Diego Montagner

A fluorescent probe for investigating the activation of anticancer platinum(IV) prodrugs based on the cisplatin scaffold.

Following the discovery of its potent antitumoral activity in 1965, the inorganic drug cisplatin has become one of the most important anticancer agents in clinical use.1 It exhibits exceptional activity against germ-cell testicular cancer, for which it is the first-line therapeutic option.2 Its success led to the development of second-generation drugs, namely carboplatin and oxaliplatin, both of which are also based on the cis-diam(m)ineplatinum(II) pharmacophore. Together, these FDA-approved PtII drugs constitute some of the most widely used chemotherapeutic agents (Figure 1).3 Yet, despite their clinical success, the use of PtII drugs is limited by their high toxicity, severe side-effects, and incidences of drug resistance.4 To address some of these limitations, researchers have developed stable PtIV carboxylate complexes as anticancer prodrugs that can be activated by intracellular reduction to release their latent cytotoxic activity.5 This versatile strategy has been used to develop PtIV prodrug complexes with highly tuned properties,6 which are capable of targeting,7 as well as delivering novel modes of action.8 The most prominent example is satraplatin, which is currently undergoing Phase III clinical trials against hormone-refractory prostate cancer (Figure 1).9 Although there is much interest in understanding and exploiting this prodrug strategy as a springboard towards the next generation of Pt drugs, tools capable of directly visualizing the uptake and accumulation of these clinically important compounds are lacking. Herein, we describe a fluorescent probe that was custom-built for the detection of PtII drugs such as cisplatin. Using confocal microscopy, we could determine the localization of the PtIV prodrugs in cancer cells in vitro after cell entry and intracellular reduction.

DNA damage and induction of apoptosis in pancreatic cancer cells by a new dinuclear bis(triazacyclonane) copper complex

The dinuclear copper(II) complex [Cu2{bcmp(-H)}(μ-OH)](NO3)2·H2O (1, bcmp=2,6-bis(1,4,7-triazacyclonon-1-ylmethyl)-4-methylphenol) has been synthesized and characterized by electrospray ionization mass spectrometry, potentiometric titration and cyclovoltammetry. The X-ray structure of the analogous perchlorate salt [Cu2{bcmp(-H)}(μ-OH)](ClO4)2·2.5H2O (2) was determined. Cytotoxicity studies showed very promising activity of 1 against various pancreatic tumor cell lines with IC50 values comparable or even lower than those of cisplatin. The Cu complex displayed low toxicity against a human non-tumor cell line (HEK 293) demonstrating selectivity for cancer cells. 1 converts supercoiled pUC19 plasmid DNA into the nicked form at micromolar concentrations in the absence of added reductants. A detailed kinetic study on the hydrolysis of the DNA model bis(2,4-dinitrophenyl) phosphate (BDNPP) has been performed. 1 hydrolyses BDNPP with a second order rate constant of 0.047 M s(-1) at pH 8 and 40 °C. Finally, single cell electrophoresis (comet assay) and fluorescence microscopy analysis showed that 1 interacts with cellular DNA and induces apoptotic cell death of Capan-1 pancreatic cancer cells. Western blotting analysis indicated that the Cu complex activates the p53 dependent pathway of apoptosis.

Synthesis, characterization and cytotoxic properties of platinum(II) complexes containing the nucleosides adenosine and cytidine

Cytidine (cyt) and adenosine (ado) react with cis-[L(2)Pt(μ-OH)](2)(NO(3))(2) (L=PMe(3), PPh(3)) in various solvents to give the nucleoside complexes cis-[L(2)Pt{cyt(-H),N(3)N(4)}](3)(NO(3))(3) (L=PMe(3), 1),cis-[L(2)Pt{cyt(-H),N(4)}(cyt,N(3))]NO(3) (L=PPh(3), 2), cis-[L(2)Pt{ado(-H),N(1)N(6)}](2)(NO(3))(2) (L=PMe(3), 3) and cis-[L(2)Pt{ado(-H),N(6)N(7)}]NO(3) (L=PPh(3), 4). When the condensation reaction is carried out in solution of nitriles (RCN, R=Me, Ph) the amidine derivatives cis-[(PPh(3))(2)PtNH=C(R){cyt(-2H)}]NO(3) (R=Me, 5a; R=Ph, 5b) and cis-[(PPh(3))(2)PtNH=C(R){ado(-2H)}]NO(3) (R=Me, 6a: R=Ph, 6b) are quantitatively formed. The coordination mode of these nucleosides, characterized in solution by multinuclear NMR spectroscopy and mass spectrometry, is similar to that previously observed for the nucleobases 1-methylcytosine (1-MeCy) and 9-methyladenine (9-MeAd). The cytotoxic properties of the new complexes, and those of the nucleobase analogs, cis-[(PPh(3))(2)PtNH=C(R){1-MeCy(-2H)}]NO(3) (R=Me, 7a: R=Ph, 7b), cis-[(PPh(3))(2)PtNH=C(R){9-MeAd(-2H)}]NO(3) (R=Me, 8a: R=Ph, 8b) have been investigated in a wide panel of human cancer cells. Interestingly, whereas the Pt(II) nucleoside complexes (1-4) did not show appreciable cytotoxicity, the corresponding amidine derivatives (7a, 7b, 8a, 8b, 5b, and 6b) exhibited a significant in vitro antitumor activity.

Role of the phosphine ligands on the stabilization of monoadducts of the model nucleobases 1-methylcytosine and 9-methylguanine in platinum(II) complexes

The addition of 1-methylcytosine (1-MeCy) or 9-methylguanine (9-MeGu) to solutions of cis-(PPh3)2P(ONO2)2 (1a), in a molar ratio of 1:1, affords the monoadducts cis-[(PPh3)2Pt(1-MeCy)(ONO2)]NO3 (2a) and cis-[(PPh3)2Pt(9-MeGu)(ONO2)]NO3 (3a) and only trace amounts of the bisadducts cis-[(PPh3)2Pt(1-MeCy)2](NO3)2 (4a) and cis-[(PPh3)2Pt(9-MeGu)2](NO3)2 (5a), respectively. The X-ray structural determination of 2a and 3a indicates a strong pi-pi stacking interaction between one of the PPh3 phenyl groups and the pyrimydinic N3-platinated cytosine or the imidazole part of the N7-coordinated guanine base. The addition of a further equiv of nucleobase to the monoadducts forms quantitatively the bisadducts that have been isolated as pure compounds 4a and 5a. Under the same experimental conditions, the dinitrato analogue cis-[(PMePh2)2Pt(ONO2)2] (1b) forms the monoadducts 2b and 3b in equilibrium with a relatively high concentration (20-30%) of the bisadducts cis-[(PMePh2)2Pt(1-MeCy)2](NO3)2 (4b) and cis-[(PMePh2)2Pt(9-MeGu)2](NO3)2 (5b), which have been structurally characterized by single-crystal X-ray analysis. The characterization of the isolated complexes by multinuclear NMR spectroscopy is also described.

Irreversible insertion of benzonitrile into platinum(II)-nitrogen bonds of nucleobase complexes. Synthesis and structural characterization of stable azametallacycle compounds.

Deprotonation of 1-methylcytosine (1-MeCy) and 9-methyladenine (9-MeAd) promoted by cis-[L(2)Pt(mu-OH)](2)(NO(3))(2) (L = PPh(3), PMePh(2), (1)/(2)dppe) in PhCN causes the irreversible insertion of a nitrile molecule into the Pt-N4 and Pt-N6 bonds of the cytosinate and adeninate ligands, respectively, to form the stable azametallacycle complexes cis-[L(2)PtNH=C(Ph){1-MeCy(-2H)}]NO(3) (L = PPh(3), 1; PMePh(2), 2; (1)/(2)dppe, 3) and cis-[L(2)PtNH=C(Ph){9-MeAd(-2H)}]NO(3) (L = PPh(3), 4; PMePh(2), 5) containing the deprotonated form of the molecules (Z)-9-N-(1-methyl-2-oxo-2,3-dihydropyrimidin-4(1H)-ylidene)benzimidamide and (Z)-N-(9-methyl-1H-purin-6(9H)-ylidene)benzimidamide. Single-crystal X-ray analyses of 2 and 4 show the metal coordinated to the N3 cytosine site [Pt-N3 = 2.112(7) A] and to the N1 site of adenine [Pt-N1 = 2.116(6) A] and to the nitrogen atom of the inserted benzonitrile [Pt-N2 = 2.043(6) and 2.010(6) A in 2 and 4, respectively], with the exocyclic nucleobase amino nitrogen bound to the carbon atom of the CN group. Complex 2, in solution, undergoes a dynamic process related to a partially restricted rotation around Pt-P bonds, arising from a steric interaction of the oxygen atom of the cytosine with one ring of the phosphine ligands. The reaction of 4 with acetylacetone (Hacac) causes the quantitative protonation of the anionic ligand, affording the acetylacetonate complex cis-[(PPh(3))(2)Pt(acac)]NO(3) and the free benzimidamide NH=C(Ph){9-MeAd(-H)}. In the same experimental conditions, complex 3 reacts with Hacac only partially.

Antitumor platinum(IV) derivatives of carboplatin and the histone deacetylase inhibitor 4-phenylbutyric acid

Five new platinum(IV) derivatives of carboplatin each incorporating the histone deacetylase inhibitor 4-phenylbutyrate in axial position were synthesized and characterized by 1H and 195Pt NMR spectroscopy, electrospray ionization mass spectrometry and elemental analysis, namely cis,trans-[Pt(CBDCA)(NH3)2(PBA)(OH)] (1), cis,trans-[Pt(CBDCA)(NH3)2(PBA)2] (2), cis,trans-[Pt(CBDCA)(NH3)2(PBA)(bz)] (3), cis,trans-[Pt(CBDCA)(NH3)2(PBA)(suc)] (4) and cis,trans-[Pt(CBDCA)(NH3)2)(PBA)(ac)] (5) (PBA=4-phenylbutyrate, CBDCA=1,1-cyclobutane dicarboxylate, bz=benzoate, suc=succinate and ac=acetate). The reduction behavior in the presence of ascorbic acid was studied by high performance liquid chromatography. The cytotoxicity against a panel of human tumor cell lines, histone deacetylase (HDAC) inhibitory activity, cellular accumulation and the ability to induce apoptosis were evaluated. The most effective complex, compound 3, was found to be up to ten times more effective than carboplatin and to decrease cellular basal HDAC activity by approximately 18% in A431 human cervical cancer cells.

Oxidative Stress Induced by Pt(IV) Pro-drugs Based on the Cisplatin Scaffold and Indole Carboxylic Acids in Axial Position.

The use of Pt(IV) complexes as pro-drugs that are activated by intracellular reduction is a widely investigated approach to overcome the limitations of Pt(II) anticancer agents. A series of ten mono- and bis-carboxylated Pt(IV) complexes with axial indole-3-acetic acid (IAA) and indole-3-propionic acid (IPA) ligands were synthesized and characterized by elemental analysis, ESI-MS, FT-IR, 1H and 195Pt NMR spectroscopy. Cellular uptake, DNA platination and cytotoxicity against a panel of human tumor cell lines were evaluated. All the complexes are able to overcome cisplatin-resistance and the most potent complex, cis,cis,trans-[Pt(NH3)2Cl2(IPA)(OH)] was on average three times more active than cisplatin. Mechanistic studies revealed that the trend in cytotoxicity of the Pt(IV) complexes is primarily consistent with their ability to accumulate into cancer cells and to increase intracellular basal reactive oxygen species levels, which in turn results in the loss of mitochondrial membrane potential and apoptosis induction. The role of the indole acid ligand as a redox modulator is discussed.

Steroid–AuI–NHC Complexes: Synthesis and Antibacterial Activity

A series of gold(I) pioneer complexes bearing N‐heterocyclic carbenes and steroid derivatives (ethynylestradiol and ethisterone) with the generic formula [Au(R2‐imidazol‐2‐ylidene)(steroid)] (where R=CH3 or CH2CH2OCH3) were synthesized, and the X‐ray structure of a rare of gold(I)–estradiol derivative is discussed. Toxicity studies reveal notable antibacterial activity of the gold‐based compounds, which is significantly increased in vivo by the presence of the estradiol unit. Toxicity profiling was estimated in vitro versus Gram‐positive (Staphylococcus aureus) and Gram‐negative (Escherichia coli) bacteria, and in vivo on Galleria mellonella larvae against E. coli.

Development of an Efficient Dual-Action GST-Inhibiting Anticancer Platinum(IV) Prodrug

The cytotoxicity of cisplatin (cDDP) is enhanced when co‐administered with ethacrynic acid (EA), a glutathione S‐transferase (GST) inhibitor. A PtIV–EA conjugate containing a cDDP core and two axial ethacrynate ligands (compound 1) was shown to be an excellent inhibitor of GST, but did not readily release a PtII species to exert a synergistic cytotoxic effect. In this study, a redesigned PtIV construct composed of a cDDP core with one axial ethacrynate ligand and one axial hydroxido ligand (compound 2) was prepared and shown to overcome the limitations of compound 1. The EA ligand in 2 is readily released in vitro together with a cytotoxic PtII species derived from cisplatin, working together to inhibit cell proliferation in cDDP‐resistant human ovarian cancer cells. The in vitro activity translates well in vivo with 2, showing effective (∼80 %) inhibition of tumor growth in a human ovarian carcinoma A2780 tumor model, while showing considerably lower toxicity than cisplatin, thus validating the new design strategy.

A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

Abstract Free radical generation is an inevitable consequence of aerobic existence and is implicated in a wide variety of pathological conditions including cancer, cardiovascular disease, ageing and neurodegenerative disorder. Free radicals can, however, be used to our advantage since their production is catalysed by synthetic inorganic molecules—termed artificial metallonucleases—that cut DNA strands by oxidative cleavage reactions. Here, we report the rational design and DNA binding interactions of a novel di-Cu2+ artificial metallonuclease [Cu2(tetra-(2-pyridyl)-NMe-naphthalene)Cl4] (Cu2TPNap). Cu2TPNap is a high-affinity binder of duplex DNA with an apparent binding constant (Kapp) of 107 M(bp)−1. The agent binds non-intercalatively in the major groove causing condensation and G-C specific destabilization. Artificial metallonuclease activity occurs in the absence of exogenous reductant, is dependent on superoxide and hydrogen peroxide, and gives rise to single strand DNA breaks. Pre-associative molecular docking studies with the 8-mer d(GGGGCCCC)2, a model for poly[d(G-C)2], identified selective major groove incorporation of the complex with ancillary Cu2+-phosphate backbone binding. Molecular mechanics methods then showed the d(GGGGCCCC)2 adduct to relax about the complex and this interaction is supported by UV melting experiments where poly[d(G-C)2] is selectively destabilized.