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Synthesis of a heterodinuclear ruthenium(II)–platinum(II) complex linked by l -cysteine methyl ester
Polyhedron 26 (2007) 318–328 www.elsevier.com/locate/poly
Synthesis of a heterodinuclear ruthenium(II)–platinum(II) complex linked by L-cysteine methyl ester Sarah J. Yousouf, Craig R. Brodie, Nial J. Wheate, Janice R. Aldrich-Wright
*
School of Biomedical and Health Sciences, University of Western Sydney, Campbelltown, NSW 2560, Australia Received 22 March 2006; accepted 2 June 2006 Available online 10 June 2006
Abstract A potential anticancer heterodinuclear ruthenium(II)–platinum(II) complex, [ruthenium(II)(4,4 0 -dimethyl-2,2 0 -bipyridine)2(5-(L-cysteine-methyl ester)-1,10-phenanthroline)-trans-chlorodiammineplatinum(II)] chloride, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3, was synthesised. L-Cysteine methyl ester was used to link the two metal centres, as more conventional straight chain diaminoalkanes and 2-mercaptoethylamine failed to couple to the phenanthroline ligand. From the precursor mononuclear ruthenium(II) complexes, which were separated into their D- and K-isomers by column chromatography, the dinuclear complex was synthesised and characterised by 1H and 13C NMR, UV–Vis, circular dichroism, fluorescence and electrospray ionisation mass spectrometry. 2006 Elsevier Ltd. All rights reserved. Keywords: Ruthenium(II); Platinum(II); Heterodinuclear; L-Cysteine methyl ester; Phenanthroline; Anticancer
1. Introduction Cisplatin, carboplatin and oxaliplatin are the only platinum-based drugs widely approved for the treatment of human cancers [1]. Over the last decade, multi-nuclear platinum drugs, like BBR3464 (a novel trinuclear complex having two mono-functional trans-[PtCl(NH3)2] units coupled by a central trans-[Pt(NH3)2(H2N(CH2)6NH2)2] linker), have demonstrated clinical potential, but are limited by toxic side-effects and in vivo degradation before reaching their cellular target, DNA [1,2]. Both mono- and multinuclear drugs derive their anticancer activity by forming coordinate covalent adducts with DNA, thereby preventing replication and transcription [3]. By changing the structure of the DNA adducts, or increasing the DNA binding affin-
*
Corresponding author. Present address: School of Biomedical and Health Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia. Tel.: +61 2 4620 3218; fax: +61 2 4620 3025. E-mail address: [email protected] (J.R. Aldrich-Wright). 0277-5387/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2006.06.004
ity, new complexes may be developed that are able to overcome drug resistance. Over the last 20 years many researchers have examined the synergistic properties of combining a DNA intercalating compound with a moiety capable of covalent binding to DNA. These new complexes include: organic intercalators linked to cisplatin-like molecules [4–21], octahedral metal intercalators coupled to cisplatin-like molecules [22–27], and organic intercalators linked to DNA-alkylating agents like mustards [28–32]. Recently, carborane cages have been attached to intercalators for use in radiation therapy [33–36]. These synergistic compounds have been developed to increase the DNA binding affinity and drug transport properties of conventional chemotherapeutic drugs and/or to modify their DNA adducts and sequence selectivity. There has been little research, however, into heteronuclear metal complexes which link platinum groups capable of forming DNA coordinate covalent adducts to other groups capable of DNA groove binding. In this paper we report the synthesis of a heterodinuclear ruthenium(II)–platinum(II) metal complex, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3,
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Table 1 Synthetic yield and characterisation data for the heterodinuclear ruthenium(II)–platinum(II): [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ and its mononuclear precursors for the racemic and D- and K-isomer solutions Metal complex
% Yield
D,K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2
77
ESI-MS (m/z)
285 (246) 285 (+301) 54
285 (1955)
285 (+1998)
78
K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3
containing a platinum moiety attached to a DNA groove binding ruthenium moiety via a modified amino acid linker. This metal complex was designed as a potential anticancer agent that may display increased DNA binding affinity compared to native mononuclear platinum complexes (Table 1). 2. Experimental 2.1. Materials and methods 2.1.1. Materials Deuterated acetonitrile (d3-CD3CN) and dimethylsulphoxide (d6-DMSO) were obtained from Cambridge Isotope Laboratories. ()-O,O 0 -dibenzoyl-L-tartaric acid and Sephadex LH20 were obtained from Fluka. Silica gel 60 was obtained from Merck. 2-Mercaptoethylamine hydrochloride, 1,6-diaminohexane, 1,3-diaminopropane, L-cysteine methyl ester (tert-butoxy)carbonate (L-cysteineMeBOC), DOWEX 550A OH anion exchange resin (DOWEX), Amberlite IRA-400(Cl) ion exchange resin (Amberlite), potassium hexafluorophosphate, SP Sephadex C25, 4,4 0 -dimethyl-2,2 0 -bipyridine (Me2bipy), 5chloro-1,10-phenanthroline (5-Cl-phen) and deuterated methanol (d4-CD3OD) were all purchased from Aldrich. Ruthenium(II) trichloride hydrate {RuCl3 Æ xH2O (Ru 40–43%)} and trans-diamminedichloroplatinum(II) (transplatin) were purchased from Precious Metals Online. [Ru(Me2bipy)2Cl2] was prepared as previously described [37,38]. All other reagents and solvents were of analytical
Fluorescence quantum yield
0.035 0.035
1028.0
K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2
D,K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3 D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3
UV–Vis kmax nm (e) 451 (8.91 · 103) 261 (4.58 · 104)
341.7
D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 D,K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2
CD kmax nm (De)
456 334 279 456 334 267
(1.11 · 103) (9.21 · 102) (7.69 · 103) (1.61 · 103) (1.38 · 103) (3.39 · 104)
456 334 257 282 206 456 334 257 282 206
(1.55 · 104) (1.25 · 104) (3.17 · 104) (3.17 · 104) (6.95 · 104) (1.45 · 104) (1.17 · 104) (2.88 · 104) (6.09 · 104) (6.03 · 104)
0.086
0.157
342.1 285 (2808)
285 (+2505)
grade and were used as received, except trifluoroacetic acid (TFA) which was redistilled before use. Aqueous solutions were prepared with Milli-Q water (Millipore, MA). 2.1.2. Nuclear magnetic resonance spectroscopy 1 H NMR spectra were obtained with a 300 MHz Varian Mercury VX spectrometer, a 400 MHz Varian Mercury VX spectrometer, or a Varian 300 MHz UnityPlus spectrometer. All spectra were recorded at 25 C, unless otherwise stated and were internally referenced to the solvent resonances. All final and precursor metal complexes were characterised using NOESY, g-DQCOSY, HMBC and HMQC experiments. Spectra were obtained over a spectral width of 4200 Hz with a mixing time of either 300 or 800 ms using 2048 points in the t2 dimension with 256 increments and a total recycle time of 1.5 s. 2.1.3. UV–Vis spectrophotometry Absorption spectra were measured with a Varian Cary 300 Bio UV–Vis double-beam spectrophotometer. Spectra were obtained over the range of 200–800 nm with samples in 1 cm quartz cells, which were referenced to water. A scan rate of 600 nm/min, data interval of 1 nm and average time of 0.1 s were utilised. Extinction coefficients were determined by repeated measurement of the absorbance of known concentrations of the metal complexes (as PF6 salts) in a 3% acetone/water solution. All absorbances were within the range 0.3–0.8, and the average of repeated measurements at kmax gave the extinction coefficient, emax.
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2.1.4. Circular dichroism spectropolarimetry CD spectra were acquired with a JASCO J-810 spectropolarimeter equipped with J-800 for Windows. Samples were prepared in either 3% acetone/water or in water and were placed in 0.1 cm cylindrical quartz cells. Data collection was performed at room temperature, between 200 and 650 nm, and at a speed of 500 nm/min. A sensitivity setting of 100 mdeg, data pitch of 0.5 nm, response time of 1 s and bandwidth of 1 s were used. Spectra are given as an average of 10 scans. From the angle of ellipticity (h, mdeg), the molar absorptivity (De, mdeg/M cm) was calculated using Eq. (1), where c is the concentration (mol/L) and l is the path length De ¼
h 32 982 c l
ð1Þ
2.1.5. Fluorescence spectroscopy Fluorescence spectra were acquired with a Varian Cary Eclipse spectrofluorimeter with Windows based software, at an excitation wavelength of 450 nm. The emission was monitored over the range 500–800 nm. Slit widths of 5 nm were used for both the excitation and emission slits, with a medium scan speed and voltage over an average of five scans. Quartz cuvettes (1 cm pathlength) were used and the metal complex samples were dissolved in acetonitrile (0.167 mg/mL) at room temperature. Fluorescence quantum yields were calculated using ratios of the integrated fluorescence intensities to that of [Ru(Me2bipy)3]2+ in acetonitrile (Ur = 0.062) [39], using Eq. (2) [40]: 2 I unk Astd gunk ð2Þ Uunk ¼ Ustd Aunk I std gstd where Uunk is the radiative quantum yield of the sample, Ustd is the radiative quantum yield of the standard, and Iunk and Istd are the integrated emission intensities of the sample and standard, respectively. Aunk and Astd are the absorbances of the sample and standard, respectively, at a common excitation wavelength (nm), and gunk and gstd are the indexes of refraction of the sample and standard solutions, respectively. Values of g corresponding to the pure solvent were used, and as all measurements were performed in the same solvent, therefore gunk/gstd = 1. 2.1.6. Electrospray ionisation mass spectrometry Mass spectra were acquired with a Micromass (Wyntheshaw, UK) Qtof 2 spectrometer with a Z-spray probe. All samples were diluted with 5 mM ammonium acetate (pH 6.8) in acetonitrile, to a final concentration of approximately 1 lM. These were injected directly into the spectrometer using a Harvard model 22 syringe pump (Natick, MA, USA) at a flow rate of 20 mL min1. Positive ion ESI mass spectra were acquired using a probe tip potential of 2500 V, a cone voltage of either 40 or 50 V, and the source block and desolvation temperatures were set to 60 and 150 C, respectively. The transport and aper-
ture were set to 2.0 and 13.0, respectively. In most experiments spectra were acquired over the range 200–2000 m/ z. Typically, 50–70 scans were summed to obtain representative spectra. The data points were calibrated against a standard CsI solution (750 mM) over the same m/z range. 2.1.7. Elemental analysis Elemental analysis (C, H, and N) were conducted using a Carlo Erba 1106 automatic analyser by the Microanalytical Unit, Research School of Chemistry, of the Australian National University. 2.2. Metal complex synthesis 2.2.1. [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 Æ H2O [Ru(Me2bipy)2Cl2] (0.30 g, 0.56 mmol) and 5-Cl-phen (0.13 g, 0.58 mmol) were dissolved in methanol (100 mL) and refluxed for 45 min. Once cooled, the solvent was removed under reduced pressure yielding the product as a red solid. Purification was achieved by applying the product, dissolved in a minimal amount of water, to an SP Sephadex C-25 column (5 · 10 cm). The metal complex was eluted with a sodium chloride gradient (0.1–0.3 M) using 0.1 M increments. KPF6 was then added to the fractions to induce precipitation. The resulting precipitate was extracted into dichloromethane (3 · 30 mL), the extracts dried over anhydrous sodium sulphate and the organic solvent removed under reduced pressure. Yield: 0.42 g (77%). Anal. Calc. for C37H34F12N6P2RuCl Æ H2O: C, 44.12; H, 3.61; N, 8.35. Found: C, 44.12; H, 3.43; N, 8.29%. kmax/nm (e/mol1 dm3 cm1) (H2O/(CH3)2CO): 451 (8.91 · 103); 261 (4.58 · 104). MS (ESMS, CH3CN, MW [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 = 974.12) m/z: 341.7 [M(1H+2PF6)]2+. U (CH3CN) = 0.035. 1H NMR (300 MHz, CD3CN) d: 8.80 (H4; 1H, dd, J1 = 1.27 Hz, J2 = 8.56 Hz), 8.51 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.24 Hz), 8.39 (H6; 1H, s), 8.38 (A/B3; 2H, s), 8.34 (A/B3; 2H, s), 8.16 (H2; 1H, dd, J1 = 1.11 Hz, J2 = 5.23 Hz), 8.09 (H9; 1H, dd, J1 = 1.11 Hz, J2 = 5.23 Hz), 7.82 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 8.57 Hz), 7.73 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.29 Hz), 7.63 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.04 (A/B5; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s). 13C NMR (400 MHz, CD3CN) d: 154.2 (C2; CH), 153.8 (C11; CH), 152.3 (C26; CH), 152.0 (C20; CH), 151.50 (C18; QC), 151.4 (C24; QC), 136.7 (C9; CH), 134.5 (C4; CH), 132.6 (C8; QC), 129.3 (C19; CH), 129.1 (C25; CH), 128.2 (C7; CH), 127.6 (C3; CH), 127.6 (C10; CH), 125.9 (C23; CH), 125.8 (C17; CH), 21.4 (C18Me; CH3), 21.3 (C24Me; CH3). 2.2.2. Resolution of isomers Rac-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 Æ H2O (0.25 g, 0.26 mmol) was converted to the chloride salt by dissolving in acetone, diluting with water, then stirring with Amberlite anion exchange beads (Cl form) for 1 h. The solution was filtered to remove the beads, and the
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resin washed with water to elute any residual metal complex. The solvent was then removed by rotary evaporation before the red coloured residue was dissolved in water. Rac-[Ru(Me2bipy)2(5-Cl-phen)]Cl2 was loaded onto an SP Sephadex column (2.6 · 100 cm). The isomers were eluted with 75 mM disodium-O,O 0 -dibenzoyl-L-tartrate (pH 8), which was prepared by neutralising O,O 0 -dibenzoyl tartaric acid with sodium hydroxide. The solution was also filtered prior to use through 0.45 lm nylon membranes. Extraction of each fraction into dichloromethane (3 · 30 mL) after the addition of KPF6 gave each isomer. Optical purity was then established by circular dichroism. D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2: Anal. Calc. for C37H34F12N6P2RuCl: C, 44.93; H, 3.46; N, 8.50. Found: C, 45.12; H, 3.69; N, 8.00%. CD kmax nm (De mdeg/M cm) (H2O/(CH3)2CO): 210 (41); 226 (+40); 264 (+118); 285 (246); 410 (+11). K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2: Anal. Calc. for C37H34F12N6P2RuCl: C, 44.93; H, 3.46; N, 8.50. Found: C, 44.67; H, 3.51; N, 8.13%. CD kmax nm (De mdeg/M cm) (H2O/(CH3)2CO): 210 (+41); 226 (56); 264 (151); 285 (+301); 410 (24). 2.2.3. Rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)] (PF6)2 [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 (0.03 g, 0.03 mmol) and L-cysteine methyl ester butoxy carbamate (L-cysteineMeBOC) (22 lL, 0.30 mmol) were dissolved in DMF (3 mL) and de-aerated for 5 min under N2, after which time a degassed solution of Na2CO3 (0.030 g, 0.30 mmol) in water (3 mL) was added via syringe. The solution was then heated to 55 C for 1.5 h. The reaction mixture was allowed to cool to room temperature and the solvent removed under reduced pressure. The product was purified by silica gel chromatography [41] with an eluent of 1–3% KNO3 in 10% water/acetonitrile. The eluted product was rotary evaporated to dryness, dissolved in a minimum amount of water then KPF6 added. The resultant precipitate was extracted into dichloromethane and dried over sodium sulphate. The solvent was removed and the residue dried under reduced pressure. Yield: 19 mg (54%). MS (ESMS, CH3CN, [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 = 1173.17) m/z: 829.1 (M{1(PF6) + C9H16NO4}+). 1H NMR (400 MHz, CD3CN) d: 8.81 (H4; 1H, dd, J1 = 1.32 Hz, J2 = 8.42 Hz), 8.50 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.39 (H6; 1H, s), 8.36 (A/B3; 2H, s), 8.32 (A/B3; 2H, s), 8.14 (H2; 1H, dd, J1 = 1.06 Hz, J2 = 5.22 Hz), 8.08 (H9; 1H, dd, J1 = 1.22 Hz, J2 = 5.22 Hz), 7.80 (H3; 1H, dd, J1 = 5.20 Hz, J2 = 8.55 Hz), 7.71 (H8; 1H, dd, J1 = 5.20 Hz, J2 = 8.36 Hz), 7.62 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.90 (D-NH; 1H, bs), 5.72 (KNH; 1H, bs), 5.42 (OCH3; 3H, s), 4.48 (D-N–CH; 1H, bs), 4.38 (K-N–CH; 1H, bs), 3.22 (D-CH2; 2H, m), 2.96 (K-CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.40 (BOC; 9H, s). 13C NMR (400 MHz, CD3CN) d: 154.2 (C2; CH), 153.8 (C11; CH), 152.3 (C26; CH), 152.0
321
(C20; CH), 151.5 (C18; QC), 151.4 (C24; QC), 136.7 (C9; CH), 134.5 (C4; CH), 132.6 (C8; QC), 129.3 (C19; CH), 129.1 (C25; CH), 128.2 (C7; CH), 127.6 (C3; CH), 127.6 (C10; CH), 125.9 (C23; CH), 125.8 (C17; CH), 56.6 (Cys; CH), 53.0 (BOC; CH3), 28.5 (BOC; CH3), 27.2 (Cys; CH2), 21.4 (C18Me; CH3), 21.3 (C24Me; CH3). 2.2.3.1. D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2. Experimental conditions were the same as those in Section 2.2.3. CD kmax nm (De mdeg/M cm) (H2O/ (CH3)2CO): 210 (254); 227.5 (+172); 271 (+716); 285 (1955); 410 (+77). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.11 · 103); 334 (9.21 · 102); 279 (7.69 · 103). U (CD3CN) = 0.086. 1H NMR (400 MHz, CD3CN) d: 8.73 (H4; 1H, dd, J1 = 1.41 Hz, J2 = 8.66 Hz), 8.43 (H7; 1H, dd, J1 = 1.21 Hz, J2 = 8.26 Hz), 8.36 (A/B3; 2H, s), 8.31 (A/B3; 2H, s), 8.08 (H2; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.94 (H9; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.91 (H6; 1H, s), 7.71 (H3; 1H, dd, J1 = 5.33 Hz, J2 = 8.54 Hz), 7.65 (H8; 1H, dd, J1 = 5.55 Hz, J2 = 8.38 Hz), 7.62 (A/B6; 2H, m), 7.32 (A/B6; 2H, m), 7.26 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.98 (NH; 1H, bs), 5.42 (OCH3; 3H, s), 4.54 (N–CH; 1H, bs), 3.20 (CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.41 (BOC; 9H, s). 2.2.3.2. K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2. Experimental conditions were the same as those in Section 2.2.3. CD kmax nm De mdeg/M cm (H2O/ (CH3)2CO): 210 (+210); 227.5 (248); 271 (726); 285 (+1999); 410 (214). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.63 · 103); 334 (1.38 · 103); 267 (3.39 · 104). U (CD3CN) = 0.157. 1H NMR (400 MHz, CD3CN) d: 8.73 (H4; 1H, dd, J1 = 1.41 Hz, J2 = 8.66 Hz), 8.43 (H7; 1H, dd, J1 = 1.21 Hz, J2 = 8.26 Hz), 8.36 (A/B3; 2H, s), 8.31 (A/B3; 2H, s), 8.08 (H2; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.94 (H9; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.91 (H6; 1H, s), 7.71 (H3; 1H, dd, J1 = 5.33 Hz, J2 = 8.54 Hz), 7.65 (H8; 1H, dd, J1 = 5.55 Hz, J2 = 8.38 Hz), 7.62 (A/B6; 2H, m), 7.32 (A/ B6; 2H, m), 7.26 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.75 (NH; 1H, bs), 5.42 (OCH3; 3H, s), 4.33 (N–CH; 1H, bs), 2.94 (CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/ BMe; 6H, s), 1.41 (BOC; 9H, s). 2.2.4. Deprotection of rac-[Ru(Me2bipy)2(5-(L-cysteineMeBOC)-phen)](PF6)2 Rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (50 mg, 0.040 mmol) was dissolved in dichloromethane (2 mL) and added to a solution of trifluoroacetic acid/ dichloromethane (1:1, 2.2 mL) containing water (40 lL) which was stirred for 1.5 h. The reaction was monitored by TLC {3% KNO3 in 10 % H2O/CH3CN, ninhydrin stain (0.2% w/v in ethanol)}. Upon completion, the solvent was removed under reduced pressure and the residue stirred with DOWEX 550A OH anion exchange resin
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(148 mg, loading capacity 3.44 mmol/g, washed with methanol). The solution was filtered to remove the resin, and the solvent removed to yield a red solid that was dried under vacuum. Yield: 46 mg (100%). 1H NMR (300 MHz, CD3CN) d: 8.81 (H4; 1H, dd, J1 = 1.32 Hz, J2 = 8.42 Hz), 8.50 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.39 (H6; 1H, s), 8.36 (A/B3; 2H, s), 8.32 (A/B3; 2H, s), 8.14 (H2; 1H, dd, J1 = 1.06 Hz, J2 = 5.22 Hz), 8.08 (H9; 1H, dd, J1 = 1.22 Hz, J2 = 5.22 Hz), 7.80 (H3; 1H, dd, J1 = 5.20 Hz, J2 = 8.55 Hz), 7.71 (H8; 1H, dd, J1 = 5.20 Hz, J2 = 8.36 Hz), 7.62 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.42 (OCH3; 3H, s), 4.48 (D-N–CH; 1H, bs), 4.38 (K-N–CH; 1H, bs), 3.22 (D-CH2; 2H, m), 2.96 (K-CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.26 (NH2; 2H, s). 2.2.5. Rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 Trans-[PtCl2(NH3)2] (0.013 g, 0.040 mmol) was suspended in water (5 mL), acidified with 1 M HCl to pH 5, then heated to 95 C to dissolve the metal complex. Over a period of 1.5 h, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]Cl2 (0.037 g, 0.040 mmol) in water (10 mL) was added drop wise to the solution, in the dark, after which time the temperature (95 C) was maintained for 5 h. The solution was then cooled to 0 C and filtered to remove any precipitate. The solution volume was reduced under vacuum and the remaining water removed by lypholysation. The red/ orange precipitate was dissolved in a minimum of methanol and applied to a Sephadex LH20 column (3 · 15 cm, 70 g packing material). Fractions (3 mL) were collected and monitored by TLC (3% saturated potassium nitrate, 10% water/acetonitrile, ninhydrin stain). Rac[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ eluted first, and fractions containing the product were combined before the solvent was removed under reduced pressure. Yield: 37 mg (78%). MS (ESMS, CH3CN, MW [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ = 1045) m/z: 342.1 (M=3 1NH3 þ , 1H+). 1H NMR (400 MHz, d6-DMSO) d: 8.84 (H4; 1H, dd, J1 = 1.02 Hz, J2 = 8.46 Hz), 8.71 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.36 (A/B3; 2H, s), 8.69 (A/B3; 2H, s), 8.73 (H6; 1H, s), 8.22 (H2; 1H, d, J = 5.22 Hz), 8.14 (H9; 1H, d, J = 4.90 Hz), 7.96 (H3; 1H, dd, J1 = 5.29 Hz, J2 = 8.42 Hz), 7.87 (H8; 1H, dd, J1 = 5.09 Hz, J2 = 8.23 Hz), 7.62 (A/B6; 2H, m), 7.40 (A/B6; 2H, m), 7.35 (A/B5; 2H, m), 7.14 (A/B5; 2H, m), 4.35 (NH2; 1H, s), 4.07 (N–CH; 1H, q, J1 = 4.93 Hz, J2 = 10.01 Hz), 3.50 (OCH3; 3H, s), 3.16 (CH2; 2H, d, J = 4.78 Hz), 2.53 (A/BMe; 6H, s), 2.45 (A/BMe; 6H, s). 2.2.5.1. D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3. Experimental conditions were the same as those in Section 2.2.5. CD kmax nm (De mdeg/M cm) (H2O): 210 (289); 228.5 (+334); 270.5 (+1044); 285 (2808); 410 (+250). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.55 · 104); 334 (1.25 · 104); 257
(3.17 · 104); 282 (7.12 · 104); 206 (6.95 · 104). 1H NMR (400 MHz, d6-DMSO) d: 8.74 (H4; 1H, dd, J1 = 1.20 Hz, J2 = 8.56 Hz), 8.72 (A/B3; 2H, s), 8.68 (A/B3; 2H, s), 8.61 (H7; 1H, dd, J1 = 1.20 Hz, J2 = 8.38 Hz), 8.14 (H2; 1H, dd, J1 = 1.16 Hz, J2 = 5.22 Hz), 8.11 (H6; 1H, s), 7.98 (H9; 1H, dd, J1 = 1.31 Hz, J2 = 5.37 Hz), 7.87 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 7.88 Hz), 7.80 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.27 Hz), 7.61 (A/B6; 2H, m), 7.38 (A/ B6; 2H, m), 7.32 (A/B5; 2H, m), 7.15 (A/B5; 2H, m), 3.95 (N–CH; 1H, d, J = 8.77 Hz), 3.89 (NH2; 1H, s), 3.73 (CH2; 2H, dd, J1 = 7.10 Hz, J2 = 14.20 Hz), 2.81 (OCH3; 3H, s), 2.53 (A/BMe; 6H, s), 2.44 (A/BMe; 6H, s). 2.2.5.2. K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3. Experimental conditions were the same as those in Section 2.2.5. CD kmax nm (De mdeg/M cm) (H2O): 210 (+289); 228.5 (243); 270.5 (895); 285 (+2505); 410 (210). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.45 · 104); 334 (1.17 · 104); 257 (2.88 · 104); 282 (6.09 · 104); 206 (6.03 · 104). 1H NMR (400 MHz, d6-DMSO) d: 8.74 (H4; 1H, dd, J1 = 1.20 Hz, J2 = 8.56 Hz), 8.72 (A/B3; 2H, s), 8.68 (A/B3; 2H, s), 8.61 (H7; 1H, dd, J1 = 1.20 Hz, J2 = 8.38 Hz), 8.14 (H2; 1H, dd, J1 = 1.16 Hz, J2 = 5.22 Hz), 8.11 (H6; 1H, s), 7.98 (H9; 1H, dd, J1 = 1.31 Hz, J2 = 5.37 Hz), 7.87 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 7.88 Hz), 7.80 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.27 Hz), 7.61 (A/B6; 2H, m), 7.38 (A/ B6; 2H, m), 7.32 (A/B5; 2H, m), 7.15 (A/B5; 2H, m), 3.95 (N–CH; 1H, d, J = 8.77 Hz), 3.89 (NH2; 1H, s), 3.73 (CH2; 2H, dd, J1 = 7.10 Hz, J2 = 14.20 Hz), 2.81 (OCH3; 3H, s), 2.53 (A/BMe; 6H, s), 2.44 (A/BMe; 6H, s). 3. Results and discussion 3.1. Metal complex synthesis The heterodinuclear metal complex was synthesised using a six step method as shown in Scheme 1 (where the proton numbering scheme is shown after step 2 and the carbon numbering scheme is shown after step 3). All reactions were initially performed with unresolved mononuclear complexes then repeated with the D- and K-isomers once the reaction conditions had been optimised. [Ru(Me2bipy)2Cl2] was synthesised using alternative methods [37,38]. The phenanthroline ligand was then coordinated to the [Ru(Me2bipy)2Cl2] complex by refluxing in methanol for approximately half an hour. Completion of the reaction was signified by a change in colour of the solution from deep purple to red. Upon removal of the solvent a red/brown solid resulted. Two methods of purifying rac-[Ru(Me2bipy)2(5-Clphen)](PF6)2 were trialed. Dissolution of the ruthenium complex as a hexafluorophosphate salt in acetonitrile and elution through an aluminum oxide column produced an impure product; however, further purification on a cation exchange column enabled isolation of the desired purified metal complex. Purification using an
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323
2+
8
N
N
Cl
Ru
RuCl3.3H2O 2 x Me2bipy
N
N
B5
5-Cl-phen
N
N N
N BMe
6
Ru
B6
Cl
7
9
N
N
Cl
A6 2
4
B3
3 A3
A5 AMe
stereoisomer resolution
Δ,Λ
2+
2+
10
N N
N N
N
N
O
Ru S
N
O
O
25
L-cysteine-MeBOC
24
7 6
N
22 16 N
20
23
O
8 13 14
N
NH
9
N
Ru
26
N
11
Cl
5
2
4 3
17
19 18
HCl/TFA
3+ 2+
N
N N
N
N O
Ru N
N N
transplatin S
N O
Ru O
N
N
S
N
O NH2
NH2 H3N
Pt
NH3
Cl
Scheme 1.
SP Sephadex C-25 column alone (sodium chloride eluent) was also trialed. Before loading onto the Sephadex column, the ruthenium complex was converted to the chloride salt by stirring with Amberlite anion exchange beads. The first band to be eluted with water from the column was a golden brown colour, followed by a yellow band that eluted with 0.1 M NaCl. The remaining deep red band eluted with 0.3 M NaCl and was the desired product. A residual brown band, thought to comprise [Ru(Me2bipy)2Cl2], remained bound to the column head. A column 10–15 cm in length and 5 cm in width was found to be sufficient to attain separation between the bands. The metal complex was isolated by the addition of potassium hexafluorophosphate and extracted into dichloromethane.
The isomers of [Ru(Me2bipy)2(5-Cl-phen)]2+ were resolved using solvent-recycled solution cation exchange chromatography [42]. Using SP Sephadex C25 and 75 mM disodium-O,O 0 -dibenzoyl-L-tartrate as the eluent, two discreet bands were observed after an effective column length of 0.9 m, indicating baseline-to-baseline separation. After collection, the metal complexes were precipitated by the addition of potassium hexafluorophosphate and extracted into dichloromethane, to remove the tartrate salt. Two diaminoalkanes of varying carbon chain lengths were protected using BOC [43], with the intention of observing the effect of linker length on the DNA binding affinity of the heterodinuclear complex. Diaminoalkanes were selected for their flexibility, which would allow the different metal centres to bind at preferential DNA structures,
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and because of their relevance in multi-nuclear platinum agents [44–46]. Attempts were made to couple the single amine BOC-protected linkers 1,6-diaminohexane, 1,3-diaminopropane and 2-mercaptoethylamine to free 5-Cl-phen before addition of the ligand to [Ru(Me2bipy)2Cl2]. Unfortunately the coupling attempts were unsuccessful as the phenanthroline-linker bond was degraded upon coordination of the ligand to the metal centre. Subsequent attempts were made to couple the linkers to phenanthroline in [Ru(Me2bipy)2(5-Cl-phen)]2+, however, degradation of the phenanthroline-linker C–N bond was again observed. The method of Hurley et al. [47] which involved the coupling of L-cysteine to 3-bromo-1,10-phenanthroline containing ruthenium complexes under mildly basic conditions was therefore attempted, but instead using BOC-protected L-cysteine methyl ester (L-cys-MeBOC) to couple to the phenanthroline ligand of [Ru(Me2bipy)2(5-Cl-phen)]2+. After the reaction, two distinct bands were observed with silica gel column chromatography, one eluting with 1% (Rf = 0.57) and the other with 3% (Rf = 0.33) saturated potassium nitrate solutions. Excess L-cys-MeBOC moved with the solvent front. 1H NMR spectra subsequently showed that the desired compound was contained within the second fraction. The BOC-protecting group was cleaved from [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 using strongly acidic conditions. Initially, de-protection was achieved using a solution of concentrated hydrochloric acid in methanol (48 h) to isolate the amine as a hydrochloride salt. Alternatively, the use of trifluoroacetic acid (TFA) in dichloromethane also achieved de-protection, in a shorter length of time (1 h) with 100% recovery of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)](PF6)2, and was thus the preferred option of the two reactions. Stirring over DOWEX Monosphere 550A (OH form) in methanol neutralised the TFA/amine salt complex. The coupling of transplatin {trans-diamminedichloroplatinum(II)} to the amine group of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]2+ proved more difficult than first anticipated. After purification on Sephadex LH20, the presence of light during the reaction was found to result in a very low yield of [Ru(Me2bipy)2(5-(L-cysteine-Me)phen)-trans-Pt(NH3)2Cl]Cl3. Repeated experiments with altered exposure to light demonstrated that formation of the heterodinuclear complex was photosensitive. Consequently, the synthesis and purification of the final metal complex were performed in the dark. The separation of the mononuclear reactant and the heterodinuclear product was achieved using Sephadex LH20 support media as the stationary phase with methanol. TLC was used to verify the contents of the fractions, with rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 displaying an Rf value of 0.67 and the unreacted rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]2+ stained purple with ninhydrin at Rf 0.38. The final product was precipitated by the addition of potassium hexafluorophosphate, and extracted into dichlo-
romethane. Conversion to the water soluble chloride salt was then achieved by stirring with Amberlite anion exchange beads, followed by filtration in order to remove the resin, and finally freeze drying, which yielded the heterodinuclear complex as a red solid. 3.2. Characterisation The heterodinuclear metal complex and the precursor mononuclear complexes were fully characterised by 1H and 13C NMR, circular dichroism, and electrospray ionisation mass spectrometry, and are described in more detail in Sections 3.2.1, 3.2.2, 3.2.3. UV–Vis spectra were measured to determine the molar extinction coefficients, fluorescence spectra were used to determine quantum yields and elemental analysis to confirm bulk purity. Unfortunately, the use of Eq. (2) for the determination of the quantum yields of fluorescent samples in comparison to a standard, at a common excitation wavelength, was not applicable for the heterodinuclear complex. This was because its excitation wavelength had shifted dramatically from the excitation wavelength of tris(bipyridine) ruthenium(II) (the standard). The method is thus limited in use, and another standard of known quantum yield is required to calculate the relative fluorescence of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3. Elemental analysis demonstrated bulk purity of Rac[Ru(Me2bipy)2(5-Cl-phen)] Æ 2PF6 with one water of crystallisation, but which is lost upon separation into its isomers. Bulk purity of the boc-protected ruthenium complex and the final heterodinuclear complex could not be obtained by elemental analysis; however, purity is indicated by both 1H and 13C NMR and high resolution mass spectrometry. Bulk and isomeric purity was also confirmed by CD with the spectrum of each isomer of all metal complexes synthesised showing peaks in almost equal proportion, in opposite directions, at the same wavelength. In addition, the CD spectra also confirm the purity of the samples through the presence of isosbestic points, where the two spectra intercept on the xaxis. 3.2.1. NMR spectroscopy All metal complexes in this study were characterised using one-dimensional 1H and 13C NMR and two-dimensional NOESY, g-DQCOSY experiments. 13C assignments were made using heteronuclear multiple quantum correlation spectroscopy (HMQC) and heteronuclear multiple bond correlation spectroscopy (HMBC) experiments. Full details of the 1H NMR spectrum of the heterodinuclear complex, rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)trans-Pt(NH3)2Cl]Cl3, including proton assignments are presented here as an example of how all the metal complexes were characterised. The 1H NMR spectrum of rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3 displays 13 resonances in the aromatic region and six resonances in the
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aliphatic region. The two singlets at 2.46 and 2.54 ppm each integrate for six protons, and are assigned as the two methyl groups on the Me2bipy rings. The NOESY spectrum shows NOE cross-peaks from the A/BMe resonance at 2.54 ppm to the singlet at 8.38 ppm and a multiplet at 7.27 ppm, which are assigned as the A/B3 and A/ B5 protons, respectively. In the g-DQCOSY spectrum, the A/B5 resonance also displays cross-peaks with a multiplet 7.63 ppm, which is assigned as the A/B6 proton (Fig. 1). Similar cross-peaks were observed from the A/ BMe resonance at 2.46 ppm, to the A/B3 resonance at 8.34 ppm, A/B5 at 7.04 ppm and from this latter resonance to the A/B6 at 7.31 ppm. It was hoped that cross-peaks might be observed in the NOESY spectrum from the A/ B6 resonances to either H2 or H9 on the phenanthroline ring, allowing the definitive assignments of the Me2bipy proton resonances to particular protons, but no NOE cross-peaks were observed. The substitution of a chloro group at the 5-position in 1,10-phenanthroline results in seven separate proton resonances for this ligand. The H6 proton resonance couples to no other proton (d 8.39 ppm), and is the only aromatic singlet in the spectrum integrating for one proton. Both
325
H3 and H8 each have two neighbouring protons and can be identified by their doublet of doublet resonances (see Fig. 1). The remaining resonances for the H2, H4, H7 and H9 protons all appear as doublets. Definitive assignments can be made using NOESY spectra. The H6 resonance gives one NOE cross-peak to the resonance at 8.50 ppm (assigned as H7), which in turn gives an NOE to H8 at 7.73 ppm, and from H8 to H9 at 8.10 ppm. The remaining doublet of doublet resonance at 7.82 is assigned as the H3 proton. The H2 and H4 resonances can be assigned based on the coupling constants. In pyridine rings, the proton nearest the nitrogen atom exhibits the smallest coupling constant (typically 5–6 Hz), whilst a proton further away exhibits a larger coupling constant (typically 7–9 Hz) [48]. On this basis, the resonance at 8.81 ppm is assigned as H4 and the resonance at 8.16 ppm as H2. The remaining unassigned proton resonances in the spectrum are the –NH2, –CH, –OCH3, and –CH2 protons of the L-cysteine methyl ester. The resonance at 4.35 ppm is assigned to –NH2, due to its broadness, the singlet at 3.50 ppm to –OCH3, the triplet at 3.16 ppm to –CH2 and the multiplet at 4.07 ppm to –CH proton. Coordination of the platinum to the cystiene-NH2 group is confirmed
Fig. 1. 1H g-DQCOSY NMR spectrum showing the assignment of the aromatic proton resonances of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]3+.
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by the large downfield movement of the proton resonance, which moves from 1.26 to 4.35 ppm, upon reaction with transplatin. It is of note that there are slight differences in the 1H NMR spectra of the D- and K-ruthenium complexes compared to that of the racemic solutions for all metal complexes synthesised in this study. For instance, a significant chemical shift change of the H6 resonance (8.29–7.91 ppm) is observed in the spectra of the resolved isomers of [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)]2+ in comparison to the spectrum of the racemic complex (Fig. 2). There is also a large movement of the –NH2 resonance (4.35–3.89 ppm). All other resonances undergo less significant chemical shift changes upon resolution of the isomers.
3000 2000 1000 0 -1000 -2000 -3000 200
250
300
350
400
450
Wavelength (nm)
Fig. 3. Circular dichroism spectra of the isomeric complexes from top to bottom (at 285 nm): K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3, K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2, K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2, D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2, D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 and D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3.
3.2.2. Circular dichroism spectrophotometry CD spectra were recorded for each of the metal complex isomers by plotting the angle of ellipticity (h mdeg) versus wavelength (nm). This was converted to the change in molar absorptivity by Eq. (1). All metal complexes synthesised in this study show four major peaks at 210, 226, 264 and 285 nm, and one broad peak at 410 nm (Fig. 3). In comparison to [Ru(Me2bipy)2(5-Cl-phen)]2+, attachment of the amino acid and then the platinum metal centre do not change either the wavelength, nor shape of the CD peaks, however, the intensities are increased.
The assignments of the absolute configuration of the metal complexes are based on previous studies of analogous compounds [42]. The metal complex exhibiting a positive value of h at 250 nm corresponds to the D-isomer, while the K-isomer exhibits a negative peak at the same wavelength. The elution profile of the isomers from the Sephadex column further supported this finding, with the D-isomer eluting prior to the K-isomer.
A H6
B
H6
C H6
8.75
8.50
8.25
8.00
7.75
7.50
7.25
ppm (f1)
Fig. 2. 1H NMR spectra of (a) rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (b) K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (c) D[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 demonstrating the chemical shift change of the phenanthroline H6 proton resonance in a racemic solution and when separated into its D- and K-isomers.
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3.2.3. Mass spectrometry The synthesis of the precursor mononuclear complexes and the final heteronuclear complexes were also confirmed by electrospray ionisation mass spectrometry. [Ru(Me2bipy)2(5-Cl-phen)]2+ shows a molecular ion at 341.7 m/z which corresponds to the metal complex with a 2+ charge and the loss of one proton and both hexafluorophosphate counter ions. For [Ru(Me2bipy)2(5-(L-cysteineMeBOC)-phen)]2+, five peaks are observed for the parent and daughter ion fragments. For instance, a molecular ion at 1028.2 m/z is consistent with the [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)]2+ cation and one PF6 counterion. Finally, the heterodinuclear complex was confirmed by the observance of a molecular ion at 342.1 m/z, corresponding to the charged parent ion minus both counterions and one ammine group. This is characteristic of charged metal complexes of this type and matched the expected isotopic pattern for a ruthenium(II)–platinum(II) complex. 4. Summary The heterodinuclear complex, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3, was synthesised using a six step method, as a potential agent to overcome platinum resistance in human cancer treatment. The heterodinuclear complex, and its mononuclear precursors were characterised by 1H and 13C NMR, UV–Vis, circular dichroism, fluorescence and electrospray ionisation mass spectrometry. The cytotoxicity and DNA binding studies of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 are currently under examination, with the results to be used to develop a family of heterodinuclear complexes with anticancer activity. Acknowledgements This work was supported by an Australian Research Council Grant (No. DP0343480) and the University of Western Sydney. We thank Assoc. Prof. S. Ralph (University of Wollongong) for assistance with the mass spectrometry and Prof. W.S. Price and M. van Holst (University of Western Sydney) and Dr. D.M. D’Alessandro (University of Sydney) for helpful discussions. References [1] N.J. Wheate, J.G. Collins, Curr. Med. Chem. – Anti-Cancer Agents 5 (2005) 267. [2] N.J. Wheate, J.G. Collins, Coord. Chem. Rev. 241 (2003) 133. [3] N. Farrell, Met. Ions Biol. Sys. 42 (2004) 251. [4] V. Murray, H. Motyka, P.R. England, G. Wickham, H.H. Lee, W.A. Denny, W.D. McFadyen, J. Biol. Chem. 267 (1992) 18805. [5] C. Cullinane, G. Wickham, W.D. McFadyen, W.A. Denny, B.D. Palmer, D.R. Phillips, Nucl. Acids Res. 21 (1993) 393. [6] L.C. Perrin, P.D. Prenzler, C. Cullinane, D.R. Phillips, W.A. Denny, W.D. McFadyen, J. Inorg. Biochem. 81 (2000) 111. [7] L.C. Perrin, C. Cullinane, W.D. McFadyen, D.R. Phillips, AntiCancer Drug Des. 14 (1999) 243.
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Synthesis of a heterodinuclear ruthenium(II)–platinum(II) complex linked by L-cysteine methyl ester Sarah J. Yousouf, Craig R. Brodie, Nial J. Wheate, Janice R. Aldrich-Wright
*
School of Biomedical and Health Sciences, University of Western Sydney, Campbelltown, NSW 2560, Australia Received 22 March 2006; accepted 2 June 2006 Available online 10 June 2006
Abstract A potential anticancer heterodinuclear ruthenium(II)–platinum(II) complex, [ruthenium(II)(4,4 0 -dimethyl-2,2 0 -bipyridine)2(5-(L-cysteine-methyl ester)-1,10-phenanthroline)-trans-chlorodiammineplatinum(II)] chloride, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3, was synthesised. L-Cysteine methyl ester was used to link the two metal centres, as more conventional straight chain diaminoalkanes and 2-mercaptoethylamine failed to couple to the phenanthroline ligand. From the precursor mononuclear ruthenium(II) complexes, which were separated into their D- and K-isomers by column chromatography, the dinuclear complex was synthesised and characterised by 1H and 13C NMR, UV–Vis, circular dichroism, fluorescence and electrospray ionisation mass spectrometry. 2006 Elsevier Ltd. All rights reserved. Keywords: Ruthenium(II); Platinum(II); Heterodinuclear; L-Cysteine methyl ester; Phenanthroline; Anticancer
1. Introduction Cisplatin, carboplatin and oxaliplatin are the only platinum-based drugs widely approved for the treatment of human cancers [1]. Over the last decade, multi-nuclear platinum drugs, like BBR3464 (a novel trinuclear complex having two mono-functional trans-[PtCl(NH3)2] units coupled by a central trans-[Pt(NH3)2(H2N(CH2)6NH2)2] linker), have demonstrated clinical potential, but are limited by toxic side-effects and in vivo degradation before reaching their cellular target, DNA [1,2]. Both mono- and multinuclear drugs derive their anticancer activity by forming coordinate covalent adducts with DNA, thereby preventing replication and transcription [3]. By changing the structure of the DNA adducts, or increasing the DNA binding affin-
*
Corresponding author. Present address: School of Biomedical and Health Sciences, University of Western Sydney, Locked Bag 1797, Penrith South DC, NSW 1797, Australia. Tel.: +61 2 4620 3218; fax: +61 2 4620 3025. E-mail address: [email protected] (J.R. Aldrich-Wright). 0277-5387/$ - see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2006.06.004
ity, new complexes may be developed that are able to overcome drug resistance. Over the last 20 years many researchers have examined the synergistic properties of combining a DNA intercalating compound with a moiety capable of covalent binding to DNA. These new complexes include: organic intercalators linked to cisplatin-like molecules [4–21], octahedral metal intercalators coupled to cisplatin-like molecules [22–27], and organic intercalators linked to DNA-alkylating agents like mustards [28–32]. Recently, carborane cages have been attached to intercalators for use in radiation therapy [33–36]. These synergistic compounds have been developed to increase the DNA binding affinity and drug transport properties of conventional chemotherapeutic drugs and/or to modify their DNA adducts and sequence selectivity. There has been little research, however, into heteronuclear metal complexes which link platinum groups capable of forming DNA coordinate covalent adducts to other groups capable of DNA groove binding. In this paper we report the synthesis of a heterodinuclear ruthenium(II)–platinum(II) metal complex, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3,
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319
Table 1 Synthetic yield and characterisation data for the heterodinuclear ruthenium(II)–platinum(II): [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ and its mononuclear precursors for the racemic and D- and K-isomer solutions Metal complex
% Yield
D,K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2
77
ESI-MS (m/z)
285 (246) 285 (+301) 54
285 (1955)
285 (+1998)
78
K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3
containing a platinum moiety attached to a DNA groove binding ruthenium moiety via a modified amino acid linker. This metal complex was designed as a potential anticancer agent that may display increased DNA binding affinity compared to native mononuclear platinum complexes (Table 1). 2. Experimental 2.1. Materials and methods 2.1.1. Materials Deuterated acetonitrile (d3-CD3CN) and dimethylsulphoxide (d6-DMSO) were obtained from Cambridge Isotope Laboratories. ()-O,O 0 -dibenzoyl-L-tartaric acid and Sephadex LH20 were obtained from Fluka. Silica gel 60 was obtained from Merck. 2-Mercaptoethylamine hydrochloride, 1,6-diaminohexane, 1,3-diaminopropane, L-cysteine methyl ester (tert-butoxy)carbonate (L-cysteineMeBOC), DOWEX 550A OH anion exchange resin (DOWEX), Amberlite IRA-400(Cl) ion exchange resin (Amberlite), potassium hexafluorophosphate, SP Sephadex C25, 4,4 0 -dimethyl-2,2 0 -bipyridine (Me2bipy), 5chloro-1,10-phenanthroline (5-Cl-phen) and deuterated methanol (d4-CD3OD) were all purchased from Aldrich. Ruthenium(II) trichloride hydrate {RuCl3 Æ xH2O (Ru 40–43%)} and trans-diamminedichloroplatinum(II) (transplatin) were purchased from Precious Metals Online. [Ru(Me2bipy)2Cl2] was prepared as previously described [37,38]. All other reagents and solvents were of analytical
Fluorescence quantum yield
0.035 0.035
1028.0
K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2
D,K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3 D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3
UV–Vis kmax nm (e) 451 (8.91 · 103) 261 (4.58 · 104)
341.7
D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 D,K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2
CD kmax nm (De)
456 334 279 456 334 267
(1.11 · 103) (9.21 · 102) (7.69 · 103) (1.61 · 103) (1.38 · 103) (3.39 · 104)
456 334 257 282 206 456 334 257 282 206
(1.55 · 104) (1.25 · 104) (3.17 · 104) (3.17 · 104) (6.95 · 104) (1.45 · 104) (1.17 · 104) (2.88 · 104) (6.09 · 104) (6.03 · 104)
0.086
0.157
342.1 285 (2808)
285 (+2505)
grade and were used as received, except trifluoroacetic acid (TFA) which was redistilled before use. Aqueous solutions were prepared with Milli-Q water (Millipore, MA). 2.1.2. Nuclear magnetic resonance spectroscopy 1 H NMR spectra were obtained with a 300 MHz Varian Mercury VX spectrometer, a 400 MHz Varian Mercury VX spectrometer, or a Varian 300 MHz UnityPlus spectrometer. All spectra were recorded at 25 C, unless otherwise stated and were internally referenced to the solvent resonances. All final and precursor metal complexes were characterised using NOESY, g-DQCOSY, HMBC and HMQC experiments. Spectra were obtained over a spectral width of 4200 Hz with a mixing time of either 300 or 800 ms using 2048 points in the t2 dimension with 256 increments and a total recycle time of 1.5 s. 2.1.3. UV–Vis spectrophotometry Absorption spectra were measured with a Varian Cary 300 Bio UV–Vis double-beam spectrophotometer. Spectra were obtained over the range of 200–800 nm with samples in 1 cm quartz cells, which were referenced to water. A scan rate of 600 nm/min, data interval of 1 nm and average time of 0.1 s were utilised. Extinction coefficients were determined by repeated measurement of the absorbance of known concentrations of the metal complexes (as PF6 salts) in a 3% acetone/water solution. All absorbances were within the range 0.3–0.8, and the average of repeated measurements at kmax gave the extinction coefficient, emax.
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2.1.4. Circular dichroism spectropolarimetry CD spectra were acquired with a JASCO J-810 spectropolarimeter equipped with J-800 for Windows. Samples were prepared in either 3% acetone/water or in water and were placed in 0.1 cm cylindrical quartz cells. Data collection was performed at room temperature, between 200 and 650 nm, and at a speed of 500 nm/min. A sensitivity setting of 100 mdeg, data pitch of 0.5 nm, response time of 1 s and bandwidth of 1 s were used. Spectra are given as an average of 10 scans. From the angle of ellipticity (h, mdeg), the molar absorptivity (De, mdeg/M cm) was calculated using Eq. (1), where c is the concentration (mol/L) and l is the path length De ¼
h 32 982 c l
ð1Þ
2.1.5. Fluorescence spectroscopy Fluorescence spectra were acquired with a Varian Cary Eclipse spectrofluorimeter with Windows based software, at an excitation wavelength of 450 nm. The emission was monitored over the range 500–800 nm. Slit widths of 5 nm were used for both the excitation and emission slits, with a medium scan speed and voltage over an average of five scans. Quartz cuvettes (1 cm pathlength) were used and the metal complex samples were dissolved in acetonitrile (0.167 mg/mL) at room temperature. Fluorescence quantum yields were calculated using ratios of the integrated fluorescence intensities to that of [Ru(Me2bipy)3]2+ in acetonitrile (Ur = 0.062) [39], using Eq. (2) [40]: 2 I unk Astd gunk ð2Þ Uunk ¼ Ustd Aunk I std gstd where Uunk is the radiative quantum yield of the sample, Ustd is the radiative quantum yield of the standard, and Iunk and Istd are the integrated emission intensities of the sample and standard, respectively. Aunk and Astd are the absorbances of the sample and standard, respectively, at a common excitation wavelength (nm), and gunk and gstd are the indexes of refraction of the sample and standard solutions, respectively. Values of g corresponding to the pure solvent were used, and as all measurements were performed in the same solvent, therefore gunk/gstd = 1. 2.1.6. Electrospray ionisation mass spectrometry Mass spectra were acquired with a Micromass (Wyntheshaw, UK) Qtof 2 spectrometer with a Z-spray probe. All samples were diluted with 5 mM ammonium acetate (pH 6.8) in acetonitrile, to a final concentration of approximately 1 lM. These were injected directly into the spectrometer using a Harvard model 22 syringe pump (Natick, MA, USA) at a flow rate of 20 mL min1. Positive ion ESI mass spectra were acquired using a probe tip potential of 2500 V, a cone voltage of either 40 or 50 V, and the source block and desolvation temperatures were set to 60 and 150 C, respectively. The transport and aper-
ture were set to 2.0 and 13.0, respectively. In most experiments spectra were acquired over the range 200–2000 m/ z. Typically, 50–70 scans were summed to obtain representative spectra. The data points were calibrated against a standard CsI solution (750 mM) over the same m/z range. 2.1.7. Elemental analysis Elemental analysis (C, H, and N) were conducted using a Carlo Erba 1106 automatic analyser by the Microanalytical Unit, Research School of Chemistry, of the Australian National University. 2.2. Metal complex synthesis 2.2.1. [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 Æ H2O [Ru(Me2bipy)2Cl2] (0.30 g, 0.56 mmol) and 5-Cl-phen (0.13 g, 0.58 mmol) were dissolved in methanol (100 mL) and refluxed for 45 min. Once cooled, the solvent was removed under reduced pressure yielding the product as a red solid. Purification was achieved by applying the product, dissolved in a minimal amount of water, to an SP Sephadex C-25 column (5 · 10 cm). The metal complex was eluted with a sodium chloride gradient (0.1–0.3 M) using 0.1 M increments. KPF6 was then added to the fractions to induce precipitation. The resulting precipitate was extracted into dichloromethane (3 · 30 mL), the extracts dried over anhydrous sodium sulphate and the organic solvent removed under reduced pressure. Yield: 0.42 g (77%). Anal. Calc. for C37H34F12N6P2RuCl Æ H2O: C, 44.12; H, 3.61; N, 8.35. Found: C, 44.12; H, 3.43; N, 8.29%. kmax/nm (e/mol1 dm3 cm1) (H2O/(CH3)2CO): 451 (8.91 · 103); 261 (4.58 · 104). MS (ESMS, CH3CN, MW [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 = 974.12) m/z: 341.7 [M(1H+2PF6)]2+. U (CH3CN) = 0.035. 1H NMR (300 MHz, CD3CN) d: 8.80 (H4; 1H, dd, J1 = 1.27 Hz, J2 = 8.56 Hz), 8.51 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.24 Hz), 8.39 (H6; 1H, s), 8.38 (A/B3; 2H, s), 8.34 (A/B3; 2H, s), 8.16 (H2; 1H, dd, J1 = 1.11 Hz, J2 = 5.23 Hz), 8.09 (H9; 1H, dd, J1 = 1.11 Hz, J2 = 5.23 Hz), 7.82 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 8.57 Hz), 7.73 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.29 Hz), 7.63 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.04 (A/B5; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s). 13C NMR (400 MHz, CD3CN) d: 154.2 (C2; CH), 153.8 (C11; CH), 152.3 (C26; CH), 152.0 (C20; CH), 151.50 (C18; QC), 151.4 (C24; QC), 136.7 (C9; CH), 134.5 (C4; CH), 132.6 (C8; QC), 129.3 (C19; CH), 129.1 (C25; CH), 128.2 (C7; CH), 127.6 (C3; CH), 127.6 (C10; CH), 125.9 (C23; CH), 125.8 (C17; CH), 21.4 (C18Me; CH3), 21.3 (C24Me; CH3). 2.2.2. Resolution of isomers Rac-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2 Æ H2O (0.25 g, 0.26 mmol) was converted to the chloride salt by dissolving in acetone, diluting with water, then stirring with Amberlite anion exchange beads (Cl form) for 1 h. The solution was filtered to remove the beads, and the
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resin washed with water to elute any residual metal complex. The solvent was then removed by rotary evaporation before the red coloured residue was dissolved in water. Rac-[Ru(Me2bipy)2(5-Cl-phen)]Cl2 was loaded onto an SP Sephadex column (2.6 · 100 cm). The isomers were eluted with 75 mM disodium-O,O 0 -dibenzoyl-L-tartrate (pH 8), which was prepared by neutralising O,O 0 -dibenzoyl tartaric acid with sodium hydroxide. The solution was also filtered prior to use through 0.45 lm nylon membranes. Extraction of each fraction into dichloromethane (3 · 30 mL) after the addition of KPF6 gave each isomer. Optical purity was then established by circular dichroism. D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2: Anal. Calc. for C37H34F12N6P2RuCl: C, 44.93; H, 3.46; N, 8.50. Found: C, 45.12; H, 3.69; N, 8.00%. CD kmax nm (De mdeg/M cm) (H2O/(CH3)2CO): 210 (41); 226 (+40); 264 (+118); 285 (246); 410 (+11). K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2: Anal. Calc. for C37H34F12N6P2RuCl: C, 44.93; H, 3.46; N, 8.50. Found: C, 44.67; H, 3.51; N, 8.13%. CD kmax nm (De mdeg/M cm) (H2O/(CH3)2CO): 210 (+41); 226 (56); 264 (151); 285 (+301); 410 (24). 2.2.3. Rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)] (PF6)2 [Ru(Me2bipy)2(5-Cl-phen)](PF6)2 (0.03 g, 0.03 mmol) and L-cysteine methyl ester butoxy carbamate (L-cysteineMeBOC) (22 lL, 0.30 mmol) were dissolved in DMF (3 mL) and de-aerated for 5 min under N2, after which time a degassed solution of Na2CO3 (0.030 g, 0.30 mmol) in water (3 mL) was added via syringe. The solution was then heated to 55 C for 1.5 h. The reaction mixture was allowed to cool to room temperature and the solvent removed under reduced pressure. The product was purified by silica gel chromatography [41] with an eluent of 1–3% KNO3 in 10% water/acetonitrile. The eluted product was rotary evaporated to dryness, dissolved in a minimum amount of water then KPF6 added. The resultant precipitate was extracted into dichloromethane and dried over sodium sulphate. The solvent was removed and the residue dried under reduced pressure. Yield: 19 mg (54%). MS (ESMS, CH3CN, [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 = 1173.17) m/z: 829.1 (M{1(PF6) + C9H16NO4}+). 1H NMR (400 MHz, CD3CN) d: 8.81 (H4; 1H, dd, J1 = 1.32 Hz, J2 = 8.42 Hz), 8.50 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.39 (H6; 1H, s), 8.36 (A/B3; 2H, s), 8.32 (A/B3; 2H, s), 8.14 (H2; 1H, dd, J1 = 1.06 Hz, J2 = 5.22 Hz), 8.08 (H9; 1H, dd, J1 = 1.22 Hz, J2 = 5.22 Hz), 7.80 (H3; 1H, dd, J1 = 5.20 Hz, J2 = 8.55 Hz), 7.71 (H8; 1H, dd, J1 = 5.20 Hz, J2 = 8.36 Hz), 7.62 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.90 (D-NH; 1H, bs), 5.72 (KNH; 1H, bs), 5.42 (OCH3; 3H, s), 4.48 (D-N–CH; 1H, bs), 4.38 (K-N–CH; 1H, bs), 3.22 (D-CH2; 2H, m), 2.96 (K-CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.40 (BOC; 9H, s). 13C NMR (400 MHz, CD3CN) d: 154.2 (C2; CH), 153.8 (C11; CH), 152.3 (C26; CH), 152.0
321
(C20; CH), 151.5 (C18; QC), 151.4 (C24; QC), 136.7 (C9; CH), 134.5 (C4; CH), 132.6 (C8; QC), 129.3 (C19; CH), 129.1 (C25; CH), 128.2 (C7; CH), 127.6 (C3; CH), 127.6 (C10; CH), 125.9 (C23; CH), 125.8 (C17; CH), 56.6 (Cys; CH), 53.0 (BOC; CH3), 28.5 (BOC; CH3), 27.2 (Cys; CH2), 21.4 (C18Me; CH3), 21.3 (C24Me; CH3). 2.2.3.1. D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2. Experimental conditions were the same as those in Section 2.2.3. CD kmax nm (De mdeg/M cm) (H2O/ (CH3)2CO): 210 (254); 227.5 (+172); 271 (+716); 285 (1955); 410 (+77). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.11 · 103); 334 (9.21 · 102); 279 (7.69 · 103). U (CD3CN) = 0.086. 1H NMR (400 MHz, CD3CN) d: 8.73 (H4; 1H, dd, J1 = 1.41 Hz, J2 = 8.66 Hz), 8.43 (H7; 1H, dd, J1 = 1.21 Hz, J2 = 8.26 Hz), 8.36 (A/B3; 2H, s), 8.31 (A/B3; 2H, s), 8.08 (H2; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.94 (H9; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.91 (H6; 1H, s), 7.71 (H3; 1H, dd, J1 = 5.33 Hz, J2 = 8.54 Hz), 7.65 (H8; 1H, dd, J1 = 5.55 Hz, J2 = 8.38 Hz), 7.62 (A/B6; 2H, m), 7.32 (A/B6; 2H, m), 7.26 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.98 (NH; 1H, bs), 5.42 (OCH3; 3H, s), 4.54 (N–CH; 1H, bs), 3.20 (CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.41 (BOC; 9H, s). 2.2.3.2. K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2. Experimental conditions were the same as those in Section 2.2.3. CD kmax nm De mdeg/M cm (H2O/ (CH3)2CO): 210 (+210); 227.5 (248); 271 (726); 285 (+1999); 410 (214). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.63 · 103); 334 (1.38 · 103); 267 (3.39 · 104). U (CD3CN) = 0.157. 1H NMR (400 MHz, CD3CN) d: 8.73 (H4; 1H, dd, J1 = 1.41 Hz, J2 = 8.66 Hz), 8.43 (H7; 1H, dd, J1 = 1.21 Hz, J2 = 8.26 Hz), 8.36 (A/B3; 2H, s), 8.31 (A/B3; 2H, s), 8.08 (H2; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.94 (H9; 1H, dd, J1 = 1.21 Hz, J2 = 5.13 Hz), 7.91 (H6; 1H, s), 7.71 (H3; 1H, dd, J1 = 5.33 Hz, J2 = 8.54 Hz), 7.65 (H8; 1H, dd, J1 = 5.55 Hz, J2 = 8.38 Hz), 7.62 (A/B6; 2H, m), 7.32 (A/ B6; 2H, m), 7.26 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.75 (NH; 1H, bs), 5.42 (OCH3; 3H, s), 4.33 (N–CH; 1H, bs), 2.94 (CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/ BMe; 6H, s), 1.41 (BOC; 9H, s). 2.2.4. Deprotection of rac-[Ru(Me2bipy)2(5-(L-cysteineMeBOC)-phen)](PF6)2 Rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (50 mg, 0.040 mmol) was dissolved in dichloromethane (2 mL) and added to a solution of trifluoroacetic acid/ dichloromethane (1:1, 2.2 mL) containing water (40 lL) which was stirred for 1.5 h. The reaction was monitored by TLC {3% KNO3 in 10 % H2O/CH3CN, ninhydrin stain (0.2% w/v in ethanol)}. Upon completion, the solvent was removed under reduced pressure and the residue stirred with DOWEX 550A OH anion exchange resin
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(148 mg, loading capacity 3.44 mmol/g, washed with methanol). The solution was filtered to remove the resin, and the solvent removed to yield a red solid that was dried under vacuum. Yield: 46 mg (100%). 1H NMR (300 MHz, CD3CN) d: 8.81 (H4; 1H, dd, J1 = 1.32 Hz, J2 = 8.42 Hz), 8.50 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.39 (H6; 1H, s), 8.36 (A/B3; 2H, s), 8.32 (A/B3; 2H, s), 8.14 (H2; 1H, dd, J1 = 1.06 Hz, J2 = 5.22 Hz), 8.08 (H9; 1H, dd, J1 = 1.22 Hz, J2 = 5.22 Hz), 7.80 (H3; 1H, dd, J1 = 5.20 Hz, J2 = 8.55 Hz), 7.71 (H8; 1H, dd, J1 = 5.20 Hz, J2 = 8.36 Hz), 7.62 (A/B6; 2H, m), 7.31 (A/B6; 2H, m), 7.27 (A/B5; 2H, m), 7.03 (A/B5; 2H, m), 5.42 (OCH3; 3H, s), 4.48 (D-N–CH; 1H, bs), 4.38 (K-N–CH; 1H, bs), 3.22 (D-CH2; 2H, m), 2.96 (K-CH2; 2H, m), 2.55 (A/BMe; 6H, s), 2.46 (A/BMe; 6H, s), 1.26 (NH2; 2H, s). 2.2.5. Rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 Trans-[PtCl2(NH3)2] (0.013 g, 0.040 mmol) was suspended in water (5 mL), acidified with 1 M HCl to pH 5, then heated to 95 C to dissolve the metal complex. Over a period of 1.5 h, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]Cl2 (0.037 g, 0.040 mmol) in water (10 mL) was added drop wise to the solution, in the dark, after which time the temperature (95 C) was maintained for 5 h. The solution was then cooled to 0 C and filtered to remove any precipitate. The solution volume was reduced under vacuum and the remaining water removed by lypholysation. The red/ orange precipitate was dissolved in a minimum of methanol and applied to a Sephadex LH20 column (3 · 15 cm, 70 g packing material). Fractions (3 mL) were collected and monitored by TLC (3% saturated potassium nitrate, 10% water/acetonitrile, ninhydrin stain). Rac[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ eluted first, and fractions containing the product were combined before the solvent was removed under reduced pressure. Yield: 37 mg (78%). MS (ESMS, CH3CN, MW [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]3+ = 1045) m/z: 342.1 (M=3 1NH3 þ , 1H+). 1H NMR (400 MHz, d6-DMSO) d: 8.84 (H4; 1H, dd, J1 = 1.02 Hz, J2 = 8.46 Hz), 8.71 (H7; 1H, dd, J1 = 1.11 Hz, J2 = 8.31 Hz), 8.36 (A/B3; 2H, s), 8.69 (A/B3; 2H, s), 8.73 (H6; 1H, s), 8.22 (H2; 1H, d, J = 5.22 Hz), 8.14 (H9; 1H, d, J = 4.90 Hz), 7.96 (H3; 1H, dd, J1 = 5.29 Hz, J2 = 8.42 Hz), 7.87 (H8; 1H, dd, J1 = 5.09 Hz, J2 = 8.23 Hz), 7.62 (A/B6; 2H, m), 7.40 (A/B6; 2H, m), 7.35 (A/B5; 2H, m), 7.14 (A/B5; 2H, m), 4.35 (NH2; 1H, s), 4.07 (N–CH; 1H, q, J1 = 4.93 Hz, J2 = 10.01 Hz), 3.50 (OCH3; 3H, s), 3.16 (CH2; 2H, d, J = 4.78 Hz), 2.53 (A/BMe; 6H, s), 2.45 (A/BMe; 6H, s). 2.2.5.1. D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3. Experimental conditions were the same as those in Section 2.2.5. CD kmax nm (De mdeg/M cm) (H2O): 210 (289); 228.5 (+334); 270.5 (+1044); 285 (2808); 410 (+250). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.55 · 104); 334 (1.25 · 104); 257
(3.17 · 104); 282 (7.12 · 104); 206 (6.95 · 104). 1H NMR (400 MHz, d6-DMSO) d: 8.74 (H4; 1H, dd, J1 = 1.20 Hz, J2 = 8.56 Hz), 8.72 (A/B3; 2H, s), 8.68 (A/B3; 2H, s), 8.61 (H7; 1H, dd, J1 = 1.20 Hz, J2 = 8.38 Hz), 8.14 (H2; 1H, dd, J1 = 1.16 Hz, J2 = 5.22 Hz), 8.11 (H6; 1H, s), 7.98 (H9; 1H, dd, J1 = 1.31 Hz, J2 = 5.37 Hz), 7.87 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 7.88 Hz), 7.80 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.27 Hz), 7.61 (A/B6; 2H, m), 7.38 (A/ B6; 2H, m), 7.32 (A/B5; 2H, m), 7.15 (A/B5; 2H, m), 3.95 (N–CH; 1H, d, J = 8.77 Hz), 3.89 (NH2; 1H, s), 3.73 (CH2; 2H, dd, J1 = 7.10 Hz, J2 = 14.20 Hz), 2.81 (OCH3; 3H, s), 2.53 (A/BMe; 6H, s), 2.44 (A/BMe; 6H, s). 2.2.5.2. K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3. Experimental conditions were the same as those in Section 2.2.5. CD kmax nm (De mdeg/M cm) (H2O): 210 (+289); 228.5 (243); 270.5 (895); 285 (+2505); 410 (210). kmax/nm (e/mol1 dm3 cm1) (H2O/ (CH3)2CO): 456 (1.45 · 104); 334 (1.17 · 104); 257 (2.88 · 104); 282 (6.09 · 104); 206 (6.03 · 104). 1H NMR (400 MHz, d6-DMSO) d: 8.74 (H4; 1H, dd, J1 = 1.20 Hz, J2 = 8.56 Hz), 8.72 (A/B3; 2H, s), 8.68 (A/B3; 2H, s), 8.61 (H7; 1H, dd, J1 = 1.20 Hz, J2 = 8.38 Hz), 8.14 (H2; 1H, dd, J1 = 1.16 Hz, J2 = 5.22 Hz), 8.11 (H6; 1H, s), 7.98 (H9; 1H, dd, J1 = 1.31 Hz, J2 = 5.37 Hz), 7.87 (H3; 1H, dd, J1 = 5.22 Hz, J2 = 7.88 Hz), 7.80 (H8; 1H, dd, J1 = 5.22 Hz, J2 = 8.27 Hz), 7.61 (A/B6; 2H, m), 7.38 (A/ B6; 2H, m), 7.32 (A/B5; 2H, m), 7.15 (A/B5; 2H, m), 3.95 (N–CH; 1H, d, J = 8.77 Hz), 3.89 (NH2; 1H, s), 3.73 (CH2; 2H, dd, J1 = 7.10 Hz, J2 = 14.20 Hz), 2.81 (OCH3; 3H, s), 2.53 (A/BMe; 6H, s), 2.44 (A/BMe; 6H, s). 3. Results and discussion 3.1. Metal complex synthesis The heterodinuclear metal complex was synthesised using a six step method as shown in Scheme 1 (where the proton numbering scheme is shown after step 2 and the carbon numbering scheme is shown after step 3). All reactions were initially performed with unresolved mononuclear complexes then repeated with the D- and K-isomers once the reaction conditions had been optimised. [Ru(Me2bipy)2Cl2] was synthesised using alternative methods [37,38]. The phenanthroline ligand was then coordinated to the [Ru(Me2bipy)2Cl2] complex by refluxing in methanol for approximately half an hour. Completion of the reaction was signified by a change in colour of the solution from deep purple to red. Upon removal of the solvent a red/brown solid resulted. Two methods of purifying rac-[Ru(Me2bipy)2(5-Clphen)](PF6)2 were trialed. Dissolution of the ruthenium complex as a hexafluorophosphate salt in acetonitrile and elution through an aluminum oxide column produced an impure product; however, further purification on a cation exchange column enabled isolation of the desired purified metal complex. Purification using an
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323
2+
8
N
N
Cl
Ru
RuCl3.3H2O 2 x Me2bipy
N
N
B5
5-Cl-phen
N
N N
N BMe
6
Ru
B6
Cl
7
9
N
N
Cl
A6 2
4
B3
3 A3
A5 AMe
stereoisomer resolution
Δ,Λ
2+
2+
10
N N
N N
N
N
O
Ru S
N
O
O
25
L-cysteine-MeBOC
24
7 6
N
22 16 N
20
23
O
8 13 14
N
NH
9
N
Ru
26
N
11
Cl
5
2
4 3
17
19 18
HCl/TFA
3+ 2+
N
N N
N
N O
Ru N
N N
transplatin S
N O
Ru O
N
N
S
N
O NH2
NH2 H3N
Pt
NH3
Cl
Scheme 1.
SP Sephadex C-25 column alone (sodium chloride eluent) was also trialed. Before loading onto the Sephadex column, the ruthenium complex was converted to the chloride salt by stirring with Amberlite anion exchange beads. The first band to be eluted with water from the column was a golden brown colour, followed by a yellow band that eluted with 0.1 M NaCl. The remaining deep red band eluted with 0.3 M NaCl and was the desired product. A residual brown band, thought to comprise [Ru(Me2bipy)2Cl2], remained bound to the column head. A column 10–15 cm in length and 5 cm in width was found to be sufficient to attain separation between the bands. The metal complex was isolated by the addition of potassium hexafluorophosphate and extracted into dichloromethane.
The isomers of [Ru(Me2bipy)2(5-Cl-phen)]2+ were resolved using solvent-recycled solution cation exchange chromatography [42]. Using SP Sephadex C25 and 75 mM disodium-O,O 0 -dibenzoyl-L-tartrate as the eluent, two discreet bands were observed after an effective column length of 0.9 m, indicating baseline-to-baseline separation. After collection, the metal complexes were precipitated by the addition of potassium hexafluorophosphate and extracted into dichloromethane, to remove the tartrate salt. Two diaminoalkanes of varying carbon chain lengths were protected using BOC [43], with the intention of observing the effect of linker length on the DNA binding affinity of the heterodinuclear complex. Diaminoalkanes were selected for their flexibility, which would allow the different metal centres to bind at preferential DNA structures,
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and because of their relevance in multi-nuclear platinum agents [44–46]. Attempts were made to couple the single amine BOC-protected linkers 1,6-diaminohexane, 1,3-diaminopropane and 2-mercaptoethylamine to free 5-Cl-phen before addition of the ligand to [Ru(Me2bipy)2Cl2]. Unfortunately the coupling attempts were unsuccessful as the phenanthroline-linker bond was degraded upon coordination of the ligand to the metal centre. Subsequent attempts were made to couple the linkers to phenanthroline in [Ru(Me2bipy)2(5-Cl-phen)]2+, however, degradation of the phenanthroline-linker C–N bond was again observed. The method of Hurley et al. [47] which involved the coupling of L-cysteine to 3-bromo-1,10-phenanthroline containing ruthenium complexes under mildly basic conditions was therefore attempted, but instead using BOC-protected L-cysteine methyl ester (L-cys-MeBOC) to couple to the phenanthroline ligand of [Ru(Me2bipy)2(5-Cl-phen)]2+. After the reaction, two distinct bands were observed with silica gel column chromatography, one eluting with 1% (Rf = 0.57) and the other with 3% (Rf = 0.33) saturated potassium nitrate solutions. Excess L-cys-MeBOC moved with the solvent front. 1H NMR spectra subsequently showed that the desired compound was contained within the second fraction. The BOC-protecting group was cleaved from [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 using strongly acidic conditions. Initially, de-protection was achieved using a solution of concentrated hydrochloric acid in methanol (48 h) to isolate the amine as a hydrochloride salt. Alternatively, the use of trifluoroacetic acid (TFA) in dichloromethane also achieved de-protection, in a shorter length of time (1 h) with 100% recovery of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)](PF6)2, and was thus the preferred option of the two reactions. Stirring over DOWEX Monosphere 550A (OH form) in methanol neutralised the TFA/amine salt complex. The coupling of transplatin {trans-diamminedichloroplatinum(II)} to the amine group of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]2+ proved more difficult than first anticipated. After purification on Sephadex LH20, the presence of light during the reaction was found to result in a very low yield of [Ru(Me2bipy)2(5-(L-cysteine-Me)phen)-trans-Pt(NH3)2Cl]Cl3. Repeated experiments with altered exposure to light demonstrated that formation of the heterodinuclear complex was photosensitive. Consequently, the synthesis and purification of the final metal complex were performed in the dark. The separation of the mononuclear reactant and the heterodinuclear product was achieved using Sephadex LH20 support media as the stationary phase with methanol. TLC was used to verify the contents of the fractions, with rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 displaying an Rf value of 0.67 and the unreacted rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)]2+ stained purple with ninhydrin at Rf 0.38. The final product was precipitated by the addition of potassium hexafluorophosphate, and extracted into dichlo-
romethane. Conversion to the water soluble chloride salt was then achieved by stirring with Amberlite anion exchange beads, followed by filtration in order to remove the resin, and finally freeze drying, which yielded the heterodinuclear complex as a red solid. 3.2. Characterisation The heterodinuclear metal complex and the precursor mononuclear complexes were fully characterised by 1H and 13C NMR, circular dichroism, and electrospray ionisation mass spectrometry, and are described in more detail in Sections 3.2.1, 3.2.2, 3.2.3. UV–Vis spectra were measured to determine the molar extinction coefficients, fluorescence spectra were used to determine quantum yields and elemental analysis to confirm bulk purity. Unfortunately, the use of Eq. (2) for the determination of the quantum yields of fluorescent samples in comparison to a standard, at a common excitation wavelength, was not applicable for the heterodinuclear complex. This was because its excitation wavelength had shifted dramatically from the excitation wavelength of tris(bipyridine) ruthenium(II) (the standard). The method is thus limited in use, and another standard of known quantum yield is required to calculate the relative fluorescence of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3. Elemental analysis demonstrated bulk purity of Rac[Ru(Me2bipy)2(5-Cl-phen)] Æ 2PF6 with one water of crystallisation, but which is lost upon separation into its isomers. Bulk purity of the boc-protected ruthenium complex and the final heterodinuclear complex could not be obtained by elemental analysis; however, purity is indicated by both 1H and 13C NMR and high resolution mass spectrometry. Bulk and isomeric purity was also confirmed by CD with the spectrum of each isomer of all metal complexes synthesised showing peaks in almost equal proportion, in opposite directions, at the same wavelength. In addition, the CD spectra also confirm the purity of the samples through the presence of isosbestic points, where the two spectra intercept on the xaxis. 3.2.1. NMR spectroscopy All metal complexes in this study were characterised using one-dimensional 1H and 13C NMR and two-dimensional NOESY, g-DQCOSY experiments. 13C assignments were made using heteronuclear multiple quantum correlation spectroscopy (HMQC) and heteronuclear multiple bond correlation spectroscopy (HMBC) experiments. Full details of the 1H NMR spectrum of the heterodinuclear complex, rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)trans-Pt(NH3)2Cl]Cl3, including proton assignments are presented here as an example of how all the metal complexes were characterised. The 1H NMR spectrum of rac-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3 displays 13 resonances in the aromatic region and six resonances in the
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aliphatic region. The two singlets at 2.46 and 2.54 ppm each integrate for six protons, and are assigned as the two methyl groups on the Me2bipy rings. The NOESY spectrum shows NOE cross-peaks from the A/BMe resonance at 2.54 ppm to the singlet at 8.38 ppm and a multiplet at 7.27 ppm, which are assigned as the A/B3 and A/ B5 protons, respectively. In the g-DQCOSY spectrum, the A/B5 resonance also displays cross-peaks with a multiplet 7.63 ppm, which is assigned as the A/B6 proton (Fig. 1). Similar cross-peaks were observed from the A/ BMe resonance at 2.46 ppm, to the A/B3 resonance at 8.34 ppm, A/B5 at 7.04 ppm and from this latter resonance to the A/B6 at 7.31 ppm. It was hoped that cross-peaks might be observed in the NOESY spectrum from the A/ B6 resonances to either H2 or H9 on the phenanthroline ring, allowing the definitive assignments of the Me2bipy proton resonances to particular protons, but no NOE cross-peaks were observed. The substitution of a chloro group at the 5-position in 1,10-phenanthroline results in seven separate proton resonances for this ligand. The H6 proton resonance couples to no other proton (d 8.39 ppm), and is the only aromatic singlet in the spectrum integrating for one proton. Both
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H3 and H8 each have two neighbouring protons and can be identified by their doublet of doublet resonances (see Fig. 1). The remaining resonances for the H2, H4, H7 and H9 protons all appear as doublets. Definitive assignments can be made using NOESY spectra. The H6 resonance gives one NOE cross-peak to the resonance at 8.50 ppm (assigned as H7), which in turn gives an NOE to H8 at 7.73 ppm, and from H8 to H9 at 8.10 ppm. The remaining doublet of doublet resonance at 7.82 is assigned as the H3 proton. The H2 and H4 resonances can be assigned based on the coupling constants. In pyridine rings, the proton nearest the nitrogen atom exhibits the smallest coupling constant (typically 5–6 Hz), whilst a proton further away exhibits a larger coupling constant (typically 7–9 Hz) [48]. On this basis, the resonance at 8.81 ppm is assigned as H4 and the resonance at 8.16 ppm as H2. The remaining unassigned proton resonances in the spectrum are the –NH2, –CH, –OCH3, and –CH2 protons of the L-cysteine methyl ester. The resonance at 4.35 ppm is assigned to –NH2, due to its broadness, the singlet at 3.50 ppm to –OCH3, the triplet at 3.16 ppm to –CH2 and the multiplet at 4.07 ppm to –CH proton. Coordination of the platinum to the cystiene-NH2 group is confirmed
Fig. 1. 1H g-DQCOSY NMR spectrum showing the assignment of the aromatic proton resonances of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]3+.
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by the large downfield movement of the proton resonance, which moves from 1.26 to 4.35 ppm, upon reaction with transplatin. It is of note that there are slight differences in the 1H NMR spectra of the D- and K-ruthenium complexes compared to that of the racemic solutions for all metal complexes synthesised in this study. For instance, a significant chemical shift change of the H6 resonance (8.29–7.91 ppm) is observed in the spectra of the resolved isomers of [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)]2+ in comparison to the spectrum of the racemic complex (Fig. 2). There is also a large movement of the –NH2 resonance (4.35–3.89 ppm). All other resonances undergo less significant chemical shift changes upon resolution of the isomers.
3000 2000 1000 0 -1000 -2000 -3000 200
250
300
350
400
450
Wavelength (nm)
Fig. 3. Circular dichroism spectra of the isomeric complexes from top to bottom (at 285 nm): K-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3, K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2, K-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2, D-[Ru(Me2bipy)2(5-Cl-phen)](PF6)2, D-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 and D-[Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3.
3.2.2. Circular dichroism spectrophotometry CD spectra were recorded for each of the metal complex isomers by plotting the angle of ellipticity (h mdeg) versus wavelength (nm). This was converted to the change in molar absorptivity by Eq. (1). All metal complexes synthesised in this study show four major peaks at 210, 226, 264 and 285 nm, and one broad peak at 410 nm (Fig. 3). In comparison to [Ru(Me2bipy)2(5-Cl-phen)]2+, attachment of the amino acid and then the platinum metal centre do not change either the wavelength, nor shape of the CD peaks, however, the intensities are increased.
The assignments of the absolute configuration of the metal complexes are based on previous studies of analogous compounds [42]. The metal complex exhibiting a positive value of h at 250 nm corresponds to the D-isomer, while the K-isomer exhibits a negative peak at the same wavelength. The elution profile of the isomers from the Sephadex column further supported this finding, with the D-isomer eluting prior to the K-isomer.
A H6
B
H6
C H6
8.75
8.50
8.25
8.00
7.75
7.50
7.25
ppm (f1)
Fig. 2. 1H NMR spectra of (a) rac-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (b) K-[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 (c) D[Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)](PF6)2 demonstrating the chemical shift change of the phenanthroline H6 proton resonance in a racemic solution and when separated into its D- and K-isomers.
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3.2.3. Mass spectrometry The synthesis of the precursor mononuclear complexes and the final heteronuclear complexes were also confirmed by electrospray ionisation mass spectrometry. [Ru(Me2bipy)2(5-Cl-phen)]2+ shows a molecular ion at 341.7 m/z which corresponds to the metal complex with a 2+ charge and the loss of one proton and both hexafluorophosphate counter ions. For [Ru(Me2bipy)2(5-(L-cysteineMeBOC)-phen)]2+, five peaks are observed for the parent and daughter ion fragments. For instance, a molecular ion at 1028.2 m/z is consistent with the [Ru(Me2bipy)2(5-(L-cysteine-MeBOC)-phen)]2+ cation and one PF6 counterion. Finally, the heterodinuclear complex was confirmed by the observance of a molecular ion at 342.1 m/z, corresponding to the charged parent ion minus both counterions and one ammine group. This is characteristic of charged metal complexes of this type and matched the expected isotopic pattern for a ruthenium(II)–platinum(II) complex. 4. Summary The heterodinuclear complex, [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-trans-Pt(NH3)2Cl]Cl3, was synthesised using a six step method, as a potential agent to overcome platinum resistance in human cancer treatment. The heterodinuclear complex, and its mononuclear precursors were characterised by 1H and 13C NMR, UV–Vis, circular dichroism, fluorescence and electrospray ionisation mass spectrometry. The cytotoxicity and DNA binding studies of [Ru(Me2bipy)2(5-(L-cysteine-Me)-phen)-transPt(NH3)2Cl]Cl3 are currently under examination, with the results to be used to develop a family of heterodinuclear complexes with anticancer activity. Acknowledgements This work was supported by an Australian Research Council Grant (No. DP0343480) and the University of Western Sydney. We thank Assoc. Prof. S. Ralph (University of Wollongong) for assistance with the mass spectrometry and Prof. W.S. Price and M. van Holst (University of Western Sydney) and Dr. D.M. D’Alessandro (University of Sydney) for helpful discussions. References [1] N.J. Wheate, J.G. Collins, Curr. Med. Chem. – Anti-Cancer Agents 5 (2005) 267. [2] N.J. Wheate, J.G. Collins, Coord. Chem. Rev. 241 (2003) 133. [3] N. Farrell, Met. Ions Biol. Sys. 42 (2004) 251. [4] V. Murray, H. Motyka, P.R. England, G. Wickham, H.H. Lee, W.A. Denny, W.D. McFadyen, J. Biol. Chem. 267 (1992) 18805. [5] C. Cullinane, G. Wickham, W.D. McFadyen, W.A. Denny, B.D. Palmer, D.R. Phillips, Nucl. Acids Res. 21 (1993) 393. [6] L.C. Perrin, P.D. Prenzler, C. Cullinane, D.R. Phillips, W.A. Denny, W.D. McFadyen, J. Inorg. Biochem. 81 (2000) 111. [7] L.C. Perrin, C. Cullinane, W.D. McFadyen, D.R. Phillips, AntiCancer Drug Des. 14 (1999) 243.
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