Structure–activity relationship investigation of coumarin–chalcone hybrids with diverse side-chains as acetylcholinesterase and butyrylcholinesterase inhibitors

Article abstract of DOI:10.1007/s11030-018-9839-y

Chalcones containing tertiary amine side-chains have potent activity as acetylcholinesterase (AChE) inhibitors. However, the effects of the location of the tertiary amine groups as well as of other groups on AChE and butyrylcholinesterase (BChE) activity have not been reported. Here, we report the synthesis and testing of 36 new coumarin–chalcone hybrids (5d–7j, 9d–11f, 12k–13m) against AChE and BChE. The nature and position of the chalcone substituents had major effects on inhibitory activity as well as selectivity for AChE over BChE. Compounds with para-substituted chalcone fragments in which the substituents were choline-like had potent activity against AChE and poor activity against BChE, while ortho-substituted analogs exhibited an opposite effect. Replacement of the terminal amine groups by amide, alkyl or alkenyl groups abrogated activity. Compound 5e showed potent inhibitory activity (IC50=0.15±0.01μmol/L) and good selectivity for AChE over BChE (ratio 27.4), and a kinetic study showed that 5e exhibited mixed-type inhibition against AChE. Computational docking results indicate that 5e binds to Trp 279, Tyr334 and Trp 84 in AChE, but only to Trp 82 in BChE. Overall, the results show that coumarin–chalcone hybrids with choline-like side-chains have promising activity and selectivity against AChE and be promising therapeutic leads for Alzheimer’s disease.

Full text of DOI:10.1007/s11030-018-9839-y

Molecular Diversity
https://doi.org/10.1007/s11030-018-9839-y
ORIGINAL ARTICLE
Structure–activity relationship investigation of coumarin–chalcone
hybrids with diverse side-chains as acetylcholinesterase and
butyrylcholinesterase inhibitors
1
2
1
1
3
Lu Kang · Xiao-Hui Gao · Hao-Ran Liu · Xue Men · Hong-Nian Wu ·
3
3
4
Pei-Wu Cui · Eric Oldfield · Jian-Ye Yan
Received: 13 November 2017 / Accepted: 5 June 2018
©
Springer International Publishing AG, part of Springer Nature 2018
Abstract
Chalcones containing tertiary amine side-chains have potent activity as acetylcholinesterase (AChE) inhibitors. However, the
effects of the location of the tertiary amine groups as well as of other groups on AChE and butyrylcholinesterase (BChE)
activity have not been reported. Here, we report the synthesis and testing of 36 new coumarin–chalcone hybrids (5d–7j,
9d–11f, 12k–13m) against AChE and BChE. The nature and position of the chalcone substituents had major effects on
inhibitory activity as well as selectivity for AChE over BChE. Compounds with para-substituted chalcone fragments in which
the substituents were choline-like had potent activity against AChE and poor activity against BChE, while ortho-substituted
analogs exhibited an opposite effect. Replacement of the terminal amine groups by amide, alkyl or alkenyl groups abrogated
activity. Compound 5e showed potent inhibitory activity (IC50 = 0.15 ± 0.01 μmol/L) and good selectivity for AChE over
BChE (ratio 27.4), and a kinetic study showed that 5e exhibited mixed-type inhibition against AChE. Computational docking
results indicate that 5e binds to Trp 279, Tyr334 and Trp 84 in AChE, but only to Trp 82 in BChE. Overall, the results show
that coumarin–chalcone hybrids with choline-like side-chains have promising activity and selectivity against AChE and be
promising therapeutic leads for Alzheimer’s disease.
Keywords Coumarin · Chalcone · Cholinesterase inhibitors · Structure–activity relationship · Tertiary amine group
Introduction
cognition and self-care ability [1]. At present, treatment of
AD is mainly provided with acetylcholinesterase (AChE)
inhibitors, compounds which increase the level of acetyl-
choline in the brain [2]. However, since there are frequent
adverse side-effects including hepatic injury, diarrhea or car-
diovascular damage [3–5], the discovery of new, selective
AChE inhibitors is of interest. In this context, many nat-
ural products or their derivatives have been found to have
AChE inhibitory activity [6–9]. Chalcones, for example,
are promising natural product scaffolds for drug develop-
ment and exhibit a wide variety of bioactivities including
anti-obesity, anti-inflammatory and anti-gout activities. For
example, the chalcones metochalcone and sofalcone, Fig. 1,
have been used in the clinic as choleretic and anti-ulcer
agents, respectively [10]. Flavokawain B, first isolated from
Piper methysticum Forst [11], is another natural product con-
taining the chalcone scaffold. In our laboratory, we found
that flavokawain B Mannich base derivatives had AChE
inhibitory effects [12] and, following this discovery, we
synthesized a series of tertiary amine chalcone derivatives,
Alzheimer’s disease (AD) is one of most common nervous
system diseases and is characterized by impaired memory,
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11030-018-9839-y) contains supplementary
material, which is available to authorized users.
B Hao-Ran Liu
pharmaliu@126.com
B Jian-Ye Yan
yanjianye201@126.com
1
College of Chemistry and Chemical Engineering, Hu’nan
University, Changsha 410082, China
2
College of Pharmacy, Changsha Medical University,
Changsha 410219, China
3
College of Pharmacy, Hu’nan University of Chinese
Medicine, Changsha 410208, China
4
Department of Chemistry, University of Illinois at
Urbana-Champaign, Urbana, IL 61801, USA
1
23

Molecular Diversity
Fig. 1 Examples of chalcones and coumarins
some of which had quite potent AChE inhibitory activity
Results and discussion
(
IC50 = 0.21−4.68 μmol/L) [13–16]. In our studies, we
investigatedAChEinhibitoryeffectsofvarioustertiaryamine
groups, the nature of the spacer between the chalcone scaf-
fold and the tertiary amine side-chain, as well as the effects
of other substituents in the chalcone scaffold, but no inves-
tigations into the influence of different substituent positions
of tertiary amine groups were reported.
Chemistry
Coumarin–chalcone hybrids were synthesized as follows.
Briefly, 3-acetyl coumarin was prepared by the condensa-
tion of salicylic acid with ethyl acetoacetate using piperidine
as catalyst [26]. Compounds 4a–4c were synthesized from
3-acetyl coumarin and hydroxybenzaldehyde via Claisen–
Schmidt condensation in the presence of piperidine [27].
Compounds 5d–7j were generated from compounds 4a–
4c with one of the following: (chloroethyl dimethylamine
hydrochloride, chloroethyl diethylamine hydrochloride,
chloroethyl piperidine hydrochloride, chloroethyl pyrroli-
dine hydrochloride, chloroethyl morpholine hydrochloride,
1-bromo-3-methylbutane, or 3,3-dimethylallyl bromide), all
in the presence of K2CO3 and NaI [28] (Scheme 1).
Compounds 9d–11f were synthesized via the routes
illustrated in Scheme 2. First, chloroacetyl chloride was
converted into compounds 8a–8c using a secondary amine
(dimethylamine, pyrrolidine or piperidine) in the presence
of anhydrous sodium acetate [29]. Next, compounds 8a–
8c were condensed with compounds 4a–4c (in the presence
of K2CO3 and NaI as catalysts) to afford compounds 9d–
11f (Scheme 2). Compounds 5 and 6 were generated by
condensing 3-acetyl coumarin with vanillic aldehyde or
syringaldehyde using p-toluenesulfonic acid as catalyst.
Then, compounds 12k–13m were synthesized from com-
pound 5 or 6, chloroethyl dimethylamine hydrochloride,
chloroethyl piperidine hydrochloride, or chloroethyl pyrroli-
A second interesting class of natural products for scaffold
building are coumarins. This class of compound includes
anticoagulants such as warfarin [17], choleretics [18] and
even antibiotics such as novobiocin, and several structures
are shown in Fig. 1. Coumarins are also used extensively
as starting materials in drug discovery, and some coumarin
derivatives have potent enzyme inhibition activity [19–21].
For example, the hybrids containing coumarin and other moi-
eties have been used as histone deacetylase inhibitors [22],
cyclo-oxygenase inhibitors [23] and monoamine oxidase
inhibitor [24]. Coumarin–chalcone hybrids, are reported as
anti-parasitics agents against Plasmodium spp. [25], malaria
parasites, as well as trypanosomatids. Such hybrids offer
multiple opportunities for derivatization on both aromatic
rings.
In this work, we synthesized and tested a broad range
of coumarin–chalcone hybrids focusing on derivatizing the
chalcone ring with both neutral and basic groups (Scheme 1).
Then, we determined IC50 values of these compounds against
AChE and BChE, evaluated AChE inhibition kinetics of the
most potent inhibitors and used computational docking to
elucidate AChE/BChE selectivity.
1
23

Molecular Diversity
Scheme1 Synthesis of compounds 5d–7j. Reagents and conditions: (a) piperidine, room temperature; (b) piperidine, hydroxybenzaldehyde, ethanol,
◦
reflux; (c) Cl(CH2)2NR/Br(CH2)2R, K2CO3, Nal, DMF, 56 C
◦
Scheme 2 Synthesis of compounds 9d–11f. Reagents and conditions: (d) HNR1R2, NaAc, DCM, 0 C to room temperature; (e) K2CO3, NaI, DMF,
◦
56 C
dine hydrochloride, again in the presence of K2CO3 and NaI
and BChE as well as the selectivity for AChE are summarized
in Table 1. All assays were conducted in triplicate. These
results indicate that many of the compounds show inhibition
activity against AChE and BChE, and some of them have
far more potent inhibitory activity than that of the control,
rivastigmine.
(Scheme 3).
The final compounds were purified by silica gel chro-
matography, and structures were confirmed by using nuclear
1
13
magnetic resonance spectroscopy ( H NMR, C NMR) and
high-resolution mass spectrometry (HRMS). Representative
1
13
H, C NMR and mass spectra are given in the supplemen-
The chemical nature of the substituents as well as their
positions in the chalcone benzene ring greatly influenced
both activity and selectivity. Compounds 5d, 5e and 5f
with IC50 values of 0.37, 0.15 and 0.69 μmol/L, respec-
tively, showed more potent activity than the positive control
rivastigmine (IC50 = 10.54 μmol/L) with compound 5e
tary materials.
In vitro inhibition of AChE and BChE
The inhibitory effects of the newly synthesized compounds
on AChE and BChE activity were evaluated by using the
Ellman method using rivastigmine as positive control. Half-
maximal inhibitory concentrations (IC50 values) for AChE
exhibiting the most potent AChE inhibitory activity (IC
=
5
0
0
.15 μmol/L) as well as higher selectivity for AChE ver-
sus BChE (an IC50 ratio of ratio 27.4:1) than rivastigmine
1
23

Molecular Diversity
◦
Scheme 3 Synthesis of compounds 12k–13m. Reagents and conditions: (f) Acetic acid, TsOH, 70 C; (g) Cl(CH2)2NR1R2, K2CO3, NaI, acetone,
reflux
(a ratio of 0.02:1). In general, the inhibitory potency of the
tion mechanism. As shown in Supplementary Table 2, the
coumarin–chalconederivativesagainstAChEwasasfollows:
para-substituted > meta-substituted > ortho-substituted.
However, the inhibitory potency against BChE was reversed:
ortho-substituted > meta-substituted > para-substituted. In
addition, the inhibitory activity against AChE was highly
dependent on the nature of the ring substituent. Com-
pounds containing a dimethylamine, pyrrolidine or piperi-
dine group (compounds 5d, 5e, 5f, 6d and 6f) possessed
much higher activity than did those compounds containing
competitive inhibition constant (Ki) of compound 5e is
0.11μmol/L, while the non-competitive constant (Ki ) is
0.19 μmol/L.
ꢀ
Molecular modeling studies
To explore the molecular mechanism of AChE and BChE
inhibition, representative compounds (5e, 6e, 7e, 5h) were
selected for a molecular docking investigation using
MOE2014 (Montreal, Quebec, Canada: Chemical Comput-
ing Group Inc., www.chemcomp.com). As shown in Supple-
mentary Fig. 3, compound 5e exhibited multiple interactions
with AChE (with a total binding energy of − 34.6 kJ/mol).
In the top of the AChE “gorge,” the phenyl ring bound to the
peripheral anionic site (PAS) via a π–π stacking interaction
with Trp279 (4.03 Å) and Tyr334 (4.32 Å). In the bottom
of the gorge, the protonated nitrogen of the pyrrolidine ring
bound to the catalytic active site (CAS) via a cation-π inter-
action with Trp84 (4.33 Å), while compound 5e bound to
isopropyl or isopropenyl groups (compounds 5i–7j) (IC5
0
>
500 μmol/L) (Table 1), while all compounds containing
amide groups (9d–11f) showed very weak inhibition activity
against both AChE and BChE. It can be observed that the
presence of choline-like dimethylethanolamine (5d) or anal-
ogous pyrrolidine species (5e) make major contributions to
activity.
Since compounds 5d, 5e and 5f had potent activity and
high selectivity in inhibiting AChE, we next investigated sev-
eral analogs of these compounds. Interestingly, compounds
13k, 13l, 13m, containing two methoxyl substituents, had
+
BChE (− 20.08 kJ/mol) via a π −H interaction in the CAS
much weaker inhibitory activity against AChE than did the
with Trp84 (3.11 Å). Compound 6e exhibited binding to mul-
tiple sites in AChE (− 35.7 kJ/mol). In the top of the gorge,
the phenyl ring bound to the PAS via a π–π stacking inter-
action with Trp279 (3.44 Å). In the bottom of the gorge, the
protonated nitrogen of the pyrrolidine ring bound to the CAS
via a cation-π interaction with Trp84 (4.06 Å). Compound 6e
bound to BChE (− 20.9kJ/mol) via a π–H interaction with
Phe329 (3.77 Å) in the PAS and a cation-π interaction with
Trp82 (3.76 Å), in the CAS, providing a partial explanation
for the similar inhibition for AChE and BChE. However,
compound 7e exhibited multiple binding interactions with
BChE (− 20.5 kJ/mol). In the bottom of the gorge, the 2-
H-chromen-2-one moiety was observed to bind to the CAS
via a π–π stacking interaction with Trp82 (3.81 Å), and a
π–H interaction with His438 (4.47 Å). Compound 7e bound
parentcompounds, 5d, 5e, 5f. However, compounds 12k, 12l,
12m, whichcontainedjustasinglemethoxylgroup, hadcom-
parable inhibitory activity to that of the parent compounds
with compound 12m (IC50 = 0.37 ± 0.02 μmol/L) actually
having slightly more potent activity that the parent compound
5
f (IC5 = 0.69 ± 0.06 μmol/L). Thus, addition of the sec-
0
ond methoxyl group clearly decreases activity, presumably
due to a steric repulsion in the AChE active site.
Compound 5e was then selected for a kinetic assay since
it had the most potent activity against AChE. A Lineweaver–
Burk analysis of the steady-state inhibition data of compound
5e is shown in Supplementary Fig. 2 and shows that increas-
ing the concentration of compound 5e results in different
slopes and intercepts. The Km values show an increase with
increasing concentrations of compound 5e, but the Vmax
values exhibit a decrease, indicating a mixed-type inhibi-
+
to AChE (− 28.9 kJ/mol) via a π − H interaction with
Trp84 (3.29 Å) in the CAS. Compound 5h exhibited very
1
23

Molecular Diversity
Table 1 Effects of new synthesized compounds on AChE and BChE
a
Selectivity for AChEb
Compounds
Position
R
IC (μmol/L)
5
0
AChE
BChE
5
6
7
9
1
1
1
1
5
6
7
9
1
1
1
1
5
6
7
9
1
1
1
1
5
6
7
5
6
7
5
6
7
5
6
7
d
Para
Meta
Ortho
Para
Meta
Ortho
Para
Para
Para
Meta
Ortho
Para
Meta
Ortho
Para
Para
Para
Meta
Ortho
Para
Meta
Ortho
Para
Para
Para
Meta
Ortho
Para
Meta
Ortho
Para
Meta
Ortho
Para
Meta
Ortho
0.37 ± 0.03
0.92 ± 0.07
16.40 ± 0.22
18.61 ± 1.06
>500
>500
>1340
16.64
0.92
>26.86
<0.09
–
d
15.32 ± 1.16
15.16 ± 0.60
>500
d
d
0d
1d
2k
3k
e
44.02 ± 2.37
>500
>500
1.65 ± 0.09
7.07 ± 0.46
0.15 ± 0.01
3.25 ± 0.14
230.34 ± 14.82
>500
8.48 ± 0.29
13.22 ± 0.32
4.11 ± 0.16
4.62 ± 0.16
3.76 ± 0.35
>500
5.15
1.87
27.40
1.42
0.02
–
e
e
e
0e
1e
2l
3l
f
>500
>500
–
>500
>500
–
1.67 ± 0.04
28.95 ± 1.68
0.69 ± 0.06
0.61 ± 0.02
29.86 ± 0.98
>500
0.46 ± 0.01
13.61 ± 0.53
1.03 ± 0.05
4.01 ± 0.14
4.50 ± 0.22
>500
0.28
0.47
1.49
6.61
0.15
–
f
f
f
0f
1f
2m
3m
g
152.49 ± 12.82
>500
222.84 ± 11.54
106.14 ± 3.09
0.52 ± 0.01
6.41 ± 0.36
6.21 ± 0.19
1.12 ± 0.11
1.96 ± 0.14
>500
1.46
0.21
1.41
0.67
1.43
0.02
0.01
–
0.37 ± 0.02
9.58 ± 0.80
4.34 ± 0.29
51.30 ± 1.04
202.70 ± 2.50
>500
g
g
h
h
>500
>500
–
h
260.95 ± 4.17
>500
28.98 ± 1.23
>500
0.11
–
i
i
>500
>500
–
i
>500
>500
–
j
>500
>500
–
j
>500
>500
–
j
>500
100.33 ± 6.84
0.26 ± 0.08
<0.20
Rivastigmin
10.54 ± 0.86
∗
e
0.02
a
IC50: 50% inhibitory concentration (means ± SD of three assays)
Selectivity for AChE is defined as IC50 (BChE)/IC50 (AChE)
Used for control
b
∗
weak binding to both AChE and BChE, consistent with its
IC50 > 500 μM against both AChE and BChE (see Table 1).
Compound 5e thus has potent inhibitory activity against
AChE together with a high selectivity over BChE; com-
pound 6e has moderate inhibitory activity against AChE with
low selectivity over BChE, while compound 7e has poor
inhibitory activity against AChE as well as poor selectivity
over BChE, and compound 5h has almost has no inhibitory
activity against either AChE or BChE, indicating that hav-
ing a hydrophilic morpholine ring abrogates essentially all
1
23

Molecular Diversity
activity, demonstrating that hydrophobic interactions in this
region are essential for activity [35].
vent was removed, and the resulting residue was dissolved in
ethanol. The target product obtained was recrystallized from
ethanol. Pale yellow solid, Yield: 90%, mp: 118−119 C.
◦
Conclusions
In summary, we synthesized a series of coumarin–chalcone
hybrids (5d–7j, 9d–11f, 12k–3m) containing amino-alkyl,
alkyl, alkenyl and amide side-chains. The nature of the side-
chain as well as its location on the chalcone phenyl ring
markedly influenced activity in inhibiting AChE and BChE.
In general, compounds with para-substituted amino-alkyl
groups had the most potent activity and were selective for
AChE. Most of compounds with amide, alkyl or alkenyl
groups had very poor activity, IC50 values of them against
AChE are more than 500 μmol/L. Addition of a single
methoxyl group into the benzene ring resulted in activity
comparable to that of the parent compounds, but addition
of two methoxyl groups again blocked activity. Compound
General procedure for the synthesis of compounds
4
a–4c
Compound 3 (5.00 mmol, 0.94 g) was added into absolute
ethanol (30 mL) under stirring, followed by the addition of
an appropriate hydroxybenzaldehyde (15.00 mmol, 1.83 g).
Then, a catalytic amount of piperidine (4 drops) was added
into the solution, and the reaction mixture was refluxed for
6–8h. The mixture was cooled, and the solid obtained by fil-
tration. The crude products were recrystallized from ethanol
to afford compounds 4a–4c with yield 82–90%.
5
e with an IC50 value of 0.15 ± 0.01 μmol/L against AChE
exhibited the best activity as well as a high selectivity for
AChE over BChE, and our kinetics results indicate that com-
pound 5e has a mixed-type inhibition against AChE. The
molecular docking results show that compound 5e can bind
to both the CAS and the PAS of AChE, contributing to its
potent activity. In conclusion, compound 5e may serve as a
potential lead for the development of novel compounds as
AD therapeutics.
General procedure for the synthesis of compounds
5
d–7j
Compounds 4a–4c (1.00 mmol, 0.292 g), together with
aminoethyl chloride or aminoethyl bromide (3.00 mmol)
and anhydrous potassium carbonate (3 mmol, 0.415 g) in
8
mL DMF, were stirred for 20 minutes. Then, a small
amount of NaI (0.005 g, 0.02 mmol) was added as cata-
◦
lyst. The reaction mixture was stirred for 10–15h at 56 C,
then cooled to room temperature and filtered. The filtrate
was poured into a saturated saline solution and filtered. The
solid obtained was purified using column chromatography
with dichloromethane/methanol (70:1) to yield compounds
Experimental
Chemistry
5
d–7j, respectively.
All chemicals and reagents were of analytical reagent grade
and were used without further purification. Melting points
were measured on a WRS-lA melting point detector (uncor-
rected). H NMR and C NMR spectra were recorded on a
Bruker 400 MHz instrument using tetramethylsilane (TMS)
as the internal standard (s = singlet, d = doublet, t =
triplet, m = multiplet). Mass spectra were obtained on
Waters XEVO-G2XSQTOF Liquid chromatography–mass
spectrometry with electrospray ionization (ESI-MS) method.
The purity of compounds was checked using a Shimadzu LC-
1
13
(E)-3-(3-(4-(2-(dimethylamino)ethoxy)phenyl)acryloyl)-2H-
chromen-2-one (5d)
◦
1
Yellow solid, yield: 85.6%, mp: 137.5–139.2 C; H NMR
(400MHz, DCCl ): δ (ppm)2.29 (6H, s, 2 × NCH ), 2.70
3
3
(2H, t, J = 8 Hz, OCH CH ), 4.06 (2H, t, J = 8 Hz,
2
2
2
0A high-performance liquid chromatography.
Preparation of 3-acetyl-2H-chromen-2-one (3)
-Hydroxybenzaldehyde (15 mmol, 1.56 mL) and ethyl ace-
OCH CH ), 6.86 (2H, d, J = 8 Hz, H phenyl), 7.26–
2
2
3,5
7.34 (2H, m, H6,8 coumarin), 7.55–7.61 (4H, m, H2,6 phenyl
and H5,7 coumarin), 7.74 (1H, d, J = 16 Hz, Hα), 7.79
1
3
(
1H, d, J = 16 Hz, H ), 8.51(1H, s, H4 coumarin) C NMR
β
2
(100MHz, CDCl3) δ: 45.54, 57.46, 65.73, 114.94, 116.68,
118.64, 121.47, 124.91, 125.60, 127.37, 129.95, 130.83,
134.04, 145.23, 147.75, 155.18, 159.36, 161.61, 186.36.
toacetate (15 mmol, 1.89 mL) were added into a flask (25.0
mL) at room temperature. A small amount of piperidine (4
drops) was added as catalyst, and the solution was stirred
continuously until the reaction was complete. Then, the sol-
+
HRMS(ESI) m/z: 364.1549 [M+H] . Purity: 98.9% by
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
1
23

Molecular Diversity
(E)-3-(3-(4-(2-(pyrrolidin-1-yl)ethoxy)phenyl)acryloyl)-
(E)-3-(3-(4-(2-morpholinoethoxy)phenyl)acryloyl)-
2H-chromen-2-one( 5e)
2H-chromen-2-one (5h)
◦
1
◦
1
Yellow solid, yield: 81.5%, mp: 135.9–137.3 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.88 (4H, m, pyrrolidine-
Yellow solid, yield: 80.8%, mp: 167.8–169.2 C; H NMR
(
(400MHz, DCCl3):δ(ppm)2.63(4H, s, morpholine-H), 2.87
H), 2.77 (4H, m, pyrrolidine-H), 3.02 (2H, t, J = 8 Hz,
(2H, s, J = 8 Hz, OCH2CH ), 3.77(4H, s, morpholine-H),
2
OCH2CH ), 4.23 (2H, t, J = 8 Hz, OCH CH2), 6.93–
4.18(2H, t, J = 8 Hz, OCH CH2), 6.94 (2H, d, J = 8 Hz,
2
2
2
6
.95 (2H, d, J = 8 Hz, H phenyl), 7.34–7.41 (2H, m,
H
3,5
phenyl), 7.34–7.41 (2H, m, H6,8 coumarin), 7.63–
3
,5
H6,8 coumarin), 7.62–7.68(4H, m, H2,6 phenyl and H
7.68 (4H, m, H2,6 phenyl and H5,7 coumarin), 7.81–7.89
5
,7
1
3
coumarin), 7.80–7.89 (2H, m, Hα and H ), 8.58(1H, s,
(2H, m, Hα and H ), 8.59 (1H, s, H4 coumarin), C NMR
β
β
1
3
H4 coumarin) C NMR (100MHz, CDCl3) δ:23.50, 54.75,
(100MHz, CDCl3) δ:54.05, 57.47, 66.76, 114.98, 116.70,
118.62, 121.79, 124.96, 125.50, 127.85, 129.99, 130.82,
134.13, 144.96, 147.89, 155.20, 159.39, 160.99, 186.29.
6
1
1
7.13, 114.99, 116.68, 118.62, 121.63, 124.93, 125.54,
27.65, 129.97, 130.81, 134.08, 145.10, 147.82, 155.18,
+
59.36, 161.21, 186.30. HRMS(ESI) m/z: 390.1698
HRMS(ESI) m/z: 406.1660 [M+H] . Purity: 98.2% by
+
[M+H] . Purity: 98.8% by HPLC (MeOH/0.1% TEA 85:15
v/v)).
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(
(E)-3-(3-(4-(isopentyloxy)phenyl)acryloyl)-
2H-chromen-2-one (5i)
◦
1
(E)-3-(3-(4-(2-(piperidin-1-yl)ethoxy)phenyl)acryloyl)-2H-
Yellow solid, yield: 80.5%, mp: 161.7–164.3 C; H NMR
chromen-2-one (5f)
(400 MHz, DCCl3): δ (ppm) 0.97 (6H, d, J = 8 Hz,
2
×CHCH ), 1.71 (2H, t, J = 8 Hz, OCH2CH ), 1.81–
3
2
◦
1
Brown solid, yield: 85.9%, mp: 144.1–144.9 C; H NMR
400 MHz, DCCl3): δ(ppm), 1.18 (5H, m, piperidine-H),
.61 (4H, m, piperidine-H), 2.89 (2H, m, OCH2CH ), 4.19
1.88(1H, m, CH(CH3)2), 4.04(2H, t, J =12 Hz, OCH CH2),
2
(
2
6.92 (2H, d, J = 8 Hz, H3,5 phenyl), 7.33–7.41 (2H, m,
H6,8 coumarin), 7.62–7.68(4H, m, H2,6 phenyl and H5,7
coumarin), 7.81 (1H, d, J = 16 Hz, Hα), 7.86 (1H, d,
2
(
H
2H, t, J = 8 Hz, OCH CH2), 6.86 (2H, d, J = 8 Hz,
2
1
3
phenyl), 7.26–7.34 (2H, m, H6,8 coumarin), 7.55–
,5
J = 16 Hz, Hβ), 8.57 (1H, s, H4 coumarin), C NMR
(100MHz, CDCl3) δ:22.58, 25.04, 37.85, 66.59, 114.91,
116.68, 118.64, 121.47, 124.91, 125.60, 127.37, 129.95,
130.83, 134.04, 145.23, 147.75, 155.18, 159.36, 161.63,
3
7.61 (4H, m, H2,6 phenyl and H5,7 coumarin), 7.73–7.81
1
3
(
2H, m, Hα and H ), 8.51(1H, s, H4 coumarin). C NMR
β
(100MHz, CDCl3) δ:24.01, 25.28, 54.63, 57.30, 65.34,
+
1
1
1
14.96, 116.68, 118.62, 121.63, 124.93, 125.54, 127.65,
29.97, 134.18, 145.16, 147.82, 155.18, 159.36, 161.21,
86.34. HRMS(ESI) m/z: 404.1860 [M+H] . Purity: 99.1%
186.30. HRMS(ESI) m/z: 363.2079 [M+H] . Purity: 98.5%
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
+
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(E)-3-(3-(4-(3-methylbut-2-enyloxy)phenyl)-prop-
2-enoyl)-2H-chromen-2-one (5j)
◦
Yellow solid, yield: 89%, mp:119–121 C. This is a known
compound but without reports regarding inhibiting
cholinesterases [27].
(
2
E)-3-(3-(4-(2-(diethylamino)ethoxy)phenyl)acryloyl)-
H-chromen-2-one (5g)
◦
1
Brown solid, yield: 85.6%, mp: 159.3–160.1 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.14 (6H, t, J = 8 Hz,
(E)-3-(3-(3-(2-(dimethylamino)ethoxy)phenyl)acryloyl)-
2H-chromen-2-one (6d)
(
2
×NCH2CH ), 2.75 (4H, m, 2×NCH CH3), 2.99 (2H, t,
3
2
◦
1
J = 8 Hz, OCH2CH ), 4.17 (2H, t, J = 8 Hz, OCH CH2),
Yellow solid, yield: 85.5%, mp:127.9–128.7 C; H NMR
2
2
6.93 (2H, d, J = 8 Hz, H
phenyl), 7.33–7.41 (2H,
(400 MHz, DCCl3): δ (ppm) 2.41 (6H, s, 2×NCH ), 2.83
3
,5
3
m, H6,8 coumarin), 7.60–7.68(4H, m, H2,6phenyl and H5
(2H, t, J = 8 Hz, OCH2CH ), 4.15 (2H, t, J = 8 Hz,
,7
2,
coumarin), 7.81 (1H, d, J = 16 Hz, Hα), 7.86 (1H, d,
OCH CH2,), 6.99 (2H, d, J = 8 Hz, H4 phenyl), 7.21
2
1
3
J = 16 Hz, H ), 8.58(1H, s, H4 coumarin) C NMR
(1H, s, H2 phenyl), 7.24–7.42 (4H, m, H phenyl and H6,8
β
5,6
(
100MHz, CDCl3) δ:11.34, 47.78, 51.45, 66.13, 114.96,
coumarin), 7.65–7.69 (2H, m, H5,7 coumarin), 7.82 (1H, d,
1
16.69, 118.62, 121.72, 124.95, 125.52, 127.76, 129.98,
J = 16 Hz, Hα), 7.92 (1H, d, J = 16 Hz, H ), 8.59 (1H, s,
β
1
1
30.83, 134.11, 145.05, 147.85, 155.18, 159.39, 161.00,
86.33. HRMS(ESI) m/z: 392.1855 [M+H] . Purity: 98.3%
H4 coumarin) 13C NMR (100MHz, CDCl3) δ:45.70, 58.10,
65.75, 114.23, 116.74, 117.38, 118.56, 122.01, 124.20,
125.01, 125.29, 129.92, 130.06, 134.28, 136.15, 144.98,
+
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
1
23

Molecular Diversity
1
48.13, 155.26, 159.02, 159.32, 186.49. HRMS(ESI) m/z:
129.90, 130.05, 134.27, 136.13, 144.97, 148.08, 155.23,
+
364.1297 [M+H] . Purity: 98.5% by HPLC (MeOH/0.1%
159.07, 159.34, 186.42. HRMS(ESI) m/z: 392.1702
+
TEA 85:15 (v/v)).
[M+H] . Purity: 98.8% by HPLC (MeOH/0.1% TEA 85:15
(
v/v)).
(E)-3-(3-(3-(2-(pyrrolidin-1-yl)ethoxy)phenyl)acryloyl)-
2
H-chromen-2-one (6e)
(E)-3-(3-(3-(2-morpholinoethoxy)phenyl)acryloyl)-
H-chromen-2-one (6h)
2
◦
1
Pale yellow solid, yield: 80.8%, mp: 136.1–138.2 C;
◦
1
H NMR (400 MHz, DCCl3): δ (ppm) 1.91 (4H, m,
pyrrolidine-H), 2.85 (4H, t,J = 8 Hz, pyrrolidine-H), 2.97
Yellow solid, yield: 80.5%, mp: 134.1–135.0 C; H NMR
(400 MHz, DCCl3): δ (ppm) 2.62 (4H, t, J = 8 Hz,
(
2H, t, J = 8 Hz, OCH2CH ), 4.23 (2H, t, J = 8 Hz,
morpholine-H), 2.85 (2H, t, J = 8 Hz, OCH2CH ), 3.76
2
2
OCH CH2), 7.03 (2H, d, J = 8 Hz, H4 phenyl), 7.21 (1H,
(4H, t, J = 8 Hz, morpholine-H), 4.18 (2H, t, J = 8 Hz,
2
s, H2 phenyl), 7.29–7.42 (4H, m, H5 phenyl and H6,8
OCH CH2), 6.967 (2H, d, J = 8 Hz, H4 phenyl),7.21
,6
2
coumarin), 7.63–7.69 (2H, m, H5,7 coumarin), 7.82 (1H, d,
(1H, s, H2phenyl), 7.28–7.42 (4H, m, H phenyl and H6,8
5,6
J = 16 Hz, Hα), 7.91 (1H, d, J = 16 Hz, H ), 8.59 (1H, s,
coumarin), 7.65–7.69 (2H, m,H5,7 coumarin), 7.82 (1H, d,
β
H4 coumarin) 13C NMR (100MHz, CDCl3) δ:23.54, 54.75,
J = 16 Hz, Hα), 7.92 (1H, d, J = 16 Hz, H ), 8.59(1H, s,
β
6
1
1
3
7.05, 114.38, 116.72, 117.34, 118.53, 121.88, 124.18,
25.00, 125.27, 129.90, 130.05, 134.28, 136.13, 144.97,
48.08, 155.23, 159.07, 159.26, 186.43. HRMS(ESI) m/z:
H4 coumarin) 13C NMR (100MHz, CDCl3) δ: 54.13, 57.72,
66.85, 114.35, 116.72, 117.34, 118.54, 121.82, 124.18,
125.00, 125.27, 129.90, 130.05, 134.27, 136.13, 144.97,
148.08, 155.23, 159.07, 159.22, 186.45. HRMS(ESI) m/z:
+
90.1704 [M+H] . Purity: 98.6% by HPLC (MeOH/0.1%
+
TEA 85:15 (v/v)).
406.1645 [M+H] . Purity: 98.9% by HPLC (MeOH/0.1%
TEA 85:15 (v/v)).
(E)-3-(3-(3-(2-(piperidin-1-yl)ethoxy)phenyl)acryloyl)-
2H-chromen-2-one (6f)
(E)-3-(3-(3-(isopentyloxy)phenyl)acryloyl)-2H-chromen-
2-one (6i)
◦
1
Pale yellow solid, yield: 85.6%, mp: 151.8–153.1 C;
◦
1
H NMR (400 MHz, DCCl3): δ (ppm) 1.47–1.67 (6H, m,
piperidine-H), 2.58 (4H, m, piperidine-H), 2.4 (2H, t, J =
Yellow solid, yield: 86.8%, mp: 150.7–152.2 C; H NMR
(400 MHz, DCCl3): δ (ppm) 0.96 (6H, d, J = 8 Hz,2×
CHCH ), 1.71 (2H, t, J = 8 Hz, OCH2CH ), 1.81–1.89
8
Hz, OCH2CH ), 4.18 (2H, t, J = 8 Hz, OCH CH2), 6.97
2
2
3
2
(2H, d, J = 8 Hz, H4 phenyl), 7.20 (1H, s, H2 phenyl),
(1H, m, CH (CH3)2), 4.17 (2H, t, J = 8 Hz, OCH CH2),
2
7.29–7.41 (4H, m, H5 phenyl and H6,8 coumarin), 7.66
6.98 (2H, d, J = 8 Hz, H4 phenyl), 7.21 (1H, s, H2 phenyl),
,6
(
(
(
2H, m, H5,7 coumarin), 7.81 (1H, d, J = 16 Hz, Hα), 7.90
7.29–7.42 (4H, m, H phenyl and H6,8 coumarin), 7.65–
5,6
1
3
1H, d, J = 16 Hz, H ), 8.58(1H, s, H4 coumarin) C NMR
7.69 (2H, m, H5,7 coumarin), 7.82 (1H, d, J = 16 Hz, Hα),
β
100MHz, CDCl3) δ: 24.05, 25.73, 55.01, 57.80, 66.85,
7.92 (1H, d, J = 16 Hz, H ), 8.52 (1H, s, H4 coumarin)
β
1
3
1
1
1
14.33, 116.72, 117.34, 118.54, 121.88, 124.18, 125.00,
25.27, 129.90, 130.05, 134.27, 136.13, 144.97, 148.08,
C NMR (100MHz, CDCl3) δ:22.91, 25.04, 37.18, 66.75,
114.23, 116.74, 117.38, 118.56, 122.01, 124.20, 125.01,
125.29, 129.92, 130.06, 134.28, 136.15, 144.98, 148.13,
155.26, 159.02, 159.32, 186.51. HRMS(ESI) m/z: 363.1548
55.23, 158.26, 159.28, 186.49. HRMS(ESI) m/z: 404.1874
+
[M+H] . Purity: 99.3% by HPLC (MeOH/0.1% TEA 85:15
v/v)).
+
(
[M+H] . Purity: 98.0% by HPLC (MeOH/0.1% TEA 85:15
(
v/v)).
(E)-3-(3-(3-(2-(diethylamino)ethoxy)phenyl)acryloyl)-
2
H-chromen-2-one (6g)
(E)-3-(3-(3-(3-methylbut-2-enyloxy)phenyl)-prop-2-enoyl)-
H-chromen-2-one (6j)
2
1
Brown viscous liquid, yield: 82.5%; H NMR (400 MHz,
◦
DCCl3): δ (ppm) 1.14 (6H, t, J = 8 Hz, 2×NCH2CH ),
Pale yellow solid, yield: 88.7%, mp: 144.4–146.3 C;
3
1
2
.72–2.77 (4H, m, 2×NCH CH3), 3.00 (2H, t, J = 8 Hz,
H NMR (400 MHz, DCCl3): δ (ppm) 1.77 (3H, s, CH ),
2
a3
OCH2CH ), 4.16 (2H, t, J = 8 Hz, OCH CH2,), 6.97 (2H,
1.81 (3H, s, CH ), 4.56 (2H, d, J = 8 Hz, OCH CH),
2
,
2
b3
2
d, J = 8 Hz, H4 phenyl), 7.20 (1H, s, H2 phenyl), 7.29–7.41
5.50 (1H, t,J = 8 Hz, OCH2CH), 6.97 (2H, d, J = 8 Hz,
H4 phenyl), 7.20 (1H, s, H2 phenyl), 7.29–7.39 (4H, m,
H5,6 phenyl and H6,8 coumarin), 7.63–7.67(2H, m, H5,7
coumarin), 7.82 (1H, d, J = 16 Hz, Hα), 7.91 (1H, d, J =
(4H, m, H5 phenyl and H6,8 coumarin), 7.64–7.67(2H, m,
,6
H5 coumarin), 7.82 (1H, d, J = 16 Hz, Hα), 7.90 (1H,
,7
1
3
d, J = 16 Hz, H ), 8.57 (1H, s, H4 coumarin) C NMR
β
1
3
(100MHz, CDCl3) δ:11.85, 47.51, 57.38, 65.90, 114.23,
16 Hz, H ), 8.56 (1H, s, H4 coumarin). C NMR (100MHz,
β
116.72, 117.34, 118.54, 121.88, 124.18, 125.00, 125.27,
CDCl3) δ: 54.13, 57.72, 66.85, 114.35, 116.72, 117.34,
1
23

Molecular Diversity
1
1
1
18.54, 121.82, 124.18, 125.00, 125.27, 129.90, 130.05,
155.15, 158.26, 159.21, 186.97. HRMS(ESI) m/z: 404.1870
[M+H] . Purity: 98.7% by HPLC (MeOH/0.1% TEA 85:15
(v/v)).
+
34.27, 136.13, 144.97, 148.08, 155.23, 159.07, 159.22,
+
86.45. HRMS(ESI) m/z:361.3237[M+H] . Purity:98.5%
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(
2
E)-3-(3-(2-(2-(dimethylamino)ethoxy)phenyl)acryloyl)-
H-chromen-2-one (7g)
(
2
E)-3-(3-(2-(2-(dimethylamino)ethoxy)phenyl)acryloyl)-
H-chromen-2-one (7d)
◦
1
Pale red solid, yield: 85.8%, mp: 135.7–136.1 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.13 (6H, t,J = 8 Hz,
2×NCH CH ), 2.72–2.77 (4H, m, 2×NCH CH ), 2.99 (2H,
◦
1
Yellow solid, yield: 81.8%, mp: 128.7–130.3 C; H NMR
(
(
400 MHz, DCCl3): δ (ppm) 2.28 (6H, s, 2×NCH ), 2.89
3
2
3
2
3
(
2H, t, J = 8 Hz, OCH2CH ), 4.18 (2H, t, J = 8 Hz,
2
t, J = 8 Hz, OCH CH ), 4.16(2H, t, J = 8 Hz, OCH CH ),
2
2
2
2
OCH CH2), 6.92–7.00 (2H, m, H
phenyl),7.32–7.39
2
3,5
6.91–7.00(2H, m, H3 phenyl), 7.32–7.39(3H, m, H phenyl
,5 4
(
3H, m, H4 phenyl and H6,8 coumarin), 7.62–7.69 (3H,
m, H6 phenyl and H5,7 coumarin), 8.00 (1H, d,J
and H6,8 coumarin), 7.61–7.69 (3H, m, H6 phenyl and H5,7
=
coumarin), 8.00 (1H, d,J = 16 Hz, Hα), 8.20 (1H, d,J =
1
6 Hz, Hα), 8.20 (1H, d,J = 16 Hz, H ), 8.54(1H, s,
13
β
1
6 Hz, H ), 8.54(1H, s, H4 coumarin) C NMR (100MHz,
β
1
3
H4 coumarin) C NMR (100MHz, CDCl3) δ:45.96, 57.98,
CDCl3) δ:11.36, 47.71, 57.44, 66.16, 112.25, 116.68, 118.61,
23.89, 124.48, 124.92, 125.75, 129.83, 129.93, 132.19,
34.03, 140.81, 147.64; 155.18, 158.29, 159.25, 186.92.
HRMS(ESI) m/z:392.4354 [M+H] . Purity: 98.1% by
6
1
1
3
6.94, 112.17, 116.64, 118.58, 120.94, 123.90, 124.46,
24.90, 125.65, 129.81, 129.94, 132.17, 134.04, 140.73,
47.66; 155.15, 158.28, 159.18, 186.91. HRMS(ESI) m/z:
1
1
+
+
64.1552 [M+H] . Purity: 98.7% by HPLC (MeOH/0.1%
HPLC (MeOH /0.1% TEA 85:15 (v/v)).
TEA 85:15 (v/v)).
(
2
E)-3-(3-(2-(2-morpholinoethoxy)phenyl)acryloyl)-
H-chromen-2-one (7h)
(
2
E)-3-(3-(2-(2-(pyrrolidin-1-yl)ethoxy)phenyl)acryloyl)-
H-chromen-2-one (7e)
1
◦
1
Brown viscous liquid, Yield:85.5%; H NMR (400 MHz,
Yellow solid, yield: 80.6%, mp: 137.7–138.5 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.82–1.85 (4H, m, pyrrolidine-
DCCl3): δ (ppm) 2.61 (4H, m, morpholine-H), 2.95 (2H, t,
(
J = 8 Hz, OCH2CH ), 3.72–3.74 (4H, m, morpholine-H),
H), 2.61–2.68 (4H, m, pyrrolidine-H), 2.94 (2H, t, J =
2
4
.24 (2H, t, J = 8 Hz, OCH CH2), 6.98–7.05 (2H, m, H
2
3,5
8
6
Hz, OCH2CH ), 4.16 (2H, t, J = 8 Hz, OCH CH2),
2
2
phenyl), 7.29–7.39 (3H, m, H4 phenyl and H6,8 coumarin),
.91–7.00 (2H, m, H3 phenyl), 7.32–7.39 (3H, m, H4
,5
7
.82–7.86 (3H, m, H6 phenyl and H5,7 coumarin), 8.01 (1H,
phenyl and H6,8 coumarin), 7.61–7.69 (3H, m, H6 phenyl
and H5,7 coumarin), 8.00 (1H, d,J = 16 Hz, Hα), 8.19 (1H,
d, J = 16 Hz, Hα),8.18 (1H, d, J = 16 Hz, H ), 8.55
β
(1H, s, H4 coumarin) 13C NMR (100MHz, CDCl3) δ: 54.94,
1
3
d,J = 16 Hz, H ), 8.51 (1H, s, H4 coumarin) C NMR
β
5
1
1
7.68, 66.68, 67.62, 112.24, 116.68, 118.61, 123.89, 124.48,
24.92, 125.75, 129.83, 129.93, 132.19, 134.03, 140.81,
47.64; 155.18, 158.29, 159.25, 186.99. HRMS(ESI) m/z:
406.1639 [M+H] . Purity: 98.9% by HPLC (MeOH/0.1%
TEA 85:15 (v/v)).
(
100MHz, CDCl3) δ:23.53, 54.72, 67.17, 112.25, 116.68,
1
1
1
18.61, 123.89, 124.48, 124.92, 125.75, 129.83, 129.93,
32.19, 134.03, 140.81, 147.85, 155.18, 158.26, 159.23,
86.94. HRMS(ESI) m/z: 390.1707 [M+H] . Purity: 99.1%
+
+
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(
2
E)-3-(3-(2-(2-(piperidin-1-yl)ethoxy)phenyl)acryloyl)-
H-chromen-2-one (7f)
(E)-3-(3-(2-(isopentyloxy)phenyl)acryloyl)-2H-chromen-
2-one (7i)
◦
1
◦
1
Brown solid, yield: 80.9%, mp: 150.3–151.6 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.45–1.64 (6H, m, piperidine-
Yellow solid, yield: 85.6%, mp: 153.4–155.2 C; H NMR
(400 MHz, DCCl3): δ (ppm) 0.98 (6H, d,J = 8 Hz, 2×
CHCH ), 1.71 (2H, t, J = 8 Hz, OCH2CH ), 1.80–1.88
(
H), 2.58–2.61 (4H, m, piperidine-H), 2.94 (2H, t, J =
3
2
8
7
Hz, OCH2CH ), 4.24 (2H, t, J = 8 Hz, OCH CH2), 6.93–
(1H, m, CH(CH3)2), 4.04 (2H, t, J = 8 Hz, OCH CH2),
2
2
2
.01(2H, m, H3 phenyl), 7.33–7.41 (3H, m, H4 phenyl
6.92–7.01(2H, m, H phenyl), 7.32–7.41 (3H, m, H4 phenyl
,5
3,5
and H6,8 coumarin), 7.63–7.69 (3H, m, H6 phenyl and
H5,7 coumarin), 8.00 (1H, d,J = 16 Hz, Hα), 8.19 (1H,
and H6,8 coumarin), 7.63–7.69 (3H, m, H6 phenyl and H5,7
coumarin), 8.00 (1H, d,J = 16 Hz, Hα), 8.19 (1H, d,J =
1
3
13
d,J = 16 Hz, H ), 8.55 (1H, s, H4 coumarin) C NMR
16 Hz, H ), 8.57 (1H, s, H4 coumarin) C NMR (100MHz,
β
β
(100MHz, CDCl3) δ:24.01, 25.82, 54.97, 57.62, 66.63,
CDCl3) δ:22.59, 25.06, 37.87, 66.62, 112.26, 116.68, 118.61,
123.89, 124.48, 124.92, 125.75, 129.83, 129.93, 132.19,
134.03, 140.81, 147.83, 155.18, 158.26, 159.28, 186.93.
1
1
12.24, 116.68, 118.61, 120.92, 123.89, 124.48, 124.92,
25.75, 129.83, 129.93, 132.19, 134.03, 140.81, 147.84,
1
23

Molecular Diversity
+
HRMS(ESI) m/z: 363.1596 [M+H] . Purity: 98.6% by
(E)-3-(3-(4-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)phenyl)acryl
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
oyl)-2H-chromen-2-one (9e)
◦
1
Yellow solid, yield: 90.3%, mp: 225.7–227.2 C; H NMR
(
2
E)-3-(3-(2-(3-methylbut-2-enyloxy)phenyl)-prop-
-enoyl)-2H-chromen-2-one (7j)
(
400 MHz, DCCl3): δ (ppm) 1.85–2.01 (4H, m, pyrrolidine-
H), 3.53 (4H, m, pyrrolidine-H), 4.68 (2H, s, OCH CO),
2
◦
6.98 (2H, d, J = 8 Hz, H3 phenyl), 7.33–7.41 (2H, m,
,5
Yellow solid, yield: 80%, mp: 92–95 C; This is a known
compound but without reports regarding inhibiting
cholinesterases [27].
H6,8 coumarin), 7.63–7.68 (4H, m, H2,6 phenyl and H
5
,7
coumarin), 7.79–7.87 (2H, m, Hα and H ), 8.58 (1H, s,
β
H4 coumarin) 13C NMR (100MHz, CDCl3) δ:23.82, 26.28,
4
1
1
4
6.02, 46.27, 67.82, 115.09, 116.69, 118.60 122.10, 124.96,
25.46, 128.40, 130.00, 130.82, 134.14, 144.76, 147.90,
55.19, 159.35, 160.31, 165.91, 186.32. HRMS(ESI) m/z:
General procedure for the synthesis of compounds
8
a–8c
+
04.1502 [M+H] . Purity: 98.9% by HPLC (MeOH/0.1%
Secondary amine (dimethylamine, pyrrolidine, piperidine,
.00 mmol) and anhydrous sodium acetate (5.5 mmol, 0.451
TEA 85:15 (v/v)).
5
g) were sequentially added into CH2Cl2 (10 mL). The reac-
tion mixture stirred for 20 min at 0 C. Chloroacetyl chloride
(
E)-3-(3-(4-(2-oxo-2-(piperidin-1-yl)ethoxy)phenyl)acryloyl)
◦
-2H-chromen-2-one (9f)
(6.00 mmol, 0.477 mL) was then added into the mixture,
and the reaction was stirred for 5–6h at ambient tempera-
ture. The resulting mixture was extracted thrice with 60 mL
CH2Cl2. The separated organic layer was dried over anhy-
drous magnesium sulfate and concentrated under reduced
pressure. Then, compounds 8a–8c were isolated. Yield: 80–
◦
1
Yellow solid, yield: 92.5%, mp: 217.4–219.6 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.56–1.65 (6H, m, piperidine-
H), 3.48–3.57 (4H, m, piperidine-H), 4.74 (2H, s, OCH CO),
(
2
6
.98 (2H, d, J = 8 Hz, H3 phenyl), 7.33–7.40 (2H,
,5
m, H6,8 coumarin), 7.62–7.68 (4H, m, H2,6 phenyl and
89%.
H5,7 coumarin), 7.78–7.86 (2H, m, Hα and H ), 8.57 (1H,
β
1
3
s, H4 coumarin) C NMR (100MHz, CDCl3) δ:24.42,
5.53, 26.50, 43.27, 46.41, 67.56, 115.09, 116.67, 118.59
22.08, 124.96, 125.45, 128.35, 130.00, 130.81, 134.14,
44.74, 147.87, 155.17, 159.33, 160.32, 165.57, 186.30.
General procedure for the synthesis of compounds
d–11f
2
1
1
9
Compounds 8a–8c (3.00 mmol) were added into a solu-
tion containing intermediate compounds 4a–4c (1.00 mmol),
K2CO3 (3.00 mmol, 0.43 g,) and NaI (0.05 mmol, 0.012 g) in
+
HRMS(ESI) m/z: 418.1854 [M+H] . Purity: 98.7% by
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
DMF (6 mL). The reaction mixture was stirred for 10–12h at
(
3
E)-N,N-dimethyl-2-(3-(3-oxo-3-(2-oxo-2H-chromen-
-yl)prop-1-en-1-yl)phenoxy)acetamide (10d)
◦
56 C, then cooled to room temperature and filtered. The fil-
trate was poured into a saturated saline solution and filtered.
The crude product was purified by column chromatography
using dichloromethane/methyl alcohol (100:1) as eluent to
give the pure product compounds 9d–11f. Yield: 90–95%.
◦
Pale yellow solid, yield: 94.6%, mp: 127.8–129.1 C;
1
H NMR (400 MHz, DCCl3): δ (ppm) 2.89 (3H, s, NCH ),
a3
3
.12 (3H, s, NCH ), 4.78 (2H, s, OCH CO), 7.03 (1H, d,
b3 2
J = 8 Hz, H4 phenyl), 7.21 (1H, s, H2 phenyl), 7.30–7.40
(4H, m, H5,6 phenyl and H6,8 coumarin), 7.65–7.69 (2H,
m,H5,7 coumarin), 7.81 (1H, d, J = 16 Hz, Hα), 7.90 (1H,
(
3
E)-N,N-dimethyl-2-(4-(3-oxo-3-(2-oxo-2H-chromen-
-yl)prop-1-en-1-yl) phenoxy)acetamide (9d)
1
3
d, J = 16 Hz, H ), 8.54(1H, s, H4 coumarin) C NMR
β
◦
1
Yellow solid, yield: 90.8%, mp: 193.7–195.1 C; H NMR
(100MHz, CDCl3) δ:35.66, 36.78, 67.65, 114.29, 116.69,
117.64, 118.55, 121.69, 124.11, 125.28, 130.04, 134.22,
136.10, 145.09, 148.03, 155.24, 158.71, 159.39, 166.25,
(
400 MHz, DCCl3): δ (ppm) 2.99 (3H, s, NCH ), 3.10 (3H,
a3
s, NCH ), 4.75 (2H, s, OCH CO), 6.98 (2H, d, J = 8 Hz,
b3
2
+
H
phenyl), 7.34–7.41 (2H, m, H6,8 coumarin), 7.63–
,5
186.45. HRMS(ESI) m/z: 378.2412 [M+H] . Purity: 98.6%
3
7
.68 (4H, m, H2,6 phenyl and H5,7 coumarin), 7.80–7.88
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
1
3
(2H, m, Hα and H ), 8.58 (1H, s, H4 coumarin) C NMR
β
(100MHz, CDCl3) δ:35.75, 36.58, 67.35, 115.11, 116.70,
(E)-3-(3-(3-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)phenyl)acryl
1
1
1
18.61, 122.13, 124.95, 125.48, 128.46, 129.99, 130.82,
oyl)-2H-chromen-2-one (10e)
34.13, 144.76, 147.90, 155.21, 159.36, 160.23, 167.29,
+
◦
86.30. HRMS(ESI) m/z: 378.2458 [M+H] . Purity: 98.6%
Pale yellow solid, yield: 93.2%, mp: 246.7–248.3 C;
1
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
H NMR (400 MHz, DCCl3): δ (ppm) 1.86–2.02 (4H, m,
1
23

Molecular Diversity
pyrrolidine-H), 3.55 (4H, t, J = 8 Hz, pyrrolidine-H), 4.68
H6 phenyl and H5,7 coumarin), 8.04 (1H, d,J = 16 Hz,
(
2H, s, OCH CO), 7.03 (2H, d, J = 8 Hz, H4 phenyl),
Hα), 8.19 (1H, d,J = 16 Hz, H ), 8.60 (1H, s, H4
2
β
7.22 (1H, s, H2 phenyl), 7.30–7.41 (4H, m, H
phenyl
,6
coumarin) 13C NMR (100MHz, CDCl3) δ:23.77, 26.32,
46.28, 46.33, 68.68, 112.54, 116.66, 118.61, 121.71, 124.01,
124.89, 124.93, 125.61, 130.01, 130.08, 132.22, 134.10,
140.20, 147.83; 155.18, 157.49, 159.18, 166.23, 186.93.
5
and H6,8 coumarin), 7.65–7.69 (2H, m, H5,7 coumarin), 7.81
(
8
1H, d, J = 16 Hz, Hα), 7.90 (1H, d, J = 16 Hz, H ),
β
1
3
.58 (1H, s, H4 coumarin) C NMR (100MHz, CDCl3)
+
δ:23.83, 26.28, 46.05, 46.27, 67.84, 114.60, 116.72, 117.24,
HRMS(ESI) m/z: 404.1311 [M+H] . Purity: 99.1% by
1
1
1
18.53, 122.37, 124.41, 125.03, 125.21, 130.08, 134.31,
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
36.29, 144.71, 148.17, 155.24, 158.41, 159.30, 166.20,
+
86.48. HRMS(ESI) m/z: 404.2765 [M+H] . Purity: 98.9%
(E)-3-(3-(2-(2-oxo-2-(piperidin-1-yl)ethoxy)phenyl)acry
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
loyl)-2H-chromen-2-one (11f)
◦
(
E)-3-(3-(3-(2-oxo-2-(piperidin-1-yl)ethoxy)phenyl)acryl
Pale yellow solid, yield: 90.5%, mp: 148.7–150.3 C;
1
oyl)-2H-chromen-2-one (10f)
H NMR (400 MHz, DCCl3): δ (ppm) 1.54–1.62 (6H, m,
piperidine-H), 3.57 (4H, m, piperidine-H), 4.82 (2H, s,
◦
1
Yellow solid, yield: 90.7%, mp: 152.6–154.3 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.56–1.65 (6H, m, piperidine-
H), 3.50–3.58(4H, m, piperidine-H), 4.73(2H, s, OCH CO),
OCH CO), 7.01–7.05 (2H, m, H
phenyl), 7.35–7.40
3,5
2
(
(3H, m, H4 phenyl and H6,8 coumarin), 7.63–7.71 (3H, m,
H6 phenyl and H5,7 coumarin), 8.02 (1H, d,J = 16 Hz,
2
7
.03 (1H, d, J = 8 Hz, H4 phenyl), 7.21 (1H, s, H2
Hα), 8.20 (1H, d,J = 16 Hz, H ), 8.58 (1H, s, H4
β
phenyl), 7.30–7.40 (4H, m, H phenyl and H6,8 coumarin),
7
1
coumarin) 13C NMR (100MHz, CDCl3) δ:24.45, 25.58,
26.51, 43.31, 46.62, 68.29, 112.48, 116.66, 118.59, 121.66,
123.92, 124.77, 124.94, 125.57, 129.90, 130.00, 132.27,
134.11, 140.15, 147.82; 155.17, 157.43, 159.20, 165.83,
5
,6
.64–7.69 (2H, m, H5,7 coumarin), 7.81 (1H, d, J =
6 Hz, Hα), 7.90 (1H, d, J = 16 Hz, H ), 8.57(1H, s,
β
1
3
H4 coumarin) C NMR (100MHz, CDCl3) δ:24.46, 25.55,
+
2
1
1
6.52, 43.25, 46.43, 67.61, 114.80, 116.70, 117.10, 118.52,
22.25, 124.42, 125.02, 125.22, 130.07, 134.29, 136.28,
186.82 HRMS(ESI) m/z: 418.1647 [M+H] . Purity: 98.5%
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
44.68, 148.12, 155.23, 158.52, 159.27, 165.78, 186.47.
+
HRMS(ESI) m/z: 418.1599 [M+H] . Purity: 98.7% by
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
General procedure for the synthesis of compounds 5
and 6
(
E)-N,N-dimethyl-2-(2-(3-oxo-3-(2-oxo-2H-chromen-3-yl)
Compound 3 (5.00 mmol, 0.94 g) was added into acetic acid
(30 mL) under stirring, followed by the addition of an appro-
priate vanillic aldehyde or syringaldehyde (5.00 mmol) and
p-toluenesulfonic acid (4.5 mmol). The reaction was main-
prop-1-en-1-yl)phenoxy)acetamide (11d)
◦
Pale yellow solid, yield: 92.3%, mp: 161.7–163.4 C;
1
◦
H NMR (400 MHz, DCCl3): δ (ppm) 3.00 (3H, s, NCH ),
tainedat70 Cfor5–7h, thencooledtoroomtemperatureand
a3
3
.15 (3H, s, NCH ), 4.82 (2H, s, OCH CO), 6.98–7.06 (2H,
filtered. The filtrate was neutralized with a sodium bicarbon-
ate solution, and the resulting mixture was extracted thrice
with 90 mL CH2Cl2. The organic layer was separated and
dried over anhydrous magnesium sulfate and concentrated
under reduced pressure. The residue was purified by silica
gel column chromatography using dichloromethane/methyl
alcohol (90:1) as eluent to give pure products 5 and 6.
b3
2
d, J = 8 Hz, H3 phenyl), 7.33–7.40 (3H, m, H4 phenyl
,5
and H6,8 coumarin), 7.64–7.71 (3H, m, H6 phenyl and H5
,7
coumarin), 8.04 (1H, d,J = 16 Hz, Hα), 8.19 (1H, d,J =
1
3
1
6 Hz, H ), 8.59 (1H, s, H4 coumarin) C NMR (100MHz,
β
CDCl3) δ:35.77, 36.91, 68.20, 112.55, 116.68, 118.61,
1
1
1
21.76, 124.06, 124.93, 125.63, 130.00, 130.12, 132.21,
34.10, 140.21, 147.83; 155.20, 157.40, 159.21, 167.55,
+
86.92. HRMS(ESI) m/z: 378.1331 [M+H] . Purity: 98.3%
General procedure for the synthesis of compounds
12k–13m
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(E)-3-(3-(2-(2-oxo-2-(pyrrolidin-1-yl)ethoxy)phenyl)acryl
Compounds 5 or 6 (1.00 mmol), together with aminoethyl
oyl)-2H-chromen-2-one (11e)
chloride (3.00 mmol) and anhydrous potassium carbonate
(
3.00 mmol, 0.415 g), were dissolved into 15 mL acetone in
◦
1
Yellow solid, yield: 92.8%, mp: 288.8–231.0 C; H NMR
400 MHz, DCCl3): δ (ppm) 1.84–1.99 (4H, m, pyrrolidine-
H), 3.52–3.59 (4H, m, pyrrolidine-H), 4.76 (2H, s,
OCH CO), 6.98–7.05 (2H, m, H phenyl), 7.35–7.40
a flask, and stirred for 20 minutes. Then, a little NaI (0.005
g, 0.02 mmol) was added into the reaction solution as a cata-
lyst. The reaction solution kept at 40 C for 10–12h, then
cooled to room temperature and filtered. The filtrate was
concentrated under reduced pressure, and the crude prod-
(
◦
2
3,5
(
3H, m, H4 phenyl and H6,8 coumarin), 7.63–7.70 (3H, m,
1
23

Molecular Diversity
+
uct was purified by silica gel column chromatography using
dichloromethane/methyl alcohol (40:1) as eluent to give the
pure product compounds 12k–3m. Yield: 80–85%.
186.27 HRMS(ESI) m/z: 434.1960 [M+H] . Purity: 98.2%
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(
E)-3-(3-(4-(2-(dimethylamino)ethoxy)-3,5-dimethoxyph
(
E)-3-(3-(4-(2-(dimethylamino)ethoxy)-3-methoxyphenyl)
enyl)acryloyl)-2-chromen-2-one (13k)
acryloyl)-2H-chromen-2-one (12k)
◦
1
Yellow solid, yield: 80.2%, mp: 183.9–185.6 C; H NMR
Brownish yellow solid, yield: 85.6%, mp: 124.9–126.3
(
400 MHz, DCCl3): δ (ppm) 2.62 (6H, s, 2×NCH ), 3.14
3
◦
1
C; H NMR (400 MHz, DCCl3): δ (ppm) 2.62 (6H, s,
(
2H, s, J = 8 Hz, OCH2CH ), 3.82 (6H, s, 2×OCH ), 4.33
2
3
2
×NCH ), 3.15 (2H, t, J = 8 Hz, OCH2CH ), 3.92 (3H,
3
2
(2H, t, J = 8 Hz, OCH CH ), 6.83 (2H, s, H phenyl),
2
2
2,6
s, OCH ), 4.34 (2H, t, J = 8 Hz, OCH CH2), 6.92 (1H, d,
3
2
,8 5,7
7.36–7.43 (2H, m, H6 coumarin), 7.65–7.70 (2H, m, H
J = 8 Hz, H phenyl), 7.20(1H, s, H2 phenyl), 7.25 (1H, d,
5
coumarin), 7.77 (1H, d, J = 16 Hz, Hα), 7.85(1H, d, J =
J = 8 Hz, H6 phenyl), 7.35–7.42 (2H, m, H6,8 coumarin),
13
1
6 Hz, H ), 8.65 (1H, s, H4 coumarin) C NMR (100MHz,
β
7.65–7.69 (2H, m, H5 coumarin), 7.78–7.86 (2H, m, Hα
,7
CDCl3) δ:45.67, 52.46, 56.12, 67.93, 105.68, 116.79, 118.54,
23.71, 125.08, 125.87, 128.73, 130.09, 134.35, 139.91,
42.35, 144.78, 148.18, 153.28, 155.29, 159.44, 186.29
HRMS(ESI) m/z: 424.1759 [M+H] . Purity: 98.8% by
1
3
and H ), 8.59 (1H, s, H4 coumarin) C NMR (100MHz,
β
1
CDCl3) δ:45.46, 55.98, 57.46, 65.77, 110.75, 112.83, 116.67,
1
1
1
1
18.59, 122.03, 123.88, 124.99, 125.47, 128.31, 130.00,
+
34.15, 145.17, 147.85, 149.46, 150.63, 155.15, 159.41,
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
+
86.25. HRMS(ESI) m/z: 394.1651 [M+H] . Purity: 98.5%
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(E)-3-(3-(3,5-dimethoxy-4-(2-(pyrrolidin-1-yl)ethoxy)phe
nyl)acryloyl)-2H-chromen-2-one (13l)
(E)-3-(3-(3-methoxy-4-(2-(pyrrolidin-1-yl)ethoxy)
phenyl)acryloyl)-2H-chromen-2-one (12l)
Brownish yellow solid, yield: 80.3%, mp: 274.1–276.4
◦
1
C; H NMR (400 MHz, DCCl3): δ (ppm) 2.22–2.26 (4H,
Brownish yellow solid, yield: 75.2%, mp: 158.9–160.7
◦
1
m, pyrrolidine-H), 3.12–3.20 (4H, m, pyrrolidine-H), 3.62
C; H NMR (400 MHz, DCCl3): δ (ppm) 2.22–2.26 (4H, m,
(
(
7
2H, s, J = 8 Hz, OCH2CH ), 3.90 (6H, s, 2×OCH ), 4.62
pyrrolidine-H), 3.15–3.20 (4H, m, pyrrolidine-H), 3.62 (2H,
2
3
2H, t, J = 8 Hz, OCH CH2), 6.88 (2H, s, H2,6 phenyl),
s, OCH2CH ), 3.92 (3H, s, OCH ), 4.65 (2H, s, OCH CH2),
2
2
3
2
.35–7.41 (2H, m, H6,8 coumarin),7.65–7.74 (2H, m, H
5,7
6
.94 (1H, d, J = 8 Hz, H phenyl), 7.20–7.24 (1H, m,
5
coumarin), 7.77 (1H, d, J = 16 Hz, Hα), 7.85(1H, d, J =
H2,6 phenyl), 7.35–7.41 (2H, m, H6,8 coumarin), 7.65–
1
3
1
6 Hz, H ), 8.57 (1H, s, H4 coumarin) CNMR(100MHz,
β
7
.70 (2H, m, H5,7 coumarin), 7.81 (2H, m, Hα and H ),
β
1
3
CDCl3) δ:23.75, 52.46, 56.05, 67.93, 105.35, 116.75, 118.54,
8.59 (1H, s, H4 coumarin) C NMR (100MHz, CDCl3)
123.71, 125.08, 125.87, 128.75, 130.09, 134.35, 139.91,
δ:23.29, 53.78, 54.60, 55.98, 64.37, 110.80, 114.18, 116.70,
1
42.35, 144.76, 148.18, 153.28, 155.20, 159.35, 186.25
1
1
1
18.57, 122.74, 123.66, 125.05, 125.34, 129.67, 130.07,
34.25, 144.69, 148.03, 149.06, 149.71, 155.18, 159.44,
86.30. HRMS(ESI) m/z: 420.1808 [M+H] . Purity: 98.9%
+
HRMS(ESI) m/z: 450.1915 [M+H] . Purity: 98.2% by
+
HPLC (MeOH/0.1% TEA 85:15 (v/v)).
by HPLC (MeOH/0.1% TEA 85:15 (v/v)).
(E)-3-(3-(3,5-dimethoxy-4-(2-(piperidin-1-yl)ethoxy)phenyl)
(E)-3-(3-(3-methoxy-4-(2-(piperidin-1-yl)ethoxy)
acryloyl)-2H-chromen-2-one (13m)
phenyl)acryloyl)-2H-chromen-2-one (12m)
◦
1
Yellow solid, yield: 78.3%, mp: 214.9–216.7 C; H NMR
◦
1
Yellow solid, yield: 80.6%, mp: 270.9–272.1 C; H NMR
(400 MHz, DCCl3): δ (ppm) 1.26 (6H, s, piperidine-H),
1.63 (4H, s, piperidine-H), 2.33 (2H, s, OCH2CH ), 3.88
(
400 MHz, DCCl3): δ (ppm) 1.26–1.52 (6H, m, piperidine-
2
H),1.74 (4H, s, piperidine-H), 3.01 (2H, t, J = 8 Hz ,
OCH2CH ), 3.92 (3H, s, OCH ), 4.31 (2H, t, J = 8 Hz,
(6H, s, 2×OCH ), 4.36 (2H, s, OCH CH2), 6.87 (2H, s,
3
2
H2,6 phenyl), 7.35–7.41 (2H, m, H6,8 coumarin), 7.65–7.70
(2H, m, H5,7 coumarin), 7.77(1H, d, J = 16 Hz, Hα),
2
3
OCH CH2), 6.91 (1H, d, J = 8 Hz, H phenyl), 7.19
2
5
(
7
1H, s, H2 phenyl), 7.25 (1H, d, J = 8 Hz, H6 phenyl),
7.85(1H, d, J = 16 Hz, H ), 8.59(1H, s, H4 coumarin)
β
1
3
.34–7.40 (2H, m, H6,8 coumarin), 7.64–7.69 (2H, m, H5
C NMR (100MHz, CDCl3) δ:21.33, 23.34, 29.70, 54.00,
,7
coumarin), 7.77–7.85 (2H, m, Hα and H ), 8.57 (1H, s,
56.11, 57.14, 67.93, 105.66, 116.71, 118.54, 123.71, 125.08,
125.87, 128.75, 130.09, 134.35, 139.91, 142.35, 144.76,
148.18, 153.28, 155.20, 159.44, 186.26. HRMS(ESI) m/z:
464.2076 [M+H] . Purity: 99.1% by HPLC (MeOH/0.1%
TEA 85:15 (v/v)).
β
H4 coumarin) 13C NMR (100MHz, CDCl3) δ:23.55, 25.01,
29.70, 54.68, 55.98, 57.12, 65.77, 110.76, 112.80, 116.67,
118.59, 122.03, 123.88, 124.99, 125.47, 128.31, 130.00,
134.15, 145.17, 147.85, 149.46, 150.60, 155.14, 159.41,
+
1
23

Molecular Diversity
AChE and BChE inhibition assay
References
1
. Association A (2017) Alzheimer’s disease facts and figures.
Alzheimer’s Dement 13:325–373. https://doi.org/10.1016/j.jalz.
The effects of newly synthesized compounds in AChE or
BChE inhibition were measured by using a modified Ell-
man method [30]. The individual compound was dissolved
in Tween 80 (final concentration was 0.06% in each reaction)
and diluted with water to different concentrations immedi-
ately before use. Five different concentrations were tested for
each compound in triplicate. The reaction mixture containing
2017.02.001
2
. Dey A, Bhattacharya R, Mukherjee A, Pandey DK (2016) Natu-
ral products against Alzheimer’s disease: pharmaco-therapeutics
and biotechnological interventions. Biotechnol Adv 35:178–216.
https://doi.org/10.1016/j.biotechadv.2016.12.005
3
. Blanco-Silvente L, Castells X, Saez M, Barceló MA, GarreOlmo
J, Vilalta-Franch J, Capellà D (2017) Discontinuation, efficacy,
and safety of cholinesterase inhibitors for Alzheimer’s disease: a
meta-analysis and meta-regression of 43 randomized clinical trials
enrolling 16106 patients. Int J Neuropsychoph 20:519–528. https://
doi.org/10.1093/ijnp/pyx012
2
1
.76 mL of Na2HPO4/NaH2PO4 buffer (pH 8.0, 0.1mol/L),
00 μL of the different concentrations of tested compounds,
and 100 μL of AChE or BChE (100 μL) was incubated for 30
◦
min (30 C). Then, the reaction was terminated by the addi-
4
. Gurbuz AS, Ozturk S, Acar E, Cagan Efe S, Akgun T, Kilicgedik A,
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◦
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