Synthesis, Cytotoxicity, and Mechanistic Investigation of Platinum(IV) Anticancer Complexes Conjugated with Poly(ADP-ribose) Polymerase Inhibitors

Article abstract of DOI:10.1021/acs.inorgchem.9b02839

Many clinical trials using combinations of platinum drugs and PARP-1 inhibitors (PARPi) have been carried out, with the hope that such combinations will lead to enhanced therapeutic outcomes against tumors. Herein, we obtained seven potential PARPi with structural diversity and then conjugated them with cisplatin-based platinum(IV) complexes. Both the synthesized PARPi ligands and PARPi-Pt conjugates [PARPi-Pt(IV)] show inhibitory effects against PARP-1's catalytic activity. The PARPi-Pt(IV) conjugates are cytotoxic in a panel of human cancer cell lines, and the leading ones display the ability to overcome cisplatin resistance. A mechanistic investigation reveals that the representative PARPi-Pt(IV) conjugates efficiently enter cells, bind to genomic DNA, disturb cell cycle distribution, and induce apoptotic cell death in both cisplatin-sensitive and-resistant cells. Our study provides a strategy to improve the cytotoxicity of platinum(IV)-based anticancer complexes and overcome cisplatin resistance by using a small-molecule anticancer complex that simultaneously damages DNA and inhibits PARP.

Full text of DOI:10.1021/acs.inorgchem.9b02839

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Synthesis, Cytotoxicity, and Mechanistic Investigation of
Platinum(IV) Anticancer Complexes Conjugated with Poly(ADP-
ribose) Polymerase Inhibitors
Zoufeng Xu,†,‡,§ Cai Li,†,‡,§ Qiyuan Zhou,† Zhiqin Deng,†,‡ Zixuan Tong,† Man-Kit Tse,†
and Guangyu Zhu*,†,‡
†Department of Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong SAR 999077, People’s Republic of
China
‡City University of Hong Kong Shenzhen Research Institute, Shenzhen 518057, People’s Republic of China
S
* Supporting Information
ABSTRACT: Many clinical trials using combinations of
platinum drugs and PARP-1 inhibitors (PARPi) have been
carried out, with the hope that such combinations will lead to
enhanced therapeutic outcomes against tumors. Herein, we
obtained seven potential PARPi with structural diversity and
then conjugated them with cisplatin-based platinum(IV)
complexes. Both the synthesized PARPi ligands and PARPi-
Pt conjugates [PARPi-Pt(IV)] show inhibitory effects against
PARP-1’s catalytic activity. The PARPi-Pt(IV) conjugates are
cytotoxic in a panel of human cancer cell lines, and the leading
ones display the ability to overcome cisplatin resistance. A mechanistic investigation reveals that the representative PARPi-
Pt(IV) conjugates efficiently enter cells, bind to genomic DNA, disturb cell cycle distribution, and induce apoptotic cell death in
both cisplatin-sensitive and -resistant cells. Our study provides a strategy to improve the cytotoxicity of platinum(IV)-based
anticancer complexes and overcome cisplatin resistance by using a small-molecule anticancer complex that simultaneously
damages DNA and inhibits PARP.
olaparib became the first Food and Drug Administration
(FDA)-approved PARP inhibitor for germline BRCA-mutated
advanced ovarian cancer, and currently it is also undergoing
phase II trials for breast, ovarian, colorectal, and advanced
prostate cancer.12 Later on, in 2016, rucaparib was granted
accelerated approval from the FDA as a PARP inhibitor used
for cancer treatment. Recently, in 2017, the FDA approved
niraparib as an oral PARP inhibitor to treat ovarian cancer. At
the same time, other PARP inhibitors, including talazoparib,
veliparib, and E7449, are all in late-phase clinical trials (Figure
1).12−14
Platinum(II)-based anticancer drugs, including cisplatin,
carboplatin, and oxaliplatin, have been approved to be used
worldwide.15 Acting as alkylating agents, these platinum(II)-
based anticancer drugs are able to effectively bind to genomic
DNA and finally lead to cell apoptosis.16 However, obvious
shortfalls, including drug resistance and several severe side
effects, limit further application of these platinum(II)-based
anticancer drugs.17 Recently, platinum(IV)-based compounds
are quite promising to become the next generation of
platinum-based anticancer prodrugs.15,18,19 The additional
two axial ligands in platinum(IV) complexes can be further
INTRODUCTION
■
Poly(ADP-ribose) polymerases (PARPs) are a family of
nicotinamide adenine dinucleotide (NAD+)-consuming en-
zymes that mediate different biological processes including
DNA repair, maintenance of the genomic integrity, and cell
death.1 Until now, 17 types of PARPs have been identified in
humans, and these PARPs are further grouped into four
subfamilies including deoxyribonucleic acid (DNA)-dependent
PARPs, tankyrases, CCCH zinc finger PARPs, and macro-
PARPs.2,3 Among the PARP family, poly(ADP-ribose)
polymerase-1 (PARP-1) is one of the most thoroughly studied
and understood enzymes and contributes to more than 90% of
the total PARP activity.4 PARP-1 plays an essential role in
DNA single-strand break repair and subsequently maintenance
of the genomic integrity.5 Lots of efforts have been devoted to
developing PARP-1 inhibitors (PARPi) as drugs for cancer
chemotherapy.4,6,7 Synthetic lethality occurs between cancer
cells with BRCA1 (breast cancer susceptibility gene 1) or
BRCA2 (breast cancer susceptibility gene 2) mutations and
PARPi.8,9 It has also been proven that certain PARPi enhance
the growth inhibition and cytotoxicity of some major
chemotherapeutics used in the clinic including temozolomide,
topotecan, irinotecan, and cisplatin.10,11 During the last 3
decades, thousands of small molecules have been synthesized
and developed as potential PARP inhibitors.4,12 In 2014,
Received: September 23, 2019
© XXXX American Chemical Society
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Figure 1. Chemical structures of some PARPi.
Figure 2. Syntheses of PARPi-L1 and the PARPi-L1-Pt(IV) complex.
functionalized with targeting moieties, and the ligands can also
control the biological properties of platinum(IV) prodrugs.16
In 2010, the specific role of PARP-1 in the recognition of
platinum−DNA damage was revealed.20 Synergistic effects of
PARP inhibitors and platinum(II) anticancer drugs have also
been observed both in vitro and in vivo.21,22 After the
combination of PARPi with platinum-based chemotherapy, a
significant decrease of cell survival has been observed
compared to single treatment.23−25 In addition, combined
olaparib and carboplatin in OLICARB nanoparticles efficiently
enhance the cytotoxicity and selectivity toward human cancer
cells.26
Herein, we rationally designed several new platinum(IV)
complexes bearing PARPi at the axial positions, aiming to
improve the anticancer activity of platinum-based drugs. We
utilized the derivatives of 4-quinazolinone and 1-(2H)-
phthalazinone, which have already been applied as potential
PARP inhibitors to conjugate with cisplatin-based platinum-
(IV) complexes. The seven PARP inhibitors and correspond-
ing PARPi-Pt(IV) conjugates were successfully obtained and
fully characterized. The physicochemical properties, PARP-1
inhibitory effects, and cytotoxicity of these platinum(IV)
conjugates were examined. A detailed investigation of the
mechanism of representative PARPi-Pt(IV) conjugates was
also carried out. Our data indicate that functionalization of
cisplatin-based platinum(IV) complexes with PARP inhibitors
leads to enhanced inhibitory activity against both the enzyme
and the proliferation of cancer cells.
RESULTS AND DISCUSSION
■
Because NAD+ is the substrate for PARP, most of the reported
PARP inhibitors are designed as analogues of nicotinamide to
compete with the binding of NAD+ in the active site. The
pharmacophore of PARP inhibitors usually contains an amide
group, which is essential for their PARP inhibitory activity.4
This necessary amide group in PARPi should contain at least
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Figure 3. Syntheses of PARPi-L2 and the PARPi-L2-Pt(IV) complex.
Figure 4. Syntheses of PARPi-L3 and the PARPi-L3-Pt(IV) complex.
one proton and always appear as a primary or secondary
amide. However, considering that the most widely applied
methods for obtaining platinum(IV) conjugates require a
conjugation reaction between the carboxyl group in bioactive
molecules, e.g., PARP inhibitors, and the axial hydroxyl groups
in platinum(IV) complexes, the primary amide in PARP
inhibitors may be too active and other side reactions may also
occur during conjugation with platinum. Hence, PARP
inhibitors containing a secondary amide together with a
carboxylate would be our first choice because of the lower
reactivity of the secondary amide group.
are the analogues of 1-(2H)-phthalazinone. The most widely
used method to synthesize 1-(2H)-phthalazinone is the
reaction between the derivatives of 2-acetylbenzoic acids
with hydrazine. First of all, 2-(p-toluoyl)benzoic acid (1) was
oxidized by KMnO4 to form compound 2. Then, compound 2
reacted with hydrazine in ethanol under reflux, and PARPi-L1
was subsequently obtained.27 The reactivity of the carboxyl
group in PARPi-L1 is too low to directly react with the OH
group in cis,cis,trans-[Pt(NH3)2Cl2(OH)2] (oxoplatin). After
activation of the carboxyl group in PARPi-L1 with 1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide/N-hydroxysuccini-
mide (EDCI/NHS) and then further reaction with oxoplatin
in dimethyl sulfoxide (DMSO), the PARPi-L1-Pt(IV)
conjugate was finally obtained (Figure 2).
Among thousands of potential PARP inhibitors, one of the
most widely adopted structures is 1-(2H)-phthalazinone. For
example, the pharmacophores of both olaparib and talazoparib
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Figure 5. Syntheses of PARPi-L4 and the PARPi-L4-Pt(IV) complex.
Figure 6. Syntheses of PARPi-L5 and the PARPi-L5-Pt(IV) complex.
The successful synthesis of the platinum(IV) complex
conjugated with a PARPi encouraged us to further expand
the scope of similar PARPi-Pt(IV) conjugates. Olaparib (also
known as Lynparza) is the first FDA-approved PARP inhibitor
for cancer treatment. Recently, lots of clinical trials involving
olaparib combined with platinum therapy are in progress. The
initial results show promising antitumor activity of olaparib in
combination with platinum-based DNA-damaging agents.28,29
The challenge to obtain a conjugate with olaparib is that, in
olaparib, there is no functional group that can be easily utilized
to directly conjugate to oxoplatin. Hence, modification of the
structure of olaparib is necessary. PARPi-L2 is the key
intermediate of olaparib and has been identified as a
moderately potent PARP inhibitor.30 The free carboxyl
group in PARPi-L2 is anticipated to easily react with the
OH groups in oxoplatin after activation. 3-Dimethoxyphos-
phoryl-3H-2-benzofuran (3) was first synthesized.30 After
mixing with 2-fluoro-5-formylbenzonitrile (4) and triethyl-
amine in tetrahydrofuran, 2-fluoro-5-[(Z/E)-(3-oxo-2-benzo-
furan-1-ylidene)methyl]benzonitrile (5) was obtained. These
isomers were directly used to further react with hydrazine in
ethanol under reflux, and 2-fluoro-5-[(4-oxo-3H-phthalazin-1-
yl)methyl]benzonitrile (6) was obtained. After hydrolysis of
the cyanide group in compound 6 in an aqueous solution of
sodium hydroxide (NaOH), PARPi-L2 was finally obtained.30
Subsequently, the PARPi-L2-Pt(IV) conjugate was synthesized
following the same method as that of the PARPi-L1-Pt(IV)
conjugate (Figure 3).
The unsubstituted compound 9 is a highly potent PARPi
(PARP-1 IC50 = 5 nM).30 This ligand was synthesized by first
conjugating compound PARPi-L2 with tert-butyl-1-piperazine-
carboxylate (7) and then obtained after deprotection of the
BOC group in compound 8 in an aqueous solution of
hydrochloric acid (HCl).30 By utilizing compound 9 as a
precursor, PARPi-L3 was designed and synthesized by reacting
it with succinic anhydride in N,N-dimethylfomamide (DMF).
PARPi-L3 has a structure similar to that of olaparib, and the
only difference is that succinic anhydride is used to substitute
the 4 position of the piperazine ring. By conjugating
compound 9 with cis,cis,trans-[Pt(NH3)2Cl2(succinato)-
(benzoato)] (10) using N,N′-dicyclohexylcarbodiimide
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Figure 7. Syntheses of PARPi-L6 and the PARPi-L6-Pt(IV) complex.
Figure 8. Syntheses of PARPi-L7 and PARPi-L7-Pt(IV) complex.
(DCC) as a coupling agent, conjugate PARPi-L3-Pt(IV) was
finally obtained (Figure 4).
their properties, hence, following a similar method, the
syntheses of PARPi-L6 and PARPi-L7 as well as the PARPi-
L6-Pt(IV) and PARPi-L7-Pt(IV) conjugates were achieved by
using ethyl 3-formylphenoxyacetate (16; Figure 7) and 2-
formylphenoxyacetic acid (17; Figure 8) as the starting
materials, respectively. All seven ligands and their correspond-
ing conjugates are well characterized (Figures S1−S42).
The stability of all of the PARPi-Pt(IV) conjugates was first
explored in phosphate-buffered saline (PBS), and they were
proven to be relatively stable in PBS buffer (pH = 7.4) at 37
°C for 24 h (Figure S43). Furthermore, the stability of these
complexes in serum (pH = 7.4, 37 °C) was tested. Except for
PARPi-L1-Pt(IV) and PARPi-L3-Pt(IV), all of the other
PARPi-Pt(IV) complexes were quite stable under the same
conditions after 12 h, and at least more than 70% of the
original platinum(IV) complexes remained after 24 h (Figure
S43). The reduction of the PARPi-Pt(IV) conjugates by
ascorbate in PBS (1 mM, pH = 7.4) at 37 °C was also tested,
and the results show that most of the conjugates can be
efficiently reduced. For example, except PARPi-L3-Pt(IV),
other PARPi-Pt(IV) complexes were completely reduced
within 12 h, and for PARPi-L3-Pt(IV), 50% was reduced
after 12 h (Figure S43). The reduction potentials of the
platinum(IV) complexes are shown in Table 1. All of the
complexes show an irreversible reduction peak due to
Besides 1-(2H)-phthalazinone, 4-quinazolinone has also
been widely adopted during the design of new PARP
inhibitors. With appropriate modifications, these 4-quinazoli-
none-based PARP inhibitors also exhibited strong inhibitory
effects against the PARP enzyme.11,31−33 PARPi-L4 was
synthesized by utilizing anthranilamide (11) and 4-cyanoben-
zoyl chloride (12).34,35 The reaction between compounds 11
and 12 easily occurred in dichloromethane (DCM). The
cyclization of compound 13 to form 4-quinazolinone was
catalyzed by NaOH in ethanol. PARPi-L4 was obtained by
hydrolyzing the cyanide group of compound 14 in an aqueous
solution of NaOH. Because of the low reactivity of the carboxyl
group in PARPi-L4, again the carboxyl group was activated by
forming its NHS ester. The final PARPi-L4-Pt(IV) conjugate
was obtained by reaction with oxoplatin in DMSO (Figure 5).
The condensation oxidation reaction between 11 and
aldehyde can also provide quinazolinone.35 Hence, 4-
formylphenoxyacetic acid (15) was chosen to react with 11
by utilizing I2 as an oxidant, and PARPi-L5 was obtained in
one step. After conjugation with oxoplatin, PARPi-L5-Pt(IV)
was obtained (Figure 6).
Considering the possibility that the different substituted
positions of the platinum moiety in PARPi might also affect
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Table 2. Cellular Accumulation and Platinum Levels in Genomic DNA upon the Treatment of Cisplatin, PARPi-L6-Pt(IV),
a
and PARPi-L7-Pt(IV) in A2780 and A2780cisR Cells
A2780 cell line
ng of Pt/μg of DNA
A2780cisR cell line
compound
cisplatin
PARPi-L6-Pt(IV)
PARPi-L7-Pt(IV)
ng of Pt/106 cells
ng of Pt/106 cells
ng of Pt/μg of DNA
13.9 3.1
145.6 32.2
37.2 0.4
0.38 0.06
0.82 0.21
0.34 0.15
5.9 0.7
220.4 2.2
53.4 7.8
0.35 0.03
0.98 0.17
0.53 0.20
a
Cells were treated with 10 μM compound for 8 h.
Figure 9. Flow cytometry analysis of Annexin V/7-AAD double-stained (A) A2780 and (B) A2780cisR cells upon the treatment of 10 μM
compounds for 24 h.
transformation from platinum(IV) to platinum(II) (Figures
S44−S50). Except for conjugate PARP-L3-Pt(IV) (Ep
−0.588 V), whose slightly higher Ep value can be attributed
to its two axial carboxylato ligands, other platinum(IV)
complexes show a similar level of reduction potentials ranging
from −0.700 to −0.744 V.
themselves can hardly enter cells efficiently to execute the
cytotoxic effect. For PARPi-Pt(IV), some of the conjugates are
active against cancer cells, showing IC50 values comparable
with those of cisplatin. For example, the IC50 values of PARPi-
L3-Pt(IV) are similar to those of cisplatin. PARPi-L5-Pt(IV),
PARPi-L6-Pt(IV), and PARPi-L7-Pt(IV) display significantly
improved cytotoxic potency compared to cisplatin. For
instance, the IC50 values of PARPi-L6-Pt(IV) are 0.21, 0.21,
and 0.13 in A549, MDA-MB-231, and A2780 cells, showing
24-, 43-, and 15-fold increases compared to cisplatin,
respectively. Notably, PARPi-L5-Pt(IV), PARPi-L6-Pt(IV),
and PARPi-L7-Pt(IV) are active in both cisplatin-resistant
lung cancer cells (A549cisR) and cisplatin-resistant ovarian
cancer cells (A2780cisR). The resistant factor (RF) in
A549cisR cells decreases from 3.2 for cisplatin to 1.9, 0.8,
and 0.1 for PARPi-L5-Pt(IV), PARPi-L6-Pt(IV), and PARPi-
L7-Pt(IV), respectively. In A2780cisR cells, the RFs for
PARPi-L5-Pt(IV), PARPi-L6-Pt(IV), and PARPi-L7-Pt(IV)
are 1.2, 1.0, and 0.7, much smaller than the RF for cisplatin
(9.6), indicating the promising ability of the conjugates to
overcome cisplatin resistance. These leading complexes are
also more cytotoxic than a mixture of cisplatin and the
corresponding PARPi, proving that such a conjugation results
in enhanced cytotoxicity (Table S1).
The impressive cytotoxic potency and negative cisplatin
cross-resistance of PARPi-L6-Pt(IV) and PARPi-L7-Pt(IV)
encouraged us to carry out additional cell-based assays to
evaluate these conjugates. The cellular accumulation levels of
cells treated with PARPi-L6-Pt(IV) and PARPi-L7-Pt(IV)
were determined in A2780 and A2780cisR cells. Cisplatin was
included as a control. Upon the treatment of 10 μM cisplatin,
A2780 and A2780cisR cells have 13.9 and 5.9 ng of Pt/106
cells, respectively. The decreased accumulation of cisplatin in
the resistant cells is one of the reasons for drug resistance.39
=
We first tested the inhibitory effects of the synthesized
PARPi ligands and PARPi-Pt(IV) conjugates against human
recombinant PARP-1. The enzymatic activity of PARP-1
without an inhibitor was set to 100%, and the IC50 values
against PARP-1 are summarized in Table 1. Ligands PARPi-L1
and PARPi-L7 display no PARPi effect in the tested
concentrations. Ligands PARPi-L4, PARPi-L2, PARPi-L5,
and PARPi-L6 have IC50 values in the micromolar range.
Among the PARPi ligands, PARPi-L3 shows the strongest
inhibitory effect with an IC50 value of 170 nM. The addition of
platinum to the PARPi ligands enhances the PARP inhibitory
effects in most cases, which is consistent with our previous
report.38 For example, the IC50 values of PARPi-L2-Pt(IV) and
PARPi-L3-Pt(IV) are 63 and 6.2 nM, showing 40- and 27-fold
increases compared to PARPi-L2 and PARPi-L3, respectively.
PARPi-L5-Pt(IV) (IC50 = 4800 nM) and PARPi-L7-Pt(IV)
(IC50 = 7200 nM) display enhanced inhibitory activity
compared to their corresponding ligands as well. The strong
inhibitory effect of the PARPi-Pt(IV) conjugates may
contribute to their anticancer activity.
The cytotoxicities of the synthesized PARPi ligands and
PARPi-Pt(IV) conjugates were then assessed against a panel of
human carcinoma cell lines including A549 (lung), A549cisR
(cisplatin-resistant lung), MDA-MB-436 (BRCA1-deficient
breast), MDA-MB-231 (breast), MCF-7 (breast), A2780
(ovarian), and A2780cisR (cisplatin-resistant ovarian). PARPi
ligands show very limited cytotoxic activity against the tested
cell lines, maybe because the negatively charged PARPi
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Figure 10. Cell cycle distribution of (A) A2780 and (B) A2780cisR cells upon the treatment of 10 μM compounds for 24 h.
The numbers increase to 145.6 and 220.4 for PARPi-L6-
Pt(IV) in A2780 and A2780cisR cells, respectively. The
treatment of PARPi-L7-Pt(IV) under the same conditions also
leads to an increased accumulation level in both the A2780 and
A2780cisR cells (Table 2). We also measured the platinum
levels in the genomic DNA of A2780 and A2780cisR cells
under the same conditions of treatment (Table 2). In most of
the cases, the increased cellular accumulation levels of PARPi-
L6-Pt(IV) and PARPi-L7-Pt(IV) lead to increased platinum
levels in the genomic DNA. For example, in A2780cisR cells,
the treatment of PARPi-L6-Pt(IV) and PARPi-L7-Pt(IV)
results in 0.98 and 0.53 ng of Pt/μg of DNA, showing 2.8-
and 1.5- fold increases compared to cisplatin, respectively.
These elevated cellular accumulation levels may contribute to
the higher cytotoxicity of PARPi-L6-Pt(IV) and PARPi-L7-
Pt(IV), especially in the drug-resistant cells.
is in agreement with previous reports.40 In A2780 cells, the
cotreatment of cisplatin and PARPi ligands leads to weak cell
cycle arrest at the S phase. The fraction of cells in the S phase
slightly increases from 9% for the control group to 11.1% and
15.7% for cisplatin + PARPi-L6 and cisplatin + PARPi-L7,
respectively. In contrast, PARPi-L6-Pt(IV) and PARPi-L7-
Pt(IV) increase the populations of the cells in the S phase to
35.7% and 21.6%, respectively, which are significantly higher
than the population of the S phase in the cisplatin-treated
group (14.2%). In A2780cisR cells, the percentages of cells in
the G0/G1, G2/M, and S phases are 71.9%, 12.2%, and 15.8%
for the control group, respectively. PARPi-L6-Pt(IV) increases
the fraction of cells in the G2/M phase to 24.3%. After the
treatment of PARPi-L7-Pt(IV), 22.7% of cells are arrested at
the S phase. These results indicate the ability of PARPi-Pt(IV)
conjugates to disturb the cell cycle distribution in both the
A2780 and A2780cisR cells.
The cell death pathway upon the treatment of PARPi-Pt(IV)
conjugates was further studied by an Annexin V/7-AAD
double-staining assay. A2780 and A2780cisR cells were treated
with 10 μM cisplatin, PARPi-L6-Pt(IV), or PARPi-L7-Pt(IV)
for 24 h. A2780 cells treated with cisplatin have 26.8% and
39.7% of apoptosis and dead fraction, respectively. The
cotreatment of cisplatin and PARPi ligands leads to similar
levels of apoptosis and dead fraction. The fractions of Annexin
V positive cells with the treatment of PARPi-L6-Pt(IV) and
PARPi-L7-Pt(IV) are 54.7% and 39.4%, respectively, confirm-
ing their impressive ability to induce apoptotic cell death
(Figures 9 and S51). In A2780cisR cells, the treatment of 10
μM cisplatin or the cotreatment of cisplatin and PARPi ligands
does not have a significant effect on the population of Annexin
V positive cells compared to the control (untreated) group.
The fraction of apoptotic cells in the control group is 1.5%,
whereas the numbers for cisplatin, cisplatin + PARPi-L6, and
cisplatin + PARPi-L7 are 1.5%, 2.7%, and 2.3%, respectively.
Notably, under the same conditions, 16.1% and 25.4% of
apoptotic cells are induced by the treatment of PARPi-L6-
Pt(IV) and PARPi-L7-Pt(IV), respectively. In addition,
A2780cisR cells treated with PARPi-L7-Pt(IV) have 42.6% of
dead cells. Thus, PARPi-L6-Pt(IV) and PARPi-L7-Pt(IV) are
able to effectively induce apoptosis, especially in cisplatin-
resistant cells.
We also determined the expression levels of PARP-1 in the
cells treated with the PARPi-Pt(IV) complexes. The leading
complexes, PARPi-L5-Pt(IV) and PARPi-L6-Pt(IV), effec-
tively induce the cleavage of PARP-1, indicating their ability to
induce apoptosis. Also, this result suggests that blocking the
enzymatic activity of PARP does not affect its expression level,
which is consistent with other studies.41,42
CONCLUSIONS
■
In conclusion, seven platinum(IV) complexes containing
PARPi at their axial positions have been designed and
synthesized. All of these complexes are proven to be stable
under physiological conditions, and the conjugates can be
activated by the reducing agent. The synthesized PARPi
ligands show a PARP inhibitory effect, with IC50 values mostly
in the micromolar range. The addition of platinum to the
ligands enhances the PARP inhibitory effect and results in
PARPi-Pt(IV) conjugates with IC50 values as low as 6.2 nM
[for PARPi-L3-Pt(IV)] against the catalytic activity of PARP-1.
Most of the PARPi-Pt(IV) conjugates show comparable or
improved cytotoxic potency in several cancer cell lines
compared to cisplatin, and the leading ones are significantly
active in cisplatin-resistant cell lines. The detailed mechanistic
studies in A2780 and A2780cisR cells show that the
representative PARPi-Pt(IV) conjugates efficiently enter cells,
bind to genomic DNA, disturb the cell cycle distribution, and
induce the apoptosis pathway. Our study proves that the
conjugation of PARP inhibitors to the platinum(IV) center can
We further studied the cell cycle distribution upon the
treatment of PARPi-Pt(IV) conjugates (Figures 10 and S52).
The treatment of 10 μM cisplatin for 24 h results in cell cycle
arrest at the S phase in both A2780 and A2780cisR cells, which
H
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H]+). Calcd for C15H17Cl2N4O4Pt: m/z 583.0 ([M + H]+). HPLC: tR
= 10.8 min; purity = 95.1% (Figure S6).
be one of the applicable approaches to developing new
platinum(IV) anticancer complexes as potential drug candi-
dates for cancer therapy.
Syntheses of Complexes PARPi-L2 and PARPi-L2-Pt(IV).
PARPi-L2 was synthesized by following a previous report.30
PARPi-L2. 1H NMR (600 MHz, DMSO-d6): δ 13.25 (s, 1H), 12.60
(s, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.90 (t, J =
7.6 Hz, 1H), 7.83 (t, J = 7.4 Hz, 2H), 7.62−7.51 (m, 1H), 7.29−7.13
(m, 1H), 4.36 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 165.4,
160.4 (d, J = 255.2 Hz), 159.8, 145.4, 135.4 (d, J = 8.7 Hz), 134.8 (d,
J = 3.6 Hz), 134.0, 132.3, 132.0, 129.5, 128.3, 126.5, 125.9, 119.61 (d,
J = 10.7 Hz), 117.47 (d, J = 22.5 Hz), 36.8. ESI-MS (positive-ion
mode). Found: m/z 299.2 ([M + H]+). Calcd for C16H12FN2O3: m/z
299.1 ([M + H]+). HPLC: tR = 12.6 min; purity = 99.6% (Figure
S12).
EXPERIMENTAL SECTION
■
Materials and Physical Measurements. Unless otherwise
noted, all of the reactions were carried out under normal atmospheric
conditions. All of the platinum-related reactions were protected from
light by aluminum foil. Cisplatin and other chemicals were purchased
from commercial suppliers. All of the solvents were used as received
without additional drying or purification.
1H, 13C, and 195Pt NMR spectra were recorded with a Bruker
AVANCE III 400 or a Bruker Ascend AVANCE II HD 600 MHz
spectrometer at room temperature. All NMR chemical shifts (δ) are
reported in parts per million (ppm) and referenced as described
below. 1H and 13C NMR spectra were referenced internally to residual
solvent peaks with the use of deuterated dimethyl sulfoxide (DMSO-
d6) as the solvent. 195Pt NMR spectra were referenced externally using
K2PtCl4 in D2O (δ = −1628 ppm vs Na2PtCl6). Electrospray
ionization mass spectrometry (ESI-MS) was performed using an
Agilent API-2000 Triple-Q MS/MS spectrometer. Elemental analysis
was carried out on a Vario Micro elemental analyzer. A Shimadzu
Prominence high-performance liquid chromatography (HPLC)
system with a Phenomenex C18 column (5 μm, 110 Å, 250 × 4.60
mm, 1 mL min−1 flow) was carried out to analyze the purity,
hydrolysis, and reduction behavior of platinum(IV) complexes. The
detectors were set at 254 and 365 nm. The platinum levels were
examined by inductively coupled plasma atomic emission spectros-
copy (PerkinElmer Optima 8000) and inductively coupled plasma
mass spectrometry (ICP-MS; PerkinElmer Nexion 2000).
The purities of all of the potential PARPi and PARPi-Pt(IV)
complexes were determined by reversed-phase HPLC (RP-HPLC)
and found to be more than 95%. Phase A is 95% H2O, 5% acetonitrile
(ACN), and 0.1% formic acid. Phase B is 95% ACN, 5% H2O, and
0.1% formic acid. Each time, a 20 μL sample (0.1 mg mL−1 in
methanol) was injected into the HPLC system. The HPLC program
was set as follows: 0% to 100% phase B (linear increase from 0 to 12
min), 100% to 0% phase B (linear decrease from 12 to 14 min), 0%
phase B (from 14 to 20 min), and then stopped at 20 min. The flow
rate was 1.0 mL min−1, and the purity was calculated based on the
results at 254 nm.
PARPi-L2-Pt(IV) was synthesized from oxoplatin (50 mg, 0.15
mmol) by using the same method as that described for PARPi-L1-
Pt(IV).
PARPi-2-Pt(IV). Yield: 69%, 64 mg (0.10 mmol). Elem anal. Calcd
for C16H17Cl2FN4O4Pt (614.32): C, 31.28; H, 2.79; N, 9.12. Found:
C, 30.96; H, 2.54; N, 8.89. 1H NMR (600 MHz, DMSO-d6): δ 12.59
(s, 1H), 8.26 (d, J = 7.0 Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.89 (t, J =
7.0 Hz, 1H), 7.83 (t, J = 7.5 Hz, 1H), 7.68 (d, J = 9.2 Hz, 1H), 7.50−
7.34 (m, 1H), 7.18−7.04 (m, 1H), 6.35−5.74 (m, 6H), 4.31 (s, 2H).
13C NMR (151 MHz, DMSO-d6): δ 171.5, 159.8, 159.6 (d, J = 252.2
Hz), 145.5, 134.0, 133.8 (d, J = 3.5 Hz), 133.1 (d, J = 8.2 Hz), 132.4,
132.0, 129.6, 128.3, 126.5, 126.0, 124.0 (d, J = 11.4 Hz), 116.8 (d, J =
22.5 Hz), 37.2. 195Pt NMR (129 MHz, DMSO-d6): δ 1042.2. ESI-MS
(positive-ion mode). Found: m/z 615.2 ([M + H]+). Calcd for
C16H18Cl2FN4O4Pt: m/z 615.0 ([M + H]+). HPLC: tR = 10.9 min;
purity = 96.0% (Figure S12).
Syntheses of Complexes PARPi-L3 and PARPi-L3-Pt(IV). The
unsubstituted compound 9 was first synthesized by exactly following
the previous report.30 Compound 9 (200 mg, 0.54 mmol) was
dissolved in 2 mL of DMF. Succinic anhydride (163 mg, 1.64 mmol)
was added to the DMF solution. The mixture was stirred at 80 °C for
12 h. After the reaction, half of DMF was removed and the residual
was added to Et2O to form a precipitate. The white precipitate was
isolated by centrifugation and further purified by column chromatog-
raphy (30:1 to 10:1 DCM/methanol).
1
PARPi-3. Yield: 50%, 128 mg (0.27 mmol). H NMR (400 MHz,
DMSO-d6): δ 12.61 (s, 1H), 12.10 (s, 1H), 8.27 (d, J = 7.7 Hz, 1H),
7.97 (d, J = 7.9 Hz, 1H), 7.90 (dd, J = 11.2 and 6.8 Hz, 1H), 7.84 (t, J
= 7.4 Hz, 1H), 7.44 (s, 1H), 7.38 (s, 1H), 7.24 (t, J = 8.6 Hz, 1H),
4.34 (s, 2H), 3.75−3.48 (m, 4H), 3.25−3.07 (m, 3H), 2.59 (s, 1H),
2.47−2.41 (m, 2H). 13C NMR (151 MHz, DMSO-d6): δ 174.4, 170.3,
164.5, 159.9, 156.8 (d, J = 240.1 Hz), 145.3, 135.3, 134.0, 132.2,
132.0, 129.6, 129.4, 128.4, 126.6, 125.9, 124.1 (d, J = 6.8 Hz), 116.4
(d, J = 21.8 Hz), 47.0, 46.7, 45.2, 44.7, 42.0, 41.8, 41.7, 41.2, 36.9,
29.4, 27.9. ESI-MS (positive-ion mode). Found: m/z 467.4 ([M +
H]+). Calcd for C24H24FN4O5: m/z 467.2 ([M + H]+). HPLC: tR =
14.1 min; purity = 96.8% (Figure S18).
Syntheses of Complexes PARPi-L1 and PARPi-L1-Pt(IV).
PARPi-L1 was synthesized by following a previous report.27
1
PARPi-L1. Yield: 77%, 342 mg (1.28 mmol). H NMR (600 MHz,
DMSO-d6): δ 13.15 (s, 1H), 12.95 (s, 1H), 8.40−8.31 (m, 1H), 8.12
(d, J = 8.3 Hz, 2H), 7.96−7.87 (m, 2H), 7.74 (d, J = 8.4 Hz, 2H),
7.72−7.64 (m, 1H). 13C NMR (151 MHz, DMSO-d6): δ 167.4, 159.7,
146.0, 139.7, 134.2, 132.3, 131.6, 130.1, 123.0, 129.2, 128.4, 126.8,
126.6. ESI-MS (positive-ion mode). Found: m/z 267.1 ([M + H]+).
Calcd for C15H11N2O3: m/z 267.1 ([M + H]+). HPLC: tR = 12.4 min;
purity = 95.5% (Figure S6).
PARPi-L1 (58 mg, 0.22 mmol) was dissolved in 2 mL of DMSO.
Then NHS (37 mg, 0.33 mmol) and EDCI (51 mg, 0.26 mmol) were
added to the DMSO solution. The mixture was stirred overnight at
room temperature. After the reaction, oxoplatin (50 mg, 0.15 mmol)
was added to the DMSO solution. The mixture was stirred at 90 °C
for 8 h. After the reaction, the unreacted oxoplatin was removed by
centrifugation, and the clear DMSO solution was added to 50 mL of
diethyl ether (Et2O). A white precipitate was formed and isolated by
centrifugation. After washing the white solid with 20 mL of Et2O
three times, PARPi-L1-Pt(IV) was obtained.
PARPi-L1-Pt(IV). Yield: 85%, 74 mg (0.13 mmol). Elem anal. Calcd
for C15H16Cl2N4O4Pt (582.30): C, 30.94; H, 2.77; N, 9.62. Found: C,
31.16; H, 2.30; N, 9.37. 1H NMR (400 MHz, DMSO-d6): δ 12.92 (s,
1H), 8.36 (d, J = 4.9 Hz, 1H), 8.05 (d, J = 7.8 Hz, 2H), 7.92 (d, J =
3.9 Hz, 2H), 7.67 (m, 3H), 6.54−5.81 (m, 6H). 13C NMR (151 MHz,
DMSO-d6): δ 173.5, 159.7, 146.5, 138.0, 135.7, 134.2, 132.2, 123.0,
129.4, 128.4, 128.2, 126.9, 126.6. 195Pt NMR (129 MHz, DMSO-d6):
δ 1038.8. ESI-MS (positive-ion mode). Found: m/z 582.9 ([M +
cis,cis,trans-[Pt(NH3)2Cl2(succinato)(benzoato)]43 (247 mg, 0.46
mmol) was dissolved in 2 mL of DMF. Compound 9 (202 mg, 0.52
mmol) and DCC (114 mg, 0.52 mmol) were then added to the DMF
solution. The mixture was stirred at room temperature for 8 h. After
the reaction, the byproduct of DCC was isolated by centrifugation
and half of DMF was vaporized in a vacuum. The residual was added
to Et2O to form a precipitate. The precipitate was collected by
centrifugation and recrystallized with methanol.
PARPi-L3-Pt(IV). Yield: 34%, 140 mg (0.16 mmol). Elem anal.
Calcd for C31H33Cl2FN6O7Pt (886.62): C, 41.99; H, 3.75; N, 9.48.
Found: C, 41.77; H, 3.68; N, 9.41. 1H NMR (600 MHz, DMSO-d6):
δ 12.60 (s, 1H), 8.27 (d, J = 2.5 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H),
7.94−7.77 (m, 4H), 7.53 (t, J = 7.2 Hz, 1H), 7.43 (d, J = 7.1 Hz, 3H),
7.39 (s, 1H), 7.25 (t, J = 9.9 Hz, 1H), 6.61 (s, 6H), 4.34 (s, 2H),
3.76−3.49 (m, 4H), 3.40 (m, 2H), 3.17 (m, 2H), 2.52 (m, 4H). 13C
NMR (151 MHz, DMSO-d6): δ 180.6, 173.8, 170.9, 164.6, 164.5,
159.9, 156.8 (d, J = 244.6 Hz), 145.3, 135.3, 134.0, 133.6, 132.2 (d, J
= 8.0 Hz), 132.1, 132.0, 129.8, 129.6, 129.4 (d, J = 2.8 Hz), 128.4,
126.6, 125.9, 124.1 (d, J = 17.9 Hz), 116.4 (d, J = 21.7 Hz), 47.0,
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DOI: 10.1021/acs.inorgchem.9b02839
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Inorganic Chemistry
Article
46.7, 45.4, 45.0, 42.0, 41.8, 41.7, 41.3, 36.9, 31.3, 29.1. 195Pt NMR
(129 MHz, DMSO-d6): δ 1212.3. ESI-MS (positive-ion mode).
Found: m/z 909.2 ([M + Na]+). Calcd for C31H33Cl2FN6O7PtNa: m/
z 909.1 ([M + Na]+). HPLC: tR = 12.7 min; purity = 97.2% (Figure
S18).
Syntheses of Complexes PARPi-L6 and PARPi-L6-Pt(IV). 2-
Aminobenzamide (200 mg, 1.47 mmol), ethyl 3-formylphenoxyace-
tate (285 mg, 1.47 mmol), iodine (447 mg, 1.76 mmol), and sodium
carbonate (330 mg, 1.76 mmol) were dissolved in 5 mL of DMF. The
mixture was stirred at 70 °C for 6 h. After that, 30 mL of a saturated
NaHSO3 solution was added to the mixture. A white precipitate
formed immediately and was isolated by centrifugation. The
precipitate was then added to a NaOH aqueous solution (2.5 M,
15 mL). The solution was heated at 60 °C for 6 h. After the addition
of an excess amount of HCl solution (6 M), the precipitate was
collected by centrifugation. The final product was obtained as a white
solid after the precipitate was dried in a vacuum.
Syntheses of Complexes PARPi-L4 and PARPi-L4-Pt(IV).
PARPi-L4 was synthesized by modification of the reported
methods.34,35 2-Aminobenzamide (200 mg, 1.47 mmol) and 4-
cyanobenzoyl chloride (267 mg, 1.62 mmol) were dissolved in 20 mL
of DCM. The mixture was stirred overnight at room temperature, and
a white precipitate formed during this period. The precipitate was
collected by centrifugation and then dissolved in 15 mL of ethanol
with NaOH (59 mg, 1.47 mmol). The ethanol solution was refluxed
for 8 h. After that, the ethanol was removed in a vacuum, and a NaOH
aqueous solution (2.5 M, 15 mL) was added to the residual. The
solution was heated for another 6 h. After neutralization with a HCl
solution (6 M), the final product was obtained as a white precipitate.
1
PARPi-L6. Yield: 50%, 220 mg (0.74 mmol). H NMR (600 MHz,
DMSO-d6): δ 13.13 (s, 1H), 12.56 (s, 1H), 8.16 (s, 1H), 7.98−7.65
(m, 4H), 7.51 (d, J = 38.4 Hz, 2H), 7.16 (s, 1H), 4.83 (s, 2H). 13C
NMR (151 MHz, DMSO-d6): δ 170.5, 162.8, 158.4, 152.3, 149.1,
135.1, 134.4, 130.3, 128.0, 127.2, 126.3, 121.5, 121.1, 118.6, 113.6,
65.2. ESI-MS (positive-ion mode). Found: m/z 297.1 ([M + H]+).
Calcd for C16H13N2O4: m/z 297.1 ([M + H]+). HPLC: tR = 14.7 min;
purity = 97.3% (Figure S36).
PARPi-L6-Pt(IV) was synthesized from oxoplatin (50 mg, 0.15
mmol) by using the same method as that described for PARPi-L1-
Pt(IV).
PARPi-L6-Pt(IV). Yield: 85%, 78 mg (0.13 mmol). Elem anal. Calcd
for C16H18Cl2N4O5Pt (612.33): C, 31.38; H, 2.96; N, 9.15. Found: C,
31.06; H, 2.93; N, 9.05. 1H NMR (600 MHz, DMSO-d6): δ 8.16 (d, J
= 7.9 Hz, 1H), 7.85 (t, J = 8.4 Hz, 1H), 7.79 (dd, J = 14.5 and 8.0 Hz,
2H), 7.74 (s, 1H), 7.53 (t, J = 8.0 Hz, 1H), 7.43 (t, J = 8.0 Hz, 1H),
7.15 (dd, J = 8.2 and 2.3 Hz, 1H), 6.31−5.73 (m, 6H), 4.67 (s, 2H).
13C NMR (151 MHz, DMSO-d6): δ 176.0, 162.8, 158.9, 152.5, 149.2,
135.1, 134.3, 130.1, 128.1, 127.1, 126.3, 121.5, 120.7, 118.7, 114.0,
66.4. 195Pt NMR (129 MHz, DMSO-d6): δ 1048.7. ESI-MS (positive-
ion mode). Found: m/z 613.0 ([M + H]+). Calcd for
C16H19Cl2N4O4Pt: m/z 613.0 ([M + H]+). HPLC: tR = 12.9 min;
purity = 96.7% (Figure S36).
1
PARPi-L4. Yield: 62%, 242 mg (0.91 mmol). H NMR (600 MHz,
DMSO-d6): δ 12.17 (s, 1H), 8.28 (d, J = 8.1 Hz, 2H), 8.17 (d, J = 7.7
Hz, 1H), 8.09 (d, J = 8.1 Hz, 2H), 7.87 (t, J = 7.4 Hz, 1H), 7.82 (d, J
= 7.9 Hz, 1H), 7.56 (t, J = 7.3 Hz, 1H). 13C NMR (151 MHz,
DMSO-d6): δ 167.1, 162.5, 152.5, 148.2, 136.4, 135.2, 133.7, 129.8,
128.7, 127.6, 127.5, 126.4, 121.5. ESI-MS (positive-ion mode).
Found: m/z 267.1 ([M + H]+). Calcd for C15H11N2O3: m/z 267.1
([M + H]+). HPLC: tR = 13.0 min; purity = 98.1% (Figure S24).
PARPi-L4-Pt(IV) was synthesized from oxoplatin (80 mg, 0.24
mmol) by using the same method as that described for PARPi-L1-
Pt(IV).
PARPi-L4-Pt(IV). Yield: 67%, 93 mg (0.16 mmol). Elem anal. Calcd
for C15H16Cl2N4O4Pt (582.30): C, 30.94; H, 2.77; N, 9.62. Found: C,
31.21; H, 2.57; N, 9.72. 1H NMR (400 MHz, DMSO-d6): δ 12.58 (s,
1H), 8.22 (d, J = 8.0 Hz, 2H), 8.18 (d, J = 7.8 Hz, 1H), 8.01 (d, J =
8.0 Hz, 2H), 7.86 (t, J = 7.6 Hz, 1H), 7.78 (d, J = 8.1 Hz, 1H), 7.55
(t, J = 7.4 Hz, 1H), 6.56−5.65 (m, 6H). 13C NMR (151 MHz,
DMSO-d6): δ 173.2, 162.6, 152.4, 149.1, 137.7, 135.4, 135.1, 129.9,
128.1, 127.8, 127.3, 126.4, 121.6. 195Pt NMR (129 MHz, DMSO-d6):
δ 1036.9. ESI-MS (positive-ion mode). Found: m/z 582.8 ([M +
H]+). Calcd for C15H17Cl2N4O4Pt: m/z 583.0 ([M + H]+). HPLC: tR
= 11.1 min; purity = 97.4% (Figure S24).
Syntheses of Complexes PARPi-L7 and PARPi-L7-Pt(IV).
PARPi-L7 was synthesized from 17 (360 mg, 2.00 mmol) by using
the same method as that described for PARPi-L5.
1
PARPi-L7. Yield: 57%, 340 mg (1.15 mmol). H NMR (600 MHz,
Syntheses of Complexes PARPi-L5 and PARPi-L5-Pt(IV). 2-
Aminobenzamide (272 mg, 2.00 mmol), 15 (360 mg, 2.00 mmol),
iodine (608 mg, 2.40 mmol), and sodium carbonate (330 mg, 2.40
mmol) were dissolved in 5 mL of DMF. The mixture was stirred at 70
°C for 6 h. After that, 30 mL of a saturated NaHSO3 solution was
added to the mixture. A white precipitate formed immediately and
was isolated by centrifugation. After washing twice with 30 mL of
water and acetone, the final product was obtained as a white solid.
DMSO-d6): δ 13.44 (s, 1H), 12.13 (s, 1H), 8.17 (d, J = 6.9 Hz, 1H),
7.92 (d, J = 6.2 Hz, 1H), 7.86 (t, J = 7.6 Hz, 1H), 7.74 (d, J = 8.0 Hz,
1H), 7.55 (t, J = 7.5 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 7.5
Hz, 1H), 4.92 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 171.2,
161.6, 156.2, 152.3, 149.2, 135.0, 133.1, 131.4, 127.7, 127.1, 126.3,
122.7, 122.1, 121.4, 114.1, 66.2. ESI-MS (positive-ion mode). Found:
m/z 319.1 ([M + Na]+). Calcd for C16H12N2O4Na: m/z 319.1 ([M +
Na]+). HPLC: tR = 18.5 min; purity = 98.5% (Figure S42).
PARPi-L7-Pt(IV) was synthesized from oxoplatin (50 mg, 0.15
mmol) by using the same method as that described for PARPi-L1-
Pt(IV).
PARPi-L7-Pt(IV). Yield: 73%, 67 mg (0.11 mmol). Elem anal. Calcd
for C16H18Cl2N4O5Pt (612.33): C, 31.38; H, 2.96; N, 9.15. Found: C,
31.01; H, 2.93; N, 9.39. 1H NMR (600 MHz, DMSO-d6): δ 8.16 (d, J
= 6.7 Hz, 1H), 7.99 (dd, J = 7.7 and 1.7 Hz, 1H), 7.86 (t, J = 8.4 Hz,
1H), 7.75 (d, J = 8.1 Hz, 1H), 7.54 (dd, J = 15.8 and 8.6 Hz, 2H),
7.34 (d, J = 8.4 Hz, 1H), 7.17 (t, J = 7.5 Hz, 1H), 6.40−5.77 (m, 6H),
4.86 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 177.2, 161.6, 156.7,
152.1, 149.5, 135.0, 133.2, 131.3, 128.0, 127.1, 126.3, 122.5, 122.1,
121.4, 115.0, 67.6. 195Pt NMR (129 MHz, DMSO-d6): δ 1051.9. ESI-
MS (positive-ion mode). Found: m/z 613.1 ([M + H]+). Calcd for
C16H19Cl2N4O4Pt: m/z 613.0 ([M + H]+). HPLC: tR = 12.0 min;
purity = 97.9% (Figure S42).
1
PARPi-L5. Yield: 71%, 420 mg (1.41 mmol). H NMR (600 MHz,
DMSO-d6): δ 13.13 (s, 1H), 12.43 (s, 1H), 8.18 (d, J = 8.9 Hz, 2H),
8.14 (dd, J = 7.8 and 0.8 Hz, 1H), 7.82 (t, J = 7.6 Hz, 1H), 7.71 (d, J
= 8.1 Hz, 1H), 7.49 (t, J = 7.5 Hz, 1H), 7.08 (d, J = 8.9 Hz, 2H), 4.81
(s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 170.4, 162.8, 160.8,
152.3, 149.4, 135.0, 129.9, 127.8, 126.6, 126.3, 125.8, 121.2, 115.0,
65.0. ESI-MS (positive-ion mode). Found: m/z 297.1 ([M + H]+).
Calcd for C16H13N2O4: m/z 297.1 ([M + H]+). HPLC: tR = 14.7 min;
purity = 96.8% (Figure S30).
PARPi-L5-Pt(IV) was synthesized from oxoplatin (50 mg, 0.15
mmol) by using the same method as that described for PARPi-L1-
Pt(IV).
PARPi-L5-Pt(IV). Yield: 76%, 70 mg (0.11 mmol). Elem anal. Calcd
for C16H18Cl2N4O5Pt (612.33): C, 31.38; H, 2.96; N, 9.15. Found: C,
1
31.16; H, 2.97; N, 8.89. H NMR (600 MHz, DMSO-d6): δ 8.22−
8.04 (m, 3H), 7.82 (t, J = 8.4 Hz, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.49
(t, J = 8.0 Hz, 1H), 7.07 (d, J = 8.9 Hz, 2H), 6.30−5.73 (m, 6H), 4.64
(s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 175.8, 162.8, 161.4,
152.4, 149.4, 135.0, 129.7, 127.8, 126.6, 126.3, 125.3, 121.1, 115.2,
66.3. 195Pt NMR (129 MHz, DMSO-d6): δ 1053.9. ESI-MS (positive-
ion mode). Found: m/z 613.1 ([M + H]+). Calcd for
C16H19Cl2N4O4Pt: m/z 613.0 ([M + H]+). HPLC: tR = 12.8 min;
purity = 96.9% (Figure S30).
HPLC Analysis of Platinum(IV) Complexes. The HPLC
physical settings, program, and conditions were exactly the same as
the descriptions in the purity test.
Stability Test. Platinum(IV) complexes (50 μM) in PBS buffer
(pH = 7.4) or fetal bovine serum (FBS, pH = 7.4) were incubated at
37 °C in the dark. The samples in PBS buffer were directly injected
into the RP-HPLC system to analyze the remaining platinum(IV)
complexes for 24 h. For samples in FBS, 400 μL of methanol was
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added to 100 μL of FBS containing the platinum(IV) complexes to
precipitate protein at the relevant time points. After centrifugation
and filtering, the supernatant was injected into the RP-HPLC system
to analyze the remaining platinum(IV) complexes. The results are
shown in Figure S43.
Reduction Test. Platinum(IV) complexes (50 μM) in PBS buffer
(pH = 7.4, with 1 mM ascorbate) were incubated at 37 °C in the dark.
Every 2 h for 12 h, samples were injected into the RP-HPLC system
to analyze the remaining platinum(IV) complexes in a PBS solution.
The results are shown in Figure S43.
colorimetric substrate was added to each well and incubated in the
dark for 15 min at room temperature. The reaction was stopped by
adding 50 μL of 0.2 M HCl. The absorbance was recorded at 450 nm.
Cellular Accumulation. A2780 and A2780cisR cells were plated
in 6-well plates and allowed to attach for 24 h. Cells were exposed to
10 μM cisplatin or PARPi-Pt for 8 h. Cells were washed with PBS
buffer three times, scraped, and resuspended in PBS buffer. The cell
number in each sample was determined by a hemocytometer. Cells
were then washed with PBS another time and digested with 65%
HNO3 at 65 °C overnight. The platinum content was determined by
ICP-MS (PE Nexion 2000).
Genomic DNA. A2780 and A2780cisR cells were plated in 6-well
plates and allowed to attach for 24 h. Cells were exposed to 10 μM
cisplatin or PARPi-Pt for 8 h. Cells were washed with PBS buffer three
times, scraped, and resuspended in PBS buffer. Cells were finally
collected by centrifugation. Genomic DNA was isolated using a
GeneJET Genomic DNA Purification Kit following a given protocol.
The DNA content was determined by NanoDrop. The platinum
content was determined by ICP-MS (PE Nexion 2000) after
digestion.
Apoptosis. A2780 and A2780cisR cells were plated in 6-well
plates and allowed to attach for 24 h. Cells were exposed to 10 μM
cisplatin, cisplatin + PARPi-L6 (1:1, mol/mol), cisplatin + PARPi-L7
(1:1, mol/mol), PARPi-L6-Pt, or PARPi-L7-Pt for 24 h. Cells were
then collected by trypsinization, washed once with PBS buffer, washed
twice with Annexin V binding buffer (10 mM HEPES, 140 mM NaCl,
2.5 mM CaCl2, pH = 7.4), and resuspended in Annexin V binding
buffer with a cell density of 1 × 106 cells mL−1. A total of 5 μL of
Annexin V FITC and 1 μL of 7-AAD were added to each sample.
Cells were incubated at room temperature for 15 min, diluted five
times with Annexin V binding buffer, and analyzed by a FACS Calibur
flow cytometer.
Cell Cycle. A2780 and A2780cisR cells were plated in 6-well plates
and allowed to attach for 24 h. Cells were exposed to 10 μM cisplatin,
cisplatin + PARPi-L6 (1:1, mol/mol), cisplatin + PARPi-L7 (1:1,
mol/mol), PARPi-L6-Pt, or PARPi-L7-Pt for 24 h. Cells were then
collected by trypsinization, washed twice with PBS buffer, and fixed
with 70% ethanol at 4 °C overnight. Cells were then washed with PBS
another time and resuspended in a propidium iodide (PI) solution
(0.1% Triton X-100, 200 μg mL−1 RNase A, 20 μg mL−1 PI). Cells
were stained for 15 min at 37 °C, and the cell cycle distribution was
analyzed by a FACS Calibur flow cytometer.
Reduction Potential. Because of the low aqueous solubility of
these PARPi-Pt(IV) conjugates, cyclic voltammetry was carried out in
a DMF solution containing 0.1 mol L−1 (n-Bu4N)PF6 as the
supporting electrolyte in a three-electrode cell at room temperature.
Ferrocene was added as an internal reference, and the scan rate was
set as 100 mV s−1. Then all of the initial results (vs Ag/Ag+) were
recalculated versus a normal hydrogen electrode and are listed in
Table 1. The original results are shown in Figures S44−S50.
Cell Lines and Cell Culture Conditions. Human lung carcinoma
A549 cells, cisplatin-resistant A549cisR cells, human breast MDA-
MB-231 cells, and MCF-7 cells were cultured in a Dulbecco’s
modified Eagle’s medium (DMEM) with 10% FBS and 100 units
penicillin/streptomycin. Human ovarian A2780 and cisplatin-resistant
A2780cisR cells were cultured in RPMI 1640 with 10% FBS and 100
units penicillin/streptomycin. Human breast MDA-MB-436 cells were
cultured in Leibovitz’s L-15 with 10% FBS, 10 μg mL−1 insulin, and
16 μg mL−1 glutathione. Human fetal lung fibroblasts WI-38 were
cultured in MEM with 10% FBS, 1% glutamine, 1% nonessential
amino acids, and 1% sodium pyruvate. For resistant cells, 5 μM
cisplatin was added into the culture medium after attachment to
maintain the resistance. All of the cells except MDA-MB-436 were
cultured at 37 °C in 5% CO2. MDA-MB-436 were cultured without
gas exchange.
Cytotoxicity Test. The cytotoxicities of the PARPi ligands and
PARPi-Pt(IV) conjugates were analyzed by MTT assay. Cells were
plated in 96-well plates and allowed to attach for 24 h. Cells were
incubated with a medium containing drugs and 1% DMF for 72 h. For
the WI-38 cell line, a medium without DMF was used because of the
high toxicity of DMF to the WI-38 cells. After exposure, the medium
was replaced by a FBS-free medium with 1 mg mL−1 MTT. Cells were
incubated for 4 h. The medium was then removed, and DMSO was
added to each well. The absorbance was measured at 570 and 630 nm.
For the MDA-MB-436 cells, a mixed medium of Leibovitz’s L-15 and
RPMI 1640 (1:1, v/v) was used.
PARP Enzyme Inhibition Assay. The ability of the ligands and
platinum(IV) compound to inhibit PARP was determined using a
Trevigen PARP Universal Colorimetric assay kit. This kit provides
recombinant human PARP-1 as the enzyme, activated DNA as the
substrate, and a histone protein-coated 96-well strip. The presence of
inhibitors blocks the PARP-catalyzed addition of poly(ADP-ribose) to
histone proteins. The incorporation amount of biotinylated poly-
(ADP-ribose) was then detected by a horseradish peroxidase substrate
TACS-Sapphire, which is readable at 630 nm (blue) or at 450 nm
(yellow) after stopping the reaction with 0.2 M HCl to indicate the
activity of the PARP-1 enzyme.
Briefly, 50 μL of 1X PARP buffer was added to each well and
incubated at room temperature for 0.5 h to rehydrate the histones.
Then a compound diluted in PARP buffer (2.5% DMF) was added,
followed by the diluted PARP enzyme. A negative control without
PARP was used to determine the background absorbance, and a
positive control without an inhibitor was used as the reference. 3-
Aminobenzamide (3-AB; provided at 200 mM in ethanol) was used as
a control inhibitor. A total of 25 μL of 1X PARP cocktail was then
added into each well, and the plate was incubated at room
temperature for 1 h. The plate was washed twice with 1X PBS +
0.1% Triton X-100, followed by two washes with 1X PBS. A total of
50 μL of diluted Strep/horseradish peroxidase was added, and the
plate was incubated at room temperature for 60 min. The plate was
washed twice with 1X PBS + 0.1% Triton X-100, followed by two
washes with 1X PBS. A total of 50 μL of a prewarmed TACS-Sapphire
Western Blotting. A2780 cells (1 × 106 cells) were cultured in 10
cm culture dishes for 24 h. Complexes were added to the culture for
12 h (1 μM for PARPi-L5-Pt(IV) and PARPi-L6-Pt(IV) complexes,
10 μM for all other complexes). After incubation, the cells were
washed with PBS twice, and then the cells were lysed and scraped.
Then a lysate solution was collected by centrifugation, and the protein
concentration in the solution was determined by BSA assay. After
that, the solution was mixed with a loading buffer [Laemmli buffer
recipe, 4% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol,
20% glycerol, 0.004% bromophenol blue, and 0.125 M Tris-HCl, pH
= 6.8]. SDS polyacrylamide gel electrophoresis was applied, and then
proteins were transferred to the poly(vinylidene difluoride)
membrane at 100 V for 90 min. Subsequently, the membrane was
blocked by 5% (w/v) nonfat milk powder in a Tris-buffered saline/
0.1% Tween 20 (TBST) buffer for 2 h at room temperature and
washed twice with TBST. The membrane was incubated with PARP-1
(CST, #9532) and β-actin (Abcam, ab8227) primary antibodies for
12 h at 4 °C, then washed with TBST three times, and incubated with
the corresponding secondary antibody for 2 h at room temperature.
After that, the membrane was washed twice with TBST and incubated
with an enhanced chemiluminescence (Bio-Rad) solution for 5 min.
Then, the membrane was imaged with a Bio-Rad ChemiDoc Touch
Imaging System.
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DOI: 10.1021/acs.inorgchem.9b02839
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02839.
■
S
(12) Wang, Y.-Q.; Wang, P.-Y.; Wang, Y.-T.; Yang, G.-F.; Zhang, A.;
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Novel, Highly Potent, and Orally Efficacious Poly(ADP-ribose)
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NMR spectra and HPLC chromatograms of all of the
newly synthesized complexes, stability and reduction of
all of the platinum(IV) complexes, reduction potentials
of all of the platinum(IV) complexes, apoptosis analysis
and cell cycle distribution of A2780 and A2780cisR cells
upon treatment with PARPi-Pt(IV) complexes, expres-
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PARPi-Pt(IV) complexes, and cytotoxicity of a mixture
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AUTHOR INFORMATION
Corresponding Author
*E-mail: guangzhu@cityu.edu.hk.
ORCID
■
Guangyu Zhu: 0000-0002-4710-7070
Author Contributions
§These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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Mechanisms of Platinum Drugs Resistance. Trends Pharmacol. Sci.
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We thank the National Natural Science Foundation of China
(Grant 21877092), the Hong Kong Research Grants Council
(CityU 11304318 and 11307419), and the City University of
Hong Kong (Project 9667148) for funding support.
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