Mono- and tetra-nuclear polypyridylruthenium(II) complexes as therapeutic agents

Antimicrobial resistance has become a worldwide problem despite of the successful development of antibiotics; therefore, there is a great need to develop new antimicrobials. A series of mononuclear ruthenium(II) complexes containing the tetradentate ligand bis[4(4′-methyl-2,2′-bipyridyl)]-1,7-heptane has been synthesised and their biological properties examined. In the synthesis of the [Ru(phen′)(bb7)]2+ complexes (where phen′ = 1,10-phenanthroline and its 5-nitro-, 4,7-dimethyl- and 3,4,7,8-tetramethyl-derivatives), both the symmetric cis-α and non-symmetric cis-β isomers were formed. However, upon standing for a number of days (or more quickly under harsh conditions) the cis-β isomer converted to the more thermodynamically stable cis-α isomer. The minimum inhibitory concentrations (MIC) and the minimum bactericidal concentrations (MBC) of the ruthenium(II) complexes were determined against six strains of bacteria: Gram-positive Staphylococcus aureus (S. aureus) and methicillin-resistant S. aureus (MRSA); and the Gram-negative Escherichia coli (E. coli) strains MG1655, APEC, UPEC and Pseudomonas aeruginosa (P. aeruginosa). The results showed that the [Ru(5-NO2phen)(bb7)]2+ complex had little or no activity against any of the bacterial strains. By contrast, for the other cis-α-[Ru(phen′)(bb7)]2+ complexes, the antimicrobial activity increased with the degree of methylation of the phenanthroline ligand. In particular, the cis-α-[Ru(Me4phen)(bb7)]2+ (designated as α-Me4phen-7) complex showed good and uniform MIC activity against all bacteria. By contrast, the MBC values for α-Me4phen-7 varied considerably across the bacteria and even within S. aureus and E. coli strains. In order to gain an understanding of the relative antimicrobial activities, the DNA-binding affinity, cellular accumulation and logarithm of water-octanol partition coefficients (log P) of the ruthenium complexes were determined. Interestingly, all the [Ru(phen′)(bb7)]2+ complexes exhibited stronger DNA binding affinity (Ka 1 × 10^7 M−1) than the well-known DNA intercalating complex [Ru(phen)2(dppz)]2+ (where dppz = dipyrido[3,2-a:2′,3′-c]phenazine). In addition, a determination of extracellular ATP concentration was carried out to examine the action of α-Me4phen-7 on bacterial membranes. The results showed that a significant increase in ATP leakage was obtained for α-Me4phen-7 at 2 × MIC compared to the cell only control, suggesting that α-Me4phen-7 could damage the membrane of UPEC. Given the good antimicrobial activity of α-Me4phen-7, the corresponding toxicity towards eukaryotic cells was also examined and compared to other ruthenium complexes. The determination of the half maximal inhibitory concentration (IC50) of the ruthenium complexes towards three eukaryotic cell lines − BHK (baby hamster kidney), Caco-2 (heterogeneous human epithelial colorectal adenocarcinoma) and HepG2 (liver carcinoma) − was carried out. The results demonstrated that α-Me4phen-7 showed little toxicity toward the three cell lines, and was considerably less toxic than cis-α-[Ru(phen)(bb12)]2+ (α-phen-12) and the dinuclear complex Rubb12 {[{Ru(phen)2}2{µ-bb12}]4+, where bb12 = bis[4(4′-methyl-2,2′-bipyridyl)]-1,12-dodecane}. Fluorescence spectroscopy was used to study the binding of the ruthenium complexes with human serum albumin (HSA). The binding of α-Me4phen-7 to the macrocyclic host molecule cucurbit[10]uril (Q[10]) was examined by NMR spectroscopy. Large upfield 1H NMR chemical shift changes were observed for the methylene protons in the bb7 ligand upon addition of Q[10], coupled with the observation of several intermolecular ROEs in ROESY spectra, indicated that α-Me4phen-7 bound Q[10] with the bb7 methylene carbons within the cavity and the metal centre positioned outside one of the portals. Simple molecular modelling confirmed the feasibility of the binding model. An α-Me4phen-7-Q[10] binding constant of 9.9 ± 0.2 × 10^6 M−1 was determined by luminescence spectroscopy. Q[10]-encapsulation decreased the toxicity of α-Me4phen-7 against the three eukaryotic cell lines and increased the binding affinity of the ruthenium complex for HSA. Confocal microscopy experiments indicated that the level of accumulation of α-Me4phen-7 in BHK cells is not significantly affected by Q[10]-encapsulation in a 1-hour incubation. Interestingly, different intracellular targets were observed for the free α-Me4phen-7 and α-Me4phen-7 administered encapsulated in Q[10] complexes after a 20-hour incubation. The mitochondria was determined to be the primary target for free α-Me4phen-7. However, staining was observed in the nucleolus, which is rich in RNA in the nucleus, for α-Me4phen-7 at higher concentrations after a 20-hour incubation. In addition, a higher proportion of nucleolus staining was observed for α-Me4phen-7 administered encapsulated in Q[10] with no significant mitochondrial staining. This observation suggests that the Q[10]-encapsulation could facilitate the ruthenium complex to localise in the nucleolus. Taken together, the combined results suggest that α-Me4phen-7 could be a good candidate as a new antimicrobial agent, and Q[10]-encapsulation could be a method to improve the pharmacokinetics of the ruthenium complex. Two tetranuclear ruthenium(II) complexes bridged by the bb7 ligand were successfully synthesised. Good antibacterial activity was obtained for Rubb7-TL (tetranuclear linear ruthenium complex [{Ru(phen)2}(µ-bb7){Ru(phen)}(µ-bb7){Ru(phen)}(µ-bb7){Ru(phen)2}]8+) and Rubb7-TNL (tetranuclear non-linear ruthenium complex [{Ru(µ-bb7)3}{Ru(phen)2}3]8+). Both tetranuclear complexes exhibited high toxicity to eukaryotic cells. Interestingly, they exhibited higher selectivity to cancer cells than towards bacteria, which is very unusual for ruthenium complexes containing a bbn (where bbn = bis[4(4′-methyl-2,2′-bipyridyl)]-1,n-alkane) ligand. Although the antimicrobial tests suggest that they are not good candidates as antimicrobial agents, Rubb7-TNL exhibited impressive antifungal activity against two fungi, Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans), with low cytotoxicity against HEK-293 (human embryonic kidney) and red blood cells. The antimicrobial activity of [Ru(bb7)(dppz)]2+ and [Ru(phen)2(dppz)]2+ was also examined in this study to help understand the importance of the bb7 ligand towards antimicrobial activity. The results showed that both of the dppz-based complexes exhibited good activity against Gram-positive bacteria, but [Ru(bb7)(dppz)]2+ showed at least an eight-fold better activity against the Gram-negative bacteria than [Ru(phen)2(dppz)]2+. Luminescence assays demonstrated that [Ru(bb7)(dppz)]2+ accumulated in a Gram-negative bacterium to the same degree as in a Gram-positive species, and assays with liposomes showed that [Ru(bb7)(dppz)]2+ interacted more strongly with model membranes than the parent [Ru(phen)2(dppz)]2+ complex. Although more toxic to eukaryotic cells than [Ru(phen)2(dppz)]2+, [Ru(bb7)(dppz)]2+ exhibited greater activity against bacteria than eukaryotic cells. This study highlights the potential of α-Me4phen-7 as a new antimicrobial agent. Moreover, the results obtained in this study regarding antimicrobial activity and eukaryotic cell toxicity can help further studies to develop ruthenium-based complex as therapeutic agents for treating specific bacteria