Dual-target platinum(IV) complexes exhibit antiproliferative activity through DNA damage and induce ER-stress-mediated apoptosis in A549 cells

Article abstract of DOI:10.1016/j.bioorg.2021.104741

Platinum(II)-based chemotherapeutics are commonly used to treat various types of solid tumors, such as lung cancers. However, these compounds can cause serious side effects, including nephrotoxicity and ototoxicity, which affect the quality of life of pat

Full text of DOI:10.1016/j.bioorg.2021.104741

Bioorganic Chemistry 110 (2021) 104741
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Dual-target platinum(IV) complexes exhibit antiproliferative activity
through DNA damage and induce ER-stress-mediated apoptosis in
A549 cells
,
,
,d,
Meng Wanga 1, Zhikun Liuc 1, Xiaochao Huanga *, Yuanhang Chena, Yanming Wanga,
Jing Konga, Yong Yanga, Chunhao Yua, Jin Lia, Xu Wangb *, Hengshan Wangd
,
d
,
,*
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 Guangxi Key Laboratory of Green Processing of Sugar Resources, College of Biological and Chemical Engineering, Guangxi University of Science and Technology,
Liuzhou 545006, China
c Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China
d State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Collaborative Innovation Center For Guangxi Ethnic Medicine, School of
Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Platinum(II)-based chemotherapeutics are commonly used to treat various types of solid tumors, such as lung
cancers. However, these compounds can cause serious side effects, including nephrotoxicity and ototoxicity,
which affect the quality of life of patients. In our work, four novel dual target platinum(IV) complexes were
designed and synthesized. In vitro results indicated that the title platinum(IV) complexes exhibited effective
antitumor activities against the tested cancer cells and had lower toxicity and resistance factors than oxaliplatin
and cisplatin. Further mechanistic experiments demonstrated that complex 11 accumulated in mitochondria and
induced an elevation in ROS and an ER stress response via mitochondrial dysfunction. Notably, complex 11
significantly modulated the expression levels of proapoptosis proteins including cleaved-Caspase-3, Bax, and
p53, and decreased the level of the prosurvival protein Bcl-2. Together, these results suggested that complex 11
might be a potential lead compound for future cancer therapy due to its potency and selectivity.
Platinum(IV) prodrugs
ER stress
Antitumor
Apoptosis
Chalcones
1. Introduction
In contrast to platinum(II) drugs, platinum(IV) scaffolds exhibit oral
availability, antitumor activity, and low toxicity [5–8]. Several
Platinum(II) complexes (Fig. 1), including cisplatin, carboplatinm
and oxaliplatin, have been widely used as chemotherapeutics against
many human solid tumors [1,2]. Despite the great achievements of
platinum(II)-based anticancer drugs, their utilization in the clinic has
been hampered by serious side effects and acquired drug resistance
[3,4]. Therefore, extensive studies aiming to identify new prominent
platinum candidates with enhanced anticancer activity but reduced
toxicity and increased stability have been undertaken to overcome the
inherent shortcomings of platinum(II) complexes.
outstanding complexes, including ormaplatin (also called tetraplatin),
the highly water-soluble iproplatin, and the orally active satraplatin
(JM216) have progressed onto clinical trials [9–12]. Notably, platinum
(IV) complexes have two axial ligands, which provide the possibility of
introducing active molecules with improved anticancer activity or tar-
geting groups (for example, a cyclic RGD tripeptide, chlorotoxin, and
biotin) to enhance functionality [13,14]. For example, in several over-
expressed CLC-3 cancer cells, chlorotoxin (CTX)-functionalized plat-
inum(IV) complexes, which can bind specifically to a chloride channel
protein (CLC-3), can enhance intracellular uptake and subsequently
induce better cytotoxicity than CTX and its platinum(II) complexes [14].
Moreover, platinum(IV) complexes are generally accepted to act as
In recent years, a vast number of studies have indicated that plat-
inum(IV) complexes, which offer great therapeutic advantages, are
promising for the development of the next generation of platinum drugs.
* Corresponding authors at: State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Collaborative Innovation Center For
Guangxi Ethnic Medicine, School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, China.
E-mail addresses: viphuangxc@126.com (X. Huang), wangxu504@126.com (X. Wang), whengshan@163.com (H. Wang).
1
Co-first author: These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104741
Received 13 October 2020; Received in revised form 30 January 2021; Accepted 8 February 2021
Available online 18 February 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
prodrugs owing to reduction by intracellular reductants, such as gluta-
thione or ascorbic acid, and high-molecular reducing agents, including
metallothionein, that are usually present at high concentrations in
tumor cells [15,16]. In addition, platinum(IV) complexes also tend to
possess high stability because of their intrinsic kinetics [7]. Therefore,
the exploration of novel platinum(IV) anticancer complexes, especially
ones with improved therapeutic outcomes, that can overcome the
drawbacks of platinum(II) drugs and possess unique action mechanisms
has become an attractive topic in recent years.
complexes 6 and 7 were obtained through the oxidative reaction of the
oxaliplatin or DACHPt with N-chlorosuccinimide (NCS) in water ac-
cording to a modified literature procedure [27,28]. Moreover, the target
compounds 8–11 obtained by the formation of ester bond between
compound 4 or 5 and 6 or 7 in the presence of TBTU/Et3N in DMF, and
the target platinum(IV) complexes 8–11 were characterized by 1H NMR,
13C NMR spectra and ESI-MS spectroscopy as well as elemental analysis.
2.2. In vitro cytotoxicity of complexes 8–11
Chalcone-based compounds that contain an α,β-unsaturated ketone
moiety have been widely reported to display various biological activ-
ities, especially anticancer activities [17–19]. Many studies have
recently demonstrated that natural or synthetic chalcone derivatives can
act as novel tubulin polymerization inhibitors by inhibiting tubulin
polymerization and combining with the colchicine binding site.
Furthermore, chalcone-based derivatives have considerable potential
antiproliferation effects against many types of solid tumors [20–22]. For
example, the chalcone derivative Millepachine (1a, Fig. 2), a novel
tubulin polymerization inhibitor isolated form Millettia pachycarpa, ex-
hibits good anticancer activities against different human cancer cells
[23]. Accordingly, as reported by Wang and co-workers, most Mil-
lepachine derivatives (such as 1b, Fig. 2) present great potential in
inhibiting tubulin polymerization and antiproliferative activities [24].
Interestingly, Schobert and co-workers reported that chalcone-based
platinum(II) complexes also present potent antiproliferative activities
and inhibit tubulin polymerization [25]. Thus, we imagined that
incorporating a tubulin-targeted chalcone fragment into platinum(IV)
complexes will be an effective strategy for developing novel platinum
(IV) anticancer drugs. Therefore, in the present study, a series of
chalcone-modified platinum(IV) compounds 8–11 were designed and
prepared (Scheme 1). All of the platinum(IV) complexes were charac-
terized via 1H NMR, 13C NMR, and HR-MS analysis. Finally, we inves-
tigated the in vitro antiproliferative activities and action mechanism of
the compounds.
The in vitro cytotoxicity of platinum(IV) complexes 8–11 were
investigated in SK-OV-3 (ovary), HCT-116 (colon), HepG-2 (hepatoma)
and MCF-7 (breast) and normal cells LO2 (Liver) with oxaliplatin,
DACHPt, and compound 3 as reference controls using MTT assay. As
depicted in Table 1, complexes 8–11 showed significant anticancer ac-
tivities against all tested human tumor cells, with IC50 values ranging
from 0.27 to 0.60
μM, which were significantly lower than that of
oxaliplatin (IC50 = 4.21 ~ 12.05
μ
M) and DACHPt (IC50 = 7.23 ~ 13.19
μ
M). Besides, complexes 8–11 also exhibited lower toxicity toward to
normal cells (LO2) as compared with the oxaliplatin and DACHPt,
respectively. Taken the toxicity of the Pt(IV) derivatives in cancer cells
in consideration, complex 11 (IC50 = 0.27 ~ 0.39
μM) exhibited
significantly more potent cytotoxicity against all tested human cancer
cell lines than oxaliplatin (IC50 = 4.21 ~ 12.05 M). Notably, complex
11 (IC50 = 0.27 ± 0.09 M), exhibited up to 37.8-fold and 2.7-fold
increased cytotoxicity against HepG-2 cells than that of oxaliplatin
(IC50 = 10.20 ± 1.02 M) and 3 (0.75 ± 0.12 M), while lower cyto-
μ
μ
μ
μ
toxicity against LO2 cells was found. A similar trend was also observed
in other human cancer cells. Moreover, we also investigated the anti-
proliferative activities of combined group (Oxa + 3, 1:1, n/n) against
these cells mentioned above. As shown in Table 1, the combined group
(Oxa + 3) exerted great potential in anti-proliferative effects against the
cancer cells tested in this manuscript, with IC50 values ranged from 0.41
to 0.83 μM, which was quite the same as that of compound 3. However,
it should be noticed that the combined group (Oxa + 3) also appeared to
be cytotoxic toward human normal cells (LO2) and shared poor tumor
cell selectivity (SI was 1.4). Taken, all these results together, we believe
that the anti-proliferative activity of complex 11 came from both com-
ponents of oxaliplatin and compound 3 rather than each of them. In sum,
these results suggested that the platinum(IV) derivatives of oxaliplatin,
or DACHPt conjugated with 3 derivative ligand in the axial position can
enhance cytotoxic activities together with decrease toxicity toward to
normal cells LO2.
2. Results
2.1. Synthesis and characterization
The synthetic route to obtain title compounds 8–11 were described
in Scheme 1. Chalcone 3 was prepared from 3,4,5-trimethoxyacetophe-
none (1) and isovanillin (2) in one step through Claisen-Schmidt
condensation reaction according to the reported procedure [26]. In
addition, the hydroxyalkyl functionalized chalcone analogues 4 and 5
were obtained by Williamson etherification of the compound 3 with
succinic anhydride or glutaric anhydride in DMF in the presence of
potassium carbonate at 50 ◦C for 2 h. The intermediate platinum(IV)
Fig. 1. FDA approved platinum(II) anticancer agents (etc., cisplatin, carboplatin and oxaliplatin), and platinum(IV) prodrugs in clinical trials (etc., iproplatin,
ormaplatin and JM216).
2

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
Fig. 2. Structures of chalcone analogues as potent inhibitors of tubulin polymerization.
Scheme 1. Synthetic pathway to target compounds 8–11. Reagents and conditions: (a) KOH (50% w/v aqueous solution), CH3OH, 0 ◦C, overnight; (b) Succinic
anhydride or glutaric anhydride, K2CO3, DMF, 50 ◦C.; (c) TBTU, Et3N, DMF, 30 ◦C.
2.3. Anti-proliferative activity of complex 11 against drug-resistant
commercial drug cisplatin (35.05 ± 1.39 and 26.81 ± 1.73 μM),
cancer cells
respectively. Notably, the complex 11 exhibited a lower RF (RF = 1.3
and 1.2) compared to the cisplatin (RF = 7.7 and 7.6, respectively)
against two cisplatin resistant cells A549/CDDP and SGC-7901/CDDP
(Table 2).
It is generally accepted that acquired resistance is a major obstacle to
drug therapy [3,5]. According to the above biological results, the cyto-
toxicity of complex 11 was further investigated in cisplatin-sensitive
(A549 and SGC-7901) and cisplatin-resistant (A549/CDDP and SGC-
7901/CDDP) cancer cell lines with cisplatin, oxaliplatin and DACHPt
as controls using the MTT method. As shown in Table 2, complex 11
showed a more potent cytotoxic effect in A549, SGC-7901 and A549/
CDDP, SGC-7901/CDDP cancer cells than that of cisplatin, and the
cytotoxicity activity of complex 11 was not obviously changed against
cisplatin resistant cells A549/CDDP and SGC-7901/CDDP, with IC50
The great potency in anti-proliferative activities against cisplatin
resistant A549/CDDP cells may raise a question that whether the incu-
bation time affected the intrinsic anti-tumor effects. With this doubt in
mind, we performed in vitro cytotoxicity assays of 24 h, 48 h and
detected the IC50 values against A549 and A549/CDDP cells. As sum-
marized in Table S1, significantly decreased anti-proliferative effects
against A549 or A549/CDDP cells were displayed after 24 h co-
incubation with various tested compound. Compound 11 showed little
cytotoxic effect in both A549 and A549/CDDP cells (IC50 values were
value of 0.31 ± 0.09 and 0.71 ± 0.18
μM as compared with the
3

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
(50
μ
M) as controls to match the peaks during the assays. As illustrated
Table 1
Cytotoxic effects of complexes 8–11 on human cancer and normal cell lines.
in Fig. S1D and S1E, compound existed stable in physiological envi-
ronment of a PBS solution (pH = 7.4) in 72 h. However, upon mixing
with the same volume of ascorbic acid in dark, compound 11 gradually
decomposed, illustrating as the decreased peaks of 11 in HPLC spectra
along with the extension of incubation time (Fig. S1C and S1F). Inter-
estingly, the decomposition of 11 led to another increased peak, which
was right at the position of compound 5, indicating that compound was
reduced to compound 5 and the reaction completed in 7 h. It should be
note that oxaliplatin appeared at the same position as that of ascorbic
acid in HPLC spectra (Fig. S1A and S1B) and only slight increased peaks
occurred at the end of the reduction (5–7 h in Fig. S1F). Taken together,
all the results revealed that complex 11 acted as a prodrug and released
corresponding oxaliplatin and compound 11 under the reduction of
ascorbic acid. Subsequently, we investigated the stability of compound
Compds.
IC50
(
μ
M) d
SK-OV-3
HCT-
116
HepG-2
MCF-7
LO2
SI e
8
0.55 ±
0.08
0.53 ±
0.03
0.39 ±
0.11
0.56 ±
0.13
10.95 ±
1.42
28.1
33.3
31.8
44.7
6.0
9
0.60 ±
0.11
0.32 ±
0.05
0.34 ±
0.05
0.48 ±
0.09
11.31 ±
1.51
10
0.47 ±
0.07
0.45 ±
0.12
0.41 ±
0.14
0.43 ±
0.10
13.05 ±
1.49
11
0.39 ±
0.06
0.29 ±
0.07
0.27 ±
0.09
0.33 ±
0.08
12.07 ±
1.63
3
0.81 ±
0.13
0.87 ±
0.21
0.75 ±
0.12
0.97 ±
0.16
4.52 ±
0.58
Oxa + 3 a
DACHPtb
Oxa c
0.41 ±
0.08
0.54 ±
0.10
0.72 ±
0.09
0.83 ±
0.14
1.03 ±
0.06
1.4
11 (50 μM) in other conditions, including DMF (containing PBS 50%),
9.48 ±
0.71
7.23 ±
0.87
12.07 ±
1.23
13.19 ±
1.71
6.35 ±
1.13
0.5
DMEM and DMEM + FBS and monitored at different time points of 0 h,
6 h, 24 h, 48 h and 72 h. As shown in Fig. S1G-S1I, compound 11
maintained stable in 72 h co-incubation of these solutions indicated
except for negligible decomposition in DMEM + FBS in 72 h (Fig. S1I).
Furthermore, we also investigated the intracellular existence form of
compound 11 via the same HPLC methods. Briefly, A549 cells were
12.05 ±
0.93
4.21 ±
0.65
10.20 ±
1.02
11.26 ±
1.22
8.06 ±
1.05
0.8
a
The combined group of oxaliplatin and 3 (1:1, n/n).
Dichloro(1R,2R-diaminocyclohexane) platinum(II).
Oxaliplatin.
b
c
d
In vitro cytotoxicity was determined by MTT assay upon incubation of the
treated with compound 11 (10 μM) for 24 h and lysate was used for
live cells with the compounds for 72 h.
HPLC analysis. The calibration curve of compound 3 was shown in
Fig. S2A while the HPLC spectra of lysate indicated in Fig. S2B. As shown
in Fig. S2B, we found four target peaks, including oxaliplatin (2.019
min), compound 11 (6.951 min), compound 3 (8.492 min) and com-
e
Selectivity Index = IC50(LO2)/IC50(HepG-2). Mean values based on three
independent experiments.
pound 5 (9.078 min). Moreover, higher concentration of 1.53
μM
Table 2
(calculated by calibration curve of compound 3) were recorded than
negelegible compound 5, indicating that the intracellular compound 11
was reduced and thereafter mainly produced compound 3 (Fig. S2B).
Anti-proliferative activities of optimal complex 11 on the cisplatin-resistant cells
A549 and SGC-7901.
Compds.
IC50
(
μ
M) d
A549
A549/
CDDP
RF
SGC-7901
SGC-7901/
CDDP
RF
2.5. Cell uptake and possible pathway of intracellular uptake
e
e
11
0.23 ±
0.07
0.31 ±
0.09
1.3
1.8
7.7
3.0
2.9
0.57 ±
0.16
0.71 ± 0.18
1.2
1.7
7.6
2.6
2.7
Many studies demonstrated that the cellular uptake is recognized as
an important factor influencing anti-proliferative performance of plat-
inum compounds [29]. Owing to the complex 11 exhibited more potent
cytotoxicity in A549 cells, thus it was used in subsequent experiments
investigating the mechanism of anticancer activity in the human lung
cancer cells A549. Thus, in order to further evaluate the cellular uptake
properties of complex 11, here we used ICP-MS to detect the levels of the
complex 11 in human lung cancer cells A549. As shown in Fig. 3, after
3
0.70 ±
0.10
1.25 ±
0.28
1.21 ±
0.08
2.05 ± 0.72
CDDPa
4.55 ±
0.82
35.05 ±
1.39
3.53 ±
0.08
26.81 ±
1.73
DACHPt
10.43 ±
1.46
31.30 ±
2.31
10.38 ±
1.02
27.05 ±
1.89
b
Oxa c
9.05 ±
1.46
25.83 ±
1.84
9.25 ±
0.88
25.36 ±
1.55
the cells were treated with complex 11 (5 and 10
μM) and oxaliplatin (5
a
Cisplatin.
and 10 M) for 12 h, the platinum(IV) complex 11 accumulate at 1.9 ~
μ
b
c
d
Dichloro(1R,2R-diaminocyclohexane)platinum(II).
Oxaliplatin.
2.4 times higher levels in whole cells than that of the positive drug
oxaliplatin at the indicate concentrations, indicating platinum(IV)
complex 11 exhibited higher platinum accumulation in A549 cells than
that of the reference drug oxaliplatin, which is probably attributed to the
high lipophilicity of complex 11. Generally, small molecules enter cells
by passive diffusion whereas macromolecules through energy-
dependent endocytosis [29]. Therefore, in order to investigate the
whether the internalization of 11 was energy-dependent, the cells were
pre-treated with sodium azide (10 mM) (which depletes intracellular
ATP) for 4 h followed with another 12 h co-incubation under the same
condition [29]. As shown in Fig. 3, in the presence of sodium azide,
In vitro cytotoxicity was determined by MTT assay upon incubation of the
live cells with the compounds for 72 h.
e
Resistant factor = (IC50 human resistance cells)/(IC50 human cancer sensi-
tive cells).
5.21 and 6.08
7.03 and 9.12
μ
M), which were almost the same as that of 3 (IC50 were
μ
M, respectively). However, when the incubation time
was extended to 48 h, sharply degrease in IC50 values were found,
especially for 11 in A549 or A549/CDDP cells. Nevertheless, the anti-
proliferative effects of 3, 11 or both against A549 and A549/CDDP
cells were weaker than that of 72 h (Table S1).
intracellular uptake of complex 11 (5 μM) changed little while that of 11
(10
μ
M)-treated group was 290 ng/106 cells, which was significantly
lower than 363 ng/106 cells of untreated group. Interestingly, with 4 h
pretreation of sodium azide, the amount of intracellular oxaliplatin was
significantly decreased as compared with untreated group. Taken all
these results, we believed that 11 entered cells mainly by passive
diffusion at lower concentration, but which was significantly affected in
an energy-dependent way at high concentration.
2.4. The release of compound 5 and stability of compound 11 in various
conditions
The stability of compound 11 in various conditions was monitored
via reversed phase high performance liquid chromatography (HPLC).
Briefly, solutions of 11 (50 μM) in PBS (150 mM) and acetonitrile (10
mL, 9.5:0.5, V/V) and ascorbic acid (500
μ
M) in PBS (pH = 7.4, 150 mM)
were prepared, using solutions of oxaliplatin (50
μ
M) and compound 5
4

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
Fig. 3. Cellular uptake of the platinum(IV) complex 11 (5 and 10
μM) and oxaliplatin (5 and 10
μ
M) in A549 cells in the absence or presence of sodium azide (10
mM). The results were obtained and analyzed with ICP-MS and showed as one of three independent experiments.
2.6. Analysis of immunofluorescence staining
especially for mitosis. Thus, it was of great significance to disturb the
microtubule dynamics in cancer cells to block mitosis or induce
apoptosis [21]. Besides, our previous reports explained that a structure
Microtubules played a crucial role in a variety of cellular process,
Fig. 4. Effects of 11 on microtubles network. A549 cells were untreated (A1-C1), incubated with 3 (A2-C2,10 μM), incubated with 11 (A3-C3, 5 μM), incubated with
11 (A4-C4, 10
μ
M) for 24 h, fixed and visionlized by treatment
α
-tubulin monoantibody and IgG FITC conjugated antibody. DAPI was used as track the cells. The
results were shown as one of three independent experiments.
5

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
of chalcone obtained inhibitory activities on microtubules polymeriza-
tion [30]. Therefore, for the purpose to confirm the mechanism of the
action of target complex 11, immunofluorescence assays on microtu-
bules polymerization were performed. As shown in Fig. 4A1-C1, the
microtubule structures in A549 cells of untreated-group were slim and
fibrous, appearing from nuclear region to the plasma membrane. How-
ever, when treated with 11, the tubulin network was altered and con-
but was obviously different from that of oxaliplatin-treated cells. In sum,
complex 11 induced A549 cells G2/M pahse arrestment in a dose-
dependent manner which contributed to apoptosis and anti-
proliferation in cancers.
2.8. Complex 11 induced apoptosis in A549 cells
densated around the nucleus. Moreover, in the presence of 11 (10 μM)
The perfect anti-proliferation activity against A549 cells in vitro
motivated us to investigate whether the inhibitory effects of 11 on A549
cells were due to the enhanced apoptosis. Therefore, A549 cells were
seeded into 6-well plates and incubated with various compounds of
the spindle microtubules turned aberrant multipolar or irregular or
complete absent (Fig. 4A4-C4), which illustrated that complex 11 effi-
ciently inhibited microtubules polymerization and thereafter block
mitosis and finally induced apoptosis. Not surprisingly, compound 3, a
ligand of 11, shared the same inhibitory effects on microtubules poly-
merization (Fig. 4A3-C3).
oxaliplatin (10
μ
M), 3 (5
μ
M), combined group (oxaliplatin, 10
μM, 3, 5
μ
M) and 11 (2.5 and 5
μM), respectively. Similarly, cells seeded in
complete culture medium and were treated with DMF as controls. After
24 h co-incubation, the cells were collected and double-stained with
Annexin V-FITC and propidium iodide (PI) and subjected to flow
2.7. Cell cycle analysis
cytometry assays. The results in Fig. 6 showed that oxaliplatin (10 μM)
or compound 3 (5
cells. However, when co-treatment with 3 (5
M) the percentage of A549 cells in apoptosis increased to 39.43%,
μM) induced apoptosis in 13.95% or 16.72% in A549
The anti-proliferation tests and apoptosis tests showed that 11 ob-
tained the best potential in inducing apoptosis in A549 cells. Therefore,
to investigate the effects on cycle, A549 cells were co-incubated with 11
μ
M) and oxaliplatin (10
μ
which revealed that the combined usage of 3 and oxaliplatin more easily
contribute to apoptosis than one of 3 and oxaliplatin. More importantly,
when A549 cells were exposed to 11 for 24 h, the cells in apoptotic state
increased from 31.95% to 42.87% in a dose-dependent manner. Alto-
gether, the results in apoptosis data illustrated that 11 was more po-
tential in inducing apoptosis in A549 cells than 3 or 11 or both at the
same concentration.
(2.5 and 5
μ
M) for 24 h, fixed with ice-cold ethanol at ꢀ 20 ◦C overnight,
stained with PI and subjected to flow cytometry assays. Similarly,
vechicle (DMF), oxaliplatin (10
μM), 3 (5 μM), combined group (oxali-
platin, 10 M, 3, 5 μM) were used as controls. As revealed in Fig. 5,
μ
oxaliplatin-treated A549 cells were mainly arrested at S phase as com-
prison with untreated cells (S pahses, 36.24%), while A549 cells treated
with compound 3 were mainly arrested at G2/M pahse (G2/M phase,
23.45%). Moreover, cells incubated with 11 (2.5 and 5 μM) for 24 h
were mainly arrested at G2/M pahse in a dose-dependent manner with
the percentage increased from 28.30% to 58.09% and could not escape
this pahse (Fig. 5). Intrestingly, when A549 cells were treated with
2.9. Mitochondrial membrane potential (MMP) assays
To further investigate whether apoptosis in A549 cells induced by 11
was executed by the collapse of mitochondrial membrane potential
(MMP). Fluorescent mitochondrial dye, 5,5,6,6′-tetrachloro-1,1,3,3-
combined group (oxaliplatin, 10 μM, 3, 5 μM), 50.44% of cells were
arrested at G2/M phase, which was not distinctive to that of 11-treate
Fig. 5. Cell cycle arrest assays induced by 11. A549 cells were incubated with vechicle (DMF), oxaliplatin (10 μM), 3 (5 μM), combined group (oxalipaltin, 10 μM, 3,
5
μ
M), 11 (2.5 and 5
μ
M) for 24 h, collected and fixed with ice-cold ethanol at ꢀ 20 ◦C overnight. The cells were stained with PI, collected and subjected to flow
cytometry. The results were shown as one of three independent experiments.
6

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
Fig. 6. Apoptosis assays induced by 11. A549 cells were co-incubated with control (DMF), oxaliplatin (10 μM), 3 (2.5 μM), combined group (oxalipaltin, 10 μM, 3,
2.5 μM), 11 (2.5 and 5 μM) for 24 h, collected and stained with Annexin-V and PI. Q1, Q2, Q3, and Q4 represent four different cell states: necrotic cells, late apoptotic
cells, living cells and apoptotic cells, respectively. The results were obtained and analyzed with flow cytometry and showed as one of three independent experiments.
Fig. 7. Complex 11 induced loss of mitochondrial membrane potential in A549 cells. A549 cells were incubated with various compounds of vechicle (DMF), 3 (5
μ
M), oxaliplatin (10 μM), combined group (oxalipaltin, 10 μM, 3, 5 μM) and 11 (5 μM) for 24 h. After that, the cells harvested for JC-1 staining and anylyzed via flow
cytometry. The results were presented as one of three indipendent experiments.
7

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
tetraethyl-imidacarbocyanine (JC-1) was used to detect the transition of
MMP. Generally, A549 cells were incubated with pretreated with vehicle
induced moderrate intracellular ROS accumulation in A549 cells.
However, a large number of A549 cells were in ROS-overexpression
(DMF), oxaliplatin (10
μ
M), 3 (5
μM), combined group (3, 5
μM, oxa-
status when treated with combined compounds of 3 (5 μM) and 11
liplatin, 10
μM), and 11 (2.5 and 5
μM) for 24 h, washed with cold PBS,
(10 μM) (Fig. S4 A4-C4) which illustrated that 3 and 11 in some degree
and collected for JC-1 staining. Generally, in healthy cells, the dye easily
entered in mitochondria and aggregated in red fluorescence, while the
MMP collapsed in apoptosis cells, the dye cannot accumulate in mito-
chondria and dispersed into cytoplasm in green fluorescence. In Fig. S3
A1-C1, the mitochondria in healthy A549 cells of control group were
stained in red florescence while the mitochondria in 3-treated or
oxaliplatin-treated were shown in more green florescence than red
florescence (Fig. S3 A2-D3), which clearly explained that 3 and oxali-
platin decrease MMP in A549 cells. It was worth noting that the com-
co-effected the accumulation of ROS in A549 cells. More intrestingly,
when treatment with 11 significantly and rapidly increased ROS levels
were induced in almost all of A549 cells (Fig. S4 A5-C6). It was worth
noting that 11 in low concentration (2.5
μM) was more preferably
induced ROS accumulation than that of 3 or oxaliplatin or both (Fig. S4).
Besides we also detected the ROS accumulation in A549 cells after 24 h
incubation with various compouns indicated above. As illustrated in
Fig. 8, moderate ROS accumulation was induced in 3 (5 μM) treated or
oxaliplatin (10
11 (5 M) for 24 h intracellular ROS level was significantly elevated,
which was more than two times of that in the combined group (oxali-
μM) treated group. However, after exposure to complex
bined group of 3 (5
μ
M) and oxaliplatin (10
μ
M) can only partly induced
μ
MMP collapse with red florescence remaining in A549 cells. Interest-
ingly, 11 induced concentration-dependent collapse of mitochondrial
membrane potential and seem to disturb all of the mitochondrial tran-
platin + 3, 10 + 5 μM). Taken all these results together, we believed that
complex was an efficient ROS inducer for intracellulsr ROS accumulaion
in cancer cells.
sition in A549 cells in concentration of 10 μM, leading to the randomly
distribution of green JC-1 monomer in cytoplasm. Herein, we performed
quantitative analysis via flow cytometry instrument to investigate the
changes induced by these compounds indicated above. As shown in
Fig. 7, 32.8 percent of cells were in the status of MMP collapse in 3 (5
Moreover, many studies demostrted that most of metal anticancer
drugs could be ability to affect endoplasmic reticulum (ER) stress, which
is an important factor in drug efficacy [33,34]. On the other hand, the
simultaneous presence of promoted the accumulation of intracellular
ROS and resulted ER stress can effectively induce the death of the tumor
cells. In order to further investigate if complex 11 induce ER stress, the
expression of three crucial ER stress markers, such as CHOP, p-eIF2a and
p-PERK was explored using western blotting assay. As depicted in
Fig. 9A and B, it was observed that complex 11 significantly promoted
the accumulation of these three proteins including CHOP, p-eIF2a and p-
PERK, indicating that the complex 11 may promote intracellular ROS
production and induce an ER stress response ultimately leading to the
death of A549 cells.
μ
M) treated group whereas that of oxaliplatin (10
μ
M) treated group was
M),
the population rose to 55.5% which was slightly better than 50.7% of
increased to 45.8%. However, after 24 h co-treatment with 11 (5
μ
combined group (oxaliplatin + 3, 10 + 5 μM). In sum, the sharply
decreased red fluorescence intensity induced by 11 clearly suggested
that 11 disturbed the function of mitochondria in A549 cells and
thereafter induced cytotoxicity in A549 cells.
2.10. Complex 11 triggered reactive oxygen species (ROS) generation
Previously, platinum(IV) complexes were reported to disturb redox
balance in cancer cells, leading to the accumulation of intracellular ROS
and apoptosis [31-33]. To investigate whether the inhibitory effeets of
11 on A549 cells cellular proliferation, intracellular ROS acuumulation
was detected. Briefly, A549 cells were indipendently treated in triplate
2.11. Complex 11-induced apoptotic pathways
The Bcl-2 family proteins, such as pro-apoptosis protein Bax and pro-
survival protein Bcl-2, are critical regulators of cell apoptosis [35,36],
therefore we further investigated the apoptosis-associated protein al-
terations, the expression of Bax and Bcl-2 were analyzed. As shown in
Fig. 9A, after treatment with oxaliplatin and complex 11, the level of
pro-apoptotic protein Bax was significantly up-regulated, and the anti-
apoptotic protein Bcl-2 was down-regulated significantly. Increasing
evidence has indicated that the p53 protein, as a crucial molecule in
DNA damage repair and a cancer suppressor protein, which is involved
with various compounds of 3 (5
μ
M), oxaliplatin (10 M), combined
μ
group (3, 5 M, oxaliplatin, 10
μ
μM), and 11 (2.5 and 5 μM). After 24 h-
coincubation with these compounds mentioned above, the cells were
washed with cold PBS, collected and stained with DCFH-DA (Beyotime)
to record and analyze the laser confocal microscope. As shown in Fig. S4
A2-C3, treatment with compcound 3 (5 μM) or oxaliplatin (10 μM) only
Fig. 8. Intracellular ROS accumulation assays induced by 11. A549 cells were incubated with various compounds of vechicle (DMF), 3 (5 μM), oxaliplatin (10 μM),
combined group (oxalipaltin, 10 μM, 3, 5 μM) and 11 (5 μM) for 24 h. After that, the cells harvested for DCFH-DA staining and anylyzed via flow cytometry. The
results were presented as one of three indipendent experiments. (Excitation wavelength, 505 nm; Emission wavelength, 535 nm).
8

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
Fig. 9. The effects of complex 11 on the apoptosis-related protein. A549 cells of 24 h incubation with complex 11 (5 μM) or oxaliplatin (10 μM) were collected for
western blotting assays. (A) Representative western blots in complex 11 treated A549 cells. (B) Percentage expression levels of CHOP, phosphor-eIF2a (p-eIF2a),
phosphor-PERK (p-PERK), Bax, Bcl-2, cleaved-Caspase-3 and p53. The GAPDH was used as internal control and the percentage values are normalized with GAPDH.
in a multitude of biological processes (such as cell cycle arrest, apoptosis
and autophagy, respectively) [37]. As depicted in Fig. 9A, treatment
concentrations. Interestingly, as shown in Fig. S1I, complex 11 main-
tained its stability in the physiological environment (pH = 7.4, DMEM,
DMEM + FBS) but was efficiently reduced into oxaliplatin and com-
pound 5 by ascorbic acid (Fig. S2B). Therefore, this complex could serve
as a multifunctional antineoplastic agent to combat cancer cells, espe-
cially cisplatin-resistant cancer cells.
with complex 11 (5 μM) resulted in higher expression of the p53 protein,
a marker of DNA damage, than that of combined group (oxaliplatin + 3,
10 + 5 M) though similar anti-proliferative activities exerted in vitro.
μ
Moreover, it is well known that the cleaved-Caspase-3 protein has been
considered as an important effector of tumor cell apoptosis, and iden-
tified to be activated in response to chemotherapy drugs [38,39],
Therefore, we also investigated the expression level of cleaved-Caspase-
3 by means of western blotting assay. As depicted in Fig. 9A, the
expression level of cleaved-Caspase-3 protein was significantly up-
regulated by complex 11, oxaliplatin and combined group (Oxa + 3)
at the indicated concentrations compared to the untreated control cells
(Fig. 9B), whereas the expression of cleaved-Caspase-3 protein was
markedly up-regulated in contrast to that of the positive drug
oxaliplatin.
Additionally, as shown in Figs. 3 and 4, complex 11 could signifi-
cantly improve the level of cellular uptake and efficiently inhibit
microtubule polymerization. Traditionally, the antineoplastic mecha-
nisms of platinum drugs have been proven to be the induction of DNA
damage and the subsequent induction of apoptosis in cancer cells. As
shown in Figs. 5 and 6, complex 11 significantly arrested the cell cycle at
the G2/M phases and induced cell apoptosis. In addition, complex 11
enhanced the expression levels of proapoptosis proteins (such as Bax,
cleaved-Caspase-3, and p53) and simultaneously suppressed the levels
of the prosurvival protein Bcl-2 (Fig. 9). Previous studies have shown
that the mitochondria play a major role in regulating cellular functions,
and a decrement in MMP has been implicated as an early event in
apoptotic cells [34]. Moreover, the death-inducing capacity of anti-
tumor agents has been related to ROS production. As shown in Figs. 7
and 8, complex 11 significantly induced MMP collapse, mitochondrial
dysfunction, and intracellular ROS accumulation. Additionally, the
capability of chemotherapeutic drugs to affect ER stress is an important
element in drug efficacy. Through further studies, we found that the
expression levels of proteins, including CHOP, p-eIF2a, and p-PERK, had
all increased (Fig. 9) after the incubation of A549 cells with complex 11.
This result indicated ER stress. Taken together, these results indicated
that complex 11 disrupted mitochondrial membrane potential balance,
induced intracellular ROS accumulation, and finally activated
mitochondrion-dependent and ER-stress-mediated apoptosis pathways.
These obvious differences might originate from the introduction of a
chalcone derivative, which caused complex 11 inherit the advantages of
chalcone and oxaliplatin to efficiently inhibit the proliferation of cancer
cells at multiple levels of pathological mechanisms. All the results
indicated that this novel series of dual-target platinum(IV) complexes
showed low toxicities and enhanced the antineoplastic effects of oxali-
platin, especially its effects against cisplatin-resistant cancer cells.
3. Discussion
Cisplatin and its analogues, such as carboplatin and oxaliplatin, are
widely used as first-line treatments in many therapeutic schemes [40].
However, their further improvement is urgently needed owing to their
innate or acquired drug resistance and serious side effects. Currently,
drug combination strategies based on multiple drugs with different
mechanisms of action are regarded as effective approaches for over-
coming the limitations of single-drug chemotherapy [41]. Given that
platinum(IV) complexes are promising tools for designing multi-
modulatory drugs, we constructed four novel platinum(IV) prodrugs
(8–11) based on tubulin polymerization inhibitor agents (chalcone de-
rivatives) and oxaliplatin or DACHPt. As shown in Tables 1 and 2,
platinum(IV) complexes 8–11 not only exhibited great potential in
inhibiting the proliferation of cisplatin-sensitive (SK-OV-3, HCT-116,
HepG-2, and MCF-7) and cisplatin-resistant (A549) cells but showed
improved selectivity indexes for human normal cells (LO2). Notably, the
target complex 11 also reversed cisplatin resistance in A549 cells,
whereas the combined group (Oxa + 3) showed potential cytotoxicity
against normal human cells (LO2) with the SI value of 1.4. In contrast to
the traditional multidrug combination used to combat resistance, com-
plex 11 was a single compound that contained two drugs with the same
pharmacokinetics that are easy to control. Furthermore, unlike oxipla-
tin, 11 entered cells mainly via passive diffusion at low concentrations
but was significantly affected in an energy-dependent manner at high
4. Conclusions
Cisplatin and its analogues carboplatin and oxaliplatin are
commonly used in chemotherapy for various human solid tumors, such
9

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
as lung cancers. However, high doses of these platinum(II)-based anti-
cancer agents cause serious side effects, such as nephrotoxicity and
ototoxicity, that affect the quality of life of patients. Here, we designed
and synthesized four novel dual-target chalcone platinum(IV) com-
plexes. We also explored their capability to be taken up by cancer cells
and their antiproliferative activity in detail. The results of in vitro ex-
periments demonstrated that the title complexes 8–11 showed more
potent antiproliferative activity than platinum(II) drugs against the
tested human cancer cell lines, including cisplatin-resistant cells (A549/
CDDP). Among them, complex 11 displayed great anticancer activity
against the tested cancer cells and reduced toxicity to normal LO2 liver
cells. Moreover, complex 11 significantly induced G2/M stage arrest and
DNA damage, inhibited microtubule polymerization, and triggered
apoptosis in A549 cells. Notably, mechanistic experiment results indi-
cated that complex 11 could cause mitochondrial dysfunction, generate
significant levels of ROS, and further induce ER stress. Thus, these re-
sults indicated that complex 11 could induce cell death mainly through
mitochondrial dysfunction and a ROS-mediated ER stress pathway.
Interestingly, complex 11 had no cross-resistance with cisplatin owing
to its dual target mechanisms of action.
:479.1109; found: 479.0948.
(E)-5-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)
phenoxy)-5-oxopentanoic acid (5). 3.2 g, 69.9% yield as a yellow solid. 1H
NMR (400 MHz, DMSO‑d6) δ 12.18 (s, 1H), 7.86 – 7.81 (m, 2H), 7.74 –
7.69 (m, 2H), 7.42 (s, 2H), 7.19 (d, J = 8.6 Hz, 1H), 3.90 (s, 6H), 3.84 (s,
3H), 3.76 (s, 3H), 2.65 (t, J = 7.3 Hz, 2H), 2.39 (t, J = 7.3 Hz, 2H), 1.91 –
1.84 (m, 2H). HR-MS (m/z) (ESI): calcd for C24H26O9 [M + Cl]ꢀ
:493.1265; found:493.1058.
5.1.3. General procedures for preparing complex 6 and 7
To a solution of oxaliplatin (1.0 g 2.56 mmol) or DACHPt (1.0 g, 2.63
mmol) in water (30 mL) was dropwise added N-chlorosuccinimide
(NCS) (534.1 mg, 4.0 mmol) in 10 mL water at room temperature. After
that, the reaction solution was heated to 50 ◦C 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.
Complex 6. 974.8 mg. Yield 86.0%. Pale yellow solid. 1H NMR (600
MHz, DMSO‑d6) δ 7.57–7.50 (m, 1H), 7.33–7.32 (m, 1H), 6.93 – 6.85 (m,
1H), 6.82–6.76 (m, 1H), 2.73–2.61 (m, 2H), 2.07– 2.02 (m, 2H),
1.50–1.47 (m, 4H), 1.39 (s, 1H), 1.05 – 0.96 (m, 2H).
5. Experimental section
All chemicals and solvents were analytical reagent grade and
commercially available, and the column chromatography was per-
formed using silica gel (200–300 mesh). The elemental analyses of C, H,
and N used a Vario MICRO CHNOS elemental analyzer (Elementary),
and 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.
Complex 7. 1.04 g. Yield 90.0%. Pale yellow solid. 1H NMR (600
MHz, DMSO‑d6) δ 7.94–7.93 (m, 1H), 7.72–7.71 (m, 1H), 7.21 – 7.12 (m,
1H), 7.05 – 6.98 (m, 1H), 2.97 (s, 1H), 2.56–2.50 (m, 2H), 2.03 (d, J =
12.5 Hz, 1H), 1.97 (d, J = 12.1 Hz, 1H), 1.55 – 1.40 (m, 4H), 1.11 – 1.03
(m, 2H).
5.1.4. General procedure for preparing compounds 8–11
To a solution of 3 (0.35 mmol) or 4 (0.35 mmol) in anhydrous DMF
(3 mL), and then TBTU (0.525 mmol), Et3N (0.525 mmol) and complex 6
or 7 (0.35 mmol) was added in reaction, respectively and the resulting
reaction was stirred at 30 ◦C for overnight. After confirming completion
of the reaction, added to CH2Cl2 (150 mL) in solution, and then
extracted twice with water (150 mL), and the organic phase was dried
over anhydrous Na2SO4. The residue was purified on silica gel column
eluted CH3OH/CH2Cl2 to obtain the desired products 8–11.
5.1. General procedure for the preparation of compounds
5.1.1. (E)-3-(3-hydroxy-4-methoxyphenyl)-1-(3,4,5-trimethoxyphenyl)
prop-2-en-1-one (3)
Compounds 1 (15.0 mmol) and 2 (15.0 mmol) was dissolved in
methanol (30 mL) and cooled to ice-water, and then and the 50% KOH
(10 mL, aq) added to dropwise to the solution, respectively, and the
solution was stirred at the same temperature for overnight. After
completion of reaction, adjusted to pH = 1 ~ 2 with 4 N HCl, and
washed with CH2Cl2. The organic phase was dried with anhydrous
Na2SO4, and the resulting crude product was purified by silica gel col-
umn chromatography with CH3OH/CH2Cl2 to obtain the final product 3
(3.4 g, 65.9%) as a yield solid.1H NMR (600 MHz, DMSO‑d6) δ 9.16 (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), 7.30 (dd, J = 8.4, 2.0 Hz, 1H), 7.00 (d, J = 8.4
Hz, 1H), 3.90 (s, 6H), 3.84 (s, 3H), 3.76 (s, 3H). HR-MS (m/z) (ESI):
calcd for C19H20O6 [Mꢀ H]ꢀ : 343.1182; found: 343.1064.
(OC-6–33)-trichlorido(cyclohexane-1R,2R-diamine)((E)-4-(2-
methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenoxy)-4-
oxobutanoicate)-platinum(IV)(8). 167 mg, 58.9% yield as a yellow solid.
1H NMR (600 MHz, DMSO‑d6) δ 9.49 – 9.46 (m, 1H), 8.10 – 7.98 (m,
1H), 7.82 (d, J = 15.4 Hz, 2H), 7.76 (d, J = 7.8 Hz, 2H), 7.69 (d, J = 15.4
Hz, 1H), 7.42 – 7.34 (m, 3H), 7.20 (d, J = 8.3 Hz, 1H), 2.86 – 2.78 (m,
2H), 2.66 – 2.56 (m, 3H), 2.14 (d, J = 10.0 Hz, 1H), 1.97 (d, J = 10.2 Hz,
1H), 1.49 – 1.17 (m, 5H), 1.01 – 0.77 (m, 2H). 13C NMR (150 MHz,
DMSO‑d6) δ 188.13, 181.60, 171.16, 153.39, 143.58, 142.41, 140.05,
133.54, 129.74, 128.13, 123.02, 120.69, 113.33, 106.62, 63.46, 62.54,
60.67, 56.80, 56.63, 32.30, 31.28, 31.24, 29.95, 24.00, 23.67. HR-MS
(m/z) (ESI): calcd for C29H37Cl3N2O9Pt [M + H]+:859.1269; found:
859.0989. Elemental analysis calcd (%) for C29H37Cl3N2O9Pt: C, 40.55;
H, 4.34; N, 3.26; found: C, 40.78; H, 4.49; N, 3.01.
5.1.2. General procedure for preparing compounds 4 and 5
Synthesis of compounds 4 and 5. The compound 3 (10.0 mmol) was
dissolved in anhydrous DMF (15 mL), and then anhydrous K2CO3 (20.0
mmol) and succinic anhydride (50.0 mmol) or glutaric anhydride (50.0
mmol) was added to the solution, respectively, and the mixture was
stirred at 50 ◦C for 2 h and monitored by TLC. After confirming
completion of the reaction, the solution was adjusted to pH = 2 ~ 3 with
4 N HCl, The resulting mixture was extracted with CH2Cl2 (3 × 100 mL),
and the organic phase was dried with anhydrous Na2SO4. The resulting
crude product was purified by silica gel column chromatography with
CH3OH/CH2Cl2 to obtain the final product 4 or 5.
(OC-6–34)-chlorido(dicarboxylate)(cyclohexane-1R,2R-diamine)((E)-
4-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phe-
noxy)-4-oxobutanoicate)-platinum(IV)(9). 182 mg, 59.5% yield as a yel-
low solid.1H NMR (600 MHz, DMSO‑d6) δ 8.40 – 8.23 (m, 3H), 7.93 (d, J
= 32.4 Hz, 1H), 7.79 – 7.66 (m, 4H), 7.44 (s, 2H), 7.19 (d, J = 6.7 Hz,
1H), 3.88 (s, 6H), 3.82 (s, 3H), 3.76 (s, 3H), 2.81 (d, J = 18.9 Hz, 2H),
2.68 – 2.55 (m, 3H), 2.08 (d, J = 6.4 Hz, 1H), 1.97 (d, J = 8.8 Hz, 1H),
1.50 – 1.23 (m, 5H), 1.09 – 0.92 (m, 2H). 13C NMR (150 MHz, DMSO‑d6)
δ 188.15, 179.56, 171.11, 163.84, 163.76, 153.38, 153.31, 143.65,
142.38, 140.09, 133.58, 130.21, 128.24, 122.67, 120.77, 113.19,
106.60, 61.86, 61.66, 60.66, 56.78, 56.60, 31.69, 31.32, 31.00,
29.81,23.96, 23.79. HR-MS (m/z) (ESI): calcd for C31H37ClN2O13Pt
[M+Na]+: 899.1524; found: 899.1266. Elemental analysis calcd (%)
(E)-4-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)
phenoxy)-4-oxobutanoic acid (4). 2.8 g, 63.1% yield as a yellow solid. 1H
NMR (600 MHz, DMSO‑d6) δ 9.16 (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 – 2.86 (m, 4H). HR-MS (m/z) (ESI): calcd for C23H24O9 [M + Cl]ꢀ
C31H37ClN2O13Pt for: C, 41.41; H, 4.15; N, 3.12; found: C, 41.68; H,
10

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
4.36; N, 2.91.
with ascorbic acid (10 equiv.) the time-independent release with
(OC-6–33)-trichlorido(cyclohexane-1R,2R-diamine)((E)-5-(2-
recorded every 1 h. Besides, a solution of complex 11 (50 μM) in DMF
methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phenoxy)-5-
oxopentanoicate)-platinum(IV)(10). 175 mg, 57.6% yield as a yellow
solid. 1H NMR (400 MHz, DMSO‑d6) δ 9.60 – 9.51 (m, 1H), 8.23 – 8.07
(m, 1H), 7.86 – 7.81 (m, 3H), 7.75– 7.69 (t, J = 12.4 Hz, 2H), 7.56 – 7.47
(m, 1H), 7.42 (s, 2H), 7.20 (d, J = 8.4 Hz, 1H), 3.90 (s, 6H), 3.84 (s, 3H),
3.76 (s, 3H), 2.76 – 2.65 (m, 3H), 2.47 – 2.37 (m, 2H), 2.19 (d, J = 10.0
Hz, 1H), 2.06 (d, J = 10.7 Hz, 1H), 1.90 – 1.86 (m, 2H), 1.56 – 1.40 (m,
3H), 1.31 – 1.02 (m, 4H). 13C NMR (100 MHz, DMSO‑d6) δ 188.14,
182.94, 171.42, 153.38, 143.58, 142.40, 140.15, 133.58, 129.90,
128.21, 122.74, 120.74, 113.30, 106.65, 63.90, 62.72, 60.67, 56.76,
56.65, 36.66, 32.88, 31.41, 31.33, 24.08, 24.03, 21.46. HR-MS (m/z)
(ESI): calcd for C30H39Cl3N2O9Pt [M+Na]+:895.1245; found:895.0971.
Elemental analysis calcd (%) for C30H39Cl3N2O9Pt: C, 40.21; H, 4.39; N,
3.13; found: C, 40.47; H, 4.62; N, 2.73.
(containing 50% PBS,pH = 7.4), DMEM and DMEM + FBS were pre-
pared and monitored at different time points of 0 h, 6 h, 24 h, 48 h and
72 h. All solutions were stored in dark at 37 ◦C. As for intracellular
analysis, A549 cells were incubated with complex 11 (10 μM) for 24 h.
After that, the cells were harvested, lysed and lysate was pre-filtered
with microporous filter membrane (0.22 m) for HPLC ananlysis.
μ
HPLC conditions: flow rate, 1.0 mL/min; detection wavelength, 254 nm;
ODS column (250 × 4.6 mm, 5 μm), volume of sample injection, 10.0 µL;
column incubator, 35 ◦C.
5.4. Cell uptake
A549 cells were seeded into 6-well plates at a density of 1 × 106
cells/mL and allowed to adhere overnight until 80% confluence. After
(OC-6–34)-chlorido(dicarboxylate)(cyclohexane-1R,2R-diamine)((E)-
5-(2-methoxy-5-(3-oxo-3-(3,4,5-trimethoxyphenyl)prop-1-en-1-yl)phe-
noxy)-5-oxopentanoicate)-platinum(IV)(11). 183 mg, 58.8% yield as a
yellow solid. 1H NMR (400 MHz, DMSO‑d6) δ 8.44 – 8.28 (m, 3H), 7.88 –
7.83 (m, 2H), 7.73 – 7.69 (m, 3H), 7.42 (s, 2H), 7.20 (d, J = 8.6 Hz, 1H),
3.89 (s, 6H), 3.82 (s, 3H), 3.76 (s, 3H), 2.63 – 2.59 (m, 3H), 2.44 (t, J =
7.1 Hz, 2H), 2.11 (d, J = 10.8 Hz, 1H), 2.03 (d, J = 11.4 Hz, 1H), 1.88 –
1.82 (m, 2H), 1.57 – 1.33 (m, 4H), 1.25 – 1.06 (m, 3H). 13C NMR (100
MHz, DMSO‑d6) δ 188.11, 180.56, 171.27, 163.75, 153.38, 153.33,
143.60, 142.38, 140.11, 133.57, 130.06, 128.22, 122.65, 120.69,
113.25, 106.60, 62.00, 61.88, 60.66, 56.75, 56.57, 35.78, 32.78, 31.38,
31.05, 24.00, 24.91, 21.23. HR-MS (m/z) (ESI): calcd for
24 h incubation, the cells were treated with oxaliplatin (10 μM) and 11
(5.0 and 10 μM) for another 12 h in the absence or presence of sodium
azide (10 mM). After that, the cells were collected, and washed twice
with ice PBS, and centrifuged at 1000 g, and resuspended in PBS (2 mL).
So, the cell numbers were counted by using 200 μL suspension, and then
the rest cells were digested with 65% HNO3 at 65 ◦C for another 12 h.
After that, the Pt level in A549 cells were analyzed by ICP-MS and the
results were shown as one of three independent experiments.
5.5. Immunocytochemistry assay
A549 cells were seeded on glass coverslips at a density of 1 × 106
C
32H39ClN2O13Pt [M+Na]+: 913.1681; found: 913.1420. Elemental
cells/mL, allowed to adhere overnight and grew with 3 (10 μM), and 11
analysis calcd (%) C32H39ClN2O13Pt for: C, 42.09; H, 4.30; N, 3.07;
found: C, 42.39; H, 4.51; N, 2.75.
(5 and 10 μM) for another 24 h, using DMF as controls. After that, the
cells were washed twice with ice PBS, fixed with paraformaldehyde
(4%) for 20 min at room temperature, permeabilized with Triton X-100
(0.5% in PBS) and finally blocked with FBS for 1 h at room temperature.
Then, the cells were labeled with antibody (a-tubulin) for 2 h, followed
with IgG FITC conjugated antibody for 1 h. In order to track the cells, the
cell nucleus stained with DAPI staining solution. The effects on micro-
tubules polymerization of various compounds were recorded and
analyzed with confocal laser microscope, and the results were shown as
one of three independent experiments.
5.2. In vitro cytotoxicity
The cytotoxicities of platinum(IV) complexes 8–11 against SK-OV-3,
HCT-116, HepG-2, MCF-7, A549, SGC-7901, A549/CDDP and SGC-
7901/CDDP were investigated by means of MTT assay, and the plat-
inum(II) complexes and compound 3 were served as positive control.
Culture medium Roswell Park Memorial Institute (RPMI-1640), phos-
phate buffered saline (PBS, pH = 7.2), fetal bovine serum (FBS), and
Antibiotice-Antimycotic came from KeyGen Biotech Company (China).
All cancer cell lines were cultivate in the supplemented with 10% FBS,
and human normal liver cells LO2 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 ◦C. After that, the tested
title compounds dissolved in DMF (Sigma), and diluted with a medium
to five different concentrations. As for the negative control, 0.4% DMF
was added. The cells were incubated in a humidified atmosphere of 5%
5.6. Cell apoptosis analysis
A549 cells were seeded into 6-well plates at a density of 1 × 106
cells/mL and allowed to adhere overnight until 80% confluence. After
24 h incubation, the cells were treated with 3 (2.5
μ
M), oxaliplatin (10
μ
M), combined group (3, 2.5 μM, oxaliplatin, 10 μM), and 11 (2.5 and 5
μ
M) for another 24 h, using DMF as controls. After that, the cells were
CO2 at 37 ◦C for 72 h. After that, added to 10
μM of MTT (5 mg/mL) in
trypsinized, washed with ice PBS, suspended into 1 × binding buffer and
doubled stained with annexin V-FITC and PI according to manufacture’s
protocol. Apoptosis results were recorded and analyzed with flow
cytometry.
the cell culture fluid and the cells were incubated for another 4 h. After
4 h incubation, the medium was thrown away and replaced by 100 μL of
DMSO (Sigma), and the O.D. value was read with a 570/630 nm
enzyme-labeling instrument. The IC50 values were calculated by SPSS
software after three parallel experiments. Similarly, we also detected the
cytotoxic effects of complex 11 (24 h and 48 h) against A549 and A549/
CDDP cells via the same method mentioned above.
5.7. Cell cycle analysis
A549 cells were seeded into 6-well plates at a density of 1 × 106
cells/mL and allowed to adhere overnight until 80% confluence. After
5.3. The stability of complex 11 in various conditions and intracellular
24 h incubation, the cells were treated with DMF, 3 (5 μM), oxaliplatin
analysis
(10 μM), combined group (3, 5 μM, oxaliplatin, 10 μM), and 11 (2.5 and
5 μM) for another 24 h. After that, the cells were collected, washed with
For the sake to investigate the stability of compound 11 in various
ice PBS and fixed with ice-cold ethanol at ꢀ 20 ◦C overnight. Thereafter,
conditions. Solutions of 11 (50
μ
M) in PBS (150 mM) and acetonitrile
the cells were washed with ice PBS, pre-incubated with RNase A (100
(10 mL, 9.5:0.5, V/V), ascorbic acid (500
μ
M) in PBS (pH = 7.4, 150
μ
g/mL) at 20 ◦C and collected to stain with propidium iodide (1 mg/mL)
mM), oxaliplatin (50
μ
M) and compound 5 (50
μ
M) were prepared. The
in dark at 4 ◦C for 30 min following with the illustrations of kits. The
results of cell cycle assays were recorded and analyzed with flow
cytometry.
experiments were carried out in dark at 37 ◦C and monitored via RP-
HPLC (waters, e2695 system; 2489 UV/Vis detector). Upon mixing
11

M. Wang et al.
Bioorganic Chemistry 110 (2021) 104741
5.8. Mitochondrial membrane potential (MMP) assay
Acknowledgements
A549 cells were seeded into 6-well plates at a density of 1 × 106
We are grateful to the National Natural Science Foundation of China
(Grant Nos. 21977021, 81760626 and 22007036), and the China Post-
doctoral Science Foundation (2020M673553XB). In addition, we would
like to thank the Natural Science Foundation of Guangxi Province
(AB17292075) and Guangxi Funds for Distinguished Experts. Moreover,
we are also very grateful to the Natural Science Foundation of Jiangsu
Province (BK20191046) and the Open Project Program of the National &
Local Joint Engineering Research Center for Mineral Salt Deep Utiliza-
tion (SF201904) and the Open Project Program of the Jiangsu Key
Laboratory of Regional Resource Exploitation and Medicinal Research
(LPRK201902 and LPRK201904). Finally, we are also grateful to the
Guangxi Natural Science Foundation of China (2019GXNSFBA245094)
and the Youth Promotion Found of Guangxi (2019KY0386) and the
Guangxi Science and Technology Base and Talents Program
(AD19110109).
cells/mL and allowed to adhere overnight until 80% confluence. After
24 h incubation, the cells were treated with DMF, 3 (5 μM), oxaliplatin
(10
μM), combined group (3, 5 μM, oxaliplatin, 10 μM), and 11 (2.5 and
5
μ
M) for another 24 h. After that, the cells were trypsinized, washed
with ice PBS and collected to stain with JC-1 fluorescent probe prepared
previously according to the instructions of test kit. Thereafter, the cells
were washed with ice PBS, collected at 2500 rpm and subjected to laser
confocal microscope. The images of MMP changes were recorded and
analyzed with laser confocal microscope with the 514, 585 and 529,
590 nm for excitation wavelength and emission wavelength, respec-
tively, while the quantitative analysis was performed via flow
cytometry.
5.9. Reactive oxygen species (ROS) assay
Appendix A. Supplementary material
A549 cells were seeded into 6-well plates at a density of 1 × 106
cells/mL and allowed to adhere overnight until 80% confluence.
Thereafter, the cells were incubated in triplicate with various com-
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104741.
pounds of vehicle (DMF), 3 (5
μM), oxaliplatin (10
μM), combined group
(3, 5 M, oxaliplatin, 10 M), and 11 (2.5 and 5 μ
μ
μ
M). After 24 h co-
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in dark. The images of different groups were recorded and obtained with
laser confocal microscope with excitation and emission wavelength of
505 nm and 535 nm, while the quantitative analysis was performed via
flow cytometry.
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