Novel benzo[b]xanthene derivatives: Bismuth(III) triflate-catalyzed one-pot synthesis, characterization, and acetylcholinesterase, glutathione S-transferase, and butyrylcholinesterase inhibitory properties

Article abstract of DOI:10.1002/ardp.202000030

In this study, 3,4-dihydro-12-aryl-1H-benzo[b]xanthene-1,6,11-(2H,12H)trione compounds were obtained through one-pot condensation of various substituted aromatic aldehydes, 2-hydroxy-1,4-naphthoquinone, and dimedone in the presence of Bi(OTf)₃ as a green and reusable catalyst. The structural characterization of these novel substituted benzo[b]xanthenes was performed by spectroscopic methods, and their inhibitory actions against butyrylcholinesterase (BChE), acetylcholinesterase (AChE), and glutathione S-transferase (GST) were investigated. GST is an enzyme responsible for removing toxic molecules during Phase II reactions in the detoxification mechanism. The AChE and BChE enzymes, which are called cholinesterases, are among the enzymes that occur especially during dementia such as brain damage or Alzheimer's disease. Inhibition effects of the benzo[b]xanthene derivatives on AChE, BChE, and GST were found at the millimolar level. The best inhibitor for GST is compound 4a (31.18 ± 6.13 mM), for AChE, it is compound 4d (28.16 ± 3.46 mM), and for BChE, it is compound 4f (36.24 ± 3.19 mM). Compound 4a inhibited the dimerization of GST subunits, and compounds 4d and 4f directly inhibited the catalytic activity by interacting with the catalytic active site or a related site of the AChE and BChE enzymes, respectively.

Full text of DOI:10.1002/ardp.202000030

Received: 13 February 2020
Revised: 29 April 2020
Accepted: 2 May 2020
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DOI: 10.1002/ardp.202000030
F U L L P A P E R
Novel benzo[b]xanthene derivatives: Bismuth(III)
triflate‐catalyzed one‐pot synthesis, characterization, and
acetylcholinesterase, glutathione S‐transferase, and
butyrylcholinesterase inhibitory properties
Kadir Turhan1
| Begüm Pektaş1 | Fikret Türkan2
| Fatma T. Tuğcu1
| Ilhami Gulcin4
|
Zuhal Turgut1 | Parham Taslimi3 | Halide S. Karaman4
1Department of Chemistry, Faculty of Art and
Sciences, Davutpasa Campus, Yildiz Technical
Abstract
University, Istanbul, Turkey
In this study, 3,4‐dihydro‐12‐aryl‐1H‐benzo[b]xanthene‐1,6,11‐(2H,12H)trione com-
pounds were obtained through one‐pot condensation of various substituted aro-
matic aldehydes, 2‐hydroxy‐1,4‐naphthoquinone, and dimedone in the presence of
Bi(OTf)3 as a green and reusable catalyst. The structural characterization of these
novel substituted benzo[b]xanthenes was performed by spectroscopic methods, and
their inhibitory actions against butyrylcholinesterase (BChE), acetylcholinesterase
(AChE), and glutathione S‐transferase (GST) were investigated. GST is an enzyme
responsible for removing toxic molecules during Phase II reactions in the detox-
ification mechanism. The AChE and BChE enzymes, which are called cholinesterases,
are among the enzymes that occur especially during dementia such as brain damage
or Alzheimer's disease. Inhibition effects of the benzo[b]xanthene derivatives on
AChE, BChE, and GST were found at the millimolar level. The best inhibitor for GST
is compound 4a (31.18 6.13 mM), for AChE, it is compound 4d (28.16 3.46 mM),
and for BChE, it is compound 4f (36.24 3.19 mM). Compound 4a inhibited the
dimerization of GST subunits, and compounds 4d and 4f directly inhibited the cat-
alytic activity by interacting with the catalytic active site or a related site of the
AChE and BChE enzymes, respectively.
2Department of Medical Services and
Techniques, Vocational School of Health
Services, Igdir University, Iğdır, Turkey
3Department of Biotechnology, Faculty of
Science, Bartin University, Bartin, Turkey
4Department of Chemistry, Faculty of Science,
Ataturk University, Erzurum, Turkey
Correspondence
Kadir Turhan, Department of Chemistry,
Faculty of Art and Sciences, Davutpasa
Campus, Yildiz Technical University, 34210
Istanbul, Turkey.
Email: kturhan@yildiz.edu.tr
Funding information
Yildiz Teknik Üniversitesi,
Grant/Award Number: FYL‐2017‐3243
K E Y W O R D S
acetylcholinesterase, enzyme inhibition, molecular docking, spectrophotometry, synthesis
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1
INTRODUCTION
Despite continued research efforts toward the development of an-
ticancer drugs, cancer remains a primary cause of death. It is well
Benzoxanthenes are tetracyclic dibenzopyrans with diverse biologi-
cal and therapeutic properties such as antibacterial, antiviral, anti‐
inflammatory, antitumor, antimalarial, and pesticidal activities.[1–9]
These heterocyclic compounds, also known as leuco dyes that are
pH‐sensitive fluorescent materials, can be used in photodynamic
therapy, polymer photo imaging systems, and laser technologies due
to the fluorescence activities of the naphthoquinone nucleus.[10–15]
established that small heterocyclic molecules are the predominant
building blocks for biologically active compounds. Xanthene, one of
these building blocks, is an important structural unit commonly found
in natural products. Molecular scaffolds of xanthene are important as
PIM1 kinase inhibitors. Epicalyx is the most potent member of
this class as an anticancer agent against human HT‐1080
fibrosarcoma and murine 26‐L5 carcinoma.[16,17]
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Arch Pharm. 2020;e2000030.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.202000030

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In recent decades, the development of a faster and more efficient
synthesis of heterocyclic compounds containing xanthene and ben-
zoxanthene scaffolds in their structures has attracted considerable
attention. A number of methodologies for the synthesis of these
compounds have been reported, which include various catalysts, such
metabolize various electrophilic compounds via a reduced glu-
tathione conjugation.[48,49]
Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)
enzymes are both expressed as cholinesterases and are very im-
portant enzymes that break down acetylcholine (ACh) into acetate
and other choline and some other choline ester molecules that
function as neurotransmitter molecules, which are considered as
drugs for Alzheimer's disease (AD).[50–52] AChE inhibitors can be
used as a therapeutic for some common diseases including AD.
Therefore, these inhibitors have been used in the treatment of some
traumatic disorders such as AD, myasthenia gravis, and glaucoma. In
addition, cholinesterase inhibitors are extensively utilized as pesti-
cides and, if misused, can produce toxic responses in humans and
mammals.[53–55]
as aqueous systems[14,16,18,19]
,
P2O5,[17] DABCO‐based ionic
liquid,[20] [NMP]H2PO4,[21] LiCl,[22] Fe3O4 nanoparticles,[23] nano‐
Fe3O4/PEG/succinic anhydride,[24] poly(4‐vinyl‐pyridinium) hydrogen
sulfate,[25] nanocatalytic Zn/MCM‐41‐SO3H,[26] CuSO4·5H2O,[27]
GaCl3,[28] AlHMS,[29] LPCAS,[30] tetrapropylammonium bromide,[31]
H‐zeolite,[32] STA,[33] and various ionic liquids.[34–36] However, most
of the catalysts in question have led researchers to search for dif-
ferent catalysts, as they have one or more drawbacks, such as being
uneconomical and useless methods, and carrying out the reaction
with lower yields.
The aim of this study was to investigate the inhibition effects of
benzo[b]xanthane derivatives, which were synthesized in the pre-
sence of a catalytic amount of Bi(OTf)3, for the first time. The com-
pounds were tested against some metabolic enzymes including AChE,
BChE, and GST enzymes, applying in vitro conditions. Inhibitors of
AChE and BChE enzymes are very important in the use of AD drugs.
GST inhibitors are emerging as promising therapeutic agents for
managing the development of resistance amongst anticancer agents.
In the field of diagnostic medicine, as well as in antiparasitic drug
development, GST inhibitors are important lead molecules.
Trifluoromethanesulfonic acids, which can be considered as su-
peracids, are one of the most acidic monoprotic organic acids, which
easily form metal triflates on reaction with most metals. In the last
few years, bismuth(III) triflate (Bi(OTf)3) has been reported as a new
and an effective catalyst. Bismuth(III) compounds showing Lewis
acidity due to poor protection of 4f electrons (lanthanide shrinkage)
have attracted great attention due to their low toxicity, low costs,
ease of use, high catalytic efficiency, and stability. Bismuth com-
pounds are also versatile reagents for the synthesis of pharmaceu-
tically interesting compounds and various organic reactions involving
the synthesis of natural products.[37–40]
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RESULTS AND DISCUSSION
The multicomponent reactions are useful methods in which three
or more reactants are combined in a single chemical step, to produce
products containing significant portions of all reactants. Reactions in
one pot are effective in forming highly functionalized small organic
molecules, which can be obtained in a single step from readily
available starting materials. In these reactions, diversity, time, labor,
cost, and waste products are minimized.[13–17,41]
2
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2.1
Chemistry
In this report, the three‐component, one‐pot condensation of lawsone
(2‐hydroxy‐1,4‐naphthoquinone), dimedone (5,5‐dimethyl‐1,3‐cyclo-
hexanedione), and aromatic aldehyde was studied in the presence of
the Bi(OTf)3 catalyst for the preparation of 3,3‐dimethyl‐12‐aryl‐3,
4‐dihydro‐1H‐benzo[b]xanthene‐1,6,11‐(2H,12H)triones (Scheme 1 and
Table 1). Bi(OTf)3 catalyst is first tried in this study in such compound
synthesis. In this study, 10 substances (4a–j) were synthesized.
Compounds 4h–j were not found in the literature.
Glutathione S‐transferase (GST; E.C.2.5.1.18) is a multifunctional
enzyme that catalyzes the first step formation of mercapturic acid
that occurs as a final product in the detoxification metabolic pro-
cess.[42,43] GST enzyme is present in the liver of mammals such as
humans, rats, and mice.[43,44] Additionally, GSTs are present in plants,
fungi, insects, and bacteria.[44–47] Mitochondrial, cytosolic, and
microsomal enzymes are GSTs' derivatives, and these enzymes
Initially, to optimize the catalytic system, we examined the effect
of the catalyst amount on the model reaction (Table 2).
SCHEME 1 The one‐pot synthesis of benzo[b]xanthene derivatives. Bi(OTf)3, bismuth(III) triflate; EtOH, ethyl alcohol

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TABLE 1 Bismuth(III) triflate‐catalyzed one‐pot synthesis of benzo[b]xanthene derivatives
Entry
Compounds
4a
R
M.p. (°C)
Yield (%)
1
263–264[9,11,13,18,20,35,37,41]
2
4b
236–237[11,13,18,35,41,42]
88
3
4
4c
4d
246–247[11,14,18,20,42]
92
85
222–225[25]
5
6
7
4e
4f
270–271[11,20,35]
86
84
89
241–242[35]
4g
256–257[13]
8
4h
249–251
81
9
4i
4j
232–233
253–254
82
80
10
Spectroscopic methods were used to elucidate the structures of all
synthesized compounds. The peaks observed between 1,682 and
1,651 cm−1 indicate the C═O stress, and the peaks formed between
3,071 and 3,000 cm−1 reveal the aromatic structure when the Fourier‐
transform infrared (FTIR) spectra are observed to clarify the structures
of the compounds obtained with analytical purity. In addition, the
singlets observed around 0.94–1.05 ppm in the 1H nuclear magnetic
resonance (NMR) spectra belong to dimedone molecule's CH3 (methyl)
protons and the peaks observed around 2.15–2.45 ppm belong to di-
medone ring's CH2 (methylene) protons. The CH proton, to which the
aromatic aldehyde is added to the ring, is observed around 5 ppm. In
addition, aromatic protons of 2‐hydroxynaphthalene‐1,4‐dione and

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TABLE 2 Effects of the catalyst amount on the reaction yield
Entry
Catalyst
Solvent
EtOH
EtOH
EtOH
Temperature/time
80°C/6 hr
Reaction medium/yield
One‐pot, reflux/89%
One‐pot, reflux/84%
One‐pot, reflux/85%
1
2
3
Bi(OTf)3, 10 mol%
Bi(OTf)3, 20 mol %
Bi(OTf)3, 30 mol %
80°C/6 hr
80°C/6 hr
Abbreviations: Bi(OTf)3, bismuth(III) triflate; EtOH, ethyl alcohol.
substituted benzaldehyde compounds involved in the formation of the
ring (4a–j) are observed at 7.56–7.80 and 8.15–8.37 ppm.
seen from the results obtained in Table 3, these compounds effec-
tively inhibited AChE, with Ki values in the range of 28.16 3.46 to
128.14 21.23 mM. However, all compounds had almost
similar inhibition profiles. The most active compound 4d showed Ki
value of 28.16 3.46 mM. For AChE, IC50 values of tacrine as a po-
sitive control and novel derivatives are expressed in the following
order: 4d (23.31 mM; r2: 0.9845) < 4f (27.36 mM; r2: 0.9763) < 4a
(49.76 mM; r2: 0.9547) < 4g (64.32 mM; r2: 0.9685) < tacrine
(116.02 mM; r2: 0.9783). For BChE, IC50 values of TAC as a positive
control of some novel benzo[b]xanthane derivatives (4a–j) are ex-
pressed in the following order: 4f (29.67 mM; r2: 0.9614) < 4a
(34.56 mM; r2: 0.9761) < 4b (36.11 mM; r2: 0.9859) < 4d (36.58 mM;
r2: 0.9487) < TAC (125.44mM; r2: 0.9815). Additionally, the novel
benzo[b]xanthane derivatives (4a–j) effectively inhibited BChE, with
Ki values in the range of 36.24 3.19 to 69.25 14.44 mM. However,
all these compounds had almost similar inhibition profiles. The most
active compound 4f showed Ki value of 36.24 3.19 mM. In the past
decade, efforts in the development of therapy for AD have focused
on improving the cholinergic neurotransmission in the brain cells.
This mechanism is largely based on the cholinergic hypothesis, which
has prompted evaluations on cholinesterase enzyme inhibitors
(ChEIs) that increase the central cholinergic neurotransmission by
inhibiting ACh degradation. Indeed, ChEI is currently considered as
We showed the possible reaction mechanism obtained from the
literature research on the general reaction equation.[13,18,43,44]
Bi(OTf)3 can activate the carbonyl groups of aldehyde 1 to decrease the
energy of transition state. Initially, there was Knoevenagel condensa-
tion of aryl aldehydes and dimedone (1,3‐dicarbonyl compound; 3), to
form 2‐[(aryl)methylene]‐5,5‐dimethylcyclohexane‐1,3‐dione (5). This
was followed by Michael addition of 2‐hydroxy‐1,4‐naphthoquinones 2
to 5. An intermediate form (6) was furnished. By keto–enol tautomer-
ization, intramolecular cyclization and dehydration gave the fused‐ring
compound 3,3‐dimethyl‐3,4‐dihydro‐12‐aryl‐2,3,4,12‐tetrahydrobenzo
[b]xanthene‐1,6,11‐trione (4a–j; Scheme 2).
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2.2
Enzyme inhibition results
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2.2.1
Cholinesterases inhibition results
All novel benzo[b]xanthane derivatives (4a–j) exhibited a significantly
higher AChE inhibitory activity than that of standard AChE inhibitors
such as tacrine. Furthermore, the Ki values of these compounds and
standard compound (tacrine) are summarized in Table 3. As can be
SCHEME 2 The plausible mechanism for the preparation of compound 4. Bi(OTf)3, bismuth(III) triflate

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TABLE 3 Inhibition results of novel benzo[b]xanthane derivatives (4a–j)
IC50 (mM)
Ki (mM)
Molecules
GST
r2
AChE
49.76
101.56
66.84
23.31
78.58
27.36
64.32
77.12
73.64
116.02
r2
BChE
34.56
36.11
41.36
36.58
38.15
29.67
44.45
54.84
42.82
125.44
r2
GST
AChE
BChE
4a
4b
4c
4d
4e
4f
28.14
42.34
44.26
42.48
16.25
58.45
51.28
31.71
48.35
–
.9652
.9756
.9512
.9825
.9841
.9645
.9785
.9687
.9618
–
.9547
.9658
.9814
.9845
.9769
.9763
.9685
.9712
.9728
.9783
.9761
.9859
.9587
.9487
.9851
.9614
.9617
.9639
.9591
.9815
31.18 6.13
58.69 11.32
49.66 9.27
45.69 7.32
43.38 10.24
66.12 13.89
66.23 9.22
51.16 8.42
50.12 9.14
–
68.47 14.12
128.14 21.23
84.21 16.24
28.16 3.46
102.25 19.43
38.41 6.33
74.38 10.42
81.42 11.16
79.46 12.38
92.34 28.12
39.14 5.66
48.56 10.94
44.28 6.28
47.63 6.24
51.65 13.02
36.24 3.19
52.16 8.42
69.25 14.44
41.68 6.66
103.28 11.26
4g
4i
4j
TAC
Note: Molecules showing the best inhibition for each enzyme are shown in bold.
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase; GST, glutathione S‐transferase.
the most promising therapy for AD, because ChEIs re‐establish the
cholinergic function in human cells by enhancing the ACh amounts in
the central neural system.[51,53]
of the compounds were poor, compared with the EA as standard, but
overall the results were good and inhibition was observed.
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2.3
Molecular docking results
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2.2.2
GST enzyme results
To understand the possible inhibition mechanism of most effective
compounds, molecular docking studies were carried out. First, drug‐
likeness properties of synthesized compounds were calculated, and
the properties are presented in Table 4. According to these scores,
all compounds were compatible with Lipinski's rule,[61] because the
compounds have a molecular weight <500 kDa, hydrogen bond
donors and octanol/water partition coefficients <5, and hydrogen
bond acceptors <10. The compound was also nontoxic and non‐
HERG blocker due to its reactive group and blockage of HERG K+
channels. Moreover, all compounds, except 4b, demonstrated ex-
cellent oral absorption and good membrane permeability. The
properties of drug similarity have shown that the compounds are
favorable to accept as a drug.
For GST enzyme, novel benzo[b]xanthane derivatives (4a–j) have IC50
values in the range of 16.25–58.45 mM and Ki values in the range of
31.18 6.13 to 66.23 9.22 mM (Table 3). The results have clearly
documented that all of these derivatives have shown the inhibitory
effects on GST. In fact, the most effective Ki values of compounds 4a
and 4e were 31.18 6.13 and 43.38 10.24 mM, respectively. For GST,
IC50 values for novel derivatives are expressed in the following order:
ethacrynic acid (EA; [2,3‐dichloro‐4‐(2‐methylenebutyryl)phenoxy]acetic
acid) as standard (0.01037 mM)[56] < 4e (16.25 mM; r2: 0.9841) < 4a
(28.14 mM; r2: 0.9652) < 4i (31.71 mM; r2: 0.9687) < 4b (42.34 mM;
r2: 0.9756) < 4d (42.48 mM; r2: 0.9825). GSTs had crucial effects in the
stage II as detoxifying enzymes that act on cytotoxic substances and
dissolve them in water and easily from the body. Many tumor cells tend
to dramatically increase GST expression, which is associated with a poor
prognosis in response to patients' anticancer treatment. The inhibition
of elevated GST levels in cells by the chronic esteric acid inhibitor
enhances the toxicity of several cancer drugs against cell lines.[57] In a
former study, newly synthesized 2‐substituted‐5‐(4‐trifluoromethyl
phenyl sulfonamido)benzoxazole derivatives were examined for their
inhibitory effects on hGST P1‐1 enzyme. According to enzyme inhibition
studies, most of the tested compounds, showed a better inhibitory ac-
tivity than the reference drug EA.[58] Recently, some studies reported
some sulfonamidobenzoxazole derivatives, which exhibit hGST P1‐1
enzyme inhibitory activities, and one of these compounds showed a
potency similar to the reference drug EA.[59,60] In this study, the results
Binding sites of the prepared enzymes were predicted and their
site scores were calculated. The scores were used to detect cataly-
tically active site, and the binding site with the highest site score was
accepted as a catalytically active site. The binding sites and cataly-
tically active sites were used for the assessment of docking hits.
The induced‐fit docking method was tested by docking the co-
crystallized ligand of the enzyme before the compounds were
docked, and the results of the tests were analyzed by root mean
square deviation (RMSD) values between the cocrystallized and re-
docked ligands. According to the results, the ligands have 0.742,
0.106, and 2.714 Å of RMSD values. The values have demonstrated
that the cocrystallized and redocked ligands are closely located in the
catalytically active site of the enzyme, as seen in Figure 1.

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TABLE 4 The drug‐likeness properties of the novel benzo[b]xanthene derivatives
Compound
rtvFGa
MWb
DHBc
AHBd
6.50
7.50
6.50
6.50
6.50
7.25
6.50
7.25
6.50
7.25
logPo/we
3.392
2.648
3.603
4.390
3.955
3.438
4.329
3.755
3.225
5.341
logHERGf
−5.519
−5.428
−5.359
−5.683
−5.498
−5.505
−5.297
−5.348
−5.480
−7.342
Cacog
logBBh
−0.475
−1.493
−0.359
−0.627
−0.314
−0.579
−0.113
−0.543
−0.542
−0.858
PMDCKi
417.988
42.157
% Hum. oral abs.j
100.000
78.437
4a
4b
4c
4d
4e
4f
0
0
0
0
0
0
0
0
0
0
384.431
429.428
402.421
426.511
463.327
414.457
453.321
479.326
390.453
490.554
0
0
0
0
0
0
0
1
0
0
855.638
102.458
868.986
847.371
863.772
847.061
1039.441
567.706
670.474
835.844
767.150
413.625
1118.988
413.461
2335.290
711.255
430.366
407.546
100.000
100.000
100.000
100.000
100.000
100.000
96.414
4g
4h
4i
4j
100.000
Abbreviation: PMDCK, Predicted apparent Madin–Darby canine kidney.
aReactive group (toxicity).
bMolecular weight.
cNumber of hydrogen bond donors.
dNumber of hydrogen bond acceptors.
eOctanol/water partition coefficient.
fIC50 value for blockage of HERG K+ channels.
gCaco‐2 cell membrane permeability.
hBrain/blood partition coefficient.
iMDCK cell permeability in nm/s.
jQualitative human oral absorption.
The values have also indicated that the docking methods were very
reliable. Then, 4a was docked into the allosteric site of GSH enzyme
due to noncompetitive inhibition, and 4d and 4f were docked into the
catalytically active site of the AChE and BChE enzymes, respectively.
The compounds demonstrated a good binding affinity against the en-
zyme. Their binding affinity is presented in Table 5. The binding affi-
nities were compatible with existing in vitro results on a large scale.
Furthermore, to understand possible inhibition mechanisms of
the molecules, their poses with best binding affinity were chosen as
the best poses, and the best poses were analyzed on the bases of
interactions with enzyme. Compound 4a, which docked into an al-
losteric site of GST, formed hydrogen bonds with Arg74 and Gln83
residues of the B subunit. In addition to this, it constructed π–cation
and π–π stacking interactions with Arg74 residue of the A subunit
and Try79 residue of the B subunit, respectively (Figures 2a and 3a).
The size of the allosteric sites was pretty small; therefore, com-
pound 4a, which was the smallest and had minimum functional
groups among the ligands, was easily located in the site. GST has
two types of ligand‐binding sites including G‐site and H‐site.
Whereas the G‐site bound GSH, H‐site bound various compounds
such as 1‐chloro‐2,4‐dinitrobenzene (CDNB).[62] The sites play a key
role in enzyme catalysis. Besides these sites, the enzyme has a di-
merization site, which is necessary for its activity.[63] Huang et al.[64]
have indicated that an electrostatic interaction between Arg70,
Arg74, Asp90, and Asp94 residues is quite important for dimer-
ization and mutation of the residues causing a loss in the catalytic
activity. Our result has shown that compound 4a caused enzyme
inhibition by blocking subunit dimerization.
FIGURE 1 The docking methodology reliability test. (a) 3LF, (b) 1YL, and (c) 92H. The cocrystallized ligands are represented in pink, blue, and
green ball‐and‐stick models, respectively. The best pose of redocked ligands is represented as gray ball‐and‐stick model

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TABLE 5 Binding affinity scores of the most effective novel benzo
[b]xanthene derivatives
stabilizing the natural substrate. The compound 4f is very closely
located to the Trp82 residue because of its OMe group. The group
has been considered to be more efficient in BChE enzyme inhibition.
Docking scores (kcal/mol)
Com-
pounds
GST
AChE
BChE
4a
−5.897
–
–
|
3
CONCLUSION
4d
–
–
−11.257
–
We aimed to contribute to the studies of the synthesis of the ben-
zoxanthene compound class. Reactions were carried out by boiling
the benzaldehyde, and benzaldehydes were substituted with 2‐
hydroxy‐1,4‐naphthoquinone and 5,5‐dimethylcyclohexane‐1,3‐dione
in a polar solvent such as ethanol using a 10 mol% bismuth triflate
catalyst. Furthermore, to carry out the reaction with heteroaromatic
aldehydes, compound 9 was synthesized (4i) using a thiophene‐2‐
carbaldehyde. Green chemistry was used during the syntheses, using
the one‐pot method and recycled Bi(OTf)3. Thus, a new development
has been introduced in the synthesis of benzoxanthene compounds.
The one‐pot method was used to contribute to green chemistry
during the syntheses by using ethanol as a solvent and recycled Bi
(OTf)3. As a result of this study, novel derivatives showed inhibition
at the millimolar levels against these enzymes. The newly synthesized
compounds were good inhibitors for GST and AChE/BChE enzymes,
respectively. Moreover, their potential inhibition mechanisms have
been revealed in this study. The results may be useful for designing
and synthesizing new metabolic enzyme inhibitors, which may fur-
ther prove as potential lead candidates for the design of novel drugs
to treat some diseases including AD and cancer in the future.
4f
–
−10.094
Abbreviations: AChE, acetylcholinesterase; BChE, butyrylcholinesterase;
GST, glutathione S‐transferase.
Compound 4d directly formed hydrogen bonds with Phe295
residue and indirectly formed hydrogen bond with Tyr72 and Ser293
residues through a water bridge. The molecule also formed π–π
stacking interactions with Trp286 and Tyr341 residues, as seen in
Figure 2b. Moreover, it formed an aromatic hydrogen bond with a
Tyr341 residue, as seen in Figure 3b. His447, Glu334, and Ser203
residues play a role in the catalytic activity of AChE enzyme. How-
ever, many AChE inhibitors inhibit the enzyme by interacting with
the peripheral site, just next to the catalytically active site.[65]
Compound 4d inhibited the enzyme by interacting with Trp286 and
Tyr341, which are key residues of the peripheral site. The peripheral
site contains many hydrophobic amino acid residues. The hydro-
phobicity tends to allow settling of a compound, including hydro-
phobic moiety, in the site. The compound exhibits more
hydrophobicity due to its isopropylphenyl moiety; therefore, it could
constitute a more favorable interaction with key residues. The mo-
lecule exhibited similar interactions with many AChE inhibitors.[66–68]
Compound 4f mainly formed a hydrophobic interaction through π–π
stacking with Trp231 and Phe329 hydrophobic residues of BChE
(Figure 2c). Besides, it directly formed an aromatic hydrogen bond
with Tyr332 and indirectly formed hydrogen bonds with Gly117 and
Thr120 via a water bridge, as seen in Figure 3c. Trp231 and Tyr332
of the interacting residues are located in the acyl‐binding site of
BChE enzyme, and the site provides a high selectivity toward the
enzyme.[69] Additionally, Košak et al.[69] have indicated that their hit
compound is more effective because it has an OMe group. The OMe
group may contribute an additional interaction with Trp82,
|
4
EXPERIMENTAL
|
4.1
Chemistry
|
4.1.1
General
All reagents and solvents were purchased from Merck, Sigma‐Aldrich,
and used without any purification. All yields refer to isolated pro-
ducts after purification. Melting points were measured by using the
FIGURE 2 A two‐dimensional interaction profile between compounds and enzymes; (a) 4a–GST, (b) 4d–AChE, and (c) 4f–BChE.
AChE, acetylcholinesterase; BChE, butyrylcholinesterase; GST, glutathione S‐transferase

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FIGURE 3 A detailed three‐dimensional interaction profile between the compounds and the enzymes. (a) 4a–GST, (b) 4d–AChE, and
(c) 4f–BChE. Enzyme structures were represented as pink‐, blue‐, and green‐colored surfaces, respectively. Amino acid residues were
represented as pink‐, blue‐, and green‐colored thick tube models, respectively. The compounds were represented as gray ball‐and‐stick model.
Hydrogen bond was represented as a black dashed line, π–π stacking interaction was represented as a blue dashed line, π–cation interaction
was represented as a green dashed line, and the aromatic hydrogen bond was represented as a turquoise dashed line. AChE,
acetylcholinesterase; BChE, butyrylcholinesterase; GST, glutathione S‐transferase
capillary tube method with an Electrothermal apparatus. Thermo-
meter correction was not performed. FTIR spectra were obtained on
a “PerkinElmer Spectrum One” FTIR spectrometer by attenuated
total reflection (ATR) technique. 1H and 13C NMR spectra were de-
termined on a “Bruker AVANCE 500 MHz” spectrometer in dimethyl
sulfoxide (DMSO)‐d6. Liquid chromatography–mass spectrometry
(LC–MS) spectra were obtained by Agilent 6200 series, TOF/6500
series, and TOF/QTOF mass spectrometer. All the chemicals,
including enzymes and other chemicals, used in the experimental
procedure were obtained from Sigma‐Aldrich Co. (Taufkirchen,
Germany).
separation of the catalyst. The crude product was purified by chro-
matographic methods.
3,4‐Dihydro‐3,3‐dimethyl‐12‐phenyl‐1H‐benzo[b]xanthene‐1,6,11‐
(2H,12H)trione (4a)
C25H20O4, MW: 384.4239 g/mol (Table 1, entry 1), yellow solid, mp.
263–264°C, yield: 87%. FTIR (ATR) ν: 3,028, 2,955, 1,739, 1,660,
.
1,506, 1,491, 1,374, 813, 764 cm−1 1H NMR (CDCl3, 500 MHz) δ:
1.05 (3H, s, CH3), 1.14 (3H, s, CH3), 2.24 (1H, d, J = 16.7 Hz, CH2),
2.30 (1H, d, J = 16.3 Hz, CH2), 2.65 (1H, d, J = 17.7 Hz, CH2), 2.73 (1H,
d, J = 17.8 Hz, CH2), 5.14 (1H, s, CH), 7.16 (1H, t, J = 7.3 Hz, Ar–H),
7.27 (2H, d, J = 7.4 Hz, Ar–H), 7.39 (2H, d, J = 7.2 Hz, Ar–H), 7.70 (2H,
dd, J = 5.7, 3.4 Hz, Ar–H), 8.00 (1H, dd, J = 5.9, 3.1 Hz, Ar–H), 8.13
(1H, dd, J = 6.0, 3.0 Hz, Ar–H) ppm. 13C NMR (CDCl3, 125 MHz) δ:
27.55, 29.26, 32.32, 32.49, 32.93, 32.97, 40.78, 50.71, 114.48,
125.51, 126.63, 126.73, 127.35, 128.65, 128.83, 130.74, 131.84,
133.85, 134.59, 142.57, 149.17, 162.91, 178.10, 183.06, 196.27 ppm.
Gas chromatography–mass spectrometry (GC–MS; EI, 70 eV):
m/z = 384 (M+).
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information Data.
|
4.1.2
General procedure for the preparation of
compounds 4a–j
A
mixture of aromatic aldehyde 1 (1 mmol), 2‐hydroxy‐1,4‐
naphthoquinone 2 (1 mmol), dimedone 3 (1 mmol), and a catalytic
amount of Bi(OTf)3 (10 mol%) in ethanol (5 ml) was refluxed for the
stipulated time at 80°C with a magnetic stirrer. The reaction was
terminated by the thin‐layer chromatography control of the crude
product, as compared with the starting materials. After completion of
the reaction, water was added to the mixture and the precipitated
solid was filtered and then washed with water and cold ethanol for
3,4‐Dihydro‐3,3‐dimethyl‐12‐(3‐nitrophenyl)‐1H‐benzo[b]xanthene‐
1,6,11‐(2H,12H)trione (4b)
C25H19NO6, MW: 429.4214 g/mol (Table 1, entry 2), yellow solid, mp.
236–237°C, yield: 88%. FTIR (ATR) ν: 3,087 2,960 1,698, 1,666,
.
1,576, 1,360, 1,322, 809, 749 cm−1 1H NMR (CDCl3, 500 MHz) δ:
1.12 (3H, s, CH3), 1.19 (3H, s, CH3), 2.28 (1H, d, J = 16.4 Hz, CH2),
2.35 (1H, d, J = 16.4 Hz, CH2), 2.71 (1H, d, J = 17.8 Hz, CH2), 2.80 (1H,

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d, J = 17.8 Hz, CH2), 5.09 (1H, s, CH), 7.45 (1H, t, J = 7.9 Hz, Ar–H),
7.63 (2H, td, J = 7.6, 1.1 Hz, Ar–H), 7.79 (2H, td, J = 7.7, 1.3 Hz, Ar–H),
7.93 (1H, d, J = 7.8 Hz, Ar–H), 7.97 (1H, dd, J = 7.8, 1.0 Hz, Ar–H), 8.03
(1H, dd, J = 8.72, 2.3 Hz, Ar–H), 8.06 (1H, brt, J = 2.0 Hz, Ar–H), 8.13
(1H, dd, J = 7.7, 1.1 Hz, Ar–H) ppm. 13C NMR (CDCl3, 125 MHz) δ:
27.67, 29.29, 29.84, 32.64, 32.67, 40.92, 50.74, 114.50, 122.71,
124.87, 129.34, 129.80, 130.12, 132.02, 135.54, 136.21, 144.67,
148.59, 156.64, 162.74, 177.54, 178.26, 196.07 ppm. LC–MS (elec-
trospray ionisation–quadrupole time‐of‐flight mass spectrometry
[ESI–QTOF]): m/z C25H19NO6 calculated 429.4214, found 430.1287
(M+H).
d, J = 17.8 Hz, CH2), 5.08 (1H, s, CH), 7.27 (2H, d, J = 8.5 Hz, Ar–H),
7.37 (2H, d, J = 8.5 Hz, Ar–H), 7.72 (2H, dd, J = 5.8, 3.3 Hz, Ar–H), 7.99
(1H, dd, J = 5.9, 3.0 Hz, Ar–H), 8.13 (1H, dd, J = 5.9, 3.0 Hz, Ar–H)
ppm. 13C NMR (CDCl3, 125 MHz) δ: 27.51, 27.53, 29.25, 29.27,
32.61, 32.63, 40.85, 50.78, 114.02, 121.35, 124.87, 126.72, 126.74,
130.57, 130.65, 131.68, 131.75, 134.01, 134.72, 141.72, 149.19,
163.06, 177.95, 183.01, 196.28 ppm. LC–MS (ESI–QTOF): m/z
C25H19BrO4 calculated 463.3196, found 465.0517 (M+2H).
3,4‐Dihydro‐3,3‐dimethyl‐12‐(4‐methoxyphenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4f)
C26H22O5, MW: 414.4498 g/mol (Table 1, entry 6), brown solid, mp.
3,4‐Dihydro‐3,3‐dimethyl‐12‐(4‐fluorophenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4c)
241–242°C, yield: 84%. FTIR (ATR) ν: 3,090, 2,933, 1,716, 1,660,
.
1,589, 1,486, 1,355, 828, 737 cm−1 1H NMR (CDCl3, 500 MHz) δ:
C25H19FO4, MW: 402.414 g/mol (Table 1, entry 3), yellow solid, mp.
0.98 (3H, s, CH3), 1.07 (3H, s, CH3), 2.16 (1H, d, J = 16.9 Hz, CH2),
2.22 (1H, d, J = 16.3 Hz, CH2), 2.56 (1H, d, J = 17.8 Hz, CH2), 2.63 (1H,
d, J = 17.8 Hz, CH2), 3.65 (3H, s, CH3), 5.01 (1H, s, CH), 6.70 (2H, d,
J = 8.8 Hz, Ar–H), 7.20 (2H, d, J = 8.8 Hz, Ar–H), 7.62 (2H, dd, J = 5.8,
3.3 Hz, Ar–H), 7.92 (1H, dd, J = 5.9, 3.1 Hz, Ar–H), 8.05 (1H, dd,
J = 5.8, 3.2 Hz, Ar–H) ppm. 13C NMR (CDCl3, 125 MHz) δ: 27.60,
27.63, 29.23, 29.26, 32.10, 32.48, 40.97, 50.97, 55.29, 114.14,
125.67, 126.60, 126.73, 129.87, 129.89, 130.85, 131.96, 133.80,
134.52, 135.01, 149.08, 158.87, 162.68, 178.14, 183.17, 196.26 ppm.
LC–MS (ESI–QTOF): m/z C26H22O5 calculated 414.4498, found
437.1362 (M+Na).
246–247°C, yield: 92%. FTIR (ATR) ν: 3,000, 2,972, 1,716, 1,651,
1,504, 1,480, 1,376, 1,157, 828, 726 cm−1
.
1H NMR (CDCl3,
500 MHz) δ: 1.04 (3H, s, CH3), 1.14 (3H, s, CH3), 2.24 (1H, d,
J = 16.3 Hz, CH2), 2.30 (1H, d, J = 16.3 Hz, CH2), 2.65 (1H, d,
J = 17.8 Hz, CH2), 2.71 (1H, d, J = 17.8 Hz, CH2), 5.11 (1H, s, CH), 6.93
(2H, t, J = 8.7 Hz, Ar–H), 7.33 (1H, d, J = 8.6 Hz, Ar–H), 7.35 (1H, d,
J = 8.6 Hz, Ar–H), 7.71 (2H, dd, J = 5.8, 3.3 Hz, Ar–H), 8.00 (1H, dd,
J = 5.7, 3.3 Hz, Ar–H), 8.13 (1H, dd, J = 5.6, 3.3 Hz, Ar–H) ppm. 13C
NMR (CDCl3, 125 MHz) δ: 27.54, 29.26, 32.29, 32.33, 32.50, 40.87,
50.84, 114.34, 115.43, 125.20, 126.70, 126.74, 130.37, 130.43,
130.71, 131.75, 133.97, 134.68, 138.59, 149.16, 162.94, 178.01,
183.08, 196.31 ppm. LC–MS (ESI–QTOF): m/z C25H19FO4 calculated
402.414, found 403.1342 (M+H).
3,4‐Dihydro‐3,3‐dimethyl‐12‐(2,4‐dichlorophenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4g)
C25H18Cl2O4, MW: 453.314 g/mol (Table 1 entry 7), yellow solid, mp.
3,4‐Dihydro‐3,3‐dimethyl‐12‐(4‐isopropylphenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4d)
256–257°C, yield: 89%. FTIR (ATR) ν: 3,000, 2,958, 1,666, 1,618,
.
1,591, 1,470, 1,373, 851, 715 cm−1 1H NMR (CDCl3, 500 MHz) δ:
C28H26O4, MW: 426.503 g/mol (Table 1, entry 4), yellow solid, mp.
1.05 (3H, s, CH3), 1.14 (3H, s, CH3), 2.22 (1H, d, J = 16.2 Hz, CH2),
2.29 (1H, d, J = 16.3 Hz, CH2), 2.63 (1H, d, J = 17.8 Hz, CH2), 2.68 (1H,
d, J = 17.9 Hz, CH2), 5.30 (1H, s, CH), 7.18 (1H, dd, J = 8.4, 2.1 Hz,
Ar–H), 7.30 (1H, d, J = 2.1 Hz, Ar–H), 7.40 (1H, d, J = 8.4 Hz, Ar–H),
7.72 (2H, dd, J = 5.8, 3.2 Hz, Ar–H), 7.99 (1H, dd, J = 5.8, 3.2 Hz,
Ar–H), 8.14 (1H, dd, J = 5.8, 3.2 Hz, Ar–H) ppm. 13C NMR (CDCl3,
125 MHz) δ: 27.76, 27.78, 29.50, 29.52, 32.16, 32.59, 41.07, 51.02,
112.86, 123.40, 127.00, 127.55, 130.45, 130.85, 131.88, 133.76,
134.06, 134.27, 134.98, 135.04, 150.12, 163.83, 178.08, 183.41,
196.67 ppm. LC–MS (ESI–QTOF): m/z C25H18Cl2O4 calculated
453.3140, found 453.0647 (M+H).
222–224°C, yield: 85%. FTIR (ATR) ν: 3,000, 2,957, 1,660, 1,617,
.
1,591, 1,378, 1,191 (C–O stretching), 836, 736 cm−1 1H NMR
(CDCl3, 500 MHz) δ: 1.00 (3H, s, CH3), 1.07 (3H, s, CH3), 1.08 (3H, s,
CH3), 1.09 (3H, s, CH3), 2.18 (1H, d, J = 16.5 Hz, CH2), 2.23 (1H, d,
J = 16.3 Hz, CH2), 2.57 (1H, d, J = 17.7 Hz, CH2), 2.66 (1H, d,
J = 17.7 Hz, CH2), 5.04 (1H, s, CH), 7.02 (1H, d, J = 8.2 Hz, Ar–H), 7.20
(2H, d, J = 8.7 Hz, Ar–H), 7.62 (2H, dd, J = 5.8, 3.3 Hz, Ar–H), 7.93 (1H,
dd, J = 5.9, 3.1 Hz, Ar–H), 8.05 (1H, dd, J = 5.9, 3.3 Hz, Ar–H) ppm. 13
C
NMR (CDCl3, 125 MHz) δ: 23.96, 27.68, 27.70, 29.21, 29.22, 32.40,
32.51, 33.76, 33.77, 40.88, 50.88, 114.54, 125.70, 126.59, 126.75,
128.61, 130.72, 131.84, 131.90, 133.81, 134.55, 139.88, 147.76,
149.02, 162.89, 178.18, 183.13, 196.44 ppm. LC–MS (ESI–QTOF):
m/z C28H26O4 calculated 426.503, found 427.187 g/mol (M+H).
3,4‐Dihydro‐3,3‐dimethyl‐12‐(2‐hydroxy‐5‐bromophenyl)‐1H‐
benzo[b]xanthene‐1,6,11‐(2H,12H)trione (4h)
C25H19BrO5, MW: 479.31936 g/mol (Table 1, entry 8), brown solid,
3,4‐Dihydro‐3,3‐dimethyl‐12‐(4‐bromophenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4e)
mp. 249–251°C, yield: 81%. FTIR (ATR) ν: 3,210, 2,955, 1,672, 1,631,
.
1,574, 1,477, 1,362, 814, 728 cm−1 1H NMR (CDCl3, 500 MHz) δ:
C25H19BrO4, MW: 463.3196 g/mol (Table 1, entry 5), yellow solid, mp.
0.96 (3H, s, CH3), 1.04 (3H, s, CH3), 2.03 (1H, d, J = 16.0 Hz, CH2),
2.24 (1H, d, J = 16.0 Hz, CH2), 2.42 (1H, d, J = 17.7 Hz, CH2), 2.54 (1H,
d, J = 17.7 Hz, CH2), 5.02 (1H, s, CH), 7.12 (1H, d, J = 2.0 Hz, Ar–H),
7.27 (1H, dd, J = 8.6, 2.4 Hz, Ar–H), 7.37 (1H, dd, J = 8.7, 2.4 Hz,
Ar–H), 7.84 (2H, dd, J = 6.9, 2.1 Hz, Ar–H), 7.93 (1H, dd, J = 6.9,
270–271°C, yield: 86%. FTIR (ATR) ν: 3,067, 2,955, 1,660, 1,619,
.
1,590, 1,466, 1,369, 829, 715 cm−1 1H NMR (CDCl3, 500 MHz) δ:
1.04 (3H, s, CH3), 1.14 (3H, s, CH3), 2.24 (1H, d, J = 16.4 Hz, CH2),
2.30 (1H, d, J = 16.3 Hz, CH2), 2.65 (1H, d, J = 17.7 Hz, CH2), 2.71 (1H,

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2.1 Hz, Ar–H), 8.03 (1H, dd, J = 6.8, 2.1 Hz, Ar–H) ppm. 13C NMR
(CDCl3, 125 MHz) δ: 27.69, 27.74, 29.58, 29.62, 32.36, 32.50, 41.27,
51.12, 115.34, 116.08, 118.33, 121.61, 126.90, 127.87, 129.51,
130.77, 133.46, 134.44, 148.25, 150.55, 164.73, 177.55, 181.80,
195.50 ppm. LC–MS (ESI–QTOF): m/z C25H19BrO5 calculated
479.3193, found 479.0573 (M+H).
enzyme activity were evaluated by using their different concentra-
tions, similar to the concentration range used in the previous
study.[57]
|
4.2.2
AChE/BChE activities
3,4‐Dihydro‐3,3‐dimethyl‐12‐thienyl‐1H‐benzo[b]xanthene‐1,6,11‐
(2H,12H)trione (4i)
The AChE/BChE inhibitory effects of benzo[b]xanthane derivatives
was measured according to the method of Ellman et al.,[70] con-
forming to the previous studies.[71] It was spectrophotometrically
measured at 412 nm using acetylthiocholine iodide and butyrylcho-
C23H18O4S, MW: 390.45162 g/mol. (Table 1, entry 9), yellow solid,
mp. 232–233°C, yield: 82%. FTIR (ATR) ν: 3,072, 2,956, 1,659, 1,616,
1,592, 1,468, 1,377, 815, 705 cm−1
.
1H NMR (CDCl3, 500 MHz) δ:
line iodide as a substrate in reactions, and 5,5′‐dithiobis(2‐
1.13 (3H, s, CH3), 1.15 (3H, s, CH3), 2.24 (1H, d, J = 16.7 Hz, CH2),
2.30 (1H, d, J = 16.3 Hz, CH2), 2.64 (1H, d, J = 17.8 Hz, CH2), 2.73 (1H,
d, J = 17.8 Hz, CH2), 5.30 (1H, s, CH), 6.86 (1H, dd, J = 5.1, 3.6 Hz,
Ar–H), 7.01 (1H, dd, J = 3.5, 0.9 Hz, Ar–H), 7.10 (1H, dd, J = 5.1,
1.1 Hz, Ar–H), 7.73 (2H, dd, J = 7.0, 2.1 Hz, Ar–H), 8.07 (1H, dd,
J = 6.9, 2.1 Hz, Ar–H), 8.14 (1H, dd, J = 6.8, 2.2 Hz, Ar–H) ppm.
13C NMR (CDCl3, 125 MHz) δ: 27.33, 27.38, 27.64, 27.66, 29.34,
29.37, 40.88, 50.85, 114.06, 124.57, 126.73, 126.81, 127.22, 130.73,
131.78, 133.98, 134.70, 145.99, 148.94, 163.35, 178.00, 182.96,
196.20 ppm. LC–MS (ESI–QTOF): m/z C23H18O4S calculated
390.4516, found 391.0988 (M+H).
nitrobenzoic acid) was used to take the activity measurements of
AChE and BChE enzymes (Table 3). In the Ki study for these enzymes,
3 separate Ki values were obtained and their averages were calcu-
lated, and the standard deviations are presented in Table 3. For IC50
,
the procedure was performed three times, and the averages are
presented in Table 3.
|
4.3
Molecular docking studies
The possible inhibition mechanism of compounds, which most ac-
tively inhibited GSH, AChE, and BChE enzymes, was detected using
induced‐fit docking method (Small Drug Discovery Suites package,
Schrodinger, LLC). Two‐dimensional structures of synthesized com-
pounds were sketched and their three‐dimensional structures were
obtained. The compounds were prepared using the LigPrep module,
according to the method described in previous studies.[72,73]
Crystal structures of GST (PDB ID: 5J41), AChE (PDB ID: 4M0E),
and BChE (PDB ID: 5NN0) enzymes obtained from RCSB Protein Data
Bank were used in molecular docking studies. The structures were
chosen due to their high resolution and cocrystallized ligand. Then, the
structures were prepared for docking studies with the protein pre-
paration wizard module.[74] The protein preparation was performed
according to the method described in previous studies.[75,76] Binding
sites of enzymes were predicted using the Sitemap module for de-
tection of catalytically active site and allosteric site.[56] Binding site
prediction and evaluation of the sites were performed according to the
method described in previous studies.[77] The induced‐fit docking
method was tested with the redocking process. Following molecular
docking studies of most effective compounds were performed using
induced‐fit docking module.[78]
3,4‐Dihydro‐3,3‐dimethyl‐12‐(4‐benzyloxyphenyl)‐1H‐benzo[b]‐
xanthene‐1,6,11‐(2H,12H)trione (4j)
C32H26O5, MW: 490.54584 g/mol (Table 1, entry 10), yellow solid, mp.
232–233°C, yield: 82%. FTIR (ATR) ν: 3,089, 3,065, 2,962, 2,935, 1,714,
1,665, 1,585, 1,482, 1,354, 826, 733 cm−1. 1H NMR (CDCl3, 500 MHz) δ:
0.99 (3H, s, CH3), 1.08 (3H, s, CH3), 2.15 (1H, d, J = 16.4 Hz, CH2), 2.20
(1H, d, J = 16.4 Hz, CH2), 2.53 (1H, d, J = 17.7 Hz, CH2), 2.60 (1H, d,
J = 17.7 Hz, CH2), 5.09 (1H, s, CH), 5.65 (2H, s, CH2), 6.70 (2H, dd,
J = 8.5, 2.1 Hz, Ar–H), 7.27 (2H, dd, J = 8.6, 2.1 Hz, Ar–H), 7.32 (1H, t,
J = 7.9 Hz, Ar–H), 7.40 (2H, dd, J = 7.9, 1.9 Hz, Ar–H), 7.51 (2H, d, J = 7.7,
2.2 Hz, Ar–H), 7.62 (2H, dd, J = 5.8, 3.3 Hz, Ar–H), 7.92 (1H, dd, J = 5.9,
3.1 Hz, Ar–H), 8.05 (1H, dd, J = 5.8, 3.2 Hz, Ar–H) ppm. 13C NMR
(CDCl3, 125 MHz) δ: 27.50, 27.53, 29.13, 29.16, 32.15, 32.58, 40.87,
50.94, 55.32, 114.24, 126.67, 126.80, 126.93, 127.12, 127.23, 127.66,
128.54, 128,76, 129.77, 129.99, 130.55, 131.76, 133.79, 134.41,
135.13, 136.71, 149.12, 157.66, 161.98, 177.04, 183.01, 195.96 ppm.
LC–MS (ESI–QTOF): m/z C23H18O4S calculated 390.4516, found
391.0988 (M+H).
|
4.2
Biochemical studies
ACKNOWLEDGMENTS
This study was supported by the Research Fund of the Yildiz Tech-
nical University (Project Number: FYL‐2017‐3243). Also, the authors
are grateful to Dr. Muhammet Karaman for providing technical gui-
dance during the docking study of this article and for supporting
small drug discovery software.
|
4.2.1
GST activity
The GST activity was measured at 25°C using CDNB as a substrate.
The assay system included a phosphate buffer (pH 6.5), GSH
(20 mM), and CDNB (25 mM). A spectrophotometer (UV mini 1240;
Shimadzu, Japan) was used to estimate the changes in absorbance at
340 nm for 3 min. The inhibitory effects of novel compounds on GST
CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.

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ORCID
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