Reprint of “Anticancer activity of hydroxy- and sulfonamide-azobenzene platinum(II) complexes in cisplatin-resistant ovarian cancer cells”

Article abstract of DOI:10.1016/j.jinorgbio.2017.07.035

The syntheses of three platinum(II) complexes bearing sulfonamide-((E)-2-(4-methylphenylsulfonamido)-2′,6′-difluoroazobenzene, HL1) and hydroxy-azo-2,6-difluorobenzene ((E)-2-((2,6-difluorophenyl)diazenyl)phenol, HL2) bidentate ligands is described. These

Full text of DOI:10.1016/j.jinorgbio.2017.07.035

JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Reprint of “Anticancer activity of hydroxy- and sulfonamide-azobenzene
platinum(II) complexes in cisplatin-resistant ovarian cancer cells”☆
Katia G. Sampera,b, Sierra C. Markera, Pau Bayónb, Samantha N. MacMillana, Ivan Keresztesa,
Òscar Palaciosb,⁎, Justin J. Wilsona,⁎
a
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, United States
Departament de Química, Facultat de Ciències, Universitat Autònoma de Barcelona, Cerdanyola del Vallès, E-08193 Barcelona, Spain
b
A R T I C L E I N F O
A B S T R A C T
Keywords:
Cisplatin
Cisplatin resistance
Antitumor drug
Ovarian cancer
Azobenzene
The syntheses of three platinum(II) complexes bearing sulfonamide-((E)-2-(4-methylphenylsulfonamido)-2′,6′-
difluoroazobenzene, HL1) and hydroxy-azo-2,6-difluorobenzene ((E)-2-((2,6-difluorophenyl)diazenyl)phenol,
HL2) bidentate ligands is described. These complexes, [Pt(L1)(DMSO)Cl] (1), [Pt(L2)(DMSO)Cl] (2), and [Pt
(L2)2] (3), were characterized by multinuclear NMR spectroscopy, mass spectrometry, and X-ray crystal-
lography. Despite bearing azobenzene functional groups, none of the three complexes undergo photo-
isomerization. The anticancer activities of these complexes were evaluated in wild-type (A2780) and cisplatin-
resistant (A2780CP70) ovarian cancer cells. All three complexes exhibited IC50 values below 10 μM and dis-
played similar activity in both A2780 and A2780CP70 cell lines, indicating that they are not cross-resistant with
cisplatin. The DNA-binding properties of 1–3 were investigated by circular dichroism spectroscopy and by
agarose gel electrophoresis. Both studies suggest that 1 and 2 form monofunctional DNA adducts.
Photodynamic therapy
1. Introduction
ligand photosubstitution reactions to form cytotoxic complexes or re-
lease biologically active ligands [14–16]. Such strategies are potentially
Cisplatin and its second-generation analogue carboplatin are FDA-
approved chemotherapeutic drugs that are used as part of the first-line
treatment for several different types of cancer [1,2]. These relatively
simple coordination complexes of platinum(II) have had a significant
impact on cancer survival [3]. Particularly, the cure rates of testicular
cancer, once a relatively fatal disease, now exceed 80% with platinum-
based chemotherapy [4,5]. While these drugs are useful for other
cancer types as well, the development of resistance to cisplatin is a
common phenomenon that severely hinders patient survival [6]. An
additional limitation of these drugs lies in the toxic side effects that they
induce [7–10], which are a consequence of their lack of cancer cell
selectivity.
The use of light to activate cytotoxic agents is an approach that is
employed clinically in the form of photodynamic therapy (PDT) [11].
This strategy imparts cytotoxicity selectively to regions of light irra-
diation, minimizing systemic toxic side effects. While most PDT agents
act as sensitizers for singlet oxygen [12,13], recent research efforts have
focused on the development of inorganic complexes that undergo
more effective than singlet oxygen sensitization because solid tumors
often exist in a state of hypoxia where the low O2 concentration makes
this process ineffective.
In this study, we aimed to explore a different strategy for the de-
velopment of photoactivated anticancer agents, one which uses light as
a means to change the ligand configuration on the platinum(II) center
to modulate the biological activity of these complexes. This approach
has been successfully implemented using platinum(II) compounds
bearing photoreactive 1,2-dithienylethene-containing ligands [17].
Here, we explored platinum(II) coordination complexes containing
azobenzene-based ligands, which are known to undergo E → Z iso-
merization upon irradiation with light [18]. We hypothesized that a
change in ligand configuration would alter the ability of the complex to
interact with biological targets such as DNA. Additionally, azobenzene-
based ligands have effectively been employed in the development of
potent anticancer complexes of platinum [19,20], ruthenium [21,22],
and osmium [23]. We prepared two bidentate azobenzene-based li-
gands, and three platinum(II) complexes. The photoreactivities of these
DOI of original article: http://dx.doi.org/10.1016/j.jinorgbio.2017.06.003
☆
A publisher's error resulted in this article appearing in the wrong issue. The article is reprinted here for the reader's convenience and for the continuity of the special issue. For
citation purposes, please use Journal of Inorganic Biochemistry 174 (2017) 102–110.
⁎
Corresponding authors.
E-mail addresses: oscar.palacios@uab.cat (Ò. Palacios), jjw275@cornell.edu (J.J. Wilson).
http://dx.doi.org/10.1016/j.jinorgbio.2017.07.035
Received 5 April 2017; Received in revised form 13 May 2017; Accepted 12 June 2017
0162-0134/©2017ElsevierInc.Allrightsreserved.
Pleasecitethisarticleas:Wilson,J.,JournalofInorganicBiochemistry(2017),http://dx.doi.org/10.1016/j.jinorgbio.2017.07.035

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
complexes and the in vitro anticancer activities in wild-type and cis-
platin-resistant ovarian cancer cell lines were investigated. Although
the compounds were found to be inert to light irradiation, we have
discovered that these complexes possess potent anticancer activities
that are not susceptible to platinum-resistance mechanisms.
flash column chromatography (SiO2, 9:1 CH2Cl2:EtOAc) to obtain a
pink solid (43.3 mg, 0.062 mmol, 26%). 1H NMR (400 MHz, CDCl3): δ
7.73 (d, J = 8.25 Hz, 2H), 7.68–7.62 (m, 2H), 7.45–7.36 (m, 2H),
7.14–7.05 (m, 5H), 3.33 (s, 3H), 3.27 (s, 3H), 2.29 (s, 3H). 13C{1H}
NMR (100 MHz, CDCl3): δ 156.6 (d, J = 202 Hz), 155.3, 148.0, 142.5,
139.2, 136.8, 132.9, 132.3, 131.2 (t, J = 9.05 Hz), 129.1, 127.9, 124.5,
124.1, 112.1 (dd, J = 20.1 Hz, J = 3.5 Hz), 45.0, 44.4, 21.6. 19F{1H}
NMR (376 MHz, CDCl3): δ −119.8, −120.5. 195Pt NMR (128 MHz,
CDCl3): δ −2378. IR (ATR, cm−1): 3014 w, 2927 w, 2853 w, 1616 w,
1596 m, 1468 m, 1423 m, 1317 m, 1279 m, 1249 m, 1141 vs, 1084 s,
1018 s, 948 s, 914 s, 856 s, 764 s. UV–Vis (CH2Cl2): λmax (ε) 260 nm
(1.26 × 104 M−1·cm−1); 328 nm (5.48 × 103 M−1·cm−1); 537 nm
(2.06 × 103 M−1·cm−1). HR-MS (positive ion mode): m/z 718.0134
2. Experimental
2.1. General considerations
Reactions were carried out under normal atmospheric conditions
with no efforts to exclude moisture or oxygen. Solvents used were HPLC
grade. The compounds (E)-2-(4-methylphenylsulfonamido)-2′,6′-di-
fluoroazobenzene (HL1) and 2,6-difluoronitrosobenzene were synthe-
sized as previously described [24] from commercially available re-
agents. The complex cis-[Pt(DMSO)2Cl2] was prepared from K2PtCl4, as
previously reported [25].
([M + Na]+
, calcd. 718.0135). Anal. Calcd. for 1·CHCl3 (C22
H21Cl4F2N3O3PtS2): C, 32.45; H, 2.60; N, 5.16. Found: C, 32.18; H,
2.46; N, 4.96.
2.3.3. Syntheses of [Pt(L2)Cl(DMSO)] (2) and [Pt(L2)2] (3)
2.2. Physical measurements
To a solution of HL2 (24.5 mg, 0.10 mmol) and NaOH (5.5 mg,
0.14 mmol) in methanol (1.6 mL) was added a suspension of cis-[Pt
(DMSO)2Cl2] (42.8 mg, 0.10 mmol) in acetone (3.2 mL). The mixture
was heated at 45 °C for 2 h. After allowing the mixture to cool to room
temperature, the solvent was removed under reduced pressure. The
crude product was purified by flash column chromatography (SiO2,
gradient of 1:5 hexane:EtOAc to 100% EtOAc) to separate complex 3,
which eluted first, as a purple crystalline solid (12 mg, 0.018 mmol,
18%) and complex 2, a red solid (47 mg, 0.086 mmol, 86%).
Elemental analyses were carried out by Atlantic Microlab, LLC
(Norcross, Georgia, USA). NMR spectra were acquired on a 500 MHz
Bruker AV III HD spectrometer equipped with a broadband Prodigy
cryoprobe or on a Varian INOVA 400 MHz spectrometer. 1H and
13C{1H} NMR spectra were referenced using residual protic solvent
peaks [26], relative to tetramethylsilane (TMS) at 0 ppm. 195Pt, 19F
{1H}, and 15N NMR chemical shift scales are referenced relative to
Na2PtCl6 in D2O, neat CFCl3, and liquid NH3, all set at 0 ppm. UV–vis
spectra were acquired using an Agilent Cary 8454 UV–visible spectro-
photometer. IR spectra were acquired on a Bruker Hyperion ATIR with
ZnSe ATR attachment for solid powders. High-resolution mass spectra
(HRMS) were obtained with an Exactive Orbitrap mass spectrometer in
positive ESI or DART mode (ThermoFisher Scientific, Waltham, MA).
Photoisomerization studies were carried out with continuous irradia-
tion using a 6-watt UVP UVGL-55 handheld UV lamp at 254 nm and
365 nm.
Complex 2: 1H NMR (500 MHz, CDCl3): δ 7.74 (d, J = 8.2 Hz, 1H),
7.63 (t, J = 7.3 Hz, 1H), 7.35–7.27 (m, 1H), 7.24 (d, J = 8.5 Hz, 1H),
7.03–6.96 (m, 2H), 6.85 (t, J = 7.3 Hz, 1H), 3.48 (s, 6H). 13C{1H} NMR
(100 MHz, CDCl3): δ 156.6 (d, J = 200 Hz), 154.6, 149.4, 140.0, 137.9,
133.3, 128.5 (t, J = 10.12 Hz), 120.6, 118.6, 110.8 (dd, J = 19.0 Hz,
J = 4.5 Hz), 46.0. 19F{1H} NMR (376 MHz, CDCl3): δ −121.8. 195Pt
NMR (128 MHz, CDCl3): δ −2430. IR (ATR, cm−1): 3060 w, 3007 w,
2920 w, 2851 w, 1610 m, 1599 m, 1477 vs, 1394 s, 1307 s, 1291 s,
1250 m, 1146 s, 1009 vs, 959 m, 786 s, 753 s. UV–Vis (CH2Cl2): λmax
(ε) 302 nm, (1.05 × 104 M−1·cm−1); 496 nm, (5.68 × 103 M−1·cm−1).
DART-MS (positive ion mode): m/z 542.0072 ([M + H]+
, calcd.
2.3. Synthesis
542.0080). Anal. Calcd for 2 (C14H13ClF2N2O2PtS): C, 31.03; H, 2.42;
N, 5.17. Found: C, 31.30; H, 2.36; N, 5.22.
2.3.1. Synthesis of (E)-2-((2,6-difluorophenyl)diazenyl)phenol (HL2)
Complex 3: 1H NMR (500 MHz, CDCl3): δ 7.70 (d, J = 8.3 Hz, 1H),
7.58 (t, J = 7.2 Hz, 1H), 7.29 (d, J = 8.3 Hz, 1H), 7.12–7.04 (m, 1H),
6.79 (t, J = 7.3 Hz, 1H), 6.69 (m, 2H). 13C{1H} NMR (100 MHz,
CDCl3): δ 154.9 (d, J = 200 Hz), 153.9, 151.7, 143.6, 137.5, 133.7,
129.0 (t, J = 9.3 Hz), 121.9, 119.1, 112.6 (dd, J = 19.9 Hz,
J = 3.64 Hz, 2C). 19F{1H} NMR (376 MHz, CDCl3): δ −117.36. 195Pt
NMR (128 MHz, CDCl3): δ −1006. IR (ATR, cm−1): 3064 w, 2923 w,
2852 w, 1734 m, 1605 s, 1593 s, 1526 m, 1471 s, 1380 s, 1308 s,
1238 s, 1180 s, 1140 s, 1015 s, 944 s, 847 s, 779 s, 747 vs. UV–Vis
(CH2Cl2): λmax (ε) 259 nm (3.69 × 104 M−1·cm−1); 322 nm
(2.11 × 104 M−1·cm−1); 426 nm (1.27 × 104 M−1·cm−1); 538 nm
(7.05 × 103 M−1·cm−1); 592 nm (7.04 × 103 M−1·cm−1). DART-MS
(positive ion mode): m/z 662.0763 ([M + H]+, calcd. 662.0779). Anal.
Calcd. for 3·0.5EtOAc (C26H18F4N4O3Pt): C, 44.26; H, 2.57; N, 7.94.
Found: C, 44.54; H, 2.31; N, 8.35.
To
a
flask containing 2,6-difluoronitrosobenzene (1.299 g,
9.08 mmol) was added acetic acid (20 mL), inducing a color change
from brown to green. Then, 2-aminophenol (991 mg, 9.08 mmol) was
added, and the mixture was stirred at room temperature for 2 d. The
solvent was evaporated under reduced pressure, and the crude product
was purified by flash column chromatography using SiO2 as the sta-
tionary phase and hexanes as the mobile phase to afford HL2 as an
orange crystalline solid. (200 mg, 0.85 mmol, 9%) 1H NMR (400 MHz,
CDCl3): δ 12.30 (s, 1H), 7.99 (d, J = 8.2 Hz, 1H), 7.44–7.32 (m, 2H),
7.12–7.03 (m, 4H). 13C{1H} NMR (100 MHz, CDCl3): δ 156.2 (dd,
J = 261.2 Hz, J = 4.1 Hz), 152.5, 138.8, 134.5, 133.6, 130.9 (t,
J = 10.94 Hz), 128.9, 120.2, 118.61, 112.1 (dd, J = 23.8 Hz,
J = 4.1 Hz). 19F{1H} NMR (376 MHz, CDCl3): δ −120.41. IR (ATR,
cm−1): 3099 w, 3066 w, 2922 w, 2852 w, 1614 s, 1589 s, 1470 vs,
1411 s, 1358 m, 1323 w, 1289 s, 1271 s, 1227 s, 1146 s, 1111 m, 1025
vs, 818 s, 785 s, 754 vs. DART-MS (positive ion mode): m/z 235.0674
([M + H]+, calcd. 235.0677). Anal. Calcd. for C12H8F2N2O: C, 61.54;
H, 3.44; N, 11.96. Found: C, 61.37; H, 3.61; N, 11.84.
2.4. X-ray crystallography
Single crystals of 1 were grown by the slow evaporation of a solu-
tion of 1:1 CH2Cl2:CHCl3, and crystals of 2 and 3 were grown by the
slow evaporation of a solution mixture of hexane and EtOAc. These
crystals were mounted on a Bruker X8 Kappa diffractometer coupled to
an ApexII CCD detector with graphite-monochromated Mo Kα radiation
(λ = 0.71073 Å), and cooled to 223 K under a nitrogen cold-stream
during data collection. The program SHELXT was used to solve the
structures via intrinsic phasing [27], which were refined on all data by
2.3.2. Synthesis of [Pt(L1)Cl(DMSO)] (1)
Triethylamine (61.8 mg, 85.2 μL, 0.61 mmol) was added to a flask
containing HL1 (118.3 mg, 0.31 mmol) in methanol (28 mL). After
stirring for 5 min, solid cis-[Pt(DMSO)2Cl2] (99.2 mg, 0.24 mmol) was
added, and the mixture was heated at 45 °C for 2 h. The solution was
then allowed to cool to room temperature, and the solvent was eva-
porated under reduced pressure. The crude material was purified by
2

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Table 1
the rb values obtained after dialysis at a given rf value. A 10 μL quantity
of each sample was mixed with 3 μL of 5× Orange DNA Loading Dye,
and 10 μL of this mixture was loaded on a slab of 1% agarose gel. The
agarose gel was subjected to electrophoresis in 0.5× TAE pH 8 buffer
at 80 V for 1 h. The gel was then removed from the electrophoresis tank
and mixed gently in a solution containing GelStar Nucleic Acid (Lonza)
stain for 1 h. The DNA bands were visualized with a ChemiDoc MP
Imaging System (BioRad) by fluorescence.
X-ray crystallographic data collection and refinement parameters for 1–3.
1
2
3
Formula
Fw
Space group
a, Å
b, Å
C23H22Cl7F2N3O3S2Pt
933.79
C14H13ClF2N2O2SPt
541.86
P21/m
10.7585(4)
23.2042(9)
10.9692(4)
90
96.571(2)
90
2720.39(18)
6
1.985
223(2)
8.025
C24H14F4N4O2Pt
661.48
Pbca
15.6646(17)
15.514(2)
18.009(2)
90
90
90
4376.6(9)
8
2.008
P
1
10.5085(6)
10.9522(7)
15.7656(10)
83.455(3)
81.522(3)
64.594(3)
1618.48(18)
2
c, Å
α, deg
β, deg
γ, deg
V, Å3
Z
2.6. Circular dichroism measurements
Circular dichroism (CD) measurements were performed using a
JASCO spectropolarimeter (model J-715, JASCO, Groß-Umstadt,
Germany) controlled with the J700 software, (JASCO, Groß-Umstadt,
Germany). Samples were analyzed in 1-cm capped quartz cuvettes, and
the temperature was maintained at 25 °C with a Peltier PTC-351S
holder (TE Technology, Traverse City, MI, USA). Spectra were pro-
cessed using the GRAMS 32 software (Thermo Fisher Scientific,
Waltham, MA, USA). Stock solutions of calf thymus DNA (3.5 mM,
Sigma Aldrich), Tris HCl (400 mM), and platinum complexes (15 mM)
were used to prepare the different samples. The DNA concentration was
determined spectrophotometrically at 260 nm by using the molar ab-
sorptivity value of 6600 M−1·cm−1 per nucleotide. Cisplatin and 1–3
were incubated at 37 °C for 16 h with 50 μM DNA in pH 7.4 10 mM Tris
HCl at platinum to nucleotide ratios (ri) of 0.2, 0.5, and 1 prior to
analysis by CD. Each sample was scanned twice in a range of wave-
lengths between 230 and 310 nm. The ellipticity values are given in
millidegrees (mdeg).
ρ
calcd, g·cm−3
T, K
1.916
223(2)
5.085
223(2)
6.477
μ(Mo Kα),
mm−1
Θ range, deg
Completeness to
Θ (%)
Total no. of data
No. of unique
data
2.062 to 30.034
99.7
1.869 to 30.033
99.9
2.166 to 28.699
99.9
36,781
9441
34,063
8132
47,612
5647
No. of
373
337
316
parameters
R1a (%)
2.77
7.48
1.082
1.888, −1.300
2.83
5.69
1.036
1.267, −1.451
1.86
3.98
1.018
0.398, −0.724
wR2b (%)
GOFc
Max, min peaks,
e·Å−3
a
R1 = Σ||Fo| − |Fc||/Σ|Fo| for I > 2σ.
wR2 = {Σ[w(Fo2 − Fc2)2] / Σ[w(Fo2)2]}1/2 for I > 2σ.
b
c
GoF = {Σ[w(Fo2 − Fc2)2] / (n − p)}1/2, where n is the number of data and p is the
number of refined parameters.
2.7. Cell culture and cytotoxicity assays
full-matrix least squares with SHELXL [28]. Non-hydrogen atoms were
refined anisotropically. Hydrogen atoms were placed at calculated po-
sitions using a riding model, and were refined isotropically with
thermal displacement parameters constrained to be either 1.2 or 1.5
(for terminal CH3) times that of the atom to which it's attached. For
compound 2, two voids with disordered electron density within the unit
cell were modeled with the SQUEEZE algorithm [29]. These voids each
occupied a volume of 170 Å3 and accounted for the scattering con-
tributions of 59 e−, which we attribute to two disordered molecules of
ethyl acetate. Additionally, two molecules of 2 were present in the
asymmetric unit. One molecule lies on the general position and the
other lies on a crystallographic mirror plane. Crystallographic data
collection and refinement parameters are collected in Table 1.
A2780 (ovarian cancer) and A2780CP70 (cisplatin-resistant ovarian
cancer) cell lines [30] were provided by the Cell Culture Facility of Fox
Chase Cancer Center (Philadelphia, PA). These cells were cultured as
monolayers with Roswell Park Memorial Institute (RPMI)-1640 culture
media supplemented with 10% fetal bovine serum in a humidified in-
cubator at 37 °C with an atmosphere of 5% CO2. Cells were passed at
80–90% confluence, using trypsin/EDTA. Cells were tested monthly for
mycoplasma contamination with the PlasmoTest™ mycoplasma detec-
tion kit from InvivoGen.
Cisplatin was dissolved in phosphate-buffered saline (PBS, pH 7.4)
to prepare a 2 mM stock solution. Stock solutions of ligands HL1 and
HL2 were prepared as 10 mM solutions in DMSO. The platinum com-
plexes 1–3 were also prepared as 10 mM stock solutions in either DMSO
or DMF. Cells were grown to 80–90% confluence, detached with
trypsin/EDTA, seeded in 96-well plates at 4000 cells/well in 100 μL of
growth media, and incubated for 24 h. The medium was removed and
replaced with fresh medium (200 μL) containing varying concentrations
of either cisplatin, HL1, HL2, or 1–3. The maximum DMSO and DMF
content of these solutions did not exceed 1% and 0.25%, respectively.
After incubating for 72 h, the medium was removed from the wells, and
3(4,5-dimethylthiazol-2-yl)-2,5-tetrazolium bromide (MTT) in RPMI
(200 μL, 1 mg/mL) was added. After 4 h, the MTT/RPMI solution was
removed, and the formazan crystals were dissolved in 200 μL of an 8:1
mixture of DMSO and pH 10 glycine buffer. The absorbance at 570 nm
in each well was measured using a BioTek Synergy HT plate reader. Cell
viability was determined by normalizing the absorbance of the treated
wells to untreated wells. The concentration of the compound versus %
viability was plotted to produce the dose-response curves, which were
analyzed using a logistic sigmoid function fit with MagicPlot Pro soft-
ware. The reported IC50 values are the average of three independent
experiments with six replicates per concentration level.
2.5. Agarose gel electrophoresis
A stock solution of the closed supercoiled pUC19 plasmid DNA
(1000 μg/mL) was used for electrophoretic mobility experiments.
Solutions of pUC19 plasmid DNA (15 μg/mL) in Tris HCl (10 mM,
pH 7.4) were mixed with solutions of 1–3 and cisplatin to final con-
centrations ranging from 0 to 125 μM (ri = 0–0.20) in a total volume of
100 μL. Complexes 1–3 were diluted from DMF solutions and cisplatin
from an aqueous solution. These samples were incubated for 16 h at
37 °C. After incubation, the samples were dialyzed using Fisher brand
dialysis membrane (regenerated cellulose) with a molecular weight
cutoff of 6000–8000 Da against 1.0 L of TE (10 mM Tris HCl, 1 mM
EDTA, pH 7.4) buffer for 2–3 h to remove unbound platinum. The
dialyzed samples were analyzed using a NanoDrop 1000 (Thermo
Scientific, Waltham, MA) to verify plasmid concentration (μg/mL) and
a Perkin Elmer PinAAcle 900z graphite furnace atomic absorption
spectrometer (GFAAS) to determine bound platinum concentration. The
ratio of platinum atoms to nucleotides from these measurements was
used to determine the rb value. Table S1 (Supplementary data) shows
3

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Scheme 1. Synthesis of HL1 and HL2.
3. Results and discussion
3.1. Synthesis and characterization
The synthetic scheme for the azobenzene ligands HL1 and HL2 is
shown in Scheme 1. The syntheses commenced from commercially
available 2,6-difluoroaniline, which was oxidized with oxone to yield
2,6-difluoronitrosobenzene. The reaction of this reagent with N-tosyl-o-
phenylenediamine in acetic acid under reflux affords HL1 [24]. The
ligand HL2 was obtained from the reaction of 2-aminophenol and 2,6-
difluoronitrosobenzene under conditions similar to those used for HL1.
These ligands were characterized by 1H, 13C{1H}, and 19F{1H} NMR
spectroscopy after purification by column chromatography.
The reactions of HL1 and HL2 with cis-[Pt(DMSO)2Cl2] were in-
vestigated (Scheme 2). The reaction with HL1 afforded primarily a
single platinum-containing product as a pink solid, 1. From the reaction
of HL2, two compounds could be isolated and separated by column
chromatography; a red solid, 2, was the major product, and a purple
solid, 3, was the minor product. Notably, when the reaction was carried
out using lower concentrations, the yield of 2 increased. Mass spec-
trometry was consistent with formulation of the complexes as [Pt(L1)Cl
(DMSO)] (1), [Pt(L2)Cl(DMSO)] (2), and [Pt(L2)2] (3), as molecular
ion peaks with the expected masses and isotopic patterns corresponding
to [M + H]+ and [M + Na]+ were observed. The 195Pt NMR chemical
shifts of complexes 1–3 are −2378, −2430, and −1006 ppm, respec-
tively, all falling within the range expected for platinum in the +2
oxidation state [31,32]. The chemical shift of 3 is consistent with an
N2O2 coordination environment [31]. The chemical shifts of 1 and 2 are
upfield of 3 by > 1000 ppm, which reflects coordination of the soft S-
donor DMSO ligand to these platinum centers [33].
Scheme 3. Five- and six-membered chelate rings possible for L1− and L2−
.
with 15N NMR spectroscopy [34]. To determine the chelate ring forms
in 2, we employed 2-D 1H,15N-HMBC and 19F,15N-HMBC NMR spec-
troscopic experiments (Fig. 1). For HL2, two cross peaks were observed
in the 1H,15N-HMBC spectrum corresponding to a correlation between
N2 and the ortho CH hydrogen (15N δ = 531 ppm) and between N1 and
the ortho OH hydrogen (15N δ = 443 ppm). The latter correlation is
most likely mediated via a hydrogen bond. Similar correlations re-
sulting from strong hydrogen bonds have been observed in 1H,31P-
HMBC spectra [35]. The 19F,15N-HMBC NMR spectra showed that the
fluorine atoms correlate to both nitrogen nuclei. Upon coordination to
platinum(II), the 1H,15N-HMBC spectrum of 2 displays a single cross-
The ligands L1− and L2− can coordinate to metal ions in two dif-
ferent manners, forming either a five-membered or six-membered
chelate ring (Scheme 3). The assignment of chelate rings for related
dihydroxydiarylazo platinum(II) compounds was previously established
peak arising from the correlation between N2 and an ortho CH (15
N
Scheme 2. Reactions of cis-[Pt(DMSO)2Cl2] with HL1 and
HL2.
4

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Fig. 1. 2-D 1H,15N-HMBC and 19F,15N-HMBC NMR spectroscopy of HL2 and 2 in CDCl3 at 25 °C.
δ = 461 ppm). The cross-peak from the OH group is no longer present
because this group is deprotonated when coordinated to platinum(II).
The 19F,15N-HMBC NMR spectrum, however, only shows a single cross-
peak as well. No correlation with N1 is observed. The loss of this cor-
relation most likely results from the increase in the relaxation rate of
this nitrogen nucleus due to direct coordination to the platinum center.
The platinum center relaxes the coordinated 15N nucleus effectively via
chemical shift anisotropy, a dominant relaxation mechanism for heavy
nuclei. Therefore, these experiments indicate that the platinum is co-
ordinated to N1, forming a six-membered ring.
3.2. X-ray crystal structures
Complexes 1–3 were characterized structurally by X-ray crystal-
lography, as shown in Figs. 2–4. For 2, two molecules are present in the
asymmetric unit; both are similar with respect to interatomic distances
and angles. All three structures reveal square planar coordination
geometries, as expected for complexes of platinum(II). As predicted by
the 2-D NMR experiments, ligands L1− and L2− form six-membered
chelate rings in all three complexes as well. By contrast, ruthenium and
rhodium complexes bearing related sulfonamide-diazo donor ligands
exclusively form five-membered chelate rings [24,36]. Complexes 1
and 2 contain a molecule of DMSO coordinated through the sulfur
donor and chloride ligand in addition to L1− and L2−, respectively. In
both structures, the chloride ligand sits trans to the coordinated diazo
nitrogen donor. The PteS(DMSO) distance is longer in complex 1
(2.237 Å) than in 2 (2.225 and 2.227 Å), presumably due to the greater
trans influence of the sulfonamide donor compared to the phenolate
donor. Additionally, the chelate ring geometry found in 1 is sub-
stantially different than that found in 2. In 2, this six-membered chelate
ring is planar, as reflected by the fact that one molecule in the asym-
metric unit resides on a crystallographic mirror plane. The NePteO
angle in complex 2 is 90°. By contrast, the chelate ring in 1 attains a
boat conformation. As a result, the N1ePteN3 angle is 85°, deviating
substantially from the ideal 90° angle for a square planar complex.
The structure of complex 3 (Fig. 4) reveals two L2− ligands co-
ordinated to the central platinum(II) ion. The two six-membered che-
late rings, like those in 2, are nearly planar and give rise to NePteO
angles of 90°. The two L2− ligands are arranged such that the nitrogen
Fig. 2. X-ray crystal structure of 1. Ellipsoids are drawn at the 50% probability level and
hydrogen atoms are omitted for clarity. Selected interatomic distances (Å): PteCl1
2.2967(9), PteS2 2.2368(7), PteN1 2.018(3), PteN3 2.037(2). Selected angles (°):
N1ePteN3 84.99(10), N1ePteS2 94.46(7), N3ePteS2 174.31(8), N1ePteCl1
175.27(7), N3ePteCl1 90.29(8), S2ePteCl1 90.27(3).
and oxygen donor atoms of the two ligands reside in a cis configuration.
This arrangement of the ligands gives rise to a π-stacking interaction
between 2,6-difluorophenyl substituents (centroid–centroid distance
3.25 Å), which may stabilize this isomer over the trans isomer. The
PteN and PteO interatomic distances are in the expected range, falling
just under 2 Å. The crystal structure of a related platinum(II) complex
of analogous ligands, where the 2,6-difluorophenyl groups are tosyl
groups, has been reported [37]. This structure also reveals the platinum
(II) complex to be in the cis configuration and shows a π-stacking
5

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Table 2
Absorbance maxima and molar absorptivities for HL2 and 1–3 in CH2Cl2.
Compound
λ )
max, nm (ε, M−1·cm−1
HL2
1
320 (18,620), 384 (8260)
328 (5480), 537 (2060)
2
302 (10,480), 496 (5680)
3
322 (21,130), 426 (12,720), 538 (7050), 592 (7040)
Fig. 3. X-ray crystal structure of one of the molecules of 2 present in the asymmetric unit.
Ellipsoids are drawn at the 50% probability level and hydrogen atoms are omitted for
clarity. Selected interatomic distances (Å): Pt1eO1 1.993(2), Pt1eN2 2.005(3), Pt1eS1
2.2248(9), Pt1eCl1 2.3105(9). Selected angles (°): O1ePt1eN2 90.31(10), O1ePt1eS1
170.58(7), N2ePt1eS1 99.04(8), O1ePt1eCl1 83.25(7), N2ePt1eCl1 173.15(8),
S1ePt1eCl1 87.46(3).
Fig. 5. UV–vis absorbance spectra of 1 (green solid), 2 (red short dash), and 3 (blue long
dash) in CH2Cl2 at 25 °C.
hydrogen bonding between the ortho-hydroxy group and a diazo ni-
trogen atom inhibits photoisomerization, giving rise to excited state
intramolecular proton transfer as an alternative photoreaction [39].
The absorbance maxima and molar absorptivities of the platinum(II)
complexes 1–3 are collected in Table 2 and shown in Fig. 5. Low-energy
features ranging from 496 to 600 nm in the complexes are assigned as
metal-to-ligand charge transfer (MLCT) transitions and high-energy
features at wavelengths < 350 nm are assigned as intraligand π–π*
transitions. Irradiation of the three complexes with 254 and 365 nm
light failed to induce photoisomerization. When related ligands, how-
ever, are coordinated to ruthenium or rhodium, photoisomerization
occurs readily [24,36,40]. A key difference between these ruthenium
and rhodium complexes and the platinum(II) complexes studied here is
the nature of the chelate ring. Five-membered rings are observed for the
ruthenium and rhodium complexes, whereas six-membered rings are
present in 1–3. The six-membered chelate rings effectively lock the
azobenzene group in the E configuration. Photoisomerization in these
complexes would require partial ligand dissociation to allow rotation
about the NeN bond.
Fig. 4. X-ray crystal structure of 3. Ellipsoids are drawn at the 50% probability level and
hydrogen atoms are omitted for clarity. Selected interatomic distances (Å): PteO2
1.9866(16), PteN4 1.9910(19), PteO1 1.9916(15), PteN2 1.9997(19). Selected angles
(°): O2ePteN4 90.32(7), O2ePteO1 80.64(7), N4ePteO1 170.70(8), O2ePteN2
170.38(7), N4ePteN2 99.16(8), O1ePteN2 89.94(7).
3.4. In vitro anticancer activity
The in vitro anticancer activities of the platinum(II) complexes and
free ligands were evaluated in wild-type (A2780) and cisplatin-resistant
(A2780CP70) ovarian cancer cell lines [30] (Table 3). Cisplatin was
used as a positive control in these studies. Consistent with the platinum-
resistant phenotype of the A2780CP70 cell lines, the IC50 value of cis-
platin in this cell line is 28.6 times larger than that found in the wild-
type cell line A2780. Although the ligand HL2 is only moderately cy-
totoxic, HL1 exhibits IC50 values in the micromolar range and is equally
effective in both resistant and wild-type cells. Because DMSO is known
to significantly impact the activity of metal-based anticancer agents
[41,42], we explored the use of both DMSO and DMF as solubilizing
agents for 1–3. Like HL1, all of the platinum complexes tested with
either DMSO or DMF give rise to IC50 values that are < 10 μM, sig-
nifying potent in vitro anticancer activity. The IC50 values measured in
interaction between the tosyl groups, indicating that this structure type
may be general for platinum(II) complexes with this class of ligands.
3.3. Photophysical properties
Because azobenzene derivatives are known to undergo photo-
isomerization reactions [38], the photophysical properties of HL2 and
1–3 were investigated. The photophysical properties of HL1 were pre-
viously reported [24]. This ligand undergoes efficient photoisomeriza-
tion from the E to Z isomer upon irradiation with light at 315 nm [24].
By contrast, HL2 showed no sign of photoisomerization when irradiated
at either 365 or 254 nm. The lack of photoreactivity of 2-hydro-
xyazobenzenes has been documented before [39]. The intramolecular
6

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Table 3
IC50 values of ligands HL1 and HL2 and 1–3 in A2780 and A2780CP70 cell lines.
Samples
Solubilizing solvent
IC50 (μM)
RFa
A2780
A2870CP70
HL1
HL2
1
1
2
2
3
3
DMSO
DMSO
DMSO
DMF
DMSO
DMF
DMSO
DMF
PBS
7
51
2
4
7.5
66
2.7
0.8
10
3.2
7.0
3.1
5
0.1
3
0.6
0.1
3
0.3
0.1
0.6
1.1
1.3
2.7
1.9
2.8
2.0
1.1
1.1
28.6
1.0
0.43
3.4
1.60
6.5
2.7
0.18
0.3
0.05
0.7
0.09
0.1
0.9
Fig. 6. Agarose (1%) gel electrophoresis of a pUC19 plasmid in the presence of 1 (left
lanes) and 2 (right lanes) at different rb values.
thymus DNA, only minor changes in the CD spectra were observed
(Fig. 7). For 1, the changes are more pronounced at similar ratios. The
changes, however, are still not as marked as expected from bifunctional
platinum DNA adducts [46]. For example, cisplatin leads to a sig-
nificant increase in the CD feature at 280 nm at the much smaller rb
value of 0.10 [47]. Therefore, these CD studies, like the plasmid studies,
are consistent with 1 and 2 binding to DNA in a monofunctional
manner.
Cisplatin
0.07
1
a
RF = resistance factor, the ratio of the IC50 values measured for the resistant and
wild-type cell lines.
the presence of DMSO were approximately 2–3 times larger than those
measured using DMF. For comparison, the IC50 value of cisplatin dis-
solved in DMSO is sixty times greater than its IC50 value in aqueous
buffer [41]. Therefore, we conclude that DMSO has only a minor effect
on the anticancer activity of these complexes. The ratio of the IC50
values in the cisplatin-resistant and wild-type cell lines, or the re-
sistance factors (RF), for 1–3 are all < 3, substantially smaller than the
value of 28.6 measured for cisplatin. These results indicate that the
azobenzene platinum(II) complexes studied here are not cross-resistant
with cisplatin. This lack of cross-resistance may signify a novel or dis-
tinct mechanism of action of these complexes relative to cisplatin and
carboplatin.
4. Conclusions
Three platinum(II) complexes bearing azobenzene-based ligands
were synthesized and characterized. Because these compounds form
six-membered chelate rings with these ligands, no photoisomerization
occurs. The ligands in this binding mode are locked into the E con-
formation by coordination to the platinum(II) centers. Nevertheless,
these non-traditional platinum(II) compounds are cytotoxic in ovarian
cancer cell lines. Importantly, they exhibit nearly equal activity against
cisplatin-resistant and wild-type ovarian cancer cell lines, suggesting
that they operate via a novel mechanism of action. DNA-binding studies
suggest that 3, which has no labile binding sites, does not interact with
and distort DNA strongly. Compounds 1 and 2, which bear chloride and
DMSO ligands, weakly distort DNA in a manner that is consistent with
the formation of monofunctional adducts. Therefore, 1 and 2 represent
new members of the growing class of potent monofunctional platinum
(II) anticancer agents [48,49].
3.5. DNA-binding studies
Because the conventional platinum-based drugs induce their antic-
ancer activity by covalently binding to DNA, we investigated the abil-
ities of the azobenzene platinum(II) complexes 1–3 to interact with
DNA. Cisplatin-DNA adducts result in unwinding of double-helical DNA
[43], a phenomenon that is reflected by the reduction of the electro-
phoretic mobility of supercoiled closed circular plasmids bearing such
adducts [44]. The degree of electrophoretic mobility reduction is re-
lated to the amount of unwinding in the double helix that is induced per
platinum-DNA adduct. This unwinding angle, which depends on the
nature of the platinum-DNA adduct, can be determined by the amount
of platinum per nucleotide (rb) required to retard the mobility of the
supercoiled form to match that of the relaxed or nicked form [44].
Bifunctional platinum-DNA intrastrand crosslinks induce unwinding
angles that range from 10 to 15°, whereas those for monofunctional
adducts are < 8%. Irreversible covalent binding of 1 and 2 to DNA was
verified by dialysis of the samples after incubation, which revealed that
significant quantities of platinum remained bound to the plasmid. By
contrast, dialysis of plasmid samples incubated with 3 led to complete
loss of the platinum from the samples, indicating that this compound
does not covalently bind to DNA. This result is expected because 3 does
not bear any labile ligands that could exchange for a nucleobase ligand
on DNA. Analysis of the plasmid samples with adducts of 1 and 2 re-
vealed only minor unwinding of the double helix (Fig. 6). For 1, un-
winding is only apparent at rb values of > 0.06, whereas for 2 no sig-
nificant unwinding is observed up to an rb of 0.2. Based on these
experiments and the known superhelical density for pUC19 [44], it is
estimated that the unwinding angle induced by 1 is < 10°, and the
angle is < 5° for 2. Both of these small unwinding angles are consistent
with a monofunctional DNA-binding mode for these complexes [45].
The changes in the CD spectrum of DNA induced by platinum
compounds reflect the nature of their interactions. Monofunctional
platinum adducts give rise to only minor changes in the CD spectra,
whereas those for bifunctional adducts are more dramatic [46]. When 2
and 3 were incubated at platinum to nucleotide ratios up to 1 with calf
Abbreviations
CD
circular dichroism
DMF
DMSO
EDTA
N,N-dimethylformamide
dimethylsulfoxide
ethylenediamine tetraacetic acid
GFAAS graphite furnace atomic absorption spectroscopy
HRMS
HL1
HL2
IC50
MLCT
MTT
high-resolution mass spectrometry
(E)-2-(4- methylphenylsulfonamido)-2′,6′-difluoroazobenzene
(E)-2-((2,6-difluorophenyl)diazenyl)phenol
50% growth inhibitory concentration
metal-to-ligand charge transfer
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
mide
bro-
PBS
PDT
RF
phosphate-buffered saline
photodynamic therapy
resistance factor
RPMI-1640 Roswell Park Memorial Institute 1640 culture media
TAE
Tris
Tris, acetic acid, EDTA
tris(hydroxymethyl)aminomethane
Acknowledgements
Cornell University is thanked for supporting this research through
start-up funding. Katia G. Samper acknowledges Departament de
Química from UAB for PIF scholarship and UAB for the grant “short stay
for research PIF-UAB 2016”. OP, PB, and KGS also thank the financial
7

K.G. Samper et al.
JournalofInorganicBiochemistryxxx(xxxx)xxx–xxx
Fig. 7. CD spectra of calf-thymus DNA in the presence of 1–3 in pH 7.4 TrisCl buffer. ri([Complex]/[nucleotide]): ri = 0 (black solid line), ri = 0.2 (solid grey line), ri = 0.5 (long dash
line), ri = 1 (short dash line).
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support from MINECO-FEDER (Projects BIO2015-67358-C2-2-P and
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10690–10701, http://dx.doi.org/10.1039/b917753a.
and the Cornell Center for Material Research (CCMR) at Cornell
University, which are supported by the NSF under award numbers CHE-
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Appendix A. Supplementary data
CCDC 1540533–1540535 contains the supplementary crystal-
lographic data for 1–3, respectively. These data can be obtained free of
charge The Cambridge Crystallographic Data Centre via www.ccdc.
cam.ac.uk/data_request/cif. Supplementary data associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.jinorgbio.2017.06.003.
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(II) catecholates showing photocytotoxicity, nuclear uptake, and glutathione-trig-
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