Synthesis and biological evaluation of new N-benzylpyridinium-based benzoheterocycles as potential anti-Alzheimer's agents

Article abstract of DOI:10.1016/j.bioorg.2018.11.010

A novel series of benzylpyridinium-based benzoheterocycles (benzimidazole, benzoxazole or benzothiazole) were designed as potent acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitors. The title compounds 4a-q were conveniently synthesiz

Full text of DOI:10.1016/j.bioorg.2018.11.010

Accepted Manuscript
Synthesis and biological evaluation of new N-benzylpyridinium-based benzo-
heterocycles as potential anti-Alzheimer’s agents
Naeimeh Salehi, Bi Bi Fatemeh Mirjalili, Hamid Nadri, Zahra Abdolahi, Hamid
Forootanfar, Alireza Samzadeh-Kermani, Tuba Tüylü Kü ç ükkılınç, Beyza
Ayazgok, Saeed Emami, Ismaeil Haririan, Mohammad Sharifzadeh, Alireza
Foroumadi, Mehdi Khoobi
PII:
S0045-2068(18)30597-2
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.010
YBIOO 2618
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
23 June 2018
25 August 2018
10 November 2018
Please cite this article as: N. Salehi, B. Bi Fatemeh Mirjalili, H. Nadri, Z. Abdolahi, H. Forootanfar, A. Samzadeh-
Kermani, T. Tüylü Kü ç ükkılınç, B. Ayazgok, S. Emami, I. Haririan, M. Sharifzadeh, A. Foroumadi, M. Khoobi,
Synthesis and biological evaluation of new N-benzylpyridinium-based benzoheterocycles as potential anti-
Alzheimer’s agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.11.010
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Synthesis and biological evaluation of new N-benzylpyridinium-based
benzoheterocycles as potential anti-Alzheimer’s agents
a
b
b
Naeimeh Salehi , Bi Bi Fatemeh Mirjalili a,*, Hamid Nadri , Zahra Abdolahi , Hamid
c
e
Forootanfar , Alireza Samzadeh-Kermani d, Tuba Tüylü Küçükkılınç , Beyza Ayazgok e, Saeed
f
g
h
i,j
Emami , Ismaeil Haririan , Mohammad Sharifzadeh , Alireza Foroumadi , Mehdi Khoobi
k,l,**
a Department of Chemistry, College of Science, Yazd University, Yazd, P.O. Box 89195-741, I.R. Iran
bFaculty of Pharmacy, Shahid Sadoughi University of Medical Sciences, Yazd, Iran
c
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Kerman University of Medical Sciences,
Kerman, Iran
d Department of Chemistry, Faculty of Sciences, University of Zabol, Zabol, Iran
e Hacettepe University, Faculty of Pharmacy, Department of Biochemistry, Ankara, Turkey
f
Department of Medicinal Chemistry and Pharmaceutical Sciences Research Center, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
g
Department of Pharmaceutical Biomaterials, Medical Biomaterials Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
h Department of Pharmacology and Toxicology, Faculty of Pharmacy, Toxicology and Poisoning Research Centre,
Tehran University of Medical Sciences, Tehran, Iran
i Neuroscience Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman,
Iran
j Department of Medicinal Chemistry, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran
k The Institute of Pharmaceutical Sciences (TIPS), Tehran University of Medical Sciences, Tehran 1417614411, Iran
l Departmnt of Pharmaceutical Biomaterials, Medical Biomaterials Research Center, Faculty of Pharmacy, Tehran
University of Medical Sciences, Tehran, Iran
* Corresponding authors. Tel/Fax: +98-21-64121510. e-mail address: Mehdi.khoobi@gmail.com (M.
Khoobi), and fmirjalili@yazd.ac.ir (B.F. Mirjalili)
1

Abstract
A novel series of benzylpyridinium-based benzoheterocycles (benzimidazole, benzoxazole or
benzothiazole) were designed as potent acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)
inhibitors. The title compounds 4a-q were conveniently synthesized via condensation reaction of 1,2-
phenylenediamine, 2-aminophenol or 2-aminothiophenol with pyridin-4-carbalehyde, followed by N-
benzylation using various benzyl halides. The results of in vitro biological assays revealed that most of
them, especially 4c and 4g, had potent anticholinesterase activity comparable or more potent than
reference drug, donepezil. The kinetic study demonstrated that the representative compound 4c inhibits
AChE in competitive manner. According to the ligand-enzyme docking simulation, compound 4c
occupied the active site at the vicinity of catalytic triad. The compounds 4c and 4g were found to be
inhibitors of Aβ self-aggregation as well as AChE-induced Aβ aggregation. Meanwhile, these compounds
could significantly protect PC12 cells against H2O2-induced injury and showed no toxicity against HepG2
cells. As multi-targeted structures, compounds 4c and 4g could be considered as promising candidate for
further lead developments to treat Alzheimer’s disease.
Keywords: Alzheimer's disease; Benzothiazole; Pyridinium; AChE; Aβ aggregation;
Neuroprotection
2

1. Introduction
Memory problems in elderly people could be symptoms of Alzheimer's disease (AD), an
irreversible neurodegenerative brain disease causing progressive deterioration of memory,
thinking and reasoning skills. AD is currently one of the top leading causes of death among older
adults. It is estimated that the number of AD patients rises by more than 11 million people per
year, which leads to a tremendous burden on the society and the family [1].
AD is a complex multifactor disease that its exact pathogenesis is not fully clear.
Neuropathological studies have demonstrated that the low levels of acetylcholine, -amyloid
plaques formation within the brain of AD patients and oxidative damage of neuronal cells are the
key factors for the development of AD [2]. Deficiency of acetylcholine is caused by the
disturbed activity of two types of cholinesterase enzymes, namely acetylcholinesterase (AChE)
and butyrylcholinesterase (BuChE), which hydrolyze acetylcholine into the inactive metabolites,
choline and acetate [3]. Cholinesterase inhibitors are employed to reduce acetylcholine (ACh)
breakdown, thereby increasing the concentration of acetylcholine neurotransmitter in the brain.
Structural studies on AChE showed that this enzyme has two binding site, catalytic anionic site
(CAS) and peripheral anionic site (PAS). It was proved that dual binding site (DBS) AChE
inhibitors were more potent inhibitors compared to the compounds that interact with only one
site of the enzyme [4, 5]. The most prescribed cholinesterase inhibitors such as donepezil,
tacrine, rivastigmine, and galantamine (Fig. 1) can improve neuropsychiatric symptoms such as
agitation or depression but are not able to delay or halt the progression of the disease [6].
<Figure 1>
3

Several studies have provided evidences that amyloid β (Aβ) and its precursor protein (APP)
have physiological role in the brain. However, progressive accumulation of Aβ protein is an
important factor in the development of AD. Abnormal accumulation of Aβ is the result of an
imbalance between Aβ production, aggregation and clearance [7]. Furthermore, the peripheral
anionic site of AChE plays a key role in accelerating Aβ plaques deposition [8]. On the other
hand, transformation of an initial benign form of senile plaque to a malignant form associated
with AD is facilitated by BuChE [9]. In initial stages of the neurodegenerative process, Aβ could
enter the mitochondria and increase the production of reactive oxygen species (ROS) by
disrupting the electron transport chain and induce oxidative stress [10]. Therefore, prevention of
Aβ aggregation and formation of free radicals or protecting the cells against oxidative stress by
neuroprotective agents will be a promising strategy for AD pharmacotherapy.
Molecular modeling and crystal structure of AChE complex with donepezil have shown that
the benzylpiperidine group of donepezil interacts with the indole ring of Trp84 of anionic subsite
and the indanone ring has a parallel - interaction with indole ring of Trp279 at the peripheral
site of AChE enzyme [11]. The pharmacological profile of donepezil has attracted the attention
of researchers on design and synthesis of donepezil-like compounds as potential anti-
Alzheimer’s agents. Many new cholinesterase inhibitors have been synthesized by replacement
of indanone ring in donepezil with different aromatic and heteroaromatic systems [12-15].
Amine-derived groups such as phenylpiperazine, benzylpyridinium and benzylamine are also
capable to interact with the catalytic site of the AChE, like benzylpiperidine moiety of donepezil
[16-18]. Considering these scaffolds, our group has previously reported some potent AChE
inhibitors such as I and II, in which the N-benzylpyridinium moiety as an essential part for
superior activity was attached to a series of heteroaromatic scaffolds (Fig. 2) [19, 20].
4

Benzothiazoles have assumed especial significance in pharmaceutical chemistry, as well as in
drug discovery. Some benzothiazole derivatives strongly interact with A peptides and can
reduce the soluble amyloid oligomers burden [21-24]. In particular, the 2-arylbenzothiazole
pharmacophore is known to have high affinity for Aβ. For example, 2-(4′-
methylaminophenyl)benzothiazole (BTA-1, III) is an uncharged derivative of thioflavin-T that
has high affinity for amyloid deposits and good blood brain barrier (BBB) permeability [21].
Also, Huang et al. have reported a series of tacrine–phenylbenzothiazole conjugates as
multifunctional anti-Alzheimer’s disease agents. Typically, compound IV was the most potent
AChE inhibitor (IC50= 0.017 μM) with Aβ aggregation inhibitory activity [22]. Thus, this type of
molecules containing a 2-arylbenzothiazole pharmacophore can be used as suitable diagnostic
imaging agents and potential therapies for Alzheimer’s disease.
<Figure 2>
Due to the importance of multi-target drugs development for AD therapy and on the basis of
the above-mentioned findings, we have designed a novel hybrid series of donepezil-like
compounds bearing benzoheterocyclic and benzylpyridinium moieties (Fig. 2). The designed
compounds were synthesized and evaluated for multifunctional biological activities including
anti-cholinesterase, Aβ1-42 protein anti-aggregating, and protective effect against H2O2-induced
PC12 cell injury, as well as docking study.
2. Results and discussion
2.1. Chemistry
5

The designed compounds 4-(1H-benzo[d]imidazol-2-yl)-1-benzylpyridinium bromide (4a), 4-
(benzo[d]oxazol-2-yl)-1-benzylpyridinium bromide (4b) and 4-(benzo[d]thiazol-2-yl)-1-
benzylpyridinium halides 4c-q were easily prepared in two steps (Scheme 1). In the first step, 2-
(pyridin-4-yl)benzo[d]imidazole (3a), 2-(pyridin-4-yl)benzo[d]oxazole (3b) and 2-(pyridin-4-
yl)benzo[d]thiazole (3c) were synthesized using different protocols.
<Scheme 1>
Several catalysts were screened for the condensation of o-phenylenediamine (1a) and pyridin-
4-carbalehyde (2) under various conditions (Table 1), but the best results were obtained using
Fe3O4@nano-cellulose–OPO3H (Fe3O4@NCs–OPO3H) in EtOH at 50 ºC. Reaction between 2-
aminophenol (1b) and pyridin-4-carbalehyde (2) in the presence of KMnO4/HOAc produced 2-
(pyridin-4-yl)benzo[d]oxazole (3b) [25]. For preparation of 2-(pyridin-4-yl)benzo[d]thiazole
(3c), 2-aminothiophenol (1c) and pyridin-4-carbalehyde (2) were condensed in the presence of
Fe3O4@NCs–OPO3H under various conditions, as listed in Table 1.
<Table 1>
It was found that the best condition is under solvent-free condition at 100 ºC. The simple
isolation of products in pure form at excellent yields without the use of chromatography and
affordable synthesis are important features of the applied protocols. At the final step, target
products 4a-q were obtained from the reaction of compounds 3a-c with benzyl halides in dry
acetonitrile under reflux conditions with excellent yields.
6

2.2. Inhibitory activity against AChE
The inhibitory activity of the synthesized compounds 4a-q was assessed in vitro against
AChE in comparison with commercially available drug donepezil as reference. The obtained
IC50 values in nM were listed in Table 2. According to the screening data, benzothiazole
derivatives 4c, 4f, 4g, and 4h have potent anti-AChE activity comparable or more potent than
standard drug donepezil (IC50 values = 14-23 nM). Among them, 4-(benzothiazol-2-yl)-1-
benzylpyridinium analogue 4c with the IC50 value of 14 nM showed the highest activity against
AChE. The comparison of unsubstituted compound 4c with substituted benzyl analogues 4d-q
revealed that introduction of different substituents diminished the anti-AChE activity of the
target compounds. The attenuation of the anti-AChE activity mainly depends on the position and
electronic features of the substituents. In the ortho-substituted series, the hydrophobic substituent
such as chlorine was more favorable than others. The movement of methyl group from ortho-
position to meta-position led to positive effect on anticholinesterase activity (compare 4g with
4d). The anticholinesterase activity of meta-halobenzyl compounds showed that flouoro group is
better than chloro and bromo substituents. In the para-substituted congeners, the compounds 4k
and 4m were less potent against AChE comparing to 4l. The electron withdrawing nitro group
more significantly decreased the activity as shown by compounds 4e and 4k, while the small
electron withdrawing groups such as fluorine atom on phenyl ring led to good inhibitory potency
against AChE (4h and 4l). The introduction of second chlorine atom at ortho or meta positions
(4o and 4p) of 2-chlorobenzyl derivative 4f resulted in a decrease in AChE inhibitory activity
whereas the insertion of fluorine at ortho position (4n) presented the same activity. Notably, the
comparison of compound 4c with its analogues 4a or 4b revealed that replacement of S by O or
NH had no favorable effect on anti-AChE activity.
7

<Table 2>
2.3. BuChE inhibitory activity
Given the increasing of BuChE activity in advanced AD patients, its inhibition as a treatment
strategy may be valuable in the management of AD [26]. Consequently, in vitro effect of target
compounds against BuChE was also assayed by the same method. As depicted in Table 2, the
anti-BuChE activity of some tested compounds was superior to standard drug donepezil (IC50
values = 182-2211 nM). In particular, compound 4c with IC50 value of 182 nM was 18-fold more
potent than donepezil. Generally, the anti-BuChE activity of substituted benzyl analogs was less
than unsubstituted counterpart 4c. Nevertheless, the 2-nitro, 2-chloro, 2-chloro-6-fluoro and 2,6-
dichloro derivatives (4e, 4f, 4n, and 4o respectively) with IC50 values ≤400 nM were at least 8
times more potent than donepezil against BuChE. Based on the results gathered in the Table 2,
the inhibitory activity of all compounds against AChE was higher over BuChE.
2.4. Inhibition of AChE-induced and self-induced Aβ aggregation
The amyloid-beta protein (Aβ1-42) anti-aggregating activity of compounds 4c and 4g, as most
potent AChE inhibitors, was determined by a thioflavin-T fluorescent method [26, 27]. Two
aforementioned compounds were tested at 100 µM concentration and were compared with
donepezil and rifampicin as reference drugs. The Aβ aggregation inhibition results were
presented in Table 3. The selected compounds exhibited a promising inhibitory potency on self-
induced Aβ1–42 aggregation. The inhibition percentage of compound 4c was 44.9%, being higher
than that of donepezil and rifampicin (22% and 27.5% respectively, Table 3). The compound 4g
having 3-methyl group produced less reduction in thioflavin-T fluorescence (28.9%) than
8

unsubstituted derivative 4c. The selected compound exhibited less activity in AChE-induced
Aβ1-42 aggregation thioflavin-T assay than self-induced Aβ1-42 aggregation.
<Table 3>
2.5. Cytotoxicity against HepG2 cells
In order to evaluate the hepatotoxicity of the selected compounds (4c and 4g), an in vitro
cytotoxicity assay was conducted on HepG2 cell line. The percentage of the viable cells was
determined at the different concentrations of compounds 4c and 4g (1-500 µM), in comparison
with reference drug tacrine. As shown in Table 4, the percentage of the viable cells was not
remarkably changed in the presence of compounds 4c and 4g even at the concentration of 25 µM
(viability >90%), while tacrine decreased the cell viability to 76.4% at this concentration. Also,
the effect of compounds 4c and 4g on cell viability was significantly lower than that of tacrine at
high concentrations.
<Table 4>
2.6. Neuroprotective activity of compounds 4c and 4g against H2O2-induced cell death in PC12
cells
The neuroprotective activity of the selected compounds 4c and 4g against H2O2-induced
injury in differentiated PC12 cells was evaluated. As illustrated in Fig. 3, exposure of PC12 cells
to H2O2 significantly reduced the cell viability to 30%, while pretreatment with various
concentrations of both 4c and 4g (0.1-20 µM) significantly increased the viability in a dose-
dependent manner.
9

<Figure 3>
2.7. Ligand-protein docking simulation
To understand the interaction mode of the tested compounds, 4c as representative compound
was selected and docked into the active site of acetylcholinesterase (1EVE). Considering the
docking data analysis, the -stacking and hydrophobic interactions are mainly responsible for the
binding process. During the docking process the ligand was prone to locate at the vicinity of
catalytic triad. The pendant benzyl moiety makes -stacking with His439 and Trp83. The central
pyridinium ring is attached to the Phe329 via -cation interaction and -stacking. Moreover, the
positively charged nitrogen appeals to Asp71 through charge interaction. The schematic binding
mode is depicted in Fig. 4 in 2D and 3D views.
<Figure 4>
2.8. Kinetics study of AChE inhibition
Kinetics study was done to figure out inhibition mechanism of compound 4c as the best active
compound toward the AChE. To obtain the Lineweaver-Burke graph, the enzyme activity was
determined in the presence of four different concentrations of compared 4c as the best inhibitor
(0, 7, 14, 28 nM) using different concentrations of substrate (ATChI, 0.14, 0.32, 0.70 µM). As
depicted in Lineweaver-Burke graph (Fig. 5), the competitive inhibition could be realized (1/V is
constant). This finding is in agreement with docking study in which the ligand particularly
occupied the catalytic site. The double reciprocal plot was constructed to achieve inhibition
constant. Hence the inhibition constant was 5.6 nM.
10

<Figure 5>
3. Conclusion
We have reported design, synthesis, molecular modeling and biological evaluation of a new
series of benzylpyridinium salts bearing benzothiazole moiety. The target compounds were
conveniently synthesized via the reaction between 2-aminothiophenol and pyridin-4-
carbalehyde, followed by N-benzylation using various benzyl halides. The results of in vitro
biological assay revealed that compounds 4c, 4f, 4g and 4h had potent anti-AChE activity
comparable or more potent than standard drug donepezil at nanomolar levels (IC50 values = 14-
23 nM). The anti-BuChE activity of all tested compounds was superior to standard drug
donepezil (IC50 values = 0.18-2.21 µM). Among the synthesized compounds, 4c and 4g, as
potent AChE inhibitors were screened for their Aβ1–42 anti-aggregating activity and
neuroprotective property in PC12 cells. The biological results indicated that these compounds
could remarkably inhibit self-induced Aβ1–42 aggregation. Furthermore, the selected inhibitors
could significantly protect PC12 cells against H2O2-induced damage. The kinetic study
demonstrated that the prototype compound inhibits AChE in competitive manner. According to
the ligand-enzyme docking simulation, compound 4c occupied the active site at the vicinity of
catalytic triad. These valuable results along with simple and affordable synthesis suggested that
4c and 4g can be used as new multi-targeted compounds toward development of effective
disease-modifying drugs to treat AD.
4. Experimental
11

4.1. General chemistry
All reagents and materials were procured from Merck, Aldrich and Fluka chemical companies
and used as received. Thin-layer chromatography (TLC) was done on silica gel 60 F254 plates
(Merck). FT-IR spectra of products were recorded by a Bruker, Equinox 55 spectrometer. All
melting points were measured by a Buchi melting point B-540 B.V.CHI apparatus. A Bruker 500
MHz NMR instrument was used to record the 1H NMR and 13C NMR spectra using DMSO-d6 as
solvent. Mass spectra of the products were obtained with an Agilent technology (HP) 5973 Mass
Selective Detector, operating at an ionization potential of 70 eV.
4.2. Synthesis of 2-(pyridin-4-yl)-1H-benzo[d]imidazole (3a)
A mixture of o-phenylenediamine (1a, 1 mmol), Fe3O4@NCs–OPO3H (0.05 g) and absolute
ethanol (2 mL) was put into a 25 mL round-bottom flask and stirred for 5 min at room
temperature. Then, a solution of pyridin-4-carbalehyde (2, 1 mmol) in ethanol (2 mL) was added
dropwise to this mixture at 50 °C under vigorous stirring for 2 h. After completion of reaction
(monitored by TLC, n-hexane/EtOAc 70:30), the catalyst was isolated from reaction mixture by
external magnet. Then, water and sodium carbonate were added to the reaction mixture and the
separated solid was collected as pure product 3a. Yield 92%; yellowish solid; mp 216-218 ºC; IR
1
(KBr, cm-1) νmax: 1610 (C=N). H NMR (DMSO-d6, 500 MHz) δ: 13.28 (s, 1H, NH), 8.76 (d,
2H, J = 5.5 Hz, Ha pyridine), 8.09 (d, 2H, J = 5.5 Hz, Hb pyridine), 7.73 (d, 1H, J = 7.8 Hz, H4
benzimidazole), 7.59 (d, 1H, J = 7.8 Hz, H7 benzimidazole), 7.30 (t, 1H, J = 7.8 Hz, H5
13
benzimidazole), 7.24 (t, 1H, J = 7.8 Hz, H6 benzimidazole). C NMR (DMSO-d6, 125 MHz) δ:
150.5, 148.8, 137.1, 124.5, 122.6, 120.3, 111.8. Anal. Calcd for C12H9N3 (195.22): C, 73.83; H,
4.65; N, 21.52. Found: C, 73.72; H, 4.43; N, 21.74.
12

4.3. Synthesis of 2-(pyridin-4-yl)benzo[d]oxazole (3b)
In a mortar, a mixture of 2-aminophenol (1b, 1 mmol), pyridine-4-carbaldehyde (2, 1 mmol),
KMnO4 (3.4 mmol) and HOAc (0.08 mL) was ground at room temperature for 10 min. After
completion of the reaction (as indicated by TLC, n-hexane/EtOAc 80:20), acetone (5 mL) was
added to the reaction mixture and the mixture was filtered. After addition of water to the filtrated
solution, the desired product 3b was separated as a pure solid. Yield 81%; cream solid; mp 129-
131 ºC; IR (KBr, cm-1) νmax: 1614 (C=N). 1H NMR (DMSO-d6, 500 MHz) δ: 8.85 (d, 2H, J = 4.5
Hz, Ha pyridine), 8.11 (d, 2H, J = 4.5 Hz, Hb pyridine), 7.90 (d, 1H, J = 7.5 Hz, H4 benzoxazole),
7.86 (d, 1H, J = 8.0 Hz, H7 benzoxazole), 7.52 (t, 1H, J = 8.0 Hz, H5 benzoxazole), 7.47 (t, 1H, J
= 7.5 Hz, H6 benzoxazole). 13C NMR (DMSO-d6, 125 MHz) δ: 160.3, 150.9, 150.4, 141.2, 133.5,
126.7, 125.4, 120.9, 120.5, 111.4. Anal. Calcd for C12H8N2O (196.20): C, 73.46; H, 4.11; N,
14.28. Found: C, 73.61; H, 4.34; N, 14.09.
4.4. Synthesis of 2-(pyridin-4-yl)benzo[d]thiazole (3c)
A mixture of 2-aminothiophenol (1c, 1 mmol), pyridine-4-carbaldehyde (2, 1 mmol) and
Fe3O4@NCs–OPO3H (0.05 g) was heated at 100 °C under solvent free condition for 90 minutes.
The progress of the reaction was monitored by TLC (n-hexane/EtOAc 80:20). After completion
of the reaction, the mixture was dissolved in hot ethanol and the catalyst was isolated by external
magnet. The solid product was appeared by addition of water to the concentrated filtrate and then
recrystallized from ethanol to give pure compound 3c. Yield 94%; cream solid; mp 132-134 ºC;
IR (ATR, cm-1) νmax: 1594 (C=N). 1H NMR (DMSO-d6, 500 MHz) δ: 8.80 (d, 2H, J = 4.5 Hz, Ha
pyridine), 8.23 (d, 1H, J = 8.0 Hz, H4 benzothiazole), 8.15 (d, 1H, J = 8.0 Hz, H7 benzothiazole),
13

8.03 (d, 2H, J = 4.5 Hz, Hb pyridine), 7.61 (t, 1H, J = 8.0 Hz, H5 benzothiazole), 7.55 (t, 1H, J =
8.0 Hz, H6 benzothiazole). 13C NMR (DMSO-d6, 125 MHz) δ: 165.0, 153.3, 151.0, 139.5, 134.8,
127.1, 126.4, 123.5, 122.7, 120.9. Anal. Calcd for C12H8N2S (212.27): C, 67.90; H, 3.80; N,
13.20. Found: C, 67.63; H, 3.71; N, 13.57.
4.5. General procedure for the preparation of compounds 4a-q
In a 25 mL round bottom flask, a mixture of compounds 3a, 3b or 3c (1 mmol), benzyl halide
derivative (1.2 mmol) and a catalytic amount of KI in dry acetonitrile (5 mL) was refluxed for 1-
4 h. After completion of the reaction (as monitored by TLC, n-hexane/EtOAc 50:50), the
reaction mixture was cooled and the solvent was removed under reduced pressure. Afterward,
the residue was triturated with diethyl ether (5 mL) to obtain the pure products 4a-q.
4.5.1. 4-(1H-benzo[d]imidazol-2-yl)-1-benzylpyridinium bromide (4a) [28]
1
Yellowish solid; mp 240-242 ºC; IR (KBr, cm-1) νmax: 1638 (C=N+), 1616 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 13.88 (s, 1H, NH), 9.32 (d, 2H, J = 6.3 Hz, Ha pyridine), 8.75 (d, 2H, J
= 6.3 Hz, Hb pyridine), 7.76 (bs, 2H, H4,7 benzimidazole), 7.59 (d, 1H, J = 7.0 Hz, H2,6 phenyl),
7.49-7.45 (m, 5H, phenyl), 7.38 (bs, 2H, H5,6 benzimidazole), 5.88 (s, 2H, CH2N+). 13C NMR
(DMSO-d6, 125 MHz) δ: 153.0, 146.3, 145.7, 145.4, 145.2, 144.4, 134.1, 129.3, 129.2, 129.1,
128.9, 125.3, 124.0, 117.9, 62.8. Anal. Calcd for C19H16BrN3 (366.25): C, 62.31; H, 4.40; N,
11.47. Found: C, 62.49; H, 4.12; N, 11.30.
4.5.2. 4-(benzo[d]oxazol-2-yl)-1-benzylpyridinium bromide (4b) [28]
14

1
Cream solid; mp 238-240 ºC; IR (KBr, cm-1) νmax: 1641 (C=N+), 1612 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.38 (d, 2H, J = 6.3 Hz, Ha pyridine), 8.80 (d, 2H, J = 6.3 Hz, Hb
pyridine), 8.00 (d, 1H, J = 8.0 Hz, H7 benzoxazole), 7.95 (d, 1H, J = 8.0 Hz, H4 benzoxazole),
7.63 (t, 1H, J = 8.0 Hz, H5 benzoxazole), 7.59 (d, 2H, J = 7.0 Hz, H2,6 phenyl), 7.55 (t, 1H, J =
8.0 Hz, H6 benzoxazole), 7.49-7.44 (m, 5H, phenyl), 5.95 (s, 2H, CH2N+). 13C NMR (DMSO-d6,
125 MHz) δ: 157.6, 150.9, 146.0, 141.2, 140.9, 134.1, 129.6, 129.3, 129.0, 128.3, 126.2, 125.3,
121.3, 111.8, 63.3. Anal. Calcd for C19H15BrN2O (367.24): C, 62.14; H, 4.12; N, 7.63. Found: C,
62.49; H, 4.18; N, 7.35.
4.5.3. 4-(benzo[d]thiazol-2-yl)-1-benzylpyridinium bromide (4c) [28]
1
Cream solid; mp 217-219 ºC; IR (KBr, cm-1) νmax: 1635 (C=N+), 1563 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.34 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 8.5 Hz, H4 benzothiazole),
7.70 (t, 1H, J = 8.5 Hz, H5 benzothiazole), 7.65 (t, 1H, J = 8.0 Hz, H6 benzothiazole), 7.59 (d,
13
2H, J = 6.5 Hz, H2,6 phenyl), 7.50-7.47 (m, 3H, H3,4,5 phenyl), 5.95 (s, 2H, -CH2N+). C NMR
(DMSO-d6, 125 MHz) δ: 161.3, 153.3, 146.6, 145.8, 136.2, 134.2, 129.4, 129.3, 128.9, 127.9,
127.8, 125.3, 124.4, 123.2, 63.0. MS m/z (%) 303 (M+, 0.4), 212 (100), 186 (13), 170 (5), 141
(3), 108 (13), 91 (92), 69 (12), 51 (7). Anal. Calcd for C19H15BrN2S (383.30): C, 59.54; H, 3.94;
N, 7.31. Found: C, 59.78; H, 3.89; N, 7.19.
4.5.4. 4-(benzo[d]thiazol-2-yl)-1-(2-methylbenzyl)pyridinium chloride (4d)
1
Cream solid; mp 200-202 ºC; IR (KBr, cm-1) νmax: 1630 (C=N+), 1563 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.15 (d, 2H, J = 6.0 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.0 Hz, Hb
15

pyridine), 8.35 (d, 1H, J = 7.5 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 7.5 Hz, H4 benzothiazole),
7.69 (t, 1H, J = 7.5 Hz, H5 benzothiazole), 7.65 (t, 1H, J = 7.5 Hz, H6 benzothiazole), 7.37-7.33
(m, 2H, H4,6 phenyl), 7.30 (t, 1H, J = 7.5 Hz, H5 phenyl), 7.25 (d, 1H, J=7.5, H3 phenyl), 5.99 (s,
2H, –CH2N+), 2.34 (s, 3H, CH3). 13C NMR (DMSO-d6, 125 MHz) δ: 161.3, 153.3, 146.7, 146.0,
145.9, 137.1, 136.1, 132.0, 130.9, 129.7, 129.2, 127.8, 126.7, 125.3, 124.4, 123.2, 61.4, 18.8. MS
m/z (%) 317 (M+, 6), 212 (95), 186 (10), 143 (3), 127 (8), 105 (100), 77 (16), 51 (12). Anal.
Calcd for C20H17ClN2S (352.88): C, 68.07; H, 4.86; N, 7.94. Found: C, 68.36; H, 4.81; N, 7.76.
4.5.5. 4-(benzo[d]thiazol-2-yl)-1-(2-nitrobenzyl)pyridinium chloride (4e)
1
Yellow solid; mp 228-229 ºC; IR (KBr, cm-1) νmax: 1630 (C=N+), 1564 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.23 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.85 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.38 (d, 1H, J = 7.5 Hz, H7 benzothiazole), 8.38 (d, 1H, J = 6.5 Hz, H4 benzothiazole),
8.28 (d, 1H, J = 7.5 Hz, H3 phenyl), 7.83 (t, 1H, J = 7.5 Hz, H5 phenyl), 7.75 (t, 1H, J = 7.5 Hz,
H4 phenyl), 7.71 (t, 1H, J = 7.5 Hz, H5 benzothiazole), 7.67 (t, 1H, J = 7.5 Hz, H6 benzothiazol),
13
7.29 (d, 1H, J= 7.5 Hz, H6 phenyl), 6.31 (s, 2H, –CH2N+). C NMR (DMSO-d6, 125 MHz) δ:
161.3, 153.4, 147.5, 147.0, 146.6, 146.5, 136.2, 134.9, 130.5, 129.2, 127.9, 125.6, 125.5, 125.3,
124.3, 123.3, 60.5. Anal. Calcd for C19H14ClN3O2S (383.85): C, 59.45; H, 3.68; N, 10.95. Found:
C, 59.66; H, 3.94; N, 10.61.
4.5.6. 4-(benzo[d]thiazol-2-yl)-1-(2-chlorobenzyl)pyridinium chloride (4f)
1
Yellow solid; mp 198-200 ºC; IR (KBr, cm-1) νmax: 1628 (C=N+), 1562 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.23 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.79 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.36 (d, 1H, J = 8.0 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 7.5 Hz, H4 benzothiazole),
16

7.69 (t, 1H, J = 7.5 Hz, H5 benzothiazole), 7.66 (d, 1H, J = 7.5 Hz, H3 phenyl), 7.62 (d, 1H, J =
8.5 Hz, H6 phenyl), 7.55-7.47 (m, 3H, H4,5 phenyl and H6 benzothiazole), 6.08 (s, 2H, –CH2N+).
13C NMR (DMSO-d6, 125 MHz) δ: 161.2, 153.3, 146.9, 146.3, 146.1, 136.2, 133.2, 131.3, 130.1,
128.1, 127.8, 125.2, 125.1, 124.4, 124.3, 123.1, 61.0. MS m/z (%) 337 (M+, 13), 301 (15), 212
(100), 186 (13), 125 (79), 89 (23), 63 (20). Anal. Calcd for C19H14Cl2N2S (373.30): C, 61.13; H,
3.78; N, 7.50. Found: C, 61.39; H, 3.67; N, 7.36.
4.5.7. 4-(benzo[d]thiazol-2-yl)-1-(3-methylbenzyl)pyridinium chloride (4g)
1
Yellow solid; mp 190-192 ºC; IR (KBr, cm-1) νmax: 1637 (C=N+), 1564 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.31 (d, 2H, J = 4.5 Hz, Ha pyridine), 8.75 (d, 2H, J = 4.5 Hz, Hb
pyridine), 8.33 (d, 1H, J = 7.5 Hz, H7 benzothiazole), 8.23 (d, 1H, J = 7.5 Hz, H4 benzothiazole),
7.66 (d, 1H, J = 7.5 Hz, H6 phenyl), 7.63 (d, 1H, J = 7.5 Hz, H4 phenyl), 7.41-7.35 (m, 3H, H5
phenyl and H5,6 benzothiazole), 7.25 (d, 1H, J = 7.5 Hz, H2 phenyl), 6.08 (s, 2H, –CH2N+), 2.32
(s, 3H, CH3). 13C NMR (DMSO-d6, 125 MHz) δ: 161.2, 153.3, 146.5, 145.8, 145.6, 138.6, 136.1,
133.9, 130.0, 129.2, 127.8, 125.9, 125.3, 125.2, 124.4, 123.2, 63.0, 20.8. MS m/z (%) 317 (M+,
6), 212 (91), 186 (8), 127 (7), 105 (100), 79 (13), 51 (10). Anal. Calcd for C20H17ClN2S (352.88):
C, 68.07; H, 4.86; N, 7.94. Found: C, 68.29; H, 4.79; N, 7.71.
4.5.8. 4-(benzo[d]thiazol-2-yl)-1-(3-fluorobenzyl)pyridinium chloride (4h)
1
Yellow solid; mp 200-202 ºC; IR (KBr, cm-1) νmax: 1634 (C=N+), 1588 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.34 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.25 (d, 1H, J = 7.8 Hz, H4 benzothiazole),
7.68 (t, 1H, J = 7.8 Hz, H5 benzothiazole), 7.64 (t, 1H, J = 7.8 Hz, H6 benzothiazole), 7.55-7.51
17

(m, 2H, H5,6 phenyl), 7.45 (d, 1H, J = 7.5 Hz, H2 phenyl), 7.30 (t, 1H, J = 8.5 Hz, H4 phenyl),
13
1
5.96 (s, 2H, –CH2N+). C NMR (DMSO-d6, 125 MHz) δ: 162.2 (d, JC-F = 237.5 Hz), 161.2,
153.3, 146.7, 145.9, 145.8, 136.4, 136.2, 131.3, 127.8, 125.3 (d, 3JC-F = 12.5 Hz), 124.3 (d, 2JC-F
= 18.7 Hz), 123.2 (d, 3JC-F = 12.5 Hz), 116.3, 116.0, 115.8 (d, 2JC-F = 18.7 Hz), 62.2. MS m/z (%)
321 (M+, 2), 212 (85), 186 (12), 159 (4), 127 (4), 109 (100), 89 (14), 69 (16), 51 (8). Anal. Calcd
for C19H14ClFN2S (356.84): C, 63.95; H, 3.95; N, 7.85. Found: C, 63.73; H, 3.76; N, 8.11.
4.5.9. 4-(benzo[d]thiazol-2-yl)-1-(3-chlorobenzyl)pyridinium bromide (4i)
1
Yellow solid; mp 216-219 ºC; IR (KBr, cm-1) νmax: 1639 (C=N+), 1565 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.35 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.25 (d, 1H, J = 7.8 Hz, H4 benzothiazole),
7.77 (s, 1H, H2 phenyl), 7.69 (t, 1H, J = 7.8 Hz, H5 benzothiazole), 7.64 (t, 1H, J = 7.8 Hz, H6
benzothiazole), 7.58 (d, 1H, J = 6.0 Hz, H6 phenyl), 7.53-7.50 (m, 2H, H4,5 phenyl), 5.95 (s, 2H,
–CH2N+). 13C NMR (DMSO-d6, 125 MHz) δ: 161.2, 153.3, 146.7, 145.9, 145.8, 136.2, 133.6,
131.0, 129.4, 128.9, 127.7, 125.4, 125.3, 124.4, 124.2, 123.1, 62.1. Anal. Calcd for
C19H14BrClN2S (417.75): C, 54.63; H, 3.38; N, 6.71. Found: C, 54.94; H, 3.20; N, 6.55.
4.5.10. 4-(benzo[d]thiazol-2-yl)-1-(3-bromobenzyl)pyridinium bromide (4j)
1
Cream solid; mp 246-248 ºC; IR (KBr, cm-1) νmax: 1635 (C=N+), 1567 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.37 (d, 2H, J = 6.0 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.0 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.25 (d, 1H, J = 7.8 Hz, H4 benzothiazole),
7.91 (s, 1H, H2 phenyl), 7.69-7.62 (m, 4H, H5 benzothiazole and H4,5,6 phenyl), 7.43 (t, 1H, J =
7.8 Hz, H6 benzothiazole), 5.95 (s, 2H, -CH2N+). 13C NMR (DMSO-d6, 125 MHz) δ: 161.3,
18

153.3, 146.7, 145.9, 136.6, 136.2, 132.3, 131.8, 131.4, 128.1, 127.9, 127.8, 125.4, 124.4, 123.2,
122.3, 62.0. Anal. Calcd for C19H14Br2N2S (462.20): C, 49.37; H, 3.05; N, 6.06. Found: C, 49.11;
H, 3.13; N, 6.21.
4.5.11. 4-(benzo[d]thiazol-2-yl)-1-(4-nitrobenzyl)pyridinium chloride (4k)
1
Orange solid; mp 240-242 ºC; IR (KBr, cm-1) νmax: 1635 (C=N+), 1610 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.40 (d, 2H, J = 6.0 Hz, Ha pyridine), 8.81 (d, 2H, J = 6.0 Hz, Hb
pyridine), 8.01 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 7.95 (d, 1H, J = 7.8 Hz, H4 benzothiazole),
7.64 (t, 1H, J = 7.8 Hz, H5 benzothiazol), 7.61 (d, 1H, J = 7.0 Hz, H3,5 phenyl), 7.56 (t, 1H, J =
7.8 Hz, H6 benzothiazole), 7.49 (d, 1H, J = 7.0 Hz, H2,6 phenyl), 5.97 (s, 2H, –CH2N+). 13C NMR
(DMSO-d6, 125 MHz) δ: 157.6, 150.8, 146.0, 145.8, 141.1, 140.8, 134.0, 129.3, 129.1, 128.2,
126.2, 125.3, 121.3, 111.8, 63.3. Anal. Calcd for C19H14ClN3O2S (383.85): C, 59.45; H, 3.68; N,
10.95. Found: C, 59.69; H, 3.55; N, 10.67.
4.5.12. 4-(benzo[d]thiazol-2-yl)-1-(4-fluorobenzyl)pyridinium chloride (4l)
Red solid; mp 242-243 ºC; IR (KBr, cm-1) νmax: 1632 (C=N+), 1601 (C=N). 1H NMR (DMSO-
d6, 500 MHz) δ: 9.32 (d, 2H, J = 6.0 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.0 Hz, Hb pyridine), 8.35
(d, 1H, J = 8.0 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 8.0 Hz, H4 benzothiazole), 7.71-7.63 (m,
4H, H5,6 benzothiazole and H2,6 phenyl), 7.33 (t, 2H, J = 8.5 Hz, H3,5 phenyl), 5.92 (s, 2H, –
1
CH2N+). 13C NMR (DMSO-d6, 125 MHz) δ: 162.62 (d, JC-F = 245.0 Hz), 161.2, 153.3, 146.6,
145.8, 145.7, 136.2, 131.5, 130.3, 127.8, 125.3 (d, 3JC-F = 11.2 Hz), 124.35. 123.17, 116.1 (d, 2JC-
F = 20.0 Hz), 62.24. MS m/z (%) 321 (M+, 4), 212 (5), 127 (30), 109 (100), 93 (4), 83 (29), 69
19

(30), 61 (21), 51 (13). Anal. Calcd for C19H14ClFN2S (356.84): C, 63.95; H, 3.95; N, 7.85.
Found: C, 63.61; H, 3.73; N, 8.08.
4.5.13. 4-(benzo[d]thiazol-2-yl)-1-(4-bromobenzyl)pyridinium chloride (4m)
1
Orange solid; mp 233-235 ºC; IR (KBr, cm-1) νmax: 1631 (C=N+), 1587 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.31 (d, 2H, J = 5.5 Hz, Ha pyridine), 8.77 (d, 2H, J = 5.5 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.5 Hz, H7 benzothiazole), 8.25 (d, 1H, J = 7.5 Hz, H4 benzothiazole),
7.69-7.64 (m, 4H, H5,6 benzothiazole and H3,5 phenyl), 7.57 (d, 2H, J = 7.5 Hz, H2,6 phenyl), 5.92
13
(s, 2H, –CH2N+). C NMR (DMSO-d6, 125 MHz) δ: 161.2, 153.3, 146.6, 145.9, 145.7, 136.2,
133.3, 132.1, 131.2, 127.8, 125.4, 124.4, 124.2, 122.9, 62.2. Anal. Calcd for C19H14BrClN2S
(417.75): C, 54.63; H, 3.38; N, 6.71. Found: C, 54.08; H, 3.16; N, 6.51.
4.5.14. 4-(benzo[d]thiazol-2-yl)-1-(2-chloro-6-fluorobenzyl)pyridinium chloride (4n)
1
Yellow solid; mp 196-198 ºC; IR (KBr, cm-1) νmax: 1632 (C=N+), 1604 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.17 (d, 2H, J = 6.2 Hz, Ha pyridine), 8.74 (d, 2H, J = 6.2 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 8.5 Hz, H4 benzothiazole),
7.69 (t, 1H, J = 7.5 Hz, H5 phenyl), 7.66-7.62 (m, 2H, H5,6 benzothiazole), 7.54 (d, 1H, J = 8.0
Hz, H3 phenyl), 7.47 (t, 1H, J = 8.5 Hz, H4 phenyl), 6.14 (s, 2H, -CH2N+). 13C NMR (DMSO-d6,
1
125 MHz) δ: 161.6 (d, JC-F = 250 Hz), 161.1, 153.4, 146.9, 146.1, 145.9, 136.2, 135.2, 133.4,
3
2
3
127.9, 126.4, 125.2 (d, JC-F = 11.2 Hz), 124.4 (d, JC-F = 17.5 Hz), 123.2 (d, JC-F = 10.0 Hz),
119.0 (d, 2JC-F = 16.2 Hz), 115.6, 55.0. MS m/z (%) 355 (M+, 0.7), 186 (12), 212 (100), 143 (89),
107 (23), 69 (15), 45 (8). Anal. Calcd for C19H13Cl2FN2S (391.29): C, 58.32; H, 3.35; N, 7.16.
Found: C, 58.64; H, 3.20; N, 7.02.
20

4.5.15. 4-(benzo[d]thiazol-2-yl)-1-(2,6-dichlorobenzyl)pyridinium chloride (4o)
1
Yellow solid; mp 213-214 ºC; IR (KBr, cm-1) νmax: 1630 (C=N+), 1577 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.12 (d, 2H, J = 6.5 Hz, Ha pyridine), 8.73 (d, 2H, J = 6.5 Hz, Hb
pyridine), 8.35 (d, 1H, J = 7.8 Hz, H7 benzothiazole), 8.27 (d, 1H, J = 7.8 Hz, H4 benzothiazole),
7.72-7.61 (m, 5H, H5,6 benzothiazole and H3,4,5 phenyl), 6.22 (s, 2H, –CH2N+). 13C NMR
(DMSO-d6, 125 MHz) δ: 161.0, 153.4, 146.9, 145.8, 136.6, 136.3, 133.2, 129.6, 128.3, 127.9,
125.1, 124.5, 124.4, 123.2, 58.7. Anal. Calcd for C19H13Cl3N2S (407.74): C, 55.97; H, 3.21; N,
6.87. Found: C, 55.62; H, 3.59; N, 6.99.
4.5.16. 4-(benzo[d]thiazol-2-yl)-1-(2,4-dichlorobenzyl)pyridinium chloride (4p)
1
Yellowish solid; mp 216-218 ºC; IR (KBr, cm-1) νmax: 1634 (C=N+), 1589 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.22 (d, 2H, J = 6.2 Hz, Ha pyridine), 8.79 (d, 2H, J = 6.2 Hz, Hb
pyridine), 8.36 (d, 1H, J = 8.0 Hz, H7 benzothiazole), 8.27 (d, 1H, J = 8.0 Hz, H4 benzothiazole),
7.83 (s, 1H, H3 phenyl), 7.70 (t, 1H, J = 8.0 Hz, H5 benzothiazole), 7.66 (t, 1H, J = 8.0 Hz, H6
benzothiazole), 7.59 (bs, 2H, H5,6 phenyl), 6.05 (s, 2H, -CH2N+). 13C NMR (DMSO-d6, 125
MHz) δ: 161.2, 153.4, 147.0, 146.3, 146.2, 136.2, 135.3, 134.4, 132.8, 130.5, 129.7, 128.2,
127.9, 125.2, 124.5, 123.3, 60.4. Anal. Calcd for C19H13Cl3N2S (407.74): C, 55.97; H, 3.21; N,
6.87. Found: C, 55.60; H, 3.40; N, 7.08.
4.5.17. 4-(benzo[d]thiazol-2-yl)-1-(3,4-dichlorobenzyl)pyridinium chloride (4q)
1
Orange solid; mp 227-229 ºC; IR (KBr, cm-1) νmax: 1632 (C=N+), 1562 (C=N). H NMR
(DMSO-d6, 500 MHz) δ: 9.31 (d, 2H, J = 6.3 Hz, Ha pyridine), 8.78 (d, 2H, J = 6.3 Hz, Hb
pyridine), 8.35 (d, 1H, J = 8.0 Hz, H7 benzothiazole), 8.26 (d, 1H, J = 8.0 Hz, H4 benzothiazole),
21

7.97 (s, 1H, H2 phenyl), 7.76 (d, 1H, J = 7.8 Hz, H5 phenyl), 7.70 (t, 1H, J = 8.0 Hz, H5
benzothiazole), 7.66 (t, 1H, J = 7.8 Hz, H6 benzothiazole), 7.61 (d, 1H, J = 8.5 Hz, H6 phenyl),
5.91 (s, 2H, –CH2N+). 13C NMR (DMSO-d6, 125 MHz) δ: 161.2, 153.3, 146.8, 146.0, 145.1,
136.2, 134.7, 132.3, 131.7, 131.3, 129.5, 127.9, 125.4, 125.3, 124.6, 123.2, 61.6. Anal. Calcd for
C19H13Cl3N2S (407.74): C, 55.97; H, 3.21; N, 6.87. Found: C, 55.66; H, 3.56; N, 6.95.
4.6. In vitro AChE/BuChE inhibition assay
The inhibition potential of the synthesized compounds toward AChE (Electrophorus
electricus) and BuChE (equine serum) was assessed using spectrophotometric method of Ellman
[29]. Donepezil was selected as reference drug. Five different concentrations of each compound
were tested to obtain 20–80% enzyme inhibition. The test medium, consisted of 2 mL phosphate
buffer (0.1 M, pH 8.0), 60 µL of 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB, 0.01 M), each
enzyme separately (20 µL of 2.5 unit/mL) and 30 µL of compound solution was incubated at 25
°C for 5 min and then, 20 µL of substrate (acetylthiocholine iodide or butyrylthiocholine iodide)
was added. As a reference, a solution containing all ingredients except the synthesized
compounds was used. Changes in absorbance were measured at 412 nm for 2 min at 25 °C and
inhibition percent of compounds was determined. The IC50 values were obtained by the method
of plotting log concentration vs. percentage of inhibition.
4.7. Determination of the inhibitory potency on self-induced and AChE-induced Aβ1-42
aggregation
22

Inhibition of self-induced and AChE-induced aggregation of amyloid β protein 1-42 was
determined using a thioflavin-T (ThT)-based fluorescence assay [27]. Aβ1-42 protein (Sigma
A9810) was dissolved in phosphate buffer saline pH 7.4 (PBS, HyClone Thermo Scientific)
containing 1% ammonium hydroxide at a concentration of 50 μM and incubated for 72 h at 37
°C for prefibrillation. Afterwards, Aβ1-42 (10 µL) ± human recombinant AChE (0.01 u/mL,
Sigma C1682) 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 inhibitors (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) and decrease in the ThT
fluorescence intensity at 448 nm (λexc( and 490 nm (λem) was measured using a SpectraMax®
Microplate Reader at 48 h. Rifampicin (100 μM, Sigma R-3501) and Donepezil (100 μM, Sigma
D-6821) were also tested as reference compounds. Self or AChE-induced aggregation percents
due to the presence of the tested compounds were determined by the following calculation:
[(IFi/IFo) × 100] where IFi and IFo are the fluorescence intensities obtained for Aβ ± AChE in the
presence and in the absence of inhibitors.
4.8. Cytotoxicity assay of the selected compounds on HepG2 cell line
Cytotoxicity of the selected compounds (4c and 4g) against HepG2 cell line (human
hepatocellular carcinoma) was determined using MTT-based assay. After culturing of the cell
line (purchased from Iranian biological resource center, IBRC, Tehran, Iran) in DMEM medium
fortified by FBS (10%) at 37 °C under 5% CO2, the cells were harvested and seeded (15000
cells/well) in 96 well microplate wells and allowed to attach for 24 h at the same conditions.
Thereafter, desired concentration (1–500 µM) of each compound (dissolved in FBS-free DMEM
medium) was added to the related well and the microplate was incubated for further 24 h. In the
23

next step, the medium of each well was replaced by MTT solution (5 mg/mL) and incubated for
4 h until the formazan crystal was formed which was then dissolved by addition of DMSO (100
µL). The related absorbance (540 nm) was consequently measured using a Synergy 2 multi-
mode plate reader apparatus (Biotek, Winooski, VT, USA) and applied to determine the viability
percent regarding to that of the control. All above mentioned experiments were performed in
triplicate and mean of the obtained results was reported.
4.9. Neuroprotection assay against H2O2-induced cell death in PC12 cells
In order to determine the neuroprotective effect of the selected compounds 4c and 4g against
H2O2-induced cell death in PC12 cells (neuron-like rat derived adrenal pheochromocytoma cell
line), the cell line (provided by Iranian biological resource center, Tehran, Iran) was cultivated in
Dulbecco’s minimal essential medium supplemented by FBS (10%), penicillin (100 U/mL), and
streptomycin (100 µg/mL) at 37 °C and 5% CO2 in a humidified incubator. The cells were
harvested at logaritmic phase and seeded (10,000 cells per well) into 96-well tissue culture
microplate and allowed to adhere to well bottom for 24 h. Thereafter, desired concentration of
compounds 4c and 4g (0.1-20 µM) were added to the related well before being exposed by H2O2
(final concentratin of 150 µM) for 6 h. The culture medium of each well was then replaced by
DMEM medium containing MTT (5 mg/mL) and incubated for 4 h. Consequently, the
precipitated formazan crystals were dissolved by 100 µL DMSO followed by recording the
related absorbance at 570 nm using a Synergy2 multi-mode microplate reader (BioTek, USA)
and calculating the viabilty percent compared to that of the control group. All experminets were
carried out in triplicates and mean of the obtained results were reported [30].
24

4.10. Docking simulations
Docking studies were performed using the Molecular Operating Environment (MOE) software
(Chemical Computing Group, Montreal, Canada). The crystal structure of acetylcholinesterase
complexed with donepezil (1EVE) was retrieved from the Protein Data Bank (PDB). The
preparation and optimization of the receptor were done by Autodock Tools (4.2) [31]. The ligand
3D structure and minimization prepared by Openbabel (2.3.1). The dimensions of active site box
were set as 15×15×15 Å, the center of grid box was placed at x = 2.023, y = 63.295, z =
67.062. The 3D molecular visualization of the strongest cluster was done by Discovery Studio
2.0 software.
Acknowledgments
This work was supported financially by a grant from The Institute of Pharmaceutical Sciences
(TIPS), Tehran University of Medical Sciences (grant number: 38526) and Iran National Science
Foundation (INSF, grant number: 94003365).
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