Synthesis and anti-acetylcholinesterase activity of scopoletin derivatives

Article abstract of DOI:10.1016/j.bioorg.2015.12.002

A series of scopoletin derivatives incorporated with the pyridinium moiety was synthesized and evaluated for their acetylcholinesterase (AChE) inhibitory activity by the colorimetric Ellman's method. A 2-fluorobenzylpyridinium derivative was the most potent among the tested compounds, with an IC₅₀ value of 0.215 ± 0.015 μM, which was greatly improved from that of scopoletin. Docking studies revealed that the scopoletin portion of the mentioned compound was bound to the peripheral anionic site of the AChE, whereas the N-benzylpyridinium residue to the catalytic anionic site.

Full text of DOI:10.1016/j.bioorg.2015.12.002

Accepted Manuscript
Synthesis and anti-acetylcholinesterase activity of scopoletin derivatives
Nisachon Khunnawutmanotham, Nitirat Chimnoi, Patchreenart Saparpakorn,
Supanna Techasakul
PII:
S0045-2068(15)30040-7
DOI:
http://dx.doi.org/10.1016/j.bioorg.2015.12.002
YBIOO 1863
Reference:
Bioorganic Chemistry
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Accepted Date:
23 September 2015
18 November 2015
6 December 2015
Please cite this article as: N. Khunnawutmanotham, N. Chimnoi, P. Saparpakorn, S. Techasakul, Synthesis and anti-
acetylcholinesterase activity of scopoletin derivatives, Bioorganic Chemistry (2015), doi: http://dx.doi.org/10.1016/
j.bioorg.2015.12.002
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Synthesis and anti-acetylcholinesterase activity of scopoletin derivatives
Nisachon Khunnawutmanotham,a Nitirat Chimnoi,b Patchreenart Saparpakorn,c and Supanna
Techasakula,c,*
aLaboratory of Organic Synthesis, Chulabhorn Research Institute, 54 Kamphaeng Phet 6,
Talat Bang Khen, Lak Si, Bangkok 10210, Thailand
bLaboratory of Natural Products, Chulabhorn Research Institute, 54 Kamphaeng Phet 6,
Talat Bang Khen, Lak Si, Bangkok 10210, Thailand
cDepartment of Chemistry, Faculty of Science, Kasetsart University, Bangkok 10900,
Thailand
*Address correspondence to Assoc. Prof. Supanna Techasakul, Department of Chemistry,
Faculty of Science, Kasetsart University, Bangkok 10900, Thailand. E-mail:
fscispt@ku.ac.th, supanna@cri.or.th; Phone: +662-553-8555 ext. 8550; Fax: +662-553-8545.
Abstract
A series of scopoletin derivatives incorporated with the pyridinium moiety was
synthesized and evaluated for their acetylcholinesterase (AChE) inhibitory activity by the
colorimetric Ellman’s method. A 2-fluorobenzylpyridinium derivative was the most potent
among the tested compounds, with an IC50 value of 0.215  0.015 µM, which was greatly
improved from that of scopoletin. Docking studies revealed that the scopoletin portion of the
mentioned compound was bound to the peripheral anionic site of the AChE, whereas the N-
benzylpyridinium residue to the catalytic anionic site.
Keywords: Scopoletin, pyridinium derivatives, acetylcholinesterase inhibitor, synthesis

1. Introduction
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the brain,
which most commonly leads to dementia among the elderly. Multiple cognitive impairments
that are characteristics of AD include the loss of memory, judgment, and learning ability. In a
recent report [1], approximately 36 million people worldwide have problems with AD; this
number is estimated to reach 115 million by 2050. Major pathological features of AD are
beta-amyloid (Aβ) plaque deposition and the presence of neurofibrillary tangles in the brain
[2]. Most of the existing treatments can only delay the development of AD and cannot cure
the disease. Among the various biochemical theories regarding the disease, the cholinergic
hypothesis is the most widely accepted. In this hypothesis, the decreased cognitive and
mental function is related to the loss of cortical cholinergic neurotransmission. Rationally, the
inhibition of acetylcholinesterase (AChE), the enzyme responsible for the degradation of the
neurotransmitter acetylcholine (ACh), can restore and enhance cholinergic
neurotransmission. To date, most of the drugs approved for treating AD are
acetylcholinesterase inhibitors (AChEi), such as galanthamine, donepezil, rivastigmine, and
tacrine (Figure 1) [3]. However, undesirable common side effects including nausea, vomiting
and weight loss were observed with these acetylcholinesterase inhibitors while tacrine was
found to be hepatotoxic [4]. Therefore, the design of novel AChEi agents is still urgently
needed for AD treatment. Based on the three-dimensional structure of the Torpedo
californica acetylcholinesterase (TcAChE) reported in 1991 [5], two binding sites were
evident, namely the active site and the peripheral anionic site (PAS). Experimental studies
have shown that the PAS of AChE interacted with Aβ and thus promoted the formation of the
amyloid fibrils [6]. It has been shown also that the molecules interacting either exclusively
with PAS or with both the active site and PAS can prevent AChE-induced Aβ aggregation
[7]. Therefore, molecules capable of binding to both PAS and the active site are potential
targets for the development of anti-AD agents.
Computational studies revealed that compounds containing planar and aromatic
scaffolds can interact with the PAS of AChE and inhibit the AChE-induced aggregation of
the Aβ peptide [8–9]. Coumarin was one of the compounds possessing this capability [10]
leading to its attractive feature to be further developed as an anti-AD agent. Thereafter, a
number of studies reported the use of coumarin as a scaffold for the AChEi synthesis [7,11-
14]. Scopoletin or 7-hydroxy-6-methoxycoumarin (1, Figure 2) is a phenolic coumarin
isolated from several different species, genera, and families of plants. Its medicinal properties
have been recently reported [15]. Scopoletin is known to possess diverse biological properties
including antimicrobial [16-18], antiviral [19], anti-inflammatory [20], and antiproliferative
[21-22] effects. Over the past decade, the pharmacophore-based virtual screening approach
indicated potent AChE inhibitory activity of scopoletin, although the in vitro tests showed a
rather high IC50 of 168 µM against AChE [23]. In this study, we aim to improve the AChE
inhibitory activity of scopoletin by incorporating different side chains which possess a
quaternary amine or basic nitrogen. We hypothesized that, with the PAS binding capability of
scopoletin and the positively charged side chain, the binding affinity of scopoletin derivatives
with AChE could be improved; the resulting compound could be an effective inhibitor for
this enzyme. Benzyl pyridinium-type side chains were of our interest due to their recently
reported promising inhibitory activity [24-26]. Therefore, scopoletin derivatives linked with
different benzyl pyridinium moiety were synthesized and their inhibitory activity against
AChE was evaluated.

2. Results and discussion
2.1 Chemistry
Scopoletin (1) was prepared from 2,4,5-trimethoxybenzaldehyde (2) in two steps [27]
as shown in Scheme 1. Treatment of 2 with 4 equiv. of aluminum (III) chloride in
dichloromethane led to the formation of an inseparable mixture of 2,4-dihydroxy-5-
methoxybenzaldehyde (3a) [28] and 2,5-dihydroxy-4-methoxybenzaldehyde (3b) [29] in an
approximate ratio of 5:1. The Wittig reaction of a mixture of 3a and 3b with
ethoxycarbonylmethylenetriphenylphosphorane in N,N-diethylaniline at 180 °C for 4 h
provided a mixture of scopoletin (1) [30] and isoscopoletin (4) [31], which were separated by
silica column chromatography in 70% and 20% yields, respectively.

The methyl pyridine moiety was attached to scopoletin by using 4-
(chloromethyl)pyridine hydrochloride in the presence of cesium carbonate as a base to
furnish compound 5. Compound 5 was converted to the corresponding N-alkyl and N-benzyl
pyridinium salts (6a–6o) by treating 5 with various alkyl and benzyl halides (Scheme 2).
2.2 Inhibition of AChE
The AChE inhibitory activity of all the synthesized scopoletin derivatives was
evaluated according to Ellman’s method [32] using Electrophorus electricus AChE;
donepezil hydrochloride was used as the reference compound. The tested compounds with
more than 50% inhibition (at concentration of 10 µM) were further evaluated for their half
maximal inhibitory concentration (IC50) values, and the results are shown in Table 1.
Scopoletin exhibited only poor inhibitory activity against AChE (23% inhibition at 10 µM),
similar to reported previously [13,23]. Introduction of the 4-pyridyl methylene moiety to the
hydroxy group at C7 via ether yielded compound 5 exhibiting AChE inhibitory activity (21%
inhibition at 10 µM) comparable to that of the parent scopoletin. According to the work by
Nadri [24] and Alipour [25], introducing a benzyl moiety into the pyridine ring to form its
pyridinium salts improved the AChE inhibitory activity. Therefore, in the present study,
different alkyl and benzyl moieties were incorporated into the pyridine ring of compound 5 to
form the corresponding pyridinium salts (6a–6o). The test assays showed that the pyridinium
derivatives with alkyl side chains (6a–6c) exhibited two- to three-fold higher activity than
scopoletin. Further improved activity was obtained when an alkyl benzene was incorporated
instead of the long-chain alkyl side chain. The derivatives 6d and 6f were approximately 7.5-

to 8.5-fold more active than 6a–6c; both derivatives were much more potent than the parent
scopoletin. For further optimization, 6d was selected as the hit compound.
Based on its promising IC50 value (1.170  0.054 µM), 6d was selected for further
modifications. Docking results in literature [25,26] revealed that introducing halogen to the
benzene ring at a proper position can modulate the binding properties of the corresponding
compound at the active site of AChE and leads to higher activity. In the current study,
chlorine and fluorine substituents were attached to the benzyl moiety of 6d at the ortho-,
meta-, and para-positions to yield derivatives 6h–6m. For the chlorine substituent, the ortho-
and meta-position substitution (6h and 6i) gave comparable IC50 values (0.360  0.024 and
0.397  0.023 µM, respectively) which were approximately 15-fold more potent than that of
chlorine substitution at the para-position. For the fluorine substituent, the activity followed
the order of ortho > meta > para. Among these substituents (6k–6m), fluorine substitution at
the ortho-position (6k) displayed the highest potency with IC50 value of 0.215  0.015 µM.
The comparison of 6k and 6d revealed that introducing the fluorine atom at the ortho-
position reduced the IC50 by five-fold, which implied that the fluorine atom enhanced the
binding efficacy in the gorge. In addition, difluorine substituents (6n and 6o) were
synthesized; however, their anti-AChE activities were not significantly different from that of
6k. Interestingly, the IC50 value of 6k was very close to that of tacrine; this value was lower
than that reported for galanthamine (approximately ten-fold) [13].
In addition to AChE assay, all synthesized compounds were subjected to the
butyrylcholinesterase (BuChE) assay. All the compounds (at 10 µM) exhibited less than 50%
inhibition, indicating good selectivity of the compounds in this series for AChE over BuChE.

Table 1. Acetylcholinesterase inhibitory activity of scopoletin derivatives
Compound
R
-
X
-
IC50 (µM) SDa,b
23%c
Scopoletin (1)
-
-
21%
5
CH3
I
I
38%
6a
6b
6c
CH2CH2CH3
CH2CH2CH2CH3
7.504  0.453
8.565  0.122
Br
Br
Br
Br
6d
6e
6f
1.170  0.054
5.048  0.345
1.022  0.147
Br
Br
6g
6h
4.281  0.167
0.360  0.024
Br
6i
0.397  0.023
Br
Br
6j
6.067  0.343
0.215  0.015
0.684  0.031
1.096  0.051
6k
Br
Br
6l
6m
Br
Br
6n
6o
0.340  0.036
0.470  0.023
Donepezil
hydrochloride
0.060  0.005
0.157  0.014
Tacrine

aConcentration of the compound that produced 50% inhibition of enzyme activity.
bResults are expressed as mean ± standard error; the average of triplicate independent experiments.
c% Inhibition at concentration of 10 µM
2.3 Molecular docking studies
Molecular docking studies were performed to study the binding mode of interaction of
the synthesized compounds with AChE. To date, in the protein data bank (PDB), there are
only 3 unliganded states of Electrophorus Electricus acetylcholinesterase (ErAChE)
structures. In addition, the resolutions of these structures are not good with the resolution of
4.20-4.50 Å. Because of the high similarity (about 88% similarity), good resolution (at 2.35
Å) and liganded state with donepezil of Human AChE structure, a crystal structure of
rhAChE in complex with donepezil (PDB code 4ey7) [33] retrieved from the Protein Data
Bank was used in this study. Molecular docking AChE via the GOLD v5.2.2 program [34]
was applied to investigate the orientation of the compounds in the rhAChE binding site.
Compounds 5, 6c, 6d, and 6k were selected as representatives for the docking study.
From the docking results, the docked orientation of donepezil showed a GoldScore of
83.67; root-mean-square deviation (RMSD) had a value of 0.481 Å as compared with the
conformation in the crystal structure, thereby indicating the appropriate method to study the
binding of compounds in the rhAChE binding site. The docked conformations of donepezil
and the synthesized compounds are shown in Figure 3. Compounds 6d and 6k had similar
GoldScores with values of 77.72 and 78.21, respectively, whereas the GoldScore of
compound 6c was relatively lower at 70.53. Compound 5 displayed poor activity and
possessed the lowest GoldScore (60.07) as compared with the other compounds. The docked
orientations of compounds 6c, 6d, and 6k had a similar pattern as the orientation of donepezil
in the binding pocket. The chromene ring of 6c, 6d, and 6k was located close to the position
of the indanone ring of donepezil, whereas the pyridine ring was located close to the position
of the piperidine ring of the same compound. The phenyl ring of these compounds was also
located close to the position of the benzyl ring of donepezil. Given its smaller size relative to
the other compounds, compound 5 aligned with only two-thirds of the compound (indanone
and piperidine rings) as compared with the binding conformation of donepezil. Therefore,
compound 5 lost the – interaction to Trp86 and some important H-bond interactions, which
may have caused the poor inhibitory activity of compound 5.
The structure of human AChE has its active site at the bottom of a deep, narrow gorge
(20Å deep) composed of several major domains. A catalytic triad (CT) that is responsible for
hydrolyzing the ester bond in ACh consists of Ser203, His 447, and Glu334 residues [35].
The catalytic anionic subsite (CAS) in the vicinity of the CT consists of Trp86, Tyr133,
Tyr337, and Phe338 [36]. CAS is responsible for the binding of the quaternary ammonium of
choline through cation– interactions. The acyl pocket which binds to the acetyl group of
ACh consists of Phe295 and Phe297 residues [36]. The oxyanion hole that interacts with the
ACh carbonyl oxygen consists of Gly121, Gly122, and Ala204 [5,37]. In addition to the
active site, another binding site called the “PAS” is located around the entrance of the active
site gorge (18Å away from the active site). The PAS consists of five amino acid residues,
namely, Tyr72, Asp74, Tyr124, Trp286, and Tyr341 [37–38]. It enhances catalytic efficiency

by binding substrate transiently on its way to the active site; thus, it could involve the
substrate inhibition characteristic of AChE [39].
In the crystal structure, donepezil revealed the key interactions of the amino acids in
the binding site as follows: (1) stack interaction between the indanone ring and the PAS
amino acid residue Trp286, (2) stack interaction between the benzyl moiety and the CAS
amino acid residue Trp86, and (3) C-H– interaction between a hydrogen atom in the
piperidine ring and the CAS amino acid residue Tyr337. The H-bond interactions to Tyr124,
Glu202, Trp286, Ser293, Phe295, Tyr 337, and His447 are shown in Figure 4a. For
compounds 6c, 6d, and 6k, the docking results revealed the similar – and H-bond
interactions to the amino acids in the binding pocket, as shown in Figures 4b–4d. Compound
6k (Fig. 4d) is the most potent compound in this study, and three – interactions were found
between (1) the chromene ring and the PAS amino acid residue Trp286, (2) the pink pyridine
ring and the CAS amino acid residue Tyr337, and (3) the phenyl ring and the CAS amino
acid residue Trp86. Two strong H-bond interactions were found between the hydrogen atoms
of the phenyl ring and the carboxylate group of Glu202, an acidic amino acid that is a part of
the hydrogen-bond network in the AChE active center [40]. In addition, the H-bond
interactions were found between the chromene ring 6k, namely, Asp74, Ser293, Val294,
Phe295, and Arg296 and the pyridine hydrogen atom for Tyr 124. The fluorine atom of 6k
was located close to His447 in CT to form H-bond interactions. For compound 6d, the –
interaction of the chromene ring to Trp286 and of the phenyl ring to Trp86 was similar to
those of 6k. The H-bond interactions of the chromene ring in 6d to Tyr124, Ser293, Val294,
Phe 295, and Arg296, as well as those of the phenyl hydrogen atom to Tyr133 and Glu202,
were observed. Compared with 6d, 6k contained a fluorine substituent at the ortho-position
of the benzyl moiety; its activity was much better than that of the unsubstituted compound
(Table 1). From the docking results, 6k possessed an additional H-bond interaction between
fluorine and His447 along with the – interaction of the pyridine ring to Tyr337. In 6d, no
interaction with His 447 was observed because of the absence of fluorine atom, causing the
pyridine ring to flip to a different orientation as shown in Figure 4c. The pyridine ring
flipping caused the loss of the – interaction to Tyr337. This phenomenon could explain the
superior activity of 6k compared with 6d.
In compound 6c, the benzyl moiety was replaced by the alkyl chain; this compound
showed less activity than compounds 6d and 6k, which highlighted the importance of the
benzyl group. Based on the docking conformation of compound 6c, the chromene and
pyridine rings were located in the same region as found in compound 6k. The alkyl chain was
found in the same region of the benzyl group as shown in compound 6k. However, the lack
of a benzyl group caused the loss of – interaction to Trp86 in CAS. Although, the alkyl
chain was also formed during the H-bond interaction to Glu202, this H-bond seemed to be
weaker than that of 6k because of its longer distance.
The docking results also depicted the plausible -cation interaction [25] between the
positively charged nitrogen of compounds 6c, 6d and 6k with Tyr337 in CAS. This
interaction might increase the affinity in inhibition process leading to the increased activity of
the compounds in this series.
The docking study showed that the docked orientation in the binding pocket of
compounds in this series was similar to that of donepezil. The result indicated the –
interaction of the chromene ring to Trp286 in PAS as well as that of the N-benzylpyridinium
moiety to Trp86 and Tyr337 in CAS. The -cation interaction between these compounds with
Tyr 337 was also depicted. The presence of a fluorine substituent at an ortho-position of the

benzyl moiety produced another additional H-bond with His447 and caused the –
interaction of the pyridine ring to Tyr337, which increased the activity in comparison with
the unsubstituted compound.
Figure 3. Docked conformation of donepezil (ball-and-stick model; pink), compounds 6c, 6d, and
6k (stick model; colored atom-type), and compound 5 (gall-and-stick model; green).

(a)
(b)
(c)
(d)
Figure 4. Binding interaction in the rhAChE binding pocket with (a) donepezil, (b) 6c, (c) 6d, and (d)
6k. Distances are shown in angstrom (Å).
3. Conclusions
A series of scopoletin derivatives incorporated with the pyridinium moiety at C-7 was
synthesized and evaluated for their AChE inhibitory activity. Substituents on the nitrogen
atom of the pyridine ring could increase the activity of scopoletin, and the presence of
aromatic substituents improved the activity more than the aliphatic substituents. A N-
benzylpyridinium derivative was selected as the target compound for further activity
improvement. Derivatives with chlorine and fluorine in the ortho-, meta-, and para-positions
were prepared and tested for their activity. Their activity was in the following order:
ortho > meta > para. Among the derivatives, the 2-fluorobenzylpyridinium derivative was
the most potent compound with an IC50 value of 0.215  0.015 µM, which was greatly
improved from that of scopoletin. Docking studies revealed that the scopoletin portion of the
compound could bind to the PAS, whereas the N-benzylpyridinium residue was bound to the
catalytic anionic site of the AChE. Our preliminary screening on BuChE inhibitory activity
showed that the compounds in the synthesized series were selective against AChE over
BuChE.

4. Experimental
4.1 Chemistry
1H- and 13C-NMR spectra were recorded on Brüker AVANCE 300 spectrometer.
Chemical shifts were reported as δ values in ppm relative to tetramethylsilane. Coupling
constants (J) are given in Hz. Mass spectra were obtained on a Finnigan POLARIS Ion Trap
Mass Spectrometer and accurate masses (HRMS) were recorded on a Brüker MicroTOF
Mass Spectrometer. The infrared (IR) spectra were determined in cm−1 with a Perkin-Elmer
Spectrum One FT-IR spectrometer. Column chromatography was performed on Merck silica
gel 60 (70–230 mesh).
2,4-Dihydroxy-5-methoxybenzaldehyde (3a and 3b). Aluminum (III) chloride (5.35 g,
40.0 mmol) was added in portions over 30 min to a stirred solution of 2,4,5-
trimethoxybenzaldehyde (2.0 g, 10.0 mmol) in dichloromethane (120 ml). After stirring at
room temperature for 24 h, the reaction mixture was placed on ice; concentrated hydrochloric
acid was added to adjust the pH to 1. The dichloromethane layer was separated and washed
with water. The organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure to produce a mixture of 3a and 3b in a ratio of 5:1 (1.5
g, 89%).
Scopoletin (1) and isoscopoletin (4). A mixture of 3a and 3b (1.00 g, 5.95 mmol) and
(ethoxycarbonylmethylene)triphenylphosphorane (2.49 g, 7.14 mmol) in N,N-diethylaniline
(40 ml) was heated at 180 °C for 6 h. After cooling, the reaction was washed with 10%
aqueous hydrochloric acid and extracted with ethyl acetate for several times. The organic
layer was separated and washed with water, dried over anhydrous sodium sulfate, filtered,
and concentrated under reduced pressure. The residue was purified by column
chromatography to yield a pale yellow solid (0.80 g, 70%) of 1 and (0.24 g, 20%) of 4.
6-Methoxy-7-(pyridine-4-yl-oxy-methyl)coumarin (5). A mixture of scopoletin (0.50 g,
2.60 mmol) and Cs2CO3 (1.75 g, 10.4 mmol) in N,N-dimethylformamide (15 ml) was
refluxed for 30 min. A solution of 4-(chloromethyl)pyridine hydrochloride (0.64 mg,
3.90 mmol) in N,N-dimethylformamide (10 ml) was then added to the reaction mixture, and
the mixture was refluxed further for 3 h. After cooling to the ambient temperature, the
reaction mixture was diluted with water and extracted with ethyl acetate. The combined
organic layer was washed with water, dried over anhydrous sodium sulfate, filtered, and
concentrated under reduced pressure. The residue was recrystallized with dichloromethane-
methanol to yield 5 as yellow crystals (0.62 g, 84%). Mp. 181.5-182.5 oC. 1H-NMR (DMSO-
d6, 300 MHz), δ: 8.60 (d, J = 3.6 Hz, 2H), 7.95 (d, J = 9.3 Hz, 1H), 7.44 (d, J = 3.6 Hz, 2H),
7.30 (s, 1H), 7.13 (s, 1H), 6.31 (d, J = 9.3 Hz, 1H), 5.29 (s, 2H), 3.85 (s, 3H); 13C-NMR
(DMSO-d6, 75 MHz), δ: 160.9, 151.3, 150.3, 149.5, 146.5, 145.7, 144.7, 122.3, 113.5, 112.2,
109.9, 101.9, 69.9, 56.5; FT-IR, νmax (cm−1): 1725, 1603, 1562, 1513, 1377, 1278, 1148,
1143, 816; MS (EI), m/z (relative intensity): 283 (M+, 100), 191 (57), 163 (39), 149 (27), 135
(30), 92 (45), 69 (39); HRMS (APCI), m/z calcd. for C16H14NO4 [M + H]+: 284.0917,
measured: 284.0912.
General procedure for alkylation (Method A). A solution of 5 (1 eq) and alkyl halide (3 eq)
in dichloromethane was stirred at room temperature for 24 h. The precipitates were then
filtered and washed with dichloromethane to give the solid salts.

General procedure for alkylation (Method B). A solution of 5 (1 eq) and alkyl halide (3 eq)
in toluene was refluxed for 24 h. Subsequently, the precipitates were filtered and washed with
dichloromethane to give the solid salts.
1-Methyl-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium iodide (6a). Prepared from 5
and methyl iodide following Method A, 6a was obtained as a brown solid (65%).1H-NMR
(DMSO-d6, 300 MHz), δ: 8.97 (d, J = 3.6 Hz, 2H), 8.09 (d, J = 3.6 Hz, 2H), 7.99 (d,
J = 9.3 Hz, 1H), 7.39 (s, 1H), 7.14 (s, 1H), 6.35 (d, J = 9.3 Hz, 1H), 5.62 (s, 2H), 4.34 (s,
3H), 3.88 (s, 3H); 13C-NMR (DMSO-d6, 75 MHz), δ: 160.3, 155.5, 150.0, 148.9, 146.1,
145.5, 144.2, 124.7, 113.5, 112.3, 109.7, 101.8, 67.8, 56.2, 47.7; FT-IR, νmax (cm−1): 1707,
1615, 1561, 1421, 1387, 1278, 1142, 1018, 825; MS (EI), m/z (relative intensity): 298 ([M]+,
3), 284 (7), 227 (18), 193 (19), 149 (41), 104 (100), 73 (81); HRMS (APCI), m/z calcd. for
C17H16NO4 [M+]: 298.1074, measured: 298.1069.
1-Propyl-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium iodide (6b). Prepared from 5
and propyl iodide following Method B, 6b was obtained as a brown solid (40%).1H-NMR
(DMSO-d6, 300 MHz), δ: 9.08 (br s, 2H), 8.13 (br s, 2H), 7.99 (d, J = 9.0 Hz, 1H), 7.39 (s,
1H), 7.17 (s, 1H), 6.36 (d, J = 9.0 Hz, 1H), 5.62 (s, 2H), 4.65–4.50 (br peak, 3H), 3.88 (s,
3H), 2.00–1.85 (br peak, 2H), 1.00–0.80 (br peak, 3H); 13C-NMR (DMSO-d6, 75 MHz), δ:
160.8, 156.5, 150.6, 149.4, 146.5, 145.2, 144.6, 125.5, 114.0, 112.8, 110.2, 102.4, 68.4, 62.3,
56.7, 24.5, 10.7; FT-IR, νmax (cm−1): 1720, 1616, 1564, 1426, 1388, 1278, 1247, 1173, 1145,
1021, 825; MS (EI), m/z (relative intensity): 326 ([M]+, 4), 284 (36), 256 (35), 213 (26), 185
(30), 149 (33), 129 (78), 73 (100); HRMS (APCI), m/z calcd. for C19H20NO4 [M+]: 326.1387,
measured: 326.1376.
1-Butyl-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6c). Prepared from
5 and n-butyl bromide following Method B, 6c was obtained as a brown solid (32%).1H-
NMR (DMSO-d6, 300 MHz), δ: 9.08 (br s, 2H), 8.12 (br s, 2H), 7.99 (d, J = 8.7 Hz, 1H), 7.39
(s, 1H), 7.17 (s, 1H), 6.36 (d, J = 8.7 Hz, 1H), 5.62 (s, 2H), 4.65–4.50 (br peak, 3H), 3.88 (s,
13
3H), 2.00–1.85 (br peak, 2H), 1.40–1.20 (br peak, 2H), 1.00–0.80 (br peak, 3H); C-NMR
(DMSO-d6, 75 MHz), δ: 160.8, 156.5, 150.6, 149.4, 146.5, 145.2, 144.6, 125.6, 114.0, 112.8,
110.3, 102.4, 68.4, 60.7, 56.7, 33.0, 19.2, 13.8; FT-IR, νmax (cm−1): 1718, 1616, 1562, 1426,
1388, 1281, 1249, 1173, 1145, 1021, 830; MS (EI), m/z (relative intensity): 339 ([M-H]+, 10),
313 (20), 284 (14), 264 (31), 236 (24), 149 (38), 129 (46), 111 (72), 97 (100); HRMS
(APCI), m/z calcd. for C20H22NO4 [M]+: 340.1543, measured: 340.1551.
1-Benzyl-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6d). Prepared from
5 and benzyl bromide following Method A, 6d was obtained as a yellow solid (56%).1H-
NMR (DMSO-d6, 300 MHz), δ: 9.21 (br s, 2H), 8.14 (br s, 2H), 7.99 (d, J = 9.0 Hz, 1H),
7.60–7.40 (m, 6H), 7.17 (s, 1H), 6.35 (d, J = 9.0 Hz, 1H), 5.88 (s, 2H), 5.62 (s, 2H), 3.87 (s,
3H);13C-NMR (DMSO-d6, 75 MHz), δ: 160.3, 156.7, 150.1, 148.9, 146.0, 144.8, 144.2,
134.2, 129.4, 129.2, 128.8, 125.4, 113.5, 112.4, 109.8, 101.9, 67.9, 62.9, 56.2; FT-IR, νmax
(cm−1): 1712, 1614, 1563, 1513, 1428, 1389, 1282, 1251, 1150, 1022, 882, 737; MS (EI), m/z
(relative intensity): 281 (14), 207 (84), 148 (27), 129 (26), 104 (100), 98 (56); HRMS
(APCI), m/z calcd. for C23H20NO4 [M+]: 374.1387, measured: 374.1379.
1-Phenylethyl-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6e). Prepared
from 5 and (2-bromoethyl)benzene following Method B, 6e was obtained as a brown solid
(92.9%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.01 (d, J = 4.8 Hz, 2H), 8.08 (d, J = 4.8 Hz,
2H), 8.00 (d, J = 9.3 Hz, 1H), 7.45–7.10 (m, 7H), 6.36 (d, J = 9.3 Hz, 1H), 4.95–4.80 (br
peak, 2H), 3.35–3.20 (br peak, 2H), 3.88 (s, 3H); 13C-NMR (DMSO-d6, 75 MHz), δ: 160.3,

156.2, 150.0, 148.9, 146.1, 144.7, 144.2, 136.1, 128.8, 128.6, 127.1, 124.9, 113.5, 112.4,
109.8, 101.9, 67.9, 61.2, 56.2, 36.3; FT-IR, νmax (cm−1): 1710, 1617, 1563, 1512, 1427, 1387,
1280, 1248, 1147, 1018, 820, 704; MS (EI), m/z (relative intensity): 368 (10), 192 (100), 177
(56), 164 (25), 149 (45), 106 (35), 69 (37), 65 (24); HRMS (ESI), m/z calcd. for C24H22NO4
[M+]: 388.1543, measured: 388.1557.
1-(3-Phenylpropyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6f).
Prepared from 5 and 1-bromo-3-phenylpropane following Method B, 6f was obtained as a
brown solid (35%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.10 (d, J = 6.3 Hz, 2H), 8.10 (d,
J = 6.3 Hz, 2H), 8.00 (d, J = 9.6 Hz, 1H), 7.40 (s, 1H),7.30–7.15 (m, 6H), 6.35 (d, J = 9.3 Hz,
1H), 5.62 (s, 2H), 4.70–4.60 (m, 2H), 3.88 (s, 3H), 2.70–2.60 (m, 2H), 2.35–2.20 (m, 2H);
13C-NMR (DMSO-d6, 75 MHz), δ: 160.3, 156.0, 150.1, 148.9, 146.1, 144.8, 144.2, 140.2,
128.4, 128.2, 126.1, 125.0, 113.5, 112.4, 109.7, 101.9, 67.9, 60.3, 56.2, 31.9, 31.6; FT-IR,
νmax (cm−1): 1727, 1424, 1387, 1280, 1249, 1176, 1151, 1023, 821, 699; MS (EI), m/z
(relative intensity): 368 (14), 192 (100), 177 (51), 164 (25), 149 (49), 57 (34); HRMS (ESI),
m/z calcd. for C25H24NO4 [M+]: 402.1700, measured: 402.1706.
1-(Naphthalen-2ylmethyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide
(6g). Prepared from 5 and 2-(bromomethyl)naphthalene following Method A, 6g was
obtained as a yellow solid (71%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.26 (d, J = 6.0 Hz,
2H), 8.15 (d, J = 6.0 Hz, 2H), 8.10 (s, 1H), 8.05–7.90 (m, 4H), 7.65–7.50 (m, 3H), 7.38 (s,
1H),7.17 (s, 1H), 6.34 (d, J = 9.6 Hz, 1H), 6.05 (s, 2H),5.26 (s, 2H), 3.87 (s, 3H); 13C NMR
(DMSO-d6, 75 MHz), δ: 160.3, 156.7, 150.0, 148.9, 146.0, 144.9, 144.1, 132.9, 132.7, 131.6,
129.1, 128.5, 128.0, 127.7, 127.1, 126.9, 125.8, 125.4, 113.5, 112.3, 109.8, 101.9, 67.9, 63.1,
56.2; FT-IR, νmax (cm−1): 1724, 1424, 1387, 1284, 1250, 1176, 1143, 1019, 825; MS (EI), m/z
(relative intensity): 368 (21), 236 (13), 192 (10), 141 (100), 115 (26), 57 (28); HRMS (ESI),
m/z calcd. for C27H22NO4 [M+]: 424.1543, measured: 424.1555.
1-(2-Chlorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6h).
Prepared from 5 and 2-chlorobenzyl bromide following Method A, 6h was obtained as a
brown solid (74%).1H-NMR (DMSO-d6, 300 MHz), δ: 9.10 (d, J = 6.6 Hz, 2H), 8.15 (d,
J = 6.6 Hz, 2H), 7.99 (d, J = 9.6 Hz, 1H), 7.62–7.45 (m, 4H), 7.39 (s, 1H), 7.18 (s, 1H), 6.35
(d, J = 9.6 Hz, 1H), 6.00 (s, 2H), 5.64 (s, 2H), 3.88 (s, 3H); 13C-NMR (DMSO-d6, 75 MHz),
δ: 160.3, 157.1, 150.0, 148.9, 146.1, 145.1, 144.1, 133.3, 131.7, 131.5, 131.3, 130.1, 128.2,
125.3, 113.5, 112.4, 109.8, 101.9, 67.9, 60.9, 56.2; FT-IR, νmax (cm−1): 1723, 1387, 1282,
1250, 1147, 1017, 827, 757; MS (EI), m/z (relative intensity): 283 (35), 192 (22), 177 (10),
149 (16), 125 (100), 89 (25); HRMS (ESI), m/z calcd. for C23H19ClNO4 [M+]: 408.0997,
measured: 408.1006.
1-(3-Chlorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6i).
Prepared from 5 and 3-chlorobenzyl bromide following Method A, 6i was obtained as a
brown solid (60%).1H-NMR (DMSO-d6, 300 MHz), δ: 9.21 (d, J = 6.6 Hz, 2H), 8.14 (d,
J = 6.6 Hz, 2H), 7.99 (d, J = 9.6 Hz, 1H), 7.70 (s, 1H), 7.54–7.46 (m, 3H), 7.39 (s, 1H), 7.17
(s, 1H), 6.35 (d, J = 9.6 Hz, 1H), 5.87 (s, 2H), 5.75 (s, 2H), 3.88 (s, 3H); 13C-NMR (DMSO-
d6, 75 MHz), δ: 160.3, 156.8, 150.0, 148.9, 146.0, 144.8, 144.1, 136.3, 133.7, 131.1, 129.4,
128.9, 127.6, 125.4, 113.5, 112.4, 109.8, 101.9, 67.9, 62.0, 56.2; FT-IR, νmax (cm−1): 1712,
1427, 1389, 1282, 1250, 1175, 1147, 1018, 828, 765; MS (EI), m/z (relative intensity): 283
(24), 192 (42), 177 (24), 149 (26), 125 (100); HRMS (ESI), m/z calcd. for C23H19ClNO4
[M+]: 408.0997, measured: 408.1007.

1-(4-Chlorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6j).
Prepared from 5 and 4-chlorobenzyl bromide following Method A, 6j was obtained as a
brown solid (47%).1H-NMR (DMSO-d6, 300 MHz), δ: 9.19 (d, J = 5.1 Hz, 2H), 8.14 (d,
J = 5.1 Hz, 2H), 7.99 (d, J = 9.3 Hz, 1H), 7.65–7.50 (m, 5H), 7.39 (s, 1H), 7.16 (s, 1H), 6.35
(d, J = 9.3 Hz, 1H), 5.88 (s, 2H), 5.62 (s, 2H), 3.87 (s, 3H); 13C-NMR (DMSO-d6, 75 MHz),
δ: 160.3, 156.7, 150.0, 148.9, 146.0, 144.8, 144.1, 133.2, 133.0, 130.9, 129.2, 125.4, 113.5,
112.4, 109.8, 101.9, 67.9, 62.0, 56.2; FT-IR, νmax (cm−1): 1715, 1385, 1280, 1249, 1146,
1094, 1017, 824; MS (EI), m/z (relative intensity): 283 (37), 192 (60), 177 (30), 164 (17), 149
(35), 125 (100), 89 (15); HRMS (ESI), m/z calcd. for C23H19ClNO4 [M+]: 408.0997,
measured: 408.0997.
1-(2-Fluorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6k).
Prepared from 5 and 2-fluorobenzyl bromide following Method A, 6k was obtained as a
brown solid (61%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.13 (d, J = 6.3 Hz, 2H), 8.15 (d,
J = 6.3 Hz, 2H), 7.99 (d, J = 9.6 Hz, 1H), 7.65–7.52 (m, 2H), 7.39–7.30 (m, 3H), 7.17 (s,
1H), 6.35 (d, J = 9.6 Hz, 1H), 5.97 (s, 2H), 5.63 (s, 2H), 3.88 (s, 3H); 13C-NMR (DMSO-d6,
75 MHz), δ: 161.0 (d, 1JF,C = 246 Hz), 160.8, 157.5, 150.5, 149.4, 146.5, 145.4, 144.6, 132.7
(d, 3JF,CH = 8.4 Hz), 132.0 (d, 4JF,CH = 3.0 Hz), 125.9, 125.8 (d, 3JF,CH = 3.6 Hz), 121.6 (d,
2JF,C = 14.5 Hz), 116.5 (d, 2JF,CH = 20.3 Hz), 114.0, 112.9, 110.3, 102.4, 68.4, 58.1 (d,
3JF,CH2 = 2.6 Hz), 56.7; FT-IR, νmax (cm−1): 1717, 1616, 1427, 1389, 1283, 1250, 1146, 1019,
829, 767; MS (EI), m/z (relative intensity): 283 (44), 192 (53), 177 (24), 149 (31), 109 (100);
HRMS (ESI), m/z calcd. for C23H19FNO4 [M+]: 392.1293, measured: 392.1295.
1-(3-Fluorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6l).
Prepared from 5 and 3-fluorobenzyl bromide following Method A, 6l was obtained as a
brown solid (55%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.19 (d, J = 5.7 Hz, 2H), 8.14 (d,
J = 5.7 Hz, 2H), 7.99 (d, J = 9.6 Hz, 1H), 7.53–7.28 (m, 5H), 7.17 (s, 1H), 6.35 (d,
J = 9.6 Hz, 1H), 5.88 (s, 2H), 5.62 (s, 2H), 3.87 (s, 3H); 13C-NMR (DMSO-d6, 75 MHz), δ:
162.2 (d, 1JF,C = 243.5 Hz), 160.3, 156.8, 150.1, 148.9, 146.0, 144.8, 144.1, 136.6 (d,
3JF,CH = 7.7 Hz), 131.4 (d, 3JF,C = 8.3 Hz), 125.4, 125.0 (d, 4JF,CH = 2.8 Hz), 116.3 (d,
2JF,CH = 20.8 Hz), 115.9 (d, 2JF,CH = 22.3 Hz), 113.5, 112.4, 109.8, 101.9, 67.9, 62.2, 56.2;
FT-IR, νmax (cm−1): 1715, 1615, 1427, 1389, 1283, 1250, 1146, 1019, 830, 767; MS (EI), m/z
(relative intensity): 283 (43), 192 (67), 177 (30), 149 (37), 109 (100); HRMS (ESI), m/z
calcd. for C23H19FNO4 [M+]: 392.1293, measured: 392.1304.
1-(4-Fluorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6m).
Prepared from 5 and 4-fluorobenzyl bromide following Method A, 6m was obtained as a
brown solid (79%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.21 (d, J = 6.6 Hz, 2H), 8.14 (d,
J = 6.6 Hz, 2H), 7.99 (d, J = 9.6 Hz, 1H), 7.66 (dd, J = 8.7, 5.4 Hz, 2H), 7.39 (s, 1H), 7.34–
7.27 (m, 2H), 7.16 (s, 1H), 6.35 (d, J = 9.6 Hz, 1H), 5.87 (s, 2H), 5.62 (s, 2H), 3.87 (s, 3H);
13C-NMR (DMSO-d6, 75 MHz), δ: 162.6 (d, 1JF,C = 245 Hz), 160.3, 156.6, 150.0, 148.9,
146.0, 144.7, 144.2, 131.5 (d, 3JF,CH = 8.6 Hz), 130.4 (d, 4JF,C = 3.0 Hz), 125.4, 116.2 (d,
2JF,CH = 21.7 Hz), 113.5, 112.4, 109.7, 101.9, 67.9, 62.0, 56.2; FT-IR, νmax (cm−1): 1728,
1507, 1427, 1389, 1283, 1251, 1148, 1020, 830, 776; MS (EI), m/z (relative intensity): 283
(31), 192 (57), 177 (29), 149 (35), 109 (100); HRMS (ESI), m/z calcd. for C23H19FNO4 [M+]:
392.1293, measured: 392.1294.
1-(2,3-Difluorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6n).
Prepared from 5 and 2,3-difluorobenzyl bromide following Method A, 6n was obtained as a
yellow solid (56%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.15 (d, J = 6.6 Hz, 2H), 8.16 (d,
J = 6.6 Hz, 2H), 7.99 (d, J = 9.3 Hz, 1H), 7.62–7.52 (m, 1H), 7.45–7.30 (m, 3H), 7.17 (s,

1H), 6.35 (d, J = 9.3 Hz, 1H), 6.03 (s, 2H), 5.64 (s, 2H), 3.88 (s, 3H); 13C-NMR (DMSO-d6,
75 MHz), δ: 160.3, 157.1, 150.0, 149.7 (dd, 1JF,C = 245.4 Hz, 2JF,C = 11.9 Hz), 148.9, 148.4
(dd, 1JF,C = 245.4 Hz, 2JF,C = 13.2 Hz), 146.0, 145.0, 144.1, 126.9 (dd, 3JF,CH = 3.4 Hz,
4JF,CH = 2.0 Hz), 125.7 (dd, 3JF,CH = 7.0 Hz, 4JF,CH = 4.6 Hz), 125.4, 123.4 (d,
2JF,C = 11.0 Hz), 119.1 (d, 2JF,CH = 16.7 Hz), 113.5, 112.4, 109.8, 101.9, 67.9, 56.9, 56.2; FT-
IR, νmax (cm−1): 1728, 1386, 1282, 1252, 1150, 1022, 824, 775; MS (EI), m/z (relative
intensity): 410 (2), 360 (11), 249 (22), 149 (70), 81 (96), 69 (100); HRMS (APCI), m/z calcd.
for C23H18F2NO4 [M+]: 410.1198, measured: 410.1207.
1-(2,6-Difluorobenzyl)-4-(((6-methoxycoumarin-7-yl)oxy)methyl)pyridinium bromide (6o).
Prepared from 5 and 2,6-difluorobenzyl bromide following Method A, 6o was obtained as a
yellow solid (47%). 1H-NMR (DMSO-d6, 300 MHz), δ: 9.08 (d, J = 6.6 Hz, 2H), 8.13 (d,
J = 6.6 Hz, 2H), 7.99 (d, J = 9.3 Hz, 1H), 7.65–7.59 (m, 1H), 7.39 (s, 1H), 7.31–7.25 (m,
2H), 7.16 (s, 1H), 6.35 (d, J = 9.3 Hz, 1H), 6.01 (s, 2H), 5.64 (s, 2H), 3.87 (s, 3H); 13C-NMR
(DMSO-d6, 75 MHz), δ: 160.9 (d, 1JF,C = 248 Hz), 160.8 (d, 1JF,C = 249 Hz), 160.3, 157.2,
150.0, 148.9, 146.0, 145.0, 144.2, 133.2 (d, 2JF,C = 21.1 Hz), 133.2, 125.4, 113.5, 112.4 (d,
2JF,CH = 24.3 Hz), 112.4, 109.8, 101.9, 67.9, 56.2, 51.7; FT-IR, νmax (cm−1): 1717, 1386, 1279,
1249, 1150, 1019, 824, 799; MS (EI), m/z (relative intensity): 410 (2), 368 (7), 236 (9), 149
(28), 136 (30), 95 (34), 81 (86), 69 (100); HRMS (APCI), m/z calcd. for C23H18F2NO4 [M+]:
410.1198, measured: 410.1208.
4.2 AChE inhibition assay
AChE (Electrophorus electricus, Type VI-S, lyophilized powder) was purchased from
Sigma-Aldrich, USA. Acetylthiocholine, 5,5′-dithiobis-(2-nitrobenzoic acid), sodium
dihydrogen phosphate, disodium hydrogen phosphate, and sodium hydrogen carbonate were
obtained from Fluka, Switzerland. AChE inhibitory activity was measured with a Molecular
Devices Spectra Max Plus 384 spectrophotometer (CA, USA) based on Ellman’s method
with slight modification. For each assay, 200 μl of the assay medium containing 100 μl of
0.1 M phosphate buffer (pH 8), 20 μl of 3.3 mM 5,5′-dithiobis(2-nitrobenzoic acid) in 0.1 M
phosphate buffer (pH 7) containing 6 mM NaHCO3, 20 μl of the test compounds at different
concentrations in methanol, and 40 μl of 0.1 unit/ml AChE in phosphate buffer (pH 8) were
incubated at 37 °C for 20 min. Subsequently, 20 μl of 5 mM acetylthiocholine iodide in water
was added, and the absorbance was measured at 412 nm every 20 s for 3 min. The rate of
each reaction was calculated by Microplate Manager software. The blank percentage of
inhibition of the tested compounds was determined by comparing the rate of the sample to the
blank. The IC50 value was determined by non-linear regression of the log inhibitor
concentration versus the percentage of inhibition using GraphPad Prism6. Donepezil
hydrochloride was applied as the standard compound. All samples were assayed in triplicate.
4.3 Molecular docking studies
The compounds were constructed in the DS Visualizer program. These structures
were optimized by the B3LYP/6-31G** method using the Gaussian 09 program [41]. To
prepare the rhAChE structure, the crystal structure of the rhAChE in complex with donepezil
(PDB code 4ey7) was retrieved from the Protein Data Bank. From the crystal structure, the
binding of donepezil indicated the presence of a binding site in rhAChE. Molecular docking
with the GOLD v5.2.2 program was applied to investigate the orientation of the compounds

in the rhAChE binding site. This binding site is defined by the center of mass of donepezil;
this expanded from the center with a radius of 10 Å. The default parameters of the automatic
settings and a genetic algorithm (GA) run of 15 were used for setting the GA parameters. The
GoldScore fitness function was used to determine the fitness score of the docked compound.
The parameters were validated by docking donepezil back into the binding site and
evaluating their effects as RMSD. Subsequently, these GA parameters were applied to dock
compounds into the rhAChE binding site. The conformation with the highest GoldScore was
selected for the analysis of the interaction between surrounding amino acids.
Acknowledgement
We gratefully acknowledged Dr. Poonsakdi Ploypradith at the Chulabhorn Research
Institute for a critical reading of the manuscript. We also thank Miss Kittiporn Trisupphakant
for recording EI-MS and IR spectra data.
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(a)
(b)
(c)
(d)

Highlights
 Scopoletin derivatives with pyridinium moiety were synthesized as AChE inhibitors.
 2-Fluorobenzylpyridinium derivative showed the strongest activity (IC50=0.215 µM).
 Docking studies showed that scopoletin portion was bound to the PAS of the AChE.
 From docking results, N-benzylpyridinium residue was bound to the CAS of enzyme.
 The compounds in the synthesized series were selective against AChE over BuChE.

IC50 = 0.215  0.015 µM