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Para-Substituted Thiosemicarbazones as Cholinesterase Inhibitors:

Synthesis, In Vitro Biological Evaluation, and In Silico Study
Momin Khan,* Hina Gohar, Aftab Alam, Abdul Wadood, Azam Shareef, Mahboob Ali, Asaad Khalid,
Ashraf N. Abdalla, and Farhat Ullah
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ABSTRACT: The current research reports the synthesis of 14 para-substituted

thiosemicarbazone derivatives in good to excellent yields using standard
procedures. Initially, 4-ethoxybenzaldehyde (1) and 4-nitrobenzaldehyde (2)
Downloaded via 196.157.107.68 on November 26, 2024 at 19:25:04 (UTC).

were refluxed with thiosemicarbazide in the presence of acetic acid in ethanol for
4−5 h. Then, various substituted phenacyl bromides were treated with the desired
thiosemicarbazones (3 and 4) in the presence of triethylamine in ethanol with
constant stirring for 5−6 h. The resulting derivatives were confirmed through
electron impact mass spectrometry and 1H NMR spectroscopy and evaluated for
anticholinesterase inhibitory activity. Among the series, four compounds, 19, 17, 7,
and 6, showed potent inhibitory activity against the acetylcholinesterase (AChE)
enzyme, having IC50 values of 110.19 ± 2.32, 114.57 ± 0.15, 140.52 ± 0.11, and
160.04 ± 0.02 μM, respectively, compared with standard galantamine (IC50 =
104.5 ± 1.20 μM). Similarly, compounds 19 (IC50 = 145.11 ± 1.03 μM), 9 (IC50 =
147.20 ± 0.09 μM), 17 (IC50 = 150.36 ± 0.18 μM), and 6 (IC50 = 190.21 ± 0.13 μM) were the most excellent inhibitors of
butyrylcholinesterase (BChE) when compared with the standard drug galantamine (IC50 = 156.8 ± 1.50 μM). In silico studies were
accomplished on the produced derivatives in order to explain the binding interface of compounds with the active sites of AChE and
BChE enzymes.

■ INTRODUCTION
Compounds possessing at least one carbon−nitrogen double
Basically, it is found in elders frequently and is considered the
most common form of dementia.26 The typical reason for AD
bond are named Schiff’s bases.1 Schiff’s bases have been known is uncertain, though the cholinergic hypothesis characterizes it
since 1864; they were first discovered by Hugo Schiff through as the loss or damage of neurotransmitters in the brain which
the condensation of primary amines with carbonyl compounds causes advanced mental impairment.27 Thus, drugs to treat AD
(aldehydes and ketones).2,3 The fundamental feature of these are based on their ability to inhibit cholinesterase enzymes,
analogues is the azomethine group, which has a general that is, acetylcholinesterase (AChE) and butyrylcholinesterase
formula of RHC = N−R1, (R and R1 may be alkyl, aryl, or
(BChE).28,29 Nowadays, various drugs are used for the
heterocyclic groups). These compounds are also known as
azomethine, imines, or anils.4−6 Schiff’s bases are the most treatment of AD, including rivastigmine, donepezil, galant-
important organic compounds with a variety of applications; amine, and tacrine (Figure 1). Synthetic organic chemists are
these compounds are extensively used as catalysts in different working on new, chief, effective, and safe drugs for AD.30,31 In
organic reactions,7 as stabilizers,8 in the polymer industry,9 as the near past, our research group has reported imine-
pesticides,10 and also as dyes.11 Likewise, these compounds containing derivatives with promising biological activities.32−35
also possess various biological or pharmacological activities, The present work is mainly focused on the synthesis of various
like anti-inflammatory,12 anti-HIV,13 antiurease,14 anticholi- thiosemicarbazone derivatives with anticholinesterase inhib-
nesterase,15 antifungal,16 antioxidant,17 and α-glucosidase
itory potential.
activities.18 One more application of imine linkage is in vision
chemistry, where the link is formed between the active vitamin
B6 (pyridoxal phosphate) and the amino acids for the purpose Received: December 20, 2022
of transamination.19−21 Imine-containing compounds have the Accepted: January 13, 2023
potential to form a defensive coating, which makes the surface Published: January 25, 2023
safe from corrosion.22
Alzheimer’s disease (AD) is an enlightened neurodegenera-
tion leading to a decline of mental and physical health.23−25
© 2023 The Authors. Published by
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5116 ACS Omega 2023, 8, 5116−5123
ACS Omega http://pubs.acs.org/journal/acsodf Article

Figure 1. Currently available marketed drugs as cholinesterase inhibitors.

■ EXPERIMENTAL SECTION
Materials and Methods. The chemicals, solvents, and
acid (DTNB), galantamine from Lycoris sp., and acetyl
thiocholine iodide, were used in the present study.
Spectrophotometric Ellman’s technique was used to carry
reagents (4-ethoxybenzaldehyde CAS no 173606 and 4-
out the AChE and BChE assays using butyryl thiocholine
nitrobenzaldehyde CAS no 130176) utilized in the current
iodide and acetyl thiocholine iodide as substrates. Two
study were of analytical grade (98−99% pure) and purchased
mixtures having 5 μL of AChE (0.03 μg/mL) and 5 μL of
from Sigma-Aldrich (St Louis, MO, USA). The analytical
BChE (0.01 μg/mL) particularly and each one added with 205
grade chemicals, solvents, and reagents utilized in the current
μL of samples (125−1000 μg/mL) and DTNB (5 μL) were
study were purchased from Sigma-Aldrich, MO, USA.
incubated for 15 min at 30 °C. After 15 min, 5 μL of substrates
Precoated aluminum sheets (silica gel 60F-254, Merck,
was added to the reaction mixture, and the reaction was
Germany) were used for thin layer chromatography (TLC),
detected for 4 min at 412 nm by using a UV−visible
and they were visualized with the help of UV light at 254 nm.
spectrophotometer. The presence of yellow color in the
A MAT312 spectrophotometer was used to obtain electron
mixture showed the creation of the 5-thio-2-nitrobenzoate
impact mass spectra, while an AVANCE AV 400 MHz was anion by reaction of thiocholines and DTNB. In the absence of
used to obtain NMR spectra of the synthesized compounds. samples and enzymes, nonenzymatic hydrolysis of substrate
General Procedure for the Synthesis of Para- was also examined. The reaction mixture was considered as a
Substituted Carbothioamides (3 and 4). In a 100 mL control in the absence of samples. Percent inhibition and
round-bottomed flask, 3 g of para-substituted benzaldehyde (4- enzyme activities were calculated through the following
nitrobenzaldehyde and 4-ethoxybenzaldehyde) was dissolved formulas
in 15 mL absolute ethanol containing a catalytic amount of
glacial acetic acid. The reaction was stirred for 20−30 min; VAbs
V=
then, 1.8 g of thiosemicarbazide was added to it and refluxed t
for 3−4 h with constant stirring. The progress of the reaction
was checked with the help of TLC in a 30% polar system of V
% age enzyme activity = × 100
ethyl acetate and n-hexane. After completion, the reaction Vmax
mixture was cooled to room temperature and poured into a
beaker containing crushed ice; formed precipitates were % age enzyme inhibition = 100 % enzyme activity
filtered, washed with excess of water, dried under vacuum, where V is the rate of reaction in the presence of an inhibitor
and collected for further reactions. The synthesized analogues and Vmax is the rate of reaction without the inhibitor.
were confirmed with the help of electron impact mass Molecular Docking Assay. Molecular docking studies are
spectrometry (EI-MS) and 1H NMR spectroscopy. mainly used to study the intermolecular interactions of a
General Procedure for the Synthesis of Carbothioa- chemical/molecule with a protein/enzyme or biological target
mide Derivatives (5−19). The resultant para-substituted to find its drug-like properties. In the current work, we have
thiosemicarbazones (3 and 4) were further treated with various studied the intermolecular interactions of synthesized com-
substituted phenacyl bromides in a basic medium using pounds with AChE and BchE via molecular docking.37 The
ethanol as a solvent. In a typical reaction, thiosemicarbazones crystal structures of human AChE and BChE with PDB codes
were dissolved in absolute ethanol containing a catalytic 4EY6 and 6QAE were downloaded from the protein data bank
amount of triethylamine, and the reaction was stirred for 30 by visiting www.rcsb.com. These proteins were prepared and
min. Then, various substituted phenacyl bromides were added minimized/stabilized in MOE 2016. The compounds 6, 7, 9,
to it and further refluxed for 4−5 h with constant stirring. The 17, and 19 having effective IC50 values against these targets,
progress of the reaction was monitored with the help of TLC along with the reference molecule galantamine, were docked
using a solvent system of ethyl acetate and n-hexane (3:7). The into the active site of these minimized proteins using the
mixture was poured into a beaker containing ice-cold water default docking protocol implemented in MOE 2016. Pymol
after completion. Precipitates formed were filtered, washed was used for ligand interaction and visualization.
with excess of water, dried under air, and recrystallized with Spectral Interpretation of the Synthesized Com-
ethanol to obtain pure products. All the synthesized analogues pounds (5−19). 2-(4-Ethoxybenzylidene)-N-(2-oxo-2-(m-
were structurally deduced with the help of EI-MS and 1H tolyl)ethyl)hydrazine-1-carbothioamide (5). White powder;
NMR spectroscopy. yield: 89%; 1H NMR (400 MHz, DMSO-d6): δ 12.05 (s, 1H,
Anti-cholinesterase Inhibition Assay. The anti-chol- −NH), 8.01 (s, 1H, −CH�N−), 7.85 (d, 1H, J = 8.0 Hz, 2H,
inesterase activity of the obtained products was accomplished Ar-H, H-2, H-6), 7.64−7.62 (m, 4H, Ar-H, H-2′, H-4′, H-5′,
according to the described procedure.36 Electric eel AChE H-6′), 7.41 (s, 1H, −NH), 7.03 (d, J = 8.0 Hz, 2H, Ar-H, H-3,
(type-VI-S), potassium phosphate buffer (pH 8.0), equine H-4), 4.13 (q, J = 8.0 Hz, 2H, −OCH2CH3), 1.39 (t, J = 8.0
BChE, butyryl thiocholine iodide, 5,5-dithio-bis-nitrobenzoic Hz, 3H, −OCH2CH3), 1.30 (s, 3H, −CH3); EI-MS m/z (rel.
5117 https://doi.org/10.1021/acsomega.2c08108
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abund, %): 355 (M+, 100), 313.9 (15), 163.9 (22), 149.9 (50), 2-(4-Nitrobenzylidene)-N-(2-oxo-2-(p-tolyl)ethyl)-
120.0 (41), 104.0 (22). hydrazine-1-carbothioamide (12). White powder; yield: 86%;
1
N-(2-(4-Bromophenyl)-2-oxoethyl)-2-(4- H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 7.2 Hz, 2H, Ar-H,
ethoxybenzylidene)hydrazine-1-carbothioamide (6). White H-3, H-5), 8.03 (s, 1H, −CH�N−), 8.01 (s, 1H, −NH), 7.97
powder; yield: 90%; 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J (d, J = 7.2 Hz, 2H, Ar-H, H-2, H-6), 7.44 (d, J = 8.0 Hz, 2H,
= 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.31 (d, J = 8.0 Hz, 2H, Ar-H, Ar-H, H-3′, H-5′), 6.72 (d, J = 8.0 Hz, 2H, Ar-H, H-2′, H-6′),
H-2′, H-6′), 7.26 (d, J = 8.0 Hz, 2H, Ar-H, H-3′, H-5′), 6.89 4.85 (s, 2H, −CH2), 2.41 (s, 3H, −CH3); EI-MS m/z (rel.
(d, J = 8.0 Hz, 2H, Ar-H, H-3, H-5), 6.82 (s, 1H, −NH), 4.12 abund, %): 356 (M+, 100), 310 (68), 221 (85), 163 (85).
(q, J = 8.0 Hz, 2H, −OCH2CH3), 2.38 (s, 2H, −CH2), 1.47 (t, 2-(4-Nitrobenzylidene)-N-(2-(4-nitrophenyl)-2-oxoethyl)-
J = 8.0 Hz, 3H, −OCH2CH3); EI-MS m/z (rel. abund, %): 419 hydrazine-1-carbothioamide (13). Orange powder; yield:
(M+, 2), 327.2 (2), 235.3 (8), 219.4 (5), 185.2 (21), 171.2 77%; 1H NMR (400 MHz, CDCl3): δ 8.40 (d, J = 8.0 Hz, 2H,
(35). Ar-H, H-3′, H-5′), 8.31 (d, J = 8.0 Hz, 2H, Ar-H, H-2′, H-6′),
N-(2-(4-Chlorophenyl)-2-oxoethyl)-2-(4- 8.27 (d, J = 7.2 Hz, 2H, Ar-H, H-3, H-5), 8.03 (s, 1H, −CH�
ethoxybenzylidene)hydrazine-1-carbothioamide (7). Off N−), 8.01 (s, 1H, −NH), 7.97 (d, J = 7.2 Hz, 2H, Ar-H, H-2,
white powder; yield: 84%; 1H NMR (400 MHz, CDCl3): δ H-6), 4.85 (s, 2H, −CH2); EI-MS: m/z (rel. abund, %): 387
7.81 (d, J = 8.0 Hz, 2H, Ar-H, H-2′, H-6′), 7.78 (s, 1H, −NH), (M+, 100), 341 (58), 252 (84), 163 (97).
7.44−7.41 (m, 4H, Ar-H, H-2, H-6, H-3′, H-5′), 6.93 (d, J = N-(2-(4-Chlorophenyl)-2-oxoethyl)-2-(4-
7.2 Hz, 2H, Ar-H, H-3, H-5), 6.87 (s, 1H, −NH), 4.13 (q, J = nitrobenzylidene)hydrazine-1-carbothioamide (14). Off
8.0 Hz, 2H, −OCH2CH3), 1.51 (t, J = 8.0 Hz, 3H, white powder; yield: 79%; 1H NMR (400 MHz, CDCl3): δ
−OCH2CH3); EI-MS m/z (rel. abund, %): 376 (M+, 99), 8.27 (d, J = 8.0 Hz, 2H, Ar-H, H-3, H-5), 8.06 (d, J = 7.2 Hz,
285.3 (4), 193.1 (53), 182.1 (49), 155.4 (8), 103.4 (3), 91.1 2H, Ar-H, H-2′, H-6′), 8.03 (s, IH, −CH�N−), 8.00 (s, 1H,
(33), 65.1 (3). −NH), 7.97 (d, J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.68 (d, J =
2-(4-Ethoxybenzylidene)-N-(2-oxo-2-phenylethyl)- 7.2 Hz, 2H, Ar-H, H-3′, H-5′), 7.00 (s, 1H, −NH), 4.85 (s,
hydrazine-1-carbothioamide (8). Yellow powder; yield: 82%; 2H, −CH2); EI-MS m/z (rel. abund, %): 422 (M2+, 98), 420
1
H NMR (400 MHz, DMSO-d6): δ 11.17 (s, 1H, −CH� (M+, 100), 375 (77), 341 (60) 223 (77).
N−), 7.88 (d, J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.46−7.35 (m, N-(2-(2-Hydroxyphenyl)-2-ozoethyl)-2-(4-
4H, Ar-H, H-2′, H-3′, H-5′, H-6′), 6.90−6.87 (m, 3H, Ar-H, nitrobenzylidene)hydrazine-1-carbothioamide (15). Yellow-
H-4′, H-3, H-5), 4.12 (q, J = 8.0 Hz, 2H, −OCH2CH3), 2.67 ish powder; yield: 80%; 1H NMR (400 MHz, DMSO-d6): δ
(s, 2H, −CH2), 1.51 (t, J = 8.0 Hz, 3H, −OCH2CH3); EI-MS 11.03 (s, 1H, −CH�N−), 9.56 (s, 1H, −OH), 7.93 (s, 1H,
m/z (rel. abund, %): 341 (M+,100), 312 (57), 163 (72), 133 −NH), 7.64 (d, J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.45 (m, 1H,
(69). Ar-H, H-4′), 7.34 (m, 1H, Ar-H, H-5′), 6.96 (d, J = 8.0 Hz,
2-(4-Ethoxybenzylidene)-N-(2-(2-hydroxyphenyl)-2- 2H, Ar-H, H-3′, H-6′), 6.40 (s, 1H, −NH), 4.14 (q, J = 8.0 Hz,
oxoethyl)hydrazine-1-carbothioamide (9). Pale yellow pow- 2H, −OCH2CH3), 1.49 (t, J = 8.0 Hz, 3H, −OCH2CH3); EI-
der; yield: 78%; 1H NMR (400 MHz, CDCl3): δ 11.23 (s, 1H, MS m/z (rel. abund, %): 358 (M+, 100), 341 (58), 223 (100).
−CH�N−), 9.66 (s, 1H, −OH), 7.91 (s, 1H, −NH), 7.63 (d, N-(2-(2,4-Dichlorophenyl)-2-ozoethyl)-2-(4-
J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.47 (m, 1H, Ar-H, H-4′), nitrobenzylidene)hydrazine-1-carbothioamide (16). White
7.24 (m, 1H, Ar-H, H-5′), 6.96 (d, J = 8.0 Hz, 2H, Ar-H, H-3′, powder; yield: 82%; 1H NMR (400 MHz, DMSO-d6): δ
H-6′), 6.40 (s, 1H, −NH), 4.14 (q, J = 8.0 Hz, 2H, 12.07 (s, 1H, −CH�N−), 7.83 (d, J = 8.0 Hz, 2H, Ar-H, H-3,
−OCH2CH3), 1.49 (t, J = 8.0 Hz, 3H, −OCH2CH3); EI-MS H-5), 7.80 (m, 2H, Ar-H, H-2, H-6), 7.71 (m, 2H, Ar-H, H-5′,
m/z (rel. abund, %): 357 (M+, 12), 327.3 (3), 311.4 (3), 297.4 H-6′), 7.70 (s, 1H, −NH), 7.69 (s, 1H, −NH), 7.24 (s, 1H,
(4), 277.3 (5), 263.3 (4), 249.3 (4), 235.5 (8), 219.3 (10), Ar-H, H-3′), 3.17 (s, 2H, −CH2); EI-MS m/z (rel. abund, %):
185.2 (66), 171.2 (58). 410 (M+2,66), 410 (M+,100), 375 (62), 341 (86), 223 (46).
N-(2-(2,4-Dichlorophenyl)-2-oxoethyl)-2-(4- N-(2-(2-Bromophenyl)-2-oxoethyl)-2-(4-
ethoxybenzylidene)hydrazine-1-carbothioamide (10). Red- nitrobenzylidene)hydrazine-1-carbothioamide (17). Yellow
dish powder; yield: 88%; 1H NMR (400 MHz, CDCl3): δ 8.03 powder; yield: 76%; 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J
(s, 1H, −CH�N−), 8.0 (s, 1H, −NH), 7.89 (d, J = 7.2 Hz, = 8.0 Hz, 2H, Ar-H, H-3, H-5), 8.03 (s, 1H, −CH�N−), 8.00
2H, Ar-H, H-2, H-6), 7.78 (d, J = 8.0 Hz, 1H, Ar-H, H-6), 7.64 (s, 1H, −NH), 7.97 (d, J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.79
(s, 1H, Ar-H, H-3′), 7.36 (d, J = 8.0 Hz, 2H, Ar-H, H-5′), 7.03 (d, J = 7.2 Hz, 1H, Ar-H, H-6′), 7.39−7.38 (m, 2H, Ar-H, H-
(d, J = 7.2 Hz, 2H, Ar-H, H-3, H-5), 7.0 (s, 1H, −NH), 4.05 4′, H-5′), 7.65 (d, J = 8.0 Hz, 1H, Ar-H, H-3′), 4.85 (s, 2H,
(q, J = 8.0 Hz, 2H, −OCH2CH3), 2.24 (s, 2H, −CH2), 1.34 (t, −CH2); EI-MS m/z (rel. abund, %): 422 (M+2, 98), 420 (M+,
J = 8.0 Hz, 3H, −OCH2CH3); EI-MS m/z (rel. abund, %): 411 100), 341 (77), 223 (61).
(M+2, 66), 409.9 (M+, 100), 374 (58), 340 (81), 163 (68). N-(2-([1,1′-Biphenyl]-4-yl)-2-ozoethyl)-2-(4-
N-(2-([1,1′-Biphenyl]-4-yl)-2-oxoethyl)-2-(4- nitrobenzylidene)hydrazine-1-carbothioamide (18). Off
ethoxybenzylidene)hydrazine-1-carbothioamide (11). white powder; yield: 68%; 1H NMR (400 MHz, CDCl3): δ
Brown powder; yield: 82%; 1H NMR (400 MHz, CDCl3): δ 7.98 (d, J = 8.0 Hz, 2H, Ar-H, H-3, H-5), 7.83 (s, 1H, −NH),
7.96 (d, J = 8.0 Hz, 2H, Ar-H, H-2′, H-6′), 7.69 (t, J = 8.0 Hz, 7.66 (d, J = 8.0 Hz, 2H, Ar-H, H-2′, H-6′), 7.59 (d, J = 8.0 Hz,
3H, Ar-H, H-3″, H-4″, H-5″), 7.62 (d, J = 8.0 Hz, 2H, Ar-H, 2H, Ar-H, H-2, H-6), 7.43 (m, 4H, Ar-H, H-3′, H-5′, H-2″, H-
H-2, H-6), 7.50 (s, 1H, −NH), 7.46 (d, J = 8.0 Hz, 2H, Ar-H, 6″), 7.19 (t, J = 8.0 Hz, 3H, Ar-H, H-3″, H-4″, H-5″), 4.85 (s,
H-3′, H-5′), 7.41 (d, J = 8.0 Hz, 2H, Ar-H, H-2″, H-6″), 6.94 2H, −CH2); EI-MS m/z (rel. abund, %): 418 (M+, 36), 376.2
(s, 1H, −NH), 6.86 (d, J = 8.0 Hz, 2H, Ar-H, H-3, H-5), 4.08 (10), 284.1 (9), 199.1 (58), 178.0 (12), 104.9 (100), 77.0
(q, J = 8.0 Hz, 2H, −OCH2CH3), 1.48 (t, J = 8.0 Hz, 3H, (30), 51.0 (8).
−OCH2CH3); EI-MS m/z (rel. abund, %): 417 (M+,100), 222 N-(2-(Chlorophenyl)-2-ozoethyl)-2-(4-nitrobenzylidene)-
(85), 195 (63), 163 (80). hydrazine-1-carbothioamide (19). Yellow powder; yield:
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Scheme 1. Thiosemicarbazone Derivatives of Para-Substituted Benzaldehydes (5−19)

Figure 2. Most active compounds of the series.

73%; 1H NMR (400 MHz, CDCl3): δ 8.27 (d, J = 8.0 Hz, 2H, washed with excess of water, dried under air, recrystallized with
Ar-H, H-3, H-5), 8.03 (s, 1H, −NH), 8.00 (s, 1H, −NH), 7.97 absolute ethanol, and collected.
(d, J = 8.0 Hz, 2H, Ar-H, H-2, H-6), 7.84 (d, J = 7.2 Hz, 1H, In Vitro Anti-cholinesterase Inhibitory Activity. All the
Ar-H, H-6′), 7.44 (t, J = 8.0 Hz, 1H, Ar-H, H-4′), 7.38−7.32 synthesized derivatives (5−19) were screened for their anti-
(m, 2H, Ar-H, H-3′, H-5′), 7.00 (s, 1H, −NH), 4.85 (s, 2H, cholinesterase inhibitory potential and compared with the
−CH2); EI-MS m/z (rel. abund, %): 378 (M+2, 37), 376 standard drug galantamine. All the compounds showed good
(100), 341 (70), 223 (51). to moderate inhibitory activity. Among the synthesized series,
four compounds 19, 17, 7, and 9 showed potent inhibitory
■ RESULTS AND DISCUSSION
Chemistry. To discover various pharmacologically/bio-
activity against the AChE enzyme, having IC50 values of 110.19
± 2.32, 114.57 ± 0.15, 140.52 ± 0.11, and 160.04 ± 0.02 μM,
logically potent cholinesterase inhibitors, efforts were made to respectively, compared with standard galantamine (IC50 =
synthesize various novel derivatives of para-substituted 104.5 ± 1.20 μM). Similarly, compounds 19 (IC50 = 145.11 ±
benzaldehydes in good to excellent yields through multistep 1.03 μM), 9 (IC50 = 147.20 ± 0.09 μM), 17 (IC50 = 150.36 ±
reactions. Initially, 4-ethoxybenzaldehyde (1) and 4-nitro- 0.18 μM), and 6 (IC50 = 190.21 ± 0.13 μM) (Figure 2) were
benzaldehyde (2) were refluxed with thiosemicarbazide in the the most excellent inhibitors of BChE when compared with
presence of acetic acid in ethanol for 4−5 h. Then, various standard drug galantamine (IC50 = 156.8 ± 1.50 μM).
substituted phenacyl bromides were treated with the desired However, compounds 15 (IC50 = 200.41 ± 0.21 μM), 8
thiosemicarbazones (3 and 4) in the presence of triethylamine (IC50 = 220.22 ± 0.14 μM), 10 (IC50 = 275.71 ± 0.12 μM), 12
in ethanol with constant stirring for 5−6 h (Scheme 1). The (IC50 = 280.24 ± 0.21 μM), and 5 (IC50 = 295.12 ± 0.12 μM)
reaction progress was monitored with the help of TLC using a showed good inhibitory potential against the AChE enzyme.
30% polar solvent system of ethyl acetate and n-hexane (3:7). Furthermore, four compounds 18 (IC50 = 212.25 ± 0.09 μM),
After completion, the reaction mixtures were cooled to room 8 (IC50 = 260.21 ± 0.23 μM), 12 (IC50 = 260.38 ± 0.25 μM),
temperature and poured into a beaker containing ice-cold and 15 (IC50 = 270.26 ± 0.19 μM) displayed good inhibitory
distilled water. Precipitates were formed, which were filtered, potential toward the BChE enzyme, while the remaining
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compounds demonstrated less enzyme inhibitory activity μM and 145.11 ± 1.03 μM, respectively. The potency of this
(Table 1). compound might be due to the attachment of a chlorine atom
The most active compound in the series against AChE and in the ortho position; the activity of compound 19 against
BChE is compound 19 having IC50 values of 110.19 ± 2.32 BChE is near that of standard galantamine. Similarly,
compound 17 was found to be the second most active
Table 1. AChE and BChE Inhibitory Activities of compound against the desired enzymes, with IC50 values of
Compounds (5−19)a 114.57 ± 0.15 and 150.36 ± 0.18 μM. The highest activity of
the compound may be due to the presence of a bromine atom
in the ortho position. Compound 3 was the third active
compound against the AChE enzyme with an IC50 value of
140.52 ± 0.11 μM, while being less active against BChE. The
activity of this compound could be due to the presence of a
chlorine atom at the para position of the benzene ring.
Galantamine was used as a standard drug for AChE and BChE
enzymes, with IC50 values of 104.5 ± 1.20 and 156.8 ± 1.50
μM, respectively.
Molecular Docking Analysis. Molecular docking was
performed in order to explore in vitro the intermolecular
interactions of para-substituted thiosemicarbazone derivatives
with human AChE and BchE enzymes. Among the series, the
most active derivatives were docked into the active site of these
targets, which had effective IC50 values in an in vitro assay
along with the reference substrate galantamine. All these
compounds had stronger and comparable interactions with
target proteins, which means that they may have the ability to
alter the chemistries of these proteins and might act as an
anticholinesterase.
For AChE, among these derivatives, 19, 17, 7, and 6 have
been found with much stronger and comparable interactions as
compared to galantamine. The docking scores of all the
compounds 5−19 against AChE are given in Table S2.
Compound 19 has stronger interactions as it has a highly
electronegative chlorine on the terminal aromatic ring, which
forms a stronger hydrogen bond with the oxygen of residue
Glu202. In addition, this compound forms two other stronger
intermolecular interactions, that is, one hydrogen bond
between sulfur of the ligand and the oxygen of residue
Ser203, followed by a π bond between the delocalized
electrons of the aromatic ring of the compound with alpha-
carbon of Tyr341 as shown in Figure 3. These interactions
made the compound a reasonable inhibitor of the enzyme.
Similarly, compound 17 has also formed a stronger bond with
AChE. Compound 17 is bonded to residues Gly120 and Asp74
of the active site of the enzyme. With the residues the
compound has formed strong hydrogen bonds, Gly120 has an
electronegative oxygen which interacts with the hydrogen of
the main chain of the compound. In the same way, the other
residue, Asp70, is linked with the sulfur of the compound via
hydrogen bonding, as shown in Figure 3. These interactions
may alter/inhibit the activity of the enzyme. Likewise, we have
compound 7 with a considerable IC50 value and good docking
interactions. This compound also forms stronger molecular
interactions with the target but is considerably weaker than the
former two, as in the former compounds there have been at
least two hydrogen bonds found in each case, but here we have
only one hydrogen with a considerably weaker π bond. Still,
the compound has shown interactions and might have the
ability to disturb the activity of the enzyme. Compound 7
forms a hydrogen bond by its chlorine with the oxygen of
residue Glu202 and the delocalized electrons of the aromatic
ring of the compound form a π−π interaction with a similar
a aromatic ring of Trp86 as shown in Figure 3. Except for these
Standard positive control galanthamine was used, and data are mean
± SEM. compounds, there is still another compound (6) with attractive
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ACS Omega http://pubs.acs.org/journal/acsodf Article

Figure 3. Protein−ligand interaction of AChE with compounds 19, 17, 7, and 6 in comparison with galantamine.

docking results and effective interactions with the active site this compound, there was still another compound which also
residues of the enzyme. The compound formed three hydrogen showed much stronger and more stable interactions during the
bonds and one H−π interaction, making this compound a course of docking, compound 17. Compound 17 also formed
possible inhibitor of the enzyme. Among the three hydrogen four bonds similar to compound 15, but they were slightly
bonds, one hydrogen bond is formed between the nitrogen of weaker than the former as the latter has two hydrogen bonds
the ligand and the oxygen of Glu202, another one is formed and two π interactions, whereas the former has three hydrogen
between the nitrogen of the ligand and the oxygen of Ser203, bonds. Anyhow, the latter is also of much importance as it has
and the third hydrogen bond is formed between the sulfur of stronger interactions than galantamine. Compound 17 has two
the ligand and the alpha carbon of His447. There is also an adjacent highly electronegative nitrogen atoms which both
H−π interaction between the carbons of the ligand and the formed hydrogen bonds with the oxygen of Asp70, Along with
aromatic ring of Tyr341, as shown in Figure 3. the terminal aromatic ring of the compound formed two π
Likewise, in AChE, the synthesized derivatives have also interactions, one π−π interaction with 5-ring of Trp82 and one
been found equally effective against BChE and showed with 6-ring of Trp82, as shown in Figure 4. There was still
considerable interactions with the enzymes. The docking another base with strong interactions with active site residues
of the enzyme; compound 7 also formed three intermolecular
scores of all the compounds 5−19 against BChE are given in
interactions, indicating the ability of the inhibition. Among
Table S3. Compound 19 has been found to be very much
these three bonds, one was a hydrogen bond formed between
effective against the enzyme; this compound might have the oxygen of the compound and His438; besides this, it has
formed the strongest interactions of this study. Compound 19 two π interactions, both formed by the aromatic ring of the
is bonded to four active site residues of the enzyme. Three compound. There was a π−H interaction formed by the
bonds were strong intermolecular hydrogen bonds, and one aromatic ring of the compound with hydrogen attached to the
was H−π interactions. Among the three hydrogen bonds, one alpha carbon of Phe329 and a π−π interaction between the
was formed between the nitrogen of the ligand and the oxygen same ring of the compound and the benzene ring of Tyr332, as
of Pro285, the second hydrogen bond formed by the sulfur of shown in Figure 4.
the compound with the hydrogen attached to the alpha carbon
of Gly117, and the third hydrogen bond formed by the sulfur
of the ligand with the nitrogen of Glu119. The H−π bond has
been formed by the carbon of the ligand with an aromatic ring
■ CONCLUSIONS
A novel series of para-substituted thiosemicarbazone deriva-
of Trp82 as shown in Figure 4. Such strong and stable linkages tives were synthesized in good to excellent yields using
may be considered as the best inhibitory source. Along with standard procedures. The resulting compounds were structur-
ally elucidated with the help of EI-MS and 1H NMR
spectroscopy and finally evaluated for their anticholinesterase
inhibitory potential. Four compounds showed excellent AChE
inhibitory activity in the range of 110.19 ± 2.32 to 160.04 ±
0.02 μM compared with the standard drug galantamine (IC50 =
104.5 ± 1.20 μM). Nevertheless, the other four compounds
displayed potent BChE inhibitory activity in the range of
145.11 ± 1.03 to 190.21 ± 0.13 μM when compared with
galantamine (156.8 ± 1.50 μM). In silico studies were
accomplished on the synthesized analogues in order to explain
the binding interface of compounds with the active sites of
AChE and BChE enzymes.

■ ASSOCIATED CONTENT
* Supporting Information
sı

Figure 4. Protein−ligand interactions of BChE with compounds 19, The Supporting Information is available free of charge at
17, and 7 in comparison with galantamine. https://pubs.acs.org/doi/10.1021/acsomega.2c08108.
5121 https://doi.org/10.1021/acsomega.2c08108
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■ AUTHOR INFORMATION
Corresponding Author
(5) Mohammed Khan, K.; Shah, Z.; Uddin Ahmad, V.; Khan, M.;
Taha, M.; Rahim, F.; Jahan, H.; Perveen, S.; Iqbal Choudhary, M.
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Momin Khan − Department of Chemistry, Abdul Wali Khan (6) Ahad, G.; Khan, M.; Khan, A.; Ibrahim, M.; Salar, U.; Khan, K.
University, Mardan 23200, Pakistan; orcid.org/0000- M.; Perveen, S. Synthesis, structural characterization, and antioxidant
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Authors Dioxide Co-Catalyzed by a Combination of Schiff Bases or Phenols
Hina Gohar − Department of Chemistry, Abdul Wali Khan and Organic Bases. Eur. J. Org. Chem. 2004, 2004, 3080−3089.
University, Mardan 23200, Pakistan (8) Terenzi, A.; Bonsignore, R.; Spinello, A.; Gentile, C.; Martorana,
Aftab Alam − Department of Chemistry, University of A.; Ducani, C.; Högberg, B.; Almerico, A. M.; Lauria, A.; Barone, G.
Malakand, Chakdara 18800, Pakistan Selective G-quadruplex stabilizers: Schiff-base metal complexes with
Abdul Wadood − Department of Biochemistry, Abdul Wali anticancer activity. RSC Adv. 2014, 4, 33245−33256.
Khan University, Mardan 23200, Pakistan; orcid.org/ (9) Antony, R.; Arun, T.; Manickam, S. T. D. A review on
0000-0002-3415-9277 applications of chitosan-based Schiff bases. Int. J. Biol. Macromol.
2019, 129, 615−633.
Azam Shareef − Department of Biochemistry, Abdul Wali
(10) Li, X.; Zhang, D.; Liu, Z.; Xu, Y.; Wang, D. Synthesis,
Khan University, Mardan 23200, Pakistan characterization of a ternary Cu(II) Schiff base complex with
Mahboob Ali − Department of Chemistry, Abdul Wali Khan degradation activity of organophosphorus pesticides. Inorg. Chim.
University, Mardan 23200, Pakistan Acta 2018, 471, 280−289.
Asaad Khalid − Substance Abuse and Toxicology Research (11) Abuamer, K. M.; Maihub, A. A.; El-Ajaily, M. M.; Etorki, A. M.;
Center, Jazan University, Jazan 45142, Saudi Arabia; Abou-Krisha, M. M.; Almagani, M. A. The role of aromatic Schiff
National Center for Research, Medicinal and Aromatic Plants bases in the dyes techniques. Int. J. Org. Chem. 2014, 4, 7−15.
and Traditional Medicine Research Institute, Khartoum (12) Rakesh, K.; Manukumar, H.; Gowda, D. C. Schiff’s bases of
11111, Sudan quinazolinone derivatives: Synthesis and SAR studies of a novel series
Ashraf N. Abdalla − Department of Pharmacology and of potential anti-inflammatory and antioxidants. Bioorg. Med. Chem.
Toxicology, College of Pharmacy, Umm Al-Qura University, Lett. 2015, 25, 1072−1077.
(13) Al-Masoudi, N. A.; Aziz, N. M.; Mohammed, A. T. Synthesis
Makkah 21955, Saudi Arabia; orcid.org/0000-0003-
and In Vitro Anti-HIV Activity of Some New Schiff Base Ligands
4770-9319 Derived from 5-Amino-4-phenyl-4H-1,2,4-triazole-3-thiol and Their
Farhat Ullah − Department of Pharmacy, University of Metal Complexes. Phosphorus, Sulfur Silicon Relat. Elem. 2009, 184,
Malakand, Chakdara, Khyber Pakhtunkhwa 18800, 2891−2901.
Pakistan (14) Aslam, M. A. S.; Mahmood, S.-u.; Shahid, M.; Saeed, A.; Iqbal,
Complete contact information is available at: J. Synthesis, biological assay in vitro and molecular docking studies of
https://pubs.acs.org/10.1021/acsomega.2c08108 new Schiff base derivatives as potential urease inhibitors. Eur. J. Med.
Chem. 2011, 46, 5473−5479.
(15) Kamalı, A.; Ç akmak, R.; Boğa, M. Anticholinesterase and
Funding antioxidant activities of novel heterocyclic Schiff base derivatives
Funding was provided by the Dean of Scientific Research at containing an aryl sulfonate moiety. J. Chin. Chem. Soc. 2022, 69,
Umm Al-Qura University under grant code: 731−743.
22UQU4331128DSR25. (16) Hamad, A.; Chen, Y.; Khan, M. A.; Jamshidi, S.; Saeed, N.;
Notes Clifford, M.; Hind, C.; Sutton, J. M.; Rahman, K. M. Schiff bases of
sulphonamides as a new class of antifungal agent against multidrug-
The authors declare no competing financial interest. resistant Candida auris. MicrobiologyOpen 2021, 10, No. e1218.

■ ACKNOWLEDGMENTS
The authors would like to thank the Deanship of Scientific
(17) Khan, K. M.; Khan, M.; Ambreen, N.; Rahim, F.; Muhammad,
B.; Ali, S.; Haider, S. M.; Perveen, S.; Choudhary, M. Bis-Schiff bases
of isatins: a new class of antioxidant. J. Pharmacol. Res. 2011, 4, 3402−
Research at Umm Al-Qura University for supporting this work. 3404.
(18) Alam, A.; Ali, M.; Rehman, N. U.; Ullah, S.; Halim, S. A.; Latif,

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