Glucose-conjugated platinum(IV) complexes as tumor-targeting agents: design, synthesis and biological evaluation

Article abstract of DOI:10.1016/j.bmc.2019.03.006

A new series of glucose-conjugated Pt(IV) complexes that target tumor-specific glucose transporters (GLUTs) was designed, synthesized, and evaluated for their anticancer activities. All six compounds, namely, A1-A6, exhibited increased cytotoxicity that w

Full text of DOI:10.1016/j.bmc.2019.03.006

Accepted Manuscript
Glucose-conjugated platinum(IV) complexes as tumor-targeting agents: design,
synthesis and biological evaluation
Haifeng Wang, Xiande Yang, Caili Zhao, Peng George Wang, Xin Wang
PII:
S0968-0896(18)32063-7
https://doi.org/10.1016/j.bmc.2019.03.006
BMC 14798
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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28 February 2019
2 March 2019
Please cite this article as: Wang, H., Yang, X., Zhao, C., Wang, P.G., Wang, X., Glucose-conjugated platinum(IV)
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Glucose-conjugated platinum(IV) complexes
as tumor-targeting agents: design, synthesis
and biological evaluation
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Haifeng Wang, Xiande Yang, Caili Zhao, Peng George Wang and Xin Wang*
College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, State Key Laboratory of Elemento-
organic Chemistry, Nankai University, Tianjin 300071 P. R. China

Bioorganic & Medicinal Chemistry
Glucose-conjugated platinum(IV) complexes as tumor-targeting agents: design,
synthesis and biological evaluation
Haifeng Wang a , Xiande Yang a , Caili Zhao a , Peng George Wang a, and Xin Wang a,

a College of Pharmacy, Tianjin Key Laboratory of Molecular Drug Research, State Key Laboratory of Elemento-organic Chemistry, Nankai University, Tianjin
300071 P. R. China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A new series of glucose-conjugated Pt(IV) complexes that target tumor-specific glucose
transporters (GLUTs) was designed, synthesized, and evaluated for their anticancer activities.
All six compounds, namely, A1-A6, exhibited increased cytotoxicity that were almost six fold
higher than that of oxaliplatin to MCF-7 cells. These Pt(IV) complexes can be reduced to release
Pt(II) complexes and cause the death of tumor cells. Simultaneously, the glycosylated Pt(IV)
complexes (30.21–91.33 μM) showed lower cytotoxicity that normal LO2 cells compared with
cisplatin (5.25 μM) and oxaliplatin (8.34 μM). The intervention of phlorizin as a GLUTs
inhibitor increased the IC50 value of the glycosylated Pt(IV) complexes, thereby indicating the
potential GLUT transportability. The introduction of glucose moiety to Pt(IV) complexes can
effectively enhance the Pt cellular uptake and DNA platination. Results suggested glucose-
conjugated Pt(IV) complexes had potential for further study as new anticancer agents.
Available online
Keywords:
Glycosylation
Platinum(IV) complexes
Antitumor
Glucose transporter
Tumor targeting
2009 Elsevier Ltd. All rights reserved.
1. Introduction
Subsequently, Pt(IV) prodrugs should be activated to Pt(II)
complexes through reduction with intracellular reductants, such
Chemotherapy can effectively control or eliminate metastasis
focus. Hence, chemotherapy plays a significant role in the
treatment of advanced malignant and refractory cancers. Since
1978, Pt-based antitumor drugs, which are represented by
cisplatin, carboplatin, and oxaliplatin, have been the first choice
of chemotherapy drugs for malignant tumors, such as testicular
cancer, colorectal cancer, nonsmall cell lung cancer, ovarian
cancer, breast cancer, head and neck cancer, and nasopharyngeal
cancer1. Pt-based antitumor drugs are also one of the most
common components in clinical treatment of multiple
malignancies. However, due to the lack of selection of cancer
cells and the structural characteristics of Pt(II) drugs in cancer
treatment, serious problems still exist with Pt(II) anticancer
drugs, which are accompanied by the toxic side effects, such as
renal toxicity, ototoxicity, neurotoxicity, and alopecia, as well as
the problems of drug and cross resistance1a. Therefore, the
exploration of targeted drugs is important in improving curative
effect and reducing toxicity.
as ascorbic acid and glutathione, which are in excess amounts in
tumor cells3. With attention given to Pt(IV) complexes, many
Pt(IV) complexes with anticancer activity were synthesized. The
most typical representatives are iproplatin, ormaplatin, and
satraplatin (JM216)4. However, most researchers stopped further
research because of their considerable toxicity or indifferent
efficacy5. Through chemical modification, the introduction of
functional molecules into the axial ligands of Pt complexes can
increase the selectivity of Pt(IV) complexes for cancer cells and
improve the pharmacological properties of the drugs6. Targeted
and synergistic sensitization can be achieved by modifying the
axial ligand of the Pt(IV) complexes. Therefore, Pt(IV)
complexes with anticancer activity can overcome the toxic side
effect and drug resistance of traditional Pt(II) anticancer drugs3, 7
.
Oncosis involves not only the removal of control over cell
proliferation but also the corresponding regulation of energy
metabolism to promote cell growth and division. Even under
sufficient oxygen supply, tumor cells mainly obtain energy
through glycolysis, which is called aerobic glycolysis or Warburg
effect6, 8. The abnormal glycolysis of tumor cells can promote
glucose uptake and lactic acid generation, thereby facilitating
tumor occurrence and development9. Therefore, this abnormal
uptake of glucose can be used as a target for cancer treatment.
Many studies have found that Pt(II) glycan complexes have
Pt(IV) complexes have attracted the attention and favor of
2
scientists . Compared with Pt(II) complexes, Pt(IV) complexes
have octahedral configuration, which is characterized by
substitutional kinetics inertia and reduced reactivity. Pt(IV) is
difficult to replace with biomolecules, such as glutathione and
p—ro—tein—in cells. Thus, Pt(IV) can reach cancer cells completely.
 Corresponding author. Tel.: + (86) 022-23501642; fax: + (86) 022-23501642; e-mail: wangxinnk@nankai.edu.cn (X. Wang)

strong antitumor activity and low toxicity and have been proven
to target glucose transporters (GLUTs)8, 10. For the first time, our
group synthesized a series of acetyl protective glycosylated
Pt(IV) complexes and tested whether they target GLUTs by using
phlorizin as a GLUT inhibitor11. Unfortunately, the results
showed that glycosylated Pt(IV) complexes do not target GLUTs.
Subsequently, the unprotected glycosylated Pt(IV) complex is
successfully synthesized and found to be transportable by
GLUT12. Meanwhile, the antitumor activity of glucose-modified
Pt(IV) is superior to that of other sugar types, such as galactose
and mannose.
types12. To improve the stability of the glycosylated portion to
the glucosidase, we selected the C-glycoside bond. C chains with
different lengths were designed to explore the appropriate linker
between the glycoligand and the Pt nucleus. Meanwhile, alkanes
with different lengths were also designed to study their effects on
the activity of glycosylated Pt(IV) complexes 7a, b, 13
.
2. Results and Discussion
2.1. Synthesis of glycosylated Pt(IV) compounds A1-A6
The synthesis of the target compounds is shown in Scheme 1.
For example, in A1, oxaliplatin was oxidized in H2O2 at 60 °C to
obtain oxoplatin, and monocarboxylic acid chain was introduced
under dicyclohexylcarbodiimide condensation by adding 1.4
equivalent of carboxylic acids. The synthesis of glycoligands was
On the basis of the results above, we designed and synthesized
a series of novel glycosylated Pt compounds and evaluated their
biological activities. Glucose was selected as a glycoligand
because its antitumor activity was superior to that of other sugar
Scheme 1. Chemical structures of glycosylated Pt(IV) complexes and synthesis of the title compounds A1 and B.

Table 1. Cytotoxicity profiles of the glycosylated Pt(IV) complexes in five human carcinoma cell lines expressed as IC50 (μM)
Compounds
Hela
HepG-2
8.13±0.07
7.41±0.08
2.75±0.06
6.39±0.10
3.79±0.07
3.02±0.08
40.68±0.34
6.31±0.07
10.90±0.07
MCF-7
3.91±0.06
4.00±0.04
3.00±0.08
4.63±0.10
2.05±0.07
1.79±0.34
68.34±0.64
8.71±0.15
10.10±0.59
A549
A549R
LO2
RFa
1.89
1.51
1.45
1.57
1.53
1.24
1.33
2.80
2.84
SIb
A1
7.36±0.05
6.30±0.07
2.06±0.11
5.42±0.05
3.68±0.08
3.53±0.08
32.68±0.12
4.60±0.58
6.60±0.15
8.23±0.16
6.68±0.08
3.12±0.28
5.44±0.11
4.74±0.16
3.97±0.06
54.65±0.76
11.90±0.08
12.69±0.24
15.63±0.17
10.14±0.11
4.55±0.20
8.59±0.39
7.29±0.14
4.93±0.32
72.56±0.15
33.40±0.32
36.07±0.11
30.21±0.21
44.46±0.09
25.56±0.16
90.34±0.12
91.33±0.07
33.12±0.34
44.32±0.27
5.25±0.23
8.34±0.15
3.72
6.00
9.29
14.14
24.10
10.90
1.09
0.83
0.77
A2
A3
A4
A5
A6
B
Cisplatin
Oxaliplatin
a RF: Resistant factor = IC50(A549R)/IC50(A549). bSI: selectivity index= IC50(LO2)/IC50(HepG-2). IC50 was evaluated by the MTT assay for 48 h. All compounds
are tested in the same batch and the experiments were performed tree times in five replicates. Hela (human cervical cancer cell line), HepG-2 (human liver cancer
cell line), MCF-7 (human breast cancer cell line)， A549 (human lung cancer cell line), LO2 (human normal liver cell line).
described in ESI.† The asymmetrically functionalized Pt(IV)
compound can be prepared by 3 and 6 in dimethyl formamide in
resistance factors of A3 and A6 can be reduced to 1.45 and 1.24,
respectively. The results above suggested that these compounds
had the potential to be further studied as new anticancer agents.
the
presence
of
O-(benzotriazol-1-yl)-N,N,N',N'-
tetramethyluronium tetrafluoroborate and triethylamine. The
removal of benzyl protective groups of 4 by using BCl3 in
anhydrous dichloromethane afforded the corresponding target
A1. B was prepared from 2 (1 equivalent) and caproic anhydride
(6 equivalent) in anhydrous dimethyl formamide at 25 °C. A2–
A6 were prepared according to the procedure described above.
The purity of A1–A6 have been determined by HPLC, which was
shown in ESI.†
2.3. LO2 cell viability
To assess the potential safety of glycosylated Pt(IV)
complexes, we used MTT assay to evaluate the cytotoxicity of
these complexes against normal LO2 cells. We compared the IC50
values of A1–A6 with cisplatin and oxaliplatin in HepG-2 and
LO2 cells (Table 1). The cytotoxicity of glycosylated Pt(IV)
complexes on normal cells was significantly reduced compared
with HepG-2 cells, and the selectivity index of A5 even attained
24.1, which was significantly higher than those of cisplatin and
oxaliplatin (0.83 and 0.77, respectively). When the concentration
range was 0–100 μM, the cell viability of LO2 cells incubated
with glycosylated Pt(IV) complexes was significantly higher than
those of cisplatin and oxaliplatin (Fig. 1). At the concentration of
8.34 μM (oxaliplatin, IC50 = 8.34 μM), the cell viability of LO2
cells corresponding to the glycosylated Pt(IV) complexes was
>70%, especially for A4 and A5, as the corresponding cell
viability of LO2 cells at this concentration was almost 100%.
These results indicated that glycosylated Pt(IV) complexes may
have enhanced the potential safety while maintaining the
antitumor activity.
2.2. Antitumor activities in vitro
The antitumor activity of A1–A6 were assessed by 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay by using five human cancer cell lines. The structure of the
synthesized targets A1–A6 contained comparable carboxyl
ligands. Therefore, di-hexanoyl-Pt(IV)-derivative B was selected
as the control. Cisplatin and oxaliplatin were also used as the
control group. The IC50 values were calculated based on the dose
dependence of surviving cells that incubation to the compounds
for 48 h (Table 1).
A1–A6 showed enhanced activities and a relatively broad
antitumor spectrum compared with cisplatin and oxaliplatin.
Compared with B, the increased activity was attributed to the
introduction of glycosylated modules, which targeted GLUT that
was overexpressed in tumor cells10c, d, 14. In particular, for MCF-7
cells (IC50 = 1.79 to 4.63 μM), all six compounds exhibited the
highest cytotoxicity, which was almost sixfold higher than that of
oxaliplatin. This result may be caused by the high GLUT1
expression in MCF-7 cells15.
The length of the C chain between glycoligand and the Pt core
had a significant influence on the antitumor activity. A4, A5, and
A6 with longer C chains were generally more cytotoxic than A1,
A2, and A3, which have the same alkyl ligands. At the same
time, the cytotoxicity of A1, A2, A3 A4, A5, and A6 enhanced
with the increase in alkane chain. A3 (IC50 = 2.05 to 5.55 μM)
and A6 (IC50 = 2.06–5.93 μM) with octanoic ligand showed
better cytotoxic for five cell lines than those with cisplatin and
oxaliplatin.
Fig. 1. Viability of LO2 cells in a 48 h incubation assay
2.4. Effects of GLUTs inhibitor phlorizin on antitumor activities
To investigate whether the Warburg effect affected the
transport of glycosylated Pt(IV) complexes to cancer cells,
Drug-resistant A549R cells were also more sensitive to Pt(IV)
glycoconjugates than cisplatin and oxaliplatin; even the

2.6. Cellular uptake and DNA platination
we studied the mechanism of A3, A5, A6, cisplatin, and
oxaliplatin in MCF-7 cells by using phlorizin as GLUT
inhibitor. As shown in Fig. 2, the IC50 values of B, cisplatin, and
oxaliplatin showed insignificant difference with and without
phlorizin, which indicated that GLUT inhibitor was unassociated
with the cytotoxicity of B, cisplatin, and oxaliplatin. However,
the IC50 values of A3, A5, and A6 in the presence of phlorizin
increased by approximately fourfold compared with those
without phlorizin. Therefore, the transport of glycosylated
Pt(IV) complexes is regulated by GLUTs.
The cellular uptake of Pt-based anticancer drugs and their
DNA-binding capacity are directly related to the antitumor
activity of the drugs. Therefore, we detected intracellular Pt
accumulation and DNA-binding capacity by using inductively
coupled plasma mass spectrometry (ICP-MS) after MCF-7 cells
was incubated with A4, A5, A6, B, cisplatin, and oxaliplatin at
20 μM for 10 h. As shown in Fig. 3A, the intracellular Pt
accumulation of the glycosylated Pt(IV) complex was 1.7-3.3
fold higher than that of di-hexanoyl-Pt(IV)-derivative B, which
did not contain the glycosylated part. DNA was extracted from
MCF-7 cells, and its Pt content was determined. The results (Fig.
3B) showed glycosylated Pt(IV) complexes exhibited 8.4- to
22.8-fold higher than B. Similarly, for cell uptake and DNA
platination, the glycosylated Pt(IV) complex was higher than
cisplatin and oxaliplatin (Fig. 3). These results indicated that the
introduction of glycosylated components to Pt(IV) complexes
can effectively increase the Pt cellular uptake and DNA
platination. Meanwhile, the cytotoxicity of Pt-based complexes
was correlated with Pt cellular uptake.
Fig. 2 In vitro antitumor activities of compounds A3, A5, A6, B, cisplatin and
oxaliplatin to MCF-7 without and with GLUTs inhibitor phlorizin
(phlorizin，100 μM； Phl: phlorizin; Cis: Cisplatin; Oxa: Oxaliplatin).
2.5. Flow cytometric analysis
To evaluate the apoptosis-inducing effect of glycosylated
Pt(IV) complexes on MCF-7 cells, we performed annexin V/PI
double staining via flow cytometry by using A3, A5, and A6
(10 μM). Pt(IV)-derivative B without saccharide ligand, cisplatin,
and oxaliplatin was used as the control (Fig. 4). As shown in
Table 2, A6 induced 51.83% of apoptosis of MCF-7 cells (9.89%
of early apoptosis, 38.6% of late apoptosis, and 3.34% of
necrosis), which was significantly higher than those of the
positive control of B with a sum of 16.87% (3.54% of early
apoptosis, 10.8% of late apoptosis, and 2.53% of necrosis). A3
and A5 also had higher apoptosis-inducing levels than B. All
glycosylated Pt(IV) complexes showed higher or comparable
apoptosis-inducing level than cisplatin and oxaliplatin. Hence,
these results indicated that with the introduction of saccharide
ligand, the apoptosis-inducing ability of Pt(IV) complexes was
enhanced.
Fig. 3 Cellular uptake and DNA platination of compounds A4, A5, A6, B,
cisplatin and oxaliplatin (20 µM, respectively) in MCF-7 cells. (A)
Platinum in whole cells. (B) Platinum in DNA.
Table 2 Quantification of apoptosis in MCF-7 cells using an
annexin V/PI assay
2.7. Reduction and DNA-binding properties
Compounds
Early
apoptosis
Late
apoptosis
Necrosis
Sum
To the best of our knowledge, Pt(IV) prodrugs should be
activated to Pt(II) complexes through the reduction by
intracellular reductants, such as ascorbic acid and glutathione,
which are in excess in tumor cells. The resulting divalent form,
generally cisplatin or a related derivative, binds to DNA, inhibits
transcription and replication, and induces apoptosis. To
investigate the mechanism underlying the title glycosylated
Pt(IV) prodrugs, we incubated A6 with or without ascorbic acid
at 37 °C (Fig. 5). The 5′-dGMP was added as a model of DNA.
After 24 h, the peak of oxaliplatin 5’-dGMP adduct appeared for
both oxaliplatin and A6 with ascorbic acid. The peak increased
after 48, 72, and 96 h. Meanwhile, when the reducing agent, that
is, ascorbic acid, was absent, the peak of the oxaliplatin 5′-dGMP
A3
A5
7.56
5.53
9.89
3.54
13.9
7.29
0.58
37.7
34.6
38.6
10.8
22.4
23.7
4.31
4.25
3.77
3.34
2.53
2.92
3.12
1.21
49.51
43.9
A6
51.83
16.87
39.22
34.11
6.1
B
Cisplatin
Oxaliplatin
Untreated

Fig. 4 Induction of apoptosis at 24 h by A3, A5, A6, B, cisplatin and Oxaliplatin in MCF-7 cells
adduct did not appear even after 96 h. Subsequently, the
adduct was isolated and identified by HRMS analysis (Fig.
S7). Following such a reductive experiment, the new peak of the
oxaliplatin 5’-dGMP (confirmed by HRMS) proved the
combination of Pt(II), which was converted from Pt(IV), with 5’-
dGMP and DNA. Therefore, Pt(IV) precursors exert their
anticancer activity by reducing to Pt(II) complexes.
3. Conclusions
Six novel glucose-conjugated Pt(IV) complexes that target
tumor-specific GLUTs were designed, synthesized, and evaluated
for their anticancer activities. All the tests on Pt(IV) complexes
showed enhanced cytotoxicity to five human cancer cell lines
compared with cisplatin and oxaliplatin due to the transport-
mediated effect of GLUTs. The effect of GLUTs on the
transportation of glycosylated Pt(IV) complexes was confirmed
by inhibitory experiment, in which phlorizin was used as an
inhibitor. The effect of alkyl ligands on cytotoxicity was greater
than that of the ligand between the glycoligand and Pt nucleus
because A3 exhibited higher cytotoxic effects than A4. These
complexes showed higher apoptosis-inducing level than
cisplatin and oxaliplatin. The transport properties of
glucose transporters increased the Pt cellular uptake and DNA
platination of the glycosylated Pt(IV) compounds. The
glycosylated Pt(IV) complexes showed lower cytotoxicity to
normal LO2 cells than cisplatin and oxaliplatin while maintaining
the antitumor activity, which may increase the potential safety of
their cancer treatment. Glycosylated Pt(IV) compounds can be
reduced in the attendance of ascorbic acid and further combined
with DNA to cause the death of tumor cells. All the results above
suggested the possibility of the development of glycosylated
Pt(IV) complexes as suitable and effective antitumor drugs and
further provided a reference for our subsequent research.
A.
B.
4. Experimental Section
4.1. Materials and general methods
Oxaliplatin and cisplatin were purchased from Yurui chemical
Co. Ltd (Shanghai, China). All other chemicals were obtained
from Aldrich, Alfa Aesar and J&K as received and were of
analytical grade. Platinum (IV) glycoconjugates were carried out
under nitrogen atmosphere and in dry solvents. 1HNMR and
13CNMR spectra were recorded on a Bruker AVANCE AV400
MHz NMR spectrometers in CDCl3 or CD3OD using TMS as
internal standard. Chemical shifts were reported as δ values
(ppm). High resolution mass spectra (HRMS) were obtained on
an IonSpec QFT mass spectrometer with ESI ionization. MTT (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide),
o
Fig. 5 A. Reaction of oxaliplatin with 5’-dGMP incubated at 37 C after 24 h.
B. Reaction of A6 with 5’-dGMP with and without ascorbic acid after 24 h,
48 h, 72 h and 96 h.

DMEM (for HeLa, MCF-7, HepG-2, A549 cells and 3T3 cells),
RPMI1640 (for A549R cells), medium containing 10% fetal
bovine serums, Fetal bovine serum (FBS), 0.25% trypsin/EDTA
solutions, penicillin-streptomycin solutions and phloretin
purchased from Invitrogen (Grand Island, NY, USA) were used
for elementary cytotoxicity evaluation. Phosphate buffered saline
(PBS) contains 2.7 mM KCl, 137 mM NaCl, 2 mM KH2PO4 and
10 mM Na2HPO4. Annexin V-FITC Apoptosis Detection Kit and
Genomic DNA mini preparation kits were purchased from
KeyGEN BioTech, China. A549 (human lung carcinoma), HeLa
(Cervical), MCF-7 (breast cancer), HepG-2 (hepatoma cells),
3T3 (mouse embryo fibroblasts) and A549R cells were obtained
from Prof. Jing Li (College of Pharmacy, Nankai University,
Tianjin, China). A549R cells were maintained with 2 mg/mL
cisplatin. ICP-MS was measured in the laboratory of Cuihong
Chen (College of environmental science and engineering, Nankai
University, Tianjin, China). Reactions involved in the preparation
of platinum compounds were carried out in the dark. RP-HPLC
analyses were performed as on a Shimadzu SPD-20A system
equipped with a Venusil MP C18 column (150 ×4.6 mm, 5 µm).
RP-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 0.8 mL/min.
incubated for 10 h at the same standard atmosphere. Then, Cells
were collected by trypsinization and PBS buffer was used to
wash the cells three times. The harvested cells were calculated as
well as concentrated and nitric acid was added. Pt concentration
in cellular DNA in MCF-7 cells was detected by ICP-MS. The
Genomic DNA Mini Preparation Kit was applied to the isolation
of DNA in MCF-7 cells.
4.6. Synthetic procedures
Synthesis of A1
Preparation of 4. To a solution of 3 (238.00 mg, 0.50 mmol,
1.0 equiv.) and 6 (0.30 g, 0.50 mmol, 1.0 equiv.) in anhydrous
DMF (30 ml) was added TBTU (242.30 mg, 0.75 mmol, 1.5
equiv.), TEA (105.20 μL, 0.75 mmol, 1.5 equiv.). The reaction
was stirred 48 h at room temperature under Argon atmosphere.
Then the reaction was concentrated and purified by column
chromatography on silica gel to give the compound 4 as a white
solid (410.00 mg, 78.00%).
Preparation of A1. To a solution of 4 (235.00 mg, 0.22 mmol,
1.0 equiv.) in anhydrous DCM (47 ml), BCl3 (1.79 mL, 1.79
mmol, 1.0 M in hexanes, 8.0 equiv.) was added dropwise at -78
°C under argon atmosphere. Then the reaction was concentrated
and purified by column chromatography on silica gel to give the
4.2. In vitro cellular cytotoxicity assays
1
compound A1 as a pale solid (38mg, 25%). H NMR (400 MHz,
Cells were seeded in a 96-well flat-bottomed microplate plates
at 2000-3000 cells per well in 100 uL of growth complete
medium in a 5% CO2 atmosphere at 37 °C for 12 h. Next 100 uL
freshly culture medium containing different concentrations drugs
was added and the microplate was incubated at 37 °C , 5 % CO2
for 48 h. After incubation, pipet directly MTT (5 mg/ mL , 20
µL) into each well of the 96-well assay plate and the plate was
placed at 37 °C in the incubator for another 4 h. Finally, the
medium was disposed completely and DMSO (150 µL) was
Methanol-d4) δ 3.87 (m, 1H), 3.80 (d, J = 11.9 Hz, 1H), 3.58 (m,
3H), 3.39 (m, 1H), 3.23 (m, 1H), 2.81 (m, 1H), 2.54 (m, 1H),
2.41 (m, 1H), 2.28 (m, 2H), 2.05 (s, 3H), 1.95 (m, 3H), 1.61 (m,
4H), 1.31 (m, 3H). 13C NMR (100 MHz, MeOD) δ 183.82,
181.79, 167.11, 76.29, 75.11, 74.66, 72.96, 72.33, 62.92, 33.35,
32.52, 31.05, 25.03, 23.03, 22.09, 21.23. HRMS: Calcd. for
C19H32N2O13Pt M+: 691.1552, found: 691.1563.
Synthesis of A2
added.
The
absorbance
resulting
was
measured
Compound A2 was synthesis according to the procedure of
A1. 1H NMR (400 MHz, Methanol-d4) δ 3.88 (m, 1H), 3.81 (dd, J
= 11.8, 2.2 Hz, 1H), 3.57 (m, 3H), 3.40 (m, 1H), 3.22 (m, 1H),
2.79 (m, 2H), 2.32 (m, 4H), 1.94 (m, 2H), 1.59 (m, 7H), 1.28 (m,
7H), 0.89 (t, J = 6.8 Hz, 3H). 13C NMR (100 MHz, MeOD) δ
184.66, 183.79, 166.89, 166.86, 76.28, 75.09, 74.64, 72.95,
72.32, 63.10, 37.30, 33.34, 32.54, 32.39, 26.66, 25.06, 25.00,
23.43, 22.08, 14.30. HRMS: Calcd. for C23H40N2O13Pt M+:
747.2178, found: 747.2217.
spectrophotometrically at the absorbance of 570 nm using a
BioRad 680 microplate reader. The IC50 values based on three
parallel experiments were counted using GraphPad Prism 6.
4.3. Apoptosis analysis by annexin V-FITC assay
The apoptosis analysis was performed using MCF-7 cells
according to the manufacturers protocol (KeyGEN Annexin V-
FITC/PI Apoptosis Detect-ion Kit). MCF-7 cells cultured in 6-
well plates were handled with and without drugs at 37 °C. Then
cells were collected with adherent cultures by trypsinization and
centrifuged at 2000 rpm for 5 min. Following PBS was used to
wash the cells and centrifuging as before. Then transfered
segmental solution to a culture tube. Then annexin V-FITC 5 µL
and propidium iodide 10 µL were added at different sample and
incubated for 15 min at room temperature keep out of the sun.
After staining, the samples were detected by flow cytometry.
Synthesis of A3
Compound A3 was synthesis according to the procedure of
A1. 1H NMR (400 MHz, Methanol-d4) δ 3.88 (m, 1H), 3.81 (dd, J
= 11.7, 2.4 Hz, 1H), 3.57 (m, 3H), 3.39 (m, 1H), 3.21 (m, 1H),
2.79 (m, 2H), 2.35 (m, 4H), 1.92 (m, 2H), 1.59 (m, 6H), 1.29 (m,
10H), 0.89 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, MeOD) δ
184.68, 183.84, 166.90, 166.87, 76.30, 75.11, 74.68, 72.97,
72.34, 63.13, 37.34, 33.35, 32.85, 32.56, 30.18, 30.15, 26.98,
25.07, 25.01, 23.69, 23.67, 22.10, 14.44. HRMS: Calcd. for
C25H44N2O13Pt M+: 775.2491, found: 775.2538.
4.4. In vitro antitumor assay with GLUTs inhibitor
MCF-7 cells were cultivated in complete medium with and
without GLUTs inhibitor phlorizin (100 μM) at 37 °C incubator.
Antitumor bioactivities of selected compounds A3, A5 and A6,
cisplatin and oxaliplatin were respectively measured according to
the procedure of in vitro cellular cytotoxicity test mentioned
above.
Synthesis of A4
Compound A4 was synthesis according to the procedure of
A1. 1H NMR (400 MHz, Methanol-d4) δ 3.86 (m, 1H), 3.80 (dd, J
= 11.9, 2.4 Hz, 1H), 3.58 (m, 3H), 3.39 (m, 1H), 3.22 (dd, J =
9.7, 8.2 Hz, 1H), 2.79 (m, 2H), 2.28 (m, 2H), 2.05 (s, 3H), 1.61
(m, 10H), 1.30 (m, 4H). 13C NMR (100 MHz, MeOD) δ 184.45,
181.74, 166.99, 77.00, 75.22, 74.31, 73.13, 72.52, 63.18, 37.33,
32.51, 26.73, 26.07, 25.07, 25.05, 23.01. HRMS: Calcd. for
C21H36N2O13Pt M+: 719.1865, found: 719.1882.
4.5. Cell uptake and DNA platination
MCF-7 cells were used to detect the cellular uptake of
cisplatin, oxaliplatin, A4, A5 and A6. MCF-7 cells were seeded
in 6-well plates and were placed at the 37 °C incubator overnight.
Next, different complexes (20 µM) were added to the cells and
Synthesis of A5

Compound A5 was synthesis according to the procedure of
A1. 1H NMR (400 MHz, Methanol-d4) δ 3.86 (m, 1H), 3.80 (d, J
= 11.8 Hz, 1H), 3.57 (m, 3H), 3.39 (m, 1H), 3.22 (t, J = 9.0 Hz,
1H), 2.76 (m, 2H), 2.33 (m, 5H), 1.59 (m, 12H), 1.28 (m, 8H),
0.89 (t, J = 6.7 Hz, 3H). 13C NMR (100 MHz, MeOD) δ 184.68,
184.48, 166.79, 166.76, 77.01, 75.24, 74.34, 73.14, 72.54, 63.20,
37.34, 37.31, 32.58, 32.42, 26.72, 26.67, 26.10, 25.09, 25.06,
23.45, 14.29. HRMS: Calcd. for C21H36N2O13Pt M+: 775.2491,
found: 775.2531.
M. Crespo, J. Quirante, J. Badia, L. Baldomà, M. Font-Bardia, M.
Cascante, Dalton Transactions. 2018, 47, 8956-8971; (r) M.
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Transactions. 2018, 47, 11491-11502; (s) Z.-Y. Ma, D.-B. Wang,
X.-Q. Song, Y.-G. Wu, Q. Chen, C.-L. Zhao, J.-Y. Li, S.-H. Cheng,
J.-Y. Xu, European Journal of Medicinal Chemistry. 2018, 157,
1292-1299.
3
4
5
M. D. Hall, H. R. Mellor, R. Callaghan, T. W. Hambley, Journal of
Medicinal Chemistry. 2007, 50, 3403-3411.
Synthesis of A6
N. J. Wheate, S. Walker, G. E. Craig, R. Oun, Dalton
Transactions. 2010, 39, 8113-8127.
Compound A6 was synthesis according to the procedure of
A1. 1H NMR (400 MHz, Methanol-d4) δ 3.86 (m, 1H), 3.80 (dd, J
= 11.9, 2.2 Hz, 1H), 3.58 (m, 3H), 3.39 (m, 1H), 3.22 (t, J = 8.9
Hz, 1H), 2.76 (m, 2H), 2.32 (m, 5H), 1.59 (m, 10H), 1.29 (m,
14H), 0.89 (t, J = 6.5 Hz, 3H). 13C NMR (100 MHz, MeOD) δ
184.65, 184.47, 166.83, 166.80, 77.00, 75.23, 74.33, 73.13,
72.52, 63.18, 37.34, 32.85, 30.18, 30.15, 26.97, 26.72, 26.09,
25.09, 25.06, 23.70, 14.44. HRMS: Calcd. for C27H48N2O13Pt M+:
803.2804, found: 803.2848.
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5. Acknowledgments
This research was supported by the National Natural Science
Foundation of China (Grants 21572106, and 21877063).
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