Design, synthesis and evaluation of benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives as COX-1/COX-2 agents against solid tumors

Article abstract of DOI:10.1039/c6ra02168a

Novel benzenesulfonamide-substituted 1,5-diarylpyrazoles containing phenylacetohydrazide derivatives have been designed, synthesized and evaluated for their biological activities as selective COX-2 inhibitors with anticancer potential. In vitro the bioass

Full text of DOI:10.1039/c6ra02168a

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Design, Synthesis and Evaluation of Benzenesulfonamide-substituted
,5-Diarylpyrazoles containing Phenylacetohydrazide Derivatives as
COX-1/COX-2 Agents Against Solid Tumors
1
†
†
Xiao-Yuan Lu , Zhong-Chang Wang *, Ting Wei, Xiao-qiang Yan,
Peng-Fei Wang, Hai-Liang Zhu*
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing
2
10023, People’s Republic of China
*
†
Corresponding author. Tel. & fax: +86ꢀ25ꢀ83592672; eꢀmail: zhuhl@nju.edu.cn,
These two authors equally contributed to this paper.

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Abstract
Novel
benzenesulfonamideꢀsubstituted
1,5ꢀdiarylpyrazoles
containing
phenylacetohydrazide derivatives have been designed, synthesized and evaluated for
their biological activities as selective COXꢀ2 inhibitor with anticancer potential. In
vitro the bioassay results revealed that some of them displayed potent inhibitory
activity in the enzymatic and cellular assays. Among them, compound 48 showed
most powerful and potent selective inhibitory activity (IC50 = 82.21 ꢀM for COXꢀ1
and IC50 = 0.37 ꢀM for COXꢀ2), comparable to the control positive compound
Celecoxib (40.29 µM, 0.15µM). Antiproliferative assay results indicated that
compound 48 possess potent antiproliferative activity against A549 cells in vitro with
IC50 value of 0.78 ꢀM. We then performed PI staining assay and cell apoptosis
analysis for compound 48 and found that it effectively causing A549 cell apoptosis.
Docking simulation was further performed to position compound 48 into the COXꢀ2
active site to determine the probable binding model. The 3DꢀQSAR models were built
for reasonable design of selective COXꢀ2 inhibitor in future.
Keywords:
Diarylpyrazoles
Benzenesulfonamide
Anticancer
COXꢀ1/COXꢀ2
Docking

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Introduction
The cyclooxygenase which to catalyze the conversion of arachidonic acid to
prostaglandins (PGs) exists in two isoforms: the constitutive COXꢀ1 and the inducible
COXꢀ2, has led to new targets for the design of mechanism based drugs in cancer
1
chemoprevention research. The two isoforms differ in regulation and expression,
depending on the tissue, which lead to the formation of various eicosanoids involved
2
3
4
5
in several disease states, such as inflammation, fever, arthritis, and more
6
recently discovered, cancer. COXꢀ1 has been known as a housekeeping enzyme
constitutively expressed in most mammalian tissues and responsible for the
production of cytoprotective prostaglandins (PGs) in the gastrointestinal (GI) tract;
whereas COXꢀ2 can be rapidly expressed during inflammatory conditions by
7, 8
proꢀinflammatory stimuli in macrophages and other cells.
The implications of
COXs have been researched in the treatment of a variety of disease states. For
example, the treatment of Alzheimer’s disease (AD), Parkinson’s disease,
inflammatory diseases and cancers, so inhibiting these enzymes seems to be even
9
, 10
more advantageous in various cardiovascular diseases and cancer therapy.
Nevertheless, interruption of COXꢀ1 activity may lead to gastrointestinal toxicity such
as ulceration, bleeding, and perforation. Selective inhibition of COXꢀ2 might be
endowed with improved blocking biosynthesis of PGs and reduced gastrointestinal
11
toxicity profiles.
Numerous studies have demonstrated that overexpression of
1
2ꢀ14
COXꢀ2 is sufficient to cause tumorigenesis in animal models.
Inhibition of the
COXꢀ2/PGE2 pathway results in reduction in tumorigenesis via a number of distinct
mechanisms: promoting tumour maintenance and progression, encouraging metastatic
15, 16
spread, and perhaps even participating in tumour initiation.
clinical, and preclinical investigations also provide compelling evidence that COXs
either COXꢀ2 or both COXꢀ1 and COXꢀ2) inhibitors could act as chemopreventive
agents, for example, Celecoxib as well as other coxibs such as Valdecoxib could
Epidemiological,
(
17
induce cancer cell growth inhibition and apoptosis.
Those COXs inhibitors may
hinder biosynthesis of prostaglandins and other pathways related to tumorigenesis and
18, 19
progression.

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Diarylheterocycles is the most widely explored pharmacophore for the
development of selective COXꢀ2 inhibitors, such as Celecoxib (1), Rofecoxib (2),
20
Valdecoxib (3), Etoricoxib (4). Extensive structureꢀactivity relationship (SAR)
studies for the diarylheterocycle class have shown that a SO NH or SO Me
substituent at the paraꢀposition of one of the aryl rings is a requirement for optimum
2
2
2
9, 21
COXꢀ2 selectivity and potency.
Furthermore, sulfonamides possess many types of
biological activities and representatives of this class of pharmacological agents are
22
23
24
widely used in clinic as antibacterial, antiꢀcarbonic anhydrase, antithyroid and
25
antitumor , devoid of the side effects of the presently available pharmacological
agents.
Celecoxib belongs to the 1,5ꢀdiarylpyrazole class of compounds and a recent study
26, 27
revealed that this class of compounds possesses promising anticancer properties,
Accordingly, in pursuance of our synthesis of various 1,5ꢀdiarylpyrazoles
heterocycles, we preferred to possess a diarylheterocyclic ring template with a central
fiveꢀmembered pyrazole ring over others for the development of novel class of
COXꢀ2 inhibitors. Acylhydrazones had been selected as pharmacological agents due
to their wideꢀranging pharmacological properties and their potential applications of
28
anticancer. The acylhydrazones group attached to 1,5ꢀdiarylpyrazoles as a
pharmacophore to explore the possibility of better antitumor potency of COXꢀ2
inhibitors.
In a preceding work, we have reported the decent inhibitory activity against
COXꢀ1/COXꢀ2 of a series of dihydropyrazole sulfonamide derivatives as a part of our
29
studies on the design and synthesis of inhibitors of COXs.
Here we extend the
earlier work of dihydropyrazole incorporating sulfonamide moieties, and now
describing the structureꢀbased design and synthesis of novel and potent inhibitors of
COXꢀ2
utilizing
a
1,5ꢀdiarylpyrazoles,
benzenesulfonamide
and
phenylacetohydrazide as new structural classes.
Thus, on the basis of this finding, we designed 44 novel benzenesulfonamideꢀ
substituted 1,5ꢀdiarylpyrazoles containing phenylacetohydrazide derivatives (21-64)
that showed significant selective COXꢀ2 inhibition as a source for new effective and

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safe antitumor drugs.
2
2
Results and Discussion
.1 Chemistry
The routes to synthesis the benzenesulfonamideꢀsubstituted 1,5ꢀdiarylpyrazoles
containing phenylacetohydrazide derivatives followed the general pathway outlined in
Scheme 1. The starting diverse substituted chalcones (6-10) were synthesized by
treating dimethyl oxalate and the substituted acetophenone in the presence of excess
sodium methoxide as catalyst in methanol. Then the reaction of cyclization of
different chalcones with 4ꢀhydrazinylbenzenesulfonamide in refluxing ethanol,
afforded the formation of benzenesulfonamideꢀsubstituted 1,5ꢀdiarylpyrazoles
containing 3ꢀcarboxylate skeleton (11-15). compounds 11-15 and the 85% hydrazine
monohydrate were dissolved in anhydrous ethanol and vigorously stirred at 80 ℃
under oil bath for 7ꢀ8 h to obtain compounds 16-20. Finally, Target compounds 21-64
were obtained from compounds 16-20 after reactions with various benzaldehyde in 70％
~
90％yield. All the compounds are reported for the first time. All of the target
1
13
compounds gave satisfactory analytical and spectroscopic data H NMR, C NMR,
ESIꢀMS, which are in accordance with their depicted structures. Furthermore, the
crystal structures of compounds 22 (CCDC: 1434485) and 40 (CCDC: 1434486) were
determined by single crystal Xꢀray diffraction analysis in Figure 2 and Table 1.
2
2
.2 Biological activity
.2.1 Antiproliferation inhibition assay
All synthesized derivatives (21-64) were evaluated for their in vitro
antiproliferation activities against four cancer cell lines, which were HepG2, A549,
HeLa, F10, with the positive contrast drugs Celecoxib. The outcomes were
summarized up in Table 2 and Table 3. As shown in Table 2 and Table 3, the results
revealed indicated that most of the target compounds can effectively inhibit the
proliferation of the four tumor cells. Especially on A549 cell antiproliferative
activities. The IC50 values of compounds 21-64 against A549 cell lines were between

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0
.78 and 35.34 µM and GI50 values were range from 0.32 to 21.55 µM. Among them,
compound 48 displayed the most potent antiꢀtumor activity in A549 with IC of 0.78
50
,
ꢀM and GI50 of 0.32 ꢀM compared to the positive control Celecoxib (IC50 =16.22 ꢀM,
GI50=10.56 ꢀM,). More potent antiꢀtumor activity for A549 together with virtual
screening results both indicated that the COXꢀ2 might be a potential target which
these
benzenesulfonamideꢀsubstituted
1,5ꢀdiarylpyrazoles
containing
phenylacetohydrazide derivatives interacted with.
2
.3.2 Enzyme inhibition assay
Compounds 21-64 were evaluated for their ability to inhibit COXꢀ1 and COXꢀ2
using human COXꢀ1/COXꢀ2 ELISA Kit and results obtained are presented in Table 3.
The results were compared with Celecoxib under identical conditions, which showed
that the majority of the synthesized compounds exhibited excellent inhibiting COXꢀ2
activities displaying IC50 values between 0.37 and 23.88 ꢀΜ, but weak to COXꢀ1.
The following structureꢀactivity relationship (SAR) studies were performed by
modification of the parent compound to determine how the substituents of the
subunits affect the COXꢀ2 inhibitory activities. Inspection of the chemical structures
of compounds 21-64 suggested that they could be divided into two subunits: A and B
rings (Scheme 1). As showed in Table 4, compound 48 was the most active and
potent selectivity with IC50 value of 0.37 ꢀM, 82.21 ꢀM which had basically similar
cyclooxygenase inhibition capability with the positive control drug Celecoxib with
IC value of 0.15 ꢀM, 40.29 ꢀM. We found that OH at position of B rings substituent
5
2
0
ꢀ
R (orthoꢀposition) possess potent antiꢀcancer effect and favorable inhibition of
1
COXꢀ2, when the substituent ꢀR is kind of halogen groups (Br, F), the compounds
gained better activity than CH , OCH , as shown in inhibition of COXꢀ2: 48
3
3
(
IC50=0.37µM) >46 (IC50=0.40 µM) >42 (IC50=0.56 µM)>45 (IC50=1.29 µM) and in
A549 cell antiproliferative activities: 48 (IC50=0.78 µM) >46 (IC50=0.86 µM) >45
IC50=1.87 µM) >42 (IC50=3.13 µM). Based on the obtained data, we also simplified
(
modifying merely substituents on the paraꢀposition of A rings and B rings. When
1
substituents Bꢀring kept immutability, the changes with different substituents ꢀR for

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COXꢀ2 inhibition activity at paraꢀposition of Aꢀring showed electronꢀ withdrawing
groups are preferable than electronꢀdonating groups. For instance, the active gradients
for compounds were 26 (IC50=8.39 µM) >27 (IC50=9.63 µM) >22 (IC50=14.79 µM),
2
5 (IC50=16.21 µM); 55 (IC50=1.17 µM) >56 (IC50=1.62 µM) >54 (IC50=17.46 µM).
In addition, we studied the same A ring and different substituents of B ring, it can be
deduced that the substituent on the paraꢀposition of the A rings is kind of potent
electronꢀwithdrawing groups rather than electronꢀdonating groups, the compounds
gained better activity, as shown in inhibition of COXꢀ2: 56 (IC50=1.62µM) >51
(
(
(
(
IC50=3.20 µM) >64 (IC50=4.58 µM)>60 (IC50=6.33 µM) >47 (IC50=7.30 µM) >27
IC50=9.63 µM)>40 (IC50=16.10 µM); 55 (IC50=1.17µM) >63 (IC50=3.78 µM) >59
IC50=5.21 µM)>26 (IC50=8.39 µM) >39 (IC50=16.72 µM); 52 (IC50=3.63µM) >49
IC =8.16 µM) >57 (IC =10.54 µM) >22 (IC =14.79 µM) >21 (IC =16.41
5
0
50
50
50
µM) >37 (IC50=17.22 µM); and the inhibitory activities increased in the following
order: NO >F > Cl > Br > OCH > CH
2
3
3
From the above mentioned analysis, it could be indicated that hydroxy at
orthoꢀposition of B ring play an important role in the COXꢀ2 inhibitory activity which
conformed to the docking of the hydroxy in compound 48 bonding to COXꢀ2 through
two hydrogen bonds. Moreover, modifying substituents on the paraꢀposition of A
rings and B rings revealed that bonds A ring and B ring in the
benzenesulfonamideꢀsubstituted
1,5ꢀdiarylpyrazoles
containing
phenylacetoꢀ
hydrazide derivatives also crucial of the COXs inhibitory activity.
In comparison, we found that these compounds with OH at position of B rings
2
substituent ꢀR (orthoꢀposition) possess remarkable inhibitory activity, with
electronꢀwithdrawing groups group on the paraꢀposition of the Aꢀring and Bꢀring
(
such as NO , F), exhibited more potent anticancer activities than those have
2
3
electronꢀdonating substituents (such as OCH3, CH ). From the aboveꢀmentioned
analysis, it could be concluded that the compounds with nitro and fluorine substituted
paraꢀposition of Aꢀring and Bꢀring were found to be the most favorable for the
anticancer activity.
2
.3.3 Apoptosis assay

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Subsequently, we investigated the effect of compound 48 on cell apoptosis. We
treated A549 cells with various concentrations of compound 48 and for 48 h and
analyzed cells for changes in apoptotic markers with a flow cytometer in vitro, as
shown in Figure 3, the percentage of apoptotic cell significantly increased after
treatment with high doses of compound 48. The percentages of cell apoptosis 6.55%,
1
2.95%, 16.23%, 27.70%, 61.46% were responding to the concentration of compound
4
8 0, 2, 4, 8, 16 ꢀM, respectively. We also measured the effect of other five
compounds (compounds 26, 40, 44, 45, 56) causing A549 cells apoptosis and results
displayed in Figure 4, compound 26 inducing the percentage of cell apoptosis 5.67％,
1
0.55％, 14.33％, 23.77％, 38.2％ was corresponding to the concentration 0, 2, 4,
, 16 ꢀM. Compound 40 (1.58％, 3.76％, 6.94％, 10.04％, 18.14％), compound 44
8
(5.67％, 11.92％, 16.17％, 21.29％, 25.34％), compound 45 (5.67％, 8.94％,
1
5.12％, 25.23％, 47.8％), compound 56 (5.67％, 12.15％, 17.41％, 31.5％, 55.87％)
separately inducing the cells apoptosis in identical concentration gradient. On the
basis of these data, these compounds which possess highꢀefficiency COXꢀ2 inhibition
activity also causing high percentages of cell apoptosis. IC50 of inducing A549 cells
apoptosis were obtained for the six compounds on the Y axis versus the corresponding
IC50 of COXꢀ2 inhibition on the X axis to create liner regression model: compound 26
(
IC of COXꢀ2 inhibition 8.39 ꢀM，IC of inducing A549 cells apoptosis 30.07 ꢀM);
50 50
compound 40 (16.10 ꢀM, 104.45 ꢀM); compound 44 (23.88 ꢀM, 174.45 ꢀM);
compound 45 (1.29 ꢀM, 18.65 ꢀM); compound 48 (0.37 ꢀM, 13.24 ꢀM); compound
2
5
6 (1.62 ꢀM, 14.22 ꢀM). As displayed in Figure 5, the correlation coefficient R
between six compounds respective IC50 of COXꢀ2 inhibition and IC50 of inducing
A549 cells apoptosis was found to be 0.948, which proved that cell apoptosis was
closely associated with COXꢀ2 inhibition activity.
2
.3.4 Cytotoxicity test
All the target compounds were evaluated for their toxicity against human kidney
epithelial cell 293T and mouse primary hepatocytes with the median cytotoxic
concentration (CC50) data of tested compounds by the MTT assay. As shown in Table

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5
, 44 compounds with the minimum CC50 of 293T cells and mouse primary
hepatocytes were 24.81 ꢀM and 43.29 ꢀM that demonstrated almost no cytotoxic
activities in vitro against human kidney epithelial cell 293T and mouse primary
hepatocytes.
2
.4 Molecular docking
To better understand the potency of compound 48 and further guide
structureꢀactivity relationship (SAR) studies, we examined the interaction of
compound 48 and Celecoxib with COXꢀ2 (PDB code: 3PGH). All amino acid
residues of COXꢀ2 that interacted with compound 48 and Celecoxib were exhibited in
pictures Figure 6, Figure 7, Figure 8 and Figure 9. The estimated interaction
energies of synthesized compounds were ranging from ꢀ41.78 to ꢀ56.84 kcal/mol, as
displayed in Figure 6 and Figure 7. In the binding mode, Celecoxib binds to COXꢀ2
with binding free energy of ꢀ49.72 kcal/mol via two hydrogen bonds with TRP387,
two Pi bonds and a SigmaꢀPi bond with HIS207. However, compound 48 had a best
estimated binding free energy of ꢀ56.84 kcal/mol and bound well to COXꢀ2 through
four hydrogen bonds, a SigmaꢀPi bond and Pi bonds. As shown, five amino acids,
ALA199, His386, GLN454, GLN203 and HIS207 were of significance in the
binding of ligand with enzyme. The ALA199 formed two hydrogen bonds with 48
(angle O · HꢀN = 105.17°, distance = 2.11 Å, angle O · HꢀN = 100.96°, distance =
2
.17 Å). Furthermore, compound 48 was also bonded with His386 (angle H · OꢀH =
47.68°, distance = 2.02 Å) and GLN454 (angle O · HꢀO = 151.20°, distance = 2.02
1
Å) by a hydrogen and GLN203 (distance = 2.49 Å) and HIS207 (distance = 3.36 Å)
by a SigmaꢀPi and Pi bond. Thus it can be concluded the sulfanilamide, pyrazole ring
and hydroxyl group are vital for their activities. According to the above, these
molecular docking results along with the biological assay data suggested that
compound 48 might be a potential inhibitor of the COXꢀ2.
2
.5 3D-QSAR model
In order to evaluate the synthesized compounds as COXꢀ2 inhibitors

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comprehensively and explore more powerful and selective inhibitors, 44 compounds
with definite IC values against COXꢀ2 were selected as the model dataset by using
50
the Create 3D QSAR protocol of Discovery Studio 3.5, all the molecular converted to
the active conformation and corresponding pIC50 (µM) values, which were converted
from the obtained IC (µM) values of COXꢀ2 inhibition. 33 compounds were chosen
5
0
as training set and rest of compounds comprising a test set.as shown in Table 6.
By default, the alignment conformation of each molecule possessed the lowest
CDOCKER_INTERACTION_ENENGY among all of the docked poses. The critical
regions (steric or electrostatic) affecting the binding affinity was gained by this
3
DꢀQSAR model. Exerting CHARMM force filed and PLS regression. The model
2
was set up with correlation coefficient R of 0.926, which indicated that this model
possessed pretty good predicting capability. Predicted pIC values and residual errors
50
for the 44 compounds by this QSAR model are given in Table 6. The good agreement
between predicted and experimental pIC50 values for both test sets and training sets
were shown in Figure 10. The molecules aligned with the isoꢀsurfaces of the
3
DꢀQSAR model coefficients on van der Waals grids and electrostatic potential grids
were also listed in Figure 11. Electrostatic map indicates red contours around regions
where high electron density (negative charge) is expected to increase activity, and
blue contours represent areas where low electron density (partial positive charge) is
expected to increase activity. Similarly, steric map indicates areas here steric bulk is
predicted to increase (green) or decrease (yellow) activity. According to the maps，A
rings and B rings were mainly surrounded by red contours, it implied the compound
with high negative charged would show higher activity, validating that most NO
2
, Br,
F substituent being a better choice than CH , OCH . Big steric bulk substituents were
3
3
unfit for A rings and B rings (surrounded by multiple yellow contours), demonstrating
that most compounds with small substituents possess better activity. This model was
accordant with the actual situation for potent compounds. Thus, this promising model
would provide a direction to design and optimize more effective COXꢀ2 inhibitors
and pave the way for the further study. It can be predicted that greater
electronegativity substituents and decreasing steric bulk might enhance the COXꢀ2

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inhibitory activity.
Conclusion
In this study, a series of novel COXꢀ2 inhibitors has been designed and synthesized;
3
their biological activities have been evaluated which suggest these compounds
possess moderate to potent antiproliferative activities against A549 cells, HepG2,
HeLa, F10 cells and COXꢀ2 inhibitory activities. According to analyzed the data of
COXꢀ2 inhibition and A549 cell apoptosis, we could find cell apoptosis was closely
associated with COXꢀ2 inhibition activity. Among these compounds, compound 48
showed the most powerful antiproliferative activity (IC50 = 0.78 µM GI50 = 0.32 µM
for A549 cell) and COXꢀ2 inhibitory activity (IC50 =0.37 µM). In addition, compound
4
8 can induce cancer cell apoptosis effectively and employing mouse primary
hepatocytes and human kidney epithelial cell 293T also indicates high safety. The
probable binding mode proposed by the docking simulation may be a good
explanation of the impressive performance of compound 48, in which 48 binds well
with COXꢀ2 through four hydrogen bonds, SigmaꢀPi and Pi bond with best estimated
binding free energy of ꢀ56.84 kcal/mol. It can be concluded that compound 48 as well
as the other benzenesulfonamideꢀsubstituted 1,5ꢀdiarylpyrazoles containing
phenylacetohydrazide derivatives are promising leads for further study as potential
anticancer agents. Moreover, A QSAR model was built to provide a reliable tool for
the rational design of novel COXꢀ2 inhibitors in the future.
4
4
Experiments
.1 Materials and measurements
1
All chemicals and reagents used in current study were analytical grade. All the H
NMR spectra were recorded on a Bruker DPX 400 model Spectrometer in DMSOꢀd
6
and chemical shifts (δ) were reported as parts per million (ppm) Elemental analyses
were performed using a CHNꢀOꢀRapid instrument and were within 0.4% of the
theoretical values. ESIꢀMS specra were recorded by Mariner System 5304 Mass
spectrometer. Melting points (uncorrected) were determined on an XT4 MP apparatus
(Taike Corp, Beijing, China). Thin layer chromatography (TLC) was performed on
silica gel plates (Silica Gel 60 GF254) and visualized in UV light (254nm).

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3
ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolium (MTT) were purchased
from Beyotime Institute of Biotechnology (Haimen, China). AnnexcinVꢀFITC cell
apoptosis assay kit (#BA11100) was purchased from BIOꢀBOX (Nanjing, China)
.
Human COXꢀ1/COXꢀ2 ELISA Kits were purchased from Cayman Chemical (catalog
number 701070/701080). SD mouse were provided by Laboratory Animal Center in
School of Life Science, Nanjing University.
4
.2 General procedure for the synthesis of compounds (6-10)
Dimethyl oxalate (100 mmol) was added to a stirred suspension of sodium
methoxide (200 mmol) in methanol (100 mL) at 0 °C. Then, a solution of
acetophenone (50 mmol) in methanol (50 mL) was added dropwise over 5 min, the
mixture was heated at reflux for 6 h. After cooling to room temperature, the solvent
was removed and the residue was taken up in water and acidified with concentrated
−1
hydrochloric acid (1 mol L ) to pH=3. The heavy precipitate formed was filtered off,
washed with cold enthanol (50 mL) for three times, and dried in vacuo. The crude
products were purified by recrystallization with ethanol, ethyl acetate and petroleum
ether (1:1:0.5) washed by iceꢀwater for three times to give pure intermediate products
(6-10).
4
.3 General procedure for the synthesis of compounds (11-15)
A mixture of 6-10 (15 mmol) and 4ꢀhydrazinylbenzenesulfonamide (15 mmol) in
methanol (40 mL) was heated at reflux for 6 h. After cooling to room temperature, the
precipitate was filtered and washed with enthanol, The solid compounds (11-15) were
crystallized from ethyl acetate, filtered and dried with Na SO .
2
4
4
.4 General procedure for the synthesis of compounds (16-20)
Hydrazine hydrate (120 mmol) were successively added to a solution of
compounds 11-15 (12 mmol) in enthanol and the mixture was stirred at 80 °C from
several hours to overnight. After being cooled, the mixture put into brine to obtain the
target compounds. Then precipitated solid was collected by filtration and washed with
2
H O and cold enthanol to afford desired compounds (16-20).

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4
.5 General procedure for the synthesis of compounds (21-64)
Compounds 16-20 (1 mmol), various benzaldehyde(1.5 mmol) and acetic acid(0.5 mL)
were dissolved in enthanol and stirred at room temperature for 12h. The precipitate
solid was collected by filtration and washed with enthanol and H
2
O. The crude
products were purified by recrystallization to obtain the target compounds (21-64).
4
.5.1 4-(3-(2-Benzylidenehydrazine-1-carbonyl)-5-(4-methoxyphenyl)-1H-
pyrazol-1-yl)benzenesulfonamide (21). White solid, yield 71.0%, m.p. 251~253 ˚C;
1
6
H NMR (DMSOꢀd , 400 MHz) δ: 11.81 (s, 1H, CONH), 8.55 (s, 1H, CHN), 7.90 (d,
J = 8.4 Hz, 2H, ArH), 7.72 (d, J = 6.8 Hz, 2H, ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH ),
.48 (t, J = 8.4 Hz, 5H, ArH and SO NH ), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (s, 1H,
7
2
2
+
13
CH), 6.98 (d, J = 8.5 Hz, 2H, ArH), 3.78 (s, 3H, OCH ). ESIꢀMS: 476.1 [M+H] . C
3
NMR δ: 160.20, 157.91, 148.56, 147.02, 145.09, 144.06, 142.13, 134.81, 130.67,
1
30.57, 129.34, 127.57, 127.17, 126.41, 121.50, 114.78, 108.86, 55.73. Anal. Calcd
for C H N O S: C, 60.62; H, 4.45; N, 14.73. Found: 60.43; H, 4.46; N, 14.76.
2
4
21
5
4
4
.5.2 4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)
-
˚
(
1H-pyrazol-1-yl)benzenesulfonamide (22). White solid, yield 70.6%, m.p. 169~171
1
C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.66 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.89
t, J = 8.5 Hz, 2H, ArH), 7.66 (d, J = 8.6 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH),
7
.49 (s, 2H, SO
2
NH
2
), 7.26 (d, J = 8.6 Hz, 2H, ArH), 7.10 (s, 1H, CH), 7.03 (d, J =
), 3.78 (s, 3H,
8
.6 Hz, 2H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH
3
1
3
OCH ). C NMR δ: 160.20, 157.95, 148.56, 147.02, 145.13, 144.06, 142.13, 134.81,
3
13
1
30.66, 129.34, 127.57, 127.17, 126.41, 121.50, 119.74, 114.78, 108.86, 55.73.
C
NMR δ: 160.18, 157.89, 148.57, 147.12, 145.16, 144.06, 143.30, 134.82, 129.37,
1
27.47, 127.17, 126.46, 121.50, 119.76, 114.77, 108.86, 58.69, 55.76. ESIꢀMS: 506.1
+
[
M+H] . Anal. Calcd for C25
H
23
N
5
O
5
S: C, 59.40; H, 4.59; N, 13.85. Found: C, 59.26;
H, 4.60; N, 13.89.
4
.5.3 4-(3-(2-(2-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)
-1H-pyrazol-1-yl)benzenesulfonamide (23). White solid, yield 82.6%, m.p. 157~160

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DOI: 10.1039/C6RA02168A
1
˚
C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.86 (s, 1H, CONH), 8.89 (s, 1H, CHN), 7.89
(
t, J = 8.5 Hz, 3H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.43 (t,
2
2
J = 8.5 Hz,1H, ArH), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (d, 2H, CH and ArH), 7.04 (t,
J = 7.4 Hz, 1H, ArH), 6.99 (d, J = 8.6 Hz, 2H, ArH), 3.87 (s, 3H, OCH ), 3.79 (s, 3H,
OCH ). C NMR δ: 160.12, 157.87, 148.57, 147.16, 145.15, 144.07, 143.31, 134.83,
3
1
3
3
1
29.38, 127.46, 127.14, 126.465, 121.51, 119.73, 114.87, 108.89, 58.67, 55.72.
+
ESIꢀMS: 506.1 [M+H] . Anal. Calcd for C25
23 5 5
H N O S: C, 59.40; H, 4.59; N, 13.85.
Found: C, 59.52; H, 4.62; N, 13.87.
4
.5.4 4-(3-(2-(3-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)
-1H-pyrazol-1-yl)benzenesulfonamide (24). Yellow solid, yield 78.0%, m.p.
1
1
50~152 ˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.81 (s, 1H, CONH), 8.52 (s, 1H,
6
CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H,
SO NH ), 7.39 (t, J = 8.0 Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 4H, ArH), 7.11 (s, 1H,
CH), 7.04~7.01 (m, 1H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH3),
2
2
1
3
3
1
5
4
3
.78 (s, 3H, OCH ). C NMR δ: 160.21, 158.86, 148.52, 147.32, 145.18, 144.09,
43.30, 134.80, 129.39, 127.48, 127.18, 126.47, 121.52, 119.77, 114.74, 108.86,
+
7.63, 55.74. ESIꢀMS: 506.1 [M+H] . Anal. Calcd for C25
23 5 5
H N O S: C, 59.40; H,
.59; N, 13.85. Found: C, 59.32; H, 4.60; N, 13.87.
4
.5.5
4-(3-(2-(4-Methoxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-
pyrazol-1-yl)benzenesulfonamide (25). White solid, yield 75.3%, m.p. 267~268 ˚C;
1
6
H NMR (DMSOꢀd , 400 MHz) δ: 11.68 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.89 (d,
J = 8.5 Hz, 2H, ArH), 7.67 (d, J = 8.6 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH),
.50 (s, 2H, SO NH ), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.13 (s, 1H, CH), 7.04 (d, J = 8.6
7
2
2
13
Hz, 2H, ArH), 3.82 (s, 3H, OCH
3
), 2.33 (s, 3H, CH
3
). C NMR δ: 159.92, 157.60,
1
1
48.88, 147.30, 145.17, 144.10, 142.11, 139.19, 129.92, 129.36, 129.12, 128.15,
+
26.31, 126.32, 125.72, 116.29, 109.06, 55.73, 21.29. ESIꢀMS: 490.1 [M+H] . Anal.
Calcd for C25
4.34.
23 5 5
H N O
S: C, 61.34; H, 4.74; N, 14.31. Found: C, 61.19; H, 4.75; N,
1

Page 15 of 50
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DOI: 10.1039/C6RA02168A
4
1
.5.6 4-(5-(4-Fluorophenyl)-3-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (26). White solid, yield 73.8%, m.p. 185~187
1
˚
C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.70 (s, 1H, CONH), 8.48 (s, 1H, CHN),
7
7
1
1
1
.91~7.87 (m, 2H, ArH), 7.67 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH),
.50 (s, 2H, SO NH ), 7.42~7.39 (m, 2H, ArH), 7.28 (t, J = 8.5 Hz, 2H, ArH), 7.18 (s,
2
2
13
3
H, CH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H, OCH ). C NMR δ: 158.85,
47.43, 147.10, 145.24, 144.14, 142.08, 139.26, 131.41, 129.92, 129.82, 129.71,
+
28.51, 127.20, 126.43, 116.62, 116.32, 109.16, 54.93. ESIꢀMS: 494.1 [M+H] . Anal.
23 5 4
Calcd for C25H N O S: C, 58.41; H, 4.08; N, 14.19. Found: C, 58.36; H, 4.12; N,
1
4.20.
4
1
.5.7 4-(5-(4-Bromophenyl)-3-(2-(4-methoxybenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (27). White solid, yield 80.3%, m.p. 264~267
1
˚
C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.75 (s, 1H, CONH), 8.48 (s, 1H, CHN), 7.91
(
t, J = 8.5 Hz, 2H, ArH), 7.67~7.60 (m, 6H, ArH), 7.52 (s, 2H, SO NH ), 7.29 (d, J =
2 2
8
.5 Hz, 2H, ArH), 7.23 (s, 1H, CH), 7.04 (d, J = 8.6 Hz, 2H, ArH), 3.82 (s, 3H,
1
3
3
OCH ). C NMR δ: 159.76, 157.92, 148.26, 147.02, 145.14, 144.09, 142.13, 134.89,
1
30.67, 129.36, 127.56, 127.19, 126.43, 121.53, 119.76, 114.79, 108.85, 55.74.
+
ESIꢀMS: 555.0 [M+H] . Anal. Calcd for C24
5 4
H20BrN O S: C, 51.99; H, 3.64; N, 12.63.
Found: C, 51.89; H, 3.62; N, 12.68.
4
.5.8
4-(5-(4-Methoxyphenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (28). Yellow solid, yield 71.3%,
1
m.p. 254~256 ˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.71 (s, 1H, CONH), 8.81 (s,
1
H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.4 Hz, 2H, ArH), 7.50 (s, 2H,
SO NH ), 7.37 (s, 1H, ArH), 7.27 (d, J = 8.5 Hz, 2H, ArH), 7.09 (s, 1H, CH), 6.98 (d,
J = 8.5 Hz, 2H, ArH), 6.76 (s, 1H, ArH ), 3.87 (s, 6H, OCH
NMR δ: 160.19, 157.60, 153.91, 152.50, 147.19, 144.88, 144.22, 143.88, 143.69,
2
2
1
3
3
3
), 3.78 (s, 6H, OCH ). C
1
5
42.15, 130.64, 127.12, 126.15, 121.61, 114.78, 114.12, 108.84, 108.11, 98.41, 57.05,
+
6.38, 56.26, 55.72. ESIꢀMS: 566.1 [M+H] . Anal. Calcd for C27
H
27
N
5
O
7
S: C, 57.34;

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DOI: 10.1039/C6RA02168A
H, 4.81; N, 12.38. Found: C, 57.46; H, 4.82; N, 12.42.
4
.5.9
4-(5-(4-Methoxyphenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (29). White solid, yield 70.8%,
1
m.p. 274~276 ˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.79 (s, 1H, CONH), 8.47 (s,
1
H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.4 Hz, 2H, ArH), 7.50 (s, 2H,
SO NH ), 7.27 (d, J = 8.5 Hz, 2H, ArH), 7.11 (s, 1H, CH), 6.99 (t, J = 6.4 Hz, 4H,
ArH), 3.85 (s, 6H, OCH
NMR δ: 157.70, 153.68, 148.73, 147.23, 144.28, 144.03, 141.75, 139.74, 132.31,
2
2
13
13
3
3 3
), 3.78 (s, 3H, OCH ), 3.72 (s, 3H, OCH ). C NMR δ: C
1
31.30, 130.27, 128.55, 127.28, 126.51, 123.17, 109.67, 104.77, 60.61, 58.47, 56.45.
+
ESIꢀMS: 566.1 [M+H] . Anal. Calcd for C27
27 5 7
H N O S: C, 57.34; H, 4.81; N, 12.38.
Found: C, 57.47; H, 4.83; N, 12.40.
4
.5.10
4-(5-(p-Tolyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)
-1H-pyrazol-1-yl)benzenesulfonamide (30). White solid, yield 80.9%, m.p. 171~173
1
˚
C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.72 (s, 1H, CONH), 8.81 (s, 1H, CHN), 7.89
(
d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.37 (s,
2 2
1
6
1
1
H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.12 (s, 1H, CH), 6.76 (s, 1H, ArH), 3.87 (s,
13
3 3 3
H, OCH ), 3.78 (s, 3H, OCH ), 2.33 (s, 3H, CH ). C NMR δ: 159.67, 157.62,
53.78, 152.51, 147.21, 144.89, 144.24, 143.89, 142.89, 142.17, 130.66, 127.14,
26.16, 121.64, 114.79, 114.06, 108.86, 106.22, 98.46, 56.40, 56.28, 55.73, 21.27.
+
ESIꢀMS: 550.1 [M+H] . Anal. Calcd for C27
27 5 6
H N O S: C, 59.01; H, 4.95; N, 12.74.
Found: C, 58.89; H, 4.93; N, 12.72.
4
.5.11 4-(5-(p-Tolyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-carbonyl)-1H
-pyrazol-1-yl)benzenesulfonamide (31). White solid, yield 74.9%, m.p. 304~307 ˚C;
1
6
H NMR (DMSOꢀd , 400 MHz) δ: 11.80 (s, 1H, CONH), 8.47 (s, 1H, CHN), 7.89 (d,
J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.23 (t, J
2
2
=
8.8 Hz, 4H, ArH), 7.14 (s, 1H, CH), 7.00 (s, 2H, ArH), 3.85 (s, 6H, OCH
3
), 3.72 (s,
1
3
3
1
1
H, OCH ), 2.33 (s, 3H, CH ). C NMR δ: 159.41, 154.63, 148.76, 147.43, 144.29,
3 3
44.13, 141.79, 139.73, 132.51, 131.32, 130.47, 128.59, 126.98, 126.53, 123.19,
+
09.66, 104.78, 60.63, 56.46, 21.28. ESIꢀMS: 550.1 [M+H] . Anal. Calcd for
27 27 5 6
C H N O S: C, 59.01; H, 4.95; N, 12.74. Found: C, 58.96; H, 4.93; N, 12.70.

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DOI: 10.1039/C6RA02168A
4
.5.12
4-(5-(4-Fluorophenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (32). Yellow solid, yield 80.5%,
1
m.p. 269~270˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.74 (s, 1H, CONH), 8.81 (s,
1
H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H,
SO NH ), 7.42~7.32 (m, 3H, ArH), 7.31~7.26 (m, 2H, ArH), 7.17 (s, 1H, CH), 6.76 (s,
2
2
1
3
1
1
1
5
5
4
3 3
H, ArH), 3.87 (s, 6H, OCH ) , 3.78 (s, 3H, OCH ). C NMR δ: 159.68, 157.42,
53.61, 152.30, 147.17, 144.84, 144.24, 143.56, 143.61, 142.02, 130.04, 127.11,
26.09, 120.67, 114.76, 114.10, 108.82, 108.02, 98.37, 56.32, 56.24, 55.73. ESIꢀMS:
+
54.1 [M+H] . Anal. Calcd for C26
5 6
H24FN O S: C, 56.41; H, 4.37; N, 12.65. Found: C,
6.38; H, 4.40; N, 12.42.
.5.13
4-(5-(4-Fluorophenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (33). White solid, yield 73.5%,
1
m.p. 259~262˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.47 (s,
1
H, CHN), 7.90 (d, J = 8.6 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H,
SO NH ), 7.43~7.39 (m, 2H, ArH), 7.29 (t, J = 8.5 Hz, 2H, ArH), 7.19 (s, 1H, CH),
2
2
1
3
7
1
1
3 3
.01 (s, 2H, ArH), 3.86 (s, 6H, OCH ) , 3.72 (s, 3H, OCH ). C NMR δ: 158.83,
54.25, 148.76, 147.26, 144.48, 144.05, 142.85, 139.76, 132.21, 131.36, 130.27,
+
28.57, 127.26, 126.53, 123.18, 109.67, 104.78, 60.62, 56.46. ESIꢀMS: 554.1 [M+H] .
Anal. Calcd for C26
N, 12.57.
5 6
H24FN O S: C, 56.41; H, 4.37; N, 12.65. Found: C, 56.28; H, 4.36;
4
.5.14
4-(5-(4-Bromophenyl)-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1
-carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (34). White solid, yield 84.1%,
1
m.p. 180~182˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.78 (s, 1H, CONH), 8.81 (s,
1
H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.65~7.59 (m, 4H, ArH), 7.52 (s, 2H,
SO NH ), 7.36 (s, 1H, ArH), 7.29 (d, J = 8.5 Hz, 2H, ArH), 7.22 (s, 1H, CH), 6.76 (s,
2
2
1
3
1
1
1
6
3 3
H, ArH), 3.86 (s, 6H, OCH ), 3.78 (s, 3H, OCH ). C NMR δ: 159.64, 158.26,
53.60, 152.31, 147.18, 145.04, 144.16, 143.52, 143.60, 142.01, 130.02, 127.06,
26.07, 120.60, 114.73, 114.12, 108.82, 108.43, 97.67, 56.31, 56.18, 55.72. ESIꢀMS:
+
15.1 [M+H] . Anal. Calcd for C26
H24BrN
5
O
6
S: C, 50.82; H, 3.94; N, 11.40. Found:
C, 50.72; H, 3.92; N, 11.36.

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DOI: 10.1039/C6RA02168A
4
.5.15
4-(5-(4-Bromophenyl)-3-(2-(3,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (35). White solid, yield 75.4%,
1
m.p. 286~288˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.87 (s, 1H, CONH), 8.46 (s,
1
H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.65~7.60 (m, 4H, ArH), 7.52 (s, 2H,
SO NH ), 7.30 (d, J = 8.5 Hz, 2H, ArH), 7.24 (s, 1H, CH), 7.00 (s, 2H, ArH), 3.85 (s,
2
2
1
3
6
1
1
3 3
H, OCH ), 3.72 (s, 3H, OCH ). C NMR δ: 157.70, 153.68, 148.73, 147.23, 144.28,
44.03, 141.75, 139.74, 132.31, 131.30, 130.27, 128.55, 127.28, 126.51, 123.17,
+
09.67, 104.77, 60.61, 56.43. ESIꢀMS: 615.1 [M+H] . Anal. Calcd for
C
26
H
24BrN
5
O
6
S: C, 50.82; H, 3.94; N, 11.40. Found: C, 50.78; H, 3.92; N, 11.36.
4
.5.16
4-(5-Phenyl-3-(2-(2,4,5-trimethoxybenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (36). White solid, yield 81.4%,
1
m.p. 243~246˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.77 (s, 1H, CONH), 8.82 (s,
6
1
H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.52 (s, 2H,
SO NH ), 7.43 (t, J = 6.0 Hz, 3H, ArH), 7.37~7.34 (m, 3H, ArH), 7.18 (s, 1H, CH),
.76 (s, 1H, ArH), 3.87 (s, 6H, OCH ), 3.78 (s, 3H, OCH ). C NMR δ: 160.06,
2
2
1
3
6
1
1
3
3
59.23, 153.69, 152.42, 147.35, 145.16, 144.32, 143.87, 143.66, 142.06, 130.04,
27.19, 126.08, 120.62, 114.74, 114.14, 108.83, 108.46, 98.96, 56.34, 56.19, 55.74.
+
ESIꢀMS: 536.1 [M+H] . Anal. Calcd for C26
25 5 6
H N O S: C, 58.31; H, 4.71; N, 13.08.
Found: C, 58.16; H, 4.70; N, 13.12.
4
.5.17
4-(5-(4-Methoxyphenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (37). White solid, yield 76.7%,
1
m.p. 272~274˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.75 (s, 1H, CONH), 8.51 (s,
6
1
H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.61 (t, J = 7.6 Hz, 4H, ArH), 7.51 (s, 2H,
SO NH ), 7.28 (t, J = 7.6 Hz , 4H, ArH), 7.11 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H,
ArH), 3.78 (s, 3H, OCH ), 2.36 (s, 3H, CH ). C NMR δ: 159.61, 157.62, 148.68,
2
2
13
3
3
1
1
47.09, 145.07, 144.11, 142.17, 134.61, 130.64, 130.53, 129.35, 127.56, 127.14,
+
26.46, 121.52, 114.73, 108.89, 55.73, 23.26. ESIꢀMS: 490.1 [M+H] . Anal. Calcd
for C25H
23
N
5
O
4
S: C, 61.34; H, 4.74; N, 14.31. Found: C, 61.20; H, 4.75; N, 14.34.
4-(3-(2-(4-Methylbenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-
pyrazol-1-yl)benzenesulfonamide (38). White solid, yield 81.9%, m.p. 300~301 ˚C;
4
.5.18

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1
H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.75 (s, 1H, CONH), 8.51 (s, 1H, CHN), 7.90 (d,
J = 8.5 Hz, 2H, ArH), 7.63~7.58 (m, 4H, ArH), 7.50 (s, 2H, SO NH ), 7.29 (d, J = 8.0
2
2
Hz, 2H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.14 (s, 1H, CH), 2.36 (s, 3H, CH
3
), 2.33
). C NMR δ: 159.82, 157.64, 148.74, 147.25, 145.20, 144.09, 142.05,
39.16, 129.92, 129.33, 129.14, 127.13, 126.52, 126.41, 125.78, 116.19, 109.04,
13
(s, 3H, CH
3
1
2
4
4
+
3.47, 21.23. ESIꢀMS: 474.1 [M+H] . Anal. Calcd for C25
23 5 3
H N O S: C, 63.41; H,
.90; N, 14.79. Found: C, 63.28; H, 4.91; N, 14.82.
.5.19
4-(5-(4-Fluorophenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-
carbonyl)-1H-pyrazol-1-yl)benzenesulfonamide (39). White solid, yield 78.6%,
1
m.p. 253~256˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.77 (s, 1H, CONH), 8.51 (s,
1
H, CHN), 7.91~7.89 (m, 2H, ArH), 7.61 (t, J = 8.0 Hz, 4H, ArH), 7.50 (s, 2H,
SO NH ), 7.42~7.39 (m, 2H, ArH), 7.31~7.26 (m, 4H, ArH), 7.19 (s, 1H, CH), 2.36 (s,
2
2
1
3
3
1
2
1
4
3
H, CH ). C NMR δ: 159.03, 147.42, 146.72, 146.27, 144.19, 142.04, 138.26,
32.43, 129.90, 129.76, 129.67, 129.14, 127.16, 126.47, 116.53, 116.30, 109.12,
+
1.26. ESIꢀMS: 478.1 [M+H] . Anal. Calcd for C H FN O S: C, 60.37; H, 4.22; N,
24
20
5
3
4.67. Found: C, 60.26; H, 4.24; N, 14.69.
.5.20 4-(5-(4-Bromophenyl)-3-(2-(4-methylbenzylidene)hydrazine-1-carbonyl)
-1H-pyrazol-1-yl)benzenesulfonamide (40). White solid, yield 87.4%, m.p.
1
3
10~312˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.51 (s, 1H,
NH ),
). C NMR δ: 159.14,
CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.65~7.60 (m, 6H, ArH), 7.52 (s, 2H, SO
2
2
13
7
1
1
.31~7.28 (m, 4H, ArH), 7.24(s, 1H, CH), 2.36 (s, 3H, CH
3
47.46, 146.73, 146.29, 143.17, 142.08, 138.27, 132.47, 129.92, 129.77, 129.68,
+
29.15, 126.86, 126.23, 116.53, 116.31, 109.14, 21.24. ESIꢀMS: 539.0 [M+H] . Anal.
5 3
Calcd for C24H20BrN O S: C, 53.54; H, 3.74; N, 13.01. Found: C, 53.56; H, 3.73; N,
1
3.04.
4
.5.21
4-(3-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-
methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (41). White solid, yield
1
8
9
8.2%, m.p. 298~301˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.59 (s, 1H, CONH),
.92 (s, 1H, OH), 8.43 (s, 1H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60~7.54 (m, 4H,
NH ), 7.26 (d, J = 8.6 Hz, 2H, ArH), 7.09 (s, 1H, CH), 6.98 (d,
ArH), 7.50 (s, 2H, SO
2
2

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13
3
J = 8.6 Hz, 2H, ArH), 6.85 (d, J = 8.4 Hz, 2H, ArH), 3.78 (s, 3H, OCH ). C NMR δ:
1
1
59.67, 157.89, 157.71, 149.14, 146.63, 145.19, 144.20, 142.11, 131.88, 130.36,
27.17, 126.44, 121.45, 119.78, 116.92, 114.78, 108.93, 55.76. ESIꢀMS: 492.1
+
[
M+H] . Anal. Calcd for C24
H
21
N
5
O
5
S: C, 58.65; H, 4.31; N, 14.25. Found: C, 58.52;
H, 4.32; N, 14.27.
4
.5.22
4-(3-(2-(2-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-
methoxyphenyl) -1H-pyrazol-1-yl)benzenesulfonamide (42). White solid, yield
1
7
9.5%, m.p. 298~301˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.14 (s, 1H, CONH),
1
1.28 (s, 1H, OH), 8.74 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5
Hz, 4H, ArH), 7.50 (d, J = 4.5 Hz, 3H, ArH and SO NH ), 7.33~7.26 (m, 3H, ArH),
.13 (s, 1H, CH), 6.99~6.91 (m, 4H, ArH), 3.78 (s, 3H, OCH
2
2
1
3
7
1
1
4
5
4
4
3
). C NMR δ: 160.23,
57.93, 157.76, 149.14, 146.61, 145.17, 144.11, 142.10, 131.88, 130.68, 130.00,
27.18, 126.43, 121.45, 119.84, 119.20, 116.90, 114.79, 108.92, 55.74. ESIꢀMS:
+
92.1 [M+H] Anal. Calcd for C24
8.54 H, 4.32 N, 14.26.
.5.23
21 5 5
H N O S: C, 58.65; H, 4.31; N, 14.25. Found: C,
-(3-(2-(3-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)
-1H-pyrazol-1-yl)benzenesulfonamide (43). White solid, yield 71.4%, m.p.
1
2
92~294˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.75 (s, 1H, CONH), 9.62 (s, 1H,
OH), 8.45 (s, 1H, CHN), 7.90 (t, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH ),
.50 (s, 2H, SO NH ), 7.26 (t, J = 8.6 Hz, 3H, ArH), 7.20 (s, 1H, CH), 7.09 (t, J = 5.4
Hz, 2H, ArH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 6.84 (d, J = 8.6 Hz, 1H, ArH), 3.78 (s,
7
2
2
13
3
3
H, OCH ). C NMR δ: 160.24, 157.93, 157.77, 149.16, 146.63, 145.20, 143.87,
1
42.12, 131.88, 130.69, 130.04, 127.18, 126.46, 120.63, 119.82, 119.21, 116.93,
+
1
5
4
14.73, 108.92, 55.75. ESIꢀMS: 492.1 [M+H] . Anal. Calcd for C H N O S: C,
24
21
5
5
8.65; H, 4.31; N, 14.25. Found: C, 58.58 H, 4.32; N, 14.27.
.5.24 4-(3-(2-(4-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-
pyrazol-1-yl)benzenesulfonamide (44). White solid, yield 70.5%, m.p. 332~336˚C;
1
H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.60 (s, 1H, CONH), 9.92 (s, 1H, OH), 8.43 (s,
1
H, CHN), 7.89 (d, J = 8.5 Hz, 2H, ArH), 7.59~7.54 (m, 4H, ArH), 7.50 (s, 2H,

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2 2
SO NH ), 7.23 (t, J = 8.8 Hz, 4H, ArH), 7.12 (s, 1H, CH), 6.85 (d, J = 8.6 Hz, 2H,
1
3
ArH), 2.33 (s, 3H, CH ). C NMR δ: 159.90, 157.59, 148.86, 147.29, 145.15, 144.07,
3
1
1
4
4
42.09, 139.18, 129.90, 129.35, 129.10, 127.15, 126.50, 126.40, 125.78, 116.19,
+
09.02, 21.28. ESIꢀMS: 476.1 [M+H] . Anal. Calcd for C24
21 5 4
H N O S: C, 60.62; H,
.45; N, 14.73. Found: C, 60.52; H, 4.45; N, 14.74.
.5.25
4-(3-(2-(2-Hydroxybenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-
pyrazol-1-yl)benzenesulfonamide (45). White solid, yield 82.3%, m.p. 304~307˚C;
1
H NMR (DMSOꢀd
H, CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.6 Hz, 2H, ArH), 7.52 (s, 1H,
ArH), 7.50 (s, 2H, SO NH ), 7.34~7.29 (m, 1H, ArH), 7.23 (t, J = 8.8 Hz, 4H, ArH),
.16 (s, 1H, CH), 6.94 (t, J = 8.6 Hz, 2H, ArH), 2.33 (s, 3H, CH
6
, 400 MHz) δ: 12.16 (s, 1H, CONH), 11.27 (s, 1H, OH), 8.74 (s,
1
2
2
1
3
7
1
1
4
6
4
1
2
3
). C NMR δ: 159.87,
57.57, 148.84, 147.29, 145.16, 144.08, 143.18, 142.08, 139.19, 139.06, 129.92,
29.34, 129.12, 127.16, 126.50, 126.31, 125.78, 116.19, 108.62, 21.29. ESIꢀMS:
+
76.1 [M+H] . Anal. Calcd for C24
21 5 4
H N O S: C, 60.62; H, 4.45; N, 14.73. Found: C,
0.47; H, 4.46; N, 14.76.
.5.26 4-(5-(4-Fluorophenyl)-3-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (46). White solid, yield 72.9%, m.p.
1
56~257˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.17 (s, 1H, CONH), 11.26 (s, 1H,
OH), 8.75 (s, 1H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH),
7
3
1
1
2 2
.51 (d, J = 6.0 Hz, 3H, ArH and SO NH ), 7.43~7.39 (m, 2H, ArH), 7.32~7.27 (m,
13
H, ArH), 7.21 (s, 1H, CH), 6.94 (t, J = 8.6 Hz, 2H, ArH). C NMR δ: 157.94,
57.57, 149.25, 146.80, 144.34, 144.12, 141.73, 132.32, 131.92, 131.32, 129.96,
28.46, 127.31, 126.53, 123.21, 119.86, 119.19, 116.91, 109.74. ESIꢀMS: 480.1
+
[
M+H] . Anal. Calcd for C23
H
18FN
5
O
4
S: C, 57.61; H, 3.78; N, 14.61. Found: C, 57.71;
H, 3.80; N, 14.64.
4
1
3
.5.27 4-(5-(4-Bromophenyl)-3-(2-(4-hydroxybenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (47). White solid, yield 76.6%, m.p.
1
04~306˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.67 (s, 1H, CONH), 9.96 (s, 1H,
OH), 8.43 (s, 1H, CHN), 7.90 (t, J = 8.5 Hz, 2H, ArH), 7.65~7.59 (m, 4H, ArH),
.56~7.50 (m, 4H, ArH and SO NH ), 7.29 (d, J = 8.6 Hz, 2H, ArH), 7.22 (s, 1H, CH),
7
2
2

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1
3
6
1
.85 (d, J = 8.5 Hz, 2H, ArH). C NMR δ: 159.24, 157.61, 149.24, 146.73, 144.34,
44.12, 140.65, 132.31, 131.92, 131.33, 129.98, 128.48, 127.28, 126.54, 119.18,
+
1
3
4
1
2
16.91, 109.74. ESIꢀMS: 541.0 [M+H] . Anal. Calcd for C23
H18BrN
5
O
4
S: C, 51.12; H,
.36; N, 12.96. Found: C, 51.06; H, 3.37; N, 13.01.
.5.28 4-(5-(4-Bromophenyl)-3-(2-(2-hydroxybenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (48). White solid, yield 76.3%, m.p.
1
89~291˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.21 (s, 1H, CONH), 11.26 (s, 1H,
OH), 8.74 (s, 1H, CHN), 7.92 (d, J = 8.5 Hz, 2H, ArH), 7.66~7.61 (m, 4H, ArH), 7.52
(t, J = 6.6 Hz, 3H, ArH and SO
2
2
NH ), 7.34~7.29 (m, 3H, ArH), 7.26 (s, 1H, CH), 6.93
1
3
(
t, J = 6.4 Hz, 2H, ArH). C NMR δ: 157.93, 157.60, 149.24, 146.78, 144.33, 144.11,
1
41.73, 132.32, 131.91, 131.32, 129.99, 128.49, 127.29, 126.52, 123.20, 119.85,
+
1
5
4
1
2
19.19, 116.91, 109.73. ESIꢀMS: 541.0 [M+H] . Anal. Calcd for C H BrN O S: C,
2
3
18
5
4
1.12; H, 3.36; N, 12.96. Found: C, 51.03; H, 3.38; N, 12.97.
.5.29 4-(3-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-
H-pyrazol-1-yl)benzenesulfonamide (49). White solid, yield 81.5%, m.p.
1
69~272˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.82 (s, 1H, CONH), 8.55 (s, 1H,
CHN), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.80~7.76 (m, 2H, ArH), 7.60 (d, J = 8.5 Hz,
2
8
1
1
H, ArH ), 7.50 (s, 2H, SO
2 2
NH ), 7.29 (m, 4H, ArH), 7.11 (s, 1H, CH), 6.98 (d, J =
1
3
.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH
3
). C NMR δ: 159.74, 157.93, 148.58, 147.04,
45.10, 144.08, 142.14, 134.82, 130.68, 130.54, 129.36, 127.56, 127.18, 126.42,
+
21.52, 114.81, 108.87, 55.74. ESIꢀMS: 494.1 [M+H] . Anal. Calcd for
C H FN O S: C, 58.41; H, 4.08; N, 14.19. Found: C, 58.26; H, 4.09; N, 14.17.
24
20
5
4
4
1
3
.5.30
4-(3-(2-(4-Fluorobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-
H-pyrazol -1-yl)benzenesulfonamide (50). White solid, yield 72.8%, m.p.
1
02~304˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.84 (s, 1H, CONH), 8.55 (s, 1H,
6
CHN), 7.91~7.89 (m, 2H, ArH), 7.80~7.76 (m, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H,
ArH), 7.50 (s, 2H, SO NH ), 7.31 (t, J = 8.8 Hz, 2H, ArH), 7.23 (t, J = 8.8 Hz, 4H,
ArH), 7.15 (s, 1H, CH), 2.33 (s, 3H, CH
2
2
13
3
). C NMR δ: 157.88, 147.45, 147.06,
1
1
45.25, 144.13, 142.06, 139.22, 131.43, 129.90, 129.80, 129.72, 129.11, 127.17,
+
26.44, 116.52, 116.31, 109.11, 21.28. ESIꢀMS: 478.1 [M+H] . Anal. Calcd for

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DOI: 10.1039/C6RA02168A
C
24
H
20FN
5
O
3
S: C, 60.37; H, 4.22; F, 3.98; N, 14.67. Found: C, 60.22; H, 4.24; N,
1
4.68.
4
.5.31
4-(5-(4-Bromophenyl)-3-(2-(4-fluorobenzylidene)hydrazine-1-
carbonyl)-1H -pyrazol-1-yl)benzenesulfonamide (51). White solid, yield 75.3%,
1
m.p. 310~312˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.90 (s, 1H, CONH), 8.55 (s,
6
1
H, CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.80~7.76 (m, 2H, ArH), 7.65~7.60 (m, 4H,
1
3
2 2
ArH), 7.52 (s, 2H, SO NH ), 7.34~7.29 (m, 4H, ArH), 7.24 (s, 1H, CH). C NMR δ:
1
59.91, 157.63, 148.85, 147.29, 145.17, 144.23, 142.06, 139.17, 129.90, 129.36,
+
1
29.12, 127.16, 126.52, 126.43, 125.78, 116.21, 109.04. ESIꢀMS: 543.0 [M+H] .
5 3
Anal. Calcd for C23H17BrFN O
S: C, 50.93; H, 3.16; N, 12.91. Found: C, 50.80; H,
3
4
1
2
.17; N, 12.94.
.5.32 4-(5-(4-Methoxyphenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (52). White solid, yield 78.2%, m.p.
1
94~298˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.14 (s, 1H, CONH), 8.66 (s, 1H,
CHN), 8.32 (d, J = 8.6 Hz, 2H, ArH), 7.98 (d, J = 8.6 Hz, 2H, ArH), 7.91 (d, J = 8.5
Hz, 2H, ArH), 7.61 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.27 (d, J = 8.6
Hz, 2H, ArH), 7.15 (s, 1H, CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH ).
2
2
3
1
3
C NMR δ: 160.21, 157.92, 148.56, 147.04, 145.08, 143.16, 142.05, 134.82, 130.63,
1
5
5
4
1
2
30.52, 129.32, 127.37, 127.04, 126.21, 116.58, 115.76, 106.36, 55.70. ESIꢀMS:
+
21.1 [M+H] . Anal. Calcd for C24
5.22; H, 3.86; N, 16.20.
20 6 6
H N O S: C, 55.38; H, 3.87; N, 16.15. Found: C,
.5.33 4-(5-(4-Methoxyphenyl)-3-(2-(2-nitrobenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (53). Yellow solid, yield 85.6%, m.p.
1
62~264˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.24 (s, 1H, CONH), 8.96 (s, 1H,
CHN), 8.15 (d, J = 8.0 Hz, 1H, ArH), 8.08 (d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.5
Hz, 2H, ArH), 7.84 (t, J = 8.0 Hz, 1H, ArH), 7.70 (t, J = 8.0 Hz, 1H, ArH), 7.60 (d, J
=
8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO
2 2
NH ), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.14 (s, 1H,
13
CH), 6.98 (d, J = 8.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH
3
). C NMR δ: 160.20, 157.87,
57.63, 149.12, 146.52, 145.16, 144.12, 142.08, 133.82, 130.60, 130.02, 127.12,
26.42, 120.42, 117.82, 117.31, 116.72, 114.75, 108.92, 55.73. ESIꢀMS: 521.1
1
1

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DOI: 10.1039/C6RA02168A
+
[
M+H] . Anal. Calcd for C24
20 6 6
H N O S: C, 55.38; H, 3.87; N, 16.15. Found: C, 55.22;
H, 3.88; N, 16.19.
4
.5.34 4-(3-(2-(4-Nitrobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-pyrazol
1
-
1-yl)benzenesulfonamide (54). Yellow solid, yield 79.7%, m.p. 297~300˚C; H
NMR (DMSOꢀd , 400 MHz) δ: 12.15 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d, J =
6
8
8
3
1
2
1
4
.8 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 7.91~7.89 (m, 2H, ArH), 7.60 (d, J =
.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.23 (s, 4H, ArH), 7.18 (s, 1H, CH), 2.33 (s,
). C NMR δ: 159.86, 157.57, 148.84, 147.28, 145.14, 144.08, 142.10,
2
2
13
H, CH
3
39.16, 129.92, 129.36, 129.12, 127.03, 126.52, 121.42, 120.08, 116.17, 109.02,
+
1.26. ESIꢀMS: 505.1 [M+H] . Anal. Calcd for C24
20 6 5
H N O S: C, 57.14; H, 4.00; N,
6.66. Found: C, 57.06; H, 4.01; N, 16.68.
.5.35 4-(5-(4-Fluorophenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)-1H
pyrazol-1-yl)benzenesulfonamide (55). White solid, yield 73.7%, m.p. 297~299˚C;
-
1
6
H NMR (DMSOꢀd , 400 MHz) δ: 12.17 (s, 1H, CONH), 8.66 (s, 1H, CHN), 8.32 (d,
J = 8.8 Hz, 2H, ArH), 7.99 (d, J = 8.8 Hz, 2H, ArH), 7.91 (t, J = 8.8 Hz, 2H, ArH),
7
7
1
1
1
4
2 2
.61 (d, J = 8.5 Hz, 2H, ArH), 7.51 (s, 2H, SO NH ), 7.43~7.39 (m, 2H, ArH),
1
3
.31~7.24 (m, 3H, ArH). C NMR δ: 157.96, 157.32, 148.84, 147.26, 145.13, 144.07,
42.13, 138.72, 129.82, 128.86, 128.12, 127.04, 126.54, 121.43, 119.08, 116.18,
+
09.02. ESIꢀMS: 509.1 [M+H] . Anal. Calcd for C23
6 5
H17FN O S: C, 54.33; H, 3.37; N,
6.53. Found: C, 54.13; H, 3.39; N, 16.55.
.5.36
4-(5-(4-Bromophenyl)-3-(2-(4-nitrobenzylidene)hydrazine-1-carbonyl)
-1H-pyrazol-1-yl)benzenesulfonamide (56). Yellow solid, yield 74.4%, m.p.
1
3
06~308˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 12.21 (s, 1H, CONH), 8.66 (s, 1H,
CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.98 (d, J = 8.8 Hz, 2H, ArH), 7.92 (d, J = 8.8
Hz, 2H, ArH), 7.63 (t, J = 8.5 Hz, 4H, ArH), 7.53 (s, 2H, SO NH ), 7.31~7.26 (m, 3H,
2
2
13
ArH and CH). C NMR δ: 159.46, 156.52, 148.82, 146.28, 145.11, 143.06, 142.12,
1
36.81, 129.62, 128.85, 128.15, 127.06, 126.06, 121.23, 119.06, 116.14, 109.02.
+
ESIꢀMS: 570.1 [M+H] . Anal. Calcd for C23
6 5
H17BrN O S: C, 48.52; H, 3.01; N, 14.76.
Found: C, 48.46; H, 3.02; N, 14.79.
4
.5.37 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-methoxyphenyl)-

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1
H-pyrazol-1-yl)benzenesulfonamide (57). White solid, yield 82.3%, m.p.
1
2
84~286˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.89 (s, 1H, CONH), 8.52 (s, 1H,
6
CHN), 7.91~7.85 (m, 3H, ArH), 7.67 (s, 4H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH),
7
8
1
2 2
.50 (s, 2H, SO NH ), 7.27 (d, J = 8.6 Hz, 2H, ArH), 7.12 (s, 1H, CH), 6.98 (d, J =
1
3
.6 Hz, 2H, ArH), 3.78 (s, 3H, OCH ). C NMR δ: 159.68, 157.84, 148.52, 146.42,
3
45.06, 144.04, 142.10, 134.80, 130.62, 130.54, 127.32, 126.40, 121.50, 120.27,
+
1
19.48, 114.72, 106.82, 55.72. ESIꢀMS: 550.0 [M+H] . Anal. Calcd for
C
24
H
20BrN
5
O
4
S: C, 51.99; H, 3.64; N, 12.63. Found: C, 51.87; H, 3.65; N, 12.66.
4
.5.38
4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H
-pyrazol-1-yl)benzenesulfonamide (58). White solid, yield 71.7%, m.p. 290~292˚C;
1
H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.90 (s, 1H, CONH), 8.53 (s, 1H, CHN),
7
.91~7.85 (m, 2H, ArH), 7.67 (s, 4H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H,
13
2 2 3
SO NH ), 7.23 (s, 4H, ArH), 7.15 (s, 1H, CH), 2.33 (s, 3H, CH ). C NMR δ: 157.86,
1
1
48.42, 147.26, 145.24, 144.12, 142.08, 139.12, 131.42, 129.82, 129.71, 129.62,
+
29.12, 127.12, 126.41, 116.41, 116.30, 109.09, 21.28. ESIꢀMS: 539.0 [M+H] . Anal.
5 3
Calcd for C24H20BrN O S: C, 53.54; H, 3.74; N, 13.01; Found: C, 53.42; H, 3.75; N,
1
4
1
1
3.05.
.5.39 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-fluorophenyl)-
H -pyrazol-1-yl)benzenesulfonamide (59). White solid, yield 79.3%, m.p.
1
73~175˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.92 (s, 1H, CONH), 8.53 (s, 1H,
CHN), 7.91~7.89 (m, 2H, ArH), 7.67 (s, 4H, ArH), 7.60 (d, J = 8.5 Hz, 2H, ArH),
.50 (s, 2H, SO NH ), 7.42~7.38 (m, 2H, ArH), 7.31~7.26 (m, 2H, ArH), 7.20 (s, 1H,
7
2
2
1
3
CH). C NMR δ: 157.94, 157.30, 148.76, 147.22, 145.10, 144.04, 142.12, 138.64,
1
29.80, 128.82, 128.10, 127.02, 126.52, 121.41, 119.06, 116.14, 109.03. ESIꢀMS:
+
5
43.0 [M+H] . Anal. Calcd for C H BrFN O S: C, 50.93; H, 3.16; N, 12.91. Found:
23
17
5
3
C, 50.84; H, 3.18; N, 12.95.
4
1
2
.5.40 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-(4-bromophenyl)-
H-pyrazol-1-yl)benzenesulfonamide (60). White solid, yield 77.4%, m.p.
1
78~281˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.97 (s, 1H, CONH), 8.52 (s, 1H,
CHN), 8.32 (d, J = 8.8 Hz, 2H, ArH), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.85 (s, 1H, ArH),

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7
1
1
6
2 2
.67~7.60 (m, 8H, ArH), 7.52 (s, 2H, SO NH ), 7.30 (d, J =8.5 Hz, 2H, ArH), 7.25 (s,
13
H, CH). C NMR δ: 159.24, 157.16, 148.64, 147.24, 145.06, 144.02, 142.08, 138.42,
29.64, 128.82, 128.06, 127.14, 125.42, 121.40, 119.04, 116.12, 109.02. ESIꢀMS:
+
00.9 [M+H] . Anal. Calcd for C23
H17Br
2
N
5
O
3
S: C, 45.79; H, 2.84; N, 11.61. Found:
C, 45.68; H, 2.85; N, 11.65.
4
.5.41 4-(3-(2-(4-Bromobenzylidene)hydrazine-1-carbonyl)-5-phenyl-1H-pyrazol
1
-
1-yl)benzenesulfonamide (61). White solid, yield 70.5%, m.p. 200~203˚C; H NMR
(
DMSOꢀd
6
, 400 MHz) δ: 11.96 (s, 1H, CONH), 8.53 (s, 1H, CHN), 7.89 (d, J = 8.5
NH ),
Hz, 2H, ArH), 7.68 (s, 4H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH), 7.52 (s, 2H, SO
2
2
1
3
7
1
1
.43 (t, J = 3.6 Hz, 4H, ArH), 7.36~7.33 (m, 2H, ArH), 7.21 (s, 1H, CH). C NMR δ:
57.84, 157.12, 147.64, 147.21, 145.02, 144.42, 142.16, 137.40, 127.64, 127.42,
+
27.06, 126.14, 125.32, 121.30, 119.02, 116.10, 109.02. ESIꢀMS: 525.0 [M+H] . Anal.
5 3
Calcd for C23H18BrN O S: C, 52.68; H, 3.46; N, 13.36. Found: C, 52.60; H, 3.48; N,
1
3.39.
4
.5.42
4-(3-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-5-(p-tolyl)-1H-
pyrazol -1-yl)benzenesulfonamide (62). White solid, yield 80.4%, m.p. 293~294˚C;
1
H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.90 (s, 1H, CONH), 8.54 (s, 1H, CHN),
.91~7.89 (m, 2H, ArH), 7.74 (d, J = 8.5 Hz, 2H, ArH), 7.59 (d, J = 8.5 Hz, 2H, ArH),
.54 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.23 (s, 4H, ArH), 7.15 (s, 1H,
). C NMR δ: 159.82, 147.42, 147.06, 145.21, 144.12, 142.07,
7
7
2
2
1
3
CH), 2.33 (s, 3H, CH
3
1
1
4
4
39.23, 130.23, 129.93, 129.45, 129.31, 129.14, 127.12, 126.42, 116.54, 116.32,
+
09.12, 21.26. ESIꢀMS:495.0[M+H] . Anal. Calcd for C H ClN O S: C, 58.36; H,
2
4
20
5
3
.08; N, 14.18. Found: C, 58.22; H, 4.10; N, 14.20.
.5.43 4-(3-(2-(4-Chlorobenzylidene)hydrazine-1-carbonyl)-5-(4-fluorophenyl)
-1H-pyrazol-1-yl)benzenesulfonamide (63). White solid, yield 76.3%, m.p.
1
1
85~187˚C; H NMR (DMSOꢀd
6
, 400 MHz) δ: 11.92 (s, 1H, CONH), 8.55 (s, 1H,
CHN), 7.90 (d, J = 8.6 Hz, 2H, ArH), 7.75 (d, J = 8.5 Hz, 2H, ArH), 7.60 (d, J = 8.5
Hz, 2H, ArH), 7.54 (d, J = 8.5 Hz, 2H, ArH), 7.50 (s, 2H, SO NH ), 7.42~7.39 (m, 2H,
ArH), 7.31~7.26 (m, 2H, ArH), 7.20 (s, 1H, CH). C NMR δ: 157.92, 157.20, 148.56,
47.20, 145.12, 144.02, 142.08, 138.62, 129.72, 128.62, 128.08, 127.21, 126.42,
2
2
13
1

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+
1
21.42, 119.04, 116.12, 109.02. ESIꢀMS: 499.0 [M+H] . Anal. Calcd for
C H ClFN O S: C, 55.48; H, 3.44; N, 14.07. Found: C, 55.39; H, 3.45; N, 14.10.
23
17
5
3
4
1
3
.5.44 4-(5-(4-Bromophenyl)-3-(2-(4-chlorobenzylidene)hydrazine-1-carbonyl)-
H-pyrazol-1-yl)benzenesulfonamide (64). White solid, yield 86.5%, m.p.
1
14~316˚C; H NMR (DMSOꢀd , 400 MHz) δ: 11.96 (s, 1H, CONH), 8.54 (s, 1H,
6
CHN), 7.91 (d, J = 8.5 Hz, 2H, ArH), 7.74 (d, J = 8.5 Hz, 2H, ArH), 7.65~7.60 (m,
4
7
1
2 2
H, ArH), 7.53 (t, J = 8.5 Hz, 4H, SO NH and ArH), 7.30 (d, J = 8.5 Hz, 2H, ArH),
13
.25 (s, 1H, CH). C NMR δ: 159.56, 157.24, 148.52, 147.23, 145.14, 144.04, 142.06,
38.64, 129.70, 128.60, 128.06, 127.10, 126.41, 121.37, 119.024, 116.10, 109.02.
+
ESIꢀMS: 558.9 [M+H] . Anal. Calcd for C23
H17BrClN
5
O
3
S: C, 49.43; H, 3.07; N,
1
2.53. Found: C, 49.26; H, 3.08; N, 12.54.
4
.6 Anti-proliferation assay
Target tumor cell lines were grown to log phase in DMEM medium supplemented
4
with 10% fetal bovine serum. After diluting to 5×10 cells/mL with the complete
medium, 100 ꢀL of the obtained cell suspension was added to each well of 96ꢀwell
culture plates and the number of viable cells was determinedat time zero (control
2
wells) then allowed to adhere for 12 h at 37 ℃, 5% CO atmosphere. Tested samples
at preꢀset concentrations (0.1, 1.0, 10, and 100 ꢀM) were added to 96 wells with
Celecoxib as positive reference. After 48 h exposure period, 20 ꢀL of PBS containing
5
mg/mL of MTT was added to each well. Plates were then incubated for further 4 h,
and then 150 ꢀL of DMSO was added to each well for coloration. The plates were
shaken vigorously to ensure complete solubilization for 10 min at room temperature.
The absorbance was measured and recorded on an ELISA reader (ELx800, BioTek,
USA) at a test wavelength of 570 nm. In all the experiments three replicate wells were
used for each drug concentration. Each assay was carried out at least three times. The
results are shown in Table 2 and Table 3.
4
.7 Cyclooxygenase inhibition assay
The ability of the test compounds (21-64) to inhibit COXꢀ1 and COXꢀ2 (IC50

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values, ꢀM) was determined using a human COXꢀ1/COXꢀ2 ELISA Kit(catalog
number 701070 and 701080, Cayman Chemical) by a modification of the method
10
described in the reference
. According to the manufacturer’s instructions,
cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid (AA) to
PGH . PGF , produced from PGH by reduction with stannous chloride, is measured
2
2α
2
by enzyme immunoassay. Stock solutions of test compounds were dissolved in a
minimum volume of DMSO. Briefly, to a series of supplied reaction buffer solutions
(
960 ꢀL, 0.1 M TrisꢀHCl pH 8.0 containing 5 mM EDTA and 2 mM phenol) with
either COXꢀ1 or COXꢀ2 (10 ꢀL) enzyme in the presence of heme (10 ꢀL) was added
0 ꢀL of various concentrations of test drug solutions (0.1, 1, 10, and 100 ꢀM in a
1
final volume of 1 mL). These solutions were incubated for a period of 5 min at 37 °C
after which 10 ꢀL of AA (100 ꢀM) solution was added and the COX reaction was
stopped by the addition of 50 ꢀL of 1 M HCl after 2 min. PGF2α, produced from PGH
2
by reduction with stannous chloride, was measured by enzyme immunoassay. This
assay is based on the competition between PGs and a PGꢀacetylcholinesterase
conjugate (PG tracer) for a limited amount of PG antiserum. The amount of PG tracer
that is able to bind to the PG antiserum is inversely proportional to the concentration
of PGs in the wells since the concentration of PG tracer is held constant while the
concentration of PGs varies. The plate is washed to remove any unbound reagents and
then Ellman’s reagent, which contains the substrate to acetylcholine esterase, is added
to the well. The product of this enzymatic reaction produces a distinct yellow color
that absorbs at 405 nm. The intensity of this color, determined spectrophotometrically,
is proportional to the amount of PG tracer bound to the well, which is inversely
proportional to the amount of PGs present in the well during the incubation:
Absorbance α [Bound PG Tracer] α1/PGs. Percent inhibition was calculated by the
comparison of compoundꢀtreated to various control incubations. The concentration of
the test compound causing 50% inhibition (IC50, ꢀM) was calculated from the
concentrationꢀinhibition response curve (duplicate determinations).
4
.8 Cell apoptosis assay
For Annexin V/PI assays, A549 cells were stained with Annexin VꢀFITC and PI and

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5
then monitored for apoptosis by flow cytometry. Briefly, 1×10 cells were seeded in
6
ꢀwell plates for 24 h and then were treated with compound 48 (0, 2, 4, 8, and 16 ꢀM)
for 48 h. After 48 h, they were trypsinized, washed in PBS and centrifuged at 2000
rpm for 5 min twice and stained with 5µL of Annexin VꢀFITC and 5 µL of PI (5
mg/mL) in binding buffer (10 mM HEPES, pH 7.4, 140 mM NaOH, 2.5 mM CaCl2)
for 30 min at room temperature in the dark. Apoptotic cells were quantified using BD
Accuri C6 Flow Cytometer (BD, USA). Statistical analysis was done using Flowjo
7
.6.1 software. Both early apoptotic (Annexin Vꢀpositive, PIꢀnegative) and late
apoptotic (double positive of Annexin V and PI) cells were detected.
4
4
.9 Cytotoxicity test
.9.1 Primary mouse hepatocyte isolation
SD Mice were anesthetized (200 mg ketamine/kg and 16 mg xylazine/kg) by
subcutaneous injection, and the livers harvested. Hepatocytes were isolated from the
30
livers by a modification of the method described previously . Briefly, the liver was
perfused, through a needle aligned along the inferior vena cava, with buffer (pH 7.5)
containing 137 mmol/L NaCl, 5.4 mmol/L KCl, 0.5 mmol/L NaH
Na HPO , 10 mmol/L HEPES, 0.5 mmol/L EGTA, 4.2 mmol/L NaHCO
mmol/L glucose; collagenase buffer, pH 7.5, contained 137 mmol/L NaCl, 5.4
mmol/L KCl, 5 mmol/L CaCl , 0.5 mmol/L NaH PO , 0.42 mmol/L Na HPO , 10
2 4
PO , 0.42 mmol/L
2
4
3
, and 5
2
2
4
2
4
mmol/L HEPES, 0.15 g/L collagenase B, 0.05 g/L trypsin inhibitor, 4.2 mmol/L
NaHCO , and 0.016 mmol/L phenol red. The collagenaseꢀperfused liver was then
3
dissected, suspended in Hanks’ solution (30 mL), and filtered through cheesecloth and
a 100 µM nylon membrane to remove connective tissue debris and cell clumps.
Hepatocytes were subjected to centrifugation (42 g, 2 min at 4 ℃) and resuspended
in Hanks solution; this was repeated 4 times. Then hepatocytes were purified using
density gradient centrifugation (45% Percoll solution, 42 g for 10 min at 4 ℃). Cell
viability, measured by trypan blue exclusion, was more than 90%. Primary mouse
hepatocytes were maintained in DMEM/F12 medium, supplemented with 10% fetal
bovine serum and antibiotics (100 U/mL penicillin and 100 µg/mL of streptomycin) at