Molekul, Vol. 20. No. 2, July 2025: 236 – 247

Articles

MOLEKUL
eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.10089

Synthesis, Molecular Docking, and In Vitro Activity Test of Thioxanthenol and Nitrothioxanthone
Derivatives As Anticancer Agents
Putri Dian Anggraeni1, Jumina Jumina1*, Chairil Anwar1, Yehezkiel Steven Kurniawan1
1

Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Gadjah Mada,
Yogyakarta 55281, Indonesia
*Corresponding author email: jumina@ugm.ac.id
Received December 18, 2023; Accepted July 09, 2025; Available online July 20, 2025

ABSTRACT. This research aimed to compare, synthesise, study molecular docking, and test the anticancer activity of
thioxanthenol, 1-hydroxythioxanthone, 4-nitrothioxanthone, and 2-nitrothioxanthone compounds through in silico and in
vitro assays, highlighting their selective cytotoxicity and potential as novel anticancer scaffolds. These four compounds were
obtained through reduction and nitration reactions of the thioxanthone. Thioxanthenol compound was obtained through the
reduction of thioxanthone using sodium borohydride. The 1-hydroxythioxanthone, 4-nitrothioxanthone, and 2nitrothioxanthone compounds were obtained from the nitration of thioxanthone compounds. The compounds were
characterised using FTIR, GC-MS, 1H-NMR, and 13C-NMR. In vitro cytotoxicity tests were performed using microtetrazolium
(MTT) assays against T47D, WiDr, and Hela cancer cell lines and the Vero cell line as normal cells. The molecular docking
process was studied to determine the in silico activity of the compounds with protein targets. The reduction reaction produced
the thioxanthenol compound as a yellowish-white solid in 40.63% yield. The nitration reaction produced 1hydroxythioxanthone, 4-nitrothioxanthone, and 2-nitrothioxanthone compounds as light-yellow solids in 33.54%; 29.27%;
and 31.71% yield, respectively. The synthesized compounds demonstrated selective anticancer activity against certain cancer
cells. Thioxanthenol compound showed an IC50 value of 17.46 µg mL-1 on the WiDr cell line and nitrotioxanthone compound
showed an IC50 value of 6.05 µg mL-1 on the T47D cell line. Molecular docking showed that the thioxanthone derivatives
might act as the anticancer agent through inhibition of epidermal growth factor receptor (EGFR), P-glycoprotein, and Erα
functions.
Keywords: anticancer, nitrothioxanthone, thioxanthenol, thioxanthone

INTRODUCTION
Pharmacology and chemistry scientists are
constantly researching potential pharmaceutical
compounds to provide new drugs against diseases
with lower toxicity effects. According to GLOBOCAN
2022 data, over 19.9 million new cancer cases and
nearly 9.7 million cancer deaths were reported
worldwide. The most common cancers include breast,
lung, and colorectal cancers, highlighting the urgent
need for effective and selective anticancer agents (Bray
et al., 2024). However, anticancer drugs often
become resistant and have low selectivity, destroying
normal cells alongside cancer cells. Thioxanthone
compounds are well-considered anticancer agents
due to their bioactivity and easy substitution, especially
hydrogen abstraction (Zhao et al., 2020).
Thioxanthenol is synthesized from thioxanthone using
sodium borohydride or lithium aluminium hydride
reagents. Reduction reactions have been carried out
using thioxanthone sulfoxide and sodium borohydride
catalysts (Ternay and Chasar, 1967). Amanatie et al.
(2017) nitrated xanthone using nitric acid, producing
a yellowish-brown solid of 2-nitroxanthone.

On the other hand, hydroxyxanthone, a potent
anticancer agent with a small IC50 value, has been
shown to be effective in treating MCF-7, WiDr, and
HeLa cell lines (Fatmasari et al., 2022).
Chlorothioxanthones
like
4-chloro-3-(4chlorophenylthio)-9H-thioxanthen-9-one and 3-(4bromophenylthio)-4-chloro-9H-thioxanthen-9-one
provide good inhibition to cancer cells (Chen et al.,
2019). These compounds inhibit P-glycoprotein
through the 4-alkoxyl or 4-hydroxyl group and amino
to the C-1 position. They can also inhibit polymer
inclusion membrane (PIM1) proteins through
hydrophobic interactions with Ile104, Leu120,
Val126, Leu174, and Ile185 amino acid residues
similar to compound HM107-g (Palmeira et al.,2012;
Brikci-Nigassa et al., 2020).
Thioxanthone derivatives can also inhibit Plateletderived growth factor receptors (PDGFR) and
epidermal growth factor receptor (EGFR) proteins. It
was reported that 4-iodo-1,3-dihydroxy-thioxanthone
has the strongest binding energy to Cys673 and
Met769 amino acid residues in PDGFR and EGFR,
respectively. The 4-iodo-1,3-dihydroxy-thioxanthone

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can also perform hydrophobic interactions at Phe723,
Val726, Ala743, Leu844, and Thr854 amino acid
residues of EGFR. Inhibition against xanthotoxol is also
achieved through hydrophobic interactions and polar
hydrogen interactions (Acharya et al., 2019;
Hermawan et al., 2020). The research aimed to
synthesize thioxanthenol and nitrothioxanthone as the
anticancer compounds. These compounds were
obtained from the reduction and nitration reactions of
thioxanthone. These compounds were tested for
anticancer activity using the MTT assay method against
various cancer cells, and then in silico investigation
was performed to understand their anticancer
mechanism.

The solvent was removed by evaporation, and the
solid was recrystallized from methanol. The product
was characterized using FTIR, NMR, and GC-MS
instruments.
Anticancer Activity Test Against Cancer Cells
Cytotoxicity test on cancer cells was carried out by
cell proliferation method. The cell lines were thawed,
added with trypsin, and counted. Cell line (100 µL)
was placed into each well on a 96-well plate and
incubated for 24 hours. Sample (5 mg) was dissolved
in DMSO (100 µL) and then samples were diluted in a
culture media with a series concentration of 7.81,
15.62, 31.25, 62.5, 125, 250, and 500 g mL-1.
Aliquots (100 µL) of each concentration were added to
each well. The cell line was incubated again for 24
hours. The culture medium was removed and cleaned
with PBS. The MTT solution (100 µL) was added to a
96-well plate and incubated for 4 hours. A stopper
solution (100 µL) was then added to each well. The
plate was incubated overnight at room temperature.
The absorbance of the solution was recorded using an
ELISA reader at a wavelength of 595 nm to calculate
IC50 values.
Molecular Docking
The molecular docking process begins by
downloading the 3D structure of a protein from the
Protein Databank website. The proteins used as the
target in this study are epidermal growth factor
receptor (EGFR) protein tyrosine kinase with PDB ID of
1M17 (Stamos el al., 2002), P-glycoprotein with PDB
ID of 2HYD (Dawson and Locher, 2006), and estrogen
receptor alpha (ERα) with PDB ID of 3ERT (Shiau et al.,
1998). Preprepared protein targets and native ligands
are processed through Autodock Vina software (Trott
& Olson, 2010), and the grid position is determined
to obtain a ligand conformation with a Root Mean
Square Deviation (RMSD) value less than 2 Å. If these
conditions are met, the molecular docking process
between the target protein and the synthesized
compound can be carried out. The used grid box size
is 20×20×20 Å. The structure of the synthesized
thioxanthone derived compound is optimized before
molecular docking with the protein target using
Gaussian 5.0 software.

EXPERIMENTAL SECTION
Materials and Instruments
The materials used in this study were 97%
thioxantone, ethanol, glacial acetic acid, nitric acid,
sulfuric acid, tetrahydrofuran, dichloromethane,
diethyl ether, anhydrous sodium sulfate (Merck), and
sodium borohydride (Loba Chemie). The materials
used for the anticancer test included T47D breast
cancer cells, HeLa cervical cancer cells, WiDr colon
cancer cells, Vero normal cells, Dulbecco's Modified
Eagle Medium (DMEM), Medium 199, Fetal Bovine
Serum (FBS), Phosphate Buffer Solution (PBS), 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT), pen strep, fungizone, doxorubicin,
cisplatin, 5-flourouracil, trypsin enzyme, and sodium
dodecyl sulfate (SDS). This study used various
equipment for synthesized products, including a rotary
evaporator Buchi type R-114, an Electrothermal
melting point device 9100, Shimadzu Prestige-21 for
infrared spectra, JEOL JNMECA 500 MHz for NMR
spectra, and GC-MS QP2010S Shimadzu for GC-MS
analysis. The anticancer assay equipment included a
Sartorius mass balance, Hirayama autoclave,
Labconco airflow, conical tube, Heraeus 5% CO2
incubator, DIVAC vacuum filter, counting device,
Axiovert 25 inverted microscope, Thermolyne vortex,
Eppendorf tube, Sorvall centrifuge, and BIO-RAD
Benchmark ELISA reader.
Synthesis of Thioxanthenol
Thioxanthone (0.32 g, 1.51 mmol) was dissolved
in 50 mL of ethanol:tetrahydrofuran (4:1) and stirred
for 15 minutes. The solution was then added to
sodium borohydride and refluxed for 2 hours. TLC was
used to monitor the reaction progress. Then, distilled
water was added to precipitate the product and the
solid was filtered and dried. The FTIR, NMR, and GCMS instruments were used to characterize the product.
Synthesis of Nitrothioxanthone
Thioxanthone of (2.45 g, 11.54 mmol) was
dissolved in glacial acetic acid and stirred at 0 °C for
10 minutes. Nitric acid (1.0 mL) and sulfuric acid (1.3
mL) were added, and the mixture was stirred for 8
hours. The reaction was monitored using TLC. The
resulting yellow solid was filtered, washed, and dried.

RESULT AND DISCUSSION
Synthesis of Thioxanthenol Through Reduction
Reaction
Thioxanthone compounds have the same structure
as xanthone compounds, but with a sulfur atom in
their cyclic ring. Both compounds have carbonyl
groups that can be reduced to hydroxyl group (Ternay
and Chasar, 1967). The reduction reaction occurs by
attacking the carbonyl group on the thioxanthone
cyclic ring, forming a C–H bond and a C–O (alkoxy
anion), and breaking this carbonyl bond to produce
an alcohol. This study used a NaBH4 reagent and a
combination of solvents with an ethanol ratio of 4:1.
The synthesis produced thioxanthenol in 40.63% yield

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as a light yellow solid with a melting point of 98.50
°C. The melting point of the synthesized thioxanthenol
in this work is in accordance with the reference
literature, which is 97.40-105 °C (Chemicalbook,
2016). Overall synthetic route is illustrated in Scheme
1. Thioxanthenol was characterized using FTIR, GCMS, 1H-NMR, and 13C-NMR spectrometers. The FTIR
spectra (Figure 1) show a shrinking carbonyl peak at
3310 cm-1, indicating a successful reduction of the
carbonyl group to a hydroxyl group.
The GC-MS characterization revealed the
formation of thioxanthenol compound as a dominant
peak at 25.70 minutes with relative abundance of
63.84% and molecular ion fragment of 214.
Thioxanthenol produced a fragmentation pattern with
abundant fragments at m/z = 197. Two other peaks,
thioxanthene and thioxanthone were also found at
retention times of 22.06 and 28.42 minutes with

percentages of 23.93% and 12.23%, respectively.
These compounds formed due to unstable
thioxanthenol, causing it to dissociate into
thioxanthene and thioxanthone. This also occurred in
the
xanthydrol
compound
which
was
disproportionated into xanthone and xanthene (Shi et
al., 2021).
The thioxanthenol compound was characterized
using 1H-NMR and 13C-NMR in DMSO-d6 solvent. The
1
H-NMR spectrum (Figure 2) revealed five types of
aromatic protons, with protons 1 and 8 appearing as
doublets, protons 2 and 7 appearing as triplets,
protons 3 and 6 appearing as triplets, and protons 4
and 5 appearing as doublets. The C–OH proton was
found as a doublets at 6.37 ppm. The 13C-NMR
spectrum (Figure 3) revealed six carbon atoms in the
aromatic ring and one carbon atom binding to
hydroxyl (–OH).

Figure 1. The 1H-NMR spectrum of thioxanthenol compound

Figure 2. The 13C-NMR spectrum of thoxanthenol compound
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Figure 3. The 13C-NMR spectrum of thioxanthenol compound
Synthesis of Nitrothioxanthone Through Nitration
Reaction
The thioxanthone was nitrated to produce 2nitrothioxanthone due to its aromatic ring at para and
ortho positions. Amanatie et al. (2017) has been
successfully synthesized 2-nitroxanthone using nitric
acid in glacial acetic acid. In this work, the
nitrothioxanthone compound was obtained in the form
of a mixture of light-yellow solids in 66.99% yield with
a melting point of 276 °C. GC-MS characterization
reveals three products, i.e., 1-hydroxythioxanthone
(33.63%), 4-nitrothioxanthone (29.46%), and 2nitrothioxanthone (31.79%). This synthetic pathway is
outlined in Scheme 2. The mass spectrum of 1hydroxythioxanthone was identified at m/z = 228,
while the nitroxanthone products were detected at 257
m/z. The fragmentation pattern of each compound is
shown in Figure 4.
The formation of 1-hydroxythioxanthone product in
nitration reactions occurs when positive species NO2+
binds with the π-aromatic system through oxygen or
H+ cation. The double bond stabilizes the intermediate
compound, and partially positively charged carbon
atoms interact with partially negatively charged
oxygen atoms to form hydroxy substituents. On the
other hand, both 4-nitrotioxanthone and 2nitrothioxanthone compounds are directly formed by
the reaction between NO2+ agent and the aromatic
ring. The FTIR spectrometer (Figure 5) reveals hydroxy
group at 3441 cm-1, indicating the 1hydroxythioxanthone compound. Nitro absorption
signals at 1574 and 1342 cm-1, correspond to the
presence
of
4-nitrothioxanthone
and
2nitrothioxanthone. The other absorption peaks at
1442, 1589, 2924, and 1172 cm-1 represent aromatic
C=C, Csp2-H, and C–S–C bonds, respectively.
The structures of nitrothioxanthone compounds
were also analyzed using 1H-NMR and 13C-NMR
spectrometers in DMSO-d6 solvent. The 1H-NMR
spectra revealed 12 types of protons from 1-

hydroxyithioxanthone, 4-nitrothioxanthone, and 2nitrothioxanthone compounds as shown in Figure 6.
Meanwhile, the 13C-NMR spectrum (Figure 7) reveals
12 aromatic carbon atoms of 1-hydroxythioxanthone,
4-nitrothioxanthone, and 2-nitrothioxanthone with 3
carbon atoms binding to hydroxy and nitro groups.
Anticancer Activity Assay of Thioxanthenol and
Nitrothioxanthone Compounds
The American National Cancer Institute (NCI)
determine the effectiveness of a compound's inhibition
activity based on the IC50 value obtained during the
MTT assay method. The category of the effectiveness
of anticancer agents is as follows: a very active
inhibitor (IC50 < 20 µg mL-1), a moderate inhibitor
(IC50 is between 21–200 µg mL-1), and an inactive
inhibitor (IC50 is between 201–500 µg mL-1).
All synthesized compounds were purified via
recrystallization prior to biological testing, and their
structures were confirmed through FTIR, NMR, and
GC-MS analyses. The study conducts an anticancer
assay on various cancer cells (T47D, HeLa, and WiDr)
and Vero normal cells and the results are listed in
Table 1. Results showed that thioxanthenol was highly
active in inhibiting WiDr cancer cells and was
moderately active against T47D cancer cells.
Meanwhile, nitrothioxanthone compounds were highly
active in inhibiting T47D cancer cells. On the other
hand, WiDr cancer cells are very well inhibited by
thioxanthone compounds with IC50 values smaller than
the 5-fluorouracil. Thioxanthone also showed
moderate inhibition to HeLa cells but was inactive
against T47D cancer cells.
The selectivity index measures a compound's
selectivity, demonstrating its effectiveness as the
anticancer agent. A score below 3 indicates nonselectivity, while a score above or equal to 3 indicates
high selectivity (Sirait et al., 2019). Thioxanthone,
thioxanthenol, and nitrothioxanthone compounds
exhibit a high selectivity index (SI≥3) on T47D,
HeLa, and/or WiDr cancer cells as shown in Table 2.

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Figure 4. Possible fragmentation pattern of (a) 1-hydroxythioxanthone, (b) 4nitrothioxanthone, and (c) 2-nitrothioxanthone

Figure 5. The FTIR spectra of nitrothioxanthone (red line) and thioxanthone (black line) compounds
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Figure 6. The 1H-NMR spectrum of nitrated thioxanthone

Figure 7. The 13C-NMR spectrum of nitrated thioxanthone
Table 1. Anticancer activity assay result
Compounds
Thioxanthone
Thioxanthenol
Nitrothioxanthone
Doxorubicin
Cisplatin
5-Fluorouracil

T47D
6496.36
70.88
6.05
0.21
-

IC50 (µg mL-1)
HeLa
WiDr
158.85
5.74
2441.00
17.46
41412.30
402.55
68.80
36.89
241

Vero
969.66
234.50
142888.00
115.42
-

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Table 2. The selectivity index of the test compounds
Compounds
Thioxanthone
Thioxanthenol
Nitrothioxanthone

Selectivity index
T47D
0.11
3.31
23637.39

HeLa
6.10
0.10
3.45

These findings indicate that the synthesized
compounds possess selective cytotoxic activity
depending on the cancer cell type, suggesting
differences in cellular sensitivity or compound uptake.
The relatively low IC₅₀ values in certain cell lines reflect
strong antiproliferative potential, especially for WiDr
and T47D. Such selectivity is important in anticancer
drug development as it may reduce toxicity to nontarget tissues. However, to fully understand the
therapeutic potential of these compounds, further
studies are required to elucidate the specific cellular
mechanisms involved in their I nhibitory effects. As
a preliminary study, we employed molecular docking
process to observe whether these synthesized
compounds against three well-known targeted protein
receptors for cancer therapies, i.e., EGFR, Pglycoprotein, and ERα. EGFR regulates cancer cells’
growth, as well as their proliferation, differentiation,
and migration while P-glycoprotein and ERα are
crucial for drug efficacy and DNA activation in cancer
cells, respectively.
Molecular
Docking
of
Thioxanthenol
and
Nitrothioxanthone Compounds
Molecular docking is a method used to predict the
binding affinity and bioactivity of a compound in silico.
It involves two stages: redocking and docking.
Redocking is used to establish a valid docking method
based on ligand conformation and RMSD value.
Docking with a proposed compound involves the
compound acting as a ligand with a protein separated
from its native ligand. A smaller binding affinity value
indicates a stronger interaction (Astalakshmi et al.,
2022). The thioxanthone derivatives in this work were
geometry-optimized using Gaussian 5.0 software. The
density functional theory (DFT) and semi-empirical
methods were used for optimization, as they don't
significantly affect the bonding of the compound with
its target molecule. Therefore, this study used the PM3
semiempirical method for the optimization of the
proposed compounds, i.e., thioxanthenol, 2nitrothioxanthone, 4-nitrothioxanthone, and 1hydroxythioxanthone.
The PM3 semiempirical method was selected due
to its superior performance in previous studies
compared to PM6 (Male et al., 2018). In this study,
PM3 was used solely for initial geometry optimization
to provide a reasonable starting structure for
molecular docking. However, it is important to note
that AutoDock Vina applies its own internal force field
and conformational sampling, and therefore does not

WiDr
168.96
13.43
354.95

strictly rely on the starting geometry during the docking
process. PM3 is faster and more accurate than ab
initio and AM1. It is particularly effective
for
compound structures with nitrogen atoms, such as –
NH2, –NHR, and –NR2 in quinoacridinium compounds
(Hadanu, 2019). The PM3 semi-empirical method
optimizes the proposed compounds' structures before
docking them with target receptors, i.e., EGFR, Pglycoprotein, and ERα proteins. It involves
understanding the formed interactions
through
hydrogen, hydrophobic, and van der Waals bonds.
Hydrogen bonds are stronger and more stable, while
hydrophobic bonds are formed between nonpolar
molecules that cannot form hydrogen bonds with
water molecules. While, van der Waals bonds are
weaker than hydrophobic bonds (Luanphaisarnnont,
2009).
EGFR Protein
A compound has been evaluated as the EGFR
inhibitor, which EGFR regulates cancer cells’ growth,
as well as their proliferation, differentiation, and
migration. A molecular docking study was conducted
to determine the interaction between the compound
and the EGFR protein. The native ligand (erlotinib)
obtained a redocking pose similar to the previous one
with RMSD value of 1.54 Å so that this redocking
method can be used to dock the proposed compound
to EGFR protein. The erlotinib ligand forms a hydrogen
bond with the Gln767 amino acid residue. This result
is in accordance with the results of previous research
conducted by Hermawan et al. in 2019. Hydrogen
bonds were formed at the redocking stage, with weak
hydrogen bonds formed by the interaction of the
oxygen atoms of Leu694, Gly772, and Gln767 with
the erlotinib compound. Additional interactions
occurred in hydrophobic and van der Waals bonds.
The thioxanthenol compound (Figure 8a and
Figure 8e) forms strong hydrogen bonds with the
EGFR's amino acid residues, demonstrating its strong
inhibitory activity. The formed interactions also involve
hydrogen bonds with Asp381 and Thr830. The van
der Waals occurs at the main amino acid residues, i.e.,
Glu738, Met742, and Thr766, which also bind to the
native ligand erlotinib. In molecular docking, the
more interactions that occur, the stronger the binding
energy (Ferreira et al., 2015). The binding affinity
value between the compound and the protein is
slightly higher than the native ligand which is -7.40
kcal mol-1, possibly due to the different hydrogen
bonds formed.

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This study also analyzed the molecular docking of
nitrated compounds of thioxanthone, resulting in 1hydroxythioxanthone, 2-nitrothioxanthone, and 4nitrothioxanthone, to determine their effectiveness in
inhibiting EGFR. The compounds interact through van
der Waals and hydrophobic bonds, with no hydrogen
bond interaction between the compounds and
proteins. The 1-hydroxythioxanthone (Figure 8b dan
Figure 8f) formed new bonds with Leu694, Met769,
and Asp831, while the 4-nitrotioxanthone (Figure 8c
dan Figure 8g) generated new bonds with Met769 and
Leu694 amino acids. The 2-nitrothioxanthone (Figure
8d dan Figure 8h) formed new bonds with Met769,
Leu764, and Glu767 amino acids. Hydrophobic
bonds form with thioxanthone-derived compounds
with nitro substitutions have smaller binding affinity
and energy production, resulting in more stable
ligand bonds and increased activity with protein (Fu et
al., 2018; Murthy and Bala Narsaiah, 2019). The 2-

nitrothioxanthone compound has a smaller binding
affinity of -8.00 kcal mol-1, indicating a stronger bond
between the protein and the compound. This differs
from the hydrogen bond found in Hermawan et al.
(2019) research. The docking data shows that 2nitrothioxanthone compounds inhibit EGFR, blocking
tyrosine-kinase from binding to the receptor, making
it inactive for cancer cell growth.
P-glycoprotein
P-glycoprotein, a crucial protein in cell entry and
exit, is crucial for drug efficacy in cancer cells.
Proposed compounds inhibit P-glycoprotein proteins
through hydrogen bonds, hydrophobic bonds, and
van der Waals bonds. Hydrophobic bonds interact
with Leu478, and van der Waals bonds form
interactions with Met375, Val532, and Gly480. The
target protein docked with a thioxanthenol compound,
forming hydrophobic and van der Waals bonds.

Figure 8. Interaction of each compound with EFGR protein: (a) thioxanthenol in 3D, (b)
1-hydroxythioxanthone in 3D, (c) 4-nitrothioxanthone in 3D, (d) 2-nitrothioxanthone in
3D, (e) thioxanthenol in 2D (f) 1-hydroxythioxanthone in 2D, (g) 4-nitrothioxanthone in
2D, and (h) 2-nitrothioxanthone in 2D.
Table 4. Binding affinity values of tested compounds and
native ligand against EGFR
Compounds
Native ligand
Thioxanthenol
1-hydroxythioxanthone
2-nitrothioxanthone
4-nitrothioxanthone

Binding affinity (kcal mol-1)
-7.40
-7.30
-7.30
-7.80
-8.00
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Hydrophobic bonds are stronger than van der Waals
bonds, with additional interactions with Pro464. The
binding affinity is -8.00 kcal mol-1, but higher than the
native ligand binding affinity of -9.30 kcal mol-1. This
indicates that the thioxanthenol compound's
hydrophobic and van der Waals bonds do not provide
a stronger bond than the native ligand. Thioxanthenol
(Figure 9a and Figure 9e) interacted through
hydrogen, hydrophobic, and van der Waals bonds.
The hydrogen bond indicates a strong bond between
the compound and the target protein. However, the
hydrogen-bonded amino acid residue differs from the
native ligand's amino acid residue (Wang et al., 2015;
Kumar et al., 2016). A compound's pharmacological
effect is more like its standard ligand if it interacts with
its major amino acid residue. Hydrophobic bonds
form new interactions with Ala113 and Pro464, while
van der Waals bonds form new interactions with

Asn350, Thr469, and Leu463. Unfortunately, the
binding affinity of 1-hydroxythioxanthone compounds
(Figure 9b and Figure 9f) is still greater than the native
ligand.
The 2-nitrothioxanthone and 4-nitrothioxanthone
compounds interact through van der Waals and
hydrophobic bonds with native ligands. The 2nitrothioxanthone forms (Figure 9d and Figure 9h)
new interactions with Ala113 and Pro464, while 4nitrothioxanthone (Figure 9c and Figure 9g) has the
largest number of interactions, resulting in similar
pharmacological effects to its standard ligand. The
binding affinity is -9.00 kcal mol-1, which closed to the
native
ligand’s
binding
energy.
The
4nitrothioxanthone compound was found to be effective
as an anticancer agent due to its smaller binding
affinity and common interactions with amino acid
residues in the ATP pocket of P-glycoprotein.

Figure 9. Interaction of each compound on P-glycoprotein: (a) thioxanthenol in 3D, (b)
1-hydroxythioxanthone in 3D, (c) 4-nitrothioxanthone in 3D, (d) 2-nitrothioxanthone in 3D,
(e) thioxanthenol in 2D (f) 1-hydroxythioxanthone in 2D, and (g) 4-nitrothioxanthone in 2D,
(h) 2-nitrothioxanthone in 2D.
Table 5. Binding affinity values of tested compounds and native ligand
against P-glycoprotein
Compounds
Native ligand
Thioxanthenol
1-hydroxythioxanthone
2-nitrothioxanthone
4-nitrothioxanthone

Binding affinity (kcal mol-1)
-9.30
-8.00
-8.30
-9.00
-9.00
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Figure 10. Interaction of each compound with ERα: (a) thioxanthenol in 3D, (b)
1-hydroxythioxanthone in 3D, (c) 4-nitrothioxanthone in 3D, (d) 2-nitrothioxanthone in 3D,
(e) thioxanthenol in 2D (f) 1-hydroxythioxanthone in 2D, (g) 4-nitrothioxanthone in 2D, and
(h) 2-nitrothioxanthone in 2D
Table 6. Binding affinity values of tested compounds and native
ligand against ERα
Compounds
Native ligand
Thioxanthenol
1-hydroxythioxanthone
2-nitrothioxanthone
4-nitrothioxanthone

Binding affinity (kcal mol-1)
-9.90
-7.80
-7.80
-7.70
-8.00

ERα Protein
ERα activates DNA by binding to estrogen
hormone, causing RNA formation, which can increase
cancer cell numbers and activity. The thioxanthenol, 2nitrothioxanthone, 4-nitrothioxanthone, and 1hydroxythioxanthone have been studied through
molecular docking against ERα. ERα protein was
redocked with its native ligand, resulting in a
molecular docking method with an RMSD value of
1.14 Å. Interactions occurred through hydrogen,
hydrophobic, and van der Waals bonds The
interacting amino acid residues are the same as the
redocking results of Narko et al. in 2017. The
thioxanthenol compound (Figure 10a and Figure 10e)
forms hydrophobic and van der Waals bonds,
resulting in greater binding affinity than the native
ligand. However, the strength of hydrophobic and van
der Waals bonds is not strong enough to inhibit
estrogen binding to ERα.

The study reveals that 1-hydroxythioxanthone
(Figure 10b and Figure 10f) interacts with the target
protein through hydrophobic and van der Waals
bonds, resulting in a binding affinity value of -7.80
kcal mol-1. Hydrophobic bonds formed with the main
amino acid residues, such as Ala350 and Leu525. The
van der Waals bonding occurs with two main amino
acid residues, namely Phe404 and Gly521.
Nitrothioxanthone compounds interact with target
protein amino acid residues through hydrophobic
and van der Waals bonds. The 4-nitrotioxanthone
(Figure 10c and Figure 10g) interacts through
hydrophobic bonds and one new residue, Leu384,
while the 2-nitrothioxanthone compound has a
hydrogen bond, making ERα inhibition stronger. The
2-nitrothioxanthone compound (Figure 10d and
Figure 10h) also binds through hydrophobic bonds at
its main amino acid residues and additional van der
Waals bonds at a new Arg394 amino acid residue.

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Molekul, Vol. 20. No. 2, July 2025: 236 – 247
Narko et al. (2017) and Acharya et al. (2019)
performed docking of their proposed compounds and
the same hydrogen bond occurred at Arg394 amino
acid residue. The 2-nitrothioxanthone compound, with
a smaller binding affinity (-8.00 kcal mol-1), may
inhibit ERα activation in cancer cells as its binding to
ERα is more difficult to release, unlike other
investigated compounds in this work.
It is important to note that molecular docking only
provides predictive binding insights and does not fully
reflect in vivo conditions. However, a significant
limitation of the current study lies in the absence of in
vivo testing. Although the compounds showed
promising in vitro and in silico results, biological
complexity in living organisms may influence
pharmacokinetics, toxicity, and therapeutic efficacy
differently. Without in vivo data, the full therapeutic
potential and safety profile of these compounds
cannot yet be conclusively determined.
Thioxanthenol
derivatives
exhibit
several
advantages that support their potential as anticancer
candidates. These include their high selectivity towards
certain cancer cell lines, ease of synthesis from readily
available thioxanthone precursors, and favorable
biological activity as observed in both in vitro
cytotoxicity and molecular docking studies. The
compounds also follow drug-likeness parameters
such as Lipinski’s Rule of Five and demonstrate
acceptable ADMET profiles, further validating their
pharmaceutical relevance.
Future research will focus on structural optimization
of the synthesized derivatives to enhance their
binding affinity and selectivity. In vivo animal
studies
will
be
conducted
to
evaluate
pharmacodynamic and pharmacokinetic behaviors,
as well as systemic toxicity. Furthermore, clinical
validation through preclinical trials in collaboration
with biological laboratories is planned to verify the
efficacy and safety of the most promising candidates,
ultimately supporting their development as viable
anticancer drugs.
CONCLUSIONS
Thioxanthenol compound has been successfully
synthesized from thioxanthone using sodium
borohydride reagent with a yield of 40.63%. The
nitration reaction of thioxanthone with nitric acid and
sulfuric acid was successfully carried out to produce 1hydroxylthioxanthone, 4-nitrothioxanthone, and 2nitrothioxanthone in 33.54%, 29.27%, and 31.71%
yield, respectively. The nitrothioxanthone compounds
had a higher IC50 value for WiDr colon cancer cells
than thioxanthenol compound, but they gave a smaller
IC50 value for T47D breast cancer cells. The nitrated
thioxanthones also selective for various cancers,
especially for HeLa cervical cancer cells. The 2nitrothioxanthone compound interacts with EGFR and
P-glycoprotein through hydrophobic bonds and van
der Waals, suggesting it could act as an anticancer

agent for HeLa cancer due to its interactions with both
receptors. To strengthen the therapeutic implications
of these findings, future studies involving in vivo
validation in collaboration with biological experiments
are planned.
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