Synthesis, docking study and neuroprotective effects of some novel pyrano[3,2-c]chromene derivatives bearing morpholine/phenylpiperazine moiety

Article abstract of DOI:10.1016/j.bmc.2017.05.043

Novel pyrano[3,2-c]chromene derivatives bearing morpholine/phenylpiperazine moiety were synthesized and evaluated against acetylcholinestrase (AChE) and butylcholinestrase (BuChE). Among the synthesized compounds, N-(3-cyano-4-(4-methoxyphenyl)-5-oxo-4,5-

Full text of DOI:10.1016/j.bmc.2017.05.043

Accepted Manuscript
Synthesis, docking study and neuroprotective effects of some novel pyrano[3,2-
c]chromene derivatives bearing morpholine/phenylpiperazine moiety
Bilqees Sameem, Mina Saeedi, Mohammad Mahdavi, Hamid Nadri, Farshad
Homayouni Moghadam, Najmeh Edraki, Muhammad Imran Khan, Mohsen
Amini
PII:
S0968-0896(17)30271-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.05.043
BMC 13761
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 February 2017
4 May 2017
18 May 2017
Please cite this article as: Sameem, B., Saeedi, M., Mahdavi, M., Nadri, H., Moghadam, F.H., Edraki, N., Khan,
M.I., Amini, M., Synthesis, docking study and neuroprotective effects of some novel pyrano[3,2-c]chromene
derivatives bearing morpholine/phenylpiperazine moiety, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.05.043
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Synthesis, docking study and neuroprotective effects of some novel pyrano[3,2-c]chromene
derivatives bearing morpholine/phenylpiperazine moiety
e
Bilqees Sameem a, Mina Saeedi b,c, Mohammad Mahdavi d, Hamid Nadri , Farshad Homayouni
Moghadam f, Najmeh Edraki g, Muhammad Imran Khan h, Mohsen Aminii,d*
a
Faculty of Pharmacy, International Campus (TUMS-IC), Tehran University of Medical
Sciences, Tehran, Iran
b
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
c
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences,
Tehran, Iran
d
Drug Design & Development Research Center, Tehran University of Medical Sciences,
Tehran, Iran
e
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
f Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran
g Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical
Sciences, Shiraz, Iran
h Department of Pharmacology, School of Medicine TUMS-IC, Tehran, Iran.
i Department of Medicinal Chemistry, Faculty of Pharmacy and Drug Design & Development
Research Center, Tehran University of Medical Sciences, Tehran, Iran
This manuscript is dedicated to the fond memory of Professor Abbas Shafiee.
1

Abstract
Novel pyrano[3,2-c]chromene derivatives bearing morpholine/phenylpiperazine moiety were
synthesized and evaluated against acetylcholinestrase (AChE) and butylcholinestrase (BuChE).
Among
the
synthesized
compounds,
N-(3-cyano-4-(4-methoxyphenyl)-5-oxo-4,5-
dihydropyrano[3,2-c]chromen-2-yl)-2-(4-phenylpiperazin-1-yl)acetamide (6c) exhibited the
highest acetylcholinestrase inhibitory (AChEI) activity (IC50 = 1.12 µM) and most of them
showed moderate butylcholinestrase inhibitory activity (BChEI). Kinetic study of compound 6c
confirmed mixed type of inhibition towards AChE which was in covenant with the results
obtained from docking study. Also, it was evaluated against β-secretase which demonstrated low
activity (inhibition percentage: 18%). It should be noted that compounds 6c, 7b, 6g, and 7d
showed significant neuroprotective effects against H2O2-induced PC12 oxidative stress.
Key words: Alzheimer’s disease, Cholinestrase inhibition, Coumarins, Neuroprotection, β-
Secretase inhibition.
1. Introduction
Alzheimer’s disease (AD) is a multifactorial, progressive neurodegenerative disorder of the
central nervous system (CNS) associated with cognitive impairment and decline in language
ability among elderly people.1 Many factors are believed to play role in the disease onset and
progression. Studies have revealed that the decrease in acetylcholine neurotransmitter,2 β-
amyloid aggregation due to the cleavage of amyloid precursor protein (APP),3 irregular
phosphorylation of tau proteins, and subsequently formation of neurofibrils tangles in the
hippocampal neurons leading to cell death.4 Furthermore, other parameters such as inflammation
conditions and oxidative stress play a critical role in the pathogenesis of AD.5 In addition to the
2

hydrolytic role of AChE, it induces pro-aggregation of amyloids and deposition into the
neurofibrils (non-catalytic role).6 Accordingly, the inhibition of this enzyme is considered as a
main approach increasing the cholinergic transmission, prevention of amyloid aggregation and
hence a vital strategy to palliate the disease symptoms.7 In normal healthy brain, AChE plays
predominant role and buthylcholinestrase (BuChE) a minor role in the hydrolysis of ACh. As
AD progresses, the level of AChE decreases or remains unchanged whereas BuChE increases in
certain regions of brain probably to offset the deficiency of AChE.8 Selective or non-selective
inhibition of BuChE represents legitimate therapeutic role in alleviating the disease symptoms as
its inhibition has shown some significant results in recent years.9 Also, compounds with
additional pharmacological effects such as neuroprotective, anti-oxidative, and β-secretase
inhibitory activity can be considered as a benefit way in AD symptomatic treatment.10,11
Pertinently, Donepezil, Rivastigmine, Tacrine, Galantamine (Fig. 1)¸ the four among five FDA
approved drugs, are acetylcholinestrase inhibitors and are used for the symptomatic treatment of
AD in the early and mild stages.12,13
Fig. 1. Structures of approved AChE inhibitors.
Coumarins are naturally occurring phytochemicals; studies have shown that both naturally and
synthetic coumarin-based scaffolds (Fig. 2) exhibit potent AChE inhibitory activity with some
additional important pharmacological features. Previous studies have demonstrated that
3

coumarin moiety primarily interacts with the peripheral anionic site (PAS) of AChE and when
linked by an appropriate spacer such as benzyl amine, phenylpiperazine, or aniline moieties,
interacts with catalytic anionic site (CAS).14
Fig. 2. Structures of some coumarin-based scaffolds and designed compounds (6a-h and 7a-h).
15
Dual binding coumarin-based compounds such as AP 2238 and Ensaculin (coumarin-based
derivative containing a phenylpiperazine moiety) are under clinical investigations (Fig. 2).16
Therefore, dual binding site coumarin-based scaffolds with some other pronounced
pharmacological effects are of vital interest in the therapeutics of the disorder. Herein, in
continuation of our work on the synthesis of anti-cholinesterase (anti-ChE) and neuroprotective
agents,17-22, 25 we report synthesis and anti-ChE activity of some novel pyrano[3,2-c]chromenes
bearing morpholine/phenylpiperazine moiety 6 and 7 (Fig. 2).
2. Results and Discussion
4

2.1. Chemistry
The synthetic route for the synthesis of target compounds 6 and 7 is shown in Scheme 1.
Scheme 1. Synthesis of desired compounds 6a-h and 7a-h, Reagents and conditions: (a)
DABCO, EtOH, rt, 24 h; (b) Chloroacetyl chloride, DMF, rt, 24 h; (c) Phenylpiperazine, KI,
MeOH, reflux 18-24 h; (d) morpholine, KI, MeOH, reflux 18-24 h.
The required starting materials 4 were prepared in high yields (85-95%) through one-pot
multicomponent reaction of 4-hydroxycoumarin (1), malanonitrile (2), and different aromatic
aldehydes 3 in the presence of DABCO in ethanol at room temperature. In the next step, reaction
of chloroacetylchloride and compound 4 in DMF at ambient temperature for 24 h led to the
formation of the corresponding amide 5. It should be noted that the later reaction was initiated at
0 ºC to avoid vigorous reaction and the reaction was left to stir at room temperature. Then,
compound 5 was treated with phenylpiperazine/morpholine to undergo nucleophilic substitution
reaction in the presence of KI in methanol under reflux conditions for 18-20 h. The Structures of
5

all target compounds were elucidated by IR and NMR spectroscopy as well as mass
spectrometry.
2.2. Anti-AChE and Anti-BuChE activities
The target compounds 6 and 7 containing phenylpiperazine and morpholine moiety, respectively
were evaluated for their AChE and BuChE inhibitory activity in vitro according to Ellman’s
modified method23 using marketed drug donepezil as standard (Table 1). Compounds in series 6
usually showed better anti-AChE activity comparing series 7. In this category, compound 6c
having strong electron donating group (OMe) at 4- position demonstrated the most potent
activity (IC50 = 1.12 µM). An additional methoxy group at 3- position led to slight decrease of
anti-AChE activity in compound 6e (IC50 = 1.42 µM). Insertion of an additional electron
donating OCH3-group at position 3 in compound 6e led to one-fold decline in inhibitory activity
apparently due to the steric hindrance. Similar SAR trend was observed in a study demonstrating
that the introduction of an additional OCH3 group on the tetrahydrochromeno pyrano [2,3-
b]quinolin scaffold led to 2.6-fold decline in activity (IC50 = 43 nM), while insertion of a third
OCH3 group showed nearly 5- fold decline in activity (IC50 = 69 nM).22 However, the
introduction of methoxy group at position 4 in compound 6d resulted in 28-fold decrease in
activity which is in analogy with the docking studies as depicted in Fig 5.
It was convinced that the position of methoxy groups played an important role in AChEI since
compound 6d possessing two methoxy groups at 2- and 4-positions of pendant aryl group
showed significant decrease in anti-AChE activity (IC50 = 31.4 µM). It is concluded that the
enzyme has good tolerance at position 3 for an extra methoxy group and not at position 2.
6

Table 1
AChE and BuChE inhibitory activity of compounds 6a-h and 7a-h.
IC50
µM
Entry
Products 6/7
Ar
IC50 µM (AChE)a
SIb
(BuChE)a
52.3±4.18
>100
1
2
3
C6H5
11.5±1.72
32.9±5.51
1.12±0.43
4.5
-
6a
6b
6c
2,4-diCH3C6H3
4-OCH3C6H4
45.7±4.32
40.8
4
2,4-diOCH3C6H3
31.4±4.72
>100
-
6d
5
3,4-diOCH3C6H3
4-FC6H4
1.42±0.23
n.dc
58.2±4.56
n.dc
41.0
6e
6
-
6f
7
4-ClC6H4
6.33±1.77
n.dc
89.2±6.97
n.dc
14.1
6g
8
4-NO2C6H4
C6H5
-
6h
9
23.9±4.60
2.05±0.58
25.6±1.11
3.25±0.55
>100
88.4±6.28
63.8±2.68
>100
3.7
7a
10
11
12
13
14
15
16
17
4-OCH3C6H4
2,4-diOCH3C6H3
3,4-diOCH3C6H3
4-FC6H4
31.1
7b
-
7c
34.2±5.74
>100
10.5
7d
-
7e
4-ClC6H4
14.6±2.50
>100
91.5±8.18
>100
6.3
7f
4-NO2C6H4
2-thiophene
-
-
7g
>100
>100
7h
Donepezil
0.031±0.007
8.73±2.27
aInhibitor concentration (mean ± SD of three experiments) required for 50% inactivation of
AChE. b Selectivity index = IC50 (\buChE)/IC50 (AChE).cThey were insoluble in buffer assay.
7

Our results showed that insertion of moderate electron donating group (Me) into 2- and 4-
positions of aryl group (6b) exhibited nearly equal anti-AChE activity (IC50 = 32.9 µM) as that of
compound 6d. However, presence of 4-Cl (6g) on the aryl group led to relatively good activity
(IC50 = 6.33 µM). Compound 6a with no substituents on aryl group showed moderate inhibitory
activity (IC50 = 11.5 µM). It should be noted that the inhibitory activity of compounds 6f and 6h
precipitated in buffer assay.
Compounds in series 6 were evaluated for their anti-BuChE activity. Totally, they showed
insignificant inhibitory activity and compound 6c was the most active BuChE inhibitor with IC50
= 45.7 µM.
From Table 1, compounds in series 7 showed moderate to good AChEI activity. Compound 7b
possessing strong electron donating group (OMe) at 4- position showed highest inhibitory
activity with IC50 = 2.05 µM. Similar to series 6, introduction of two methoxy groups into
positions 3- and 4- of aryl group 7d led to the decrease of anti-AChE activity as IC50 was
obtained 3.25 µM. Also, insertion of methoxy group to positions 2- and 4- afforded much lower
AChEI activity (7c, IC50 = 25.6 µM). Here again, the similar reason could be justified for
antithetical activity of compounds 7c and 7d that mentioned for 6c and 6d.
Pertinent to mention, the presence of electron withdrawing group (NO2, 7g) as well as
heterocyclic ring (thiophene, 7h) led to the loss of inhibitory activity (IC50 >100 µM).
Unsubstituted phenyl ring 7a showed moderate anti-AChE activity (IC50 = 23.9 µM). Another
point is related to halogenated aryl groups which showed different activities toward AChE. 4-F
substituted derivative 7e did not show activity, whereas 4-Cl substituted derivative 7f exhibited
relatively good AChEI activity (IC50 = 14.6 µM). BuChE inhibitory activity of compounds in
series 7 except 7d (IC50 = 34.2 µM) indicated low inhibitory activity.
8

Overall, our results confirmed that 4-OMe phenyl group played an imperative role in anti-AChE
activity since 6c and its analogue 7b possessing phenylpiperazine and morpholine, respectively
showed the best activities. Also, similar results were observed for compounds having two
methoxy groups (6d, 6e, 7c, and 7d). Clearly, presence of two methoxy groups at 3- and 4-
positions induced much higher activity (Table 1). Although presence of chlorine in two series 6g
and 7f led to relatively good activity, compound 6g exhibited higher inhibitory activity more
than two fold confirming the significant role of phenylpiperazine. In the same manner, in the
case of compounds possessing phenyl moieties 6a and 7a, presence of phenylpiperazine
increased AChEI activity by two fold.
As mentioned above, no remarkable anti-BuChE activity was displayed by compounds 6 and 7.
Only, compound 7d showed moderate anti-BuChE activity among the synthesized compounds 6
and 7.
Finally, the best selectivity (BuChE/AChE) was related to compound 6e by the selectivity index
(SI) = 41.0. Also, the most potent anti-AChE compound 6c showed almost similar SI by 40.8.
2.3. Kinetic study of AChE inhibition
The most active compound 6c was subjected to kinetic studies to obtain the kinetic mode of
AChE inhibition. For this purpose, the rate of enzyme activity was measured in the presence of
four different concentrations of inhibitor (0.62, 1.23, and 2.47 µM) and different concentrations
of acetylthiocholine as substrate (ATCh, [S]). For each inhibitor concentration, the initial
velocity was measured at different substrate concentrations and the reciprocal of the initial
velocity (1/v) was plotted vs the reciprocal of substrate concentration (1/[S]). As shown in Fig. 3,
the obtained double reciprocal (Lineweaver-Burk) plot represents a mixed-type inhibition pattern
9

for compound 6c. The Ki value was also calculated using the secondary plot (Ki = 3.19 µM)
(Fig. 3). All experiments were performed based on the Ellman’s method as described for in vitro
AChEI evaluation.
Fig. 3. Left: Lineweavere-Burk plot for the inhibition of AChE by compound 6b; Right:
Secondary plot for calculation of steady-state inhibition constant (Ki) of compound 6b.
2.4. Neuroprotective activity against H2O2 induced apoptosis in PC12 neuron cells
The neuroprotective effects of compounds 6c, 6e, 6g, 7b, and 7d against H2O2 induced oxidative
stress and apoptosis in PC12 neuron cells were evaluated using Methylthiazol Tetrazolium
(MTT) assay (Fig. 4). The differentiated cells were pretreated with different concentrations of
compounds 6c, 6e, 6g, 7b, and 7d at 10, 50, and100 µM. As shown in Fig. 4, the percent of cell
viabilities were calculated in comparison to control group. Compounds 6c, 6e, 6g, 7b, and 7d
demonstrated good neuroprotectivity at different concentration and showed cell viability of 84.7,
76.0, 75.0, 78.5, and 84.7 %, respectively at 10 µM with querectin as internal standard.
10

Statistical analysis of the data was performed using One-way ANNOVA ordinary method,
significance of the results was determined by Tukey’s test.
Fig. 4. Neuroprotective activity of compounds 6c, 6e, 6g, 7b, and 7d on PC12 cells against
H2O2 induced cells death. (*** P<0.001) represents the significance with respect to blank.
2.5. BACE-1 inhibitory activity of compound 6c
The BACE-1 inhibitory activity of 6c was evaluated by fluorescence resonance emission transfer
(FRET) method. The kit consists of enzyme BACE-1 and APP peptide based substrate (Rh-
EVNLDAEFK-quencher) comparing with OM99-2 (IC50 = 0.014 µM) as the reference.
Compound 6c displayed demonstrated low activity (inhibition percentage: 18%).
11

2.6. Docking
To clarify the mode of interaction of the most potent compound 6c in the active site of AChE,
docking study was conducted using Auto dock Vina 1.1.2. For this purpose, the ligand-bonded
crystallographic structure of AChE with PDB ID: 1eve was retrieved from RCSB protein data
bank. The best docked pose of each ligand in terms of the free energy of binding was extracted.
The best docked pose of compound 6c was subjected to further studies of binding mode. As
depicted in Fig. 5, the phenylpiperazine moiety oriented towards the bottom of active site where
the phenyl ring interacted with Trp84 (in the CAS) through a π-π stacking. In this orientation, the
positively charged nitrogen of piperazine ring placed in the vicinity of negative area of
carboxylic side chain of Asp72 (in the PAS) residue forming an electrostatic bond. The aromatic
pyrano coumarin moiety oriented towards the AChE via T-shaped π-π interaction with Trp279
residue in the PAS of AChE. Finally, a hydrogen bond between amide nitrogen of 6c and
hydroxyl group of Tyr121 (in the PAS) led to more stabilized interaction.
It is worthwhile mentioning that the obtained results were in good agreement with those obtained
in kinetic study.
12

Fig. 5. Left: Two dimensional mode of interaction of compound 6c with AChE. Right: Three dimensional
mode of interaction of compound 6c with AChE. Hydrogen bond and hydrophobic as well as ionic
interactions are shown by green, pink, and yellow, respectively.
3. Conclusion
Considering the fact cholinesterase inhibitors have shown the best evidence to alleviating the AD
symptoms to date, as they play a key role in cholinergic signaling. In this respect, AChEI’s and
BuChEI’s may pronounce diversity in beneficial effects as far as the severity of the disorder is
concerned. Also, selective AChE inhibitors can slow down the amylodgenic product formation
(non-catalytic function of AChE) besides increasing the levels of ACh.
In this work, a novel series of pyrano [3,2-c]chromen bearing morpholine/phenylpiperazine were
synthesized and evaluated against AChE/BuChE using modified Ellman’s method. Results
showed that among the sixteen synthesized compounds, five exhibited good, selective AChE
inhibitory activity. Compound 6c bearing a phenylpiperazine moiety and 4-methoxypheny
moieties showed the highest inhibitory activity (IC50 = 1.12 µM) with significant selectivity
towards AChE (SI = 40.8) and good neuroprotective activity against H2O2 induced oxidative
stress. Also, docking study of compound 6c confirmed those valuable interactions with both the
PAS and CAS of AChE. The results of the current research shows that pyrano[3,2-c] chromone
skeleton may be a new probe for development new agents with potential anti Alzheimer activity.
4. Experimental
4.1. General
All commercially available materials and reagents were purchased from Merck and Aldrich with
no further purification. Thin layer chromatography was performed using silica gel 250 micron
13

and F254 plates. Melting points were obtained using Koftler hot stage apparatus. The IR spectra
1
13
were recorded on Nicolet FT magna 550 spectrograph (KBr pellets). H- and C-NMR spectra
were recorded on Bruker 500Hz and the chemical shifts, coupling constants (J) are expressed in
parts per millennium (ppm) and hertz (Hz), respectively. Mass spectra were acquired on an
Agilent Technology (HP) mass spectrometer operating at an ionization potential of 70 eV.
4.2. General procedure for the synthesis of 6a-h and 7a-h.
A mixture of 4-hydroxycoumarin (1) (10 mmol), malanonitrile (2) (10 mmol), different aromatic
aldehydes (10 mmol) 3, and DABCO (9 mmol) in EtOH (25 mL) was stirred overnight at room
temperature. The precipitate formed was filtered off and washed with EtOH to furnish the pure
compound 4 (85-95%). In the next step, Chloroacetyl chloride (12 mmol) was added to the
solution of compound 4 (6 mmol) in DMF (30 mL) at 0 ºC. Then, the reaction was left to stir at
room temperature for 24 h. Reaction progress was monitored by TLC and after completion of the
reaction, the mixture was poured into ice cold water and the precipitate was filtered off, dried,
and purified by washing with CH2Cl2 to afford compounds 5 (35-60%). However, they could be
recrystallized from MeOH. Finally, the desired compounds were prepared by reacting
intermediate 5 (2 mmol) with phenylpiperazine/morpholine (4 mmol) in methanol in presence of
KI under reflux for 18-20 h. The reaction progress was monitored by TLC and the precipitate
formed was filtered off and washed with MeOH to remove KI. Finally, the crude products were
purified by plate chromatography to afford the desired product in good to high yields (70-85%).
4.2.1.
N-(3-Cyano-5-oxo-4-phenyl-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6a)
14

1
White solid; yield 77%; mp = 243 - 245 ºC; IR (KBr): 3379, 2192, 1711, 1675, 1605 cm-1. H
NMR (500 MHz, CDCl3): 8.01 (d, J = 8.0 Hz, 1H, H5), 7.73 (t, J = 8.0 Hz, 1H, H7), 7.52-7.47
(m, H6, H8), 7.33 (d, J = 7.0 Hz, 2H, H2', H6'), 7.26 (t, J = 7.0 Hz, 2H, H3' H5'), 7.22-7.17 (m,
3H, H4', phenylpiperazine), 6.92 (d, J = 7.5 Hz, 2H, phenylpiperazine), 6.77 (t, J = 7.5 Hz, 1H,
phenylpiperazine), 4.90 (s, 1H, CH), 3.47 (s, 2H, CH2), 3.15 (t, J = 4.0 Hz, 4H, 2×CH2), 2.66-
13
2.64 (m, 4H, 2×CH2). CNMR (125 MHz, CDCl3): 160.7, 159.8, 159.7, 157.6, 154.2, 152.2,
141.1, 132.0, 128.7, 128.2, 127.9, 126.9, 123.9, 123.5, 122.4, 120.0, 116.3, 116.0, 113.0, 105.3,
103.4, 59.1, 52.8, 48.7, 34.4. Anal. Calcd for C31H26N4O4: C, 71.80; H, 5.05; N, 10.80. Found: C,
71.49; H, 5.47; N, 10.55.
4.2.2. N-(3-Cyano-4-(2,4-dimethylphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6b)
1
White solid; yield 80%; mp > 250 ºC; IR (KBr): 3283, 2214, 1729, 1676, 1628 cm-1. HNMR
(500 MHz, DMSO-d6): 7.97 (d, J = 7.5 Hz, 1H, H5), 7.69 (t, J = 7.5 Hz, 1H, H7), 7.48-7.42 (m,
2H, H6, H8), 7.19 (t, J = 7.0 Hz, 2H, phenylpiperazine), 6.91-6.82 (m, 5H, H3', H5', H6',
phenylpiperazine), 6.76 (t, J = 7.0 Hz, 1H, phenylpiperazine), 5.01 (s, 1H, CH), 3.49 (s, 2H,
13
CH2), 3.15-3.13 (m, 4H, 2×CH2), 2.69-2.65 (m, 7H, 2×CH2, CH3), 2.15 (s, 3H, CH3). CNMR
(125 MHz, DMSO-d6): 161.7, 159.8, 158.9, 158.5, 154.0, 151.8, 150.9, 138.8, 136.8, 135.6,
132.7, 130.2, 128.8, 128.3, 126.8, 124.8, 122.6, 118.8, 116.4, 115.4, 113.2, 106.3, 103.6, 59.6,
52.5, 48.1, 29.9, 20.4, 19.3. Anal. Calcd for C33H30N4O4: C, 72.51; H, 5.53; N, 10.25. Found: C,
72.34; H, 5.47; N, 10.40.
4.2.3.
N-(3-Cyano-4-(4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6c)
15

1
White solid; yield 85%; mp >250 ºC; IR (KBr): 3419, 2194, 1734, 1666 cm-1. HNMR (500
MHz, DMSO-d6): 7.99 (d, J = 8.0 Hz, 1H, H5), 7.72 (td, J = 8.0, 1.5 Hz, 1H, H7), 7.50-7.46 (m,
2H, H6, H8), 7.23-7.18 (m, 4H, phenylpiperazine), 6.91 (d, J = 8.5 Hz, 2H, H3', H5' ), 6.81 (d, J
= 8.5 Hz, 2H, H2′, H6′), 6.76 (t, J = 7.0 Hz, 1H, phenylpiperazine), 4.83 (s, 1H, CH), 3.68 (s,
13
3H, OCH3), 3.44 (s, 2H, CH2), 3.15 (m, 4H, 2 ×CH2), 2.62 (m, 4H, 2 ×CH2). CNMR (125
MHz, DMSO-d6): 161.7, 159.8, 159.0, 158.8, 158.2, 154.2, 152.0, 150.9, 134.4, 132.8, 129.6,
128.9, 124.9, 122.7, 118.9, 116.6, 115.4, 113.5, 113.3, 105.3, 102.7, 59.7, 55.0, 52.5, 48.1, 33.6.
Anal. Calcd for C32H28N4O5: C, 70.06; H, 5.14; N, 10.21. Found: C, 69.84; H, 5.37; N, 10.40.
N-(3-Cyano-4-(2,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6d)
1
White crystals; yield 70%; mp > 250 ºC; IR (KBr): 3281, 2219, 1731, 1681 cm-1. HNMR (500
MHz, CDCl3): 8.08 (d, J = 7.5 Hz, 1H, H5), 7.58 (td, J = 7.5, 1.0 Hz, 1H, H7), 7.52 (d, J = 8.5,
1H, H6′), 7.38-7.34 (m, 2H, H6, H8), 7.28-7.26 (t, J = 7.9 Hz, 2H, phenylpiperazine), 6.94-6.90
(m, 4H, phenylpiperazine, H3′), 6.48 (dd, J = 8.5, 2.2 Hz 1H, 5′), 5.15 (s, 1H, CH), 3.74 (s, 3H,
OCH3), 3.65 (s, 2H, CH2), 3.61 (s, 3H, OCH3), 3.27-3.25 (m, 4H, 2×CH2), 2.82-2.80 (m, 4H,
13
2×CH2). CNMR (125 MHz, CDCl3): 161.5, 160.8, 160.6, 160.5, 159.1, 157.4, 155.1, 152.7,
133.0, 132.0, 129.2, 124.2, 122.6, 120.7, 120.4, 120.3, 116.7, 116.4, 113.8, 104.2, 104.1, 102.4,
99.3, 59.7, 55.3, 55.2, 53.2, 49.2, 32.5. Anal. Calcd for C33H30N4O6: C, 68.50; H, 5.23; N, 9.68.
Found: C, 68.44; H, 5.36; N, 9.40.
4.2.5. N-(3-Cyano-4-(3,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6e)
16

Yellow crystals; yield 70%; mp = 225 - 227 ºC; IR (KBr): 3325, 3404, 2196, 1710, 1670 cm-1.
1HNMR (500 MHz, CDCl3): 8.05 (d, J = 8.0 Hz, 1H, H5), 7.59 (t, J = 8.0 Hz, 1H, H7), 7.38-
7.33 (m, 2H, H6, H8), 7.26 (t, J = 7.5 Hz, 2H, phenylpiperazine), 7.11 (s, 1H, H2′), 6.92-6.62 (m,
4H, phenylpiperazine, H5′), 6.75 (d, J = 8.5 Hz, 1H, H6′), 5.15 (s, 1H,CH), 3.87 (s, 3H, OCH3),
3.86 (s, 3H, OCH3), 3.62 (s, 2H, CH2), 3.23 (t, J = 4.0 Hz, 4H, 2×CH2), 2.77 (t, J = 4.0 Hz, 4H,
13
2×CH2). CNMR (125 MHz, CDCl3): 161.3, 160.4, 160.2, 158.4, 154.5, 152.7, 150.7, 148.8,
148.5, 134.5, 132.4, 129.2, 124.4, 122.8, 120.4, 120.3, 116.8, 116.4, 113.6, 113.0, 111.2, 105.9,
103.8, 59.8, 56.0, 55.9, 53.4, 49.2, 34.3. Anal. Calcd for C33H32N4O6: C, 68.26; H, 5.56; N,
9.65. Found: C, 68.34; H, 5.45; N, 9.92.
4.2.6.
N-(3-Cyano-4-(4-flourophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazine-1-yl)acetamide (6f)
1
Yellow solid; yield 70%; mp = 238 - 240 ºC; IR (KBr): 3427, 2195, 1727, 1668 cm-1. HNMR
(500 MHz, CDCl3): 8.05 (d, J = 7.5 Hz, 1H, H5), 7.58 (t, J = 7.5 Hz, 1H, H7), 7.39-7.31 (m, 4H,
H6, H8, phenylpiperazine), 7.25 (t, J = 7.5 Hz, 2H, H3', H5'), 6.94-6.83 (m, 4H, H2', H6',
phenylpiperazine), 6.83 (d, J = 7.2 Hz, 1H, phenylpiperazine), 5.14 (s, 1H, CH), 3.62 (s, 2H,
13
CH2), 3.22 (t, J = 4.5 Hz, 4H, 2×CH2), 2.76 (t, J = 4.5 Hz, 4H, 2×CH2). C-NMR (125 MHz,
CDCl3): 161.9 (d, JC-F = 245.0 Hz), 161.6, 160.2, 158.8, 154.6, 152.7, 150.7, 137.4, 132.5, 130.3,
130.2, 129.1, 124.4, 122.8, 120.1, 116.7, 116.2, 115.1 (d, JC-F = 25.0 Hz), 113.4, 105.5, 103.3,
59.8, 53.3, 49.0, 34.2. MS (m/z, %): 536 (M+, 9), 376 (13), 280 (40), 161 (29), 132 (100), 104
(40), 77 (27), 56 (36). Anal. Calcd for C31H25FN4O4: C, 69.39; H, 4.70; N, 10.44. Found: C,
69.60; H, 4.44; N, 10.30.
17

4.2.7.
N-(4-(4-Chlorophenyl)-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6g)
1
White crystals; yield 70%; mp > 250 ºC; IR (KBr): 3467, 2225, 1729, 1678 cm-1. HNMR (500
MHz, CDCl3): 8.01 (d, J = 7.6 Hz, 1H, H5), 7.74 (t, J = 7.5 Hz, 1H, H7), 7.52-7.44 (m, 2H, H6,
H8), 7.37 (d, J = 8.3 Hz, 2H, H3′, H5′), 7.32 (d, J = 8.3 Hz, 2H, H2′, H6′), 7.20 (t, J = 7.8 Hz,
2H, phenylpiperazine), 6.92 (d, J = 7.8 Hz, 2H, phenylpiperazine), 6.77 (t, J = 7.8 Hz, 1H
phenylpiperazine), 4.8 (s, 1H, CH), 3.68 (s, 2H, CH2), 3.27 (s, 4H, 2×CH2), 2.84 (s, 4H, 4H,
13
2×CH2). CNMR: 161.1, 160.3, 160.2, 158.4, 154.8, 152.8, 150.5, 140.1, 133.3, 132.7, 130.1,
129.3, 128.5, 124.5, 122.9, 120.5, 116.9, 116.5, 113.4, 105.3, 103.4, 59.6, 53.3, 49.1, 34.5 MS
(m/z, %): 554 ([M+2]+, 0.3), 552 (M+, 1), 125 (100), 366 (7), 338 (99), 235 (13), 207 (11), 143
(21), 178 (62), 161 (12), 221 (62), 113 (5), 77 (21). Anal. Calcd for C31H25ClN4O4: C, 67.33; H,
4.56; N, 10.13. Found: C, 67.20; H, 4.82; N, 9.82.
4.2.8.
N-(3-Cyano-4-(4-nitrophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-(4-
phenylpiperazin-1-yl)acetamide (6h)
Yellow crystals; Yield 77%; mp > 250 ºC; IR (KBr): 3427, 3266, 2222, 1720, 1681 cm-1.
1HNMR (500 MHz, DMSO-d6): 8.12 (d, J = 8.5 Hz, 2H, H3′, H5′), 8.00 (d, J = 7.7 Hz, 1H, H5),
7.75 (t, J = 7.7 Hz, 1H, H7), 7.66 (d, J = 8.5 Hz, 2H, H2′, H6′), 7.53-7.48 (m, 2H, H6, H8), 7.20
(t, J = 7.7 Hz, 2H, phenylpiperazine), 6.91 (d, J = 7.7 Hz, 2H, phenylpiperazine), 6.77 (t, J = 7.7
Hz, 1H, phenylpiperazine), 5.03 (s, 1H, CH), 3.52 (d, J = 14.0 Hz, 1H, CH2), 3.49 (d, J = 14.0
13
Hz, 1H, CH2), 3.18-3.05 (m. 4H, 2 ×CH2), 2.74-2.61 (m, 4H, 2 ×CH2). CNMR (125 MHz,
DMSO-d6): 161.7, 159.8, 159.6, 159.2, 154.9, 152.1, 150.9, 149.6, 146.4, 133.1, 130.2, 128.9,
124.9, 123.1, 122.8, 118.8, 116.6, 115.4, 113.1, 103.9, 101.3, 59.7, 52.5, 48.1, 35.0. MS (m/z,
18

%): 564 (M+, 3), 93 (100), 120 (95), 162 (81), 281 (84), 218 (20). Anal. Calcd for C31H25N5O6:
C, 66.07; H, 4.47; N, 12.43. Found: C, 65.80; H, 4.61; N, 12.64.
4.2.9.
N-(3-Cyano-5-oxo-4-phenyl-4,5-dihydropyrano[3,2-c]chromen-2-yl)-
morpholinoacetamide (7a)
1
Pale yellow solid; yield 80%; mp > 250 ºC; IR (KBr): 3379, 2192, 1711, 1675 cm-1. H NMR
(500 MHz, DMSO-d6): 8.01 (d, J = 7.7 Hz, 1H, H5), 7.73 (t, J = 7.7 Hz, 1H, H7), 7.52-7.47 (m,
2H, H6, H8), 7.32 (d, J = 7.2 Hz, 2H, H2', H6'), 7.26 (t, J = 7.2 Hz, 2H, H3', H5') , 7.18 (t, J =
7.2 Hz, 1H, H4'), 4.88 (s, 1H, CH), 3.59-3.57 (m, 6H, 3 ×CH2), 3.40-3.38 (m, 4H, 2 ×CH2).
13CNMR (125 MHz, DMSO-d6): 154.7, 153.1, 142.5, 139.2, 138.6, 137.6, 132.9, 128.6, 128.2,
126.9, 125.1 124.9, 124.4, 122.7, 118.2, 116.6, 113.4, 66.1, 60.3, 53.1, 34.5. Anal. Calcd for
C25H21N3O5: C, 67.71; H, 4.77; N, 9.48. Found: C, 67.64; H, 4.50; N, 9.74.
4.2.10
N-(3-Cyano-4-(4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholineacetamide (7b)
White solid; yield 80%; mp = 242 - 245 ºC; IR (KBr): 3417, 2204, 1734, 1665 cm-1cm-1.
1HNMR (500 MHz, DMSO-d6) : 7.97 (dd, J = 7.7, 1.5 Hz, 1H, H5), 7.72 (td, J = 7.7, 1.5 Hz, 1H,
H7), 7.50- 7.46 (m, 2H, H6, H8), 7.21 (d, J = 6.5 Hz, 2H, H2', H6'), 6.80 (d, J = 7.5 Hz, 2H, H3',
H5'), 4.82 (s, 1H, CH), 3.68 (s, 3H, OCH3), 3.57 (s, 2H, CH2), 3.45-3.41 (m, 8H, 4×CH2).
13CNMR (125 MHz, DMSO-d6): 161.8, 159.9, 159.1, 158.8, 158.2, 154.3, 152.1, 134.5, 133.0,
129.6, 124.9, 122.7, 116.6, 113.6, 113.3, 105.3, 102.7, 66.1, 60.1, 55.1, 53.1, 33.6. Anal. Calcd
for C26H23N3O6: C, 65.95; H, 4.90; N, 8.87. Found: C, 65.69; H, 4.67; N, 8.74.
4.2.11 N-(3-Cyano-4-(2,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7c)
19

Yellow crystals; yield 75%; mp > 250 ºC; IR (KBr): 3281, 3001, 2968, 2944, 2219, 1735, 1681,
1611 cm-1. 1H NMR (500 MHz, DMSO-d6): 8.01 (d, J = 7.5 Hz, 1H, H5), 7.69 (t, J = 7.5 Hz, 1H,
H7), 7.49-7.43 (m, 2H, H6, H8), 7.25 (d, J = 8.5 Hz, 1H, H6', 1H), 6.46 (dd, J = 8.5, 1.5 Hz, 1H,
13
H5'), 6.41 (s, 1H, H3'), 4.83 (s, 1H,CH), 3.69-3.52 (m, 16 H, 5×CH2, 2×OCH3). CNMR (125
MHz, DMSO-d6): 160.1, 159.7, 158.9, 155.0, 152.0, 151.1, 132.7, 132.5, 124.8, 132.5, 122.5,
121.7, 121.6, 116.5, 113.6, 104.6, 103.8, 103.6, 99.1, 66.1, 61.3, 55.7, 55.1, 53.1, 32.0. Anal.
Calcd for C27H25N3O6: C, 64.41; H, 5.00; N, 8.35. Found: C, 64.61; H, 4.87; N, 8.54.
4.2.12 N-(3-Cyano-4-(3,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7d)
Yellow crystals; yield 75%; mp = 235 - 237 ºC; IR (KBr): 3385, 3005, 2978, 2205, 1717, 1665
1
cm-1. HNMR (500 MHz, CDCl3): 8.04 (d, J = 7.0 Hz, 1H, H5), 7.58 (td, J = 7.0, 1.0 Hz, 1H,
H7), 7.38-7.34 (m, 2H, H6, H8), 7.12 (d, J = 1.5 Hz, 1H, H2'), 6.83-6.80 (dd, J = 7.7, 1.5 Hz,
1H, H5'), 6.75 (d, J = 7.7 Hz, 1H, H6'), 5.14 (s, 1H, CH), 3.87 (s, 3H, OCH3), 3.79 (s, 3H,
OCH3), 3.75-3.73 (t, J = 4.5 Hz, 4H, 2×CH2), 3.56 (d, J = 15.0 Hz, 1H, CH2), 3.54 (d, J = 15.0
Hz, 1H, CH2), 2.61-2.59 (t, J = 4.5 Hz, 4H, 2×CH2). 13CNMR (125 MHz, CDCl3): 161.7, 160.4,
160.3, 158.4, 154.4, 152.6, 148.7, 148.4, 134.4, 132.4, 124.3, 122.8, 120.4, 116.7, 113.5, 112.8,
111.1, 105.8, 103.5, 66.5, 60.3, 56.0, 55.8, 53.8, 53.5, 53.3, 34.2. Anal. Calcd for C27H25N3O6: C,
64.41; H, 5.00; N, 8.35. Found: C, 64.40; H, 5.17; N, 8.39.
4.2.13
N-(3-Cyano-4-(4-flourophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7e)
White solid; yield 70%; mp > 255 ºC; IR (KBr): 3421, 3018, 2978, 2185, 1717, 1667 cm-1.
1HNMR (500 MHz, DMSO-d6): 8.55 (s, 1H, NH), 7.99 (d, J = 7.7 Hz, 1H, H5), 7.71 (t, J =
7.7 Hz, 1H, H7), 7.50-7.45 (m, 2H, H6, H8), 7.34 (dd, J = 8.0, 5.5 Hz, 2H, H2', H6'), 7.15 (t, J =
20

8.0 Hz, 2H, H3', H5'), 4.58 (s, 1H, CH), 3.40-3.38 (m, 4H, 2×CH2), 3.25 (s, 2H, CH2), 3.04-3.02
13
(m, 4H, 2×CH2). CNMR (125 MHz, DMSO-d6): 161.0 (d, JC-F = 240.0 Hz), 160.6, 159.5,
154.9, 152.1, 146.8, 146.6, 139.2, 132.8, 130.4 (d, JC-F = 8.7 Hz), 124.8, 122.8, 116.6, 114.7 (d,
JC-F = 21.2 Hz), 113.5, 105.2, 100.7, 66.1, 61.6, 53.2, 34.0. Anal. Calcd for C25H20FN3O5: C,
65.07; H, 4.37; N, 9.11 Found: C, 65.41; H, 4.17; N, 9.37.
4.2.14
N-(4-(4-Chlorophenyl)-3-cyano-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7f)
White crystals; yield 70%; mp > 250 ºC; IR (KBr): 3467, 3272, 2225, 1729, 1678, cm-1. 1HNMR
(500 MHz, CDCl3): 8.05 (dd, J = 8.0, 1.5 Hz, 1H, H5), 7.60 (td, J = 7.7, 1.5 Hz, 1H, H7), 7.39-
7.34 (m, 4H, H6, H8, H3', H5'), 7.23 (d, J = 9.0 Hz, 2H, H2', H6'), 5.15 (s, 1H, CH), 3.74 (t, J =
4.5 Hz, 4H, 2×CH2), 3.56 (d, 2H, J = 14.0 Hz, 1H, CH2), 3.53 (d, 2H, J = 14.0 Hz, 1H, CH2),
13
2.60 (t, 4H, J = 4.5 Hz, 4H, 2×CH2). C-NMR (125 MHz, CDCl3): 160.9, 160.3, 158.6, 154.8,
152.8, 140.2, 133.3, 132.6, 130.1, 128.5, 126.0, 124.5, 122.9, 116.9, 113.5, 105.4, 103.4, 66.6,
60.3, 53.7, 34.5. MS (m/z, %): 479 ([M+2]+, 2), 477 (M+, 6), 86 (100), 392 (44), 281 (86), 240
(55), 121 (32). Anal. Calcd for C25H20ClN3O5: C, 62.83; H, 4.22; N, 8.79. Found: C, 62.64; H,
4.40; N, 8.44.
4.2.15
N-(3-Cyano-4-(4-nitrophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7g)
White crystals; yield 70%; mp = 216 - 218 ºC, white crystals; IR (KBr): 3265, 2223, 1703, 1682
cm-1. 1H NMR (500 MHz, CDCl3): 10.20 (bs, 1H, NH), 8.14- 8.05 (m, 3H, H5, H3', H5'), 7.61-
7.59 (m, 3H, H7, H2', H6'), 7.40-7.35 (m, 2H, H6, H8), 5.27 (s, 1H,CH), 3.74-3.73 (m, 4H,
2×CH2), 3.60 (s, 2H, CH2), 2.60-2.59 (m, 4H, 2×CH2).13C-NMR (125 MHz, CDCl3): 160.7,
160.5, 160.1, 159.2, 155.3, 152.9, 148.6, 147.3, 133.1, 129.8, 124.7, 123.7, 123.1, 117.0, 113.3,
21

104.5, 102.6, 66.6, 60.2, 53.7, 35.2. Anal. Calcd for C25H20N4O7: C, 61.48; H, 4.13; N, 11.47.
Found: C, 61.65; H, 4.28; N, 11.47.
6.2.16
N-(3-Cyano-5-oxo-4-(thiophene-2-yl)-4,5-dihydropyrano[3,2-c]chromen-2-yl)-2-
morpholinoacetamide (7h)
Light pink crystals; yield 70%; mp > 250 ºC; IR (KBr): 3233, 3192, 2221, 1717, 1677 cm-1.
1HNMR (500 MHz, DMSO-d6): 7.97 (d, J = 8.0 Hz, 1H, H5), 7.75 (t, J = 8.0 Hz, 1H, H7), 7.51-
7.47 ( m, 2H, H6, H8), 7.34 (d, J = 4.5 Hz, 1H, thiophene), 6.98 (d, J = 4.5 Hz, 1H, thiophene),
6.92 (t, J = 4.5 Hz, 1H, thiophene), 5.22 (s, 1H, CH), 3.61 (t, J = 4.3 Hz, 4H, 2×CH2), 3.46-3.44
(m, 6H, 3×CH2). 13CNMR (125 MHz, DMSO-d6): 161.7, 159.8, 159.2, 159.1, 154.5, 152.1,
145.5, 133.1, 126.9, 125.7, 125.5, 125.0, 122.7, 116.7, 113.1, 104.7, 102.0, 66.0, 60.1, 53.0, 29.1.
MS (m/z, %): 449 (M+, 20), 86 (100), 364 (89), 280 (59), 240 (63), 121 (35). Anal. Calcd for
C23H19N3O5S: C, 61.46; H, 4.26; N, 9.35. Found: C, 61.32; H, 4.42; N, 9.52.
4.3. In vitro AChE and BuChE inhibition assays
To evaluation of the biological activity, all synthesized compounds 6 and 7 were subjected to
AChE/BuChE inhibition assays using Ellman’s method.23,24 The tested compounds were
dissolved in a mixture of 1 mL DMSO and 9 mL ethanol and then four different concentrations
of each compound were tested to obtain the range of 20% to 80% enzyme inhibition for AChE
and BuChE. For this purpose, in a 24 well plate, 2 mL phosphate buffer (0.1M, pH = 8.0), 65 µL
of DTNB 0.1 M, 35 µL of enzyme [2 U/mL of AChE ( E.C. 3.1.1.7, Type V-S, lyophilized
powder, from electric eel) or BuChE (E.C. 3.1.1.8, from equine serum)] and 35 µL of inhibitor
solution or DMSO:EtOH mixture (1:9) was added. Then, 10 µL of substrate 0.15 M
(acetylthiocholine iodide or butyrylthiocholine iodide) was added to each well and the change of
22

absorbance was measured at 412 nm for 6 min. The IC50 values were determined graphically
from inhibition curves (log inhibitor concentration vs. percent of inhibition). All experiments
were performed on a synergy HTX microplate reader (kinetic mode) in quadruplicates.
4.4. Kinetic study
To study the kinetic mode of mechanism of compound 6c, reciprocal plots of 1/velocity vs. 1/[S]
(substrate) were drawn at three concentrations of substrate acetylthiocholine employing Ellman’s
method (0.62, 1.23 and 2.47 µM). Initial velocity in each case, was achieved at different
concentration of substrate (acetylthiocholine) and for each experiment after adding
acetylthiocholine, progress curves were monitored at 420 nm for 2 min. The reciprocal of the
velocity was plotted against the reciprocal of substrate.
4.5. Neuroprotection study
PC12 cell line was obtained from Pasteur institute (Tehran, IRAN) and all culture media as well
as supplements were purchased from Gibco. Cells were cultivated in DMEM supplemented with
10% fetal calf serum, 5% horse serum and antibiotics (100 units/mL penicillin, 100 μg/mL
streptomycin). To induce neuronal differentiation, PC12 cells were re-suspended using
trypsin/EDTA (0.25% ) and seeded in 96 well culture plate (3000 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 compounds 6c, 6e, 6g, 7b, and 7d on survival rate of
neurons, the culture medium was changed to NGF free medium and different concentrations of
the above mentioned compounds (10, 50, 100 μM) were applied on cells. Quercetin (50 μM) was
used as a positive control. Compounds were diluted into DMEM and added 10 μL to each well.
23

Three hours later induction of ROS mediated apoptosis was initiated by adding the H2O2 (400
μM) to their medium and after 12 hours, MTT assay was performed. MTT solution (5 mg/mL)
was added to each well in a volume of 10 μL, and 3 h later culture medium replaced with 100 μL
of DMSO. Absorbance at 545 nm was measured using an ELISA reader. Each experimental was
performed in four replicates.
4.6. BACE1 enzymatic assay
A FRET-based BACE1 enzyme assay kit was purchased from Invitrogen (former Pan Vera
Corporation, Madison, WI) and the assay was carried out according to the manufacturer’s
instructions (Invitrogen. http://tools.invitrogen.com/content/sfs/manuals/L0724.pdf).24
4.7. Statistical Analysis
The statistical analysis was carried out by graph pad prism 6. Data is expressed as mean ± SD.
One-way analysis of variance (ANOVA) was used to analyze the data in conjugation with
tukey’s test to determine the data significance by single- step multiple comparisons.
4.8. Docking simulation method
The pdb file of the receptor (1EVE) complexed with E2020 (donepezil) was retrieved from the
Brookhaven protein database http://www.rcsb.org/pdb/home/home.do. Subsequently, the co-
crystallized ligand, water molecules were removed and polar hydrogen were added using
Discovery Studio 4.1. The structures of compound 6c were sketched and saved in pdb format
using Marvin Sketch 15.6.8 www.chemaxon.com. The ligand molecule 6c was saved in pdbqt
format after adding Kollman charges. The pdbqt file of ligands was prepared in Auto Dock tools
24

1.5.6rc The active site of receptor pdb file for docking was created with dimensions 60, 60, 60
with co-ordinates x, y, z of grid box was set as default. The results were visualized in Discovery
Studio 4.1 as 2D and 3D diagrams.
3D structure of AChE in complex with E2020 (Donepezil) with ID number: 1eve was retrieved
from Brook-haven Protein data bank (www.rcsb.org). To prepare protein for docking simulation,
all co-crystallized and water molecules was removed and the protein was converted to required
pdbqt format using Auto dock Tools package (1.5.6).26 The 2D structure of ligand was prepared
using Marvin Sketch 5.8.3, 2012, Chem Axon(www.chemaxon.com); then, 2D structure was
converted to 3D format by Openbabel (ver 2.3.1)27 and finally, pdbqt format of ligand was
prepared using Auto dock Tools python script, prepare_ligand4.py
Docking simulations were performed with Autodock Vina28 (1.1.2) within a docking box
defined by following parameters: size_x=15, size_y=15, size_z=15 Å, center_x=2.023,
center_y=63.295, center_z=67.062. The exhaustiveness was set to 80 and the other docking
parameters were set as default. At the end of docking simulations, the best docking solutions
were selected for further analysis of enzyme-inhibitor interactions. The graphics are depicted
using chimera 1.10.2 and Biovia discovery studio visualizer.29
Acknowledgement
This work was financially supported by Research Council of Tehran University of Medical
Sciences, International Campus (TUMS-IC) vide grant No. 93-01-103-25020. Late Prof. Abbas
Shafiee is highly acknowledged for providing me insight and all over support in this work.
References
25

1. Walsh DM, Selkoe DJ. Deciphering the molecular basis of memory failure in Alzheimer's
disease. Neuron. 2004;44:181-193.
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