Thiourea derivatives, simple in structure but efficient enzyme inhibitors and mercury sensors

Article abstract of DOI:10.3390/molecules26154506

In this study six unsymmetrical thiourea derivatives, 1-isobutyl-3-cyclohexylthiourea (1), 1-tert-butyl-3-cyclohexylthiourea (2), 1-(3-chlorophenyl)-3-cyclohexylthiourea (3), 1-(1,1-dibutyl)-3-phenylthiourea (4), 1-(2-chlorophenyl)-3-phenylthiourea (5) an

Full text of DOI:10.3390/molecules26154506

molecules
Article
Thiourea Derivatives, Simple in Structure but Efficient Enzyme
Inhibitors and Mercury Sensors
Faizan Ur Rahman 1, Maryam Bibi 2, Ezzat Khan 1,2,
* ,
, Abdul Bari Shah 3 , Mian Muhammad 1
Muhammad Nawaz Tahir 4 , Adnan Shahzad 5 , Farhat Ullah 6 , Muhammad Zahoor 7 , Salman Alamery 8
and Gaber El-Saber Batiha 9
1
Department of Chemistry, University of Malakand, Lower Dir 18800, Pakistan;
faizan.uom98@gmail.com (F.U.R.); mianchem@uom.edu.pk (M.M.)
Department of Chemistry, College of Science, University of Bahrain, Sakhir 32038, Bahrain;
2
maryamdua55@gmail.com
Division of Applied Life Science (BK21 Plus), IALS, Gyeongsang National University, Jinju 52828, Korea;
3
abs.uom28@gmail.com
4
Department of Physics, University of Sargodha, Sargodha 40100, Pakistan; dmntahir_uos@yahoo.com
Institute of Chemical Sciences, University of Swat, Swat 19150, Pakistan; adnanshahzad09@gmail.com
Department of Pharmacy, University of Malakand, Chakdara, Lower Dir 18800, Pakistan;
5
6
farhataziz80@hotmail.com
Department of Biochemistry, University of Malakand, Lower Dir 18800, Pakistan;
7
mohammadzahoorus@yahoo.com
Department of Biochemistry, College of Science, King Saud University, P.O. Box 22452,
8
Riyadh 11451, Saudi Arabia; Salamery@ksu.edu.sa
Department of Pharmacology and Therapeutics, Faculty of Veterinary Medicine, Damanhour University,
Damanhour 22511, Egypt; gaberbatiha@gmail.com
9
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
*
Correspondence: ekhan@uom.edu.pk or ezkhan@uob.edu.bh
Citation: Rahman, F.U.; Bibi, M.;
Khan, E.; Shah, A.B.; Muhammad, M.;
Tahir, M.N.; Shahzad, A.; Ullah, F.;
Zahoor, M.; Alamery, S.; et al. Thiourea
Derivatives, Simple in Structure but
Efficient Enzyme Inhibitors and
Mercury Sensors. Molecules 2021, 26,
4506. https://doi.org/10.3390/
molecules26154506
Abstract: In this study six unsymmetrical thiourea derivatives, 1-isobutyl-3-cyclohexylthiourea (
1-tert-butyl-3-cyclohexylthiourea ( ), 1-(3-chlorophenyl)-3-cyclohexylthiourea ( ), 1-(1,1-dibutyl)-3-
phenylthiourea ( ), 1-(2-chlorophenyl)-3-phenylthiourea ( ) and 1-(4-chlorophenyl)-3-phenylthiourea
) were obtained in the laboratory under aerobic conditions. Compounds and are crystalline
and their structure was determined for their single crystal. Compounds is monoclinic system with
space group P21/n while compound ) were tested
is trigonal, space group R3:H. Compounds (
1),
2
3
4
5
(6
3
4
3
4
1–6
for their anti-cholinesterase activity against acetylcholinesterase and butyrylcholinesterase (hereafter
abbreviated as, AChE and BChE, respectively). Potentials (all compounds) as sensing probes for
determination of deadly toxic metal (mercury) using spectrofluorimetric technique were also inves-
Academic Editor: Takashiro Akitsu
tigated. Compound
AChE and BChE with docking score of
showed moderate sensitivity during fluorescence studies.
3
exhibited better enzyme inhibition IC50 values of 50, and 60
µg/mL against
Received: 30 May 2021
Accepted: 11 July 2021
Published: 27 July 2021
−
10.01, and 8.04 kJ/mol, respectively. The compound also
−
Keywords: thiourea derivatives; enzyme inhibition; docking studies; mercury sensing; X-ray structure
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Thiourea derivatives play a pivotal role due to numerous applications in the field of
nanoparticles, starting precursors in a large number of chemical reactions and therefore,
remained focus of several review articles [
derivatives is an open challenge [ ]. Several researchers have made thiourea derivatives un-
der different conditions and many of them are now commercially available [ ]. Thiourea
derivatives such as cyclohexyl thiourea or phenylthiourea are attractive model compounds
for the studies in solid-state chemistry due to their tendency for the formation of intra-
and inter molecular hydrogen bonding wherein the N-H proton-donor groups and pre-
dominantly sulfur atoms are engaged. The presence of C=S groups in the same molecule
1–5]. Efficient and easy accessibility of thiourea
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
6
7,8
Molecules 2021, 26, 4506. https://doi.org/10.3390/molecules26154506
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4506
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make them more attractive for further chemistry [9]. These derivatives have an active role
in pharmaceuticals as potential therapeutic agents against HIV, HCV [10
anticonvulsant [14 15], antihyperlipidemic, antiallergic, antiparasitic, antiproliferative,
antioxidant and antidiabetic and anticholinestrase [16 17]. Cholinesterases are various
isolated enzymes which are classified as acetyl and butyryl associated with hydrolases
enzymes [18 19]. Acetylcholinesterase (AChE) provides a signaling action following degra-
dation of acetylcholine (ACh) [20 21], whereas butyrylcholinesterase (BChE) plays a pivotal
–13], anticancer,
,
,
,
,
role in the metabolism of different molecules. Hence, AChE inhibitors are considered to
be one of the most efficient approaches for the treatment of Alzheimer’s disease (AD) by
enhancing cholinergic objectives in AD patients. In a normal healthy brain, ACh metabolic
degradation is also caused by BChE, hence BChE enhances progression of AD. Therefore,
synchronous inhibition achieved by AChE and BChE may provide additional benefits
for AD treatment [22]. Several thiourea derivatives such as isobutylphenylthiourea, and
tert-butylphenylthiourea and their coordination complexes have recently been reported by
our research group as enzyme inhibitors [23]. Several studies have been conducted with
respect to thiourea derivatives which highlight the role of this versatile class of compounds
in environmental applications particularly heavy metal sensing [24–30]. Heavy metals are
expected to be one of the reasons of the AD and their timely control in the body of an
individual is utmost necessary [31,32].
Fluorescence is one of the most important physical phenomena in which a particular
chemical compound (thiourea for instance) emits electromagnetic radiations of longer
wavelength after being excited by radiations with shorter wavelength. Fluorescence based
detection, is a homogeneous system that allows direct measurement of binding in solution
and has played a significant role at the forefront of the bio-analytical area, because of
ultrahigh sensitivity and selectivity. One of the most important applications of fluorescence
technology is the decreased size of a sample down to the single-molecule detection level,
and this fact provides an opportunity for miniaturization and high throughput screen-
ing. In addition, fluorescence has enabled the elucidation of many biological processes,
determination of structure and trace amounts of biological macromolecules. Fluorescence
sensors employ a fluorescent signal for the detection of analytes. In order to detect different
organics at an earlier stage of a disease development, a low detection limit of the organics
is desirable [33]. Thiourea derivatives possess the ability to detect both anions and cations
and are therefore attracting the attention to be versatile detectors in coming decades [5].
Thiourea derivatives are used as fluorescent detectors for heavy metal ions including Hg2+
and various anions in aqueous media [4,34–36].
The AD is greatly influenced by heavy metal ions concentration and can be controlled
either by inhibition of specific enzymes or quenching of responsible metal ions. In the
current paper, we present the dual function of thiourea derivatives for the same purpose to
control AD in an efficient way. Literature review reveals that solid state structure of these
compounds is not yet reported. We hereby report solid state structure of compounds
, enzyme inhibition and fluorescence potentials of thiourea derivatives which are already
in field (Figure 1). It is notable that most of the compounds are potent inhibitors with respect
to the standard, galantamine. Molecular docking study is also carried out for compound
and in order to investigate their inhibitory action. The synthesized compounds were also
3 and
4
3
4
investigated as a sensing probe for determination of mercury with the intent to use these
biologically active molecules in removal technology in order to eliminate complications
of bioacceptance and accessibility. The fluorescent properties of compounds are very
interesting and the compound
2 was employed in the determination of mercury, as sensing
probe. These studies invite further research in the field of single molecule detectors for
determination of heavy metal ions of biological concerns in controlling a variety of diseases.

Molecules 2021, 26, 4506
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S
S
S
N
H
N
H
N
H
N
H
N
H
N
H
Cl
2
3
1
Cl
S
S
S
N
H
N
H
N
H
N
H
N
H
N
Cl
6
4
5
Figure 1. Structure of unsymmetrical thiourea derivatives, 1-cyclohexyl-3-(iso-butyl)thiourea (
1
), 1-cyclohexyl-3-
), 1-phenyl-3-(2-
(tert-butyl)thiourea ( ), 1-cyclohexyl-3-(3-chlorophenyl)thiourea ( ), 1-phenyl-3-(1,1-dibutyl)thiourea (4
2
3
chlorophenyl)thiourea (5) and 1-phenyl-3-(4-chlorophenyl)thiourea (6) used in this study.
2. Results and Discussion
Compounds
can also be obtained in laboratory under ambient conditions [37]. Compounds structurally
analogous to are good corrosion inhibitors in acidic medium [38] and show excited
results in affording metal sulfides for useful applications [39]. Compound has been used
1–6 are structurally very simple; they are accessible in the market and
1–6
5
as starting precursors for the preparation of benzothiazole derivatives under catalyst free
conditions [40]. There are several other reports wherein simple thiourea molecules after
certain modifications have shown efficiency as bioactive compounds [41]. Compound
already been used for its inhibitory activity against melanin B16 cells and mushroom tyrosi-
nase and its synthesis has been reviewed [ 42]. The IC50 value as melanin B16 inhibitor
was promising, 3.4 M, while it exhibited moderate potency as mushroom tyrosinase
inhibitor. The enzyme inhibition (AChE and BChE) and metal sensing capability of com-
pounds (Figure 1) have not been reported so far. Since they are already in the field of
6 has
6,
µ
1–6
bioorganic chemistry therefore these studies will help exploring their multidimensionality
applications within a single system or organism whatever the case may be.
2.1. Structural Description of Compound 3
Compound
room temperature, which allowed for collection of X-ray diffraction data. The structure of
compound is depicted in Figure 2, along with a dimer unit held together with the help of
H-bonding. The refinements and crystal structure solution parameters are summarized
3 was crystalline in nature and good quality crystals were obtained at
3
in Table 1, Bond angles and lengths of structural importance are summarized in Table 2
and the experimental data are compared with theoretical. Both data sets were in close
agreement with each other. The surrounding of C7 is trigonal planar with expected bond
lengths and angles and the data also revealed the sp2 hybridized environment of the
carbon. The C=S bond length was 1.695 Å, which fall within the expected limit for the said
derivative [43]. The C7-N1 and C7-N2 distances were 1.351 and 1.326 Å, where the effect
of substituent on the N was very clear, the lone pair of N1 was delocalized towards Ph ring
while that of N2 was attracted towards C7 imparting partial double bond characters to
N2-C7 bond (Table 2). This difference in delocalization of electron pair on both the N atoms
provided insights to identify suitable H-bonding sites. The 3-ClPh-N1 was comparatively
electron rich and is suitable donor, the hydrogen bonding was thus established making a
dimer as given in Figure 2. Short ranged interactions were not observed in molecules of
compound 3. See Table 3 for data pertaining to hydrogen bond parameters.
,

Molecules 2021, 26, 4506
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Figure 2. X-ray structure of Compound
3
(left), ellipsoids are drawn at 50% probability level, H atoms
are omitted, selected carbon atoms and all non-carbon atoms are numbered. Dimer of compound
3
(right) stabilized through N1H–S1 interactions. Other secondary interactions in molecules were
not detected. For data related to structural determination and refinements and selected structural
parameters (bond lengths and angles) see Tables 1 and 2, respectively.
Table 1. Structure solution and refinements parameters of compound 3 and 4.
Compound 3
Compound 4
CCDC No *
Chemical formula
Mr,
2050647
C13H17ClN2S
268.79
2050648
C15H24N2S
264.42
Temperature (K)
Crystal system, space group
296
296
Trigonal, R3:H
25.550 (2), 25.550 (2),
13.1763(11),
90.00
Monoclinic, P21/n
5.5080 (4), 22.1068 (17),
11.4404 (8)
99.967 (4)
a, b, c (Å)
◦
β ( )
V (Å3)
1372.01 (17)
4
7448.8(13)
18
Z
Radiation type
Mo Kα
0.41
Mo Kα
0.18
−1
µ (mm
)
Crystal size (mm)
Diffractometer
Absorption correction
Tmin, Tmax
0.40 × 0.32 × 0.26
0.44 × 0.38 × 0.36
Bruker Kappa APEXII CCD
Multi-scan
0.815, 0.925
Multi-scan
0.875, 0.955
No. of measured,
independent and observed
[I > 2σ(I)] reflections
9399, 3283, 2340
12267, 3614, 1852
Rint,
0.048
0.660
0.048, 0.135, 1.02
3283
0.074
0.641
0.056, 0.168, 1.02
3614
−1
(sin θ/λ) max (Å
)
R[F2 > 2σ(F2)], wR(F2), S
No. of reflections
No. of parameters/restrains
154/0
188/214
H-atom treatment
H-atom parameters constrained
0.34, −0.32
−3
0.18, −0.16
∆ρmax, ∆ρmin (e Å
)
* Crystallographic information files pertaining to compounds
3
and
4
can be obtained free of charge at www.
ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; Fax: +44-1223-336-033; e-mail: deposit@ccdc.cam.ac.uk).

Molecules 2021, 26, 4506
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Table 2. Experimental and theoretical (B3LYP/3-21G level) selected structural parameters, bond
lengths and angles of compounds 3 and 4.
Compound
Atoms
Exp.
Cal.
Atoms
Exp.
Cal.
Bond length
1.695(19)
1.351(2)
1.423(2)
1.326(2)
1.464(2)
1.686(2)
1.352(3)
1.426(3)
1.344(3)
Bond angle
127.52(17)
124.61(16)
117.68(17)
123.18(15)
119.14(15)
127.84(19)
115.86(18)
123.03(18)
123.6(17)
S1-C7
N1-C7
N1-C1
N2-C7
N2-C8
S1-C7
Ni-C7
N1-C1
N2-C7
1.730
1.383
1.434
1.342
1.483
1.731
1.390
1.421
1.351
C7-N1-C1
C7-N2-C8
N2-C7-N1
N2-C7-S1
N1-C7-S1
C7-N1-C1
N2-C7-N1
N2-C7-S1
C7-N2-C8B
128.41
124.81
117.29
123.53
118.73
128.23
116.10
124.34
123.59
3
4
Table 3. Hydrogen-bond geometries (Å) of compounds 3 and 4.
Compound
3
D—H···A
D—H
H···A
D···A
D—H···A
Symmetry Codes
N1—H1···S1
C2—H2···Cl1
C9—H9B···S1
N1—H1···S1
0.86
0.93
0.97
0.86
0.97
0.97
0.97
0.97
0.97
0.97
2.53
2.96
2.91
2.70
2.96
2.75
2.95
2.62
2.65
2.56
3.343 (18)
3.878 (2)
3.483 (3)
3.484 (2)
3.840 (12)
3.711 (11)
3.84 (4)
3.53 (4)
3.179 (14)
3.00 (10)
157.4
167.6
118.9
151.5
151.5
172.2
152.3
155.3
114.8
107.6
−x, −y, −z + 2
−x, −y, −z + 1
-
x−y + 2/3, x + 1/3, −z + 4/3
−x + y − 1/3, −x + 1/3, z + 1/3
x−y + 2/3, x + 1/3, −z + 4/3
−x + y − 1/3, −x + 1/3, z + 1/3
x−y + 2/3, x + 1/3, −z + 4/3
-
C8A—H8A ··S1
C8A—H8B ··S1
C8B—H8C ··S1
C8B—H8D ··S1
C9B—H9C ··N1
C12B—H12D ··S1
4
-
2.2. Structural Description of Compound 4
Molecular structure of thiourea derivative
4
is given in Figure 3 (left), structure
determination and refinements were carried out as summarized in Table 1, and selected
bond lengths and angles are summarized in Table 2. The bond angles and bond lengths
around the central carbon, C7 were very similar to compound 3. The sum of all bond
angles is 360◦ indicating that C7 was sp2 hybridized with characteristic trigonal planar
geometry. The C7=S1 bond length was 1.686 Å, which is slightly shorter than the same
bond in compound
different from each other revealing the effect of substituent on N atom. The difference
was small as compared to compound , indicating that lone pair of electrons at both N
3. The C7-N1 and C7-N2 distances were 1.352 and 1.344 Å were slightly
3
atoms were almost equally involved in delocalization through the NCN fragment. The
NH and CH2hydrogen atoms in molecules played an important role in stabilizing the
supramolecular structure in a 3D manner. The H8B and NH1 were attached to S1 of one
molecule with H–S separation distance of 2.748 and 2.704 Å, respectively and the H8A to
S atom of the other molecule (separation distance 2.960 Å). The difference in separation
distance was obvious and it can be clearly observed that the former interaction combined
molecule in hexameric ring form and the latter one helped to keep chains together in a 3D
fashion. Non-covalent interactions between molecules of a hexameric ring made an R12(7)
type loop in a cyclic arrangement and further extended in a 3D manner as discussed earlier.
Different views of supramolecular structure of compound
hydrogen bonding data are presented in Table 3.
4 are shown in Figure 4, and the

Molecules 2021, 26, 4506
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Figure 3. Single crystal structure (left hand) of compound 4, ellipsoids are drawn at 50% probability level, H-atoms except
NH and CH2 are omitted and selected atoms have been numbered. A cyclic hexamer (right hand) is shown, molecules
are linked with the help of S–H interactions making a macrocyclic ring containing six thiourea molecules (See Table 2 for
selected structural parameters).
Figure 4. Different view of supramolecular structure of compound 4, single layer of mutually
connected hexameric units (top), mutually connected layers (middle and bottom), the molecular
architecture extends in a 3D manner.

Molecules 2021, 26, 4506
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Optimized structure of compound
3
and
4, are given in Figure 5, a comparison of
experimental and theoretical bond parameters showed a close relationship, with negligible
variation. The data suggested that gas phase and solid-state structure did not vary from
each other.
Figure 5. Optimized structures of compounds 3 and 4, selected hydrogen atoms are omitted.
2.3. Enzyme Inhibition Studies
The effectiveness of the synthesized compounds 1–6 as inhibitors of the selected
cholinesterase was tested as discussed in experimental section, vide supra. The observed
activities are summarized in Table 4, The IC50 value of most of the compounds was
high > 100 µg/mL, except two compounds. Compound
lent activity 50 and 60 g/mL against AChE and BChE, respectively. Compound
exhibited very close IC50 values 58 and 63 g/mL, respectively. The IC50 value observed
for the standard was 15 g/mL for inhibition of both the enzymes in a respective manner.
Further derivatization of compound , can be useful in future studies to achieve the target
properties. The efficiency of this compound can be linked to the intermolecular interactions
3
was the most potent with excel-
µ
4
also
µ
µ
3
as discussed above. Lack of secondary interactions in other molecules like
reason of poor biological efficiency.
4
is the probable
Table 4. Efficiency of compounds 1–6 as inhibitors against AChE and BChE.
%AChE
Inhibition
%BChE
Inhibition
No
Conc. (µg/mL)
IC50 (µg/mL)
IC50 (µg/mL)
1000
500
250
69.47 ± 0.22
63.94 ± 0.45
57.61 ± 1.70
53.64 ± 0.16
46.52 ± 0.38
63.34 ± 0.98
56.32 ± 1.06
48.05 ± 0.75
44.70 ± 1.25
38.74 ± 0.68
73.39. ± 0.60
67.39 ±0.49
61.36 ± 0.49
57.34 ± 0.55
51.90 ± 1.16
74.4 ± 0.68
66.2 ± 0.73
61.0 ± 0.33
56.4 ± 0.63
46.9 ± 0.42
65.17 ± 0.72
57.85 ± 0.97
51.37 ± 1.65
46.73 ± 0.78
41.34 ± 1.01
76.7 ± 0.66
71.3 ± 1.11
65.5 ± 1.04
57.2 ± 0.57
49.9 ± 0.65
1
89
84
125
62.5
1000
500
250
125
62.5
1000
500
250
2
3
300
50
185
60
125
62.5

Molecules 2021, 26, 4506
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Table 4. Cont.
%AChE
Inhibition
%BChE
Inhibition
No
Conc. (µg/mL)
IC50 (µg/mL)
IC50 (µg/mL)
1000
500
250
93.58 ± 1.12
85.40 ± 0.20
77.85 ± 2.26
71.80 ± 1.50
47.90 ± 0.47
39.01 ± 0.88
66.79 ± 0.63
59.67 ± 0.61
41.69 ± 0.77
35.54 ± 0.50
29.00 ± 0.30
69.58 ± 1.12
61.65 ± 1.34
47.90 ± 0.96
39.03 ± 0.48
31.90 ± 0.48
83.19 ± 0.73
77.54 ± 0.91
71.93 ± 0.14
65.72 ± 0.49
61.67 ± 0.94
98.00 ± 0.00
94.40 ± 0.50
89.80 ± 1.50
77.40 ± 1.70
53.50 ± 0.90
47.30 ± 0.70
71.62 ± 0.74
63.86 ± 0.60
44.48 ± 0.64
37.54 ± 0.50
31.74 ± 0.61
71.33 ± 0.49
63.03 ± 0.23
49.00 ± 0.58
42.67 ± 0.89
33.00 ± 1.15
89.64 ± 0.62
81.83 ± 0.37
74.29 ± 0.73
67.92 ± 0.98
64.93 ± 0.87
4
58
63
125
62.5
31.5
1000
500
250
125
62.5
1000
500
250
125
62.5
1000
500
250
125
5
345
285
15
310
265
15
6
Galantamine
62.5
Molecular Docking Studies of Compounds 3 and 4
The acetylcholinesterase, responsible for hydrolysis of neurotransmitter acetylcholine
(ACh) in neuromuscular junctions, consisted of two active sites, namely peripheral site
and catalytic site. To corelate the obtained inhibition results in experiments, the molecular
docking investigations were carried out against the selective and potent inhibitors with
AChE and BChE, by following the literature procedure. The in vitro study showed that
the compound
experimental data of enzyme inhibition given in Table 4. To study the binding extent and
binding poses of selective thiourea derivatives and 4, the active pockets were selected for
3 is the excellent and selective inhibitor of AChE and BChE supporting the
3
binding interactions. The binding interaction of compound
are given in Figure 6.
3
and
4
with AChE and BChE
The complete analysis of co-crystalized thiourea derivative
showed that tyr-124, Ser-203 and Tyr-337 are the most potent residues showing binding in-
teractions with inhibitor compound with in the active pocket of protein. Our synthesized
compound forms strong hydrogen bonding with Tyr-124 with calculated length 2.4 Å and
with binding score of 10.01 kJ/mol. In addition to strong hydrogen bonding, some weak
polar interactions and hydrophobic interactions were also observed with Tyr-337, Tyr-341
and Ser-203 given in Figure 6a. The compound forms hydrogen bond with Trp-532
with a separation distance of 2.7 Å with binding score of 8.031 kJ/mol and hydrophobic
interactions with His-405 and Pro-537 given Figure 6b. The binding energy shows that
compound exhibited strong binding interactions as compared with compound , thus
supporting the experimental inhibition data of structurally analogous compounds [44].
During the docking studies of compound and with BChE, it was observed that
established interactions with Asn-83, Asn-85 and His-77, while compound was efficient
in establishing interactions with Tyr-128 and Trp-82. These interactions are responsible
to make a compound more potent against BChE. The co-crystalized compound formed
strong hydrogen bonding with Asn-83 and His-126 with a distance of 2.2 and 3.0 Å,
respectively and the binding score of 8.04 kJ/mol. In addition to this, compound
also established weak hydrophobic interactions with same residues of BChE given in
Figure 6c, while compound formed weak hydrogen bonding with Tyr-456 (2.4 Å) and
weak interaction with Lys-427 residues (3.2 Å) with binding score of 6.98 kJ/mol [45].
3 and AChE inhibitor
3
3
−
4
−
3
4
3
4
3
4
3
−
3
4
−

Molecules 2021, 26, 4506
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The comparison of binding energy shows that compound
3
was also a potent inhibitor of
BChE as compared to compound
Figure 6d.
4, thus our calculations support the experimental data,
Figure 6. 3D binding interaction modes of compounds
teraction posing of compound with AChE; ( ) interaction of compound
of compound 3 with BChE and (d) interaction of compound 4 with BChE.
3
and
4
as inhibitor of AChE and BChE. (
a) In-
3
b
4
with AChE; ( ) interaction
c
2.4. Fluorescence and UV-Visible Characteristics of the Compounds 1–3, 5 and 6
The UV-visible absorption spectra of compounds were measured for a 2 ppm solution
in the range 200–800 nm. The absorption maxima of all the tested compounds appeared
between 200–400 nm as shown in Figure S1, Supplementary Materials. The methanol
solution of compounds exhibited
λ
max
296, 294, 298, 294 and 295 nm, respectively. The
fluorescence characteristics of the synthesized compounds (1–3, 5, 6) were investigated
against the solvent blank by scanning the and in the range of 250–750 nm. The
λ
ex
λ
em
fluorescence spectra of compounds are shown in Figure 7 (for two selected compounds) and
respective data are tabulated in Table 5. It was observed that compound and showed
excellent fluorescence intensity, compound at low sensitivity level while compounds and
show excellent instrumental sensitivity. The large stokes shift (>300 nm) for compounds
and could possibly be attributed to the intramolecular charge transfer characteristic
3,
5
6
3
1
2
3
5
(ICT) due to the electron-accepting nature of the moiety because of its well-defined dipole
moment. Compounds which undergo ICT, exhibited different properties both in their
ground and excited states, which was caused by an electronic rearrangement after photo
excitation. This temporary dipole alternation led to larger changes in dipole moments and
to the relaxation of the structure. The energy difference between the Frank–Condon state
and the ICT state was the predominant reason for the large Stokes shift, which is ideal in
fluorescence methods [46
all the compounds at concentration as low as 10
,
47]. The fluorescence behavior of these compounds shows that
µ
g mL−1 could be used for fabrication
of fluorescence-based sensing tools for detection of environmental pollutants especially
pesticides, toxic heavy metals and drugs. The analyte molecules could interact effectively
with the compounds causing fluorescence enhancement or fluorescence quenching. The

Molecules 2021, 26, 4506
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interaction could be easily employed for development of sensitive analytical methods for
determination of the subject analytes which is the future perspective of this work.
Figure 7. Fluorescence spectra of 3 and 5 as two representative examples.
Table 5. Fluorescence characteristics of compounds 1–3, 5 and 6.
Concentration
Scan Range
(nm)
Mode of
Sensitivity
Fluorescence
Intensity
Compound
λ
(nm)
λ
(nm)
em
ex
−1
(µg mL
)
1
2
3
5
6
350–700
220–750
220–650
220–600
220–600
312
304.0
329
337
311
613
328.0
661
662
624
High
High
Low
Low
Low
276.483
16.189
212.958
180.234
275.143
10
2.5. Application of Compounds 1–3, 5, 6 as Sensing Probe for Determination of Mercury
In order to know the interaction between Hg(II) and the subject compounds, variable
concentration of Hg(II) solutions was added to known concentration of each compound
and FI at the respective excitation and emission of each compound was measured at low
and high sensitivity mode. The results are given in Table 6 and for two selected compounds
3
and 5 are shown in Figure 8. As evident from the data, FI of the mixtures increased

Molecules 2021, 26, 4506
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linearly with increase in the concentration of Hg(II) up to the maximum FI level. The
reason behind this interaction of the compounds with Hg(II) was based on the soft and
hard acid base concept [36]. Mercury (Hg(II)) is a soft center with high polarizability
and sulfur in the thiourea molecule is soft center too. The only compound that does
not show this sort of enhancement interaction with Hg(II) is 6. Though 6 also bore a
sulfur atom the probable reason of negligible interactions could be due to inappropriate
cavity size and orientation for binding of Hg(II) ions. It happened due to diminishing of
photoinduced electron transfer phenomenon between receptor and fluorophore [36]. This
fluorescence enhancement could be successfully applied for trace level determination of
Hg(II) in complex environmental samples including water, soil, vegetables and fruits. The
same could also be applied for remediation of Hg(II) ion in real biological samples in order
to develop efficient reagent/medicine to combat AD.
Figure 8. Representative fluorescence spectra of 3 and 5 as a function of concentration of Hg(II) ion.

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Table 6. Hg(II) sensing studies of compounds
1
–3
,
5
and
6, FI in the Table stands for fluorescence
intensity.
Concentration
(ppm)
λex/
λ
em(nm)
Concentration
of Hg(II) ppm
Fluorescence
Intensity
Compound
Fluorescence Intensity
0.1
0.2
0.3
608.132
901.543
1015.790
329/661
FI = 212.958
Curve:e
3
10
10
0.1
0.2
0.3
0.5
460.580
646.740
876.296
1015.790
312/613
FI = 276.483
Curve:e
1
2
5
6
0.01
0.02
0.03
0.04
60.193
81.624
117.136
139.836
264/284
FI = 16.189
Curve:e
0.01
5.0
10
0.05
0.1
0.2
197
220
400
550
337/662
FI = 180
Curve:e
0.5
0.05
0.1
0.2
311/624
FI = 275
Curve:e
No interaction
1.0
3. Materials and Methods
3.1. General Consideration
All reactions were carried out under aerobic atmosphere and no special precautions
were taken to exclude air or moisture during experiments. All chemicals were purchased
from Sigma Aldrich, TCI or Across Organics and were used without further purification.
Thiourea derivatives described in this study are also commercially available in the marked,
we reproduced them in the laboratory, they were obtained as crystalline material. The
formation of compounds was confirmed with the help of available spectroscopic techniques,
such as ATR-FTIR spectrometer and 1H- and 13C NMR spectra (in order to assist readers
these data is provided in supplementary file). Single crystal X-ray analysis was carried out
at room temperature by using a Bruker Kappa APEXII CCD diffractometer. Absorption of
solution for determination of enzyme inhibition was measured by Thermoelectron corporation,
USA and florescence studies were carried out using fluorescence spectrophotometer model RF-
5301 Schimadzu, Japan, for their potential use in sensing environmental pollutants. Thiourea
derivatives were prepared by reactions as previously reported [44,48].
3.2. Anticholinesterase Evaluation
Anticholinesterase activity of compounds
two enzymes, AChE and BChE and following our previously reported protocol without
further modifications [49 51]. The enzyme AChE is derived from Electric eel and BChE
from equine serum [52]. Buffer of phosphate, 0.1 M at pH 8.0 was prepared to whom varied
concentration (31.625–1000 g/mL) of the respective compound was added. The solution
1–6 was evaluated by Ellman’s assay using
–
µ
of AChE was diluted to 0.03 U/mL from 518 U/mg, similarly BChE from 7–16 U/mg to
0.01 U/mL at the given pH condition. Distilled water was used as solvent, for preparation
of 0.5 mM acetylthiocholine iodide (ATchI), 0.2273 mM 5,50-dithiobis-2-nitrobenzoic acid
◦
(DTNB) and 0.5 mM butyrylthiocholine iodide (BTchI). All solutions were stored at 8 C in
a refrigerator prior to performing the experiment.
Enzyme solution (5
µ
L), corresponding compound (205
µ
L), and DTNB reagent (5
µ
L)
◦
were mixed in a cuvette. The solution was incubated for 15 min at 30 C followed by
addition of 5 µL of the substrate solution. Absorbance of the incubated sample was

Molecules 2021, 26, 4506
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measured at 412 nm by a double-beam spectrophotometer (Thermo Electron Corporation,
USA) for 4 min along with the reaction time at 30 ◦C. The well-established Galanthamine
was used as positive control and all experiments were performed in triplicate. The percent
enzyme activity and enzyme inhibition by control and test samples were calculated from the
rate of absorption with respect to change in time (V =
as reported previously [49–51].
∆Abs/∆t) using the same procedure
3.3. Molecular Docking Study
Molecular docking was carried out to determine the binding interaction mode of
the synthesized compounds for the competitive and noncompetitive inhibition with the
mark enzymes, AChE and BChE [53,54]. The crystal structure of the said enzymes was
obtained from protein databank. Proteins were theoretically prepared by using auto dock
mgl tools [55]. and was optimized by removing all water molecules, heteroatoms and
co-factors, for simplicity in calculations. The structures of the synthesized compound
3 and
4
were obtain from CIFs using Avogadro and respective CCDC software. Protein-ligands
images were developed by using pymol [56,57].
3.4. Fluorescence Measurements
Spectroflourophotometer (RF-5301 PC, Shimadzu, Japan) having 150 W Xenon lamp
as excitation source with 1.0 cm quartz cell was used throughout the experimental work
for fluorescence measurements. The emission and excitation slit width was 4 nm for
fluorometric operation. The thiourea derivatives (1–3, 5, 6) of variable concentration
were prepared in acetone and incubated for 60 min at room temperature. All the tested
thiourea derivatives, were found to show excellent fluorescence behavior at low as well as
at high sensitivity levels. These compounds were employed to fabricate a sensing probe
for determination of mercury in water samples.
4. Conclusions
Thiourea derivatives
compounds and was determined with the help of X-ray diffraction for single crystal.
All compounds were tested for their enzyme inhibitory efficiency where compounds
was found more efficient. The activity of this compound (IC50 50 and 60 g/mL against
1–6 were reproduced in the laboratory and structure of two
3
4
3
µ
AChE and BChE, respectively) is very close to the efficiency of the standard drug and is
worth studying in future investigations. The experimental data particularly inhibitory
efficiency of compound 3, were validated through its docking studies and both data sets
were found in close agreement with each other. Further investigations to know their
sensing capacity against heavy metals ions, such as Hg(II) were carried out and the results
are promising, compound
3 came out to be among other compounds with better sensitivity
level. This study reveals that thiourea derivatives possess manifold useful characteristics
and may be able to control AD by inhibiting AChE, BChE or controlling the concentration
of heavy metal ion. These compounds are better sensors to determine specific metals in
real biological samples.
Supplementary Materials: Figure S1: UV absorption spectra of compounds
UV-Visible spectroscopic data of compounds and and experimental procedure for preparation
of compounds, their FTIR and NMR characterization.
1–3, 5 and 6; Table S1:
1
–3,
5
6
Author Contributions: Experimental work, data interpretation, manuscript writing (F.U.R.), Exper-
imental work, data interpretation of compound 4 (M.B.), funds managements, conceptualization,
data interpretation, manuscript writing, supervision (E.K.), NMR data collection (A.B.S.), experi-
mental work, enzyme inhibition (F.U.), sensing applications of compounds and manuscript writing
(M.M.), data collection, data interpretation, X-ray structure (M.N.T.), theoretical calculations and
manuscript writing (A.S.), manuscript writing, manuscript revision (M.Z.), funds managements,
conceptualization, data interpretation, manuscript writing, manuscript revision (S.A.), data interpre-
tation, manuscript writing, manuscript revision (G.E.-S.B.). All authors have read and agreed to the
published version of the manuscript.

Molecules 2021, 26, 4506
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Funding: This research was partially funded by King Saud University, RSP 2021/241.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article and its supplementary materials.
Acknowledgments: The authors would like to extend their gratitude to the King Saud University
(Riyadh, Saudi Arabia) for the funding of this research through Researchers Supporting Project
number (RSP-2020/241). We are highly indebted to anonymous reviewers for their professional
remarks and efforts in improving the manuscript.
Conflicts of Interest: The authors have no potential conflict of interest to declare.
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