly promotes pro-aggregation activity of Aβ
protein into amyloid fibrils, and stabilizes the AChE–Aβ complex, which could cause
neurotoxicity. This action likely happens through the interaction of Aβ with the PAS amino acid
residues of AChE. Therefore, the inhibition of the PAS of AChE can help in prevention of Aβ
aggregation and may turn AChEIs into potential disease modifying hybrids. The kinetic study of
14

AChE inhibition and molecular modeling studies have demonstrated that our designed
compounds could interact with the PAS as well as CAS of AChE. To further study of the dual
action of tacrine-isatin Schiff base hybrid derivatives, the amyloid-beta protein (Aβ1-40) anti-
aggregating activity of 7k and 7m, as the most potent AChE inhibitors, was examined by a
thioflavin-T fluorescent (ThT) assay. Two mentioned compounds were tested at 10 μM
concentration and they were compared with donepezil as reference drug. The Aβ aggregation
inhibition results are shown in Table 5.
Table 5. Inhibition of AChE-induced and self-induced Aβ1-40 aggregation by compounds 7k and 7m using ThT
method.
Compound
Inhibition of Aβ aggregation (%)
Self-induceda
n. d.c
AChE-inducedb
± 1.3
± 4.6
± 1.2
7k***
79.1
70.8
72.7
± 3.3
7m***
84.6
± 5.2
Donepezil***
25.6
a Inhibition of self-induced Aβ1-40 aggregation (10 μM) produced by the tested compound at 10 μM concentration. Values are expressed as means
±
SEM of three experiments (***p < 0.0001, compared to control).
b Co-aggregation inhibition of Aβ1-40 and AChE by the tested compound at 10 μM concentration. Values are expressed as means
SEM of three
±
experiments (***p < 0.0001, compared to control).
c n. d. : not determined
From the results, it can be seen that both synthesized compounds exhibit a good inhibitory
activity on AChE-induced Aβ1-40 aggregation as well as donepezil. The results indicate that 7m
(84.6 % at 10 μM) is the potent inhibitor of Self-induced Aβ aggregation, which is 3.3-fold more
potent than donepezil. The selected compounds exhibit very good activity in AChE-induced Aβ1-
40 aggregation thioflavin-T assay as well as self-induced Aβ1-40 aggregation.
3. Conclusion
In conclusion, a series of tacrine–isatin Schiff base hybrid compounds were designed,
synthesized and evaluated as novel multifunctional potent ChE inhibitors. Most of these
compounds were potent ChEIs with IC50 values in the nanomolar range. Compounds 7k, 7m and
7p were the most potent inhibitors, which were 92-, 62- and 41-fold, respectively, more active
than that of the reference compound tacrine toward AChE. 7k also showed a potent BuChE
inhibition but 7m and 7p exhibited selectivity for AChE over BuChE with the ratio of
BuChE/AChE equal to 5.68 and 6.37, respectively. Amongst the hybrids, 7k with a chloro group
in meta-position of the isatin Schiff base moiety and a six carbon linker between the two
pharmacophores, delivered the best results with an IC50 value of 0.42 nM for AChE and 0.57 nM
for BuChE. Kinetic studies of 7k and molecular modeling studies of all three compounds
indicated that they were mixed-type inhibitors that simultaneously bind to active and peripheral
sites of AChE. The molecular modeling studies further supported that 7m and 7p not only bind to
15

CAS and PAS but also to the mid-gorge site of AChE. Meanwhile, 7k and 7m both exhibit a
good inhibitory activity on AChE-induced and self-induced Aβ1-40 aggregation, which confirmed
the dual action of these compounds to bind to CAS and PAS of the enzyme. In addition, all three
compounds represented good metal-chelating property in comparison with EDTA as the
reference agent. Based on the results, compound 7k is a promising candidate to target ChEs with
metal chelation activity for AD treatment. Furthermore, anti-aggregation activity of 7k and 7m
qualified them as a potential multifunctional anti-AD drug candidate for further research. These
candidate compounds have better cholinesterase inhibitory activity in compare with other studies
including isatin Mannich bases [38], tacrine-melatonin derivatives [33], donepezil like indol
derivatives [48] and benzylpyridinium-based benzoheterocycles [58].
4. Experimental section
4.1. Chemistry
Sixteen derivatives of tacrine-isatin Schiff base derivatives 7a-p were synthesized, shown
in scheme 2. All solvents and reagents were purchased either from Sigma or Merck Chemical
Companies. All reaction progress was monitored by analytical thin layer chromatography (TLC)
on precoated silica gel 60 GF254 sheets and the spots were detected under UV light (254 nm).
Compound purification was performed by preparative chromatographic methods on silica gel,
using either column chromatography (silica gel 60) or glass coated silica gel 60 GF254 plates. The
1H NMR and 13C NMR spectra were recorded in CDCl3, and using TMS as the internal standard
on a Brucker Avance DPX 500, 400, 300, 250 MHZ instrument. Chemical shifts are reported in
ppm (δ), coupling constants reported in hertz (Hz), and all splitting patterns designed as s,
singlet; d, doublet; t, triplet; m, multiplet; brs, broad singlet. Mass spectra were measured on
Agilent Technology (HP) MS. Melting point was obtained on electrothermal 9200 instrument
and uncorrected.
4.2. Synthesis and characterization
4.2.1. Synthesis of tacrine 2 (1,2,3,4-tetrahydroacridin-9-amine)
Tacrine 2 was prepared by the previously reported method with a slight modification.
AlCl3 (10 mmol) was added to the mixture of 2-aminobenzonitrile (10 mmol) and cyclohexanone
(10 mmol) in toluene (50 mL) placed in a round bottom flask connected to a Dean-Stark water
separator. The mixture was heated at reflux for 24 h under stirring. After cooling to room
temperature, the suspension was filtered and the remaining solids were treated with NaOH
solution (2 mol L-1, 80 mL). Again, this mixture was heated at reflux for 24 h. On cooling to
room temperature, the reaction mixture were extracted with ethylacetate (3 × 8 mL), the organic
layers were combined and dried over anhydrous Na2SO4. The solvent was evaporated under
16

reduced pressure to produce the desired product as a yellowish solid with 93% yield (mp: 180-
185 ºC).
1H NMR (CDCl3, 500 MHz) δ: 7.91 (d, 1H, J=8.5 Hz), 7.70 (d, 1H, J=7.5 Hz), 7.57 (t, 1H, J=7.5
Hz), 7.36 (t, 1H, J=7.5 Hz), 4.70 (s, 2H), 3.04 (t, 2H, J=6.0 Hz), 2.61 (d, 1H, J=6.3 Hz), 1.96-
1.92 (m, 4H); 13C NMR (CDCl3, 500 MHz) δ: 158.76, 146.70, 146.58, 128.96, 128.63, 124.02,
119.81, 117.29, 110.56, 34.27, 23.92, 23.03, 22.94.
4.2.2. General procedure for the synthesis of 3a (N-(5-bromopentyl)-1,2,3,4-
tetrahydroacridin-9-amine) and 3b (N-(6-bromohexyl)-1,2,3,4-tetrahydroacridin-9-amine)
A solution of dibromopentane or dibromohexane (15 mmol) in dry DMF (10 mL) was
added to an ice-cold solution of tacrine 2 (1.0 g, 5.0 mmol) and NaH (15 mmol) in dry DMF (10
mL). Then the solution was stirred for 5 h at room temperature. When the reaction was
completed (monitored by TLC), it was extracted with ethylacetate and water, followed by brine
solution. The organic layer was dried over anhydrous Na2SO4 and concentrated under reduced
pressure. Then the residue was purified by silica gel column chromatography using petroleum
ether and ethyl acetate (100:50) as eluent to produce compounds 3a and 3b as brownish oil with
69.4% (3a) and 77.6% (3b) yields.
1
3b: H NMR (CDCl3, 300 MHz) δ: 8.62 (d, 1H, J=8.7 Hz), 8.18 (d, 1H, J=8.7 Hz), 7.73 (t, 1H,
J=7.7 Hz), 7.48 (t, 1H, J=7.7 Hz), 4.10 (t, 1H, J=6.5 Hz), 3.94 (t, 2H, J=6.9 Hz), 3.45 (t, 1H,
J=6.5 Hz), 3.37 (t, 2H, J=5.6 Hz), 2.62 (t, 2H, J=5.3 Hz), 1.99-1.82 (m, 8H), 1.56-1.54 (m, 4H).
4.2.3. General procedure for the synthesis of isatin Schiff base derivatives 6a-6h
Compounds 6a-h were prepared by reacting a mixture of isatin (25 mmol) and different
substituted aromatic amines (25 mmol) in 30 mL of absolute ethanol containing 1.5 mL of
glacial acetic acid. The reaction mixture was refluxed for about 3-5 hours. The progress was
monitored by TLC using petroleum ether/ ethyl acetate (2:1). The solvent was evaporated in
vacuum and the yellow product was collected, and on recrystallization with petroleum ether gave
the desired product 6a-6h in reasonable yields.
6a: 1H NMR (CDCl3, 500 MHz) δ: 7.45 (t, 2H, J=7.8 Hz), 7.31-7.25 (m, 2H), 7.05 (d, 2H, J=7.5
13
Hz), 6.98 (d, 1H, J=7.5 Hz), 6.74 (t, 1H, J=7.8 Hz), 6.66 (d, 1H, J=7.5 Hz); C NMR (CDCl3,
500 MHz) δ: 165.80, 154.92, 150.09, 145.96, 134.42, 129.45, 128.58, 126.28, 125.49, 122.74,
119.14, 117.93, 116.13, 112.07.
6f: 1H NMR (CDCl3, 500 MHz) δ: 7.43 (d, 2H, J=8.5 Hz), 7.36 (t, 1H, J=7.5 Hz), 7.02 (d, 2H,
13
J=8.5 Hz), 6.96 (d, 1H, J=7.5 Hz), 6.82 (t, 1H, J=7.8 Hz), 6.77 (d, 1H, J=7.5 Hz); C NMR
(CDCl3, 500 MHz) δ: 165.04, 154.96, 148.38, 145.75, 134.75, 131.00, 129.66, 128.73, 126.34,
122.96, 120.79, 119.53, 116.00, 111.91.
4.2.4. General procedure for the preparation of 7a-7p
17

Isatin Schiff base derivatives 6a-6h (3 mmol) were added to a stirred solution of
anhydrous K2CO3 (6 mmol) in dry DMF (10 mL) under N2 atmosphere. Then, 3a or 3b (2.55
mmol) dissolved in dry DMF (10 mL), was added dropwise and the resulting dark red solution
was stirred overnight at room temperature. The DMF was eliminated by washing it with water
and extracted with ethyl acetate (50 mL 3). The combined organic extracts were washed with
brine solution and dried over anhydrous Na2SO4. The solvent was evaporated under reduced
pressure and the residue was prepurified by silica gel column chromatography using petroleum
ether/ ethyl acetate (100:25) as the mobile phase. To obtain purified target compounds 7a-p,
plate chromatography was performed using CHCl3/MeOH (150:25) as eluent mixtures of
solvents.
4.2.4.1. 3-(phenylimino)-1-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)indolin-2-one
(7a)
Compound 3a was treated with 3-(phenylimino)indolin-2-one (6a) according to general
procedure to produce the desired product 7a as an orange solid (Yield: 65.8%, mp 74-84 ºC).
1H NMR (CDCl3, 250 MHz) δ: 8.00-7.94 (m, 2H), 7.51 (t, 1H, J=7.6 Hz), 7.36 (t, 2H, J=7.8 Hz),
7.29-7.22 ( m, 2H), 7.20-7.18 ( m, 1H), 6.92 (d, 2H, J=8.3 Hz), 6.75 (d, 1H, J=8 Hz), 6.68 (t, 1H,
J=7.6 Hz), 6.55 (d, 1H, J=7.8 Hz), 3.76 (t, 2H, J=6.9 Hz), 3.59 (t, 2H, J=7.1 Hz), 3.04 (brs, 2H),
13
2.59 (brs, 2H), 1.81-1.79 (m, 4H),1.75-1.69 (m, 4H), 1.52-1.43 (m, 2H); C NMR (CDCl3, 400
MHz) δ: 163.41, 155.04, 154.36, 152.95, 150.27, 147.20, 143.15, 134.20, 130.08, 129.48,
129.27, 126.37, 125.39, 124.70, 124.33, 123.52, 122.70, 118.15, 117.70, 115.75, 115.11, 113.54,
109.49, 48.65, 39.74, 31.07, 30.85, 27.07, 24.54, 24.11, 24.316, 22.54, 21.75; MS m/z (%):
488.6 [M+] (83.74), 413.6 (41.51), 398.6 (1.81), 291.4 (1.37), 277.4 (0.79), 267.5 (11.00), 263.5
(1.90), 225.4 (36.60), 221.4 (8.87), 211.4 (100), 197.4 (31.94), 93.3 (44.01), 77.3 (34.11).
4.2.4.2.
3-((2-chlorophenyl)imino)-1-(5-((1,2,3,4-tetrahydroacridin-9
yl)amino)pentyl)indolin-2-one (7b)
Compound 3a was treated with 3-((2-chlorophenyl)imino)indolin-2-one (6b) according to
general procedure to produce the desired product 7b as an orange solid (Yield: 75.8%; mp: 78-89
ºC). 1H NMR (CDCl3, 300 MHz) δ: 8.23 (d, 1H, J=8.1 Hz), 8.13 (d, 1H, J=8.4 Hz), 7.53 (t, 1H,
J=7.4 Hz), 7.41 (d, 1H, J=7.8 Hz), 7.36-7.25 (m, 3H), 7.14-7.09 (t, 1H, J=7.4 Hz), 6.88 ( d, 1H,
J=7.8 Hz), 6.81 (d, 1H, J=7.8 Hz), 6.71 (t, 1H, J=7.5 Hz), 6.43 (d, 1H, J=7.5 Hz), 6.00 (brs, 1H,
HNH), 3.79-3.74 (m, 4H), 3.12 (brs, 2H), 2.63 (brs, 2H), 1.87-1.77 (m, 8H), 1.50-1.48 (m, 2H);
13C NMR (CDCl3, 300 MHz) δ: 163.07, 155.88, 154.54, 152.75, 147.29, 147.16, 140.68, 134.81,
131.28, 130.32, 129.52, 127.82, 126.14, 124.82, 124.10, 123.75, 123.07, 122.29, 118.83, 116.84,
116.06, 112.22, 109.63, 48.43, 39.67, 30.39, 29.68, 27.00, 24.28, 24.00, 22.23, 21.14;
MS m/z (%): 522.6 [M+] (43.62), 487.6 (1.16), 413.5 (28.26), 396.5 (1.04), 326.5 (3.82), 311.3
(0.51), 297.4 (0.51), 267.5 (10.43), 255.3 (3.35), 225.4 (27.74), 211.4 (100), 197.3 (30.50), 127.3
(16.69), 111.3 (6.68).
18

4.2.4.3.
3-((3-chlorophenyl)imino)-1-(5-((1,2,3,4-tetrahydroacridin-9-
yl)amino)pentyl)indolin-2-one (7c)
Compound 3a was treated with 3-((3-chlorophenyl)imino)indolin-2-one (6c) according to
general procedure to produce the desired product 7c as a yellow solid (Yield: 77.3%, mp: 100-
1
110 ºC). H NMR (CDCl3, 300 MHz) δ: 8.23 (d, 1H, J=6.0 Hz), 8.04 (d, 1H, J=7.2 Hz), 7.59-
7.56 (m, 1H), 7.37-7.28 ( m, 3H), 7.20-7.18 (m, 1H), 6.93 (s, 1H), 6.80-6.72 (m, 3H), 6.58 (d,
1H, J=6.9 Hz), 5.05 (brs, 1H, HNH), 3.78-3.74 (m, 4H), 3.14 (brs, 2H), 2.59 (brs, 2H), 1.82 (m,
8H), 1.51-1.47 (m, 2H); 13C NMR (CDCl3, 300 MHz) δ: 172.86, 169.40, 157.64, 154.86, 153.70,
151.35, 147.32, 142.03, 137.55, 135.25, 134.61, 131.47, 130.72, 128.05, 126.51, 125.31, 124.78,
123.59, 122.93, 117.84, 115.91, 115.52, 109.58, 48.78, 44.95, 39.59, 30.57, 30.24, 26.98, 24.17,
23.98, 22.33; MS m/z (%): 522.6 [M+] (100), 487.6 (0.52), 411.6 (7.11), 398.6 (1.68), 325.5
(0.71), 311.4 (1.02), 297.4 (0.58), 267.5 (11.06), 255.3 (5.20), 225.4 (31.75), 211.4 (82.30),
197.4 (25.23), 127.3 (56.41), 111.3 (15.84).
4.2.4.4. 1-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-3-(m-tolylimino)indolin-2-one
(7d)
Compound 3a was treated with 3-(m-tolylimino)indolin-2-one (6d) according to general
procedure to produce the desired product 7d as a yellow solid (Yield: 81.2%, mp: 98-104 ºC).
1H NMR (CDCl3, 300 MHz) δ: 8.07 (d, 1H, J=8.4 Hz), 8.02 (d, 1H, J=8.4 Hz), 7.51 (t, 1H, J=6.3
Hz), 7.31 (t, 1H, J=7.7 Hz), 7.23 (t, 2H, J=7.5 Hz), 6.98 (d, 1H, J=7.2 Hz), 6.77 (d, 1H, J=7.5
Hz), 6.71 (s, 1H), 6.71-6.66 (m, 2H), 6.57 (d, 1H, J=7.5 Hz), 5.16 (brs, 1H, HNH), 3.77-3.73 (brs,
2H), 3.648 (brs, 2H), 3.06 (brs, 2H), 2.62 (brs, 2H), 2.29 (s, 3H), 1.80-1.69 (m, 8H), 1.48-1.46
(m, 2H); 13C NMR (CDCl3, 300 MHz) δ: 163.45, 154.98, 154.16, 153.01, 150.29, 147.10,
143.27, 143.24, 139.41, 134.06, 130.15, 129.27, 126.37, 126.09, 124.81, 124.41, 123.57, 122.67,
118.15, 115.78, 114.57, 113.75, 109.42, 48.75, 39.66, 31.35, 30.72, 27.03, 24.51, 24.06, 22.53,
21.77, 21.48; MS m/z (%): 502.6 [M+] (100), 413.5 (13.35), 305.4 (2.45), 267.4 (10.07), 237.4
(9.19), 225.4 (29.50), 211.4 (86.74), 197.3 (25.29), 106.3 (15.48), 91.3 (29.47).
4.2.4.5.
3-((3-nitrophenyl)imino)-1-(5-((1,2,3,4-tetrahydroacridin-9-
yl)amino)pentyl)indolin-2-one (7e)
Compound 3a was treated with 3-((3-nitrophenyl)imino)indolin-2-one (6e) according to
general procedure to produce the desired product 7e as an orange solid (Yield: 64.2%, mp: 65-70
1
ºC). H NMR (CDCl3, 300 MHz) δ: 8.23-8.21 (brs, 1H), 8.11 (d, 1H, J=8.4 Hz), 8.04 (d, 1H,
J=7.8 Hz), 7.79 (s, 1H), 7.57-7.54 (m, 2H), 7.42 (t, 1H, J=7.8 Hz), 7.33 (t, 1H, J=7.4 Hz), 7.26
(d, 1H, J=7.5 Hz), 6.85 (d, 1H, J=8.1 Hz), 6.70 (t, 1H, J=7.1 Hz), 6.45 (d, 1H, J=7.2 Hz), 5.80
(brs, 1H, HNH), 3.79-3.59 (m, 4H), 3.12 (brs, 2H), 2.62 (brs, 2H), 1.77 (brs, 8H), 1.50 (brs, 2H);
13C NMR (CDCl3, 300 MHz) δ: 162.89, 155.74, 154.33, 151.12, 149.08, 147.69, 135.27, 134.85,
131.18, 130.66, 126.22, 125.06, 124.79, 124.12, 123.60, 122.97, 119.97, 117.01, 115.21, 112.94,
112.38, 110.04, 109.30, 48.43, 39.78, 30.53, 29.87, 26.97, 24.29, 24.01, 22.26, 21.21; MS m/z
19

(%): 533.6 [M+] (65.45), 413.5 (18.08), 399.5 (1.44), 322.4 (0.47), 267.2 (13.93), 252.3 (11.49),
225.4 (27.27), 211.4 (100), 197.4 (28.88), 138.3 (59.42).
4.2.4.6.
3-((4-chlorophenyl)imino)-1-(5-((1,2,3,4-tetrahydroacridin-9-
yl)amino)pentyl)indolin-2-one (7f)
Compound 3a was treated with 3-((4-chlorophenyl)imino)indolin-2-one (6f) according to
general procedure to produce the desired product 7f as a red solid (Yield: 76.4%, mp: 79-85 ºC).
1H NMR (CDCl3, 250 MHz) δ: 8.03-8.00 (m, 2H), 7.59 (t, 1H, J=7.6 Hz), 7.33 (d, 1H, J=6.8
Hz), 7.39 (t, 2H, J=7.6 Hz), 6.98 (d, 2H, J=6.4 Hz), 6.86-6.81 (m, 2H), 6.74 (d, 1H, J=6.8 Hz),
3.84 (t, 2H, J=7.0 Hz), 3.60 (t, 2H, J=7.0 Hz), 3.12 (brs, 2H), 2.72 (brs, 2H), 1.94-1.92 (m, 4H),
13
1.82-1.78 (m, 4H), 1.59-1.52 (m, 2H); C NMR (CDCl3, 400 MHz) δ: 163.13, 157.36, 154.73,
151.43, 148.62, 147.43, 145.94, 134.49, 130.84, 129.67, 129.03, 128.71, 127.43, 126.35, 124.03,
122.96, 122.75, 120.71, 119.39, 116.23, 115.60, 109.56, 108.92, 49.08, 39.87, 33.00, 31.16,
27.11, 24.72, 24.21, 22.88, 22.43; MS m/z (%): 522.6 [M+] (81.04), 413.6 (23.00), 399.6 (1.25),
325.4 (0.63), 311.4 (0.74), 297.4 (0.81), 267.5 (12.11), 225.3 (5.39), 225.4 (36.41), 211.4 (100),
197.4 (31.05), 127.3 (81.14), 111.3 (13.79).
4.2.4.7.
(7g)
1-(5-((1,2,3,4-tetrahydroacridin-9-yl)amino)pentyl)-3-(p-tolylimino)indolin-2-one
Compound 3a was treated with 3-(p-tolylimino)indolin-2-one (6g) according to general
procedure to produce the desired product 7g as an orange solid (Yield: 78.6% , mp: 116-122 ºC).
1H NMR (CDCl3, 500 MHz) δ: 8.04 (d, 1H, J=8.5 Hz), 8.00 (d, 1H, J=8.5 Hz), 7.52 (t, 1H, J=7.5
Hz), 7.35-7.30 (m, 2H), 7.20 (d, 2H, J=8.0 Hz), 6.89 (d, 2H, J=8.0 Hz), 6.82 (d, 1H, J=8.0 Hz),
6.74-6.74 (m, 2H), 3.79 (t, 2H, J=7.0 Hz), 3.65 (t, 2H, J=7.0 Hz), 3.058 (brs, 2H), 2.643 (brs,
2H), 2.379 (s, 3H),1.83-1.74 (m, 8H), 1.53-1.47 (m, 2H); 13C NMR (CDCl3, 500 MHz) δ:
163.44, 155.39, 154.06, 152.63, 147.55, 147.09, 143.66, 135.19, 134.00, 129.95, 129.78, 129.09,
126.16, 125.10, 124.20, 123.44, 122.56, 118.41, 117.92, 115.81, 115.21, 113.88, 109.41, 48.64,
39.70, 31.43, 30.86, 27.05, 24.60, 24.10, 22.57, 21.86, 21.05; MS m/z (%): 502 [M+] (83.37),
413 (47.83), 225 (35.05), 211 (100), 197 (32.02), 183 (19.72), 106 (76.36).
4.2.4.8.
3-((4-methoxyphenyl)imino)-1-(5-((1,2,3,4-tetrahydroacridin-9-
yl)amino)pentyl)indolin-2-one (7h)
Compound 3a was treated with 3-((4-methoxyphenyl)imino)indolin-2-one (6h) according
to general procedure to produce the desired product 7h as a red solid (Yield: 80.7%, mp: 78-83
ºC). 1H NMR (CDCl3, 400 MHz) δ: 8.25 (d, 1H, J=8.4 Hz), 8.12 (d, 1H, J=8.4 Hz), 7.63 (t, 1H,
J=7.2 Hz), 7.42 (t, 1H, J=7.2 Hz), 7.37 (t, 1H, J=7.6 Hz), 7.06-6.98 (m, 5H), 6.87 (d, 1H, J=8.0
Hz), 6.82 (t, 1H, J=7.2 Hz), 3.89 (s, 3H), 3.87-3.85 (m, 2H), 3.78 (t, 2H, J=6.4 Hz), 3.20(t, 2H,
J=6.0 Hz), 2.69 (t, 2H, J=6.0 Hz), 1.93-1.80 (m, 8H), 1.61-1.53 (m, 2H); 13C NMR (CDCl3, 400
MHz) δ: 163.67, 157.90, 154.29, 153.71, 153.52, 147.02, 142.93, 142.44, 133.97, 130.54,
20

125.82,
124.54,
124.09,
123.74,
123.23,
122.62,
120.16,
109.47, 48.69, 39.62, 30.79, 30.65, 29.71, 27.04, 24.41, 24.05, 22.45, 21.58; MS m/z (%): 518.7
[M+] (94.84), 413.6 (24.88), 265.5 (10.95), 253.5 (9.19), 251.4 (10.66), 225.4 (31.44), 211.4
(100), 197.4 (26.33), 123.3 (27.44), 108.3 (38.71).
4.2.4.9.
(7i)
3-(phenylimino)-1-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)indolin-2-one
Compound 3b was treated with 3-(phenylimino)indolin-2-one (6a) according to general
procedure to produce the desired product 7i as a dark orange solid (Yield: 77.1%, mp: 97-102
ºC). 1H NMR (CDCl3, 500 MHz) δ: 7.93 (d, 1H, J=8.5 Hz), 7.88 (d, 1H, J=8.0 Hz), 7.50 (t, 1H,
J=8.0 Hz), 7.39 (t, 2H, J=8.5 Hz), 7.32-7.27 (m, 2H), 7.21 (t, 1H, J=7.5 Hz), 6.97 (d, 2H, J=7.5
Hz), 6.78 (d, 1H, J=8.0 Hz), 6.70 (t, 1H, J=7.5 Hz), 6.59 (d, 1H, J=7.5 Hz), 3.74 (t, 2H, J=7.3
Hz), 3.45 (t, 2H, J=7.3 Hz), 3.03 (brs, 2H), 2.68 (brs, 2H), 1.88-1.87 (m, 4H),1.72-1.66 (m, 2H),
13
1.66-1.61 (m, 2H), 1.42-1.40 (m, 4H); C NMR (CDCl3, 500 MHz) δ: 163.18, 158.42, 154.39,
150.70, 150.30, 147.40, 147.33, 134.07, 129.403, 128.62, 128.51, 128.24, 126.28, 125.28,
123.60, 122.81, 122.51, 120.24, 119.01, 117.70, 115.98, 115.71, 109.42, 49.26, 39.93, 33.97,
31.60, 27.23, 26.61, 26.56, 24.81, 23.03, 22.76; MS m/z (%): 502.7 [M+] (83.00), 427.6 (13.6),
225.4 (29.44), 211.4 (100), 197.4 (29.09), 182.4 (17.34), 77.4 (30.31).
4.2.4.10.
3-((2-chlorophenyl)imino)-1-(6-((1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)indolin-2-one (7j)
Compound 3b was treated with 3-((2-chlorophenyl)imino)indolin-2-one (6b) according
to general procedure to produce the desired product 7j as a yellow solid (Yield: 72.5%, mp: 106-
110 ºC). 1H NMR (CDCl3, 400 MHz) δ: 8.24 (d, 1H, J=8.4 Hz), 8.12 (d, 1H, J=8.4 Hz), 7.64 (t,
1H, J=7.6 Hz), 7.52 (d, 1H, J=7.6 Hz), 7.45-7.34 (m, 3H), 7.22 (t, 1H, J=7.6 Hz), 7.00 ( d, 1H,
J=8.8 Hz), 6.89 (d, 1H, J=8.0 Hz), 6.81 (t, 1H, J=7.6 Hz), 6.55 (d, 1H, J=7.6 Hz), 3.84 (t, 2H,
J=7.0 Hz), 3.78 (t, 2H, J=7.0 Hz), 3.21 (t, 2H, J=6.0 Hz), 2.69 (t, 2H, J=6.0 Hz), 1.93-1.88 (m,
13
4H), 1.83-1.78 (t, 4H), 1.54-1.50 (m, 4H); C NMR (CDCl3, 400 MHz) δ: 162.96, 155.91,
147.34, 134.70, 134.50, 130.72, 130.35, 129.52, 127.80, 127.05, 126.18, 126.12, 125.37, 124.59,
123.85, 123.61, 123.46, 122.99, 122.38, 118.89, 116.14, 109.56, 109.14, 48.68, 39.83, 31.29,
29.73, 27.14, 26.36, 26.20, 24.17, 22.40, 21.55; MS m/z (%): 536.6 [M+] (54.98), 427.6 (30.58),
297.4 (1.59), 281.5 (6.01), 255.3 (3.69), 225.4 (25.47), 211.4 (100), 197.4 (27.77), 127.3 (49.76),
111.3 (7.21).
4.2.4.11.
3-((3-chlorophenyl)imino)-1-(6-((1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)indolin-2-one (7k)
Compound 3b was treated with 3-((3-chlorophenyl)imino)indolin-2-one (6c) according to
general procedure to produce the desired product 7k as a yellow solid (Yield: 78.9%, mp: 90-95
ºC). 1H NMR (CDCl3, 300 MHz) δ: 8.21 (d, 1H, J=8.4 Hz), 8.05 (d, 1H, J=8.7 Hz), 7.56 (t, 1H,
J=7.4 Hz), 7.36-7.27 (m, 3H), 7.17-7.14 (m, 1H), 6.93 (s, 1H), 6.80 (d, 2H, J=7.8 Hz), 6.73 (t,
21

1H, J=7.7 Hz), 6.56 (d, 1H, J=7.2 Hz), 5.16 (brs, 1H, HNH), 3.76-3.68 (m, 4H), 3.13 (brs, 2H),
13
2.60 (brs, 2H), 1.82 (brs, 4H), 1.75-1.64 (m, 4H), 1.43-1.42 (m, 4H); C NMR (CDCl3, 300
MHz) δ: 163.073, 154.991, 153.778, 151.407, 147.489, 135.199, 134.596, 131.20, 130.85,
130.70, 129.66, 126.47, 125.25, 124.75, 124.65, 123.68, 123.53, 122.81, 118.77, 117.86, 115.95,
115.50, 109.65, 48.61, 39.78, 31.20, 30.43, 27.10, 26.33, 26.18, 24.14, 22.34, 21.43; MS m/z
(%): 536.6 [M+] (71.79), 427.6 (11.63), 413.6 (0.76), 281.5 (4.91), 255.3 (3.96), 225.4 (24.58),
211.4 (100), 197.4 (24.22), 127.3 (10.83), 111.3 (12.26).
4.2.4.12. 1-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(m-tolylimino)indolin-2-one
(7l)
Compound 3b was treated with 3-(m-tolylimino)indolin-2-one (6d) according to general
procedure to produce the desired product 7l as an orange solid (Yield: 79.6%, mp: 94-101 ºC).
1H NMR (CDCl3, 300 MHz) δ: 8.23 (d, 1H, J=7.8 Hz), 8.15 (d, 1H, J=8.4 Hz), 7.61-7.56 (t, 1H,
J=7.5 Hz), 7.41-7.25 ( m, 3H), 7.03 (d, 1H, J=7.5 Hz), 6.84 (d, 1H, J=7.8 Hz), 6.78 (s, 1H), 6.78-
6.71 (m, 2H), 6.62 (d, 1H, J=7.5 Hz), 3.78 (t, 2H, J=6.8 Hz), 3.16 (brs, 2H), 2.69 (brs, 2H), 2.35
(s, 1H), 1.86-1.71 (m, 8H), 1.47 (m, 4H); 13C NMR (CDCl3, 300 MHz) δ: 163.33, 154.23,
153.95, 153.64, 150.30, 147.20, 141.78, 139.38, 134.04, 130.78, 129.25, 126.33, 126.05, 124.56,
123.89, 123.43, 122.59, 118.15, 117.40, 115.76, 114.57, 112.77, 109.46, 48.42, 39.75, 31.08,
30.37, 27.13, 26.35, 26.21, 24.30, 22.35, 21.46, 21.41; MS m/z (%): 516.7 [M+] (87.20), 427.6
(5.83), 413.7 (0.74), 319.5 (2.50), 306.5 (0.90), 291.5 (0.42), 281.5 (3.95), 267.5 (22.28), 237.5
(7.71), 225.5 (22.21), 211.5 (100), 197.5 (24.95), 106.4 (36.33), 91.4 (36.09).
4.2.4.13.
3-((3-nitrophenyl)imino)-1-(6-((1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)indolin-2-one (7m)
Compound 3b was treated with 3-((3-nitrophenyl)imino)indolin-2-one (6e) according to
general procedure to produce the desired product 7m as an orange solid (Yield: 67.8%, mp: 74-
80 ºC). 1H NMR (CDCl3, 300 MHz) δ: 8.32 (brs, 1H), 8.22 (d, 1H, J=8.4 Hz), 8.15-8.07 (m, 1H),
7.83 (s, 1H), 7.62-7.59 (m, 2H), 7.48-7.30 (m, 3H), 6.90 (d, 1H, J=8.1 Hz), 6.75-6.73 (m, 1H),
6.49 (d, 1H, J=6.6 Hz), 6.27 (brs, 1H, HNH), 3.87-3.62 (m, 4H), 3.19 (brs, 2H), 2.70-2.61 (m,
2H), 1.84-1.66 (m, 8H), 1.48 (brs, 4H); 13C NMR (CDCl3, 300 MHz) δ: 162.75, 155.82, 154.79,
151.13, 149.03, 147.79, 146.36, 140.34, 135.25, 131.45, 130.65, 129.43, 126.18, 124.79, 124.14,
122.87, 119.91, 116.66, 115.16, 113.87, 112.94, 111.94, 110.07, 48.22, 39.91, 30.96, 29.44,
27.08, 26.33, 26.16, 24.18, 22.17, 21.06; MS m/z (%): 547.7 [M+] (10.69), 427.6 (38.43), 413.6
(1.44), 336.5 (0.59), 281.5 (4.50), 267.5 (12.06), 225.4 (17.91), 211.4 (83.48), 197.4 (31.34),
138.3 (100), 122.3 (1.02), 46.3 (6.71).
4.2.4.14.
3-((4-chlorophenyl)imino)-1-(6-((1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)indolin-2-one (7n)
Compound 3b was treated with 3-((4-chlorophenyl)imino)indolin-2-one (6f) according to
general procedure to produce the desired product 7n as an orange solid (Yield: 78.4%, mp: 96-
22

104 ºC). 1H NMR (CDCl3, 500 MHz) δ: 7.97 (d, 1H, J=8.5 Hz), 7.94 (d, 1H, J=8.5 Hz), 7.55 (t,
1H, J=7.5 Hz), 7.40 (d, 2H, J=8.5 Hz), 7.38-7.33 (m, 2H), 6.96 (d, 2H, J=8.5 Hz), 6.84 (d, 1H,
J=8.0 Hz), 6.79 (t, 1H, J=7.5 Hz), 6.70 (d, 1H, J=7.5 Hz), 3.78 (t, 2H, J=7.0 Hz), 3.52 (t, 2H,
J=7.0 Hz), 3.08 (brs, 2H), 2.70 (brs, 2H), 1.91-1.90 (m, 4H), 1.76-1.66 (m, 4H), 1.47-1.43 (m,
13
4H); C NMR (CDCl3, 500 MHz) δ: 163.00, 157.87, 154.78, 151.08, 148.63, 147.53, 146.65,
134.43, 130.74, 129.61, 128.66, 128.62, 128.01, 126.28, 123.77, 123.24, 122.90, 122.63, 120.65,
119.86, 119.37, 115.56, 109.58, 49.19, 39.97, 33.47, 31.59, 27.22, 26.59, 26.53, 24.75, 22.95,
22.59; MS m/z (%): 536.6 [M+] (85.80), 427.6 (8.86), 281.4 (5.53), 267.5 (25.23), 225.4 (27.19),
211.4 (100), 197.4 (27.90), 182.4 (18.69), 127.3 (92.26).
4.2.4.15. 1-(6-((1,2,3,4-tetrahydroacridin-9-yl)amino)hexyl)-3-(p-tolylimino)indolin-2-one
(7o)
Compound 3b was treated with 3-(p-tolylimino)indolin-2-one (6g) according to general
procedure to produce the desired product 7o as an orange solid (Yield: 79.2%, mp: 96-101 ºC).
1H NMR (CDCl3, 500 MHz) δ: 7.99-7.96 (m, 2H), 7.56 (t, 1H, J=7.8 Hz), 7.37-7.31 (m, 2H),
7.23 (d, 2H, J=8.0 Hz), 6.92 (d, 2H, J=8.5 Hz), 6.83 (d, 1H, J=8.0 Hz), 6.77-6.76 (m, 2H), 4.93
(brs, 1H, HNH), 3.79 (t, 2H, J=7 Hz), 3.55 (t, 2H, J=7.25 Hz), 3.09 (brs, 2H), 2.70 (brs, 2H), 2.41
(s, 3H), 1.92-1.91 (m, 4H), 1.77-1.66 (m, 4H), 1.47-1.42 (m, 4H); 13C NMR (CDCl3, 500 MHz)
δ: 163.33, 157.5, 157.46, 154.09, 151.38, 147.62, 147.27, 146.17, 135.13, 133.86, 129.93,
129.11, 128.82, 127.52, 126.18, 123.83, 122.97, 122.44, 119.61, 117.96, 115.88, 115.22, 109.33,
49.13, 39.88, 33.05, 31.56, 27.22, 26.57, 26.51, 24.70, 22.88, 22.47, 21.06; MS m/z (%): 516.6
[M+] (100), 425.5 (4.84), 413.5 (6.32), 281.5 (4.36), 267.4 (18.10), 235.4 (7.32), 225.4 (21.70),
211.4 (75.92), 197.3 (23.00), 182.3 (14.98), 167.3 (9.19), 106.3 (41.80), 91.3 (22.37).
4.2.4.16.
3-((4-methoxyphenyl)imino)-1-(6-((1,2,3,4-tetrahydroacridin-9-
yl)amino)hexyl)indolin-2-one (7p)
Compound 3b was treated with 3-((4-methoxyphenyl)imino)indolin-2-one (6h) according
to general procedure to produce the desired product 7p as a red solid (Yield: 74.8%, mp: 92-103
1
ºC). H NMR (CDCl3, 300 MHz) δ: 8.20 (d, 1H, J=7.2 Hz), 8.06 (d, 1H, J=8.4 Hz), 7.54 (brs,
1H), 7.35-7.20 (m, 2H), 6.96-6.86 (m, 5H), 6.79 (d, 1H, J=7.5 Hz), 6.71 (t, 1H, J=7.5 Hz), 3.79
(s, 3H), 3.73 (brs, 2H), 3.71 (brs, 2H), 3.12 (brs, 2H), 2.61 (brs, 2H), 1.80 (m, 4H), 1.70-1.68 (m,
13
4H), 1.41 (m, 4H); C NMR (CDCl3, 300 MHz) δ: 168.71, 163.54, 157.86, 153.85, 153.82,
153.77, 149.11, 147.14, 142.98, 133.88, 130.84, 128.77, 125.80, 124.63, 123.80, 122.51, 120.12,
117.43, 115.90, 114.56, 113.59, 112.78, 109.45, 48.52, 39.69, 31.15, 30.40, 29.68, 27.14, 26.32,
26.18, 24.23, 22.33, 21.42; MS m/z (%): 532.6 [M+] (56.49), 427.5 (22.37), 334.4 (1.82), 321.5
(0.57), 307.4 (0.36), 281.4 (4.75), 267.4 (21.35), 251.3 (5.70), 225.3 (25.47), 211.3 (100), 197.3
(25.40).
23

4.3. In vitro inhibition studies on AChE and BuChE
Acetylcholinesterase (AChE, E.C. 3.1.1.7, from electric eel), butyrylcholinesterase
(BuChE, E.C. 3.1.1.8, from equine serum), 5, 5'-dithiobis-(2-nitrobenzoic acid) (Ellman's
reagent, DTNB), acetylthiocholine iodide (ATI), butylthiocholine iodide (BTI), were purchased
from Sigma Aldrich company. The inhibition capacity of the tested compounds 7a-p against
AChE/BuChE was performed using colorimetric method assessed by Ellman et al. Tacrine and
synthesized derivatives were dissolved in DMSO (40mM). In a 24-well plate, 2000μl of
NaH2PO4/Na2HPO4 buffer (0.1 M, pH = 8), 60 μl of DTNB (0.01 M), 20 μl of AChE (1ku
prepared in NaH2PO4/Na2HPO4 buffer (0.1 M) and glycerine 25%, pH = 8.0, 2 units/mL in 0.5
mL aliquots) or 20 μl of BuChE (1ku prepared in NaH2PO4/Na2HPO4 buffer (0.1 M) and
glycerine 25%, pH = 8.0, 2 units/mL in 0.5 mL aliquots) were incubated with 30 μL in various
concentrations of tested compounds (0.026 - 400 μM) at room temperature for 5 min. Afterward,
20 μl of the corresponding substrate acetylthiocholine iodide (0.15 M) or S-butyrylthiocholine
iodide (0.15 M) was added and the absorbance was measured at 2 minutes intervals at a
wavelength of 412 nm. The compound concentration produced 50% of enzyme activity
inhibition (IC50), was calculated by nonlinear regression analysis of the response–concentration
(log) curve. Results are expressed as the mean ± SD of at least three different experiments
performed in triplicate. All experiments were performed on a Synergy HTX Multi-Mode Reader-
BioTek.
4.4. Kinetic study of AChE inhibition
Kinetic mechanism of AChE was carried out by Ellman’s method in various
concentrations (1.13, 2.22 and 4.32 nM) of selected compound 7k. In the Lineweaver–Burk plot
(or double reciprocal plot), 1/ [velocity] was plotted against 1/ [substrate] at different
concentrations of the substrate acetylthiocholine iodide (0.69, 1.37 and 3.38 mM). The plots
were assessed by a weighted least-squares analysis that assumed the variance of velocity (v) to
be a constant percentage of v for the entire data set. Slopes of these reciprocal plots were then
plotted against the concentration of 7k in a weighted analysis, and Ki was determined as the
intercept on the negative x-axis. Data analysis was performed with GraphPad Prism 4.03
software (GraphPad Software Inc.).
4.5. Metal chelating assay
The ferrous ion chelating power of the synthesized compounds was measured by the
method of Dinis with a slight modification [56, 59]. All experiments were performed in 24-well
microplates. In each well, 100 µl of each synthetic compound (4 mM) prepared in methanol was
incubated with 100 μl of FeCl2 (0.6 mM) and 700 μl of methanol. The reaction was initiated by
adding 100 ??l of ferrozine (5 mM), which was shaken at room temperature for 10 min.
24

Afterward, the absorbance at 562 nm was measured. Methanol, Fe2+ and ferrozine were used as a
blank sample. For comparison of iron-chelating activity, EDTA was used as a standard iron
chelator.
4.6. Inhibition of self-induced and AChE-Induced Aβ1-40 Aggregation Assay
Inhibitory properties of compounds on self induced and AChE induced aggregation of
amyloid β protein 1-40 was determined using a thioflavin T (ThT)-based fluorescence assay [60].
The ThT excitation/emission was measured at 448 nm/490 nm at 48 hours using a multimode
plate reader (BMG Labtech Omega FLUOstar). Amyloid β protein 1-40 (Sigma A1075) was
dissolved in Phosphate Buffer Saline pH 7.4 (PBS, HyClone Thermo Scientific) containing 1%
ammonium hydroxide. 12.5 μM Amyloid β protein 1-40 was incubated for 72 hours at 37 °C for
prefibrillation. For the assay Aβ1-40 (10 µl) ± human recombinant AChE (0.01 u/ml, Sigma
C1682) were added to 0.05 M KP buffer pH 7.4 and incubated at 37C for 48 h in the absence
and presence of compounds (10 μM). Incubated mixture (100 µL) was mixed with 50 μL of
thioflavin T (ThT, 200 μM) in 50 mM glycine-NaOH buffer (pH 8.5). Donepezil (10 μM, Sigma
D-6821) were also tested as reference agent.
Inhibition percent of self or AChE-induced aggregation due to the presence of the tested
compounds were determined by the following calculation: 100-[(IFi/IFo) × 100] where IFi and
IFo are the fluorescence intensities obtained for Aβ ± AChE in the presence and in the absence
of inhibitors. Statistical analysis was performed using GraphPad Prism software using one-way
ANOVA analysis (Bonferroni’s test) and p-Values less than 0.05 were considered statistically
significant.
4.7. Docking simulation method
Since different 3D-structures were released for AChE and BuChE, docking validation
was used to select a proper x-ray structure for docking simulation. From these complexes, 19
PDB codes were selected from Protein Data Bank (http://www.rcsb.org). Five 3D X-ray crystal
structures of the TcAChE, hAChE and fourteen 3D X-ray crystal structures of hBuChE were
retrieved from Protein Data Bank (PDB) based on the similarity of cognate ligands with our
compound structures in order to find the starting model of AChE and BuChE. The retrieved PDB
codes for AChE were 2CKM, 2CMF, 1EVE, 4EY7 and 1E66. All the retrieved PDB codes were
subjected to self-docking validation test. In this step, the cognate ligands were re-docked on their
corresponding 3D structures and the best pose of docking was superimposed with the native
conformation of the ligand at crystallographic state. Amongst the pdb files of the receptors, 1E66
complexed with huprine, 1EVE and 4EY7 complexed with donepezil for AChE and 4XII
complexed with nitroxoline for BuChE were selected based on root-mean-square deviation
(RMSD) values as shown in tables 2 and 3. The compound structures of 7k, 7m, 7p were
sketched, energy minimized and the three ligand molecules were saved in pdbqt format. To
prepare protein for docking simulation, all co-crystallized ligands and water molecules were
25

removed and the missing hydrogens were added. Non-polar hydrogens were merged with their
corresponding carbons and then the protein was converted into the required pdbqt format. All
preparation was done using Auto Dock Tools package (1.5.6).
Cross-docking simulations were performed using bash scripting in linux operating
system. Autodock Vina (1.1.2) was used for docking within a box defined by following
parameters. The grid box with the size of 30
was determined and the box was
× 30 × 30
centered on co-crystalized ligand. The center of grid box was determined as [x = 3.62, y = 69.11,
z = 64.75] for 1E66, [x = 5.16, y = 66.92, z = 66.42] for 1EVE, [x = -11.19, y = -46.35, z =
28.86] for 4EY7 and [x = 6.88, y = -15.86, z = -8.45] for 4XII. The exhaustiveness was set to
100 and the other docking parameters were set as default. At the end of cross-docking
simulations, the best docking poses were selected for further analysis of enzyme-inhibitor
interactions.
Acknowledgment
This article was extracted from the PhD dissertation (grant number: 94-7565) written by
Elham Riazimontazer and approved by the Research Vice-Chancellor at Shiraz University of
Medical Sciences. The authors wish to thank Mr. H. Argasi at the Research Consultation Center
(RCC) of Shiraz University of Medical Sciences for his invaluable assistance in editing this
manuscript.
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