Dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties selectively and potently inhibit COX-2: Design, synthesis, and anti-colon cancer activity evaluation

Article abstract of DOI:10.3390/molecules24091685

Cyclooxygenase-2 (COX-2) as a rate-limiting metabolism enzyme of arachidonic acid has been found to be implicated in tumor occurrence, angiogenesis, metastasis as well as apoptosis inhibition, regarded as an attractive therapeutic target for cancer therapy. In our research, a series of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties were designed as highly potent and selective COX-2 inhibitors by computer-aided drug analysis of known COX-2 inhibitors. A total of 26 compounds were synthesized and evaluated COX-2 inhibition and pharmacological efficiency both in vitro and in vivo with multi-angle of view. Among them, compound 4b exhibited most excellent anti-proliferation activities against SW620 cells with IC50 of 0.86 ± 0.02 μM than Celecoxib (IC₅₀ = 1.29 ± 0.04 μM). The results favored our rational design intention and provides compound 4b as an effective COX-2 inhibitor available for the development of colon tumor therapeutics.

Full text of DOI:10.3390/molecules24091685

molecules
Article
Dihydropyrazole Derivatives Containing Benzo
Oxygen Heterocycle and Sulfonamide Moieties
Selectively and Potently Inhibit COX-2: Design,
Synthesis, and Anti-Colon Cancer Activity Evaluation
Xiao-Qiang Yan 1,2,†, Zhong-Chang Wang 1,*,†, Bo Zhang 1, Peng-Fei Qi 1, Gui-Gen Li 2,* and
Hai-Liang Zhu 1,
*
1
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210023, China;
18120176083@163.com (X.-Q.Y.); zhangbo_nju@163.com (B.Z.); qipengfeinju@163.com (P.-F.Q.)
Jiangsu Key Laboratory of Advanced Organic Materials, School of Chemistry and Chemical Engineering,
Nanjing University, Nanjing 210023, China
2
*
Correspondence: wangzhongchang2006@163.com (Z.-C.W.); guigenli@nju.edu.cn (G.-G.L.);
zhuhl@nju.edu.cn (H.-L.Z.); Tel./Fax: +86-025-89682572 (H.-L.Z.)
†
These authors contributed equally to this paper.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 March 2019; Accepted: 18 April 2019; Published: 30 April 2019
Abstract: Cyclooxygenase-2 (COX-2) as a rate-limiting metabolism enzyme of arachidonic acid has
been found to be implicated in tumor occurrence, angiogenesis, metastasis as well as apoptosis
inhibition, regarded as an attractive therapeutic target for cancer therapy. In our research, a series
of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties
were designed as highly potent and selective COX-2 inhibitors by computer-aided drug analysis of
known COX-2 inhibitors. A total of 26 compounds were synthesized and evaluated COX-2 inhibition
and pharmacological efficiency both in vitro and in vivo with multi-angle of view. Among them,
compound 4b exhibited most excellent anti-proliferation activities against SW620 cells with IC50
of 0.86
±
0.02
µ
M than Celecoxib (IC50 = 1.29
±
0.04 µM). The results favored our rational design
intention and provides compound 4b as an effective COX-2 inhibitor available for the development
of colon tumor therapeutics.
Keywords: dihydropyrazole derivatives; sulfonamide; COX-2 inhibitors; docking simulation;
pharmacological efficiency; colon tumor therapeutics
1. Instruction
Cyclooxygenase (COX), also known as prostaglandin oxidase reductase, is a bifunctional enzyme
with cyclooxygenase and catalase activities, and plays a key rate-limiting role in the conversion of
arachidonic acid into prostaglandin. According to the current research, there are three isozymes for
cox-oxidase, such as COX-1, COX-2, and COX-3 [
great differences in intracellular quantity and distribution area, but also in physiological functions [
1
,
2
]. However, three isozyme types not only have
].
3
COX-1 is a structural primary enzyme, which mainly exists in gastrointestinal tract, kidney and
other parts. Its function is to promote the synthesis of physiological prostaglandin and regulate the
physiological activities of normal tissue cells by protecting the digestive tract mucosa or changing the
vascular tension. COX-3 is a spliced variant of COX-1 isozyme, which is only found in the brain of dogs,
but not in the human body [4,5]. Cox-2, by contrast, is an induced isozyme, of which the expressions
are generally quite low in normal tissue cells [
6]. Only when being in cancer cells or cells are stimulated
by inflammatory factors, the expression level of COX-2 can be significantly increased, which can be
Molecules 2019, 24, 1685; doi:10.3390/molecules24091685
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1685
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increased to about 80 times of the normal level, leading to the increase of the content of PEG2, PGI2,
and PGE1 in the inflammatory site, and then leading to inflammatory reaction and tissue damage [ ].
7,8
The differences of COX-2 and other isozymes in structure, function, expression and distribution area
lay a theoretical foundation for the design of selective COX-2 inhibitors, which will not affect the
original physiological function of COX as much as possible [
to treat pain and inflammation [12]. With the deepening and development of drugs in recent years, the
research on selective COX-2 inhibitors as specific anti-tumor drugs is increasing dramatically [13 14].
9–11]. In fact, COX-2 has long been used
,
Many studies have confirmed that the overexpression of COX-2 in various malignant tumor cells is
closely related to the occurrence, growth, metastasis, and inhibition of apoptosis of cancer [15]. In
other words, the selective COX-2 inhibitors as a targeted therapeutic drug can prevent the occurrence
and growth of malignant tumors [16,17]. Considering the large gap between the supply and demand
of anticancer drugs, it is of great significance to improve the availability of all kinds of anticancer
drugs [18]. Therefore, this study attempts to design a class of selective COX-2 inhibitors and evaluate
their potential in cancer drug development.
According to the clinical trial database, hundreds of clinical trials have tested the anti-cancer
potential of known COX-2 inhibitors such as Celecoxib [19–21]. Similar structure–activity relationships
and modification strategies summarized by different known COX-2 inhibitors should be applicable
to the design of new COX-2 inhibitors, which provides a design basis for the study of anticancer
drugs based on cox-2 inhibitors [22
been known as common COX-2 inhibitors drug skeletons to be widely used and studied [24
addition, para-sulfamoylphenyl moiety in sulfonamides plays a crucial role in the selection of COX-2,
and many COX-2 inhibitors, such as Celecoxib, Valerian and Parecoxib [27 29]. (Figure 1) Besides, the
dihydropyrazole skeleton also appeared frequently in our previously published studies on COX-2
inhibitors such as A-16A B-48, and C-4d 30 32]. On this basis, we developed a new type of diaryl
,23]. Generally, diacyl heterocyclic with a five-membered core has
–26]. In
–
,
[ –
heterocyclic sulfanilamide scaffolds with dihydropyrazole as the five-membered core ring. The scaffold
was replaced with a small molecular library where the molecules were sorted by docking score using
virtual screening. Better active molecules were then synthesized and tested in vivo and in vitro to
describe their pharmacological properties. The results suggest that we have identified a new class of
COX-2 inhibitors available for the development of colon tumor therapeutics.

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Figure 1. The design pathway for novel dihydropyrazole derivatives containing benzo oxygen
heterocycle and sulfonamide moieties.
2. Results and Discussion
2.1. Chemistry
The routes to synthesizing the novel dihydropyrazole derivatives containing benzo oxygen
heterocycle and sulfonamide moieties 4a–4z are outlined in Scheme 1. According to Experimental
Section, the target compounds can be obtained through the three-step reaction. All synthesized
compounds structures were exhibited in Table 1, which were reported for the first time and characterized
by melting test, 1H NMR, ESI-MS and elemental analysis. The structural characterization parameters
fully conform to the designed structure, and the related spectral data have been proposed in the
Supplementary Materials. Meanwhile, single crystal X-ray diffraction structure analysis further
identified representative compound to determine the specific type of skeleton structure. These crystal
data and corresponding Cambridge Crystallographic Data Centre (CCDC) number were presented
in Figure 2 and Table 2, giving a perspective view of these compounds together with the atomic
labeling system.

Molecules 2019, 24, 1685
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Scheme 1. Synthesis routes of 4a–4z. Reagents and conditions: (i) 1.5 equiv dibromomethane or
1,2-dibromoethane, 0.5 equiv anhydrous potassium carbonate, DMF, 70 ◦C, (ii) 0.5 equiv 40% potassium
hydroxide solution, ethanol, 1.0 equiv different substituted acetophenone, (iii) 0.5 acetic acid, ethanol,
1.2 equiv 4-sulfamoylphenylhydrazine hydrochloride.
Table 1. Structures of compounds 4a–4z.
Compounds
R1
R2
R3
R4
R5
Compounds
R1
R2
R3
R4
R5
4a
4b
4c
4d
4e
4f
4g
4h
4i
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
H
H
H
H
H
H
H
H
CH3
F
H
H
H
H
CH3
OCH2CH3
I
H
CH3
H
H
CH3
H
H
H
H
H
H
H
H
H
H
H
4k
4l
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
CH2CH2
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
CH3
CH3
OCH3
F
F
Cl
H
CH3
OCH3
OCH2CH3
F
Cl
Br
I
CH3
F
H
H
H
H
H
H
H
H
H
H
H
H
F
4m
4n
4o
4p
4q
4r
H
CH3
CH3
OCH3
F
H
H
4s
4t
4j
4u
4v
4w
4x
4y
4z
H
H
H
Cl
H
H
H
H
H
H
F
F
Figure 2. Crystal structure diagrams of compound 4b.

Molecules 2019, 24, 1685
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Table 2. Crystal data for compound 4b.
Compound
4b
Empirical formula
Formula weight
Temperature (◦C)
Crystal system
Space group
a (Å)
C23H21N3O4S
435.5
161–162
Monoclinic
P21
5.3096(11)
19.638(4)
10.704(2)
90.00
91.907(5)
90.00
b (Å)
c (Å)
α (◦)
β (◦)
γ (◦)
1115.9(4)
2
1.322
2.82–27.74
444
V (Å)
Z
Dcalcd/g cm−3
θ rang (degree)
F (000)
10926
Reflections collected
(Rint = 0.0815)
4715/1/273
0.176
Data/restraints/parameters
Mu (mm−1
R1
)
0.1049
wR2
0.1385
GOOF
1.118
0.140/−0.469
Larg.peak/hole (e.Å)
CDCC number
1900586
2.2. Biological Activity
2.2.1. Selective COX-2 Inhibitory Activities
Human COX-1/COX-2 ELISA kit was respectively, used to evaluate the COX-1 and COX-2
inhibitory of 26 synthetic dihydropyrazole derivatives containing benzo oxygen heterocycle and
sulfonamide moieties 4a–4z, with Celecoxib as a positive control drug. As shown in Table 3, most
of the tested compounds showed prominent COX-2 inhibitory activity, while the inhibitory activity
of these compounds on COX-1 was not obvious. This indicated that the series of compounds had
better selectivity in inhibiting COX-2. Among them, compound 4b showed the best inhibitory activity
of COX-2 with IC50 values of 0.35
±
0.02 µM, even better than the positive control drug Celecoxib
(IC50 = 0.41 0.03 M). Besides, Compound 4b and Celecoxib, exhibited an equal COX-2 selectivity index
±
µ
(IC50, COX-1/IC50, COX-2). Not only that, compound 4b also exhibited an equal COX-2 selectivity index
(IC50, COX-1/IC50, COX-2 = 137.3) compared with Celecoxib ((IC50, COX-1/IC50, COX-2 = 145.8). Furthermore,
the compounds with 1,3-dioxolane 4a
–4j generally exhibited more potent anticancer activities than
compounds having 1,4-dioxane 4k
–4z. It indicated benzo 1,3-dioxolane could benefit to enhance
COX-2 inhibitory activities.
Table 3. COX-1/COX-2 inhibition of the compounds 4a–4z and Celecoxib. COX: cyclooxygenase.
IC50 ± SD, µM a
Selectivity Indexb (SI) b
Compounds
COX-1
COX-2
4a
4b
4c
>60
1.35 ± 0.08
0.35 ± 0.02
2.02 ± 0.13
1.20 ± 0.05
>44.44
137.3
10.45
21.53
48.06 ± 0.35
21.11 ± 0.19
25.84 ± 0.21
4d

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Table 3. Cont.
IC50 ± SD, µM a
Selectivity Indexb (SI) b
Compounds
COX-1
COX-2
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
29.77 ± 0.23
58.14 ± 0.47
>60
1.55 ± 0.06
1.73 ± 0.11
3.18 ± 0.26
1.28 ± 0.07
1.98 ± 0.15
1.81 ± 0.09
1.82 ± 0.16
1.94 ± 0.05
1.52 ± 0.09
2.44 ± 0.19
1.53 ± 0.12
1.41 ± 0.07
1.27 ± 0.03
1.87 ± 0.01
1.8 ± 0.05
19.20
33.61
>18.87
41.33
>30.30
>33.15
>32.97
46.67
>39.47
>24.59
25.10
52.91 ± 0.39
>60
>60
>60
43.87 ± 0.37
>60
>60
38.41 ± 0.34
37.32 ± 0.18
26.64 ± 0.26
41.18 ± 0.17
>60
26.46
20.98
47.33
>33.33
>40.54
>22.73
>31.09
36.44
>33.71
>27.40
>33.52
145.8
>60
>60
>60
1.48 ± 0.02
2.64 ± 0.17
1.93 ± 0.08
1.51 ± 0.09
1.78 ± 0.16
2.19 ± 0.21
1.79 ± 0.13
0.41 ± 0.03
4w
4x
4y
55.02 ± 0.36
>60
>60
>60
4z
Celecoxib
59.78 ± 0.27
a
The concentration of test compound required to produce 50% inhibition of COX-1/COX-2 is the mean of four
determinations. In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
b
2.2.2. In Vitro Antiproliferative Activities
All synthetic dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide
moieties 4a–4z were evaluated for antiproliferation activities against 5 cancer cell lines (SW620, MCF-7,
HeLa, A549 and HepG2) and one noncancer cell line, NCM460 by the MTT assay, in comparison
with the reference Celecoxib. As shown in Table 4, the antiproliferative activities of the tested
compounds were more pronounced against human colorectal carcinoma SW620 cells than the other
cell lines. Compound 4b exhibited the best antiproliferative activity among the tested compounds,
with IC50 values of 0.86
drug Celecoxib (1.29 0.04
potent antiproliferative activities than compounds with 1,4-dioxane 4k
was performed on a non-cancer line NCM460 to assess the cytotoxicity of the test compound to normal
colon cells. The results showed that compound 4a 4z and Celecoxib had similar low cytotoxic effect
±
0.02
M). As for SW620, compounds with 1,3-dioxolane 4a
4z. Besides, the MTT assay
µ
M, especially against SW620 cells, compared with positive control
±
µ
–4j behaved more
–
–
on NCM460, so these series of compounds had desirable safety.
Table 4. In vitro antiproliferation inhibitory activities of compounds 4a–4z and Celecoxib.
IC50 ± SD (µM) a
CC50 ± SD (µM) a
Compounds
SW620
MCF-7
HeLa
A549
HepG2
NUM460
4a
4b
4c
4d
4e
4f
3.38 ± 0.27
0.86 ± 0.02
5.07 ± 0.26
3.04 ± 0.17
3.88 ± 0.36
4.34 ± 0.33
7.99 ± 0.56
5.77 ± 0.39
2.99 ± 0.13
8.31 ± 0.67
5.26 ± 0.42
6.52 ± 0.46
7.21 ± 0.62
6.32 ± 0.55
2.98 ± 0.17
9.37 ± 0.90
5.71 ± 0.47
7.22 ± 0.65
8.05 ± 0.77
3.54 ± 0.19
1.94 ± 0.06
6.93 ± 0.17
2.86 ± 0.06
4.54 ± 0.18
5.49 ± 0.44
9.42 ± 0.74
7.64 ± 0.57
2.96 ± 0.14
13.74 ± 0.93
6.42 ± 0.25
9.44 ± 0.74
11.15 ± 1.03
18.22 ± 1.15
88.62 ± 3.96
134.33 ± 7.85
159.38 ± 11.36
74.47 ± 5.23
109.54 ± 7.71
129.34 ± 8.96
211.35 ± 18.15
4g
12.68 ± 0.89 14.61 ± 0.96

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Table 4. Cont.
IC50 ± SD (µM) a
CC50 ± SD (µM) a
Compounds
SW620
MCF-7
HeLa
A549
HepG2
NUM460
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
4v
4w
3.24 ± 0.18
4.96 ± 0.38
4.54 ± 0.29
4.56 ± 0.14
2.35 ± 0.11
3.88 ± 0.37
6.12 ± 0.49
3.83 ± 0.27
3.53±0.16
5.56 ± 0.38
8.14 ± 0.69
7.51 ± 0.57
7.54 ± 0.42
4.22 ± 0.16
6.52 ± 0.33
9.88 ± 0.86
6.44 ± 0.37
5.99 ± 0.38
5.47 ± 0.29
3.95 ± 0.18
7.51 ± 0.47
6.31 ± 0.39
6.07 ± 0.41
9.16 ± 0.37
8.41 ± 0.29
8.44 ± 0.48
4.46 ± 0.36
7.22 ± 0.19
11.25 ± 0.86
7.12 ± 0.69
6.58 ± 0.49
5.96 ± 0.39
4.14 ± 0.21
8.41 ± 0.17
6.97±0.51
3.26 ± 0.29
6.71 ± 0.57
5.86 ± 0.36
5.92 ± 0.31
2.58 ± 0.18
4.54 ± 0.29
6.81 ± 0.55
4.43 ± 0.33
3.83 ± 0.26
3.14 ± 0.25
3.12 ± 0.08
5.86 ± 0.47
4.26 ± 0.19
7.81 ± 0.37
6.47 ± 0.29
4.34 ± 0.28
5.75 ± 0.44
7.83 ± 0.58
5.78 ± 0.16
1.93 ± 0.09
7.14 ± 0.66
13.32 ± 0.89
11.82 ± 0.96
11.88 ± 0.91
5.92 ± 0.39
9.44 ± 0.77
13.52 ± 0.59
9.24 ± 0.74
8.16 ± 0.63
6.92 ± 0.28
3.28 ± 0.17
11.82 ± 0.59
8.94 ± 0.67
15.32 ± 1.09
12.92 ± 0.99
9.08 ± 0.71
11.52 ± 0.92
15.37 ± 0.84
11.68 ± 0.86
3.12 ± 0.27
82.82 ± 7.59
154.51 ± 12.66
137.11 ± 12.74
137.88±10.96
68.67 ± 5.28
109.54 ± 7.42
156.83 ± 9.98
107.18 ± 9.21
94.65 ± 3.65
80.27 ± 8.01
38.04 ± 1.19
137.11 ± 3.98
103.74 ± 4.74
177.71 ± 10.69
149.87 ± 6.94
105.32 ± 8.87
133.63 ± 9.48
178.29 ± 12.69
135.48 ± 9.63
136.19 ± 9.86
3.18 ± 0.19
2.17 ± 0.05
4.54 ± 0.31
3.74 ± 0.12
6.63 ± 0.42
4.84 ± 0.27
3.78 ± 0.22
4.46 ± 0.19
5.51 ± 0.37
4.48 ± 0.24
1.29 ± 0.04
10.64 ± 0.67 12.16 ± 0.85
7.96 ± 0.55
6.37 ± 0.39
7.39 ± 0.55
8.96 ± 0.74
7.42 ± 0.63
2.63 ± 0.09
8.95 ± 0.39
7.04 ± 0.47
8.26 ± 0.65
10.15 ± 0.72
8.34 ± 0.69
2.56 ± 0.15
4x
4y
4z
Celecoxib
a
Antiproliferation activity and cytotoxicity of the synthetic compounds were measured using the MTT assay. Data
displayed are the mean ± SD of three independent experiments (n = 3).
2.2.3. Compound 4b Induced SW620 Cell Apoptosis
To confirm that antiproliferation activities of SW620 were associated with apoptosis, SW620 cell
apoptosis induced by compound 4b was determined using flow cytometry with Annexin V-FITC/PI
Apoptosis Detection Kit. The outcome was shown in Figure 3, which indicated that the percentage
of apoptotic cells was significantly increased in a dose-dependent manner. The percentages of cell
apoptosis 7.9%, 19.9%, 45.9%, and 65.0% were responding to the concentration of compound 4b
2.0 µM, 4.0 µM, and 8.0 µM, respectively.
0 µM,
Figure 3.
and 8.0
(
A
) Compound 4b induced apoptosis in SW620 cells with the density of 0
µ
M, 2.0
SD, n = 3, p < 0.05
B) apoptosis histogram of SW620
µM, 4.0 µM,
µ
M. SW620 cells were treated with for 24 h. Values represent the mean
±
versus control. The percentage of cells in each part was indicated. (
induced by compound 4b.

Molecules 2019, 24, 1685
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2.2.4. Compound 4b Weakened the Adhesion of SW620 Cells
The decisive factor of tumor metastasis is the cell adhesion to fibronectin and laminin. Weak
cell adhesion is beneficial to tumor metastasis inhibition, so cell adhesion to fibronectin and laminin
assay was used to evaluate the effects of different concentrations of compound 4b and Celecoxib on
the adhesion ability of SW620 cells after 24 h treatment. Results as shown in Figure 4, compound 4b
exhibited a similar ability to Celecoxib to reduce the adhesion of SW620 cells to fibronectin and laminin.
Figure 4. Influence of compound 4b and Celecoxib on SW620 cell adhesion to fibronectin and laminin.
(
A
) Influence of compound 4b on SW620 cell adhesion to fibronectin; (B) influence of compound 4d
on SW620 cell adhesion to laminin; ( ) influence of Celecoxib on SW620 cell adhesion to fibronectin;
C
(D) influence of Celecoxib on SW620 cell adhesion to laminin.
2.2.5. Xenograft Model In Vivo
In view of potent cox-2 selective inhibitory activity and anti-colon cancer proliferation activity
in vitro, compound 4b was further evaluated for anti-colon cancer activity in vivo. SW620 cells
(5
×
106) were subcutaneously injected into the rightwing nude mice to establish a xenograft model.
When the tumor volume grows to the macroscopic size of about 100 mm3, 15 tumor-bearing mice
were randomly divided into vehicle, Celecoxib (20 mg/kg) and compound 4b (20 mg/kg) groups.
Intraperitoneal administration was performed every 2 days and tumor volume changes were recorded
for 12 consecutive days. As shown in Figure 5B, tumor volume increased rapidly in the vehicle group,
whereas tumor growth was significantly inhibited in two treatment groups. Among them, the tumor
inhibition effect of compound 4b (20 mg/kg) group was better than that of Celecoxib (20 mg/kg) group.
After 12 days of treatment, the tumor volumes of the two treatment groups were 43.71 mm3 and
51.69 mm3, respectively. Finally, the tumors of each group were removed and weighed to calculate the
ratio of tumor weight to body weight. The specific results are shown in Figure 5A,D. Compared to the
vehicle group with an average ratio of tumor weight to body weight of 0.34, the other two treatment
groups showed significant reduction, with compound 4b (20 mg/kg) indicating a lighter average ratio
of tumor weight to body weight (0.46). At the same time, no significant weight change was observed
in the treatment group, suggesting that the compounds in these mice were nontoxic. In contrast, body

Molecules 2019, 24, 1685
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weight increased slightly in the vehicle group at the later stage of treatment (Figure 5C). From the
above, these results suggested that compound 4b had potent anti-colon cancer activity in vivo. This
series of representative compounds 4b, as a selective COX-2 inhibitor for the targeted therapy of colon
cancer, had prominent research prospect.
Figure 5. Antitumor activity of compound 4b in SW620 xenografts tumor model. (
of each group of tumor resection. ( ) The tumor volume changes after drug administration with
Celecoxib (20 mg/kg), 4b (20 mg/kg) or vehicle. Data were measured every other day by using a Vernier
caliper and calculated as 0.5 length ) Relative weight changes were monitored
width2 (mm3). (
) Average ratio of tumor weight to body weight from
A) Physical photos
B
×
×
C
and recorded every two days in each group. (
D
each group; ** p < 0.01.
2.3. Molecular Docking
In order to better study the binding mode and interaction, molecule docking of dihydropyrazole
derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a 4z and known COX-2
–
inhibitors Celecoxib about the COX-2 (PDB ID: 3LN1) enzymes were performed together. All
simulations were performed around the central region of the already known COX-2 (PDB ID:
3LN1) site where Celecoxib bound. The judgment criterion is to compare the predicted combined
interaction energy. The estimated value of the interaction energy of each compound were ranged
from
linear fitting was performed by comparing the binding energy and COX-2 inhibition. It behaved
approximate linearity (y = 46.82 0.86x, R2 = 0.72) exhibited in the Figure 7. The R value is
−
45.96 to
−
33.41 kcal/mol, as displayed in the histogram of Table 5 and Figure 6. Besides,
−
approximately equal to 0.85 and approaches to 1, which not only indicates that the fitting curve has a
good credibility, but also indicates that our design idea is reasonable. Compared Celecoxib (energy
value of 42.57 kcal/mol), compound 4b was relatively superior with predicted combined interaction
−
energy value of
in Figure 7.
−
45.96 kcal/mol. And their concrete combination of computer simulation was depicted

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Table 5. The CDOCKER_INTERACTION_ENERGY from the docking of compounds 4a–4z.
CDOCKER_INTERACTION
_ENERGY (−kcal/mol)
CDOCKER_INTERACTION
Compounds
Compounds
_ENERGY (−kcal/mol)
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
41.58
45.96
38.59
42.23
40.66
40.77
33.41
41.89
38.77
39.53
39.46
43.38
40.88
36.71
4o
4p
4q
4r
4s
4t
4u
4v
40.76
41.29
42.83
43.71
39.57
40.98
35.82
39.12
41.74
39.66
37.81
39.63
42.57
4w
4x
4y
4z
Celecoxib
Figure 6. The histogram about CDOCKER_INTERACTION_ENERGY of compounds 4a–4z
and Celecoxib.
As illustrated in Figure 8A,B, Celecoxib could effectively bind at this site (XYZ axis: 30.99,
−
22.28
and
−
16.51; radius: 6.96 Å) through five hydrogen bond receptors and eleven Pi bonds, with the
stronger interactional amino acids for Arg-106, Ser-339, Arg-499, Gln-178 and Leu-338. Compared with
Celecoxib, compound 4b interacts more with proteins in this region and binds better, as shown in the
Figure 4C,D. Compound 4b could effectively bind through nine hydrogen bond receptors and fourteen
Pi bonds. In addition to the stronger interactional amino acids bond with Celecoxib, there are stronger
interactions with His-75, Phe-504, and Val-102, which bound to the sulfa and 1,3-dioxolane domains,
respectively. In general, these simulation results were consistent with the actual activity detection, and
compound 4b as a COX-2 inhibitor should be a promising effective drug. The difference in simulation
between compound 4b and Celecoxib could explain that compound 4b was a better COX-2 inhibitor.

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Figure 7. The fitting correlation between the binding energy and COX-2 inhibition.
Figure 8. Characterization of molecular docking with COX-2 (PDB ID: 3LN1). (
2D spatial modeling of Celecoxib with the COX-2 binding site. ( ) Molecular docking 3D spatial
modeling of Celecoxib with the COX-2 binding site. ( ) Molecular docking 2D spatial modeling of
compound 4b with the COX-2 binding site. ( ) Molecular docking 3D spatial modeling of compound
4b with the COX-2 binding site.
A) Molecular docking
B
C
D

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3. Experimental Section
3.1. Materials and Measurements
All commercial reagents and solvents were analyzed using products produced by USA Energy
Chemical company and Aladdin reagent (Shanghai, China). The melting points of all compounds
1
were determined by an x4mp instrument. All H NMR spectra were recorded in DMSO-d6 with
Bruker AM 600 MHz made by Rhenistetten Forchheim Company in Berlin, Germany as the internal
standard. Published 1H NMR chemical shift in
δ/PPM and coupling constant in Hertz. The thin layer
chromatography (TLC) plate was coated with Merck silica gel 60 GF254, and the chemical reaction
and purification process of the spots were observed under the light of 254/365 nm. The synthetic
compounds were evaluated in vitro in the state key laboratory of pharmaceutical biotechnology of
Nanjing university (Nanjing, China).
COX-1 (human) inhibitor screening kit (catalog number 70117) andCOX-2 (human) inhibitor
screening kit (catalog number 701180) were purchased from Cayman Chemical (Ann Arbor, MI, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) were purchased from Beyotime Institute
of Biotechnology (Haimen, China). Annexcin V-FITC cell apoptosis assay kit (catalog No.BA11100)
was purchased from BIO-BOX (Nanjing, China). Fibronectin (#F1056) and laminin (#L2020) were
purchased from Sigma-Aldrich (St. Louis, MO, USA).
3.2. Chemistry
3.2.1. General Procedure for the Synthesis of Compound 2a,2b
The protocatechuic aldehyde 1a (1 mmol) was dissolved in anhydrous DMF (5 mL), anhydrous
potassium carbonate (0.5 mmol) added. The solution was then heated to 70 ◦C and then dibromomethane
or 1,2-dibromoethane was added dropwise. The reaction was stirred for 4 h and poured into ice
water. A white precipitate (compound 2a, 2b) was formed which was filtered and recrystallized from
ethanol [33].
3.2.2. General Procedure for the Synthesis of Compounds 3a–3z
Compound 2a 2b (1 mmol) was added to 15 mL absolute ethanol solution with acetophenone
,
derivative (1 mmol) and 40% sodium hydroxide solution (0.5 mmol). The reaction was stirred at room
temperature for 10 h, and then filtered to afford compound 3a–3z without further purification [34].
3.2.3. General Procedure for the Synthesis of Compounds 4a–4z
The compounds 3a–3z (1 mmol), 4-sulfamethylhydrazine hydrochloride (1.2 mmol) and glacial
acetic acid (0.5 mmol) were refluxed overnight in ethanol (15 mL). The reaction mixture was diluted
with 50 mL water and extracted with ethyl acetate. Saturated sodium bicarbonate was used to wash the
organic anhydrous layer, which was then filtered to dry by anhydrous sodium sulfate and concentrated
in vacuum. The desired products 4a–4z were purified by flash chromatography.
3.2.4. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4a
)
White crystal, yield: 66.4%. m.p. 212–214 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.80 (d, J = 7.4 Hz,
2H, ArH), 7.62 (d, J = 8.6 Hz, 2H, ArH), 7.48–7.40 (m, 3H, ArH), 7.11 (d, J = 8.6 Hz, 2H, ArH), 7.04 (s,
2H, NH2), 6.87 (d, J = 7.9 Hz, 1H, ArH), 6.78–6.75 (m, 2H, ArH), 5.90 (s, 2H, CH2), 5.56 (dd, J1 = 4.7,
J2 = 4.7 Hz, 1H, 5-H), 3.91 (d, J1 = 12.4, J2 = 12.0 Hz, 1H, 4-Hb), 3.19 (dd, J1 = 4.7, J2 = 4.6 Hz, 1H, 4-Ha).
ESI-MS: m/z Calcd for C22H20N3O4S [M + H]+, 422.1; Found: 422.1. Anal. Calcd for C22H19N3O4S: C,
62.70; H, 4.54; N, 9.97%. Found: C, 62.92; H, 4.42; N, 9.86%.

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3.2.5. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4b
)
White crystal, yield: 76.3%. m.p. 200–201 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.73 (d, J = 8.8 Hz,
2H, ArH), 7.59 (d, J = 8.7 Hz, 2H, ArH), 7.08–7.01 (m, 6H, ArH and NH2), 6.87 (d, J = 7.9 Hz, 1H, ArH),
6.77–6.73 (m, 2H, ArH), 5.98 (s, 2H, CH2), 5.51 (dd, J1 = 5.0, J2 = 5.1 Hz, 1H, 5-H), 3.88 (dd, J1 = 12.0,
J2 = 11.9 Hz, 1H, 4-Hb), 3.80 (s, 3H, OCH3), 3.15 (dd, J1 = 5.1, J2 = 5.0 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd
for C23H22N3O4S [M + H]+, 452.1; Found: 452.1. Anal. Calcd for C23H21N3O4S: C, 62.43; H, 4.86; N,
9.97%. Found: C, 62.83; H, 4.92; N, 9.82%.
3.2.6. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4c)
White crystal, yield: 77.6%. m.p.182–184 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.71 (d, J = 8.6 Hz,
2H, ArH), 7.60 (d, J = 8.7 Hz, 2H, ArH), 7.08–6.98 (m, 6H, ArH), 6.86 (d, J = 7.8Hz, 1H, ArH), 6.74 (s, 2H,
NH2), 5.98 (s, 2H, CH2), 5.50 (dd, J1 = 4.9, J2 = 5.0 Hz, 1H, 5-H), 4.05 (q, J = 6.9 Hz, 2H, CH2), 3.87 (dd,
J1 = 12.1, J2 = 11.8 Hz, 1H, 4-Hb), 3.13 (dd, J1 = 5.0, J2 = 4.8 Hz, 1H, 4-Ha), 1.34 (t, J = 6.9 Hz, 3H, CH3).
ESI-MS: m/z Calcd for C24H24N3O5S [M + H]+, 466.1; Found: 466.1. Anal. Calcd for C24H23N3O5S: C,
61.92; H, 4.98; N, 9.03%. Found: C, 61.06; H, 4.84; N, 9.32%.
3.2.7. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(4-iodophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4d)
White crystal, yield: 58.9%. m.p. 212–214 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.82 (d, J = 7.9 Hz,
2H, ArH), 7.62–7.54 (m, 4H, ArH), 7.16 (d, J = 8.5 Hz, 2H, ArH), 7.04 (s, 2H, NH2), 6.86 (d, J = 7.9 Hz,
1H, ArH), 6.54 (d, J = 4.2 Hz, 2H, ArH), 5.98 (s, 2H, CH2), 5.57 (dd, J1 = 4.7, J2 = 5.1 Hz, 1H, 5-H), 3.91
(dd, J1 = 12.2, J2 = 12.0 Hz, 1H, 4-Hb), 3.16 (dd, J1 = 4.9, J2 = 5.0 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for
C22H19IN3O4S [M + H]+, 548.0; Found: 548.0. Anal. Calcd for C22H18IN3O4S: C, 48.28; H, 3.31; N,
7.68%. Found: C, 48.75; H, 3.38; N, 7.52%.
3.2.8. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(m-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)benzenesulfonamide (4e
)
White crystal, yield: 75.8%. m.p. 187–188 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.64–7.56 (m, 4H,
ArH), 7.34 (t, J = 7.6 Hz, 1H, ArH), 7.23 (d, J = 7.4 Hz, 1H, ArH), 6.10 (d, J = 8.6 Hz, 2H, ArH), 7.04
(s, 2H, NH2), 6.87 (d, J = 7.8 Hz, 1H, ArH), 6.75 (d, J = 9.9 Hz, 2H, ArH), 5.98 (s, 2H, CH2), 5.55 (dd,
J1 = 4.7, J2 = 4.8 Hz, 1H, 5-H), 3.91 (dd, J1 = 12.1, J2 = 12.1 Hz, 1H, 4-Hb), 3.17 (dd, J1 = 4.8, J2 = 4.7 Hz,
1H, 4-Ha), 2.37 (s, 3H, CH3), ESI-MS: m/z Calcd for C23H22N3O4S [M + H]+, 436.1; Found: 436.1. Anal.
Calcd for C23H21N3O4S: C, 63.43; H, 4.86; N, 9.65%. Found: C, 63.69; H, 4.91; N, 9.71%.
3.2.9. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(3,4-dimethylphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4f)
White crystal, yield: 73.8%. m.p. 179–180 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.60 (d, J = 8.0 Hz,
3H, ArH), 7.49 (d, J = 7.8 Hz, 1H, ArH), 7.21 (d, J = 7.8 Hz, 1H, ArH), 7.08 (d, J = 8.3 Hz, 2H, ArH),
7.02 (s, 2H, NH2), 6.86 (d, J = 7.8 Hz, 1H, ArH), 6.75 (s, 2H, ArH), 5.98 (s, 2H, CH2), 5.53 (dd, J1 = 4.4,
J2 = 4.6 Hz, 1H, 5-H), 3.88 (dd, J1 = 12.1, J2 = 24.0 Hz, 1H, 4-Hb), 3.14 (dd, J1 = 4.5, J2 = 4.4 Hz, 1H, 4-Ha).
ESI-MS: m/z Calcd for C24H24N3O4S [M + H]+, 450.1; Found: 450.1. Anal. Calcd for C24H23N3O4S: C,
64.13; H, 5.16; N, 9.35%. Found: C, 64.47; H, 5.19; N, 9.44%.
3.2.10. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4g)
White crystal, yield: 76.6%. m.p. 219–221 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.61 (d, J = 8.7 Hz,
2H, ArH), 7.37–6.99 (m, 8H, ArH), 6.87 (d, J = 7.9 Hz, 1H, ArH), 6.76 (s, 2H, NH2), 6.00 (s, 2H, CH2),
5.56 (dd, J1 =4.9, J2 = 5.0 Hz, 1H, 5-H), 3.91 (dd, J1 = 12.2, J2 = 12.0 Hz, 1H, 4-Hb), 3.82 (s, 3H, CH3),

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3.18 (dd, J1 = 5.0, J2 = 4.8 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for C23H22N3O5S [M + H]+, 452.1; Found:
452.1. Anal. Calcd for C23H21N3O5S: C, 61.19; H, 4.69; N, 9.31%. Found: C, 60.87; H, 4.82; N, 9.26%.
3.2.11. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4h)
Yellow crystal, yield: 69.8%. m.p. 192–193 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.95 (t, J = 7.6 Hz,
1H, ArH), 7.63 (d, J = 8.6 Hz, 2H, ArH), 7.46 (q, J = 7.2 Hz, 1H, ArH), 7.28 (q, J = 8.1 Hz, 2H, ArH), 7.11
(d, J=8.5 Hz, 2H, ArH), 7.06 (s, 2H, NH2), 6.87 (d, J = 7.9 Hz, 1H, ArH), 6.77 (t, J = 7.0 Hz, 2H, ArH),
5.99 (s, 2H, CH2), 5.56 (dd, J1 = 5.0, J2 = 5.0 Hz, 1H, 5-H), 4.01 (dd, J1 = 12.2, J2 = 12.2 Hz, 1H, 4-Hb),
3.20 (dd, J1 = 4.6, J2 = 4.6 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for C22H19FN3O4S [M + H]+, 440.1;Found:
440.1. Anal. Calcd for C22H18FN3O4S: C, 60.13; H, 4.13; N, 9.56%. Found: C, 60.75; H, 4.79; N, 9.58%.
3.2.12. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(2,4-dimethylphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4i)
White crystal, yield: 72.9%. m.p. 180–182 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.60 (d, J = 8.6 Hz,
2H, ArH), 7.35 (d, J = 7.9 Hz, 1H, ArH), 7.17 (s, 1H, ArH), 7.10–7.03 (m, 5H, ArH and NH2), 6.87 (d,
J = 7.7 Hz, 1H, ArH), 6.77–6.74 (m, 2H, ArH), 6.98 (s, 2H, CH2), 5.48 (dd, J1 = 4.8, J2 = 4.8 Hz, 1H, 5-H),
3.98 (dd, J1 = 12.0, J2=11.8 Hz, 1H, 4-Hb), 3.20 (dd, J1 = 4.9, J2 = 4.8 Hz, 1H, 4-Ha), 2.67 (s, 3H, CH3),
2.31 (s, 3H, CH3). ESI-MS: m/z Calcd for C24H24N3O4S [M + H]+, 450.1; Found: 450.1. Anal. Calcd for
C24H23N3O4S: C, 64.13; H, 5.16; N, 9.35%. Found: C, 64.72; H, 5.19; N, 9.51%.
3.2.13. 4-(5-(Benzo[d][1,3]dioxol-5-yl)-3-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4j)
White crystal, yield: 66.5%. m.p. 180–181 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.95 (t, J = 7.6 Hz,
1H, ArH), 7.63 (d, J = 8.6 Hz, 2H, ArH), 7.46 (q, J = 7.2 Hz, 1H, ArH), 7.28 (q, J = 8.1 Hz, 2H, ArH), 7.11
(d, J = 8.5 Hz, 2H, ArH), 7.06 (s, 2H, NH2), 6.87 (d, J = 7.9 Hz, 1H, ArH), 6.77 (t, J = 7.0 Hz, 2H, ArH),
5.99 (s, 2H, CH2), 5.56 (dd, J1 = 5.0, J2 = 5.0 Hz, 1H, 5-H), 4.01 (dd, J1 = 12.2, J2 = 12.2 Hz, 1H, 4-Hb),
3.20 (dd, J1 = 4.6, J2 = 4.6 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for C22H19FN3O4S [M + H]+, 440.1; Found:
440.1. Anal. Calcd for C22H18FN3O4S: C, 60.13; H, 4.13; N, 9.56%. Found: C, 60.61; H, 4.59; N, 9.53%.
3.2.14. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4k)
White crystal, yield: 73.5%. m.p. 156–157 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.79 (d, J = 6.8 Hz,
2H, ArH), 7.61 (d, J = 8.9 Hz, 2H, ArH), 7.47–7.40 (m, 3H, ArH), 7.09 (t, J = 8.9 Hz, 2H, ArH), 6.99 (s,
2H, NH2), 6.82 (d, J = 8.4 Hz, 1H, ArH), 6.72–6.99 (m, 2H, ArH), 5.54 (dd, J1 = 4.8, J2 = 4.8 Hz, 1H, 5-H),
4.18 (s, 4H, CH2), 3.91 (dd, J1 = 12.1, J2 = 12.0 Hz, 1H, 4-Hb), 3.17 (dd, J1 = 4.9, J2 = 3.6 Hz, 1H, 4-Ha).
ESI-MS: m/z Calcd for C23H22N3O4S [M + H]+, 435.1; Found: 435.1. Anal. Calcd for C23H21N3O4S: C,
63.43; H, 4.86; N, 9.65%. Found: C, 63.52; H, 4.97; N, 9.71%.
3.2.15. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4l)
White crystal, yield: 75.6%. m.p. 153–154 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.68 (d, J = 8.1 Hz,
2H, ArH), 7.59 (d, J = 8.9 Hz, 2H, ArH), 7.26 (d, J = 8.0 Hz, 2H, ArH), 7.06 (t, J = 8.9 Hz, 1H, ArH), 6.99
(s, 2H, NH2), 6.82 (d, J = 8.6 Hz, 1H, ArH), 6.70-6.68 (m, 2H, ArH), 5.51 (dd, J1 = 4.7, J2 = 4.7 Hz, 1H,
5-H), 4.18 (s, 4H, CH2), 3.88 (dd, J1 = 12.0, J2 = 11.8 Hz, 1H, 4-Hb), 3.13 (dd, J1 = 4.9, J2 = 4.8 Hz, 1H,
4-Ha), 2.34 (s, 3H, CH3). ESI-MS: m/z Calcd for C24H24N3O4S [M + H]+, 450.1; Found: 450.1. Anal.
Calcd for C24H23N3O4S: C, 64.13; H, 5.16; N, 9.35%. Found: C, 64.59; H, 5.27; N, 9.48%.

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3.2.16. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4m)
White crystal, yield: 75.6%. m.p. 207–208 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.73 (d, J = 8.8 Hz,
2H, ArH), 7.59 (d, J = 8.7 Hz, 2H, ArH), 7.08–7.01 (m, 6H, ArH and NH2), 6.87 (d, J = 7.9 Hz, 1H, ArH),
6.77–6.73 (m, 2H, ArH), 5.49 (dd, J1 = 5.0, J2 = 5.1 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.87 (dd, J1 = 12.0,
J2 = 11.9 Hz, 1H, 4-Hb), 3.81(s, 3H, OCH3), 3.13 (dd, J1 = 5.1, J2 = 5.0 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd
for C24H24N3O5S [M + H]+, 466.1; Found: 466.1. Anal. Calcd for C24H23N3O5S: C, 64.13; H, 5.16; N,
9.35%. Found: C, 64.59; H, 5.27; N, 9.48%.
3.2.17. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-ethoxyphenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4n)
White crystal, yield: 59.8%. m.p. 193–194 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.71 (d, J = 8.6 Hz,
2H, ArH), 7.60 (d, J = 8.7 Hz, 2H, ArH), 7.07–6.98 (m, 6H, ArH), 6.81 (d, J = 8.0Hz, 1H, ArH), 6.70 (s,
2H, NH2), 5.48 (dd, J1 = 5.0, J2 = 4.7 Hz, 1H, 5-H), 4.18 (s, 4H, CH2), 4.05 (q, J = 6.8 Hz, 2H, CH2), 3.86
(dd, J1 = 12.0, J2 = 11.7 Hz, 1H, 4-Hb), 3.12 (dd, J1 = 4.7, J2 = 4.6 Hz, 1H, 4-Ha), 1.34 (t, J = 6.9 Hz, 3H,
CH3). ESI-MS: m/z Calcd for C25H26N3O5S [M + H]+, 480.1. Anal. Calcd for C25H25N3O5S: C, 62.62;
H, 5.25; N, 8.76%. Found: C, 63.53; H, 5.49; N, 8.98%.
3.2.18. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4o)
Yellow crystal, yield: 67.8%. m.p. 195–196 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.84 (q, J = 5.6 Hz,
2H, ArH), 7.60 (d, J = 8.9 Hz, 2H, ArH), 7.29 (t, J = 8.8 Hz, 2H, ArH), 7.08 (t, J = 8.9 Hz, 2H, ArH), 6.99
(s, 2H, NH2), 6.82 (q, 2, J = 8.1 Hz, 2H, ArH), 6.70 (t, J = 8.0 Hz, 2H, ArH), 5.54 (dd, J1 = 4.8, J2 = 4.8 Hz,
1H, 5-H), 4.19 (s, 4H, CH2), 3.91 (dd, J1 = 12.1, J2 = 11.9 Hz, 1H, 4-Hb), 3.16 (dd, J1 = 5.0, J2 = 4.8 Hz,
1H, 4-Ha). ESI-MS: m/z Calcd for C23H21FN3O4S [M + H]+, 454.1; Found: 454.1. Anal. Calcd for
C23H20FN3O4S: C, 60.92; H, 4.45; N, 9.27%. Found: C, 61.63; H, 4.58; N, 9.13%.
3.2.19. 4-(3-(4-Chlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4p)
Yellow crystal, yield: 73.9%. m.p. 146–148 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.79 (d, J = 8.6 Hz,
2H, ArH), 7.61 (d, J = 8.8 Hz, 2H, ArH), 7.50 (d, J = 8.6 Hz, 2H, ArH), 7.11–7.08 (m, 3H, ArH), 6.82 (d,
J = 8.0 Hz, 2H, ArH), 6.99 (s, 2H, NH2), 5.55 (dd, J1 = 4.8, J2=4.8 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.89
(dd, J1 = 7.0, J2 = 12.0 Hz, 1H, 4-Hb), 3.16 (dd, J1 = 5.0, J2 = 4.9 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for
C23H21ClN3O4S [M + H]+, 470.1; Found: 470.1. Anal. Calcd for C23H20ClN3O4S: C, 58.78; H, 4.29; N,
8.94%. Found: C, 59.97; H, 4.48; N, 9.18%.
3.2.20. 4-(3-(4-Bromophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4q)
White crystal, yield: 63.5%. m.p. 153–154 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.72 (d, J = 8.6 Hz,
2H, ArH), 7.61 (q, J = 8.8 Hz, 2H, ArH), 7.09 (t, J = 8.8 Hz, 2H, ArH), 6.99 (s, 2H, NH2), 6.81 (d, J = 8.0 Hz,
2H, ArH), 5.55 (dd, J1 = 4.9, J2 = 4.9 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.91(dd, J1 = 12.2, J2 = 12.0 Hz,
1H, 4-Hb), 3.16 (dd, J1 = 5.0, J2 = 4.9 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for C23H21BrN3O4S [M + H]+,
Found: 514.0. Anal. Calcd for C23H20BrN3O4S: C, 53.70; H, 3.92; N, 8.17%. Found: C, 53.54; H, 3.18;
N, 8.43%.
3.2.21. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-iodophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4r)
Black crystal, yield: 72.3%. m.p. 187–189 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.79 (d, J = 8.1 Hz,
2H, ArH), 7.63 (d, J = 7.6 Hz, 2H, ArH), 7.55 (d, J = 8.0 Hz, 2H, ArH), 7.11 (d, J = 8.6 Hz, 2H, ArH), 6.82
(d, J = 8.1 Hz, 2H, ArH), 6.70 (s, 2H, NH2), 5.53 (dd, J1 = 4.6, J2 = 4.7 Hz, 1H, 5-H), 4.18 (s, 4H, CH2),

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3.87(dd, J1 = 12.4, J2 = 12.0 Hz, 1H, 4-Hb), 3.13 (dd, J1 = 4.7, J2 = 4.5 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd
for C23H21IN3O4S [M + H]+, 562.0; Found: 562.0. Anal. Calcd for C23H20IN3O4S: C, 49.21; H, 3.59; N,
7.49%. Found: C, 49.88; H, 3.95; N, 7.36%.
3.2.22. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(3,4-dimethylphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4s)
White crystal, yield: 72.3%. m.p. 166–168 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.60 (d, J = 8.7 Hz,
3H, ArH), 7.48 (d, J = 7.8 Hz, 2H, ArH), 7.20 (d, J = 7.9 Hz, 1H, ArH), 7.07 (t, J = 8.7 Hz, 3H, ArH), 6.81
(d, J = 8.4 Hz, 1H, ArH), 6.90 (s, 2H, NH2), 5.50 (dd, J1 = 4.5, J2=4.6 Hz, 1H, 5-H), 4.19 (s, 4H, CH2),
3.86 (dd, J1 = 12.0, J2 = 11.8 Hz, 1H, 4-Hb), 3.12 (dd, J1 = 4.6, J2 = 4.5 Hz, 1H, 4-Ha), 2.27 (s, 3H, CH3),
2.26 (s, 3H, CH3). ESI-MS: m/z Calcd for C25H26N3O4S [M + H]+, 464.1; Found: 464.1. Anal. Calcd for
C25H25N3O4S: C, 64.78; H, 5.44; N, 9.07%. Found: C, 65.92 H, 5.69; N, 9.34%.
3.2.23. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(4-fluoro-3-methylphenyl)-4,5-dihydro-1H-
pyrazol-1-yl)benzenesulfonamide (4t)
Yellow crystal, yield: 71.4%. m.p. 159–161 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.78 (q, J = 8.9 Hz,
1H, ArH), 7.64–7.60 (m, 3H, ArH), 7.48–7.40 (m, 1H, ArH), 7.11(t, J = 8.9 Hz, 2H, ArH), 7.09 (s, 2H,
NH2), 6.82 (t, J = 8.2 Hz, 2H, ArH), 6.72–6.69 (m, 2H, ArH), 5.54 (dd, J1 = 4.9, J2 = 5.4 Hz, 1H, 5-H), 4.19
(s, 4H, CH2), 3.86 (dd, J1 = 12.1, J2 = 11.9 Hz, 1H, 4-Hb), 3.15 (dd, J1 = 5.0, J2 = 4.8 Hz, 1H, 4-Ha), 2.35
(s, 3H, CH3), ESI-MS: m/z Calcd for C24H23FN3O4S [M + H]+, 468.1; Found: 468.1. Anal. Calcd for
C24H22FN3O4S: C, 61.66; H, 4.74; N, 8.99%. Found: C, 62.42; H, 4.58; N, 9.29%.
3.2.24. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(3-methoxyphenyl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4u)
White crystal, yield: 70.1%. m.p. 146–147 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.60 (d, J = 8.9 Hz,
2H, ArH), 7.34 (d, J = 4.9 Hz, 2H, ArH), 7.11–7.00 (m, 3H, ArH), 6.99 (s, 2H, NH2), 6.82 (d, J = 8.4 Hz,
2H, ArH), 6.71–6.69 (m, 2H, ArH), 5.54 (dd, J1 = 4.7, J2 = 4.9 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.89 (dd,
J1 = 12.1, J2 = 12.0 Hz, 1H, 4-Hb), 3.80 (s, 3H, OCH3), 3.17 (dd, J1 = 4.9, J2 = 4.8 Hz, 1H, 4-Ha). ESI-MS:
m/z Calcd for C24H24N3O5S [M + H]+, Found: 466.1. Anal. Calcd for C24H23N3O5S: C, 61.92; H, 4.98;
N, 9.03%. Found: C, 62.37; H, 5.16; N, 9.31%.
3.2.25. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(3-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4v)
Yellow crystal, yield: 68.7%. m.p. 200–201 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.84 (q, J = 5.6 Hz,
2H, ArH), 7.60 (d, J = 8.9 Hz, 2H, ArH), 7.29 (t, J = 8.8 Hz, 2H, ArH), 7.08 (t, J = 8.9 Hz, 2H, ArH), 6.99
(s, 2H, NH2), 6.82 (q, 2, J=8.1 Hz, 2H, ArH), 6.70 (t, J = 8.0 Hz, 2H, ArH), 5.54 (dd, J1 = 4.8, J2 = 4.8 Hz,
1H, 5-H), 4.19 (s, 4H, CH2), 3.91 (dd, J1 = 12.1, J2 = 11.9 Hz, 1H, 4-Hb), 3.16 (dd, J1 = 5.0, J2 = 4.8 Hz,
1H, 4-Ha). ESI-MS: m/z Calcd for C22H21FN3O4S [M + H]+, 454.1; Found: 454.1. Anal. Calcd for
C22H20FN3O4S: C, 60.92; H, 4.45; N, 9.27%. Found: C, 62.21; H, 4.69; N, 9.44%.
3.2.26. 4-(3-(3,5-Difluorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4w)
White crystal, yield: 63.4%. m.p. 182–183 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.62 (q, J = 8.9 Hz,
2H, ArH), 7.50–7.47 (m, 2H, ArH), 7.31–7.26 (m, 1H, ArH), 7.15 (t, J = 8.9 Hz, 2H, ArH), 7.10 (s, 2H,
NH2), 6.82 (t, J = 8.2 Hz, 2H, ArH), 6.72–6.69 (m, 2H, ArH), 5.60 (dd, J1 = 4.9, J2 = 5.4 Hz, 1H, 5-H),
4.19 (s, 4H, CH2), 3.88 (dd, J1 = 12.1, J2 = 11.9 Hz, 1H, 4-Hb), 3.19 (dd, J1 = 5.0, J2 = 4.8 Hz, 1H, 4-Ha).
ESI-MS: m/z Calcd for C23H20F2N3O4S [M + H]+, 472.1; Found: 472.1. Anal. Calcd for C23H19F2N3O4S:
C, 58.59; H, 4.06; N, 8.91%. Found: C, 58.96; H, 4.74; N, 8.88%.

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3.2.27. 4-(3-(2,3-Dichlorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4x)
Yellow crystal, yield: 73.5%. m.p. 171–173 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.97 (d, J = 1.9 Hz,
2H, ArH), 7.76–7.61 (m, 5H, ArH), 7.24–7.06 (m, 4H, ArH), 6.82–6.68 (m, 3H, ArH and NH2), 5.59 (dd,
J1 = 4.8, J2 = 5.0 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.89 (dd, J1 = 12.0, J2 = 24.0 Hz, 1H, 4-Hb), 3.19 (dd,
J1 = 5.0, J2 = 4.9 Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for C23H20Cl2N3O4S [M + H]+, 504.1; Found: 504.1.
Anal. Calcd for C23H19Cl2N3O4S: C, 54.77; H, 3.80; N, 8.33%. Found: C, 54.96; H, 3.95; N, 8.53%.
3.2.28. 4-(5-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-3-(2-fluorophenyl)-4,5-dihydro-1H-pyrazol-1-yl)
benzenesulfonamide (4y)
Yellow crystal, yield: 69.4%. m.p. 191–192 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.94 (t, J = 7.6 Hz,
1H, ArH), 7.63 (d, J = 8.6 Hz, 2H, ArH), 7.45 (q, J = 7.1 Hz, 1H, ArH), 7.27 (q, J = 8.7 Hz, 2H, ArH), 7.11
(d, J = 9.0 Hz, 4H, ArH), 7.82 (d, J = 8.1 Hz, 4H, ArH), 6.72 (s, 2H, NH2), 5.53 (dd, J1 = 4.7, J2 = 4.8 Hz,
1H, 5-H), 4.19 (s, 4H, CH2), 3.99 (dd, J1 = 12.1, J2 = 12.1 Hz, 1H, 4-Hb), 3.17 (dd, J1=2.5, J2 = 2.2 Hz,
1H, 4-Ha). ESI-MS: m/z Calcd for C23H21FN3O4S [M + H]+, 454.1; Found: 454.1. Anal. Calcd for
C23H20FN3O4S: C, 60.92; H, 4.45; N, 9.27%. Found: C, 61.39; H, 4.69; N, 9.31%.
3.2.29. 4-(3-(2,4-Difluorophenyl)-5-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,5-dihydro-1H-pyrazol-1-
yl)benzenesulfonamide (4z)
White crystal, yield: 65.3%. m.p. 168–169 ◦C; 1H NMR (DMSO-d6, 600 MHz)
δ: 7.97 (q, J = 7.6 Hz,
1H, ArH), 7.61 (d, J = 7.9 Hz, 2H, ArH), 7.36 (q, J = 9.7 Hz, 1H, ArH), 7.20 (t, J = 8.4 Hz, 2H, ArH), 7.10
(d, J = 9.0 Hz, 2H, ArH), 7.08 (s, 2H, NH2), 6.81(d, J = 8.4 Hz, 1H, ArH), 6.70 (d, J = 10.0 Hz, 2H, ArH),
5.53 (dd, J1 = 4.5, J2 = 4.7 Hz, 1H, 5-H), 4.19 (s, 4H, CH2), 3.98 (dd, J1 = 12.3, J2 = 12.2Hz, 1H, 5-H), 3.44
(dd, J1 = 6.8, J2 = 7.4 Hz, 1H, 4-Hb), 3.14 (dd, J1 = 2.5, J2 = 4.3Hz, 1H, 4-Ha). ESI-MS: m/z Calcd for
C23H20F2N3O4S [M + H]+, 472.1; 472.1. Anal. Calcd for C23H19F2N3O4S: C, 58.59; H, 4.06; N, 8.91%.
Found: C, 59.67; H, 4.52; N, 9.06%.
3.3. Biological Assays
3.3.1. COX-1/COX-2 Inhibition Assay
The ability of the synthetic compounds to inhibit COX-1 and COX-2 was evaluated by COX-1/COX-2
ELISA kit (no. 701170/701180, Cayman Chemistry). COX-1 or COX-2 enzyme was pre-incubated with
test compounds at 0
pH 8.0, 5 mM EDTA, 2 mM phenol and 1
adding arachidonic acid to a final concentration of 100
µ
M, 1
µ
M, 10
µ
M and 100
M heme) for 10 min at 37 ◦C. The reactions were initiated by
M and incubated 2 min at 37 ◦C for. Then 1 M
µM in the supplied reaction buffer (0.1 M Tris–HCl,
µ
µ
HCl was added to the reaction mixture to stop the reaction, and one-tenth of the volume of saturated
stannous chloride (50 mg/mL) was added. The reaction mixture was incubated at room temperature
for 5 min, and PGF2 and PGH2 were produced.
3.3.2. Cell Culture
MCF-7 (human breast cell line), HeLa (human cervical cell line), A549 (human lung cell line),
HepG2 (human liver cell line), SW620 (human colorectal cell line) and a non-cancer cell line, NCM460
(Human colonic epithelial cells) were preserved at the State Key Laboratory of Pharmaceutical
Biotechnology, Nanjing University (Nanjing, China). Cells were maintained in Dulbecco0s modified
Eagle0s medium (DMEM) (HyClone, Logan, UT, USA) with 10% fetal bovine serum (FBS, BI) (HyClone,
Logan, UT, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (Hyclone, Logan, UT, USA), and
incubated at 37 ◦C in a humidified atmosphere containing 5% CO2.

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3.3.3. Cell Proliferation Assay
The antiproliferative activity of the synthetic compounds against the SW620, MCF-7, HeLa, A549,
HepG2, and NUM460 cell lines was evaluated by a colorimetric assay based on a modified standard
(MTT). Test cell lines were seeded at a density of 1
37 ◦C in DMEM supplemented with 10% fetal bovine serum. All test compounds dissolved in DMSO
were then treated to cells at 0 M, 0.1 M, 1 M, 10 M, and 100
M and incubated for 48 h at 37 ◦C in
5% CO2. Thereafter, MTT (5 mg/mL in PBS) was added to each well and incubated for 4 h. 150 L of
×
104/well in 96-well plates and incubated for 12 h at
µ
µ
µ
µ
µ
µ
DMSO was added to each well and shaken with a shaker. The absorbance (OD 570 nm) was read at
630 nm on an ELISA reader (ELx800, BioTek, montpelier, VT, USA). IC50 values for the compounds
were calculated by comparison to DMSO treated control wells. Three replicate wells were used for
each drug concentration. Each assay was performed three times.
3.3.4. Cell Apoptosis Assay
SW620 cells were seeded in the 6-well plate (2
induce apoptosis at final concentrations of 0, 2, 4, and 8
extracted with trypsin and washed with cold PBS twice. Cells are then bound to 500
then annexin-v/FITC (5 L) and PI (5 L) were added to the medium for another 10 min to incubate
×
105 cells/m2) and treated with compound 4b to
M for 24 h. After incubation, the cells were
L buffer and
µ
µ
µ
µ
darkness. Staining cells were analyzed by flow cytometry (Becton Dickinson, Franklin, NJ, USA).
Flowjo 7.6.1 software (Emerald Biotech Co. Ltd., Hangzhou, China) was used for statistical analysis.
3.3.5. Cell Adhesion Assay
In the cell adhesion experiment, the 96-well plate was coated with 50 L fibrin and laminin (10 g/mL)
(Sigma-Aldrich, St. Louis, MI, USA) at a constant temperature of 4 ◦C for 12 h, closed with 0.2% BSA
for 2 h, and washed 3 times at room temperature. Afterwards, A549 cells treated with 4 days 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. SW620 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 g/mL MTT in complete medium at
37 ◦C for 4 h. Then, mtt-treated cells were treated in DMSO, and the absorbance was measured by
ELISA reader.
3.3.6. Xenograft Model In Vivo
Nude mice aged 6-8 weeks were purchased from the model animal research center of nanjing
university and bred in a specific non-pathogenic environment. A xenograft model was established
by subcutaneous injection of SW620 cells (5 × 108)100 L DMEM into the right wing (18–22 g) of nude
mice. When the tumor mass was visible and the tumor size was close to 100 mm3.Tumor-bearing
nude mice were randomly divided into 3 groups (5 mice/group), vehicle-mounted treatment group,
compound 4b (20 mg/kg) treatment group and Celecoxib (20 mg/kg) treatment group. Vehicle treatment
groups were treated with polyethylene glycol (containing 1% DMSO). The administration method
was intraperitoneal injection every 2 days for consecutive 12 days. After administration, weighed
every two days and measured tumor volume with a vernier caliper. Animal welfare and experimental
procedures were carried out in accordance with Laboratory animal-Guideline for ethical review
of animal welfare (China national standardization management committee, GB/T 35892-2018) and
Laboratory animals-General requirements for animal experiment (China national standardization
management committee, GB/T 35823-2018). All the animal experiments were approved by Nanjing
University Animal Care and Use Committee (NJU-ACUC) and made to minimize suffering and to
reduce the number of animals used.

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3.4. Docking Simulations
The crystal structure of the protein complex was retrieved from the RCSB protein database (PDB
code: 3LN1). Then, the three-dimensional structure of the compound was constructed using Chem
3D Ultra 14.0 software (Cambridge Software, Boston, MA, USA), and then energy minimization was
performed using MMFF94, iterating 5000 times with a minimum RMS gradient of 0.10. Compound 4b
and Celecoxib molecules were docked into a three-dimensional complex structure of COX-2 using the
Discovery Studio (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China) software via the graphical
user interface DS-CDOCKER protocol. All bound water and ligand were removed from the protein
and polar hydrogen was added to the protein. Briefly, we defined the COX-2 complex as a receptor
and then replaced the Celecoxib molecule with compound 4b, mimicking the entry of the compound
into the site where COX-2 binds to Celecoxib.
4. Conclusions
Overexpression of COX-2 was clearly associated with the development of various types of cancer,
and the concept of COX-2 as a possible target was a promising therapeutic strategy. Therefore, a
series of novel dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide
moieties 4a
strong selective and inhibitory activity, and compound 4b exhibited the most potent inhibitory activity
against COX-2 and antiproliferative activity on SW620 cells with IC50 values of 0.86 M. In further
–4z were designed to be COX-2 inhibitors. Most of the synthetic compounds exhibited
µ
research, representative compound 4b induced apoptosis in SW620 cells in a dose-dependent manner
and attenuated its adhesion. Finally, the in vivo anti-colon cancer activity of compound 4b was verified
in SW620 xenograft mouse models. In conclusion, it was hoped that this study would be beneficial to
the research of COX-2 inhibitors in the treatment for colorectal carcinoma.
Supplementary Materials: The following are available online, Figure S1: The design pathway for novel
dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties., Table S1: Structures
of compounds 4a–4z, etc.
Author Contributions: All authors contributed to conceptualization, methodology, and writing. X.-Q.Y. conducted
data processing. X.-Q.Y. performed data analysis and simulation.
Funding: The work was financed by Public Science and Technology Research Fund Project of Ocean (No. 201505023).
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compound 4b are available from the authors.
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