Synthesis, in vitro assays, molecular docking, theoretical ADMET prediction, and evaluation of 4-methoxy-phenylthiazole-2-amine derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s00044-019-02405-6

Based on the cholinergic hypothesis of the reported compound, N-(4-(4-methoxy-phenyl)thiazol-2-yl)-3-(pyrrolidin-1-yl)propionamide, which had a good inhibitory activity to acetylcholinesterase (AChE), the new 4-methoxy-phenylthiazole-2-amine derivatives as AChE inhibitors (AChEIs) have been designed and synthesized in this study. Their chemical structures were confirmed by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, mass spectrometry, and infrared. Furthermore, their inhibitory activities against AChE in vitro were also tested by Ellman spectrophotometry, and the inhibitory activity test results showed that most of the compounds of 4-methoxy-phenylthiazole-2-amine derivatives had a certain AChE inhibitory activity in vitro, and the IC₅₀ (half-maximal inhibitory concentration) value of compound 5g was 5.84 μmol/L, which was higher than that of the reference compound, rivastigmine. Moreover, it had almost no inhibitory effect on butyrylcholinesterase. In addition, compound 5g was subjected to enzyme inhibition kinetics experiments, and the result of Lineweaver–Burk’s V^?1–[S]^?1 double-reciprocal plot showed that the acting type of compound 5g was mixed inhibition type. Furthermore, the AChE inhibitory activity mechanism of compound 5g was explored by the conformational analysis and molecular docking, which was based on the principle of the four-point pharmacophore model necessary for AChE inhibition. Finally, in silico molecular property and ADMET (absorption, distribution, metabolism, excretion, and toxicity) of the synthesized compounds were predicted by using Molinspiration and PreADMET online servers, respectively. It can be concluded that the lead AChEI compound 5g presented satisfactory drug-like characteristics and ADME properties.

Full text of DOI:10.1007/s00044-019-02405-6

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research
https://doi.org/10.1007/s00044-019-02405-6
ORIGINAL RESEARCH
Synthesis, in vitro assays, molecular docking, theoretical ADMET
prediction, and evaluation of 4-methoxy-phenylthiazole-2-amine
derivatives as acetylcholinesterase inhibitors
Xiao-Zhong Zhang1 Yuan Xu1 Meng-Meng Jian1 Kan Yang1 Zheng-Yue Ma1
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Received: 21 February 2019 / Accepted: 17 July 2019
© Springer Science+Business Media, LLC, part of Springer Nature 2019
Abstract
Based on the cholinergic hypothesis of the reported compound, N-(4-(4-methoxy-phenyl)thiazol-2-yl)-3-(pyrrolidin-1-yl)
propionamide, which had a good inhibitory activity to acetylcholinesterase (AChE), the new 4-methoxy-phenylthiazole-2-
amine derivatives as AChE inhibitors (AChEIs) have been designed and synthesized in this study. Their chemical structures
were confirmed by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, mass spectrometry, and
infrared. Furthermore, their inhibitory activities against AChE in vitro were also tested by Ellman spectrophotometry, and
the inhibitory activity test results showed that most of the compounds of 4-methoxy-phenylthiazole-2-amine derivatives had
a certain AChE inhibitory activity in vitro, and the IC50 (half-maximal inhibitory concentration) value of compound 5g was
5.84 μmol/L, which was higher than that of the reference compound, rivastigmine. Moreover, it had almost no inhibitory
effect on butyrylcholinesterase. In addition, compound 5g was subjected to enzyme inhibition kinetics experiments, and the
result of Lineweaver–Burk’s V−1–[S]−1 double-reciprocal plot showed that the acting type of compound 5g was mixed
inhibition type. Furthermore, the AChE inhibitory activity mechanism of compound 5g was explored by the conformational
analysis and molecular docking, which was based on the principle of the four-point pharmacophore model necessary for
AChE inhibition. Finally, in silico molecular property and ADMET (absorption, distribution, metabolism, excretion, and
toxicity) of the synthesized compounds were predicted by using Molinspiration and PreADMET online servers, respectively.
It can be concluded that the lead AChEI compound 5g presented satisfactory drug-like characteristics and ADME properties.
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Keywords Acetylcholinesterase (AChE) Phenylthiazole Molecular docking AutoDock ADMET
Introduction
cognitive deficit have been witnessed. Several factors such
as, both genetic and environmental, reduced levels of
acetylcholine, amyloid β-protein aggregation, tau-hyper-
phosphorylation, hypoxia, and so on have been considered
to play a crucial role in cognition decline (Zhang et al.
2016; Grozeva et al. 2019). Despite several attempts made
on the development of new AD therapies, efficacious
therapeutic interventions for the suppression of AD have
not yet been established. AD is characterized by the dis-
turbance of forebrain cholinergic system and the dramatic
decrease of acetylcholine (ACh), choline acetyltransferase,
and a severe loss of cholinergic neurons (Pepeu et al. 2017).
Acetylcholinesterase (AChE) belongs to the class of
serine hydrolase family of enzymes and is responsible for
catalyzing the hydrolysis of neurotransmitter ACh into
choline and acetic acid, thereby causing the termination of
cholinergic neurotransmission (Lopes et al. 2014; Kaushik
et al. 2018). The inhibition of AChE would prevent the
Alzheimer’s disease (AD), the most common cause of
dementia, is a neurodegenerative disorder characterized by
progressive deterioration of memory and cognition (Hir-
emathad et al. 2018). AD is thought to be the leading cause
of senile dementia affecting nearly 50 million people
worldwide (Schelke et al. 2018). In the past two decades,
considerable research efforts devoted to unraveling the
molecular, biochemical, and cellular mechanisms of
* Zheng-Yue Ma
mazhengy@126.com
1
Key laboratory of Drug Quality Control of Hebei Province,
College of pharmaceutical science, Hebei University, 071002
Baoding, China

Medicinal Chemistry Research
Fig. 1 Acetylcholinesterase
(AChE) inhibitors clinically
used for Alzheimer’s disease
(AD) treatment
hydrolysis of ACh, so that the level of ACh in cholinergic
synapses is increased.
Nowadays, many AChE inhibitors have been approved
for the therapeutic treatment of AD (Fig. 1), which include
tacrine, huperzine-A, donepezil, rivastigmine, physos-
tigmine, and galantamine, and so on (Atri et al. 2018).
However, none of these drugs is able to combat or reverse
the progression of the disease. Because of the limited
number of available clinical AChEIs, the search for new
AChEI is gaining wide attention.
In recent years, substantial attention has been given to
the chemistry of thiazole and its fused heterocyclic deriva-
tives due to biological importance. Cholinesterase inhibition
activity of compounds bearing thiazole scaffold have been
reported (Ibrar et al. 2018; Tripathi et al. 2018). Based on
the structural characteristics of donepezil, Yurttaş et al.
(2013) designed a series of new thiazole-piperazine deri-
vatives, and IC50 (half maximal inhibitory concentration)
value of their AChE inhibitory activity was up to
Fig. 2 Structural hypothesis of 4-methoxythiazole-2-amine derivatives
showing the shared pharmacophoric features essential for acet-
ylcholinesterase (AChE) inhibition
docking was conducted to investigate the potential binding
mode of compounds with the catalytic domain of AChE
(PDB: 4EY7) (Cheung et al. 2012), which was performed
using Autodock 4.2.
Moreover, in order to predict whether these compounds
have the potential to become clinical drugs, and get insights
into their pharmacokinetics profile, the compounds were
subjected to in silico molecular property and ADMET
(absorption, distribution, metabolism, excretion, and toxi-
city) predictions by using Molinspiration and PreADMET
online servers, respectively.
0.011 μmol/L, which is higher than donepezil (IC50
=
0.054 μmol/L) as a reference compound. A series of new N-
acyl-4-phenylthiazole-2-amine derivatives were designed
by Ma et al. (2014) and Tian et al. (2015), and the IC50
value of their AChE inhibitory activity was up to 0.51 μmol/
L, better than that of rivastigmine and huperzine-A as
reference compounds.
Based on these results (Ma et al. 2014), 4-methoxy-
phenylthiazole-2-amine derivatives as AChE inhibitors
(AChEI) were designed and synthesized in this paper.
Interestingly, three pharmacophoric features out of four-
point pharmacophore model for AChEIs, which was
reported by Bag et al. (2013), namely aromatic ring(s), H-
bond acceptor, and H-bond donor, are identified in 4-
methoxy-phenylthiazole-2-amine derivatives (Fig. 2).
Based on these evidences, the structure–activity relation-
ships (SAR) for AChE inhibition by 4-methoxy-phe-
nylthiazole-2-amine derivatives in vitro was explored.
Further, in order to better understand the action
mechanism of AChE inhibitory activity, the molecular
Materials and methods
Instrumentation and chemicals
All experiments were carried out under air atmosphere; the
reagent materials were commercially available analytically
pure or chemically pure, unless stated otherwise. High-
resolution mass spectrometry was determined by Bruker
Maxis 4G Mass Spectrometer, and the nuclear magnetic
resonance spectrum was measured by Bruker Avance III
600 MHz nuclear magnetic resonance spectrometer. The
melting point was determined by SGW X-4 micro melting

Medicinal Chemistry Research
point apparatus (Shanghai Precision Scientific Instrument
Co., Ltd). The infrared (IR) spectra was obtained by FTIR-
8400S Fourier transform infrared splitting Photometer (KBr
disks). The 96-well plate was read by 1420 Victor Micro-
plate Reader. AChE was purchased from Sigma, and
huperzine-A and rivastigmine were purchased from
Shanghai Yuanye Biotechnology Co., Ltd.
5-Bromo-N-(4-(4-methoxyphenyl)thiazol-2-yl)pentanamide
(4d)
It was obtained as a white solid; yield: 83%; mp 182–184 °C;
1
IR (KBr), ν (cm−1): 3143, 1683, 1588, 1270, 915, 893; H
NMR (600 MHz, DMSO-d6) δ = 12.09 (s, 1H, -CONH-),
7.55 (d, J = 7.6 Hz, 2H, -ArH), 7.36 (s, 1H, =CHS-), 7.03 (d,
J = 7.6 Hz, 2H, -ArH), 3.88 (s, 3H, -OCH3), 3.74 (t, J =
6.8 Hz, 2H, -CH2Br), 2.71 (t, J = 6.8 Hz, 2H, -CH2-), 1.82 (t,
Syntheses
J = 6.8 Hz, 2H, -CH2-), 1.53. (t, J = 6.8 Hz, 2H, -CH2-). 13
C
General procedure for the synthesis of (2-amino-4-
phenylthiazole) amide derivatives (4a–4e)
NMR (151 MHz, DMSO-d6) δ = 171.07, 158.49, 157.68,
148.61, 129.25, 126.95, 114.05, 105.81, 55.11, 34.58, 31.86,
23.32, 21.38; ESI-MS calcd for C15H18BrN2O2S 369.0272
[M + H]+, found 369.0264 [M + H]+.
Compounds 1, 2, and 3 (Ma et al. 2014) were obtained
according to the previously described method. Chlor-
oacetyl chloride (1.45 mmol) in CH2Cl2 (15 mL) was
added dropwise to a cooled solution (0 °C) of 4-(4-
methoxyphenyl)thiazol-2-amine 3 (1.21 mmol) and dii-
sopropylethylamine (DIEA) (1.24 mmol) in CH2Cl2
(25 mL). The completion of the reaction was assessed by
thin-layer chromatography (TLC). The organic layer was
separated, washed with saturated NaHCO3 solution
(100 mL), water, and brine, the aqueous phase was
extracted with CH2Cl2 (60 mL), and the organic phases
were collected, dried over anhydrous MgSO4, and eva-
porated in vacuo. The product was separated and purified
by column chromatography on silica gel using an ethyl
acetate/petroleum ether mixture (1/4) as the eluent to
afford pure compound 4a (Scheme 1).
The same procedure was performed with 3-
chloropropionyl chloride, 4-bromobutyryl chloride, 5-
bromopentanoyl chloride, and 6-bromohexanoyl chloride
to afford compounds 4b–4e.
2-Chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)acetamide
4a, 3-chloro-N-(4-(4-methoxyphenyl)thiazol-2-yl)propana-
mide 4b characterization data were consistent with that in
the previous literature (Ma et al. 2014).
6-Bromo-N-(4-(4-methoxyphenyl)thiazol-2-yl)hexanamide
(4e)
It was obtained as a white solid; yield: 85%; mp 186–188 °C;
1
IR (KBr), ν (cm−1):3145, 1683, 1590, 1275, 920, 890; H
NMR (600 MHz, DMSO-d6) δ = 12.17 (s, 1H, -CONH-),
7.82 (d, J = 8.5 Hz, 2H, -ArH), 7.43 (d, J = 4.6 Hz,
1H, =CHS-), 7.01 (dd, J = 33.5, 8.6 Hz, 2H, -ArH), 3.80
(d, J = 11.9 Hz, 3H, -OCH3), 3.54 (t, J = 6.7 Hz, 2H,
-CH2Br), 3.17 (s, 2H, -CH2-), 2.46 (t, J = 7.5 Hz, 2H, -CH2-),
1.86–1.79 (m, 2H, -CH2-), 1.64 (dt, J = 15.2, 7.4 Hz, 2H,
-CH2-). 13C NMR (151 MHz, DMSO-d6) δ = 171.28,
158.92, 157.72, 148.60, 127.17, 126.94, 114.05, 105.78,
55.11, 34.92, 34.68, 31.90, 27.07, 23.82; ESI-MS calcd for
C16H20BrN2O2S 383.0429 [M + H]+, found 383.0425
[M + H]+.
General procedure for the synthesis of N-(4-(4-
methoxyphenyl)thiazol-2-yl)-2-(pyridin-4-ylthio)amide and
N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-((1-benzylpiperidin-
4-yl)amino)-amide (5a–5j)
To a solution of (2-amino-4-phenylthiazole) amide deriva-
tives 4a–4e (5 mmol) in acetonitrile (5 mL), 4-
mercaptopyridine (6 mmol), DIEA (10 mmol), and potas-
sium iodide (0.5 mmol) were added, and the resulting
solution was stirred at 110 °C for 12 h. The completion of
the reaction was assessed by TLC. The organic layer was
separated, washed with saturated NaHCO3 solution
(100 mL), water, and brine, the aqueous phase was extrac-
ted with CH2Cl2 (60 mL), and the organic phases were
collected, dried over anhydrous MgSO4, and evaporated in
vacuo. The product was separated and purified by column
chromatography on silica gel using an ethyl acetate/petro-
leum ether mixture (1/2) as the eluent to afford pure com-
pounds 5a–5e.
4-Bromo-N-(4-(4-methoxyphenyl)thiazol-2-yl)butanamide
(4c)
It was obtained as a white solid; yield: 86%; mp 178–180 °C;
ν (cm−1): 3147, 1683, 1581, 1261, 912, 885; proton nuclear
magnetic resonance (1H NMR) (600 MHz, DMSO-d6): δ =
12.13 (s, 1H, -CONH-), 7.53 (d, J = 7.6 Hz, 2H, -ArH),
7.38 (s, 1H, = CHS-), 7.03 (d, J = 7.6 Hz, 2H, -ArH), 3.87
(s, 3H,-OCH3), 3.74 (t, J = 6.8 Hz, 2H, -CH2Br), 2.71 (t, J
= 6.8 Hz, 4H, -CH2-). Carbon-13 nuclear magnetic reso-
nance (13C NMR) (151 MHz, DMSO-d6): δ = 171.36,
158.49, 157.41, 148.61, 129.25, 127.37, 114.13, 105.72,
55.11, 31.66, 27.88, 21.70; ESI-MS calcd for
C14H16BrN2O2S 357.0095 [M + H]+, found 357.0090
[M + H]+.
The same procedure was performed with 4-amino-1-
benzylpiperidine using
a
methanol/dichloromethane

Medicinal Chemistry Research
Scheme 1 Synthetic routes of target compound
mixture (1/20) as the eluent to afford pure compounds 5f–5j
(Scheme 1).
J = 5.9 Hz, 2H, -ArH), 7.82 (d, J = 8.5 Hz, 2H, -ArH), 7.45
(s, 1H, =CHS-), 7.33 (d, J = 6.0 Hz, 2H, -ArH), 6.99 (d, J =
8.6 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.35 (t, J = 7.0 Hz,
2H, -CH2-), 2.88 (t, J = 7.0 Hz, 2H, -CH2-). 13C NMR
(151 MHz, DMSO-d6) δ = 169.22, 158.91, 157.09, 149.09,
147.91, 127.09, 126.95, 120.61, 114.07, 106.01, 55.12, 34.11,
24.95; ESI-MS calcd for C18H18N3O2S2 372.0840 [M + H]+,
found 372.0839 [M + H]+.
N-(4-(4-methoxyphenyl)thiazol-2-yl)-2-(pyridin-4-ylthio)
acetamide (5a)
It was obtained as a white solid; yield: 50%; mp 208–210 °C;
IR (KBr), ν (cm−1): 3112, 1674, 1580, 1260, 885; 1H NMR
(600 MHz, DMSO-d6) δ = 12.61 (s, 1H, -CONH-), 8.41 (d,
J = 5.8 Hz, 2H, -ArH), 7.83 (d, J = 8.6 Hz, 2H, -ArH), 7.49
(s, 1H, =CHS-), 7.36 (d, J = 6.0 Hz, 2H, -ArH), 7.00 (d, J
= 8.7 Hz, 2H, -ArH), 4.17 (s, 2H, -CH2-), 3.79 (s, 3H,
-OCH3). 13C NMR (151 MHz, DMSO-d6) δ = 166.59,
159.02, 157.39, 149.20, 147.44, 126.99, 125.30, 120.49,
114.10, 106.36, 55.13, 33.39; ESI-MS calcd for
C17H16N3O2S2 358.0684 [M + H]+, found 358.0675 [M +
H]+.
N-(4-(4-methoxyphenyl)thiazol-2-yl)-4-(pyridin-4-ylthio)
butanamide (5c)
It was obtained as a white solid; yield: 51%; mp 220–222 °C;
IR (KBr), ν (cm−1): 3168, 1681, 1582, 1262, 887; 1H NMR
(600 MHz, DMSO-d6) δ = 12.27 (s, 1H, -CONH-), 8.38 (d,
J = 4.2 Hz, 2H, -ArH), 7.83 (d, J = 8.5 Hz, 2H, -ArH), 7.43
(s, 1 H, =CHS-), 7.30 (d, J = 5.8 Hz, 2H, -ArH), 6.99
(d, J = 8.5 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.12 (t, J =
7.3 Hz, 2H, -CH2-), 2.65 (t, J = 7.2 Hz, 2H, -CH2-), 1.98 (p,
J = 7.3 Hz, 2H, -CH2-). 13C NMR (151 MHz, DMSO-d6)
δ = 170.65, 158.93, 157.74, 149.17, 148.13, 127.16,
126.95, 120.57, 114.05, 105.84, 55.11, 33.65, 28.90, 23.67;
ESI-MS calcd for C19H20N3O2S2 386.0997 [M + H]+,
found 386.0997 [M + H]+.
N-(4-(4-methoxyphenyl)thiazol-2-yl)-3-(pyridin-4-ylthio)
propanamide (5b)
It was obtained as a white solid; yield: 47%; mp 214–216 °C;
1
IR (KBr), ν (cm−1): 3110, 1681, 1583, 1265, 890; H NMR
(600 MHz, DMSO-d6) δ = 12.31 (s, 1H, -CONH-), 8.40 (d,

Medicinal Chemistry Research
N-(4-(4-methoxyphenyl)thiazol-2-yl)-5-(pyridin-4-ylthio)
pentanamide (5d)
NMR (600 MHz, DMSO-d6) δ = 7.83 (d, J = 8.7 Hz, 2H,
-ArH), 7.45 (s, 1H, =CHS-), 7.34–7.22 (m, 5H, -ArH), 6.99
(d, J = 8.7 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.03 (t, J =
6.5 Hz, 2H, -CH2-), 2.84–2.66 (m, 5H, -CH2-), 2.00–1.83
(m, 6H, -CH2-), 1.46 (dd, J = 20.7, 10.6 Hz, 2H, -CH2-).
13C NMR (151 MHz, DMSO-d6) δ = 169.12, 158.93,
157.49, 148.62, 138.01, 128.72, 126.84, 125.33, 124.91,
114.05, 105.88, 61.85, 55.12, 54.89, 51.34, 33.36, 29.98,
22.01; ESI-MS calcd for C25H31N4O2S 451.2168 [M + H]+,
found 451.2165 [M + H]+.
It was obtained as a white solid; yield: 49%; mp 225–227 °C;
IR (KBr), ν (cm−1): 3163, 1681, 1581, 1266, 887; 1H NMR
(600 MHz, DMSO-d6) δ = 12.21 (s, 1H, -CONH-), 8.35 (d,
J = 5.9 Hz, 2H, -ArH), 7.82 (d, J = 8.7 Hz, 2H, -ArH), 7.43
(s, 1H, =CHS-), 7.26 (d, J = 6.0 Hz, 2H, -ArH), 6.99 (d,
J = 8.7 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.09 (t, J =
7.3 Hz, 2H, -CH2-), 1.81-1.74 (m, 2H, -CH2-), 1.71–1.63
(m, 4H, -CH2-). 13C NMR (151 MHz, DMSO-d6) δ =
171.14, 158.50, 157.41, 149.13, 148.36, 127.16, 126.95,
120.53, 114.05, 105.82, 55.11, 34.23, 29.06, 27.52, 23.83;
ESI-MS calcd for C20H22N3O2S2 400.1153 [M + H]+,
found 400.1155 [M + H]+.
6-((1-Benzylpiperidin-4-yl)amino)-N-(4-(4-methoxyphenyl)
thiazol-2-yl)hexanamide (5j)
It was obtained as a white solid; yield: 40%; mp 235–237 °C;
IR (KBr), ν (cm−1): 3147, 1679, 1582, 1259, 888; 1H NMR
(600 MHz, DMSO-d6) δ = 7.82 (d, J = 8.5 Hz, 2H, -ArH),
7.41 (s, 1H, =CHS-), 7.34–7.20 (m, 5H, -ArH), 6.98 (t, J =
8.1 Hz, 2H, -ArH), 3.78 (s, 3H, -OCH3), 3.45 (d, J =
8.5 Hz, 2H, -CH2-), 2.79 (dd, J = 18.4, 10.5 Hz, 4H, -CH2-),
2.43 (dt, J = 41.0, 13.9 Hz, 2H, -CH2-), 1.97–1.81 (m, 4H,
-CH2-), 1.67–1.19 (m, 8H, -CH2-), 1.03 (dt, J = 21.0,
7.0 Hz, 1H, -CH-). 13C NMR (151 MHz, DMSO-d6) δ =
171.35, 158.92, 157.76, 148.22, 138.02, 128.69, 128.12,
126.94, 126.86, 114.05, 105.68, 61.74, 55.12, 54.88, 51.20,
34.68, 34.52, 28.97, 28.89, 25.81, 24.23; ESI-MS calcd for
C28H37N4O2S 493.2637 [M + H]+, found 493.2637 [M +
H]+.
N-(4-(4-methoxyphenyl)thiazol-2-yl)-6-(pyridin-4-ylthio)
hexanamide (5e)
It was obtained as a white solid; yield: 47%; mp 230–232 °C;
1
IR (KBr), ν (cm−1): 3112, 1681, 1585, 1260, 889; H NMR
(600 MHz, DMSO-d6) δ = 12.18 (s, 1H, -CONH-), 8.35 (d,
J = 6.1 Hz, 2H, -ArH), 7.82 (d, J = 8.7 Hz, 2H, -ArH), 7.42
(s, 1H, =CHS-), 7.25 (dd, J = 4.9, 1.3 Hz, 2H, -ArH), 6.99 (d,
J = 8.8 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.06 (t, J =
7.3 Hz, 2H, -CH2-), 2.46 (t, J = 7.3 Hz, 2H, -CH2-), 1.69–1.62
(m, 2H, -CH2-), 1.48–1.41 (m, 4H, -CH2-). 13C NMR
(151 MHz, DMSO-d6) δ = 171.31, 158.92, 157.41, 149.12,
148.50, 127.18, 126.94, 120.51, 114.05, 105.77, 55.11, 34.72,
29.30, 27.78, 27.66, 24.18; ESI-MS calcd for C21H24N3O2S2
414.1310 [M + H]+, found 414.1306 [M + H]+.
General procedure for the synthesis of N-(4-(4-
methoxyphenyl)thiazol-2-yl)-2-(pyridin-4-ylthio)amide
derivative (6a–6e)
2-((1-Benzylpiperidin-4-yl)amino)-N-(4-(4-methoxyphenyl)
thiazol-2-yl)acetamide (5f)
A solution of 5a–5e (5 mmol) and benzyl chloride (5 mmol)
in acetonitrile reacted at 110 °C for 24 h. Subsequently, the
precipitates were filtered and washed with dichloromethane
to give the solid salts 6a–6e (Scheme 1).
It was obtained as a white solid; yield: 47%; mp 232–234 °C;
IR (KBr), ν (cm−1): 3307, 1699, 1581, 1261, 890; H NMR
1
(600 MHz, DMSO-d6) δ = 7.83 (d, J = 8.6 Hz, 2H, -ArH),
7.45 (s, 1H, =CHS-), 7.33–7.21 (m, 5H, -ArH), 6.99 (d, J =
8.7 Hz, 2H, -ArH), 3.78 (s, 3H, -OCH3), 3.47 (s, 2H, -CH2-),
2.74 (d, J = 11.3 Hz, 2H, -CH2-), 2.43 (dd, J = 11.8, 8.0 Hz,
1H, -NH-), 1.98–1.90 (m, 5H, -CH2-), 1.77 (d, J = 11.4 Hz,
2H, -CH2-), 1.34–1.24 (m, 2H, -CH2-). 13C NMR (151 MHz,
DMSO-d6) δ = 170.91, 158.96, 157.08, 148.70, 138.61,
128.06, 126.75, 125.40, 125.20, 114.04, 105.97, 62.29, 55.11,
51.58, 48.86, 31.53, 21.05; ESI-MS calcd for C24H29N4O2S
437.2011 [M + H]+, found 437.2008 [M + H]+.
1-Benzyl-4-((2-((4-(4-methoxyphenyl)thiazol-2-yl)amino)-2-
oxoethyl)thio)pyri- din-1-ium chloride (6a)
It was obtained as a white solid; yield: 40%; mp 282–284 °C;
1
IR (KBr), ν (cm−1):3103, 1679, 1579, 1263, 889; H NMR
(600 MHz, DMSO-d6) δ = 12.87 (s, 1H, -CONH-), 8.99 (d,
J = 6.5 Hz, 2H, -ArH), 8.09 (d, J = 6.5 Hz, 2H, -ArH), 7.84
(d, J = 8.6 Hz, 1H, =CHS-), 7.56–7.49 (m, 5H, -ArH),
7.47–7.39 (m, 2H, -ArH), 7.00 (d, J = 8.7 Hz, 2H, -ArH),
4.53 (s, 4H, -CH2-), 3.80 (s, 3H, -OCH3). 13C NMR
(151 MHz, DMSO-d6) δ = 165.53, 161.86, 159.04, 157.09,
148.63, 142.41, 134.70, 129.15, 128.20, 127.01, 126.86,
125.31, 123.25, 114.10, 106.45, 61.77, 55.15, 33.97; ESI-
MS calcd for C24H22ClN3O2S2 448.1148 [M-Cl]+, found
448.1155 [M-Cl]+.
3-((1-Benzylpiperidin-4-yl)amino)-N-(4-(4-methoxyphenyl)
thiazol-2-yl)propan- amide (5g)
It was obtained as a white solid; yield: 40%; mp 235–237 °C;
IR (KBr), ν (cm−1): 3392, 1681, 1585, 1263, 886; 1H

Medicinal Chemistry Research
1-Benzyl-4-((3-((4-(4-methoxyphenyl)thiazol-2-yl)amino)-3-
oxopropyl)thio)pyridin-1-ium chloride (6b)
1-Benzyl-4-((6-((4-(4-methoxyphenyl)thiazol-2-yl)amino)-6-
oxohexyl)thio)pyridin-1-ium chloride (6e)
It was obtained as a white solid; yield: 35%; mp 286–288 °C;
IR (KBr), ν (cm−1): 3392, 1687, 1583, 1260, 885; 1H
NMR (600 MHz, DMSO-d6) δ = 12.41 (s, 1H, -CONH-),
8.98 (d, J = 6.9 Hz, 2H, -ArH), 8.05 (d, J = 7.0 Hz, 2H,
-ArH), 7.82 (d, J = 8.7 Hz, 1H, =CHS-), 7.54 (d, J =
6.9 Hz, 5H, -ArH), 7.49–7.38 (m, 2H, -ArH), 6.99 (d, J =
8.8 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3), 3.59 (t, J = 6.9 Hz,
4H, -CH2-), 2.97 (t, J = 6.9 Hz, 2H, -CH2-). 13C NMR
(151 MHz, DMSO-d6) δ = 168.92, 162.20, 158.97, 157.37,
148.71, 142.53, 134.69, 129.14, 128.31, 127.11, 126.96,
125.40, 123.18, 114,07, 106.08, 61.59, 55.14, 33.43, 25.97;
ESI-MS calcd for C25H24ClN3O2S2 462.1304 [M-Cl]+,
found 462.1307 [M-Cl]+.
It was obtained as a white solid; yield: 25%; mp 300–302 °C;
1
IR (KBr), ν (cm−1): 3107, 1677, 1589, 1266, 892; H NMR
(600 MHz, DMSO-d6) δ = 12.22 (s, 1H, -CONH-), 8.91 (d,
J = 7.0 Hz, 2H, -ArH), 7.97 (d, J = 7.0 Hz, 2H, -ArH), 7.82
(d, J = 8.6 Hz, 1H, =CHS-), 7.52 (d, J = 6.9 Hz, 5H,
-ArH), 7.46–7.39 (m, 2H, -ArH), 6.99 (d, J = 8.7 Hz, 2H,
-ArH), 3.79 (s, 3H, -OCH3), 3.28 (t, J = 7.3 Hz, 2H, -CH2-),
2.48 (t, J = 7.3 Hz, 2H, -CH2-), 1.74–1.64 (m, 4H, -CH2-),
1.47 (dd, J = 15.0, 7.9 Hz, 4H, -CH2-). 13C NMR
(151 MHz, DMSO-d6) δ = 171.30, 162.84, 158.93, 157.69,
148.22, 142.41, 134.28, 129.13, 128.56, 127.15, 126.89,
125.42, 123.11, 114.06, 105.79, 61.55, 55.13, 34.66, 30.29,
27.54, 27.15, 24.11; ESI-MS calcd for C28H30ClN3O2S2
504.1774[M-Cl]+, found 504.1773 [M-Cl]+.
1-Benzyl-4-((4-((4-(4-methoxyphenyl)thiazol-2-yl)amino)-4-
oxobutyl)thio)pyridin-1-ium chloride (6c)
Results and discussion
AChE and BuChE inhibition assay
In vitro AChE inhibition test
It was obtained as a white solid; yield: 30%; mp 290–293 °C;
IR (KBr), ν (cm−1): 3112, 1679, 1585, 1265, 891; 1H
NMR (600 MHz, DMSO-d6) δ = 12.35 (s, 1H, -CONH-),
8.98 (d, J = 6.6 Hz, 2H, -ArH), 8.04 (d, J = 6.4 Hz, 2H,
-ArH), 7.83 (d, J = 8.7 Hz, 1H, =CHS-), 7.55 (d, J =
7.6 Hz, 5H, -ArH), 7.43 (ddd, J = 13.3, 10.9, 5.5 Hz, 2H,
-ArH), 6.99 (d, J = 8.7 Hz, 2H, -ArH), 3.79 (s, 3H, -OCH3),
3.33 (d, J = 7.5 Hz, 4H, -CH2-), 2.69 (t, J = 7.1 Hz, 2H,
-CH2-), 2.03 (p, J = 7.2 Hz, 2H, -CH2-). 13C NMR
(151 MHz, DMSO-d6) δ = 170.53, 162.55, 158.94, 157.61,
148.63, 142.01, 134.69, 129.12, 128.42, 127.13, 126.86,
125.40, 123.17, 114.06, 105.88, 61.52, 55.13, 33.46, 29.87,
23.01; ESI-MS calcd for C26H26ClN3O2S2 476.146 [M-Cl]+,
found 476.1468 [M-Cl]+.
The inhibitory activity of 4-methoxy-phenylthiazole-2-
amine derivatives 5a–6e against electroporated AChE was
determined using Ellman’s test (Abbas-Mohammadi et al.
2018) with huperzine-A and rivastigmine as reference
compounds. The compounds (5a–6e) were dissolved in
dimethyl sulfoxide. ACTI (acetylthiocholine iodide) was
used as a substrate, and 5,5′-dithiobis [2-nitrobenzoic acid]
(DTNB) was used for the measurement of AChE activity.
Electroporated AChE was used as a source of AChE
enzyme. Tris buffer’s pH was 7.2, and the concentrations of
substrate ACTI, chromogenic reagent DTNB, and AChE
were 0.6 mmol/L, 3 mmol/L, and 0.35 U/mL, respectively.
The concentrations of compounds 5a–6e were configured as
1 × 10−4, 1 × 10−5, 1 × 10−6, 1 × 10−7, and 1 × 10−8 μmol/L,
respectively. Tris buffer B, AChE, and each concentration
of compounds were taken as 20 μL, 10 μL, and 10 μL,
respectively, and were then added to a 96-well plate reader.
The mixtures were incubated for 12 min at 37 °C. The
hydrolysis of acetylthiocholine was monitored by the for-
mation of a yellow-colored enzymatic product, 5-thio-2-
nitrobenzoate anion, which was determined by the variation
in absorbance measured at 405 nm, with a 96-well plate
reader. The blank group without the substrate and com-
pounds 5a–6e was added to 10 μL Tris buffer B two times,
respectively. The standard group without compounds 5a–6e
was added to 10 μL Tris buffer B. Each compound was
tested two times at different concentrations; then, with the
1-Benzyl-4-((5-((4-(4-methoxyphenyl)thiazol-2-yl)amino)-5-
oxopentyl)thio)pyridin-1-ium chloride (6d)
It was obtained as a white solid; yield: 28%; mp 296–298 °C;
IR (KBr), ν (cm−1): 3109, 1679, 1583, 1265, 886; 1H
NMR (600 MHz, DMSO-d6) δ = 12.26 (s, 1H, -CONH-),
8.95 (dd, J = 17.7, 6.8 Hz, 2H, -ArH), 8.01 (t, J = 11.0 Hz,
2H, -ArH), 7.82 (d, J = 8.3 Hz, 1H, =CHS-), 7.51 (dd, J =
21.0, 7.5 Hz, 5H, -ArH), 7.48–7.36 (m, 2H, -ArH), 6.99 (d,
J = 8.4 Hz, 2H, -ArH), 3.78 (s, 3H, -OCH3), 3.32 (t, J =
7.0 Hz, 4H,-CH2-), 2.54 (d, J = 6.8 Hz, 2H, -CH2-), 1.82-
1.69 (m, 4H, -CH2-). 13C NMR (151 MHz, DMSO-d6) δ =
171.11, 162.78, 158.93, 157.64, 148.22, 142.43, 134.69,
129.13, 128.53, 127.13, 126.88, 125.42, 123.13, 114.05,
105.83, 61.55, 55.13, 34.14, 30.15, 26.90, 23.66; ESI-MS
calcd for C27H28ClN3O2S2 490.1617 [M-Cl]+, found
490.1627 [M-Cl]+.

Medicinal Chemistry Research
inhibition rate I [I = 100 × (Astandard − Aexperiment)/(Astandard
−
inhibitory activity with IC50 = 5.84 μmol/L, and the activity
was higher than that of the reference compound, rivas-
tigmine, but lower than that of huperzine-A. From Tables 1
and 2 it can be seen that the compound having a better
inhibitory effect on acetylcholinesterase had almost no
inhibitory effect on BuChE.
A careful insight and analysis of the AChE inhibition
activity results presented in Tables 1 and 2 led to two
eventual SAR. By comparing the structure and bioactivity
of the compounds, it was found that: (1) the difference of RX
(X = 1, 2) and R3 had an important effect on the activity of
compounds. When R2 was 4-amino-1-benzylpiperidinyl, the
activity is better than other groups and that (2) the differ-
ence of -(CH2)n- (n = 1, 2, 3, 4, 5) had an important effect
on the activity of compounds. When carbon chain length
was -(CH2)2-, the activity is better than others. It was shown
that the group of (-NH-CO-CH2-CH2-NH-) was necessary
for the active compound.
Ablank)%] was plotted against inhibitor concentration C to
perform an S-curve linear fit to obtain the IC50 corre-
sponding to the concentration Cx.
In order to get an accurate IC50, select the nearest 1 × 10−x
μmol/L from 1 × 10−5 to 1 × 10−9 μmol/L, set 1 × 10−x
μmol/L as less than Cx. The compound for which the
activity was best was configured at concentrations 1 × 10−x,
3 × 10−x, 5 × 10−x, 7 × 10−x, and 9 × 10−x μmol/L; the
enzyme inhibition experiment chosen was the same method.
In order to find the accurate IC50, the inhibition rate I was
plotted against the inhibitor concentration C to a perform
linear fit (Table 1) and the results were expressed as mean
standard error mean (SEM).
In vitro BuChE inhibition test
Five milliliters of venous blood was collected from a rat and
kept at room temperature for 1 h and then at 4 °C for 2 h.
The supernatant obtained after centrifugation (2000 r/min
for 10 min) was used as the butyrylcholinesterase (BuChE)
enzyme. The method used used was the same as that used
for determining the activity of AChE enzyme, except that
the substrate acetylthiocholine iodide was replaced by
butyrylthiocholine iodide in the AChE activity measure-
ment (Table 2).
Kinetic analysis of lead AChEI 5g
To determine the inhibition type of these compounds
against electroporated AChE, a kinetic study was carried
out with inhibitor 5g as the representative AChEI. In this
process, the concentrations of inhibitors 0.5 × IC50, IC50,
and 2 × IC50 were 2.92, 5.84, and 11.68 μmol/L, respec-
tively. This type of inhibition was established from the
analysis of Lineweaver–Burk reciprocal plots (Fig. 3).
It can be found from V (reaction rate)–[S] (substrate con-
centration) (Fig. 3a) that at the same inhibitor concentration,
V increased as the [S] increased; at the same [S], V decreased
as the concentration of the inhibitor increased, which indi-
cated that there was a reversible mode of enzyme inhibition.
According to the characteristics of enzymatic kinetics, the
double-reciprocal curve of the competitive inhibition type is
on the Y-axis; the double-reciprocal curve of the non-
competitive inhibition type is on the negative semi-axis of
the X-axis; the double-reciprocal curve of the mixed inhibition
type is assigned to the second quadrant. The double-reciprocal
V−1–[S]−1 curve of compound 5g intersected in the second
quadrant, which indicated that there was a mixed inhibition
type (Cavdar et al. 2019) (Fig. 3b).
From Table 1, it can be seen that 10 of 13 target com-
pounds had certain acetylcholinesterase inhibitory activity
in vitro. Among them, compound 5g showed better AChE
Table 1 Inhibition of AChE by synthesized compounds 5a–6e
Compound IC50 (μmol/L) SD Compound
IC50 (μmol/L) SDa
5a
5b
5c
5d
5e
5f
49.02
48.28
NA
1.40 6a
3.21 6b
NA 6c
NA 6d
NA 6e
5.94
42.01
32.50
31.23
31.10
0.36
0.91
2.31
4.21
3.26
0.53
0.28
NA
NA
6.01
5.84
25.01
0.51 Rivastigmine 11.80
0.81 Huperzine-A 4.91
2.21
5g
5j
AChE acetylcholinesterase, IC50 half-maximal inhibitory concentra-
tion, SD standard deviation, NA not active
In silico molecular property analysis and ADMET prediction
Unfavorable ADME and toxicity properties have been
identified as the major cause of failure for new drug
molecules moving towards the higher phases of drug
development (Zhong et al. 2018). Thus, with the objective
of increasing the success rate of compounds reaching
development, in silico molecular property analysis and
ADMET prediction studies were performed using Molin-
spiration software 2015 (www.molinspiration.com) online
Table 2 Inhibition of BuChE by synthesized compounds 5f–6a
Compound IC50 (μmol/L) SD Compound
IC50 (μmol/L) SD
5f
1080
1340
1185
3.54 Rivastigmine 1.90 0.21
5g
6a
4.50 Huperzine-A >10,000.00
2.23
BuChE butyrylcholinesterase, IC50 half-maximal inhibitory concentration

Medicinal Chemistry Research
Fig. 3 Compound 5g V–[S] and
double-reciprocal V−1–[S]−1
Table 3 In silico prediction of molecular properties for title
compounds 5a–6e
property calculation toolkit and PreADMET online server
(www.preadmet.bmdrc.org), respectively.
Code MW
Rule <500
MiLogP HBA HBD nviolation TPSA
Volume
All the structures reported herein show suitable MW
values (MW < 500) necessary for a successful penetration
to central nervous system, with the exception of compounds
6c, 6d, and 6e. All compounds exhibit moderate lipophili-
city (expressed as MiLogP (octanol–water partition coeffi-
cient)), except 5j, 6c, 6d, and 6e. The number of hydrogen
bond acceptors and hydrogen bond donors for all the
compounds were in accordance with the Lipinski’s rule of
five (Lipinski et al. 2012). Moreover, the topological polar
surface area for all the compounds was observed to fall
within the range. In addition, the predicted values for all
compounds fall into the appropriate range, indicating good
bioavailability of the ligand molecules, except 5j, 6c, 6d,
and 6e (Table 3). Thus, it was predicted that most of the
compounds are likely to be orally active.
In addition, the results of in silico ADMET properties
were listed in Tables 4 and 5 (Lipinski et al. 2012).
Human intestinal absorption (HIA) property is the
determinant for those drugs that purport oral administration.
All the compounds expressed >70% HIA values, indicating
good permeation across the membrane, which exhibited
moderate permeation.
≤5
≤10
≤5
≤1
<160 Å2
5a
5b
5c
5d
5e
5f
357.46 2.67
371.49 2.94
385.51 3.21
399.54 3.72
413.57 4.22
436.58 3.87
450.61 3.77
492.69 5.05
484.05 4.72
498.07 4.99
512.10 5.26
526.13 5.77
540.15 6.27
5
5
5
5
5
6
6
6
5
5
5
5
5
1
1
1
1
1
2
2
2
1
1
1
1
1
0
0
0
0
0
0
0
1
0
0
2
2
2
64.12
64.12
64.12
64.12
64.12
66.49
66.49
66.49
51.22
51.22
51.22
51.22
51.22
301.12
317.92
334.73
351.53
368.33
402.33
419.13
469.54
409.53
426.34
443.14
459.94
476.74
5g
5j
6a
6b
6c
6d
6e
MW molecular weight, MiLogP octanol–water partition coefficient,
HBA number of hydrogen bond acceptor, HBD number of hydrogen
bond donor, nviolations violations from Lipinski’s rule, TPSA topological
polar surface area
The Caco-2 cell permeability in vitro is an important
parameter to assess intestinal absorption of the drug. The
results indicated that all compounds exhibited moderate
permeation (Table 4).
The MDCK cell permeability test in vitro utilizes canine
kidney cells for the analysis of permeability. All the com-
pounds 5a–6e showed permeation <25 nm/s, indicating low
permeability.
The skin permeability is an important factor for the
delivery of drug via transdermal administration. All the
compounds 5a–6e exhibited negative permeability values,
which indicated that transdermal mode of administration
was not the suitable means to administer these drugs.
In addition, apart from the compounds 5a, 5e, 5f, 5g, 5j,
and 6a, all the compounds were predicted to possess >90%
plasma protein binding (PPB), indicating decreased excre-
tion and increased half-life.
The Ames test assesses mutagenicity of the compounds
and all the compounds were predicted as mutagen. More-
over, carcinogenicity test was performed to identify the
tumerogenic possibility of compounds in animals (mouse
and rat). Analyzing carcinogenicity in animals (mouse and
rat), all the compounds were predicted as negative. Human
ether-a-go-go-related gene (hERG) encodes potassium
channels, which are responsible for normal repolarization of
cardiac action potentials (Macdonald et al. 2018). Blockage
or other impairment of these channels in the heart can lead
to fatal cardiac problems, so it is a major concern with
regard to drug-induced blockage of potassium channels.
Compounds 6b, 6c, and 6d presented low risk; 5a, 5b, 5c,

Medicinal Chemistry Research
Table 4 In silico ADME prediction for the title compounds 5a–6e
Code Absorption
Distribution
Human intestinal
absorption (%)
In vitro Caco-2 cell
permeability (nm/s)
In vitro MDCK cell
permeability (nm/s)
In vitro skin permeability
(log Kp, cm/h)
In vitro PPB (%)
Rule 0–20 (poor)
<4 (low)
4–70 (moderate)
>70 (high)
<25 (low)
25–500 (moderate)
>500 (high)
>90 (strongly bound)
<90 (weakly bound)
20–70 (moderate)
70–100 (well)
5a
5b
5c
5d
5e
5f
98.10
98.16
97.25
98.12
97.62
95.32
95.39
95.69
96.89
96.91
96.94
96.98
97.03
27.02
32.29
32.41
57.40
44.60
48.26
49.96
51.99
34.51
37.87
36.31
57.76
39.35
4.41
1.24
9.28
1.11
0.49
5.29
0.23
0.33
0.85
0.16
1.65
0.37
0.40
−3.50
−3.30
−2.83
−2.98
−3.36
−3.52
−3.40
−2.90
−2.86
−2.66
−2.52
−2.37
−2.24
88.16
91.66
91.76
90.12
89.94
26.70
44.04
54.57
89.60
96.50
100.00
93.04
93.61
5g
5j
6a
6b
6c
6d
6e
ADME absorption, distribution, metabolism, excretion
Table 5 In silico toxicity prediction for the title compounds 5a–6e
Code
Ames test
Carcinogenicity (mouse)
Carcinogenicity (rat)
hERG inhibition
5a
5b
5c
5d
5e
5f
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Mutagen
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Medium risk
Medium risk
Medium risk
Medium risk
Medium risk
High risk
5g
5j
High risk
Medium risk
Medium risk
Low risk
6a
6b
6c
6d
6e
Low risk
Low risk
Medium risk
hERG human ether-a-go-go-related gene
5d, 5e, 5j, 6a, and 6e presented medium risk; compounds 5f
and 5g presented high risk (Table 5).
In summary, although other compounds showed good
characteristics, it can be concluded that the lead AChEI
compound 5g presented satisfactory drug-like character-
istics and ADME properties.
the molecular docking was conducted to investigate the
potential binding mode of compound 5g with the catalytic
domain of AChE (PDB: 4EY7) using Autodock 4.2. The
complex of protein with best docked pose of inhibitor
showed that the docked ligand was almost superimposed on
the native co-crystallized ligand donepezil (Cheung et al.
2012) (Fig. 4). In this position, the 4-methoxythiazole ring
moiety oriented toward the bottom of the active site similar
to the benzylpiperidine moiety of ligand donepezil. As
represented in Fig. 4, a closer look at the binding mode
showed that the 4-methoxyphenylthiazole ring adjusted in
Docking studies
In order to achieve the binding mode of the synthesized
compounds and provide more insights into the interactions,

Medicinal Chemistry Research
selectivity, and structural modifications can be further made
on the basis of compound 5g to seek compounds having
higher inhibitory activity against acetylcholinesterase. These
results encourage medicinal chemists to explore more
structures based on 4-methoxy-phenylthiazole-2-amine
derivatives for high-efficiency cholinesterase inhibitor.
Acknowledgements This work was supported by grants from Natural
Science Foundation of Hebei Province (No. H2017201075).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Fig. 4 Three-dimensional representation of best pose of compound 5g
obtained from docking simulation in the active site of AChE
Publisher’s note: Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional affiliations.
parallel with indole ring of Trp-286 and made π–π inter-
action. The amide group also fitted in the midgorge of
AChE active site and bound to the active site via hydrogen
bonding interaction with the amino group of Phe-295 resi-
due. Moreover, the benzylpiperidine amino ion and benzyl
moiety of the compound 5g also established amino ion and
π of benzene ring interaction with Tyr-337 and another π–π
stacking interaction with Trp-86 in the peripheral anionic
site, respectively.
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Conclusion
The designed 4-methoxy-phenylthiazole-2-amine deriva-
tives as AChEIs were successfully synthesized, and their
inhibitory activity test results showed that most of 4-meth-
oxy-phenylthiazole-2-amine derivatives had a certain acet-
ylcholinesterase inhibitory activity in vitro; moreover, their
IC50 values were in micromolar range. Specially, 3-((1-
benzylpiperidin-4-yl)amino)-N-(4-(4-ethoxyphenyl)-thiazol-
2-yl)propanamide (5g) showed a strong inhibitory activity
against AChE (IC50 = 5.84 0.01 μmol/L), which was found
to be ~0.83-fold active compared to huperzine-A and ~2.02-
fold stronger compared to rivastigmine. Kinetic studies
revealed that compound 5g exhibited a mixed type and
reversible mode of enzyme inhibition. Molecular docking
stimulation of the representative compound 5g performed
using Autodock 4.2 achieved the binding mode of the syn-
thesized compounds with AChE and provided more insights
into the interactions. Furthermore, the representative com-
pound 5g basically conformed to four-point pharmacophore
model, which is essential for the AChE inhibition. Finally, it
can be concluded that the lead AChEI compound 5g pre-
sented satisfactory drug-like characteristics and ADME
properties by in silico molecular property analysis and
ADMET prediction. In summary, the compound 5g having
excellent acetylcholinesterase inhibitory activity in vitro was
found in this study, while the compound had good
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