DNA binding studies of a series of cis-[Pt(Am)2X2] complexes (Am = inert amine, X = labile carboxylato ligand)

Article abstract of DOI:10.1016/j.ica.2012.07.018

A series of platinum(II) complexes of formulae cis-[Pt(Am) ₂X₂] (where Am represents an inert amine and X a labile (carboxylato) ligand) have been prepared and characterized by elemental analysis, ESI-MS, IR and ¹H NMR spectroscopy. The single-crystal molecular structures were determined for cis-[Pt(opea)(cbdca-2H)], cis-[Pt(hmpy)(cbdca-2H)], cis-[Pt(NH₃)₂(bzmal-2H)] and cis-[Pt(hmpy)(μ-dcch-2H)₂] (where opea is picolylamine, hmpy represents 4-hydroxymethylpyridine, cbdca-2H, is 1,1-cyclobutanedicarboxylate anion, bzmal-2H stands for benzylmalonate anion and dcch-2H is trans-1,2-cyclohexanedicarboxylate anion). The interaction of all compounds with DNA was investigated with different techniques: viscosity measurements and emission fluorescence spectroscopy were used to investigate the changes induced by the binding of the platinum compounds to calf-thymus DNA, while atomic force microscopy and electrophoretic mobility allowed evaluating the potential alterations of pBR322 plasmid DNA. The cytotoxic behavior of the platinum compounds on human leukemia HL-60 tumor cell lines was also examined.

Full text of DOI:10.1016/j.ica.2012.07.018

Inorganica Chimica Acta 394 (2013) 65–76
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Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
DNA binding studies of a series of cis-[Pt(Am)2X2] complexes (Am = inert amine,
X = labile carboxylato ligand)
Esther Escribano a, Mercè Font-Bardia b,c, Teresa Calvet b, Julia Lorenzo d, Patrick Gamez a,e
,
Virtudes Moreno a,
⇑
a Departament de Química Inorgànica, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain
b Departament de Cristalꢀlografia, Mineralogia i Dipòsits Minerals, Martí i Franquès s/n, 08028 Barcelona, Spain
c Centre Científic i Tecnològic de la Universitat de Barcelona (CCITUB), Unitat de Difracció de RX, Solé i Sabarís 1-3, 08028 Barcelona, Spain
d Institut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain
e Institució Catalana de Recerca i Estudis Avançats (ICREA), Passeig Lluís Companys, 23, 08010 Barcelona, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 May 2012
Received in revised form 16 July 2012
Accepted 17 July 2012
Available online 1 August 2012
A series of platinum(II) complexes of formulae cis-[Pt(Am)2X2] (where Am represents an inert amine and
X a labile (carboxylato) ligand) have been prepared and characterized by elemental analysis, ESI-MS, IR
and 1H NMR spectroscopy. The single-crystal molecular structures were determined for cis-[Pt(o-
pea)(cbdca-2H)], cis-[Pt(hmpy)(cbdca-2H)], cis-[Pt(NH3)2(bzmal-2H)] and cis-[Pt(hmpy)(l-dcch-2H)2]
(where opea is picolylamine, hmpy represents 4-hydroxymethylpyridine, cbdca-2H, is 1,1-cyclobutan-
edicarboxylate anion, bzmal-2H stands for benzylmalonate anion and dcch-2H is trans-1,2-cyclohexan-
edicarboxylate anion). The interaction of all compounds with DNA was investigated with different
techniques: viscosity measurements and emission fluorescence spectroscopy were used to investigate
the changes induced by the binding of the platinum compounds to calf-thymus DNA, while atomic force
microscopy and electrophoretic mobility allowed evaluating the potential alterations of pBR322 plasmid
DNA. The cytotoxic behavior of the platinum compounds on human leukemia HL-60 tumor cell lines was
also examined.
Keywords:
Platinum dicarboxylate compounds
DNA binding
Atomic force microscopy
Platinum drugs
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
come these limitations [14,15]. From the several thousand plati-
num compounds synthesized, only about 35 have entered clinical
The anti-proliferative activity of cis-diamminedichloridoplati-
num(II) (cisplatin; Fig. 1) was discovered serendipitously by
Rosenberg and co-workers in 1965 [1]. A few years later, the anti-
tumor activity of cisplatin was revealed [2], and in 1978 the US
Food and Drug Administration (FDA) approved this compound for
clinical use. Since then, this drug has been applied extensively in
cancer chemotherapy, in the treatment of a number of solid tumors
including genitourinary, colorectal, and non-small cell lung cancers
[3–5]. Cisplatin is especially efficient against testicular cancer with
a cure rate of over 90% and nearly 100% when tumors are diag-
nosed early [6]. However, the clinical use of cisplatin is restricted
by several drawbacks. Serious potential side effects including nau-
sea, vomiting, ototoxicity, neuropathy, myelosupression and neph-
rotoxicity limit its usefulness [7–10]. In addition, many tumor cells
display intrinsic or acquired resistance to platinum-based drugs,
which further limits their practicality [11–13]. Hence, for over
three decades, continuous research efforts have been dedicated
to the development of improved platinum-based drugs to over-
trials [16–18]. At present, besides cisplatin, two platinum drugs
(oxaliplatin, carboplatin) are used clinically worldwide while
nedaplatin was approved in Japan (Fig. 1) [11,19,20].
Most of the anticancer-active platinum coordination com-
pounds exhibit the following general formula cis-[Pt(Am)2X2], Am
being an inert amine and X a labile ligand (leaving group). For in-
stance, cis-diammine(1,1-cyclobutanedicarboxylato)platinum(II)
(carboplatin; Am = ammonia and X = 1,1-cyclobutanedicarboxyla-
to) shows a mechanism of action that is comparable to that of cis-
platin. However, carboplatin is less reactive than cisplatin [21].
Actually, the rate of aquation of the leaving group of carbo-
platin, namely the 1,1-cyclobutanedicarboxylato ligand, is nearly
twice lower than that of cisplatin, owing to the chelating effect.
Consequently, clinical treatment with carboplatin causes less se-
vere side effects compared to cisplatin, even though higher doses
are required [26]. Together with carboplatin, cis-diammine(glyco-
lato)platinum(II) [27] (nedaplatin; Am = ammonia and X = glycola-
to) are members of the second-generation platinum drugs, for
which the leaving groups X have been optimize to reduce the inci-
dence of side effects. Both carboplatin and nedaplatin are
cross-resistant to cisplatin, therefore indicating that their mecha-
⇑
Corresponding author. Tel.: +34 93 4021274; fax: +34 93 4907725.
E-mail address: virtudes.moreno@qi.ub.es (V. Moreno).
0020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.07.018

66
E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
aimed at prospecting for new molecules with potential DNA-bind-
ing/cytotoxic properties.
The platinum(II) compounds obtained have been fully charac-
terized and their DNA-interaction abilities have been studied by
gel electrophoresis, viscosity and fluorescence measurements,
atomic-force microscopy (AFM) and cytotoxicity assays.
2. Experimental
2.1. Materials and methods
Fig. 1. Platinum(II) coordination compounds used in cancer chemotherapy:
cis-diamminedichloridoplatinum(II) (cisplatin;[1]); [(1R,2R)-cyclohexane-1,2-
diamine](oxalato)platinum(II) (oxaliplatin; [22,23]); cis-diammine(1,1-cyclob-
utanedicarboxylato)platinum(II) (carboplatin; [24]); cis-diammine(glycolato)plat-
inum(II) (nedaplatin; [25]).
Solvents and chemicals were commercially available as A.R.
grade and used as received. All reactions and purifications were
performed in air. Proton magnetic resonance (1H NMR) spectra
were recorded on a VARIAN UNITY (300 MHz) spectrometer at
25 °C. Proton chemical shifts are expressed in parts per million
(ppm, d scale) and are referenced to the solvent peak. Infrared
spectra were recorded using a NICOLET-5700 FT-IR (in the range
4000–400 cmꢁ1), and data are represented as the frequency of
absorption (cmꢁ1); the solid samples were prepared as KBr disks
while liquids were deposited on a NaCl support. C, H, N analyses
were carried out using an automatic Carlo Erba EA 1108 analyzer
(analytical services of the Universitat de Barcelona). ESI Mass Spec-
troscopy was carried out using a LC/MSD-TOF Spectrometer from
Agilent Technologies, equipped with an electrospray ionization
(ESI) source (analytical services of the Universitat de Barcelona).
The melting points or decomposition temperatures of the com-
pounds were determined with a Stuart Scientific SMP3 apparatus,
using a capillary.
nisms of action are analogous. In the third-generation platinum
drugs, the ammine ligands (Am = ammonia) are replaced by pri-
mary or secondary amine ligands. For example, in [(1R,2R)-cyclo-
hexane-1,2-diamine](oxalato)platinum(II) [22] (oxaliplatin;
Am = (1R,2R)-cyclohexane-1,2-diamine and X = oxalato), the NH3
groups have been substituted with (1R,2R)-cyclohexane-1,2-dia-
mine. Interestingly, the higher lipophilicity of the cyclohexanedi-
amine ligand allows an enhanced uptake in cancer cells
compared to cisplatin [28]. Thus, oxaliplatin displays a different
spectrum of activity and is capable of circumventing cisplatin
resistance [29], although the structure of its DNA adduct is similar
to that of cisplatin [30,31].
In the present study, different families of cis-[Pt(Am)2X2] com-
pounds have been developed, which combine a dicarboxylate li-
gand (as leaving group X) with distinct monoamine or diamine
ligands (as inert Am ligands). Hence, 1,1-cyclobutanedicarboxylate
(cbdca-2H), oxalate (ox-2H), meso-2,3-dibromosuccinate (mdbs-
2H), trans-1,2-cyclohexanedicarboxylate (dcch-2H), benzylmalo-
nate (bzmal-2H) have been used in combination with ammonia,
4-hydroxymethylpyridine (hmpy), ethylenediamine (en), tetra-
methylenediamine (temed), (1R,2R)-cyclohexane-1,2-diamine
(dach), 2-picolylamine (opa) and 2-(2-pyridyl)ethylamine (opea)
(Fig. 2). The preparation of such mixed X/Am PtII compounds is
2.2. Preparation of platinum(II) coordination compounds
All cis-[Pt(Am)2X2] compounds described in the present study
have been obtained using Dhara’s method [32], as follows:
K2½PtI4ꢂ
440.7 mg of K2[PtCl4] (1.06 mmol) were dissolved in 15.6 mL of
distilled water at 35 °C, under an inert atmosphere of nitrogen.
1.08 g of KI (6.5 mmol) were dissolved in 1.5 mL of distilled water.
Fig. 2. Carboxylic acid (H2X) and inert ligands (Am) used to prepare cis-[Pt(Am)2X2] compounds.

E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
67
The resulting aqueous solution of potassium iodide was added to
the red solution of potassium tetrachloridoplatinate. The resulting
dark-green mixture was stirred for 20 min before utilization in the
next synthetic step.
cis ꢁ ½PtðAmÞ2I2ꢂ
In a three-necked round-bottomed flask of 100 mL equipped
with two dropping funnels and a Liebig condenser were introduced
13.4 mL of distilled water. A solution of 1.08 mmol of the amine
Am in 17.8 mL of distilled water was introduced in one of the
two dropping funnels. The second dropping funnel contained
1.08 mmol of K2[PtI4] in 17.8 mL of distilled water. After purging
with nitrogen, the flask was warmed to 60 °C and the two solutions
were simultaneously and slowly added under stirring to the round-
bottomed flask (during a period of approx. 60 min). A yellow pre-
cipitate appeared during the addition of the two reactants. The
reaction mixture was subsequently cooled down slowly to room
temperature, and the solid material was isolated by filtration on
a glass filter, and washed with ethanol (2 ꢃ 5 mL) and diethyl ether
(6 mL). The yellow solid obtained was stored in a desiccator.
2.2.5. (2-(Aminoethyl)pyridine)(1,1-cyclobutanedicarboxylato)
platinum(II), [Pt(opea)(cbdca-2H)] (5)
Yield = 36%. Mw = 459.36 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 1.78 (2, m, 2H, 3J = 8 Hz), d 2.39 (9, t, 2H, 3J = 5 Hz), d
2.77 (1 and 3, t, 4H, 3J = 8 Hz), d 3.03 (8, t, 2H, 3J = 5 Hz), d 7.27
(5, t,1H, 3J = 6.8 Hz), d 7.36 (7, d, 1H, 3J = 8 Hz), d 7.86 (6, dd, 1H,
3J = 8 Hz), d 8.44 (4, d,1H, 3J = 6.5 Hz) ppm. IR (KBr pellet, cmꢁ1):
m
= 3200 (NH2), 1640 and 1615 (C = O + NH2 + C = N), 1371 (C–O).
ESI-MS (m/z, positive mode): ([M+H]+) 460, ([M+Na]+) 482,
([2 M+Na]+) 942, ([2 M+H]+) 920. Anal. Calc. for (%) for PtN2H16C13-
O4ꢀ2H2O: C, 31.52; H, 4.07; N, 5.65. Found: C, 31.62; H, 3.87; N,
5.52%. Compound 5 is partially soluble in water and chloroform,
and soluble in DMSO, DMF and methanol. The molecular structure
of 5 could be determined by single-crystal X-ray diffraction (see
text and SI for details).
Ag2X
An aqueous solution of 2 mmol of NaOH (or NaHCO3 in the case
of meso-2,3-dibromosuccinate; mdbs-2H) was added to 1 mmol of
de dicarboxylic acid (H2X) dissolved in the minimum of distilled
water. The mixture was kept in the dark, and 2 mmol of silver(I) ni-
trate were added which yielded a white precipitate. The heteroge-
neous mixture was stirred for 15–30 min and the silver salt, i.e.
Ag2X, was isolated by filtration and stored in a desiccator.
cis ꢁ ½PtðAmÞ2Xꢂ
A one-to-one mixture of cis-[Pt(Am)2I2] and Ag2X in 15 mL of
distilled water was stirred in the dark during 2–3 days. The result-
ing precipitate, i.e. AgI, was removed by filtration over Celite and
the subsequent filtrate was concentrated to dryness using a rotary
evaporator. The solid obtained, namely cis-[Pt(Am)2X] was washed
with cold distilled water and diethyl ether.
2.2.6. Bis-(4-pyridinemethanol)(1,1-cyclobutanedicarboxylato)
2.2.1. ethylenediamine(1,1-
cyclobutanedicarboxylato)platinum(II),[Pt(en)cbdca] (1)
This compound was earlier described by Rochon and co-work-
ers [33]. View supporting info.
platinum(II), [Pt(hmpy)2(cbdca-2H)] (6)
Yield = 59%. Mw = 555.45 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 1.87 (2, m, 1H, 3J = 7.8 Hz), d 2.90 (1, t, 4H, 3J = 8.0 Hz), d
4.60 (6, s, 4H, overlapped with the solvent), d 7.37 (5, d, 4H,
3J = 5.7 Hz), d 8.40 (4, d, 4H, 3J = 5.7 Hz) ppm. IR (KBr pellet,
2.2.2. N,N,N0,N0-tetramethylethylenediamine(1,1-
cyclobutanedicarboxylato)platinum(II), [Pt(temed)(cbdca-2H)] (2)
This compound was before reported by Rochon and co-workers
[34]. View supporting info.
cmꢁ1):
m = 1648 (C = O), 1619 (C = N), 1368 (C–O), 476 (Pt–N).
ESI-MS (m/z, positive mode): ([M+H]+) 556, ([M+Na]+) 578,
([2 M+Na]+) 1134, ([2 M+H]+) 1112. Anal. Calc. for (%) for PtN2H20-
C18O6.1.5H2O: C, 38.92; H, 3.63; N, 5.04. Found: C, 37.09; H, 3.65;
N, 4.39%. Compound 6 decomposes above 168 °C. 6 is soluble in
water, DMSO and DMF, and not soluble in chloroform and metha-
nol. The molecular structure of 6 could be determined by single-
crystal X-ray diffraction (see text and SI for details).
2.2.3. (Trans-1,2-cyclohexanediamine)(1,1-
cyclobutanedicarboxylato)platinum(II), [Pt(dach)(cbdca-2H)] (3)
This compound was before synthesized by Pierpont and co-
workers [35]. View supporting info.
2.2.4. (2-(Aminomethyl)pyridine)(1,1-
cyclobutanedicarboxylato)platinum(II), [Pt(opa)(cbdca-2H)] (4)
Yield = 22%. Mw = 445.33 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 1.75 (2, m, 2H, 3J = 8 Hz), d 2.29 (3, t, 2H, 3J = 8 Hz), d
2.72 (1, t, 2H, 1J = 8 Hz), d 4.13 (8, s, 2H), d 7.28 (5, t, 1H,
3J = 6.6 Hz), d 7.39 (7, d, 1H, 3J = 8 Hz), d 7.92 (6, t, 1H, 3J = 8 Hz),
d
8.23 (4, d, 1H, d, 3J = 6 Hz) ppm. IR (KBr pellet, cmꢁ1):
= 3450–3200 (NH2), 1635 (C = O + NH2 + C = N). ESI-MS (m/z, po-
m
sitive mode): ([M+H]+) 446, ([M+Na]+) 468, ([2 M+Na]+) 914. Anal.
Calc. for (%) for PtN2H14C12O4ꢀ1.5H2O: C, 30.51; H, 3.63; N, 5.93.
Found: C, 31.12; H, 3.27; N, 6.21%. Compound 4 is partially soluble
in water, soluble in DMF and methanol, but not in chloroform.

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E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
ESI-MS (m/z, positive mode): ([M+H]+) 584. Anal. Calc. for (%) for
PtN2H16C10O4Br2: C, 20.60; H, 2.77; N, 4.80. Found: C, 21.33; H,
2.89; N, 4.92%. Compound 10 decomposes above 170 °C. 10 is par-
tially soluble in water, soluble in DMSO and DMF, and not soluble
in chloroform and methanol.
2.2.7. Diammine(meso-2,3-dibromosuccinato)platinum(II),
[Pt(NH3)2(mdbs-2H)] (7)
Yield = 27%. Mw = 503.01 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 3.99–4.14 (2, br, 6H), d 4.44 (1, s, 2H) ppm. IR (KBr pellet,
cmꢁ1):
m = 3202 (NH), 1684 (C = O + NH2). ESI-MS (m/z, positive
mode): ([M+H]+) 504, ([M+Na]+) 526. Anal. Calc. for (%) for PtN2H8-
C4O4Br2: C, 9.55; H, 1.60; N, 5.57. Found: C, 9.58; H, 1.82; N, 5.58%.
Compound 7 decomposes above 193 °C. 7 is soluble in water and
DMSO, and not soluble in chloroform, DMF and methanol.
2.2.8. Ethylenediamine(meso-2,3-dibromosuccinato)platinum(II),
[Pt(en)(mdbs-2H)] (8)
2.2.11. Bis-(4-pyridinemethanol)(meso-2,3-dibromosuccinato)-
Yield = 24%. Mw = 529.04 g molꢁ1 1H NMR (D2O, 300 MHz,
.
platinum(II), [Pt(hmpy)2 (mdbs-2H)] (11)
25 °C): d 2.53 (s, 4H), d 4.39 (s, 2H) d 5.13 (br, 4H) ppm. IR (KBr pel-
Yield = 89.5. Mw = 687.20 g molꢁ1 1H NMR (D2O, 300 MHz,
.
let, cmꢁ1):
m = 3240 (NH2), 1719 (C = O), 1617 (NH2), 1052 (C–O),
25 °C): d 4.37 (1, s, 2H), d 4.62 (4, s, 4H), d 7.33 (3, br, 4H), d 8.53
540 (Pt–N). ESI-MS (m/z, positive mode): ([M+H]+/2) 265. Anal.
Calc. for (%) for PtN2H10C4O4Br2: C, 13.60; H, 2.09; N, 5.29. Found:
C, 13.26; H, 2.04; N, 6.12%. Compound 8 decomposes above 152 °C.
8 is soluble in water, DMSO and DMF, and not soluble in chloro-
form and methanol.
(2, br, 4H) ppm. IR (KBr pellet, cmꢁ1):
m
= 1622 (C = O + C = N),
1053 (C–O), 807 (C–H), 491 (Pt–N). ESI-MS (m/z, positive mode):
([M+H]+) 688, ([M+Na]+) 710. Anal. Calc. for (%) for PtN2H16C16O6-
Br2.3H2O: C, 25.93; H, 2.99; N, 3.78. Found: C, 25.56; H, 2.92; N,
4.32%. Compound 11 decomposes above 142 °C. 11 is partially sol-
uble in water, soluble in DMSO and DMF, and not soluble in chlo-
roform and methanol.
2.2.9. N,N,N0,N0-tetramethylethylenediamine(meso-2,3-
dibromosuccinato)platinum(II), [Pt(temed)(mdbs-2H)] (9)
Yield = 56%. Mw = 585.15 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 2.32 (12H, br), d 2.75 (4H, br), d 4.34 (2H, br) ppm. IR
(KBr pellet, cmꢁ1):
m
= 1734 and 1653 (C = O), 1341 (C–O), 548
(Pt–N). ESI-MS (m/z, positive mode): ([M+H]+) 586, ([MꢁBr]+)
505, ([M+Na]+) 608, ([M+NH4]+) 603. Anal. Calc. for (%) for PtN2H18-
C10O4Br2: C, 20.53; H, 3.10; N, 4.79. Found: C, 20.39; H, 3.09; N,
4.71%. Compound 9 decomposes above 147 °C. 9 is partially soluble
in water, soluble in DMSO and DMF, and not soluble in chloroform
and methanol.
2.2.12. Diammine(benzylmalonato)platinum(II), [Pt(NH3)2(bzma-2Hl]
(12)
This compound was earlier described by Ye et al. [36]. View
supporting info.
2.2.13. Ethylenediamine(benzylmalonato)platinum(II), [Pt(en)(bzmal-
2H)] (13)
This compound was earlier described by Rochon and co-work-
ers [33]. View Supporting info.
2.2.14. N,N,N0,N0-tetramethylethylenediamine(benzylmalonato)
platinum(II), [Pt(temed)(bzma-2H)l] (14)
Yield = 28.5%. Mw = 503.46 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 2.75 (7, s, 12H), d 2.79 (6, s, 4H), d 3.68 (2, d, 2H,
3J = 10 Hz), d 3.78 (1, t, 1H 3J = 10 Hz), d 7.26–7.37 (3, 4, 5, 5H)
ppm. IR (KBr pellet, cmꢁ1):
m = 1640 (C = O), 1380 (C = O). ESI-MS
2.2.10. (Trans-1,2-cyclohexanediamine)(meso-2,3-dibromosuccinato)-
(m/z, positive mode): ([M+H]+) 504, ([M+Na]+) 526, ([2 M+H]+)
1008, ([2 M+Na]+) 1030. Anal. Calc. for (%) for PtN2H24C16O4: C,
38.17; H, 4.80; N, 5.56. Found: C, 37.51; H, 4.56; N, 5.54%. Com-
pound 14 decomposes above 82.5 °C. 14 is partially soluble in
water and methanol, soluble in DMSO and DMF, and not soluble
in chloroform.
platinum(II), [Pt(dach)(mdbs-2H)] (10)
Yield = 8%. Mw = 583.14 g molꢁ1
.
1H NMR (D2O, 300 MHz,
25 °C): d 1.06 (5, br, 2H), d 1.21 (4, br, 2H), d 1.49 (50, br, 2H), d
1.96 (40, br, 2H), d 2.33 (3, br, 2H), d 4.38 (1, s, 2H) ppm. IR (KBr pel-
let, cmꢁ1):
m = 3253 (NH2), 1719 (C = O), 1617 (NH2), 1065 (C–O).

E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
69
2.2.15. (2-(Aminoethyl)pyridine)(benzylmalonato)platinum(II),
structure of 17 could be determined by single-crystal X-ray diffrac-
[Pt(opea)(bzmal-2H)] (15)
tion (see text and SI for details).
Yield = 6%. Mw = 485.40 g molꢁ1
.
1H NMR (D2O, 300 MHz,
25 °C): d 2.56 (11, br, 2H), d 2.73 (10, br, 2H), d 3.16 (2, br, 2H), d
3.32 (1, br, 1H), d 7.13–7.23 (3, 4, 5, br, 5H), d 7.36 (7, t, 1H,
3J = 8 Hz), d 7.55 (9, d, 1H, 3J = 8 Hz), d 7.85 (8, t, 1H, 3J = 9 Hz), d
8.78 (6, d, 1H, 3J = 8 Hz) ppm. IR (KBr pellet, cmꢁ1):
m = 3235
(NH), 1636 and 1617 (C = O + NH2 + C = N), 1384 (C = O). ESI-MS
(m/z, positive mode): ([M+H]+) 486, ([2 M+H]+) 973. Anal. Calc.
for (%) for PtN2H24C16O4: C, 37.12; H, 3.74; N, 5.77. Found: C,
36.68; H, 3.62; N, 5.03%. Compound 15 is partially soluble in water,
soluble in DMSO and DMF, and not soluble in chloroform and
methanol.
2.3. Gel electrophoresis of platinum-pBR322 complexes
The stock solutions of the platinum(II) compounds were pre-
pared as above in TE containing 2% DMSO. pBR322 plasmid DNA
aliquots (0.25 lg/mL) were incubated in TE (Tris-H4edta,
Tris(hydroxymethyl)aminomethaneethylendiaminetetracetic acid)
buffer (50 mM NaCl, 10 mM Tris.HCl, 1 mM H4edta, pH = 7.5) at
molar ratio ri = 0.50 for electrophoresis study. Incubation was car-
ried out at 37 °C for 24 h. The charge (4
lL) marker were added to
2.2.16. Bis-(4-pyridinemethanol)(benzylmalonato)platinum(II),
20 L aliquot parts of the compound-DNA adduct. The resulting
l
[Pt(hmpy)2(bzmal-2H)] (16)
mixture was electrophoretized on agarose gel (0.5% in TBE buffer,
Tris–Borate-EDTA) for 4 h at 1.5 V/cm. Subsequently, the DNA
was dyed with an ethidium bromide solution (0.5
for 20 min. Samples of free DNA and of cisplatin–DNA complex
were used as controls. The experiment was carried out in an ECO-
GEN horizontal tank connected to a PHARMACIA GPS 200/400 var-
iable potential power supply and the gel was photographed with a
thermal imager FUJIFILM FTI-500.
Yield = 75%. Mw = 605.51 g molꢁ1 1H NMR (D2O, 300 MHz,
.
25 °C): d 2.92 (2, d, 2H, 3J = 8 Hz), d 3.27 (1, t, 1H, 3J = 8 Hz), d
7.19–7.26 (3, 4, 5, br, 5H), d 8.18 (7, br, 4H), d 8.37 (6, br, 4H)
lg/mL in TBE)
ppm. IR (KBr pellet, cmꢁ1):
m = 1656 and 1629 (C = O), 1384.5
(C = O), 1054 (C–O). ESI-MS (m/z, positive mode): ([M+H]+) 606,
([M+Na]+) 628, ([2 M+H]+) 1212, ([2 M+Na]+) 1234. Anal. Calc. for
(%) for PtN2H22C22O6: C, 43.64; H, 3.66; N, 4.63. Found: C, 43.07;
H, 3.12; N, 4.11%. Compound 16 decomposes above 184 °C. 16 is
partially soluble in water, soluble in DMSO and DMF, and not sol-
uble in chloroform and methanol.
2.4. Atomic force microscopy (TMAFM)
pBR322 DNA was stirred gently at room temperature for 15 min
to obtain a homogeneous distribution of DNA topoisomers. Pt com-
plex stock solutions (1 mg/mL) were prepared freshly in 40 mM
HEPES buffer ((HEPES = N-2-hydroxyethyl piperazine-N0-2-ethane-
sulfonic acid), 10 mM MgCl2, pH = 7.4) containing 2% DMSO. These
solutions were diluted 1000 times with HEPES to give 2000
the Pt solutions. Each sample was prepared by addition of 1
l
l
L of
L of
pBR322 DNA (0.25
lg/lL) to 2
lL of complex stock solution, and
the final volume was adjusted to 50
l
L with HEPES. The different
solutions as well as Milli-Q water were passed through 0.2 nm
FP030/3 filters (Scheicher & Schueell GmbH, Germany) to provide
a clear background when they were imaged by AFM. All samples
were incubated at 37 °C for 24 h. The AFM samples were prepared
by casting a 2-lL drop of test solution onto freshly cleaved Musco-
vite green mica disks as the support. The drop was allowed to stand
undisturbed for 3 min to favor the adsorbate/substrate interaction.
Each DNA-laden disk was rinsed with Milli-Q water and was blown
dry with clean compressed argon gas directed normal to the disk
surface. The samples were stored over silica prior to AFM imaging.
All Atomic Force Microscopy (AFM) observations were made with a
Nanoscope III Multimode AFM (Digital Instrumentals, Santa Bar-
bara, CA). Nano-crystalline Si cantilevers of 125-nm length with a
spring constant of 50 N/m average ended with conical-shaped Si
probe tips of 10-nm apical radius and cone angle of 35° were uti-
lized. High-resolution topographic AFM images were obtained in
air at room temperature (relative humidity <40%) on different
2.2.17. Tetrakis-(4-pyridinemethane)-bis-(
edicarboxylato)diplatinum(II), [Pt2(hmpy)4(
Yield = 83%. Mw = 583.50 g molꢁ1 1H NMR (D2O, 300 MHz,
l
l
-trans-1,2-cyclohexan-
-dcch-2H)2] (17)
.
25 °C): d 4.59 (6, s, 4H), d 7.26 (5, 4H), d 8.35 (4, 4H) ppm. The
hydrogens H1, H2 and H3 are not well resolved. IR (KBr pellet,
cmꢁ1):
m = 1622 (C = O + C = N), 1059 (C–O), 497 (Pt–N). ESI-MS
(m/z, positive mode): ([M+H]+) 584, ([2 M+H]+) 1168, ([2 M+Na]+)
1190. Anal. Calc. for (%) for Pt2N4H48C40O12: C, 41.17; H, 4.15; N,
4.80. Found: C, 40.24; H, 4.31; N, 4.69%. Compound 17 decomposes
above 210.5 °C. 17 is partially soluble in DMF, soluble in water,
DMSO and methanol, and not soluble in chloroform. The molecular

70
E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
Table 1
specimen areas of 2 ꢃ 2
lm operating in intermittent contact
[Pt(Am)2X2] compounds prepared from inert ligands (Am) and Labile ligands (X)
depicted in Fig. 2.
mode at a rate of 1–3 Hz.
X ligand
Am ligand
Pt compound
2.5. Viscosity measurements
cbdca-2H
ox-2H
NH3
dach
en
temed
dach
opa
opea
hmpy
NH3
carboplatin
oxaliplatin
The viscosity experiments were carried out at a constant tem-
perature of 25 °C with an AND-SV-1 viscometer in a water bath
using a water jacket accessory. A range of 20–100 lL of 10 mM
solutions (in TE containing 2% DMSO) of the different platinum
compounds were added to 2 mL of a 100 mM ct-DNA solution in
TE. The flow time was measured by a digital stop watch. Kinetic
studies were performed over a time period of 24 h.
cbdca-2H
cbdca-2H
cbdca-2H
cbdca-2H
cbdca-2H
cbdca-2H
mdbs-2H
mdbs-2H
mdbs-2H
mdbs-2H
mdbs-2H
bzmal-2H
bzmal-2H
bzmal-2H
bzmal-2H
bzmal-2H
dccp-2H
cis-[Pt(en)(cbdca-2H)] (1)
cis-[Pt(temed)(cbdca-2H)] (2)
cis-[Pt(dach)(cbdca-2H)] (3)
cis-[Pt(opa)(cbdca-2H)] (4)
cis-[Pt(opea)(cbdca-2H)] (5)
cis-[Pt(hmpy)2(cbdca-2H)] (6)
cis-[Pt(NH3)2(mdbs-2H)] (7)
cis-[Pt(en)(mdbs-2H)] (8)
cis-[Pt(temed)(mdbs-2H)] (9)
cis-[Pt(dach)(mdbs-2H)] (10)
cis-[Pt(hmpy)2(mdbs-2H)] (11)
cis-[Pt(NH3)2(bzmal-2H)] (12)
cis-[Pt(en)(bzmal-2H)] (13)
cis-[Pt(temed)(bzmal-2H)] (14)
cis-[Pt(opea)(bzmal-2H)] (15)
cis-[Pt(hmpy)2(bzmal-2H)] (16)
en
temed
dach
hmpy
NH3
2.6. Fluorescence measurements
A 50
l
M solution of Calf Thymus DNA (ct-DNA) in a TE buffer
L of a 5 mM solution of ethidium bro-
en
solution was prepared. 30
l
temed
opea
hmpy
hmpy
mide were added to 3 mL aliquots of the ct-DNA solution. The
resulting mixtures were incubated at 37 °C for 30 min or 24 h.
[Pt2(hmpy)4(l-dccp-2H)2] (17)
Next, 20, 40, 60, 80 and 100
no more than 2% DMSO) were added to the 3 mL aliquots, giving
concentrations of 10, 20, 30, 40 and 50 M, respectively. Each Pt
lL of Pt complex solutions (containing
l
3.2. Single crystal X-ray diffraction
compound of the present study was investigated with incubation
times of 30 min and 24 h. The emission spectra were recorded in
the range 530–670 nm, with an excitation wavelength of 502 nm.
The fluorescence measurements were performed with a KONTRON
SFM 25 spectrofluorometer.
The molecular structures of compounds 5, 6, 12 and 17 have
been determined by single-crystal X-ray diffraction (see SI for the
X-ray crystallographic analysis and data collection). Selected bond
lengths and angles for these coordination compounds are given as
Supporting information.
2.7. Growth-inhibition assays
3.2.1. Crystal structure of {[Pt(opea)(cbdca-2H)]}2(H2O)3 (5)2ꢀ3H2O
The two-day reaction of [Pt(opea)I2] with Ag2(cbdca-2H) in
water yields, after filtration of the AgI salt produced, single crystals
of (5)2ꢀ3H2O after two months. (5)2ꢀ3H2O crystallizes in the mono-
clinic space group P21/c (Table S1). The single-crystal X-ray struc-
ture of (5)2 is depicted in Fig. 3.
2.7.1. Tumor cell lines and culture conditions
The cell line used for the cytotoxic tests was the human acute
promyelocytic leukemia cell line HL-60 (American Type Culture
Collection (ATCC)). The cells were routinely maintained in RPMI-
1640 medium supplemented with 10% (v/v) heat inactivated fetal
bovine serum, 2 mM glutamine, 100 U/mL penicillin, and 100 lg/
mL streptomycin (Gibco BRL, Invitrogen Corporation, Netherlands)
The unit cell contains two mononuclear PtII molecules that are
almost identical (Fig. S1 and Table S2). Therefore, only one of them
will be described in detail. The metal center Pt1 is tetracoordinated
by one cbdca-2H ligand (carboxylato oxygen atoms O11 and O13)
and one opea ligand (pyridine nitrogen atom N11 and amino nitro-
gen atom N12), generating a nearly perfect square plane (the
angles vary from 88.3(3) to 92.0(3)°; Table S2). The Pt–N and
Pt–O bond distances are in normal range for this type of coordina-
tion environment [38,39]. The cyclobutyl ring of cbdca-2H is
perpendicular to the platinum square plane, as is observed for car-
boplatin [40].
in a highly humidified atmosphere of 95% air with 5% CO2 at 37 °C.
2.7.2. Cytotoxicity assays
The potential growth-inhibitory effect of the platinum com-
plexes on the leukemia HL-60 cell line was estimated using the
MTT assay [37]. Cells growing in the logarithmic phase were
seeded in 96-well plates (104 cells per well), and subsequently
treated with varying doses of the platinum complexes and the ref-
erence drug, i.e. cisplatin, at 37 °C for 24 or 72 h. For each of the
variations tested, four wells were used. Aliquots (20 lL) of a MTT
solution were then added to each well. After 3 h, the colored for-
mazan formed was quantified by a spectrophotometric plate read-
er at 490 nm wavelength. The percentage of cell viability was
calculated by dividing the average absorbance of the cells treated
with the complex by that of the control; IC50 values (drug concen-
tration at which 50% of the cells are viable relative to the control)
were obtained using the GraphPad Prism software, version 4.0.
Each experiment was repeated at least three times, and each con-
centration tested in at least six replicates.
3. Results and discussion
3.1. Syntheses of the platinum(II) coordination compounds
A series of seventeen [Pt(Am)2X2] complexes have been pre-
pared through the combination of various inert Am ligands and la-
bile X ligands (see Fig. 2), applying Dhara’s synthetic procedure
(see Section 2) [32]. The compounds obtained are listed in Table 1.
Fig. 3. Representation of the molecular structure of [Pt(opea)(cbdca-2H)] (5).
Hydrogen atoms and lattice water molecules are omitted for clarity. Only one of the
two slightly different cis-platinum(II) molecules present in the unit cell is shown
(see Fig. S1).

E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
71
Fig. 5. Representation of the molecular structure of [Pt(NH3)2(bzmal-2H)] (12).
Hydrogen atoms are omitted for clarity.
bzmal-2H ligand. The resulting slightly distorted square plane is
characterized by coordination angles varying from 83.9(8) to
97.2(9)°. The N11–Pt–N12 angle of 97.2(9)° reflects the repulsion
between the ammine ligands, which is a feature that has been ob-
served for related platinum complexes [42,43]. As a result, all other
coordination angles of the square plane are below 90°. The Pt–N
and Pt–O bond distances are in normal ranges for such coordina-
tion compounds [33].
In addition, the crystal structure of 12 exhibits a remarkable
network of supramolecular interactions. Molecules of 12 are
hydrogen-bonded to each other via N–HꢀꢀꢀO contacts, which gener-
ate a 1D chain (Fig. S4; green dotted lines). Two 1D chains inter-
Fig. 4. Representation of the molecular structure of [Pt(hmpy)2(cbdca-2H)] (6).
Hydrogen atoms are omitted for clarity.
The crystal structure of (5)2ꢀ3H2O exhibits an intricate intermo-
lecular network of O–HꢀꢀꢀO and N–HꢀꢀꢀO bonds involving lattice
water molecules and the amino and carboxylato groups of the plat-
inum units. Hence, eight molecules of 5 are arranged around ele-
ven water molecules. This supramolecular organization results in
the formation of 1D channels that are filled with water molecules
(Fig. S2).
mingle through p–p interactions between the phenyl rings of the
bzmal-2H ligands (Fig. S4; red dotted lines) that give rise to the
formation of a zipper-like architecture. The ammine ligands and
the carboxylato H-acceptor groups are further involved in H-bond-
ing contacts, which produce a 3D supramolecular framework
(Fig. S5).
3.2.2. Crystal structure of [Pt(hmpy)2(cbdca-2H)] (6)
The reaction of [Pt(hmpy)2I2] with Ag2(cbdca-2H) in water
solution yields, after filtration of the AgI salt produced, single crys-
tals of compounds 6 after a two months period. Single-crystal X-
ray studies revealed that 6 crystallizes in the monoclinic space
group P21/c. A perspective view of the molecular structure of 6 is
represented in Fig. 4.
Details for the structure solution and refinement are summa-
rized in Table S3, and selected bond distances and angles are listed
in Table S4. The molecule is a mononuclear platinum(II) compound
consisting of a tetracoordinated metallic center in a square-planar
coordination environment (Fig. 4). The square plane is formed by
the carboxylato oxygen atoms O3 and O5 belonging to one biden-
tate cbdca-2H ligand, and two pyridine nitrogen atoms (N1 and
N2) from two monodentate hmpy ligands. The almost perfect
square-planar geometry is characterized by coordination angles
ranging from 87.9(3) to 92.1(3)° (Table S4). The bond lengths and
angles are in normal ranges for this type of coordination com-
pounds [38,41].
3.2.4. Crystal structure of [Pt2(hmpy)4(l-dcch-2H)2 (17)
The reaction between [Pt(hmpy)2I2] and Ag2(dcch-2H) in water
solution for 24 h yielded, after filtration of the AgI salt produced,
single crystals of 17ꢀ2H2O after several months. Single-crystal X-
ray studies revealed that 17ꢀ2H2O crystallizes in the orthorhombic
space group Pbcn. A perspective view of the molecular structure of
17 is represented in Fig. 6.
Details for the structure solution and refinement are summa-
rized in Table S7, and selected bond distances and angles are listed
in Table S8. The molecule is a dinuclear platinum(II) compound
consisting of tetracoordinated metallic centers in a square-planar
coordination environment. The square planes (for Pt1 and Pt1a)
are formed by two nitrogen atoms belonging to two hmpy ligands
and two carboxylato oxygen atoms from two different dcch-2H li-
gands, which are connecting the two platinum atoms through a
double bridge (Fig. 6). The Pt–N and Pt–O bond lengths are compa-
rable to those of compounds previously reported in the literature
that exhibit similar environment features [44,45]. The coordination
angles, varying from 86.92(19) to 92.51(18)°, illustrate a slight dis-
tortion of the square-planar coordination geometry, which is most
likely due to some steric constraints arising from the bridging
binding mode of the dcch-2H ligand. The angle between the coor-
dination plane of the platinum dimer is 23.30°.
The crystal structure of 6 exhibits an intricate intermolecular
network of O–HꢀꢀꢀO bonds, which connects the alcoholic O–H group
of the hmpy ligand to two neighboring platinum molecules, result-
ing in a supramolecular 3D framework (Fig. S3).
3.2.3. Crystal structure of [Pt(NH3)2(bzmal-2H)] (12)
The reaction of [Pt(NH3)2I2] with Ag2(bzmal-2H) in water gave
single crystals of 12 after two months. 12 crystallizes in the mono-
clinic space group Pc (Table S5). The single-crystal X-ray structure
of 12 is depicted in Fig. 5.
Selected bond distances and angles are listed in Table S6. The
platinum(II) ion of the mononuclear compound is tetracoordinated
by two ammine ligands (nitrogen atoms N11 and N12) and two
oxygen atoms (O11 and O13) belonging to one carboxylato
The crystal structure of 17ꢀ2H2O exhibits an intricate intermo-
lecular network involving C–OHꢀꢀꢀOC=O OHwaterꢀꢀꢀOC=O and C–
,
OHꢀꢀꢀOwater hydrogen bonds (Fig. S6). Hence, each molecule of 17
is connected to four neighbors via C–OHꢀꢀꢀOC=O contacts
(Fig. S6A), and to two other adjacent molecules of 17 through four
water molecules, by means of OHwaterꢀꢀꢀOC=O and C–OHꢀꢀꢀOwater
bonds (Fig. S6B). This supramolecular organization of the dinuclear

72
E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
Fig. 6. Representation of the molecular structure of [Pt2(hmpy)4(l-dcch-2H)2] (17). Hydrogen atoms and lattice water molecules have been omitted for clarity. Symmetry
operation: a = ꢁx, y, ꢁz + 1/2.
platinum(II) compounds in the lattice gives rise to a 3D framework
(Fig. S6C).
carboplatin and oxaliplatin and to the complexes 7–17 in
Figs. S7–S10.
Compound 1 generates a number of crossing points and DNA
supercoiling is clearly observed (Fig. 7A). This modification of the
morphology of the plasmid DNA is indicative of a strong interac-
tion of 1 with pBR322 [46]. The AFM image illustrating the forma-
tion of 2-DNA adducts is depicted in Fig. 7B. Supercoiled forms of
DNA are present in small quantity, and some crossing points are
detected; hence, compound 2 does not show strong interaction
with DNA. A comparable behavior is observed for compound 4
(Fig. 7D). In contrast, compound 3 exhibits a distinct comportment.
A significant aggregation of DNA is noticed (Fig. 7C), which results
3.3. Tapping mode atomic force microscopy (TMAFM)
Direct visualization of three conformers of plasmid DNA can be
achieved using tapping mode atomic force microscopy (TMAFM)
and thus allows graphically evaluating the interaction of plasmid
DNA with for instance platinum coordination compounds. The
AFM images of pBR322 plasmid DNA incubated with the platinum
compounds of the series [Pt(Am)2(cbda-2H)] (complexes 1–6;
Table 1) are depicted in Fig. 7 and the corresponding to cisplatin,
Fig. 7. TMAFM images of pBR322 plasmid DNA incubated at 37 °C for 24 h with A) [Pt(en)(cbdca-2H)] (1), B) [Pt(temed)(cbdca-2H)] (2), C) [Pt(dach)(cbdca-2H)] (3), D)
[Pt(opa)(cbdca-2H)] (4), E) [Pt(opea)(cbdca-2H)] (5) and F) [Pt(hmpy)2(cbdca-2H)] (6).

E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
73
from the abundant Pt-mediated crossing points that produce
supercoiled forms, in high percentage. Compound 5 does not nota-
bly modify the morphology of pBR322 plasmid DNA, and mainly
relaxed forms (OC) are present (Fig. 7E). Similarly, compound 6
does not seem to affect the morphology of DNA, therefore suggest-
ing poor interaction between this complex and DNA (Fig. 7F).
From the series of compounds 7–11, the compound 8 is the
platinum(II) complex that affects most the DNA strands
(Fig. S8B). Compound 7 modifies the morphology of pBR322 DNA,
and CCC forms are predominantly seen in the corresponding pic-
ture (Fig. S8A). The aggregation of the forms observed for com-
pound 9, in Fig. S8C, may be due to the presence of a water layer
over the mica surface. Complex 11 does not seem to modify signif-
icantly the morphology of pBR322 plasmid DNA.
Fig. 9. Agarose gel electrophoretic mobility of DNA pBR322 (lane 1) and treated
with cisplatin (c, lane 2); compound 6 (lane 3); compound 11 (lane 4); compound
16 (lane 5) and compound 17 (lane 6).
The AFM images of pBR322 plasmid DNA incubated with the
platinum compounds of the series [Pt(Am)2(bzmal-2H)] (com-
plexes 12–16; Table 1) are depicted in Figs. S9 and S10. The inter-
action between compound 12 and DNA results in broken DNA
strands, as a consequence of severe tensions caused by the coordi-
nation of the platinum complex. Compound 15 induces clear alter-
ations of the DNA structure, with crossing points, kinks and broken
strands with microfolds (Fig. S9D). The AFM image of pBR322 plas-
mid DNA incubated with the platinum(II) complex [Pt2(hmpy)4(l-
dcch-2H)2] (complex 17; Table 1) is depicted in Fig. S10. This figure
clearly evidences that 17 does not affect notably the tertiary struc-
ture of DNA.
3.4. Electrophoretic mobility
The influence of compounds 1–17 on the tertiary structure of
DNA was investigated further by their ability to modify the electro-
phoretic mobility of the covalently closed circular (CCC) and open
(OC) forms of pBR322 plasmid DNA. The results are shown in
Figs. 8–10. In the pattern corresponding to the pBR322 plasmid
DNA (Fig. 8, lanes 1 and 15; Fig. 9, lane 1 and Fig. 10, lane 1) a clean
difference between the OC and CCC bands is noticed. In lanes 2 of
Figs. 8–10 and lane 14 of Fig. 8, the typical coalescence of the two
bands resulting from the action of cisplatin is evident.
Fig. 10. Agarose gel electrophoretic mobility of DNA pBR322 (lane 1) and treated
with cisplatin (c, lane 2); compound 12 (lane 3); compound 13 (lane 4); compound
14 (lane 5) and compound 15 (lane 6).
Actually, slight modifications of the tertiary structure of pBR322
plasmid DNA have been noted by AFM for these complexes. Com-
pound 8, similarly to cisplatin, induces the complete coalescence of
the CCC and OC bands. The formation of OC forms has been ob-
served by AFM (Fig. S8B). Finally, compound 12 clearly affects
the DNA electrophoretic mobility, since the CCC form is not de-
tected (Fig. 10). Interestingly, AFM studies have revealed the pres-
ence of broken DNA strands with 12 (Fig. S9A). In summary, it
appears that [Pt(en)(mdbs-2H)] (8) and [Pt(NH3)2(bzmal-2H)]
(12) exhibit the strongest interaction with DNA.
For compounds 1–17, the electrophoresis data largely comple-
ment those obtained from the AFM studies (see above).
Compounds 2, 5–7, 9, 11, 13–17 do not affect the DNA mobility
on the agarose gel and it has been observed by AFM that these PtII
complexes do not notably modify the morphology of pBR322 plas-
mid DNA (see Figs. S8–S10). For compounds 3, 4 and 10 (Fig. 8), the
CCC and OC bands start to merge, therefore indicating an alteration
of the DNA mobility.
3.5. Viscosimetry measurements
The potential interaction between the platinum(II) complexes
and DNA was examined further by viscosimetry measurements.
Such studies give information about the possible way(s) the coor-
dination compounds interact with the DNA helix. Hence, the inter-
calation of the metal compound will induce a length change since
base pairs will have to separate to accommodate the binding of the
ligand; this will result in an increase in DNA viscosity [47]. In con-
trast, partial or non-intercalation may lead to a bend (or kink) of
the DNA helix, giving rise to a diminution of its effective length
and therefore its viscosity [48].
1/3
The values of relative specific viscosity (
g
/g0)
versus the
molecular ratio, i.e. ri, in the absence and in the presence of the
complexes (1)–(6) are plotted in Fig. 11.
Fig. 8. Agarose gel electrophoretic mobility of DNA pBR322 (lanes 1 and 15) and
treated with cisplatin (c, lane 2); compound 1 (lane 3); compound 2 (lane 4);
compound 3 (lane 5); compound 4 (lane 6); compound 5 (lane 7); compound 7
(lane 8); compound 8 (lane 9); compound 9 (lane 10); compound 10 (lane 11);
oxaliplatin (o, lane 12); carboplatin (ca, lane 13) and cisplatin (c, lane 14).
For carboplatin (Fig. 11), the viscosity decreases with increasing
ri, thus suggesting conformational changes of ct-DNA, due to the
coordination of the platinum moiety. Compound 1 induces an
abrupt decrease of the viscosity at ri = 0.1, which is ascribed to

74
E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
decreases, thus suggesting that this compound experiences com-
petitive DNA interactions through covalent or groove binding.
Compound 11 (Fig. S11) displays behavior similar to that of 5,
hence implying that this complex interacts with DNA mostly
through H bonds.
The values of relative specific viscosity (g/g0)
1/3 versus ri for the
series cis-[Pt(Am)2(bzmal-2H)] (complexes 12–16; Table S5) are
plotted in Fig. S12, Supporting information.
Compounds 12 and 13 show similar behaviors, that is a de-
crease of the viscosity up to ri = 0.3, followed by an increase at
1/3
ri = 0.5. The diminution of (
g
/g0)
may be ascribed to the forma-
tion of interstrand adducts that alter the secondary structure of
DNA. The subsequent increase of viscosity is most likely due to
Fig. 11. Viscosimetry data, at ri = 0.1, 0.3 and 0.5, for carboplatin and the
coordination compounds [Pt(en)(cbdca-2H)] (1), [Pt(temed)(cbdca-2H)] (2),
[Pt(dach)(cbdca-2H)] (3), [Pt(opa)(cbdca-2H)] (4), [Pt(opea)(cbdca-2H)] (5) and
[Pt(hmpy)2(cbdca-2H)] (6).
groove binding, by means of hydrogen-bonding contacts involving
1/3
the NꢁH groups. The (
g
/g0
)
versus ri trend for [Pt(temed)(bz-
mal-2H)] (14) (Fig. S12) is related to those of [Pt(temed)(cbdca-
2H)] (2) (Fig. 11) and [Pt(en)(mdbs-2H)] (8) (Fig. S11). Hence, a
significant decrease of viscosity is detected at ri = 0.1, which may
be explained by the bend (or kink) of the DNA helix induced by
the creation of cis-bifunctional intrastrand DNA-complex adducts.
The successive increase (at ri = 0.3) and decrease of the viscosity
(at ri = 0.5) are indicative of the occurrence of different types of
interactions between the Pt moiety and the DNA molecule, appar-
ently due to the potential ability of the complexes to generate both
the formation of cis-bifunctional DNA intrastrand adducts that
leads to a bend of the DNA molecule, lessening its viscosity. At
1/3
ri = 0.5 (
g
/g0)
increases (Fig. 11), which may be explained by
the aggregation of DNA molecules (as observed by AFM; see above
and Fig. 7A) that results in an increase of viscosity. At ri = 0.1 and
0.3, compound 2 exhibits a behavior similar to that of 1; however,
1/3
at ri = 0.5 (
g/g0
)
still decreases, therefore suggesting that the li-
gand temed (contrary to en) does not allow DNA aggregation
(Fig. 11). The comportment of complexes 3 and 4 is related to that
of 1, albeit their effect on the conformational changes is minor
(compared to that of 1). Compound 5 does not appear to signifi-
cantly affect the structure of the ct-DNA helix (Fig. 11). The slight
decrease noticed may be attributed to the weak binding of the
complex through hydrogen bonds, for instance with the phosphate
backbone. Lastly, the binding between compound 6 and ct-DNA
shows characteristics that are comparable to those of 2 (Fig. 11),
which most likely arise from the formation of interstrand adducts
that causes a conformation change, from the B-form to the Z-form.
hydrogen bonds (groove binding) and/or covalent bonds. The (g/
1/3
g0)
versus ri values for compounds 15 and 16 do not indicate
any major conformational changes. Actually, ligands that bind
exclusively in the DNA grooves (like netropsin or distamycin), un-
der the same conditions, typically cause less pronounced changes
(positive or negative) or no changes in DNA solution viscosity.
Therefore, these results suggest that 15 and 16 are mostly groove
binders.
For [Pt2(hmpy)4(l-dcch-2H))2] (17), a slight diminution of the
viscosity is noticed up to ri = 0.3 (Fig. S13), which is ensued by a
minor growth at ri = 0.5. Again, this behavior is indicative of non-
covalent binding to DNA through hydrogen-bonding contacts (with
Compounds 7, 9 and 10 (Fig. S11, Supporting information) pres-
1/3
ent (g
/g0)
versus ri trends that resemble those of compounds 2
the phosphate backbone) or/and
tween bases).
p–p stacking interactions (be-
and 6 (Fig. 11). Most likely, these complexes bind DNA covalently
via the nitrogen N7 of guanine, producing a local opening of double
helix that reduces the viscosity. Compound 8 (Fig. S11) shows the
major effects in this series. The clear diminution of the viscosity at
ri = 0.1 is probably due to the creation of interstrand adducts that
modifies the DNA conformation. The subsequent increase of the
viscosity, from ri = 0.1 to ri = 0.3 may be an indication of groove
3.6. Competitive binding studies using fluorescence spectroscopy
Ethidium bromide (EtBr), a cationic dye that can interact with
DNA through intercalation is used commonly in spectroscopic
studies [49–52]. Upon binding to DNA, EtBr shows characteristic
changes in absorbance, reflected by an enhancement in
binding (surface binding) at higher loading of complex [47]. Final-
1/3
ly, at very high complex loading, namely at ri = 0.5 (g/g0)
Fig. 12. Emission spectra of the EtBr–DNA complex (EtBr stands for ethidium bromide) in the presence of increasing concentrations of (A) acridine and (B) cisplatin, at room
temperature, after an incubation time of 30 min.

E. Escribano et al. / Inorganica Chimica Acta 394 (2013) 65–76
75
fluorescence intensity by about one order of magnitude, as com-
pared to free dye in solution [53].
after an incubation time of 72 h for [Pt(dach)(cbdca-2H)] (3), the
corresponding value for cisplatin (control) being 2.15 0.1
der the same experimental conditions.
lM, un-
Hence, when a platinum compound is added to the EtBr-DNA
complex, any quenching of the fluorescence will indicate the
replacement of EtBr by the coordination molecule, by intercalation.
Fig. 12 illustrate the competitive binding between EtBr and
Acridine or cisplatin. Acridine is better intercalator than EtBr;
therefore, its addition gives rise to a release of EtBr in the solution
associated with a diminution of the emission (Fig. S15A). In con-
trast, cisplatin cannot displace EtBr, which is indicated by the ab-
sence of fluorescence quenching (Fig. S15B).
Competitive binding studies have been carried out for com-
pounds 3, 4, 5, 6, 8, 11, 12, 15, 16 and 17, and the corresponding
emission spectra (recorded after incubation times of 30 min and
24 h) are depicted in Figs. S14–S23 (Supporting information). The
compounds have been selected on the basis of the results obtained
with other characterization techniques (described above) or to
clarify the outcome of the viscosimetry measurements (see previ-
ous section). As is clearly evidenced in Figs. S11–S20, the PtII com-
pounds investigated do not induce a significant quenching of the
fluorescence resulting from the release of EtBr, thus suggesting
that they cannot displace the cationic dye and efficiently interca-
late into DNA. These fluorescence studies thus suggest that all
the platinum(II) compounds reported herein are not effective
DNA intercalators. Consequently, the interactions observed using
other characterization techniques indicate that they occur via dif-
ferent mechanisms (such as covalent or hydrogen bonding).
4. Conclusions
A series of platinum(II) complexes have been synthesized and
the molecular structures of four of them have been determined
by X-ray diffraction studies. The interaction between DNA and
the coordination compounds have been appraised using different
techniques, namely viscosity measurements, emission fluores-
cence spectroscopy and atomic force microscopy (AFM), which
show that indeed they bind to the biological macromolecule. For
instance, the AFM images of samples of the compounds incubated
with plasmid pBR322 DNA reveal very strong DNA/compound
interactions of different types, attributed predominantly to super-
coiling, stacking and fragmentation. Surprising, only one of the
complexes prepared, namely [Pt(dach)(cbdca-2H)] (3), presents
an interesting IC50 value, in the same order of magnitude as that
of cisplatin. Compound 3 contains the inert amine (trans-cyclohex-
anediamine; dach) found in oxaliplatin and the labile dicarboxyla-
to ligand (1-cyclobutanedicarboxylate; cbdca-2H) present in
carboplatin. These results clearly demonstrate the difficulty to ob-
tain potential platinum-based anti-cancer agents, even through the
combination of ligands that have generated efficient systems (de-
picted in Fig. 1). The lack of cytotoxicity exhibited by most of the
compounds may be explained by difficulties in the internalization
processes into the cells or a more inert character of the carboxylate
ligands compared to its chloride counteparts.
3.7. Cytotoxic activity
The potential cytotoxicity of the platinum complexes 1–11 and
cisplatin (for comparison) on human leukemia cancer cells (HL-60)
was examined using the MTT assay, a colorimetric determination
of cell viability during in vitro treatment with a drug. Compounds
12–17 could not be assayed due to their insolubility under the
experimental conditions used. The assay, developed as an initial
stage of drug screening, measures the amount of MTT reduction
by mitochondrial dehydrogenase and assumes that cell viability
(corresponding to the reductive activity) is proportional to the pro-
duction of purple formazan that is measured spectrophotometri-
cally. Low IC50 values will be indicative of cytotoxicity or
antiproliferation at low drug concentrations. Cells were incubated
with each compound for a period of 72 h, and were assayed subse-
quently for growth using the MTT endpoint assay. The IC50 values
characterizing the growth inhibition of HL-60 cells induced by
complexes 1–11 and cisplatin are summarized in Table 2. Most
platinum complexes presented herein exhibit poor cytotoxic prop-
erties against HL-60 cells; therefore, apoptosis studies were not
Acknowledgments
Financial support from the Spanish Ministry of Ciencia e Inno-
vación, Contracts CTQ2008-02064 and BIO2010-22321-C02-01
are kindly acknowledged. Esther Escribano thanks the University
of Barcelona for granting her a Ph.D. scholarship.
Appendix A. Supplementary material
CCDC 865068–865071 contains the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2012.07.018.
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