Novel Kiteplatin Pyrophosphate Derivatives with Improved Efficacy

Jun 21, 2017 - The pH titration curves were fitted to the Henderson–Hasselbalch equation using the program KaleidaGraph (version 3.5, Synergy Softwa...
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Novel Kiteplatin Pyrophosphate Derivatives with Improved Efficacy Alessandra Curci,† Valentina Gandin,‡ Cristina Marzano,‡ James D. Hoeschele,§ Giovanni Natile,† and Nicola Margiotta*,† †

Department of Chemistry, University of Bari Aldo Moro, Via E. Orabona 4, 70125 Bari, Italy Department of Pharmaceutical and Pharmacological Sciences, University of Padua, Via Marzolo 5, 35131 Padova, Italy § Department of Chemistry, Eastern Michigan University, 48197 Ypsilanti, Michigan, United States ‡

S Supporting Information *

ABSTRACT: Two new Pt(II) derivatives of kiteplatin ([PtCl2(cis-1,4-DACH)]) with pyrophosphate as carrier ligand, one mononuclear (1) and one dinuclear (2), were synthesized with the aim of potentiating the efficacy of kiteplatin. Complex 1 resulted to be remarkably stable at physiological pH, but it undergoes a fast hydrolysis reaction at acidic pH releasing free pyrophosphate and (aquated) kiteplatin. The dinuclear compound 2 resulted to be less stable than 1 at both neutral and acidic pH forming 1 and (aquated) kiteplatin as first step. Both compounds (1 and 2) do not react as such with 5′-GMP, whereas their hydrolysis products readily form adducts with the nucleotide. The in vitro cytotoxicity assays against a panel of six human cancer cell lines showed that complex 2 affects cancer cell viability even at nanomolar concentrations. The cytotoxic activity of 2 is greater (up to 2 orders of magnitude) than that of cisplatin, oxaliplatin, and kiteplatin, whereas the mononuclear complex 1 has shown a cytotoxic activity comparable to that of oxaliplatin and kiteplatin, but higher than cisplatin. The latter result is not surprising, since the presence of two negative charges reduces the uptake of 1 into the tumor cells as compared to the neutral compound 2. The remarkable activity of 2 against the pancreatic cell line BxPC3 (average IC50 = 0.07 μM) deserves further investigation.

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nancies.12−14 The affinity for bone apatite is a property of the P−C−P motif, because bisphosphonates (similarly to the P− O−P motif of pyrophosphate) can chelate calcium ions by bidentate coordination through the oxygen atoms of the phosphonate groups. BPs have been used to deliver therapeutic agents with different activity (antibacterial, anticancer, antiosteoporosis, etc.) specifically to bones15 and, in the context of Drug Targeting and Delivery (DTD) of platinum drugs to bone, platinum(II) complexes with phosphonate ligands were reported by different groups.16−20 Also some of the authors of this paper have been involved, in the past decade, in the preparation of bone-targeted platinum anticancer drugs. A series of four water-soluble platinum(II) complexes (compounds 3by, 3bz, 3cx, and 3cz in Figure 1. We used number 3 having indicated with 1 and 2 the new compounds in the Abstract),21,22 containing the bisphosphonates 2-ammonium-1-hydroxyethane-1,1-diyl-bisphosphonate (y) or 3-ammonium-1-hydroxpropane-1,1-diyl-bisphosphonate (z), was prepared. In these complexes the bisphosphonate acts as a bridging ligand between two platinum moieties of the type cis-[Pt(NH3)2]2+, directly related to cisplatin, and [Pt(cis-1,4DACH)]2+, related to kiteplatin ([PtCl2(cis-1,4-DACH)]; DACH = diaminocyclohexane),23 this latter known to be able to overcome both the cisplatin and the oxaliplatin-resistance.

INTRODUCTION Currently, cis-diamminedichloridoplatinum(II) (cisplatin; CDDP) is one of the most potent drugs widely used in cancer chemotherapy.1 Since 1978 it has been used, alone or in combination with other chemotherapeutics, in the treatment of testicular,2 ovarian, head, neck, cervical, and bladder carcinomas.3−5 Despite the tremendous success of cisplatin, severe side effects, including nephro-, neuro-, and oto-toxocities as well as myelosuppression,6,7 limit its application. Additionally, a significant percentage of patients develop resistance to the treatment with platinum drugs. To overcome adverse toxic effects and resistance or to expand the clinical efficacy of cisplatin, many new platinum complexes have been synthesized and tested for their anticancer activity. Among these, carboplatin and oxaliplatin are the only complexes that received the Food and Drug Administration (FDA) approval for the clinical worldwide use.8 The systemic side effects of cisplatin and related Pt- drugs can be reduced by several ways. In this regard, a promising strategy consists in the use of carrier ligands able to promote the specific accumulation of the drug in target organs or cells.9−11 One class of such carrier ligands is represented by bisphosphonates (BPs). BPs are synthetic analogues of natural pyrophosphate and are potent inhibitors of bone resorption and have become an important class of drugs used to treat bone disorders such as Paget’s disease, postmenopausal osteoporosis, tumor-induced osteolysis, and hypercalcemia due to malig© 2017 American Chemical Society

Received: April 11, 2017 Published: June 21, 2017 7482

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry

Figure 2. Molecular structures of [Pt(dihydrogen pyrophosphate)(cis1,4-DACH)] (1) and [{Pt(cis-1,4-DACH)}2(pyrophosphate)] (2).

1,4-diaminocyclohexane (cis-1,4-DACH) ligand, an isomeric form of the carrier ligand present in oxaliplatin, with the aim of taking advantage of the unique-features of (cis-1,4-DACH)bearing Pt-complexes and potentiating their activity by using the pyrophosphate ligand.23 The new complexes, one mononuclear (1) and one dinuclear (2), were synthesized and fully characterized. Interestingly, the mononuclear compound (1, Figure 2) bears both a chelating pyrophosphate ligand and a net negative charge at neutral pH, which could be useful for adsorption on hydroxyapatite. In the perspective of a possible in vivo application, the stability of the newly synthesized Pt(II)-pyrophosphate complexes in a physiological-like environment as well as in acidic conditions (to simulate the hypoxic and low-pH environment surrounding a tumor mass) was investigated. The two complexes were also tested in vitro to assess their cytotoxicity against a panel of human tumor cell lines.

Figure 1. Sketches of bisphosphonates-platinum derivatives. The first letter indicates the platinum-bonded amine ligands (ethylenediamine, a; 2(NH3), b; cis-1,4-diaminocyclohexane, c), while the second letter indicates the bisphosphonate ligand (methylenebisphosphonate, w; 1hydroxy-1-(pyridin-4-yl)-methane-1,1-bisphosphonate, x; 2-ammonium-1-hydroxyethane-1,1-diyl-bisphosphonate, y; 3-ammonium-1hydroxpropane-1,1-diyl-bisphosphonate, z).

The four dinuclear complexes were evaluated for their in vitro cytotoxicity against a set of 13 human tumor cell lines including two pairs of cisplatin-sensitive and -resistant cell lines and three pairs of multidrug-sensitive and -resistant cell lines, and the results showed that, on the average, the compounds containing the cis-[Pt(NH3)2]2+ moiety (b) were less active than those containing the kiteplatin residue (c). This finding was ascribed to an intrinsic feature of the Pt(cis-1,4-DACH)2+ moiety, which could give more effective adducts with the target DNA.24,25 The local treatment of the tumor by the implantation of a biomaterial in which the drug has been embedded could represent another interesting perspective. In this context, some of us already reported the conjugation of the Pt-bisphpsphonate complexes 3ay and 3aw with inorganic silica xerogels or hydroxyapatite nanocrystals with the aim of using these matrices for the local treatment of bone tumors.22,26−31 The osteotropic properties as well as the inhibitory activity on osteoclast functions are the main features that have been considered in the development of osteotropic platinum-based drugs with bisphosphonate ligands. However, Bose and colleagues have discovered another class of platinum compounds somehow related to bisphosphonate derivatives. In this case the platinum ion is coordinated to two cis N-donors and a single bidentate pyrophosphate ligand. 32 These compounds, generally named phosphaplatins, possess highly desirable physiochemical properties, including excellent water solubility and stability under physiological conditions.33 Furthermore, phosphaplatins show enhanced cytotoxicity and greater effectiveness than conventional anticancer drugs, sometimes even on human tumors resistant to cisplatin and carboplatin.34−36 These superior properties have been ascribed to a different mechanism of action of phosphaplatins, with respect to cisplatin, based on the observation that they neither bind covalently to DNA nor enter into the nucleus of the cell in significant quantity, thereby nullifying DNA-repair-based resistance.34,36,37 In this context, we designed and fully characterized two new Pt(II)-pyrophosphate complexes (Figure 2) containing the cis-

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EXPERIMENTAL SECTION

Material and Methods. Commercial reagent-grade chemicals and solvents were used as received without further purification. 1 H NMR, 31P NMR, and 195Pt NMR spectra were recorded on Bruker Avance DPX 300 MHz, Bruker Avance II 600 MHz, and Bruker Avance III 700 MHz instruments. Standard pulse sequences were used for 1H and 31P{1H} (121.5 and 242.9 MHz) and 195Pt{1H} (64.5 MHz) one-dimensional (1D) spectra. Chemical shifts (1H) were referenced using the internal residual peak of the solvent (D2O: 4.80 ppm). Chemical shifts (31P) were referenced to external H3PO4 (85% w/w; 0 ppm). Chemical shifts (195Pt) were referenced to external K2[PtCl4] in D2O fixed at −1628 ppm. Electrospray ionization mass spectrometry (ESI-MS) was performed with a dual electrospray interface and a quadrupole time-offlight mass spectrometer (Agilent 6530 Series Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC-MS). Elemental analyses were performed with an Eurovector EA 3000 CHN instrument. A Crison Micro-pH meter Model 2002, equipped with Crison microcombination electrodes (5 and 3 mm diameter) and calibrated with Crison standard buffer solution at pH 4.01, 7.02, and 10.00, was used for pH measurements. The pH readings from the pH meter for D2O solutions are indicated as pH* values and are uncorrected for the effect of deuterium on glass electrodes.38 Kiteplatin ([PtCl2(cis-1,4-DACH)]) was prepared according to already reported procedure.39 Preparation and Characterization of Platinum Complexes. [Pt(ONO2)2(cis-1,4-DACH)]. [PtCl2(cis-1,4-DACH)], kitplatin, (0.100 g, 0.264 mmol) was suspended in 12 mL of H2O, and the suspension was treated with a solution of AgNO3 (0.087 g, 0.515 mmol in 3 mL of water) and stirred at room temperature for 20 h. The suspension was filtered through a plug of Celite to remove AgCl, and the solvent was evaporated to dryness under reduced pressure, yielding the desired compound [Pt(ONO2)2(cis-1,4-DACH)] as a white residue. Obtained 0.098 g (86% yield). Anal. Calcd. for [Pt(ONO2)2(cis-1,4-DACH)] 7483

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry (C6H14N4O6Pt, Mw = 433.28 g mol−1): C, 16.63%; H, 3.26%; N, 12.93%. Found: C, 16.40%; H, 3.40%; N, 12.80%. [Pt(Dihydrogen pyrophosphate)(cis-1,4-DACH)] (1). This complex was prepared by adapting an already reported procedure.40 Tetrasodium pyrophosphate decahydrate (0.200 g, 0.447 mmol) was dissolved in 12.5 mL of Milli-Q water at 50 °C, and the pH of the solution was adjusted to 8.0 with 1.0 M HNO3. The solution was kept under magnetic stirring at 50 °C for 15 min, and then [PtCl2(cis-1,4DACH)] (0.050 g, 0.131 mmol) was slowly added fractionated in small quantities over 2 h. The reaction mixture was incubated at 40 °C for 15 h. Following the incubation period, the solution was filtered to remove any unreacted starting material and was concentrated to minimum volume (∼2 mL) by rotary evaporation. The pH was lowered to ∼1.0 by the addition of 1.0 M HNO3, and the temperature was lowered to 4 °C to induce the precipitation of the product as a light-yellow powder. Precipitation was completed by cooling on ice for 10 min, and the product was then isolated by filtration and washed with little ice-cold water and acetone, and dried under vacuum. Obtained: 0.035 g (55% yield). Anal. Calcd. for [Pt(dihydrogenpyrophosphate)(cis-1,4-DACH)] (C6H16N2O7P2Pt, Mw = 485.23 g mol−1): C, 14.84%; H, 3.32%; N, 5.77%. Found: C, 14.96%; H, 3.32%; N, 5.77%. ESI-MS: calc. for [Pt(HP2O7)(C6H14N2)]− ([C6H15N2O7P2Pt]−) = 484.22; found: m/z (% relative to the base peak) [M-H]− = 484.02 (100). 1H NMR (D2O, pH* = 8.0 due to NaOD): 3.14 (2H, CHa), 1.79 (8H, CHb/c) ppm (see Figure 3 for numbering of protons). 31P NMR (D2O, pH* = 8.0): 1.60 ppm. 195PtNMR (D2O, pH* = 8.0): −1679.32 ppm.

concentrated to minimum volume (3 mL) and kept at 5 °C overnight. The obtained white precipitate was isolated by filtration, washed with small amounts of ice-cold water and acetone, and dried under vacuum. Obtained: 0.073 g (89% yield). Anal. Calcd. for [{Pt(cis-1,4DACH)}2(pyrophosphate)]·5H2O (C12H28N4O7P2Pt2·5H2O, Mw = 882.12 g mol−1): C, 16.32%; H, 4.34%; N, 6.34%. Found: C, 16.21%; H, 4.11%; N, 6.59%. ESI-MS: calcd. for [{Pt(cis-1,4-DACH)}2(pyrophosphate)]+Na+ ([C12H28N4O7P2Pt2Na]+) = 815.48; found: m/z (% relative to the base peak) [M + Na]+ = 815.25 (100). 1H NMR (D2O): 5.35 (4H, NH2), 5.05 (4H, NH2), 3.12 (4H, CHa), 1.87 (16H, CHb/c) ppm (see Figure 4 for numbering of protons). 31P NMR (D2O): 14.66 ppm; 195Pt NMR (D2O): −1585.88 ppm.

Figure 4. 1H (top; 300 MHz), 31P{1H} (middle; 121.5 MHz), and 195 Pt{1H} (bottom; 64.5 MHz) NMR spectra of 2 in D2O (pH* = 6.0). The spectra were recorded soon after dissolution of the sample within 1 h time. The asterisk indicates the residual solvent peak. NMR Experiments at Different pH and Calculation of pKa Values. Acidity constants for complex 1 were determined from chemical shift/pH 31P NMR titrations. Mononuclear compound 1 (0.0046 mmol) was dissolved in 1.0 mL of D2O and transferred into an NMR tube. The pH of the sample was adjusted to the required value by addition of DClO4 (10, 1.0, 0.5, 0.1, or 0.05 M) or NaOD (1.0, 0.5, 0.1, or 0.05 M) solutions, and the pH* value was measured by using a 3 mm diameter electrode for NMR tube. No control of the ionic strength was performed. The pH titration curves were fitted to the Henderson−Hasselbalch equation using the program KaleidaGraph (version 3.5, Synergy Software, Reading, PA).41 Stability of Compounds in Physiological or Acidic Conditions. The stability of compounds 1 and 2 in buffered solutions at 37 °C was assessed by 31P NMR spectroscopy. Each compound (∼2 mg) was dissolved in 0.8 mL of D2O containing (i) 50 mM 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer (pH* = 7.4) and 120 mM NaCl or (ii) 50 mM MES buffer (pH* = 4.5) and 120 mM NaCl. The resulting four solutions were transferred into

Figure 3. 1H (top; 700 MHz), 31P{1H} (middle; 242.9 MHz) and 195 Pt{1H} (bottom; 64.5 MHz) NMR spectra of 1 in D2O (pH* = 8). The asterisk indicates the residual solvent peak. [{Pt(cis-1,4-DACH)}2(pyrophosphate)] (2). Tetrasodium pyrophosphate decahydrate (0.046 g, 0.103 mmol) was dissolved in 2 mL of water, and the pH of the resulting solution (pH = 9) was brought to ∼6.0 by addition of HNO3 (1.0 M). The acid pyrophosphate solution was added to a dilute solution of [Pt(ONO2)2(cis-1,4-DACH)] (0.100 g, 0.231 mmol in 38 mL of water) kept under magnetic stirring at 40 °C. The pH of the mother solution was monitored and maintained at a constant value of ∼6.0 by addition of NaOH (0.1 M), when required. The end of the reaction was evidenced by pH stability (after ∼4 h). The reaction was then stopped, and the white suspension was 7484

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry Scheme 1. Synthesis of Complex 1

NMR tubes and maintained at 37 °C. 31P NMR spectra were recorded from time to time over a period of three weeks. The relative concentrations of the individual species in solution were deduced from integration of the 31P signals and plotted against time using Origin 8.1 (OriginLab, Northampton, MA)42 or SigmaPlot 12.0 (Systat Software, San Jose, CA) software.43 The equations used to fit the curves are reported in the electronic Supporting Information. Reactivity of Compounds toward 5′-GMP. Complexes 1 (2 mg, 0.0041 mmol) or 2 (3 mg, 0.0034 mmol) were dissolved in 0.8 mL of D2O containing 50 mM HEPES buffer (pH* = 7.4) and 120 mM NaCl. Subsequently, 5.3 mg (0.0103 mmol) and 7.83 mg (0.0153 mmol) of guanosine 5′-monophosphate sodium salt (5′-GMP-Na· 6H2O) were added into the NMR tubes containing the solutions of 1 and 2, respectively, and the obtained solutions were maintained at 37 °C. Both reactions were monitored for three weeks by recording 1H and 31P NMR spectra at defined time intervals. Cell Culture Studies. Human lung (H157), pancreatic (BxPC-3), and colon (HCT-15) carcinoma cell lines along with osteosarcoma (U-2 OS) and melanoma (A375) cell lines were obtained by American Type Culture Collection (ATCC, Rockville, MD). The human ovarian 2008 adenocarcinoma cells were kindly provided by Prof. G. Marverti (Dept. of Biomedical Science of Modena University, Italy). Human colon (LoVo) carcinoma cells were obtained by European Collection of Cell Culture (ECACC, Salisbury, UK). Cell lines were maintained using the following culture media containing 10% fetal calf serum (Euroclone, Milan, Italy), antibiotics (50 units/mL penicillin and 50 μg/mL streptomycin), and L-glutamine (2 mM): (i) RPMI for BxPC3, HCT-15, H157, and 2008 cells; (ii) F-12 HAM’S for LoVo cells; (iii) DMEM medium for A375 cells; (iv) McCoy’s for U-2 OS cells. Cytotoxicity Assays. The growth inhibitory effect toward tumor cell lines was evaluated by means of the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, (3−8) × 103 cells/well, depending upon the growth characteristics of the cell line, were seeded in 96-well microplates in growth medium (100 μL) and then incubated at 37 °C in a 5% carbon dioxide atmosphere. After 24 h, the medium was removed and replaced with a fresh one containing the compound to be tested at the appropriate concentration. Triplicate cultures were established for each treatment. After 48 h, each well was first treated with 10 μL of a 5 mg/mL MTT saline solution and incubated for five additional hours and then treated with 100 μL of a sodium dodecyl sulfate (SDS) solution in 0.01 M HCl. After an overnight incubation, the inhibition of cell growth induced by the tested compound was detected by measuring the absorbance of each well at 570 nm using a BioRad 680 microplate reader. Mean absorbance for each drug dose was expressed as a percentage of the control and plotted versus drug concentration. Dose−response curves were fitted and IC50 values were calculated with 4-PL model (P < 0.05). IC50 values represent the drug concentrations that reduce the mean absorbance at 570 nm to 50% of those of the untreated control wells. Cellular Uptake. HCT-15 cells (2 × 106) were seeded in 75 cm2 flasks in growth medium (20 mL). After overnight incubation, the

medium was replaced, and the cells were treated with tested compounds for 24 h. Cell monolayers were washed twice with cold phosphate-buffered saline (PBS), harvested, and counted. Samples were then subjected to three freezing/thawing cycles at −80 °C and then vigorously vortexed. The samples were treated with highly pure nitric acid (Pt ≤ 0.01 μg kg−1, TraceSELECT Ultra, Sigma Chemical Co.) and transferred into a microwave Teflon vessel. Subsequently, samples were submitted to standard procedures using a speed wave MWS-3 Berghof instrument (Eningen, Germany). After they cooled, each mineralized sample was analyzed for platinum by GF-AAS using a Varian AA Duo graphite furnace atomic absorption spectrometer (Varian, Palo Alto, CA) at the wavelength of 324.7 nm. The calibration curve was obtained using known concentrations of standard solutions purchased from Sigma Chemical Co.

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RESULTS AND DISCUSSION Synthesis and NMR Characterization. The mononuclear complex 1 was readily obtained by reacting kiteplatin with an excess of sodium pyrophosphate at pH = 8.0 (Scheme 1).40 The precipitation of the desired product (1) was obtained by lowering the pH of the mother solution to 1.0 with nitric acid, taking advantage of the different solubility of the neutral (protonated) and anionic forms of the complex. Compound 1 was characterized by elemental analysis, ESIMS, and multinuclear NMR. The ESI-MS spectrum showed the presence of a peak at m/z = 484.18 corresponding to [1-H]−, and the experimental isotopic pattern of the peak was in good agreement with the theoretical one (data not shown). Both the elemental analysis and ESI-MS indicated the formation of a complex having Pt and pyrophosphate in 1:1 ratio. The 1H NMR spectrum of compound 1 in D2O is reported in Figure 3 (top). The singlet with Pt satellites (3JPt−H = 74 Hz; the satellites are not visible in the spectrum reported in Figure 3 because of the high field of the instrument) and the multiplet integrating for eight protons, falling at 3.14 and 1.79 ppm, respectively, were assigned to the methynic and methylenic groups of coordinated DACH. The presence of a single set of 1 H NMR signals indicates the formation of a symmetric platinum(II) complex having both phosphate groups involved in the coordination to the metal (see following discussion). The 31P NMR of 1 in D2O (pH* = 8.0) is reported in Figure 3 (middle). It displays a single peak with unresolved platinum satellites (platinum satellites usually broaden with an increase in molecular size and chemical shift anisotropy)44 falling at 1.60 ppm and assigned to two phosphorus atoms of the pyrophosphate group. This chemical shift value is 8.44 ppm downfield compared to the free pyrophosphate ligand (−6.84 7485

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry

falling at 3.12 ppm was assigned to the methynic groups of coordinated DACH, while the broad multiplet falling at 1.87 ppm was assigned to the methylenic protons of cis-1,4 DACH. The 31P NMR of 2 in D2O is reported in Figure 4 (middle). It displays a single peak with unresolved platinum satellites located at 14.67 ppm and assigned to two phosphorus atoms of the pyrophosphate group, which are magnetically equivalent because of the presence of a plane of symmetry perpendicular to the coordination planes and passing through the two platinum atoms and the P−O−P oxygen atom. The 195Pt NMR of 2 in D2O is reported in Figure 4 (bottom). It shows a single peak falling at −1585.88 ppm. The presence of a single signal for the two Pt atoms suggests the presence of a plane of symmetry passing through the P−O−P atoms and parallel to the coordination plane, which renders the two platinum atoms equivalent. Our overall data indicate that complex 2 has a dinuclear structure with the pyrophosphate bridging two platinum moieties in a W conformation. A similar structure was found for analogous complexes with bisphosphonate ligands prepared by our group.21,22,26−28 Determination of pKa Values via 31P NMR pH Titration. Pyrophosphate, being a tetrabasic acid, has four ionization equilibria with pKa values corresponding to pKa1 = 0.85, pKa2 = 1.49, pKa3 = 5.77, and pKa4 = 8.22.47 The pH titration of complex 1 was performed by recording 31P NMR spectra at different pH* (Figure 5). The plot displays the

ppm) measured at the same pH*. The presence of a single peak in the 31P NMR spectrum is a further evidence in support of the formation of a symmetric complex with both phosphate groups coordinated to Pt. The 195Pt NMR spectrum of 1 in D2O is also reported in Figure 3 (bottom). It shows a single peak falling at −1679.32 ppm at pH* = 8.0 in agreement with the 195Pt chemical shift obtained for [Pt(dihydrogen pyrophosphate)(1R,2RDACH)].34,36 This signal is in the range typical for a Pt(II) atom in an O2N2 coordination environment. The synthesis of the dinuclear complex 2 was performed by treating a solution of sodium pyrophosphate in H2O (pH 6.0 by addition of HNO3 1 M) with a solution containing 2.2 equiv of [Pt(ONO2)2(cis-1,4-DACH)]. The reaction mixture was left under magnetic stirring for a shorter reaction time (4 h) at a higher temperature (40 °C) with respect to the procedure adopted for compound 1. A careful control of the pH of the mother solution (maintained at 6.0) allowed the coordination of sodium pyrophosphate to two units of the Pt precursor and the formation of the neutral dinuclear species 2 (see Scheme 2 for details). Scheme 2. Synthesis of Complex 2

Figure 5. Plot of 31P chemical shift vs pH* for compound 1.

The compound was characterized by elemental analysis, ESIMS, and NMR. The elemental analysis of the resulting compound was in accordance with the presence of one pyrophosphate unit per two Pt(cis-1,4-DACH) residues (2, Scheme 2). The ESI-MS spectrum showed the presence of a peak at m/z = 815.25 corresponding to [2+Na]+, and the experimental isotopic pattern of the peak was in good agreement with the theoretical one (data not shown). The 1H NMR spectrum of compound 2 in D2O is reported in Figure 4 (top). The broad singlets falling at 5.35 and 5.05 ppm were assigned to the aminic protons of coordinated cis-1,4DACH. The geminal protons of each aminic group are made unequivalent by the lack of symmetry with respect to the coordination plane.45,46 The slight acid conditions (pH* = 6.0) slow the exchange of the aminic protons with the deuterium of the solvent, allowing detection of the aminic protons in water solution. The singlet, with Pt satellites (3JPt−H = 90.30 Hz),

change in 31P chemical shift as a function of pH* with the titration curve showing changes in the slope (inflection points) corresponding to the various deprotonation steps (Scheme 3). As the pH* of the sample was increased from ca. 1 to 3, we observed a shift to lower field of the 31P chemical shift with an inflection point (calculated by fitting the points in this pH range to the Henderson−Hasselbach equation) falling at pH* = 2 (Figure 5). This pH* corresponds to the first deprotonation step of complex 1 (pKa1 = 2.03). By further increasing the pH from 3 to 6 we observed a deshielding of the phosphorus nuclei with a second inflection point falling at pH* 4.47. This corresponds to the second deprotonation of the coordinated pyrophosphate (pKa2 = 4.47, Scheme 3). Further increase of the pH* until ca. 13 had no effect on the 31P chemical shift. The pKa values relative to complex 1 are comparable to those 7486

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry Scheme 3

obtained for [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)], for which a similar biphasic profile, corresponding to two protonation/deprotonation steps, was observed (pKa1 = 2.06 and pKa2 = 4.40).34 Stability in Physiological Conditions. To investigate the stability of compounds 1 and 2 in physiological-like conditions, we monitored their solutions in D2O containing HEPES buffer (50 mM, pH = 7.4) and NaCl (120 mM) at 37 °C by 31P NMR spectroscopy. As can be seen from Figure 6, at pH = 7.4 and 37

Figure 6. 31P NMR (121.5 MHz) spectra at different times of 1 in physiological-like conditions (D2O, HEPES buffer 50 mM, pH* = 7.4, 120 mM NaCl, 37 °C).

Figure 7. (A) Relative percentages of 2 in physiological-like conditions (D2O, 50 mM HEPES buffer, pH* = 7.4, 120 mM NaCl, 37 °C), obtained by integration of the 31P NMR signals, plotted as a function of time; (B) 31P NMR (242.9 MHz) spectra of 2 at pH* = 7.4 recorded at different times.

°C, the mononuclear compound 1 resulted to be very stable even after one week of incubation. This result is in line with the data reported in the literature for the analogous complex [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)], which also resulted to be remarkably stable.34 Only after 9 d (216 h), the 31 P NMR spectrum showed the appearance of a new signal falling at −6.83 ppm corresponding to free pyrophosphate. This new peak reaches a stable value of ∼19% after ca. 24 d. Differently from the mononuclear complex, the dinuclear compound 2 undergoes a more rapid transformation in physiological-like conditions. The 31P spectrum showed a decrease in intensity of the signal of the starting compound (14.43 ppm at pH = 7.4) with the simultaneous formation of a singlet at 1.60 ppm that, after 21 h, accounts for nearly 92% of the total phosphorus (Figure 7B). The singlet at 1.60 ppm can be assigned to the monomeric derivative 1, obtained by release of a Pt(cis-1,4-DACH) moiety, by comparison with the chemical shift obtained for complex 1 in the same conditions. Hence, complex 2 undergoes a hydrolysis process with a half-

life (Figure 7A) of ca. 6 h with formation of 1 and kiteplatin or its solvato species (Scheme 4). No signal assignable to free pyrophosphate was detected (the free ligand gives a singlet at −6.8 ppm at pH* = 7.4). Stability in Acidic Environment. To simulate the acidic environment surrounding cancer tissues, the stability of compounds 1 and 2 was also investigated at pH* = 4.5 at 37 °C by 31P NMR spectroscopy. Differently from what observed in physiological conditions, complex 1 resulted to be unstable at acidic pH, analogously to what reported in the literature for [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)].34 Indeed, release of the pyrophosphate ligand (and formation of kiteplatin or its solvato derivatives; Scheme 5) was evidenced by the appearance, soon after dissolution of the sample at pH* 4.5, of the free pyrophosphate signal at −10.1 ppm (Figure 8B). The 31P spectrum showed a decrease of the intensity of the 7487

DOI: 10.1021/acs.inorgchem.7b00931 Inorg. Chem. 2017, 56, 7482−7493

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Inorganic Chemistry Scheme 4. Stability of 2 at pH* = 7.4

Scheme 5. Stability of 1 at pH* = 4.5

signal of the starting compound 1 (1.4 ppm at pH = 4.5) with the simultaneous increase of the singlet at −10.1 ppm that, after 72 h, accounts for nearly 61% of the total phosphorus (t1/2 ≈ 37 h; Figure 8A). After 7 d, a new peak at 0.5 ppm (see Figure 8B) appeared in the 31P NMR spectrum that remains stable up to 528 h accounting for nearly 5% of total phosphorus (Figure 8A). Most likely, the decomposition product is free phosphate obtained by hydrolysis of the pyrophosphate ion. Similarly to the mononuclear complex 1, also compound 2 was unstable at acidic pH. After 24 h, only 8% of initial compound 2 was still present in solution (Figure 9A), while most of the compound underwent a decomposition process (t1/2 ≈ 14 h) with simultaneous formation of the monomeric species 1 (31P NMR signal at 1.6 ppm at pH* = 4.5) and kiteplatin (Scheme 6). As soon as it is formed also 1 undergoes a hydrolysis process as evidenced by the appearance of the peak of free pyrophosphate (singlet at −10.1 ppm). After 49 h, the 31 P spectrum showed a further decrease of the intensity of the signal of compound 1 and an increase of free pyrophosphate, which reaches 71% of the total phosphorus after 240 h (Figure 9B). Similarly to the case of compound 1 at pH* 4.5, significant formation of phosphate ion is observed after ca. 336 h (peak falling at 0.5 ppm, Figure 9B). In summary, the investigation of the stability of the two Ptpyrophosphate complexes at acidic pH confirms that both complexes are unstable allowing the release of the active species [PtCl2(cis-1,4-DACH)] or its solvato derivatives. This issue is important, since it could be considered an activation process of the complexes with formation of the active species kiteplatin specifically at the acidic tumor site. Reaction with 5′-GMP. A peculiar feature of phosphaplatins is a pharmacological activity that is retained independent from reaction with DNA,37 the generally accepted target of clinically used Pt drugs. To simulate the reaction of compounds 1 and 2 with DNA, we monitored their reactions with 5′-GMP (used as a model of DNA) by 31P and 1H NMR at 37 °C in 50 mM HEPES buffer (pH* = 7.4) and 120 mM NaCl. The experiments were performed by treating 1 and 2 with an excess of 5′-GMP (2.5 and 4.5 equiv, respectively). The 31P spectrum recorded soon after mixing of the reactants (1 and 5′-GMP, Figure 10A) showed only the peaks of the starting substrates

Figure 8. (A) Relative percentages of 1 at pH* = 4.5 (D2O, MES buffer 50 mM, pH*=4.5, 120 mM NaCl, 37 °C), obtained by integration of 31P NMR signals, plotted as a function of time; (B) 31P NMR (121.5 MHz) spectra of 1 at pH* = 4.5 recorded at different times (PP = free pyrophosphate).

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Figure 10. (A) 31P NMR spectra at different times of 1 (5 mM) in the presence of 5′-GMP (12.5 mM) at pH* = 7.4 (D2O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C). (B) Relative percentages of the species formed in a solution of compound 1 and 5′-GMP at pH* = 7.4, obtained by integration of 31P NMR (242.9 MHz) signals, plotted as a function of time.

Figure 9. (A) Relative percentages of the species formed in a solution of compound 2 at pH* = 4.5 (D2O, MES buffer 50 mM, pH* = 4.5, 120 mM NaCl, 37 °C), obtained by integration of 31P NMR signals, plotted as a function of time; (B) 31P NMR (121.5 MHz) spectra of 2 at pH* = 4.5 recorded at different times (PP = free pyrophosphate).

Scheme 6. Stability of 2 at pH* = 4.5

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Inorganic Chemistry falling at 1.34 ppm (complex 1) and at 3.61 ppm (5′-GMP). After incubation for 94 h at 37 °C, two new peaks, falling at −7.3 and 3.76 ppm, were observed in the NMR spectrum. The first peak can be assigned to free pyrophosphate, while the second peak at 3.76 ppm can be assigned to the adduct [PtCl(cis-1,4-DACH)(5′-GMP)] formed by reaction of kiteplatin (or its solvato species), formed from 1 by dissociation of the pyrophosphate, with 5′-GMP. The peak at 3.76 ppm increases as complex 1 decreases. Fifty percent reaction of 1, calculated by integration of the signal of free pyrophosphate, was observed over a period of 26 d (633 h; Figure 10B). The hydrolysis of complex 1 in the presence of 5′-GMP appears to be faster than that of pure 1 at pH 7.4, where signs of degradation were observed only after 20 d at 37 °C (Figure 6). The reaction was also monitored by 1H NMR, focusing our attention on the H8 signal of 5′-GMP (data not shown). The spectrum, recorded immediately after the addition of 5′-GMP, showed the signals of the free nucleotide with a sharp singlet at 8.23 ppm (H8 proton of guanine). Coordination of 5′-GMP to Pt is generally accompanied by a deshielding of the H8 signal; therefore, the reaction progress could be monitored by following the new H8 signal at 8.55 ppm (0.32 ppm downfield with respect to free 5′-GMP). The latter signal grew in intensity with time and, after 28 d of reaction, its integral was comparable to that of unreacted 5′-GMP. The signal at 8.55 ppm belongs to H8 of [PtCl(cis-1,4-DACH)(5′-GMP)], an adduct that had already been characterized in a previous work.39,48 From the 31P NMR investigation, we conclude that complex 1 does not react with 5′-GMP, but a reaction occurs as soon as it hydrolyzes with release of the active species [PtCl2(cis-1,4-DACH)] (or its solvato derivatives). This result is in line with the data reported by Bose et al.35 for [Pt(dihydrogen pyrophosphato)(1R,2R-DACH)], which did not exhibit any detectable binding to DNA after 7 d of incubation. We also monitored the reaction between complex 2 and 5′GMP at 37 °C (pH* = 7.4) by recording 31P NMR spectra. The 31P spectrum recorded soon after mixing of the reactants (Figure 11A) showed only the peaks of the starting substrates at 14.29 (2) and 3.62 ppm (5′-GMP). The stability study at pH* = 7.4 (Figure 7B) evidenced that complex 2 undergoes a rather fast hydrolysis releasing complex 1 (detectable at 1.35 ppm) and kiteplatin, [PtCl2(cis-1,4-DACH)], the latter capable to react with GMP. Indeed, after 4 h, a new weak peak is observed at 3.76 ppm that can be assigned to [PtCl(cis-1,4DACH)(5′-GMP)]. The peak increases in intensity as complex 2 hydrolyzes. The half-life of this first hydrolysis process is of 3 h, ca. half the value obtained in the experiment of stability of complex 2 at neutral pH (Figure 7A). It seems that the presence of 5′-GMP facilitates the degradation of complex 2 to 1. After 18 h, complex 2 had completely turned into complex 1 and kiteplatin (Figure 11B, left). Formed compound 1, in turn, hydrolyzes releasing free pyrophosphate, which is detectable at −7.33 ppm. The peak of the Pt-GMP adduct grew with time as complex 1 hydrolyzed. Fifty percent reaction of 1 was observed over a period of 586 h (Figure 11B, right) as monitored by the release of free pyrophosphate. In our experiments there was no evidence of formation of intermediate species bearing both GMP and monocoordinated pyrophosphate; hence, both compounds 1 and 2 do not appear to react directly with GMP, whereas their degradation products

Figure 11. (A) 31P NMR spectra at different times of 2 (4.25 mM) in the presence of 5′-GMP (19.12 mM) at pH* = 7.4 (D2O, 50 mM HEPES buffer, 120 mM NaCl, 37 °C). (B) Relative percentages of the species formed in a solution of compound 2 and 5′-GMP at pH* = 7.4, obtained by integration of the 31P NMR (242.9 MHz) signals, plotted as a function of time.

(kiteplatin or its solvato species) can form adducts with the nucleotide. Cytotoxicity Assay. The in vitro antitumor activity of the Pt-pyrophosphate complexes 1 and 2 was evaluated on a panel of human cancer cell lines and compared to that of cisplatin, oxaliplatin, and kiteplatin. Cell lines representative of colon (HCT-15 and LoVo), pancreatic (BxPC3), melanoma (A375), ovarian (2008) carcinoma cells as well as of osteosarcoma (U-2 OS) were included. The cytotoxicity was evaluated by means of the MTT test for 72 h of incubation with different concentrations of the tested compounds. IC50 values, calculated from dose-survival curves, are reported in Table 1. The two pyrophosphate derivatives of kiteplatin were found to be much more effective than the reference compound cisplatin (CDDP) in most of the cell lines. In particular, the dinuclear complex 2 (average IC50 = 0.39 μM) resulted to possess an activity up to 2 orders of magnitude greater than that of CDDP (average IC50 = 10.12 μM). Complex 2 resulted 7490

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Inorganic Chemistry Table 1. In Vitro Antitumor Activity IC50 (μM) ± SD compounda 1 2 CDDP OXP kiteplatin

ovarian 2008 2.45 0.41 2.22 1.53 1.89

± ± ± ± ±

0.30 0.15 1.02 0.88 1.04

melanoma A375 1.26 0.22 2.41 2.06 1.87

± ± ± ± ±

0.11 0.03 0.62 0.88 1.25

osteosarcoma U-2 OS

colon HCT-15

± ± ± ± ±

1.88 ± 0.76 0.89 ± 0.45 15.28 ± 2.63 1.15 ± 0.43 2.66 ± 0.95

2.43 0.50 5.12 1.22 3.95

0.95 0.08 1.08 0.48 1.11

colon LoVo 2.07 0.26 9.52 1.01 1.11

± ± ± ± ±

0.24 0.11 2.82 0.34 0.45

pancreas BxPC3

average

± ± ± ± ±

2.03 ± 0.95 0.39 ± 0.45 10.12 ± 2.82 1.85 ± 1.07 2.57 ± 1.25

2.06 0.07 6.17 4.15 3.93

0.78 0.03 1.37 1.07 0.84

a 1: [Pt(dihydrogenpyrophosphate)(cis-1,4-DACH)], 2: [{Pt(cis-1,4-DACH)}2(pyrophosphate)], CDDP: cisplatin, OXP: oxaliplatin. SD = standard deviation. Cells ((3−5) × 104 mL−1) were treated for 72 h with different concentrations of tested compounds. Cytotoxicity was assessed by the MTT test. IC50 values were calculated by a four-parameter logistic model (P < 0.05).

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CONCLUSIONS Two new Pt(II) derivatives of kiteplatin with pyrophosphate as carrier ligand, one mononuclear (1) and one dinuclear (2), have been synthesized and fully characterized with the aim of potentiating the activity of kiteplatin. 31P{1H} NMR experiments at different pH values have allowed the determination of the acidity constants of complex 1 and an estimation of the stability of both compounds in different conditions. Analogously to what reported in the literature for [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)],34 compound 1 resulted to be remarkably stable at physiological pH, but it undergoes hydrolysis at acidic pH releasing free pyrophosphate. On the contrary, the dinuclear compound 2 resulted to be less stable than 1 at both neutral and acidic pH. The reactivity at acidic pH could be exploited to activate the complexes with formation of the species kiteplatin specifically at the acidic tumor sites. Our preliminary NMR studies on the reactivity of 1 and 2 toward 5′-GMP (used as a model of nucleic acids) comply with what was previously observed by Bose et al. for [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)].35 Both compounds (1 and 2) do not react as such with 5′-GMP, whereas their hydrolysis products readily form adducts with the nucleotide. Moreover, the hydrolysis of the complexes in the presence of 5′-GMP appears to be faster than in the absence of the nucleobase under the same experimental conditions (pH 7.4). The in vitro cytotoxicity assays, performed against a panel of six human cancer cell lines, have shown that complex 2 affects cancer cell viability even at nanomolar concentrations. The cytotoxic activity of 2 is greater (up to 2 orders of magnitude) than that of cisplatin, oxaliplatin, and kiteplatin, whereas the mononuclear complex 1 has shown a cytotoxic activity comparable to that of oxaliplatin and kiteplatin, but higher than that of cisplatin. This result is not surprising, since complex 1 has only one Pt atom and results more stable than the dinuclear complex 2. In addition, the two negative charges of 1 could reduce its uptake into tumor cells as compared to the neutral compound 2. A striking result of this investigation is the nanomolar activity of 2 against the osteosarcoma U-2-OS cell line (IC50 = 0.50 μM) and the pancreatic tumor cell line BxPC3 (IC50 = 0.07 ± 0.03 μM). The search for new Pt drugs to be used in combination with gemcitabine against pancreatic cancer is urgent, since this gastrointestinal tumor represents the fourth most common cause of cancer mortality with a five-year survival rate lower than 5%.49 A future perspective of this investigation will be to test the activity of compounds 1 and 2 after their embodiment in

to be also more cytotoxic than oxaliplatin (OXP average IC50 = 1.85 μM) and kiteplatin (average IC50 = 2.57 μM). In general, the dinuclear complex 2 showed a better in vitro antitumor activity (ca. 5 times greater) than the mononuclear complex 1 (average IC50 = 2.03 μM). Our results indicate also that the cytotoxicity potency of mononuclear complex 1 at the double concentration with respect to the dinuclear complex 2 is not equivalent (see a representative dose−response curve reported in Figure S1 in Supporting Information). Platinum Uptake by Cancer Cells. To correlate the cytotoxicity potential of the pyrophosphate complexes with their ability to enter cancer cells, uptake experiments were performed on human HCT-15 colon cancer cells. As depicted in Figure 12, cells treated for 24 h with 1 showed a platinum

Figure 12. Intracellular accumulation (24 h) of platinum complexes detected by GF-AAS analysis.

intracellular content similar to that detected with kiteplatin but ∼3 times lower than that obtained by treating cancer cells with 2. Hence the superior activity of 2 compared with 1 is related to the higher drug uptake in the case of 2 (which is neutral) than in the case of 1 (which is dianionic). A similar behavior to 1 was observed by Bose et al.35,36 for the analogous compound [Pt(dihydrogen pyrophosphate)(1R,2R-DACH)], which showed a reduced cellular accumulation compared to cisplatin. The greater cytotoxic activity of the dinuclear complex 2 could also be attributed to the intracellular hydrolysis of this compound, which resulted to be quite unstable releasing directly the active species kiteplatin together with 1. 7491

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(11) Gabano, E.; Ravera, M.; Osella, D. The Drug Targeting and Delivery Approach Applied to Pt-Antitumour Complexes. A Coordination Point of View. Curr. Med. Chem. 2009, 16 (34), 4544−4580. (12) Russell, R. G.; Rogers, M. J.; Frith, J. C.; Luckman, S. P.; Coxon, F. P.; Benford, H. L.; Croucher, P. I.; Shipman, C.; Fleisch, H. a. The Pharmacology of Bisphosphonates and New Insights into Their Mechanisms of Action. J. Bone Miner. Res. 1999, 14 (S2), 53−65. (13) Ezra, A.; Golomb, G. Administration Routes and Delivery Systems of Bisphosphonates for the Treatment of Bone Resorption. Adv. Drug Delivery Rev. 2000, 42 (3), 175−195. (14) Lin, J. H. Bisphosphonates: A Review of Their Pharmacokinetic Properties. Bone 1996, 18 (2), 75−85. (15) Zhang, S.; Gangal, G.; Uludağ, H. Magic Bullets” for Bone Diseases: Progress in Rational Design of Bone-Seeking Medicinal Agents. Chem. Soc. Rev. 2007, 36 (3), 507−531. (16) Appleton, T. G.; Hall, J. R.; McMahon, I. J. Multinuclear NMR Study of Reactions of Methylphosphonic Acid, CH3PO3H2, and (Aminoalkyl)phosphonic Acids, NH2(CH2)nPO3H2 (N = 1−3), with the Cis-diamminediaquaplatinum(II) Cation and Cisdiamminedihydroxoplatinum(II). Inorg. Chem. 1986, 25 (6), 720−725. (17) Klenner, T.; Valenzuela-Paz, P.; Keppler, B. K.; Angres, G.; Scherf, H. R.; Wingen, F.; Amelung, F.; Schmähl, D. Cisplatin-Linked Phosphonates in the Treatment of the Transplantable Osteosarcoma in Vitro and in Vivo. Cancer Treat. Rev. 1990, 17 (2), 253−259. (18) Galanski, M.; Slaby, S.; Jakupec, M. A.; Keppler, B. K. Synthesis, Characterization, and in Vitro Antitumor Activity of Osteotropic Diam(m)ineplatinum(II) Complexes Bearing a N,N-Bis(phosphonomethyl)glycine Ligand. J. Med. Chem. 2003, 46 (23), 4946−4951. (19) Xue, Z.; Lin, M.; Zhu, J.; Zhang, J.; Li, Y.; Guo, Z. Platinum(ii) Compounds Bearing Bone-Targeting Group: Synthesis, Crystal Structure and Antitumor Activity. Chem. Commun. 2010, 46 (8), 1212. (20) Nakatake, H.; Ekimoto, H.; Aso, M.; Ogawa, A.; Yamaguchi, A.; Suemune, H. Dialkyl Bisphosphonate Platinum(II) Complex as a Potential Drug for Metastatic Bone Tumor. Chem. Pharm. Bull. 2011, 59 (6), 710−713. (21) Margiotta, N.; Capitelli, F.; Ostuni, R.; Natile, G. A New Dinuclear Platinum Complex with a Nitrogen-Containing Geminal Bisphosphonate as Potential Anticancer Compound Specifically Targeted to Bone Tissues. J. Inorg. Biochem. 2008, 102 (12), 2078− 2086. (22) Margiotta, N.; Ostuni, R.; Gandin, V.; Marzano, C.; Piccinonna, S.; Natile, G. Synthesis, Characterization, and Cytotoxicity of Dinuclear Platinum-Bisphosphonate Complexes to Be Used as Prodrugs in the Local Treatment of Bone Tumours. Dalton Trans. 2009, No. 48, 10904−10913. (23) Margiotta, N.; Marzano, C.; Gandin, V.; Osella, D.; Ravera, M.; Gabano, E.; Platts, J. A.; Petruzzella, E.; Hoeschele, J. D.; Natile, G. Revisiting [PtCl2(cis-1,4-DACH)]: An Underestimated Antitumor Drug with Potential Application to the Treatment of OxaliplatinRefractory Colorectal Cancer. J. Med. Chem. 2012, 55 (16), 7182− 7192. (24) Kasparkova, J.; Suchankova, T.; Halamikova, A.; Zerzankova, L.; Vrana, O.; Margiotta, N.; Natile, G.; Brabec, V. Cytotoxicity, Cellular Uptake, Glutathione and DNA Interactions of an Antitumor LargeRing PtII Chelate Complex Incorporating the Cis-1,4-Diaminocyclohexane Carrier Ligand. Biochem. Pharmacol. 2010, 79 (4), 552−564. (25) Brabec, V.; Malina, J.; Margiotta, N.; Natile, G.; Kasparkova, J. Thermodynamic and Mechanistic Insights into Translesion DNA Synthesis Catalyzed by Y-Family DNA Polymerase across a Bulky Double-Base Lesion of an Antitumor Platinum Drug. Chem. - Eur. J. 2012, 18 (48), 15439−15448. (26) Palazzo, B.; Iafisco, M.; Laforgia, M.; Margiotta, N.; Natile, G.; Bianchi, C. L.; Walsh, D.; Mann, S.; Roveri, N. Biomimetic Hydroxyapatite-Drug Nanocrystals as Potential Bone Substitutes with Antitumor Drug Delivery Properties. Adv. Funct. Mater. 2007, 17 (13), 2180−2188.

hydroxyapatite matrices to be used for the local treatment of bone tumor after implantation. In the latter case the calcium of the hydroxyapatite matrix might favor the displacement of the pyrophosphate, allowing the release of the platinum drug in its active form.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.7b00931. Equations used in the fitting of curves reported in the Figures 7−11 and Figure S1 (dose−response curve for compounds 1 and 2 tested against A375 cells) (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: +39 080 5442759. E-mail: [email protected]. ORCID

Nicola Margiotta: 0000-0003-4034-875X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the Univ. of Bari (Italy), the Italian Ministero dell’Università e della Ricerca, the Inter-Univ. Consortium for Research on the Chemistry of Metal Ions in Biological Systems, and the European Union (COST CM1105: Functional metal complexes that bind to biomolecules) for support. We are grateful to Dr. N. Ditaranto (Univ. of Bari) for assistance in the preparation of kinetic plots.

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REFERENCES

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