Platinum(IV) complexes conjugated with phenstatin analogue as inhibitors of microtubule polymerization and reverser of multidrug resistance

Article abstract of DOI:10.1016/j.bmc.2017.07.011

Pt(IV) complexes comprising a phenstatin analogue, as dual-targeting Pt(IV) prodrug, were designed and synthesized. They were found not only to carry the DNA binding platinum warhead into the tumor cells, but also to have a small molecular unit to inhibit tubulin polymerization. In vitro evaluation results revealed that Pt(IV) complexes showed better and more potent activity against the test human cancer cells including cisplatin resistant cell lines than their corresponding Pt(II) counterparts. In addition, the Pt(IV) derivative of cisplatin, complex 10, exhibited highly selective inhibition in human cancer cells and displayed no obvious toxicity to two human normal cell lines, respectively. Mechanism study suggested that complex 10 induced cell-cycle arrest at the G2/M phase and caused apoptotic cell death of human lung cancer NCI-H460 cells through the mitochondrial mediated pathway. Moreover, complex 10 effectively inhibited the tumor growth in the NCI-H460 xenograft model.

Full text of DOI:10.1016/j.bmc.2017.07.011

Accepted Manuscript
Platinum(IV) complexes conjugated with phenstatin analogue as inhibitors of
microtubule polymerization and reverser of multidrug resistance
Xiaochao Huang, Rizhen Huang, Shaohua Gou, Zhimei Wang, Zhixin Liao,
Hengshan Wang
PII:
S0968-0896(17)31089-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.011
BMC 13847
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 May 2017
4 July 2017
6 July 2017
Please cite this article as: Huang, X., Huang, R., Gou, S., Wang, Z., Liao, Z., Wang, H., Platinum(IV) complexes
conjugated with phenstatin analogue as inhibitors of microtubule polymerization and reverser of multidrug
resistance, Bioorganic & Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.07.011
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Platinum(IV) complexes conjugated with phenstatin analogue as inhibitors of
microtubule polymerization and reverser of multidrug resistance
a, b, 1
a, b, 1
a , b
Xiaochao Huang,
Rizhen Huang,
Shaohua Gou,a, b, * Zhimei Wang,a, b Zhixin Liao
and Hengshan Wang c,*
a
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering and b
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing 211189, China.
c State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
(Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi
Normal University, Guilin 541004, China.
ABSTRACT: Pt(IV) complexes comprising a phenstatin analogue, as dual-targeting Pt(IV)
prodrug, were designed and synthesized. They were found not only to carry the DNA binding
platinum warhead into the tumor cells, but also to have a small molecular unit to inhibit tubulin
polymerization. In vitro evaluation results revealed that Pt(IV) complexes showed better and more
potent activity against the test human cancer cells including cisplatin resistant cell lines than their
corresponding Pt(II) counterparts. In addition, the Pt(IV) derivative of cisplatin, complex 10,
exhibited highly selective inhibition in human cancer cells and displayed no obvious toxicity to
two human normal cell lines, respectively. Mechanism study suggested that complex 10 induced
cell-cycle arrest at the G2/M phase and caused apoptotic cell death of human lung cancer
NCI-H460 cells through the mitochondrial mediated pathway. Moreover, complex 10 effectively
inhibited the tumor growth in the NCI-H460 xenograft model.
Keywords: Antitumor activity; Pt(IV) complex; Tubulin polymerization; 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.

1. Introduction
Cisplatin (Fig. 1), representing one of the most powerful anticancer drugs, has been widely
used for the treatment of a variety of cancers since it was approved for clinical use in 1978.1-4
Although cisplatin is effective, the side effects including nephrotoxicity, neurotoxicity and toxicity
have largely limited its clinical applications.5-8 In addition, drug resistance, both inherent and
acquired, is another important reason for treatment failure of Pt(II) drugs.9-11 Thus, the
development of novel and more effective platinum based drugs to overcome the shortcomings of
cisplatin is still an important issue of today’s research.
Recent studies indicated that combination of platinum anticancer agents with tubulin inhibitors
including paclitaxel and docetaxel can lead to the improvement of chemotherapeutic efficacy.12 Up
to now, the natural products including vincristine, paclitaxel, vinblastine, combretastatin-A 4 and
docetaxel, as inhibitor of tubulin, are forceful as anti-mitotic drugs that can effectively induce
tumor cell apoptosis and obviously inhibit tumor cell proliferation and angiogenesis.13
Microtubules are cytoskeletal filaments comprising α- and β-tubulin that polymerise parallel to a
cylindrical axis.14 Microtubules, which plays an important role in cellular processes involved in
the formation of the mitotic spindle, cell proliferation, intracellular transport and cell signaling,
have been considered as a fascinating molecular target for antitumor agents in recent years.15-17
Anti-mitotic agents attack microtubules by interfering with the dynamics of tubulin
polymerization or dissociation, leading to mitotic arrest.18 So far two major types of antitumor
drugs have presented their effectiveness in this mechanism, defined as microtubule stabilizing
agents (e.g. paclitaxel, discodermolide) and microtubule destabilizing agents (e.g. vinca alkaloids,
colchicine and combretastatin-4).19, 20 Combretastatin-A 4 (CA-4, Fig. 2) as a natural cis-stilbene
product, isolated by Pettit and co-workers in 1988 from the bark of African willow tree
Combretum caffrum, was found to inhibit strong tubulin polymerization by binding to the
colchicine binding site.21, 22 Besides, CA-4 has been known to exert potent cytotoxicity against a
large number of human cancer cells including multidrug resistant ones.23-27
It is generally considered that CA-4 has three major pharmacophone components: two
hydrophobic rings (rings A and B) and a linking bridge between the aromatic rings.18 Research on
CA-4 to increase its in vivo activity has led to the discovery of phenstatin (Fig. 3). Phenstatin, a

benzophenone type CA-4 analogue, synthesized by Pettit’s group with a carbonyl moiety in place
of an ethylene (CH2=CH2) bridge between the two rings, was studied to be a tubulin
polymerization inhibitor which binded to the colchicine site and exhibited potent cytotoxicity
against a variety of human cancer cells including multidrug resistant cancer cell lines.28-30
Particularly, Schobert and co-workers reported that the Pt(II) complexes conjugating with chalcone
exhibited moderate anti-proliferative activities, due to the different mode of actions such as
DNA-targeting Pt(II) complexes and tubulin-targeting chalcone moieties.31 Therefore, a
combination of cytotoxic DNA damaging platinum-based drugs with tubulin inhibitors can be an
attractive strategy for targeting microtubules and DNA, and at least in theory, can improve the
anticancer activity of platinum drugs and overcome their adverse side effects. Pt(IV) complexes as
prodrugs, showing kinetic inertness compared with their Pt(II) counterparts, are expected to
exhibit great promise in the search for the next generation of platinum drugs, because they can be
effectively reduced to Pt(II) equivalents once inside the cells. Recently, there have been several
reports about Pt(IV) prodrugs conjugated with phenylbutyrate, chalcone derivatives or
dichloroacetate as the axial ligands in the octahedral geometry of Pt(IV) complexes, in which
Pt(IV) species were easily reduced to their Pt(II) equivalents inside the tumor cells in the presence
of ascorbic acid or glutathione and released other molecular fragments to activate p53, inhibit
histone deacetylases (HDACs), suppress efficacy of pyruvate dehydrogenase kinase (PDK) and
induce cell cycle arrest at the S and G2/M phases and cell death through apoptosis,
respectively.32-39
In our previous work, we reported three Pt(IV) complexes containing a CA-4 analogue as
dual-targeting Pt(IV) prodrug. Among them, the Pt(IV) derivative of cisplatin comprising a CA-4
analogue displayed potent antitumor activities against the test cancer cell lines and effectively
inhibit the tumor growth in the HepG-2 xenograft model.40 However, the in vivo activity of the
reported complex was lower than that of cisplatin. In order to search for novel Pt(IV) complex
prodrugs with higher antitumor activities against the human cancer lines and lower toxicity toward
human normal cells, dual-targeting Pt(IV) complexes containing a phenstatin analogue were
studied herein as anticancer prodrugs with the ability to release Pt(II) equivalents and the
phenstatin analogue for their respective biological actions. Apart from their effective in vitro
anti-proliferative activities, in vivo tumor inhibition of a representative Pt(IV) complex together

with its mechanism of apoptotic pathway was also investigated.
Fig. 1. FDA approved Pt(II) anticancer agents.
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
H3CO
OCH3
OCH3
NHAc
OPO3Na2
OCH3
OH
OCH3
O
O
CA-4P
CA-4
Colchicine
H3CO
H3CO
O
H3CO
H3CO
H3CO
H3CO
OH
O
O
OCH3
OCH3
NH2
OCH3
N
OCH3
H
OCH3
NH2
OH
AEV8063
Phenstatin
AEV8062
Fig. 2. Chemical structures of natural products and clinical trial agents that bind at the
colchicine-binding site of tubulin.
Fig. 3. Chemical structures of a few Pt(IV) prodrugs
2. Results and discussion

2.1 Design and synthesis.
Compounds 1-4 were synthesized according to the reported procedures.41 Pt(IV) complexes
7-9 were prepared in agreement with the literature method.42 Compound 4 upon etherification with
α-bromoethyl acetate, K2CO3 and KI in the presence of DMF gave ester 5 followed by its
hydrolysis with aluminum hydroxide to afford the acid 6. The synthesis of target compounds was
achieved by the formation of ester bond between 6 and Pt(IV) complexes 7-9, respectively, in the
presence of TBTU/Et3N. The target compounds were characterized by microanalysis, 1 H, and13
C
NMR spectra together with HR-MS spectrometry. The synthetic profiles of the compounds and
their chemical structures are listed in Scheme 1.
Scheme 1. Synthetic Pathway to Prepare Target Compounds 10-12. Reagents and Conditions:
(a)THF, n-BuLi, -78 oC; (b) PDC, CH2Cl2, rt; (c) 1N HCl/CH3OH, 50 oC; (d) K2CO3, DMF, rt.; (e)

LiOH.H2O, THF/H2O, rt.; (f) TBTU, Et3N, DMF, rt.
2.2. HPLC analyses on the released ability of the resulting Pt(IV) complexes.
To evaluate whether our Pt(IV) complexes can be reduced to their Pt(II) equivalents or not,
complex 10 in a solution of acetonitrile and water (1.5:1) to release compound 6 and cisplatin in
the presence of ascorbic acid was examined by HPLC. As illustrated in Fig. S1, complex 10
gradually released compound 6 as time passed, accompanied by the falling down peak of complex
10 and the rising peak of compound 6, respectively. It was found in the HPLC chromatograms that
cisplatin was not observed due to its weak chromophore in the ultraviolet detecting condition.
Under the same condition, the behavior of complex 12 was also examined for comparison,
because oxaliplatin could be observed in HPLC chromatograms. As shown in Fig. S1, similar
results for complex 12 were observed in HPLC chromatograms except the presence of oxaliplatin.
Taken together, these data demonstrated that the Pt(IV) complexes were easily reduced to their
Pt(II) equivalents under the ascorbic acid at the room temperature, releasing the corresponding
active drug species.
2.3 In vitro cytotoxicity assay.
The in vitro cytotoxicity of the target Pt(IV) complexes 10-12 were investigated using MTT
assay on a panel of human cancer cell lines including HepG-2 (hepatoma), Bel-7404 (hepatoma),
NCI-H460 (lung), MGC-803 (gastric), and human normal cell lines such as HL-7702 (human
normal liver cell line) and NCM460 (human normal colon mucosal epithelial cell line). Phenstatin,
CDDP, OXP and DACHPt were used as the positive controls. The corresponding IC50 values,
obtained after 72 h exposure, are given in Table 1. In vitro evaluation results revealed that
phenstatin analogue 6 exhibited lower cytotoxicity against the tested tumor cell lines than
phenstatin. However, complexes 10-12, the Pt(IV) derivative of cisplatin, oxaliplatin, or DACHPt
with one phenstatin analogue ligand in the axial position, possessed higher cytotoxicity against all
test cancer cell lines than the positive control. Especially, complex 10, the Pt(IV) derivative of
cisplatin with one phenstatin analogue ligand in the axial position, possessed better antitumor
activities against all test cancer cell lines than that of cisplatin, with IC50 values in the range of
0.55–1.97 µM, and synchronously displayed lower cytotoxicity toward NCM460 and HL-7702
with IC50 values of 35.13 2.41 and 45.03 2.55 µM than cisplatin (15.21 0.35 and 6.30 0.32 µM)

and phenstatin (2.01 0.54 and 1.41 0.16 µM), respectively. Pt(IV) complexes 11 and 12 also
exhibited better anti-proliferative activity against the tested cell lines than their corresponding
Pt(II) counterparts, DACHPt and oxaliplatin, with IC50 values in the range of 0.61–1.30 and
0.98–4.07 µM, respectively. Interestingly, complex 10 exhibited up to 7.7-fold increased
cytotoxicity compared with cisplatin in HepG-2 cells and showed significantly more effective
anticancer activity than phenstatin. The similar trend was also observed in Bel-7404 and
NCI-H460 cells. Notably, all Pt(IV) complexes revealed lower cytotoxicity than their
corresponding positive references (CDDP, OXP and DACHPt) against two human normal cell
lines, indicating that these complexes have a selective toxicity for the cancer cells over the normal
cell.
Table 1. Biological activity of Pt(IV) complexes 10-12.
Compd.
IC50 (µM) d
NCI-H460 MGC-803
HepG-2
Bel-7404
NCM460
HL-7702
19.82 1.27 25.37 2.03
10.21 2.03
0.55 0.06
0.91 0.21
1.75 0.33
0.70 0.16
3.21 0.42
4.23 0.39
3.51 0.42
25.54 2.35 55.23 3.02 65.27 2.19
6
10
0.62 0.04
0.78 0.07
2.08 0.28
0.69 0.05
4.76 0.41
5.06 0.20
4.06 0.17
1.97 0.45
1.30 0.22
4.07 0.39
2.25 0.57
4.38 0.52
7.90 0.71
6.21 0.32
0.64 0.11
0.61 0.18
0.98 0.20
0.31 0.08
6.90 0.75
5.24 0.31
4.49 0.14
35.13 2.41 45.03 2.55
37.42 1.58 37.42 2.59
39.63 3.27 49.63 2.01
11
12
Phenstatin
CDDP a
OXP b
2.01 0.54
15.21 2.35
16.52 0.93
13.65 1.04
1.41 0.16
6.30 0.32
8.23 0.52
5.65 0.74
DACHPt c
a
b
c
Cisplatin, Oxaliplatin, Dichloro(1R,2R-diaminocyclohexane)platinum(II). d IC50 is the drug
concentration effective in introducing 50% of cell death measured by MTT assay after 72 h drug
exposure and mean values standard deviation depend on three independent experiments.
2.4. Antitumor activity against cisplatin resistant cell lines.
According to the above biological result, we further investigated sensitivity of the Pt(IV)
complexes (10-12) against cisplatin resistant and the corresponding normal cancer cells (human
ovarian cancer cell SK-OV-3, human lung epithelial cell A549). As shown in Table 2, the IC50
value of cisplatin toward SK-OV-3 and A549 resistant cell lines were increased to 15.61 and 29.05
µM, respectively, compared with those of cisplatin toward the normal cancer cell lines.

Interestingly, the activity of complex 10 did not significantly vary for both these two cisplatin
resistant cancer cell lines and the non-resistant ones. Its IC50 values toward cisplatin resistant
SK-OV-3 and A549 cell lines were 1.41 and 1.95 µM, respectively. Notably, complex 10 had a
much lower resistance index (1.88 for resistant SK-OV-3 cells and 1.93 for resistant A549 cells)
than cisplatin (18.15 for resistant SK-OV-3 cells and 6.92 for resistant A549 cells). The similar
tendency was also observed in complexes 11 and 12, suggesting that these complexes might be
effective in the treatment of drug refractory tumors resistant to other Pt(II) drugs.
Table 2. In vitro growth inhibitory effect of Pt(IV) complexes on cisplatin-resistant cancer cells.
Compd.
IC50 (µM)
resistant
factor d
1.76
IC50 (µM)
A549/CDDP
26.16 2.13 32.11 2.07
resistant
factor d
1.22
SK-OV-3
SK-OV-3/CDDP
21.32 2.13
1.41 0.19
A549
12.11 0.95
0.75 1.09
0.95 0.12
2.63 0.49
0.86 0.31
0.86 0.31
5.61 0.25
6
10
1.88
1.01 0.24
1.25 0.37
1.86 0.68
3.26 0.33
4.20 0.61
5.36 0.70
5.21 0.47
1.95 0.12
1.86 0.19
2.67 0.66
5.66 0.77
29.05 1.55
23.21 2.01
1.93
11
1.72 0.21
1.81
1.49
3.53 2.59
1.34
1.44
12
Phenstatin
CDDP a
OXP b
1.61 0.46
1.87
1.74
15.61 0.46
16.21 2.31
18.15
2.88
6.92
4.33
DACHPt c 4.12 0.38
17.04 1.55
4.41
19.54 0.32
3.75
a
b
c
d
Cisplatin,
Oxaliplatin,
Dichloro(1R,2R-diaminocyclohexane)platinum(II), The values
express the ratio between IC50 determined in resistant and nonresistant cell lines.
2.5. Cell uptake and cell viability.
Since complex 10 showed potent anti-proliferative effects, it was selected to carry out the
cellular uptake test in NCI-H460 cells by using the inductively coupled plasma mass spectrometry
(ICP-MS). As shown in Fig. 4 A and Table 3, treating NCI-H460 cells with complex 10 (5.0 and
10.0 µM) for 24 h resulted in a substantial increase in the content of cellular platinum in a dose
dependent manner, suggesting facile internalization of complex 10 within 24 h. Especially, the
uptake of this complex was remarkably higher than those of cisplatin. After exposure to 10.0 µM
of complex 10 for 24 h, the concentration of cellular platinum rose to 459 ng/106 cells, which is
nearly up to 2.1 times as much as that of cisplatin. According to the corresponding experimental

results from the cytotoxicity assay and cellular uptake tests, it seems that the improvement of
cellular uptake can result in the increase of the antitumor activity.
The effect of complex 10 on cell proliferation after 72 h treatment was further assessed in
NCI-H460 cancer cell line using MTT assay. Our results found that the complex 10 was
significantly inhibited cell proliferation in human lung cancer cells compared with cisplatin under
the same conditions (Fig. 4 B).
Table 3. Cellular uptake of complex 10 in NCI-H460 cells.
Complex
Pt content (ng/106 cells) b
NCI-H460
10 (5 µM)
10 (10 µM)
175 20
459 46
105 12
219 22
CDDP (5 µM)
CDDP (10 µM)
b
a Cisplatin. Incubation for 24 h. Results are expressed as the mean SD for three independent
experiments.
Fig. 4. (A) Intracellular accumulation of complex 10, and CDDP (5, 10µM) in NCI-H460 cells
after 24 h. Each value is in nanograms of platinum per106 cells. (B) In vitro cytotoxicity of
complex 10, and CDDP toward NCI-H460 cancer cells at the same concentration for 72 h. Results
are expressed as the mean SD of three independent experiments. P < 0.05
2.6. Competitive binding to colchicine binding site of tubulin.
Compounds 6, 10, phenstatin and CA-4 were examined for inhibitory effects on tubulin
polymerization and for binding effects on [3H] colchicine to tubulin in determining whether their
antitumor effects might be caused by an interaction with microtubules. For comparison, CA-4 and

phenstatin were evaluated in contemporaneous experiments. The result is given in Table 4. In the
colchicine binding studies, both 6 and 10 are capable of inhibiting tubulin polymerization with
calculated IC50 values of 12.5 µM and 13.9 µM, respectively. In addition, 10 showed the ability to
compete with [3H] colchicine in binding to tubulin. The binding potency of 10 (42.5%) to the
tubulin colchicine binding site, comparable to 6 (45.0%), was much stronger than that of cisplatin
but less than that of CA-4 (98%) and phenstatin (99%), respectively.
Table 4. Inhibition of tubulin polymerization and colchicine binding by complexes 6, 10, CDDP,
CA-4 and Phenstatin.
Compd.
(10 µM)
6
Tubilin assembly a
IC50 (µM)
12.5 1.3
13.9 1.5
>100
Colchicines binding b
(% inhibition)
45.0 1.2
42.5 1.3
10
CDDP c
<5
CA-4
1.1 0.2
98.0 0.5
Phenstatin
0.6 0.3
99.0 0.3
a
b
Inhibition of tubulin polymerization. Tubulin was at 10 µM. Inhibition of [3H] colchicine
c
binding. Tubulin, colchicine and the tested compound were at 1, 5, and 5 µM, respectively.
Cisplatin.
2.7 Complex 10 inhibited the polymerization of tubulin in vitro.
It is generally known that the mechanism of tubulin-binding agents can be divided into two
types: stimulating agents and inhibiting agents.20 In order to determine which type complex 10
belongs to, its effect on the assembly kinetics of tubulin with paclitaxel, CA-4 and phenstatin as
references was evaluated. As illustrated in Fig. 5, paclitaxel (10 µM) was found to stimulate
tubulin polymerization, while CA-4 (10 µM) or phenstatin (10 µM) almost inhibited tubulin
polymerization as expected. For complex 10, an obvious inhibition of polymerization was
observed at two concentrations, and the rate of assembly as well as the final amount of
microtubules was lower than the control group.

0.5
0.4
0.3
0.2
0.1
0.0
Control
Paclitaxel (10 µM)
CA-4 (10 µM)
Phenstatin (10 µM)
10 (10 µM)
10 (20 µM)
0
10
20
30
40
50
60
Time (min)
Fig. 5. Effects of complex 10 on microtubule dynamics. Polymerization of tubulin at 37 °C in the
presence of paclitaxel (10 µM), CA-4 (10 µM), phenstatin (10 µM) and complex 10 (10 µM and
20 µM) were monitored continuously by recording the absorbance at 340 nm over 60 min. The
reaction was initiated by the addition of tubulin to a final concentration of 3.0 mg/mL.
2.8. Molecular modeling.
To determine the possible binding mode of the Pt(IV) complexes, compounds 6 and 10 were
chosen to perform a molecular docking to ensure that the Pt(IV) complexes have the same binding
site as colchicine and CA-4. The binding modes of compounds 6 and 10 in the colchicine binding
site of tubulin are depicted in Fig. 6. The Surflex docking scores are 12.06 for 10 and 8.57 for 6,
where higher scores indicate greater binding affinity (data not shown). Biological activity studies
suggested that complex 10 potentially inhibited tubulin polymerization, hence, the docking detail
of complex 10 was examined to compare with those of 3E22-colchicine and CA-4 to evaluate
binding modes.
Fig. 6 C and D show that the interacting mode of CA-4 with 3,4,5-trimethoxyphenyl rings in
the binding site is surrounded by Ser178, Ala180, Ala316, Val315, Asn350, Lys352, Phe377 and
Gly379. In particular, CA-4 forms three hydrogen bonds with the polar amino acids Asn249, Ala
250 and Asn258, suggesting a probable strong electrostatic interaction with the protein. In
addition, the hydrophobic moiety of the 3E22-colchicine is well embedded in a pocket interacting
with several hydrophobic residues making 3E22-colchicine bind tightly to tubulin. Not
surprisingly, the accommodation of compound 6 in the binding site is similar to colchicine (Fig. 6
F). Also in this case, docking simulations showed that the 3,4,5-trimethoxy-phenyl rings of

compound 6 like colchicine can also be accommodated in the same hydrophobic groove, adopting
an energetically stable conformation. Moreover, the methoxy group in compound 6 as an acceptor
establishes three hydrogen bonds with Asn101, Asn249 and ALA250, which is consistent with the
observation that colchicine stabilizes the tubulin heterodimer and further confirms that this moiety
is also crucial for binding (Fig. 6 A and B). As shown in Fig. 6 E, in comparison with CA-4 or
colchicine, complex 10 inserts a deep cave and formed hydrogen bonding with VAL315 and
ASN350, respectively. In addition, the 3,4,5-trimethoxy-phenyl ring of complex 10 also
establishes three hydrogen bonds with ASN101, ASN249 and ALA250, which is similar to CA-4
and compound 6. It is interesting to note that the crucial electrostatic interactions between the
methoxy group of the 3,4,5-trimethoxy-phenyl unit and residues Ser178 and Asn101 of the
neighboring α-subunit were observed in the binding pocket, demonstrating a plausible competitive
mechanism of action at the colchicine site.
Fig. 6. Molecular modeling of CA-4, phenstatin, 3E22-colchicine and compound 6 in complex
with tubulin. Illustrated are the proposed binding mode and interaction between tubulin and
selected compounds, (A) CA-4, (B) phenstatin, (C) compound 6, (D) 3E22-colchicine, (E)
complex 10. (F) Comparison of the crystallographic structure of colchicines (in green), CA-4 (in
purple), compound 6 (in orange) in complex with tubulin (Protein Data Bank Code 3E22) and the
energetically most favorable pose of complex 10 (in gray) obtained by molecular docking
simulation. Hydrogen atoms are omitted.

2.9. Effect on cell cycle arrest.
To evaluate the effect of the synthetic compound 10 on cell cycle arrest, we used flow
cytometry to examine the cell cycle distribution of NCI-H460 cells following a 24 h treatment
with complex 10 at different concentrations. Untreated cells were used as a negative control, and
cells treated with cisplatin were used as a positive control. It is noted from Fig. 7 A that in the G2
phase, the percentage of cells treated with complex 10 increased to 30.30% (5 µM) and 65.87%
(10 µM) compared with the control. But cells treated with cisplatin 10 µM for 24 h, the percentage
of cells in the S phase increased to 35.23% compared with untreated cells. These results showed
that complex 10 may cause cell accumulation in the G2 phase of the cell cycle by either delaying
or inhibiting progression to the next phase.
Furthermore, the association between the induced G2 arrest by complex 10 and alterations in
expression of various proteins that regulate cell division were evaluated. Cyclin B in its complex
with CDK1, which triggers entry into mitosis, is considered to be a major regulator of G2/M
checkpoint.43, 44 Protein p53 and p21 play a key role in G2 arrest through accumulation of inactive
cyclin B1/p34cdc2 complex.45-47 Thus, the levels of regulatory proteins implicated in G2 arrest,
including p21, p53, CDK1 and cyclin B1, were investigated. As shown in Fig. 7 B, the expression
of CDK1 and Cyclin B1 were significantly down-regulated, but p21 and p53 levels were
significantly up-regulated in NCI-H460 cells after complex 10 treatment. These results suggesting
that the cells are effectively arrested at G2 phase of the cell cycle.

Fig. 7. (A) Investigation of cell cycle distribution by flow cytometry analysis. Untreated
NCI-H460 cells as a control. NCI-H460 cells were treated with complex 10 (5 and 10 µM) and
CDDP (10 µM) as positive control for 24 h, respectively. (B) Western blot analysis of p21, p53,
CDK1 and Cyclin B1 in NCI-H460 cells treated with complex 10 (10 µM) for 48 h. CDDP (10
µM) was used as a positive control, β-actin was used as the internal control. Data are expressed as
the mean SEM of three independent experiments.
2.10. Complex 10 induced apoptotic cell death.
The mode of cell death induced by complex 10 was investigated with the
annexin-V/propidium iodide (PI) assay and the number of apoptotic cells was evaluated by flow
cytometry. NCI-H460 cells were treated with complex 10 with cisplatin served as a positive
control. Q1, Q2, Q3, and Q4 represent four different cell states: necrotic cells, late apoptotic or
necrotic cells, apoptotic cells and living cells, respectively. A dose-dependent improvement in the
percentage of apoptotic cells was evident after the cells were treated with complex 10 for 24 h at
the concentrations of 5 µM and 10 µM, respectively. As shown in Fig. 8, the percentage of cell
apoptosis was 6.51% for the control, and cell apoptosis was added up to 10.36% and 26.5% at 5
and 10 µM of cisplatin; while treated with complex 10 at 5 and 10 µM, the percentage of cell
apoptosis was increased to 17.59% and 69.8%, respectively. Taken together, the results clearly
demonstrated that complex 10 effectively induced apoptosis in NCI-H460 cells.

Fig. 8. Representative flow cytometric histograms of apoptotic NCI-H460 cells after 24 h
treatment with complex 10 (5, 10 µM) with cispatin (5, 10 µM) 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.
2.11. Complex 10 induces mitochondrial membrane Permeability in NCI-H460 cells.
Mitochondria play a key role in cell apoptosis by changing its intracellular and extracellular
membrane permeability and resulting in cytochrome c release and caspases activation.48, 49 Thus,
compound 10 was further examined to check whether it induced an alteration of the mitochondrial
transmembrane potential (MMP). NCI-H460 cells were treated with compound 10 (5, 10 µM) for
24 h with cisplatin (5, 10 µM) as a positive control, changes in the mitochondrial membrane
potential were monitored using the fluorescent dyes JC-1 as described in materials and methods.49
As shown in Fig. 9, compound 10 induced a dose concentration-dependent increase in the
proportion of cells compared with the control, indicating that compound 10 treatment induced
mitochondrial membrane permeability in NCI-H460 cells. These results are direct evidence that
mitochondrial function is extremely impaired in complex 10 induced apoptosis in NCI-H460 cells.

Fig. 9. Assessment of mitochondrial membrane potential (MMP) after treatment of NCI-H460
cells with cisplatin (5, 10 µM) or complex 10 (5, 10 µM). Cells were treated with the indicated
concentration of the compound for 24 h and then stained with the fluorescent. Data are expressed
as the mean SEM of three independent experiments.
2.12. Complex 10 induces intracellular ROS generation in NCI-H460 cells.
Mitochondrial membrane depolarization is associated with mitochondrial generation of
reactive oxygen species (ROS).50 ROS production has been implicated as an early event in cell
apoptosis. In order to determine whether complex 10 inducing apoptosis is ROS-dependent,
NCI-H460 cells were treated with complex 10 (5 and 10 µM) for 24 h and cisplatin (5 and 10 µM)
as a positive control, using the fluorescent probe 2,7-dichlorofluorescein diacetate (DCF-DA) by
flow cytometry, respectively. As shown in Fig. 10 A, a good deal of ROS induced by complex 10
was effectively produced compared with control cells, in agreement with the dissipation of the
mitochondrial trans-membrane potential. In addition, the effect of complex 10 on the ROS
generation was found similar to that of compound 6 as shown in Fig. 10 A indicating that the
ability of complex 10 to generate ROS originated from its precursor (compound 6). In short, these
data indicate that complex 10 induced apoptosis through the mitochondrial pathway.

Fig.10. (A) Assessment of the ROS production in NCI-H460 cells. After 24 h incubations with
complex 10 and compound 6, cells were stained with DCF-DA and analyzed by flow cytometry.
(B) Western blot analysis of Cyt c, Bax, Bcl-2, Apaf-1, caspase-9, caspase-3 and PARP after
treatment of NCI-H460 cells with complex 10 at the indicated concentrations and for the indicated
times. β-actin antibody was used as reference control.Data are expressed as the mean SEM of
three independent experiments.
2.13. Complex 10 induced apoptosis via the regulation of apoptosis-related protein expression.
Activation of apoptotic pathways is a major way by which antitumor agents kill tumor cells.51
It was reported that anticancer drugs can stimulate apoptotic signaling through two major
pathways. One is via the extrinsic pathway involved in death receptor and death ligand interaction
including Fas receptor and other members of the tumor-necrosis factor (TNF) receptor family; the
other is via the intrinsic (mitochondrial) pathway involved in changing the ratio of Bcl-2 family
protein, causing loss of mitochondrial membrane integrity and the release of cytochrome c in
cytosol, leading to the activation of the caspase cascade and the induction of apoptotic cell
death.52-54 So, the mechanism of action of the newly synthesized complex was further investigated.
The mitochondrial related apoptotic proteins of cytochrome c, Bax, Bcl-2, Apaf-1, caspase-9,
caspase-3 and PARP were tested in NCI-H460 cells treated with complex 10 (5 and 10 µM) for 48
h using the western blot method. Apoptosis associated protein levels are shown in Fig. 10 B,
which indicated that complex 10 significantly promoted expression of Bax, cytochrome c, Apaf-1,
caspase-9, caspase-3 and cleavage-PARP, but reduced levels of Bcl-2 protein that could contribute
to cell apoptosis. The above results displayed that the complex 10 inducing apoptotic cell death in
NCI-H460 cells occurs via an intrinsic signaling pathway.
2.14. Anti-tumor effect of complex 10 in vivo.
To further investigate the antitumor activity of complex 10, the NCI-H460 tumor xenograft

nude mice were used. As shown in Fig. 11, complex 10 inhibited tumor growth in the NCI-H460
tumor xenograft at a dose-dependent manner, and the tumor inhibitory rates on day 28 after
treatment were 52.11% and 60.23%, comparable to that of cisplatin, after iv administration at 5
mg/kg ( equal weighting dose to cisplatin) and 12 mg/kg ( equal molar dose to cisplatin),
respectively. Interestingly, complex 10 exhibited a little lower toxicity than cisplatin as time
passed, which was evidenced by changes in the weight of the mice after iv administration at 5
mg/kg or 12 mg/kg of complex 10 in the fourth week. The results proved that complex 10
displayed potent anticancer activity which was comparable to that of cisplatin with the equal
molar dosage without obvious toxicity in vivo, indicating that it might be used as a drug candidate
for further research on the therapy of lung carcinoma.
Fig. 11. In vivo antitumor activity of complex 10 in mice (BALB/c nude mice) bearing NCI-H460
xenograft. (A-B) After administered with complex 10 at the dose of 5 and 12 mg/kg i.v. once a
week for 28 days, cisplatin at the dose of 5 mg/kg for i.v. once a week for 28 days, the mice were
sacrificed and weighed the tumors. (C) The tumor volume of the mice in each group during the
observation period. (D) The body weight of the mice from each group at the end of the
observation period. (E) The weight of the excised tumors of each group. The data were presented

as the mean SEM. *P < 0.05.
3. Conclusion
In this investigation, three Pt(IV) prodrugs derived cisplatin, oxaliplatin and DACHPt,
containing a phenstatin analogue as a potent inhibitor of tubulin, were designed and synthesized.
All resulting Pt(IV) complexes not only exhibited better antitumor activities than their Pt(II)
counterparts against the tested cancer cell lines including cisplatin resistant ones, but also
displayed less toxic than all the corresponding Pt(II) complexes against two normal human cell
lines. Further mechanistic evaluation on complex 10 revealed that it can effectively enter cells,
strongly inhibit tubulin polymerization, arrest the cell cycle at G2/M phases, and markedly
enhance the cell apoptosis level. More importantly, complex 10 displayed potent activity against
SK-OV-3 and A549 cancer cell lines that were resistant to cisplatin, and the resistance index of
complex 10 was much lower than cisplatin. Molecular mechanism studies showed that complex 10
caused apoptotic cell death of human non-small lung cancer cell line NCI-H460 through the
mitochondrial mediated pathway by releasing mitochondrial cytochrome c, activating Apaf-1,
down-regulating Bcl-2, up-regulating Bax, which in turn proteolytically activated downstream
caspase-9, caspase-3, and PARP cleavage. It is proved that complex 10 had a potent inhibitory
effect on tumor growth in the NCI-H460 xenograft mouse model in vivo. Our study suggested that
Pt(IV) anticancer prodrugs containing a small molecule fragment that can stimulate or inhibit
tubulin polymerization may be a promising approach for multiple targeted cancer therapy.
4. Experimental section
All chemicals and solvents were of analytical reagent grade and provided by Energy Chemical,
Shanghai of China and used without further purification, unless noted specifically. Compounds
42
7-9 were prepared as described previously.
The purity of all target compounds used in the
biophysical and biological studies was ≥95%. Column chromatography was performed using silica
gel (200–300 mesh). The Acitn, Bax, Bcl-2, cytochrome c, caspase-9, caspase-3, Apaf-1, cyclin
B1, CDK1, p21 and p53 antibodies were purchased from Imgenex, USA. All tumor cell lines were
obtained from the Shanghai Institute for Biological Science (China). 1 H NMR and 13 C NMR
spectra were recorded in CDCl3 or d6-DMSO with a Bruker 300 or 400 MHz spectrometer and 195
Pt NMR spectra were recorded in d6-DMSO with a Bruker 600 spectrometer. 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).
4.1 General procedure for the preparation of target compounds.
Synthesis of compounds 1-6 and target complexes 10-12.
Synthesis of compound 1. To a solution of 3-hydroxy-4-methoxybenzaldehyde (1.52 g, 10.0
mmol) and Et3N (2.02 g, 20.0 mmol) in dry CH2Cl2 (25 mL) was added TBDMSCl (2.25 g, 15.0
mmol) in reaction at 0 °C and the mixture was stirred at room temperature for 2 h. After
completion of reaction, the reaction mixture was poured into ice water (150 mL) and extracted
with CH2Cl2 (2×100 mL). The combined organic phase was washed with saturated NaCl solution,
dried over anhydrous Na2SO4 and concentrated under reduced pressure to give the desired product
as yellow oil (2.5 g, yield 94%) which was used directly without further purification. 1 H NMR
(300 MHz, CDCl3) δ 9.81 (s, 1H), 7.48 – 7.45 (m, 1H), 7.36 (d, J = 1.7 Hz, 1H), 6.95 (d, J = 8.3
Hz, 1H), 3.88 (s, 3H, -OCH3), 1.00 (s, 9H, 3×-CH3), 0.17 (s, 6H, 2×-CH3). HR-MS (m/z) (ESI):
calcd for C14H22O3Si [M+H+]: 267.1417; found: 267.1423. Elem Anal. Calcd (%) for C, 62.88; H,
8.67; found: C, 62.60; H, 8.52.
Synthesis of compound 2. Bromo-3,4,5-trimethoxybenzene (2.47g, 10 mmol）in three-necked
round bottomed flask was added dry THF (30 mL) and cooled to -78℃ with a dry ice-acetone
bath under a steady stream of dry N2 gas. Then, 8.3 mL of 1.6 M n-BuLi in hexanes was slowly
added into the reaction and the reaction mixture was stirred at the same temperature for 2 h. After
for 2 h, the compound 1 (3.2 g, 12 mmol）in dry THF (15 mL) was added to dropwise in portions
and the mixture was stirred at room temperature for overnight. TLC showed the reaction was
complete. The reaction mixture was poured in ice water (100 mL) and extracted with CH2Cl2
(2×100 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduce
pressure. The residue was purified by silica gel column chromatography eluted with petroleum
1
ether/ethyl acetate to give the desired product (1.5 g, 35%) as a yellow oil. H NMR (300 MHz,
CDCl3) δ 6.92 – 6.89 (m, 1H), 6.86 (d, J = 1.9 Hz, 1H), 6.81 (d, J = 8.2 Hz, 1H), 6.59 (s, 2H), 5.68
(s, 1H), 3.82 (s, 9H, 2×-OCH3), 3.79 (s, 3H, -OCH3), 2.14 (d, J = 16.5 Hz, 1H), 0.97 (s, 9H,
3×-CH3), 0.13 (s, 6H, 2×-CH3). HR-MS (m/z) (ESI): calcd for C23H34O6Si [M+Na+]: 457.2022;
found: 457.2029. Elem Anal. Calcd (%) for C, 63.56; H, 7.89; found: C, 63.42; H, 7.73.
Synthesis of compound 3. To a solution of compound 2 (1.3 g, 3.0 mmol) in dry DCM (30 mL)
was added compound PDC (2.3 g, 6.0 mmol) in portions and the reaction mixture was stirred at

room temperature for overnight. TLC showed the reaction was complete. After completion of
reaction, the solid was removed by filtration and the solution was added water (50 mL) and
extracted with CH2Cl2 (100 mL). The organic phase was dried over anhydrous Na2SO4 and
concentrated under reduce pressure. The residue was purified by silica gel column
chromatography eluted with petroleum ether/ethyl acetate to give the desired product (1.0 g, 83%)
as a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 7.46 – 7.42 (m, 1H), 7.37 (d, J = 1.7 Hz, 1H), 7.02
(s, 2H), 6.90 (d, J = 8.4 Hz, 1H), 3.93 (s, 3H, -OCH3), 3.89 (s, 3H, -OCH3), 3.88 (s, 6H,
2×-OCH3), 1.00 (s, 9H, 3×-CH3), 0.18 (s, 6H, 2×-CH3). HR-MS (m/z) (ESI): calcd for
C23H32NaO6Si [M+H+]: 433.2046; found: 433.2053. Elem Anal. Calcd (%) for C, 63.86; H, 7.46;
found: C, 63.63; H, 7.22.
Synthesis of compound 4. To a solution of compound 3 (1.2 g, 2.78 mmol) in dry MeOH (20
mL), 1N MeOH/HCl (8 mL) was added. The reaction mixture was stirred at 50 °C for 2 h, and
then cooled to the room temperature. After the solvent was removed by evaporation, the resulting
residue was diluted with water (50 mL) and extracted with CH2Cl2 (100 mL). The organic phase
was dried over anhydrous Na2SO4 and concentrated under vacuum. The residue was purified on a
silica gel column eluted with petroleum ether/ethyl acetate to give the desired product (850 mg,
1
1
yield 96.2%) as a white solid. H NMR (300 MHz, CDCl3): H NMR (300 MHz, CDCl3) δ 7.44
(d, J = 1.9 Hz, 1H), 7.41 – 7.38 (m, 1H), 7.03 (s, 2H), 6.93 (d, J = 8.3 Hz, 1H), 5.69 (s, 1H, -OH),
3.99 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3), 3.88 (s, 6H, 2×-OCH3). HR-MS (m/z) (ESI): calcd for
C17H19O6 [M+ H+]: 319.1182, found: 319.1196. Elem Anal. Calcd (%) for C, 64.14; H, 5.70;
found: C, 64.01; H, 5.86.
Synthesis of compound 5. To a solution of compound 4 (800 mg, 2.52 mmol) in DMF (10 mL),
α-bromoethyl acetate (546 mg, 3.27 mmol), K2CO3 (695 mg, 5.03 mmol) and KI (42 mg, 0.252
mmol) were added in portions. The mixture reaction was stirred at room temperature for overnight
and the reaction was monitored by TLC. After completion of reaction, the mixture was diluted
with water (200 mL) and extracted with CH2Cl2 (2×100 mL). The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel
column eluted with petroleum ether/ethyl acetate to give the desired product (1.0 g, yield 98%) as
a yellow oil. 1 H NMR (300 MHz, CDCl3) δ 7.49 – 7.46 (m, 1H), 7.32 (d, J = 6.9 Hz, 1H), 7.00 (s,
2H), 6.95 (d, J = 8.4 Hz, 1H), 4.73 (s, 2H), 4.28 – 4.21 (m, 2H), 3.97 (s, 3H, -OCH3), 3.92 (s, 3H,

-OCH3), 3.88 (s, 6H, 2×-OCH3), 1.27 (t, J = 7.1 Hz, 3H, -CH3). HR-MS (m/z) (ESI): calcd for
C21H24O8 [M+H+]: 405.1549, found: 405.1542. Elem Anal. Calcd (%) for C, 62.37; H, 5.98; found:
C, 62.13; H, 6.21.
Synthesis of compound 6. To a solution of compound 5 (900 mg, 2.23 mmol) in THF (15 mL)
lithium hydroxide (280 mg, 6.69 mmol) was added and stirred at room temperature for 2 h. The
reaction mixture was adjusted pH = 2 with 1 N HCl solution, the mixture was added water (50 mL)
and extracted with CH2Cl2 (100 mL), then the organic phase was dried over anhydrous Na2SO4
and concentrated under reduced pressure to give the target product (800 mg, yield 95%) as a white
1
solid. H NMR (300 MHz, CDCl3): δ 7.51 – 7.48 (m, 1H), 7.44 (d, J = 1.6 Hz, 1H), 7.00 (s, 2H),
6.96 (d, J = 8.4 Hz, 1H), 4.78 (s, 2H), 3.98 (s, 3H, -OCH3), 3.93 (s, 3H, -OCH3), 3.87 (s, 6H,
2×-OCH3). HR-MS (m/z) (ESI): calcd for C19H20O8 [M+H+]: 377.1236, found: 377.1243. Elem
Anal. Calcd (%) for C, 60.64; H, 5.36; found: C, 60.51; H, 5.18.
Synthesis of complexes 10-12. To a solution of compound 6 (150 mg, 0.399 mmol), TBTU (192
mg, 0.599 mmol) and Et3N (60 mg, 0.599 mmol) in dry DMF (4 mL), compound 10, 11 or 12
(0.399 mmol) was added in reacion. The mixture was stirred at room temperature for overnight.
After completion of reaction, the whole mixture was added CH2Cl2 (150 mL), then extracted two
times with water (150 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated
under reduced pressure. The residue was purified on silica gel column eluted CH2Cl2/ CH3OH
(40:1) to give the desired product as a yellow solid.
Compound 10. Yield: 46%. 1 H NMR (400 MHz, DMSO-d6) δ 7.38 – 7.36 (m, 2H), 7.10 (d, J =
8.9 Hz, 1H), 6.99 (s, 2H), 6.42 – 5.97 (m, 6H), 4.68 (s, 2H), 3.87 (s, 3H, -OCH3), 3.81 (s, 6H,
2×-OCH3), 3.77 (s, 3H, -OCH3). 13 C NMR (100 MHz, DMSO-d6) δ 193.7, 175.9, 153.4, 153.0,
147.8, 141.5, 133.2, 123.0, 125.6, 115.1, 111.6, 107.6, 66.7, 60.6, 56.5, 56.3. 195 Pt NMR (129
MHz, DMSO-d6) δ 540.6. HR-MS (m/z) (ESI): calcd for C19H25Cl3N2O8Pt [M+H+]: 710.0402,
found: 710.0417. Elem Anal. Calcd (%) for C, 32.10; H, 3.55; N, 3.94; found: C, 32.01; H, 3.33;
N, 3.62.
Compound 11. Yield: 66%. 1 H NMR (400 MHz, DMSO-d6) δ 8.98 – 8.12 (m, 2H), 7.95 – 7.45
(m, 2H), 7.39 – 7.36 (m, 1H), 7.29 (d, J = 1.6 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.97 (s, 2H), 4.73
(s, 2H), 3.88 (s, 3H, -OCH3), 3.81 (s, 6H, 2×-OCH3), 3.77 (s, 3H, -OCH3), 2.15 – 2.03 (m, 2H),
1.63 – 1.26 (m, 4H), 1.25 – 0.80 (m, 4H). 13 C NMR (100 MHz, DMSO-d6) δ 193.6, 177.7, 153.2,

153.0, 147.6, 141.5, 133.2, 129.9, 125.6, 114.3, 111.5, 107.6, 66.9, 63.9, 62.9, 60.6, 56.5, 56.3,
31.4, 31.2, 24.0, 23.8. 195 Pt NMR (129 MHz, DMSO-d6) δ 401.9. HR-MS (m/z) (ESI): calcd for
C25H33Cl3N2O8Pt [M+H+]: 790.1028, found: 790.1067. Elem Anal. Calcd (%) for C, 37.96; H,
4.21; N, 3.54; found: C, 37.73; H, 4.34; N, 3.31.
Compound 12. Yield: 63%. 1 H NMR (400 MHz, DMSO-d6) δ 8.37 – 8.27 (m, 2H), 8.00 – 7.95
(m, 2H), 7.67 – 7.24 (m, 1H), 7.37 – 7.35 (m, 1H), 7.08 (d, J = 8.5 Hz, 1H), 6.99 (s, 2H), 4.75 (s,
2H), 3.87 (s, 3H), 3.81 (s, 6H), 3.77 (s, 3H), 2.09 – 1.98 (m, 2H), 1.71 – 0.94 (m, 8H). 13 C NMR
(100 MHz, DMSO-d6) δ 193.7, 174.9, 163.6, 163.6, 153.3, 152.9, 147.7, 141.4, 133.3, 129.7,
125.9, 113.7, 111.5, 107.6, 65.4, 62.2, 61.5, 60.6, 56.5, 56.3, 31.3, 31.0, 24.0, 23.9. 195Pt NMR
(129 MHz, DMSO-d6) δ 1014.6. HR-MS (m/z) (ESI): calcd for C25H33Cl3N2O8Pt [M+H+]:
808.1448, found: 808.1483. Elem Anal. Calcd (%) for C, 40.13; H, 4.12; N, 3.47; found: C, 39.90;
H, 4.22; N, 3.25.
4.2.The released ability of Pt(IV) complexes under reduction with ascorbic acid.
The released ability of Pt(IV) complexes in a solution of acetonitrile/water (60:40, v:v) was
examined by HPLC. The test compounds were made by the addition of ascorbic acid, compound 6,
complexes 10 and 12, respectively, to a solution containing 60.0% acetonitrile and 40.0% water.
The incubation was generated by adding test compounds to a solvent containing 60% acetonitrile
and 40% water, which was carried out for 0 h, 1 h and 2 h at 25 °C, respectively. Reversed-phase
HPLC was performed with a 250×4.5 mm ODS column. HPLC profiles were recorded on UV
detection at 210 nm. Mobile phase comprised of acetonitrile /Water (60:40, v/v), and flow rate was
1.0 mL/min. The samples were taken for HPLC analysis after filtered by 0.45 µm filter.
4.3. Cell culture and maintenance.
All cancer cell lines and two human normal cell lines in this study 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 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.
4.4 Cytotoxicity assay (MTT).
The antitumor activity of the target compounds (6, 10-12) as well as the positive drugs were

dissolved in DMF and evaluated in five human cancer cell lines (HepG-2, Bel-7404, NCI-H460,
MGC-803 and A549), two human normal cell lines (NCM460 and HL-7702) and two cisplatin
resistant cell lines (SK-OV-3/CDDP and A549/CDDP), respectively. About 5×104 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 (2.5, 5, 10, 20 and 50 µM)
were then added to the test well and 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 examined 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.
4.5. Cellular uptake test.
NCI-H460 cells were seeded in each well of 96-well plates. After the cells reached about 80%
confluence, NCI-H460 cells were treated with different concentrations of the test complex 10 for
24 h, respectively. After completion of 12 h incubation, cells were collected and washed three
times with ice-cold PBS, then centrifuged at 1000×g for 10 min and resuspended in 1 mL PBS. A
volume of 100 µL was taken out to examine the cell density. The remaining cells were digested by
HNO3 (200 µL, 65%) at 65 oC for 12 h. The Pt level in cells were examined by ICP-MS.
4.6. Tubulin polymerization assay in vitro and competitive inhibition assays.
Tubulin polymerization assay was monitored by the change in optical density at 340 nm using
a modification of methods described by Jordan et al.55 Purified brain tubulin polymerization kit
was purchased from Cytoskeleton (BK006P, Denver, CO). The final buffer concentrations for
tubulin
polymerization
contained
80.0
mM
piperazine-N,N′-bis(2-ethanesulfonic
acid)sequisodium salt (pH 6.9), 2.0 mM MgCl2, 0.5 mM ethylene glycol bis(β-aminoethyl
ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM GTP, and 10.2% glycerol. Test compounds were
added in different concentrations, and then all components except the purified tubulin were
warmed to 37 °C. The reaction was initiated by the addition of tubulin to a final concentration of
3.0 mg/mL. Paclitaxel, CA-4 and phenstatin were used as positive controls under the same
conditions. The optical density was measured for 1 h at 1 min intervals in BioTek’s Synergy 4
multifunction microplate spectrophotometer with a temperature controlled cuvette holder. Assays
were performed according to the manufacturer’s instructions and under conditions similar to those

employed for the tubulin polymerization assays described above.56, 57 To further evaluated whether
the competitive binding activity of inhibitors was investigated using a [3H] colchicine competition
scintillation proximity (SPA) assay.58 The test compounds were pre-incubated with tubulin in
different concentrations (5, 10, 15, 20, and 25 µM, respectively), which used to compete with
colchicine originally bound to tubulin. After completion of incubation, the filtrate was obtained as
described.59 The ability of the test compounds to inhibit colchicine binding to tubulin was
measured as described previously 60 except that the reaction mixtures contained 1 µM tubulin, 5
µM [3 H] colchicine, and 5 µM test compound, respectively.
4.7. Molecular modeling.
All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation. The
crystal structure of the tubulin in complex with colchicine was retrieved from the RCSB Protein
62
Data Bank (PDB: 3E22.pdb).61, The synthetic phenstatin analogues, including the parent
compound CA-4 and phenstatin, were selected for the docking studies. The 3D structures of these
selected compounds were first built using Sybyl-X 2.0 sketch followed by energy minimization
using the MMFF94 force field and Gasteiger-Marsili charges. We employed Powell’s method for
optimizing the geometry with a distance dependent dielectric constant and a termination energy
gradient of 0.005 kcal/mol. All the selected compounds were automatically docked into the
colchicine binding pocket of tubulin by an empirical scoring function and a patented search engine
in the Surflex docking program. Before the docking process, the natural ligand was extracted; the
water molecules were removed from the crystal structure. Subsequently, the protein was prepared
by using the Biopolymer module implemented in Sybyl. The polar hydrogen atoms were added.
The automated docking manner was applied in the present work. Other parameters were
established by default to estimate the binding affinity characterized by the Surflex-Dock scores in
the software. Surflex-Dock total scores, which were expressed in -log10 (Kd) units to represent
binding affinities, were applied to estimate the ligand-receptor interactions of newly designed
molecules. A higher score represents stronger binding affinity. The optimal binding pose of the
docked compounds was selected based on the Surflex scores and visual inspection of the docked
complexes.
4.8. Flow cytometry analysis of cell cycle distribution.
The NCI-H460 cells were grown on 6-well plates and treated with complex 10 (5, 10 µM) and

CDDP (10 µM) 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 twice 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 twice ice-cold PBS, and finally stained with 1
mg/ml propidium iodide (PI) in the dark at 4 °C for 1 h. Analysis was performed with the system
software (Cell Quest; BD Biosciences).
4.9. Apoptosis analysis.
Apoptosis was examined by flow cytometric analysis of annexin V/PI staining. NCI-H460 cells
were seeded in each well of 96-well plates at the density of 1×105 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 different concentrations of the test compound 10 and CDDP 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 5× 105 cells /ml. cells were then incubated with 5 µL of annexin
V-FITC and 5 µL of PI for 30 minutes at room temperature in the dark. The cells were analyzed by
system software (Cell Quest; BD Biosciences).
4.10. Determination of mitochondrial membrane potential.
The mitochondrial membrane potential was measured with the lipophilic cation probe JC-1
(Beyotime, Haimen, China, Molecular Probe), as previously described.50 NCI-H460 cells were
treated with different concentrations of the test compounds for 24 h. After for 24 h, the JC-1
fluorescent probe was added 20 minutes after replacing with fresh medium. Cells were collected at
2500 rpm and washed twice with ice-cold PBS and mitochondrial membrane potential were
analyzed by flow cytometer. 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.
The production of ROS was measured by flow cytometry using DCFH-DA (Molecular Probe,
Beyotime, Haimen, China), as previously described.50 NCI-H460 cells were seeded into six-well
plates and subjected to various treatments. On the following treatment, cells were harvested at
2000 rpm and washed twice with ice-cold PBS, and then resuspend cells in 10mM DCFH-DA
dissolved in cell free medium at 37 °C for 30 min in dark, and then washed twice with PBS.
Cellular fluorescence was evaluated by flow cytometry at an excitation of 485 nm and an emission

of 538 nm.
4.12. Western blot analysis.
Western blot analysis was performed as described previously.50 NCI-H460 cells were treated
with different concentrations of the test complex 10 for 48 h. After for 48 h, cells were collected,
centrifuged, and washed twice 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 10 min.
The protein concentration in the supernatant was evaluated 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, Bax, Apaf-1, Cyt c, PARP, caspase-9,
caspase-3, p21, p53, cyclin B 1, CDK1, or β-actin for overnight at 4 °C. Membranes were next
incubated with peroxidase labeled secondary antibodies for 2 h. Then all membranes were washed
with TBST four times for 20 minutes and the protein blots were examined 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 antitumor activity of compound 10 was evaluated using a human lung carcinoma
cell line NCI-H460 in BALB/c nude mice. Five week-old male BALB/c nude mice were housed
purchased from Shanghai Ling Chang biotechnology company (China), tumors were induced by a
subcutaneous injection in their dorsal region of 1 × 107 cells in 100 µL of sterile PBS. Animals
were casually divided into four groups, and starting on the second day. When the tumors reached a
−150 mm3
volume of 100
in all mice on day 15, the first group was injected with an equivalent
volume of 5% dextrose injection via a tail vein injection as the vehicle control mice. The second
group was treated with cisplatin at the doses of 5 mg/kg body weight once a week for four weeks,
respectively. No.3 and No.4 groups were treated with complex 10 at the doses of 5 mg/kg
(equal weighting dose to cisplatin) or 12 mg/kg (equal molar dose to cisplatin) body weight once a
week for four weeks, respectively. All compounds were dissolved in vehicle. Tumor volume and
body weights were recorded every other day after drug treatment. All mice were sacrificed after

four weeks of treatment and the tumor volumes were measured with electronic digital calipers and
examined by measuring length (A) and width (B) to calculate volume (V = AB2/2).
4.14. 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 No. 21571033)
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). Huang is grateful to the Innovation
Program of Jiangsu Province postgraduate education (Project KYLX16_0263). The research was
also supported by the Scientific Research Foundation of Graduated School of Southeast University
(YBJJ1677). Jiangsu KeyGen Biotech Company (China) was appreciated for completing the in
vivo tests.
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