Pt(IV) prodrugs containing microtubule inhibitors displayed potent antitumor activity and ability to overcome cisplatin resistance

Article abstract of DOI:10.1016/j.ejmech.2018.07.016

It is well-known that cisplatin exhibited a broad spectrum of anticancer activities against many solid tumors, but its severe toxicity and drug resistance have largely limited wider clinical applications. Various strategies have been tried to discover new Pt (II) drugs with at least equal activity as well as low toxicity compared to cisplatin, but the inherent problem remains unsolved. Here we report that Pt (IV) complexes comprising a CA-4 analogue, as dual-targeting Pt (IV) prodrug, were synthesized and evaluated for anti-proliferative activity using MTT assay. Among them, complex 19 displayed most potent activity against the tested cancer cell lines, and simultaneously exhibited better cell selectivity between cancer cells and normal cells than that of cisplatin. Mechanism studies revealed that complex 19 effectively induced cell cycle arrest at the G2/M phase and dramatically disrupted the microtubule organization. Moreover, complex 19 significantly induced cell apoptosis and decreased MMP. Importantly, complex 19 significantly inhibited tumor growth in SK-OV-3 xenograft model in vivo without apparent toxicity.

Full text of DOI:10.1016/j.ejmech.2018.07.016

Accepted Manuscript
Pt(IV) prodrugs containing microtubule inhibitors displayed potent antitumor activity
and ability to overcome cisplatin resistance
Lingxue Li, Xiaochao Huang, Rizhen Huang, Shaohua Gou, Zhimei Wang, Hengshan
Wang
PII:
S0223-5234(18)30572-5
10.1016/j.ejmech.2018.07.016
EJMECH 10551
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 24 October 2017
Revised Date: 2 July 2018
Accepted Date: 6 July 2018
Please cite this article as: L. Li, X. Huang, R. Huang, S. Gou, Z. Wang, H. Wang, Pt(IV) prodrugs
containing microtubule inhibitors displayed potent antitumor activity and ability to overcome cisplatin
resistance, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.016.
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Graphical abstract
Pt(IV) prodrugs containing microtubule inhibitors displayed potent
antitumor activity and ability to overcome cisplatin resistance
a 1
a 1
a
a
Lingxue Li,
Xiaochao Huang,
Rizhen Huang, Shaohua Gou, a * Zhimei Wang, and
Hengshan Wang b *

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Pt(IV) prodrugs containing microtubule inhibitors displayed potent
antitumor activity and ability to overcome cisplatin resistance
a 1
a 1
a
a *
a
Lingxue Li,
Xiaochao Huang,
Rizhen Huang, Shaohua Gou,
Zhimei Wang, and
Hengshan Wang b *
a
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing 211189, China.
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004,
China.
Abstract
It is well-known that cisplatin exhibited a broad spectrum of anticancer activities against many
solid tumors, but its severe toxicity and drug resistance have largely limited wider clinical
applications. Various strategies have been tried to discover new Pt(II) drugs with at least equal
activity as well as low toxicity compared to cisplatin, but the inherent problem remains unsolved.
Here we report that Pt(IV) complexes comprising a CA-4 analogue, as dual-targeting Pt(IV)
prodrug, were synthesized and evaluated for anti-proliferative activity using MTT assay. Among
them, complex 19 displayed most potent activity against the tested cancer cell lines, and
simultaneously exhibited better cell selectivity between cancer cells and normal cells than that of
cisplatin. Mechanism studies revealed that complex 19 effectively induced cell cycle arrest at the
G2/M phase and dramatically disrupted the microtubule organization. Moreover, complex 19
significantly induced cell apoptosis and decreased MMP. Importantly, complex 19 significantly
inhibited tumor growth in SK-OV-3 xenograft model in vivo without apparent toxicity.
Keywords: Pt(IV) complex; Antitumor activity; Tubulin; Apoptosis
* Corresponding author.
Corresponding author: E-mail addresses: sgou@seu.edu.cn (S. Gou), whengshan@163.com (H.
-S. Wang).
1 Co-first author: These authors contributed equally to this work.

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1. Introduction
Ptlatinum(II)-based anticancer drugs, such as cisplatin (CDDP), carboplatin and oxaliplatin
(OXP) are widely used for the treatment of many solid tumors as first-line chemotherapeutic
agents in clinic. [1-5] Mechanism studies indicated that they induce apoptosis in cancer cells
mainly through DNA damage. [6-8] However, non-specificity of these drugs toward cancer cells
has limited their applications. [9-11] In the last two decades, various Pt(II) complexes have been
synthesized and screened for antitumor activity, but, unfortunately, only a few of compounds
entered clinical trials and most of them failed. [12] Trying to get rid of the embarrassment,
researchers focused on inert Pt(IV) complexes as prodrugs that can be activated by intracellular
reduction and reduced to Pt(II) equivalents following cellular uptake. [13, 14] For example,
satraplatin, which used to be a promising Pt(IV) prodrug, entered phase III clinical trials for an
orally active drug against prostate cancer due to better stability, lower toxicity, and higher blood
circulation time than cisplatin. [15, 16] In addition, several research groups reported that Pt(IV)
prodrugs containing biologically active units in the axial position could significantly improve the
antitumor activities and overcome the side effect of Pt(II) drugs (Fig.1). [17-22]
The chemotherapeutic agent combretastatin A-4 (CA-4, Fig. 2), a naturally occurring
cis-stilbene from the bark of the South African Cape Bushwillow (Combretum caffrum), was
found to show potent antitumor activity against many types of human cancer cells, own capability
to bind the colchicine binding site of tubulin, and interfere with the polymerization of tubulin.
[23-25] Its derivatives, compounds 7 and 17, also exhibited excellent anticancer activities against
a number of human cancer cells including multidrug resistant cancer cell lines in addition to
strongly inhibit tubulin polymerization via combining the colchicine binding site [26, 27].
Moreover, combretastatin A-4-phosphate (1a, Fig. 2) and serine amino acid analogue of CA-4
(AVE8062, Fig. 2) in combretastatin family are currently under clinical trials as a single drug or in
combination for anticancer therapy. [28, 29] To overcome resistance of cisplatin and reduce its
side effect, researchers now prefer to use combination chemotherapy treating the patients with
other therapeutics drugs that act by different mechanisms. [13] Recent studies indicated that the
combination of platinum anticancer agents with tubulin inhibitors, primarily with paclitaxel and
docetaxel, could result in improved therapeutic profiles. [30] Schobert and co-workers recently
reported Pt(II) complexes containing chalcone derivatives 2a and 3a (Fig. 2), which displayed

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moderate antitumor activities against certain types of solid tumors mainly due to the DNA-damage
Pt(II) agents and tubulin-targeting chalcone moieties. [31] Therefore, a combination of platinum
anticancer agents with tubulin inhibitors can be an effective strategy to overcome drug resistance
and enhance therapeutic effects.
Based on the above background, we have designed a new series of dual-targeting Pt(IV)
prodrugs, which are expected not only to carry the DNA damaging platinum-based agent warhead
into the tumor cells, but also have a CA-4 derivative 7 or 17 to inhibit tubulin polymerization.
Herein, we report the synthesis and biological activities of these compounds as well as their
underlying mechanism of actions.
2. Results and discussion
2.1. Design and synthesis
The synthetic route to prepare compounds 12-13 and 19-20 are depicted in Schemes 1 and 2.
Firstly, the CA-4 derivatives 7 and 17 were obtained according to the reported procedures. [26, 28]
On one hand, compound 8 was achieved by the formation of amide bond between 7 and
mono-methyl glutarate in the presence of HOBT/EDCI, which was followed by hydrolysis of 8
with aluminum hydroxide in the presence of THF/H2O to obtain the acid 9. On the other hand,
compound 18 was achieved by treatment of compound 17 with succinic anhydride in the presence
of Et3N in DMF. Meanwhile, Pt(IV) complexes 10 and 11 were prepared through the oxidative
chlorination of the corresponding Pt(II) complexes (cisplatin or oxaliplatin) with
N-chlorosuccinimide (NCS) in water according to the former methods. [32] Finally, target
compounds 12-13 and 19-20 were obtained by esterification between 9 or 18 and 10 or 11 in the
presence of TBTU/Et3N. The resulting target compounds were characterized by 1H NMR and 13
NMR spectra together with ESI-MS spectroscopy.
C
2.2. In vitro cytotoxicity.
The in vitro cytotoxicity of complexes 12-13 and 19-20 were evaluated by MTT assays on
three human cancer cell lines, HCT-116 (colorectal), HepG-2 (hepatocellular), MGC-803 (gastric)
and two normal cells NCM460 (colon) and HL-7702 (liver), using cisplatin, oxaliplatin, 7, 17 and
CA-4 as the positive controls, respectively. The IC50 values (concentration required to reduce
viability to 50%) were summarized in Table 1. In vitro evaluation results revealed that compounds
7 and CA-4 displayed excellent antitumor activities against the tested three human cancer cells as

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expected, but CA-4 analogues 9 and 13 exhibited lower cytotoxicity than the positive drugs 7 and
17. It was noted that complexes 12-13 and 19-20, the Pt(IV) derivatives of cisplatin or oxaliplatin
with one CA-4 analogous ligand in the axial position, possessed higher cytotoxicity against all test
cancer cell lines than cisplatin and oxaliplatin. Especially, complexes 12 and 19 possessed better
antitumor activities against all test cancer cell lines than cisplatin, with IC50 values in the range of
0.62–0.97 µmol/L (12) and 0.39–0.71 µmol/L (19), which showed 8.6~14.4 (12) and 11.8~21.8
(19) fold increase in activity than those of cisplatin. In addition, both 12 and 19 synchronously
exhibited lower cytotoxicity toward two normal cells NCM460 and HL-7702 than cisplatin and
CA-4, respectively. Moreover, Pt(IV) complexes 13 and 20 significantly exhibited better
anti-proliferative activity against the tested cancer cell lines than oxaliplatin, with IC50 values in
the range of 0.75–1.21 and 0.52–1.06 µmol/L, respectively. Interestingly, complex 19 showed
significantly more effective anticancer activity than compound 17 against the tested three human
cancer cells, and exhibited up to 1.7~2.6 fold increased cytotoxicity compared with compound 17.
More importantly, the selectivity index of complexes 12 and 19 were calculated out as 36.7, 20.9
and 43.6, 49.6, respectively, much higher than that of cisplatin (1.4 and 1.3). The similar results
were also observed in complexes 13 and 20. In short, the in vitro evaluation results suggested that
these Pt(IV) complexes showed better cell selectivity between cancer cells and normal liver cells
than those of cisplatin and oxaliplatin.
2.3. Antitumor activity of target complexes against cisplatin resistant cancer cell line.
Drug resistance is an important therapeutic problem that confined the efficacies of cisplatin for
some human cancer cells. Thus, we further evaluated the cytotoxicity of complexes 12-13 and
19-20 against cisplatin sensitive and resistant cancer cells (human ovarian cancer cells SK-OV-3
and SK-OV-3/CDDP)). As shown in Table 2, the IC50 value of cisplatin against SK-OV-3/CDDP
resistant cells was increased to 30.57 µmol/L. While in contrast, the corresponding IC50 values of
complexes 12 and 19 against SK-OV-3/CDDP resistant cells did not obviously change compared
with those against cisplatin sensitive cells. It was much significant to observe that complexes 12
and 19 had much lower resistance factors (1.25 and 1.26) compared with cisplatin (5.99).
Interestingly, other Pt(IV) complexes 13 and 20 were also found to exhibit obvious anticancer
activities against cisplatin resistant SK-OV-3/CDDP cells comparable to oxaliplatin with small
resistance factors. The results indicated that these Pt(IV) complexes with almost equal potent

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activity against cisplatin-resistant cells might be useful in the treatment of drug refractory cancer
resistance to other platinum drugs.
2.4 HPLC analysis of releasing ability of the typical target compound 19.
In order to investigate whether compound 9 or 18 and divalent Pt(II) species can be released
from target compounds (12-13 or 19-20) by biomolecular reducing agents such as ascorbic acid,
we incubated a typical compound, 19, in a solution of acetonitrile and water (13:7) in the presence
of ascorbic acid (0.5 mmol/L) at 37 , and analyzed the liberation of compound 19 and cisplatin
over a period of 12 h by HPLC chromatograms. As shown in Fig. S1, compound 19 can be easily
reduced to compound 18 and the corresponding Pt(II) equivalent under the test condition as time
passed, accompanied by the falling down peak of compound 19 and the rising up peak of
compound 18 in a time-dependent mode. Notably, cisplatin was not observed in the HPLC
chromatograms due to its weak chromophore in the ultraviolet detecting condition. In short, these
results suggested that the target compound can be easily reduced to release the corresponding Pt(II)
equivalent and compound 9 or 18 under reduction, indicating their potential biological activity.
2.5. Complex 19 induced apoptosis in SK-OV-3 cells.
Studies suggested that most metal complex anticancer drugs generally kill cancer cells by
activating apoptosis. [33] Because complex 19 was found to exhibit broad spectrum antitumor
activity against all tested human cancer cells and the best activity against SK-OV-3 cancer cells in
vitro, it was chosen to be further investigated on the mechanism of action. The apoptosis of
SK-OV-3 cells induced by complex 19 was studied using FITC-Annexin V staining and propidium
iodide (PI) staining method, in which Q1, Q2, Q3, and Q4 represent four different cell states:
necrotic cells, late apoptotic or necrotic cells, apoptotic cells and living cells, respectively. The
percentages of apoptotic cells were determined by flow cytometry. SK-OV-3 cells were treated
with complex 19 and cispaltin at the indicated concentrations for 24 h, respectively. As shown in
Fig. 3, SK-OV-3 cells treated with complex 19 exhibited a concentration-dependent increase of
apoptosis by 29.4% and 44.5% at 5 and 10 µmol/L, respectively. In contrast, the percentage of
apoptosis cells was 23.98% after incubation with cisplatin at 10 µmol/L for 24 h, which was
obviously lower than that of complex 19 under the same condition. These results clearly revealed
that complex 19 could effectively induce apoptosis in SK-OV-3 cancer cells at the indicated
concentrations.

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2.6. Hoechst 33258 staining.
The ability of complex 19 to induce apoptosis was further investigated by analyzing the
nuclear morphology of the exposed SK-OV-3 using Hoechst 33258 staining. SK-OV-3 cells were
treated with complex 19 and cisplatin at the indicated concentrations for 24 h, and then stained by
Hoechst 33258 to detect apoptosis morphologically under the fluorescence microscope. As shown
in Fig. 4, the control cells exhibited weak blue fluorescence and appeared as regular round
contours. Following treatment with complex 19, most cells emitted brilliant blue fluorescence, and
the nuclei of SK-OV-3 cells appeared hyper condensed (brightly stained). The number of apoptotic
nuclei containing condensed chromatin obviously increased when SK-OV-3 cells were treated
with complex 19 (10 µmol/L) for 24 h (Fig. 4), suggesting that apoptosis of SK-OV-3 cells was
significantly induced by complex 19 in a concentration-dependent manner, consistent with the
results for FITC-Annexin V staining and propidium iodide staining.
2.7. Cellular uptake.
It is well known that the cytotoxicity of current Pt(II)-based anticancer agents, such as
cisplatin and oxaliplatin, was associated with the uptake of platinum, hence the cellular uptake
measurement of complex 19 was carried out. The platinum content in SK-OV-3 cells was analyzed
using ICP-MS after 24 h of incubation with complex 19 and cisplatin. As shown in Table 3, the
whole uptake of complex 19 in the SK-OV-3 cells was 335±31 (5 µmol/L) and 709±70 ng Pt per
106 cells (10 µmol/L), respectively, obviously higher than those of cisplatin under the same
conditions. Interestingly, the cellular platinum of compound 19 was up to 2.2-fold increase at high
dose in SK-OV-3 cells compared to cisplatin under the same concentration. In addition, it was
notable that the data was completely in accordance with the MTT assay (IC50 value in SK-OV-3:
19 > cisplatin), which suggested that introduction of a CA-4 analogue in the axial position of the
related Pt(IV) complex can effectively improve the cellular uptake and eventually result in the
increase of its antitumor activity.
2.8. TUNEL staining.
Many studies indicated that the cytotoxicity of platinum-based drugs are able to induce DNA
damage [34], thus, we further investigated the effect of complex 19 on the integrity of DNA in
SK-OV-3 cells using TUNEL staining according to a method reported previously [35]. As shown
in Fig. 5, the cells with increased green florescence intensity (in the web version) suggested the

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degree of DNA damage induced by cisplatin at the indicated concentration as expected.
Interestingly, complex 19-treated SK-OV-3 cells significantly exhibited enhanced green
florescence intensity (in the web version) in a concentration-dependent manner compared to the
control cultures, suggesting the presence of terminal DNA damage caused by complex 19.
2.9. Cell cycle analysis.
In order to further understand the effect of the synthetic Pt(IV) complex 19 on cell cycle arrest,
we used flow cytometry to detect the cell cycle distribution of SK-OV-3 cells following a 24 h
treatment with complex 19 at the indicated concentrations. Untreated cells were served as a
negative control, and cells treated with cisplatin were acted as a positive control. As shown in Fig.
6 A and B, complex 19 were found to be as effective in arresting the cell cycle at G2/M phase in a
concentration-dependent manner compared with the control. With the untreated SK-OV-3 cells,
the percentage of cells in the G0/G1 phase was at 75.37% with only 5.05% in the G2/M phase.
After treatment with cisplatin at 10 µmol/L, the percentage of cells in the G2/M phase only
increased to 11.88%, while in the cells treated with complex 19 at 5 and 10 µmol/L, the percentage
of cells in the G2/M phase increased to 27.11% and 67.84%, respectively. These trends were in
accordance with the current anti-mitotic agents, suggesting that tubulin might probably be an
effective target for complex 19.
2.10. Complex 19 regulated the expression of G2/M-related proteins.
In order to further investigate the molecular mechanism of complex 19 induced G2/M phase
block, the expression of cell division regulated proteins (major Cdc2, cyclin B1, and Cdc25c)
were confirmed by immunoblotting analyses. As shown in Fig. 6 C and D, compared with the
vehicle treated control, complex 19 caused a significant decrease in Cdc2, cyclin B1, and Cdc25C
expression in a concentration-dependent manner. These results, which were consistent with the
cell cycle analysis assay, further suggested the mechanism of the cell cycle arrest effect.
2.9. Analysis of immunofluorescence staining.
It is well-known that the biological activity of CA-4 and its analogues has been reported to be
bound up with tubulin [24, 26]. In order to further evaluate whether the antitumor activity of
complex 19 was derived from an interaction with tubulin, SK-OV-3 cells were treated with
complex 19 and CA-4 at the indicated concentrations for 24 h to analyze the cellular microtubule
network
by
immunohistochemistry
followed
by
nuclear
staining
with
4,

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6-diamidino-2-phenylindole (DAPI). As shown in Fig. 7, a well-organized microtubule network
throughout the cells was observed in the control group, while cells treated with CA-4 exhibited
severely disrupted microtubule organization compared with the control group as expected.
Importantly, it was noted that cells treatment with complex 19 obviously disrupted microtubule
organization compared with untreated cells. These results demonstrated that complex 19
dramatically disrupted the microtubule organization in a concentration-dependent manner, and
further revealed that tubulin was an effective target for its anticancer activity.
2.11. Complex 19 triggered mitochondrial pathway dependent apoptosis.
Many recent researches demonstrated that mitochondria play an important role in the
progression of apoptosis. [36, 37] The loss of mitochondrial membrane potential (MMP) has been
implicated as an early event in apoptotic cells. In order to learn whether apoptosis induced by
complex 19 was related to mitochondrial dysfunction, cells treated with complex 19 at the
indicated concentrations for 24 h and the MMP were examined by fluorescence microscope using
JC-1 and Hoechst 33258 staining. Untreated cells were used to serve as a negative control, and
cells treated with cisplatin were used to act as a positive control. In the control group (untreated
cells), JC-1 existed as aggregates in the mitochondria (red), while the apoptotic and necrotic cells,
JC-1 existed as a monomer in the cytosol (green). As shown in Fig. 8, treatment of SK-OV-3 cells
with complex 19 and cisplatin, respectively, caused a decreased MMP level at the indicated
concentration for 24 h.
2.12. Complex 19 triggered ROS generation.
Because mitochondrial dysfunction is associated with mitochondrial production of reactive
oxygen species (ROS),[38] we further evaluated whether ROS production increased after
treatment with complex 19 at the indicated concentrations. The fluorescent probe 2,
7-dichlorofluorescein diacetate (DCF-DA) was used to detect by flow cytometry after cells treated
with the test compounds for 24 h. Untreated cells served as a negative control, and cells treated
with cisplatin acted as a positive control. As shown in Fig. 9, both complex 19 and cisplatin
induced the production of significant amounts of ROS in comparison with control cells, in
agreement with the dissipation of MMP. Interestingly, after exposure to 10 µmol/L of complex 19
for 24 h, the ROS level was 75.2%, which showed up to 3.3-fold increase in SK-OV-3 cells
compared to cisplatin under the same concentration. Altogether, these results proved that complex

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19 induced apoptosis through the mitochondrial pathway.
2.13. Complex 19 induced apoptosis via the activation of caspases and regulated
apoptosis-related protein expression.
Caspases, in general, are a family of cysteine proteases that play an important role in apoptosis.
[39] In order to investigate whether complex 19 induced caspase-dependent cell death, we
performed an immunoblot analysis of the activation of two caspases, namely caspase-3 and -9,
which are involved in the apoptotic mitochondrial pathway. As shown in Fig.10 A and B, exposure
of SK-OV-3 cells to complex 19 resulted in the activation of caspase-3 and caspase-9 in a
concentration-dependent manner. In addition, many studies indicated that Bcl-2 family proteins
are crucial components of mitochondrial stress-induced cellular apoptosis [40, 41], hence, the
expression of apoptosis-related proteins (Bax, pro-apoptotic protein and Bcl-2, anti-apoptotic
protein) was also investigated. Western blot analysis suggested that complex 19 significantly
up-regulated the expression of Bax and correspondingly down-regulated the expression of Bcl-2.
2.14. Antitumor effect of complex 19 in vivo.
To evaluate the efficacy of complex 19 to inhibit tumor growth in vivo, we established the
nude mouse SK-OV-3 tumor xenograft models by subcutaneously injecting SK-OV-3 cells in the
logarithmic phase into the right armpit of the mice. When the model was well-established, mice
with tumors at the volume of 100-150 mm3 were randomly divided into six groups (n=5/group): (1)
vehicle treated group (5% dextrose injection), (2) complex 19 (5 mg/kg) treated group, (3)
complex 19 (13 mg/kg) treated group, (4) cisplatin (5 mg/kg) treated group; (5) CA-4 (5 mg/kg)
treated group; (6) equal mass mixture of cisplatin (5 mg/kg) + CA-4 (5 mg/kg) treated group. As
shown in Fig. 11 A and D, the growth of SK-OV-3 tumor xenograft was effectively suppressed by
53.1% and 60.5% (percentage of inhibition rate [IR] values) after treatment groups were injected
with complex 19 at two doses (5 and 13 (mg/kg)/7 days) for 21 days in the SK-OV-3 tumor model.
Interestingly, complex 19 exhibited better antitumor activity than CA-4 (IR, 51.6%) in vivo in the
entire observation period, as shown in Fig. 11 B. It was noticed that complex 19 exhibited better
antitumor activity in vitro, while displayed a little lower value of IR in vivo than that of cisplatin
(IR, 65.8%) and the combination of cisplatin+CA-4 (IR, 69.7%), respectively. Although the
cisplatin effectively caused tumor growth inhibition, its toxicity was conspicuously obvious, as
evidenced by loss of body weight compared with complex 19 in the entire observation period (Fig.

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11 C). Notably, it was found that conjugation of cisplatin with CA-4 not only improved
therapeutic effects, but also reduced the toxicity of cisplatin as evidenced by the inhibition of
tumor growth and less decline of body weight than cisplatin, as shown in Fig. 11. Furthermore, the
toxicity was further investigated by histological images in major organs (liver, heart, lung, kidney
and spleen) through H&E staining. It was noted that there was no obvious damage to major organs
after treatments with complex 19 in three weeks (Fig.12). In contrast, serious damage to the major
organ was observed in mice treated with cisplatin, consistent with the loss of body weight (Fig. 11
C). In short, complex 19 showed high antitumor activity and low toxicity both in vitro and in vivo,
and deserved further study.
3. Conclusions
In summary, four Pt(IV) prodrugs derived from cisplatin and oxaliplatin with two CA-4
analogues as an inhibitor of tubulin were designed and synthesized. In vitro assays revealed that
all the resulting Pt(IV) complexes not only exhibited higher anticancer activities than their mother
Pt(II) counterparts against tested human cancer cells, but also indicated less toxic than their
corresponding Pt(II) complexes against two normal human cells. Among them, complex 19, the
Pt(IV) derivative from cisplatin with one CA-4 analogue ligand in the axial position, possessed
better antitumor activities against three cancer cell lines (HCT-116, HepG-2 and MGC-803) than
cisplatin with a 11.8-21.8 fold increase in activity. Interestingly, the activity of complex 19 was
also sensitive to cisplatin resistant cancer cells (SK-OV-3/CDDP, IC50=0.35±0.12) when compared
with cisplatin sensitive cells (SK-OV-3, IC50=0.28±0.16 µmol/L). Importantly, complex 19
significantly caused cell cycle arrest at G2/M phase and obviously disrupted the microtubule
organization, and altered the expression of cell cycle-related proteins. Moreover, complex 19
effectively induced cell apoptosis and decreased MMP. Molecular mechanism studies indicated
that complex 19 caused apoptotic cell death of human cancer cells SK-OV-3 through the
mitochondrial mediated pathway by producing reactive oxygen species (ROS), down-regulating
Bcl-2, up-regulating Bax, and further proteolytically activating downstream caspase-9 and -3.
More importantly, the antitumor efficacy of complex 19 was verified in ovarian cancer xenograft
mouse models with almost no toxicity. Complex 19 significantly suppressed the tumor volume and
reduced tumor weight by 53.1% and 60.5% at doses of 5 mg/kg and 13 mg/kg/7 days (iv) in an
SK-OV-3 ovarin cancer xenograft nude mice model, which was greater than that of the positive

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control, CA-4 (51.6%). Our study indicated that complex 19 displayed significant antitumor
efficacy both in vitro and in vivo, which has the potential to be further developed as a promising
antitumor agent. Consequently, Pt(IV) anticancer prodrugs containing a tubulin inhibitor moiety
can effectively inhibit tubulin polymerization that may be a promising approach for multiple
targeted cancer therapy.
4. Experimental section
All chemicals and solvents were analytical reagent grade and commercially available, and used
without further purification unless noted specifically. Column chromatography was performed
using silica gel (200–300 mesh). Mass spectra were measured on an Agilent 6224 TOF LC/MS
instrument. Elemental analyses of C, H, and N used a Vario MICRO CHNOS elemental analyzer
(Elementary). 1H NMR and 13C NMR spectra were recorded in CDCl3 or d6-DMSO with a Bruker
300, 400 or 600 MHz NMR spectrometer.
4.1 The released ability of compound 19 under reduction with ascorbic acid
The ability of compound 19 to release the active platinum species and compound 13 under
reducing condition was studied. Reduction of compound 19 (0.1 mmol/L) was carried out using
ascorbic acid (0.5 0.1 mmol/L) in a solution of acetonitrile/water (65:35, V/V) and the resulting
product was detected by HPLC and peak detection at 254 nm under UV. The incubation was
generated by adding the test compounds to a solvent containing 65% acetonitrile and 35% water,
which was carried out for 0 h, 2 h, 4 h, 6 h, 8 h, 10 h and 12 h at 37 , respectively. Pure
compound 19 was served as control to match the compound 18 peak in the HPLC chromatogram.
Reversed-phase HPLC was performed with a 250 4.5 mm ODS column. Mobile phase comprised
of acetonitrile/water (V/V ¼ 65:35, 0.1% TFA), and flow rate was 1.0 mL/min. The samples were
taken for HPLC.
4.2. Cell culture and maintenance.
All human cancer cells and two human normal cells were purchased from China Life Science
Collage (Shanghai, PRC). Culture medium Dulbecco’s modified Eagle medium (DMEM), fetal
bovine serum (FBS), phosphate buffered saline (PBS, pH=7.2), and Antibiotice-Antimycotic came
from Jiangsu KeyGen Biotech Company (China). Cell lines were grown in the supplemented with
10% FBS, 100 units/ml of penicillin and 100 g/ml of streptomycin in a humidified atmosphere of
5% CO2 at 37 oC.

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4.3. Cytotoxicity assay.
The target complexes were dissolved in DMF and evaluated in three human cancer cells
(HCT-116, HepG-2 and MGC-803) and two human normal cells (NCM460 and HL7702) as well
as cisplatin-sensitive and resistant cells (SK-OV-3 and SK-OV-3/CDDP), respectively. About
5.0×104 cells/mL cells, which were in the logarithmic phase, were seeded in each well of 96-well
plates and incubated for 12 h at 37 °C in 5% CO2. Complexes at five different concentrations
(1.25, 2.5, 5, 10 and 20 µmol/L) were added to the test well and then the cells were incubated at
37 °C in a 5% CO2 atmosphere for 72 h. An enzyme labeling instrument was used to read
absorbance with 570/630 nm double wavelength measurement. Cytotoxicity was detected on the
percentage of cell survival compared with the negative control. The final IC50 values were
calculated by the Bliss method (n = 5). All of the tests were repeated in triplicate.
4.4. Apoptosis analysis.
Apoptosis was investigated by flow cytometric analysis of annexin V/PI staining. SK-OV-3
cells were seeded in each well of six-well plates at the density of 5.0×104 cells/mL of the DMEM
medium with 10% FBS to the final volume of 2 mL. The plates were incubated for overnight and
treated with complex 19 and cisplatin at the indicated concentrations for 24 h. Briefly, cells were
harvested and washed with twice ice-cold PBS, and then suspended cells in the annexin-binding
buffer at a concentration of 2× 105 cells /ml. Cells were then incubated with 5 µL of annexin
V-FITC and 5 µL of PI in the dark at 4 °C for 20 minutes. The cells were examined by system
software (Cell Quest; BD Biosciences).
4.5. Hoechst 33258 staining.
SK-OV-3 cells (5.0×104 cells) were seeded in six-well tissue culture plates and exposed to
different concentrations (5 and 10 µmol/L) of complex 19 for 24 h. Untreated cells was served as
negative control and cells treated with cisplatin (10 µmol/L) was acted as positive control. The
cells were fixed in 4% paraformaldehyde for 10 min, and the cells were then washed twice with
cold PBS and incubated with 0.5 mL of Hoechst 33258 at dark for 10 min. After 10 min
incubation, the cells were washed twice with cold PBS and the results were analysis by a Nikon
ECLIPSETE2000-S fluorescence microscope using 350 nm excitation and 460 nm emissions.
4.6. Cell uptake.
SK-OV-3 cells were seeded in each well of 96-well plates. After the cells reached about 80%

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confluence, 5 and 10 µmol/L of complex 19 and cisplatin were added and the plates were also
incubated for 24 h at 37 °C in 5% CO2, respectively. After completion of 24 h incubation, cells
were collected and washed three times with ice-cold PBS, then centrifuged at 1000×g for 10 min
and resuspended in 2 mL PBS. A volume of 100 µL was taken out to examine the cell density. The
o
remaining cells were digested by HNO3 (200 µL, 65%) at 65 C for 12 h, and then the Pt level in
cells were examined by ICP-MS.
4.7. TUNEL assay.
TUNEL assay was performed as described previously. [35] The TUNEL method was
performed to label 3-end of fragmented DNA of the apoptotic SK-OV-3 cells. SK-OV-3 cells
(5.0×104 cells) were seeded in six-well tissue culture plates and exposed to different concentrations
(5 and 10 µmol/L) of complex 19 for 24 h. Untreated cells was served as negative control and cells
treated with cisplatin (10 µmol/L) was acted as positive control. After 24 h, the cells were washed
two times with ice-PBS and then fixed with 4% paraformaldehyde.
The staining was carried out
according to the manufacturer’s protocol. The FITC-labeled TUNEL-positive cells were imaged
under a fluorescent microscopy by using 488 nm excitation and 530 nm emission.
4.8. Cell cycle analysis.
SK-OV-3 cells were seeded in 6-well plates and treated with complex 19 and cisplatin at the
indicated concentrations and maintained with of the proper culture medium in 5% CO2 at 37 °C
for 24 h. After completion of incubation, cells were harvested and washed two times with ice-cold
PBS, fixed with ice-cold 70% ethanol at -20 °C for overnight. The cells were treated with
100 µg /mL RNase A for 30 minutes at 37 °C after washed with two times ice-cold PBS, and
finally stained with 1 mg/ml propidium iodide (PI) in the dark at 4 °C for 30 minutes. Analysis
was performed with the system software (Cell Quest; BD Biosciences).
4.9. Immunofluorescence assay.
SK-OV-3 cells (5.0×104 cells) were seeded in 6-well plates and treated with complex 19 and
CA-4 at the indicated concentrations and maintained with the proper culture medium in 5% CO2 at
37 °C for 24 h. After being washed three times with ice-PBS and fixed in 4% paraformaldehyde at
37 °C for 20 minutes, then the cells were permeabilized with 0.5% Triton X-100 for 15 min and
blocked for 30 minutes in 10% goat serum. The cells were incubated overnight with primary
antibody (α-tubulin) at 4 °C. In the next day the cells were washed three times with ice-PBS, and

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cells were incubated with corresponding fluorescence-conjugated secondary antibody for 1 h.
After the nuclei of cells were stained with DAPI in the dark at room temperature for 30 minutes.
Cells were visualized using a fluorescence microscopy.
4.10. Mitochondrial membrane potential (MMP) assay.
SK-OV-3 cells were seeded in six-well plates at the density of 5.0×104 cells/mL of the DMEM
medium with 10% FBS to the final volume of 2 mL. The cells were treated with or without tested
compounds at the indicated concentrations for 24 h. After incubation for 24 h, the cells were then
stained with 2 µmol/L JC-1 at room temperature for 30 min and washed three times with ice-PBS,
and the cell nuclei were stained with Hoechst 33258 for 20 minutes at room temperature in the
dark. The emission fluorescence for JC-1 was monitored at 530 and 590 nm, under the excitation
wavelength at 488 nm, respectively.
4.11. ROS assay
SK-OV-3 cells were seeded in six-well plates and treated with complex 19 and cisplatin at the
indicated concentrations and maintained with of the proper culture medium in 5% CO2 at 37 °C
for 24 h. After completion of incubation, cells were harvested at 2000 rpm and washed three times
with ice-cold PBS, and then resuspend cells in 10 mM DCFH-DA (Molecular Probe, Beyotime,
Haimen, China) dissolved in cell free medium at 37 °C for 30 min in dark, and then washed twice
with PBS. Cellular fluorescence was detected by flow cytometry at an excitation of 485 nm and an
emission of 538 nm, respectively.
4.12. Western blot analysis
Western blot analysis was performed as described previously. [36] SK-OV-3 cells were treated
with complex 19 and cisplatin at the indicated concentrations for 24 h, respectively. After
incubation for 24 h, cells were collected, centrifuged, and washed three times with ice-cold PBS.
The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min,
lysates were centrifuged at 20000g at 4 °C for 5 min. The protein concentration in the supernatant
was examined using the BCA protein assay reagents (Imgenex, USA). Equal amounts of protein
per line were was separated on 12% SDS polyacrylamide gel electrophoresis and transferred to
PVDF Hybond-P membrane (GE Healthcare). Membranes were incubated with 5% skim milk in
Tris-buffered saline with Tween 20 (TBST) buffer for 1 h and then the membranes being gently
rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against Bcl-2,

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Bax, caspase-9, caspase-3 or GAPDH for overnight at 4 °C. Membranes were next incubated with
peroxidase labeled secondary anti-bodies for 2 h, and then all membranes were washed with TBST
three times for 15 minutes and the protein blots were detected with chemiluminescence reagent
(Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and fixed with
fixer solution.
4.13. Antitumor Activity in vivo.
The in vivo cytotoxic activity of complex 19 was evaluated using human ovarian cells
(SK-OV-3) in BALB/c nude mice. Five-week-old female BALB/c nude mice were purchased from
Shanghai Ling Chang biotechnology company (China); tumors were induced by a subcutaneous
injection in their dorsal region of 1.0×107 cells in 100 µL of sterile PBS. Animals were randomly
divided into six groups, and started on the second day. When the tumors reached a volume of
100-150 mm3 in all mice on day 18, the first group was injected with an equivalent volume of 5%
dextrose via a tail vein as the vehicle control mice. No. 2 and No. 3 groups were treated with
complex 19 at doses of 5 mg/kg and 13 mg/kg once a week for three weeks, respectively. No. 4
group was treated with cislatin at the dose of 5 mg/kg. No. 5 group was treated with CA-4 at the
dose of 5 mg/kg. No. 6 group was treated with cisplatin/CA-4 mixture at the dose of 5 mg/kg + 5
mg/kg once a week for three weeks. All tested compounds were dissolved in vehicle. Tumor
volume and body weight were recorded every other day after drug treatment. All mice were
sacrificed after three weeks of treatment and the tumor volumes were measured with electronic
digital calipers and determined by measuring length (A) and width (B) to calculate volume (V =
AB2/2).
4.14. H&E staining.
Tumor samples were shown in routine histopathological examination using Hematoxylin and
Eosin (H&E) staining. Mouse organs (liver, heart, lung, kidney and spleen) were collected in 4%
paraformaldehyde for proper fixation and then embedded in paraffin using tissue embedding
machine. Sections were cut and stained with H&E staining. Briefly, sections were prepared
orderly by dewaxing, stainingm and dehydration. After staining in Harris hematoxylin solution,
sections were stained in eosin-phloxine solution for 1 min and then dehydrated and mounted with
neutral resin. The tissue morphology was then observed under fluorescence microscopy using an
Eclipse E800 Nikon (Nikon, Tokyo, Japan).

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4.15. Statistical analysis.
All statistical analysis was performed with SPSS Version 10. Data was analyzed by one-way
ANOVA. Mean separations were performed using the least significant difference method. Each
experiment was replicated thrice, and all experiments yielded similar results. Measurements from
all the replicates were combined, and treatment effects were analyzed.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (Grant Nos. 21571033,
21431001 and 81760626) and the New Drug Creation Project of the National Science and
Technology Major Foundation of China (Grant No. 2015ZX09101032) for financial aids to this
work. The authors would also like to thank the Fundamental Research Funds for the Central
Universities (Project 2242016K30020) for supplying basic facilities to our key laboratory. We also
want to express our gratitude to the Priority Academic Program Development of Jiangsu Higher
Education Institutions for the construction of fundamental facilities (Project 1107047002). The
research was also supported by the Scientific Research Foundation of Graduated School of
Southeast University (YBJJ1677).
Appendix A. Supplementary data
General procedure for the preparation of complexes 12-13 and 19-20 and figures.
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Schemes
Scheme 1. Synthetic pathway to target complexes 12 and 13. Reagents and conditions: (a) NaBH4,
o
o
o
CH3OH, 0 C; (b) PBr3, CH2Cl2, 0 C; (c) PPh3, toluene, 110 C; (d) NaH, CH2Cl2, room
o
o
temperature; (e) Fe, NH4Cl, EtOH, 85 C; (f) EDCI, HOBT, Et3N, DMF, 25 C; (g) LiOH·H2O,
THF/H2O, 0 oC; (h) TBTU, Et3N, DMF, 30 oC.

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H3CO
H3CO
CHO
H3CO
H3CO
CHO
a
PPh3Br
b
H3CO
H3CO
OCH3
OTBDMS
OCH3
OTBDMS
OCH3
OH
14
16
15
4
O
H3CO
H3CO
H3CO
H3CO
H3CO
OH
O
e
d
c
H3CO
OH
OCH3
OCH3
O
OCH3
O
O
OH
OCH3
OCH3
OCH3
CA-4
17
18
H3CO
H3CO
H2
N
OH
O
OH
Pt
Cl
Cl
H3N
H3N
O
or
O
O
OCH3
O
O
O
Pt
Cl
Cl
H3N
OCH3
O
O
N
Pt
Cl
Cl
H2
Cl
11
19
H3N
10
H3CO
H3CO
f
O
OCH3
O
H2
O
O
N
O
O
O
OCH3
O
Pt
Cl
20
O
N
H2
Scheme 2. Synthetic pathway to target complexes 19 and 20. Reagents and conditions: (a)
o
o
o
TBDMSCl, Et3N, CH2Cl2, 0 C; (b) NaH, CH2Cl2, 25 C; (c) 1N HCl/CH3OH, 50 C; (d)
2-bromoethanol, K2CO3, DMF, 50 oC; (g) succinic anhydride, Et3N, DMF, 50 oC; (h) TBTU, Et3N,
DMF, 30 oC.

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Table 1. Cytotoxic effects of complexes 12-13 and 19-20 on human cancer and normal cell lines.
Compd.
IC50 (µmol/L) a
MGC-803
HCT-116
1.82±0.51
0.62±0.27
0.75±0.34
1.05±0.41
0.41±0.18
0.52±0.41
0.13±0.05
0.82±0.47
0.12±0.09
8.95±1.08
HepG-2
NCM460
14.29±1.89
22.74±2.58
24.46±2.17
15.16±1.55
17.86±2.07
20.24±2.16
7.37±1.36
10.61±1.77
5.21±1.06
15.82±1.93
9.23±1.44
HL-7702
11.21±1.76
20.24±1.52
25.46±2.04
12.07±1.13
19.35±1.87
23.28±2.61
6.27±1.08
9.95±1.71
3.21±0.66
10.85±0.91
12.63±1.52
SI b
7.9
SI c
3.5
3.24±0.89
0.97±0.38
1.21±0.27
2.25±0.36
0.71±0.23
1.06±0.55
0.14±0.06
1.84±0.69
0.11±0.05
8.36±0.89
1.95±0.60
0.74±0.52
0.53±0.21
1.61±0.44
0.39±0.15
0.73±0.59
0.24±0.15
0.65±0.58
0.09±0.08
7.81±1.03
10.24±1.14
9
12
36.7
32.6
14.4
43.6
38.9
56.7
12.9
43.4
1.8
20.9
21.0
5.4
13
18
49.6
21.9
44.8
5.4
19
20
7
17
CA-4
Cisplatin
29.2
1.3
Oxaliplatin 4.07±0.82 10.19±1.76
2.3
1.2
a In vitro cytotoxicity was determined by MTT assay upon incubation of the live cells with the
b
c
compounds for 72 h. Selectivity Index = IC50 (HCT-116) /IC50 (NCM460). Selectivity Index =
IC50(HL-7702)/IC50(HepG-2). Mean values based on three independent experiments.
Table 2. Biological activity of complexes 12-13 and 19-20 against cisplatin sensitive and resistant
cancer cells (SK-OV-3 and SK-OV-3/CDDP).
Comp.
IC50 (µmol/L) a
SK-OV-3/CDDP
3.66±0.83
SK-OV-3
3.01±1.03
0.42±0.31
0.85±0.43
1.88±0.81
0.28±0.16
0.75±0.61
0.49±0.58
RF b
1.22
1.26
1.15
1.36
1.25
1.19
1.24
9
0.53±0.25
12
13
18
19
20
7
0.98±0.63
2.56±0.93
0.35±0.12
0.89±0.37
0.61±0.28

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0.94±0.72
0.17±0.19
5.10±1.21
9.37±1.39
1.06±0.65
0.21±0.11
30.57±1.95
23.05±2.13
1.13
1.24
5.99
2.46
17
CA-4
Cisplatin
Oxaliplatin
a In vitro cytotoxicity was determined by MTT assay upon incubation of the live cells with the
compounds for 72 h. b RF (resistant factor) is defined as IC50 in SK-OV-3CDDP/IC50 in SK-OV-3.
Mean values based on three independent experiments, and the results of the representative
experiments are shown.
Table 3. Cellular uptake of complex 19 in SK-OV-3 cells after 24 h of incubation.
Pt content (ng/106 cells)
Comp.
SK-OV-3
335±31
709±70
198±20
323±31
19 (5 µmol/L)
19 (10 µmol/L)
Cisplatin (5 µmol/L)
Cisplatin (10 µmol/L)
The experiments were performed three times, and the results of the representative experiments are
shown.

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Fig. 1. Chemical structures of some Pt(IV) complexes as prodrugs.
Fig. 2. Some tubulin interacting agents and lead structures.
Fig. 3. Representative flow cytometric histograms of apoptotic SK-OV-3 cells after 24 h treatment

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with complex 19 (5 and 10 µmol/L) and cispatin (10 µmol/L) as positive control. The cells were
harvested and labeled with annexin-V-FITC and PI, and analyzed by flow cytometry. Data are
expressed as the mean ± SEM for three independent experiments.
Fig. 4. Morphological changes in the nuclei (typical of apoptosis) of SK-OV-3 cells treated with
complex 19 and cisplatin at the indicated concentrations for 24 h, respectively. Then, the cells
were harvested, and stained by Hoechst 33258. The cells were analyzed by the Nikon Te2000
deconvolution microscope (magnification 200×). The experiments were performed three times,
and the results of the representative experiments are shown.
Fig. 5. TUNEL assay of complex 19 in SK-OV-3 cells. Cells treated with complex 19 and
cisplatin at the indicated concentrations for 24 h, respectively. Selected fields illustrating
condensed chromatin (white arrow heading) as occurrence of cell apoptosis were shown. Images
were visualized by fluorescence microscope. The experiments were performed three times, and the
results of the representative experiments are shown.

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Fig. 6. (A) Cell cycle arrest effect of complex 19. SK-OV-3 cells treated with complex 19 and
cisplatin at the indicated concentrations for 24 h. Then, the cells were trypsinized, harvested and
washed three times with ice-PBS for PI-stained DNA content detected by flow cytometry. (B)
Quantitative analysis of the percentage of cells in each cell cycle phase. (C) The total proteins
were harvested for the Western blot analysis to detect the expression of Cdc2, Cdc25c and cyclin
B1. (D) Expression levels of (C) shown as percentages. The experiments were performed three
times, and the results of the representative experiments are shown. P<0.05

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Fig. 7. Effect of complex 19 on the organization of cellular microtubule network. Untreated cells
served as the negative control, and CA-4 as the positive drug. SK-OV-3 cells treated with complex
19 were fixed and stained with a-tubulin, and then counterstained with 4,
6-diamidino-2-phenylindole (DAPI). Microtubules and unassembled tubulin are shown in red.
DNA, stained with DAPI, is shown in blue. The experiments were performed three times, and the
results of the representative experiments are shown.