Platinum(IV) complexes conjugated with chalcone analogs as dual targeting anticancer agents: In vitro and in vivo studies

Article abstract of DOI:10.1016/j.bioorg.2020.104430

For the sake to develop novel platinum(IV) complexes to reverse cisplatin (CDDP) resistence, four multifunctional platinum(IV) prodrugs via conjugating chalcones with the related platinum(IV) complexes derived from cisplatin were designed and evaluated fo

Full text of DOI:10.1016/j.bioorg.2020.104430

Journal Pre-proofs
Platinum(IV) complexes conjugated with chalcone analogs as dual targeting
anticancer agents: in vitro and in vivo studies
Xiaochao Huang, Zhikun Liu, Meng Wang, Xiulian Yin, Yanming Wang,
Lumei Dai, Hengshan Wang
PII:
S0045-2068(20)31728-4
DOI:
https://doi.org/10.1016/j.bioorg.2020.104430
YBIOO 104430
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To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
15 May 2020
22 September 2020
23 October 2020
Please cite this article as: X. Huang, Z. Liu, M. Wang, X. Yin, Y. Wang, L. Dai, H. Wang, Platinum(IV)
complexes conjugated with chalcone analogs as dual targeting anticancer agents: in vitro and in vivo studies,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104430
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Platinum(IV) complexes conjugated with chalcone analogs as dual targeting
anticancer agents: in vitro and in vivo studies
Xiaochao Huang a, b 1, Zhikun Liu c 1, Meng Wang a *, Xiulian Yin a, Yanming Wang a, Lumei Dai d
* and Hengshan Wang b *
a Jiangsu Key Laboratory of Regional Resource Exploitation and Medicinal Research, and National
& Local Joint Engineering Research Center for Mineral Salt Deep Utilization, Huaiyin Institute of
Technology, Huaian 223003, 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.
c
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing 211189, China.
d
Key Laboratory of Tropical Marine Bio-resources and Ecology, South China Sea Institute of
Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China.
Abstract: For the sake to develop novel platinum(IV) complexes to reverse cisplatin (CDDP)
resistence, four multifunctional platinum(IV) prodrugs via conjugating chalcones with the related
platinum(IV) complexes derived from cisplatin were designed and evaluated for anti-tumor
actyivities in vitro and in vivo. Among them, complex 9 exhibited excellent anticancer activities in
vitro with IC50 values at the submicromolar level against the tested human cancer cells, whereas
showed low cytotoxicity towards human normal liver cells HL-7702. Further mechanistic studies
indicated that complex 9 induced G2/M phase arrest and apoptosis in A549 cells, which was
associated with a collapse of the mitochondrial membrane potential (MMP), alterations in the
expression of some apoptosis-related proteins, and enhanced level of the intracellular reactive
oxygen species (ROS). More importantly, complex 9 significantly suppressed the tumor growth in
the A549 xenograft model without obvious hints of toxicity.
Key words: Platinum(IV) complexes; anti-tumor activity; tubulin; chalcones; apoptosis
* Corresponding author.
E-mail addresses: vipmengwang@126.com; dlmei610@163.com; whengshan@163.com.
1 Co-first author: These authors contributed equally to this work.

1. Introduction
The uncontrolled and rapid proliferation property of cancer and the high cost of its treatment
were main cause encouraging the development for novel and effective chemotherapeutic agents as
well as low cytotoxicity to normal cells [1]. Among chemotherapeutic agent targets, tubulin
polymerization is an attractive choice owing to the microtubule system plays an important role in
essential cellular processes, such as intracellular transport, cell signaling and cell shape organization,
respectively [2-4]. Up to now, sever microtubule targeting agents including paclitaxel, docetaxel,
and vinblastine have been successfully used chemotherapeutically against various human solid
tumors and additional candidates such as combretastatin-A4 (CA-4) and its derivatives were also in
clinical trials [5-9]. Moreover, it has been found that chalcone-derived compound 1a (Fig. 1)
exhibits strongly inhibition on the growth of cancer cell lines including multi-drug resistance cells
and tubulin polymerization by binding to the colchicine site of tubulin [10, 11]. However, the
utilization of microtubule targeting agents in clinic has been hampered by drug resistance, low
bioavailability and serious side effects [12-14].
Platinum(II) complexes (Fig. 2), such as cisplatin, carboplatin and oxaliplatin, which usually
considered as a class of alkylating agents, are indispensable choices for various solid tumors in
clinic due to its high efficiency [15-17]. Until now, CDDP, which mainly interacts with DNA,
induces the blockage of replication and transcription and ultimately causes death of tumor cells,
have been widely used in the clinical treatment of different solid tumors [18, 19]. However, all these
platinum(II)-based agents are associated with multi-drug resistance and severe side effects including
nephrotoxicity, myelosuppression, and neurotoxicity during the clinical chemotherapy [20-22],
which has further driving the search for new and effective platinum complexes with satisfactory
antitumor activities and low toxicity to normal cells.
In recent years, the potential therapeutic effects of platinum(IV) complexes for cancer treatment
have attracted broad attention [23]. Notably, platinum(IV) complexes possess several advantages
compared to their platinum(II) complexes, such as their better toxicological profile and possibility
of oral administration as well as acting as prodrugs of their platinum(II) counterparts, respectively
[23, 24]. More importantly, platinum(IV) complexes cloud offer two valuable axial positions in
comparison to their platinum(II) counterparts, which afford a higher molecular diversity and more
potential for drug optimization, resulting in various hybrid compounds (e.g., multi-target

compounds) [25, 26]. Until now, such platinum(IV) hybrid compounds (Fig. 2) as Asplatin,
Mitaplatin, NERi-Pt(IV) and Cx-platin exhibited remarkable anticancer activities against human
cancer cells including cisplatin-resistant cell lines owing to the introduction of the bioactive moiety
to the axial position [27-30]. Thus, it can be imagined that the combination of platinum(IV)
complexes derived from cisplatin with tubulin inhibitor 1a could remarkably improve therapeutic
profiles and overcome the side effect as well as multi-drug resistance of cisplatin.
Previously, we reported a dual-targeting platinum(IV) prodrugs derived from CA-4, proving
that microtubule inhibitor-derived platinum(IV) complexes enhanced anti-tumor activities in vitro
and reverse cisplatin resistance in lung cancer [30b]. Taken the advantages of both microtubule
inhibitor and platinum complexes in consideration, four dual-targeting platinum(IV) prodrugs,
which made structural modifications to 1a by introducing platinum(IV) complexes derived from
cisplatin, were synthesized and prepared as shown in Scheme 1. These platinum(IV) complexes
were characterized and evaluated for antitumor activities in vitro and in vivo, and their underlying
cytotoxic mechanisms were also investigated.
2. Results and discussion
2.1. Synthesis and characterization.
As shown in scheme 1, compound 1a was obtained by Claisen-Schmidt condensation of 1 and
2 in the presence of KOH (50% w/v aqueous solution) in methanol at 0 °C for overnight. Then
esterification reaction of compound 1a with succinic anhydride or glutaric anhydride in the presence
of potassium carbonate in N,N-Dimethylformamide (DMF) gave compound 4 and 5. In addition,
the synthesis of two platinum(IV) complexes 5 and 6 were prepared through the oxidative reaction
of the cisplatin with H2O2 or N-chlorosuccinimide (NCS) in water according to the reported
procedure [31]. Finally, the target platinum(IV) complexes 7-10 were obtained by esterification
between 3 or 4 and 5 or 6 in the presence of O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium
tetrafluoroborate (TBTU) and Et3N in DMF. Meanwhile, the resulting title target platinum(IV)
1
complexes 7-10 were characterized by H NMR, 13C NMR, 195 Pt spectra and elemental analysis
together with ESI-MS spectroscopy.
2.2. In vitro cytotoxicity.
In present work, the cytotoxicity of the synthesized target platinum(IV) complexes 7-10 were
evaluated their anti-proliferative activities against HeLa (cervical), MGC-803 (gastric), HepG-2

(hepatocellular), NCI-H460 (non-small-cell lung carcinoma) and human normal liver cells HL-7702
using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, using
compound 1a and cisplatin as positive drugs. As shown in Table 1, the positive drug 1a exhibited
excellent anticancer activities against the tested human cancer cells, with IC50 values at
submicromolar level as expected, which urged us to search for potential compounds with better
activities. We then evaluated the anti-proliferative activity of platinum(IV) complexes 7-10, for
which were attached to an inhibitor of tubulin 1a at one axial position of platinum(IV) complexes
octahedral coordination sphere via a linker. Similarly, compounds 3 and 4 were performed as
controls. The results listed in Table 1 indicated that these platinum(IV) complexes 7-10 displayed
excellent antitumor activities toward tested human cancer cell lines, possessed higher antitumor
activity and lower cytotoxicity than that of 1a and cisplatin. Among them, complex 9 exhibited very
good activity with IC50 values that ranged from 0.26 to 0.37 μM against the four human cancer cell
lines, which exhibited a 21.2~29.4 fold increase in activity than that of cisplatin, respectively.
Notably, complexes 9 (IC50 value of 12.35 μM) also exhibited lower cytotoxicity against human
normal liver cells HL-7702 that of cisplatin (IC50 value of 10.36 μM) and 1a (IC50 value of 3.07
μM), and the selectivity index (SI) of complex 9 (SI of 42.6) synchronously exhibited much higher
than that of the positive drugs cisplatin (SI of 1.3) and 1a (SI of 7.3), respectively. Not surprisingly,
compounds 3 and 4, derivatives of compound 1a, inherited efficient antitumor activity with IC50
values ranged from 0.52 to 1.35 M against cancer cells tested in this work, while showed high SI
to HL-7702 (SI values of 11.4 and 14.8, respectively) More importantly, complexes 7 and 9
exhibited better antitumor activities compared to complexes 8 and 10, indicating that the axial ligand
from hydroxyl group to chloride can obviously result in different activity, respectively.
2.3. Anti-proliferative activity of complex 9 against drug-resistant cancer cells.
Previous studies indicated that development of multidrug resistance to first-line chemotherapy
drugs, such as cisplatin and doxorubicin (DOX), was an importantly reason for the failure of most
first round chemotherapy [20, 32]. Accordingly, it will be interesting to further evaluate if our
synthesized target compounds, which were platinum(IV) complexes derived from cisplatin, could
also overcome the multidrug resistance. Therefore, the most active complex 9 was selected to test
the cytotoxicity against cisplatin-resistant cells (A549/CDDP) together with doxorubicin-resistant
cells (MCF-7/DOX), using compound 1a, cisplatin and compound 4 as controls. As shown in Table

2, cisplatin exerted poor anti-proliferation activity against A59/CDDP cells (IC50 value of 35.06 μM
and resistant factor (RF) of 5.5, respectively), while DOX against MCF-7/CDDP cells (IC50 value
of 42.71±2.01 μM and RF of 14.1, respectively) was also obviously decreased compared with
sensitive cancer cell lines. Interestingly, it was noted that complex 9 could remarkably inhibit the
proliferation of the cell lines A549/CDDP (IC50 value of 0.23 μM and RF of 1.2, respectively) and
MCF-7/DOX (IC50 value of 0.42 μM and RF of 1.1, respectively) with very small resistant factor
values. Similarly, compound 4 showed great potential anti-tumor activity against A549 and
A549/CDDP cells (IC50 values were 0.65±0.13 M and 0.83±0.11 M), and exhibited moderate
anti-proliferation activity against MCF-7 and MCF-7/DOX cells (IC50 values were 1.76 ±0.27M
and 2.33 ±0.39 M), respectively. Moreover, the combined group (compound 4 and CDDP) seemed
to reverse cisplatin resistance in A549/CDDP cells with RF value of 2.0, which was higher than that
of complex 9 but lower than cisplatin. In shorts, these results indicated that complex 9 exhibited
potent anti-MDR potential and deserved further study, which might be useful in the treatment of
drug refractory cancer resistance to cisplatin.
2.4. Antitumor effect of complex 9 in vivo.
Compound 9 was chosen to evaluate the in vivo antitumor efficacy owing to its excellent
anticancer activity against the tested human cancer cells including multidrug-resistant cells. Thus,
to further evaluate the antitumor potency of complex 9 in vivo, we established xenograft models by
subcutaneously injecting A549 cells at the logarithmic growth phase into the right armpit of mice.
When the model was well-established, mice with tumors at the volume of 100-150 mm3 were
randomly divided into four groups (n=5/group): (1) vehicle treated group (5% dextrose injection),
(2) cisplatin (5 mg/kg) treated group, (3) compound 1a (5 mg/kg) treated group, (4) compound 9 (5
mg/kg) treated group, (5) compound 9 (13 mg/kg) treated group, respectively. We collected and
weighed the tumors and calculated the inhibition rate of tumor growth (IRT) at the end of treatment.
As exhibited in Fig. 3 B and D, the growth of A549 tumor xenograft was effectively suppressed by
58.7% and 68.7% (IRT) after treatment groups were injected with compound 9 at two doses (5 and
13 (mg/kg)/7days) for three weeks in the A549 tumor model, respectively. Notably, compound 9
displayed better in vivo antitumor activity than those of 1a (IRT, 62.5%) and cisplatin (IRT, 65.7%)
after administration of compound 9 at high dosage (13mg/kg), respectively. More importantly,
compound 9 displayed lower toxicity compared to the positive drugs cisplatin and 1a in vivo, as

evidenced by changes in the weight of the mice after iv administration at 13 mg/kg of compound 9
in the entire observation period (Fig. 3 C).
2.5. Anticancer mechanism of complex 9.
2.5.1 The release of compound 4 under the reduction of vitamin C.
Based on the concept of prodrugs, platinum(IV) complexes could release compound 4 and
corresponding platinum(II) complexes under reducing condition (such as vitamin C) [23, 24].
Therefore, complex 9 was chosen for in vitro release assays via reversed phase high performance
liquid chromatography (RP-HPLC), using vitamin C (2.211 min), compound 4 (12.785 min) as
controls to match the peaks during the release process of complex 9. Firstly, we investigated the
stability of complex 9 in physiological environment (PBS, pH = 7.4), and the merged
chromatograms in Fig. S1E showed that complex 9 was stable and was not decomposed in 72 h.
However, when co-incubated with vitamin C (3.0 equiv.), the peak at 9.101 min of complex 9
gradually decreased along with another increasing peak (12.785 min), which appeared right at the
position of compound 4, indicating that complex 9 successfully decomposed compound 4 and
cisplatin. Not surprisingly, the cisplatin was not be detected under this condition of liquid conditions
due to its weak UV absorption, which had been reported previously [11,18, 20, 24]. To investigate
whether complex 9 can be reduced to compound 4 in equivalents, standard curve of compound 4
was established. As illustrated in Fig. S1H, after 3 h co-incubation of vitamin C, 1.8 M of complex
9 can release almost the same concentration of compound 4 (1.78 M), indicating that the target
complex 9 could be acted as platinum(II) prodrug. Furthermore, 4.5 h co-treatment with
carboxylesterase only led 60 % of complex 9 decomposition to compound 1a, whereas 100 % of
complex 9 was deducted within 3 h under reduction of vitamin C, showing that the anticancer
activities induced by complex 9 was mainly contributed to the strategy of platinum(IV) prodrug
rather esterase.
2.5.2 Analysis of immunofluorescence staining.
Many studies indicated that tubulin was an essential eukaryotic protein that plays an important
role in cell division and basic cellular functions [6, 7]. In order to investigate whether the activity
of complex 9 was related to the interaction with microtubule systems, here, we determined the effect
of optimal compounds on the microtubule cytoskeleton of A549 cells using immunofluorescence
assay. As exhibited in Fig.4, in the control groups, the microtubule network in the A549 cells

showed normal arrangement and organization, characterized by regularly assembled in the absence
of drug treatment. After treatment with compound 1a at a concentration of 10 μM, cells displayed
obviously disrupted microtubule organization as expected (Fig.4). Interestingly, cells after exposure
to complex 9 at 5 and 10 μM for 24 h, the spindle microtubule organization was significantly
deranged, and the nucleus of the cells narrowed sharply and the spindle microtubules were heavily
disrupted microtubule organization, respectively (Fig.4).
2.5.3 Complex 9 induced apoptosis in A549 cells.
In order to investigate the capacity of complex 9 to induce apoptosis in human lung cancer cells
A549, thus, a FITC conjugated annexin-V/PI assay was performed with A549 cells. As shown in
Fig.5, after A549 cells were exposed to10 μM of the cisplatin for 24 h, the total fractions of early
apoptotic cells (Q4) and late apoptotic cells (Q2) was 11.93% compared with 2.74%. Notably, after
A549 cells were exposed to 5.0 or 10 μM of the complex 9 for 24 h, the total fractions of early
apoptotic cells (Q4) and late apoptotic cells (Q2) were 14.8% and 23.06%, respectively.
2.5.4 Cell uptake.
Since compound 9 exhibited the most potent antitumor activity in the cellular assays, according
to the former reported, which may be partly attributed to the increased cellular uptake [20]. In order
to this hypothesis, here, we selected inductively coupled plasma-mass spectrometry (ICP-MS) to
carry out the intracellular platinum content in A549 cells. Notably, as exhibited in Table 3, after
A549 cells exposure to 10 µM of compound 9 for 12 h, the concentration of cellular platinum
increase to 263 ng/106 cells, which was nearly up to 2.24-fold as much as that of cisplatin (263
ng/106 cells). In brief, the outcomes of this experimental seem to exhibit that the improvement of
cellular uptake could result in enhanced therapeutic profiles.
2.5.5 Cell cycle analysis.
To gain deeper insight on the mode of action, the most valid compound 9 was examined for its
effect on cell cycle progression of A549 cells by flow cytometry. As shown in Fig. 6, incubation of
compound 9 at the indicated concentrations for 24 h led to a gradual accumulation of A549 cells in
the G2/M phase of the cell cycle in a dose-dependent manner, but the control group cells were
primarily in the G1 and S stage. Notably, the cell cycle was obviously changed after treatment with
compound 9 at 5.0 and 10 μM for 24 h: the percentage of G2/M phase increases to 72.79% and
86.42% compared to the control group cells, with a concomitant decrease of cells in the G1 and S

phase cells. Moreover, the percentage of A549 cells in the G2/M and S phase increased to 16.09%
and 54.21%, respectively, after incubation with cisplatin at 10 μM for 24 h (Fig. 6). Thus, these
results indicated that compound 9 caused arrest in the G2/M phase in human lung cancer cells A549.
2.5.6 Complex 9 inhibited the migration of A549 cells in vitro
Increasing evidence indicated that cancer cell migration was one of the important causes resulting
to the death of tumor patients [33]. Thus, in order to further investigate the inhibitory ability of
complex 9 to cell motility, a well-established wound-healing assay was conducted to investigate
whether complex 9 could inhibit the migration of A549 cells. As exhibited in Fig. 7, in comparison
to the control group, the wound closure of the migration was remarkably suppressed for complex 9
treated groups. Notably, the inhibition rate of migration was in the order of complex 9 treated groups
> cisplatin treated groups > control groups. In shorts, these results suggested that complex 9 showed
a significant inhibitory effect on migration of human lung cancer cells A549, and may be a
promising candidate lead compound for chemotherapy of metastatic cancer.
2.5.7 Complex 9 triggered mitochondrial pathway dependent apoptosis.
Many studies indicated that decreased MMP has been implicated as an important event in
apoptotic cells [34-36]. To further investigate the contribution of mitochondria in complex 9
induced cell apoptosis, here we utilized a quantitative MMP assay to measure the activities of JC-1
stained mitochondria and detected by flow cytometry analysis in A549 cells. As presented in Fig.
8, the MMP level in A549 cells was decreased to 88.20% compared to control group cells (97.41%)
after treatment with cisplatin at 10 μM for 24 h. In comparison of control cells, it was noted that the
MMP level in A549 cells was decreased from 84.83% to 72.77%, respectively, after incubation of
complex 9 at 5.0 and 10 μM for 24 h (Fig. 8), suggesting that complex 9 decreased the MMP of
A549 cells during the apoptosis process.
2.5.8 Complex 9 triggered reactive oxygen species (ROS) generation.
According to previous reported, compromised mitochondria could significantly induce an
increase in ROS levels [37, 38], which can in turn lead to cell apoptosis [39, 40]. In order to further
investigate the deeper mechanism of complex 9 for inhibiting tumor cells, therefore, we evaluated
the ability of complex 9 to induce ROS in human lung cancer cells A549 using 2′, 7′-
dichlorofluoresceindiacetate (DCFH-DA) fluorescent probe. As the results illustrated in Fig. 9, the
generation of ROS level was enhanced to 14.81% compared to the control cells (2.32%) after

treatment with cisplatin at 10 μM for 24 h. Moreover, complex 9 induced the generation of ROS
level in a concentration-dependent manner, and the generation of ROS level in human lung cancer
cells A549 was increased from 27.93% to 46.57%, respectively. More importantly, as presented in
Fig. 9, it was worth notated that the generation of ROS level was nearly up to 3.14-fold as much as
that of cisplatin after cells exposure to 10 μM of complex 9 for 24 h, respectively. In shorts, these
results seem to indicate that ROS may mediate A549 cells apoptosis in the presence of complex 9.
2.5.9. Complex 9 induced apoptosis via the activation of caspases and regulated apoptosis-related
protein expression.
Increasing evidence has indicated that the regulation of the Bcl-2 family of proteins was involved
in the signaling pathways [41]. Accordingly, several apoptotic-related proteins such as Bcl-2 (anti-
apoptotic protein) and Bax (pro-apoptotic protein) were investigated using western blot assay. As
the results illustrated in Fig. 10, in comparison of control cells, A549 cells after incubation of
complex 9 at the indicated concentrations (2.5, 5.0 and 10 μM), the level of anti-apoptotic protein
Bcl-2 was efficiently down-regulated, whereas the pro-apoptotic protein Bax was up-regulated
significantly. Moreover, according to previous reported, the caspases protein family including
caspase-3 and caspase-9, were known as an important executioner caspases in cell apoptosis [42].
Therefore, we further investigated whether the caspase-3 and -9 was activated by complex 9 using
western blot assay. As presented in Fig.10, complex 9 remarkably up-regulated the expression level
of caspase-3 and -9 at the indicated concentrations, whereas cisplatin had little influence on them.
3. Conclusions
Many strategies have been developed to overcome the shortcomings of cisplatin, however the
inherent problems were still not resolved well. In the present study, here four tubulin-targeting
platinum(IV) prodrugs were successfully synthesized and evaluated for anti-proliferative activity
using MTT assay. The results of MTT activity test in vitro indicated that these four platinum(IV)
complexes 7-10 not only showed greater potency in anti-proliferative activity compared to the
positive drugs cisplatin and 1a against tested human cancer cells including multidrug-resistant
cancer cell lines, but also exhibited lower cytotoxicity than that of positive drugs (cisplatin and 1a)
toward human normal liver HL-7702, respectively. Especially, complex 9 displayed the most potent
anti-proliferative activities against tested human cancer cell lines with IC50 values of 0.19~0.37 μΜ.
Moreover, complex 9 significantly induced cell apoptosis, caused cell cycle arrest at G2/M phase

and obviously disrupted the microtubule organization, respectively. Cellular studies revealed that
the induction of apoptosis by complex 9 was associated with a collapse of the MMP, and enhancing
the intracellular ROS level, and alterations in the expression of apoptosis related proteins (e.g., Bax
and Bcl-2, respectively). In addition to these mechanism studies, we further investigated the
antitumor potency of complex 9 in vivo. In vivo experiment results revealed that complex 9 could
significantly inhibit tumor growth in the A549 xenograft model, and had no observable toxic effect
during the test. Taken together, these in vitro and in vivo results indicated that complex 9 may be
promising lead compounds for the development of new anticancer drugs.
4. Experimental section
All chemicals and solvents were analytical reagent grade and commercially available, and the
column chromatography was performed using silica gel (200–400 mesh). 1H NMR and 13C NMR
spectra were recorded in CDCl3 or DMSO-d6 with a Bruker 400 or 600 MHz NMR spectrometer,
and the mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. In addition, the
elemental analyses of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementary)
4.1. General procedure for the preparation of compounds.
4.1.1. (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (1a)
To a solution of compound 1 (2.1 g, 10.0 mmol) and 2 (1.52 g, 10.0 mmol) in methanol (20 mL)
was cooled to ice-water, and the 50% KOH (10 mL, aq) added to dropwise to the reaction, and then
the mixture stirred at ice-water for overnight. After completion of reaction, the mixture was poured
into ice-water (200 mL), and adjusted to pH = 1~2 with 4 N HCl. The precipitated was filtered,
washed with water and dried to offer the crude product which was purified by chromatography on
silica gel to obtain 1a as a yellow solid (yield: 2.6 g, 75.6%). 1H NMR (600 MHz, CDCl3) δ 7.73
(d, J = 15.5 Hz, 1H), 7.34 (d, J = 15.5 Hz, 1H), 7.29 (d, J = 2.1 Hz, 1H), 7.26 (s, 2H), 7.13 – 7.11
1
(m, 1H), 6.86 (d, J = 8.3 Hz, 1H), 5.81 (s, 1H), 3.94 (s, 6H), 3.93 (s, 3H), 3.92 (s, 3H). H NMR
(600 MHz, CDCl3) δ 7.74 (d, J = 15.5 Hz, 1H), 7.35 (d, J = 15.5 Hz, 1H), 7.30 (d, J = 2.1 Hz, 1H),
7.27 (m, 2H), 7.13 – 7.11 (m, 1H), 6.87 (d, J = 8.3 Hz, 1H), 5.82 (s, 1H), 3.94 (s, 6H), 3.93 (s, 3H),
3.93 (s, 3H). HR-MS (m/z) (ESI): calcd for C19H20O6 [M-H]-: 343.1182; found:343.1060.
4.1.2. General procedure for preparing compounds 3-4.
Synthesis of compounds 3 and 4. To a solution of 1a (2.5 g, 7.3 mmol) and anhydrous K2CO3
(2.0 g, 14.6 mmol) in DMF (15 mL), succinic anhydride (35.6 mmol) or glutaric anhydride (35.6

mmol) was added, and the mixture reaction was stirred at 50 °C for 1 h and monitored by TLC.
After completion of reaction, the mixture was adjusted to pH = 2~3 with 2 N HCl, and then the
mixture was added dichloromethane (150 mL), washed with water (3×300 mL), and the combined
organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified on silica gel column eluted methanol/dichloromethane to obtain the desired
compound 3 or 4.
(E)-4-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenoxy)-4-oxobutanoic
acid (3). 2.2 g, 67.9% yield as a yellow solid. 1H NMR (600 MHz, DMSO-d6) δ 9.15 (s, 1H), 7.71
(d, J = 15.4 Hz, 1H), 7.63 (d, J = 15.4 Hz, 1H), 7.40 (s, 2H), 7.38 (d, J = 2.1 Hz, 1H), 7.31 – 7.29
(m, 1H), 7.00 (d, J = 8.4 Hz, 1H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.92 – 2.87 (m, 4H). HR-
MS (m/z) (ESI): calcd for C23H24O9 [M+Cl]- :479.1109; found: 479.0947.
(E)-5-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenoxy)-5-
oxopentanoic acid (4). 2.4 g, 72.7% yield as a yellow solid. 1H NMR (600 MHz, DMSO-d6) δ 12.19
(s, 1H), 7.84 (d, J = 15.5 Hz, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.75 – 7.73 (m, 1H), 7.71 (d, J = 15.4
Hz, 1H), 7.43 (s, 2H), 7.21 (d, J = 8.6 Hz, 1H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.65 (t, J =
7.4 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 1.90 – 1.85 (m, 2H). HR-MS (m/z) (ESI): calcd for C24H26O9
[M+Cl]- :493.1265; found:493.1075.
4.1.3. General procedure for preparing compounds 5, 6.
H2O2 (30 wt%, 33.3 mmol) or N-chlorosuccinimide (NCS) (534.1 mg, 4.0 mmol) in 10 mL
water was added dropwise to a solution of cisplatin (1.0 g, 3.33 mmol) in water (30 mL) at
temperature. After that, the reaction solution was heated to 50 oC for 10 h. Process of the reaction
was monitored with TLC and visualized with iodine in silica gel. Part of the solution was removed
under reduced pressure, after completion of the reaction. The yellow crystal solid obtained was
filtered, washed with water, cold ethanol and ether. Final target product was dried at dark in vacuum
to give yellow solid.
cis,cis,trans-[Pt(NH3)2Cl2(OH)2] (5). Yellow solid (812 mg). Yield 72.5 %. IR (KBr): 3512 (s,
OH stretch), 1036 (s, Pt-OH bend), 549 (m, Pt-N(O) stretch). Elemental Analysis (%):H, 2.41; N,
8.39; found H, 2.43; N, 8.47.
cis,cis,trans-[Pt(NH3)2Cl2((OH)(Cl))] (6). Yellow solid (600 mg). Yield 51.2 %. 1H NMR (600
MHz, DMSO-d6) δ 5.76 – 5.50 (m, 6H), 1.50 (s, 1H).

4.1.4. General procedure for preparing compounds 7-10.
To a solution of 3 (150 mg, 0.327 mmol) or 4 (150 mg, 0.33 mmol), TBTU (158 mg, 0.491
mmol), and Et3N (50 mg, 0.491 mmol) in dry DMF (3 mL), complex 5 or 6 (0.327 mmol) was added
in reaction. The reaction was stirred at 30 °C for overnight and monitored by TLC. The mixture
reaction was added to CH2Cl2 (150 mL), and then extracted twice with water (150 mL), and the
organic layer was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The
residue was purified on silica gel column eluted methanol/dichloromethane to obtain the desired
product 7, 8, 9 or 10.
cis,cis,trans-[Pt(NH3)2Cl2((E)-2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-
yl)phenyl succinate (OH)] (7). 100 mg, 40.5% yield as a yellow solid. 1H NMR (600 MHz, DMSO-
d6) δ 7.84 – 7.81 (m, 2H), 7.75 – 7.73 (m, 1H), 7.71 (d, J = 15.4 Hz, 1H), 7.42 (s, 2H), 7.20 (d, J =
8.6 Hz, 1H), 6.06 – 5.81 (m, 6H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.76 (t, J = 7.1 Hz, 2H),
2.57 (t, J = 7.1 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 188.15, 179.42, 171.18, 153.42, 153.38,
143.65, 142.38, 140.13, 133.58, 129.86, 128.12, 122.99, 120.68, 113.27, 106.63, 60.67, 56.82, 56.62,
31.64, 30.37. 195Pt NMR (129 MHz, DMSO-d6): δ/ppm 1229.10; HR-MS (m/z) (ESI): calcd for
C23H30Cl2N2O10Pt [M+Cl]-:794.0614; found: 795.0308. Elemental analysis calcd (%) for
C23H30Cl2N2O10Pt: C, 36.33; H, 3.98; N, 3.68; found: C, 36.46; H, 4.03; N, 3.39.
cis,cis,trans-[Pt(NH3)2Cl2((E)-2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-
yl)phenyl succinate)(Cl)] (8). 125 mg, 49.2% yield as a yellow solid. 1H NMR (600 MHz, DMSO-
d6) δ 7.84 – 7.81 (m, 2H), 7.75 – 7.73 (m, 1H), 7.70 (d, J = 15.4 Hz, 1H), 7.42 (s, 2H), 7.20 (d, J =
8.6 Hz, 1H), 6.10 – 5.80 (m, 6H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.76 (t, J = 7.1 Hz, 2H),
2.57 (t, J = 7.2 Hz, 2H). 13C NMR (150 MHz, DMSO-d6) δ 188.17, 179.44, 171.18, 153.42, 153.39,
143.64, 142.41, 140.15, 133.59, 129.84, 128.13, 123.00, 120.71, 113.29, 106.67, 60.67, 56.83, 56.63,
31.67, 30.38. 195Pt NMR (129 MHz, DMSO-d6): δ/ppm 548.03; HR-MS (m/z) (ESI): calcd for
C23H29Cl3N2O9Pt [M+H]+:778.0665; found: 778.0673. Elemental analysis calcd (%) for
C23H29Cl3N2O9Pt: C, 35.47; H, 3.75; N, 3.60; found: C, 35.68; H, 3.61; N, 3.39.
cis,cis,trans-[Pt(NH3)2Cl2((E)-2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-
yl)phenyl glutarate)(OH)] (9). 105 mg, 41.7% yield as a yellow solid. 1H NMR (400 MHz, DMSO-
d6) δ 7.86 – 7.81 (m, 2H), 7.74 – 7.68 (t, J = 11.0 Hz, 2H), 7.42 (s, 2H), 7.20 (d, J = 8.5 Hz, 1H),
6.25 – 5.76 (m, 6H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.65 (t, J = 7.2 Hz, 2H), 2.33 (t, J = 7.0

Hz, 2H), 1.87 – 1.81 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 188.15, 180.71, 171.64, 153.38,
143.61, 142.40, 140.19, 133.59, 129.94, 128.20, 122.72, 120.72, 113.28, 106.66, 60.67, 56.76, 56.65,
35.69, 33.15, 21.58. 195Pt NMR (129 MHz, DMSO-d6): δ/ppm 1228.24; HR-MS (m/z) (ESI): calcd
for C24H32Cl2N2O10Pt [M+H]+:774.1160; found:775.0909. Elemental analysis calcd (%) for
C24H32Cl2N2O10Pt: C, 37.22; H, 4.16; N, 3.62; found: C, 37.46; H, 4.31; N, 3.31.
cis,cis,trans-[Pt(NH3)2Cl2((E)-2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-
yl)phenyl glutarate)(Cl)] (10). 123 mg, 47.6% yield as a yellow solid. 1H NMR (400 MHz, DMSO-
d6) δ 7.86 – 7.81 (m, 2H), 7.74 – 7.68 (m, 2H), 7.42 (s, 2H), 7.20 (d, J = 8.6 Hz, 1H), 6.43 – 6.02
(s, 6H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H), 2.67 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.1 Hz, 2H),
1.89 – 1.82 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 188.15, 180.21, 171.59, 153.38, 143.59,
142.40, 140.18, 133.58, 129.93, 128.21, 122.72, 120.73, 113.29, 106.66, 60.67, 56.76, 56.65, 35.49,
33.04, 21.42. 195Pt NMR (129 MHz, DMSO-d6): δ/ppm 547.76; HR-MS (m/z) (ESI): calcd for
C24H31Cl3N2O9Pt [M+Na]+:814.0641; found:815.0348. Elemental analysis calcd (%) for
C24H31Cl3N2O9Pt: C, 36.35; H, 3.94; N, 3.53; found: C, 36.57; H, 4.01; N, 3.21.
4.2. In vitro cytotoxicity.
All human cancer cell lines (HeLa, NCI-H460, HepG-2, MCF-7, MGC-803, A549, A549/CDDP
and MCF-7/DOX) and human normal liver cells (HL-7702) were purchased from the Shanghai Cell
Bank of the Chinese Academy of Sciences. Culture medium Roswell Park Memorial Institute
(RPMI-1640), phosphate buffered saline (PBS, pH=7.2), fetal bovine serum (FBS), and Antibiotice-
Antimycotic came from KeyGen Biotech Company (China). In this study, the cytotoxicity of target
compounds was tested using MTT methods. All cancer cell lines were cultivate in the supplemented
with 10% FBS, and human normal liver HL-7702 cells were cultivate in the supplemented with 20%
FBS, 100 units/ml of penicillin and 100 g/ml of streptomycin in a humidified atmosphere of 5%
CO2 at 37 oC. After that, target compounds and dissolved in DMF (Sigma) and diluted to different
concentrations with medium were added to those different cells culture medium, respectively. After
72 h incubation, added to 10 μM of MTT (5 mg/mL) in the cell culture fluid, and the cells were
incubated for another 4 h. After 4 h treatment, the medium was replaced with DMSO (100 μL), and
the O.D. The value was read with a 570/630 nm enzyme-labeling instrument. All data were
independently tested repeated in triplicate.
4.3. The release of compound 4 under the reduction of vitamin C.

In order to investigate the stability of complex 9 in various solutions, RP-HPLC (waters, e2695
system; 2489 UV/Vis detector) assays were carried out. As for the release of compound 4 assays,
the stored solutions of complex 9 (2.0 mM, acetonitrile : water= 50 : 50,V/V, 1.0 mL) and vitamin
C (60 mM, waster, 100 L) were prepared and were incubated at 37 oC in dark. The time-dependent
release of compound 4 was monitor with HPLC (UV/Vis, 254 nm). The stability of complex 9 (10.0
mM) in PBS was detected with HPLC (UV/Vis, 254 nm). A solution of complex 9 (1.0 mM, PBS
solution with 5% acetonitrile) and carbonate esterase (2.0 unit/mL) was incubated at 37 C in dark
and was monitored via HPLC (UV/Vis, 210 nm). Similarly, compound 4, vitamin C and compound
1a were recorded as controls. The standard curves of compound 4 (0.025-2.5 mM, acetonitrile) and
compound 1a (0.06-3.0 mM, acetonitrile) were recorded via HPLC and analyzed with graphpad
prism 8.0. HPLC conditions: flow rate, 1.0 mL/min; ODS column (250×4.6 mm, 5 μm), volume of
sample injection, 20.0 µL; column incubator, 35 oC.
4.4. Cell uptake.
The content of cellular uptake of complex 9 was measured on A549 cells, and the cisplatin was
served as positive drug. Firstly, A549 cells were seeded in each well of 24-well plates until reached
about 80% confluence, and 2.5 and 5.0 μM of complex 9 and cisplatin were added. The plates were
also incubated at an atmosphere of 5% CO2 and 95% air at 37.0 °C for 12 h. After completion of 12
h incubation, the cells were harvested, washed with ice-cold PBS, centrifuged at 1000 g, and
resuspended in PBS (2 mL). The cell numbers were counted by using 100 μL suspension, and the
rest cells were digested with 65% nitric acid at 65 oC for 12 h, and then the Pt level in cells were
detected by ICP-MS.
4.5. Cell apoptosis analysis.
A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the
RPMI-1640 medium with 10% FBS to the final volume of 2 mL. The plates were also incubated at
an atmosphere of 5% CO2 and 95% air at 37.0 °C for overnight, and incubated in the presence or
absence of complex 9 and cisplatin at the indicated concentrations for 24 h to induce cell apoptosis.
After 24 h treatment, cells were harvested, washed with ice-cold PBS, resuspended in PBS (2 mL)
and incubated with 5 μL of Annexin-V/FITC 5 μL of PI in the dark at 4 °C for 30 minutes. Finally,
the cells analyzed by flow cytometry (Cell Quest; BD Biosciences).
4.6. Cell cycle analysis.

Firstly, A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL
of the RPMI-1640 medium with 10% FBS to the final volume of 2 mL. The plates were also
incubated at an atmosphere of 5% CO2 and 95% air at 37.0 °C for overnight, and incubated in the
presence or absence of complex 9 and cisplatin at the indicated concentrations for 24 h. Secondly,
cells were harvested by centrifugation and fixed in ice-cold 70% ethanol at -20 °C for overnight.
After the ethanol was removed the next day, the cells were resuspended in the ice-cold PBS and
incubated with 100 μg /mL RNase A at 37 °C for 30 min, followed by incubated with the DNA
staining solution propidium iodide (PI) in the dark at 4 °C for another 30 minutes detected by flow
cytometry, and the results analysis was performed with the system software (Cell Quest; BD
Biosciences).
4.7. Immunofluorescence assay.
A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the
RPMI-1640 medium with 10% FBS to the final volume of 2 mL. The plates were also incubated at
an atmosphere of 5% CO2 and 95% air at 37.0 °C for overnight, and incubated in the presence or
absence of complex 9 and cisplatin at the indicated concentrations for 24 h. After that, cells were
fixed with 4% paraformaldehyde at 37 °C for 30 minuntes, and then permeabilized with 0.5% Triton
X-100/PBS for 15 minuntes. After blocking for 30 minuntes in 5% BSA/PBS, cells were washed
with ice-cold PBS and treated with a-tubulin for 2 h, and then tubulin was immunostained with
monoclonal antibody to a-tubulin followed by fluorescence antibody. Finally, cells were visualized
by fluorescence microscope after the nuclei of cells were labeled with DAPI.
4.8. Cell migration assay.
A549 cells were seeded in 6-well plates and allowed to grow to ≥ 95% confluent, and then
wounds were created perpendicular to the lines by 20 μL tips, and unattached cells were removed
by washing with thrice in PBS. Then the calcium AM (1 mM stock solution in DMSO with 1:1500
dilution in PBS) was used to stain cells. After that, cells incubated in the presence or absence of
complex 9 and cisplatin at the indicated concentrations for 24 h. After 24 h incubation, the cells
were washed with ice-cold PBS, and then photographed to mark the final scratched tracks. The
migration rates analyzed by Equation 1: Migration rate (%) = (d1-d2)/d1. The d1 and d2 represented
the width of wound at 0 and 24 h, respectively.
4.9. Mitochondrial membrane potential (MMP) assay.

A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the
RPMI-1640 medium with 10% FBS to the final volume of 2 mL. The plates were also incubated at
an atmosphere of 5% CO2 and 95% air at 37.0 °C for overnight, and incubated in the presence or
absence of complex 9 and cisplatin at the indicated concentrations for 24 h. After 24 h incubation,
cells were then stained with 2 μM 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine (JC-
1) in the dark at 4 °C for another 30 minutes. After that, cells were harvested at 2000 rpm and
washed ice-cold PBS analyzed by flow cytometry.
4.10. Reactive oxygen species (ROS) assay.
A549 cells were grown in each well of six-well plates at the density of 5×104 cells/mL of the
RPMI-1640 medium with 10% FBS to the final volume of 2 mL. The plates were also incubated at
an atmosphere of 5% CO2 and 95% air at 37.0 °C for overnight, and incubated in the presence or
absence of complex 9 and cisplatin at the indicated concentrations for 24 h. After 24 h incubation,
cells were then incubated with DCFH-DA in the dark at 4 °C for another 30 minutes. After that,
cells were harvested at 2000 rpm and washed ice-cold PBS analyzed by flow cytometry.
4.11. Western blot assay.
Western blot analysis was performed as described previously [43, 44]. A549 cells were grown
in each well of six-well plates at the density of 5×104 cells/mL of the RPMI-1640 medium with 10%
FBS to the final volume of 2 mL. The plates were also incubated at an atmosphere of 5% CO2 and
95% air at 37.0 °C for overnight, and incubated in the presence or absence of complex 9 and cisplatin
at the indicated concentrations for 24 h. After for 24 h treatment, cells were collected, centrifuged,
and washed ice-cold PBS. The pellet was then re-suspended in lysis buffer containing 150 mM NaCl,
50 mM Tris (pH 7.4), 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100, 0.1% (w/v) SDS, and
1 mM EDTA (Beyotime, China), and then the lysates were incubated at 37 °C for another 30 minutes,
and centrifuged at 20000g at 4 °C for 10 minutes. The protein concentration in the supernatant was
analyzed by the BCA protein assay reagents. 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, aspase-3 and caspase-9 or GAPDH
for overnight at 4 °C. After that, washed thrice in TBST, and then the membranes were next

incubated with peroxidase labeled secondary antibodies for another 2 h. Finally, all membranes
were washed with TBST four times for 20 minutes and then the protein blots were detected by
chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with
developer and fixed with fixer solution.
4.12. Anti-tumor activity in vivo.
The in vivo cytotoxic activity of complex 9 was further investigated by human lung cells (A549)
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 mL of sterile PBS. Animals were randomly divided into
five 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 group was treated with cisplatin at the dose of 5 mg/kg once
a week for three weeks. No. 3 group was treated with 1a at the dose of 5 mg/kg every two days for
three weeks. No. 4 and No. 5 groups were treated with complex 9 at doses of 5 mg/kg (equal mass
dose of cisplatin) and 13 mg/kg (equal molar dose to cisplatin) once a week for three weeks,
respectively. All tested compounds were dissolved in vehicle. Tumor volume and body weight were
recorded every other day after drug treatment. We collected and weighed the tumors and calculated
the inhibition rate of tumor growth (IRT) at the end of 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).
Notes
The authors declare no competing financial interest.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (Grant Nos. 21977021,
81760626 and 22007036) and the China Postdoctoral Science Foundation (2020M673553XB), and
the Ministry of Education Innovation Team Fund (IRT_16R15, 2016GXNSFGA380005). In
addition, we would like to thank the Natural Science Foundation of Guangxi Province
(AB17292075) and Guangxi Funds for Distinguished Experts. Finally, we are also very grateful to
the Open Project Program of the National & Local Joint Engineering Research Center for Mineral
Salt Deep Utilization (SF201904), and the Major projects of natural science research in Colleges

and universities of Jiangsu Province (18KJA530002), and the Jiangsu Industry-University-Research
Cooperative Program (BY2019169).
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Schemes:

O
O
O
O
O
O
OH
O
OH
O
ii
i
O
O
O
1a
2
1
O
OH
O
O
O
OH
H3N
H3N
Cl
Cl
iii
n
O
+
Target complexes
Pt
R
O
O
O
5
6
, R = OH
, R = Cl
7-10
3
4,
, n = 2
n = 3
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H3N
H3N
Cl
Cl
H3N
H3N
Cl
Cl
O
O
Pt
Cl
Pt
O
O
OH
8
7
9
O
O
O
O
O
O
O
O
O
O
O
O
O
O
H3N
H3N
Cl
Cl
O
O
O
O
H3N
H3N
Cl
Pt
Pt
Cl
OH
Cl
10
Scheme 1. Synthetic pathway to target compounds 7-10. Reagents and conditions: (i) KOH (50%
w/v aqueous solution), CH3OH, 0 °C, overnight; (ii) Succinic anhydride or glutaric anhydride,
K2CO3, DMF, 50 ℃.; (iii) TBTU, Et3N, DMF, 30 ℃.
Table 1. Cytotoxic effects of complexes 7-10 on human cancer and normal cell lines.

Compds.
IC50 (μM) a
HeLa
MGC-803
HepG-2
NCI-H460
HL-7702
SI b
11.4
3
0.780.14
1.350.19
0.810.23
0.970.13
9.241.53
4
14.8
35.9
29.3
42.6
37.4
7.3
0.520.12
0.41±0.09
0.50±0.12
0.32±0.03
0.43±0.09
0.65±0.05
9.41±1.09
1.160.18
0.30±0.07
0.45±0.09
0.26±0.06
0.39±0.10
0.55±0.14
6.56±1.18
0.691.46
0.33±0.08
0.41±0.05
0.29±0.04
0.35±0.07
0.42±0.09
8.01±1.24
0.780.16
0.45±0.12
0.61±0.10
0.37±0.09
0.55±0.13
0.72±0.08
7.85±1.32
10.251.34
11.85±1.37
12.03±1.41
12.35±1.83
13.08±1.56
3.07±0.72
7
8
9
10
1a
Cisplatin
10.36±1.43
1.3
a In vitro cytotoxicity was determined by MTT assay upon incubation of the live cells with the
compounds for 72 h. b Selectivity Index = IC50(HL-7702)/IC50(HepG-2). Mean values based on three
independent experiments.
Table 2. Anti-proliferative activities of optimal complex 9 on the cisplatin-resistant cells A549 and
the doxorubicin-resistant cells MCF-7.
Compds.
IC50 (μM) b
RF c
A549
A549/CDDP
0.23±0.06
0.72±0.14
0.830.11
1.090.17
5.24±0.53
35.06±2.31
MCF-7
MCF-7/DOX
0.42±0.11
1.52±0.54
2.330.39
1.770.22
42.71±2.01
9.05±1.33
RF c
1.1
9
1a
0.19±0.04
0.43±0.08
0.650.13
0.540.14
2.15±0.26
6.43±1.15
1.2
1.7
1.3
2.0
2.4
5.5
0.38±0.09
0.95±0.08
1.760.27
1.150.16
3.03±0.08
6.38±1.02
1.6
4
1.3
4+CDDP
DOX a
CDDP
1.5
14.1
1.4
a Doxorubicin; b In vitro cytotoxicity was determined by MTT assay upon incubation of the live cells
with the compounds for 72 h; c Resistant factor = (IC50 human resistance cells)/(IC50 human cancer
sensitive cells).
Table 3. Cellular uptake of complex 9 in A549 cells.

Compounds
Pt content (ng/106 cells) b
A549
9 (5 μM)
9 (10 μM)
126 ± 15
263 ± 32
72 ± 9
Cisplatin (5 μM)
Cisplatin (10 μM)
117 ± 13
The experiments were performed three times, and the results of the representative experiments are
shown.

OH
H3CO
H3CO
H3CO
N
O
O
O
O
O
N
H
H
OH
H
N
O
N
H
H
NHAc
OH
HO
MeO2C
H
O
O
O
O
O
N
O
OH
O
H3CO
O
O
O
O
H
O
Colchicine
Paclitaxel
Vincristine
H3CO
H3CO
O
O
O
H3CO
H3CO
OH
OCH3
OPO3Na2
OCH3
OCH3
H3CO
OCH3
OCH3
1a
CA-4P
Millepachine
Fig.1. Structures of natural products and chalcone analogues as potent inhibitors of tubulin
polymerization.
OH
H3N
H3N
Cl
Cl
H2
N
O
O
O
O
O
O
Pt
O
O
H3N
H3N
H3N
H3N
Cl
Cl
Pt
Pt
Pt
N
OH
H2
Carboplatin
Oxaliplatin
Oxoplatin
Cisplatin
H
N
N
Cl
Cl
N
N
O
O
O
N
Cl
O
O
O
O
O
O
O
H3N
H3N
Cl
H3N
H3N
Cl
Pt
H3N
H3N
Cl
H3N
Cl
Cl
Pt
Cl
Cl
Pt
Pt
Cl
O
H3N
Cl
OH
O
Cl
Mitaplatin
OH
Cx-platin
OH
NERi-Pt(IV)
Asplatin
Fig. 2. FDA approved platinum(II) anticancer agents and chemical structures of some
platinum(IV) complexes as prodrugs.

Fig. 3. In vivo anticancer effect of complex 9 on A549 xenograft models. Fifteen mice were
randomly divided into five groups, No. 1 group was injected with equal dose of saline was used as
control group, No. 2 group was treated with cisplatin at the dose of 5 mg/kg once a week for three
weeks, No. 3 group was treated with 1a at the dose of 5 mg/kg every two days for three weeks, No.
4 and No. 5 groups were treated with complex 9 at doses of 5 mg/kg and 13 mg/kg once a week for
three weeks, respectively. (A) Tumor images obtained from mice of various groups. (B) The tumor
volume of the mice in each group during the observation period. (C) The body weight of the mice
from each group at the end of the observation period. (D) The weight of the excised tumors of each
group. The data were presented as the mean ± SEM. IR=Inhibitory rate. *P < 0.05

Fig. 4. Effects of complex 9 on the microtubule network of cells. After 24 h co-treatment with
complex 9 (5 and 10 μM), A549 cells were fixed in paraformaldehyde (4 %), stained with α-tubulin
and DAPI, and the images were recorded via laser confocal microscope. Microtubules and
unassembled tubulin are shown in green, and DNA stained in blue. The results represent one of
three independent experiments.

Fig. 5. Apoptosis in A549 cells induced by complex 9. Upon treatment with complex 9 (5, 10 μM)
or cisplatin (10 M) for 24 h. The cells were harvested, labeled with annexin-V-FITC and PI, and
analyzed by flow cytometry. Data are expressed as the mean ± SEM for three independent
experiments.