Mono-functionalized glycosylated platinum(IV) complexes possessed both pH and redox dual-responsive properties: Exhibited enhanced safety and preferentially accumulated in cancer cells in?vitro and in?vivo

Article abstract of DOI:10.1016/j.ejmech.2017.01.032

A serious of carbohydrate-conjugated platinum(IV) complexes in the form Pt(L2)(A2)(OH)R based on the clinical drug cisplatin and oxaliplatin were designed, synthesized and evaluated as antitumor agents in?vitro and in?vivo. The conjugates possessing both pH and redox dual-responsive properties exhibited more potent cytotoxicity in seven different human cancer cell lines and lower toxicity to the normal 3T3 cells than cisplatin, oxaliplatin and even the reported bis-functionalized glycosylated platinum(IV) complexes indicating the enhanced safety of the sugar conjugates. Cellular drug uptake and DNA platination were also superior to cisplatin, oxaliplatin and the reported bis-functionalized ones. Peak current of B7 and B8 with the scan rate of 200mv/s at the concentration of 0.08?mM was 5-fold higher at pH 6.4 than the pH 7.4, indicating that carbohydrate-conjugated mono-functionalized platinum(IV) complexes possessed both pH and redox dual-responsive properties in the cancer cells. The in?vivo assays demonstrated that the Pt(IV) compounds could inhibit the growth of MCF-7 tumour and exert more safety than oxaliplatin.

Full text of DOI:10.1016/j.ejmech.2017.01.032

European Journal of Medicinal Chemistry 128 (2017) 45e55
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Mono-functionalized glycosylated platinum(IV) complexes possessed
both pH and redox dual-responsive properties: Exhibited enhanced
safety and preferentially accumulated in cancer cells in vitro and
in vivo
, b, *
Jing Ma a, Xiande Yang a, Wenpei Hao a, Zhonglv Huang a, Xin Wang a
,
, b, **
Peng George Wang a
a College of Pharmacy, State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin 300071, PR China
b Tianjin Key Laboratory of Molecular Drug Research, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University,
Tianjin 300071, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A serious of carbohydrate-conjugated platinum(IV) complexes in the form Pt(L2)(A2)(OH)R based on the
clinical drug cisplatin and oxaliplatin were designed, synthesized and evaluated as antitumor agents
in vitro and in vivo. The conjugates possessing both pH and redox dual-responsive properties exhibited
more potent cytotoxicity in seven different human cancer cell lines and lower toxicity to the normal 3T3
cells than cisplatin, oxaliplatin and even the reported bis-functionalized glycosylated platinum(IV)
complexes indicating the enhanced safety of the sugar conjugates. Cellular drug uptake and DNA
platination were also superior to cisplatin, oxaliplatin and the reported bis-functionalized ones. Peak
current of B7 and B8 with the scan rate of 200mv/s at the concentration of 0.08 mM was 5-fold higher at
pH 6.4 than the pH 7.4, indicating that carbohydrate-conjugated mono-functionalized platinum(IV)
complexes possessed both pH and redox dual-responsive properties in the cancer cells. The in vivo assays
demonstrated that the Pt(IV) compounds could inhibit the growth of MCF-7 tumour and exert more
safety than oxaliplatin.
Received 14 December 2016
Received in revised form
9 January 2017
Accepted 21 January 2017
Available online 23 January 2017
Keywords:
Antitumor
Glycosylation
Metal complexe
Platinum
Cancer
© 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction
afford unique opportu-nities for the design of platinum(IV) pro-
drugs, which are believed to be activated inside the cancer cells by
Many of the limitations existing in the platinum(II)-based che-
motherapies have been overcame by the platinum(IV)-based anti-
cancer compounds. Due to the special inert of platinum(IV)
complexes, the unwanted side reactions with biological nucleo-
philes are resisted to reduce the undesired side-effects and increase
the lifetime in biological fluids [1]. The reductants like ascorbic acid
and glutathione used to reduce the platinum(IV) complexes to
regain their cytotoxicity present higher concentrations in the
tumour cells than in blood and normal tissues. This characteristics
reduction to cytotoxic platinum(II) complexes [2]. Many attempts
have been done to tune the chemical properties of platinum(IV)
complexes to enhance the anticancer efficacy of platinum agents
[3]. The axial ligands play an important role in conferring the
pharmacological properties and enhancing the target therapy [4,5].
Recent years, various cell-responsive prodrugs, such as redox
active, pH dependent, and enzymes, have been intensively inves-
tigated. The nature of the axial ligands can influence the reduction
potentials of the platinum(IV) prodrugs. Therefore, it is imperative
to choose axial ligands that will minimize the chance of reduction
in the blood stream before the prodrugs reach the cancer cells, but
are amenable to reduction once they get inside these cells [6]. The
intracellular pH (pHi) is about 7.2 and the extracellular pH (pHe)
about 7.4 in normal cells. In cancer cells, the pHe value is lower than
7.1. Some cellular environments, such as endosomes (pH 5.0) and
lysosomes (pH 4.5), are relatively acidic [7]. Due to tumour hypoxia,
* Corresponding author. College of Pharmacy, State Key Laboratory of
Elemento-organic Chemistry, Nankai University, Tianjin 300071, PR China.
** Corresponding author. College of Pharmacy, State Key Laboratory of
Elemento-organic Chemistry, Nankai University, Tianjin 300071, PR China.
E-mail addresses: wangxinnk@nankai.edu.cn (X. Wang), pwang@nankai.edu.cn
(P.G. Wang).
http://dx.doi.org/10.1016/j.ejmech.2017.01.032
0223-5234/© 2017 Elsevier Masson SAS. All rights reserved.

46
J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
lower pH, elevated levels of GSH [8,9], tumours behave an altered
redox state compared with normal tissues. Therefore, one of the
important strategies to enhance drug efficacy and specificity is to
improve the capacity of reduction at pH < 7.1, under which the
platinum(IV) prodrugs possesses both pH and redox dual-
responsive properties in the cellular environment.
pH and cellular reductants in cancer cells, it would behave an
enhanced the cellular drug uptake and DNA platination (Scheme 1).
2. Results and discussion
Currently, functionalization is most commonly carried out using
anhydrides and acyl chlorides, which undergo direct carboxylation
reactions with platinum(IV) axial hydroxido ligands [22,23]. And
therefore, succinic anhydride was introduced to the platinum(IV)
axial hydroxido ligands. As we know, different glycosyl receptors
are over expressed in different cancer cells. In order to evaluate
their impact on antitumor potency, different glycosyl moieties
including galactose, mannose and rhamnose with variable aliphatic
chains in different lengths were introduced to these serial com-
plexes. Two clinical Pt(II) drugs cisplatin and oxaliplatin were
selected for construction of Pt(IV) complexes B1-B10 to investigate
the influence of different platinum cores on antitumor activities
(Scheme 2).
Recently, the liposomes [10], polymers [11], inorganic nano-
materials [12e14], metaleorganic frameworks [15,16], and calcium
phosphate (CaP) nanoparticles [17] were used to synthesize the
platinum(IV) prodrugs with pH or redox responsive properties. The
platinum(IV) complexes platin-A with mono-functionalized ligands
of aspirin were reported to have a positive shift of 42 mV at pH 6.4
and a negative shift of ꢀ563 mV at pH 7.4 [18,19a]. The reduction
properties indicated that the reduced pHe in the cancer microen-
vironment would facilitate reduction of the platinum(IV) com-
plexes with mono-functionalized ligands to release the active
platinum(II) complexes. And also the mono-functionalized plati-
num(IV) complexes would behave a low toxicity to the normal cells
as the result of the negative shift of reduction potential. And the use
of the monohydroxido monocarboxylato framework was also re-
ported to have advantages over the use of either the dihydroxido or
the dicarboxylato framework due to its intermediate properties in
the design of targeted Pt(IV) derivatives of oxaliplatin with
reasonable rates of reduction [19b]. And thus we designed and
synthesized a serious of carbohydrate-conjugated platinum(IV)
complexes in the form Pt(L2)(A2)(OH)R based on the clinical drug
cisplatin and oxaliplatin.
2.1. Antitumor activities in vitro
We evaluated the cytotoxicity of B1-B10 against a panel of hu-
man cancer cells of different origin using the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
The cytotoxicity of cisplatin, oxaliplatin, B13 and B14 were also
determined as control. Inhibitory effect (IC50 in mM) of carbohy-
drate platinum(IV) complexes on cancer cells are summarized in
Table 1.
In the mitochondria of healthy non-cancerous cells, D-glucose is
metabolized oxidatively and produces 36 mol ATP per mole of
glucose. However, cancer cells metabolize glucose by aerobic
glycolysis, which is energetically very inefficient and provides only
4 mol ATP from one mole of glucose. And therefore it is proved that
carbohydrate uptake of cancer cells significantly increased with the
unrestricted proliferation, which is explained by the “Warburg ef-
fect”. Therefore, the design of glycoconjugations can increase the
specificity of the complex towards cancer cells whose receptors are
over expressed in cancer cells [20]. Recently, many works have been
devoted to the investigation of novel glycosylated Pt(II) derivatives,
which indicated that the incorporation of sugar to platinum com-
plex significantly reduced their toxicity and improved antitumor
efficiency [21aed]. And our group firstly synthesized the
carbohydrate-conjugated platinum(IV) complexes and glyco-
conjugation is a promising strategy for specific targeting of cancer
[21e,f]. The carbohydrate-conjugated platinum(IV) complexes in
the form Pt(L2)(A2)(OH)R designed to possess both pH and redox
dual-responsive properties are totally different from the reported
carbohydrate-conjugated platinum complexes. In response to low
As shown in Table 1, the cytotoxicity of the carbohydrate plati-
num(IV) complexes are greater than that of oxoplatin B13 and B14
for MCF-7, HeLa and A549. It is worth mentioning that the cyto-
toxicity of mono-functionalized glycosylated platinum(IV) com-
plexes B1-B10 (RF 0.86
of bis-functionalized glycosylated platinum(IV) complexes A1-A5
(IC50 > 100 M) for A549R. And also for A549 and HepG-2, B1 with
IC50 9.38 M and 8.96 M, is more effective than A1-A5 with IC50
11.71 Me117.00 M (A549) and 19.92 M (HepG-2),
M ꢀ51.12
respectively. For MCF-7, B1, B5, B6, B7, B8 and B10 (IC50 ¼ 4.86 M-
9.34 M), also show more prominent efficacy compared with A1-
A5. (IC50 ¼ 18.18 M ꢀ130.98 M).
mM-3.22 mM) are generally superior to that
m
m
m
m
m
m
m
m
m
m
m
It is also observed that different glycosyl fragments behave
different cytotoxicity to different cancer cells. And meanwhile the
carbohydrate platinum(IV) complexes with cisplatin and oxalipla-
tin core display different antitumor efficacy. For the tested Pt(IV)
compounds B1-B10, It is demonstrated that cisplatin derivatives B1,
B3, B5 and B7 exhibit relatively more effective antitumor activities
in comparison with corresponding oxaliplatin ones B2, B4, B6 and
B8 for HeLa and A549. For PC3 and HepG-2, cisplatin derivatives B1,
B5, B7 and B9 are also more effective than corresponding oxali-
platin ones B2, B6, B8 and B10. And different structural carbohy-
drates and linkers also show great influences on the bioactivities of
target compounds.
Noticeably, the rhamnose oxaliplatin derived compounds B2
and B4 show important cytotoxicity against the PC3 and HepG-2
than the mannosylated and galactosylated compounds B6, B8 and
B10. When the carbohydrate platinum(IV) complexes are replaced
by mannose and galactose to yield B6, B8 and B10, the antitumor
activities dramatically decreased. Furthermore, the cisplatin
derived compounds B1, B3, B5, B7, B9, exhibit relatively more
effective antitumor activities against PC3 and HepG-2. The man-
nosylated and galactosylated compounds B5 and B9 are even more
potent than cisplatin and oxaliplatin against PC3. Rhamnose con-
taining propyl amino linker cisplatin derivative B1 displays com-
parable IC50 value to that of cisplatin, but rhamnosylated cisplatin
derivative (B3) with ethyl amino linker has higher IC50 value in
Scheme 1. Schematic illustration of the synthesis carbohydrate-conjugated with
mono-functionalized platinum(IV) complexes and the release of platinum(II) in
response to low pH and cellular reductants.

J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
47
Scheme 2. Synthetic route of target compounds. Conditions and reagents: (a) H2O2 (30% w/v)/H2O; (b) Succinic anhydride, DMSO; (c) HATU, DIPEA, DMF, r.t.; (d) Succinic an-
hydride, DMF.
Table 1
Inhibitory effect (IC50 in mM) of carbohydrate platinum(IV) complexes on cancer cells.
HeLa
MCF-7
LNCaP
PC3
HepG-2
A549
A549R
RFa
B1
B2
B3
B4
B5
B6
B7
B8
1.32 1.01
4.59 0.87
6.16 0.54
24.28 0.53
11.53 0.34
14.14 0.45
1.20 0.78
26.96 0.65
24.99 0.67
12.59 0.78
35.98 0.89
34.80 1.12
10.32 1.34
6.18 1.70
21.23 0.91
2.81 0.75
8.36 0.91
7.18 0.89
4.96 0.96
7.55 1.44
22.00 1.87
22.61 1.56
18.99 0.78
9.34 0.12
4.86 0.45
9.20 0.79
8.60 0.66
13.36 0.77
7.40 0.56
72.49 1.34
>100
5.52 0.38
8.23 0.89
54.91 0.78
14.09 1.34
42.90 1.45
5.08 1.78
8.68 0.45
5.06 0.89
>100
20.45 1.45
28.90 1.49
44.34 0.45
40.73 0.78
15.23 0.56
>100
33.69 1.89
>100
13.76 1.78
>100
8.96 1.58
28.75 1.49
55.20 1.67
19.67 1.49
20.90 1.79
>100
21.08 1.46
>100
73.00 1.38
>100
38.73 1.39
>100
>100
80.89 1.67
>100
46.85 0.78
67.42 1.03
7.70 1.89
23.54 1.38
9.38 1.39
25.76 1.47
23.53 1.49
30.9 1.48
26.39 0.89
31.44 0.78
38.17 2.31
40.59 2.98
38.93 2.78
18.44 1.78
>100
>100
42.31 1.21
>100
35.13 1.35
11.71 1.20
24.71 1.35
10.47 1.68
16.07 2.90
16.83 1.90
>100
1.79
/
3.22
0.99
1.68
1.29
0.86
/
1.09
/
/
/
/
/
/
/
75.70 1.78
30.69 1.79
44.45 1.67
40.51 1.69
32.76 1.69
>100
42.24 1.23
>100
>100
24.80 2.57
>100
>100
>100
>100
B9
B10
B13
B14
A1
A2
A3
20.10 1.66
>100
58.08 1.45
>100
18.90 0.79
11.37 2.24
65.76 1.78
30.07 2.25
4.82 0.45
1.90 0.67
11.54 1.77
20.45 1.55
94.64 2.23
>100
19.92 0.67
51.12 1.12
36.86 0.69
26.35 0.76
41.53 1.56
28.00 1.56
42.93 1.38
72.07 2.95
30.44 2.56
18.18 1.15
10.00 0.78
11.60 1.56
A4
A5
>100
38.94 1.57
39.99 2.67
/
cisplatin
Oxaliplatin
3.72
2.49
a
RF: Resistant factor ¼ IC50(A549R)/IC50(A549).
comparison to that of cisplatin. In addition, glycosylated Pt(IV)
compounds succeed to overcome the drug resistance of A549R cells
with the RF from 0.86 to 1.79 (Table 1).
cisplatin and oxali-platin. The results obtained enable these com-
pounds to be of much potential for further investigations as new
antitumor agents.
Furthermore, the rhamnose derived compounds B1 show
prominent efficacy against HepG-2, A549 and A549R than the
galactosylated and mannosylated compounds. Compound B1 ex-
It is obvious that when the linkage of compound B1 is replaced
by shorter ones to yield B3, the antitumor activities decreased
largely. We can also see the same phenomenon for B2-B4 to HeLa,
LNCap, PC3, A549 and B6-B8 to HeLa, MCF-7, A549 and A549R.
HeLa and MCF-7 were more sensitive to the carbohydrate
hibits competent activities with IC50 values lower than 20.45
mM to
all tested cell lines which are comparable or even more potent than

48
J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
platinum(IV) complexes than the others. B1 showed 5-fold and 4-
The inhibitory effects (IC50 in mM) of B1, B6, B7 and B8 on MCF-
fold cytotoxic increase compared with clinical drug cisplatin and
oxaliplatin against HeLa with the IC50 value of 1.32 mM.
In summary, to different cancer cells, different glycoside
including rhamnoside, mannoside and galcatosde shows different
antitumor abilities. This maybe enable these compounds to be of
much potential for further investigations with target therapy.
7 cells are also almost 6-fold superior to the bis- functionalized
prodrugs A4 and A5. But to the normal cells, B1, B6 and B8 also
behaved an enhanced safety, which indi-cates its more sensitive to
low pH and cellular reductants in cancer cells than the normal cells
performing totally different properties from the reported
carbohydrate-conjugated platinum complexes.
2.2. Cell viability of 3T3
2.4. Cellular uptake and DNA platination
An ideal anticancer compounds should be selective for cancer
cells over normal healthy cells, thereby mitigating undesired toxic
side effects associated with chemotherapy. We therefore evaluated
the selectivity of B1-B10 using normal 3T3 cell (mouse embryo fi-
broblasts obtained from Prof. Yanming Wang, Nankai University).
We evaluated the cytotoxicity of B1-B10 against 3T3 cell using the
MTT assay. Strikingly, as presented in Fig. 1, the cell viability of 3T3
was significantly higher after incubation with B1, B6, B7, B8 as
compared with cisplatin and oxaliplatin. Notably, at the concen-
Subsequently, the mechanism was explored to investigate the
correlation between intracellular platinum accumulation and
antitumor activity by inductively-coupled plasma mass spe trom-
etry (ICP-MS) using 196Pt detection after 10 h incubation (Fig. 3).
For HeLa, A4 (IC50 ¼ 2.81
(IC50 ¼ 1.20 M) are more effective than the others. And meanwhile,
A5 (IC50 ¼ 1.90 M)
mM), B1 (IC50 ¼ 1.32
mM) and B7
m
m
M), B6 (IC50 ¼ 5.08
m
M) and B8 (IC50 ¼ 5.06
m
exhibit enhanced cytotoxicity to LNCaP. So we compared cellular
drug uptake for A4, B1, B7 in HeLa cell line and A5, B6 and B8 in
LNCaP cell line.
tration of 17.8
B6, B7, B8 and 25%, 65% for cisplatin and oxaliplatin, respectively.
Compared with cisplatin (IC50 ¼ 8 M), B1 (IC50 > 200 M), B6
(IC50 > 200 M), B8 (IC50 > 100 M) showed
M), B7 (IC50 ¼ 60
obvious lower toxicities. And compared with A4 (IC50 ¼ 84 M) and
M), B1, B6, B8 also behaved a greater advantage
mM, cell viability of 3T3 is 100%,100%, 70%, 72% for B1,
In contrast to the comparable activity of B7 and B8 observed in
the 48 h incubation MTT assay, the accumulations of B1, B6, B7 and
B8 in HeLa and LNCaP cells are higher than cisplatin and oxaliplatin.
(2.5e18 times) And also the DNA was extracted and its platination
was evaluated. B7 exhibit 5.4 and 1.33 folds higher than oxaliplatin
and cisplatin. Moreover, cellular drug uptake of B7 in HeLa cells and
DNA platination are 2.3 and 1.5 folds higher than A4. For LNCaP
cells, B6 and B8 show 1.67 and 1.75 times higher than A5.
These results indicated that the carbohydrate-conjugated
mono-functionalized platinum(IV) prodrugs are superior to the
platinum(II) complexes and bis-functionalized platinum(IV) pro-
drugs in cell uptake and DNA platination. Simultaneously, cellular
uptake studies by ICP-MS confirmed a good correlation between
intracellular platinum accumulation and antitumor activity.
m
m
m
m
m
m
A5 (IC50 ¼ 169
m
indicating an enhanced safety of mono-carbohydrate platinum(IV)
complexes. Compared with A4 and A5, B5 and B7 with the same
core, the same sugar and the same linker displaying comparable
IC50 values to 3T3, exhibited more effective to MCF-7.
2.3. Cell viability of MCF-7 cancer cells and the normal 3T3 cells
As shown in Fig. 2, cell viability of the normal 3T3 cells for B1,
B6, B7 and B8 is better than that of MCF-7 cancer cells after incu-
bation for 48 h at the concentration of 100
7.5 M, 3.2 M, 1.3 M, 0.6 M and 0.2 M IC50 values of B1, B6, B7
and B8 to the normal 3T3 cells are almost 50 folds higher than the
most sensitive MCF-7 cells (IC50 ¼ 7.55 M, 4.86 M, 9.20 M and
8.60 M respectively) (Table 2). This results indicate that B1-B10
mM, 42.2 mM, 17.8 mM,
m
m
m
m
m
2.5. Flow cytometric analysis
m
m
m
In order to investigate the incidence of cell cycle arrest (LNCaP),
the DNA-flow cytometric studies were conducted following treat-
ment with B6 and B8. A sum of 11.09% and 12.43% accumulated at
m
are much more potent in breast carcinoma MCF-7 cells compared
with normal 3T3.
the G2/M after 30 h incubation at 1 mM with B6 and B8, indicating
Cell viability of 3T3
100%
80%
60%
40%
20%
0%
μM
100
B1
42.2
B6
17.8
7.5
3.2
B8
1.3
Cisplatin
0.6
0.2
Oxaliplatin
B7
Fig. 1. Cell viability of 3T3.

J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
49
B1,3T3
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
B6,3T3
B7,3T3
B8,3T3
B1,MCF-7
B6,MCF-7
B7,MCF-7
B8,MCF-7
Blank
μM
100
42.2
17.8
7.5
3.2
1.3
0.6
0.2
Fig. 2. Cell viability of MCF-7 cells and the normal 3T3 cells for B1, B6, B7 and B8 after incubation for 48 h at the concentration of 100
0.6 M and 0.2 M.
mM, 42.2 mM, 17.8 mM, 7.5 mM, 3.2 mM, 1.3 mM,
m
m
Table 2
Inhibitory effect (IC50 in
A5, cisplatin and oxaliplatin on MCF-7 cells and 3T3 cells.
identified by ESI-MS analysis. After reduction by Vc, oxaliplatin was
formed and then combined with 5’-dGMP to generate the bis-
substituted products. Simultaneously, in the absence of Vc we
cannot observe the same peak, which indicate that the glycosylated
Pt(IV) compounds exert their anticancer activity via the released
Pt(II) complexes.
And we can see that in Fig. 4A and B, the adduct formation
between oxaliplatin and 5’-dGMP is faster and more than B8. The
reason is that firstly the platinum(IV) complexe (B8) need to be
reduced by the reductants like ascorbic acid and glutathione and
then bind with 5’-dGMP to form the adduct. But the Pt(II) drug
oxaliplatin can bind with 5’-dGMP directly.
m
M) of carbohydrate platinum(IV) complexes B1-B10, A4,
MCF-7
3T3
B1
B2
B3
B4
B5
B6
B7
B8
7.55
>200
>100
88.16
>100
85.06
>200
60.00
>100
55.21
>100
84
22.00
22.61
18.99
9.34
4.86
9.20
8.60
13.36
7.40
30.44
18.18
10.00
11.60
B9
B10
A4
A5
cisplatin
Oxaliplatin
169
8
71
2.7. Antitumor activities in vivo
We also conducted the antitumor activities in vivo to know
whether the target compounds sensitive to low pH and cellular
reductants in cancer cells in vitro can also perform the properties of
decreased toxicity. The acute toxicity study was done to evaluate
the potential safety of the glycosylated Pt(IV) compounds. Kunming
mice were administered intravenously (i.v.) and the experiment
was proceeded for 2 weeks. The maximum tolerated dose (MTD)
and the lethal dosage values (LD50) were confirmed and the results
that B6 and B8 arrested the cell cycle at the G2/M phases in a time-
dependent manner. Meanwhile, quantification of apoptosis in HeLa
cells using an annexin V/PI assay is showed in Table 3. Annexin V/PI
coupled flow cytometric analysis at 10 mM after 30 h incubation in
HeLa showed that B7 can efficiently induce apoptosis by prompting
a larger population of cells to undergo early apoptosis (23.7%), late
apoptosis (42.3%) and necrosis (12.8%) with the sum of 78.8%
compared with cisplatin (35.5%) and A4 (59.8%) as shown in Table 3,
respectively. And also the quantification of 51.4% was observed after
incubation with B1 in HeLa cells. As for the apoptosis-inducing
properties, the carbohydrate platinum(IV) complexes are also su-
perior to cisplatin at the same concentration for 30 h.
were summarized in Table 4. The ratios of the cytotoxicity (IC50
)
toward MCF-7 to animal lethal dosage values (LD50) of the studied
complexes were used as a measure for the therapeutic index.
The MTD and LD50 for B6, B7 and B8 are nearly 5-fold higher
than that of oxaliplatin indicating that the glycosylated plati-
num(IV) complexes may have significantly enhanced the feasibility
and potential safety of high-dose treatment. The results show that
B6 (TI ¼ 37.04) B7 (TI ¼ 20.22) and B8 (TI ¼ 21.86) exhibit a ther-
apeutic index over 5e11 folds higher than that of clinical drug
oxaliplatin (TI ¼ 6.51) and the reported bis-functionalized plati-
num(IV) prodrugs A4 (TI ¼ 3.02) and A5 (TI ¼ 5.37).
2.6. The reduction of Pt(IV) complexes
As far as we know, the Pt (IV) compounds are converted to Pt(II)
in the existence of ascorbic acid (Vc) or GSH and then combined
with DNA. The combination of DNA and Pt(II) complexes then
induce the death of cancer cells. In order to investigate the mech-
anism of the glycosylated Pt(IV) compounds, B8 was incubated with
and without Vc in the existence of 5’-dGMP (Fig. 4). After 24 h, a
new peak was observed for both oxaliplatin and B8 with Vc and the
peak became clear at 48 h and 72 h. The adduct was isolated and
After the acute toxicity study, we also did the further evaluations
in vivo with oxaliplatin as control to investigate the antitumor ac-
tivity of the glycosylated Pt(IV) complexes. The in vivo activities
were conducted on MCF-7 tumour mice models. NOD/SCID mice
bearing MCF-7 tumours were treated with B6, B7, B8 and oxali-
platin at the dose of 5 mg Pt/kg. PBS was also used as negative
control. Tumour growth as a function of time and the tumour

50
J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
Table 3
Quantification of apoptosis in HeLa cells using an annexin V/PI assay.
Compd.
Early apoptosis
Late apoptosis
Necrosis
Sum
B1
B7
A4
Cisplatin
Untreated
6.1
23.7
8.3
34.6
0.2
9.1
36.2
12.8
39.6
0.3
51.4
78.8
59.8
35.5
0.5
42.3
11.9
0.6
0.2
0.1
weight in each group at the end of the experiment were recorded.
The bodyweight during the treatments and the images of tumours
at the end of the experiment were also shown in Fig. 5. The results
indicated that the glycosylated Pt(IV) complexes B6, B7 and B8
show significant inhibition to the growth of tumour compared with
the negative control and the tumour control rates of B6, B7 and
oxaliplatin are 41.2%, 52.9% and 64.7%. And it is important that the
bodyweight of mice in Fig. 5 indicated that the glycosylated Pt(IV)
compounds exhibited the deduced toxicity compared with oxali-
platin. The results reveal that the glycosylated Pt(IV) complexes B7
exhibited the important antitumor activities in vivo with a higher
MTD, LD50, TI and comparative tumour control rates compared with
oxaliplatin.
e c l l )
2 * g 1 n / 0 ( t e P a k u p t
5
2.8. Cyclic voltammograms
Subsequently, we want to know whether the carbohydrate-
conjugated platinum(IV) complexes with mono-functionalized li-
gands would possess both pH and redox dual-responsive properties
in the cancer cells, the cyclic voltammograms of B1-B10 in phos-
phate buffer-0.1 M KCl were done (Fig. 6). By mimicking the narrow
pH range of cancer cells, it was found that the reduction potential of
B1-B10 behaved relative differences at pH 6.4 with the scan rate of
200 mv/s as shown in Fig. 6.
Importantly, the peak current and peak potential of B1, B6, B7
and B8 with the scan rate of 200mv/s at pH 6.4 for the microen-
vironment of cancer cells and 7.4 for microenvironment of the
normal cells made great differences. As shown in Table 5, the peak
current of B1, B6, B7 and B8 with the scan rate of 200mv/s at the
concentration of 0.08 mM was 5-fold higher at pH 6.4 than the pH
7.4 and the peak potential also made differences. But the peak
current of the bis-functionalized platinum(IV) prodrugs A4 and A5
at pH 6.4 is.
e c l l )
5 * 1 n 0 ( g t / e P a k u p t
5
almost the same with that of 7.4. And also the peak current of
mono-functionalized platinum(IV) prodrugs B1, B6, B7 and B8 at
pH 6.4 is 2.3e3.2 times higher than that of the bis-functionalized
platinum(IV) prodrugs A4 and A5, indicating the pH and redox
dual-responsive properties in the cancer cells. The results revealed
that the glycosylated Pt(IV) complexes may be easier to be reduced
at pH 6.4 than pH 7.4, which is consistent with the results that the
carbohydrate-conjugated platinum(IV) complexes with mono-
functionalized ligands exhibited more potent cytotoxicity in
seven different human cancer cell lines and low toxicity to 3T3. All
of the results indicate the potential safety of the mono-sugar
conjugates.
3. Conclusions
Ten novel carbohydrate-conjugated platinum(IV) complexes
with mono-functionalized ligands were designed, synthesized and
evaluated the antitumor activity in vitro and in vivo. Pt(IV) com-
plexes conjugated with different carbohydrate behaved different
antitumor activities to different cancer cells. Importantly, the
mono-functionalized carbohydrate-conjugated platinum(IV) com-
plexes also showed more safety and more active to cancer cells than
e c l l )
5 * g 1 n / 0 ( t P e k a u p t
5

J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
51
Fig. 4. A. Reaction of oxaliplatin with 5’-dGMP with and without ascorbic acid incubated at 370C after 24 h, 48 h and 72 h. B. Reaction of B8 with 5’-dGMP after 24 h, 48 h and 72 h.
Table 4
In vivo maximum tolerated dose and lethal dosage values, and calculated therapeutic indices (LD50/IC50) of complexes B6 and oxaliplatin.
Complexes
MTD (mg/kg)
LD50 (mg/kg)
LD50
(
mM/kg)
48 h Avg IC50
(m
M)
LD50/IC50
B6
B7
B8
85
75
90
16
165
150
170
30
180
186
188
4.86
9.20
8.60
11.60
37.04
20.22
21.86
6.51
oxaliplatin
75.56
the bis-functionalized ones in vitro and in vivo, which supported its
clinical development to become a new class of Pt(IV) antitumor
agent.
The platinum(IV) complexes with mono-functionalized ligands
also displayed a more positive shift of reduction properties at PH
6.4 than the bis-functionalized platinum(IV) prodrugs, which
indicated that the reduced pHe in the cancer microenvironment
would facilitate reduction of the platinum(IV) complexes to release
the active platinum(II) complexes. Thus, when designing new
Pt(IV) pro-drugs with axial ligands, the attractive biological per-
formance of glycosylated Pt(IV) complexes with mono-
functionalized ligands should be considered and could prove to
be of much prime importance in future development of platinum
antitumor drugs. The report of platinum(IV) anticancer prodrugs-
hypotheses and facts pointed out that the bioactive axial ligands
of the “dual action” prodrugs often performed the expected tasks
inside the cells. More in vivo studies are needed in order to gain a
better perspective on this approach [24].

52
J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
Fig. 5. In vivo antitumor activities of compounds B6, B7, B8 and oxaliplatin in MCF-7. (A) Tumour growth as a function of time. (B) The tumour weight in each group at the end of the
experiment. (C) The bodyweight of mice during the treatments. (D) The images of tumours at the end of the experiment.
Fig. 6. Cyclic voltammograms of B1-B10 in phosphate buffer-0.1 M KCl at two different pH values. (a). The cyclic voltammograms of B1-B10 with the scan rate of 200mv/s at pH 6.4.
(b).The cyclic voltammograms of B1, B2, B6, B7 and B8 with the scan rate of 200mv/s at pH 6.4 and 7.4.
Table 5
4. Experimental
The peak current (1e-5A) and peak potential (mV) of B1, B6, B7, B8, A4 and A5 with
the scan rate of 200 mv/s at the concentration of 0.08 mM.
4.1. Materials and methods
B1
B6
B7
B8
A4
A5
Cisplatin and oxaliplatin was purchased from Yurui chemical Co.
Ltd (Shanghai, China). All other chemicals obtained from com-
mercial suppliers were used as received and were of analytical
grade. If necessary, the reactions were carried out in dry solvents
and under argon atmosphere. 1H NMR and 13C NMR spectra were
recorded on a Bruker AVANCE AV400 (400 MHz and 100 MHz).
High resolution mass spectra (HRMS) were obtained on an IonSpec
Peak current
pH ¼ 7.4 (1e-5A)
ꢀ6.7
ꢀ5.5
ꢀ5.2
ꢀ5.3
ꢀ7.5
ꢀ9.9
ꢀ610
ꢀ563
ꢀ7.6
ꢀ9.8
ꢀ610
ꢀ563
Peak current
pH ¼ 6.4 (1e-5A)
Peak potential
pH ¼ 7.4 (mV)
Peak potential
pH ¼ 6.4 (mV)
ꢀ32.1
ꢀ604
ꢀ500
ꢀ26.7
ꢀ600
ꢀ540
ꢀ23.9
ꢀ590
ꢀ540
ꢀ25.5
ꢀ590
ꢀ540

J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
53
QFT mass spectrometer with ESI ionization.
amide bond from peracetyl rhamnose, mannose and galactose with
a propyl amino or ethyl amino linker at the reducing end and a
carboxylic function in the Pt(IV) complexes (B11 and B12) (Scheme
2). The key step involved the preparation of oxoplatin B13 and B14
which was reacted successively in water (12 mL) with cisplatin
(0.2 g) or oxaliplatin (0.2 g) and H2O2 (20 mL) at 60 ꢂC with the
yield of 60% and 55% respectively. B11 and B12 were prepared from
oxoplatin B13 (1equiv) or B14 (1equiv) and succinic anhydride
(1equiv) in anhydrous DMSO at room temperature overnight.
DMSO was removed under vacuum to afford a yellow oil and the
product was washed with acetone and diethylether, and dried in
vacuum. The target compounds B11 and B12 were obtained as a
pale yellow solid after purification by recrystallization with the
yield of 75% and 78% respectively. Finally, to a solution of B11 or B12
(1equiv) in DMF was added a DMF solution containing HATU. The
mixture was stirred for 10 min at room temperature. A DMF solu-
tion containing amines and DIPEA was added to the resulting so-
lution to obtain B1 to B10 in yields of 20e50%, respectively. The
mixture was stirred at room temperature for 24 h in the dark. The
product was characterized using 1H-NMR, 13C-NMR, ESI-MS spec-
trometry and elemental analysis (see ESIþ). And meanwhile, the
synthesis methods of A4 and A5 which have been reported by our
group recently with an increasing antitumor activities and a po-
tential safety compared with cisplatin and oxaliplatin are also listed
in Scheme 2.
3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazoliumbromide
(MTT), ascorbic acid (AsA), 50-GMP (purity > 98.0%), DMEM (for 3T3
cells and HepG-2 cells) and RPMI1640 (for A549, A549 R, HeLa,
LNCaP, MCF-7 and PC3 cells) medium containing 10% fetal bovine
serums were purchased from GL Biochem Ltd. Genomic DNA Mini
Preparation Kit from Beyotime, China was used for cellular drug
uptake and DNA platination and Annexin V-FITC Apoptosis Detec-
tion Kit from KeyGEN Biothch, China was used for Annexin V/PI
coupled flow cytometric analysis. Phosphate buffered saline (PBS)
contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 2 mM
KH2PO4. Fetal bovine serum (FBS), 0.25% trypsin/EDTA solutions,
and penicillin-streptomycin solutions were purchased from Invi-
trogen (Grand Island, NY, USA). HeLa (Cervical), MCF-7 (breast
cancer), A549 (lung carcinoma), HepG-2 (hepatoma cell), LNCaP
(Prostate), PC3 (Prostate) cells and 3T3 (mouse embryo fibroblasts)
were obtained from Prof. Yanming Wang (College of Pharmacy,
Nankai University, Tianjin, China). A549R cells were maintained
with 2 mg/mL cisplatin.
HPLC analyses were performed as on an Waters E2695-2998
system equipped with a Venusil MP C18 column (150 ꢁ 4.6 mm,
5 mm). HPLC profiles were recorded by UV detector at 273 nm at
room temperature. The mobile phase consisted of MeOH and H2O
was used and the flow rate of 1 mL/min.
4.2. In vitro cellular cytotoxicity assays
4.4.1. Preparation of B1
Cells seeded in 96-well plates were incubated in a 5% CO2 at-
The complexes B11 was synthesized as previously described. To
a solution of B11 (1equiv) in DMF was added a DMF solution con-
taining HATU (1.5equiv). This mixture was stirred for 10 min at
room temperature. To the resulting solution was added a DMF so-
lution containing S10 (1.2equiv) and DIPEA (2.4equiv). The mixture
was stirred at room temperature for 24 h in the dark. The DMF was
then removed under vacuum to afford a yellow oil. Compound B1
was purified by silica gel column chromatography as yellow solid in
mosphere in 100
100 L freshly prepared culture medium containing drugs at
different concentrations were added and incubated for another
48 h. MTT (5 mg/mL, 20 L) was added and incubated for 3 h.
Finally, the medium was removed and DMSO (150 L) was added.
m
L of complete medium at 37 ꢂC for 24 h. Then
m
m
m
The absorbance was measured at 570 nm using a Bio-Rad 680
microplate reader. The IC50 values were calculated using GraphPad
Prism software, which were based on three parallel experiments.
yield of 20%. 1H NMR (400 MHz, MeOD)
d 5.30e5.13 (m, 1H), 5.00
(dd, J ¼ 13.8, 5.8 Hz, 1H), 4.76 (q, J ¼ 8.8 Hz, 1H), 3.93e3.68 (m, 2H),
3.62e3.19 (m, 4H), 2.73e2.40 (m, 4H), 2.27e1.91 (m, 9H), 1.82 (ddd,
J ¼ 13.7, 10.7, 6.7 Hz, 2H), 1.42e1.02 (m, 3H). 13C NMR (100 MHz,
4.3. In vivo antitumor assay
Kunming mice (28-42d) for the acute toxicity study were pur-
chased from Laboratory Animal Center, Academy of Military Med-
ical Science (Beijing, China). All animals received care in compliance
with the guidelines outlined in the Guide for the Care and Use of
Laboratory Animals. The maximum tolerated dose (MTD) evaluated
by calculating body weight loss (mean weight loss < 15% and <15%
toxic deaths) and the lethal dosage values (LD50) were determined.
BALB/c-nu mice (28-42d) were purchased from Laboratory Animal
Center, Academy of Military Medical Science (Beijing, China). All
animals received care in compliance with the guidelines outlined in
the Guide for the Care and Use of Laboratory Animals.
MeOD) d 183.04, 175.74, 171.92, 171.90, 171.82, 98.96, 72.33, 71.19,
71.00, 67.78, 67.00, 37.91, 37.86, 33.07, 30.31, 20.89, 20.81, 20.75,
17.92. HRMS: Calcd. for C19H35Cl2N3O12Pt (Mþ): 762.1246, found:
0.762.1291. Elemental analysis, found C 29.67%; H 4.68%; N 5.62%,
calcd for C19H35Cl2N3O12 Pt C 29.89%; H 4.62%; N 5.50%.
4.4.2. Preparation of B2
B2 was synthesized according to B1 with the yield of 35%. 1H
NMR (400 MHz, MeOD)
d
5.17 (ddd, J ¼ 13.4, 6.7, 2.5 Hz, 1H), 4.97
(dd, J ¼ 18.6, 8.7 Hz, 1H), 3.97e3.36 (m, 5H), 3.00e2.15 (m, 8H), 1.98
(dd, J ¼ 56.4, 18.1 Hz, 9H), 1.83e1.00 (m, 13H). 13C NMR (100 MHz,
The MCF-7 single-cell suspension in PBS (5 ꢁ 106 per mouse)
was injected subcutaneously into the buttock of the mouse. When
the tumour grew to a size of 80e150 mm3 at 14 days after cell
implantation, the mice were randomly divided into five groups
(PBS, oxaliplatin 5 mg Pt/kg, B6 5 mg Pt/kg, B7 5 mg Pt/kg and B8
5 mg Pt/kg), with all drugs administered intravenously via tail vein.
The drugs were given 5 times at 3-days intervals. Tumour growth
was monitored by measuring the perpendicular diameter of the
tumour using calipers every two days. Five days after the last
treatment, animals were sacrificed and tumours were excised.
MeOD) d 182.17, 174.11, 170.36, 170.34, 170.26, 165.51, 165.43, 97.47,
70.80, 69.44, 66.26, 65.47, 61.65, 36.34, 31.72, 31.12, 30.91, 28.78,
23.77, 23.68, 19.31, 19.29, 19.23, 16.41. HRMS: Calcd. for
C
27H43N3O16Pt (Mþ): 860.2291, found: 0.860.2323. Elemental
analysis, found
C
37.63%;
H
5.12%;
N
4.83%, calcd for
C27H43N3O16 Pt C 37.68%; H 5.04%; N 4.88%.
4.4.3. Preparation of B3
B3 was synthesized according to B1 with the yield of 25%. 1H
NMR (400 MHz, MeOD)
d
5.18 (dtd, J ¼ 12.4, 3.4, 1.7 Hz, 2H),
5.05e4.91 (m,1H), 3.93e3.80 (m,1H), 3.78e3.66 (m,1H), 3.61e3.36
(m, 2H), 3.26 (dt, J ¼ 3.3, 1.6 Hz, 2H), 2.59e2.53 (m, 2H), 2.48e2.37
(m, 2H), 2.13e1.81 (m, 9H), 1.18e1.06 (m, 3H). 13C NMR (100 MHz,
4.4. Synthesis of target compounds
The carbohydrate-conjugated mono-functionalized plati-
MeOD)
d 183.69, 176.07, 172.03, 171.92, 171.89, 98.89, 72.45, 71.08,
num(IV) prodrugs (B1-B10) were prepared by the reaction of an
70.99, 67.82, 67.56, 40.36, 32.98, 27.23, 20.92, 20.87, 20.82, 17.96.

54
J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
HRMS: Calcd. for C18H33Cl2N3O12Pt (Mþ): 748.1089, found:
748.1138. Elemental analysis, found C 28.88%; H 4.57%; N 5.67%,
calcd for C18H33Cl2N3O12 Pt C 28.85%; H 4.44%; N 5.61%.
4.4.9. Preparation of B9
B9 was synthesized according to B1 with the yield of 30%. 1H
NMR (400 MHz, MeOD)
d 5.40e5.02 (m, 2H), 4.29e4.09 (m, 2H),
3.93 (d, J ¼ 14.9 Hz, 1H), 3.75 (d, J ¼ 11.7 Hz, 2H), 3.51 (d, J ¼ 4.7 Hz,
4.4.4. Preparation of B4
4H), 2.47 (s, 4H), 2.20e1.89 (m, 12H), 1.79 (d, J ¼ 6.0 Hz, 2H). 13
C
B4 was synthesized according to B1 with the yield of 35%. 1H
NMR (100 MHz, MeOD) d 172.24, 172.19, 171.69, 171.63, 171.60,
NMR (400 MHz, MeOD)
d
5.55e5.22 (m, 4H), 4.36 (d, J ¼ 7.0 Hz,1H),
171.52, 102.14, 72.53, 72.24, 70.55, 70.44, 69.02, 64.92, 62.73, 46.47,
40.70, 40.39, 20.94, 20.89, 20.74, 20.63. HRMS: Calcd. for
C
4.24e4.11 (m, 2H), 3.87 (d, J ¼ 4.1 Hz, 2H), 3.71 (s, 4H), 3.57 (s, 2H),
2.90 (d, J ¼ 10.4 Hz, 2H), 2.44e2.13 (m, 9H), 1.89 (d, J ¼ 69.2 Hz, 5H),
1.49 (dd, J ¼ 27.8, 10.6 Hz, 3H), 1.37e1.02 (m, 1H). 13C NMR
21H37Cl2N3O14Pt (Mþ): 820.1297, found: 820.2892. Elemental
analysis, found
C
30.75%;
H
4.61%;
N
5.19%, calcd for
(100 MHz, MeOD)
d
170.96, 170.60, 170.40, 162.64, 97.38, 70.41,
C21H37Cl2N3O14 Pt C 30.70%; H 4.54%; N 5.12%.
69.39, 69.28, 66.74, 66.20, 44.43, 38.87, 28.16, 19.27, 19.23, 16.40.
HRMS: Calcd. for C26H41N3O16Pt (M þ NHþ4 ): 864.2481, found:
864.2424. Elemental analysis, found C 36.86%; H 4.79%; N 4.93%,
calcd for C26H41N3O16 Pt C 36.88%; H 4.88%; N 4.96%.
4.4.10. Preparation of B10
B10 was synthesized according to B1 with the yield of 38%. 1H
NMR (400 MHz, MeOD) 5.41e4.96 (m, 3H), 4.34e4.02 (m, 3H),
d
3.93e3.36 (m, 4H), 2.98 (s, 3H), 2.66e2.20 (m, 4H), 2.02 (ddd,
J ¼ 32.0, 25.6, 22.2 Hz, 12H), 1.80e1.02 (m, 8H). 13C NMR (100 MHz,
4.4.5. Preparation of B5
B5 was synthesized according to B1 with the yield of 40%. 1H
MeOD) d 183.67,175.22,172.27,172.14,171.68,171.66,167.02,166.95,
NMR (400 MHz, MeOD)
d
5.25 (dd, J ¼ 7.9, 4.4 Hz, 2H), 4.46e3.97
102.33, 72.51, 71.95, 70.62, 69.05, 68.82, 62.73, 40.60, 37.59, 32.70,
30.64, 25.30, 25.22, 20.93, 20.75, 20.67, 20.65, 14.60. HRMS: Calcd.
for C29H45N3O18Pt (Mþ): 918.2346, found: 918.2398. Elemental
(m, 4H), 3.87e3.42 (m, 6H), 3.01 (d, J ¼ 2.9 Hz, 1H), 2.82e2.32 (m,
3H), 2.24e1.90 (m, 12H), 1.82 (dd, J ¼ 12.0, 5.3 Hz, 2H). 13C NMR
(100 MHz, MeOD)
d 183.69, 176.07, 172.03, 171.92, 171.89, 98.89,
analysis, found
C
37.95%;
H
4.99%;
N
4.64%, calcd for
72.45, 71.08, 70.99, 67.82, 67.56, 40.36, 32.98, 27.23, 20.92, 20.87,
20.82, 17.96. HRMS: Calcd. for C21H37Cl2N3O14Pt (Mþ): 820.1300,
found: 0.820.2892. Elemental analysis, found C 30.74%; H 4.63%; N
5.19%, calcd for C21H37Cl2N3O14 Pt C 30.70%; H 4.54%; N 5.12%.
C
29H45N3O18 Pt C 37.91%; H 4.94%; N 4.57%.
4.5. Cellular platinum uptake and DNA platination
The cellular uptake of cisplatin, oxaliplatin, the compounds
were measured on HeLa and LNCaP cells. HeLa and LNCaP cells
were seeded in 6-well plates overnight and then incubated with
4.4.6. Preparation of B6
B6 was synthesized according to B1 with the yield of 48%. 1H
NMR (400 MHz, MeOD)
d
5.27 (dd, J ¼ 9.8, 6.8 Hz, 3H), 4.59 (m, 1H),
50 mM drugs in standard culture conditions for 10 h. Then the cells
4.29 (dd, J ¼ 12.3, 4.8 Hz,1H), 4.20e4.03 (m, 2H), 3.90e3.77 (m, 2H),
were washed with PBS buffer for three times, and harvested by
trypsinization. The harvested cells were concentrated and digested
by nitric acid for the ICP-MS. The cell numbers were counted before
the digested. Genomic DNA Mini Preparation Kit was used for the
isolation of DNA in Hela cells and Pt concentration in cellular DNA
in HeLa cells digested by nitric acid was also measured by ICP-MS.
3.56 (td, J ¼ 10.1, 6.1 Hz, 2H), 3.19e2.64 (m, 4H), 2.56e2.42 (m, 2H),
2.27 (d, J ¼ 11.3 Hz, 2H), 2.21e1.94 (m, 12H), 1.93e1.26 (m, 8H). 13
C
NMR (100 MHz, MeOD)
d 176.98, 175.32, 172.59, 171.91, 171.83,
171.78, 171.70, 99.17, 71.00, 70.90, 70.00, 67.47, 67.24, 63.81, 37.88,
37.82, 32.65, 32.45, 30.34, 25.31, 25.24, 20.83, 20.79, 20.76, 20.73.
HRMS: Calcd. for C29H45N3O18Pt (Mþ): 918.2343, found: 918.2380.
Elemental analysis, found C 37.94%; H 4.98%; N 4.64%, calcd for
4.6. Flow cytometric analysis
C
29H45N3O18 Pt C 37.91%; H 4.94%; N 4.57%.
HeLa and LNCaP cells cultured in 6-well plates were treated with
and without drugs at 37 ꢂC. Cells were harvested from adherent
cultures by trypsinization and centrifuged at 1000 rpm for 5 min.
PBS was added to wash the cells. Fixed cells with 70% ethanol in PBS
were collected by centrifugation at 2500 rpm for 3 min, washed
with PBS, and centrifuged as before. Cellular pellets were resus-
4.4.7. Preparation of B7
B7 was synthesized according to B1 with the yield of 37%. 1H
NMR (400 MHz, MeOD)
d 5.44e5.12 (m, 2H), 4.39e3.95 (m, 3H),
3.95e3.71 (m, 2H), 3.55 (ddd, J ¼ 61.9, 21.5, 4.1 Hz, 2H), 3.27e2.78
(m, 2H), 2.75e2.37 (m, 3H), 2.05 (ddd, J ¼ 34.4, 29.6, 1.9 Hz, 12H),
1.31 (t, J ¼ 7.3 Hz, 1H). 13C NMR (100 MHz, MeOD)
d 172.65, 172.59,
pended in 50
mg/mL propidium iodide in PBS for nucleic acids
171.84, 171.77,171.73, 171.70, 99.08, 70.95, 70.83, 70.06, 67.94, 67.49,
63.78, 49.15, 40.37, 32.80, 32.56, 20.82, 20.76, 20.72, 9.38. HRMS:
Calcd. for C20H35Cl2N3O14Pt (Mþ): 806.1140, found: 806.1208.
Elemental analysis, found C 29.79%; H 4.49%; N 5.26%, calcd for
staining and treated with 100
mg/mL RNaseA. Apoptotic cells were
detected by flow cytometry after staining with Annexin V and
Propidium Iodide (PI) using the AnnexinV-FITC apoptosis detection
kit.
C
20H35Cl2N3O14 Pt C 29.75%; H 4.37%; N 5.20%.
4.4.8. Preparation of B8
4.7. Reduction of Pt(IV) complexes
B8 was synthesized according to B1 with the yield of 35%. 1H
NMR (400 MHz, MeOD)
d
5.35e5.18 (m, 2H), 4.24 (dt, J ¼ 8.6, 4.3 Hz,
To investigate the binding properties of DNA with Pt(II) com-
plexes, 50-GMP was selected as a model of DNA. Oxaliplatin was
incubated with 50-GMP at 37 ꢂC for 24 h, 48 h and 72 h. Further
experiments were designed to test the reduction potential of Pt(IV)
complexes B8 with and without ascorbic acid after 24 h, 48 h and
72 h. Then, the Pt(II) compounds combined with 50-GMP to form
Oxp-Pt(II)-GMP, which were confirmed by HRMS.
1H), 4.16e4.05 (m, 1H), 3.81e3.69 (m, 2H), 3.66e3.54 (m, 2H),
3.52e3.36 (m, 2H), 3.04 (d, J ¼ 21.8 Hz, 1H), 2.92e2.73 (m, 2H),
2.69e2.40 (m, 4H), 2.23 (d, J ¼ 8.9 Hz, 2H), 2.03 (dt, J ¼ 46.6,
22.3 Hz, 12H), 1.80e1.16 (m, 6H). 13C NMR (100 MHz, MeOD)
d
183.67, 181.75, 175.40, 172.59, 171.80, 171.77, 171.70, 99.09, 70.93,
70.03, 67.91, 67.46, 63.76, 63.20, 62.28, 40.34, 33.22, 32.58, 25.30,
25.21, 20.88, 20.85, 20.81, 20.75. HRMS: Calcd. for C28H43N3O18Pt
(Mþ): 904.2187, found: 0.904.2205. Elemental analysis, found C
37.19%; H 4.81%; N 4.71%, calcd for C28H43N3O18 Pt C 37.17%; H
4.79%; N 4.64%.
HPLC analyses were performed on Waters E2695-2998 equip-
ped with a Venusil MP C18 column (150 ꢁ 4.6 mm, 5
mm).
HPLC profiles were recorded by UV detector at 273 nm. Flow
rate: 1 mL/min.

J. Ma et al. / European Journal of Medicinal Chemistry 128 (2017) 45e55
55
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analytical system model CHI 920c potentiostat from CH In-
struments, Inc using a glassy carbon working electrode, platinum
wire auxiliary electrode and an Ag/AgCl (3 M KCl used as a sup-
porting electrolyte) reference electrode. B1-B10 solutions were
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The authors declare no competing financial interest.
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This research was supported by the National Natural Science
Foundation of China (Grants 21102076, 91013013, 31100587 and
21572106) and the Natural Science Foundation of Tianjin (Grant
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