DNA interaction of a fluorescent, cytotoxic pyridinimino platinum(II) complex

Article abstract of DOI:10.1016/j.jinorgbio.2019.110874

New pyridinimino complexes of platinum(II) [PtCl₂(N^N-R)] (N^N = 2-pyridylmethanimino, R = -(CH₂)₂O(CH₂)₂OH, -(CH)₂O(CH₂)₂OCH₂Pyr), Pyr = pyren-1-yl) have been prepared. They are characterized by a dioxygenated alkyl side chain and, in one case, by a fluorescent terminal 1-pyrenyl residue. The complexes were characterized by elemental analysis, IR, ¹H–, ¹³C–and ¹⁹⁵Pt NMR spectroscopies. For [PtCl₂(N^N-(CH₂)₂O(CH₂)₂OH] the molecular structure was determined by single crystal X-ray diffraction. The complexes are soluble and stable in DMSO/H₂O (80/20, v/v). The pyrenyl terminated compound was tested as antiproliferative agent against selected human cancer cell lines. Comparable cytotoxic effect was obtained on human ovarian carcinoma A-2780 and A-2780cis cells, thus suggesting a certain ability to circumvent cisplatin resistance. The interaction of this complex with DNA was investigated by linear flow dichroism and by spectrophotometric (absorbance and fluorescence) titrations. Both techniques enlightened the presence of a complex mode of interaction with DNA, involving both groove binding and intercalation.

Full text of DOI:10.1016/j.jinorgbio.2019.110874

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DNA interaction of a fluorescent, cytotoxic pyridinimino
platinum(II) complex
Riccardo Bondi, Tarita Biver, Lisa Dalla Via, Federica Guarra,
Mariafrancesca Hyeraci, Claudia Sissi, Luca Labella, Fabio
Marchetti, Simona Samaritani
PII:
S0162-0134(19)30488-X
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110874
JIB 110874
Reference:
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
24 July 2019
5 September 2019
28 September 2019
Please cite this article as: R. Bondi, T. Biver, L.D. Via, et al., DNA interaction of
a fluorescent, cytotoxic pyridinimino platinum(II) complex, Journal of Inorganic
Biochemistry (2019), https://doi.org/10.1016/j.jinorgbio.2019.110874
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© 2019 Published by Elsevier.

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Journal of Inorganic Biochemistry
DNA interaction of a fluorescent, cytotoxic pyridinimino platinum(II)
complex
Riccardo Bondi,a Tarita Biver,b,c Lisa Dalla Via,c,d Federica Guarra,a Mariafrancesca
Hyeraci,d Claudia Sissi,d Luca Labella,a,c Fabio Marchetti,a Simona Samaritani*a,c
a) Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Giuseppe Moruzzi 13,
56124 Pisa (Italy)
b) Dipartimento di Farmacia, Università di Pisa, Via Bonanno 6, 56126 Pisa (Italy)
c) Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici
(C.I.R.C.M.S.B.)
d) Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, Via F. Marzolo 5, Padova,
I-35131
Abstract
New pyridinimino complexes of platinum(II) [PtCl2(N^N-R)] (N^N= 2-
pyridylmethanimino, R =-(CH2)2O(CH2)2OH, -(CH2)2O(CH2)2OCH2Pyr), Pyr = pyren-1-
yl) have been prepared. They are characterized by a dioxygenated alkyl side chain and, in
one case, by a fluorescent terminal 1-pyrenyl residue. The complexes were characterized
1
by elemental analysis, IR, H-, 13C- and 195Pt NMR spectroscopies. For [PtCl2(N^N-
(CH2)2O(CH2)2OH] the molecular structure was determined by single crystal X-ray
diffraction. The complexes are soluble and stable in DMSO/H2O (80/20, v/v). The pyrenyl
terminated compound was tested as antiproliferative agent against selected human cancer
* Corresponding Author. email: simona.samaritani@unipi.it
Phone number: +39 0502219261

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cell lines. Comparable cytotoxic effect was obtained on human ovarian carcinoma A-2780
and A-2780cis cells, thus suggesting a certain ability to circumvent cisplatin resistance.
The interaction of this complex with DNA was investigated by linear flow dichroism and
by spectrophotometric (absorbance and fluorescence) titrations. Both techniques
enlightened the presence of a complex mode of interaction with DNA, involving both
groove binding and intercalation.
Keywords: pyridinimino platinum(II) complexes; antiproliferative properties; DNA
interaction; groove binding; intercalation
Introduction
Cisplatin anticancer properties were discovered serendipitously in the Sixties[1] and the
complex was accepted by FDA as a chemotherapeutic drug at the end of the Seventies.[2]
It is nowadays well known that cisplatin main target is DNA.[3][4] After crossing the
cellular membrane, the complex can be activated by hydrolysis, affording positively
charged species that react with DNA coordinating sites and modify the bio macromolecule
structure. This causes various grades of DNA damages that initiate a process that leads to
cellular death. Despite the still wide use of cisplatin in the treatment of cancer, two main
problems remain unsolved and strongly limit the drug efficacy: cisplatin is scarcely
tolerated by treated patients and it is inactive toward resistant cancer cell lines. Resistance
is a multifactorial event generally ascribed also to the ability developed by a cell to repair
DNA damages, or to side reactions occurring before the drug reaches its target. For this
reason, many efforts are ongoing in the attempt to prepare more effective compounds
changing the ligand nature. In particular, steric hindrance on the metal can be enhanced as
in picoplatin,[5][6] to slow down side reactions, while additional functional groups can be
added to favor synergistic effects. Over the last fifteen years, the pyridinimino scaffold

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(Figure 1) has been exploited,[7,8,9,10,11,12,13,14,15] using a steric hindrance on the
metal center similar to picoplatin and changing the side residue R nature through an easy
synthetic route, the reaction of pyridine-2-carboxaldehyde with the suitable amine. The
reported complexes have side residues R, decorated by different functional groups, from
simple alkyl-[9,15] or aryl[8,13,14] substituents, to modified carbohydrates,[12] with
antiproliferative profiles against selected cancer cells, changing with the nature of R.
Mechanisms of action have not always been elucidated, but the reported data seem to
indicate a correlation between the observed cytotoxicity and the side chain flexibility. As a
matter of fact, a relatively low (IC50 ≥ 20 μM) cytotoxicity can be observed in systems
where side functional groups are connected to the pyridinimino scaffold through short and
rigid spacers,[10,12,13] while structural analogues characterized by longer spacers proved
to be more active.[10,16]
Figure 1
Herein the preparation and the antiproliferative activity of a Pt(II) pyridinimino complex,
bearing a fluorescent pyrenyl side substituent is described. A flexible, dioxygenated alkyl
chain has been used as a spacer to link the pyrenyl group to the pyridinimino scaffold. The
interactions of the fluorescent complex with DNA were investigated, by both linear flow
dichroism and spectroscopic (UV-vis and fluorescence) titrations.
Experimental
General
All operations were carried out under dry dinitrogen, if not otherwise stated. Solvents were
purified according to reported methods[17] and stored over dry molecular sieves. 2-(2-
aminoethoxy)ethanol, 2-pyridinecarboxaldehyde, 1-pyrenecarboxaldehyde (Sigma-

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Aldrich) were used without further purification. Cis-[PtCl2(DMSO)2] was prepared
according to a reported procedure.[18]
1H-, 13C- and 195Pt NMR spectra were recorded on a Bruker “Avance DRX 400”
spectrometer, in CDCl3 solution, if not otherwise stated. Chemical shifts (δ ppm) are
13
referred to tetramethylsilane (1H- and C-) and H2PtCl6 in D2O solution (195Pt-, external
standard). When non deuterated solvents were used, a sealed capillary containing C6D6 was
used to lock the instrument. Following abbreviations were used to describe signals: s
(singlet), d (doublet), t (triplet), m (multiplet), br (broad).
IR spectra were recorded at 298 K by an FT-IR Perkin Elmer “Spectrum One”
spectrometer, equipped with ATR accessory. The position of absorption bands were
expressed in cm-1 and the following abbreviations were used to indicate the bands relative
intensities: w (weak), m (medium), s (strong), vs (very strong), sh (shoulder), br (broad).
Elemental analyses were expressed as C, H, N % content and were carried out with an
Elementar “vario MICRO cube” instrument.
When necessary, products were purified by flash chromatography (column diameter 2.5
cm); 60 Å silica gel (40-63 μm pore size) was used as stationary phase.
As for the DNA binding studies, stock solutions of calf thymus DNA were done by
dissolving the sodium salt (Sigma-Aldrich) in water, sonicated (producing short fragments
of ca. 500 base pairs) and standardized spectrophotometrically (ε = 13200 M-1 cm-1 at 260
nm, I = 0.10 M, pH = 7.0 ).[19] Sodium cacodylate (CH3)2AsO2Na 2.5 mM is the pH = 7.0
buffer. UV-Vis absorption spectra were recorded with a double ray Shimazdu “UV-2450”
spectrophotometer. Measurements were carried out using 1.0 cm quartz cuvettes and the
temperature was controlled by a ± 0.1 °C precision thermostat. Spectrofluorimetric
measurements were carried out with a Perkin Elmer “LS55” spectrofluorimeter, equipped
with a pulsed xenon lamp. Quartz cuvettes (1.0 cm) were used and the temperature was

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controlled by a ± 0.1 °C precision thermostat. Solutions were prepared in ultrapure water
(Sartorius “Arium pro®” purification system). In these experiments CD and CP will be used
to indicate respectively the total complex (D, dye) molar concentration and the total DNA
(P, polynucleotide) molar concentration in base pairs.
Linear dichroism (LD) measurements were performed on a Jasco “J500A” circular
dichroism spectropolarimeter, converted for LD, and equipped with an IBM PC and a
Jasco J interface. Linear dichroism was defined as:
LD(λ) = A//(λ) − A(λ)
where A// and A correspond to the absorbances of the sample when polarized light was
oriented parallel or perpendicular to the flow direction, respectively. The orientation was
produced by a device designed by Wada and Kozawa[20] at a shear gradient of 500–700
rpm. Salmon testes DNA was purchased from Sigma-Aldrich. The concentration was
determined by using (see above) an extinction coefficient of 13200 M-1cm-1 at 260 nm for
DNA molar concentration in base pairs.
Volumes of concentrated solutions in DMSO of the examined compounds were added to a
solution of salmon testes DNA (1.0×10-3 M) in milliQ water. Each spectrum was
accumulated twice and recorded at room temperature at [drug]/[DNA] = CD/CP = 0, 0.04,
0.08 and 0.16.
Synthesis of ligands
For the synthesis of compounds 1-5, [21,[22] see Supplementary Information.
Synthesis of platinum(II) complexes. General procedure.[23]
A suspension of [PtCl2(DMSO)2] (162 – 440 mg), in CH3NO2 (10 mL) was treated with the
suitable ligand (ligand/Pt = 1 in moles), at 25 °C, under vigorous stirring. A dark orange
1
solution formed and, after 1-12 h, the complete conversion was checked by H NMR. As

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indicated below, the products precipitated out of the mixture spontaneously or upon
addition of a mixture of solvents, were filtered, washed with Et2O and dried under vacuum.
Dichloro-[N-{2-[2'-(hydroxyethoxy)]ethyl}-pyridyl-2-methanimino]platinum(II), [PtCl2(1)]
(6).
Orange solid (76 % yield). Found: C 25.3, H 3.5, N 5.5 %. C10H14Cl2N2O2Pt∙H2O requires:
C 25.1, H 3.4, N 5.8 %. IR: 3479m, 3084m, 3041m, 2935m, 2903m, 2866m, 1593w,
1562w 1475m, 1432m, 1133s, 1125s, 1070s, 1034s, 1016s, 895m, 883m, 773vs. 1H NMR:
3
3
9.38 (d, 1H, JH-Pt = 37 Hz, NCHCH); 9.11 (s, 1H, JH-Pt = 94 Hz, CHNCH2); 8.17 (d, 1H,
HPy); 7.93 (m, 1H, HPy); 8.40 (m, 1H, HPy); 4.60 (s, 1H, OH); 3.83-4.17 (2 m, 4H,
OCH2CH2OH); 3.47 (m, 4H, CHNCH2CH2). 13C{1H} NMR:172.4, 163.2, 149.5, 141.3,
129.6, 129.0, 72.6, 68.3, 60.5, 59.0. 195Pt{1H} NMR: -2358. Crystals of 6 obtained from
ethanol were suitable for the determination of the structure by single crystal x-ray
diffraction.
Dichloro-[N-{2-[2'-(pyren-1''-ylmethoxy)ethoxy]ethyl}-pyridyl-2-methanimino]platinum(II),
[PtCl2(5)] (7)
EtOH/Et2O (1/5 v/v) was added at 0 °C, under vigorous stirring. Orange solid (87 %).
Found: C 46.3; H 3.6; N 3.9 %. C27H24Cl2N2O2Pt∙H2O requires: C 46.6; H 3.8; N 4.0 %.
IR: 3040m, 2861m, 1595m-br, 1560w, 1474m, 1446m, 1344m, 1305m, 1263w, 1251w,
1236w, 1218w, 1180w, 1125s, 1113s, 1094s, 1033m, 914m, 891w, 877w, 843vs, 838vs,
759vs, 708vs. 1H NMR: 9.05 (d, 1H, 3JHH = 4.9 Hz, 3JHPt = 34.6 Hz, -CHCHNPt-); 8.24 (m,
3
4H); 8.09 (m, 5H); 7.97 (d, 1H, J HH = 7.3 Hz); 6.74 (m, 1H); 6.64 (m, 1H); 5.67 (d, 1H,
3J
= 7.3 Hz); 5.14 (s, 2H, -OCH2Ar); 4.26 (m, 2H, -CHNCH2-); 3.95 (m, 2H, -
HH
NCH2CH2-); 3.83 (m, 2H, -OCH2CH2O-); 3.76 (m, 2H, -OCH2CH2O-). 13C{1H} NMR:
169.2; 156.1, 149.0, 137.9, 131.3, 131.1, 130.6, 129.4, 128.1, 127.7, 127.3 (2C), 126.9,

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126.4 (2C), 125.8, 125.7, 125.5, 124.8, 124.5 (2C), 123.5; 72.0, 70.2, 69.7, 67.9, 59.1. 195Pt
{1H} NMR: - 2355.
Solubility and stability of platinum(II) complexes.
Complexes 6-7 are not soluble in water nor ethanol. They are all well soluble in DMSO
and in DMSO/water mixtures (DMSO/water = 80/20 v/v). Their stability was checked by
1H NMR. In a typical experiment, an NMR test tube was charged with a 2×10-2 M solution
of the complex in DMSO/H2O (80/20 v/v) and 1H NMR spectrum was registered at 30 °C.
1
Temperature was gradually increased to 70 °C and H NMR spectra were registered at 40,
1
50 and 70 °C, showing no significant modification of the signals pattern. The set of H
NMR measurements was repeated after 48h.
Inhibition growth assay
HeLa (human cervix adenocarcinoma cells) were grown in Nutrient Mixture F-12 [HAM]
(Sigma Chemical Co.); A2780 (human ovarian carcinoma wild type) and A2780cis (human
ovarian carcinoma cisplatin-resistant) were grown in RPMI 1640 (Sigma Chemical Co.).
MSTO-211H (human mesothelioma cells) were grown in RPMI 1640 (Sigma Chemical
Co.), Hepes (2.38 g/L), sodium piruvate (0.11 g/L) and glucose (2.5 g/L). 1.5g/L NaHCO3,
10 % heat-inactivated fetal calf serum (Invitrogen), 100 U/mL penicillin, 100 μg/mL
streptomycin, and 0.25 μg/mL amphotericin B (Sigma Chemical Co.) were added to the
media. The cells were cultured at 37 °C in a moist atmosphere of 5 vol.% carbon dioxide in
air. The cells (2.5-3·104), were seeded into each well of a 24 well cell culture plate. After
incubation for 24 h, various concentrations of the test agents were added to the complete
medium and incubated for a further 72 h. Stock solutions of new complexes were made in
DMSO at 20 mM concentration and then diluted with complete medium in such a way that

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the final amount of solvent in each well did not exceed 0.5 vol.%. Cisplatin was dissolved
in 0.9 wt.% NaCl. A Trypan blue assay was performed to determine cell viability.
Cytotoxicity data were expressed as GI50 values, i.e., the concentration of the test agent
inducing 50 % reduction in cell number compared with control cultures.
X-Ray study
Crystals of 6 were selected at room temperature (296 K), glued to glass fibers and analyzed
with a Bruker “Smart Breeze” CCD diffractometer. Table 1 summarizes the lattice parame-
ters and the space group. Intensity data were collected in the range of 2θ angles reported in
Table 1. After correction for Lorentz and polarization effects and for absorption, the struc-
ture solution was obtained using the direct methods contained in SHELXS program.[24]
The final reliability factors of the refinement procedure, done using SHELXL program,[25]
are listed in Table 1. Other control calculations were performed with the programs con-
tained in the WINGX suite.[26]
Supplementary crystallographic data for compound 6 have been deposited with the Cam-
bridge Crystallographic Data Centre (see Tab. 1 for deposition number). These data can be
obtained free of charge by quoting the present paper.
Table 1
Results and Discussion
Synthesis
Chelating pyridinimino ligands were obtained by condensation of 2-
pyridinecarboxaldehyde with the suitable primary amines in refluxing ethanol. While 2-(2-
aminoethoxy)ethanol
is
commercially
available,
2-[2'-(pyren-1''-
ylmethoxy)ethoxy]ethylamine (4) was synthesized from 1-pyrenecarboxaldehyde,
according to the steps reported in Scheme 1 and purified by gradient flash chromatography.

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Scheme 1
The condensation of amines with 2-pyridinecarboxaldehyde afforded the pure chelating
pyridinimino ligands.
The following reaction between the iminopyridine chelating ligands and cis-
[PtCl2(DMSO)2] afforded the corresponding platinum(II) dichloro complexes (Scheme 2),
with very good yields (69-87%).
Scheme 2
Complex 6 was crystallized from a very diluted 80/20 EtOH/Et2O v/v solution and its
structure was determined by single crystal X-ray diffraction (Figure 2). The coordination
geometry around platinum is square planar, with bond lengths and angles in good
agreement with those reported for similar compounds.[11,15,27,28,29,30] The main
contributions to the crystal packing are given by - stacking interactions acting in the a
direction and to OHCl interactions (O(2)Cl(2') 3.354 Å) acting between molecules
organized in head-to-tail rows.
Figure 2
In order to investigate the antiproliferative properties and the interaction with DNA of the
prepared compounds, their solubility was checked. Complexes 6 and 7 are not water
soluble, slightly soluble in ethanol, but well soluble in DMSO/water mixtures. Since it is
known that the presence of water can enhance platinum complexes reactivity towards

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DMSO,[31] the stability of the platinum derivatives in DMSO/water mixtures was checked
by 1H NMR spectroscopy. Representatively, a fresh 2×10-2 M solution of complex 7 in d6-
1
DMSO/D2O (80/20 v/v) was prepared into an NMR tube and its H NMR spectrum was
registered at 30 °C. Spectra were recorded also at 40, 50 and 70 °C. While no new signals
attributable to hydrolysis/substitution products appeared with heating, small (0.01 ≤ Δδ ≤
0.10 ppm) signal displacements were observed throughout the spectra, well ascribable to
temperature changes.[32] Spectra at 30 °C, repeated soon after the heating cycle and after
1
48 h, were superimposable with the H NMR spectrum of the freshly prepared solution
(Fig. S1), proving the stability of the complex in 20 mM DMSO/H2O solutions, the usual
concentration of the stock solutions used for biological tests.
The stability of complex 7 was checked spectroscopically (UV) in more diluted solutions,
for concentrations similar to those used for biological studies (Fig. S2). Stock DMSO
solutions of complex 7 were diluted (final 7 concentration = 1.8×10-5 M) with an aqueous
solution of sodium cacodilate ([NaCac] = 2.5 mM, pH 7.0), with DMSO total amount <
1%. In order to overcome precipitation and/or aggregation phenomena, the working
solutions were heated (50 °C) and then cooled (25 °C). UV spectra (Figure S2A) were
registered at 25 °C on the freshly prepared solution (curve a), at 50 °C (curve b) and at
25 °C after the thermal treatment (curve c). In curve c the spectrum profile is much more
definite, with peaks shape and baseline resembling those obtained for an analogous
solution of 7 in chloroform (Figure S2B), where the complex is well soluble and stable.
The heating procedure, followed by a spectrophotometric check, was repeated for all
working solutions of 7, before titrations.
DNA binding tests

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The possible interaction of 7 with calf thymus DNA was analysed by means of
spectrophotometric and spectrofluorometric titrations. Note that the pyrene fluorophore
both enables the use of these optical techniques and favours the presence of hydrophobic π-
π interactions with DNA base pairs, i.e. an intercalation binding mode which could have
pharmacological significance. The titrations were done by adding increasing amounts of
DNA directly into the cuvette containing the complex solution and were repeated at
different temperatures. Fig. 3 shows an example of spectrophotometric titration (for others
see Fig. S3) and enlightens the presence of hypochromic and bathochromic effects which
confirm that a strong binding does indeed take place. The presence of a not-well defined
isosbestic point would suggest the presence of multiple equilibria.
Figure 3
The binding process can be better inspected by plots of binding isotherms (ΔA/CD vs. CP,
Figure S4) and by using equation (1) to try to evaluate the binding constant between P and
D.
CD/ΔA = 1/Δε + 1/ΔεK×1/[P]
(1)
Equation (1) needs an iterative calculation for [P] (the concentration of free DNA base
pairs) which is first set as equal to CP and then evaluated using i-th step K estimates.
However, in the present case, no convergence is reached indicating quantitative binding
(1:1 stoichiometry, see Fig. S4).

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Spectrofluorometric titrations were done similarly to spectrophotometric experiments and
confirmed a fluorescence emission variation occurring upon DNA binding (Figure S5).
Very interestingly, the amplitude of the binding isotherm turns out to be a function of
temperature, being it high and negative at 10.1°C, very small at 26.6°C and high and
positive at 44.8°C (Fig. 4A). Under highly diluted conditions (Fig. 4B and S6) a biphasic
trend is visible.
Figure 4
This finding confirms the presence of two different, overlapping and very strong, binding
modes as suggested by the non-perfect UV-vis isosbestic point. The data analysis
according to equation (1) (where signal change ΔA is simply replaced by ΔF) renders again
quantitative binding with the exception of the binding mode related to signal decrease at
10.1°C (K = 2.4×106 M-1) and that related to fluorescence increase at 44.8.°C (K = 3.2×106
M-1). Therefore, the first phase, occurring in the presence of dye excess conditions and
producing fluorescence emission decrease, is related to a binding constant that increases
with temperature (ΔH > 0): these features are indicative of external/groove binding. On the
contrary, the second phase, occurring in the presence of DNA excess conditions and
producing fluorescence emission increase, is related to a binding constant that decreases
upon temperature increase (ΔH < 0): these features are indicative of intercalation.
On the whole, it can be concluded that the affinity of 7 for DNA is very high, likely due to
the presence of two flexibly linked aromatic moieties. The possible binding modes are both
groove and intercalation, the prevailing one being dependent on temperature and reagents
concentrations. This dual behaviour was already found for other small molecule /DNA

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systems.[19,33,34,35] Literature data also confirm that pyrene derivatives can either bind
in the DNA minor groove producing a fluorescence emission decrease,[36] or intercalate
producing a fluorescence emission increase.[37]
DNA interaction geometry
To clarify the geometry of complexation between 7 and DNA, linear flow dichroism (LD)
experiments were performed. The LD spectra of aqueous solutions of DNA alone (trace a)
and in the presence of complex 7 at different [7]/[DNA] ratios (traces b-d) are shown in
Figure 5.
The LD spectrum of the macromolecule shows, as expected, a strong negative signal at 260
nm, due to the contribution of purine and pyrimidine base pairs (trace a). Following the
addition of 7 (traces b-d) a concentration-dependent induced dichroic signal appears at
higher wavelengths (300-450 nm), which is a spectral region where no contribution from
DNA base pairs can be detected and only the chromophore of the added complex can
absorb. Since small molecules cannot be oriented in the flow field, the occurrence of this
latter signal is indicative that 7 forms a molecular complex with the macromolecule and
becomes oriented.
Figure 5
Furthermore, the sign of this induced dichroic signal depends on the orientation of the
bound chromophore, relative to the axis of the DNA helix. In particular, for planar
aromatic scaffolds, it is possible to assume that the strong -* transitions are generally
polarised in the plane of the chromophore. Then, a negative signal is ascribable to a
parallel orientation to the plane of DNA bases, which is an intercalative mode of binding,

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where the aromatic plane of the drug and DNA bases are essentially coplanar. Otherwise,
for compounds bound in the groove of DNA, an induced dichroic signal of positive sign is
expected.[38] In the spectra of the DNA solution incubated with 7 (traces b-d) the induced
signal shows both positive and negative contribution suggesting a heterogeneity of binding.
This behaviour can be explained by the presence of drug molecules that both intercalate
between base pairs and accommodate along the groove.
Cytotoxicity studies
The antiproliferative activity of 7 was assayed on four human tumor cell lines and the
obtained results, expressed as GI50 values, i.e. the concentration (M) of test complex able
to induce a 50% reduction in cell number in comparison with the control culture, were
shown in Table 2. The well-known drug cisplatin was taken into account as reference.
Table 2
The obtained results indicate the ability of 7 to induce a significant antiproliferative effect
on most of the human tumor cell lines taken into consideration, with GI50 values in the
micromolar range. Notwithstanding these values are higher with respect to those calculated
for the reference drug, indicating a lower efficacy on cells, the capacity of 7 to exert a
comparable cytotoxicity on both resistant (A2780cis) and sensitive (A2780wt) ovarian
carcinoma cells, suggests for this chemical scaffold the ability to overcome the resistance
phenomenon and renders it worthy for further development.

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Conclusions
A new pyridinimino platinum(II) complex has been prepared, bearing a terminal pyrene
group linked to the chelating scaffold through a dioxygenated, flexible spacer (complex 7).
The antiproliferative activity of 7 was checked in vitro against both sensitive and resistant
cancer cell lines. Complex 7 showed an interesting capacity to circumvent resistance
towards A2780 cis cells, and its interaction with DNA was investigated by two independent
spectroscopic techniques (UV-vis/fluorescence titrations and linear flow dichroism). A
very strong and complex interaction with DNA was enlightened in both cases. In particular,
collected data sets show that both groove binding and intercalation take place, with one
interaction becoming more evident with respect to the other according to the involved
species molar ratio and the temperature. The aromatic pyrene moiety is most likely
responsible for intercalation through - interactions, common for this kind of residues,
while groove binding could be ascribed to DNA-templated pyrene-pyrene interactions,
favoured also by the metal containing residue and/or to the spacer. It has to be underlined
that, due to the flexibility of the linking alkyl chain, cooperative effects cannot be excluded
a priori, with two different interaction points of the same molecule acting at the same time.
The collected data suggest that aryl-terminated pyridinimino complexes are a versatile
class of antiproliferative complexes. Studies are in progress to investigate structural
analogues of complex 7.
Declaration of interests
None.
Acknowledgements

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This research was funded by Ministero dell'Università e della Ricerca (MIUR) for grant PRIN
2015, “Design of Innovative Metal-Ligand Systems for Catalysis and Energy Applications”;
Università di Pisa (Fondi di Ateneo 2017 and Progetti di Ricerca di Ateneo 2017—PRA_2017_25);
Dipartimento di Scienze del Farmaco-Università di Padova-(Progetti di Ricerca di Dipartimento
PRID 2017, DALL_SID17_02).
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R = Alkyl, Aryl, modified carbohydrate...
Figure 1. Platinum(II) pyridinimino complexes
Table 1. Crystal data and structure refinement for complex 6.
Compound
CCDC number
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
6
1938455
C10H14Cl2N2O2Pt
460.22
Triclinic
P
1
7.6622(2)
8.2251(3)
10.7771(3)
84.6900(14)
85.8650(12)
80.1320(12)
665.18(4)
2
b (Å)
c (Å)
 (°)
 (°)
 (°)
Volume (Å3)
Z
ρcalc (g cm−1)
μ (mm−1)
2.298
10.940
F(000)
432
θ range (°)
2.7 to 36.0
18913
Reflections collected
Independent reflections
Goodness-of-fit on F2
Final R1 [I ≥ 2σ(I )]
Final wR2 [I ≥ 2σ(I )]
5603 [Rint = 0.0251]
0.993
0.0258
0.0694

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Final R1 [all data]
0.0311
Final wR2 [all data]
Largest peak/hole (e Å−3)
0.0724
2.155, 1.437
b
c
a
2
3
4
Scheme 1. Synthesis of amine 4: a) NaBH4, THF, 0 °C; b) PBr3, THF, 0 °C; c) 1) H2N(CH2CH2O)2H, NaH,
THF, 0 °C; 2) gradient flash chromatography
[PtCl2(DMSO)2]
MeNO2
R = -(CH2CH2O)2H
(6)
-(CH2CH2O)2CH2Pyr (7)
Scheme 2. Synthesis of complexes 6-7.

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Figure 2. Molecular structure of 6. Most significant bond lengths (Å): Pt(1)N(2) 2.001(3); Pt(1)N(1)
2.009(3); Pt(1)Cl(1) 2.2782(12); Pt(1)Cl(2) 2.2918(10) and angles ( °): N(2)Pt(1)N(1) 80.37(13);
N(2)Pt(1)Cl(1) 95.98(10); N(1)Pt(1)Cl(1) 175.30(9); N(2)Pt(1)Cl(2) 174.50(9); N(1)Pt(1)Cl(2)
94.40(10); Cl(1)Pt(1)Cl(2) 90.27(6).
0.2
0.1
0.0
300
350
400
450
 (nm)
Figure 3. Spectrophotometric titration for the 7/DNA system; CD = 1.8×10-5 M, CP from 0 M to
5.3×10-5 M (arrow follows increased DNA content), [NaCac] = 2.5 mM, pH 7.0, DMSO = 1%, T = 25.7 °C.

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0.0
-0.5
-1.0
-1.5
-2.0
-2.5
A
B
1
0
-1
-2
0.0
0.5
CP (10-5 M)
1.0
0.0
0.5
CP (10-5 M)
1.0
Figure 4. Spectrofluorometric binding isotherms for the 7/DNA system, [NaCac] = 2.5 mM, pH 7.0,
DMSO = 0.4%, λex = 310 nm, λem = 378 nm; (A) CD = 7.1×10-6 M, T = 10.1°C (circles), 26.6°C (open
triangles), 44.8°C (squares), (B) CD = 1.6×10-6 M, T = 21.6 °C.
Figure 5: Linear flow dichroism (LD) spectra of 7 at different [drug]/[DNA] ratios: trace (a), 0;
trace (b), 0.04; trace (c) 0.08 and trace (d) 0.16. [DNA]= CP = 1.0×10-3 M.

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Table 2. Human cell growth inhibition in the presence of 7 and cisplatin, taken as
reference.a
Cell Line GI50b (μM)
Complex
HeLa
A2780wt
5.30.4
1.10.4
A2780cis
6.81.5
6.60.8
MSTO-211H
17.42.3
>20
7
cisplatin
1.50.2
1.40.3
a Complex 6 was inactive towards the tested cell lines; b Values are the mean ± SD of at least three
independent experiments.

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GRAPHICALABSTRACT
DNA interaction of a fluorescent, cytotoxic pyridinimino platinum(II)
complex.
Riccardo Bondi, Tarita Biver, Lisa Dalla Via, Federica Guarra, Mariafrancesca
Hyeraci, Claudia Sissi, Luca Labella, Fabio Marchetti and Simona Samaritani
A versatile pyridinimino Pt(II) complex, bearing a pyrenyl-terminated side chain was
synthesized. The complex circumvents cisplatin resistance towards A2780cis cells and is
able to interact with DNA by both groove binding and intercalation.

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Journal of Inorganic Biochemistry
DNA interaction of a fluorescent, cytotoxic pyridinimino
platinum(II) complex
Highlights
 A fluorescently tagged, cytotoxic pyridinimino platinum(II) complex was prepared.
 The complex circumvents cisplatin resistance against A2780cis cancer cell line.
 Complex/DNA interaction was studied by independent spectroscopic techniques.
 DNA interaction takes place by both groove binding and intercalation.