Rollover Cyclometalated Bipyridine Platinum Complexes as Potent Anticancer Agents: Impact of the Ancillary Ligands on the Mode of Action

Article abstract of DOI:10.1021/acs.inorgchem.7b03210

Platinum-based anticancer coordination compounds are widely used in the treatment of many tumor types, where they are very effective but also cause severe side effects. Organoplatinum compounds are significantly less investigated than the analogous coordination compounds. We report here rollover cyclometalated Pt compounds based on 2,2′-bipyridine which are demonstrated to be potent antitumor agents both in vitro and in vivo. Variation of the co-ligands on the Pt(2,2′-bipyridine) backbone resulted in the establishment of structure-activity relationships. They showed that the biological activity was in general inversely correlated with the reaction kinetics to biomolecules as shown for amino acids, proteins, and DNA. The less stable compounds caused higher reactivity with biomolecules and were shown to induce p53-dependent DNA damage. In contrast, the presence of bulky PTA and PPh₃ ligands was demonstrated to cause lower reactivity and increased antineoplastic activity. Such compounds were devoid of DNA-damaging activity and induced ATF4, a component of the endoplasmic reticulum (ER) stress pathway. The lead complex inhibited tumor growth similar to oxaliplatin while showing no signs of toxicity in test mice. Therefore, we demonstrated that it is possible to fine-tune rollover-cyclometalated Pt(II) compounds to target different cancer pathways and be a means to overcome the side effects associated with cisplatin and analogous compounds in cancer chemotherapy.

Full text of DOI:10.1021/acs.inorgchem.7b03210

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Rollover Cyclometalated Bipyridine Platinum Complexes as Potent
Anticancer Agents: Impact of the Ancillary Ligands on the Mode of
Action
Maria V. Babak,*,†,‡,§ Martin Pfaffeneder-Kmen,∥ Samuel M. Meier-Menches,⊥ Maria S. Legina,†
Sarah Theiner,⊥,# Cynthia Licona,∇ Christophe Orvain,∇ Michaela Hejl,† Muhammad Hanif,§
Michael A. Jakupec,†,# Bernhard K. Keppler,†,# Christian Gaiddon,∇ and Christian G. Hartinger*,§
†Institute of Inorganic Chemistry, University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria
‡Department of Chemistry, National University of Singapore, 3 Science Drive 2, 117543 Singapore
§School of Chemical Sciences, University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
∥Institute of Physical Chemistry, University of Vienna, Waehringer Str. 42, 1090 Vienna, Austria
⊥Department of Analytical Chemistry, University of Vienna, Waehringer Str. 38, 1090 Vienna, Austria
#Research Cluster Translational Cancer Therapy Research, University of Vienna, 1090 Vienna, Austria
∇Inserm UMR_S1113, Signalisation molec
́ ́
ulaire du stress cellulaire et pathologies, Universite de Strasbourg, 67200 Strasbourg,
France
S
* Supporting Information
ABSTRACT: Platinum-based anticancer coordination com-
pounds are widely used in the treatment of many tumor types,
where they are very effective but also cause severe side effects.
Organoplatinum compounds are significantly less investigated
than the analogous coordination compounds. We report here
rollover cyclometalated Pt compounds based on 2,2′-
bipyridine which are demonstrated to be potent antitumor
agents both in vitro and in vivo. Variation of the co-ligands on
the Pt(2,2′-bipyridine) backbone resulted in the establishment
of structure−activity relationships. They showed that the
biological activity was in general inversely correlated with the
reaction kinetics to biomolecules as shown for amino acids,
proteins, and DNA. The less stable compounds caused higher reactivity with biomolecules and were shown to induce p53-
dependent DNA damage. In contrast, the presence of bulky PTA and PPh3 ligands was demonstrated to cause lower reactivity
and increased antineoplastic activity. Such compounds were devoid of DNA-damaging activity and induced ATF4, a component
of the endoplasmic reticulum (ER) stress pathway. The lead complex inhibited tumor growth similar to oxaliplatin while showing
no signs of toxicity in test mice. Therefore, we demonstrated that it is possible to fine-tune rollover-cyclometalated Pt(II)
compounds to target different cancer pathways and be a means to overcome the side effects associated with cisplatin and
analogous compounds in cancer chemotherapy.
tigation of Pt(II) complexes with 2,2′-bipyridine (bipy) and its
derivatives.9−11 The presence of bipy might result in DNA
intercalation of the complex in addition to the covalent
platination of guanine residues at DNA, establishing a dual
DNA-binding mode.12,13 Another advantage of bipy is its high
lipophilicity which facilitates and improves the cellular uptake
and accumulation as compared to cisplatin. Introduction of a
2,2′-bipyridine ligand resulted in significantly increased levels of
cell lethality in prostate cancer and melanoma cell lines.14,15 In
addition, various PtII complexes with bipy derivatives induced
INTRODUCTION
■
Since cisplatin was introduced into oncological practice in
1978, it has made a significant contribution to the successful
treatment of cancer patients. However, its severe side-effects
prompted the development of platinum- and other transition-
metal-based compounds with improved chemotherapeutic
potential.1−6 Nowadays, there are numerous chemical libraries
of antiproliferative Pt compounds available but only a few drug
candidates made their way from bench to clinic.7 As the
structure of cisplatin contains ammine ligands as nonleaving
groups, numerous complexes with ligands with nitrogen donors
were prepared, and structure−activity relationships were
established.8 Several research groups focused on the inves-
Received: December 28, 2017
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
a
20
Scheme 1. Synthesis of Cyclometalated Pt(II) Complexes with 2,2′-Bipyridine from K2PtCl4
a
(i) DMSO, H2O, RT; (ii) Sn(CH3)4, DMSO, 80 °C; (iii) 2,2′-bipyridine, toluene, 110 °C; (iv) HCl, acetone, DMSO, RT.
apoptosis in cancer cell lines,14,15 while the encapsulation of
perfluoroalkylated bipy Pt complexes into liposomes did not
result in enhanced cytotoxicity.16 The advantages of using bipy
have also been illustrated with other metals, such as Ru-based
complexes displaying DNA intercalation17 or with Os-based
complexes inducing higher cytotoxicity that correlates with
higher lipophilicity.18
Typically bipy coordinates to metal centers through the two
N donor atoms in a N,N′-bidentate fashion.19 However, under
certain conditions, the C−H bond of one pyridine ring might
be activated leading to the formation of Pt(II) cyclometalated
complexes, where bipy is coordinated in N,C-mode (Scheme
1).20 These so-called rollover Pt complexes were discovered by
Minghetti et al. in 1999,21 and within the past decade they were
extensively investigated by Zucca et al.22−28
The research on cyclometalated Pt(II) complexes with
anticancer properties has focused on N,C-coordinated bipyr-
idine,29 phenylpyridine,30−35 COD,36 and other li-
gands.12,13,37−42 These complexes displayed high cytotoxicity
in cisplatin-resistant cancer models, interacted with DNA, and
several examples showed promising in vivo activity.43 N,C-
coordination seems to have beneficial effects on the biological
activity of Pt(II) complexes, probably due to their improved
stability and strong Pt−C σ-bond. However, in case of the
tridentate N,C,N′- and C,N,N′-coordinated ligands 1,3-di(2-
pyridyl)benzene and 6-phenyl-2,2′-bipyridine, correspondingly,
the biological activity was strongly dependent on the
coordination pattern at the Pt center and the trans effect of
the carbon donors.44 Based on these observations, we expected
that the flipped N,C-coordinated bipy ligand in Pt(II)
complexes would open the possibility to fine-tune the
mechanism of action of the complexes and control their
biological activity through the positioning of different ligands
trans to the carbon or nitrogen atoms of N,C-coordinated
bipyridine. The release of these ligands would cause a change in
charge state and alter the physiochemical properties of the
complexes as well as their affinity to biomolecules. Therefore,
cyclometalated rollover Pt(II) complexes of N,C-coordinated
bipyridine were prepared, and studies on their stability,
cytotoxicity, cellular accumulation, interactions with biomole-
cules, effects on protein expression as well as in vivo
experiments in mouse tumor models are reported.
chem and L-methionine from Sigma-Aldrich, methanol (HPLC grade)
from Fisher, formic acid, L-glutamic acid, and L-cysteine from Fluka,
and L-histidine from Merck. Milli-Q water was taken from an
Advantage A10 (18.2 MΩ, 185 UV Ultrapure water system, Millipore,
Molsheim, France). Elemental analyses were performed by the
Microanalytical Laboratory of the Faculty of Chemistry of the
1
University of Vienna. The H, 31P, 195Pt NMR spectra were recorded
at 500.10, 202.44, and 107.33 MHz, respectively, on a Bruker FT
NMR spectrometer Avance II 500 MHz. Chemical shifts are given in
parts per million (ppm) relative to the residual solvent peak.
The X-ray intensity data were measured on Bruker D8 Venture
diffractometer equipped with multilayer monochromator, Mo−Kα
INCOATEC micro focus sealed tube and Kryoflex cooling device. The
structure was solved by direct methods and refined by full-matrix least-
squares techniques. Non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were inserted
at calculated positions and refined with a riding model. The following
software was used: Bruker SAINT software package48 using a narrow-
frame algorithm for frame integration, SADABS49 for absorption
correction, OLEX250 for structure solution, refinement, molecular
diagrams, and graphical user-interface, Shelxle51 for refinement and
graphical user-interface SHELXS-201352 for structure solution,
SHELXL-201353 for refinement, and Platon54 for symmetry check,
molecular diagrams, Mercury 3.0. Experimental data and CCDC codes
can be found in Table S1.
Cell Lines and Culture Conditions. CH1(PA-1) cells (identified
via STR profiling as PA-1 ovarian teratocarcinoma cells by Multi-
plexion, Heidelberg, Germany; compare Korch et al.55) were obtained
from Lloyd R. Kelland, CRC Centre for Cancer Therapeutics, Institute
of Cancer Research, Sutton, U.K. A549 (human non-small cell lung
cancer) and SW480 (human colon carcinoma) cells were supplied by
Brigitte Marian (Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Austria). HCT116 human
colon cancer cells were obtained from ATCC. All cell culture media
and reagents were purchased from Sigma-Aldrich, Austria, unless
noted otherwise, and plastic ware was obtained from Starlab, Germany.
Adherent cell cultures were grown in 75 cm2 culture flasks in complete
medium [i.e., minimal essential medium (MEM) supplemented with
10% heat-inactivated fetal bovine serum (Invitrogen), 1 mM sodium
pyruvate, 4 mM L-glutamine, and 1% v/v nonessential amino acids
from 100× ready-to-use stock]. HCT116 cell line was cultured in
Dulbecco’s modified minimal essential medium (DMEM) containing
10% fetal bovine serum (Dominique Dutcher) and 1% penicillin +
streptomycin (Sigma). All cell lines were grown at 37 °C in a
humidified atmosphere of 95% air and 5% CO2. Experiments were
performed on cells within 20 passages.
Cytotoxicity Assay in Cancer Cell Lines. The cytotoxicity of the
compounds was determined by means of a colorimetric microculture
assay (MTT assay, MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide). The cells were harvested from culture flasks
by trypsinization and seeded in densities of 1 × 103 [CH1(PA-1)], 2 ×
103 (SW480), 3 × 103 (A549), and 5 × 103 cells (for HCT116) in 100
μL/well aliquots in 96-well microculture plates. After the cells were
allowed to resume exponential growth for 24 h, the test compounds
were dissolved in DMSO, then diluted in MEM, and added to the
plates where the final DMSO content did not exceed 0.5%. After
exposure for 96 h (48 h in case of HCT116), drug solutions were
replaced with 100 μL/well of a 1/7 MTT/RPMI 1640 solution (MTT
solution, 5 mg/mL of MTT reagent in phosphate-buffered saline;
RPMI 1640 medium, supplemented with 10% heat-inactivated fetal
EXPERIMENTAL SECTION
■
Materials and Methods. Materials from chemical suppliers were
used as received, and all reactions were carried out under argon
atmosphere in anhydrous solvents in darkness. The products were
isolated without special precautions. 1,3,5-Triaza-7-phosphaadaman-
tane (PTA),45 cis-[Pt(Me)2(DMSO)2],46 1−3, 7, and 820 were
prepared according to literature procedures. Dichloromethane was
dried and distilled over CaH2 under argon atmosphere. Toluene was
distilled under argon atmosphere.47 2,2′-Bipyridine was purchased
from Alfa Aesar, dimethyl sulfoxide, guanosine-5′-triphosphate,
ubiquitin (bovine erythrocytes) and horse heart cytochrome C from
Sigma, tert-butyl isocyanide, adenosine-5′-triphosphate from Calbio-
B
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
bovine serum and 4 mM L-glutamine). All manipulations with
HCT116 cells were performed in DMEM. After incubation for 4 h
at 37 °C, the MTT/RPMI 1640 or MTT/DMEM mixtures were
removed, and the formazan crystals formed in viable cells were
dissolved in 150 μL of DMSO per well. Optical densities were
measured at 550 nm with a microplate reader (Biotek ELx808), by
using a reference wavelength of 690 nm to correct for unspecific
absorption. The quantity of viable cells was expressed in terms of
treated/control (T/C) values by comparison to untreated control
microcultures, and 50% inhibitory concentrations (IC50) were
calculated from concentration−effect curves by interpolation. Evalua-
tion was based on means from at least three independent experiments,
each comprising three replicates per concentration level.
nitrocellulose membrane. Equal loading of protein was confirmed by
comparison with actin. Immunoblotting was done with anti-p53
(rabbit anti-p53, FL-393, Santa Cruz, CA), anti-ATF4 (anti-CREB-2
(B-3): sc-390063), and antiactin (rabbit anti- β-actin, Sigma)
antibodies. The protein bands were visualized via enhanced
chemiluminescence imaging (PXi, Syngene).
Animal Studies. The toxicity studies were performed in female
Balb/C mice. Complex 1 was administered intraperitoneally to three
groups of mice (3 mice per group) every 4 days over 12 days, and after
that the mice were observed for 8 days. Mice in group 1 were injected
with a drug dose of 4 μmol/kg on days 1 and 4 and 15 μmol/kg on
days 8 and 12. Mice in group 2 were injected with a drug dose of 8
μmol/kg on days 1 and 4 and 12 μmol/kg on days 8 and 12. Mice in
group 3 were injected with 10 μmol/kg only on days 1, 4, and 8.
Mouse survival and body weight variations were monitored daily for
20 days in all groups. The maximum tolerated dose (MTD) was
defined as the highest dose that induced no more than 15% weight loss
vs control, caused no toxic death, and was not associated with
remarkable changes in vital signs within a week after administration.
The in vivo antitumor effect of the drug of interest was evaluated in
the Balb/C syngeneic CT26 tumor model. CT26 mouse colon cancer
cells (1 × 105 cells) were implanted subcutaneously in 8 weeks-old
Balb/C mice (n = 8). When tumors reached 150 mm3, mice were
injected intraperitoneally with oxaliplatin (25 μmol/kg) or compound
1 (18 μmol/kg) twice a week (on days 1 and 4). Tumor sizes were
monitored using a caliper, and body weight was measured daily for
toxicity evaluation. The stock solution of 1 was prepared in DMSO
and subsequently diluted with hydrogenated castor oil Cremophor and
PBS. The solubility of complexes 3, 4, and 5 in Cremophor was not
sufficient for the in vivo studies. Statistical analysis was done by the
two-tail ANOVA test with Bonferroni post-test using GraphPad Prism
software (GraphPad Software Inc., CA) with P < 0.05 considered as
significant (* p < 0.05, *** p < 0.001)
NMR- and MS-Based Biomolecule Interaction Studies. NMR
and mass spectrometric (MS) methods were employed to characterize
the adduct formation of the Pt complexes with biomolecules such as
amino acids, nucleotides, and proteins. For NMR experiments, a
solution of 4 (1 mg/mL) in [D6]DMSO/D2O (5/95) was treated
with 2 equiv of Cys, His and Met, and 1H and 31P {1H} NMR spectra
were recorded after 30 min, 1, 3, 24, 48, and 72 h on a Bruker DRX
400 MHz NMR spectrometer at measurement frequencies of 400.13
(1H), and 161.98 MHz (31P{1H}) at ambient temperature. For MS
experiments, stock solutions of 2 (1% DMSO, 100 μM), 1 and 4 (1%
DMSO, 400 μM) and mixtures of amino acids (Met, His, Cys and Glu,
each 400 μM), nucleic acids (ATP and GTP, each 400 μM) and
proteins (ub and cyt, each 400 μM) were prepared in water. The
complexes were incubated with the proteins in water in the dark at 37
°C at a metal concentration of 50 μM. The reaction mixtures with the
amino acids (1:1:1:1:1 metal-to-amino acid ratio) and the nucleic acids
(1:1:1 metal-to-NTP ratio) were prepared analogously. Additionally,
the same compounds were incubated with a mixture of ubiquitin (ub)
and cytochrome C (cyt) at a 1:1:1 metal-to-protein ratio. Mass spectra
of the incubation mixtures were recorded after 2 and 24 h on an
electrospray ionization-ion trap-mass spectrometer (ESI-IT-MS) and
additionally on an ESI-time-of-flight (TOF) MS for the protein
incubation after 48 h. Samples were diluted to 5 μM metal or protein
content with water or water:methanol:formic acid (50:50:0.2),
respectively.
Cellular Accumulation. Studies on the cellular accumulation of
complexes 1−4 were performed in comparison to cisplatin according
to a previously described procedure.56 SW480 cells were seeded in 6-
well plates at densities of 1.2 × 105 cells per well in aliquots of 2.5 mL
MEM. Accumulation experiments and corresponding adsorption/
desorption controls were located on the same plate, and an additional
plate included controls for cell counting. Plates were kept at 37 °C for
24 h prior to addition of the respective complex. Cells were incubated
with the compounds at concentrations of 50 μM for 2 h at 37 °C.
Afterward, the medium was removed, and cells were washed three
times with PBS and lyzed with 0.5 mL subboiled HNO3 per well for 1
h at room temperature. Lysates were diluted with Milli-Q water
resulting in nitric acid concentrations lower than 3% and platinum
concentrations lower than 15 μg/g. The total platinum content was
determined with an ICP-quadrupole MS Agilent 7500ce (Agilent
Technologies, Waldbronn, Germany). The adsorption/desorption
blank data were subtracted from the data for the corresponding
accumulation sample, and the platinum content was referred to the cell
number. The results are based on at least three independent
experiments, each consisting of three replicates. The ICP-MS
instrument was equipped with a CETAX ASX-520 autosampler
(Nebraska, USA) and a MicroMist nebulizer operating at a sample
uptake rate of approximately 0.25 mL/min. The instrument was tuned
on a daily base in order to achieve maximum sensitivity. Platinum and
rhenium standards were obtained from CPI International (Amsterdam,
The Netherlands). Rhenium served as the internal standard for
platinum to account for instrumental fluctuations and matrix effects.
Quantification was done using the isotopes 185Re and 195Pt with a
dwell time of 0.3 s and 10 replicates. The ICP-MS instrument was
equipped with nickel cones and was operated at an RF power of 1550
W. Argon was used as plasma (15 L/min) and carrier gas with a flow
rate of ∼1.1 L/min. The Agilent MassHunter software package
(Workstation Software, Version B.01.01, 2012) was used for data
processing.
Interactions with DNA. A 500 ng portion of plasmid DNA
pUC19 (2686 bp; Fermentas Life Sciences) was incubated with 50 μM
of the test compounds in a 0.1× Tris-EDTA (TE) buffer for time
intervals of 5, 15, 30, 60, 120, and 240 min at 37 °C. Electrophoresis
was performed in a 1% agarose gel (Sigma-Aldrich) in 1× Tris-borate-
EDTA (TBE) buffer for 90 min at 80 V. Gels were stained with
ethidium bromide in 1× TBE (0.75 μg/mL) for 20 min. Images were
taken with the detection system Fusion SL (Vilber Lourmat). Three
independent experiments were performed.
Protein Extraction and Western Blot. HCT116 cells were
grown on Cellstar 6-well plates (Greiner Bio-One) and treated at 37
°C and 5% CO2 for 24 h with 1, 3−5 at their respective IC50 and IC75
concentrations as well as oxaliplatin and tunicamycin at their IC50
concentrations. The cells were lysed with lysis buffer [100 μL, 1%
NP40, 150 mM NaCl, 50 mM Tris-HCl (pH 8.0), protease inhibitor].
The cell lysate was transferred to individual 2 mL tubes and sonicated
for 10 s. The samples were then centrifuged at 13,000 rpm and 4 °C
for 15 min. The supernatant liquid containing the proteins was
collected, and the total protein content of each sample was quantified
via Bradford’s assay. Forty μg of protein from each sample were
reconstituted in loading buffer (5% DDT, 1× protein loading dye) and
heated at 95 °C for 5 min. The protein mixtures were resolved on a
10% SDS-PAGE gel by electrophoresis and transferred to a
ESI-IT-mass spectra were recorded on an AmaZon SL ion trap mass
spectrometer (Bruker Daltonics GmbH, Bremen, Germany) by direct
infusion at a flow rate of 4−5 μL/min. The instrument parameters
were as follows: 77% RF level, average accumulation time 3 ms (ESI+)
and 14 ms (ESI−), scan range m/z 100−2200, 64.7 trap drive, −4.5 kV
capillary voltage, −0.5 kV end plate offset, 180 °C dry temperature, 8
psi nebulizer, 6 L/min dry gas. Mass spectra were recorded over 0.5
min and averaged in positive and negative ion modes. Data were
processed using DataAnalysis 4.0 (Bruker Daltonics GmbH, Bremen,
Germany). The protein spectra were deconvoluted using the
maximum entropy deconvolution algorithm with automatic data
point spacing and 0.5 instrument peak width. Samples containing
C
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
a
Scheme 2. Synthesis of Mono- And Dinuclear Cyclometalated 2,2′-Bipyridine Pt(II) Complexes
a
(i−iv) L, toluene, 110 °C; (v−vii) L, dichloromethane, RT; (viii) HCl, acetone, RT.
2
proteins were additionally analyzed on a Maxis UHR-TOF (Bruker
Daltonics GmbH, Bremen, Germany) in positive ion mode. The
instrument was equipped with a Triversa nanomate (Advion
Biosystems Inc., Ithaca, New York, USA) using the ChipSoft 8.3
software for Chip control (Advion Biosystems Inc.). The instrument
parameters were as follows: Ion cooler RF 280 Vpp, ion cooler transfer
time 150 μs, scan range m/z 100−2000, gas flow 0.1 psi, dry heater
180 °C, HV capillary −1.8 kV. Data was processed using DataAnalysis
4.0 (Bruker Daltonics GmbH, Bremen, Germany). Protein spectra
were deconvoluted using the maximum entropy deconvolution
algorithm with automatic data point spacing and 30,000 instrument
resolving power.
Synthesis of Complexes. [Pt(bipy)(PTA)(Me)] (4). Method 1.
The synthetic method was adapted from a literature procedure.20 PTA
(16 mg, 0.1 mmol, 1 equiv) was added to a solution of
[Pt(DMSO)(bipy)(Me)] (46 mg, 0.1 mmol) in anhydrous CH2Cl2
(20 mL). The pale yellow solution was stirred for 2 h, filtered, and
concentrated under reduced pressure to ca. 2 mL. The product was
precipitated by slow addition of cold hexane (ca. 50 mL), filtered,
washed with hexane, and dried in vacuo to get 27 mg of yellow powder.
Yield: 52%.
1.8 Hz, H5), 4.55 (d, 3H, JH−H = 12.6 Hz, P(CHH)3), 4.45 (d, 3H,
2JH−H = 12.6 Hz, P(CHH)3),4.26 (s, 6H, (NCH2)3), 0.74 (d with 195Pt
satellites, 3H, JP−H = 7.6 Hz, JPt−H = 83.0 Hz, CH3); 31P NMR
3
2
(202.44 MHz, [D6]DMSO): δ = −67.0 (s with 195Pt satellites, 1JPt−P
=
1964 Hz, P(CH2)3); 195Pt NMR (107.33 MHz, [D6]DMSO): δ =
1
−2536 ppm (d, JPt−P = 1948 Hz).
[Pt(bipy)(PTA)Cl] (5). The synthetic method was adapted from a
literature procedure.20 PTA (16 mg, 0.1 mmol, 1 equiv) was added to
a solution of [Pt(DMSO)(bipy)Cl] (46 mg, 0.1 mmol) in anhydrous
CH2Cl2 (20 mL). The pale yellow solution was stirred for 2 h, filtered,
and concentrated under reduced pressure to ca. 2 mL. The product
was precipitated by slow addition of cold hexane (ca. 50 mL), filtered,
washed with hexane, and dried in vacuo to get 31 mg of light yellow
powder. Yield: 57%.
Elemental analysis (%) calcd for C16H19N5PPtCl (542.86 g mol−1):
C 35.40, H 3.53, N 12.90, P 5.71; found: C 35.24, H 3.39, N 12.83, P
5.68; H NMR (500.10 MHz; [D6]DMSO): δ = 9.44 ppm (m, 1H,
1
3
4
H6′), 8.36 (dd, 1H, JH−H = 4.6 Hz, JH−H = 1.2 Hz, H6), 8.23−8.17
(m, 2H, H3′, H4′), 7.91 (d with 195Pt satellites, 1H, JH−H = 7.6 Hz,
3
3JPt−H = 54 Hz, H4), 7.71 (td, 1H, JH−H = 5.6 Hz, JH−H = 2.7 Hz,
3
4
H5′), 7.14 (dd, 1H, 3JH−H = 7.8 Hz, 3JH−H = 4.6 Hz, H5), 4.61 (d, 3H,
2JH−H = 12.5 Hz, P(CHH)3), 4.47 (d, 3H, JH−H = 12.5 Hz,
2
Method 2. The synthetic method was adapted from a literature
procedure.25 2,2′-Bipyridine (94 mg, 0.6 mmol) and cis-[Pt-
(Me)2(DMSO)2] (127 mg, 0.3 mmol) were dissolved in anhydrous
toluene (10 mL) and heated to reflux for 3 h. The color of the solution
slowly changed from red to yellow. PTA (56 mg, 0.4 mmol,1.2 equiv)
was added to the solution, and the color of the solution immediately
changed to pale yellow. The solution was refluxed for 1 h and then
cooled down to room temperature. The reaction mixture was
concentrated to a small volume, the residue was extracted with
water, and organic phase was quickly separated and dried with
Na2SO4. The solution was filtered and concentrated to a small volume.
The product was precipitated by slow addition of cold hexane (ca. 50
mL), filtered, washed with hexane, and dried in vacuo to get 96 mg of
yellow microcrystals. Yield: 61%. The product can be additionally
purified by slow diffusion of diethyl ether into dichloromethane
solution, resulting in the formation of yellow crystals.
P(CHH)3), 4.45 (s, 6H, (NCH2)3). 31P NMR (202.44 MHz,
[D6]DMSO): d = −66.6 (s with 195Pt satellites, JPt−P = 3780 Hz,
1
P(CH2)3); 195Pt NMR (107.33 MHz, [D6]DMSO): δ = −2528 ppm
1
(d, JPt−P = 3794 Hz).
[Pt(bipy)(tBuNC)(Me)] (6). BuNC (11 μL, 0.1 mmol, 1 equiv) was
t
added to a solution of [Pt(DMSO)(bipy)(Me)] (46 mg, 0.1 mmol) in
anhydrous CH2Cl2 (20 mL). The pale yellow solution was stirred for 2
h, filtered, and concentrated under reduced pressure to ca. 2 mL. The
product was precipitated by slow addition of cold hexane (ca. 50 mL),
filtered, washed with hexane, and dried in vacuo to get light yellow film
(3 mg). Yield: 15%.
Elemental analysis (%) calcd for C16H19N3Pt (448.42 g mol−1): C
1
42.86, H 4.27, N 9.37; found: C 42.74, H 4.33, N 9.28; H NMR
(500.10 MHz; [D6]DMSO): δ = 8.77 ppm (d with 195Pt satellites, 1H,
3JH−H = 5.4 Hz, JPt−H = 15 Hz, H6′), 8.29 (dd, 1H, JH−H = 4.6 Hz,
3
3
4JH−H = 1.6 Hz, H6), 8.21 (dd, 1H, JH−H = 7.8 Hz, JH−H = 1.0 Hz,
3
4
Elemental analysis (%) calcd for C17H22N5PPt (522.44 g mol−1): C
H3′), 8.16 (td, 1H, 3JH−H = 8.0 Hz, 4JH−H = 1.2 Hz, H4′3), 7.91 (dd with
1
39.08, H 4.24, N 13.41; found: C 38.93, H 4.30, N 13.15; H NMR
195Pt satellites, 1H, JH−H = 7.4 Hz, JH−H = 1.7 Hz, JPt−H = 47 Hz,
3
4
(500.10 MHz; [D6]DMSO): δ = 8.70 ppm (d with broad 195Pt
satellites, 1H, 3JH−H = 5.4 Hz, 3JPt−H = 14 Hz, H6′), 8.30 (d, 1H, 3JH−H
= 4.8 Hz, H6), 8.23 (dd, 1H, 3JH−H = 7.9 Hz, 4JH−H = 0.9 Hz, H3′), 8.15
3
3
4
H4), 7.55 (ddd, 1H, JH−H = 7.2 Hz, JH−H = 5.6 Hz, JH−H = 1.8 Hz,
3
3
H5′), 7.19 (dd, 1H, JH−H = 7.5 Hz, JH−H = 4.7 Hz, H5), 1.61 (s, 6H,
tBuNC), 0.85 (s with 195Pt satellites, 3H, 2JPt−H = 86.0 Hz, CH3); 195Pt
NMR (107.33 MHz, [D6]DMSO): δ = −2381 ppm (s).
3
4
(td, 1H, JH−H = 8.3 Hz, JH−H = 0.9 Hz, H4′), 7.96 (ddd with 195Pt
satellites, 1H, 3JH−H = 7.7 Hz, 4JH−H = 5.4 Hz, 4JP−H = 1.6 Hz, 3JPt−H
48 Hz, H4), 7.53 (ddd, 1H, JH−H = 7.7 Hz, JH−H = 5.6 Hz, JH−H
1.6 Hz, H5′), 7.21 (ddd, 1H, JH−H = 7.7 Hz, JH−H = 4.6 Hz, JP−H
=
=
=
3
3
4
[(bipy)(Pt(PTA)(Me))2] (9). The synthetic method was adapted from
a literature procedure.20 PTA (20 mg, 0.128 mmol, 2 equiv) was added
3
3
5
D
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
spectra of 4 revealed strong P−Pt coupling (1JPt−P ≈ 1948 Hz;
compare the 31P and 195Pt NMR signals for 1−9 listed in Table
S2).
to a solution of [(bipy)(Pt(DMSO)(Me))2] (47 mg, 0.064 mmol) in
anhydrous CH2Cl2 (20 mL). Several minutes after PTA addition, a
yellow precipitate formed. The suspension was stirred for 2 h and
filtered. The solid was washed with diethyl ether and dried in vacuo to
give 37 mg of pale yellow solid. Yield: 65%
The stability of 3 and 4 in chloroform, dimethylformamide,
and dimethyl sulfoxide was monitored by NMR spectroscopy.
Elemental analysis (%) calcd for C24N8H36P2Pt2·H2O·0.5CH2Cl2
1
(888.70 g mol−1): C 31.00, H 4.14, N 11.81; found: C 31.30, H 4.05,
While H NMR spectra of the complexes in [D6]DMSO and
1
N 11.60. H NMR (500.10 MHz; CDCl3): δ = 8.19 (ddd with 195Pt
[D7]DMF remained unchanged for 24 h of measurements, in
CDCl3 gradual decomposition of the complexes was observed
after several hours with the appearance of additional sets of
satellites, 2H, 3JH−H = 7.7 Hz, 4JH−H = 2.1 Hz, 4JP−H = 6.4 Hz, 2JPt−H
45 Hz, H4/4′), 8.10 (d, 2H, JH−H = 5.2 Hz, H6/6′), 7.11 (ddd, 2H,
=
3
3
5
3JH−H = 7.6 Hz, JH−H = 5.4 Hz, JP−H = 2.1 Hz, H5/5′), 4.63 (d, 6H,
1
1
signals in H and 31P NMR spectra. The H NMR spectrum
recorded in CDCl3 of the dinuclear PTA complex 9 is similar to
those of 7 and 8 but markedly different from that of the
mononuclear analogue 4. The aromatic region featured only
three signals which supports the symmetric dinuclear structure
of the complex. Similarly to mononuclear 4, the PTA P−CH2−
N protons were detected as two doublets at 4.63 and 4.58 ppm,
whereas the PTA N−CH2−N protons were identified as a
singlet at 4.33 ppm. The methyl groups were observed at 0.84
ppm as a doublet flanked by 195Pt satellites (3JP−H = 8 Hz, 2JPt−H
= 80 Hz).
2JH−H = 13.1 Hz, P(CHH)3), 4.58 (d, 6H, JH−H = 13.1 Hz,
2
P(CHH)3), 4.33 (s, 12H, (NCH2)3), 0.84 (d with 195Pt satellites, 3H,
2
3JP−H = 7.6 Hz, JPt−H = 80.0 Hz, CH3); 31P NMR (202.44 MHz,
CDCl3): δ = −65.4 (s with 195Pt satellites, JPt−P = 2050 Hz,
1
1P(CH2)3); 195Pt NMR (107.33 MHz, CDCl3): δ = −2448 ppm (d,
1JPt−P = 2095 Hz).
RESULTS AND DISCUSSION
■
The C−H bond in bipy may be activated upon interaction with
cis-[Pt(Me)2(DMSO)2] at 110 °C in anhydrous toluene under
inert atmosphere.20 Depending on the ratio of Pt complex and
bipyridine, the mononuclear complex 1 (1:1 ratio) or dinuclear
7 (2:1 ratio) can be obtained (Scheme 2).20 Both complexes
allow for substitution of the S-bound DMSO ligand, which
occupies a trans position to the C3 atom coordinated to the Pt
center, with triphenylphosphine (complexes 3 and 8) and other
2e− donors. The structurally related mono- and dinuclear 1,3,5-
triaza-7-phosphaadamantane (PTA) complexes 4 and 9 were
prepared in moderate yields of 52 and 65%, respectively, by
stirring 1 and 7 in dichloromethane with PTA for 2 h. Complex
6 with a C-coordinated tert-butyl isocyanide was synthesized in
a similar way, but the yield was unsatisfactory (15%).
Alternatively, complexes 4 and 9 were obtained from one-pot
reactions starting from cis-[Pt(Me)2(DMSO)2] and bipy in
toluene,25 which does not require tedious isolation of
intermediate DMSO complexes. It should be noted that PTA
was added to the reaction mixture in a strictly stoichiometric
amount, because addition of PTA in excess resulted in the
formation of poorly soluble Pt species with three PTA ligands
(as detected by mass spectrometry, data not shown), which
could not be separated from the target product.
The methylato ligand in cyclometalated bipy complexes can
be substituted by a chlorido ligand, and such Me/Cl exchange
is accompanied by displacement of DMSO from trans to a cis
position with respect to the coordinated carbon atom of bipy (1
and 2, Scheme 1).58 Thus, contrary to 4 and 9, the PTA ligand
in 5 was found in trans position to the coordinated nitrogen
atom of bipy. As a result, the H6 proton resonated in NMR
spectra ([D6]DMSO) downfield at 9.44 vs 8.70 ppm, and the
1JP−Pt coupling constant was significantly higher at 1948 and
3794 Hz for 4 and 5, correspondingly, which was previously
observed for structurally related triphenylphosphine com-
plexes.20
The molecular structures of complexes 4·CH2Cl2 and 5 were
confirmed by X-ray diffraction analysis (Figure 1, selected bond
distances and angles are listed in Table 1) and compared to that
of 359 and [Pt(bipy)(PPh3)Cl].20
Complexes 1−9 were characterized by NMR spectroscopy
1
and elemental analysis. The aromatic regions in the H NMR
spectra ([D6]DMSO) of the mononuclear complexes 4 and 5
revealed seven well-separated resonances with two proton
signals flanked by 195Pt satellites (3JPt−H ≈ 47 Hz for H4 and
3JPt−H ≈ 14 Hz for H6). This is in agreement with the
cyclometalated nature of the Pt complex and literature data.20
The chemical shifts of the bipy protons do not depend on the
chemical nature of the ligand in trans position with the
exception of H4 and H6′, which are proximate to the Pt center.
The methylate group in 4 is represented by a doublet with 195Pt
Figure 1. Molecular structures of 4·CH2Cl2 (left) and 5 (right) drawn
at 50% probability level. Solvent molecules and hydrogen atoms were
omitted for clarity.
3
2
satellites (δ 0.74 ppm, JP−H = 8 Hz, JPt−H = 83 Hz) which is
consistent with its trans position to the nitrogen atom and
being cis to the phosphorus atom of PTA. Due to the absence
of a phosphorus atom in 6, the methyl group is represented by
a singlet with 195Pt satellites at δ 0.85 ppm (2JPt−H = 86 Hz). In
contrast to PTA complexes,39,57 where the PTA ligand is
usually represented by two singlets (CH2 groups of upper and
lower rims of adamantane), three signals were assigned to PTA
in 4, that is, two signals for the diastereotopic P−CH2−N
protons at 4.55 and 4.45 ppm and one signal for the N−CH2−
N protons at 4.26 ppm. Additionally, 31P and 195Pt NMR
The coordination geometry around the metal center in the Pt
complexes is slightly distorted from a square-planar environ-
ment, which is in agreement with previously published
structural data on Pt cyclometalated complexes.20,40 The angles
around the Pt center deviate from 90° with the smallest values
for the C7−Pt1−N1 angle (79.61(9)° in 3,59 79.66(7)° in 4,
and 80.55(9)° in 5) and the biggest values found between the
phosphorus-containing ligand and the pyridine ring (97.91(6)°
and 103.95(5)° for N1−Pt1−P1 in 3 and 4, correspondingly,
and 96.57(7)° for C7−Pt1−P1 in 5). This may be explained by
E
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 1. Key Bond Lengths (Å) and Angles (deg) Observed in the Molecular Structures of 4 and 5 in Comparison with 359 and
[Pt(bipy)(PPh3)Cl]20
bond lengths (Å) and angles (deg)
3
4
5
[Pt(bipy)(PPh3)Cl]
Pt1−C7
Pt1−P1
Pt1−N1
Pt1−CH3
2.043(3)
2.2904(7)
2.130(2)
2.077(4)
2.049(2)
1.997(2)
2.2123(6)
2.100(2)
2.002(3)
2.228(1)
2.091(2)
−
2.2772(5)
2.1183(17)
2.062(2)
−
Pt1−Cl1
−
−
2.3908(6)
2.379(1)
C7−Pt1−CH3
C7−Pt1−N1
H3C−Pt1−N1
C7−Pt1−P1
H3C−Pt1−P1
N1−Pt1−P1
C7−Pt1−Cl1
N1−Pt1−Cl1
P1−Pt1−Cl1
91.65(11)
79.61(9)
169.26(1)
173.67(7)
91.41(8)
97.91(6)
−
90.08(8)
79.66(7)
169.74(7)
175.92(6)
86.30(6)
103.95(5)
−
−
−
80.55(9)
80.82(9)
−
94.68(8)
−
96.57(7)
−
174.71(6)
173.53(7)
93.23(6)
89.77(2)
−
171.86(5)
171.59(7)
91.51(6)
93.35(2)
−
−
−
−
the constraint of the five-membered ring and the steric
demands of the bulky PTA and PPh3 ligands. The C−N
distances in bipy are shorter than the C−C bonds and do not
significantly vary from each other in all complexes. The longest
distance in bipy was assigned to C5−C6 (1.477(3), 1.476(3),
and 1.471(3) Å in 3, 4, and 5, correspondingly) and the
shortest bond length to N2−C10 (1.336(3), 1.337(3) and
1.339(3) Å in 3, 4 and 5, respectively). However, due to the
shift of PTA from a trans position to C7 in 4 to trans to N1 in 5
and the stronger trans influence of the carbon atom than that of
the nitrogen atom, the Pt1−P1 distance in 4 (2.2772(5) Å) is
significantly longer than in 5 (2.2123(6) Å). For the same
reason, the Pt1−Cl1 distance (2.3908(8) Å) is markedly longer
than Pt1−CH3 (2.062(2) Å). In the structurally related
complex dichloro(N-methyl-2,2′-bipyridylium)platinum(II)
Pt−Cl bond lengths of 2.301(3) and 2.396(3) Å were reported
for the chlorido ligands in trans position to the N and C atoms
of N-methyl-2,2′-bipyridylium, respectively.60 The values for
complex 5 are in good agreement with the values reported for
complex Pt(bipy)(PPh3)Cl.20 Notably, in all structures the
rotation of one pyridine ring brings N2 close to H4, with N2···
H4 distances of 2.574, 2.576, and 2.617 Å in 3−5,
correspondingly.
complexes by hydrolyzing the Pt−Cl bond and forming the
aqua complex [(bipy-κ2N,C)Pt(DMSO) + H2O]+ (m/z 446.06,
Figure 2). In addition, release of DMSO with a concurrent
Figure 2. ESI-IT mass spectra of 2 in water after 2 and 24 h.
retention of the chlorido ligand was also observed by the
formation of [(bipy-κ2N,C)PtCl + H]+ (m/z 387.19) and
[(bipy-κ2N,C)PtCl + H2O + H]+ (m/z 405.00). The hydrolysis
pathway seems to terminate in a dimeric species corresponding
to an aqua-bridged dimer of the form [(bipy-κ2N,C)Pt(μ-
2+
H2O)]2 (m/z 368.02), implying that both the chlorido and
Stability and Identification of Reactive Species in
Solution. Pt(II) anticancer drugs usually act as prodrugs and
require activation through ligand exchange reactions in the
biological medium. In cyclometalated Pt(II) complexes, the
stronger Pt−L bond is known to be associated with higher
biological activity.12 It was shown that neutral ligands which
remain bound to a Pt center in aqueous solution (i.e., PPh3,
P(OMe)3, 2,6-dimethylpyridine) enhance the antiproliferative
effect of the complexes in comparison with compounds where
L is quickly released (i.e., S-DMSO, NH3).12 In order to
evaluate the relationship between the behavior of the
complexes in aqueous media and their biological activity,
stability studies of complexes 1, 2, and 4 were performed by
means of ESI-MS (see Table S3 for the experimental and
theoretical mass signals).
the DMSO ligands are ultimately exchanged by aqua ligands.
Hydrolysis may therefore occur either via release of the
chlorido or the DMSO ligands for 2.
Replacing the chlorido by a methylato ligand as in 1 and 4
leads to increased resistance to hydrolysis. No significant
changes were observed in their mass spectra during the 24 h
incubation period, as was also shown for 4 by NMR
spectroscopy (Figure S1). The most abundant signal of 4 was
assigned to [4 + H]+ (m/z 523.10), while [1 − CH3 + OCH3]+
(m/z 460.01) was most abundant for 1. The mass signal
assigned to [4 + OH]+ was also detected for 4 (Figure S2).
This suggests that the reaction pathways of the methylato
complexes may occur via an associative pathway. In contrast to
2, no cleavage of the DMSO ligand was detected for 1.
Additionally, 4 features a small mass signal corresponding to [4
− CH3]+ (m/z 507.05), which may indicate that the reaction
pathway for this compound occurs via hydrolysis of the
methylate and not the PTA ligand. Tandem mass spectrometric
investigation by collisional activation of [1 + OH]+ indicated
that H2O, MeOH, and DMSO cleaved off and supports the
After 2 h of dissolution in water, the mass spectra recorded
for the chlorido complex 2 featured several signals in addition
to the most abundant peak assigned to the pseudo molecular
ion [2 + H]+ (m/z 464.99), which remained the most abundant
signal for up to 24 h. The complex seems to be activated
according to the classical scheme of square-planar PtII
F
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
notion of an associative pathway or even migratory insertion
into the Pt−Me bond (Figure S2).
(m/z 499.05) and [(bipy-κ2N,C)Pt + His]+ (m/z 505.07).
Furthermore, the coordination of Met or Cys to a Pt-
(bipyridine) fragment may lead to dimerization as observed,
for example, as [(bipy)2Pt2(μ-S-Cys)]2+ (m/z 410.50) and
most probably involves thiolate or thioether bridging of two Pt
centers.
Reactivity toward Biomolecules. The main mode of
action of Pt anticancer drugs responsible for their therapeutic
effect is DNA targeting.61 However, even the very well-studied
mechanism of action of cisplatin seems not to be fully
established yet due to the numerous potential reaction partners
inside a cell.62 Therefore, understanding the interactions of
metal-based drugs with biomolecules, such as amino acids,
proteins, and nucleotides, on a molecular level is of particular
interest to elucidate the reaction pathways of metal-based
anticancer agents. The determination of drug−biomolecule
adducts can be performed by a number of analytical methods,63
and ESI-MS proved to be a reliable method for monitoring
these interactions.64−66 The reactivity of 1, 2, and 4 toward
selected amino acids (L-cysteine [Cys], L-histidine [His], L-
methionine [Met], and L-glutamic acid [Glu]) and the model
proteins ub and cyt was evaluated by means of ESI-MS (Figures
S2 and S3, Tables S4−S6).
Compounds 1 and 4 demonstrated comparable aqueous
chemistry and were less reactive toward amino acids than the
kinetically more labile 2. Interestingely, their preferences for
adduct formation with amino acids were markedly different
(Figure 4). Compound 1 mainly formed adducts with Cys by
cleavage of the Pt−DMSO bond to yield [(bipy-κ2N,C)Pt(Me)
+ Cys + H]+ (m/z 485.02) with a subsequent cleavage of the
Pt−Me bond forming a bidentate adduct [(bipy-κ2N,C)Pt +
Cys]+ (m/z 471.01). However, in the mass spectra of the
reaction mixture with the PTA complex 4, the most abundant
signal was attributed to adducts with His (Figure S2), which
were obtained solely by cleavage of the Pt−CH3 bond, for
example, [(bipy-κ2N,C)Pt(PTA) + His]+ (m/z 662.13) and
[(bipy-κ2N,C)Pt(PTA) + His + H2O]+ (m/z 680.18), which is
in accordance with the hydrolysis studies, while neither adducts
with Glu nor Met were detected. Although possessing the same
chelating ligand, the three complexes formed quite distinct
adducts with amino acids depending on the nature of the
monodentate ligands, that is, 2 forms adducts with Met and
Cys, while 1 binds preferentially to Cys and 4 to His and Cys.
This implies that the choice of the leaving group and the
additional monodentate ligand influences the selectivity of the
metallodrug toward certain biological nucleophiles, that is,
imines, thiolates, or thioether.
Amino Acids. Compounds 1, 2, and 4 were incubated with
an equimolar mixture of the amino acids Met, Cys, His and Glu
in water (Figure 3, Table S4). Compound 2 preferentially
The reactivity of 4 toward His and Met was also studied by
31P{1H} NMR spectroscopy. For this purpose, 2 equiv of the
respective amino acid were added to a 5% [D6]DMSO/D2O
solution of 4, and the reaction progress was determined after 30
min, 3, 24, 48 h, and 8 d. Within the first 3 h of incubation, 4
did not react with the amino acids, but after 24 h a small
percentage (ca. 5%) of complex underwent ligand exchange
reactions with Met, while no reaction was observed with His
during this time period as shown in both 1H and 31P{1H} NMR
spectra. After 24 h of reaction of 4 with Met and His (Figures
S4 and S5), the 31P{1H} NMR spectrum showed a signal at ca.
−3 ppm in addition to a signal for PTA in the intact complex at
−61 ppm. However, these species account for <10% of the
original compound within the first 72 h, indicating high stability
of 4. The adduct formation kinetics in these experiments was
higher for Met than for His.
Proteins. Compounds 1, 2, and 4 were incubated with a
mixture of ub and cyt, and their reactions were monitored by
ESI-IT-MS (Figure 5, Table S5) and ESI-TOF-MS (for a
discussion of the latter compare the Supporting Information;
Table S6, Figure S3). The chlorido complex 2 forms aqua
species, and this resulted in the detection of pronounced
Pt(bipy) adducts with both ub and cyt. In contrast, the
methylate analogue 1 demonstrated some selectivity toward cyt
([cyt + (bipy)Pt(OCH3)]+, m/z 12737.7), while adducts with
ub were not observed. On the other hand, 4 featured only an ub
adduct corresponding to [ub + (bipy-κ2N,C)Pt(PTA)]+ (m/z
9071.9). It seems that modulation of the monodentate ligands
from chlorido to methylato influences the selectivity of binding
toward model proteins similarly to amino acids. In general, 2
was most reactive toward ub and cyt, followed by the
methylato-containing 1 and 4. A bidentate binding mode to
proteins leads to metalation of both proteins and also bis-
Figure 3. ESI-IT mass spectra of 1 (A) and 2 (B) after 2 h incubation
in water and in the presence of a mixture of Cys/His/Met/Glu (AA)
at an equimolar ratio.
formed adducts with Met and Cys, whereas His adducts were
detected only after 24 h and no adducts with Glu were
observed over the time of the experiment. The formation of
Met and Cys adducts occurred via exchange with DMSO as
indicated by the detection of the ions [(bipy-κ2N,C)PtCl + Met
+ H]+ (m/z 536.03) and [(bipy-κ2N,C)PtCl + His + H]+ (m/z
542.03). This is followed by the cleavage of the Pt−Cl bond
and subsequent formation of complexes with amino acids
bound in a bidentate manner, that is, [(bipy-κ2N,C)Pt + Met]+
G
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Figure 4. Reaction pathways of 1 and 2 with amino acids are displayed showing the detected ions during ESI-IT-MS analysis of the incubation
mixtures.
Figure 5. Deconvoluted ESI-IT mass spectra recorded in the binding studies of 1 (A), 2 (B), and 4 (C) after incubation with a ub/cyt mixture in
aqueous solution for 24 h.
adducts, while monodentate binding yields only monoadducts
and a certain selectivity depending on the monodentate ligand.
Consequently, 1 forms adducts with cyt, while the PTA-
containing 4 yields mainly ub adducts.
assay. The IC50 values are listed in Table 2. The yield of 6 was
unsatisfactory, and the dinuclear compounds 7−9 were not
sufficiently soluble in aqueous media to include them into
biological assays.
Cytotoxicity in Human Cancer Cell Lines. The rollover
cyclometalated Pt(II) complexes were screened for their
antiproliferative activity in ovarian teratocarcinoma CH1(PA-
1), colon carcinoma SW480 and HCT116, and non-small cell
lung cancer A549 cells by means of the colorimetric MTT
Cisplatin was more cytotoxic than complexes 1−5 in all cell
lines with the exception of SW480 where 5 was slightly more
active. The antiproliferative activity of the investigated
complexes in HCT116 cells decreased in the order 3 ≥ 4 >
5 > 1 > 2, whereas in other cell lines it was found to decrease in
H
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 2. Cytotoxicity of Pt Complexes 1−5 and Cisplatin Given as 50% Inhibitory Concentrations (IC50) in CH1(PA-1)
(ovarian teratocarcinoma), A549 (non-small cell lung cancer), and SW480 and HCT116 (colon carcinoma) Cells, Determined
by Means of the MTT Assay after Exposure for 48 h (HCT116) or 96 h and Their Cellular Accumulation in SW480 Cells Upon
a
2 h of Exposure
IC50 values SD, μM
Cellular uptake, fg Pt/cell
SW480
A549
CH1(PA-1)
SW480
44
HCT116
1
129 19
287 20
27
1
7
88 21
357 76
66 14
2
76 12
4.2 0.9
3.4 0.3
3.0 0.3
0.14 0.03
88 13
202 45
3
15
104
13
3
8
1
5.0 0.2
25
28
2
9
266 63
160 32
4
14
1
b
5
1.4 0.2
3.3 0.4
51 13
11
n.d.
cisplatin66,67
1.3 0.4
2
12
1
a
Note that no direct comparison should be drawn between HCT116 and the other cell lines because of the different exposure times. Values are
b
means standard deviations obtained from at least three independent experiments. n.d., not determined.
Figure 6. Electropherograms of dsDNA plasmid pUC19 after exposure to a 50 μM solution of 1 (lanes 2−7), 2 (lanes 10−15), 3 (lanes 18−23), 4
(lanes 26-31), and 5 (lanes 34−39) for different exposure times (5 min up to 4 h) compared to untreated controls C0 (lanes 1, 9, 17, 25 and 33) and
C4h (lanes 8, 16, 24, 32 and 40). SC and OC indicate supercoiled and open circular DNA, respectively. The gels for compounds 1/4 and 2/3 were
run together, and the untreated controls C0 or C4h were added to the left or right of the gels, separated by a white vertical line if not directly analyzed
on the adjacent lane.
the order 5 ≥ 3 ≥ 4 > 1 > 2. As the incubation time for
HCT116 cancer cells was only 48 h (96 h for the other cell
lines), the results cannot be directly compared. Complex 2,
which was most reactive to amino acids and proteins, was the
least active in all cancer cell lines and complexes with more
thermodynamically stable Pt−P bonds (3 and 4) demonstrated
higher antiproliferative activity. In SW480 and A549 cells, 5 was
markedly more active than the other investigated complexes,
while the PTA complexes 4 and 5 were similarly potent in the
chemosensitive CH1(PA-1) cells. The biological activity of 1, 2,
and 4 was inversely related with their reactivity toward
biomolecules (4 > 1 > 2, where 4 was the most active and
the least reactive). As observed previously for other complexes,
the faster the reaction kinetics in amino acid interaction studies,
the less potent are the compounds with respect to their
antiproliferative activity.66,67 This can be compared with
observations made for cisplatin, for which reactions with
serum proteins have been suggested to lead to inactivation of
the drug and possibly contribute to side effects experienced by
patients.53 However, clinical trials with cisplatin−protein
adducts resulted in similar acitivity as cisplatin alone.68
Interestingly, even extensive formation of Pt adducts with
cellular DNA does not necessarily lead to an increase in
cytotoxicity, since these adducts may be quickly removed by the
repair systems, as is the case for transplatin which was shown to
be at least 2.5-fold more reactive than cisplatin with no increase
of cytotoxicity.54
Cellular Accumulation. The cellular accumulation of Pt in
SW480 cells was determined by ICP-MS upon exposure to
compounds 1−4 in comparison to cisplatin (Table 2). SW480
cells were chosen for the experiments because A549 cells
showed the lowest sensitivity toward the tested complexes, and
CH1(PA-1) cells are known to detach easily during the washing
steps required in the cellular uptake experimental protocol.56
The cellular accumulation of all tested complexes is markedly
(up to ∼30-fold) higher than for cisplatin, decreasing in the
order 1 > 3 > 4 > 2. With the exception of 1, there is a
correlation between the antiproliferative activity and cellular
accumulation of the complexes. The cellular accumulation of 2
was significantly lower than that of the other compounds, which
is in line with its lowest activity in this cell line (IC50 = 88 μM).
The triphenylphosphine complex 3 demonstrated enhanced
intracellular accumulation (and higher cytotoxicity except in
CH1/PA-1 cells) in comparison with the structurally similar
PTA complex 4 (266 63 fg Pt/cell and 160 32 fg Pt/cell,
correspondingly) which might be related to the higher
lipophilicity of triphenylphosphine.
I
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
DNA Binding. DNA is considered the critical cellular target
for Pt complexes, and changes in the tertiary DNA structure are
determined by the type of adducts formed. In order to estimate
the influence of the DNA adducts formed with the investigated
complexes on the tertiary structure, DNA unwinding experi-
ments were conducted. The time-dependent ability of 1−5 to
modify the electrophoretic mobility of the supercoiled pUC19
plasmid DNA was determined by means of an agarose gel
mobility shift assay (Figure 6).69 The complexes were
incubated at a concentration of 50 μM with pUC19 plasmid
DNA at 37 °C for 5 min up to 4 h and analyzed by
electrophoresis in native agarose gels. The mobility of DNA
fragments migrating in the gel is influenced by the
conformation of the DNA molecule. Supercoiled DNA (SC)
is smaller in size, and therefore it experiences less resistance
from the gel and migrates faster than open circular DNA (OC).
Adducts that induce lower mobility of SC DNA relieve
torsional stress by unwinding and relaxing the compact SC
form. In contrast, higher mobility of OC DNA indicates the
formation of adducts causing DNA condensation.49
Based on the structures of the Pt complexes and the amino
acid/protein reactivity studies, we anticipated that the Me
complexes 1, 3, and 4 would be less reactive toward DNA
plasmid than the kinetically more labile chlorido complexes 2
and 5. Indeed, complexes 3 and 4 induced no or minor changes
in the DNA mobility within the incubation period, while 1, 2,
and 5 significantly affected the electrophoretic mobility of
pUC19 plasmid DNA, suggesting conformational changes and
the alteration of super helicity of the DNA molecules.
Complexes 1, 2, and 5 induced relaxation (unwinding) of the
SC form, and at least complexes 1 and 2 induced mobilization
(condensation) of the OC form of the DNA plasmid within 5
min of incubation (lanes 2 and 10). The less reactive Me/
DMSO complex 1 converted plasmid DNA into a “relaxed-like”
form with an apparent maximum after 15 min, which may
indicate counter-coiling in the opposite direction (i.e., positive
instead of negative supercoils) at later time points. It should be
noted that in case of 5, the maximum unwinding may have
happened too quickly to notice in this assay. Longer incubation
times with 2 and 5 resulted in a higher mobility of the plasmid
than for the respective controls, which probably indicates
enhanced coiling. Similar patterns were observed for other Cl/
DMSO−PtII complexes.37,39,70 It was reported that relaxation of
SC form of DNA by Pt complexes was associated with local
untwisting at the sites of adducts consistent with the formation
of monofunctional adducts.52 On the contrary, condensation of
the OC form was associated with the formation of multiple
rigid or flexible DNA bends, caused by bifunctional intra-
strand50,71 or interstrand adducts (two covalent bonds),51 or
pseudobifunctional adducts (one covalent bond plus hydro-
phobic/electrostatic interactions),72 and was described for
cisplatin,49 transplatin,49 and other Pt complexes.52,72 Hence,
the observed differences in plasmid migration upon treatment
with 1, 2, and 5 might indicate the formation of mono- and/or
bifunctional DNA adducts; however, this hypothesis has to be
confirmed by other experimental methods.
oxaliplatin (Oxa) led to a marked increase in p53 protein
levels (Figure 7). Complexes 1, 4, and 5 also caused an increase
Figure 7. Protein levels for the DNA damage-induced transcription
factor p53 and the ER stress-induced transcription factor ATF4.
Proteins were extracted from HCT116 cells treated for 24 h using
sample buffer with complexes 1, 3, 4, and 5 at their respective IC50 and
IC75 values from MTT assay. Western blot analysis revealed p53,
ATF4, and actin expression. Oxaliplatin (Oxa) and tunicamicin (Tun)
were used at their IC50 as positive control for DNA damage and ER
stress, respectively. Ct (control) cells were not treated with any
compounds.
in p53 protein level, although significantly less than oxaliplatin,
while complex 3 did not affect p53 protein levels. These
observations correlate with the results of DNA binding studies
and suggest that complexes 1, 4, and 5 might induce DNA
damage in a p53-dependent manner. Subsequently, we chose to
investigate the effects of Pt(II) compounds on alternative cell
death pathways not related to DNA damage, namely the
endoplasmic reticulum (ER) stress pathway. The ER stress
pathway is a part of the proteostasis mechanisms that control
protein homeostasis of the cells in response to various stressful
conditions, such as depletion in glucose, ROS production, and
misfolding of proteins.76 ER stress-inducing compounds are
known to target nonapoptotic cell death pathways and might
overcome multidrug resistance;77 however, there are only few
Pt-based compounds that can trigger this pathway to induce
cell death.78−80 We chose to follow the protein levels of the
transcription factor ATF4, which regulates antistress responses
and is involved in PERK-eIF2α-ATF4 pathway of ER stress
process.81,82
Complexes 1, 3, and 4 induced protein levels of ATF4
(Figure 7), and the strongest ATF4 upregulation was observed
in cells treated with complex 3. The results suggest that
complex 3 might induce p53-independent ER stress-mediated
cell death. In contrast, complex 5 caused a significant
diminution of ATF4 protein levels, similar to oxaliplatin,
which suggests p53-dependent DNA damage as a main mode of
action. Interestingly, complexes 4 and 5, that only differ by a Cl
vs a Me ligand, showed drastic differential response in cancer
cells. It is yet to be established whether this difference can be
correlated to the stability of these two complexes and their
ability to interact with biomolecules. Complex 1, which induced
the strongest uncoiling of the DNA plasmid, also induced the
upregulation of ATF4. These observations suggest that complex
1 is involved in more than one type of cancer cell death
pathway. To conclude, despite minor variations of the ligands
in complexes 1−5, they seem to display significant differences
in their modes of action, as illustrated by differential impact on
Changes in Protein Expression. To further investigate
the mode of action of the complexes, we analyzed their impact
on the protein levels of signaling markers involved in cell
growth arrest and cell death. We assayed the protein levels of
p53, a well-known transcription factor induced by DNA
damage and Pt-based drugs in cancer cells and healthy
cells.73−75 As expected, treatment of cancer cells with
J
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
purified DNA as well as on molecular signaling pathways
responding to DNA damage or ER stress.
CONCLUSIONS
■
The treatment of many widespread tumor types relies heavily
on cytotoxic Pt complexes. Despite their side effects and the
emergence of novel therapeutic approaches (i.e., targeted
therapies, immunotherapies), they remain essential parts of
many therapy schemes (e.g., colon, gastric, head and neck,
testicular, ovarian cancers) due to their high efficacy and low
cost for health care agencies. In order to reduce the side effects
of Pt compounds, a variety of strategies have been investigated.
One of those is to render the complexes less labile, as for
example in Pt(IV) complexes, and thereby reduce their
reactivity with biomolecules, which is thought at least in part
to be responsible for the side effects. In this approach, we have
introduced a molecular platform based on organoplatinum
compounds with a C,N-coordinated rollover 2,2′-bipyridine
ligand, which were equipped with co-ligands that formed more
or less stable bonds with the Pt(II) center. The small
modifications of the ligand scaffold affected the overall stability
of the complexes and their interactions with biomolecules. This,
in turn, impacted the ability of the complexes to induce distinct
pathways, such as DNA damage response and ER stress, via
upregulation of p53 and ATF4, respectively. In particular,
complex 3, being the most cytotoxic derivative of the series,
seems to have a completely different mode of action
characterized by no impact on purified DNA or p53 protein
level while presenting the strongest ability to induce ER stress
by up-regulating ATF4. Excitingly, complex 1, which induced
both p53-dependent DNA-damage and a component of the ER
stress response, showed similar tumor-inhibiting potency as
oxaliplatin against CT26 tumors with no signs of side-effects, as
indicated by no weight loss in test mice. This study illustrates
how the choice of specific ligands around the metal core
impacts drastically not only on the physicochemical properties
of complexes in terms of reactivity but also on the molecular
mechanisms triggered inside the cells, as in this case DNA
damage response and ER stress.
In Vivo Tests in Mouse Tumor Models. The novel Pt
complexes demonstrated a mode of action drastically different
from the classical Pt drug oxaliplatin. Therefore, complex 1 was
subjected to in vivo studies with Balb/C mice. To explore its
MTD, Balb/C mice were treated with 1, and the dose of the
compound was gradually increased from 4 to 15 μmol/kg. The
body weight changes of the mice upon treatment are shown in
Figure S6. None of the regimes caused deaths or any weight
loss during the course of toxicity studies. During the whole
experiment, all mice were bright, alert, and responsive.
Subsequently, the antitumor effect of the most soluble
compound 1 was evaluated in comparison with oxaliplatin in
the Balb/C syngeneic CT26 tumor model (Figure 8), which
Figure 8. Growth curves of CT26 tumors starting from day 12 when
tumors reached a volume of 150 mm3, and mice were subsequently
injected with drugs of interest twice a week. Curves are means and
standard deviations (n = 8) of tumor volume in % of tumor volume
measure at day 12. Statistical analysis was carried out by the two-tail
ANOVA test with Bonferroni post-test using GraphPad Prism software
(GraphPad Software Inc., CA) with P < 0.05 considered as significant
(* p < 0.05, *** p < 0.001).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b03210.
respects the immune system integrity. Mouse colon cancer cells
were implanted subcutaneously, and when tumors reached a
size of 150 mm3, mice were treated intraperitoneally with
oxaliplatin (25 μmol/kg) or compound 1 (18 μmol/kg) twice a
week. In general, intraperitoneal administration of substances is
characterized by pharmacokinetics more similar to that
observed after oral administration.6,48 The drugs administered
intraperitoneally may undergo hepatic metabolism before
reaching systemic circulation, possibly resulting in a lower
systemic toxicity.
■
S
Crystallographic and in vivo data, as well as further NMR
and mass spectra for stability and biomolecule interaction
studies (PDF)
Accession Codes
CCDC 1585871 and 1585872 contain the supplementary
crystallographic data for this paper. These data can be obtained
free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by
emailing data_request@ccdc.cam.ac.uk, or by contacting The
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
Compound 1 demonstrated significant inhibition of tumor
growth of about 33% compared to the control. Importantly, the
effects on the tumor inhibition were similar to those caused by
oxaliplatin; however, the injected dose of compound 1 (18
μmol/kg) was lower than the dose of oxaliplatin (25 μmol/kg).
About half of the mice treated with compound 1 displayed
signs of incontinence at the end of the treatment. Otherwise, no
signs of toxicity were detected. To conclude, the in vivo results
demonstrated that complex 1 which was suggested to inhibit
cancer cell growth via several different mechanistic pathways,
including DNA damage and ER stress, was shown to
significantly inhibit tumor growth similar to oxaliplatin with
no signs of severe toxicity. We believe the optimal therapeutic
regimen is yet to be established and will be the subject of our
future studies.
AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: babak.maria@nus.edu.sg.
*E-mail: c.hartinger@auckland.ac.nz; http://hartinger.
auckland.ac.nz.
ORCID
Samuel M. Meier-Menches: 0000-0002-8930-4574
Sarah Theiner: 0000-0001-5301-0139
K
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
palladium complexes incorporated into liposomes. Eur. J. Med. Chem.
1993, 28, 235−242.
Muhammad Hanif: 0000-0002-2256-2317
Christian G. Hartinger: 0000-0001-9806-0893
Notes
(17) Klajner, M.; Hebraud, P.; Sirlin, C.; Gaiddon, C.; Harlepp, S.
DNA Binding to an Anticancer Organo-Ruthenium Complex. J. Phys.
Chem. B 2010, 114, 14041−14047.
The authors declare no competing financial interest.
(18) Boff, B.; Gaiddon, C.; Pfeffer, M. Cancer Cell Cytotoxicity of
Cyclometalated Compounds Obtained with Osmium(II) Complexes.
Inorg. Chem. 2013, 52, 2705−2715.
ACKNOWLEDGMENTS
■
We thank COST actions CM1105 (short term scientific
mission to M.V.B.) and BM1307, and the University of Vienna
(travel award to M.V.B.) for financial support. This project was
supported by the Centre National pour la Recherche
Scientifique (CNRS, France; C.G.), ARC, Ligue contre le
Cancer, European action. We thank Prof. Markus Galanski for
providing Pt precursors.
(19) Kaes, C.; Katz, A.; Hosseini, M. W. Bipyridine: The Most
Widely Used Ligand. A Review of Molecules Comprising at Least Two
2,2′-Bipyridine Units. Chem. Rev. 2000, 100, 3553−3590.
(20) Zucca, A.; Petretto, G. L.; Stoccoro, S.; Cinellu, M. A.;
Manassero, M.; Manassero, C.; Minghetti, G. Cyclometalation of 2,2′-
Bipyridine. Mono- and Dinuclear C,N Platinum(II) Derivatives.
Organometallics 2009, 28, 2150−2159.
(21) Minghetti, G.; Doppiu, A.; Zucca, A.; Stoccoro, S.; Cinellu, M.
A.; Manassero, M.; Sansoni, M. Palladium(II) and platinum(II)-C(3)-
substituted 2,2′-bipyridines. Chem. Heterocycl. Compd. 1999, 35, 992−
1000.
(22) Minghetti, G.; Stoccoro, S.; Cinellu, M. A.; Soro, B.; Zucca, A.
Activation of a C-H Bond in a Pyridine Ring. Reaction of 6-Substituted
2,2′-Bipyridines with Methyl and Phenyl Platinum(II) Derivatives:
N′,C(3)-″Rollover″ Cyclometalation. Organometallics 2003, 22, 4770−
4777.
(23) Cocco, F.; Cinellu, M. A.; Minghetti, G.; Zucca, A.; Stoccoro, S.;
Maiore, L.; Manassero, M. Intramolecular C(sp2)-H Bond Activation
in 6,6′-Dimethoxy-2,2′-Bipyridine with Gold(III). Crystal and
Molecular Structure of the First N′,C(3) ″Rollover″ Cycloaurated
Derivative. Organometallics 2010, 29, 1064−1066.
(24) Petretto, G. L.; Rourke, J. P.; Maidich, L.; Stoccoro, S.; Cinellu,
M. A.; Minghetti, G.; Clarkson, G. J.; Zucca, A. Heterobimetallic
Rollover Derivatives. Organometallics 2012, 31, 2971−2977.
(25) Maidich, L.; Zuri, G.; Stoccoro, S.; Cinellu, M. A.; Masia, M.;
Zucca, A. Mesoionic Complexes of Platinum(II) Derived from
″Rollover″ Cyclometalation: A Delicate Balance between Pt-C(sp3)
and Pt-C(sp2) Bond Cleavage as a Result of Different Reaction
Conditions. Organometallics 2013, 32, 438−448.
(26) Zucca, A.; Cordeschi, D.; Maidich, L.; Pilo, M. I.; Masolo, E.;
Stoccoro, S.; Cinellu, M. A.; Galli, S. Rollover Cyclometalation with 2-
(2′-Pyridyl)quinoline. Inorg. Chem. 2013, 52, 7717−7731.
(27) Maidich, L.; Zuri, G.; Stoccoro, S.; Cinellu, M. A.; Zucca, A.
Assembly of symmetrical and unsymmetrical platinum(II) rollover
complexes with bidentate phosphine ligands. Dalton Trans. 2014, 43,
14806−14815.
(28) Maidich, L.; Dettori, G.; Stoccoro, S.; Cinellu, M. A.; Rourke, J.
P.; Zucca, A. Electronic and Steric Effects in Rollover C-H Bond
Activation. Organometallics 2015, 34, 817−828.
(29) Fereidoonnezhad, M.; Niazi, M.; Shahmohammadi Beni, M.;
Mohammadi, S.; Faghih, Z.; Faghih, Z.; Shahsavari, H. R. Synthesis,
biological evaluation, and molecular docking studies on the DNA
binding interactions of platinum(II) rollover complexes containing
phosphorus donor ligands. ChemMedChem 2017, 12, 456−465.
(30) Esmaeilbeig, A.; Samouei, H.; Abedanzadeh, S.; Amirghofran, Z.
Synthesis, characterization and antitumor activity study of some
cyclometalated organoplatinum(II) complexes containing aromatic N-
donor ligands. J. Organomet. Chem. 2011, 696, 3135−3142.
(31) Fereidoonnezhad, M.; Niazi, M.; Ahmadipour, Z.; Mirzaee, T.;
Faghih, Z.; Faghih, Z.; Shahsavari, H. R. Cyclometalated platinum(II)
complexes comprising 2-(diphenylphosphino)pyridine and various
thiolate ligands: synthesis, spectroscopic characterization, and bio-
logical activity. Eur. J. Inorg. Chem. 2017, 2017, 2247−2254.
(32) Samouei, H.; Rashidi, M.; Heinemann, F. W. Cyclometalated
platinum(II) complexes containing monodentate phosphines: anti-
proliferative study. J. Iran. Chem. Soc. 2014, 11, 1207−1216.
(33) El-Mehasseb, I. M.; Kodaka, M.; Okada, T.; Tomohiro, T.;
Okamoto, K.-i.; Okuno, H. Platinum (II) complex with cyclo-
metalating 2-phenylpyridine ligand showing high cytotoxicity against
cisplatin-resistant cell. J. Inorg. Biochem. 2001, 84, 157−158.
REFERENCES
■
(1) Jakupec, M. A.; Galanski, M.; Arion, V. B.; Hartinger, C. G.;
Keppler, B. K. Antitumor metal compounds: more than theme and
variations. Dalton Trans. 2008, 183−194.
(2) Kelland, L. The resurgence of platinum-based cancer chemo-
therapy. Nat. Rev. Cancer 2007, 7, 573−584.
(3) van Zutphen, S.; Reedijk, J. Targeting platinum anti-tumour
drugs: Overview of strategies employed to reduce systemic toxicity.
Coord. Chem. Rev. 2005, 249, 2845−2853.
(4) Hartinger, C. G.; Metzler-Nolte, N.; Dyson, P. J. Challenges and
Opportunities in the Development of Organometallic Anticancer
Drugs. Organometallics 2012, 31, 5677−5685.
(5) Barry, N. P. E.; Sadler, P. J. Exploration of the medical periodic
table: towards new targets. Chem. Commun. (Cambridge, U. K.) 2013,
49, 5106−5131.
(6) Turner, P. V.; Brabb, T.; Pekow, C.; Vasbinder, M. A.
Administration of substances to laboratory animals: routes of
administration and factors to consider. J. Am. Assoc. Lab. Anim. Sci.
2011, 50, 600−613.
(7) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The status of
platinum anticancer drugs in the clinic and in clinical trials. Dalton
Trans. 2010, 39, 8113−8127.
(8) Johnstone, T. C.; Park, G. Y.; Lippard, S. J. Understanding and
Improving Platinum Anticancer Drugs − Phenanthriplatin. Anticancer
Res. 2014, 34, 471−476.
(9) Newkome, G. R.; Panteleo, D. C.; Puckett, W. E.; Ziefle, P. L.;
Deutsch, W. A. Complexes of palladium(II), platinum(II), copper(II),
cobalt(II), and zinc(II) chlorides with 6,6′-dimethyl-2,2′-dipyridyl. J.
Inorg. Nucl. Chem. 1981, 43, 1529−1531.
(10) Garelli, N.; Vierling, P. Incorporation of new amphiphilic
perfluoroalkylated bipyridine platinum and palladium complexes into
liposomes: stability and structure-incorporation relationships. Biochim.
Biophys. Acta, Lipids Lipid Metab. 1992, 1127, 41−48.
(11) Garelli, N.; Vierling, P. Synthesis and characterization of
amphiphilic platinum and palladium complexes linked to perfluor-
oalkylated side-chain disubstituted bipyridines. Inorg. Chim. Acta 1992,
194, 247−253.
(12) Edwards, G. L.; Black, D. S. C.; Deacon, G. B.; Wakelin, L. P. G.
In vitro and in vivo studies of neutral cyclometallated complexes
against murine leukaemias. Can. J. Chem. 2005, 83, 980−989.
(13) Edwards, G. L.; Black, D. S. C.; Deacon, G. B.; Wakelin, L. P. G.
Effect of charge and surface area on the cytotoxicity of cationic
metallointercalation reagents. Can. J. Chem. 2005, 83, 969−979.
(14) Vo, V.; Kabuloglu-Karayusuf, Z. G.; Carper, S. W.; Bennett, B.
L.; Evilia, C. Novel 4,4′-diether-2,2′-bipyridine cisplatin analogues are
more effective than cisplatin at inducing apoptosis in cancer cell lines.
Bioorg. Med. Chem. 2010, 18, 1163−1170.
(15) Elwell, K. E.; Hall, C.; Tharkar, S.; Giraud, Y.; Bennett, B.; Bae,
C.; Carper, S. W. A fluorine containing bipyridine cisplatin analog is
more effective than cisplatin at inducing apoptosis in cancer cell lines.
Bioorg. Med. Chem. 2006, 14, 8692−8700.
(16) Garelli, N.; Vierling, P.; Fischel, J. L.; Milano, G. Cytotoxic
activity of new amphiphilic perfluoroalkylated bipyridine platinum and
L
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(34) Okada, T.; El-Mehasseb, I. M.; Kodaka, M.; Tomohiro, T.;
Okamoto, K.; Okuno, H. Mononuclear platinum(II) complex with 2-
phenylpyridine ligands showing high cytotoxicity against mouse
sarcoma 180 cells acquiring high cisplatin resistance. J. Med. Chem.
2001, 44, 4661−4667.
(35) Fereidoonnezhad, M.; Kaboudin, B.; Mirzaee, T.; Babadi
Aghakhanpour, R.; Golbon Haghighi, M.; Faghih, Z.; Faghih, Z.;
Ahmadipour, Z.; Notash, B.; Shahsavari, H. R. Cyclometalated
Platinum(II) Complexes Bearing Bidentate O,O′-Di(alkyl)-
dithiophosphate Ligands: Photoluminescence and Cytotoxic Proper-
ties. Organometallics 2017, 36, 1707−1717.
(51) Huang, H.; Zhu, L.; Reid, B. R.; Drobny, G. P.; Hopkins, P. B.
Solution structure of a cisplatin-induced DNA interstrand cross-link.
Science 1995, 270, 1842−1845.
(52) Zorbas-Seifried, S.; Jakupec, M. A.; Kukushkin, N. V.; Groessl,
M.; Hartinger, C. G.; Semenova, O.; Zorbas, H.; Kukushkin, V. Y.;
Keppler, B. K. Reversion of structure-activity relationships of
antitumor platinum complexes by acetoxime but not hydroxylamine
ligands. Mol. Pharmacol. 2007, 71, 357−365.
(53) Reedijk, J. Why does cisplatin reach guanine-N7 with competing
S-donor ligands available in the cell? Chem. Rev. (Washington, DC, U.
S.) 1999, 99, 2499−2510.
(36) Frik, M.; Fernandez-Gallardo, J.; Gonzalo, O.; Mangas-Sanjuan,
V.; Gonzalez-Alvarez, M.; Serrano del Valle, A.; Hu, C.; Gonzalez-
Alvarez, I.; Bermejo, M.; Marzo, I.; Contel, M. Cyclometalated
Iminophosphorane Gold(III) and Platinum(II) Complexes. A Highly
Permeable Cationic Platinum(II) Compound with Promising
Anticancer Properties. J. Med. Chem. 2015, 58, 5825−5841.
(37) Ruiz, J.; Cutillas, N.; Vicente, C.; Villa, M. D.; Lopez, G.;
Lorenzo, J.; Aviles, F. X.; Moreno, V.; Bautista, D. New Palladium(II)
and Platinum(II) Complexes with the Model Nucleobase 1-
Methylcytosine: Antitumor Activity and Interactions with DNA.
Inorg. Chem. 2005, 44, 7365−7376.
(38) Ruiz, J.; Lorenzo, J.; Vicente, C.; Lopez, G.; Lopez-de-Luzuriaga,
J. M.; Monge, M.; Aviles, F. X.; Bautista, D.; Moreno, V.; Laguna, A.
New Palladium(II) and Platinum(II) Complexes with 9-Amino-
acridine: Structures, Luminescence, Theoretical Calculations, and
Antitumor Activity. Inorg. Chem. 2008, 47, 6990−7001.
(39) Ruiz, J.; Rodriguez, V.; Cutillas, N.; Espinosa, A.; Hannon, M. J.
Novel C,N-chelate platinum(II) antitumor complexes bearing a
lipophilic ethisterone pendant. J. Inorg. Biochem. 2011, 105, 525−531.
(40) Ruiz, J.; Rodriguez, V.; Cutillas, N.; Lopez, G.; Bautista, D.
Acetonimine and 4-Imino-2-methylpentan-2-amino Platinum(II)
Complexes: Synthesis and in Vitro Antitumor Activity. Inorg. Chem.
2008, 47, 10025−10036.
(41) Ma, D.-l.; Che, C.-M. A bifunctional platinum(II) complex
capable of intercalation and hydrogen-bonding interactions with DNA:
Binding studies and cytotoxicity. Chem. - Eur. J. 2003, 9, 6133−6144.
(42) Omae, I. Applications of five-membered ring products of
cyclometalation reactions as anticancer agents. Coord. Chem. Rev.
2014, 280, 84−95.
(54) Farrell, N.; Kelland, L. R.; Roberts, J. D.; Van Beusichem, M.
Activation of the trans geometry in platinum antitumor complexes: a
survey of the cytotoxicity of trans complexes containing planar ligands
in murine L1210 and human tumor panels and studies on their
mechanism of action. Cancer Res. 1992, 52, 5065−5072.
(55) Korch, C.; Spillman, M. A.; Jackson, T. A.; Jacobsen, B. M.;
Murphy, S. K.; Lessey, B. A.; Jordan, V. C.; Bradford, A. P. DNA
profiling analysis of endometrial and ovarian cell lines reveals
misidentification, redundancy and contamination. Gynecol. Oncol.
2012, 127, 241−248.
(56) Egger, A. E.; Rappel, C.; Jakupec, M. A.; Hartinger, C. G.;
Heffeter, P.; Keppler, B. K. Development of an experimental protocol
for uptake studies of metal compounds in adherent tumor cells. J. Anal.
At. Spectrom. 2009, 24, 51−61.
(57) Allardyce, C. S.; Dyson, P. J.; Ellis, D. J.; Heath, S. L. [Ru(η6-p-
cymene)Cl2(pta)] (pta = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]-
decane): a water soluble compound that exhibits pH dependent
DNA binding providing selectivity for diseased cells. Chem. Commun.
2001, 1396−1397.
(58) Black, D. S. C.; Deacon, G. B.; Edwards, G. L. Observations on
the mechanism of halogen-bridge cleavage by unidentate ligands in
square planar palladium and platinum complexes. Aust. J. Chem. 1994,
47, 217−227.
(59) Zucca, A.; Maidich, L.; Canu, L.; Petretto, G. L.; Stoccoro, S.;
Cinellu, M. A.; Clarkson, G. J.; Rourke, J. P. Rollover-Assisted C(sp2)-
C(sp3) Bond Formation. Chem. - Eur. J. 2014, 20, 5501−5510.
(60) Snow, M.; Tiekink, E. Crystal structure of dichloro(N-methyl-
2,2′-bipyridylium)platinum(II), C11H10Cl2N2Pt. Z. Kristallogr. 1992,
198, 148−150.
(61) Allardyce, C. S.; Dyson, P. J. Metal-based drugs that break the
rules. Dalton Trans. 2016, 45, 3201−3209.
(62) Wexselblatt, E.; Yavin, E.; Gibson, D. Cellular interactions of
platinum drugs. Inorg. Chim. Acta 2012, 393, 75−83.
(63) Groessl, M.; Hartinger, C. G. Anticancer metallodrug research
analytically painting the ″omics″ picture-current developments and
future trends. Anal. Bioanal. Chem. 2013, 405, 1791−1808.
(64) Hartinger, C. G.; Casini, A.; Duhot, C.; Tsybin, Y. O.; Messori,
L.; Dyson, P. J. Stability of an organometallic ruthenium-ubiquitin
adduct in the presence of glutathione: Relevance to antitumor activity.
J. Inorg. Biochem. 2008, 102, 2136−2141.
(43) Frezza, M.; Dou, Q. P.; Xiao, Y.; Samouei, H.; Rashidi, M.;
Samari, F.; Hemmateenejad, B. In Vitro and In Vivo Antitumor
Activities and DNA Binding Mode of Five Coordinated Cyclo-
metalated Organoplatinum(II) Complexes Containing Biphosphine
Ligands. J. Med. Chem. 2011, 54, 6166−6176.
(44) Vezzu, D. A. K.; Lu, Q.; Chen, Y.-H.; Huo, S. Cytotoxicity of
cyclometalated platinum complexes based on tridentate NCN and
CNN-coordinating ligands: Remarkable coordination dependence. J.
Inorg. Biochem. 2014, 134, 49−56.
(45) Daigle, D. J.; Pepperman, A. B.; Vail, S. L. Synthesis of a
monophosphorus analog of hexamethylenetetramine. J. Heterocycl.
Chem. 1974, 11, 407−408.
(65) Meier, S. M.; Tsybin, Y. O.; Dyson, P. J.; Keppler, B. K.;
Hartinger, C. G. Fragmentation methods on the balance: unambiguous
top-down mass spectrometric characterization of oxaliplatin-ubiquitin
binding sites. Anal. Bioanal. Chem. 2012, 402, 2655−2662.
(66) Babak, M. V.; Meier, S. M.; Legin, A. A.; Adib Razavi, M. S.;
Roller, A.; Jakupec, M. A.; Keppler, B. K.; Hartinger, C. G. Am(m)ines
Make the Difference: Organoruthenium Am(m)ine Complexes and
Their Chemistry in Anticancer Drug Development. Chem. - Eur. J.
2013, 19, 4308−4318.
(67) Meier, S. M.; Hanif, M.; Kandioller, W.; Keppler, B. K.;
Hartinger, C. G. Biomolecule binding vs. anticancer activity: Reactions
of Ru(arene)[(thio)pyr(id)one] compounds with amino acids and
proteins. J. Inorg. Biochem. 2012, 108, 91−95.
(46) Romeo, R.; Scolaro, L. M. (2,2′:6′,2″-Terpyridine)-
methylplatinum(II) chloride and (1,10-phenanthroline)-
methylchloroplatinum(II). Inorganic Syntheses; John Wiley & Sons,
Inc.: Hoboken, NJ, 1998; Vol. 32, pp 153−158.
(47) Becker, H. Organicum: Organic Chemistry Basic Laboratory
Course, 15th ed.; Deutscher Verlag der Wissenschaften: Berlin, 1977; p
880 pp.
(48) Lukas, G.; Brindle, S. D.; Greengard, P. Route of absorption of
intraperitoneally administered compounds. J. Pharmacol. Exp. Ther.
1971, 178, 562−566.
(49) Cohen, G. L.; Bauer, W. R.; Barton, J. K.; Lippard, S. J. Binding
of cis- and trans-dichlorodiammineplatinum(II) to DNA: Evidence for
unwinding and shortening of the double helix. Science 1979, 203,
1014−1016.
(50) Takahara, P. M.; Rosenzweig, A. C.; Frederick, C. A.; Lippard, S.
J. Crystal structure of double-stranded DNA containing the major
adduct of the anticancer drug cisplatin. Nature 1995, 377, 649−652.
(68) Holding, J. D.; Lindup, W. E.; van Laer, C.; Vreeburg, G. C.;
Schilling, V.; Wilson, J. A.; Stell, P. M. Phase I trial of a cisplatin-
albumin complex for the treatment of cancer of the head and neck. Br.
J. Clin. Pharmacol. 1992, 33, 75−81.
M
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(69) Wehner, J.; Horneck, G.; Buecker, H. Plasmids as test system for
the detection of DNA strand breaks. NATO ASI Ser., Ser. A 1993,
243A, 49−52.
(70) Ruiz, J.; Vicente, C.; de Haro, C.; Espinosa, A. Synthesis and
Antiproliferative Activity of a C,N-Cycloplatinated(II) Complex with a
Potentially Intercalative Anthraquinone Pendant. Inorg. Chem. 2011,
50, 2151−2158.
(71) Recio Despaigne, A. A.; Da Silva, J. G.; da Costa, P. R.; dos
Santos, R. G.; Beraldo, H. ROS-mediated cytotoxic effect of copper(II)
hydrazone complexes against human glioma cells. Molecules 2014, 19,
17202−17220.
(72) Zakovska, A.; Novakova, O.; Balcarova, Z.; Bierbach, U.; Farrell,
N.; Brabec, V. DNA interactions of antitumor trans-[PtCl2(NH3)-
(quinoline)]. Eur. J. Biochem. 1998, 254, 547−557.
(73) Benosman, S.; Gross, I.; Clarke, N.; Jochemsen, A. G.;
Okamoto, K.; Loeffler, J. P.; Gaiddon, C. Multiple neurotoxic stresses
converge on MDMX proteolysis to cause neuronal apoptosis. Cell
Death Differ. 2007, 14, 2047−2057.
(74) Licona, C.; Spaety, M.-E.; Capuozzo, A.; Ali, M.; Santamaria, R.;
Armant, O.; Delalande, F.; Dorsselaer, A. V.; Cianferani, S.; Spencer, J.;
Pfeffer, M.; Mellitzer, G.; Gaiddon, C. A ruthenium anticancer
compound interacts with histones and impacts differently on
epigenetic and death pathways compared to cisplatin. Oncotarget
2017, 8, 2568−2584.
(75) von Grabowiecki, Y.; Abreu, P.; Blanchard, O.; Palamiuc, L.;
Benosman, S.; Meriaux, S.; Devignot, V.; Gross, I.; Mellitzer, G.;
́
Gonzalez de Aguilar, J. L.; Gaiddon, C. Transcriptional activator
TAp63 is upregulated in muscular atrophy during ALS and induces the
pro-atrophic ubiquitin ligase Trim63. eLife 2016, 5, e10528.
(76) Cubillos-Ruiz, J. R.; Bettigole, S. E.; Glimcher, L. H.
Tumorigenic and immunosuppressive effects of endoplasmic reticulum
stress in cancer. Cell (Cambridge, MA, U. S.) 2017, 168, 692−706.
(77) Chow, M. J.; Licona, C.; Pastorin, G.; Mellitzer, G.; Ang, W. H.;
Gaiddon, C. Structural tuning of organoruthenium compounds allows
oxidative switch to control ER stress pathways and bypass multidrug
resistance. Chem. Sci. 2016, 7, 4117−4124.
(78) Wong, D. Y. Q.; Ong, W. W. F.; Ang, W. H. Induction of
Immunogenic Cell Death by Chemotherapeutic Platinum Complexes.
Angew. Chem., Int. Ed. 2015, 54, 6483−6487.
(79) Wang, X.; Guo, Q.; Tao, L.; Zhao, L.; Chen, Y.; An, T.; Chen,
Z.; Fu, R. E platinum, a newly synthesized platinum compound,
induces apoptosis through ROS-triggered ER stress in gastric
carcinoma cells. Mol. Carcinog. 2017, 56, 218−231.
(80) Zou, T.; Lok, C.-N.; Fung, Y. M. E.; Che, C.-M. Luminescent
organoplatinum(II) complexes containing bis(N-heterocyclic carbene)
ligands selectively target the endoplasmic reticulum and induce potent
photo-toxicity. Chem. Commun. (Cambridge, U. K.) 2013, 49, 5423−
5425.
(81) Ye, J.; Koumenis, C. ATF4, an ER stress and hypoxia-inducible
transcription factor and its potential role in hypoxia tolerance and
tumorigenesis. Curr. Mol. Med. 2009, 9, 411−416.
(82) Zheng, Q.; Ye, J.; Cao, J. Translational regulator eIF2α in tumor.
Tumor Biol. 2014, 35, 6255−6264.
N
DOI: 10.1021/acs.inorgchem.7b03210
Inorg. Chem. XXXX, XXX, XXX−XXX