Synthesis, molecular docking and anticancer activity of diflunisal derivatives as cyclooxygenase enzyme inhibitors

Article abstract of DOI:10.3390/molecules23081969

Cyclooxygenase enzymes play a vital role in inflammatory pathways in the human body. Apart from their relation with inflammation, the additional involvement of COX-2 enzyme with cancer activity was recently discovered. In some cancer types the level of COX-2 enzyme is increased indicating that this enzyme could be a suitable target for cancer therapy. Based on these findings, we have synthesized some new diflunisal thiosemicarbazides and 1,2,4-triazoles and tested them against androgen-independent prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines. Specifically, the diflunisal and thiosemicarbazide functionality are combined during the synthesis of original compounds anticipating a potency enhancement. Compounds 6, 10, 15 and 16 did not show cytotoxic effects for the HEK293 cell line. Among them, compounds 15 and 16 demonstrated anticancer activity for the breast cancer cell line T47D, whereas compounds 6 and 10 which are thiosemicarbazide derivatives displayed anti-tumourigenic activity against the PC-3 cell line, consistent with the literature. However, no activity was observed for the HCT-116 cancer cell line with the tested thiosemicarbazide derivatives. Only compound 16 displayed activity against the HCT-116 cell line. Therefore, it was speculated that the diflunisal and thiosemicarbazide functionalities potentiate anticancer activity on prostate cancer and the thiosemicarbazide functionality decreases the anticancer activity of diflunisal on colon cancer cell lines. In order to gain insight into the anticancer activity and COX-2 inhibition, molecular docking studies were carried out for COX-1 and COX-2 enzymes utilizing the newly synthesized compounds 15, and 16. Both 15 and 16 showed high selectivity and affinity toward COX-2 isozyme over COX-1, which is in agreement with the experimental results.

Full text of DOI:10.3390/molecules23081969

molecules
Article
Synthesis, Molecular Docking and Anticancer
Activity of Diflunisal Derivatives as Cyclooxygenase
Enzyme Inhibitors
Göknil Pelin Cos¸kun 1, Teodora Djikic 2, Taha Bartu Hayal 3, Nezaket Türkel 3
,
ID
Kemal Yelekçi 2, Fikrettin S¸ahin 3 and S¸. Güniz Küçükgüzel 4,
*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cumhuriyet University, Sivas 58140, Turkey;
goknilpelincoskun@gmail.com
2
3
Department of Bioinformatics and Genetic, Faculty of Engineering and Natural Sciences,
Kadir Has University, Istanbul 34083, Turkey; teodorica.djikic@gmail.com (T.D.); kyelekci@gmail.com (K.Y.)
Department of Genetics and Bioengineering, Faculty of Engineering and Architecture, Yeditepe University,
Kayıs¸dag˘ı, Istanbul 34755, Turkey; taha.bartu.hayal@gmail.com (T.B.H.); nezaket.turkel@gmail.com (N.T.);
fsahin@yeditepe.edu.tr (F.S¸.)
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Istanbul 34668, Turkey
Correspondence: gkucukguzel@marmara.edu.tr; Tel.: +90-216-414-29-62
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 2 July 2018; Accepted: 31 July 2018; Published: 6 August 2018
Abstract: Cyclooxygenase enzymes play a vital role in inflammatory pathways in the human body.
Apart from their relation with inflammation, the additional involvement of COX-2 enzyme with
cancer activity was recently discovered. In some cancer types the level of COX-2 enzyme is increased
indicating that this enzyme could be a suitable target for cancer therapy. Based on these findings,
we have synthesized some new diflunisal thiosemicarbazides and 1,2,4-triazoles and tested them
against androgen-independent prostate adenocarcinoma (PC-3), colon carcinoma (HCT-116), human
breast cancer (T47D), breast carcinoma (MCF7) and human embryonic kidney (HEK-293) cell lines.
Specifically, the diflunisal and thiosemicarbazide functionality are combined during the synthesis
of original compounds anticipating a potency enhancement. Compounds
show cytotoxic effects for the HEK293 cell line. Among them, compounds 15 and 16 demonstrated
anticancer activity for the breast cancer cell line T47D, whereas compounds and 10 which
6, 10, 15 and 16 did not
6
are thiosemicarbazide derivatives displayed anti-tumourigenic activity against the PC-3 cell line,
consistent with the literature. However, no activity was observed for the HCT-116 cancer cell line with
the tested thiosemicarbazide derivatives. Only compound 16 displayed activity against the HCT-116
cell line. Therefore, it was speculated that the diflunisal and thiosemicarbazide functionalities
potentiate anticancer activity on prostate cancer and the thiosemicarbazide functionality decreases
the anticancer activity of diflunisal on colon cancer cell lines. In order to gain insight into the
anticancer activity and COX-2 inhibition, molecular docking studies were carried out for COX-1 and
COX-2 enzymes utilizing the newly synthesized compounds 15, and 16. Both 15 and 16 showed
high selectivity and affinity toward COX-2 isozyme over COX-1, which is in agreement with the
experimental results.
Keywords: diflunisal; thiosemicarbazide; 1,2,4-triazole-3-thione; anticancer; COX-2; docking
1. Introduction
Cyclooxygenase (COX) enzymes play an important role in the synthesis of prostaglandins. So far,
three isoenzymes of COX have been discovered; COX-1, COX-2 and COX-3. The constitutive COX-1
isoenzyme which can be found throughout the body, where it is responsible for the protection of the
Molecules 2018, 23, 1969; doi:10.3390/molecules23081969
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1969
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gastrointestinal track, whereas COX-2 is an inducible isoenzyme responsible for inflammation [
COX-3 enzyme is a variation of COX-1, encoded by the same gene [ ]. Recently, it was suggested that
COX-3 is the main target of paracetamol [ ]. The important discovery of the role of COX-2 isoenzyme,
led researchers to investigate the potential involvement of these enzymes in pain [ ]. Recent studies
1,2].
3
4
5
have been carried out to obtain some selectivity between COX-1 and COX-2 enzymes. Since COX-1
isoenzyme is responsible for the protection of the gastrointestinal system and COX-2 is responsible for
pain, the selective inhibition of COX-2 is extremely important. Therefore, development of non-steroid
anti-inflammatory drugs (NSAIDs), has received the attention of researchers seeking to find potent
and selective COX-2 inhibitors [6]. Up-regulated expression of COX-2 isoenzyme under pathological
conditions was proved to be involved in inflammation and various cancers types. The COX-2 is
commonly over-expressed in several cancers including esophageal, gastric, pancreatic, colorectal and
prostate cancers [7]. Thus, studies on the anti-cancer effects of COX-2 selective NSAIDs drugs have
opened the ways for new research areas. [
8
]. Due to the noxious side-effects of various anti-cancer
agents on the market, it is important to develop potent anti-cancer agents with less adverse effects.
Diflunisal (20,40-difluoro-4-hydroxybiphenyl-3-carboxylic acid), a salicylic acid derivative, is known
as a NSAID. It is a selective inhibitor of COX-2 isoenzyme over COX-1 [9]. Diflunisal and its derivatives
have been reported to possess diverse biological effects, including anti-cancer activities [10
–15]. In order to
increase the suppressive effect of diflunisal on the proliferation of cancer cells, some diflunisal derivatives
were synthesized by adding new functional groups to the diflunisal scaffold. For this purpose, medicinal
chemists use different tools including fragment-based drug design to optimize the potency, unwanted
property and selectivity of a given lead drug structure toward a given targeted cancer cells. The obtained
structures were tested to check their biological activities for cancer cell lines. Thiosemicarbazides, which are
intermediate products of the synthesis of many biological active compounds, have attracted the attention of
researchers due to of their clinical use and diverse biological activities [16]. 1,2,4-Triazole-3-thiones were
reported to possess different biological activities [17]. In addition, Süzgün et al. reported that etodolac
triazole possesses anticancer effect [18].
Some of the known anti-cancer agents are not very effective, as many of them have inadequate ADMET
properties and easily induce resistance. There is also a considerable diversity in resistance of various cells
within the same tumor. Thus, development of safe and effective new agents with less side effects and reduced
drug resistance for long-term treatment of cancer is highly required [19]. Diflunisal and diflunisal derivatives
are associated with cancer prevention [15,20,21]. There is a meaningful correlation between the degree of
their COX-2 selectivity and anti-cancer activity [22]. The mechanism underlying the chemo-preventative
actions of NSAIDs are not clear, though suppression of prostaglandin E2 synthesis, particularly via inhibition
of cyclooxygenase (COX)-2 activity, has been suggested to be important reason [23]. On the other hand,
selective COX-2 inhibitors have been reported to have serious cardiovascular adverse effects, so their usage
is limited for chemo-prevention of cancer [24]. Therefore, we have mainly focused on developing more
selective and potent anti-cancer agents with less adverse effects and decreased drug resistance.
In this study, we hypothesized that the conversion of carboxylic acid group of diflunisal
into thiosemicarbazides and 1,2,4-triazole-3-thiones would enhance the selectivity of the drug
towards cancer cells. Accordingly, we have some synthesized new thiosemicarbazides and
1,2,4-triazole-3-thiones of diflunisal and elucidated their structures by FT-IR, 1H-NMR and 13C-NMR.
Their purity was proven by TLC, HPLC and elemental analysis. We have also investigated their
anticancer activity on PC-3, HCT-116, T47D and MCF-7 cancer cell lines using cell proliferation assays.
Among the tested compounds; compounds 6, 10, 15 and 16 showed anticancer activity against prostate
adenocarcinoma (PC-3), colon carcinoma (HCT-116), and human breast cancer (T47D) cancer cell lines.
None of the tested compounds were found active against the MCF-7 cell line. In order to correlate the
anticancer activity and selectivity with COX-2 inhibition, molecular docking studies were performed
utilizing both COX-1 and COX-2 as target enzymes with the newly synthesized compounds. These in
silico studies, suggest that compounds 15 and 16 show high selectivity toward COX-2 isoenzyme over
COX-1, agreeing with the experimental results.

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2. Results and Discussion
2.1. Chemistry
Diflunisal (20,40-difluoro-4-hydroxybiphenyl-3-carboxylic acid) was converted into its corresponding
diflunisal ester ( ) and diflunisal hydrazide ( ) according to a reported procedure [10]. In brief, diflunisal
1
2
hydrazide was converted into substituted thiosemicarbazides with overall yields ranging between 65–93%
by treatment with suitable aryl isothiocyanates in ethanol. Different types of solvents were used in
the thiosemicarbazide preparation such as dioxane, acetonitrile, benzene, methanol, tetrahydrofuran,
isopropyl, pyridine, however, the solvent of choice was ethanol, as it is safe and easy to use in synthesis.
These reactions were all monitored by TLC (mobile phase: petroleum benzene/ethyl acetate 50:50, v/v).
After two crystallizations from ethanol, the purities of the compounds were checked by HPLC.
Compounds
synthesized thiosemicarbazides
3
,
4
, and
5
were reported in our previously study [25]. The structures of the newly
1
6–
10 were confirmed by FT-IR, H-NMR, 13C-NMR and HR-MS.
The results gained from the spectral data were all compatible with the proposed structures. The FT-IR
data showed that hydrazide C=O stretching bands had shifted from 1641 cm−1 to 1622–1651 cm−1
which confirmed the formation of an amide functionality. Further, C=S stretching bands were detected
between 1238–1274 cm−1, which also proved the presence of characteristic thiosemicarbazide C=S
1
stretching bands. The H-NMR spectra showed the chemical shifts of the N1, N2 and N4 protons
which also confirmed the formation of the thiosemicarbazide functionality. The thiosemicarbazide
protons were observed between 13.71–11.99; 12.03–10.65 and 8.17–11.96 ppm, respectively. Formation
of thiosemicarbazides was also confirmed by 13C-NMR studies. The presence of a thiosemicarbazide
functionality produces certain C=O and C=S peaks. In this study, these C=O and C=S peaks were
observed between 166.00–169.00 and 180 ppm, respectively. For further evaluation, high resolution
mass spectrometry (HR-MS) was performed for three thiosemicarbazide derivatives (compounds
4, 6 and 9). These HR-MS studies confirmed the molecular weights and empirical formula within
8 mmu. The molecular ion peaks were observed as expected. Mass fragments of the compounds also
supported the expected structures. The main fragment of the two compounds formed by the loss of
isothiocyanate part of the compound, which was detected as [M-RNCS]+. Beside the main fragment,
OH radical and halogen losses were detected in both compounds.
In order to prepare the 1,2,4-triazole-3-thione derivatives 11–16, ring closures were carried out
with 2 N NaOH for 4 h using the reported method [18]. Adding one more step for triazole compounds
instead of trying to synthesize them from hydrazides was safer and improved the overall yield
(65–98%). However, attempts to cyclize compounds
8 and 10 failed. There are not many different
methods for the synthesis of 1,2,4-triazole-3-thiones except for synthesizing them from corresponding
thiosemicarbazides. We tried using 2% NaOH and 4 N NaOH medium instead of 2 N NaOH for the
the cyclization but all three methods failed. Diflunisal 1,2,4-triazole-3-thione derivatives 11–16, were
neutralized with concentrated hydrochloric acid after monitoring the reaction with TLC. The purity of
the compounds were confirmed with both TLC and HPLC as performed for the thiosemicarbazides
using the same methods. FT-IR spectra of the newly synthesized diflunisal triazoles supported the
cyclization. The disappearance of the amide C=O and formation of C=N stretching bands proved
the triazole synthesis. The C=N and C=S stretching bands of the 1,2,4-triazole moiety were detected
−1
between 1620–1633 cm and 1240–1269 cm−1, respectively. The C=S stretching bands also confirmed
that all the synthesized 1,2,4-triazoles are in their thione form as no stretching bands corresponding
1
to –SH groups were observed between 2550–2600 cm−1. H-NMR spectral data also confirmed the
formation of triazole compounds. The N1, N2 and N4 protons of the thiosemicarbazide were not
detected in the 1,2,4-triazole compounds. Instead, N-H peaks of 1,2,4-triazole were observed between
13.76–14.16 ppm. There was no peak between 1.0–3.7 ppm which might be attributed to a -SH function
and this finding also supported the presence of the thione form of the 1,2,4-triazole compounds 11–16.
All the other aromatic protons of the compounds were observed in the expected regions. In 13C-NMR
studies, all the carbons were observed in the expected regions. The C=N and C=S carbons that belong

Molecules 2018, 23, 1969
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to triazole functionality were observed between 155.47–155.75 and 167.58–168.19 ppm, respectively.
No carbon peaks of thiosemicarbazide C=O and C=S were observed; and this was accepted as a proof
of the synthesis of 1,2,4-triazoles. In 13C-NMR studies, the structure of compounds was confirmed
to present in thione form with the observation of C=S peaks. HR-MS studies were performed on
two selected triazole compounds, compounds 14 and 16. HR-MS studies confirmed the molecular
weights and empirical formulas within 8 mmu. The molecular ion peaks were observed as expected.
The anticipated compound fragments were observed in the mass spectra. The main observed fragments
of the two compounds were their [M-S]+, [M-OH]+ and [M-RNCS]+ peaks (Figure 1).
Figure 1. Synthetic route to newly synthesized diflunisal thiosemicarbazides (3–10) and 1,2,4-triazole-
3-thiones 11–16.
2.2. Biological Evaluation
2.2.1. Anticancer Activity
A number of studies have reported the association between COX-2 overexpression and cancer
progression. Expression of the COX-2 isoenzyme is upregulated in many human cancers, including
gastric, breast, lung, colon, esophageal, prostate and hepatocellular carcinomas [26]. It has been
shown that diflunisal selectively inhibits the COX-2 enzyme [15]. Furthermore, diflunisal derivatives
have been reported to possess anticancer activity against leukemia [14
cancer cell lines. In the relevant studies with diflunisal, thiosemicarbazide and 1,2,4-triazole-3-thione
functionalities were correlated with anticancer activity [22 27 32]. Therefore, in this study, we
selected to study diflunisal thiosemicarbazides 10 and diflunisal 1,2,4-triazole-3-thiones 11 16 to
,20] and hepatocellular [14]
,
–
3
–
–
investigate their cytotoxic effect against to cancer cells. Four different cancer cell lines, namely, prostate
adenocarcinoma (PC-3), colon carcinoma (HCT-116), human breast cancer (T47D), breast carcinoma
(MCF7) and a normal human embryonic kidney cell line (HEK293) were used to evaluate the anticancer
activity of newly synthesized compounds.
Firstly, the half maximal inhibitory concentration (IC50) values of all the synthesized compounds
were determined for all the cell lines used in this study by a MTS assay. Compounds for which the
IC50 value against HEK293 cell line was twice as high as the IC50 value for at least one cancer cell line
(compound 6, 10, 15, 16) were considered active (Table 1) and included into structure-activity analysis.

Molecules 2018, 23, 1969
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Further experiments were performed by using selected active compounds. There are reliable
studies about the anti-colon cancer activity of diflunisal and the anti-prostate cancer activity of
the thiosemicarbazide functionality [22]. Therefore, here prostate cancer (PC-3) and colon cancer
(HCT-116) cell lines were selected to study the anticancer activity of diflunisal and thiosemicarbazide.
We combined the diflunisal and thiosemicarbazide functionalities in order to increase the anticancer
activity of the resulting compounds. The expected results were obtained for the PC-3 cancer cell
line (the compounds showed activity, with IC50 values as 41.8 µM for compound 6 and 11.7 µM
for compound 10), however, no activity was observed for the HCT-116 cancer cell line with
the thiosemicarbazide derivatives. Therefore, we assumed that diflunisal and thiosemicarbazide
functionality enhance anti-prostate cancer activity but the thiosemicarbazide functionality decreases
the activity of diflunisal on colon cancer cell lines [15]. Comparing the differences between the active
thiosemicarbazide molecules, alkyl substitution increased the activity; whereas halogen substitution
showed more decreased activity than compound 10.
Similar to the anticancer activities of thiosemicarbazides, 1,2,4-triazole-3-thiones were reported to
have diverse biological activities, including anticancer activity [17]. There are several studies reporting
the activity of the triazole functionality against breast cancer. In fact, letrozole and anastrozole are
anticancer drugs (aromatase inhibitors) in breast cancer treatment on the market. In this study, we used
the MCF7 and T47D breast cancer cell lines. Both cancer cell lines were isolated from tumours located
in breast, but they differ from each other in a mutation in the p53 gene. The wild-type form of the
p53 gene is expressed in the MCF7 cancer cell line, whereas in the T47D cancer cell line the p53
gene is mutated [33]. Among the tested compounds, compounds 15 and 16 which possess a triazole
functionality showed activity against the T47D cell line, whereas for the MCF7 cancer cell line none of
the compounds displayed any activity. These contrasting effects could be related to differential p53
expression levels. The presence of the wild-type form of p53 in the MCF7 cell line may promote its
resistance against all of the compounds tested in this study. Furthermore, it is well established in the
literature that the MCF7 cell line has multidrug resistance compared to the T47D cell line because of
excessive expression of P-glycoprotein drug transporter gene and having an estrogen receptor subunit
alpha rather than estrogen receptor subunit beta, unlike T47D [34–36]. It can be deduced from the
results that compounds bearing a methoxy substituent are more important for T47D activity than
halogen substituted ones. A compound bearing bromo substitution showed activity, with an IC50
value of 43.4
µM (compound 15) and a compound bearing methoxy substitution showed activity with
an IC50 value of 27.3
µM (compound 16) for the T47D cancer cell line. Thus, it can be concluded that
electron donating groups increase the anticancer activity on the T47D cancer cell line. When comparing
the activity of these triazole compounds on other cancer cell lines, compound 16 showed activity
against the HCT116 cancer cell line with an IC50 value of 6.2 µM and compound 15 did not show any
activity to the other cancer lines (PC-3 and HCT116). The differences between these results could be
related with the cancer cell types. The HCT116 cell line is known to have K-RAS activation and PIK3CA
mutation [37]. Because of that, this cancer cell line is resistant to a number of anticancer drugs and even
epidermal growth factor inhibitors [38,39]. The PC-3 cancer cell line differs from other prostate cancer
cell lines in its synthesis of androgen receptor (AR) and prostate specific antigen (PSA), indicating
that survival does not depend on androgen [40]. Furthermore, PC-3 is a CD44 positive cancer cell line
and therefore, it is thought to have stem cell features [41]. Like the HCT116 cancer cell line, PC-3 is
also resistant to many anticancer drugs. However, we still found immensely significant activity from
compound 16 against HCT116 cancer cell line. In addition to that, the triazole functionality did not
possess any activity against the PC-3 cancer cell line., and theIC50 values of the PC-3-active compounds
demonstrated that the thiosemicarbazide functionality is more significant for this cell line.
In conclusion, the comparison of the compounds 6 and 10 indicates that alkyl substitution increases
the anticancer activity on the PC-3 cell line. On the other hand, only bromophenyl derivatives showed
activity in this study; which proved the importance of the bromine atom instead of chlorine or fluorine.
Compound 16 has a triazole functionality and has methoxyphenyl substitution. Taking everything said

Molecules 2018, 23, 1969
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into account, regarding the activity against colon cancer, small molecules are fundamental and electron
donating groups are central for the anticancer activity. Compounds 15 and 16 showed the best activity
on the T47D breast cancer cell line as these compounds have triazole functionalities. When the triazole is
replaced by a thiosemicarbazide, the activity decreases as was observed in compound 6. For the anticancer
activity against breast cancer line, the triazole functionality has significant importance compared to the
thiosemicarbazide one.
Table 1. IC50 values (µM) of compounds.
Compound
PC-3
HCT116
T47D
MCF7
HEK
3
4
ND
PCV
ND
PCV
ND
PCV
ND
PCV
ND
617 + 35
5
6
7
8
9
10
11
12
67,2 ± 4.5
41.8 ± 3.4
11.7 ± 4.2
2.85 ± 3.6
1.575 ± 1.4
11.7 ± 2.6
PCV
PCV
PCV
28,2 ± 2.5
100 ± 2.3
16.73 ± 2.8
8.07 ± 2.0
3.86 ± 0.7
NSC
PCV
PCV
PCV
PCV
44,4 ± 1,7
93 ± 3.43
3.17 ± 1.06
5.04 ± 1.5
3.02 ± 1.9
94 ± 3.74
11.52 ± 0.2
3.68 ± 2.1
1.83 ± 0.92
PCV
3.92 ± 0.27
PCV
174.13
±
6.3
NSC
NSC
NSC
PCV
10.05
±
1.57
22.18 ± 2.3
15.03 ± 1.3
33.95 ± 2.1
34.09 ± 3.7
PCV
9.24 ± 1.01
5.55 ± 2.7
11.47 ± 1.8
43.4 ± 2.72
27.3 ± 2.28
* 0.836 µM
10.66
±
0.96
13
14
15
12.883
±
2.1
8.7 ± 1.79
12,8 ± 2.1
145 ± 2.3
53 ± 2.39
NSC
PCV
PCV
145 ± 2.8
PCV
16
168.86
* 39.9 µM
±
3.9
6.2 ± 3.1
Cisplatin
* 1.25 µM
PCV: Promoting cell viability; NSC: No significant change; ND: Not detected, experiment could not be performed,
compound was precipitated; * Lit [42,43].
2.2.2. Effect of the Synthesized Compounds on DNA Synthesis
Determination of IC50 values of the compounds was subsequently followed by observation
of newly synthesized DNA in the cells. It is well-known that DNA synthesis is correlated with
cell proliferation [44] and 5-ethynyl-20-deoxyuridine (EdU) staining enables one to visualize newly
synthesized DNA to determine cell proliferation. EdU is a thymidine analogue that integrates DNA
during DNA synthesis, therefore here it stained only newly synthesized DNA during the experimental
0
procedure (in green fluorescence, Figure 2). 4 ,6-Diamidino-2-phenylindole (DAPI) stain binds strongly
to A-T rich areas in DNA and passes through cell membranes easily, thereby it stains nucleus of the all
cells regardless of whether the DNA is newly synthesized or not (in blue, Figure 2). It was revealed that
COX-2 enzyme inhibition via the newly synthesized compounds decreases the rate of DNA synthesis
in the HCT116, PC-3 and T47D cell lines, whereas the DNA synthesis in HEK293 cells did not appear
to be affected by the treatment of any of the compounds found active based on IC50 values (Figure 2).
These results indicated that the rate of DNA synthesis decreased dramatically in the presence of our
newly synthesized compounds in cancer cell lines compared to the healthy cell line HEK293.
2.2.3. Effect of Synthesized Compounds on Apoptosis
Evaluation of the effect of the synthesized compounds on apoptosis was determined by the
FITCH-AnnexinV/PI protocol. Damaged cell membrane of necrotic cell enables propidium iodide
(PI) to bind DNA and induces a red fluorescence, although it is excluded by non-damaged cells, select
live and apoptotic cells. FITCH-Annexin V has the ability to bind to phosphatidylserine which is
a specific marker of apoptosis. Figure 3 demonstrates the results of FITCH-Annexin V/PI of PC-3,
T47D, HCT116 and HEK293 cell lines after treatment with the COX-2 inhibitors synthesized in this
study. The lower left panel of the quadrant of the cytograms indicates live cells due to exclusion of PI
and non-bound Annexin V.

Molecules 2018, 23, 1969
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Figure 2. DAPI and EdU staining patterns of HEK293, PC-3, HCT116 and T47D cell lines. DAPI (in blue,
left panel) shows all the cells, EdU shows newly synthesized DNA (in green, middle panel and merged
image, right panel). As shown, when treated with compounds, the rate of DNA synthesis decreased
compared to the negative control group of each cell line individually, except for the non-cancer cell line
HEK293. All images captured at 20× magnification.
The lower right quadrant shows early apoptotic cells and the upper right quadrant shows late
apoptotic cells by virtue of no PI inclusion but positive Annexin V binding. The upper left panel of the
cytogram represents necrotic cells which are positive for PI. Each cytogram of each cell was analyzed
and a histogram graph was drawn for further visual information. Overall, treatment of HEK293
with active compounds (compounds 6, 10, 15, 16) did not result in apoptosis (Figure 3A). Although
slightly more necrotic and late apoptotic cells were observed with the treatment of HEK293 with
compound 10, it was not significant when compared to its effect on PC-3 cancer cells. PC-3 cancer cells
undergo apoptosis highly with the treatment of compound 6 and compound 10 when it is compared to
non-treated negative control (Figure 3B). Compound 16 with a triazole functionality had an extensive
apoptotic effect on HCT116 cancer cell line (Figure 3C). Most of the HCT116 cells went through early
and late apoptosis when treated with this compound (Figure 3C), whereas it resulted in necrosis in the
T47D cancer cell line. Furthermore, compound 15 led to cell death in T47D cell line largely through late
apoptosis following early apoptosis indicating that these cells went through programmed cell death
quite rapidly and thereby started to lose their cell membrane integrity. To sum up, all the compounds

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that are tested against to different cancer cell lines that were found to be active according to their IC50
values demonstrated higher cytotoxicity in tumor cells than normal cells (HEK293).
Figure 3. Determination of apoptosis and necrosis potential of synthesized compounds on HEK293,
PC-3, HCT116 and T47D cell lines. (
cytotoxic effects, except compound 10 with a slight insignificant increase in necrosis; (
PC-3 cells with compounds and 10 resulted in increase of late apoptosis markers; ( ) Compound 16
displayed extensive apoptotic effects (both early and late markers were elevated) on the HCT116 cancer
cell line; ( ) T47D cancer cells underwent vast late apoptosis through the treatment with compound 15
A
) Compounds tested on HEK293 cell line did not exhibit any
B
) Treatment of
6
C
D
,
whereas compound 16 caused necrosis of T47D cancer cells (* p < 0.05, ** p < 0.01, *** p < 0.001).
2.2.4. Docking Studies
In an attempt to explain the biological activity and the difference in selectivity profiles of the
newly synthesized 1,2,4-triazole-3-thiones toward COX subtypes based on their orientation and
binding patterns, the molecules were docked into the active sites of COX-1 and COX-2, respectively,
using AutoDock 4.2 [45].

Molecules 2018, 23, 1969
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Most of the compounds were found to be potential inhibitors of both COX-1 and COX-2 enzymes
(Table 2). As expected, all of the tested compounds showed more affinity to COX-2, rather than COX-1
enzyme. The COX-2 enzyme activities of the compounds correlate their anticancer activity results.
According to the docking studies, compounds 15 and 16 showed good binding affinity on COX-2
enzyme with free energies of binding,
∆
G =
−
10.57 kcal/mol and
−
9.60 kcal/mol, respectively (Table 2).
Halogen substitution appeared to have considerable importance but only flouro substitution affected
the results in a positive way. When fluorine substitution is changed for chlorine or bromine atoms,
the selectivity decreased (compound 14). Interestingly, alkyl substitution also favored COX-2 selectivity.
The best COX-2 selectivity was shown by compound 13. For comparison, 3D representation of
predicted binding mode and protein-ligand interactions of diflunisal in the active site of COX-2 enzyme
is given in Figure 4. The 3D picture of compound 15 and 16 in the active site COX-2 enzymes are shown
in Figure 5A,B. The COX-2 inhibitors were entering hydrophobic pocket consisted of Phe205, Phe381,
Tyr348, Tyr385, Trp387 and Phe518. Other important interactions are halogen interactions, H-bonds
interactions with His90, Glu198, Tyr355, Ser530 and Pi-Sulphur interactions with Met522 amino acid
side chains, depending on the substituents of the phenyl group. The obtained computational modeling
studies and binding energies support that the compounds 15 and 16 are potent and selective COX-2
inhibitors, agreeing with the experimental results.
Table 2. Docking study results of synthesized compounds.
Compound
COX-1
COX-2
13
14
15
16
∆G = −10.07 kcal/mol
∆G = −10.33 kcal/mol
∆G = −10.11 kcal/mol
∆G = −9.41 kcal/mol
∆G = −11.20 kcal/mol
∆G = −10.66 kcal/mol
∆G = −10.57 kcal/mol
∆G = −9.60 kcal/mol
Figure 4. 3D representation of predicted binding mode and protein-ligand interactions of diflunisal to
COX-2 enzyme. H-bonds occur between hydroxyl groups of hydroxybenzoic acid part of diflunisal
and TYR355 and ARG120 amino acid side chains and HIS90 monocation with the fluoro substituent of
the fluorophenyl ring.

Molecules 2018, 23, 1969
10 of 19
(A)
(B)
Figure 5. 3D representations of the predicted binding mode and protein-ligand interactions of
compounds 15 ) and 16 ) for COX-2. Halogen interactions of fluoro substituents with LEU352 are
(
A
(B
shown in blue dashed lines; hydrophobic interactions of aromatic rings with side chains of Phe205,
Val344, Tyr348, Phe381, Tyr355, Trp387, Ala527 and Leu534 are shown in pink. Two hydrogen bonds
of compound 16
and the triazole ring are represented with green dashed lines. Yellow lines represent S/
of Met522 and with the aromatic rings of compounds. Compound 15 ) showed one more S/
interaction between the sulphur of the 1,2,4-triazole-3-thion ring with the aromatic ring of Phe381.
(
B
): HIS90 monocation with fluoro substituent, and the hydroxyl group of SER530
π
interactions
(A
π

Molecules 2018, 23, 1969
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3. Materials and Methods
3.1. General
All the chemicals were purchased from Merck (Darmstadt, Germany) or Sigma-Aldrich
(St. Louis, MO, USA). Reactions were monitored by TLC on silica gel plates purchased from
Merck. Melting points of the synthesized compounds were determined in a IA 9300 melting
point apparatus (ThermoScientific, Staffordshire, UK) and are uncorrected. The purity of the
compounds was verified by TLC, HPLC and elemental analysis. Elemental analysis was performed on
a CHNS-932 apparatus (LECO, St. Joseph MI, USA). FT-IR spectra were recorded on a FT-IR-8400S
spectrophotometer (Shimadzu, Columbia, MD, USA)
. NMR spectra were recorded on a Bruker
1
spectrometer (Billerica, MA, USA) (300 MHz for H-NMR and 75 MHz for 13C-NMR, decoupled).
Data are reported as follows: chemical shift, multiplicity (b.s.: broad singlet, d: doublet; m: multiplet,
s: singlet, and t: triplet), coupling constants (Hz), integration. An Agilent 1100 LC-MS system
(Agilent, Santa Clara, CA, USA) using the EI technique was employed for MS analysis. Rf
×
100
◦
values were obtained with petroleum ether/ethyl acetate (50:50 v/v) mobile phase at 21 C. An Agilent
1100 series instrument equipped with a G1315A DAD detector, a pump (1311A Quat pump) and
manual sampler was used for the identification of Rt (retention time) values; using a Zorbax SB
C8 5
µ
m, 250
×
4.6 mm column. Operating conditions were performed by an isocratic HPLC
grade acetonitrile/phosphate buffer (pH:3.7) (70:30 v/v) mobile phase at a flow rate of 1 mL/min.
ChemStation A-08.03 (847)2000 software program with HP Compaq d330 DT computer was used to
analyze the spectral data.
3.2. Synthesis of Methyl 20,40-Difluoro-4-hydroxybiphenyl-3-carboxylate (Diflunisal Ester, 1, CAS Number:
55544-00-8) and 20,40-Difluoro-4-hydroxybiphenyl-3-carbohydrazide (Diflunisal Hydrazide, 2)
These compounds were prepared and synthesized as described previously in our studies [10].
3.3. General Procedure for the Synthesis of 2-[(20,40-Difluoro-4-hydroxybiphenyl-3-yl)carbonyl]-N-
(substituted)hydrazinocarbothioamides 3–10
Diflunisal hydrazide (2, 0.01 mol) was dissolved in ethanol (25 mL) and an equimolar amount
of a substituted isothiocyanate was added to the reaction mixture which was refluxed for 2 to 6
h. The crude product was precipitated, filtered and washed with distilled water. The compounds
were recrystallized from ethanol or DMF [12]. Compounds
our study [25].
3, 4 and 5 were previously reported in
N-(3-Bromophenyl)-2-[(20,4◦0-difluoro-4-hydroxybiphenyl-3-yl)carbonyl]hydrazine carbothioamide (
(cm−1): 3244 (N-H), 1626 (Amide C=O), 1274 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS)
6
): White
max.
ppm:
solid. Yield 83%; m.p. 199 C; MW: 478.309 g/mol; Rf
×
100 value: 21; Rt value: 4.82 min. FT-IR ν
δ
7.09 (d, 1 H, Ar-H; J = 8.4 Hz), 7.22 (dt, 1H, Ar-H; J = 2.2 Hz), 7.24–7.40 (m, 3 H, Ar-H), 7.48–7.74 (m, 4 H,
Ar-H), 8.06 (s, 1 H, Ar-H), 9.94 (s, 1 H, Ar-OH), 10.02 (s, 1 H, CSNH), 10.82 (s, 1 H, CONHNH), 12.01 (s,
1 H, CONH). 13C-NMR (DMSO-d6/TMS)
δ ppm: 104.45 (C-9), 111.98 (C-11), 115.37 (C-5), 117.46 (C-3),
120.43 (C-17,18), 123.97 (C-7), 125.03 (C-1), 129.95 (C-13), 129.18 (C-14,16), 131.58 (C-12), 127.57 (C-2),
134.28 (C-6), 140.74 (C-15), 159.00 (C-4), 157.32–160.62 (C-8), 159.82–163.09 (C-10), 169.00 (C=O), 180.00
(C=S). HR-MS (EI, Calculated/Found m/z): 478.9937 (M++2), C20H14BrF2N3O2S 476.9952/476.9952
(M+), 383.0898/383.0902, 306.0295/306.0296, 264.0704/264.0700233.0403/233.0408. Anal. Cald.
C20H14BrF2N3O2S Calculated/Found (%): C:50.22/50.22 H:2.95/3.27 N:8.79/8.77 S:6.70/6.60.
N-(4-Bromophenyl)-2-[(20,40-difluoro-4-hydroxybiphenyl-3-yl)carbonyl]hydrazine carbothioamide (
solid. Yield 93%; m.p. 196 ◦C; MW: 551.502 g/mol; Rf
100 value: 62; Rt value: 5.02 min. FT-IR
max. (cm−1): 3242 (N-H), 1637 (Amide C=O), 1238 (C=S), 1138 (Ar-F). 1H-NMR (DMSO-d6/TMS)
7): White
×
ν
δ
ppm: 2.73 (s, 3 H, DMF methyl protons), 2.89 (s, 3 H, DMF(2) methyl protons), 7.08 (d, 1 H, Ar-H;
J = 9 Hz), 7.21 (dt, 1 H, Ar-H; J = 1.8 Hz), 7.23 (dt, 1 H, Ar-H; J = 2.4 Hz), 7.35–7.63 (m, 6 H, Ar-H),

Molecules 2018, 23, 1969
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7.95 (s, 1 H, Ar-H), 8.07 (s, 1 H, CHO proton), 9.89 (s, 1 H, Ar-OH), 9.97 (s, 1 H, CSNH-Ar), 10.82
(s, 1 H, NHNHCS), 12.02 (s, 1 H, CONH). 13C-NMR (DMSO-d6/TMS)
ppm: 104.45 (C-9), 111.98
δ
(C-11), 115.35 (C-5), 117.44 (C-3), 123.97 (C-7), 125.01 (C-1), 127.64 (C-14,15,17,18), 129.18 (C-13), 130.32
(C-16), 131.58 (C-12), 134.27 (C-2), 138.51 (C-6), 158.87 (C-4), 157.35–160.65 (C-8), 159.88–163.09 (C-10),
167.00 (C=O), 180.00 (C=S). Anal. Cald. C20H14BrF2N3O2S.DMF Calculated/Found (%): C:50.10/50.03
H:3.84/3.96 N:10.16/10.12 S:5.82/5.98.
0
0
N-({2-[(2 ,4 -Difluoro-4-hydroxybiphenyl-3-yl)carbonyl]hydrazinyl}carbonothioyl)benzamide (8): White solid.
◦
Yield 91%; m.p. 233 C; MW: 427.424 g/mol; Rf
max. (cm−1): 3254 (N-H), 1676 (Benzoyl C=O) 1651 (Amide C=O), 1263 (C=S), 1139 (Ar-F). 1H-NMR
×
100 value: 16; Rt value: 4.90 min. FT-IR ν
(DMSO-d6/TMS) ppm: 7.13 (d, 1 H, Ar-H; J = 8.4 Hz), 7.19 (dd, 1 H, Ar-H; J = 6.6 Hz), 7.35 (dt,
δ
1 H, Ar-H; J = 2.4 Hz), 7.52–7.69 (m, 6 H, Ar-H), 7.98 (d, 1 H, Ar-H; J = 7.2 Hz), 8.12 (t, 1 H, Ar-H;
J = 2.1 Hz), 11.96 (s, 2 H, Ar-OH and CONHNH), 12.03 (s, 1 H, CSNH), 13.71 (s, 1 H, CONH). 13C-NMR
(DMSO-d6/TMS) δ ppm: 104.48 (C-9), 112.08 (C-11), 115.74 (C-5), 117.32 (C-3), 123.82 (C-7), 125.71 (C-1),
128.50 (C-2), 129.69 (C-16), 130.15 (C-14,18), 130.39 (C-13), 131.48 (C-12), 131.73 (C-17,18), 134.20 (C-6),
140.74 (C-15), 159.75 (C-4), 157.32–160.62 (C-8), 159.96–163.09 (C-10), 168.45 (C=O), 171.50 (Benzoyl
C=O), 180.00 (C=S). Anal. Cald. C21H15F2N3O3S Calculated/Found (%): C:59.01/58.38 H:3.54/3.98
N:9.83/9.61 S:7.50/7.27.
0
0
2-[(2 ,4 -Difluoro-4-hydroxybiphenyl-3-yl)carbonyl]-N-(2-methoxyphenyl)hydrazine carbothioamide (
solid. Yield 82%; m.p. 195 ◦C; MW: 502.532 g/mol; Rf
100 value: 68; Rt value: 4.48 min. FT-IR
max. (cm−1): 3198 (N-H), 1645 (DMF C=O), 1633 (Amide C=O), 1240 (C=S), 1138 (Ar-F). 1H-NMR)
(DMSO-d6/TMS) ppm: 2.73 (s, 3 H, DMF methyl protons), 2.88 (s, 3 H, DMF(2) methyl protons), 3.39
9): White
×
ν
δ
(water in solvent and O-CH3 protons), 6.95 (t, 1 H, Ar-H; J = 7.5 Hz), 7.08 (d, 1 H, Ar-H; J = 9 Hz), 7.16
(t, 1 H, Ar-H; J = 6.9 Hz), 7.21 (t, 1 H, Ar-H; J = 7.8 Hz), 7.36 (dt, 1 H, Ar-H; J = 2.4 Hz), 7.57–7.69 (m,
4 H, Ar-H), 7.95 (s, 1 H, Ar-H), 8.07 (s, 1 H, CHO protons), 9.36 (s, 1 H, Ar-OH), 9.84 (s, 1 H, CSNH-Ar),
10.85 (s, 1 H, NHNHCS), 11.44 (s, 1 H, thiol proton), 11.99 (s, 1 H, CONH). 13C-NMR (DMSO-d6/TMS)
δ
ppm: 55.63 (O-CH3), 104.38 (C-9), 111.44 (C-16), 112.00 (C-11), 115.83 (C-5), 117.32 (C-3), 119.79
(C-14), 123.94 (C-7), 125.17 (C-1), 126.12–126.72 (C-15,17,18), 127.51 (C-13), 131.55 (C-12), 129.42 (C-2),
134.01 (C-6), 159.50 (C-4), 157.31–160.58 (C-8), 159.78–162.06 (C-10), 166.20 (C=O), 180.00 (C=S). HR-MS
(EI, Calculated/Found m/z): C21H17F2N3O3S 429.0953/429.0953, C21H18FN3O2S 395.1098/395.1098,
C13H10F2N2O2 264.0704/264.0704, C13H7F2O2 233.0403/233.0408 Anal. Cald. C21H17F2N3O3S.DMF
Calculated/Found (%): C:57.36/56.66 H:4.81/4.77 N:11.15/10.95 S:6.38/6.57.
2-[(20,40-Difluoro-4-hydroxybiphenyl-3-yl)carbonyl]-N-(4-ethylphenyl)hydrazine carbothioamide (10): White
◦
solid. Yield 89%; m.p. 185–186 C; MW: 473.5454 g/mol; Rf
ν
×
100 value: 38; Rt value: 4.84 min. FT-IR
max. (cm−1): 3234 (N-H), 1635 (Amide C=O), 1263 (C=S), 1139 (Ar-F). 1H-NMR) (DMSO-d6/TMS)
δ
ppm: 1.05 (t, ethanol CH3), 1.17 (t, 3 H, C6H4-CH2-CH3 J = 7.5 Hz), 2.57 (q, 2 H, C6H4-CH2-CH3
J = 7.5 Hz), 3.40 (water in solvent and Ethanol CH2), 4.39 (s, Ethanol OH), 7.06 (d, 1 H, Ar-H; J = 8.7 Hz),
7.15–7.25 (m, 2 H, Ar-H), 7.32–7.40 (m, 2 H, Ar-H), 7.55–7.64 (m, 4 H, Ar-H), 8.06 (s, 1 H, Ar-H), 9.82 (s,
2 H, Ar-OH ve CSNH-Ar), 10.85 (s, 1 H, NHNHCS), 12.04 (s, 1 H, CONH). 13C-NMR) (DMSO-d6/TMS)
δ
ppm: 15.55–27.61 (CH2CH3) 104.38 (C-9), 111.93 (C-11), 115.59 (C-5), 117.38 (C-3), 123.96 (C-7), 125.04
(C-1), 127.44 (C-14,18), 128.03 (C-15,17), 129.19 (C-13), 131.56 (C-12), 134.14 (C-2), 136.55 (C-6), 139.17
(C-16), 158.50 (C-4), 157.32–160.60 (C-8), 159.81–163.07 (C-10), 168.00 (C=O), 180.00 (C=S). Anal. Cald.
C22H19F2N3O2S. C2H5OH Calculated/Found (%): C:60.81/60.24 H:5.27/5.23 N:8.86/8.91 S:6.75/6.87.
3.4. General Procedure for the Synthesis of 5-(20,40-Difluoro-4-hydroxybiphenyl-3-yl)
-4-(substituted)-2,4-dihydro-3H-1,2,4-triazole-3-thiones 11–16
Compounds 3–9 except for compound 8 and 10 (0.01 mol) were dissolved in sodium hydroxide
solution (2 N, 25 mL) and heated under reflux for 4 to 6 h. The solution then was neutralized with
concentrated hydrochloric acid. The crude product was precipitated, filtered and washed with distilled
water. The compounds recrystallized twice from ethanol or DMF [12].

Molecules 2018, 23, 1969
13 of 19
5-(20,40-Difluoro-4-hydroxybiphenyl-3-yl)-4-propyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (11): White solid.
Yield 69%; m.p. 177–179 ◦C; MW: 347.3823 g/mol; Rf
max. (cm−1): 3190 (N-H), 1626 (triazole C=N), 1257 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS)
×
100 value: 35; Rt value: 3.81 min. FT-IR
ν
δ
ppm: 0.66 (t, 3 H, CH2-CH2-CH3 J = 7.3 Hz), 1.54 (m, 2 H, CH2-CH2-CH3), 3.84 (t, 2 H, CH2-CH2-CH3
J = 7.3 Hz), 7.12 (d, 1 H, Ar-H; J = 8.7 Hz), 7.17 (dd, 1 H, Ar-H; J = 2.1 Hz), 7.32 (dt, 1 H, Ar-H;
J = 2.4 Hz), 7.47–7.61 (m, 3 H, Ar-H), 13.76 (bs, 1 H, triazole NH). AR-OH exchanged with deuterium of
solvent. Anal. Cald. C17H15F2N3OS Calculated/Found (%): C:58.78/58.28 H:4.35/4.50 N:12.10/11.99
S:9.23/9.56.
5-(20,40-Difluoro-4-hydroxybiphenyl-3-yl)-4-(3-fluorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12): White
◦
solid. Yield 98%; m.p. 244 C; MW: 399.3889 g/mol; Rf
×
100 value: 37; Rt value: 3.81 min. FT-IR
ν
max. (cm−1): 3250 (N-H), 1633 (triazole C=N), 1255 (C=S), 1139 (Ar-F). 1H-NMR (DMSO-d6/TMS)
δ
ppm:
6.84 (d, 1 H, Ar-H; J = 8.7 Hz), 7.12–7.18 (m, 2 H, Ar-H), 7.19–7.31 (m, 2 H, Ar-H), 7.32–7.53 (m, 5 H, Ar-H),
10.32 (s, 1 H, Ar-OH), 14.14 (bs, 1 H, triazole NH). 13C-NMR) (DMSO-d6/TMS)
δ
ppm: 104.43 (C-9), 111.94
(C-11), 113.27 (C-5), 115.46 (C-17) 115.75 (C-16), 115.99 (C-3), 123.77 (C-7), 124.20 (C-14), 124.75 (C-1), 130.17
(C-13), 132.54 (C-2), 131.36 (C-12), 131.73 (C-18), 135.77 (C-6), 149.20 (C-4), 155.62 (C=N), 157.22–160.57
(C-8), 159.71–162.97 (C-10), 159.62 (C-15), 167.68 (C=S). Anal. Cald. C20H12F3N3OS Calculated/Found (%):
C:60.15/59.85 H:3.03/3.27 N:10.52/10.46 S:8.03/8.16.
4-(3-Chlorophenyl)-5-(20,40-difluoro-4-hydroxybiphenyl-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
White solid. Yield 95%; m.p. 243–244 ◦C; MW: 415.8435 g/mol; Rf
100 value: 36; Rt value: 3.94 min.
FT-IR
max.(cm−1): 3188 (N-H), 1620 (triazole C=N); 1269 (C=S), 1139 (Ar-F). 1H-NMR (300 MHz)
(DMSO-d6/TMS) ppm: 6.84 (d, 1 H, Ar-H; J = 8.4 Hz), 7.15 (dt, 1 H, Ar-H; J = 2.1 Hz), 7.24–7.31 (m,
1 H, Ar-H), 7.34 (t, 1 H, Ar-H; J = 2.2 Hz), 7.35 (d, 1 H, Ar-H; J = 2.7 Hz), 7.39–7.56 (m, 4 H, Ar-H), 7.57
(s, 1 H, Ar-H), 10.34 (s, 1 H, Ar-OH), 14.16 (bs, 1 H, triazole NH). 13C-NMR (DMSO-d6/TMS)
ppm:
(13):
×
ν
δ
δ
104.44 (C-9), 111.93 (C-11), 113.22 (C-5), 115.99 (C-3), 123.77 (C-7), 124.80 (C-1), 126.66–128.14–128.89
(C-14,16,17), 130.18 (C-13), 132.45 (C-2), 131.38 (C-12), 131.81 (C-18), 132.65 (C-15), 135.62 (C-6),
149.19 (C-4), 155.54 (C=N), 157.24–160.53 (C-8), 159.71–162.98 (C-10), 167.63 (C=S). Anal. Cald.
C20H12ClF2N3OS Calculated/Found (%): C:57.77/57.54 H:2.91/3.25 N:10.10/10.13 S:7.71/8.03.
4-(3-Bromophenyl)-5-(20,40-difluoro-4-hydroxybiphenyl-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
White solid. Yield 84%; m.p. 232–233 ◦C; MW: 460.2945 g/mol; Rf
100 value: 36; Rt value: 4.18 min.
FT-IR
max.(cm−1): 3201 (N-H), 1620 (triazole C=N), 1240 (C=S), 1139 (Ar-F). 1H-NMR (300 MHz)
(DMSO-d6/TMS) ppm: 6.85 (d, 1 H, Ar-H; J = 8.7 Hz), 7.14 (dt, 1 H, Ar-H; J = 2.1 Hz), 7.28–7.59
(m, 8 H, Ar-H), 10.42 (s, 1 H, Ar-OH), 14.12 (bs, 1 H, triazole NH). 13C-NMR (DMSO-d6/TMS)
(14):
×
ν
δ
δ
ppm: 104.43 (C-9), 111.93 (C-11), 113.19 (C-5), 116.05 (C-3), 123.81 (C-7), 124.69 (C-1), 127.09 (C-14,16),
130.44 (C-13), 130.96 (C-17,18), 131.36 (C-12), 131.68 (C-15), 132.46 (C-2), 135.91 (C-6), 149.21 (C-4),
155.69 (C=N), 157.22–160.52 (C-8), 159.69–162.95 (C-10), 167.59 (C=S). HR-MS (EI, Calculated/Found
m/z): 460.9822 (M+2), C20H12BrF2N3OS 458.9846/458.9847 (M+), C20H12BrF2N3O 427.0126/427.0126,
347.0853/347.0856, 231.0490/231.0492. Anal. Cald. C20H12BrF2N3OS Calculated/Found (%):
C:52.19/51.31 H:2.63/3.01 N:9.13/9.06 S:6.97/6.62.
4-(4-Bromophenyl)-5-(20,40-difluoro-4-hyd◦roxybiphenyl-3-yl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
(15):
White solid. Yield 69%; m.p. 263–264 C; MW: 460.294 g/mol; Rf
×
100 value: 60; Rt value: 4.38
1
min. FT-IR
(DMSO-d6/TMS)
2 H, Ar-H, o-Br, J = 6.6 Hz), 7.29–7.35 (m, 2 H, Ar-H), 7.43–7.60 (m, 2 H, Ar-H), 7.61 (d, 2 H, Ar-H,
m-Br, J = 6.6 Hz), 10.27 (s, 1 H, Ar-OH), 14.13 (s, 1 H, NH). 13C-NMR (DMSO-d6/TMS)
ppm:
ν
max. (cm−1): 3196 (N-H), 1622 (triazole C=N), 1242 (C=S), 1139 (Ar-F). H-NMR
δ
ppm: 6.84 (d, 1 H, Ar-H; J = 8.7 Hz), 7.14 (dt, 1 H, Ar-H; J = 2.4 Hz), 7.28 (d,
δ
104.45 (C-9), 111.90 (C-11), 113.30 (C-5), 116.01 (C-3), 121.95 (C-14,18), 123.75 (C-7), 124.82 (C-1),
130.02 (C-15,17), 131.40 (C-12), 131.57 (C-13), 131.80 (C-16), 132.62 (C-2), 133.76 (C-6), 149.25 (C-4),
155.47 (C=N), 157.24–160.53 (C-8), 159.70–162.97 (C-10), 167.58 (C=S). Anal. Cald. C20H12BrF2N3OS
Calculated/Found (%): C:52.19/52.27 H:2.63/2.91 N:9.13/8.80 S:6.97/6.77.

Molecules 2018, 23, 1969
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5-(20,40-Difluoro-4-hydroxybiphenyl-3-yl)-4-(2-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (16):
◦
White solid. Yield 65%; m.p. 196 C; MW: 457.493 g/mol; Rf
ν
×
100 value: 60; Rt value: 3.90 min. FT-IR
max. (cm−1): 3198 (N-H), 1626 (triazole C=N), 1257 (C=S), 1141 (Ar-F). 1H-NMR (DMSO-d6/TMS)
δ
ppm: 1.05 (ethanol CH3), 3.43 (Ethanol CH2), 3.55 (s, 3 H, Ar-O-CH3), 3.90 (ethanol OH), 6.85 (d, 1 H,
Ar-H; J = 8.4 Hz), 6.80–7.04 (m, 2 H, Ar-H), 7.13 (dt, 1 H, Ar-H; J = 2.1 Hz), 7.25–7.39 (m, 6 H, Ar-H),
10.31 (s, 1 H, Ar-OH), 14.02 (bs, 1 H, NH). 13C-NMR (DMSO-d6/TMS)
δ ppm: 55.41 (O-CH3), 104.42
(C-9), 111.93 (C-11), 112.28 (C-15), 116.02 (C-5), 120.14 (C-17) 122.68 (C-16,18), 123.80 (C-7), 124.39
(C-1), 130.34 (C-13), 130.80 (C-2), 131.23 (C-12), 132.14 (C-6), 133.50 (C-3), 149.89 (C-4), 154.21 (C-14),
155.75 (C=N), 157.19–160.48 (C-8), 159.64–162.90 (C-10), 168.19 (C=S). HR-MS (EI, Calculated/Found
m/z): C21H15F2N3O2S 411.0847/411.0847 (M+), C21H14F2N3O2 378.1043/378.1048, 231.0490/231.0478.
Anal. Cald. C21H15F2N3O2S. C2H5OH Calculated/Found (%): C:60.32/60.06 H:4.59/4.05 N:9.18/9.63
S:6.99/7.26.
3.5. Cell Culture Conditions
In this project, the colorectal cancer cell line HCT116 (ATCC® CCL-247™), androgen-dependent
prostate cancer cell line PC-3 (ATCC® CRL-1435
™
), breast cancer cell lines T47D (ATCC® HTB-133
™
),
) and finally, epithelial embryonic kidney cell line HEK293 (ATCC®
) were used. All media were completed with %10 fetal bovine serum (FBS) and %1
MCF7 (ATCC® HTB-22
CRL-1573
™
™
penicillin streptomycin amphotericin (PSA). All cells were cultured periodically at 37 ◦C, 5% (v/v)
CO2 and 95% (v/v) air in T-75 flasks. Different media were used for the different cell lines based
on ATCC standards: MCF7 and HEK293 cell lines were cultured in DMEM High Mmedium (ATCC,
30-2003); T47D and PC-3 cell lines were cultured in RPM 1640 medium (ATCC, 30-2001); HCT116 cell
line was cultured in DMEM F12 medium (ATCC, 30-2006). RPM 1640 medium was further completed
with 0.2 unit/mL bovine insulin for the T47D cell line. Cells were passaged when they reached %80
confluency in order to support proliferation.
3.5.1. Cytotoxicity Assay and Determination of IC50 Values of Synthesized Compounds
The effects of the synthesized compounds on the cell viability were tested. Stock solutions for each
compound were prepared by measuring 20 mg of each compound and dissolving it in 1 mL DMSO
to a final concentration of 20 mg/10 mL, followed by filtration through a 0.2-µm filter (Sartorius AG,
Gottingen, Germany). Then stock solutions were diluted as 1/10, 1/100 and 1/1000 in the designated
media and applied on the cell lines. In order to determine the effect of DMSO itself, the same dilutions
were performed by using DMSO, only and applied on the cell lines. Cell densities used for each cell
line in 96 well plate were as follows: for PC-3, HCT116 and HEK293 cell lines 5000 cells/well; for T47D
cell lines 20,000 cells/well. Cell viability was measured by the MTS cytotoxicity assay (CellTiter96
AqueousOne Solution; Promega, Southampton, UK) according to the manufacturer’s instructions.
3-(4,5-Dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS)
is a yellow tetrazolium salt which is catabolized to formazan by dehydrogenase enzyme in the
mitochondria of the living cells. Formazan is a purple compound and the detection in this assay is
based on the measurement of formazan compounds by an ELISA plate reader. After incubating the
cells for 72 h, 10 µL MTS reagent and 100 µL designated medium was given to the cells followed by 2 h
of incubation at 37 ◦C. Thereafter, the absorbance at 490 nm was measured by an ELISA plate reader.
(BioTek Instruments, Inc., Winooski, VT, USA). The activity doses of the compounds were determined
according to 50% cell death rate when compared to DMSO, only after 72 h of incubation. Compounds
that do not affect cell viability of healthy cell line (HEK293) but deathly to cancer cell lines were further
tested with narrower test conditions for MTS assay to determine IC50 values. IC50 values are calculated
by the GraphPad prism software.

Molecules 2018, 23, 1969
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3.5.2. Annexin V
Determination of IC50 values was followed by the investigation of effect of the synthesized
compounds on apoptosis. Cells were seeded into six well plates at densities of 15,000 cells/well (PC-3,
HCT116 and HEK293 cell lines) and 60,000 cells/well (T47D cell lines). After the treatment cells were
harvested and washed with ice cold PBS. 1
were being washed. Working solution of PI (100
1 mg/mL PI stock solution in 45 L 1 Annexin-binding buffer. After the precipitation of the cells,
they were resuspended in 500 L 1 Annexin-binding buffer. Resuspended cells were separated into
four groups (Annexin V, PI, Annexin V + PI and NC) each containing 100 L of suspension. Five L of
the Annexin V conjugate was added to the proper groups. Samples were incubated for 15 min at room
temperature. The incubation step was followed by adding 2 L of the 100 g/mL PI working solution
×
Annexin-binding buffer was prepared while the cells
µ
g/mL) was prepared as well by diluting 5 L of the
µ
µ
×
µ
×
µ
µ
µ
µ
to the proper groups then the fluorescence was observed by flow cytometry.
3.5.3. 5-Ethynyl-20-deoxyuridine (EdU) Staining
The EdU staining protocol directly measures the level of active DNA synthesis of the cell cycle.
This measurement enables one to see dividing cells under a fluorescence microscope. In order
to perform EdU staining cells were seeded on the coverslips which were located in the wells of
six well plates. As in the Annexin V procedure cells were seeded into six well plates at densities
of 15,000 cells/well (PC-3, HCT116 and HEK293 cell lines) and 60,000 cells/well (T47D cell lines).
After drug treatment, cells that are on the coverslips were incubated with 10 µM EdU. The incubation
step is followed by cell fixation and permeabilization. Fixation occurred with 3.7% formaldehyde
in PBS for 15 min then cells were washed with 3% BSA in PBS. Permeabilization was performed by
incubating cells in 0.5% Triton® X-100 in PBS for 20 min at room temperature. Click-iT® reaction
cocktail (0.5 mL) was added to the cells after washing with 3% BSA solution. Cells were incubated for
30 min at room temperature, then DNA synthesis was observed under a confocal microscope.
3.6. Docking Studies
The preparation of the protein, as well as the compounds were performed in BIOVIA Discovery Studio
4.5. The crystal structures of the enzymes COX-1 and COX-2 were downloaded from the PDB database
(accession code: 1EQG for COX-1 [46] and 3NT1 for COX-2 [47] (http://www.rcsb.org/pdb/). All protein
structures were cleaned of inhibitors, water molecules and non-interacting ions, while cofactors were retained.
All hydrogens were added and proteins minimized using the “Clean Geometry” toolkit. For more complete
optimization, proteins were submitted to the “Prepare Macromolecule” protocol. Missing hydrogen atoms
were added based on the protonation state of the titratable residues at pH 7.4. Compounds were prepared
and minimized in the same force field. After preparation, docking was performed in AutoDock 4.2 program
(http://autodock.scripps.edu). All the compounds were set to be flexible, while the proteins were kept rigid.
AutoDock adds a free-energy scoring function created from a linear regression analysis, the AMBER force
field, and a large set of diverse protein-ligand complexes with known inhibition constants. The coordinates
of alpha carbon of Tyr355 were used for grid-centering for COX enzymes, grid box was set to be 70 grid
points (each grid point is 0.375 Å). For the docking studies the Lamarkian genetic algorithm was used as
search algorithm. For visualization of non-bonded interactions, we also used BIOVIA Discovery Studio 4.5.
4. Conclusions
Diflunisal and its derivatives have been widely studied and different biological activities have
been reported for these compounds. The correlation between the COX-2 selectivity and anticancer
activity, had become the basic subject of our research. Thiosemicarbazides and 1,2,4-triazole-3-thiones
have been related with anticancer activity, therefore, we have designed some novel compounds
combining these active pharmacophores and a NSAID.

Molecules 2018, 23, 1969
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In this study, we have synthesized new diflunisal thiosemicarbazides and 1,2,4-triazole-3-thiones
in order to determine their possible anticancer activity. The synthesis, structure characterization and
antibacterial activity against Helicobacter pylori of thiosemicarbazides, which are the prototypes of
the 1,2,4-triazoles 11
against the PC-3 cancer cell line, with IC50 values of 41.8 and 11.7
was found active against the HCT-116 cancer cell line with a 6.2 M IC50 value. Compounds 15 and 16
were found active against the T47D cancer cell line with IC50 values of 43.4 and 27.3 M, respectively.
–
13, are given previously study [25]. Compounds
6 and 10 were found active
µM, respectively. Compound 16
µ
µ
The in silico and in vitro studies that we performed lead us to the conclusion that there is a correlation
between COX-2 enzyme activity and anticancer activity in vitro. In the docking studies, compounds
12 and 13 appeared to show the most affinity towards COX-2 enzyme however, compounds
15 (∆G = −10.11 kcal/mol) and 16
(
∆
G =
−
9.41 kcal/mol) showed high inhibition on COX-2 enzyme.
The highest selectivity towards COX-2 was shown by compound 13. Further experiments are still
needed in order to improve our understanding of how these compounds elicit their anticancer activity.
Author Contributions: S¸.G.K. designed the research; G.P.C. synthesized the compounds and elucidated their
structures. T.D. and K.Y. performed the docking studies. T.B.H., N.T. and F.S¸. performed the activity tests.
S¸.G.K. was also responsible for the correspondence of the manuscript. All authors read and approved the
final manuscript.
Funding: This research received no external funding.
Acknowledgments: This work was supported by The Scientific and Technical Research Council of Turkey
˙
(TÜBITAK), Research Fund Project Number: 114S966. The authors are grateful to Jürgen Gross from the Institute
of Organic Chemistry, University of Heidelberg, for his generous help on obtaining HR-EI/FAB mass spectra
˙
of the synthesized compounds. Diflunisal was supplied by Sanovel Pharmaceutical Industry Inc, Istanbul,
Turkey. Teodora Djikic kindly acknowledges “Training in Neurodegeneration, Therapeutics, Intervention and
Neurorepair” project number 608381 funded by Marie Skłodowska-Curie action, funding scheme: FP7-MC-ITN.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).