Inorganica Chimica Acta 412 (2014) 88–93

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Inorganica Chimica Acta

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Synthesis, characterization and cytotoxicity of novel Pt(II)j2O,O0-acetylacetonate complexes with nitrogen ligands

0020-1693/$ - see front matter � 2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.ica.2013.12.018

⇑ Corresponding author. Tel.: +39 (0)832 298 867; fax: +39 (0)832 298 626.E-mail address: fp.fanizzi@unisalento.it (F.P. Fanizzi).

Sandra A. De Pascali, Antonella Muscella, Carla Vetrugno, Santo Marsigliante, Francesco Paolo Fanizzi ⇑Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali, Università del Salento, prov.le Lecce/Monteroni, I-73100 Lecce, Italy

a r t i c l e i n f o

Article history:Received 10 October 2013Received in revised form 11 December 2013Accepted 18 December 2013Available online 31 December 2013

Keywords:Platinum(II)AcetylacetonateNMR spectroscopyCytotoxicityCancer cells

a b s t r a c t

Synthetic procedures to obtain the new b-diketonate compounds [Pt(acac-j2O,O0)(acac-j-C3)(NH3)] (1)and [Pt(acac-j2O,O0)(acac-j-C3)(py)] (2) and their characterization by multinuclear, multidimensionalNMR spectroscopy are reported. The new complexes have been designed and synthetized in order toestablish the role of the sulphur ligand (DMSO/DMS) in the cytotoxic activity of [Pt(acac-j2O,O0)(acac-j-C3)(DMSO)] and [Pt(acac-j2O,O0)(acac-j-C3)(DMS)]. These complexes, already reported by our researchgroup, were extensively studied for their outstanding biological activities. [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] showed high cytotoxicity on cisplatin resistant cell lines and selective cytotoxic effects againstbreast cancer cells in primary cultures at concentrations lower than cisplatin. The new b-diketonate com-plexes (1 and 2), containing a nitrogen ligand (NH3 or pyridine) in place of DMSO/DMS ligand, have beenobtained and isolated as stable products by cleavage of binuclear [{PtI2(NH3)}2] or starting with cis-[PtI2(-py)2] monomer in the presence of excess acac. In this respect the used synthetic pathways were com-pletely different from those previously used in the case of analogues containing sulphur ligands(DMSO/DMS). Preliminary in vitro cytotoxic effects (MTT and clonogenic assays) of complex 1 on cisplatinresistant MCF-7 breast cancer cell line are reported and compared with the [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] activity.

� 2013 Elsevier B.V. All rights reserved.

1. Introduction

After more than 30 years since its first clinical use, cisplatin isstill one of the most widely used drugs in anticancer chemotherapy[1] and its action mechanism has been explained in all essentialaspects related to interaction with DNA. Nevertheless, some keychemical processes, taking place before cisplatin reaches DNA,generally considered its final target, are still to be clarified [2,3].Cisplatin is particularly active against testicular cancer and, iftumors are discovered early, an impressive cure rate of nearly100% is achieved [4]. Its clinical use against this and other malig-nancies is, however, severely limited by dose limiting side effectssuch as neuro-, hepato- and nephro-toxicity. On the other hand,the need to improve the cisplatin clinical therapy drives muchresearch into better understanding of its antitumor activitymechanism [5]. Moreover, in order to overcome acquired cellularresistance to cisplatin, much effort is currently devoted to discovernew Pt anticancer drugs. In the last years several Pt(II) and Pt(IV)complexes have been synthesized but only a few compounds, suchas carboplatin and oxaliplatin [1,6], are actually used in clinicaltherapy. Our research group has long been involved in the

synthesis and biological studies of Pt(II) complexes containing atleast one chelate acetylacetonate (acac-j2O,O0) and a sulphurligand (dimethylsulfoxide, DMSO, or dimethylsulfide, DMS) [7,8].Although every metal has been found to give acetylacetonatecomplexes, few studies describe their potential utility inplatinum-based therapeutic agents [9]. Among our complexes[Pt(acac-j2O,O0)(acac-j-C3)(DMS)] containing both an acac-j2O,O0

and a r-bonded acac (acac-j-C3) resulted the most effective [10],showing in vitro and in vivo antitumoral activity higher thancisplatin, also on cisplatin-resistant cell lines [11] and breastcancer cells in primary culture [12]. This compound not only wasable to induce apoptosis in endometrial cancer cells (HeLa) [10]with activity up to about 100 times higher than that of cisplatin,but also showed high cytotoxicity in cisplatin-resistant breastcancer cell lines (MCF-7) and other breast cancer cell lines, butnot in MCF-10A, considered to be normal and non-cancerousbreast cells [11]. Recently, to confirm that [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] has the potentiality for therapeutic interven-tion, its effects in primary cultured epithelial breast cells obtainedfrom cancers and also from the corresponding histologicallyproven nonmalignant tissue adjacent to the tumor were studied.[Pt(acac-j2O,O0)(acac-j-C3)(DMS)] showed potent and selectivecytotoxic effects against breast cancer cells in primary cultures atconcentrations lower than cisplatin, whose undesirable side effects

S.A. De Pascali et al. / Inorganica Chimica Acta 412 (2014) 88–93 89

continue to limit its effectiveness. Therefore, [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] has been recently suggested as new Pt(II) anticancerdrug candidate with selective cytotoxicity for breast cancer cells[12]. In vivo experiments using a xenograft model of breast cancerdeveloped by injection of MCF-7 cells in the flank of BALB/c nudemice also confirmed this potential therapeutic use [13]. Moreover,the complex rapidly produces a sustained apoptotic response char-acterized by: mitochondrial depolarization, cytosol accumulationof cytochrome c, mithocondrial translocation of proapoptotic pro-teins (Bax and truncated form of Bid), activation of caspase-7 and -9, chromatin condensation, DNA fragmentation and generation ofreactive oxygen [14]. [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] was alsoable to increase the level of free [Ca2+]i in MCF-7 cells, alteringthe homeostasis of Ca2+, an effect that is likely to be linked to itsability to trigger rapid apoptosis [15]. Moreover, [Pt(acac-j2-

O,O0)(acac-j-C3)(DMS)] passed the blood–brain barrier and reachesthe central nervous system in amounts much higher than cisplatin[16,17]. Nevertheless, [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] displayeda low neurotoxicity in normal tissue, certainly less important thanthat caused by cisplatin [18]. Further studies on the cytotoxic ef-fects and intracellular transduction apoptosis mechanism of [Pt(a-cac-j2O,O0)(acac-j-C3)(DMS)] on brain cancer cells (SH-SY5Yhuman neuroblastoma cell line) showed cytotoxicity on neuroblas-toma cells with effects ca. 10-fold greater than that observed forcisplatin [19]. Besides its specific biological activity, the b-diketo-nate complex gave selective substitution of DMS with soft biolog-ical nucleophiles, such as l-methionine, and negligible reactivitywith nucleobases (Guo and 50-GMP) [20,21]. Interestingly, totalcellular and nuclear Pt contents for cisplatin and [Pt(acac-j2-

O,O0)(acac-j-C3)(DMS)] in vitro in HeLa and MCF-7 cells, deter-mined by atomic absorption spectrometry using doses that givethe same intracellular Pt content, arisen in a similar distributionin the nucleus with respect to cisplatin but resulting unable to linkDNA [10,11]. However, at the same doses and for the same incuba-tion times, [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] retained its cyto-toxic effect on cancer cells higher than cisplatin. Then, differentlyfrom cisplatin, whose activity appears to be associated with cellu-lar accumulation and DNA linking, the cytotoxicity of the newcompounds is related to intracellular accumulation only. All thesedata suggest that the cytotoxicity mechanisms of [Pt(acac-j2-

O,O0)(acac-j-C3)(DMS)] may not necessarily require interactionwith DNA and that its biological activity is connected to the reac-tion with non genomic biological targets. In this context, in orderto ascertain the role of the sulphur ligand, new b-diketonate com-plexes have been synthetized containing nitrogen ligand (NH3 orpyridine) in place of DMSO/DMS ligand: [Pt(acac-j2O,O0)(acac-j-C3)(NH3)], 1, and [Pt(acac-j2O,O0)(acac-j-C3)(py)], 2. (Scheme 1).Synthetic procedures to obtain the new b-diketonate compounds,chemical characterization by multinuclear, multidimensionalNMR spectroscopy and biological activity (cytotoxicity and clono-genicity assays) of complex 1 on MCF-7 breast cancer cell line withrespect to the [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] are reported.

O

O

HC

CH3

H3C

Pt

LO

O

H3C

H3C

H

L = DMSO, DMS, NH3, py

Scheme 1. Chemical structure of complexes [Pt(acac-j2O,O0)(acac-j-C3)(DMSO)],[Pt(acac-j2O,O0)(acac-j-C3)(DMS)], [Pt(acac-j2O,O0)(acac-j-C3)(NH3)], 1, and [Pt(a-cac-j2O,O0)(acac-j-C3)(py)], 2.

2. Experimental

2.1. Physical measurements

Elemental analyses were performed using a Carlo-Erba elemen-tal analyser, model 1106. 1H NMR, 195Pt NMR, 1H COSY, 1H NOESY,1H–13C HSQC and 1H–13C HMBC two-dimensional experimentswere recorded on a Bruker Avance III 400 MHz. CDCl3 was usedas solvent and chemical shifts were referenced to TMS by the resid-ual protic solvent peaks as internal references (d(H) = 7.24 ppm,d(C) = 77.0 ppm). 195Pt chemical shifts were referenced to Na2[-PtCl6] (d(Pt) = 0 ppm) in D2O as an external reference [22].

2.2. Starting materials

Acetylacetone (2,4-pentanedione) > 99%, pyridine > 99%,ammonium hydroxide solution 33% and deuterated solvents pur-chased by Sigma–Aldrich were used without further purification.cis-[PtI2(NH3)2] and cis-[PtI2(py)2] were prepared according to Dha-ra method [23]. The dimer complex [{PtI2(NH3)}2] was prepared byRochon and Kong procedure [24].

2.3. Biological assays

2.3.1. Cell linesMCF-7 cells from breast carcinoma cells were grown in Dul-

becco’s modified eagle’s medium (DMEM, Euroclone). The culturemedium was supplemented with 10% heat-inactivated fetal bovineserum (Euroclone), 0.2 mg mL�1 streptomycin, 200 IU mL�1 peni-cillin. Cells were cultured routinely at 37 �C and 5% CO2 in a humid-ified incubator.

2.3.2. MTT assayThe MTT assay, already described by Mosmann [25], is a cyto-

toxicity test based on the metabolic reduction of a soluble tetrazo-lium salt (3-[4,5-dimethyllthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide) (Sigma) by a mitochondrial enzyme of cultured cells intoan insoluble colored formazan product. After harvesting, the cellswere counted and diluted appropriately with culture medium;100 mL containing 5000 (MCF-7) cells were seeded in each wellof a 96-well microtiter plate (Corning). After 24 h of incubation,cisplatin and its analogues, previously solubilized in PBS (Phos-phate Buffer Solution) were administered to each well in appropri-ate concentrations (from 1 to 200 lM). The toxicity of thesecompounds was tested for 24, 48, and 72 h. At the end of everyincubation MTT was added at a final concentration of500 mg mL�1. After 4 h of incubation, the amount of formazanwas spectrophotometrically measured at 550 nm wavelength inisopropanol. Optical density was used to calculate cell growth inhi-bition, as % with respect to the control. For the statistical analysisof the data the Bonferroni–Dunn test was used and a p value<0.05 was considered significant. All the results are the mean ofthree, separately performed, experiments.

2.3.3. Sulphorhodamine B assayThe sulphorhodamine B (SRB) assay was carried out as previ-

ously described [26]. Briefly, 70 ml 0.4% (w v_1) SRB in 1% aceticacid solution was added to each well and left at room temperaturefor 20 min. SRB was removed and the plates washed five timeswith 1% acetic acid before air-drying. Bound SRB was dissolved in200 ml of 10 mM unbuffered Tris–base solution and plates wereleft on a plate shaker for at least 10 min. Absorbance was measuredin a 96-well plate reader at 492 nm. The test optical density valuewas defined as the absorbance of each individual well minus theblank value (‘blank’ is the mean optical density of the background

I

Pt

I

Pt

I

NH3

H3N

IEtOH / 40 °C

3 Hacac /2 KOHEtOH / 40 °C, 6h3 Hacac /2 KOH

25°C,12 hO

O

HC

H3C

CH3

Pt

H3N O

O

CH3

CH3

H

1

Scheme 2. Scheme reaction of [Pt(acac-j2O,O0)(acac-j-C3)(NH3)] (1).

90 S.A. De Pascali et al. / Inorganica Chimica Acta 412 (2014) 88–93

control wells, n1=48). The percentage survival was calculated as theabsorbance ratio of treated to untreated cells. The data presentedare means ± s.d. from eight replicate wells per microtitre plate,repeated four times.

2.4. Syntheses of complexes

2.4.1. Syntheses of [Pt(acac-j2O,O0)(acac-j-C3)(NH3)] (1)An ethanol solution (20 mL) of Hacac (0.044 mL, 0.216 mmol)

and KOH (0.012 g, 0.108 mmol) was dropwise added to ethanolsuspension (30 mL) of [{PtI2(NH3)}2] (0.100 g, 0.108 mmol) understirring and at 313 K. Slowly the colour of mixture changed to am-ber. After 5 h other 20 mL of Hacac (0.022 mL, 0.108 mmol) andKOH (0.012 mg, 0.108 mmol) in ethanol were dropwise added, atthe same temperature. After ca. 6 h the solution became yellowand the temperature was then set to 298 K and left under stirringfor further 12 h. Then, the solution was cooled at room tempera-ture and filtered through Celite to remove traces of Pt metal. Thefiltered solution was then concentrated to 2 mL, added of H2O(�15 mL) and extracted with 20 mL of CH2Cl2. The extract was re-duced to 5 mL, added pentane (15 mL) and kept at 278 K overnight.The yellow–brown powder precipitated was isolated, washed withpentane and dried under vacuum. (Yield 0.022 g, 0.054 mmol, 25%)Anal. Calc. for C10H20NO4Pt (413.354): C, 29.06; H, 4.88; N, 3.39.Found: C, 28.91; H, 5.03; N, 3.11%.

2.4.2. Syntheses of [Pt(acac-j2O,O0)(acac-j-C3)(py)] (2)An ethanol solution (5 mL) of Hacac (2.039 mL, 19.86 mmol)

and KOH (0.074 g, 1.324 mmol) was dropwise added to ethanolsuspension (5 mL) of cis-[PtI2(py)2] (0.201 g, 0.331 mmol) understirring and at 343 K. Slowly the colour of the reaction mixturechanged to amber. After 2 days the stirring was stopped, the mix-ture was cooled and filtered to remove residual starting complex.The filtered solution was concentrated to 2 mL, added H2O(�15 mL) and extracted with 20 mL of CH2Cl2. The extract waschromatographed on SiO2 using as eluent a mixture of CH2Cl2:(CH3)2CO (10:1). The fraction was then evaporated to drynessand the resulted brown solid collected. (Yield 0.034 g, 0.089 mmol,27%). Anal. Calc. for C15H22NO4Pt (475.423): C, 37.89; H, 4.66; N,2.95. Found: C, 37.71; H, 4.90; N, 2.85%.

1 NMR data in CDCl3 of [PI(acac-j2O,O’)(py)]. d(1H): 1.86, 1.92 (Me/acac); 546 (CHc/cac); 8.84[3JH–Pt = 38 Hz], 7.26, 7.76 (H2/6, H3/5 and H4/py). d(13C): 25.8, 25.9 (Me/cac); 100.9 (CHc/acac),182.3, 184.5 (C@O/acac); 155.1, 125.0, 137.8 (C2/6, C3/5, C4/

py).

3. Results and discussion

3.1. Synthesis and NMR spectroscopy characterization of complexes

The complex [Pt(acac-j2O,O0)(acac-j-C3)(NH3)] (1) was synthe-tized by reacting [{PtI2(NH3)}2] with excess Hacac and stoichiome-tric (twofold with respect to the metal) KOH in ethanol. The[{PtI2(NH3)}2] dimer, obtained according to the Rochon and Kongprocedure [24], gave the final product in reasonable yield. Theslight Hacac excess and KOH were added to the dimer in two stepsas reported in the experimental section. The first Hacac and KOHaddition produces the iodide dimer bridge splitting followed prob-ably by the coordination of O-monodentate acac and subsequentO,O0-acac chelate formation to give the [PtI(acac-j2O,O0)(NH3)]

species. The second Hacac and KOH addition leads to the r-bondedcoordination of a further acac by I� substitution with formation ofthe final [Pt(acac-j2O,O0)(acac-j-C3)(NH3)] complex (scheme 2). Itshould be noted that 1 complex could also be obtained by reactingcis-[PtI2(NH3)]2 with excess Hacac and stoichiometric KOH inethanol but in a much lower yield and purity due to formation ofseveral by products.

On the other hand the [Pt(acac-j2O,O0)(acac-j-C3)(py)] (2) com-plex was obtained in a single step reacting cis-[PtI2(py)2] with avery large Hacac excess and KOH in stoichiometric amount withrespect to the Hacac to coordinate. The use of a large excess Hacac(60-folds with respect to the Pt moles) favors protonation and re-moval of one pyridine from cis-[PtI2(py)2] but results in a loweryield of the final product [Pt(acac-j2O,O0)(acac-j-C3)(py)] (seeScheme 3). As expected, by modulating the excess of Hacac andthe KOH amount the intermediate species [PI(acac-j2O,O0)(py)]has been observed in solution and characterized by 1H and 13CNMR spectroscopy1.

1 and 2 complexes were characterized by 1H, 13C and 195Pt NMRspectroscopy. Relevant NMR data for all complexes are reported inTable 1 (1H, 195Pt), Table 2 (13C). The 1H NMR spectra in CDCl3 of 1and 2 complexes show the characteristics signals of one acetylace-tonate chelate through the two oxygens and one acetylacetonater-bonded by the j-C3 methine. Due to the asymmetry of the com-plexes, the signals of acac-j2O,O0 methyl groups show differentresonances.

The two singlets at lower frequencies (1.81 and 1.86 ppm for 1and 1.81 and 1.90 ppm for 2, respectively), integrating for threeprotons, and the singlet at 5.41 and 5.45 ppm for 1 and 2, respec-tively, integrating for one proton, were assigned to the methylgroups and the methine of the acac-j2O,O0. The two singlets at2.18 and 5.19 ppm (2JH–Pt = 112 Hz) for 1 complex and 2.18 ppmand 4.99 ppm (2JH–Pt = 131 Hz) for 2, integrating for six and oneprotons, respectively, were attributed to the methyl groups andthe j-C3 of the r-bonded acac. In the 1H NMR spectrum of 1 thebroad signal at 3.37 ppm with a 2JH–Pt of 70 Hz was assigned tothe coordinated NH3 ligand, whereas in the 1H NMR spectrum of2 the higher frequency resonances at 8.69 (3JH–Pt = 42 Hz), 7.75,and 7.31 ppm were attributed to the H2/6, H4 and H3/5 aromaticprotons of pyridine. 2D 1H NOESY spectra in CDCl3 were acquiredto assign the methyl group of acac-j2O,O0 in cis to the nitrogenligand (NH3 or py) of 1 and 2. In both complexes the more shieldedsinglet of O,O0-acac (1.81 ppm for 1 and 2) shows a correlationcross peak with the broad signal of NH3 and H2/6 protons of pyri-dine, respectively, whereas the more deshielded singlet (1.86 and1.90 ppm for 1 and 2, respectively) shows correlation with themethine of c-acac (see Fig. 1). Also in the cases of [Pt(acac-j2-

O,O0)(acac-j-C3)(L)] (L = DMSO, DMS, PPh3, ethylene, and CO) themore shielded singlet was in cis to the L [8]. This is in accord witha lower deshielding effect for the chelate acac moiety trans to ther-carbon ligand and therefore more loosely bound to the metal

aa

N

IPt

N

I

60Hacac, 4KOHEtOH, 70°C, 2 days

IPt

O

O

CH3

CH3

HN

O

O

CH3

CH3

PtN

HHC

CH3

H3C

OO

2

Scheme 3. Scheme reaction of [Pt(O,O0-acac)(acac-j-C3)(py)] (2).

Table 11H NMR and 195Pt chemical shifts of complexes.

H/j-C3 Me/acac L = NH3, py d195Pt

1 5.41 (j2O,O0-bonded)5.19 [112]⁄ (j-C3-bonded) 1.86; 1.81# (j2O,O0-bonded)2.18 (j-C3-bonded) 3.37 [70] �21192 5.41 (j2O,O0-bonded)4.99 [131] (j-C3-bonded) 1.90; 1.81# (j2O,O0-bonded)2.18 (j-C3-bonded) 8.69 [42], 7.75, 7.31 �2024

# Indicates the acac methyl group cis to the L ligand. JH–Pt (⁄) are reported in square brackets [Hz] where measurement was possible.

Table 213C NMR chemical shifts of complexes.

C/(acac-j2O,O0) C/(acac-j-C3) C/L ligand

1 26.3 (Me),101.9 (j-C3)183.4 (C@O)

30.7 (Me), 39.9(j-C3)206.5 (C@O)

2 27.0*,26.6 (Me),101.8 (j-C3),184.5*, 183.6 (C@O)

30.5 (Me), 41.4(j-C3)207.8 (C@O)

153.4 (H2,6),137.3 (H4)125.3 (H3,5)

* Indicates the acac methyl group and C@O group cis to the L ligand.

S.A. De Pascali et al. / Inorganica Chimica Acta 412 (2014) 88–93 91

as generally observed. The 13C NMR spectra of 1 and 2 complexesconfirmed the structures assigned on the basis of 1H NMR spectro-scopic data. One bond and long range 2D 1H–13C HSCQ and HMBCexperiments allowed correct assignments for all the 13C reso-nances. In particular, j2O,O0-chelated and j-C3-bonded acacgroups show very different chemical shifts for the j-C3 atom. The

Fig. 1. Expansion of 1H NOESY spectrum in CDCl3 o

pseudo-aromatic j-C3 carbons of the acac chelates resonate athigher frequencies (d = 101.9 and 101.8 for 1 and 2, respectively)with respect to the corresponding carbon in the r-bonded acac(d = 39.9 and 41.4 for 1 and 2, respectively). Carbonyls of the acacchelate, involved in the pseudo-aromatic metallacycle, resonate atlower frequencies (d = 183.4 for 1 complex and 184.5 and 183.6 for2 complex) with respect to the r-bonded acac ones (d = 206.5 and207.8 for 1 and 2, respectively). The long range 2D 1H–13C HMBCspectra, together with the above mentioned NOESY data, wereused to correlate the carbonyls and the methyl groups belongingto the same half of the asymmetric chelate acac. The 195Pt NMRspectra show only one 195Pt signal at �2119 and �2024 ppm for1 and 2, respectively. By comparison with [Pt(acac-j2O,O0)(acac-j-C3)(L)] (L = DMSO, DMS), the substitution of sulphur ligand(DMSO/DMS) by nitrogen ligand (NH3/py) in 1 and 2 complexesresults in a deshielding of �1000 ppm of the 195Pt.

f complex [Pt(acac-j2O,O0)(acac-j-C3)(py)] (2).

T

Pt-compounds (μM)

0

20

40

60

80

100

120

12 h

a a

T

c

b T

c c

0

20

40

60

80

100

120

T

b

T

c

T T

cdd

24 h

0 1 10 100 200 0 1 10 100 200

0

20

40

60

80

100

120

48 h

a a

T

c

b

T

T T cd d

0

20

40

60

80

100

120

T

b

72 h

0 1 10 100 200 0 1 10 100 200

b

a

T c cd d

T T T b

c

c c

b

Pt-compounds (μM)

Pt-compounds (μM) Pt-compounds (μM)

Viab

lece

llnu

mbe

r(%

ofc

ontr

ol)

Viab

lece

llnu

mbe

r(%

ofc

ontr

ol)

Fig. 2. The sensitivity of MCF-7 cells to [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] and 1 complexes. Cells were treated with and without increasing concentrations of Pt-compounds;viable cell number was determined 12 h (a), 24 h (b), 48 h (c) and 72 h (d) later by MTT assay. The data are means ± s.d. of four different experiments, with eight replicates ineach, and are presented as % of control. Values with shared letters are not significantly different according to Bonferroni/Dunn post hoc tests.

Table 3The IC50 e IC90 values after a 72 h exposure to [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] and1 complex.

Pt-compounds IC50 IC90

[Pt(acac-j2O,O0)(acac-j-C3)(DMS)] 0.65 ± 0.046 4.77 ± 0.065[Pt(acac-j2O,O0)(acac-j-C3)(NH3)] (1) 1.22 ± 0.09 5.28 ± 0.032Cisplatin 55.19 ± 1.8 n.d.

92 S.A. De Pascali et al. / Inorganica Chimica Acta 412 (2014) 88–93

3.2. Biological effects

The cytotoxicity data shown here were obtained by MTT meta-bolic assay [25] and confirmed by SRB assay [26] to rule out poten-tial effects of Pt compounds on mitochondrial enzymes. Indeed,comparable results were obtained when cell number was directly

0

20

40

60

80

100

120 a

T

bc

b T

T

T d d

0 1 2 3 4 5 6 12 24

Viab

le c

ell n

umbe

r (%

of c

ontr

ol)

T T

T T

Time (h)

a

b c

cd

c cd

d

d d

e

e ef

f

A

Fig. 3. (A) The sensitivity of MCF-7 cells to [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] and 1 comnumber was determined at the indicated time by MTT assay. The data are means ± s.d. ocontrol. Values with shared letters are not significantly different according to Bonferronj2O,O0)(acac-j-C3)(DMS)] and 1 complexes cells were exposed to Pt-compounds the indthan 50 cells were scored. The percentage survival shown represents the mean ± s.d. of tware not significantly different according to Bonferroni/Dunn post hoc tests.

determined by cell counting. Consequently, we used the MTT assayin the combined experiments described. We previously demon-strated that [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] was significantlymore cytotoxic than cisplatin in MCF-7 human breast cancer cells[11].

Exposure of the MCF-7 cells to complex 1 at concentrationsranging from 1 to 200 lM resulted in a dose dependent inhibitionof cell survival (MTT assay) (Fig. 2). IC50 and IC90 values of 1 and[Pt(acac-j2O,O0)(acac-j-C3)(DMS)] after 72 h of treatment are cal-culated and reported in Table 3. [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] (IC50 0.65 ± 0.046 lM) and 1 (IC50 1.22 ± 0.09 lM)showed similar potencies, approximately 90-fold greater than thatobserved for cisplatin (IC50 55.19 ± 1.8 lM) (Fig. 2, Table 3).

A phenotypic hallmark of cancer cell is the ability to grow undernon-adhesive or anchorage-independent conditions. The ability ofMCF-7 cells to grow in soft agar correlates with their tumourigenic

T

T

T

Pt-compounds (µM)

T

T T

0

20

40

60

80

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120 a

b

d

0 0.10 0.5 1.0 2.5 5.0

T c

T b

b

b

c

d e

e

(% o

f con

trol

)

B

plexes. Cells were treated with and without 100 lM of Pt-compounds; viable cellf four different experiments, with eight replicates in each, and are presented as % ofi/Dunn post hoc tests. (B) Clonogenic survival assay in cells treated with [Pt(acac-icated amounts of for 2 h, and after 15 days of growth, colonies consisting of moreo independent experiments each performed in duplicate. Values with shared letters

S.A. De Pascali et al. / Inorganica Chimica Acta 412 (2014) 88–93 93

potential. When cultured on soft agar (an anchorage-independentgrowth), MCF-7 cells formed more than 150 colonies greater than50 mm in size per well at day 10. As shown in Fig. 3, very low, non-cytotoxic concentrations (0.5–5 lM) of compound 1 and [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] decreased dramatically and to the sameextent the ability of MCF-7 cells to grow in soft agar (Fig. 3b).While the biological data showed in this paper are limited, the sig-nificance of these two very similar effects of 1 and [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] on cell death and clonogenicity is twofold.

First, likely both compounds initiated overlapping apoptoticprocesses and second, both compounds interfered with the abilityof cells to form clones, i.e. decreased the number of neoplastic cellscapable of repopulation hence diminishing their tumorigenic po-tential. This should mean that in at least these two importantand desirable biological effects, the replacement of DMS withNH3 is not influential.

4. Conclusions

We have reported for the first time the synthesis and chemicalcharacterization by NMR spectroscopy of new b-diketonate com-plexes and nitrogen ligand (NH3 or pyridine) [Pt(acac-j2O,O0)(acac-j-C3)(NH3)], 1, and [Pt(acac-j2O,O0)(acac-j-C3)(py)], 2. Thenew b-diketonate complexes (1 and 2) have been obtained andisolated as stable products by cleavage of binuclear [{PtI2(NH3)}2]or starting with cis-[PtI2(py)2] monomer in the presence of excessacac. These complexes, which have been obtained and isolated asstable products, were designed in order to understand the role ofthe ligand other than acac in their biological activity (includingin vitro antitumor) of the analogous compounds [Pt(acac-j2O,O0)(acac-j-C3)(DMSO)] and [Pt(acac-j2O,O0)(acac-j-C3)(DMS)]. In thisrespect the synthetic pathways of 1 and 2 complexes were almostdifferent from those previously used in the case of analogues con-taining sulphur ligands (DMSO/DMS). In particular [Pt(acac-j2O,O0)(acac-j-C3)(DMS)], extensively studied by our research group, re-sulted more cytotoxic than cisplatin on cisplatin resistant cell linesand also in primary culture cells. However, it is not able to linkDNA and exerts its cytotoxic effect interacting with non genomicbiological target. According with the results of the present workthe presence of a sulphur ligand in [Pt(acac-j2O,O0)(acac-j-C3)(DMSO)] and, in particular, in the [Pt(acac-j2O,O0)(acac-j-C3)(DMS)] complex may not be the key requirement for their biolog-ical activity. The cytotoxicity results here presented suggest thatthe presence of a sulphur ligand, easy replaceable by another softligand (methionine, cysteine), may not play a key role with respectto the overall structure of such complexes. Indeed substitution ofsulphur ligand with the much more inert ammonia gave almostthe same in vitro cytotoxicity results on cisplatin resistant MCF-7cells exhibited by [Pt(acac-j2O,O0)(acac-j-C3)(DMS)]. On the other

hand, further work is needed in order to envisage a possibleinvolvement of the overall structure of these complexes in somemolecular recognition mechanism.

Acknowledgements

This work was supported by the University of Lecce and theMinistero dell’Istruzione, dell’Università e della Ricerca (MIUR),Cofin. 2009 (No. 2009ZFPSPW) and by the Consorzio Interuniversi-tario di Ricerca in Chimica dei Metalli nei Sistemi Biologici(CIRCMSB), Bari, Italy.

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