Multinuclear ruthenium( II ) complexes as anticancer agents

A series of dinuclear ruthenium( II ) complexes that contain labile chlorido ligands, [{Ru(tpy)Cl} 2 { m -bb n }] 2+ {designated Cl-Rubb n ; tpy = 2,2 0 :6 0 ,2 00 -terpyridine, bb n = bis[4(4 0 -methyl-2,2 0 -bipyridyl)]-1, n -alkane ( n = 7, 10, 12, 14 or 16)} and derivatives containing nitro substituents on the tpy ligand and/or secondary amines within the bb n linking chain have been synthesised and their potential as anticancer agents examined. Some of the Cl-Rubb n species showed good anticancer activity against MCF-7 and MDA-MB-231 breast cancer cell lines, with the Cl-Rubb 12 complex being four-times more active than cisplatin. Inclusion of nitro substituents on the tpy ligands of Cl-Rubb 12 resulted in significantly decreased anticancer activity. The incorporation of amine groups into the linking ligand did not increase the anticancer activity of the Cl-Rubb n complexes. The Cl-Rubb n complexes and those containing amine groups in the linking chain aquated at approximately the same rate, with 50% aquation within 120 minutes. By comparison, the complexes containing nitro substituents on the tpy ligand aquated extremely slowly, with 60% of the chlorido complex remaining 24 hours after they were dissolved in water. Cyclic voltammetry with the model mononuclear complex [Ru{(NO 2 ) 3 tpy}(Me 2 bpy)Cl] + {(NO 2 ) 3 tpy = 4,4 0 ,4 00 - trinitro-2,2 0 :6 0 ,2 00 -terpyridine} showed that the nitro substituents exerted a strong eﬀect on the ruthenium centre, with the anodic peak corresponding to the Ru( III / II ) couple shifted positively by 300 mV compared to that from the non-nitrated parent complex [Ru(tpy)(Me 2 bpy)Cl] + . 1 H NMR studies of the reaction of the Cl-Rubb n complexes with GMP indicated that the ruthenium complexes covalently bound the nucleotide slowly, with 33% bound in 24 hours. However, the results of this study suggest that the cytotoxicity of the dinuclear ruthenium complexes is a combination of covalent and reversible binding with DNA.


Introduction
Although cisplatin has been in clinical use for over 30 years, its toxicity and natural/acquired resistance to many cancers has considerably limited its application. 1While some secondgeneration platinum complexes are less toxic than cisplatin, and others can partially overcome acquired resistance, there has been little success in developing drugs that are active in cancer cell lines resistant to cisplatin.[4][5][6][7] Multinuclear platinum complexes, where two or more platinum coordination units are linked by a variety of organic ligand bridges, represent a genuinely new class of anticancer drug. 2 While complexes with bi-functional platinum centres have been reported, those containing mono-functional coordinating spheres on the terminal platinum atoms (e.g.][10][11] Furthermore, complexes bearing a cationic charge and hydrogen-bonding capacity (e.g.3][14][15][16][17][18][19][20] The trinuclear complex BBR 3464, [trans-{PtCl(NH 3 ) 2 } 2 -{m-trans-Pt(NH 3 ) 2 (H 2 N(CH 2 ) 6 NH 2 ) 2 }] 4+ , has undergone Phase II clinical trials, [21][22][23] while dinuclear complexes linked by spermidine (BBR 3571, see Fig. 1) and spermine (BBR 3610 and BBR 3611) are cytotoxic at nanomolar concentrations. 24][25][26] Furthermore, upon administration they bind thiol-containing plasma proteins in the bloodstream, and are subsequently degraded to non-active metabolites.Although BBR 3464 has been withdrawn from This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 clinical trials, there has been recent interest in ''transferring the concept of multinuclearity to ruthenium complexes''. 27Mendoza-Ferri et al. synthesised a series of dinuclear ruthenium(II)-arene compounds containing a bis(pyridinone)alkane linking ligand that incorporated 3, 6 or 12 methylene groups in the alkane chain. 27The ruthenium-arene complexes showed good activity in a variety of cancer cell lines, with the activity increasing with the length of the alkane linker, and were more active than a similar mononuclear analogue.In addition, Yamada et al. synthesised [{Ru(bpy) 2 Cl} 2 {m-BL}] 2+ complexes {where bpy = 2,2 0 -bipyridine and BL = 1,6-diaminohexane or 1,12-diaminododecane} and examined their cytotoxicity. 28While the chlorido complexes showed little activity, replacement of the chlorido ligand by DMSO in the 1,12-diaminododecane-bridged complex resulted in good activity against L1210 cells.

Synthesis
The synthesis of the mononuclear [Ru(tpy)(bpy)Cl] + and the dinuclear complexes [{Ru(tpy)Cl} 2 (m-bb n )] 2+ (Cl-Rubb n for n = 7, 10, 12, 14 and 16) have been previously reported. 30,31In this study, we have extended the family of dinuclear complexes through the synthesis of Cl-Rubb n NO 2 , Cl-RubbN n , and Cl-RubbN n NO 2 complexes, as shown in Schemes 1-3.For the Cl-RubbN n complexes, the procedure used for the synthesis of the Cl-Rubb n complexes resulted in poor yield and purity for the Cl-RubbN n complexes.To obtain satisfactory yields the bbN n ligand was dissolved in ethanol-water and heated to 60 1C before the [Ru(tpy)Cl 3 ] was added, and then the mixture refluxed for a longer time period than was necessary for the synthesis of Cl-Rubb n .[Ru{(NO 2 ) 3 tpy}Cl 3 ] was prepared in a similar manner to that previously reported for [Ru(tpy)Cl 3 ], 32 and upon addition of 4,4 0 -dimethyl-2,2 0 -bipyridine yielded [Ru{(NO 2 ) 3 tpy}(Me 2 bpy)Cl]Cl in good yield.The synthesis of the new chlorido-containing dinuclear complexes Cl-Rubb n NO 2 and Cl-RubbN n NO 2 were achieved using similar procedures.

Cytotoxicity
The in vitro cytotoxicities of the ruthenium complexes and the control platinum complexes cisplatin and carboplatin were determined against the MCF-7 and MDA-MB-231 breast cancer cell lines, and the results are summarised in Table 1.Cisplatin showed moderate cytotoxicity against both cell lines, while carboplatin was essentially inactive.][35] The dinuclear ruthenium complexes Cl-Rubb n , for n = 10, 12 and 14 were more active than cisplatin against both cell lines.Interestingly, Cl-Rubb 12 was the most active, with the ruthenium complexes having the shortest linking chain (Cl-Rubb 7 ) and longest linking chain (Cl-Rubb 16 ) being the least active.Addition of nitro substituents onto the tpy rings of Cl-Rubb 12 and Cl-Rubb 16 decreased the activity of the ruthenium complexes, particularly in the case of the highly active Cl-Rubb 12 .The replacement of two methylene groups by two amine groups in the ligand bridge for Cl-Rubb 7 (giving Cl-RubbN 7 ) and Cl-Rubb 16 (Cl-RubbN 16 ) decreased the activity of the former but had no effect on the latter complex that contained the longer linking chain.However, it was also noted that the replacement of the Me 2 bpy ligand in [Ru{(NO 2 ) 3 tpy}-(Me 2 bpy)Cl] + by the bbN 16 ligand to form the mononuclear complex Cl-RubbN 16 NO 2 -mono did significantly increase the activity in both cancer cell lines.In the one example examined, the combination of amine groups in the linking ligand and nitro substituents on the tpy ligands for Cl-RubbN 16 NO 2 had little Scheme 1 This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 effect on the cytotoxicity with the MCF-7 cells but decreased the activity against the MDA-MB-231 cell line.

Aquation and GMP binding
Previous studies with mononuclear ruthenium(II) complexes that contain a chlorido ligand have shown that the first step in the binding to GMP, a simple model for DNA, is aquation.Consistent with previous studies, 30 aquation of [Ru(tpy)(Me 2 bpy)Cl] + was found to be relatively fast, with 50% of the ruthenium complex being converted to the corresponding aqua form in approximately 60 minutes.Similarly, 50% aquation of each ruthenium centre in the dinuclear complexes Cl-Rubb n and Cl-RubbN n was shown by 1 H NMR spectroscopy to occur in approximately 120 minutes (see Fig. 3).The aquation then proceeds to equilibrium, where approximately 90% of the ruthenium complex exists in the aqua form.The inclusion of amine groups into the linking ligand had no significant effect on the rate or equilibrium position of aquation.
Fig. 4 shows the 1 H NMR spectrum of Cl-RubbN 16 as a function of time after dissolution in D 2 O and the addition of 2 equivalents of GMP.After 120 minutes, the spectrum of the Cl-RubbN 16 is essentially identical to that in the absence of GMP, as shown in Fig. 3, with approximately 50% of the dinuclear complex aquated but with no covalent binding to GMP observed.As evidenced by the increasing intensity of the resonance at 5.36 ppm, assigned to the sugar H1 0 of GMP bound to a ruthenium centre, the aquated form of Cl-RubbN 16 slowly reacts with GMP, reaching an equilibrium of approximately 33% bound in 24 hours.Similar results were obtained with the Cl-Rubb n complexes (results not shown).
Fig. 5 shows the 1 H NMR spectrum of [Ru{(NO 2 ) 3 tpy}-(Me 2 bpy)Cl] + at various time points after the ruthenium complex Table 1 The IC 50 values of the metal complexes against the MCF-7 and MDA-MB-231 breast cancer cell lines, defined as the concentration (mM) of the complex required to inhibit cell growth by 50%    36 it is proposed that one of the three nitro substituents on the tpy ligand is reduced to an amine.This new ''(NO 2 ) 2 (NH 2 )-tpy'' complex then slowly aquates.
The electrochemical response of the [Ru(tpy)(Me 2 bpy)Cl] + complex as a hexafluorophosphate salt has previously been investigated; 37 the results here are consistent with that report: two ligand-based reductions are observed in the cathodic region (tpy/tpy À followed by Me 2 bpy/Me 2 bpy À ), while the anodic region shows a reversible Ru(III/II) peak at +0.90 V.In the present case, an irreversible peak is also seen at +1.28 V, corresponding to oxidation of the chloride counter-ion.The [Ru{(NO 2 ) 3 tpy}(Me 2 bpy)Cl] + complex shows several important changes compared to the non-nitrated parent complex.Three closely-spaced reductions appear at low potentials in the cathodic region (À0.4 to À0.7 V), followed by further irreversible peaks at more negative potentials.Previous work on the electrochemical behaviour of nitrated bipyridines and their platinum complexes has shown analogous cathodic behaviour: for example [Pt{4,4 0 -(NO 2 ) 2 bpy}Cl 2 ] displayed two closely-spaced reductions, and the LUMOs for that complex were shown to be localised largely on the ''NO 2 -py'' units. 38Further reduction of the complex occurred at À1.05 V, 39 very close to the potential of À1.06 V observed for the first reduction (bpy/bpy À ) of the non-nitrated complex [Pt(bpy)Cl 2 ] under the same conditions. 38Based on these observations, the first three cathodic peaks for [Ru{(NO 2 ) 3 tpy}(Me 2 bpy)Cl]Cl are assigned here to reductions involving the NO 2 -py moieties.The next two peaks are assigned to further reduction of the (NO 2 ) 3 tpy ligand and reduction of the Me 2 bpy ligand, probably in that order.
Most importantly, the nitro substituents are observed to exert a strong effect on the ruthenium centre, as the anodic  peak corresponding to the Ru(III/II) couple is shifted positively by at least 300 mV, to the point where it coincides with oxidation of the chloride counter-ion (and is irreversible).This large positive shift indicates that the nitro substituents cause a significant decrease in the electron density on the ruthenium centre, making oxidation to Ru(III) more difficult.

Discussion
The results of this study show that the dinuclear ruthenium(II) complexes Cl-Rubb n have potential as drugs against breast cancer.The most active complex, Cl-Rubb In previous studies with chlorido-containing dinuclear ruthenium(II) complexes, 27,28,30,40 the cytotoxicity has always increased as the number of methylene groups in the flexible alkane chain increased.Interestingly, in the present study the Cl-Rubb 16 complex was the least active of the Cl-Rubb n complexes.The decreased activities of Cl-Rubb 7 and Cl-Rubb 16 , compared to Cl-Rubb 12 suggest two competing factors govern the anticancer activity.While it is yet to be confirmed, it is assumed that the major mechanism of anticancer activity is related to DNA binding, analogous to the corresponding dinuclear platinum complexes.Increasing the number of methylene groups in the linking chain should increase the lipophilicity of the dinuclear complex, and hence the ease with which it can pass through the cellular membrane.While aquation is the necessary first step in DNA binding, as determined by the GMP binding experiments, all the Cl-Rubb n complexes exhibited similar rates of aquation and percentage of the aqua form at equilibrium.Consequently, the relative cytotoxicity results could imply that the range of possible DNA cross-linked adducts formed have significantly different biological outcomes, and/or the anticancer activity is controlled by both covalent and reversible binding to DNA.For the corresponding inert Rubb n complexes, the DNA binding affinity decreases with increasing methylene groups in the linking chain. 41Furthermore, based purely upon polycation condensation of polyanionic DNA, it would also be expected that the cytotoxicity of the Cl-Rubb n complexes would decrease with increasing chain length.
The inclusion of three nitro substituents on the tpy ligand significantly increased the IC 50 value for the more cytotoxic Cl-Rubb 12 but had a relatively small effect with the less cytotoxic Cl-Rubb 16 .It was determined that the [Ru{(NO 2 ) 3 tpy}-(Me 2 bpy)Cl] + complex aquated significantly more slowly than the non-nitrated parent complex [Ru(tpy)(Me 2 bpy)Cl] + .This observation is consistent with the results from the cyclic voltammetry study, from which it was concluded that there was a significant reduction in the electron density on the ruthenium centre for the trinitrated complex, compared to the non-nitrated parent complex.The reduced electron density on the ruthenium centre of [Ru{(NO 2 ) 3 tpy}(Me 2 bpy)Cl] + would increase the energy barrier for the removal of the chlorido ligand from the metal centre, thereby decreasing the rate of the aquation reaction.Aquation was shown to be the first step in the coordination of the ruthenium complexes with DNA.Consequently, the Cl-Rubb n NO 2 complexes would not form as many covalent adducts with DNA over the time period of the cytotoxicity assays, compared to their non-nitrated parent complexes.This suggests that the observed cytotoxicity of the Cl-Rubb n NO 2 complexes would largely be due to their reversible, non-covalent, binding to DNA.Furthermore, it is reasonable to expect that the chlorido form of the complex would more easily cross a cellular membrane than the more highly positivelycharged aquated species.Based upon these assumptions, it could be tentatively concluded that the activity of Cl-Rubb 16 was predominantly due to reversible binding to DNA, while the activity of Cl-Rubb 12 was due to a combination of covalent and reversible binding to DNA.
Although the inclusion of one or more secondary amines into the bridging ligand of multinuclear platinum complexes significantly increases their cytotoxicity, 2 the incorporation of amine groups into the ligand bridge of Cl-Rubb n did not increase the cytotoxicity.For the multinuclear platinum complexes, incorporation of an amine group or an inert am(m)ineplatinum(II) centre into the bridge enhances cellular accumulation and increases the affinity for DNA. 2,14,42The corresponding inert Rubb n dinuclear ruthenium complexes (that do not contain labile chlorido ligands) enter L1210 murine leukaemia cells by passive diffusion, with a minor contribution from proteinmediated active transport. 41Consequently, incorporation of amine groups into the ligand bridge could decrease the cellular uptake of the Cl-Rubb n complexes, and thereby result in the observed lower activity for Cl-RubbN 7 relative to Cl-Rubb 7 .However, it was also noted that Cl-RubbN 16 was equally as active (albeit weakly) as Cl-Rubb 16 .This could suggest that the inclusion of an amine in the bridging ligand of a Cl-Rubb n complex does increase the reversible binding affinity for DNA, thereby compensating for the lower cellular uptake.

Conclusions
In conclusion, the results of this study support the idea of developing a new class of anticancer agent by transferring from platinum to ruthenium the concept of gaining advantages in efficacy through the use of multinuclear complexes, as proposed by Mendoza-Ferri et al. 27

Dinuclear ruthenium complexes -containing
This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 a single chlorido ligand on each metal centre -were synthesised and found to be significantly more active than cisplatin against two breast cancer cell lines.The anticancer activity appears to be due to a combination of covalent and reversible binding with DNA.The IC 50 results indicated that the Cl-Rubb 12 complex was the most active of the dinuclear complexes.The superior activity of Cl-Rubb 12 might be due to the best compromise between lipophilicity (for cellular uptake) and the cytotoxic effects of the covalent adducts formed with DNA.Given the vast array of ligands that can be utilised for the Cl-Rubb n complexes, it should be possible to optimise cellular uptake and the kinetics of DNA binding, and thereby produce dinuclear ruthenium(II) complexes with significant clinical potential.

Cyclic voltammetry
Cyclic voltammetry was carried out using an eDAQ EA161 potentiostat operated via an eDAQ ED401 e-corder.A glassy carbon working electrode, platinum wire counter electrode and Ag/AgCl reference electrode were used.HPLC grade acetonitrile was used as solvent and the supporting electrolyte was 0.1 mol L À1 tetra-n-butyl ammonium hexafluorophosphate (Aldrich).

Cytotoxicity assays
Cytotoxicity data was obtained using the mitochondrialdependent reduction of 3-(3,4-dimethylthiazol-2yl)-5-diphenyl tetrazolium bromide (MTT) to formazan as described by Guh et al. 43 Metal complex solutions, including the control platinum complexes cisplatin and carboplatin, were made to the required concentrations in warm Milli-Q water.Growth inhibition assays were carried out over a 72 h continuous exposure period.
Anal.The chloride salts were obtained by stirring the PF 6 salt in water using Amberlite IRA-400 (chloride form) anion-exchange resin.The resin was removed by filtration, and the solution was freeze-dried to obtain a fluffy dark violet-purple powder of pure [{Ru(NO 2 terpy)(Cl)} 2 (m-bb n )]Cl 2 in 30-35% yield.
[{Ru(tpy)Cl} 2 (m-bbH 2 N n )]Cl 4 .To the bbN 7 ligand (53 mg, 0.122 mmol) dissolved in EtOH/H 2 O (4 : 1; 15 mL), solid [Ru(tpy)Cl 3 ] (108 mg, 0.245 mmol) was added at 60 1C and the reaction mixture was refluxed under an N 2 atmosphere for 5-6 h.After cooling, half of the solvent was evaporated from the reaction mixture and saturated aqueous NH 4 PF 6 was added to obtain the PF 6 À salt as a dark purple-brown material, which was filtered and washed with ethanol (2 Â 20 mL) followed by diethyl ether (2 Â 20 mL).The crude product was dissolved in a minimum amount of acetone and loaded onto a column of Sephadex LH-20 (2 cm diam.Â 30 cm); and eluted with acetone, the major first band (dark purple coloured) was collected, the acetone evaporated to obtain [{Ru(tpy)Cl} 2 (m-bbH 2 N n )]Cl 4 complex as a dark purple-brown material.

Fig. 1
Fig. 1 Cisplatin, and the structure of a generic dinuclear platinum complex (top right) with the linking ligands (Y) shown for BBR 3464, BBR 3005 and BBR 3571.

Fig. 2
Fig. 2 Chlorido-containing dinuclear ruthenium(II) complexes, top Cl-Rubb n for X = H and Cl-Rubb n NO 2 for X = NO 2 , and bottom Cl-RubbN n for X = H and Cl-RubbN n NO 2 for X = NO 2 .

Fig. 3
Fig. 3 Aromatic region of the 1 H NMR spectrum of Cl-RubbN 16 in D 2 O as a function of time, after 5 minutes (A), 120 minutes (B) and 27 hours (C).The asterisk indicates the decrease in the H6-Me 2 bpy resonances of the Cl-RubbN 16 complex, while the arrow shows the increase in the H6-Me 2 bpy resonances from the D 2 O-RubbN 16 complex.

Fig. 4
Fig. 4 Aromatic region of the 1 H NMR spectrum of the Cl-RubbN 16 + GMP in D 2 O as a function of time, after 10 minutes (A), 120 minutes (B), 450 minutes (C), 25 hours (D) and 76 hours (E).The asterisk indicates the decrease in H6-Me 2 bpy resonances of the Cl-RubbN 16 complex, while the arrows shows the increase of the peak for the H6-Me 2 bpy protons of the GMP bound ruthenium complex (8.76 ppm) and the sugar H1 0 of the bound GMP (5.36 ppm).

Table 2 Electrode
potentials for [Ru(L)(Me 2 bpy)Cl]Cl in acetonitrile (in V vs. Ag/AgCl; working electrode = glassy carbon) This journal is © The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2014 New J. Chem., 2014, 38, 4049--4059 | 4055 27,29as almost fourtimes more active than cisplatin.Furthermore, Cl-Rubb 12 is more active than the mononuclear [Ru(apy)(tpy)Cl] + and dinuclear [{Ru(bpy) 2 Cl} 2 {m-BL}] 2+ complexes previously reported by other groups,28,29and of similar activity to the most active dinuclear ruthenium-arene complex linked by a bis(pyridinone)alkane chain reported by Mendoza-Ferri et al.27Interestingly, the Cl-Rubb n complexes with the shortest (Cl-Rubb 7 ) or the longest linking chain (Cl-Rubb 16 ) were the least active against both breast cancer cell lines.Insertion of three nitro substituents onto the tpy ligand of Cl-Rubb 12 significantly decreased the activity against both breast cancer cell lines.Incorporation of amine groups into the linking bridging ligand of Cl-Rubb 7 decreased the activity, whereas it had little effect on the activity of Cl-Rubb 16 .