Naphthalimide Platinum(IV) Compounds as Antitumor Agents with Dual DNA Damage Mechanism to Overcome Cisplatin Resistance

Article abstract of DOI:10.1002/ejic.201800799

A new series of naphthalimide platinum(IV) compounds with dual DNA damage mechanism were designed, synthesized and evaluated for antitumor activities. The platinum(IV) compounds could combine with DNA and cause DNA damage via naphthalimide fragment. Then the platinum(II) complexes released in reductive microenvironment would cause remarkable secondary DNA lesions. Some title compounds exhibit good antitumor activities and are of great potential in overcoming the drug resistance of cisplatin. Furthermore, naphthalimide platinum(IV) complexes could effectively combine with HSA by electrostatic force, which would influence the drug distribution and bioactivities in vivo. Moreover, the accumulation of the tested platinum(IV) compounds in whole cells and DNA is remarkably enhanced in comparison with cisplatin and oxaliplatin.

Full text of DOI:10.1002/ejic.201800799

A Journal of
Accepted Article
Title: Naphthalimide Platinum(IV) Compounds as Antitumor Agents
with Dual DNA Damage Mechanism to Overcome Cisplatin
Resistance
Authors: Qingpeng Wang, Guoshuai Li, Zhifang Liu, Xiaoxiao Tan,
Zhuang Ding, Jing Ma, Lanjie Li, Dacheng Li, Jun Han, and
Bingquan Wang
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
FULL PAPER
Naphthalimide Platinum(IV) Compounds as Antitumor Agents
with Dual DNA Damage Mechanism to Overcome Cisplatin
Resistance
Qingpeng Wang,*[a] Guoshuai Li,[a] Zhifang Liu,[a] Xiaoxiao Tan,[a] Zhuang Ding,[a] Jing Ma,[b] Lanjie Li,[a]
Dacheng Li,*[a] Jun Han,[a] and Bingquan Wang[a]
Dedication ((optional))
Abstract: A new series of naphthalimide platinum(IV) compounds
with dual DNA damage mechanism were designed, synthesized and
evaluated for antitumor activities. The platinum(IV) compounds could
combine with DNA and cause DNA damage via naphthalimide
fragment. Then the platinum(II) complexes released in reductive
microenvironment would cause remarkable secondary DNA lesions.
Some title compounds exhibit good antitumor activities and are of
great potential in overcoming the drug resistance of cisplatin.
Furthermore, naphthalimide platinum(IV) complexes could effectively
combine with HSA by electrostatic force, which would influence the
drug distribution and bioactivities in vivo. Moreover, the
accumulation of the tested platinum(IV) compounds in whole cells
and DNA is remarkably enhanced in comparison with cisplatin and
oxaliplatin.
the development of new platinum complexes with novel DNA
binding modes hitting one or more therapeutic targets to
overcome the drug resistance has become an urgent task for
pharmaceutical researchers.[7,8]
Platinum(IV) compounds are of great potential to be
developed as the new generation of platinum anticancer drugs
and display many attracting pharmaceutical superiorities in
contrast to the platinum(II) drugs. [9–12] It is widely accepted that
platinum(IV) complexes as pro-drugs of platinum(II) drugs
exhibit antitumor activities via reduction to bivalence by
intracellular reductants including high molecular reducing
biomolecules and low molecular ascorbic acid (AsA), glutathione
(GSH) etc. which occur at higher concentrations in
neoplasm[13,14]. Consequently, platinum(IV) compounds tend to
possess high stabilities, low toxicities and good targeting
properties.[15–19] The investigation on platinum(IV) antitumor
complexes has become a more and more attractive topic in
recent years.
Introduction
Naphthalimide, an effective DNA intercalator with large π-
conjugated system, could easily combine with DNA double helix
by diverse weak interactions such as π-π stacking, hydrogen
bonds, Van der Waals force etc., and display remarkable
Platinum compounds as the most prominent metallic drugs are
widely applied in the treatment of various malignancies in clinic
including testicular, ovarian, bladder, head and neck cancers.[1,2]
Platinum(II) drugs account for approximately 50% of total
expenditures of anticancer drugs and play a vital role in
anticancer treatments. It has been fully demonstrated that
platinum(II) compounds achieve their therapeutic effects by
forming DNA lesions, which interfere with the genomic activities
and ultimately trigger apoptosis.[3] However, with the wide use of
platinum drugs for decades, the undesirable side effects have
rather limited their clinical efficiency. Especially the increasingly
serious drug resistance which mainly arise from the increased
DNA repair and/or DNA damage tolerance etc., has badly
reduced the cancer-cure effects of platinum drugs.[4–6] Therefore,
anticancer
properties.[20,21]
Naphthalimide
platinum(II)
compounds I-VI (Figure 1) exhibited interesting antitumor
properties. The naphthalimide was proven a suitable “carrier”
that favored DNA targeting and decreased the drug resistance of
target compounds.[22–24] Notably, the conversion of compounds V
and VI into platinum(IV) form by chlorination in the axial
positions led to remarkably enhanced antitumor activities and
robust stability in contrast to their platinum(II) precursors.[25]
Therefore, the incorporation of DNA targeted naphthalimide
fragment into platinum(IV) system is a promising strategy for
exploration of novel chemotherapeutic drugs.
Inspired by the facts mentioned above and in continuation of
our ongoing interest in the development of new antitumor
compounds,[26–29] a series of new naphthalimide platinum(IV)
complexes with potential dual DNA damage mechanism were
designed in this work (Scheme 1). It is expected that the DNA
targeted naphthalimide would enable target platinum(IV)
compounds to conjugate with DNA before reduction, and cause
DNA lesions to tumor cells. The further degradation of
platinum(IV) complexes would release platinum(II) drugs and
induce a secondary damage to DNA. This dual DNA damage
mechanism is rather different from the traditional platinum(II)
drugs such as cisplatin and oxaliplatin, as well as the promising
platinum(IV) complex satraplatin (JM216), and would shade
positive effects on the overcoming of the drawbacks of clinical
[a]
Dr. Q. Wang, G Li, Z. Liu, X. Tan, Dr. Z. Ding, Dr. L. Li, Prof. D. Li,
Prof. J. Han, Prof. B. Wang
Institute of Biopharmaceutical Research
Liaocheng University
Liaocheng 252059, P.R. China
E-mail: lywqp@126.com (Q. Wang)
http://ibpr.lcu.edu.cn/index.html
lidacheng62@163.com (D. Li)
http://ibpr.lcu.edu.cn/content/163.html
Dr. J. Ma
[b]
Institute of Chemical Biology, College of Pharmacy
Henan University
Kaifeng 475004, P.R. China
Supporting information for this article is given via a link at the end of
the document.((Please delete this text if not appropriate))
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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platinum drugs. Herein, the target naphthalimide platinum(IV)
compounds 1–5 with cisplatin as mother nucleus were designed
and prepared. In order to get suitable linker between
naphthalimide and platinum core, compounds 1–3 with variable
aliphatic chains were prepared. Moreover, it has been revealed
in literatures that different substituents especially the nitrogen
containing ones on the naphthalene ring of naphthalimide
compounds had great impacts on their antitumor activities.[30]
Therefore, compounds 4 and 5 bearing nitro and amino groups
were designed. The antitumor activities of the title compounds
were evaluated in vitro and in vivo, and their likely antitumor
mechanism was also investigated.
Figure 1. Chemical structures of naphthalimide platinum complexes.
Scheme 1. Synthetic route for naphthalimide platinum(IV) compounds. Conditions and reagents: (a) HNO3/H2SO4, rt, 1.5 h; (b) H2, Pd/C, DMF, rt, 4h; (c) Amino
acid, DMF, 80 °C, 9h; (d) H2O2/H2O, 60 °C, 4 h; (e) TBTU, TEA, DMF, 50 °C, 48 h.
The target compounds were prepared according to the synthetic
route
presented
in
Scheme
1.
Nitro
naphthalenedicarboxylicanhydride
nitration reaction of starting material 6. The following
hydrogenization of compound 7 with Pd/C afforded the amino
7
was obtained by the
Results and Discussion
Synthesis of naphthalimide platinum(IV) compounds
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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derivate 8. Compounds 9–11 could be prepared by the reaction
of naphthalimide compounds 6–8 with various amino acids.
Oxoplatin 13 was synthesized via the oxidation of cisplatin by
hydrogen peroxide. Subsequently, the condensation of acids 9–
11 with oxoplatin 13 by TBTU afforded the target naphthalimide
platinum(IV) compounds 1–5 in yields of 32–62%.
Different linkages between the naphthalimide and platinum(IV)
core significantly affect the bioactivities of target compounds,
while the incorporation of nitro and amino moieties to
naphthalimide fragment also shades much influence on their
antitumor properties. For compounds 1–3, compound 2 with
three carbon chain exhibits more prominent activities than
compounds 1 and 3 with longer or shorter ones. Thus,
naphthalimide platinum(IV) complex with three carbon linkage
was selected for further modification. The following introduction
of amino group onto naphthalimide to afford compound 5
increases the anticancer efficacy in comparison with compound
2 especially toward A549 and Hela/DDP. Meanwhile the nitro
derivative 4 exhibits decreased activities. Compound 5 exerts
the most prominent activities with half-maximal inhibitory
concentrations to all tested cell lines below 6.53 μM, especially
to A549 (IC50 = 3.99 μM), A549R (IC50 = 6.53 μM) and Hela/DDP
(IC50 = 3.98 μM) which are over 2-folds more potent than
cisplatin and oxaliplatin. Notably, naphthalimide platinum(IV)
complexes almost fully overcome the drug resistance of cisplatin,
particularly compound 5 which decreases the drug resistant
factors of A549R and Hela/DDP to 1.64 and 0.76 respectively.
Summarily, the introduction of naphthalimide to platinum(IV)
system to yield title compounds causes enhanced activities
against tumor cells in contrast to their precursors which is
probably attributed to their lipophilicity and easier reduction
potential. The linkage between naphthalimide and platinum core
influences their antitumor effects dramatically, and the three
carbon linker seems the most favorable linkage for the tested
serial compounds. Furthermore, the modification with an amino
moiety at 3-position of naphthalimide ring leads positive effects
on the antitumor abilities. Besides, the target compounds are of
great potential to overcome drug resistance of cisplatin. All these
facts enable naphthalimide platinum(IV) complexes to be worthy
for further evaluation as new anticancer agents.
Antitumor activities in vitro
The antitumor activities of naphthalimide platinum(IV)
compounds 1–5 and reference platinum compounds cisplatin,
oxaliplatin and oxoplatin 13 in vitro were evaluated using an
MTT assay toward five human cancer cell lines, including
ovarian cancer (SKOV-3), lung cancer (A549) and cervical
cancer (HeLa) as well as two cisplatin resistant cells A549R and
Hela/DDP. With the intention of validating the combination of
naphthalimide with platinum(IV) groups leads positive influence
on their activities, the antitumor effects of acids 9–11 and
oxoplatin 13 as well as the mixture of acids with oxoplatin 9–
11&13 were also tested. The IC50 values were calculated based
on three parallel experiments and the results were presented in
Table 1.
As demonstrated, the acids 9–11 and oxoplatin 13 exert
negligible cytotoxicity to tumor cells with high IC50 values. The
mixtures of oxoplatin with acids 9–11&13 give no enhanced
activities in contrast to compounds 9–11 and 13 alone. Then, the
naphthalimide platinum(IV) complexes 1–5 display superior
activities and broaden antitumor spectrum in comparison with
their precursors which are probably ascribed to the enhanced
lipophilicity of the title complexes[31-33]. Moreover, the complexes
1–5 with ester moieties in axial position may reduce faster than
the oxoplatin bearing hydroxyl axial ligands which undergoes
slowly reduction in reducing condition[14], and further enhance
activities. Accordingly, it is reasonable to infer that the
conjunction of naphthalimide with platinum(IV) core to construct
the title complexes leads to better antitumor bioactivities than
the precursory acids and oxoplatin as well as their mixtures.
Table 1. Cytotoxicity profiles of naphthalimide platinum(IV) complexes with five human carcinoma cell lines expressed as IC50 (μM).
Compounds
SKOV-3
A549
A549R
RF(A549R)[a]
0.68
0.48
1.42
1.13
1.64
NC
NC
NC
NC
NC
HeLa
Hela/DDP
13.09±1.86
10.27±2.25
71.74±10.65
21.17±3.31
3.98±0.92
≥100
RF(Hela/DDP)[a]
1
2
3
4
5
9
9.98±0.65
1.47±0.11
3.1±0.43
2.49±0.30
1.28±0.28
≥100
44.47±12.44
14.94±1.20
17.49±2.56
18.17±1.37
3.99±0.98
86.32±12.84
68.61±10.21
≥100
43.30±15.21
≥100
57.98±4.64
31.33±3.62
9.49±1.51
11.73±3.84
30.02±9.32
7.21±2.14
24.82±6.57
20.45±4.07
6.53±2.90
≥100
64.28±15.51
≥100
46.71±6.82
≥100
8.64±1.41
3.6±0.53
43.9±3.22
35.35±2.51
5.26±1.35
≥100
30.29±3.56
≥100
39.71±7.51
≥100
1.52
2.85
1.63
0.60
0.76
NC
NC
NC
NC
NC
9&13[b]
10
33.54±6.50
≥100
45.31±8.72
≥100
10&13[b]
11
20.58±4.54
89.14±10.21
62.54±22.29
20.42±7.10
1.28±0.15
2.77±0.48
52.48±6.54
75.68±15.23
45.35±9.25
29.24±4.57
12.19±3.84
19.54±1.61
11&13[b]
13
81.08±14.54
50.56±15.38
32.72±8.81
17.61±5.17
NC
NC
3.45
1.50
40.02±5.58
26.19±6.51
2.98±0.76
10.42±3.54
NC
NC
4.09
1.88
Cisplatin
Oxaliplatin
[a] RF: Resistant factor. RF(A549) = IC50(A549R)/IC50(A549); RF(HeLa) = IC50(Hela/DDP)/IC50(HeLa). [b] 9–11&13: mixture of 2 equivalent of compound 9/10/11
mixed with 1 equivalent of oxoplatin compound 13.
UV-vis and fluorescence analyses for binding studies of
compound 5 with DNA
Naphthalimide as an effective DNA intercalator was incorporated
to the platinum(IV) compounds. It was as an objective to study
whether the naphthalimide platinum(IV) could combine with DNA
via naphthalimide fragment before reduction, while most
platinum(IV) compounds could not interact with DNA double
helix. Herein, the DNA binding properties of platinum(IV)
compound
5 was evaluated by UV-vis and fluorescence
spectrum taking CT-DNA as a model of dsDNA.
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The UV-vis absorption spectra of DNA with naphthalimide
platinum(IV) compound 5 as shown in Figure 2 illustrate that the
absorption at 260 nm improves linearly with increasing
concentrations of compound 5. Moreover a hypochromic effect
is revealed by the observation that the absorption intensities of
platinum(IV)-DNA complex (Compd. 5-DNA) is relatively weaker
than the sum intensities of free compound 5 and DNA (Compd.
5 + DNA) which is probably ascribed to the interaction of
naphthalimide platinum(IV) compound with DNA (Inset of Figure
2).
To further testify the combination of naphthalimide platinum(IV)
complex with DNA, the fluorescence spectra of CT-DNA with
compound 5 were tested. Ethidium bromide (EB) as a planar
cationic dye could effectively intercalate with DNA and induce
strong fluorescence emission at 594 nm under the excitation of
510 nm. The fluorescence displacement experiments of EB-DNA
are widely used to investigate the intercalation of DNA
intercalators. The emission of EB-DNA is quenched with the
increasing concentration of compound 5 as demonstrated in
Figure 3, which is probably due to the competitive DNA
conjunction of naphthalimide platinum(IV) complex and EB. The
quenching trend of the EB-DNA system by the complex 5 is in
agreement with the linear Stern-Volmer equation, and the Stern-
Volmer constant KSV = 7449 M-1 is obtained. The calculated
quenching rate constant Kq = 7.45 × 1011 M-1S-1 is much greater
than the maximum scatter collision quenching constant of the
biomolecule (2 × 1010 M-1S-1) indicating the probable static
quenching procedure. The detailed calculation procedures are
presented in ESI (Figure S1, S2).
Figure 3. Fluorescence spectra of EB-DNA system in the absence and
presence of the platinum(IV) complex 5 (λex = 510 nm). a-g: c(EB) = 7.5 μM,
c(DNA) = 7.5 μM, c(Compound 5) = 0.0, 8.0, 16.0, 24.0, 32.0, 50.0 and 66.0
μM. Inset: the plot of F0/F vs [C] × 10-6.
Electrophoretic dsDNA plasmid assay
The electrophoretic DNA plasmid assay is a straightforward
method to detect the interaction of compounds with DNA. To
further confirm the combination of naphthalimide platinum(IV)
compounds with DNA, the pAUR101 plasmid was selected as
the model of dsDNA. The cell-free electrophoretic DNA assay
was carried out in the presence of compound 5 with cisplatin
and oxaliplatin as reference drugs. Moreover, the reduced
system of complex 5 in the presence of AsA (Comp. 5/AsA) was
also prepared and evaluated to judge the influence of the
reduction of platinum(IV) compounds on the DNA binding
properties.
The UV-vis and fluorescence analyses results manifest that
naphthalimide platinum(IV) compound 5 could combine with CT-
DNA before reduction which is probably ascribed to the DNA
targeted naphthalimide moiety.
1.5
1.2
Comp+DNA
Comp-DNA
i
1.0
1.2
0.8
a
0.6
0.9
0.4
Figure 4. Electropherograms of dsDNA plasmid pAUR101 incubated with 50
μM of cisplatin, oxaliplatin, platinum(IV) compound 5 or its deduction system
Comp. 5/AsA for different incubation times (1h or 4 h) at 37 °C.
0.6
0
5
10
15
20
25
6
10 [M]
0.3
0.0
It is manifested in Figure 4 that the dsDNA plasmid is
structural stable in 4h in PBS buffer (band a and b), while AsA
exerts no significant influence on the mobility of DNA as
revealed by bands k and l. Cisplatin causes total untwisting of
the supercoiled form of the plasmid to the open circular form in 1
h (band c and d), and oxaliplatin also induces shift of covalently
closed circular band of DNA in 4 h (band e and f). These facts
indicate the effective interactions of platinum(II) drugs with
dsDNA. As for compound 5, a slight shift of the maximum DNA
band is observed after 1 h incubation (band g), meanwhile the
supercoiled form of plasmid totally untwists after 4 h incubation
(band h). Thus, it is proved that the naphthalimide platinum(IV)
complex could interact with DNA in solution. In the reducing
250
300
350
400
450
500
Wavelength (nm)
Figure 2. UV-vis absorption spectra of CT-DNA with different concentrations
of compound 5. a-i: c(DNA) = 43.0 μM, c(compound 5) = 0.0–24.0 μM, at
increments of 3.0 μM. Inset: comparison of absorptions at 260 nm between the
compound 5-DNA complex (Compd. 5-DNA) and the sum values of free DNA
and free compound 5 (Compd. 5 + DNA).
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system (Comp. 5/AsA), platinum(IV) compound 5 is converted to
platinum(II) complex and releases two equivalents of
naphthalimide acid. The interaction of the reducing system with
DNA (band i and j) is significantly more efficient than that of
platinum(IV) compound 5, and the total untwisting form of the
plasmid is observed in 1 h which is similar to cisplatin.
Therefore, the electrophoretic dsDNA plasmid assay
evidences that naphthalimide platinum(IV) complex exhibits dual
DNA damage mechanism. The naphthalimide platinum(IV)
compound in tetravalent form could bind with DNA supercoiling
to some extent before reduction. Furthermore, the platinum(II)
complex released after reduction could combine with DNA to
form DNA adducts effectively and cause remarkable DNA
structural damage.
CD spectra for binding studies of compound 5 with DNA
CD spectroscopy as a sensitive optical rotation spectrum is
widely applied in the monitor of the variation of DNA structure.
The CD spectrum of CT-DNA in the absence and presence of
the platinum(IV) compound 5 were recorded. The CT-DNA
displays two absorption peaks at 245 and 275 nm respectively.
The positive band at about 275 nm is mainly due to the base
stacking of DNA double helix, and the negative band around 245
nm is attributed to the helicity. As displayed in Figure 5, both
absorption peaks decrease with the addition of compound 5
demonstrating the change of DNA base stacking and right-
handed helicity which are probably ascribed to the intercalation
of compound 5 with DNA double helix via naphthalimide
fragment. It is reasonable to ascertain that naphthalimide
platinum(IV) complex could combine with DNA and these results
are consistent with the conclusion obtained above.
concentration as in cancer tissues[34]). The emergence of acid
peaks confirms the reduction of compound 5, following by the
release of an equivalent of platinum(II) complex. The adduct of
cisplatin with 5’-GMP in the spectra reveals the interaction of the
reduced platinum(II) complex with DNA. Summarily,
naphthalimide platinum(IV) compounds could undergo reduction
in reductive milieu and release platinum(II) compounds which
would cause remarkable DNA damage by the formation of
covalent adduct.
UV-vis and fluorescence analyses for binding studies of
compound 5 with HSA
HSA as the most abundant protein in blood plays key roles in
the transport and storage of exogenous drugs, and thus
influencing their metabolism, toxicities and clearance in vivo.[35,36]
In view of this, the binding properties of naphthalimide
platinum(IV) complex 5 with HSA were tested by UV-vis and
fluorescence spectrum.
The UV-vis spectra (Figure 6) demonstrate that the
subtraction spectrum (Curve D) of the absorption of compound
5-HSA complex and compound 5 ranging from 250 to 300 nm
could not be superposed with the absorption spectrum of HSA
(Curve A). These facts indicate the combination of
naphthalimide platinum(IV) complex with HSA in solution
qualitatively.[37,38]
1.2
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
0.24
0.20
0.16
0.12
0.08
0.04
0.00
D
A
250 260 270 280 290 300
A
B
C
D
240 260 280 300 320 340 360
Wavelength (nm)
Figure 6. UV-vis spectra of HSA with and without compound 5: (A) absorption
spectrum of HSA, c(HSA) = 10 μM; (B) absorption spectrum of compound 5-
HSA (1:1), c(HSA) = c(compound 5) = 10 μM; (C) absorption spectrum of
compound 5, c(compound 5) = 10 μM; (D) subtracting spectrum of (B) and (C).
Inset: the curve (A) and (D) for the wavelength ranging from 250 to 300 nm.
Figure 5. CD spectra of CT-DNA in the absence and presence of the
platinum(IV) complex 5. a-c: c(DNA) = 80.0 μM and c(complex 5) = 0, 20.0,
and 40.0 μM.
Further fluorescence spectra evaluation (Figure 7) indicates
that a gradual quenching of the fluorescence intensity of HSA is
caused by the increase of compound 5 at 298 K, accompanied
with a slight blue shift of maximum emission wavelength from
343 to 337 nm in the albumin spectrum. These observations
further demonstrate the binding of complex 5 with HSA.
Reduction of platinum(IV) compound 5 and the DNA binding
properties of the reduced platinum(II) complex
The reduction potential of naphthalimide platinum(IV) compound
5 with and without AsA were investigated to detect its inherent
proficiency as platinum pro-drug. The 5’-GMP was selected and
added as the model of DNA base to investigate the DNA binding
properties of the reduced platinum(II) product. The HPLC results
(ESI Figure S3) manifest that compound 5 keeps stable for at
least 48 h in PBS solution, and could not interact with 5’-GMP
until it undergoes reduction in the presence of 1 mM AsA (same
To study the interaction mode, the emission spectra at
different temperatures (291 K, 298 K, 305K) are detected. The
Stern-Volmer constant KSV, binding constant Kb and the number
of binding sites n for platinum(IV) compound 5-HSA system are
calculated and presented in Table 2. The detailed calculation
procedures are presented in ESI. It is revealed that the KSV
decreases with the increasing temperature, and the quenching
rate constant Kq at each temperature is much greater than the
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10.1002/ejic.201800799
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maximum scatter collision quenching constant of the
apoptosis of SKOV-3. Compound 5 exhibits more effective
biomolecule indicating the probable static quenching mechanism. apoptosis-inducing ability than oxaliplatin at the concentration of
Furthermore, the decreasing trend of Kb with increasing
temperatures is also observed, and the binding site n is
approximately equal to 1. Thus, platinum(IV) complex could
combine with HSA via one binding site at the interface of protein.
The decreasing binding constant between metal complex and
HSA with increasing temperature indicates the formation of a
conjugated complex between platinum(IV) compound and
protein supporting that the fluorescence quenching is a static
process.
20 μM for 24 h incubation, meanwhile it prompts relatively lower
population of cells to undergo apoptosis than that of cisplatin.
And these facts are in accordance with the trend of the in vitro
antitumor activities.
Table 3. Quantification of apoptosis in SKOV-3 cells using an annexin V/PI
assay.
It has been manifested that the sign and magnitude of the
thermodynamic parameter are associated with various individual
kinds of interaction that may take place in protein association
processes. From the thermodynamic standpoint, ΔH > 0 and ΔS
> 0 imply a hydrophobic interaction, ΔH < 0 and ΔS < 0 reflect
the van der Waals force or hydrogen bond formation, and ΔH <
0 and ΔS > 0 are characteristics for electrostatic interactions[39,40]
The thermodynamic parameters ΔH, ΔG and ΔS in Table 2 were
calculated by van’t Hoff equation. The negative values of Gibbs
free energy change ΔG suggest the spontaneous combination of
platinum(IV) compound with HSA, meanwhile the positive
entropy change ΔS and negative enthalpy change ΔH values
prove that the electrostatic force is involved in the HSA
interaction.
.
Early
apoptosis
Late
apoptosis
Compound
Necrosis
Sum
5
9.75
4.24
2.98
2.86
23.64
36.60
17.88
6.74
15.78
20.64
11.74
0.14
49.17
61.48
32.6
Cisplatin
Oxaliplatin
Untreated
9.74
Cellular uptakes and DNA platination
The cellular uptake and DNA platination are concerned as
crucial factors influencing antitumor competence of platinum
compounds with DNA damage mechanism. Therefore, the
cellular uptake and DNA platination of the selected active
compounds 2 and 5 as well as the reference drugs cisplatin and
oxaliplatin were tested in SKOV-3 cells using ICP-MS. Results
were presented as the mean value of three determinations for
each experiment.
Figure 7. Fluorescence spectrum of HSA in the absence and presence of the
platinum(IV) complex 5 (λex = 280 nm, T = 298 K). a-k: c(HSA) = 4.0 μM,
c(complex 5) = 0.0–12.5 μM, at increments of 1.25 μM; l: fluorescence
spectrum of free complex 5, c(complex 5) = 12.5 μM.
Table 4. Cellular uptake and DNA platination of naphthalimide platinum(IV)
complexes 2, 5, cisplatin and oxaliplatin in SKOV-3 cells. (A) Platinum in
whole cells; (B) Platinum in DNA.
(A)
(B)
A
B
14
12
10
8
400
350
300
250
200
150
100
50
Table 2. Thermodynamic parameters of compound 5-HSA interaction at
different temperatures.
6
4
ΔS
(J/mol
K)
10-12Kq
10-4Ksv
10-5Kb
ΔH
(kJ/mol)
ΔG
(kJ/mol)
2
T
(K)
n
(M-1S-1)
(M-1)
(M-1)
0
0
2
5
CisplatinOxaliplatin
2
5 CisplatinOxaliplatin
Compounds
Compounds
291
298
305
10.01
8.79
8.03
10.01
8.79
8.03
2.187
1.936
1.685
1.07
1.07
1.06
-29.80
-30.16
-30.52
-14.81
51.5
Ratio of Pt-DNA/Pt-Cell
Pt-Cell
Pt-DNA
Compound
(ng/106 Cells)
(ng/106 Cells)
(%)
2.5
3.4
2.6
3.2
Apoptosis experiments
348±32
296±24
68±11
75±27
8.6±1.8
10.1±1.5
1.8±0.4
2.4±0.7
2
5
The apoptosis induced by platinum(IV) complex 5 was quantified
using an annexin V-FITC and propidium iodide (PI) strained flow
cytometric assay in SKOV-3 cells with cisplatin and oxaliplatin
as reference drugs. The results in Table 3 demonstrate that the
tested naphthalimide platinum(IV) compound could induce cell
Cisplatin
Oxaliplatin
It is indicated (Table 4) that naphthalimide platinum(IV)
complexes 2 and 5 accumulate at 3.9–5.1 times higher levels in
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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a standard of K2PtCl4 in D2O (δ = -1628 ppm). All coupling constants J
were quoted in Hz. High resolution mass spectra (HRMS) were obtained
on an IonSpec QFT mass spectrometer with ESI ionization. The HPLC
analyses were performed on a Thermo Ultimate 3000 RS equipped with
an Agilent Eclipse XDB-C18 column (250×4.6 mm, 5 μm). All cells were
kindly provided by Dr Jing Li (College of Pharmacy, Nankai University,
Tianjin, China). The plasmid pAUR101 was obtained from Takala.
Genomic DNA mini preparation kit was purchased from Beyotime, China.
The annexin V-FITC/PI apoptosis detection kit was purchased from
KeyGen BioTech, China. UV-vis spectra were measured on a Shimadzu
UV-2550 spectrophotometer. Fluorescence spectroscopic data were
obtained on a Perkin-elmer LS55 spectrofluorometer. CD spectra were
recorded on a Jasco J-810 spectropolarimeter.
whole cells (Pt-Cell) than cisplatin and oxaliplatin, meanwhile the
platination in DNA (Pt-DNA) are also 3.6–5.6 folds higher than
that of the reference drugs. It can be concluded that
naphthalimide platinum(IV) compounds display higher platinum
accumulation in tumor cells than platinum(II) drugs which is
probably attributed to the high lipophilicity. Then the ratio of Pt-
DNA to Pt-Cell was calculated. It was observed that, the ratio of
compounds 2 and 5 are almost same to that of platinum(II)
drugs cisplatin and oxaliplatin. Therefore, the higher DNA
platination of the title platinum(IV) complexes are likely due to
the high uptake.
Cell culture
Conclusions
Dulbecco’s Modified Eagle Medium (DMEM), RPMI1640, 0.25%
trypsin/EDTA solutions, fetal bovine serum (FBS) and penicillin-
streptomycin solutions were purchased from Gibco. MTT was purchased
from Sigma. The cells were maintained in either DMEM (for HeLa,
Hela/DDP cells) or RPMI1640 (for SKOV-3, A549, A549R cells) medium
containing 10% FBS in a humidified atmosphere containing 5% CO2 at
37 °C. The A549R and Hela/DDP cells were maintained with 2 μg/mL
cisplatin. Phosphate buffered saline (PBS) contained 137 mM NaCl, 2.7
mM KCl, 10 mM Na2HPO4 and 2 mM KH2PO4. The solution pH was 7.4.
MTT solution with concentration of 5 mg·mL-1 for MTT assays was
prepared before it used.
In summary,
a new series of naphthalimide platinum(IV)
complexes were designed, synthesized and evaluated for
antitumor activities. The in vitro results demonstrate that some
naphthalimide platinum(IV) compounds display remarkable
antitumor activities especially compounds 2 and 5 which are
comparable or even superior to the positive controls cisplatin
and oxaliplatin and are of great potential in overcoming drug
resistance of platinum(II) drugs. The linkages between
naphthalimide and platinum core significantly affect the
antitumor activities and the substituents on naphthalimide also
shade much influences on their antitumor effects. The three
carbon linker is screened out as the most favorable linkage for
the target compounds, and the amino moiety shows positive
effects on antitumor activities. The mechanism investigation
reveals that naphthalimide as DNA targeting functional group
endows naphthalimide platinum(IV) complexes with dual DNA
interaction capacities. Naphthalimide platinum(IV) complex could
combine with DNA via naphthalimide fragment and induce DNA
damage before reduction. Subsequently, the platinum(II)
complex released by platinum(IV) compounds in reductive
microenvironment would rapidly interact with DNA and cause a
secondary remarkable DNA damage of tumor cells. Furthermore,
naphthalimide platinum(IV) complex could combine with HSA via
electrostatic interaction which might influence their drug
distribution and bioactivities in vivo. Additionally, cellular uptake
and DNA platination of the tested platinum(IV) compounds are
dramatically higher than platinum(II) drugs. All these facts
enable naphthalimide platinum(IV) complexes to be worthy for
further development as new anticancer agents.
In vitro cellular cytotoxicity assays
Cells were seeded in 96-well plates at 5000 cells per well in 100 μL of
complete medium, and incubated in a 5% CO2 atmosphere at 37 °C for
24 h. Then 100 μL of freshly prepared culture medium containing drugs
at different concentrations were added. The cells were further incubated
for another 48 h, and then freshly prepared MTT solution (5 mg·mL-1) 20
μL was added. The resultant mixtures were incubated for 4 h to allow
viable cells to reduce the yellow tetrazolium salt into dark blue formazan
crystals. After removal of the medium, formazan was dissolved in DMSO
(150 μL) and quantified by a microplate reader (570 nm). The IC50 values
were calculated using GraphPad Prism 6 based on three parallel
experiments.
DNA binding experiments
UV-vis spectra assays: CT-DNA was selected as a model of DNA for
UV-vis spectra. The experiments were carried out in Tris-HCl buffer (10
mM Tris-HCl/10 mM NaCl, pH 7.4). The tested solutions were prepared
by addition of increasing amount of complex
5 (0.0–24.0 μM, at
increments of 3 μM) to the DNA solutions (43.0 μM). The UV-vis profiles
were measured from 200 to 800 nm using a quartz cell of path length 1
cm at room temperature. The UV-vis spectra of free complex 5 at
different concentrations were also tested.
Experimental Section
Materials and general methods
Fluorescence spectra assays: The binding experiments of platinum(IV)
complex with CT-DNA were performed in buffer solution (10 mM Tris-
HCl/10 mM NaCl, pH 7.4). Fluorescence quenching spectra were
recorded by adding increasing amount of complex 5 (0.0, 8.0, 16.0, 24.0,
32.0, 50.0 and 66.0 μM) to the EB-DNA system (c(EB) = 7.5 μM, c(DNA)
= 7.5 μM). The well-mixed solutions were holding for 15 min for
equilibrium before evaluation, and the fluorescence emission spectra
were then recorded from 520 to 720 nm (λex = 510 nm) at scan rate of
300 nm·min-1 with widths of both the excitation and emission slits setting
as 5.0 nm.
All reactions were carried out under an atmosphere of nitrogen in flame-
dried glassware with magnetic stirring unless otherwise indicated.
Cisplatin and oxaliplatin were purchased from Yurui chemical Co. Ltd.
(Shanghai, China). Other reagents were purchased from Alfa Aesar,
Sigma, J&K, and GL Biochem Ltd. 1H NMR, 13C NMR and 195Pt NMR
spectra were recorded on a Varian 400 MHz. The 1H and 13C NMR
chemical shifts were referenced to residual solvent peaks or to TMS as
an internal standard. 195Pt NMR spectra were referenced externally using
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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Electrophoretic dsDNA plasmid experiments: The pAUR101 plasmid
dsDNA (6687 bp) purchased from Takala was used as a dsDNA model
for electrophoretic assay. The DNA interaction properties of platinum(IV)
complex 5 was evaluated to confirm its combination with DNA double
helix taking cisplatin and oxaliplatin as references. The DNA interaction
reactions were performed in PBS buffer. A well-mixed solution of plasmid
DNA and platinum complexes 20 μL were incubated at 37 °C by thermo
cycler. After 1 h, solution 10 μL was extracted and the remained 10 μL
solution was allowed to react for 4 h. The solution containing compound
5 (1 mM) and AsA (5 mM) was incubated at 37 °C for 48 h, and the
reducing system of platinum(IV) complex (Compd. 5/AsA) was obtained
after dilution of 20-folds. To investigate the impact of the reduction of
platinum(IV) complex on the DNA binding competence, the Compd.
5/AsA was tested. The influence of AsA (250 μM) to plasmid dsDNA was
also evaluated. Then the samples were loaded in the agarose gel (1.0%
w/v) and electrophoresis was carried out for a period of 30 min at
approximately 150 V in TAE 1 × (Tris-acetate-EDTA) buffer. After
electrophoresis, the gel was stained with Gel Red for 30 min. Finally, the
stained gel was imaged with a Bio-Rad Universal Hood II instrument.
solutions were holding for 15 min for equilibrium, and the fluorescence
emission spectra were then tested from 290 to 600 nm (λex = 280 nm) at
scan rate of 300 nm·min-1. The widths of both the excitation and emission
slits were 5.0 nm. The fluorescence spectra of HSA in the absence and
presence of the platinum(IV) complex 5 at different temperatures (291 K,
298 K and 305 K) were tested.
Apoptosis experiments–Annexin V/PI assay
The experiment was performed according to the manufacture’s protocol
(KeyGEN Annexin V-FITC/PI Apoptosis Detection Kit). SKOV-3 cells
were incubated with or without the test compounds for 24 h at 37 °C
(Negative control: no compound; Compound 5: 20 μM; Cisplatin: 20 μM;
Oxaliplatin: 20 μM). Then cells were harvested from adherent cultures by
trypsinization with no EDTA. Following centrifugation at 2000 rpm for 5
min, cells were washed with PBS twice and suspended in binding buffer
500 µL. Subsequently, the samples were stained with Annexin V-FITC 5
µL and PI 5 µL for 15 min at room temperature and tested by flow
cytometric assay.
CD spectrum Assay: The solution of CT-DNA (80 μM) with increasing
concentrations of platinum(IV) complex (0, 20, 40 μM) in buffer (10 mM
Tris-HCl/10 mM NaCl, pH 7.4) were incubated for 48 h in dark. The CD
spectrum were recorded at room temperature in the range of 220 and
320 nm with a scan speed of 100 nm·min−1 and 1 s response time. Each
CD spectrum was recorded as an average of three scans. The final
spectra were background-corrected by subtracting the corresponding
buffer spectra.
Cell uptake and DNA platination
To measure the cellular uptake of the platinum complexes, ca. 1 million
SKOV-3 cells were seeded on 60 mm × 10 mm petri dishes and
°
incubated for 3 h at 37 C. Then cells were treated with 100 µM of the
°
complexes at 37 C for 10 h. Cells were harvested after the removal of
media. The cells were washed with PBS solution (1 mL × 3) and counted,
and then mineralized with 70% HNO3. The samples were analyzed using
ICP-MS. Cellular platinum levels were expressed as ng Pt per million
cells. Results were presented as the mean of 3 determinations for each
data point.
Reduction of platinum(IV) complexes by AsA
HPLC analyses were performed on Thermo Ultimate 3000 RS equipped
with an Agilent Eclipse XDB-C18 column (250×4.6 mm, 5 μm). HPLC
profiles were recorded by UV detector with flow rate of 1.0 mL/min with
follow linear gradient (Table 5).
For the DNA platination, 1 million SKOV-3 cells were treated in the same
way as that for cellular uptake. The DNA was extracted using genomic
DNA mini preparation kit and mineralized with 70% HNO3. The platination
of DNA were determined using ICP-MS and the results were presented
as the mean of 3 determinations for each data point.
Table 5. The linear gradient.
Time (min)
A (0.01% aqueous TFA)
B (Methol)
10
10
100
100
Purity assessment
0
5
35
45
90
90
0
The final purities of the analogues was assessed on a Thermo Ultimate
3000 RS equipped with a Agilent Eclipse XDB-C18 column (250 × 4.6
mm, 5 μm) using the same analytical method as mentioned in the
“Reduction of platinum(IV) complexes by AsA” section.
0
To investigate the binding properties of DNA with platinum(II) complexes,
5’-GMP was selected as a model. It has been proved that cisplatin could
conjunct with 5’-GMP at 37 °C to form new peak of cis-Pt(II)-GMP (the
adduct of cisplatin with 5’-GMP)[26]. The degradation of compound 5 and
the following combination with 5’-GMP with and without AsA were
evaluated.
Synthetic procedures
The synthetic procedures of acids 9–11 and oxoplatin 13 were
established in ESI according to procedures mentioning in literatures.
HSA binding experiments
Preparation of compound 1: To a solution of compound 9a (192 mg,
0.75 mmol) and TBTU (241 mg, 0.75 mmol) in dry DMF 5 mL was
injected TEA (104 μL, 0.75 mmol), and the mixture was allowed to
intensively stir for 10 min at room temperature. Then oxoplatin 13 (100
mg, 0.30 mmol) was added and the reaction mixture was stirred for
another 48 h at 50 °C. The solvent was evaporated, and the residue was
purified by silica gel column chromatography to afford compound 1 as
white solid (82 mg, 32%). 1H NMR (400 MHz, DMSO-d6) δ(ppm) 8.58 –
8.45 (m, 4H), 8.37 (d, J = 8.2 Hz, 4H), 7.80 (t, J = 7.8 Hz, 4H), 6.52 (br,
6H), 4.29 – 4.23 (m, 4H), 2.66 – 2.54 (m, 4H). 13C NMR (100 MHz,
DMSO-d6) δ(ppm) 183.42, 177.63, 168.61, 168.37, 139.37, 139.17,
136.41, 136.36, 136.11, 135.95, 135.65, 132.65, 132.04, 131.94, 127.18,
UV-vis spectra assays: The experiments were carried out in Tris-HCl
buffer (10 mM Tris-HCl/10 mM NaCl, pH 7.4). The UV-vis spectra were
measured from 200 to 380 nm using a quartz cell of path length 1 cm at
room temperature. The complex 5-HSA solutions with complex 5 (10.0
μM) and HSA (10.0 μM) were prepared and evaluated. The spectra of
free complex 5 and HSA were also tested.
Fluorescence spectra assays: The binding experiments of platinum(IV)
complex with HSA were performed in buffer solution (10 mM Tris-HCl/10
mM NaCl, pH 7.4). Fluorescence quenching spectra were recorded by
adding increasing concentrations of complex
5 (0.0–12.5 μM, at
increments of 1.25 μM) to the HSA solution (4.0 μM). The well-mixed
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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127.14, 41.89, 40.92, 39.09, 37.45. 195Pt NMR (86 MHz, DMSO-d6) δ
(ppm) 1188. 96.1% purity determined by HPLC: tR = 27.2 min.
Acknowledgements
The work was supported by Natural Science Foundation of
Shandong (No. ZR2017BH092, ZR2014HL105), National
Natural Science Foundation of China (No. 21473085), Doctoral
Foundation of Liaocheng University (No. 318051635), Open
Project of Shandong Collaborative Innovation Center for
Antibody Drugs (No. CIC-AD1836, CIC-AD1835) and Taishan
Scholar Research Foundation.
Preparation of compound 2: To a solution of compound 9b (202 mg,
0.75 mmol) and TBTU (241 mg, 0.75 mmol) in dry DMF 5 mL was
injected TEA (104 μL, 0.75 mmol), and the mixture was allowed to stir for
10 min at room temperature. Then oxoplatin 13 (100 mg, 0.30 mmol) was
added and the reaction mixture was stirred for 48 h at 50 °C. The solvent
was evaporated, and the residue was purified by silica gel column
chromatography to afford compound 2 as white solid (160 mg, 62%). 1H
NMR (400 MHz, DMSO-d6) δ(ppm) 8.64 – 8.47 (m, 4H), 8.39 (d, J = 7.5
Hz, 4H), 7.83 (t, J = 7.8 Hz, 4H), 6.56 (br, 6H), 4.15 (t, J = 7.0 Hz, 4H),
2.38 (t, J = 7.6 Hz, 4H), 1.97 – 1.87 (m, 4H). 13C NMR (100 MHz, DMSO-
d6) δ(ppm) 180.30, 174.46, 163.99, 163.93, 134.80, 134.67, 131.73,
131.72, 131.26, 131.13, 127.88, 127.82, 127.64, 127.60, 122.56, 122.42,
33.55, 31.79, 24.46. 195Pt NMR (86 MHz, DMSO-d6) δ (ppm) 1193.
HRMS: Calcd. for C32H30Cl2N4O8Pt (M+H)+: 864.1167, found: 864.1145.
97.6% purity determined by HPLC: tR = 27.4 min.
Keywords: platinum • DNA damage • antitumor agents •
naphthalimide • prodrugs
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European Journal of Inorganic Chemistry
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10.1002/ejic.201800799
European Journal of Inorganic Chemistry
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Antitumor agents: This article
describes the design and synthesis of
a new series of platinum(IV)
Qingpeng Wang,* Guoshuai Li, Zhifang
Liu, Xiaoxiao Tan, Zhuang Ding, Jing
Ma, Lanjie Li, Dacheng Li,* Jun Han,
Bingquan Wang
compounds with dual DNA damage
mechanism. Target compounds
exhibited good antitumor activities and
were of great potential in overcoming
drug resistance. Moreover, they could
effectively combine with HSA and
further influence the drug distribution
and bioactivities in vivo.
Page No. – Page No.
Apoptosis
Naphthalimide Platinum(IV)
Compounds as Antitumor Agents
with Dual DNA Damage Mechanism to
Overcome Cisplatin Resistance
.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.