Development of novel bis-pyrazole derivatives as antitumor agents with potent apoptosis induction effects and DNA damage

Article abstract of DOI:10.1016/j.ejmech.2017.11.098

A series of bis-pyrazole derivatives were designed and synthesized, and their antitumor effects in vitro and in vivo were investigated. Several compounds displayed good antiproliferative activity with IC₅₀ values in low-micromolar range against three human cancer cell lines in vitro, superior to 5-FU. The most potent compound 10M selectively inhibited human hepatocellular carcinoma cells but not non-tumor liver cell proliferation in vitro, and significantly triggered SMMC-7721 cell apoptosis by cleavage of both PARP and caspase-3 in a concentration-dependent manner. Further study revealed that the potent activity in the cell growth inhibition and apoptosis induction effects of 10M were related to DNA damage and activation of the p53 signaling pathway. Moreover, 10M showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo.

Full text of DOI:10.1016/j.ejmech.2017.11.098

Accepted Manuscript
Development of novel bis-pyrazole derivatives as antitumor agents with potent
apoptosis induction effects and DNA damage
Hong Dai, Shushan Ge, Jin Guo, Shi Chen, Meiling Huang, Jiaying Yang, Siyu Sun,
Yujun Shi, Yong Ling
PII:
S0223-5234(17)31001-2
10.1016/j.ejmech.2017.11.098
EJMECH 9973
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 October 2017
Revised Date: 27 November 2017
Accepted Date: 29 November 2017
Please cite this article as: H. Dai, S. Ge, J. Guo, S. Chen, M. Huang, J. Yang, S. Sun, Y. Shi, Y.
Ling, Development of novel bis-pyrazole derivatives as antitumor agents with potent apoptosis
induction effects and DNA damage, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2017.11.098.
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ACCEPTED MANUSCRIPT
Development of novel bis-pyrazole derivatives as antitumor agents
with potent apoptosis induction effects and DNA damage
Hong Dai a,b, Shushan Ge a,b, Jin Guo b,c, Shi Chen b,c, Meiling Huang b, Jiaying Yangb, Siyu Suna,
Yujun Shi a,*, and Yong Ling a,b,c,
*
a College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, People’s
Republic of China
b School of Pharmacy, Nantong University, Nantong 226001, People’s Republic of China
c Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong
University, Nantong 226001, China.
Graph Abstract:

ACCEPTED MANUSCRIPT
European Journal of Medicinal Chemistry
journal homepage: www.elsevier.com
Development of novel bis-pyrazole derivatives as antitumor agents with potent
apoptosis induction effects and DNA damage
Hong Dai a,b, Shushan Ge a,b, Jing Guo b,c, Shi Chen b,c, Meiling Huang b, Jiaying Yang b, Siyu Sun a, Yujun
Shi a,*, Yong Ling a,b,c,
a College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, People’s Republic of China
b School of Pharmacy, Nantong University, Nantong 226001, People’s Republic of China
c Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226001, China.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A series of bis-pyrazole derivatives were designed and synthesized, and their antitumor effects
in vitro and in vivo were investigated. Several compounds displayed good antiproliferative
activity with IC50 values in low-micromolar range against three human cancer cell lines in vitro,
superior to 5-FU. The most potent compound 10M selectively inhibited human hepatocellular
carcinoma cells but not non-tumor liver cell proliferation in vitro, and significantly triggered
SMMC-7721 cell apoptosis by cleavage of both PARP and caspase-3 in a concentration-
dependent manner. Further study revealed that the potent activity in the cell growth inhibition
and apoptosis induction effects of 10M were related to DNA damage and activation of the p53
signaling pathway. Moreover, 10M showed low acute toxicity to mice and significant growth
inhibition of the hepatoma tumor in vivo.
Available online
Keywords:
Synthesis
Antitumor agent
Bis-pyrazole derivatives
Apoptosis
2009 Elsevier Ltd. All rights reserved
.
DNA damage
1. Introduction
Like as energy and environmental crisis, cancer has become one of the most important things which people living on earth have to
face today [1]. Although great efforts from worldwide researchers have been devoted in this field, the mechanism and treatment method
of cancer remain very limited. Chemotherapy has established a new era of molecularly targeted therapeutics, the efficacy of existing
drugs for the treatment of cancers is rather limited [2], and it is necessary to develop potent and effective novel therapeutic agents to
overcome limitations of current therapy including the development of drug resistance.
For their fruitful applications in pharmaceutical and agrochemical fields, heterocyclic compounds have built a reputation in
bioactive molecules [3]. The majority of anticancer drugs currently used in clinical are derived from heterocyclic compounds [4-6]. As
an important branch of heterocycles, pyrazole have been widely involved in the development of various bioactive molecules such as
antibacterial, anti-inflammatory, antifungal, antiviral, and antimicrobial agents [7-16]. Apart from that, pyrazole-containing compounds
also show great potentials to be used as anticancer agents. As shown in Fig. 1, pyrazole-containing compounds a and b synthesized by
Ganga et al [17] exhibit good growth inhibitory activities against MIAPaCa-2, MCF-7 and HeLa human cancer cell lines. Effective
anti-proliferative activities against human lung cancer cell lines A549 has been observed by Sun et al [18] for compound c. Hassan and
the coworkers [19] had reported the wonderful bioactivities to colorectal cancer HCT116 and prostate adenocarcinoma PC-3 human
cancer cell lines of compounds d and e. Ding et al [20] also reported a significant effect of compound f to promote cell apoptosis with
an IC50 value of 15.22 µM.
Among various pyrazole derivatives, pyrazole oxime compounds have shown broad spectrum of antitumor, antibacterial,
insecticidal, antimicrobial, and antifungal activities and attracted increasing attention [21-32]. For instance, Park et al [33] reported an
excellent pyrazole oxime lead compound g (Fig. 1) delivering potential antitumor activities against XF498 and HCT15, with IC50 values
of 0.02 and 0.01 µg/mL, respectively. Stronger anti-colonic carcinoma activities superior to 5-FU and cisplatin have also been found in
our previously studies with pyrazole oxime derivatives bearing a 1,2,3-thiadiazole moiety [34]. Recently, Gomha [35] indicated that
increasing the number of pyrazole rings in the molecule structure can be in favor of their bioactivities. On the basis of above-mentioned
cases, incorporating substituted pyrazole groups with pyrazole oximes to form novel bis-pyrazole derivatives would facilitate its further
enhancement of anticancer activity. We herein report the design and syntheses of a series of novel bis-pyrazole derivatives (10a–n and
———
Corresponding author. Tel.: +86-513-85051892; fax: +86-513-85051892; e-mail: Lyyy111@sina.com, yjshi2015@163.com.

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European Journal of Medicinal Chemistry
10A-N), as well as the investigation on their in vitro and in vivo antitumor activities, selectivity, and apoptotic mechanism in human
ACCEPTED MANUSCRIPT
carcinoma cell lines.
O
F
O
S
Cl
O
O
N
N
N
H
N
N
N
N
H
H
N
N
N
N
N
H
N
N
N
N
O
Cl
O
O
c
a
b
H
O
Cl
HO
N
OH
N
N
N
HN
N
N
N
N
H
N
N
N
N
O
NH2
HN
Cl
O
O
O
O
d
f
e
Cl
N
H3C
N
N
O
R1
O
N
H3C
O
O
Cl
O
N
N
H3C
N
R2
10a-n, 10A-N
N
O
g
Fig. 1. Chemical structures of pyrazole derivatives a~g as antitumor agents and new designed compounds 10a–n and 10A-N
.
2. Results and discussions
2.1. Chemistry
R1
N
R1
N
O
O
O
O
i
ii
iii
O
O
R1
+
O
O
O
R1
N
N
O
O
O
H
O
2a-c
O
CH3
3a-c
1a-c
R1
N
R1
Cl
R1
Cl
Cl
vi
iv
v
N
Cl
N
OH
N
6a R1=C2H5
6b R1=4-FC6H4
N
N
O
CH3
CH3
CH3
6a-c
6c R1=4-ClC6H4
4a-c
5a-c
H3C
N
N
OH
R1
N
O
H3C
CHO
Cl
H3C
CHO
N
H3C
ix
H3C
R2
vii
viii
Cl
R2
N
N
N
O
N
N
N
O
N
CH3
R2
N
CH3
O
H3C
CH3
9
10a-n, 10A-N
7
8
R2
4-F
R2
4-F
Compd.
10a
10b
10c
10d
10e
R1
Compd.
10A
10B
10C
10D
10E
10F
10G
10H
10I
R1
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
C2H5
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-FC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
2-F
4-Cl
4-Br
4-I
4-CH3
4-OCH3
4-OCF3
4-F
4-Cl
3-Cl
2-Cl
4-Br
4-I
4-CH3
3-CH3
2-CH3
10f
10g
10h
10i
4-Cl
4-Br
10j
10J

4-OCH3
2-OCH3
4-OCF3
2-OCF3
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-ClC6H4
4-I
C2H5
C2H5
C2H5
C2H5
10k
10l
10m
10n
10K
ACCEPTED MANUSCRIPT
4-CH3
4-OCH3
4-OCF3
10L
10M
10N
Scheme 1. General procedure for the synthesis of compounds 10a-n and 10A-N. Reagents and conditions: (i) EtONa, EtOH, 0 ,12 h, 72-80%; (ii)
hydrazine hydrate, HOAc, EtOH, rt, 12 h, or 80% hydrazine hydrate, concentrated hydrochloric acid, EtOH, 85 , 5-8 h, 65-70%; (iii) Me2SO4,
Na2CO3, CH2Cl2, reflux, 24 h, 68-75%; (iv) SO2Cl2, ClCH2CH2Cl, reflux, 4 h, 75-83%; (v) LiAlH4, THF, 0 , 1 h, 63-69%; (vi) SOCl2, CH2Cl2, 0
,
20 min, rt, 1 h, 55-62%; (vii) substituted phenols, NaOH, EtOH, reflux, 2-3 h, DMSO, 105 , 10-18 h, 56-71%; (viii) hydroxylamine hydrochloride,
KOH, MeOH, reflux, 6-17 h, 59-68%; (ix) compounds 6a-6c, K2CO3, Cs2CO3, MeCN, reflux, 12-18 h, 75-86%.
The synthetic route for the target compounds 10a-n and 10A-N was summarized in Scheme 1. Claisen condensation of different
substituted ketones with diethyl oxalate afforded ethyl 2,4-dioxo substituted butanoates 1a-c, which were further reacted with hydrazine
to obtain ethyl 3-phenyl-1H-pyrazole-5-carboxylates 2a-c [36]. Compounds 2a-c were treated with dimethyl sulfate to afford ethyl 1-
methyl-3-ethyl(or aryl)-1H-pyrazole-5-carboxylates 3a-c. Then the reaction of compounds 3a-c with sulfuryl chloride gave compounds
4a-c conveniently, which were transformed into intermediates 5a-c by the addition of LiAlH4. The reaction of compounds 5a-c with
thionyl chloride produced the key intermediates 6a-c. Meanwhile, the condensation of 1-methyl-3-methyl-5-chloro-1H-pyrazole-4-
carbaldehyde 7 [37] with various substituted phenols under basic condition to form 1-methyl-3-methyl-5-aryloxy-1H-pyrazole-4-
carbaldehyde 8. Intermediate 8 was reacted with hydroxylamine hydrochloride under basic condition to provide 1-methyl-3-methyl-5-
aryloxy-1H-pyrazole-4-carbaldehyde oxime 9 [38]. Finally, compound 9 and the key intermediates 6 were mixed in acetonitrile and
heated to reflux for 12-18 h using potassium carbonate as alkali and cesium carbonate as catalyst, and a series of novel bis-pyrazole
derivatives 10a-n and 10A-N were obtained in good yields.
2.2. MTT assay of compounds 10a-n and 10A-N
All the synthesized compounds 10a-n and 10A-N were preliminarily screened for their cancer cell growth inhibitory activity against
human hepatocellular carcinoma cells (SMMC-7721) using MTT assay, and 5-Fluorouracil (5-FU) and adriamycin (ADM) were used
as the positive compounds. As shown in Fig. 2, ADM significantly inhibited the proliferation of the cells. It was observed that most
compounds strongly inhibited cell growth of these cancer cell lines at 25 µM, superior to 5-FU. Interestingly, compounds 10A-N with
an aryl group showed greater inhibition of cancer cells than compounds 10a-n with an ethyl group at the R1 positions, especially, 10A,
10B, 10E-J, and 10L-N comparable to ADM with the inhibition by 80%.
100
80
60
40
20
0
Compounds (25 µM)
Fig. 2. Inhibition of cell proliferation (%) of target compounds against SMMC-7721 cell lines after incubation for 72 h at a concentration of 25
µM. Data are expressed as means + SD of each compound from three separate measurements.
These active compounds 10A, 10B, 10E-J, and 10L-N were further assayed for their antiproliferative activity against three cancer
cell lines including hepatocellular carcinoma cells (SMMC-7721), human colonic carcinoma cells (HCT116), and human gastric cancer
cells (SGC-7901) by MTT assay. IC50 values of the tested compounds against three tumor cell lines above were listed in Table 1. All
tested compounds displayed antiproliferative potency in the low micromolar range in all three cells, which were much better than those
of 5-FU. Especially, compounds 10E-G, 10L, and 10M exhibited significant antiproliferative effects with the IC50 values of 0.76-5.84
µM, which were comparable to or better than ADM (IC50 = 2.31-4.12 µM). Notably, 10M exhibited the most potent inhibitory activity
with IC50 values of 0.76-2.01 µM against all tested cancer cells, which were nearly two- to three-fold better than ADM and seventeen-
to fifty-fold more potent than 5-FU (IC50 = 29.5-37.8 µM).
Table 1. IC50 values of compounds 10A, 10B, 10E-J, and 10L-N against three human cancer cell linesa
In vitro antiproliferative activity (IC50a, µM)
Compd.
SGC7901
SMMC-7721
6.64 ± 0.72
HCT116
7.08 ± 0.65
8.02 ± 0.67
10A

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European Journal of Medicinal Chemistry
8.11 ± 0.76
7.97 ± 0.9
A
1
CCEPTED MANUSCRIPT
6.03 ± 0.55
2.91 ± 0.37
1.18 ± 0.23
2.31 ± 0.26
7.25 ± 0.66
4.67 ± 0.49
5.91 ± 0.65
2.85 ± 0.33
1.26 ± 0.25
3.02 ± 0.29
29.5 ± 3.76
2.31 ± 0.30
10B
10E
10F
10G
10H
10I
10J
10L
10M
4.62 ± 0.52
5.23 ± 0.48
2.05 ± 0.33
5.21 ± 0.65
3.23 ± 0.29
6.82 ± 0.77
7.13 ± 0.68
5.84 ± 0.65
0.76 ± 0.11
8.67 ± 0.56
37.8 ± 4.62
2.67 ± 0.25
2.84 ± 0.36
2.97 ± 0.41
4.43 ± 0.52
7.51 ± 0.90
5.96 ± 0.49
3.73 ± 0.38
2.01 ± 0.31
3.22 ± 0.44
10N
5-Fluorouracil
ADM
34.4 ± 4.15
4.12 ± 0.38
a The inhibitory effects of individual compounds on the proliferation of cancer cell lines were determined by the MTT assay. The data are expressed as the mean
± SD of three independent experiments data.
Given the strong growth inhibitory activity of 10M in vitro, the selectivity profile was investigated by examining its inhibitory
effects on the growth of both cancer cells SMMC-7721 and normal liver cells LO2. It was found that treatment with increased dose of
10M had no significant effect on the survival of non-tumor LO2 cells while the same treatment induced majority of SMMC-7721 cell
death (Fig. 3), suggesting 10M might possess selective antiproliferative activity against tumor cells.
Fig. 3. Inhibitory effects of 10M on the proliferation of SMMC-7721 and LO2 cells. Cells were incubated with the indicated concentrations of
10M for 72 h. Cell proliferation was assessed using the MTT assay. Data are means ± SD of the inhibition (%) from three independent
experiments.
The Structure–activity relationship (SAR) studies of designed compounds revealed that the R1 and R2 groups have significant
impacts on inhibiting cell growth. As for R1 substituent, compounds 10A-N with an aryl ring displayed stronger anti-proliferative
activity against tested cancer cells than the corresponding compounds 10a-n carrying an ethyl moiety. As for R2 group, compounds
10E-G and 10L-N containing methyl, methoxy, or trifluoromethoxy group indicated more potent activity than the corresponding
compounds 10A-D and 10H-K containing halogen group.

Fig. 4. Compound 10M induces SMMC-7721 cell apoptosis in vitro. SMMC-7721 cells were incubated with the indicated concentrations of
ACCEPTED MANUSCRIPT
10M, or ADM (3.2 µM) for 72 h, and the cells were stained with FITC-Annexin V/PI, followed by flow cytometry analysis. (A) Flow
cytometry analysis. (B) Quantitative analysis of apoptotic cells. Data are expressed as means ± SD of the percentages of apoptotic cells from
three independent experiments. *P< 0.01 vs control.
2.3. Compound 10M induces apoptosis in SMMC-7721 cells
To determine whether the inhibitory effects of 10M on hepatocellular carcinoma cell proliferation are accompanied by enhanced
cancer cell apoptosis, SMMC-7721 cells were incubated with different concentrations of 10M, or ADM for 72 h, and the percentages of
apoptotic cells were determined by FITC-Annexin V/PI staining and flow cytometry. As shown in Fig. 4, the percentage of annexin V +
apoptotic SMMC-7721 cells gradually increased for those cells exposed to the increasing concentrations of 10M (29.6% for 0.2 µM;
49.2% for 0.8 µM; and 63.2% for 3.2 µM), which demonstrating that the incubation with 10M induced SMMC-7721 cell apoptosis in a
dose-dependent manner. In contrast, incubations performed using ADM (3.2 µM) only induced apoptosis of 47.5% SMMC-7721 cells.
To further investigate the apoptosis induction of 10M, we examined the expression of apoptotic proteins Bax, Bcl-2, and the
cleavage states of caspase-3 and PARP, in response to 10M treatment (Fig. 5). Sub-confluent SMMC-7721 hepatocellular carcinoma
cells were treated with or without 10M for 72 h, and then lysed and analyzed by western blot. β-Actin expression was used as an
internal control. It indicated that treatment with 10M dramatically increased pro-apoptotic Bax expression in the relative levels, but
reduced the anti-apoptotic Bcl-2 expression in a dose-dependent manner. Furthermore, compound 10M resulted in more significant
cleavage of both PARP and caspase-3 than the control group (Fig. 5). Importantly, 10M treatment also induced more cleavage of PARP
and caspase-3 than the AMD treated group at the same concentration of 3.2 µM. Taken together, these results confirmed that 10M
treatment induced apoptosis in SMMC-7721 cells.
Fig. 5. (A) The expression of Bax, Bcl-2, cleaved Caspase 3 and PARP, and β-actin was examined by western blot analysis. SMMC-7721 cells
were incubated with, or without, 10M and ADM at the indicated concentrations for 72 h and the levels of protein expression were detected
using specific antibodies. Data shown are representative images of each protein for three separate experiments. (B) Quantitative analysis of
Bax, Bcl-2, and cleaved Caspase 3 and PARP. The relative levels of each protein compared to control β-actin were determined by densimetric
scanning. Data are expressed as means ± SD from three separate experiments. *P < 0.01 vs respective control.
Fig. 6. Immunoblot analysis of the expression of DNA damage and DNA-damage response p53 activation in vitro. (A) SMMC-7721 cells were
treated with vehicle (control), different doses of 10M, or ADM were homogenized, and their lysates were subjected to immunoblot analysis
using anti-H2AXS139ph, antiphospho-p53 (ser15), anti-p53, and anti-β-actin antibodies, respectively. β-Actin was used as the control. (B)
Quantitative analysis. The relative levels of each signaling event to control β-actin were determined by densimetric scanning. The data are
expressed as means ± SD from three separate experiments. *P < 0.01 vs respective control.
2.4. Compound 10M induces DNA damage and activates the p53 signaling pathway

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European Journal of Medicinal Chemistry
It is well-known that DNA damage is one of the molecular events associated with cell apoptosis [39]. Histone H2A.X, a variant
ACCEPTED MANUSCRIPT
form of histone H2A, is required for checkpoint-mediated DNA repair following double-stranded DNA breaks [40]. To verify whether
the anti-cancer activities of bis-pyrazole derivatives were partly from DNA damage, we examined the DNA damage extent and the
change of the DNA-damage response p53 signaling pathway caused by 10M in HCC cells. In the present study, we used histone H2AX
phosphorylation as DNA damage markers. SMMC-7721 cells were treated with vehicle or 10M for 72 h, using ADM as positive
controls. H2AXS139ph and p-p53 (ser15) were then detected using western blot analysis. As shown in Fig. 6, treatment with 10M
dose-dependently increased the levels of H2AX phosphorylation in SMMC-7721 cells. Importantly, H2AXS139ph level in both 0.8 µM
and 3.2 µM 10M treatment groups was significantly higher than that in 0.8 µM ADM group.
p53 is an important tumor suppressor protein that plays a major role in cellular response to DNA damage. Ser 15, a common site for
a variety of kinases such as ATM/ATR/DNA-NK, is activated during the DNA repair pathway. p53 acts as a nuclear transcription factor
and transactivates multiple genes involved in apoptosis progress [41]. To evaluate the effect of new compounds on p53 activation,
SMMC-7721 cells were cultured in the presence of 10M for 72 h at 0.2, 0.8, and 3.2 µM concentrations, respectively. Western blotting
results showed a significantly increased p53 phosphorylation at Ser15 in response to 10M treatment in dose-dependent manner (Fig. 6).
These results demonstrated that the activation of p53 as downstream signaling molecules in response to DNA damage induced by new
bis-pyrazole derivatives.
2.5. In vivo anti-cancer activity of 10M
To evaluate the safety of 10M in vivo, groups of ICR mice were injected intraperitoneally with a single dose of 10M or vehicle
control, respectively. The survival of mice was monitored up to 14 days after injection. Only one mice that had been treated with 10M
at the highest dose (488.3 mg/kg) survived (shown in Table 2). In contrast, injection with 10M at the lowest dose (200 mg/kg) did not
cause any death and abnormality in eating, drinking, body weight, and activity throughout the observation period. As a result, the LD50
value of 10M was calculated to be 349.6 mg/kg for this train of mice.
Next, we established a BALB/c nude mice model which was inoculated subcutaneously with SMMC-7721 cells to evaluate the in
vivo antitumor activity of 10M. After the establishment of solid tumor, BALB/c nude mice were randomly intraperitoneally
administered with 10M, ADM, and vehicle, respectively. The changes in tumor volumes and weights were measured over 19 days.
Compared with the vehicle control, treatment with 10M significantly reduced the tumor volume growth rate in a dose-dependent
manner (Fig. 7). It was observed that treatment with 10M (30 mg/kg) strongly inhibited the growth of the tumor cells in vivo similar to
ADM at the dose of 15 mg/kg. Importantly, the tumor weights (0.61 ± 0.04, 0.38 ± 0.02 g) in the mice treated with 10M at 15 and 30
mg/kg, respectively, were significantly reduced by 44.00% and 65.35% compared to the vehicle-treated controls (1.09 ± 0.02 g, p <
0.01). Besides, there was no statistical difference in body weight in postinoculation among the four groups of mice. Together, our data
clearly demonstrated that 10M could evidently inhibit the growth of tumor in vivo.
Table 2 The acute toxicity of 10M in mice
Number of dead mice
1h 4h 1d 2d 3d 4d 5-14d
Dose (mg/kg) Number of mice
Total death Survival (%) on day 14
488.3
390.6
312.5
250.0
200.0
10
10
10
10
10
0
0
0
0
0
0
0
0
0
0
1
1
1
1
0
1
1
1
1
0
2
2
0
1
0
2
1
2
0
0
3
1
0
0
0
9
6
4
3
0
10
40
60
70
100
Fig. 7. Inhibitory effects of 10M on the growth of an implanted SMMC-7721 xenograft in nude mice. BALB/c nude mice were subcutaneously
inoculated with SMMC-7721 cells. After establishment of solid tumor, the mice were randomized and treated with solvent control, ADM, or
10M at the indicated doses, respectively. The growth of tumors was measured longitudinally. Data are expressed as means (SD of tumor
volumes at each time point for each group of mice (n = 6 per group)). *P < 0.01 vs control group.

3. Conclusions
ACCEPTED MANUSCRIPT
In summary, we designed and synthesized a series of new bis-pyrazole compounds and investigated their antitumor effects in vitro
and in vivo. Some compounds displayed potent cell growth inhibitory activity with IC50 values in low-micromolar range against three
human cancer cells in vitro. The most potent compound 10M possessed stronger antiproliferative activity of cancer cells than ADM,
and did not affect proliferation of non-tumor LO2 cells in the effective dose against cancer cells. In addition, 10M exhibited a
significant induction of cell apoptosis through marked cleavage of both PARP and caspase-3. Furthermore, our study demonstrated that
the potent activity in the cell growth inhibition and apoptosis induction effects of 10M may be related to DNA damage and activation of
the p53 signaling pathway. Finally, 10M displayed low acute toxicity and significant growth inhibition of cancer cells in vivo.
4. Experimental section
4.1. General methods and materials
1H NMR and 13C NMR spectra were obtained on a Bruker AV400 spectrometer (Bruker Co., Switzerland) in CDCl3 solution with
tetramethylsilane as the internal standard. Chemical shift values (δ) are given in parts per million. The melting points were determined
on an X-4 binocular microscope melting point apparatus (Beijing Tech Instrument Co., Beijing, China) and are uncorrected. High
resolution mass spectra (HRMS) were recorded on Shimadzu LCMS-IT-TOF. Compounds 1a-c, 2a-c, and 7-9 were synthesized
according the methods of literatures [36-38]. The reagents used were of analytical grade, where the anhydrous reagents were used after
pretreatment. All anhydrous solvents were dried and purified by standard techniques.
4.1.1. General procedure for the preparation of compounds 3a-c
To a solution of compound 2 (77 mmol) in dichloromethane (100 mL) was added sodium carbonate (107 mmol), the resulting mixture
was heated to 40 . To the above solution was added dimethyl sulfate (107 mmol). The mixture was refluxed for 24 h, and then the
reaction mixture was poured in water and extracted with dichloromethane. The combined extracts were dried over anhydrous sodium
sulfate and filtered, the crude product was concentrated and was purified by silica gel column chromatography (hexane-EtOAc, 20:1
elution) to give compounds 3a-c.
Ethyl 3-ethyl-1-methyl-1H-pyrazole-5-carboxylate (3a). Yellow oil; yield, 75%; 1H NMR (400 MHz, CDCl3) δ: 6.64 (s, 1H,
Pyrazole-H), 4.34 (q, J=7.2 Hz, 2H, OCH2), 4.12 (s, 3H, N-CH3), 2.64 (q, J=7.6 Hz, 2H, CH2), 1.37 (t, J=7.2 Hz, 3H, CH3), 1.24 (t,
J=7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 160.0, 153.1, 132.9, 108.9, 60.9, 39.1, 21.2, 14.3, 13.8.
1
Ethyl 3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-carboxylate (3b). White solid; yield, 68%; mp: 72-74 °C; H NMR (400 MHz,
CDCl3) δ: 7.73 (d, J=6.8 Hz, 2H, Ar-H), 7.37 (d, J=8.4 Hz, 2H, Ar-H), 7.09 (s, 1H, Pyrazole-H), 4.38 (q, J=7.2 Hz, 2H, OCH2), 4.12 (s,
3H, N-CH3), 1.40 (t, J=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 159.7, 148.6, 133.9, 133.8, 131.1, 128.9, 126.7, 107.8, 61.2,
39.7, 14.3.
1
Ethyl 3-(4-chlorophenyl)-1-methyl-1H-pyrazole-5-carboxylate (3c). White solid; yield, 71%; mp: 77-79 °C; H NMR (400 MHz,
CDCl3) δ: 7.73 (d, J=8.4 Hz, 2H, Ar-H), 7.37 (d, J=8.4 Hz, 2H, Ar-H), 7.10 (s, 1H, Pyrazole-H), 4.39 (q, J=7.2 Hz, 2H, OCH2), 4.23 (s,
3H, N-CH3), 1.41 (t, J=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 159.7, 148.6, 134.0, 133.9, 130.9, 128.9, 126.8, 107.8, 61.2,
39.7, 14.3.
4.1.2. General procedure for the preparation of compounds 4a-c
To a solution of compound 3 (40 mmol) in 1,2-dichloroethane (100 mL) was added dropwise sulfuryl chloride (44 mmol). The
reaction mixture was refluxed for 4 h. After the completion of reaction, the mixture was added water (100 mL), the product was
extracted from water with ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate, filtered, and evaporated to
produce compounds 4a-c.
1
Ethyl 4-chloro-3-ethyl-1-methyl-1H-pyrazole-5-carboxylate (4a). Yellow oil; yield, 83%; H NMR (400 MHz, CDCl3) δ: 4.41 (q,
J=7.2 Hz, 2H, CH2), 4.09 (s, 3H, N-CH3), 2.66 (q, J=7.6 Hz, 2H, CH2), 1.41 (t, J=7.2 Hz, 3H, CH3), 1.24 (t, J=7.6 Hz, 3H, CH3). 13C
NMR (100 MHz, CDCl3) δ: 159.2, 150.4, 129.1, 113.1, 61.3, 40.6, 19.2, 14.2, 12.8.
1
Ethyl 4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-carboxylate (4b). White solid; yield, 75%; mp: 83-85 °C; H NMR (400
MHz, CDCl3) δ: 7.83 (d, J=8.4 Hz, 2H, Ar-H), 7.41 (d, J=8.8 Hz, 2H, Ar-H), 4.45 (q, J=7.2 Hz, 2H, CH2), 4.19 (s, 3H, N-CH3), 1.44 (t,
J=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 159.0, 145.7, 134.4, 130.4, 129.5, 128.9, 128.7, 112.5, 61.6, 41.2, 14.2.
1
Ethyl 4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazole-5-carboxylate (4c). White solid; yield, 78%; mp: 76-78 °C; H NMR (400
MHz, CDCl3) δ: 7.82 (d, J=8.4 Hz, 2H, Ar-H), 7.41 (d, J=8.4 Hz, 2H, Ar-H), 4.45 (q, J=7.2 Hz, 2H, CH2), 4.18 (s, 3H, N-CH3), 1.44 (t,
J=7.2 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 159.0, 145.7, 134.4, 130.4, 129.5, 128.7, 112.5, 61.6, 41.2, 14.2.
4.1.3. General procedure for the preparation of compounds 5a-c
To a cold solution of compound 4 (40 mmol) in THF (100 mL) was added LiAlH4 (80 mmol) in portions. After addition, the reaction
mixture was stirred at room temperature for 1 h. After completion of the reaction, the mixture was cooled by ice-water bath as added
water slowly. Then the mixture was filtered, the filtrate was extracted with ethyl acetate. The combined extracts were dried over
anhydrous sodium sulfate, filtered and evaporated to afford compounds 5a-c.
(4-Chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methanol (5a). White solid; yield, 69%; mp: 125-127 °C; 1H NMR (400 MHz, CDCl3) δ:
4.62 (s, 2H, CH2), 3.83 (s, 3H, N-CH3), 2.59 (q, J=7.6 Hz, 2H, CH2), 1.21 (t, J=7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 149.5,
137.7, 107.4, 53.0, 37.1, 19.4, 13.0.

8
European Journal of Medicinal Chemistry
1
[4-Chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-yl]methanol (5b). White solid; yield, 63%; mp: 112-114 °C; H NMR (400
ACCEPTED MANUSCRIPT
MHz, CDCl3) δ: 7.82 (d, J=8.8 Hz, 2H, Ar-H), 7.39 (d, J=8.4 Hz, 2H, Ar-H), 4.72 (s, 2H, CH2), 3.96 (s, 3H, N-CH3). 13C NMR (100
MHz, CDCl3) δ: 144.7, 139.0, 134.0, 130.1, 129.0, 128.7, 128.4, 115.5, 107.1, 53.2, 37.7.
1
[4-Chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-yl]methanol (5c). White oil; yield, 65%; H NMR (400 MHz, CDCl3) δ: 7.81
(d, J=8.8 Hz, 2H, Ar-H), 7.39 (d, J=8.8 Hz, 2H, Ar-H), 4.71 (s, 2H, CH2), 3.95 (s, 3H, N-CH3). 13C NMR (100 MHz, CDCl3) δ: 144.7,
139.0, 134.0, 130.1, 128.7, 128.4, 107.0, 53.2, 37.7.
4.1.4. General procedure for the preparation of compounds 6a-c
To a cold solution of compound 5 (35 mmol) in dichloromethane (100 mL) was added dropwise thionyl chloride (70 mmol). The
resulting mixture was stirred at room temperature for another 1 h. The mixture was cooled by ice-water bath and water was slowly
added thereto. The product was extracted with ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate, filtered
and evaporated to obtain compounds 6a-c.
1
4-Chloro-5-(chloromethyl)-3-ethyl-1-methyl-1H-pyrazole (6a). White solid; yield, 62%; mp: 67-69 °C; H NMR (400 MHz, CDCl3)
δ: 4.58 (s, 2H, CH2), 3.87 (s, 3H, N-CH3), 2.63 (q, J=7.6 Hz, 2H, CH2), 1.24 (t, J=7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ:
149.7, 134.5, 108.6, 37.2, 32.6, 19.4, 12.8.
4-Chloro-5-(chloromethyl)-3-(4-fluorophenyl)-1-methyl-1H-pyrazole (6b). White solid; yield, 58%; mp: 56-58 °C; 1H NMR (400 MHz,
CDCl3) δ: 7.83 (d, J=8.4 Hz, 2H, Ar-H), 7.39 (d, J=8.8 Hz, 2H, Ar-H), 4.65 (s, 2H, CH2), 3.97 (s, 3H, N-CH3). 13C NMR (100 MHz,
CDCl3) δ: 164.0, 145.0, 136.0, 134.2, 129.9, 129.0, 128.7, 115.6, 108.1, 37.7, 32.5.
1
4-Chloro-5-(chloromethyl)-3-(4-chlorophenyl)-1-methyl-1H-pyrazole (6c). White oil; yield, 55%; H NMR (400 MHz, CDCl3) δ:
7.83 (d, J=8.4 Hz, 2H, Ar-H), 7.39 (d, J=8.4 Hz, 2H, Ar-H), 4.65 (s, 2H, CH2), 3.97 (s, 3H, N-CH3). 13C NMR (100 MHz, CDCl3) δ:
145.0, 136.0, 134.2, 129.9, 128.7, 128.3, 108.1, 37.7, 32.5.
4.1.5. General procedure for the preparation of the target compounds 10a-n and 10A-N
To a solution of compound 6 (12 mmol) and compound 9 (10 mmol) in acetonitrile (40 mL) was added potassium carbonate (20
mmol) and cesium carbonate (5 mmol). The resulting reaction mixture was refluxed for 12-18 h. On completion of the reaction, the
mixture was filtered and the solution was evaporated to give the crude product, which was recrystallized from a mixture of ethyl acetate
and petroleum ether to afford the title compounds 10a-n and 10A-N.
5-(4-Fluorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10a). White solid; yield, 79%; mp: 72-74 °C; 1H NMR (400 MHz, CDCl3) δ: 7.74 (s, 1H, CH=N), 7.00 (t, J = 8.6 Hz, 2H, Ar-H), 6.86-
6.83 (m, 2H, Ar-H), 4.97 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.59 (s, 3H, N-CH3), 2.61 (q, J = 7.6 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 1.22
(t, J = 7.6Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 158.8, 152.6, 149.4, 147.9, 147.1, 141.3, 135.2, 116.7, 116.6, 116.5, 109.1,
99.7, 63.8, 37.3, 34.3, 19.4, 14.8, 13.0. HRMS (ESI) m/z calcd for C19H22ClFN5O2, 406.1446; found, 406.1441 [M + H]+.
5-(2-Fluorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
1
(10b). White solid; yield, 78%; mp: 58-60 °C; H NMR (400 MHz, CDCl3) δ: 7.75 (s, 1H, CH=N), 7.19 (m, 1H, Ar-H), 7.06 (m, 2H,
Ar-H), 6.76 (m , 1H, Ar-H). 4.95 (s, 2H, CH2), 3.77 (s, 3H, N-CH3), 3.64 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H, CH2), 2.34 (s, 3H,
CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 151.9, 149.4, 147.5, 147.0, 141.1, 135.2, 124.7, 124.6, 124.5,
117.3, 117.1, 116.6, 109.1, 99.6, 63.3, 37.4, 34.3, 19.4, 14.6, 13.0. HRMS (ESI) m/z calcd for C19H22ClFN5O2, 406.1446; found,
406.1443 [M + H]+.
5-(4-Chlorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10c). White solid; yield, 80%; mp: 74-75 °C; 1H NMR (400 MHz, CDCl3) δ: 7.75 (s, 1H, CH=N), 7.28 (d, J = 9.2 Hz, 2H, Ar-H), 6.83
(d, J = 8.8 Hz, 2H, Ar-H), 4.95 (s, 2H, CH2), 3.77 (s, 3H, N-CH3), 3.64 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H, CH2), 2.34 (s, 3H,
CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 155.1, 149.4, 147.4, 147.1, 141.2, 135.2, 130.0, 128.9, 116.6,
109.1, 99.9, 63.4, 37.4, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for C19H22Cl2N5O2, 422.1151; found, 422.1141 [M + H]+.
5-(3-Chlorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10d). White solid; yield, 78%; mp: 77-78 °C; 1H NMR (400 MHz, CDCl3) δ: 7.76 (s, 1H, CH=N), 7.25 (t, J = 8.0 Hz, 1H, Ar-H), 7.10
(d, J = 8.0 Hz, 1H, Ar-H), 6.90 ( s, 1H, Ar-H), 6.79-6.76 ( m, 1H, Ar-H), 4.97 (s, 2H, CH2), 3.78 ( s, 3H, N-CH3), 3.58 (s, 3H, N-CH3),
2.60 (q, J = 7.6 Hz, 2H, CH2), 2.36 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 157.1, 149.4, 147.1,
147.0, 141.2, 135.52, 135.2, 130.8, 124.1, 115.9, 113.5, 109.1, 100.0, 63.4, 37.4, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for
C19H22Cl2N5O2, 422.1151; found, 422.1141 [M + H]+.
5-(2-Chlorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10e). White solid; yield, 79%; mp: 73-75 °C; 1H NMR(400 MHz, CDCl3) δ: 7.74 (s, 1H, CH=N), 7.45 (m, 1H, Ar-H), 7.16 (m, 1H, Ar-
H), 7.07 (m, 1H, Ar-H), 6.68 (m, 1H, Ar-H), 4.95 (s, 2H, CH2), 3.77 (s, 3H, N-CH3), 3.62 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H,
CH2), 2.35 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 152.1, 149.4, 147.3, 147.1, 141.1, 135.1, 131.0,
128.1, 124.7, 122.7, 115.5, 109.0, 99.9, 63.4, 37.4, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for C19H22Cl2N5O2, 422.1151; found,
422.1149 [M + H]+.
5-(4-Bromophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10f).White solid; yield, 77%; mp: 79-81 °C; 1H NMR (400 MHz, CDCl3) δ: 7.75 (s, 1H, CH=N), 7.42 (d, J = 8.8 Hz, 2H, Ar-H), 6.78
(d, J = 9.2 Hz, 2H, Ar-H), 4.96 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.59 (s, 3H, N-CH3), 2.61 (q, J = 7.6 Hz, 2H, CH2), 2.35 (s, 3H,
CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 155.7, 149.4, 147.3, 147.1, 141.2, 135.2, 132.9, 117.0, 116.3,
109.1, 99.9, 63.3, 37.4, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for C19H22BrClN5O2, 466.0645; found, 466.0635 [M + H]+.

5-(4-Iodophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
ACCEPTED MANUSCRIPT
1
(10g). White solid; yield, 78%; mp: 127-129 °C; H NMR (400 MHz, CDCl3) δ: 7.74 (s, 1H, CH=N), 7.61 (d, J = 8.8 Hz, 2H, Ar-H),
6.66 (d, J = 8.8 Hz, 2H, Ar-H), 4.96 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.58 (s, 3H, N-CH3), 2.61 (q, J = 7.6 Hz, 2H, CH2), 2.34 (s, 3H,
CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 156.5, 149.4, 147.2, 147.1, 141.2, 138.9, 135.2, 117.5, 109.1, 99.9,
86.6, 63.4, 37.4, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for C23H21IClFN5O2, 514.0507; found, 514.0497 [M + H]+.
5-(4-Methylphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10h). White solid; yield, 77%; mp: 52-53 °C; 1H NMR (400 MHz, CDCl3) δ: 7.75 (s, 1H, CH=N), 7.10 (d, J = 8.4 Hz, 2H, Ar-H), 6.77
(d, J = 8.4 Hz, 2H, Ar-H), 4.96 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.58 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H, CH2), 2.35 (s, 3H,
CH3), 2.31 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 154.6, 149.4, 148.3, 146.9, 141.6, 135.3, 133.3,
130.4, 115.1, 109.0, 99.8, 63.3, 37.4, 34.2, 20.6, 19.4, 15.0, 13.0. HRMS (ESI) m/z calcd for C20H25ClN5O2, 402.1697; found, 402.1693
[M + H]+.
5-(3-Methylphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
1
(10i). Yellow solid; yield, 79%; mp: 75-76 °C; H NMR(400 MHz, CDCl3) δ: 7.76 ( s, 1H, CH=N ), 7.18 ( t, J = 7.8 Hz, 1H, Ar-H ),
6.91 (d, J = 7.2 Hz, 1H, Ar-H), 6.67 (d, J = 8.4 Hz, 2H, Ar-H), 4.98 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.58 (s, 3H, N-CH3), 2.61 (q, J =
7.6 Hz, 2H, CH2), 2.37 (s, 3H, CH3), 2.32 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 156.7, 149.4,
148.1, 146.9, 141.6, 140.4, 135.3, 129.7, 124.5, 1115.8, 112.2, 109.0, 99.9, 63.3, 37.4, 34.3, 21.4, 19.4, 15.0, 13.0. HRMS (ESI) m/z
calcd for C20H25ClN5O2, 402.1697; found, 402.1693 [M + H]+.
5-(2-Methylphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]oxime
(10j). White solid; yield, 78%; mp: 92-94 °C; 1H NMR (400 MHz, CDCl3) δ: 7.69 (s, 1H, CH=N), 7.21 (d, J = 7.2 Hz, 1H, Ar-H), 7.10-
6.99 (m, 2H, Ar-H), 6.52 (d, J = 8.0 Hz, 1H, Ar-H), 4.96 (s, 2H, CH2), 3.76 (s, 3H, N-CH3), 3.58 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz,
2H, CH2), 2.38 (s, 3H, CH3), 2.36 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 154.9, 149.4, 148.4,
146.9, 141.6, 135.2, 131.6, 127.3, 126.5, 123.7, 113.4, 109.0, 99.6, 63.3, 37.4, 34.1, 19.4, 16.0, 14.9, 13.0. HRMS (ESI) m/z calcd for
C20H25ClN5O2, 402.1697; found, 402.1695 [M + H]+.
5-(4-Methoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]
1
oxime (10k). White solid; yield, 76%; mp: 56-58 °C; H NMR (400 MHz, CDCl3) δ: 7.73 (s, 1H, CH=N), 6.82 (s, 4H, Ar-H), 4.98 (s,
2H, CH2), 3.79 (s, 3H, OCH3), 3.78 (s, 3H, N-CH3), 3.59 (s, 3H, N-CH3), 2.61 (q, J = 7.6 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 1.22 (t, J =
7.4 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 155.8, 150.6, 149.4, 148.7, 146.9, 141.6, 135.3, 116.3, 114.9, 109.1, 99.5, 63.3, 55.7,
37.4, 34.2, 19.4, 15.0, 13.0. HRMS (ESI) m/z calcd for C20H25ClN5O3, 418.1646; found, 418.1642 [M + H]+.
5-(2-Methoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-yl)methyl]
oxime (10l). White solid; yield, 78%; mp: 85-87 °C; 1H NMR (400 MHz, CDCl3) δ: 7.71 (s, 1H, CH=N), 7.11-7.07 (m, 1H, Ar-H), 6.98
(d, J = 7.2 Hz, 1H, Ar-H), 6.86-6.82 (m, 1H, Ar-H), 6.68 (d, J = 8.0 Hz, 1H, Ar-H), 4.96 (s, 2H, CH2), 3.90 (s, 3H, OCH3), 3.78 (s, 3H,
N-CH3), 3.61 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H, CH2), 2.34 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz,
CDCl3) δ: 149.3, 139.0, 146.7, 145.5, 141.5, 135.3, 124.9, 121.0, 116.0, 112.7, 108.9, 99.3, 63.3, 56.0, 37.4, 34.2, 19.4, 14.9, 13.0.
HRMS (ESI) m/z calcd for C20H25ClN5O3, 418.1646; found, 418.1641 [M + H]+.
5-(4-Trifluoromethoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-
1
yl)methyl]oxime (10m). Yellow solid; yield, 75%; mp: 49-51 °C; H NMR(400 MHz, CDCl3) δ: 7.76 (s, 1H, CH=N), 7.18 (d, J = 8.8
Hz, 2H, Ar-H), 6.90 (d, J = 9.2 Hz, 2H, Ar-H), 4.95 (s, 2H, CH2), 3.78 (s, 3H, N-CH3), 3.60 (s, 3H, N-CH3), 2.60 (q, J = 7.6 Hz, 2H,
CH2), 2.35 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 154.9, 149.4, 147.3, 147.1, 144.8, 141.2, 135.2,
122.9, 120.4, 116.3, 109.1, 99.9, 63.3, 37.3, 34.3, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for C20H22ClF3N5O3, 472.1363; found,
472.1356 [M + H]+.
5-(2-Trifluoromethoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-[(4-chloro-3-ethyl-1-methyl-1H-pyrazol-5-
1
yl)methyl] oxime (10n). Yellow solid; yield, 78%; mp: 127-129 °C; H NMR(400 MHz, CDCl3) δ: 7.73 (s, 1H, CH=N), 7.36 (m, 1H,
Ar-H), 7.20 (m, 1H, Ar-H). 7.13 (m, 1H, Ar-H ). 6.71 (m, 1H, Ar-H), 4.94 (s, 2H, CH2), 3.77 (s, 3H, N-CH3), 3.60 (s, 3H, N-CH3), 2.60
(q, J = 7.6 Hz, 2H, CH2), 2.35 (s, 3H, CH3), 1.22 (t, J = 7.6 Hz, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 149.4, 148.7, 147.1, 146.6,
141.0, 137.5, 135.1, 128.3, 124.2, 123.9, 120.6, 115.5, 109.1, 100.0, 99.9, 63.3, 37.4, 34.2, 19.4, 14.7, 13.0. HRMS (ESI) m/z calcd for
C20H22ClF3N5O3, 472.1363; found, 472.1364 [M + H]+.
5-(4-Fluorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10A). White solid; yield, 85%; mp: 133-134 °C; H NMR (400 MHz, CDCl3) δ: 7.85 (m, 2H, Ar-H), 7.77 (s, 1H,
CH=N), 7.10 (m, 2H, Ar-H), 7.00 (m, 2H, Ar-H), 6.84 (m, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.59 (s, 3H, CH3), 2.36 (s,
3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 162.6, 158.8, 152.6, 152.5, 148.0, 147.1, 145.0, 141.6, 136.7, 128.9, 127.9, 116.6, 116.5,
116.4, 115.4, 108.4, 99.7, 63.3, 37.8, 34.3, 14.8. HRMS (ESI) m/z calcd for C23H21ClF2N5O2, 472.1352; found, 472.1348 [M + H]+.
5-(4-Chlorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10B). White solid; yield, 85%; mp: 155-157 °C; H NMR (400 MHz, CDCl3) δ: 7.85 (m, 2H, Ar-H), 7.77 (s, 1H,
CH=N), 7.26 (m, 2H, Ar-H), 7.10 (t, J =8.8 Hz, 2H, Ar-H), 6.82 (m, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.58 (s, 3H, CH3),
2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 162.6, 155.1, 147.5, 145.0, 141.4, 136.6, 130.0, 128.9, 116.6, 115.5, 108.4, 99.9,
63.3, 37.8, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21Cl2FN5O2, 488.1056; found, 488.1043 [M + H]+.
5-(4-Bromophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10C). White solid; yield, 83%; mp: 167-168 °C; H NMR (400 MHz, CDCl3) δ: 7.85 (m, 2H, Ar-H), 7.77 (s, 1H,
CH=N), 7.41 (d, J = 8.6 Hz, 2H, Ar-H), 7.11 (m, 2H, Ar-H), 6.78 (d, J = 8.6 Hz, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.58

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European Journal of Medicinal Chemistry
(s, 3H, CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 162.6, 155.7, 147.3, 147.1, 145.0, 141.4, 136.6, 132.9, 128.9, 127.9,
ACCEPTED MANUSCRIPT
127.8, 117.0, 116.3, 115.4, 108.4, 99.9, 63.3, 37.8, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21BrClFN5O2, 532.0551; found,
532.0538 [M + H]+.
5-(4-Iodophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10D). White solid; yield, 84%; mp: 163-165 °C; H NMR (400 MHz, CDCl3) δ: 7.85 (q, J = 5,4 Hz, Ar-H), 7.77 (s,
1H, CH=N), 7.60 (d, J = 8.8 Hz, 2H, Ar-H), 7.11 (t, J = 8.8 Hz, 2H, Ar-H), 6.66 (d, J = 8.8 Hz, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.88 (s,
3H, CH3), 3.58 (s, 3H, CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 162.6, 156.5, 147.3, 147.1, 145.0, 141.4, 138.9, 136.6,
128.9, 127.9, 127.9, 117.5, 115.4, 108.4, 99.9, 86.6, 63.3, 37.8, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21IClFN5O2, 580.0412;
found, 580.0393 [M + H]+.
5-(4-Methylphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10E). White solid; yield, 86%; mp: 136-138 °C; H NMR (400 MHz, CDCl3) δ: 7.84 (m, 2H, Ar-H), 7.78 (s, 1H,
CH=N), 7.10 (m, 4H, Ar-H), 6.77 (d, J = 8.8 Hz, 2H, Ar-H), 5.05 (s, 2H, CH2), 3.88 (s, 3H, CH3), 3.57 (s, 3H, CH3), 2.37 (s, 3H, CH3),
2.30 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 162.6, 154.6, 148.4, 146.9, 145.0, 141.9, 136.8, 133.3, 30.4, 128.9, 115.4, 115.1,
108.4, 99.7, 63.3, 37.8, 34.3, 20.6, 14.9. HRMS (ESI) m/z calcd for C24H24ClFN5O2, 468.1603; found, 468.1592 [M + H]+.
5-(4-Methoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10F). White solid; yield, 84%; mp: 127-129 °C; H NMR (400 MHz, CDCl3) δ: 7.84 (m, 2H, Ar-H), 7.76 (s, 1H,
CH=N), 7.10 (t, J = 8.8 Hz, 2H, Ar-H), 6.82 (s, 4H, Ar-H), 5.06 (s, 2H, CH2), 3.89 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.60 (s, 3H, CH3),
2.36 (s, 3H, CH3). 13C NMR (100MHz, CDCl3) δ: 162.6, 155.8, 150.6, 148.7, 146.9, 145.0, 141.9, 136.8, 128.9, 128.0, 127.9, 116.3,
115.4, 115.0, 108.4, 99.5, 63.2, 55.7, 37.8, 34.3, 15.0. HRMS (ESI) m/z calcd for C24H24ClFN5O3, 484.1552; found, 484.1544 [M + H]+.
5-(4-Trifluoromethoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-fluorophenyl)-1-methyl-1H-
pyrazol-5-yl]methyl}oxime (10G). White solid; yield, 86%; mp: 77-78 °C; 1H NMR (400 MHz, CDCl3) δ: 7.85 ( m, 2H, Ar-H ), 7.79 ( s,
1H, CH=N ), 7.17 ( d, J = 8.8 Hz, 2H, Ar-H ), 7.10 ( t, J = 8.8 Hz , 2H, Ar-H ), 6.90 ( m, 2H, Ar-H ), 5.03 ( s, 2H, CH2 ), 3.88 ( s, 3H,
CH3 ), 3.60 ( s, 3H, CH3 ), 2.36 ( s, 3H, CH3 ). 13C NMR (100 MHz, CDCl3) δ: 162.6, 154.9, 147.4, 147.2, 145.0, 144.9, 141.4, 136.6,
128.9, 127.9, 127.8, 122.9, 120.4, 116.3, 115.4, 108.4, 99.9, 63.3, 37.8, 34.3, 14.7. HRMS (ESI) m/z calcd for C24H21ClF4N5O3,
538.1269; found, 538.1256 [M + H]+.
5-(4-Fluorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10H). White solid; yield, 84%; mp: 126-128 °C; H NMR (400 MHz, CDCl3) δ: 7.83 (d, J = 8.4 Hz, 2H, Ar-H), 7.77
(s, 1H, CH=N), 7.39 (d, J = 8.4 Hz, 2H, Ar-H), 7.01 (m, 2H, Ar-H), 6.85 (m, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.89 (s, 3H, CH3), 3.59 (s,
3H, CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 158.8, 152.6, 148.0, 147.1, 144.7, 141.6, 136.8, 133.9, 130.3, 128.6,
128.3, 116.7, 116.6, 116.5, 116.5, 108.6, 99.7, 62.4, 37.9, 34.3, 14.8. HRMS (ESI) m/z calcd for C23H21Cl2FN5O2, 488.1056; found,
488.1048 [M + H]+.
5-(4-Chlorophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10I). White solid; yield, 86%; mp: 100-102 °C; H NMR (400 MHz, CDCl3) δ: 7.83 (d, J = 8.4 Hz, 2H, Ar-H), 7.77
(s, 1H, CH=N), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 7.26 (t, J = 4.4 Hz, 2H, Ar-H), 6.82 (d, J = 9.2 Hz, 2H, Ar-H), 5.04 (s, 2H, CH2), 3.88
(s, 3H, CH3), 3.58 (s, 3H, CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 155.1, 147.4, 147.1, 144.6, 141.5, 136.8, 133.9,
130.3, 130.0, 128.9, 128.6, 128.3, 116.6, 108.6, 99.8, 63.3, 37.9, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21Cl3N5O2, 504.0761;
found, 504.0751 [M + H]+.
5-(4-Bromophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
yl]methyl}oxime (10J). White solid; yield, 85%; mp: 116-118 °C; 1H NMR (400 MHz, CDCl3) δ: 7.83 ( d, J = 8.4 Hz, 2H, Ar-H ), 7.77
( s, 1H, CH=N ), 7.40 ( m, 4H, Ar-H ), 6.77 ( d, J = 8.4 Hz, 2H, Ar-H ), 5.04 ( s, 2H, CH2 ), 3.88 ( s, 3H, CH3 ), 3.58 ( s, 3H, CH3 ), 2.35
( s, 3H, CH3 ). 13C NMR (100 MHz, CDCl3) δ: 155.7, 147.3, 147.1, 144.7, 141.4, 136.8, 133.9, 133.0, 130.3, 128.6, 128.3, 117.0, 116.3,
108.6, 99.9, 63.3, 37.9, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21BrCl2N5O2, 548.0256; found, 548.0242 [M + H]+.
5-(4-Iodophenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10K). White solid; yield, 83%; mp: 134-136 °C; H NMR (400MHz, CDCl3) δ: 7.83 (d, J = 8.8 Hz, 2H, Ar-H), 7.77
(s, 1H, CH=N), 7.60 (d, J = 8.8 Hz, 2H, Ar-H), 7.39 (d, J = 8.8 Hz, 2H, Ar-H), 6.66 (d, J = 9.2 Hz, 2H, Ar-H), 5.03 (s, 2H, CH2), 3.88
(s, 3H, N-CH3), 3.58 (s, 3H, N-CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 156.5, 147.3, 147.1, 144.7, 141.4, 138.9, 136.8,
133.9, 130.3, 128.6, 128.3, 117.5, 108.6, 99.9, 86.6, 63.3, 37.9, 34.3, 14.7. HRMS (ESI) m/z calcd for C23H21ICl2N5O2, 596.0117;
found, 596.0105 [M + H]+.
5-(4-Methylphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
1
yl]methyl}oxime (10L). White solid; yield, 84%; mp: 111-112 °C; H NMR (400 MHz, CDCl3) δ: 7.82 (d, J = 8.8 Hz, 2H, Ar-H), 7.77
(s, 1H, CH=N), 7.38 (d, J = 8.4 Hz, 2H, Ar-H), 7.09 (d, J = 8.8 Hz, 2H, Ar-H), 6.76 (d, J = 8.4 Hz, 2H, Ar-H), 5.05 (s, 2H, CH2), 3.88
(s, 3H, CH3), 3.57 (s, 3H, CH3), 2.36 (s, 3H, CH3), 2.30 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 154.6, 148.4, 146.9, 144.6, 141.9,
136.9, 133.8, 133.3, 130.4, 130.3, 128.6, 128.3, 115.1, 108.6, 99.7, 63.2, 37.9, 34.2, 20.6, 14.9. HRMS (ESI) m/z calcd for
C24H24Cl2N5O2, 484.1307; found, 484.1306 [M + H]+.
5-(4-Methoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-pyrazol-5-
yl]methyl}oxime (10M). White solid; yield, 83%; mp: 109-111 °C; 1H NMR (400 MHz, CDCl3) δ: 7.82 (d, J = 8.4 Hz, 2H, Ar-H), 7.76
(s, 1H, CH=N), 7.38 (d, J = 8.8 Hz, 2H, Ar-H), 6.82 (s, 4H, Ar-H), 5.05 (s, 2H, CH2), 3.89 (s, 3H, CH3), 3.76 (s, 3H, OCH3), 3.58 (s,
3H, CH3), 2.35 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 155.8, 150.6, 148.7, 146.9, 144.6, 141.9, 136.9, 133.8, 130.3, 128.6,
128.4, 116.3, 115.0, 108.6, 99.5, 63.2, 55.7, 37.9, 34.2, 14.9. HRMS (ESI) m/z calcd for C24H24Cl2N5O3, 500.1256; found, 500.1247 [M
+ H]+.

5-(4-Trifluoromethoxyphenoxy)-1-methyl-3-methyl-1H-pyrazole-4-carbaldehyde-O-{[4-chloro-3-(4-chlorophenyl)-1-methyl-1H-
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1
pyrazol-5-yl]methyl}oxime (10N). White solid; yield, 85%; mp: 85-86 °C; H NMR (400 MHz, CDCl3) δ: 7.83 (d, J = 8.8Hz, 2H, Ar-
H), 7.78 (s, 1H, CH=N), 7.38 (d, J = 8.4 Hz, 2H, Ar-H), 7.17 (d, J = 8.8 Hz, 2H, Ar-H), 6.90 (d, J = 9.2 Hz, 2H, Ar-H), 5.03 (s, 2H,
CH2), 3.88 (s, 3H, CH3), 3.60 (s, 3H, CH3), 2.36 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ: 154.8, 147.4, 147.1, 144.7, 141.4, 136.7,
133.9, 130.3, 128.6, 128.3, 122.9, 116.3, 108.6, 99.9, 63.3, 37.8, 34.8, 14.7. HRMS (ESI) m/z calcd for C24H21Cl2F3N5O3, 554.0974;
found, 554.0962 [M + H]+.
4.2. Cell culture and reagents
Human hepatocellular carcinoma cells (SMMC-7721), human gastric cancer cells (SGC7901), human colon cancer cells (HCT116)
and human liver normal cells (LO2) were maintained in DMEM supplemented with 10% fetal bovine serum (Invitrogen), 100 units/ml
of penicillin, and 0.1 µg/ml of streptomycin in a humid atmosphere incubator with 5% CO2 at 37 °C. All cell lines were originally from
the Shanghai Institute of Cell Biology (Shanghai, China). Cells were routinely subcultured twice weekly. 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), monoclonal anti-actin antibody, and goat peroxidase-conjugated anti-rabbit IgG antibody, and
goat peroxidase-conjugated anti-mouse IgG antibody were purchased from Sigma-Aldrich (St. Louis, MO). FITC-Annexin V and PI are
from (BioVision). Antibodies such as Bax, Bcl-2, p-H2AX, cleaved-PARP, cleaved-caspase 3, and p-p53 (S15) are from Cell Signaling
Technology (Danvers, MA).
4.3. MTT assay
Anti-proliferative activities of synthesized compounds were evaluated in vitro against three human cancer cell lines (SMMC-7721,
SGC7901, and HCT116) and human normal cell (LO2). Briefly, 100 µL of different cells were plated in a 96-well flat bottom tissue
culture plate at a density of 104 cells/mL, respectively, in DMEM medium and 10% fetal bovine serum and allowed to adhere overnight
at 37 °C in 5% CO2. The cells were incubated in triplicate with, or without, different concentrations of each test compound for 72 h.
During the last 4 h incubation, 30 µL of tetrazolium dye (MTT) solution (5 mg/mL) was added to each well. The resulting MTT-
formazan crystals were dissolved in 150 µL DMSO, and absorbance was measured spectrophotometrically at 570 nm using an ELISA
plate reader. The inhibition induced by each test compound at the indicated concentrations was expressed as a percentage. The
concentration required for 50% inhibition (IC50) was calculated using the software (GraphPadPrism Version 4.03).
4.4. Flow cytometry assay of cell apoptosis
SMMC-7721 cells were cultured overnight and incubated in triplicate with different concentrations of 10M, ADM, or vehicle for 72
h. The cells were harvested and stained with APC-Annexin V and 7-AAD at room temperature for 15 min. The percentage of apoptotic
cells was determined by flow cytometry (Calibur, BD, USA) analysis. The APC signal detector (FL4) (Ex = 633 nm; Em = 660 nm) and
7-AAD staining signal detector (FL3) (Ex = 488 nm; Em =647 nm) were used to detect the cells with the flow cytometer. Ten thousand
cells were counted for three independent experiments. The data were analyzed using WinList 3D (version7.1) and the histogram was
plotted using Excel 2010.
4.5. Western blot analysis
SMMC-7721 cells with or without ADM, or 10M treatment at indicated time and doses were washed with PBS and lysed on ice for
30 minutes in PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mg/ml PMSF, and 20 mM leupeptin. The
protein concentrations were determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Up to 50 µg
of total protein were separated onto an SDS-PAGE and transferred to polyvinylidenedifluoride membranes. After blocking with 5% fat
free milk for 2 h, membranes were incubated overnight at 4 °C with a primary antibody in TBS-T and then reacted with a peroxidase-
conjugated secondary antibody for 1 h. Immuno reactive proteins were detected with the ECL Western Blotting Detection System.
4.6. Maximum tolerated dose determination
All in vivo studies were approved by the Nantong University Animal Care and Use Committee. The maximum tolerated dose (MTD)
of compound 10M in female ICR mice were determined by intraperitoneal injection of a single dose of 10M (488.3, 390.6, 312.5, 250,
and 200 mg/kg) or vehicle control (n = 10 per group), respectively. The MTD was set below the dose that caused severe loss of body
weight (>20% of original weight), or death of one or more animals of a dose group. MTD was defined as the highest dose that could be
given resulting in no drug-related moribund state or death, while temporary body weight loss was within 20%.
4.7. In vivo tumor growth inhibition
Female BALB/c nude mice at the age of 5 to 6-week-old were inoculated subcutaneously with 106 SMMC-7721 cells. When tumor
volumes reached 50-100 mm3, the mice were randomly administered with 10M, ADM, and vehicle, respectively. The body weight of
all animals was monitored throughout the study and animals were euthanized if they incurred 20 % weight loss between observations.
Two axes (mm) of a tumor (L, longest axis; W, shortest axis) were measured with a Vernier caliper. Tumor volume (mm3) was
calculated using a formula of “tumor volume = ½ (L×W2)”. Progression of tumors was monitored every 3 days up to 19 days post
treatment. At the end of the experiment, the mice were sacrificed, and their tumors were dissected out and weighed.
Acknowledgments
We gratefully acknowledge the financial support by the Natural Science Foundation of China (Grant Nos. 21372135 and
81302628), the Project of “Jiangsu Six Peaks of Talent” (2013-SWYY-013, 2014-SWYY-044, and 2016-SWYY-CXTD-008),
China Pharmaceutical University for the Open Project Program of State Key Laboratory of Natural Medicines
(SKLNMKF201611), China and Jiangsu Province Postdoctoral Science Foundation (2016M590488 and 1601136B), the Project

12
European Journal of Medicinal Chemistry
of “Jiangsu 333 high-level talents”, Jiangsu Government Scholarship for Overseas Studies (JS-2014-212), Applied Research
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Projects of Nantong City (MS22015020, MS12015060), and also thank a project funded by the Priority Academic Programs
Development of Jiangsu Higher Education Institutions (PAPD).
References and notes
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ACCEPTED MANUSCRIPT
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ACCEPTED MANUSCRIPT
Development of novel bis-pyrazole derivatives as antitumor agents
with potent apoptosis induction effects and DNA damage
Hong Dai a,b, Shushan Ge a,b, Jin Guo b,c, Shi Chen b,c, Meiling Huang b, Jiaying Yangb, Siyu Suna,
Yujun Shi a,*, and Yong Ling a,b,c,
*
a College of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, People’s
Republic of China
b School of Pharmacy, Nantong University, Nantong 226001, People’s Republic of China
c Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong
University, Nantong 226001, China.
Highlights
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New bis-pyrazole derivatives were designed and synthesized.
Compound 10M selectively inhibited cancer cells but not non-tumor cell.
10M trigged cancer cell apoptosis by cleavage of caspase 3 and PARP.
10M induced DNA damage by activation of the p53 signaling pathway.
10M showed low acute toxicity and significant tumor growth inhibition in vivo.