Glycosylated platinum(IV) prodrugs demonstrated significant therapeutic efficacy in cancer cells and minimized side-effects

Article abstract of DOI:10.1039/c6dt02207c

Conjugates (A1-A5) of the Pt(iv) derivative (A6) with amino groups from peracetyl glucose, rhamnose and mannose with a propyl amino or ethyl amino linker at the reducing end were synthesized and exhibited significant therapeutic efficacy in tumour cells, especially for prostate cancer (PCa). The antitumor activities are greatly affected by glycosyl groups. Cytotoxic experiments in vitro indicated that the antitumor activities were increased by 5-fold when its Pt(iv) derivative was conjugated to S18 (IC₅₀ = 4.82 ± 0.45 μM) and by 12-fold when conjugated to S21 (IC₅₀ = 1.9 ± 0.67 μM). The mannose substituted Pt(iv) complexes A4 and A5 were also over an order of magnitude more potent towards HeLa, A549, MCF-7 and PC3 than cisplatin and oxaliplatin. Importantly, the glycosylated Pt(iv) derivatives A4 and A5 displayed potential safety for clinical therapeutic exposure with IC₅₀ of 84 μM and 169 μM compared with cisplatin (IC₅₀ = 8 μM) to 3T3. Cellular uptake and DNA platination are higher than cisplatin and oxaliplatin. ESI-MS analysis of A5 binding to 5′-dGMP revealed that bifunctional DNA lesions were formed. The antitumor activities in vivo showed that the MTD and LD₅₀ for A4 and A5 are nearly 4-fold higher than that of oxaliplatin indicating the potential safety for the glycosylated Pt(iv) complexes.

Full text of DOI:10.1039/c6dt02207c

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The glycosylated platinum(IV) prodrugs demonstrated significant
therapeutic efficacy in cancer cells and minimized side‐effects
a,b,c
a,b,c
a,c
a,c
a,c
Jing Ma,
Qingpeng Wang,
Xiande Yang, Wenpei Hao, Zhonglv Huang, Jiabao Zhang,
a,c
a,b,c
a,b,c
Xin Wang*
and Peng George Wang*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Conjugates (A1‐A5) of the Pt(IV) derivative (A6) with amino group from peracetyl glucose, rhamnose and mannose with a
propyl amino or ethyl amino linker at the reducing end were synthesized and exhibited significant therapeutic efficacy in
tumour cells, especially for prostate cancer (PCa). The antitumor activities are greatly affected by glycosyl groups.
Cytotoxic experiments in vitro indicated that the antitumor activities were increased by 5‐fold when its Pt(IV) derivative
was conjugated to S18 (IC50 = 4.82 ± 0.45 µM) and by 12‐fold when conjugated to S21 (IC50 = 1.9 ± 0.67 µM). The mannose
substituted Pt(IV) complexes A4 and A5 were also over an order of magnitude more potent towards Hela, A549, MCF‐7
and PC3 than cisplatin and oxaliplatin. Importantly, the glycosylated Pt(IV) derivatives A4 and A5 displayed a potential
safety for clinical therapeutic exposure with the IC50 84 µM and 169 µM compared with cisplatin (IC50 = 8 µM) to 3T3.
Cellular uptake and DNA platination are higher than cisplatin and oxaliplatin. ESI‐MS analysis of A5 binding to 5’‐dGMP
revealed that the bifunctional DNA lesions were formed. The antitumor activities in vivo showed that the MTD and LD50 for
A4 and A5 are nearly 4‐fold higher than that of oxaliplatin indicating the potential safety for the glycosylated Pt(IV)
complexes.
can improve the stability and facilitate the intravenous‐to‐oral
switch in cancer chemotherapy. Recently, more and more
1
1
Introduction
favourable Pt(IV) prodrugs have been developed. As shown
in Figure 1, JM216 (Satraplatin) (1), Ormaplatin (2) and JM9 (3)
(
1
2
Iproplatin), have stepped into the clinical trials. Satraplatin,
The platinum‐based drugs cisplatin, carboplatin and oxaliplatin
are widely used in the clinical treatment of cancer.
1,
2
designed to reduce toxicity and overcome acquired resistance
of cisplatin, was effective against various cancer cells in vitro
and orally absorbed through intestinal membrane via
passive diffusion with a slower aquation rate, more
13‐
Nedaplatin, lobaplatin and heptaplatin are only approved in 16
Asia. Cisplatin is one of the most favourable antitumor drugs
17
3, 4
for a variety of solid cancers, such as ovarian, lung, head and
5
potentials in improving stability, decreasing toxicity, increasing
neck. Despite great medical successes of platinum(II)‐based
18‐23
blood‐circulation time than that of cisplatin.
It is also
cytostatics, severe dose‐limiting side effects appeared due to
the accumulation of platinum(II) in healthy tissues such as
nephrotoxicity, myelosuppression, ototoxicity. The applicati‐
on of platinum(II) drugs is limited by the intrinsic or acquired
drug resistance resulting from DNA repair/damage tolerance,
reduced cellular drug uptake and intensive way of
administration. The cisplatin‐based therapeutic strategy for
hormone refractory disease failed because of the resistance
to apoptotic death of the advanced prostate cancer (PCa).
Therefore, we should devote more efforts to find novel
platinum agents with high anti‐tumour potency.
One of the most promising strategies is to develop a series
of platinum(IV) complexes with bioactive ligands. Compared
considered that Pt(IV) compounds regain antitumor activities
via reduction to Pt(II) complexes by reductants like ascorbic
acid and glutathione, which present higher concentrations in
the tumour cells than in blood and normal tissues. Therefore,
the Pt(IV) compounds will undergo fewer substitution
reactions in blood and normal tissues than in cancer cells and
thus improve its targeting properties. The activation order of
platinum drugs by dissociation the leaving group ligands is
dichloride (cisplatin) > oxalate (oxaliplatin) > malonate
6, 7
8
9
10
(
carboplatin), indicating the high cytotoxicity for the Pt(IV)
prodrugs modified by cisplatin. As shown in Figure 1,
Mitaplatin (4) combining the orphan drug dichloroacetate
(DCA) with cisplatin alters the mitochondrial membrane
potential gradient in cancer cells. Moreover, the Pt(IV)
6
with Pt(II) complexes, Pt(IV) complexes have a low‐spin d
electron configuration and exhibit octahedral geometry, which
[
[
[
a] College of Pharmacy, Nankai University, Tianjin 300071, PR China. E‐mail: wangxinnk@nankai.edu.cn,pwang@nankai.edu.cn
b] State Key Laboratory of Elemento‐organic Chemistry, Nankai University, Tianjin 300071,PR China.
c] Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, PR China.
1
13
Electronic Supplementary Information (ESI) available: Details characterization data, H and C NMR spectra of new compounds.
J. Name., 2013, 00, 1‐3 | 1
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compounds (5) and asplatin (6) also show high a series of platinum(IV) prodrugs instead of the Pt(II)
2
4, 25
cytotoxicity.
complexes to improve the stability, increase the lifetime in
It is well known that carbohydrates play a key role in various biological fluids and disfavour the reactions with biological
2
6
biological processes. It was proved that carbohydrate uptake nucleophiles, (2) adjusting the length and steric hindrance of
of cancer cells significantly increased as the reason of their axial ligands to accommodate the reduction potential with a
unrestricted proliferation, known as the “Warburg effect”. positive shift in cancer cells but not the normal cells such as
Dysfunctional mutations may lead to GLUT1 deficiency 3T3, (3) altering their lipophilicity which can affect the ability
syndrome and whereas the overexpression of GLUT1 is a of entering tumour cells before being reduced to the active
prognostic indicator for cancer. Conjugating 2‐deoxy glucose Pt(II) compound to explore the platinum(IV) prodrugs with a
with therapeutic or diagnostic agents was successfully high tumour cellular uptake and DNA platination, (4)
2
7, 28
achieved as the GLUT‐targeted tumour drug delivery.
Sugar–platinum conjugates as enzyme activating compounds for hormone refractory disease.
have been reported playing key roles in the antibody‐directed Based on these observations, we report, for the first time, a
As shown in Figure 1, novel series of sugar conjugated platinum(IV) analogous (A1
our group firstly reported the synthesis, characterization and A5) and evaluate their bioactivities in vitro and in vivo. (Figure
cytotoxicity of novel carbohydrate Pt(II) analogue 2). In order to evaluate the impacting potency of different
compound 7), but the glycosylated platinum(IV) cancer glycosyl moieties to tumour cells, glucose, mannose and
overcoming the failure of cisplatin‐based therapeutic strategy
2
9, 30
enzyme prodrug therapy approach.
‐
a
1
3
(
32
chemotherapy has remained virtually unexplored.
rhamnose were introduced to cisplatin, respectively. To the
In order to overcome the severe side‐effects of cisplatin‐, best of our knowledge, the length and steric hindrance of axial
carboplatin‐ and oxaliplatin‐based chemotherapy, the ligands play an important role in the cytotoxicity to cancer
3
3‐38
glycosylation of platinum(IV) prodrugs were developed. The cells.
And therefore variable aliphatic chains in different
following points should be taken into consideration during the lengths were designed with the aim of exploring suitable linker
development of more potent cisplatin analogues: (1) exploring between sugar and platinum.
O
O
Pt
O
Cl
Pt
Cl
OH
Pt
H2
N
H2
N
H2
N
Cl
Cl
Cl
Cl
Cl
Cl
H N
N
N
3
H
H2
2
OH
O
1
. JM216 (Satraplatin)
2.
Ormaplatin
O
3. JM9 (Iproplatin)
O
AcO
O
O
CHCl2
Cl
O
N
H
n
H3N
H3N
H3N
Cl
O
Pt
Pt
O
H3N
H3N
Cl
Cl
Cl
H3N
Cl
O
Pt
O
CHCl2
OH
O
OH
O
4
. Mitaplatin
5
6
H2
N
HO
HO
HO
Cl
Cl
O
O
Pt
N
2
OH
H
7
Figure 1. Pt(IV) compounds with bioactive ligands: (1) JM216 (Satraplatin), (2) Ormaplatin, (3) JM9 (Iproplatin), (4) Mitaplatin, (5)
Pt(IV) compounds with variation of the length of the aliphatic tail, (6) Asplatin, (7) A carbohydrate‐linked cisplatin analogue.
J. Name., 2013, 00, 1‐3 | 2

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Figure 2. Chemical structures of the glycosylated platinum(IV) prodrugs (A1‐A6), cisplatin, oxoplatin, and oxaliplatin.
Results and Discussion
Synthesis of target compounds
Scheme 1. Synthetic route of target compounds. Conditions and reagents: (a) H
c) HATU, DIPEA, DMF.
2
O
2
(30% w/v) /H
2
O; (b) Succinic anhydride, DMF;
(
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A novel series of sugar conjugated platinum(IV) analogous therapeutic strategy for hormone refractory disease as the
(
A1‐A5) were synthesized via the formation of an amide reason of resistance to apoptotic death to the advanced PCa
bond from peracetyl glucose, rhamnose and mannose with a with the IC50 from 1.9 μM to 30.07 μM, especially the
propyl amino or ethyl amino linker at the reducing end and a compounds A4 and A5 with the IC50 4.82 μM and 1.9 μM
carboxylic function in the Pt(IV) complex. In our case, we respectively. The results enable these compounds to be of
used cis,cis,trans‐[PtCl
2
(NH
3
)
2
(succinate)] (A6) as the much potential for further investigations as new antitumor
precursor for the synthesis of the conjugates. A6 was agents.
prepared from oxoplatin (1equiv) and succinic anhydride
(
4equiv) in anhydrous DMF at 75℃. After 4h reaction, DMF
Cell viability of 3T3
was removed under vacuum to afford a yellow oil. And then
the oil was washed with dichloromethane and diethylether,
and dried in vacuum. The target complex A6 was obtained as
a pale yellow solid after purification by recrystallization. The
key step involved the preparation of oxoplatin which was
reacted successively in water (12 mL) with cisplatin (0.2 g)
and H O (20 mL) at 60℃. Finally, to a solution of A6 (1equiv)
2 2
in DMF was added a DMF solution containing HATU. The
mixture was stirred for 10 min at room temperature. A DMF
The antitumor activities in vitro indicated that the
glycosylated Pt(IV) compounds A4 and A5 behaved enhanced
cytotoxicity to a variety of cancer cells. To evaluate the
potential safety of the sugar conjugates A4 and A5, the
inhibitory effect (IC50 in µM) of A4 and A5 on normal cell
(
3T3) was done. Compared with cisplatin (IC50 = 8 µM) and
oxaliplatin (IC50 = 71 µM), A4 (IC50 = 84 µM) and A5 (IC50
69 µM) showed an obvious lower toxicities to 3T3. As to A4
=
1
solution containing S5
added to the resulting solution to obtain A1
A5 in yields of 20‐30%, respectively. The mixture was stirred
,
S10
,
S13
,
S18 or S21 and DIPEA was
A2 A3 A4 and
and A5, cell viability of 3T3 is superior to the control group
cisplatin and oxaliplatin (Figure 3).
,
,
,
at room temperature for 24 in the dark. The product was
1
13
characterized using H‐NMR, C‐NMR, HRMS spectrometry
Cell viability of 3T3
and elemental analysis (see ESI).
1
20%
00%
80%
Antitumor activities in vitro
1
Five human cancer cell lines, including lung carcinoma cells
(
A549), cervical cancer (HeLa), prostatic carcinoma (LNCaP
and PC3) and breast carcinoma (MCF7) were used to
evaluated the in vitro antitumor activities of glycosylated
Pt(IV) compounds to determine their potential as anticancer
agents using the MTT assay. The cytotoxicities of cisplatin,
oxoplatin and oxaliplatin were also determined as control. As
listed in Table 1, the cancer cells were exposured to the
6
4
2
0%
0%
0%
platinum(IV) (A1
precursory oxoplatin, the introduction of glycosyl fragments
to Pt(IV) complexes to yield compounds A1 A5 leads rather
–A5) complexes for 48h. In contrast to their
0
%
1
00 42.2 17.8 7.5
3.2
1.3
0.6
0.2
μM
‐
broaden antitumor spectrum and enhanced activities.
Especially, the introduction of mannoside fragments to Pt(IV)
complexes to yield compounds A4 and A5 leads an more
positive antitumor spectrum and activities of these target
compounds in contrast to glucoside and rhamnoside (Table
A4
A5 Cisplatin
Oxaliplatin
Figure 3. Cell viability of 3T3.
1
). These results manifest that different structural Antitumor activities in vivo
carbohydrates show great influence on the bioactivities of
target compounds. Moreover, linkers between glycoside and
platinum core also shade much effects on the antitumor
potency of target compounds.
To evaluate the potential safety of A4 and A5, we also
conducted the antitumor activities in vivo (Table 2). An acute
toxicity study using Kunming mice administered
intravenously (i.v.) was proceeded for 2 weeks. The
maximum tolerated dose (MTD) and the lethal dosage values
Noticeably, conjugate A4 displayed significantly high
cytotoxicity against the five cancer cells, especially for Hela
and LNCaP. A4 showed an IC50 value of 4.82±0.45 μM, which
represents a 5‐fold cytotoxic increase of the oxaliplatin and
about 2‐fold increase of the cisplatin to LNCaP. In addition,
the cytotoxicity against Hela, A549 and PC3 cell lines was
increased by 2‐fold when compared with oxaliplatin. A5 also
showed obvious cytotoxicity to A549, MCF‐7, LNCaP and PC3.
Moreover, A5 showed 12‐fold and 6‐fold cytotoxic increase
compared with clinical drug oxaliplatin and cisplatin against
LNCaP with the IC50 value of 1.9±0.67 μM.
(
LD50) were confirmed and the results were summarized in
Table 2. The MTD and LD50 for A4 and A5 are nearly 4‐fold
higher than that of oxaliplatin indicating that the glycosylated
platinum(IV) complexes may have significantly enhanced the
feasibility and potential safety of high‐dose treatment.
The ratios of the cytotoxicity (IC50) toward LNCaP to animal
lethal dosage values (LD50) of the studied complexes were
used as a measure for the therapeutic index. The results
show that A5 is the safest complex with a therapeutic index
over 16‐fold higher than that of clinical drug oxaliplatin
In addition, glycosylated Pt(IV) compounds (A1
, A3, A4 and
A5) almost fully overcame the failure of cisplatin‐based
4
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Table 1. Inhibitory effect (IC50 in µM) of carbohydrate platinum(IV) complexes on cancer cells.
IC50
(
µM)
A1
A2
A3
A4
A5
Hela
10.32±1.34
6.18±1.70
21.23±0.91
2.81±0.75
8.36±0.91
1.83±0.14
4.96±1.56
25.89±1.09
A549
42.31±1.21
>100
MCF‐7
94.64±2.23
>100
LNCaP
11.37±2.24
65.76±1.78
30.07±2.25
4.82±0.45
1.90±0.67
10.72±1.65
22.76±1.78
>100
PC3
19.92±0.67
51.12±1.12
36.86±0.69
26.35±0.76
41.53±1.56
28.00±1.45
42.93±1.55
50.08±1.61
35.13±1.35
11.71±1.2
24.71±1.35
15.23±1.14
26.07±1.83
>100
72.07±2.95
30.44±2.56
18.18±1.15
28.02±1.01
11.60±0.89
>100
cisplatin
oxaliplatin
oxoplatin
effectively induced cell damage and thus led to cell cycle
indicating that it may potentially allow an adequate margin of arrest and apoptosis in cancer cells.
safety over the in vivo and clinical therapeutic exposure.
Table 3. Quantification of apoptosis in Hela cells using an
Table 2. In vivo maximum tolerated dose and lethal dosage annexin V/PI assay.
values, and calculated therapeutic indices (LD50 / IC50) of
complexes A4, A5 and oxaliplatin.
Early
apoptosis
Late
apoptosis sis
Necro
Compd.
Sum
Complexes MTD
LD50
LD50
(µM
48h
Avg
IC50
LD50
IC50
/
A4
8.3
11.9
0.6
39.6
59.8
35.5
0.5
(
mg/kg) (mg/kg)
Cisplatin
Untreated
34.6
0.2
0.3
0.1
/kg)
(
µM)
0.2
A4
60
61
16
120
125
30
91.79 4.82
19.04
51.43
A5
97.71 1.90
Cellular uptake and DNA platination
oxaliplatin
75.56 22.76 3.32
For the cytotoxicity of metal‐based anticancer drugs, the
investigation of the cellular uptake efficiency is of high
relevance to determine the potential activity of the
compounds as well as to understand their mechanism of
Annexin V/PI coupled flow cytometric analysis
The DNA‐flow cytometric studies were conducted to identify action. Subsequently, the mechanism was explored to
the incidence of cell cycle arrest following treatment with A4
investigate the correlation between intracellular platinum
.
A4 arrests the cycle in LNCaP cells in a time‐dependent accumulation and antitumor activity. Intracellular levels of
manner. A large population of cells (24.8%) accumulated at platinum in Hela and LNCaP cells and the platinum in DNA
the G2/M after 30h incubation at 1µM. We found that the were quantified by inductively‐coupled plasma mass
196
glycosylated Pt(IV) compounds A4 effectively entered cancer spectrometry (ICP‐MS) using
cells and arrested the cell cycle at the G2/M phases, exposure to the compounds at 50 µM as shown in Figure 4.
distinctive of that from cisplatin.
The accumulation of platinum in Hela cells after the
Pt detection after a 10 h
Annexin V/PI coupled flow cytometric analysis at 10 µM incubation with A4 was 7.9‐fold and 2.8‐fold higher than that
after 30 h incubation in Hela showed that A4 can efficiently of cisplatin and oxaliplatin, respectively. The platinum in DNA
induce apoptosis by prompting a larger population of cells to isolated from Hela cells were also measured, which showed
undergo early apoptosis (8.3 %), late apoptosis (11.9 %) and 3.6‐fold and 3.1‐fold higher than oxaliplatin and cisplatin,
necrosis (39.6 %) with the sum of 59.8 % compared with respectively. The results indicated that the presence of sugar
cisplatin (35.5 %) as show in Table 3, respectively. All these fragment in Pt(IV) complexes has a positive effect on the
cell‐based experiments clearly indicated that A4 can intracellular accumulation of the Pt(IV) pro‐drug. Under the
same incubation conditions, the evaluated compound A5 also
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C. Cellular drug uptake
LNCaP)
1
000
A. Cellular drug uptake
(Hela)
B. DNA platination
(
3
0
350
00
250
(Hela)
9
8
7
6
5
4
3
2
1
00
00
00
00
00
00
00
00
00
0
3
25
2
0
2
1
00
50
15
1
0
5
0
100
50
0
A5
cisplatin Oxaliplatin
A4
cisplatin Oxaliplatin
A4
cisplatin Oxaliplatin
Figure 4. Cellular drug uptake and DNA platination. Platinum in HeLa and LNCaP cells were determined using ICP‐MS at the 50
µM platinum complex. (A) Platinum in Hela cells, (B) Platinum in DNA (Hela), (C)Platinum in LNCaP cells.
showed 1.9‐fold and 1.4‐fold higher accumulation in LNCaP
cells compared with cisplatin and oxaliplatin, respectively. Figure 5. Cyclic voltammograms of A1
‐
A5 in phosphate
buffer‐0.1 M KCl at two different pH values. (a). The cyclic
voltammograms of A1 A5 with the scan rate of 200 mv/s at
The properties of better DNA targeting efficiency of A4 and
A5 may account for the higher cytotoxicity. Simultaneously,
cellular uptake studies by ICP‐MS confirmed a good
correlation between intracellular platinum accumulation and
antitumor activity.
‐
pH 6.4. (b).The cyclic voltammograms of A4 and A5 with the
scan rate of 200 mv/s at pH 6.4 and 7.4.
The reduction of Pt(IV) complexes
Cyclic voltammograms
The important anticancer activity of cisplatin resulted from
the inherent proficiency to bind with the N7 position of
guanine bases, thus leading to DNA damage and inducing cell
To investigate the conceivable reasons why the introduction
of mannoside fragments to Pt(IV) complexes to yield
compounds A4 and A5 leads an more positive antitumor
spectrum and activities in contrast to glucoside and
rhamnoside, cyclic voltammograms were employed. At the
same time, A4 and A5 also behaved lower toxicity to the
normal cells such as 3T3 compared with the cisplatin and
oxaliplatin. Generally, the intracellular pH is about 7.2,
whereas the extracellular pH about 7.4 in normal cells.
However, the extracellular pH is lower than 7.1 in cancer
cells. And therefore we studied the reduction properties of
A1 to A5 at pH 6.4 to mimic the reduced extracellular pH of
the cancer cells. Simultaneously, the cyclic voltammograms
of A4 and A5 at pH 7.4 were studied to imitate the pH value
of the normal cells.
At pH 6.4, a relative positive shift of the peak current for
A1, A2, A4 and A5 was observed at the concentration of 0.08
mM (Figure 5). Compared with the reduction properties of
A4 and A5 at pH 6.4, the relative negative shift of the peak
current at pH 7.4 was observed. The results indicated that
the peak current from A1 to A5 did not show much
difference. The mechanism of the glycosylated platinum(IV)
3
9
death. In these experiments 5’‐dGMP was applied as a
model of DNA. The reactivity of platinum(IV) pro‐drugs A5
towards 5’‐dGMP was investigated by treatment with
ascorbic acid for RP‐HPLC analysis at 37 °C in presence of
4
0‐41
reducing ascorbic acid (Vc). The adducts (Figure 6)
were
observed after the incubation of A5, Vc, and 5’‐dGMP for 24h
and 72h (other HPLC results are given in supporting
information). The unknown peak fractions were identified by
ESI‐MS analysis. No visible reaction was observed in the
absence of ascorbic acid, which indicated that the pro‐drugs
were activated upon the reduction by ascorbic acid and
meanwhile glycosylated Pt(IV) compounds exerted their
anticancer activity via the released Pt(II) complexes. Reaction
of A5 in the presence of ascorbic acid was also showed in
Figure 6. After reduction by Vc, cisplatin was formed and
then combined with 5′‐dGMP to generate the bis‐substituted
products. Therefore, the glycosylated Pt(IV) complexes could
be reduced by ascorbic acid. Following such reduction,
glycosylated Pt(IV) compounds exert their anticancer activity
via the released Pt(II) complexes.
3
6
prodrugs (A1‐A5) need to be further studied.
6
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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 Detection Kit from KeyGEN Biothch,
China was used for Annexin
analysis. Phosphate buffered saline (PBS) contains 137 mM
NaCl, 2.7 mM KCl, 10 mM Na HPO and 2 mM KH PO . Fetal
Ⅴ/PI coupled flow cytometric
2
4
2
4
bovine serum (FBS), 0.25% trypsin/EDTA solutions, and
penicillin‐streptomycin solutions were purchased from
Invitrogen (Grand Island, NY, USA). Hela (Cervical), MCF‐7
Figure 6. Reaction of A5 with 5′‐dGMP in the presence of
ascorbic acid incubated at 37 °C after 24h.
(
breast cancer), A549 (lung carcinoma), LNCaP (Prostate),
PC3 (Prostate) cells were obtained from Prof. Yanming Wang
(
College of Pharmacy, Nankai University, Tianjin, China).
HPLC analyses were performed as on an Waters E2695‐
Conclusions
2
4
998 system equipped with a Venusil MP C18 column (150 ×
.6 mm, 5 μm). HPLC profiles were recorded by UV detector
In summary, five novel glycosylated platinum(IV) prodrugs
were designed and synthesized. And the biological
evaluations of the glycosylated platinum(IV) prodrugs
indicated that the mannose conjugated platinum(IV)
complexes A4 and A5 exhibited high potent cytotoxicity to
cancer cells, especially to LNCaP. Furthermore, the high
cytotoxic platinum(IV) complexes A4 and A5 showed low
toxicities to the normal cell (3T3). It was worth mentioning
that A5 with a shorter chain compared with A4, exhibited a
better cytotoxicity to LNCaP (IC50 = 1.9 µM). Compared with
cisplatin and oxaliplatin, IC50 value of A5 is higher than 100
µM against 3T3 indicated a low toxicity to normal cells. The
cellular platinum in Hela and LNCaP cells and the DNA
platinum of A4 and A5 indicated that the higher cytotoxicity
could be correlated with its higher DNA targeting efficiency
upon the reduction by ascorbic acid.
at 273 nm at room temperature. The mobile phase consisted
of MeOH and H2O was used and the flow rate of 1 mL/min.
In Vitro Cellular Cytotoxicity Assays
Cells seeded in 96‐well plates were incubated in a 5% CO
atmosphere in 100 µL of complete medium at 37 for 24 h.
Then 100µL freshly prepared culture medium containing
drugs at different concentrations was added and incubated
for another 48 h. MTT (5 mg/mL, 20 µL) was added and
incubated for 3 h. Finally, the medium was removed and
DMSO (150µL) was added. The absorbance was measured at
2
℃
5
70 nm using a Bio‐Rad 680 microplate reader. The IC50
values were calculated using GraphPad Prism software,
which were based on three parallel experiments.
Interestingly, A4 and A5 overcame the failure of cisplatin‐
based therapeutic strategy for hormone refractory disease,
which probably resulted from the different cellular responses
of the mannose substituted platinum(IV) complexes relative
to cisplatin. The favourable biological results of the mannose
conjugated platinum(IV) complexes supports its clinical
development to become a new class of Pt(IV) antitumor
agent. These studies provide new insights into the potential
of the glycosylated Pt(IV) anticancer prodrugs, particularly on
how multivalency can improve both the selectivity and
potency of such metallodrugs by increasing cellular
accumulation in tumour tissues.
In vivo antitumor assay
Kunming mice (28‐42 d) 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. 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. The value of TI was calculated
according to the literature.
4
2
Flow cytometric analysis
Experimental Details
Materials
HeLa and LNCaP cells cultured in 6‐well plates were treated
with and without drugs at 37 °C. 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
Cisplatin and oxaliplatin was purchased from Yurui chemical
Co. Ltd (Shanghai, China). All other chemicals obtained from
commercial suppliers were used as received and were of
analytical grade. If necessary, the reactions were carried out
2
500 rpm for 3 min, washed with PBS, and centrifuged as
before. Cellular pellets were resuspended in 50 µg/mL
propidium iodide in PBS for nucleic acids staining and treated
with 100 µg/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.
1
13
in dry solvents and under argon atmosphere. H NMR and
C
NMR spectra were recorded on a Bruker AVANCE AV400 (400
MHz and 100 MHz). High resolution mass spectra (HRMS)
were obtained on an IonSpec QFT mass spectrometer with
ESI ionization. 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetra‐
zolium bromide (MTT), ascorbic acid (AsA), 5’‐GMP (purity ≥
Cellular Platinum Uptake and DNA Platination
9
8.0%), DMEM (for 3T3 cells) and RPMI1640 (for A549, Hela,
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DOI: 10.1039/C6DT02207C
The cellular uptake of cisplatin, oxaliplatin, A4 and A5 was 69.86, 69.63, 66.65, 66.23, 37.60, 32.47, 29.89, 28.85, 22.82,
+
measured on HeLa and LNCaP cells. HeLa and LNCaP cells
were seeded in 6‐well plates overnight and then incubated
with 50 µM drugs in standard culture conditions for 10 h.
Then the cells 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.
2
1
3
1.14, 20.99, 17.49. Calcd. for C38
191.2878, found: 1191.2777. Elemental analysis, found % C,
8.33; H, 5.30; N, 4.60. calcd. for C38 22Pt C, 38.26 ;
2 4
H62Cl N O22Pt (M ):
H
2 4
62Cl N O
H, 5.24; N, 4.70.
Preparation for A3
Compound A3 was prepared according to the procedure
described for compound A1. H NMR (400 MHz, CDCl ) δ 6.58
1
3
(
4
d, J = 274.5 Hz, 6H), 5.37 – 4.64 (m, 6H), 3.62 (dd, J = 133.1,
8.2 Hz, 9H), 2.58 (d, J = 55.1 Hz, 6H), 2.33 – 1.50 (m, 18H),
Cyclic Voltammetry
13
Electrochemical measurements were made at 25 °C on an 1.02 (d, J = 136.3 Hz, 13H). C NMR (100 MHz, CDCl ) δ
3
analytical system model CHI 920c potentiostat from CH
Instruments, Inc using a glassy carbon working electrode,
platinum wire auxiliary electrode and an Ag/AgCl (3 M KCl
1
6
2
81.94, 173.50, 170.43, 170.29, 170.02, 97.32, 70.74, 69.51,
9.32, 66.46, 54.03, 53.53, 42.31, 39.09, 29.64, 20.94, 20.86,
2 4
0.80, 18.62, 17.42, 17.35, 12.17. Calcd. for C36H58Cl N O22Pt
used as a supporting electrolyte) reference electrode. A1
A3 A4 and A5 solutions were prepared in phosphate
buffered saline (PBS) of pH 6.4. A4 and A5 were also
, A2,
+
(
M ): 1163.2565, found: 1163.2526. Elemental analysis,
,
found % C, 37.22; H, 5.08; N, 4.90. calcd. for C36
2 4
H58Cl N O22Pt
prepared for PH 7.4 and cyclic voltammograms were C, 37.12; H, 5.02; N, 4.81.
recorded at the scan rate of 200 mv/s.
Preparation for A4
Synthetic procedures
Compound A4 was prepared according to the procedure
Preparation for A1
1
described for compound A1. H NMR (400 MHz, CDCl
(d, J = 157.4 Hz, 6H), 5.39 – 5.03 (m, 5H), 4.81 (br, 2H), 4.25
br, 2H), 4.10 (d, J = 11.7 Hz, 2H), 3.97 (br, 2H), 3.75 (br, 2H),
3
) δ 6.39
[
43]
The complexe A6 was synthesized as previously described.
(
To a solution of A6 (0.38 mmol) in DMF (10 mL) was added a
DMF solution (0.5 mL) containing HATU (1.52 mmol). This 3.61 – 3.11 (m, 5H), 2.55 (d, J = 37.4 Hz, 8H), 2.23 – 1.49 (m,
1
3
mixture was stirred for 10 min at room temperature. To the
resulting solution was added a DMF solu tion containing S5 (3
mmol) and DIPEA (1.9 mmol). 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 A1
2
7H), 1.19 (dd, J = 15.9, 9.0 Hz, 5H). C NMR (100 MHz,
CDCl3) δ 182.41, 173.59, 171.01, 170.49, 169.92, 97.70, 69.58,
6
2
1
9.45, 68.62, 66.49, 66.17, 62.72, 37.32, 32.35, 32.17, 29.07,
+
1.07, 20.95, 20.90. HRMS: Calcd. for C42
307.2988, found: 1307.3002. Elemental analysis, found % C,
26Pt C, 38.54;
2 4
H66Cl N O26Pt (M ):
was purified by silica gel column chromatography as yellow
1
solid in yield of 20%. H NMR (400 MHz, CDCl
3
) δ 6.41 (d, J = 38.50; H, 5.10; N, 4.29. calcd. for C42
H66Cl
2
N
4
O
1
–
=
46.8 Hz, 4H), 5.47 – 4.79 (m, 14H), 4.64 – 4.08 (m, 5H), 4.02 H, 5.08; N, 4.28.
3.52 (m, 4H), 3.39 (d, J = 69.4 Hz, 3H), 2.55 (s, 5H), 1.96 (t, J
1
3
61.2 Hz, 26H), 1.26 (dd, J = 167.1, 94.1 Hz, 5H). C NMR
) δ 173.56, 173.52, 171.00, 170.38, 170.15,
69.59, 100.82, 90.43, 72.70, 72.00, 71.47, 71.25, 70.00,
8.56, 68.52, 67.37, 62.07, 61.95, 60.49, 37.40, 32.30, 29.85, Compound A5 was prepared according to the procedure
Preparation for A5
(
100 MHz, CDCl
3
1
6
2
1
8.85, 21.01, 20.98, 20.94, 20.91, 20.88, 20.83, 20.75, 14.35.
): 1307.2988, found:
307.2979. Elemental analysis, found % C, 38.58; H, 5.06; N,
.12% calcd. for C42 26 Pt C,38.54 ; H, 5.08; N, 4.28.
described for compound A1. H NMR (400 MHz, CDCl
3
) δ 6.42
2 4 +
HRMS: Calcd. for C42H66Cl N O26Pt (M
1
4
(
d, J = 190.6 Hz, 6H), 5.28 (dd, J = 16.8, 9.1 Hz, 6H), 4.87 (br,
2
3
2
0
1
6
2
C
H), 4.26 (dd, J = 12.2, 4.8 Hz, 2H), 4.14 (d, J = 10.4 Hz, 2H),
.99 (br, 2H), 3.78 (br, 2H), 3.58 (d, J = 9.3 Hz, 3H), 3.41 (br,
H), 2.58 (d, J = 38.5 Hz, 8H), 2.08 (dd, J = 39.8, 21.5 Hz, 24H),
.88 (dd, J = 14.7, 8.2 Hz, 3H). C NMR (100 MHz, CDCl
70.95, 170.82, 170.49, 170.36, 169.77, 97.64, 69.37, 68.68,
7.08, 66.19, 66.02, 62.59, 39.42, 29.67, 29.33, 20.94, 20.88,
0.80, 20.75, 20.70, 20.64, 20.58, 20.53. HRMS: Calcd. for
H
2 4
66Cl N O
Preparation for A2
13
3
) δ
Compound A2 was prepared according to the procedure
described for compound A1. H NMR (400 MHz, CDCl
1
3
) δ 5.28
+
(br, 6H), 5.22 – 4.89 (m, 5H), 4.72 (br, 1H), 3.78 (d, J = 34.5 Hz,
40
H62Cl
2
N
4
O
26Pt (M ): 1279.2675, found: 1279.2678.
4
H), 3.37 (d, J = 54.1 Hz, 8H), 2.63 (d, J = 73.3 Hz, 5H), 1.99
Elemental analysis, found % C, 37.66; H, 4.89; N, 4.40. calcd.
1
3
(
dd, J = 77.6, 46.1 Hz, 21H), 1.21 (d, J = 11.5 Hz, 12H).
C
for C40
H62Cl
2
N
4
O
26Pt C, 37.51; H, 4.88; N, 4.37.
3
NMR (100 MHz, CDCl ) δ 170.83, 170.76, 170.20, 97.45, 70.91,
Supporting Information
8
| J. Name., 2012, 00, 1‐3
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Experimental details for the characterization of the Acta Oncol 2011; 50 (7):1105–1110.
compounds studied are provided in the Supporting 22. Cetnar J, Wilding G, McNeel D, LoConte NK, McFarland TA,
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