Synthesis, biological evaluation, and docking of dihydropyrazole sulfonamide containing 2-hydroxyphenyl moiety: A series of novel MMP-2 inhibitors

Article abstract of DOI:10.1111/cbdd.12604

In this study, we synthesized a series of dihydropyrazole sulfonamide derivatives containing 2-hydroxyphenyl moiety as antitumor agents to target the matrix metalloproteinase-2 (MMP-2). All of the synthesized compounds were examined by bioactivity assays, in which compound 4c turned out as a potential antagonist of MMP-2 along with potent anticancer activity against four tumor cell lines. Structure-activity relationship analysis was also performed to examine how structural changes impacted the bioactivity. Suggested to be caused by the induction of apoptosis, the antitumor mechanism of 4c was further confirmed by PI combining with annexin V-FITC staining assay using flow cytometry analysis. These new findings along with molecular docking observations suggested that compound 4c could be developed as a potential anticancer agent. A series of dihydropyrazole sulfonamide derivatives containing 2-hydroxyphenyl moiety were designed and synthesized as antitumor agent to target the matrix metalloproteinase-2 (MMP-2). All of the synthesized compounds were examined by bioactivity assays, and structure-activity relationship analysis was also performed to discuss how structural changes could impact the bioactivity.

Full text of DOI:10.1111/cbdd.12604

Chem Biol Drug Des 2015; 86: 1405–1410
Research Article
Synthesis, Biological Evaluation, and Docking of
Dihydropyrazole Sulfonamide Containing
2-hydroxyphenyl Moiety: A Series of Novel MMP-2
Inhibitors
Peng-Fei Wang1, Han-Yue Qiu1, Shahla Karim
proteases, and hence play a pleiotropic role in diverse bio-
processes (4). In normal circumstances, MMPs are tightly
regulated and controlled in activities, and atypical alterations
may lead to a range of pathologies; for example, aberrant
Baloch1,2, Hai-Bin Gong1,3,*, Zhong-Chang
Wang1,* and Hai-Liang Zhu1,*
1State Key Laboratory of Pharmaceutical Biotechnology,
Nanjing University, Nanjing 210093, China
increase gives rise to several diseases such as osteoarthritis
(5), rheumatoid arthritis (6), periodontal disease (7), multiple
sclerosis (8), and tumor metastasis (9). In particular, patho-
logical level of MMPs has a close link with cancer in virtually
all aspects of cancer progression and metastasis, although
the mechanism is not fully understood yet. Still, MMPs have
proven to be potential antitumor target, and their inhibitors
possess the potency to serve as effective anticancer agents
(10). For the last two decades or so, many research studies
within academia and industries have been focused on the
design and screening for potent MMPs inhibitors (11). In
general, the MMPs inhibitors can be divided into two types
according to whether they are binding to the activity site S1’
cavities or chelating with the zinc ion (12,13). Zinc-binding
groups (ZBGs) are essential to form strong co-ordination
bond with the zinc cation, and by far, various groups have
been validated as ZBGs including phenol hydroxy, second-
ary amine, amide, imine, imidazole, carboxylate, aminocarb-
oxylate, sulphydryl, hydroxamate, phosphonate, and
phosphinate moieties (14–18). However, although many
efforts have been made, they appeared to be insufficient
due to the constraints of toxicity and dose-limiting efficacy
which impeded the clinical use of these inhibitors (19). Thus,
there remains a considerable need for the optimization of
oral absorption, drug processes, and action duration while
maintaining potency and selectivity. To address these short-
falls, new MMPs inhibitor templates should be elaborated
and applied into the design.
2Department of Biotechnology, FCPD, Sindh Agriculture
University, Tandojam, Sindh, Pakistan
3Xuzhou Central Hospital, Xuzhou 221009, China
*Corresponding authors: Hai-Bin Gong, ghbxzh@126.com;
Zhong-Chang Wang, wangzhongchang2006@163.com;
Hai-Liang Zhu, zhuhl@nju.edu.cn
In this study, we synthesized a series of dihydropyrazole
sulfonamide derivatives containing 2-hydroxyphenyl
moiety as antitumor agents to target the matrix metallo-
proteinase-2 (MMP-2). All of the synthesized com-
pounds were examined by bioactivity assays, in which
compound 4c turned out as a potential antagonist of
MMP-2 along with potent anticancer activity against
four tumor cell lines. Structure–activity relationship
analysis was also performed to examine how structural
changes impacted the bioactivity. Suggested to be
caused by the induction of apoptosis, the antitumor
mechanism of 4c was further confirmed by PI combining
with annexin V-FITC staining assay using flow cytometry
analysis. These new findings along with molecular dock-
ing observations suggested that compound 4c could be
developed as a potential anticancer agent.
Key words: anticancer, dihydropyrazole sulfonamide, extra-
cellular matrix enzyme, flow cytometry analysis, MMP-2
Received 18 January 2015, revised 27 May 2015 and
accepted for publication 3 June 2015
In our previous work, compounds bearing the benzene-
sulfonate sulfonamide skeleton were synthesized and
investigated for their bioactivities (20). To our delight, some
showed potent activity against cancer cells and high safety
to non-cancer cells. The aforementioned progress laid the
foundation and encouraged us to carry out further study.
From the previous docking mode of the most potent com-
pound, benzenesulfonamide and appropriate stereo-space
are found to be favorable, along with alteration in enzyme-
active site caused by the binding. Our recent work
focused on the design of new inhibitors on the basis of
The matrix metalloproteinases (MMPs) represent a super-
family of zinc-dependent enzymes functioning as metallo-
proteases, whose proteolytic activity on the substrates of
extracellular matrix (ECM) is crucial for physiological pro-
cesses of tissue remodeling and repairing, homeostatic reg-
ulation, innate immunity control, and so on (1–3). The
proteases are also capable to cleave non-matrix substrates,
including growth factors, peptidase inhibitors, cytokines,
receptors, adhesion molecules, clotting factors, and other
ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12604
1405

Wang et al.
previous study, and the structure of dihydropyrazole was
employed as it is less flexible than the -C-N- structure,
offering more preferable stereo-space. Meanwhile, the
benzenesulfonamide was retained as zinc-binding group
and the result turned out to support our assumption.
Based on the results, we concluded that all the newly syn-
thesized compounds showed enhanced potential against
different tumor cell lines and MMP-2 enzyme.
compound 4x with atomic labeling system is presented in
Figure 1, and also the data are listed on Table S1.
Biological activity
MMP-2 inhibitory activity
All the synthesized compounds (4a–4y) were evaluated for
their in vitro bioactivity against the MMP-2 enzyme inhibition
compared to the control group CMT-1. The results are sum-
marized on Table 1 and indicate that compound 4e has the
same effects as the positive control CMT-1 with the IC50 of
1.81 and 1.13 lM, respectively, while compound 4c has
Results and Discussion
Chemistry
Scheme 1 illustrates the general synthetic route to obtain
the target compounds (4a–4y). The starting chalcones
(3a–3y) were directly condensed by equivalent salicylic
aldehydes (1a–1c) and acetophenones (2a–2g). Catalyzed
by 40% potassium hydroxide, the Claisen–Schmidt reac-
tions were obtained and gave precipitates in ethanol
cooled to the temperature of 0 °C. The precipitates were
then filtered and washed by cold ethanol, and purified
chalcones (3a–3y) were obtained. Concomitantly, target
chalcones were dissolved in ethanol with 4-sulfamoylphe-
nyl hydrazine hydrochloride, whereafter several drops of
acetic acid were added and the mixture was heated to
reflux to furnish compounds (4a–4y). All the compounds
gave satisfactory analytical and spectroscopic data, and
23 compounds (4a–4w) were reported for the first time.
Elemental analyses, 1H NMR, and ESI-MS spectra were
consistent with the assigned structures.
Crystal structures of compound 4x
Through recrystallization, the single crystal structure of
compound 4x was obtained and subsequently tested by
X-ray diffraction analysis. The perspective view of
Figure 1: Crystal structure diagram of compound 4x.
Scheme 1: General synthesis of derivatives (4a–4y). Reagents and conditions: (i) 40% aqueous potassium hydroxide solution, ethanol,
ice bath; (ii) 4-sulfamoylphenyl hydrazine hydrochloride, glacial acetic acid, reflux, 6–8 h.
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Design and evaluation of new MMP-2 inhibitors
Table 1: Antiproliferation and enzyme inhibitory activities (IC50a, lM) of target compounds (4a–4y)
a
IC50 (lM)
Compounds
R1
R2
R3
R4
A549
MCF-7
Hela
1.70
HepG2
MMP-2b
4a
4b
H
H
H
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Br
Br
Br
Br
Br
Br
H
H
H
H
H
H
H
OCH3
CH3
Cl
H
H
H
H
H
H
Cl
H
OCH3
H
OCH3
H
H
H
H
Cl
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
–
3.97
12.0
1.93
2.88
5.24
6.18
13.32
6.49
7.35
19.4
5.87
7.39
14.03
24.67
9.52
4.12
14.03
7.39
24.67
9.51
2.25
4.12
5.24
36.8
8.93
2.83
2.26
–
2.68
5.96
4.37
6.31
4.27
5.30
5.69
3.14
1.67
1.85
3.35
1.87
1.62
2.45
22.94
6.95
2.39
2.40
3.73
2.40
3.73
2.40
13.96
18.5
12.6
6.76
6.89
–
4.56
2.31
4.21
7.05
5.51
9.54
10.02
7.21
4.21
9.64
4.45
8.73
6.77
5.20
11.38
6.78
10.03
8.59
7.66
4.13
1.01
5.69
4.51
7.05
11.51
–
9.83
11.51
0.33
18.76
1.81
4.65
12.87
8.83
16.43
13.54
11.28
4.62
18.90
16.49
3.84
2.13
12.78
3.23
17.25
14.96
15.84
8.73
16.24
24.66
1.83
–
7.45
3.17
4.68
6.63
8.64
12.15
4.51
8.13
16.52
5.99
8.53
9.87
11.74
10.05
5.13
7.21
6.09
3.54
6.89
1.74
4.95
14.98
7.61
7.85
1.43
7.61
–
4c
4d
OCH2CH3
Br
H
CH3
Cl
F
H
H
Cl
OCH3
Br
Cl
OCH2CH3
H
H
H
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
F
H
4t
4u
Cl
Br
Cl
F
OCH3
–
4v
4w
H
Cl
H
H
–
4x
4y
H
Gefitinib -
Celecoxib -
CMT-1 -
–
–
–
–
–
–
0.73
–
–
1.13
aValues are the average of three independent experiments run in triplicate. Variation was generally 5–10%.
bHuman recombinant enzymes, by the esterase assay (4-nitrophenylacetate as substrate).
stronger MMP-2 enzyme-suppressing capability than that of
the positive control CMT-1, possessing IC50 of 0.33 lM. By
analyzing the relationship between the structure and activity of
the compounds, it can be inferred that the changing of substi-
tuent salicylaldehyde exhibited less effect on SAR, whereas
the maximum impacts were observed from the substituents
on the benzene ring of acetophenone. In details, when the
substituents on the para-position of the benzene ring are
potent electron-donating groups rather than electron-with-
drawing groups, the compounds gained better activity, for 4c
(IC50 = 0.33 lM) > 4a (IC50 = 9.8 lM) > 4d (IC50 = 18.76 lM);
the substituents on meta-position also mattered, for 4p
(IC50 = 2.13 lM) > 4j (IC50 = 13.54 lM). However, substitu-
ent salicylaldehydes had minor effect on the inhibitory varia-
tions of these compounds, indicated by the order 4c
(IC50 = 0.33 lM) < 4e (IC50 = 1.81 lM) < 4p (IC50 = 2.13
lM) < 4r (IC50 = 3.23 lM).
groups in enhancing pharmaceutical potency on the
whole.
Antiproliferation assay
All the synthesized compounds (4a–4y) were evaluated for
their antiproliferative activities against different tumor lines
of MCF-7, HepG2, Hela, and A549, whereas two classical
clinical drugs gefitinib and celecoxib were used as positive
control. The bioactive data summarized on Table 1 dem-
onstrated that all the compounds possessed moderate to
good restraint abilities. Against MCF-7 cell line,several
compounds were comparable to the positive group drugs,
such as 4b, 4d, 4f, 4g and 4p, while inspiringly most of
the rest compounds were more potent than the control
groups. Also against the other cancer cell lines, most
compounds exhibited impressive potency. Taking all the
results into consideration, compounds 4a, 4c and 4u
revealed comprehensive inhibition abilities, and for all, the
corresponding IC50 was below 5 lM.
In comparison, we found that these compounds, with
electron-donating substituents on the benzene ring (such
as CH3, OCH3), exhibited more potent anticancer activi-
ties than those having bulky or electron-withdrawing
group (such as F). From the aforementioned analysis, it
could be concluded that the electron-donating substitu-
ents were more ideal groups than electron-withdrawing
Cytotoxicity
All the target compounds were evaluated for their cytotox-
icity against human kidney epithelial cell 293T with the
Chem Biol Drug Des 2015; 86: 1405–1410
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Wang et al.
Figure 2: Compound 4c induced apoptosis in Hela cells with the density of 32, 8, 2 and 0 lM. Hela cells were treated with compounds
for 24 h. Values represent the mean Æ SD, n = 3. p < 0.05 versus control. The percentage of cells in each part was indicated.
median cytotoxic concentration (CC50) data of tested com-
pounds by the MTT assay. These compounds were tested
at multiple doses to study the viability of 293T cells, and
as showed on Table S2, all the compounds demonstrated
low cytotoxic activities in vitro against human kidney epi-
thelial cell 293T.
revealed that the amino acid residues TYR155, PHE157,
and PHE180 located in the binding cavity are important in
binding with compound 4c. Two hydrogen bonds and two
pi–pi interactions could be found in the 2D model, while
the 3D model revealed that two co-ordination bonds were
also formed between zinc cation and 4c. The docking
results suggested that the sulfanilamide and salicylalde-
hyde backbone are of vital importance, while the substitute
acetophenones provide the compounds with admirable
steric stabilization.
Apoptosis assay
To verify whether the inhibition of cell growth of Hela was
related to cell apoptosis, apoptosis of Hela cells induced
by compound 4c was determined using flow cytometry.
The result is shown in Figure 2. According to the data
annotated, the percentage of apoptotic cells elevated
directly and markedly increased in a dose-dependent
manner The percentages of cell apoptosis 3.26%, 8.35%,
16.4% and 94.2% were responding to the concentration
of compound 4c 0, 2, 8 and 32 lM.
Conclusion
To summarize, on the basis of previous study, a series of
novel MMP-2 inhibitors (4a–4y) bearing sulfonamide skele-
ton have been synthesized and examined for their biologi-
cal activities. The results suggested that these inhibitors
possess improved activities, exhibiting moderate to potent
antiproliferative potency against MCF-7, Hela, HepG2, and
A549 cells and enzyme MMP-2. Among them, 4a, 4c and
4u showed the most potent inhibitory activities against all
the cell lines with IC50 values smaller than 5 lM. Besides,
compound 4c inhibited the MMP-2 with IC50 of 0.33 lM.
The probable binding models were obtained by docking
simulation, suggesting that the sulfanilamide and salicylal-
Molecular docking
Docking study was performed to fit these compounds into
the active site of the matrix metalloproteinases MMP-2
(PDB code: 1QIB). The probable binding mode of com-
pound 4c which showed the most potent enzymatic inhibi-
tion is presented in Figures S1 and S2. This model
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Design and evaluation of new MMP-2 inhibitors
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bonds and hydrogen bonds which provide considerable
steric binding stabilization.
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online version of this article:
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Wang et al.
Appendix S1. Experimental details, NMR, MS and ele-
4c with the MMP-2 binding site: for clarity, only interacting
mental analyses data for the synthesized compounds.
residues are displayed.
Figure S1. Molecular docking 2D modeling of compound
4c with MMP-2.
Table S1. Crystal data for compound 4x.
Table S2. The median cytotoxic concentration (CC50) data
of all compounds (4a–4y).
Figure S2. Molecular docking 3D modeling of compound
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