Polyamine-Based Pt(IV) Prodrugs as Substrates for Polyamine Transporters Preferentially Accumulate in Cancer Metastases as DNA and Polyamine Metabolism Dual-Targeted Antimetastatic Agents

Article abstract of DOI:10.1021/acs.jmedchem.9b01641

Diverse platinum drug candidates have been designed to improve inhibitory potency and overcome resistance for orthotopic tumors. However, the antimetastatic properties have rarely been reported. We herein report that homospermidineplatin (4a), a polyamine-Pt(IV) prodrug, can potently inhibit tumor growth in situ and reverse cisplatin resistance as expected, and more importantly, 4a displays remarkably elevated antimetastatic activity in vivo (65.7%), compared to those of cisplatin (27.0%) and oxaliplatin (19.6%). The underlying molecular mechanism indicates that in addition to targeting nuclear DNA, 4a can modulate polyamine metabolism and function in a manner different from that of cisplatin. By upregulating SSAT and PAO, 4a downregulates the concentrations of Put, Spd, and Spm, which favors the suppression of fast-growing tumor cells. Moreover, the p53/SSAT/β-catenin and PAO/ROS/GSH/GSH-Px pathways are involved in the inhibition of 4a-induced tumor metastasis. Our study implies a promising strategy for the design of platinum drugs for the treatment of terminal cancer.

Full text of DOI:10.1021/acs.jmedchem.9b01641

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Article
Polyamine-based Pt(IV) Prodrugs as Substrates for Polyamine
Transporters Preferentially Accumulates in Cancer Metastases as
DNA and Polyamine Metabolism Dual-Targeted Antimetastatic Agents
hanfang liu, Jing Ma, Yingguang Li, Kexin Yue, Linrong Li, Zhuoqing Xi, Xiao Zhang, Jianing
Liu, Kai Feng, Qi Ma, Sitong Liu, Shudi Guo, Peng G. Wang, Chaojie Wang, and Songqiang Xie
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01641 • Publication Date (Web): 25 Nov 2019
Downloaded from pubs.acs.org on November 26, 2019
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Polyamine-based Pt(IV) Prodrugs as Substrates for Polyamine
Transporters Preferentially Accumulates in Cancer
Metastases as DNA and Polyamine Metabolism Dual-Targeted
Antimetastatic Agents
Hanfang Liu,†# Jing Ma,†#* Yingguang Li,† Kexin Yue,† Linyong Li,† Zhuoqing Xi,†II Xiao Zhang,§
Jianing Liu,I Kai Feng,I Qi Ma,† Sitong Liu,† Shudi Guo,† Peng George Wang,III Chaojie Wang,§* and
Songqiang Xie,&*
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† School of Pharmacy, Institute for Innovative Drug Design and Evaluation, Henan University, N. Jinming Ave, 475004
Kaifeng, China
& School of Pharmacy, Institute of Chemical Biology, Henan University, N. Jinming Ave, 475004 Kaifeng, China.
§The Key Laboratory of Natural Medicine and Immuno-Engineering, Henan University, Kaifeng, 475004, China.
I School of medicine, Henan University Minsheng College, Kaifeng, 475004, China.
II Henan University of Science and Technology Second Affiliated Hospital, Luoyang, 471000, China.
III The State Key Laboratory of Microbial Technology and National Glycoengineering Research Center, Shandong University,
Qingdao, 266237, China.
# The authors contribute equally to this work.
KEYWORDS: Platinum drugs, Cancer, Polyamine Metabolism and Function, Anticancer and Antimetastatic Activity
ABSTRACT: Diverse platinum drug candidates have been designed to improve inhibitory potency and overcome resistance for
orthotopic tumor. However, the antimetastatic properties are rarely reported. We herein report that homospermidineplatin (4a), a
polyamine-Pt(IV) prodrug, can potently inhibit tumor growth in situ and reverse cisplatin resistance as expected. And more
importantly, 4a displays remarkably elevated antimetastatic activity in vivo (65.7%), compared to that of cisplatin (27.0%) and
oxaliplatin (19.6%). The underlying molecular mechanism indicates that in addition to targeting nuclear DNA, 4a can modulate the
polyamine metabolism and function in a different way from that of cisplatin. By up-regulating SSAT and PAO, 4a down-regulates
the concentrations of Put, Spd and Spm, which are in favor of suppressing fast-growing tumor cells. Moreover, the p53/SSAT/β-
catenin and PAO/ROS/GSH/GSH-Px pathways are involved in the 4a-induced tumor metastasis inhibition. Our study implies a
promising strategy for the design of platinum drugs to treat terminal cancer.
1. INTRODUCTION
targeting Pt(IV) complexes. Unfortunately, both Pt(II) and
Pt(IV) complexes failed to provide simultaneous anticancer
and antimetastatic activity in vitro and in vivo.
Advances in our understanding of polyamine metabolism
and function and their alterations in cancer have led to
resurgence in the interest of targeting polyamine metabolism
as an anticancer strategy. [13a-13c] The requirements for
polyamine metabolism are dysregulated in cancer, thus
making polyamine function and metabolism attractive targets
One of the major causes of death in cancer patients and a
major obstacle in present chemotherapy are tumour metastasis.
Approximately 90% of cancer patient deaths are due to
metastatic cancer. [1, 2] Current anticancer agents can cure
well-defined primary tumour, but they have limited impacts on
metastatic cells. [3-5] Pt(II) drugs and new Pt(IV) agents
prevail in the treatment of cancer, but they are less effective in
the treatment of metastatic tumour. [6] Thus, the design of
novel antimetastatic therapeutic agents, including new Pt
agents represents an area in need of urgent attention. Recently,
a surge in the activity of Pt drugs, based on a great deal of
mechanistic information, has been aimed at developing
nonclassical Pt(IV) complexes that operate via mechanisms of
action distinct from those of the approved drugs. [7-10] Our
projects [11] and others [12] previously focused on using
bioactive moieties to achieve enhanced pharmacological
effects, such as the modification of axial ligands to tune
lipophilicity or kinetics and the generation of dual- or multi-
for therapeutic intervention. Natural products with
a
polyamine moiety have been found to prevent tumour cell
invasion. [13d-13f] Our group [14] has provided significant
evidence that some polyamine conjugates could potently
inhibit tumour growth and metastasis in vivo. The polyamines
spermidine (Spd), spermine (Spm) and their diamine precursor
putrescine (Put) are naturally occurring polycationic
alkylamines that are essential for eukaryotic cell growth. [13]
The intracellular pathway of mammalian polyamine
catabolism is a two-step process, the
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Journal of Medicinal Chemistry
Table 1. IC50 (the concentration of inhibition cell proliferation by 50%) values (μM) of Pt(IV) prodrugs. Cells were treated for 48 h,
and the cell viability was determined by MTT assays[d].
1
2
3
4
5
6
7
8
9
SMMC7721 Hela
MDA-MB-231 MCF-7
1.32±0.14 0.50±0.09
10.98±1.09 ND
ND >30
15.02±1.23 32.48±1.49
15.65 24.61
14.17±1.29 17.72±0.89
14.76 13.42
HCT-116 A549cisR
0.99±0.19 1.41±0.19
A549
RF[a]
4a
0.99±0.14
15.48±1.90
>20
0.96±0.19
2.70±0.25
ND
0.52
ND
ND
6a
14.58±1.89 6.96±0.69 ND
8a
>30
>20
>50
>30
cisplatin
FI[b]
23.37±1.26
23.51
9.60±0.60
19.20
5.30±0.14 42.89±1.89 10.20±0.89 4.01
5.35 30.42 3.78 7.71
Oxaliplatin
FI[c]
11.77±1.88
11.89
11.62±0.78 5.89±0.29 38.97±3.23 10.00±1.23 3.90
23.24 5.95 27.64 3.70 7.50
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[a] The RF (resistance factor) is defined as the IC50 value in A549cisR cells/IC50 value in A549 cells.
[b] FI (fold increase) is defined as IC50(cisplatin)/IC50(4a).
[c] FI (fold increase) is defined as IC50(oxaliplatin)/IC50(4a).
[d] An average of three measurements. ND = not determined.
rate of which is controlled by the activity of the inducible
enzymes spermidine/spermine N1-acetyltransferase (SSAT)
and N1-acetylpolyamine oxidase (APAO). Moreover,
polyamine transporters (PTs) are also overexpressed in most
types of cancer cells, the identity of which is still unknown.
[13-14] Therefore, polyamine conjugates have become an
appealing strategy for the targeted delivery of anticancer drugs
with antimetastatic activity.
selected for the construction of Pt(IV) complexes 4-7 to
investigate the influence of different Pt cores on antitumor
activity. Compounds 6a and 7a, without a polyamine
modification, were also designed to investigate the targeting
properties of polyamine. We also synthesized the released
polyamine-ligand 8a to study how the effects come about.
Feasible routes to 4-8 were established in Scheme S1-S3.
Moreover, recent advances in p53 regulation of ammonia
metabolism through the urea cycle controlling polyamine
biosynthesis and catabolism indicate that polyamine
metabolism and function are closely associated with cisplatin
resistance. [15] Targeting polyamine metabolism and function
may imply a practical strategy for the design of Pt drugs to
overcome cisplatin resistance in a totally new field.
2. RESULTS AND DISCUSSION.
2.1 Synthesis and characterization of polyamine-Pt(IV)
conjugates.
We established feasible routes to 4-5 (Scheme S1-S3) given
in detail in the supporting information. The asymmetrically
functionalized Pt(IV) compounds e and z can be obtained by
the reaction of oxide-cisplatin and oxide-oxaliplatin with
palmitic anhydride and then succinic anhydride. [12a] In this
series, compounds 4-5 bear a hydrophobic polyamine chain
varying in connecting formats from d (4+4), q (3+3+3) to y
(3+3+3). The solid was filtered, washed several times with
absolute ethanol, and dried under vacuum to give the pure
target compound 4-5. All new compounds were characterized
by 1H, 13C, 195Pt NMR spectroscopy, ESI-MS (Figures S2-S29)
and CHN elemental analysis.
O
O
O
14
H3N
H3N
Cl
O
14
Pt
O
H3N
H3N
Cl
Cl
O
Cl
Pt
R1
R1=
H2N
O
H
N
OH
N
H
4a
Homospermidineplatin (
)
6a)
Platinum(IV) Conjugate (
O
H
H2N
N
. 2HCl
OH
N
H
2.2 The stability of 4a.
O
8a
Figure 1. Chemical structures of homospermidineplatin (4a),
Pt(IV) prodrugs without polyamine modified 6a and the
released polyamine-ligand 8a.
The stability in water was evaluated using compound
homospermidineplatin (4a). We observed that 4a is highly
stable in water, as evidenced by no change in the HPLC
chromatogram after 24 h. (Figure S30) [12] And
approximately 70% of 4a remained unchanged even after 48 h
in RPMI 1640, suggesting that the compound is stable in
biological media. Pt levels in A549 and A549cisR cells with
no dependence on aminoguanidine (AG) indicated improved
metabolic stability. (Figure S31) [13f]
Based on the key role of polyamine in mediating antitumor
and antimetastatic efficacy, we rationally designed multi-
action Pt prodrugs targeting polyamine function and
metabolism. We firstly designed and synthesized polyamine-
Pt(IV) conjugates 4-5 with the classical unsymmetrically-
substituted polyamine analogs 1-3, some of which have
entered Phase III clinical trials. [13a-13c] (Figure 1 and Figure
S1) Two clinical Pt(II) drugs, cisplatin and oxaliplatin, were
2.3 In vitro cytotoxicity effects.
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A.
B.
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5
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Control
***
6a (4.8 mg Pt/kg)
**
Oxp. (5 mg/kg, 2.46 mg Pt/kg)
Cis. (5 mg/kg, 3.26 mg Pt/kg)
4a1 (5 mg/kg, 1.20 mg Pt/kg)
4a2 (10 mg/kg, 2.40 mg Pt/kg)
4a3 (20 mg/kg, 4.8 mg Pt/kg)
500
17.28%
ns
19.65%
400
300
200
100
0
27.03%
100
90
80
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50
30.13%
42.27%
65.78%
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l
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p
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C
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O
o
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C
C. Control
6a
Oxp.
Cis.
4a1
4a2
4a3
Figure 2. In vivo antimetastatic activity of 4a, 6a, cisplatin and oxaliplatin in 4T1 breast carcinoma tumors every two days for a
total of four treatments. A) Body weight of mice during treatment with 4a, 6a, cisplatin, oxaliplatin, and control group for 14 days.
B) Statistics of lung metastasis nodules from mice after treatment with 4a, 6a, cisplatin, oxaliplatin, and control group for 14 days.
C) Representative images of pulmonary metastasis from mice treated with control, 6a (14 mg/kg, 4.8 mg Pt/kg), oxaliplatin (5
mg/kg, 2.46 mg Pt/kg), cisplatin (5 mg/kg, 3.26 mg Pt/kg), 4a1 (5 mg/kg, 1.20 mg Pt/kg), 4a2 (10 mg/kg, 2.40 mg Pt/kg), 4a3 (20
mg/kg, 4.80 mg Pt/kg) by intravenous delivery once every two days (n = 8 mice per group). **P< 0.01; ***P< 0.001.
The in vitro anticancer activity of these newly synthesized
polyamine-Pt(IV) compounds was assessed by using the MTT
assay (Table 1 and Table S3). [15c-15d] The oxaliplatin-based
Pt(IV) compound 5a is less active than the corresponding
cisplatin-based analog 4c. For cisplatin-based scaffolds (4a-
4c), 4a with the homospermidine moiety is more potent than
its alkynyl (4b) and cyclopropyl (4c) counterparts. 4a is
significantly more active than cisplatin and oxaliplatin in all of
the tested cancer cells with some potencies in the nanomolar
range, IC50 values of which is almost 30 folds lower than
cisplatin and oxaliplatin. The RF (resistance factor) values of
polyamine-platinum(IV) conjugates 4 and 5 (RF 0.24-0.84)
are 5-17 folds lower than that of cisplatin (RF=4.01),
indicating its better ability to overcome cisplatin resistance.
Moreover, polyamine-platinum(IV) conjugates 4 and 5 (SI
1.47-4.52) show lower cytotoxicity in normal cells HL-7702,
with selectivity index (SI) values 3-12 folds higher than those
of cisplatin and oxaliplatin. (Table S4) A similar tendency was
observed by ICP-MS (Figure S32).
To evaluate if 4a with a polyamine motif can prevent the
tumor metastasis, the in vitro transwell assay was conducted.
Compared with cisplastin, 4a could inhibit breast cancer
migration more potently in a dose-dependent manner as
illustrated in Figure S33. This prompted us to further test the
in vivo antitumor and antimetastatic activities of 4a.
As shown in Table S3, the IC50 of 4a in MCF-7 cells is 0.50
μM, which is the highest in all tested cancer cells, indicating a
better therapeutic effect in breast cancer. Cellular drug uptake
and DNA platination of 4a in the lung carcinoma cell lines
A549 and A549cisR, the hepatocellular carcinoma cell line
SMMC-7721 and the breast cancer cell line MCF-7 were also
tested by ICP-MS (Figure S34). The highest Pt levels in MCF-
7 suggest a potential therapy for breast cancer. Therefore, we
tested the in vivo antitumor and antimetastatic activities of 4a
in the breast cancer model.
Metastasis is the main factor affecting mortality in cancer
patients, and the lung is one of the most common sites. [16]
Therefore, we injected 4T1 breast cancer cells via the tail vein
to assess whether 4a was effective against pulmonary
metastasis in vivo. Surprisingly, 4a significantly reduced the
number of tumour metastatic nodules (65.78%) as shown in
Figure 2, which was 3-fold higher than that of cisplatin
(27.03%) and oxaliplatin (19.65%). In addition, the body
weight of the mice after treatment with 4a showed no
significant difference with the control group (Figure 2A).
Moreover, the variations in the organ weight indices implied
that 4a had no obvious patholog-
Additionally, polyamine-platinum(IV) conjugate 4a
exhibited better cell-killing effects than the Pt(IV) prodrugs 6a,
7a without polyamine modified, and especially the released
polyamine-ligand 8a (Table 1), indicating that polyamine
plays an important role in the synthesized platinum(IV)
conjugates. Among these compounds, 4a displays the highest
cytotoxicity. Therefore, we focus on 4a for the following tests.
2.4 In vivo antitumor and antimetastatic activities.
Our group [14] and others [13] emphasize the importance of
the potential antimetastatic activities of polyamine conjugates.
And cisplatin is well known to be a poor metastasis inhibitor.
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Journal of Medicinal Chemistry
A.
B.
1
2
2.5
PBS
Polyomine ligand
ns
2.0
3
4
5
6
7
8
9
6a (14 mg/kg 4.80 mg Pt/kg)
Oxp.(5 mg/kg 2.46 mg Pt/kg)
Cis. + Polyomine ligand
Cis.(5 mg/kg 3.26 mg Pt/kg)
4a (5 mg/kg 1.20 mg Pt/kg)
4a (10 mg/kg 2.40 mg Pt/kg)
4a(20 mg/kg 4.80 mg Pt/kg)
1.5
51.13%
54.22%
56.99%
57.06%
60.74%
**
1.0
0.5
0.0
**
**
***
64.28%
75.44%
**
120
110
100
90
***
***
.
p
.
)
)
)
)
g
d
d
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S
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m
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m
0
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e
4
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n
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(
(
m
i
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a
m
a
m
a
4
1
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(
6
a
o
80
y
y
l
a
l
4
o
o
P
P
+
.
70
s
i
.
.
.
.
.
.
.
3
C
0
2
4
6
8
0
1
1
compounds
Days
C.
D.
PBS
Polyomine ligand
6a (4.8 mg Pt/kg)
Oxp.(5 mg/kg 2.46 mg Pt/kg)
Cis. + Polyamine ligand
Cis.(5 mg/kg 3.26 mg Pt/kg)
4a (5 mg/kg 1.2 mg Pt/kg)
4a (10mg/kg 2.4 mg Pt/kg)
4a (20mg/kg 4.8 mg Pt/kg)
1500
1000
500
0
**
***
.
.
.
.
.
.
3
2
4
6
8
0
1
1
Days
Figure 3. In vivo antitumor activity of 4a, polyamine ligand 8a, 6a without polyamine modified, cisplatin and oxaliplatin in 4T1
breast carcinoma tumors. Eight mice each received the vehicle, cisplatin (5 mg/kg, 3.26 mg Pt/kg), oxaliplatin (5 mg/kg, 2.46 mg
Pt/kg), cisplatin + Polyamine ligand (1:1, 5 mg/kg cisplatin + 5 mg/kg Polyamine ligand), 4a (5 mg/kg, 1.20 mg Pt/kg; 10 mg/kg,
2.40 mg Pt/kg; 20 mg/kg, 4.80 mg Pt/kg) 6a (14 mg/kg, 4.8 mg Pt/kg) or 8a (20 mg/kg) intravenously (i.v.) every two days for a
total of four treatments. A) Body weight of the mice during treatment. B) Tumor weight in each group at the end of the experiment.
C) Tumor growth as a function of time. D) Images of the tumors at the end of the experiment. First horizontal line, control group;
Second line, polyamine ligand 8a group; Third line, 6a group; Fourth line, oxaliplatin group (5 mg/kg, 2.46 mg Pt/kg); Fifth line,
cisplatin + Polyamine ligand group; Sixth line, cisplatin group (5 mg/kg, 3.26 mg Pt/kg); Seventh line, 4a group (5 mg/kg, 1.20 mg
Pt/kg); Eighth line, 4a group (10 mg/kg, 2.40 mg Pt/kg); Ninth line, 4a group (20 mg/kg, 4.80 mg Pt/kg). **, P < 0.01; ***, P <
0.001.
ical changes compared with the control group in the in vivo
toxicological profile experiments (Figure S35).
activity in vivo. Upon 4a treatment, the tumour weight
decreased significantly to 29% of that in the control group. 4a,
with the inhibition ratio of 75.44%, was better than that of
cisplatin (57.06%) and oxaliplatin (54.22%). Furthermore, the
acceptable toxicity of 4a was confirmed by measuring the
change in body weight (Figure 3A). After the last injection,
the body weight of 4a returned to 107% and cisplatin down to
80% of the initial weight. These results indicate that 4a is
highly efficient in inhibiting tumour growth in vivo. Moreover,
the variations in the organ weight indices of 4a had no obvious
pathological changes compared with the control group in the
in vivo toxicological profile experiments (Figure S36).
To further evaluate the potential safety of the polyamine
conjugates in vivo, we conducted an acute toxicity study of 4a,
6a, cisplatin and oxaliplatin using BALB/c mice aged 5 weeks.
Both the MTD and LD50 values of 4a are nearly 6-10 folds
higher than those of 6a, cisplatin and oxaliplatin in Table S5.
The ratios of the cytotoxicity (IC50) to MCF-7 cells to the
animal lethal dose values (LD50) of 4a were used as a measure
of the therapeutic index. [12a] The results show that 4a is the
safest complex with a therapeutic index over 33-fold higher
than that of the clinical drug cisplatin and oxaliplatin.
We also tested the in vivo antitumor activity of 4a in the
breast cancer model. (Figure 3) On day 13, the average tumour
volume was 1025 mm3 for the control group and 400 mm3 for
4a, which was 61% and 36% lower than that of the control and
oxaliplatin-treated groups, indicating marked antitumor
2.5 4a is transported by PTs.
For the first time, we found the rational discovery and
evaluation of homospermidineplatin (4a), a polyamine-Pt(IV)
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prodrug providing simultaneous enhanced anticancer and bind at the N7 position of guanine bases. [11] The fractions
1
2
3
4
5
6
7
8
antimetastatic activity in vitro and in vivo. More efforts were
thus conducted to illustrate the underlying molecular
mechanism.
that contained unknown peaks were separated and identified
by ESI-MS analysis, observing the same products as cisplatin.
(Figure S40 and Table S6) The higher FI of inhibition ratio for
4a/cisplatin with the same Pt levels in genomic DNA in
A549cisR cells (Figure S41) indicated that the mechanism of
4a was different from that of cisplatin. (Table S7)
We further examined the expression levels of phospho-
histone H2A-X (Ser139) by immunoblotting (Figure S42) to
determine whether 4a is able to cause DNA damage. [17] We
can see a slight increase of phospho-histone H2A-X (Ser139)
after treatment with 4a compared with cisplatin and 6a.
One crucial question in polyamine-conjugate chemistry is
whether 4a is actively transported by PTs. [13, 14] Spd, a
natural PTs ligand, is usually used to compete with the
polyamine conjugate for the PTs. The reduced cellular
concentration of the polyamine conjugate by the added Spd
indicated that the polyamine conjugate enters the cell by PTs.
Cellular uptake and platination of nuclear DNA were first
monitored in the absence and presence of Spd (Figure S37). A
68% reduction in cellular uptake and a 50% reduction in
nuclear DNA were observed in the presence of 20 μM Spd.
Under similar conditions, the cellular uptake of 6a and
cisplatin did not significantly change. The same results can be
observed in MCF-7 cells in Figure S38. It is reasonable that an
increase in the cellular viability (23%) of 4a with Spd,
accompanied by the reduced drug accumulation was also
observed in Figure S37C. In summary, the uptake assays
support the hypothesis that PTs were at least partially involved
in the cellular entrance of 4a.
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A.
B.
2.6 The cellular accumulation and distribution of Pt in
A549 and A549cisR cells.
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Platination of nuclear DNA
Whole cell uptake
A549
***
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A549cisR
A549
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Concentration (μM)
0
Figure 5. Effects of different concentrations of 4a and cisplatin on p53
protein and gene expression in A549cisR cells. A) Effect of 4a and
cisplatin on p53 protein in A549cisR cells after 24 h treatment. B) qRT-
PCR analysis of the p53 transcriptional level was performed with total
RNAs purified from A549cisR cells treated with 4a and cisplatin after 24h.
.
a
4
a
s
i
.
6
a
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Figure 4. Cellular uptake and distribution following 8 h incubation with
10 μM 4a in A549 and A549cisR cells. A) Pt accumulation in A549 and
A549cisR cells upon 10 μM treatment for 8 h. B) Platination levels of
nuclear DNA extracts from A549 and A549cisR cells after 8 h incubation
with 10 μM tested compounds. ***, P< 0.001.
Due to the weak influence of 4a on phospho-histone H2A-X
(Ser139), we tested the change of p53 gene and p53 protein,
which also plays an important role in cell apoptosis and
cisplatin resistance. We observed after treatment with 4a, the
p53 protein and p53 gene were 3 folds higher levels than
cisplatin (Figure 5). Recently, it is reported ureagenesis and
ammonia metabolism including SSAT have a great influence
on p53. [15] Markedly higher levels of the polyamines
including Put, Spd, and Spm (Figure 5B) and a lack of p53
function (Figure 4) in A549cisR cells treated by cisplatin were
observed for the first time, which was totally in contrary to 4a,
indicating that polyamine metabolism and function are good
targets to overcome cisplatin resistance and targeting
polyamine metabolism and function is a practical strategy for
the design of Pt drugs to overcome cisplatin resistance in a
totally new field. We next examined polyamine metabolism
and function because such properties maybe the main factors
significantly affecting the activity of 4a.
Next, the cellular accumulation and distribution of Pt in
A549 and A549cisR cells were measured (Figure 4). The Pt
accumulation of 4a in A549cisR cells was almost 23 times
greater than cisplatin and 6a. (Figure 4A) And also the Pt
levels in A549cisR and A549 genomic DNA were 19.74 and
18.78 ng Pt per 2×105 cells, respectively, which was 2-6 folds
higher than cisplatin and 6a. (Figure 4B)
2.7 Apoptosis induced by DNA damage and DNA binding
properties after reduction.
To further determine if apoptosis was induced by DNA
damage, flow cytometry experiments using Annexin V/PI
double staining were conducted. (Figure S39) Treatment with
4a (5 μM) resulted in A549cisR cell apoptosis with early
apoptotic cells (33.00%) and late apoptotic cells (37.50%),
which was higher than that of cisplatin (15 μM). We also
investigated the reduction and DNA binding properties of
cisplatin and 4a with ascorbic acid using 5’-dGMP as the
DNA model for the inherent proficiency of the Pt prodrugs to
2.8. By promoting p53/SSAT/β-catenin and PAO/ROS/
GSH/GSH-Px pathway, 4a significantly inhibits tumour
metastasis and overcomes cisplatin resistance.
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Journal of Medicinal Chemistry
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D.
A549
GSH
A549cisR
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Figure 6. Effects of different concentrations of 4a (A) and
cisplatin (B) on SSAT expression in A549cisR cells after 24 h
treatment.
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Next, two key catabolism enzymes SSAT and PAO in
polyamine cycle were determined to explore the detailed
reason. We found 4a markedly up-regulated the expression of
SSAT in A549cisR cells (Figure 6A), which is totally opposite
to cisplatin (Figure 6B). Our group also found that the β-
catenin signalling pathway is vital in SSAT-regulated cell
migration and invasion [14]. Next, β-catenin was also
determined. The down-regulation of β-catenin after treatment
with 4a indicates significant antitumor migration activity,
which also cannot be observed in cisplatin group (Figure 7).
E.
A549
GSH-Px
A549cisR
3000
2000
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Figure 8. 4a affects polyamine metabolism and function by
upregulating PAO and ROS and downregulating GSH and
GSH-Px. (A) Effect of 4a, 8a, cisplatin and 6a on PAO
expression in A549cisR cells with 10 µM after 24 h treatment.
(B) Effect of 4a, cisplatin and 6a on PAO expression in A549
cells with 10 µM after 24 h treatment. (C). Effect of 4a,
cisplatin and 6a on ROS expression in A549cisR cells with 10
µM after 24 h treatment. (Yellow, homospermidineplatin 4a;
Green, cisplatin; Red, control group) (D, E) GSH and GSH-Px
expression in A549 and A549cisR cells after 24 h treatment by
4a, 8a, 6a and cisplatin, respectively.
Figure 7. Effect of 4a (A) and cisplatin (B) on β-catenin
expression in A549cisR cells with 10 µM after 24 h treatment.
In addition to SSAT, PAO is another critical catabolism
enzyme. Moreover, one of the crucial reasons for Pt drug
resistance is that Pt drugs are easily poisoned by S-containing
proteins. [6, 7a] Cisplatin bound to a S-containing protein is
immediately excreted through multidrug resistance protein. [6,
7a, 18] By raising the levels of PAO, polyamine conjugates
can also up-regulate oxidizing substances ROS and down-
regulate reducing substances such as GSH and GSH-Px to
overcome cisplatin resistance. Surprisingly, we found that the
relative PAO activity upon 4a treatment is almost 2-10 folds
higher than 6a, 8a and cisplatin (Figure 8A and 8B). Our
group previously reported [19] a significant accumulation of
ROS is closely related to PAO activity.
The ROS content measured by FCM (flow cytometry)
(Figure 8C) in A549cisR cells after treatment with 4a was also
higher than that of cisplatin. High levels of oxidizing ROS can
decrease reducing substances, such as GSH and GSH-Px. GSH
and GSH-Px content in A549cisR cells upon 10 μM 4a
treatment were 3-fold lower than the control group in Figure
8D and 8E. The significantly elevated cellular levels of PAO
are believed to be among the major reasons for the marked
cytotoxicity of 4a to overcome cisplatin resistance. We also
found the P-gp is also partially involved in the mechanism of
4a to overcome cisplatin resistance. (Figure S43)
A.
B.
A.
B.
Put
Put
Spd
Spm
A549
Spd
Spm
A549cisR
1500
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1200
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A549cisR (PAO)
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100
A549 (PAO)
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Figure 9. Effect of 4a, 6a, and cisplatin on polyamine
metabolism and function. Polyamine (Put, Spd, and Spm)
concentrations in A549 (A) and A549cisR cells (B) after 24 h
treatment with or without 4a were determined. Cisplatin and
6a are the reference drugs. ***P< 0.001.
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Figure 10. Proposed mechanism of action for polyamine-Pt(IV) prodrugs.
A549cisR cells imply that polyamine metabolism and function
are good targets to overcome cisplatin resistance, which is
totally opposite to polyamine-Pt(IV) prodrug. This evidence
may be beneficial to the discovery of new Pt drugs to
overcome cisplatin resistance in a totally new field and the
treatment of terminal cancer.
4a significantly upregulated two key catabolism enzymes
SSAT and PAO in polyamine cycle. The upregulated SSAT
and PAO can decrease the concentrations of Put, Spd and Spm
to promote polyamine metabolism and suppress fast-growing
tumour cells. Next, the contents of endogenous polyamine,
Spm, Spd, and Put in A549 and A549cisR cells were measured,
which plays an important role in polyamine metabolism and
function (Figure 9). [13] Compared with 6a, cisplatin and the
control group, 4a preferentially down-regulated the
concentrations of Put, Spd and Spm in A549 and A549cisR
cells. Moreover, the significant down-regulated SSAT and p53,
and up-regulated Put, Spd and Spm (Figure 9B) after treated
with cisplatin in A549cisR cells, which is totally opposite to
4a, imply that targeting polyamine metabolism and function is
a practical strategy for the design of Pt drugs to overcome
cisplatin resistance in a totally new field.
4. EXPERIMENTAL
4.1 HPLC Studies.
The purities of all target compounds were determined by
HPLC (Waters E2695-2998 equipped with a Venusil MP C18
column (150 × 4.6 mm, 5 μm). The purity of the platinum
complexes (4–7) were confirmed to be ≥95% by analytical
HPLC. Method for determining the purity of target
compounds and the purities results were shown in Table S1
and S2, respectively.
3. CONCLUSIONS
4.2 General Procedure for the Synthesis of Compound
Taken together, our findings provide the first example of
polyamine-platinum(IV) prodrugs, targeting polyamine cata-
bolic enzyme SSAT and PAO by PTs to regulate tumour high
polyamine microenvironment and overcome cisplatin
resistance, providing simultaneous enhanced anticancer and
antimetastatic activity. (Figure 10) Prodrug 4a induces
apoptosis and causes DNA damage by up-regulating the p53
gene and p53 protein. The possible mechanism of polyamine-
Pt(IV) complexes is targeting SSAT and PAO to down-
regulate Put, Spd and Spm in A549cisR cells, which is totally
opposite to cisplatin. The β-catenin signalling pathway is vital
in 4a regulated cell migration and invasion in vitro and in vivo.
By raising the levels of PAO, polyamine conjugates up-
regulate oxidizing substances ROS and down-regulate
reducing substances such as GSH and GSH-Px to overcome
cisplatin resistance.
The discovery of the potential role of homospermidineplatin,
a polyamine-Pt(IV) prodrug, broadens our strategy for the
design of more potential antimetastatic agents, although the
details of the homospermidineplatin antimetastatic mechanism
require further exploration. Markedly higher levels of the
polyamines Put, Spd, and Spm, the down-regulation of SSAT
and a lack of p53 function after treatment with cisplatin in
4a1.
To a solution of e (0.38 mmol) in DMF (10 mL) was added
a DMF solution (0.5 mL) containing HATU (0.57 mmol). This
mixture stirred for 10 min at room temperature. A DMF
solution containing d (1.5 mmol) and DIPEA (0.92 mmol) was
added to the resulting solution. 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 4a1
was purified by silica gel column chromatography as a yellow
solid in a yield of 50%. 1H NMR (300 MHz, MeOD) δ = 3.17
– 3.10 (m, 2H), 3.11 – 2.78 (m, 6H), 2.71 – 2.53 (m, 2H), 2.46
(s, 2H), 2.38 – 2.20 (m, 2H), 1.55 (t, J=16.9, 8H), 1.43 (d,
J=7.2, 28H), 1.27 (s, 16H), 0.91 (t, J=16.6, 3H). 13C NMR
(300 MHz, MeOD) δ = 175.08, 174.30, 164.74, 158.27,
157.17, 80.47, 80.10, 55.67, 40.87, 40.39, 40.08, 37.00, 32.95,
30.71, 30.37, 28.77, 28.18, 27.51, 26.90, 23.63, 14.51.
4.3 General Procedure for the Synthesis of Compound 4a.
Intermediate 4a1 was dissolved in EtOH (10 mL) and
stirred at 0 °C for 10 min. Then, 4 M HCl was added dropwise
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Journal of Medicinal Chemistry
at 0 °C. The reaction mixture stirred at room temperature for added to the resulting solution. The mixture was stirred at
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5
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8
24–48 h and monitored by TLC. The solution typically gave a
faint yellow solid as a precipitate. The solid was filtered,
washed several times with absolute ethanol, and dried under
room temperature for 24 h in the dark. The DMF was then
removed under vacuum to afford a yellow oil. Compound 4c1
was purified by silica gel column chromatography as a yellow
solid with a yield of 40%. 1H NMR (300 MHz, CDCl3) δ =
3.74 (d, J=6.1, 2H), 3.22 (s, 16H), 2.60 (d, J=32.1, 7H), 1.77
(m, 5H), 1.40 (m, 54H), 0.92 (s, 2H), 0.78 (s, 2H), 0.63 (s,
3H).13C NMR (300 MHz, CDCl3) δ = 182.08, 173.24, 156.65,
156.21, 155.48, 79.68, 79.42, 79.33, 54.39, 46.85, 45.26,
44.92, 43.95, 42.61, 32.20, 31.96, 31.87, 29.68, 29.63, 29.31,
28.71, 28.45, 27.73, 25.72, 22.64, 18.01, 14.08, 12.35, 8.02.
1
vacuum to give the pure target compound 4a. H NMR (300
MHz, MeOD) δ = 3.19 (d, J=20.4, 6H), 2.95 (dd, J=17.3, 7.4,
6H), 2.53 (t, J=6.2, 2H), 2.40 (d, J=6.6, 2H), 2.28 (t, J=7.6,
2H), 1.88 – 1.38 (m, 10H), 1.21 (s, 24H), 0.82 (t, J=6.2, 3H).
13C NMR (300 MHz, MeOD) δ = 184.14, 182.29, 175.55,
40.18, 39.29, 37.07, 33.08, 32.87, 32.73, 30.81, 30.68, 30.57,
30.48, 30.37, 27.36, 27.02, 25.65, 24.44, 24.28, 23.74, 14.45.
195Pt NMR (86 MHz, DMSO-d6): δppm 1233. ESI-MS
(positive ion mode): m/z [M]+: calcd: 812.4297; obsd:
812.4259 Calcd for C28H63Cl4N5O5Pt: C 37.93%, H 7.16%, N
7.90%. Found: C 38.10%, H 7.12%, N 7.92%.
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4.7 General Procedure for the Synthesis of Compound 4c.
Intermediate 4c1 was dissolved in EtOH (10 mL) and
stirred at 0 °C for 10 min. Then, 4 M HCl was added dropwise
at 0 °C. The reaction mixture was stirred at room temperature
for 24–48 h and monitored by TLC. The solution typically
gave a faint yellow solid as a precipitate. The solid was
filtered, washed several times with absolute ethanol, and dried
4.4 General Procedure for the Synthesis of Compound
4b1.
To a solution of e (0.38 mmol) in DMF (10 mL) was added
a DMF solution (0.5 mL) containing HATU (0.57 mmol). This
mixture stirred for 10 min at room temperature. A DMF
solution containing q (1.5 mmol) and DIPEA (0.92 mmol) was
added to the resulting solution. 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 4b1
was purified by silica gel column chromatography as a yellow
solid with a yield of 47%. 1H NMR (300 MHz, MeOD) δ =
3.28 – 3.16 (m, 2H), 3.09 (d, J=11.2, 11H), 2.63 – 2.42 (m,
2H), 2.35 (d, J=7.5, 2H), 2.23 (s, 2H), 1.96 – 1.40 (m, 8H),
1.35 (s, 27H), 1.28 – 0.87 (m, 22H), 0.78 (t, J=6.1, 3H). 13C
NMR (300 MHz, MeOD) δ = 175.19, 174.36, 157.16, 156.55,
81.57, 80.94, 73.18, 46.24, 46.04, 45.81, 37.99, 37.01, 33.02,
32.73, 32.50, 30.76, 30.63, 30.51, 30.43, 30.29, 30.19, 28.83,
26.97, 25.99, 23.69, 19.34, 14.51.
1
under vacuum to give the pure target compound 4c. H NMR
(300 MHz, D2O) δ = 4.10 – 3.98 (m, 1H), 3.10 (m, 21H), 2.59
(d, J=28.9, 4H), 2.42 (s, 3H), 2.24 – 1.58 (m, 12H), 1.31 –
0.97 (m, 6H). 13C NMR (300 MHz, D2O) δ = 179.71, 179.55,
49.15, 48.99, 48.92, 48.75, 40.24, 40.09, 34.46, 34.42, 34.35,
33.74, 30.00, 29.97, 29.90, 27.00, 26.78, 17.62, 7.70. 195Pt
NMR
(86
MHz, DMSO-d6): δppm 1234. ESI-MS (positive ion mode):
m/z [M+Na]+: calcd: 893.4276; obsd: 893.4278. Calcd for
C32H71Cl5N6O5Pt: C 38.73%, H 7.21%, N 8.47%. Found: C
38.65%, H 7.32%, N 8.42%.
4.8 General Procedure for the Synthesis of Compound
5a1.
To a solution of z (0.38 mmol) in DMF (10 mL) was added
a DMF solution (0.5 mL) containing HATU (0.57 mmol). This
mixture was stirred for 10 min at room temperature. A DMF
solution containing y (1.5 mmol) and DIPEA (0.92 mmol) was
added to the resulting solution. The mixture stirred at room
temperature for 24 h in the dark. The DMF was then removed
under vacuum to afford a yellow oil. Compound 5a1 was
purified by silica gel column chromatography as a yellow
4.5 General Procedure for the Synthesis of Compound 4b.
Intermediate 4b1 was dissolved in EtOH (10 mL) and
stirred at 0 °C for 10 min. Then, 4 M HCl was added dropwise
at 0 °C. The reaction mixture stirred at room temperature for
24–48 h and monitored by TLC. The solution typically gave a
faint yellow solid as a precipitate. The solid was filtered,
washed several times with absolute ethanol, and dried under
vacuum to give the pure target compound 4b. 1H NMR (300
MHz, MeOD) δ = 3.73 (m, 1H), 3.28 – 2.83 (m, 18H), 2.43 (d,
J=22.4, 4H), 2.25 – 1.39 (m, 8H), 0.93 (s, 24H), 0.78 (s, 3H).
13C NMR (300 MHz, MeOD) δ = 176.16, 170.19, 152.41,
87.01, 73.92, 46.42, 46.24, 34.97, 34.79, 33.13, 33.03, 31.27,
30.71, 30.63, 30.52, 30.43, 30.32, 30.15, 30.03, 26.09, 24.34,
23.69, 19.41, 14.40. 195Pt NMR(86 MHz, DMSO-d6): δppm
1236. ESI-MS (positive ion mode): m/z [M]+: calcd: 881.4134;
obsd: 881.4167. Calcd for C32H69Cl5N6O5Pt: C 38.81%, H
7.02%, N 8.49%. Found: C 38.89%, H 7.12%, N 8.38%.
1
solid in a yield of 39%. H NMR (300 MHz, CDCl3) δ = 3.05
(dd, J=17.1, 18H), 2.49 (d, J=8.4, 7H), 1.89 – 1.00 (m, 65H),
0.84 (d, J=6.8, 2H), 0.71 (d, J=5.7, 2H), 0.55 (s, 3H). 13C
NMR (300 MHz, CDCl3) δ = 175.65, 172.54, 165.32, 156.77,
155.60, 79.44, 46.93, 45.38, 44.92, 36.23, 32.01, 31.95, 29.81,
29.76, 29.67, 29.50, 29.44, 29.31, 28.84, 28.56, 27.94, 27.82,
25.85, 25.80, 22.76, 14.19, 8.14.
4.9 General Procedure for the Synthesis of Compound 5a.
Intermediate 5a1 was dissolved in EtOH (10 mL) and
stirred at 0 °C for 10 min. Then, 4 M HCl was added dropwise
at 0 °C. The reaction mixture was stirred at room temperature
for 24–48 h and monitored by TLC. The solution typically
gave a faint yellow solid as a precipitate. The solid was
filtered, washed several times with absolute ethanol, and dried
4.6 General Procedure for the Synthesis of Compound 4c1.
To a solution of e (0.38 mmol) in DMF (10 mL) was added
a DMF solution (0.5 mL) containing HATU (0.57 mmol). This
mixture was stirred for 10 min at room temperature. A DMF
solution containing y (1.5 mmol) and DIPEA (0.92 mmol) was
1
under vacuum to give the pure target compound 5a. H NMR
(300 MHz, CDCl3) δ = 3.02 (m, 13H), 2.33 (m, 5H), 1.38 (s,
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32H), 1.19 (s, 11H), 0.74 (m, 4H), 0.52 (m, 2H). 13C NMR
agents in the A549 and A549cisR cellular assays were also
measured by microplate reader according to the procedure as
described in our previous projects. [14]
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5
6
7
8
(300 MHz, CDCl3) δ = 171.73, 171.42, 155.65, 154.48, 45.81,
44.31, 43.99, 42.65, 35.23, 30.89, 30.83, 28.69, 28.64, 28.55,
28.38, 28.32, 28.19, 27.72, 27.44, 26.75, 24.68, 21.64, 13.07,
7.02.
195Pt
NMR
(86
4.13 In vivo antitumor and antimetastasis assays
MHz, DMSO-d6): δppm 1611. ESI-MS (positive ion mode):
m/z [M+Na]+: calcd: 994.5522; obsd: 994.5533. Calcd for
C40H79Cl3N6O9Pt: C 44.10%, H 7.31%, N 7.71%. Found: C
44.18%, H 7.29%, N 7.79%.
In compliance with the Guide for the Care and Use of
Laboratory Animals, we chose healthy female BALB/c mice
(from the Laboratory Animal Center, Academy of Military
Medical Science, Cat. SCXK 2016-0006, Beijing, China) aged
5 weeks, weighing 18−22 g. 4T1 cells (1 × 106 cells per mouse)
were injected via the tail vein for the determination of lung
metastasis and anticancer activity. 4a was dissolved in glucose
injection and immediately used after preparation. All animal
procedures were performed following the protocol approved
by the Institutional Animal Care and Use Committee at Henan
University. The projects in vivo were approved by the ethical
committee with the number of the approved ethical vote
HUSOM-2016-316.
For antimetastatic activity in vivo, the tumor cells were
inoculated for 7 days to ensure the growth of pulmonary
metastasis before drug treatment. On day 8, we injected via the
tail vein compound 4a (5.0 mg/kg, 10 mg/kg, or 20 mg/kg),
oxaliplatin (5 mg/kg, positive control), or normal saline
(negative control) every two days for a total of four treatments
with the randomly grouped mice (n = 8 mice per group). On
day 15, we anesthetized and euthanized the mice and removed
the lungs. We counted the lung metastasis nodules of each
mouse after fixation with 4% paraformaldehyde for 1 day.
For the solid tumor study, tumors were 80-120 mm3 after 1
week. Then, mice were injected via the tail vein with 4a (5.0
mg/kg, 10 mg/kg, or 20 mg/kg), oxaliplatin (5 mg/kg, positive
control), or physiological saline (negative control) every two
days for a total of four treatments. The mice were weighed
every two days, and the tumor volume was measured by a
Vernier caliper.
9
4.10 General Procedure for the Synthesis of Compound 6a
and 7a
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The synthetic routes of 6a and 7a have been reported by our
group. [14]
4.11 General Procedure for the Synthesis of Compound 8a.
Our group has reported the synthetic route of d. [14] After
reaction with succinic anhydride overnight, the intermediate
was dissolved in EtOH (10 mL) and stirred at 0 °C for 10 min
1
to give the target compound 8a-1. 8a-1 H NMR (300 MHz,
MeOD) δ = 3.17 (s, 3H), 2.98 (m, 9H), 2.61 – 2.32 (m, 4H),
1.85 (m, 10H), 1.55 (d, J=6.6, 4H), 1.23 (s, 8H). 13C NMR
(300 MHz, CDCl3) δ = 174.85, 172.29, 155.53, 155.39, 81.15,
80.59, 58.04, 50.24, 29.49, 28.68, 28.40, 28.35, 28.25, 28.10.
Then, 4 M HCl was added dropwise at 0 °C. The reaction
mixture stirred at room temperature for 24–48 h and
monitored by TLC. The solution typically gave a faint yellow
solid as a precipitate. The solid was filtered, washed several
times with absolute ethanol, and dried under vacuum to give
the pure target compound 8a. 1H NMR (300 MHz, MeOD) δ =
3.44 – 3.24 (m, 2H), 3.15 – 2.92 (m, 10H), 1.78 (dt, J=44.0,
22.1, 11H). 13C NMR (300 MHz, MeOD) δ = 173.01, 171.47,
46.50, 28.29, 28.23, 27.81, 27.75, 27.51.
4.12 In Vitro Cellular Cytotoxicity Assays
The mice were sacrificed by anesthesia and the tumor
tissues were removed and weighed. We calculated the
inhibition rate as follows: inhibition rate (%) = [(average
tumor weight of negative control group − average tumor
weight of the drug treated or positive control group)/average
tumor weight of control group] × 100. Moreover, on the last
day, we removed and weighed the organs (heart, liver, kidney,
lung, and spleen) of the mice. We calculated the organ weight
index as follows: organ index (%) = (organ weight/body
weight) × 100%
Cells seeded in 96-well plates were incubated in a 5% CO2
atmosphere in 100 µL of complete medium at 37 °C for 24 h.
Then, 100 µL of 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 (150 µL) was added. The absorbance was measured at
570 nm using a Bio-Rad 680 microplate reader. The IC50
values were calculated using GraphPad Prism software.
Technical repetitions and independent experiments were based
on three parallel experiments.
For cytotoxicity assays using the PAT inhibitor Spd, a
similar procedure as described above was followed, except
that A549cisR cells (5000 cells/well) were seeded on a 96-
well plate in 100 µL of RPMI and incubated for 24 h at 37 °C.
Spd-containing RPMI medium was used for serial dilution of
the platinum compound-containing concentrated solutions, and
100 µL/well was added. The cytotoxicity profiles of the
compounds were evaluated using the MTT assay.
4.14 In vivo MTD and LD50 antitumor assays
The maximum tolerated dose (MTD) (n = 10 mice per
group) was evaluated by calculating body weight loss (mean
weight loss <15% and <15% toxic deaths) and the lethal dose
values (LD50) (n = 10 mice per group) were determined. The
TI value was calculated according to the procedure as previous
described. [12b]
4.15 Transwell Migration Assay
A PAO, GSH and GSH-Px Preparation Kit (containing
reduced and oxidized glutathione) was used for the isolation of
PAO, GSH and GSH-Px in A549 and A549cisR cells, and
PAO, GSH and GSH-Px concentrations treated with several
Transwell migration assays were performed by using modified
Boyden’s chamber in 24-well cell culture plate with a 8 μm
pore. Chambers were washed with PBS for three times. Then
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the medium with the tested compound was placed in the lower Total PAO level was measured using GSH assay kit
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chamber, and cells were seeded in the top chamber. Cells were
treated with 4a or cisplatin at 37 °C in a humidified
atmosphere of 5% CO2. After incubation for 24 h, non-
migrated cells on the top surface of the membrane were gently
scraped away with cotton swab. The migrated-cells was fixed
with 4% paraformaldehyde for 20 min and stained with 0.2%
crystal violet. Images were recorded using a Leika inverted
microscope.
according to the manufacturer ’ s instructions ((Hepeng
Biotechnology, Cat. HEPENGBIO156). Briefly, after
treatment with 4a (10 μM) for 24 h, cells were washed twice
with Reagent A (3 mL) and then collected the cells by cell
scraper. Pellets were resuspended in Reagent A (3 mL) by
centrifugation at 300g for 5 min. Supernate were resuspended
in Reagent B (500 μL) and vortexed immediately followed by
being lysed by three freeze−thaw cycles. After cell lysates
were centrifuged at 16,000g for 5 min at 4 °C, supernatant
containing total PAO was transferred to another pre-cooling
tube. For quantifying total PAO level, 10 μL of supernatant
was mixed with 240 μL (200 μL Reagent C, 25 μL Reagent D
and 15μL Reagent E) of total PAO assay buffer for 3 min at
37 °C, and then 10 μL of Reagent F was added. Absorbance
was read at 440 nm by using the microplate reader.
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4.16 Cellular Platinum Uptake and DNA Platination
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Cellular uptake was measured in A549, A549cisR, SMMC-
7721, and MCF-7 cells. A549, A549cisR, SMMC-7721, and
MCF-7 cells were seeded in 6-well plates overnight and then
incubated with 10 µM homospermidineplatin 4a or cisplatin
for 8 h. Subsequently, the cells were washed with PBS buffer
three times and harvested by trypsinization. The harvested
cells were concentrated and digested with nitric acid for ICP-
MS. The cell numbers were counted before digestion. A
Genomic DNA mini preparation kit was used for the isolation
of DNA in A549, A549cisR, SMMC-7721, and MCF-7 cells,
and Pt concentrations in the cellular DNA of A549, A549cisR,
SMMC-7721, and MCF-7 cells digested by nitric acid were
also measured by ICP-MS.
4.20 Reactive Oxygen Species Assay
Reactive oxygen species (ROS) were examined with the
fluorescent probe DCFH-DA and detected by flow cytometry
as our previously described . [14]
4.21 Detection of total glutathione (GSH)
4.17 Annexin V-FITC/Propidium Iodide Staining
Total glutathione (containing reduced and oxidized
glutathione) level was measured using GSH assay kit
according to the manufacturer’s instructions (Solarbio
Biochemical Assay Division, Cat. 20180226, Beijing, China).
Briefly, after treatment with 4a (10 μM) for 24 h, cells were
washed twice with PBS by centrifugation at 600g for 5 min.
Pellets were resuspended in protein removal buffer and
vortexed immediately followed by being lysed by three
freeze−thaw cycles. After cell lysates were centrifuged at
12,000g for 10 min at 4 °C, supernatant containing total
glutathione was transferred to another tube. For quantifying
total glutathione level, 10 μL of supernatant was mixed with
150 μL of total glutathione assay buffer for 5 min, and then 50
μL of NADPH (0.16 mg/mL) was added. Absorbance was
read at 405 nm by using the microplate reader.
Apoptosis of A549cisR cells was evaluated using Annexin
V-FITC/PI staining detected by flow cytometry. Cells were
plated into six-well plates at 1 × 105 cells/well. After cultured
for 24 h, cells were hatched with 4a (5 or 10 μM), cisplatin (15
μM) for 24 h. Then cells were harvested, washed three times
with PBS to remove unbound dyes, and stained according to
the manufacturer’s protocol. Analysis was performed by flow
cytometry (BD Biosciences, San Jose, CA, USA).
4.18 Western Blot Assay
To detect the expression of the related proteins phospho-
histone H2A-X (Ser139), p53, ODC and SSAT, Western blot
analysis was performed. Cells were treated with 4a at
concentrations of 5, 10, and 15 μM for 24 h and were then
harvested and centrifuged. The cell pellets were washed three
times with ice-cold PBS and then lysed with RIPA buffer
(Beyotime, China). Using a BCA assay kit (Beyotime, China),
we determined the total concentration of protein. After that,
we denatured the total lysates in 5 × SDS-loading buffer at
100 °C for 10 min. Using 12% SDS-PAGE for 2 h, we
separated equal amounts of total proteins and then transferred
the proteins onto PVDF membranes. The membranes were
blocked with 5% dried skimmed milk in TBST at room
temperature for 1 h. After incubation with the corresponding
primary antibodies overnight at 4 °C, the membranes were
washed three times with TBST and incubated with the
appropriate HRP-conjugated secondary antibody. Protein
expression was detected using the ECL plus reagents
(Beyotime, Jiangsu, China).
4.22 Detection of total glutathione peroxidase (GSH-Px)
Total GSH-Px level was measured using GSH-Px assay kit
according to the manufacturer ’ s instructions (Leagene
Biotechnology, Cat. 0309A18, Beijing, China). Briefly, after
treatment with 4a (10 μM) for 24 h, cells were washed twice
with PBS by centrifugation at 12, 000g for 10 min. Pellets
were resuspended in protein removal buffer and vortexed
immediately followed by being lysed by three freeze−thaw
cycles. After cell lysates were centrifuged at 12,000g for 10
min at 4 °C, supernatant containing total GSH-Px was
transferred to another tube. For quantifying total GSH-Px level,
0.2 mL of supernatant was mixed with 0.4 mL of total GSH-
Px assay buffer for 5 min at 37 °C, and then 0.25 mL of
benzoic acid was added. Absorbance was read at 422 nm by
using the microplate reader.
4.19 Detection of polyamine oxidase (PAO)
4.23 Polyamines Contents Assay
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Coupled with
a G1321A fluorescence detector, we Science and Technology in University of Henan Province)
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quantified the polyamine (putrescine, spermidine, and
spermine) content in A549cisR cells by using HPLC (Agilent
1260, Agilent Technologies, USA) according to our previous
reported. [14]
(Grant No. 19IRTSTHN004), Project of Innovation and
Entrepreneurship Support Program for College Students of
Henan University Minsheng College (Grant No.
MSCXCY2018058) and Henan University (Grant No.
The polyamines were separated on a C18 chromatographic
column (25 × 4.6 mm, 5 μm) with dansyl chloride as the
derivation reagent and 1,6-diaminohexane as the internal
standard substance using methanol−water (65:35−100:0, 30
min gradient elution) (excitation = 340 nm and emission = 515
nm). The polyamines (putrescine, spermidine, and spermine)
were converted to the corresponding dansyl derivatives and
separated on a C18 analytical column by eluting with an ethyl
acetate/water gradient. 1,6-Diaminohexane was used as the
internal standard.
201910475012 and 2019102004), Scientific
Research
Cultivating Program for Young Talents in Henan Medical
School (Grant No. 2019006) and the key scientific research
projects of universities in Henan province (No13A350095).
9
REFERENCES
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16
17
18
19
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22
23
24
25
26
27
28
29
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[1] Forner, A.; Llovet, J. M.; Bruix, Hepatocellular carcinoma.
J. Lancet 2012, 379, 1245−1255.
[2] Dou, C.; Liu, Z.; Xu, M.; Jia, Y.; Wang, Y.; Li, Q.; Yang,
W.; Zheng, X.; Tu, K.; Liu, Q. miR-187-3p inhibits the
metastasis and epithelial-mesenchymal transition of
hepatocellular carcinoma by targeting S100A4. Cancer Lett.
2016, 381, 380−390
[3] Valastyan, S.; Weinberg, R. A. Tumor metastasis:
molecular insights and evolving paradigms. Cell 2011, 147,
275−292.
[4] Sonnenschein, C.; Soto, A. M. Cancer metastases: so close
and so far. J. Natl. Cancer Inst. 2015, 107, djv236.
[5] Ran, S. The role of TLR4 in chemotherapy-driven
metastasis. Cancer Res. 2015, 75, 2405−2410.
[6] Johnstone, T.C.; Suntharalingam, K.; Lippard, S.J. The
Next generation of platinum drugs: targeted Pt(II) agents,
nanoparticle delivery, and Pt(IV) prodrugs. Chem Rev. 2016,
116, 3436-3486.
[7] a) Kenny RG, Marmion CJ. Toward multi-targeted
platinum and ruthenium drugs-a new paradigm in cancer drug
treatment regimens? Chem Rev. 2019, 119, 1058-1137; b)
Wang, D.; Lippard, S. J. Cellular processing of platinum
anticancer drugs. Nat. Rev. Drug Discovery 2005, 4, 307 –
320; c) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The
status of platinum anticancer drugs in the clinic and in clinical
trials. Dalton Trans. 2010, 39, 8113-8127. d) L. Kelland, The
resurgence of platinum-based cancer chemotherapy. Nat. Rev.
Cancer 2007, 7, 573-584.
[8] Wang, X.; Wang, X.; Guo, Z. Functionalization of
platinum complexes for biomedical applications. Acc Chem
Res. 2015, 48, 2622-2631.
[9] Basu, U.; Banik, B.; Wen, R.; Pathak, R.K.; Dhar, S. The
platin-X series: activation, targeting, and delivery. Dalton
Trans. 2016, 45, 12992-3004.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website.
Full experimental details, NMR data, and bioassay
information (PDF)
Molecular formula strings(CSV)
AUTHOR INFORMATION
Corresponding Author *Phone: (86) 0371-23880680. Fax:
(86) 0371-23880680. E-mail: majing@henu.edu.cn;
wcjsxq@henu.edu.cn; xiesq@vip.henu.edu.cn
Author Contributions
#The authors contribute equally to this work.
Abbreviations
PTs, Polyamine Transporters; SSAT, spermidine/spermine
N1-acetyltransferase; PAO, N1-acetylpolyamine oxidase;
ROS, Reactive Oxygen Species; GSH-Px, glutathione
peroxidase; GSH, glutathione; Spd, spermidine; Spm,
spermine; Put, putrescine. LD50, lethal dose of 50%; MTD,
maximum tolerated dose; IC50, half maximal inhibitory
concentration; ICP-MS, Inductively Coupled Plasma Mass
Spectrometry; DMF, dimethyl formamide; DIPEA,
diisopropylethylamine; HATU, 2-(7-Azabenzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium hexafluorophosphate.
[10] Han, X.; Sun, J.; Wang, Y.; He, Z. Recent advances in
platinum (IV) complex-based delivery systems to improve
platinum (II) anticancer therapy. Med Res Rev. 2015, 35,
1268-1299.
Notes
The authors declare no competing financial interest.
[11] a) Chen, Y.; Heeg, M. J.; Braunschweiger, P. G.; Xie, W.;
Wang, P. G. A carbohydrate-linked cisplatin analogue having
antitumor activity. Angew. Chem., Int. Ed. 1999, 38: 1768; b)
Ma, J.; Wang, Q.; Yang, X.; Hao, W.; Huang, Z.; Zhang, J.;
Wang, X.; Wang, P. Glycosylated platinum(iv) prodrugs
demonstrated significant therapeutic efficacy in cancer cells
and minimized side-effects. Dalton Trans. 2016, 45:
11830−11838. c). Wang, Q.; Huang, Z.; Ma, J.; Lu, X.; Zhang,
L.; Wang, X.; Wang, P. G. Design, synthesis and biological
evaluation of a novel series of glycosylated platinum(iv)
complexes as antitumor agents. Dalton Trans. 2016, 45:
ACKNOWLEDGMENTS
This work was supported by National Natural Science
Foundation of China (Grant No. 21807025, 81772832 and
81573465), China Postdoctoral Science Foundation (Grant No.
2018M640673), Postdoctoral Research Grant in Henan
Province (Grant No. 001802020), Key Scientific Research
Projects in Henan Colleges and Universities (Grant No.
19A350002), Program for Innovative Research Team (in
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Page 13 of 16
Journal of Medicinal Chemistry
10366−10374. d) Ma, J.; Wang, Q.; Huang, Z.; Yang, X.; Nie, mutated cancer cell lines. Angew. Chem. Int. Ed. 2017, 56,
1
2
3
4
5
6
7
8
Q.; Hao, W.; Wang, P.G.; Wang, X. Glycosylated platinum(IV)
complexes as substrates for glucose transporters (GLUTs) and
organic cation transporters (OCTs) exhibited cancer targeting
and human serum albumin binding properties for drug delivery.
J Med Chem. 2017, 60: 5736-5748. e) Ma, J.; Yang, X.; Hao,
W.; Huang, Z.; Wang, X.; Wang, P.G. 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. Eur J Med Chem. 2017, 128: 45-55. f). Ma J, Liu H, Xi
Z, Hou J, Li Y, Niu J, Liu T, Bi S, Wang X, Wang C, Wang J,
Xie S, Wang PG. Protected and de-protected platinum(IV)
glycoconjugates with GLUT1 and OCT2-mediated selective
cancer targeting: demonstrated enhanced transporter-mediated
cytotoxic properties in vitro and in vivo. Front Chem. 2018, 6,
386. g). Wang H, Yang X, Zhao C, Wang PG, Wang X.
Glucose-conjugated platinum(IV) complexes as tumor-
targeting agents: design, synthesis and biological evaluation.
Bioorg Med Chem. 2019, 27, 1639-1645.
[12] a). Dhar S, Daniel WL, Giljohann DA, Mirkin CA,
Lippard SJ. Polyvalent oligonucleotide gold nanoparticle
conjugates as delivery vehicles for platinum(IV) warheads. J
Am Chem Soc. 2009, 131, 14652-14653. b). Liu P, Lu Y, Gao
X, Liu R, Zhang-Negrerie D, Shi Y, Wang Y, Wang S, Gao Q.
Highly water-soluble platinum(II) complexes as GLUT
substrates for targeted therapy: improved anticancer efficacy
and transporter-mediated cytotoxic properties. Chem
Commun. 2013, 49, 2421-2423. c). Y.-R. Zheng, K.
Suntharalingam, T. C. Johnstone, H. Yoo, W. Lin, J. G.
Brooks, S. J. Lippard, Pt(IV) prodrugs designed to bind non-
covalently to human serum albumin for drug delivery. J. Am.
Chem. Soc. 2014, 136, 8790-8798; d). Q. Cheng, H. Shi, H.
Wang, Y. Min, J. Wang, Y. Liu, The ligation of aspirin to
cisplatin demonstrates significant synergistic effects on tumor
cells. Chem. Commun. 2014, 50, 7427-7430; e). R. K. Pathak,
S. Marrache, J. H. Choi, T. B. Berding, S. Dhar, The prodrug
platin-A: simultaneous release of cisplatin and aspirin. Angew.
Chem. Int. Ed. 2014, 53, 1963-1967; f). S. G. Awuah, Y. R.
Zheng, P. M. Bruno, M. T. Hemann, S. J. Lippard, A Pt(IV)
pro-drug preferentially targets indoleamine-2,3-dioxygenase,
providing enhanced ovarian cancer immuno-chemotherapy. J.
Am. Chem. Soc. 2015, 137, 14854-14857; g). J. Kasparkova,
H. Kostrhunova, O. Novakova, R. Krˇikavov J. Vancˇo, Z.vn
cˇek, V. Brabec, A photoactivatable platinum(IV) complex
targeting genomic DNA and histone deacetylases. Angew.
Chem. Int. Ed. 2015, 54, 14478-14482; h). R. Raveendran, J.
P. Braude, E. Wexselblatt, V. Novohradsky, O. Stuchlikova, V.
Brabec, V. Gandin, D. Gibson, Pt(iv) derivatives of cisplatin
and oxaliplatin with phenylbutyrate axial ligands are potent
cytotoxic agents that act by several mechanisms of action.
Chem. Sci. 2016, 7, 2381-2391; i). G. Thiabaud, R. McCall, G.
He, J. F. Arambula, Z. H. Siddik, J. L. Sessler, Activation of
platinum(IV) prodrugs by motexafin gadolinium as a redox
mediator. Angew. Chem. Int. Ed. 2016, 55, 12626-12631; j).
N. Muhammad, N. Sadia, C. Zhu, C. Luo, Z. Guo, X. Wang,
Biotin-tagged platinum(iv) complexes as targeted cytostatic
agents against breast cancer cells. Chem. Commun. 2017, 53,
9971 – 9974; k). E. Petruzzella, J. P. Braude, J. R.
AldrichWright, V. Gandin, D. Gibson, A quadruple-action
platinum(IV) prodrug with anticancer activity against KRAS
11539 – 11544; l). Ma L, Wang N, Ma R, Li C, Xu Z, Tse MK,
Zhu G. Monochalcoplatin: an actively transported, quickly
reducible, and highly potent Pt(IV) anticancer prodrug.
Angew. Chem. Int. Ed. 2018, 57, 9098-9102. m). Petruzzella
E, Sirota R, Solazzo I, Gandin V, Gibson D. Triple action
Pt(iv) derivatives of cisplatin: a new class of potent anticancer
agents that overcome resistance. Chem Sci. 2018, 9, 4299-
4307. n). Wang K, Zhu C, He Y, Zhang Z, Zhou W,
Muhammad N, Guo Y, Wang X, Guo Z. Restraining cancer
cells by dual metabolic inhibition with a mitochondrion-
targeted platinum(II) complex. Angew. Chem. Int. Ed. 2019,
58, 4638-4643; o). Zhu Z, Wang Z, Zhang C, Wang Y, Zhang
H, Gan Z, Guo Z, Wang X. Mitochondrion-targeted platinum
complexes suppressing lung cancer through multiple pathways
involving energy metabolism. Chem Sci. 2019, 10, 3089-3095;
p). Babak MV, Zhi Y, Czarny B, Toh TB, Hooi L, Chow EK,
Ang WH, Gibson D, Pastorin G. Dual-targeting dual-action
platinum(IV) platform for enhanced anticancer activity and
reduced nephrotoxicity. Angew. Chem. Int. Ed. 2019, 58,
8109-8114. q). Chen F, Huang X, Wu M, Gou S, Hu W. A
CK2-targeted Pt(IV) prodrug to disrupt DNA damage
response. Cancer Lett. 2017, 385, 168-178. r). Chun W, Ke-
Jia W, Jin-Biao L, Xiao-Ming Z, Chung-Hang L and Dik-
Lung M. A dual-functional molecular strategy for in situ
suppressing and visualizing of neuraminidase in aqueous
solution using iridium(iii) complexes. Chem. Commun., 2019,
55, 6353. s). Guan-Jun Y, Wanhe W, Simon Wing FM, Chun
W, Betty Yuen KL, Xiang-Min M, Ke-Jia W, Hai-Jing Z,
Chun-Yuen W, Vincent Kam W, DikLung M and Chung-
Hang L. Selective inhibition of lysine-specific demethylase 5A
(KDM5A) using a rhodium(III) complex for triple-negative
breast cancer therapy. Angew. Chem. Int. Ed. 2018, 57,
13091. t). Huaiyi H, Samya B, Kangqiang Q, Pingyu Z,
Olivier B, Thomas M, Martin J. P, Guy J. C, Michael S,
Vasilios G. S, Gilles G, Hui C and Peter J. S. Targeted
photoredox catalysis in cancer cells. Nat. Chem., 2019, DOI:
10.1038/s41557-019-0328-4.
[13] a). Casero RA Jr, Murray Stewart T, Pegg AE. Polyamine
metabolism and cancer: treatments, challenges and
opportunities. Nat Rev Cancer. 2018, 18, 681-695. b).
Phanstiel O 4th. An overview of polyamine metabolism in
pancreatic ductal adenocarcinoma. Int J Cancer. 2018, 142,
1968-1976. c). Casero RA Jr, Marton LJ. Targeting polyamine
metabolism and function in cancer and other
hyperproliferative diseases. Nat Rev Drug Discov. 2007, 6,
373-90. d). Muth, A.; Pandey, V.; Kaur, N.; Wason, M.; Baker,
C.; Han, X.; Johnson, T. R.; Altomare, D. A.; Phanstiel, O.,
4th Synthesis and biological evaluation of antimetastatic
agents predicated upon dihydromotuporamine C and its
carbocyclic derivatives. J. Med. Chem. 2014, 57, 4023−4034.
e). Skruber, K.; Chaplin, K. J.; Phanstiel, O., 4th. Synthesis
and bioevaluation of macrocycle-polyamine conjugates as cell
migration inhibitors. J. Med. Chem. 2017, 60, 8606−8619. f).
Muth A, Kamel J, Kaur N, Shicora AC, Ayene IS, Gilmour
SK, Phanstiel O 4th. Development of polyamine transport
ligands with improved metabolic stability and selectivity
against specific human cancers. J Med Chem. 2013, 56,
5819-5828.
9
10
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17
18
19
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25
26
27
28
29
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31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
[14] a). Chaojie, W.; Jean-Guy, D.; Laura, C.; Fanta, K.;
Horacio, C.; John, B.; Richard Andrew, G.; Phanstiel O 4th.
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Defining the molecular requirements for the selective delivery [18] Ishikawa T. The ATP-dependent glutathione S-conjugate
1
2
3
4
5
6
7
8
of polyamine conjugates into cells containing active
polyamine transporters. J Med Chem. 2003, 46, 5129-5138.
b). Chaojie, W.; Jean-Guy, D.; John, B.; Phanstiel O 4th.
Molecular requirements for targeting the polyamine transport
system. Synthesis and biological evaluation of polyamine-
anthracene conjugates. J Med Chem. 2003, 46, 2672-2682. c).
Chaojie, W.; Jean-Guy, D.; John, B.; Phanstiel O 4th.
Synthesis and biological evaluation of N1-(anthracen-9-
ylmethyl)triamines as mo
export pump. Trends Biochem Sci. 1992, 11, 463-468.
[19] Yang, L.; Zhao, J.; Zhu, Y.; Tian, Z.; Wang, C. Reactive
oxygen species (ROS) accumulation induced by
mononaphthalimide-spermidine leads to intrinsic and AIF-
mediated apoptosis in HeLa cells. Oncol. Rep. 2011, 25 (4),
1099−1107.
9
lecular recognition elements for the polyamine transporter. J
Med Chem. 2003, 46, 2663-2671. d). Xie, S.; Wang, J.;
Zhang, Y.; Wang, C. Antitumor conjugates with polyamine
vectors and their molecular mechanisms. Expert Opin Drug
Deliv. 2010, 7, 1049-1061. e). Wang, Y. X.; Zhang, X.; Zhao,
J.; Xie, S. Q.; Wang, C. J. Nonhematotoxic naphthalene
diimide modified by polyamine: synthesis and biological
evaluation. J. Med. Chem. 2012, 55, 3502−3512. f). Li, M.;
Li, Q.; Zhang, Y. H.; Tian, Z. Y.; Ma, H. X.; Zhao, J.; Xie, S.
Q.; Wang, C. Antitumor effects and preliminary systemic
toxicity of ANISpm in vivo and in vitro. Anti-Cancer Drugs
2013, 24, 32−42. g). Li, Q.; Zhai, Y.; Luo, W.; Zhu, Z.;
Zhang, X.; Xie, S.; Hong, C.; Wang, Y.; Su, Y.; Zhao, J.;
Wang, C. Synthesis and biological properties of polyamine
modified flavonoids as hepatocellular carcinoma inhibitors.
Eur. J. Med. Chem. 2016, 121, 110 −119. h). Dai, F. J.; Li,
Q.; Wang, Y. X.; Ge, C. C.; Feng, C.; Xie, S. Q.; He, H.; Xu,
X.; Wang, C. J. Design, synthesis, and biological evaluation of
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
mitochondria-targeted
flavone-naphthalimide-polyamine
conjugates with antimetastatic activity. J. Med. Chem. 2017,
60, 2071−2083. i). Li J, Tian R, Ge C, Chen Y, Liu X, Wang
Y, Yang Y, Luo W, Dai F, Wang S, Chen S, Xie S, Wang C.
Discovery of the polyamine conjugate with benzo[ cd]indol-
2(1 H)-one as a lysosome-targeted antimetastatic agent. J Med
Chem. 2018, 61, 6814-6829. j). Cong W, Ping R, Ying Z,
Xiaomin L, Jun W, Xiaoxiao W, Tong L, Shasha W, Jiuzhou
H, Wei L, Qian L, Jinghua L, Fujun D, Dong F, Chaojie W,
Songqiang X. Spermidine/spermine N1-acetyltransferase
regulates cell growth and metastasis via AKT/β-catenin
signaling pathways in hepatocellular and colorectal carcinoma
cells. Oncotarget, 2017, 8, 1092-1109.
[15] a) Li L, Mao Y, Zhao L, Li L, Wu J, Zhao M, Du W, Yu
L, Jiang P. p53 regulation of ammonia metabolism through
urea cycle controls polyamine biosynthesis. Nature. 2019, 567,
253-256. b) Ou Y, Wang SJ, Li D, Chu B, Gu W. Activation
of SAT1 engages polyamine metabolism with p53-mediated
ferroptotic responses. Proc Natl Acad Sci U S A. 2016, 113,
E6806-E6812. c). Eastman A. Improving anticancer drug
development begins with cell culture: misinformation
perpetrated by the misuse of cytotoxicity assays. Oncotarget.
2017, 8, 8854-8866. d). Mirzayans R, Andrais B, Murray D.
Do Multiwell Plate High Throughput Assays Measure Loss of
Cell Viability Following Exposure to Genotoxic Agents? Int J
Mol Sci. 2017, 18, 1679.
[16] Nguyen, D. X.; Bos, P. D.; Massague, J. Metastasis: from
dissemination to organ-specific colonization. Nat. Rev.
Cancer 2009, 9, 274−284.
[17] W. M. Bonner, C. E. Redon, J. S. Dickey, A. J. Nakamura,
O. A. Sedelnikova, S. Solier, Y. Pommier, GammaH2AX and
cancer. Nat. Rev. Cancer 2008, 8, 957 – 967.
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