Design, synthesis, biological evaluation, and molecular modeling studies of chalcone-rivastigmine hybrids as cholinesterase inhibitors

Article abstract of DOI:10.1016/j.bmc.2016.11.002

A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and tested in vitro for their ability to inhibit human acetylcholinesterase and butyrylcholinesterase. Most of the target compounds showed hBChE selective activity in the micro- a

Full text of DOI:10.1016/j.bmc.2016.11.002

Accepted Manuscript
Design, synthesis, biological evaluation, and molecular modeling studies of
chalcone-rivastigmine hybrids as cholinesterase inhibitors
Ling Wang, Yu Wang, Yiguang Tian, Jinling Shang, Xiaoou Sun, Hongzhuan
Chen, Hao Wang, Wen Tan
PII:
S0968-0896(16)31102-6
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.002
BMC 13368
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 August 2016
31 October 2016
1 November 2016
Please cite this article as: Wang, L., Wang, Y., Tian, Y., Shang, J., Sun, X., Chen, H., Wang, H., Tan, W., Design,
synthesis, biological evaluation, and molecular modeling studies of chalcone-rivastigmine hybrids as cholinesterase
inhibitors, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.002
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Design, synthesis, biological evaluation, and molecular modeling studies of
chalcone-rivastigmine hybrids as cholinesterase inhibitors
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Ling Wang a,‡,*, Yu Wang b,‡, Yiguang Tian a, ‡,1, Jinling Shang a, Xiaoou Sun a,
Hongzhuan Chen b, Hao Wang b,*, Wen Tan a,*
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aGuangdong Provincial Key Laboratory of Fermentation and Enzyme Engineering, Pre-Incubator
for Innovative Drugs & Medicine, School of Bioscience and Bioengineering, South China
University of Technology, Guangzhou 510006, China
bDepartment of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University,
School of Medicine, 280 South Chongqing Road, Shanghai 200025, China
‡Equal contribution
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*Corresponding author:
lingwang@scut.edu.cn (L. Wang)
went@scut.edu.cn (W. Tan);
angela_wanghao@hotmail.com (H. Wang);
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Abstract
A series of novel chalcone-rivastigmine hybrids were designed, synthesized, and
tested in vitro for their ability to inhibit human acetylcholinesterase and
butyrylcholinesterase. Most of the target compounds showed hBChE selective activity
in the micro- and submicromolar ranges. The most potent compound 3 exhibited
comparable IC50 to the commercially available drug (rivastigmine). To better
understand their structure activity relationships (SAR) and mechanisms of
enzyme-inhibitor interactions, kinetic and molecular modeling studies including
molecular docking and molecular dynamics (MD) simulations were carried out.
Furthermore, compound 3 blocks the formation of reactive oxygen species (ROS) in
SH-SY5Y cells and shows the required druggability and low cytotoxicity, suggesting
this hybrid is a promising multifunctional drug candidate for Alzheimer’s disease
(AD) treatment.
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Keywords
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Cholinesterase inhibition, chalcone-rivastigmine hybrids, Alzheimer’s disease,
antioxidative, molecular docking
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1. Introduction
Alzheimer’s disease (AD) is a debilitating and fatal progressive neurodegenerative
disorder always resulting in death over a course that varies from 3 to 20 years.1
Although the etiology of AD is not yet entirely known, some factors are reported to
play key roles in the pathogenesis of this disease, including protein misfolding and
aggregation, oxidative stress, mitochondrial abnormalities, neuroinflammation,
neuronal toxicity, and low levels of neurotransmitter acetylcholine (ACh).2-4
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According to the cholinergic hypothesis for AD pathogenesis, the cognitive
impairments in AD correlated with cholinergic deficits such as reduced choline
acetyltransferase (ChAT) activity and synaptic acetylcholine synthesis.5 These
observations were made just as the role of the cholinergic system in memory
processing and learning began to be more widely appreciated.6 At the neuronal level,
ACh can be degraded by two types of cholinesterase enzymes: acetylcholinesterase
(EC 3.1.1.7; AChE) and butyrylcholinesterase (EC 3.1.1.8; BChE). AChE is mainly
expressed by neurons and its activity is dominant in the healthy brain (80%), while
BChE activity is mainly associated with glial cells and seems to play only a
supportive role in the healthy brain.7 Due to the extraordinary efficiency and the
classical relationship between AChE and the neurotransmitter acetylcholine, AChE
has been a drug target for the treatment of AD. Over the past 20 years, several AChE
inhibitors have been launched on the market for AD treatment, including tacrine,
donepezil, rivastigmine, and the alkaloid galanthamine (Figure 1). These drugs had
made an important contribution to the management and well-being of early stage AD
patients. However, significant scientific and clinical questions about AChE inhibitors
remain unsolved, e.g., lacking the efficacy for late stage AD patients,8 peripheral side
effects,9 and the rapid emergence of drug resistance.10, 11
In contrast to AChE, BChE is found ubiquitously throughout the human body and
BChE knockout mice show no physiological Alterations.12 The characterization of
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AChE nullizygote (AChE -/-) mouse with a clear phenotype appears to have normal
CNS function, demonstrating that BChE can rescue the cholinergic function in the
absence of AChE.13 This finding is consistent with previous studies that exhibited an
increase of BChE expression in the brain to compensate the loss of AChE activity
during AD progression. Furthermore, silent BChE mutations in humans show a slower
rate of cognitive decline.14 Selective BChE inhibitors, cymserine analogs, could
elevate extracellular ACh levels and augment long-term potentiation and learning in
AD model rats.15 A notable feature of cymserine analogs was the absence of
peripheral side effects associated with AChE inhibitors.15 Increasing evidence for the
involvement of BChE in AD supports it as a promising drug target for AD treatment.
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Figure 1. Chemical structures of ChE ligands and chalcone.
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Rivastigmine (Figure 1), a newer-generation inhibitor approved in 2000 under the
name of Exelon, with a dual inhibitory action on both AChE and BChE, has
demonstrated broad benefits across the severity of AD and also in patients with Lewy
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body variant of AD.16,
Compared with other commercial AChE inhibitors,
rivastigmine is a relative weak AChE and BChE inhibitor because of its specific
inhibitory mechanism of the carbamate structure encoded in rivastigmine as slow
substrate reacting covalently with the active site of the AChE and BChE.18 Previous
experimental and computational studies demonstrate that the carbamate structure of
rivastigmine is a necessary pharmacophore for in vitro and in vivo peculiar
pharmacological profiles which are benefited from the ability to inhibit BChE in
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addition to AChE.19, 20 Chalcones (trans-1,3-diphenyl-2-propen-1-ones, Figure 1), the
biogenetic precursors of flavonoids and isoflavonoids, are abundant in edible plants21.
They display a broad spectrum of pharmacological activities such as antioxidative,
anticancer, anti-inflammatory, antimalarial, antifungal, antilipidemic, and antiviral
activities.21-24 Some chalcone derivatives also exhibit potent activity against
amyloid-β aggregation and 5-lipoxygenase, suggesting that chalcone derivatives can
be considered as multifunctional agents for AD treatment.25
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In the present study, combining both the rivastigmine and chalcones, we designed,
synthesized, and evaluated chalcone-rivastigmine hybrids as inhibitors of AChE and
BChE. Their structure activity relationships (SAR) and mechanisms of
enzyme-inhibitor interactions were studied through molecular modeling technology.
Furthermore, the druggability of chalcone-rivastigmine hybrids was evaluated using
in silico ADMET prediction and SH-SY5Y cell line assay.
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2. Materials and methods
2.1. Chemistry
The high-resolution mass spectra were generated on an Bruker Agilent1290 /maXis
mass spectrometer with an ESI mass selective detector. NMR spectra were recorded
using TMS as the internal standard on a Bruker Avance III spectrometer at 400 (1H
NMR) and 101 or 151 (13C NMR) MHz. Melting points were determined on an
Stuart-smp40 Melt automated melting point instrument. The reactions were followed
by thin-layer chromatography (TLC) on glass-packed precoated silica gel plates and
visualized with a UV lamp. Flash column chromatography was performed using silica
gel (200–300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The purity
(≥ 95%) of the synthesized compounds was determined by HPLC (Table S1).
2.2. General procedure for the synthesis of intermediate compounds (M1-M14)
A suspension of ketone or aldehyde (0.044 mol), K2CO3/1.5H2O (0.785 eq, 0.035
mol) and K2CO3 (0.215 eq, 0.009 mol) in appropriate ethyl acetate was added pyridine
(0.144 eq, 0.006 mol). The mixture was stirred and heated to 70 ºC. Acyl chloride (1.5
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eq, 0.066 mol) in appropriate ethyl acetate was added dropwise. The reaction was
stirred at 70 ºC, and was monitored by TLC. Upon completion, an equal volume of
water was added to the mixture, stirred at 70 ºC for 1.5 h. The reaction was cooled
and the aqueous layer was separately washed 2x with 2% H2SO4 and water, then the
combined organics were dried over MgSO4, filtered, and concentrated in vacuo to
provide the target products ( M1-M14, Yield: 85%-98%).
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Scheme 1. Synthesis of compounds 1-8, 9-12, 21-24. Reagents and conditions: (a)
N,N-Dimethylcarbamoyl chloride (b) N-Ethyl-N-methylcarbamoyl Chloride (c)
Diethylcarbamyl Chloride (d) Diisopropylcarbamyl chloride, K2CO3, K2CO3/1.5H2O,
Pyridine, ethyl acetate, 70 ºC, 12-36 h; (e) 37% HCl, ethyl alcohol, 70 ºC,24-36 h; (f)
8% KOH, CH3OH, 70 ºC, 24-36 h.
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Scheme 2. Synthesis of compounds 13-20. Reagents and conditions: (a) N,N-
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Dimethylcarbamoyl chloride (b) N-Ethyl-N-methylcarbamoyl Chloride, K2CO3,
K2CO3/1.5H2O, pyridine, ethyl acetate, H2O, 70 ºC, 12-36 h; (c) 37% HCl, ethyl
alcohol, 70 ºC, 24-36 h.
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2.3. General procedure for the synthesis of B1 and B2
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M4 or M6 (2 mmol) was dissolved in 5 mL methanol, 4 mL 8% KOH solution was
added dropwise at room temperature. P-anisaldehyde in appropriate methanol was
added to the mixture slowly. The reaction was stirred at room temperature for 72 h.
After the completion of the reaction, excess solvent was evaporated in vacuo. Then
the solution was partitioned between 90 mL water and 90 mL methylene chloride. The
organic layer was washed 3x with water, and the combined organics were dried over
MgSO4, filtered, and concentrated in vacuo to provide a crude product which was
purified by flash column chromatography on silica gel (eluent: Ethyl
acetate/petroleum) to afford the desired product.
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2.3.1. (E)-1-(3-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (B1)
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Pale yellow solid, 45.9% Yield. M.P. 126.5–128.3 ºC. H NMR (400 MHz,
DMSO-d6) δ 9.76 (s, 1H), 7.84 (d, J = 8.7 Hz, 2H), 7.70 (s, 2H), 7.60 (d, J = 7.7 Hz,
1H), 7.44 (s, 1H), 7.36 (t, J = 7.9 Hz, 1H), 7.08-6.97 (m, 3H), 3.83 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 189.42, 161.81, 158.16, 144.30, 139.76, 131.23, 130.25,
127.77, 120.52, 120.14, 119.93, 115.02, 114.88, 55.84. HRMS: calcd for C16H14O3,
[M + Na]+, m/z 277.0835; found, m/z 277.0835; HPLC purity: 95.44%.
2.3.2. (E)-1-(4-hydroxyphenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (B2)
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Pale yellow solid, 35.4% Yield. M.P. 181.5–183.4 ºC. H NMR (400 MHz,
DMSO-d6) δ 10.35 (s, 1H), 8.05 (d, J = 8.7 Hz, 2H), 7.82 (d, J = 8.7 Hz, 2H), 7.77 (d,
J = 15.5 Hz, 1H), 7.65 (d, J = 15.5 Hz, 1H), 7.01 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.7
Hz, 2H), 3.82 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.50, 162.48, 161.58,
143.16, 131.51, 131.01, 129.81, 127.99, 120.02, 115.79, 114.82, 55.80. HRMS: calcd
for C16H14O3, [M + Na]+, m/z 277.0835; found, m/z 277.0836; HPLC purity: 98.04%.
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2.4. General procedure for the synthesis of compound 1-8
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M1~M8 (2 mmol) and p-hydroxybenzaldehyde (2.4 mmol) was dissolved in 10 mL
ethanol, 10 drops concentrated hydrochloric acid was added dropwise at room
temperature. The reaction was stirred at room temperature. The reaction was
monitored by TLC. After the completion of the reaction, excess solvent was
evaporated in vacuo. Then the solution was partitioned between 90 mL water and 90
mL methylene chloride. The organic layer was dried over MgSO4, filtered, and
concentrated in vacuo to provide a crude product which was purified by flash column
chromatography on silica gel (eluent: Ethyl acetate/petroleum) to afford the desired
product.
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2.4.1. (E)-5-(3-(4-hydroxyphenyl)acryloyl)-1,3-phenylene-bis(dimethylcarbamate)
(1)
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Yellow solid, 12.8% Yield. M.P. 170–172 ºC. H NMR (400 MHz, DMSO-d
6) δ 10.12 (s, 1H), 7.78 (d, J = 8.7 Hz, 2H), 7.76 (d, J = 2.2 Hz, 2H), 7.72
(s, 2H), 7.26 (s, 1H), 6.84 (d, J = 8.6 Hz, 2H), 3.01 (d, J = 56.5 Hz, 12H).
13C NMR (101 MHz, DMSO-d6) δ 187.90, 161.05, 154.05, 152.28, 145.97, 139.
64, 131.79, 126.29, 120.66, 118.63, 117.97, 116.14, 36.92, 36.50. HRMS: calcd
for C21H22N2O6, [M + H]+, m/z 399.1551; found, m/z 399.1555; HPLC purity:
98.98%.
2.4.2. (E)-5-(3-(4-hydroxyphenyl)acryloyl)-1,3-phenylene-bis(ethyl(methyl)-
carbamate) (2)
Yellow solid, 22.9% Yield. M.P. 150–152 ºC. 1H NMR (400 MHz, DMSO-d6) δ
10.14 (s, 1H), 7.77 (dd, J = 15.3, 9.2 Hz, 6H), 7.27 (t, J = 2.1 Hz, 1H), 6.85 (d, J = 8.6
Hz, 2H), 3.46 (dd, J = 13.8, 6.8 Hz, 2H), 3.37–3.30 (m, 2H), 2.99 (d, J = 52.8 Hz, 6H),
1.22 (t, J = 7.0 Hz, 3H), 1.12 (t, J = 7.0 Hz, 3H). 13C NMR (101 MHz, DMSO-d6) δ
187.72, 160.83, 157.75, 153.68, 152.34, 145.84, 139.89, 131.79, 129.79, 126.17,
120.89, 119.07, 118.38, 116.26, 115.67, 44.04, 34.42, 34.07, 13.52, 12.71. HRMS:
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calcd for C23H26N2O6, [M + H]+, m/z 427.1864; found, m/z 427.1864; HPLC purity:
97.76%.
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2.4.3. (E)-3-(3-(4-hydroxyphenyl)acryloyl)phenyl dimethylcarbamate (3)
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Yellow solid, 9% Yield. M.P. 153–155 ºC. H NMR (400 MHz, DMSO-d6) δ 10.10
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(s, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.88–7.84 (m, 1H), 7.76 (d, J = 8.7 Hz, 2H), 7.71 (d,
J = 1.1 Hz, 2H), 7.57 (t, J = 7.9 Hz, 1H), 7.41 (ddd, J = 8.0, 2.3, 0.9 Hz, 1H), 6.84 (d,
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J = 8.6 Hz, 2H), 3.08 (d, J = 9.3 Hz, 3H), 2.93 (d, J = 4.2 Hz, 3H). C NMR (101
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MHz, DMSO-d6) δ 188.48, 160.73, 154.36, 152.10, 145.41, 139.61, 131.64, 130.09,
126.91, 126.20, 125.59, 122.18, 118.69, 116.28, 36.81, 36.61. HRMS: calcd for
C18H17NO4, [M + H]+, m/z 312.1230; found, m/z 312.1235; HPLC purity: 99.63%.
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2.4.4. (E)-3-(3-(4-hydroxyphenyl)acryloyl)phenyl ethyl(methyl)carbamate (4)
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Pale yellow solid, 12.8% Yield. M.P. 118.9–121.5 ºC. H NMR (400 MHz,
DMSO-d6) δ 10.10 (s, 1H), 7.99 (d, J = 7.7 Hz, 1H), 7.84 (s, 1H), 7.76 (d, J = 8.5 Hz,
2H), 7.71 (s, 2H), 7.57 (t, J = 7.9 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 6.84 (d, J = 8.5
Hz, 2H), 3.47 (d, J = 6.8 Hz, 1H), 2.99 (d, J = 56.4 Hz, 3H), 1.26–1.09 (m, 4H). 13C
NMR (101 MHz, DMSO-d6) δ 188.31, 160.79, 153.92, 152.06, 145.41, 139.62,
132.95, 131.64, 130.08, 126.89, 126.19, 125.59, 122.11, 118.69, 116.28, 44.00, 34.40,
34.07, 13.55, 12.73. HRMS: calcd for C19H19NO4, [M + Na]+, m/z 348.1206; found,
m/z 348.1213; HPLC purity: 99.87%.
2.4.5. (E)-4-(3-(4-hydroxyphenyl)acryloyl)phenyl dimethylcarbamate (5)
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Yellow solid, 30% Yield. M.P. 188.5–170.3 ºC. H NMR (400 MHz, DMSO-d6) δ
10.10 (s, 1H), 8.17 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 3.3 Hz, 4H), 7.73 (d, J = 3.3 Hz,
2H), 7.70 (s, 1H), 7.29 (t, J = 8.7 Hz, 2H), 6.86 (t, J = 12.1 Hz, 2H), 3.07 (s, 3H), 2.94
(s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 188.26, 160.61, 155.29, 153.87, 144.91,
135.27, 131.48, 130.29, 126.25, 122.39, 118.83, 116.28, 36.81, 36.63. HRMS: calcd
for C18H17NO4, [M + Na]+, 334.1055; found, m/z 334.1053; HPLC purity: 99.83%.
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2.4.6. (E)-4-(3-(4-hydroxyphenyl)acryloyl)phenyl ethyl(methyl)carbamate (6)
Yellow solid, 59.7% Yield. M.P. 145.9–146.5 ºC. 1H NMR (400 MHz, DMSO-d6) δ
10.10 (s, 1H), 8.17 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 2.8 Hz, 4H), 7.73 (d, J = 4.0 Hz,
2H), 7.68 (d, J = 15.5 Hz, 1H), 7.30 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H),
3.50–3.34 (m, 2H), 2.99 (d, J = 50.0 Hz, 3H), 1.23–1.09 (m, 3H). 13C NMR (101 MHz,
DMSO-d6) δ 188.30, 160.61, 155.28, 153.40, 144.92, 135.25, 131.48, 130.29, 126.25,
122.38, 118.82, 116.28, 44.04, 34.41, 34.11, 13.53, 12.69. HRMS: calcd for
C19H19NO4, [M + H]+, m/z 326.1387; found, m/z 326.1388; HPLC purity: 98.37%.
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2.4.7. (E)-2-(3-(4-hydroxyphenyl)acryloyl)phenyl dimethylcarbamate (7)
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Yellow liquid, 11.1% Yield. H NMR (400 MHz, DMSO-d6) δ 10.13 (s, 1H), 7.66
(dd, J = 7.6, 1.6 Hz, 1H), 7.58 (dd, J = 10.7, 5.0 Hz, 3H), 7.47–7.32 (m, 2H), 7.23 (s,
1H), 7.11 (d, J = 15.9 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 2.92 (s, 3H), 2.77 (s, 3H). 13C
NMR (101 MHz, DMSO-d6) δ 191.34, 160.78, 157.77, 153.97, 149.48, 145.05,
131.15, 129.80, 125.99, 125.78, 124.19, 122.63, 119.23, 116.37, 36.70, 36.45. HRMS:
calcd for C18H17NO4, [M + Na]+, m/z 334.1050; found, m/z 334.1055; HPLC purity:
99.91%.
2.4.8. (E)-2-(3-(4-hydroxyphenyl)acryloyl)phenyl ethyl(methyl)carbamate (8)
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Pale yellow solid, 15.7% Yield. M.P. 109.5–111.6 ºC. H NMR (400 MHz,
DMSO-d6) δ 10.12 (s, 1H), 7.64 (t, J = 7.6 Hz, 1H), 7.58 (d, J = 8.1 Hz, 3H), 7.43 (d,
J = 15.9 Hz, 1H), 7.36 (t, J = 7.3 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.09 (dd, J = 15.9,
8.9 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H), 3.34–3.28 (m, 1H), 3.17 (q, J = 7.0 Hz, 1H),
2.84 (d, J = 47.8 Hz, 3H), 0.98 (dt, J = 55.8, 7.0 Hz, 3H). 13C NMR (151 MHz,
DMSO-d6) δ 191.23, 160.70, 153.50, 149.49, 145.18, 145.02, 133.66, 132.60, 131.18,
129.83, 125.97, 124.09, 122.57, 116.27, 43.81, 34.24, 33.94, 13.28, 12.50. HRMS:
calcd for C19H19NO4, [M + Na]+, m/z 348.1206; found, m/z 348.1207; HPLC purity:
99.92%.
2.5. General procedure for the synthesis of compound 9-12, 22, and 23
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A suspension of B1 or B2 (0.47 mmol), K2CO3/1.5H2O (0.369 mmol) and K2CO3
(0.101 mmol) in appropriate ethyl acetate was added 4 drops pyridine. The mixture
was stirred and heated to 70 ºC. Acyl chloride (0.705 mmol) in appropriate ethyl
acetate was added dropwise. Then the reaction was stirred at 70 ºC, the reaction was
monitored by TLC. Upon completion, an equal volume of water was added to the
mixture, stirred at 70 ºC for 1.5 h. The reaction was cooled and the aqueous layer was
separately washed 2x with 2% H2SO4 and water, then the combined organics were
dried over MgSO4, filtered, and concentrated in vacuo to give the desired product.
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2.5.1. (E)-3-(3-(4-methoxyphenyl)acryloyl)phenyl dimethylcarbamate (9)
Pale yellow solid, 98% Yield. M.P. 60–62 ºC. 1H NMR (400 MHz, DMSO-d6) δ
8.01 (d, J = 7.8 Hz, 1H), 7.87 (t, J = 11.6 Hz, 3H), 7.81–7.70 (m, 2H), 7.58 (t, J = 7.9
Hz, 1H), 7.42 (dd, J = 8.0, 1.5 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 3.83 (s, 3H), 3.09 (s,
3H), 2.94 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 188.57, 161.99, 154.35, 152.12,
144.92, 139.48, 131.42, 130.11, 127.72, 127.03, 125.67, 122.23, 119.74, 114.86,
55.85, 36.81, 36.60. HRMS: calcd for C19H19NO4, [M + H]+, m/z 326.1387; found,
m/z 326.1387; HPLC purity: 97.81%.
2.5.2. (E)-4-(3-(4-methoxyphenyl)acryloyl)phenyl dimethylcarbamate (10)
1
Pale yellow solid, 72.4% Yield. M.P. 110.2–112.1 ºC. H NMR (400 MHz,
DMSO-d6) δ 8.19 (d, J = 8.6 Hz, 2H), 7.85 (dd, J = 9.7, 6.1 Hz, 4H), 7.83–7.79 (m,
0H), 7.74 (t, J = 9.7 Hz, 0H), 7.31 (d, J = 8.6 Hz, 2H), 7.02 (t, J = 7.2 Hz, 2H), 3.83
(d, J = 2.9 Hz, 3H), 3.07 (s, 3H), 2.94 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ
188.30, 161.85, 155.38, 153.86, 144.42, 135.15, 131.28, 130.37, 127.78, 122.42,
119.88, 114.87, 55.85, 36.81, 36.64. HRMS: calcd for C19H19NO4, [M + Na]+, m/z
348.1206; found, m/z 348.1206; HPLC purity: 97.03%.
2.5.3. (E)-3-(3-(4-methoxyphenyl)acryloyl)phenyl ethyl(methyl)carbamate (11)
1
Yellow liquid, 81.5% Yield. H NMR (400 MHz, DMSO-d6) δ 8.01 (d, J = 7.8 Hz,
1H), 7.87 (t, J = 11.3 Hz, 5H), 7.80 (s, 0H), 7.75 (d, J = 15.5 Hz, 0H), 7.58 (t, J = 7.9
10

1
2
Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 8.7 Hz, 2H), 3.83 (s, 3H), 3.53–3.34 (m,
1H), 3.00 (d, J = 56.7 Hz, 3H), 1.23–1.11 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ
188.57, 161.94, 153.89, 152.08, 144.92, 139.49, 131.43, 130.12, 127.72, 127.07,
125.68, 122.17, 119.75, 114.87, 55.86, 44.00, 34.41, 34.07, 13.54, 12.73. HRMS:
calcd for C20H21NO4, [M + Na]+, m/z 362.1363; found, m/z 362.1365; HPLC purity:
95.99%.
3
4
5
6
7
8
2.5.4. (E)-4-(3-(4-methoxyphenyl)acryloyl)phenyl ethyl(methyl)carbamate (12)
Yellow liquid, 81.5% Yield. 1H NMR (400 MHz, DMSO-d6) δ 8.19 (d, J = 8.7 Hz,
2H), 7.90–7.83 (m, 2H), 7.81 (s, 1H), 7.73 (d, J = 15.5 Hz, 1H), 7.30 (t, J = 9.6 Hz,
2H), 7.03 (d, J = 8.8 Hz, 2H), 3.52–3.40 (m, 1H), 3.35 (d, J = 7.1 Hz, 1H), 3.06–2.90
(m, 3H), 1.23–1.10 (m, 3H). 13C NMR (101 MHz, DMSO-d6) δ 188.30, 161.85,
155.36, 153.51, 144.43, 135.14, 131.28, 130.38, 127.79, 122.41, 119.88, 114.87,
55.84, 44.04, 34.41, 34.11, 13.53, 12.69. HRMS: calcd for C20H21NO4, [M + H]+, m/z
340.1543; found, m/z 340.1548; HPLC purity: 95.47%.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.5.5. (E)-3-(3-(4-methoxyphenyl)acryloyl)phenyl diethylcarbamate (22)
1
Yellow liquid, 67% Yield. H NMR (400 MHz, DMSO-d6) δ 8.03 (d, J = 7.7 Hz,
1H), 7.89 (d, J = 8.6 Hz, 5H), 7.85 (s, 0H), 7.81 (s, 0H), 7.76 (d, J = 15.5 Hz, 0H),
7.58 (t, J = 7.9 Hz, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.03 (d, J = 8.6 Hz, 2H), 3.84 (s,
3H), 3.45 (dd, J = 13.1, 6.2 Hz, 2H), 3.26 (dd, J = 16.2, 7.5 Hz, 2H), 1.25 (d, J = 6.9
13
Hz, 6H), 1.14 (t, J = 7.1 Hz, 1H). C NMR (151 MHz, DMSO-d6) δ 188.61, 161.97,
153.65, 152.08, 144.95, 139.52, 131.45, 130.14, 127.74, 127.07, 125.71, 122.12,
119.77, 114.88, 55.87, 42.24, 13.54. HRMS: calcd for C21H23NO4, [M + H]+, m/z
354.1700; found, m/z 354.1700; HPLC purity: 96.00%.
2.5.6. (E)-3-(3-(4-methoxyphenyl)acryloyl)phenyl diisopropylcarbamate (23)
1
Brown liquid, 91.7% Yield. H NMR (400 MHz, DMSO-d6) δ 8.04 (d, J = 7.8 Hz,
1H), 7.89 (d, J = 8.8 Hz, 5H), 7.83 (d, J = 15.5 Hz, 2H), 7.76 (d, J = 15.5 Hz, 1H),
7.58 (t, J = 7.9 Hz, 1H), 7.40 (dd, J = 8.0, 1.6 Hz, 1H), 7.03 (d, J = 8.8 Hz, 2H), 4.03
11

13
1
2
(dt, J = 13.3, 6.8 Hz, 2H), 3.83 (s, 3H), 1.28 (d, J = 20.4 Hz, 14H). C NMR (101
MHz, DMSO-d6) δ 188.68, 161.97, 153.05, 151.96, 144.95, 139.55, 131.46, 130.15,
127.73, 127.03, 125.66, 121.93, 119.82, 114.89, 55.87, 46.76, 21.76, 20.65. HRMS:
calcd for C23H27NO4, [M + H]+, m/z 382.2013; found, m/z 382.2011; HPLC purity:
95.12%.
3
4
5
6
7
2.6. General procedure for the synthesis of 21 and 24
8
M9 or M10 (4 mmol) and p-hydroxybenzaldehyde (4 mmol) was dissolved in 20
mL ethanol, 15 drops concentrated hydrochloric acid was dropwised at room
temperature. The reaction was stirred at room temperature. The reaction was
monitored by TLC, after the completion of the reaction, excess solvent was
evaporated in vacuo. Then the solution was partitioned between 105 mL ethyl acetate
and 180 mL water. The organic layer was washed 2x with water, and the combined
organics were dried over MgSO4, filtered, and concentrated in vacuo to provide a
crude product which was purified by flash column chromatography on silica gel
(eluent: Ethyl acetate/petroleum) to afford the desired product.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.6.1. (E)-3-(3-(4-hydroxyphenyl)acryloyl)phenyl diethylcarbamate (21)
Brown solid, 16% Yield. M.P. 110.5–111.9 ºC. 1H NMR (400 MHz, DMSO-d6) δ
10.13 (s, 1H), 8.01 (d, J = 7.8 Hz, 1H), 7.84 (s, 1H), 7.74 (dd, J = 23.9, 12.5 Hz, 4H),
7.63–7.53 (m, 1H), 7.41 (dd, J = 8.0, 1.7 Hz, 1H), 6.85 (d, J = 8.5 Hz, 2H), 3.45 (dd, J
= 13.3, 6.4 Hz, 2H), 3.33 (dd, J = 13.9, 6.9 Hz, 2H), 1.23 (dd, J = 15.1, 8.4 Hz, 3H),
13
1.15 (dd, J = 14.8, 7.9 Hz, 3H). C NMR (101 MHz, DMSO-d6) δ 188.56, 160.76,
153.66, 152.06, 145.44, 139.65, 131.67, 130.11, 126.95, 126.22, 125.62, 122.07,
118.73, 116.30, 42.25, 42.00, 14.67, 13.74. HRMS: calcd for C20H21NO4, [M + H]+,
m/z 340.1543; found, m/z 340.1546; HPLC purity: 98.37%.
2.6.2. (E)-3-(3-(4-hydroxyphenyl)acryloyl)phenyl diisopropylcarbamate (24)
Yellow solid, 7.8% Yield. M.P. 153.1–155.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ
10.14 (s, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.74 (dd, J = 24.2, 12.6 Hz, 5H), 7.63–7.49 (m,
12

1
2
1H), 7.39 (dd, J = 8.0, 1.6 Hz, 1H), 6.85 (d, J = 8.6 Hz, 2H), 4.01 (dq, J = 13.3, 6.6
Hz, 2H), 1.25 (t, J = 21.9 Hz, 13H). 13C NMR (101 MHz, DMSO-d6) δ 188.63,
160.76, 153.06, 151.95, 145.45, 139.67, 131.68, 130.13, 126.92, 126.20, 125.58,
121.88, 118.75, 116.30, 46.75, 21.72, 20.60. HRMS: calcd for C22H25NO4, [M + H]+,
m/z 368.1856; found, m/z 368.1860; HPLC purity: 99.34%.
3
4
5
6
7
2.7. General procedure for the synthesis of 13-20
8
M11~M14 (4 mmol) and 3'-Hydroxyacetophenone or 4'-Hydroxyacetophenone
(4mmol) was dissolved in 20 mL ethanol, 20 drops concentrated hydrochloric acid
was added dropwise at room temperature. The reaction was stirred at room
temperature. The reaction was monitored by TLC, after the completion of the reaction,
excess solvent was evaporated in vacuo. Then the solution was partitioned between
100 mL water and 150 mL ethyl acetate. The organic layer was dried over MgSO4,
filtered, and concentrated in vacuo to provide a crude product which was purified by
flash column chromatography on silica gel (eluent: Ethyl acetate/petroleum) to afford
the desired product.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.7.1. (E)-4-(3-(3-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl ethyl(methyl)
carbamate (13)
Yellow-green solid, 9% Yield. M.P. 117.9–199.6 ºC. 1H NMR (400 MHz, DMSO-d6)
δ 9.82 (d, J = 9.3 Hz, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.83 (d, J = 15.6 Hz, 1H), 7.72 (d,
J = 15.6 Hz, 1H), 7.64 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 1.9 Hz, 1H), 7.38 (t, J = 7.9
Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.06 (dd, J = 7.9, 2.2 Hz, 1H), 3.44 (dd, J = 14.1,
7.0 Hz, 1H), 3.31 (d, J = 7.0 Hz, 1H), 2.97 (d, J = 49.5 Hz, 3H), 1.23–1.08 (m, 3H).
13C NMR (101 MHz, DMSO-d6) δ 189.01, 157.71, 153.25, 152.99, 142.96, 139.01,
131.61, 129.90, 129.79, 122.28, 121.89, 120.24, 119.54, 114.60, 43.50, 33.89, 33.57,
13.03, 12.21. HRMS: calcd for C19H19NO4, [M + H]+, m/z 326.1387; found, m/z
326.1390; HPLC purity: 96.48%.
2.7.2. (E)-4-(3-(3-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl dimethyl
13

1
2
carbamate (14)
Yellow solid, 9% Yield. M.P. 144.2–146.1 ºC. 1H NMR (400 MHz, DMSO-d6) δ
9.80 (s, 1H), 7.88 (s, 1H), 7.70 (d, J = 15.9 Hz, 4H), 7.48 (s, 2H), 7.38 (t, J = 7.9 Hz,
1H), 7.21 (dd, J = 7.8, 2.0 Hz, 1H), 7.07 (dd, J = 8.1, 2.4 Hz, 1H), 3.09 (s, 3H), 2.94
(s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 189.48, 158.21, 154.12, 153.49, 143.48,
139.49, 132.14, 130.43, 130.31, 122.81, 122.36, 120.76, 120.07, 115.08, 36.79, 36.61.
HRMS: calcd for C18H17NO4, [M + Na]+, m/z 334.1050; found, m/z 334.1056; HPLC
purity: 98.14%.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.7.3. (E)-3-(3-(3-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl dimethyl
carbamate (15)
1
Yellow liquid, 9% Yield. H NMR (400 MHz, DMSO-d6) δ 9.83 (s, 1H), 7.90 (d, J
= 15.6 Hz, 1H), 7.71 (d, J = 16.2 Hz, 4H), 7.48 (s, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.21
(d, J = 7.8 Hz, 1H), 7.11–7.02 (m, 1H), 3.54–3.37 (m, 2H), 2.99 (d, J = 54.8 Hz, 3H),
1.23–1.12 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 189.52, 158.22, 154.43, 152.28,
143.31, 139.37, 136.54, 130.31, 130.17, 126.78, 124.66, 123.49, 121.96, 120.86,
120.15, 115.15, 36.81, 36.61. HRMS: calcd for C18H17NO4, [M + Na]+, m/z 334.1050;
found, m/z 334.1056; HPLC purity: 97.42%.
2.7.4. (E)-3-(3-(3-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenylethyl-(methyl)
carbamate (16)
Yellow solid, 7.6% Yield. M.P. 90.1–91.9 ºC. 1H NMR (400 MHz, DMSO-d6) δ
9.81 (s, 1H), 7.91 (d, J = 8.6 Hz, 2H), 7.84 (d, J = 15.6 Hz, 1H), 7.72 (d, J = 15.6 Hz,
1H), 7.64 (d, J = 7.7 Hz, 1H), 7.49–7.45 (m, 1H), 7.38 (t, J = 7.9 Hz, 1H), 7.22 (d, J =
8.6 Hz, 2H), 7.08–7.05 (m, 1H), 3.06 (s, 4H), 2.92 (d, J = 8.1 Hz, 4H). 13C NMR (101
MHz, DMSO-d6) δ 189.48, 158.22, 153.96, 152.22, 143.36, 139.34, 136.53, 130.32,
130.17, 126.85, 124.70, 123.41, 122.00, 120.87, 120.19, 115.13, 43.97, 34.39, 34.06,
13.55, 12.76. HRMS: calcd for C19H19NO4, [M + Na]+, m/z 348.1206; found, m/z
348.1206; HPLC purity: 97.18%.
14

1
2
2.7.5. (E)-3-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl dimethyl
carbamate (17)
1
3
Yellow-green solid, 12.8% Yield. M.P. 164.7–166.5 ºC. H NMR (400 MHz,
4
DMSO-d6) δ 10.44 (s, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 15.6 Hz, 1H), 7.71 (d,
J = 14.0 Hz, 2H), 7.45 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 5.6 Hz, 1H), 6.90 (d, J = 8.6
Hz, 2H), 3.08 (s, 3H), 2.94 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 187.46, 162.76,
154.45, 152.24, 142.25, 136.75, 131.77, 130.13, 129.49, 126.75, 124.45, 123.32,
121.80, 115.85, 36.78, 36.59. HRMS: calcd for C18H17NO4, [M + Na]+, m/z 334.1050;
found, m/z 334.1055; HPLC purity: 97.39%.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.7.6. (E)-3-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl-ethyl(methyl)
carbamate (18)
Pale green solid, 8.3% Yield. M.P. 149.5–151.3 ºC. 1H NMR (400 MHz, DMSO-d6)
δ 10.43 (s, 1H), 8.10 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 15.6 Hz, 1H), 7.74–7.64 (m, 3H),
7.45 (t, J = 7.9 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 6.90 (d, J = 8.7 Hz, 2H), 3.46 (d, J =
13
7.2 Hz, 1H), 3.35 (s, 1H), 3.07–2.90 (m, 3H), 1.13 (t, J = 7.1 Hz, 3H). C NMR (101
MHz, DMSO-d6) δ 187.48, 162.76, 154.09, 152.21, 142.27, 136.76, 131.77, 130.13,
129.49, 126.73, 124.51, 123.32, 121.81, 115.85, 43.97, 34.38, 34.06, 13.56, 12.76.
HRMS: calcd for C19H19NO4, [M + Na]+, m/z 348.1206; found, m/z 348.1209; HPLC
purity: 96.53%.
2.7.7. (E)-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl-dimethyl
carbamate (19)
Pale green solid, 23% Yield. M.P. 154.2–155.8 ºC. 1H NMR (400 MHz, DMSO-d6)
δ 10.43 (s, 1H), 8.09 (d, J = 8.6 Hz, 2H), 7.98–7.82 (m, 3H), 7.69 (d, J = 15.6 Hz, 1H),
7.21 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.6 Hz, 2H), 3.06 (s, 3H), 2.93 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 187.87, 162.66, 154.15, 153.29, 142.35, 132.36, 131.64,
130.22, 129.67, 122.75, 122.33, 115.85, 36.80, 36.61. HRMS: calcd for C18H17NO4,
[M + Na]+, m/z 334.1050; found, m/z 334.1053; HPLC purity: 98.54%.
15

1
2
2.7.8. (E)-4-(3-(4-hydroxyphenyl)-3-oxoprop-1-en-1-yl)phenyl-ethyl(methyl)
carbamate (20)
1
3
Yellow-green solid, 14.9% Yield. M.P. 150.5–152.0 ºC. H NMR (400 MHz,
4
DMSO-d6) δ 10.43 (s, 1H), 8.07 (t, J = 13.0 Hz, 2H), 7.88 (dd, J = 15.6, 10.6 Hz, 3H),
7.69 (d, J = 15.5 Hz, 1H), 7.21 (d, J = 8.1 Hz, 2H), 7.06–6.82 (m, 2H), 3.63–3.14 (m,
3H), 2.95 (dd, J = 46.9, 17.1 Hz, 3H), 1.30–0.96 (m, 4H). 13C NMR (101 MHz,
DMSO-d6) δ 187.54, 162.66, 153.90, 153.28, 142.36, 132.35, 131.64, 130.22, 129.63,
122.75, 122.32, 115.85, 34.40, 34.09, 13.54, 12.73. HRMS: calcd for C19H19NO4, [M
+ Na]+, m/z 348.1206; found, m/z 348.1209; HPLC purity: 98.57%.
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.8. Biology
2.8.1. In vitro inhibition of hAChE and hBChE
Acetylcholinesterase (from human recombinant, C1682,), butyrylcholinesterase
(from human serum, B4186), 5,5’-dithiobis (2-nitrobenzoic acid) (DTNB, D8130),
acetylthiocholine iodide (ATC, A5751,), and S-Butyrylthiocholine iodide (BTC,
B3253) were purchased from Sigma Chemical Co.
Ellman assay was employed to measure the inhibition effect of designed
compounds on hAChE and hBChE activity.26 Compounds were diluted with reaction
buffer (phosphate buffer, 0.08 M Na2HPO4 and 0.02 M NaH2PO4, pH 8.0). 0.004
U/mL hAChE or 0.002 U/mL hBChE was incubated with each compound at 37 ºC for
15 min. Then 5,5’-dithiobis (2-nitrobenzoic acid) was added to a final concentration
of 50 µM. After mixing with ATC for hAChE, or BTC for hBChE at a final
concentration of 50 µM, the complex was scanned immediately at 412 nm using
Thermo Scientific Varioskan Flash. The average absorbance variation for 5
consecutive minutes was calculated. The inhibitory rate for each compound was
calculated by comparing the average absorbance of samples containing compounds
with that absence of test compound. The dose-response curve and concentration
needed to inhibit 50% enzyme activity (IC50) was calculated in Graph Pad version
6.01 by fitting replicates with the log (inhibitor) vs normalized response variable
slope equation.
16

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2
2.8.2. Kinetic characterization of hAChE and hBChE inhibition
3
The kinetic studies of compound 3 against hAChE and hBChE were performed.
After incubating 0.004 U/mL hAChE or 0.002 U/mL hBChE with diluted compound
at three concentrations range from 100 nM to 900 nM at 37 ºC for 15 min. Then
DTNB was added as usual, and substrate was added finally with six serial diluted
concentrations range from 0.125 mM to 1 mM. Enzyme activities were determined
immediately at 412 nm using Thermo Scientific Varioskan Flash for five minutes at
one min interval. Kinetic characterizations of the hydrolysis of substrate by hAChE
and hBChE were analyzed by estimating kinetic parameters Km, Vmax in
Michaelis-Menten plot in Graph Pad. Then the data were plotted on a
Lineweaver–Burk diagram to reveal the mechanism of inhibition.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.8.3. In vitro cell viability assay
Cytotoxicity was measured with an MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diph
enyl-2-H-tetrazolium bromide) assay protocol.27, 28 5 × 103 SH-SY5Y cells were
seeded in a 96-well plate with 100 µL of culture medium. After incubation f
or 16-18 hours, the culture medium were changed to 100 µL indicated medium
in the presence of compounds at the concentration of 6.25 µM, 12.5 µM, 25
µM, 50 µM and 100 µM, respectively. After incubation for 48 hours, the cells
were further incubated with 20 µL of 2.5 mg/mL MTT for 4 hours at 37 ºC
in a humidified incubator with 5% CO2. After dissolution of the formazan dy
e in 120 µL of DMSO was added to the cells, and the absorbance was measu
red at 490 nm by using EnSpire-2300 Multimode Reader (PerkinElmer).
2.8.4. Antioxidation activity assay
Intracellular ROS was stimulated by tert-Butyl hydroperoxide (t-BHP) and
measured with the fluorescent probe (2’,7’-dichlorofluorescein diacetate
(DCFH-DA).28 For assays, SH-SY5Y cells were cultured in 24-well plates at a
seeding density of 5×104 cells per well. After 16-18 h, the cells were treated with
17

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2
compound 3 and curcumin. After 24 h of treatment with the compounds, the cells
were washed with PBS and then incubated with 5 µM DCFH-DA and 100 µM t-BHP
in PBS at 37 ºC in 5% CO2 for 30 min. After the cells were washed with PBS and
resuspended, the fluorescence of the cells from each well was measured at 488 nm
excitation, and 525 nm emission, with a Flow Cytometer (CytoFLEX, Beckman
Coulter). Results are expressed as a percentage of the sample average divided by the
control group data, calculated as follows: (FIsample-FIblank) / (FIcontrol-FIblank) ×
100%.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.9. Molecular modeling
2.9.1. Molecular docking
The crystal structures of the hAChE (PDB ID: 4EY7)29 and hBChE (PDB ID:
4TPK)30 were derived from the RCSB Protein Data Bank. The resolution of hAChE
and hBChE structures was 2.35 and 2.7 Å, respectively. The structures of compound 3,
donepezil, and 3F9 were drawn using ISIS draw and optimized based on MMFF94
force field using MOE 2010.10 (Chemical Computing Group Inc.).31 Two protein
structures were preprocessed (i.e., protonated, removed water, and then minimized
with the CHARMm force field, etc.) with the “Protein Preparation Protocol” in the
Discovery Studio 3.5 (DS 3.5, Accelrys Inc.). The native ligand in the crystal structure
was used to define the binding site. The docking program CDOCKER encoded in DS
3.5 was applied to identify the potential binding of compound 3 to the hAChE and
hBChE. Other CDOCKER parameters were set to default values. During the docking
process, 20 poses were retained. After running CDOCKER, the poses were visually
inspected, and the most suitable docking pose was selected on the basis of the score
and interactions with key residues of the active site of hAChE and hBChE. The
complexes of hAChE and hBChE and compound 3 acquired from the docking
experiments were used as the initial coordinates for the subsequent molecular
dynamics (MD) simulations.
2.9.2. Molecular dynamics simulations
18

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2
The proteins were assigned with the AMBER ff99SB force field32 Parameters for
compound 3 were obtained from the ANTECHAMBER module using the Generalized
Amber Force Field (GAFF)33 with the RESP charge-fitting procedure with input from
HF/6-31G* calculations made with the Gaussian 09 package.34 All hydrogen atoms of
the protein were added using the tleap module, considering ionizable residues set at
their default protonation states at a neutral pH. The systems were solvated in a TIP3P
water box with a minimum distance of 10 Å between any protein atom and the edge
of the box. 20 ns MD simulations were performed in AMBER 12 and the processes of
MD simulations were taken from our previously studies.34-36 After MD simulation,
100 snapshots were extracted from the last 2 ns of the trajectory at intervals of 15 ps.
The MM-GBSA method in the AMBER 12 suite was used to calculate the binding
free energies.37, 38
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
2.9.3. In silico pharmacokinetic properties of the active compounds
ADME/T properties, including the absorption, solubility, BBB, hepatotoxicity, CYP
2D6, alogP, and PSA of the 13 actives, have been evaluated in silico through DS 3.5.
3. Results and discussion
3.1. Chemistry
A series of chalcone-rivastigmine hybrids were prepared by using previously
published methods.39-41 The synthetic routes for the target compounds are listed in
Schemes 1 and 2. Various corresponding hydroxyphenylcetone (A1, A2, A3, and A4)
or hydroxy benzaldehyde (C1 and C2) were first acylated by reacting with
carbamoylchloride in the presence of K2CO3, K2CO3/1.5H2O, and catalyst pyridine to
afford the intermediate compounds (M1~M14), then the target compounds were
obtained from M1-M14 by using Claisene-Schmidt condensation.
As shown in Scheme 1, M1~M10 reacted with p-hydroxy benzaldehyde to provide
the target compounds 1-8, 21, and 24. M4 and M6 reacted with
p-methoxybenzaldehyde to generate B1 and B2, which was further acylated to afford
the target compounds 9-12, 22, and 23. Target compounds 13-20 were synthesized
19

1
2
with M11~M14, corresponding hydroxyphenylacetone by the same aldol
condensation procedure (Scheme 2).
3
4
3.2. Inhibition of hAChE and hBChE
5
All targeted compounds were evaluated in vitro for their inhibition of hBChE and
hAChE using Ellman assay.26 The results are depicted in Supplementary Figure S1.
The 13 of 26 compounds showed > 50% inhibition activity against hBChE at 10 µM.
The IC50 values of the 13 active compounds were further characterized (Table 1 and
Figure S2). Rivastigmine was used as a positive control with an IC50 value of 0.38 µM.
Compound 3 is the most potent inhibitor of hBChE with an IC50 of 0.36 µM, which is
comparable or slightly better than that of rivastigmine.
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Analyzing the SAR, it is well established that several structural elements determine
the hBChE inhibitory activity of chalcone-rivastigmine hybrids as follows: (i) the
introduction of 3-substituted carbamate in A ring could increase inhibitory activity
against hBChE, while 4- or 5-substituted carbamate will lose or decrease the activity
(1, 2, 5, 6, 11, and 12, Table 1); (ii) the 5- or 6-substituted carbamate in B ring will
keep the activity against hBChE (15-18); (iii) simultaneously 3- and 5-substituted
carbamates in A ring of chalcone will lose the inhibitory activity (1-2); and (iv) the
alkyl substituent on the carbamoyl nitrogen strongly affects the affinity profile
(dimethyl > bis(ethyl(methyl)) > diethyl), and the most potent inhibitors were methyl
derivatives (3, 9, 15, 17, and 19). Moreover, compared with 9 and 11, compounds B1
and B2 (not chalcone-rivastigmine hybrids analogues) did not exhibit inhibition
against hBChE, demonstrating that carbamate in our designed compounds is
necessary for hBChE.
The results at hAChE had a potency trend dissimilar to that observed at hBChE.
Only 3-substituted dimethylcarbamate in A ring of chalcone exhibited inhibitory
activity against hAChE (3 and 9). The bis(ethyl(methyl) carbamate and diethyl
carbamate derivatives will lose the potency, which are good in agreement with
previous studies. Similar with hBChE, simultaneously 3- and 5-substituted
carbamates in A ring of chalcone lost the inhibitory activity against hAChE (1-2). The
20

1
2
3
4
5
6
7
most potent hAChE inhibitor is compound 3 with an IC50 of 0.872 µM, which is
approximately 3-fold better than that of rivastigmine.
Consequently, similar with rivastigmine, the new compound 3 with a dual
inhibitory action on both hAChE and hBChE might show a better therapeutic profile
in AD and related dementia.19
Table 1. Inhibitory activity of compounds against hAChE and hBChE.
8
IC50 (µM)a
hAChE
Compounds
R1
R2
R3
R4
R5
R6
hBChE
ND
—H
—H
—H
—H
—H
—H
—H
—H
—H
—H
—OH
—OH
—OH
—OH
—OH
—OH
—H
—H
—H
—H
—H
—H
ND
ND
1
2
3
4
5
6
ND
0.36 0.01
4.37 0.38
ND
—H
—H
—H
—H
0.87 0.19
ND
—H
—H
ND
ND
ND
—H
—H
—H
—H
—H
—OH
—OH
—H
—H
ND
ND
ND
7
8
—H
—H
5.97 0.08
—H
—H
—H
—H
—H
—H
—OCH3
—OCH3
—OCH3
—H
—H
—H
8.05 0.54
ND
3.37 0.50
ND
9
—H
10
11
—H
ND
3.93 0.56
21

—H
—H
—H
—H
—H
—OCH3
—H
—H
ND
ND
7.36 0.98
5.60 0.37
12
13
—OH
—H
—H
—H
—H
—H
—H
—OH
—OH
—OH
—H
—H
—H
—H
—H
—H
—H
—H
—H
ND
ND
ND
ND
ND
ND
14
15
16
17
18
—H
—H
—H
—H
1.91 0.27
2.87 0.19
2.19 0.55
2.52 0.54
—H
—OH
—OH
—H
—H
—H
—H
—H
—OH
—OH
—H
—H
—H
—H
ND
ND
4.48 0.50
6.08 0.78
19
20
—H
—H
—H
—H
—H
—H
—OH
—H
—H
ND
ND
ND
ND
21
22
—OCH3
—H
—H
—H
—H
—OCH3
—H
ND
ND
23
—H
—H
—OH
—H
ND
ND
24
—H
—H
—OH
—H
—H
—H
—H
—OCH3
—OCH3
—H
—H
ND
ND
ND
ND
B1
B2
—OH
Rivastigmine
3.12 0.46
0.38 0.02
b
1
2
3
4
5
aIC50 values are indicated as the mean±SD (standard error) of three independent experiments. bRivastigmine:
a positive control compound. ND: not determined (because the inhibitory activities were too weak to permit
an IC50 determination).
3.3. Kinetic evaluation of compound 3
22

1
2
3
4
5
6
7
8
To obtain the mechanism of hAChE and hBChE inhibition, kinetic experiments
were performed. As shown in Figure 2A and 2C, the lines intersect at the negative
x-axis into a same point, and Vmax decreases as the concentration of compound 3
increases. The Lineweaver–Burk plots suggest that 3 is a typically non-competitive
for hAChE and hBChE. The mechanism of action was also confirmed by plotting
substrate-velocity curves in the presence of several concentrations of compound 3
(Figure 2B and 2D). The kinetic constant (Ki) of hAChE and hBChE were estimated
to be 0.52 0.04 and 0.16 0.01 µM.
9
10
Figure 2. Kinetic studies on the mechanism of hAChE and hBChE inhibition by
compound 3. Overlaid Lineweaver−Burk reciprocal plots of the hAChE (A) and
hBChE (C) initial velocity at increasing substrate in the absence and presence of
compound 3. Substrate-velocity plots in the presence of several concentrations of
compound 3 (B) for hAChE and (D) for hBChE.
11
12
13
14
15
16
17
18
19
3.5. Characteristic binding patterns of compound 3
The binding modes of compound 3 with hAChE and hBChE were investigated by
molecular docking. The native ligands, donepezil and 3F9, were redocked back to the
binding sites of hAChE and hBChE , and the root-mean-square deviations (RMSD) of
23

1
2
the redocked and native ligand poses were 0.39 and 0.46 Å (Figure S3), indicating
that CDOCKER was a capable docking method for our complex systems. The
docking scores of compound 3 with hAChE and hBChE are –26.7 and –36.4 kcal/mol,
respectively. Subsequently, the stability and energy profile of the docking poses of
compound 3 were investigated through MD simulations and the calculations of
binding free energies. Figure 3A shows the time dependence of the RMSD values for
structure backbone atoms of two enzymes and compound 3 over the production phase
of simulation. The RMSD values of simulation converged after ~2 ns, indicating that
the system is stable and equilibrated. The RMSD values of ligand compared docking
pose is swinging within 0.75 Å, illustrating that the docking pose is reliable.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
The detailed binding modes of compound 3 and hAChE and hBChE were revealed
from the conformation clustering analysis. As shown in Figure 3B and 3C, the
carbamate ester of compound 3 inserted the catalytic triad of hAChE (SER203 and
HIS447) and hBChE (SER198 and HIS438). Similar with rivastigmine, this binding
direction of the carbamate ester will be hydrolyzed, resulting in hAChE and hBChE
inhibition.18, 42 Compared with hBChE, the compound 3 was surrounded with big and
hydrophobic side chains of the binding pocket of hAChE (e.g., PHE338, PHE295,
PHE297, TRP86, and TRP286, Figure 3B), suggesting that steric hindrance is not
negatively affected during lead optimizations. The observed results from the binding
pockets of hAChE and hBChE are consistent with our SAR analysis results. For
hBChE, compound 3 can form two hydrogen bonds with the side chains of GLY117
and THR120 (Figure 3C). Superimposition of compound 3 and hAChE and hBChE
suggests that compound 3 occupies the catalytic binding pocket in a pattern similar to
that of rivastigmine (Figure 3D).18
24

1
2
Figure 3. Stability properties of the simulation systems and binding model of
compound 3 with hAChE and hBChE. (A) RMSD plots for the backbone atoms and
compound 3 during 20 ns MD simulations; the detailed bind modes of compound 3
with (B) hAChE and (C) hBChE; (D) superposition of compound 3 in the binding
pockets of hAChE (cyan) and hBChE (green). Hydrogen bonds are shown by red
dashed lines. The cutoff value for the formation of a hydrogen bond is 3.5 Å.
3
4
5
6
7
8
9
The MM-GBSA method was used to calculate the binding free energies and to gain
information on the different components of interaction energy that contribute to
compound 3 binding for hAChE and hBChE. Detailed results are shown in Table 2.
Table 2 indicates that van der Waals (∆Evdw) and electrostatic components (∆Eele) play
key roles in compound 3 binding for both hAChE and hBChE. The van der Waals
contribution is approximately 1.6- and 1.7-fold greater than the electrostatic
component for hAChE and hBChE, respectively. Non-polar component to solvation
(∆Enonpol,solv) values are also favorable for compound 3 binding (e.g., –5.42
kcal/molfor hAChE and –8.38 kcal/mol for hBChE), while polar component to
solvation (∆Epol,solv) does not favor compound 3 binding (Table 2). The binding free
10
11
12
13
14
15
16
17
18
19
energy of compound 3-hBChE is higher than that of compound 3-hAChE, which is
25

1
2
consistent with our bioassay results (Table 1).
Table 2. Binding free energy for compound 3 based on MM-GBSA method.
Binding free energy (Kcal/mol) (SEM)
Energy terms
compound 3-hAChE
–40.99 (4.95)
–25.60 (4.81)
37.26 (4.64)
compound 3-hBChE
–38.33 (4.23)
–22.68 (3.79)
29.82 (3.49)
a
∆Evdw
b
∆Eele
c
∆Epol,solv
d
–5.42 (0.58)
–8.38 (0.31)
∆Enonpol,solv
e
–66.59 (8.06)
31.84 (4.20)
–57.01 (7.28)
24.43 (2.97)
∆Ggas
f
∆Gsolv
g
–34.75 (5.03)
–36.57 (5.17)
∆Gbind
3
4
aNon-bonded van der Waals; bNon-bonded electrostatics; cPolar component to solvation; dNon-polar
e
f
component to solvation; Total gas phase energy; Sum of nonpolar and polar contributions to solvation;
gFinal estimated binding free energy calculated from the terms above. Standard errors of the mean are given
in parentheses.
5
6
7
8
Energy decomposition analysis was conducted to identification of key residues
contributing to binding affinity. Generally, if the interaction energy between the
residue and the ligand is lower than –1 kcal/mol, those residues are considered to be
hot residues.2, 27 As shown in Figure 4, the hot residues of hAChE are TRP86,
TYR124, VAL294, PHE338, and TYR341, while the hot residues of hBChE are
TRP82, GLY116, GLY17, SER198, TRP231 TYR332, and PHE329. The hot residues
from decomposition results are consistent with the binding mode analyses (Figure 3B
and 3C), which provides a theoretical basis for further lead optimization.
9
10
11
12
13
14
15
26

1
2
3
4
5
6
−residue interaction energies from MM
Figure 4. Ligand
-GBSA energy
decomposition for compound 3-hAChE (A) and compound 3-hBChE (B). Delta
Gsubtotal represents total estimated binding free energy for each residue. Delta Gele
represents non-bonded electrostatics interactions. Delta Gvdw represents non-bonded
van der Waals interactions.
7
8
3.6. Cell viability assay on the SH-SY5Y cell Line
9
A preliminary toxicity of the 13 chalcone-rivastigmine hybrids was assessed using
cell viability assays on the SH-SY5Y cell line. The results are listed in Supplementary
Table S2. The most active compound 3 exhibited 96.66% and 94.62% cell viability at
12.5 and 25 µM, suggesting that compound 3 did not significantly reduce cell viability.
In general, the cell toxicity profile of compound 3 roughly matches that of
rivastigmine, with higher cytotoxicity at the highest tested concentration (50 and 100
µM, Figure 5). Compared to the drug rivastigmine, the similar potency, lack of
toxicity, and new chemotype of compound 3 suggests that it is worthy of further
development.
10
11
12
13
14
15
16
17
27

1
2
3
Figure 5. Cell viability in SH-SY5Y cells for compound 3 and rivastigmine. Data
correspond to the mean SEM of different experiments performed in triplicate. *p <
0.05, **p < 0.01, and ***p < 0.001 versus control.
4
5
6
3.7. Compound 3 reduces the production of oxidative stress in SH-SY5Y cell lines
SH-SY5Y cells were incubated with compound 3 in 5 µM and 1 µM concentrations.
The cells were also incubated with curcumin (a known antioxidant) in the same
concentration series. Figure 6 shows that the ROS is significantly inhibited by
compound 3 (1 and 5 µM, respectively), which is superior to curcumin in the same
concentration.
7
8
9
10
11
12
13
Figure 6. Compound 3 inhibits ROS in SH-SY5Y cell lines. The results are
represented in the percentage of control cells. The untreated cells were used as
negative control, and curcumin was used as a positive control.
14
15
28

1
2
3.8. In silico ADME/T properties of active compounds
3
The ADME/T properties of 13 hBChE inhibitors were assessed using DS 3.5.
Compared with rivastigmine, most of these inhibitors satisfied ADMET rules defined
in ADMET module of DS 3.5, especially for compound 3. The detailed results and
comparisons can be found in Table 3. As shown in Table 3, compound 3 may cross the
blood-brain barrier (BBB). Moreover, active compounds don’t contain problematic
substructures suggested from Pan Assay Interference Compounds (PAINS).43 All of
these results suggested that the compound 3 designed in the present study provided a
valuable alternative for AD and related dementia treatment.
4
5
6
7
8
9
10
11
Table 3. In silico predicted ADMET properties of the 13 compounds.a
Compounds
Absorptionb Solubilityc
BBBd
-0.165
-0.058
-0.058
0.092
CYP2D6e Hepatotoxicf AlogP98g
PSAh
67.699
67.699
67.699
55.814
55.814
55.814
67.699
67.699
67.699
67.699
67.699
67.699
67.699
32.935
0
0
0
0
0
0
0
0
0
0
0
0
0
0
-3.763
-3.972
-4.009
-4.257
-4.459
-4.417
-3.929
-3.758
-3.966
-3.758
-3.966
-3.726
-3.929
-3.171
-4.182
-5.317
-5.016
-6.217
-6.346
-6.333
-5.619
-3.811
-4.946
-3.509
-4.645
-4.168
-5.304
-2.499
-3.025
-3.174
-2.444
-3.381
-3.875
-2.891
-3.070
-2.520
-2.669
-2.335
-2.484
-2.041
-2.191
-1.324
3.429
3.778
3.778
3.655
4.003
4.003
3.778
3.429
3.778
3.429
3.778
3.429
3.778
2.599
3
4
8
9
0.200
11
12
13
15
16
17
18
19
20
0.200
-0.058
-0.165
-0.058
-0.165
-0.058
-0.165
-0.058
0.128
Rivastigminei
b
12
13
14
15
16
17
18
19
aADMET properties were calculated using DS 3.5. Predicted human intestinal absorption level (0: Good
absorption; 1: Moderate absorption; 2: Low absorption; 3: Very low absorption). cPredicted aqueous
solubility level (< -8.0: Extremely low; -8.0 to -6.0: No, very low, but possible; -6.0 to -4.0: Yes, low; -4.0 to
-2.0: Yes, good; -2.0 to 0.0: Yes, optimal; 0.0 < : No, too soluble). dPredicted Blood brain barrier (BBB, ≥ 0.7:
Very high penetrant; 0 to 0.7: High penetrant; -0.52 to 0: Medium penetrant; ≤ -0.52: Low penetrant).
ePredicted cytochrome P450 2D6 enzyme inhibition (CYP2D6, < 0.162: Non-inhibitor; ≥ 0.162: Inhibitor).
fPredicted hepatotoxicity (< -0.409: Nontoxic; ≥ -0.409: Toxic). AlogP98: Atom-based LogP. PSA: Fast
polar surface area. iRivastigmine: a positive control compound.
g
h
20
21
22
4. Conclusion
In conclusion, a novel series of chalcone-rivastigmine hybrids were designed,
29