Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2 and 5-LOX

Article abstract of DOI:10.1016/j.bmcl.2017.07.020

In our previous study, we designed a series of pyrazole derivatives as novel COX-2 inhibitors. In order to obtain novel dual inhibitors of COX-2 and 5-LOX, herein we designed and synthesized 20 compounds by hybridizing pyrazole with substituted coumarin w

Full text of DOI:10.1016/j.bmcl.2017.07.020

Accepted Manuscript
Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2
and 5-LOX
Fa-Qian Shen, Zhong-Chang Wang, Song-Yu Wu, Shen-Zhen Ren, Ruojun
Man, Bao -Zhong Wang, Hai-Liang Zhu
PII:
S0960-894X(17)30715-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.07.020
BMCL 25133
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 May 2017
5 July 2017
6 July 2017
Please cite this article as: Shen, F-Q., Wang, Z-C., Wu, S-Y., Ren, S-Z., Man, R., -Zhong Wang, B., Zhu, H-L.,
Synthesis of novel hybrids of pyrazole and coumarin as dual inhibitors of COX-2 and 5-LOX, Bioorganic &
Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.07.020
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Synthesis of novel hybrids of pyrazole and coumarin
as dual inhibitors of COX-2 and 5-LOX
Fa-Qian Shena,b,†, Zhong-Chang Wang*,†,a,b, Song-Yu Wua,b, Shen-Zhen Rena,b,
a,b
Ruojun Man a,b, Bao -Zhong Wang*, , Hai-Liang Zhu*,
a,b
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
210046, People’s Republic of China
b
Elion Nature Biological Technology Co., Ltd 16 Hengtong Road, Nanjing 210038, 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
In our previous study, we designed a series of pyrazole derivatives as novel
COX-2 inhibitors. In order to obtain novel dual inhibitors of COX-2 and 5-LOX,
herein we designed and synthesized 20 compounds by hybridizing pyrazole with
substituted coumarin who was reported to exhibit 5-LOX inhibition to select potent
compounds using adequate biological trials sequentially including selective inhibition
of COX-2 and 5-LOX, anti-proliferation in vitro, cells apoptosis and cell cycle.
Among them, the most potent compound 11g (IC50 = 0.23 0.16 µM for COX-2, IC50
= 0.87
0.07 µM for 5-LOX, IC50 = 4.48
0.57 µM against A549) showed
preliminary superiority compared with the positive controls Celecoxib (IC50 = 0.41
0.28 µM for COX-2, IC50 = 7.68 0.55 µM against A549) and Zileuton (IC50 = 1.35
0.24 µM for 5-LOX). Further investigation confirmed that 11g could induce human
non-small cell lung cancer A549 cells apoptosis and arrest the cell cycle at G2 phase
in a dose-dependent manner. Our study might contribute to COX-2, 5-LOX dual
inhibitors thus exploit promising novel cancer prevention agents.
Key words: COX-2; 5-LOX; Pyrazole; Coumarin; Cancer

The incidence rate of malignant tumor which is the leading reason of death
worldwide is still increasing 1. To improve this situation, developing agents aiming at
anti-inflammation could be a potential approach for treatment of carcinoma due to the
link between inflammation and carcinoma 2. It is recognized that several
inflammatory mediators like chemokines, cytokines and growth factors, activated
chronically to promote the development of tumors by regulating the tumor
microenvironment. Targeting cancer mediators linked to inflammation in cancer cells,
especially regulating vascular programming and microenvironment of solid tumors,
might be a good strategy 3.
Prostaglandin E2 (PGE2), exerting major effects on the inflammation, is a
mediator with profound biological functions on cell proliferation, angiogenesis,
apoptosis and metastasis. Therefore, it is necessary to inhibit the biosynthesis of PGE2
to reduce the adverse effects of long-term use of common nonsteroidal
anti-inflammatory drugs (NSAIDs) 4, 5. As documented, Cyclooxygenases (COXs)
pathway, one of the two main metabolic pathways of Rarchidonic acid (AA),
contributing to the production of PGE2, consists of three subtypes: COX-1, COX-2
6
and COX-3 . COX-1 exists in most normal tissues or cells and COX-3 primarily in
the central nervous system, while COX-2, almost undetectable in most normal cells or
tissues, in turn, is found up-regulated in various types and stages of tumor. Further
study has showed that the peroxidase activity of COX-2 actually promotes the
7,
deterioration of tumors
8. Currently, a number of clinical trials, testing the
anti-cancer activity of selective COX-2 inhibitors are under conduction as said in
database of clinical trials (www.clinicaltrials.gov) due to the inhibition of COX-1
could cause serious side effects like ulcers 9, 10
.
Lipoxygenases (LOXs) pathway is the other main pathway in the AA cascade. To
be specific, AA is transformed into hydroxyeicosatetraenioc acids (HETEs) or
leukotrienes (LTs), reported to play a major role in the development and progression
of human cancers by LOXs 11-14. 5-LOX, a member of LOXs and pro-inflammatory
enzyme, works with COXs jointly to dominate in inflammation and foster tumor
development 15, 16. Emerging evidence suggested that the high expression of 5-LOX

and its products, particularly leukotriene B4 (LTB4) were found in some kinds of
human cancer 17. Similar to COX-2, the overexpression of 5-LOX is significantly
18
associated with cell cycle, blocking apoptosis and stimulating angiogenesis
.
However, the inhibition of either COX-2 or 5-LOX was reported to promote the other
pathway of AA metabolism as compensatory mechanism 19. Considering the
similarities of the two enzymes, the dual inhibition of them to get more efficiency and
safer agents for human cancer treatment is promising, albeit the exact mechanism has
not been fully illustrated.
Most selective COX-2 inhibitors whose side effects include gastric ulceration and
gastrointestinal hemorrhage, comprising with a five-membered core, can be
characterized as diarylheterocycles such as Celecoxib, Valdecoxib, Parecoxib and
Rofecoxib 20, 21. Meanwhile, as recent study showed, coumarin derivatives, possessing
biological activities like antibacterial, antioxidant, anti-inflammatory and anticancer,
existing in tonka beans, Kosteletzkya virginica, etc, is the redox-active inhibitor of
5-LOX 22, 23. The previous research has shown the more potent effects and less
complexity of the prediction of pharmacodynamic and pharmacokinetic relationships
by integrating two drugs together, compared with using the two drugs independently
24. As reported, however, patients, receiving coumarin alone as a single daily oral dose,
caused many side effects, including insomnia, nausea, vomiting, diarrhea, and
dizziness. Based on the above, we designed a series of compounds to seek for the
most potent and safe agent on treating cancer, beginning with the famous COX-2
selective inhibitors, Celecoxib, and integrating with substituted coumarin.
In this study, we modified the linker between pyrazole ring and substituted
coumarin to increase the flexibility and polarity of the synthesized compounds to
develop better fitting into the binding pockets of target proteins. Moreover, we used a
series of substituted acetophenone considering factors as the same substitution on
ortho, meta, and para-position, different substitutes on the same position and the
number of substitutes on the same phenyl ring, and a trio of substituted coumarin
(3-carboxy coumarin, 4-hydroxycoumarin and 7-hydroxycoumarin) for the integration
with a series of pyrazole sulfonamides to select the most effective and safe drug for

screening in vitro as dual inhibitors of COX-2 and 5-LOX (Fig.1).
The synthesis route of the intermediate products 3a-3j has been described in our
5
previous
research
(Scheme
1).
4-(5-(4-fluorophenyl)-3-(hydroxymethyl)-1H-pyrazol-1-yl) benzenesulfonamide (3b)
25
was reduced by lithium aluminium hydride to give the intermediate product (4b)
,
which was linked with coumarin-3-carboxylic acid through esterification reaction to
yield target compound
(5-(4-fluorophenyl)-1-(4-sulfamoylphenyl)-1H-pyrazol-3-yl)methyl
2-oxo-2H-chromene-3-carboxylate (5b)(Scheme 2). Meanwhile, 3a was hydrolyzed
by KOH into pyrazolesulfonamide carboxylic acids 6a, which was directly integrated
with 7-hydroxycoumarin directly after activated to get target compound 7a (Scheme
3).

Fig.1 Pyrazole coumarin derivatives as dual inhibitors of COX-2 and 5-LOX
Scheme 1 Synthesis of compounds 3a-3j
Reagents and conditions: (i) dimethyl oxalate, methanol, reflux
6
h; (ii)

4-hydrazinylbenzenesulfonamide, methanol, reflux, 6 h;
Scheme 2 Synthesis of compounds 5b
Reagents and conditions: (i) LAH, tetrahydrofuran, r.t., 2 h; (ii) EDC·HCl, HOBt, DMAP,
coumarin-3-carboxylic acid, dichloromethane, r.t., overnight.
Scheme 3 Synthesis of compounds 7a
Reagents and conditions: (i) KOH, methanol, reflux, 2 h; (ii) EDC·HCl, HOBt, DMAP,
7-hydroxycoumarin, dichloromethane, r.t., overnight;
Scheme 4 Synthesis of compounds 9a, b, c, e, g, h, i, j, 11a~11i

Reagents and conditions: (i) KCO3, KI, 2-bromoethyl alcohol, DMF, 65 ˚C overnight; (ii)
EDC·HCl, HOBt, 6a, b, c, e, g, h, i, j, DMAP, dichloromethane, r.t., overnight; (iii) KCO3, KI,
2-bromoethyl alcohol, DMF, 65 ˚C overnight; (iv) KCO3, KI, 6a ~ 6i, DMF, 65 ˚C, overnight;
Scheme 5 Synthesis of compounds 13a
Reagents and conditions: (i) KCO3, KI, 2-bromoethylamine, DMF, 65 ˚C, overnight; (ii)
EDC·HCl, HOBt, DMAP, 6a, dichloromethane, r.t., overnight.
The synthesis of pyrazolesulfonamide esters was outlined in Scheme 4. Like
Scheme 3, the compounds 3b-3j was hydrolyzed into the corresponding
pyrazolesulfonamide carboxylic acids 6b-6j in the presence of KOH. The
construction of coumarin esters 8, 10 were achieved by etherification reaction of

either 4-hydroxycoumarin or 7-hydroxycoumarin with 2-bromoethyl alcohol in the
presence of KOH. Next, following the acid activation in the presence of EDC·HCl,
HOBt and DMAP, 6a, b, c, e, g, h reacted with 8 through esterification condensation
reaction gave the corresponding target compounds 9 a, b, c, e, g, h while 6a-6h were
transformed into 11a-11h by condensing with 10 respectively.
And the synthesis route of coumarin amide derivative is shown in Scheme 5.
Compound 12a could be directly prepared by treating 7-hydroxycoumarin with
2-bromoethylamine in the presence of KOH in Dimethyl Formamide, by the
successive reaction with pyrazolesulfonamide carboxylic acids (6a) in the presence of
EDC·HCl, HOBt and DMAP in dichloromethane, gave compound 13a.
Table 1 Structure of target compound
Compds.
7a
R
n
0
2
2
2
2
2
2
2
2
2
X
O
O
O
O
O
O
O
O
O
O
R1
H
H
H
H
H
H
H
H
H
H
R2
H
R3
H
R4
H
7-coumarin
4-coumarin
4-coumarin
4-coumarin
4-coumarin
4-coumarin
4-coumarin
4-coumarin
4-coumarin
7-coumarin
H
H
H
9a
H
F
H
9b
H
CH3
H
H
9c
Cl
H
9e
OCH3
OCH3
F
OCH3
H
OCH3
H
9g
9h
H
H
9i
H
NO2
H
H
9j
11a
H
H

7-coumarin
7-coumarin
7-coumarin
7-coumarin
7-coumarin
7-coumarin
7-coumarin
7-coumarin
7-coumarin
2
2
2
2
2
2
2
2
2
O
O
O
O
O
O
O
O
N
H
H
H
H
Cl
H
H
H
H
H
F
H
11b
11c
11d
11e
11f
H
CH3
Cl
H
H
H
H
Cl
H
H
H
H
OCH3
OCH3
F
OCH3
H
OCH3
H
11g
11h
11i
H
H
13a
H
H
H
The ability of the tested compounds to inhibit COX-2 and 5-LOX was evaluated in
vitro, compared with standard compounds Celecoxib (COX-2 inhibitor) and Zileuton
(5-LOX inhibitor). The inhibitory ability of all tested compounds exhibits in Table 2.
All of the tested compounds showed inhibition of COX-2 and 5-LOX while the
majority exhibits little COX-1 inhibitory activities in excess of 40 µM. Among them,
the most potent agent, compound 11g, substituted with a trio of methoxy groups on
the phenyl ring, exhibited potential COX-2 inhibitory activity (IC50 = 0.23 0.16 µM)
in vitro, compared with the standard COX-2 selective inhibitor, Celecoxib (IC50 =
0.41
0.28 µM). Noting that electron-withdrawing groups substituted on
para-position exhibited better COX-2 inhibition activities than electron-donating
groups when comparing compound 9b (0.44 0.09 µM), 9c (0.49 0.08 µM) and 9j
(0.33 0.43 µM). According to the different inhibitory COX-2 activity of compound
11d (0.91 0.29 µM), 11e (0.49 0.12 µM) and 11f (0.77 0.76 µM), the substitution
on para-position were more efficient than other positions of the same phenyl ring. As
the phenyl ring substituted by more groups might form more hydrogen bonds with
amino acid residues of target protein, the COX-2 inhibition could be more active,
comparing compound 11g (0.23 0.16 µM) and 11h (0.82 0.26 µM). In this study,
we found that the linkage could enhance the inhibitory activity, but the amide linkage
showed no extra efficacy than the ester linkage obviously while comparing 13a and
11a. Meanwhile, the substitution of 7-hydroxycoumarin, compared with

4-hydroxycoumarin, exhibited more potent 5-LOX inhibitory activity. Compounds
11g, 9e and 9j (IC50 = 0.87-1.07 µM) exhibited potent 5-LOX inhibitory activity by
the standard of Zileuton (IC50 = 1.35 0.24 µM).
Table 2 Inhibition activities of target compounds against COX-1/COX-2 and 5-LOX
COX-1/COX-2
Compounds
IC50a, µM
ratio
COX-1
COX-2
3.36 0.31
5-LOX
6.32 0.19
43.28 2.89
43.61 2.61
59.23 3.81
30.41 3.23
>60
5b
～4
2.50 0.95
0.54 0.30
0.44 0.08
0.49 0.09
1.43 0.27
0.29 0.12
0.43 0.49
0.30 0.02
0.33 0.10
0.78 0.48
0.53 0.64
0.40 0.16
0.91 0.29
0.49 0.12
0.77 0.76
0.23 0.16
0.82 0.26
0.86 0.64
0.49 1.08
0.41 0.28
-
2.05 0.67
2.11 0.45
2.24 0.39
2.81 0.57
1.46 0.61
1.02 0.52
2.32 0.40
1.38 0.84
1.07 0.41
2.27 0.11
1.63 0.81
1.40 0.67
1.17 0.92
1.40 0.59
1.32 1.01
0.87 0.07
1.61 1.73
1.56 0.99
1.11 0.61
-
7a
～3
9a
～119
～62
>300
～12
～163
～173
～134
～137
～65
～79
>300
>300
~28
9b
9c
41.46 5.47
47.55 4.78
57.26 1.92
40.45 2.80
59.06 1.22
51.39 1.76
41.98 2.47
>60
9e
9g
9h
9i
9j
11a
11b
11c
11d
11e
11f
>60
56.52 3.51
45.35 1.42
53.08 3.47
52.67 2.72
36.14 2.51
26.25 3.74
36.44 2.59
-
~58
11g
11h
11i
～230
～64
~42
13a
Celecoxib
Zileuton
～53
～88
-
1.35 0.24

In general, the target compounds, forming more hydro-keys with target proteins
resulted by the substitution of the phenyl ring, could exhibit more inhibitory activities
against COX-2 and 5-LOX.
In order to investigate the binding mode and interaction with the binding sites of
target enzymes, docking study of compound 11g that exhibited most potent inhibitory
activity of COX-2 was performed. A model of compound 11g, docked with COX-2
(PDB code: 1CX2), was displayed in (Fig.2 a). Compound 11g showed a highest
docking score of 64.38 and was well inserted into the active pocket of the COX-2 by
two hydrogen bonds with THR 212 (angle O…H–N = 171.01°, distance = 2.27 Å,
angle O…H–N = 120.10°, d = 2.31 Å), one with HIS 386 (angle O…H–N = 135.85°,
d = 2.14 Å), two with TYR 385 (angle O…H–C = 124.04°, d = 2.08 Å, angle O…
H–N = 90.97°, d = 2.50 Å), and one with TRP 387 (angle O…H–C = 124.04°, d =
2.02 Å). Meanwhile, sorts of other weaker interactions, like Van Der Waals and
carbon-hydrogen bonds, enhanced the binding affinity of compound with COX-2 as
well. As illustrated in (Fig.2 b), the 3D models suggests the bulge of compound 11g
fits neatly into COX-2’s dent.
As documented 26, in 2011, the solution of the crystal structure of human 5-LOX
enzyme did great favor in identifying efficient agents in pharmacological screening.
Likewise, we also carried out docking study to know the most potent compound 11g,
binding with 5-LOX (PDB code: 3V99), interacted with amino acid residues
surrounding the binding domain (Fig.2 c). Compound 11g formed four H-bond with
5-LOX enzyme. Oxygen atoms in phenyl ring and sulfanilamide, H-bond acceptors,
formed H-bond with HIS 372 (angle O…H–N = 108.34°, d = 2.38 Å) and GLN 363
(angle N…H–N = 102.08°, d = 3.04 Å), respectively. The carbonyl in the linker and
hydrogen atom in the pyrazol ring formed two H-bonds with ASN 180 (angle O…
H–N = 127.50°, d = 2.11 Å, angle O…H–N = 100.70°, d = 2.94 Å). Moreover,
weaker interactions such as Carbon Hydrogen Bond, Pi-Cation, Pi-Pi Stacked,
Pi-Sigma, Pi-Pi T-shaped contribute to the lowest interaction energy of -66.62
kcal/mol (Fig.2 d).
(a)
(b)

(c)
(d)
Fig.2 Binding mode of compound 11g with the COX-2 (PDB code: 1CX2) and 5-LOX (PDB code:
3V99). (a) 2D diagram of the interaction between compound 11g and COX-2; (b) 3D models of
compound 11g binding with the active site ; (c) 2D diagram of the interaction between compound
11g and 5-LOX (PDB code: 3V99); (d) 3D models of compound 11g in the 5-LOX pocket. Green
lines represent hydrogen bonds.
In summary, the trimethoxy acylhydrazone, benzenesulfonamide, pyrazole ring,
coumarin moieties and the carbonyl linkage of compound 11g contributed to its
inhibitory activities of COX-2 and 5-LOX. As the docking studies shown, it might be
a promising way to develop potent agents that the hybrids of coumarin andpyrazole
sulphonamide act as COX-2/5-LOX dual inhibitors.
All synthesized compounds were evaluated for anti-proliferative activities by
MTT assay against four different cancer cell lines (A549: human lungs cell line, HeLa:
human cervix cell line, SMMC-7721: human liver cell line, HT-29: human colorectal
cell line) and one non-cancer cell line, 293T: human kidney epithelial cell) which

indicates a considerable safety profile, compared with intermediate products (6a),
7-hydroxycoumarin (7-C) and Celecoxib. As shown in Table 3, most target
compounds, superior to the parent compounds (7-C and 6a), exhibit anti-proliferative
activities and low cytotoxic effects through the safety trial on 293T. Moreover, it has
been demonstrated that the introduction of substituted coumarin was very necessary to
enhance the anti-proliferative activity and safety profile, and it was the same as
linkage while comparing others with compound 7a. The reason that compound 5b
showed the lowest anti-cancer effects may be the short linkage which decreased the
polarity of the compound, affecting the solubleness in water. Among them, the
compound 11g, comparing with the positive control Celecoxib (IC50 = 7.68 µM),
exhibited the most potent activity against A549 with IC50 of 4.48 µM for the reason
that a trio of methoxy groups would increase the hydrogen bonding of the compound,
affecting the affinity of drugs and protein.
Table 3 Proliferation inhibitory activities of compounds and Celecoxib against cancer cells
Compound
IC50,a,b µM SD
A549
HT29
Hela
SMMC-7721
16.82 0.34
24.11 0.92
22.21 0.31
20.27 0.76
16.54 0.66
11.48 1.23
12.30 0.85
5.90 0.88
16.29 0.90
12.21 0.52
9.60 0.13
15.76 0.61
9.82 0.84
293T
9.64 1.20
16.76 1.06
17.27 1.25
11.30 0.38
13.66 0.32
7.43 0.73
17.60 0.17
12.99 1.02
17.83 1.06
9.61 1.11
7.29 0.28
11.73 0.46
12.60 0.91
5.13 0.50
16.21 0.88
6.83 0.53
7.20 0.43
6.26 1.80
5.65 0.71
2.31 0.94
5.61 1.55
7.29 1.28
4.31 0.37
8.73 0.69
6.40 1.17
9.69 0.68
28.50 0.48
14.67 0.34
9.72 0.71
7.94 0.77
15.83 0.70
25.78 1.07
15.65 0.61
8.45 1.05
22.54 0.17
5.52 1.22
21.95 0.92
15.21 0.81
18.83 0.63
115.54 0.67
134.08 0.56
145.8 1.18
114.4 0.64
225.44 0.86
93.06 0.69
126.44 0.93
122.9 0.61
168.57 1.36
199.8 0.89
207.06 0.97
188.76 1.46
227.52 0.62
5b
7a
9a
9b
9c
9e
9g
9h
9i
9j
11a
11b
11c

8.85 0.16
11.37 1.27
11.76 1.71
8.46 0.85
11.20 0.71
6.82 1.21
16.04 1.62
21.98 1.03
22.67 1.36
8.47 0.31
7.08 0.54
3.53 1.24
10.41 0.72
5.51 1.28
6.28 1.33
14.69 0.33
15.22 0.48
51.26 1.24
42.35 0.59
11.06 0.93
9.28 0.51
7.31 0.34
8.42 0.71
4.48 0.57
26.96 0.16
16.84 0.89
37.66 1.33
14.31 0.25
21.11 0.51
7.68 0.55
9.96 1.83
13.31 0.83
15.66 0.59
9.62 0.35
6.87 1.59
8.92 0.85
9.15 0.47
25.51 1.13
19.90 0.92
5.96 0.76
>300
11d
11e
205.95 1.55
189.63 0.27
121.86 0.39
220.17 1.94
195.66 0.89
179.4 0.96
205.95 0.59
189.63 0.82
111.86 1.51
11f
11g
11h
11i
13a
6a
7-C
Celecoxib
a IC 50 : Concentration inhibits 50% of cell growth
b Data are shown as the mean SD of three independent experiments (n = 3) run in triplicate
Flow cytometry analysis was applied into verifying whether the potent
anti-proliferative and COX-2/5-LOX inhibitory activity of compound 11g related to
the apoptosis of cancer cell like A549 cell line. The results of Fig.3 indicated that
compound 11g could induce apoptosis of A549 cells in a varying concentrations (0
µM, 1.25 µM, 2.5 µM, 5 µM and 10 µM) for 24 h and the percentage of apoptotic cells
increased significantly in a dose-dependent manner. Compound 11g, obviously, could
induce apoptosis in A549 cells in a dose-dependent manner.
Fig.3 Different concentrations of compound 11g induces apoptosis in A549 cells for
24 h. (a) Flow cytometry analysis of apoptotic A549 cells; (b) Apoptotic ratio.
(a)
(b)

We conducted further study to evaluate the cytostatic effects of compound 11g
on A549 cells. After treated with different concentrations (0 µM, 1.25 µM, 2.5 µM, 5
µM and 10 µM) of compound 11g for 24 h, G2-block arrest of A549 was observed in
a dose-dependent manner (Fig.4). As illustrated in previous studies, EGFR,
co-overexpressing and co-localizing with COX-2 in cancer cells, decreasing along
with the inhibition of COX-2, induced cell cycle arrest in G2 27. In summary,
increasing concentration of compound 11g could enhance the accumulation of cells in
G2/M through the mechanism need to be further understood.
Fig.4 Influence of compound 11g on the cell cycle distribution of A549 cells in a dose-dependent
(a)
(b)
As a promising strategy for cancer therapy, dual inhibitors of COX-2 and 5-LOX,
however, have yet to be fully illustrated. Therefore, for further exploration, we
designed a series of novel hybrids of pyrazole and coumarin as dual inhibitors of
COX-2 and 5-LOX for the treatment of cancer. After tested in initial experiments, all
the synthesized compounds, exhibiting inhibitory activities of COX-2 and 5-LOX

with satisfying selectivity, have potential to be effective drugs, especially
representative compound 11g. As the docking studies revealed, these compounds has
relatively high affinity to both COX-2 and 5-LOX. Most compounds showed potent
anti-proliferative activity with high safety by MTT bioassay. For further researches of
the anti-proliferative mechanism, compound 11g could induce apoptosis and G2 phase
cell cycle arrest of A549 in a dose-dependent manner. In general, we designed and
evaluated a series of hybrids of pyrazole and coumarin so that they could benefit the
study to find novel anti-tumor drugs as dual inhibitors of COX-2 and 5-LOX.
Acknowledgments
We thank Miss Bing-Jie Zhang in China Pharmaceutical University and Miss
Xiao-Xuan Liu in Nanjing Foreign Languages School for the synthesis of compounds
3a, 5b and 13a of this paper. The work was financed by the National Science &
Technology Supporting Project No.2015BAD16B07, by Public Science and
Technology Research Fund Project of Ocean (No. 201505023), and by the Project (No.
CXY1412) from the Science & Technology Bureau of Lianyungang City of Jiangsu
Province.
References and Notes
1. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA: a cancer
journal for clinicians. 2011;61(2): 69-90.
2. Mantovani A. Cancer: Inflaming metastasis. Nature. 2009;457(7225): 36-37.
3. Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting chronic inflammation: a
magic bullet? Science. 2013;339(6117): 286-291.
4. Elkady M, Niess R, Schaible AM, et al. Modified Acidic Nonsteroidal Anti-Inflammatory Drugs as
Dual Inhibitors of mPGES-1 and 5-LOX. J Med Chem. 2012;55(20): 8958-8962.
5. Lu XY, Wang ZC, Ren SZ, Shen FQ, Man RJ, Zhu HL. Coumarin sulfonamides derivatives as potent
and selective COX-2 inhibitors with efficacy in suppressing cancer proliferation and metastasis. Bioorg
Med Chem Lett. 2016;26(15): 3491-3498.
6. Khan YS, Gutierrez-de-Teran H, Boukharta L, Aqvist J. Toward an Optimal Docking and Free Energy
Calculation Scheme in Ligand Design with Application to COX-1 Inhibitors. J Chem Inf Model.
2014;54(5): 1488-1499.
7. Tavolari S, Bonafe M, Marini M, et al. Licofelone, a dual COX/5-LOX inhibitor, induces apoptosis in
HCA-7 colon cancer cells through the mitochondrial pathway independently from its ability to affect
the arachidonic acid cascade. Carcinogenesis. 2008;29(2): 371-380.
8. Cai H, Huang XJ, Xu ST, et al. Discovery of novel hybrids of diaryl-1,2,4-triazoles and caffeic acid as

dual inhibitors of cyclooxygenase-2 and 5-lipoxygenase for cancer therapy. Eur J Med Chem. 2016;108:
89-103.
9. Qiu HY, Wang PF, Li Z, et al. Synthesis of dihydropyrazole sulphonamide derivatives that act as
anti-cancer agents through COX-2 inhibition. Pharmacol Res. 2016;104: 86-96.
10. Pal M, Madan M, Padakanti S, et al. Synthesis and cyclooxygenase-2 inhibiting property of
1,5-diarylpyrazoles with substituted benzenesulfonamide moiety as pharmacophore: Preparation of
sodium salt for injectable formulation. J Med Chem. 2003;46(19): 3975-3984.
11. Soumaoro LT, Iida S, Uetake H, et al. Expression of 5-lipoxygenase in human colorectal cancer.
World journal of gastroenterology. 2006;12(39): 6355-6360.
12. Steele VE, Holmes CA, Hawk ET, et al. Lipoxygenase inhibitors as potential cancer
chemopreventives. Cancer Epidem Biomar. 1999;8(5): 467-483.
13. Ghosh J, Myers CE. Inhibition of arachidonate 5-lipoxygenase triggers massive apoptosis in
human prostate cancer cells. P Natl Acad Sci USA. 1998;95(22): 13182-13187.
14. Hong SH, Avis I, Vos MD, Martinez A, Treston AM, Mulshine JL. Relationship of arachidonic acid
metabolizing enzyme expression in epithelial cancer cell lines to the growth effect of selective
biochemical inhibitors. Cancer Res. 1999;59(9): 2223-2228.
15. Jang Y, Park NY, Rostgaard-Hansen AL, Huang J, Jiang Q. Vitamin
E metabolite
13'-carboxychromanols inhibit pro-inflammatory enzymes, induce apoptosis and autophagy in human
cancer cells by modulating sphingolipids and suppress colon tumor development in mice. Free radical
biology & medicine. 2016;95: 190-199.
16. Matsuyama M, Yoshimura R, Tsuchida K, et al. Lipoxygenase inhibitors prevent urological cancer
cell growth. Int J Mol Med. 2004;13(5): 665-668.
17. Ye YN, Wu WK, Shin VY, Bruce IC, Wong BC, Cho CH. Dual inhibition of 5-LOX and COX-2
suppresses colon cancer formation promoted by cigarette smoke. Carcinogenesis. 2005;26(4):
827-834.
18. Romano M, Claria J. Cyclooxygenase-2 and 5-lipoxygenase converging functions on cell
proliferation and tumor angiogenesis: implications for cancer therapy. FASEB journal : official
publication of the Federation of American Societies for Experimental Biology. 2003;17(14): 1986-1995.
19. Asako H, Kubes P, Wallace J, Gaginella T, Wolf RE, Granger DN. Indomethacin-Induced Leukocyte
Adhesion in Mesenteric Venules - Role of Lipoxygenase Products. Am J Physiol. 1992;262(5):
G903-G908.
20. Abouzid KAM, Khalil NA, Ahmed EM, El-Latif HAA, El-Araby ME. Structure-based molecular
design, synthesis, and in vivo anti-inflammatory activity of pyridazinone derivatives as nonclassic
COX-2 inhibitors. Med Chem Res. 2010;19(7): 629-642.
21. Steinbach G, Lynch PM, Phillips RKS, et al. The effect of celecoxib, a cyclooxygenase-2 inhibitor, in
familial adenomatous polyposis. New Engl J Med. 2000;342(26): 1946-1952.
22. Ribeiro D, Freitas M, Tome SM, et al. Inhibition of LOX by flavonoids: a structure-activity
relationship study. Eur J Med Chem. 2014;72: 137-145.
23. Bai B, Gu XW, Chen Y, Guan FQ, Shan Y, Feng X. Virginicin, a New Naphthalene from Kosteletzkya
virginica (Malvaceae). J Brazil Chem Soc. 2015;26(4): 723-728.
24. Morphy R, Rankovic Z. Designed multiple ligands. An emerging drug discovery paradigm. J Med
Chem. 2005;48(21): 6523-6543.
25. Rogez-Florent T, Meignan S, Foulon C, et al. New selective carbonic anhydrase IX inhibitors:
synthesis and pharmacological evaluation of diarylpyrazole-benzenesulfonamides. Bioorganic &

medicinal chemistry. 2013;21(6): 1451-1464.
26. Gilbert NC, Rui Z, Neau DB, et al. Conversion of human 5-lipoxygenase to a 15-lipoxygenase by a
point mutation to mimic phosphorylation at Serine-663. Faseb Journal. 2012;26(8): 3222-3229.
27. Li YB, Wang ZQ, Yan X, et al. IC-4, a new irreversible EGFR inhibitor, exhibits prominent
anti-tumor and anti-angiogenesis activities. Cancer Lett. 2013;340(1): 88-96.
Scheme 1 Reagents and conditions: (i) dimethyl oxalate, methanol, reflux 6 h; (ii)
4-hydrazinylbenzenesulfonamide, methanol, reflux, 6 h;
Scheme 2 Reagents and conditions: (i) LAH, tetrahydrofuran, r.t., 2 h; (ii) EDC·HCl, HOBt,
DMAP, coumarin-3-carboxylic acid, dichloromethane, r.t., overnight.
Scheme 3 Reagents and conditions: (i) KOH, methanol, reflux, 2 h; (ii) EDC·HCl, HOBt, DMAP,
7-hydroxycoumarin, dichloromethane, r.t., overnight;
Scheme 4 Reagents and conditions: (i) KCO3, KI, 2-bromoethyl alcohol, DMF, 65 ˚C overnight;
(ii) EDC·HCl, HOBt, 6a, b, c, e, g, h, i, j, DMAP, dichloromethane, r.t., overnight; (iii) KCO3, KI,
2-bromoethyl alcohol, DMF, 65 ˚C overnight; (iv) KCO3, KI, 6a~6i, DMF, 65 ˚C, overnight;
Scheme 5 Reagents and conditions: (i) KCO3, KI, 2-bromoethylamine, DMF, 65 ˚C, overnight; (ii)
EDC·HCl, HOBt, DMAP, 6a, dichloromethane, r.t., overnight.
Table 1 Structure of target compounds
Table 2 Inhibition activities of target compounds against COX-1/COX-2 and 5-LOX
Table 3 Proliferation inhibitory activities of compounds and Celecoxib against cancer cells
Fig.1 Pyrazole coumarin derivatives as dual inhibitors of COX-2 and 5-LOX
Fig.2 Binding mode of compound 11g with the COX-2 (PDB code: 1CX2) and 5-LOX (PDB code:
3V99). (a) 2D diagram of the interaction between compound 11g and COX-2; (b) 3D models of
compound 11g binding with the active site ; (c) 2D diagram of the interaction between compound
11g and 5-LOX (PDB code: 3V99); (d) 3D models of compound 11g in the 5-LOX pocket. Green
lines represent hydrogen bonds.
Fig.3 Different concentrations of compound 11g induces apoptosis in A549 cells for 24 h. (a)
Flow cytometry analysis of apoptotic A549 cells; (b) Apoptotic ratio.
Fig.4 Influence of compound 11g on the cell cycle distribution of A549 cells in a dose-dependent

Synthesis of novel hybrids of pyrazole and coumarin as
dual inhibitors of COX-2 and 5-LOX
Fa-Qian Shena,b,†, Zhong-Chang Wang*,†,a,b, Song-Yu Wua,b, Shen-Zhen Rena,b
,
a,b
Ruojun Man a,b, Bao -Zhong Wang*, , Hai-Liang Zhu*,
a,b
a
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210046,
People’s Republic of China
b
Elion Nature Biological Technology Co., Ltd 16 Hengtong Road, Nanjing 210038, People’s
Republic of China
Corresponding author email: zhuhl@nju.edu.cn
(a)
(b)
(c)
(d)

As docking study exhibited, target compound 11g showed the lowest interaction
energy of 64.38 kcal/mol and -66.62 kcal/mol after interacting with COX-2 (PDB code:
1CX2, a, b) and 5-LOX (PDB code: 3V99, c, d), respectively.