Platinum(IV) prodrugs multiply targeting genomic DNA, histone deacetylases and PARP-1

Article abstract of DOI:10.1016/j.ejmech.2017.09.074

Several Pt(IV) prodrugs containing SAA, a histone deacetylases inhibitor, were designed and prepared for multiply targeting genomic DNA, histone deacetylases and PARP-1. The resulting Pt(IV) prodrug had significantly strong antiproliferative activity against the tested cancer cell lines, especially SAA1, derived from the conjugation of cisplatin and SAA, had potent ability to overcome cisplatin resistance. Under the combined action of DNA platination and inhibition of HDACs and PARP-1 activity, the cytotoxic activity of SAA1 was 174-fold higher than cisplatin against cisplatin-resistant SGC7901/CDDP cancer cells. The mechanism of action of SAA1 was preliminarily investigated, in which cellular uptake, cell apoptosis and cell cycle arrest as well as western blot analysis were made by treating SAA1 with SGC7901/CDDP cells. Besides, HDACs inhibition activity and PARP-1 enzyme inhibition of SAA1 were also studied.

Full text of DOI:10.1016/j.ejmech.2017.09.074

Accepted Manuscript
Platinum(IV) prodrugs multiply targeting genomic DNA, histone deacetylases and
PARP-1
Zichen Xu, Weiwei Hu, Zhimei Wang, Shaohua Gou
PII:
S0223-5234(17)30789-4
10.1016/j.ejmech.2017.09.074
EJMECH 9789
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 4 August 2017
Revised Date: 17 September 2017
Accepted Date: 29 September 2017
Please cite this article as: Z. Xu, W. Hu, Z. Wang, S. Gou, Platinum(IV) prodrugs multiply targeting
genomic DNA, histone deacetylases and PARP-1, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.09.074.
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Graphical Abstract
Platinum(IV) prodrugs multiply targeting genomic DNA, histone
deacetylases and PARP-1
Zichen Xu a, b, 1, Weiwei Hu a, b, 1, Zhimei Wang a, b, Shaohua Gou a, b,
*

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Platinum(IV) prodrugs multiply targeting genomic DNA,
histone deacetylases and PARP-1
Zichen Xu a, b, 1, Weiwei Hu a, b, 1, Zhimei Wang a, b, Shaohua Gou a, b,
*
a
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering,
Southeast University, Nanjing 211189, China
b
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing 211189, China
1 These authors contributed equally.
* Corresponding author
E-mail: sgou@seu.edu.cn
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ABSTRACT: Several Pt(IV) prodrugs containing SAA, a histone deacetylases inhibitor, were
designed and prepared for multiply targeting genomic DNA, histone deacetylases and PARP-1.
The resulting Pt(IV) prodrug had significantly strong antiproliferative activity against the tested
cancer cell lines, especially SAA1, derived from the conjugation of cisplatin and SAA, had
potent ability to overcome cisplatin resistance. Under the combined action of DNA platination
and inhibition of HDACs and PARP-1 activity, the cytotoxic activity of SAA1 was 174-fold
higher than cisplatin against cisplatin-resistant SGC7901/CDDP cancer cells. The mechanism of
action of SAA1 was preliminarily investigated, in which cellular uptake, cell apoptosis and cell
cycle arrest as well as western blot analysis were made by treating SAA1 with SGC7901/CDDP
cells. Besides, HDACs inhibition activity and PARP-1 enzyme inhibition of SAA1 were also
studied.
Keywords: multi-target, Pt(IV) prodrug, HDAC inhibitor, PARP-1 inhibitor, overcoming drug-
resistance.
1. Introduction
Histone deacetylases (HDACs), a class of zinc metalloenzymes that deacetylate core histone
lysine residues, have attracted increased attention for the critical role in the regulation of gene
expression owing to governing the acetylation state of lysine residues [1, 2]. The finding that
overexpression of HDACs transforms the function and expression of tumor-related proteins,
chiefly involved in cell proliferation, migration, metastasis and angiogenesis, has promoted
histone deacetylase inhibitors (HDACIs) as a new class of anticancer agents [3-5].
Generally HDAC inhibitors consist of three domains: a chelator refers to the zinc binding
group (ZBG); a cap component fits into the active site pocket, and a linker domain connects the
ZBG and the cap group [6]. So far, a series of diverse HDACIs have been found to be effective
antitumor agents via multiple mechanisms, such as autophagic cell death, cell-cycle arrest,
mitotic cell death and so on [7, 8]. Moreover, HDACIs have been found to have few adverse
effects on normal cells. In the nucleus, DNA is noncovalently related to histone to form the
nucleosomes which constitute chromatin subunits. HDACIs, inducing hyperacetylation of
histone proteins, could enhance the accessibility of DNA within chromatin [9, 10] and
consequently reinforce the antitumor activities of platinum and other DNA-damaging drugs [11,
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12]. These facts indicate that cancer cell death caused by simultaneous HDAC inhibition and
DNA damage has emerged as a highly promising alternative approach in cancer therapy.
SAA, a derivative of the drug SAHA, has been reported to exert HDAC inhibitory activity
[13] by carboxylic acid groups partially chelating with zinc ions and the aromatic groups
occupying the tubular pocket. In addition, it is also found to possess PARP inhibitory activity
with benzamide, a major pharmacophoric group in many PARP inhibitors [14], to form multiple
hydrogen bonds with two critical amino acid residues in the PARP active site, Gly-863 and Ser-
904 [15-17]. PARP-1 is regarded as a valuable target in the prospecting cancer treatment due to
its key role in the repair of DNA strand breaks [18, 19]. As a DNA repair enzyme [20], PARP
has been observed overexpressed in a number of hematological and solid tumors as compared to
normal cells [14]. Besides, it has the catalytic function to transfer ADP-ribose units from
nicotinamide adenine dinucleotide (NAD+) to a variety of receptor proteins [21, 22], and is also
responsible for the formation of ADP-ribose polymers, which is crucial in the repair of damaged
DNA caused by chemotherapeutic agents [23]. Thus, PARP-1 contributes to the diminished
cancer therapies effectiveness and the resistance that often develops after cancer therapy [24-26].
Moreover, PARP-1 is playing an important role in maintaining the integrity of the structure of
chromosomes [27] and participating in DNA replication, transcription [28], and cellular death
processes [29]. Therefore, inhibition of PARP-1 activity can enhance the effect of chemotherapy
drugs [30] and overcome drug resistance to improve the result of the treatment of tumor [31-38].
Cisplatin (CDDP) has been widely used in cancer therapy as a classic cytotoxic drug for its
brilliant antitumor effects on numerous cancers. However, drug resistance is one of the
drawbacks to limit its chemotherapeutic efficacy in tumors, which is partly due to DNA damage
repair in addition to the other effects [39-41]. Recently, a strategy of applying Pt(IV) complexes
with two axial bioactive groups has been adopted to promote the efficacy of cisplatin and
overcome its resistance [11, 12, 42-45]. According to the literature reports, divalent platinum
chemotherapy drugs have shortcomings such as serious side effects, low bioavailability and so
on.[46, 47] Compared to Pt(II), Pt(IV) prodrugs are more kinetically inert, resulting in less
toxicity. In addition, Pt(IV) prodrugs are easy to be reduced to liberate axially connected
bimolecules and divalent platinum species, which may play a joint action to overcome drug
resistance and improve the pharmacological activity of platinum-based anticancer drugs. In our
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recent research on prodrugs derived from the conjugation of Pt(IV) complexes with different
biological units, Pt(IV) complexes with only one bioactive ligand in an axial position and a
chlorine atom or hydroxyl group in the opposite axial position were involved, because they were
found more likely to be reduced than those with two bioactive species in two axial positions and
subsequently to release the pharmacophore [48-52].
Based on the above, SAA was chosen as a bioactive ligand (HDACs and PARP-1 dual
inhibitor) to conjugate with four Pt(IV) complexes derived from cisplatin, oxaliplatin, DN603
and DN604 [53], respectively (Fig. 1). The resulting compounds were expected to
simultaneously target genomic DNA, HDACs and PARP-1, hypothesizing that the incorporation
of SAA into platinum drug might produce new multifunctional Pt(IV) prodrugs by maintaining
the effective Pt(II) species to bind nuclear DNA and getting ability to overcome drug resistance
(Fig. 2).
2. Results and discussion
2.1. Chemistry.
The synthetic procedure is listed in Scheme 1. SAA was obtained according to a literature
procedure [54]. Meanwhile, Pt(IV) precursors 3-6 were synthesized through the oxidative
chlorination of the corresponding Pt(II) complexes (cisplatin, oxaliplatin, DN603 and DN604)
with N-chlorosuccinimide (NCS) in water [52, 55]. Finally, four Pt(IV) prodrugs, SAA1-4, were
obtained via SAA coupling with the corresponding Pt(IV) intermediate in the presence of O-
(benzotriazol-1-yl)-N,N,N’,N’- tetramethyluronium tetrafluoroborate (TBTU) as the coupling
agent [48]. The products were purified by column chromatography, whose structures were
confirmed by 1H, 13C, 195Pt NMR and IR spectra together with ESI-MS spectroscopy.
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Scheme 1. Preparation of Pt(IV) prodrugs SAA1-4. Reagents and conditions: (a) 180 °C; (b)
TBTU, Et3N, DMF, room temperature.
2.2. HPLC analysis on the stability and reduction of complex SAA1.
The stability of SAA1 in a solution of PBS/DMF was examined by HPLC technique at
different time. As shown in Fig. S1 (see Supporting Information), SAA1 was stable in a period
of 48 h under the physiological condition. In order to verify whether SAA1 could be reduced to
its Pt(II) equivalent and release SAA in accordance with our design, SAA1 was studied by HPLC
in a solution of acetonitrile/water (25:75, v:v) in the presence of ascorbic acid. As shown in Fig.
S2 (see Supporting Information), SAA1 was gradually reduced to release SAA as time passed,
accompanied by the decreasing peak of SAA1 and the rising peak of SAA. It was noted that
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cisplatin was not observed owing to its weak chromophore under the ultraviolet detecting
condition.
2.3. In vitro antiproliferative activity.
SAA1-4 and their parent divalent Pt(II) complexes were screened against four different
human cancer cell lines including breast (MCF-7 and MDA-MB-231), colon (HCT-116) and
gastric carcinoma (SGC7901) along with normal liver cells LO2 using MTT assay. Parent Pt(II)
complexes as well as SAHA and SAA were used as positive controls. The corresponding
cytotoxic data are given in Table 1.
It was noted that SAHA presented potent antitumor activities comparable to cisplatin and
oxaliplatin, while SAA displayed negligible cytotoxicity against all the tested tumor cell lines,
with IC50 values greater than 200 µM. In addition to HCT-116, cytotoxicity of cisplatin is
generally stronger than that of oxaliplatin, and the two clinical drugs were more effective than
DN603 and DN604. It is pleasing to find that by introducing SAA, complexes SAA1-4, the
Pt(IV) derivatives of cisplatin, oxaliplatin, DN603 and DN604, exhibited significant ameliorative
antitumor activity, especially against breast (MCF-7) and gastric (SGC7901) cancer cell lines, in
which SAA1 and SAA2, as the trend with their parent compounds, had particularly better
cytotoxic activity. Compared to oxaliplatin, SAA2 has a slight increased antitumor activity for
MCF-7, but for SGC7901 cancer cells, it was 1.5 times more effective than oxaliplatin.
Regrettably, SAA2 showed higher toxicity to normal cells LO2 compare to oxaliplatin at the
same time. Encouragingly, SAA1 exhibited 13.4 times more effective than cisplatin against
gastric carcinoma (SGC7901) cell lines, with IC50 value of 0.95 µM in comparison to 12.69 µM
of cisplatin, but showed lower cytotoxicity against normal liver cells LO2 compared with the
positive drugs cisplatin and SAHA, prompting it to be a good drug candidate.
2.4. Antitumor activity against drug-resistant cancer cell lines.
Cisplatin has been used as a first-line chemotherapy agent for human gastric cancer, while
drug resistance is a key factor that restricts the efficacy of cisplatin in treatment. Upon the
cytotoxic data in Table 1, we further tested the typical complex, SAA1, against cisplatin-resistant
SGC7901/CDDP cells. The corresponding data for cisplatin were also determined for
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comparative purposes. The cytotoxic effect of cisplatin was significantly influenced in cisplatin-
sensitive SGC7901. As shown in Table 2, the IC50 value of cisplatin was increased to 67.88 µM
with the resistance factor (RF) value of 5.35. In contrast, the cytotoxicity of SAA1 was markedly
promoted against SGC7901/CDDP while compared with that of SGC7901. SAA1 was nearly
174 times as effective as cisplatin against cisplatin-resistant gastric (SGC7901/CDDP) cell lines.
The RF value of SAA1 was surprisingly 0.41, which was 13-fold superior to cisplatin,
demonstrating its strong sensitivity to cisplatin resistance cancer cells. It was noted that SAA1
showed much potent cytotoxicity toward both cisplatin sensitive and resistant cancer cell lines.
As far as we know, only a limited number of Pt(IV) complexes targeting both genomic DNA and
histone deacetylases have been reported to achieve favorable activity in the past a few years [42,
56], in which resistance factors against cisplatin-resistant cancer cell lines were approximately 1.
As postulated, the mechanism of action of SAA1 was not only involved in the process associated
with platination of DNA like cisplatin, but also involved in other processes not affected by the
mechanisms of resistance developed by cells towards DNA damaging cisplatin. In addition, we
conducted two critical control tests. The results indicated that, obviously, mechanically
equimolar mixed CDDP and SAA (1:1) could not overcome the resistance of cisplatin. The Pt(IV)
drug molecule used in SAA1 was not as potent as SAA1 against SGC7901 cells and showed a
strong drug resistance in SGC7901/CDDP cells. Based on the above two control tests, the
rationality of using tetravalent platinum strategy is also proved
2.5.Cellular uptake.
Since SAA1 showed better cytotoxic activity, it was selected for cell uptake assays in gastric
cancer cells using inductively coupled plasma mass spectrometry (ICP-MS). As displayed in Fig.
3 and Table S1, SGC7901/CDDP cells were treated with the complex (12.0 µM) for 12 hours,
resulting in the higher uptake of SAA1 than that of cisplatin and ctc-[Pt(NH3)2(OH)Cl3]. In
particular, the intracellular platinum concentration of SAA1-treated cells reached 269 ng/106
cells, which was 2.36-fold and 1.93-fold higher than that of cisplatin (114 ng/106 cells) and ctc-
[Pt(NH3)2(OH)Cl3] (139 ng/106 cells), respectively. In addition, we had run the blank test with
cells washed immediately after treatment with platinum complexes without incubation. The level
of Pt in cells was almost negligible compared with that of incubation group. According to the
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results of cell uptake tests and cytotoxic assay, they appeared to have a positive correlation,
implying enhanced cellular uptake can lead to increased cytotoxicity.
2.6.HDACs inhibition activity.
HDACs have received increasing attention in recent years because of their close association
with cancer. HDACIs as a class of antineoplastic drugs can alter gene transcription and
anticancer, such as cell growth inhibition, differentiation, apoptosis and tumor angiogenesis. And
several studies suggested that HDACIs have a synergistic effect on DNA damage agent cisplatin.
Thus, our complexes were designed to release SAA inside the cells so as to exert HDACs
inhibition. In the test, Hela cell nuclear extract, a rich source of HDACs, was used to efficiently
evaluate the most active Pt(IV) prodrug SAA1. As expected, cisplatin lacked the activity to
inhibit HDACs with IC50 value exceeding 100 µM. As shown in Fig.4 and Table S2, SAA1 had
potent HDACs inhibitory activity with IC50 value of 26.83 µM in the nuclear extract, but showed
weaker HDACs inhibitory ability than the positive drug SAHA (IC50= 0.13 µM) and SAA (IC50=
16.77 µM). This observation is basically in agreement with the well-known HDACs inhibitory
effect of SAA [13], indicating that the addition of SAA to the axial position of a Pt(IV) atom is a
useful way to introduce HDAC activity into the Pt(IV) prodrug.
2.7.PARP-1 enzyme inhibition.
According to the previous reports [42, 56], the axial ligands concerned only had a single
HDAC inhibitory effect. While SAA, as an axial ligand in our complexes, was reported to not
only have a HDAC inhibitory activity, but also have a PARP-1 inhibition effect. It has been
indicated that PARP-1 plays an important role in DNA single strand breaks repair (SSB) by
implementing basic excision repair (BER) pathway. And DNA damage caused by agents like
cisplatin is usually repaired by BER pathway. Thus inhibition of PARP-1 may interfere with
BER pathway thereby enhancing the cytotoxicity of these agents and sensitizing cancer cells to
them. Based on the above, SAA1 as well as SAA and cisplatin was evaluated in vitro for their
PARP-1 enzyme inhibition activity (Fig.5 and Table S3). As expected, SAA1 indicated activity
with IC50 value of 38.26 µM against PARP-1 comparable to SAA, revealing that addition of
SAA into a Pt(IV) moiety resulted in a significant increase in enzyme potency, while cisplatin
had hardly inhibitory activity with IC50 beyond 100 µM. Consistent with the HDAC result above,
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SAA1 had a strong PARP-1 inhibition activity, which may explain the better cytotoxic activity
of SAA1 against gastric cancer cells and the ability to overcome cisplatin resistance.
2.8.Effect on cell cycle arrest.
In order to investigate the effect of our Pt (IV) complexes on cell cycle arrest, the cycle
distribution of SGC7901/CDDP cells treated with different concentrations of SAA1 after 24
hours was analyzed by flow cytometry with untreated cells as negative control and cisplatin-
treated cells as positive control. As shown in Fig.6, SAA1 was as effective as cisplatin in
blocking the cell cycle in G2/M phase. For the untreated cells, the percentage of cells in G0/G1
phase was 67.47% and in G2/M phase was only 6.66%. It was noted that SAA did not have
much effect on the cell cycle, the percentage of cells in G0/G1 and G2/M phase was comparable
to that of the untreated group. (Fig. S25) After treatment with 2 µM of SAA1, the percentage of
cells in G2/M phase increased to 18.74%, while the percentage in S phase did not change
significantly. At 4 µM of SAA1, 42.61% of cells were arrested in G2/M phase, which was higher
than the percentage of cells treated with high-dose of cisplatin (24.90%). These results showed
that SAA1 evidently arrested the G2/M phase of the cell cycle in a dose-dependent manner.
2.9.Induced apoptotic cell death and western blot analysis.
To confirm whether the inhibitory effect of SAA1 on cancer cell proliferation was
accompanied by enhanced apoptosis, we performed a FITC-Annexin V/propidium iodide (PI)
staining and flow cytometry assay. The compounds tested were co-cultured with
SGC7901/CDDP cells at increasing concentrations for 24 hours with cisplatin as a positive
control. As illustrated in Fig.7 and Fig. S26, only a few apoptotic cells were present in the
control group (6.68%) and SAA essentially had little effect on apoptosis. Gratifyingly, a dose-
dependent increase in the percentage of apoptotic cells was observed after being treated with
SAA1. The population rose to 15.44% after treatment with 2 µM of SAA1 for 24 h and further
increased to 37.42% after treatment with 4 µM of SAA1, which was significantly higher than
that after treatment with 4 µM of cisplatin (15.79% apoptotic cells at the same concentration).
Overall, the results above evidently verified that complex SAA1 effectively induced apoptosis in
SGC7901/CDDP cells.
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In addition, the promotion of apoptosis induced by platinum complexes was closely related to
induction of tumor suppressor p53 [57-59]. Since p53 plays such a pro-apoptotic role in
apoptosis that we studied its pathway involved in the mechanism of action of SAA1 in
SGC7901/CDDP cells. As depicted in Fig.8, treatment of SGC7901/CDDP cells with SAA1
resulted in an increase in p53 activation, which was consistent with cisplatin. These results
indicated that activation of the p53 pathway was involved in SAA1-induced cancer cell death.
3. Conclusion
In summary, we successfully developed several multifunctional Pt(IV) prodrugs containing a
HDACs and PARP-1 dual inhibitor for the first time. Notably, SAA1 is the most prominent one,
whose cytotoxicity against MDA-MB-231 breast cancer and SGC7901 gastric cancer cells is
superior to its parent complex, cisplatin. Noteworthy, the markedly improved ability of SAA1 to
overcome cisplatin resistance was found in cisplatin-resistant SGC7901 cells. The resistance
factor was 0.41 for SAA1, much lower than that for cisplatin. This suggests that the mechanism
underlying the biological action of SAA1 is different from that of cisplatin, allowing SAA1 to
successfully overcome the resistance mechanisms operating in the case of cisplatin. The
biological results also indicates that the mechanism also comprises inhibitory effect towards
HDACs and PARP-1, leading to increasing the accessibility of DNA in chromatin and DNA
damage caused by the platinum moiety and hereafter hindering the DNA repair process to
sensitize cancer cells. In addition, through the combined action, the cell cycle of cancer cells was
successfully blocked in the G2/M phase and the apoptosis was elevated in cisplatin-resistant
cancer cells compared to the same dose of cisplatin. Consequently, our study provided a new and
promising platform for constructing multi-target platinum drugs as well as a broad implication
for multi-target chemotherapy in cancer treatment.
4. Experimental Section
4.1. Materials and measurements.
All chemicals and solvents were obtained from commercial sources and used directly without
further purification, except where noted. Compounds 3, 4, 5 and 6 were prepared according to
literature reports [55]. The purity of all compounds used in the biological studies was ≥95%. β-
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acitn and p53 antibodies were purchased from Imgenex, USA. All tumor cell lines were
1
13
purchased from Nanjing KeyGEN BioTech company (China). H NMR and C NMR spectra
were recorded in DMSO-d6 on a Bruker 300 MHz spectrometer. 195Pt NMR spectra were
measured in DMSO-d6 with a Bruker 400 MHz spectrometer using a solution of 10 mM
potassium hexachloroplatinate in 95% H2O/5% D2O as an external standard. IR spectra of the
complexes were detected by Thermo fisher Nicolet iS10 spectrometer. Decomposition points
were measured by SGW X-4 Microscopic melting point meter (Shanghai Precision Scientific
Instrument Co. Ltd). Platinum contents were determined by inductively coupled plasma-mass
spectrometer (ICP-MS, Optima 5300DV, PerkinElmer, USA). High solution mass spectra were
measured by an Agilent 6224 ESI/TOF MS instrument. The stability and released ability of
complex was measured by Waters 1525 binary HPLC instrument.
4.2. Chemistry
4.2.1. Synthesis of compound SAA.
A mixture of suberic acid (5.73 g, 55.0mmol) and aniline (5.12 g, 55.0 mmol) was stirred at
180 °C for 1 h. After cooling, the mixture was diluted with AcOEt–THF and filtered. The filtrate
was washed with saturated aqueous NaHCO3, and the aqueous layer was acidified with
concentrated HCl. The precipitated crystals were collected by filtration to give SAA as a white
solid.
4.2.2. Synthesis of compounds SAA1-4.
To a solution of SAA (0.30 g, 1.2 mmol), TBTU (0.39 g, 1.2 mmol), and Et3N (0.12 g, 1.2
mmol) in dry DMF (15 mL), and complex 4, 5, 6 or 7 was added, respectively. The mixture was
stirred at room temperature overnight. After completion of reaction, the whole mixture was
added to CH2Cl2 (120 mL), and then extracted twice with water (100 mL). The organic phase
was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was
purified on silica gel column eluted DCM/MeOH (50:1) to give the desired product as a yellow
solid.
4.2.3. Compound SAA.
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1
Yield, 66.7%; H NMR (300 MHz, methanol-d4) δ 7.53 (d, J = 8.2 Hz, 2H), 7.29 (t, J = 7.8
Hz, 2H), 7.07 (t, J = 7.4 Hz, 1H), 2.33 (dt, J = 21.8, 7.4 Hz, 4H), 1.67 (dt, J = 23.8, 7.1 Hz, 4H),
1.49 – 1.30 (m, 4H). ESI-MS (m/z): calcd for C14H19NO3 [M-H]-: 249.13649, found: 248.12759.
4.2.4. Compound SAA1.
Yield, 56.9%; IR (KBr, cm-1): 3261, 3173, 2933, 2854, 1659, 1597, 1498, 1464, 831, 793,
722, 690. 1H NMR (300 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.58 (d, J = 7.8 Hz, 2H), 7.28 (t, J = 7.6
Hz, 2H), 7.02 (d, J = 8.1 Hz, 1H), 6.18 (s, 6H), 2.35 – 2.18 (m, 4H), 1.53 (d, J = 28.7 Hz, 4H),
13
1.31 (s, 4H). C NMR (75 MHz, DMSO) δ 181.20, 171.73, 139.83, 129.08, 123.36, 119.52,
36.91, 36.78, 29.02, 28.89, 25.84, 25.53. 195Pt NMR (129 MHz, DMSO-d6) δ 552.41 ppm. ESI-
MS (m/z): calcd for C14H24Cl3N3O3Pt [M-H]-: 583.79800, found: 582.05684.
4.2.5. Compound SAA2.
Yield, 62.5%; IR (KBr, cm-1): 3182, 2936, 2857, 1705, 1666, 1625, 1597, 1532, 1498, 1440,
1
753, 693. H NMR (300 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.58 (d, J = 7.7 Hz, 2H), 7.33 – 7.23
(m, 2H), 7.01 (t, J = 7.4 Hz, 1H), 2.54 (s, 2H), 2.29 (t, J = 7.5 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H),
2.01 (dd, J = 35.2, 12.3 Hz, 2H), 1.61 – 1.39 (m, 8H), 1.30 (p, J = 3.7 Hz, 4H), 1.07 (d, J = 8.5
Hz, 2H).13C NMR (75 MHz, DMSO) δ 174.94, 171.67, 164.37, 164.35, 139.79, 129.09, 123.37,
119.48, 61.94, 60.97, 36.82, 34.09, 31.18, 30.72, 28.86, 28.79, 25.45, 24.85, 24.23, 24.10. 195Pt
NMR (129 MHz, DMSO-d6) δ 1221.83 ppm. ESI-MS (m/z): calcd for C22H32ClN3O7Pt [M-H]-:
680.15765, found: 680.06525.
4.2.6. Compound SAA3.
Yield, 65.9%; IR (KBr, cm-1): 3136, 3055, 2933, 2857, 1793, 1659, 1598, 1535, 1498, 1441,
857, 756, 693. 1H NMR (300 MHz, DMSO-d6) δ 9.79 (s, 1H), 7.57 (d, J = 7.9 Hz, 2H), 7.26 (t, J
= 7.8 Hz, 2H), 7.00 (t, J = 7.3 Hz, 1H), 3.53 (s, 2H), 3.39 (s, 2H), 2.63 (dd, J = 22.2, 11.0 Hz,
13
2H), 2.27 (t, J = 7.2 Hz, 4H), 2.16 – 1.93 (m, 2H), 1.62 – 1.01 (m, 14H). C NMR (75 MHz,
DMSO) δ 203.34, 180.35, 176.01, 175.93, 171.17, 139.31, 128.57, 122.86, 119.02, 45.74, 37.68,
36.56, 36.35, 35.21, 35.12, 30.88, 30.48, 28.44, 28.36, 25.17, 24.98, 23.56, 23.42. 195Pt NMR
(129 MHz, DMSO-d6) δ 1195.47 ppm. ESI-MS (m/z): calcd for C26H36ClN3O8Pt [M-H]-:
749.12100, found: 748.17727.
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4.2.7. Compound SAA4.
Yield, 61.7%; IR (KBr, cm-1): 3198, 2934, 2854, 1791, 1624, 1597, 1541, 1498, 1442, 857,
1
755, 693. H NMR (300 MHz, DMSO-d6) δ 9.82 (s, 1H), 7.58 (d, J = 7.5 Hz, 2H), 7.27 (t, J =
7.3 Hz, 2H), 7.02 (d, J = 6.8 Hz, 1H), 6.30 (s, 6H), 3.50 (s, 4H), 2.27 (d, J = 7.4 Hz, 4H), 1.52 (d,
13
J = 28.7 Hz, 4H), 1.26 (d, J = 13.6 Hz, 4H). C NMR (75 MHz, DMSO) δ 203.48, 179.21,
175.86, 175.65, 171.20, 139.32, 128.57, 122.85, 119.02, 45.48, 37.57, 36.36, 35.51, 28.49, 28.38,
25.16, 24.98. 195Pt NMR (129 MHz, DMSO-d6) δ 1337.74 ppm. ESI-MS (m/z): calcd for
C20H28ClN3O8Pt [M-H]-: 668.99100, found: 668.11416.
4.3. The stability of complex SAA1 in PBS/DMF buffer.
The stability of Pt(IV) complexes in a PBS/DMF (99:1, v:v) was investigated by HPLC. The
incubation was generated by adding SAA1 to PBS/DMF buffer, which was performed at 25 °C
for 0, 6, 12, 24 and 48 h, separately. Reversed phase HPLC was implemented on a 250 × 4.5 mm
ODS column and the HPLC profiles were recorded on UV detection at 210 nm. Mobile phase
consisted of acetonitrile/water (25:75, v/v), and flow rate was 1.0 mL/min. The samples were
taken for HPLC analysis after filtration by 0.45 µm filter.
4.4. Released ability of complex SAA1 under reduction with ascorbic Acid.
The released ability of SAA1 in a solvent comprising acetonitrile/water (25:75, v:v) was
investigated by HPLC. The standard compounds were made by adding ascorbic acid, SAA, and
SAA1, respectively, to a solvent containing 25% acetonitrile and 75% water. The incubation was
generated by adding test compounds (10 mM) to a solvent containing 25% acetonitrile and 75%
water in the presence of 15 mM ascorbic acid, which was performed at 25 °C for 0, 1, and 2 h,
separately. Reversed phase HPLC was implemented on a 250 × 4.5 mm ODS column and the
HPLC profiles were recorded on UV detection at 210 nm. Mobile phase consisted of
acetonitrile/water (25:75, v/v), and flow rate was 1.0 mL/min. The samples were taken for HPLC
analysis after filtration by 0.45 µm filter.
4.5. Cell culture.
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Breast (MCF-7 and MDA-MB-231), colon (HCT-116), gastric (SGC7901 and cisplatin
resistant SGC7901) carcinoma cells along with normal liver cells LO2 were cultured at 37 °C in
a 5% CO2 atmosphere using the following monolayer culture media containing 10% fetal bovine
serum (FBS), 100 mg/mL of penicillin and 100 mg/mL of streptomycin.
4.6. Cytotoxicity analysis.
The growth inhibitory effect towards human cell lines was evaluated by means of MTT
assay. Briefly, 1 × 105 cells/well, MCF-7, MDA-MB-231, HCT-116, SGC7901,
SGC7901/CDDP and LO2 cell lines, were separately seeded in 96-well microplates in DMEM
medium with 10% FBS and then incubated 24 h at 37 °C in a humidified atmosphere of 5%
CO2/95% air. The medium was removed and the cells were then exposed to a fresh one
containing the compounds to be studied at the appropriate concentration. Triplicate cultures were
established for each treatment. After incubated for 72 h, each well was treated with 10 µL of a 5
mg·mL-1 MTT. And after 5 h additional incubation, the medium was replaced by 100 µL DMSO.
The inhibition of cell growth induced by the tested complexes was detected by measuring the
absorbance of each well at 570/630 nm using enzyme labeling instrument.
4.7. HDACs inhibition activity assay.
HDACs inhibition activity assays were conducted as reported previously [60, 61]. At first,
various concentrations of tested compound (50 µL) was mixed with 10 µL of HeLa nuclear
extract solution and incubated at 37 °C for 10 min. Then 40 µL of fluorogenic substrate Boc-Lys
(acetyl)-AMC was added. After incubation at 37 °C for 30 min, 100 µL of developer, containing
trypsin and Trichostatin A (TSA), was added to stop the reaction. After 20 min incubation,
fluorescence intensity was measured using a microplate reader at excitation and emission
wavelengths of 390 and 460 nm, respectively. The inhibition ratios were calculated by the
fluorescence intensity readings of tested wells deducting those of control wells, and the IC50
values were calculated using the software GraphPad Prism (version 5.0).
4.8. PARP-1 inhibition assay.
The inhibition of the target compounds on PARP-1 enzymatic assay was determined by
ELISA in 96-well plates as previously reported [62]. Histone (20 µg/mL) was diluted in 100 µL
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PBS buffer prepared by 10 mM Na2HPO4, 10 mM NaH2PO4 and 150 mM NaCl. The mixture
was precoated in each well and incubated at 4 °C overnight. 100 µΜ NAD+, 200 nM slDNA and
25 µM biotinylated NAD+ diluted in 30 µL of reaction buffer (2 mM MgCl2, 50 mM Tris) were
added into each well, and then 5 µL of solvent control or various concentrations of test
compound was added. 20 µL PARP-1 (50 ng/well) was added to initiate the reaction at 30 °C.
After 1 h, 50 µL streptavidin conjugated HRP was added and maintain temperature at 30 °C for
30 min. At last, 100 µL mixture of luminol and H2O2 in citrate buffer (0.1 M) was added.
Luminescent signal was measured by spectrophotometer (Molecular Devices SpectraMax M5
microplate reader) and the concentration required for 50% inhibition of PARP-1 enzymatic
activity (IC50) was calculated using the software GraphPad Prism (version 5.0).
4.9. Cellular uptake test.
SGC7901/CDDP cells were seeded in 6-well plates in grow medium. When the cells
achieved about 80% confluence, SGC7901/CDDP cells were treated with 12 µM of ctc-
[Pt(NH3)2(OH)Cl3], cisplatin and SAA1 for 12 h, respectively. Cells were collected and washed
three times with cold PBS, harvested and counted, then centrifuged at 1000 × g for 8 min and
resuspended in 1 mL PBS. A volume of 100 µL was taken out to determine the cell density.
Finally, the cells were digested in 200 µL 65% HNO3 for 10 h at 65 °C. The Pt level in cells was
measured by ICP-MS.
4.10. Cell cycle analysis.
SGC7901/CDDP cells were treated with suitable concentration of SAA, SAA1 and cisplatin,
separately. After 24 h of incubation, cells were collected by trypsinization, washed three times
with ice-cold PBS, fixed, permeabilized with ice-cold 70% ethanol and stored at -20 °C
overnight. After washed with ice-cold PBS, the cells were incubated with 100 µg/mL RNase A
for 30 min at 37 °C, stained with 1 mg/mL propidium iodide (PI) for 30 min in the dark at 4 °C.
Finally, analysis was performed by flow cytometry using a fluorescence-activated cell sorting
(FACS) Calibur (Becton Dickinson, Palo Alto, CA).
4.11. Apoptosis analysis.
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SGC7901/CDDP cells were seeded at the density of 2 × 106 cells/mL of the DMEM medium
with 10% FBS on 6-well plates to the final volume of 2 mL. Cisplatin was positive control at a
concentration of 4 µM. The plates were incubated for overnight and then treated with different
concentrations compound SAA, SAA1 and cisplatin for 24 h. Briefly, after incubation 24 h, cells
were collected and washed with PBS twice, and then resuspend cells in 1 × Binding Buffer (0.1
M Hepes/NaOH (pH 7.4), 1.4 M NaCl, 25 mM CaCl2) at a concentration of 1 × 106 cells/mL.
The cells were stained with 5 µL of FITC Annexin V (BD, Pharmingen) and 5 µL propidium
iodide (PI) staining using annexin-V FITC apoptosis kit followed; 100 µL of the solution was
transferred to a 5 mL culture tube and incubated for 30 min at room temperature (25 °C) in the
dark. The apoptosis ratio was quantified by system software (Cell Quest; BD Biosciences).
4.12. Western blot analysis.
Cisplatin-resistant SGC7901 cells were treated with tested compounds at the indicated
concentrations for 24 h. Afterwards, cells were harvested and lysed in cell in RIPA buffer (0.1%
SDS, 0.5% sodium deoxycholate, 1% NP40), and were centrifuged at 13000 rpm for 20 min at 4
°C. Proteins from cell lysates was electrophoresed on a 12% SDS-PAGE and blotted to a
nitrocellulose membrane. The membrane was blocked with PBST containing 5% non-fat dry
milk for 1 h and another 1 h incubation with primary antibodies at 37 °C. The membrane was
further stained with the corresponding secondary antibodies for 1 h at room temperature (25 °C).
Protein blots were detected by ECL according to the manufacturer’s protocol (GE). β-actin was
used as a loading control.
Notes
The authors declare that they have no conflicts of interest.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (Grant No. 21571033)
and the New Drug Creation Project of the National Science and Technology Major Foundation
of China (Grant No. 2015ZX09101032) for financial aids to this work. The authors would also
like to thank the Fundamental Research Funds for the Central Universities (Project
2242016K30020) for supplying basic facilities to our key laboratory. We also want to express
our gratitude to the Priority Academic Program Development of Jiangsu Higher Education
16

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Institutions for the construction of fundamental facilities (Project 1107047002). We would also
like to thank shiyanjia lab for the support of relevant analysis.
Appendix A. Supplementary data
1
Compounds’ characterization by H, 13C and 195Pt-NMR spectra together with ESI mass
spectroscopy and HPLC chromatograms. Cellular uptake of measured samples on the tested
cancer cell line and biological assays such as HDAC and PARP-1 inhibitory activity.
Supplementary data related to this article can be found at ****.
Abbreviations List
HDAC
HDACIs
PARP-1
ZBG
Histone deacetylase
Histone deacetylase inhibitors
Poly (ADP-ribose) polymerase 1
Zinc binding group
NAD+
CDDP
NCS
Nicotinamide adenine dinucleotide
Cisplatin
N-chlorosuccinimide
TBTU
O-(benzotriazol-1-yl)-N,N,N’,N’-
tetramethyluronium tetrafluoroborate
RF
Resistance factor
ICP-MS
Inductively
coupled
plasma
mass
spectrometry
SSB
BER
PI
Single strand breaks
Basic excision repair
Propidium iodide
Dichloromethane
Ultraviolet
DCM
UV
HPLC
High performance liquid chromatography
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FBS
Fetal bovine serum
DMEM
FBS
Dulbecco's modified eagle medium
Fetal bovine serum
TSA
Trichostatin A
HRP
Horseradish peroxidase
Fluorescence-activated cell sorting
Radio immunoprecipitation assay
FACS
RIPA
SDS-PAGE
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
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TABLES
Platinum(IV) prodrugs multiply targeting genomic
DNA, histone deacetylases and PARP-1
Zichen Xu a, b, 1, Weiwei Hu a, b, 1, Zhimei Wang a, b, Shaohua Gou a, b,
*
Table 1.
Effect of target compounds against cell viability of different cell linesa
IC50 values (µM)
Complex
Cisplatin
HCT-116
MCF-7
MDA-MB-231 SGC7901
LO2
7.44 ± 0.72
3.75 ± 0.29
7.96 ± 0.64
12.69 ± 0.82 8.68 ± 0.35
26.82 ± 2.16 16.95 ± 1.17
Oxaliplatin 8.67 ± 0.33
18.35 ± 1.18 14.33 ± 0.55
43.15 ± 3.36 30.45 ± 2.17 14.25 ±1.28
DN603
DN604
SAHA
SAA
8.14 ± 0.64
8.20 ± 0.57
7.43 ± 0.54
>200
14.11 ± 1.27
35.84 ± 3.12
2.48 ± 0.18
>200
32.50 ± 2.59 9.36 ± 0.82
40.99 ± 3.23
3.18 ± 0.25
>200
6.98 ± 0.52
>200
3.11 ± 0.16
>200
SAA1
SAA2
SAA3
SAA4
16.27 ± 1.21 6.70 ± 0.58
5.72 ± 0.36
0.95 ± 0.07
16.87 ± 0.35
15.43 ± 1.08 15.07 ± 1.10 18.17± 0.29
45.96 ± 4.35 15.68 ± 1.23 17.57 ± 1.69
32.90 ± 3.08 10.25 ± 0.95 46.46 ± 2.58
17.39 ± 0.16 6.93 ± 0.32
14.75 ± 1.12 6.71 ± 0.54
6.84 ± 0.49
82.88 ± 6.25
a Results are expressed as the mean ± SD from three independent experiments.

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Table 2.
In vitro growth inhibitory effect of SAA1 against cisplatin-resistant cell lines
SGC7901a
IC50 values (µM)
Complex
Cisplatin
SAHA
SGC7901
12.69 ± 0.82
7.43 ± 0.54
>200
SGC7901/CDDP LO2
RF
67.88 ± 2.16
7.18 ± 0.48
>200
8.68 ± 0.35
5.35
0.96
2.48 ± 0.18
>200
SAA
SAA1
0.95 ± 0.07
1.37 ± 0.09
0.39 ± 0.01
13.82 ± 1.12
13.46 ± 0.34
16.87 ± 0.35
nd*
0.41
10.08
3.9
SAA : CDDP =1:1
ctc-[Pt(NH3)2(OH)Cl3] 3.44 ± 0.25
nd*
a Results are expressed as the mean ± SD from three independent experiments.
nd* means not detected

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Figures
Platinum(IV) prodrugs multiply targeting genomic
DNA, histone deacetylases and PARP-1
Zichen Xu a, b, 1, Weiwei Hu a, b, 1, Zhimei Wang a, b, Shaohua Gou a, b,
*
Fig. 1. Chemical structure of SAHA, SAA and platinum(II) complexes.

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Fig. 2. Design of a novel platinum(IV) prodrug derived from cisplatin, for example,
as a potential HDAC inhibitor, PARP inhibitor and antitumor agent.
Fig. 3. Intracellular accumulation of ctc-[Pt(NH3)2(OH)Cl3], cisplatin and SAA1 (12
µM) in SGC7901/CDDP cells after 12 h. Each value shown in the table is in
nanograms of platinum per 106 cells. Results are expressed as the mean ± SD for three
independent experiments. *P < 0.05 was calculated relative to cisplatin.