Dinuclear ruthenium ( II ) complexes containing one inert metal centre and one coordinatively-labile metal centre : syntheses and biological activities

A series of non-symmetric dinuclear polypyridylruthenium(II) complexes (Rubbn-Cl) that contain one inert metal centre and one coordinatively-labile metal centre, linked by the bis[4(4’-methyl-2,2’-bipyridyl)]1,n-alkane ligand (“bbn” for n = 7, 12 and 16), have been synthesised and their potential as antimicrobial agents examined. The minimum inhibitory concentrations (MIC) of the ruthenium(II) complexes were determined against four strains of bacteria − Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA), and Gram-negative Escherichia coli (E. coli) and Pseudomonas aeruginosa (P. aeruginosa). The Rubbn-Cl complexes displayed good antimicrobial activity, with Rubb12-Cl being the most active complex against both Gram-positive and Gram-negative strains. Interestingly, Rubb7-Cl was found to be eightand sixteen-fold more active towards E. coli than against S. aureus and MRSA, respectively. The cytotoxicities of the Rubbn-Cl complexes against three eukaryotic cell lines – two kidney cell lines (BHK and HEK-293) and one liver cell line (HepG2) – were examined. The Rubbn-Cl complexes were found to be considerably less toxic towards eukaryotic cells than S. aureus, MRSA and E. coli, with Rubb12-Cl being thirtyto eighty-times more toxic to the bacteria than to BHK, HEK-293 or HepG2 cells. Unexpectedly, Rubb7-Cl was far more toxic to HepG2 cells (24 h-IC50 = 3.7 μM) and far less toxic to BHK cells (24 h-IC50 = 238 μM) than the Rubb12-Cl and Rubb16-Cl complexes. In order to understand the unexpected large differences in the cytotoxicities of the Rubbn-Cl complexes towards eukaryotic cells, a confocal microscopic study of their intracellular localisation was undertaken. The results suggest that the observed cytotoxicity might be related to the extent of DNA binding.


Introduction
The emergence of drug-resistant populations of microorganisms has become a serious worldwide health issue. 1 There is clearly a need for new antimicrobials; however more impor-tantly, new classes of antimicrobials are needed rather than drugs based upon analogues of known scaffolds.
Traditionally, the design and development of new antimicrobial drugs has centred upon organic chemistry.0][11][12][13][14] Dwyer and co-workers were the first to report the biological activity of mononuclear tris(bidentate) inert polypyridyl metal complexes, in particular complexes with 1,10-phenanthroline ligands. 9,10[Ru( phen) 3 ] 2+ was found to be inactive; however, the introduction of methyl substituents on the phen ligands dramatically increased the activity against all bacteria. 9,10More recently, it has been demonstrated that polypyridylruthenium(II) complexes which bind DNA by intercalation have significant bactericidal activity, particularly against Gram-positive strains. 11,12While DNA binding is generally thought to be responsible for the antimicrobial activity of polypyridylruthenium(II) complexes, Lam et al. suggested that the antimicrobial activity of a bis(bipyridine)ruthenium(II) complex containing a N-phenyl-substituted diazafluorene ligand might be due to DNA damage caused by the formation of reactive oxygen species. 15In addition, a range of labile ruthenium(II) and ruthenium(III) complexes have shown antimicrobial activity. 16,179][20][21] These ruthenium complexes were highly active against a range of pathogenic bacteria, particularly Gram-positive strains, 18 and maintained the activity against drug-resistant bacteria, including strains that are of considerable current concern, e.g.methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE).Furthermore, preliminary toxicity experiments indicated the dinuclear Rubb n complexes were significantly less toxic to eukaryotic cells. 22,23Based upon the good antimicrobial activity and cell selectivity of the Rubb n complexes, corresponding tri-and tetra-nuclear inert ruthenium complexes were subsequently synthesised. 24These complexes generally showed better activities than the dinuclear analogues and were more active against Gram-positive species. 24n another approach, we have also examined the effects of incorporating labile chlorido groups into dinuclear ruthenium (II) complexes linked by the bb n ligand, [{Ru(tpy)Cl} 2 {μ-bb n }] 2+ (Cl-Rubb n -Cl; where tpy = 2,2′:6′,2″-terpyridine), 25 see Fig. 2. The symmetrical Cl-Rubb n -Cl complexes showed good activity against both Gram-positive bacteria and Gram-negative species.However, incorporation of the chlorido groups did significantly affect the relative activities of the ruthenium complexes, compared to the corresponding inert Rubb n complexes.Whereas the order of activities for the inert complexes was Rubb 16 ≥ Rubb 12 > Rubb 7 , it was found that Cl-Rubb 12 -Cl was the most active of the Cl-Rubb n -Cl complexes and Cl-Rubb 16 -Cl was slightly less active than the Cl-Rubb 7 -Cl complex.Taken together, the combined results highlight the balance between cationic charge and lipophilicity; however, it is not yet clear where the optimal charge/lipophilicity balance lies.In order to help clarify this issue, we aimed to synthesise and examine the antimicrobial activities of dinuclear ruthenium complexes that contained one inert metal centre and one metal centre that incorporated a chlorido ligand (Rubb n -Cl complexes, see Fig. 2).
In this study, the synthesis and the antimicrobial properties of the non-symmetrical Rubb n -Cl complexes (for n = 7, 12 and 16) against Gram-positive S. aureus and MRSA, and Gramnegative Escherichia coli and Pseudomonas aeruginosa were examined.As the clinical potential of any new drug is determined by both the antimicrobial activity and the associated toxicity towards eukaryotic cells, the cytotoxicities of the Rubb n -Cl complexes against three eukaryotic cell lines were also examined.In order to understand the unexpected large differences in the cytotoxicities of the Rubb n -Cl complexes towards eukaryotic cells, a confocal microscopic study of their intracellular localisation was also undertaken.
The mononuclear ruthenium complex [Ru(tpy)(Me 2 bpy)Cl] Cl was synthesised as previously described. 26The synthesis of the bridging ligands bb n (n = 7, 12 and 16; A) were performed in a similar manner to that reported in the literature. 27[29] Synthesis of [Ru( phen) 2 (μ-bb n )Ru(tpy)Cl]Cl 3 (Rubb n -Cl, n = 7, 12 and 16, G) For [Ru( phen) 2 (μ-bb 7 )Ru(tpy)Cl]Cl 3 , solid [Ru(tpy)Cl 3 ] (10 mg, 0.032 mmol) and [Ru( phen) 2 (bb 7 )](PF 6 ) 2 (3.7 mg, 0.032 mmol) were refluxed in ethanol/water (4 : 1, 10 mL) for 3 h.After cooling, excess NH 4 PF 6 was added to precipitate a dark-brown material which was filtered and washed with ethanol.The crude product was then loaded onto a Sephadex LH20 sizeexclusion column and eluted with acetone.The [Ru( phen) 2 (μ-bb 7 )Ru(tpy)Cl](PF 6 ) 3 fraction was obtained as the major dark-brown band which was isolated and evaporated to dryness.The PF 6 − salt was converted to the chloride (metathesis) by dissolving the solid in the minimum amount of acetone followed by the dropwise addition of a saturated solution of tetraethylammonium chloride in acetone with stirring for 30 min.The resulting fluffy precipitate was centrifuged, decanted, washed several times with cold acetone and dried under reduced pressure to afford [Ru( phen) 2 (μ-bb 7 )Ru(tpy)Cl] Cl 3 (yield 70-80%).Separation of the possible geometric isomers (the chlorido ligand in the coordinatively-labile metal centre can potentially be either cis or trans to the pyridine ring of the bb n ligand bearing the methyl groupthe trans disposition is shown in Fig. 2) was not attempted.

Aquation of Rubb n -Cl complexes
The ruthenium complexes [Ru(tpy)(Me 2 bpy)Cl]Cl and Δ-Rubb 7 -Cl were dissolved in D 2 O (650 µL) to give 1.0 mM solutions. 1 H NMR spectra were then recorded as a function of time at 25 °C.

Reaction of ruthenium complexes with nucleotides
Reactions with nucleotides were carried out after the aquation of the chlorido-containing ruthenium complexes had reached equilibrium.The nucleotides 5′-GMP, 5′-AMP, 5′-CMP, and 5′-UMP were dissolved in D 2 O and separately added to the ruthenium complexes (1 mM) dissolved in D 2 O at the desired [Ru complex] : [nucleotide] ratio. 1 H NMR spectra were recorded as a function of time after mixing the sample thoroughly.NOESY experiments were conducted using the method of States et al., 30 with 1024 data points in t2 for 256 t1 values, a pulse repetition delay set to 1.5 s and mixing times from 100 to 500 ms.Correlation spectroscopy experiments (DQFCOSY) were recorded using the same t1, t2 and pulse repetition values.
Molecular modelling was performed using HyperChem. 31nergy minimisation by Polak-Ribiere conjugate-gradient refinement was carried out with the metal complex treated as a rigid group.The ruthenium complex was manually docked to the GMP to reflect observed intermolecular NOEs.

Bacterial strains
Note: the bacterial strains used in this study are classified as risk group 2 according to the Australian/New Zealand Standard (AS/NZS 2243.4:2010) and accordingly were manipulated in a PC2 class laboratory.Gram-positive isolates {a methicillin-susceptible S. aureus strain (ATCC 25923) and a clinical, multidrug-resistant, MRSA strain (JCU culture collection)}, and two Gram-negative isolates {E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853)}, were used for antimicrobial studies.

Determination of minimum inhibitory concentration (MIC)
MIC values were determined in duplicate by standard microdilution methodology in CAMHB, 32 using gentamicin as the positive control, as previously described. 18,19

Determination of minimum bactericidal concentration (MBC)
The MBC tests were performed in duplicate according to a standard microbiological techniques protocol, 33 as previously described. 18,19ll culture Two kidney cell lines {BHK (baby hamster kidney) and HEK-293 (embryonic kidney)}, and one liver cell line {HepG2 (liver carcinoma)} were used in this study.All cell lines were generously supplied by the Australian Army Malaria Institute (AMI, Enoggera, QLD, Australia), and originated from the American Type Culture Collection (ATCC, Manassas, USA).Cells were cultured as previously described. 22Cells used in the study were in the logarithmic growth phase and were grown to 70% confluence, and then trypsinised with 0.25% trypsin-0.02%EDTA (Sigma-Aldrich) for detachment and used in the assays described below.

Aquation of chlorido-containing ruthenium complexes
As the Rubb n -Cl complexes are activated towards covalent bond formation with intracellular targets by aquation, a study of the aquation of Rubb 7 -Cl and the parent complex [Ru(tpy)-(Me 2 bpy)Cl] + was carried out.The rate of aquation of [Ru(tpy)-(Me 2 bpy)Cl] + has been previously determined by 1 H NMR spectroscopy, 26 where the concomitant disappearance of the resonance from the H6 of the Me 2 bpy ligand from the chlorido complex and the emergence of the corresponding resonance from the aqua species were monitored as a function of time.By following the disappearance of the H6 resonance of the Me 2 bpy ligand for [Ru(tpy)(Me 2 bpy)Cl] + (9.71 ppm) and Rubb 7 -Cl (9.52 ppm) and the emergence of the corresponding resonance of the aqua complexes (9.52 and 9.24 ppm, respectively), it was determined that 50% aquation is achieved   NOESY spectra (data not shown).In the NOESY spectrum of the aromatic to sugar H1′ region, strong NOE cross peaks were observed between the tpy H6 and H6″, H5 and H5″, H4 and H4″, H3 and H3″ and H3′ and H5′, indicating the tpy protons were in slow exchange (on the NMR time scale) between two forms.Due to rotation around the Ru-N7 bond, it is possible that [Ru(tpy)(Me 2 bpy)GMP] complex may exist in two conformers that are in slow exchange.An NOE cross peak between the H8 and sugar H1′ of the bound GMP at 6.56/5.50ppm provided further support for the assignment of the H8 of the bound GMP.Based on the NMR analysis, the binding site of ruthenium complex is most likely at the N7 of the GMP, given the large shift observed for the GH8 resonance.
A molecular model of the ruthenium complex-GMP adduct is shown in Fig. 6.In the model, the GMP H8 is positioned directly below the tpy aromatic rings, which may explain the unusual upfield shift observed for the resonance upon N7-metallation.The time-course 1 H NMR spectra of the reaction between the dinuclear complex Rubb 7 -Cl and GMP also showed the emergence of resonances for the ruthenium complex-GMP adduct (data not shown).A singlet at 6.65 ppm could be assigned to the H8 from bound GMP, and new broad peaks in the 8.7-8.9 ppm region were possibly from the bound metal complex.These observations were similar to those from the reaction between [Ru(tpy)(Me 2 bpy)Cl] + and GMP, described above, suggesting that Rubb 7 -Cl interacts with GMP by forming a covalent bond.

Antimicrobial activity
The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values for the Rubb n -Cl complexes against S. aureus, MRSA, E. coli and P. aeruginosa were determined and are summarised in Table 1.All Rubb n -Cl complexes showed significant activity against both the Gram-negative and Gram-positive strains, with the Gram-negative strain P. aeruginosa being the least susceptible.Overall, Rubb 12 -Cl showed the best activity across the Rubb n -Cl series.Compared to Rubb 12 -Cl, Rubb 16 -Cl was similarly active against the two Gram-positive strains, but showed considerably lower activity against the Gram-negative strains.On the other hand, Rubb 7 -Cl was less active than Rubb 12 -Cl against the Gram-positive strains, but equally as active as Rubb 12 -Cl against the Gramnegative bacteria.As the MBC values were ≤2 × MIC, it is concluded that all the Rubb n -Cl complexes are bactericidal, rather than bacteriostatic.The Rubb n -Cl complexes generally showed similar or better activity than the Rubb n and Cl-Rubb n -Cl complexes, with Rubb 12 -Cl exhibiting as good an activity profile as any of the other previously-reported ruthenium complexes linked by the bb n ligand, including the tri-and tetra-nuclear inert complexes. 18,24ver the four bacteria examined in this study, Rubb 7 -Cl and Rubb 12 -Cl showed slightly better antimicrobial activities compared to their inert analogues, Rubb 7 and Rubb 12 .Conversely, Rubb 16 -Cl displayed slightly lower activity than Rubb 16 .Interestingly, Rubb 7 -Cl and Rubb 12 -Cl show better activities against the two Gram-negative species E. coli and P. aeruginosa than Rubb 7 and Rubb 12 , respectively.Particularly noteworthy is the activity of Rubb 7 -Cl against E. coli compared to the Grampositive species S. aureus and MRSA: Rubb 7 -Cl is eight-and sixteen-fold more active towards E. coli than against S. aureus and MRSA, respectively.This preferential activity towards a Gram-negative species is very unusual for metal-based antimicrobial agents.

Cytotoxicity against eukaryotic cells and selective activity
To further evaluate the potential of the ruthenium complexes as antimicrobial agents, an understanding of their cytotoxicity towards mammalian cells is necessary.The cytotoxicities of the ruthenium complexes against the kidney cell lines BHK and HEK-293 were determined for incubation times of 24 hours.As there were no differences in the cytotoxicities (within experimental error) between the Δ-Rubb n -Cl and the rac-Rubb n -Cl complexes in preliminary experiments, the rac-Rubb n -Cl complexes were used to determine the 24-hour IC 50 values.The 24 h-IC 50 values of the Rubb n -Cl and Rubb n complexes are summarised in Table 2.
Generally, the complexes with a longer linking chain were more toxic to the cells.Among the rac-Rubb n -Cl complexes, Rubb 16 -Cl was the most toxic complex towards BHK and HEK-293 cells.In contrast, Rubb 7 -Cl was nontoxic (>200 µM) towards BHK but showed moderate cytotoxicity towards HEK-293, while Rubb 12 -Cl showed similar cytotoxicity towards both cell lines.These observations suggest that the linking chain length plays an important role in their cytotoxicities.The inert complexes ΔΔ-Rubb 12 and ΔΔ-Rubb 16 also showed the same trend with ΔΔ-Rubb 16 being more toxic than ΔΔ-Rubb 12 .
The good antimicrobial activity of the ruthenium complexes suggests they may have potential as antimicrobial agents.However, to be clinically useful as antimicrobial agents, it is desirable that the compounds exhibit significantly greater toxicity towards bacterial cells than mammalian cells.Table 2 also summarises the selectivity indices (SI, 24 h-IC 50 /MIC) between the BHK and HEK-293 cell lines and the Gram-positive bacterium S. aureus and the Gram-negative species E. coli.
In general, the SI values demonstrated that all the ruthenium complexes were more toxic against bacterial cells than eukaryotic cells, with the SI values ranging from 8-340.Noticeably, Rubb 7 -Cl displayed the highest SI values for both BHK and HEK-293 cells against E. coli.Of particular note, the SI value of 340 between BHK and E. coli was at least five-times higher than with the other complexes.By contrast, Rubb 12 -Cl showed similar selectivity towards S. aureus and E. coli, compared to both kidney cell lines.The inert complex Rubb 12 displayed a better selectivity for S. aureus than E. coli against both kidney cell lines.Taken together, Rubb 12 -Cl exhibited the best overall selectivity (but only marginally better than Rubb 12 ), while Rubb 7 -Cl showed the best selectivity towards the Gramnegative E. coli.The liver cell line HepG2 was chosen to determine the cytotoxicities of the ruthenium complexes in liver cells.The cytotoxicities of the ruthenium complexes against HepG2 cells were determined after a 24-hour incubation, and the results are summarised in Table 3.The HepG2 cells were more susceptible than the kidney cells to Rubb 7 -Cl and Rubb 12 -Cl, but were more resistant to Rubb 16 -Cl.Interestingly, and unlike what was observed with the BHK and HEK-293 cells, the cytotoxicities of the Rubb n -Cl complexes decreased with increasing chain length in the bb n ligand.Surprisingly, Rubb 7 -Cl was the most cytotoxic of all the ruthenium complexes assayed against the HepG2 cells, with the 24 h-IC 50 being 3.7 µM.The comparative selectivities between the two kidney cell lines and the HepG2 cell line were calculated for each of the ruthenium complexes and the results are summarised in Table 3. Rubb 7 -Cl clearly exhibited significantly higher toxicity towards the HepG2 cell line − compared to the two kidney cell lines − than did the other ruthenium complexes.Although the relative difference was much smaller, Rubb 12 -Cl was the only other complex to show some preferential toxicity to the HepG2 cells.

Cellular localisation of rac-Rubb n -Cl complexes in HepG2 cells
Cellular localisation patterns are important in unravelling the mechanism of cytotoxicity for any drug.Therefore, the intrinsic phosphorescence properties of the ruthenium complexes were used to study their cellular localisation by laser-scanning confocal microscopy.To achieve this, the ruthenium complex phosphorescence patterns were overlaid with the fluorescence patterns of DNA/RNA/mitochondria-specific stains in colabelling experiments.
As shown in Fig. 7, the rac-Rubb n -Cl complexes showed similar nucleolus localisation to that previously reported for the inert Rubb n complexes. 22The nucleolus of HepG2 cells were stained by Rubb n -Cl and SYTO 9.In addition to SYTO 9-stained rRNA, the most toxic complex Rubb 7 -Cl also showed localisation with DAPI-stained DNA.By contrast, Rubb 12 -Cl and Rubb 16 -Cl overlaid more with SYTO 9-stained rRNA in the nucleolus, and less with DAPI-stained DNA.In particular, Rubb 12 -Cl was exclusively localised in the nucleolus.These results suggest that Rubb 12 -Cl and Rubb 16 -Cl have greater selectivity for rRNA, compared to DNA, than does Rubb 7 -Cl.
BHK cells incubated with Rubb 7 -Cl at a concentration of 100 μM (approx.50% of the IC 50 ) for 20 hours are shown in Fig. 8.The Rubb 7 -Cl phosphorescence is observed throughout the cytoplasm and in the nucleus; however, within the nucleus the phosphorescence was mainly in the nucleolus, with little overlay with the DAPI-stained DNA.When BHK cells were incubated with Rubb 7 -Cl at a concentration of 50 μM for 20 hours, almost no phosphorescence was observed in the nucleus, with only relatively weak accumulation of the ruthenium complex in the cytoplasm (see Fig. 9).

Discussion
We have previously demonstrated that the dinuclear ruthenium complexes Rubb n and Cl-Rubb n -Cl have good antimicrobial activity. 18,25As would be expected, significant differences in activity are observed upon changes to the total charge, charge separation and lipophilicity of the ruthenium complexes.To further investigate the interplay between these para-meters, we have in this study synthesised and analysed the antimicrobial activities of the Rubb n -Cl series of complexes.
The results demonstrate that the Rubb n -Cl complexes also have good antimicrobial activity and are bactericidal, with the most active complex against both Gram-positive and Gramnegative strains being Rubb 12 -Cl.Similarly to what was observed for the Rubb n complexes, the Rubb n -Cl complexes were considerably more toxic to bacterial cells than towards  The preferential activity of Rubb 7 -Cl towards Gram-negative E. coli over the Gram-positive species S. aureus and MRSA is very unusual for a metal-based antimicrobial agent.A few metal complexes have shown higher activity against Gramnegative than Gram-positive bacteria.For example, two helicate-like dinuclear iron complexes showed two-fold higher activity against E. coli than S. aureus, 35 and a [Ru 2 L 3 ] 4+ triplystranded helicate complex also showed higher activity towards E. coli than S. aureus, but the activities were modest. 36][20][21][22][23][24][25]37,38 The Rubb n -Cl complexes also showed a different pattern of activities to the Rubb n and Cl-Rubb n -Cl complexes.Against the Gram-positive bacteria, Rubb 12 -Cl and Rubb 16 -Cl were of equal activity and considerably more active than Rubb 7 -Cl.However, for the Gram-negative species, Rubb 7 -Cl and Rubb 12 -Cl were of equal activity and were considerably more active than Rubb 16 -Cl.For the Rubb n and Cl-Rubb n -Cl complexes, the same pattern of activities was observed for all bacteria.For the Rubb n complexes, the order of the activities across the four bacteria was Rubb 16 ≥ Rubb 12 > Rubb 7 ; while for the Cl-Rubb n -Cl series, Cl-Rubb 12 -Cl > Cl-Rubb 7 -Cl ≥ Cl-Rubb 16 -Cl across the bacteria.][20][21][22][23][24][25] Cytotoxicity assays against the kidney cell lines BHK and HEK-293 and the liver cell line HepG2 were carried out to estimate the toxicity of the Rubb n -Cl complexes towards mammalian organ cells.In BHK and HEK-293 cells, the 24 h-IC 50 values for the Rubb n -Cl series decreased with the increasing number of methylene groups in the bb n ligand.A similar trend was observed for the Rubb n complexes.These results suggest that cellular uptake is the key parameter, at least to a first approximation, with the uptake being correlated to the lipophilicity of the ruthenium complex.However, in HepG2 cells the least lipophilic complex Rubb 7 -Cl exhibited the highest cytotoxicity while the most lipophilic complex Rubb 16 -Cl was the least toxic.Hence, it is probable that while lipophilicity is important, at least in terms of cellular uptake, there are other factors to be considered.0][41] In contrast, in non-cancerous cells the membrane is largely occupied by zwitterionic phospholipids, with negligible or weak negative charge. 42,43In addition, the increased number of microvilli on cancer cells, which lead to an increase in cell surface area, may also enhance their susceptibility. 44,45Therefore, the cellular uptake of the polycationic ruthenium complexes may be less affected by lipophilicity in cancer cell lines compared with healthy cells.
Intracellular localisation could also be an important aspect of cytotoxicity.Confocal microscopy was used to determine the cellular localisation of the Rubb n -Cl complexes in HepG2 cells.The Rubb n -Cl complexes preferentially accumulated in the nucleolus, while significant DNA binding was also observed at higher concentrations.The preference for rRNA is consistent with our previous studies on the localisation of Rubb 12 in BHK cells 22 and Rubb 16 in the ribosomes of E. coli. 21However, the Rubb n -Cl complexes showed differences in nuclear localisation in HepG2 cells based upon the length of the alkyl chain in the bb n linking ligand.The least lipophilic complex Rubb 7 -Cl appeared to co-localise to a higher degree with DNA, while the other two complexes, particularly Rubb 12 -Cl, showed greater accumulation in the nucleolus.The effect of lipophilicity on the cellular localisation of ruthenium complexes has been previously reported.Lincoln, Nordén and co-workers found the length of an alkyl chain in a dppz-based complex (dppz = dipyrido[3,2-a:2′,3′-c]phenazine) had a significant effect on the localisation patternthe least lipophilic complex was found to stain nuclear DNA, the most lipophilic complex preferably stained cellular membranes, whereas the derivative of intermediate lipophilicity selectively stained the RNA-rich nucleoli. 46Furthermore, Thomas and co-workers demonstrated that the dinuclear complex [{Ru( phen) 2 } 2 {μ-tpphz}] 4+ (where tpphz = tetrapyridophenazine) localised in the nucleus, while the more lipophilic 4,7-diphenyl-1,10-phenanthroline analogue [{Ru(DIP) 2 } 2 {μ-tpphz}] 4+ localised in the endoplasmic reticulum. 47,48While other factors are yet to be examined, the greater level of DNA localisation in HepG2 by Rubb 7 -Cl, compared to Rubb 12 -Cl and Rubb 12 , could be related to its higher cytotoxicity against this cell line.Consistent with this proposal was the observed low level of DNA binding, compared to that in the nucleolus and cytoplasm, by Rubb 7 -Cl in BHK cells at concentrations considerably higher than the 24 h-IC 50 determined against HepG2 cells.
In conclusion, a new class of dinuclear ruthenium complexes, Rubb n -Cl, has been synthesised and characterised.These ruthenium complexes exhibited good antimicrobial activities; and interestingly, showed relatively better activity towards Gram-negative bacteria, (compared to Gram-positive species) than previously reported ruthenium complexes linked by the bb n ligand.In addition, the Rubb n -Cl complexes were considerably less toxic to eukaryotic cells, compared to bacteria, with Rubb 7 -Cl showing striking differences in cytotoxicity between the eukaryotic cell lines.It is possible that Rubb 7 -Cl could become a new lead compound for metal-based anticancer or antimicrobial agents.
n -Cl (n = 7, 12 and 16) complexes The syntheses of the symmetric oligonuclear polypyridine ruthenium complexes Rubb n , Tri-Rubb n , Tetra-Rubb n and Cl-Rubb n -Cl have been previously reported and their biological properties have been thoroughly studied. 18-25To obtain a better structure-activity relationship, a new class of nonsymmetric dinuclear ruthenium complexes Rubb n -Cl was designed and synthesised.The Rubb n -Cl complexes were prepared from [Ru( phen) 2 ( py) 2 ] 2+ (E), [Ru(tpy)Cl 3 ] (B) and the bb n ligand (A) following the synthetic route shown in Scheme 1, with the final dinuclear product Rubb n -Cl (G) obtained through reaction of [Ru( phen) 2 bb n ] 2+ (F) with [Ru(tpy)Cl 3 ].[Ru( phen) 2 ( py) 2 ] 2+ and [Ru( phen) 2 bb n ] 2+ were purified by cation-exchange chromatography on an SP Sephadex C-25 column, whereas the final Rubb n -Cl complexes were purified by size-exclusion on a Sephadex LH20 column.The Rubb n -Cl complexes were characterised by 1 H and 13 C NMR, microanalysis and ESI-MS.

Scheme 1 in
Scheme 1

Fig. 3 (
Fig. 3 (A) The relative proportions of [Ru(tpy)(Me 2 bpy)Cl] + and [Ru(tpy)(Me 2 bpy)(D 2 O)] 2+ as a function of time after dissolving the chlorido species in D 2 O; (B) the relative proportions of Rubb 7 -Cl and Rubb 7 -D 2 O as a function of time after dissolving the chlorido form in D 2 O.

Fig. 4 1 H
Fig. 4 1 H NMR spectra of [Ru(tpy)(Me 2 bpy)(D 2 O)] 2+ and either UMP, CMP, AMP and GMP, at a ruthenium complex to nucleotide ratio of 2 : 1, in D 2 O after a 24 hour incubation.In the spectrum of [Ru(tpy)(Me 2 bpy)(D 2 O)] 2+ with GMP the arrows indicate the new peaks due to the covalent adduct.

Fig. 5 1 H
Fig. 5 1 H NMR spectra of the reaction between [Ru(tpy)(Me 2 bpy)(D 2 O)] 2+ and GMP.A, B and C are time-course experiments with the [Ru] : [GMP] = 1 : 1 at 25 °C at 10 minutes, 6 hours and 64 hours after starting the reaction, respectively; D after the addition of three more equivalents of GMP to give a [Ru] : [GMP] = 1 : 4; E spectrum after D was heated at 50 °C for 16 hours, black arrow indicates GH8 in the bound adduct.

Table 1
MIC and MBC values (µM)for the Rubb n -Cl complexes and the corresponding dinuclear inert Rubb n complexes.MIC values, after 14-16 hours of incubation, were determined as mg L −1 but converted to μM for direct comparison with the IC 50 data obtained with eukaryotic cells

Table 2
24 h-IC 50 values (µM) of the ruthenium complexes against BHK and HEK-293 cells, and selectivity indices SI = 24 h-IC 50 /MIC, of the ruthenium complexes between kidney cells (BHK and HEK-293) and two bacterial strains (S. aureus and E. coli)

Table 3
24 h-IC 50 values (µM) of the ruthenium complexes against HepG2 cells, and selectivity indices (SX) of the ruthenium complexes compared to the kidney cell lines.SX is defined as the ratio of the IC 50 against BHK or HEK-293 cells divided by the IC 50 against the HepG2 cell line