Dihydropyrazothiazole derivatives as potential MMP-2/MMP-8 inhibitors for cancer therapy

Article abstract of DOI:10.1016/j.bmcl.2018.05.004

MMP-2/MMP-8 is established as one of the most important metalloenzymes for targeting cancer. A series of dihydropyrazothiazole derivatives (E1–E18) bearing a salicylaldehyde group linked to Pyrazole ring were designed, synthesized, and evaluated for their pharmacological activity as MMP-2/MMP-8 inhibitors. Among them, compound E17 exhibited most potent inhibitory activity (IC₅₀ = 2.80 μM for MMP-2 and IC₅₀ = 5.6 μM for MMP-8), compared to the positive drug CMT-1 (IC₅₀ = 1.29 μM). Compounds (E1–E18) were scrutinized by CoMFA and CoMSIA techniques of Three-dimensional quant. structure-activity relationship (3D-QSAR), as well as a docking simulation. Moreover, treatment with compound E4 could induce MCF-7 cell apoptosis. Overall, the biological profile of E1–E18 may provide a research basis for the development of new agents against cancer.

Full text of DOI:10.1016/j.bmcl.2018.05.004

Accepted Manuscript
Dihydropyrazothiazole derivatives as potential MMP-2/MMP-8 inhibitors for
cancer therapy
Zhong-Chang Wang, Fa-Qian Shen, Meng-Ru Yang, Ling Xia-You, Li-Zhi
Chen, Hai-Liang Zhu, Ya-Dong Lu, Fan-Lei Kong, Ming-Hua Wang
PII:
S0960-894X(18)30394-9
https://doi.org/10.1016/j.bmcl.2018.05.004
BMCL 25821
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
11 February 2018
17 April 2018
2 May 2018
Please cite this article as: Wang, Z-C., Shen, F-Q., Yang, M-R., Xia-You, L., Chen, L-Z., Zhu, H-L., Lu, Y-D.,
Kong, F-L., Wang, M-H., Dihydropyrazothiazole derivatives as potential MMP-2/MMP-8 inhibitors for cancer
therapy, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/10.1016/j.bmcl.2018.05.004
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Dihydropyrazothiazole derivatives as potential
MMP-2/MMP-8 inhibitors for cancer therapy
Zhong-Chang Wanga,b, Fa-Qian Shenc, Meng-Ru Yangc, Ling Xia-Youb, Li-Zhi
Chenb, Hai-Liang Zhuc, Ya-Dong Lud*, Fan-Lei Kongb*, Ming-Hua Wanga*
aDepartment of Pesticide Science, College of Plant Protection, Nanjing Agricultural University,
Nanjing 210095, China
bJiangsu Agrochem Laboratory Co.,Ltd. Changzhou 213000,
cState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
China
dNeonatal Medical Center, Children’s Hospital of Nanjing Medical University, Nanjing 210008,
China;
*Corresponding author. Tel. & fax: +86 25 84395479; e-mail: wangmha@njau.edu.cn (Minghua
Wang); kongfanlei@jsmone.com (Fan-Lei Kong); lydawq@sina.com(Ya-Dong Lu)
1

Abstract
MMP-2/MMP-8 is established as one of the most important metalloenzymes for
targeting cancer. A series of dihydropyrazothiazole derivatives (E1-E18) bearing a
salicylaldehyde group linked to Pyrazole ring were designed, synthesized, and
evaluated for their pharmacological activity as MMP-2/MMP-8 inhibitors. Among
them, compound E17 exhibited most potent inhibitory activity (IC50 =2.80 μM for
MMP-2 and IC50=5.6 μM for MMP-8), compared to the positive drug CMT-1 (IC50
=1.29 μM). Compounds (E1-E18) were scrutinized by CoMFA and CoMSIA
techniques of Three-dimensional quant. structure-activity relationship (3D-QSAR), as
well as a docking simulation. Moreover, treatment with compound E4 could induce
MCF-7 cell apoptosis. Overall, the biological profile of E1-E18 may provide a
research basis for the development of new agents against cancer.
Keywords:
Dihydropyrazothiazole
MMP-2/MMP-8
Synthesis
3D-QSAR
Docking simulation
2

Malignant tumor, also known as cancer, is a common and frequent malignant
disease that seriously threatens human health. Its incidence is the second highest in
the global cancer prevalence rate1. Although much efforts has been made in
developing new drugs to cure cancer, improving survival, the number of deaths
caused by cancer is still very high2, due to lack of selectivity and high cytotoxicity in
traditional medicine, metastasis of cancer cell, the diversity of cancer and drug
3
resistance . Therefore, it is imminent for researchers to find selective inhibitory
anticancer drugs with high efficiency and low toxicity.
Matrix metalloproteinases (MMPs) are a family of extracellular zinc and
calcium-dependent neutral endopeptidases structurally and functionally related,
playing a crucial role in degradation of extracellular matrix components, tissue
remodeling and the pathogenesis of major diseases4. MMPs are involved in a wide
range of physiological processes, including ovulation, embryonic development,
angiogenesis, cellular variation, and wound healing5. Furthermore, they also play a
key role in degrading the major components of the extracellular matrix, inflammation,
tumor cells proliferation, and some other typical diseases6. MMPs play a crucial role
in the remodeling and repair of physiological tissues, but their overexpression has led
to a wide variety of diseases7, such as cancer8, arthritis9, osteoporosis10, and
cirrhosis11. Therefore, inhibiting MMPs is an attractive approach which can treat
multitude of diseases. Much important progress has been made in the mechanism of
MMPs worldwide from the early 90s of the last century12, For example, many
inhibitors of MMP-2 have been proved to prevent cancer tumor growth,13 some of
which had reached an advanced clinical trial. Representative examples include 1-5
had been tested against cancers;14 MMP inhibitors 6 (cipemastat)15 and 7
(illomastat)16 were tried in the clinics for inflammation (Chart1).
Unfortunately, however, there had been no clinically useful inhibitor to be
demonstrated until now. This is mainly because the serious side effects, like
musculoskeletal syndrome, which would fail the clinical trials17. Meanwhile, there are
some other reasons, such as MMPs involving in promotion of some pathological
process, converse in other pathological process18. In addition, previous attempts to
study targeting MMPs ended in failure due to a lack of understanding of the role of
MMPs in physiology and pathology19.
3

Chart1
On the other hand, all of the first generation and most of the second generation
matrix metalloproteinase inhibitors (MMPIs) failed in application and promotion due
to lack of specificity20. Therefore, at present, selective inhibitors without zinc ions or
phosphate groups are considered as both targets and antitargets for cancer therapy21.
Specifically, MMP-2, MMP-3, MMP-8, MMP-9, MMP-12 and MMP-14 are
considered as antitargets whereas MMP-2/MMP-8, the focus of our study, is taken
into account as a target and play a role in blocking the proliferation of tumor cells22.
In the past few decades, highly efficient inhibitors of MMP has opened up a new
horizon for researchers23.
So far, tetracycline and its derivatives were mostly found as natural inhibitiors of
MMPs. With the in-depth study, tetracyclines or structural modifications of
tetracycline derivatives can selectively inhibit MMPs24, like structurally modified
compound CMT-1. Based on the reported three-dimensional crystal structure
information of MMPs and their small molecule ligand complexes, we use
computer-aided drug design technology to screen a series of dihydropyrazothiazole
derivatives E1-E18 as potential MMP-2 inhibitors according to the size and shape of
4

the receptor binding pocket, the binding site of the binding region and the
transformable space of non-binding region. This study may contribute to discover
potential and selective MMP-2 inhibitors for the treatment of cancer.
All of the novel dihydropyrazothiazoles containing salicylaldehyde moieties
followed the synthetic pathway was depicted in Scheme 1a. The starting diverse
substituted chalcones (1a-1c) were synthesized by the cross-condensation reaction of
salicylaldehyde with the substituted acetophenone in the presence of 40% KOH
solution, acting as catalyst, in ethanol, then cyclized by thiosemicarbazide at room
temperature to afford the formation of Salicylaldehyde dihydropyrazolamide (2a-2c).
Subsequently, Compounds (2a-2c) and 2-bromo-1-phenyl were dissolved in
dimethylformamide (DMF) to obtain target compounds (E1-E18). All of the target
compounds are reported for the first time, and gave satisfactory analytical and
1
spectroscopic data H NMR and ESI-MS, which are in full accordance with their
depicted structures.
All target compounds (E1-E18) were evaluated for their antiproliferatve activities
in vitro against three cancer cell lines, including MCF-7, HepG2, HeLa, compared
with the positive contrast drugs Gefitinib and Celecoxib. As shown in Table 1,
MCF-7 was effectively suppressed by most of the target compounds, which showed
better anti-proliferative activities than positive contrast drugs, especially E4 (IC50 up
to 1 µM).
Generally, the halogen substituent could enhance anti-cancer activity of synthesized
compounds, while the strong electron-donating group failed. To be specific, when R1,
R3 is substituted by F, Br, respectively, they showed more potent anti-proliferative
activity on MCF-7 than other substituents. In order to verify our research results more
deeply, compounds (E1-E18) were evaluated for their inhibitory activities on
MMP-2/MMP-8 in our further study, with positive control drug CMT-1. The results
5

were compared with CMT-1 under identical conditions, which showed that the
majority of the synthesized compounds exhibited excellent inhibiting MMP-2
activities displaying IC50 values between 2.8 and 58.8 μΜ, but weak to MMP-8. The
following structure-activity relationship (SAR) studies were performed by
modification of the parent compound to determine how the substituents of the
subunits affect the MMP-2 inhibitory activities. When substituents R2, R3 remained
unchanged, selection of different substituents R1 against MMP-2 indicated strong
electron-donating groups are preferable than electron-withdrawing groups. For
instance, E17 (IC50=2.80 µM) > E11 (IC50=6.70 µM). R1, conversely, was kept
unaltered, alteration of R2, R3 showed that electron-donating groups could contribute
to the increase of anti-cancer activity. Furthermore, compounds E1-E18 exhibited
moderate inhibitory activity against MMP-2 but failed to achieve the effects of
positive control, even the most effective compound E8 could just close to the half
effect of CMT-1. After mentioned analysis, it could be concluded that
dihydropyrazothiazole derivatives acted as MMP-2 inhibitors for cancer therapy.
Table 1 Data from in vitro anti-proliferative and MMP-2/MMP-8 inhibitory activities
(IC50, µM) of target compounds (E1-E18) (µM)
IC50a (µM)
Compounds
R1
R2
R3
MCF-7 HepG2
Hela
5.05
4.88
5.51
8.64
2.73
2.25
13.14
2.70
10.85
16.97
9.73
11.65
18.15
28.1
13.02
16.31
13.70
29.26
1.48
7.55
-
MMP-2
21.64
34.76
28.33
11.23
8.31
MMP-8
62.5
75.6
86.5
48.9
35.8
>100
39.7
48.8
60.1
28.2
36.4
100.1
30.5
36.8
48.1
65.2
25.6
>100
E1
E2
H
H
H
Br
2.08
2.14
1.07
1.03
5.3
9.90
17.58
21.68
11.94
10.87
8.85
H
H
E3
H
Br
H
E4
H
H
OCH3
H
E5
H
H
F
F
F
F
OCH3
H
E6
CF3
3.5
48.16
9.65
E7
H
3.24
1.47
8.01
4.56
4.01
2.89
2.39
20.14
4.42
3.17
3.72
11.13
6.77
6.96
-
25.00
15.04
18.41
18.22
17.75
10.49
23.32
9.52
E8
H
Br
H
12.32
16.24
10.81
6.70
E9
Br
H
E10
E11
OCH3
H
F
OCH3
H
E12
E13
E14
E15
E16
E17
E18
Gefitinib
Celecoxib
CMT-1
F
CF3
H
58.43
12.86
16.87
13.96
11.72
2.80
OCH3
OCH3
OCH3
OCH3
H
H
Br
H
Br
H
7.55
OCH3
H
15.28
12.55
12.00
-
OCH3 OCH3
OCH3
H
-
CF3
-
58.8
-
-
-
-
-
-
0.78
-
-
-
-
1.29
21.9
6

a Values are the average of three independent experiments run in triplicate. Variation was generally 5–10%.
The method of compound E4 on MCF-7 apoptosis was studied by Annexin
V-FITC/PI double staining. MCF-7 cells were treated with different concentrations (0
µM, 2 µM, 8 µM and 32 µM) of E4 for 48 h. As illustrated in Fig. 1, the percentage of
apoptotic cells (7.87%, 26.84%, 46.14% and 63.19%) increased in a dose-dependent
manner, corresponding to concentrations of compound E4 on 0 µM, 2 µM, 8 µM and
32 µM, respectively. In conclusion, compound E4 could effectively induce MCF-7
cells apoptosis in a dose-dependent manner.
Fig. 1. MCF-7 cells treated with 0, 2, 8 and 32 µM 4d for 48 h were collected and
analysed. Images are representative of three independent experiments.
Compounds E1-E18 were evaluated for their toxicity against human kidney
epithelial cell 293T/LO2, comparing with positive control drug Celecoxib, and the
median cytotoxic concentration (CC50) data (Table 2) of tested compounds was
achieved using the MTT assay. As shown in Table 2, some of the target compounds
exhibited less cytotoxicity against 293T/LO2 than the positive control drug. Hence,
we were inspired by the discovery and conducted further investigation.
7

Table 2 The median cytotoxic concentration (CC50) data of compounds E1-E18
against 293T/LO2
CC50, μmola
293T
CC50, μmola
Compounds
Compounds
LO2
88.3
65.2
42.3
72.4
57.9
42.8
85.6
48.2
59.4
85.6
293T
39.15
34.78
64.07
71.94
24.50
63.04
35.22
45.43
55.83
LO2
55.10
52.77
41.77
57.12
46.63
25.38
50.73
52.01
70.09
59.42
43.8
66.1
75.2
>100
68.7
49.7
56.2
45.6
78.2
E1
E2
E3
E4
E5
E6
E7
E8
E9
E10
E11
E12
E13
E14
E15
E16
E17
E18
Celecoxib
a Values are the average of three independent experiments run in triplicate. Variation was generally 5-10%.
From preliminary virtual and in vitro MMP-2 inhibitory activities of salicylidene
dihydropyrazole thiazide derivatives screening results showed that, the IC50 values of
E15, E16 against MMP-2 were 13.96 and 11.72 μM, respectively. As shown in the
docking model of E16 and MMP-2 (Fig. 2), E16 had functional groups complexing
with Zn2+. Besides, N-grafted thiazole skeleton in the dihydropyrazole ring could
penetrate into the S1 binding pocket, and p-methoxybenzene ring grafted at the C-3
position could penetrate into the S2 'and S3' binding cavities. Therefore, compound
E16 fitted each pocket cavity of MMP-2 well and stabilized its binding conformation.
Meanwhile, a strong π bond was formed between the phenyl ring at the 2-position of
thiazole and amino acid PHE. Thus it can be speculated that salicylaldehyde
containing dihydropyrazole thiazole derivatives can fit into each pocket of MMP-2
well. We retained the dihydropyrazole thiazole derivative skeleton with thiazole at the
N-1 position, followed by further structure optimization, and explored the possibility
and the structure-activity relationship of this compound as a lead compound of
MMP-2 inhibitor.
8

Fig. 2 (a) Interactions between compound E16 and MMP-2 in a two-dimensional plan
(b) Interactions between compound E16 and MMP-2 in a three dimentional
stereogram
In order to acquire a systematic and visual SAR profile on dihydropyrazothiazole
derivatives bearing a salicylaldehyde group as MMP-2 inhibitors and to explore more
clues which led to further optimization of inhibitory molecular structure, 3D-QSAR
model of compounds E1-E18 was built, and hence we can choose activity
conformation of the designed molecular and reasonably evaluate the designed
molecules using the corresponding pIC50 values, converted from the obtained IC50
(µM) values of MMP-2 inhibition via built-in QSAR software of DS 3.5 (Discovery
Studio 3.5, Accelrys, Co. Ltd). The training and test set was divided in diverse
molecules module of DS 3.5, in which the test set includes four compounds (E5, E8,
E10, E15) while other 14 compounds were classified into the training set. The
3D-QSAR model defined the critical regions (steric or electrostatic) affecting the
binding affinity. It was a PLS model set up 400 independent variables (conventional
R2 = 0.92) which proved the reliability of this model.
9

Table 3 Data based on molecular simulation of 3D-QSAR of compounds E1-E18
MMP-2
Compound a
Residual error
Actual pIC50
Predicted pIC50
E1
E2
4.67
4.46
4.55
4.95
5.08
4.32
5.02
4.91
4.79
4.97
5.17
4.23
4.89
4.77
4.86
4.93
5.55
4.68
4.46
4.55
4.95
4.795
4.325
5.02
4.72
4.79
4.73
5.16
4.23
4.89
4.77
4.87
4.93
5.55
-0.0061
0.0042
0.00013
0.00038
0.289
-0.0046
0.0012
0.19
E3
E4
E5
E6
E7
E8
E9
-0.0032
0.24
E10
E11
E12
E13
E14
E15
E16
E17
0.0051
0.0018
0.0014
-0.0015
-0.013
0.0013
-0.0024
E18
4.24
4.24
0.0022
a
Underlined for the test set, the rest belonging to the training set.Practical test and the 3D-QSAR
model predicted pIC50 MMP-2 values of compounds E1-E18, and the residual error
values were summarized in Table 3. Based on the linear relationship between them in
Fig. 3a, this model had a good forecasting ability. A contour plot of the electrostatic
field region, enhancing anticancer activity, was in blue which means that the electron
density increases, but red converse Fig. 3b. As shown in Fig. 3c, the energy grids
corresponding to the favorable (in green) or unfavorable (yellow) steric effects for the
MMP-2 affinity. As illustrated in these pictures, this QSAR model predicted
structure-activity relationship of the ring substituents on R2 was consistent with the
actual value. It also offers the possibility to optimize the molecular structure of the
inhibitor, like introducing large volume of electron-donating substituents on the
benzene ring to which R1 is attached and small volume of electron-donating
substituents of R2. This promising model would give directions to design and
optimize more potent MMP-2 inhibitors based on the dihydropyrazothiazole bearing
salicylaldehyde groups and contributed to our further study in future.
10

Fig. 3. (a) The pIC50 regression curve based on the predictive and experimental value
of MMP-2 inhibitor (b) Isosurface of the 3D-QSAR model coefficients on
electrostatic potential grids. (c) Isosurface of the 3D-QSAR model coefficients on Van
der Waals grids.
In this study, a series of salicylaldehyde containing dihydropyrazole thiazole
derivatives E1-E18 have been designed, synthesized and evaluated for their
anti-proliferative activities against MCF-7, HeLa and HepG2 cells. Most of them
exhibited more anti-proliferative activities than positive control drugs Gefitinib and
Celecoxib. We also evaluated these target compounds for their selective MMP-2
inhibitory activities. Among them, compound E17 showed the most potent MMP-2
inhibition activities (IC50 = 2.80 μM). The probable binding models were obtained by
docking simulation, suggesting that the dihydropyrazole thiazole and salicylaldehyde
backbone are favorable for the zinc-chelating bonds and π-cation bond which provide
considerable steric binding stabilization. But beyond that, 3D-QASR models were
built with previous activity data and binding conformations to begin our work in this
paper as well as to provide a reliable tool for reasonable design and synthesis of
potent MMP-2 inhibitors. Furthermore, we will continue to optimize these
compounds in the hope of obtaining drugs with highly effective.
11

Acknowledgments
This work was supported by the National Key Research and Development Program
of China (2016YFD0200207).
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14

Dihydropyrazothiazole
derivatives
as
potential
MMP-2/MMP-8 inhibitors for cancer therapy
Zhong-Chang Wanga,b, Fa-Qian Shenc, Meng-Ru Yangc, You-Ling Xiab, Li-Zhi Chenb,
Hai-Liang Zhuc, Ya-Dong Lu d*, Fan-Lei Kongb*, Ming-Hua Wang a*
a
Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural University,
Nanjing 210095, China
bJiangsu Agrochem Laboratory Co.,Ltd. Changzhou 213000,
cState Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093,
China
d
Neonatal Medical Center, Children’s Hospital of Nanjing Medical University, Nanjing 210008,
China;
Interactions between compound E16 and MMP-2 in a three dimentional stereogram
15

> 18 dihydropyrazothiazole derivatives had been synthesized.
> Most of the compounds showed low toxicity to 293T/LO2 cells.
> Compound E17 showed the most potent and best selective MMP-2 inhibition.
> We built a 3D-QASR model for the further explanation of SAR about these
compounds.
16