Synthesis and cholinesterase inhibitory activity of new 2-benzofuran carboxamide-benzylpyridinum salts

Article abstract of DOI:10.1016/j.bioorg.2018.06.006

A series of benzofuran-2-carboxamide-N-benzyl pyridinium halide derivatives (6a-o) are synthesized as new cholinesterase inhibitors. The synthetic pathway involves the reaction of salicylaldehyde derivatives and ethyl bromoacetate, followed by hydrolysis and amidation with 3- and 4-picolyl amine. Subsequently, N-((pyridin-4-yl) methyl) benzofuran-2-carboxamide and substituted N-((pyridin-3-yl) methyl) benzofuran-2-carboxamides reacts with benzyl halides to afford target compounds (6a-o). The chemical structures of all derivatives were confirmed by spectroscopic methods. The studies reveal that some of the synthesized compounds are potent butyrylcholinesterase inhibitors with IC₅₀ values in the range of 0.054–2.7 μM. In addition, good inhibitory effects on Aβ self-aggregation are observed for 6h and 6k (33.1 and 46.4% at 100 μM, respectively).

Full text of DOI:10.1016/j.bioorg.2018.06.006

Accepted Manuscript
Synthesis and anticholinesterase inhibitory activity of new 2-benzofuran car-
boxamide-benzylpyridinum salts
Fahimeh Abedinifar, S. Morteza F. Farnia, Mohammad Mahdavi, Hamid Nadri,
Alireza Moradi, Jahan B. Ghasemi, Tuba Tüylü Kü ç ükkılınç, Loghman
Firoozpour, Alireza Foroumadi
PII:
S0045-2068(18)30312-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.06.006
YBIOO 2386
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
1 April 2018
22 May 2018
3 June 2018
Please cite this article as: F. Abedinifar, S. Morteza F. Farnia, M. Mahdavi, H. Nadri, A. Moradi, J.B. Ghasemi, T.
Tüylü Kü ç ükkılınç, L. Firoozpour, A. Foroumadi, Synthesis and anticholinesterase inhibitory activity of new 2-
benzofuran carboxamide-benzylpyridinum salts, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.06.006
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Synthesis and anticholinesterase inhibitory activity of new 2-benzofuran
carboxamide-benzylpyridinum salts
a
a
b
c
Fahimeh Abedinifar, S. Morteza F. Farnia, * Mohammad Mahdavi, Hamid Nadri, Alireza
c
a
d
e
Moradi, Jahan B. Ghasemi, Tuba Tüylü Küçükkılınç, Loghman Firoozpour, Alireza
f,g
Foroumadi*
a
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
b
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
d
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
e
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences
(TIPS), Tehran University of Medical Sciences, Tehran, Iran
f
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
g
Department of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center,
Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
*Corresponding authors
E-mail addresses: aforoumadi@yahoo.com (A. Foroumadi); mfarnia@khayam.ut.ac.ir (S.
Morteza F. Farnia)
This paper is dedicated to the memory of the late Professor Abbas Shafiee at Tehran
University of Medical Sciences.
1

Abtract: A series of benzofuran-2-carboxamide-N-benzyl pyridinium derivatives (6a-o) are
synthesized as new cholinesterase inhibitors. The synthetic pathway involves the reaction of
salicylaldehyde derivatives and ethyl bromoacetate, followed by hydrolysis and amidation
with 3- and 4-picolyl amine. Subsequently, N-((pyridin-4-yl) methyl) benzofuran-2-
carboxamide and substituted N-((pyridin-3-yl) methyl) benzofuran-2-carboxamides reacts
with benzyl halides to afford target compounds (6a-o). The chemical structures of all
derivatives are confirmed by spectroscopic methods. The studies revealed that some of the
synthesized compounds exhibited potent butyrylcholinesterase inhibitory activities in the
range of 0.054-2.7 µM. In addition, good inhibitory effects on Aβ self-aggregation are
observed for 6h and 6k (33.1 and 46.4 % at 100 µM, respectively).
Keywords:
Alzheimer’s
disease,
Benzofuran-2-carboxamide,
Benzylpyridinium,
Cholinesterase inhibitor.
2

1
. Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease, classified as the
fourth leading cause of illness and death in people at old ages in developed countries.
Scientists predict that the number of people, suffering from this disease will be nearly tripled
by 2050 [1]. Alzheimer is a progressive disorder, characterized by loss of cognitive abilities,
severe behavioral disorders and ultimately death. Low levels of acetylcholine (ACh) [2, 3], β-
amyloid (Aβ) deposits [4], metal-ion imbalance [5, 6] and many other factors have been
considered as important pathogenesis in AD [7-9].
The cognitive impairment in AD patients is due to the loss of cholinergic neurons and
subsequently reduction in acetylcholine levels in the specific regions of the brain [10].
Therefore, according to cholinergic hypothesis, the reduced levels of acetylcholine in the
brain have been introduced as the most important target among the treatment strategies [11].
The structures of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are very
similar, expressing 65% identity in amino acid sequence [12]. The inhibition of
acetylcholinesterase and butyrylcholinesterase enzymes led to the alleviation of the
Alzheimer’s symptoms, including cognitive level and short-term memory, by restoring ACh
level. The increased levels of BuChE at the later stages of AD in the hippocampus and
temporal cortex highlighted the importance of selective BuChE inhibitors in regulating the
ACh level in cholinergic neurons. This fact was also proved by Mesulam’s experiments with
nullizygous and wild-type mice, exhibiting the constitutive function of BuChE in the
hydrolysis of ACh in the normal brain [13]. Relied on multifactorial nature of AD, the
synchronous inhibition, which is achieved by AChE and BChE inhibitors may be beneficial
for AD treatment [14-16].
Another crucial hypothesis for AD suggested that the aggregation and accumulation of β-
amyloid peptide in a brain could lead to toxic fibrils and consequently neuronal cell death
[
17-20]. Many strategies are currently being assessed for preventing the formation of
amyloid and toxic oligomers. Several classes of small molecules have been reported in the
literature, capable of preventing or reversing fibrillization [21-22], while none of them are
successful to be moved to clinical phase.
3

Therefore, design and synthesis of new compounds with dual inhibitory activity may be
promising in AD treatment [23]. Benzofurans are one of the most significant heterocyclic
categories with wide range of bioactivities [24]. In addition, benzofuran-2-carboxamide
derivatives are also known as antitumor [25-28], antimicrobial [29], anti-hyperlipidemic [30],
AChE inhibitor [31], antibacterial [32] and suppressing agent in allergic rhinitis [33].
Recently, various benzofuran-based compounds were reported as potent acetylcholinesterase
inhibitors. Considering the importance of heterocyclic compounds by our research team [34-
4
0], herein we report the synthesis and anticholinesterase inhibitory activity of a series of
derivatives bearing N-benzylpyridinium and benzofuran backbone which providing
interactions with catalytic active site (CAS) and peripheral anionic site (PAS) of AChE,
respectively (Figure 1) [41, 42]. It is expected that the intrinsic dual mechanism of action
could result in efficient anticholinesterase agent discoveries.
Figure 1. Structure of donepezil hydrochloride as a ChE inhibitor and designed compounds 6a-o as selective
ChE inhibitors
2
. Results and discussion
2.1 Chemistry
The synthetic pathway toward benzofuran-2-carboxamide bearing pyridinium moiety is
shown in scheme 1. The condensation reaction between salicylaldehyde derivatives 1 and
ethyl bromoacetate in the presence of K
2
CO afforded ethyl benzofuran-2-carboxylates [43].
3
Subsequent hydrolysis in aqueous ethanol/KOH afforded 3a-b [44]. The amidation reaction
of 3a-b with 3-(methylamino) pyridine or 4-(methylamino) pyridine in the presence of
4

hydroxybenzotriazole (HOBt) and N-(3-dimethylaminopropyl)-N´-ethylcarbodiimide
hydrochloride (EDC) as coupling agents resulted in 4, 5a-b [45]. The N-benzylation of the
latter compounds with appropriate benzyl halides in acetonitrile at reflux temperature resulted
in benzyl pyridinium halide salts of desired products 6a-6o in good yields [46]
.
Scheme 1. Synthesis of benzofuran-2-carboxamide 6a-o: (a) Ethyl bromoacetate, K
h; (b) KOH, Ethanol: H O (2:1), reflux; (c) (Pyridin-3-yl)methanamine or (pyridin-4-yl)methanamine; HOBT,
EDC, Dry CH CN, r.t., 24-48 h; (d) substituted benzyl halides, CH CN, reflux, 2-4 h.
2 3
CO , Dry DMF, 90 ˚C, 4-6
2
3
3
The presence of electron-donating groups including Me or OMe at the ortho, meta or para
positions of benzyl moiety decreased the reaction yield. However, the difference in electronic
5

characters of substitutions, electron-donating and electron-withdrawing groups, had no
distinguishing influence on the isolated yields.
2.2 Cholinesterase activity evaluation
Inhibitory activities of 6a-o series of benzofuran-carboxamide benzyl pyridinium derivetives
towards AChE and BChE were measured by modified Ellman’s protocol [47]. The results are
summarized in Table 1 and are reported as IC50 and the percent of inhibition at 56 µM for
target compounds (Table 1).
Table 1. Cholinesterase inhibitory activity of the synthesized compounds (6a-6o)
R1
1
7
O
1
7
O
O
X
6
O
a
c
6
5
b
N
a
N
N
H
5
H
3
d
R2
3
4
b
4
d
N
c
R2
6a-6m
6n-6o
X
a
Compound
R
1
R
2
X
AChE inhibition
BChE inhibition
6
6
6
a
b
c
H
H
H
H
H
H
H
H
OMe
OMe
OMe
OMe
OMe
H
H
3-Me
4-Me
2-NO
4-NO
4-F
2,4-Cl
2-F-6-NO
H
3-OMe
2-Me
2,4-Cl
Br
Br
Br
Br
Br
Br
Cl
Br
Cl
Cl
Br
Cl
Br
Br
Br
5.5±0.3
13.8±0.5
19.8±0.8
3.5±0.3
(40.0±4.0)
12.3±0.3
4.4±0.3
33.8±1.2
4.7±0.4
(2.0±0.8)
2.1±0.1
0.29±0.01
0.11±0.01
0.65±0.04
0.15±0.01
0.76±0.05
0.37±0.01
0.37±0.01
0.054±0.002
0.87±0.07
9.6±0.3
6d
2
6
6
e
f
g
2
6
6
2
h
2
6
6
i
j
6k
0.45±0.05
0.18±0.01
2.45±0.30
2.7±0.2
6
l
2
14.5±1.0
29.3±2.8
61.0±1.6
(44.0±5.0)
6
m
4-Br
2-Br
4-Br
6n
6
o
H
4.3±0.4
Donepezil
-
-
-
0.031±0.005
5.4±0.1
a
IC50 (µM) or inhibition % at 56.3 µM concentration (in parentheses). Values were the means of three replicates
standard deviation (SD)
±
None of the compounds exhibited higher AChE inhibition compared to donepezil, but all of
them except 6j are better BChE inhibitors. Among the target compounds, 6k showed the best
6

inhibitory activity against AChE and 6h was more potent than donepezil against BChE,
exhibiting IC50 value of 0.054 µM, which is 100 times stronger than positive control.
As depicted in Table 1, in both series, the ortho and meta substituted derivatives exhibit
better inhibition towards AChE and BChE than the para substituted ones. The presence of
strong electron withdrawing groups at para position in 6e decreases the inhibitory activity
against AChE in 3-pyridinium series. As shown in Table 1, the presence of methoxy group at
7
-position of benzofuran leads to the reduced butyrylcholinesterase inhibitory activity (6a vs.
i). The 4-pyridinium series display the weakest inhibitory activity against both AChE and
6
BChE compared to 3-pyridinium series.
To predict BBB penetration and intestinal absorption the admetSAR server was used [48].
Calculated logP and tPSA were retrieved from chembiodraw ultra 14.0 (PerkinElmer). Based
on the obtained data from the server all compounds were predicted as CNS active with
satisfactory probability (Table 2).
Table 2. BBB and HIA prediction. All prediction was performed by admetSAR server.
Blood-Brain
Barrier
probability
0.9915
0.9887
0.9870
0.9564
0.9648
0.9920
0.9824
0.9569
0.9865
0.9656
0.9781
0.9744
0.9806
0.9904
0.9934
0.9953
Human Intestinal
Absorption
probability
0.9791
*
2 a
Compound
ClogP
tPSA(Å )
6
6
a
b
0.226
0.725
0.725
-0.111
-0.031
0.369
1.65
41.34
41.34
41.34
93.15
93.15
41.34
41.34
93.15
50.57
59.8
50.57
50.57
50.57
41.34
41.34
38.77
0.9828
0.9877
0.9837
0.9850
0.9824
0.9810
0.9866
0.9879
0.9483
0.9844
0.9892
0.9857
0.9549
0.9584
0.9966
6
c
6
d
6
e
6
f
6
g
6
h
0.031
0.356
0.275
0.805
1.78
1.21
1.09
1.09
4.59
6
i
6
j
6
k
6
l
6
m
6
6
n
o
Donepezil
a
ClogP and tPSA were calculated by chembiodraw ultra (14.0)
7

2.3 Docking studies
Docking studies were carried out to provide insight into the binding mode of the synthesized
compounds 6h and 6k. In order to validate the docking reliability, root-mean-square distance
(
RMSD) values of 0.774 Å and 0.615 Å were obtained between bounded ligands and the re-
docked ligands tacrine and donepezil, which shows the high reliability of the GOLD method
to reproduce the known binding mode.
According to the docking results, the best docked pose of molecule 6h shows a hydrogen
bond between oxygen atom of benzofuran and Ser198. The positively charged nitrogen
induces the π-cation interactions with aromatic residues Phe329 and Trp82. Moreover, the
benzyl pyridinium section of 6h involves in the - interaction with Tyr332 (Figure 2).
Figure 2. The interacting mode of the most active compound 6h with the active site of BChE.
8

Figure 3. The interacting mode of the most active compound 6k with the active site of AChE.
Amongst the synthetic compounds, 6k (IC50 = 2.1 µM) with methoxy substitution as R and
1
methyl as R showed the most potent compounds toward AChE comparing to other
2
congeners of the series. As depicted in Figure 3, four -cation interactions between
quaternary nitrogen of pyridine ring with Trp86 and Tyr337 of AChE lead to the stabilization
of the ligand in the active site of enzyme. In addition, benzofuran part of the molecule
involves in a - interaction with Trp286 and Tyr341.
There are two hydrogen bonding interactions of nitrogen atom of amide group and methoxy
moiety of the benzofuran with Asp74. The same binding mode and interactions were also
observed for the reference drug (Donepezil).
2.4 Inhibitory Effects on the Amyloid-β self-aggregation
The potential of compounds 6h and 6k to inhibit amyloid-beta aggregation was evaluated
using thioflavin T (ThT) assay. Results indicated that compounds 6h and 6k emerged as
potent inhibitors of Aβ fibrillization (33.1 and 46.4 % inhibition, respectively) in comparison
with donepezil (22 %) and rifampicin (27.5 %) (Table 2). Compound 6k is two-fold more
potent than reference drugs in inhibitory activity against Aβ aggregation. The superiority of
the 6k inhibitory activity on 6h, confirmed the role of methoxy group in the benzofuran
framework for Aβ binding.
Table 2. Inhibitory effects of compounds 6h and 6k on Aβ self-aggregation.
a
compound
Donepezil
Rifampicin
Inhibition of Aβ self-aggregation (%)
22.0±5.4
27.5±4.3
33.1±11.2
46.4±2.2
6
h
6
k
a
Inhibition of self-induced Aβ(1-42) aggregation (10 µM) produced by the tested compound at 100 µM
concentration. Values are expressed as means ± SEM of three experiments.
3
. Conclusion
Regarding the pivotal role of BuCh in the treatment of AD, herein, we presented the novel
benzofuran carboxamide-based derivatives with significant anti butyrylcholinesterase
activity. It was revealed that all the synthesized compounds are better BChE inhibitors rather
than AChE. The 2-fluoro-6-nitro containing derivative, compound 6h, along with compound
9

6
k was subjected to docking studies and Aβ fibrillization inhibitory activity. On the other
side, based on ClogP and tPSA calculation, all compounds were predicted as CNS active with
satisfactory probability. Therefore, compound 6h with such multifunctional properties could
introduce as a candidate for further modification in the development of anti-AD drugs.
4
. Materials and Methods
4.1 Chemistry
All the chemical compounds were purchased from Merck, Aldrich and Acros chemical and
used without further purification. Melting points were determined with a Kofler hot stage
apparatus and are uncorrected. The IR spectra were taken using Nicolet FT-IR Magna 550
spectrometer (KBr disks). Mass spectra were recorded on an Agilent Technologies (HP) 5973
1
13
mass spectrometer operating at an ionization potential of 70 eV. The H and C nuclear
magnetic resonance (NMR) were recorded in DMSO-d and/or CDCl on a Bruker FT-500
6
3
MHz spectrometer with tetramethyl silane (TMS) as the internal standard. Coupling constants
were reported in Hertz (Hz) and chemical shifts are given as δ value (ppm) relative to TMS as
internal standard. To express spin multiplicities s (singlet), d (doublet), t (triplet), dd (doublet
of doublet) and m (multiplet) were used. The atom numbering of products used for NMR
interpretation is shown in Figure 4.
Figure 4. Atom numbering of compounds 6a-o, used for NMR spectra interpretation
4.1.1 General Procedure for the Synthesis of Ethyl benzofuran-2-carboxylate (2a, 2b)
A mixture of 2-hydroxybenzaldehyde 1a or 1b (0.05 mol), ethyl bromoacetate (0.05 mol),
∘
anhydrous potassium carbonate (0.075 mol) and dried DMF (70 mL) were heated at 90 C for
4
hours [28]. The solution was decanted into ice water. The precipitate was filtered off and
washed with cold water.
.1.1.1. Ethyl benzofuran-2-carboxylate (2a)
4
10

1
∘
∘
Pale yellow powder; yield: 75 %; mp 32 C [mp lit. 30 C][49]; H NMR (CDCl
3
, 500 MHz):
-benzofuran),
-benzofuran), 7.59 (d, J = 8.5 Hz, 1H,
1
7
.43 (m, 3H, -CH
.44 (t, J = 7.5 Hz, 1H, H
-benzofuran), 7.67 (d, J = 8 Hz, H
3
), 4.45 (q, 2H, J = 7 Hz, -CH
-benzofuran), 7.53 (s, 1H, H
-benzofuran).
2
), 7.30 (t, J = 7.5 Hz, 1H, H
5
6
3
H
7
4
4
.1.1.2. Ethyl 7-methoxybenzofuran-2-carboxylate (2b)
1
∘
∘
Pale yellow powder; yield: 78 %; mp: 73 C [mp lit. 73-75 C][49]; H NMR (CDCl
3
, 500
), 6.90 (d, J = 7
-benzofuran), 7.50 (s, 1H, H
MHz): 1.41(m, 3H, -CH
Hz, 1H, H -benzofuran), 7.20-7.24 (m, 2H, H
benzofuran).
3
), 4.00 (s, 3H, -OCH
3
), 4.42 (q, 2H, J = 7 Hz, -CH
2
6
4
-benzofuran, H
5
3
-
4
.1.2 General Procedure for the Synthesis of Benzofuran-2-carboxylic acid (3a, 3b)
To ethyl-1-benzofuran-2-carboxylate (2) (1 mmol) containing ethanol was added water (2:1,
0 mL) and potassium hydroxide (2 mmol). The mixture refluxed for 2 hrs. The reaction was
3
monitored by thin layer chromatography. After completion of the reaction, the reaction
mixture was decanted into ice water and extracted the ethyl acetate layer to give target
compounds 3a and 3b [42].
4.1.2.1.Benzofuran-2-carboxylic acid (3a)
1
∘
∘
White crystal; yield: 85 %; mp 190 C [mp lit. 191-192 C][50]; H NMR (500 MHz, DMSO-
d
6
): 7.35 (t, J = 7.5 Hz, 1H, H
5
-benzofuran), 7.50 (t, J = 7.5 Hz, 1H, H -benzofuran), 7.66 (s,
6
1
H, H -benzofuran), 7.69 (d, J = 8.5 Hz, 1H, H
3
7
-benzofuran), 7.79 (d, J = 7.5 Hz, H -
4
benzofuran), 13.5 (bs, 1H, -OH).
4
.1.2.2. 7-MethoxyBenzofuran-2-carboxylic acid (3b)
1
∘
∘
Pale yellow crystal; yield: 85 %; mp 224 C [lit. mp 220-222 C][50]; H NMR (500 MHz,
DMSO-d
benzofuran), 7.30 (d, J = 7.5 Hz, 1H, H
H, OH).
6
): 3.94 (s, 1H, -CH
3
), 7.06 (d, J = 7.5 Hz, 1H, H
6
-benzofuran), 7.24 (t, 1H, H
5
-
4
-benzofuran), 7.63 (s, 1H, H
3
-benzofuran) 13.5 (bs,
1
4.1.3 General Procedure for the Synthesis of N-(pyridine-N-ylmethyl) Benzofuran-2-
carboxamide (4, 5a-b)
A mixture of benzofuran-2-carboxylic acid ethyl ester (3a or 3b) (1.5 mmol), 1-hydroxy-
benzotriazole (1.7 mmol) and N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (1.7 mmol) in dried acetonitrile (10 mL) were stirred for 30 minutes at ambient
temperature. The 3 or 4-(methylamino) pyridine was added and the mixture was stirred for
11

another 24-48 hours. After completion, the crude product was extracted with chloroform,
solution of sodium hydrogen carbonate (10%) and citric acid (10%). The organic layer was
separated, dried and evaporated under vacuum to give 4, 5a or 5b [51].
4.1.4 General Procedure for the Synthesis of pyridinium halide salts derivatives )6a-o(
4
a or 5a-b (1 mmol) was dissolved in dried acetonitrile (7 mL) and heated under reflux.
Then, appropriate benzyl halide (1.8 mmol) was added dropwise to the mixture. Upon
completion (checked by TLC), the mixture was cooled down to room temperature. The
precipitated solid was separated by filtration, washed with diethyl ether and dried to afford
the corresponding compounds 6a-o[46].
4.1.4.1 3-((Benzofuran-2-carboxamido)methyl)-1-benzylpyridin-1-ium bromide (6a)
Cream powder; 0.180 g, yield: 42 %; mp 178–180 ˚C; IR (KBr): 3027 (N-H), 1657
-
1
1
(
C=O) cm ; H NMR (DMSO-d
CH N), 7.36 (t, J = 7 Hz, 1H, H
-benzofuran), 7.66 (d, J = 8.5 Hz, 1H, H
-benzofuran), 8.15 (t, J = 7 Hz, 1H, H -pyridine), 8.58 (d, J = 8 Hz, 1H, H
pyridine), 9.12 (d, J = 6 Hz, 1H, H -pyridine), 9.24 (s, 1H, H -pyridine), 9.48 (t, J = 6 Hz,
, 125 MHz) 63.19, 110.18, 110.25, 111.78, 122.82, 123.73,
6
, 500 MHz) 4.69 (d, J = 6 Hz, 2H, CH
-benzofuran), 7.41-7.53 (m, 6H, H
-benzofuran), 7.80 (d, J =
2
-NH), 5.88 (s, 2H,
-
2
5
6
-benzofuran, H
aromatic), 7.62 (s, 1H, H
3
7
7
.5 Hz, 1H, H
4
c
d
-
b
a
1
3
1
1
1
H, N-H); C NMR (DMSO-d
6
27.02, 128.02, 128.22, 128.66, 129.23, 134.22, 140.51, 143.23, 143.44, 144.63, 148.42,
54.26, 158.61.
4.1.4.2 3-((Benzofuran-2-carboxamido)methyl)-1-(3-methylbenzyl)pyridin-1-ium bromide
(
6b)
White powder; 0.182 g, yield: 41 %; mp 178–180 ˚C; IR (KBr): 3277 (N-H), 2943,
-
1 1
1
655 (C=O), 1504, 1288 cm ; H NMR (DMSO-d
6 Hz, 2H, CH -NH), 5.81 (s, 2H, -CH N), 7.21 (d, J=6.5 Hz, 1H, H aromatic), 7.29-7.37
m, 4H, H -benzofuran, H aromatic), 7.49 (t, J = 8 Hz, 1H, H -benzofuran), 7.62 (s, 1H, H
benzofuran), 7.66 (d, J = 8 Hz, 1H, H -benzofuran), 7.79 (d, J =8 Hz, 1H, H -benzofuran),
-pyridine), 8.57 (d, J = 7.5 Hz, 1H, H -pyridine), 9.10 (d, J = 6 Hz,
-pyridine), 9.21 (s, 1H, H -pyridine), 9.51 (1H, bs, N-H); C NMR (DMSO-d
6
, 500 MHz) 2.28 (s, 3H, -CH ), 4.68 (d, J
3
=
2
2
(
5
6
3
-
7
4
8
1
.14 (t, J = 6 Hz, 1H, H
H, H
c
d
1
3
b
a
6
, 125
MHz) 20.83, 63.26, 110.23, 111.74, 122.84, 123.72, 125.79, 127.97, 128.20, 129.92, 134.08,
+
1
38.52, 140.49, 143.41, 143.43, 144.60, 148.42, 154.26, 158.59; MS m/z (%): 357 (M , <1),
12

2
6
52.2 (49.5), 235.2 (22.5), 189.2 (9.3), 145.1 (43.9), 127.0 (8.6), 105.2 (100), 89.1 (34.5),
3.1 (13.8)
4.1.4.3 3-((Benzofuran-2-carboxamido)methyl)-1-(4-methylbenzyl)pyridin-1-ium bromide
(
6c)
White powder; 0.174 g, yield: 40 %; mp 180-182 ˚C; IR (KBr): 3176 (N-H), 3089,
-
1
1
3
024, 1653 cm (C=O); H NMR (DMSO-d
-NH), 5.81 (s, 2H, -CH N), 7.23 (d, J = 7 Hz, 2H, H aromatic), 7.36 (dd, J = 6,
.5 Hz, 1H, H -benzofuran), 7.42 (d, J = 7 Hz, 2H, H aromatic), 7.49 (dd, J = 7.5, 8 Hz, 1H,
-benzofuran), 7.64 (s, 1H, H -benzofuran), 7.67 (d, J = 8 Hz, 1H, H -benzofuran), 7.80 (d,
-benzofuran), 8.13 (dd, J = 6.5, 7 Hz, 1H, H -pyridine), 8.57 (d, J = 7.5
-pyridine), 9.10 (d, J = 6 Hz, 1H, H -pyridine), 9.21 (s, 1H, H -pyridine), 9.56 (bs,
, 125 MHz): 20.66, 63.05, 110.20, 111.75, 122.80, 123.72,
6
, 500 MHz): 2.28 (s, 3H, -CH ), 4.68 (d, J = 5.5
3
Hz, 2H, CH
2
2
7
5
H
6
3
7
J = 7.5 Hz, 1H, H
4
c
Hz, 1H, H
d
b
a
1
3
1
1
1
H, NH); C NMR (DMSO-d
6
27.03, 127.94, 128.12, 128.77, 128.89, 129.60, 131.26, 135.24, 138.92, 140.52, 143.33,
44.50, 148.87, 154.26, 158.23, 158.59.
4.1.4.4 3-((Benzofuran-2-carboxamido)methyl)-1-(2-nitrobenzyl)pyridin-1-ium bromide
(
6d)
White powder; 0.25 g; yield: 53%; mp 226-228 ˚C ; IR (KBr): 3239, 3027 (N-H), 1657
-
1
1
cm (C=O); H NMR (DMSO-d
6
, 500 MHz) 4.71 (d, J = 6 Hz, 2H, CH
-benzofuran), 7.36 (t, J = 7.5 Hz, 1H, H
-benzofuran), 7.60 (s, 1H, H -benzofuran), 7.66 (d, J = 8.5 Hz, 1H, H
-pyridine), 8.26
-pyridine), 9.01 (d, J = 5.5 Hz, 1H,
2
-NH), 6.24 (s, 2H,
-
(
CH
2
N), 7.14 (d, J = 8 Hz, 1H, H
7
5
-benzofuran), 7.49
t, J = 7.5 Hz, 1H, H
6
3
4
-
benzofuran), 7.71-7.82 (m, 3H, H aromatic), 8.21 (dd, J = 6.5, 7 Hz, 1H, H
d, J = 8 Hz, 1H, H aromatic), 8.66 (d, J = 8 Hz, 1H, H
-pyridine), 9.07 (s, 1H, H -pyridine), 9.49 (1H, bs, N-H); C NMR (DMSO-d , 125 MHz)
c
(
d
1
3
H
b
a
6
6
1
0.61, 110.14, 111.79, 122.88, 123.74, 125.44, 127.01, 127.94, 128.18, 128.93, 134.78,
40.46, 143.83, 144.12, 145.19, 147.51, 148.41,154.25, 158.60.
4.1.4.5 3-((Benzofuran-2-carboxamido)methyl)-1-(4-nitrobenzyl)pyridin-1-ium bromide (6e)
-
1
White crystal; 0.21 g, yield: 44 %; mp 195-198 ˚C; IR (KBr): 3493 (N-H), 1646 cm
1
(
C=O); H NMR (DMSO-d
CH N), 7.35 (t, J =7.5 Hz, 1H, H
.64-7.67 (m, 2H, H - benzofuran, H
benzofuran), 8.20 (t, J = 7 Hz, 1H, H -pyridine), 8.28 (d, J = 8.5 Hz, 2H, H aromatic), 8.62
6
, 500 MHz) 4.70 (d, 2H, J = 6 Hz, CH
2
-NH), 6.06 (s, 2H, -
2
5
-benzofuran), 7.5 (dd, 1H, J = 7.5 , 8 Hz, H
6
-benzofuran),
7
3
7
-benzofuran), 7.77-7.80 (m, 3H, H aromatic, H -
4
c
13

(
d, J = 7.5 Hz, 1H, H
d
-pyridine), 9.18 (d, J = 6 Hz, H
b
-pyridine), 9.27 (s, 1H, H -pyridine),
a
+
9
.55 (t, J = 5.5 Hz, 1H, N-H); MS m/z (%): 388 (M , <1), 252 (51.3), 145.1 (57.4), 136.1
(
68.4), 118 (29.6), 108.1 (3.8), 89.1(100), 63.1(39.5), 51.1(15.7),
4.1.4.6 3-((Benzofuran-2-carboxamido)methyl)-1-(4-fluorobenzyl)pyridin-1-ium bromide
(
6f)
-1
Cream powder; 0.28 g, yield: 62 %; mp 149-151 ˚C; IR (KBr): 3423 (N-H), 1662 cm
1
(
C=O); H NMR (DMSO-d
6
, 500 MHz) 4.69 (d, J = 6 Hz, 2H, CH
-benzofuran), 7.49 (t, J
- benzofuran, H -benzofuran and H
-benzofuran), 8.15 (t, 1H, J = 6 Hz, H -pyridine), 8.58
-pyridine), 9.15 (d, J = 6 Hz, 1H, H -pyridine), 9.27 (s, 1H, H
, 125 MHz) 62.26, 110.28, 111.77,
15.96 (d, J C-F = 19 Hz), 122.85, 123.84, 127.01, 128.01, 128.23, 130.34, 131.48, 140.48,
43.25, 143.37, 144.4, 148.4, 154.27, 158.62, 162.56 (d, JC-F = 245 Hz).
2
-NH), 5.89 (s, 2H, -
CH
2
N), 7.28 (t, J = 8 Hz, 2H, H aromatic), 7.35 (t, J = 7.5 Hz, 1H, H
-benzofuran), 7.63-7.68 (m, 4H, H
aromatic), 7.80 (d, J = 7.5 Hz, 1H, H
d, J = 7.5 Hz, 1H, H
pyridine), 9.51 (t, J = 6 Hz, 1H, NH); C NMR (DMSO-d
5
=
7.5 Hz, 1H, H
6
3
2
4
c
(
d
b
a
-
1
3
6
1
1
4.1.4.7 3-((Benzofuran-2-carboxamido)methyl)-1-(2,4-dichlorobenzyl)pyridin-1-ium
chloride (6g)
-1
Cream powder; 0.322 g, yield: 66 % ; mp 210-212 ˚C; IR (KBr): 3418(N-H), 1654 cm
1
(
C=O); H NMR (DMSO-d
6
, 500 MHz) 4.69 (d, J = 6 Hz, 2H, CH
-benzofuran), 7.48 (dd, J = 6.5, 8.5 Hz, 1H, H
-benzofuran), 7.69 (s,
-benzofuran, H aromatic), 8.16 (t, J = 6 Hz, 1H,
-pyridine), 9.02 (d, J = 6 Hz, 1H, H -pyridine), 9.14
, 125 MHz) 60.48, 110.20,
10.91, 111.76, 122.82, 123.69, 127.00, 127.86, 128.09, 129.52, 130.44, 133.04, 134.43,
2
-NH), 6.02 (s, 2H, -
CH
2
N), 7.35 (dd, J = 6, J = 7.5 Hz, 1H, H
5
6
-
benzofuran), 7.60-7.64 (m, 2H, H aromatic), 7.66 (d, J = 8.5 Hz, 1H, H
H, H -benzofuran), 7.76-7.80 (m, 2H, H
-pyridine), 8.64 (d, J = 7.5 Hz, 1H, H
s, 1H, H -pyridine), 9.71 (bs, 1H, NH); C NMR (DMSO-d
7
1
3
4
H
c
d
b
1
3
(
a
6
1
1
+
35.19, 140.45, 143.77, 145.15, 148.42, 154.26, 158.59; MS m/z (%): 411 (M , <1), 252.2
(
83.4), 235.2 (37.3), 196 (19), 159.1 (100), 145 (64), 107.1 (44.4), 89.1 (62.4), 63.1 (26.8),
1.1 (8.9).
5
4.1.4.8 3-((Benzofuran-2-carboxamido)methyl)-1-(2-fluoro-6-nitrobenzyl)pyridin-1-ium
bromide (6h)
-1
White powder; 0.20 g; yield: 41%; mp 230 ˚C (dec.); IR (KBr): 3239 (N-H), 1657 cm
1
(
C=O); H NMR (DMSO-d
6
, 500 MHz) 4.68 (d, J = 6 Hz, 2H, CH -NH), 6.16 (s, 2H, -
2
14

CH
s, 1H, H
benzofuran), 7.85-7.88 (m, 2H, H aromatic), 8.12-8.15 (m, 2H, H
.60 (d, J = 8 Hz, 1H, H -pyridine), 8.99 (d, J = 6 Hz, 1H, H -pyridine), 9.01 (s, 1H, H
2
N), 7.36 (t, J = 6 Hz, 1H, H
5
-benzofuran), 7.50 (t, J = 7.6 Hz, 1H, H
6
-benzofuran), 7.59
(
3
-benzofuran), 7.66 (d, J = 8.5 Hz, 1H, H
7
-benzofuran), 7.80 (d, J = 7.5 Hz, 1H, H
4
-
c
-pyridine, H aromatic),
8
d
b
a
-
1
3
pyridine), 9.46 (t, J = 6 Hz, 1H, N-H); C NMR (DMSO-d ,125 MHz) 53.86, 110.27,
6
2
1
1
11.76, 115.00, 122.03, 123.86, 127.01, 127.75, 127.85(d, JC-F = 25), 133.17, 140.22,
1
43.13, 143.46, 144.94, 148.39, 149.49, 154.26, 158.58, 161.45( d, JC-F = 250).
4.1.4.9 1-Benzyl-3-((7-methoxybenzofuran-2-carboxamido)methyl)pyridin-1-ium chloride
(
6i)
-1
White powder; 0.140 g; yield: 31 %; mp 174-176 ˚C; IR (KBr): 3591 (N-H), 1652 cm
1
(
C=O); H NMR (DMSO-d
6
, 500 MHz): 3.96 (s, 3H, -OCH
N), 7.10 (t, J = 8 Hz, 2H, H -benzofuran, H aromatic), 7.23-7.33 (m,
-benzofuran and H aromatic), 7.58 (s, 1H, H -benzofuran), 8.16 (t, J =
-pyridine), 8.61 (d, 1H, J = 8 Hz, H -pyridine), 8.94 (d, J = 6 Hz, 1H, H
-pyridine), 9.42 (t, J = 6 Hz, 1H, NH); C NMR (DMSO-d
MHz) 16.74, 55.78, 61.55, 108.92, 110.6, 114.48, 124.45, 126.66, 128.02, 128.21, 128.58,
3
), 4.68 (d, J = 6 Hz, 2H, CH -
2
NH), 5.93 (s, 2H, -CH
2
5
6
7
H, H
4
- benzofuran, H
6
3
Hz, 1H, H
c
d
b
-
1
3
pyridine), 9.04 (s, 1H, H
a
6
, 125
1
1
28.93, 129.32, 130.88, 132.17, 136.89, 140.51, 143.58, 143.70, 144.83, 145.23, 148.46,
58.48.
4.1.4.10 3-((7-Methoxybenzofuran-2-carboxamido)methyl)-1-(3-methoxybenzyl) pyridin-1-
ium chloride (6j)
-1
White crystal; 0.183 g; yield: 42 %; mp>230 ˚C; IR (KBr): 3424 (N-H), 1622 cm
1
(
C=O); H NMR (DMSO-d
6
, 500 MHz) 2.28 (s, 3H, -OCH
3
), 3.95 (s, 3H, -OCH
3
benzofuran), 4.68 (d, J = 5.5 Hz, 2H, CH
-benzofuran), 7.25-7.31 (5H, m, H
.17 (bs, 1H, H -pyridine), 8.61 (d, J = 5.5 Hz, 1H, H
.08 (s, 1H, H -pyridine), 9.49 (bs, 1H, N-H); C NMR (DMSO-d
2
-NH), 5.96 (s, 2H, -CH
2
N), 7.09-7.12 (2H, bs, H ,
4
H
6
5
-benzofuran, H aromatic ), 7.61 (s, 1H, H
-pyridine), 8.99 (bs, 1H, H
, 125 MHz) 18.83, 55.80,
3
-benzofuran),
8
9
6
1
c
d
b
-pyridine),
1
3
a
6
1.49, 108.89, 110.67, 114.50, 114.80, 124.65, 126.66, 128.13, 128.57, 128.91, 128.94,
29.31, 130.88, 132.30, 136.91, 140.48, 143.49, 143.63, 144.80, 145.22, 148.44, 158.50.
4.1.4.11 3-((7-Methoxybenzofuran-2-carboxamido)methyl)-1-(2-methylbenzyl)pyridin-1-ium
bromide (6k)
15

White powder; 0.200 g; yield: 43 % ; mp 174-176 ˚C; IR (KBr): 3592 (N-H), 1653
-
1
1
cm (C=O); H NMR (DMSO-d
6
, 500 MHz) 2.29 (s, 3H, -CH
-NH), 5.97 (s, 2H, -CH N), 7.08-7.13 (m, 2H, H
-benzofuran, H aromatic) ,7.62 (s, 1H, H -benzofuran), 8.18 (t, J = 7 Hz,
-pyridine), 8.62 (d, J = 8 Hz, 1H, H -pyridine), 8.99 (d, J = 6 Hz, 1H, H -pyridine),
.09 (s, 1H, H -pyridine), 9.49 (bs, 1H, N-H); C NMR (DMSO-d , 125 MHz) 18.77, 55.80,
3
), 3.96 (s, 3H, -OCH
3
), 4.69
(
d, J = 5.5 Hz, 2H, CH
.27-7.33 (m, 5H, H
H, H
2
2
4
, H -benzofuran),
6
7
1
9
6
1
5
3
c
d
b
1
3
a
6
1.50, 108.91, 110.57, 110.66, 114.48, 124.59, 126.63, 128.0, 128.20, 128.57, 128.95,
29.30, 130.86, 132.19, 136.90, 140.47, 143.58, 143.70, 144.83, 145.22, 148.44, 158.48.
4.1.4.12 1-(2,4-Dichlorobenzyl)-3-((7-methoxybenzofuran-2-carboxamido)methyl) pyridin-1-
ium chloride (6l)
White crystal; 0.190 g; yield: 40 %; mp>230 ˚C; IR (KBr): 3277 (N-H), 1654 (C=O)
-
1 1
cm ; H NMR (DMSO-d
NH), 5.83 (2H, s, -CH N), 6.97 (d, J =7.8 Hz, 1H, H
-benzofuran), 7.16 (s, 1H, H -benzofuran), 7.49 (1H, t, J = 8 Hz, H
H, H aromatic), 7.68 (d, J = 8 Hz, 1H, H aromatic), 7.80 (d, J = 8 Hz, 1H), 8.16 (t, J = 6 Hz,
H, H -pyridine), 8.58 (d, J = 7.9 Hz, 1H, H -pyridine), 9.14 (d, J = 5.7 Hz, 1H, H -pyridine),
.24 (s, 1H, H , 125 MHz) 55.25, 63.21,
6
, 500 MHz) 3.74 (3H, s, -OCH
-benzofuran), 7.09 (d, J = 7.4 Hz, 1H,
-benzofuran), 7.61 (s,
3
), 4.69 (d, J = 5.5 Hz, 2H, CH -
2
2
6
H
4
3
5
1
1
9
1
1
c
d
b
1
3
a
-pyridine), 9.50 (1H, t, NH); C NMR (DMSO-d
6
10.27, 111.81, 114.55, 114.77, 120.74, 122.93, 123.87, 127.07, 127.17, 128.16, 130.43,
30.53, 135.57, 140.52, 143.25, 143.41, 144.63, 148.46, 154.28, 158.64, 159.62.
4.1.4.13 3-((7-Methoxybenzofuran-2-carboxamido) methyl) 1-(4-bromobenzyl)-pyridin-1-ium
bromide (6m)
-1
Yellow crystal; 0.238 g; yield: 44 %; 127-130 ˚C; IR (KBr): 3609 (N-H), 1656 cm
1
(
C=O); H NMR (DMSO-d
NH), 5.86 (s, 2H, -CH N), 7.10 (d, J = 7.5 Hz, 1H, H
benzofuran), 7.32 (d, J = 7.5 Hz, 1H, H
.58 (s, 1H, H3-benzofuran), 7.65 (d, J = 8 Hz, 2H, H aromatic), 8.15 (t, J = 6 Hz, 1H, H
pyridine), 8.58 (d, J = 8 Hz, 1H, H -pyridine), 9.11 (d, J = 6.5 Hz, 1H, H -pyridine), 9.20 (s,
, 125 MHz) 15.11,
6
, 500 MHz): 3.96 (s, 3H, -OCH
3
), 4.67 (d, J = 5 Hz, 2H, CH
2
-
2
4
-benzofuran), 7.26 (m, 1H, H
5
-
6
-benzofuran), 7.50 (d, J = 8 Hz, 2H, H aromatic),
7
c
-
d
b
1
3
1
5
1
H, H
a
-pyridine), 9.39 (t, J = 6.5 Hz, 1H, N-H); C NMR (DMSO-d
6
5.78, 62.47, 64.84, 108.93, 110.57, 110.68, 114.54, 122.86, 124.66, 128.06, 128.26, 128.57,
30.98, 131.12, 132.09, 133.49, 140.58, 143.45, 143.69, 144.76, 145.52, 148.47, 158.48; MS:
m/z (%) 43.1 (100), 53.1 (75.3), 63.1 (74.1), 69.1 (30.2), 77.1 (37.2), 83.1 (20.6), 89.1 (81.3),
16

9
7.1 (14.6), 107.1 (43.8), 119.1 (19.1), 127 (18.4), 169 (86.9), 175.1 (25.6), 192 (3.9), 282
(
21.9).
4.1.4.14 4-((Benzofuran-2-carboxamido) methyl)-1-(2-bromobenzyl)pyridin-1-ium bromide
(
6n)
White powder; 0.198 g; yield: 47%; mp 175-178 ˚C ; IR (KBr): 3372 (N-H), 1650
-
1
1
(
C=O), 1599 cm (C=C); H NMR (DMSO-d
.82 (2H, s, -CH N), 7.36 (dd, J = 6, 7.5 Hz, 1H, H
benzofuran, H aromatic), 7.62-7.71 (m, 4H, H
.81 (d, J = 8 Hz, 1H, H
d, J = 6 Hz, 2H, H -pyridine, H
6
, 500 MHz) 4.76 (d, 2H, J = 5 Hz, CH
2
-NH),
5
2
5
-benzofuran), 7.53-7.49 (m, 3H, H -
7
3
-benzofuran, H
6
-benzofuran and H aromatic),
-pyridine, H -pyridine), 9.08
-pyridine), 9.80-9.83 (m, 1H, N-H); C NMR (DMSO-d
7
4
-benzofuran), 8.09 (d, J = 6 Hz, 2H, H
b
d
1
3
(
a
c
6
,
1
1
1
25 MHz) 41.78, 61.82, 110.57, 111.79, 122.82, 123.90, 126.02, 126.08, 126.22, 127.01,
27.10, 130.99, 131.11, 132.12, 132.23, 133.58, 144.43, 144.51, 148.30, 154.30, 158.69,
59.68.
4
.1.4.15 4-((Benzofuran-2-carboxamido)methyl)-1-(4-bromobenzyl)pyridin-1-ium bromide
6o)
Cream powder; 0.246 g; yield: 49 %; mp 165-167 ˚C; IR (KBr): 3375 (N-H), 1657
(
-
1
1
(
C=O), 1600 cm (C=C); H NMR (DMSO-d
.84 (s, 2H, -CH N), 7.35 (t, J = 8 Hz, 1H, H
H aromatic), 7.64-7.69 (m, 4H, H -benzofuran, H
.5 Hz, 1H, H -benzofuran), 8.09 (d, J = 6 Hz, 2H, H
Hz, 2H, H -pyridine, H -pyridine), 9.62-9.64 (m, 1H, N-H); C NMR (DMSO-d
6
, 500 MHz) 4.75 (d, J = 5.5 Hz, 2H, CH
-benzofuran), 7.47-7.52 (m, 3H, H -benzofuran,
-benzofuran and H aromatic), 7.80 (d, J =
-pyridine, H -pyridine), 9.14 (d, J = 7.5
, 125 MHz)
2
-NH),
5
2
5
7
3
6
7
4
b
d
1
3
a
c
6
4
1
1.76, 61.74, 110.38, 111.79, 122.81, 123.73, 123.76, 126.02, 126.21, 127.00, 127.12,
31.15, 131.18, 132.09, 133.58, 144.44, 145.28, 148.28, 148.31, 154.29, 158.69, 159.66.
4.2 Molecular modeling
The crystal structures of BChE enzyme complexed with tacrine (PDB Code: 4BDS) and
AChE enzyme complexed with donepezil (PDB Code: 4EY70) were taken from RCSB
protein databank (http://www.pdb.org). Since BChE and AChE in PDB are not complexed
with any of the understudy molecules, in docking step their original ligands BChE and AChE
were removed and then compounds in this study data set were docked in the active sites of
BChE and AChE one by one. Ligands were sketched and minimized in SYBYL 7.3
molecular modeling package (Tripos Inc., St. Louis, USA) running on a Red Hat Linux
17

workstation 4.7. The resultant structures were imported into Discovery Studio 4.1 (Accelrys
Inc, San Diego, CA, USA), CHARMm force field was applied and Momany-Rone option
was used to calculate partial charges [52]. The 1000 steps of steepest descent minimization
with a RMS gradient tolerance of 3, followed by conjugate gradient minimization was
performed. To prepare the structure of BChE and AChE, protein preparation protocol was
used. In this step, CHARMm force field was applied, hydrogen atoms were added, all water
molecules were removed and pH of protein was adjusted to near neutral 7.4. All molecules
were again minimized in-situ with Smart Minimizer option [53]. The binding sites of proteins
were defined as a sphere with a radius of 10Å around the bounded ligand which confirms the
movement of the ligand atoms and the side-chains of the residues of the protein within 10 Å
from the center of the active site. Then bounded ligands BChE and AChE were removed from
the active site. Other parameters were set by default protocol settings. GOLD program was
used to dock the compounds into receptors [54].
4.3 Anticholinesterase assay
According to the spectrophotometric method of Ellman [47], the target compounds (6a-6o)
revealed anticholinesterase activity against butyrylcholinesterase from equin serum and
acetylcholinestrase from electrophorus electricus (AChE, eel). Each compound was tested in
five different concentrations against the enzyme to achieve a range of inhibition between 20-
8
0 %. After five minutes incubation of a mixture containing phosphate buffer (0.1 M,
pH=8.0, 2 mL), acetylcholinesterase or butyrylcholinesterase (20 µL), 5,5-dithio-bis-2-
nitrobenzoic acid (DTNB, 60 µL) and compounds solution (30 µL), acetylthiocholine iodide
or butyrylthiocholine iodide (20 µL) was added as substrate and the change of absorbance
was recorded at 412 nm for two minutes using a Synergy Biothech® multiplate reader. The
IC50 values were obtained from Log concentration vs. percent of inhibition curves. To
guarantee the optimum result, each experiment was accomplished in triplicate.
4.4 Determination of the inhibitory potency on Aβ1-42 self-aggregation
Inhibitory properties of compounds on self-aggregation of amyloid β protein 1-42 was
determined using a thioflavin T (ThT)-based fluorescence assay [55]. Amyloid β protein 1-42
(
Sigma A9810) was dissolved in Phosphate Buffer Saline pH 7.4 (PBS, HyClone Thermo
Scientific) containing 1% ammonium hydroxide. 50 µM Amyloid β protein 1-42 was
incubated for 72 hours at 37 °C for prefibrillation .
18

For the assay, Aβ1-42 (10 µl) were added to 0.05 M KP buffer pH 7.4 and incubated at 37˚C
for 48 h in the absence and presence of compounds (100 μM). Incubated mixture (100 µL)
was mixed with 50 μL of thioflavin T (ThT, 200 μM) in 50 mM glycine-NaOH buffer (pH
8
.5). The ThT excitation/emission was measured at 448 nm/490 nm at 48 hours using a
SpectraMax® Microplate Reader. Rifampicin (100 μM, Sigma R-3501) and Donepezil (100
μM, Sigma D-6821) were also tested as reference agents .
Self-aggregation due to the presence of the tested compounds (6h and 6k) were determined
by the following calculation: [(IFi/IFo) × 100] where IFi and IFo are the fluorescence
intensities obtained for Aβ in the presence and in the absence of inhibitors.
Acknowledgements
This work was supported and funded by Tehran University of Medical Sciences (TUMS);
Grant no. 96-02-33-34229.
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24

Graphical Abstract
Synthesis and anticholinesterase inhibitory activity of new 2-benzofuran
carboxamide-benzylpyridinum salts
a
a
b
c
Fahimeh Abedinifar, S. Morteza F. Farnia, * Mohammad Mahdavi, Hamid Nadri, Alireza
c
a
d
e
Moradi, Jahan B. Ghasemi, Tuba Tüylü Küçükkılınç, Loghman Firoozpour, Alireza
f,g
Foroumadi*
a
School of Chemistry, College of Science, University of Tehran, Tehran, Iran
b
Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical
Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran
c
Department of Medicinal Chemistry, Faculty of Pharmacy, Shahid Sadoughi University of
Medical Sciences, Yazd, Iran
d
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
e
Drug Design and Development Research Center, The Institute of Pharmaceutical Sciences
(TIPS), Tehran University of Medical Sciences, Tehran, Iran
f
Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
g
Department of Medicinal Chemistry, Faculty of Pharmacy and Neuroscience Research Center,
Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman, Iran
*Corresponding authors
E-mail addresses: aforoumadi@yahoo.com (A. Foroumadi); mfarnia@khayam.ut.ac.ir (S.
Morteza F. Farnia)
25

Highlights



Benzofuran-2-carboxamide-pyridinium salts were evaluated as
anticholinesterase agents.
Compound 6h showed good BChE and Aβ-self aggregation inhibitory
activity.
Docking studies indicate the interactions of 6h resulted in stabilization of
it.
26