Antitumoral Gold and Silver Complexes with Ferrocenyl-Amide

Oct 1, 2013 - ... Emilia Bigaeva , Paul Kavanagh , Michel Picquet , Pierre Le Gendre ... T. Hutton , Bruno Therrien , Patrick J. Bednarski , and Grego...
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Antitumoral Gold and Silver Complexes with Ferrocenyl-Amide Phosphines Helen Goitia,† Yolanda Nieto,† M. Dolores Villacampa,† Cornelia Kasper,‡ Antonio Laguna,† and M. Concepción Gimeno*,† †

Departamento de Quı ́mica Inorgánica, Instituto de Sı ́ntesis Quı ́mica y Catálisis Homogénea (ISQCH), CSIC-Universidad de Zaragoza, E-50009 Zaragoza, Spain ‡ Department of Biotechnology, Institute for Applied Microbiology, University of Material Resource and Life Science, Muthgasse 18, 1180 Vienna, Austria S Supporting Information *

ABSTRACT: The monosubstituted ferrocenyl-amide phosphine ligands PPh2CH2CH2NHCOFc and (PPh2CH2CH2)2NCOFc and the disubstituted ferrocenyl-amide diphosphine (PPh2CH2CH2NHCO)2Fc have been synthesized and used to prepare the gold chloride derivatives [AuCl(PPh2CH2CH2NHCOFc)], [Au2Cl2{(PPh2CH2CH2)2NCOFc}], and [Au2Cl2{(PPh2CH2CH2NHCO)2Fc}] or the silver species [Ag(OTf)(PPh2CH2CH2NHCOFc)2] and [Ag(OTf)(PPh3)(PPh2CH2CH2NHCOFc)]. In the gold complexes the chloro ligands can be easily substituted by several biologically relevant thiolates such as 2-mercaptonicotinic acid, 2-thiocytosine, 2-thiouracil, 2-mercaptopurine, and 2,3,4,6-tetra-6-acetyl-1-thiol-β-D-glucopyranosato, affording the gold phosphine thiolate derivatives. In addition, the gold phosphine thiolates of the closely related 1,1′-bis(diphenylphosphine)ferrocene ligand have been prepared in order to compare their biological activities. The antiproliferative activity of these compounds has been tested by the MTT viability assay in two murine cell lines, NIH-3T3 (mouse embryonic fibroblasts) and PC-12 (pheochromocytoma of the rat adrenal medulla), and two human cell lines, A-549 (adenocarcinomic human alveolar basal epithelial cells) and Hep-G2 (hepatocellular carcinoma). The amide-phosphine ligands are not active, whereas the chloro-gold derivatives have good antiproliferative activity in the murine cell lines and very low activity in the human cell lines. The silver complexes are less active than the gold derivatives. The gold thiolate complexes have moderate to very good cytotoxic activity for all of the ligands, showing excellent IC50 values for the thiolate complexes of the amidephosphines PPh2CH2CH2NHCOFc and (PPh2CH2CH2N)2COFc and dppf.

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INTRODUCTION The antitumoral properties of metallic complexes started with the great success of cisplatin and the second- or third-generation drugs carboplatin, paraplatin, and oxaliplatin, which open a new field for exploring the biological applications of metal-based drugs.1 Although platinum drugs are largely used in cancer treatment, their toxicity and the development of platinum drug resistance in cell lines has led to a greater research in other metallic complexes, as for example ruthenium, for which two complexes have been successfully entered into clinical trials.2 Gold complexes, which are largely known as good antiarthritic drugs, have attracted much attention because of their strong antiproliferative activity, together with other biological properties found for these complexes such as antimicrobial activity, anti-VIH, antimalaria, etc.3 Several gold compounds, including gold(I)4 and gold(III)5 derivatives, have shown promising cytotoxic activity, in some cases overcoming cisplatin resistance to specific cancer cells, and in addition they have shown a mechanism of action clearly different from that of platinum drugs. Several targets have been identified for gold complexes, © 2013 American Chemical Society

the inhibition of thioredoxine reductase being the most important to date.6 Organometallic complexes have also been proven to be excellent candidates as antitumoral agents; among them ferrocene and its derivatives have aroused great interest because of their potential biological applications.7 Ferrocene itself and its derivatives have been used as cytotoxic and antianemic agents within the area of medicinal applications.8 The ferrocenyl group has been incorporated into the structure of a number of biologically active molecules such as antibiotic,9 anticancer,10 and malaria drugs,11 resulting in an increase of the activity. Thus, the ferrocene moiety has proved to be a successful addition to penicillin and cephalosporine, enhancing their antibacterial activity, to tamoxifen, increasing its antitumor properties, or to known malaria therapeutics, increasing their efficacy toward chloroquine-resistant strains of Special Issue: Ferrocene - Beauty and Function Received: June 29, 2013 Published: October 1, 2013 6069

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amide-phosphine ligands, and also in order to compare their activity with that of other ferrocenyl phosphines, we have added 1,1′-bis(diphenylphosphino)ferrocene as a comparison (Chart 1). The monosubstituted ferrocenyl-amide phosphines have been obtained by reaction of (chlorocarbonyl)ferrocene with NH2CH2CH2PPh2 or NH(CH2CH2PPh2)2 to give ligand L1 or L2, respectively (Scheme 1). We have previously synthesized the disubstituted ligand L3 by a similar procedure,14 starting from 1,1′-dichlorocarbonylferrocene. In both cases the 1H NMR spectra for L1 and L2 show a singlet for the protons of the unsubstituted cyclopentadienyl ligand, two multiplets for the α and β protons of the substituted cyclopentadienyl units, and two multiplets for the methylene protons. The 31P{1H} NMR spectra gives only one resonance at −20.9 for L1, where only one phosphorus atom is present, and two resonances at −20.1 and −21.5 ppm for L2, which indicates that both phosphorus atoms are inequivalent because of the conformational rigidity imposed on the molecule by the CpCO unit. The IR spectra show absorptions arising for the amide groups at 1539 (N−CO), 1601 (CO), and 3420 (NH) cm−1 for L1 and 1574 (N−CO) and 1598 (CO) cm−1 for L2. In the mass spectra molecular peaks at m/z 442 (100%) and 654 (50%) are observed. The reactions of L1 with gold(I) and silver(I) complexes such as [AuCl(tht)], Ag(OTf), and [Ag(OTf)(PPh3)] afford the linear gold(I) species [AuCl(PPh2CH2CH2NHCOFc)] (1) and the three-coordinated silver(I) complexes [Ag(OTf)(PPh2CH2CH2NHCOFc)2] (2) and [Ag(OTf)(PPh3)(PPh2CH2CH2NHCOFc)] (3), shown in Scheme 2. Complexes 1−3 have been characterized by means of 1H, 31 1 P{ H}, and 13C{1H} NMR spectroscopy. The 1H NMR spectra for all of the complexes show mainly the resonances arising from the ferrocene amide-phosphine ligand displaced after the coordination of the gold and silver centers, together with the resonances for the phenylic protons. The 31P{1H} NMR spectra are indicative of coordination of the metal. A low-field displacement is observed at 24.3 ppm in the case of gold and in the range 2−12 ppm for the silver complexes. In the 31 1 P{ H} NMR spectrum of complex 2 a broad signal appears at room temperature that splits into two doublets at −60 °C, because of the coupling of the phosphorus atoms with the two different silver nuclei, 107Ag and 109Ag. For the spectrum of complex 3 also a broad signal appears; at low temperature (−60 °C) the spectrum is complex because usually these P−Ag−P′ systems should appear as an AB system, which are further coupled to both silver nuclei; this means that each of the four resonances in the AB spectrum splits into two doublets. However, an equilibrium with the homoleptic species [Ag(OTf)(PPh3)2] and [Ag(OTf)(L1)2] (2) is observed, and it is possible to see the resonances arising for all of the compounds. For all compounds 1−3 the mass spectrum shows the molecular ion, while the base peaks correspond to the fragments

the parasite. Furthermore, the antiproliferative activity of compounds based on polyphenols is dramatically activated by the introduction of the ferrocene group.12 We have previously reported on the synthesis of some ferrocene conjugates with amino acid esters, the formation of the corresponding gold complexes, and the evaluation of their antiproliferative activity.13 The complexes showed moderate to significant cytotoxic activity, and a synergic effect between ferrocene and gold was observed. As the structure−activity relationship in gold complexes shows that the gold atom exerts the cytotoxic activity, that a phosphine ligand enhanced their activity, and that other auxiliary ligands are important in taking the gold center to its biological target, we believe that a combination between a phosphine ligand with ferrocene moieties and relevant biological thiolates would be a good combination for gold-based drugs. Therefore, here we report on the synthesis of ferrocenyl amide-phosphine ligands, which could also be important because of their ability to form hydrogen bonds, their coordination to the gold chloride fragment, and further substitution for several thiolate ligands. An evaluation of their antiproliferative activity has been carried out in two murine and two human cell lines.

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RESULTS AND DISCUSSION Synthesis and Structural Characterization. As phosphine ligands are important in gold complexes in improving the cytotoxic activity of the complex, probably because they allow the gold compound to better cross the plasma membrane, we have prepared several ferrocenyl-amide phosphines in order to form the gold complexes and test their antiproliferative activity. Monosubstituted species such as PPh2CH2CH2NHCOFc and (PPh2CH2CH2)2NCOFc and the disubstituted derivative (PPh2CH2CH2NHCO)2Fc have been chosen as suitable ferrocenyl Chart 1. Ferrocene-Based Phosphines Used

Scheme 1. Synthesis of the Ferrocenyl Amide-Phosphine Ligandsa

a

Legend: (i) NH2CH2CH2PPh2, (ii) NH(CH2CH2PPh2)2. 6070

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Scheme 2. Synthesis of Metal Complexes with the Ligand PPh2CH2CH2NHCOFca

a

Legend: (i) [AuCl(tht)]; (ii) 1/2 Ag(OTf); (iii) [Ag(OTf)(PPh3)].

Scheme 3. Synthesis of Thiolate Gold Complexes with the Ligand PPh2CH2CH2NHCOFca

a

Legend: (i) 2-mercaptonicotinic acid + K2CO3; (ii) 2-thiocytosine + K2CO3; (iii) 2-thiouracil + K2CO3; (iv) 2-mercaptopurine + K2CO3.

[M − Cl]+ (m/z 638), [M − OTf]+ (m/z 840) and [M − PPh3]+ (m/z 548) for 1−3, respectively. Substitution reactions have been carried out with complex 1, and therefore, treatment with thiols such as 2-mercaptonicotic acid, 2-thiocytosine, 2-thiouracil, and 6-mercaptopurine in the presence of K2CO3 produces the corresponding thiolate derivatives 4−7 (see Scheme 3). The NMR data for these complexes agree with the substitution of the chlorine ligand for the corresponding thiolate; in the 1H and 13C NMR spectra the resonances for both the amidephosphine and the thiolate ligands appear. In the 31P{1H} NMR the resonance for the phosphorus atom is displaced downfield.

The reaction of ligand L2 with [AuCl(tht)] in a 1:2 molar ratio affords the dinuclear gold derivative [Au2Cl2{(PPh2CH2CH2)2NCOFc}] (8). The chloro ligand can be easily substituted by reactions with thiols such as 2-thiocytosine and 2,3,4,6-tetra-6acetyl-1-thiol-β-D-glucopyranosato in the presence of K2CO3 (see Scheme 4). The 1H and 13C{1H} spectra of complex 8 show all of the resonances arising from the ligand with a different chemical shift after coordination to the gold center. The 31P{1H} spectrum shows two singlets at 25.7 and 22.2 ppm with a higher chemical shift displacement in comparison to the free phosphine. The mass spectrum presents the molecular peak at m/z 1117 (45%). For complexes 9 and 10 the 1H and 13C{1H} 6071

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Scheme 4. Synthesis of Gold Complexes with the Ligand (PPh2CH2CH2)2NCOFca

a

Legend: (i) 2 [AuCl(tht)]; (ii) 2-thiocytosine + K2CO3; (iii) 2,3,4,6-tetra-6-acetyl-1-thiol-β-D-glucopyranosato + K2CO3.

The Au−P distances are 2.232(2) and 2.228(2) Å, and the Au−Cl distances are 2.294(2) and 2.292(2) Å, which are similar to those found in complexes such as [Au2Cl2(dppf)].15 In the crystal the molecules associated through intermolecular aurophilic interactions of 3.2816(5) Å, forming a chain polymer (see Figure 2). The reaction of the ligand L3 or dppf with [AuCl(tht)] (molar ratio 1:2) gives the dinuclear gold derivative [Au2Cl2{(PPh2CH2CH2NHCO)2Fc}] or [Au2Cl2(dppf)], previously reported by us.14,16 These complexes further react with thiols such as 2thiocytosine, 2-thiouracil, and 2,3,4,6-tetra-6-acetyl-1-thiol-β-Dglucopyranosato (HTG) in the presence of K2CO3 to afford the thiolate gold species (see Figure 3). The 1H NMR spectra of complexes 11−14 show the resonances for the disubstituted ferrocene as two multiplets for the equivalent α and β protons of both cyclopentadienyl groups, two multiplets for the methylene protons in complexes 11 and 12, and resonances arising from the phenyl protons and the corresponding thiolate ligand. The 31P{1H} NMR spectra present a unique resonance in all cases, corresponding to equivalent phosphorus atoms. The structure of complex 13 has been established by X-ray diffraction studies, and the molecule is shown in Figure 4. The compound crystallizes in the orthorhombic noncentrosymmetric P212121 space group. The molecule is chiral, and the original configuration of the D isomer (2R,3R,4S,5R,6R) is retained. The gold centers are found in a linear geometry with angles of 177.81(15) and 177.44(16) Å. The Au−S bond distances are 2.319(5) and 2.301(4) Å, and the Au−P distances are 2.280(4) and 2.266(4) Å, which are on the same order as those found in thiolate gold phosphine complexes. The ferrocenyl rings are planar and adopt a rotated anticlinal conformation. In the structure short intermolecular aurophilic interactions of 3.010(3) Å are observed (see Figure 5), and a polymeric chain is formed, in a way similar to that for complex 8. There

spectra show all of the resonances arising from the ligand with a different chemical shift after coordination to the gold center. The 31P{1H} spectra at room temperature show a broad resonance for 9 and two broad singlets for 10. The low-temperature spectra at −85 °C for 9 and −60 °C for 10 show the expected two singlets for each compound. The molecular structure of complex 8 has been established by X-ray diffraction, and the molecule is shown in Figure 1.

Figure 1. Molecular structure for compound 8 with 50% probability ellipsoids. Hydrogen atoms are omitted for clarity. Selected bond lengths (Å) and angles (deg): Au(1)−P(1) = 2.233(2), Au(1)−Cl(1) = 2.294(2), Au(2)−P(2) = 2.228(2), Au(2)−Cl(2) = 2.292(2); P(1)− Au(1)−Cl(1) = 174.35(9), P(2)−Au(2)−Cl(2) = 173.17(9).

The gold(I) centers are linearly coordinated with angles P(1)−Au(1)− Cl(1) = 174.35(9)° and P(2)−Au(2)−Cl(2) = 173.17(9)°. 6072

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Figure 2. Polymer chain of complex 8 though intermolecular aurophilic interactions. Selected bond lengths (Å) and angles (deg): Au(1)−Au(2) = 3.2816(5); P(1)−Au(1)−Au(2) = 110.76(6), P(2)−Au(2)−Au(1) = 91.64(6).

Figure 3. Thiolate gold complexes prepared from L3 and dppf.

are also several secondary bonds between the oxygen atoms of the thiolate and some of the protons that can be considered as hydrogen bonds, the shortest being C(45)−H(45)···O(9)#3 (symmetry transformation: (#3) x + 1/2, −y + 3/2, −z + 1) of 2.33 Å, with an angle of 156.6°. Biological Evaluation. The ferrocenyl-amide phosphine ligands and the complexes synthesized with them were tested for cytotoxicity against four tumor cell lines: the two murine cell lines NIH-3T3 (mouse embryonic fibroblasts) and PC-12 (pheochromocytoma of the rat adrenal medulla) and the two human cell lines A-549 (adenocarcinomic human alveolar basal epithelial cells) and Hep-G2 (hepatocellular carcinoma). Cells were exposed to each compound for a total of 48 h. Using the calorimetric mitochondrial function-based MTT viability assay, the IC50 values (final concentration ≤0.5% DMSO) were calculated from dose−response curves obtained by nonlinear regression analysis. IC50 values are concentrations of drugs required to inhibit tumor cell proliferation by 50% in comparison to the control viability.

As demonstrated by the IC50 values collected in Table 1, neither of the ligands L1 and L2 showed significant antiproliferative activity. For the gold chloride and silver complexes higher cytotoxic activity is found for the chloro-gold compounds, for which the smallest values are found for [AuCl(PPh2CH2CH2NHCOFc)] (1) in the murine cells, with a very low value for NIH-3T3 cells of 0.1 μM, and for the previously reported [Au2Cl2(μ-dppf)] in the human cell lines with values of 8.2 and 10.7 μM. The silver complexes 2 and 3 have only a moderate activity in all of the cell lines. The antiproliferative activity of all the thiolate gold derivatives of L1−L3 and dppf have been measured under the same conditions (Table 2). The general observation is that very good values for the activity are found in the murine cell lines NIH3T3 and PC-12 and moderate to good values are found in the human cell lines A-549 and Hep-G2. For the murine cell lines lower values are found for the gold thiolates of L1, with the nicotinic acid thiolate (4), thiocytosine (5), and thiouracil (6), with values even lower than those for auranofin. Surprisingly 6073

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Table 1 IC50 (μM) compd

NIH-3T3

PC-12

A-549

Hep-G2

L1 L2 1 2 3 8 [Au2Cl2(μ-L3)] [Au2Cl2(μ-dppf)]

>100 >100 0.1 23.3 42.3 11.4 14.5 9.8

>100 >100 4.2

>100 >100 >100 21 26.4 29.9 >100 8.2

>100 >100 68 70.7 >100 29.1 >100 10.7

Table 2 IC50 (μM)

Figure 4. Molecular structure of complex 13 with atom-labeling scheme. Hydrogen atoms have been omitted. Radii are arbritrary. Selected bond lengths (Å) and angles (deg): Au(1)−P(1) = 2.280(4), Au(1)−S(1) = 2.319(5), Au(2)−P(2)#1 = 2.266(4), Au(2)−S(2) = 2.301(4); P(2)#1−Au(2)−S(2) = 177.44(16), P(2)#1−Au(2)−Au(1) = 104.31(8). Symmetry transformation: (#1) −x + 1/2, −y + 2, z + 1/2.

compd

NIH-3T3

PC-12

A-549

Hep-G2

4 5 6 7 9 10 11 12 13 14 auranofin cisplatin

0.2 0.8 2.0 13.7 4.7 4.0 5.4 14.7 5.9 5.4 2.5

2.1 2.4 1.0

12.6 95.1 15.0 21.0 13.1 14.5 30.3 74.9 7.0 12.0 3.4 29.2

7.8 >100 20.0 70.7 12.4 11.1 17.6 72.9 13.7 14.8 2.0 15

or thiolate as auxiliary ligands. These values are always higher than those obtained for auranofin but much lower than those for cisplatin. With all these data it is not easy to establish a structure− activity relationship. It seems that among the ferrocenyl amide phosphines good to excellent values are found for the complexes with the monosubstituted ferrocenyl phosphines, and with the monophosphine L1 excellent values are found in the murine cells for all of the complexes, but only for the

the values for the purine thiolate are higher, although this thiol has biological activity by itself. For the human cell lines the best values are found for compound 4, [Au(SNic)(PPh2CH2CH2NHCOFc)] (SNic = thionicotinic acid), again with L1 and the thioniconitic acid, and for the dppf derivatives. In all the cases the activity obtained with the thiolate ligands is much higher than that for the corresponding chloro derivatives with the same amide-phosphine ligand, with the exception of the diphosphine dppf, which possesses similar values among chloro

Figure 5. Formation of a polymer chain through aurophilic interactions. Selected bond lengths (Å) and angles (deg): Au(1)−Au(2) = 3.010(3); P(1)−Au(1)−Au(2) = 97.61(9), S(1)−Au(1)−Au(2) = 80.92(10), P(2)#1−Au(2)−Au(1) = 104.31(8), S(2)−Au(2)−Au(1) = 75.74(9). Symmetry transformation: (#1) −x + 1/2, −y + 2, z + 1/2. 6074

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were separated, and anhydrous Na2SO4 was added to the organic phase for drying. The solvent was evaporated to ca. 5 mL, and hexane (20 mL) was added to obtain a yellow solid: 75% yield (0.066 g). 1H NMR (CDCl3) δ (ppm): 7.47 (m, 4H, Ph), 7.36 (m, 6H, Ph), 5.92 (m, br, 1H, NH), 4.58 (m, 2H, β-C5H4), 4.31 (m, 2H, α-C5H4), 4.19 (s, 5H, Cp), 3.54 (m, 2H, −CH2N−), 2.40 (t, J = 7.1 Hz, 2H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 170.34 (CO), 137.70 (d, 1JP−C = 11.4 Hz, ipso-C6H5), 132.91 (d, 2JP−C = 18.7 Hz, o-C6H5), 129.07 (p-C6H5), 128.82 (d, 3JP−C = 6.9 Hz, m-C6H5), 76.15 (Cipso), 70.48, 68.18 (C5H4), 69.89 (Cp), 36.99 (d, 2JC−P = 18.8 Hz, −CH2N−), 28.93 (d, 1JC−P = 12.6 Hz, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): −20.93. MS (FAB+): m/z 442 (100%). Anal. Calcd for C25H24FeNOP (441.3): C, 68.04; H, 5.48; N, 3.17. Found: C, 67.82; H, 5.45; N, 3.29. Synthesis of (PPh2CH2CH2)2NCOFc (L2). To a solution of bis[2(diphenylphosphino)ethyl]amine (0.4777 g, 1 mmol) in CH2Cl2 (50 mL) was added triethylamine (300 μL, 2.1 mmol); the solution was stirred for 10 min at room temperature and then poured into an ice bath, a solution of chlorocarbonylferrocene in CH2Cl2 (20 mL) was added dropwise, and the mixture was then stirred overnight. An aqueous solution of NaHCO3 (30 mL) was added, the two phases were separated, and anhydrous Na2SO4 was added to the organic phase. The solvent was evaporated to ca. 15 mL, and hexane (40 mL) was added to obtain a white solid: 85% yield (0.555 g). 1H NMR (CDCl3) δ (ppm): 7.38 (m, 20H), 4.37 (m, 2H, β-C5H4), 4.18 (m, 2H, α-C5H4), 4.13 (s, 5H, Cp), 3.51 (m, 4H, −CH2N−), 2.33 (m, 4H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 170.33 (CO), 132.88 (d, 2JP−C = 18.8 Hz, o-C6H5), 128.77 (d, 3JP−C = 6.4 Hz, m-C6H5), 70.43, 69.77 (C5H4), 69.84 (Cp). 31P{1H} NMR (CDCl3) δ (ppm): −20.11, −21.54. MS (FAB+): m/z 654 (50%). Anal. Calcd for C39H37FeNOP2 (653.5): C, 71.68; H, 5.71; N, 2.14. Found: C, 71.76; H, 5.27; N, 1.96. Synthesis of [AuCl(L1)] (1). To a solution of L1 (0.066 g, 0.15 mmol) in CH2Cl2 (20 mL) was added [AuCl(tht)] (0.048 g, 0.15 mmol), and the mixture was stirred for 1 h. Then, the solution was evaporated to ca. 5 mL and hexane (15 mL) was added to obtain a yellow solid: 88% yield (0.088 g). 1H NMR (CDCl3) δ (ppm): 7.71 (m, 4H, Ph), 7.48 (m, 6H, Ph), 6.23 (m, br, 1H, NH), 4.67 (m, 2H, β-C5H4), 4.37 (m, 2H, α-C5H4), 4.21 (s, 5H, Cp), 3.68 (m, br, 2H, −CH2N−), 2.87 (m, br, 2H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 171.21 (CO), 133.34 (d, 2JP−C = 13.3 Hz, o-C6H5), 132.37 (p-C5H6), 129.63 (d, 3JP−C = 11.7 Hz, m-C6H5), 128.76 (d, 1JP−C = 61.7 Hz, ipso-C6H5), 75.13 (Cipso), 70.95, 68.39 (C5H4), 69.90 (Cp), 36.38 (d, JP−C = 5.6 Hz, −CH2N−), 28.72 (d, JP−C = 37.7 Hz, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): 24.32. MS (FAB+): [M]+ m/z 673 (48.18%), [M − Cl]+ m/z 638 (100%). Anal. Calcd for C25H24AuClFeNOP (673.7): C, 44.57; H, 3.59; N, 2.08. Found C, 44.73; H, 3.54; N, 2.19. Synthesis of [Ag(OTf)(L1)2] (2). Caution! Reactions with silver salts should be protected from the light. To a solution of L1 (0.1058 g, 0.24 mmol) in CH2Cl2 (20 mL) was added [AgOTf] (0.0308 g, 0.12 mmol), and the mixture was stirred for 1 h. Afterward the solution was evaporated to ca. 5 mL and hexane was added (15 mL) to obtain a yellow solid: 73% yield (0.0997 g). 1H NMR (CDCl3) δ (ppm): 7.59− 7.23 (m, 22H, Ph, NH), 4.80 (m, 4H, β-C5H4), 4.29 (m, 4H, α-C5H4), 4.18 (s, 10H, Cp), 3.54 (m, br, 4H, −CH2N−), 2.72 (m, br, 4H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 132.88 (o-C6H5), 131.12 (p-C6H5), 129.47 (m-C6H5), 70.96, 68.67 (C5H4), 69.87 (Cp), 36.43 (−CH2N−), 27.29 (−CH2P−). 31P{1H} NMR (CDCl3, −60 °C) δ (ppm): 2.42 (2d, JP−Ag = 577.0, 501.1 Hz). 19F{1H}-NMR (CDCl3) δ (ppm): −77.74. MS (FAB+): [M]+ m/z 989 (35%). Anal. Calcd for C55H48AgF3Fe2N2O5P2S (1139.5): C, 53.76, H, 4.25, N, 2.46; S, 2.81. Found: C, 53.46, H, 4.28, N, 2.42; S, 3.09. Synthesis of [Ag(OTf)(L1)(PPh3)] (3). Caution! Reactions with silver salts should be protected from the light. To a solution of L1 (0.044 g, 0.1 mmol) in CH2Cl2 (20 mL) was added [Ag(OTf)(PPh3)] (0.0518 g, 0.1 mmol), and the mixture was stirred for 1 h. Afterward the solution was evaporated to ca. 5 mL and hexane (15 mL) was added to obtain a yellow solid: 87% yield (0.0835 g). 1H NMR (CDCl3) δ (ppm): 7.73−7.29 (m, 26H, Ph, NH), 4.75 (m, 2H, β-C5H4), 4.15 (m, 7H, α-C5H4, Cp), 3.66 (m, 2H, −CH2N−), 2.83

nicotinic acid thiolate in the human cell lines. However, compounds with the monosubstituted ferrocenyl diphosphine ligand L2 have good values in all of the cell lines. The complexes with the disubstituted ferrocenyl amide diphosphine (L3) present the poorest antiproliferative values. The IC50 values for the complexes with dppf are good but are of the same order as that of the complexes with L2.

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CONCLUSIONS Several ferrocenyl amide phosphines such as PPh2CH2CH2NHCOFc (L1), (PPh2CH2CH2)2NCOFc (L2), and (PPh2CH2CH2NHCO)2Fc (L3) have been chosen in order to prepare different gold(I) and silver(I) complexes and measure their antiproliferative activities. As a comparison, complexes with the well-known dppf ligand have been prepared. The gold chloride derivatives [AuCl(PPh2CH2CH2NHCOFc)], [Au2Cl2{(PPh2CH2CH2)2NCOFc}], and [Au2Cl2{(PPh2CH2CH2NHCO)2Fc}] have been synthesized in which the chloro ligand can be easily substituted by several biologically relevant thiolates such as 2-mercaptonicotinic acid, 2-thiocytosine, 2-thiouracil, 2-mercaptopurine, and 2,3,4,6-tetra-6-acetyl-1-thiol-β-D-glucopyranosato, affording the gold phosphine thiolate species. The antiproliferative activity of these compounds have been tested by the MTT viability assay in two murine cell lines, NIH-3T3 (mouse embryonic fibroblasts) and PC-12 (pheochromocytoma of the rat adrenal medulla), and two human cell lines, A-549 (adenocarcinomic human alveolar basal epithelial cells) and Hep-G2 (hepatocellular carcinoma). The ferrocenyl amide phosphine ligands are not active, whereas the metal complexes are. The silver complexes are less active than the gold species. Among the ferrocenyl amide phosphines good to excellent values are found for the gold complexes with the monosubstituted ferrocenyl phosphines. With the monophosphine L1 excellent values are found in the murine cells for all the complexes, but only for the nicotinic acid thiolate species in the human cell lines. However, the complexes with the monosubstituted ferrocenyl diphosphine ligand L2 have good values in all the cell lines. The complexes with the disubstituted ferrocenyl amide diphosphine (L3) present the poorest antiproliferative values. The IC50 values for the compounds with dppf are good but are of the same order as those with L2.

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EXPERIMENTAL SECTION

Instrumentation. C, H, and N analysis were carried out with a Perkin-Elmer 2400 microanalyzer. Mass spectra were recorded on a Bruker Esquire 3000 Plus instrument, with the electrospray (ESI) technique, and on a Bruker Microflex (MALDI-TOF) instrument. 1H, 13 C{1H}, and 19F NMR spectra, including 2D experiments, were recorded at room temperature on a Bruker Avance 400 spectrometer (1H, 400 MHz; 13C, 100.6 MHz; 19F, 376.5 MHz) or on a Bruker Avance II 300 spectrometer (1H, 300 MHz; 13C, 75.5 MHz; 19F, 282.3 MHz), with chemical shifts (δ, ppm) reported relative to the solvent peaks of the deuterated solvent.17 Starting Materials. The starting materials FcCOCl, 18 (PPh2CH2CH2NHCO)2Fc,14 [AuCl(tht)],19 and [Ag(OTf)(PPh3)]20 were prepared according to published procedures. All other reagents were commercially available. Solvents were used as received without purification or drying or dried with a SPS solvent purification system. Synthesis of PPh2CH2CH2NHCOFc (L1). To a solution of 2-(diphenylphosphino)ethylamine (46 μL, 0.2 mmol) in CH2Cl2 (20 mL) was added triethylamine (60 μL, 0.43 mmol); the solution was stirred for 10 min at room temperature, and then it was poured into an ice bath, a solution of chlorocarbonylferrocene in CH2Cl2 (20 mL) was added dropwise, and the mixture was stirred overnight. An aqueous solution of NaHCO3 (30 mL) was added, the two phases 6075

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(m, 2H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 171.85 (C O), 134.08 (o-C6H5), 133.13 (o-C6H5), 131.24 (p-C6H5), 131.13 (p-C6H5), 129.50 (m-C6H5), 129.38 (m-C6H5), 75.45 (Cipso), 70.63, 68.59 (C5H4), 69.89 (Cp), 36.19 (−CH2N−), 27.78 (−CH2P−). 31 1 P{ H} NMR (CDCl3) δ (ppm): 12.89 (2d, JP−Ag = 571.03, 495.04 Hz), 11.32 (m), 2.46 (2d, JP−Ag = 577.11, 500.17 Hz), 1.25 (2d, JP−Ag = 572.59, 495.6 Hz). 19F NMR (CDCl3) δ (ppm): −77.68. MS (FAB+): [M]+ m/z 810 (30.4%), [M − PPh3]+ m/z 548 (100%). Anal. Calcd for C44H39AgF3FeNO4P2S (960.5): C, 55.02; H, 4.09; N, 1.46; S, 3.34. Found C, 55.16; H, 4.09; N, 1.46; S, 3.44. General Procedure for the Synthesis of Phosphinegold(I) Thiolates and Thionucleobase Analogues. A suspension of thiol or thionucleobase (0.2 mmol) and K2CO3 (1 mmol) in CH2Cl2 (15 mL) was stirred for 30 min. Then, complex 1 (0.2 mmol) was added and this mixture was stirred overnight. The solution was filtered, the solvent was evaporated to ca. 5 mL, and hexane was added (15 mL) to obtain a solid. Synthesis of [Au(2-thionicotinicacid)(L1)] (4): yellow solid, 65% yield (0.103 g). 1H NMR (CDCl3) δ (ppm): 8.51 (d, J = 7.2 Hz, 1H, H6), 8.40 (d, J = 4.8 Hz, 1H, H4), 7.73 (m, 4H, Ph), 7.49 (m, 6H, Ph), 7.13 (dd, J = 7.8, 4.9 Hz, 1H, H5), 6.65 (m, br, 1H, NH), 4.69 (m, 2H, β-C5H4), 4.34 (m, 2H, α-C5H4), 4.21 (s, 5H, Cp), 3.71 (m, 2H, −CH2N−), 3.10 − 2.68 (m, 2H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 171.35 (CO), 133.33 (d, 2JP−C = 13.0 Hz, o-C6H5), 132.27 (p-C6H5), 129.60 (d, 3JP−C = 11.4 Hz, m-C6H5), 129.00 (d, 1JP−C = 59.9 Hz, ipso-C6H5), 75.23 (Cipso), 70.81, 68.39 (C5H4), 69.91 (Cp), 36.46 (−CH2N−), 28.78 (d, J = 35.6 Hz, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): 25.47. Anal. Calcd for C31H28AuFeN2O3PS (792.4): C, 46.99; H, 3.56; N, 3.54, S 4.05. Found: C, 46.64; H, 3.49; N, 3.41; S, 3.79. Synthesis of [Au(2-thiocytosine)(L1)] (5): yellow solid, 73% yield (0.111 g). 1H NMR (CDCl3) δ (ppm): 7.93 (s, 1H, NH), 7.84 (d, J = 5.7 Hz, 1H, H6), 7.73 (m, 4H, Ph), 7.45 (m, 6H, Ph), 6.07 (d, J = 5.8 Hz, 1H, H5), 4.97 (m, br, 2H, NH2), 4.71 (m, 2H, β-C5H4), 4.26 (m, 2H, α-C5H4), 4.13 (s, 5H, Cp), 3.71 (m, 2H, −CH2N−), 2.92 (m, 2H, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): 28.81. Anal. Calcd for C29H28AuFeN4OPS (764.4): C, 45.57; H, 3.69; N, 7.33; S, 4.19. Found: C, 45.75; H, 3.83; N, 6.97; S, 3.89. Synthesis of [Au(2-thiouracil)(L1)] (6): yellow solid, 67% yield (0.102 g). 1H NMR (CDCl3) δ (ppm): 10.42 (s, 1H, NH), 7.71 (m, 4H, Ph), 7.63 (d, J = 6.7 Hz, 1H, H6), 7.48 (m, 6H, Ph), 6.14 (d, J = 6.6 Hz, 1H, H5), 4.69 (m, 2H, β-C5H4), 4.29 (m, 2H, α-C5H4), 4.14 (s, 5H, Cp), 3.69 (m, 2H, −CH2N−), 2.93 (m, 2H, −CH2P−). 13 C{1H} NMR (CDCl3) δ (ppm): 171.52 (CO), 133.36 (d, 2JP−C = 13.2 Hz, o-C6H5), 132.13 (p-C6H5), 129.63 (d, 4JP−C = 57.8 Hz, ipsoC6H5), 129.58 (d, 3JP−C = 11.4 Hz, m-C6H5), 110.13 (C5), 75.55 (Cipso), 70.74, 68.46 (C5H4), 69.84 (Cp), 36.51 (−CH2N−), 29.06 (d, J = 36.8 Hz, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): 28.82. Anal. Calcd for C29H27AuFeN3O2PS (765.4): C, 45.51; H, 3.56; N, 5.49; S, 4.19. Found: C, 45.47; H, 3.68; N, 5.20; S, 3.98. Synthesis of [Au(6-thiopurine)(L1)] (7): yellow solid. 58% yield (0.092 g). 1H NMR (CDCl3) δ (ppm): 8.64 (s, 1H, H9), 8.13 (m, br, 1H, NH), 8.03 (s, 1H, H5), 7.70 (m, 4H, Ph), 7.42 (m, 6H, Ph), 4.69 (m, 2H, β-C5H4), 4.16 (s, 2H, α-C5H4), 4.11 (s, 5H, Cp), 3.71 (m, 2H, −CH2N−), 2.87 (m, 2H, CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 171.55 (CO), 133.43 (d, 2JP−C = 13.1 Hz, o-C6H5), 131.97 (p-C6H5), 129.50 (d, 3JP−C = 11.4 Hz, m-C6H5), 75.64 (Cipso), 70.64, 68.54 (C5H4), 69.86 (Cp). 31P{1H} NMR (CDCl3) δ (ppm): 25.64. Anal. Calcd for C30H27AuFeN5OPS (789.4): C, 45.64; H, 3.45; N, 8.87; S, 4.06; found. C 45.50; H, 3.40; N, 8.67; S, 3.85. Synthesis of [Au2Cl2(L2)] (8). To a solution of L2 (0.1771 g, 0.15 mmol) in CH2Cl2 (20 mL) was added [AuCl(tht)] (0.1282 g, 0.30 mmol), and the mixture was stirred for 1 h. Afterward, the solution was evaporated to ca. 5 mL and hexane was added (15 mL) to obtain a yellow solid: 93% yield (0.094 g). 1H NMR (CDCl3) δ (ppm): 7.73 (m, 8H, Ph), 7.54 (m, br, 12H, Ph), 4.59 (m, 2H, β-C5H4), 4.40 (m, 2H, α-C5H4), 4.24 (s, 5H, Cp), 3.73 (m, br, 4H, −CH2N−), 2.80 (m, br, 4H, −CH2P−). 13C{1H} NMR (CDCl3) δ (ppm): 133.37 (d, 2JP−C = 13.6 Hz, o-C6H5), 132.65 (p-C6H5), 129.89 (d, 3JP−C = 10.4 Hz, m-C6H5), 70.92, 70.47 (C5H4), 70.17 (Cp).

P{1H} NMR (CDCl3) δ (ppm): 25.67, 22.24. MS (FAB+): [M]+ m/z 1117 (45%). Anal. Calcd for C39H37Au2Cl2FeNOP2 (673.7): C, 41.88; H, 3.33; N, 1.25. Found: C, 41.76; H, 3.47; N, 1.26. [Au2(2-thiocytosine)2(L2)] (9): yellow solid, 70% yield (0.182 g). 1 H NMR (CDCl3) δ (ppm): 7.85 (d, J = 5.7 Hz, 2H, H6), 7.74 (m, 8H, Ph), 7.47 (m, 12H, Ph), 5.83 (d, J = 5.5 Hz, 2H, H5), 5.13 (m, br, 4H, NH2), 4.46 (m, 2H, β-C5H4), 4.21 (m, 2H, α-C5H4), 4.19 (s, 5H, Cp), 3.90 (m, br, 4H, −CH2N−), 2.80 (m, br, 4H, −CH2P−). 13 C{1H} NMR (CDCl3) δ (ppm): 171.73 (CO), 162.13 (C4), 155.69 (C6), 133.47 (d, J = 13.7 Hz, o-C6H5), 132.04 (p-C6H5), 129.58 (d, J = 11.4 Hz, m-C6H5), 100.45 (C5), 70.46, 70.35 (C5H4), 70.13 (Cp). 31P{1H} NMR (acetone, −85 °C) δ (ppm): 29.36, 27.97. Anal. Calcd for C47H45Au2FeN7OP2S2 (1299.8): C, 43.43; H, 3.49; N, 7.54; S, 4.93. Found: C, 43.17; H, 3.32; N, 7.39; S, 4.67. [Au2(TG)2(L2)] (10): yellow solid, 75% yield (0.265 g). 1H NMR (CDCl3) δ (ppm): 7.74 (m, 8H, Ph), 7.49 (m, 12H, Ph), 5.13 (m, 8H, H1, H4, H5, H6), 4.49 (m, 1H, β-C5H4), 4.36 (m, 1H, β-C5H4), 4.27 (m, 1H, α-C5H4), 4.24 (m, 1H, α-C5H4), 4.20 (dd, J = 12.3, 4.8 Hz, 1H, −CH2−), 4.16 (s, 5H, Cp), 4.09 (dd, J = 12.2, 2.4 Hz, 1H, −CH2−), 3.76 (m, br, 6H, H3, −CH2N−), 2.75 (m, br, 4H, −CH2P−), 2.01 (s, 6H, −CH3), 1.96 (s, 6H, −CH3), 1.92 (s, 6H, −CH3), 1.86 (s, 6H, −CH3). 13C{1H} NMR (CDCl3) δ (ppm): 171.20 (N−CO), 170.88, 170.40, 169.86, 169.75 (O−CO), 133.43 (t, 2JP−C = 14.2 Hz, o-C6H5), 131.69 (p-C6H5), 129.55 (d, 3JP−C = 9.4 Hz, m-C6H5), 129.50 (d, 3JP−C = 10.1 Hz, m-C6H5), 83.42 (C1), 77.83 (C6), 75.86 (C3), 74.26 (C5), 70.47, 70.44 (C5H4), 70.00 (Cp), 69.09 (C4), 62.96 (−CH2−), 21.26, 20.88, 20.83, 20.79 (−CH3). 31 1 P{ H} NMR (CDCl3, −60 °C) δ (ppm): 29.44, 27.11. Anal. Calcd for C67H75Au2FeNO19P2S2 (1774.2): C, 45.36; H, 4.26; N, 0.79; S, 3.61. Found: C, 45.25; H, 4.21; N, 0.96; S, 3.84. [Au2(TG)2(μ-L3)] (11): yellow solid, 70% yield (0.253 g). 1H NMR (CDCl3) δ (ppm): 7.76 (m, 8H, Ph), 7.46 (m, 12H, Ph), 5.19 (m, 8H, H5, H6, H4, H1), 4.53 (m, 2H, Cp), 4.45 (m, 2H, Cp), 4.32 (m, 2H, Cp), 4.26 (m, 2H, Cp), 4.23 (m, 2H, −CH2−), 4.12 (m, 2H, −CH2−), 3.72 (m, 6H, H3, −CH2N−), 2.91 (m, 4H, −CH2P−), 2.08 (s, 6H, COCH3), 2.03 (s, 6H, COCH3), 1.98 (s, 6H, COCH3), 1.88 (s, 6H, COCH3). 13C{1H} NMR (CDCl3) δ (ppm): 170.95, 170.89, 170.48, 170.38, 169.83 (CO), 133.44 (t, 2JP−C = 13.3 Hz, o-C6H5), 131.72 (d, 4JP−C = 3.1 Hz, p-C6H5), 129.48 (d, 3JP−C = 10.9 Hz, mC6H5), 129.42 (d, 3JP−C = 11.0 Hz, m-C6H5), 83.51 (C1), 78.12 (C6), 75.90 (C3), 74.31 (C5), 71.59, 71.44, 69.94 (C5H4), 69.02 (C4), 62.86 (−CH2−), 36.68 (−CH2N−), 28.39 (d, JP−C = 31.2 Hz, −CH2P−), 21.46, 20.89, 20.83 (−CH3−). 31P{1H} NMR (CDCl3) δ (ppm): 25.54. Anal. Calcd for C68H76Au2FeN2O20P2S2 (1817.2): C 44.94; H, 4.22; N, 1.54; S, 3.53. Found: C, 44.93; H, 3.90; N, 1.46; S, 3.59. [Au2(2-thiocytosine)2(μ-L3)] (12): yellow solid, 74% yield (0.198 g). 1 H NMR (CDCl3) δ (ppm): 8.30 (m, br, 2H, NH), 7.84 (d, J = 5.8 Hz, 2H, H6), 7.74 (m, 8H, Ph), 7.44 (m, 12H, Ph), 6.06 (d, J = 5.8 Hz, 2H, H5), 4.71 (m, 2H, β-C5H4), 4.19 (m, 2H, α-C5H4), 3.71 (m, 4H, −CH2N−), 2.90 (m, 4H, −CH2P−). 13C{1H}-NMR (CDCl3) δ (ppm): 178.77 (C2), 170.92 (CO), 162.61 (C4), 155.28 (C6), 133.49 (d, 2JP−C = 11.2 Hz, o-C6H5), 131.84 (p-C6H5), 129.46 (d, 3 JP−C = 8.1 Hz, m-C6H5), 100.65 (C5), 72.25, 70.24 (C5H4), 36.53 (−CH2N−), 28.80 (d, J = 29.8 Hz, −CH2P−). 31P{1H} NMR (CDCl3) δ (ppm): 28.20. Anal. Calcd for C48H46Au2FeN8O2P2S2 (1342.8): C, 42.93; H, 3.45; N, 8.34; S, 4.78. Found: C, 43.07; H, 3.68; N, 8.22; S, 4.61. [Au2(TG)2(μ-dppf)] (13): yellow solid, 83% yield (0.277 g). 1H NMR (CDCl3) δ (ppm): 7.48 (m, 10H, Ph), 5.16 (m, 4H, H5, H4), 4.91 (m, 2H, Cp), 4.85 (m, 2H, Cp), 4.33 (m, 2H, Cp), 4.27 (m, 3H, Cp, −CH2−), 4.11 (m, 1H, −CH2−), 3.79 (m, 1H, H3), 2.09, 2.01, 1.97, 1.87 (s, 12H, COCH3). 13C{1H} NMR (CDCl3) δ (ppm): 170.82, 170.37, 169.92, 169.69 (CO), 133.89 (d, 2JP−C = 13.9 Hz, o-C6H5), 133.73 (d, 2JP−C = 13.8 Hz, o-C6H5), 131.66 (d, 4JP−C = 2.5 Hz, p-C6H5), 131.61 (d, 4JP−C = 2.4 Hz, p-C6H5), 131.53 (d, 1JP−C = 57.9 Hz, ipso-C6H5), 131.35 (d, 1JP−C = 58.1 Hz, ipso-C6H5), 129.16 (d, 3JP−C = 11.5 Hz, m-C6H5), 129.14 (d, 3JP−C = 11.5 Hz, m-C6H5), 83.62 (C1), 78.14 (C6), 76.03 (C3), 75.27 (m, C5H4), 74.41 (C5), 71.96 (d, J = 65.1 Hz, ipso-C5H4), 69.18 (C4), 63.05 (−CH2−), 21.31, 31

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20.93, 20.86, 20.78 (CO−CH3). 31P{1H} NMR (CDCl3) δ (ppm): 33.46. Anal. Calcd for C62H66Au2FeO18P2S2 (1675.03): C, 44.46; H, 3.97; S, 3.83. Found: C, 44.39; H, 3.75; S, 3.59. [Au2(2-thiouracil)2(μ-dppf)] (14): yellow solid, 85% yield (0.204 g). 1 H NMR (CDCl3) δ (ppm): 7.77 (d, J = 6.3 Hz, 2H, H6), 7.66 (m, 8H, Ph), 7.47 (m, 12H, Ph), 6.22 (d, J = 6.3 Hz, 2H, H5), 4.63 (m, br, 4H, Cp), 4.12 (m, br, 4H, Cp). 13C{1H} NMR (CDCl3) δ (ppm): 154.41 (C6), 133.91 (d, 2JP−C = 13.9 Hz, o-C6H5), 131.82 (d, 4JP−C = 2.3 Hz, p-C6H5), 131.16 (d, 1JP−C = 61.0 Hz, ipso-C6H5), 129.17 (d, 2 JP−C = 11.7 Hz, m-C6H5), 110.42 (C5), 73.41 (d, 3JP−C = 8.4 Hz, Cp), 71.44 (d, 1JP−C = 66.3 Hz, ipso-Cp). 31P{1H} NMR (CDCl3) δ (ppm): 27.34. Anal. Calcd for C42H34Au2FeN4O2P2S2 (1202.6): C, 41.95; H, 2.85; N, 4.66; S, 5.33. Found: C, 41.90; H, 2.92; N, 4.63; S, 5.30. Cell Culture. Four different cell lines were cultivated: HEP-G2 cells, NIH-3T3 cells, PC-12 cells, and A-549 cells. HEP-G2 cells are human hepatocellular carcinoma cells (DMSZ no.: ACC 180). NIH3T3 cells are mouse fibroblasts (DMSZ no.: ACC 59). PC-12 cells are rat adrenal pheochromocytoma cells (DSMZ no.: ACC 159). A-549 cells are human lung carcinoma cells (DSMZ no.: ACC 107). All cell lines were cultivated in Dulbecco’s Modified Eagle’s medium (DMEM, D7777 Sigma-Aldrich, Steinheim, Germany) supplemented with the according serum (Table 3) and 1% antibiotics (penicillin/ 100U/mL and streptomycin/100 μg/mL) in a humidified environment at 37 °C/5% CO2. After 3−4 days the cells had grown to confluence and were then detached with trypsin and cultured in a new cell culture flask. Cultivation with Suspensions. Wells of a 96-well plate were seeded with a defined number of cells (see Table 3), depending on the

refined using a riding model. Refinements were carried out by fullmatrix least squares on F2 for all data.

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S Supporting Information *

CIF files giving X-ray crystallographic data for compounds 8 and 13. This material is available free of charge via the Internet at http://pubs.acs.org.

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DMEM additive

A-549 10% FCS (fetal calf serum) NIH-3T3 10% FCS PC-12 10% HOS, 5% NCS (newborn calf serum), 1% sodiumpyruvate, 1% L-glutamine HEP-G2 10% FCS

AUTHOR INFORMATION

Corresponding Author

*E-mail for M.C.G.: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Ministerio de Economı ́a y CompetitividadFEDER (CTQ2010-20500-C02-01) and DGA-FSE (E77) for financial support. This paper is dedicated to Prof. Dr. Marı ́a Pilar Garcı ́a for her valuable talent as teacher and as chemist. Thanks for being a wonderful person beyond the chemistry and now beyond the stars.

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REFERENCES

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Table 3. DMEM Additives and the Cell Numbers Per Well Used for the Experiments cell line

ASSOCIATED CONTENT

cell number per well 8000 6000 10000 10000

doubling times, and incubated for 3 days. At day 4 the medium of each well was removed and 200 μL of the different gold and silver complexes in varying concentrations in the cell culture medium was added to the wells (1−100 μM). The cells were cultivated for 3 days more, and then the viability of the cells was determined by the MTT assay. MTT. The cells were cultivated in 96-well plates. The viability of the cells was analyzed by the MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay (Sigma-Aldrich, Steinheim, Germany). This assay is based on the hydrolysis of the tetrazonium ring by mitochondrial dehydrogenase enzymes to an insoluble blue reaction product. To perform the MTT assay, at first medium had to be removed from each well. Afterward, 100 μL of fresh medium and 10 μL of MTT solution (5 mg/mL PBS, sterile) were added to each well and incubated for 4 h at 37 °C/5% CO2. Subsequently, 100 μL of 10% SDS (sodium dodecyl sulfate) in 0.01 M HCl was added and the plates were further incubated for 24 h. The transmission signals at 570/630 nm were determined using a microplate reader (Bio-Rad, München, Germany). Crystallography. Crystals were mounted in inert oil on glass fibers and transferred to the cold gas stream of a SMART APEX CCD diffractometer equipped with a low-temperature attachment. Data were collected using monochromated Mo Kα radiation (λ = 0.71073 Å) (scan type ω). Absorption corrections based on multiple scans were applied with the program SADABS.21 The structures were solved by direct methods and refined on F2 using the program SHELXL-97.22 All non-hydrogen atoms were refined anisotropically. In all cases, hydrogen atoms were included in calculated positions and 6077

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