Palladium(II), platinum(II) and gold(I) complexes containing chiral diphosphines of the Josiphos and Walphos families – Synthesis and evaluation as anticancer agents

Polyhedron 36 (2012) 97–103

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Palladium(II), platinum(II) and gold(I) complexes containing chiral diphosphines of the Josiphos and Walphos families – Synthesis and evaluation as anticancer agents Tebogo V. Segapelo a, Stacy Lillywhite a, Ebbe Nordlander b, Matti Haukka c, James Darkwa a,⇑ a b c

Department of Chemistry, University of Johannesburg, Auckland Park Kingsway Campus, P.O. Box 524, Johannesburg 2006, South Africa Inorganic Chemistry Research Group, Chemical Physics, Center for Chemistry and Chemical Engineering, Lund University, Box 124, SE-221 00 Lund, Sweden Department of Chemistry, University of Joensuu, Box 111, FI-80101 Joensuu, Finland

a r t i c l e

i n f o

Article history: Received 29 September 2011 Accepted 27 January 2012 Available online 2 February 2012 Keywords: Chiral ligands Diphosphine ligands Palladium complexes Platinum complexes Anticancer agents

a b s t r a c t A series of palladium(II) and platinum(II) complexes ([PdCl2(J003)] (1), [PdCl2(W001)] (2), [PtCl2(J003)] (3) and [PtCl2(W001)] (4), where J003 = the Josiphos ligand (R)-1-[(S)-2-diphenylphosphino)ferrocenyl]ethyldicyclohexylphosphine and W001 = the Walphos ligand (R)-1[(R)-2-(20 -diphenylphosphinyl) ferrocenyl]ethyldo(bis-3,5-trifluoromethylphenyl)phosphine), were prepared from the reaction of the diphosphine ligands with [PdCl2(NCMe)2] or [PtCl2(cod)] and characterised by multinuclear NMR spectroscopy, mass spectrometry and elemental analyses. Single crystal X-ray structures were used to confirm the proposed structures. Attempts to use the same ligands to prepare isoelectronic d8 Au(III) analogues of the palladium and platinum complexes resulted in the reduction of Au(III) to Au(I) and isolation of the Au(I) complexes [AuCl(J003)] (5), [Au2Cl2(J003)] (6) and [Au2Cl2(W001)] (7). The cytotoxicity of the four chiral, bidentate ferrocenylphosphine palladium and platinum complexes was investigated against HeLa cells and were found to have low to moderate cytotoxicity. In general, the two Josiphos complexes showed better cytotoxicity compared to the Walphos complexes, irrespective of the metal used. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction The importance of ferrocene and its applications in several areas of chemistry have been well documented and extensively reviewed [1]. One such application is its functionalization to diphosphine compounds. Due to their abundance and easy functionalization it is no surprise that diphosphine derivatives are the auxiliary ligands of choice in many coordination compounds. In recent years, the use of chiral diphoshinoferrocenes in areas of chemistry such as asymmetric catalysis has gained prominence [2]. Chiral diphosphine ligands of the Josiphos and Walphos structural families (Fig. 1) have been widely used and are known to impart very high degrees of enantio selectivity to several transition-metal-catalyzed reactions [3]. Furthermore, it is easy to vary the nature of the two ligating moieties attached to the same cyclopentadienyl ring independently from each other [4]. Another area where diphosphinoferrocene ligands have been used is in preparing gold(I) anticancer agents [5]. In this regard, emphasis has been on non-ferrocenyl diphoshines and disubstituted ferrocenyl phosphines such as diphenylphoshinoferrocene (dppf). Gold(III), being isoelectronic to platinum(II), has always ⇑ Corresponding author. E-mail address: [email protected] (J. Darkwa). 0277-5387/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2012.01.027

been seen as a potential source of anticancer agents similar to cisplatin. In a project where we sought to prepare palladium(II), platinum(II) and gold(III) complexes, with chiral phosphines, viz. Josiphos ligand (R)-1-[(S)-2-diphenylphosphino)ferrocenyl]ethyldicyclohexylphosphine (J003) and the Walphos ligand (R)-1 [(R)-2-(20 -diphenylphosphinyl)ferrocenyl]ethyldo(bis-3,4-trifluoromethylphenyl)phosphine) (W001), we reacted palladium(II), platinum(II) or gold(III) starting materials with these diphosphines, with the knowledge that chiral phosphines can be discriminate between healthy and diseased cells. Here we report the synthesis and structures of Josiphos and Walphos diphosphine complexes of the above-mentioned group 10 and 11 metals, as well as their antitumor activity on HeLa cells. 2. Results and discussion 2.1. Synthesis Complexation of the chiral Josiphos and Walphos diphosphine ligands with palladium were performed in dichloromethane using [PdCl2(NCMe)2] as the metal precursor (Scheme 1). Using the Josiphos ligand J003, [PdCl2(J003)] (1) was obtained as an orange solid in 86% yield, while the Walphos ligand gave [PdCl2(W001)] (2) as a red solid in 60% yield. Complexation of the same chiral

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R1

R2 R1

R2

P P

Fe

R1

R2

P

P

Me

Fe

R1

Josiphos (J-) family

R2

Me

Walphos (W-) family

Fig. 1. Framework structures of the Josiphos and Walphos families of ferrocenebased chiral diphosphine ligands; J003: R1 = R2 = cyclohexyl; W001: R1 = phenyl; R2 = 3,5-trifluoromethylphenyl.

diphosphine ligands with platinum were performed using [PtCl2(cod)] as the metal precursor (Scheme 1). In both instances the complexes were obtained as orange solids, with a 61% yield for [PtCl2(J003)] (3) and 65% yield for [PtCl2(W001)] (4). Single crystals of 1 and 3 suitable for X-ray crystallographic studies were obtained by slow evaporation of dichloromethane solutions of the complexes at room temperature. The molecular structure of [PdCl2(J003)] (1) is shown in Fig. 2 and that of [PtCl2(J003)] (3) in Fig. 3. Their crystal data are presented in Table 1 and selected bond distances and angles are given in Table 2. In 1, the geometry around the Pd atom is distorted square planar with two cis chlorines and two phosphorus atoms. The structure is similar to that of the analogous palladium complex [PdCl2(J002)] (J002 = FcCHMePtBu2(PCy2), cf. Fig. 1) in which the two phosphorus atoms carry tert-butyl (tBu) and cyclohexyl (Cy) substituents [6]. The P(2)–Pd(1)–P(1) and Cl(2)–Pd(1)–Cl(1) bond angles of 96.23(3)° and 88.44(3)° in 1 are in the same range as those observed for [PdCl2(J002)] (97.03° and 86.48°, respectively). However, the P(2)–Pd(1)–P(1) bond angles in 1 and [PdCl2(J002)] are larger than those found in [FcCHMePCy2(PPh2)PdMe2(J001)] (93.26(9)°) (J001 = FcCHMePCy2(PPh2), cf. Fig. 1) [7]. The Pd–P bond distances in 1 for Pd(1)–P(1) and Pd(1)–P(2) are 2.2717(7)

and 2.2702(7) Å, respectively. These Pd–P bond distances are in agreement with the expected donor ability of the two PCy2 groups on the palladium atom. The Pd(1)–P(2) bond distances in 1 is similar to 2.276 Å reported for [PdCl2(J002)] [6] where P(2) is also directly attached to the cyclopentadienyl ring. In contrast, the Pd(1)–P(1) distance of 2.327(3) Å in [PdMe2(J001)] is significantly longer than in 1. Despite the similar Pd–P bond distances in 1, the Pd–Cl distances are different. For Pd(1)–Cl(1) the distance is 2.3781(7) Å while for Pd(1)–Cl(2) it is 2.3416(7) Å. Generally an increase of the P–Pd–P angle in achiral ferrocenyl diphosphine compounds results in lengthening of Pd–P bonds and a decrease of the Cl–Pd–C1 angle and Pd–C1 bond distances [8]. The discrepancies observed between 1 and [PdMe2(J001)] can thus be ascribed to the electron-withdrawing effect of the chlorine in the former, relative to the donor effects of the methyl groups in the latter. Similarly, the structure of [PtCl2(J003)] (3) can be described as a distorted square planar geometry with two cis chlorine and two cis phosphorus atoms. The P(2)–Pt(1)–P(1) and Cl(2)–Pt(1)–Cl(1) bond angles of 96.41(6)° and 85.76(6)° are in the same range as observed by Ghent et al. [6] for [PtCl2(J002) (97.33(3)° and 83.54(3)°, respectively). The Pt(1)–P(1) and Pt(1)–P(2) bond distances in 3 are 2.2641(18) and 2.2517(17) Å, respectively, and the Pt(1)–Cl(1) and Pt(1)–Cl(2) distances are 2.3713(17) and 2.3669(16) Å, respectively. The Pt–Cl bond trans to the PCy2 directly attached to the cyclopentadienyl ring in 3 (2.3669(16) Å) and in [PtCl2(J002)] (2.3696(9) Å) are also similar. Attempts to prepare the corresponding d8 gold(III) complexes resulted in reduction of the gold(III) precursor, H[AuCl4]4H2O, to produce gold(I) complexes, as has also been observed for reactions with the achiral diphsophine ligands bis(diphenylphosphino)ferrocene (dppf) and bis(diisopropylphosphino)ferrocence (dippf) recently been reported by us [9]. The gold(I) complexes could thus be prepared from either [AuCl(tht)] (tht = tetrahydrothiophene) or H[AuCl4]4H2O (Schemes 2 and 3). The 1H NMR spectra of these chiral diphosphine gold complexes are complicated by their

Cl

Me P

Cl M

PdCl2(NCMe)2 or PtCl2(cod)

P

P

P

CH2Cl2 Fe

Fe

J003

M = Pd (1), Pt (3) F3C CF3

Ph2P

Me

P

P

Cl

Cl

CF3

M

CF3

P

CF3 Me CF3

Fe F3C

PdCl2(NCMe)2 or PtCl2(cod)

Fe

CH2Cl2

W001

M = Pd (2), Pt (4) Scheme 1. Schematic description of synthetic routes to complexes 1–4.

CF3

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99

Fig. 2. The molecular structure of 1 shown with 50% ellipsoids (for clarity, hydrogen atoms have been omitted).

Fig. 3. The molecular structure of 3 shown with 50% ellipsoids (for clarity, hydrogen atoms have been omitted).z

rotational behaviour and could therefore not provide reliable structural information. Rotational averaging arises because of the linear mode of binding around the gold atom in these complexes, thus allowing free rotation about the stereogenic centre. Better insight into the nature of the gold compounds was therefore obtained from 31P NMR spectroscopic data and mass spectrometry. Reaction of J003 with one equivalent of [AuCl(tht)] in dichloromethane afforded [AuCl(J003)] (5) as a brick red solid in 64% yield (Scheme 2). The formation of the product depends on the stoichiometry of the reagents. Interestingly, when one equivalent of [AuCl(tht)] was used, a three-coordinate gold(I) complex in which one gold atom is attached to the two phosphine moieties is formed.

The 31P NMR spectrum of the complex shows peaks at d 39.11 and 63.45 ppm corresponding to the two phosphorus atoms (Fig. S1), indicating coordination of both phosphine groups to the gold atom, and the absence of fluxional behaviour strongly supports the chelating binding mode of the J003 diphosphine ligand. Addition of one more equivalent of [AuCl(tht)] to 5 did not have any effect as no changes in the 31P NMR spectrum were observed. Formation of three coordinate gold(I) complexes has been observed in the literature with basic ferrocenyl-phosphine ligands such as [Fe(g5C5Me4PPh2)2] (dppomf) [10]. Given the general donor and basic nature of J003, the formation of a three-coordinate complex is not surprising. Further confirmation of the nature of 5 was

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obtained from mass spectrometry data, in which a parent ion corresponding to [Au(J003)]+ was observed at m/z = 804 (100%). The results from mass spectrometry were also backed by elemental analysis data (cf. Section 4). When two equivalents of [AuCl(tht)] were used, the expected dinuclear gold complex [Au2Cl2(J003)] (6), in which each gold atom is separately bound to one phosphine group, was formed (Scheme 2). When J003 was reacted with H[AuCl4]4H2O in a 1:1 mixture of dichloromethane and ethanol, it afforded an intractable red material. In contrast, the reaction of W001 with H[AuCl4]4H2O under similar reaction conditions led to the precipitation of [Au2Cl2(W001)] (7) as a beige solid (Scheme 3). This complex is proposed to consist of the diphosphine ligand coordinated by each of its phosphine moiety to a separate Au–Cl moiety. The reason for the formation of this dinuclear complex rather than a structure analogous to that of 5 is most likely due to the rigidity of the Walphos framework relative to the Josiphos framework. Although the chelating coordination mode has been demonstrated for W001 in compounds 2 and 4 (vide supra), this is expected to be a more strained coordination mode for the Walphos ligand when compared to analogous Josiphos ligands. Thus, the less strained and open conformation is adopted in 7. Also for this compound, rotational averaging was detected by 1H NMR spectroscopy at ambient temperature. The 31P NMR spectrum of 7 showed only one broad peak at 23.8 ppm at room temperature but variable temperature 31 P NMR studies revealed a second peak at 39.5 ppm at 243 K. The mass spectrum of compound 7 showed a peak corresponding to the loss of one chloride ligand at m/z = 1359 (30%) and elemental analysis was in good agreement with the proposed formula.

Table 1 Crystal data and structure refinement parameters for 1 and 3. Complex

1

3

Empirical formula Formula weight Temperature (K) Wavelength (Å) Crystal system Space group a (Å) b (Å) c (Å) a (°) b (°) c (°) Volume (Å3) Z DCalc (mg/m3) l(Mo Ka) (mm1) Crystal size (mm3) Theta range for data collection (°) Reflections collected Unique reflections Absorption correction

C36H56Cl2FeP2Pd 783.90 120(2) 0.71073 tetragonal P43 14.7718(8) 14.7718(8) 16.6620(18) 90 90 90 3635.7(5) 4 1.432 1.152 0.15  0.09  0.08 2.30–27.52

C39H63Cl2FeP2Pt 915.67 100(2) 0.71073 tetragonal P41212 15.4968(2) 15.4968(2) 34.7033(6) 90 90 90 8334.0(2) 8 1.460 3.931 0.27  0.09  0.04 2.94–25.34

62 732

45 275 7616 Semi-empirical from equivalents 1.046 R1 = 0.0340, wR2 = 0.0771

2

Goodness-of-fit on F Final R indices (I P 2r) a b

Semi-empirical from equivalents 1.066 R1a = 0.0298, wR2b = 0.0444

R1 = R||Fo|  |Fc||/R|Fo|. wR2 = [R[w(Fo2  Fc2)2]/R[w(Fo2)2]]1/2.

Table 2 Selected bond lengths [Å] and angles [°] for 1 and 3.

2.2. Biological studies

Bond lengths [Å]

1

3

M(1)–P(2) M(1)–Cl(2) M(1)–P(1) M(1)–Cl(1)

2.2702(7) 2.3416(7) 2.2717(7) 2.3781(7)

2.2517(17) 2.3669(16) 2.2641(18) 2.3713(17)

Bond angles [°]

1

3

P(2)–M(1)–P(1) P(1)–M(1)–Cl(2) P(1)–M(1)–Cl(1) P(2)–M(1)–Cl(2) P(2)–M(1)–Cl(1) Cl(2)–M(1)–Cl(1)

96.23(3) 85.29(3) 173.41(3) 171.65(3) 90.27(3) 88.44(3)

96.41(6) 88.21(6) 173.17(6) 170.04(6) 90.05(6) 85.76(6)

Complexes 1–4 were screened for antitumour activity against human cervix epithelial carcinoma (HeLa), using cisplatin as a comparative standard. The complexes were added to sterile 96well culture plates containing HeLa cells in 100 lL of medium/well, and the cytotoxicity was assayed by spectrophotometry, following the decomposition of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by living cells. The complexes containing the Josiphos ligand J003 were found to show better cytotoxicity, which is a reflection of their good solubility and stability in a mixture of DMSO and water. The IC50 values obtained for the Josiphos complexes [PdCl2(J003)] (1) and [PtCl2(J003)] (3) were six to seven times higher than observed for cisplatin, while Cl P

[AuCl(tht)] (1 eq.)

Au

P Me

Fe Me P

(5) P

Fe

Cl Me P

J003 [AuCl(tht)] (2 eq.)

Au P

Au Cl Fe

(6) Scheme 2. Schematic description of synthetic routes to complexes 5 and 6.

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CF3

Cl

Me

Me

P

P

CF3 CF3

Fe

HAuCl4 EtOH/CH2Cl2

F3C

Au P

P Au

(7)

CF3 CF3

Fe

Cl

CF3

F3C

W001 Scheme 3. Schematic description of synthetic route to complex 7.

phine (J003) and (R)-1-[(R)-2-(20 -diphenyl-phosphinophenyl)ferrocenyl]ethyl-di(bis-3,5-trifluoromethylphenyl) phosphine (W001) were generously donated from Solvias, Basel, Switzerland, and used as received. NMR spectra were recorded on a Varian Unity INOVA 500 spectrometer working at 499.77 MHz (1H). Chemical shifts are reported in d (ppm) downfield from TMS using residual solvent peaks (1H, 13C NMR) or using H3PO4 as an external reference (31P). Microanalyses were carried out at the University of Cape Town (South Africa) using a Fisons EA 1108 CHNS elemental analyzer. Mass spectrometry was performed at the University of Stellenbosch (South Africa) on a Waters API Quatro Micro at a cone voltage of 15 V.

50

IC50 (µM)

40

30

20

4.2. Preparation of Josiphos and Walphos metal complexes

10

0 4

2

1

3

Cisplatin

Compound tested Fig. 4. IC50 values for complexes 1–4 and the reference compound cisplatin.

those observed for the Walphos complexes [PdCl2(W001)] (2) and [PtCl2(W001)] (4) were 35–38 times higher than cisplatin (Fig. 4). Although the palladium complexes were found to decay extensively in solution compared to the platinum complexes, they showed activity comparable to their platinum analogues. Despite this similar cytotoxic effect, the platinum complex 3 was found be three times more specific in its action towards deceased cells compared to 1. 3. Conclusions Cytotoxicity studies performed on the palladium and platinum complexes revealed that complexes containing Josiphos have better cytotoxicity than their Walphos counterparts, but the selectivity between deceased and normal cells for complexes from both ligands is low. 4. Experimental 4.1. Materials and instrumentation All manipulations were performed under a dry, deoxygenated nitrogen atmosphere using standard Schlenk techniques. Hexane, benzene, toluene, diethyl ether, and tetrahydrofuran (THF) were dried by refluxing and distilling over sodium diphenyl ketyl under a nitrogen atmosphere. All other solvents were of reagent grade and were used without further purification, unless oxygen-free solvent was needed, in which case the solvent was purged with nitrogen for ca. 15 min. The diphosphine ligands (R)-1-[(S)-2-(dicyclohexylphosphino)-ferrocenyl]ethyldicyclo-hexylphos

4.2.1. Preparation of dichloro-{(R)-1-[(S)-2-(dicyclohexylphosphino) ferrocenyl]ethyldi-cyclohexylphosphine}palladium (1) To a solution of the ligand J003 (0.17 g, 0.28 mmol) in dry dichloromethane (10 mL) was added [PdCl2(NCMe)2] (0.071 g, 0.28 mmol). The solution was stirred for 18 h and then filtered, and solvent evaporated under reduced pressure. The product was recrystallized from a 1:2 mixture of dichloromethane and hexane and was obtained as an orange solid. Yield = 0.19 g, 86%. 1H NMR (CDC13): d 4.84 (s 1H, g5-C5H3), 4.52 (s 2H, g5-C5H3), 4.27 (s 5H, g5-C5H5), 3.66 (t, 3H, CHMePCy2), 2.34 (q, 1H, CHMePCy2), 1.53 (m, 20H, PCy2). 31P{1H} NMR (CDCl3): d 33.68 (s), 91.99 (s). MS (ESI): m/z (%) 749 (M+Cl, 100). Anal. Calc. for C36H56Cl2FeP2Pd: C, 55.15; H, 7.20. Found: C, 56.07; H, 7.34%. Complexes 2–4 were prepared using similar procedures as described for the synthesis of 1, but using the respective reagents shown for each complex. 4.2.2. Preparation of dichloro-{(R)-1-[(R)-2-(20 -diphenylphosphino phenyl)ferrocenyl]-ethyldi(bis-3,5-trifluoromethylphenyl)phosphine} palladium (2) Complex 2 was prepared from W001 (0.11 g, 0.12 mmol) and [PdCl2(NCMe)2] (0.030 g, 0.12 mmol). A red solid was isolated. Yield = 77.4 mg, 60%. 1H NMR (CDC13): d 8.14 (m, 4H), 8.04 (d, 3H, 3JHH = 10.0 Hz), 7.65 (br, 3H), 7.38 (m, 4H), 7.21 (m, 2H), 6.85 (q, 1H, 3JHH = 7.5 Hz), 4.23 (s 5H, g5-C5H5), 4.09 (s 1H, g5-C5H3), 3.90 (s 1H, g5-C5H3), 3.04 (q, 1H, CHMeP, 3JHH = 8.5 Hz), 2.96 (dd, 3H, CHMeP{C6H3(CF3)2}2, 3JHH = 7.5 Hz, 4JHH = 1.2 Hz). 31P{1H} NMR (CDCl3): d 46.52 (s), 25.17 (s). MS (ESI): m/z (%) 1073 (M+Cl, 95). Anal. Calc. for C46H32Cl2F12FeP2Pd: C, 49.87; H, 2.91. Found: C, 50.54; H, 2.81%. 4.2.3. Preparation of dichloro-{(R)-1-[(S)-2(dicyclohexylphosphino) ferrocenyl]ethyldi-cyclohexylphosphine}platinum (3) Complex 3 was prepared from J003 (0.17 g, 0.28 mmol) and [PtCl2(cod)] (0.10 g, 0.28 mmol). The product was an orange solid. Yield = 0.15 g, 86%). 1H NMR (CDC13): d 4.79 (s 1H, g5-C5H3), 4.49 (s 1H, g5-C5H3), 4.44 (s 1H, g5-C5H3), 4.24 (s 5H, g5-C5H5), 3.64 (t, 3H, CHMePCy2), 2.34 (q, 3H,CHMePCy2, 3JHH = 8.4 Hz),

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1.66 (m, 20H, PCy2). 31P{1H} NMR (CDCl3): d 9.33 (s), 55.07 (s). MS (ESI) m/z (%) 837 (M+Cl, 100). Anal. Calc. for C36H56Cl2FeP2Pt0.5Et2O: C, 50.17; H, 6.76. Found: C, 50.44; H, 6.38%. 4.2.4. Preparation of dichloro-{(R)-1-[(R)-2-(20 -diphenylphosphino phenyl)ferrocenyl]-ethyldi(bis-3,5-trifluoromethylphenyl)phosphine} platinum (4) Complex 4 was prepared from W001 (0.10 g, 0.11 mmol) and [PtCl2(cod)] (0.040 g, 0.11 mmol) and product isolated as an orange solid. Yield = 83 mg, 65%. 1H NMR (CDC13): d 8.12 (m, 4H), 8.04(q, 1H, 3JHH = 11.5 Hz), 7.99 (d, 2H, 3JHH = 10.0 Hz), 7.62 (t, 1H, 3 JHH = 7.5 Hz), 7.51 (t, 1H, 3JHH = 7.5 Hz), 7.36 (m, 6H), 7.22 (m, 2H), 7.18 (m, 2H), 6.85 (q, 1H, 3JHH = 7.5 Hz), 4.22 (s 5H, g5C5H5), 4.05 (s 1H, g5-C5H3), 3.88 (s 1H, g5-C5H3), 3.01 (q, 1H, CHMePCy2, 3JHH = 8.5 Hz), 2.94 (dd, 3H, CHMeP{C6H3(CF3)2}2, 3 JHH = 7.5 Hz, 4JHH = 1.2 Hz). 31P{1H} NMR (CDCl3): d 6.99 (s), 28.42 (s). MS (ESI): m/z (%) 1161 (M+Cl, 100). Anal. Calc. for C46H32Cl2F12FeP2Pt2Et2O: C, 48.23; H, 3.89. Found: C, 48.26; H, 3.07%. 4.2.5. Preparation of dichloro-{(R)-1-[(S)-2(dicyclohexylphosphino) ferrocenyl]ethyldi-cyclohexylphosphine}gold(I) chloride (5) To a solution of the ligand J003 (0.17 g, 0.28 mmol) in dry dichloromethane (10 mL) was added [AuCl(tht)] (0.088 g, 0.28 mmol). The solution was stirred for 2 h and then filtered, and solvent evaporated under reduced pressure. The product was obtained as a brick red solid. Yield = 0.19 g, 81%. 31P{1H} NMR (CDCl3): d 39.11 (s), 63.45 (s). MS (ESI): m/z (%) 804 (M+Cl, 100). Anal. Calc. for C36H56AuClFeP2CH2Cl2: C, 48.09; H, 6.32. Found: C, 48.39; H, 6.56%. 4.2.6. Preparation of {(R)-1-[(S)-2(dicyclohexylphosphino) ferrocenyl]ethyldi-cyclohexylphosphine}bis(gold(I)) dichloride (6) To a solution of the ligand J003 (0.021 g, 0.07 mmol) in dry dichloromethane (5 mL) was added [AuCl(tht)] (0.022 g, 0.07 mmol). The solution was stirred for 1.5 h and then filtered, and solvent evaporated under reduced pressure. The product was obtained as an orange sticky solid. Yield = 0.05 g, 70%. 31P{1H} NMR (CDCl3): d 34.25 (br s), 58.82 (s). MS (ESI): m/z (%) 1156.14 (M+Cl, 100). Anal. Calc. for C36H56Au2Cl2FeP2CH2Cl2: C, 38.43; H, 5.06. Found: C, 38.26; H, 5.37%. 4.2.7. Preparation of dichloro-{(R)-1-[(R)-2-(20 -diphenylphosphino phenyl)ferrocenyl]-ethyldi(bis-3,5-trifluoromethylphenyl)phosphine} digold(I) chloride (7) To a solution of W001 (90 mg, 0.097 mmol) in dry dichloromethane (5 mL), a solution of [HAuCl4]4H2O (40 mg, 0.097 mmol) in ethanol (5 mL) was added drop wise. The solution changes from orange to dark brown upon addition of the auric acid. A beige precipitate started forming within 5 min. The solution was stirred for 12 h and then filtered, and the precipitate dried. The product was obtained as beige solid. Yield = 56 mg, 42%. 31P{1H} NMR at 243 K (CDCl3): d 39.41 (vbr s), 24.10 (br s). MS (ESI) m/z (%) 1359 (M+Cl, 30). Anal. Calc. for C46H32Au2Cl2F12FeP2: C, 39.60; H, 2.31. Found: C, 39.37; H, 2.17%.

correction (SADABS [15] or Xprep in SHELXTL [16] was applied to all data. Structural refinements were carried out using SHELXL-97. In 2, hexane of crystallization was partially lost and therefore it was refined with occupation 0.5. The carbon atoms of the hexane molecule were restrained with effective standard deviation 0.01–0.02 so that their Uij components approximate to isotropic behavior. Also, one of the cyclohexyl groups (C7–C12) was disordered over two sites with occupancies 0.60/0.40. The anisotropic displacement parameters of the adjacent carbons in the disordered cyclohexyl group as well as in another cyclohexyl group (C1–C6) were restrained to be similar. Hydrogens were positioned geometrically and constrained to ride on their parent atoms, with C–H = 0.95–1.00 Å and Uiso = 1.2–1.5 Ueq (parent atom). The crystallographic details are summarized in Table 1 and selected bond lengths and angles in Table 2.

4.4. Anticancer evaluation 4.4.1. Biological reagents and instrumentation All commercial reagents were used as received. Phosphate Buffered Saline (PBS), Eagle’s RPMI-1640 medium, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) kit, phytohemagglutinin-protein form (PHA-P) and the 96-well flatbottomed culture plates were all purchased from BD Biosciences Ltd. Cisplatin was purchased from Sigma Aldrich. Eagle’s medium with 0.1 mM non-essential amino acids was prepared by adding 2 mM L-glutamine, 1.0 mM sodium pyruvate and 5% bovine foetal calf serum to the pure Eagle’s RPMI-1640 medium. Five compounds (cisplatin and 1–4) were screened for their antitumour activities against Human cervix epithelial carcinoma (HeLa) by the Department of Pharmacology, University of Pretoria, South Africa. The absorbance values were recorded on a Whittaker Microplate Reader 2001 spectrophotometer at 570 nm and the reference wavelength of 630 nm.

4.4.2. Cell culture and drug treatment HeLa cells were cultured in Eagle’s medium with 0.1 mM nonessential amino acids, 2 mM L-glutamine, 1.0 mM sodium pyruvate and 5% bovine fetal calf serum, at 37 °C in an atmosphere of 5% CO2. Cells were plated in 96-well sterile plates at a density of 1  104 cells/well in 100 lL of medium and incubated for 1 h. Subsequently, compounds were added with concentrations from 0 to 50 lM. Cytotoxicity was determined by using MTT to stain treated HeLa cells after 7 days according to literature methods [17]. MTT dye is reduced by living cells to yield a soluble formazan product that can be assayed colourimetrically [17]. A 20 lL volume of freshly prepared MTT (5 mg/mL) was added to each well and the cells incubated for another 4 h. Cell survival was evaluated by measuring absorbance at 570 nm, using a Whittaker Microplate Reader 2001. The IC50 values were calculated with the Graphpad programme. All experiments were performed in triplicate.

4.3. X-ray structure determinations Acknowledgements The crystals of 1 and 3 were immersed in cryo-oil, mounted in a Nylon loop, and measured at a temperature of 100–120 K. The Xray diffraction data were collected on a Nonius KappaCCD diffractometer using Mo Ka radiation (k = 0.71073 Å). The Denzo-Scalepack program package [11] was used for cell refinements and data reductions. The structures were solved by direct methods using the SIR2002, SIR2004 [12] or SHELXS-97 [13] programs with the WINGX [14] graphical user interface. A semi-empirical absorption

This research has been supported by grants from Mintek (South Africa) and National Research Foundation (NRF) (South Africa) (J.D.) and the Swedish Research Council (V.R. to E.N.). Exchange between the South African and Swedish groups has been funded by a grant from the Swedish International Development Agency (SIDA), administered by V.R. and N.R.F. We thank Ahmed Fawzy for the preparation of Figures 2 and 3.

T.V. Segapelo et al. / Polyhedron 36 (2012) 97–103

Appendix A. Supplementary data A plot of the 31P NMR spectrum of [AuCl(J003)] (5). CCDC numbers 845977 and 845976 contains the supplementary crystallographic data for (1) and (3), respectively. These data can be obtained free of charge via http://www.ccdc.cam.ac.uk/conts/ retrieving.html, or from the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: (+44) 1223 336 033; or e-mail: [email protected]. Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.poly.2012.01.027. References [1] (a) A. Togni, T. Hayashi, Ferrocenes: Homogeneous Catalysis, Organic Synthesis and Materials Science, VCH, Weinheim, 1995; b) N.J. Long, Metallocenes: An Introduction to Sandwich Complexes, Blackwell Science, Oxford, 1998; c) G. Bandoli, A. Dolmella, Coord. Chem. Rev. 209 (2000) 161. [2] P. Barbaro, C. Bianchini, G. Giambastiani, S.L. Parisel, Coord. Chem. Rev. 248 (2004) 2131. [3] H.-U. Blaser, W. Brieden, B. Pugin, F. Spindler, M. Studer, A. Togni, Top Catal 19 (2002) 3.

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