Design, synthesis and biological evaluation of dihydro-2-quinolone platinum(iv) hybrids as antitumor agents displaying mitochondria injury and DNA damage mechanism

Article abstract of DOI:10.1039/d0dt03194a

The design of novel platinum(iv) complexes with mitochondria injury competence, besides the DNA damage mechanism, is a promising way to develop new platinum drugs. Herein, dihydro-2-quinolone (DHQLO) as a mitocan was incorporated into the platinum(iv) system for the first time to prepare a new series of DHQLO platinum(iv) compounds. Complex 1b could effectively inhibit the proliferation of tumor cells in vitro and in vivo. It accumulated at higher levels in both whole cells and DNA, and easily underwent intercellular reduction to release platinum(ii) and DHQLO moieties. The released platinum(ii) complex caused serious DNA damage by covalent conjunction with the DNA duplex, and remarkably increased the expression of the γ-H2AX protein. Moreover, 1b also caused serious mitochondria injury to induce mitochondrial membrane depolarization and increase ROS generation. Such actions upon DNA and mitochondria activate the p53 apoptotic pathway synergetically in tumor cells by upregulating the protein p53 and apoptotic proteins caspase9 and caspase3, which efficiently promoted the apoptotic death of tumor cells. Compound 1b with such synergic mechanism exhibited great potential in reversing cisplatin resistance and improving antitumor efficacies.

Full text of DOI:10.1039/d0dt03194a

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Design, synthesis and biological evaluation of
dihydro-2-quinolone platinum(IV) hybrids as
antitumor agents displaying mitochondria injury
and DNA damage mechanism†
Cite this: DOI: 10.1039/d0dt03194a
a
a
b
a
a
a
Zhifang Liu, Zuojie Li, Tao Du, Yan Chen, Qingpeng Wang, * Guoshuai Li,
Min Liu,
a
a
a,c
a
Ning Zhang, * Dacheng Li* and Jun Han
The design of novel platinum(IV) complexes with mitochondria injury competence, besides the DNA
damage mechanism, is a promising way to develop new platinum drugs. Herein, dihydro-2-quinolone
DHQLO) as a mitocan was incorporated into the platinum(IV) system for the first time to prepare a new
series of DHQLO platinum(IV) compounds. Complex 1b could effectively inhibit the proliferation of tumor
cells in vitro and in vivo. It accumulated at higher levels in both whole cells and DNA, and easily under-
went intercellular reduction to release platinum(II) and DHQLO moieties. The released platinum(II)
complex caused serious DNA damage by covalent conjunction with the DNA duplex, and remarkably
increased the expression of the γ-H2AX protein. Moreover, 1b also caused serious mitochondria injury to
induce mitochondrial membrane depolarization and increase ROS generation. Such actions upon DNA
and mitochondria activate the p53 apoptotic pathway synergetically in tumor cells by upregulating the
protein p53 and apoptotic proteins caspase9 and caspase3, which efficiently promoted the apoptotic
death of tumor cells. Compound 1b with such synergic mechanism exhibited great potential in reversing
cisplatin resistance and improving antitumor efficacies.
(
Received 13th September 2020,
Accepted 24th November 2020
DOI: 10.1039/d0dt03194a
rsc.li/dalton
other drugs in clinic, especially the ones with rather different
mechanisms. Thus, they display a synergistic function in
1
. Introduction
Platinum-based drugs are the mainstay of chemotherapy regi- cancer treatment. However, the different pharmacokinetic pro-
1
,2
mens for cancer treatment. In particular, cisplatin, carbopla- files of the individual drugs make it hard to predict their total
tin and oxaliplatin (Fig. 1) are used worldwide for the treat- therapeutic effects in organisms. Excitingly, platinum(IV) com-
ment of various tumors, such as testicular, lung, breast, and plexes as prodrugs of platinum(II) drugs supply a promising
3
,4
8–10
ovarian cancers. They are regarded to rapidly induce apopto- strategy to deal with such uncertain risks.
The axial ligands
sis of tumor cells by covalently binding with DNA in the in the octahedral platinum(IV) system allow the combination of
5
,6
nucleus. However, the intrinsic or acquired drug resistance desired synergistic moieties with the platinum core to form a
and severe side effects badly hinder their applications in single molecule. Furthermore, the platinum(IV) complex dis-
7
clinic. It is an urgent task for researchers to explore novel plays many advantages over platinum(II) drugs. Platinum(IV)
structural platinum complexes with the potential to defeat the complexes are more kinetically inert in blood plasma than
inherent drawbacks of classical platinum drugs.
platinum(II) analogues, thus undergoing fewer off-target reac-
With the aim of reducing the serious toxicities and acquired tions and reducing side effects that are typical of the more
resistance, platinum(II) drugs are widely co-administered with reactive platinum(II) progenitors. Moreover, the platinum(IV)
compound could be activated after reduction to cytotoxic diva-
lent metabolites, which happens more likely in hypoxia and
a
Institute of Biopharmaceutical Research, Liaocheng University, Liaocheng 252059,
P.R. China. E-mail: lywqp@126.com, zhangning1111@126.com,
lidacheng62@163.com
b
Henkel Loctite China Corp.Ltd, Yantai 264006, P.R. China
c
Shandong Provincial Key Laboratory of Chemical Energy Storage and Novel Cell
Technology, Liaocheng University, Liaocheng 252059, PR China
†
Electronic supplementary information (ESI) available: Detailed characterization
1
13
data, H and C NMR spectra of new compounds. See DOI: 10.1039/d0dt03194a
Fig. 1 Platinum(II) drugs approved for clinical use.
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Fig. 2 Structures of DHQLO platinum(IV) compounds 1a–c and 2a–c.
reducing tumor microenvironment (TME), and would be the platinum core. Three mono DHQLO platinum(IV) complexes
greatly in favor of the tumor selectivity. Thereby, the design of 1a–c and three dual DHQLO coordinated complexes 2a–c were
platinum(IV) complexes with multi-targeted mechanism has prepared (Fig. 2). All of the title complexes were characterized
become a hot topic in medicinal chemistry.
and evaluated for antitumor activities in vitro, and the primary
Recently, mitochondria have emerged as a major target in SAR were summarized. Subsequently, the proposed mecha-
tumor chemotherapy. “Mitocans” as drugs targeting mitochon- nisms for the title complexes targeting both DNA and mito-
dria or mitochondrial proteins have become an attractive class chondria were confirmed via multiple strategies. Moreover, the
1
1,12
of anticancer agents.
Some new platinum(IV) complexes antitumor activities in vivo were determined in BALB/c mice
bearing mitochondrial sensitizers such as dichloroacetate bearing CT26 colon carcinoma.
1
3
14
15,16
17
(
DCA), α-TOS, phenylbutyrate (PHB),
and Lonidamine
have been prepared. They displayed multiple antitumor
mechanisms that seriously disturb the mitochondrial func-
tion, mitochondrial membrane potential (MMP, ΔΨ
m
) and 2. Results and discussion
morphology, besides causing DNA damage. The synergistic
mechanisms do much favor in reducing the toxicities and over-
coming the drug resistance of platinum(II) drugs. The plati-
num(IV) complex bearing a mitochondria damage ligand is an
appealing scaffold in the design of multitalented platinum-
based anticancer drugs.
2.1 Chemistry
The synthetic route of the DHQLO platinum(IV) complexes is
shown in Scheme 1. The DHQLO acids A1–A3 were synthesized
conveniently starting from material DHQLO (see ESI†).
Oxoplain O1 was prepared by the oxidation of oxaliplatin in
the presence of N-chloro succinimide (NCS), while oxoplatin
O2 was prepared by the oxidation of oxaliplatin with hydrogen
peroxide. The condensation of oxoplatins O1 and O2 with
acids A1–A3 in dry N,N-dimethylformamide (DMF) in the pres-
ence of N,N,N′,N′-tetramethyl-O-(benzotriazol-1-yl)uronium
Dihydro-2-quinolone (DHQLO) is an important scaffold that
is present in a number of drugs, such as the FDA-approved
drugs cilostazol, carteolol and aripiprazole, which display
1
8
rather fascinating clinical performances. In view of its mod-
erate lipophilicity, good stability and broad spectrum of activi-
ties (e.g., antiplatelet, antidepressant, anticonvulsant), new
DHQLO derivatives were also recently exploited as antitumor
1
9,20
agents,
and exerted great potential in curing hematological
cancers by inhibiting BCL-6. It has been proven that BCL-6
2
1
directly targeted the anti-apoptotic proto-oncogene BCL-2,
which was crucial in the mitochondrial apoptotic pathway and
further interfering with mitochondrial function.
Accordingly, as a continuation of our interest in developing
2
2,23
new platinum(IV) drugs,
herein DHQLO was recognized as a
potential mitocan and incorporated with the platinum(IV)
system for the first time to prepare a new series of DHQLO plati-
num(IV) compounds as antitumor agents. Additionally, oxalipla-
tin (as the third generation of platinum drug) displays excellent
antitumor activities in clinic, especially to colorectal cancers. It
is regarded to overcome cisplatin resistance and reduce side
effects through forming different DNA adducts with the stable
24
cyclohexanediamine ligand. Thus, oxaliplatin was selected as Scheme 1 Synthetic route of the DHQLO platinum(IV) compounds.
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Table 1 Cytotoxic profiles of DHQLO platinum(IV) complexes toward five carcinoma cell lines, and one normal human cell line. Cells were treated
with drugs at different concentrations for 48 h, and the results are given as IC50 values (μM) based on three parallel experiments
a
b
c
Compound
CT26
SKOV-3
HeLa
A549
A549R
RF
Average IC50
293T
SI
1
1
1
2
2
2
O1
O2
A2
a
b
c
a
b
c
5.8 ± 0.3
11.7 ± 1.8
9.2 ± 0.4
19.2 ± 0.7
>50
>50
12.6 ± 2.7
>50
4.0 ± 0.4
2.7 ± 0.2
3.5 ± 0.5
>50
>50
>50
11.3 ± 0.9
43.6 ± 3.7
>50
3.2 ± 0.5
2.6 ± 0.4
2.5 ± 0.6
11.27 ± 2.0
6.7 ± 1.1
11.3 ± 1.4
6.7 ± 0.7
48.4 ± 6.8
>50
9.8 ± 1.7
8.6 ± 0.9
6.1 ± 0.5
49.5 ± 8.9
27.9 ± 3.3
44.8 ± 7.2
28.0 ± 2.5
>50
23.4 ± 4.2
8.3 ± 1.4
8.9 ± 0.6
38.2 ± 5.3
20.1 ± 2.1
34.2 ± 1.9
43.8 ± 8.3
>50
2.4
1.0
1.5
0.8
0.7
0.8
1.6
—
9.2
6.8
6.0
>33.6
>30.9
>38.1
20.48
—
3.8 ± 0.4
10.2 ± 1.7
8.5 ± 0.3
14.0 ± 1.2
37.2 ± 2.1
17.7 ± 1.9
15.6 ± 0.7
>50
0.4
1.5
1.4
—
—
—
0.8
—
d
>50
>50
>50
—
—
>50
—
Cisplatin
Oxaliplatin
5.3 ± 1.9
3.9 ± 0.8
2.4 ± 0.2
5.3 ± 0.4
2.4 ± 0.5
7.4 ± 1.0
13.5 ± 2.1
26.8 ± 3.8
22.6 ± 3.2
22.2 ± 1.8
1.7
0.8
9.2
13.1
9.4 ± 1.0
9.9 ± 0.6
1.0
0.8
a
b
c
RF: resistant factor. RF = IC50(A549R)/IC50(A549). Average IC50: average IC50 values for five tumor cell lines. SI: selectivity index. SI =
d
IC50(293T)/Average IC50
. —: Not tested or not calculated.
tetrafluoroborate (TBTU) and N,N,N-triethylamine (TEA) platinum(IV) system shows much more positive influence on
afforded complexes 1a–c and 2a–c in yields of 23–59%.
improving antitumor activities and overcoming the drug resis-
tance of cisplatin. To judge the tumor-selective properties of the
DHQLO platinum(IV) complexes, the tumor selectivity indexes
2
.2 Antitumor activities in vitro
The antitumor activities of DHQLO platinum(IV) compounds
–2, oxoplatins O1–O2, and DHQLO acid A2 were tested toward
(
SI) defined as the ratio of IC50 for normal cell 293T to the
average IC50 for tumor cells are afforded in Table 1. The mono
DHQLO platinum(IV) compounds 1b and 1c with potent antitu-
mor activities exerted higher tumor selectivity (SI = 1.5 and 1.4)
than cisplatin (SI = 1.0) and oxaliplatin (SI = 0.8), as well as the
oxoplatin O1 (SI = 0.8). However, complex 1a exhibits a low SI of
1
five tumor cells and a normal cell using the MTT assay with cis-
platin and oxaliplatin as reference drugs. The cell lines are
human ovarian cancer (SKOV-3), human cervical cancer cell
(
(
HeLa), human lung cancer (A549), murine colon cancer
CT-26), cisplatin resistant cell line A549R, and normal human
0.4, indicating its poor tumor targeting competence.
The SAR analysis demonstrates that mono DHQLO plati-
embryonic kidney cell line 293T. The results are given as IC50
values in Table 1. It is manifested that DHQLO platinum(IV)
complexes 1–2 display more effective antitumor activities than
oxoplatins O1, O2 and DHQLO acid A2. In addition, mono
DHQLO hybrids 1a–c exhibited significantly potent antitumor
competence to the tested tumor cells (average IC50 = 9.2, 6.8
and 6.0 μM), which are comparable or even more effective than
cisplatin and oxaliplatin (average IC50 = 9.2 and 11.3 μM).
Furthermore, complexes 1a–c are at least 3.6 folds more potent
than the corresponding dual DHQLO modified platinum(IV)
complexes 2a–c (average IC50 > 33.6, 30.9, 38.06 μM), which is
probably ascribed to their better lipid–water partition. As for the
num(IV) complexes are more effective than the dual DHQLO
derived ones. The linker in the DHQLO platinum(IV) system
remarkably influences the properties of overcoming drug resis-
tance and targeting tumor cells. The action mechanism for
such molecules and the activities in vivo are worthy of further
exploration. Complex 1b with prominent antitumor efficacy,
promising competence in overcoming cisplatin resistance and
targeting tumor cells was screened out as a superior candidate
over cisplatin and oxaliplatin for the following study.
2.3 Reduction and DNA binding properties
mono DHQLO platinum(IV) hybrids, 1b and 1c with butyryl and It was widely accepted that the platinum(IV) complexes
valeryl linkers, respectively, give relatively lower IC50 values than regained antitumor activities via reduction to platinum(II) com-
compound 1a with a shorter linker acetyl group. Notably, 1b pounds. Therefore, the reduction potentials of the DHQLO
and 1c display superior activities to the lung cancers A549 and platinum(IV) hybrids, as well as the DNA binding properties of
A549R, which are 1.6–2.7 and 2.5–4.4 times more potent than the platinum(II) metabolites, play pivotal roles in killing tumor
cisplatin and oxaliplatin, respectively. To evaluate the compe- cells. It was demonstrated that compound 1b (500 μM) mainly
tence in overcoming the cisplatin resistance, the resistance keeps stable for at least 4 h in PBS at 37 °C, and only a little
factor (RF) values of the title complexes were calculated as a portion was reduced in 15 h (Fig. S1†). Then, compound 1b
ratio of IC50 values for A549R to A549. Compound 1b exhibited could totally be reduced in 4 h (Fig. S2†) in the presence of
great potential in overcoming drug resistance by reducing the reducing ascorbic acid (AsA, 1 mM, same concentration as in
RF value from 1.7 for cisplatin to 1.0, whereas compound 1c TME) to release the oxaliplatin and DHQLO moieties, which
showed a high RF value of 1.5, indicating its poor ability to over- are evidenced by the emergence of peaks of oxaliplatin and
come the resistance of cisplatin. Interestingly, the dual DHQLO acid A2 in the HPLC spectra during the reduction process. To
platinum(IV) complexes 2a–c possessed low RF values of 0.7–0.8, further detect its DNA binding properties, guanosine-5′-mono-
although their antitumor activities were not so potent in vitro. phosphate (5′-GMP, 3 mM) was added as a model of the DNA
These facts indicate that the conjunction of DHQLO into the base to the solution of 1b (500 μM) and AsA (1 mM). Results in
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Fig. S3† show that a new peak of platined-GMP (adduct of oxa-
25
liplatin with 5′-GMP) appears, along with the decrease of the
b peak. These facts prove the effective DNA binding potency
1
of the liberated platinum(II) moieties following the reduction
of the DHQLO platinum(IV) complex.
2
.4 Cellular uptake and subcellular distribution
Cellular uptake is tightly associated with the antitumor efficacy
of chemotherapeutics. Furthermore, DNA is the typical cellular
target for platinum drugs, the cytotoxic effects of which are
mediated by an irreversible DNA lesion in nucleus. Thus, the
detection of the cellular uptake and localization of the DHQLO
platinum(IV) complex in tumor cells, especially in DNA, makes
great sense in understanding its antitumor competence.
Fig. 4 Quantification of apoptosis in SKOV-3 cells using an annexin
V-FITC/PI staining assay. SKOV-3 cells were incubated with and without
platinum complexes for 24 h at 37 °C: (a) blank; (b) 1b (10 μM); (c) oxali-
platin (10 μM); (d) 1b (20 μM); (e) oxaliplatin (20 μM); (f) the stacking
columns.
Results in Fig. 3 reveal that 1b accumulates in whole cells at a
6
1
.8-fold higher level (538.8 ng-Pt per 10 cells) than the refer-
6
ence drug oxaliplatin (292.2 ng-Pt per 10 cells). Moreover, the
subcellular distribution was tested by fractionating the
different cellular components. It was observed that only 16.6%
and 17.8% of the drugs accumulate in the membrane for
complex 1b and oxaliplatin, respectively, which reflects that
the drugs could effectively penetrate the cellular membrane to
enter the tumor cells. Subsequently, 1b induces higher platina-
tion in both cytosol and DNA than oxaliplatin. The higher
accumulation of 1b in DNA than oxaliplatin would lead to
more effective DNA damage and potent antitumor activities.
Ultimately, in view of the 2-fold lower IC50 value for 1b
concentrations of 10 μM and 20 μM, using an annexin V-FITC/
propidium iodide (PI) double staining assay. It is demon-
strated in Fig. 4 that the platinum drugs cause apoptosis of
SKOV-3 cells in a concentration dependent manner. Complex
1b and oxaliplatin induce apoptosis in a small proportion of
cells at 10 μM. Meanwhile, the SKOV-3 cells undergo signifi-
cant apoptosis (76.85%) after treatment with compound 1b at
a concentration of 20 μM for 24 h in contrast to the blank
group (3.71%), which is higher than oxaliplatin at same con-
centration (67.79%). This trend is in accordance with the IC50
values for the tested compounds. Furthermore, these results
are reflected by the fluorescence microscopic images in
Fig. S5.† A larger portion of cells with green and red fluo-
rescence were observed in the compound 1b treated group
than in the blank group, which is comparable to the oxalipla-
tin group. These facts demonstrate the effective apoptosis-
inducing properties of the DHQLO platinum(IV) complex.
(2.7 μM) than oxaliplatin (5.3 μM), the improved uptakes of the
DHQLO platinum(IV) complex in both whole cells and DNA are
positively correlated with the antitumor activities in this work.
2
.5 Apoptosis
To further study the mechanism of the DHQLO platinum(IV)
complexes, the apoptosis-inducing properties of 1b were deter-
mined in SKOV-3 cells with oxaliplatin as a reference drug at
2.6 Mitochondrial membrane depolarization
The apoptosis induced by the title platinum(IV) complex is con-
sidered to be associated with mitochondrial injury for the
introduction of the DHQLO moiety. The decrease of MMP is
an early indicator for mitochondria injury, which would lead
2
6,27
to following downstream events in the apoptosis cascade.
Herein, SKOV-3 cells were treated with platinum drugs, and
stained with the mitochondrial dyes JC-1 and Rhodamine 123
(
Rho 123).
JC-1 could be taken up into the normal mitochondria and
formed red aggregates. Conversely, in cells with mitochondrial
damage, the dye remained as green monomers in the cyto-
plasm. The results were analyzed by flow cytometry, and is
shown in Fig. 5. Complex 1b was tested at concentrations of
Fig. 3 Intracellular accumulation of 1b and oxaliplatin in whole cells,
membrane, cytosol and DNA. The SKOV-3 cells were treated with plati-
num compounds (50 μM) for 12 h. The platinum in whole cells, mem-
brane, cytosol and DNA were determined by ICP-MS. *P < 0.05, **P <
10 μM and 20 μM. It is considered that the DHQLO platinum
(
IV) complex displays antitumor activities after reduction to
oxaliplatin and DHQLO moiety. Therefore, oxaliplatin and
0
.01, ***P < 0.001, ns: no significant difference compared with the
control group.
DHQLO acid A2 were evaluated to compare with 1b at the
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SKOV-3 cells, which is superior to that of oxaliplatin. It could
be observed that DHQLO acid A2 also influences the MMP of
cancer cells to some extent in both assays. Subsequently, the
fluorescence microscopic images of SKOV-3 cells treated with
compound 1b also indicate significant MMP loss, which is evi-
denced by the decrease of red fluorescence in JC-1 stained
cells (Fig. S6†) and the reduction of green mitochondrial stain-
ing in Rho 123 stained cells (Fig. S7†). Summarily, the effective
abilities of 1b in decreasing MMP of tumor cells are probably
attributed to the synergistic functions of the released DHQLO
and oxaliplatin.
2.7 ROS generation
Reactive oxygen species (ROS) are an inevitable product of cell
metabolism, which are mainly produced by the mitochondria.
The level of ROS in cells is always changing with mitochon-
drial function disruption, and further promoting apoptosis of
2
6,28
cells.
To evaluate the generation of ROS induced by
complex 1b, ROS accumulation was determined using 2′,7′-
dichlorofluorescein diacetate (DCFH-DA). DCFH-DA could be
taken up into cells by passive diffusion, and then be quickly
hydrolyzed by esterases to yield non-fluorescent dichlorofluor-
Fig. 5 Mitochondrial membrane potential assay analyzed by flow cyto-
metry. SKOV-3 cells were treated with and without platinum complexes
for 24 h at 37 °C, and stained with JC-1. (a) 1b (20 μM); (b) 1b (10 μM); (c)
oxaliplatin (20 μM); (d) oxaliplatin (10 μM); (e) A2 (20 μM); (f) A2 (10 μM); escein (DCFH). The following oxidization by ROS would gene-
(
g) CCCP (10 μM); (h) blank; (i) statistical analysis of the decrease of rate fluorescent dichlorofluorescein (DCF), and the mean fluo-
MMP.
rescence intensities were analyzed to reflect an overall index of
ROS (Fig. 7). Intracellular ROS levels for the DHQLO platinum
(
IV) complex 1b treated group increased significantly compared
same concentrations. Compound 1b effectively causes the
MMP loss of SKOV-3 cells at a high rate of 43.18% in contrast
to that of oxaliplatin (33.72%) at the same concentration of
to the untreated group (P < 0.001). Moreover, 1b induced 1.4-
fold more ROS generation than oxaliplatin at the same concen-
tration (P < 0.01), which positively correlates with the mito-
chondrial membrane depolarization in SKOV-3 cells, as well as
the IC50 values. Thus, ROS production caused by the DHQLO
platinum(IV) drugs showed much influence on causing the
apoptosis of tumor cells.
2
0 μM, which is in tune with their IC50 values in vitro.
Furthermore, 1b with a DHQLO ligand causes mitochondrial
membrane depolarization in dose-dependent manner,
a
wherein a low concentration of 10 μM leads to a significantly
low rate of 27.06% in comparison with that of a high dosage
2
0 μM (43.18%). To reconfirm these results, the influence of 2.8 Expression of proteins γH2AX, p53, capase9 and capase3
the DHQLO platinum(IV) complex on the mitochondrial mem-
brane potential was monitored with Rho 123 by flow cytome-
try. The Rho 123 assay (Fig. 6) displays similar results in that
complex 1b (20 μM) causes a dramatic decrease of MMP in
Phosphorylated histone H2AX (γH2AX) is the earliest detect-
able indicator of DNA double strand breaks (DSBs) caused by
Pt–DNA conjugates. Furthermore, p53 displays a profound role
in response to Pt–DNA damage in the induction of apoptosis.
The mitochondrial injury and oxidative stress also tightly
associate with the expression of p53 protein. Moreover, the
lack of p53 function is an important factor in causing cisplatin
2
9
resistance. The activation of caspase proteases is fundamen-
3
0
tal to apoptotic pathway.
The injury of mitochondria,
decrease of MMP and ROS production could activate caspase
3
1
proteins, and further trigger apoptotic response. To further
determine the action mechanism of the DHQLO platinum(IV)
complexes in the tumor cells, the expression of proteins
γH2AX, p53, caspase9 and caspase3 in 1b treated SKOV-3 cells
were tested by Western blot with oxaliplatin as a control.
Results in Fig. 8 show that γ-H2AX is remarkably evoked by
1
b and oxaliplatin in the tumor cells, which indicated the
Fig. 6 Mitochondrial membrane potential assay analyzed by flow cyto-
metry. SKOV-3 cells were treated with and without platinum complexes
serious Pt–DNA damage caused by 1b and oxaliplatin.
Subsequently, complex 1b effectively elevated the expression of
p53 in SKOV-3 cells in comparison with the blank group.
(
1b: 20 μM, 10 μM; oxaliplatin: 20 μM, 10 μM; A2: 20 μM, 10 μM; blank)
for 24 h at 37 °C and stained with Rho 123.
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Fig. 8 Western blot analysis of p53 and γ-H2AX expression. SKOV-3
cells were incubated with and without compound 1b and oxaliplatin for
24 h at 37 °C (blank: untreated group; oxaliplatin, compound 1b: 20 μM).
(a) Blots; (b) relative gray intensity analysis (relative gray intensity = (gray
intensity of indicated protein)/(gray intensity of GAPDH)).
Fig. 7 Staining of ROS with DCFH-DA in SKOV-3 cells treated by plati-
num complexes. SKOV-3 cells were incubated with and without com-
pound 1b and oxaliplatin for 24 h at 37 °C, and stained by DCFH-DA.
(
Blank: untreated group; compound 1b and oxaliplatin: 20 μM). (a)
Representative micrographs. (b) Quantified data of the ROS production.
Results are presented as the mean ± SD (n = 5). *P < 0.05, **P < 0.01,
*
**P < 0.001, ns: no significant difference compared with the control
group. The IC50 values of the compounds against SKOV-3 cells were
depicted in red above the columns.
Notably, the expression level of p53 in the 1b-treated cells is
much higher than that for oxaliplatin, although 1b causes a
lower expression of γH2AX than oxaliplatin. The higher
expression of p53 is probably attributed to the synergetic func-
tions of mitochondria injury and ROS production (see section
2
.6 and 2.7), besides the Pt–DNA damage of 1b. Moreover, the
activation of the p53 pathway might be beneficial for com-
pound 1b in overcoming cisplatin resistance.
Fig. 9 Western blot analysis of the cell apoptotic pathway induced by
To determine whether the apoptotic pathway is mediated platinum complexes. SKOV-3 cells were incubated with and without
compound 1b, cisplatin and oxaliplatin for 24 h at 37 °C (blank:
untreated group; cisplatin, oxaliplatin, compound 1b: 20 μM). (a) Blots;
through caspase proteases, caspase9 and cleaved caspase9 as
key initiators of the caspase cascade were evaluated by western
blot (Fig. 9). Usually, the activated caspase-9 would further
(b) relative gray intensity analysis (relative gray intensity = (gray intensity
of indicated protein)/(gray intensity of β-actin)).
cleave downstream effector caspases, such as caspase-3, and
3
2
was attributed to subsequent apoptotic cell death. Thus, the
expressions of caspase-3 and cleaved caspase3 were also inves-
tigated. Caspase9 overexpresses at a remarkably higher level in caspase3 and cleaved caspase3 as the downstream proteins of
b-treated cells than cisplatin and oxaliplatin, as well as the caspase9 are up-regulated by 1b compared with the blank
1
blank groups, which is probably ascribed to the serious mito- group, which are obviously higher than the oxaliplatin and cis-
chondrial damage caused by the DHQLO platinum(IV) complex platin groups. Accordingly, these facts show that the apoptosis
to the tumor cells. Subsequently, the higher expression of the caused by the DHQLO platinum(IV) complex is tightly related
cleaved caspase9 is also observed in the 1b group. Moreover, with caspase9 and caspase3 in the tumor cells.
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.9 HSA binding studies
Paper
2
negligible fluorescence quenching of HSA in comparison with
1
b (A log P = 2.424). Additionally, complex 2b with dual
Human serum albumin (HSA) is the most abundant protein in
blood plasma. The remarkable binding properties of HSA with
exogenous drugs play a critical role in influencing their storage
DHQLO ligands and higher A log P value (3.187) gives even
higher K (2.128 × 10 M ) than mono DHQLO complex 1b
b
5
−1
5
−1
3
3–35
(1.213 × 10 M ). The interaction of the DHQLO platinum(IV)
complexes with HSA is primarily attributed to the introduction
of the DHQLO ligand to the platinum(IV) system, which leads
to enhanced hydrophobicity.
To further study the quenching mechanism, the fluo-
rescence spectra of 1b–HSA at 301 K and 304 K were also evalu-
ated (Fig. S9†). The quenching procedures at different temp-
eratures are in accordance with the Stern–Volmer equation.
in blood and delivery.
Moreover, the complexations of
large protein HSA with small molecular drugs possess many
properties of nanoparticles, and would favor the drugs target-
ing tumor cells via the enhanced permeability and retention
3
6,37
effects (EPR).
Therefore, the investigation on the inter-
action of drugs with HSA is of fundamental and rational
importance. Herein, the HSA binding properties of DHQLO
platinum(IV) complexes and platinum(II) drugs were deter-
mined by UV-Vis and fluorescence spectra.
The fluorescence emission spectra of HSA with increasing
concentrations of 1b and 2b at 298 K are shown in Fig. 10 and
S7.† It is manifested that the intrinsic fluorescence of HSA at
The KSV, K
q b
, K and number of binding sites n of 1b–HSA
system were calculated, and are given in Table 3. Both KSV and
K
K
b
q
are inversely correlated with the temperature. Moreover, the
1
2
−1
values at three different temperatures (2.71–2.77 × 10
M
−
1
S
) are significantly larger than the maximum scatter col-
3
30 nm could be steadily quenched with the increasing con-
1
0
−1
lision quenching constant of the biomolecule (2.0 × 10
M
centrations of 1b and 2b in solution. These facts confirm the
conjunction of complexes 1b and 2b with HSA. The quenching
trends of these two complexes are in accordance with the
Stern–Volmer equation, and the Stern–Volmer constants KSV,
quenching constants K , and binding constants K are calcu-
−
1
S
). In addition, the change of temperature leads to no dra-
matic variation of the values of K . These results show that a
b
static quenching procedure is involved in the interaction of 1b
with HSA. The number of binding sites n per HSA molecule is
approximately equal to 1, suggesting the presence of one main
binding site in the 1b–HSA interaction. Then, the thermo-
dynamic parameters including enthalpy change ΔH, entropy
change ΔS, and Gibbs free energy change ΔG were calculated
by the van’t Hoff equation. The ΔG < 0 demonstrates a spon-
taneous process for the binding of 1b with HSA. The negative
ΔH and ΔS indicate that the process is primarily driven by
enthalpy. The van der Waals force and hydrogen bond are
involved in the 1b–HSA binding interaction.
q
b
lated and given in Table 2 (the detailed calculation procedure
is given in ESI†). The platinum(II) drugs cisplatin and oxalipla-
tin were also evaluated. It seems that the hydrophobicity plays
a key role in influencing the HSA binding properties. Cisplatin
and oxaliplatin with low A log P values (0.556, 0.691) induce
Subsequently, to qualitatively determine the combination
of 1b with HSA, a quick UV-Vis test was employed and the
results are shown in Fig. 11. The results demonstrated that the
UV-Vis absorption spectrum of HSA (Curve A) at a range of
2
50–300 nm could not be superposed with the subtraction
spectrum (Curve D) between the compound 1b–HSA solution
Curve B) and free compound 1b (Curve C) at the same concen-
trations, and these facts provide evidences for the interaction
(
3
8,39
of HSA with compound 1b,
which are coincident with the
results of the fluorescence spectra above.
Accordingly, all facts reveal that 1b could effectively
combine with HSA in plasma. Furthermore, the encapsulation
of the DHQLO platinum(IV) drugs by HSA would largely prevent
them from reduction and complexation by the reducing pro-
Fig. 10 (a–k) Fluorescence spectra of HSA in the absence and presence
of platinum(IV) complex 1b (λex = 280 nm, T = 298 K), c(HSA) = 4.0 μM,
c(complex 1b) = 0.0–37.5 μM, at increments of 3.75 μM. (l) Fluorescence
spectrum of free 1b, c(complex 1b) = 37.5 μM.
q b
Table 2 The A log P, Stern–Volmer constants KSV, quenching rate constants K and binding constants K of platinum complexes 1b, 2b, cisplatin
and oxaliplatin to HSA at 298 K
a
10−4
sv (M−1
10−12
(M−1 S−1
10−4
(M−1
Compound
A log P
K
)
K
q
)
R
K
b
)
R
1
2
b
b
2.424
3.187
0.556
0.691
2.77 ± 0.06
3.37 ± 0.07
0.28 ± 0.02
0.50 ± 0.02
2.77 ± 0.06
3.37 ± 0.07
0.28 ± 0.02
0.50 ± 0.02
0.9908
0.9940
0.9748
0.9894
12.13 ± 0.07
21.28 ± 0.13
—
0.9994
0.9968
—
b
Cisplatin
Oxaliplatin
—
—
a
b
The values of A log P were predicted by Discovery Studio. —: Not calculated.
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Table 3 Thermodynamic parameters of compound 1b–HSA interactions at different temperatures
10−
4
K
SV (M−1
)
10−12
K
q
(M−1 S−1
)
10−4
K
b
(M−1
)
n
ΔH (kJ mol−1
−86.88 ± 3.32
)
ΔG (kJ mol−1
)
ΔS (J mol−1 K−1
−194.27 ± 11.47
)
T (K)
298
301
304
2.77 ± 0.06
2.73 ± 0.06
2.71 ± 0.04
2.77 ± 0.06
2.73 ± 0.06
2.71 ± 0.04
12.13 ± 0.07
8.35 ± 0.07
6.07 ± 0.08
1.14
1.11
1.08
−28.99 ± 0.10
−28.41 ± 0.13
−27.82 ± 0.16
29.3% of the saline group (Fig. 12B), and the TGI for 1b
(
70.7%) was relatively higher than oxaliplatin (TGI = 62.5%),
which indicates that 1b is highly efficient in inhibiting tumor
growth in vivo. Subsequently, these facts are also evidenced in
the hematoxylin and eosin (H&E) staining of tumor slices
(
Fig. 12C). Severe degeneration/necrosis with the dispersion of
the majority of the nucleus was observed in the 1b slice, indi-
cating the apoptosis of tumor cells induced by the drug, which
is similar to that of oxaliplatin.
The systematic toxicities of DHQLO platinum(IV) compound
1b were assessed during the treatment in vivo. Complex 1b-
treated mice showed no apparent signs of locomotor impair-
ment, anorexia, dehydration, muscle loss, or other symptoms
associated with toxicity during the treatment. However,
obvious anorexia and dyskinesia were observed in the oxalipla-
Fig. 11 UV-Vis spectra of HSA with and without compound 1b. (A)
Absorption spectrum of HSA, c(HSA) = 10 μM; (B) absorption spectrum tin-treated group in about 4 h after administration. The results
of compound 1b–HSA (1 : 1), c(HSA) = c(compound 1b) = 10 μM; (C) in Fig. 13A show that 1b leads to a slight loss of body weight in
absorption spectrum of compound 1b, c(compound 1b) = 10 μM; (D)
subtracting spectrum of (B) and (C). Inset: the lines (A) and (D) at the
Meanwhile, oxaliplatin also causes little weight loss of mice.
range of 250 to 300 nm.
comparison with the saline group during the experiment.
The histopathological evaluations of the major organs, includ-
ing the heart, liver, spleen, lung, and kidney were carried out,
and are shown in Fig. 13B. Encouragingly, no significant
appreciable histological differences are apparent in the slices
of the 1b-treated groups in comparison with the saline-treated
ones. It has been proven in many publications that there are
significant differences between the results in vivo and in vitro.
In this work, the results in vivo are not in accordance with the
results of experiments in vitro. The IC50 values for 1b to CT-26
teins like GSH and other S-containing proteins in blood in
comparison with their precursor platinum(II) drugs, which
would benefit for improving stability and reducing off-target
effects and further decreasing toxicities and side effects
in vivo.
2
.10 Antitumor activities in vivo
(
11.7 μM) is obviously higher than oxaliplatin (3.9 μM) in vitro.
To further determine the antitumor activity of the DHQLO
platinum(IV) drugs in vivo, complex 1b was evaluated with its
precursor oxaliplatin as a reference drug. As is well known,
oxaliplatin has great superiority in the clinical treatment of
colorectal cancer, whereas cisplatin and carboplatin exhibit
minor activities. Moreover, the immune system is an impor-
tant factor for oxaliplatin, as well as its derivatives during
Meanwhile, 1b displays potent tumor growth inhibitory com-
petence as oxaliplatin in vivo without causing serious toxic
effects.
3
3
. Experimental
.1 Chemistry
4
0–42
cancer treatment.
Therefore, the immunocompetent
BALB/c mice bearing CT26 syngeneic murine colon carcinoma
were selected as models for our experiments. Compound 1b
General. All reactions were carried out under an atmosphere
and oxaliplatin were administrated at the same dosage of 5 mg of nitrogen in flame-dried glassware with magnetic stirring,
Pt per kg (i.p.) for five times at three-day intervals, taking unless otherwise indicated. Cisplatin and oxaliplatin were pur-
saline as the negative reference.
chased from Boyuan Pharmaceutical Co., Ltd (Jinan, China).
Results in Fig. 12A reflect that the tumors of the saline Other reagents were obtained from Alfa Aesar, Sigma, Aladdin,
3
1
13
group grew rapidly to 1002 mm at day 18 post-tumor inocu- and J&K Scientific Ltd. H NMR and C NMR spectra were
195
lation. Meanwhile, the volume of the 1b-treated group was sig- recorded on a Varian (400 MHz and 100 MHz).
nificantly inhibited to about 458 mm , which is as effective as spectra were recorded on a Bruker Avance NEO (86 MHz). All
oxaliplatin (330 mm , P: ns). After sacrificing the mice, all coupling constants J were quoted in Hz. The HPLC analyses
Pt NMR
3
3
tumor tissues were collected and are shown in Fig. 12C. Upon were performed on a Thermo Ultimate 3000 RS equipped with
treatment with 1b, the tumor weight decreased remarkably to an Agilent Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm).
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Fig. 12 In vivo antitumor activities of compound 1b and oxaliplatin to CT-26 tumors in BALB/c mice. Drugs were administered (i.p.) on days 6, 9, 12,
5 and 18 days post-tumor inoculation, as indicated by the arrows in A. Results are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001,
1
ns: no significant difference compared with the control group. (A) Tumor growth as a function of time. (B) Tumor weight in each group at the end of
the experiment. The TGI of the tested drugs in comparison with the saline group are depicted in red above the columns (TGI = (1 − tumor weight of
the drug-treated group/tumor weight of the saline group) × 100%). (C) The images of the tumors and H&E staining slices from tumor tissues at the
end of the experiment.
Fig. 13 In vivo toxicities of compound 1b, and oxaliplatin to BALB/c mice. (A) Body weight of the mice during treatment. Results are presented as
the mean ± SD. (B) H&E staining of slices from the heart, liver, spleen, lung, and kidney.
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HSA was purchased from Sigma. The genomic DNA mini Then, oxoplatin O1 (100 mg, 0.20 mmol) was added, and the
preparation kit, membrane and cytosol protein extraction kit reaction mixture was stirred for another 48 h at 50 °C. The
and annexin V-FITC/PI apoptosis detection kit were purchased solvent was evaporated, and the residue was purified by silica
from Beyotime Biotechnology, China. The mitochondrial gel column chromatography to afford compound 1c as a white
membrane potential assay kit (JC-1) was purchased from solid (36 mg, 23%) with 99.1% purity, as determined by HPLC.
1
Solarbio, China. UV-Vis spectra were measured on a Scinco
6
H NMR (400 MHz, DMSO-d ) δ 9.93 (s, 1H), 8.53–7.65 (m,
S-3100 UV-Vis spectrophotometer. Fluorescence spectroscopic 4H), 7.01 (d, J = 8.2 Hz, 1H), 6.48–6.38 (m, 2H), 3.86–3.81 (m,
data were recorded on a Hitachi F-7000 spectrofluorometer. 2H), 2.75 (t, J = 7.4 Hz, 2H), 2.60–2.53 (m, 1H), 2.38 (t, J = 7.5
Flow cytometry was performed on a Millipore Guava easyCyte Hz, 2H), 2.30 (dd, J = 16.4, 9.7 Hz, 2H), 2.05 (dd, J = 33.0, 12.4
1
3
8
8
HT flow cytometer. MS were detected on a Shimadzu LCMS Hz, 2H), 1.72–1.38 (m, 8H), 1.37–1.00 (m, 3H). C NMR
040 with ESI ionization. HRMS were recorded on Thermo Q (100 MHz, DMSO-d ) δ 181.4, 170.7, 163.6, 158.3, 139.6, 128.8,
6
Exactive Focus. The fluorescence microscopic images were 115.9, 108.0, 102.2, 67.5, 62.4, 62.1, 61.9, 36.6, 33.8, 31.2, 28.6,
+
taken by an inverted fluorescence microscope (Olympus IX73).
28.5, 24.4, 22.5, 21.7. MS-ESI: calcd for [M + Na] : 717 (M =
+
Synthetic procedures. The synthetic procedures of the acids C H ClN O Pt), found: 717. HRMS: calcd for [M] : 694.1364
2
2
30
3 8
A1–A3 and oxoplatin O1 and O2 are established in the ESI,† (M = C22
H
30ClN
3 8
O Pt), found: 694.1270.
4
3,44
according to procedures mentioned in the literature.
Preparation of compound 2a. To a solution of compound A1
Preparation of compound 1a. To a solution of compound A1 (112 mg, 0.51 mmol) and TBTU (162 mg, 0.51 mmol) in dry
64 mg, 0.29 mmol) and TBTU (93 mg, 0.29 mmol) in dry DMF DMF (5 mL) was injected TEA (71 μL, 0.51 mmol), and the
5 mL) was injected TEA (40 μL, 0.29 mmol), and the mixture mixture was allowed to intensively stir for 10 min at room
(
(
was allowed to intensively stir for 10 min at room temperature. temperature. Then, oxoplatin O2 (100 mg, 0.23 mmol) was
Then, oxoplatin O1 (100 mg, 0.20 mmol) was added and the added, and the reaction mixture was stirred for another 48 h at
reaction mixture was stirred for another 48 h at 50 °C. The 50 °C. The solvent was evaporated, and the residue was puri-
solvent was evaporated, and the residue was purified by silica fied by silica gel column chromatography to afford compound
gel column chromatography to afford compound 1a as a pale 2a as a white solid (74 mg, 38%) with 99.0% purity as deter-
1
yellow solid (33 mg, 23%) with 98.3% purity, as determined by mined by HPLC. H NMR (400 MHz, DMSO-d
6
) δ 9.88 (s, 2H),
1
HPLC. H NMR (400 MHz, DMSO-d ) δ 9.97 (s, 1H), 7.02 (d, J = 7.85–8.31 (m, 4H), 6.98–7.02 (d, J = 8.9 Hz, 2H), 6.37–6.41 (m,
6
7
2
2
.9 Hz, 1H), 6.46–6.38 (m, 2H), 4.54 (s, 2H), 2.76 (t, J = 7.5 Hz, 4H), 4.58 (s, 4H), 2.75 (t, J = 7.4 Hz, 4H), 2.37–2.45 (m, 6H),
1
3
H), 2.50–2.46 (m, 1H), 2.39 (t, J = 7.5 Hz, 2H), 2.08–1.84 (m, 2.00–2.05 (m, 2H), 1.53–0.89 (m, 6H). C NMR (100 MHz,
1
3
H), 1.42–1.51 (m, 3H), 1.37–0.91 (m, 4H). C NMR (100 MHz, DMSO-d ) δ 175.6, 170.7, 164.0, 157.5, 139.6, 128.8, 116.5,
6
DMSO-d
34 4
07.8, 102.3, 65.1, 62.4, 61.8, 31.1, 24.4, 24.1. MS-ESI: calcd for H] : 839 (M = C30H N O12Pt), found: 839. HRMS: calcd for [M
6
) δ 170.7, 170.5, 163.5, 157.5, 139.6, 128.8, 116.6, 107.6, 102.4, 65.2, 61.4, 31.1, 24.5, 23.9. MS-ESI: calcd for [M +
+
1
[
+
+
M + Na] : 676 (M = C H ClN O Pt), found: 676. HRMS: calcd + H] : 838.1894 (M = C H N O Pt), found: 838.1748.
1
9
24
3
8
30 34 4 12
+
for [M + H] : 653.0972 (M = C19
H
24ClN
3
O
8
Pt), found: 653.0975.
Preparation of compound 2b. To a solution of compound A2
Preparation of compound 1b. To a solution of compound A2 (127 mg, 0.51 mmol) and TBTU (162 mg, 0.51 mmol) in dry
72 mg, 0.29 mmol) and TBTU (93 mg, 0.29 mmol) in dry DMF DMF (5 mL) was injected TEA (71 μL, 0.51 mmol), and the
5 mL) was injected TEA (40 μL, 0.29 mmol), and the mixture mixture was allowed to intensively stir for 10 min at room
(
(
was allowed to intensively stir for 10 min at room temperature. temperature. Then, oxoplatin O2 (100 mg, 0.23 mmol) was
Then, oxoplatin O1 (100 mg, 0.20 mmol) was added and the added, and the reaction mixture was stirred for another 48 h at
reaction mixture was stirred for another 48 h at 50 °C. The 50 °C. The solvent was evaporated, and the residue was puri-
solvent was evaporated, and the residue was purified by silica fied by silica gel column chromatography to afford compound
gel column chromatography to afford compound 1b as a white 2b as a white solid (122 mg, 59%) with 98.5% purity as deter-
1
solid (34 mg, 23%) with 98.6% purity, as determined by HPLC. mined by HPLC. H NMR (400 MHz, DMSO-d
6
) δ 9.92 (s, 2H),
1
H NMR (400 MHz, DMSO-d
H), 7.03 (d, J = 8.2 Hz, 1H), 6.50–6.38 (m, 2H), 3.87 (t, J = 6.2 4H), 3.85 (t, J = 6.3 Hz, 4H), 2.75 (t, J = 7.4 Hz, 4H), 2.39 (dd, J
Hz, 2H), 2.73–2.79 (m, 2H), 2.38–2.45 (m, 4H), 2.05 (dd, J = = 15.4, 8.6 Hz, 8H), 2.07 (d, J = 11.1 Hz, 2H), 1.84 (p, J = 6.7 Hz,
6
) δ 9.93 (s, 1H), 8.33–7.65 (m, 8.27 (d, J = 64.2 Hz, 4H), 7.01 (d, J = 8.3 Hz, 2H), 6.47–6.36 (m,
4
1
3
3
5
1
3
5.9, 11.0 Hz, 2H), 1.88 (dd, J = 13.8, 6.9 Hz, 2H), 1.62–1.31 (m, 4H), 1.46–1.03 (m, 8H). C NMR (100 MHz, DMSO-d
H), 1.28–0.97 (m, 3H). C NMR (100 MHz, DMSO-d ) δ 180.6, 170.8, 164.0, 158.2, 139.6, 128.9, 116.1, 108.0, 102.2, 67.0, 61.5,
70.7, 163.7, 158.2, 139.6, 128.8, 116.0, 108.0, 102.2, 67.0, 61.9, 32.6, 31.4, 31.2, 25.5, 24.5, 23.9.
d ) δ (ppm) 1588. MS-ESI: calcd for [M + Na] : 916 (M =
6
DMSO-d ) δ (ppm) 974. MS-ESI: calcd for [M + K] : 719 (M = C H N O Pt), found: 916. HRMS: calcd for [M + H] :
6
) δ 180.6,
1
3
6
1
95
Pt NMR (86 MHz, DMSO-
1
95
+
3.4, 31.4, 31.2, 31.0, 25.5, 24.4, 23.9.
Pt NMR (86 MHz,
+
+
6
34 42 4 12
+
C
21
H
28ClN
3
O
8
Pt), found: 719. HRMS: calcd for [M + H] : 894.2520 (M = C34
28ClN Pt), found: 681.1565. Preparation of compound 2c. To a solution of compound A3
Preparation of compound 1c. To a solution of compound A3 (133 mg, 0.51 mmol) and TBTU (162 mg, 0.51 mmol) in dry
42 4
H N O12Pt), found: 894.2488.
6
81.1285 (M = C21
H
3 8
O
(
(
76 mg, 0.29 mmol) and TBTU (93 mg, 0.29 mmol) in dry DMF DMF (5 mL) was injected TEA (71 μL, 0.51 mmol), and the
5 mL) was injected TEA (40 μL, 0.29 mmol), and the mixture mixture was allowed to intensively stir for 10 min at room
was allowed to intensively stir for 10 min at room temperature. temperature. Then, oxoplatin O2 (100 mg, 0.23 mmol) was
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added, and the reaction mixture was stirred for another 48 h at Table 4 The linear gradient for HPLC
5
0 °C. The solvent was evaporated, and the residue was puri-
fied by silica gel column chromatography to afford compound
c as a white solid (98 mg, 46%) with 99.2% purity as deter-
Time (min)
A (0.1% aqueous TFA)
B (methol)
2
0
5
90
90
0
10
10
100
100
1
mined by HPLC. H NMR (400 MHz, DMSO-d ) δ 9.93 (s, 2H),
6
3
4
5
5
8
.34 (d, J = 29.0 Hz, 4H), 7.01 (d, J = 8.3 Hz, 2H), 6.46–6.36
0
(
(
(
m, 4H), 3.83 (t, J = 6.0 Hz, 4H), 2.75 (t, J = 7.4 Hz, 4H), 2.35
dt, J = 13.9, 7.4 Hz, 8H), 2.10 (d, J = 11.4 Hz, 2H), 1.66–1.54
m, 8H), 1.48–1.00 (m, 8H). C NMR (100 MHz, DMSO-d )
6
1
3
δ 181.3, 170.7, 163.9, 158.3, 139.6, 128.9, 116.0, 108.0, 102.1,
The stability of the compound 1b solution (500 μM) in PBS
6
7.5, 65.4, 61.6, 35.8, 31.2, 28.5, 24.5, 24.0, 22.4. MS-ESI: was monitored for 15 h. The reduction potential of 1b was
+
46 4
calcd for [M + Na] : 945 (M = C36H N O
12Pt), found: 945. evaluated using a 1b solution (500 μM) in the presence of AsA
+
HRMS: calcd for [M + H] : 922.2833 (M = C36
found: 922.2876.
H
46
N
4
O
12Pt), (1 mM, similar concentration as in tumor cells), and the
release of the platinum(II) and DHQLO moieties were also
examined. Then, 5′-GMP (3 mM) was added as a model of DNA
base to the solution of 1b solution (500 μM) and AsA (1 mM)
3
.2 Biological evaluation
In vitro cellular cytotoxicity assays. All of the DHQLO plati- to determine the DNA binding properties of the released plati-
num(IV) derivatives 1a–c and 2a–c, oxoplatins O1–O2 and num(II) counterpart.
DHQLO acid A2 were evaluated for antitumor activities toward
Cellular uptake and subcellular distribution. The cellular
five tumor cell lines, including human ovarian cancer uptake was measured, as reported in our previous work. The
SKOV-3), human cervical cancer cell (HeLa), human lung SKOV-3 cells were seeded in a φ-10 cm culture plate and incu-
4
6
(
cancer (A549), murine colon cancer (CT-26), cisplatin-resistant bated for 12 h, and then platinum drugs 1b and oxaliplatin
cell line A549R, and a normal human embryonic kidney cell (50 µM) were added. The cells were then treated with drugs for
line 293T by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- 12 h. Subsequently, the cells were harvested, washed with PBS
lium bromide (MTT) assay. The RPMI1640, Dulbecco’s for three times, counted and mineralized with 70% HNO
Modified Eagle Medium (DMEM), fetal bovine serum (FBS), (LC). The cellular uptakes of the drugs were measured using
.25% trypsin/EDTA solutions and penicillin–streptomycin ICP-MS. To determine the cellular distribution of 1b and oxali-
3
0
solutions were purchased from Gibco. The MTT and platin, cells were treated with drugs in the same way as men-
Rhodamine 123 were purchased from Sigma. The cells were tioned above for cellular uptake. After the cells were harvested
maintained in either DMEM (for HeLa, 293T cells) or and counted, the membrane and cytosol were isolated with a
RPMI1640 (for SKOV-3, A549, A549R, CT-26 cells) medium con- membrane and cytosol protein extraction kit (Beyotime
taining 10% FBS in a humidified atmosphere containing 5% Biotechnology, China). The DNA of the tumor cells was
−
1
CO
platin. The MTT solution (5 mg mL ) was prepared before Biotechnology, China). Then, the platinum in the membrane,
use.
cytosol and DNA was determined by ICP-MS after mineraliz-
Cells (5000/well) were seeded in 96-well plates in 100 μL of ation with 70% HNO (LC). The results were calculated based
complete medium RPMI1640 or DMEM, and incubated for on 3 determinations for each data point.
2 h in a 5% CO atmosphere at 37 °C. Then, 100 μL freshly
Apoptosis experiment. The apoptosis experiments were
2
at 37 °C. The A549R was maintained with 2 μg mL cis- extracted with a genomic DNA mini preparation kit (Beyotime
−1
3
1
2
prepared culture medium containing tested drugs at serial carried out using an Annexin V-FITC/PI apoptosis detection kit
concentrations were added, and cells were further incubated (Beyotime Biotechnology) according to the manufacturer’s pro-
for 48 h. Subsequently, freshly prepared MTT solution (20 μL) tocol. The SKOV-3 cells were seeded in a 6-well cell culture
5
was added. The resultant mixtures were incubated for another plate (2 × 10 cells per well) and incubated for 12 h. Then, the
4
h in an incubator. Finally, DMSO (150 μL) was added to dis- drugs (blank: no compound; 1b: 20 μM; oxaliplatin: 20 μM)
solve the purple crystals after removal of the medium. The were added and the cells were further incubated for 24 h in an
absorbance was quantified by a microplate reader at 570 nm. incubator. After that, the cells were harvested with trypsiniza-
The IC50 values were obtained based on three parallel experi- tion with no EDTA, and washed twice with PBS. Subsequently,
ments using GraphPad Prism 6.
the samples were stained with annexin V-FITC and PI, and
Reduction by AsA and DNA binding properties. The plati- tested by flow cytometric assay. Simultaneously, the samples
num(IV) compound as the pro-drug of the platinum(II) complex were also imaged with a fluorescence microscope under visible
is regarded to activate after the reduction to the divalent form. light, blue light and green light.
To study the reduction potential of the DHQLO platinum(IV)
complex, a HPLC assay was employed using Thermo Ultimate
Mitochondrial membrane depolarization detection
JC-1 assay. The mitochondrial membrane depolarization was
3
(
000 RS equipped with an Agilent Eclipse XDB-C18 column detected using a mitochondrial membrane potential assay kit
250 × 4.6 mm, 5 μm) with a flow rate of 1 mL min− . HPLC (JC-1) (Solarbio, China), according to the manufacturer’s proto-
1
profiles were recorded by a diode array detector. The linear gra- col. Briefly, the SKOV-3 cells were treated with different drugs
4
5
dient is given in Table 4.
(blank: no compound; 1b: 10 μM, 20 μM; oxaliplatin: 10 μM,
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(
0 μM) for 24 h in an incubator. Cells treated with CCCP the excitation and emission slits were both 5.0 nm. The well-
10 μM) for 20 min were set as the positive reference of MMP mixed solutions were kept for 15 min for equilibrium, and the
loss. After that, the cells were suspended in 0.5 mL culture fluorescence emission spectra under an excitation of 280 nm
−1
medium, and stained with JC-1 for 20 min. Then, the cells were tested from 290 to 500 nm at a scan rate of 300 nm min
were washed twice with washing buffer solution and tested The fluorescence quenching spectra of HSA by compounds 1b,
using a flow cytometric assay. 2b, cisplatin and oxaliplatin were recorded by the gradual
.
Rhodamine 123 assay. The mitochondrial membrane poten- addition of compounds from 0.0 μM to 37.5 μM at increments
tial (ΔΨ ) was also monitored by the fluorescent dye Rho 123, of 3.75 μM to the HSA solution (4.0 μM) at 298 K. The quench-
m
4
7,48
as described in the literature.
The cells were treated with ing spectra of 1b to HSA at 301 K and 304 K were also recorded.
tested compounds, just as mentioned above, in JC-1 assay.
For the UV-Vis assay, three solutions were prepared and
−
1
Then, the cells were harvested, washed twice with PBS and tested from 200 to 600 nm at a scan rate of 600 nm min in
stained with Rhodamine 123 (5 μM) for 30 min at 37 °C. After Tris-HCl buffer (10 mM Tris-HCl/10 mM NaCl, pH 7.4) at room
washing twice with PBS, the samples were analyzed using flow temperature using a quartz cell of path length 1 cm. The
cytometry.
absorption spectrum of free HSA (10 μM) was recorded as line
Fluorescence microscopic images. The SKOV-3 cells in a six- A. The spectrum of compound 1b–HSA solution with HSA:
well plate were treated with different drugs (blank: no com- 10 μM and compound 1b: 10 μM was line B. The absorption
pound; 1b: 10 μM, 20 μM; oxaliplatin: 10 μM, 20 μM) for 24 h spectrum of free compound 1b (10 μM) was line C. Then, line
in an incubator. Then, the cells were washed twice with PBS D was drawn as a subtracting spectrum of (B) and (C).
and stained with JC-1 (20 min) or Rhodamine 123 (30 min).
Antitumor activities in vivo. The antitumor activities in vivo
After that, the dyes were removed and the cells were gently were carried out on male BALB/c mice (18–20 g), which were
washed twice with PBS. The images were taken by an inverted purchased from the Laboratory Animal Center, Shandong
fluorescence microscope.
University (Shandong, China). This study was performed in
Measurement of ROS generation. The production of ROS strict accordance with the NIH guidelines for the care and use
was measured in SKOV-3 cells using DCFH-DA based on the of laboratory animals (NIH Publications No. 8023, revised 1978),
ROS-dependent oxidation of DCFH to DCF. After cells were and was approved by the Institute Animal Ethics Committee of
exposed to drugs (blank: untreated group; compound 1b and Liaocheng University (Liaocheng, China). Murine CT26 cells (5
5
oxaliplatin: 20 μM) for 24 h, they were gently washed twice × 10 ) were injected subcutaneously into the right flank of the
with cold PBS. Then, the cells were stained with DCFH-DA at a mice. The mice were divided into three groups randomly after
concentration of 10 μM in FBS-free culture medium for 20 min tumor nodules were palpable on day 6. Then, compound 1b
at 37 °C, and imaged with a fluorescence microscope under and oxaliplatin were administrated at the same dosage of 5 mg
visible light and blue light, respectively. The ROS production Pt per kg i.p., and saline was used as the negative control.
was determined by quantifying the mean fluorescence inten- Drugs were given 5 times on days 6, 9, 12, 15 and 18. The tumor
sity of five random fields.
volume and body weight of all mice were monitored during the
Measurement of expressions of p53, γH2AX, capase9 and experiment. The tumor volume (V) was determined as V = L ×
2
capase3. After exposure to drugs (blank: untreated group; cis- W /2, where the length (L) and width (W) were measured. The
platin, oxaliplatin, compound 1b: 20 μM) for 24 h, SKOV-3 mice were euthanized 24 h after the last drug administration on
cells were harvested. The proteins were extracted with lysis day 19, and the tumors were excised and weighed. The tumors
buffer, and their concentrations were measured using a BCA and tissues (including liver, heart, spleen, lung, and kidney)
protein assay kit (Servicebio). The proteins (20 mg per Lane) were collected, formalin fixed in 4% formaldehyde overnight,
were separated by 10% sodium dodecyl sulfate-polyacrylamide paraffin-embedded, and cut with a microtome (5 μm sections)
gel electrophoresis (SDS-PAGE) (Servicebio), and transferred for hematoxylin and eosin (H&E) staining detection.
onto polyvinylidene difluoride (PVDF) Immobilon-P mem-
brane (Millipore). The β-actin or GAPDH was used as a loading
control. The blots were blocked with 5% nonfat milk in TBST
4
. Conclusion
(
Tris buffered saline plus 0.1% Tween 20) for 1 h. The mem-
brane was incubated with primary antibodies overnight at Taken together, a series of DHQLO platinum(IV) hybrids target-
°C. Then, the membrane was washed with PBS three times, ing both DNA and mitochondria were designed, synthesized
4
and incubated with a secondary antibody for 1 h at 37 °C. for the first time and evaluated for antitumor activities in vitro
After that, the membrane was treated with an ECL western and in vivo. Mono DHQLO ligand platinum(IV) complexes are
blotting substrate kit (Servicebio), and detected by a scanning more effective than the corresponding dual ligands ones, and
system (EPSON).
the alkyl chain linking DHQLO and platinum core displays
HSA binding experiments. The HSA binding properties were remarkable influence on the antitumor activities. The mono
evaluated by UV-Vis and fluorescence spectra assays. The fluo- DHQLO platinum(IV) complex 1b with the butyryl linker is
rescence experiments were performed in buffer solution screened out as a candidate, which displays comparable or
(10 mM Tris-HCl/10 mM NaCl, pH 7.4) on a Hitachi F-7000 even more potent antitumor activities than cisplatin and oxali-
spectrofluorometer using a quartz cell of 1.00 cm. The widths of platin. Furthermore, 1b could overcome the drug-resistance of
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Project of Shandong Collaborative Innovation Center for
Antibody Drugs (No. CIC-AD1836, CIC-AD1835), National
Science and Technology Major Project (No. 2017ZX09201-003)
and Taishan Scholar Research Foundation. This work was also
technically supported by the Shandong Collaborative
Innovation Center for Antibody Drugs and Engineering
Research Center for Nanomedicine and Drug Delivery Systems.
Fig. 14 The proposed antitumor mechanism for DHQLO platinum(IV)
complexes targeting both DNA and mitochondria.
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