Investigation of potent inhibitors of cholinesterase based on thiourea and pyrazoline derivatives: Synthesis, inhibition assay and molecular modeling studies

Article abstract of DOI:10.1016/j.bioorg.2019.103036

Owing to the desperate need of new drugs development to treat Alzheimer's ailment the synthesis of 1-aroyl-3-(5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2–6) starting from (4-amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol) (1) and synt

Full text of DOI:10.1016/j.bioorg.2019.103036

Accepted Manuscript
Investigation of potent inhibitors of cholinesterase based on thiourea and pyra-
zoline derivatives: Synthesis, inhibition assay and molecular modeling studies
Amara Mumtaz, Abdul Majeed, Sumera Zaib, Shafiq Ur Rahman, Saba
Hameed, Aamer Saeed, Hummera Rafique, Ehsanullah Mughal, Aneela Maalik,
Izhar Hussain, Jamshed Iqbal
PII:
S0045-2068(19)30473-0
https://doi.org/10.1016/j.bioorg.2019.103036
103036
DOI:
Article Number:
Reference:
YBIOO 103036
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 March 2019
27 May 2019
3 June 2019
Please cite this article as: A. Mumtaz, A. Majeed, S. Zaib, S. Ur Rahman, S. Hameed, A. Saeed, H. Rafique, E.
Mughal, A. Maalik, I. Hussain, J. Iqbal, Investigation of potent inhibitors of cholinesterase based on thiourea and
pyrazoline derivatives: Synthesis, inhibition assay and molecular modeling studies, Bioorganic Chemistry (2019),
doi: https://doi.org/10.1016/j.bioorg.2019.103036
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Investigation of potent inhibitors of cholinesterase based on thiourea and
pyrazoline derivatives: Synthesis, inhibition assay and molecular modeling
studies
Amara Mumtaz1#*, Abdul Majeed1#, Sumera Zaib2, Shafiq Ur Rahman2, Saba Hameed1, Aamer
Saeed3, Hummera Rafique4, Ehsanullah Mughal4, Aneela Maalik1, Izhar Hussain5, Jamshed
Iqbal2,5*
1Department of Chemistry; 2Centre for Advanced Drug Research, COMSATS University Islamabad,
Abbottabad Campus, Abbottabad-22060, Pakistan.
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Department of Chemistry, University of Gujrat, Gujrat, Pakistan.
5Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-22060,
Pakistan.
*Corresponding author: Dr. Amara Mumtaz, Department of Chemistry, COMSATS University
Islamabad, Abbottabad Campus Ph No. +923005316570, email: amaramumtaz@ciit.net.pk. Prof.
Dr. Jamshed Iqbal, Centre for Advanced Drug Research, COMSATS University Islamabad,
Abbottabad Campus, Abbottabad-22060, Pakistan, Ph No. +923365101651, Email:
drjamshed@ciit.net.pk; jamshediqb@gmail.com. #equal contribution: Dr Amara Mumtaz, Abdul
Majeed, Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus Ph
No. +923319590957, email: majeed9211@gmail.com
1

Investigation of potent inhibitors of cholinesterase based on thiourea and pyrazoline
derivatives: Synthesis, inhibition assay and molecular modeling studies
Amara Mumtaz1#*, Abdul Majeed1#, Sumera Zaib2, Shafiq Ur Rahman2, Saba Hameed1, Aamer
Saeed3, Hummera Rafique4, Ehsanullah Mughal4, Aneela Maalik1, Izhar Hussain5, Jamshed
Iqbal2,5*
1Department of Chemistry; 2Centre for Advanced Drug Research, COMSATS University Islamabad,
Abbottabad Campus, Abbottabad-22060, Pakistan.
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Department of Chemistry, University of Gujrat, Gujrat, Pakistan.
5Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad-22060,
Pakistan.
Abstract
Owing to the desperate need of new drugs development to treat Alzheimer's ailment the
synthesis of 1-aroyl-3-(5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2-6) starting
from (4-amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol) (1) and synthesis of 1-(3-(4-
aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (7-9)
starting from 2-(4-isobutylphenyl)propanehydrazide (a) with the cyclization with substituted
chalcones (c-e) was carried out. To check the biological potential of the synthesized compounds,
all were subjected to acetylcholinesterase (AChE) and butrylcholinesterase (BChE) inhibition
assays. The most potent and selective inhibitor for the acetylcholinesterase was compound 7
having an inhibitory concentration of 123 ± 51 nM, whereas, compound 6 was found as selective
inhibitor of butyrylcholinesterase (BChE) with an IC50 value of 201 ± 80 nM. However, the
compounds 1 and 2 were found as dual inhibitors i.e. active against both acetylcholinesterase as
well as butyrylcholinesterase.
Keywords: Triazole, Pyrazolines, Ibuprofen, Chalcones, Alzheimer disease, Enzyme inhibition
2

Graphical Abstract
The overlap of all the docked inhibitors (1 (brown), 2 (red), 5 (pink) and 7 (yellow)) inside the active site
of acetylcholinesterase
Introduction
Despite the way that it has been more than a century since the dynamic, neurodegenerative
turmoil known as Alzheimer's ailment (AD) was first officially portrayed in the scientific
writing; still there is much more we need to know about this overwhelming malady [1]. The
actual reason and succession of occasions prompting sickness and development has escaped
scientists regardless of the recognizable proof of various elements which are reason of AD [2-4].
Current pharmacological medicines for AD may offer some help for the outstanding
psychological indications. Over a century has gone since the first report of Alzheimer, and the
infection has developed up to great extent [5]. Alone in the United States that it was consider that
AD is assessed to influence an aggregate of 5.2 million Americans among them 96% are age 65
and more. Globally, it is evaluated that more than 24 million individuals are experiencing AD
[6]. According to world health organization WHO, AD is sixth driving reason for death over all
ages in the United States. With the time this number is increasing [7]. Generally, AD is supposed
to be the reason for neurodegeneration and neuronal decay in the brain. Several neurotic signs of
AD have been distinguished and observation of these characteristics has prompted a few theories
3

in endeavors to clarify the basic reason for this disease. In any case, the correct reason and time
allotment of occasions prompting AD stays unclear, and it is likely multifactorial and includes an
unpredictable cluster of components [8-16].
Acetylcholine (ACh) is a neurotransmitter that under normal and neurodegenerative conditions
helps in the modulation of function of memory while cholinesterases (ChEs) is a class of
enzymes that play a significant role in the cholinergic signaling and regulation of Ach. [17-18]
AChE and BChE simultaneously play a vital role in the regulation of ACH level, its synaptic
hydrolysis and in terminating its neurotransmitter action. In order to re-establish the
neurotransmitter level is done by using inhibitors of cholinesterase that function by suppressing
the ChE enzymes which results in both the level increase and duration of the neurotransmitter
action [19]. The ChE inhibitors (ChEIs) are the first generation of drugs for the treatment of
Alzheimer’s disease, glaucoma and myasthenia gravis and being used for the treatment of many
other neuromuscular disorders. It has a limitation, with the increase in the level of Ach,
symptoms of these diseases increase. Thus, search for new ChEIs is highly appreciated for
further advancement in the treatment of such diseases [5, 20].
In our efforts to contribute to the development of novel compounds that may be useful in the
treatment of neurodegenerative disorders such as Parkinson’s disease (PD) or Alzheimer’s
disease (AD), we are focusing on the synthesis of derivatives of thioureas and pyrazolines.
Thioureas derivatives are famous for their broad spectrum of activities which include antifungal,
antibacterial, anticancerous, insecticidal activities [21-22]. Coumarin substituted thioureas [23]
and 1-butanoyl-3-arylthiourea [24] showed moderate to excellent activity as cholinesterase
inhibitor. On the other hand, derivatives of pyrazolines are famous for their analgesic, anti-
inflammatory activities [25]. Pyrazoline substituted sulfonamide derivatives showed inhibition
against AChE at the nanomolar concentrations [26]. 1-N-substituted thiocarbamoyl-3-phenyl-5-
thienyl-2-pyrazoline derivatives were synthesized by Gulberk et al and on exposure to
acetylcholinesterase and butrylcholinesterase enzymes they were found very active [27].
Similarly, indolopyrazoline derivatives were synthesized by Sridevi et al and found excellent
inhibitors of acetylcholinesterase [28]. Keeping in mind the inhibitory activities of thioureas and
pyrazolines as acetylcholinesterase inhibitors, we are focusing on the synthesis of ibuprofen
4

substituted pyrazolines and thioureas of 5(4-chlorophenyl)-1,2,4-triazole-3-thioneamine as new
compounds with potential activity as cholinesterase inhibitors.
Results & Discussion
Chemistry
Synthesis of 1-aroyl-3-5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2-6) and 1-(3-
(4-aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (7-
9) were carried out staring from their common precursor hydrazide. For the synthesis of 1-aroyl-
3-5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2-6) reaction of 1 [29] with
substituted benzoylisothiocyanate prepared in situ was carried out to get 2-6 (Scheme 1).
O
N
N
NH2
R"
7-9
O
Cl
R"
H2N
N
S
c-e
NH
N
S
O
NH2
N
HN
HN
O
Cl
1. ROH/H
S
1.CS2,KOH
2.NH2NH2
R
OH
R
NH.NH2
benzoylisothiocyanate
acetone
2. NH2NH2
MeOH
HN
N
O
a-b
R'
1
R:
2-6
a.
R'=
R"=
2. 2-methoxy
3. 4-Cl
7. 2,4-dimethoxy
8. 3-hydroxy-4-methoxy
4. 2,4-dimethoxy
5. 4-pyridiyl
9. 4-hydroxy-3,5-dimethoxy
b.
6. 3,4-dimethoxy
Cl
Scheme 1: Synthesis of thiourea (2-6) and pyrazoline (7-9) derivatives
In the FT-IR data of the compound (2-6) structures were confirmed by the appearance of a
stretching band at 2923-3001cm-1 indicating the presence of C-H group, the stretching bands at
1578-1673, 1584-1600 cm-1 showed the presence of the aromatic rings while the stretching band
at 3409-3489 cm-1 showed the presence of N-H groups. In 1H NMR data of the compound (2-6)
presence of two singlets at 10.32-10.75 ppm and 9.98-10.19 ppm confirmed the presence of two
5

NH groups which are the characteristic signal and confirm the structure of thiourea group. In 13C
NMR peaks at 165.0-195.0 ppm and signal at 147.1-164.7 ppm confirmed the presence of C=O
and C=S groups respectively. [30]
Synthesis
of
1-(3-(4-aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-
isobutylphenyl)propan-1-one (7-9) was carried out by condensation of ibuprufen hydrazide (a)
with substituted chalcones (b-d) [31] (Scheme 1). In the FT-IR data of the compound (7-9) a
stretching band at 2955-3110 cm-1 confirmed the presence of C-H group, the stretching bands at
1428-1442, 1551cm-1 for the aromatic rings while the stretching band at 3288 cm-1 showed the
presence of C-N groups and stretching at 698-1711 cm-1 confirmed the presence of C-O bond. In
1H NMR data of the compound (7-9) presence of chiral center of pyrazoline ring and NH2 groups
were confirmed by the presence of doublet of doublet at a range of 3.87-3.96 ppm and a singlet
at 2.73-4.23 ppm respectively. Similarly, in 13C NMR peaks 173.3-194.8 ppm confirmed the
presence of carbonyl carbon. [31]
Table 1: Physical data of compounds (2-9)
R a
Sr. No.
Yield (%)
M.P (℃)
f
1
2
3
4
5
6
7
8
9
198-200
190-192
204-206
220-222
201-203
207-209
203-205
227-229
Semisolid
0.2
0.6
0.5
0.4
0.5
0.4
0.5
0.4
0.5
81
83
72
78
83
82
77
76
80
Biological Assay
Cholinesterase inhibitory activity
6

The thiourea moiety with different derivatives like butanoyl and acetyl arylthioureas [32-33],
were screened against cholinesterases. Moreover, the similar studies were carried out against
coumarin linked thioureas [34] and halogenated thioureas [35] and results suggested that
synthesized analogues were potent inhibitors of cholinesterases. Therefore, 1,3,4-triazole-2-
thioneaminylthioureas were designed and synthesized (1-8) and tested for their potential
inhibition against two isozymes of cholinesterase family, acetylcholinesterase and
butyrylcholinesterase. Dual inhibitors of both the isozymes were also identified among screened
compounds. The key objective of the study was to explore the most potent and selective
inhibitors of acetylcholinesterase and butyrylcholinesterase. The inhibition data obtained from
the enzymatic analysis was presented in Table 2. The donepezil and galanthamine were taken as
reference standards during the assay. The inhibitory concentration obtained for the donepezil was
32.1 ± 3.1 nM for the acetylcholinesterase and 6414 ± 342 nM for butyrylcholinesterase,
whereas, IC50 value of galanthamine against acetylcholinesterase was 622 ± 11 nM and that of
butyrylcholinesterase was found as 878 ± 37 nM.
Structure-Activity Relationship
The most potent and selective inhibitor for the acetylcholinesterase was 7 having an inhibitory
concentration of 123 ± 51 nM, however, compound 6 was found as selective inhibitor of
butyrylcholinesterase with an IC50 value of 201 ± 80 nM. However, the compound 1 and 2 were
chosen as dual inhibitors of acetylcholinesterase as well as butyrylcholinesterase. These
compounds exhibited significant inhibition against acetylcholinesterase along with good
inhibitory activity against butyrylcholinesterase and our findings were like previously reported
data [34]. Figure 1 and 2 represented the detail structure activity relationship of active scaffolds
and substituents attached. From the Table 2, it was noticed that compounds 4 and 8 showed no
significant inhibition against any of cholinesterase. From the substituted pyrazolines, the
compound 8, 1-(3-(4-aminophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-
(4-isobutylphenyl)propan-1-one showed very less inhibition towards acetyl and
bytyrylcholinesterase. Similar pattern was observed by compound 4, N-((3-(4-chlorophenyl)-5-
mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)-2,4-dimethoxybenzamide, which belongs to 5-4-
chlorphenyl-1,2,4-triazole-2-thione amine substituted thioureas. However, rest of the derivatives
were found as either selective inhibitors of acetylcholinesterase/ butyrylcholinesterase or have
7

shown dual inhibition towards both the enzymes. The Figure 1 showed the detail structure
activity relationship of 5-(4-chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione amine, 5-4-
chlorphenyl-1,3,4-triazole-2-thione amine substituted thioureas, in which 1, 2 and 5 were dual
inhibitors, however, the selective inhibition towards the butyrylcholinesterase was exhibited by
6. Figure 2 presented the substituted pyrazolines derivatives and their inhibition potential
towards both the enzymes.
SH
SH
N
N
N
N
S
O
OCH3
S
O
N
N
N
H
N
N
H
N
H
H
N
OCH3
Cl
Cl
5
4
AChE (IC50 ± SEM) = 513 ± 48 nM
BChE (IC50 ± SEM) = 255 ± 19 nM
AChE = 24.3%
BChE = 23.6%
N
SH
N
N
NH2
Cl
1
AChE (IC50 ± SEM) = 139 ± 12 nM
BChE (IC50 ± SEM) = 377 ± 21 nM
SH
SH
N
N
N
N
S
O
OCH3
S
O
N
OCH3
OCH3
N
N
H
N
N
H
N
H
H
Cl
Cl
2
6
AChE (IC50 ± SEM) = 375 ± 55 nM
BChE (IC50 ± SEM) = 148 ± 33 nM
AChE = 27.1%
BChE (IC50 ± SEM) = 201 ± 80 nM
8

Figure 1. Structure activity relationship of synthetic compounds
The results depicted that the parent compound of thioureas, 1 (4-amino-5-(4-chlorophenyl)-4H-
1,2,4-triazole-3-thiol) [29] exhibited dual inhibition towards AChE and BChE with an IC50
values of 139 ± 12 and 377 ± 21 nM, respectively. Upon addition of substituent on the main
compound, different patterns were observed in the inhibition. The addition of 2-methoxy
benzamide group exhibited similar pattern like parent compound and showed inhibition for both
the enzymes. However, upon substitution of similar group at position 3 and 4 give rise to a
selective and potent inhibitor of butyrylcholinesterase. But when the substitution of same group
(methoxy) at position 2 and 4 was noticed, the compound has not shown any inhibition towards
acetylcholinesterase and butyrylcholinesterase. Therefore, it maybe suggested that the
attachment of methoxy group at position 3 was the contributing factor for inhibition. The only
compound with different substituent in thioureas was 5, having isonicotinamide group attached
to the parent ring. The substituent gave rise to a dual inhibitor and showed inhibitory activity of
513 ± 48 and 255 ± 19 nM, for acetylcholinesterase and butyrylcholinesterase, respectively.
NH2
NH2
N
N
N
N
O
O
OCH3
H3CO
OH
H3CO
7
8
AChE = 17.9%
BChE = 12.2%
AChE (IC50 ± SEM) = 123 ± 51 nM
BChE = 26.4%
Figure 2. Structure activity relationship of synthetic compounds
When the structures of substituted pyrazolines, (7 and 8) were investigated, it was observed that
3-hydroxy-4-methoxyphenyl group in compound 7 was the important factor towards the
inhibition profile of the compound. The compound was noted as potent and selective inhibitor of
AChE. The compound 8, having 2 and 4-methoxy phenyl was found as inactive and showed less
than 50% inhibition towards acetylcholinesterase and butyrylcholinesterase. The results showed
9

that the attachment of different substituents at ortho, para and meta position at the main moiety
has a significant impact and contribution towards inhibitory profile of acetylcholinesterase and
butyrylcholinestase. The red and yellow colors in Figure 2 are the positions at which the groups
vary, and these groups were found to be responsible for the inhibitory activity of the synthesized
derivatives.
Table 2. Cholinesterase inhibition efficacy and anti-proliferative activity of synthetic derivatives
Code
AChE
BChE
Anti-proliferative activity (HeLa)
IC50 (nM) ± SEM/% inhibition
1
139 ± 12
375 ± 55
26.2%
377 ± 21
148 ± 33
5.8%
62.5%
2
36.8%
32.2%
28.5%
19.7%
21.1%
21.2%
18.6%
-
3
4
24.3%
23.6%
5
513 ± 48
27.1%
255 ± 19
201 ± 80
26.4%
6
7
123 ± 51
17.9%
8
12.2%
Donepezil
Galanthamine
Cisplatin
32.1 ± 3.1
622 ± 11
-
6414 ± 342
878 ± 37
-
-
89.3%
Molecular Docking Analysis
To justify the experimental results, the docking analysis was carried out against the potent and
selective inhibitors of acetylcholinesterase as well as butyrylcholinesterase. The in vitro results
clarify that compound 7 was potent and selective inhibitor of acetylcholinesterase, whereas, 6
was selective as well as potent inhibitor of butyrylcholinesterase. However, some dual inhibitors
were found and docking analysis was carried out against those dual inhibitors, 1 and 2 in the
active site of both the receptors. To investigate the binding poses of selected compounds, the
protein structures were selected from protein databank. In case of acetylcholinesterase, the
crystal structure of Tetronarce californica was selected and downloaded [36] because electric eel
10

structures for AChE are only available at low crystallographic resolutions (>4 Å) and the
vesicular acetylcholine transporter of electric ray (Torpedo californica) is structurally related to
the vesicular monoamine transporters of mammals, therefore structure of electric ray was chosen.
For butyrylcholinesterase, the x-ray structure of human butyrylcholinesterase was selected and
downloaded [37] because structures of equine BChE are currently not available. Figure 3 (a, b)
depicts the overlap of all the selected compounds docked inside the active site of AChE and
BChE, respectively.
(b)
(a)
Figure 3. The overlap of all the docked inhibitors (7 (yellow), 2 (red), 5 (pink) and 1 (brown)) inside
the active site of acetylcholinesterase (a) and inhibitors (6 (orange), 2 (purple), 5 (pink) and 1 (green))
inside the butyrylcholinesterase (b)
To validate the docking studies, the co-crystallized ligands of acetylcholinesterase and
butyrylcholinesterase were docked inside the active site after extraction from the respective
receptor. The re-docking of co-crystallized ligands (E2020 for 1EVE and THA for 4BDS) were
carried out by root mean square deviation of 1.32 and 1.04 Å for acetylcholinesterase and
butyrylcholinesterase, respectively. The active pockets were selected after analysis of binding
interactions of co-crystallized ligands within the active site. The 3D interaction diagrams of co-
crystallized ligand, selective inhibitor and dual inhibitors of acetylcholinesterase were shown in
Figure 4. While, the 3D interaction diagrams of co-crystallized ligand, selective inhibitor and
dual inhibitors of butyrylcholinesterase was shown in Figure 5.
11

The detailed analysis of acetylcholinesterase co-crystallized ligand and the inhibitors showed
that Tyr334, Trp114 and Trp84 are the most important residues showing interaction with the
inhibitors within the active site. The cognate ligand E20, 1-benzyl-4-[(5,6-dimethyoxy-1-
indanon-2-yl)methyl]piperidine presented the same interactions and our selective inhibitor, 7 was
making π-π interactions with Tyr84. In addition to π-π interactions, the hydrogen bonds were
noticed between active site amino acid residues and compound. The dual inhibitors, 2 and 1 were
making the hydrogen bonds and π-π interactions with important residues. The interactions
analysis suggested that the selected compounds showed best fit interaction inside the pocket of
acetylcholinesterase. However, the bond between the selective inhibitor of acetylcholinesterase,
compound 7 was noticed as hydrogen bonding in addition to π-π interactions with Phe330.
Tyr334 showed similar π-π interactions with isobutylphenyl group of the compound 7.
Moreover, His440 was hydrogen bonded with methoxy group. Trp279 was showing pi-alkyl
linkage with isobutyl moiety. The bond distance showed by Trp84 with E2020 was 2.78 Å and
that of our compound 7 was 2.93 Å. Similarly, the dual inhibitors showed the interactions with
active site residues. In the compound 2 having 2-methoxy group, the phenyl ring was showing π-
π interactions with amino acid Phe330, His440, Phe331 and 5-mercapto group was showing π-π
interactions. Similar interactions were noticed, when compound 1 was considered. The residue
Trp84 showed hydrogen bonds along with π-π interactions with the 4-chlorophenyl part of the
compound. The selected compounds showed the binding interactions necessary for the inhibition
of acetylcholinesterase within the active site.
12

(a)
(b)
(c)
(d)
Figure 4. 3D interaction diagrams of (a) cognate ligand (E2020) (b) selective inhibitor 7, of AChE along
with dual inhibitors (c) compound 2 and (d) compound 1 inside the 1EVE
When butyrylcholinesterase active site residues and interaction analysis of selected compounds
were investigated, it was noticed that the compounds exhibited most of the interactions with
Trp82, along with His438 and Tyr332 and are responsible for the inhibitory behavior towards
butyrylcholinesterase that were coherent with previous studies [34]. However, the bond distance
of some of the important residues and their type of interactions are explained here. The co-
crystallized ligand, tacrine, showed the hydrogen bonding with Trp82 at the bond distance of
2.98 Å. The tacrine showed π-π interactions with Trp82 and hydrogen bond with Ala328.
However, the selective inhibitor of butyrylcholinesterase, compound 6 showed the same
13

interaction with Trp82 at bond distance of 3.14 Å. The dual inhibitors, 2 and 1 exhibited 3.64
and 3.99 Å, respectively with Trp82. In addition to π-π interactions, hydrogen bonding was
noticed, and the residue involved in the formation of hydrogen bond with docked compounds
were Phe398, Trp231, Gly116, Ala328, Leu286, Val288 and Tyr332. All the compounds showed
hydrogen bonds. The formation of strong hydrogen bonding suggested that these contribute well
towards the inhibition profile and our selected compounds showed these bonding. The
interaction analysis suggested that the compounds were found in best fitted poses inside the
active site of butyrylcholinesterase and therefore, the investigated interactions are responsible for
the inhibition profile of the compounds.
(a)
(b)
14

(c)
(d)
Figure 5. 3D interaction diagrams of (a) cognate ligand (THA) (b) selective inhibitor compound 6, of
BChE along with dual inhibitors (c) compound 2 and (d) compound 1 inside the 4BDS
The physicochemical properties of synthesized derivatives
In silico physicochemical properties and drug-likeness of the screened derivatives were
evaluated by MedChem Designer 3.0 [38]. Different characteristics are responsible for
categorizing the synthetic compound into either a drug or a chemical. The parameter used to
check out the drug-likeness was Lipinski’s rule of 5, according to it, Mol. Wt. must be less than
499 Dalton, calculated octanol/water partition coefficient not more than 5 (lipophilicity), the no.
of H-bond donors must be less than 5 and the no. of H-bond acceptors can be equal of less than
10 [39]. The literature reports that drugs for central nervous system must have lower level of
lipophilicity and Mol. Wt. should be lower than 500. Moreover, low hydrogen bond and little
number of rotatable bonds are also required for a compound to act as a drug [40]. Therefore, we
evaluated the compounds for these parameters and evaluated the distribution of octanol-water
and partition coefficients (S + log D), coefficient of octanol-water partition (S + log P), and
number of hydrogen bond donors (HBD), hydrogen bond acceptor (MNO) and topological polar
surface area (TPSA). Any drug with the polar surface area of 60-140 Å2, is supposed to be
sufficiently bioavailable [41]. Octanol-water partition helps to estimate the level of
hydrophobicity and hydrophilicity of molecule. The synthetic derivatives showed significant
contribution towards the physicochemical parameters. All the compounds exhibited less than 100
15

Å2. Therefore, it may be suggested that all the derivatives can be penetrated easily if used as
drugs. The compounds with Mol. Wt. less than 500, M log P value lesser than 5 are considered
as orally bioavailable with favorable absorption, distribution, metabolism and excretion (ADME)
profile [42-43]. The derivatives have Mol. Wt. less than 500 and bear lesser than 10 rotatable
bonds. The physicochemical parameters of the synthetic derivatives are given in Table 3.
Table 3. Physicochemical parameters of synthetic compounds
Codes
1
M log P S + log P S + log D Mol. Wt. MNO TPSA HBDH
2.389
1.997
1.348
226.688
4
56.730
2
2
4
3.670
3.154
3.216
3.221
1.803
1.887
419.914
449.940
7
8
81.070
90.300
2
2
5
6
7
8
2.678
2.643
4.854
4.533
2.662
3.329
5.831
6.004
1.223
1.943
5.828
6.004
390.875
449.940
485.630
499.657
7
8
6
6
84.730
90.300
88.150
77.150
2
2
3
2
In vitro anti-proliferative activity against HeLa cervical carcinoma cells
The anti-proliferative activity of (1-8) against cervical carcinoma cells (HeLa) was tested by
MMT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay [44-45]. The %age
inhibition of the synthetic derivatives was assessed at 100 µM (Table 2). The positive standard
used was cisplatin and it exhibited 89.3% inhibition against cells at same concentration. The
screened compounds presented lower cytotoxicity than that of cisplatin and these derivatives
revealed lower anti-proliferative activity as compared to cisplatin towards the cervical HeLa
cells. The compounds exhibited %age inhibition against cancer cells in the range of 18.6 –
62.5%. The compound 1, (4-amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol) was found to
have more anti-proliferative activity than other derivatives with the percentage inhibition of
62.5%. All the other compounds presented less than 50% inhibition towards HeLa cell lines.
16

Materials & Methods:
Capillary tubes were used for recording melting point by using electrothermal melting point
apparatus, model SMP 10 digital melting point apparatus OE/Digital (08-09) 169/1 DMPA 09-
01. 1H NMR was recorded on 300 MHz spectrophotometer by using deuterated DMSO as
solvent, while the tetramethylsilane (TMS) was used as internal reference. The chemicals and
reagents used in the study were of analytical grade and purchased from Merck (Germany) and
Sigma Aldrich (USA).
Experimental:
General Procedure for the synthesis of 5-(4-chlorphenyl)-1,2,4-triazole-3-thione amine
substituted thioureas (2-6)
For the synthesis of thiourea (2-6), acid chloride of substituted acid was prepared by refluxing
the substituted benzoic acid with thionyl chloride (1.5 eq) for 2 hrs. In another two neck round
bottom flask potassium thiocyanate (KSCN) (1 eq) was suspended in 5 mL of acetone. Acid
chloride was added in it and stirred for 35 min. After adding triazole amine (1) [29] dissolved in
5 mL of acetone (dry) into reaction mixture and refluxed for one hour. Upon completion, the
reaction mixture was poured in ice cold water; obtained solid precipitates were passed through
filter paper, washed and purified by using suitable solvent [30].
N-((3-(4-chlorophenyl)-5-mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)-2-methoxybenzamide
(2)
M.P (℃): 190-192; Color: yellow; yield (%): 83; Rf (n-Hexane: Ethyl acetate, 7:3): 0.6; FT-IR
(ύ, cm-1): 3489 (NH), 2923 (C-H), 1564, 1496 (C-H arom), 1423 (C-H), 1145 (C-O),744 cm-1;
1H NMR (300 MHz, DMSO-d6)δ: 10.75 (s, 1H, NH), 10.08 (s, 1H, NH), 7.95 (d, J = 8.4 Hz,
2HAr), 7.76 (dd, J = 7.6 Hz, 1HAr), 7.60 (d, J = 8.7 Hz, 2HAr), 7.54 (td, J = 3.5 Hz, 1HAr), 7.53
13
(td, J = 3.0 Hz, 1HAr), 7.19 (d, J = 7.6 Hz, 1HAr), 3.91 (s, 3H, OCH3 ), C NMR (75 MHz,
DMSO-d6) 165.0, 164.7, 157.5, 137.1, 133.2, 131.7, 130.9, 129.9, 129.8, 122.1, 120.0, 112.5,
56.3.
17

4-Chloro-N-((3-(4-chlorophenyl)-5-mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)benzamide
(3)
M.P (℃): 204-206; Color: yellow; yield (%): 72; Rf (n-Hexane: Ethyl acetate, 7:3): 0.5; FTIR (ύ,
cm-1): 3201, 3167 (=C-H), 3001 (C-H), 1733 (C=O), 1707, 1689(C=O), 1649, 1592 (C-H arom),
1
1500 (C=C), 1496 (C-H arom), 1101 ( C-O) cm-1; H NMR (300 MHz, DMSO-d6) δ: 10.73 (s,
1H, NH), 9.98 (s, 1H, NH), 8.16(d, J = 8.7 Hz, 2HAr), 7.95 (d , J = 8.4 Hz, 2HAr), 7.72 (d, J = 8.7
13
Hz, 2HAr), 7.58 (d, J = 7.6 Hz, 2HAr); C NMR (75 MHz, DMSO-d6) δ: 166.9, 161.6, 140.6,
138.2, 132.7, 131.6, 130.0, 129.9, 129.8, 129.2, 128.7, 127.3.
N-((3-(4-chlorophenyl)-5-mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)-2,4-
dimethoxybenzamide (4)
M.P (℃): 220-222; Color: yellow; yield (%): 78; Rf (n-Hexane: Ethyl acetate, 7:3): 0.4; FTIR (ύ,
cm-1): 3167 (=C-H), 3201, 3001 (C-H), 1707, 1689(C=O), 1649, 1592 (C-H arom), 1500 (C=C),
1
1496 (C-H arom), 1101 ( C-O), cm-1; H NMR (300 MHz, DMSO-d6) δ: 10.76 (s, 1H, NH),
10.03 (s, 1H, NH), 7.95 (d , J = 8.4 Hz, 2HAr), 7.74 (d, J = 7.8, 2HAr), 7.62 (d, J = 7.6 Hz, 1HAr),
7.10 (d, J = 8.7 Hz, 1HAr), 6.92 (s, 1HAr), 3.91 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 13C NMR (75
MHz, DMSO-d6) δ: 195.5, 158.8, 155.0, 147.5, 134.9, 131.2, 125.3, 124.8, 118.6, 116.5, 65.5.
N-((3-(4-chlorophenyl)-5-mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)isonicotinamide (5)
M.P (℃): 201-203; Color: yellow; yield (%): 83; Rf (n-Hexane: Ethyl acetate, 7:3): 0.5; FTIR (ύ,
cm-1): 3201, 3167 (=C-H), 3001 (C-H), 1707, 1649, 1689(C=O), 1592 (C-H)arom, 1500 (C=C),
1496 (C-H)arom, 1101 ( C-O) cm-1; 1H NMR (300 MHz, DMSO-d6) δ: 10.34 (s, 1H, NH), 10.19
(s, 1H, NH), 7.95 (d , J = 8.4 Hz, 2HAr), 7.76 (d, J = 7.6 Hz, 2HAr), 7.60 (d, J = 8.7 Hz, 2HAr),
7.49 (d, J = 7.6 Hz, 2HAr); 13C NMR (75 MHz, DMSO-d6) δ: 185.5, 160.0, 156.8, 137.5, 132.9,
129.2, 127.2, 123.1.
N-((3-(4-chlorophenyl)-5-mercapto-4H-1,2,4-triazol-4-yl)carbamothioyl)-3,4-
dimethoxybenzamide (6)
M.P (℃): 207-209; Color: yellow; yield (%): 82; Rf (n-Hexane: Ethyl acetate, 7:3): 0.4; FTIR (ύ,
cm-1): 3201, 3167 (=C-H), 3001 (C-H), 1698, 1688 (C=O), 1623, 1592 (C-H arom), 1500 (C=C),
1496 (C-H arom),, 1101 (C-O) cm-1; 1H NMR (300 MHz, DMSO-d6) δ: 10.32 (s, 1H, NH), 10.02
18

(s, 1H, NH), 7.89 (d , J = 8.4 Hz, 2HAr), 7.71 (d, J = 7.6 Hz, 1HAr), 7.60 (d, J = 8.7 Hz, 2HAr),
7.49 (d, J = 7.0 Hz, 1HAr), 7.19 (d, J = 2.8 Hz, 1HAr), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3);
13C NMR (75 MHz, DMSO-d6) δ: 197.6,147.7, 147.1, 137.0, 127.6, 125.8, 124.0, 122.3, 119.4,
65.2, 56.5.
General procedure for the synthesis of 1-(3-(4-aminophenyl)-5-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (7-9):
Prepared chalcones [31] were dissolved in ethanol and refluxed for 7 hours with ibuprofen
hydrazide in the presence of piperidine as a catalyst.
1-(3-(4-aminophenyl)-5-(2,4-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-
isobutylphenyl)propan-1-one (7)
M.P (℃): 203-205; Color: brown; yield (%): 77; Rf (n-Hexane: Ethyl acetate, 7:3): 0.5; FTIR (ύ,
cm-1): 3444, 3378, 3110, 1677, 1655, 1595, 1499, 1428, 1163, 1H NMR (300 MHz, DMSO-d6) δ:
7.97 (d, J = 8.1 Hz, 2HAr), 7.80 ( d, J = 7.5 Hz, 2HAr), 7.53 (s, 1HAr), 7.51 (d, J = 8.1Hz, 2HAr),
7.37 (d , J = 7.5 Hz, 2HAr), 7.13 (d, J = 8.4 Hz, 1HAr), 6.95 (d, J = 8.1 Hz, 1HAr), 6.09 (d, J = 4.7
Hz, 2H, CH), 4.01 (s, 2H, NH2), 3.81 (dd, J = 4.7 Hz, 1H, CH), 3.78 (s, 3H, OCH3), 1.84 (d, J =
4.7, 3H, CH3CH), 1.49 (m, J = 5.1, CHCH3), 0.81 (d, J = 6.2, CH3); 13C NMR (75 MHz, DMSO-
d6) δ: 173.3, 171.4, 169.1, 168.4, 140.3, 139.7, 139.2, 129.6, 129.1, 127.5, 127.4, 127.3, 56.2,
44.7, 30.1, 25.6, 24.4, 22.6, 19.9, 18.7.
1-(3-(4-aminophenyl)-5-(3-hydroxy-4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-
isobutylphenyl)propan-1-one (8)
M.P (℃):227-229; Color: white; yield (%): 76; Rf (n-Hexane: Ethyl acetate, 7:3): 0.4; FTIR (ύ,
1
cm-1): 3355, 2995, 1509, 1438, 1155; H NMR (300 MHz, DMSO-d6) δ: 7.97 (d, J = 8.1 Hz,
2HAr), 7.80( d, J = 7.5 Hz, 2HAr), 7.68 (d, J = 7.1Hz, 1HAr), 7.60 (s, 1HAr), 7.59 (d, J = 2.4 Hz,
1HAr), 7.51 (d, J = 8.1 Hz, 2HAr), 7.37 (d, J = 7.5 Hz, 2HAr), 6.61 (d, J = 4.7 Hz, 2H, CH),
4.01(q, 1H, J = 6.7 Hz, CHCH3), 3.96 (dd, J = 4.7 Hz, 1H, CH), 3.78 (s, 3H, OCH3), 2.73 (s, 2H,
NH2), 1.83 (d, J = 5.5, 3H, CH3CH), 1.31 (m, J = 5.6, CHCH3), 0.84 (d, J = 6.1, CH3); 13C NMR
(75 MHz, DMSO-d6) δ: 173.3, 171.4, 169.1, 168.4, 159.9, 159.6, 154.0, 140.3, 144.1, 139.7,
139.2, 136.6, 132.5, 130.1, 129.6, 129.1, 129.0, 127.5, 127.4, 127.3, 126.1, 56.1, 55.8, 46.1, 30.1,
23.6, 22.6.
19

1-(3-(4-aminophenyl)-5-(4-hydroxy-3,5-dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-
isobutylphenyl)propan-1-one (9)
M.P (℃):oil; Color: darkbrown; yield (%): 80; Rf (n-Hexane: Ethyl acetate, 7:3): 0.5; FTIR (ύ,
1
cm-1): 3341, 3288, 3009, 1551, 1465, 1128; H NMR (300 MHz, DMSO-d6) δ: 8.32 (bs, IH,
OH), 7.98 (d, J = 8.3 Hz, 2HAr), 7.84( d, J = 7.7 Hz, 2HAr), 7.57 (d, J = 7.9 Hz, 2HAr), 7.43 (d , J
= 7.4 Hz, 2HAr), 7.11 (s, 2HAr), 6.55 (d, J = 4.9 Hz, 2H, CH), 4.23 (s, 2H, NH2), 4.00 (q, 1H, J =
6.7 Hz, CHCH3), 3.88(s, 6H, OCH3), 1.46 (d, J = 5.3, 3H, CH3), 1.33(m, 3H), 0.81 (d, J = 4.7,
6H, CH3); (75 MHz, DMSO-d6) δ: 195.3, 186.5, 162.9, 159.9, 149.7, 149.2, 139.5, 136.6, 132.5,
131.2, 131.0, 130.2, 129.1, 127.5, 127.4, 127.3, 126.1, 119.7, 116.5, 116,3, 56.1, 55.8, 46.1, 39.3,
27.6, 25.2, 23.4, 22.9, 21.7.
Cholinesterase Inhibition Assay
In vitro inhibition potencies of synthesized derivatives were determined by spectrophotometric
method established by Ellman [46] with little modifications [47]. A mixture consisting of 60 μL
phosphate buffer (KH2PO4/ KOH, having pH 7.7), a 10 μL test compounds (end concentration of
DMSO was 1%) and 10 μL enzyme (0.015 unit/ well for AChE & BChE). The mixture was
o
incubated at 37 C for 10 min for preincubation. After this, 10 μL substrate (acetylthiocholine
chloride or butyrylthiocholine chloride, 1 mM) were added to respective AChE or BChE reaction
mixtures to start the reaction. Later, 10 µL of 0.5 mM DTNB was added as coloring reagent.
After this incubation at 37 oC for 20 min was carried out and later on measurments were taken at
405 nm with plate reader (Bio-Tek ELx 800TM, USA). All the experiments were carried out in
triplicate and the results were calculated as percentage inhibition values. The compounds which
exhibited above 50% inhibition against cholinesterases were further checked by 7-8 dilutions to
get the IC50 values by GraphPad PRISM (USA).
Molecular Docking Studies
The crystal structures of AChE (1EVE) [36] and BChE (4BDS) [37] were taken from Protein
databank. The structures were prepared by MOE builder tool [48]. Afterwards the energy
minimization and protonation of downloaded proteins was carried out by MOE [48]. The
structures of compounds were prepared by MOE builder tool and minimization was done using
20

MMFF94x forcefield [49]. LeadIT (BioSolveIT GmbH, Germany) [50] was used to perform the
docking studies of prepared ligands inside the respective receptors. After docking studies, the
HYDE assessment was used to inspect the possible interactions of ligands with proteins [51].
Visualization was carried out by Discovery Studio Visualizer [52].
Cell Viability Assay
The cytotoxic potential of the test derivatives was investigated in human cervical
adenocarcinoma cells HeLa by MTT based cell viability assay as described earlier [44-45].
Briefly, the cells were cultured (2.5 × 104/mL) in a volume of 90 µL in each well of a 96-well
o
plate and kept in incubator at 37 C in 5% CO2. After an overnight incubation, the dosing of
compounds (100 µM) was done and further incubated for overnight on the same conditions. The
media was poured out and 10 µL MTT reagent was added to well to crystallize the viable cells.
The plate was kept at 37 oC and 5% CO2, for 4 hours followed by the addition of 100 µL of the
reagent (1:1 solution of 50% isopropanol and 10% SDS in solution). The plate was further
incubated for 30 minutes at RT and the OD was observed at 570 nm wavelength using
microplate reader (Bio-Tek ELx 800TM, Winooski, USA). The assay was carried out thrice and
the results were calculated as percentage inhibition.
Acknowledgement
A.M. greatly acknowledge the higher education commission of Pakistan for funding under IPFP
startup research grant and CIIT research grant. J.I. is thankful to the Organization for the
Prohibition of Chemical Weapons (OPCW), The Hague, The Netherlands and the Higher
Education Commission of Pakistan for the financial support through Project No. 20-
3733/NRPU/R&D/14/520.
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24

Investigation of potent inhibitors of cholinesterase based on thiourea and pyrazole
derivatives: Synthesis, inhibition assay and molecular modeling studies
Amara Mumtaz1#*, Abdul Majeed1#, Sumera Zaib2, Shafiq Ur Rahman2, Saba Hameed1, Aamer
Saeed3, Hummera Rafique4, Ehsanullah Mughal4, Aneela Maalik1, Izhar Hussain5, Jamshed
Iqbal2,5*
1Department of Chemistry; 2Centre for Advanced Drug Research, COMSATS University Islamabad,
Abbottabad Campus, 22060, Pakistan.
3Department of Chemistry, Quaid-i-Azam University Islamabad, Pakistan.
4Department of Chemistry, University of Gujrat, Gujrat, Pakistan.
5Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, 22060, Pakistan.
Graphical Abstract
The overlap of all the docked inhibitors (1 (brown), 2 (red), 5 (pink) and 7 (yellow)) inside the
active site of acetylcholinesterase
25

Research highlights
1. Synthesis of 1,2,4-triazole-3-thione amine substituted thioureas
2. Synthesis of 1-4-aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-
isobutylphenyl)propan-1-one
3. Investigation of acetylcholinesterase (AChE) and butrylcholinesterase (BChE) inhibition
assays
26