Hetero-annulated coumarins as new AChE/BuChE inhibitors: synthesis and biological evaluation

Article abstract of DOI:10.1007/s00044-016-1626-7

A series of chromene-fused coumarins known as 10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-diones 4a–o were synthesized through one-pot reaction of appropriate benzaldehydes, dimedone, and 4-hydroxycoumarin in the presence of nano-silica sulfuric acid

Full text of DOI:10.1007/s00044-016-1626-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-016-1626-7
ORIGINAL RESEARCH
Hetero-annulated coumarins as new AChE/BuChE inhibitors:
synthesis and biological evaluation
Seyed Esmaeil Sadat Ebrahimi1 Pegah Ghadirian1 Hamideh Emtiazi2
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Azadeh Yahya-Meymandi1 Mina Saeedi3,4 Mohammad Mahdavi5 Hamid Nadri6
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Alireza Moradi6 Bilqees Sameem1 Mohsen Vosooghi7 Saeed Emami8
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Alireza Foroumadi9 Abbas Shafiee9
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Received: 10 January 2015 / Accepted: 27 June 2016
© Springer Science+Business Media New York
Abstract A series of chromene-fused coumarins known
as 10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-
3-hydroxyphenyl analog 4e showed the highest inhibitory
activity against both acetylcholinesterase and butyr-
ylcholinesterase, possessing IC50 values of 3.28 and 2.19 µM,
respectively. The structure-activity relationships study
demonstrated that the selectivity for acetylcholinesterase
over butyrylcholinesterase could be modulated by introdu-
cing second hydroxyl or methoxy substituent on the para-
position of the 3-hydroxyphenyl pendent group. The
docking study of compound 4e with acetylcholinesterase
confirmed π–π stacking interaction between the coumarin
moiety and Trp279 as well as the formation of hydrogen
bonding between hydroxyl group and Asn85.
diones 4a–o were synthesized through one-pot reaction
of appropriate benzaldehydes, dimedone, and 4-
hydroxycoumarin in the presence of nano-silica sulfuric
acid under solvent-free condition in good yields.
The in vitro anticholinesterase assay revealed that the
Electronic supplementary material The online version of this article
(doi:10.1007/s00044-016-1626-7) contains supplementary material,
which is available to authorized users.
* Abbas Shafiee
ashafiee@ams.ac.ir
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Keywords Alzheimer’s disease Acetylcholinesterase
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Coumarin-fused derivatives One-pot synthesis
Multicomponent reactions
1
School of Pharmacy, Tehran University of Medical Sciences,
International Campus (TUMS- IC), Tehran, Iran
2
Faculty of Pharmacy, Shahid Sadoughi University of Medical
Sciences, Yazd, Iran
3
Medicinal Plants Research Center, Faculty of Pharmacy, Tehran
Introduction
University of Medical Sciences, Tehran, Iran
4
Persian Medicine and Pharmacy Research Center, Tehran
Acetylcholinesterase (AChE) is a serine hydrolase enzyme
mainly found in peripheral and central nervous system. It
catalyzes the hydrolysis of the ACh to choline in order to
terminate nerve impulses (Čolović et al., 2013). Hence, cho-
linergic neurotransmission is involved in a variety of patho-
physiological conditions; AChE is a known pharmacological
target for the treatment of several pathologies particularly
Alzheimer’s disease (AD) (Orhan et al., 2011). The patho-
genesis of AD is related to the deficiency of ACh in the brain
and in this respect, AChE inhibitors may be used to treat some
symptoms of AD (Hong-Qi et al., 2012). Moreover, AChE
inhibitors have been used in the treatment of glaucoma
(McKinnon et al., 2008), myasthenia gravis, and neuromuscular
blockade (Meriggioli and Sanders, 2009; Bevan et al., 1992).
University of Medical Sciences, Tehran, Iran
5
Drug Design and Development Research Center, Tehran
University of Medical Sciences, Tehran, Iran
6
Department of Medicinal Chemistry, Faculty of Pharmacy and
Neurobiomedical Research Center, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
7
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
8
Department of Medicinal Chemistry and Pharmaceutical Sciences
Research Center, Faculty of Pharmacy, Mazandaran University of
Medical Sciences, Sari, Iran
9
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, Tehran, Iran

Med Chem Res
Fig. 1 Structures of hetero-
annulated coumarins (decursinol
and mesuagenin B) with AChE
inhibitory activity and new
designed compounds 4a–o
CH3
H3C
H3C
CH3
O
H3C
HO
O
O
HO
O
O
H3C
R
O
O
O
H3C
H3C
O
O
O
H3C
Decursinol
Mesuagenin B
4a-o
Besides AChE, butyrylcholinesterase (BuChE) plays a
crucial role in ACh hydrolysis, especially when selective
anticholinesterases inhibit AChE. However, the neurobio-
logical role of BuChE has not yet been fully understood
(Alves-Amaral et al., 2010).
Recently, we designed and synthesized different series
of hetero-annulated comarins namely tetrahydro-
chromeno[3′,4′:5,6]pyrano[2,3-b]quinolin-6(7H)-ones and
5-oxo-4,5-dihydropyrano[3,2-c]chromene derivatives as
AChE and BuChE inhibitors (Khoobi et al., 2013a, b).
Herein, in continuation of our research program on the
synthesis of bioactive compounds (Mahdavi et al., 2015;
Mohammadi-Khanaposhtani et al., 2015a, b; Rahmani-
Nezhad et al., 2015; Rayatzadeh et al., 2015), we describe
synthesis, anti-cholinesterase activity, and docking study of
10,11-dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-diones
4a–o (Fig. 1).
The X-ray crystallographic structures of AChE–ligand
complexes revealed that there are two main binding sites in
the enzyme: (i) the catalytic binding site, comprising the
Ser-His-Glu catalytic triad, and (ii) the peripheral anionic
site (PAS), connected by a deep, hydrophobic gorge (Mis-
hra and Sasmal, 2013). Previous studies demonstrated that
AChE could also play an important role in increasing
amyloid β-peptide (Aβ) plaques deposition in the brain
(Hardy and Selkoe, 2002). It seems that AChE binds to Aβ
via a pool of amino acids located in the proximity of PAS
and promotes amyloid fibril formation (De Ferrari et al.,
2001). It has been shown that compounds interacting with
PAS or both PAS and catalytic anionic site (CAS) can
prevent pro-aggregation activity of AChE toward Aβ
(Inestrosa et al., 1996).
Coumarin derivatives are chemically synthesized com-
pounds or naturally occurring phytochemicals with a broad
spectrum of bioactivities such as antimicrobial, antifungal,
antiviral, anti-HIV-1, anti-cancer, anti-inflammatory, anti-
allergic, antiasthmatic, antioxidant, antinociceptive, and hepa-
toprotective effects (Anand et al., 2012). Previous studies have
demonstrated that coumarin derivatives have shown potent
cholinesterase inhibitory activity. Moreover, anti-amnestic
activity and memory restorative effect of coumarins have
been documented in the literature (Wu et al., 2007). The
importance of coumarin moiety from both chemical and bio-
logical points of view have made coumarins useful and
interesting scaffold for drug discovery research (Riveiro et al.,
2010). Youkwan et al. (2010) reported that furanocoumarin
derivatives found in Citrus hystrix fruits acted as AChE
inhibitors. Decursinol (Fig. 1), a dihydropyranocoumarin iso-
lated from Angelica gigas (Umbelliferae), exhibited potent
AChE inhibition (IC50 = 0.28 µM) (Kang et al., 2001). Also,
mesuagenin B (Fig. 1), derived from pyranocoumarin was
found to be a potent AChE inhibitor possessing IC50 = 0.7 µM
(Awang et al., 2010).
Materials and methods
Melting points were taken on a Kofler hot stage apparatus
and are uncorrected. 1H and 13C nuclear magnetic resonance
(NMR) spectra were recorded on Bruker FT-400 MHz,
using TMS as an internal standard. The infrared spectra
were obtained on a Nicolet Magna FTIR 550 spectro-
photometer (in KBr). Mass spectra were determined on an
Agilent Technology (HP) mass spectrometer operating at an
ionization potential of 70 eV. The elemental analysis was
performed on an Elementar Analysensystem GmbH
VarioEL.
General procedure for the synthesis of chromeno[4,3-b]
chromenederivatives 4a–o
A mixture of appropriate aldehyde 1 (1 mmol), dimedone 2
(1 mmol, 0.140 g), 4-hydroxycoumarine 3 (1 mmol, 0.162
g), and nano-silica sulfuric acid (nano-SSA) (0.015 g) was
heated under solvent free conditions at 100 °C for 2–3 h.
After completion of the reaction (monitored by thinb layer
chromatography), the mixture was dissolved in hot
dichloromethane and was filtered to remove the catalyst.
The solvent was evaporated under vacuum and the pure
product 4 was obtained by recrystallization from ethanol.

Med Chem Res
10,10-Dimethyl-7-phenyl-7,9,10,11-tetrahydro-6H,8H-
chromeno[4,3-b]chromene-6,8-dione (4a)
mp 203-205 °C; IR (KBr) νmax2980, 2870, 1724, 1665 cm−1;
1H NMR (CDCl3, 400 MHz): δ = 7.87 (1H, dd, J = 8.0, 1.2
Hz, H1), 7.58 (1H, td, J = 8.0, 1.2 Hz, H3), 7.38–7.33 (2H,
m, H4, H5′), 7.23–7.19 (2H, m, H2, H2′), 7.05–7.02 (1H, m,
H6′), 6.89–6.83 (1H, m, H4′), 4.98 (1H, s, CH, H7), 2.73
(1H, d, J = 17.6 Hz, CH2b′), 2.65 (1H, d, J = 17.6 Hz,
CH2b), 2.34 (1H, d, J = 16.4 Hz, CH2a′), 2.28 (1H, d, J =
16.4 Hz, CH2a), 1.17 (3H, s, Me), 1.10 (3H, s, Me); 13C
NMR (CDCl3, 100 MHz): δ = 196.0 (C=O, C-8), 162.7 (d,
JC-F = 245.0 Hz, C-3′), 162.2 (C=O, C-6), 160.6 (C-O, C-
O12), 154.2 (C-O, C11-C-O12), 152.6 (C-O, C-O5), 145.0
(C-1′), 144.9 (C-5′), 132.5 (C-3), 129.9 (C-2), 129.7 (C-6′),
124.5 (C-1), 124.4 (C-4), 122.5 (C, C-C1), 116.9 (C, C7-C-
C8), 115.5 (d, JC-F = 21.7 Hz, C-2′), 114.1 (d, JC-F = 20.9
Hz, C-4′), 106.2 (C, C6-C-C7), 50.7 (CH2), 40.8 (CH2),
33.2 (C-7), 32.3 (C-10), 29.1 (Me), 27.6 (Me); anal. calcd.
for C24H19FO4: C, 73.84; H, 4.91. Found: C, 74.00;
H, 5.09.
Yellow solid (This compound was prepared by the reaction
of benzaldehyde, dimedone, and 4-hydroxycoumarin. It was
obtained as yellow solid); mp 215–216 °C; IR (KBr)
1
νmax2960, 1718, 1666 cm−1; H NMR (CDCl3, 400 MHz):
δ = 7.89 (1H, dd, J = 8.0, 1.6 Hz, H1), 7.58 (1H, td, J = 8.0,
1.6 Hz, H3), 7.41–7.37 (3H, m, Ph), 7.34 (1H, dd, J = 8.0,
1.6 Hz, H4), 7.29–7.25 (2H, m, Ph), 7.16 (1H, td, J = 8.0,
1.6 Hz, H2), 4.99 (1H, s, CH, H7), 2.74 (1H, d, J = 18.0 Hz,
CH2b′), 2.67 (1H, d, J = 18.0 Hz, CH2b), 2.35 (1H, d, J =
16.0 Hz, CH2a′), 2.28 (1H, d, J = 16.0 Hz, CH2a), 1.19 (3H,
s, Me), 1.11 (s, 3H, Me); 13C NMR (CDCl3, 100 MHz): δ =
196.0 (C=O, C-8), 161.9 (C=O, C-6), 160.6 (C-O, C-O12),
153.9 (C-O, C11-C-O12), 152.6 (C-O, C-O5), 142.5 (C-1′),
132.2 (C-3′, C-5′), 128.6 (C-3), 128.3 (C-2′, C-6′), 127.1
(C-2), 124.2 (C-4′), 122.4 (C-1), 116.9 (C-4), 115.1 (C, C-
C1), 113.7 (C, C7-C-C8), 106.8 (C, C6-C-C7), 50.7 (CH2),
40.8 (CH2), 33.4 (C-7), 32.4 (C-10), 29.2 (Me), 27.6 (Me);
anal. calcd. for C24H20O4: C, 77.40; H,5.41. Found: C,
77.63; H, 5.57.
10,10-Dimethyl-7-(3-nitrophenyl)-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4d)
White solid (This compound was prepared by the
reaction of 3-nitrobenzaldehyde, dimedone, and
4-hydroxycoumarin. It was obtained as white solid); mp
228–230 °C; IR (KBr) νmax2959, 1740, 1682, 1545, 1341
7-(3-Bromophenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4b)
1
Pale yellow solid (This compound was prepared by the
reaction of 3-bromobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 233–235 °C; IR (KB) νmax3063, 2977, 1729, 1663
cm−1. 1H NMR (CDCl3, 400 MHz): δ = 7.90 (1H, dd,
J = 8.0, 1.2 Hz, H1), 7.61 (1H, t, J = 8.0 Hz, H3), 7.43–7.41
(3H, m, H4′, H5′, H6′), 7.39 (1H, d, J = 4.0 Hz, H2′), 7.33
(1H, d, J = 8.0 Hz, H4), 7.16 (1H, t, J = 8.0 Hz, H2), 4.95
(1H, s, CH, H7), 2.71 (1H, d, J = 17.0 Hz, CH2b′), 2.68 (1H,
d, J = 17.0 Hz, CH2b), 2.35 (1H, d, J = 16.5 Hz, CH2a′),
2.30 (1H, d, J = 16.5 Hz, CH2a), 1.19 (3H, s, Me), 1.13 (3H,
s, Me); 13C NMR (CDCl3, 100 MHz): δ = 195.9 (C=O,
C-8), 162.3 (C=O, C-6), 160.5 (C-O, C-O12), 154.1 (C-O,
C11-C-O12), 149.9 (C-O, C-O5), 144.7 (C-1′), 132.5
(C-2′), 131.3 (C-5′), 130.3 (C-4′), 129.8 (C-6′), 127.9
(C-2), 124.4 (C-1), 122.5 (C-3), 122.4 (C-3′), 116.9 (C-4),
114.6 (C, C-C1), 113.6 (C, C7-C-C8), 106.1 (C, C6-C-C7),
60.7 (CH2), 40.8 (CH2), 33.3 (C-7), 32.4 (C-10), 29.1 (Me),
27.6 (Me); anal. calcd. for C24H19BrO4: C, 63.87; H, 4.24.
Found: C, 64.01; H, 4.38.
cm−1; H NMR (CDCl3, 400 MHz): δ = 8.08–8.03 (2H, m,
H2′, H4′), 7.94–7.90 (2H, m, H1, H6′), 7.61 (1H, t, J = 8.0
Hz, H3), 7.47 (1H, t, J = 8.0 Hz, H5′), 7.38 (1H, t, J = 8.0
Hz, H2), 7.35 (1H, d, J = 8.0 Hz, H4), 5.06 (1H, s, CH, H7),
2.78 (1H, d, J = 17.6 Hz, CH2b′), 2.70 (1H, d, J = 17.6 Hz,
CH2b), 2.34 (1H, d, J = 16.4 Hz, CH2a′), 2.27 (1H, d, J =
16.4 Hz, CH2a), 1.19 (3H, s, Me), 1.10 (3H, s, Me); 13C
NMR (CDCl3, 100 MHz): δ = 195.0 (C=O, C-8), 162.8
(C=O, C-6), 160.5 (C-O, C-O12), 154.5 (C-O, C11-C-
O12), 152.7 (C-O, C-O5), 148.4 (C-1′), 144.6 (C-3′), 136.7
(2′), 132.8 (C-3), 129.1 (C-2), 124.6 (C-1), 122.8 (C-5′),
122.7 (C6′), 122.3 (C-4′), 117.0 (C-4), 114.1 (C, C-C1),
113.3 (C, C7-C-C8), 105.4 (C, C6-C-C7), 50.6 (CH2), 40.8
(CH2), 33.6 (C-7), 32.4 (C-10), 29.1 (Me), 27.5 (Me); EIMS
m/z 417 [M]+ (33), 400 (100), 370 (27), 295 (98), 239 (50),
211 (14), 165 (5), 139 (15), 92 (8), 76 (8); anal. calcd. for
C24H19NO6: C, 69.06; H,4.59; N, 3.36. Found: C, 69.30; H,
4.40; N, 3.22.
7-(3-Hydroxyphenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4e)
7-(3-Fluorophenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4c)
Pale yellow solid (This compound was prepared by the
reaction of 3-hydroxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 202–204 °C; IR (KBr) νmax3441, 2990, 1715, 1665 cm−1;
1H NMR (CDCl3, 400 MHz): δ = 9.29 (1H, s, OH), 7.93
Pale yellow solid (This compound was prepared by the
reaction of 3-fluorobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);

Med Chem Res
(1H, d, J = 8.0 Hz, H1), 7.69 (1H, t, J = 8.0 Hz, H3),
7.46–7.43 (2H, m, H2, H4), 7.03 (1H, t, J = 8.0 Hz, H5′),
6.72 (1H, s, H2′), 6.66 (1H, d, J = 8.0 Hz, H6′), 6.55 (1H, d,
J = 8.0 Hz, H4′), 4.63 (1H, s, CH, H7), 2.75–2.80 (2H, m,
CH2), 2.32 (1H, d, J = 16.4 Hz, CH2a′), 2.26 (1H, d, J =
16.4 Hz, CH2a), 1.10 (3H, s, Me), 1.01 (3H, s, Me); 13C
NMR (CDCl3, 100 MHz): δ = 196.7 (C=O, C-8), 163.4
(C=O, C-6), 160.1 (C-O, C-O12), 157.9 (C-O, C11-C-
O12), 154.5 (C-3′), 152.8 (C-O, C-O5), 144.9 (C-1′), 133.6
(C-5′), 129.9 (C-3), 125.6 (C-2), 123.4 (C-1), 119.7 (C-6′),
117.4 (C-2′), 116.4 (C-4), 114.8 (C-4′), 114.7 (C, C-C1),
113.9 (C, C7-C-C8), 106.8 (C, C6-C-C7), 50.9 (CH2), 39.9
(CH2), 33.6 (C-7), 32.9 (C-10), 29.4 (Me), 27.6 (Me); anal.
calcd. for C24H20O5: C, 74.21; H, 5.19. Found: C, 74.48;
H, 5.31.
2.64 (1H, d, J = 17.6 Hz, CH2b), 2.32 (1H, d, J = 16.0 Hz,
CH2a′), 2.26 (1H, d, J = 16.0 Hz, CH2a), 1.16 (3H, s, Me),
1.09 (3H, s, Me); 13C NMR (CDCl3, 100 MHz): δ = 196.2
(C=O, C-8), 161.8 (C=O, C-6), 160.7 (C-O, C-O12), 158.5
(C-4′), 153.7(C-O, C11-C-O12), 152.6 (C-O, C-O5), 134.9
(C-1′), 132.2 (C-2′, C-6′), 129.6 (C-3), 129.3 (C-2), 124.3
(C-1), 122.4 (C-4), 116.9 (C, C-C1), 115.3 (C-4′), 113.7 (C,
C7-C-C8), 106.9 (C, C6-C-C7), 55.2 (OCH3), 50.7 (CH2),
40.8 (CH2), 32.5 (C-7), 29.7 (C-10), 29.2 (Me), 27.6 (Me);
anal. calcd. for C25H22O5: C, 74.61; H, 5.51. Found: C,
74.48; H, 5.62.
10,10-Dimethyl-7-(p-tolyl)-7,9,10,11-tetrahydro-6H,8H-
chromeno[4,3-b]chromene-6,8-dione (4h)
Pale yellow solid (This compound was prepared by the
reaction of 4-methylbenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
7-(4-Hydroxyphenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4f)
1
mp 174–176 °C; IR (KBr) νmax2982, 1730, 1660 cm−1; H
Pale yellow solid (This compound was prepared by the
reaction of 4-hydroxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 203–204 °C; IR (KBr) νmax3440, 2990, 1715, 1665,
NMR (CDCl3, 400 MHz): δ = 7.86 (1H, dd, J = 8.0, 1.6 Hz,
H1), 7.56 (1H, t, J = 8.0 Hz, H3), 7.36–7.31 (2H, m, H2,
H4), 7.25 (2H, d, J = 8.0 Hz, H3′, H5′), 7.05 (2H, d, J = 8.0
Hz, H3′, H5′), 4.92 (1H, s, CH, H7), 2.72 (1H, d, J = 17.6
Hz, CH2b′), 2.65 (1H, d, J = 17.6 Hz, CH2b), 2.32 (1H, d, J
= 16.4 Hz, CH2a′), 2.26 (3H, s, Me), 2.22 (1H, d, J = 16.4
Hz, CH2a), 1.16 (3H, s, Me), 1.09 (3H, s, Me); 13C NMR
(CDCl3, 100 MHz): δ = 196.1 (C=O, C-8), 161.8 (C=O, C-
6), 160.6 (C-O, C-O12), 152.6 (C-O, C11-C-O12), 139.6
(C-O, C-O5), 136.6 (C-1′), 132.1 (C-4′), 129.0 (C-2′, C-6′),
128.5 (C-3′, C-5′), 128.2 (C-3), 124.2 (C-2), 122.4 (C-1),
116.9 (C-4), 115.2 (C, C-C1), 113.7 (C, C7-C-C8), 106.9
(C, C6-C-C7), 50.7 (CH2), 40.8 (CH2), 32.9 (C-7), 31.4
(Me), 29.1 (C-10), 27.5 (Me), 21.1 (Me); anal. calcd. for
C25H22O4: C, 77.70; H, 5.74. Found: C, 77.61; H, 5.55.
1
1606, 1362, 1189 cm−1; H NMR (CDCl3, 400 MHz): δ =
7.87 (1H, dd, J = 8.0, 1.6 Hz, H1), 7.56 (1H, t, J = 8.0 Hz,
H3), 7.31–7.37 (2H, m, H2, H4), 7.19 (2H, d, J = 8.5 Hz,
H2′, H6′), 6.63 (2H, d, J = 8.5 Hz, H3′, H5′), 4.88 (1H, s,
CH, H7), 2.72 (1H, d, J = 18.0 Hz, CH2b′), 2.64 (1H, d, J =
18.0 Hz, CH2b), 2.33 (1H, d, J = 16.4 Hz, CH2a′), 2.27 (1H,
d, J = 16.4 Hz, CH2a), 1.16 (3H, s, Me), 1.10 (3H, s, Me);
13C NMR (CDCl3, 100 MHz): δ = 196.8 (C=O, C-8), 162.1
(C=O, C-6), 160.9 (C-O, C-O12), 155.0 (C-O, C11-C-
O12), 153.8 (C-4′), 152.5 (C-O, C-O5), 134.4 (C-1′), 132.2
(C-2′, C-6′), 129.7 (C-3), 124.3 (C-2), 122.4 (C-1), 116.9
(C-4), 115.4 (C-3′, C-5′), 115.3 (C, C-C1), 113.7 (C, C7-C-
C8), 106.5 (C, C6-C-C7), 50.7 (CH2), 40.8 (CH2), 32.5 (C-
7), 29.7 (C-10), 29.1 (Me), 27.6 (Me); EIMS m/z 388 [M]+
(6), 295 (6), 256 (69), 239 (5), 211 (2), 192 (17), 160 (36),
128 (48), 96 (26), 64 (98); anal. calcd. for C24H20O5: C,
74.21; H, 5.19. Found: C, 74.08; H, 5.05.
10,10-Dimethyl-7-(4-nitrophenyl)-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4i)
Pale yellow solid (This compound was prepared by the
reaction of 4-nitrobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 233–235 °C; IR (KBr) νmax3100, 2957, 1731, 1665,
7-(4-Methoxyphenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4g)
1
1657, 1550, 1345 cm−1; H NMR (CDCl3, 400 MHz): δ =
8.12 (2H, d, J = 8.8 Hz, H3′, H5′), 7.91 (1H, dd, J = 8.0, 1.6
Hz, H1), 7.62 (1H, td, J = 8.0, 1.6 Hz, H3), 7.57 (2H, d, J =
8.8 Hz, H2′, H6′), 7.41 (1H, t, J = 8.0 Hz, H2), 7.35 (1H, dd,
J = 8.0, 0.8 Hz, H4), 5.06 (1H, s, CH, H7), 2.79–2.69 (2H,
m, CH2), 2.36 (1H, d, J = 16.0 Hz, CH2a′), 2.28 (1H, d, J =
16.0 Hz, CH2a), 1.20 (3H, s, Me), 1.10 (3H, s, Me); 13C
NMR (CDCl3, 100 MHz): δ = 195.9 (C=O, C-8), 162.6
(C=O, C-6), 160.4 (C-O, C-O12), 154.5 (C-O, C11-C-
O12), 152.7 (C-O, C-O5), 149.7 (C-1′), 146.8 (C-4′), 132.8
(C-3), 129.7 (C-2′, C-6′), 124.6 (C-2), 123.6 (C-3′, C-5′),
Pale yellow solid (This compound was prepared by the
reaction of 4-methoxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 188–190 °C; IR (KBr) νmax3447, 2989, 1728, 1658
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 7.86 (1H, dd, J =
8.0, 1.2 Hz, H1), 7.56 (1H, td, J = 8.0, 1.2 Hz, H3),
7.31–7.31 (2H, m, H2, H4), 7.29 (2H, d, J = 8.5 Hz, H2′,
H6′), 6.78 (2H, d, J = 8.5 Hz, H3′, H5′), 4.91 (1H, s, CH,
H7), 3.74 (3H, s, OMe), 2.71 (1H, d, J = 17.6 Hz, CH2b′),

Med Chem Res
122.6 (C-1), 117.3 (C-4), 114.1 (C, C-C1), 113.2 (C, C7-C-
C8), 105.3 (C, C6-C-C7), 50.5 (CH2), 40.8 (CH2), 33.7 (C-
7), 32.4 (C-10), 29.1 (Me), 27.4 (Me); anal. calcd. for
C24H19NO6: C, 69.06; H,4.59; N, 3.36. Found: C, 68.87; H,
4.41; N, 3.22.
(CH2), 33.3 (C-7), 32.2 (C-10), 29.2 (Me), 27.5 (Me); EIMS
m/z 408 [M+2]+ (2), 406 [M]+ (6), 388 (52), 371 (100), 349
(16), 295 (76), 239 (45), 211 (15), 139 (13), 121 (13), 65
(11); anal. calcd. for C24H19ClO4: C, 70.85; H,4.71. Found:
C, 70.72; H, 4.58.
7-(4-Fluorophenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4j)
7-(2,4-Dichlorophenyl)-10,10-dimethyl-7,9,10,11-
tetrahydro-6H,8H-chromeno[4,3-b]chromene-6,8-dione
(4l)
Pale yellow solid (This compound was prepared by the
reaction of 4-fluorobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 250–252 °C; IR (KBr) νmax3020, 2921, 1730, 1674
Pale yellow solid (This compound was prepared by the
reaction of 2,4-dichlorobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 207–208 °C; IR (KBr) νmax2983, 1729, 1668 cm−1;
1H NMR (CDCl3, 400 MHz): δ = 7.89 (1H, dd, J = 8.0, 1.2
Hz, H1), 7.84 (1H, d, J = 8.0 Hz, H5′), 7.60 (1H, td, J = 8.0,
1.2 Hz, H3), 7.46 (1H, d, J = 4.4 Hz, H3′), 7.39–7.29 (3H,
m, H2, H4, H6′), 5.83 (1H, s, CH, H7), 2.71 (1H, d, J = 17.6
Hz, CH2b′), 2.64 (1H, d, J = 17.6 Hz, CH2b), 2.30 (1H, d, J
= 16.0 Hz, CH2a′), 2.23 (1H, d, J = 16.0 Hz, CH2a), 1.16
(3H, s, Me), 1.07 (3H, s, Me); 13C NMR (CDCl3, 100
MHz): δ = 196.2 (C=O, C-8), 162.9 (C=O, C-6), 160.4 (C-
O, C-O12), 154.4 (C-O, C11-C-O12), 152.7 (C-O, C-O5),
147.3 (C-1′), 145.7 (C-2′), 134.7 (C-4′), 134.4 (C-6′), 132.7
(C-3′), 127.1 (C-3), 124.5 (C-5′), 122.6 (C-2), 116.9 (C-1),
114.5 (C-4), 114.1 (C, C-C1), 113.3 (C, C7-C-C8), 105.3
(C, C6-C-C7), 50.6 (CH2), 40.8 (CH2), 32.2 (C-7), 31.7 (C-
10), 29.1 (Me), 27.4 (Me); anal. calcd. for C24H18Cl2O4: C,
65.32; H,4.11. Found: C, 65.10; H, 4.28.
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 7.87 (1H, dd, J =
8.0, 1.6 Hz, H1), 7.58 (1H, td, J = 8.0, 1.6 Hz, H3),
7.38–7.32 (4H, m, H2, H4, H2′, H6′), 6.93 (2H, t, J = 8.5 Hz,
H3′, H5′), 4.94 (1H, s, CH, H7), 2.72 (1H, d, J = 17.6 Hz,
CH2b′), 2.66 (1H, d, J = 17.6 Hz, CH2b), 2.33 (1H, d, J =
16.4 Hz, CH2a′), 2.26 (1H, d, J = 16.4 Hz, CH2a), 1.17 (3H,
s, Me), 1.09 (3H, s, Me); 13C NMR (CDCl3, 100 MHz): δ =
196.0 (C=O, C-8), 162.9 (d, JC-F = 237.2 Hz, C-4′), 161.9
(C=O, C-6), 153.9 (C-O, C-O12), 152.6 (C-O, C11-C-
O12), 138.4 (C-O, C-O5), 132.3 (C-1′), 129.8 (C-3), 130.2
(d, JC-F = 8.2 Hz, C2′, C-6′), 124.3 (C-2), 122.4 (C-1),
116.9 (C-4), 114.9 (C, C-C1), 115.1 (d, JC-F = 21.3 Hz, C-
3′, C-5′), 113.5(C, C7-C-C8), 106.5 (C, C6-C-C7), 50.7
(CH2), 40.8 (CH2), 32.7 (C-7), 32.4 (C-10), 29.1 (Me), 27.5
(Me); EIMS m/z 390 [M]+. (27), 368 (28), 295 (37), 273
(38), 256 (88), 239 (16), 192 (26), 160 (52), 128 (68), 96
(36), 64 (100); anal. calcd. for C24H19FO4: C, 73.84;
H,4.91. Found: C, 73.70; H, 5.12.
7-(3,4-Dihydroxyphenyl)-10,10-dimethyl-7,9,10,11-
tetrahydro-6H,8H-chromeno[4,3-b]chromene-6,8-dione
(4m)
7-(2-Chlorophenyl)-10,10-dimethyl-7,9,10,11-tetrahydro-
6H,8H-chromeno[4,3-b]chromene-6,8-dione (4k)
Pale yellow solid (This compound was prepared by the
reaction of 3,4-dihydroxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid); mp
232–234 °C; IR (KBr) νmax3443, 2989, 1715, 1665 cm−1;
1H NMR (CDCl3, 400 MHz): δ = 8.84 (1H, s, OH), 8.74
(1H, s, OH), 7.93 (1H, d, J = 8.0 Hz, H1), 7.69 (1H, t, J =
8.0 Hz, H3), 7.45–7.49 (2H, m, H2, H4), 6.68 (1H, d, J =
1.6 Hz, H2′), 6.57 (1H, d, J = 8.4 Hz, H5′), 6.47 (1H, dd, J
= 8.4, 1.6 Hz, H6′), 4.54 (1H, s, CH, H7), 2.74 (2H, s, CH2),
2.34 (1H, d, J = 16.4 Hz, CH2a′), 2.19 (1H, d, J = 16.4 Hz,
CH2a), 1.10 (3H, s, Me), 1.01 (3H, s, Me); 13C NMR
(CDCl3, 100 MHz): δ = 195.9 (C=O, C-8), 162.1 (C=O, C-
6), 159.9 (C-O, C-O12), 153.2 (C-O, C11-C-O12), 151.8
(C-O, C-O5), 144.7 (C-3′), 144.1 (C-4′), 133.8 (C-1′),
132.6 (C-3), 124.7 (C-2), 122.4 (C-1), 118.9 (C6′), 116.5
(C2′), 115.9 (C-4), 115.2 (C-5′), 114.2 (C, C-C1), 113.2 (C,
C7-C-C8), 106.3 (C, C6-C-C7), 50.1 (CH2), 40.1 (CH2),
39.6 (C-7), 31.9 (C-10), 28.5 (Me), 26.7 (Me); anal. calcd.
for C24H20O6: C, 71.28; H, 4.98. Found: C, 71.37; H, 5.18.
Pale yellow solid (This compound was prepared by the
reaction of 2-chlorobenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 230–232 °C; IR (KBr) νmax3062, 2978, 1728, 1663
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 7.89 (1H, dd, J =
8.0, 1.6 Hz, H1), 7.59 (1H, td, J = 8.0, 1.6 Hz, H3), 7.55
(1H, dd, J = 7.0, 1.6 Hz, H3′), 7.39–7.34 (2H, m, H2, H4),
7.26 (1H, dd, J = 7.0, 1.6 Hz, H6′), 7.22 (1H, td, J = 7.0,
1.6 Hz, H4′), 7.15 (1H, td, J = 7.0, 1.6 Hz, H5′), 5.24 (1H, s,
CH, H7), 2.71 (1H, d, J = 17.6 Hz, CH2b′), 2.66 (1H, d, J =
17.6 Hz, CH2b), 2.33 (1H, d, J = 16.4 Hz, CH2a′), 2.27 (1H,
d, J = 16.4 Hz, CH2a), 1.19 (3H, s, Me), 1.12 (3H, s, Me);
13C NMR (CDCl3, 100 MHz): δ = 196.2 (C=O, C-8), 162.7
(C=O, C-6), 160.4 (C-O, C-O12), 154.6 (C-O, C11-C-
O12), 152.7 (C-O, C-O5), 139.5 (C-1′), 138.4 (C-2′), 138.3
(C-3′), 133.4 (C-3), 132.4 (C-4′), 130.3 (C-5′), 128.5 (C6′),
126.6 (C-2), 124.3 (C1), 122.6 (C-4), 116.9 (C, C-C1),
113.4 (C, C7-C-C8), 104.6 (C, C6-C-C7), 50.7 (CH2), 40.8

Med Chem Res
7-(3-Hydroxy-4-methoxyphenyl)-10,10-dimethyl-7,9,10,11-
tetrahydro-6H,8H-chromeno[4,3-b]chromene-6,8-dione
(4n)
In vitro inhibition assay
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyo-
philized powder, from electric eel, 1000 unit), butyr-
ylcholinesterase (BuChE, E.C. 3.1.1.8, from equine serum),
and butyrylthiocholine iodide were purchased from Sigma-
Aldrich. 5,5′-Dithiobis-(2-nitrobenzoic acid) (DTNB) and
acetylthiocholine iodide were obtained from Fluka. Briefly,
five different concentrations of the compounds were ana-
lyzed to obtain 20 to 80 % enzyme inhibition. The assay
solution was comprised of 3 mL phosphate buffer (0.1 M,
pH = 8), 100 µL DTNB, 50 µL test compound and 50 µL of
5 IU/mL acetylcholinesterase solution. The above mixture
was pre-incubated for 10 min accompanied by the addition
of enzyme substrate. Blank assays were also carried out
comprising all ingredients excluding enzyme to consider the
non-enzymatic reaction. The absorbance changes were
scored at 412 nm for 6 min and the IC50 values were cal-
culated. All experiments were performed in triplicate at
25 °C using a Unico UV spectrophotometer. The same
method was used for BuChE inhibition assay (Khoobi et al.,
2013a, b).
Pale yellow solid (This compound was prepared by the
reaction of 3-hydroxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid);
mp 249–251 °C; IR (KBr) νmax3447, 1713, 1665, 1514
1
cm−1; H NMR (CDCl3, 400 MHz): δ = 7.86 (1H, dd, J =
7.8, 1.5 Hz, H1), 7.59 (1H, td, J = 7.8, 1.5 Hz, H3),
7.37–7.32 (2H, m, H2, H4), 7.13 (1H, d, J = 2.0 Hz, H2′),
6.75 (1H, d, J = 8.4 Hz, H5′), 6.62 (1H, dd, J = 8.4, 2.0 Hz,
H6′), 4.88 (1H, s, CH, H7), 3.92 (3H, s, OMe), 2.72 (1H, d,
J = 17.0 Hz, CH2b′), 2.65 (1H, d, J = 17.0 Hz, CH2b), 2.33
(1H, d, J = 16.4 Hz, CH2a′), 2.28 (1H, d, J = 16.4 Hz,
CH2a), 1.17 (3H, s, Me), 1.11 (3H, s, Me); 13C NMR
(CDCl3, 100 MHz): δ = 196.2 (C=O, C-8), 161.8 (C=O,
C-6), 160.8 (C-O, C-O12), 153.7 (C-O, C11-C-O12), 152.5
(C-O, C-O5), 150.8 (C-4′), 146.1 (C-3′), 144.6 (C-1′),
134.8 (C-3), 132.1 (C-2), 124.3 (C-1), 122.4 (C-6′), 120.2
(C-4), 116.9 (C, C-C1), 115.2 (C-2′), 114.1 (C, C7-C-C8),
112.5 (C-5′), 106.9 (C, C6-C-C7), 55.9 (OCH3), 50.7
(CH2), 40.8 (CH2), 32.8 (C-7), 32.4 (C-10), 29.2 (Me), 27.5
(Me); EIMS m/z 418 [M]+ (100), 387 (20), 295 (80), 239
(70), 211 (28), 165 (13), 139 (28), 108 (25), 77 (54); anal.
calcd. for C25H22O6: C, 71.76; H, 5.30. Found: C, 71.88; H,
5.49.
Docking simulation
Autodock Vina 1.1.2 was used for docking studies (Trott
and Olson, 2010). The AChE enzyme complexed with
donepezil (1EVE, RCSB Protein Data Bank, http://www.
rcsb.org/pdb/home/home.do, 2014) was chosen for docking
study. The receptor was prepared according to the following
procedure. Initially, the PDB heteroatom including cofac-
tors and bound ligands were manually removed from the
coordinate file and then the missing atom types were cor-
rected by Swiss pdb Viewer. Afterwards, the receptor was
protonated and the Kollman charge was added using
Autodock tools (Sanner, 1999). The two-dimension coor-
dinate of the ligand was prepared by MarvinSketch 5.8.3,
2012, ChemAxon (http://www.chemaxon.com) and then
converted to three-dimension using Openbabel (ver. 2.3.1)
(O’Boyle et al., 2011). The geometries of the ligands were
optimized for 1000 steepest descent steps using Hyperchem
7.5 (Hypercube Inc.). Hydrogen atoms were added to ligand
and Gasteiger charges were assigned using Autodock tools
and then saved as pdbqt format. Acceptable docking results
were obtained by setting the following parameters for
Autodock Vina. The grids were centered at the aromatic
gorge of the enzyme, with dimensions of 25 Å × 25 Å × 25
Å and a grid spacing of 0.5 Å. The number of calculated
models and exhaustiveness were 15 and 80, respectively.
Finally, the best conformations in terms of the lowest free
energy of binding were chosen for analyzing the interac-
tions with the enzyme in details. All molecular
7-(3,4-Dimethoxyphenyl)-10,10-dimethyl-7,9,10,11-
tetrahydro-6H,8H-chromeno[4,3-b]chromene-6,8-dione
(4o)
Pale yellow solid (This compound was prepared by the
reaction of 3,4-dimethoxybenzaldehyde, dimedone, and 4-
hydroxycoumarin. It was obtained as pale yellow solid); mp
181–183 °C; IR (KBr) νmax3448, 2989, 1728, 1658 cm−1;
1H NMR (CDCl3, 400 MHz): δ = 7.88 (1H, d, J = 8.0 Hz,
H1); 7.59 (1H, t, J = 8.0 Hz, H3), 7.39–7.34 (2H, m, H2,
H4), 7.06 (1H, s, H2′), 6.82 (1H, d, J = 8.0 Hz, H5′), 6.75
(1H, d, J = 8.0 Hz, H6′), 4.94 (1H, s, CH, H7), 3.90 (3H, s,
OMe), 3.81 (3H, s, OMe), 2.74 (1H, d, J = 17.6 Hz, CH2b′),
2.68 (1H, d, J = 17.6 Hz, CH2b), 2.36 (1H, d, J = 16.0 Hz,
CH2a′), 2.30 (1H, d, J = 16.0 Hz, CH2a), 1.20 (3H, s, Me),
1.14 (3H, s, Me); 13C NMR (CDCl3, 100 MHz): δ = 196.2
(C=O, C-8), 161.9 (C=O, C-6), 160.8 (C-O, C-O12), 153.7
(C-O, C11-C-O12), 152.6 (C-O, C-O5), 148.6 (C-3′), 148.0
(C-4′), 135.4 (C-1′), 132.2 (C-3), 124.3 (C-2), 122.4 (C-1),
120.2 (C-6′), 116.9 (C-4), 115.2 (C, C-C1), 113.7 (C-2′),
112.6 (C, C7-C-C8), 110.9 (C-5′), 106.9 (C, C6-C-C7),
55.9 (OCH3), 55.8 (OCH3), 50.7 (CH2), 40.8 (CH2), 32.8
(C-7), 32.3 (C-10), 28.5 (Me), 26.7 (Me); anal. calcd. for
C26H24O6: C, 72.21; H, 5.59. Found: C, 72.46; H, 5.71.

Med Chem Res
Scheme 1 One-pot
multicomponent reaction of
benzaldehyde (1), dimedone (2),
and 4-hydroxycoumarin (3)
H
O
OH
O
+
+
O
O
O
1
2
3
O
O
O
O
O
OH
O
OH
O
+
+
O
O
O
4a
5a
6a
Possible Products
visualizations were carried out in DS Viewer Pro (Accelrys,
Inc., San Diego, CA).
when the reaction was carried out in a solvent, compound
6a was formed as the main product. Finally, the generality
of this reaction was investigated by preparing a wide variety
of substituted chromeno[4,3-b]chromene derivatives 4
using various aldehydes, 4-hydroxycoumarin and dimedone
under the optimized conditions (Table 1). As shown in
Table 1, using aldehydes bearing both electron-donating
and electron-withdrawing substituents afforded the corre-
sponding products 4a–o in good to excellent yields.
Results and discussion
Chemistry
Currently, the use of reusable heterogeneous catalysts has
received considerable attention in organic synthesis due to
their environmental, economic, and industrial benefits
(Yassaghi et al., 2012). Particularly, the application of silica
sulfuric acid (SSA) or nano-silica sulfuric acid (nano-SSA)
has been recognized as a stable and efficient heterogeneous
catalyst. It has showed excellent activity and selectivity.
Also, it can be recovered from a reaction mixture and reused
(Emtiazi et al., 2013).
In this work, we report one-pot synthesis of 10,11-
dihydrochromeno[4,3-b]chromene-6,8(7H,9H)-diones 4 in
the presence of nano-SSA under solvent-free conditions.
Primarily, the model reaction was conducted by the reaction
of benzaldehyde (1), dimedone (2), and 4-hydroxycoumarin
(3) using different solvents such as DMF, THF, H2O, EtOH,
CHCl3, and MeCN as well as solvent-free conditions in the
presence of different amounts of nano-SSA (Scheme 1).
The best result was obtained using 0.015 g of catalyst under
solvent-free conditions at 100 °C. It should be noted that a
trace amount of 5a was produced through the reaction
which was removed by recrystallization from ethanol
leading to the desired product 4a. Also, it was found that
Cholinesterase inhibitory activity
The inhibitory activity of synthesized compounds 4a–o was
evaluated against AChE and BuChE, in comparison to
tacrine as the reference drug. The IC50 values of tested
compounds were listed in Table 2. Most compounds
showed satisfactory anti-AChE activity with IC50 values of
3.28–52.6 µM. Compound 4e followed by compounds 4m
and 4o were the most potent compounds against AChE
(IC50s ≤ 3.67 µM).
The results obtained from IC50 values for compounds 4a
and 4b–e revealed that substituents at 3-position of aryl ring
improved the anti-AChE activity. The comparison of 3-
substituted derivatives with the corresponding 4-substituted
analogs, demonstrated that 3-regioisomers depicted better
AChE inhibitory activity. As observed by compound 4c,
fluorine was found as an effective substituent comparing
with the bromine and chlorine. Different para-substituted
counterpart of compound 4a showed different anti-AChE
effects. Although insertion of methyl and chlorine groups

Med Chem Res
Table 1 Synthesis of compound 4a–o under solvent-free conditions
Entry
Ar
Product
Yield (%)
Mp (°C)
1
C6H5
4a
4b
4c
4d
4e
4f
78
65
61
64
62
60
60
66
60
75
65
62
65
60
62
215–216
233–235
203–205
228–230
202–204
203–204
188–190
174–176
233–235
250–252
230–232
207–208
232–234
249–251
181–183
2
3-Br C6H4
3
3-F C6H4
4
3-NO2 C6H4
3-OH C6H4
4-OH C6H4
4-OMe C6H4
4-Me C6H4
4-NO2 C6H4
4-F C6H4
5
6
7
4g
4h
4i
8
9
10
11
12
13
14
15
4j
2-Cl C6H4
4k
4l
2,4-Cl2C6H3
3,4-(OH)2C6H3
3-OH-4-OMeC6H3
3,4-(OCH3)2C6H3
4m
4n
4o
diminished the AChE inhibitory activity, hydroxyl, meth-
oxy, and fluorine substituents increased the anti-AChE
activity. Comparing 4-hydroxy and 4-methoxy derivatives
(4f and 4g) with compounds 4m and 4n showed that the
introduction of 3-hydroxy group resulted in more potent
compounds, respectively. In general, the compounds 4e and
4m–o bearing 3-hydroxy or 3-methoxy group showed
superior activity against AChE (IC50 < 4.5 µM). Thus, anti-
AChE activity was directly affected by the oxygenated
functionality at 3-position of aryl ring.
In the terms of anti-BuChE activity, compound 4e
bearing 3-hydroxy group was the most potent compound
(IC50 = 2.19 µM). Besides 4e, compounds 4b, 4i, 4k, and 4l
with IC50 values < 15 µM showed potent activity against
BuChE. It is worthwhile mentioning that compounds
bearing 3-OH or 3-OMe (4e and 4m–o) showed statistically
similar anti-AChE activity whereas their anti-BuChE was
very different. Introduction of the second hydroxyl or
methoxy group into the para-position (compounds 4m and
4n) led to the almost same anti-AChE and less anti-BChE
activities. It shows that the selectivity against AChE can be
regulated through the second substituent on the 3-
hydroxyphenyl moiety. On the other hand, compounds
4b, 4e, 4f, 4h, 4i, and 4l showed more selectivity for
BuChE over AChE.
Docking study
To determine the plausible enzyme interaction of synthe-
sized compounds, molecular docking studies were per-
formed. All the compounds were docked into the AChE
enzyme (1EVE) active site and the analysis of the results
showed that they have similar binding modes in the active
site. In this regard, the most active compound 4e was
chosen for further analysis. The important residues involved
in the interaction of compound 4e and the type of interac-
tions are summarized in Table 3. Ligand–receptor contacts
are based on distance at ≤ 4.5 Å that was calculated by web-
based software ContPro (http://procarb.org/contpro/).
As can be seen in Fig. 2, compound 4e has been fitted
into the active site covering the space between CAS and
PAS. Making hydrogen bonding between Asn85 and

Med Chem Res
Table 2 The inhibitory activity
(IC50, μM) of synthesized
compounds 4a–o against AChE
and BuChE
Compound
Ar
AChE IC50 (μM)
BuChE IC50 (μM)
Selectivity for AChE
4a
C6H5
52.60
20.41
10.03
13.42
3.28
272
5.17
0.58
—
4b
4c
3-Br C6H4
3-F C6H4
11.8
NAa
4d
4e
3-NO2 C6H4
3-OH C6H4
4-OH C6H4
4-OMe C6H4
4-Me C6H4
4-NO2 C6H4
4-F C6H4
53.64
2.19
4
0.66
0.74
5.44
0.5
4f
27.80
23.50
157
20.58
128
4g
4h
4i
78.80
10.83
508
43.50
28.53
NA
0.25
17.8
—
4j
4k
4l
2-Cl C6H4
10.65
14.07
35.84
56.58
NA
2,4-Cl2C6H3
3,4-(OH)2C6H3
3-OH-4-OMeC6H3
3,4-(OCH3)2C6H3
—
37.32
3.67
0.375
9.76
12.6
—
4m
4n
4o
Tacrine
4.49
3.38
0.250
0.40
1.6
a
No activity
3-hydroxy enabled the compound to anchor the active site at
the vicinity of the CAS. In this orientation, cyclohexenone
moiety surrounded by Phe330, Phe331, and Trp84 made
hydrophobic interactions. Moreover, carbonyl of cyclo-
hexenone participated in π–π interaction with Phe330. Also,
the pyran ring showed H–π interaction with Tyr121. In this
position, coumarin moiety binds to Trp279 via π–π stacking
interaction enabling compound 4e to interact with the
receptor more tightly. It was worth notifying that both R and
S enantiomers were subjected for docking studies however,
S-enantiomer showed no eminent interactions with the
enzyme active site.
Table 3 Ligand–enzyme interaction summary;key residues, type of
interactions and distances are presented
Residue Position Heteroatom Distance (Å) Type of interaction
Tyr
Trp
Asn
Tyr
Phe
Trp
70
lig
lig
lig
lig
lig
lig
4.10
3.50
3.23
3.86
3.65
4.50
hydrophobic
hydrophobic
Hydrogen bond
H–π
84
85
121
330
279
π–π
π–π
In order to compare the mode of interaction of compound
4e to that of standard drug tacrine, we also docked tacrine in
the active site of AChE using the applied parameters for
compound 4e. As illustrated in Fig. 3, while both of them
occupied the same area in the active site, they showed
different interactions with the enzyme. Indeed, tacrine made
π–π interaction with Tyr334 that is different from com-
pound 4e.
Conclusions
Based on the importance of coumarin moiety as a phar-
macophoric scaffold, we designed a series of chromene-
fused coumarins known as 10,11-dihydrochromeno[4,3-b]
chromene-6,8(7H,9H)-diones as potent cholinesterase inhi-
bitors. The target compounds were synthesized through
one-pot reaction of appropriate benzaldehydes, dimedone,

Med Chem Res
Fig. 2 Binding interaction of
best ranked most active
compound 4e in the binding site
of AChE
a hydrogen bond is formed between hydroxyl group and
Asn85. Further studies on the structural requirements and
investigation on the other aspects of biological activity of
prototype 4e can introduce new lead compound for anti-
AChE activity and potential agent for treating Alzheimer’s
disease.
Acknowledgments This research was supported by grants from the
Research Council of Tehran University of Medical Sciences Interna-
tional Campus (TUMS- IC) and the Iran National Science Foundation
(INSF).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
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