S-Propargylthiopyridine Phosphane Derivatives As Anticancer Agents

Article pubs.acs.org/Organometallics

S‑Propargylthiopyridine Phosphane Derivatives As Anticancer Agents: Characterization and Antitumor Activity Elena García-Moreno,† Sonia Gascón,‡ Ma Jesus Rodriguez-Yoldi,‡ Elena Cerrada,† and Mariano Laguna*,† †

Departamento de Química Inorgánica, Instituto de Síntesis Química y Catálisis Homogénea, Universidad de Zaragoza-CSIC, E-50009 Zaragoza, Spain ‡ Departamento de Farmacología y Fisiología, Unidad de Fisiología, Facultad de Veterinaria, Universidad de Zaragoza, 50013, Zaragoza, CIBERobn, Spain S Supporting Information *

ABSTRACT: S-Propargylthiopyridine phosphane gold(I) derivatives with the water-soluble phosphanes PTA (1,3,5-triaza7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5phosphabicyclo[3.3.1]nonane), and TPPTS (sodium triphenylphosphane trisulfonate) are described and used as metalloligands by coordination to copper(I). New heteronuclear gold(I) and copper(I) complexes of the type [Au2Cu(C CCH2SC5H4N)2L2] (L = PTA, DAPTA, PPh3, TPPTS) and [AuCu(CCCH 2 SC 5 H 4N)(L)(PPh 3 ) 2 ]NO 3 (L = PTA, DAPTA) are reported. The X-ray crystal structure of [Au(CCCH2SC5H4N)(PTA)] (1), which confirms the coordination of the metallic center to the alkyne unit, displays a zigzag polymeric chain with short gold−gold contacts of 3.2680(16) Å. Strong antiproliferative effects are found for most of the new complexes in human colon cancer cell lines (Caco-2, PD7, and TC7 clones), with these effects being more pronounced than for the reference drugs cisplatin and auranofin, especially for the heterodimetallic derivatives, which are markedly more active than the corresponding mononuclear precursors. Apoptosis-induced cell death is found for all compounds, as shown by an annexin-V/propidium iodide double-staining assay.

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INTRODUCTION Cisplatin is one of the most widely used metallic complexes for cancer treatment, especially for cancers of the ovary, head and neck, bladder, and cervix, melanoma, and lymphomas.1 However, the effectiveness of cisplatin is hindered by toxic side-effects and the occurrence of tumor resistance.2−5 In an attempt to overcome these drawbacks, new platinum-based anticancer agents similar to cisplatin have been developed and entered human clinical trials, although only a few of these have reached routine clinical use.6 As a consequence, there is considerable interest in the search for new metallic complexes for use in cancer treatment. Among the new non-platinum compounds with potential as anticancer drugs, gold derivatives have gained increasing attention due to their generally strong tumor cell growth inhibiting effects and the observation that many such compounds inhibit the enzyme thioredoxin reductase (TrxR).7−10 This enzyme is relevant for the proliferation of tumor tissues and has emerged as a new target for drug development.8,11 Several reviews12−23 concerning gold(I) and gold(III) derivatives with anticarcinogenic activity have been published in the past few years. One of the most widely studied such gold(I) derivatives is (1-thio-β-Dglucopyranose-2,3,4,6-tetraacetato-S)(triethylphosphane) gold© 2013 American Chemical Society

(I), also known as auranofin, which was found to possess antiarthritic activity24 and subsequently similar, or even better, anticancer properties than cisplatin.25 However, although this derivative has been successfully tested in vitro in different cancer cell lines,26,27 it has shown good in vivo antitumor activity only in murine models against P388 leukemia.28 The mechanism of action of auranofin appears to differ from that of cisplatin since the former does not damage DNA.24,26,27 Instead, auranofin and related derivatives induce cell death as a result of their effects on mitochondrial integrity.27 In this context, we have recently reported a series of thiolate29−31 or alkyne32 gold(I) compounds that exhibit potent cytotoxicity against different cancer cell lines, with this activity being higher than cisplatin in several cases. Additionally, alkynyl gold(I) derivatives with biological activity have been recently published.33−35 The presence of the water-soluble phosphanes PTA (1,3,5-triaza-7-phosphaadamantane), DAPTA (3,7-diacetyl-1,3,7-triaza-5-phosphabicyclo[3.3.1]nonane), TPPMS (sodium triphenylphosphane monosulfonate), or TPPTS (sodium triphenylphosphane trisulfonate) confers a moderate to high Received: April 19, 2013 Published: June 21, 2013 3710

dx.doi.org/10.1021/om400340a | Organometallics 2013, 32, 3710−3720

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Scheme 1. (i) [AuCl(PR3)]/KOEt, (ii) 1/2[Cu(MeCN)4]PF6, and (iii) [Cu(NO3)(PPh3)2]

Table 1. Selected Bond Lengths and Angles for [Au(CCCH2SC5H4N)(PTA)] (1)

a

bond

bond length (Å)

Au(1)−C(1) Au(1)−P(1) Au(1)−Au(1)a Au(1)−Au(1)b S(2)−C(4) S(2)−C(3) C(2)−C(1) P(1)−C(9) P(1)−C(11) P(1)−C(10)

1.970(17) 2.272(4) 3.2680(16) 3.2680(16) 1.782(18) 1.845(17) 1.24(3) 1.80(2) 1.87(2) 1.87(2)

bond angle (deg) C(1)−Au(1)−P(1) C(1)−Au(1)−Au(1)a P(1)−Au(1)−Au(1)a C(1)−Au(1)−Au(1)b P(1)−Au(1)−Au(1)b Au(1)#1−Au(1)−Au(1)b

176.9(5) 92.6(5) 89.59(11) 89.9(5) 89.74(11) 142.67(4)

−x+y+1, −x+1, z+1/3. b−y+1, x−y, z−1/3.

improve their antitumor activity in comparison with their mononuclear precursor probably as a result of interaction of both metals with different biological targets. Working with this idea, here we report on the synthesis and characterization of new propargylthiopyridine gold(I) derivatives with the water-soluble phosphanes PTA and DAPTA and the sulfonated triphenylphosphane derivative TPPTS, as well as their reaction with copper(I) derivatives to give heteropolynuclear compounds. An evaluation of the cytotoxic activity of the resulting compounds toward two human colon cancer cell lines has also been undertaken. In addition, as apoptosis is the predominant mechanism by which cancer cells die, we have also evaluated the apoptic activity of these new complexes and their implication in cell-cycle progression. The results of this study suggested that S-propargylthiopyridine phosphane derivatives could find a use as therapeutic drugs for colon cancer.

water solubility on these complexes that could facilitate drug administration and transport in the body. The occurrence of anticancer activity in both thiolato phosphane and alkyne phosphane gold(I) derivatives encouraged us to synthesize new phosphane complexes with the ligand 2-propargylthiopyridine, which was previously described by some of us,36 by combining the thiol and alkyne units in the same molecule. Moreover, we also evaluated their behavior as metalloligands for group 11 metal ions, such as copper, via the N and S atoms of the thiol unit. Our interest in the synthesis of copper derivatives is not casual, as copper is a crucial trace element in redox chemistry that is present in all living organisms. Many copper complexes have been investigated as therapeutic agents.37 As examples, copper thiosemicarbazones have demonstrated therapeutic potential in Alzheimer’s disease,38,39 copper aspirinate is used in cardiovascular dysfunction,40 and derivatives with nonsteroidal anti-inflammatory drugs have exhibited increased antiinflammatory properties and reduced toxicity.41 In addition, metabolic changes occurring in cancer cells have been associated, among other factors, with copper, which plays an important role in angiognesis42 and in proteasome inhibition.43 Consequently, many examples of Cu(I and II) complexes have recently been investigated for their antitumor activity.44−56 A combination of two different cytotoxic metals in the same molecule is scarcely represented in the literature. Only examples of bimetallic Ti−Ru,57 Ti−Au,58 Ti−Pd,59 and Ti− Pt59 have recently been described. Most of these derivatives

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RESULTS AND DISCUSSION 2-Propargylthiopyridine reacts with the chlorophosphane gold(I) derivatives [AuCl(PR3)] (PR3 = PTA, DAPTA, PPh3, TPPTS) in the presence of KOH in MeOH as deprotonating agent to give air-stable alkynylgold(I) derivatives with the formula [Au(CCCH2SC5H4N)(PR3)] (PR3 = PTA, 1; DAPTA, 2; PPh3, 3; TPPTS, 4; Scheme 1, i). The absence of the CC−H signal in the 1H NMR spectra of the four derivatives confirmed the presence of the anionic form of the ligand and subsequent coordination to the metallic center. The 1 H NMR spectra exhibit multiplets in the aromatic region 3711


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confirms the coordination of gold to the alkyne unit, consists of a molecule associated with two additional symmetry-related units in the unit cell. The molecules are connected in a head-totail arrangement, with a torsion angle of 116.24°, via short intermolecular gold−gold contacts of 3.2680(16) Å to give a zigzag polymeric chain (Figure 2). These contacts are longer than those observed in gold(I) derivatives with PTA (3.047− 3.1164 Å)32,60−62 but shorter than in recently reported thiolatogold(I) complexes with PTA (ca. 3.35 Å).29,31 A similar polymeric chain has been found in [Au(PTA)4][Au(CN2)],63 although with a longer separation between the metallic units (3.45 Å). The independent molecule displays a typical linear geometry around the metal, with a C1−Au1−P1 angle of 176.9(5)°. Only two examples of alkyne gold(I) with PTA, namely, rac[Au{CCC(Et)(Me)OH}(PTA)] 60 and [Au(CC-3SC4H3)(PTA)],32 have been characterized by X-ray analysis. In the former, three molecules are connected by intermolecular gold−gold contacts to form a trinuclear chain, with an additional single molecule, which is laterally connected, affording an infinite polymeric chain with gold side-chains. Only two units are connected to each other in the case of [Au(CC-3-SC4H3)(PTA)]. Additional differences are observed for the Au−C and C−C distances in the alkyne unit, with the former being slightly shorter in complex 1 (Au1−C1 1.970(17) Å) than in previously reported alkyne derivatives (1.997−2.079 Å)32,60 and the latter (C1−C2 1.24 Å) longer than in the same complexes (1.20 and 1.21 Å on average). The presence of uncoordinated nitrogen and sulfur atoms in these new derivatives means that they could act as metalloligands and form heterodimetallic complexes. Thus, the reaction of two equivalents of 1−4 with [Cu(MeCN)4]PF6 affords the cationic gold(I)/Cu(I) compounds [Cu{Au(C CCH2SC5H4N)(L)}2]PF6 (L = PTA, 5; DAPTA, 6; PPh3, 7; TPPTS, 8) (Scheme 1, ii). The presence of the new metallic center in these heterodimetallic derivatives was confirmed by the high-field shift in the 1H NMR spectra of the PTA and DAPTA derivatives and the downfield shift found for their PPh3 and TPPTS counterparts, with respect to complexes 1−4. Furthermore, downfield shifts in the signals due to the thiopyridine moiety are observed in the 1H NMR spectra of all complexes. In addition, a septuplet at about −144 ppm is also observed in the 31P{1H} NMR spectra due to the presence

corresponding to the pyridine fragment, together with the characteristic pattern of the phosphanes PTA and DAPTA in 1 and 2 and additional multiplets due to the triphenyl phosphanes PPh3 and TPPTS. The 31P{1H} NMR spectra of the complexes show only one resonance, which is shifted downfield by about 50 ppm with respect to that for the free phosphanes. Slow diffusion of a dichloromethane solution of [Au(C CCH2SC5H4N)(PTA)] (1) into diethyl ether gave crystals suitable for X-ray diffraction. Selected bond lengths and angles are listed in Table 1. The crystal structure (Figure 1), which

Figure 1. ORTEP plot of the X-ray structure of complex 1. View of the three symmetry-related units. H atoms have been omitted for clarity. Thermal ellipsoids are drawn at 50% probability.

Figure 2. Polymeric structure of [Au(CCCH2SC5H4N)(PTA)]. 3712


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Table 2. IC50 Values of Metallic Complexes against PD7 and TC7 Colon Cancer Cell Lines Compared with Auranofin and Cisplatin IC50 (μM)a

a

compound

log D7.4

PD7

TC7

[Au(CCCH2SC5H4N)(PTA)] (1) [Au(CCCH2SC5H4N)(DAPTA)] (2) [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5) [Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6) [AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9) [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) [Cu(NO3)(PPh3)] cisplatin auranofin

−0.07 −0.03 −0.01 −0.40 +0.10 +0.07

3.65 ± 0.34 13.12 ± 1.65 2 × 10−4 ± 10−5 0.02 ± 9.9 × 10−3 0.47 ± 0.06 1.17 ± 0.12 1.15 ± 0.34 37.24 ± 5.15 1.8 ± 0.1

4.53 ± 0.18 14.91 ± 0.74 0.03 ± 5.6 × 10−3 0.02 ± 2.9 × 10−3 0.6 ± 2 × 10−2 3.4 × 10−3 ± 10−4 2.91 ± 0.06 45.6 ± 8.08 2.1 ± 0.4

Mean ± SE of at least three determinations.

same as the remaining two doublets, thus indicating that the former signal is due to two of the four possible isomers. In the absence of an X-ray structure, we can only propose a hypothetical structure for these dinuclear derivatives, which is depicted in Scheme 1. Cytotoxicity of the S-Propargylthiopyridine Complexes. The cellular effects of some of these new derivatives have been tested against human colon cancer cell lines. Two clones were selected for further analysis: Caco-2/PD7 (from early passage) and Caco-2/TC7 (from late passage). These clones exhibited a well-organized brush border at late confluence and no differences in the levels of dipeptidylpeptidase IV activity. Expression of the brush border-associated hydrolate sucrase-isomaltase was shown to increase from early to late passages of Caco-2 cells, concomitant with a decrease in the rates of glucose consumption.72 The results are expressed in terms of IC50 values (Table 2), which were obtained after exposure to the drug for 72 h, using the well-established MTT assay (see Experimental Section for details). We also evaluated the cell proliferation inhibition induced by auranofin, cisplatin, and the copper starting material [Cu(NO3)(PPh3)2] used to prepare complexes 9 and 10 under the same experimental conditions. Exposure of PD7 cell lines to increasing concentrations (0−20 μM) of complexes 1−10 led to a remarkable inhibition of cell growth. Thus, complex [Au(C CCH2SC5H4N)(PTA)] (1) displayed a similar IC50 value to that found for auranofin, and its counterpart complex 2, which contains DAPTA, exhibited an almost 4-fold higher value than 1. Both complexes show markedly lower values than that found for cisplatin. Notably, the heteronuclear gold and copper derivatives 5−10 exhibited particularly potent cytotoxicity, with IC50 values ranging from 10−4 to 1.17 μM, much lower than the values observed for the reference drugs and for the mononuclear Au and Cu precursors. [AuCu(C CCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) exhibited a similar cytotoxicity to the precursor [Cu(NO3)(PPh3)2], whereas the related complex 9, which contains PTA as ligand, exhibited higher cytotoxicity. Complexes [Cu{Au(CCCH2SC5H4N)L}2]PF6 (L = PTA, 5; DAPTA, 6) showed the highest antitumor efficacy, with IC50 values of around 10−2 and 10−4 for the PD7 cell line and 10−2 for TC7 clones. Generally speaking, similar or slightly higher IC50 values were observed for the TC7 cell lines than for the PD7 lines, except in the case of complex 5, which had a value 2 orders of magnitude higher for the former than for the latter, and complex 10, where the lowest value (by 3 orders of magnitude) was found for TC7 cell lines.

of the PF6 anion, which is confirmed by the appearance of two intense bands at ca. 840 and 555 cm−1 in the IR spectra, assignable to the stretching vibration of this anion. Since attempts to grow suitable crystals for X-ray analysis failed, we can only propose the structure of complexes 5−8. Considering the presence of both uncoordinated N and S atoms, and the tendency of copper(I) to prefer N-ligand coordination and a tetrahedral geometry,64 we suggest the structure depicted in Scheme 1, in which both [Au(CCCH2SC5H4N)(L)] units are coordinated to the Cu(I) center. The presence of low-field displacement in the NMR protons of the thiopyridine unit in complexes 5−8 in comparison with the starting gold derivatives 1−4 and similar ν(CC) vibration values in the IR spectra for all the complexes should be in accordance with our hypothesis. The reaction of 1 and 2 with [Cu(NO3)(PPh3)2] in a 1:1 molar ratio affords air-stable solids with the stoichiometry [AuCu(CCCH2SC5H4N)L(PPh3)2]NO3 (L = PTA, 9; DAPTA, 10). Their 1H NMR spectra are in accordance with the presence of the phosphane PPh3 and the propargylthiopyridine unit, together with the water-soluble phosphane PTA or DAPTA, in a relative 2:1 proportion. The 31P{1H} NMR spectra show three broad signals at room temperature. At −60 °C these signals split into a singlet at about 40 ppm, which has also been observed in gold(I) acetylides with triphenylphosphine,65−68 two doublets at ca. 0 ppm, one of which has a lower intensity (in 9), in a similar region to that observed for the CuPPh3 starting material, and three doublets in the region of −90 ppm in 9 and −69 ppm in 10, which are characteristic of Cu-PTA69−71 or Cu-DAPTA units, respectively. The two sets of doublets centered at 0 and −90 ppm, respectively, which have the same coupling constants, are coupled, as confirmed by P,P-COSY NMR experiments. Integration of the three sets of resonances gives a 1:1:1 ratio, thus pointing to the presence of one Au-PPh3 unit and a copper(I) center simultaneously coordinated to one PPh3 and one PTA or DAPTA molecule as the result of PPh3 migration from the Cu(I) to the Au(I) center. This fact was corroborated by the detection of a peak at m/z 608 in the mass spectrum, which corresponds to a [M − (CuLPPh3)]+, in this case [Au(CCCH2SC5H4N)(PPh3)]+, unit and indicates the presence of AuPPh3 coordinated to the propargylthiopyridine anion. The two doublets in the region of 0 ppm in 9 agree with the presence of two epimers, since the copper center has an asymmetric environment. The occurrence of further doublets in the Cu−L (L = PTA or DAPTA) region could be due to the presence of other isomers arising from the relative position of the Au-PPh3 unit, in addition to their corresponding epimers. One of these doublets integrates the 3713


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Figure 3. Quantitative flow cytometry analyses using propidium iodide (PI) uptake and annexin V staining in PD7 and TC7 colon cancer cells treated with DMSO, 20 μM of complexes 1−10, cisplatin, auranofin, and [Cu(NO3)(PPh3)] after 72 h. [Au(CCCH2SC5H4N)(PTA)] (1); [Au(CCCH2SC5H4N)(DAPTA)] (2); [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5); [Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6); [AuCu(CCCH2SC5H4N)L(PPh3)}]NO3 L = PTA (9) and L = DAPTA (10).

bis(diphenylphosphane)ethane) caused myocardial lesions83 and severe hepatotoxicity84 attributed to alteration in mitochondrial function.85 The lipophilicity/hydrophilicity of our derivatives covers a narrow range, with n-octanol/water (log D7.4) distribution coefficients of between −0.4 and +0.1 (Table 2), thus indicating a balanced relationship between their lipophilic and hydrophilic character. Apoptosis Studies. As cancer is characterized by uncontrolled cellular proliferation, there is considerable interest in chemotherapy-induced apoptosis.86,87 In light of this, we tested the ability of our metal derivatives to inhibit human tumor cell proliferation by inducing apoptosis using the annexin-V/propidium iodide double-staining assay, which are well-established biomarkers of cell death,88 over 72 h (Figure 3). We also measured the effects of cisplatin and auranofin for comparison purposes. During the early stages of apoptosis, the cell membrane loses asymmetry and the membrane phospholipid phosphatidylserine (PS) is translocated from the cytoplasmic to the extracellular side. Annexin-V (FITC conjugated) specifically binds to PS exposed on the cell surface and can be detected by flow cytometry, thereby allowing the identification of early stage apoptic cells. Viable cells with intact membranes and cells undergoing early apoptosis but with relatively intact membranes exclude propidium iodide (PI),

Cisplatin and its derivatives are among the most effective anticancer agents and, as such, are used to treat different types of tumors, including colon cancer.73−75 Indeed, the combination of oxaliplatin, a third-generation platinum derivative, with 5-fluorouracil and leuvocorin, as standard adjuvants, is among the most effective chemotherapies for advanced76 or metastatic colorectal cancer.77,78 It should be noted that our new derivatives exhibit higher cytotoxicities than cisplatin in both cell lines, with a difference of up to 5 orders of magnitude in the best case, [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5) (IC50 of 2 × 10−4 μM). Drug delivery, including absorption, distribution, and elimination, can be favored by a balanced relationship between lipophilicity and hydrophilicity since the drug must be transported through multiple mixed water−lipid layers. As poor water solubility may lead to poor absorption and bioavailability following oral dosing, solubility is an important physicochemical property in drug discovery.79 Furthermore, a direct relationship between hepatocyte uptake and lipophilicity has been reported recently in vitro.80 As well, it has also been shown that gold derivatives with a highly lipophilic character provoke severe adverse effects as a result of drug accumulation in the mitochondria.81,82 Thus, intravenous administration of the highly liphophilic compound [Au(dppe)2]Cl (dppe = 1,23714


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Table 3. Summary of the Effects of Treating PD7 and PC7 Cell Lines with the Metallic Compounds 1−10, Cisplatin, and Auranofin cells

complex

live (%)

early apoptic (%)

late apoptic (%)

necrotic (%)

PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7 PD7 TC7

DMSO DMSO [Au(CCCH2SC5H4N)(PTA)] (1) [Au(CCCH2SC5H4N)(PTA)] (1) [Au(CCCH2SC5H4N)(DAPTA)] (2) [Au(CCCH2SC5H4N)(DAPTA)] (2) [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5) [Cu{Au(CCCH2SC5H4N)(PTA)}2]PF6 (5) [Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6) [Cu{Au(CCCH2SC5H4N)(DAPTA)}2]PF6 (6) [AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9) [AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9) [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) [Cu(NO3)(PPh3)2] [Cu(NO3)(PPh3)2] cisplatin cisplatin auranofin auranofin

89.9 88.1 0.8 1.6 68.9 87.6 0.6 0.7 1.3 1.7 0.9 1.6 1.6 0.4 2.0 3.0 55.4 82.0 37.4 0.0

3.2 2.7 60.7 61.5 18.8 4.9 0.7 1.2 0.5 0.6 1.9 3.7 34.8 31.6 6.2 6.1 7.6 3.3 7.7 18.3

5.8 5.7 36.6 32.7 9.1 4.9 94.8 92.9 85.7 92.3 94.2 91.4 61.0 67.3 88.0 86.5 22.4 8.8 32.2 81.5

1.1 3.5 1.9 4.2 3.2 2.5 3.6 5.1 12.5 5.1 3.0 3.3 2.6 0.7 3.7 4.4 14.6 6.0 22.7 0.2

Figure 4. Cell-cycle analysis after treatment with the metal complexes for 72 h. Cell cycle and DNA fragmentation were determined by propidium iodide staining. Percentages of G1-, S-, and G2-phase cells are shown when possible.

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cisplatin, auranofin, and [Cu(NO3)(PPh3)2]. Cell-cycle progression could be determined only for complex [Au(C CCH2SC5H4N)(DAPTA)] (2) and cisplatin. In contrast, treatment of both cell lines with complexes 1, 3, 5−10, [Cu(NO3)(PPh3)2], and auranofin led to a markedly perturbed flow cytometry profile, most likely due to extensive cell death (see Table 3), with values of less than 2% for live cells. These disturbances in the cell cycle could inhibit the cell proliferation; therefore these compounds have an anticancer effect by stimulating apoptosis, as is shown in Table 3 and Figure 3. Nonsignificant alterations in cell-cycle progression are observed after complex treatment with complex 2 and cisplatin. Although classical Pt(II) complexes are known to induce alterations in the cell cycle, particularly an important increase in the G2/M cell fraction,90 both complex 2 and cisplatin barely induce cellcycle changes for TC7 cells and only insignificant changes for PD7 cells.

whereas the membranes of dead and damaged cells are permeable to PI and are also stained with annexin-V. The data reported in Table 3 and Figure 3 reveal that treatment with complexes 1−10 for 72 h produces much higher amounts of early and late apoptosis than in cells treated with DMSO for both cancer cell lines (PD7 and TC7), thus indicating their ability to induce cell death by an apoptotic pathway. In particular, compound [Au(CCCH2SC5H4N)(PTA)] (1) is the only example that induces a significantly higher degree of early apoptosis (approximately 10-fold in PD7 and 22-fold in TC7 cell lines) compared with DMSO treatment, whereas [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10) causes a 11-fold increase in both early and late apoptosis in PD7 and a 12-fold increase in early and late apoptosis in TC7 cell lines. Complex [Au(C CCH2SC5H4N)(DAPTA)] (2) increases the degree of early apoptosis 6-fold, with a similar population of live cells to that found in DMSO medium in both cell lines, which is in accordance with the high IC50 value measured. Complexes 5, 6, and 9 exhibit similar behavior, with most of the cell population (values ranging from 85% to 94%) in late-stage apoptosis. Similar values were found for the [Cu(NO3)(PPh3)2] starting material. Treatment of the cells with auranofin produces different behavior in the two clones. Thus, the percentage of live cells with PD7 is much higher than with TC7, where no living cells remain after 72 h and only apoptotic cells are detected. For cisplatin, similarly to complex 2, most of the population remains as live cells and only a small degree of apoptosis is observed. In addition, the lack of PI uptake for all complexes supports the conclusion that they do not cause necrosis. The only exception to this is complex [Cu{Au(C CCH2SC5H4N)(DAPTA)}2]PF6 (6), with 12.5% of necrosis for the PD7 cell line. In contrast, both cisplatin and auranofin produce similar percentages or even twice that of DMSO, except in the case of auranofin with TC7 cells, where no necrosis is detected and live cells remain after 72 h. The above findings highlight the fact that these new derivatives are able to induce cell death by activating apoptic pathways, thereby reducing their ability to nonselectively react with biological targets to cause necrosis and its related sideeffects.89 A comparison of all the complexes studied shows that [Au(CCCH2SC5H4N)(PTA)] (1) displays a markedly different behavior since most of the population is found at an early apoptotic stage. Similarly high population values are found for complex [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10), although in this case for late apoptosis. We subsequently analyzed the effects of the metal complexes on cell-cycle progression in both cancer cell lines after 72 h of treatment. Cell-cycle analysis was performed by using flow cytometry to assess the DNA content of cells stained with propidium iodide. The fluorescence intensity of the stained cells at certain wavelengths correlates with the amount of DNA contained therein. As the DNA content of cells doubles during the S phase of the cell cycle, the relative amount of cells in the G0 phase and G1 phase (before the S phase), in the S phase, and in the G2 phase and M phase (after the S phase) can be determined, as the fluorescence of cells in the G2/M phase will be twice as high as that of cells in the G0/G1 phase. Cell-cycle anomalies can indicate various kinds of cell damage, for example DNA damage, which cause the cell to interrupt the cell cycle at certain checkpoints to prevent transformation into a cancer cell (carcinogenesis). Figure 4 shows a histogram of PD7 and TC7 cells treated with DMSO, complexes 1−10,

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CONCLUSION The reaction of chlorophosphane gold(I) derivatives bearing water-soluble phosphanes with 2-propargylthiopyridine leads to the formation of mononuclear complexes that can act as metalloligands upon coordination to copper(I). Trinuclear Au− Cu compounds are obtained by using starting copper derivatives containing labile ligands. However, in addition to PPh3 migration, a mixture of different possible isomers is detected when triphenylphosphane copper(I) is employed as starting material. Most of the compounds synthesized were tested for their antiproliferative activity against the human colon cancer cell line Caco-2 (PD7 and TC7 clones). Although all derivatives showed a better cytotoxicity than cisplatin, the corresponding heteronuclear compounds displayed the lowest IC50 value, with a value of 2 × 10−4 μM being detected in the best case, thus indicating remarkably more activity than the mononuclear precursors. A balanced relationship between lipophilicity and hydrophilicity, which is one of the prerequisites for the synthesis of an anticancer drug in order to facilitate its administration and transport in the body and avoid host toxicity, is found for these derivatives. Apoptosisrelated cell death is activated in Caco-2 cells after treatment with all complexes. The present study provides a better understanding of the mechanisms underlying the effect of propargylthiopyridine phosphane derivatives on cancer cells. These advances may lead to therapeutic strategies to protect and improve cancer treatment. Further studies in this respect are currently under way in our group.

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EXPERIMENTAL SECTION

1

H, 31P{1H}, and 13C{1H} NMR spectra were recorded on 400 or 300 MHz Bruker Avance spectrometers. Chemical shifts are quoted relative to external TMS (1H, 13C) or 85% H3PO4 (31P); coupling constants are reported in Hz. MALDI mass spectra were measured on a Micromass Autospec spectrometer in positive ion mode using DCTB as matrix. IR spectra were recorded on a Perkin-Elmer Spectrum 100 FTIR (far-IR) spectrometer. Elemental analyses were obtained inhouse using a LECO CHNS-932 microanalyzer. The starting materials phosphanes PTA91 and DAPTA,92,93 C5H4NSCH2CCH,36 [AuCl(PR′3)],93,94 and auranofin95 were prepared according to published methods. A sample of TPPTS was kindly provided by European Oxo GmbH. Cisplatin was purchased from Sigma-Aldrich. Synthesis of the [Au(CCCH2SC5H4N)(L)] Complexes. To a solution of KOH (0.014 g, 0.25 mmol) in MeOH (ca. 10 mL) containing 2-propargylthiopiridine (0.2 mmol) was added [AuCl(L)] 3716


Organometallics

Article

CH2−SC5H4N)PTA)]+. Anal. Calcd (%) for C28H36Au2CuF6N8P3S2 (1212.02): C 27.72, H 2.99, N 9.24. Found: C 27.41, H 3.03, N 9.59. [Cu{Au(CCCH2SC5H4N)2(DAPTA)}2]PF6 (6): 63% yield, brown solid. 1H NMR (400 MHz, CD2Cl2, 25 °C): δ (ppm) 1.98 (s, 12H, −CH3CO), 3.56−3.62 (m, 2H, NCH2P), 3.76−3.93 (m, 8H, NCH2P + −SCH2), 4.08−4.01 (m, 2H, NCH2P), 4.15−4.22 (m, 2H NCH2N) 4.55−4.71 (m, 4H, NCH2N + NCH2P), 4.83−4.87 (m, 2H, NCH2N), 5.38−5.53 (m, 2H, NCH2P), 5.64 (d, 2H, J = 14.4 Hz, NCH2N), 7.25−7.33 (m, 2H, H5), 7.46−7.52 (m, 2H, H3), 7.68−7.72 (m, 2H, H4), 8.47−8.54 (m, 2H, H6). 31P{1H} NMR (161.97 MHz, dmso-d6): δ −26.6 (s), −144.2 (sept, PF6−) ppm. IR (KBr): 2163 cm−1 ν(C C), 833, 556 cm−1 ν(PF6−). MALDI MS: m/z (%) 637 (47) [M-(C C−CH2−SC5H4N)DAPTA]+; 575 (100) [M − (Cu(CC−CH2− SC5H4N)DAPTA)]+. Anal. Calcd (%) for C34H44Au2CuF6N8O4P3S2 (1356.07): C 30.09, H 3.27, N 8.26. Found: C 30.30, H 3.23, N 8.32. [Cu{Au(CCCH2SC5H4N)2(PPh3)}2]PF6 (7): 75% yield, brown solid. 1 H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.75 (s, 4H, −SCH2), 7.20−7.59 (m, 34H, PPh3+ H5 + H3), 7.74 (td, 2H, J = 7.9, 1.8 Hz, H4), 8.71 (dd, 2H, J = 6, 1.8 Hz, H6). 31P{1H} NMR (161.97 MHz, dmso-d6): δ 45 (s), −144.2 (sept, PF6−) ppm. 13C{1H} NMR (75.4 MHz, CDCl3): δ 139.4 (s, C6), 134.2, 134, 132.2 (s, Ph), 130.1 (s, C4), 129.7 (s, C3), 129.3 (s, C5), 77.0 (s, CC), 22.8 (s, CH2) ppm. IR (KBr): 2158 cm−1 ν(CC). IR (KBr): 2151 cm−1 ν(CC), 832, 557 cm−1 ν(PF6−). MALDI MS: m/z (%) 670 (50) [M − (CC−CH2− SC5H4N)PPh3]+; 607 (100) [M − (Cu(CC−CH2−SC5H4N)PPh3)]+. Anal. Calcd (%) for C52H42Au2CuF6N2P3S2 (1423.42): C 43.88, H 2.97, N 1.97. Found: C 43.59, H 3.21, N 2.00. [Cu{Au(CCCH2SC5H4N)2(TPPTS)}2]PF6 (8): 73% yield, brown solid. 1H NMR (400 MHz, CD2Cl2, 25 °C): δ (ppm) 3.75 (s, 4H, −SCH2), 7.15−7.29 (m, 2H, H5), 7.27−7.32 (m, 2H, H3), 7.52−7.77 (m, 24H, TPPTS), 7.89 (ddd, 2H, J = 8.4, 7.8, 1.6 Hz, H4), 8.54−8.65 (m, 2H, H6). 31P{1H} NMR (161.97 MHz, dmso-d6): δ 45 (s), −144.2 (sept, PF6−) ppm. 13C{1H} NMR (100 MHz, MeOD): δ (ppm) 134.2 (TPPTS); 134.0 (s, C6), 132.0 (s, C4), 129.5 (s, C3), 116.7 (s, C5), 77.0 (m, CC), 20 (s, SCH2). IR (KBr): 2157 cm−1 ν(CC), 832, 556 cm−1 ν(PF6−). MALDI MS: m/z (%) 975 (63) [M − (CC−CH2− SC5H4N) TPPTS]+; 912 (100) [M − (Cu(CC−CH2−SC5H4N)TPPTS]+. Anal. Calcd (%) for C52H36Au2CuF6N2Na6P3S8 (2035.69): C 30.68, H 1.78, N 1.38. Found: C 30.35, H 1.91, N 1.30. [AuCu(CCCH2SC5H4N)(PTA)(PPh3)2]NO3 (9): 70% yield, brown solid. 1H NMR (400 MHz; CDCl3; 25 °C): δ (ppm) 3.77 (s, 6H, NCH2N), 3.87 (s, 2H, −SCH2), 4.34 and 4.12 (AB system, 6H, JAB = 14.2 Hz, NCH2P), 7.01 (t, 1H, J = 3 Hz, H3), 7.19−7.34 (m, 31H, PPh3 + H5), 7.53 (td, 1H, J = 5.4, 1.2 Hz, H4), 8.51 (d, 1H, J = 3.3 Hz, H6). 31P{1H} NMR (121.45 MHz, CDCl3, −60 °C): δ 40.3 (s, AuPPh3), 0.5 (d, minor isomer, Cu-PPh3), 0.16 (d, mayor isomer, CuPPh3), −89.9 (d, Cu-PTA), −85.4 (d, Cu-PTA), −96.2 (d, Cu-PTA) (JPP = 85 Hz) ppm. 13C{1H} NMR (100 MHz, MeOD): δ 142.4 (s, C6), 138.7 (s, PPh3 + C4), 134.1 (s, PPh3 + C3), 126.1 (s, C5), 77.0 (s, CC), 70.1 (s, CC), 44.7 (m, NCH2N + PCH2N), 25 (s, SCH2). IR ν: 2160 (CC), 1098, 1014, 691 (NO3−) cm−1. MALDI MS (m/ z): [M − CuPTAPPh3]+ 608 (100%). Anal. Calcd (%) for C50H48AuCuN5O3P3S (1152.45): C 52.11, H 4.88, N 6.08. Found: C 52.30, H 4.57, N 6.30. [AuCu(CCCH2SC5H4N)(DAPTA)(PPh3)2]NO3 (10): 70% yield, brown solid. 1H NMR (400 MHz; CDCl3; 25 °C): δ (ppm) 1.55 (s, 3H, CH3CO), 1.94 (s, 3H, CH3CO), 3.15 (m, 1H, PCH2N), 3.40 (m, 1H, PCH2N), 3.50 (m, 1H, NCH2N), 3.71 (m, 2H, PCH2N), 3.84 (s, 2H, SCH2), 3.88 (m, 1H, NCH2N), 4.33 (m, 1H, NCH2N), 4.73 (d, 1H, J = 14 Hz, PCH2N), 5.10 (d, 1H, J = 15.6 Hz, PCH2N), 5.57 (d, 1H, J = 14.4 Hz, NCH2N), 7.07 (t, 1H, J = 5.6 Hz H5), 7.20−7.41 (m, 31H, PPh3 + H3), 7.52 (td, 1H, J = 1.8 Hz, J = 7.8 Hz, H4), 8.58 (d, 1H, J = 4.3 Hz, H6). 31P{1H} NMR (121.45 MHz, CDCl3, −60 °C): δ 39.7 (s, Au-PPh3), 0.17 (d, Cu-PPh3), −64.5 (d, Cu-DAPTA), −68.9 (d, Cu-DAPTA), −69.6 (d, Cu-DAPTA) ppm (JPP = 83 Hz). 13 C{1H} NMR (100 MHz, MeOD): δ 170.0 (s, CO), 133.7 (s, C6); 129.2 (s, C4), 125.7 (s, C3), 119.7 (s, C5), 77.1 (s, CC); 70.0 (s, NCH2N); 45.2 (s, PCH2N), 21.0 (s, CH3), 20.1 (s, SCH2). MALDI MS (m/z): [M − Cu(DAPTA)PPh3] 608 (66%), [M − (DAPTA)PPh3] 671 (12%). IR ν: 1098, 1027, 692 (NO3−) cm−1. Anal. Calcd

(L = PTA, DAPTA, PPh3, TPPTS) (0.2 mmol). Light brown solids precipitated in the ethanolic solution, which were isolated by filtration after 20 h of stirring at room temperature, washed with ethanol and diethyl ether, and dried in vacuo. Using this method the following complexes were prepared: [Au(CCCH2SC5H4N)(PTA)] (1): 75% yield, pale brown solid. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.98 (s, 2H, −SCH2), 4.21 (s, 6H, PCH2N), 4.48 and 5.54 (AB system, 6H, JAB = 13.2 Hz, NCH2N), 6.98 (dd, 1H, J = 6.9, 5.4 Hz, H5), 7.16 (d, 1H; J = 8 Hz, H3), 7.42 (ddd, 1H, J = 8, 7.1, 1.8 Hz, H4), 8.34(d, 1H, J = 4.4 Hz, H6). 31 1 P{ H} NMR (161.97 MHz, CDCl3): δ −51.2 (s) ppm. 13C{1H} NMR (100 MHz, MeOD): δ 149.4 (s, C6); 136.1 (s, C4); 121.6 (s, C3); 119.4 (s, C5); 77.1 (s, CC); 73.1 (s, NCH2N); 52.1 (s, PCH2N); 20.2 (s, SCH2). IR (KBr): 2158 cm−1 ν(CC). MALDI MS: m/z (%) 503 (40.45) [M]+. Anal. Calcd (%) for C14H18AuN4PS (502.07): C 33.47, H 3.61, N 11.15. Found: C 33.08, H 3.42, N 10.85. S25°,H2O: 83 g/L. [Au(CCCH2SC5H4N)(DAPTA)] (2): 79% yield, pale brown solid. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 2.01 (s, 6H, −CH3CO), 3.48 (d, 1H, J = 13.2 Hz, NCH2P), 3.82 (s, 2H, NCH2P), 3.96−4.08 (m, 3H, NCH2P + −SCH2), 4.12 (d, 1H, J = 11.3 Hz, NCH2N), 4.51− 4.59 (m, 2H NCH2N + NCH2P), 4.83 (d, 1H, J = 13.23 Hz, NCH2N), 5.67 (d, 1H, J = 13.2 Hz, NCH2P), 5.45 (dd, 1H, J = 11.1 Hz, J = 1.9 Hz, NCH2N), 6.91 (dd, 1H, J = 6.8, 5.3 Hz, H5), 7.14 (d, 1H, J = 8 Hz, H3), 7.42 (ddd, 1H; J = 7.5, 7.2, 1.6 Hz, H4), 8.34 (d, 1H, J = 4.1 Hz, H6). 31P{1H} NMR (161.97 MHz, CDCl3): δ −25.03 (s) ppm. 13 C{1H} NMR (100 MHz, MeOD): δ 170.1 (s, CO); 149.5 (s, C6); 136.2 (s, C4); 121.7 (s, C3); 119.7 (s, C5), 77.1 (s, CC), 67.1 (s, NCH2N), 61.8 (s, NCH2N), 49.4 (s, PCH2N), 44.9 (s, PCH2N), 39.3 (s, PCH2N), 21.4 (s, CH3), 20.2 (s, SCH2). IR (KBr): 1646 ν(CO), 2160 cm−1 ν(CC). Anal. Calcd (%) for C17H22AuN4O2PS (574.09): C 35.55, H 3.88, N 9.75. Found: C 35.80, H 3.58, N 10.02. S25°,H2O < 0.1 g/L. [Au(CCCH2SC5H4N)(PPh3)] (3): 68% yield, pale brown solid. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 4.14 (s, 2H, −SCH2), 6.97 (ddd, 1H, J = 8, 5.2, 0.8 Hz, H5), 7.28 (t, 1H; J = 1.8 Hz, H3), 7.45− 7.66 (m, 16H, H4 + PPh3), 8.45 (d, 1H, J = 1.7 Hz, H6). 31P{1H} NMR (161.97 MHz, CDCl3): δ 42.1 (s) ppm. 13C{1H} NMR (75.4 MHz, CDCl3): δ 149.4 (s, C6), 135.9, 134.3, 131.5 (s, Ph), 129.1 (s, C4), 121.7 (s, C3), 119.3 (s, C5), 99.2 (s, CC), 20.2 (s, CH2) ppm. IR (KBr): 2158 cm−1 ν(CC). MALDI MS: m/z (%): 608 (90) [M]+. Anal. Calcd (%) for C26H21AuNPS (607.08): C 51.41, H 3.48, N 2.31. Found: C 50.97, H 3.32, N 2.55. S25°,H2O < 0.1 g/L. [Au(CCCH2SC5H4N)(TPPTS)] (4): 89% yield, pale brown solid. 1H NMR (400 MHz, CDCl3, 25 °C): δ (ppm) 3.96 (s, 2H, −CH2), 7.12 (dd, 1H, J = 7.2, 54.9 Hz, H5), 7.33 (d, 1H; J = 8.1 Hz, H3), 7.43−7.61 (m, 12H, TPPTS), 7.69−7.67 (m, H4), 8.44 (d, 1H, J = 4.7 Hz, H6). 31 1 P{ H} NMR (161.97 MHz, CDCl3): δ 41.7 (s) ppm. IR (KBr): 2161 cm−1 ν(CC). Anal. Calcd (%) for C26H18AuNNa3O9PS4 (912.9): C 34.18, H 1.99, N 1.53. Found: C 34.30, H 2.11, N 1.75. S25°,H2O < 0.1 g/L. Synthesis of the [Cu{Au(CCCH2SC5H4N)2(L)}2]PF6 Complexes. To a solution of [Au(CCCH2SC5H4N)(L)] (L = PTA, DAPTA, PPh3, TPPTS) (0.2 mmol) in dichloromethane/acetonitrile (5/5 mL) under argon atmosphere was added [Cu(MeCN)4]PF6 (0.1 mmol). The solution was concentrated after 3 h of stirring at room temperature. Addition of diethyl ether gave pale brown solids, which were isolated by filtration. Using this method the following complexes were prepared. [Cu{Au(CCCH2SC5H4N)2(PTA)}2]PF6 (5): 78% yield, pale brown solid. 1H NMR (400 MHz, dmso-d6, 25 °C): δ (ppm) 3.94 (s, 4H, −SCH2), 4.27 (s, 12H, PCH2N), 4.51 and 4.36 (AB system, 12H, JAB = 4 Hz, NCH2N), 7.36−7.37 (m, 2H, H5), 7.57−7.58 (d, 2H, H3), 7.83− 7.87 (m, 2H, H4), 8.51 (s,br, 2H, H6). 31P{1H} NMR (161.97 MHz, CD2Cl2): δ −37 (s) ppm, −144.2 (sept, PF6−). 13C{1H} NMR (100 MHz, MeOD): δ 142.4 (s, C6), 138.7 (s, C4), 134.1 (s, C3), 126.1 (s, C5), 77.0 (s, CC), 70.1 (s, CC), 44.7 (m, NCH2N + PCH2N), 25.1 (s, SCH2). IR (KBr): 2163 cm−1 ν(CC), 830, 555 cm−1 ν(PF6−). MALDI MS: m/z (%): 1067 (10) [M]+; 565 (63%) [M − (CC−CH2−SC5H4N)PTA]+; 503 (100%) [M − (Cu(CC− 3717


Organometallics

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Measurements of Apoptosis. Human Caco-2 cell line PD7 and TC7 clones were exposed for 72 h with 20 μM of the metallic compounds, collected, and stained with Annexin V-FTIC according to the manufacturer’s recommendation. A negative control was prepared by unreacted cells, which was used to define the basal level of apoptic and necrotic or dead cells. After incubation, cells were transferred to flow-cytometry tubes, washed twice with temperate phosphatebuffered saline (PBS), and resuspended in 100 μL of annexin V binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), 5 μL of the annexin V-FITC, and 5 μL of PI to each 100 μL of cell suspension. After incubation for 15 min at room temperature in the dark, 400 μL of 1× annexin binding buffer was added and analyzed by flow cytometry within one hour. The signal intensity was measured using a FACSARIA BD and analyzed using FASCDIVA BD. Cell-Cycle Analyses. Human Caco-2 cell line PD7 and TC7 clones were exposed for 72 h with 20 μM of the metallic compounds. Cells were fixed in 70% ice-cold ethanol and stored at 4 °C for 24 h. After centrifugation, cells were rehydrated in PBS and stained in PI (50 μg/ mL) solution containing RNase A (100 μg/mL). PI-stained cells were analyzed for DNA content in a FACSARRAY BD equipped with an argon ion laser. The red fluorescence emitted by PI was collected by 620 nm longer pass filter, as a measure of the amount of DNA-bound PI, and displayed on a linear scale. Cell-cycle distribution was determined on a linear scale. The percentage of cells in cycle phases was determined using MODIFIT 3.0 verity software.

(%) for C53H52AuCuN5O5P3S (1224.51): C 51.99, H 4.28, N 5.72. Found: C 51.60, H 4.31, N 5.40. X-ray Structure Determination of 1. Pale brown crystals of 1, suitable for X-ray diffraction, were grown by slow diffusion of diethyl ether over dichloromethane solution. A crystal was mounted on a glass fiber with inert oil and centered in a Bruker-Smart CCD area-detector diffractometer with graphite-monochromated Mo Ka radiation (λ = 0.7107 Å) at 100 K. The diffraction frames were corrected for absorption with SADABS.96 The structure was solved by sir97.97 Fullmatrix least-squares refinement was carried out using SHELXL98 minimizing w(Fo2* − Fc2)2. Hydrogen atoms were included by using a riding model. Weighted R factors (Rw) and all goodness of fit S values are based on F2; conventional R factors are based on F. The complete crystallographic data have been deposited with the Cambridge Crystallographic Data Centre [CCDC 934115]. A copy of this material can be obtained free of charge from CCDC via www.ccdc. cam.ac.uk/data-request/cif or e-mail: [email protected]. Principle crystal and refinement data: empirical formula, C14H18AuN4PS; formula weight, 502.32; crystal system and space group, hexagonal, P3(2); a = 12.357(5) Å, b = 12.357(5) Å, c = 9.110(5) Å; volume, 1204.7(10) Å3; Z, 3; density (calculated), 2.077 Mg/m3; absorption coefficient, 9.386 mm−1; theta range for data collection, 1.90° to 24.97°; reflections collected, 6533; independent reflections, 2764 [R(int) = 0.1282]; R1(F2 > 2σ(F2)), 0.0720; wR2(all data), 0.1873; S(all data), 1.086. Distribution Coefficients (log D7.4). The n-octanol−water coefficients of the complexes were determined as previously reported99 using a shake-flask method. PBS-buffered distilled water (100 mL, phosphate buffer [PO43−] = 10 μM, [NaCl] = 0.15 M, pH 7.4) and noctanol (100 mL) were shaken together for 72 h to allow saturation of both phases. A 1 mg sample of the complexes was mixed in 1 mL of the aqueous and organic phase, respectively, for 10 min. The resultant emulsion was centrifuged to separate the phases. The concentrations of the compound in each phase were determined using UV absorbance spectroscopy. Log D7.4 was defined as [compound(organic)]/[compound(aqueous)]. Antiproliferative Assays. Human Caco-2 cell line PD7 and TC7 clones were kindly provided by Dr. Edith Brot-Laroche (Université Pierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers, France). Caco-2 cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Cells (passages 50−80) were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco Invitrogen, Paisley, UK) supplemented with 20% fetal bovine serum (FBS), 1% nonessential amino acids, 1% penicillin (1000 U/mL), 1% streptomycin (1000 μg/ mL), and 1% amphoterycin (250 U/mL). Experiments were performed 24 h postseeding. Stock solutions of the complexes (saline solution or DMSO) were diluted in complete medium to the required concentration. DMSO at similar concentrations did not shown any effects on cytotoxicity. For cytotoxicity screening cells were seeded in 96-well plates at a density of 4 × 103 cells/well. The culture medium was replaced with fresh medium (without FBS) containing the complexes at concentrations varying from 0 to 20 μM, with an exposure time of 72 h. Thereafter, the cell survivals were measured using the MTT test as previously described.100 The assay is dependent on the cellular reduction of 3(4,5-dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT, Merck) by the mitochondrial dehydrogenase of viable cells to a blue formazan product, which can be measured spectrophotometrically. Following appropriate incubation of cells, with or without the metallic complexes, MTT was added to each well in an amount equal to 10% of the culture volume and gentle stirring in a gyratory shaker, which enhances dissolution, and incubation was continued at 37 °C for 4 h. Thereafter the medium and MTT were removed, and DMSO was added in each well. At the end, the results are obtained by measuring absorbance with a scanning multiwell spectrophotometer (BIOTEX SINERGY HT SIAFRTD) at a wavelength of 560/670 nm and compared to the values of control cells incubated in the absence of complexes. Experiments were conducted in quadruplicate wells and repeated at least three times.

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ASSOCIATED CONTENT

* Supporting Information S

This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*(M. Laguna) E-mail: [email protected]. Fax: (+34) 976761187. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This article is dedicated to Prof. Dr. Ma Pilar Garcia, in memoriam. We thank Centro de Investigación Biomédica de Aragón (CIBA), España (http://www.ics.aragon.es), for the technical assistance and Torrecid S.A. for a generous donation of H[AuCl4].

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