Design and synthesis of garlic-related unsymmetrical thiosulfonates as potential Alzheimer's disease therapeutics: In vitro and in silico study

Article abstract of DOI:10.1016/j.bmc.2021.116194

Garlic contains a wide range of organosulfur compounds, which exhibit a broad spectrum of biological activities. Amongst the sulfur-containing compounds in garlic, the thiosulfonates are considerably popular in various fields. In light of this, we decided

Full text of DOI:10.1016/j.bmc.2021.116194

Bioorg. Med. Chem. 40 (2021) 116194
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of garlic-related unsymmetrical thiosulfonates as
potential Alzheimer’s disease therapeutics: In vitro and in silico study
,
Kani Zilbeyaza *, Aykut Oztekinb, Emine Gunbatar Kutluanaa
a Department of Chemistry, Agri Ibrahim Cecen University, Agri, Turkey
b Department of Medical Services and Techniques, Agri Ibrahim Cecen University, Agri, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
Garlic contains a wide range of organosulfur compounds, which exhibit a broad spectrum of biological activities.
Amongst the sulfur-containing compounds in garlic, the thiosulfonates are considerably popular in various fields.
In light of this, we decided to investigate the enzyme inhibition ability of thiosulfonates. In this paper, the
synthesis and biological activity of a small library of unsymmetrical thiosulfonates as inhibitors of acetylcho-
linesterase (AChE) and butyrylcholinesterase (BChE) are described. The activity evaluation revealed nanomolar
IC50 and Ki values against both enzymes tested. Furthermore, molecular docking studies allowed for the deter-
mination of possible binding interactions between the thiosulfonates and AChE.
Acetylcholinesterase
Butyrylcholinesterase
Enzyme inhibition
Garlic
Organosulfur compounds
Thiosulfonate
1. Introduction
decided to synthesize and screen a small library of thiosulfonates against
two cholinesterase enzymes, namely acetylcholinesterase (AChE) and
Garlic (Allium sativum L.) is globally consumed as a food and has been
used as a traditional medicine for many centuries. It is superabundant in
organosulfur compounds that possess many interesting biological ac-
tivities including antibacterial,1 antifungal,2 antiviral,3 antiparasitic,4
anti-inflammatory,5 antithrombotic,6 antihypertensive,7 antioxidant,8
antiatherosclerotic,9 and anticarcinogenic.10–12 Some of the biologically
active compounds isolated from garlic are shown in Fig. 1.
butyrylcholinesterase (BChE).
AD is an irreversible, progressive neurodegenerative disorder that
slowly eradicates memory. The principle cause of AD according to the
cholinergic hypothesis is a decline in the levels of acetylcholine (ACh),
which is hydrolytically degraded by two cholinesterases namely AChE
and BChE.24,25 These cholinesterases are therefore key therapeutic tar-
gets for AD. Inhibition of these enzymes by cholinesterase inhibitors
(ChEIs) result in higher concentrations of ACh and thus temporary
improving the symptoms of AD.26,27 Unfortunately, there is no cure for
AD, the current treatments have unfavorable side effects and the ther-
apeutic strategies are not permanent solutions.28,29 AD is a serious
medical problem and therefore developing new drugs targeting this
disease continues to attract interest. Screening various functional scaf-
folds is a good starting point to scan for potential new lead compounds
and thus we report the synthesis and screening results of a library of
thiosulfonates as ChEIs. Furthermore, molecular docking studies were
undertaken to model the interaction between the thiosulfonates and the
enzyme (only AChE was investigated) in order to characterize the
behavior of the thiosulfonates in the enzyme binding site.
These compounds have been extensively studied and each compound
is reported to possess more than one pharmacological activity, repre-
sentative examples are given in Table 113 . However, many of these
compounds are rapidly transformed into other organosulfur compounds
under various conditions. Propyl-propane thiosulfonate is one example
of a stable secondary compound from garlic, it is formed by the
decomposition of the primary sulfur-containing constituents naturally
present in the bulb and is proven to be biologically active.14–17
Over the past two decades due to their stability the interest in thio-
sulfonates as therapeutics has rapidly increased. Thiosulfonates reported
to date have been shown to possess anticancer,18–21 antiparasitic,22 and
antifungal properties,23 however, to the best of our knowledge, the
cholinesterase inhibition properties of thiosulfonates remains unex-
plored. In light of this and given that the search for new small molecules
as potential inhibitors of the cholinesterases has become a topic of in-
terest due to their association with Alzheimer’s disease (AD) it was
* Corresponding author at: Agri Ibrahim Cecen University, Faculty of Art and Science, Department of Chemistry, Agri 04100, Turkey.
E-mail address: kbeyaz25@yahoo.com (K. Zilbeyaz).
https://doi.org/10.1016/j.bmc.2021.116194
Received 17 March 2021; Received in revised form 23 April 2021; Accepted 26 April 2021
Available online 1 May 2021
0968-0896/© 2021 Elsevier Ltd. All rights reserved.

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
2. Results and discussion
Table 1
Biological activities of a few compounds isolated from garlic.
2.1. Synthesis of thiosulfonate derivatives 3–12
Compound
Activities
Alliin
Anticancer, Antioxidant, Antidiabetic
Antibacterial, Antiviral, Antiprotozoal, Anticancer,
Immunomodulatory, Antioxidant, Anti-inflammatory,
Antidiabetic
A library of unsymmetrical thiosulfonates was prepared by keeping
the p-tolylsulfonyl group constant and varying the substituent (R group)
on the benzyl group attached to the sulfur (II) atom. The various R
groups were chosen to address the influence of both the electronic and
steric effects on the enzyme inhibition study. The unsymmetrical thio-
sulfonate derivatives 3–12 were synthesized from potassium p-tolue-
nethiosulfonate and various substituted benzyl halides in DMF at room
temperature for 3 h as outlined in Scheme 1.30 Compounds 3–12 were
obtained in good yields (%) and characterized by infrared (IR), high
resolution mass spectrometry (HRMS), proton and carbon nuclear
magnetic resonance (1H and 13C NMR) spectroscopy.
Allicin
Ajoene
Antiprotozoal, Anticancer, Anti-obesity
Anticancer, Antioxidant, Anti-inflammatory
Antifungal, Anticancer, Antioxidant
Diallyl Sulfide
Diallyl Disulfide
Diallyl Trisulfide
Antiviral, Antifungal, Antiprotozoal, Antioxidant
2.3. Inhibition of AChE
The inhibition of AChE was assessed for 3–12 and the results are
reported in Table 2. The various electron donating (ED, R = OCH3, i-Pr,
t-Bu) and electron withdrawing (EW, R = F, Cl, NO2, CN, CF3, OCF3)
groups attached to the benzyl ring of compounds 3–12 had different
effects on the inhibition of AChE. The compounds displayed IC50 values
ranging from 11.9 nM to 488.2 nM and Ki values ranging from 25.6 ±
2.7 nM to 714.4 ± 82.5 nM. The decreasing order of AChE inhibition by
the thiosulfonates according to IC50 values is as follow: 11 (i-Pr) > 8
(CF3) > 9 (OCF3) > 12 (t-Bu) > 7 (CN) > 6 (NO2) > 4 (F) > 5 (Cl) > 10
(OCH3) > 3 (H). Compound 11 with an ED alkyl group (R = i-Pr) showed
the most potent inhibitory activity (IC50 = 11.9 nM and Ki = 25.6 ± 2.7
nM) amongst the library of thiosulfonates. Comparing 11 with 12 we
observe that the activity (IC50 = 176.6 nM and Ki = 157.3 ± 28.5 nM for
12) of the inhibitor decreases as the ED alkyl substituent (R = t-Bu)
becomes more bulky. The weakest inhibitors in the series according to
its IC50 values were identified as the parent thiosulfonate 3 (R = H, IC50
= 517.4 nM) and 10 (IC50 = 667.6 nM) with a methoxy R group.
Replacing the methoxy group with a trifluoromethoxy group as in
compound 9 (IC50 = 129.4 nM) resulted in a fivefold improvement in the
inhibition potential. The same effect is observed when comparing 3 (R
= H, IC50 = 517.4 nM) and 4 (R = F, IC50 = 312.5 nM), the fluorine
substituent resulted in a 1.7 times better inhibitory activity compared to
a hydrogen substituent.
The 1H and 13C NMR spectra displayed the correct number of reso-
nances as expected for each thiosulfonate derivative 3–12. In the 1H
NMR spectra, the presence of the benzylic CH2 protons of each com-
pound in the series was confirmed by a singlet signal observed in the
range δ 4.19–4.32 ppm. In agreement with this the benzylic CH2 carbon
for each compound resonated in the range δ 38.7–40.4 ppm in the 13
C
NMR spectra. Furthermore, the correct number of proton signals for the
benzene rings were observed in the aromatic region in the 1H and 13
NMR spectra.
C
2.2. Inhibition assay
The synthesized unsymmetrical thiosulfonate compounds 3–12 were
evaluated as potential inhibitors of human AChE and BChE using a
modified version of the Ellman method.31 To determine the influence of
the various substituents at the para position of the benzyl ring on the
potency of the compounds a structure–activity analysis was performed.
The study revealed that the compounds exhibited good inhibitory po-
tential against the enzymes, as evident from their half maximal inhibi-
tory concentration (IC50 values) and inhibitory constants (Ki values).
Rivastigmine, a clinically used cholinesterase inhibitor, was included in
the study for comparison purposes. The IC50 and Ki values for each of the
target compounds are summarized in Table 2 and Table 3. The results
indicated that the potency of the unsymmetrical thiosulfonates 3–12 is
varied but pleasingly all the compounds inhibited both AChE and BChE
in the nanomolar range.
Considering the compounds that contain fluorine atoms (4 vs 8 vs 9)
it is observed that 8 (R = CF3, IC50 = 86.6 nM) is the more effective
inhibitor and the second most potent among the series. Many reports
have shown that the presence of fluorine atoms in biologically active
molecules can dramatically improve their pharmacological effect.32,33
Three main reasons have been reported for introducing fluorine atoms
O
S
S
S
S
S
S
S
S
Diallyl Disulfide
Diallyl Sulfide
Diallyl Trisulfide
Allicin
O
NH2
COOH
O
S
S
S
S
SH
S
SH
Allyl persulfide
Alliin
Ajoene
Allyl mercaptan
S
S
S
S
2-Vinyl-4H-1,3-dithiin
3-Vinyl-4H-1,2-dithiin
Fig. 1. Chemical structures of a few biological active compounds isolated from garlic.
2

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
R
O
S
X
DMF
+
SK
R
O
rt, 3 h
O
S
S
O
1
2
3-12
Scheme 1. Synthesis of unsymmetrical thiosulfonates 3–12 (X = Cl or Br; R = 3: H, 4: F, 5: Cl, 6: NO2, 7: CN , 8: CF3, 9: OCF3, 10: OCH3, 11: i-Pr, 12: t-Bu).
Table 2
Table 3
Inhibition of AChE by 3–12 and standard compound rivastigmine.
Inhibition of BChE by 3–12 and standard compound rivastigmine.
Compound
IC50 (nM)
r2
Ki (nM)
Compound
IC50 (nM)
r2
Ki (nM)
R
R
3
4
5
6
517.4
312.5
478.2
212.3
0.94
0.98
0.93
0.97
389.7 ± 62.8
326.9 ± 22.8
714.4 ± 82.5
251.4 ± 30.2
3
4
5
6
318.5
302.7
314.5
214.3
0.95
0.93
0.98
0.97
217.6 ± 25.6
207.6 ± 9.7
198.7 ± 21.8
302.5 ± 51.4
H
H
F
F
Cl
Cl
NO2
CN
CF3
NO2
CN
CF3
7
8
195.7
86.6
0.98
0.94
165.5 ± 11.3
147.9 ± 27.5
7
8
202.5
117.9
0.96
0.96
201.2 ± 31.6
102.6 ± 12.7
9
129.4
488.2
0.95
0.95
198.2 ± 33.0
218.7 ± 27.67
9
53.3
0.95
0.96
17.5 ± 1.0
OCF3
OCF3
10
10
201.5
251.5 ± 17.3
OCH3
OCH3
i-Pr
11
11.9
176.6
60.0
0.97
0.99
0.98
25.6 ± 2.7
157.3 ± 28.5
–
11
84.5
159.4
14.3
0.96
0.98
0.99
37.9 ± 2.8
102.1 ± 19.3
–
i-Pr
12
12
t-Bu
t-Bu
Rivastigmine
Rivastigmine
into molecules: (i) for improving metabolic stability, (ii) for altering
physicochemical properties and (iii) for increasing binding affinity.32 In
light of this, it is not surprising that the fluorinated thiosulfonates were
better inhibitors compared to their protonated counterparts. The
remaining thiosulfonates, namely 5 (R = Cl, IC50 = 478.2 nM), 6 (R =
NO2, IC50 = 212.3 nM) and 7 (R = CN, IC50 = 195.7 nM) were all more
active compared with the parent thiosulfonate 3 (R = H, IC50 = 517.4
nM), indicating that the presence of a para functional group generally
increases the inhibition potential of the compounds. As a final point, it
should be noted that besides being the most effective inhibitor, 11 was
the only compound more active than the clinically approved reference
drug rivastigmine (IC50 = 60.0 nM), showing a fivefold improvement
compared with the standard.
2.4. Inhibition of BChE
The screening of 3–12 against BChE showed that the enzyme was
inhibited by all the compounds with IC50 and Ki values ranging from
53.3 to 318.5 nM and 17.5 ± 1.0–302.5 ± 51.4 nM, respectively. As for
AChE the various para substituents on the benzyl ring presented similar
trends in the inhibition activity against BChE. The results are shown in
Table 3. The decreasing order of BChE inhibition by the thiosulfonates
according to the IC50 values is as follow: 9 (OCF3) > 11 (i-Pr) > 8 (CF3)
> 12 (t-Bu) > 10 (OCH3) > 7 (CN) > 6 (NO2) > 4 (F) > 5 (Cl) > 3 (H). In
3

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
this case, all the compounds in the series were better inhibitors than the
parent thiosulfonate 3 (IC50 = 318.5 nM), the compound with the
unsubstituted benzyl ring, making 1 the weakest inhibitor in the library.
Addition of hydrophobic EW halogen substituents slightly improved the
inhibition activity, 4 (R = F, IC50 = 302.7 nM) and 5 (R = Cl, IC50
=
314.5 nM). Adding strong EW groups such as nitro (6, IC50 = 214.3 nM)
and cyano (7, IC50 = 202.5 nM) further enhanced the inhibition activity.
Once again, the decrease in inhibition activity going from 11 (IC50
=
84.5 nM and Ki = 37.9 ± 2.8 nM) to 12 (IC50 = 159.4 nM and Ki = 102.1
± 19.3 nM) demonstrates the steric hindrance effect of the t-butyl sub-
stituent. Similarly, replacing the hydrogen atoms with fluorine atoms
(compare: 3 and 4, 9 and 10) saw an increase in inhibition potency of
the thiosulfonates.
Compound 9 (IC50 = 53.3 nM and Ki = 17.5 ± 1.0 nM) with a tri-
fluoromethoxy substituent exhibited the strongest inhibition followed
by 11 (R = i-Pr) in which the ED character of the isopropyl group
possibly plays a big role. Despite the fact that nanomolar range IC50 and
Ki values were found in this investigation, the unsymmetrical thiosul-
fonates were less potent compared with rivastigmine. However, these
promising results may be useful in the design of potent cholinesterase
inhibitors.
The results revealed that generally all the thiosulfonate compounds
screened against AChE and BChE showed relatively good inhibition
activity. Four of the thiosulfonates (6, 7, 8 and 11) were better AChE
inhibitors, while the remaining six thiosulfonates (3, 4, 5, 9, 10 and 12)
were better BChE inhibitors. Although the thiosulfonates, except 11
against AChE, were less potent at inhibiting AChE and BChE compared
to rivastigmine, the IC50 and Ki values of compounds 8, 9, 11 and 12
demonstrate the superior inhibition potency of the thiosulfonates.
2.5. Molecular docking
Molecular docking studies were performed on AChE complexed with
the unsymmetrical thiosulfonate compounds 3–12 to understand the
differences in their inhibition activity, by analyzing the interactions of
the thiosulfonates within the enzyme active site. For the computational
method, human AChE crystal structures were used and docking calcu-
lations were carried out using the programs AutoDock and AutoDock
Vina.34 The most suitable docking poses of the compounds were used to
calculate the potential binding energies and their estimated Ki values,
which indicated a behaviour in compliance with the experimental data.
These results together with the amino acid residues involved in binding
are summarized in Table 4. As presented in the table of results, the
binding energies of the compounds were in the range ꢀ 8.1 to ꢀ 9.5 kcal/
mol for AutoDock and ꢀ 7.64 to ꢀ 9.60 kcal/mol for AutoDock Vina.
Interactions between the compounds and the binding cavity of AChE
included both bonded and non-bonded interactions. Interpretation of
the results discloses that the thiosulfonate moiety play a crucial role in
enzyme-ligand binding. Generally both sulfur atoms formed π-sulfur
interactions while the oxygen atoms of majority of the thiosulfonates
formed hydrogen bonds with various amino acid residues. It is note-
worthy to mention that four amino acid residues, namely Tyr 341, Trp
286, Phe 338, and Tyr 124, were involved in each of the complexes
between the thiosulfonates and AChE. A visualisation of compounds 6,
7, 8, 9, 11, and 12 in the active site of AChE from the molecular docking
study is presented in Fig. 2 and Fig. 3. Assessment of the docked com-
plexes revealed that thiosulfonates 8 (ΔGAutoDockVina = ꢀ 8.42 kcal/mol),
11 (ΔGAutoDockVina = ꢀ 9.60 kcal/mol) and 12 (ΔGAutoDockVina = ꢀ 9.44
kcal/mol) showed the lowest binding energies and significant interac-
tion patterns. X-ray crystallography studies revealed that AChE consists
of two ligand binding sites; a catalytic active site (CAS) and a peripheral
anionic site (PAS).35 The unsymmetrical thiosulfonates bound mainly to
PAS as evident from the numerous interactions with amino acid residues
(Tyr 72, Tyr 124, Trp 286, and Tyr 341) found around the entrance to
the active site gorge, where PAS is located. It has been reported that PAS
comprises of Tyr 72, Asp 74, Tyr 124, Trp 286, and Tyr 341 and that
4

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
Fig. 2. Binding interactions of 6, 7, 8, 9, 11, and 12 with AChE.
inhibitors bound to PAS block the enzyme catalytic activity by limiting
substrate traffic.36–38 The CAS includes a catalytic triad of three amino
acids, namely Ser 203, Glu 334, and His 447 at the bottom of the gorge.
From the docking results, van de Waals interactions with the amino acid
residues (His 447 and Ser 203) in CAS were observed for three ligands,
thiosulfonates 9, 11 and 12. The results reveal that the thiosulfonate
compounds may have potential to be designed as dual binding
inhibitors.
between the alkyl groups of the docked thiosulfonate and Trp 86 and Trp
286. The increase in activity observed by 11 and 12 may be attributed to
the hydrophobic group attached to the benzyl ring as it has been re-
ported that hydrophobic substituents positively affect inhibition of
AChE inhibitors.40,41 The aromatic moiety forms
π-π stacking with Tyr
337, Tyr 341 and Trp 286, which results in stabilization of the complex.
In addition, the ligand makes numerous weak Van der Waals in-
teractions with the side chains of various amino acids.
Compound 11 displayed the most potent inhibition with a binding
energy of ꢀ 9.6 kcal/mol (AutoDock Vina) and the lowest estimated Ki
value (92 nM). These results are in agreement with the in vitro studies
which also revealed the lowest Ki value for 11 (25.6 ± 2.7 nM) in the
series of thiosulfonates tested. The best docked pose of 11 within the
active site of AChE is presented in Fig. 4, which shows the interacting
amino acid residues of the complex are Trp 86, Tyr 337, Tyr 341, Trp
286, Tyr 124, Phe 338, Phe 297, and Tyr 72. Four of these residues (Trp
86, Tyr 341, Trp 286, and Phe 338) were reported to be crucial targets
for designing new AChE inhibitors due to their importance in enzyme
catalytic activity.39 The critical role of these residues is confirmed by the
low Ki value obtained for inhibitor 11.
Comparing the binding patterns of the AChE-11 complex with the
AChE-rivastigmine complex (Supplementary Data, Fig. S11) revealed
that both compounds form close interactions with important amino acid
residues, forming hydrogen bonds, π-σ and Van der Waals interactions,
Fig. 4. In the comparison study a few common interactions were
observed, these include interactions with the following amino acid
residues: Tyr 124, Tyr 72, Trp 86, Tyr 337. Interestingly rivastigmine
bound to only one of the important residues (Trp 86) mentioned before,
which may explain the five-fold improvement in activity for 11 (IC50
=
11.9 nM) compared with the reference standard (IC50 = 60.0 nM). Both
compounds form π-σ interactions with Trp 86 which is situated in the
choline binding site and has been reported to be crucial for inhibition of
human AChE.36 These important enzyme-compound interactions can be
exploited to design better inhibitors for AD, which is a destroying lives
and haunting society.
For compound 11 both the sulfur atoms form π-sulfur interactions
with the following amino acids: Tyr 341, Tyr 124, Phe 338 and Phe 297,
while the sulfur (II) atom forms a hydrogen bond with the hydroxyl
substituent of Try 337. Hydrophobic π-σ interactions were observed
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K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
Fig. 3. Two-dimensional docking poses of 6, 7, 8, 9, 11, and 12 with AChE.
6

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
Fig. 4. A: Three-dimensional representation for the interactions of 11 with human AChE. B: Docking of rivastigmine (red) and 11 (green) in the active site of human
AChE (the amino acids that interacted with rivastigmine are shown in yellow, with 11 are shown in pink and common interactions are shown in blue).
3. Conclusion
dissolved in DMF (1 M) was added dropwise a solution of benzyl halides
(1.0 equiv) dissolved in DMF. The solution was allowed to stir at room
temperature for 3 h. The reaction was controlled by TLC. After
completion the reaction was quenched with saturated aqueous NaHCO3
and the resulting mixture was extracted with CH2Cl2 (3 × 15 mL). The
combined organic extracts were dried over MgSO4, filtered and the
solvent was removed under reduced pressure. The crude residue was
purified by silica-gel column chromatography using hexane/ethyl ace-
tate mixtures to afford the desired pure compound.
Biological activities of thiosulfonates have been well documented,
however, their cholinesterase inhibitory activity has not been reported.
In this study, a range of unsymmetrical thiosulfonates were investigated
for their effect on AChE and BChE. The screening results revealed
nanomolar inhibition rendering the thiosulfonates as potential in-
hibitors of the enzymes. One candidate, S-4-isopropylbenzyl 4-methyl-
benzenthiosulfonate, presented excellent IC50 and Ki values and was
more effective than the clinically available ChEI rivastigmine. Molecular
docking simulations of S-4-isopropylbenzyl 4-methylbenzenthiosulfo-
nate in the active site of AChE provided valuable information
regarding the binding interactions of the complex. Overall, these results
provide valuable information for the design of potent ChEIs and we trust
that S-4-isopropylbenzyl 4-methylbenzenthiosulfonate may be a lead
candidate for finding new drugs for the treatment of AD.
4.3. S-benzyl 4-methylbenzenthiosulfonate (3)
Obtained as a white solid (75%); m.p. 58–59 ◦C, lit. m.p. 57–59 ◦C.42
IR (cmꢀ 1): 3288, 2931, 1590, 1556, 1507, 1418, 1327, 1275, 1139,
1074, 1016, 809, 764, 698, 654. 1H NMR (400 MHz, CDCl3): δ = 2.46
(3H, s, CH3), 4.27 (2H, s, CH2), 7.19–7.27 (5H, m, Ar-H), 7.30 (2H, d, J
= 8.4 Hz, Ar-H), 7.75 (2H, d, J = 8.4 Hz, Ar-H). 13C NMR (100 MHz,
CDCl3): δ = 21.8, 40.4, 127.1, 128.1, 128.9, 129.2, 129.9, 133.8, 142.1,
144.8. HRMS m/z (ESI): calculated for C14H18NO2S2 [M+NH4]+:
296.0773; found: 296.0763.
4. Experimental section
4.1. General procedure: chemicals
All the chemicals and reagents for the synthesis of the unsymmetrical
thiosulfonates were purchased from Sigma Aldrich (St. Louis, MO, USA)
and used as received without further purification. All solvents were
purchased from Merck (Darmstadt, Germany). Thin layer chromatog-
raphy (TLC) was used to monitor the reactions using aluminium-backed
plates coated with silica-gel F254, with visualization using ultra-violet
(UV) light. Column chromatography was carried out using silica-gel
60 mesh and petroleum ether:ethyl acetate mixtures as eluent. The un-
symmetrical thiosulfonates were stored at ꢀ 18 ◦C after they were syn-
thesized. The 1H and 13C NMR spectra were recorded on a Varian Unity
400 spectrometer at 400 MHz for 1H and 100 MHz for 13C using deu-
teriochloroform (CDCl3) as solvent. All chemical shifts are reported in
parts per million (ppm) and all coupling constants are quoted in hertz
(Hz). Melting points were measured in open glass capillary tubes on a
Stuart SMP30 Melting Point Meter and are uncorrected. Infrared spectra
were recorded on a Thermo Nicolet iS10 spectrometer. Electrospray
ionization (ESI) mass spectra were recorded using an Agilent 6200 series
TOF/6500 series instrument.
4.4. S-4-fluorobenzyl 4-methylbenzenthiosulfonate (4)
Obtained as a white solid (73%); m.p. 44–45 C. IR (cmꢀ 1): 3172,
◦
2928, 1594, 1507, 1417, 1317, 1301, 1290, 1223, 1142, 1073, 1015,
837, 813, 755, 688, 649. 1H NMR (400 MHz, CDCl3): δ = 2.40 (3H, s,
CH3), 4.19 (2H, s, CH2), 6.87 (2H, t, J = 8.4 Hz, Ar-H), 7.12 (2H, t, J =
8.4 Hz, Ar-H), 7.24 (2H, d, J = 8.4 Hz, Ar-H), 7.67 (2H, d, J = 8.4 Hz, Ar-
H). 13C NMR (100 MHz, CDCl3): δ = 20.7, 38.7, 114.8 (d, JC-F = 21.4
Hz), 126.1, 128.9, 129.9 (d, JC-F = 8.4 Hz), 141.2, 141.2, 143.9, 161.5
(d, JC-F = 244.9 Hz). HRMS m/z (ESI): calculated for C14H17FNO2S2
[M+NH4]+: 314.0679; found: 314.0670.
4.5. S-4-chlorobenzyl 4-methylbenzenthiosulfonate (5)
Obtained as a colourless oil (75%). IR (cmꢀ 1): 3401, 3062, 3031,
2985, 2931, 1604, 1496, 1450, 1373, 1303, 1211, 1072. 1H NMR (400
MHz, CDCl3): δ = 2.42 (3H, s, CH3), 4.20 (2H, s, CH2), 7.06 (2H, d, J =
8.8 Hz, Ar-H), 7.14 (2H, d, J = 8.8 Hz, Ar-H), 7.25 (2H, d, J = 8.0 Hz, Ar-
H), 7.67 (2H, d, J = 8.0 Hz, Ar-H). 13C NMR (100 MHz, CDCl3): δ = 21.8,
39.9, 127.1, 128.9, 129.9, 130.6, 132.6, 134.0, 142.0, 144.9. HRMS m/z
(ESI): calculated for C14H17ClNO2S2 [M+NH4]+: 330.0384; found:
330.0377.
4.2. General procedure for the synthesis of compounds 3–12
To a stirred solution of potassium p-toluenethiosulfonate (1.3 equiv)
7

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
4.6. S-4-nitrobenzyl 4-methylbenzenthiosulfonate (6)
4.12. S-4-tert-butylbenzyl 4-methylbenzenthiosulfonate (12)
Obtained as a white solid (75%); m.p. 118–119 ◦C lit. m.p. 120 ◦C.43
IR (cmꢀ 1): 3178, 2939, 1576, 1558, 1447, 1320, 1293, 1139, 1072,
1013, 885, 811, 705. 1H NMR (400 MHz, CDCl3): δ = 2.41 (3H, s, CH3),
4.32 (2H, s, CH2), 7.23 (2H, d, J = 8.4 Hz, Ar-H), 7.35 (2H, d, J = 8.4 Hz,
Obtained as a white solid (76%); m.p. 65–66 C. IR (cmꢀ 1): 3163,
◦
2952, 1595, 1558, 1472, 1418, 1319, 1138, 1076, 1017, 1015, 837, 807,
728, 753, 702, 651. 1H NMR (400 MHz, CDCl3): δ = 1.30 (9H, s, CH3),
2.45 (3H, s, CH3), 4.26 (2H, s, CH2), 7.13 (2H, d, J = 8.0 Hz, Ar-H),
7.27–7.29 (4H, m, Ar-H), 7.74 (2H, d, J = 8.4 Hz, Ar-H). 13C NMR
(100 MHz, CDCl3): δ = 21.8, 31.4, 34.7, 40.1, 125.9, 127.1, 129.0,
129.8, 130.7, 142.3, 144.6, 151.2. HRMS m/z (ESI): calculated for
Ar-H), 7.65 (2H, d, J = 8.4 Hz, Ar-H), 8.05 (2H, d, J = 8.4 Hz, Ar-H). 13
C
NMR (100 MHz, CDCl3): δ = 21.7, 39.4, 123.9, 127.1, 129.9, 130.0,
141.9, 142.0, 145.3, 147.5. HRMS m/z (ESI): calculated for
C
14H13NNaO4S2 [M+Na]+: 346.0178; found: 346.0171.
C
18H22NaO2S2 [M+Na]+: 357.0953; found: 357.0957.
4.7. S-4-cyanobenzyl 4-methylbenzenthiosulfonate (7)
4.13. Cholinesterase activity assay
Obtained as a white solid (78%); m.p. 78–79 C. IR (cmꢀ 1): 3566,
◦
The cholinesterase inhibition activities of the unsymmetrical thio-
sulfonates compounds were measured by the assay described by Ellman
and co-workers with some modifications applied.31 The activity assays
were conducted using human serum butyrylcholinesterase (BChE) and
recombinant human acetylcholinesterase (AChE). Acetylthiocholine io-
dide and butyrylthiocholine iodide (both purchased from Sigma-
Aldrich, USA) were used as substrates in the reactions catalyzed by
AChE and BChE, respectively. Hydrolysis of the substrates by the en-
zymes leads to the formation of thiocholine, which reacts with
5,5′dithiobis-2-nitrobenzoic acid (DTNB: Ellman’s reagent, Sigma-
Aldrich) to form the 5-thio-2-nitrobenzoate anion which exhibits
maximum absorbance at 412 nm. Briefly, the reaction mixture was
prepared in microplate wells and consisted of the test compounds 3–12
(varied concentrations in the range 1–500 nM), 0.1 M of Tris/HCl buffer
(pH 8), 25 µM of DTNB and 10 µL of enzyme solution (0.20 units/mL of
AChE and 0.22 units/mL of the BChE). The mixture was incubated at
25 ◦C for 10 min, which was followed by addition of 0.5 mM acetylth-
iocholine/butyrylthiocholine iodide to start the reaction. The total vol-
ume of the reaction mixture added up to 250 µL. Inhibitory activity was
determined by measuring the increase in absorbance at 412 nm by a
Multiskan™ GO UV/Vis microplate reader (Thermo Fisher Scientific
Spectrophotometer). The IC50 values were calculated from activity %
versus concentrations graphs, while the Ki values were obtained from
Lineweaver–Burk graphs (three different concentrations of the com-
pounds and five different concentrations of each substrate). Riva-
stigmine was used as a reference standard.
3064, 2959, 2239, 1647, 1558, 1457, 1328, 1259, 1136, 1073, 1053,
1018, 810, 742, 701, 668, 651. 1H NMR (400 MHz, CDCl3): δ = 2.44 (3H,
s, CH3), 4.28 (2H, s, CH2), 7.25 (2H, d, J = 8.4 Hz, Ar-H), 7.30 (2H, d, J
= 8.4 Hz, Ar-H), 7.50 (2H, d, J = 8.4 Hz, Ar-H), 7.66 (2H, d, J = 8.4 Hz,
Ar-H). 13C NMR (100 MHz, CDCl3): δ = 21.4, 39.5, 111.3, 118.2, 126.8,
129.7, 129.7, 132.2, 139.8, 141.6, 145.0. HRMS m/z (ESI): calculated
for C15H13NNaO2S2 [M+Na]+: 326.0280; found: 326.0268.
4.8. S-4-(trifluoromethyl) benzyl 4-methylbenzenthiosulfonate (8)
Obtained as a white solid (79%); m.p. 55–56 C. IR (cmꢀ 1): 3243,
◦
2930, 1590, 1507, 1489, 1419, 1316, 1137, 1106, 1065, 1015, 887, 813,
753, 702, 651. 1H NMR (400 MHz, CDCl3): δ = 2.32 (3H, s, CH3), 4.22
(2H, s, CH2), 7.19 (2H, d, J = 8.4 Hz, Ar-H), 7.28 (2H, d, J = 8.0 Hz, Ar-
H), 7.43 (2H, d, J = 8.4 Hz, Ar-H), 7.61 (2H, d, J = 8.0 Hz, Ar-H). 13
C
NMR (100 MHz, CDCl3): δ = 21.7, 39.8, 124.0 (q, JC-F = 270.9 Hz),
125.7 (m), 127.0, 129.5, 129.8, 130,0, 138.4, 142.2, 145.0. HRMS m/z
(ESI): calculated for C15H17F3NO2S2 [M+NH4]+: 364.0647; found:
364.0628.
4.9. S-4-(trifluoromethoxy) benzyl 4-methylbenzenthiosulfonate (9)
Obtained as a colourless oil (74%). IR (cmꢀ 1): 3155, 2931, 1594,
1559, 1507, 1457, 1325, 1255, 1211, 1136, 1075, 1018, 810, 701, 650.
1H NMR (400 MHz, CDCl3): δ = 2.41 (3H, s, CH3), 4.26 (2H, s, CH2), 7.05
(2H, d, J = 8.4 Hz, Ar-H), 7.19–7.24 (4H, m, Ar-H), 7.66 (2H, d, J = 8.0
Hz, Ar-H). 13C NMR (100 MHz, CDCl3): δ = 21.7, 39.6, 120.5 (q, JC-F
=
4.14. Molecular modeling studies
255.5 Hz), 121.3, 127.0, 129.8, 130.6, 133.0, 142.3, 145.0, 148.9.
HRMS m/z (ESI): calculated for C15H17F3NO3S2 [M+NH4]+: 380.0596;
found: 380.0589.
The 3D crystal structure of human AChE (PDB ID: 4M0E) was used,44
which was downloaded from the Protein Data Bank (https://www.rcsb.
org). The macromolecule preparation protocol for the docking analyses
was followed on AutoDockTools 1.5.6. Water molecules were removed,
non polar hydrogens were merged and Gasteiger charges were added.
The chemical 2D structures of the compounds (3–12) were drawn using
ChemDraw 19.1. The structures were then transferred to the Avogadro
program for optimizing the 3D conformations. After preparation of the
ligands and receptor, structural based computational analysis were
separately performed with both AutoDock and AutoDock Vina at the
PyRx platform.34 Discovery Studio Visualizer was utilized to analyze and
visualize the 3D models of the docked conformations and interactions.45
4.10. S-4-methoxybenzyl 4-methylbenzenthiosulfonate (10)
Obtained as a white solid (79%); m.p. 59–60 C. IR (cmꢀ 1): 3214,
◦
2936, 1653, 1540, 1507, 1457, 1318, 1247, 1174, 1137, 1075, 808, 747,
701, 649. 1H NMR (400 MHz, CDCl3): δ = 2.44 (3H, s, CH3), 3.77 (3H, s,
OCH3), 4.21 (2H, s, CH2), 6.76 (2H, d, J = 8.8 Hz, Ar-H), 7.10 (2H, d, J
= 8.8 Hz, Ar-H), 7.29 (2H, d, J = 8.2 Hz, Ar-H), 7.74 (2H, d, J = 8.2 Hz,
Ar-H). 13C NMR (100 MHz, CDCl3): δ = 21.8, 40.0, 55.4, 114.3, 125.4,
127.1, 129.9, 130.5, 142.2, 144.7, 159.5. HRMS m/z (ESI): calculated
for C15H20NO3S2 [M+NH4]+: 326.0879; found: 326.0860.
Declaration of Competing Interest
4.11. S-4-isopropylbenzyl 4-methylbenzenthiosulfonate (11)
Obtained as a colourless oil (75%). IR (cmꢀ 1): 3567, 2961, 1647,
1594, 1457, 1323, 1138, 1076, 1053, 1018, 810, 737, 701, 650, 621. 1H
NMR (400 MHz, CDCl3): δ = 1.21 (6H, d, J = 6.8 Hz, CH3), 2.43 (3H, s,
CH3), 2.85 (1H, sep, J = 6.8 Hz, CH), 4.24 (2H, s, CH2), 7.08–7.12 (4H,
m, Ar-H), 7.27 (2H, d, J = 7.8 Hz, Ar-H), 7.73 (2H, d, J = 7.8 Hz, Ar-H).
13C NMR (100 MHz, CDCl3): δ = 21.8, 24.0, 33.9, 40.3, 126.9, 127.1,
129.2, 129.8, 131.0, 142.3, 144.6, 149.0. HRMS m/z (ESI): calculated
for C17H20NaO2S2 [M+Na]+: 343.0797; found: 343.0782.
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors would like to thank Agri Ibrahim Cecen University Sci-
entific Research Council for funding (Project No: FEF.20.005).
8

K. Zilbeyaz et al.
Bioorganic & Medicinal Chemistry 40 (2021) 116194
20 Lubenets VI, Parashchyn ZD, Vasylyuk SV, Novikov VP. The S-methyl-(2-
methoxycarbonylamino-benzimidazole-5) thiosulfonate as a potential anticancer
agents. Global J Pharm Pharm Sci. 2017;3(2), 555607. https://doi.org/10.19080/G
JPPS.2017.03.555607.
Ethical statement
There is no need to provide ethical approval in terms of content and
method of this study.
21 Khodyuk RGD, Bai R, Hamel E, et al. Diaryl disulfides and thiosulfonates as
combretastatin A-4 analogues: Synthesis, cytotoxicity and antitubulin activity. Bioorg
Chem. 2020;101, 104017. https://doi.org/10.1016/j.bioorg.2020.104017.
22 Dmitryjuk M, Szczotko M, Kubiak K, et al. S-methyl-(2-methoxycarbonylamino-
benzimidazole-5) thiosulfonate as a potential antiparasitic agent - Its action on the
development of Ascaris suum eggs in vitro. Pharmaceuticals. 2020;13:332. https://doi.
org/10.3390/ph13110332.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmc.2021.116194.
23 Baerlocher FJ, Baerlocher MO, Chaulk CL, Langler RF, MacQuarrie SL. Antifungal
thiosulfonates: potency with some selectivity. Aust J Chem. 2000;53:399–402.
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