Heteronuclear gold(I)–silver(I) sulfanylcarboxylates: Synthesis, structure and cytotoxic activity against cancer cell lines

Journal of Inorganic Biochemistry 131 (2014) 68–75

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Heteronuclear gold(I)–silver(I) sulfanylcarboxylates: Synthesis, structure and cytotoxic activity against cancer cell lines Elena Barreiro a, José S. Casas a, María D. Couce b,⁎, Agustín Sánchez a, José Sordo a,⁎, Ezequiel M. Vázquez-López b a b

Departamento de Química Inorgánica, Facultade de Farmacia, Universidade de Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain Departamento de Química Inorgánica, Facultade de Química, Universidade de Vigo, 36310 Vigo, Galicia, Spain

a r t i c l e

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Article history: Received 18 July 2013 Received in revised form 24 October 2013 Accepted 25 October 2013 Available online 1 November 2013 Keywords: Gold(I) complexes Silver(I) complexes Heteronuclear complexes Sulfanylpropenoic acids Cytotoxic activity

a b s t r a c t Heteronuclear complexes of the type [AgAu(PPh3)2(xspa)] [H2xspa = 3-(aryl)-2-sulfanylpropenoic acids; (x = 3-phenyl-; 3-(2-chlorophenyl)-; 3-(o-methoxyphenyl)-; 3-(p-methoxyphenyl)-; 3-(p-hydroxyphenyl)-; 3-(2-furyl)-; 3-(2-thienyl)-; spa = 2-sulfanylpropenoate)] were prepared by reacting the appropriate [Au(PPh3)(Hxspa)] precursor with Ag(PPh3)NO3. The compounds were characterized by spectroscopic methods, (IR; 1H, 13C and 31P NMR) and mass spectrometry and the structures of the phenyl and p-methoxyphenyl derivatives were determined by X-ray diffraction. The in vitro antitumor activity against the HeLa-229, A2780 and A2780cis cell lines was determined and compared with that of cisplatin and the equivalent homonuclear gold(I) complexes. © 2013 Elsevier Inc. All rights reserved.

1. Introduction Cisplatin and other platinum metallo-drugs are considerably effective for the treatment of cancer. However, side effects, the limited spectrum of tumors against which these drugs are active and the frequent development of drug resistance are negative and limiting factors for its clinical use [1–3]. At the same time as new platinum drugs and new delivery vehicles that are able to minimize these adverse effects are investigated, new alternatives based on other metals are defining a particularly interesting and active field [4]. Recent developments in cadmium [5], copper [6], palladium [7], ruthenium [8], silver [9], tin [10] or zinc [11] based drugs are significant examples of this activity. In addition, gold [Au(III) and Au(I)]-based drugs have been extensively studied [12,13]. Due to its electronic and structural similarity with those of Pt(II), Au(III) compounds have received predictable and significant attention [13,14]. But, on the other hand, Au(I) complexes exhibit cytotoxic activity toward cells from several tumor cell lines, some of which are resistant to cisplatin [12,13] thus the effect on the enzyme thioredoxin reductase is increasingly considered as the origin of this activity [15–20]. Among these gold(I) complexes the thiolate derivatives containing the S–Au–P fragment – where the antiarthritic drug Auranofin, triethylphosphine (2,3,4,6-tetra-o-acetyl-β,1-D-thiopyranosato-S)

⁎ Corresponding authors. Tel.: +34 981528074; fax: +34 981547102. E-mail addresses: delfi[email protected] (M.D. Couce), [email protected] (J. Sordo). 0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jinorgbio.2013.10.022

gold(I), which also shows significant cytotoxic activity, is included – were widely studied [21] and the modulation of its biological activity was related to the replacement of the thiolate ligand [22,23]. In previous papers we have explored the interaction of gold(I) with 3(aryl)-2-sulfanylpropenoic acids, R–CH–C(SH)–COOH, H2xspa, a class of sulfanylcarboxylic acids which, once deprotonated, have, besides the S-donor atom, the O-atoms of the carboxylic group as potential donor atoms capable of reinforcing the gold–sulfur bond. As a result of this study, mononuclear compounds of the types R–CH–C(SAuPPh3)– COOH, [Au(PPh3)(Hxspa)] [24], [HQ][R–CH–C(SAuPPh3)–COO], [HQ] [Au(PPh3)(xspa)], and [HP][R–CH–C(SAuPPh3)–COO], [HP][Au(PPh3) (xspa)] [25], (HQ = diisopropylammonium; HP = triethylammonium) and dinuclear compounds of the type {R–C[S(AuPPh3)2]–COO}, [(AuPPh3)2(xspa)] [26], were prepared and structurally characterized. All of these compounds but particularly the latter dinuclear ones showed promising activity against the cell lines of human cervix carcinoma HeLa229 and human ovarian carcinoma A2780, and also with its cisplatinresistant mutant A2780cis. The presence of different metallic centers in heterodi- or polynuclear complexes can give rise to an improved biological activity with respect to the equivalent homonuclear compounds. This can be due to their ability to interact with multiple biological targets or to their favorable physicochemical properties [27,28]. Promising heteronuclear Ti–Ru [27], Ti–Au [28], Ti–Au2 [29], Ag2Au [30] and Co–Sn [31] compounds have been recently described. In this line, we try to study the effect that the substitution of a gold(I) atom for a silver(I) atom would have on the structure and on the biological activity of the active homo-dinuclear [(AuPPh3)2(xspa)] complexes.

E. Barreiro et al. / Journal of Inorganic Biochemistry 131 (2014) 68–75

In a first attempt we synthesized the complex [AgAu(PPh3)2(cpa)] (H2cpa = 2-cyclopentilidene-2-sulfanylacetic acid) [32]. The structure of this compound proved to be dinuclear, resembling those of the [(AuPPh3)2(xspa)] complexes and containing S-bonded Ag(I) and Au(I) atoms placed at a short distance of 3.0463(10) Å, which can be compared to the Au–Au distance, 3.0338(10) Å, found in the equivalent dinuclear complex [(AuPPh3)2(cpa)]. The biological activity of this heteronuclear compound, even though better than that of cisplatin against the A2780 and A2780cis cell lines, was slightly worse than the equivalent homo-dinuclear [(AuPPh3)2(cpa)] compound. However, the situation could be different for complexes prepared from sulfanylcarboxylate ligands with a different R fragment due to their ability to modulate the hydro/ lipophilicity of the compounds, a property of great importance for drug action [22,23,33]. This fact, together with the lack of biological data for similar heteronuclear compounds led us to select the H2xspa sulfanylcarboxylic acids depicted in Scheme 1 and to prepare their derived heteronuclear [AgAu(PPh3)2(xspa)] complexes. The structural study of some of them revealed significant differences with the cpa derivative as the carboxylate group is now being involved in the formation of Ag\O bonds, which led to the formation of tetranuclear units, resembling those present in homonuclear silver(I) complexes previously described. This enables a comparative study on the structural changes caused by the replacement of silver(I) atoms for gold(I) atoms. The cytotoxic activity of these compounds against the HeLa-229, A2780 and A2780cis cell lines was investigated and compared with that of cisplatin and the equivalent homo-dinuclear [(AuPPh3)2(xspa)] complexes.

2. Experimental

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sulfanylcarboxylic acid and KOH in ethanol [24]. Ag(PPh3)NO3 was prepared in accordance to the literature [34]. Elemental analyses were performed with a Fisons 1108 microanalyzer. Melting points were determined with a Büchi apparatus. Mass spectra (MS) were recorded on a Kratos MS50TC spectrometer connected to a DS90 system and operating in FAB (fast atom bombardment) mode (m-nitrobenzyl alcohol, Xe, 8 eV; ca. 1.28 × 10−15 J), and positive electrospray ionization (in methanol) on a Hewlett-Packard 1100 LC/MSD spectrometer. Ions were identified by DS90 software and the data characterizing the metallated peaks were calculated using the isotope 197Au and assuming the Ag isotope to be 107Ag. IR spectra (KBr pellets or Nujol mulls) were recorded on a Bruker IFS66V FT-IR spectrometer and are reported in the Synthesis section using the following abbreviations: vs = very strong, s = strong, and m = medium. Fluorescence spectra were recorded in a trace-element X-ray fluorescence spectrometer (XRF1) with a sealed tube of 2.2 kW with a molybdenum anode. 1H and 13C NMR spectra in solution were recorded in DMSO-d6, at room temperature on a Bruker AMX 300 operating at 300.14 and 75.40 MHz, respectively, using 5 mm o.d. tubes; chemical shifts are reported relative to TMS (tetramethylsilane) using the solvent signal (δ1H = 2.50 ppm; δ13C = 39.50 ppm) as reference. The 1H–1H COSY (correlated spectroscopy) NMR spectra, 1 H–13C HMBC (heteronuclear multiple bond correlation) and HMQC (heteronuclear multiple quantum coherence) experiments were measured using a Varian Inova 400 spectrometer. 31P NMR spectra were recorded at 202.46 MHz on a Bruker AMX 500 spectrometer using 5 mm o.d. tubes and are reported relative to external H3PO4 (85%). NMR data were obtained from freshly prepared concentrated solutions. All the physical measurements were carried out by the RIAIDT services of the University of Santiago de Compostela.

2.1. Materials and methods Triphenylphosphine (from Riedel-de-Haën) and silver nitrate (from Prolabo) were used as supplied. Complexes of type [Au(PPh3)(Hxspa)] (x = p = 3-phenyl-; Clp = 3-(2-chlorophenyl)-; mp = 3-methoxyphenyl-; hp = 3-hydroxyphenyl-; f = 3-(2-furyl)-; t = 3-(2-thienyl)-; spa = 2-sulfanylpropenoate) were prepared by adding Au(PPh3)Cl in 1:1 mole ratio to a solution of the appropriate

2.2. Synthesis Complexes were prepared by adding solid Ag(PPh3)NO3 in 1:1 molar ratio to a methanol solution of the corresponding [Au(PPh3) (xspa)] complex, to which the appropriate amount of aqueous concentrated NaOH was previously added. After stirring the mixture in the

Scheme 1.

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E. Barreiro et al. / Journal of Inorganic Biochemistry 131 (2014) 68–75

dark at room temperature for 24 h the solution was reduced to about a quarter of its initial volume under vacuum and the solid formed was filtered, washed with water and dried under vacuum. 2.2.1. [AgAu(PPh3)2(pspa)] (1) [Au(PPh3)(Hpspa)] (0.10 g, 0.16 · 10−4 mol), Ag(PPh3)NO3 (0.068 g, 0.16 · 10−4 mol), NaOH (6.23 · 10−3 g, 0.16 · 10−4 mol), methanol (20 cm3), White solid. Yield 76%. M.p.: 180 °C. Anal.: found, C 53.9, H 3.6, S 3.1%. Calc. for C45H36O2SP2AuAg: C 53.6, H 3.6, S 3.2%. MS (FAB): m/z 1288 (2%), [(AgPPh3)3pspa]+; 1007 (5), [M]+; 919 (31), [(AgPPh3)2(pspa)]+; 721 (88), [(PPh3)2Au]+; 639 (2), [Au(PPh3) (Hpspa)]+; 633 (100), [(PPh3)2Ag]+; 550 (2), [(AgPPh3)(Hpspa)]+; 459 (21), [(PPh3)Au]+ and 369 (59), [(PPh3)Ag]+. IR (cm−1): 1537 vs, νas(COO); 1348 vs, νsym(COO); Δν 189; 1480 s, 1435 vs, ν(PPh3). NMR (DMSO-d6): 1H, δ 7.72 (s, 1H, C(3)H), 7.91 (d, 2H, C(5)H, C(9) H), 7.18 (d, 1H, C(7)H), 7.32–7.48 (m, 32H,C(6)H, C(8)H, H(PPh3)); 13 C, δ 169.6 C(1), 128.8 C(2), 136.9 C(3), 135.1 C(4), 133.9 C(5) and C(9), 127.1 C(6), C(8), 129.4 C(7), 133.4 (d, Co(Ph3), J = 14.4), 129.1 (d, Cm(Ph3), J = 10.4), 131.4 Cp(Ph3); 31P {1H}: δ 19.1 (s); 40.0 (s). Suitable crystals for X-ray diffraction were obtained from an acetone solution. 2.2.2. [AgAu(PPh3)2(Clpspa)] (2) [Au(PPh3)(HClpspa)] (0.10 g, 0.15 · 10−4 mol), Ag(PPh3)NO3 (0.064 g, 0.15 · 10−4 mol), NaOH (6.23 · 10−3 g, 0.15 · 10−4 mol), methanol (15 cm3), pale yellow solid. Yield: 61%. M.p.: 165 °C. Anal.: found, C 52.0, H 3.4, S 3.9%. Calc. for C45H35O2SP2AuAgCl: C 51.8, H 3.3, S 3.1%. MS (FAB): m/z 1042 (11%), [M]+; 721 (100), [(PPh3)2Au]+; 633 (1), [(PPh3)2Ag]+; 459 (28), [(PPh3)Au]+ and 369 (5), [(PPh3) Ag]+. IR (cm−1): 1539 s, νas(COO); 1356 vs, νsym(COO); Δν 189; 1478 s, 1434 vs, ν(PPh3). NMR (DMSO-d6): 1H, δ 7.78 (s, 1H, C(3)H), 7.17 (d, 1H, C(6)H), 7.07 (st, 1H, C(7)H), 7.30 (m, 1H, C(8)H), 8.22 (d, 1H, C(9)H), 7.37–7.56 (m, 30H, H(PPh3)); 13C, δ 169.6 C(1), 126.1 C(2), 132.9 C(3), 136.0 C(4), 130.0 C(5), 128.8 C(6), 129.5 C(7), 128.3 C(8), 128.4 C(9), 133.6 (d, Co(Ph3), J = 15.2), 129.2 (d, Cm(Ph3), J = 10.7), 131.4 Cp(Ph3); 31P {1H}: δ 19.0 (s), 33.5 (s). Although the elemental analysis value for “S” was somewhat unsatisfactory, the MS (FAB) result and other spectroscopic analysis data (IR, 1H and 13C NMR) reasonably support the formula.

1

H, δ 7.72 (s, 1H, C(3)H), 7.96 (d, 1H, C(5)H, C(9)H), 6.90 (d, 1H, C(6)H, C(8)H), 3.71 (s, 3H, OCH3), 7.30–7.56 (m, 30H, H(PPh3)); 13 C, δ 170.6 C(1), 129.7 C(2), 135.2 C(3), 130.0 C(4), 131.7 C(5) and C(9), 113.3 C(6) and C(8), 158.5 C(7), 55.1 C(OCH3), 133.6 (d, Co(Ph3), J = 15.5), 129.3 (d, Cm(Ph3), J = 11.6), 131.4 Cp(Ph3); 31P {1H}: δ 27.2 (s), very broad signal. Crystals of [AgAu(PPh3)2p-mpspa]·2/ 3C3H6O (4·2/3C3H6O) suitable for X-ray diffraction were obtained by slow evaporation of an acetone solution of 4. 2.2.5. [AgAu(PPh3)2(p-hpspa)] (5) [Au(PPh3)(H-p-hpspa)] (0.10 g, 0.15 · 10−4 mol), Ag(PPh3)NO3 (0.065 g, 0.15 · 10−4 mol), NaOH (6.23 · 10−3 g, 0.15 · 10−4 mol), methanol (15 cm3), pale yellow solid. Yield: 55%. M.p.: 190 °C. Anal.: found, C 52.4, H 3.6, S 2.9%. Calc. for C45H36O3SP2AuAg: C 52.7, H 3.5, S 3.1%. MS (FAB): m/z 934 (3%), [(AgPPh3)2-p-hpspa]+; 721 (43), [(PPh3)2Au]+; 633 (4), [(PPh3)2Ag]+; 459 (10), [(PPh3)Au]+ and 369 (4), [(PPh3)Ag]+. IR and Raman (cm− 1): 1569 m, νas(COO); 1355 vs, νsym(COO); Δν 214; 1480 m, 1436 s, ν(PPh3). NMR (DMSOd6): 1H, δ 7.62 (s, 1H, C(3)H), 7.86 (d, 1H, C(5)H, C(9)H), 6.76 (d, 1H, C(6)H, C(8)H), 9.66 (s, 1H, C(7)OH), 7.33–7.53 (m, 30H, H(PPh3)); 13C and 31P NMR spectra were not recorded due to the low solubility of the compound. 2.2.6. [AgAu(PPh3)2(fspa)] (6) [Au(PPh3)(Hfspa)] (0.09 g, 0.14 · 10−4 mol), Ag(PPh3)NO3 (0.065 g, 0.14 · 10−4 mol), NaOH (5.60 · 10−3 g, 0.14 · 10−4 mol), methanol (15 cm3), brown solid. Yield: 64%. M.p.: 150 °C. Anal.: found, C 52.0, H 3.8, S 3.0%. Calc. for C43H34O3SP2AuAg: C 51.8, H 3.5, S 3.2%. MS (FAB): m/z 1457 (2%), [AgAu2(PPh3)3fspa]; 1409 (1), [S(AuPPh3)3]+; 1087 (1), [(AuPPh3)2fspa]+; 998 (10), [M]+; 909 (2), [(AgPPh3)2fspa]+; 721 (100), [(PPh3)2Au]+; 633 (2), [(PPh3)2Ag]+; 459 (34), [(PPh3)Au]+ and 369 (9), [(PPh3)Ag]+. IR (cm−1): 1548 m, νas(COO); 1350 m, νsym(COO); Δν 198; 1480 s, 1435 vs, ν(PPh3). NMR (DMSO-d6): 1H, δ 7.68 (s, 1H, C(3) H), 6.60 (t, 1H, C(6)H), 7.69 (d, 1H, C(7)H), 7.36–7.56 (m, 31H, C(5)H, H(PPh3)); 13C, δ 169.1 C(1), 128.6 C(2), 128.7 C(3), 152.8 C(4), 112.1 C(5), 111.7 C(6), 142.5 C(7), 133.6 (d, Co(Ph3), J = 15.3), 129.3 (d, Cm(Ph3), J = 10.7), 131.5 Cp(Ph3); 31P {1H}: δ 12.7 (s), 35.0 (bs). 2.2.7. [AgAu(PPh3)2(tspa)] (7) [Au(PPh3)(Htspa)] (0.10 g, 0.15 · 10−4 mol), Ag(PPh3)NO3 (0.067 g, 0.15 · 10−4 mol), NaOH (6.20 · 10−3 g, 0.15 · 10−4 mol), methanol (15 cm3), yellow solid. Yield: 70%. M.p.: 165 °C. Anal.: found, C 50.3, H 3.5, S 6.6%. Calc. for C43H35O2S2P2AuAg: C 50.1, H 3.5, S 6.3%. MS (FAB): m/z 1473 (2%), [AgAu2(PPh3)3tspa]; 1409 (1), [S(AuPPh3)3]+; 1103 (3), [(AuPPh3)2tspa]+; 1014 (3), [M]+; 721 (100), [(PPh3)2Au]+; 459 (41), [(PPh3)Au]+ and 369 (6), [(PPh3)Ag]+. IR (cm−1): 1546 vs, νas(COO); 1356 vs, νsym(COO); Δν 190; 1479 s, 1435 vs, ν(PPh3). NMR (DMSO-d6): 1H, δ 8.04 (s, 1H, C(3)H), 7.11 (t, 1H, C(6)H), 7.34–7.60 (m, 32H, C(5)H, C(7)H, H(PPh3)); 13C, δ 169.5 C(1), 126.3 C(2), 130.8 C(3), 141.4 C(4), 129.8 C(5), 127.5 C(6), 129.0 C(7), 133.7 (d, Co(Ph3), J = 14.2), 129.4 (d, Cm(Ph3), J = 10.3), 131.7 Cp(Ph3); 31P {1H}: δ 16.3 (s), 35.5 (s).

2.2.3. [AgAu(PPh3)2(o-mpspa)] (3) [Au(PPh3)(H-o-mpspa)] (0.10 g, 0.15 · 10−4 mol), Ag(PPh3)NO3 (0.065 g, 0.15 · 10−4 mol), NaOH (6.23 · 10−3 g, 0.15 · 10−4 mol), methanol (15 cm3), pale orange solid. Yield: 66%. M.p.: 183 °C. Anal.: found, C 53.8, H 3.9, S 3.0%. Calc. for C46H38O3SP2AuAg: C 53.2, H 3.7, S 3.1%. MS (FAB): m/z 1127 (1%), [(AuPPh3)2-o-mpspa]+; 1038 (14), [M]+; 949 (2), [(AgPPh3)2-o-mpspa]+; 721 (100), [(PPh3)2Au]+; 633 (3), [(PPh3)2Ag]+; 459 (37), [(PPh3)Au]+ and 369 (12), [(PPh3)Ag]+. IR (cm−1): 1542 s, νas(COO); 1354 vs, νsym(COO); Δν 188; 1480 s, 1435 vs, ν(PPh3). RMN (DMSO-d6): 1H, δ 7.89 (s, 1H, C(3)H), 8.24 (d, 1H, C(6)H), 7.18 (t, 1H, C(7)H), 6.94 (d, 1H, C(9)H), 3.72 (s, 3H, OCH3), 7.30–7.56 (m, 31H, C(8)H, H(PPh3)); 13C, δ 170.2 C(1), 128.6 C(2), 133.8 C(3), 126.1 C(4), 157.2 C(5), 110.6 C(6), 130.4 C(7), 119.4 C(8), 129.9 C(9), 55.3 C(OCH3), 133.5 (d, Co(Ph3), J = 15.5), 129.2 (d, Cm(Ph3), J = 11.6), 131.3 Cp(Ph3); 31P {1H}: δ 15.2 (s), 37.7 (s).

2.3. Crystallography

2.2.4. [AgAu(PPh3)2(p-mpspa)] (4) [Au(PPh3)(H-p-mpspa)] (0.10 g, 0.15 · 10−4 mol), Ag(PPh3)NO3 (0.065 g, 0.15 · 10−4 mol), NaOH (6.23 · 10−3 g, 0.15 · 10−4 mol), methanol (15 cm3), pale orange solid. Yield: 63%. M.p.: 165 °C. Anal.: found, C 52.9, H 3.9, S 2.9%. Calc. for C46H38O3SP2AuAg: C 53.2, H 3.7, S 3.1%. MS (FAB): m/z 1127 (1%), [(AuPPh3)2-p-mpspa]+; 1038 (11), [M]+; 949 (2), [(AgPPh3)2-p-mpspa]+; 721 (100), [(PPh3)2Au]+; 633 (4), [(PPh3)2Ag]+; 459 (33), [(PPh3)Au]+ and 369 (11), [(PPh3)Ag]+. IR and Raman (cm− 1): 1568 m, 1568 s (R), νas(COO); 1350 vs, νsym(COO); Δν 218; 1480 m, 1436 s, ν(PPh3). NMR (DMSO-d6):

2.3.1. X-ray data collection and reduction Single crystals of [AgAu(PPh3)2(pspa)] (1) and [AgAu(PPh3)2 (p-mpspa)]·2/3C3H6O (4·2/3C3H6O) were mounted on glass fibers for data collection in a Bruker Smart CCD automatic diffractometer at 293 K using Mo-Kα radiation (λ = 0.71073 Å). Table 1 summarizes the crystal data, experimental details and refinement results. Corrections for Lorentz effects, polarization [35] and absorption [36] were made. The structures were solved by direct methods and refined using SHELX-97 [37]. Atomic scattering factors and corrections for anomalous dispersion for all atoms were made based on Ref. [38].

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Table 1 Crystal data for [AgAu(PPh3)2(pspa)] (1), [AgAu(PPh3)2(p-mpspa)]·2/3C3H6O (4·2/3C3H6O). Compound

[AgAu(PPh3)2(pspa)] (1)

[AgAu(PPh3)2(p-mpspa)]·2/3C3H6O (4·2/3C3H6O)

Empirical formula M T/K Crystal system Space group a/Å b/Å c/Å α/° β/° γ/° V/Å3 Z Dc/mg·m−3 μ/mm−1 Crystal size/mm−3 θ range for data collection/Ί Index ranges Reflections collected Unique reflections, R Goodness-of-fit on F2 Final R1, wR2 [I N 2σ(I)] (All data)

C45H36AuAgO2P2S 1007.57 293(2) Monoclinic P2(1)/c 14.2309(14) 16.2755(15) 18.5868(18)

C46H38AgAuO3P2S 1037.60 293(2) Triclinic P-1 12.742(2) 13.678(3) 15.548(3) 107.639(3) 98.522(4) 113.571(4) 2251.5(7) 2 1.531 3.843 0.09 × 0.13 × 0.22 1.44–28.36 −16 ≤ h ≤13, −17 ≤ k ≤17, −20 ≤ l ≤20 12197 8761, [R(int) = 0.1451] 0.851 0.0791, 0.1872 0.1653, 0.2193

111.578(2) 4003.3(7) 4 1.672 4.318 0.16 × 0.11 × 0.10 1.54–28.06 −18 ≤ h ≤15, −21 ≤ k ≤15, −22 ≤ l ≤24 21998 9002, [R(int) = 0.0903] 0.790 0.0622, 0.1107 0.2013, 0.1368

The ΔF map in 4·2/3C3H6O showed a diffuse electron density area of around 341 Å3 and high values of the R-factors (mainly the R1 values with 0.094 and 0.187 for Fo N 4σ(Fo) and all data, respectively). Attempts to introduce different models of disordered solvent (acetone) were unsuccessful. Consequently, reflection data for this compound were corrected for diffuse scattering by means of the program SQUEEZE [39]. The correction of electron count per cell (47 e) besides the void volume aforementioned, agrees with the presence of 2/3 acetone per asymmetric unit. The contribution of the disordered acetone was also omitted from the data included in Table 1.

was able to inhibit cell growth by 50% with respect to controls, IC50, was then determined by semi-logarithmic dose–response sigmoid curves using GraphPad Prism Ver. 2.01 software (GraphPad Software Inc.). The cytotoxicity of the free ligands and that of cisplatin (dissolved in water) was evaluated for comparison purposes under the same experimental conditions. All compounds were tested in three independent experiments with quadruplicate points. Results were expressed as mean ± S.E. In order to test for differences between the appropriate complexes a Student's T test was used (P b 0.05). The in vitro studies were performed in the Unit for the evaluation of pharmacological activities of chemical compounds of the USC.

2.4. In vitro antitumor activity 2.4.1. Cell line and growth conditions The human cervix carcinoma cell line HeLa-229 used in this study was kindly provided by Dr. Guadalupe Mengod (CSIC-IDIBAPS of Barcelona, Spain), human ovarian cancer cell line A2780 and its cisplatin-resistant mutant A2780cis were obtained from the European Collection of Cell Cultures through Sigma-Aldrich. The cells were grown in Dulbecco's modified Eagle's medium (DMEM, HeLa-229) or RPMI 1640 medium (A2780, A2780cis) supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine. Cells were maintained in continuous logarithmic culture in a humidified atmosphere of 5% CO2 at 37 °C and were harvested using trypsin-ethylenediaminetetraacetic acid. All media and supplements were purchased from Sigma-RBI, Spain. 2.4.2. In vitro chemosensitivity assay The cells were seeded into 96-well plates (Beckton-Dickinson, Spain) in a volume of 100 μL in a number of 4000 cells/well and were incubated for 4–6 h (HeLa-229) or 24 h (A2780, A2780cis) prior to dosage. Solutions of the heteronuclear complexes in ethanol were added to the cells using the same concentration of ethanol per well (1%). After the appropriate incubation time, i.e. 48 h for HeLa-229 and 96 h for A2780 and A2780cis, the cells were fixed by adding 10 μL of 11% glutaraldehyde per well for 15 min. The fixative was then removed and the wells were washed four times with distilled water. Cell biomass was determined by a crystal violet staining technique [40] and the optical density was measured at 595 nm with a Tecan Ultra Evolution microplate reader. Each complex was tested using six or seven consecutive dilutions ranging from 50 μM to 0.025 μM. The compound concentration that

3. Results and discussion In order to confirm that the formation of heteronuclear Au–Ag complexes takes place, the reaction between [Au(PPh3)(Hpspa)] and Ag(PPh3)NO3 was monitored by ESI/MS. Ag(PPh3)NO3 was added to a solution of [Au(PPh3)(Hpspsa)] and NaOH in methanol. Electrospray spectra were recorded at different times (1 h after the mixture, 3, 24, 48, 72 h, 6 days and one week) at 20, 40, 60, 80 and 100 V in order to identify new species in the reaction media. A buffer solution containing 1% of formic acid was used. All the spectra show the signal of the [AgAu(PPh3)2(pspsa)] complex, and, also, different signals of fragments containing Ag, Au, or both. Having proved that the reaction takes place under these conditions, the synthesis of [AgAu(PPh3)2(xspa)] complexes was accomplished as described in the Experimental section and the presence of both metals in the isolated solids was confirmed by X-ray fluorescence and chemical analysis. The complexes are soluble in acetone, ethanol, methanol, DMSO and chloroform, but are insoluble in ether and water, a behavior similar to that previously found for the equivalent di-homonuclear [(AuPPh3)2(xspa)] complexes [26]. The structural study of [AgAu(PPh3)2(pspa)] (1) and [AgAu(PPh3)2 (p-mpspa)]·2/3C3H6O (4·2/3C3H6O) showed the presence of tetranuclear units, which was in contrast with the presence of dinuclear units in the previously described [AgAu(PPh3)2(cpa)] complex [32] but in line with the results obtained for similar silver(I) sulfanylcarboxylates, among which, the silver(I) p-mpspa derivative [(AgPPh3)2(p-mpspa)] was included [41]. The existence of this complex enables a structural comparison among the heteronuclear gold(I)–silver(I) and the homonuclear silver(I) derivatives of the p-mpspa ligand.

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P Au

S

O

P Ag

OO

Ag P

O S

Au P Scheme 2.

Fig. 1. Structure of [AgAu(PPh3)2(pspa)] (1), (for clarity, hydrogen atoms were omitted and triphenylphosphine phenyl rings are represented by their P-bound ipso carbons).

3.1. Structural characterization Fig. 1 shows the structure and numbering scheme of the complex [AgAu(PPh3)2(pspa)] (1) and Table 2 lists their most significant structural parameters (phenyl rings of the PPh3 ligand have been omitted Table 2 Selected interatomic distances (Å) and angles (°) for (1) and (4·2/3C3H6O). 1

4·2/3C3H6O

(a). Au and Ag environment Au(1)–P(1) Au(1)–S(1) Au(1)–O(1) Au(1)–O(2)#1 Au(1)–Ag(1)#1 Ag(1)–O(1) Ag(1)–P(2) Ag(1)–O(2) S(1)–C(2) S(1)–Ag(1)#1 P(1)–Au(1)–S(1) P(1)–Au(1)–O(1) S(1)–Au(1)–O(1) O(1)–Ag(1)–P(2) O(1)–Ag(1)–O(2) P(2)–Ag(1)–O(2) O(1)–Ag(1)–S(1)#1 P(2)–Ag(1)–S(1)#1 O(2)–Ag(1)–S(1)#1 C(2)–S(1)–Au(1) C(2)–S(1)–Ag(1)#1 Au(1)–S(1)–Ag(1)#1 C(1)–O(1)–Ag(1) C(1)–O(1)–Au(1) Ag(1)–O(1)–Au(1) C(1)–O(2)–Ag(1)

2.251(3) 2.337(3) 2.570(7) 3.322(7) 3.2610(10) 2.664(7) 2.365(3) 2.486(7) 1.779(11) 2.445(3) 164.42(11) 117.83(17) 77.18(17) 111.61(16) 50.5(2) 105.48(18) 99.49(16) 143.15(11) 109.52(17) 104.2(4) 101.2(3) 85.97(9) 88.0(6) 116.4(7) 155.1(3) 97.0(7)

2.282(4) 2.364(3) 2.485(8) 3.046(10) 3.4293(14) 2.337(8) 2.375(3) 2.558(9) 1.744(12) 2.558(4) 164.01(13) 114.4(2) 78.0(2) 128.8(3) 52.1(3) 116.9(2) 101.3(2) 128.69(12) 101.3(2) 103.9(5) 101.0(4) 88.23(11) 99.4(8) 116.3(8) 142.3(4) 88.2(8)

(b). Ligand O(1)–C(1) O(2)–C(1) C(1)–C(2) C(2)–C(3) C(3)–C(4) O(1)–C(1)–O(2) O(1)–C(1)–C(2) O(2)–C(1)–C(2) C(3)–C(2)–S(1) C(1)–C(2)–S(1) Symmetry transformations used to generate equivalent atoms: #1=

1.263(11) 1.232(11) 1.494(14) 1.338(12) 1.463(15) 123.7(10) 118.2(10) 118.1(10) 119.5(9) 122.0(8) −x + 1, −y + 1,−z

1.235(16) 1.260(15) 1.518(18) 1.341(17) 1.497(19) 119.9(13) 121.0(11) 119.1(12) 125.1(11) 118.9(9) −x,−y, −z + 2

for clarity). The crystal contains tetranuclear units, [AgAu(PPh3)2(pspa)]2, the core of which can be described as based on an eight membered ring Ag2Au2O2S2 in which each Ag and Au atoms are additionally coordinated by a P atom and the Ag atom by a second O atom (Scheme 2). In each unit the gold atom and the silver atom have different coordinative environments. The gold atom is three coordinated, to P [Au–P(1): 2.251(3) Å], to S [Au–S(1): 2.337(3) Å] and to one of the carboxylate O atoms of the same pspa fragment to which the S belongs [Au–O(1): 2.570(7) Å]. Furthermore, an O atom of the carboxylate group from the other pspa fragment is placed at a distance of 3.322(7) Å. Whereas in the case of P, S and O(1) the bond distance is slightly shorter or only slightly exceeds, [O(1)], the sum of the covalent radii of the involved atoms [42]. In this last case the value significantly exceeds the sum and also slightly exceeds the sum of the van der Waals radii of both atoms, (3.20 Å) [43], thus suggesting the non-existence of Au– O(2)#1 interaction. Additional support for this proposition is the planarity of the AgP(1)SO(1) fragment; this fragment is quasi-planar (r.m.s.: 0.0311) and the Au atom is only at 0.0524 Å of the best least-square plane. The Ag atom is four coordinated to P [Ag–P(2): 2.365(3) Å], to the S atom coordinated to Au [Ag–S: 2.445(3) Å], and to both the carboxylate O atoms from the other pspa moiety in the unit [Ag–O(2): 2.486(7) Å; AgO(1): 2.664(7) Å]. As in the case of the Au atom, the Ag–P and Ag–S bond distances are shorter than the sum of the covalent radii, but the Ag–O distance slightly exceeds this sum. The Ag–Au distance is 3.2610(10) Å, shorter than the sum of the van der Waals radii for both atoms (3.40 Å) [43]. Fig. 2 shows the structure and numbering scheme for [AgAu(PPh3)2 (p-mpspa)]·2/3C3H6O (4·2/3C3H6O) and Table 2 lists their most significant structural parameters (phenyl rings of the PPh3 ligand have again been omitted for clarity). As in 1, the crystal contains tetranuclear units, [AgAu(PPh3)2(p-mpspa)]2 in this case. The structure of this unit is similar to that described for 1, with the Au and Ag atoms placed in similar coordinative environments, although there are slight differences in the bond distances and angles as Table 2 shows. The distances have been included in Scheme 3 to be illustratively compared; as can be seen in the Au environment, the Au–S bond distances are similar in both structures but AuO(1) is shorter in 4·2/3C3H6O than in 1. Furthermore, in 4·2/3C3H6O, the O(2) atom is placed at a shorter distance from the Au atom than in 1, making it now lower than the sum of the van der Waals radii of both atoms and indicative of a weak interaction. Accordingly, the planarity of the AuPSO(1) plane (r.m.s.: 0.0767, the Au atom is now located at 0.1289 Å from the best least-square plane) shows the slight influence of this O atom on the Au environment. In the Ag environment, the significant differences refer to the Ag–S and Ag–O(2) distances, which are longer in 4·2/3C3H6O, whereas Ag–O(1) is longer in 1. The Ag–Au distance in 4·2/3C3H6O is longer than in 1, and exceeds the sum of the van der

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Fig. 2. Structure of [AgAu(PPh3)2(p-mpspa)]·2/3C3H6O (4·2/3C3H6O), (for clarity, hydrogen atoms were omitted and triphenylphosphine phenyl rings are represented by their P-bound ipso carbons).

Waals radii for both atoms. Although not included in Scheme 3, there are also differences in the interatomic angles between the two structures. These differences are more significant in the Ag environment. Note, for example, the remarkable difference in the P(2)–Ag(1)–S(1)#1 angle (143.15(11) in 1 vs. 128.69(12)° in 4·2/3C3H6O) (Table 2). The structures of [AgAu(PPh3)2(pspa)] (1) and [AgAu(PPh3)2 (p-mpspa)]·2/3C3H6O (4·2/3C3H6O) can be illustratively compared to that of [AgAu(PPh3)2(cpa)] complex [32], a previously described example of S-supported Ag–Au heteronuclear complex, to analyze the effect of the change of the sulfanylcarboxylate ligand. The main difference between the structures refers to the nuclearity. The crystal of [AgAu(PPh3)2(cpa)] contains dinuclear units with the Ag and Au placed in different coordinative environments. Au is essentially S- and P-bonded, whereas Ag shows, besides equivalent S- and P-bonds, additional O-bonds with one of the O atoms of the carboxylate group of the cpa ligand. The tetranuclear unit found in this case can be seen as formed by two mutually inverted [AgAu(PPh3)2(pspa)] or [AgAu(PPh3)2 (p-mpspa)] dinuclear units which, by using the two O-donor atoms (Ag-coordinated and uncoordinated) of the carboxylate group to coordinate the silver of the other unit, collapse to form the tetranuclear unit. A second relevant comparison for these structures can be made with those complexes containing the same ligand but two Ag atoms instead of a Ag and Au atom. This comparison enables us to analyze the effect

of the substitution of a Ag atom for a Au atom. Thus, a comparison of the structures of [AgAu(PPh3)2(p-mpspa)]·2/3C3H6O with that of [(AgPPh3)2(p-mpspa)] [41] reveals that the more significant changes are those of the fragment close to the point where the Ag/Au substitution was produced. Thus, whereas the Ag–P(2), Ag–S, Ag–O(1) and Ag–O(2) distances are similar, Ag–P(1) and Ag–S are longer than Au–P(1) and Au–S, though Ag–O(1) is shorter than Au–O(1). In the IR spectra of the complexes, the SH bands disappear and the bands due to the CO2H group in the free ligand are strongly modified due to deprotonation and coordination. The asymmetric carboxylate band lies around 1550 cm−1 and the symmetric band lies around 1350 cm−1, the parameter νas(COO)–νsym(COO) being close to 200. The position of these two bands is close in all the complexes and also close to those found in similar tetranuclear silver(I) sulfanylcarboxylates for which a carboxylate group coordinated as in this case was also identified by X-ray diffraction [41]. Therefore, and on this basis, we suggest a similar tetranuclear structure for all the complexes described in this paper. In the 1H NMR spectra of the complexes, the CO2H and SH signals of the free ligands are absent according to their deprotonation and the shift of the C(3)H signal to higher field is in keeping with the S-coordination found in the solid state for the heteronuclear complexes 1 and 4·2/3C3H6O. The 13C spectra show the signal of C(3) to be more shielded in the complexes than in the free ligands, which corroborates the persistence of the S-coordination in solution. The position of C(1) suggests that the O-coordination also persists in solution. However, previous results on silver(I) sulfanylcarboxylates [41] advise against a definite conclusion regarding the permanence of the tetranuclear unit in this media. The 31P NMR spectra of the heteronuclear complexes, except in 4 where a very broad signal is present, consist of two single

Table 3 In vitro cytotoxicity against the HeLa-229 cell line expressed as IC50 (μM) for [(AuPPh3)2 (xspa)] and [AgAu(PPh3)2(xspa)] complexes.

Scheme 3.

Ligand (H2xspa)

[(AuPPh3)2(xspa)]

[AgAu(PPh3)2(xspa)]

H2pspa H2Clpspa H2-o-mpspa H2-p-mpspa H2-p-hpspa H2fspa H2tspa Cisplatin

1.5(0.2) 2.7(0.4) 1.5(0.2) 2.4(0.4) 1.5(0.3) 2.6(0.3) 2.4(0.3) 0.53(0.06)

9.5(0.1)a 13.0(1.0)a 2.5(0.02) 1.1(0.01) 29.0(3.0)a 2.5(0.01) 13.0(1.0)a

a

Significant with respect to the [(AuPPh3)2(xspa)] complexes (P b 0.05).

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Table 4 In vitro cytotoxicity against the A2780 and A2780cis cell line expressed as IC50 (μM) for [(AuPPh3)2(xspa)] and [AgAu(PPh3)2(xspa)] complexes. The resistance factor, RF, is expressed as IC50(A2780cis)/IC50(A2780). Ligand (H2xspa)

H2pspa H2Clpspa H2-o-mpspa H2-p-mpspa H2-p-hpspa H2fspa H2tspa Cisplatin a

[(AuPPh3)2(xspa)]

[AgAu(PPh3)2(xspa)]

A2780

A2780cis

RF

A2780

A2780cis

RF

0.62(0.08) 1.06(0.08) 1.01(0.06) 1.3(0.4) 1.9(0.5) 1.1(0.3) 0.42(0.06) 0.44(0.06)

0.77(0.09) 2.8(0.5) 1.2(0.5) 1.6(0.5) 3.6(0.7) 1.5(0.4) 0.91(0.1) 3.6(0.5)

1.24 2.64 1.18 1.23 1.89 1.36 2.16 8.18

1.6(0.02) 3.3(0.1)a 0.5(0.01) 0.8(0.01) 0.8(0.06)a 1.4(0.07) 5.1(0.02)a

6.5(0.1)a 1.0(0.04)a 0.9(0.01) 0.8(0.01) 2.7(0.02) 0.5(0.03) 4.1(0.07)a

4.06 0.3 1.8 1.0 3.3 0.3 0.8

Significant with respect to the [Au(PPh3)2(xspa)] complexes (P b 0.05).

signals placed around 14 and 35 ppm. Both signals, on the basis of their position in other silver(I)- [41], gold(I)- [26] and heteronuclear gold(I)– silver(I) sulfanylcarboxylates [32] were attributed, respectively, to the PPh3 unit of the Ag-PPh3 and Au-PPh3 fragments.

3.2. Cytotoxicity in human cervix carcinoma and human ovarian carcinoma Human HeLa-229 cervix carcinoma cells and human ovarian carcinoma A2780 cells, together with their cisplatin-resistant mutant A2780cis, were used to assess the effect of the exchange of a AuPPh3 group for a AgPPh3 group on the previously studied [26] cytotoxicity of [(AuPPh3)2(xspa)] complexes. As reported previously [24], the ligands are inactive apart from (H2tspa), which has a very low activity against the HeLa culture cell. The IC50 values for the complexes against the three lines are shown in Tables 3 and 4. These values are represented in Figs. 3, 4 and 5. For comparative purposes, the values for the homonuclear equivalent gold(I) complexes were also included. It can be seen that the effect of the exchange of a AuPPh3 group for a AgPPh3 group on the activity of the dinuclear gold(I) complexes against the three cell cultures is different. Against the HeLa cells, in general the least sensitive of the three, the IC50 values of the [AgAu(PPh3)2(xspa)] complexes, irrespective of the R substituent, are equivalent or even worse than those of the [(AuPPh3)2(xspa)] complexes and also significantly worse than the value for cisplatin. The [AgAu(PPh3)2(xspa)] complexes showed significant activity against the A2780 cell line but only for the p-hpspa derivative was the IC50 value significantly reduced with respect to the equivalent gold(I) homonuclear complex. Similar or worse values were found in the other cases which also showed no increased activity when compared with cisplatin.

Fig. 3. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [AgAu(PPh3)2(xspa)] against the HeLa-229 cell line.

Fig. 4. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [AgAu(PPh3)2(xspa)] against the A2780 cell line.

Against the A2780cis cell line, the IC50 values for the [(AuPPh3)2 (xspa)] complexes are equivalent (p-hpspa derivative) or better than that of cisplatin. In this case, only the [AgAu(PPh3)2(Clpspa)] complex proved to be significantly more active than the equivalent homonuclear gold(I) complex. However, among the seven [AgAu(PPh3)2(xspa)] complexes assayed against this cell line, four (the Clpspa, o-mpspa, p-mpspa and fspa derivatives) are significantly more active than cisplatin as they are able to avoid the multifactorial resistance mechanism [44] shown by this line against cisplatin and other platinum anticancer agents. The resistance factor (RF = IC50A2780cis/IC50A2780) values listed in Table 4 are evidence of this ability. In most of the cases, these values are better than those of the [(AuPPh3)2(xspa)] complexes and in all of them the values are significantly better than that of cisplatin. In summary, the introduction of a AgPPh3 group in substitution of a AuPPh3 group in the highly active [(AuPPh3)2(xspa)] complexes leads to an increase in the activity only in some specific cases. Further structural modifications involving the phosphine ligand are being carried out on these homo and heteronuclear complexes, in search of a better behavior against these and other cellular lines. 4. Conclusions We have synthesized several heteronuclear compounds involving Ag(I) and Au(I) centers. The complexes are of the type [AgAu(PPh3)2 (xspa)], where xspa is a 3(aryl)-2-sulfanylpropenoato fragment. The molecular structures show the tetranuclear nature of the compounds and the presence of Au(I) centers, essentially S,O,P tricoordinated and Ag(I) centers, essentially S,O,O,P tetracoordinated. The screening of the cytotoxic activity against the HeLa-229, A2780 and A2780cis lines shows that the compounds are highly effective, particularly against

Fig. 5. The in vitro cytotoxicity of complexes [(AuPPh3)2(xspa)] and [AgAu(PPh3)2(xspa)] against the A2780cis cell line.

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the A2780cis line, showing an ability to circumvent the cellular resistance to cisplatin. When this ability is compared to that of the equivalent homodinuclear [(AuPPh3)2(xspa)] complexes, only in some cases do we observe a better behavior of the heteronuclear complexes. Acknowledgments We thank the Dirección Xeral de I + D, Xunta de Galicia, Spain, (IN845B-2010/121) for the support. Appendix A. Supplementary data Crystallographic data for the structures of [AgAu(PPh3)2(pspa)] (1) and [AgAu(PPh3)2(p-mpspa)]·2/3C3H6O (4·2/3C3H6O) have been deposited at the Cambridge Crystallographic Data Centre as supplementary publication Nos. CCDC 948797–948798 respectively. Copies of the data can be obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-1223-336-033; e-mail: deposit@ ccdc.cam.ac.uk or http://www.ccdc.cam.ac.uk). References [1] In: B. Lippert (Ed.), Cisplatin. Chemistry and Biochemistry of a Leading Anticancer Drug, Wiley-VCH, Weinheim, 1999. [2] V. Brabec, J. Kasparkowa, Drug Resist. Updat. 8 (2005) 131–146. [3] N.J. Wheate, S. Walker, G.E. Craig, R. Oun, Dalton Trans. 39 (2010) 8113–8127. [4] G. Sava, A. Bergamo, P.J. Dyson, Dalton Trans. 40 (2011) 9069–9075. [5] T. Karmakar, Y. Kuang, N. Neamati, J.B. Baruah, Polyhedron 54 (2013) 285–293. [6] J.L. García-Giménez, J. Hernández-Gil, A. Martínez-Ruiz, A. Castiñeiras, M. Liu-González, F.V. Pallardó, J. Borrás, G. Alzuet Piña, J. Inorg. Biochem. 121 (2013) 167–178. [7] A.I. Matesanz, I. Leitao, P. Souza, J. Inorg. Biochem. 125 (2013) 26–31. [8] G.E. Büchel, A. Gavriluta, M. Novak, S.M. Meier, M.A. Jakupec, O. Cuzan, C. Turta, J.B. Tommasino, E. Jeanneau, G. Novitchi, D. Luneau, V.B. Arion, Inorg. Chem. 52 (2013) 6273–6285. [9] M.A. Carvalho, R.E.F. de Paiva, F.R.G. Bergamini, A.F. Gomes, F.C. Gozzo, W.R. Lustri, A.L.B. Formiga, S.M. Shishido, C.V. Ferreira, P.P. Corbi, J. Mol. Struct. 1031 (2013) 125–131. [10] H. Wang, D. Li, M. Hong, J. Dou, J. Organomet. Chem. 740 (2013) 1–9. [11] T.F.S. Silva, P. Smolenski, L.M.D.R.S. Martins, M.F.C. Guedes da Silva, A.R. Fernandes, D. Luis, A. Silva, S. Santos, P.M. Borralho, C.M.P. Rodrigues, A.J.L. Pombeiro, Eur. J. Inorg. Chem. (2013) 3651–3658. [12] E.R.T. Tiekink, Inflammopharmacology 16 (2008) 138–142. [13] I. Ott, Coord. Chem. Rev. 253 (2009) 1670–1681. [14] A. Casini, G. Kelter, Ch. Gabbiani, A.A. Cinellu, G. Minghetti, D. Fregona, H.H. Fiebig, L. Messori, J. Biol. Inorg. Chem. 14 (2009) 1139–1149. [15] P.J. Barnard, S.J. Berners-Price, Coord. Chem. Rev. 251 (2007) 1889–1902.

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[16] R. Rubbiani, I. Kitanovic, H. Alborzinia, S. Can, A. Kitanovic, L.A. Onambele, M. Stefanopoulou, Y. Geldmacher, W.S. Sheldrick, G. Wolber, A. Prokop, S. Wölfl, I. Ott, J. Med. Chem. 53 (2010) 8608–8618. [17] K. Yan, C.-N. Lok, K. Bierla, C.-M. Che, Chem. Commun. 46 (2010) 7691–7693. [18] C. Wetzel, P.C. Kunz, M.U. Kassack, A. Hamacher, P. Böhler, W. Watjen, I. Ott, R. Rubbiani, B. Spingler, Dalton Trans. 40 (2011) 9212–9220. [19] J.A. Lessa, J.C. Guerra, L.F. de Miranda, C.F.D. Romeiro, J.G. Da Silva, I.C. Mendes, N.L. Speziali, E.M. Souza-Fagundes, H. Beraldo, J. Inorg. Biochem. 105 (2011) 1729–1739. [20] G. Lupidi, L. Avenali, M. Bramucci, L. Quassinti, R. Pettinari, H.K. Khalife, H. Gali-Muhtasib, F. Marchetti, C. Pettinari, J. Inorg. Biochem. 124 (2013) 78–87. [21] E.R.T. Tiekink, Bioinorg. Chem. Appl. 1 (2003) 53–67. [22] C.F. Shaw III, Chem. Rev. 99 (1999) 2589–2600. [23] K. Nomiya, S. Yamamato, R. Noguchi, H. Yokoyama, N.Ch. Kasuga, K. Oyamarand, C. Kato, J. Inorg. Biochem. 95 (2003) 208–220. [24] E. Barreiro, J.S. Casas, M.D. Couce, A. Sánchez, A. Sánchez-Gonzalez, J. Sordo, J.M. Varela, E.M. Vázquez-López, J. Inorg. Biochem. 102 (2008) 184–192. [25] E. Barreiro, J.S. Casas, M.D. Couce, A. Sánchez, A. Sánchez-Gonzalez, J. Sordo, J.M. Varela, E.M. Vázquez-López, J. Inorg. Biochem. 103 (2009) 1023–1032. [26] E. Barreiro, J.S. Casas, M.D. Couce, A. Sánchez, A. Sánchez-Gonzalez, J. Sordo, J.M. Varela, E.M. Vázquez-López, J. Inorg. Biochem. 104 (2010) 551–559. [27] F. Pelletier, V. Comte, A. Massard, M. Wenzel, S. Toulot, P. Richard, M. Picquet, P. Le Gendre, O. Zava, F. Edafe, A. Casini, P.J. Dyson, J. Med. Chem. 53 (2010) 6923–6933. [28] M. Wenzel, B. Bertrand, M.J. Eymin, V. Comte, J.A. Harvey, P. Richard, M. Groessl, O. Zava, H. Amrouche, P.D. Harvey, P. Le Gendre, M. Picquet, A. Casini, Inorg. Chem. 50 (2011) 9472–9480. [29] J.F. González-Pantoja, M. Stern, A.A. Jarzecki, E. Royo, E. Robles-Escajeda, A. Varela-Ramírez, R.J. Aguilera, M. Contel, Inorg. Chem. 50 (2011) 11099–11110. [30] M. Frik, J. Jiménez, I. García, L.R. Falvello, S. Abi-Habib, K. Suriel, T.R. Muth, M. Contel, Chem. Eur. J. 18 (2012) 3659–3674. [31] S. Tabassum, G.C. Sharma, A. Asim, A. Azam, R.A. Khan, J. Organomet. Chem. 713 (2012) 123–133. [32] E. Barreiro, J.S. Casas, M.D. Couce, A. Laguna, J.M. López-de Luzuriaga, M. Monge, A. Sánchez, J. Sordo, J.M. Varela, E.M. Vázquez-López, Eur. J. Inorg. Chem. (2011) 1322–1332. [33] J. McKeage, L. Maharaj, S.J. Berners-Price, Coord. Chem. Rev. 232 (2002) 127–135. [34] R.A. Stein, C. Knobler, Inorg. Chem. 16 (1977) 242–245. [35] Bruker Analytical Instrumentation, SAINT: SAX Area Detector Integration1996. [36] G.M. Sheldrick, SADABS, Version 2.03, University of Göttingen, Germany, 2002. [37] G.M. Sheldrick, SHELXS97 and SHELXL97, Programs for the Solution and Refinement of Crystal Structures, University of Göttingen, Germany, 1997. [38] International Tables for Crystallography, Kluwer, Dordrecht, The Netherlands, 1992. [39] A.L. Spek, Acta Crystallogr. Sect. A 46 (1990) 194–201. [40] W. Kueng, E. Silber, U. Eppenberger, Anal. Biochem. 182 (1989) 16–19. [41] E. Barreiro, J.S. Casas, M.D. Couce, A. Sánchez, J. Sordo, J.M. Varela, E.M. Vázquez-López, Dalton Trans. (2005) 1707–1715. [42] B. Cordero, V. Gómez, A.E. Platero-Prats, M. Revés, J. Echevarría, E. Cremades, F. Barragán, S. Alvarez, Dalton Trans. (2008) 2832–2838. [43] J.E. Huheey, E.A. Keiter, R.L. Keiter, Inorganic Chemistry, Principles of Structure and Reactivity, 4th ed. Harper Collins, New York, 1993. [44] J.M. Pérez, L.R. Kelland, E.I. Montero, F.E. Boxall, M.A. Fuertes, C. Alonso, C. Navarro-Ranniger, Mol. Pharm. 63 (2003) 933–944(and references therein).