New tacrine-derived AChE/BuChE inhibitors: Synthesis and biological evaluation of 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates

Article abstract of DOI:10.1016/j.ejmech.2017.01.042

A series of poly-functionalized tacrine-derived compounds namely 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates were designed and synthesized as cholinesterases inhibitors. The in?vitro inhibition assay against AChE and BuChE demonstrated that most of compounds had potent AChE inhibitory with reserving potential of BuChE inhibition. Among them, compound 6i bearing a 4-(3-bromophenyl) moiety showed the most potent activity against AChE/BuChE (IC₅₀s values of 0.069 and 1.35?μM, respectively). The anti-AChE activity of 6i was five times more than that of tacrine. The SAR study revealed that chloro/bromo substituent at ortho or meta position of the 4-phenyl ring can improve the anticholinesterase activity.

Full text of DOI:10.1016/j.ejmech.2017.01.042

Accepted Manuscript
New tacrine-derived AChE/BuChE inhibitors: Synthesis and biological evaluation of 5-
amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates
Mohammad Eghtedari, Yaghoub Sarrafi, Hamid Nadri, Mohammad Mahdavi, Alireza
Moradi, Farshad Homayouni Moghadam, Saeed Emami, Loghman Firoozpour, Ali
Asadipour, Omid Sabzevari, Alireza Foroumadi
PII:
S0223-5234(17)30060-0
10.1016/j.ejmech.2017.01.042
EJMECH 9193
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2016
Revised Date: 24 January 2017
Accepted Date: 28 January 2017
Please cite this article as: M. Eghtedari, Y. Sarrafi, H. Nadri, M. Mahdavi, A. Moradi, F.H. Moghadam,
S. Emami, L. Firoozpour, A. Asadipour, O. Sabzevari, A. Foroumadi, New tacrine-derived AChE/
BuChE inhibitors: Synthesis and biological evaluation of 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-
carboxylates, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.01.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New tacrine-derived AChE/BuChE inhibitors: synthesis and biological evaluation of 5-
amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates
Mohammad Eghtedaria, Yaghoub Sarrafia**, Hamid Nadrib, Mohammad Mahdavic, Alireza
Moradib, Farshad Homayouni Moghadamd, Saeed Emamie, Loghman Firoozpourc, Ali
Asadipourf, Omid Sabzevarig, Alireza Foroumadif,h*
a Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
c Drug Design and Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
d Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran.
e Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
f Department of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center,
Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
g Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
h Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
Br
O
NH2
N
NH2
EtO
O
N
Tacrine
6i
A series of tacrine-derived compounds were synthesized as cholinesterases inhibitors. Among
them, compound 6i with 4-(3-bromophenyl) moiety showed the most potent activity against
AChE/BuChE (IC50s of 0.069 and 1.35 µM, respectively).

ACCEPTED MANUSCRIPT
New tacrine-derived AChE/BuChE inhibitors: synthesis and biological evaluation of 5-
amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-carboxylates
Mohammad Eghtedaria, Yaghoub Sarrafia**, Hamid Nadrib, Mohammad Mahdavic, Alireza
Moradib, Farshad Homayouni Moghadamd, Saeed Emamie, Loghman Firoozpourc, Ali
Asadipourf, Omid Sabzevarig, Alireza Foroumadif,h*
a Faculty of Chemistry, University of Mazandaran, Babolsar, Iran
b Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
c Drug Design and Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
d Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran.
e Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of
Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran
f Department of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center,
Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
g Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
h Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research
Center, Tehran University of Medical Sciences, Tehran, Iran
Abstract: A series of poly-functionalized tacrine-derived compounds namely 5-amino-2-phenyl-4H-
pyrano[2,3-b]quinoline-3-carboxylates were designed and synthesized as cholinesterases inhibitors.
The in vitro inhibition assay against AChE and BuChE demonstrated that most of compounds had
potent AChE inhibitory with reserving potential of BuChE inhibition. Among them, compound 6i
bearing a 4-(3-bromophenyl) moiety showed the most potent activity against AChE/BuChE (IC50s
values of 0.069 and 1.35 µM, respectively). The anti-AChE activity of 6i was five times more than
that of tacrine. The SAR study revealed that chloro/bromo substituent at ortho or meta position of the
4-phenyl ring can improve the anticholinesterase activity.
Keywords: Acetylcholinesterase; butyrylcholinesterase; Alzheimer’s disease; tacrine
Corresponding authors: E-mail address: ysarrafi@umz.ac.ir (Y. Sarrafi); E-mail address:
aforoumadi@yahoo.com (A. Foroumadi), Tel.: +98 21 66954708; fax: +98 21 66461178.
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1. Introduction
Alzheimer’s disease (AD) is a complex neurodegenerative disorder of the brain, affecting a
large number of elderly populations. AD is the most common cause of dementia and death in
aging peoples [1,2]. The estimated number of patients with dementia in the world is currently
expected to be 46 million rising to 131.5 million by 2050 [3].
The molecular basis of AD is yet to be determined but this disease is associated with a
deficiency in cholinergic neurotransmission in the brain areas related to memory and learning
[4]. Furthermore, β-amyloid plaques and neurofibrillary tangles are the major pathological
signs in the brain of AD patients [5].
Currently, most of the drugs approved for the AD therapy are acetylcholinesterase (AChE)
inhibitors including donepezil, rivastigmine, and galantamine [6-8]. These drugs are able to
enhance cholinergic neurotransmission by increasing acetylcholine (ACh) level in the central
nervous system [9]. Recent studies in AD patients indicated that while the AChE activity is
greatly reduced in specific regions of the brain, the butyrylcholinesterase (BuChE) activity
increases. Due to the involvement of BuChE in the hydrolysis of ACh, it seems the inhibition
of both types of cholinesterase enzymes is essential for successful treatment of AD [10-12].
Besides normal hydrolyzing function, AChE also acts as a promoter of Aβ fibril formation
[13,14] It has been demonstrated AChE inhibitors that interact with the peripheral site of the
.
enzyme could inhibit the aggregation of β-amyloid proteins [15]. However, the clinical
usefulness of AChE inhibitors is limited, mainly due to their adverse effects. In particular,
tacrine which was the first AChE inhibitor approved by FDA, has been removed from the
market because of hepatotoxicity [16-18]. Accordingly, a part of attentions is focused on
developing new AChE inhibitors derived from tacrine with improved activity and reduced
toxicity.
Several reports have considered design of tacrine-like compounds by replacing or annulating
the benzene ring in tacrine with different heterocyclic systems. Recently we introduced some
pyrazolo[4′,3′:5,6]pyrano[2,3-b]quinolines (I) as new tacrine-based AChE inhibitors [19,20].
Maalej et al. synthesized a series of benzopyran-derived tacrines namely 7-aryl-9,10,11,12-
tetrahydro-7H-benzo[7,8]chromeno[2,3-b]quinolin-8-amines (II) as multi-targeted AChE
inhibitors. Based on their biological results, the compounds had potent and selective
inhibitory activity against AChE in the micromolar and submicromolar ranges [21,22].
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Moreover, numerous tacrine-based compounds (III) bearing 4H-pyrano[2,3-b]quinoline-3-
carboxylate scaffold have been synthesized and evaluated by Marco team as
acetylcholinesterase inhibitors with neuroprotective activity [23].
As seen in Fig. 1, all described compounds I-III share in the same scaffold namely
tetrahydro-pyrano[2,3-b]quinoline resembling the framework of well-known drug tacrine. In
continuation of our works on hetero-annulated pyrans [24, 25], the tetrahydro-pyrano[2,3-
b]quinoline structure was used as a pharmacophoric moiety in the design of new AChE
inhibitors. Thus, we report here the synthesis and biological evaluation of ethyl 5-amino-2-
phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-carboxylates 6a-p. Structurally, the
designed compounds 6a-p are derived from compounds III by replacing the methyl group
with phenyl ring which enables the molecule to make additional π-π interaction with aromatic
or heteroaromatic amino acid residues of enzyme binding site. Furthermore, the attached
phenyl ring in the designed compounds 6a-p is matched to benzene moiety annulated to the
tetrahydro-pyrano[2,3-b]quinoline in the lead compounds II.
<Fig. 1>
2. Chemistry
The target compounds 6a-p were synthesized in two steps as illustrated in Scheme 1. Firstly,
a multi-component reaction between ethyl 3-oxo-3-phenylpropanoate (1), appropriate
benzaldehyde derivative 2 and malononitrile (3) in the presence of piperdine as catalyst
afforded corresponding ethyl 6-amino-5-cyano-4H-pyran-3-carboxylate derivatives 4a-p.
Cyclization of compounds 4a-p with cyclohexanone by using Friedländer reaction in the
presence of AlCl3 gave the final compounds 6a-p.
<Scheme 1>
3. Results and discussion
The anti-cholinesterase activity of the synthesized compounds 6a-p was evaluated in vitro
against AChE and BuChE. The obtained results were summarized as IC50 values of tested
compounds in Table 1. The well-known cholinesterase inhibitor tacrine was used as reference
drug.
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3.1. Anti-AChE activity
A survey on the IC50 values against AChE revealed that all compounds had significant
inhibitory activity at micro-molar and submicro-molar levels (IC50= 0.069-6.22 µM). The 3-
bromo- derivative 6i with IC50 value of 0.069 µM was found to be the most potent compound
against AChE. This compound was 5-fold more potent than reference drug tacrine. It should
be noted that the 2-Br, 2-Cl and 3-Cl analogs (compounds 6m, 6n and 6j, respectively) were
also more potent than tacrine against AChE. Furthermore, the anti-AChE activity of
compounds 6a, 6c, 6f, and 6o was comparable to that of tacrine. The comparison of para-
substituted compounds 6b-g with unsubstituted congener 6a demonstrated that the
introduction of substituent at the para position of 4-phenyl ring cannot improve the anti-
AChE activity. As observed in compound 6b, the 4-methyl substituent significantly
decreased the activity. In contrast, the insertion of bromo or chloro group at the ortho or meta
position increased the inhibitory activity. Thus, compounds 6i, 6j, 6m and 6n showed
superior activity respect to the parent compound 6a. The best result was obtained by bromo
substituent at meta position. The 2,4-dichloro analog 6p was less active than 2-chloro and 4-
chloro derivatives (compounds 6n and 6e). Therefore, the concurrent 2,4-substitution was not
favorable.
3.2. Anti-BuChE activity
Based on IC50 values listed in Table 1, BuChE enzyme was less susceptible to the target
compounds compared to AChE. However, the tested compounds showed remarkable anti-
BuChE activity with IC50 values in the range of 1.35-74.25 µM. Interestingly, the most potent
compound against AChE (compound 6i) displayed the highest activity towards BuChE.
Moreover, potent compounds against AChE (6j and 6m-o) significantly inhibited the BuChE
enzyme (IC50s= 2.14-3.63 µM). From the SAR point of view, there was similarity between
BuChE and AChE. While the para-substitution on the 4-phenyl moiety diminished the
activity, the presence of halogen (Cl or Br) at the ortho or meta position had positive effect
on BuChE inhibition. Again best result was obtained by compound 6i bearing a bromo group
at the meta position.
<Table 1>
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3.3. Kinetic study of AChE inhibition
To determine the mechanism of enzyme inhibition the kinetic study was performed using 6i
as representative compound. The relative velocity of the enzyme was determined on three
increasing concentrations of the substrate ATChI (acetylthiocholine iodide). To construct the
Lineweaver-Burk plot, the enzyme velocity was measured in the presence of inhibitor (6i) at
following concentrations: 0, 35, 70 and 140 nM. The Lineweaver-Burke plot was then
schemed using the reciprocal of velocity and substrate concentration (Fig. 2a). Based on the
obtained plot, a mixed type of inhibition by compound 6i was established. Using the
Lineweaver-Burk secondary plot (Fig. 2b), a Ki value equal to 48.5 nM was obtained for
compound 6i.
<Fig. 2>
3.4. Molecular Docking
3.4.1. Docking study with AChE
The ligand-protein docking was guided by AutoDock Vina (1.1.2) [26] to predict the binding
poses of the ligand in the active site of AChE. The 3D coordinate of the AChE (PDB ID:
1acj) in complex with tacrine was retrieved from Protein Data Bank (PDB) at
http://www.rcsb.org/pdb/home/home.do. For protein preparation, all of the non-protein atoms
were removed and then minimized using OPLS3 force field (RMSD= 0.3 Å). The grid box
with the size of 25×25×25 was determined and the center of box was fixed on co-crystal
ligand. After docking the best pose was selected for further analysis.
The molecular docking results for representative compound 6i showed that the ligand was
well accommodated in the active site. The ligand was laid at the bottom of the active site
gorge near catalytic triad. Since the chemical nature of the binding site was hydrophobic
therefore the hydrophobic interactions played an important role in binding affinity. Tyr70,
Asp72, Trp84, Phe290, Phe330, Tyr334, Trp432, Ile439 and Tyr442 are responsible for
hydrophobic interaction with hydrophobic fragments of the ligand as depicted in Fig. 3 (All
figures was prepared using Discovery Studio Visualizer 4.5 client). The central pyridine ring
was stacked between two aromatic residues Trp84 and Phe330 by making π-π interaction.
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This orientation of the ligand enables it to make a hydrogen bond with His440 via amino
moiety. In this position the cyclohexyl ring was well fitted in the hydrophobic pocket that
located in the vicinity of catalytic triad. At the other side of the ligand that has more
hydrophobic nature, the hydrophobic interactions are dominant. The 2-phenyl moiety
attached to pyran ring involved in T-shaped π-π interaction and π-anion interaction with
Tyr131 and Asp72, respectively. The pendant ethyl carboxylate was fixed by three π-alkyl
interactions accompanied by hydrogen bond with Ser122. Finally the 3-bromophenyl moiety
made another T-shaped π-π interaction with Trp84.
<Fig. 3>
3.4.2. Docking study with BuChE
Due to the significant potency of target compounds on BuChE, a ligand-protein docking was
also conducted to predict the binding poses of the representative compound 6i in the active
site of BuChE. The 3D coordinate of the human BuChE (PDB ID: 5LKR) in complex with N-
propargylpiperidine was retrieved from PDB. For protein preparation, all of the non-protein
atoms were removed and then minimized using OPLS3 force field (RMSD= 0.3 Å). The grid
box with the size of 30×30×30 was determined and the center of box was fixed on co-
crystalized ligand. After docking the best pose was selected for further analysis.
As depicted in Fig. 4, compound 6i was well fitted in the active site cavity of BuChE. The
hydrophobic and hydrogen bonds were predominant in ligand binding. The nitrogen atom of
the core pyridine ring contributed in hydrogen-binding with Ser198, the key residue in the
catalytic triad. The fused cyclohexane ring oriented to hydrophobic pocket composed by
Val288, Leu286 and Trp231. On the other side of molecule, the π-π interactions were
predominant. Two phenyl rings on position 2 and 4 were involved in π-π interactions with
Trp82 and Tyr332 respectively.
<Fig. 4>
3.5. Prediction of blood-brain barrier (BBB) permeability
The BBB penetration of synthesized compounds 6a-p was predicted by using admetSAR
server (http://lmmd.ecust.edu.cn:8000/predict/) [27]. The predicted data were listed in Table
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2. As seen, all compounds with the exception of nitro- derivatives 6g and 6l are BBB
permeable.
<Table 2>
3.6. Neuroprotective effect against ROS-mediated cell apoptosis in differentiated PC12 cells
The neuroprotective potential of the promising compounds 6i, 6j, 6m and 6n against H2O2-
induced cell death in differentiated PC12 cells was evaluated in comparison to quercetine and
tactine. The differentiated cells from PC12 neurons were pretreated with different
concentrations (1, 10 and 100 µM) of the compounds for 3h, before treatment with H2O2 (300
µM). Addition of H2O2 induced apoptosis in PC12 cells and then cell viability was measured
after 24 h by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)
colorimetric assay.
The obtained results for compounds 6i, 6j, 6m and 6n in the concentrations of 1, 10 and 100
µM were compared with control group, quercetine and tacrine (10 µM) and depicted in Fig.
5. As shown in Fig. 5, H2O2 significantly reduced the cell viability compared with negative
control. The pretreatment of PC12 cells with the test compounds protected neurons against
cell death. In particular, compound 6n could significantly protect cells at the concentration of
10 µM (P<0.0001). All compounds were effective on prevention of ROS mediated cell
apoptosis at concentration of 100 µM (P<0.01).
<Fig. 5>
3.7. Cytotoxicity on Hepatocyts
The cytotoxic activity of selected compounds 6i, 6j, 6l, 6m and 6n was evaluated against
HepG2 cell line by using MTT cell viability assay. The IC50 values of compounds were
determined and compared with that of reference drug tacrine (Table 3). While compounds 6l-
n showed no significant cytotoxic activity against HepG2 cells (IC50s >100 µg/mL) but
compounds 6i and 6j with IC50 values of 72.8 and 50.4 µg/mL showed moderate cytotoxicity.
However, the cytotoxicity of the promising compound 6i was several times less than that of
reference drug tacrine (IC50= 17.9 µg/mL).
<Table 3>
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4. Conclusion
In order to find new potential entities for treatment of AD, a series of poly-functionalized
tacrine-derived compounds namely 5-amino-2-phenyl-4H-pyrano[2,3-b]quinoline-3-
carboxylates were designed and synthesized as cholinesterase inhibitors. The in vitro
cholinesterases inhibition assay demonstrated that most of compounds had potent AChE
inhibitory with reserving potential of BuChE inhibition. Among them, compound 6i bearing a
3-bromophenyl moiety at 4-position of the 4H-pyrano[2,3-b]quinoline ring showed the most
potent activity against AChE/BuChE (IC50s values of 0.069 and 1.35 µM, respectively). The
anti-AChE activity of 6i was five times more than that of tacrine. Furthermore, the promising
compound 6i showed less cytotoxicity on HepG2 cells compared to tacrine. The SAR study
by the modification of substituent on the 4-phenyl ring revealed that while the electron
donating and electron withdrawing group are not favorable for para-position, the electron
withdrawing group such as chloro and bromo at ortho or meta can improve the cholinesterase
inhibition potential of compounds.
5. Experimental
All commercially available reagents were used without further purification. TLC was
conducted on silica gel 250 micron, F254 plates. Melting points were measured on a Kofler
hot stage apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR
Magna 550 spectrograph (KBr discs). All NMR spectra were recorded on Brucker 400 MHz
NMR instruments. The chemical shifts (δ) and coupling constants (J) are expressed in parts
per million and Hertz, respectively. The atoms numbering of the target compounds used for
1H NMR data are shown in Scheme 1. Mass spectra of the products were obtained with an HP
(Agilent technologies) 5937 Mass Selective Detector. Elemental analyses were carried out by
a CHN-Rapid Heraeus elemental analyzer. The results of elemental analyses (C, H, N) were
within ±0.4% of the calculated values.
5.1. General procedure for the preparation of ethyl 6-amino-5-cyano-2,4-diphenyl-4H-pyran-
3-carboxylate derivatives 4a-p
A mixture of ethyl 3-oxo-3-phenylpropanoate (1, 1 mmol), aromatic aldehyde 2 (1 mmol),
malonoitrile (3, 1 mmol), piperdine (1 drop) in ethanol (5 mL) was refluxed for 3 h. After
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completion of the reaction, the mixture was cooled to 0-5 °C. The precipitated solid was
filtered, washed with cold ethanol and dried to give compounds 4a-p.
5.2. General procedure for the preparation of ethyl 5-amino-2,4-diphenyl-6,7,8,9-tetrahydro-4H-
pyrano[2,3-b]quinoline-3-carboxylate derivatives 6a-p
Aluminum chloride (1.5 mmol) was suspended in dry 1,2-dichloroethane (5 mL) at room
temperature under nitrogen atmosphere. After stirring the suspension for a few minutes, the
corresponding 6-amino-5-cyano-4H-pyran 4a-p (1 mmol) and cyclohexanone (5, 1.5 mmol)
were added to the mixture and the reaction mixture was heated under reflux for 3-6 h.
Progress of the reaction was monitored by TLC. After completion, water was added and the
mixture was basified with 10% sodium hydroxide solution to pH= 8-9. The layers were
separated and the aqueous layer was extracted with dichloromethane (3 × 15 mL). The
combined extracts were dried (Na2SO4) and concentrated. Purification by column
chromatography on silica gel afforded the title compounds 6a-p.
5.2.1.
Ethyl
5-amino-2,4-diphenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-
carboxylate (6a)
1
Yield 80%; yellow solid; mp 154-156 °C. H NMR (400 MHz, CDCl3) δ: 7.49–7.47 (m, 2H,
Hphenyl), 7.44-7.39 (m, 2H, Hphenyl), 7.38-7.35 (m, 3H, Hphenyl), 7.34–7.31 (m, 2H, Hphenyl),
7.25–7.21 (m, 1H, Hphenyl), 4.98 (s, 1H, H4), 4.12 (br s, 2H, NH2), 3.89–3.84 (m, 2H, CH2),
2.78–2.75 (m, 2H, 2H9), 2.37–2.32 (m, 1H, H6), 2.24–2.20 (m, 1H, H6), 1.82–1.79 (m, 4H,
2H7 and 2H8), 0.84 (t, 3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 167.14, 157.87,
154.72, 154.16, 150.48, 143.48, 134.90, 129.35, 128.84, 128.45, 127.58, 127.29, 113.79,
107.98, 99.26, 60.29, 39.53, 32.48, 22.92, 22.58, 22.36, 13.46. Anal. Calcd for C27H26N2O:
C, 76.03; H, 6.14; N, 6.57; Found C, 76.17; H, 6.29; N, 6.71.
5.2.2. Ethyl 5-amino-2-phenyl-4-(p-tolyl)-6,7,8,9-tetrahydro-4H-pyrano[2,3-b]quinoline-3-
carboxylate (6b)
Yield 77%; yellow solid; mp 114-116 °C. IR (KBr, cm-1) νmax: 3461, 3330, 3211, 2942, 1691,
1
1633, 1449, 1278, 1084. H NMR (400 MHz, CDCl3) δ: 7.49-7.47 (m, 2H,Hphenyl), 7.37–7.33
(m, 3H, Hphenyl), 7.32–7.28 (m, 2H, Hphenyl), 7.14–7.10 (m, 2H, Hphenyl), 4.94 (s, 1H, H4), 4.13
(bs, 2H, NH2), 3.90–3.84 (m, 2H, CH2), 2.78–2.74 (m, 2H, H9), 2.36–2.32 (m, 1H, H6), 2.31
(s, 3H, CH3), 2.28–2.20 (m, 1H, H6), 1.82–1.79 (m, 4H, 2H7 and 2H8), 0.85 (t, 3H, CH3, J=
7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 167.19, 157.75, 154.69, 154.01, 150.48, 140.50,
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136.94, 134.95, 129.56, 129.32, 128.85, 128.30, 127.58, 113.76, 108.11, 99.43, 60.27, 39.08,
32.43, 22.92, 22.59, 22.37, 21.11, 13.47. Anal. Calcd for C28H28N2O: C, 76.34; H, 6.41; N,
6.36; Found C, 76.51; H, 6.68; N, 6.49.
5.2.3.
Ethyl
5-amino-4-(4-bromophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6d)
1
Yield 70%; off-white solid; mp 180-182 °C. H NMR (400 MHz, CDCl3) δ: 7.46-7.42 (m,
4H, Hphenyl), 7.38–7.34 (m, 3H, Hphenyl), 7.30–7.28 (m, 2H, Hphenyl), 4.95 (s, 1H, H4), 4.12 (br
s, 2H, NH2), 3.86 (q, 2H, CH2, J= 7.2Hz), 2.71–2.78 (m, 2H, H9), 2.37–2.33 (m, 1H, H6),
13
2.26–2.23 (m, 1H, H6), 1.83–1.78 (m, 4H, 2H7 and 2H8), 0.83 (t, 3H, CH3, J= 7.2 Hz). C
NMR (100 MHz, CDCl3) δ: 167.04, 158.45, 154.64, 154.37, 150.35, 142.52, 134.70, 131.93,
130.11, 129.51, 128.84, 127.62, 121.21, 113.90, 107.47, 98.76, 60.42, 38.84, 32.39, 22.94,
22.52, 22.31, 13.43. Anal. Calcd for C27H25BrN2O3: C, 64.16; H, 4.99; N, 5.54; Found C,
64.26; H, 5.18; N, 5.74.
5.2.4.
Ethyl
5-amino-4-(4-chlorophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6e)
1
Yield 74%; off-white solid; mp 185-187 °C. H NMR (400 MHz, CDCl3) δ: 7.48–7.45 (m,
2H, Hphenyl), 7.39–7.33 (m, 5H, Hphenyl), 7.29–7.27 (m, 2H, Hphenyl), 4.97 (s, 1H, H4), 4.13 (br
s, 2H, NH2), 3.90–3.84 (m, 2H, CH2), 2.79–2.77 (m, 2H, H9), 2.38–2.33 (m, 1H, H6), 2.27–
13
2.24 (m, 1H, H6), 1.84–1.82 (m, 4H, 2H7 and 2H8), 0.84 (t, 3H, CH3, J= 7.2 Hz). C NMR
(100 MHz, CDCl3) δ: 167.05, 158.35, 154.62, 154.32, 150.39, 141.96, 134.69, 133.09,
129.75, 129.52, 129.00, 128.84, 127.63, 113.89, 107.58, 98.83, 60.42, 38.79, 32.36, 22.93,
22.51, 22.31, 13.43. Anal. Calcd for C27H25ClN2O3: C, 70.35; H, 5.47; N, 6.08; Found C,
70.57; H, 5.54; N, 6.27.
5.2.5.
Ethyl
5-amino-4-(4-fluorophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6f)
1
Yield 85%; off-white solid; mp 178-179 °C. H NMR (400 MHz, CDCl3) δ: 7.50–7.49 (m,
2H, Hphenyl), 7.41–7.35 (m, 5H, Hphenyl), 7.00–6.95 (m, 2H, Hphenyl), 6.42 (br s, 2H, NH2), 5.07
(s, 1H, H4), 3.91–3.88 (m, 2H, CH2), 3.02–2.99 (m, 2H, H9), 2.54–2.48 (m, 1H, H6), 2.38–
13
2.34 (m, 1H, H6), 1.79–1.72 (m, 4H, 2H7 and 2H8), 0.85 (t, 3H, CH3, J= 7.2 Hz). C NMR
(100 MHz, CDCl3) δ: 166.11, 163.51, 161.05, 155.83, 154.12, 150.10, 147.04, 136.45,
131.84, 130.48, 130.16, 130.08, 128.78, 127.89, 116.17, 115.95, 113.96, 110.02, 98.46,
10

ACCEPTED MANUSCRIPT
61.22, 37.39, 27.29, 22.66, 21.29, 20.90, 13.45. Anal. Calcd for C27H25FN2O3: C, 72.96; H,
5.67; N, 6.30; Found C, 72.84; H, 5.45; N, 6.54.
5.2.6.
Ethyl
5-amino-4-(4-nitrophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6g)
Yield 83%; yellow solid; mp 184-186 °C; IR (KBr, cm-1) νmax: 3467, 3328, 3205, 2933, 1698,
1
1643, 1451, 1298, 1238, 1090. H NMR (400 MHz, CDCl3) δ: 8.20–8.17 (m, 2H, Hphenyl),
7.63–7.59 (m, 2H, Hphenyl), 7.48–7.45 (m, 2H, Hphenyl), 7.43–7.39 (m, 1H, Hphenyl), 7.38–7.34
(m, 2H, Hphenyl), 5.11 (s, 1H, H4), 4.11 (br s, 2H, NH2), 3.92–3.84 (m, 2H, CH2), 2.79-2.78
(m, 2H, H9), 2.40–2.35 (m, 1H, H6), 2.30–2.23 (m, 1H, H6), 1.85–1.80 (m, 4H, 2H7 and 2H8),
0.83 (t, 3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 166.88, 159.65, 154.98, 154.66,
150.84, 150.10, 147.04, 134.47, 129.76, 129.27, 128.87, 127.68, 124.09, 114.10, 106.63,
98.16, 60.59, 39.21, 32.52, 22.98, 22.49, 22.27, 13.40. Anal. Calcd for C27H25N3O5: C, 68.78;
H, 5.34; N, 8.91; Found C, 68.94; H, 5.54; N, 8.77.
5.2.7.
Ethyl
5-amino-4-(3-methoxyphenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6h)
Yield 85%; yellow solid; mp 142-145 °C. IR (KBr, cm-1) νmax: 3461, 3322, 3214, 2942, 1690,
1
1635, 1450, 1291, 1241, 1084. H NMR (400 MHz, CDCl3) δ: 7.49-7.47 (m, 2H, Hphenyl),
7.39-7.32 (m, 3H, Hphenyl), 7.25-7.21 (m, 1H, Hphenyl), 7.02–7.00 (m, 1H, Hphenyl), 6.94–6.96
(m, 1H, Hphenyl), 6.78–6.76 (m, 1H, Hphenyl), 4.97 (s, 1H, H4), 4.16 (br s, 2H, NH2), 3.87 (q,
2H, J= 7.2 Hz, CH2), 3.76 (s, 3H, OCH3), 2.76–2.75 (m, 2H, H9), 2.36–2.33 (m, 1H, H6),
13
2.25–2.21 (m, 1H, H6), 1.82–1.79 (m, 4H, 2H7 and 2H8), 0.85 (t, 3H, CH3, J= 7.2 Hz). C
NMR (100 MHz, CDCl3) δ: 167.11, 160.00, 157.80, 154.65, 154.10, 150.62, 145.02, 134.88,
129.78, 129.36, 128.82, 127.60, 124.84, 114.48, 113.77, 112.36, 107.89, 99.12, 60.32, 55.21,
39.52, 32.42, 29.69, 22.92, 22.56, 22.36, 13.49. Anal. Calcd for C28H28N2O4: C, 73.66; H,
6.18; N, 6.14; Found C, 73.84; H, 6.24; N, 6.08.
5.2.8.
Ethyl
5-amino-4-(3-bromophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6i)
1
Yield 85%; off-white solid; mp 218-220 °C. H NMR (400 MHz, CDCl3) δ: 7.52–7.49 (m,
2H, Hphenyl), 7.41–7.40 (m, 1H, Hphenyl), 7.40–7.37 (m, 4H, Hphenyl), 7.34–7.31 (m, 1H,
Hphenyl), 7.26–7.20 (m, 2H, Hphenyl), 4.97 (s, 1H, H4), 4.08 (br s, 2H, NH2), 3.91–3.87 (m, 2H,
CH2), 2.82–2.79 (m, 2H, H9), 2.40–2.33 (m, 1H, H6), 2.32–2.48 (m, 1H, H6), 1.87–1.84 (m,
11

ACCEPTED MANUSCRIPT
4H, 2H7 and 2H8), 0.90 (t, 3H, J= 7.2Hz, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.85,
158.68, 153.46, 152.34, 151.42, 147.51, 133.91, 135.17, 130.45, 129.68, 127.97, 128.41,
126.74, 126.14, 113.40, 106.14, 97.89, 60.75, 38.87, 31.54, 22.74, 22.12, 22.02, 13.74. MS,
m/z (%): 504 [M+] (10.7), 424 (100), 349 (85.3), 297 (76.8), 77 (26.2). Anal. Calcd for
C27H25BrN2O3: C, 64.16; H, 4.99; N, 5.54; Found C, 64.21; H, 5.14; N, 5.49.
5.2.9.
Ethyl
5-amino-4-(3-chlorophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6j)
Yield 78%; yellow solid; mp 214-217 °C; IR (KBr, cm-1) νmax: 3416, 2860, 1691, 1640, 1451,
1
1243, 1089. H NMR (400 MHz, CDCl3) δ: 7.50– 7.47 (m, 2H, Hphenyl), 7.41–7.40 (m, 1H,
Hphenyl), 7.39–7.36 (m, 4H, Hphenyl), 7.33–7.30 (m, 1H, Hphenyl), 7.26–7.21 (m, 2H, Hphenyl),
4.98 (s, 1H, H4), 4.10 (br s, 2H, NH2), 3.93–3.87 (m, 2H, CH2), 2.80–2.78 (m, 2H, H9), 2.40–
2.35 (m, 1H, H6), 2.30–2.48 (m, 1H, H6), 1.87–1.83 (m, 4H, 2H7 and 2H8), 0.87 (t, 3H, CH3,
13
J= 7.2 Hz). C NMR (100 MHz, CDCl3) δ: 166.93, 158.53, 154.66, 153.53, 150.32, 145.47,
134.72, 134.68, 130.06, 129.51, 128.57, 127.63, 127.60, 126.64, 113.91, 107.35, 98.59,
60.45, 39.20, 32.48, 22.95, 22.53, 22.33, 13.46. MS, m/z (%): 460 [M+] (1.8), 349 (100), 321
(48.5), 273 (22.7), 105 (33.6), 77 (11.2). Anal. Calcd for C27H25ClN2O3: C, 70.35; H, 5.47; N,
6.08; Found C, 70.52; H, 5.51; N, 6.29.
5.2.10.
Ethyl
5-amino-4-(3-nitrophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6l)
1
Yield 87%; yellow solid; mp 194-196 °C. H NMR (400 MHz, CDCl3) δ: 8.33-8.31 (m, 3H,
Hphenyl), 8.13-8.10 (m, 1H, Hphenyl), 7.76–7.73 (m, 1H, Hphenyl), 7.52–7.47 (m, 3H, Hphenyl),
7.43–7.35 (m, 3H, Hphenyl), 5.11 (s, 1H, H4), 4.12 (br s, 2H, NH2), 3.93–3.87 (m, 2H, CH2),
2.81–2.78 (m, 2H, H9), 2.37–2.34 (m, 1H, H6), 2.29–2.25 (m, 1H, H6), 1.88–1.81 (m, 4H, 2H7
and 2H8), 0.85 (t, 3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 166.77, 159.61,
154.99, 154.69, 150.04, 148.38, 145.69, 134.45, 129.89, 129.73, 128.88, 127.69, 123.17,
122.44, 114.07, 106.77, 98.10, 60.62, 39.10, 32.51, 22.98, 22.48, 21.27, 13.45. Anal. Calcd
for C27H25N3O5: C, 68.78; H, 5.34; N, 8.91; Found C, 68.97; H, 5.50; N, 8.71.
5.2.11.
Ethyl
5-amino-4-(2-bromophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6m)
Yield 82%; off-white solid; mp 207-208 °C. IR (KBr, cm-1) νmax: 3456, 3320, 3200, 2931,
1
1687, 1642, 1466, 1291, 1233, 1084. H NMR (400 MHz, CDCl3) δ: 7.54–7.52 (m, 2H,
12

ACCEPTED MANUSCRIPT
Hphenyl), 7.47–7.44 (m, 1H, Hphenyl), 7.39–7.36 (m, 4H, Hphenyl), 7.28–7.24 (m, 1H, Hphenyl),
7.20–7.18 (m, 1H, Hphenyl), 5.41 (s, 1H, H4), 4.47 (bs, 2H, NH2), 3.85 (q, 2H, CH2, J= 7.2
Hz), 2.83–2.75 (m, 2H, H9), 2.42–2.37 (m, 1H, H6), 2.24–2.20 (m, 1H, H6), 1.85–1.79 (m,
4H, 2H7 and 2H8), 0.91 (t, 3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 168.54,
160.71, 155.41, 153.97, 151.54, 142.41, 134.78, 132.84, 131.89, 130.43, 129.95, 129.14,
128.74, 128.95, 128.47, 113.85, 108.14, 99.14, 60.54, 35.68, 32.51, 22.98, 22.64, 22.52,
13.61. MS, m/z (%): 506 [M+] (2.8), 349 (100), 321 (51.9), 105 (43.6), 77 (45.5). Anal. Calcd
for C27H25BrN2O3: C, 64.16; H, 4.99; N, 5.54; Found C, 64.20; H, 5.14; N, 5.51.
5.2.12.
Ethyl
5-amino-4-(2-chlorophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6n)
Yield 85%; white solid; mp 118-121 °C; IR (KBr, cm-1) νmax: 3406, 2929, 1695, 1637, 1520,
1
1346, 1244, 1086. H NMR (400 MHz, CDCl3) δ: 7.52–7.50 (m, 2H, Hphenyl), 7.48–7.45 (m,
1H, Hphenyl), 7.40–7.33 (m, 4H, Hphenyl), 7.26–7.22 (m, 1H, Hphenyl), 7.18–7.14 (m, 1H,
Hphenyl), 5.42 (s, 1H, H4), 4.51 (br s, 2H, NH2), 3.91 (q, 2H, CH2, J= 7.2 Hz), 2.80–2.68 (m,
2H, H9), 2.36–2.32 (m, 1H, H6), 2.23–2.19 (m, 1H, H6), 1.81–1.78 (m, 4H, 2H7 and 2H8),
0.89 (t, 3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 166.72, 159.13, 154.58, 153.91,
150.60, 141.51, 134.66, 131.92, 131.20, 129.56, 129.20, 128.81, 128.44, 128.12, 127.71,
113.47, 107.42, 98.93, 60.43, 35.55, 32.31, 22.94, 22.51, 22.33, 13.56. MS, m/z (%): 460
[M+] (25), 349 (100), 321 (34.8), 105 (29.7), 77 (14.3). Anal. Calcd for C27H25ClN2O3: C,
70.35; H, 5.47; N, 6.08; Found C, 70.53; H, 5.57; N, 6.27.
5.2.13. Ethyl 5-amino-4-(2,4-dichlorophenyl)-2-phenyl-6,7,8,9-tetrahydro-4H-pyrano[2,3-
b]quinoline-3-carboxylate (6p)
1
Yield 80%; off-white solid; mp 134-137 °C. H NMR (400 MHz, CDCl3) δ: 7.50–7.48 (m,
2H, Hphenyl), 7.38–7.32 (m, 4H, Hphenyl), 7.31–7.25 (m, 1H, Hphenyl), 7.16–7.12 (m, 1H,
H
phenyl), 5.90 (s, 1H, H4), 4.39 (br s, 2H, NH2), 3.87–3.79 (m, 2H, CH2), 2.75–2.72 (m, 2H,
H9), 2.34–2.28 (m, 1H, H6), 2.24–2.20 (m, 1H, H6), 1.80–1.78 (m, 4H, 2H7 and 2H8), 0.79 (t,
3H, CH3, J= 7.2 Hz). 13C NMR (100 MHz, CDCl3) δ: 166.81, 159.94, 155.28, 154.07,
150.65, 137.10, 135.69, 135.63, 135.17, 131.34, 129.30, 128.83, 128.45, 128.15, 127.68,
113.46, 103.11, 95.73, 60.26, 36.23, 32.25, 22.92, 22.50, 22.36, 13.44. Anal. Calcd for
C27H24Cl2N2O3: C, 65.46; H, 4.88; N, 5.65; Found C, 65.54; H, 4.68; N, 5.89.
13

ACCEPTED MANUSCRIPT
5.3. Cholinesterases inhibition assay
AChE from Electrophorus electricus (electric eel, type V-s, lyophilized powder, ≥1000
units/mg protein), human BuChE (recombinant, expressed in goat, ≥500 units/mg protein),
acetylthiocholine iodide, butyrylthiocholine iodide, DTNB [5,5’-dithiobis(2-nitrobenzoic
acid)], and tacrine were purchased from Sigma-Aldrich. The inhibitory activity of test
compounds 6a-p against AChE/BuChE was assessed by Ellman’s method [28].
Ethanol/DMSO (9:1, 1 mL) was used as a solvent system for dissolving test compounds. The
stock solution was then diluted using phosphate buffer to achieve final concentrations. A
mixture of phosphate buffer 0.1 M, pH=8.0 (2000 µL), DTNB 0.1 M (65 µL), corresponding
enzyme 0.2 U/ml (35 µL) and inhibitor (50 µL) was incubated for 5 min at room temperature.
Then, acetylthiocholine iodide 0.01M (10 µL) was added and the change of the absorbance
was measured at 412 nm for 1 min. The same protocol was applied for determination of
BuChE activity by using butyrylthiocholine iodide as substrate. All experiments were
performed on a Synergy HTX Multi-Mode Reader-BioTek.
5.4. H2O2-induced cell death in PC12 cells
All culture media and supplements were purchased from Gibco. PC12 cell line was purchased
from Pasteur institute. Cells were cultivated in DMEM supplemented with 10% fetal calf
serum, 5% horse serum and antibiotics (100 units/mL penicillin, 100 ꢀg/mL streptomycin).
To induce neuronal differentiation, PC12 cells were re-suspended using trypsin/EDTA
(0.25% ) and seeded in 96 well culture plate (4000 cells/well) and cultured for 1 week in
differentiation medium [DMEM + 2% horse serum+ NGF (100 ng/mL) + penicillin &
streptomycin]. To evaluate the effect of drugs on survival rate of neurons, the culture medium
was changed to NGF free medium and different concentrations of test compounds (1, 10, 100
ꢀM) were applied on cells. Quercetin and tacrine (10 ꢀM) were used as positive controls.
Drugs were diluted into DMEM and added to each well in the volume of 10 ꢀL. Then, three
hours later induction of ROS mediated apoptosis was initiated by adding the H2O2 (300 ꢀM)
to the medium and after 12 h, MTT assay was performed. MTT solution (5 mg/mL) was
added to each well in a volume of 10 ꢀL, and 3 h later 100 ꢀL of the solubilization solution
[10% SDS in 0.01 M HCl (w/v)] was added into each well. The plates were allowed to stand
overnight in the incubator in a humidified atmosphere. Absorbance at 570 nm was
14

ACCEPTED MANUSCRIPT
determined for each well using an ELISA reader. Each experimental was performed in
triplicates.
5.5. Cytotoxicity assay on HepG2 cells
The HepG2 cell line was provided from Pasture institute, Tehran, Iran. Cells were cultured at
25 mL flask in RPMI 1640 medium (Biowest, France) containing 10 % Fetal Bovine Serum
(Gibco, USA) and 1% pen/strep (PAA, Austria) in saturated humidity and 5% CO2 incubator.
The cytotoxicity of compounds was studied by MTT assay. The cells were seeded in 96 well
plate (6 × 106 cell per well). After 20 h, the cells were treated with various concentrations of
compounds (500, 250, 125, 60 and 30 µg dissolved in DMSO). The concentration of DMSO
was 2% of medium culture. After 24 h incubation, the RPMI 1640 medium of each well
replaced with 100 µl MTT solution (0.5 mg/mL dissolved in PBS) and the plate was
incubated for 4 h in saturated humidity and 5% CO2 incubator. Then, 100 µl DMSO was
added to each well, gently shaken and the absorbance was read with ELISA reader at 570 nm.
Acknowledgments
This research has been supported by grants from the Research Council of Tehran University
of Medical Sciences (Grant No. 95-03-92-33171) and Iran National Science Foundation
(INSF).
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Captions:
Figure 1. The structures of reported tacrine-derived AChE inhibitors I-III and newly
designed compounds 6a-p
Figure 2. Kinetic of AChE inhibition by compound 6i: Left) Lineweaver-Burk plot, Right)
Lineweaver-Burk secondary plot
Figure 3. (a) The best pose of the most active compound (6i) in the active site of AChE; (b)
residues of the active site involved in ligand binding.
Figure 4. Illustration of compound 6i binding mode in the active site of BuChE
Figure 5. Neuroprotective activity of selected compounds 6i, 6j, 6m and 6n against ROS-
mediated cell apoptosis in PC12 cells. The PC12 cells were treated with different
concentrations (1-100 µM) of the compounds for 3 h. After 24 h, cell viability was
determined by MTT assay in the presence H2O2. Quercetine (Q) and tacrine (T) were used as
reference drugs.
Scheme 1. Synthesis of target compounds 6a-p. Reagents and conditions: (a) piperdine,
EtOH, reflux; (b) AlCl3 (1.5 equiv.), dry 1,2-dichloroethane, reflux.
19

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Table 1. The IC50 values of compounds 6a-p against cholinesterases in comparison with
tacrine
R
O
NH2
N
EtO
O
Compound
R
IC50 AChE (µM)
0.423 ± 0.011
4.16 ± 0.33
IC50 BuChE (µM)
5.49 ± 0.61
54.1 ± 0.67
5.76 ± 0.38
74.25 ± 0.50
17.6 ± 0.35
6.1 ± 0.51
H
6a
4-Me
4-OMe
4-Br
4-Cl
4-F
6b
6c
0.480 ± 0.010
6.22 ± 0.36
6d
6e
1.10 ± 0.13
0.404 ± 0.007
0.583 ± 0.011
0.510 ± 0.010
0.069 ± 0.005
0.228 ± 0.006
0.778 ± 0.009
2.73 ± 0.15
6f
4-NO2
3-OMe
3-Br
3-Cl
3-F
9.91 ± 0.42
7.14 ± 0.47
1.35 ± 0.07
3.10 ± 0.45
11.67 ± 0.43
40.95 ± 0.64
2.76 ± 0.27
2.14 ± 0.33
3.63 ± 0.39
22.68 ± 0.56
0.014
6g
6h
6i
6j
6k
6l
3-NO2
2-Br
2-Cl
2-F
0.118 ± 0.005
0.153 ± 0.006
0.370 ± 0.014
1.62 ± 0.09
6m
6n
6o
2,4-Cl
-
6p
Tacrine
0.356
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Table 2. The BBB permeability of compounds predicted
by online admetSAR server
Compound
Permeability
(BBB–/+)
+
Probability
0.84
0.82
0.59
0.80
0.82
0.85
0.65
0.59
0.80
0.82
0.85
0.65
0.80
0.85
0.85
0.82
0.98
6a
+
+
+
+
+
–
6b
6c
6d
6e
6f
6g
+
+
+
+
–
6h
6i
6j
6k
6l
+
+
+
+
+
6m
6n
6o
6p
Tacrine
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Table 3. In vitro cytotoxicity of selected compounds
on HepG2 cells in comparison to tacrine.
Compound
IC50 (µg/mL)
72.8
50.4
>100
>100
>100
17.9
6i
6j
6l
6m
6n
Tacrine
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NH2
N
Tacrine
R
R
R
NH2
O
NH2
N
NH2
N
R1
EtO
Me
R2
N
N
O
N
O
O
I
II
III
R
O
NH2
N
EtO
O
6a-p
Figure 1. The structures of reported tacrine-derived AChE inhibitors I-III and newly
designed compounds 6a-p
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Figure 2. Kinetic of AChE inhibition by compound 6i: Left) Lineweaver-Burk plot, Right)
Lineweaver-Burk secondary plot
24

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(a)
(b)
Figure 3. (a) The best pose of the most active compound (6i) in the active site of AChE; (b)
residues of the active site involved in ligand binding.
25

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Figure 4. Illustration of compound 6i binding mode in the active site of BuChE
26

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Figure 5. Neuroprotective activity of selected compounds 6i, 6j, 6m and 6n against ROS-
mediated cell apoptosis in PC12 cells. The PC12 cells were treated with different
concentrations (1-100 µM) of the compounds for 3 h. After 24 h, cell viability was
determined by MTT assay in the presence H2O2. Quercetine (Q) and tacrine (T) were used as
reference drugs.
27

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R
O
O
CN
O
O
EtO
a
H
+
NC
CN
OEt
R
+
O
NH2
R
1
3
2a-p
4a-p
R
O
O
NH2
O
4
6
9
5
CN
3
2
EtO
b
7
EtO
+
8
O
NH2
O
N
5
4a-p
6a-p
Scheme 1. Synthesis of target compounds 6a-p. Reagents and conditions: (a) piperidine,
EtOH, reflux; (b) AlCl3 (1.5 equiv.), dry 1,2-dichloroethane, reflux.
28