Synthesis, kinetics and biological assay of some novel aryl bis-thioureas: A potential drug candidates for Alzheimer's disease

Article abstract of DOI:10.1016/j.molstruc.2021.131136

A new series of bis-thioureas (4a-4j) was synthesized and characterized through spectroscopic and elemental analysis. The synthesized compounds 4a-4j were subjected to acetylcholinesterase enzyme (AChE) inhibition activity and free radical scavenging activity. The results of AChE inhibition assay were found to be active in inhibiting the target enzyme with different IC₅₀ values. Among all derivatives, the 4 g showed highly potent inhibition potential against AChE enzyme with IC₅₀ value of 0.1761±0.00768 μM, which is several times better than the reference inhibitor neostigmine methylsulfate IC₅₀ 2.469±0.069 μM. The initial structure-activity relationship (SAR) of 4 g revealed dual hydrogen bonding ability (donor and acceptor). Moreover, the electronic environment around the aromatic ring also greatly influenced the enzyme inhibition of AChE. To further explore the newly synthesized AChE inhibitors, kinetic studies were carried out to determine the mode of inhibition and it was found to be competitive inhibition. Pharmacokinetic predictions (ADMET parameters) were also evaluated and compounds showed good lead-like potential with little hepatotoxic and no skin-sensitive effects. The molecular docking studies delineated the binding affinity of the ligands with target protein and showed docking scores in the range of -10.3 to -7.6 kcal/mol.

Full text of DOI:10.1016/j.molstruc.2021.131136

Journal of Molecular Structure 1246 (2021) 131136
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Synthesis, kinetics and biological assay of some novel aryl
bis-thioureas: A potential drug candidates for Alzheimer’s disease
Rabail Ujana, Pervaiz Ali Channarb, Ali Bahadurb,c,∗, Qamar Abbasd, Mazloom Shahe,
S.G. Rashidf, Shahid Iqbalg,∗, Aamer Saeedb,∗, Hisham S.M. Abd-Rabbohh,i, Hussain Razaj,
Mubashir Hassank, Ali Nawaz Siyala, Parvez Ali Mahesarl, Bhajan Lall,
Kashif Ali Channarm, Bilal Ahmad Khann, Muhammad Nawazo,
Muhammad Shahid Riaz Rajokap, Jung Min Kimc
a Dr. M. A. Kazi Institute of Chemistry, University of Sindh, Jamshoro, Pakistan
b Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
c Department of Transdisciplinary Studies, Graduate School of Convergence Science and Technology, Seoul National University, Seoul, 08826, South Korea
d Department of Biology, College of Science, University of Bahrain, Sakhir, Kingdom of Bahrain
e Department of Chemistry, Abbottabad University of Science and Technology (AUST) Abbottabad, Pakistan
f Department of Chemistry, School of Natural Sciences (SNS), National University of Science and Technology (NUST), H-12, Islamabad, 46000, Pakistan
g School of Chemistry and Materials Engineering, Huizhou University, Huizhou 516007, Guangdong, China
h Chemistry Department, Faculty of Science, King Khalid University, P.O. Box 9004, Abha 61413, Saudi Arabia
i Department of Chemistry, Faculty of Science, Ain Shams University, Abbassia, Cairo 11566, Egypt
j Department of Biological Sciences, College of Natural Sciences, Kongju National University, 56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of
Korea
k Institute of Molecular Biology and Biotechnology/(IMBB), The University of Lahore, 1-KM, Defence Road, Bhubtian Chowk, Lahore, Pakistan
l Institute of Chemistry, Shah Abdul Latif University, Khairpur, 66020, Pakistan
m Department of Oral and Maxillofacial Surgery Institute of Dentistry Liaquat University, Jamshoro, Pakistan
n Department of Chemistry, University of Azad Jammu and Kashmir, Muzaffarabad, 13100, Pakistan
o Department of Chemistry, Balochistan University of Information Technology, Engineering and Management Sciences, Quetta, Pakistan
p Food and Feed Immunology Group, Laboratory of Animal Food Function, Graduate School of Agricultural Science, Tohoku University, Sendai 980-8572,
Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of bis-thioureas (4a-4j) was synthesized and characterized through spectroscopic and ele-
mental analysis. The synthesized compounds 4a-4j were subjected to acetylcholinesterase enzyme (AChE)
inhibition activity and free radical scavenging activity. The results of AChE inhibition assay were found to
be active in inhibiting the target enzyme with different IC50 values. Among all derivatives, the 4 g showed
highly potent inhibition potential against AChE enzyme with IC50 value of 0.1761 0.00768 μM, which is
several times better than the reference inhibitor neostigmine methylsulfate IC50 2.469 0.069 μM. The
initial structure-activity relationship (SAR) of 4 g revealed dual hydrogen bonding ability (donor and ac-
ceptor). Moreover, the electronic environment around the aromatic ring also greatly influenced the en-
zyme inhibition of AChE. To further explore the newly synthesized AChE inhibitors, kinetic studies were
carried out to determine the mode of inhibition and it was found to be competitive inhibition. Pharma-
cokinetic predictions (ADMET parameters) were also evaluated and compounds showed good lead-like
potential with little hepatotoxic and no skin-sensitive effects. The molecular docking studies delineated
the binding affinity of the ligands with target protein and showed docking scores in the range of -10.3 to
-7.6 kcal/mol.
Received 5 May 2021
Revised 23 June 2021
Accepted 16 July 2021
Available online 23 July 2021
Keywords:
Bis-thiourea
AChE inhibitors
Antioxidant
Molecular docking
Pharmacokinetics ADMET parameters
Kinetic Mechanism
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
Alzheimer’s disease (AD) is a disease that initially damages the
memory, but ultimately causes loss of consciousness [1-10]. AD
∗
Corresponding authors.
E-mail addresses: alibahadur138@gmail.com (A. Bahadur), shahidiqbal@
is a neurological disease and is characterized in part by the ac-
hzu.edu.cn (S. Iqbal), aamersaeed@yahoo.com (A. Saeed).
https://doi.org/10.1016/j.molstruc.2021.131136
0022-2860/© 2021 Elsevier B.V. All rights reserved.

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
Fig. 1. Some selected potent AChE inhibitors.
cumulation of amyloid protein, which causes cognitive decline in
the brain [3]. This vastly common neurodegenerative disease af-
fects more than 35 million worldwide, and this number is expected
to increase drastically as the population ages and the human
body loses the internal immune system to battle life-threatening
diseases [11-14]. Acetylcholine a key neurotransmitter produced
by presynaptic chlonergic terminals is metabolically degraded by
acetylcholinesterase. Therefore, for the prevention of AD, acetyl-
cholinesterase inhibitors (AChEIs) are usually administered, which
enhance the level of acetylcholine in the synaptic cleft.
contain groups that are capable of effectively binding in the ac-
tive site of the target protein. In continuation of ongoing research
on the development and design of new and effective inhibitors for
AChE, we describe herein, our efforts to explore the bis-thiourea
compounds as a potent inhibitor of AChE and their structure-
activity relationship, kinetic studies, pharmacokinetics, and molec-
ular docking studies.
2. Experimental
Current therapeutic approaches are directed at (i) halting the
oxidative damage of nerve cells; (ii) ceasing or reducing the pro-
duction of the amyloid protein; (iii) compensating cholinergic func-
tion loss; (iv) increasing or restoring the level of acetylcholine [15,
16]. To cope with the slowly progressing damages of AD, several
AChE inhibitors have been approved including donepezil (Aricept®,
Pfizer, Inc., and Eisai Corp.), rivastigmine (Exelon®, Novartis), and
galanthamine (Reminyl® renamed Razadyne®, Janssen, and John-
son and Johnson). There are few adverse effects connected with
marketed drugs for AD, such as nausea and diarrhea [17].
2.1. Materials and methods
Melting points were recorded using
a digital Gallenkamp
(SANYO) model MPD BM 3.5 apparatus and are uncorrected. 1H
NMR and 13C NMR spectra were determined in DMSO–d6 solutions
at 300 MHz and 75.4 MHz respectively using Bruker AM-300 spec-
trophotometer. FTIR spectra were recorded using an FTS 3000 MX
spectrophotometer and elemental analyses with a LECO-183 CHNS
analyzer.
In addition to catalytic action, AChE improves non-cholinergic
functions associated with the peripheral anionic site (PAS) and me-
diates the accumulation and progression of amyloid protein [18,
19]. The effective treatment and to inhibit the onset progress of AD
is still a challenging task for medicinal chemists. However, the lit-
erature review suggested that there are several small organic nat-
ural and synthetic molecules have been reported to show high in-
hibition potential against acetylcholinesterase (Fig. 1) [20-26].
Thiourea derivatives are blessed with a spacious spectrum of
biological activities including, antibacterial, antimicrobial, antiox-
idant, anticancer, anti-tuberculosis, anti-HIV, anti-parasitic, and
enzyme inhibition activities such as urease inhibition, acetyl-
cholinesterase butyrylcholinesterase inhibition, and tyrosinase in-
hibition [27-38]. Thioureas are also considered as a valuable or-
ganic synthon for the synthesis of various heterocyclic motifs [39].
Thioureas possess unique and simple structures and display in-
triguing chemical reactivity. Bisthioureas possess three key struc-
2.2. General procedure for the synthesis of derivatives (4a-4j)
The aromatic acid chlorides were synthesized by reacting suit-
able aromatic carboxylic acids (10 mmol) with thionyl chloride
(10 mmol) to afford 10 mmol of freshly prepared aromatic acid
chlorides. A freshly prepared solution of aromatic substituted acid
chlorides was synthesized from aromatic carboxylic acids by treat-
ing the reaction mixture with thionyl chloride. The acid chloride
synthesized (10 mmol) in anhydrous acetone (50 mL) was added
dropwise to a suspension of potassium thiocyanate (10 mmol) in
acetone (30 mL) at room temperature. The reaction mixture was
refluxed at 140 °C for 3–4 h and cooled to room temperature. A
solution of the substituted anilines (10 mmol) in anhydrous ace-
tone (20 mL) was added dropwise and the reaction mixture was
refluxed further for 2–3 h. On completion of the reaction, the mix-
ture was poured into cold water and the precipitated thioureas
were filtered and recrystallized from methanol (Fig. 2).
–
tural units such as hydrogen bonding area (N H moiety), com-
plementary (C = S), and auxiliary 1,3-disubstituted pattern. Bis-
thioureas have more lipophilic character than monothiourea and
urea congener which paves the way for penetration into cells.
Aryl rings provide an opportunity to incorporate various groups on
phenyl rings which greatly influence biological activity. These ex-
citing structural features play a significant role in the enhancement
of biological activity and designing lead molecules for drug devel-
opment. We envisioned exploring the behave of the bis motif of
thioureas as acetylcholinesterase inhibitors because bis-thioureas
2.3. Acetylcholinesterase inhibition assay
The inhibitory activities of synthesized compounds were de-
termined spectrophotometrically using acetylthiocholine iodide as
substrate by following the method of Ellman et al. [40]. Briefly, the
assay solution consisted of 180 μL of 50 mM Tris–HCl buffer, pH
7.5, containing 0.1 M sodium chloride and 0.02 M MgCl2, and 20
μL of enzyme (AChE, EC 3.1.1.7, acetylcholinesterase from human
erythrocytes) solution (50 U per well); increasing concentrations of
2

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
Fig. 2. Synthesis of new bis-thioureas having variously substituted aryl rings (4a-4j).
test compounds (10 μL) were added to the assay solution and pre-
2.5. Free radical scavenging assay
ꢀ
incubated for 30 min at 4 °C. After that 5,5 Dithiobis (2nitroben-
zoic acid) (0.3 mM, 20 μL) and acetylthiocholine iodide (1.8 mM,
20 μL) was added to the reaction mixture and incubated at 37 °C
for 10 min, followed by the measurement of absorbance at 412 nm.
For nonenzymatic reaction, the assays were carried out with a
blank containing all components except acetylcholinesterase. The
assay measurements were measured at 412 nm using a microplate
reader (OPTI Max, Tunable). Wavelength range 340–850 nm; for
96 well plates). The reaction rates were compared and the percent
inhibition due to the presence of tested inhibitors was calculated.
Neostigmine methylsulfate was used as a reference inhibitor. Each
concentration was analyzed in three independent experiments run
in triplicate. IC50 values were calculated by nonlinear regression
using GraphPad Prism 5.0 (GraphPad Software, Sand Diego, CA).
The% of Inhibition of acetylcholinesterase was calculated as fol-
lows Inhibition (%) = [(B-S)/B] × 100. Here, the B and S are the
absorbances for the blank and samples.
Radical scavenging activity was determined by modifying the
already reported method by 2, 2-diphenyl-1 picrylhydrazyl
a
(DPPH) assay [41]. The assay solution consisted of 100 μL of DPPH
(150 μM), 20 μL of increasing concentration of test compounds and
the volume was adjusted to 200 μL in each well with DMSO. The
reaction mixture was then incubated for 30 min at room temper-
ature. Ascorbic acid (Vitamin C) was used as a reference inhibitor.
The assay measurements were carried out by using a microplate
reader (OPTI Max, Tunable) at 517 nm. The reaction rates were
compared, and the percent inhibition caused by the presence of
tested inhibitors was calculated. Each concentration was analyzed
in three independent experiments run in triplicate.
2.6. Computational methodology
2.6.1. Retrieval of human acetylcholinesterase structure from PDB
The crystal structure of human acetylcholinesterase (PDBID:
4PQE) was retrieved from the Protein Data Bank (PDB) (http://
www.rcsb.org). The target protein was energy minimized by em-
ploying the conjugate gradient algorithm and amber force field us-
ing UCSF Chimera 1.10.1 [42]. The protein Ramachandran graph was
accessed through PDB. The basic structural protein architecture of
helices, beta-sheets, coils, and turns was accessed by VADAR 1.8
[43]. The Discovery Studio 2.1 Client was used to view the 3D
structure of the target protein [44].
2.4. Kinetic study
Kinetic analysis was carried out to determine the mode of inhi-
bition. Compound 4 g was selected based on the most potent IC50
values. Kinetics were carried out by varying the concentration of
acetylthiocholine iodide in the presence of different concentrations
of compound 4 g (0.00, 0.05, 0.1, and 0.2 μM). Briefly, the acetylth-
iocholine iodide concentration was changed from 4, 2, 1, 0.5, 0.25,
and 0.125 mM for acetylthiocholine iodide kinetics studies and the
remaining procedure was the same for all4 kinetic studies as de-
scribed in acetylcholine esterase inhibition assay protocol. Maximal
initial velocities were determined from the initial linear portion of
absorbances up to 5 min after the addition of enzyme at 30-second
intervals. The inhibition type on the enzyme was determined by
the Lineweaver-Burk plot of inverse velocities (1/V) versus the in-
verse of substrate concentration 1/ [S] mM−1. The EI dissociation
constant Ki was determined by the secondary plot of 1/V versus in-
hibitor concentration. The results (change in absorbance per min)
were processed by using SoftMaxPro (Molecular Devices, Sunny-
vale, CA).
2.7. Candidate structures
The
synthesized
ligands
(4a-4j)
were
sketched
in
ACD/ChemSketch. The basic biochemical properties and Lip-
inski’s rule of five (RO5) of synthesized compounds (4a-
4j) were justified through online computational tools such
as Molsoft (http://www.molsoft.com/) and Molinspiration
(http://www.molinspiration.com/),
respectively.
Furthermore,
the pharmacokinetic properties such as Absorption, Distribution,
Metabolism, Excretion, and Toxicity (ADMET) of synthesized
ligands were evaluated through the pkCSM online server [45].
3

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
Table 1
position of phenyl rings. In contrast to 4b, which contains two
electron-donating groups (methoxy group) showed poor inhibition
against AChE. The disubstituted phenyl rings in the case of com-
pounds 4c and 4d showed good inhibition. The compound 4f also
showed more inhibition potential compared to the standard, this
can be attributed to the F atoms attached at the para position of
two phenyl rings. Compound 4c contains two chlorine atoms at or-
tho and para position of phenyl ring while compound 4d contains
two electron-withdrawing groups (nitro group) at ortho and para
position concerning phenyl ring and showed significant inhibition
against AChE. The compound 4 h was found to be the least potent
derivative in the series 4a-4j, and it showed less minimum inhibi-
tion against the AChE enzyme.
Acetylcholinesterase (human erythrocytes inhibitory activity and free radical% scav-
enging of derivatives (4a-4j).
Compound
Acetylcholine esterase
IC50 SEM (μM)
Free radical% scavenging
(100 μg/mL)
4.757 0.352
94.478 7.405
10.997 0.745
96.896 8.569
14.0722 0.854
17.293 0.985
18.338 1.143
16.359 1.209
21.785 1.577
26.899 6.501
————
4a
0.413 0.0162
23.373 0.981
1.6775 0.0683
1.9230 0.0849
17.649 0.687
0.4693 0.0182
0.1761 0.00768
25.2063 1.586
2.3967 0.8547
4.0471 0.581
2.469 0.069
4b
4c
4d
4e
4f
4 g
4h
4i
4j
Neostigmine methylsulfate
Vitamin C
————
95.899 7.519
3.2. Kinetic mechanism
Values are expressed as mean
SEM; SEM= Standard Error of Mean; — = not
To understand the inhibitory mechanism of synthetic com-
pounds on acetylcholine esterase, an inhibition kinetic study was
performed. Based upon IC50 results, the most potent compound
4 g was used to determine the inhibition type and inhibition con-
stant. The kinetic results of the enzyme by the Lineweaver-Burk
plot of 1/V versus acetylthiocholine iodide in the presence of dif-
ferent inhibitor concentrations gave a series of straight lines, the
result of the Lineweaver-Burk plot of compound 4 g showed that
Vmax remains the same while Km increases with increasing com-
pound concentrations. This behavior indicates that the 4 g com-
pound inhibits the AChE in a competitive manner (Fig. 3a). The
plot of the slope against the concentration of 4 g determined the
reversible dissociation constant (Ki) of 4 g be 0.0086 μM (Fig. 3b).
determined; for radical scavenging, all compound concentrations were 100 μg/mL.
2.8. Molecular docking
A docking experiment was performed on all synthesized com-
pounds (4a-4j) against targeted protein through the PyRx docking
tool [46]. All synthetic compounds were docked separately against
the crystal structure of human acetylcholinesterase. In the dock-
ing experiment, the grid box dimension values were adjusted as
X=−25.27, Y = 22.43, and Z = 0.665, respectively, with by default
exhaustiveness = 8 value. The docked complexes were evaluated
based on the lowest binding energy (kcal/mol) values and hydro-
gen/ hydrophobic interactions. The bonding interaction pattern be-
tween ligands and receptor were keenly observed by using Dis-
covery Studio (2.1) and UCSF Chimera 1.10.1, respectively. The 2D
graphical depiction of all other docked complexes was evaluated
by LIGPLOT [47].
3.3. Free radical scavenging
All the synthesized bis-thioureas 4a-4j series compounds were
evaluated for DPPH free radical scavenging ability. The compounds
4b and 4d showed good% scavenging potency, other compounds
did not show significant radical scavenging potential even at high
concentrations (100 μg/mL) Table 1. Free radicals are highly dan-
gerous factors in producing cell death and other kinds of irrepara-
ble damage. Neurons are especially susceptible to attack by free
radicals because of some reasons these are, a. The neurons glu-
tathione content and if their natural antioxidant is in lesser quan-
tity b. their covering have a higher concentration of polyunsatu-
rated fatty acids c. oxygen is required to the brain for metabolism
and other very vital factor is age is the main component in
Alzheimer’s disease. The free radical hypothesis can be very impor-
tant for the widely different nature of Alzheimer’s disease. These
general concerns indicate that free radicals are participating in
Alzheimer’s disease, therefore, inhibition of these radical play vi-
tal role in formulating drugs against Alzheimer’s disease (Christen
2000)
3. Results and discussion
The synthesis of bis-thiourea derivatives (4a-4j) is outlined in
Fig. 2. Bis-thioureas were synthesized by using substituted aro-
matic carboxylic acid as starting materials. The variously substi-
tuted aromatic carboxylic acids were converted into correspond-
ing acid chlorides using thionyl chloride which was further reacted
with potassium thiocyanate to afford isothiocyanate 2 as a key in-
termediate which was not further isolated. The key intermediate
2 was reacted with aromatic aniline 3 to afford desired products.
The yield, melting Point, FTIR peak values, 1H NMR, and 13C NMR
values are provided in the supporting informations
The synthesized compounds (4a-4j) were characterized through
1H NMR and 13C NMR spectroscopy (Figure S11-S30). The tenta-
tive signal assignments of protons revealed the formation of the
desired products. The two separate signals that appeared at an 11–
–
12 ppm value were assigned to two distinct N H protons. The ap-
3.4. ADMET assessment of synthesized compounds
pearance of signals at around 7–8 ppm value was attributed to an
aromatic ring. In 13C NMR spectroscopy, the most deshielded sig-
nal appeared at 180 ppm value while the carbon of carbonyl group
appeared at around 175–170 ppm value. The sp2 carbons of the
aromatic ring appeared at around 140–120 ppm value.
The pharmacokinetics properties (ADMET) of newly synthesized
compounds were considered a major hallmark to predict the lead
structures. Table 2 showed the pharmacokinetic properties of syn-
thesized compounds (4a-4j). The absorption properties like water
absorption (log mol/L), intestinal solubility (% absorbed), and skin
permeability (log Kp) values justified the strong therapeutic poten-
tial of synthetic compounds. The literature report depicted those
ligands having good absorption value have more chance to cross
the gut barrier by passive penetration. The water solubility results
predict justified those compounds (4a-4j) showed good absorption
prediction. Furthermore, all compounds (4a-4j) exhibited good in-
testinal solubility results which are comparable to the standard
value (>30% abs). The skin permeability values of all compounds
3.1. Acetylcholinesterase assay
The synthesized compounds 4a-4j were screened against AChE.
All the prepared compounds are biologically active against the
studied enzyme with different IC50 values. The compound 4 g was
found to be the most potent derivative with an IC50 value several
folds better than the reference inhibitor neostigmine methylsul-
fate (Table 1). Compound 4 g bears two methyl groups at para-
4

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
Fig. 3. Line weaver-Burk plot of compound 4 g (a) at constant Vmax and (b) different concentrations.
Table 2
Pharmacokinetic assessment of synthesized compounds.
ADMET Properties
4a
4b
4c
4d
4e
4f
4 g
4h
4i
4j
Absorption
WS (logS)
IS(%abs)
−3.641
83.166
−2.735
−0.296
−1.519
−1.814
Yes
−4.13
84.777
−2.735
−0.519
−1.304
−2.505
No
−3.632
80.762
−2.735
−0.229
−1.886
−1.595
Yes
−3.017
74.449
−2.735
−1.516
−1.766
−3.012
No
−3.627
83.625
−2.735
−0.271
−1.554
−1.816
Yes
−4.07
85.79
−2.73
−0.67
−1.33
−2.22
No
−4.091
86.951
−2.735
−0.461
−0.947
−2
−3.62
83.49
−2.73
−0.24
−1.59
−1.77
Yes
−3.624
80.494
−2.735
−0.182
−1.968
−1.503
Yes
−3.531
80.458
−2.735
−1.131
−1.334
−2.543
No
SP(log Kp)
Distribution VDss (logL/kg)
BBBP (logBB)
CNSP (logPS)
Metabolism CYP1A2 (uM)
(Inhibitors) CYP2C19 (uM)
CYP2C9 (uM)
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
CYP3A4 (uM)
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Excretion
Toxicity
TC (log ml/min/kg)
−0.319
No
−0.53
No
−0.472
No
0.017
Yes
−0.851
No
−0.89
No
−0.709
No
−0.89
No
−1.007
No
−0.559
Yes
AMES
Max. Tolerat. Dose (log mg/kg/day)
0.616
2.117
Yes
0.302
2.485
Yes
0.674
2.095
Yes
0.392
2.453
Yes
0.652
2.113
Yes
0.367
2.491
Yes
0.331
2.575
Yes
0.651
2.113
Yes
0.671
2.095
Yes
0.241
2.392
Yes
ORAT (log mg/kg_bw/day)
HT
SS
No
No
No
No
No
No
No
No
No
No
∗Abbreviation: WS=Water solubility (log mol/L), IS=intestinal solubility, SP=skin permeability, Volume of Distribution at Steady State (VDss; log L/kg) BBBP=blood brain
barrier permeability (Log BB), CNSP=CNS permeability (LogPS), TC=Total clearance (log ml/min/kg), ORAT=Oral Rat Acute Toxicity, HT=Hepatotoxicity, SS=Skin Sensitization.
were also comparable with standard values (−2.5 log Kp) which
justified their drug-likeness behavior. Moreover, the Blood-Brain
Barrier (BBB) and Central Nervous System (CNS) permeability val-
ues of all synthesized compounds were also comparable with the
standard values (>0.3 to <−1 log BB and >−2 to <−3 logPS), re-
spectively. It has been observed that compounds with greater than
0.3 log BB value have the potential to cross BBB, while with less
than −1 value are poorly distributed to the brain.
3.6. Molecular docking analyses
The docked complexes of synthesized compounds (4a-4j) were
analyzed based on the lowest binding energy values (kcal/mol) and
hydrogen/hydrophobic bonding analyses. Results showed that 4f,
4a, and 4d were the most active compounds with good binding en-
ergy values (−10.30, −9.70, and −9.70 kcal/mol), respectively. The
docking energy values of all the docking complexes were calcu-
lated by using Eq. (1).
Similarly, the compounds that have > −2 log PS value are con-
sidered to penetrate the CNS, while with <−3 are difficult to move
in the CNS. Results showed that compounds did not display a sig-
nificant potential to cross the barriers and maybe target directly to
a receptor molecule. Moreover, their computational metabolic be-
havior was confirmed with different inhibitors, which are isoforms
of cytochrome P450. The excretion and toxicity predicted values
were also justified the drug-likeness behavior of these compounds
based on total clearance (log ml/min/kg), AMES toxicity, maximum
tolerated dose (MTD), and LD50 values. The non-mutagenic and
non-toxic behavior was also observed from AMES toxicity predic-
tion. The values indicate the toxic effect of compounds on life if
the value is higher it means the compounds are more toxic. In
our study, all compounds showed hepatotoxic behavior, while no
one with skin-sensitive behavior. The ADMET properties result jus-
tified that these novel synthesized compounds showed good lead-
like potential with little hepatotoxic and no skin-sensitive effects.
࢞G binding = ࢞Ggauss + ࢞Grepulsion + ࢞Ghbond + ࢞Ghy-
drophobic + ࢞Gtors
(1)
Here, ࢞G gauss: attractive term for dispersion of two gaus-
sian functions, ࢞Grepulsion: square of the distance if closer than
a threshold value, ࢞Ghbond: ramp function - also used for inter-
actions with metal ions, ࢞Ghydrophobic: ramp function, ࢞Gtors:
proportional to the number of rotatable bonds. The standard error
for Autodock is reported as 2.5 kcal/mol. However, Vina achieves
a comparatively low standard error of 2.85 kcal/mol [27]. How-
ever, all the synthesized compounds have no more than standard
docking energy value difference. The docking study explores the
actual binding pattern within the active region of the target pro-
tein. The best IC50 value compound (4 g) was further analyzed
through structure-activity relationship (SAR) analysis. The gener-
5

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
Fig. 4. The graphical depiction of the 4 g docking complex.
Table 3
tional and interactive behavior (Fig. 4(A)). A common pattern of
interaction revealed that all ligands showed good interactive be-
havior against binding pocket residues. The binding pocket and all
the other docked complexes for the other compounds can be found
in supplementary material (Figure S2-S10).
Docking energies of the synthesized derivatives 4a-4j.
Compounds
Docking energy
(kcal/mol)
Compounds
Docking energy
(kcal/mol)
4a
4b
4c
4d
4e
−9.7
−8.5
−9.1
−9.7
−8.9
4f
−10.3
−8.7
−7.9
−7.6
−8.8
4 g
4h
4i
4. Conclusion
4j
In summary, we have synthesized substituted aryl bis-thioureas
(4a-4j). The synthesized compounds show an alternative as effec-
tive inhibitors of AChE. The initial structural insights revealed that
bis-thioureas possess thiourea nucleus as core pharmacophore and
substituted phenyl rings also influence the results of inhibition. Ki-
netic studies were carried out through the Lineweaver-Burk plot
which showed competitive behavior of inhibition against AChE. The
pharmacokinetic studies guided those compounds possess good
lead-like properties with little toxicity. Molecular docking studies
delineated the binding affinity of the most potent derivative 4 g
with the AChE.
ated results also showed a good correlation with in vitro data,
4 g is the most active compound in inhibition analysis and pos-
sessed 0.1761 0.00768 (μM), whereas in silico results exhibited
−8.7 kcal/mol energy value. It has been reported that docking en-
ergy values possessed The standard error of VINA docking results
is 2.85 kcal/mol [27]. Therefore, among all the compounds (4a-4j)
the energy value differences were less than 2.85 kcal/mol, which
ensures the predicted competitive behavior of all ligands against
the target protein. Therefore, based on in silico and in vitro re-
sults, 4 g is a proposed good candidate molecule for the designing
of new drugs against AChE associated diseases Table 3.
Credit author statement
3.7. Structure-activity relationship (SAR) analyses between 4 g and
AChE
Rabail Ujan: Performed major experimental works, writing-
original draft preparation. Pervaiz Ali Channar: Writing-original
draft preparation and editing. Ali Bahadur: Conception, design
of study, conducted photoluminescence analysis, writing-original
draft preparation. Qamar Abbas: Material synthesis, visualization
of data. Mazloom Shah: Drafting the revised manuscript and crit-
ical revision. S.G. Rashid: Visualization of data, writing-reviewing
and editing. Shahid Iqbal: Conception, design of study, performed
some experimental works, writing-original draft preparation, su-
pervision. Aamer Saeed: Conception, design of study, Interpret
the data. supervision. Hisham S.M. Abd-Rabboh: Interpretation
of data, writing-reviewing and editing. Hussain Raza: Visualiza-
tion of data, writing-reviewing and editing. Mubashir Hassan:
Visualization of data, writing-reviewing and editing. Ali Nawaz
Siyal: Reviewed revised manuscript and critical revision. Parvez
Ali Mahesar: Interpret the revised data, help a lot in revising the
manuscript, supervision. Bhajan Lal: Reviewed revised manuscript
and critical revision. Kashif Ali Channar: Drafting the revised
All synthesized compounds directly interact within the active
region with different conformational poses. Based on in-vitro and
in-silico analyses 4 g compound was selected for SAR analysis
to view conformational pose against AChE. The SAR analysis re-
vealed that the carbonyl sulfur atom of 4 g interacts with aromatic
Tyr124 to form a strong hydrogen bond with bonding distances
3.09, respectively. Another hydrogen bond was observed between
the amino group and aromatic Tyr337 having a bond length of
˚
1.35 A. In both interactions, hydrogen bond length was compara-
˚
ble with standard value (<3 A) which gives the stability of dock-
ing complexes. The literature study also justified that these inter-
acted residues are significant in downstream signaling pathways
(Hassan et al., 2019; Ujan et al., 2019). The graphical depiction of
the 4 g docking complex is illustrated in Fig. 4. The binding of all
ligands inside the active region of AChE having similar conforma-
6

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
manuscript and critical revision. Bilal Ahmad Khan: Reviewed re-
vised manuscript and critical revision. Muhammad Nawaz: Visual-
ization of data, writing-reviewing and editing. Muhammad Shahid
Riaz Rajoka: Visualization of data, writing-reviewing and editing.
Jung Min Kim: Interpretation of data, writing-reviewing and edit-
ing.
[17] A. Bahadur, S. Iqbal, R. Ujan, et al., Effect of organic solvents on solvatochromic,
fluorescence, and electrochemical properties of synthesized thiazolylcoumarin
derivatives, Luminescence (2021) 1–9.
[18] S.L. Rogers, M.R. Farlow, R.S. Doody, R. Mohs, L.T. Friedhoff, A 24-week, dou-
ble- blind, placebo-controlled trial of donepezil in patients with Alzheimer’s
disease. Donepezil Study Group, Neurology 50 (1998) 136–145.
[19] J. Marco-Contelles, M. do Carmo Carreiras, C. Rodriguez, M. Villarroya, A.G. Gar-
cia, Synthesis and pharmacology of galantamine, Chem. Rev. 106 (2006)
116–133.
[20] M. Mohammadi-Khanaposhtani, M. Saeedi, N.S. Zafarghandi, M. Mahdavi,
R. Sabourian, E.K. Razkenari, H. Alinezhad, M. Khanavi, A. Foroumadi,
A. Shafiee, T. Akbarzadeh, Potent acetylcholinesterase inhibitors: design, syn-
thesis, biological evaluation, and docking study of acridone linked to 1, 2, 3-tri-
azole derivatives, Eur. J. Med. Chem. 92 (2015) 799–806.
[21] A. Saeed, S. Zaib, S. Ashraf, J. Iftikhar, M. Muddassar, K.Y. Zhang, J. Iqbal, Syn-
thesis, cholinesterase inhibition and molecular modelling studies of coumarin
linked thiourea derivatives, Bioorg. Chem. 63 (2015) 58–63.
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgment
[22] H. Mehfooz, A. Saeed, A. Sharma, F. Albericio, F.A. Larik, F. Jabeen, P.A. Channar,
U. Flörke, Dual Inhibition of AChE and BChE with the C-5 substituted deriva-
tive of Meldrum’s acid: synthesis, structure elucidation, and molecular docking
studies, Crystals 7 (2017) 211.
[23] M.L. Bolognesi, V. Andrisano, M. Bartolini, R. Banzi, C. Melchiorre, Propidi-
um-based polyamine ligands as potent inhibitors of acetylcholinesterase and
acetylcholinesterase-induced amyloid-β aggregation, J. Med. Chem. 48 (2005)
24–27.
The authors gratefully acknowledge the support by the pro-
gram of environmental analysis and pollution treatment innova-
tive research team, the Huizhou University Foreign staff project
(HZU202036), the Deanship of Scientific Research at King Khalid
University for funding this work through General Research Project
under grant number (RGP.2/145/42), the Major Project of Funda-
mental and Application Research of the Department of Education
of Guangdong Province (2015KTSCX132 and 2017KZDXM080), the
Science and Technology Foundation of Huizhou (2019 × 0705016),
the support received for this research work from the National Nat-
ural Science Foundation of China (51162026) and the Korean Re-
search Fellowship Program through the National Research Founda-
tion of Korea (NRF), Korea, funded by the Ministry of Science and
ICT (Grant no. NRF-2019H1D3A1A01102931).
[24] S. Darvesh, K.V. Darvesh, R.S. McDonald, D. Mataija, R. Walsh, S. Mothana,
O. Lockridge, E. Martin, Carbamates with differential mechanism of inhibition
toward acetylcholinesterase and butyrylcholinesterase, J. Med. Chem. 51 (2008)
4200–4212.
[25] E. Jameel, P. Meena, M. Maqbool, J. Kumar, W. Ahmed, S. Mumtazuddin, M. Ti-
wari, N. Hoda, B. Jayaram, Rational design, synthesis and biological screen-
ing of triazine-triazolopyrimidine hybrids as multitarget anti-Alzheimer agents,
Eur. J. Med. Chem. 136 (2017) 36–51.
[26] T. Sauvaître, M. Barlier, D. Herlem, N. Gresh, A. Chiaroni, D. Guenard, C. Guillou,
New potent acetylcholinesterase inhibitors in the tetracyclic triterpene series,
J. Med. Chem. 50 (2007) 5311–5323.
[27] A. Saeed, H. Rafique, A. Hameed, S. Rasheed, Synthesis and antibacterial ac-
tivity of some new 1-aroyl-3-(substituted-2-benzothiazolyl) thioureas, Pharm.
Chem. J. 42 (2008) 191–195.
Supplementary materials
[28] A. Saeed, U. Shaheen, A. Hameed, S.H. Naqvi, Synthesis, characterization
and antimicrobial activity of some new 1-(fluorobenzoyl)-3-(fluorophenyl)
thioureas, J. Fluor. Chem. 130 (2009) 1028–1034.
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.molstruc.2021.131136.
[29] F.A. Larik, A. Saeed, P.A. Channar, H. Ismail, E. Dilshad, B. Mirza, New 1-oc-
tanoyl-3-aryl thiourea derivatives: solvent-free synthesis, characterization and
multi-target biological activities, Bangladesh. J. Pharmacol. 11 (2016) 894–902.
[30] S. Saeed, N. Rashid, P.G. Jones, M. Ali, R. Hussain, Synthesis, characterization
and biological evaluation of some thiourea derivatives bearing benzothiazole
moiety as potential antimicrobial and anticancer agents, Eur. J. Med. Chem. 45
(2010) 1323–1331.
References
[1] J. Hardy, D.J. Selkoe, The amyloid hypothesis of Alzheimer’s disease: progress
and problems on the road to therapeutics, Science 297 (2002) 353–356.
[2] J.L. Cummings, Treatment of Alzheimer’s disease, Clin. Cornerstone 3 (2001)
27–39.
[3] D.J. Selkoe, Preventing Alzheimer’s disease, Science 337 (2012) 1488–1492.
[4] D.H. Small, S.S. Mok, J.C. Bornstein, Alzheimer’s disease and Abeta toxicity:
from top to bottom, Nat. Rev. Neurosci. 2 (2001) 595–598.
[31] S. Karakus¸ , S. Rollas, Synthesis and antituberculosis activity of new
ꢀ
N-phenyl-N -[4-(5-alkyl/arylamino-1, 3, 4-thiadiazole-2-yl) phenyl] thioureas,
Il Farmaco 57 (2002) 577–581.
[32] I. Küçükgüzel, E. Tatar, S¸ .G. Küçükgüzel, S. Rollas, E. De Clercq, Synthe-
sis of some novel thiourea derivatives obtained from 5-[(4-aminophenoxy)
methyl]-4-alkyl/aryl-2, 4-dihydro-3H-1, 2, 4-triazole-3-thiones and evaluation
as antiviral/anti-HIV and anti-tuberculosis agents, Eur. J. Med. Chem 43 (2008)
381–392.
[5] S. Darvesh, D. Hopkins, A.C. Geula, Neurobiology of butyrylcholinesterase, Nat.
Rev. Neurosci. 4 (2003) 131–138.
[6] C. Mattes, R. Bradley, E. Slaughter, S. Browne, Cocaine and butyryl-
cholinesterase (BChE): determination of enzymatic parameters, Life Sci. 58
(1996) 257–261.
[33] T. Stringer, D. Taylor, C. de Kock, H. Guzgay, A. Au, S.H. An, B. Sanchez,
R. O’Connor, N. Patel, K.M. Land, P.J. Smith, Synthesis, characterization, antipar-
asitic and cytotoxic evaluation of thioureas conjugated to polyamine scaffolds,
Eur.J. Med. Chem 69 (2013) 90–98.
[34] A. Saeed, R. Sajid, P.A. Channar, F.A. Larik, A. Qamar, Hussain Mubashir, U. Raza,
U. Flörke, Y.S Sung, Long chain 1-Acyl-3-arylthioureas as jack bean urease in-
hibitors, synthesis, kinetic mechanism and molecular docking studies, J. Tai-
wan. Institute. Chem. Engineers 77 (2017) 54–63.
[35] A. Saeed, M.S. Shah, F.A. Larik, S.U. Khan, P.A. Channar, U. Flörke, J. Iqbal, Syn-
thesis, computational stu dies and biological evaluation of new 1-Acetyl-3-aryl
thiourea derivatives as potent cholinesterase inhibitors, Med. Chem. Res 26 (8)
(2017) 1–12.
[7] Y. Shen, J. Zhang, R. Sheng, X. Dong, Q. He, B. Yang, Y. Hu, Synthesis and
biological evaluation of novel flavonoid derivatives as dual binding acetyl-
cholinesterase inhibitors, J. Enzyme Inhib. Med. Chem. (2009) 372–380.
[8] P. Zelik, A. Lukesova, L.N. Voloshko, D. Stys, J. Kopecky, Screening for acetyl-
cholinesterase inhibitory activity in cyanobacteria of the genus Nostoc, J. En-
zyme Inhib. Med. Chem. 24 (2009) 531–536.
[9] R.M. Lane, S.G. Potkin, A. Enz, Targeting acetylcholinesterase and butyryl-
cholinesterase in dementia, Int. J. Neuropsychopharmacol. 9 (2006) 101–124.
[10] R. Léon, A.G. Garcia, J. Marco-Contelles, Recent advances in the multitarget-
directed ligands approach for the treatment of Alzheimer’s disease, Med. Res.
Rev. 33 (2013) 139–189.
[11] H.O. Tayeb, H.D. Yang, B.H. Price, F.I. Tarazi, Pharmacotherapies for Alzheimer’s
disease: beyond cholinesterase inhibitors, Pharmacol. Ther. 134 (2012) 8–25.
[12] N.H. Greig, D.K. Lahiri, K. Sambamurti, Butyrylcholinesterase: an important
new target in Alzheimer’s disease therapy, Int. Psychogeriatr. 14 (2002) 77–91.
[13] T. Hikida, Y. Kitabatake, I. Pastan, S. Nakanishi, Acetylcholine enhancement in
the nucleus accumbens prevents addictive behaviors of cocaine and morphine,
Proc. Natl. Acad. Sci. USA 100 (2003) 6169–6173.
[14] F. Bergmann, I.B. Wilson, D. Nachmansohn, The inhibitory effect of stilbami-
dine, curare and related compounds and its relationship to the active groups
of acetylcholine esterase; action of stilbamidine upon nerve impulse conduc-
tion, Biochim. Biophys. Acta 6 (1950) 217–224.
[36] A. Saeed, R. Sajid, F.A. Larik, P.A. Channar, A. Qamar, Hussain Mubashir, U. Raza,
U. Flörke, Y.S. Sung, Jack bean urease inhibitors, and antioxidant activity based
on palmitic acid derived 1-Acyl-3-Arylthioureas: synthesis, kinetic mechanism
and molecular docking studies, Drug Re 11 (2017) 381–388.
[37] J. Iqbal, S. Zaib, A. Saeed, M. Muddassar, Biological evaluation of halogenated
thioureas as cholinesterases inhibitors against Alzheimer’s disease & molecular
modeling studies, Lett. Drug Des. Discov. 12 (2015) 488–49.
[38] F.A. Larik, A. Saeed, P.A. Channar, U. Muqadar, Q. Abbas, M. Hassan, S.Y. Seo,
M. Bolte, Design, synthesis, kinetic mechanism and molecular docking studies
of novel 1-pentanoyl-3-arylthioureas as inhibitors of mushroom tyrosinase and
free radical scavengers, Eur. J. Med.Chem 141 (2017) 273-28.
[15] L. Austin, W.K. Berry, Two selective inhibitors of cholinesterase, Biochem. J. 54
(1953) 695–700.
[39] A. Saeed, U. Flörke, M.F. Erben, A review on the chemistry, coordination, struc-
ture and biological properties of 1-(acyl/aroyl)-3-(substituted) thioureas, J. Sul-
fur Chem 35 (2014) 318–355.
[16] K. Musilek, R. Pavlikova, J. Marek, M. Komloova, O. Holas, M. Hrabinova, M. Po-
hanka, V. Dohnal, M. Dolezal, F.G. Moore, K. Kuca, The preparation, in vitro
screening and molecular docking of symmetrical bisquaternary cholinesterase
inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage, J. Enzyme In-
hib. Med. Chem. 26 (2011) 245–253.
[40] G.L. Ellman, K.D. Courtney, V. Andres, R.M.A. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activity, Biochem. Pharma-
col. 7 (1961) 88–95.
7

R. Ujan, P.A. Channar, A. Bahadur et al.
Journal of Molecular Structure 1246 (2021) 131136
[41] Q. Abbas, Z. Ashraf, M. Hassan, H. Nadeem, M. Latif, S. Afzal, S.Y. Seo, Develop-
ment of highly potent melanogenesis inhibitor by in vitro, in vivo and compu-
tational studies, Drug Des. Devel. Ther. 11 (2017) 2029.
[45] D.E.V. Pires, T.L. Blundell, Ascher DB, pkCSM: predicting small-molecule phar-
macokinetic properties using graph-based signatures, J. Med. Chem 58 (9)
(2015) 4066–4072.
[42] E.F. Pettersen, T.D. Goddard, C.C. Huang, G.S. Couch, D.M. Greenblatt, E.C. Meng,
T.E. Ferrin, UCSF Chimera – a visualization system for exploratory research and
analysis, J. Comput. Chem. 25 (13) (2006) 1605–1612.
[46] S. Dallakyan, A.J. Olson, Small-molecule library screening by docking with
PyRx, Method. Mol. Biol. 1263 (2015) 243–250.
[47] A.C. Wallace, R.A. Laskowski, J.M. Thornton, LIGPLOT:
a program to gener-
[43] L. Willard, A. Ranjan, H. Zhang, H. Monzavi, R.F. Boyko, B.D. Sykes, D.S. Wishart,
VADAR: a web server for quantitative evaluation of protein structure quality,
Nucleic Acids Res. 31 (2003) 3316–3319.
ate schematic diagrams of protein-ligand interactions, Protein Eng. 8 (1996)
127–134.
[44] R. Ujan, A. Bahadur, G. Shabir, et al., Facile synthesis of novel fluorescent thi-
azole coumarinyl compounds: electrochemical, time resolve fluorescence, and
solvatochromic study, J. Mol. Struct. (2021) 1227.
8