Novel Tacrine-Based Pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones: Synthesis and Cholinesterase Inhibitory Activity

Article abstract of DOI:10.1002/ardp.201600123

In order to develop effective anti-cholinesterase compounds, a novel series of pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones were designed, synthesized, and evaluated in vitro against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). All derivati

Full text of DOI:10.1002/ardp.201600123

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Arch. Pharm. Chem. Life Sci. 2016, 349, 1–10
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Full Paper
Novel Tacrine-Based Pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones:
Synthesis and Cholinesterase Inhibitory Activity
Roshanak Hariri1, Zahra Afshar1, Mohammad Mahdavi2, Maliheh Safavi3, Mina Saeedi4,5, Zahra Najafi6,
Reyhaneh Sabourian5, Elahe Karimpour-Razkenari5, Najmeh Edraki7, Farshad Homayouni Moghadam8,
Abbas Shafiee1, Mahnaz Khanavi9, and Tahmineh Akbarzadeh1,5
1
Faculty of Pharmacy, Department of Medicinal Chemistry, Tehran University of Medical Sciences,
Tehran, Iran
2
Drug Design and Development Research Center, Tehran University of Medical Sciences, Tehran, Iran
Department of Biotechnology, Iranian Research Organization for Science and Technology, Tehran, Iran
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran,
3
4
Iran
5
Persian Medicine and Pharmacy Research Center, Tehran University of Medical Sciences, Tehran, Iran
Faculty of Pharmacy, Department of Medicinal Chemistry, Hamadan University of Medical Sciences,
6
Hamadan, Iran
7
Medicinal and Natural Products Chemistry Research Center, Shiraz University of Medical Sciences, Shiraz,
Iran
8
Department of Cellular Biotechnology at Cell Science Research Center, Royan Institute for
Biotechnology, ACECR, Isfahan, Iran
Faculty of Pharmacy, Department of Pharmacognosy, Tehran University of Medical Sciences, Tehran,
9
Iran
In order to develop effective anti-cholinesterase compounds, a novel series of pyrano[3’,4’:5,6]-
pyrano[2,3-b]quinolinones were designed, synthesized, and evaluated in vitro against acetylcholines-
terase (AChE) and butyrylcholinesterase (BChE). All derivatives showed very good AChE inhibitory
(AChEI) activity (IC50 ¼ 0.37–5.62 mM) compared with rivastigmine (IC50 ¼ 11.07 mM). Among them,
11-amino-12-(2,3-dichlorophenyl)-3-methyl-7,8,9,10-tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-
1(12H)-one (6f) displayed the best inhibitory activity. However, most of the synthesized compounds
showed no anti-BChE activity and compounds 6b and 6f were found to be only moderate inhibitors.
The most potent anti-AChE compound 6f had low and moderate inhibitory activity and neuro-
protective effects against beta-secretase (BACE1) and oxidative stress-induced cell death, respectively.
Also, kinetic and molecular docking studies of binding interactions elucidated that compound 6f
bound to both the catalytic anionic site (CAS) and peripheral anionic site (PAS) of AChE.
Keywords: Acetylcholinesterase / Alzheimer’s disease / Butyrylcholinesterase / Fused heterocycles /
Pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones
Received: April 25, 2016; Revised: October 12, 2016; Accepted: October 13, 2016
DOI 10.1002/ardp.201600123
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
Correspondence: Dr. Tahmineh Akbarzadeh, Faculty of Pharmacy,
Department of Medicinal Chemistry, Tehran University of Medical
Sciences, Tehran, Iran.
E-mail: akbarzad@tums.ac.ir
Fax: þ98-21-66461178
Alzheimer’s disease (AD) has emerged as the most common
age-related neurodegenerative disorder [1]. It is categorized
as one of the top ten causes of death in the United States.
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Figure 1. Structures of four approved ChEI drugs.
Although several reasons such as genetic, environmental,
and endogenous factors are associated with AD, decrease
of acetylcholine (ACh) is the main cause of AD [2] leading to
cognitive and memory symptoms [3].
tacrine-based AChE inhibitors [14–20]. Herein, in continuation
of our research program on the synthesis of anti-ChE
compounds [19–25], we designed and synthesized novel
tacrine analogs, pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinone
derivatives (Fig. 2).
Acetylcholinesterase (AChE) and butyrylcholinesterase
(BChE) are two enzymes which lyse choline-based esters.
Thus, inhibition of AChE and BChE improves the symptoms
in patients with AD. In a healthy brain, AChE activity
is significant and predominates over BChE activity (as a
pseudocholinesterase or nonspecific cholinesterase). Also, it
has been revealed that AChE is mainly found in neurons while
BChE is generally present in plaques and tangles in glial
cells [4]. Although most research on brain cholinesterases
have been conventionally associated with AChE, important
role of BChE in cholinergic neurotransmission and different
nervous system dysfunctions has been well documented in
the literature [5, 6]. In this regard, effective ChE inhibitors
have been the center of attention as the key agents to
enhance cholinergic transmission at cholinergic autonomic
synapses as well as neuromuscular junction [7].
It has been proved that neurodegenerative diseases are
also due to the accumulation of uncharacteristically folded
b-amyloid peptide (Ab) in the brains of patients, described
by “Amyloid Cascade Hypothesis” theory [8]. It seems that
the deposition of Ab is the first pathological event which
causes formation of senile plaques (SPs) followed by
neurofibrillary tangles (NFTs) and death of neurons leading
to AD. Thus, it is clear that inhibition of Ab aggregation has
been the masterwork for the treatment of AD [9]. Apart from
these facts, noncholinergic role of AChE has been considered
as a versatile reason for inducing SPs via accelerating Ab
deposition. It is supposed that AChE constructs a stable
complex with SP components through its peripheral anionic
site (PAS) [10, 11]. Therefore, AChEIs may increase not
only cholinergic function, but also affect the generation of
b-amyloid.
Results and discussion
Chemistry
Synthetic route for the preparation of the desired pyrano-
[3’,4’:5,6]pyrano[2,3-b]quinolinones 6a–o is shown in
Scheme 1. For this purpose, various 2-amino-4-aryl-7-methyl-5-
oxo-4,5-dihydropyrano[4,3-b]pyran-3-carbonitriles 4a–o were
prepared by the reaction of 4-hydroxy-6-methyl-2H-pyran-2-
one (1), aromatic aldehydes 2, and malononitrile (3). Screening
various commercially available basic reagents such as DABCO,
NEt3, K2CO3, and piperidine as well as different solvents
(EtOH, MeOH, CH3CN, PhCH3, and CH2Cl2) in a model reaction
revealed that using DABCO in EtOH at room temperature led
to the formation of the corresponding products 4a–o in
good yields (80–85%). Then, reaction of compounds 4a–o and
cyclohexanone (5) was conducted in the presence of AlCl3
in ClCH2CH2Cl (DCE) under reflux conditions for 12–18 h to
obtain the desired products 6a–o in good yields (Table 1).
The structure of all compounds was elucidated using IR, 1H,
and 13C NMR spectroscopy as well as chemical analysis.
Pharmacology
Cholinesterase inhibitory activity
All synthesized pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones
6a–o were evaluated for their cholinesterase inhibitory
There are four approved ChEI drugs, tacrine, donepezil,
rivastigmine, and galantamine (Fig. 1) and among them,
tacrine (1,2,3,4-tetrahydroacridine-9-amine) is known as an
effective AChEI and BChEI [12]. However, extensive use of
tacrine was restricted since various side effects such as
hepatoxicity were reported [13]. Hence, recent studies have
been devoted to the design and synthesis of new and effective
Figure 2. Structure of pyrano[3’,4’:5,6]pyrano[2,3-b]quinoli-
nones 6.
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Scheme 1. Synthesis of pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinone derivatives 6.
activity based on Ellman’s modified procedure [20] (Table 1).
All compounds showed very good anti-AChE activity
(0.370–5.620 mM) comparing with rivastigmine as the refer-
ence drug (IC50 ¼ 11.070 mM). They were also compared with
tacrine (IC50 ¼ 0.048 mM) in Table 1. Among the prepared
derivatives, 11-amino-12-(2,3-dichlorophenyl)-3-methyl-
7,8,9,10-tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6f) was the most potent compound toward AChE. Also,
compounds 6n, 6d, and 6e were found to be potent anti-AChE
derivatives and IC50 values were obtained as 0.720, 0.800, and
0.820 mM, respectively. As can be seen in Table 1, compounds
6j, 6k, 6g, and 6h depicted good anti-AChE activity with
IC50 ¼ 1.170, 1.122, 1.234, and 1.259, respectively. Also, our
results demonstrated that moderate activities were obtained
Table 1. Synthesis and IC50 values of pyrano[3’,4’:5,6]pyrano[2,3-b]quinolinones 6 against AChE and BChE.a)
AChE inhibition IC50
BChE inhibition IC50
Entry
Compound 6
(mM)
(mM)
1
2
3
4
5
6
7
8
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
Tacrine
C6H5
2-F-C6H4
4-F-C6H4
2-Cl-C6H4
4-Cl-C6H4
1.940 ꢀ 0.028
1.870 ꢀ 0.014
4.100 ꢀ 0.223
0.800 ꢀ 0.078
0.820 ꢀ 0.021
0.370 ꢀ 0.014
1.234 ꢀ 0.099
1.259 ꢀ 0.049
1.970 ꢀ 0.042
1.170 ꢀ 0.042
1.122 ꢀ 0.042
1.970 ꢀ 0.049
2.970 ꢀ 0.035
0.720 ꢀ 0.007
5.620 ꢀ 0.332
0.048 ꢀ 0.013
11.070 ꢀ 0.010
>50
40.420 ꢀ 0.019
>50
>50
>50
2,3-diCl-C6H3
2-NO2-C6H4
3-NO2-C6H4
2-Me-C6H4
3-Me-C6H4
4-Me-C6H4
4-MeO-C6H4
3,4,5-triMeO-C6H2
2-NO2-5-Cl-C6H3
2-Thiophenyl
17.670 ꢀ 0.429
>50
>50
>50
>50
>50
>50
>50
>50
>50
9
10
11
12
13
14
15
16
21
0.014 ꢀ 0.009
Rivastigmine
7.720 ꢀ 0.020
a) Data are expressed as mean ꢀ SE (three independent experiments).
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by compounds 6b, 6a, 6l, and 6m (IC50 ¼ 1.870–2.970 mM).
Compounds 6c and 6o were found to be the weakest
inhibitors (IC50 ¼ 4.100 and 5.620 mM) in comparison to other
derivatives.
activity in comparison to compounds 6a and 6b. Compound 6o
possessing 2-thienyl moiety was found to be the weakest
anti-AChE compound (IC50 ¼ 5.620 mM).
Anti-BChE evaluation indicated that most of synthesized
compounds 6a–o induced no inhibitory activity toward BChE.
Only compounds 6b and 6f showed BChEI activity with IC50
values of 40.420 and 17.670 mM, respectively. It is clear that
compound 6f was the most potent anti-ChE agent.
According to our results in Table 1, electronic properties of
substituents as well as their positions on the aryl group at
12-position of compounds 6 played important role in AChEI
activity. Compound 6f having two chlorine atoms on the aryl
ring showed the best inhibitory activity. Decreasing the
number of chlorine atoms led to the reduction of inhibitory
activity, which was confirmed by IC50 values obtained for
compounds 6d and 6e. Nevertheless, compounds 6d and 6e
did not show significant difference, indicating that the
number of chlorine atoms was more important than their
positions. It should be noted that the presence of an electron
withdrawing group (NO2) along with chlorine (compound 6n)
led to a potent anti-AChE activity; however, it was not as
potent as compound 6f having two chlorine atoms. Also,
introduction of methyl (compounds 6j and 6k) or NO2
(compounds 6g and 6h) groups into the various positions of
aryl group induced good inhibitory activity. Comparing the
corresponding results did not show remarkable anti-AChE
activity since IC50 values were obtained in the range of
1.170–1.259 mM. It should be noted that the presence of
2-methylphenyl group did not induce potent inhibitory
activity in compound 6i (IC50 ¼ 1.970 mM) comparing with
meta and para Me-substituted derivatives (6j and 6k). Also,
presence of strong electron donating group (OMe) on the aryl
group of compound 6l induced weaker inhibitory activity
(IC50 ¼ 1.970 mM). Moreover, increasing the number of
methoxy groups led to much weaker activity. Compounds
6a and 6b having phenyl and 2-flurophenyl groups showed
activity with IC50 ¼ 1.940 and 1.870 mM, respectively. It seems
that the presence of fluorine at 2-pition of aryl group does not
play important role in the inhibitory activity and introduction
of fluorine into 4-position of aryl group induced much weaker
Kinetic studies
In order to reveal the inhibition mode of AChE induced
by the most potent compound 6f, a kinetics study was
performed based on the modified Ellman’s method [20]. As it
is clear in Fig. 3, graphical analysis of the Lineweaver–Burk
reciprocal plots demonstrated a mixed type of inhibition,
confirming the fact that compound 6f can bind to both the
CAS and PAS of AChE. The inhibition constant (Ki ¼ 0.918 mM)
was calculated using secondary replots of the slope versus
concentrations of compound 6f.
BACE1 enzyme inhibitory activity of compound 6f
Considering the fact that b-secretase (BACE1) plays significant
role in AD pathogenesis, BACE-1 inhibitory activity of the
most potent AChEI 6f was evaluated via a fluorescence
resonance energy transfer (FRET) based BACE-1 kit including
BACE-1 enzyme and specific APP-based peptide substrate
(Rh-EVNLDAEFK-quencher). Experiments were repeated for
three times and compared with the reference compound
OM99-2 (Table 2). Compound 6f demonstrated low BACE1
inhibitory activity with percentage inhibition ¼ 13.20 and
10.35 at 50 and 10 mM of 6f, respectively.
Neuroprotective effect of compound 6f against
H2O2-induced cell death in PC12 neurons
The neuroprotective activity of compound 6f was evaluated
using PC12 cell line protocol to study neuronal
Figure 3. Left: Lineweaver–Burk plot for the inhibition of AChE by compound 6f at different concentrations of acetylthiocholine
(ATCh). Right: Steady-state inhibition constant (Ki) of compound 6f.
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Table 2. Inhibitory activity of compound 6f against BACE1
comparing with OM99-2.a)
this respect, both (R)- and (S)-enantiomers were studied in
docking simulations (Fig. 5). As can be seen in Fig. 6, the
(R)-enantiomer occupied the peripheral anionic site (PAS) of
AChE whereas the (S)-enantiomer was placed at the mid of
the gorge of AChE interacting with both the PAS and CAS.
The interaction between (R)-enantiomer and AChE demon-
strated that 2,3-dichloroaryl moiety has oriented toward
Leu282 and makes hydrophobic interaction (Fig. 6) whereas
other parts of compound have oriented toward Tyr70,
Tyr121, Tyr334, and Trp279 residues of the PAS. Also, it made
p–p stacking interaction with Trp279 and hydrophobic
interactions between cyclohexyl ring and Tyr334. The hydro-
gen bond between oxygen of pyran moiety and Tyr70 is
another important interaction. All results confirmed that
the compound has interacted with the PAS of AChE.
Similarly (S)-enantiomer as depicted in Fig. 7, has located
in the mid of the gorge of AChE. There are two p–p T-
shaped interactions between heterocyclic moieties and Trp84
and His440 of the CS. It is clear that there are two p–p T-
shaped interactions between pyridine moiety and Tyr334 and
Phe331 of the PAS. Also, 2,3-dichloroaryl moiety has oriented
toward Asp72 of the PAS and made anion-p interaction.
However, hydrogen bond between nitrogen of pyridine
moiety and Tyr121 of the PAS is significant.
Inhibition at
Inhibition at
Compound 6
6f
50 mM (%)b)
10 mM (%)b)
13.20 ꢀ 1.94
10.35 ꢀ 6.50
a) OM99-2 was tested at 10, 1, and 0.1 nM (IC50
¼
0.014 ꢀ 0.001 mM).
b) Values represent means ꢀ standard error (S.E.) of three
independent experiments.
differentiation [20]. In this respect, differentiated PC12
cells and H2O2 were considered as in vitro model and
oxidative agent, respectively. Neuroprotective activity of
compound 6f was evaluated by subjecting PC12 cells to
H2O2-induced damage comparing with quercetin. As
depicted in Fig. 4, the percentage of cell viability was
calculated in comparison to control group. Compound 6f
showed moderate neuroprotectivity at 100 mM (cell viability
¼ 59.77% and P < 0.0001 vs. H2O2 treatment alone). It
should be noted that compound 6f demonstrated neuro-
protectivity at 50 mM (cell viability ¼ 54.25% with P < 0.05 vs.
H2O2 treatment alone).
Molecular docking study
Conclusion
Docking study of the most active anti-AChE compound (6f)
was performed to clarify the possible interactions between
that compound and AChE. Compound 6f possessing a chiral
center at 12-position was prepared as a racemic mixture. In
In conclusion, we designed, synthesized, and evaluated
novel anti-ChE compounds, pyrano[3’,4’:5,6]pyrano[2,3-b]-
quinolinones. The in vitro inhibitory activity revealed that
11-amino-12-(2,3-dichlorophenyl)-3-methyl-7,8,9,10-tetra-
hydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-one (6f)
having two chlorine atoms on the aryl moiety showed the
best inhibitory activity toward both AChE and BChE.
Although most of the synthesized compounds depicted good
anti-AChE activity, the corresponding anti-BChE activities
were not remarkable. Also, compound 6f showed low and
Figure 4. Neuroprotective effect of compound 6f on cell viability
of PC12 cells in H2O2-induced damage. Data are expressed as
mean ꢀ SD, and one-way analysis of variance (ANOVA) followed
by Tukey’s multiple comparisons test which was carried out to
determine the level of significance. ###P < 0.0001 vs. control,
Figure 5. The proposed orientation of (R)- (green) and
(S)-enantiomers (orange) of compound 6f in the AChE.
ꢁ
ꢁꢁꢁꢁP < 0.0001 vs. H2O2 and P < 0.05 vs. H2O2.
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Figure 6. The binding mode of (R)-enantiomer
(green) of compound 6f in the active site of
AChE.
moderate inhibitory activity and neuroprotective effects
against beta secretase (BACE1) and oxidative stress-induced
cell death, respectively. It should be noted that docking and
kinetic studies confirmed that the inhibitory activity of the
most potent compound 6f spans both the CAS and PAS of
AChE.
Experimental
Chemistry
General
Melting points were taken on a Kofler hot stage apparatus
and are uncorrected. H and 13C NMR spectra were recorded
1
Figure 7. Representation of (S)-enantiomer
(orange) of compound 6f interacting with
residues in AChE.
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on Bruker FT-400 (Germany), using TMS as an internal
standard. The IR spectra were obtained on a Nicolet Magna
FTIR 550 spectrometer (KBr disks). The elemental analysis was
performed with an Elementar Analysensysteme GmbH Vario
EL (Germany). The InChI codes and representative spectra of
the investigated compounds are provided as Supporting
Information.
129.1 (d, JC–F ¼ 8.6 Hz), 130.6 (d, JC–F ¼ 3.7 Hz), 150.7, 153.4,
154.2, 160.3 (d, JC–F ¼ 243.8 Hz), 161.0, 162.1, 162.8. Anal.
calcd. for C22H19FN2O3: C, 69.83; H, 5.06; N, 7.40. Found: C,
69.62; H, 5.21; N, 7.28.
11-Amino-12-(4-fluorophenyl)-3-methyl-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6c)
Procedure for the synthesis of 2-amino-4-aryl-7-methyl-5-
oxo-4,5-dihydropyrano[4,3-b]pyran-3-carbonitriles 4a–o
A mixture of 4-hydroxy-6-methyl-2H-pyran-2-one (1) (1 mmol),
appropriate aldehyde 2 (1 mmol), malononitrile (3) (1 mmol),
and DABCO (20 mol%) in EtOH (10 mL) was stirred at
room temperature for 12 h. After completion of reaction
(checked by TLC), the mixture was filtered off, washed
with cold EtOH, and dried at 50–60°C to afford pure
compounds 4.
Yield: 53%; white solid; m.p. > 250°C. IR (KBr): 3422, 3344,
2937, 2859, 1716, 1657 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.84–
1.85 (m, 4H, 2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.26–2.39 (m, 2H, CH2),
2.78–2.81 (m, 2H, CH2), 4.11 (s, 2H, NH2), 4.89 (s, 1H, H12), 6.05
(s, 1H, H4), 6.98 (d, J ¼ 8.5 Hz, 2H, H30, H50), 7.39 (dd, J ¼ 8.5,
5.5 Hz, 2H, H20, H60). 13C NMR (CDCl3, 100 MHz): 19.9, 22.2,
22.5, 22.9, 32.5, 32.3, 98.8, 99.4, 101.5, 114.5, 115.6 (d,
JC–F ¼ 22.4 Hz), 130.2 (d, JC–F ¼ 21.5 Hz), 137.4 (d, JC–F ¼ 3.4 Hz),
150.8, 153.7, 154.6, 160.2, 160.4 (d, JC–F ¼ 233.3 Hz), 161.9,
163.2. Anal. calcd. for C22H19FN2O3: C, 69.83; H, 5.06; N, 7.40.
Found: C, 70.00; H, 4.81; N, 7.51.
Procedure for the synthesis of pyrano[3’,4’:5,6]pyrano[2,3-
b]quinolinones 6a–o
A mixture of compound 4 (1 mmol) and cyclohexanone (5)
(2 mmol) was added to the suspension of AlCl3 in dry DCE
(2 mmol in 30 mL) and heated at reflux for 12–18 h. After
completion of reaction (checked by TLC), mixture of H2O/THF
(1:1, 100 mL) was added to the mixture and it was basicified
with NaOH (10%). The mixture was stirred at room tempera-
ture for 30 min, the crude product was extracted using CH2Cl2
(2 ꢂ 50 mL) and washed with brine (2 ꢂ 50 mL). The organic
phase was dried (Na2SO4) and the solvent was evaporated
under vacuum. All compounds were recrystallized from EtOH
to obtain pure products 6.
11-Amino-12-(2-chlorophenyl)-3-methyl-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6d)
Yield: 79%; white solid; m.p. > 250°C. IR (KBr): 3481, 3369,
2924, 1712, 1644, 1612 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.80–
1.82 (m, 4H, 2 ꢂ CH2), 2.24 (s, 3H, CH3), 2.28–2.40 (m, 2H, CH2),
2.75–2.77 (m, 2H, CH2), 4.45 (s, 2H, NH2), 5.33 (s, 1H, H12), 6.07
(s, 1H, H4), 7.35 (dd, J ¼ 7.5, 2.0 Hz, 1H, H30), 714 (td, J ¼ 7.5,
2.0 Hz, 1H, H40), 7.19 (td, J ¼ 7.5, 2.0 Hz, 1H, H50), 7.26 (dd,
J ¼ 7.5, 2.0 Hz, 1H, H60), 7.35 (dd, J ¼ 7.5, 2.0 Hz, 1H, H30).
13C NMR (CDCl3, 100 MHz): 20.0, 22.2, 22.4, 22.9, 32.4, 32.5,
98.6, 99.2, 101.0, 114.1, 127.7, 128.6, 129.5, 131.3, 133.1, 139.5,
151.0, 153.5, 154.3, 160.8, 162.0, 162.7. Anal. calcd. for
11-Amino-3-methyl-12-phenyl-7,9,10,12-tetrahydro-
1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1-one (6a)
Yield: 69%; white solid; m.p. > 250°C. IR (KBr): 3422, 3344,
C
22H19ClN2O3: C, 66.92; H, 4.85; N, 7.09. Found: C, 66.78;
H, 4.63; N, 6.91.
2933, 1722, 1657 cmꢃ1 1H NMR (CDCl3, 400 MHz): 1.84–1.87
.
(m, 4H, 2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.27–2.40 (m, 2H, CH2), 2.78–
2.81 (m, 2H, CH2), 4.15 (s, 2H, NH2), 4.89 (s, 1H, H12), 6.05 (s,
1H, H4), 7.21–7.33 (m, 3H, H30, H40, H50), 7.42 (dd, J ¼ 7.5,
1.6 Hz, 2H, H20, H60). 13C NMR (CDCl3, 100 MHz): 19.9, 22.3,
22.5, 22.9, 32.5, 36.1, 99.1, 99.4, 101.6, 114.4, 127.5, 128.6,
128.7, 141.7, 150.9, 153.7, 154.4, 160.2, 161.7, 163.1. Anal.
calcd. for C22H20N2O3: C, 73.32; H, 5.59; N, 7.77. Found: C,
73.11; H, 5.38; N, 7.61.
11-Amino-12-(4-chlorophenyl)-3-methyl-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6e)
Yield: 51%; white solid; m.p. > 250°C. IR (KBr): 3415, 3341,
2930, 1719, 1660 cmꢃ1 1H NMR (CDCl3, 400 MHz): 1.84–1.85
.
(m, 4H, 2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.28–2.39 (m, 2H, CH2), 2.78–
2.80 (m, 2H, CH2), 4.10 (s, 2H, NH2), 4.88 (s, 1H, H12), 6.05
(s, 1H, H4), 7.26 (d, J ¼ 8.4 Hz, 2H, H30, H50), 7.35 (d, J ¼ 8.4 Hz,
2H, H20, H60). 13C NMR (CDCl3, 100 MHz): 20.0, 22.2, 22.4, 22.9,
32.5, 35.4, 98.5, 99.4, 101.2, 114.5, 128.9, 130.0, 133.3, 140.1,
150.8, 153.6, 154.7, 160.3, 162.0, 163.0. Anal. calcd. for
11-Amino-12-(2-fluorophenyl)-3-methyl-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6b)
C
22H19ClN2O3: C, 66.92; H, 4.85; N, 7.09. Found: C, 67.18;
Yield: 87%; white solid; m.p. > 250°C. IR (KBr): 3446, 3369,
H, 4.71; N, 7.23.
2946, 1722 cmꢃ1
.
1H NMR (CDCl3, 400 MHz): 1.85–1.86
(m, 4H, 2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.26–2.40 (m, 2H, CH2),
2.78–2.81 (m, 2H, CH2), 4.18 (s, 2H, NH2), 4.85 (s, 1H,
H12), 6.05 (s, 1H, H4), 7.07–7.31 (m, 4H, H3’, H4’, H5’, H6’).
13C NMR (CDCl3, 100 MHz): 20.0, 22.2, 22.4, 220.9, 29.4
(d, JC–F ¼ 2.9 Hz), 32.4, 98.2, 99.3, 100.4, 114.2, 115.5
(d,JC–F ¼ 22.4Hz),124.8(d,JC–F ¼ 3.2 Hz),128.8(d,JC–F ¼ 13.0 Hz),
11-Amino-12-(2,3-dichlorophenyl)-3-methyl-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6f)
Yield: 62%; white solid; m.p. > 250°C. IR (KBr): 3487, 3372,
2940, 1710, 1645 cmꢃ1 1H NMR (CDCl3, 400 MHz): 1.82–1.83
.
(m, 4H, 2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.25–2.41 (m, 2H, CH2),
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Archiv der Pharmazie
2.71–2.81 (m, 2H, CH2), 4.40 (s, 2H, NH2), 5.37 (s, 1H, H12), 6.07
(s, 1H, H4), 7.13 (t, J ¼ 7.6 Hz, 1H, H50), 7.20 (d, J ¼ 7.6 Hz, 1H,
H60), 7.34 (dd, J ¼ 7.6, 1.6 Hz, 1H, H40). 13C NMR (CDCl3,
100 MHz): 20.0, 22.2, 22.4, 22.9, 32.4, 35.3, 98.5, 99.1, 101.1,
114.2, 127.8, 129.5, 129.6, 131.5, 133.5, 141.9, 151.0, 153.4,
154.5, 160.9, 162.3, 162.6. Anal. calcd. for C22H18Cl2N2O3:
C, 61.55; H, 4.23; N, 6.53. Found: C, 61.38; H, 4.40; N, 6.71.
2.76–2.79 (m, 2H, CH2), 4.16 (s, 2H, NH2), 4.74 (s, 1H, H12), 6.05
(s, 1H, H4), 7.02 (d, J ¼ 7.0 Hz, 1H, H60), 7.15–7.22 (m, 2H, H40,
H50), 7.28 (s, 1H, H20). 13C NMR (CDCl3, 100 MHz): 19.9, 21.4,
22.3, 22.5, 22.9, 32.4, 36.0, 99.3, 99.4, 101.7, 114.4, 125.8,
128.3, 128.5, 129.2, 138.5, 141.7, 151.0, 153.6, 154.3, 160.1,
161.6, 163.1. Anal. calcd. for C23H22N2O3: C, 73.78; H, 5.92;
N, 7.48. Found: C, 73.60; H, 5.78; N, 7.24.
11-Amino-3-methyl-12-(2-nitrophenyl)-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6g)
11-Amino-3-methyl-12-(p-tolyl)-7,9,10,12-tetrahydro-
1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1-one (6k)
Yield: 66%; white solid; m.p. > 250°C. IR (KBr): 3419, 3341,
2937, 2853, 1716, 1660 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.82–
1.84 (m, 4H, 2 ꢂ CH2), 2.21 (s, 3H, CH3), 2.27–2.38 (m, 5H, CH2,
CH3), 2.77–2.80 (m, 2H, CH2), 4.16 (s, 2H, NH2), 4.74 (s, 1H, H12),
6.04 (s, 1H, H4), 7.10 (d, J ¼ 8.0 Hz, 2H, H30, H50), 7.30 (d,
J ¼ 8.0 Hz, 2H, H20, H60). 13C NMR (CDCl3, 100 MHz): 19.9, 21.1,
22.3, 22.5, 22.9, 32.5, 35.6, 99.3, 99.4, 101.8, 114.4, 128.5,
129.5, 137.2, 138.8, 150.9, 153.7, 154.3, 160.0, 161.6, 163.1.
Anal. calcd. for C23H22N2O3: C, 73.78; H, 5.92; N, 7.48. Found: C,
73.94; H, 6.11; N, 7.62.
Yield: 75%; white solid; m.p. > 250°C. IR (KBr): 3462, 3363,
2933, 1713, 1651, 1608, 1527, 1351 cmꢃ1 1H NMR (CDCl3,
.
400 MHz): 1.83–1.86 (m, 4H, 2 ꢂ CH2), 2.20 (s, 3H, CH3), 2.24–
2.41 (m, 2H, CH2), 2.79–2.80 (m, 2H, CH2), 5.10 (s, 2H, NH2), 5.73
(s, 1H, H12), 6.02 (s, 1H, H4), 7.26 (d, J ¼ 7.5 Hz, 1H, H60), 7.33
(t, J ¼ 7.5 Hz, 1H, H40), 7.35 (t, J ¼ 7.5 Hz, 1H, H50), 7.79
(d, J ¼ 7.5 Hz, 1H, H30). 13C NMR (CDCl3, 100 MHz): 20.0, 22.2,
225, 23.0, 30.2, 32.5, 96.9, 99.2, 101.2, 114.1, 123.7, 128.0,
131.2, 133.3, 136.8, 150.2, 151.5, 154.0, 154.8, 160.0, 162.4,
162.8. Anal. calcd. for C22H19N3O5: C, 65.18; H, 4.72; N, 10.37.
Found: C, 65.30; H, 4.58; N, 10.51.
11-Amino-12-(4-methoxyphenyl)-3-methyl-7,9,10,12-
tetrahydro-1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-
1-one (6l)
11-Amino-3-methyl-12-(3-nitrophenyl)-7,8,9,10-
tetrahydropyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1(12H)-
one (6h)
Yield: 74%; white solid; m.p. > 250°C. IR (KBr): 3434, 3351,
3159, 2943, 2841, 1726, 1654, 1611 cmꢃ1 1H NMR (CDCl3,
.
Yield: 69%; white solid; m.p. > 250°C. IR (KBr): 3483, 3391,
3091, 2937, 1707, 1530, 1302 cmꢃ1. 1H NMR (CDCl3, 400 MHz):
1.85–1.86 (m, 4H, 2 ꢂ CH2), 2.24 (s, 3H, CH3), 2.27–2.40 (m, 2H,
CH2), 2.80–2.82 (m, 2H, CH2), 4.15 (s, 2H, NH2), 5.03 (s, 1H, H12),
6.09 (s, 1H, H4), 7.48 (t, J ¼ 7.6 Hz, 1H, H50), 7.81 (dt, J ¼ 7.6,
2.0 Hz, 1H, H60), 8.09 (dt, J ¼ 7.5, 2.0 Hz, 1H, H40), 8.22
(t, J ¼ 2.0 Hz, 1H, H20). 13C NMR (CDCl3, 100 MHz): 20.0, 22.2,
22.4, 22.9, 32.5, 35.8, 97.1, 99.5, 100.5, 114.7, 122.6, 123.4,
129.5, 134.8, 143.7, 148.6, 150.6, 153.6, 155.2, 160.7, 162.5,
163.0. Anal. calcd. for C22H19N3O5: C, 65.18; H, 4.72; N, 10.37.
Found: C, 65.27; H, 4.88; N, 10.18.
400 MHz): 1.81–1.85 (m, 4H, 2 ꢂ CH2), 2.20 (s, 3H, CH3), 2.25–
2.39 (m, 2H, CH2), 2.77–2.80 (m, 2H, CH2), 3.76 (s, 3H, OCH3),
4.13 (s, 2H, NH2), 4.84 (s, 1H, H12), 6.04 (s, 1H, H4), 6.82
(d, J ¼ 8.4 Hz, 2H, H30, H50), 7.33 (d, J ¼ 8.4 Hz, 2H, H20,
H60). 13C NMR (CDCl3, 100 MHz): 19.9, 22.3, 22.5, 22.9, 32.5,
35.2, 55.2, 99.3, 99.4, 101.8, 114.1, 114.4, 129.7, 133.8, 150.9,
153.7, 154.3, 158.8, 159.9, 161.6, 163.1. Anal. calcd. for
C
23H22N2O4: C, 70.75; H, 5.68; N, 7.17. Found: C, 70.57; H, 5.81;
N, 6.94.
11-Amino-3-methyl-12-(3,4,5-trimethoxyphenyl)-
7,9,10,12-tetrahydro-1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-
b]quinolin-1-one (6m)
11-Amino-3-methyl-12-(o-tolyl)-7,9,10,12-tetrahydro-
1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1-one (6i)
Yield: 69%; white solid; m.p. > 250°C. IR (KBr): 3514, 3419,
2940, 1710 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.80–1.82 (m, 4H,
2 ꢂ CH2), 2.22 (s, 3H, CH3), 2.24–2.35 (m, 2H, CH2), 2.65 (s, 3H,
CH3), 2.75–2.77 (m, 2H, CH2), 4.06 (s, 2H, NH2), 5.04 (s, 1H, H12),
6.05 (s, 1H, H4), 7.12–7.25 (m, 4H, H30, H40, H50, H60). 13C NMR
(CDCl3, 100 MHz): 19.5, 20.0, 22.3, 22.5, 22.8, 32.3, 33.2, 99.2,
100.0, 101.4, 114.5, 126.8, 127.3, 130.3, 131.0, 136.1, 140.0,
151.1, 153.4, 154.2, 160.4, 161.6, 163.0. Anal. calcd. for
Yield: 79%; white solid; m.p. > 250°C. IR (KBr): 3474, 3381,
2940, 1722 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.85–1.86 (m, 4H,
2 ꢂ CH2), 2.23 (s, 3H, CH3), 2.26–2.40 (m, 2H, CH2), 2.77–2.80
(m, 2H, CH2), 3.79–3.80 (s, 9H, 3 ꢂ OCH3), 4.18 (s, 2H, NH2), 4.85
(s, 1H, H12), 6.05 (s, 1H, H4), 6.62 (s, 2H, H20, H60). 13C NMR
(CDCl3, 100 MHz): 19.9, 22.3, 22.5, 22.9, 32.5, 36.3, 56.3, 60.7,
98.9, 99.5, 101.5, 105.9, 114.4, 137.3, 137.4, 151.2, 153.4, 153.7,
154.5, 160.1, 161.7, 163.2. Anal. calcd. for C25H26N2O6: C,
66.65; H, 5.82; N, 6.22. Found: C, 66.48; H, 5.66; N, 6.50.
C
23H22N2O3: C, 73.78; H, 5.92; N, 7.48. Found: C, 73.56; H, 6.21;
N, 7.61.
11-Amino-12-(5-chloro-2-nitrophenyl)-3-methyl-
7,9,10,12-tetrahydro-1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-
b]quinolin-1-one (6n)
Yield: 54%; white solid; m.p. > 250°C. IR (KBr): 3483, 3394,
2943, 1716, 1648, 1515, 1345 cmꢃ1. 1H NMR (CDCl3, 400 MHz):
1.86–1.88 (m, 4H, 2 ꢂ CH2), 2.22 (s, 3H, CH3), 2.27–2.42 (m, 2H,
CH2), 2.80–2.82 (m, 2H, CH2), 5.06 (s, 2H, NH2), 5.75 (s, 1H, H12),
11-Amino-3-methyl-12-(m-tolyl)-7,9,10,12-tetrahydro-
1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-1-one (6j)
Yield: 80%; white solid; m.p. > 250°C. IR (KBr): 3487, 3443,
2933, 2859, 1731 cmꢃ1 1H NMR (CDCl3, 400 MHz): 1.81–1.83
.
(m, 4H, 2 ꢂ CH2), 2.21 (s, 3H, CH3), 2.27–2.37 (m, 5H, CH2, CH3),
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Archiv der Pharmazie
6.05 (s, 1H, H4), 7.18 (d, J ¼ 2.0 Hz, 1H, H60), 7.30 (dd, J ¼ 8.8,
2.0 Hz, 1H, H40), 7.77 (d, J ¼ 8.8 Hz, 1H, H30). 13C NMR (CDCl3,
100 MHz): 20.0, 22.2, 22.4, 22.9, 30.4, 32.5, 96.2, 99.3, 100.6,
114.3, 125.1, 128.4, 131.1, 138.9, 139.8, 146.9, 148.7, 151.4,
153.8, 155.2, 160.2, 162.7. Anal. calcd. for C22H18ClN3O5: C,
60.07; H, 4.12; N, 9.55 Found: C, 59.84; H, 4.32; N, 9.34.
Kinetic studies of AChE inhibition
Estimates of the inhibition model and inhibition constant
Ki, reciprocal plots of 1/V versus 1/[S] were obtained using
different concentrations of the substrate, acetylthiocholine.
For this purpose, experiments were performed similar to
enzyme inhibition assay [20]. The rate of enzymatic reaction
was obtained with different concentrations of inhibitor (0,
0.24, 0.47, and 0.93 mM). For each experiment, reaction was
started by adding substrate (acetylthiocholine) and progress
curves were recorded at 405 nm over 2 min. Next, double
reciprocal plots (1/v vs. 1/[s]) were made using the slopes of
progress curves to obtain the type of inhibition. Slopes of
these reciprocal plots were then plotted against the
concentration of compound 6f in a weighted analysis, and
Ki was determined as the intercept on the negative x-axis. All
rate measurements were performed in triplicate and data
analysis was performed with Microsoft Excel 2013.
11-Amino-3-methyl-12-(thiophen-2-yl)-7,9,10,12-
tetrahydro-1H,8H-pyrano[3’,4’:5,6]pyrano[2,3-b]quinolin-
1-one (6o)
Yield: 55%; white solid; m.p. > 250°C. IR (KBr): 3425, 3338,
2940, 1713, 1660, 1611 cmꢃ1. 1H NMR (CDCl3, 400 MHz): 1.84–
1.88 (m, 4H, 2 ꢂ CH2), 2.25 (s, 3H, CH3), 2.31–2.43 (m, 2H, CH2),
2.79–2.81 (m, 2H, CH2), 4.26 (s, 2H, NH2), 5.29 (s, 1H, H12), 6.05
(s, 1H, H4), 6.92 (dd, J ¼ 5.2, 3.5 Hz, 1H, H40), 7.15 (d, J ¼ 3.5 Hz,
1H, H50), 7.18 (dd, J ¼ 5.2, 1.2 Hz, 1H, H30). 13C NMR (CDCl3,
100 MHz): 20.0, 22.2, 22.5, 22.9, 30.7, 32.5, 98.7, 99.5, 100.2,
114.4, 125.2, 125.8, 126.5, 126.7, 145.2, 151.2, 154.8, 160.3,
161.9, 163.1. Anal. calcd. for C20H18N2O3S: C, 65.55; H, 4.95;
N, 7.64. Found: C, 65.41; H, 5.18; N, 7.47.
BACE1 enzymatic assay
Compound 6f was evaluated for the related BACE-1 inhibitory
activity using
a fluorescence resonance energy transfer
Pharmacology
(FRET) assay with recombinant human BACE-1 and quenched
fluorescent peptide substrate based on the Swedish mutant
APP sequence (SEVNLDAEFK). FRET-based enzymatic kit
was purchased from Invitrogen (former Pan Vera corporation,
Madison, WI) and the assay was carried out according
to the manufacturers’ instructions (Invitrogen, http://tools.
invitrogen.com/content/sfs/manuals/L0724.pdf) using a multi-
plate spectrofluorometer instrument (recording fluorescence
at 544 nm excitation and 590 nm emission wavelengths)
(BMG LABTECH, Polar star, Germany) [20].
Reagents and chemicals
Acetylcholinesterase (AChE, E.C. 3.1.1.7, type V-S, lyophilized
powder, from electric eel, 1000 units), butylcholinesterase
(BChE, E.C. 3.1.1.8, from equine serum), acetylthiocholine
iodide (ATCI), and 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)
were purchased from Sigma–Aldrich. Potassium dihydrogen
phosphate, dipotassium hydrogen phosphate, potassium
hydroxide, and sodium hydrogen carbonate were obtained
from Fluka. We followed our previous procedure for the
evaluation of AChEI and BChEI activity based on Ellman’s
method. For this purpose, a solution of compound 6 in a
mixture of DMSO (5 mL) and methanol (5 mL) was diluted
in 0.1 M KH2PO4/K2HPO4 buffer (pH 8.0) to obtain final
assay concentrations. All tests were conducted at 25°C
and four different concentrations were evaluated for
each compound in triplicate to obtain the range of 20–
80% inhibition for AChE. In vitro anti-AChE activity was
performed using a 96-well plate reader (BioTek ELx808).
Each well included 50 mL potassium phosphate buffer
(KH2PO4/K2HPO4, 0.1 M, pH 8), 25 mL sample dissolved in
50% methanol and 50% DMSO and 25 mL enzyme (final
concentration 0.22 U/mL in buffer). They were preincubated
for 15 min at room temperature and 125 mL DTNB (3 mM in
buffer) was added to each plate. Characterization of the
hydrolysis of ATCI catalyzed by AChE was performed
spectrometrically at 405 nm followed by the addition of
substrate (ATCI 3 mM in water). The change in absorbance
was measured at 405 nm after 15 min. The IC50 values were
determined graphically from inhibition curves (log inhibitor
concentration vs. percent of inhibition). A control experi-
ment was performed under the same conditions without
inhibitor and the blank contained buffer, water, DTNB, and
substrate. The described method was also used for BChE
inhibition assay.
Neuroprotection assay
PC12 cell line was purchased from the Pasteur Institute of Iran.
Cells were cultivated in DMEM supplemented with 10% fetal
calf serum, 5% horse serum, and antibiotics (100 units/mL
penicillin, 100 mg/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 þ 2% horse serum þ NGF (100 ng/mL) þ penicillin
and 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
candidate drugs (10, 50, 100 mM) were applied on cells.
Quercetin (50 mM) was used as a positive control. Drugs
were diluted into DMEM and added to each well in the
volume of 10 mL. Three hours later, induction of ROS
mediated apoptosis was initiated by adding the H2O2
(400 mM) to their medium and after 12 h, MTT assay was
performed. MTT solution (5 mg/mL) was added to each well
in a volume of 10 mL, and 3 h later the culture medium was
replaced with 100 mL of DMSO. Absorbance at 545 nm
was determined for each well using an ELISA reader. Each
experiment was performed in four replicates. All culture
media and supplements were purchased from Gibco.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Archiv der Pharmazie
Docking study
[12] E. Heilbronn, Acta Chem. Scand. 1961, 15, 1386–1390.
[13] Tacrine. Drugs in Clinical Trials. Alzheimer Research
Forum, http://www.alzforum.org/drg/drc/detail.asp?
id ¼ 90.
Docking simulations for compound 6f were carried out by
Autodock Vina (AV) 1.1.1 program [26]. For this purpose, the
pdb structure of 1EVE was retrieved from the Brookhaven
protein database (http://www.rcsb.org). Then, the water
molecules and inhibitor were removed and enzyme.pdbqt
was prepared by Autodock Tools (ver 1.5.4) with default
parameters. The compound 6f and its 3D structure were
generated by MarvineSketch 5.8.3, 2012 and then ligand.
pdbqt provided by Autodock Tools (ver 1.5.4). The Autodock
ꢀ
ꢀ
[14] M. I. Rodrıguez-Franco, M. I. Fernandez-Bachiller,
ꢀ
ꢀ
ꢀ
C. Perez, B. Hernandez-Ledesma, B. Bartolome, J. Med.
Chem. 2006, 49, 459–462.
[15] S. Butini, E, Guarino, G. Campiani, M. Brindisi,
S. S. Coccone, I. Fiorini, E. Novellino, T. Belinskaya,
A. Saxena, S. Gemma, Bioorg. Med. Chem. Lett. 2008, 18,
5213–5216.
[16] E. Nepovimova, J. Korabecny, R. Dolezal, K. Babkova,
A. Ondrejicek, D. Jun, V. Sepsova, A. Horova, M.
Hrabinova, O. Soukup, N. Bukum, P. Jost, L. Muckova,
J. Kassa, D. Malinak, M. Andrs, K. Kuca, J. Med. Chem.
2015, 58, 8985–9003.
̊
Vina parameters were set as follow; box size: 40 ꢂ 40 ꢂ 40 A,
the center of box: x ¼ 2.576, y ¼ 65.277, z ¼ 67.033 (geomet-
rical center of co-crystallized ligand), the exhaustiveness:
10, and the other parameters were left unchanged. The
calculated geometries were ranked in terms of free
energy of binding and the best pose was selected for
further analysis. Molecular visualizations were performed by
Discovery Studio 4.0 Client software (Accelrys, Inc., San
Diego, CA) [18].
[17] A. Romero, R. Cacabelos, M. J. Oset-Gasque, A. Samadi,
J. Marco-Contelles, Bioorg. Med. Chem. Lett. 2013, 23,
1916–1922.
[18] M. Khoobi, F. Ghanoni, H. Nadri, A. Moradi, M. P.
Hamedani, F. H. Moghadam, S. Emami, M. Vosooghi,
R. Zadmard, A. Foroumadi, A. Shafiee, Eur. J. Med. Chem.
2015, 89, 296–303.
This research has been supported by a grant from the
Research Council of Tehran University of Medical Sciences
with project no. 93-03-92-26151.
[19] M. Mahdavi, M. Saeedi, L. Gholamnia, S. A. B. Jeddi,
R. Sabourian, A. Shafiee, A. Foroumadi, T. Akbarzadeh,
J. Heterocyclic Chem., in press. DOI: 10.1002/jhet.2594
[20] Z. Najafi, M. Saeedi, M. Mahdavi, R. Sabourian,
M. Khanavi, M. B. Tehrani, F. H. Moghadam, N. Edraki,
E. Karimpor-Razkenari, M. Sharifzadeh, A. Foroumadi,
A. Shafiee, T. Akbarzadeh, Bioorg. Chem. 2016, 67,
84–94.
[21] M. Mohammadi-Khanaposhtani, M. Mahdavi, M. Saeedi,
R. Sabourian, M. Safavi, M. Khanavi, A. Foroumadi,
A. Shafiee, T. Akbarzadeh, Chem. Biol. Drug Des. 2015,
86, 1425.
[22] M. Mohammadi-Khanaposhtani, M. Saeedi, N. S.
Zafarghandi, M. Mahdavi, R. Sabourian, E. K. Razkenari,
H. Alinezhad, M. Khanavi, A. Shafiee, A. Foroumadi,
T. Akbarzadeh, Eur. J. Med. Chem. 2015, 92, 799–806.
[23] M. Saeedi, M. Golipoor, M. Mahdavi, A. Moradi, H. Nadri,
S. Emami, A. Foroumadi, A. Shafiee, Arch. Pharm. Chem.
Life Sci. 2016, 349, 293–301.
The authors have declared no conflicts of interest.
References
[1] M. Prince, J. Jackson, World Alzheimer Disease Report,
Alzheimer’s Disease International, London 2009,
pp. 25–46.
[2] E. J. Mufson, S. E. Counts, S. E. Perez, S. D. Ginsberg,
Expert. Rev. Neurother. 2008, 8, 1703–1718.
[3] R. T. Bartus, R. L. Dean, B. Beer, A. S. Lippa, Science 1982,
217, 408–414.
[4] S. Darvesh, D. A. Hopkins, C. Geula, Nature Rev. Neurosci.
2003, 4, 131–138.
[5] S. Darvesh, D. A. Hopkins, C. Geula, Nat. Rev. Neursci.
2003, 4, 131–138.
[6] H. Tago, T. Maeda, P. L. McGeer, H. Kimura, J. Comp.
Neurol. 1992, 325, 301–312.
[7] E. Giacobini, Neurochem. Int. 1998, 32, 413–419.
[8] R. A. Armstrong, Int. J. Alzheimers Dis. 2011, 2011,
630865.
[9] G. B. Irvine, O. M. El-Agnaf, G. M. Shankar, D. M. Walsh,
Mol. Med. 2008, 14, 451–464.
[24] A. Rayatzadeh, M. Saeedi, M. Mahdavi, Z. Rezaei,
R. Sabourian, M. H. Mosslemin, T. Akbarzadeh,
A. Foroumadi, A. Shafiee, Monatsh. Chem. 2015, 146,
637–643.
[25] M. Saeedi, S. Ansari, M. Mahdavi, R. Sabourian,
T. Akbarzadeh, A. Foroumadi, A. Shafiee, Synth.
Commun. 2015, 45, 2311–2318.
ꢀ
[10] N. C. Inestrosa, A. Alvarez, M. C. Dinamarca, T. Perez-Acle,
M. Colombres, Curr. Alzheimer Res. 2005, 2, 301–306.
[11] D. J. Selkoe, Nature 1999, 399, A21–A31.
[26] O. Trott, A. J. Olson, J. Comput. Chem. 2010, 31, 455–461.
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