Synthesis and evaluation of chromone-2-carboxamido-alkylamines as potent acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-020-02508-5

Alzheimer’s disease (AD) is considered one of the greatest global public burdens. Pathophysiology of AD is proposed to be associated with reduced levels of the neurotransmitter acetylcholine (ACh). Cholinesterase enzymes, namely acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) cleave ACh via hydrolysis. Cholinesterase inhibitors (ChEIs) are the main group of drugs currently used for the treatment of AD. Novel chromone-2-carboxamido-alkylamines (7–18) were designed, synthesized, and evaluated for cholinesterase inhibitory activity. The compounds exhibited potent AChE inhibitory activities at micromolar range (IC₅₀ 0.09–9.16 μM) and demonstrated weak BChE inhibitory activities (IC₅₀ 12.09–44.56 μM). Compound 14 (IC₅₀ 0.09 ± 0.02 μM) was the most potent AChEI in this series; it showed higher activity than the clinical used drug tacrine. Enzyme kinetic study suggested that 14 was an uncompetitive inhibitor. Molecular docking study revealed that 14 was a dual-binding site inhibitor. Compound 14 did not induce any concentration-related cytotoxic effect against SH-SY5Y cells. It also showed neuroprotective effect in the cell line. Chromone-2-carboxamido-alkylamines can be promising lead compounds for development of anti-Alzheimer’s agents.

Full text of DOI:10.1007/s00044-020-02508-5

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-020-02508-5
ORIGINAL RESEARCH
Synthesis and evaluation of chromone-2-carboxamido-alkylamines
as potent acetylcholinesterase inhibitors
1
Paptawan Suwanhom1 Teerapat Nualnoi2 Pasarat Khongkow3 Vannajan Sanghiran Lee4 Luelak Lomlim
●
●
●
●
Received: 1 August 2019 / Accepted: 13 January 2020
© Springer Science+Business Media, LLC, part of Springer Nature 2020
Abstract
Alzheimer’s disease (AD) is considered one of the greatest global public burdens. Pathophysiology of AD is proposed to be
associated with reduced levels of the neurotransmitter acetylcholine (ACh). Cholinesterase enzymes, namely
acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) cleave ACh via hydrolysis. Cholinesterase inhibitors
(ChEIs) are the main group of drugs currently used for the treatment of AD. Novel chromone-2-carboxamido-alkylamines
(7–18) were designed, synthesized, and evaluated for cholinesterase inhibitory activity. The compounds exhibited potent
AChE inhibitory activities at micromolar range (IC50 0.09–9.16 µM) and demonstrated weak BChE inhibitory activities (IC50
12.09–44.56 µM). Compound 14 (IC50 0.09 0.02 µM) was the most potent AChEI in this series; it showed higher activity
than the clinical used drug tacrine. Enzyme kinetic study suggested that 14 was an uncompetitive inhibitor. Molecular
docking study revealed that 14 was a dual-binding site inhibitor. Compound 14 did not induce any concentration-related
cytotoxic effect against SH-SY5Y cells. It also showed neuroprotective effect in the cell line. Chromone-2-carboxamido-
alkylamines can be promising lead compounds for development of anti-Alzheimer’s agents.
●
●
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Keywords Chromone Acetylcholinesterase inhibitors Neuroprotective Molecular docking
Introduction
Papageorgiou 2013). As the average lifespan of the global
population increases, neurodegenerative diseases become
more prevalent. Pathogenesis of AD is still unclear. Low
levels of acetylcholine (ACh) in various regions in the brain,
β-amyloid aggregation (Hardy and Selkoe 2002) and oxida-
tive stress are proposed to be key factors in AD pathophy-
siology (Baptista et al. 2014; Tang and Kumar 2008).
Alzheimer’s disease (AD) is a neurodegenerative disorder
characterized by progressive memory loss and personality
change (Mayeux and Sano 1999). AD is considered one of
the greatest global public burdens, currently affecting more
than 46 million people worldwide (Yiannopoulou and
Drug therapy of AD mainly depends on acet-
ylcholinesterase inhibitors (AChEIs) such as tacrine (Ahmed
et al. 2006), galanthamine (Greenblatt et al. 1999), donepezil
(Sugimoto et al. 2000), and rivastigmine. These drugs
increase ACh levels at cholinergic brain synapses through
inhibition of the ACh degrading enzyme, AChE (Mehta et al.
2012). AChE resides in the serine protease family. X-ray
crystallographic analysis of AChE from Torpedo californica
(TcAChE) revealed the active site of the enzyme as a narrow,
20 Å deep gorge. Several aromatic amino acids lining the
entrance of the gorge form the “peripheral anionic site”
(PAS). The catalytic triad that functions in ACh hydrolysis is
located at the bottom of the narrow gorge. “Catalytic anionic
site” (CAS) and acyl pocket support ACh binding during the
hydrolytic degradation process (Bourne et al. 1995; Chatonnet
and Lockridge 1989). Several AChEIs exert their activity via
binding at CAS or both CAS and PAS sites, as so called
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02508-5) contains supplementary
material, which is available to authorized users.
* Luelak Lomlim
luelak.l@psu.ac.th
1
Department of Pharmaceutical Chemistry, Faculty of
Pharmaceutical Sciences, Prince of Songkla University, Hat Yai,
Songkhla 90112, Thailand
2
Department of Pharmaceutical Technology, Faculty of
Pharmaceutical Sciences, Prince of Songkla University, Hat Yai,
Songkhla 90112, Thailand
3
Institute of Biomedical Engineering, Faculty of Medicine, Prince
of Songkla University, Hat Yai, Songkhla 90112, Thailand
4
Department of Chemistry, Faculty of Science, University of
Malaya, 50603 Kuala Lumpur, Malaysia

Medicinal Chemistry Research
“dual-binding site inhibitors” and block the hydrolysis of
ACh. While tacrine and galanthamine have high affinity
toward the CAS, donepezil acts as a dual-binding site inhi-
bitor and leads to higher potency and selectivity for AChE
(Castro and Martinez 2001).
of AD. Up to 45% of AChE may be lost in certain brain
regions during progression of AD while levels of BChE
activity conversely increase by up to 90%. Thus, both
enzymes have been identified as potential targets in the
treatment of AD (Greig et al. 2002). In this research, we
describe design, synthesis, and evaluation of AChE and
BChE inhibitory activities of chromone-2-carboxamido-
alkylamine derivatives. The mode of AChE inhibition was
explored. Molecular docking was employed to predict inter-
actions between the inhibitor and the enzymes. Cytotoxicity
of the synthesized compounds was evaluated in SH-
SY5Y cells.
Chromone is a scaffold that has attracted much attention
due to diverse biological properties such as anti-inflamma-
tory, metal chelating and neuroprotective effects (Grazul
and Budzisz 2009). Mimicking the indanone ring (red) of
donepezil, chromone moiety (red) was used as a scaffold for
the design of the target compounds. Several reported
AChEIs contain at least one basic amino group in their
molecular structure, either in the form of N-aryl or N-aralkyl
moieties. In physiological pH, the protonated amino group
binds with aromatic amino acid residues at the CAS
via cation–π interaction. The aromatic residue in N-aryl or
N-aralkyl moieties was predicted to form π–π interaction in
the CAS region and supported the binding of the molecule
to the active site gorge of the enzyme (Liu et al. 2015).
However, N-aryl or N-aralkyl moieties were not found to be
essential for AChE inhibitory activity. Some AChEIs
bearing chromone moiety with side chains containing N-
alkyl or alicyclic amine also exhibited potent AChE inhi-
bitory activity (Sang et al. 2017); (Pourshojaei et al. 2015;
Luo et al. 2016). These findings inspired us to design new
chromone-2-carboxamido-alkylamines as AChEIs using
chromone as a PAS binding unit (red). Piperidine ring or
dimethylamine moieties bearing a tertiary amino group
(blue) were incorporated in the structure. These amine
moieties were anticipated to bind with the CAS. The
chromone and tertiary amino groups were connected via a
methylene chain with various lengths (n = 2, 3) in order to
explore the optimal distance between the two units. As the
structure of donepezil consists of a dimethoxyl indanone
ring, positions 6- and 7- of the chromone ring system of our
designed compounds were replaced with mono- or di-
methoxyl groups to explore structure-activity relationships.
Figure 1 illustrates the idea in the design of the chromone-2-
carboxamido-alkylamines in this work.
Materials and methods
Experimental
All chemical and solvents required for synthesis were pur-
chased from Sigma-Aldrich. All reaction was checked by
thin layer chromatography and visualized by UV light.
Column chromatography was performed on silica gel. IR
spectra were performed on a Perkin Elmer spectrum and
principal absorptions were given in cm−1. 1H-NMR and 13C-
NMR spectra were measured on a Varian Unity Inova
500 MHz. NMR spectrometer using deuterated chloroform
(CDCl3) or dimethylsulfoxide (d6-DMSO) as solvent. The
coupling constants are reported in Hertz (Hz). Proton cou-
pling patterns were designed as s, singlet; d, doublet; t, tri-
plet; m, multiplet. Melting points were recorded using the
Mel-TEMP II LABORATORY DEVICES, USA. ESI-MS
spectra were recorded on a Thermo Finnigan MAT 95XL.
The Power Wave x, Biotele was used as a microplate reader.
Chemistry
Pathway for the synthesis of the desired chromone-2-
carboxamido-alkylamines can be illustrated in Scheme 1.
BChE is also able to hydrolyze ACh and is known as
pseudocholinesterase. This enzyme is found primarily in the
liver, intestine, heart, lung, and the brain (Cokugras 2003). In
a normal human brain, AChE accounts for major cholines-
terase activity. There is growing evidence that both AChE and
BChE may be important in the development and progression
General procedure for the preparation of compounds 4–6
Sodium hydride (65 mmol) was dissolved in tetra-
hydrofuran (THF, 10 ml) and then diethyl oxalate
(35 mmol) solution in THF (20 ml) was added. The reaction
mixture was stirred overnight at room temperature under
PAS
PAS
8
CAS
Fig. 1 Design strategy of
chromone derivatives
O
O
1
n
CAS
H3CO
H3CO
2
R1
R2
7
O
R3
N
H
N
6
4
R4
3
N
5
O
chromone-2-carboxamido-alkylamines (7-18)
Donepezil

Medicinal Chemistry Research
Scheme 1 Synthesis of
chromone derivatives: (a)
diethyl oxalate, NaH/THF, room
temperature; (b) amine/CH2Cl2/
reflux, then CH3COOH, 70 °C
nitrogen. It was then poured into iced-water (70 ml), HCl
(6 N, 20 ml), and extracted with diethylether (3 × 80 ml).
The precipitated solid was dried in a vacuum to give a
yellow solid. The product was purified by chromatography
on the silica gel by using ethyl acetate:hexane (40:60) as
mobile phase and then recrystallized in ethyl acetate to give
ethyl-4-oxo-4H-chromene-2-carboxylate (4–6).
(DMSO-d6, 125 MHz) 14.38, 56.35, 57.11, 63.04, 101.31,
103.90, 113.79, 117.73, 148.51, 151.99, 152.07, 155.69,
160.52, and 176.39.
General procedure for the preparation of compounds 7–18
Ethyl-4-oxo-4H-chromene-2-carboxylate derivatives (4–6,
0.46 mmol) and N, N-dimethylenediamine (1.38 mmol)
were dissolved in dichloromethane (5 ml), added to a round
bottom flask and allowed to reflux for 20 min. Then, glacial
acetic acid (5 ml) was added and the solution was stirred at
75 °C under a nitrogen atmosphere for 24 h. The mixture
was extracted with ethyl acetate (3 × 80 ml). The organic
layer was dried by vacuum. The product was purified by
chromatography on the silica gel by using dichloromethane:
methanol (80:20) as mobile phase and then recrystallized in
ethyl acetate to give chromone-2-carboxamido-alkylamine
derivatives (7–18)
Ethyl-4-oxo-4H-chromene-2-carboxylate (4) Pale yellow
solid (82% yield), m.p. 100–102 °C; IR (KBr): ν (cm−1
)
1
3448, 2984, 1740, 1658, 1465, 1258, and 772. H-NMR
(DMSO-d6, 500 MHz) 1.32 (t, J = 7.11 Hz, 3H), 4.37 (q,
J = 7.11 Hz, 2H), 6.92 (s, 1H), 7.51 (ddd, J = 1.04, 7.15,
8.11 Hz, 1H), 7.71 (m, 1H), 7.85 (ddd, J = 1.72, 7.15,
8.57 Hz, 1H), and 8.02 (dd, J = 1.59,8.11 Hz, 1H). 13C-
NMR (DMSO-d6, 125 MHz) 14.03, 62.87, 113.91, 119.04,
123.94, 125.12, 126.38, 135.49, 152.44, 160.15, and
177.54.
Ethyl-7-methoxy-4-oxo-4H-chromene-2-carboxylate (5)
Pale yellow solid (78% yield), m.p. 113–115 °C; IR (KBr):
ν (cm−1) 3462, 2919, 1743, 1439, 1257, and 778. 1H-NMR
(DMSO-d6, 500 MHz) 1.34 (t, J = 7.11 Hz, 3H), 3.92 (s,
3H), 4.39 (quin, J = 7.11 Hz, 2H), 6.88 (s, 1H), 7.10 (dd,
J = 2.40, 8.88 Hz, 1H), 7.23 (d, J = 2.39 Hz, 1H), and 7.94
(d, J = 8.88 Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz)
14.04, 56.49, 62.79, 101.16, 114.12, 115.90, 117.83,
126.53, 152.11, 157.51, 160.15, 164.81, and 176.48.
N-(2-(dimethylamino)ethyl)-4-oxo-4H-chromene-2-carboxa-
mide (7) Yellow liquid (64% yield), 100% HPLC purity,
m.p. 97–100 °C; IR (KBr): ν (cm−1) 3851, 3311, 2947,
1652, 1532, 1464, 1387, 1018, and 758. 1H-NMR (DMSO-
d6, 500 MHz) 2.21 (s, 6H), 2.48 (m, 2H), 3.39 (m, 2H), 6.81
(s, 1H), 7.53 (m, 1H), 7.72 (m, 1H), 7.88 (ddd, J = 1.72,
7.11, 8.50 Hz 1H), 8.04 (dd, J = 1.89, 7.83 Hz, 1H), and
9.03 (t, J = 5.51 Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz)
34.40, 45.20, 57.79, 110.57, 118.89, 123.70, 124.98,
126.14, 135.03, 155.20, 155.76, 159.06, and 177.42. ESI-
MS: (m/z, [M+H]+) (Calcd: 261.1236. Found: 261.1234).
Ethyl-6,7-dimethoxy-4-oxo-4H-chromene-2-carboxylate
(6) Pale yellow solid (86% yield), m.p. 116–117 °C; IR
(KBr): ν (cm−1) 3606, 2983, 1742, 1632, 1510, 1255, and
4-oxo-N-(2-(piperidin-1-yl)ethyl)-4H-chromene-2-carboxa-
mide (8) Pale yellow solid (62% yield), 100% HPLC
purity, m.p. 110–113 °C; IR (KBr): ν (cm−1) 3518, 3303,
2936, 1654, 1464, 1385, 1130, and 757. 1H-NMR (DMSO-d6,
1
865. H-NMR (DMSO-d6, 500 MHz) 1.34 (t, J = 7.11 Hz,
3H), 3.86 (s, 3H), 3.94 (s, 3H), 4.39 (quin, J = 7.11 Hz,
2H), 6.89 (s, 1H), 7.29 (s, 1H), 7.35 (s, 1H). 13C-NMR

Medicinal Chemistry Research
1
500 MHz) 1.51 (m, 6H), 2.68 (m, 6H), 3.50 (m, 2H), 6.83
(s, 1H), 7.53 (ddd, J = 1.06, 7.16, 8.07 Hz, 1H), 7.74 (dd,
J = 0.60, 8.62 Hz, 1H), 7.88 (ddd, J = 1.71, 7.16, 8.62 Hz,
1H), 8.04 (dd, J = 1.54, 8.07 Hz, 1H), and 9.21 (s, 1H). 13C-
NMR (DMSO-d6, 125 MHz) 24.27, 25.82, 37.18, 54.36,
57.45, 110.90, 119.19, 124.11, 125.43, 126.50, 135.48,
155.52, 156.09, 159.42, and 177.75. ESI-MS: (m/z, [M+H]+)
(Calcd: 301.1549. Found: 301.1547).
2936, 2839, 1649, 1609, 1439, 1386, 1024, and 835. H-
NMR (DMSO-d6, 500 MHz) 1.45 (m, 6H), 2.53 (m, 6H),
3.43 (m, 2H), 3.91 (s, 3H), 6.74 (s, 1H), 7.11 (dd, J = 2.41,
8.87 Hz, 1H), 7.16 (d, J = 2.41 Hz, 1H), 7.94 (d,
J = 8.87 Hz, 1H), and 9.01 (t, J = 5.60 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 23.97, 25.43, 36.85, 54.17, 56.54,
57.29, 101.34, 110.95, 115.71, 117.94, 126.86, 155.69,
157.35, 159.47, 172.48, and 176.87. ESI-MS: (m/z,
[M+H]+) (Calcd: 331.1666, Found: 331.1644).
N-(3-(dimethylamino)propyl)-4-oxo-4H-chromene-2-carbox-
amide (9) Pale yellow solid (68% yield), 100% HPLC
purity, m.p. 99–112 °C; IR (KBr): ν (cm−1) 3867, 3400,
N-(3-(dimethylamino)propyl)-7-methoxy-4-oxo-4H-chro-
mene-2-carboxamide (13) Pale yellow solid (72% yield),
1
2943, 1654, 1532, 1464, 1386, 1019, and 758. H-NMR
100% HPLC purity, m.p. 120–123 °C; IR (KBr): ν (cm−1
)
(DMSO-d6, 500 MHz) 1.69 (quin, J = 6.95 Hz, 2H), 2.16
(s, 6H), 2.30 (t, J = 6.90 Hz, 2H), 3.30 (m, 2H), 6.81 (s,
1H), 7.53 (m, 1H), 7.71 (ddd, J = 0.45, 1.04, 8.48 Hz, 1H),
7.89 (ddd, J = 1.73, 7.12, 8.48 Hz, 1H), 8.05 (dd, J = 1.70,
7.94 Hz, 1H), and 9.22 (t, J = 5.55 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 26.92, 38.50, 45.54, 57.35, 110.79,
119.16, 124.08, 125.40, 126.46, 135.46, 155.52, 156.20,
159.25, and 177.76. ESI-MS: (m/z, [M+H]+) (Calcd:
275.1398. Found: 275.1383).
3447, 2943, 2863, 1649, 1613, 1440, 1388, 1021, and 836.
1H-NMR (DMSO-d6, 500 MHz) 1.68 (m, 2H), 2.14 (s, 6H),
2.27 (t, J = 6.95 Hz, 2H), 2.49 (m, 2H), 3.92 (s, 3H), 6.73
(s, 1H), 7.11 (dd, J = 2.41, 8.86 Hz, 1H), 7.15 (d, J =
2.41 Hz, 1H), 7.94 (d, J = 8.86 Hz, 1H), and 9.13 (t, J =
4.51 Hz, 1H). 13C-NMR (DMSO-d6, 125 MHz) 26.80,
38.05, 45.28, 56.23, 56.96, 100.98, 110.53, 115.41, 117.63,
126.56, 155.55, 157.06, 158.95, 164.38, and 176.55. ESI-
MS: (m/z, [M+H]+) (Calcd: 305.1509. Found: 305.1496).
4-oxo-N-(3-(piperidin-1-yl)propyl)-4H-chromene-2-carboxa-
mide (10) Pale yellow solid (63% yield), 100% HPLC
purity, m.p. 108–112 °C; IR (KBr): ν (cm−1) 3748, 3429,
7-methoxy-4-oxo-N-(3-(piperidin-1-yl)propyl)-4H-chro-
mene-2-carboxamide (14) Pale yellow solid (71% yield),
100% HPLC purity, m.p. 123–126 °C; IR (KBr): ν (cm−1
)
1
2947, 1652, 1606, 1464, 1388, 1019, and 758. H-NMR
3421, 2945, 2686, 1650, 1609, 1439, 1386, 1022, and 839.
1H-NMR (DMSO-d6, 500 MHz) 1.89 (m, 8H), 2.66 (m,
6H), 3.35 (m, 2H), 3.91 (s, 3H), 6.76 (s, 1H), 7.11 (dd, J =
2.42, 8.88 Hz, 1H), 7.20 (d, J = 2.42 Hz, 1H), 7.95 (d, J =
8.88 Hz, 1H), and 9.25 (t, J = 5.77 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 21.83, 22.83, 23.75, 36.97, 52.31,
54.07, 56.27, 101.18, 110.65, 115.41, 117.64, 126.55,
155.35, 157.09, 159.34, 164.41, and 176.60. ESI-MS: (m/z,
[M+H]+) (Calcd: 345.1823. Found: 345.1804).
(DMSO-d6, 500 MHz) 1.67 (m, 6H), 1.99 (m, 2H), 2.99 (m,
4H), 3.30 (m, 4H), 6.84 (s, 1H), 7.54 (ddd, J = 1.04, 7.20,
8.01 Hz, 1H), 7.75 (dd, J = 0.49, 8.60 Hz, 1H), 7.90 (ddd,
J = 1.71, 7.20, 8.60 Hz, 1H), 8.05 (dd, J = 1.67, 8.01 Hz,
1H), and 9.34 (t, J = 6.06 Hz, 1H). 13C-NMR (DMSO-d6,
125 MHz) 21.57, 22.52, 23.48, 36.82, 52.11, 53.77, 110.62,
118.99, 123.77, 125.08, 126.22, 135.17, 155.25, 155.69,
159.39, and 177.50. ESI-MS: (m/z, [M+H]+) (Calcd:
315.1560. Found: 315.1558).
N-(2-(dimethylamino)ethyl)-6,7-dimethoxy-4-oxo-4H-chro-
N-(2-(dimethylamino)ethyl)-7-methoxy-4-oxo-4H-chro-
mene-2-carboxamide (15) Pale yellow solid (64% yield),
mene-2-carboxamide (11) Pale yellow solid (74% yield),
100% HPLC purity, m.p. 115–118 °C; IR (KBr): ν (cm−1
)
100% HPLC purity, m.p. 117–119 °C; IR (KBr): ν (cm−1
)
3446, 2923, 2863, 1642, 1598, 1536, 1273, 1021, and 863.
1H-NMR (DMSO-d6, 500 MHz) 2.22 (s, 6H), 2.50 (m, 2H),
3.41 (m, 2H), 3.86 (s, 3H), 3.93 (s, 3H), 6.75 (s, 1H), 7.23
(s, 1H), 7.34 (s,1H), and 9.00 (t, J = 5.68 Hz, 1H). 13C-
NMR (DMSO-d6, 125 MHz) 37.70, 45.55, 56.27–56.75,
58.22, 100.95, 103.96, 110.37, 117.40, 148.24, 151.51,
155.21, 155.32, 159.46, and 176.52. ESI-MS: (m/z,
[M+H]+) (Calcd: 321.1456. Found: 321.1450).
3315, 2947, 2823, 1649, 1609, 1439, 1386, 1022, and 833.
1H-NMR (DMSO-d6, 500 MHz) 2.25 (s, 6H), 2.52 (m, 2H),
3.41 (m, 2H), 3.91 (s, 3H), 6.74 (s, 1H), 7.10 (dd, J = 2.43,
8.88 Hz, 1H), 7.17 (d, J = 2.43 Hz, 1H), 7.94 (d, J =
8.88 Hz, 1H), and 9.01 (t, J = 5.47 Hz, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 36.71, 44.61, 56.58, 57.38, 101.38,
111.01, 115.82, 117.86, 126.91, 155.62, 157.36, 159.77,
164.81, and 177.04. ESI-MS: (m/z, [M+H]+) (Calcd:
291.1351. Found: 291.1338).
6,7-dimethoxy-4-oxo-N-(2-(piperidin-1-yl)ethyl)-4H-chro-
mene-2-carboxamide (16) Pale yellow solid (78% yield),
7-methoxy-4-oxo-N-(2-(piperidin-1-yl)ethyl)-4H-chromene-
2-carboxamide (12) Pale yellow solid (66% yield), 100%
HPLC purity, m.p. 119–121 °C; IR (KBr): ν (cm−1) 3323,
100% HPLC purity, m.p. 121–123 °C; IR (KBr): ν (cm−1
)
3422, 2935, 2863, 1643, 1608, 1506, 1270, 1019, and 828.
1H-NMR (DMSO-d6, 500 MHz) 1.49 (m, 6H), 2.58 (m,

Medicinal Chemistry Research
6H), 3.46 (m, 2H), 3.86 (s, 3H), 3.93 (s, 3H), 6.76 (s, 1H),
7.23 (s, 1H), 7.34 (s, 1H), and 9.09 (s, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 23.65, 25.03, 36.46, 53.97, 56.74,
57.09, 100.95, 103.94, 110.34, 117.40, 148.25, 151.51,
155.21, 159.60, 172.51, and 176.52. ESI-MS: (m/z,
[M+H]+) (Calcd: 361.1763. Found: 361.1754).
(%Inhibition) was calculated by the following expression:
ðMean velocity of blank À Mean velocity of sampleÞ Â 100
%Inhibition ¼
Mean velocity of blank
Determination of 50% inhibition of the AChE activity
(IC50) values was performed graphically from an inhibition
curve (log inhibitor concentration vs. percent of inhibition)
and done under the same protocol as above. Eight difference
concentrations of the inhibitor (50 − 1.6 × 10−3 µM) were
used. The response curve was created using Graphpad Prism
2.01.
Enzyme from horse serum was used in the in vitro BChE
assay and BTCI was used as a substrate. This assay was
performed using a method similar to that described above.
Donepezil and tacrine were used as positive controls.
Selectivity of acetylcholinesterase inhibitory activity of the
compounds can be evaluated by the ratio between IC50 of
horse BChE with IC50 of eeAChE and shown as Selectivity
index (SI) (Liston et al. 2004; Rogers and Friedhoff 1998).
N-(3-(dimethylamino)propyl)-6,7-dimethoxy-4-oxo-4H-chro-
mene-2-carboxamide (17) Pale yellow solid (67% yield),
100% HPLC purity, m.p. 119–123 °C; IR (KBr): ν (cm−1
)
3447, 2955, 2714, 1643, 1600, 1510, 1275, 1021, and 865.
1H-NMR (DMSO-d6, 500 MHz) 1.90 (m, 2H), 2.70 (s, 6H),
3.02 (m, 2H), 3.35 (m, 2H), 3.87 (s, 3H), 3.94 (s, 3H), 6.77
(s, 1H), 7.22 (s, 1H), 7.36 (s, 1H), and 9.20 (t, J = 5.84 Hz,
1H). 13C-NMR (DMSO-d6, 125 MHz) 24.67, 36.88, 42.82,
55.11, 56.35–56.82, 100.92, 104.02, 110.44, 117.43,
148.34, 151.56, 155.25, 155.33, 159.89, and 176.58.
ESI-MS: (m/z, [M+H]+) (Calcd: 335.1615. Found:
335.1605).
6,7-dimethoxy-4-oxo-N-(3-(piperidin-1-yl)propyl)-4H-chro-
Kinetic study
mene-2-carboxamide (18) Pale yellow solid (70% yield),
100% HPLC purity, m.p. 122–125 °C; IR (KBr): ν (cm−1
)
The kinetic study for the characterization of AChE inhibition
was evaluated with the same protocol of inhibitory activity by
using AChE from an electric eel (Nochi et al. 1995); (Kamal
et al. 2000). In this study, compound 14 had the highest
inhibitory activity and was chosen. The assay solution
(250 µL) consists of 125 µL of 3 mM DTNB, 25 µL of
1.5 mM of ATCI, 50 µL of 50 mM Tris/HCl (pH = 8), 25 µL
of enzyme, and 25 µL of three different concentrations (2.5,
7.5, and 15 µM) of inhibitor were added to the assay solution.
Kinetic study of the hydrolysis of ATCI catalyzed by
EeAChE was done spectrometrically at 405 nm every 11 s for
2 min and can be evaluated by the Michaelis–Menten model
constructed by plotting velocity (V) opposite to the con-
centration of substrate (S) at varying concentrations of the
substrate acetylthiocholine iodide (5, 10, 25, 50, 125, and
250 µM). Michaelis–Menten model was converted to
Lineweaver–Burk model into a straight line by plotting 1/
velocity opposite to 1/(S) by Prism5 program. The type of
inhibition (competitive, noncompetitive, uncompetitive, and
mixed-type inhibition) can be determined from the variation
of Km and Vmax at different inhibitor concentrations.
3423, 2939, 2871, 1643, 1606, 1474, 1276, 1013, and 826.
1H-NMR (DMSO-d6, 500 MHz) 1.80 (m, 8H), 2.94 (m,
6H), 3.35 (m, 2H), 3.12 (s, 3H), 3.93 (s, 3H), 6.77 (s, 1H),
7.23 (s, 1H), 7.35 (s, 1H), and 9.20 (s, 1H). 13C-NMR
(DMSO-d6, 125 MHz) 21.55, 22.95, 23.97, 36.92, 52.60,
54.18, 56.26–56.75, 100.82, 103.89, 110.36, 117.30,
148.27, 151.51, 155.15, 155.30, 159.88, and 176.64. ESI-
MS: (m/z, [M+H]+) (Calcd: 375.1934. Found: 375.1915).
Enzyme inhibition assay
AChE and BChE activities were measured by Ellman’s
method (Ellman et al. 1961) with slight modification. We
used AChE both from a human source (HuAChE) and an
electric eel (eeAChE) and BChE from horse serum. The
5,5′-dithiobis-(2-nitrobenzoic acid) (Ellman’s reagent,
DTNB), acetylthiocholine iodide (ATCI) and butyr-
ylthiocholine iodide (BTCI) were acquired from Sigma-
Aldrich.
All the in vitro AChE assays were carried out on a 96-
well plate. Overall, 25 µl of 100 µM sample was dissolved
in ethanol, 125 µl of 3 mM DTNB, 25 µl of 1.5 mM of
ATCI, an analog substrate of ACh, 50 µl of 50 mM Tris/
HCl (pH = 8), and followed by 25 µl of enzyme. This
reaction resulted in the development of a yellow color. The
color of the product was measured at 405 nm every 11 s for
2 min. Each experiment was repeated in triplicate. The
result of the measurements showed mean velocity which
was obtained from graphs plotted between times and
absorbance. The percentage of AChE inhibitory activity
Cytotoxicity
The cytotoxicity assay of chromone derivatives against SH-
SY5Y cells was measured using a sulforhodamine B (SRB)
assay. Human neuroblastoma cell line SH-SY5Y (ATCC
−2266) was purchased from ATCC. This cell line is the
most appropriate in vitro model for therapeutic testing of
neurodegenerative diseases (Xie et al. 2010). SH-SY5Y
cells were cultured in DMEM: F12 supplemented with 10%

Medicinal Chemistry Research
fetal bovine serum (Gibco, USA) and grown at 37 °C in a
humid atmosphere containing 5% CO2. Cells (8 × 103cells/
well) were seeded in a 96-well plate and incubated at 37 °C,
5% CO2 for 24 h. The cells were then treated with or
without various concentrations (0, 0.4, 0.8, 1.5, 3.125, 6.25,
12.5, 25, 50 µM) of each compound for 48 h. After an
incubation period, 40% (w/v) Trichloroacetic acid (TCA)
was added to the cells and the cells were then incubated at
4 °C for 1 h. The medium was removed and the cells were
rinsed with slow running tap water. 0.4% (w/v) SRB
solution (100 μL) was added to each well and the cells were
incubated for 1 h at room temperature. The SRB solution
was removed and then the cells were washed 3 times with
1% (v/v) acetic acid and were allowed to dry at room
temperature. The protein-bound dye was dissolved with
10 mM Tris base solution and the absorbance was measured
at 492 nm using a microplate reader.
50 Å × 50 Å. For HuBChE, the center of the grid box was
determined at x = 135.117, y = 119.222, z = 40.667, and the
dimensions of the active site box were set at 34 Å × 46 Å ×
34 Å. After that, ligands are put in the grid box to calculate
free energy of binding (ΔG). Ligands are arranged by the
calculated ΔG value; lower ΔG values correspond to more
desirable ligand binding, while higher ΔG values are less
desirable (favorable) (Trott and Olson 2010). The docking
score is the predicted binding affinity in kcal/mol. Finally, the
complex structures were evaluated using Discovery Studio
2.5 program.
Results and discussion
Chemistry
The chromone derivatives were prepared via 2-step synthesis.
In the first step, ethyl-4-oxo-4H-chromene-2-carboxalate
derivatives (4–6) were prepared from Claisen condensation
(Walenzyk et al. 2005) between 2-hydroxyacetophenone (1)
or substituted 2-hydroxyacetophenone (2–3) and diethyl
oxalate using sodium hydride as a base. The resulting esters
were subjected to aminolysis (Bera et al. 2011) with N-alkyl
dimethylamines or N-alkyl piperidine to yield the corre-
sponding chromone derivatives (7–18, Scheme 1). Com-
pounds 9 and 13 are known compounds (Walenzyk et al.
2005) but the AChE inhibitory activity of these compounds
has not been reported. Structure of the synthesized com-
Neuroprotective studies against H2O2-induced stress
The SH-SY5Y cells were seeded into 96-well plates (8 ×
103 cells/well) and allowed to adhere for 24 h. Cells were
then pretreated with 5 µM of the tested compound, and then
freshly prepared H2O2 (from 30 % stock) was added at a
concentration of 100 µM and further cultured for 24 h. To
examine potential effects, the medium was removed fol-
lowing treatments, and the SRB assay was performed.
Molecular docking
1
pounds was characterized with IR, H-NMR, 13C-NMR and
In this study, AutoDock Vina 1.1.2 (Trott and Olson 2010);
(Forli et al. 2016) and Discovery Studio 2.5 programs were
used for molecular docking and visualization. The protocol
included three main steps: the optimization of compounds, the
minimization of enzymes and the molecular docking study to
predict the structures of enzyme-ligand complexes (Lee
2008). The best compound from results of inhibitory activity
was selected for this docking experiment. The structure of the
compound was optimized using density functional theory at
B3LYP/6-311 G (d, p) level while the enzymes were mini-
mized using AMBER software. The protein was prepared
under the protein preparation protocol achieved in Discovery
Studio 2.5. PROKA was used to assign protonation state at
neutral pH (pH = 7). The enzyme structures from Torpedo
californica acetylcholinesterase (TcAChE, PDB ID: 1EVE)
and human butyrylcholinesterase (HuBChE, PDB ID: 4BDS)
were achieved from protein data bank (PDB) and the mole-
cular docking studies were carried out where the binding site
covered the PAS and CAS of the enzyme. The grid box center
was set at the center of the active site of enzyme. For
TcAChE, the center of the grid box was placed at the bottom
of the active site gorge (x = 2.023, y = 63.295, z = 67.062),
and the dimensions of the active site box were set at 50 Å ×
Mass spectrometry. The target compounds were successfully
synthesized in high yield. Spectroscopic data and high-
resolution mass spectrometry confirmed that the desired pro-
ducts were obtained.
In vitro inhibitory activity against AChE and BChE
In vitro AChE and BChE inhibitory activities were screened
with electric eel AChE (eeAChE) and equine BChE enzymes
using the colorimetric Ellman’s method. All compounds
exhibited potent acetylcholinesterase inhibitory activity with
IC50 values in micromolar level and showed moderate BChE
inhibitory activities. Ellman’s assay was further performed
with HuAChE in order to infer acetylcholinesterase inhibitory
activity of the compounds in human (Table 1).
From the Ellman’s assay using eeAChE, compounds with
unsubstituted chromone moiety (7–10) showed activities with
IC50 values ranging from 1.75 to 2.55 µM. Compound 8
(IC50 = 1.75 0.19 µM) was the most potent compound in
this series. Compounds with 7-methoxyl chromone moiety
(11–14, R1 = OCH3) showed activities with IC50 values
between 0.09–9.16 µM. Compound 14 (IC50 = 0.09
0.0003 µM) exerted the highest potency in this series and

Medicinal Chemistry Research
Table 1 Inhibition concentration
at 50% to AChE and BChE of
chromone derivatives
Cpd
R1
R2
NR3R4
n
IC50 SD (µM)
SIa
eeAChE
HuAChE
BChE
7
H
H
a
b
a
b
a
b
a
b
a
b
a
b
2
2
3
3
2
2
3
3
2
2
3
3
7.58 0.91
1.75 0.19
3.42 0.32
2.55 0.06
1.05 0.22
0.88 0.08
9.16 0.24
0.09 0.0003
0.14 0.03
0.41 0.04
2.56 0.06
0.78 0.04
0.13 0.02
0.006 0.001
4.10 0.66
6.38 0.67
5.34 0.43
1.61 0.12
2.15 0.16
1.73 0.07
3.16 0.06
0.09 0.02
2.78 0.46
1.35 0.07
3.15 0.49
0.22 0.02
0.31 0.02
0.004 0.001
22.95 0.04
28.32 1.60
14.51 0.45
13.74 0.96
17.82 0.92
12.23 0.76
13.72 0.48
27.91 0.34
44.56 0.46
39.62 0.06
12.09 1.23
32.3 1.13
0.01 0.001
1.06 0.02
3.03
8
H
H
16.18
4.24
9
H
H
10
H
H
5.38
11
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
H
16.97
13.89
1.49
12
H
13
H
14
H
310.11
318.29
96.63
4.72
15
OCH3
OCH3
OCH3
OCH3
16
17
18
41.41
0.86
Tacrine
Donepezil
176.67
aSI = (IC50 Horse BChE)/(IC50 eeAChE)
among all synthesized compounds in this work. Compounds
with 6,7-dimethoxyl chromone moiety (15–18, R1 = R2 =
OCH3) showed activities with IC50 values in the range of
0.14–2.56 µM. Compound 15 (IC50 = 0.14 0.03 µM)
exerted the best activity among compounds in this series.
Changing the amino group moiety from N,N-dimethyl
group to piperidine ring generally resulted in higher
potency. For example, in the unsubstituted chromones
(7–10) with the same methylene chain length, 8 (n = 2, IC50
1.75 0.19 µM) showed higher potency than 7 (n = 2, IC50
7.58 0.91 µM). This trend was also observed among most
compounds in the 7-methoxyl and 6,7-dimethoxyl chro-
mone series. However, effect from varying the length of the
methylene chain joining the chromone and amine moiety to
AChE activity was not observed. From these findings,
compound 14 (IC50 = 0.09 0.0003 µM) with 7-methoxyl
chromone moiety and piperidine moiety was the most
potent compound among the whole series. A similar trend
was observed when the compounds were tested using
human AChE. Compound 14 (IC50 = 0.09 0.02 µM) was
still the most potent acetylcholinesterase inhibitor in this
series. This supported the idea that compound 14 showed
high potential for development for the treatment of AD in
human. Most of the synthesized chromone derivatives were
found to be moderately or non-selective to AChE. In con-
trast, compounds 14 (SI = 310.11) and 15 (SI = 318.29)
were highly selective to AChE, almost two times more
selective than donepezil (SI = 176.67), the dual-binding site
inhibitor. Molecular docking was used to suggest insights in
difference of binding energy and binding interaction
between the compounds with AChE and BChE that leaded
to the high selectivity to AChE.
Kinetic study
A kinetic study was carried out on the most potent com-
pound in the series (14) with AChE from electric eel at three
different concentrations (2.5, 7.5 and 15 µM) of the inhi-
bitor. Lineweaver–Burk plot (Fig. 2) showed a decrease in
V
max and Km as the concentration of the inhibitor increased,
which showed uncompetitive inhibition.
Compound 14 demonstrated uncompetitive inhibition to
AChE. This mode of inhibition is different from donepezil
which was indicated as a mixed-type AChE inhibitor. The
mixed-type inhibitors were proposed to be able to bind with
both the CAS and the PAS of the enzyme (8, 14). N-benzyl
moiety in the structure of donepezil might facilitate binding at
the CAS via π-π interaction resulting in dual-binding site
inhibition. The uncompetitive inhibitor binds only with
enzyme-substrate [ES] complex and gives enzyme-substrate-
inhibitor [ESI] complex without binding to the free enzyme.
Compound 14, lack of N-benzyl group, might bind with the
CAS with less affinity than donepezil. The smaller piperidine
moiety of 14 might allow larger room at the bottom of the
active site gorge for substrate binding, then 14 might bind
with the [ES] complex to give enzyme-substrate [ES] com-
plex and thus block the action of AChE.
Cytotoxicity and neuroprotective effect studies
To gain insight into the therapeutic potential of synthesized
compounds, the cytotoxicity and neuroprotective capacity
against oxidative stress were investigated in the human SH-
SY5Y neuroblastoma cell line. First, we examined the
cytotoxic effects of the selected compounds, including 10,

Medicinal Chemistry Research
Fig. 2 Lineweaver–Burk plot illustrating uncompetitive types of
eeAChE inhibition for compound 14. The inverse initial velocity (V−1
)
)
Fig. 4 Neuroprotective effect of synthesized compounds on H2O2-
induced cytotoxicity in SH-SY5Y cells. Cells were pretreated with
5 µM of the tested compound for 24 h and then 100 µM of freshly
prepared H2O2 was added and further cultured for 24 h. Cell viability
was determined by SRB assay. Data are means SD of three inde-
pendent experiments and %cell viability was calculated relative to
nontreated control; **p < 0.05
as a function of the inverse of the substrate concentration ([ATCI]−1
was plotted for different concentrations of inhibitor (0.0, 2.5, 7.5 and
15 µM)
the tested compound followed by exposure to 100 µM
H2O2. The result showed that among all the compounds, 14
and 18 exhibited neuroprotective activities against H2O2-
induced damage at 5 μM, restoring cell viability at 7.1% and
15.5%, respectively. This result suggested effectiveness of
compounds 14 and 18 in preventing oxidative stress to
neuronal cells. Compounds 14 and 18 showed neuropro-
tective effects against H2O2-induced cell death. The
methoxyl substituent(s) may play a role in the anti-oxidative
effect of the compounds.
Fig. 3 Cytotoxic effect of synthesized compounds on SH-SY5Y cells
as determined by SRB assay. Cells were treated with the synthesized
compounds at different concentrations for 48 h, and then SRB assay
was performed. Data are means SD of three independent experiments
and %cell viability was calculated relative to nontreated control
Molecular docking
The most potent chromone-2-carboxamido-alkylamine
derivative (14) was further studied for binding interactions
and binding affinity using molecular docking. Compound
14 showed selectivity to AChE (SI = 310.11) that is
approximately two times higher than donepezil (SI =
176.67, Table 1). In order to explain this high selectivity of
14 over donepezil, binding affinity with AChE and BChE
enzymes of the two molecules were compared.
14, 18, on the cell viability of SH-SY5Y cells by using SRB
assay. The cells were treated with varying concentrations
ranging from 0.4 to 50 µM of the tested compounds for
48 h. As shown in Fig. 3, results revealed that all of the test
compounds did not show significant toxicity at any tested
concentrations to the neuron cells (LC50 > 50 µM) when
compared with the reference compound, donepezil, (LC50
=
The initial structures of TcAChE and HuBChE were
retrieved from protein data bank with PDB ID: 1EVE and
4BDS, respectively. A molecular modeling study was per-
formed using the docking program by AutoDock Vina 1.1.2
and Discovery studio 2.5. The result showed affinity of
binding energy between the enzyme and the compound as
noted in Table 2. The molecular docking studies of com-
pound 14 and donepezil resulted in the low-negative bind-
ing energy which indicated strong favorable binding for
both enzymes (TcAChE and HuBChE). The lowest binding
energy conformer of each ligand was selected to investigate
the residue interaction. Furthermore, distance values which
43.13 7.9 µM). This result suggested safety of the chro-
mone derivatives when used with the neuronal cell line at
the effective lower micromolar level.
Oxidative stress plays an important role in the progres-
sion of AD. Therefore, in this study, we also investigated
the neuroprotective effect against H2O2-induced cell death
of chromone derivatives. Exposure of cells to 100 µM H2O2
induced significant oxidative stress leading to the reduction
of cell viability, ~20% of control after 24 h exposure
(Fig. 4). In order to test the neuroprotective effect of
selected chromone series, cells were pretreated with 5 µM of

Medicinal Chemistry Research
measured the distance between amino acid residues in the
active sites of enzyme and functional groups on the ligands
were calculated and illustrated in Fig. 5.
Compound 14-TcAChE complex showed affinity of
binding energy of −9.5 kcal/mol (Table 2). Compound 14
occupied the entire enzymatic PAS, mid-gorge, and CAS
Table 2 Binding energy of the
chromone derivative in
comparison with donepezil
Compound
IC50 SD (µM)
Binding energy (kcal/mol)
EeAChE
Horse BChE
HuAChE
TcAChE
HuBChE
14
0.09 0.0003
0.006 0.001
27.91 0.34
1.06 0.02
0.09 0.02
−9.5
−7.9
−9.1
Donepezil
0.004 0.001
−10.9
Fig. 5 Molecular docking study of 14-TcAChE (a), Donepezil-TcAChE (b), 14-HuBChE (c), and Donepezil-HuBChE (d) complexes

Medicinal Chemistry Research
regions (Fig. 5a). The chromone moiety showed interaction
with PAS of active site of enzyme and showed π–π inter-
action with Trp278 (distance of 3.53 Å), methylene chain
showed hydrophobic interaction with Phe330 in the mid-
gorge and piperidine ring of compound showed cation–π
interaction with Phe329 (distance of 4.17 Å).
and showed high selectivity to AChE (SI = 310.11). Kinetic
study suggested that 14 is an uncompetitive AChE inhibitor.
Results from molecular docking supported the enzyme inhi-
bitory results and high selectivity to AChE of the compound.
As proposed, the chromone nucleus showed affinity to the
PAS of AChE and the piperidine nitrogen showed affinity
with the CAS of the enzyme. Cytotoxicity of the chromone
derivatives was further determined in SH-SY5Y cell line and
the compounds showed higher cell viability than donepezil
at the same concentration. The selected chromone-2-
carboxamido-alkylamines demonstrated a neuroprotective
effect. These findings supported that chromone derivatives are
promising for further evaluation and development as agents for
the treatment of AD.
In 14-HuBChE complex, the affinity of binding energy
was calculated as −7.9 kcal/mol (Table 2) demonstrating
weak affinity to BChE. The tertiary amino group of the
compound interacted only with Trp79 (distance of 4.20 Å)
with cation–π interaction (Fig. 5c). These findings might
reflect the higher selectivity of 14 toward AChE over
BChE. Donepezil-TcAChE (Fig. 5b) complex showed the
binding affinity of −10.9 kcal/mol. Strong interactions
between donepezil and the enzyme were observed in
several areas of the active site gorge. The indanone ring of
donepezil was observed to bind to the PAS via a π–π
interaction with Trp278 at the distance of 3.62 Å. The
long chain of methylene was observed to bind to the mid-
gorge via a hydrophobic interaction with Phe330. The
piperidine ring was observed to bind to the CAS via a
cation-π interaction with Phe329 at the distance of 3.96 Å.
The benzyl group of donepezil additionally interacted
with Trp83 at the bottom of the gorge by π–π interaction
at the distance of 4.15 Å. This might facilitate affinity of
the drug to the active site gorge, both at the PAS and CAS
sites, and explain the higher potency in AChE inhibition
and the mixed-type inhibition of donepezil. On the other
hand, compound 14 could bind at an upper part of the
CAS and allow some space for substrate binding at the
bottom of the gorge. This might allow the compound to
bind with (ES) complex and explain uncompetitive inhi-
bition of 14.
Acknowledgements We would like to thank Prince of Songkla Uni-
versity (Grant No. PHA590412S) and the University of Malaya
(UMRG: RP037D-17AFR) for financial support of this study. The
authors would like to thank Miss Maria Mullet for providing linguistic
proofreading for the manuscript.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Publisher’s note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
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