Antiviral Aromatic

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Platinum(II) Complexes with Antitumoral/Antiviral Aromatic Heterocycles: Effect of Glutathione upon in Vitro Cell Growth Inhibition Paride Papadia,†,# Nicola Margiotta,† Alberta Bergamo,‡ Gianni Sava,‡ and Giovanni Natile*,† Dipartimento Farmaco-Chimico, Universita` degli Studi di Bari, Via E. Orabona 4, 70125 Bari, Italy, and Fondazione Callerio, Via A. Fleming 22, 34127 Trieste, Italy Received January 17, 2005

The compounds [Pt(Me2phen)(acy)2](NO3)2 (1), [Pt(Me2phen)(pen)2](NO3)2, [Pt(phen)(acy)2](NO3)2 (2), and [Pt(phen)(pen)2](NO3)2, containing the bidentate 1,10-phenanthroline (phen) or 2,9dimethyl-1,10-phenanthroline (Me2phen, neocuproine) and the antiviral agents acyclovir (acy) or penciclovir (pen), show different in vitro toxicity, the Me2phen complexes being appreciably more toxic than the phen complexes. To explain the different behavior, we investigated the reaction of complexes 1 and 2 with glutathione (γ-glutamylcysteinylglycine, GSH), a peptide believed to play an important role in driving the cellular effects of platinum drugs. The reaction led to different products, the phen complexes forming a stable binuclear µ-thiol-bridged species still containing the phenanthroline and the Me2phen complexes releasing the neocuproine ligand and forming an insoluble material. In vitro tests confirmed that the greater cell toxicity of complex 1 is due to the displacement of the neocuproine ligand by GSH. The results highlight the great dependence of the glutathione reactivity upon relatively small changes in the platinum coordination sphere. Introduction The ability of the antiviral drug acyclovir to coordinate to metal centers has been widely exploited in recent years. Complexes of acyclovir with Cd(II), Co(II), and Cu(II)1 have been synthesized, and in some cases, their antiviral properties have also been investigated.2,3 The structural analogies of acyclovir with guanosine and the propensity of platinum compounds to bind to purine bases suggested the possibility of its use as ligand for new platinum drugs. The new compounds would incorporate into the same molecule the pharmacological properties of the antiviral guanosine derivative and the antitumoral activity of platinum species.4-8 Very promising antiviral and anticancer properties were first reported for cis-[PtCl(NH3)2(acy)](NO3).7 Good antiviral activity and in vitro cell growth inhibition were later reported for platinum complexes containing 1,10phenanthroline (phen) or 2,9-dimethyl-1,10-phenanthroline (neocuproine, Me2phen), and the antiviral agents acyclovir (acy) or penciclovir (pen), of formulas [Pt(Me2phen)(acy)2]I2, [Pt(Me2phen)(acy)2](NO3)2 (1), [Pt(Me2phen)(pen)2](NO3)2, [Pt(phen)(acy)2](NO3)2 (2), and [Pt(phen)(pen)2](NO3)2 (Chart 1).9,10 The main cellular target of antitumoral platinum complexes is DNA, on which they form inter- and intrastrand cross-links by coordinating to N(7) of purine bases.11,12 However, other nucleophiles compete with purine bases for binding to platinum, particularly the sulfur-containing amino acids cysteine and methionine.13 Thiol containing ligands, like glutathione (γglutamylcysteinylglycine, GSH; for metal-coordinated * To whom correspondence should be addressed. Phone: +39-0805442774. Fax: +39-080-5442230. E-mail: [email protected]. † Universita ` degli Studi di Bari. # Present address: Di.S.Te.B.A., Universita ` degli Studi di Lecce, Prov.le Monteroni/Lecce 73100 Lecce, Italy. ‡ Fondazione Callerio.

Chart 1. Platinum Complexes with Neocuproine or 1,10-Phenanthroline and Acyclovir

glutathione, the abbreviation GS will be used), exist in relevant concentrations in extracellular fluids and in cells, and it has been estimated that the intracellular concentration of sulfidrilic groups varies in the range of 10-20 mM.14-16 Glutathione is involved in physiologically relevant metabolic functions and in cell protection17 and is considered to be one of the most important factors in cell resistance toward platinum drugs. It appears that in cisplatin-resistant cancer cells only a reduced quantity of drug is able to reach DNA because of an ATPdependent efflux mediated by a glutathione S conjugate

10.1021/jm0500471 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/08/2005

Effect of Glutathione

Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3365

export pump, which is also dependent on a high level of GSH. Under these conditions Pt(GS)2 complexes (platinum/glutathione molar ratio of 1:2) have been isolated from tumor cells after treatment with cisplatin.18 Conversely a low level of GSH is known to sensitize cells to cytotoxic agents, such as cisplatin, by activating sphingomyelinase (SMase), which increases ceramide levels and leads to SMase-induced apoptosis.19 In vitro tests indicated that complexes containing Me2phen (i.e., compound 1) had a greater cell growth inhibition activity against some types of lymphoma and carcinoma than the analogous compounds containing unsubstituted phenanthroline (i.e., compound 2).10 One possible explanation for the different biological activity of 1 and 2 could be that the platinum complex containing Me2phen is more stable in physiological medium because of the steric hindrance of the methyl substituents protecting the metal from substitution reactions. To verify this hypothesis, we have investigated the ability of glutathione to react with complexes 1 and 2 under physiologically relevant conditions. The results have been just the opposite of what we expected. GSH displaces the phenanthroline from compound 1 but not from compound 2, and the greater cell growth inhibition of 1 appears to stem from an intrinsic toxicity of the Me2phen ligand. Results and Discussion Analysis of Reaction Products. The reaction of the aqua species [Pt(H2O)2(phen)]2+ with glutathione performed at pH 3.1 and 298 K or at pH 7.1 and 310 K gave in both cases a dark-yellow product isolated by lyophilization of the solution and formed in greater than 90% yield. The elemental analysis was in agreement with a Pt2+/phen/GS2- ratio of 1:1:1. Moreover, the disappearance of the characteristic SH stretching band in the IR spectrum of the isolated product (2524 cm-1 in free GSH) indicated the involvement of the sulfur in the coordination of glutathione to platinum. The 1H NMR spectrum of the product is shown in Figure 1B. The most deshielded signal in the aliphatic region is assigned to the cysteine CRH proton (triplet, δ ) 4.56 ppm, Table 1). The CRH2 protons of glycine give a singlet at 3.58 ppm, while the CRH proton of the glutamic acid gives a triplet at 3.47 ppm. The CβH2 protons of cysteine exhibit a diastereotopic splitting of 0.27 ppm (δ ) 3.61 and 3.34 ppm), comparable to that of the CγH2 protons of the glutamic acid (δ ) 2.16 and 1.91 ppm). The last multiplet centered at 1.70 ppm belongs to the CβH2 protons of the glutamic acid. The 13C spectra for the free and complexed GSH are shown in Figure 2, and the corresponding chemical shifts are reported in Table 1. As a consequence of coordination to platinum, the Cβ carbon of cysteine undergoes a slight broadening and a strong shift to lower field (11.1 ppm). All other carbons remain unchanged or undergo a slight shift to higher field (highest shift for cysteine CR of 2.2 ppm). Although the elemental analysis indicated a platinum:GS ratio of 1:1, which could be in accord with the formation of a platinum complex with a chelated GS2- anion, the 13C NMR data clearly showed that only the sulfur atom of cysteine was involved in coordination to platinum, thus causing a significant downfield shift of the cysteine Cβ

Figure 1. The tripeptide glutathione and its 1H NMR spectrum (A) together with the 1H NMR spectrum of its reaction product with [Pt(H2O)2(phen)]2+ (B) in D2O. The symbol # marks impurities. Table 1. 13C and 1H Chemical Shifts (ppm) for GSH and Its Complex with [Pt(H2O)2(phen)]2+ CdO 13C

174.6, 173.3, 172.1

1H 13C

1H

R-Cys R-Glu R-Gly 55.3 4.53

173.8, 172.6, 170.3

53.1 4.56

53.5 3.78

γ-Glu

GSH 41.3 30.9 3.93 2.48

[Pt2(phen)2(GS)2] 52.8 41.1 30.5 3.47

3.58 2.16/1.91

β-Glu 25.7

β-Cys 25.1

2.13 2.88 25.2

36.2

1.70 3.61/3.34

carbon. Moreover, the [1H-15N]-HSQC spectrum (Figure 3) of the reaction product shows cross-peaks for both amidic NH groups, 15N/1H at 100.2/8.70 ppm for cysteine and at 88.5/8.42 ppm for glycine. Since both amidic groups are protonated, the involvement of amidic nitrogen in coordination to platinum can be excluded. The coordination to platinum of carboxylic groups could already be excluded on the basis of constant chemical shifts of carboxylic carbons in free and complexed GSH (Table 1).

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Chart 2. Sulfur-Bridged Dinuclear Complex [Pt2(µ-GS)2(phen)2]

Figure 2. Portions of the 13C NMR spectra of free GSH (A) and of its reaction product with [Pt(H2O)2(phen)]2+ (B) in D2O. Arrows show peaks that undergo significant shift after coordination.

Chart 3. Syn and Anti Configurations for [Pt2(µ-GS)2(phen)2]

Figure 3. [1H-15N]-HSQC spectrum of the reaction product of GSH with [Pt(H2O)2(phen)]2+. The spectrum was recorded in H2O/D2O (90:10, v/v). The asterisk (/) indicates NH crosspeaks for unreacted GSH (glycine NH cross-peaks for free and coordinated GSH overlap).

Since all data point to a glutathione coordinated to platinum only through the sulfur atom, it is most likely that the sulfur atom acts as a bridge between two platinum units. In this way each platinum can attain the four-coordination by linking the two nitrogens of a phen ligand and the sulfur atoms of two GS2- molecules. A drawing of the dimeric species is shown in Chart 2. This mode of coordination with the sulfur atoms of two cysteines bridging two platinum units has already been reported for the reaction of [PtCl2(2,2′-bipyridine)] with N-acetyl-L-cysteinato (AC).20,21 Dimeric complexes of platinum with bridging thiols can have syn or anti configuration, depending on the disposition of the S-substituents with respect to the platinum coordination plane (the two substituents on one side in syn and on opposite sides in anti; Chart 3). In our case, the NMR spectra are in accord with the presence of only one isomer (either the syn or the anti) because there is only one set of signals for the GSH and the phen ligands. Moreover, it has been possible to distinguish between the syn and the anti configurations on the basis of the multiplicity of the 1H NMR signals in the aromatic region (Figure 4; signal assignment

carried out with the help of COSY, NOESY, and TOCSY 2D experiments; data not shown). In the syn configu-


Figure 4. Aromatic region of the 1H NMR spectrum of the product of the reaction between GSH and [Pt(H2O)2(phen)]2+ in D2O with the assignment of the phenanthroline protons (see Chart 3 for the numbering of the atoms).

ration the two phenanthrolines are equivalent within the dimer but each of them has unequivalent halves. In contrast, in the anti configuration the two phenanthrolines are different within the dimer but each of them is symmetrical (equivalent halves). Eight resonances are expected for both the syn or anti configurations; however, the H5 and H6 protons (which are not coupled with other protons) are expected to have the multiplicity of an AB system only in the syn form (the two protons unequivalent within the same phen molecule), while in the case of the anti form, two separate singlets are expected for these two protons (the two protons equivalent within each phen molecule). The presence of a welldefined AB system for the H5 and H6 protons assigns the syn configuration to the isolated product. It is worth noting the formation of only one conformer (syn). Moreover, the X-ray crystal structure of [Pt2(µ-AC)2(bpy)2] also revealed the presence of only the syn form (both S-substituents on the same side of the coordination plane). Many binuclear complexes with bridging sulfur atoms have been reported with a number of metals,22,23 including platinum,24 and in quite a few cases the two conformations (syn and anti) were found to coexist in solution.25-27 Why only the syn form appears to be favored with AC and GS is not clear at the moment. However, the X-ray structure of [Pt2(µ-AC)2(bpy)2] has revealed a small but significant bending of the Pt2S2 four-membered metallacycle, the dihedral angle between the planes of the two PtS2 moieties being ca. 168° compared to a theoretical value of 180°. It is conceivable that such a bending (possible only in the syn configuration) can reduce the steric interactions between the bulky sulfur substituents and the two platinum units leading to a more stable configuration. In the case of less bulky sulfur substituents, there is less need for such a bending and both syn and anti configuration can cohexist in solution. Sadler and co-workers have investigated the reactions of [PtCl2(en)],28 [PtCl2(DACH)], and oxaliplatin29 (en ) ethylendiamine and DACH ) 1,2,-diaminocyclohexane) with GSH and GSSG. They also reported evidence for the formation of either a bridged dimer or an unusual macrochelate with a 2:1 Pt/GS ratio; however, no details for the 1:1 bridged dimer were given. The reported chemical shift of [Pt2(µ-AC)2(bpy)2] 195Pt (-2909 ppm) is very close to that observed for our [Pt2(GS)2(phen)2] complex (-2871 ppm) and can be considered typical for a platinum atom in a PtN2(µ-S)2 environment. The characterization of the reaction product between [Pt(NO3)2(phen)] and GSH was helpful for the investigation of the reaction of the more complex [Pt(phen)(acy)2](NO3)2 (2) species with GSH. The reaction was


monitored by 1H NMR spectroscopy and showed the same pattern already observed for the diaqua complex. The NMR spectrum of the starting complex 2 (Figure S1 in Supporting Information) exhibits in the region of aromatic protons one set of four signals characteristic of a symmetric phenanthroline plus one H(8) signal for the two coordinated acyclovir ligands. The 1H NMR spectrum of the reaction mixture, after 2 days at 310 K, showed the appearance of the signals of free acyclovir in addition to a complete set of signals superimposable onto that of the reaction products described above for the reaction of the diaqua complex with GSH. Therefore, we can conclude that the reaction of glutathione with [Pt(phen)(acy)2](NO3)2 proceeds through substitution of the two acyclovir moieties and formation of the [Pt2(GS)2(phen)2] dimer. The reaction of the [Pt(NO3)2(Me2phen)] complex with GSH was investigated under the experimental conditions similar to those used in the case of [Pt(NO3)2(phen)] (pH* 3.50 and 298 K; pH* ) pH in D2O solutions). The 1H NMR spectrum, recorded after 1 h of reaction time, showed the disappearance of the sharp signals of free GSH and the appearance, in the same aliphatic region, of very broad signals. In contrast, in the aromatic region, the characteristic rather broad signals of coordinated phenanthroline for [Pt(H2O)2(Me2phen)]2+ disappeared and a set of very sharp signals coincident with those of free Me2phen appeared. The reaction was accompanied by formation of a precipitate that was insoluble in common organic solvents. Elemental analyses were performed on several samples, but the results obtained were not reproducible, indicating that a polymeric material of variable composition was formed in the reaction (data not reported). An analogous reaction performed at pH* 7.1 and 310 K gave similar results. The reaction of [Pt(Me2phen)(acy)2](NO3)2 (1) with GSH was also performed at pH* 7.10 and 310 K. The signals of complex 1 and free GSH disappeared in about 30 min. At the same time, broad signals in place of the sharp aliphatic signals of GSH and sharp signals of uncoordinated acyclovir and Me2phen appeared. Similar to the reaction with GSH of the corresponding aqua species, a precipitate insoluble in most common organic solvents separated from the solution. In conclusion, the most striking difference between the behavior of the compounds with coordinated phenanthroline and those with dimethyl-substituted phenanthroline is that the latter complexes react more quickly with GSH and the reaction leads to substitution not only of Acy but also of the Me2phen ligand. Cell Growth Inhibition. The different reactivities of compounds 1 and 2 toward GSH (only compound 1 loses the phenanthroline ligand) suggested the possible involvement of this reaction in determining the different toxicities of the two compounds toward tumor cell lines (good toxicity observed only for compound 1). Therefore, a series of biological tests were undertaken using compounds 1 and 2 in combination with GSH. A preliminary experiment showed that TS/A adenocarcinoma cells, cultured in the presence of increasing concentrations of GSH (72 h incubation time), did not show any significant alteration of the cell growth profile in comparison to untreated controls (cell growth always

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Papadia et al.

Figure 5. Cell growth under the combined action of platinum compound (1 or 2) and GSH. TS/A adenocarcinoma cells were exposed for 72 h to equimolar concentrations of compound 1 or 2 and GSH. Cell growth was measured by MTT test. Results are expressed as percent of cell growth in treated groups versus controls.

Figure 6. Effects on cell growth of platinum compounds 1 or 2 pretreated with GSH. Compounds 1 or 2 were reacted with equimolar concentrations of GSH for 30 min at 310 K and added to TS/A adenocarcinoma culture. After 72 h of incubation, cell growth was measured by MTT test. Results are expressed as percent of cell growth in treated groups versus controls. Analysis of variance (ANOVA) and Tukey-Kramer post-test were done: (§) p < 0.001 vs the same concentration of 1 + GSH.

higher than 90% in all treated groups for a range of GSH concentrations between 1 and 100 µM; data not shown). When TS/A adenocarcinoma cells were treated for the same time (72 h) with equimolar concentrations of platinum compound (1 or 2) and GSH, a significant concentration-dependent decrease of cell growth was observed (Figure 5), which was identical to that of cells receiving only the platinum compound.10 In an additional experiment the platinum compound was first allowed to react for 30 min at physiological pH and 310 K with an equimolar amount of GSH (conditions mimicking those used for investigating the chemical reactivity of 1 or 2 toward GSH) and then added to the tumor cell culture (Figure 6). Under these conditions the cell growth inhibition of compound 1 increases significantly while that of compound 2 remains unchanged. A more detailed investigation of the role of glutathione was performed in the case of compound 1 by increasing the concentration of GSH with respect to that of the platinum complex (Table 2). An increase of cell growth inhibition is observed at high concentrations of compound 1 (5 and 10 µM) and GSH (10, 50, and 100 µM) for a 24 h incubation time (left-hand side of Table 2). If the incubation time is increased from 24 to 72 h, the activating effect of added GSH is completely lost and the activity remains the same for compound 1 alone and for the combination compound 1/GSH. Moreover, the

values of cell growth inhibition for a 72 h incubation time are comparable to those obtained for the highest concentration of GSH in the experiments performed at 24 h of incubation time. Therefore, compound 1 increases the cell growth inhibition if subject to a preincubation with equimolar GSH at 310 K for 0.5 h (conditions that have been shown to promote displacement of the Me2phen from platinum). In the absence of a preincubation (compound 1 and GSH added directly to the cell culture), an increase of toxicity is observed only at high GSH concentrations that again favor displacement of Me2phen from the platinum complex. Finally, no effect of GSH concentration is observed at long incubation time (72 h) because under these conditions the naturally occurring concentration of GSH is probably sufficient to promote the displacement of Me2phen from platinum. Finally, TS/A cells were treated with free Me2phen. It was found that after a 72 h treatment at 10 µM, compound 1 and Me2phen give the same inhibition of cell proliferation (Table 2, right-hand side). Moreover, the cell growth inhibition of free Me2phen was comparable to that of compound 1 pretreated with GSH at 310 K for 0.5 h. Contrary to Me2phen, free acyclovir and penciclovir did not inhibit cell proliferation (data not shown). Taken together, these data suggest that the cell



Table 2. Cell Growth under the Combined Action of 1 and GSHa compd 1 µM 1 1 1 1 5 5 5 5 10 10 10 10

+

cell growth, % of controls GSH µM

+ + +

1 5 10

+ + +

5 25 50

+ + +

10 50 100

24 h

72 h

100 ( 2.34 96.3 ( 3.71 96.4 ( 2.33 99.9 ( 2.99 96.8 ( 4.22 86.8 ( 3.54 82.0 ( 0.80 81.6 ( 3.12 62.0 ( 5.63** 70.0 ( 3.71 59.0 ( 2.80** 47.7 ( 1.32** 54.3 ( 2.00**

100 ( 0.97 92.1 ( 1.11 86.8 ( 1.34* 85.5 ( 0.25** 84.6 ( 1.39** 62.1 ( 1.32 57.8 ( 0.74 63.9 ( 1.72 58.5 ( 1.33 27.8 ( 2.77 49.3 ( 2.97 29.2 ( 1.10 27.7 ( 0.38

cell growth, % of controls

compd Me2phen µM

24 h

72 h

1

100 ( 2.34 52.5 ( 3.10***

100 ( 0.97 45.4 ( 4.73***

5

41.2 ( 0.01***

33.1 ( 0.02***

10

36.5 ( 3.89***

28.3 ( 8.40***

a TS/A adenocarcinoma cells were treated for 24 and 72 h with compound 1 (concentrations 1, 5, and 10 µM) and GSH (equimolar, 5, or 10-fold the concentration of complex) (left-hand side of the table) or with Me2phen (1, 5, and 10 µM) (right-hand side of the table). Cell growth was measured by MTT test. Results are expressed as percent of untreated controls. The values are the mean ( SE. One-way analysis of variance (ANOVA) and Dunnett post-test were performed: left-hand side of the table, (/) p < 0.05, (//) p < 0.01 vs the same concentration of complex 1 without GSH; right part of the table, (///) p < 0.001 vs controls.

growth inhibition of compound 1 depends on the ability of GSH (and other thiol-containing biomolecules) to displace the toxic Me2phen from the platinum complex. Conclusions It has been shown that complexes 1 and 2 react with GSH at physiological pH and temperature in a fundamentally different way. While complex 2 containing 1,10-phenanthroline forms a stable binuclear µ-thiolbridged species, releasing only the acyclovir ligands, in contrast, complex 1 containing neocuproine loses both the acyclovir and the Me2phen ligand, forming an insoluble precipitate. The binuclear [Pt2(GS)2(phen)2] complex has been shown to have exclusively the syn configuration, which ensures lower steric interaction between bulky S substituents and platinum coordination moieties. The release of Me2phen appears to be responsible for the greater cell growth inhibition of 1 compared to 2. GSH appears to be very efficient in displacing Me2phen from compound 1 in cell-free medium, while in the cell culture higher concentrations of GSH are required. It appears that for long exposure times (72 h) the physiological concentration of GSH is sufficient to ensure complete displacement of Me2phen from the platinum substrate. Thus, complex 1 can be considered as a prodrug while Me2phen can be considered the active species. When the investigation was started, we did not expect that platinum compounds with phen and Me2phen could have such a different chemical reactivity; however, this investigation has allowed us to find a reasonable explanation for the rather puzzling different biological activity of two platinum compounds differing only in the type of phenanthroline ligand. Materials and Methods Physical Measurements. Elemental analyses were performed using a Carlo Erba elemental analyzer model 1106 instrument. IR spectra were recorded on a Perkin-Elmer Spectrum One spectrophotometer using KBr as solid support for pellets. A Crison micro-pH meter model 2002 equipped with Crison microcombination electrodes (5 and 3 mm diameter) and calibrated with Crison standard buffer solutions at pH 4.00 and 7.02 was used for all pH measurements. The pH

readings for D2O solutions are indicated as pH* values and are uncorrected for the effect of deuterium on glass electrodes.30 NMR spectra were recorded on a Bruker wide bore Avance DPX 300 MHz instrument. 1H and 13C chemical shifts were referenced to internal TSP or 1,4-dioxane (66.3 ppm for 13C and 3.70 ppm for 1H). 195Pt chemical shift were referenced to external K2[PtCl4] in D2O placed at -1615 ppm. 15N chemical shift were referenced to 1 N NH4Cl in 1 N HCl. Standard pulse sequences were used for 1H, 13C{1H}, and 195Pt{1H} 1D spectra. The COSY, TOCSY, and [1H-15N]-HSQC experiments were carried out using gradient-selected versions of the standard Bruker pulse programs. Starting Materials. Commercial reagent grade chemicals 2,9-Me2-1,10-phenanthroline (Me2phen, neocuproine), 1,10phenanthroline (phen), glutathione (γ-glutamylcysteinylglycine, GSH; for coordinated glutathione we will use the abbreviation GS), and dimethyl sulfoxide (DMSO) we used without further purification. [Pt(phen)(acy)2](NO3)2 (2), [Pt(Me2phen)(acy)2](NO3)2 (1), [Pt(NO3)2(phen)], and [Pt(NO3)2(Me2phen)] were prepared according to previously reported procedures.9,10 Reactions of Phen Complexes with GSH. [Pt(NO3)2(phen)]. [Pt(NO3)2(phen)] (50 mg, 0.10 mmol) was suspended in H2O (100 mL) and kept under stirring for 1.5 h at 313 K to obtain complete dissolution with formation of the corresponding aqua species. GSH (30.73 mg, 0.10 mmol, pH 3.2) was added to the solution, and the mixture was left under magnetic stirring for 5 days under argon atmosphere. After filtration, the yellow solution was frozen at 253 K and lyophilized. A dark-yellow product was obtained in 90% yield. Elemental analysis results are reported in Table S1 of Supporting Information. IR data: 3400-3200 cm-1, O-H and N-H stretching; 1661 cm-1, amide CdO stretching; 1520 cm-1, N-H bending. [Pt(phen)(acy)2](NO3)2. [Pt(phen)(acy)2](NO3) (5 mg, 0.0053 mmol) in D2O (1.0 mL) was reacted in a NMR tube with GSH (1.6 mg, 0.0053 mmol) under argon atmosphere. The reaction was first performed at pH* 3.5 and 298 K and then repeated at pH* 7.1 and 310 K. The reaction products were characterized by NMR spectroscopy. NMR spectral data are reported in Table 1. Reactions of 2,9-Dimethyl-1,10-phenanthroline Complexes with GSH. [Pt(NO3)2(Me2phen)]. [Pt(NO3)2(Me2phen)] (5 mg, 0.0095 mmol) in D2O (1.0 mL) was reacted in a NMR tube with GSH (2.8 mg, 0.0095 mmol) under argon atmosphere. The reaction was monitored by NMR spectroscopy under two different sets of conditions: (A) pH* 7.0 and 310 K and (B) pH* 3.5 and 293 K. The NMR spectra revealed that the reaction course was the same in both cases: complete displacement of Me2phen from platinum and precipitation of an insoluble material made of platinum and GS.

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[Pt(Me2phen)(acy)2](NO3)2. [Pt(Me2phen)(acy)2](NO3)2 (10 mg, 0.010 mmol) in D2O (1.0 mL) was reacted in a NMR tube with a stoichiometric amount of GSH (3.1 mg, 0.010 mmol) under argon atmosphere at pH* 7.1 and 310 K. The reaction was monitored by NMR spectroscopy acquiring spectra at different time intervals. Displacement of platinum ligands (Me2phen and acy) and precipitation of an insoluble material were also observed in this case. Cell Growth Inhibition Evaluation. The influence of GSH on the biological activity of complexes 1 and 2 was investigated in the murine adenocarcinoma TS/A cell line. TS/A murine adenocarcinoma cells were maintained in RPMI 1640 medium (EuroClone, Whetherby, U.K.) supplemented with 10% fetal bovine serum, FBS (Invitrogen Italia, Milano, Italy), 2 mM L-glutamine (EuroClone, Whetherby, U.K,), and 50 µg/mL gentamicine (EuroClone, Whetherby, U.K.) in a humidified atmosphere with 5% CO2 at 310 K. Cells from confluent monolayers were removed from flasks by 0.05% trypsin solution (Sigma, St. Louis, MO), and the cell viability was determined by the trypan blue exclusion test. For experimental purposes, cells were seeded onto 96-well plates in 100 µL of complete medium with 5% of FBS. Twentyfour hours after the sowing, cells were exposed to equimolar concentrations of the platinum drug and GSH. The treatment was performed in two different ways: (i) direct addition of platinum drug and GSH to the cell culture; (ii) digestion of platinum drug and GSH for 30 min at 310 K and then addition to the cell culture. In the case of compound 1 the former experiment (direct addition of drug and GSH to cells) was also performed using 5- and 10-fold greater concentrations of GSH. All treatments were performed for 24, 48, and 72 h. Control experiments in which the cells were treated with GSH without addition of platinum drug or with free Me2phen (concentrations in the range 0.1-100 µM) were also performed. Each experiment was performed in quadruplicate and repeated twice. At the end of the treatment, the cell growth was evaluated by MTT, a colorimetric assay based on the ability of the viable cells to reduce a soluble yellow tetrazolium salt 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to blue formazan.31,32 Briefly, an amount of 10 µL of a 5 mg/mL solution of the tetrazolium salt was added per 100 µL of medium, and plates were incubated for 4 h at 310 K. Optical density was measured after dissolving the blue formazan crystals with DMSO, and the absorbance was measured at 570 nm using a SpectraCount Packard Bell (Meriden, CT) spectrophotometer.

Acknowledgment. The authors thank the University of Bari, the Italian “Ministero dell’Istruzione, Universita` e Ricerca (MIUR)” (PRIN 2004 no. 2004059078_006), and the EC (COST Chemistry Projects D20/ 0001/2000 and D20/0003/01) for support. Work was done with the contribution of LINFA (Laboratorio per l’Identificazione di Nuovi Farmaci Antimetastasi) under the MADE project. Supporting Information Available: Figure S1 showing the 1H NMR spectra of [Pt(phen)(acy)2]2+ (complex 2) in D2O and its reaction product with GSH and Table S1 listing the results from elemental analysis. This material is available free of charge via the Internet at http://pubs.acs.org.

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