Potent CDC25B and PTP1B phosphatase inhibitors: 2′,4′,6′-trihydroxylchalcone derivatives

Article abstract of DOI:10.1007/s00044-014-1264-x

In this study, we examined a series of 2′,4′,6′-trihydroxychalcone derivatives for their inhibitory activity as inhibitors of CDC25B and PTP1B. The pharmacological results showed that all of the tested compounds significantly inhibited CDC25B and PTP1B phosphatase in vitro. Among them, three compounds 2, 6, and 7 had the best inhibition activity, with inhibition rates against CDC25B were 99.56, 99.68, and 99.63 %, respectively, and with inhibition rates against PTP1B were 98.99, 99.37, and 98.08 %, respectively, which is similar to reference drugs Na₃VO₄ and Oleanolic acid, respectively. Cytotoxic activity assays showed compounds 2, 6, and 7 are potent against HeLa and HCT116. Moreover, compound 6 delayed the potent antitumor inhibitory activity in a colo205 xenograft model in vivo.

Full text of DOI:10.1007/s00044-014-1264-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1264-x
ORIGINAL RESEARCH
Potent CDC25B and PTP1B phosphatase inhibitors:
20,40,60-trihydroxylchalcone derivatives
•
Shui-Lian Zhao Zhou Peng Xing-Hua Zhen Hong-Guo Jin
•
•
•
•
Yan Han You-Le Qu Li-Ping Guan
•
Received: 19 April 2014 / Accepted: 14 September 2014
Ó Springer Science+Business Media New York 2014
Abstract In this study, we examined a series of 20,40,60-
trihydroxychalcone derivatives for their inhibitory activity
as inhibitors of CDC25B and PTP1B. The pharmacological
results showed that all of the tested compounds signifi-
cantly inhibited CDC25B and PTP1B phosphatase in vitro.
Among them, three compounds 2, 6, and 7 had the best
inhibition activity, with inhibition rates against CDC25B
were 99.56, 99.68, and 99.63 %, respectively, and with
inhibition rates against PTP1B were 98.99, 99.37, and
98.08 %, respectively, which is similar to reference drugs
Na3VO4 and Oleanolic acid, respectively. Cytotoxic
activity assays showed compounds 2, 6, and 7 are potent
against HeLa and HCT116. Moreover, compound 6
delayed the potent antitumor inhibitory activity in a
colo205 xenograft model in vivo.
Introduction
Chalcone (trans-1,3-diphenyl-2-propen-1-ones) are the
biogenetic precursors of all known flavonoids and are
abundant in edible plants (Go et al., 2005). Chemically,
they consist of open-chain flavonoids in which the two
aromatic rings are joined by a three-carbon a,b-unsaturated
carbonyl system. Chalcone moieties are common sub-
structures in numerous natural products belonging to the
flavonoid family (Akihisa et al., 2006; Narender et al.,
2005; Zhang et al., 2006). Chalcone derivatives are very
versatile as physiologically active compounds and sub-
strates for the evaluation of various organic syntheses.
These compounds have been reported to possess several
biological activities, such as anti-inflammatory (Lee et al.,
2006; Nowakowska, 2007), antimalarial (Dominguez et al.,
2005; Alain Valla et al., 2006), cytotoxic (Modzelewska
et al., 2006; Hsu et al., 2006), antileishmanial (Nielsen
et al., 1998; Boeck et al., 2006), antifungal (Svetaz et al.,
2004), anti-HIV (Cheenpracha et al., 2006), and as tyrosine
kinase inhibitors (Nerya et al., 2004). Having such varied
pharmacological activities, these molecules have attracted
medicinal chemists and therefore several strategies have
been developed to synthesize them.
Keywords 20,40,60-Trihydroxychalcone ꢀ
CDC25B ꢀ PTP1B ꢀ Phosphatase inhibitors
Cell division cycle 25(CDC25)B is a dual-specificity
phosphatase that plays pivotal role in controlling cell cycle
progression by catalyzing removal of covalently attached
contiguous phosphates and the subsequent activation of
cyclin-dependent kinase 1 (Rudolph, 2007). Among the
CDC25 family members, CDC25B has been proposed to
regulate reentry into mitosis after DNA damage (Boutros
et al., 2006; Vugt et al., 2004; Bugler et al., 2006). Impor-
tantly, CDC25B is oncogenic (Galaktionov et al., 1995) and
its overexpression has been documented in a variety of
human cancers, including head and neck, colon, and
S.-L. Zhao ꢀ Z. Peng ꢀ X.-H. Zhen ꢀ Y. Han ꢀ Y.-L. Qu ꢀ
L.-P. Guan (&)
Zhejiang Provincial Key Engineering Technology Research
Center of Marine Biomedical Products and Food and Pharmacy
College, Zhejiang Ocean University, Zhejiang Provincial,
Zhoushan 316022, People’s Republic of China
e-mail: glp730@163.com
H.-G. Jin (&)
Department of Neurology, Zhoushan Hospital of Zhejiang
Province, Zhoushan 316021, Zhejiang,
People’s Republic of China
e-mail: jhgyj2003@163.com
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Med Chem Res
Fig. 1 Structure of reported
CDC25B and PTP1B inhibitors
OCH3
OH
HO
OH
O
HO
O
O
CF3
OH
OH
I
II
OH
OH
HO
OH
HO
O
OCH3
OH
O
III
IV
Table 1 Inhibition rates of compounds 1–12 against recombinant
human CDC25B and PTP1B
non-small cell lung cancer (Gasparotto et al., 1997; Tak-
emasa et al., 2000; Sasaki et al., 2001). CDC25B also
interacts with the steroid receptors (Ma et al., 2001), sug-
gesting that some Cdk-independent sites might contribute to
the oncogenic potential of CDC25B. In addition, transgenic
mice that overexpress CDC25B in mammary epithelium
exhibit mammary gland hyperplasia and increased suscep-
tibility to 9,10-dimethyl-1,2-benzanthracene-induced
mammary tumorigenesis (Ma et al., 1999; Yao et al., 1999).
Recently, several chalcones derived from synthesis and
their derivatives have been identified as CDC25B and
PTP1B inhibitors (Fig. 1). For example, Sun et al. reported
the chalcone compound I and II synthesized exhibited
moderate inhibitory effects against CDC25B (Sun et al.,
2013). Yoon et al. reported that several chalcones (III)
isolated from Glycyrrhiza inflata and semisynthetic lic-
ochalcone A derivatives also exhibited moderate inhibitory
effects against PTP1B (Yoon et al., 2009). Chen et al. also
reported that brousso-chalcone A (IV) isolated from
Broussonetia papyrifera exhibited moderate inhibitory
effects against PTP1B (Fig. 1) (Chen et al., 2002).
Compounds R
Dose
(lg/
mL)
CDC25B PTP1B
inhibition rates inhibition rates
(%)
(%)
1
2
H
20
20
20
20
20
20
20
20
20
20
99.03 0.02
99.56 0.44
98.03 0.01
95.34 0.23
97.72 0.08
99.68 0.15
99.63 0.37
98.55 0.99
99.19 0.23
98.34 0.99
97.25 1.98
98.99 0.68
97.97 2.37
97.31 1.91
98.31 0.71
99.37 0.91
98.08 0.95
92.00 7.67
92.86 10.85
98.37 1.29
2-F
3
3-F
4
4-F
5
2-Cl
3-Cl
2,4-Cl2
2-Br
4-CH3
6
7
8
9
10
3,4-
(CH3)2
11
3,4-
20
99.30 0.23
94.34 8.74
(OCH3)2
12
4-OH
20
20
20
95.05 0.78
98.00 0.06
–
97.25 0.02
–
Na3VO4
–
–
Oleanolic
acid
98.46 0.11
Although chalcones, which are widely expressed in
plants, exert various biological activities, including
CDC25B and PTP1B inhibition, the structure–activity rela-
tionship of chalcones against CDC25B and PTP1B inhibi-
tion has not been fully examined because of an insufficient
number of molecular compounds to test. Therefore, in the
present study, a series of 20,40,60-trihydroxychalcone com-
pounds evaluated their inhibitory effects on CDC25B and
PTP1B, and then elucidated the structure–activity relation-
ship. Selected compound was also subjected to kinetics study
and selectivity analyses to determine whether their biologi-
cal properties made them suitable for further development.
The pharmacological results showed that all of the tested
compounds significantly inhibited CDC25B phosphatase
and PTP1B phosphatase in vitro (Table 1). The mechanism
of inhibition was further studied.
Materials and methods
Chemicals and reagents
20,40,60-Trihydroxychalcone compounds (1–12) (Fig. 2)
were synthesized in our laboratory as described previously
(Sui et al., 2012). All compounds were confirmed by IR
spectra (FT-IR1730, Bruker), 1H-NMR and 13C-NMR
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Med Chem Res
O
O
O
OH
O
CH3
R
R
O
O
O
OH
O
B
HO
OH
O
3 M HCl
KOH, EtOH
A
MeOH
O
OH
O
O
CH
1-12
R
R
1=-H
2=2-F
3=3-F
4=4-F
5=2-Cl
6=3-Cl
7=2,4-Cl2
12=4-OH
8=2-Br 9=4-CH3
10=3,4-(CH3)2
11=3,4-(OCH3)2
Fig. 2 Synthesis routes of target compounds 1–12
spectra (AV-300, Bruker, Switzerland), Mass spectra
(HP1100LC/MS, Agilent Technologies, USA), and Ele-
mental analysis (Perkin Elmer 204Q CHN). 3-O-methyl-
fluorescein phosphate(OMFP), 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide (MTT), HeLa and
HCT-116 cells were purchased from Sigma Aldrich (St
Louis, MO, USA). All other reagents and solvents used in
experiments were the analytical grade.
Sciences, Boston, MA, USA). Assays were performed in a
total volume of 100 lL containing 50 mM 3-[N-morpho-
lino]propanesulfonicacid (MOPs), pH 6.5, 2 Mm pNPP,
and 20 nM recombinant PTP1B, as well as the indicated
concentrations of the inhibitor. The production of pNP was
continuously monitored, and the initial rate of the hydro-
lysis was determined from the early linear region of the
enzymatic reaction kinetic curve.
Enzyme kinetic analysis
Biological activity
The enzyme kinetic assays of compound 6 was performed
as previously (McGovern et al., 2003). Assays were per-
formed in a total volume of 100 lL containing 5 lmol/L
OMFP, pH 8.0, 20 nmol/L recombinant CDC25B, OMFP
in two-fold dilutions from 80 mmol/L, and different con-
centrations of the inhibitor. In the presence of the com-
petitive inhibitor, the Michaelis–Menten equation is
CDC25B activity assay
The enzymatic activities of the CDC25B catalytic domain
were determined by monitoring the dephosphorylation of
OMFP. Dephosphorylation of OMFP generates product
OMF, which was detected at a 485 nm excitation/535 nm
emission. In a typical 100 lL assay mixture containing
50 mmol/L Tris–HCl, 50 mmol/L NaCl, pH 8.0, 5 lmol/L
OMFP, 20 nmol/L recombinant CDC25B, 1 % glycerin,
1 mmol/L DTT, in presence or absence of 2 lL inhibitor in
dimethylsulfoxide (DMSO), activities were continuously
monitored and the initial rate of the dephosphorylation was
determined using the early linear region of the enzymatic
reaction kinetic curve. Continuous kinetic monitoring was
performed in clear 96-well plates (Corning, Lowell, MA).
described as 1/v = (Km/(Vmax[S])) (1 ? [I]/Ki) ? 1/Vmax
,
where v = initial rate, Vmax = maximum rate, and
[S] = substrate concentration. The Ki value was deter-
mined using a linear replot of the apparent Km/Vmax slope
from the primary reciprocal plot versus inhibitor concen-
tration [I] according to the equation Km/Vmax = 1 ? [I]/Ki.
Cell culture
HeLa and HCT116 cells were kept at logarithmic growth in
5 % CO2 at 37 °C with HG-DMEM, McCoy’s 5A and F12
medium, respectively, supplemented with 10 % FBS and
100 units/mL each of penicillin G and streptomycin.
Biological assays for PTP1B
The enzymatic assays for PTP1B were performed as
described elsewhere (Shi et al., 2008). Briefly, the enzy-
matic activity of the PTP1B catalytic domain was deter-
mined at 30 °C by monitoring the hydrolysis of pNPP.
Dephosphorylation of pNPP generates the product pNP,
which was monitored at an absorbance of 405 nm using an
EnVision multilabel plate reader (PerkinElmer Life
Cytotoxicity
Cytotoxicity assays were performed on human colon can-
cer (HCT116) and cervical carcinoma cell lines. Cells
(6,000–10,000) in 100 lL culture medium per well were
123

Med Chem Res
seeded onto a 96-well microtest plate (Falcon, CA). Cells
were treated in trplicate with a gradient concentration of
tested compounds were added and the plate was incubated
at 37 °C for 72 h. For two cell lines, MTT assay was
performed to measure the cytotoxic effects. The com-
pounds concentration required for IC50 of tumor cells was
determined from the dose–response curve.
We next analyzed the structure–activity relationships of
compounds 1–12. The substituted compounds 2–12, which
contained electron-donating or electron-withdrawing
groups on the B ring, showed potent CDC25B inhibitory
activity. The halogen substituted derivatives (2–8) with
electron-withdrawing groups on the B ring, showed more
potent activity (95.34–99.68 %). We also found that the
position of the substituent on the B ring significantly
influenced CDC25B inhibition activity, with an activity in
the order of 2-F [ 3-F [ 4-F for fluorinated compounds,
3-Cl [ 2-Cl [ 2,4-Cl2 for chlorinated compounds. For
brominated compound (2-Br), also possessed more potent
activity, which was similar to that of Na3VO4. We also
found that the same position of the different halogen atoms
substituent (F, Cl, and Br) on the B ring influenced
CDC25B inhibition, with an activity in the order of
2-F [ 2-Br [ 2-Cl. Among them, compounds 6 and 7 had
better inhibition activity and significantly inhibits CDC25B
with an inhibition rates against CDC25B was 99.68 and
99.63 %, respectively, which is more potent to reference
drugs Na3VO4.
Antitumor activities in vivo
The antitumor activity of compound 6 was evaluated and
Irinotecan (IRT) was used as a reference drug in vivo.
BALB/C nude male mice (weighting 18–20 g) were
obtained from Shanghai weasley grams of laboratory ani-
mal co., LTD. Colo205 cancer cell suspensions were
implanted subcutaneously into the right axillary region in
mice. Treatment was begun when implanted tumor had
reached a volume of *100–300 mm3 (after 17 days). The
animals were randomized into appropriate groups (6 ani-
mals per treatment and 8 animals for the control group) and
administered by gavage once daily for five consecutive
days from day 17 after implantation of the cells. Tumor
volumes were monitored by caliper measurement of the
length and width and calculated using the formula of
TV = 1/2 9 a 9 b2, where a is the tumor length and b is
the width. Tumor volumes and body weights were moni-
tored every 4 days over the course of treatments.
We investigated the influence of electron-donor groups
(1, 9–12) on CDC25B activity. The order of contribution
was 3,4-(OCH3)2 [ -H [ 3,4-(CH3)2 [ 4-CH3 [ 4-OH.
Compound 11 showed the most inhibition activity, its
inhibition rates against CDC25B was 99.30 %, which is
similar to reference drugs Na3VO4 (Table 1).
PTP1B is one member of the PTP family, and several
groups have demonstrated that the overexpression of
PTP1B is sufficient to drive tumorigenesis in mice, pro-
viding additional support for the use of PTP1B inhibitors as
therapeutic agents against cancer (Easty et al., 2006; Tonks
and Muthuswamy, 2007; He et al., 2011; Yang et al., 2011;
He et al. 2012). The inhibitory activity of the synthesized
compounds (1–12) against PTP1B was measured using
pNPP as a substrate. The results are summarized in Table 1.
All compounds showed potent PTP1B inhibitory activity at
a concentration of 20 lg/mL, with the percentage inhibition
values ranging from 92.00 to 99.37 %. After analyzing the
inhibitory activity of the synthesized compounds 1–12, the
following SARs were obtained. The substitutions (R) with
electron-withdrawing groups on the B ring in compounds
2–8, the position of the substituent (Br, F, and Cl) on the B
ring significantly influenced the PTP1B inhibitory activity,
with an activity in the order of 2-F [ 3-F [ 4-F for the
fluorinated compounds, and 3-Cl [ 2-Cl [ 2,4-Cl2 for the
chlorinated compounds. In all cases, the 2-F containing
provided higher levels of inhibitory activity toward PTP1B
than the corresponding Cl and Br containing compounds.
The presence of an electron-donating group at the 4-posi-
tion on the B ring led to moderate levels of activity against
PTP1B. The order of contribution was 3,4-(CH3)2 [
4-OH [-H [3,4-(O CH3)2 [4-CH3. Compound 10 showed
Results and discussion
The CDC25 phosphatases are considered potential targets
for the development of new cancer therapeutic agents. The
collective drug discovery efforts being directed toward
identifying novel CDC25 inhibitors that work in vitro and
that may even be active against human tumors in vivo have
been reported (Feng et al., 2008; Contour-Galcera et al.,
2007). To find novel small-molecule inhibitors, a high
throughput screening (HTS) with recombinant CDC25B
was developed (Feng et al., 2008).
The inhibitory activities of the tested compounds (1–12)
against CDC25B were measured using 3-OMFP as a sub-
strate. Na3VO4, an inhibitor, was used as a positive control.
We first examined the inhibitory effects of the tested
compounds against CDC25B, the results are summarized in
Table 1. All of the tested compounds showed potent
CDC25B inhibitory activity at a concentration of 20 lg/
mL. The actual percentage inhibition values ranged from
95.05 to 99.68 %. Among them, three compounds 2, 6, and
7 showed excellent inhibition activity, the percentage
inhibition values against CDC25B were 99.56, 99.68, and
99.63 %, respectively, which is more potent than that of
reference drug Na3VO4.
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Med Chem Res
Fig. 3 Characterization of 6 to
CDC25B. a At various fixed
concentrations of 6 the initial
velocity was determined with
various concentrations of
OMFP. b Typical competitive
inhibition of 6 shown by
Lineweaver–Burkplot. c Ki
determination
70
60
50
40
30
20
10
0
0.5
1.5
1.0
2.5
2.0
[l]µM
10
8
6
4
2
0
0.5
2.5
1.0
1.5
2.0
3.0
[l]µM
A
0.08
0.06
0.08
0.06
0.04
0.02
y=0.0301x+0.012
0.04
0.02
R2=0.976
-1.0
-2.0
0.5
0.0
1.0
2.0
-1.0
-2.0
1.0
0.0
2.0
3.0
-0.02
-0.02
-0.04
-0.04
B
C
l/[S]
[l]uM
Table 2 The anticancer activity of the target compounds in vitro
the most inhibition activity, which was similar to the ref-
erence drug oleanolic acid.
Compounds
IC50 (lmol/L)
A kinetic study was performed to shed light on the
inhibitory mechanism of compound 6 (McGovern et al.,
2003). As shown in Fig. 3a, we determined the inhibition
modality of compound 6 on CDC25B that exhibited char-
acteristics typical of a competitive inhibitor, including
increased Km values and unchanged Vmax values following
increased inhibitor concentration. The Lineweaver–Burk
plot also indicated that compound 6 is a competitive
inhibitor of CDC25B because a nest of lines for increasing
inhibitor concentrations intersect on the y-axis (Fig. 3b).
The results indicate that compound 6 binds to the catalytic
pocket of CDC25B and behaves as a competitor to the
physiological substrate. The Ki value calculated from
Fig. 3c was 0.34 lM.
HCT116
HeLa
2
2.03
1.16
1.27
1.66
1.98
1.49
1.69
1.58
6
7
IRT
Cancer cells, HCT116 colon cancer, HeLa cervical carcinoma cell
In order to determine the antiproliferative activity, we
also investigated the inhibition by the best compounds 2, 6,
and 7 of the proliferation of cancer cell lines. As shown in
Table 2. Three compounds were tested against different
human tumor cell lines (HCT116 and HeLa) based on MTT
assays. The results showed that two compounds revealed
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Med Chem Res
Foundation of Zhejiang Province of China (No. LY14C190001). We
would like to express our gratitude to the National Center for Drug
Screening for consultation in inhibition CDC25B and PTP1B activity
testing.
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In conclusion, twelve 20,40,60-trihydroxychalcone com-
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