Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-norcantharidin hybrid drugs

Article abstract of DOI:10.1016/j.bmcl.2016.05.063

In this study, we designed and synthesized eighteen podophyllotoxin-norcantharidin hybrid drugs which could exhibit more potent anti-cancer activity than the parent drugs. Through the anti-proliferation assay, the most potent anti-cancer agent was screened out, namely Q9 (IC₅₀?=?0.88?±?0.18?μM against MCF-7 cell line), and it showed lower cytotoxicity against non-cancer cells, human embryonic kidney cells (293T) (IC₅₀?=?54.38?±?3.78?μM). Additionally, based on the flow cytometry analysis result, it can cause a remarkable cell cycle arrest at G2/M phase and induce apoptosis in MCF-7 cells more significantly than podophyllotoxin or norcantharidin per se. Moreover, the expression of cell cycle relative protein CDK1 was up regulated while a protein required for mitotic initiation, Cyclin B1 was down regulated. Furthermore, according to the confocal microscopy observation results, it was shown that Q9 was a potent tubulin polymerization inhibitor and the effect is comparable to that of colchicine. For further investigation on the aforementioned mechanisms, we performed western blot experiments, thus finding the increase of the cleavage of PARP. Consistent with these new findings, molecular docking observations suggested that compound Q9 could be developed as a potential anticancer agent.

Full text of DOI:10.1016/j.bmcl.2016.05.063

Accepted Manuscript
Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-nor-
cantharidin hybrid drugs
Hong-Wei Han, Han-Yue Qiu, Cui Hu, Wen-Xue Sun, Rong-Wu Yang, Jin-
Liang Qi, Xiao-Ming Wang, Gui-Hua Lu, Yong-Hua Yang
PII:
S0960-894X(16)30560-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.05.063
BMCL 23922
Reference:
Bioorganic & Medicinal Chemistry Letters
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Received Date:
Revised Date:
Accepted Date:
29 March 2016
14 May 2016
21 May 2016
Please cite this article as: Han, H-W., Qiu, H-Y., Hu, C., Sun, W-X., Yang, R-W., Qi, J-L., Wang, X-M., Lu, G-H.,
Yang, Y-H., Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-norcantharidin hybrid drugs,
Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.05.063
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Bioorganic & Medicinal Chemistry Letters
Design, synthesis and anti-cancer activity evaluation of podophyllotoxin-
norcantharidin hybrid drugs
Hong-Wei Han,a,b,† Han-Yue Qiu,a,b,† Cui Hu,a,b Wen-Xue Sun,a,b Rong-Wu Yang,a,b Jin-Liang Qi,a,b Xiao-Ming
Wang,a,b,* Gui-Hua Lua,b,*and Yong-Hua Yanga,b,
*
aState Key Laboratory of Pharmaceutical Biotechnology, NJU-NJFU Joint Institute of Plant Molecular Biology, Nanjing University, Nanjing, 210023,
China
bCo-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing, 210037, China
† These Two authors equally contribute to this paper. E-mail: wangxm07@nju.edu.cn,Guihua.lu@nju.edu.cn,Yangyh@nju.edu.cn
ABSTRACT
In this study, we designed and synthesized eighteen podophyllotoxin-norcantharidin hybrid
drugs which could exhibit more potent anti-cancer activity than the parent drugs. Through the
anti-proliferation assay, the most potent anti-cancer agent was screened out, namely Q9 (IC50=
0.88±0.18 μM against MCF-7 cell line), and it showed lower cytotoxity against non-cancer cells,
human embryonic kidney cells (293T) (IC50=54.38±3.78 μM). Additionally, based on the flow
cytometry analysis result, it can cause a remarkable cell cycle arrest at G2/M phase and induce
apoptosis in MCF-7 cells more significantly than podophyllotoxin or norcantharidin per se.
Moreover, the expression of cell cycle relative protein CDK1 was up regulated while a protein
Keywords:
required for mitotic initiation, Cyclin B1 was down regulated. Furthermore, according to the
confocal microscopy observation results, it was shown that Q9 was a potent tubulin
polymerization inhibitor and the effect is comparable to that of colchicine. For further
investigation on the aforementioned mechanisms, we performed western blot experiments, thus
finding the increase of the cleavage of PARP. Consistent with these new findings, molecular
docking observations suggested that compound Q9 could be developed as a potential anticancer
agent.
Natural product
Podophyllotoxin
Norcantharidin
anti-tubulin
2009 Elsevier Ltd. All rights reserved.
Natural products play a major role in the development of
Podophyllotoxin (1) Fig. 1, an active ingredient of
1-3
4
drugs, especially for the treatment of cancer
and infections ,
pedophilia species, is a lignan useful in the semi-synthesis of
5
as well as immunosuppressive compounds . However, there is
limited number of organic products, whereas millions of hybrids
as combinations of parts of different organic products can be
prepared. This new approach appears to be promising for the
development of new and more potent anti-cancer drugs, for that
the biological activity of some new hybrids would exceed that of
the parent compounds 6-8. The advantage of this concept over a
combinatorial chemistry approach is the extreme diversity, as
well as the inherent biological activity of the hybrids.
commercially, which can be employed in chemotherapy for
10
various cancer types 9, including cervical carcinoma
osteosarcoma, nasopharyngeal carcinoma 11, colon cancer
,
12
,
breast cancer, prostate cancer 13, testicular carcinoma, small cell
lung cancer and lymphoma 14,15. However, due to some
unacceptable toxic side-effects, it is unable to be used in human
clinical trials 16,17. To overcome the limitations, numerous
researchers have been conducted with the focus on the structural
modification at the 4 position of cycloparaffin (C ring). It could
thus generate some derivatives with superior pharmacological

2
Bioorganic & Medicinal Chemistry Letters
profiles 18-20, such as NK611 (2), etoposide (3) teniposide (4) and
4-azapodophyllotoxin (5), which retains cytotoxicity as well as
inhibition of tubulin polymerization comparable to that of
podophyllotoxin 21. In previous studies, we also synthesized some
podophyllotoxin derivatives. On this basis, we found that the
cytotoxic effects of the derivatives mentioned above in different
types of tumors have been attributed to their ability to bind the
tubulin during mitosis, which would thus inhibit microtubule
all the obtained compounds showed higher IC50 against the
three cell lines compared with podophyllotoxin. As shown
in Table 2 (in the Supplementary data) the anti-
proliferative activities of Q6 (IC50=1.17±1.26 µM), Q9
(IC50= 0.88±0.18 µM), Q16 (IC50=1.22±1.04 µM) and Q17
(IC50=1.17±0.76 µM) are better than podophyllotoxin
(IC50=4.21±1.11 µM) and the positive control etoposide
(IC50=1.74±0.35 µM). Interestingly, anti-proliferative effects
of Q9 (IC50=7.82±3.60 µM) were much lower than that of
podophyllotoxin (IC50=5.32±1.36 µM) against A549 cells.
In case of HeLa cells, the anti-proliferative properties of Q9
(IC50=3.21±0.99 µM) is superior to podophyllotoxin (IC50=
6.56±1.45 µM) and etoposide (IC50= 4.39±1.09 µM).
Meanwhile, as shown from the anti-proliferation results,
these compounds (Q1-Q18) exhibited low toxicity towards
293T cell line. According to the structure–activity
relationship, we clearly found that cyclohexanol anhydride
and NCTD substituted amino acids derivatives can
significantly improve the anti-proliferation activity of
podophyllotoxin and NCTD themselves. Previous
experiments confirmed that podophyllotoxin and NCTD
derivatives have better application prospects than the parent
drugs in cancer chemotherapy. Moreover, it demonstrated
that NCTD significantly suppressed the growth of highly-
assembly and induce apoptosis 22-24
.
Fig. 1 Chemical structures of podophyllotoxin and some
podophyllotoxin derivatives
Inspired by the hybrid concept, we would like to design a
series of podophyllotoxin hybrid molecules, with the presence of
the available natural product norcantharidin (NCTD) or its
analogues, such as ring oxalic acid anhydride and noticeable. It is
well known that as a premedicant for tumor treatment, NCTD is
well used for cancer chemotherapy 25. Previous studies reported
that NCTD can interrupt cell mitosis and cause cell cycle arrest
in M phase 26. Given the same anti-cancer mechanism of
podophyllotoxin and NCTD, we report the design and synthesis
of three groups of hybrid molecules with NCTD or its two
analogues based on podophyllotoxin scaffold, concerning which
amino acid is used as the bridges. It is hoped that the new hybrids
can achieve better effects than the original two parent drugs,
namely podophyllotoxin and NCTD.
metastatic human breast cancer cell in vitro and ex vivo 28,29
.
Taken together, we selected Q9, the best candidate for breast
cancer cell lines MCF-7 in this study, for further
investigation.
In the preliminary study, we synthesized the
intermediate acid, as shown in Scheme 1 and
2
27. (in the
Supplementary data) In this study, we use the intermediate
carboxylic acid to prepare a series of podophyllotoxin
derivatives (Scheme 3). These synthetic compounds were
1
presented in Table 1 and their H NMR and ESI-MS results
Scheme 3 The synthetic routes for Q1-Q18
were consistent with the assigned structures, which were
reported in the Supplementary data. In addition, these
compounds were accounted for and characterized for the
To investigate whether Q9 has anti-mitotic effects on
MCF-7 cells, cell cycle analysis was performed by flow
cytometry in which podophyllotoxin and NCTD are
employed as positive controls. The results were shown in
Fig. 2, 69.51% cells were arrested in G2/M phase while
cells were treated with 8 μM Q9 for 24 h. By contrast, the
percentage of the cells that arrested in G2/M with NCTD or
podophyllotocin is 24.89% or 15.77%, respectively. These
indicating that Q9 can potently arrest MCF-7 cells in G2/M
phase while the effect was far better than NCTD and
podophyllotoxin. Moreover, the expression of cell cycle
related proteins CDK1 was at the same time up regulated,
1
first time by H NMR, elemental analysis, melting test, and
mass spectroscopy, with the results in accordance with the
depicted structures.
All the synthesized podophyllotoxin derivatives Q1-
Q18 were evaluated for their anti-proliferation activities
against three human cancer lines, namely human breast cell
line (MCF-7), human lung cancer cells (A549) and human
cervical carcinoma cells (HeLa), as well as one non-cancer
cell line, namely human embryonic kidney 293T cells
(293T) by MTT (3-[4, 5-di-methylthiazol-2-yl]-2, 5-
diphenyltetrazolium bromide) assay with etoposide as the
positive control. It was observed that after modification not
along with the down regulation of Cyclin B1. (Fig. 3
)

Table 1 Chemical structures of Q1-Q18
R
R
R
R
Compd
Compd
Compd
Compd
Q1
Q6
Q10
Q15
Q2
Q7
Q11
Q16
Q3
Q4
Q5
Q8
Q9
Q12
Q13
Q14
Q17
Q18
Fig. 3 Immuno-detection of cell cycle related proteins
CDK1 and cycin B induced cell cycle arrest at G2/M phase
in MCF-7 cells. Each band (left to right) represents the
treatment using different concentrations (0, 2, 4, 8 μM) of
Q9. GAPDH served as a loading control.
Fig. 2 Effects of Q9, norcantharidin and podophyllotoxin on
the cell cycle distribution of MCF-7 cells. Cells treated with
0, 2, 4 and 8 μM Q9, norcantharidin and podophyllotoxin
for 24 h were collected and processed for analysis.
In order to verify whether Q9 can induce apoptosis in
MCF-7 cells, we further performed apoptosis detection in a
dose-dependent manner. (Fig. 4) We first treated MCF-7
cells with varying concentrations (0, 2, 4, and 8 μM) of Q9
and also using NCTD and podophyllotoxin as the positive
controls. After treatment, AnnexinV-FITC/PI assay was
performed with cells, after which we found that MCF-7 cells
showed considerable sensitivity to Q9 in a dose-dependent
manner. When the drug concentration rose to 2 μM, the
proportion of the apoptotic cells was up to 64.50%.
Additionally, we also found that NCTD could induce
apoptosis in MCF-7 cells, but the effect was weaker than
Fig. 4 AnnexinV/PI dual-immuno-fluorescence staining
after treatment with different concentrations of Q9
,
norcantharidin and podophyllotoxin for 24 h revealed
significantly increased number of apoptotic and necrotic
cells (measured with Annexin V+/PI+ cells). Cells treated
with 0, 1, 2, 4 and 8 μM Q9, norcantharidin and
podophyllotoxin for 24 h were collected and processed for
analysis. The percentage of early apoptotic cells in the lower
right quadrant (annexin V-FITC positive/PI negative cells),
as well as late apoptotic cells located in the upper right
quadrant (annexin V-FITC positive/PI positive cells).
Images are representative of three independent experiments.
Data are mean±S.E.M. of three independent experiments.
(**, P < 0.01).
Q9
.
However, with the other positive control,
podophyllotoxin showed no apoptosis inducing effect in
MCF-7 cells and even at highest concentration, it only lead
to 8.21% cells apoptosis. Thus, it can be concluded that Q9
could significantly induce apoptosis of MCF-7 cells in a
dose dependent manner.
To investigate the effect of Q9 on cytoskeleton
architecture, we performed confocal microscopy observation

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Bioorganic & Medicinal Chemistry Letters
on MCF-7 cells processed by Q9 at indicated
concentrations, as shown in Fig. 5. In the control group, we
observed a typical array of radial organization and
accumulation of microtubules at the microtubule-organizing
center. However, in 4 μM Q9 treated group, the architecture
of the interphase microtubule network was deemed to be
altered, accompanied by the destruction of tubulin
cytoskeleton. Through the comparison of the two controls,
colchicine and paclitaxel, we found that the effect of Q9 on
cytoskeleton architecture was analogous to that of colchicine
and contrary to paclitaxel. The results confirmed that Q9 can
destroy the cytoskeleton structure and perturb mitosis in
MCF-7 cells, and were in accordance to the cell cycle
distribution consequences.
Fig. 6 Immuno-detection of apoptosis related proteins PARP
and C-PARP induced apoptosis in MCF-7 cells. Each band
(left to right) represents the treatment using different
concentrations (0, 2, 4, 8 μM) of Q9. GAPDH served as a
loading control.
In this study, we designed and synthesized a series of
novel podophyllotoxin-norcantharidin hybrid drugs (Q1-
Q18) in which phenylalanine and alanine are used as the
bridges, and also evaluated their biological activities against
three different cancer cell lines and one non-cancer cell line.
Generally, introduction of NCTD or its analogues could
really reduce the cytotoxicity side effect of podophyllotoxin
toward non-cancer cell. Furthermore, Q9 displayed the best
anti-proliferation activities among all the compounds for
breast cancer cell lines MCF-7. As indicated by the cell
apoptosis and cell cycle analysis results, Q9 could
effectively prevent mitosis in cancer cells, thus resulting in
cell cycle arrest and eventually inducing cell apoptosis. In
order to confirm that Q9 would exhibit better anti-cancer
effect than the parent drugs, namely podophyllotoxin and
NCTD, we used them as references in each assay and the
results were in accordance with our prediction. Confocal
microscopy results further confirmed that Q9 can interrupt
cancer cell mitosis, and cause cell cycle arrest in G2/M
phase through inhibiting microtubules polymerization.
Meanwhile, the expression of CDK1, G2/M phase check
point regulatory protein which plays an important role on
cell proliferation regulation, was also up regulated. In
addition, Cyclin B1 is the regulatory subunit of the CDK1,
while reduction of Cyclin B1 can stop cells in the G2 phase
of the cell cycle and trigger cell death by preventing the
chromosomes from condensing and aligning. The ability of
Q9 up- regulating intracellular Cyclin B1 levels suggests
that the Cyclin B1-CDK1 plays a role in the events of
mitosis. These data prove pro-apoptotic activities of Q9 in
MCF-7 cells and PARP is involved in the apoptosis process
induced by Q9. In addition, the CDOCK interaction energy
values of compounds bind to tubulin (PDB code: 1SA0)
were performed. The results obtained are presented in the
Table 3.( in the Supplementary data ) In view of the
interaction energy, Q9 had a best estimated binding free
energy of -74.3204 kcal/mol, which meant that the binding
of Q9 with tubulin was more stable than that of others and
Fig. 5 Effect of Q9 on interphase microtubules of MCF-7
cells using colchicine and paclitaxel as references.
Microtubules tagged with rhodamine (red) and nuclei tagged
with DAPI (blue) were observed under a confocal
microscope.
Finally, on the basis of the above studies, we conducted
a further detailed interpretation on the effect of inducing
cells to apoptosis by Q9. We detected poly-ADP ribose
polymerase (PARP), a family of proteins involved in DNA
repair and programmed cell death. Compared with the
control group, when the concentration of drug reached 8
μM, results showed that the expression of cleaved of poly-
ADP ribose polymerase (PARP) were markedly increased
(Fig. 6).

this effect was more potent than that of colchicine. Thus,
biological activity data along with molecular docking
observations suggested that compound Q9 could be
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The authors are grateful to the Program for Changjiang
Scholars and Innovative Research Team in University
(IRT_14R27), the fund for University Ph.D. Program from the
Ministry of Education of China (20120091110037), the
National Natural Science Foundation of China (NSFC) (Nos.
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3
Graphical Abstract
It proves pro-apoptotic activities of Q9
in MCF-7 cells and PARP is involved in
the apoptosis process induced by Q9.
.
Design, synthesis and anti-cancer activity
evaluation of podophyllotoxin-
norcantharidin hybrid drugs
Hong-Wei Han, Han-Yue Qiu, Cui Hu, Wen-Xue
Sun, Rong-Wu Yang, Jin-Liang Qi,a,b Xiao-Ming
Wang, Gui-Hua Lu and Yong-Hua Yang
aState Key Laboratory of Pharmaceutical
Biotechnology, NJU-NJFU Joint Institute of Plant
Molecular Biology, Nanjing University, Nanjing,
210023, China
bCo-Innovation Center for Sustainable Forestry in
Southern China, Nanjing Forestry University,
Nanjing, 210037, China