In vitro cytotoxicity evaluation of thiourea derivatives bearing Salix sp. constituent against HK-1 cell lines

Article abstract of DOI:10.1080/14786419.2018.1517120

In searching for drugs from natural product scaffolds has gained interest among researchers. In this study, a series of twelve halogenated thiourea (ATX 1-12)via chemical modification of aspirin (a natural product derivative) and evaluated for cytotoxic a

Full text of DOI:10.1080/14786419.2018.1517120

Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
In vitro cytotoxicity evaluation of thiourea
derivatives bearing Salix sp. constituent against
HK-1 cell lines
Norsyafikah Asyilla Nordin, Vannessa Lawai, Zainab Ngaini, Ainaa Nadiah
Abd Halim, Siaw San Hwang, Reagan Entigu Linton, Boon Kiat Lee & Paul
Matthew Neilsen
To cite this article: Norsyafikah Asyilla Nordin, Vannessa Lawai, Zainab Ngaini, Ainaa Nadiah Abd
Halim, Siaw San Hwang, Reagan Entigu Linton, Boon Kiat Lee & Paul Matthew Neilsen (2018): In
vitro cytotoxicity evaluation of thiourea derivatives bearing Salixꢀsp. constituent against HK-1 cell
lines, Natural Product Research, DOI: 10.1080/14786419.2018.1517120
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NATURAL PRODUCT RESEARCH
https://doi.org/10.1080/14786419.2018.1517120
In vitro cytotoxicity evaluation of thiourea derivatives
bearing Salix sp. constituent against HK-1 cell lines
a,b
b
b
Norsyafikah Asyilla Nordin , Vannessa Lawai , Zainab Ngaini , Ainaa Nadiah
b
c
c
c
Abd Halim , Siaw San Hwang , Reagan Entigu Linton , Boon Kiat Lee and
d
Paul Matthew Neilsen
a
b
Faculty of Pharmacy, Universiti Sultan Zainal Abidin, Besut Campus, Terengganu, Malaysia; Faculty of
c
Resource Science and Technology, Universiti Malaysia Sarawak, Sarawak, Malaysia; Faculty of Engineering,
Computing and Science, Swinburne University of Technology Sarawak Campus, Sarawak, Malaysia;
d
School of Health Medical and Applied Sciences, Central Queensland University, Norman Gardens, Australia
ABSTRACT
ARTICLE HISTORY
In searching for drugs from natural product scaffolds has gained inter-
est among researchers. In this study, a series of twelve halogenated
thiourea (ATX 1-12) via chemical modification of aspirin (a natural
product derivative) and evaluated for cytotoxic activity against naso-
pharyngeal carcinoma (NPC) cell lines, HK-1 via MTS-based colorimetric
assay. The cytotoxicity studies demonstrated that halogens at meta pos-
ition of ATX showed promising activity against HK-1 cells (IC50 value
Received 7 May 2018
Accepted 24 August 2018
KEYWORDS
Aspirin; thiourea;
cytotoxicity; molecular
docking; cyclooxygenase-2
ꢀ
15 mM) in comparison to cisplatin, a positive cytotoxic drug (IC50 value
8.9 ± 1.9 mM). ATX 11, bearing iodine at meta position, showed robust
¼
cytotoxicity against HK-1 cells with an IC50 value of 4.7 ± 0.7 mM.
Molecular docking interactions between ATX 11 and cyclooxygenase-2
demonstrated a robust binding affinity value of ꢁ8.1 kcal/mol as com-
pared to aspirin’s binding affinity value of ꢁ6.4 kcal/mol. The findings
represent a promising lead molecule from natural product with excel-
lent cytotoxic activity against NPC cell lines.
CONTACT Zainab Ngaini
nzainab@unimas.my
Supplemental data for this article can be accessed https://doi.org/10.1080/14786419.2018.1517120.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group

2
N. A. NORDIN ET AL.
1. Introduction
Willow tree (Salix sp.) has been reported as an effective traditional remedy to treat
fever and pain (Desborough and Keeling 2017). Willow tree is a Salicaceae family
which comprised of several species including Salix alba L., Salix pentandra L. and Salix
purpurea L (Mahdi 2010). Salicylic acid is an active metabolite extracted from willow
bark. Chemical modification of salicylic acid into acetylsalicylic acid or aspirin has been
reported for gastrointestinal study (Mahdi et al. 2006).
Aspirin is a type of non-steroidal anti-inflammatory drug which commonly used to
relieve pain, reduce fever and prevent diseases related to aging (Vane and Botting
2003). Prolonged use of aspirin, however, has caused gastrointestinal bleeding and
ulceration. Several efforts in modifying aspirin has been reported to increase its
pharmacological properties (Turnbull et al. 2008; Ngaini and Mortadza 2018). Phospho-
aspirin is an example of structural modification of aspirin that exhibits less gastrointes-
tinal toxicity and enhanced anti-inflammatory effect compared to the conventional
aspirin (Huang et al. 2011). Other aspirin modifications were also reported to augment
its antifungal (Chavan et al. 2012) and anticancer activities (Huang et al. 2014).
Synthesis of new drugs and studies on their efficacy requires higher cost and longer
times. Many new drugs fail in the clinical trials due to poor bioavailability or pharma-
cokinetic properties. Medicinal chemistry approaches to modify known drugs seems to
be more viable and practical option. Our preliminary natural product modification of
aspirin via thiourea moieties bearing amino acids showed excellent antibacterial activ-
ities (Ngaini et al. 2012, Wan Zullkiplee et al. 2014). The potential of thiourea moieties
as an effective drug has been widely reported via production of several commercial
drug-based thioureas such as carbimazole, propylthiouracil and methylthiouracil
(Ansari et al. 2014). Thiourea is an organic compound which consist of thiocarbonyl
and amine groups and known to display broad range of biological properties such as
antibacterial (Abd Halim and Ngaini 2017; Nordin et al. 2017), larvicidal (Kaymakcioglu
et al. 2013) and anticancer (Yahyazadeh and Ghasemi 2013). The presence of substitu-
ents such as halogens has been reported for excellent antibacterial (Zhou et al. 2006;
Prabhashankar et al. 2012), antifungal (Feng et al. 2014), cytotoxic (El-Gohary and
Shaaban 2013) and antitumor properties (Jain et al. 2012).
Herein, we report the synthesis of halogenated thiourea moieties (ATX 1-12) bear-
ing aspirin, a natural product-derived molecule, by reacting acetoxybenzoyl thiocyan-
ate with a series of halogenated anilines. All 12 compounds were evaluated for
potential cytotoxic agent against NPC cell line, HK-1. The interaction of the com-
pounds with the targeted enzyme was evaluated via molecular docking analysis
against COX-2 protein.
2. Results and discussion
2.1. Chemistry
Series of novel aspirin analogues (ATX 1-12) bearing halogenated thiourea moieties
were prepared from the reaction of aspirin, an active scaffold of natural product
derivative, and oxalyl chloride in the presence of potassium thiocyanate (Ngaini et al.

NATURAL PRODUCT RESEARCH
3
2
012) to form acetoxybenzoyl isothiocyanate as intermediates. Further synthesis of the
intermediates with a series of halogenated anilines afforded ATX 1-12 in 30.7-42.0%
yield (Scheme 1). The synthesis of thiourea also form urethane as side product which
contributed to the low product yield (Katritzky et al. 2004).
1
13
The structural elucidation of ATX 1-12 was performed using FTIR, H NMR and
C
NMR spectroscopy. The FTIR spectra showed sharp peaks corresponded to v(NH) group
ꢁ
1
ꢁ1
at 3356–3382 cm . The disappearance of peak at 2000–2500 cm for –NCS indicated
the successful conversion of intermediates into thioureas (Fathalla et al. 2001). The
ꢁ
1
ꢁ1
absorption peaks at 1774–1785 cm and 1663–1675 cm were assigned to v(C¼O)
ester and amide, respectively. The absorption peak attributed to aryl groups was
observed at 1524–1561 cm . The presence of v(C–N) was attributed to the peaks at
142–1169 cm (Saeed et al. 2009) while peaks at 749–863 cm were assigned to
ꢁ
1
ꢁ
1
ꢁ1
1
v(C¼S) (Bielenica et al. 2015).
1
H NMR spectra of ATX 1-12 indicated the presence of CH at d 2.31–2.32 ppm
3
H
and aromatic groups at d 7.13–8.16 ppm. The resonance at d 11.65–11.86 ppm and
H
H
dH 12.27–60 ppm were attributed to CSNH and CONH, respectively. The deshielding
effect of carbonyl and thiocarbonyl groups has shifted the signal to downfield region
13
(
Saeed et al. 2010). C NMR spectra also gave resonance peaks corresponded to the
proposed structures. The peaks at dC 20.7–21.4 ppm were attributed to the methyl
group in aspirin. The resonance at d 91.7–163.0 ppm were attributed to aromatic car-
C
bons, while signals at d 166.3–169.4 ppm and d 178.8–179.9 ppm were corresponded
C
C
to C¼O and C¼S, respectively (Rauf et al. 2009).
2.2. Anticancer assay
The application of chemically modified natural product compounds of aspirin bearing
thioureas moieties were evaluated for in vitro cytotoxic activity against NPC cell line,
HK1 at different concentrations ranging from 0.39 to 50mM using the 3-(4,5-dimethylth-
iazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. The
relative influence of the aspirin-thiourea derivatives on the viability of the NPC cell line
was determined via concentration-response curves using regression analysis to deter-
mine the IC50 (concentration causing 50% cell growth inhibition) (Figure 1). The IC50 of
ATX 1-12 is summarized in Table S1 with values ranging from 4.7 to 14.6mM.
Scheme 1. Synthesis of ATX 1-12.

4
N. A. NORDIN ET AL.
Figure 1. Cell viability of NPC cells, HK1 treated with various concentrations of ATX 11 for 7 h. The
cell viability was measured by the MTS assay.
All compounds with the exception of ATX 1, 3, 4, 7, 8 and 10 showed promising
activities against HK1 with IC50 values ꢀ10 mM. This is encouraging compared to the
parental aspirin, which showed no anticancer activity at the same concentrations on
HK1 cells (IC50 > 50 mM) and a reference drug cisplatin (IC50 ꢀ 8.9 mM). Cisplatin is the
chemotherapeutic drug used as a standard for treating NPC (Al-Sarraf et al. 1998).
ATX with halogens (except bromine) introduced at the meta position in thiourea
moieties displayed excellent activities (IC ꢀ 8.0 mM) as compared to ortho and para
50
positions. Compound with substituents at meta position was reported to be more
selective to bind on specific protein (Vaz and Klabunde 2008), thus giving excellent
activities by affecting the cell’s survival functions.
ATX 11, with iodine at meta position, showed the most potent cytotoxic activity
against HK1 amongst the tested aspirin derivatives, with an IC50 of 4.7 mM.
Importantly, neither the halogenated aniline used to synthesise ATX 11 (with the iod-
ine at the meta position) nor aspirin itself, showed activity against HK1 cells
(IC50 > 50 mM). In other words, the cytotoxic activity of ATX 11 is likely to be a result
of the modification to aspirin, rather than an independent cytotoxicity associated with
the halogenated aniline. The presence of iodine has increased lipophilicity of the
aspirin-thiourea compared to the other halogens (Shirasaka et al. 1990) and more lipo-
philic character resulted in greater cytotoxicity (Modi et al. 2011). The presence of hal-
ogens at ortho position in thiourea moieties showed a noticeably reduced cytotoxicity
(IC50 10.8 to 13.9 mM) (P < 0.05) as compared to meta and para positions. While in ATX
9
, the introduction of bromine at para position has displayed good cytotoxicity with
an IC50 value of 5.7 mM. Based on the IC50 values in this study, it can be concluded
that the presence of iodine and chlorine at meta position as well as bromine at para
position of the phenyl ring has improved the cytotoxicity profiles against NPC cells,
HK1 (P < 0.05).

NATURAL PRODUCT RESEARCH
5
In comparison to aspirin, the presence of thiourea moiety in the molecular structure
of ATX 1-12 provides important functional groups for a promising anticancer activity.
High electronegative sulfur atom presence could incorporate with protein through
hydrogen bonding in cancer cell and subsequently inhibited the cells’ growth (Zhao
et al. 2015).
2.3. Molecular docking
Cyclooxygenase-2 (COX-2) enzyme is an inducible protein which expressed abnormally
in many types of cancers (Chen et al 2010). The suppression of viability of the NPC
cell line, HK1 could be due to the inhibition of COX-2 (Chen et al 2010). In this regard,
the binding interaction between ATX 11 (the most potent cytotoxic activity against
HK1) to the active site of COX-2 was performed via molecular docking for further esti-
mation of the binding energy in comparison to aspirin. The aspirin-thiourea derivative
of ATX 11 demonstrated robust molecular docking in the binding pockets of COX-2
and displayed strong electrostatic interaction with Trp388, Leu392, His389, His208, Tyr
3
86, Phe211, Gln204, Val296 and Val448 (Adinarayana et al. 2012) (Figure 2). ATX 11
showed good binding affinity value of -8.1 Kcal/mol. In contrast, aspirin showed weak
interaction with the residue Trp388, Leu391, Leu392 and Gln204 and a binding energy
value of -6.4 Kcal/mol (Khan et al. 2015) (Figure 3). These interaction further explained
and supported the anticancer activity of ATX 11 against COX-2 by significant
Figure 2. Binding interaction of ATX 11 at the active site of COX-2.

6
N. A. NORDIN ET AL.
Figure 3. Binding interaction of aspirin at the active site of COX-2.
interaction with specific amino acid residues with higher binding affinity compared
to aspirin.
3. Experimental
3.1. General
Aspirin, potassium thiocyanate and halogenated anilines series were obtained from
Merck. All other reagents were used without further purification nevertheless the acet-
one was distilled before used. Melting points were determined on Stuart SMP3 using
ꢁ
1
open tube capillary method. FTIR spectra (v/cm ) were recorded as KBr pellets on
1
13
Perkin Elmer 1605 FTIR Spectrophotometer. H-NMR and C-NMR spectra were
1
13
recorded on JEOL ECA 500 at 500 MHz ( H) and 125 MHz ( C) with the chemical shift
reported relative to DMSO-d as the standard reference and chemical shift values were
6
expressed in d ppm.
3.2. General synthesis of ATX 1-12
Aspirin (0.1802 g, 1.0 mmol) was reacted with oxalyl chloride and DMF was added to
initiate the reaction. Acetylsalicyloyl chloride formed was added drop wise into a solu-
tion of KSCN (0.0972 g, 1.0 mmol) in dry acetone (10 mL). The mixture was stirred for

NATURAL PRODUCT RESEARCH
7
3
0 min at room temperature and white precipitate (KCl) was filtered off from the mix-
ture. A series of halogenated aniline (1.0 mmol) in 10 mL dry acetone was added into
the filtrate and the mixture was stirred at room temperature for 24 h to form precipi-
tate. Purification by column chromatography (silica gel, 1:6 ethyl acetate/hexane)
afforded ATX 1-12 as shown in Supplementary Materials (Data S1).
3.3. Cell culture and cell viability assay
MTS based colorimetric assay was used to evaluate the cytotoxic activity of ATX 1-12
ꢂ
on NPC cell line, HK1 (Huang et al. 1980). The cells were maintained at 37 C in a
humidified atmosphere of 5% CO in RPMI 1640 (GIBCO) supplemented with 10% fetal
2
bovine serum (FBS; Sigma-Aldrich) and 1% penicillin-streptomycin (GIBCO). The cells
3
were seeded onto a 96 well plate at 5x10 cells/100 mL. ATX 1-12 were dissolved in
DMSO at different concentrations of which 100 mL of the diluted mixtures were added
onto designated wells of the 96-well plate; with final concentration ranging from 0.39
to 50 mM. Cells added with the equivalent concentration of DMSO vehicle were ana-
ꢂ
lysed as controls. The 96-well plate was incubated for 72 hours at 37 C and culture
media was carefully removed after the incubation. Subsequently, 10 mL of MTS (Cell
Titer 96@ Aqueous Non-Rad Cell Proliferation Assay Kit, Promega) and 50 mL of fresh
ꢂ
media were added to each well, followed by an incubation of 90 minutes at 37 C. The
absorbance was read at 490 nm using microplate reader (Synergy HT, BioTek, USA).
3.4. Molecular docking
Binding interaction of the most active compound ATX 11 and binding site of cycloox-
ygenase-2 (COX-2) was performed using AutodockTools 1.5.6 and the binding energy
was further estimated using Autodock Vina programme (Nguyen et al. 2016). The crys-
tal structure of COX-2 was obtained from RCSB Protein Data Bank with PDB ID: 5IKR
(
Orlando and Malkowski, 2016). A grid box with 60 ꢃ 60 ꢃ 60 Å dimension with a spac-
ing of 0.375 Å was centered at active site of the protein. Aspirin was also docked into
active site of COX-2 as reference.
3.5. Statistical analysis
Statistical analysis was performed using t-test (SPSS version 21). A value of P < 0.05
was considered as significant.
4. Conclusion
Twelve aspirin-thiourea derivatives (ATX 1-12) have been successfully prepared and
demonstrated as potential anticancer agents. Among the 12 compounds in this study,
ATX 11 showed excellent effect towards cell viabilities of nasopharyngeal cancer cell
line compared to aspirin alone. ATX 11 (with iodine at meta position) showed the
most potent cytotoxic/inhibitory effect on NPC cell viability with IC50 of 4.7 mM.
Molecular docking demonstrated good binding interaction of ATX 11 against COX-2

8
N. A. NORDIN ET AL.
compared to aspirin, in correlation to the IC50 values. This study offers significant
breakthrough in producing a potential chemotherapeutic agent in NPC treatment
using natural product derivatives as a reactive scaffold in the presence of thiourea
moieties. In vitro study of ATX 11 as enzyme inhibitor can be further investigated for
potential pharmaceutical applications.
Acknowledgements
We acknowledge support from Universiti Malaysia Sarawak and Malaysia Ministry of Science,
Technology and Innovation for the financial support through FRGS/ST02(01)/1115/2014(01) and
FRGS/ST01(01)/1298/2015(15). We also acknowledge Universiti Sultan Zainal Abidin for grant
support through UniSZA/2017/DPU/02. We thank Universiti Teknologi MARA for providing CHN
elemental analysis services and Corine Neilsen for outstanding editing and proof reading of
the manuscript.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
We acknowledge support from Universiti Malaysia Sarawak and Malaysia Ministry of Science,
Technology and Innovation for the financial support through FRGS/ST02(01)/1115/2014(01) and
FRGS/ST01(01)/1298/2015(15). We also acknowledge Universiti Sultan Zainal Abidin for grant
support through UniSZA/2017/DPU/02. We thank Universiti Teknologi MARA for providing CHN
elemental analysis services and Corine Neilsen for outstanding editing and proof reading of
the manuscript.
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