Design, synthesis, docking study and biological evaluation of some novel tetrahydrochromeno [3′,4′:5,6]pyrano[2,3-b]quinolin-6(7H)-one derivatives against acetyl- and butyrylcholinesterase

Article abstract of DOI:10.1016/j.ejmech.2013.07.045

Novel hybrid derivatives of two known scaffolds; tetrahydroaminoquinoline and coumarin were synthesized and evaluated for both acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities. By means of an efficient nanocatalyst, the reaction time for the syntheses of the target compounds was reduced. Subsequently, Ellman's modified method was used to evaluate the enzyme inhibitory activity of the synthesized structures. It was observed that most hybrid structures were moderate to potent inhibitors of AChE compared to Tacrine as the reference drug among which 7f with 4-fluorophenyl substituent was the most active compound (IC₅₀ = 5 nM).

Full text of DOI:10.1016/j.ejmech.2013.07.045

Accepted Manuscript
Design, synthesis, docking study and biological evaluation of some novel
tetrahydrochromeno [3’,4’:5,6]pyrano[2,3-b]quinolin-6(7H)-one derivatives against
acetyl- and butyrylcholinesterase
Mehdi Khoobi, Masoumeh Alipour, Alireza Moradi, Amirhossein Sakhteman, Hamid
Nadri, Seyyede Faeze Razavi, Mehdi Ghandi, Alireza Foroumadi, Abbas Shafiee
PII:
S0223-5234(13)00495-9
10.1016/j.ejmech.2013.07.045
EJMECH 6334
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 April 2013
Revised Date: 23 June 2013
Accepted Date: 1 July 2013
Please cite this article as: M. Khoobi, M. Alipour, A. Moradi, A. Sakhteman, H. Nadri, S.F. Razavi,
M. Ghandi, A. Foroumadi, A. Shafiee, Design, synthesis, docking study and biological evaluation
of some novel tetrahydrochromeno [3’,4’:5,6]pyrano[2,3-b]quinolin-6(7H)-one derivatives against
acetyl- and butyrylcholinesterase, European Journal of Medicinal Chemistry (2013), doi: 10.1016/
j.ejmech.2013.07.045.
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ACCEPTED MANUSCRIPT
Graphical abstract
Design, synthesis, docking study and biological evaluation of some novel
tetrahydrochromeno [3’,4’:5,6]pyrano[2,3-b]quinolin-6(7H)-one derivatives against acetyl-
and butyrylcholinesterase.
Mehdi Khoobi, Masoumeh Alipour, Alireza Moradi, Amirhossein Sakhteman, Hamid Nadri,
Seyyede Faeze Razavi, Mehdi Ghandi, Alireza Foroumadi, Abbas Shafiee*
7f
A
novel series of hybrid structures, composed of two known scaffolds including
tetrahydroaminoquinoline and coumarin were synthesized and evaluated for both acetylcholinesterase
(AChE) and butyrylcholinesterase (BuChE) inhibitory activities.

ACCEPTED MANUSCRIPT
Research highlights
• In this study the synthesis of some tetrahydrochromeno[3’,4’:5,6]pyrano[2,3-b]quinolin-
6(7H)-one derivatives is reported.
• We examine the anticholinesterase activity of the target compounds.
• It was revealed that all compounds exhibited high anticholinesterase activity.
• The docking study showed that all compounds were dual binding site inhibitors.
• According to the kinetic study the mixed-type of inhibition was revealed.

ACCEPTED MANUSCRIPT
Design, synthesis, docking study and biological evaluation of some novel
tetrahydrochromeno [3’,4’:5,6]pyrano[2,3-b]quinolin-6(7H)-one derivatives
against acetyl- and butyrylcholinesterase
a
b
c
c
Mehdi Khoobi , Masoumeh Alipour , Alireza Moradi , Amirhossein Sakhteman ,
c
a
b
a,d
Hamid Nadri , Seyyede Faeze Razavi , Mehdi Ghandi , Alireza Foroumadi
Abbas Shafiee a,*
,
a
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical
Sciences Research Center, Tehran University of Medical Sciences, Tehran 14176,
Iran
b School of Chemistry, University College of Science, University of Tehran, P.O. Box
14155-6455, Tehran, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi
University of Medical Sciences-Yazd, Iran
d
Neuroscience Research Center, Kerman University of Medical Sciences, Kerman,
Iran
Abstract
Novel hybrid derivatives of two known scaffolds; tetrahydroaminoquinoline and
coumarin were synthesized and evaluated for both acetylcholinesterase (AChE) and
butyrylcholinesterase (BuChE) activities. By means of an efficient nanocatalyst, the
reaction time for the syntheses of the target compounds was reduced. Subsequently,
Ellman’s modified method was used to evaluate the enzyme inhibitory activity of the
synthesized structures. It was observed that most hybrid structures were moderate to
potent inhibitors of AChE compared to Tacrine as the reference drug among which 7f
with 4-fluorophenyl substituent was the most active compound (IC50 = 5 nM).
Keywords: Alzheimer’s disease; Coumarin; Tacrine; Cholinesterase inhibitor;
Docking study
* Corresponding author. Tel: +98 21 66406757; Fax: +98 21 66461178.
E-mail address: ashafiee@ams.ac.ir (A. Shafiee).
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1. Introduction
Alzheimer’s disease (AD) is the most common form of dementia [1]. The principle
pathological hallmarks of the disease are senile plaques that consist of amyloid β, and
neurofibrillary tangles. The formation of these plaques in hippocampal cholinergic
neurons results in neuronal cell death via apoptotic pathway [2]. In view of the fact
that, most of the AD symptoms were related to loss of cholinergic function in basal
forebrain, cholinergic hypothesis was developed [3]. Since Acetylcholinesterase
(AChE) is responsible for the hydrolysis of acetylcholine (Ach) in the synaptic cleft,
inhibition of this enzyme can increase the synaptic level of Ach and might lead to the
improvement of cognition and some behavioral problems [4]. Moreover, the role of
Butyrylcholinesterase (BuChE) in progression of AD has been proved. Furthermore,
BuChE inhibitors could recover cholinergic activity through restoring the
AChE/BuChE activity ratios as seen in healthy brain [5]. As a result, investigations
have been focused on dual AChE/BuChE inhibitors [6]. Tacrine (Fig. 1) (THA, 9-
amino-1,2,3,4-tetrahydroacridine), the first AChE inhibitor approved for treatment of
AD, has shown selectivity towards BuChE instead of AChE [7]. In this context
several Tacrine-like derivatives (such as compounds I [8], II [9], III [10] and IV
[11]) have been synthesized in which tetrahydroaminoquinoline moiety present in
tacrine was fused with different ring systems (Figure 1).
On the other hand, coumarin (2H-chromen-2-one) scaffold has recently gained
interest for the study of its biological activities. Compounds tethering coumarin
scaffold have shown wide range of biological activities such as anti-inflammatory,
anti-tumor, hepatoprotective, anti-allergic, anti-HIV-1, antiviral, antifungal,
antimicrobial, antiasthmatic, anti-oxidant, antinociceptive, anti-diabetic and
antidepressant effects [12]. Moreover, synthetic coumarins have exhibited potent
AChE inhibitory activity [13]. Further studies have been focused on modification of
coumarin ring that led to development of new analogs of coumarin with superior
activity (Figure 2) [14].
In continuation of the previous efforts in order to find more potent compounds [15],
new series of 8-amino-tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one
derivatives were designed and their BuChE/AChE inhibitor activities were
determined (Figure 3).
2. Results and discussion
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2.1. Chemistry
The synthesis of a novel series of Tacrine analogues 7a–r in a rather convenient way
is described in this work. As outlined in Scheme 1, these compounds could be easily
prepared by the AlCl3 promoted Friedländer reaction [16] between the corresponding
known dihydropyrano[c]chromenes and cyclohexanone. Several methods have been
reported for the synthesis of dihydropyrano[c]chromenes. Recently, the successful
usage of magnetic nanocatalyst including (2-aminomethyl)phenol moiety on the
surface of hydroxyapatite encapsulated maghemite (γ-Fe2O3) in aqueous medium for
the synthesis of 2-amino-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitriles
has been reported [17a]. In refluxing water and in the presence of magnetic
nanocatalyst, the addition of 4-hydroxycoumarin to the stirring mixture of various
aldehydes, 1a-r, and malonitrile 2 yielded 7-substituted 2-amino-5-oxo-4,5-
dihydropyrano[3,2-c]chromene-3-carbonitriles 5a-r. This ‘one-pot’ reaction protocol
proceeds through formation of alkylidene or arylidenemalononitrile intermediate 3.
Treatment of pyranochromene 5a-r, with cyclohexanone 6, in the presence of
aluminum trichloride and in refluxing 1,2-dicholorethane as solvent, afforded the
target molecules 7a-r. A notable feature of this procedure is the straightforward
product isolation without formation of any side-products. All products were
characterized using spectral data. For example, compound 7m was fully characterized
1
by IR, H and 13C NMR spectra and MS. The mass spectrum of 7m displayed a
molecular ion signal at m/z 426 and an ion signal at m/z 319 indicating the loss of the
1
4-methoxyphenyl group. In the H-NMR spectrum of compound 7m, in addition to
the aromatic protons of coumarin ring and those assigned to the phenyl ring (δ= 8.01–
6.78 ppm), a sharp singlet due to hydrogen in the pyrano moiety on 7 position (5.16
ppm) was observed. A broad singlet, exchangeable with D2O, at 5.80 ppm was also
assigned to amine group. In addition, the aliphatic protons of tetrahydroquinoline
moiety were observed at δ= 2.64-1.62 ppm. The most important absorption band of
7m in the IR spectrum is detected at 3475 and 3374 cm−1 and it is attributed to the
amine group stretching frequency. Absorption bands at 1709 cm−1 were associated to
1
13
the carbonyl group. The H decoupled C NMR spectrum of 7m showed 23 distinct
signals. In this spectrum, the methine of the pyrano moiety on 7 position resonated at
δ = 33.6 and the signal for the carbonyl was observed at δ = 157.9. In addition, six
methines and eleven quaternary carbons, all in the aromatic region were in agreement
with the structure.
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2.2. Cholinesterase inhibition study
Compounds 7a-r were evaluated as AChE/BuChE inhibitors using colorimetric
Ellman’s method [18,19]. Tacrine was used as the reference drug. The inhibitory
activity of the target compounds was summarized in Table 1. It was demonstrated that
7f was the most active compound against eelAChE (IC50 = 5 nM) and compound 7p
was the most potent inhibitor of BuChE (IC50 = 570 nM). It was also found that most
of the synthesized structures were more selective against AChE. On the other hand,
the compounds 7a, 7b, and 7e could be considered as dual inhibitors of both
enzymes.
According to the table values, replacement of the aromatic substituents at position 7
by aliphatic methyl group (7a) led to an increase in the activity towards BuChE but
the AChE inhibition potency was diminished. The resulting compound could be
therefore considered as a dual inhibitor. Meanwhile, elongation of aliphatic side chain
in case of compound 7b and 7c increased the activity due to the more lipophilic
property of these substituents. Insertion of aromatic groups at position 7 with the
ability to form π-π interaction in the hydrophobic cavity caused significant increase in
activity. However, the substituent position on the aromatic ring could largely affect
the activity of the target compounds. Substitution at positions 2 and 4 was therefore
more tolerated while substitution at position 3 was not favorable as seen in compound
7e, 7h and 7k with the IC50 values of 67, 288 and 141 nM, respectively. The order of
activity in 4-substituted compounds was F>CH3=OCH3=H>Cl. The electron
withdrawing substituents such as fluoro and chloro at position 2 and 4 increased the
inhibitory activity probably through reinforcing the π-π interactions as seen in
compounds 7f, 7g and 7i. On the other hand, electron donating groups such as methyl
and methoxy induce their enhancing effects through lipophilic interaction with the
active site as seen in 7l, 7m and 7p.
In order to verify the validity of our hypothesis on the efficacy of compounds with
hybrid structures of tetrahydroaminoquinoline and coumarin, all synthesized
intermediates 5a-r, with only one scaffold of coumarin were evaluated for their
inhibitory activity. It was observed that the intermediates were significantly less
potent than their corresponding target compounds. This finding has therefore
confirmed our hypothesis for design of hybrid structure using the two aforementioned
scaffolds.
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To determine the kinetic type of AChE inhibition in case of the most active
compound 7f, a kinetic study was carried out. The inhibition type was established
through analysis of Lineweaver-Burk reciprocal plots (Figure 4). A mixed-type
inhibition was concluded during the study. This finding proposed that the target
compounds interact with enzyme in both peripheral anionic site (PAS) and central
anionic site (CAS) and was in good agreement with docking data. Ki value of 22.4
nM was obtained from the slopes of the double reciprocal plots versus compound 7f
concentrations.
2.3. Docking studies
In order to find out the binding mode for the synthesized compounds, docking
simulation studies were conducted using Autodock vina 1.1.1 [20]. To verify the
predictive ability of vina scoring function for this target, a set of 107 AChE inhibitors
together with 1097 decoys were retrieved from DUD database and docked with
Autodock vina. The two common metrics of virtual ligand screening including
enrichment factor (EF) and the area under the curve of ROC (AUCROC) were
calculated for the docking results [21]. As depicted in (Figure 5) both values of EFmax
and AUCROC were within reasonable range (EFmax=5.63 and AUCROC=0.671)
verifying that the used parameters and scoring function are able to discriminate
between active and inactive compound. Being satisfied with the predictive ability of
the docking procedure, the target compounds were subjected to vina as described in
the experimental section.
The binding mode of the most active compounds 7f as depicted in Figure 6 revealed a
proper fitting of the compound in the gorge of eelAChE. The phenyl ring at position 7
is oriented towards the hydrophobic pocket of the binding cavity composed of
Phe330, Tyr334 and Phe331. A π-π stacking between the phenyl side chain of Tyr334
and the phenyl moiety in compound 7f takes role in stabilization of the ligand-
receptor complex. It seems that the observed decrease in the activity of the
compounds with aliphatic methyl group, 7a, might be due to lack of this π-π
interaction. On the other hand, in compound 7c the possible hydrophobic interaction
of butyl group with the mentioned hydrophobic pocket might lead to an increase in
the activity compared to 7a. Another π-π stacking interaction between pyridine ring
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and indole side chain of Trp279 is a common feature of all target compounds. This
interaction is able to donate specific conformation to the compounds so that the
lipophilic cyclohexane ring is fitted in the hydrophobic packet composed by Phe290,
Leu282, Phe288, Ile287 and Ser286. Other notable interactions of the synthesized
hybrid structures were as follow: a hydrogen bonding of coumarin carbonyl moiety
with hydroxyl of Tyr121 and CH-π stacking between side chain of Gln74 and phenyl
ring of coumarin scaffold.
3. Conclusions
New hybrid structures of tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-
one were synthesized and evaluated for AChE and BuChE inhibitory activity. By
using magnetic nanocatalyst in water medium, a very efficient method for the
preparation of the intermediate compounds with short reaction times was obtained.
Most of the synthesized compounds revealed significant AChE inhibitory activity
among which 7f was the most active compound (IC50 = 5 nM). Based on the docking
studies it was suggested that the complex of ligand protein in the active site is
stabilized through π-π stacking and hydrogen bond interactions. Finally, it was
concluded that the hybrid of tetrahydroaminoquinoline and coumarin scaffold could
be proposed as promising lead structure for further modifications.
4. Experimental
4.1. General
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 spectrographs (KBr discs). 1H NMR spectra were recorded
on a Varian 400 or Brucker 500 MHz NMR instruments. The atoms numbering of the
intermediate and target compounds used for 1H NMR data are depicted in Scheme 1.
The chemical shifts (δ) and coupling constants (J) are expressed in parts per million
and hertz, respectively. Mass spectra of the products were obtained with an HP
(Agilent technologies) 5937 Mass Selective Detector. Elemental analyses were
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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.
4.2. General procedure for the synthesis of 2-amino-4-alkyl or aryl-5-oxo-4,5-
dihydropyrano[3,2-c]chromene-3-carbonitriles 5a-r
A stirring mixture of an appropriate aldehyde (1 mmol), malononitrile (1.2 mmol),
magnetic catalytic system (1.5 mol %) and water (5 mL) were heated under refluxing
conditions for a few minutes. To this stirred mixture, 4-hydroxycoumarin (1 mmol)
was added. The reaction mixture was refluxed for 10 to 30 minutes. The progress of
the reaction was monitored by TLC. After completion of the reaction, it was allowed
to cool at room temperature and the reaction mixture was diluted with ethyl acetate
and the catalyst was easily separated from the reaction mixture with an external
magnet and washed twice with ethyl acetate. The combined organic layers were
concentrated in vacuum and the resulting residue was purified by recrystallization
from ethanol.
4.2.1.
(5a)
2-amino-4-methyl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile
Yield 70%, white solid, mp 227-229 ºC,17b IR υmax/cm-1 (KBr): 3390 and 3314 (NH2),
1
2192 (CN), 1708 (C=O). H NMR (DMSO-d6, 400 MHz) δ 7.81 (d, 1H, J = 7.5 Hz,
H10), 7.70 (t, 1H, J = 7.5 Hz, H8), 7.49-7.41 (m, 2H, H7,9), 7.25 (s, 2H, NH2), 4.34 (q,
1H, J = 7.0 Hz, H4), 1.30 (d, 3H, CH3, J = 7.0 Hz).
4.2.2. 2-amino-4-ethyl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (5b)
Yield 88%, white solid, mp 239-241 ºC, IR υmax/cm-1 (KBr): 3395 and 3316 (NH2),
2191 (CN), 1710 (C=O). 1H NMR (DMSO-d6, 500 MHz) δ 7.82 (dd, 1H, J = 1.4 and
7.7 Hz, H10), 7.71 (dt, 1H, J = 1.4 and 7.7 Hz, H8), 7.50-7.44 (m, 2H, H7,9), 7.32 (s,
2H, NH2), 3.45 (t, 1H, J = 7.0 Hz, H4), 1.83-1.56 (m, 2H, CH2), 0.77 (t, 3H, J = 7.5
Hz, CH3). Anal. Calcd for C15H12N2O3 C, 67.16; H, 4.51; N, 10.44. Found C, 67.35,
H, 4.39, N, 10.73.
4.2.3. 2-amino-4-butyl-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile (5c)
Yield 70%, white solid, mp 202-205 ºC, IR υmax/cm-1 (KBr): 3405 and 3291 (NH2),
1
2192 (CN), 1723 (C=O). H NMR (DMSO-d6, 400 MHz) δ 7.81 (d, 1H, J = 7.5 Hz,
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H10), 7.70 (t, 1H, J = 7.5 Hz, H8), 7.49-7.44 (m, 2H, H7,9), 7.31 (s, 2H, NH2), 3.43 (t,
1H, J = 7.0 Hz, H4), 1.74-1.56 (m, 2H, CH2CH2CH2CH3), 1.27-1.14 (m, 4H,
CH2CH2CH2CH3), 0.83 (t, 3H, J = 7.0 Hz, CH2CH2CH2CH3). Anal. Calcd for
C17H16N2O3: C, 68.91; H, 5.44; N, 9.45. Found: C, 69.15, H, 5.18, N, 9.73.
4.2.4.
(5d)
2-amino-5-oxo-4-phenyl-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile
Yield 78%, white solid, mp 256-258 ºC,17a IR υmax/cm-1 (KBr): 3378 and 3286 (NH2),
1
2196 (CN), 1709 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.90 (d, 1H, J = 7.5 Hz,
H10), 7.70 (t, 1H, J = 7.5 Hz, H8), 7.52-7.44 (m, 2H, H7,9), 7.41 (s, 2H, NH2), 7.35-
7.23 (m, 5H, Hphenyl), 4.45 (s, 1H, H4).
4.2.5.
2-amino-4-(3-fluorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5e)
Yield 76%, pale yellow solid, mp 234-236 ºC,17c IR υmax/cm-1 (KBr): 3384 and 3315
1
(NH2), 2199 (CN), 1701 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.91 (d, 1H, J =
7.5 Hz, H10), 7.73 (t, 1H, J = 7.5 Hz, H8), 7.54-7.44 (m, 4H, H7,9 and NH2), 7.38-7.34
(m, 1H, Hphenyl), 7.14-7.07 (m, 3H, Hphenyl), 4.52 (s, 1H, H4).
4.2.6.
2-amino-4-(4-fluorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5f)
Yield 79%, yellow solid, mp 250-252 ºC,17d IR υmax/cm-1 (KBr): 3380 and 3293
1
(NH2), 2192 (CN), 1715 (C=O). H NMR (DMSO-d6, 400 MHz) δ 7.90 (d, 1H, J =
7.5 Hz, H10), 7.71 (t, 1H, J = 7.5 Hz, H8), 7.52-7.45 (m, 2H, H7,9), 7.43 (s, 2H, NH2),
7.35-7.31 (m, 2H, Hphenyl), 7.16-7.11 (m, 2H, Hphenyl), 4.48 (s, 1H, H4).
4.2.7.
2-amino-4-(2-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5g)
Yield 70%, pale yellow solid, mp 273-275 ºC,17c IR υmax/cm-1 (KBr): 3407 and 3282
1
(NH2), 2200 (CN), 1718 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.92 (d, 1H, J =
7.5 Hz, H10), 7.73 (t, 1H, J = 7.5 Hz, H8), 7.51 (t, 1H, J = 7.5 Hz, H9), 7.48 (d, 1H, J =
7.5 Hz, H7), 7.44-7.37 (m, 3H, 1Hphenyl and NH2), 7.33-7.24 (m, 3H, Hphenyl), 4.98 (s,
1H, H4).
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4.2.8.
2-amino-4-(3-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5h)
Yield 74%, yellow solid, mp 253-255 ºC,17d IR υmax/cm-1 (KBr): 3384 and 3323
1
(NH2), 2204 (CN), 1697 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.91 (d, 1H, J =
7.5 Hz, H10), 7.73 (t, 1H, J = 7.5 Hz, H8), 7.52-7.49 (m, 4H, H7,9), 7.48-7.43 (m, 3H,
1Hphenyl and NH2), 7.39-7.33 (m, 3H, Hphenyl), 4.74 (s, 1H, H4).
4.2.9.
2-amino-4-(4-chlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5i)
Yield 89%, white solid, mp 266-268 ºC,17e IR υmax/cm-1 (KBr): 3380 and 3312 (NH2),
1
2192 (CN), 1713 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.90 (d, 1H, J = 7.5 Hz,
H10), 7.72 (t, 1H, J = 7.5 Hz, H8), 7.52-7.43 (m, 4H, H7,9 and NH2), 7.37 (d, 2H, J =
8.5 Hz, Hphenyl), 7.31 (d, 2H, J = 8.5 Hz, Hphenyl), 4.49 (s, 1H, H4).
4.2.10. 2-amino-4-(2,4-dichlorophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5j)
Yield 78%, white solid, mp 244-246 ºC,17e IR υmax/cm-1 (KBr): 3459 and 3414 (NH2),
2198 (CN), 1719 (C=O). 1H NMR (DMSO-d6, 500 MHz) δ 7.91 (dd, 1H, J = 7.7 and
1.4 Hz, H10), 7.74 (dt, 1H, J = 1.4 and 7.7 Hz, H8), 7.59 (d, 1H, J = 1.7 Hz, Hphenyl),
7.53-7.48 (m, 4H, H7,9 and NH2), 7.41-7.34 (m, 2H, Hphenyl), 4.98 (s, 1H, H4).
4.2.11.
2-amino-4-(3-nitrophenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5k)
Yield 84%, white solid, mp 262-264 ºC,17a IR υmax/cm-1 (KBr): 3404 and 3322 (NH2),
1
2202 (CN), 1703 (C=O), 1527 and 1345 (NO2). H NMR (DMSO-d6, 500 MHz) δ
8.14-8.10 (m, 2H, Hphenyl), 7.92 (d, 1H, J = 7.5 Hz, H10), 7.80 (d, 1H, J = 7.2 Hz,
Hphenyl), 7.72 (t, 1H, J = 7.5 Hz, H8), 7.63 (t, 1H, J = 7.2 Hz, Hphenyl), 7.54 (s, 2H,
NH2), 7.50 (t, 1H, J = 7.5 Hz, H9), 7.45 (d, 1H, J = 7.5 Hz, H7), 4.72 (s, 1H, H4).
4.2.12. 2-amino-5-oxo-4-p-tolyl-4,5-dihydropyrano[3,2-c]chromene-3-carbonitrile
(5l)
Yield 78%, white solid, mp 227-229 ºC,17d IR υmax/cm-1 (KBr): 3387 and 3312 (NH2),
1
2192 (CN), 1713 (C=O). H NMR (DMSO-d6, 400 MHz) δ 7.90 (d, 1H, J = 7.5 Hz,
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H10), 7.70 (t, 1H, J = 7.5 Hz, H8), 7.52-7.43 (m, 2H, H7,9), 7.37 (s, 2H, NH2), 7.16-
7.08 (m, 4H, Hphenyl), 4.40 (s, 1H, H4), 2.28 (s, 3H, CH3).
4.2.13. 2-amino-4-(4-methoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5m)
Yield 78%, white solid, mp 240-242 ºC,17a IR υmax/cm-1 (KBr): 3365 and 3280 (NH2),
1
2202 (CN), 1707 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.89 (d, 1H, J = 7.5 Hz,
H10), 7.69 (t, 1H, J = 7.5 Hz, H8), 7.50-7.42 (m, 2H, H7,9), 7.35 (s, 2H, NH2), 7.16 (d,
2H, J = 7.2 Hz, Hphenyl), 6.85 (d, 2H, J = 7.2 Hz, Hphenyl), 4.39 (s, 1H, H4), 3.72 (s, 3H,
OCH3).
4.2.14. 2-amino-4-(3,4-dimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-c]chromene-
3- carbonitrile (5n)
Yield 84%, white solid, mp 234-236 ºC, IR υmax/cm-1 (KBr): 3370 and 3323 (NH2),
1
2191 (CN), 1723 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.90 (d, 1H, J = 7.5 Hz,
H10), 7.71 (t, 1H, J = 7.5 Hz, H8), 7.51-7.46 (m, 2H, H7,9), 7.36 (s, 2H, NH2), 6.88 (d,
1H, J = 8.3 Hz, Hphenyl), 6.51 (s, 1H, Hphenyl), 6.75 (d, 1H, J = 8.3 Hz, Hphenyl), 4.41 (s,
1H, H4), 3.72 (s, 6H, 2×OCH3). Anal. Calcd for C21H16N2O5 C, 67.02; H, 4.28; N,
7.44. Found C, 66.83, H, 4.46, N, 7.64.
4.2.15.
2-amino-4-(3,4,5-trimethoxyphenyl)-5-oxo-4,5-dihydropyrano[3,2-
c]chromene-3- carbonitrile (5o)
Yield 82%, white solid, mp 218-220 ºC, IR υmax/cm-1 (KBr): 3433 and 3303 (NH2),
2192 (CN), 1717 (C=O). 1H NMR (DMSO-d6, 500 MHz) δ 7.90 (dd, 1H, J = 1.4 and
8.2 Hz, H10), 7.72 (dt, 1H, J = 1.4 and 8.2 Hz, H8), 7.52-7.47 (m, 2H, H7,9), 7.38 (s,
2H, NH2), 6.53 (s, 2H, Hphenyl), 4.44 (s, 1H, H4), 3.72 (s, 6H, 2×OCH3), 3.63 (s, 3H,
OCH3). Anal. Calcd for C22H18N2O6 C, 65.02; H, 4.46; N, 6.89. Found C, 65.31, H,
4.69, N, 6.63.
4.2.16.
2-amino-4-(benzo[d][1,3]dioxol-5-yl)-5-oxo-4,5-dihydropyrano[3,2-
c]chromene-3 carbonitrile (5p)
Yield 79%, white solid, mp 253-255 ºC, IR υmax/cm-1 (KBr): 3399 and 3317 (NH2),
1
2190 (CN), 1721 (C=O). H NMR (DMSO-d6, 400 MHz) δ 7.89 (d, 1H, J = 7.5 Hz,
H10), 7.70 (t, 1H, J = 7.5 Hz, H8), 7.52-7.41 (m, 2H, H7,9), 7.37 (s, 2H, NH2), 6.85-
10

ACCEPTED MANUSCRIPT
6.79 (m, 2H, Hphenyl), 6.73 (d, 1H, J = 7.2 Hz, Hphenyl), 5.98 (s, 2H, OCH2O), 4.39 (s,
1H, H4). Anal. Calcd for C20H12N2O5: C, 66.67; H, 3.36; N, 7.77. Found: C, 66.89, H,
3.13, N, 8.02.
4.2.17.
2-amino-5-oxo-4-(pyridin-4-yl)-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5q)
Yield 70%, white solid, mp 270-272 ºC,17a IR υmax/cm-1 (KBr): 3375 and 3283 (NH2),
1
2192 (CN), 1707 (C=O). H NMR (DMSO-d6, 500 MHz) δ 8.51 (d, 2H, J = 4.5 Hz,
Hpyridin), 7.91 (d, 1H, J = 7.5 Hz, H10), 7.72 (t, 1H, J = 7.5 Hz, H8), 7.53 (s, 2H, NH2),
7.51-7.45 (m, 2H, H7,9),7.33 (d, 2H, J = 4.5 Hz, Hpyridin), 4.51 (s, 1H, H4).
4.2.18.
2-amino-5-oxo-4-(thiophen-2-yl)-4,5-dihydropyrano[3,2-c]chromene-3-
carbonitrile (5r)
Yield 75%, white solid, mp 256-258 ºC,17a IR υmax/cm-1 (KBr): 3370 and 3272 (NH2),
1
2200 (CN), 1709 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.87 (d, 1H, J = 7.5 Hz,
H10), 7.68 (t, 1H, J = 7.5 Hz, H8), 7.52 (s, 2H, NH2), 7.49-7.37 (m, 3H, 2H7,9 and
1HThiophen), 7.16 (d, 1H, J = 4.5 Hz, HThiophen), 6.95 (t, 1H, J = 4.5 Hz, HThiophen), 4.81
(s, 1H, H4).
4.3. General method for the Friedländer reaction
Aluminium chloride (1.5 equiv) was suspended in dry 1,2-dichloroethane (10 mL) at
ambient temperature under argon atmosphere. The suspension was stirred for a few
minutes. Then the corresponding pyranochromene (1 equiv) and cyclohexanone (1.5
equiv) were added. The reaction mixture was heated under reflux (24 h). After
completion of the reaction (monitored by TLC), an aqueous solution of sodium
hydroxide (10%) was added drop wise to the mixture until the aqueous solution
became basic. After stirring for 30 min, the precipitate was filtered and washed with
water. For further purification the solid was crystallized from ethanol or acetonitrile to
give corresponding products.
4.3.1.
8-amino-7-methyl-9,10,11,12-tetrahydrochromeno[3',4':5,6]pyrano[2,3-
b]quinolin-6(7H)-one (7a)
11

ACCEPTED MANUSCRIPT
Yield 90%, white solid, mp 250-252 ºC, IR υmax/cm-1 (KBr): 3425 and 3375 (NH2),
1
1706 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.93 (d, 1H, J = 7.6 Hz, H1), 7.77-
7.74 (m, 3H, H3 and NH2), 7.55-7.52 (m, 2H, H2,4), 4.16 (q, 1H, J = 6.2 Hz, H7), 2.82-
2.75 (m, 2H, H12), 2.445-2.32 (m, 2H, H9), 1.82-1.71 (m, 4H, 2H10 and 2H11), 1.33 (d,
3H, J = 6.2 Hz, CH3). 13C NMR (DMSO-d6, 125 MHz) δ 160.0, 156.2, 153.5, 151.9,
148.7, 145.6, 133.0, 124.8, 122.1, 116.6, 113.7, 112.8, 106.4, 99.2, 26.8, 23.2, 22.3,
20.8, 20.5, 19.3. MS m/z (%) 334 (M+, 7), 319 (100), 303 (9), 291 (13), 121 (18), 77
(13). Anal. Calcd for C20H18N2O3 C, 71.84, H, 5.43, N, 8.38. Found C, 72.06, H, 4.62,
N, 8.46.
4.3.2.
8-amino-7-ethyl-9,10,11,12-tetrahydrochromeno[3',4':5,6]pyrano[2,3-
b]quinolin-6(7H)-one (7b)
Yield 93%, white solid, mp 260-262 ºC, IR υmax/cm-1 (KBr): 3428 and 3385 (NH2),
1
1706 (C=O). H NMR (CDCl3, 500 MHz) δ 8.17 (dd, 1H, J = 1.4, 7.5 Hz, H1), 7.58
(dt, 1H, J = 1.4, 7.5 Hz, H3), 7.39-7.33 (m, 2H, H2,4), 4.40 (s, 2H, NH2), 4.18 (t, 1H, J
= 4.0 Hz, H7), 2.84-2.82 (m, 2H, H12), 2.45 (t, 2H, J = 6.1 Hz, H9), 2.04-1.82 (m, 6H,
2H10, 2H11 and CH2CH3), 0.74 (t, 3H, J = 7.5 Hz, CH3). 13C NMR (CDCl3, 125 MHz)
δ 161.9, 157.9, 153.7, 152.6, 150.4, 140.4, 132.0, 124.2, 123.3, 116.5, 114.2, 114.1,
102.8, 97.8, 32.3, 30.3, 24.5, 23.1, 22.4, 22.2, 8.8. MS m/z (%) 348 (M+, 2), 319
(100), 303 (10), 291 (13), 121 (10), 77 (5). Anal. Calcd for C21H20N2O3 C, 72.40; H,
5.79; N, 8.04. Found C, 72.66, H, 5.51, N, 7.81.
4.3.3.
8-amino-7-butyl-9,10,11,12-tetrahydrochromeno[3',4':5,6]pyrano[2,3-
b]quinolin-6(7H)-one (7c)
Yield 90%, white solid, mp 242-244 ºC, IR υmax/cm-1 (KBr): 3410 and 3355 (NH2),
1
1709 (C=O). H NMR (DMSO-d6, 500 MHz) δ 7.93 (d, 1H, J = 7.3 Hz, H1), 7.67 (t,
1H, J = 7.3 Hz, H3), 7.47-7.42 (m, 2H, H2,4), 6.04 (s, 2H, NH2), 4.28-4.2 (m, 1H, H7),
2.65-2.57 (m, 2H, H12), 2.39-2.33 (m, 2H, H9), 1.79-1.62 (m, 4H, 2H10 and 2H11),
1.11-1.06 (m, 2H, CH2CH2CH2CH3), 1.06-0.83 (m, 4H, CH2CH2CH2CH3), 0.68 (t,
3H, J = 6.7 Hz, CH2CH2CH2CH3). 13C NMR (DMSO-d6, 125 MHz) δ 160.6, 156.8
(2C), 154.1, 152.0, 151.5, 132.3, 124.5, 122.3, 116.4, 113.7, 113.6, 103.6, 96.7, 31.8,
31.2, 28.7, 26.1, 23.0, 22.3, 22.2, 22.0, 13.8. MS m/z (%) 376 (M+, 38), 319 (100),
303 (40), 291 (77), 121 (31), 77 (19), 57 (27). Anal. Calcd for C23H24N2O3 C, 73.38,
H, 6.43, N, 7.44. Found C, 73.57, H, 6.68, N, 7.35.
12

ACCEPTED MANUSCRIPT
4.3.4.
8-amino-7-phenyl-9,10,11,12-tetrahydrochromeno[3',4':5,6]pyrano[2,3-
b]quinolin-6(7H)-one (7d)
Yield 95%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3409 and 3359 (NH2), 1710
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.02 (d, 1H, J = 7.5 Hz, H1), 7.67 (t, 1H, J
= 7.5 Hz, H3), 7.48-7.39 (m, 4H, H2,4 and 2Hphenyl), 7.22 (t, 2H, J = 7.2 Hz, Hphenyl),
7.14 (t, 1H, J = 7.2 Hz, Hphenyl), 5.84 (s, 2H, NH2), 5.23 (s, 1H, H7), 2.64-2.62 (m, 2H,
H12), 2.39-2.17 (m, 2H, H9), 1.85-1.65 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6,
125 MHz) δ 160.1, 155.2, 153.1, 152.6, 152.0, 151.5, 142.8, 132.5, 128.3, 128.0,
126.7, 124.6, 122.6, 116.4, 113.9, 113.8, 104.8, 97.9, 34.4, 31.8, 23.0, 22.1, 21.9. MS
m/z (%) 396 (M+, 18), 319 (100), 121 (14), 92 (15), 77 (70), 51 (42). Anal. Calcd for
C25H20N2O3 C, 75.74, H, 5.08, N, 7.07. Found C, 75.91, H, 4.83, N, 7.21.
4.3.5.
8-amino-7-(3-fluorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4'5,6]pyrano[2,3-b]quinolin-6(7H)-one (7e)
Yield 92%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3436 and 3363 (NH2), 1708
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.01 (d, 1H, J = 7.5 Hz, H1), 7.68 (t, 1H, J
= 7.5 Hz, H3), 7.48-7.37 (m, 3H, 1Hphenyl and 2H2,4), 7.27-7.23 (m, 1H, Hphenyl), 7.11
(d, 1H, J = 7.4 Hz, Hphenyl), 6.98 (t, 1H, J = 7.4 Hz, Hphenyl), 5.93 (s, 2H, NH2), 5.28 (s,
1H, H7), 2.65-2.61 (m, 2H, H12), 2.39-2.15 (m, 2H, H9), 1.73-1.65 (m, 4H, 2H10 and
2H11). 13C NMR (DMSO-d6, 125 MHz) δ 162.6, 160.6, 160.1, 155.4, 153.0, 152.1,
151.6, 145.6, 132.6, 130.1, 124.6, 124.1, 122.7, 116.4, 115.7, 115.5, 114.0, 113.7,
113.6, 113.4, 104.6, 97.7, 34.2, 31.8, 23.0, 22.1, 21.9. MS m/z (%) 414 (M+, 50), 319
(75), 162 (44),121 (38), 109 (100), 95 (58), 77 (60), 62 (83). Anal. Calcd for
C25H19FN2O3 C, 72.45, H, 4.62, N, 6.76. Found C, 72.36, H, 4.95, N, 6.91.
4.3.6.
8-amino-7-(4-fluorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4'5,6]pyrano[2,3-b]quinolin-6(7H)-one (7f)
Yield 92%, orange solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3373 and 3230 (NH2),
1
1709 (C=O). H NMR (DMSO-d6, 500 MHz) δ 8.01 (d, 1H, J = 7.6 Hz, H1), 7.68 (t,
1H, J = 7.6 Hz, H3), 7.48-7.43 (m, 4H, 2Hphenyl and H2,4), 7.07-7.04 (m, 2H, Hphenyl),
5.87 (s, 2H, NH2), 5.26 (s, 1H, H7), 2.624-2.62 (m, 2H, H12), 2.39-2.15 (m, 2H, H9),
1.79-1.70 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125 MHz) δ 161.8, 160.1,
159.9, 155.2, 153.0, 152.8, 152.0, 151.5, 138.9, 132.5, 130.2 (2C), 124.6, 122.6,
13

ACCEPTED MANUSCRIPT
116.4, 114.8, 114.6, 114.0, 113.8, 104.6, 97.7, 33.6, 31.8, 23.0, 22.1, 21.9. MS m/z
(%) 414 (M+, 70), 319 (100), 121 (18), 95 (15), 77 (10). Anal. Calcd for C25H19FN2O3
C, 72.45, H, 4.62, N, 6.76. Found C, 72.67, H, 4.85, N, 6.93.
4.3.7.
8-amino-7-(2-chlorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7g)
Yield 90%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3489 and 3383 (NH2), 1710
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.02 (d, 1H, J = 7.5 Hz, H1), 7.69 (t, 1H, J
= 7.5 Hz, H3), 7.57 (d, 1H, J = 7.1 Hz, Hphenyl), 7.50-7.42 (m, 2H, H2,4), 7.34 (d, 1H, J
= 7.1 Hz, Hphenyl), 7.27-7.22 (m, 2H, Hphenyl), 5.48 (s, 2H, NH2), 5.37 (s, 1H, H7), 2.65-
2.60 (m, 2H, H12), 2.39-2.15 (m, 2H, H9), 1.78-1.65 (m, 4H, 2H10 and 2H11). 13C NMR
(DMSO-d6, 125 MHz) δ 159.8, 155.7, 153.0, 152.8, 152.1, 151.5, 138.7, 132.7 (2C),
132.3, 129.6, 128.8, 127.3, 124.6, 122.8, 116.4, 114.1, 113.5, 102.4, 96.6, 34.0, 31.7,
22.9, 22.1, 21.8. MS m/z (%) 432 (M++2, 19), 430 (M+, 55), 395 (42), 319 (100), 175
(47), 121 (41), 111 (41), 77 (30). Anal. Calcd for C25H19ClN2O3 C, 69.69, H, 4.44, N,
6.50. Found C, 69.58, H, 4.71, N, 6.39.
4.3.8.
8-amino-7-(3-chlorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7h)
Yield 92%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3423 and 3375 (NH2), 1710
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.00 (d, 1H, J = 6.4 Hz, H1), 7.68-7.62 (m,
2H, H3 and Hphenyl), 7.45-7.39 (m, 2H, H2,4), 7.27-7.18 (m, 3H, Hphenyl), 5.93 (s, 2H,
NH2), 5.26 (s, 1H, H7), 2.69-2.53 (m, 2H, H12), 2.42-2.15 (m, 2H, H9), 1.75-1.61 (m,
4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125 MHz) δ 160.1, 155.5 (2C), 152.9,
152.1, 151.6, 145.1, 132.6, 132.4, 130.1, 128.5, 126.7 (2C), 124.6, 122.7, 116.4,
114.0, 113.7, 104.1, 97.2, 34.1, 31.8, 23.0, 22.1, 21.8. MS m/z (%) 432 (M++2, 1),
430 (M+, 3), 319 (100), 121 (27), 111 (50), 92 (36), 77 (59), 62 (90), 55 (89). Anal.
Calcd for C25H19ClN2O3 C, 69.69, H, 4.44, N, 6.50. Found C, 69.90, H, 4.70, N, 6.83.
4.3.9.
8-amino-7-(4-chlorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7i)
Yield 93%, white solid, mp 248-250 ºC, IR υmax/cm-1 (KBr): 3497 and 3386 (NH2),
1707 (C=O). 1H NMR (CDCl3, 500 MHz) δ 8.23 (d, 1H, J = 7.7 Hz, H1), 7.57 (t, 1H,
J = 7.7 Hz, H3), 7.37 (t, 1H, J = 7.7 Hz, H2), 7.31 (d, 1H, J = 7.7 Hz, H4), 7.28-7.25
14

ACCEPTED MANUSCRIPT
(m, 4H, Hphenyl), 5.04 (s, 1H, H7), 4.17 (s, 2H, NH2), 2.89-2.86 (m, 2H, H12), 2.39-2.30
(m, 2H, H9), 1.89-1.87 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125 MHz) δ
161.2, 155.7, 154.7, 153.6, 152.6, 150.8, 139.9, 133.3, 132.2, 130.0, 128.9, 124.2,
123.5, 116.5, 114.7, 114.1, 103.8, 98.4, 35.9, 32.4, 22.9, 22.4, 22.1. MS m/z (%) 432
(M++2, 3), 430 (M+, 9), 350 (23), 319 (100), 283 (22), 249 (46), 239 (98), 121 (33),
92 (26). Anal. Calcd for C25H19ClN2O3 C, 69.69; H, 4.44; N, 6.50. Found C, 69.41,
H, 4.63, N, 6.28.
4.3.10.
8-amino-7-(2,4-dichlorophenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7j)
Yield 93%, white solid, mp >300 ºC, IR υmax/cm-1 (KBr): 3485 and 3388 (NH2), 1718
1
(C=O). H NMR (CDCl3, 500 MHz) δ 8.25 (d, 1H, J = 7.8 Hz, H1), 7.59 (t, 1H, J =
7.8 Hz, H3), 7.41-7.36 (m, 2H, H2 and Hphenyl), 7.33 (d, 1H, J = 8.2 Hz, Hphenyl), 7.23
(d, 1H, J = 8.2 Hz, Hphenyl), 7.15 (d, 1H, J = 7.8 Hz, H4), 5.45 (s, 1H, H7), 4.49 (s, 2H,
NH2), 2.85-2.80 (m, 2H, H12), 2.40-2.26 (m, 2H, H9), 1.86-1.83 (m, 4H, 2H10 and
2H11). 13C NMR (CDCl3, 125 MHz) δ 160.8, 156.3, 15.9, 153.2, 152.8, 151.0, 137.9,
133.8, 133.6, 132.5, 132.1, 131.0, 129.2, 128.2, 124.3, 123.6, 116.6, 114.4, 113.9,
98.0, 33.2, 32.3, 22.9, 22.3, 22.1. MS m/z (%) 468 (M++4, 2), 466 (M++2, 12), 464
(M+, 19), 429 (33), 396 (18), 319 (100), 291 (20), 264 (13), 121 (15), 77 (16). Anal.
Calcd for C25H18Cl2N2O3 C, 64.53; H, 3.90; N, 6.02. Found C, 64.74, H, 4.21, N,
6.37.
4.3.11.
8-amino-7-(3-nitrophenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7k)
Yield 91%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3419 and 3363 (NH2), 1708
(C=O), 1531 and 1350 (NO2). 1H NMR (DMSO-d6, 500 MHz) δ 8.47 (s, 1H, Hphenyl),
8.05-8.01 (m, 2H, H1 and Hphenyl), 7.77-7.67 (m, 2H, H3 and Hphenyl), 7.54-7.42 (m,
3H, 2H2,4 and Hphenyl), 6.04 (s, 2H, NH2), 5.46 (s, 1H, H7), 2.66-2.61 (m, 2H, H12),
2.41-2.13 (m, 2H, H9), 1.79-1.62 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125
MHz) δ 160.1, 155.6, 153.0, 152.9, 152.1, 151.8, 147.2, 144.7, 134.8, 132.7, 129.7,
124.6, 123.3, 122.7, 121.9, 116.5, 114.2, 113.7, 103.8, 96.9, 34.1, 31.7, 22.9, 22.0,
21.8. MS m/z (%) 441 (M+, 6), 401 (11), 319 (100), 212 (31), 177 (20), 159 (85), 120
(20), 89 (21), 77 (17). Anal. Calcd for C25H19N3O5 C, 68.02, H, 4.34, N, 9.52. Found
C, 68.25, H, 4.13, N, 9.84.
15

ACCEPTED MANUSCRIPT
4.3.12.
8-amino-7-(p-tolyl)-9,10,11,12-tetrahydrochromeno[3',4':5,6]pyrano[2,3-
b]quinolin-6(7H)-one (7l)
Yield 95%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3474 and 3375 (NH2), 1708
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.01 (d, 1H, J = 6.9 Hz, H1), 7.66 (t, 1H, J
= 6.9 Hz, H3), 7.46-7.40 (m, 2H, H2,4), 7.27 (d, 2H, J = 6.5 Hz, Hphenyl), 7.02 (d, 2H, J
= 6.5 Hz, Hphenyl), 5.80 (s, 2H, NH2), 5.17 (s, 1H, H7), 2.64-2.59 (m, 2H, H12), 2.39-
2.11 (m, 5H, 2H9 and CH3), 1.77-1.62 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6,
125 MHz) δ 160.1, 155.0, 153.0, 152.6, 152.0, 151.5, 139.8, 135.8, 132.4, 128.6,
128.2, 124.6, 122.6, 116.4, 113.9, 113.8, 104.9, 98.0, 34.0, 31.8, 23.0, 22.1, 21.9,
20.5. MS m/z (%) 410 (M+, 81), 319 (100), 121 (18), 91 (26), 65 (33). Anal. Calcd for
C26H22N2O3 C, 76.08, H, 5.40, N, 6.82. Found C, 76.29, H, 5.73, N, 7.03.
4.3.13.
8-amino-7-(4-methoxyphenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7m)
Yield 93%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3475 and 3374 (NH2), 1709
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.01 (d, 1H, J = 7.5 Hz, H1), 7.68 (t, 1H, J
= 7.5 Hz, H3), 7.48-7.40 (m, 2H, H2,4), 7.31 (d, 2H, J = 7.8 Hz, Hphenyl), 6.78 (d, 2H, J
= 7.8 Hz, Hphenyl), 5.80 (s, 2H, NH2), 5.16 (s, 1H, H7), 3.66 (s, 3H, OCH3), 2.64-2.62
(m, 2H, H12), 2.37-2.20 (m, 2H, H9), 1.75-1.62 (m, 4H, 2H10 and 2H11). 13C NMR
(DMSO-d6, 125 MHz) δ 160.1, 157.9, 154.9, 153.0, 152.5, 152.0, 151.4, 134.8, 132.4,
129.3, 124.6, 122.6, 116.4, 113.8 (2C), 113.4, 105.0, 98.1, 54.9, 33.6, 31.8, 23.0, 22.2,
21.9. MS m/z (%) 426 (M+, 40), 319 (33), 98 (30), 64 (98), 62 (100), 49 (70). Anal.
Calcd for C26H22N2O4 C, 73.23, H, 5.20, N, 6.57. Found C, 73.46, H, 5.51, N, 6.81.
4.3.14.
8-amino-7-(3,4-dimethoxyphenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7n)
Yield 91%, white solid, mp 171-173 ºC, IR υmax/cm-1 (KBr): 3471 and 3342 (NH2),
1720 (C=O). 1H NMR (CDCl3, 500 MHz) δ 8.22 (d, 1H, J = 7.5 Hz, H1), 7.56 (t, 1H,
J = 7.5 Hz, H3), 7.35 (t, 1H, J = 7.7 Hz, H2), 7.31 (d, 1H, J = 7.7 Hz, H4), 7.02 (d, 1H,
J = 1.7 Hz, Hphenyl), 6.96 (dd, 1H, J = 1.7, 8.3 Hz, Hphenyl), 6.78 (d, 1H, J = 8.3 Hz,
Hphenyl), 5.02 (s, 1H, H7), 4.22 (s, 2H, NH2), , 3.83 (s, 3H, OCH3), 3.82 (s, 3H, OCH3),
2.88-2.84 (m, 2H, H12), 2.42-2.30 (m, 2H, H9), 1.90-1.82 (m, 4H, 2H10 and 2H11). 13C
NMR (CDCl3, 125 MHz) δ 159.9, 154.6, 152.8, 152.2, 151.8, 151.0, 145.9, 143.8,
16

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136.1, 133.2, 124.2, 123.1, 122.3, 115.8, 114.1, 113.1, 109.8, 108.2, 105.4, 98.5, 59.5,
55.7, 34.5, 32.1, 23.3, 22.2, 22.0. MS m/z (%) 456 (M+, 27), 319 (100), 291 (19), 137
(16), 121 (24), 92 (17), 77 (31). Anal. Calcd for C27H24N2O5 C, 71.04; H, 5.30; N,
6.14. Found C, 71.37, H, 5.17, N, 6.46.
4.3.15.
8-amino-7-(3,4,5-trimethoxyphenyl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7o)
Yield 95%, white solid, mp 262-264 ºC, IR υmax/cm-1 (KBr): 3476 and 3334 (NH2),
1726 (C=O). 1H NMR (CDCl3, 500 MHz) δ 8.23 (d, 1H, J = 7.5 Hz, H1), 7.57 (t, 1H,
J = 7.5 Hz, H3), 7.37 (t, 1H, J = 7.5 Hz, H2), 7.31 (d, 1H, J = 7.5 Hz, H4), 6.66 (s, 2H,
Hphenyl), 5.02 (s, 1H, H7), 4.31 (s, 2H, NH2), 3.80 (s, 6H, 2×OCH3), 3.79 (s, 3H,
OCH3), 2.90-2.85 (m, 2H, H12), 2.43-2.29 (m, 2H, H9), 1.92-1.86 (m, 4H, 2H10 and
2H11). 13C NMR (CDCl3, 125 MHz) δ 161.3, 153.4 (3C), 152.6, 137.4 (2C), 136.8,
132.3, 124.3, 123.6, 116.5, 114.6, 114.1, 105.9 (2C), 104.0, 98.8, 60.7, 56.2, 36.8,
29.6, 22.8, 22.3, 22.1. MS m/z (%) 486 (M+, 7), 319 (100), 291 (5), 167 (3), 121 (18),
97 (35), 85 (42), 71 (64), 57 (98). Anal. Calcd for C28H26N2O6 C, 69.12; H, 5.39; N,
5.76. Found C, 69.38, H, 5.57, N, 5.54.
4.3.16.
8-amino-7-(benzo[d][1,3]dioxol-5-yl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7p)
Yield 94%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3432 and 3355 (NH2), 1720
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.01 (d, 1H, J = 7.5 Hz, H1), 7.68 (t, 1H, J
= 7.5 Hz, H3), 7.48-7.41 (m, 2H, H2,4), 7.03 (s, 1H, Hphenyl), 6.78-6.75 (m, 2H, Hphenyl),
5.92 (s, 2H, OCH2O), 5.85 (s, 2H, NH2), 5.15 (s, 1H, H7), 2.65-2.60 (m, 2H, H12),
2.40-2.18 (m, 2H, H9), 1.79-1.63 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125
MHz) δ 160.1, 155.0, 153.0, 152.6, 152.0, 151.5, 146.7, 145.9, 136.7, 132.5, 124.6,
122.6, 121.4, 116.4, 113.9, 113.8, 109.1, 107.9, 104.8, 100.8, 97.9, 34.0, 31.8, 23.0,
22.1, 21.9. MS m/z (%) 440 (M+, 75), 319 (100), 121 (24), 65 (35). Anal. Calcd for
C26H20N2O5 C, 70.90, H, 4.58, N, 6.36. Found C, 70.68, H, 4.79, N, 6.11.
4.3.17.
8-amino-7-(pyridin-4-yl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7q)
Yield 89%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3453 and 3374 (NH2), 1712
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.42 (d, 2H, J = 5.1 Hz, Hpyridine), 8.03 (d,
17

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1H, J = 7.5 Hz, H1), 7.70 (t, 1H, J = 7.5 Hz, H3), 7.50-7.40 (m, 4H, H2,4 and
2Hpyridine), 5.94 (s, 2H, NH2), 5.32 (s, 1H, H7), 2.68-2.54 (m, 2H, H12), 2.61-2.18 (m,
2H, H9), 1.77-1.64 (m, 4H, 2H10 and 2H11). 13C NMR (DMSO-d6, 125 MHz) δ 160.1,
155.9 (2C), 153.1, 152.1, 151.7, 151.0, 149.4, 132.7, 124.7, 123.5, 122.7, 116.5,
114.1, 113.7, 103.4, 96.5, 33.9, 31.8, 23.0, 22.1, 21.8. MS m/z (%) 397 (M+, 73), 319
(100), 121 (35), 79 (40). Anal. Calcd for C24H19N3O3 C, 72.53, H, 4.82, N, 10.57.
Found C, 72.29, H, 5.14, N, 10.78.
4.3.18.
8-amino-7-(thiophen-2-yl)-9,10,11,12-
tetrahydrochromeno[3',4':5,6]pyrano[2,3-b]quinolin-6(7H)-one (7r)
Yield 90%, white solid, mp >260 ºC, IR υmax/cm-1 (KBr): 3413 and 3367 (NH2), 1710
1
(C=O). H NMR (DMSO-d6, 500 MHz) δ 8.00 (d, 1H, J = 7.6 Hz, H1), 7.70 (t, 1H, J
= 7.6 Hz, H3), 7.49-7.45 (m, 2H, H2,4), 7.26 (d, 1H, J = 4.7 Hz, Hthiophen), 7.15 (d, 1H,
J = 2.2 Hz, Hthiophen), 6.87 (dd, 1H, J = 4.7 and 2.2 Hz, Hthiophen), 6.07 (s, 2H, NH2),
5.65 (s, 1H, H7), 2.71-2.60 (m, 2H, H12), 2.42-2.23 (m, 2H, H9), 1.69-1.75 (m, 4H,
2H10 and 2H11). 13C NMR (DMSO-d6, 125 MHz) δ 160.1, 155.3, 152.8, 152.0, 151.8,
146.5, 132.7, 126.4, 125.8, 124.7(2C), 122.6, 116.5, 113.9, 113.7, 104.4, 97.5, 31.9,
29.7, 23.0, 22.1, 21.9. MS m/z (%) 402 (M+, 80), 319 (100), 121 (50), 84 (43), 77
(41). Anal. Calcd for C23H18N2O3S C, 68.64, H, 4.51, N, 6.96. Found C, 68.85, H,
4.74, N, 7.18.
4.4. Determination of anticholinesterase activity
The inhibitory potency of target compounds on AChE and BuChE was determined
using colorimetric Ellman’s method [18,19]. Acetylcholinesterase (AChE, E.C.
3.1.1.7, Type V-S, lyophilized powder, from electric eel, 1000 unit),
butyrylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum), butyrylthiocholine
iodide (BTC), were obtained from Sigma–Aldrich. 5, 5′-Dithiobis-(2-nitrobenzoic
acid) (DTNB), potassium dihydrogen phosphate, dipotassium hydrogen phosphate,
potassium hydroxide, sodium hydrogen carbonate, and acetylthiocholine iodide were
purchased from Fluka. In brief, 50 µl of five different concentration of the test
compounds was added to the mixture of 3 ml phosphate buffer 0.1M (pH=8.0), 100 µl
of DTNB solution (0.1M), and 50 µl of AChE (2.5 IU/ml). After 10 minutes
incubation at 25oC, 10 µl solution of acetylthiocholine iodide (0.15M) as substrate
was added. The change of absorbance was measured at 412 nm for 6 minutes. The
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IC50 values were determined graphically from inhibition linear curves (log inhibitor
concentration Vs percent of inhibition). UV-2100 Rayleigh Double Beam
Spectrophotometer was used for spectrophotometric measurements. The same method
was used for BuChE inhibition assay.
4.5. Kinetic study
To obtain estimates of the inhibition model and inhibition constant Ki, reciprocal
plots of 1/V versus 1/[S] were constructed at different concentrations of the substrate
acetylthiocholine (0.14-0.69 mM) by using Ellman’s method. For this purpose,
experiments were performed similar to enzyme inhibition assay. The rate of
enzymatic reaction was measured with different inhibitor concentrations and without
inhibitor for proposed substrate concentrations. For each experiment, reaction was
initiated by adding acetylthiocholine as substrate. Progress curves were monitored at
412 nm over 2 min. Then, double reciprocal plots (1/v vs. 1/[s]) were constructed
using the slopes of progress curves to obtain the type of inhibition. Slopes of the
reciprocal plots were then plotted against the concentration of inhibitor, to evaluate Ki
data. Data analysis was performed with Microsoft Excel 2003. All rate measurements
were performed in triplicate.
4.6. Docking studies
The pdb structure of 3I6Z (Torpedo californica acetylcholinesterase) was
downloaded from the Brookhaven protein database [22] in complex with the inhibitor
N-sacharinohexyl-galantamine. Afterwards, the co-crystallized ligand and the water
molecules were removed from the protein. The structures of the target compounds
were
drawn
using
MarvineSketch
5.8.3,
2012,
ChemAxon
(http://www.chemaxon.com) and converted to 3D by means of Openbabel 2.3.1 [23].
Autodock Tools 1.5.4 [24] was finally used to prepare the proper pdbqt format of the
protein and ligands with default parameters. For docking purpose Autodock vina
1.1.1 was taken. The exhaustiveness was set to 10 and other parameters were
remained unchanged. The box size was set to the dimensions of 50, 50, 50, to cover
all binding sites occurred in the active site of the enzyme. The center of grid box was
19

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set to x=6.231, y = 67.871, z = 58.888 and the lowest energy conformations were
selected for analyzing the interactions between the AChE and inhibitor. PosViewWeb
1.97.035 [25] and Chimera 1.6 [26] were used for visual representation of the ligand
protein complex. Calculation and plot of ROC and EF metrics were done using our
application implemented in vb.net.
Acknowledgments
This work was financially supported by Grants from the Research Council of Tehran
University of Medical Sciences, Iran National Elite Foundation (INEF) and Iran
National Science Foundation (INSF). Molecular graphics and analyses were
performed with the UCSF Chimera package. Chimera is developed by the 25
Resource for Biocomputing, Visualization, and Informatics at the University of
California, San Francisco.
20

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Figure captions:
Figure 1. Tacrine and Tacrine-like derivatives as dual AChE/BuChE inhibitors.
Figure 2. Some potent anticholinesterase structures bearing coumarin moiety.
Figure 3. Design strategy of the target compounds 7a–r by juxtaposition of coumarin
and tetrahydroaminoquinoline.
Figure 4. Steady-state inhibition of AChE by compound 7f. (left) Lineweaver–Burk
plot of reciprocal of initial velocities versus reciprocal of acetylthiocholine iodide
concentrations (0.14-0.69 mM) in the absence and presence of 7f at 2 nM, 5 nM and
10 nM; (right) secondary plots of the Lineweaver-Burk plot, slope versus various
concentrations of 7f.
Figure 5. (left) ROC plot of virtual screening step, (right) enrichment factor plot in
logarithmic scale.
Figure 6. (left) residues involved in the interactions with compound 7f, (right) 2D
representation of binding mode of 7f in the gorge of TcAChE.
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Table 1. Inhibition of AChE from electrophorus electricus (eelAChE) and horse
serum butyrylcholinesterase (eqBuChE) by compounds 7a-r.
O
R
NH2
N
O
O
IC50 (nM)
AChE
Selectivity
Compounds
R
Index(S.I.)a
BuChE
1220
5600
23000
1700
890
7a
7b
methyl
ethyl
432
266
57
2.8
21
400
71
13
3140
140
-
7c
n-butyl
7d
24
Phenyl
7e
3-fluorophenyl
67
7f
4-fluorophenyl
2-chlorophenyl
3-chlorophenyl
4-chlorophenyl
2,4-dichlorophenyl
3-nitrophenyl
5
15700
4900
NAb
NA
7g
35
7h
288
93
7i
-
7j
50
NA
-
7k
141
14
18300
NA
130
-
7l
4-methylphenyl
4-methoxyphenyl
3,4-dimethoxyphenyl
3,4,5-trimethoxyphenyl
benzodioxole-5-yl
4-pyridyl
7m
7n
16
840
52.5
49
47
44
-
43
2100
3260
570
7o
69
7p
13
7q
53
NA
7r
2-thienyl
84
6700
32
80
0.08
Tacrine
-
410
a Selectivity for AChE
b No activity (IC50 > 30 µM)
26

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Figure 1
Figure 2
27