Synthesis, in vitro and in vivo anticancer activities of novel 4-substituted 1,2-bis(4-chlorophenyl)-pyrazolidine-3,5-dione derivatives

Article abstract of DOI:10.1039/c5md00240k

To develop potent and selective anticancer agents, a series of novel 4-substituted 1,2-bis(4-chlorophenyl)-pyrazolidine-3,5-dione derivatives were designed and synthesized. All the compounds were evaluated for their antiproliferative activities against a panel of four human cancer cell lines. Among them, compound 4u is the most potent, exhibiting IC₅₀ values ranging from 5.1 to 10.1 μM. Flow cytometry and western blot analyses revealed that treatment of MGC-803 cells with compound 4u induces early cellular apoptosis via activation of caspases-9/3. Furthermore, compound 4u effectively reduced the tumor growth exhibited by human gastric cancer cells in vivo without obvious adverse side effects. Our findings indicate that compound 4u may serve as a lead compound to target solid tumors.

Full text of DOI:10.1039/c5md00240k

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Synthesis, in vitro and in vivo anticancer activities of novel 4ꢀsubstituted
1,2ꢀbis(4ꢀchlorophenyl)ꢀpyrazolidineꢀ3,5ꢀdione derivatives
XuꢀYao Zhang, YiꢀFei Gu, Ting Chen, DongꢀXiao Yang, XiꢀXin Wang, BaiꢀLing Jiang,
KunꢀPeng Shao, Wen Zhao, Cong Wang, JunꢀWei Wang, QiuꢀRong Zhang∗, HongꢀMin Liu∗
Key Laboratory of Advanced Pharmaceutical Technology, Ministry of Educaction of China;
Coꢀinnovation Center of Henan Province for New Drug R & D and Preclinical Safety; School of
Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan 450001,
China
Abstract: To develop potent and selective anticancer agents, a series of novel
4ꢀsubstituted 1,2ꢀbis (4ꢀchlorophenyl)ꢀpyrazolidineꢀ3,5ꢀdione derivatives were
designed and synthesized. All the compounds were evaluated for their
antiproliferative activities against a panel of four human cancer cell lines. Among
them, compound 4u is the most potent, exhibiting IC50 values ranging from 5.1 to
10.1 µM, respectively. Flow cytometry analysis and western blot analysis revealed
that treatment of compound 4u inducing MGCꢀ803 cells cellular early apoptosis via
activation of caspasesꢀ9/3. Furthermore, compound 4u effectively reduced the tumor
growth bared by human gastric cancer cells in vivo without obvious adverse side effects.
Our findings indicate that compound 4u may serve as a leading compound to target
solid tumors.
Keywords: Pyrazolidineꢀ3,5ꢀdione; Antitumor; In vitro; In vivo; Caspasesꢀ9/3;
Apoptosis
Cancer is a worldwide lifeꢀthreatening disease, the cancer patients and the death cases
are continually increasing in recent years, but the development of anticancer drugs
with high efficiency and minimal sideꢀeffect remain to be a challenge.1 Pyrazolidine
∗ Corresponding authors: (1) Tel.: +86 371 67781739. Eꢀmail: liuhm@zzu.edu.cn (H.ꢀM. Liu);
(2) Tel.: +86 371 67781896. Eꢀmail: zqr406@sina.com (Q.ꢀR. Zhang).
1

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diketones are a class of important nitrogenous heterocyclic compounds, which exhibit
diverse biological activities in living organisms.2ꢀ4 The biological properties of
modified pyrazolidine diketones depend upon structural features of the heterocyclic
ring system and side chains, providing a way for chemical modifications.5, 6
Since the phenylbutazone, a nonsteroidal antiꢀinflammatory agent had been
synthetized in 1946, a series of improved agents have been discovered, such as
oxyphenbutazone, γꢀketophenylbutazone, sulfinpyrazone (Figure 1). Moreover,
pyrazolidineꢀ3,5ꢀdione derivatives have generated considerable interest lately due to
their diverse biological activities, including the effect of antiꢀcardiovascular diseases,
antihyperglycemic,5 antiꢀtumour,7 antiꢀHIV,3
antiꢀinflammatory.
Recently,
pyrazolineꢀ3,5ꢀdione derivatives against COXꢀ2 protein as well as marked inhibition
of tumor progression and metastasis8ꢀ12 have been reported, based on a close
relationship between inflammation and cancer.13
Regarding to the pharmacological importance of pyrazolineꢀ3,5ꢀdione functional
group and continuation of our previous work14 in developing novel anticancer
derivatives on the side chains, we designed
a
series of 4ꢀsubstituted
1,2ꢀbis(4ꢀchlorophenyl)ꢀpyrazolidineꢀ3,5ꢀdiones derivatives by an efficient synthetic,
using different kinds of substituents R1 and R2, and investigated their cytotoxic
activities in vitro and in vivo. We found that compound 4u significantly inhibited
tumor growth in vitro and in vivo via inducing cell apoptosis.
O
O
S
R1
R2
O
O
O
O
O
O
O
O
N N
N N
N N
N N
Cl
Cl
Ketophenylbutazone
Oxyphenbutazone
Sulfinpyrazone
the Representative Compound
Figure 1. Three classical pyrazolidineꢀ3,5ꢀdiones derivatives and representative
2

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compound.
The preparations of 4ꢀsubstitutedꢀ1,2ꢀbis (4ꢀchlorophenyl)ꢀpyrazolidineꢀ3,5ꢀdiones
were outlined in Scheme 1. Dimerization of pꢀchloroaniline, followed by Znꢀmediated
reduction gave hydrazine 2. Condensation of 2 with diethyl malonate in the presence
of MeONa afforded compound 3. Condensation of compound 3 with aldehyde or
ketone gave compounds 4aꢀw. It is worth noting that no catalyst was needed for the
condensation between 3 and aldehyde or ketone. All the synthesized compounds were
fully characterized by 1H NMR, 13C NMR and high resolution mass spectra.
Scheme 1. Synthesis of compounds 4aꢀw. Reagents and conditions: (a) MnO2,
toluene, reflux; (b) Zn, NH4Cl, acetone, H2O, r.t; (c) NaOMe, diethylmalonate, EtOH,
50ꢀ140℃(dryness); (d) CH3OH, reflux.
All synthesized compounds were evaluated for their cytotoxic activities against four
human cancer cell lines, including MGCꢀ803 (gastric cancer), ECꢀ109 (esophageal
cancer), MCFꢀ7 (breast cancer) and SMMCꢀ7721 (hepatic cancer) and compared with
the positive control 5ꢀFu (5ꢀfluorouracil) by MTT assay15, 16. The IC50 values of the
tested compounds were listed in Table 1. As shown in Table 1, the cytotoxic activity
of the target compounds against four human cancer cell lines with the IC50 values
ranging from 5.1 to 80.2 µM. Among them, the most potent compound was 4u,
exhibiting IC50 values ranging from 5.1 to 10.1 µM, respectively.
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During the structureꢀactivity relationship (SAR) analysis, we found that aromatic ring
on the side chain of pyrazolidineꢀ3,5ꢀdione was important for antiproliferative
acitivity: the aromatic ring derivative compounds 4s, 4t, 4v and 4w were more potent
than the alicyclic hydrocarbon derivative compounds 4a and 4b.
Table 1. Antitumor activity of compounds 4aꢀw.
IC50(µM)a
Compound
R1
R2
MGCꢀ803
ECꢀ109
MCFꢀ7
SMMCꢀ7721
4a
4b
4c
4d
4e
4f
CH3
CH3
H
CH3
39.0 ± 1.1
66.5 ± 1.3
19.5 ± 0.9
80.2 ± 1.4
26.0 ± 1.0
32.6 ± 1.2
16.9 ± 1.4
17.3 ± 1.4
21.8 ± 1.1
24.3 ± 1.2
> 128
42.1 ± 1.6
41.1 ± 1.6
34.2 ± 1.1
> 128
39.6 ± 1.1
41.6 ± 1.8
23.2 ± 0.8
> 128
45.6 ± 1.2
49.2 ± 1.7
20.6 ± 1.2
> 128
CH2CH3
4ꢀOHꢀC6H4
4ꢀN(CH3)2ꢀC6H4
H
H
4ꢀFꢀC6H4
35.6 ± 0.6
17.3 ± 1.2
12.3 ± 0.7
10.1 ± 0.7
7.1 ± 0.5
39.0±1.1
31.4 ± 1.1
28.5 ± 0.9
30.4 ± 1.1
16.2 ± 1.3
22.8 ± 1.0
26.7 ± 1.0
> 128
23.3 ± 1.2
30.9 ± 0.9
16.6 ± 1.3
29.5 ± 1.1
64.9 ± 1.4
64.9 ± 1.4
> 128
H
4ꢀClꢀC6H4
4g
4h
4i
H
4ꢀBrꢀC6H4
H
3,4,5ꢀ(OCH3)3ꢀC6H2
3ꢀOCH3ꢀ4ꢀOHꢀC6H3
3ꢀOCH3ꢀ2ꢀOHꢀC6H3
H
4j
H
4k
4l
H
4ꢀOCH3ꢀC6H4
3ꢀClꢀC6H4
> 128
H
14.4 ± 1.2
26.6 ± 1.0
42.8 ± 1.2
> 128
22.6 ± 0.7
29.0 ± 1.2
55.6 ± 1.3
> 128
18.3 ± 1.0
18.1 ± 1.5
76.5 ± 1.1
> 128
20.2 ± 1.0
30.2 ± 1.3
57.0 ± 1.4
> 128
4m
4n
4o
4p
4q
4r
4s
H
3ꢀOCH3ꢀC6H4
4ꢀOHꢀC6H4
CH3
H
3,4ꢀ(OCH3)2ꢀC6H3
2,3ꢀ(OCH3)2ꢀC6H3
2ꢀOCH3ꢀC6H4
H
29.6 ± 0.9
28.6 ± 1.3
> 128
6.8 ± 0.2
30.4 ± 1.5
60.2 ± 1.3
13.1 ± 0.7
14.3 ± 0.5
9.9 ± 0.4
23.4 ± 1.0
32.7 ± 0.7
15.9 ± 0.9
11.9 ± 0.7
26.3 ± 1.3
> 128
14.1 ± 0.8
25.8 ± 1.0
> 128
H
H
2,4ꢀ(Cl)2ꢀC6H3
3,4ꢀ(F)2ꢀC6H3
H
35.7 ± 1.2
12.8 ± 1.1
5.1 ± 0.7
21.8 ± 1.1
27.2 ± 1.1
9.2 ± 0.7
22.0 ± 0.9
20.3 ± 1.0
9.4 ± 0.6
34.2 ± 0.9
20.2 ± 1.2
13.5 ± 1.0
35.8 ± 1.2
19.5 ± 1.0
10.1 ± 0.2
19.6 ± 1.2
26.2 ± 1.1
14.2 ± 1.3
4t
H
2ꢀfuranyl
styryl
4u
4v
4w
5ꢀFu
H
H
3ꢀindolyl
C6H5
H
a Inhibitory activity was assayed by exposure for 72 h to substances. Data are presented as the
means ± SDs of three independent experiments.
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To evaluate the importance of the electronegativity and position of substituent groups
on the aromatic ring for the antiproliferative acitivity, compounds 4f, 4g, 4l, and 4w
were synthesized. Introduction of a weak electronꢀwithdrawing group, a paraꢀchlorine
atom (4f), decreased the activity relative to 4w on three cancer cell lines, except
ECꢀ109 cells. Moving the chlorine from the paraꢀposition (4f) to the metaꢀposition (4l)
enhanced cytotoxicity activity. In addition, adding halogen size from chlorine (4f) to
bromine (4g) increased cytotoxicity activity on three cancer cell lines, except MCFꢀ7
cells.
The importance of the substitution pattern and the number of methoxy groups on the
phenyl ring of our compounds was also explored. The results showed that trimethoxy
substitution on the 3’4’ and 5’ꢀpositions of the phenyl ring (4h) was well tolerated.
And comparsion to the unsubstituted benzene derivative 4w, the trimethoxy
substitution increased cytotoxicity activity substantially. Further, moving methoxy
group from the paraꢀposition (4k) to the ortho-position (4m and 4p) produced an
increase of the antiproliferative activity on the four tested cell lines.
The cytotoxicity of compound 4u against human normal gastric epithelial cell (GESꢀ1)
and the cell viability, using different concentrations of compound 4u, was also
evaluated. As demonstrated in Figure 2, the cell viability decreased significantly with
the increasing concentrations of compound 4u in cancer cells. However, in the normal
gastric epithelial cell, the viability dropped slowly. The viability of human normal
gastric epithelial cell stood at around 75% even treated with 20 µM of 4u, indicating
the selectivity of compound 4u against the tested cancer cells.
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Figure 2. Selectivity of 4u on the cancer cell lines and human normal gastric
epithelial cell line. (A) Cell viability was measured by MTT assay. (B) Value of IC50
was summarized with histogram graphs. The results were calculated by Graphpad
prism software. Data are means ± SDs of three independent experiments.
Apoptosis is considered a major way of most of the anticancer drugs. On the basis of
the strong cytotoxicity of compound 4u in MGCꢀ803, then compound 4u was chosen
to further explore its mechanism of action. After treatment with 4u at the indicated
concentrations for 12 h, morphology changes of MGCꢀ803 cells were recorded using
an inverted microscope (Figure 3A). Besides, nuclear morphological changes at the
corresponding concentration were also recorded and qualitatively evaluated by means
of Hoechst 33258 fluorescent staining (Figure 3B) after incubation for 12 h at the
indicated concentrations. Typical apoptotic markers, including cell rounding, nuclear
condensation, nuclear fragmentation and apoptotic bodies were detected, especially at
the highest concentrations of 4u.
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0
4 µM
8 µM
12 µM
A
B
Figure 3. MGCꢀ803 cells treated by 4u showed typical apoptotic morphologies. (A)
Morphology changes in MGCꢀ803 cells treated with 4u at the indicated
concentrations. (magnification 200×). (B) Effects of 4u on the nuclear morphology of
MGCꢀ803 cells were assayed by Hoechst 33258 staining. (magnification 200×).
In order to better characterize the mode of cell death induced by compound 4u,
Annexin VꢀFITC/PI double staining was also applied, and a flow cytometer was used
for quantitive analysis for apoptotic cells. As shown in Figure 4, when the cells were
exposed to compound 4u for 12 h and 24 h, early apoptosis rates were extended from
6.3% (control) to 30.1%, and from 6.0% (control) to 52.7%, respectively. The results
indicate that compound 4u markedly increased the cellular apoptosis in a
concentration and time dependent manner.
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Figure 4. Apoptosis effect on human MGCꢀ803 cell line induced by compound 4u.
(A) Incubated for 12 h; (B) Incubated for 24 h. (C and D) Percentages of cells with
apoptosis were summarized with histogram graphs. **P <0.01 was considered
statistically highly significant.
To further illustrate the mechanism of compound 4u induced apoptosis, we examined
the effect of 4u on the activation of Caspaseꢀ9/3. Figure 5 showed that 4u
significantly increased the expression of cleaved Caspasesꢀ9/3 and decreased the
expression of their proenzymes. Meanwhile, the key proteins in the
mitochondriaꢀrelated apoptotic pathway were investigated. We found that the
proapoptotic protein, Bax, was upregulated and the antiꢀapoptotic protein, Bclꢀ2, was
downregulated in a concentration dependent manner after 24 h treatment of
compound 4u (Figure 6). Our findings indicate that compound 4u may be involved in
the mitochondriaꢀrelated apoptosis.
A
B
C
0 µM
4 µM
8 µM
12 µM
Proꢀcaspaseꢀ9
Cleavedꢀcaspaseꢀ9
Proꢀcaspaseꢀ3
Cleavedꢀcaspaseꢀ3
GAPDH
Figure 5. Expression of Cleavedꢀcaspasesꢀ9/3 and Proꢀcaspaseꢀ9/3 in MGCꢀ803 cells
after treatment by compound 4u for 24h. (A) Expression level of
Cleavedꢀcaspasesꢀ9/3 and Proꢀcaspaseꢀ9/3 were determined by western blot. (B)
Densitometry quantitation of Cleavedꢀcaspasesꢀ9 with indicated treatment. (C)
Densitometry quantitation of Cleavedꢀcaspasesꢀ3 with indicated treatment. Total
levels of GAPDH were used as loading control. *P <0.05 was considered statistically
significant. **P <0.01 was considered statistically highly significant.
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A
B
12 µM
C
4 µM
8 µM
0 µM
Bax
Bclꢀ2
GADPH
Figure 6. Expression of Bax and Bclꢀ2 in MGCꢀ803 cells after treatment by
compound 4u for 24h. (A) Expression level of Bax and Bclꢀ2 were determined by
western blot. (B) Densitometry quantitation of Bax with indicated treatment. (C)
Densitometry quantitation of Bclꢀ2 with indicated treatment. Total levels of GAPDH
were used as loading control. *P <0.05 was considered statistically significant. **P
<0.01 was considered statistically highly significant.
In vivo inhibitory effect of compound 4u on tumor growth was examined in a
xenograft model. Xenograft tumors were generated by subcutaneous implantation of
MGCꢀ803 cells into nude mice. After the treatment of compound 4u by oral
administration, the mouse body weights were monitored and the tumor sizes were
measured and recorded every 4 days (Figure 7A). After 28 days of treatment,
compound 4u significantly delayed the growth of tumor and reduced average tumor
weight by 60.4%, which is very close to the effect of the positive control capecitabine
(reduced average tumor weight by 62.7%) at the dose of 30mg/kg/d (Figure 7A, B and
D). And there was no apparent body weight loss during the treatment (Figure 7C).
These data indicate that compound 4u was efficacious in inhibiting the growth of
gastric tumor in vivo, but no obvious global toxicity.
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Figure 7. In vivo antitumor effects of compound 4u in MGCꢀ803 bearing nude mice.
(A) Tumor volume with the indicated treatment. (B) Represented tumors with the
indicated treatment. (C) Body weight with the indicated treatment. (D) Tumor weight
with the indicated treatment. *P <0.05 was considered statistically significant. **P
<0.01 was considered statistically highly significant. Data are mean ± SDs.
In conclusion, twentyꢀthree novel 4ꢀalkylidene(arylidene)ꢀ1,2ꢀbis(4ꢀchlorophenyl)ꢀ
pyrazolidineꢀ3,5ꢀdione derivatives were designed and synthesized from commercially
available pꢀchloroaniline. All synthesized compounds were evaluated for their
antitumor activities against four human cancer cell lines. Among them, the most
potent compound was 4u, exhibiting IC50 values ranging from 5.1 to 10.1 µM,
respectively. Further investigation showed that compound 4u caused the cellular
apoptosis via the activation of capspaseꢀ9/3 in a time and concentration dependent
manner. Meanwhile, compound 4u showed efficacious activity in inhibiting the
growth of gastric tumor in vivo with no obvious global toxicity. Synthesis of more
analogs, SAR studies and further mechanism investigations are under way and will be
reported in due course.
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Acknowledgements
This work was supported by National Natural Science Foundation of China (Project
No. 81430085 and Project No. 81172937 for H.ꢀM.L.; Project No.81270270 for W.Z.;
Project No.81272371 for G.ꢀZ.J.); the Basic Research Project of Henan Province (No.
142300410328 for Q.ꢀR.Z.) and the Research Project of Science and Technology
Bureau of Zheng Zhou City (No. 141PQYJS554 for Q.ꢀR. Z.).
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