Identification of novel ROS inducer by merging the fragments of piperlongumine and dicoumarol

Article abstract of DOI:10.1016/j.bmcl.2016.08.016

A series of novel ROS inducers were designed by merging the fragments of piperlongumine and dicoumarol. Most of these derivatives showed potent in vitro activity against three cancer cell lines and good selectivity towards normal lung cells. The most pote

Full text of DOI:10.1016/j.bmcl.2016.08.016

Accepted Manuscript
Identification of novel ROS inducer by merging the fragments of piperlongu-
mine and dicoumarol
Xiaojuan Xu, Xia Fang, Jun Wang, Hong Zhu
PII:
S0960-894X(16)30837-X
http://dx.doi.org/10.1016/j.bmcl.2016.08.016
BMCL 24142
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
20 May 2016
14 July 2016
6 August 2016
Please cite this article as: Xu, X., Fang, X., Wang, J., Zhu, H., Identification of novel ROS inducer by merging the
fragments of piperlongumine and dicoumarol, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://
dx.doi.org/10.1016/j.bmcl.2016.08.016
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Identification of novel ROS inducer by
merging the fragments of piperlongumine
and dicoumarol
Leave this area blank for abstract info.
a, 
a
a
a
Xiaojuan Xu , Xia Fang , Jun Wang and Hong Zhu

Bioorganic & Medicinal Chemistry Letters
Identification of novel ROS inducer by merging the fragments of
piperlongumine and dicoumarol
a, 
a
a
a
Xiaojuan Xu , Xia Fang , Jun Wang and Hong Zhu
a
School of Chemistry and Chemical Engineering, Yancheng Teachers University, Yancheng, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of novel ROS inducers were designed by merging the fragments of piperlongumine and
dicoumarol. Most of these derivatives showed potent in vitro activity against three cancer cell
lines and good selectivity towards normal lung cells. The most potent and selective compound
3e was proven to exhibit obvious ROS elevation and excellent in vivo antitumor activity with
suppressed tumor growth by 48.46% at the dose of 5 mg/kg. Supported by these investigation,
these findings encourage further investigation around this interesting antitumor chemotype.
Keywords:
Piperlongumine
Dicoumarol
2
009 Elsevier Ltd. All rights reserved.
antitumor
Reactive oxygen species
Biological activity
—
——

Corresponding author. Tel.: +86 0515 8823 3186; fax: +86 0515 8823 3186; e-mail: xxjyctu@163.com.

Cancer remains one of the most threatening diseases in the
world. Lots of financial resources have been devoted to discover
On careful review at PL and DIC, it was noticed that these two
natural products may share same skeleton. In view of this, we
initially merged the two natural products into a new structure of
3a that retained the key pharmacophores for antitumor activity.
In order to expand and identify novel PL derivatives, we
designed a series of new redox-modulating agents (3a-3g) which
were based on the merged structure (Fig. 1). Eight compounds
have been synthesized and tested for antitumor activity both in
vitro and in vivo.
1-2
cancer-related drugs. Over the past decades notable progress
has been achieved on developing new antitumor active drugs, but
there is still urgent to develop new and safe drugs to target tumor
cell specific mechanisms. There is a new strategy to target
emerging hallmark of cancer cells such as oxidative stress which3
is a key bioenergetics alterations observed in cancer cells.
Cancer cells are usually exposed to elevated levels of reactive
oxygen species (ROS) and excessive ROS levels irreversibly
damage DNA and lipids which would ultimately lead to
The synthesis of the target compounds 3a-3h were shown in
Scheme 1. Compound 5 was synthesized via the Vilsmeier Haack
reaction by refluxing substituted 4-hydroxy-2-oxo-2H-1-
benzopyrans (4) with dimethyl formamide and phosphorous
oxychloride. Condensation substituted 4-hydroxy-2-oxo-2H-
chromene-3-carbaldehyde (5) with malonic acid in pyridine
yielded the intermediate substituted (E)-3-(4-hydroxy-2-oxo-2H-
chromen-3-yl)acrylic acids (6). Treatment of 6 with oxalyl
4-5
apoptosis of cancer cells.
Compared with cancer cells, normal cells seem to have much
lower ROS levels of endogenous oxidative stress in culture and
6
in vivo. Actually, developing compounds that exploit ROS in
cancers is a novel and promising therapeutic approach in drug
6
-7
discovery. A number of small molecule ROS inducers have
been discovered with diverse scaffolds and exert their anticancer
activity dependent on the high basal ROS levels uniquely present
chloride in anhydrous toluene under N
2
gave the acyl chlorides 7
which were reacted with piperidin-2-ones to afford PL
8-10
in cancer cells.
Among them, piperlongumine (PL), a small
derivatives 9 using anhydrous THF as solvent and Et
catalyst. Subsequently, treatment of compound
3
N as
with
molecule ROS generator was found to selectively kill cancer cells
9
11-14
in recent years.
The tumor specific effect of PL on
phenylselenyl chloride gave the α-phenylseleno imide 10 which
then subject to oxidation with hydrogen peroxide to obtain the
target compounds 3a-3g in 70~80% yields. In addition,
palladium-carbon catalyzed hydrogenation of the double bond of
3a to provide the target compound 3h in 56% yield. The
cytotoxicity and ROS shows a new strategy for selective
10-11
targeting of cancer cells.
Since the natural product has been
reported to be efficient ROS inducers, many derivatives of PL
have been synthesized to explore the structure and biological
10-11, 15-16
1
activity relationship.
Several key pharmacophores, such
structures of all target compounds were confirmed by H NMR
as C2-C3 and C7-C8 double bonds, have been regarded as
and electrospray ionization mass spectrometry (ESI-MS). Before
these compounds were used in biological experiments, they were
purified by silica gel column chromatography and HPLC was
used to determine their purity (all > 95%).
1
1
necessary for PL’s toxicity to cancer cells. Meanwhile, another
natural product dicoumarol (DIC), chemically termed as 3,3’-
methylenebis (4-hydroxycoumarin) shows cytotoxic effects on
several malignant cell types as previously reported in various in
vitro and in vivo studies and is considered as an anticancer
17
agents.
Figure 1. Representative structures of known inhibitors of NQO1.
Cytotoxicity studies were first performed on all of the PL
derivatives (3a-3h). Cell survival was measured by the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT
assay. We utilized four cancer cell lines (non-small cell lung
cancinoma A549, human colorectal carcinoma HCT116,
hepatocellular carcinoma HepG2) and human lung normal cells
MRC-5 to compare the cytotoxicity of these compounds. As
illustrated in Table 1, most of these PL derivatives showed potent
activities against the three cancer cell lines. In particular, the
their toxicity towards the human lung normal cells MRC-5, a
superior safety profile of most compounds (3a-3e) was
confirmed. Due to the selective toxic towards cancer cell lines, 3e
was selected for further evaluation.
It is supported that these PL derivatives exerted their
antitumor activity by accumulating ROS in cancer cells, we next
determined the effect the representative compound 3e on cellular
ROS level in A549 cancer cells by fluorescence microscopy.
2
’,7’-Dichlorfluorescein diacetate (DCFH-DA) is a specific
6,7,8-methoxy substituted derivative 3e exhibited IC50 values less
oxidation-sensitive fluorescent probe to detect total intracellular
production of ROS. After being uptaken by cells, DCFH-DA is
hydrolyzed by cellular esterases to dichlorodihydrofluorescin
than 10 μM against all the cancer cell lines. Reduction or
replacement of the methoxy group of the most active compound
3e with mono-methoxy, methyl or halogen groups resulted in less
(DCFH), which is trapped within the cell. The nonfluorescent
potent derivatives 3a-3d and 3f-3g respectively. Saturated
derivative 3h was not potent against all cell lines, indicated the
DCFH is then oxidized to fluorescent dichlorofulorescent by
action of cellular ROS. As shown in Fig. 2A, after treating A549
cells with 5 μM or 10 μM 3e for 1 h, a marked increase in ROS
levels was caused. Besides, remarkable elevation of the ROS
necessity of the presence of C
3 4 7 8
-C and C -C double bonds in PL
derivatives. The target compounds were further evaluated for

production was also observed in long time treatment (24 h and 48
h) with 2 μM 3e (Fig. 2). The results indicated that 3e induced
ROS production in dose- and time-dependent manners in cancer
cells. It was speculated that the induced ROS production of 3e in
cancer cell lines may relate to the interaction with recombinant
thioredoxin reductase (TrxR) and alters the antioxidant system.
IC50 (μM)
HCT116
Cpds
R
H
A549
Hep G2
6.7 ± 0.5
7.8 ± 0.3
7.1 ± 1.3
12 ± 1.2
5.7 ± 0.9
15 ± 1.3
9.7 ± 0.9
> 90
MRC-5
22 ± 2.2
35 ± 1.9
53 ± 4.1
31 ± 5.9
57 ± 1.2
19 ± 1.1
8.9 ± 2.3
58 ± 8.1
37 ± 5.4
3a
3b
3c
3d
3e
12 ± 2.2
15 ± 1.3
9.8 ± 1.2
11 ± 2.4
4.2 ± 0.1
6.5 ± 2.0
18 ± 0.3
> 90
15 ± 2.1
5.1 ± 1.1
10 ± 0.6
18 ± 0.2
3.2 ± 0.1
12 ± 1.0
11 ± 1.6
> 90
C
6
,C
7
-Me
C
6
-OCH
3
C - OCH
7
3
1
8-23
Further investigation is underway by our laboratory.
In
C ,C ,C -OCH
3
6
7
8
addition, the increased ROS levels might be exerted in A549
cancer cell lines mediated apoptosis.
7
C -Cl
3
f
3
3
g
7
C -F
h
-
Table 1
PL
-
10 ± 2.1
9.2 ± 3.5
8.9 ± 1.8
In vitro antitumor activity of derivatives
o
Scheme 1. Synthesis of the derivatives of PL. Reagents and conditions: (i) DMF/POCl
3
, 45 C, 8 h, 72%; (ii) malonic acid, pyridine, benzene, reflux, 6 h, 60%;
o
o
o
(
iii) SOCl
vii) H
2
, toluene, 80 C, 4 h; (iv) THF, Et
3
N, rt, 12 h, 60-70%. (v) PhSeCl, LDA, THF, -50~-78 C, 5-7 h, 20-30%; (vi) H
2
O
2
, THF, 0 C, 30-60 min, 70-80%.
(
2
, Pd/C, r.t., 10 h, 56%.
Figure 3. Compound 3e induced apoptosis in A549 cells in a dose-dependent
manner.
To verify the potential drug-like property of compound 3e,
then the compound was further selected to determine its
physicochemical property before in vivo evaluation. As shown in
Table 2, compound 3e exhibited acceptable Log D7.4 value and
much improved solubility than PL. In addition, 3e also displayed
-
6
Figure 2. Induction of ROS in A549 cells. (A) The A549 cells were treated
with different concentration of 3e for 1 h followed by the incubation with
DCFH-DA (10 μM) for 30 min. (B) The A549 cells were treated with a fixed
concentration of 3e (2 μM) for 24 h and 48 h followed by the incubation with
DCFH-DA (10 μM) for 30 min. The fluorescence images were obtained by
inverted fluorescence microscopy.
acceptable membrane permeability, with Pe value of 20.21 × 10
cm/s. Permeability is an important property reflecting the
ability of molecules to diffuse through the cell membrane.
Taking together, compound 3e possessed balanced properties
between hydrophilicity and hydrophobicity. This compound
could be selected for further evaluation in vivo due to its
potent in vitro activity and proper physicochemical property.
We next examined apoptosis in A549 cells following
treatment with 3e. When A549 cells were incubated with
different concentration of 3e for 24 h followed by DAPI staining,
an increasing number of cells displayed condensed nuclei, a
characteristic morphology of cells undergoing apoptosis (Fig. 3).
Table 2
Physicochemical properties of compound 3e.
instrinsic
A
specific analysis revealed nuclei with dose-dependent
Pe, pH = 7.4
Cpds
Log D, pH = 7.4
solubility
μg/mL)
-
6
a
chromatin condensation and characteristic morphological
changes of apoptosis, in cell cultures with 3e.
(
10 cm/s)
(
3
e
0.42
2.84
315.8
0.75
20.21
24.56
PL

a
−6
−6
Ketoprofen (1.98 × 10 cm/s) and propranolol (117.21 × 10 cm/s) are
3
Ai, Y.; Zhu, B.; Ren, C.; Kang, F.; Li, J.; Huang, Z.; Lai, Y.; Peng, S.;
Ding, K.; Tian, J.; Zhang, Y. J. Med. Chem. 2016, 59, 1747−1760.
Dickinson, B. C.; Chang, C. J. Nat. Chem. Biol. 2011, 7, 504−511.
Szatrowski, T. P.; Nathan, C. F. Cancer Res. 1991, 51, 794−798.
Kawanishi, S.; Hiraku, Y.; Pinlaor, S.; Ma, N. Biol. Chem. 2006, 387,
internal standards in permeability determinations.
Having obtained the favorable in vitro cellular activity,
selectivity for normal cells and proper physicochemical property,
compound 3e was selected for further in vivo antitumor activity
studies in A549 tumor xenografts mouse model (Fig. 4). Nude
mice bearing established A549 tumor xenografts were injection
with compound 3e (5 mg/kg daily over a 21-day period). PL was
employed as a positive control drug. As shown in Fig. 4, the
results demonstrated that significant antitumor effects were
observed in mice treated with 3e which was administered
intraperitoneally (ip) every other day for three weeks. Compound
4
5
6
3
65−372.
7
8
Wondrak, G. T.; Antioxid. Redox Signal. 2009, 11, 3013−3069.
Adams, D. J.; Boskovic, Z. V.; Theriault, J. R.; Wang, A. J.; Stern, A.
M.; Wagner, B. K.; Shamji, A. F.; Schreiber, S. L. ACS Chem. Biol.
2
013, 8, 923−929.
9
Trachootham, D.; Alexandre, J.; Huang, P. Nat. Rev. Drug Discov.
2009, 8, 579−591.
3e (tumor growth inhibition = 48.46%) showed the same level of
in vivo antitumor potency with the control PL (tumor growth
inhibition = 41.23%). Furthermore, in the mice treated by 3e, no
significant body weight loss was observed compared with the
vehicle control group. Therefore, the results indicated a low toxic
compound 3e which would be promising anticancer therapeutic
for the treatment of cancer.
10
Adams, D. J.; Dai, M.; Pellegrino, G.; Wagner, B. K.; Stern, A. M.;
Shamji, A. F.; Schreiber, S. L. Proc. Natl. Acad. Sci. U. S. A. 2012,
1
09, 15115−15120.
1
1
Wu, Y.; Min, X.; Zhuang, C.; Li, J.; Yu, Z.; Dong, G.; Yao, J.; Wang,
S.; Liu, Y.; Wu, S.; Zhu, S.; Sheng, C.; Wei, Y.; Zhang, H.; Zhang, W.;
Miao, Z. Eur. J. Med. Chem. 2014, 82, 545−551.
1
1
1
1
2
3
4
5
Bezerra, D. P.; Pessoa, C.; de Moraes, M. O.; Saker-Neto, N.; Silveira,
E. R.; Costa-Lotufo, L. V. Eur. J. Pharm. Sci. 2012, 48, 453−463.
Boskovic, Z. V.; Hussain, M. M.; Adams, D. J.; Dai, M.; Schreiber, S.
L. Tetrahedron, 2013, 69.
Randhawa, H.; Kibble, K.; Zeng, H.; Moyer, M. P.; Reindl, K. M.
Toxicol. In Vitro 2013, 27, 1626−1633.
Wang, Y.; Wang, J.; Li, J.; Zhang, Y.; Huang, W.; Zuo, J.; Liu, H.;
Xie, D.; Zhu, P. Chem. Biol. Drug Des. 2015, Doi:
1
0.1111/cbdd.12714.
1
6
Punganuru, S. R.; Madala, H. R.; Venugopal, S. N.; Samala, R.;
Mikelis, C.; Srivenugopal, K. S. Eur. J. Med. Chem. 2016, 107, 233-
2
44.
Figure 4. Compound 3e retards the tumor growth in vivo in A549 tumor
xenografted nude. The tumor volume measurement. PL (5 mg/kg), 3e (5
mg/kg) (p < 0.05).
1
1
1
2
2
7
8
9
0
1
Aras, D.; Cinar, O.; Cakar, Z.; Ozkavukcu, S.; Can, A. Mol. Hum.
Reprod. 2016, 22, 57-67.
Cai, W.; Zhang, B.; Duan, D.; Wu, J.; Fang, J. Toxicol. Appl.
Pharmacol. 2012, 262, 341-348.
In this present work, a key scaffold of 3-(4-hydroxy-2-oxo-
2H-chromen-3-yl)acryloyl)-5,6-dihydropyridin-2(1H)-one was
Duan, D.; Zhang, B.; Yao, J.; Liu, Y.; Fang, J. Free Radic. Biol. Med.
merged from two natural products, PL and DIC. Based on the
scaffold, a series of novel PL derivatives were designed,
synthesized and evaluated. These derivatives were characterized
and evaluated as efficient antitumor agents in vitro and showed
modest selectivity for human lung normal cells MRC-5. Among
them, the most potent compound 3e also showed superior safety
profile than the control PL in vitro. Furthermore, this
highlighting compound 3e was proven to exhibit obvious ROS
elevation and excellent in vivo antitumor potency. Supported by
these investigation, these findings encourage further investigation
around this interesting antitumor chemotype.
2
014, 70, 182-193.
Duan, D.; Zhang, B.; Yao, J.; Liu, Y.; Sun, J.; Ge, C.; Peng, S.; Fang,
J. Free Radic. Biol. Med. 2014, 69, 15-25.
Zhang, B.; Duan, D.; Ge, C.; Yao, J.; Liu, Y.; Li, X.; Fang, J. J. Med.
Chem. 2015, 58, 1795-1805.
22
23
Duan, D.; Zhang, J.; Yao, J.; Liu, Y.; Fang, J. J. Biol. Chem. 2016,
291, 10021-10031.
Zhang, J.; Li, Y.; Duan, D.; Yao, J.; Gao, K.; Fang, J. Biochem.
Pharmacol. 2016, 102, 34-44.
Acknowledgments
We are thankful for the financial support of the National
Natural Science Foundation of China (no. 21102125).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at
References and notes
1
2
Available online: www.who/int/cancer/en (accessed 02.09.2015).
Wiemann, J.; Heller, L.; Csuk, R. Bioorg. Med. Chem. Lett. 2016, 26,
9
07−909.