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

Inorganica Chimica Acta 394 (2013) 65–76

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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,

⇑

^aDepartament de Química Inorgànica, University of Barcelona, Martí i Franquès 1-11, 08028 Barcelona, Spain

^bDepartament de Cristalꢀlograﬁa, Mineralogia i Dipòsits Minerals, Martí i Franquès s/n, 08028 Barcelona, Spain

^cCentre Cientíﬁc i Tecnològic de la Universitat de Barcelona (CCITUB), Unitat de Difracció de RX, Solé i Sabarís 1-3, 08028 Barcelona, Spain

^dInstitut de Biotecnologia i Biomedicina, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain

^eInstitució 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

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).

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 cm^ꢁ1), and data are represented as the frequency of

absorption (cm^ꢁ1); 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 Pt^IIcompounds 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 mol^ꢁ^{1 1}H 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, cm^ꢁ1):

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 PtN₂H₁₆C_13-

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 NaHCO₃in 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 mol^ꢁ^{1 1}H 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.

cm^ꢁ1):

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 PtN₂H_20-

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 mol^ꢁ^{1 1}H 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, cm^ꢁ1):

= 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.

68

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 mol^ꢁ^{1 1}H NMR (D₂O, 300 MHz,

.

25 °C): d 3.99–4.14 (2, br, 6H), d 4.44 (1, s, 2H) ppm. IR (KBr pellet,

cm^ꢁ1):

m = 3202 (NH), 1684 (C = O + NH₂). ESI-MS (m/z, positive

mode): ([M+H]⁺) 504, ([M+Na]⁺) 526. Anal. Calc. for (%) for PtN₂H_8-

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 mol^ꢁ^{1 1}H 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 mol^ꢁ^{1 1}H NMR (D₂O, 300 MHz,

.

let, cm^ꢁ1):

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, cm^ꢁ1):

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 PtN₂H₁₆C₁₆O_6-

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 mol^ꢁ^{1 1}H 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, cm^ꢁ1):

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 PtN₂H_18-

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 mol^ꢁ^{1 1}H 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, cm^ꢁ1):

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 mol^ꢁ1

.

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, cm^ꢁ1):

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 mol^ꢁ1

.

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, cm^ꢁ1):

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 r_i= 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 mol^ꢁ^{1 1}H 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, cm^ꢁ1):

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 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 mol^ꢁ^{1 1}H NMR (D₂O, 300 MHz,

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, H²and H³are not well resolved. IR (KBr pellet,

cm^ꢁ1):

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

mdbs-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

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)₂is 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 Pt^IImolecules 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% CO₂at 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 (10⁴cells 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; IC₅₀values (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–OHꢀꢀꢀO_C=OOH_waterꢀꢀꢀO_C=Oand C–

,

OHꢀꢀꢀO_waterhydrogen bonds (Fig. S6). Hence, each molecule of 17

is connected to four neighbors via C–OHꢀꢀꢀO_C=Ocontacts

(Fig. S6A), and to two other adjacent molecules of 17 through four

water molecules, by means of OH_waterꢀꢀꢀO_C=Oand C–OHꢀꢀꢀO_water

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 Pt^II

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. r_i, 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

r_i, 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 r_i= 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)

^1/3versus r_ifor 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 r_i= 0.3, followed by an increase at

1/3

r_i= 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 r_i= 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 r_itrend 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 r_i= 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 r_i= 0.3) and decrease of the viscosity

(at r_i= 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

r_i= 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 r_i= 0.1 and

0.3, compound 2 exhibits a behavior similar to that of 1; however,

1/3

at r_i= 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 r_ivalues 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 r_i= 0.3 (Fig. S13), which is ensued by a

minor growth at r_i= 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 r_itrends 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

r_i= 0.1 is probably due to the creation of interstrand adducts that

modiﬁes the DNA conformation. The subsequent increase of the

viscosity, from r_i= 0.1 to r_i= 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 r_i= 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 Pt^IIcom-

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 IC₅₀value, 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 IC₅₀values 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 IC₅₀values

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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Article Doi

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

DOI: 10.1016/j.ica.2012.07.018

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