Coumarin sulfonamides derivatives as potent and selective COX-2 inhibitors with efficacy in suppressing cancer proliferation and metastasis

Article abstract of DOI:10.1016/j.bmcl.2016.06.037

Cyclooxygenase-2 is frequently overexpression in malignant tumors and the product PGE₂promotes cancer cell progression and metastasis. We designed novel series of coumarin sulfonamides derivatives to improve biological activities of COX-2 inhib

Full text of DOI:10.1016/j.bmcl.2016.06.037

Accepted Manuscript
Coumarin sulfonamides derivatives as potent and selective COX-2 inhibitors
with efficacy in suppressing cancer proliferation and metastasis
Xiao-Yuan Lu, Zhong-Chang Wang, Shen-Zhen Ren, Fa-Qian Shen, Ruo-Jun
Man, Hai-Liang Zhu
PII:
S0960-894X(16)30648-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.06.037
BMCL 23990
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 April 2016
12 June 2016
15 June 2016
Please cite this article as: Lu, X-Y., Wang, Z-C., Ren, S-Z., Shen, F-Q., Man, R-J., Zhu, H-L., Coumarin
sulfonamides derivatives as potent and selective COX-2 inhibitors with efficacy in suppressing cancer proliferation
and metastasis, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.06.037
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Coumarin sulfonamides derivatives as potent and selective
COX-2 inhibitors with efficacy in suppressing cancer
proliferation and metastasis
Xiao-Yuan Lu†, Zhong-Chang Wang†*, Shen-Zhen Ren, Fa-Qian
Shen, Ruo-Jun Man, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210023, People’s Republic of China
*Corresponding author. Tel. & fax: +86-25-89682572; e-mail: zhuhl@nju.edu.cn,
†These two authors equally contributed to this paper.

Abstract
Cyclooxygenase-2 is frequently overexpression in malignant tumors and the
product PGE2 promotes cancer cell progression and metastasis. We designed novel
series of coumarin sulfonamides derivatives to improve biological activities of COX-2
inhibition and anticancer. Among them, compound 7t showed most powerful selective
inhibitory and antiproliferative activity (IC50 = 0.09 μM for COX-2, IC50 = 48.20 μM
for COX-1, IC50 = 0.36 μM against HeLa cells), comparable to the control positive
compound Celecoxib (0.31 µM, 43.37 µM, 7.79 µM). Cancer cell apoptosis assay
were performed and results indicated that compound 7t effectively fuels HeLa cells
apoptosis in a dose and time-dependent manner. Moreover, 7t could significantly
suppress cancer cell adhesion, migration and invasion which were essential process of
cancer metastasis. Docking simulations results was further indicated that compound
7t could bind well to the COX-2 active site and guided a reasonable design of
selective COX-2 inhibitor with anticancer activities in future.
Keywords:
Coumarin
Sulfonamides
COX-2 inhibitor
Anticancer
Docking simulation

Inflammation had emerged as a dominating factor promoting cancer progression.
1, 2
Inflammatory mediators including cytokines, chemokines, and growth factors
3, 4
promoted cancer cell growth.
Prostaglandin E2 which acted as inflammatory
mediators influence many mechanisms that played a significant role in tumorigenesis
5, 6
such as cell proliferation, angiogenesis, apoptosis, and metastasis.
Prostaglandins
including PGE2 were derived from arachidonic acid catalyzed by two isoforms
cyclooxygenase: the constitutive COX-1 and the inducible COX-2. 7 COX-1 protein
was ubiquitously expressed a wide range of tissues, whereas COX-2 was an inducible
enzyme that could promptly up-regulated by mediator of triggering inflammation and
8, 9
thus was in the charge of acute increases in PGs synthesis.
COX-2 overexpression
was a characteristic feature of most malignant tumor and contributed to poor
outcomes in multiple malignancies.10-12
Recent studies have demonstrated that COXs inhibitor Aspirin combined with
immunotherapies promoted much more rapid tumor regression and eradication of
4
cancer cells than immunotherapies alone. These researches caused mechanisms of
cyclooxygenase-dependent tumor and COXs inhibitors have been taken seriously.
COXs inhibitors have been therapy in many clinical diseases including atherosclerosis,
endothelial dysfunction, neuroinflammation, preterm labor, Parkinson’s disease, and
13-15
cancer.
In fact, the interruption of COX-1, particularly in the gastrointestinal
system, may lead to dangerous side effects such as ulcers. 16 COX-2-dependent PGE2
biosynthesis has been identified promoting cell progression, metastasis and survival in
a variety of tumors. 17-19 COX-2 selective inhibitors necessarily represent a potentially
useful class of anticancer drugs, such as Celecoxib have been shown to decrease
mammary and colorectal tumor burden in animal models by increasing tumor cell
apoptosis, decreasing proliferation, and suppressing metastasis. 10, 20
Most of selective COX-2 inhibitors were characterized by diarylheterocycle
often substituting SO2NH2 or SO2Me moiety at para-position of one of the aryl rings,
such as Celecoxib (1), SC-558 (2), Valdecoxib (3), Etoricoxib (4). COX-2 selective

inhibition potency of these coxibs were verified by multiple researches and docking
21-23
simulations.
Celecoxib exhibited most potent chemopreventive activity in these
COX-2 inhibitors. It owned 1,5-diarylpyrazole structure containing sulfanilamide
group and should be an ideal lead COX-2 inhibitors with potent anticancer activity. 24,
25
Based on the structure of Celecoxib, 1,5-diarylpyrazole class of analogs were
designed and synthesized in this study. 4-chlorocoumarin and acylhydrazones
moieties were selected as pharmacophore to explore more powerful and
high-efficiency COX-2 inhibitor with anticancer activities. Coumarins were chosen
due to their derivatives are extensively found in different natural products and
possessed compelling wide-ranging biological activities, such as antibacterial,
antioxidant, anti-inflammatory and anticancer. 26-28 Coumarin derivatives were linked
by introduction of acylhydrazones group which has been widely used in the design of
new bioactive compounds with prominent pharmacological profiles. 29
On the basis of this finding, we designed 20 novel acylhydrazone and 4
-chlorocoumarin-substituted 1,5-diarylpyrazoles benzenesulfonamide derivatives
(7a-7t) to explore efficacy in COX-2 inhibition and cancer suppression. 7t was found
and showed significant selective COX-2 inhibition as effective anticancer drugs
which could accelerate cancer cells apoptosis, inhibit proliferation, migration and
invasion.

Figure 1. Selective COX-2 inhibitors
The routes to synthesis the acylhydrazone and 4-chlorocoumarin-substituted
1,5-diarylpyrazoles benzenesulfonamide derivatives according to the general pathway
outlined in Scheme 1. And the structures of the target compounds are shown in Table
1. The various substituted chalcones (2a-2t) were carried out using dimethyl oxalate
and the substituted acetophenone under vigorous stirring with excessive sodium
methoxide in refluxing methanol. Then (3a-3t) was prepared according to the reaction
of different chalcones with 4-hydrazinylbenzenesulfonamide in methanol.
Compounds 3a-3t was added a solution of hydrazine monohydrate and ethanol under
vigorously stirred at 80 °C from several hours to overnight to obtain compounds 4a-4t.
4-hydroxycoumarin was transformed to 4-chloro-coumarin- 3-aldehyde in mixture of
phosphoric trichloride and dimethyl formamide. Finally, compounds 4a-4t were
converted into the corresponding target compounds 7a-7t by reacting with
4-chloro-coumarin-3-aldehyde (6) in 60 ％ ~90 ％ yield. All the compounds are
reported for the first time. All of the target compounds gave satisfactory analytical
1
and spectroscopic data H NMR, 13C NMR, EI-MS, which are in accordance with
their depicted structures.

Scheme 1a.
a General synthesis of compounds (7a-7t). Reagents and conditions: (i) 2.0 equiv
dimethyl oxalate, MeOH, MeONa, reflux, 6 h; (ii) 1.0 equiv 4-hydrazinyl
benzenesulfonamide, MeOH, reflux, 6 h; (iii) 10.0 equiv hydrazine hydrate, EtOH,
reflux, 8 h; (iv) POCl3, DMF, 0 °C to rt, 2h, then 60 °C, 6h; (v) EtOH, AcOH, rt, 12 h.
Table 1. Chemical structure of compounds 7a-7t.
R1
R2
R3
R4
Compounds
H
H
H
H
H
CH3
H
H
H
H
H
H
H
7a
7b
7c
7d
7e
7f
H
CH3
H
CH3
H
CH3
CH3
OCH3
CH3
H
H

H
H
OCH3
OCH3
OCH3
H
H
H
H
7g
7h
7i
OCH3
H
OCH3
OCH3
H
H
OCH2CH3
7j
OH
H
H
H
OH
H
H
7k
7l
H
H
Cl
H
H
H
7m
7n
7o
7p
7q
7r
7s
Cl
H
H
H
Cl
Cl
H
F
H
H
H
H
F
H
H
H
H
F
H
H
H
NO2
CF3
H
H
H
H
7t
All synthesized derivatives (7a-7t) were evaluated for their in vitro
antiproliferation activities against four cancer cell lines (HeLa, HepG2, F10, A549)
and two non-cancer cell lines (293T, L02) with the positive control drugs Celecoxib.
The outcomes were summarized up in Table 2. The results indicated that most of the
target compounds can powerfully inhibit the proliferation of the HeLa and HepG2
cells. The IC50 values of compounds 7a-7t against HeLa cell lines were between 0.36
and 16.19 µM and HepG2 cell lines were range from 0.85 to 21.19 µM. Furthermore,
all compounds had lower toxicity against the non-cancer cells 293T and L02 than
Celecoxib with these compounds minimum IC50 of 293T and L02 cells were 101.24
μM and 104.57 μM. Among them, compound 7t displayed the most potent anti-tumor
activity in HeLa with IC50 of 0.36 μM, compared to the positive control Celecoxib
(IC50 =7.79 μM). Moreover, 7t possess weak toxicity toward 293T and L02 cells with
IC50 of 293T and L02 cells were 234.46 μM and 267.28 μM, compared to the toxicity
of Celecoxib (95.26, 98.15 μM). Thus it can be seen that introduction of
acylhydrazone and chlorocoumarin moieties obviously promoted the activities of
antiproliferation and attenuated toxicity activities, the results demonstrated the two
moieties in synthesized compounds were significant for anticancer activities.

Table 2. Inhibition of cell proliferation against HeLa, HepG2, F10, A549, 293T, L02 cells by
target compounds (7a-7t)
IC50a (µM)
Compounds
HeLab
HepG2b
F10b
A549b
293Tb
L02b
9.86±0.76
5.76±0.42
9.53±0.65 33.52±1.64 179.99±4.57 182.52±4.64
7a
7b
8.81±0.73 11.27±1.21 6.22±0.53 22.14±1.21 188.63±5.21
11.08±0.86 5.98±0.51 5.28±0.48 20.26±0.98 >300
>300
199.27±4.82
7c
7.95±0.51
6.92±0.54
6.70±0.57
4.41±0.45
4.68±0.34 28.46±1.32 175.00±4.67 206.23±4.96
7d
6.93±0.72 24.23±1.41 232.64±5.39
>300
>300
>300
7e
12.02±1.02 14.68±1.26 7.31±0.84 31.48±1.82
7f
7.58±0.63 15.66±1.34 9.58±0.87 22.41±1.07 106.62±2.43 104.57±2.73
13.12±1.12 21.19±1.41 19.80±1.36 40.14±1.76 174.96±3.68 187.14±3.92
16.19±1.26 20.04±1.29 26.24±1.57 26.14±1.13 101.24±2.27 116.35±2.69
8.76±0.82 17.03±1.08 10.33±1.05 24.34±1.09 105.46±2.76 112.25±2.85
7g
7h
7i
7j
11.67±0.98 19.94±1.24 20.92±1.49 17.16±0.97
>300
159.26±3.43
7k
12.43±1.14 18.23±1.05 21.32±1.61 26.42±1.57 138.52±3.08 163.34±3.68
7l
5.09±0.35
5.46±0.39
4.91±0.51
2.85±0.27
3.43±0.42
0.94±0.12
0.52±0.09
0.36±0.05
6.10±0.40
9.12±0.86 12.28±1.03 253.72±5.04 214.09±4.65
7m
7n
6.75±0.51 15.62±1.17 14.66±1.10 151.54±3.26 168.32±3.89
5.12±0.36
5.94±0.39
4.74±0.24
3.53±0.16
1.63±0.12
0.85±0.08
7.45±0.61 23.46±1.17 153.07±3.59
>300
7o
6.41±0.52 14.34±1.08 107.68±2.39 147.75±3.76
8.79±0.85 16.17±1.22 155.06±4.07 205.87±5.26
9.76±1.02 11.24±0.87 181.36±4.63 192.27±5.03
7p
7q
7r
4.04±0.31
2.27±0.17
3.85±0.42 112.87±2.84 133.41±3.34
2.56±0.34 234.46±4.52 267.28±4.87
7s
7t
7.79±0.84 10.03±0.84 14.36±0.96 15.64±1.23 95.26±2.28
98.15±2.39
Celecoxib
a
Antiproliferation activity and cytotoxicity was measured using the MTT assay. Data shown are the mean ±SD of
three independent experiments (n=3) run in triplicate.
b Cancer and non-cancer cells kindly supplied by State Key Laboratory of Pharmaceutical Biotechnology, Nanjing
University.
Compounds 7a-7t was evaluated for their ability to inhibit COX-1 and COX-2
using human COX-1/COX-2 ELISA Kit and results were showed in Table 3 indicated
that the majority of the synthesized compounds exhibited excellent selectivity
inhibiting COX-2 activities displaying IC50 values between 0.09 and 21.93 μM.
Among them, 7t was the most active and significant selectivity with IC50 value of
inhibition COX-2/ COX-1 were 0.09, 48.20 μM, which owned better cyclooxygenase
selective inhibition capability with the positive control drug Celecoxib with IC50

value of 0.31 μM, 43.37 μM.
The subsequent structure-activity relationship (SAR) studies were performed to
determine how the substituents affect the COX-2 inhibitory activities. We found
change of substituents -R3 (para-position) could greatly affect the activities of COX-2
inhibition. When substituents -R1, -R2, -R4 kept immutability, the changes with
different substituents -R3 for COX-2 inhibition activity showed electron-withdrawing
groups on para-position are preferable than electron-donating groups. For instance,
the active gradients for compounds were 7t (0.09 μM), 7s (0.13 μM) >7r (0.17 μM)>
7a (4.53 μM), 7b (6.94 μM)> 7j (8.83 μM), 7l (9.97 μM), 7f (10.36 μM). The
inhibitory activities increased in the following order: CF3, NO2 >F >H, CH3> OCH3,
OCH2CH3, OH. While -R2 or -R1 were modified, the inhibition activity result
indicated that electron-withdrawing groups performed better than electron-donating
groups in the same way. As shown in inhibition of COX-2: 7q (0.26 μM) >7n (0.54
μM) > 7a (4.53 μM), 7c (4.79 μM), 7g (6.09 μM); 7p (0.47 μM)> 7m (1.06 μM)> 7a
(4.53 μM), 7k (7.57 μM).
From the above mentioned analysis, it could be concluded that change for
substituent -R3 caused remarkable variation of inhibitory activity, with the substituent
-R3 (such as CF3, NO2), exhibited more potent COX-2 inhibition and anticancer
activities than those have electron-donating substituents (such as OCH3, CH3, OH).
This promising finding would provide a direction to design and optimize more
effective COX-2 inhibitors with anticancer activity.
Table 3. Inhibition activities of compounds (7a-7t) against COX-1/COX-2
IC50, μMa
COX-1/COX-2
Compounds
ratio
>22
12.9
10.0
5.5
COX-1
>100
COX-2
4.53±0.53
6.94±0.72
4.79±0.60
7.13±0.81
6.25±0.66
10.36±0.94
6.09±0.75
10.41±0.79
7a
7b
7c
7d
7e
7f
89.53±2.06
48.21±1.42
39.45±1.33
>100
>16
8.1
84.27±2.21
>100
>16
6.6
7g
7h
69.25±1.83

49.76±1.52
78.54±2.10
>100
21.93±1.26
8.83±0.83
7.57±0.67
9.97±1.23
1.06±0.21
0.54±0.15
0.30±0.09
0.47±0.13
0.26±0.10
0.17±0.06
0.13±0.04
0.09±0.01
0.31±0.12
2.7
7i
8.89
7j
>13
7k
>100
>10
7l
86.24±2.47
>100
81.35
>185
322.9
>212
285.7
314.3
324.1
>500
139.9
7m
7n
96.59±2.61
>100
7o
7p
74.30±1.96
53.43±1.74
42.16±1.25
48.20±1.30
43.37±1.44
7q
7r
7s
7t
Celecoxib
aCOX-1/COX-2 inhibitory activity was measured using the human COX-1/COX-2 assay kit. Values shown are the
mean ±SD of three independent experiments (n=3) run in triplicate.
Subsequently, we investigated the effect of compound 7t on HeLa cell apoptosis.
We treated HeLa cells with different concentrations (1, 2, 4, 8 μM) of compound 7t
for 24 h and treated with 3 μM 7t for different times (0, 12, 24, 36 h) and analyzed
cells for changes in apoptotic markers with a flow cytometer in vitro, as shown in
Figure 2, the percentage of apoptosis cell were 4.1％, 5.7％, 13.9％, 35.8％, 49.2％,
corresponding to the concentration of 0, 1, 2, 4, 8 μM, that revealed apoptosis cells
significantly increased after treatment with high doses of compound 7t. Meanwhile,
7t also causing cell apoptosis in a time-dependent manner that the percentage of
apoptosis cell were 3.8％, 6.8％, 23.6％, 31.8％ when treated with 3 μM for 0, 12,
24, 36 h. From the above, 7t could effectively causing HeLa cell apoptosis in a dose
and time-dependent manner.

Figure 2. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 7t for different times revealed significantly increased number of apoptotic cells.
(A) Compound 7t induced apoptosis in HeLa cells with the density of 0, 1, 2, 4, 8 μM for 24 h
were collected and processed for analysis. (B) Cells treated with 3 μM 7t for different times (0, 12,
24, and 36 h) was collected and analyzed. The percentage of cells in each part was indicated.
Images are representative of three independent experiments. Data are mean ±S.E.M. of three
independent experiments (n=3). *P < 0.05, **P < 0.01.
Cell adhesion plays a significant role in cancer progression and metastasis.
Herein, we investigated the adhesive ability of HeLa cells as affected by 7t and
positive control drug Celecoxib. HeLa cells (2×105 per well) were treated with
different doses of 7t and Celecoxib for the adhesion assay to detect their inhibition of
adhesive ability. The data shown in Figure 3 indicated that 7t could effectively reduce
the attachment of HeLa cells but the effects of Celecoxib were not obvious in low
dose. Indeed, at low dose of 2 µM 7t, the attachment decreased about 60%.

Figure 3. Compound 7t caused dose-dependent decrease of HeLa cell attachment. (A) Influence
of 7t on HeLa cell adhesion. (B) Influence of Celecoxib on HeLa cell adhesion. Data shown are
the mean ±S.E.M. of three independent experiments (n=3). *P < 0.05, **P < 0.01.
Metastasis is multistage process that results from the convergence of a variety of
cellular processes. Some of these processes include cell migration, invasion, adhesion
into the surrounding stroma and through vessel walls, and survival in a foreign
environment. Cancer cell migration and invasion are the initial steps in metastasis. So
we investigated the effect of 7t on migration and invasion of HeLa cell using 8 μm
24-well transwell. As shown in Figure 4, a significant decrease in cell migration was
observed with increased concentration of 7t, compared with control. Furthermore, we
conducted transwell invasion assays to evaluate the potency of 7t on cancer cell
invasion. The results showed that HeLa cancer cells significantly lost their ability to
invade through the matrigel matrix and the number of cells that invaded through the
matrigel matrix was observably decreased. These data suggest that 7t could
effectively suppress cancer cell migration and invasion.

Figure 4. Compound 7t significantly inhibits HeLa cells migration and invasion. (A) The images
(×100) of cells that migrated outside of transwell insert membrane in transwell migration assay. (B)
The images (×100) of cells that invaded through the matrigel to outside of transwell insert
membrane in transwell invasion assay. The number of migrated and invaded cells was counted in
16 random fields per chamber of three independent experiments (n=3). *P < 0.05, **P < 0.01.
We determined the secretion of PGE2 in HeLa cells after treatment with LPS,
Celecoxib and 7t for 24 h. The result shown in Figure 5 indicated that 7t significantly
reduced synthesis and secretion of PGE2 in HeLa cells. Compared to Celecoxib, the
effect of 7t on PGE2 secretion was more pronounced. In order to verify whether 7t
effects on anticancer were mediated by COX-2/PGE2 pathway, we investigated the
effects of PGE2, COX-2 inhibitor 7t and 7t+PGE2 on cellular proliferation, adhesion,

apoptosis, migration, and invasion under identical experimental conditions in HeLa
cells using method described previously with some modifications. As shown in
Figure 6A, PGE2 promoted proliferation of HeLa cells in a certain extent. 7t was able
to completely reverse PGE2 induced cancer cell proliferation. Likewise, PGE2 caused
cancer cell adhesion and apoptosis could be inverted by 7t (Figure 6B and 6C).
Based on these encouraging results, we subsequently determined whether PGE2 could
increase migration and invasion of cancer cell in vitro and 7t could s uppress this
tendency caused by PGE2. Fortunately, migration and invasion of HeLa cells was
reasonably stimulated by PGE2, 7t was capable of reversing PGE2 induced cancer cell
migration and invasion. These data suggested that PGE2 indeed mediate proliferation,
adhesion, apoptosis, migration, and invasion of HeLa cells. 7t participated in the
COX-2/PGE2 pathway acted as COX-2 inhibitor and thus exert efficacy in
suppressing cancer proliferation and metastasis.
Figure 5. The effects of 7t (0.1 μM), Celecoxib (0.1 μM), LPS (0.1 μg/ml) on PGE2 secretion in
HeLa cells. Data shown are the mean ±S.E.M. of three independent experiments (n=3). *P < 0.05.

Figure 6. The effects of PGE2 (1 μM), 7t (2μM) and 7t (2 μM)+PGE2 (1 μM) on HeLa cell
proliferation, adhesion, apoptosis, migration, and invasion. (A) The effects of PGE2, 7t and
7t+PGE2 on cell proliferation were determined using MTT assay. (B) The effects of PGE2, 7t and
7t +PGE2 on cell adhesion. (C) The effects of PGE2, 7t (3 μM) and 7t (3 μM) +PGE2 on cell
apoptosis. (D) The effects of PGE2, 7t and 7t +PGE2 on cell migration. (E) The effects of PGE2, 7t
and 7t +PGE2 on cell invasion. Data shown are the mean ±S.E.M. of three independent
experiments.
Docking study was performed to gain a better understand the potency of
compound 7t and further guide structure-activity relationship (SAR) research, we
proceeded to examine the interaction of 7t and Celecoxib with COX-2 (PDB code:

3PGH). All amino acid residues of COX-2 that interacted with compound 48 and
Celecoxib were exhibited in pictures Figure 7, Figure 8 and Figure 9. The estimated
interaction energies of synthesized compounds were ranging from -46.85 to -66.41
kcal/mol, as displayed in Figure 7 and Figure 8. In the binding mode, Celecoxib
binds to COX-2 with binding free energy were -49.72 kcal/mol via two hydrogen
bonds with TRP387, two Pi bonds and a Sigma-Pi bond with HIS207. However,
compound 7t had a best estimated binding free energy of -66.41 kcal/mol and bound
superiorly to COX-2 through four hydrogen bonds, two Pi bonds and a Sigma-Pi bond.
As shown in Figure 8, three amino acids, GLN203, HIS207, ASN382 significantly
bound with 7t. The GLN203, HIS207, ASN382 formed four hydrogen bonds with 7t
(angle O · H-N = 161.98°, distance = 1.96 Å, angle O · H-N = 140.07°, distance =
1.98 Å, angle H · O-C = 145.75°, distance = 2.23 Å, angle H · N-N = 136.30°,
distance = 2.36 Å). Furthermore, compound 7t was also bonded with His207 by two
Pi bonds (distance = 3.59 Å, distance = 3.94 Å) and a Sigma-Pi bond (distance = 2.70
Å). The results indicated that acylhydrazone, benzenesulfonamide, pyrazole ring,
chlorocoumarin moieties are crucial for activities of compound and these with
biological assay data suggested that 7t could be a potential inhibitor of the COX-2.

Figure 7. The histogram about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of
compounds (7a-7t) and Celecoxib for COX-2.
Figure 8. Molecular docking analysis of compound 7t and Celecoxib, showing 7t owned
remarkable binding mode in COX-2. (A) Molecular docking 2D modeling of compound 7t with
COX-2: for clarity, only interacting residues are displayed. Three amino acids, GLN203, HIS207,
ASN382 was significant in the binding of ligand and formed four hydrogen bonds, two Pi bonds
and a Sigma-Pi bond (B) Molecular docking 2D modeling of Celecoxib with COX-2: Three

amino acids HIS207, HIS386, TRP387 in enzyme formed two Pi bonds, a Sigma-Pi bonds and
two hydrogen bonds with Celecoxib.
Figure 9. Molecular docking 3D modeling of compound 7t with the COX-2 binding site: for
clarity, only interacting residues are displayed.
In summary, the tumorigenic effects and metastatic potential of COX-2 in the
tissue are largely determined to be attributed to its role in producing PGE2. It is worth
noting that COX-2/PGE2 pathway inhibition was useful in the prevention of
malignant tumors. In our study, a series of novel COX-2 inhibitors possess
extraordinarily potent antiproliferative activities against HeLa, HepG2, A549, F10
cells and COX-2 selectivity inhibition activities. Among these compounds, compound
7t showed the most powerful antiproliferative activity (IC50 = 0.36 µM for HeLa cells)
and COX-2 inhibitory activity (IC50 =0.09 µM). In addition, 7t can induce cancer cell
apoptosis in a dose and time-dependent manner. Inhibition of cancer cell adhesion,
migration and invasion which were pivotal processes in cancer progression and
metastasis were also detected and results indicated that 7t possess high-efficiency
performance on characteristic feature of malignant tumor. We investigated the effects
of PGE2, 7t and 7t+PGE2 in vitro cellular experiment to verify PGE2 involved in
proliferation and metastasis of HeLa cells and 7t could completely reverse PGE2
induced progression of cancer cell by operating in COX-2/PGE2 pathway.
The probable binding mode proposed by the docking simulation might be a

convincing explanation of the impressive performance of compound 7t, in which 7t
binds eminently to COX-2 through four hydrogen bonds, two Pi bonds and a
Sigma-Pi bond with best estimated binding free energy of -66.41 kcal/mol. Celecoxib
binds to COX-2 was less stable than that of 7t and possess undesirable anticancer
activities.
This
result
indicated
acylhydrazone,
chlorocoumarin
and
electron-withdrawing moieties are crucial for anticancer activities of synthesized
derivatives. It can be concluded that compound 7t as well as the other coumarin
sulfonamides derivatives are promising leads for further study as potential anticancer
COX-2 inhibitor.
Acknowledgement
The work was financed by a grant (No.J1103512) from National Natural Science
Foundation of China and the Fundamental Research Funds for the Central
Universities (No.020814380011) and the Projects (Nos.CXY1409 & CG1305) from
the Science & Technology Burea of Lianyungang City of Jiangsu Province.
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Figure Captions
Table 1. Chemical structure of compounds 7a-7t.
Table 2. Inhibition of cell proliferation against HeLa, HepG2, F10, A549, 293T, L02 cells by
target compounds (7a-7t)
Table 3. Inhibition activities of compounds (7a-7t) against COX-1/COX-2
Figure 1. Selective COX-2 inhibitors
Figure 2. Annexin V-FITC/PI dual-immuno-fluorescence staining after treatment with different
concentrations of 7t for different times revealed significantly increased number of apoptotic cells.
(A) Compound 7t induced apoptosis in HeLa cells with the density of 0, 1, 2, 4, 8 μM for 24 h
were collected and processed for analysis. (B) Cells treated with 3 μM 7t for different times (0, 12,
24, and 36 h) was collected and analyzed. The percentage of cells in each part was indicated.
Images are representative of three independent experiments. Data are mean ±S.E.M. of three
independent experiments (n=3). *P < 0.05, **P < 0.01.

Figure 3. Compound 7t caused dose-dependent decrease of HeLa cell attachment. (A) Influence
of 7t on HeLa cell adhesion. (B) Influence of Celecoxib on HeLa cell adhesion. Data shown are
the mean ±S.E.M. of three independent experiments (n=3). *P < 0.05, **P < 0.01.
Figure 4. Compound 7t significantly inhibits HeLa cells migration and invasion. (A) The images
(×100) of cells that migrated outside of transwell insert membrane in transwell migration assay. (B)
The images (×100) of cells that invaded through the matrigel to outside of transwell insert
membrane in transwell invasion assay. The number of migrated and invaded cells was counted in
16 random fields per chamber of three independent experiments (n=3). *P < 0.05, **P < 0.01.
Figure 5. The effects of 7t (0.1 μM), Celecoxib (0.1 μM), LPS (0.1 μg/ml) on PGE2 secretion in
HeLa cells. Data shown are the mean ±S.E.M. of three independent experiments (n=3). *P < 0.05.
Figure 6. The effects of PGE2 (1 μM), 7t (2μM) and 7t (2 μM)+PGE2 (1 μM) on HeLa cell
proliferation, adhesion, apoptosis, migration, and invasion. (A) The effects of PGE2, 7t and
7t+PGE2 on cell proliferation were determined using MTT assay. (B) The effects of PGE2, 7t and
7t +PGE2 on cell adhesion. (C) The effects of PGE2, 7t (3 μM) and 7t (3 μM) +PGE2 on cell
apoptosis. (D) The effects of PGE2, 7t and 7t +PGE2 on cell migration. (E) The effects of PGE2, 7t
and 7t +PGE2 on cell invasion. Data shown are the mean ±S.E.M. of three independent
experiments.
Figure 7. The histogram about CDOCKER_INTERACTION_ENERGY (-kcal/mol) of
compounds (7a-7t) and Celecoxib for COX-2.
Figure 8. Molecular docking analysis of compound 7t and Celecoxib, showing 7t owned
remarkable binding mode in COX-2. (A) Molecular docking 2D modeling of compound 7t with
COX-2: for clarity, only interacting residues are displayed. Three amino acids, GLN203, HIS207,
ASN382 was significant in the binding of ligand and formed four hydrogen bonds, two Pi bonds
and a Sigma-Pi bond (B) Molecular docking 2D modeling of Celecoxib with COX-2: Three
amino acids HIS207, HIS386, TRP387 in enzyme formed two Pi bonds, a Sigma-Pi bonds and
two hydrogen bonds with Celecoxib.
Figure 9. Molecular docking 3D modeling of compound 7t with the COX-2 binding site: for
clarity, only interacting residues are displayed.
Scheme 1a.

Coumarin sulfonamides derivatives as potent and selective
COX-2 inhibitors with efficacy in suppressing cancer
proliferation and metastasis
Xiao-Yuan Lu†, Zhong-Chang Wang†*, Shen-Zhen Ren, Fa-Qian Shen, Ruo-Jun Man,
Hai-Liang Zhu*
Novel Coumarin sulfonamides derivatives have been synthesized. Among them,
compound 7t showed most powerful selective inhibitory and antiproliferative activity.
7t could effectively causing HeLa cells apoptosis in a dose and time-dependent
manner and significantly suppress cancer cell adhesion, migration and invasion.