Synthesis, Biological Evaluation, and Molecular Docking of 8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene Analogs: New Dual AChE Inhibitors as Potential Drugs for the Treatment of Alzheimer's Disease

Article abstract of DOI:10.1111/cbdd.12732

Alzheimer's disease onset and progression are associated with the dysregulation of multiple and complex physiological processes, and a successful therapeutic approach should therefore address more than one target. In line with this modern paradigm, a series of 8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene analogs (4a–q) were synthesized and evaluated for their multitarget-directed activity on acetylcholinesterase, butyrylcholinesterase (BuChE), 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical, and amyloid-β peptide (Aβ) specific targets for Alzheimer's disease therapy. Most of the synthesized compounds showed remarkable acetylcholinesterase inhibitory activities in low nm concentrations and good ABTS radical scavenging activity, however, no evidence of BuChE inhibitory activity. Among them, 3-bromobenzylamide derivative 4m exhibited the best acetylcholinesterase inhibitory activity with IC₅₀ value of 13 ± 1.4 nm which is 51-fold superior to galantamine, a reference drug. Kinetic and molecular docking studies indicated 4m as mixed-type inhibitor, binding simultaneously to catalytic active and peripheral anionic sites of acetylcholinesterase. Five compounds 4e, 4f, 4g, 4j, and 4k have shown 1.4- to 2.5-fold of higher antioxidant activities than trolox. Interestingly, the most active compound 4m demonstrated dosage-dependent acceleration of Aβ_1?42 aggregation, which may reduce toxicity of oligomers. Overall, these results lead to discovery of fused tricyclic coumarins as promising dual binding site inhibitors of acetylcholinesterase and afford multifunctional compounds with potential impact for further pharmacological development in Alzheimer's therapy.

Full text of DOI:10.1111/cbdd.12732

Chem Biol Drug Des 2016; 88: 43–53
Research Article
Synthesis, Biological Evaluation, and Molecular
Docking of 8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene
Analogs: New Dual AChE Inhibitors as Potential Drugs
for the Treatment of Alzheimer’s Disease
Jeelan Basha Shaik1, Bhagath Kumar Palaka2,
binding site inhibitors of acetylcholinesterase and
Mohan Penumala1, Siddhartha Eadlapalli3,
afford multifunctional compounds with potential impact
Manidhar Darla Mark4, Dinakara Rao Ampasala2,
for further pharmacological development in Alzheimer’s
therapy.
Ramakrishna Vadde3 and Damu Amooru
Gangaiah1,*
Key words: acetylcholinesterase, Alzheimer’s disease,
antioxidant, Ab1À42 aggregation, butyrylcholinesterase, cou-
1Department of Chemistry, Yogi Vemana University,
Kadapa, India
marin, docking, Kinetic study
2Centre for Bioinformatics, School of life Sciences,
Pondicherry Central University, Puducherry, India
3Department of Biotechnology and Bioinformatics, Yogi
Vemana University, Kadapa, India
Received 6 August 2015, revised 18 December 2015 and
accepted for publication 10 January 2016
4Department of Chemistry, University College of Sciences,
Sri Venkateswara University, Tirupati, India
*Corresponding author: Damu Amooru Gangaiah,
agdamu01@gmail.com
Neurodegenerative diseases (NDs) are, along with cancer
and cardiovascular diseases, the most important cause
of illness and death in developed countries. The high
social and clinical costs of health care and assistance for
ND suffering patients are exasperated by the lack of dis-
ease-modifying therapies. Despite the wide efforts of both
academic and industrial researchers, there are still only a
few pharmacological treatments for NDs (1). In the vast
plethora of NDs, Alzheimer’s disease (AD) stands out as
the most common form of dementia in the elderly popu-
lation with an insidious onset and a chronic progression.
With the progression of disease, the prominent indica-
tions are the continuous memory loss, confusion, petu-
lance, anger and the lack of vigor in body to function
evenly which eventually become the ultimate cause of
death (2). According to the World Alzheimer Report 2015,
currently, over 46 million people around the world live
with dementia conditions and the number is expected to
nearly double every 20 years, resulting in more than
131.5 million in 2050 if no efficient treatment is discov-
ered (3).
Alzheimer’s disease onset and progression are associ-
ated with the dysregulation of multiple and complex
physiological processes, and a successful therapeutic
approach should therefore address more than one tar-
get. In line with this modern paradigm, a series of
8-imino-2-oxo-2H,8H-pyrano[2,3-f]chromene
analogs
(4a–q) were synthesized and evaluated for their
multitarget-directed activity on acetylcholinesterase,
butyrylcholinesterase (BuChE), 2,2’-azino-bis(3-ethyl-
benzthiazoline-6-sulfonic acid) (ABTS) radical, and
amyloid-b peptide (Ab) specific targets for Alzheimer’s
disease therapy. Most of the synthesized compounds
showed remarkable acetylcholinesterase inhibitory
activities in low nM concentrations and good ABTS
radical scavenging activity, however, no evidence of
BuChE inhibitory activity. Among them, 3-bromobenzy-
lamide derivative 4m exhibited the best acetyl-
cholinesterase inhibitory activity with IC50 value of
13 Æ 1.4 nM which is 51-fold superior to galantamine, a
reference drug. Kinetic and molecular docking studies
indicated 4m as mixed-type inhibitor, binding simulta-
neously to catalytic active and peripheral anionic sites
of acetylcholinesterase. Five compounds 4e, 4f, 4g, 4j,
and 4k have shown 1.4- to 2.5-fold of higher antioxi-
dant activities than trolox. Interestingly, the most
active compound 4m demonstrated dosage-dependent
acceleration of Ab1À42 aggregation, which may reduce
toxicity of oligomers. Overall, these results lead to dis-
covery of fused tricyclic coumarins as promising dual
In the past decade, treatment strategies for AD have
mainly been aimed at improving cholinergic neurotransmis-
sion in the brain, which were mostly based on the ‘cholin-
ergic hypothesis’. Concerning the cholinergic hypothesis,
one of the rational and effective approaches to treat the
AD’s symptoms is raising the acetylcholine (ACh) through
inhibition of acetylcholinesterase (AChE) that is responsible
for hydrolysis of ACh in presynaptic areas (4). Although
AChE inhibitors have become the mainstays for treating
ª 2016 John Wiley & Sons A/S. doi: 10.1111/cbdd.12732
43

Shaik et al.
AD, the non-selectivity of these drugs, poor bioavailability,
adverse cholinergic side-effects in the periphery, narrow
therapeutic ranges and hepatotoxicity are some of the
severe limitations to their therapeutic success (5).
represents a widely occurring, nature friendly privileged
structure, whose functionalization is straightforward. These
facts have made coumarins as interesting molecules for
drug discovery in the field of AChE inhibitors.
Recent research has demonstrated that oxidative stress
plays a key role in initiating the aggregation of Ab and tau
protein hyperphosphorylation, involved in the early stage of
the pathologic cascade of AD (6). Therefore, drugs aimed
at clearing or preventing the formation of the free radicals
may be useful for either the prevention or effective treat-
ment of AD. Because AChE as well as oxidative stress are
important targets for the treatment of AD, some studies
have been devoted to searching multifunctional agents
that act on both AChE and oxidative stress and proved
that the combination of AChE inhibition and antioxidant is
an effective strategy for developing new multifunctional
drugs for AD treatment (7).
Following these reasons, a series of seventeen new fused
tricyclic coumarin derivatives bearing varied amide moieties
were synthesized. The inhibitory effects on AChE and
BuChE, kinetics of inhibition and molecular docking study
of compounds, were investigated. In addition, an in vitro
ABTS radical scavenging activity and accelerating activity
of Ab aggregation process was tested.
Methods and Materials
For the synthesis, starting materials and reagents were com-
mercially purchased and used without further purification. All
synthetic compounds were confirmed by ESI mass analysis,
FT-IR, 1H and 13C NMR spectra. Acetylcholinesterase (EC
3.1.1.7, from Electric eel), butyrylcholinesterase (EC 3.1.1.8,
from Horse serum), 2,2’-azino-bis(3-ethylbenzthiazoline-
6-sulfonic acid) (ABTS), b-amyloid1–42 (Ab1–42), thioflavin
T, 5,5’-dithio-bis(2-nitrobenzoic acid) (DTNB), acetylthio-
choline iodide, butyrylthiocholine iodide, galantamine and
trolox were purchased from Sigma-Aldrich (St. Louis, MO,
USA).
The 4-kDa (40–42-amino acid) amyloid-b peptide (Ab) is
derived from the amyloid precursor protein (APP) through
sequential proteolysis (8). According to amyloid cascade
hypothesis, progressive accumulation of Ab aggregates is
widely believed to be fundamental to the initial develop-
ment of neurodegenerative pathology and to trigger a cas-
cade of events such as neurotoxicity, oxidative damage,
and inflammation that contribute to the progression of AD
(9–12). The aggregation of soluble Ab monomer or oligo-
mers into insoluble plaques or amyloid fibrils is a crucial
step. A fibrillization involves formation of dimers and small
oligomers followed by growth into protofibrils and fibrils via
a complex multistep-nucleated polymerization. It is also
reported that the oligomers of amyloid peptides are more
toxic to neurons in comparison with the fibril aggregates.
To reduce the cytotoxicity of these peptides, two kinds of
strategies, that is, inhibiting aggregation and disassembling
aggregates of amyloid peptides by a variety of designed
molecules such as peptides, antibodies, metal ion chela-
tors, small molecules, and nano particles are applied (13–
17). Currently, smart self-assembling molecules have been
identified that associate and promote the peptide–peptide
and peptide–organic interactions converting the Ab mono-
mers and oligomers into non-toxic forms, which is a differ-
ent non-destructive approach as compared to inhibiting
the aggregation of peptides (18,19).
General
Reaction progress was monitored using analytical thin layer
chromatography (TLC) on precoated silica gel 60 F254 alu-
minum plates, and the spots were detected under UV light
(254 nm). Melting points were measured on an electrically
heated melting point apparatus and were uncorrected. The
IR spectra were taken as KBr disks using Perkin Elmer
Spectrum 2 spectrophotometer (Singapore). The NMR
spectra were recorded on a Bruker spectrometer (Avance
II, Switzerland) operating at 400 MHz (1H) and at 100 MHz
(
13C). The chemical shifts (d) and coupling constants (J) are
expressed in parts per million and hertz, respectively. Split-
ting patterns are designated as s, singlet; d, doublet; t, tri-
plet; m, multiplet. The atoms numbering of target
compounds used for NMR data are depicted in Scheme 1.
Mass spectra were determined with a 70 eV (ESI probe) on
an Agilent LC/MSD trap SL 1100 series spectrometer (Agi-
lent Technology, California, USA).
Structural development of AChE inhibitors resulted in intro-
ducing naturally occurring as well as the synthetic cou-
marin analogs as potent AChE inhibitors (20,21). Previous
studies have demonstrated that coumarin motif can bind
primarily to the PAS of AChE and amine functional moi-
eties linked to coumarin using appropriate spacer interact
with CAS of AChE and thus act as dual binding site inhibi-
tors (22–24). Furthermore, functionalization of the aromatic
center of coumarins has led to development of novel ana-
logs that are capable of inhibiting Ab aggregation and pro-
vide protection to neurons against Ab-induced oxidative
stress and free radicals (25). Actually, coumarin scaffold
Procedure for the preparation of 7-hydroxy
coumarin (1)
To a mixture of resorcinol 3 g (27 mmol) and DL-Malic
acid 2.46 g (18 mmol) in a round-bottom flask (100 mL),
sulfuric acid (6.1 mL) was added slowly at room tempera-
ture. The reaction mixture was stirred at 120 °C for 2 h,
cooled to room temperature, and then, it was slowly
poured into crushed ice. The resulting solid was separated
by filtration and washed successively with water (26). The
44
Chem Biol Drug Des 2016; 88: 43–53

Fused Tricyclic Coumarins as Dual AChE Inhibitors
Scheme 1: Synthetic pathway of 8-imino-2-
oxo-2H,8H-pyrano[2,3-f]chromene
derivatives (4a–q) Reagents and conditions:
(i) DL-malic acid, H2SO4, 120 °C, 2 h (ii)
HMTA, glacial acetic acid, 90 °C, reflux, 6 h
(iii) corresponding amine (iv) Et3N, EtOH.
product was dried and recrystallized with ethanol to get
compound 1.
8-imino-N-methyl-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4a)
Orange red solid; yield: 71%; mp: 215–217 °C; IR (KBr): v
3435, 3308 (N–H), 2939, 2856, 1746 (C=O), 1683 (C=N),
1
Synthetic procedure for 7-hydroxy-8-
1617 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 9.92 (brs,
formylcoumarin (2)
1H, 12-NH), 9.01 (s, 1H, 10-H), 7.74 (brs, 1H, 8-NH), 7.67 (d,
1H, J = 9.2 Hz, 4-H), 7.53 (d, 1H, J = 8.4 Hz, 5-H), 7.03 (d,
1H, J = 8.4 Hz, 6-H), 6.41 (d, 1H, J = 9.2 Hz, 3-H), 3.00 (d,
3H, J = 4.8 Hz, 1’-CH3); ESI-MS (m/z): 271 (M+H)+.
7-Hydroxycoumarin 1 (20.0 g, 0.123 mol) and hexam-
ethylenetetramine (40.0 g, 0.285 mol) were added to gla-
cial acetic acid (150 mL) and stirred at 90 °C for 6 h.
Thereafter, aqueous solution of HCl (300 mL, conc. HCl/
H2O = 84:100, v/v) was added and heated under reflux at
70 °C for 30 min. After cooling, mixture was poured into
ice water (1500 mL) and exhaustively extracted with ethyl
acetate. The combined organic extracts were dried over
Na2SO4, and the solvent was removed under reduced
pressure (27). The crude solid was purified by crystalliza-
tion from ethanol.
N-ethyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4b)
Orange red solid; yield: 62%; mp: 174–176 °C; IR (KBr): v
3454, 3320 (N–H), 2925, 1745 (C=O), 1672 (C=N), 1615
(C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d 9.97 (brs, 1H,
;
12-NH), 8.91 (s, 1H, 10-H), 7.77 (brs, 1H, 8-NH), 7.66 (d,
1H, J = 9.6 Hz, 4-H), 7.52 (d, 1H, J = 8.4 Hz, 5-H), 7.01
(d, 1H, J = 8.4 Hz, 6-H), 6.37 (d, 1H, J = 9.6 Hz, 3-H),
3.46 (m, 2H, 1’-CH2), 1.24 (t, 3H, J = 7.2 Hz, 2’-CH3);
ESI-MS (m/z): 285 (M+H)+.
General procedure for the synthesis of N-
substituted cyano acetamide derivatives (3a–q)
A solution of different amines (1 mmol) and ethylcyanoac-
etate (1.2 mmol) was stirred at room temperature in an
easily available screw cap bottle (28,29). The amide, in
most cases, typically precipitated after some minutes to
hours. Formed product was filtered and washed several
times with ether to obtain pure N-substituted cyanoac-
etamide derivatives 3a–q.
N-dodecyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4c)
Brick red solid; yield: 60%; mp: 165–167 °C; IR (KBr): v
3427, 3212 (N–H), 2956, 2921, 2851, 1714 (C=O), 1678
(C=N), 1619 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d
;
10.00 (s, 1H, 12-NH), 9.01 (s, 1H, 10-H), 7.75 (brs, 1H,
8-NH), 7.66 (d, 1H, J = 9.6 Hz, 4-H), 7.52 (d, 1H,
J = 8.8 Hz, 5-H), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.40 (d,
1H, J = 9.6 Hz, 3-H), 3.43 (q, 2H, J = 7.2 Hz, 1’-CH2),
1.25 (brs, 20H, 2’–11’-CH2), 0.87 (t, 3H, J = 4.6 Hz, 12’-
H); ESI-MS (m/z): 425 (M+H)+.
General synthetic procedure for preparation of
final product: (4a–q)
To a solution of 7-hydroxy-8-formylcoumarin 2 (1.5 mmol)
and (3a–q) (3.1 mmol) in ethanol (10 mL) was added Et3N
(0.1 mmol) drop wise at room temperature. The resulting
mixture was heated to reflux temperature for 2–3 h and
then allowed to cool to room temperature (30). The pro-
duct precipitated from the reaction mixture was collected
by filtration and washed with cold methanol (2–3 mL) to
yield corresponding final product (4a–q).
N-cyclopropyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4d)
Off white solid; yield: 77%; mp: 196–198 °C; IR (KBr): v
3452, 3314 (N–H), 3065, 2924, 1735 (C=O), 1677 (C=N),
Chem Biol Drug Des 2016; 88: 43–53
45

Shaik et al.
1619 (C=O) cmÀ1
;
1H NMR (400 MHz, CDCl3): d 10.04
8-imino-2-oxo-N-(1-phenylethyl)-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4h)
(brs, 1H, 12-NH), 8.93 (s, 1H, 10-H), 7.69 (brs, 1H, 8-
NH), 7.66 (d, 1H, J = 9.6 Hz, 4-H), 7.52 (d, 1H,
J = 8.4 Hz, 5-H), 7.01 (d, 1H, J = 8.4 Hz, 6-H), 6.38 (d,
1H, J = 9.6 Hz, 3-H), 2.95 (m, 1H, 1’-H), 0.83 (m, 2H,
2’,3’-H), 0.61 (m, 2H, 2’,3’-H); 13C NMR (100 MHz,
CDCl3): d162.7 (C-2), 159.1 (C-8), 156.3 (C-11), 155.5
(C-6a), 151.4 (C-1a), 142.9 (C-4), 134.2 (C-5), 131.1 (C-
10), 121.2 (C-10a), 115.6 (C-9), 114.6 (C-3), 111.9 (C-
4a), 108.6 (C-6), 23.0 (C-1’), 6.6 (C-2’,3’); ESI-MS (m/z):
297 (M+H)+.
Brick red solid; yield: 70%; mp: 174–176 °C; IR (KBr): v
3449, 3272 (N–H), 3067, 2932, 1738 (C=O), 1674 (C=N),
1617 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d 9.42 (s,
;
1H, 10-H), 9.16 (d, 1H, J = 7.6 Hz, 12-NH), 7.76 (s, 1H,
8-NH), 7.73 (d, 1H, J = 9.6 Hz, 4-H), 7.39–7.27 (m, 7H,
5-H, 6-H, 2’-H, 3’-H, 4’-H, 5’-H and 6’-H), 6.49 (d, 1H,
J = 9.6 Hz, 3-H), 5.33–5.29 (m, 1H, 13-H), 1.61 (d, 3H,
J = 6.8 Hz, 14-CH3); 13C NMR (100 MHz, CDCl3):
d
160.5 (C-1’), 160.4 (C-2), 158.8 (C-8) 156.0 (C-11),
151.6 (C-6a), 142.8 (C-1a), 142.7 (C-4), 142.2 (C-5),
132.8 (C-10), 128.9 (C-3’,5’), 127.6 (C-4’), 126.2
(C-2’,6’), 118.6 (C-10a), 116.5 (C-9), 115.2 (C-3), 113.0
(C-4a), 108.6 (C-6), 49.9 (C-13), 22.4 (C-14); ESI-MS (m/
z): 361 (M+H)+.
N-cyclohexyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4e)
Pale yellow solid; yield: 85%; mp: 151–154 °C; IR (KBr): v
3535, 3228 (N–H), 3066, 2928, 2855, 1717 (C=O), 1675
(C=N), 1632 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d
;
10.01 (d, 1H, J = 6.8 Hz, 12-NH), 8.85 (s, 1H, 10-H), 7.73
(s, 1H, 8-NH), 7.66 (d, 1H, J = 9.6 Hz, 4-H), 7.51 (d, 1H,
J = 8.8 Hz, 5-H), 7.00 (d, 1H, J = 8.8 Hz, 6-H), 6.35 (d,
1H, J = 9.6 Hz, 3-H), 3.96 (m, 1H, 1’-H), 1.95–1.22 (m,
10H, 2’–6’-CH2); 13C NMR (100 MHz, CDCl3): d 160.2
(C-2), 159.2 (C-8), 156.3 (C-11), 155.5 (C-6a), 151.3
(C-1a), 143.0 (C-4), 134.0 (C-5), 131.0 (C-10), 121.5 (C-
10a), 115.4 (C-9), 114.5 (C-3), 111.9 (C-4a), 108.5 (C-6),
48.3 (C-1’), 32.8 (C-2’), 32.7 (C-6’), 25.7 (C-4’), 24.7
(C-3’), 24.5 (C-5’); ESI-MS (m/z): 339 (M+H)+.
N-benzhydryl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4i)
Off white solid; yield: 82%; mp: 197–199 °C; IR (KBr): v
3449, 3314 (N–H), 3063, 2925, 1734 (C=O), 1681 (C=N),
1
1619 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 11.02 (s,
1H, 12-NH), 9.03 (s, 1H, 10-H), 7.75 (s, 1H, 8-NH), 7.65
(d, 1H, J = 9.6 Hz, 4-H), 7.53 (d, 1H, J = 8.6 Hz, 5-H),
7.33 (m, 10H, 2’-H, 3’-H, 4’-H, 5’-H, 6’-H and 2’’-H, 3’’-
H, 4’’-H, 5’’-H, 6’’-H), 7.05 (d, 1H, J = 8.6 Hz, 6-H), 6.45
(d, 1H, J = 8.4 Hz, 13-H), 6.40 (d, 1H, J = 9.6 Hz, 3-H);
ESI-MS (m/z): 423 (M+H)+.
8-imino-9-(morpholine-4-carbonyl)-2H,8H-pyrano
[2,3-f]chromen-2-one (4f)
8-imino-N-(1-(4-methoxyphenyl)ethyl)-2-oxo-2H,8H-
pyrano[2,3-f]chromene-9-carboxamide (4j)
Pale yellow solid; yield: 87%; mp: 143–146 °C; IR (KBr): v
3454, 3281 (N–H), 2984, 2925, 2855, 1740 (C=O), 1661
Pale yellow solid; yield: 65%; mp: 158–161 °C; IR (KBr): v
3451, 3318, 3279 (N–H), 3063, 1734 (C=O), 1679 (C=N),
(C=N), 1621 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d
;
1
8.50 (s, 1H, 10-H), 7.81 (s, 1H, 8-NH), 7.68 (d, 1H,
J = 9.6 Hz, 4-H), 7.49 (d, 1H, J = 8.4 Hz, 5-H), 7.02 (d,
1H, J = 8.4 Hz, 6-H), 6.39 (d, 1H, J = 9.6 Hz, 3-H), 3.78
(t, 4H, 3’-H2, 5’-H2), 3.72 (m, 2H, 2’-H, 6’-H), 3.47 (m,
2H, 2’-H, 6’-H); ESI-MS (m/z): 327 (M+H)+.
1615 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.43 (s,
1H, 12-NH), 8.94 (s, 1H, 10-H), 7.74 (brs, 1H, 8-NH), 7.64
(d, 1H, J = 9.6 Hz, 4-H), 7.50 (d, 1H, J = 8.8 Hz, 5-H),
7.31 (d, 2H, J = 8.4 Hz, 2’-H, 6’-H), 7.01 (d, 1H,
J = 8.8 Hz, 6-H), 6.87 (d, 2H, J = 8.4 Hz, 3’-H, 5’-H),
6.37 (d, 1H, J = 9.6 Hz, 3-H), 5.23 (m, 1H, 13-H), 3.78 (s,
3H, 4’-OCH3), 1.55 (d, 3H, J = 6.8 Hz, 14-CH3); 13C NMR
(100 MHz, CDCl3): d 160.4 (C-2), 159.1 (C-8), 158.7 (C-
4’), 156.4 (C-11), 155.5 (C-6a), 151.4 (C-1a), 142.9 (C-4),
135.8 (C-1’), 134.4 (C-5), 131.1 (C-10), 127.4 (C-2’,6’),
121.3 (C-10a), 115.6 (C-9), 114.6 (C-3), 114.0 (C-3’,5’),
111.9 (C-4a), 108.6 (C-6), 55.4 (4’-OCH3), 49.0 (C-13),
22.6 (C-14); ESI-MS (m/z): 391 (M+H)+.
N-benzyl-8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-9-carboxamide (4g)
Pale yellow solid; yield: 83%; mp: 217–219 °C; IR (KBr): v
3452, 3305 (N–H), 3193, 3067, 1735 (C=O), 1677 (C=N),
1
1618 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.43 (s,
1H, 12-NH), 9.02 (s, 1H, 10-H), 7.73 (s, 1H,
8-NH), 7.65 (d, 1H, J = 9.6 Hz, 4-H), 7.52 (d, 1H,
J = 8.8 Hz, 5-H), 7.35 (m, 5H, 2’-H, 3’-H, 4’-H, 5’-H and
6’-H), 7.03 (d, 1H, J = 8.8 Hz, 6-H), 6.40 (d, 1H,
J = 9.6 Hz, 3-H), 4.65 (d, 2H, J = 6.0 Hz, 13-CH2); 13C
NMR (100 MHz, CDCl3): d 160.8 (C-2), 158.7 (C-8), 156.1
(C-11), 153.5 (C-6a), 142.7 (C-1a), 142.0 (C-4), 137.8 (C-
5), 132.6 (C-10), 128.8 (C-3’,5’), 127.9 (C-1’), 127.8
(C-2’,6’), 127.6 (C-4’), 119.0 (C-10a), 116.5 (C-9), 115.2
(C-3), 113.0 (C-4a), 108.7 (C-6), 44.1 (C-13); ESI-MS (m/
z): 347 (M+H)+.
N-(2,4-dimethoxybenzyl)-8-imino-2-oxo-2H,8H-
pyrano[2,3-f]chromene-9-carboxamide (4k)
Pale yellow solid; yield: 89%; mp: 143–145 °C; IR (KBr):
v 3449, 3272 (N–H), 3067, 2932, 1738 (C=O), 1674
(C=N), 1617 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d
;
10.34 (s, 1H, 12-NH), 8.97 (s, 1H, 10-H), 7.72 (brs, 1H,
8-NH), 7.63 (d, 1H, J = 9.6 Hz, 4-H), 7.49 (d, 1H,
J = 8.8 Hz, 5-H), 7.25 (d, 1H, J = 8.4 Hz, 6’-H), 7.00 (d,
46
Chem Biol Drug Des 2016; 88: 43–53

Fused Tricyclic Coumarins as Dual AChE Inhibitors
1H, J = 8.8 Hz, 6-H), 6.46 (s, 1H, 3’-H), 6.44 (d, 1H,
J = 8.4 Hz, 5’-H), 6.37 (d, 1H, J = 9.6 Hz, 3-H), 4.56 (d,
2H, J = 5.6 Hz, 13-CH2), 3.84 (s, 3H, 2’-OCH3), 3.79 (s,
3H, 4’-OCH3); 13C NMR (100 MHz, CDCl3): d 160.5 (C-
2), 159.2 (C-8), 158.7 (C-4’), 156.3 (C-11), 155.6 (C-2’),
151.4 (C-6a), 143.0 (C-1a), 142.9 (C-4), 134.0 (C-5),
130.9 (C-6’), 130.2 (C-10), 121.6 (C-1’), 118.5 (C-10a),
115.6 (C-9), 114.6 (C-3), 111.9 (C-4a), 108.5 (C-6),
103.9 (C-5’), 98.7 (C-3’), 55.5 (2’,4’-OCH3), 39.1 (C-13);
ESI-MS (m/z): 407 (M+H)+.
N-(3-chlorobenzyl)-8-imino-2-oxo-2H,8H-pyrano
[2,3-f]chromene-9-carboxamide (4o)
Pale yellow solid; yield: 65%; mp: 183–185 °C; IR (KBr): v
3438, 3310 (N–H), 3065, 2923, 1740 (C=O), 1682 (C=N),
1
1620 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.49 (s,
1H, 12-NH), 9.03 (s, 1H, 10-H), 7.75 (s, 1H, 8-NH), 7.67
(d, 1H, J = 9.6 Hz, 4-H), 7.54 (d, 1H, J = 8.4 Hz, 5-H),
7.34 (s, 1H, 2’-H), 7.24 (m, 3H, 4’-H, 5’-H and 6’-H), 7.04
(d, 1H, J = 8.4 Hz, 6-H), 6.40 (d, 1H, J = 9.6 Hz, 3-H),
4.63 (d, 2H, J = 5.6 Hz, 13-CH2); 13C NMR (100 MHz,
CDCl3): d 161.7 (C-2), 159.1 (C-8), 156.4 (C-11), 155.6
(C-6a), 151.5 (C-1a), 142.8 (C-1’), 140.5 (C-4), 135.0
(C-3’), 134.6 (C-2’), 131.2 (C-6’), 130.0 (C-5), 127.8
(C-10), 127.6 (C-4’), 125.9 (C-5’), 121.1 (C-10a), 115.7
(C-9), 114.7 (C-3), 111.9 (C-4a), 108.6 (C-6), 43.4 (C-13);
ESI-MS (m/z): 381 (M+H)+.
N-(2-bromobenzyl)-8-imino-2-oxo-2H,8H-pyrano
[2,3-f]chromene-9-carboxamide (4l)
Brick red solid; yield: 82%; mp: 219–221 °C; IR (KBr): v
3437, 3304 (N–H), 2924, 1755, 1736 (C=O), 1682 (C=N),
1
1617 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.55 (s,
1H, 12-NH), 9.03 (s, 1H, 10-H), 7.79 (s, 1H, 8-NH), 7.66
(d, 1H, J = 9.6 Hz, 4-H), 7.56 (d, 1H, J = 8.8 Hz, 3’-H),
7.53 (d, 1H, J = 8.4 Hz, 5-H), 7.47 (d, 1H, J = 9.2 Hz, 6’-
H), 7.30 (t, 1H, J = 8.8 Hz, 4’-H), 7.13 (t, 1H, J = 8.8 Hz,
5’-H), 7.03 (d, 1H, J = 8.4 Hz, 6-H), 6.40 (d, 1H,
J = 9.6 Hz, 3-H), 4.72 (d, 2H, J = 6 Hz, 13-CH2); ESI-MS
(m/z): 427 (M+H)+.
N-(4-(tert-butyl)benzyl)-8-imino-4-methyl-2-oxo-
2H,8H-pyrano[2,3-f]chromene-9-carboxamide (4p)
Pale yellow solid; yield: 68%; mp: 210–212 °C; IR (KBr): v
3438, 3310 (N–H), 3065, 2923, 1740 (C=O), 1682 (C=N),
1
1620 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.39 (s,
1H, 12-NH), 8.99 (s, 1H, 10-H), 7.72 (s, 1H, 8-NH), 7.64
(d, 1H, J = 9.6 Hz, 4-H), 7.51 (d, 1H, J = 8.4 Hz, 5-H),
7.37 (d, 2H, J = 8.4 Hz, 2’-H, 6’-H), 7.30 (d, 2H,
J = 8.4 Hz, 3’-H, 5’-H), 7.02 (d, 1H, J = 8.4 Hz, 6-H),
6.38 (d, 1H, J = 9.6 Hz, 3-H), 4.62 (d, 2H, J = 5.6 Hz,
13- CH2), 1.31 (s, 9H, 4’-C(CH3)3); 13C NMR (100 MHz,
CDCl3): d 161.5 (C-2), 159.1 (C-8), 156.3 (C-11), 155.6
(C-6a), 151.5 (C-1a),150.3 (C-4’), 142.9 (C-4), 135.3 (C-
1’), 134.6 (C-5), 131.1 (C-10), 127.5 (C-2’,6’), 125.6 (C-
3’,5’), 121.3 (C-10a), 115.6 (C-9), 114.7 (C-3), 111.9 (C-
4a), 108.6 (C-6), 43.7 (C-13), 34.6 (3’-C(CH3)3), 31.4 (3’-C
(CH3)3); ESI-MS (m/z): 403 (M+H)+.
N-(3-bromobenzyl)-8-imino-2-oxo-2H,8H-pyrano
[2,3-f]chromene-9-carboxamide (4m)
Orange red solid; yield: 72%; mp: 182–184 °C; IR (KBr):
v 3443, 3310 (N–H), 3068, 2924, 1741 (C=O), 1678
(C=N), 1618 (C=O) cmÀ1 1H NMR (400 MHz, CDCl3): d
;
10.49 (s, 1H, 12-NH), 9.02 (s, 1H, 10-H), 7.75 (s, 1H,
8-NH), 7.66 (d, 1H, J = 9.4 Hz, 4-H), 7.54 (d, 1H,
J = 8.4 Hz, 5-H), 7.49 (s, 1H, 2’-H), 7.39 (d, 1H,
J = 8.1 Hz, 4’-H), 7.29 (d, 1H, J = 8.1 Hz, 6’-H), 7.20 (t,
1H, J = 8.1 Hz, 5’-H), 7.03 (d, 1H, J = 8.4 Hz, 6-H),
6.40 (d, 1H, J = 9.4 Hz, 3-H), 4.62 (d, 2H, J = 6 Hz,
13-CH2); 13C NMR (100 MHz, CDCl3): d 161.7 (C-2),
159.1 (C-8), 156.4 (C-11), 155.6 (C-6a), 151.5 (C-1a),
142.8 (C-1’), 140.8 (C-4), 135.0 (C-2’), 131.3 (C-4’),
130.7 (C-5), 130.5 (C-5’), 130.3 (C-10), 126.4 (C-6’),
122.8 (C-3’), 121.1 (C-10a), 115.7 (C-9), 114.7 (C-3),
111.9 (C-4a), 108.6 (C-6), 43.3 (C-13); ESI-MS (m/z):
427 (M+H)+.
8-imino-N-(2-(2-methoxyphenoxy)ethyl)-2-oxo-
2H,8H-pyrano[2,3-f]chromene-9-carboxamide (4q)
Off white solid; yield: 87%; mp: 145–148 °C; IR (KBr): v
3452, 3313 (N–H), 3067, 2922, 1729 (C=O), 1681 (C=N),
1
1618 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.41 (s,
1H, 12-NH), 8.99 (s, 1H, 10-H), 7.80 (s, 1H, 8-NH), 7.67 (d,
1H, J = 9.6 Hz, 4-H), 7.53 (d, 1H, J = 8.4 Hz, 5-H), 7.03 (d,
1H, J = 8.4 Hz, 6-H), 6.94 (m, 4H, 3’-H, 4’-H, 5’-H, 6’-H),
6.40 (d, 1H, J = 9.6 Hz, 3-H), 4.22 (t, 2H, 14-H), 3.89 (m,
2H, 13-H), 3.87 (s, 3H, 2’-OCH3); ESI-MS (m/z): 407 (M+H)+.
N-(2-chlorobenzyl)-8-imino-2-oxo-2H,8H-pyrano
[2,3-f]chromene-9-carboxamide (4n)
Pale yellow solid; yield: 61%; mp: 222–224 °C; IR (KBr): v
3451, 3311 (N–H), 3064, 1756, 1737 (C=O), 1682 (C=N),
Determination of AChE and BuChE inhibitory
activities
1
1617 (C=O) cmÀ1; H NMR (400 MHz, CDCl3): d 10.54 (s,
1H, 12-NH), 9.02 (s, 1H, 10-H), 7.78 (s, 1H, 8-NH), 7.66
(d, 1H, J = 9.6 Hz, 4-H), 7.52 (d, 1H, J = 8.8 Hz, 5-H),
7.46 (dd, 1H, J = 7.4, 2.0 Hz, 3’-H), 7.37 (dd, 1H,
J = 7.4, 2.0 Hz, 6’-H), 7.25–7.21 (m, 2H, 4’-H, 5’-H), 7.03
(d, 1H, J = 8.8 Hz, 6-H), 6.40 (d, 1H, J = 9.6 Hz, 3-H),
4.73 (d, 2H, J = 6.0 Hz, 13-CH2); ESI-MS (m/z): 381
(M+H)+.
Inhibitory potency of target compounds against AChE and
BuChE were assayed using spectrophotometric method
developed by Ellman et al. (31) with slight modification. In
brief, reaction mixture composed of 10 lL of test sample
of five different concentrations, 145 lL phosphate buffer
200 mM (pH 7.7), 80 lL of dithiobisnitrobenzoic acid
(DTNB) (18.5 mg of DTNB dissolved in 10 mL phosphate
Chem Biol Drug Des 2016; 88: 43–53
47

Shaik et al.
buffer pH 7.7), and 10 lL of enzyme (0.4 U/mL). The mix-
ture was incubated at 25 °C for 5 min. Subsequently, the
enzymatic reaction was initiated by addition of 15 lL of
1 mM of acetylthiocholine iodide or butyrylthiocholine
iodide (according to the respective enzyme), and the mix-
ture was again incubated for 5 min at 25 °C. The rate of
absorbance change was measured at 412 nm for 6 min
using a microplate reader (Bio-Rad 680, Germany). Galan-
tamine was applied as positive drug.
tions of inhibitors (5, 10, and 30 nM) were added to the
assay solution and preincubated for 5 min at 25 °C with
the AChE followed by the addition of substrate in different
concentrations. The parallel control experiments were per-
formed without inhibitor in the assay. Progress curves
were monitored at 412 nm over 6 min. Then, double
reciprocal plots (1/v versus 1/[s]) were constructed using
GraphPad prism version 5 (34). The re-plots of the slopes
and intercepts of the double reciprocal plots against inhibi-
tor concentrations gave the inhibitor constants (Ki1 and
Ki2, for the binding to free enzyme and enzyme substrate
complex) as the intercepts on the x-axis. Data analysis
was performed using Microsoft Excel.
In vitro antioxidant activity assay
The radical scavenging activity of the test compounds was
measured by the ABTS method (32). The ABTS was dissolved
in water to obtain an 8 mM concentration of ABTS stock solu-
tion. ABTS radical cation (ABTS•+) was generated by adding
3 mM potassium persulfate to the ABTS stock solution and
keeping it in the dark at room temperature for 12–18 h. The
ABTS•+ solution was diluted with ethanol to give an absor-
bance of 0.48 Æ 0.07 at 734 nm. The 10 lL of the test com-
pounds were allowed to react with 290 lL of ABTS•+ solution.
The absorbance was taken 30 min after initial mixing. Trolox
was used as a standard.
Molecular Docking study
€
Molecular docking studies were performed using SCHRODIN-
€
GER MAESTRO software (version 9.2; Schrodinger LLC, New-
York, NY, USA). The protein used for docking is the crystal
structure of electric eel acetylcholinesterase (EeAChE, PDB
ID: 1C2O) after eliminating the original inhibitors and water
molecules. The ligands used as inputs for docking were
designed using CHEMBIOOFFICE 2010 software and prepared
€
€
using LIGPREP [version 2.5, Schrodinger Inc., Schrodinger
€
maestro software (version 9.2; Schrodinger LLC, New York,
ThT fluorescence assay
USA)]. GLIDE (grid-based ligand docking with energetics)
module of Schrodinger was used for the docking studies. A
grid was generated around the active site of the enzyme by
specifying the key active site residues: D74, T83, W86,
G120, G121, G122, Y124, Y133, E202, S203, W286, F295,
F297, Y337, and Y341. The poses generated per ligand
were set to 10 000, and the pose chosen for energy mini-
mization was set to 10. Glide docking score was used to
determine the best docked structure from the output (35).
The acceleration activity of synthetic analogs on Ab1À42
aggregation was determined using ThT fluorescence assay
(33). The accelerating Ab1À42 (Sigma) was dissolved in DMSO
to make a 200 lM stock solution. The stock solution was cen-
trifuged at the speed of 13 500 9 g for 10 min. The above
supernatant was used for experiments. The most active com-
pound 4m was dissolved in DMSO at concentrations of 0.02,
0.06, 0.1 mM. Further, the binding and modulating ability of
4m was ascertained by the most widely used method thiofla-
vin T (ThT) assay, to identify Ab fibrils with high sensitivity. Test
compound 4m (2 lL) (in concentrations of 0.02–0.1 mM) and
2 lL of 200 mM Ab42 were added into 76 lL of phosphate-
buffered saline (PBS at pH 7.4). After incubation for 24 h at
room temperature, 80 lL of 5 lM ThT solution (in 50 mM gly-
cine-NaOH at pH 8.5) was added to the reaction solution. Flu-
orescence emission spectra measurements were carried out
on Perkin Elmer LS55 fluorescence spectrometer within the
range from 410 to 600 nm, with an excitation wavelength of
390 nm. Identical spectra were recorded by performing the
independent experiments thrice.
Results and Discussion
Chemistry
As illustrated in Scheme 1, the target compounds 4a–q
could be prepared in economical way using inexpensive
materials in four steps starting from resorcinol. At first stage,
7-hydroxycoumarin (1) was prepared by using Pechmann
condensation between resorcinol and DL-malic acid in the
presence of conc. H2SO4 (26). Then, the obtained 1 was
treated with HMTA in acetic acid to gain 7-hydroxy-8-for-
mylcoumarin (2) by Duff formylation (27). Readily available
ethylcyanoacetate was reacted with a series of appropriate
amines to give the corresponding N-substituted cyanoac-
etamide derivatives (3a–q) according to reported proce-
dures (28,29). Finally, the different N-substituted
cyanoacetamides and 2 underwent intramolecular cycliza-
tion with Et3N to yield desired prototypes (30). All synthe-
sized compounds 4a–q were characterized by mass
analysis and IR, 1H, and 13C NMR spectroscopy. The IR
spectra of compounds showed absorption bands of =N–H
and C=N stretching vibrations at 3535–3427 cmÀ1 and
1661–1683 cmÀ1, respectively. The characteristic strong
Kinetic study
To obtain estimates of the inhibition model and inhibition
constant Ki, reciprocal plots of 1/V versus 1/[S] were con-
structed at different concentrations of the substrate
acetylthiocholine iodide (0.1–0.5 mM) using Ellman’s
method. The assay solution (250 lL) consists of 145 lL of
200 mM phosphate buffer (pH 7.7), with the addition of
80 lL of DTNB (18.5 mg of DTNB dissolved in 10 mL
phosphate buffer pH 7.7), 10 lL of 0.4 units/mL AChE,
and 15 lL of substrate (ATCh). Three different concentra-
48
Chem Biol Drug Des 2016; 88: 43–53

Fused Tricyclic Coumarins as Dual AChE Inhibitors
Table 1: Acetylcholinesterase inhibitory and ABTS radical scav-
enging activities of synthetic 8-imino-2-oxo-2H,8H-pyrano[2,3-f]
chromene-based compounds 4a–q and reference compounds
bands appeared for C=O stretching in coumarin at 1714–
1746 cmÀ1. The 1H NMR spectra of all the compounds
showed singlet of the imino group proton resonated at
7.69–7.81 ppm and the heterocyclic ring protons of cou-
marin resonated as two doublets between 6.35–6.49 and
7.63–7.73 ppm. In the 13C NMR spectra, the carbon reso-
nance frequencies of the C=O of coumarin was at 160.2–
162.7 ppm and C=O of amide at 156.0–156.4 ppm. These
data confirmed the formation of extended iminopyran ring
on coumarin moiety by the reaction between –CHO, –OH,
and –CN groups. Moreover, their ESI-MS spectra showed
molecular ion (M+H)+ peaks further confirming their struc-
ture. All spectroscopic measurements confirmed the struc-
ture and high purity of synthesized compounds.
IC50 Æ SEMa
ABTS radical scavenging
activity (lm)
Compounds
AChEb (nm)
4a
4b
415 Æ 30.44
259 Æ 9.71
83 Æ 3.2
>50
29.82 Æ 0.74
4c
4d
>50
799 Æ 31.88
169 Æ 15.5
226 Æ 16.74
497 Æ 40.50
462 Æ 36.03
669 Æ 47.89
390 Æ 24.34
300 Æ 25.38
27 Æ 2
24.35 Æ 1.64
19.59 Æ 1.23
11.19 Æ 0.98
10.79 Æ 1.05
>50
26.24 Æ 1.59
14.81 Æ 0.81
15.24 Æ 1.41
>50
4e
4f
4g
4h
4i
4j
4k
In vitro inhibition of cholinesterase
To determine the therapeutic potential of target tricyclic
fused coumarin derivatives in the treatment of AD, the inhi-
bitory activities of compounds 4a–q together with refer-
ence compound galantamine against both AChE and
BuChE was evaluated according to Ellman’s protocol by
measuring the rate of acetylthiocholine or butyrylthio-
choline hydrolysis in the presence of the inhibitor. All com-
pounds proved to be remarkably active toward AChE in
the low nanomolar range of IC50 values and almost inac-
tive on BuChE revealed an extremely selective inhibitory
profile for this series of compounds. The anti-AChE activi-
ties of the target compounds are summarized in Table 1
as IC50 values. From the data, it is notable that among the
seventeen compounds tested, six analogs exhibited 4- to
51-fold higher inhibitory activity toward AChE than galan-
tamine, a standard drug. The other ten analogs have
shown the better to comparable potency to that of galan-
tamine, while only 4d was less active. Although the activity
of compound 4d was less than standard drug galan-
tamine, but it had a fairly good inhibitory activity. Com-
pound 4m, which has N-(3-bromobenzyl)amide moiety,
exhibited superior anti-AChE activity with IC50 value of
13 Æ 1.4 nM in this series which is 51-fold more potent
than galantamine. The changes of substituents on the ter-
minal amide moiety evidently influence AChE inhibitory
effect of target compounds. Elongation of aliphatic chain
on N of amide increased the activity due to the more lipo-
philic property of these substituents. It is worthwhile to
note that 4d with cyclopropyl amide moiety showed the
most dramatic activity loss relative to compounds with
straight chain alkyl groups on N of amide as in 4a, 4b,
and 4c. Mutation of this moiety from cyclopropylamido to
cyclohexylamido improved the activity by 4.7 times. Mean-
while, changing alkyl amide to alkylaryl amide moiety led
to a huge decrease in activity. However, the substituent
position on the aromatic ring could largely affect the activ-
ity of the target compounds. The electron withdrawing
substituents such as bromo and chloro at position 2 and
3 increased the inhibitory activity by 5.6- to 38.2-fold as
seen in compounds 4l, m, n, and o. On the other hand,
electron donating groups such as methoxy at positions 2
4l
4m
13 Æ 1.4
>50
4n
4o
30 Æ 2
48.16 Æ 1.48
41.40 Æ 3.75
22.34 Æ 1.58
28.35 Æ 0.7
–
88 Æ 7.5
4p
4q
77 Æ 4.5
440 Æ 31
665 Æ 20.20
–
Galantamine
Trolox
27.35 Æ 1.34
aConcentration required to produce 50% inhibition of enzyme
activity. IC50 values are given as the mean of three independent
determinations.
bAChE from electric eel.
and 4 induce their enhancing effects as seen in 4j and k.
Comparing to the unsubstituted counterpart 4g, analog
bearing 4-ter-butyl-substituted benzylamido (4p) displayed
approximately four-fold stronger activity.
Enzyme Kinetic studies
The mechanism involved in the AChE inhibition by these
fused coumarins was investigated using compound 4m,
the most potent EeAChE inhibitor (Table 1). For this pur-
pose, the rate of the enzyme activity was measured at
four different concentrations of compound 4m (0, 5, 10,
and 30 nM) using different concentration of the substrate
acetylthiocholine (ATCh). In each case, the initial velocity
was measured at different concentrations of the substrate
(S), and the reciprocal of the initial velocity (1/v) was plot-
ted against the reciprocal of [ATCh]. The graphical pre-
sentation of steady state inhibition data of 4m for AChE
is shown Figure 1. The results revealed that there was an
increasing slope and increasing intercept at higher inhibi-
tor concentrations, indicating a mixed-type inhibitory abil-
ity for 4m. This behavior was also indicated by
intersection of double reciprocal lines in the upper left
quadrant of Lineweaver–Burk plot. Generally, this type of
inhibition indicates dual binding site mode of interactions
by binding to both the CAS and PAS of AChE. The inhi-
bition constants Ki1 and Ki2 obtained were 11.84 nM and
25.91 nM.
Chem Biol Drug Des 2016; 88: 43–53
49

Shaik et al.
A
B
Figure 1: Kinetic study on the mechanism of EeAChE inhibition by compound 4m. (A) Lineweaver–Burk plot of reciprocal of initial
velocities versus reciprocal of acetylthiocholine iodide concentrations (0.1–0.5 mM) in the absence and presence of 4m at 5, 10, and
30 nM; (B) secondary plots of the Lineweaver–Burk plot, slope versus various concentrations of 4m.
4c
4l
4m
4n
Figure 2: Best ranked binding orientation of most active compounds 4c, 4l, 4m, and 4n (in green color) in the binding site of AChE
enzyme. The hydrogen bonds and p-p interactions are shown as bold black and orange dashed lines, respectively.
Molecular docking study
the enzyme. An overall view of docking poses of 4c, 4l,
4m, and 4n with respect to the key residues in the binding
site is depicted in Figure 2.
To further study the interaction mode of fused tricyclic
coumarin derivatives with respect to AChE, ligand docking
studies were performed on active compounds using
€
€
Schrodinger maestro software (version 9.2; Schrodinger
LLC, NewYork) based on the 3D structure of the electric
eel enzyme complex (EeAChE, PDB ID: 1C2O). The best
docked poses in terms of glide energy of binding were fur-
ther analyzed to clarify interactions between ligands and
The results of docking revealed that all of these com-
pounds exhibit multiple binding modes with AChE. At the
entrance of the enzyme, p-p stacking interactions
between fused tricyclic coumarin moiety and indole ring
of PAS residue Trp286 is a common feature of all docked
50
Chem Biol Drug Des 2016; 88: 43–53

Fused Tricyclic Coumarins as Dual AChE Inhibitors
compounds. This feature of the molecules takes part in
recognition and orientation of the ligands in the active
site. This interaction is able to attribute two orientations
to these compounds so that the substituted benzyl amide
or alkyl amide moiety at position 9 is extended either into
the CAS or out from the active site gorge. It was there-
fore concluded that the coumarin parts of the molecules
are accommodated in the gorge rim, while amide frag-
ment is oriented toward the CAS or outwards the active
site.
In vitro antioxidant activity
The reduction of the oxidative stress is another crucial
aspect in designing agents for AD treatment. The antioxi-
dant capacity of coumarin derivatives 4a–q were examined
using the well-established ABTS (2,2’-azino-bis(3-ethyl-
benzthiazoline-6-sulfonic acid)) radical scavenging method
in comparison with trolox, a water-soluble vitamin E ana-
log. The ability of compounds to scavenge ABTS radicals
was shown as IC50, the test compound’s concentration
resulting in 50% inhibition of free radical (Table 1). The
data showed that ten synthetic derivatives demonstrated
potent ABTS radical scavenging capacities in the range of
10.79–29.82 lM. Among them, five compounds had higher
antioxidant activities ranging from 1.4- to 2.5-fold of trolox.
Among all seventeen analogs, only five displayed poor
antioxidant activities, with IC50 values >50 lM.
The binding poses of 4l and 4p places the ligand PAS
region and sandwiched the fused tricyclic coumarin moiety
between Trp286 and Tyr341 residues by p-p stacking with
ꢀ
the ring–ring distance in the range of 4.3–4.7 A similar to
that observed in known inhibitor tacrine. Oxygen of the
carbonyl group of coumarin moiety forms a hydrogen
bond interaction with the backbone -NH group of Phe295.
Consequently, 2-bromobenzylamide (4l) and 4-t-butylben-
zylamide (4p) moieties extended out from the active gorge
by forming hydrophobic interactions with Leu289, His287,
Ser293, and Glu292 at gorge rim.
Effect on the Ab aggregation
Reducing the neuronal cytotoxicity of amyloid peptides by
accelerating the aggregation process, which is different
from prevalent methods via inhibiting the aggregation of
peptides, is another crucial aspect in the development of
anti-AD agents (18). Thioflavin T (ThT) assay is one of the
most widely used methods to identify Ab aggregates
with high sensitivity (36). The emission band at 450 nm
is expected to be directly proportional to the amount of
Ab aggregates present; consequently, the formation of
Ab aggregates can be easily followed by measuring
ThT fluorescence enhancement by organic molecules in
concentration-dependent manner (19). To investigate the
effect of the synthesized analogs on Ab aggregation, com-
pound 4m (0.02, 0.06, 0.1 mM), which showed good activ-
ity in former tests, and galantamine were selected to
perform thioflavin T (ThT) assay. After the addition of 4m
The most energetically favored binding mode for com-
pounds 4c, 4m, and 4o inverse to that of 4l and places
the ligand in the PAS with the fused tricyclic coumarin
moiety stacking with Trp286 residue and the substituted
benzyl or alkyl amide moiety leaning toward the CAS by
forming hydrophobic interactions with Trp86, Glu121,
His447, Ser203, and Tyr337 residues which resembles
with those observed for known inhibitor donepezil. In this
orientation, the nitrogen of the iminopyran moiety of 4m
and 4o establishes hydrogen bond interaction with the -
NH group of Phe295 and Arg296, respectively. In case of
4c, the carbonyl group of coumarin moiety is involved in
ꢀ
bifurcated hydrogen bond interactions with -NH (2.14 A)
ꢀ
and -OH (2.15 A) of Ser 293. The long aliphatic chain in
4c was found to be entered deep into the gorge showing
interactions with the catalytic triad residues as that
observed in galantamine and rivastigmine. This binding
mode is in agreement with mixed-type inhibition pattern of
4c, 4m, and 4o in which ligand simultaneously bind to
CAS and PAS of AChE. From the above observations, it is
understood that the compound 4m could act as a potent
dual binding site inhibitor of AChE.
A
close examination into the docking mode of 4n
reveals hydrogen bond interactions between the carbonyl
group of coumarin moiety and -NH group of Arg296,
and from the carbonyl and -NH groups of amide frag-
ment to Ser 293 and Trp286, respectively. The tricyclic
coumarin moiety interacts with Trp286 by means of a p-
p stacking as in 4o and the 3-chlorobenzylamide moiety
extended out from the active site gorge as in 4l. There-
fore, the higher potency of these tricyclic coumarin com-
pounds with N-benzyl/alkyl carboxamide could be due
to the more favorable interactions of compounds with
the target enzyme.
Figure 3: Detection of Ab aggregates using ThT assay;
fluorescence enhancement spectra (kex = 390 nm, kem = 450 nm)
of 80 lL of 5 lM ThT solution (in 50 mM glycine-NaOH at pH 8.5)
mixed in 2 lL of 200 mM Ab1À42, with different concentrations
(0.02, 0.06, 0.1 mM) of 4m was measured after 24 h of incubation
time.
Chem Biol Drug Des 2016; 88: 43–53
51

Shaik et al.
to Ab1À42 aqueous solution and incubation for 24 h, a
gradual enhancement in the fluorescence intensity of
ThT at 450 nm was observed which is indicative of
dosage-dependent manner of the acceleration of Ab1À42
aggregation (Figure 3). As for galantamine, no significant
enhancement in fluorescence was observed no matter low
or high concentration of galantamine was used. Clearly, in
comparison with galantamine, compound 4m showed
additional acceleration of Ab1À42 aggregation process,
which may alleviate the Ab-induced toxicity and eventually
benefit the treatment of AD.
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Appendix S1. Spectra for selected compounds.
Chem Biol Drug Des 2016; 88: 43–53
53