Michele Benedetti

Water-soluble organometallic analogues of oxaliplatin with cytotoxic and anticlonogenic activity.

that determine the antitumor activity of platinum complexes have been outlined, such as the neutrality of the compounds. 7] For this reason, the majority of cisplatin analogues that have been tested for anticancer activity are neutral compounds of the type cis-[PtA2X2] [8, 9] and cis-[PtA2X4], [10–13] in which A is an amine ligand, and X is an anionic leaving group. Moreover, it was found that the effectiveness of cisplatin and its analogues could be greatly improved by substituting the labile chlorido ligands with other leaving groups. This approach led to the development of second-generation platinum drugs such as carboplatin, cis-[diammine(1,1-cyclobutanedicarboxylato-O,O’)platinum(II)] and nedaplatin, cis-[diammineglycoloato-O,O’-platinum(II)] , which are now in clinical use. Members of a third generation of platinum-based drugs, characterized by replacement of both the ammonia carrier ligands and the chlorido leaving groups of cisplatin, are now undergoing clinical trials. Many such third-generation platinum drugs bear the (R,R)-1,2-diaminocyclohexane (R,R-chxn) carrier ligand, and a successful example is oxaliplatin, [Pt(R,R-chxn)(oxalatoO,O’)] (Figure 1), which was recently approved for clinical use. Importantly, oxaliplatin is currently the only clinically approved cisplatin analogue that has shown potential for use in cisplatin-resistant tumors under preclinical evaluations. The discovery of antitumor and/or mutagenic activity in cationic Pt complexes has resulted from the pursuit of pharmacological advantages by violating some of the classical structure–activity relationships of platinum-based drugs. This idea has recently gained strength, as favorable interactions between aquated cationic species and human organic cation transporters (hOCT) have demonstrated an improvement in the antitumor activity of cisplatin and oxaliplatin. In this context, our interest has been focused on the unstable cationic complexes of the type [PtCl(h-C2H4)(N N)] , in which N N is a dinitrogen ligand. In particular, we synthesized new cationic complexes of the type [PtCl(h-C2H4)(R,R-chxn)] + (1) (Figure 1) and [PtCl(h-C2H4)(S,S-chxn)] + (2), organometallic analogues of oxaliplatin and hOCT substrates, and determined their cytotoxicity in terms of potential antitumor activity. In aqueous solution, complexes 1 and 2 are readily hydrolyzed into well-known antitumor-active oxaliplatin metabolites (i.e. , [PtCl2(R,R-chxn)] (3), [PtCl(H2O)(R,R-chxn)] + (5), and [Pt(H2O)2(R,R-chxn)] + (7) ; Figure 1) or the corresponding enantiomers (i.e. , [PtCl2(S,S-chxn)] (4), [PtCl(H2O)(S,S-chxn)] + (6), and [Pt(H2O)2(S,S-chxn)] 2 + (8)). Compounds 1 and 2 were synthesized by a method similar to that previously reported by Maresca and co-workers by allowing the lithium derivative of Zeise’s salt, Li[PtCl3(h -C2H4)] , to react with the appropriate diamine in methanol at low temperature (0 8C). Under these conditions, all reactants are soluble in the reaction medium, and products 1 and 2 can be obtained as white precipitates and isolated in high yields (~90 %) within a few hours (~3 h). The H NMR spectra of the enantiomeric complexes 1 and 2 in D2O show coupled multiplets (COSY) in the range of 1.2–2.7 ppm. These are attributed to the aliphatic protons of the coordinated cyclohexanediamine. A sharp singlet at 4.71 ppm flanked by two satellites due to coupling with the Pt nucleus (J(Pt H) = 56 Hz) can be assigned to the h-olefin on the basis of the olefin H chemical shift range observed for analogous cationic compounds. 44–47] Moreover, the presence of only one signal for all the olefin protons at room temperature in D2O indicates that the rotation around the platinum–olefin bond is fast on the NMR time Figure 1. Comparison of the structures of cisplatin, oxaliplatin, and [PtCl(hC2H4)(R,R-chxn)] + (1), and the hydrolysis products formed from both 1 and oxaliplatin: [PtCl2(R,R-chxn)] (3), [PtCl(H2O)(R,R-chxn)] + (5), and [Pt(H2O)2(R,Rchxn)] + (7) ; the latter of which are responsible for the antitumor activity of oxaliplatin.

Modulation of properties in analogues of Zeise's anion on changing the ligand trans to ethene. X-Ray crystal structures of trans-[PtCl2(OH)(η2-C2H4)]− and trans-[PtCl2(η1-CH2NO2)(η2-C2H4)]−

To get further insight in the reaction of nucleophilic substitution upon changing the ligand trans to a η(2)-olefin, the reactivity of some monoanionic platinum(II) complexes (trans-[PtCl(2)X(η(2)-C(2)H(4))](-), X = Cl(-), 1, OH(-), 2, and CH(2)NO(2)(-), 3) towards pyridines with different steric hindrance (py, 4-Mepy, and 2,6-Me(2)py) has been tested. All crystallographic (2 and 3 reported for the first time) and spectroscopic data are in accord with a platinum-olefin interaction decreasing in the order 2 > 1 > 3, paralleling the decreasing electronegativity of the donor atom (O > Cl > C). Not only the platinum-olefin bond but also the bond between platinum and the ligand trans to the olefin appear to be strongest in 2 (Pt-O distance at the lower limit for this type of bond). In the reaction with py, the ligand trans to the olefin is displaced in 1 and 2. Moreover the reaction is in equilibrium in the case of sterically hindered 2,6-Me(2)py, the equilibrium being shifted moderately or prevalently toward the reagents in the case of 1 and 2, respectively. In the case of 3, the reaction with pyridines leads to substitution of the olefin instead of the carbanion. This is in accord with the observation that carbanions strongly weaken the trans Pt-olefin bond.

Adsorption of the cis-[Pt(NH3)2(P2O7)]2 − (phosphaplatin) on hydroxyapatite nanocrystals as a smart way to selectively release activated cis-[Pt(NH3)2Cl2] (cisplatin) in tumor tissues

The relevant adsorption of cis-[Pt(NH3)2(P2O7)](2-) (phosphaplatin) on hydroxyapatite nanocrystals (nHAP) was observed and studied in water suspension. Phosphaplatin cytotoxicity, which is very low for HeLa, MCF-7 and HS-5 cell lines could be enhanced, reaching that of cisplatin, by interaction with solid nHAP. This effect stems from nHAP ability to catalyze the phosphaplatin hydrolysis, producing the same hydrolytic species responsible for cisplatin antitumor activity.

Possible Incorporation of Free N7-Platinated Guanines in DNA by DNA Polymerases, Relevance for the Cisplatin Mechanism of Action

Cisplatin, cis-diamminedichloroplatinum (II), is one of the most widely used anticancer drugs. The main cellular target of cisplatin is DNA, where the platinum atom is able to form covalent bonds with the N7 of purines. It is commonly accepted that there is a direct attack of cisplatin on DNA. But it should be noted that, inside cells, free purine bases, which can react with cisplatin, are also available. Free bases have many functional roles, not least the constitution of building blocks for the synthesis of new DNA and RNA molecules. For this reason, under physiological conditions, the erroneous insertion of platinated bases in the synthesized nucleic acids could compete with direct DNA/RNA platination. Moreover, due to the lower sterical hindrance offered by single nucleobases with respect to nucleic acids, platination is expected to be even easier for free purines with respect to DNA and RNA. We have recently shown, for the first time, that platinated DNA can be formed in vitro by Taq DNA polymerase promoted incorporation of platinated purines. Cytotoxicity tests with [Pt(dien)(N7-G)], dien = diethylenetriamine, G = 5′-dGTP, 5′-dGDP, 5′-GMP, 5′-dGMP, GUO, dGUO, complexes on HeLa cancer cells support this hypothesis of the relative cytotoxicity of [Pt(dien)(N7-G)] derivatives being clearly related to their bioavailability. In vivo platination of free purines before their incorporation in nucleic acids therefore opens new perspectives in platinum based antitumour drugs, for a better understanding of both the action mechanism and the new molecular design.

Metalated nucleotide chemisorption on hydroxyapatite

The experiments here reported evidence on the importance of the residual charge of a nucleotide derivative, for the adsorption on nHAP (hydroxyapatite nanocrystals), in water solution. We found that the simple presence of phosphates on the nucleotide derivative does not guarantee adsorption on nHAP. On the other hand, we demonstrated that a cationic or neutral charge on a nucleotide derivative produces a strongly reduced chemical adsorption (chemisorption) whereas, in the presence of a net negative charge, relevant adsorption on nHAP is observed. The number of phosphates can only modulate the adsorption efficiency of a molecule provided that this latter bears an overall negative charge. The neutral zwitterionic nucleotide Pt(II) complexes, bearing negatively charged phosphates, are unable to give stable chemisorption. Previous considerations are important to model the binding ability of phosphate bearing nucleotide derivatives or molecules on hydroxyapatite. The findings reported in the present paper could be relevant in bone tissue targeting or nHAP mediated drug delivery.

The unexpected reactivity of Zeise's anion in strong basic medium discloses new substitution patterns at the platinum centre

Zeises anion in strongly basic hydroxylated solvents undergoes unprecedented nucleophilic addition of OR- (R = H, Me, Et) to the eta2-ethene giving trans-[PtCl2(eta1-C2H4OR)(OR)]2- which readily reacts with bidentate nitrogen donors N-N to give Cl- and OR- substitution and formation of [PtCl(CH2CH2OR)(N-N)]. Protonolysis of this stable organometallic species offers a versatile route to cationic [PtCl(eta2-C2H4)(N-N)]+ complexes.

Chiral discrimination in the formation reaction and at equilibrium for N,N,N′,N′-tetramethyl-1,2-diaminocyclohexane–PtG2 complexes

The effect of an extremely bulky carrier ligand, such as N,N,N′,N′-tetramethyl-1,2-diaminocyclohexane (Me4DACH, R,R and S,S configurations of the asymmetric carbon atoms of the chelate ring), upon the rate of formation and stability of Me4DACH–PtG2 derivatives has been investigated (G = 9-EtG, 3′-GMP, and 5′-GMP). Of the three possible rotamers, two head-to-tail (ΔHT and ΛHT) and one head-to-head (HH), only the former two are found in solution. The very small difference in H8 chemical shifts between ΔHT and ΛHT rotamers indicates that the relationship between the two guanines is very similar in the two conformers which are canted to the same degree but in opposite directions. A smaller interligand steric clash appears to favour the HT rotamer in which the H8 atoms of the guanines are on the same side of pseudo-axial N–Mes with respect to the platinum coordination plane. In the case of 5′-GMP, because of the absence of aminic protons on the carrier ligand, the 5′-phosphate directs its H-bonding potential towards the second ligand in cis-position (the coordinated H2O molecule in the mono adduct, [Me4DACH–Pt(H2O)(5′-GMP)]+, and the cis-G in the bis-adduct). As a consequence the rotamer in which such an interaction is possible, with the nucleotide keeping its favoured anti conformation, is more stable than expected. In contrast with the behaviour of 5′-GMP, 3′-GMP is found to increase the reactivity of the mono adduct in which the phosphate is directed towards the cis-water molecule and to give a negligible contribution to the stability of the HT rotamer in which the 3′-phosphate is directed towards the cis-nucleotide.

Response of Cisplatin Resistant Skov-3 Cells to [Pt(O,O′-Acac)(γ-Acac)(DMS)] Treatment Revealed by a Metabolomic 1H-NMR Study

The novel [Pt(O,O′-acac)(γ-acac)(DMS)], Ptac2S, Pt(II) complex has recently gained increasing attention as a potential anticancer agent for its pharmacological activity shown in different tumor cell lines, studied both in vitro and in vivo. The mechanism of action of Ptac2S, operating on non-genomic targets, is known to be very different from that of cis-[PtCl2(NH3)2], cisplatin, targeting nucleic acids. In this work, we evaluated the cytotoxicity of Ptac2S on the cisplatin resistant Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells, by the MTT assay. A 1H-NMR metabolomic approach coupled with multivariate statistical analysis was used for the first time for Ptac2S to figure out the biological mechanisms of action of the complex. The metabolic variations of intracellular metabolites and the composition of the corresponding extracellular culture media were compared to those of cisplatin (cells were treated at the IC50 doses of both drugs). The reported comparative metabolomic analysis revealed a very different metabolic profile between Ptac2S and cisplatin treated samples, thus confirming the different mechanism of action of Ptac2S also in the Epithelial Ovarian Carcinoma (EOC), SKOV-3 cells line. In particular, higher levels of pyruvate were observed in Ptac2S treated, with respect to cisplatin treated, cells (in both aqueous and culture media). In addition, a very different lipid expression resulted after the exposure to the two drugs (Ptac2S and cisplatin). These results suggest a possible explanation for the Ptac2S ability to circumvent cisplatin resistance in SKOV-3 cells.

Pauling electronegativity on-off effects assessed by 13C and 29Si NMR spectroscopy

In carbon and silicon tetrahalide compounds, the experimental 13 C and 29 Si NMR chemical-shift values are known to increase or decrease on increasing the overall sum of the ionic radii of the bonded halides Σ(rh ) (normal and inverse halogen dependence (NHD and IHD, respectively)). Herein, we extrapolate the main factors responsible for such NMR chemical shifts. Intriguingly, we found a characteristic value for the overall sum of the Pauling electronegativities of the bonded halides Σ(χh ), which works as a triggering factor to determine the transition from the NHD to IHD. Below this Σ(χh ) value, the chemical shift of the central atom was strictly related to only the Σ(rh ) value, thus producing a NHD trend. Conversely, above this value, the chemical shift of the central atom was dependent on both the Σ(rh ) and Σ(χh ) values, thus producing a IHD trend. A simple model, in which the effect of the Σ(χh ) value on 13 C and 29 Si NMR chemical shifts is related to an apparent increase in the Σ(rh ) value, is deduced.