Synthesis of dihydropyrazole sulphonamide derivatives that act as anti-cancer agents through COX-2 inhibition

Article abstract of DOI:10.1016/j.phrs.2015.12.025

COX-2 has long been exploited in the treatment of inflammation and relief of pain; however, research increasingly suggests COX-2 inhibitors might possess potential benefits to thwart tumour processes. In the present study, we designed a series of novel COX-2 inhibitors based on analysis of known inhibitors combined with an in silico scaffold modification strategy. A docking simulation combined with a primary screen in vitro were performed to filter for the lead compound, which was then substituted, synthesized and evaluated by a variety of bioassays. Derivative 4d was identified as a potent COX-2 enzyme inhibitor and exerted an anticancer effect through COX-2 inhibition. Further investigation confirmed that 4d could induce A549 cell apoptosis and arrest the cell cycle at the G2/M phase. Moreover, treatment with 4d reduced A549 cell adhesive ability and COX-2 expression. The morphological variation of treated cells was also visualized by confocal microscopy. Overall, the biological profile of 4d suggests that this compound may be developed as a potential anticancer agent.

Full text of DOI:10.1016/j.phrs.2015.12.025

Accepted Manuscript
Title: Synthesis of dihydropyrazole sulphonamide derivatives
that act as anti-cancer agents through COX-2 inhibition
Author: Han-Yue Qiu Peng-Fei Wang Zhen Li Jun-Ting Ma
Xiao-Ming Wang Yong-Hua Yang Hai-Liang Zhu
PII:
S1043-6618(15)00292-3
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.phrs.2015.12.025
YPHRS 3018
To appear in:
Pharmacological Research
Received date:
Revised date:
Accepted date:
9-8-2015
20-12-2015
20-12-2015
Please cite this article as: Qiu Han-Yue, Wang Peng-Fei, Li Zhen, Ma
Jun-Ting, Wang Xiao-Ming, Yang Yong-Hua, Zhu Hai-Liang.Synthesis of
dihydropyrazole sulphonamide derivatives that act as anti-cancer agents through COX-2
inhibition.Pharmacological Research http://dx.doi.org/10.1016/j.phrs.2015.12.025
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Synthesis of dihydropyrazole sulphonamide derivatives that
act as anti-cancer agents through COX-2 inhibition
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Han-Yue Qiu†, Peng-Fei Wang†, Zhen Li, Jun-Ting Ma, Xiao-Ming Wang*,
Yong-Hua Yang*, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210046, People’s Republic of China
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*Corresponding author. Tel. & fax: +86-25-83592672; e-mail: zhuhl@nju.edu.cn,
yangyh@nju.edu.cn
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†These two authors contributed equally to this paper.
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Aseries of novel COX-2 inhibitors was designed and synthesized as antitumor agents
based on the knowledge of known COX-2 inhibitors and in silico scaffold modification
strategy.
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Abstract
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COX-2 has long been exploited in the treatment of inflammation and relief of pain;
however, research increasingly suggests COX-2 inhibitors might possess potential
benefits to thwart tumour processes. In the present study, we designed a series of novel
COX-2 inhibitors based on analysis of known inhibitors combined with an in silico
scaffold modification strategy. A docking simulation combined with a primary screen
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in vitro were performed to filter for the lead compound, which was then substituted,
synthesized and evaluated by a variety of bioassays. Derivative 4d was identified as a
potent COX-2 enzyme inhibitor and exerted an anticancer effect through COX-2
inhibition. Further investigation confirmed that 4d could induce A549 cell apoptosis
and arrest the cell cycle at the G2/M phase. Moreover, treatment with 4d reduced A549
cell adhesive ability and COX-2 expression. The morphological variation of treated
cells was also visualized by confocal microscopy. Overall, the biological profile of 4d
suggests that this compound may be developed as a potential anticancer agent.
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Keywords:
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Dihydropyrazole
COX-2 selectivity
Antitumor
Diarylheterocycle
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1 Introduction
Cyclooxygenase (COX), which is known for its rate-limiting role in the conversion
of arachidonic acid to prostaglandin (PG), is generally classified into three subtypes:
COX-1, COX-2 and COX-3.(1) The isoforms differ not only in expression but also in
physiological and pathological functions. COX-1 is stimulated continuously in most
normal tissues and COX-3 primarily in the central nervous system, while COX-2 is
inducible. The amount of COX-2 often reaches a relatively high level when induced by
stimuli such as pro-inflammatory cytokines, growth factors and tumour promoters, but
it remains undetectable in most normal cells or tissues.(2-4) The significantly up-
regulated expression of COX-2 in pathological processes revealed its involvement in
diseases such as inflammation and various types of cancer. These characteristic
variances in expression and distribution between COX-2 and other isoenzymes laid the
foundation for designing COX-2-selective drugs that minimally disturb normal COX
function.(5, 6) In practice, COX-2 has long been exploited to treat inflammation and
relieve pain,(7-10) and along with continued research, interest in developing COX-2-
specific antitumor medicaments is increasing considerably.(11-14) According to the
database of clinical trials (www.clinicaltrials.gov), hundreds of clinical trials have been
or are being conducted to test the anti-cancer potential of COX-2 inhibitors, mostly
celecoxib (celebrex). The same repurposing strategy also applies to other known COX-
2 inhibitors, providing a reference basis for COX-2-inhibitor-based anti-cancer drug
development.(13, 15, 16) Given that there is a major discrepancy between supply and
demand of anti-cancer agents, it is important to expand the availability of various types
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of anti-cancer medicines. Therefore, in this study we attempted to design a class of
COX-2-selective agents and assess their potential in anti-cancer agent development.
In general, selective COX-2 inhibitors can be categorized as diarylheterocycles or
non-diarylheterocycles, and the largest proportion of selective COX-2 inhibitors
comprises diarylheterocycles with a five-membered core.(17-20) As illustrated in Fig.
1, the most famous Coxibs (COX-2 selective inhibitors) are unexceptionally composed
of various five-membered heterocyclic cores and 1, 2-diarylsubstitution. Based on this
knowledge, we exploited a new diarylheterocycle scaffold with a dihydropyrazole
group as the five-membered core ring. Additionally, a sulphonamide group was
attached to the p-position of one aryl ring, as many studies have indicated that this
pharmacophore plays a crucial role in COX-2 selectivity.(21, 22) The scaffold was
substituted to obtain a library of small molecules. Using virtual screening, the
molecules were ranked according to the docking score. The best hits were selected and
preliminarily screened in vitro. We then validated the lead compound, which possessed
good COX-2 inhibitory activity and comparable binding energy with celecoxib in
docking with the COX-2 enzyme (Table 1). Derivatives of the lead compound (4a-4t)
were synthesized and evaluated by additional bioassays.
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Fig. 1 Some well-known COX-2 inhibitors (coxibs) and the design pathway for novel
dihydropyrazole sulphonamide derivatives.
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Table 1. Interaction energy and AlogP of compounds 4a – 4t
Interaction energy
AlogPa
Compd
R
ΔGb (kcal / mol))
Lead
4a
-58.44
3.736
3.942
3.942
- 62.97
- 60.15
4b
- 59.01
- 63.11
- 62.60
3.942
4.147
4.147
4c
4d
4e
- 60.26
- 57.77
4.401
4.401
4f
4g
- 57.38
- 62.73
- 62.68
4.401
5.065
5.729
4h
4i
4j
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- 61.72
- 60.38
4.485
4.485
4k
4l
- 52.23
4.485
4m
- 61.09
- 52.51
4.314
4.314
4n
4o
- 57.92
4.314
4p
4q
- 61.42
- 49.20
- 48.91
4.222
4.678
3.720
4r
4s
4t
- 58.87
- 56.13
3.631
5.946
Celecoxib
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a Calculated using Discovery Studio 3.5.
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2 Materials and methods
2.1 Materials
All chemicals (reagent grade) used were purchased from Nanjing Chemical Reagent
Co. Ltd. (Nanjing, China). Celecoxib was purchased from Sigma-Aldrich (St. Louis,
MO). All the 1H NMR spectra were recorded on a Bruker DPX 400 model spectrometer
in DMSO-d6, and chemical shifts (δ) are reported as parts per million (ppm). ESI-MS
spectra were recorded by a Mariner System 5304 Mass spectrometer. Melting points
were determined on a XT4 MP apparatus (Taike Corp, Beijing, China). Thin layer
chromatography (TLC) was performed on silica gel plates (Silica Gel 60 GF254) and
visualized in UV light (254 nm and 365 nm). Column chromatography was performed
using silica gel (200-300 mesh) and eluting with ethyl acetate and petroleum ether (bp
30-60 °C).
The COX-1 (human) Inhibitor Screening Assay Kit (#701070) and COX-2 (human)
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Inhibitor Screening Assay Kit (#701080) were purchased from Cayman Chemical, (MI,
USA). The PTGS2 small interfering RNA kit was purchased from Ribobio
(GuangZhou, China). RNase A (#EN0531) was purchased from Thermo Scientific,
Fermentas (USA). The AnnexcinV-FITC cell apoptosis assay kit (#BA11100) was
purchased from BIO-BOX (Nanjing, China). Fibronectin (#F1056) and laminin
(#L2020) were purchased from Sigma-Aldrich (St. Louis, MO). COX-2 anti-body
(#12282P) was purchased from Cell Signalling Technology (Beverly, MA, USA).
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2.2 General procedure for the synthesis of compounds 4a-4t
A mixture of compound 3a-3t (5 mmol), 4-hydrazinylbenzenesulfonamide (5 mmol)
and glacial acetic acid (2 mL) in ethanol (20 mL) was refluxed for 8 h. Afterwards, the
cooled reaction contents were poured into ice water, and the products were filtered and
washed carefully with ice water and cool ethanol. The crude products were
recrystallized from methanol to obtain pure compounds 4a-4t.
2.3 Cell Culture
A human hepatoma cell line (HepG2), human lung adenocarcinoma epithelial cell
line (A549), carcinoma of cervix cell line (HeLa) and human kidney epithelial cell
(293T) were purchased from Nanjing Keygen Technology (Nanjing, China). Cells were
maintained in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone) (High Glucose)
with L-glutamine supplemented with 10% foetal bovine serum (FBS, BI), 100 U/mL
°
penicillin and 100 mg/mL streptomycin (Hyclone), and incubated at 37 C in a
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humidified atmosphere containing 5% CO2.
2.4 COX inhibitor screening assay
The ability of compounds 4a-4t to inhibit COX-1 and COX-2 was determined using
COX inhibitor screening assay kits according to the instruction manual.(23) In brief,
COX-1 or COX-2 enzyme was pre-incubated with test compounds at 0 μM, 1 μM, 10
μM and 100 μM in reaction buffer (0.1 M Tris-HCl, pH 8.0, 5 mM EDTA, 2 mM phenol
and 1 μM heme) at 37 °C for 10 min. The reactions were initiated by adding arachidonic
acid to a final concentration of 100 μM and incubated at 37 °C for 2 min. Afterwards, 1
M HCl was added to the reaction mixtures to stop the reaction, followed by one tenth
the volume of saturated stannous chloride (50 mg/mL). The reaction mixtures were
incubated for 5 min at room temperature, and the amount of prostaglandin E2 formed
during the reaction was measured by enzyme immunoassay.(24)
2.5 Anti-proliferation assay
The anti-proliferative activities of the prepared compounds against the A549, Hela,
HepG2, and 293T cell lines were evaluated using a standard (MTT)-based colorimetric
assay with some modification.(25) Cell lines were grown to log phase in DMEM
supplemented with 10% foetal bovine serum. Cell suspensions were prepared and 100
μL/well dispensed into 96-well plates to give 104 cells/well. The subsequent incubation
was performed at 37 °C, 5% CO2 atmosphere for 24 h to allow the cells to reattach.
Subsequently, cells were treated with the target compounds at 0 μM, 1 μM, 10 μM and
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100 μM in the presence of 10% FBS for 24 h. Afterwards, cell viability was assessed
by the conventional 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
(MTT) reduction assay carried out strictly according to the manufacturer’s instructions
(Sigma). The absorbance (OD570) was read on an ELISA reader (Tecan, Austria). In all
experiments, three replicate wells were used for each drug concentration. Each assay
was performed at least three times.
2.6 Cell adhesion assay
For the cell adhesion assay, 96-well flat-bottom plates were coated with 50 μL
fibronectin and laminin (10 μg/mL) at 4 °C for 12 h and then blocked with 0.2% BSA
for 2 h at room temperature followed by washing three times. Afterwards, A549 cells
treated with 4d and celecoxib for 24 h each were plated to the coated wells (104 per
well) and incubated at 37 °C, 5% CO2 for 40 min. A549 cells were allowed to adhere
to the coated surface, washed intensively with PBS three times to remove non-adherent
cells, and then incubated in 5 μg/mL MTT in complete medium at 37 °C for 4 h. Next,
MTT-treated cells were lysed in DMSO, and absorbance was measured on an ELISA
reader (Tecan, Austria). Each assay was performed at least three times.
2.7 Cell apoptosis assay
Approximately 105 cells/well were plated in a 24-well plate and allowed to adhere.
Subsequently, the medium was replaced with fresh culture medium containing
compound 4d at final concentrations of 0, 1, 3 and 10 μM. Non-treated wells received
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an equivalent volume of ethanol (<0.1%). After 24 h, cells were trypsinized, washed in
PBS and centrifuged at 2000 rpm for 5 min. The pellet was resuspended in 500 μL
staining solution (containing 5 μL AnnexinV-FITC and 5 μL PI in Binding Buffer),
mixed gently and incubated for 15 min at room temperature in dark. The samples were
then analysed by a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA,
USA).
2.8 Cell cycle analysis
Cells were plated in 6-well plates (106 cells per well) and incubated at 37 °C for 24
h. Exponentially growing cells were then incubated with compound 4d at different
concentrations (0, 1, 3 and 10 μM). After 24 hours, cells were centrifuged at 1500 rpm
at 4 °C for 5 minutes, fixed in 70% ethanol at 4 °C for at least 12 hours and subsequently
resuspended in phosphate buffered saline (PBS) containing 0.1 mg/mL RNase A and 5
mg/mL propidium iodide (PI). The cellular DNA content was measured by flow
cytometry for cell cycle distribution analysis, plotting at least 10000 events per sample.
The percentage of cells in the G0/G1, S and G2/M phases of the cell cycle were
determined using Flowjo 7.6.1 software.
2.9 Confocal microscopy assay
A549 cells were incubated with 5 μM compound 4d for 30 min at 37 °C and washed
three times with Hepes buffer, then scanned under microscopy. Subsequently, cells were
seeded on a cover glass-bottom confocal dish and allowed to adhere for at least 24 h.
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After treatment with compound 4d at 1, 3, or 10 μM for 8 h, A549 cells were washed
with cold PBS three times and fixed in 4% paraformaldehyde for 20 min. Fixed cells
were stained with 1 μg/mL DAPI for 15 min. Before scanning by microscopy, the
staining solution was removed, the dishes were washed with methanol three times, and
glycerin was added to the dishes for imaging. The nuclear morphology of A549 cells
was observed under an Olympus confocal microscope.
2.10 Western blot analysis
The A549 cells on 6-well plates were rinsed twice with cold PBS and lysed in RIPA
lysis buffer containing a protease inhibitor mixture at 1:100 dilution on ice for 30 min.
The insoluble components of cell lysates were removed by centrifugation (4 °C, 12000
× g, 10 min), and protein concentrations were measured using a Pierce BCA protein
assay kit. Proteins were separated by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF)
membranes. Membranes were blocked using skim milk and then incubated with diluted
anti-COX-2 primary antibody (1:1000 dilution) at 4 °C with gentle shaking overnight.
After washing five times, membranes were incubated with secondary antibody (1:1000-
1:3000 dilution) for 1 h at room temperature.
2.11 siRNA transfection
Transfections of siRNA (nonsense siRNA and PTGS2 siRNA) into A549 cells were
carried out using Opti-MEM and Lipo2000 (Invitrogen, Carlsbad, CA) according to the
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manufacturer’s instructions. Lipo2000 and siRNA were diluted separately in Opti-
MEM, mixed and incubated for 5 min. The mixture was then added to A549 cells
cultured in 6-well plates or 96-well plates. The treated cells were cultured at 37 °C, 5%
CO2 atmosphere for 24 h. Subsequently, A549 cells in 6-well plates were harvested for
Western blotting, and the transfected cells in 96-well plates were treated with drugs for
another 24 h. Cell viability was determined by the MTT assay in 96-well plates as
previously described.
2.12 Scaffold modification
Scaffold modification was achieved with the module Grow Scaffold in Discovery
Studio (version 3.5). Following the protocol, the scaffold was docked into COX-2 (PDB
code: 3LN1) binding site beforehand. The 3-position of the dihydropyrazole core and
the unsubstituted aryl ring were then marked as sites to be substituted. After calculation,
modified molecules were produced to generate a library of small molecules.
2.13 Docking simulation
Molecular docking of the compounds binding the three-dimensional X-ray structure
of COX-2 (PDB code: 3LN1) was carried out using Discovery Studio (version 3.5) as
implemented through the graphical user interface DS-CDOCKER protocol. The
aforementioned compounds were constructed, minimized and prepared. The crystal
structures of the protein complex were retrieved from the RCSB Protein Data Bank
(http://www.rcsb.org/pdb/home/home.do). All bound waters and ligands were
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eliminated from the protein. Molecular docking was performed by inserting molecules
into the binding pocket of COX-2 based on the binding mode. The types of interactions
between the docked protein with ligand-based pharmacophore model were analysed
after the end of molecular docking.
3. Results
3.1 Chemistry
The synthesis of compounds 4a-4t followed the general pathway outlined in Scheme
1. The target compounds were obtained in three steps as described in the experimental
section. All of the synthetic compounds 4a-4t are being reported for the first time
(Table 1) and gave satisfactory analytical and spectroscopic data. 1HNMR and ESI-MS
spectra were in full accordance with the assigned structures, which are presented in the
Supplementary Material.
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Scheme 1 (a) 1.0 equiv, 4-fluorobenzaldehyde, DMSO, 120 °C, 4 h; (b) 1.0 equiv, acetophenones,
CH3CH2OH, 0 °C, 2 h; (c) 1.2 equiv, 4-hydrazinylbenzenesulfonamide, CH3CH2OH, 80 °C, 8 h.
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3.2 Compound 4d selectively inhibited COX-2 activity
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The ability of the tested compounds to inhibit human COX-1 and COX-2 was
determined using COX inhibitor screening assay kits. The efficacies of the tested
compounds were determined as the concentration causing 50% enzyme inhibition
(IC50) (Table 2). The majority of the tested compounds showed no inhibition of COX-
1 up to 60 μM. However, a reasonable in vitro COX-2 inhibitory activity was observed
with compounds 4d with IC50 0.08 ± 0.03 μM. The selectivity indices (COX-1/COX-
2) were calculated and compared with that of the standard COX-2 selective inhibitor,
celecoxib.
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Table 2. Data from the in vitro COX-1/COX-2 enzyme inhibition assay of the designed
compounds
Selectivityb
IC50±SD (μM)
Compd
Index (SI)
~ 401
~ 26
COX-1
44.12± 0.26
31.62 ± 0.13
46.73 ± 0.01
> 60
36.11 ± 0.56
40.09 ± 0.19
> 60
COX-2
0.11 ± 0.11
1.23 ± 0.16
2.67 ± 0.33
6.32 ± 0.68
0.08 ± 0.03
0.11 ± 0.07
3.21 ± 0.18
3.92 ± 0.36
4.83 ± 0.44
0.17 ± 0.09
0.31 ± 0.05
0.92 ± 0.51
1.68 ± 1.01
0.22 ± 0.24
2.23 ± 0.59
3.12 ± 0.51
5.23 ± 1.13
1.12 ± 0.17
0.24 ± 0.09
0.19 ± 0.11
0.14 ± 0.50
0.07 ± 0.01
Lead
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
~ 18
> 9
~ 451
~ 364
> 19
> 15
> 12
~ 196
~ 94
~ 48
~ 28
> 273
> 27
> 19
> 11
> 60
> 60
33.32 ± 1.06
29.15 ± 0.29
44.13 ± 0.73
47.10 ± 0.51
> 60
> 60
> 60
> 60
> 60
> 54
36.23 ± 0.06
30.16 ± 0.34
29.12 ± 0.53
29.1 ± 0.12
~ 151
~ 159
~ 208
~ 415
4r
4s
4t
Celecoxib
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b In vitro COX-2 selectivity index (IC50 COX-1 / IC50 COX-2).
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3.3 Compound 4d inhibited cancer cell proliferation in a dose-dependent manner
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All the compounds 4a-4t were evaluated for their anti-proliferation activities against
three cancer cell lines, HeLa (human cervix cell line), HepG2 (human liver cell line),
A549 (human lungs cell line), and one non-cancer cell line, 293T (human kidney
epithelial cell) by the MTT assay, and the calculated IC50 values are listed in Table 3.
All compounds showed moderate to excellent anti-proliferative effects on A549 and
Hela cancer cell lines but not on HepG2. Against the A549 cell line, compound 4d
showed the best anti-proliferative activity with lower IC50 value (1.63 ± 0.97 μM)
compared with celecoxib (2.21 ± 1.31 μM). Subsequently, the MTT assay against one
non-cancer cell line, 293T, was performed to test the cytotoxicity of the obtained
compounds. As shown in Table 3, all the compounds have low cytotoxic effects on
293T, indicating a considerable safety profile.
Compound 4d and celecoxib were further tested in COX-2 knockdown A549 cells
with nonsense siRNA treated A549 cells used as control. Compared to the control
group, the knockdown of COX-2 in A549 cells led to remarkable reduction of cell
viability after drug treatment. The results are summarized in Fig. 2 and demonstrate
that the inhibitory action of compound 4d against cancer cell proliferation is related to
the level of COX-2.
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Table 3. Proliferation inhibitory activities of compounds 4a-4t against three cancer cell lines and
cytotoxicity towards non-cancer cells
IC50±SD (μM)
CC50±SD (μM)
Compd
A549
HeLa
HepG2
293T
3.18 ± 0.24
4.43 ± 0.31
6.29 ± 0.43
9.87 ± 1.01
1.63 ± 0.97
9.12± 0.32
10.37 ± 0.49
11.44 ± 0.34
22.7 ± 1.15
6.12 ± 0.84
18.15 ± 0.25
14.88 ± 0.87
20.51 ± 0.66
27.38 ± 1.34
10.21 ± 0.45
>100
>100
>100
>100
>100
Lead
4a
4b
4c
4d
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1.80 ± 0.25
5.18 ± 0.37
11.51 ± 1.05
13.65 ± 1.30
4.21 ± 0.32
3.25 ± 0.19
6.38 ± 0.54
6.80 ± 0.29
10.22 ± 0.14
10.47 ± 0.91
11.61 ± 0.28
15.11 ± 0.47
20.84 ± 0.13
4.43 ± 0.45
8.29 ± 0.19
6.87 ± 0.12
2.21 ± 1.31
6.98 ± 0.21
13.51 ± 0.33
15.88 ± 0.65
18.47 ± 0.53
13.43 ± 0.21
10.3 ± 0.34
8.69 ± 0.88
9.06 ± 1.54
15.82 ± 0.58
14.24 ± 0.82
12.55 ± 0.35
17.30 ± 0.51
15.35 ± 0.32
11.44 ± 0.69
22.70 ± 2.09
10.37 ± 0.21
7.51 ± 1.28
18.92 ± 0.67
20.92 ± 0.34
19.99 ± 0.85
23.75 ± 0.44
15.91 ± 0.98
14.26 ± 0.25
9.54 ± 1.24
11.38 ± 2.08
13.28 ± 0.71
11.54 ± 1.90
11.93 ± 1.36
22.96 ± 0.98
38.53 ± 0.67
14.50 ± 2.71
31.39 ± 2.28
13.11 ± 0.82
0.68 ± 3.14
>100
>100
4e
4f
>100
4g
>100
4h
>100
4i
>100
4j
4k
>100
>100
4l
>100
4m
>100
>100
4n
4o
>100
4p
>100
4q
>100
4r
4s
>100
>100
4t
65.34 ± 0.18
Celecoxib
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Fig. 2 Knockdown of COX-2 in A549 cells led to remarkable reduction of cell viability after drug
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treatment.
3.4 Compound 4d reduced the adhesive ability of A549 cells.
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Cell adhesion plays a significant role in cancer progression and metastasis, and
decreased cell adhesion benefits cancer therapy. In this study, a cell adhesion assay was
employed to assess the effects of treatment with compound 4d and celecoxib at different
concentrations for 24 h on the ability of A549 cells to adhere. After harvesting the cells,
their adhesive ability to fibronectin and laminin was measured. The results shown in
Fig. 3 indicated that compound 4d could significantly reduce the adhesive ability of
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A549 cells to fibronectin and laminin while the effects of celecoxib were not as striking.
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Fig. 3 Influence of compound 4d and celecoxib on A549 cell adhesion to fibronectin and laminin
(A) Influence of compound 4d on A549 cell adhesion to fibronectin; (B) Influence of compound 4d
on A549 cell adhesion to laminin; (C) Influence of celecoxib on A549 cell adhesion to fibronectin;
(D) Influence of celecoxib on A549 cell adhesion to laminin.
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3.5 Compound 4d caused cell apoptosis in A549 cells in a dose-dependent manner
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To verify whether compound 4d could inhibit the growth of A549 by inducing
apoptosis, flow cytometry was applied, and the results indicated that after treating A549
cells with varying concentrations (0 μM, 1 μM, 3 μM and 10 μM) of 4d for 24 h, the
percentage of apoptotic cells was markedly elevated in a dose-dependent manner.
Likewise, A549 cells were treated with corresponding doses of celecoxib (0 μM, 1 μM,
3 μM and 10 μM). As shown in Fig. 4, increased concentrations of compound 4d
yielded an increased apoptotic rate of A549 cells. The percentages of cell apoptosis of
8.30%, 19.86%, 34.52% correspond to treatment concentrations of compound 4d of 1,
3 and 10 μM, respectively. Clearly, compound 4d can cause cell apoptosis more
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effectively than celecoxib. In conclusion, compound 4d could induce apoptosis in A549
cells in a dose-dependent manner.
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Fig. 4 A549 cells treated with 0, 1, 3 and 10 μM 4d and celecoxib for 24 h were collected and
analysed. The percentage of early apoptotic cells is shown in the lower right quadrant (Annexin V-
FITC positive/PI negative cells), and late apoptotic cells are located in the upper right quadrant
(Annexin V-FITC positive/PI positive cells). Images are representative of three independent
experiments.
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3.6 Compound 4d induced cell-cycle arrest in A549 cells in a dose-dependent manner
We further assessed the effect of compound 4d on the cell cycle to ascertain whether
A549 cells are blocked in mitosis. A549 cells were treated with different concentrations
(0 μM, 1 μM, 3 μM and 10 μM) of compound 4d for 24 hours. As illustrated in Fig. 5,
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treatment of A549 cells with compound 4d led to G2/M arrest in a dose-dependent
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manner. Incubation of the cells with 3 μM compound 4d caused 35.38% of cells to
arrest at the G2/M phase. When the concentration of compound 4d increased to 10 μM,
85.93% of cells were arrested in the G2/M phase. In summary, the accumulation of cells
in G2/M phase increased with increased concentration of compound 4d.
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Fig. 5 Effect of compound 4d on the cell cycle distribution of A549 cells in a dose-dependent
manner (0, 1, 3 and 10 μM). Images are representative of three independent experiments. (G1 phase,
green; S phase, yellow and G2/M phase, blue).
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3.7 Compound 4d was able to permeate cell membrane and affect cell morphology
Many studies have applied the strategy of conjugating a fluorescent probe with
agents to illustrate the location of these compounds in cells and verify their membrane
permeability. Given that our target compounds are generally fluorescent under certain
excitations, the conjugation with probe was actually inessential in this assay. Under
two-photon excitation, we imaged compound 4d in A549 cells as shown in Fig. 6. No
fluorescence was observed in normal A549 cells; however, intense blue fluorescence
emerged after incubating A549 cells with 5 μM compound 4d for 30 min at 37 °C and
washed three times with Hepes buffer. The results reflected the ability of4d to penetrate
cell membrane and its location in cells. Additionally, morphological alterations between
A549 cells treated with different concentrations (0 μM, 1 μM, 3 μM and 10 μM) of
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compound 4d for 8 h were observed. DAPI staining was exploited because it can
partially penetrate the nucleus to stain nuclear DNA. As shown in Fig. 7, while non-
treated A549 cells exhibited normal morphology, cells undergoing apoptosis induced
by compound 4d were morphologically varied, with condensed or fragmented nuclei.
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Fig. 6 Confocal microscopy images ofA549 live cells visualizing changes in the level of florescence
using compound 4d (5 μM). Images represent emission intensities collected in optical windows
between 500 and 600 nm upon excitation at 450 nm for compound 4d. (a) Two-photon image of
A549 cells. λex = 450 nm (emission wavelength from 500 to 600 nm); (b) Bright-field image of
A549 cells; (c) Overlay of (a) and (b); (d) Two-photon image of A549 cells incubated with 5 μM
compound 4d after 30 min of incubation, washed with Hepes buffer. λex = 450 nm (emission
wavelength from 500 to 600 nm); (e) Bright-field image of A549 cells; (f) Overlay of (d) and (e).
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Fig. 7 Morphological changes of DAPI-stained A549 cells after treatment with compound 4d (0, 1,
3 and 10 μM) observed under confocal microscope.
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3.8 Compound 4d reduced the expression of COX-2 in A549 cells.
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Western blot analysis was performed to determine whether compound 4d could
suppress the expression of COX-2 in A549 cells. Total proteins were extracted from
A549 cells treated with compound 4d at different concentrations (0 μM, 1 μM, 3 μM
and 10 μM). The results were analysed by Image J and indicated that the COX-2
expression of treated cells was inferior to that of untreated cells. As pictured in Fig. 8,
the expression of COX-2 was reduced as the concentration of compound 4d increased.
In summary, compound 4d could reduce the protein expression of COX-2 in A549 cells
in a dose-dependent manner.
We also performed a Western blot to estimate the transfection efficacy of control
siRNA and COX-2 siRNA into A549 cells. The results shown in Fig. 2 indicated that
the interference was performed effectively, and the expression of COX-2 decreased
sharply in COX-2 siRNA-mediated cells while it varied undetectably in the control
cells.
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Fig. 8 A549 cells were treated with compound 4d (0.2, 1, 5 and 10 μM) for 24 h, and the protein
level of COX-2 was observed in A549 cells by immunoblotting assay.
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3.9 Scaffold modification
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Because the scaffold was constructed based on analysis of known COX-2-selective
inhibitors, modifications were made to establish a library of small molecules in order
to screen for potential anticancer compounds. The members in the library were filtered
by Lipinski's “Rule of 5” to ensure good drug characteristics, complementing the
docking screening. In addition, the complexity of the synthesis route for these
candidates was taken into consideration to ensure synthetic feasibility. A primary
screen was conducted to identify the best hits in an in vitro COX-2 inhibition assay.
Finally, the lead compound with good activity was validated (Fig. 1). It possessed
better AlogP (with value of 3.736) and binding potential with COX-2 (with binding
energy of - 58.44 kcal/mol) than celecoxib (with AlogP of 5.946 and binding energy
of - 56.13 kcal/mol). It was then substituted to generate the class of compounds
investigated in this study.
3.10 Molecular docking
A docking study was performed iteratively in this study, and the general workflow
was described in the method section. In modifying the diarylheterocylic scaffold in
silico, molecular docking was carried out to fit the scaffold molecules into the activity
pocket of COX-2. Afterwards, docking screening was employed to filter the candidates
for better binding potential. When the lead compound was finally validated and
concomitantly modified to produce the target compounds, docking simulation was also
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performed to explore the probable binding modes of these compounds. The latter
docking results are summarized in Table 1, and a model of compound 4d docked with
COX-2 is depicted in Fig. 9(A). In the binding model, compound 4d is nicely bound to
the active site of the cyclooxygenase-2 by five hydrogen bonds with GLN 178 (angle
O · H-N = 125.41°, distance = 2.30 Å), LEU 338 (angle O · H-N = 144.42°, distance =
2.48 Å), SER 339 (angle O · H-N = 146.24°, distance = 2.40 Å), ARG 499 (angle O · H-
N = 159.09°, distance = 2.30 Å), and PHE 504 (angle O · H-N = 149.07°, distance =
2.32 Å), and one Pi-Sigma bond with SER 339. Furthermore, other weak interactions,
such as van der Waals and carbon-hydrogen bonds, also contributed to the binding
affinity of 4d with COX-2. In Fig. 9(B), 3D models of the interaction between
compound 4d and cyclooxygenase-2 are depicted. The molecular docking suggests that
compound 4d may be a potential COX-2 ligand.
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Fig. 9 Binding mode of compound 4d with COX-2 (PDB code: 3LN1). (A) 2D diagram of the
interaction between compound 4d and amino acid residues of the nearby active site. (B) 3D diagram
of compound 4d inserted in the COX-2 binding site: for clarity, only interacting residues are
displayed.
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4. Conclusions
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Aberrant expression of COX-2 has been found to be closely related to various types
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of cancer, and it is generally accepted that targeting COX-2 is a promising treatment
strategy in cancer therapies that has yet to be fully realized. However, the corresponding
research is not very elaborate; to further explore the anti-proliferative potential of COX-
2 inhibitors, we designed a series of novel dihydropyrazole sulphonamide derivatives
with high COX-2 selectivity. These compounds were tested in a series of experiments,
and the results indicated that some compounds may be promising in drug development.
Compound 4d is the most impressive, with a notable biological profile. Analysis
revealed that it has a lower AlogP value compared with the famous COX-2 agent
celecoxib, along with a higher binding affinity to COX-2. The advantages of 4d were
also highlighted by bioassays. Against COX-2 and the A549 cancer cell line, 4d
demonstrated better inhibitory potency than celecoxib. Assisted by siRNA-mediated
COX-2 knockdown, 4d was shown to exert its anticancer effect through COX-2
inhibition. The initial investigation also suggested satisfying selectivity and safety.
While no overt adhesive reduction was observed in the celecoxib-treated group, 4d was
found able to decrease the adhesive ability of A549 cells. Compound 4d could induce
apoptosis of A549 cell and cell cycle arrest in G2/M phase in a dose-dependent manner.
The morphological alterations of A549 cells affected by compound 4d can be directly
perceived by confocal microscopy observation. The western blot results also
demonstrated that compound 4d could reduce the expression of COX-2 in A549 cells,
hinting that a more complicated mechanism is involved in its inhibitory activities. In
summary, we reported a series of dihydropyrazole sulphonamide derivatives, along
with their design and bioactivities. We hope that this study will benefit the study of
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cancer treatment through COX-2 inhibition.
Conflict of interest
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The authors have no relevant affiliations or financial involvement with any organization
or entity with a financial interest in or financial conflict with the subject matter or
materials discussed in the manuscript.
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Acknowledgment
The work was financed by a grant (No. J1103512) from National Natural Science
Foundation of China.
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References
454
455
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457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
1.
2.
3.
Y. Shamsudin Khan, H. Gutiérrez-de-Terán, L. Boukharta, and J. Åqvist. Toward an optimal
docking and free energy calculation scheme in ligand design with application to COX-1
inhibitors. Journal of chemical information and modeling. 54:1488-1499 (2014).
Q. Peng, S. Yang, X. Lao, W. Tang, Z. Chen, H. Lai, J. Wang, J. Sui, X. Qin, and S. Li. Meta-
analysis of the association between COX-2 polymorphisms and risk of colorectal cancer based
on case–control studies. PloS one. 9:e94790 (2014).
R.H. Tolba, N. Fet, K. Yonezawa, K. Taura, A. Nakajima, K. Hata, Y. Okamura, H. Uchinami,
U. Klinge, and T. Minor. Role of preferential cyclooxygenase-2 inhibition by meloxicam in
ischemia/reperfusion injury of the rat liver. European Surgical Research. 53:11-24 (2014).
E. Yıldırım, O. Sağıroğlu, F.S. Kılıç, and K. Erol. Effects of nabumetone and dipyrone on
experimentally induced gastric ulcers in rats. Inflammation. 36:476-481 (2013).
A. Greenhough, H.J. Smartt, A.E. Moore, H.R. Roberts, A.C. Williams, C. Paraskeva, and A.
Kaidi. The COX-2/PGE2 pathway: key roles in the hallmarks of cancer and adaptation to the
tumour microenvironment. Carcinogenesis. 30:377-386 (2009).
4.
5.
6.
7.
8.
9.
N. Ghosh, R. Chaki, V. Mandal, and S.C. Mandal. COX-2 as a target for cancer chemotherapy.
Pharmacological reports. 62:233-244 (2010).
L. Minghetti. Cyclooxygenase‐2 (COX‐2) in inflammatory and degenerative brain
diseases. Journal of Neuropathology & Experimental Neurology. 63:901-910 (2004).
L.M. Jacksonand C.J. Hawkey. COX-2 selective nonsteroidal anti-inflammatory drugs. Drugs.
59:1207-1216 (2000).
J.J. Talley, D.L. Brown, J.S. Carter, M.J. Graneto, C.M. Koboldt, J.L. Masferrer, W.E. Perkins,
R.S. Rogers, A.F. Shaffer, and Y.Y. Zhang. 4-[5-Methyl-3-phenylisoxazol-4-yl]-
benzenesulfonamide, valdecoxib: a potent and selective inhibitor of COX-2. Journal of
medicinal chemistry. 43:775-777 (2000).
10.
C.A. Birbara, A.D. Puopolo, D.R. Munoz, E.A. Sheldon, A. Mangione, N.R. Bohidar, G.P.
Geba, and E.P.S. Group. Treatment of chronic low back pain with etoricoxib, a new cyclo-
oxygenase-2 selective inhibitor: improvement in pain and disability—a randomized, placebo-
25

481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
524
controlled, 3-month trial. The journal of pain. 4:307-315 (2003).
11.
12.
S.-J. Cho, N. Kim, J.S. Kim, H.C. Jung, and I.S. Song. The anti-cancer effect of COX-2
inhibitors on gastric cancer cells. Digestive diseases and sciences. 52:1713-1721 (2007).
L. Tao, S. Wang, Y. Zhao, X. Sheng, A. Wang, S. Zheng, and Y. Lu. Phenolcarboxylic acids
from medicinal herbs exert anticancer effects through disruption of COX-2 activity.
Phytomedicine. 21:1473-1482 (2014).
13.
A. Kirane, J.E. Toombs, K. Ostapoff, J.G. Carbon, S. Zaknoen, J. Braunfeld, R.E. Schwarz,
F.J. Burrows, and R.A. Brekken. Apricoxib, a novel inhibitor of COX-2, markedly improves
standard therapy response in molecularly defined models of pancreatic cancer. Clinical Cancer
Research. 18:5031-5042 (2012).
14.
15.
M. Vosooghiand M. Amini. The discovery and development of cyclooxygenase-2 inhibitors as
potential anticancer therapies. Expert opinion on drug discovery. 9:255-267 (2014).
K. Ng, J.A. Meyerhardt, A.T. Chan, K. Sato, J.A. Chan, D. Niedzwiecki, L.B. Saltz, R.J.
Mayer, A.B. Benson, and P.L. Schaefer. Aspirin and COX-2 Inhibitor Use in Patients With
Stage III Colon Cancer. Journal of the National Cancer Institute. 107:dju345 (2015).
A. Okamoto, T. Shirakawa, T. Bito, K. Shigemura, K. Hamada, A. Gotoh, M. Fujisawa, and
M. Kawabata. Etodolac, a selective cyclooxygenase-2 inhibitor, induces upregulation of E-
cadherin and has antitumor effect on human bladder cancer cells in vitro and in vivo. Urology.
71:156-160 (2008).
16.
17.
18.
A. Zarghiand S. Arfaei. Selective COX-2 inhibitors: a review of their structure-activity
relationships. Iranian journal of pharmaceutical research: IJPR. 10:655 (2011).
F. Wuest, T. Kniess, R. Bergmann, and J. Pietzsch. Synthesis and evaluation in vitro and in
vivo of a 11 C-labeled cyclooxygenase-2 (COX-2) inhibitor. Bioorganic & medicinal
chemistry. 16:7662-7670 (2008).
19.
20.
21.
K.A. Abouzid, N.A. Khalil, E.M. Ahmed, H.A.A. El-Latif, and M.E. El-Araby. Structure-
based molecular design, synthesis, and in vivo anti-inflammatory activity of pyridazinone
derivatives as nonclassic COX-2 inhibitors. Medicinal chemistry research. 19:629-642 (2010).
A. Pacelli, J. Greenman, C. Cawthorne, and G. Smith. Imaging COX‐2 expression in cancer
using PET/SPECT radioligands: current status and future directions. Journal of Labelled
Compounds and Radiopharmaceuticals. 57:317-322 (2014).
S. Ovais, S. Yaseen, R. Bashir, P. Rathore, M. Samim, S. Singh, V. Nair, and K. Javed.
Synthesis and anti-inflammatory activity of celecoxib like compounds. Journal of enzyme
inhibition and medicinal chemistry. 28:1105-1112 (2013).
22.
23.
S. Pericherla, J. Mareddy, D. Rani, P.V. Gollapudi, and S. Pal. Chemical modifications of
nimesulide. Journal of the Brazilian Chemical Society. 18:384-390 (2007).
N. Handler, W. Jaeger, H. Puschacher, K. Leisser, and T. Erker. Synthesis of novel curcumin
analogues and their evaluation as selective cyclooxygenase-1 (COX-1) inhibitors. Chemical
and pharmaceutical bulletin. 55:64-71 (2007).
24.
25.
P.A. Connolly, M.M. Durkin, A.M. LeMonte, E.J. Hackett, and L.J. Wheat. Detection of
histoplasma antigen by a quantitative enzyme immunoassay. Clinical and Vaccine
Immunology. 14:1587-1591 (2007).
M. Oka, S. Maeda, N. Koga, K. Kato, and T. Saito. A modified colorimetric MTT assay
adapted for primary cultured hepatocytes: application to proliferation and cytotoxicity assays.
Bioscience, biotechnology, and biochemistry. 56:1472-1473 (1992).
26

525
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