Design, synthesis, biological evaluation, and docking study of novel dual-acting thiazole-pyridiniums inhibiting acetylcholinesterase and β-amyloid aggregation for Alzheimer's disease

Article abstract of DOI:10.1016/j.bioorg.2020.104186

New compounds containing thiazole and pyridinium moieties were designed and synthesized. The potency of the synthesized compounds as selective inhibitors of acetylcholinesterase (AChE), and β-amyloid aggregation (Aβ) was evaluated. Compounds 7d and 7j sho

Full text of DOI:10.1016/j.bioorg.2020.104186

Bioorganic Chemistry 103 (2020) 104186
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, biological evaluation, and docking study of novel dual-
acting thiazole-pyridiniums inhibiting acetylcholinesterase and β-amyloid
aggregation for Alzheimer’s disease
a,b,c
d
e
f
Golaleh Ghotbi
, Mohammad Mahdavi , Zahra Najafi , Farshad Homayouni Moghadam ,
b,c c,g
b,c,h,⁎
Maryam Hamzeh-Mivehroud , Soodabeh Davaran , Siavoush Dastmalchi
a
b
c
d
e
f
g
h
Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Biotechnology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran
Endocrinology & Metabolism Research Institute (EMRI), Tehran University of Medical Sciences, Tehran, Iran
Department of Medicinal Chemistry, School of Pharmacy, Hamadan University of Medical Sciences, Hamadan, Iran
Department of Cellular Biotechnology, Cell Science Research Center, Royan Institute for Biotechnology, ACECR, Isfahan, Iran
Drug Applied Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
Faculty of Pharmacy, Near East University, POBOX:99138, Nicosia, North Cyprus, Mersin 10, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
New compounds containing thiazole and pyridinium moieties were designed and synthesized. The potency of the
synthesized compounds as selective inhibitors of acetylcholinesterase (AChE), and β-amyloid aggregation (Aβ)
was evaluated. Compounds 7d and 7j showed the best AChE inhibitory activities at the submicromolar con-
centration range (IC50 values of 0.40 and 0.69 μM, respectively). Most of the novel compounds showed moderate
to low inhibition of butyrylcholinesterase (BChE), which is indicative of their selective inhibitory effects towards
AChE. Kinetic studies using the most potent compounds 7d and 7j confirmed a mixed-type of AChE inhibition
mechanism in accordance with the docking results, which shows their interactions with both catalytic active
Alzheimer's disease
Acetylcholinesterase
Docking study
Thiazole-pyridinium
Neuroprotection
β-amyloid self-aggregation
(
CAS) and peripheral anionic (PAS) sites. The specific binding of 7a, 7j, and 7m to PAS domain of AChE was also
confirmed experimentally. In addition, 7d and 7j were able to show β-amyloid self-aggregation inhibitory effects
20.38 and 42.66% respectively) stronger than donepezil (14.70%) assayed at 10 μM concentration. Moreover,
(
compounds 7j and 7m were shown to be effective neuroprotective agents in H
2
O -induced oxidative stress on
2
PC12 cells almost similar to those observed for donepezil. The ability of 7j to pass blood-brain barrier was
demonstrated using the PAMPA method. The results presented in this work provide useful information about
designing novel anti-Alzheimer agents.
1
. Introduction
[3,4], β-amyloid (Aβ) deposits [5,6], and metal-ion imbalance [7] just
to name a few [8-11]. Studies had revealed that the increased levels of
The most common neurodegenerative disease and the major cause
ACh upon inhibition of acetylcholinesterase (AChE) improve the cog-
nitive function and memory in AD patients [12]. Based on the choli-
nergic hypothesis, the leading cause of cognitive impairment in AD is
attributed to the reduced levels of ACh in the brain due to the loss of
cholinergic neurons [13]. Thus, the most important treatment strategy
is the elevation of ACh function in CNS via limiting its enzymatic
of death among the elderly population is Alzheimer’s disease (AD) [1].
This multifactorial illness is characterized by various mental symptoms
such as a progressive decline in cognitive function, and extensive
neuronal and memory loss [2]. The pathological findings associated
with AD can be exemplified by reduced acetylcholine (ACh) levels
Abbreviations: AD, Alzheimer’s disease; ACh, Acetylcholine; AChE, Acetylcholinesterase; BChE, Butyrylcholinesterase; DTNB, (5,5-dithiobis-(2-nitrobenzoic acid));
Aβ, β-amyloid; CAS, Catalytic active site; PAS, Peripheral anionic site; MTT, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); NFT, Neurofibrillary
tangle; PHF, Paired helical filaments; MAP, Microtubule associated protein; ThT, Thioflavin T; HFIP, Hexafluoroisopropanol; PDB, Protein data bank; DMSO,
Dimethyl sulfoxide; SI, Selectivity index; BBB, Blood-brain barrier; PAMPA, Parallel artificial membrane permeability assay
⁎
Corresponding author at: Department of Medicinal Chemistry, School of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran.
E-mail address: dastmalchi.s@tbzmed.ac.ir (S. Dastmalchi).
https://doi.org/10.1016/j.bioorg.2020.104186
Received 29 February 2020; Received in revised form 15 July 2020; Accepted 12 August 2020
Available online 26 August 2020
0045-2068/ © 2020 Elsevier Inc. All rights reserved.

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
biodegradation by AChE and its evolutionary related enzyme butyr-
ylcholinesterase (BChE) [14]. Formation of neurofibrillary tangles
Based on the above-mentioned evidence, it seemed reasonable to
design and synthesize novel thiazole and pyridinium containing com-
pounds as potential anticholinesterase inhibitors effective in AD.
Current work reports the synthesis and cholinesterase inhibitory ac-
tivities of a novel series of thiazole-pyridinium derivatives proposed
using molecular hybridization-based drug design concept. The strategy
was to connect the phenylthiazole segment via a diamide linker to
benzylpyridinium moiety to provide a scaffold with two pharmaco-
phoric groups capable of interacting with the peripheral anionic site
(PAS) and catalytic active site (CAS) of AChE, respectively. The mole-
cular hybridization design strategy used in this study was schematically
shown in Fig. 1. All of the template structures used in hybridization
strategy, i.e., coumarin [55], benzofuran [56,57], and benzothiazole
derivatives [49,58], contain a substituted benzylpyridinium segment.
Hence, in the proposed structures, the benzene ring of this segment was
substituted with different functional groups with varying physico-che-
mical properties in terms of electronic, lipophilicity, and steric effects to
provide structural diversity required to draw rational structure–activity
relationship. Moreover, in the in silico component of the design strategy,
the proposed hybrid structures were docked into the binding site of
AChE and the resulting fitness scores were compared to that of done-
pezil as an early stage evaluation of the designed compounds. It is ex-
pected that the intrinsic dual mechanism of AChE inhibition by inter-
acting with both CAS and PAS, and prevention of Aβ aggregation could
result in the discovery of efficient anti-Alzheimer agents.
(
NFTs) [15] composed of bundles of paired helical filaments (PHF)
mainly made of the microtubule-associated protein (MAP) tau is an-
other main cause of AD [16]. Another well-documented AD hypothesis
is the aggregation and accumulation of Aβ peptide in the brain [17].
The plaques formed outside of the neural cells cause neurotoxicity and
ultimately lead to cell death. Therefore, various strategies are currently
explored to prevent the formation of Aβ plaques and eradicate toxic
oligomers [18–20]. Numerous studies are supporting the association
between AChE and Aβ aggregation into senile plaques observed in AD
[
21-24]. In vitro studies using purified or recombinant Aβ proteins
showed that AChE accelerates but BChE attenuates aggregation of Aβ
and formation of fibrils [25]. It has been shown that these recombinant
proteins adopt different conformations and the conformer with a high
β-strand secondary structure content, called amyloidogenic, is resistant
to proteases and has high propensity to form Aβ aggregate. In contrast,
the other conformer, i.e., non-amyloidogenic, adopted a random coil or
α-helix conformation and was protease sensitive. Such behavior of Aβ
peptides is used for in vitro evaluation of anti-Aβ aggregation activity of
anti-Alzheimer drug candidates [17,26]. Co-localization of AChE with
Aβ plaques in the brain provides substantial evidence for the interplay
of AChE and Aβ in the pathology of AD. It is proposed that AChE binds
to the Aβ non-amyloidogenic form through the peripheral site and then
converts it into amyloidogenic form with the subsequent formation of
amyloid fibrils [27]. The mechanism of the AChE association with
amyloid plaques is not fully understood. Still, the experimental studies
showed that upon the association, AChE changes its enzymatic and
pharmacological properties, and prevention of such association may
have beneficial effects in the treatment of AD patients [28,29]. The
development of compounds capable of binding to the catalytic site of
AChE, as well as its peripheral site, can be a very promising strategy
2. Results and discussion
2.1. Chemistry
To prepare compounds 7a–u shown in Scheme 1, first, compound 3
was reacted with pyridin-4-ylmethanamine 4a or pyridin-3-ylmetha-
namine 4b in the presence of 1-ethyl-3-(3-dimethylaminopropyl) car-
[
30]. The complex pathophysiology of AD, as mentioned above, re-
quires combination therapy [31,32], and one way of getting around this
is to develop multi-targeted agents, which are effective at multiple
pathways simultaneously [33-36].
bodiimide (EDCI) and hydroxybenzotriazole (HOBt) in dry CH CN at
3
room temperature for 24 h to obtain compounds 5a and b. Then, these
products were reacted with appropriate benzyl halides 6 by refluxing in
acetonitrile for 2–3 h to afford compounds 7a–u. The structures of all
target compounds were confirmed by IR and NMR spectroscopy, as well
as MS spectrometry.
Proposing novel compounds capable of acting on multiple targets
was investigated in the hope of developing promising agents for the
treatment of AD [37]. Donepezil, a clinically used anti-AD, belongs to
the class of anti-cholinesterase which contain piperidine ring. Nu-
merous other examples exist in the literature, where piperidine con-
taining compounds with different anti-cholinesterase potency were
designed and synthesized. The range of concentration at which they
inhibit cholinesterases can be quite broad from nanomolar up to a few
hundred micromolar [38–41]. It was shown by both experimental and
theoretical methods that the piperidine moiety of such compounds, like
donepezil, is involved in π–cation interaction with the binding site of
AChE [41,42]. Various studies have shown that the piperidine ring can
be replaced with other groups such as pyridinium [43–45]. For ex-
ample, Lan et al. have reported a series of pyridinium analogs with
potent anti-cholinesterase activity [44]. Thiazoles are an important
class of heterocyclic compounds with many biological effects [46–48]
including anticholinesterase activity [49,50]. For example, acotiamide
hydrochloride, a thiazole based selective AChEI, is in the advanced
stages of clinical studies to treat functional dyspepsia [51]. Based on
this observation, Sun et al. have reported a series of thiazole acetamide
derivatives showing anticholinesterase activity ranging from 3.14 to
2.2. AChE and BChE inhibitory activity
In vitro inhibitory activities of the synthesized compounds 7a–u
against AChE and BChE enzymes were evaluated using Ellman’s method
[59] (Table 1). As reported in the table, the synthesized compounds
7a–u can be classified into two categories based on the position of the
diamide linker on the pyridinium ring. In compounds 7a–p (series i),
the linker is connected to para position, while in compounds 7q–u
(series ii) the linker is located in meta position relative to the positively
charged nitrogen atom of pyridinium ring. The results illustrated in
Table 1 indicate that all designed derivatives were able to inhibit AChE
with IC50 values in the range of 0.40 to 54.58 µM. The results were
compared with the inhibitory effect of donepezil (IC50 of 0.03 µM) used
as the reference drug. The compounds demonstrated much weaker in-
hibitory effects (almost an order of magnitude) on BChE with the
minimum IC50 values of 25.40 µM observed for compound 7a. Based on
the inhibitory effects on AChE and BChE, a selectivity index was cal-
culated for those compounds demonstrating IC50 values less than
100 µM. The most potent AChE inhibitor was 2-fluoro compound 7d
(IC50 = 0.40 μM) and the second most potent inhibitor was 2-Br sub-
stituted 7j (IC50 = 0.69 μM), both belonging to class i derivatives. In
both of these most active compounds, the F and Br substituents are
located in ortho position on the benzyl ring. Moving these functional
groups in 7d and 7j to meta (7e and 7k) and para (7f and 7l) positions
led to a gradual decrease in inhibitory potency. This trend of activity
3
2.45 nM for a possible role in the treatment of AD [52]. Yadav and co-
workers (2016) reported a series of compounds containing both pi-
peridine and thiazole rings with the most active analog inhibiting AChE
with an IC50 value of 0.3 μM [41]. In recent years, inspired by the
structure of donepezil, some benzyl pyridinium derivatives with cho-
linesterase inhibitory activity were designed and synthesized
[
43,53,54]. The designed compounds showed potent cholinesterase
inhibition.
2

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Fig. 1. The molecular hybridization strategy used for the design of novel AChE inhibitors presented in this work. The structural similarities of donepezil hydro-
chloride as a FDA-approved AChE inhibitor and related structures, N-benzylpyridinium chalconoids (derived from coumarin and benzofuran and benzothiazoles,
reported as potent anti-AChE agents), and the newly designed thiazole-pyridinium AChE inhibitors were color coded in different molecules.
can also be seen for all other substitutions in series i. According to the
results shown in Table 1, the ortho and meta substituted compounds
exert stronger inhibition towards AChE than the para substituted deri-
vatives (the order of substitution position is ortho > meta > para). As
stated above, among the ortho substituted derivatives, 2-fluorobenzyl
observable correlation. But, considering just halogen-substituted com-
pounds, it seems that there is an inverse correlation between π values of
the substituents and AChE inhibitory potency. It is known that the π
lipophilicity constant may also represent other features of a substituent,
such as size, and therefore the overall dissimilar pattern observed for
the lipophilicity effect of the substituents in different positions may be
realized. The steric feature of the substituents (evaluated by Taft’s Es
steric parameters, Table S1) shows overall a direct correlation to the
inhibitory potency of ortho, meta and para-substituted derivatives,
which means less steric effects leads to higher potencies [62]. The
overall trend of AChE inhibition potency for the halogenated com-
pounds is F > Cl > Br and they convey higher potency than methyl
and methoxy substitutions. In general, the diversity of 3-pyridinium
series (5 derivatives) is much restricted compared to the 4-pyridinium
series (16 derivatives) and all of the substitutions are limited to the para
position. The correlation observed between the physicochemical nature
7
d from class i showed the highest activity. The substitution of strong
electron-withdrawing groups at para position diminished the inhibitory
activities of 4-pyridinium compounds against AChE. For para-nitro 7c
(
IC50 = 40.80 μM) and para-halogen derivatives 7f, 7i, and 7 l, as the
electron-withdrawing effect decreases (based on Hammett electronic
constant [60], σ, supporting materials, Table S1), the activity increases.
Although such a correlation between activity and electronic effect
cannot be extrapolated for the methyl group, it may indicate the gen-
eral pattern observed for the studied substitutions. The lipophilicity of
the substituents (Hansch π constants [61], Table S1) affects the in-
hibitory activities of the studied compounds differently without any
3

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Scheme 1. General route for the synthesis of thiazole-pyridinium hybrids 7a–u.
Table 1
Cholinesterase inhibitory activity of the synthesized compounds 7a-u.
Compounds
R
X
(AChE) inhibition
IC50 μM ± SE
(BChE) inhibition
IC50 μM ± SE
SI
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
H
Br
Br
Br
Cl
Br
Cl
Cl
Br
Cl
Br
Br
Br
Br
Cl
Cl
Cl
Br
Br
Br
Cl
Br
1.79 ± 0.08
14.37 ± 0.33
40.80 ± 2.82
0.40 ± 0.04
1.29 ± 0.17
6.48 ± 0.89
0.77 ± 0.09
0.78 ± 0.16
21.49 ± 1.98
0.69 ± 0.06
6.33 ± 0.73
30.49 ± 7.02
2.67 ± 0.24
18.24 ± 1.68
33.87 ± 3.12
9.40 ± 1.51
1.64 ± 0.15
54.58 ± 12.57
1.95 ± 0.27
28.73 ± 6.62
35.05 ± 8.07
0.03 ± 0.003
25.40 ± 2.92
56.54 ± 6.51
> 100
14.19
2-NO
4-NO
2-F
2
2
3.93
–
> 100
–
3-F
> 100
–
4-F
> 100
–
g
h
i
2-Cl
3-Cl
4-Cl
2-Br
3-Br
4-Br
> 100
–
> 100
–
35.29 ± 5.69
50.90 ± 9.38
> 100
1.64
j
73.77
k
l
–
> 100
–
m
n
o
p
q
r
2-CH
3-CH
4-CH
3
3
3
> 100
–
> 100
–
> 100
–
3-OCH
3
> 100
–
H
> 100
–
4-NO
2
> 100
–
s
4-F
> 100
–
t
4-Cl
4-Br
> 100
–
u
88.43 ± 8.15
5.22 ± 0.48
2.52
174
Donepzil
Data are expressed as Mean ± SE for three independent experiments. X is the counterion present in the final compound.
SI means Selectivity Index and is defined by IC50 (BChE)/IC50 (AChE)
.
of the substituents in series ii derivatives and their AChE inhibitory
potency is more or less the same as that explained above for series i
steric hindrance associated with the substitution at the para position.
Comparing the AChE inhibitory potency of the compounds introduced
in this study with that of the previously reported inhibitors containing
piperidine, pyridinium, or combined piperidine-thiazole moieties re-
vealed that these structures may demonstrate different potencies based
on their defined structure and no single structural element can promise
improved activity. For instance, some piperidin containing AChE in-
hibitors exert stronger activity than the pyridinium based inhibitors and
vice versa [38,39,44,63].
(
para-substituted) derivatives. Here again, the p-nitro compound is less
potent (IC50 = 54.58 μM) than the halogenated derivatives, and among
the different halogen groups, F grants the highest AChE inhibitory effect
to compound 7s (IC50 = 1.95 μM). However, the best anti-AChE ac-
tivity in class ii compounds was shown for 7q, unsubstituted compound
(
IC50 = 1.64 μM). Generally, in series ii derivatives, similar to para-
substituted compounds in series i, the introduction of substitution has
led to the decreased activity. This may indicate that there may be a
4

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Table 2
using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay (Fig. 2). The differentiated cells from PC12 neurons were
pretreated with different concentrations (1, 10, and 100 μM) of the
Inhibitory effects of compounds 7a, 7d, 7g, 7j, 7m on Aβ self-aggregation.
a
Compounds
Inhibition of Aβ self-aggregation (%)
synthesized compounds for 3 h, before treatment with H
2
2
O (400 μM).
7
7
7
7
7
a
d
g
j
9.69 ± 1.76
20.38 ± 1.51
14.34 ± 2.04
42.66 ± 1.33
10.89 ± 1.18
14.70 ± 2.35
58.18 ± 0.88
After the addition of H
2
O to induce apoptosis in PC12 cells, the cell
2
viability was measured by using MTT colorimetric assay. The protective
effects obtained for the studied compounds at the above-stated con-
centrations were compared to the control group (where the cells were
m
Donepezil
Curcumin
subjected to H
O , but not incubated with any compound), and done-
2
2
pezil (1, 10 and 100 μM), and the results depicted in Fig. 2. Compounds
7
j, the second most active AChE inhibitor, and 7m demonstrated rea-
a
Inhibition of self-induced Aβ1-42 aggregation (10 µM) produced by the
tested compounds at 10 µM concentration. Values are expressed as
means ± SE of three experiments.
sonable neuroprotective activity akin to that seen for donepezil [66] at
different studied concentrations. Compound 7d, the most active AChE
inhibitor, did not show neuroprotective activity at the used con-
centrations.
2
.3. Inhibitory effects on β-amyloid self-aggregation
The 7a and ortho-substituted compounds 7d, 7g, 7j and 7m with
2.6. Evaluation of passive BBB penetration of 7j
potent AChE inhibitory activities were evaluated for their ability to
inhibit self-aggregation of Aβ1-42 peptide assessed by the thioflavin T
Determination of brain permeability is one of the essential pre-
requisites to target the compounds for the treatment of AD. PAMPA
(parallel artificial membrane permeability) assay is a technique to ob-
serve the permeability of drug molecules across the blood–brain barrier
(BBB) [67,68]. This method is used to measure the effective perme-
(
ThT) fluorescence method [64]. According to the results presented in
Table 2, the most potent derivative, compound 7j, shows
(
42.66% ± 1.33) inhibition of Aβ self-aggregation, three folds higher
than that of donepezil (14.70 ± 2.35%). The level of inhibition of Aβ
self-aggregation observed for 7j is almost comparable to that of cur-
cumin (58.18 ± 0.88%). The derivative 7d was the second most po-
tent inhibitor of Aβ fibrillization (20.38 ± 1.51%).
ability (P , cm/s) of the artificial lipid membrane and predict the rate of
e
transcellular passive diffusion of test compounds through BBB. Com-
pound 7j with good activities in different AD-related bioassays (i.e.,
inhibition of Aβ self-aggregation, AChE inhibition, specific binding to
PAS domain of AChE, and neuroprotective activity) was selected for
PAMPA-BBB assay, and the results were compared with that of control
drugs donepezil, diazepam and fexofenadine. Diazepam and fex-
ofenadine were used as the high and low permeability standards, re-
2
.4. Evaluation of PAS binding ability of thiazole-pyridiniums
Propidium iodide (PI) is a known ligand, which binds specifically at
the PAS domain of AChE, and upon binding, its fluorescence intensity
spectively. The equation used to determine permeability rates (P ) is as
e
increases up to eight folds [65]. The affinity of compounds 7a, 7j and
follows:
7
m, the most potent inhibitors of AChE, for binding to the PAS site of
AChE was evaluated by PI displacement assay (Table 5). As reported in
the table, compound 7j, the most potent derivative, exhibited the most
reduction in the fluorescent intensity of PI and hence possessed the
highest displacement potency among the studied compounds
[
drug]acceptor
Pe = C × ln 1
, where C
[
drug]equilibrium
VD × VA
=
(
VD + VA) × Area × time
(
15.5 ± 1.1%). Taking into account that the compound 7j also inhibits
Aβ self-aggregation, one may deduce that in the presence of AChE, it
can very efficiently inhibit Aβ aggregation, which in turn may have a
pronounced effect on amelioration of AD. Also, molecular docking for
this derivative suggested its significant interaction with PAS residues.
VD, VA, Area, time, [drug]acceptor, and [drug]equilibrium are the volume of
donor compartment, the volume of acceptor compartment, the active
surface area of membrane, incubation time (expressed in seconds), the
concentration of the compound in acceptor compartment at the com-
pletion of the assay, and concentration of the compound at theoretical
equilibrium, respectively.
2
.5. Neuroprotective activity of the synthesized compounds
The determined P
e
value for 7j is 5.9 × 10−6 cm/s indicating ac-
The neuroprotective effects of compounds 7a, 7d, 7g, 7j and 7m
ceptable CNS penetration for this derivative. Based on the results pre-
against apoptosis induced in PC12 neuron cells by H
2
O
2
were evaluated
sented in Table 6, the effective permeability of 7j is lower than that of
Fig. 2. Neuroprotective activity of compounds
7
a, 7d, 7g, 7j, and 7m on PC12 cells against
H
2
O
2
induced cells death. Intact cells were not
exposed to either designed compounds or H
2
O
2
2
2
,
while the control cells were treated just by H
O
.
The cells treated with donepezil were used as
positive control. The asterisk indicates statisti-
cally significant difference compared to the
control group with p-value of < 0.001 based on
one-way ANOVA analysis performed by Prism
software.
5

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Fig. 3. Lineweavere-Burk plot, secondary plot, and enzyme kinetics profile for the inhibition of AChE by compound 7j at 0, 0.9 and 1.8 µM concentrations to
determine the inhibition mechanism as well as the steady-state inhibition constant (Ki) of the studied compound.
donepezil (7.8 × 10−6 cm/s) and diazepam (12.0 × 10
−6
cm/s),
1
v
Ks
Vmax
[I]
Ki [S]
1
1
Vmax
[I]
Ki
=
1 +
+
1 +
which are regarded as BBB permeable drugs. However, its P
e
is higher
(1)
(2)
(3)
−
6
than fexofenadine (3.8 × 10
cm/s), an antihistamine with minor
1
v
1
1
[I]
1
Vmax
CNS side effects. According to the results, it can be deduced that 7j may
pass the BBB and exert its in vivo anti-AD effects.
axis intercept =
×
+
Vmax
Ki
Ks
Vmax Ki
1
Ks
Vmax
Slope =
×
+
[I]
2.7. Investigation of AChE inhibition mechanism
where v, Vmax, K
s
, K
i
, and α are initial velocity, maximum enzyme ve-
To reveal the mechanism by which the synthesized compounds in-
locity without inhibitor, enzyme-substrate dissociation constant, en-
zyme-inhibitor dissociation constant, and α factor.
hibit AChE, the kinetics of inhibition was studied for the most active
compounds 7d and 7j using Ellman’s method [59]. To this end, the rate
of enzyme activity in the presence of different concentrations of in-
hibitors ([I] equal to 0.0, 0.45, 0.9, 1.8 µM) and substrate (acet-
ylthiocholine, [S] equal to 0.33, 0.67, 1 and 2 mM) was measured. For
each inhibitor concentration [I], the initial velocity (v) was measured at
different substrate concentrations [S], and the reciprocal of the initial
velocity (1/v) was plotted against the reciprocal of the substrate con-
centration (1/[S]) to construct the Lineweaver-Burk (LWB) curve as
shown in Figs. 3 and 4. The slopes and (1/v)-axis intercepts of the ob-
tained lines in LWB plot were used to generate secondary plots, slope
The data were also analyzed using the nonlinear regression method
implemented in GraphPad Prism program (version 6.01, GraphPad Inc,
2012). The obtained LWB plot represented a non-competitive mixed-
type inhibition pattern for both compounds 7d and 7j. In non-compe-
titive inhibition, the lines in the LWB plot intersect the horizontal axis.
However, the intersection point may also lie above or below the axis
[69]. In such cases, which happen most of the time, the inhibition is
called mixed-type inhibition. A non-competitive inhibitor binds to both
free enzyme and enzyme-substrate complex affecting both the slope and
the vertical intercept of a Lineweaver–Burk plot [69,70]. The K values
i
and y-intercept replots, where one can estimate the K
i
and α factor (the
calculated using the secondary plots and nonlinear method were
0.79 μM and 0.77 μM for 7d and 0.64 μM and 0.50 μM for 7j, respec-
tively (Figs. 3 and 4). The kinetic parameters of AChE inhibition by 7d
and 7j are shown in Table 3.
factor by which K changes in the presence of inhibitor) values from the
s
abscissa intercepts of the lines. The following equations show the bases
of the LWB, slope and y-intercept plots:
6

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Fig. 4. Lineweavere-Burk plot, secondary plot, and enzyme kinetics profile for the inhibition of AChE by compound 7d (the inhibitor) at 0, 0.45 and 0.9 µM
concentrations to determine the inhibition mechanism as well as the steady-state inhibition constant (Ki) of studied compound.
Table 3
low resolution of experimental structures (PDB codes: 1C2O, 1C2B,
1EEA) for eel AChE [71] and much higher resolution of hAChE struc-
ture (4EY7) in PDB, (ii) minor differences between human and eel AChE
structures, (iii) lack of difference between residues forming the binding
site of human and eel AChEs, and (iv) presence of a ligand (donepezil)
in the binding site of hAChE structure, [42,72] it seemed appropriate to
perform the docking calculations of the selected compounds using ex-
perimental hAChE structure. To optimize the parameters for the
docking experiments, first donepezil structure present in 4EY7 was re-
moved, and then a new structural file for donepezil was prepared and
energy optimized, and subsequently was used for the docking calcula-
tion using the GOLD program. The residues within the 6 Å from the co-
crystallized donepezil were defined as the active residues in the binding
site. Furthermore, three water molecules present in the binding pocket
of the experimental structure and known to be involved in hydrogen-
bonds with donepezil and the enzyme were retained. Different scoring
functions available in the GOLD program were used, and then the
RMSD value between the experimental and docked donepezil was cal-
culated. The minimum RMSD (0.55 Å) was obtained using ASP [73]
scoring function. All experimentally observed interactions for done-
pezil-AChE complex (4EY7 structure) were present in the complex ob-
tained by docking calculations (see. Fig. S1 of the supporting materials).
Using the same parameter set identified for the optimum docking of
donepezil, compounds 7j and 7d were docked in the binding site of
human AChEs experimental structure (4EY7). The modes of interaction
for the most promising compounds 7d and 7j were discussed below for
the docking calculations performed using 4EY7 structure.
The kinetic parameters of AChE inhibition by 7d and 7j.
−1
Compounds
V
max Abs min
K
m mM
K
i
μM
α
7
7
d
j
0.390 (0.380)
0.040 (0.039)
0.32 (0.29)
0.20 (0.19)
0.79 (0.77)
0.64 (0.50)
1.7 (1.5)
5.9 (5.6)
The values in the parentheses are from Prism GraphPad calculation.
2.8. Docking studies
At the initial stages of the study, docking calculations were used to
evaluate the binding of the designed compounds to AChE. First, it was
noted that the best docking condition (scoring function and genetic
algorithm (GA) search parameters) to reproduce the experimentally
observed binding pose of donepezil could be achieved using the ASP
scoring function as outlined in the experimental section. Then, the same
docking strategy was applied for the docking of designed compounds,
and the results were compared to that of donepezil. The docking fitness
scores for the designed compounds ranged from 65 to 73. Comparing
these scores to that calculated for donepezil (i.e., ~70) indicated that
the proposed compounds may show AChE inhibition potency close to
that of donepezil, and some may even have higher potencies. With this
initial in silico assessment on the binding, the synthesis and biological
evaluation of the designed compounds were carried out as outlined in
the previous sections. At each step, more promising compounds have
been selected for further investigations in the next steps. In this way,
two most promising derivatives 7j and 7d were selected to investigate
their AChE inhibition mechanisms and more detailed docking studies.
There are many experimental structures available for AChE from dif-
ferent sources in the protein data bank (PDB). The enzyme inhibition
potency of the synthesized derivatives in this study was determined
using eel AChE. Therefore, it seems more appropriate to do the docking
of the derivatives using the structure of the same enzyme. Due to (i) the
The results of docking calculations indicated a high similarity be-
tween the interactions seen for the most potent compounds 7d and 7j.
Fig. 5 shows the docking pose for compound 7d. The more detailed
visual illustrations of the interactions revealed by docking results for
this compound are shown in Fig. 6, panels A, B, and C. According to the
results, in compound 7d, benzyl moiety plays a vital role in ligand re-
cognition via π-π stacking interaction with Trp86 residue. In addition,
7

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
Fig. 5. Three-dimensional illustration of 7d
docked into the binding site of human AChE.
The enzyme is depicted in transparent
carton representation while the residues in-
teracting with 7d are shown in stick mode.
Compound 7d is shown in line representa-
tion for clarity. Distance between heavy
atoms for H-bind observed between amid
2
95
nitrogen atom of Phe
amide group proximal to pyridinium ring in
d is illustrated by dashed red line. The
image was generated using PyMol (Version
and oxygen atom of
7
1
.5.X, Open-Source PyMol, Schrodinger,
LLC). (For interpretation of the references to
color in this figure legend, the reader is re-
ferred to the web version of this article.)
Fig. 6. Three- and two-dimensional illustrations of the binding of compound 7d with human AChE. Three dimensional representations of the residues interacting
with 7d are shown in panels A and B from two different angles for more clarity. Hydrogen-bond and π-π/π-cation interactions are shown by red and yellow dotted
lines. The images were generated using PyMol (Version 1.5.X, Open-Source PyMol, Schrodinger, LLC). Panel C shows 2D representation of the key interactions
between 7d docked into the binding site of human AChE prepared using PoseView program. The residues involved in hydrophobic interactions are shown in green
and the corresponding areas in 7d are indicated by green curved lines. The side chain of residues form AChE and the corresponding functional group in 7d involved in
π-π/π-cation interactions are indicated by green dots linked by green dotted lines. H-bond interaction was indicated by black dotted line. Panel D shows the relative
positions of 7d (green) and 7j (cyan), the two most potent derivatives, while docked into the binding site of human AChE. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
8

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
pyridinium moiety interacts with Tyr337, Phe , and Tyr , via π-π
338
341
Table 4
stacking/π-cation interactions. Furthermore, the thiazole ring also in-
Physicochemical properties for the compounds 7d and 7j.
7
2
teracts with Tyr via π-π stacking interaction. There is a hydrogen
bonding interaction formed between the oxygen atom of the carbonyl
group proximal to the pyridinium ring of 7d with amide hydrogen of
Compounds
MW
C logP
HBA
HBD
tPSA
RBC
7
d
475.5
536.4
−1.23 ± 0.60
−0.51 ± 0.60
4
3
2
2
103.21
103.21
11
11
2
95
86
286
337
341
7j
Phe
residue. Trp , Trp , Tyr , and Tyr
residues stabilize 7d in
the binding pocked through hydrophobic interactions. Docking-based
identified interactions between 7d and AChE are mostly in common
with those reported experimentally for donepezil (i.e., shown in crystal
Clog P: Calculated n-octanol–water partition coefficient, HBA: H-bond accep-
tors, HBD: H-bond donors, tPSA: topological polar surface area, RBC: Rotatable
bond count.
8
6
286
86
structure 4EY7). Donepezil shows π-π stacking with Trp and Trp
,
,
2
95
H-bonding with Phe , and hydrophobic interactions with Trp
2
86
337
338
341
Trp , Tyr , Phe
and Tyr . The identified commonalities be-
Table 5
tween the mode of interactions observed for compound 7d and done-
pezil may indicate similar AChE inhibition mechanism by this series of
compounds. Compound 7j, the other most potent derivative, illustrates
very close modes of interactions with hAChE predicted by docking
calculations. The binding mode involves π-π stacking interaction be-
The results of propidium iodide displacement assay for compounds 7a, 7j
and7m.
Compounds
Propidium iodide displacement from PAS-AChE (% inhibition)
7
a
11.6 ± 1.5
15.5 ± 1.1
5.3 ± 0.8
20.2 ± 1.3
2
86
7j
tween thiazole and indol side chain of Trp , π-cation interactions
7
m
3
,
37
338
286
between quaternary nitrogen of pyridinium ring with Tyr
Phe
,
,
Donepezil
3
41
86
and Tyr
residues, and hydrophobic interactions with Trp , Trp
341
3
37
338
Tyr , Phe , and Tyr
residues. Also, the benzyl moiety interacts
Values are reported as mean
±
SEM for three independent experiments.
8
6
Propidium iodide displacement assay was performed on AChE to test the ability
of compounds to displace propidium from PAS-AChE. Donepezil, an anti-AD
drug with known PAS binding property, was used as positive control.
with Trp residue via π-π stacking interaction. Furthermore, a hy-
drogen bonding interaction was observed between the oxygen atom of
the carbonyl group of compound 7j close to pyridinium ring with the
2
95
backbone amide hydrogen of Phe
residue similar to that seen for
compounds 7d and donepezil. Fig. 6 (panel D) shows the relative po-
sition of 7d and 7j compounds while docked into the binding site of
human AChE. As can be seen in the figure, the corresponding molecular
moieties in two compounds are positioned very close to each other and
therefore making similar interactions with the enzyme. In agreement
with the results of kinetic studies, docking investigation confirmed the
mixed-type of AChE inhibition mechanism for compounds 7d and 7j
due to their interaction with both CAS and PAS sites of the enzyme as
described above. Such an inhibition mechanism was also shown for
donepezil and other donepezil like derivatives. The experimental and in
silico structural studies revealed that donepezil and similar compounds
occupy both CAS and PAS binding sites of AChE [42,43,74], which are
in accordance with their mixed-type non-competitive inhibition me-
chanisms proposed based on analyzing the kinetics of enzyme inhibi-
tion using LWB and other methods [54]. In contrast to mixed-type in-
hibitors, pure non-competitive inhibitors only bind to allosteric PAS site
of the enzyme and lines in the LWB plot intersect on the x-axis (i.e., 1/S
axis). For example, it was shown that bromotyrosine-derived alkaloids
inhibit AChE in a non-competitive manner deduced from the LWB and
docking analyses [75]. On the other hand, in the presence of compe-
titive inhibitors, the Vmax of enzymatic reaction does not change, but
Table 6
The results of PAMPA-BBB assay for compound 7j and Donepezil, Diazepam
and Fexofenadine were used as controls.
Compounds
(10−6cm.s−1
)
PAMPA-BBB permeability P
e
7
j
5.9 ± 0.1
7.8 ± 0.3
12.0 ± 1.5
3.8 ± 0.3
Donepezil
Diazepam
Fexofenadine
All the results are expressed as means ± SE of three experiments.
(RBC) for 7d and 7j exceed maximum acceptable numbers defined by
Ghose [80] and Veber [81]. Collectively, it is anticipated that 7d and 7j
may show reasonable ADMET properties, but is yet to be determined
experimentally.
3. Conclusion
Cholinesterase inhibitors play a crucial role in cholinergic signaling,
and hence, they are regarded as the first-line therapeutics in alleviating
the symptoms of Alzheimer’s disease. Such inhibitors, exemplified by
donepezil, can also slow down the amyloidogenic product formation,
apart from increasing the levels of ACh. In the current study, a novel
series of thiazole-pyridinium hybrids were synthesized, and evaluated
against AChE and BChE using Ellman’s method. Results showed that all
derivatives were selective for AChE, and among them, compounds 7d
and 7j demonstrated the highest AChE inhibitory effects with IC50 va-
lues of 0.40 and 0.69 μM, respectively. In addition, 7j was three times
more potent than donepezil, the reference drug, in preventing Aβ ag-
gregation. Based on docking studies, it was proposed that compounds
7d and 7j form key interactions with both CAS and PAS regions of
AChE. The specific interaction with the latter site was evaluated using
the PAS site marker displacement technique further confirming the
ability of these derivatives acting as Aβ aggregation inhibitors.
Furthermore, compound 7j showed very good neuroprotective activity
the apparent K value increases leading to a LWB plot where the lines
m
intersect on the y-axis (1/v axis). For example, using docking studies
and enzyme kinetic assay, Rizvi et al. showed that glimepiride interacts
with the substrate binding site of AChE (i. e., CAS) and in the obtained
LWB plot, lines intersect on the y-axis indicating a pure competitive
inhibition [76]. In summary, based on the results of docking calcula-
tions and kinetic studies of AChE inhibition by compounds 7d and 7j a
mixed non-competitive inhibition mechanism is proposed for the stu-
died compounds.
2.9. Prediction of physicochemical properties
Some physicochemical properties related to the drug-likeness of
compounds 7d and 7j including octanol/water partition coefficients
(
(
Clog P), number of H-bond acceptors (HBA), number of H-bond donors
HBD), polar surface area (tPSA), and number of rotatable bonds (RBC)
against H
2
O induced oxidative stress. The results of in vitro PAMPA
2
were calculated and shown in Table 4. Based on the results, these
compounds pass the drug-likeness rules proposed by Lipinski [77], Egan
assay showed that 7j is capable of passing BBB and exerting its anti-AD
effects. Collectively, although the more promising derivatives show less
AChE inhibitory potency than the standard drug donepezil, however,
they are better inhibitors of Aβ aggregation while exerting similar
[
78], and Muegge [79], while violating one or two criteria in other
drug-likeness systems. For example, the number of rotatable bonds
9

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
neuroprotective activity. The results obtained in this work showed that
the thiazole-pyridinium structure may represent a new useful multi-
targeted scaffold for the development of novel anti-Alzheimer agents.
134.9, 144.8, 147.6, 148.7, 157.8, 160.9, 170.9 and 172.1 (2C]O). MS
(m/z, %): 502.3 (M+, 100). Purity: 90%.
4
.2.3. 1-(4-Nitrobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
4
. Experimental section
butanamido)methyl) pyridin-1-ium bromide (7c)
−1
Yield: 72%; mp: 214–216 °C; IR (KBr) (νmax/cm ): 3232 and 3046
4.1. Chemistry
1
(
(
2NH), 1670 and 1605 (2C]O). H NMR(DMSO‑d
6
, 500 MHz) :δ
ppm) = 2.61 and 2.75 (t, J = 6.7 Hz, 4H, CH
2
eCH ), 4.56 (d,
2
All of the melting points were measured using Kofler hot stage ap-
+
1
13
J = 5.7 Hz, 2H, CH eNH), 5.98 (s, 2H, eCH N ), 7.32–7.42 (m, 3H, H
paratus. For all derivatives, H and C nuclear magnetic resonance
2
2
aromatic), 7.58 (s, 1H, H
a
thiazole), 7.76 (d, J = 8.0 Hz, 2H, H aro-
(
NMR) spectra were obtained on a Bruker FT-500 spectrometer, using
matic), 7.88 (d, J = 7.5 Hz, 2H, H aromatic), 8.04 (d, J = 6.5 Hz, 2H,
TMS as an internal standard. The IR spectra were recorded by Nicolet
Magna FTIR 550 spectrophotometer using KBr disk sample preparation
method. Mass spectra were acquired on Agilent 6410 triple quadrupole
liquid chromatography-mass spectrometer equipped with an electro-
spray ionization (+ESI) system. The purity of the compounds was as-
sessed by HPTLC analysis. All reagents were purchased from Sigma
Aldrich, Merck and Fluka unless otherwise stated.
H
c
,Hc' pyridine), 8.26 (d, J = 8 Hz, 2H, H aromatic), 8.80 (t,
J = 5.8 Hz, 1H, NeH ), 9.14 (d, J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.26
). C NMR (DMSO‑d , 125 MHz): δ (ppm) = 29.4, 30.0,
1.7, 61.4, 107.8, 124.2, 125.8, 126.0, 127.7, 128.6, 129.9, 134.2,
1
b
1
3
(
s, 1H, NeH
2
6
4
1
41.2, 144.5, 147.8, 148.7, 158.0, 160.8, 170.9 and 172.0 (2C]O). MS
(
m/z, %): 502.3 (M+, 100). Purity: 97%.
4
.2. The general procedure for the synthesis of thiazole-pyridinium hybrids
4
.2.4. 1-(2-Fluorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
7a–u
butanamido)methyl) pyridin-1-ium chloride (7d)
−1
Yield: 80%; mp: 196–198 °C; IR (KBr) (νmax/cm ): 3332 and 3171
To the solution of compound 3 (1 mmol) in dry acetonitrile (10 mL)
1
(
(
2NH), 1673 and 1604 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
was added EDCI (1 mmol) and HOBt (1 mmol) and the mixture was
stirred at room temperature (RT) for 30 min. Then, the mixture was
added pyridin-4-ylmethanamine 4a (or pyridin-3-ylmethanamine 4b)
ppm) = 2.57 and 2.74 (t, J = 6.7 Hz, 4H, CH
2
eCH ), 4.55 (d,
2
+
J = 5.7 Hz, 2H, CH
2
eNH), 5.91 (s, 2H, eCH
2
N
), 7.25 (d, J = 5.6 Hz,
1
H, H aromatic), 7.30–7.44 (m, 5H, H aromatic), 7.59–7.61 (m, 2H, H
thiazole, H aromatic), 7.90 (d, J = 7.3 Hz, 2H, H aromatic), 8.01 (d,
a
(
1 mmol) and the reaction was continued at room temperature for 24 h.
After completion of reaction, the solvent was reduced under vacuum
and the residue was dissolved in dichloromethane and washed with
J = 6.5 Hz, 2H, H
c
,Hc' pyridine), 8.57 (t, J = 5.8 Hz, 1H, NeH
d, J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.28 (s, 1H, NeH ). C NMR
DMSO‑d , 125 MHz): δ (ppm) = 29.7, 30.2, 41.7, 61.6, 107.8, 116.2
1
), 9.03
13
(
(
(
b
2
sodium carbonate (10%). The organic phase was dried using Na
SO
2 4
6
and evaporated under vacuumed condition at RT. Finally, to 10 mL of
dry acetonitril were added compound 5a or 5b (1 mmol) and appro-
priate benzyl halides 6 (1.2 mmol) and the reaction mixture was re-
fluxed for 2–3 h. After completion of the reaction assessed by TLC, the
solid product 7 was collected by filtration and purified using re-
crystallization in ethanol-petroleum ether mixture.
J
C–F = 20.0 Hz), 121.9, 125.6, 125.7, 127.7, 128.7, 129.3, 131.8,
1
31.9 134.3, 144.8, 148.6, 157.9, 160.7, 162.2 (JC–F = 249.7 Hz),
70.9, 171.4 (2C]O). MS (m/z, %): 475.3 (M+, 100). Purity: > 99%.
1
4
.2.5. 1-(3-Fluorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
butanamido)methyl) pyridin-1-ium bromide (7e)
4
.2.1. 1-Benzyl-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)butanamido)
−1
Yield: 80%; mp: 226–228 °C; IR (KBr) (νmax/cm ): 3196 and
methyl)pyridin-1-ium bromide (7a)
1
3
042(2NH), 1672 and 1639 (2C]) H NMR (DMSO‑d
6
, 500 MHz): δ
eCH ), 4.55 (d,
), 7.25 (t, J = 8.0 Hz,
thia-
−
1
Yield: 85%; mp: 232–234 °C; IR (KBr) (νmax/cm ): 3197 and 3046
(
ppm) = 2.61 and 2.75 (t, J = 6.7 Hz, 4H, CH
2
2
1
(
(
2NH), 1672 and 1639 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
+
J = 5.7 Hz, 2H, CH
2
eNH), 5.81 (s, 2H, eCH N
2
ppm) = 2.60 and 2.74 (t, J = 6.7 Hz, 4H, CH
2
eCH ), 4.54 (d,
2
1
H, H aromatic) 7.31–7.49 (m, 6H, H aromatic), 7.60 (s, 1H, H
a
+
J = 5.8 Hz, 2H, CH
2
eNH), 5.79 (s, 2H, eCH
2
N
), 7.33 (t, J = 7.5 Hz,
zole), 7.90 (d, J = 7.5 Hz, 2H, H aromatic), 8.02 (d, J = 6.5 Hz, 2H,
1
2
H, H aromatic), 7.39–7.45 (m, 5H, H aromatic), 7.50 (d, J = 6.5 Hz,
H, H aromatic), 7.61 (s, 1H, H thiazole), 7.90 (d, J = 7.5 Hz, 2H, H
,Hc' pyridine), 8.78 (t, J = 5.8 Hz,
), 9.09 (d, J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.29 (s, 1H,
H
c
,Hc' pyridine), 8.79 (t, J = 5.8 Hz, 1H, NeH ), 9.11 (d, J = 6.5 Hz,
13
1
a
2
1
H, H
,Hb' pyridine), 12.30 (s, 1H, NeH
). C NMR (DMSO‑d ,
b
2
6
aromatic), 8.00 (d, J = 6.5 Hz, 2H, H
c
25 MHz):
δ
(ppm) = 29.4, 30.0, 41.6, 61.8, 107.7, 115.8
1
H, NeH
1
b
(
J
C–F = 22.4 Hz), 116.2 (JC–F = 20.7 Hz), 124.9, 125.6, 127.7, 128.7,
1
3
NeH
2
). C NMR (DMSO‑d , 125 MHz): δ (ppm) = 29.4, 30.0, 41.6,
6
1
31.3, 131.4, 134.3, 136.7, 144.1, 148.7, 157.8, 160.5, 162.1
6
1
2.5, 107.9, 125.7, 125.8, 127.7, 128.6, 128.9, 129.1, 129.5, 134.2,
+
(
J
C–F = 243.8 Hz), 170.9, 172.0 (2C]O). MS (m/z, %): 475.3 (M ,
34.3, 144.1, 148.7, 157.8, 160.3, 170.8 and 171.9 (2C]O). MS (m/z,
1
00). Purity: 96%.
%
): 457.3 (M+, 100). Purity: > 99%.
4
.2.2. 1-(2-Nitrobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
4.2.6. 1-(4-Fluorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
butanamido)methyl) pyridin-1-ium bromide (7b)
butanamido)methyl) pyridin-1-ium chloride (7f)
−
1
−1
Yield: 70%; mp: 224–226 °C; IR (KBr) (νmax/cm ): 3189 and
Yield: 85%; mp: 188–190 °C; IR (KBr) (νmax/cm ): 3333 and
1
1
3
058(2NH), 1688 and 1654 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
3183(2NH), 1674 and 1603 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
(
ppm) = 2.63 and 2.77 (t, J = 6.7 Hz, 4H, CH
2
eCH
2
), 4.60 (d,
(ppm) = 2.57 and 2.74 (t, J = 6.7 Hz, 4H, CH
2
eCH ), 4.54 (d,
2
+
+
J = 5.7 Hz, 2H, CH
2
eNH), 6.17 (s, 2H, eCH
2
N
), 7.15 (d, J = 8.0 Hz,
J = 5.7 Hz, 2H, CH
2
eNH), 5.78 (s, 2H, eCH
2
N
), 7.25–7.33 (m, 3H, H
1
H, H aromatic), 7.32 (t, J = 7.5 Hz, 1H, H aromatic), 7.43 (t,
aromatic), 7.42 (t, J = 7.5 Hz, 2H, H aromatic), 7.60–7.62 (m, 3H, H
thiazole, H aromatic), 7.89 (d, J = 7.5 Hz, 2H, H aromatic), 7.99 (d,
J = 6.5 Hz, 2H, H ,Hc' pyridine), 8.57 (t, J = 6 Hz, 1H, NeH ), 9.09 (d,
J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.28 (s, 1H, NeH ). C NMR
(DMSO‑d
a
J = 7.5 Hz, 2H, H aromatic), 7.57 (s, 1H, H
J = 8.0 Hz, 1H, H aromatic), 7.80 (t, J = 8.0 Hz, 1H, H aromatic), 7.89
d, J = 7.5 Hz, 2H, H aromatic), 8.06 (d, J = 6.5 Hz, 2H, H ,Hc' pyr-
idine), 8.26 (d, J = 8.0 Hz, 1H, H aromatic), 8.83 (t, J = 5.8 Hz, 1H,
NeH ), 8.98 (d, J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.31 (s, 1H, NeH ).
C NMR (DMSO‑d , 125 MHz): δ (ppm) = 29.6, 30.2, 41.7, 59.9,
07.8, 125.6, 127.7, 128.3, 128.7, 129.0, 130.5, 130.6, 131.5, 134.2,
a
thiazole,), 7.73 (t,
c
1
1
3
(
c
b
2
6
, 125 MHz): δ (ppm) = 29.4, 30.0, 41.0, 61.7, 107.8, 116.2
1
b
2
(JC–F = 21.1 Hz), 122.0, 125.6, 127.7, 128.7, 131.4, 131.6, 134.3,
144.6, 148.7, 157.9, 160.2, 162.2 (JC–F = 251.3 Hz), 170.9, 171.4
(2C]O). MS (m/z, %): 475.3 (M+, 100). Purity: > 99%.
1
3
6
1
10

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
+
4
.2.7. 1-(2-Chlorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
J = 5.7 Hz, 2H, CH
aromatic), 7.60–7.63 (m, 2H, H
aromatic), 7.90 (d, J = 7.5 Hz, 2H, H aromatic), 8.00 (d, J = 6.5 Hz,
2H, H ,Hc' pyridine), 8.77 (t, J = 5.8 Hz, 1H, NeH ), 9.10 (d,
J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.30 (s, 1H, NeH
2
eNH), 5.78 (s, 2H, eCH
2
N
), 7.32–7.53 (m, 5H, H
butanamido)methyl) pyridin-1-ium chloride (7g)
a
thiazole, H aromatic), 7.82 (s, 1H, H
−
1
Yield: 80%; mp: 190–192 °C; IR (KBr) (νmax/cm ): 3333 and 3170
1
(
(
2NH), 1673 and 1603 (2C]O). H NMR(DMSO‑d
6
, 500 MHz) :δ
c
1
1
3
ppm) = 2.56 and 2.74 (t, J = 6.9 Hz, 4H, CH
2
eCH
2
), 4.58 (d,
b
2
). C NMR
+
J = 5.9 Hz, 2H, CH
2
eNH), 5.94 (s, 2H, eCH
2
N
), 7.25 (d, J = 5.7 Hz,
(DMSO‑d , 125 MHz) :δ (ppm) = 29.6, 30.2, 41.9, 61.9, 108.0, 122.5,
6
1
H, H aromatic), 7.30–7.44 (m, 5H, H aromatic), 7.59–7.61 (m, 2H, H
thiazole, H aromatic), 7.90 (d, J = 7.1 Hz, 2H, H aromatic), 8.03 (d,
J = 6.5 Hz, 2H, H ,Hc' pyridine), 8.57 (t, J = 6 Hz, 1H, NeH ), 9.0 (d,
J = 6.5 Hz, 2H, H ,Hb' pyridine), 12.28 (s, 1H, NeH ). C NMR
, 125 MHz): δ (ppm) = 29.4, 30.0, 41.0, 61.0, 107.8, 122.0,
a
125.8, 126.1, 128.0, 128.1, 129.0, 131.6, 131.8, 132.5, 134.4, 136.9,
144.3, 148.9, 158.1, 160.8, 171.2, 172.4 (2C]O). MS (m/z, %): 537.2
+
c
1
(M , 100). Purity: > 99%.
1
3
b
2
(
DMSO‑d
6
4.2.12. 1-(4-Bromobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
1
1
25.6, 127.7, 128.1, 128.7, 129.3, 130.1, 131.4, 131.6, 133.8, 134.3,
butanamido)methyl) pyridin-1-ium bromide (7l)
−
1
44.6, 148.7, 157.9, 160.9, 170.9, 171.3 (2C]O). MS (m/z, %): 491.2
Yield: 82%; mp: 221–223 °C; IR (KBr) (νmax/cm ): 3180 and 3057
+
1
(
M
, 100). Purity: 97%.
(2NH), 1670 and 1637 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
(
ppm) = 2.60 and 2.75 (t, J = 6.5 Hz, 4H, CH
2
eCH ), 4.54 (d,
2
+
4
.2.8. 1-(3-Chlorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
J = 5.7 Hz, 2H, CH
2
eNH), 5.79 (s, 2H, eCH
2
N
), 7.31–7.50 (m, 5H, H
butanamido)methyl) pyridin-1-ium bromide (7h)
aromatic), 7.59–7.63 (m, 3H, H
J = 7 Hz, 2H, H aromatic), 8.00 (d, J = 5.0 Hz, 2H, H
8.80 (t, J = 5.8 Hz, 1H, NeH ), 9.10 (d, J = 5.0 Hz, 2H, H
idine), 12.26 (s, 1H, NeH ).
a
thiazole, H aromatic), 7.89 (d,
,Hc' pyridine),
,Hb' pyr-
, 125 MHz): δ
−
1
Yield: 82%; mp: 225–227 °C; IR (KBr) (νmax/cm ): 3194 and
c
1
3
065(2NH), 1672 and 1640 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
1
b
1
3
(
ppm) = 2.61 and 2.75 (t, J = 6.5 Hz, 4H, CH
2
eCH
), 7.33 (t, J = 7.0 Hz,
thia-
2
), 4.55 (d,
2
C NMR (DMSO‑d
6
+
J = 5.7 Hz, 2H, CH
2
eNH), 5.82 (s, 2H, eCH
2
N
(ppm) = 29.4, 30.0, 41.7, 61.8, 107.7, 122.8, 125.7, 125.9, 127.7,
128.8, 131.0, 132.1, 133.6, 134.3, 144.1, 148.7, 157.8, 160.5, 170.9,
172.0 (2C]O). MS (m/z, %): 537.2 (M+, 100). Purity: 92%.
1
H, H aromatic) 7.41–7.50 (m, 5H, H aromatic), 7.59 (s, 1H, H
a
zole), 7.69 (s, 1H, H aromatic), 7.90 (d, J = 7.5 Hz, 2H, H aromatic),
8
.02 (d, J = 6.5 Hz, 2H, H
c
,Hc' pyridine), 8.78 (t, J = 5.8 Hz, 1H,
NeH
1
), 9.13 (d, J = 6.5 Hz, 2H, H
b
,Hb' pyridine), 12.27 (s, 1H, NeH ).
2
4.2.13. 1-(2-Methylbenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
1
3
C NMR (DMSO‑d
6
, 125 MHz): δ (ppm) = 29.4, 30.0, 41.6, 61.7,
butanamido)methyl) pyridin-1-ium bromide (7m)
−
1
1
1
07.8, 125.7, 125.9, 127.7, 128.7, 128.9, 129.4, 129.6, 131.1, 133.6,
Yield: 70%; mp: 224–226 °C; IR (KBr) (νmax/cm ): 3176 and 3028
1
34.2, 136.5, 144.2, 148.7, 157.8, 160.5, 170.9, 172.0 (2C]O). MS
(2NH), 1697 and 1654 (2C]O). H NMR(DMSO‑d
6
, 500 MHz) :δ
+
(
m/z, %): 491.2 (M , 100). Purity: > 99%.
(ppm) = 2.27 (s, 3H, eCH
CH eCH
3
), 2.61 and 2.75 (t, J = 6.7 Hz, 4H,
+
2
2
), 4.57 (d, J = 5.8 Hz, 2H, CH
2
eNH), 5.86 (s, 2H, eCH
2
N
),
4
.2.9. 1-(4-Chlorobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
7.11 (d, J = 7.5 Hz, 1H, H aromatic), 7.25e7.33 (m, 4H, H aromatic),
7.43 (t, J = 7.7 Hz, 2H, H aromatic), 7.59 (s, 1H, H thiazole), 7.90 (d,
J = 7.7 Hz, 2H, H aromatic), 8.01 (d, J = 6.5 Hz, 2H, H ,Hc' pyridine),
8.81 (t, J = 5.8 Hz, 1H, NeH ), 8.92 (d, J = 6.5 Hz, 2H, H ,Hb' pyr-
, 125 MHz): δ
butanamido)methyl) pyridin-1-ium chloride (7i)
a
−
1
Yield: 85%; mp: 208–210 °C; IR (KBr) (νmax/cm ): 3174 and
c
1
3
047(2NH), 1671and 1639 (2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
1
).
b
1
3
(
ppm) = 2.61 and 2.75 (t, J = 6.7 Hz, 4H, CH
2
eCH
2
), 4.54 (d,
idine), 12.30 (s, 1H, NeH
2
C NMR (DMSO‑d
6
+
J = 5.7 Hz, 2H, CH
2
eNH), 5.82 (s, 2H, eCH
2
N
), 7.32 (t, J = 7.5 Hz,
(ppm) = 18.7, 29.5, 30.1, 41.6, 61.4, 108.4, 125.6, 125.7, 126.7,
127.7, 128.7, 129.0, 129.1, 129.3, 130.9, 132.3, 136.8, 144.8, 148.7,
1
H, H aromatic), 7.43 (t, J = 7.5 Hz, 2H, H aromatic), 7.49 (d,
+
J = 8.5 Hz, 2H, H aromatic), 7.57 (d, J = 8.5 Hz, 2H, H aromatic), 7.61
s, 1H, H thiazole), 7.90 (d, J = 7.5 Hz, 2H, H aromatic), 8.01 (d,
,Hc' pyridine), 8.80 (t, J = 5.8 Hz, 1H, NeH ), 9.12
157.9, 160.7, 170.9, 172.0 (2C]O). MS (m/z, %): 471.3 (M , 100).
(
a
Purity: > 99%.
J = 6.5 Hz, 2H, H
c
1
1
3
(
(
d, J = 6.5 Hz, 2H, H
b
,Hb' pyridine), 12.30 (s, 1H, NeH
2
). C NMR
4.2.14. 1-(3-Methylbenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
DMSO‑d
6
, 125 MHz): δ (ppm) = 29.6, 30.2, 41.6, 61.6, 107.8, 125.6,
butanamido)methyl) pyridin-1-ium chloride (7n)
−1
1
1
25.8 127.7, 128.7, 129.2, 130.8, 133.3, 134.1, 134.2, 144.1, 148.7,
Yield: 68%; mp: 196–198 °C; IR (KBr) (νmax/cm ): 3333 and 3178
57.8, 160.5, 170.9, 172.0 (2C]O). MS (m/z, %): 491.2 (M+, 100).
(2NH), 1673 and 1606 (2C]O). H NMR(DMSO‑d
1
6
, 500 MHz): δ
Purity: 90%.
(ppm) = 2.25 (s, 3H, eCH
3
), 2.57 and 2.74 (t, J = 6.7 Hz, 4H,
+
CH eCH
2
2
), 4.58 (d, J = 5.9 Hz, 2H, CH
2
eNH), 5.75 (s, 2H, eCH
2
N
),
4
.2.10. 1-(2-Bromobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
7.25 (d, J = 5.0 Hz, 1H, H aromatic), 7.29–7.44 (m, 6H, H aromatic),
7.60 (s, 1H, H thiazole), 7.90 (d, J = 7.8 Hz, 2H, H aromatic), 8.01 (d,
J = 6.5 Hz, 2H, H ,Hc' pyridine), 8.57 (t, J = 5.8 Hz, 1H, NeH ), 9.11
(d, J = 6.5 Hz, 2H, H
(DMSO‑d
butanamido)methyl) pyridin-1-ium bromide (7j)
a
−
1
Yield: 80%; mp: 223–225 °C; IR(KBr) (νmax/cm ): 3185 and 3052
c
1
1
13
(
(
2NH), 1655 and 1639(2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
b
,Hb' pyridine), 12.28 (s, 1H, NeH ). C NMR
2
ppm) = 2.62 and 2.76 (t, J = 6.7 Hz, 4H, CH
2
2
eCH ), 4.58 (d,
6
, 125 MHz): δ (ppm) = 21.6, 29.6, 30.2, 41.0, 61.5, 107.8,
+
J = 5.7 Hz, 2H, CH
2
eNH), 5.92 (s, 2H, eCH
2
N
), 7.31–7.50 (m, 6H, H
122.0, 125.6, 125.8, 127.7, 128.4, 128.7, 129.5, 129.7, 131.6, 134.3,
136.2, 144.6, 148.7, 157.9, 160.3, 170.9, 171.3 (2C]O). MS (m/z, %):
471.3 (M+, 100). Purity: 92%.
aromatic), 7.59 (s, 1H, H
a
thiazole), 7.75 (d, J = 7.9 Hz, 1H, H aro-
matic), 7.90 (d, J = 7.5 Hz, 2H, H aromatic), 8.03 (d, J = 6.5 Hz, 2H,
H
c
,Hc' pyridine), 8.83 (t, J = 5.7 Hz, 1H, NeH ), 8.98 (d, J = 6.5 Hz,
1
1
3
2
1
1
1
1
H, H
b
,Hb' pyridine), 12.31 (s, 1H, NeH
2
). C NMR (DMSO‑d
6
,
4.2.15. 1-(4-Methylbenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
25 MHz): δ (ppm) = 29.4, 30.0, 41.6, 62.3, 107.9, 123.5, 125.7,
butanamido)methyl) pyridin-1-ium chloride (7o)
−1
27.6, 128.7, 128.8, 129.5, 131.4, 131.6, 133.2, 133.5, 134.3, 144.6,
Yield: 75%; mp: 208–210 °C; IR (KBr) (νmax/cm ): 3333 and 3170
+
1
48.8, 157.9, 160.9, 171.0, 172.2 (2C]O). MS (m/z, %): 537.2 (M
,
(2NH), 1673 and 1639 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
00). Purity: > 99%.
(ppm) = 2.24 (s, 3H, eCH
CH eCH
3
), 2.60 and 2.74 (t, J = 6.3 Hz, 4H,
+
2
2
), 4.53 (d, J = 5.5 Hz, 2H, CH
2
eNH), 5.76 (s, eCH
2
N
), 7.21
4
.2.11. 1-(3-Bromobenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
(d, J = 7.5 Hz, 2H, H aromatic), 7.24–7.45 (m, 5H, H aromatic), 7.61
(s, 1H, H thiazole), 7.91 (d, J = 7.0 Hz, 2H, H aromatic), 7.99 (d,
J = 6.0 Hz, 2H, H ,Hc' pyridine), 8.91 (t, J = 5.5 Hz, 1H, NeH ), 9.12
(d, J = 6.0 Hz, 2H, H
(DMSO‑d , 125 MHz): δ (ppm) = 20.7, 29.4, 30.2, 41.6, 62.4, 107.8,
butanamido)methyl) pyridin-1-ium bromide (7k)
a
−
1
Yield: 75%; mp: 210–212 °C; IR (KBr) (νmax/cm ): 3197 and 3043
c
1
1
13
(
(
2NH), 1671and 1641(2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
b
,Hb' pyridine), 12.30 (s, 1H, NeH ). C NMR
2
ppm) = 2.61 and 2.75 (t, J = 6.6 Hz, 4H, CH
2
2
eCH ), 4.55 (d,
6
11

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
1
1
1
22.0, 125.6, 125.7, 127.7, 128.7, 129.7, 131.4, 134.3, 138.9, 144.0,
(JC–F = 21.6 Hz), 125.8, 127.5, 127.7, 128.9, 129.5, 130.9, 131.5,
133.6, 134.4, 141.8, 144.4, 149.0, 157.8, 161.3 (JC–F = 251.3 Hz),
171.0, 172.0 (2C]O). MS (m/z, %): 475.3 (M+, 100). Purity: > 99%.
+
48.7, 157.8, 160.3, 170.9, 171.3 (2C]O). MS (m/z, %): 471.3 (M
,
00). Purity: 93%.
4
.2.16. 1-(3-Methoxybenzyl)-4-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
4.2.20. 1-(4-Chlorobenzyl)-3-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
butanamido)methyl) pyridin-1-ium chloride (7p)
butanamido)methyl) pyridin-1-ium bromide chloride (7t)
−
1
−1
Yield: 70%; mp: 199–201 °C; IR (KBr) (νmax/cm ): 3333 and 3178
Yield: 68%; mp: 194–196 °C; IR (KBr) (νmax/cm ): 3246 and 3065
1
1
(
(
2NH), 1674 and 1637 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
(2NH), 1659 and 1599 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
ppm) = 2.60 and 2.74 (t, J = 6.5 Hz, 4H, CH
), 4.54 (d, J = 5.5 Hz, 2H, CH
2
eCH
2
), 3.70 (s, 3H,
(ppm) = 2.58 and 2.75 (t, J = 6.0 Hz, 4H, CH
2
eCH ), 4.47 (d,
2
+
+
eOCH
3
2
eNH), 5.75 (s, 2H, eCH
2
N
),
J = 4.5 Hz, 2H, CH
2
eNH), 5.87 (s, 2H, eCH
2
N
), 7.32 (t, J = 7.5 Hz,
6
.97 (d, J = 8.0 Hz, 1H, H aromatic), 7.07 (d, J = 8.0 Hz, 1H, H
aromatic), 7.16 (s, 1H, H aromatic), 7.25–7.45 (m, 4H, H aromatic),
.61 (s, 1H, H thiazole), 7.90 (d, J = 7.5 Hz, 2H, H aromatic), 8.00 (d,
,Hc' pyridine), 8.85 (t, J = 5.0 Hz, 1H, NeH ), 9.12
d, J = 6.0 Hz, 2H, H ,Hb' pyridine), 12.30 (s, 1H, NeH ). C NMR
DMSO‑d , 125 MHz): δ (ppm) = 29.5, 30.2, 41.6, 55.0, 62.5, 107.8,
1H, H aromatic), 7.42 (t, J = 7.5 Hz, 2H, H aromatic), 7.51 (d,
J = 7.8 Hz, 2H, H aromatic) , 7.59–7.63 (m, 3H, H thiazole, H aro-
matic), 7.88 (d, J = 7.5 Hz, 2H, H aromatic), 8.13 (t, J = 6.5 Hz, 1H,
pyridine), 8.50 (d, J = 6.5 Hz, 1H, H pyridine), 8.88 (t, J = 5.0 Hz,
1H, NeH ), 9.13–9.15 (m, 2H, H ,H pyridine), 12.33 (s, 1H, NeH ).
C NMR (DMSO‑d
a
7
a
J = 6.0 Hz, 2H, H
c
1
H
d
c
1
3
(
(
b
2
1
b
e
2
1
3
6
6
, 125 MHz): δ (ppm) = 29.4, 30.0, 41.1, 62.4,
1
1
4
12.7, 114.6, 120.5, 125.6, 125.7, 128.6, 128.8, 129.8, 131.6, 135.7,
107.7, 125.6, 127.7, 127.9, 128.6, 129.1, 130.7, 133.0, 134.1, 134.2,
141.1, 143.0, 143.1, 144.3, 148.7, 157.7, 170.9, 171.9 (2C]O). MS
(m/z, %): 491.2 (M+, 100). Purity: > 99%.
44.1, 148.7, 157.5, 159.6, 160.8, 171.7, 172.6 (2C]O). MS (m/z, %):
+
87.3 (M , 100). Purity: > 99%.
4
.2.17. 1-Benzyl-3-((4-oxo-4-((4-phenylthiazol-2-yl)amino)butanamido)
4.2.21. 1-(4-Bromobenzyl)-3-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
methyl)pyridin-1-ium bromide (7q)
butanamido)methyl) pyridin-1-ium bromide bromide (7u)
−
1
−1
Yield: 75%; mp: 158–160 °C; IR (KBr) (νmax/cm ): 3392 and 3193
Yield: 65%; mp: 138–140 °C; IR (KBr) (νmax/cm ): 3306 and 3187
1
1
(
(
2NH), 1672 and 1557 (2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
(2NH), 1699 and 1654 (2C]O). H NMR(DMSO‑d
6
, 500 MHz): δ
ppm) = 2.57 and 2.75 (t, J = 6.5 Hz, 4H, CH
2
eCH
2
), 4.48 (d,
(ppm) = 2.58 and 2.75 (t, J = 6.4 Hz, 4H, CH
2
eCH ), 4.47 (d,
2
+
+
J = 5.5 Hz, 2H, CH
2
eNH), 5.85 (s, 2H, eCH
2
N
), 7.32 (t, J = 7.5 Hz,
J = 5.3 Hz, 2H, CH
2
eNH), 5.84 (s ,2H, eCH
2
N
), 7.32 (t, J = 7.5 Hz,
1
H, H aromatic), 7.41–7.45 (m, 5H, H aromatic), 7.52 (d, J = 7 Hz, 2H,
H aromatic), 7.59 (s, 1H, H thiazole), 7.89 (d, J = 7.5 Hz, 2H, H
aromatic), 8.13 (t, J = 7.5 Hz, 1H, H pyridine), 8.49 (d, J = 7.5 Hz,
H, H pyridine), 8.76 (t, J = 5.8 Hz, 1H, NeH ), 9.09–9.11 (m, 2H,
,H ). C NMR (DMSO‑d , 125 MHz): δ
1H, H aromatic), 7.43 (t, J = 7.5 Hz, 2H, H aromatic), 7.49 (d,
J = 8.0 Hz, 2H, H aromatic) 7.59 (s, 1H, H thiazole), 7.65 (d,
a
a
d
J = 8.0 Hz, 2H, H aromatic), 7.89 (d, J = 7.5 Hz, 2H, H aromatic), 8.13
1
c
1
(t, J = 7.5 Hz, 1H, H
d
pyridine), 8.49 (d, J = 7.5 Hz, 1H, H
), 9.08–9.10 (m, 2H, H ,H
c
e
pyridine),
pyridine),
1
3
H
b
e
pyridine), 12.32 (s, 1H, NeH
2
6
8.75 (t, J = 5.7 Hz 1H, NeH
1
b
1
3
(
ppm) = 29.4, 30.0, 41.1, 63.3, 107.7, 125.6, 127.7, 127.9, 128.6,
12.31 (s, 1H, NeH
2
). C NMR (DMSO‑d , 125 MHz): δ (ppm) = 29.5,
6
1
1
28.7, 129.1, 129.3, 134.1, 134.2, 141.1, 142.9, 143.1, 144.2, 148.7,
30.0, 42.3, 62.6, 107.8, 122.9, 125.6, 127.8, 128.1, 128.7, 129.6,
131.0, 132.1, 133.5, 134.3, 141.2, 143.2 144.3, 148.7, 157.8, 171.0,
172.0 (2C]O). MS (m/z, %): 537.2 (M+, 100). Purity: > 99%.
+
57.8, 170.9, 171.9 (2C]O). MS (m/z, %): 457.3 (M
, 100).
Purity: > 99%.
4
.2.18. 1-(4-Nitrobenzyl)-3-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
4.3. Cholinesterase inhibition assay
butanamido)methyl) pyridin-1-ium bromide (7r)
−
1
Yield: 65%; mp: 203–205 °C; IR (KBr) (νmax/cm ): 3347 and 3241
Acetylcholinesterase (AChE, E.C. 3.1.1.7, Type V-S, lyophilized
powder, from electric eel, 1000 units), butyrylcholinesterase (BChE,
E.C. 3.1.1.8, from equine serum), and acetylthiocholine iodide were
from Sigma-Aldrich. Reagent 5,5-dithiobis-(2-nitrobenzoic acid)
(DTNB) from Sigma-Aldrich was kindly donated by Dr Y. Azarmi
(Department of Pharmacology, Tabriz University of Medical Sciences,
Tabriz, Iran). Disodium hydrogen phosphate was obtained from Fluka.
Donepezil hydrochloride (Merck) used as the reference drug was kind
gift from Darou Pakhsh Pharma Chem co, Tehran, Iran. The AChE and
BChE inhibitory activity of synthesized compounds were determined by
the modified spectroscopic method introduced by Ellman using acet-
ylthiocholine iodide as the substrate in 96-well plates [59]. Derivatives
and donepezil (the positive control) were dissolved in methanol (or a
mixture of dimethyl sulfoxide (DMSO) and methanol) and then diluting
in phosphate buffer adjusted at pH 8 to prepare the stock solutions.
(Total amount of organic phase (DMSO and methanol) in each well was
0.5% at most.) Each compound was tested to inhibit the enzyme at
different concentrations to achieve a range of inhibition between 20
and 80%. The assay solution consisted of 100 μL phosphate buffer
(0.1 M, pH = 8), 30 μL DTNB (3.5 mM), 30 μL test compound solution
and 20 μL of 2.5 U/mL AChE or BChE solution. The reaction was then
initiated by adding 30 μL of acetylthiocholine iodide (7 mM) as the
substrate to each well. The hydrolysis rate of acetylthiocholine was
monitored at 412 nm by measuring the formation of yellow 5-thio-2-
nitrobenzoate anion produced due to enzyme catalysis. The obtained
1
(
(
2NH), 1658 and 1607 (2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
ppm) = 2.57 and 2.75 (t, J = 7.0 Hz, 4H, CH
2
eCH ), 4.48 (d,
2
+
J = 5.5 Hz, 2H, CH
2
eNH), 6.02 (s, 2H, eCH
2
N
), 7.32 (t, J = 7.5 Hz,
1
H, H aromatic), 7.42 (t, J = 7.5 Hz, 2H, H aromatic), 7.58 (s, 1H, H
a
thiazole), 7.74 (d, J = 8.3 Hz, 2H, H aromatic), 7.89 (d, J = 7.5 Hz, 2H,
H aromatic), 8.16 (t, J = 7.5 Hz, 1H, H
H, H aromatic), 8.52 (d, J = 7.5 Hz, 1H, H
), 9.10–9.12 (m, 2H, H ,H
d
pyridine), 8.28 (d, J = 8.3 Hz,
2
c
pyridine), 8.77 (t,
pyridine), 12.31 (s,
J = 5.5 Hz, 1H, NeH
1
b
e
1
3
1
4
1
H, NeH
2
). C NMR (DMSO‑d , 125 MHz): δ (ppm) = 29.4, 30.0,
6
2.7, 62.3, 107.9, 124.1, 125.6, 127.8, 128.2, 128.7, 129.9, 131.6,
33.7, 134.3, 141.1, 141.3, 144.5, 147.6, 148.7, 157.8, 171.0, 172.1
+
(
2C]O). MS (m/z, %): 502.3 (M , 100). Purity: > 99%.
4
.2.19. 1-(4-Fluorobenzyl)-3-((4-oxo-4-((4-phenylthiazol-2-yl)amino)
butanamido)methyl) pyridin −1-ium bromide (7s)
−
1
Yield: 62%; mp: 134–136 °C; IR (KBr) (νmax/cm ): 3365 and 3203
1
(
(
2NH), 1657 and 1604 (2C]O). H NMR (DMSO‑d
6
, 500 MHz): δ
ppm) = 2.57 and 2.75 (t, J = 6.5 Hz, 4H, CH
2
eCH ), 4.47 (d,
2
+
J = 5.0 Hz, 2H, CH
2
eNH), 5.83 (s, 2H, eCH
2
N
), 7.27–7.44 (m, 5H, H
aromatic), 7.59–7.63 (m, 3H, H
J = 7.6 Hz, 2H, H aromatic), 8.12 (t, J = 7.5 Hz, 1H, H
d, J = 7.5 Hz, 1H, H pyridine), 8.76 (t, J = 5.0 Hz, 1H, NeH
.08–9.10 (m, 2H, H ,H
DMSO‑d , 125 MHz): δ (ppm) = 29.5, 30.0, 42.6, 62.4, 107.8, 116.3
a
thiazole, H aromatic), 7.89 (d,
pyridine), 8.47
),
). C NMR
d
(
c
1
1
3
9
b
e
pyridine), 12.31 (s, 1H, NeH
2
(
6
12

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
data were used to calculate IC50 values (mean ± S.E.) by GraphPad
Prism (Version 6.01, 2012, GraphPad Software, Inc. USA). Each data
point was the average of experiments in triplicate.
reader mode with excitation at 535 nm and emission at 595 nm. Each
compound was assayed in triplicate. Background intensity was sub-
tracted from all the readings. The percent of fluorescence emission in-
hibition was calculated using the following formula: 100 – (IF /
i
4.4. Kinetic studies of AChE inhibition
IF
0
× 100), where IF
i
and IF are the fluorescence intensities with and
0
without inhibitor, respectively.
The reciprocal plots of 1/v versus 1/[S] were obtained by per-
forming the AChE inhibition experiments at various concentrations of
acetylthiocholine [S] and test compounds 7d and 7j using Ellman’s
method. The initial velocity (v) was determined based on the progress
of the hydrolysis reaction monitored by absorbance change (ΔA) at
4.7. Cell culture and MTT assay
PC12 cells were obtained from Pasteur Institute, Tehran, Iran, and
cultured in DMEM supplemented with 10% fetal calf serum, 60 μg/mL
penicillin, and 100 μg/mL streptomycin. To induce neuronal differ-
entiation, the cells grown to 70% confluency were harvested by
trypsin/EDTA (0.25%) solution and seeded in 96 well culture plate
(4000 cells/well) and then cultured for one week in differentiation
medium (DMEM + 2% horse serum + Nerve growth factor (NGF)
(100 ng/ml) + penicillin and streptomycin). The effect of compounds
7a, 7d, 7g, 7j and 7m on the survival rate of PC12 neural cells were
evaluated by changing the culture medium to NGF free medium and
applying different concentrations of the compounds (1, 10 and 100 μg/
mL) on cells. Donepezil (1, 10 and 100 μM) was used as the positive
control. To apply the studied compounds at the required concentra-
tions, their methanolic stock solutions were diluted in DMEM and a
10 μL volume was added to each well. Three hours later, apoptosis was
4
12 nm for 2 min and was expressed by ΔA/min. The slopes and (1/v)-
axis intercepts of the reciprocal plots for each compound at different
concentrations were used to generate secondary plots (slope and y-in-
tercept replots). In slope replot, the slops of 1/v versus 1/[S] lines in the
LWB plot were drawn against inhibitor concentration. In this replot, the
slope is K
the K
slope (i.e., the x-axis intercept equals – K
m
/(Vmax × K
i
) and vertical intercept is K /Vmax from which
m
i
value was determined by vertical-axis intercept divided by the
i
). The K values were also
i
determined using the nonlinear regression method implemented in
GraphPad Prism (Version 6.01, 2012, GraphPad Software, Inc. USA) by
analyzing the variation of velocity as a result of changes in the con-
centrations of both substrate and inhibitor. The factor α for the in-
hibition was determined using the y-intercept replot (also called (1/v)-
intercept replot) where the abscissa intercept equals –αK
i
.
induced by adding H
2
2
O (400 μM) to the medium followed by per-
forming MTT assay after 12 h incubation. To each well was added a
10 μL MTT solution (5 mg/mL), and after 3.5 h, the medium was re-
moved gently and 100 μL of the formazan solubilization solution con-
taining 10% SDS in 0.01 M HCl (w/v) was added into each well. Then,
the absorbance was measured at 570 nm with a reference wavelength of
630 nm using a plate reading spectrophotometer. The experiments were
performed in triplicates. Culture media and supplements were from
Gibco.
4
.5. Inhibition of Aβ1–42 self-induced aggregation
Inhibition of self-induced Aβ1–42 aggregation of compounds 7a, 7d,
g, 7j and 7m were measured using a thioflavin T (ThT)-based
7
fluorometric assay method [64]. The Aβ sample (Anaspec Inc) as HFIP
(
hexafluoroisopropanol) pretreated Aβ1–42 was dissolved in DMSO to
give a 200 µM stock solution and then diluted in phosphate buffer
(
pH = 7.4, 50 mM) to obtain a 20 µM solution of Aβ1–42 for the use in
the assay. Compounds were dissolved in DMSO and diluted in the
phosphate buffer to a final concentration of 20 μM. Then, the incuba-
tion of the peptide (final Aβ concentration = 10 µM, 10 µL) with and
without inhibitor (final concentration = 10 µM, 10 µL) was performed
at 30 °C for 48 h. After incubation, the prepared samples were diluted
by 180 μL of 5 mM ThT in 50 mM glycine-NaOH buffer adjusted at pH
4.8. In vitro PAMPA-BBB assay
The in vitro brain permeability of the compounds was assessed by
PAMPA-BBB assay described in literature with some modifications
[67,68]. Test compounds were dissolved in DMSO at ~5 to 10 mg/mL
and the stock solutions were diluted in PBS at pH 7.4 to get the final
concentrations ~50 to 125 μg/mL. To each donor well was added
200 μL of the final solution. The filter membrane of the acceptor mi-
croplate (MultiScreen PAMPA filter plate, Millipore, kindly donated by
Professor M. Foroutan from Faculty of Pharmacy, Shahid Beheshti
University of Medical Sciences) was hydrated overnight in EtOH:PBS
(3:7 V/V) and then was coated with 5 μL of soy lecithin lipid (lecithin,
Carl Roth, kindly donated by Professor H. Valizadeh from Faculty of
Pharmacy, Tabriz University of Medical Sciences) in dodecane (20 mg/
mL). Subsequently, the acceptor well was filled with 200 μL of pH 7.4
PBS. The acceptor filter plate was placed on top of the donor plate to
form a “sandwich”, which then was incubated for 4 h to allow the
diffusion of the test compound from the donor well into the acceptor
well via lipid membrane. After incubation, the drug concentrations in
acceptor and donor plates were determined by UV or fluorescent
spectroscopy and the results were used to determine effective perme-
ability (Pe) of the compounds. The experiment for each compound was
performed in triplicates. Appropriate controls with known BBB per-
meability were also tested to validate the method. The integrity of the
lipid membrane was evaluated using phenol red, a highly charged co-
lored compound.
8
.5 to the final volume of 200 μL. The fluorescence intensity was re-
corded on a microplate reader (Synergy HTX Multi-Mode Reader,
BioTek Instruments) with excitation and emission wavelengths set at
4
40 nm and 485 nm, respectively. Each measurement was run in tri-
plicates. Background fluorescence for a 5 mM ThT solution was sub-
tracted from the intensities of the samples and the percent of ag-
gregation inhibition was calculated according to equation (1- IF /
i
IF
o
) × 100% in which IF
i
and IF are the fluorescence intensities ob-
o
tained for Aβ in the presence and absence of inhibitor, respectively. The
data were expressed as the average of measurements in triplicates.
4.6. Propidium iodide displacement assay
Propidium iodide is a compound that binds to PAS of AChE speci-
fically. Being attached to the PAS of the enzyme, its fluorescence in-
tensity increases up to eight folds. The compounds which are able to
bind to the PAS could displace propidium iodide in a competitive
manner leading to a decrease in fluorescence. This phenomenon implies
that the compounds are PAS binders. 150 μL of 250 μM concentration of
test compounds (final concentration = 93.75 μM) were added to 200 μL
of 10U/mL of AChE (final concentration = 5U/mL) followed by 20 h
incubation at 25 °C. Afterward, 50 μL propidium iodide 8 μM (final
concentration = 1 μM) was added and the assay mixture was further
incubated for 15 min. To justify the background signal, control wells
containing all reagents except AChE were used as the blank solution.
The decrease in fluorescence intensity was measured on a well plate
4.9. Docking studies
The 3D structures of the studied compounds were generated using
Hyperchem software (version 8.0.8) and energy minimization using
molecular mechanics (MM+) and semiempirical AM1 methods
13

G. Ghotbi, et al.
Bioorganic Chemistry 103 (2020) 104186
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in Alzheimer's disease, Neurotherapeutics. 12 (2015) 3–11.
The authors declare that they have no known competing financial
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pitfalls and promise, Biol. Psychiatry. 83 (2018) 311–319.
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The authors would like to dedicate this paper to the memory of the
late Professor Abbas Shafiee from Tehran University of Medical
Sciences who contributed to the design of the project at the initial
stages. We are grateful to Professor H. Nadri from Shahid Sadoughi
University of Medical Sciences for his valuable assistance in performing
propidium iodide displacement assay. We also would like to thank the
Research Office and Biotechnology Research Center at Tabriz
University of Medical Sciences for providing financial support under
Postgraduate Research Scheme to the PhD thesis of GG (Grant number:
[23] A. Tripathi, P.K. Choubey, P. Sharma, A. Seth, P.N. Tripathi, M.K. Tripathi,
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Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
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