Discovery and characterization of novel imidazopyridine derivative CHEQ-2 as a potent CDC25 inhibitor and promising anticancer drug candidate

Article abstract of DOI:10.1016/j.ejmech.2014.05.063

Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and proliferation via the interactions with CDK/Cyclin complexes. Overexpression of CDC25 proteins is frequently observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus, inhibiting CDC25 activity in cancer treatment appears a good therapeutic strategy. In this article, refinement of the initial hit XDW-1 by synthesis and screening of a focused compound library led to the identification of a novel set of imidazopyridine derivatives as potent CDC25 inhibitors. Among them, the most potent molecule was CHEQ-2, which could efficiently inhibit the activities of CDC25A/B enzymes as well as the proliferation of various different types of cancer cell lines in vitro assay. Moreover, CHEQ-2 triggered S-phase cell cycle arrest in MCF-7, HepG2 and HT-29 cell lines, accompanied by generation of ROS, mitochondrial dysfunction and apoptosis. Besides, oral administration of CHEQ-2 (10 mg/kg) significantly inhibited xenografted human liver tumor growth in nude mice, while demonstrated extremely low toxicity (LD₅₀ > 2000 mg/kg). These findings make CHEQ-2 a good starting point for further investigation and structure modification.

Full text of DOI:10.1016/j.ejmech.2014.05.063

European Journal of Medicinal Chemistry 82 (2014) 293e307
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Discovery and characterization of novel imidazopyridine derivative
CHEQ-2 as a potent CDC25 inhibitor and promising anticancer drug
candidate
,
, *
Yu'ning Song a, Xiaoqian Lin a, Dongwei Kang b, Xiao Li b, Peng Zhan b *, Xinyong Liu b
,
,
*
Qingzhu Zhang a
a Department of Pharmacology, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
44 West Culture Road, 250012, Jinan, Shandong, PR China
b Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University,
44 West Culture Road, 250012, Jinan, Shandong, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cell division cycle (CDC) 25 proteins are key phosphatases regulating cell cycle transition and prolifer-
ation via the interactions with CDK/Cyclin complexes. Overexpression of CDC25 proteins is frequently
observed in cancer and is related to aggressiveness, high-grade tumors and poor prognosis. Thus,
inhibiting CDC25 activity in cancer treatment appears a good therapeutic strategy. In this article,
refinement of the initial hit XDW-1 by synthesis and screening of a focused compound library led to the
identification of a novel set of imidazopyridine derivatives as potent CDC25 inhibitors. Among them, the
most potent molecule was CHEQ-2, which could efficiently inhibit the activities of CDC25A/B enzymes as
well as the proliferation of various different types of cancer cell lines in vitro assay. Moreover, CHEQ-2
triggered S-phase cell cycle arrest in MCF-7, HepG2 and HT-29 cell lines, accompanied by generation
of ROS, mitochondrial dysfunction and apoptosis. Besides, oral administration of CHEQ-2 (10 mg/kg)
significantly inhibited xenografted human liver tumor growth in nude mice, while demonstrated
extremely low toxicity (LD50 > 2000 mg/kg). These findings make CHEQ-2 a good starting point for
further investigation and structure modification.
Received 18 March 2014
Received in revised form
6 May 2014
Accepted 26 May 2014
Available online 27 May 2014
Keywords:
CDC25 inhibitor
Anticancer agents
Cell cycle arrest
Apoptosis
Reactive oxygen species
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
could activate CDKs, which in turn regulate progression through
the cell division cycle.
As well known, different types of cancers may have different
pathogenesis and show diverse clinical symptoms, but one of the
most prominent features shared in cancer cells is deregulated
growth [1,2]. The uncontrolled proliferation is mostly attributed to
cell cycle deregulation [3,4]. Cell cycle is a tightly controlled pro-
cedure, which needs a delicate signaling network to determine the
proper progression [4,5]. Cyclin-dependent kinases (CDKs) are
foremost cell cycle regulators that phosphorylate and activate
downstream players like retinoblastoma (Rb) protein to promote
cell cycle progression [6]. The cell division cycle 25 (CDC25) family
of proteins are highly conserved dual specificity phosphatases that
CDC25 dual phosphatase family can be found in all eukaryotic
organisms except plants. In mammalian cells, it has three mem-
bers: CDC25A, CDC25B, and CDC25C [7]. CDC25A acts on the control
of G1-to-S and G2-to-S transitions in cell cycle whereas CDC25B
and CDC25C are mainly responsible for regulating the progression
at the G2-to-M transition. CDC25B is proposed to be responsible for
the initial activation of CDK1eCyclin B at the centrosome during
the G2eM transition [8e10], which is then followed by a complete
activation of CDK1eCyclin B complexes by CDC25C in the nucleus
at the onset of mitosis [11]. Owing to such an important contribu-
tion to the cell cycle regulation, CDC25 phosphatases have been
considered to be involved in oncogenic transformations and human
cancers. CDC25A and CDC25B overexpression are frequently found
in many cancers, such as breast cancer, colorectal cancer, hepato-
cellular carcinoma, non-Hodgkin lymphoma etc., and are often
associated with high-grade tumors and poor prognosis. Therefore,
the inhibition of CDC25 phosphatases may represent a novel
* Corresponding authors. School of Pharmaceutical Sciences, Shandong Univer-
sity, 44 West Culture Road, 250012, Jinan, Shandong, PR China.
E-mail addresses: zhanpeng1982@163.com (P. Zhan), xinyongllab@163.com
(X. Liu), zhangqzh@sdu.edu.cn (Q. Zhang).
http://dx.doi.org/10.1016/j.ejmech.2014.05.063
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

294
Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
approach for the development of anticancer therapeutics, although
more details about the involvement of CDC25A and CDC25B over-
expression in tumorigenesis remain to be clarified.
3. Results and discussions
3.1. Evaluation of CHEQ-1eCHEQ-6 in vitro CDC25 inhibition and
Many compounds with CDC25 phosphatase inhibitory activities
are currently being developed. The existing structural types include
quinonoids, phosphate surrogates, or electrophilic derivatives [12].
Starting from the structure of vitamin K published in 1993 as a
potent CDC25 inhibitor [13], numerous compounds were derived
from a para-quinonoid structure. And many other quinonoid de-
rivatives were able to inhibit CDC25 activity and tumor cell pro-
liferation. The most potent quinonoid-based compounds identified
so far are active on xenografted tumor models. To date, there is no
suitable inhibitor for further drug development, and thus innova-
tive compounds are required.
cell growth suppression
The six newly synthesized compounds (CHEQ-1eCHEQ-6) were
evaluated for their inhibitory effects against the recombinant full
length CDC25A and CDC25B, and the growth of colon carcinoma
HT-29 cells and hepatoma HepG2 cells. The results, expressed as
IC50 values are summarized in Table 1 together with those of lead
compound XDW-1 and a classic positive compound vitamin K3
(VK3) as the references.
As listed in Table 1, the six imidazopyridine derivatives showed
notable activity against CDC25A/B with IC50 values ranged from 0.3
Recently, 1,2,4-triazole derivative XDW-1 (compound 4,
Scheme 1) was identified as a novel inhibitor of CDC25 phos-
phatase via a virtual screening approach [14]. This compound was
structurally original unlike the existing para-quinonoid structure
to 30
(IC50 ¼ 0.39
ble inhibitory potential with two reference compound VK3
M) against CDC25A.
Regarding the CDC25B inhibition, the majority of them showed
comparable activity with two reference compounds (IC50 values in
m
M. Compound CHEQ-1 (IC50
¼
0.30
mM), CHEQ-2
m
M), and CHEQ-6 (IC50 ¼ 0.64
mM) showed compara-
(IC50 ¼ 0.80
mM) and XDW-1 (IC50 ¼ 0.28
m
and exhibited
a
significant potency in micromolar range,
providing a new scaffold for further development as anticancer
drugs by structureeactivity relationships (SARs) studies or in-
depth druggability investigations.
the range of 1.26e6.67
mM), except for CHEQ-6 (IC50 ¼ 16.61
mM).
Especially, compound CHEQ-2 was approximately 3-fold and 2-fold
more effective against CDC25B compared to XDW-1 and VK3,
respectively. Interestingly, compared with CDC25B, CDC25A
demonstrated more sensitive profiles towards nearly all the tested
compounds including two reference ones (except for CHEQ-5). It is
not hard to find that the position of nitrogen atom in imidazopyr-
idine and the nature of the substituent groups linked to
“eSCH2COe” essentially influenced the inhibitory activity and
selectivity of CDC25A/B.
To obtain the structural insight into the inhibitory actions and to
underline the potential of this new type of CDC25 phosphatase
inhibitor, herein, optimization of the initial hit XDW-1 by synthesis
and preliminary screening of a focused compound library with led
to the identification of a set of novel imidazopyridine derivatives as
potent CDC25 inhibitors. Further in vitro and in vivo investigations
led to the identification of compound CHEQ-2 as a promising
anticancer drug candidate with remarkable antiproliferative
effects.
After analyzing their biochemical actions on CDC25A/B, we then
focused our attention on the evaluation of their ability to block
proliferation of two human tumor cells in culture. Except for CHEQ-
2. Chemistry
4 (IC50 > 100 mM in HT-29) and CHEQ-6 (IC50 > 100 mM in HepG2),
other compounds displayed equivalent or more potent anti-
proliferative activities in HT-29 and HepG2 cell lines. Notably,
CHEQ-2 and CHEQ-5 were the most potent inhibitors of cell pro-
liferation, especially for HepG2 with IC50 values of 11.25 and
The lead compound 2-(4-(4-chlorophenyl)-5-p-tolyl-4H-1,2,4-
triazol-3-ylthio)-1-(3,4-dihydroxyphenyl)ethanone (4, XDW-1) was
obtained by the alkylation reaction of 4-(4-chlorophenyl)-5-p-tolyl-
4H-1,2,4-triazole-3-thiol (3) (CAS No. 451501-95-4) [15] with 2-
chloro-1-(3,4-dihydroxyphenyl)ethanone (2) in the presence of
K2CO3 in an easy, rapid and profitable way (Scheme 1). Similarly, the
newly designed compounds (CHEQ-2 (9a), CHEQ-1 (9b), CHEQ-3
(13a), CHEQ-4 (13b), CHEQ-5 (16a) and CHEQ-6 (16b)) were syn-
thesized by alkylation reaction of intermediates 1-mesityl-1H-imi-
dazo[4,5-c]pyridine-2-thiol (8a) or 3-mesityl-3H-imidazo[4,5-b]
pyridine-2-thiol (8b) with 2-chloro-1-(3,4-dihydroxyphenyl)etha-
none (2), N-(2-aminophenyl)-2-chloroacetamide (12), or N-(2-
hydroxyphenyl)-2-chloroacetamide (15) in good yields (Schemes 2
and 3). The key intermediates 8a and 8b were synthesized accord-
ing to the reported literature with minor modification [16]. Both
analytical and spectral data of all the newly synthesized compounds
are in full agreement with the proposed structures.
11.77 mM, respectively.
All in all, of all the tested compounds, CHEQ-2 exhibited the
most potent inhibition against CDC25A/B as well as cell growth
among all the compounds. Comparing with XDW-1 and VK3,
CHEQ-2 showed comparable inhibition potential on enzymes (both
CDC25A and B) and much stronger suppression on cell growth.
These preliminary results demonstrated that the isosteric
replacement of imidazopyridine for 1,2,4-triazole core ring in
XDW-1 was gratifying and afforded a set of potent imidazopyr-
idines with CDC25A/B and tumor inhibiting activity in vitro.
Further, the inhibitory effects of CHEQ-2 on a mass variety of
different tumor cells, e.g. colon carcinoma, mammary carcinoma,
hepatoma, microglioma, etc., were investigated. Encouragingly, as
highlighted in Table 2, CHEQ-2 led to a decreased viability in these
different tumor cells. And most of the IC50 values were below
20 mM. In contrast, XDW-1 and VK3 showed much weaker inhibi-
tion on most of the tested cell lines. The IC50 values were much
higher, approximately two or three times of CHEQ-2 in most cases.
However, The IC50 value of CHEQ-2 in neuroblastoma cells SH-SY5Y
and drug-resistant mammary carcinoma MCF-7/A cells was a little
higher, approximately 35 mM, demonstrating that these cells were
less sensitive to CHEQ-2. Normal hepatocyte L-02 cells and rat
primary cultured neurocytes were also found less sensitive to
CHEQ-2 according to our results, indicating CHEQ-2 had relatively
low toxicity toward these normal cells.
According to these results, CHEQ-2, as the most promising
molecule was selected and further investigated, both in vitro and
Scheme 1. Reagents and conditions: (i) 2-chloroacetyl chloride, AlCl3, ClCH2CH2Cl; (ii)
4-(4-chlorophenyl)-5-p-tolyl-4H-1,2,4-triazole-3-thiol (3), K2CO3, acetone.

Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
295
Scheme 2. Reagents and conditions: (i) 2,4,6-trimethylbenzenamine, NaHCO3, EtOH, reflux; (ii) 2,4,6-trimethylbenzenamine, KF, 120 ꢀC; (iii) H2, Pd/C; (iv) NaHCO3, EtOH, H2O,
CH3CH2OCS2K, reflux, acidification; (v) 2-chloro-1-(3,4-dihydroxyphenyl)ethanone (2), K2CO3, acetone.
Scheme 3. Reagents and conditions: (i) 2-chloroacetyl chloride, DCM, Et3N; (ii) Fe, NH4Cl, AcOH, DMF, H2O; (iii) 2-chloroacetyl chloride, chloroacetic acid, 1,2-dichloroethane; (iv)
K2CO3, DMF.
in vivo inhibitory potential effects. As described above, CDC25
proteins reported to be overexpressed in many tumor tissues,
especially in the samples from patients with breast, colorectal and
hepatocellular cancers etc [17]. Therefore, three different CDC25
overexpression human tumor cell lines MCF-7 of breast cancer,
HepG2 of hepatocellular cancer and HT-29 of colorectal cancer
were applied in our research. In consistent with previous result, cell
viability study displayed that CHEQ-2 displayed much stronger
inhibitory potential than VK3 and XDW-1 at nearly all doses against
MCF-7, HepG2 and HT-29 cell lines (As shown in Fig. 1).
cell cycle. Regardless of the initial insult, the net result is an inhi-
bition of CDKeCyclin complexes in order to stop cell-cycle pro-
gression. It has been illustrated that CDC25 mainly activates the
CDK2eCyclin E and CDK2eCyclin A complexes during the G1eS
transition [18e20], but also has a role in the G2eM transition
[21,22] by activating CDK1eCyclin B complexes. Moreover, it has
been proved that CDK2eCyclin A complex is also necessary for cells
progression through the S phase [23], and CDK2 is the primary
downstream substrate of CDC25A which activates CDK2/Cyclin A
complex [24]. Cyclin B is also indispensable in the control of the
onset of mitosis. As remarkable S-phase cell cycle arrest was found
in CHEQ-2 treated cells, we speculated that CHEQ-2 could affect
these cell cycle proteins, including CDK2, Cyclin A, B and E, as well
as CDC25A and B. In consistent with our speculation, it was showed
that CHEQ-2 not only suppressed the expression of CDC25A and B,
but also inhibited the expression of CDK2, Cyclin A, B and E (See
Fig. 2B, D, F), which mediated the S-phase cell cycle arrest. How-
ever, the expression of Cyclin A and B didn't show any reduction in
CHEQ-2 treated HT-29 cells (Fig. 2F), which could help to explain
the relative insensitivity of HT-29 (IC50 value was nearly twice that
of MCF-7 and HepG2 cells).
3.2. CHEQ-2 induced S-phase cell cycle arrest by inhibiting CDC25
expression
Cells were treated with different doses of CHEQ-2 (7.5, 15,
30 mM) for 12, 24 and 48 h. The distribution in different phases of
the cell cycle is illustrated in Fig. 2. The results demonstrated CHEQ-
2 treatment could trigger significant reduction of G0/G1 phase cells
in all three different tumor cell lines MCF-7 (Fig. 2A), HepG2
(Fig. 2C) and HT-29 (Fig. 2E). And this reduction was accompanied
by an apparent increase in the proportion of cells in the S phase in
concentration-dependent manner. And the maximum increases of
S-phase cell fraction reached 40.0% (MCF-7), 38.1% (HepG2) and
27.7% (HT-29) respectively. However, the changes of G2/M cells
were varied by different cell lines, which can be explained by tiny
distinguishment existed in cell-type specific molecular mecha-
nisms of CHEQ-2.
3.3. CHEQ-2 induced apoptosis cell death
The induction of apoptosis by CHEQ-2 was first examined by
Hoechst staining under fluorescence microscopy. The changes in
cell nuclear morphology were shown in Fig. 3A. After treatment
with CHEQ-2 for 48 h, cells of all three cell lines showed bright
fluorescent nuclei blebbing and DNA fragmentation, comparing
Our choice of the cell cycle related proteins tested in this study is
partly related to the importance of their role in the control of the

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Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
Table 1
mitochondrial outer membrane permeabilization and subsequent
activation of caspase 3 and PARP [25e27].
In vitro IC50 values (mM) of CHEQ1e6 against CDC25 (A and B) and two cell lines (HT-
29 and HepG2) in MTT assay.
Compounds
IC50(mM)
3.4. CHEQ-2 induced generation of reactive oxygen species and loss
of mitochondrial membrane potential
Enzymes
CDC25A
Cell lines
HT-29
CDC25B
HepG2
21.93
11.25
22.05
99.44
11.77
>100
21.06
42.11
ROS is associated with many crucial factors in apoptosis signal
pathways, and abnormally high concentration of ROS is an impor-
tant cause of cell apoptosis. Therefore, we examined whether there
is more ROS production upon CHEQ-2 administration. To this end,
we performed DCFH-DA-based fluorescence detection by both
fluorescence microscopy and flow cytometry in MCF-7, HepG2 and
HT-29 cells. The results showed that CHEQ-2 treatment elevated
ROS levels in all three cell lines in a dose-dependent manner (As
shown in Fig. 4A).
CHEQ-1
CHEQ-2
CHEQ-3
CHEQ-4
CHEQ-5
CHEQ-6
XDW-1
VK3
0.30
0.39
2.58
3.64
29.03
0.64
0.28
0.80
4.30
1.26
4.91
3.84
6.75
16.61
3.48
2.58
28.87
24.20
23.76
>100
23.26
39.67
37.26
31.30
Moreover, oxidative stress is always closely linked with mito-
chondrial dysfunction. Mitochondrial membrane potential is the
hallmark of the status of mitochondrial membrane, and a mito-
chondrial potential sensor JC-1 was used to determine the mito-
chondria function. It forms J-aggregates in intact mitochondria
that result in emission of red/orange fluorescence, whereas it
forms monomers upon the mitochondrial membrane depolariza-
tion that emit green fluorescence [28]. As shown in Fig. 4B, CHEQ-2
treatment resulted in a dose-dependent loss of mitochondrial
potential in all three cell lines, as evidenced by the shift of fluo-
Table 2
IC50 values (
lines in MTT assay.
m
M) of VK3, XDW-1 and CHEQ-2 against different kinds of tumor cell
Tumor
Cell line
IC50
(m
M)
XDW-1
VK3
CHEQ-2
11.40
Colon carcinoma
SW620
SW480
HCT-116
MCF-7
H-7402
BV2
23.79
47.51
29.35
22.76
47.88
4.79
20.61
30.43
23.59
28.84
64.19
5.82
15.47
15.47
14.35
27.17
7.98
Mammary carcinoma
Hepatoma
Microglioma
Neuroblastoma
Drug-resistant mammary
carcinoma
Human normal fetal hepatocyte L-02
cell line
rescence. And 30 mM of CHEQ-2 caused an increase (31.1% (MCF-7),
SH-SY5Y
MCF-7/A
26.13 >100
34.12
37.17
67.6% (HepG2) and 29.1% (HT-29)) of cells with green fluorescence
(See Fig. 4C), indicating that CHEQ-2 treatments led to reduction of
mitochondrial membrane potential and dysfunction of
mitochondria.
39.32
69.36
19.11
46.81
35.29
Rat primary cultured neurocytes Primary cells 30.26 >100
>1000
3.5. NAC blocked the CHEQ-2-induced ROS generation and
mitochondrial damage, and attenuated the CHEQ-2-induced cell
growth inhibition
with non-treated control. An increase of apoptotic cells accompa-
nied by a drop of cell density could be observed in a dose-
dependent manner. These changes are in accordance with the
characteristics of apoptosis cell death.
CHEQ-2-induced apoptosis was also confirmed by flow cyto-
metric analysis of MCF-7, HepG2 and HT-29 cells stained with
annexin V and PI (Fig. 3B). The results demonstrated that CHEQ-2
caused a dose-dependent increase in both early and late stage of
apoptosis in all three cell lines, and the apoptosis rate increased to
49.5% (MCF-7), 45.7% (HepG2), and 52.8% (HT-29).
Evaluation of apoptosis protein confirmed that CHEQ-2 treat-
ment could cause obvious apoptosis associated with down-
regulation of Bcl-2 expression, and elevate Bax protein levels in
MCF-7, HepG2 and HT-29 cells (Fig. 3C). It was also found that
CHEQ-2 dramatically induced activation of caspase 3 and PARP. It is
thought that Bcl-2 and Bax are involved in the regulation of
The antioxidant NAC was employed to determine the contri-
bution of ROS in the anti-cancer effects of CHEQ-2. As shown in
Fig. 5A, cellular ROS level in MCF-7, HepG2, and HT-29 cells was
significantly increased after the treatment of 30 mM CHEQ-2 for
48 h. As anticipated, 5 mM NAC significantly suppressed CHEQ-2-
induced ROS generation in all three cell lines. Moreover, NAC
could also abrogate the loss of mitochondrial membrane potential
in all three cell lines. As shown in Fig. 5B, NAC treatment attenuated
CHEQ-2 induced elevation of percentages of cells with green fluo-
rescence. Meanwhile, it was noteworthy that CHEQ-2-mediated
inhibition on MCF-7, HepG2 and HT-29 cell viability was also
compromised by the treatment of 5 mM NAC (Fig. 5C), further
suggesting the contribution of CHEQ-2-induced ROS accumulation
in its anti-cancer properties.
Fig. 1. Evaluation of cell viability of MCF-7, HepG2 and HT-29 cells treated with DMSO, VK3, XDW-1 or CHEQ-2 1, 5, 10, 25, 50
(n ¼ 5).
mM for 48 h. The data were expressed as mean SD

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297
Fig. 2. CHEQ-2 induced a strong cell accumulation in S phase in MCF-7 (A), HepG2 (B) and HT-29 (C) cells. Cells were incubated with or without various concentrations of CHEQ-2
for 12 h, 24 h and 48 h. Then the cells were collected and analyzed by flow cytometry. The photographs are representatives of three independent experiments. *P < 0.05 vs. control
of the same time spot. CHEQ-2 played an important role in modulating the expression of cell cycle related proteins in MCF-7 (D), HepG2 (E) and HT-29 (F) cells. CHEQ-2 treatment
caused dose-dependent downregulation of both CDC25A and CDC25B. The representative blots were shown on the left, and statistical analysis of bands intensity was shown on the
right. Data were expressed as mean SD from three independent experiments.
However, the source of ROS formation is still remain unclear as
the polyphenol structure of CHEQ-2 is most likely undergoes
oxidation reaction rather than reduction action. We speculated that
ROS accumulation might be a result of damaged mitochondria and
disordered cell cycle, which helped to explain the reason why ROS
formation in 48 h CHEQ-2 treated cells was much greater than 24 h
or 12 h CHEQ-2 treated cells according to our preliminary experi-
ment (data not shown). The generated ROS could also directly affect

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Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
Fig. 2. (continued).

Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
299
Fig. 2. (continued).

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Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
Fig. 3. CHEQ-2 induced a dramatic apoptosis in MCF-7, HepG2 and HT-29 cells. (A) Effect of CHEQ-2 on the morphology of cell nuclei. Cells were treated with or without various
concentrations of CHEQ-2 for 48 h. Then the cells were collected, stained with Hoechst 33342, and the images were captured by fluorescence microscopy using identical exposure
settings. Original magnification, 40ꢁ. The arrows point to the condensed and/or fragmented cell nuclei. (B) Flow cytometric analysis of the cell-surface phosphoserine. Cells were
treated with or without various concentrations of CHEQ-2 for 48 h, co-stained with Annexin VeFITC and PI and analyzed by flow cytometry (FACSCalibur, BD Biosciences, USA).
Early apoptotic (low right quadrant: annexin Vþ/PIꢂ), late apoptotic or necrotic (upper right quadrant: annexin Vþ/PIþ) and viable cells (low left quadrant: annexin Vꢂ/PIꢂ) was
determined and analyzed. The corresponding statistical analysis of apoptosis cells were showed at the bottom. (C) Effect of CHEQ-2 on caspase-3, cleaved PARP, bax and bcl-2
expression in MCF-7, HepG2 and HT-29 cells. All photographs are representatives of at least three independent experiments. *P < 0.05 vs. control.
CDC25 structure, as the thiolate groups of catalytic cysteines of
CDC25 phosphatases were highly reactive to oxidation [29,30]. This
reaction step allowed the formation of a disulfide bond to a back-
door cysteine, preventing the binding of substrate to the activesite
[30,31], and therefore caused CDC25 inhibition. The duel effect of
ROS highlighted the possibility of a direct link between apoptosis
and cell cycle.
3.7. Xenografted human liver tumors in nude mice
Subcutaneous inoculation of 106 HepG2 cells into the left flank
of nude mice resulted in the development of tumors at the site of
inoculation. Daily oral administration of CHEQ-2 (10 mg/kg) was
given when the tumor was well-grown. CHEQ-2-treatment resul-
ted in apparent decrease of tumor volumes in HepG2 cells (Fig. 7A),
and after a 14-day treatment with 10 mg/kg of CHEQ-2, the tumor
volumes were decreased by 42.1% (Fig. 7B). As for control mice, the
tumor volumes maintained steady growth momentum in the
whole experiment period. The body weight of mice was consistent
within 14 days of treatment with and without CHEQ-2, and no
significant difference was observed (Fig. 7C). All animals showed no
signs of weight loss or severe adverse action and none of the mice
died during this study, suggesting that oral administration of
CHEQ-2 is safe in this mouse model. The autopsy of mice did not
reveal any metastatic site in both vehicle and CHEQ-2 treatment
group. Compared to that of the vehicle group, the tumor weight
was reduced by 43.0% in CHEQ-2-treated mice (Fig. 7D).
3.6. Acute toxicity experiment of oral administration and
abdominal injection of CHEQ-2 in female BALB/c mice
There were no animal deaths in oral administration groups and
only
1 death in abdominal injection CHEQ-2-treated group.
Accordingly, the approximate lethal doses of CHEQ-2 in female
mice are higher than 2000 mg/kg. Observed clinical signs included
loss of appetite and energy in 3/4 animals after dosing for 1 or 2
days in CHEQ-2 2000 mg/kg group. The symptoms were found
more serious in the abdominal injection groups, and there was also
a temporary body weight drop on the second day of dosing (day 1).
Other than that, normal body weight gains were observed, no
significant differences were found between the vehicle control and
CHEQ-2 treatment groups (Fig. 6). No abnormal gross findings were
observed in any animals.
The expression of CDC25A, B, CDK2, and Cyclin A, B, E in the
tumor tissues was further studied by western blotting. Compared
with the vehicle (Fig. 7E), the CDC25A and B expression was
significantly lower in the tissue from the CHEQ-2 treated mice,

Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
301
Fig. 4. CHEQ-2 increased ROS formation and triggers mitochondrial dysfunction in MCF-7, HepG2 and HT-29 cells. (A) Effect of CHEQ-2 on the formation of ROS. Cells were treated
with or without various concentrations of CHEQ-2 for 48 h. The ROS levels were determined by assaying the fluorescent product 20,70-dichlorofluorescein (DCF) from 20,70-
dichlorofluorescin diacetate (DCFH-DA) in viable cells. The images were captured by fluorescence microscopy using identical exposure settings. Original magnification, 20ꢁ. The
statistical analysis was made on the flow cytometer. (B) Effect of CHEQ-2 on the mitochondrial membrane potential. Cells were treated with or without various concentrations of
CHEQ-2 for 48 h and subsequently stained with a fluorescent dye JC-1. The cells were visualized for their green and red emission components by using optical filters designed for
fluorescein and tetramethylrhodamine fluorescence microscopy (Original magnification, 20ꢁ) and statistical analysis was made by using the flow cytometer. *P < 0.05 vs. control.
(For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
suggesting that the CDC25 expression was also inhibited in vivo by
CHEQ-2. Additionally, the expression of Cyclin B was also down-
regulated in the tumor tissue from the mice treated with CHEQ-2.
These results suggested that the inhibition of tumor growth could
be indeed attributed to the suppression of CDC25A and B expres-
sion and subsequent inhibition of Cyclin B.
It was showed in this experiment that CHEQ-2 is very efficient in
slowing the tumor growth. It works at a dose as low as 10 mg/kg,
while LD50 of CHEQ-2 is over 2000 mg/kg in mice according to the
acute toxicity evaluation study. It proved CHEQ-2 could be applied
into use at a wide range of dosage with low toxicity. Another
remarkable property of CHEQ-2-mediated tumor growth inhibition
is that it remains efficient in oral dosage form, which could be
enormously beneficial to its potential to be developed into a future
drug. Moreover, the effect of CHEQ-2 in reducing tumor volume and
weight was also observed with other doses (100 mg/kg and
1000 mg/kg). But higher doses of CHEQ-2 didn't demonstrate
stronger inhibition effect on the growth of HepG2 tumor (data not
shown), which may related to the drug distribution and meta-
bolism in the animals.
4. Conclusion
In summary, the present study described the discovery and
characterization of a novel CDC25A/B inhibitor, CHEQ-2, by efficient
synthesis and screening of a focused compound library of the initial
hit XDW-1. We provide evidence that CHEQ-2 demonstrated
improved inhibitory potential compared to XDW-1 and VK3, and
showed favorable inhibition on the proliferation of tumor cells.
Moreover, CHEQ-2 could cause dramatic S-phase cell cycle arrest by
suppressing CDC25A/B expression, eventually leads to activation of
apoptosis. The CHEQ-2-treated cells also exhibited much higher
ROS level and apparent decline in membrane potential, which may
attribute to the cell apoptosis and could be abolished by NAC
treatment. The in vivo approach further proved CHEQ-2 could
inhibit tumor growth in nude mice with low toxicity. Over all, the
data revealed that CHEQ-2 is a novel CDC25 inhibitor with
remarkable tumor inhibition activities that induces cell cycle arrest,
apoptosis, ROS production and mitochondrial dysfunction. With
robust anticancer potencies and lower toxicity, CHEQ-2 is more
effective on multiple cancer cell death than its prototype XDW-1

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Fig. 5. ROS production, mitochondrial dysfunction and cytotoxicity effect induced by CHEQ-2 are diminished by a ROS scavenger, N-acetylcysteine (NAC). (A) NAC abrogated
ROSproductioninduced by CHEQ-2 in MCF-7, HepG2 and HT-29 cells. (B) NAC abolished mitochondrial dysfunction induced by CHEQ-2 in MCF-7, HepG2 and HT-29 cells. (C) NAC
could attenuate the cytotoxicity effect of CHEQ-2 in MCF-7, HepG2 and HT-29 cells. Cells were treated with or without CHEQ-2 (30 mM) at the presence or absence of NAC (5 mM) for
48 h. Data were expressed as mean SD from at least three independent experiments. *P < 0.05 vs. control. #P < 0.05 vs. CHEQ-2 alone.
Fig. 6. CHEQ-2 showed extremely low acute toxicity in BALB/c mice (female). (A) Changes of body weights in the tested animals after one time I.G. injection of CHEQ-2 (2000 mg/
kg). (B) Changes of body weights in the tested animals after one time I.P. injection of CHEQ-2 (2000 mg/kg). *P < 0.05 vs. control.
and positive compound VK3. Therefore, this compound deserves
further investigation in the hope of providing new alternatives to
treat cancer patients.
chromatography was performed on column packed with Silica
Gel60 (230e400 mesh). Solvents were reagent grade, when
necessary, were purified and dried by standard methods. Concen-
tration of the reaction solutions involved the use of rotary evapo-
rator at reduced pressure.
5. Experimental section
5.1. Synthesis procedure
5.1.1. Preparation of 2-(4-(4-chlorophenyl)-5-p-tolyl-4H-1,2,4-
triazol-3-ylthio)-1-(3,4-dihydroxyphenyl)ethanone (4, XDW-1)
To a cooled solution of 1,2-dichloroethane (20 mL) at about
5e10 ꢀC, aluminum chloride (6.0 g, 45 mmol) was added, then the
mixture solution was stirred at 5e10 ꢀC for about 30 min. Then
catechol (1) (2.0 g,18.1 mmol) was added portion wise to the stirred
reaction mixture within about 10 min and the reaction mixture was
further stirred for 20 min. To the above solution, chloroacetyl
chloride (2.2 g, 19.3 mmol) was added at 5e10 ꢀC. Then the tem-
perature was raised to room temperature and with further stirring
All melting points were determined on a micromelting point
apparatus and are uncorrected. 1H NMR and 13C NMR spectra were
obtained on a Bruker Avance-400NMR-spectrometer in the indi-
cated solvents. Chemical shifts are expressed in
d units and TMS as
internal reference. Mass spectra were taken on a LC Autosampler
Device: Standard G1313A instrument. TLC was performed on Silica
Gel GF254 for TLC and spots were visualized by iodine vapors or by
irradiation with UV light
(
l
¼
254 nm). Flash column

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303
Fig. 7. CHEQ-2 inhibited tumor growth from hepatocellular carcinoma cells HepG2 in nude mice. (A) Photographs of tumor removed from nude mice after 14 days of vehicle or
CHEQ-2 treatment. 1 ꢁ107 HepG2 cells were injected s.c. into the left flanks of 6-week-old female nude mice (n ¼ 6). Vehicle or CHEQ-2 (10 mg/kg) were given orally once a day for
2 weeks when the tumor were grown (length ꢁ width > 100 mm2). Tumor volume (B) was measured on day 0, 3, 7, 10, 14 and body weight (C) was measured every single day. (D)
The tumors were weighed after sacrifice and the inhibitory effect was defined as a percentage of vehicle control tumor weight. All the data were obtained from 6 Balb/c nude mice.
*P < 0.05 vs. vehicle control. (E) CHEQ-2 treatment induced downregulation of CDC25A, CDC25B and Cyclin B in xenograft tumors. A small piece (approximately 0.05 g) was taken
from each collected tumor and homegenated to form a mixture. Then the protein was extracted and assayed by western blotting. Data were expressed as mean SD. *P < 0.05 vs.
control.
for about 20 h. After completion of the reaction, the reaction was
quenched with dilute hydrochloric acid solution (40 mL) at 5e10 ꢀC
and stirred for 2e3 h at room temperature. The obtained solid was
filtered and washed with water. The wet solid was suspended in
dilute acetic acid and heated to get a clear solution. To this clear
solution, carbon (0.1 g) was added and stirred for 30 min. The re-
action mixture was filtered in hot. The filtrate was cooled and the
formed solid was filtered and washed with water. It was dried to
obtain the product 2-chloro-1-(3,4-dihydroxyphenyl)ethanone (2).
Yield: 2.5 g, 74%; M.P. 175e177 ꢀC.
To a solution of the 4-(4-chlorophenyl)-5-p-tolyl-4H-1,2,4-
triazole-3-thiol (3) (CAS No. 451501-95-4) (0.6 g, 1.99 mmol) in
acetone (30 mL) was added K2CO3 (0.3 g, 2.17 mmol) followed by 2-
chloro-1-(3,4-dihydroxyphenyl)ethanone (2) (0.35 g, 1.88 mmol).
The resulting solution was stirred at room temperature for 14 h,
diluted with water and extracted with EtOAc. The organic layer
was washed with water, dried on anhydrous Na2SO4, then filtered
and evaporated under reduced pressure to afford white solid as
crude product. Then recrystallization of the crude product in
ethanol/petroleum(60e90) gave the purified product 2-(4-(4-
chlorophenyl)-5-p-tolyl-4H-1,2,4-triazol-3-ylthio)-1-(3,4-
dihydroxyphenyl)ethanone (XDW-1, 4) as white solid. Yield: 0.20 g,
23.5%; Rf ¼ 0.50 (petroleum 60e90/EtOAc, 1:1). 1H NMR (400 MHz,
DMSO-d6, ppm)
d
: 9.71 (2H, brs, 2 ꢁ OH), 7.62 (2H, d, J ¼ 8.6 Hz),
7.46 (2H, d, J ¼ 8.7 Hz), 7.44 (1H, dd, J ¼ 8.7, 2.4 Hz), 7.36 (1H, d,
J ¼ 2.4 Hz), 7.23 (2H, d, J ¼ 8.2 Hz), 7.17 (2H, d, J ¼ 8.2 Hz), 6.84 (1H,
d, J ¼ 8.2 Hz), 4.80 (2H, s, SeCH2), 2.28 (3H, s, Me). ESI-MS: m/z
452.3 (Mþ1), 454.4 (Mþ3). C23H18ClN3O3S (451.076).
5.1.2. Preparation of 1-(3,4-dihydroxyphenyl)-2-((1-mesityl-1H-
imidazo[4,5-c] pyridin-2-yl)thio)ethanone (CHEQ-2, 9a)
Compound 4-chloro-3-nitropyridine (5a) (1.58 g, 10 mmol),
NaHCO3 (2.52 g, 30 mmol) and 2,4,6-trimethylbenzenamine
(1.50 g, 11 mmol), were dissolved in alcohol (25 mL). The reaction

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Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
mixture was heated to reflux temperature with stirring and kept
for 10 h and then continued the reaction at room temperature for
another 12 h (monitored by TLC). The reaction liquid was evapo-
rated under reduced pressure. Then, the residue was dissolved
with a small amount DCM (CH2Cl2) and chromatographed on silica
gel using ethyl acetateepetroleum ether system. Pure fractions
were collected and concentrated to give 6a as a yellow solid, yield:
59.2%, mp: 133e139 ꢀC. MS (ESI): m/z 258.12 (Mþ1). C14H15N3O2
(257.12).
The reaction intermediate 6a (3 g, 12 mmol), Pd/C (0.1 g) and
25 mL alcohol was vacuumed and filled with excess hydrogen gas.
The reaction mixture was kept for 15 h and then filtered and
evaporated under reduced pressure. The obtained crude product
was washed with petroleum ether and evaporated under reduced
pressure again to give the purified compound 7a. Brown powder,
yield: 81%. mp: 187e192 ꢀC. MS (ESI): m/z 228.6. C14H17N3 (227.14).
Intermediate 7a (0.6 g, 2.6 mmol), EtOCS2K (0.5 g, 3.1 mmol) and
NaHCO3 (0.05 g, 0.6 mmol) were dissolved in alcohol (10 mL) and
water (3 mL), then the solution was refluxed for 5 h. The reaction
liquid was cooled to room temperature and then 20 mL water and
8 mL NaOH solution (2 M) was added. The formed precipitate was
removed by suction filtration and the filtrate was modulated to PH
7 by adding acetic acid. 1-Mesityl-1H-imidazo[4,5-c]pyridine-2-
thiol (8a) was precipitated, filtered by suction filtration and air-
dried to yield a yellowish-white powder. Yield: 83.2%. mp:
247e250 ꢀC. MS (ESI): m/z 270.7. C15H15N3S (269.1).
procedure described for the preparation of 8a. Gray powder, yield:
63%. mp: 247e253 ꢀC. MS(ESI): m/z 270.7. C15H15N3S (269.1).
To a solution of compound 8b (0.27 g, 1.0 mmol) in acetone
(20 mL) was added K2CO3 (0.14 g, 1.0 mmol) followed by 2-chloro-
1-(3,4-dihydroxyphenyl)ethanone (2) (0.18 g, 0.96 mmol). The
resulting solution was stirred at room temperature for 10.5 h,
diluted with water and extracted with EtOAc. The organic layer was
washed with water, dried on anhydrous Na2SO4, then filtered and
evaporated under reduced pressure to afford the crude product as
white solid. Then recrystallization of the crude product in ethanol
gave the purified product1-(3,4-dihydroxyphenyl)-2-(3-mesityl-
3H-imidazo[4,5-b]pyridin-2-ylthio)ethanone (CHEQ-1, 9b) as
white solid. Yield: 0.27 g, 67.0%; Rf ¼ 0.6 (petroleum 60e90/EtOAc,
1:1). 1H NMR (400 MHz, DMSO-d6, ppm)
d: 10.00 (1H, s, OH), 9.44
(1H, s, OH), 8.13 (1H, dd, J ¼ 4.1 Hz, C7-imidazo[4,5-b]pyridine-H),
7.96 (1H, dd, J ¼ 7.76 Hz, C5-imidazo[4,5-b]pyridine-H), 7.49 (1H, d,
J ¼ 7.88 Hz, C6-Ph0-H), 7.42 (1H, s, C2-Ph0-H), 7.24 (1H, dd, J ¼ 2.4,
7.3 Hz, C6-imidazo[4,5-b]pyridine-H), 7.14 (2H, s, C3,5-Ph-H), 6.86
(1H, d, J ¼ 7.0 Hz, C5-Ph0-H), 4.99 (2H, s, SeCH2), 2.37 (3H, s, Me),
1.87 (6H, s, 2 ꢁ Me). MS (ESI): m/z 420.3 (Mþ1). C23H21N3O3S
(419.13).
5.1.4. Preparation of N-(2-aminophenyl)-2-((1-mesityl-1H-imidazo
[4,5-c]pyridin-2-yl) thio)acetamide (CHEQ-3, 13a) (Scheme 3)
The starting material 2-nitroaniline (10) (2.0 g, 14.5 mmol) was
dissolved in DCM (15 mL), then Et3N (2.5 mL, 17.4 mmol) was added
and stirred for 10 min. Then the mixed solution of 2-chloroacetyl
chloride (1.38 mL, 17.4 mmol) in DCM (10 mL) was slowly added
to the above solution within 40 min. The reaction mixture was
stirred for 3 h. Ice water (40 mL) was added to the reaction solution,
then the solution was extracted with DCM (3 ꢁ 15 mL). Combined
organic phase was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to give the product 2-chloro-
N-(2-nitrophenyl) acetamide (11) as a yellow crystal with a yield of
83.6%. Then Fe (0.56 g, 10 mmol), NH4Cl (0.06 g, 1 mmol), HOAc
(0.12 mL, 2 mmol) were added into 5 mL H2O and stirred at 50 ꢀC for
15 min. A solution of 2-chloro-N-(2-nitrophenyl)acetamide (0.21 g,
1 mmol) in DMF (5 mL) was added into the above solution quickly,
and stirring was continued at 50 ꢀC for 15 min. Then the reaction
solution was alkalized to pH ¼ 9 with aqueous sodium carbonate
solution. The reaction mixture was filtered, the formed solid was
washed with water and ethyl acetate. The combined filtrate was
extracted with ethyl acetate. Then the combined organic layers
were washed with brine, dried over anhydrous Na2SO4, filtered,
concentrated under reduced pressure. Purification of the crude
product by flash chromatography on silica gel (petroleum ether/
ethyl acetate, 1/1) to give the N-(2-aminophenyl)-2-
chloroacetamide (12) as a gray solid. Yield: 72.5%.
Compound 8a (0.269 g, 1.0 mmol), K2CO3 (0.138 g, 1.0 mmol),
with 2-chloro-1-(3,4-dihydroxyphenyl)ethanone (2) (0.186 g,
1.0 mmol) were dissolved in acetone (10 mL). The reaction mixture
was stirred at room temperature for 7 h and then evaporated under
reduced pressure. Then the residue was poured into water (30 mL)
at room temperature and a large amount of gray precipitation was
obtained. Filtration and recrystallization the precipitation in
ethanol yielded the purified product1-(3,4-dihydroxyphenyl)-2-
((1-mesityl-1H-imidazo[4,5-c]pyridin-2-yl)thio)ethanone (CHEQ-
2, 9a) as gray power. Yield: 0.283 g, 67%; mp: 256e258 ꢀC. 1H NMR
(400 MHz, DMSO-d6, ppm) d: 9.78 (2H, brs), 8.88 (1H, s, C4-imidazo
[4,5-c]pyridine-H), 8.26 (1H, d, J ¼ 4.8 Hz, C6-imidazo[4,5-c]pyri-
dine-H), 7.49 (1H, dd, J ¼ 1.5, 8.2 Hz, C6-Ph0-H), 7.41 (1H, s, C2-Ph0-
H), 7.19 (2H, s, C3,5-Ph-H), 6.97 (1H, d, J ¼ 4.8 Hz, C7-imidazo[4,5-c]
pyridine-H), 6.86 (1H, d, J ¼ 8.2 Hz, C5-Ph0-H), 5.03 (2H, s, SeCH2),
2.38 (3H, s, Me), 1.88 (6H, s, 2 ꢁ Me). 13C NMR (100 MHz, DMSO-d6,
ppm) d: 190.1, 155.1, 152.2, 146.0, 142.5, 141.2, 140.8, 140.5, 140.1,
136.7 (2 ꢁ C), 130.1 (2 ꢁ C), 129.0, 127.4, 122.4, 115.7, 115.5, 105.0,
21.2, 17.4 (2 ꢁ C). MS (ESI): m/z 420.3 (Mþ1). C23H21N3O3S (419.13).
5.1.3. Preparation of 1-(3,4-dihydroxyphenyl)-2-(3-mesityl-3H-
imidazo[4,5-b] pyridin-2-ylthio)ethanone (CHEQ-1, 9b)
The reaction mixture of 2-chloro-3-nitropyridine (5b) (0.79 g,
5 mmol), 2,4,6-trimethylben-zenamine (2.7 g, 20 mmol), and KF
(0.47 g, 8 mmol) was slowly heated to reflux temperature with
stirring and continued for another 12 h (monitored by TLC). The
suspension was cooled to room temperature and then poured into
80 mL water and 10 mL ammonia. The mixture was then extracted
with CH2Cl2 (15 mL), washed successively with water (3 ꢁ 10 mL).
The organic layer was dried over anhydrous Na2SO4, filtered and
concentrated under reduced pressure to afford crude product 6b as
brown oil. And then the crude product was purified by recrystal-
lization from alcoholepetroleum ether to give 6b as yellow needle
crystals, yield: 67%, MS (ESI): m/z 258.12 (Mþ1). C14H15N3O2
(257.12). Then, compound N2-mesitylpyridine-2,3-diamine (7b)
was prepared from 6b using a similar procedure described for the
preparation of 7a. Gray powder, yield: 69%. mp: 181e184 ꢀC. MS
(ESI): m/z 228.6. C14H17N3 (227.14). Compound 3-mesityl-3H-imi-
dazo[4,5-b] pyridine-2-thiol (8b) was prepared using a similar
Compound 8a (0.15 g, 0.5 mmol), K2CO3 (0.14 g, 1.0 mmol), and
the intermediate 12 (0.12 g, 0.6 mmol) were dissolved in DMF
(10 mL). The reaction mixture was stirred at room temperature for
5 h and then evaporated under reduced pressure. The residue was
washed by water, then extracted with ethyl acetate (3 ꢁ 50 mL).
Combined organic phase was dried over anhydrous Na2SO4, filtered
and chromatographed on silica gel using ethyl acetateepetroleum
ether system. Pure fractions were collected and concentrated and
recrystallized from EtOAc/petroleum 60e90 to give the desired
compound N-(2-aminophenyl)-2-((1-mesityl-1H-imidazo[4,5-c]
pyridin-2-yl)thio) acetamide (CHEQ-3, 13a) as white crystal. Yield
65.5%, mp: 154e158 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 9.60
(1H, s, NH), 8.92 (1H, s, C4-imidazo[4,5-c]pyridine-H), 8.28 (1H, d,
J ¼ 5.4 Hz, C6-imidazo[4,5-c]pyridine-H), 7.18 (2H, s, C3,5-Ph-H),7.09
(1H, dd, J ¼ 7.72, 0.92 Hz, C6-Ph0-H), 6.97 (1H, d, J ¼ 5.44 Hz, C7-
imidazo[4,5-c]pyridine-H), 6.92 (1H, td, J ¼ 7.92, 1.2 Hz, C4-Ph0-H),
6.71 (1H, dd, J ¼ 7.92, 0.8 Hz, C3-Ph0-H), 6.52 (1H, td, J ¼ 7.68,

Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
305
0.92 Hz, C5-Ph0-H), 5.08 (2H, s, NH2), 4.33 (2H, s, SeCH2), 2.36 (3H, s,
mp: 181e184 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 9.92 (1H, s,
Me), 1.85 (6H, s, 2 ꢁ Me). 13C NMR (100 MHz, DMSO-d6, ppm)
d:
NH), 9.82 (1H, s, OH), 8.16 (1H, dd, J ¼ 4.80,1.28 Hz, C7-imidazo[4,5-
b]pyridine-H), 8.05 (1H, dd, J ¼ 7.96, 1.28 Hz, C5-imidazo[4,5-b]
pyridine-H), 7.93 (1H, dd, J ¼ 8.04, 1.24 Hz, C6-Ph0-H), 7.31 (1H, dd,
J ¼ 7.96, 4.84 Hz, C6-imidazo[4,5-b]pyridine-H), 7.93 (2H, s, C3,5-Ph-
H), 6.92 (1H, td, J ¼ 8.20, 1.40 Hz, C4-Ph0-H), 6.85 (1H, dd, J ¼ 7.88,
1.20 Hz, C3-Ph0-H), 6.75 (1H, td, J ¼ 8.12,1.28 Hz, C3-Ph0-H), 4.34 (2H,
s, SeCH2), 2.36(3H, s, Me), 1.85 (6H, s, 2 ꢁ Me). 13C NMR (100 MHz,
166.1, 155.2, 143.4, 142.6, 141.1, 140.7, 140.6, 140.0, 136.7 (2 ꢁ C),
130.1 (2 ꢁ C), 128.9, 127.0, 126.6, 122.9, 116.3, 115.8, 105.1, 36.1, 21.2,
17.4 (2 ꢁ C). ESI-MS: m/z 418.5 (Mþ1), 420.3 (Mþ3), 440.5 (MþNa).
C
23H23N5OS (417.53).
5.1.5. Preparation of N-(2-aminophenyl)-2-((3-mesityl-3H-imidazo
[4,5-b]pyridin- 2-yl)thio)acetamide (CHEQ-4, 13b)
DMSO-d6, ppm) d: 166.3, 154.4, 149.4, 147.7, 143.4, 140.1, 136.9
Compound CHEQ-4 (13b) was synthesized from N-(2-
aminophenyl)-2-chloroacetamide (12) and compound 8b accord-
ing to the procedure of CHEQ-3 (13a). White crystal, yield 71.3%,
(2 ꢁ C), 135.4, 129.7 (2 ꢁ C), 129.3, 126.7, 125.7, 124.8, 121.5, 119.4,
118.9, 115.5, 35.4, 21.2, 17.6 (2 ꢁ C). ESI-MS: m/z 419.4 (Mþ1), 422.6
(Mþ3), 441.4 (MþNa). C23H22N4O2S (418.51).
mp: 165e167 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 9.57 (1H, s,
NH), 8.16 (1H, dd, J ¼ 4.63, 0.8 Hz, C7-imidazo[4,5-b]pyridine-H),
8.01 (1H, dd, J ¼ 7.92, 0.92 Hz, C5-imidazo[4,5-b]pyridine-H), 7.29
(1H, dd, J ¼ 7.96, 6.05 Hz, C6-Ph0-H), 7.13 (2H, s, C3,5-Ph-H), 7.09(1H,
d, J ¼ 7.72, 0.92 Hz, C6-imidazo[4,5-b]pyridine-H), 6.92 (1H, td,
J ¼ 8.04, 1.04 Hz, C4-Ph0-H), 6.70 (1H, d, J ¼ 7.24 Hz, C3-Ph0-H), 6.52
(1H, td, J ¼ 7.68, 0.52 Hz, C5-Ph0-H), 5.04 (2H, s, NH2), 4.33 (2H, s,
SeCH2), 2.36 (3H, s, Me), 1.85 (6H, s, 2 ꢁ Me). 13C NMR (100 MHz,
5.2. Biological evaluations
5.2.1. Drugs and biochemicals
VK3 was purchased from Sigma Chemical Company (Sigma,
USA). Stock solutions (5 mM) of VK3, XDW-1 and CHEQ-1~CHEQ-6
were prepared in DMSO and stored at ꢂ20 ꢀC for less than 2 weeks.
All chemicals were of the purest reagent grade commercially
available. All cell culture reagents were from Gibco BRL (Gibco,
USA). Monoclonal anti-CDC25A, anti-Cyclin E, anti-caspase-3, anti-
Bax and anti-Bcl-2 antibodies were purchased from Cell Signaling
Technology Inc. (CST, USA) and anti-CDC25B, anti-CDK2 anti-
Cyclins (A and B) and anti-cleaved-PARP antibodies were pur-
chased from Abcam (Abcam, USA). Protein concentrations were
determined using a bicinchoninic acid (BCA) protein determination
kit from Thermo Scientific Pierce (Thermo, USA).
DMSO-d6, ppm) d: 166.2, 154.7, 149.3, 143.3, 143.2, 140.1, 136.9 (2C),
135.5, 129.7 (2C), 129.4, 126.9, 126.4, 125.4, 123.1, 118.8, 116.4, 115.9,
35.6, 21.2, 17.6 (2C). ESI-MS: m/z 418.5 (Mþ1), 420.3 (Mþ3), 440.5
(MþNa). C23H23N5OS (417.53).
5.1.6. Preparation of N-(2-hydroxyphenyl)-2-((1-mesityl-1H-
imidazo[4,5-c] pyridin- 2-yl)thio)acetamide (CHEQ-5, 16a)
The starting material 2-aminophenol (14) (1.0 g, 9.16 mmol) was
dissolved in 1,2-dichloroethane (8 mL). Then the mixed solution of
2-chloroacetyl chloride (1.53 mL, 19.2 mmol) and chloroacetic acid
(0.06 mL, 0.96 mmol), 1,2-dichloroethane (5 mL) was slowly added
to the above solution in 50 min. Then the mixture was refluxed for
3 h, and then cooled to room temperature. A large amount of
precipitation of crystallization was formed, filtered and recrystal-
lized from 1,2-dichloroethane to give the N-(2-hydroxyphenyl)-2-
chloroacetamide (15) as brown crystal. Yield: 90.3%.
5.2.2. Cell lines and cell culture
Human colon cancer HT-29 cells, breast cancer MCF-7 cells and
hepatocellular carcinoma HepG2 cells and all the other cells used in
the experiment were purchased from the Type Culture Collection of
the Chinese Academy of Sciences, Shanghai, China. HT-29 cells were
cultured in McCOY's 5A medium, while MCF-7 and HepG2 cells
were cultured in MEM medium. The other cell lines were main-
tained in the culture recommended by American type culture
collection (ATCC), see Supplementary data Table 1. All the mediums
were supplemented with 10% fetal bovine serum (FBS) and 1%
penicillin/streptomycin. Cultures were maintained at 37 ꢀC in a CO2
incubator with a controlled humidified atmosphere composed of
95% air and 5% CO2.
Compound 8a (0.15 g, 0.5 mmol), K2CO3 (0.14 g, 1.0 mmol), and
compound 15 (0.12 g, 0.6 mmol), was dissolved in DMF (10 mL). The
reaction mixture was stirred at room temperature for 5 h and then
evaporated under reduced pressure. The residue was washed by
water, then extracted with ethyl acetate (3 ꢁ 50 mL). Combined
organic phase was dried over anhydrous Na2SO4, filtered and
chromatographed on silica gel using ethyl acetateepetroleum ether
system. Pure fractions were collected, concentrated, and recrys-
tallized from EtOAc/petroleum 60e90 to give N-(2-
hydroxyphenyl)-2-((1-mesityl-1H-imidazo[4,5-c]pyridin-2-yl)
thio)acetamide (CHEQ-5, 16a) as white crystal. Yield 68.9%, mp:
5.2.3. In vitro enzyme assays
The enzyme inhibition activity of CHEQ-1~CHEQ-6 was
measured according to the method reported previously. Briefly,
10 mL of enzyme (300 ng of CDC25A or 200 ng of CDC25B) was
preincubated for 20 min with the different concentrations of
compounds or with DMSO, as a vehicle control. The reaction
231e233 ꢀC. 1H NMR (400 MHz, DMSO-d6, ppm)
d: 9.96 (1H, s, NH),
9.84(1H, s, OH), 8.96 (1H, s, C4-imidazo[4,5-c]pyridine-H), 8.28 (1H,
d, J ¼ 5.44 Hz, C6-imidazo[4,5-c]pyridine-H), 7.93(1H, dd, J ¼ 8.0,
1.12 Hz, C6-Ph0-H), 7.18 (2H, s, C3,5-Ph-H), 6.98 (1H, d, J ¼ 5.44 Hz,
C7-imidazo[4,5-c]pyridine-H), 6.92 (1H, td, J ¼ 8.12, 1.36 Hz, C4-Ph0-
H), 6.85 (1H, dd, J ¼ 7.92, 1.24 Hz, C3-Ph0-H), 6.74 (1H, td, J ¼ 7.12,
1.28 Hz, C5-Ph0-H), 4.35 (2H, s, SeCH2), 2.36 (3H, s, Me), 1.85 (6H, s,
mixture including 5
40 mM NaCl, 1 mM DTT, and 20% glycerol) and 10
m
L of reaction buffer (100 mM TriseHCl, pH 8.2,
L of substrate
m
assay solution (0.5 mM OMFP, 3-O-methylfluorescein phosphate)
were added to initiate the reaction. The reaction was performed in a
water bath at 37 ꢀC for 30 min. Then 100
mL of BIOMOL Green Re-
2 ꢁ Me). 13C NMR (100 MHz, DMSO-d6, ppm)
d
: 166.2, 155.0, 147.8,
agent (BioMol, USA) were added and the samples were incubated at
room temperature for 30 min to allow development of the green
color. The absorbance of the reaction solution was measured in a
spectrophotometer at 630 nm by a microplate reader. IC50 values
were then estimated at least three times and the averaged values
were selected.
142.7, 141.3, 140.6, 140.5, 140.3, 136.6 (2 ꢁ C), 130.1 (2 ꢁ C), 128.8,
126.7, 124.8, 121.4, 119.3, 115.5, 105.1, 36.1, 21.2, 17.4 (2 ꢁ C). ESI-MS:
m/z 419.4 (Mþ1), 422.6 (Mþ3), 441.4 (MþNa).
C23H22N4O2S
(418.51).
5.1.7. Preparation of N-(2-hydroxyphenyl)-2-((3-mesityl-3H-
imidazo[4,5-b] pyridine-2-yl)thio)acetamide (CHEQ-6, 16b)
Compound CHEQ-6 (16b) was synthesized from N-(2-
hydroxyphenyl)-2-chloroacetamide (15) and compound 8b ac-
cording to the procedure of CHEQ-5 (16a). White crystal, yield 69.7%,
5.2.4. Cell viability assay
The effect of CHEQ-1~CHEQ-6 on cellular viability was assessed
by using the MTT assay. Cells were seeded at an initial density of
3000 cells per well in 96-well culture plates (Costar, USA) 24 h prior

306
Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
to the experiment. Then the cells were treated with various con-
centrations of XDW-1, VK3 or CHEQ-1~CHEQ-6 for 48 h. At the end
of the time point, MTT was added to the medium (0.5 mg/mL) and
incubated at 37 ꢀC for 4 h. The resulting insoluble formazan was
dissolved with DMSO and measured at 570 nm using a spectro-
photometer. IC50 values were then estimated at least three times
and the averaged values were selected.
Annexin VeFITC (20
mL) for 30 min in the dark room, and then diluted with an addi-
tional 300 L of binding buffer. The apoptotic index was deter-
mined by flow cytometry (BD FACScan™, USA), and the form scatter
diagram was analyzed.
mg/mL) and 5 mL of propidium iodide (PI, 50 mg/
m
5.2.10. Western blot analysis
Western blot analysis was performed as described previously
[32]. Briefly, total protein from the cells was extracted in RIPA lysis
buffer (Beyotime, China) supplemented with protease inhibitor
cocktail tablets (complete ULTRA Tablets, Roche) and quantified
using a BCA Protein Assay Kit (Thermo Fisher Scientific, USA). A total
5.2.5. Cell cycle analysis
The ratio of HT29, HepG2 and MCF-7 cells in the G0/G1, S and
G2/M phases of cell cycle was determined by their DNA content.
Cells were treated with or without various concentrations of CHEQ-
2 for 48 h and then harvested, washed twice with cold PBS, and
fixed with 75% ice-cold ethanol overnight. Fixed cells were washed
of 30 mg of protein was separated using 10% SDS-PAGE and elec-
trophoretically transferred to a PVDF membrane (Millipore, USA).
The membrane was blocked with 5% nonfat milk and incubated at
twice with cold PBS and incubated with 5
for 30 min at 37 ꢀC. After incubation, the cells were stained with
50 g/mL PI for 15 min in the dark room and analyzed by flow
mL of 100 mg/mL RNase A
4
ꢀC overnight with the desired primary antibody. After washing
m
three times with PBS containing 0.05% Tween-20 (PBST), the
membrane was incubated for 70 min with horseradish peroxidase-
conjugated secondary antibody diluted in PBST. Protein bands were
visualized using enhanced chemiluminescence (Millipore, USA) and
detected using a ChemiDoc XRS þ Imaging System (Bio-rad, USA).
cytometry. Untreated cells were used as a control.
5.2.6. ROS production assay
Intracellular ROS levels were analyzed using the ROS-sensitive
dye, 20,70-Dichlorofluorescin diacetate (DCFH-DA, Sigma, USA).
DCFH-DA is a cell-permeable non-fluorescent probe, which can be
de-esterified intracellularly and turned into highly fluorescent
20,70-dichlorofluorescein (DCF) upon oxidation. DCFH-DA dissolved
in absolute ethanol (10 mM), was used at a final concentration of
5.2.11. Acute toxicity experiment
Mortality and clinical signs were continually observed for 0, 1, 2,
3, 4, 5, and 6 h after oral administration or abdominal injection of a
single dose (2000 mg/kg) on day 0 and at one time per day from day
1 to day 14. The animals (10 mice per group) were weighed before
dosing (day 0, justified as 100%) and after dosing (day 1 to day 7,
once a day) using an electronic balance (Sartorius Co., Germany).
After 7 days, the body weights of animals were no longer recorded
and clinical signs were continued observed. On day 14, all animals
were euthanized by cervical vertebra luxation and examined for
internal organ abnormalities.
10 mM. Cells were treated with or without various concentrations of
CHEQ-2 for 48 h until culture medium was replaced with serum-
free medium. ROS probes were then added and incubated for
30 min at 37 ꢀC. The cells were harvested for image capture by
fluorescence microscope or flow cytometry analysis.
5.2.7. Mitochondrial membrane potential (Dj) assay
Mitochondrial membrane potential was determined using the
Beyotime Mitochondrial Membrane Sensor kit (Beyotime, China) by
measuring the potential-dependent accumulation of 5,50,6,60-tet-
rachloro-1,10,3,30tetraethylbenzimidazolylcarbocyanine iodide (JC-
1). JC-1 aggregates in the mitochondria of healthy cells and emits
red fluorescence against a green monomeric cytoplasmic back-
ground staining. However, in cells with a collapsed mitochondrial
membrane potential, the dye is not able to accumulate in the
mitochondria and remains as monomers throughout the cells
emitting green fluorescence [15]. After 48 h of CHEQ-2 treatment,
cells were washed with PBS once, then incubated with JC-1 staining
solution according to the manufacturer's specification for 20 min at
37 ꢀC. After staining, the cells were harvested for image capture by
fluorescence microscope or flow cytometry analysis.
5.2.12. Tumor xenograft experiment
All nude mice were housed in sterile cages within laminar air
flow hoods under specific pathogen-free conditions with sterile
food and water ad libitum. Exponentially growing HepG2 cells were
harvested, washed with PBS, and resuspended at a concentration of
1 ꢁ 107/mL. The left flank of mice was sterilized with iodophor and
then inoculated subcutaneously with 0.1 mL of cell suspension
(approximately 1 ꢁ106 cells). Tumor development was followed by
caliper measurements in 2 dimensions (L and W), and the volume
(V) of the tumor was calculated by the formula for a prolate ellip-
soid (V ¼ L ꢁ W2 ꢁ 3.14/6) as reported [33]. The treatment started
when the average volume of tumor reached 150 mm2. For treat-
ment with CHEQ-2, the mice were given 10 mg/kg of CHEQ-2 daily
by oral administration, while the control mice received vehicle
solvent. After 14 days of treatment, the animals were euthanized.
And the tumors were harvested, weighed, and prepared for further
examination. All animal experiments were performed according to
a protocol approved by the Institutional Animal Care and Use
Committee.
5.2.8. Hoechst 33342 staining
Fluorescent dye Hoechst 33342 was used to observe the Chro-
matin condensation of the cells. HT-29, HepG2 and MCF-7 cells
were seeded into 6-well plate 24 h prior to the experiment. The
cells were then treated with different concentrations of CHEQ-2 for
the next 48 h. The cells were washed with PBS once and incubated
with 2
m
g/mL Hoechst 33342 at 37 ꢀC for 30 min. Chromatin
5.2.13. Statistical analysis
condensation was observed by fluorescence microscopy.
Data are expressed as the means S.D. Statistical comparisons
were performed by two-way ANOVA followed by Fisher's protected
least significance difference (PLSD) test. A probability value <0.05
was considered statistically significant.
5.2.9. Annexin VeFITC/PI staining experiment
Apoptosis was assessed by annexin Vefluorescence isothiocya-
nate (FITC)/PI apoptosis detection kit (Beyotime, China). The
externalization of phosphatidylserine, an early apoptotic event, was
analyzed by flow cytometry-based annexin V binding assays after
treatment with different concentrations of CHEQ-2 for 48 h. After
treatment, cells were digested and washed twice with PBS. The
Acknowledgments
The financial support from the National Natural Science Foun-
dation of China (NSFC No. 81102320), Research Fund for the
Doctoral Program of Higher Education of China (No.
cells were re-suspended in 200 mL of binding buffer, stained with

Y. Song et al. / European Journal of Medicinal Chemistry 82 (2014) 293e307
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China Postdoctoral Science Foundation funded project (Nos.
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The authors have declared no conflict of interest.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.05.063.
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