AU2011265513A1 - Antimicrobial Agents - Google Patents

Abstract

C.\NfPobrbDCC\DAR\4O61M95 .DOC-21/121201 I The present invention relates to ruthenium complexes, and in particular di- and multi nuclear ruthenium complexes which may be used as antimicrobial agents. The invention also relates to pharmaceutical compositions comprising such complexes, and methods for their use in treating or preventing microbial infections.

Description

Australian Patents Act 1990 - Regulation 3.2 ORIGINAL COMPLETE SPECIFICATION STANDARD PATENT Invention Title "Antimicrobial Agents" The following statement is a full description of this invention, including the best method of performing it known to us: P/00/01 I C :\Ronbl\DCC DARt06195_l.DOC.21/12/2011 ANTIMICROBIAL AGENTS FIELD OF THE INVENTION 5 The present invention relates to ruthenium complexes, and in particular di- and multi nuclear ruthenium complexes which may be used as antimicrobial agents. The invention also relates to pharmaceutical compositions comprising such complexes, and methods for their use in treating or preventing microbial infections. 10 BACKGROUND OF THE INVENTION The development of antimicrobial agents has been one of the major advances in medical science. However, a consequence of their widespread use has been the development of drug-resistant populations of bacteria. Infection by these organisms is emerging as an 15 important cause of morbidity and mortality worldwide. In a recent update from the Infectious Diseases Society of America, Enterococcusfaecium, Staphylococcus aureus (S. aureus), Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa (P. aeruginosa) and Enterobacter species were identified as the pathogens of most current concern. In particular, methicillin-resistant S. aureus (MRSA), fluoroquinolone-resistant 20 P. aeruginosa and vancomycin-resistant Enterococcus (VRE) show rapidly increasing rates of infection. There is clearly a need for the development of new antimicrobial agents, but perhaps more importantly, there is the need for the development of new classes of antimicrobials that may not be as susceptible to the bacterial mechanisms of resistance developed against the current range of drugs. 25 SUMMARY OF THE INVENTION It has now been found that a particular class of ruthenium complexes having two or more ruthenium centres is effective in the treatment and/or prevention of microbial infections. 30 Accordingly in one aspect the present invention provides a compound of the following C:NRPorbthDCC\DAR\4061H95_1 DOC-21/12/2011 -2 formula: b+ L L L I-L L L Ru(II) Ru(II) [ZIb L ' L- -Q-L-- -- H L L a wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or 5 different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and 10 each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; wherein when the compound does not contain a labile ligand and a = I then Q contains at 15 least one -NH-, -N(alkyl)- or -0- group. In another aspect the present invention provides a pharmaceutical composition comprising a compound of the following formula: b+ L L L L L L Ru(II) Ru(II) [Zib L- L- -Q-L L--' I' -- H L L a 20 wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 2 to 8; C:WRPonbWlCC\DAR\46 1895_ DOC-21/12/2011 -3 Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl 5 ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; wherein when the compound does not contain a labile ligand and a = 1 then Q contains at least one -NH-, -N(alkyl)- or -0- group; 10 or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically acceptable carrier or diluent. In another aspect the present invention provides a method of preventing or treating a microbial infection comprising administering to a subject in need thereof an effective 15 amount of a compound of the following formula: L L L 1 L L L Ru(II) Ru(il) [ZIb L L- -Q- L L- -H L L -a wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; 20 b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl 25 ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-.

C:WRPonbrDCC\DARuA06 1M95_1DOC-21112/2011 -4 In another aspect the present invention provides a compound of the following formula: b+ L L L L L L Ru(II) Ru(il) [Z]b L L- -Q- L ' L-- H L L a wherein: 5 a is an integer from 1 to 3, wherein when a is greater than I each Q may be the same or different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or 10 labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; 15 for use in preventing or treating a microbial infection. In another aspect the present invention provides use of a compound of the following formula: b+ L L L L L L Ru(II) Ru(II) iZib L I L- Q- L - -- H L L -a 20 wherein: a is an integer from I to 3, wherein when a is greater than I each Q may be the same or different; b is an integer from 2 to 8; C \NRPorbl\DCC\DAR M61895I.DOC.2/2/2011 -5 Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl 5 ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; in the manufacture of a medicament for preventing or treating a microbial infection. 10 These compounds and uses thereof have been shown to be active against a range of organisms, including antibiotic resistant bacteria. In some embodiments of the invention one or more of the following definitions apply: 15 a is an integer from 1 to 3, preferably a is 1; Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-, preferably Q is a C 2 . 1 6 alkylene linking group wherein any one or more methylene moieties in alkylene is 20 optionally independently replaced with -NH-, -N(alkyl)- or -0-, more preferably Q is a C 2

-

16 alkylene linking group; b is an integer from 2 to 8, typically when a is I then b is an integer from 2 to 4, preferably when a is 1 then b is 2 or 3, more preferably when a is I then b is 3, typically when a 25 is 2 then b is an integer from 3 to 6, preferably when a is 2 then b is an integer from 3 to 5, more preferably when a is 2 then b is 4 or 5 such as when one terminal ruthenium centre does not complex a labile ligand, typically when a is 3 then b is an integer from 4 to 8, preferably when a is 3 then b is an integer from 4 to 7, more preferably when a is 3 then b is an integer from 5 to 7 such as when one terminal 30 ruthenium centre does not complex a labile ligand; C:\NRPorbrDCCDARW61895_LDOC-21/12201 1 -6 Z represents one or more counteranions, preferably halide (such as fluoride, chloride, bromide or iodide), acetate, succinate, maleate, trifluoromethanesulfonate (triflate) or hexafluorophosphate and mixtures thereof; and 5 each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom, preferably pyridyl is selected from optionally substituted bipyridine (bipy or bpy), optionally substituted terpyridine 10 (terpy) and optionally substituted phenanthroline (phen), such as methylbipyridine, terpyridine, phenanthroline and tetramethylphenanthroline, preferably labile ligand is selected from halide (such as iodide, bromide and chloride, preferably chloride) and water. 15 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 depicts the results of 24 h haemolysis assays for AA-Rubb 7 , AA-Rubbio, AA Rubb 12 and AA-Rubbi 6 with freshly-collected human red blood cells, fitted as logarithmic curves. 20 Figure 2 depicts haemolytic dose-response curves of AA-Rubb 16 (a) and AA-Rubb 12 (b) for different incubation times (2, 4, 8 and 24 h). Figure 3 depicts a comparison of the antimicrobial activity, haemolytic activity and 25 cytotoxicity against the THP-1 cell line after 24 h incubation with AA-Rubb 7 , AA-Rubbio, AA-Rubb 12 and AA-Rubb 16 . The ratio of HC 50 and ICso of the complexes to their own MIC is shown. DETAILED DESCRIPTION OF THE INVENTION 30 As defined herein, each ligand (L) that complexes the ruthenium centres of the compounds C:\NRPonbl\DCC\DAR\4061895I DOC-21112/2011 -7 of the invention is selected from pyridyl ligand or labile ligand: b+ L L L I L L L Ru(II) Ru(II) [Zib L - 2 - Q- L2 I L-- H L L -a In some embodiments the compounds of the invention are symmetric with respect to the 5 types of ligands on the ruthenium centres, such as [{Ru(phen) 2

}

2 (p (bpy(Me)CH 2

CH

2 bpy(Me)))](PF) 4 where each ruthenium centre complexes the same group of pyridyl ligands, and [{Ru(terpy)CI} 2 (p-(bpy(Me)CH 2

CH

2 bpy(Me)))]Cl 2 where each ruthenium centre complexes the same group of pyridyl and labile ligands (choride). 10 In some embodiments the complexes are non-symmetric with respect to the types of ligands on the ruthenium centres, such as [{Ru(phen) 2 }(p-(bpy(Me)CH 2

CH

2 bpy(Me))){Ru(phen)(bpy)}]Cl 4 where each ruthenium centre complexes a different group of pyridyl ligands and [{Ru(terpy)Cl}(p-(bpy(Me)CH 2

CH

2 bpy(Me))){Ru(phen)(Me 2 bpy)}]C1 3 where each 15 ruthenium centre complexes a different group of pyridyl and/or labile ligands (chloride). In this latter example, one ruthenium centre complexes a labile ligand and the other ruthenium centre does not complex a labile ligand. Such non-symmetric compounds wherein one terminal ruthenium centre complexes a labile ligand and one terminal ruthenium centre does not complex a labile ligand are believed to be particularly 20 advantageous as compounds of the invention and in the prevention or treatment of a microbial infection as defined herein. Accordingly in one aspect the present invention provides a compound of the following formula: C-1NRPonbr\DCC\DAR\4061895_ DOC-21U12/20 -8 b+ LL L L 1 Ru(II) Ru(II) [Zib

L

1 I 'L 1 -- Q- L< 'L1--H L2 L1 - -a wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; 5 b is an integer from 2 to 8; Z represents one or more counteranions; each L, may be the same or different and is independently selected from pyridyl ligand such that each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; 10 L 2 is a labile ligand; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-. The compound may be used in the prevention or treatment of a microbial infection. 15 As used herein the term "pyridyl ligand" takes its standard meaning in the art and refers to the class of ligands which comprise one or more pyridyl groups (such as derivatives of pyridine) as well as pyridine itself. Typically complexation of the ligand to the ruthenium nucleus in the compounds of the invention occurs though the nitrogen atom within the or 20 each pyridine ring. The pyridyl ligands of the present invention form a polydentate ligand together with one or more other pyridyl ligands on the same ruthenium atom. In this respect the pyridyl ligands may be referred to as polypyridyl ligands. As used herein the term "polydentate ligand" takes its standard meaning in the art and refers to ligands which may be bidentate (or didentate), tridentate, tetradentate, etc. The polypyridyl ligands may 25 be optionally substituted with suitable groups including alkyl groups such as methyl groups. Examples of optionally substituted polypyridyl ligands according to the invention include optionally substituted bipyridine (bipy or bpy), optionally substituted terpyridine C:NRPorbl\DCC\DAR\461895_I.DOC-21/2/2011 -9 (terpy) and optionally substituted phenanthroline (phen), such as methylbipyridine, terpyridine, phenanthroline and tetramethylphenanthroline. By way of example, in the compound represented by the following structure: 3+ N MMe Me N N N N Ru(ll) Ru(II) N 'N N N N CI .3 Cl I (CH 2 )n 5 the skilled worker shall appreciate that the ruthenium centre on the left of the structure coordinates two phenanthroline ligands and one substituted bipyridine ligand, whereas the ruthenium centre on the right of the structure coordinates one terpyridine ligand, one substituted bipyridine ligand and one chloride ligand. 10 As used herein the term "labile ligand" takes its standard meaning in the art and refers to the class of ligands which may readily dissociate from the ruthenium centre to which the ligand is complexed. The labile ligand may be charged or uncharged. Examples of labile ligands according to the invention are halide (such as iodide, bromide and chloride, preferably chloride) and water. By way of example, in the compound represented by the 15 following structure: 3+ Me Me N N N N _N Ru(II) Ru(II) N N N I--, IN1 N N CI .3 Cl I N(CH 2 )n the skilled worker shall appreciate that the ruthenium atom on the right of the structure coordinates a labile chloride ligand. When contacted with water, under suitable conditions for a sufficient time, a water molecule may substitute for the labile chloride ligand. In this 20 event the overall charge of the cationic portion of the compound shall be increased to 4* C:\NRPortbhDCC\DAR"W61895_1.DOC-2112/2011 - 10 thereby leading to an association with four chloride counteranions as shown below: 4+ N- Me Me N N N N N N Ru(Il) Ru(II) N N N ,(1) N H H N 1 H'O H . .4Cl

(CH

2 )n Compounds of the invention bearing at least one labile ligand, and uses of those 5 compounds in the prevention and treatment of microbial infection, are in some circumstances preferred. Without wishing to be bound by theory, it is believed that the labile ligand typically dissociates from the ruthenium nucleus under physiological conditions, which may occur in the presence of other ligands such as water, or heteroatoms present in nucleic acids (eg DNA, RNA) or proteins. In some proposed mechanisms, the 10 labile ligand possibly dissociates through stepwise combinations of ligands such as substitution with water followed by substitution with a phosphate group of a nucleic acid followed by substitution with N7 of a nucleic acid (eg GMP). In one aspect the present invention provides a compound of the following formula: b+

-

R -R 'N-Rn R-' N N ' NN n N NNN N nN, N N " Ru(II) Ru(II) Ru(Il) N 'NN N N N [Z]b fl N -Q N _N_' Q 'N N ' 'N ' RN Rn 1 R 15 a-1 wherein: a is an integer from I to 3, wherein when a is greater than I each Q may be the same or different; b is an integer from 4 to 8; C:\NRPorbMlCC\DARW16 I895_lDOC-21112/201I - 11 Z represents one or more counteranions; Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; each R may be the same or different and is independently selected from an alkyl group, 5 such as methyl; each n is independently selected from 0, 1 or 2. The compound may be used in the prevention or treatment of a microbial infection. 10 In some embodiments, when a = 1 then Q contains at least one -NH-, -N(alkyl)- or -0 group. In further embodiments when a = 1 then Q is not selected from 1,2-ethylene, 1,5 pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16 hexadecylene, or any one of: H H N N,,\ H H N ' N N N H H 15 and when a = 2 or 3 then Q is not selected from 1,7-heptylene. In still further embodiments when a = 1, 2 or 3 then Q is not selected from 1,2-ethylene, 1,5-pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16-hexadecylene, or any one of: H H N N H H /,-N N N H H H N N N N H H 20 or any one of: C:\NRPotbl\DCC\DAR4O61895_ .DOC-211121201 I - 12 In another aspect the present invention provides a compound of the following formula: 1;1 b+ Me Me Me N N N N N N Ru (11) Ru(II) Ru(1l) N NN N N N, [Zlb x N QN N -Q 0 ' N ' "' N N' Me _ a-1 5 wherein: a is an integer from I to 3, wherein when a is greater than I each Q may be the same or different; b is an integer from 4 to 8; Z represents one or more counteranions; 10 Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-. The compound may be used in the prevention or treatment of a microbial infection. 15 In some embodiments, when a = I then Q contains at least one -NH-, -N(alkyl)- or -0 group. In further embodiments when a = I then Q is not selected from 1,2-ethylene, 1,5 pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16 hexadecylene, or any one of: C:\NRPonblDCCDAR\4061895_1 DOC-21112201 I - 13 H H N N H H N "-- N -' N -" N > H H and when a = 2 or 3 then Q is not selected from 1,7-heptylene. In still further embodiments when a = 1, 2 or 3 then Q is not selected from 1,2-ethylene, 1,5-pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16-hexadecylene, 5 or any one of: H H N N H H N N ~N H H H N N N H H or any one of: 10 In one aspect the present invention provides a compound of the following formula: b+ R-R R- RnL -R R- N N C R-j~ R Rn R R N N N R I - I NI N R-'I Ru(II) Ru(II) Ru(II) N N N N NN N [Z]b R, L2 N -Q L2 R Ra ~~a-1 C :NRPorb\DCODAR606I952 IDOC-21/12/2011 - 14 wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; each L 2 may be the same of different and is independently selected from a labile ligand; 5 b is an integer from 2 to 8; Z represents one or more counteranions; Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; each R may be the same or different and is independently selected from an alkyl group, 10 such as methyl; each n is independently selected from 0, 1 or 2. The compound may be used in the prevention or treatment of a microbial infection. 15 In some embodiments, when a = 1 then Q contains at least one -NH-, -N(alkyl)- or -0 group. In further embodiments when a = 1 then Q is not selected from 1,7-heptylene, 1,10 decylene, 1,1 2-dodecylene and 1,1 4-tetradecylene. In another aspect the present invention provides a compound of the following formula: "b+ Me Me N Me N N NN N N N Ru(II) Ru(II) Ru(II) N N N N N N- (Zb L2 N-Q L Me 20 a-1 wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; each L 2 may be the same of different and is independently selected from a labile ligand; C:\NRPorb\DCC\DAR\4061895_L DOC-21/12/2011 - 15 b is an integer from 2 to 8; Z represents one or more counteranions; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-. 5 The compound may be used in the prevention or treatment of a microbial infection. In further embodiments when a = I then Q is not selected from 1,7-heptylene, 1,10 decylene, 1,1 2-dodecylene and 1,1 4-tetradecylene. 10 In one aspect the present invention provides a compound of the following formula: R -R R - R R -R N N n N N,, N R- I r-RR~ RI -Rn ~'~N ~N. R-N ~ N- 'N ~ ZN Ru(II) Ru(II) Ru(II) N N N N N [Z] Rn~~h<1 Rn ' L2 Q N -Q N -R Rn Rn -a-1 wherein: a is an integer from 1 to 3, wherein when a is greater than I each Q may be the same or 15 different; each L 2 may be the same of different and is independently selected from a labile ligand; b is an integer from 2 to 8; Z represents one or more counteranions; Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is 20 optionally independently replaced with -NH-, -N(alkyl)- or -0-; each R may be the same or different and is independently selected from an alkyl group, such as methyl; each n is independently selected from 0, 1 or 2.

C:NRPotbl\DCC\DAR\4061K95_ .DOC-21112/2011 - 16 The compound may be used in the prevention or treatment of a microbial infection. In another aspect the present invention provides a compound of the following formula: Me Me Me N N NN N N N Ru(ll) Ru(I ) Ru(II) N N - N 'N - N ~ N - [Z]b I -- , I "I ".,( " Il

L

2 N N - Q N Me

-

-~j a-1 5 wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different;

L

2 is a labile ligand; b is an integer from 2 to 8; 10 Z represents one or more counteranions; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-. The compound may be used in the prevention or treatment of a microbial infection. 15 The skilled person will recognise that a number of atoms, such as the complexed ruthenium centres, within the compounds of the invention may exist in more than one stereoisomeric form. The present invention contemplates within its scope compounds of all possible absolute configurations about all such atoms. For example, the compounds of 20 the invention may exist as mixtures of A- and A-stereoisomeric forms about any one or more of the ruthenium centres, including racemic mixtures or enantioenriched mixtures, or may exist in enantiopure form wherein each ruthenium centre exists as the A- or the A stereoisomeric form. In some embodiments, each ruthenium centre within the compound has the same absolute configuration such that for a compound bearing two ruthenium C:\NRPotbl\DCC\DAR\4061895_ IDOC-21/12/2011 - 17 centres the stereochemical configuration is either AA- or AA-. In those compounds bearing more than two ruthenium centres, the non-terminal ruthenium centres may exist as racemic mixtures whereas the terminal ruthenium centres may be in either the A- or A stereoisomeric form. 5 The skilled person will appreciate that there are a range of techniques available to produce the compounds of the invention in racemic, enantioenriched or enantiopure forms. For example, enantioenriched or enantiopure forms of the compounds may be produced through stereoselective synthesis and/or through the use of chromatographic or selective 10 recrystallisation techniques. Accordingly in some embodiments the present invention provides a compound of the formula: R,-R, Rn Rn Rn Rn Rn R,,, Rn Rn-iQN N YQ0 nN ~ R, N , 9 9Rn Ru(II) Ru(II) Ru(II) 11 " "L.1 [Zib. R~- r J 0o a N INo O0N Rn -N N0 Q 0 N -Q R n Rn a-1 15 wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 4 to 8; Z represents one or more counteranions; 20 Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; each R may be the same or different and is independently selected from an alkyl group, such as methyl; each n is independently selected from 0, 1 or 2.

C:NRPoNb\DCCDAR\4061895.I.DOC.21/12/2011 - 18 The compound may be used in the prevention or treatment of a microbial infection. In some embodiments, when a = 1 then Q contains at least one -NH-, -N(alkyl)- or -0 5 group. In further embodiments when a = 1 then Q is not selected from 1,2-ethylene, 1,5 pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16 hexadecylene, or any one of: H H N N H H N N N H H and when a = 2 or 3 then Q is not selected from 1,7-heptylene. In still further 10 embodiments when a = 1, 2 or 3 then Q is not selected from 1,2-ethylene, 1,5-pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16-hexadecylene, or any one of: H H N ., N \ H H N N N H H H N N N N H H or any one of: 15 In some embodiments the compounds of the present invention have the following formula: C:NRPonbl\DCC\DAR\4061895_1. OC-21/12/20I - 19 b+ N Me Me 0 0 Me,,. 0 0 0 N,,,, NO ON,, ,~ Ru(II) Ru(II) Ru(II) N N N 0 N 0 00 0 00 Me - -a-I wherein: a is an integer from 1 to 3, wherein when a is greater than 1 each Q may be the same or different; 5 b is an integer from 4 to 8; Z represents one or more counteranions; Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-. 10 The compound may be used in the prevention or treatment of a microbial infection. In some embodiments, when a = I then Q contains at least one -NH-, -N(alkyl)- or -0 group. In further embodiments when a = I then Q is not selected from 1,2-ethylene, 1,5 pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16 15 hexadecylene, or any one of: H H N N N '/ H H and when a = 2 or 3 then Q is not selected from 1,7-heptylene. In still further embodiments when a = 1, 2 or 3 then Q is not selected from 1,2-ethylene, 1,5-pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14-tetradecylene and 1,16-hexadecylene, 20 or any one of: C:\NRPonbrDCC\DARU 6895_.DOC.211j2/20II - 20 H H N NN H H H H H N-' N N N or any one of: 5 In some embodiments a is 2 and the compound has the stereochemistry shown below: 6+ Me Me Me N Q N Q1 N, .Ru(IuI .,I N N N -N"u( 1 Ru(II)MeR(I N N MeI

'N

1 N wherein: each Q may be the same or different and is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, 10 N(alkyl)- or -0-; and Z represents one or more counteranions. The compound may be used in the prevention or treatment of a microbial infection. 15 In some embodiments Q contains at least one -NH-, -N(alkyl)- or -0- group. In further C:\NRPorb\DCC\DAR\4061895_ .DOC-21/12/2011 -21 embodiments Q is not selected from 1,7-heptylene. In some embodiments a is 3 and the compound has the stereochemistry shown below: Me Me Me Me 8+ N Q N Ru(1 Iu1) N (Zla 5 wherein each Q may be the same or different and is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with NH-, -N(alkyl)- or -0-; and Z represents one or more counteranions 10 The compound may be used in the prevention or treatment of a microbial infection. In some embodiments Q contains at least one -NH-, -N(alkyl)- or -0- group. In further embodiments Q is not selected from 1,7-heptylene. 15 As used herein, the term "alkylene" is intended to denote the divalent form of "alkyl" as herein defined. The term "alkyl" denotes straight chain, branched or cyclic alkyl, for example C 1 4 0 alkyl, or CI-20 or C 2

-

1 6 . Examples of straight chain and branched alkyl include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, i-butyl, n-pentyl, 1,2 dimethylpropyl, 1, 1 -dimethyl-propyl, hexyl, 4-methylpentyl, I -methylpentyl, 2 20 methylpentyl, 3-methylpentyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, heptyl, 5-methylhexyl, I methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2 dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl, 1,1,2 trimethylbutyl, 1,1,3-trimethylbutyl, octyl, 6-methylheptyl, 1-methylheptyl, 1,1,3,3 25 tetramethylbutyl, nonyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-methyloctyl, 1-, 2-, 3-, 4- or 5-ethylheptyl, 1-, 2- or 3-propylhexyl, decyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-methylnonyl, 1-, 2-, 3-, 4-, 5- or C:NRPorb\DCC DAR4061R95_ .DOC-21/12/2011 -22 6-ethyloctyl, 1-, 2-, 3- or 4-propylheptyl, undecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9 methyldecyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-ethylnonyl, 1-, 2-, 3-, 4- or 5-propyloctyl, 1-, 2- or 3 butylheptyl, 1-pentylhexyl, dodecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-methylundecyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-ethyldecyl, 1-, 2-, 3-, 4-, 5- or 6-propylnonyl, 1-, 2-, 3- or 4 5 butyloctyl, 1,2-pentylheptyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonoadecyl, eicosyl and the like. Examples of cyclic alkyl include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like. 10 In preferred embodiments the alkylene linking group "Q" is a flexible alkylene linking group. Examples of flexible alkylene linking group include linear alkylene groups, such as methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, nonylene, decylene, undecylene, dodecylene, tridecylene, tetradecylene, pentadecylene, and hexdecylene. Preferably the alkylene linking group links through the terminal carbon 15 atoms of the group, namely through the a,to-carbon atoms of the group. Specific examples include 1,2-ethylene, 1,5-pentylene, 1,7-heptylene, 1,10-decylene, 1,12-dodecylene, 1,14 tetradecylene and 1,1 6-hexadecylene as shown below: 20 As herein defined Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-, such as -NH- or -0-. It will be understood that the replacement of a methylene group in an alkylene group with -NH- or -N(alkyl)- (such as -NMe-, -N(ethyl)-, -N(propyl)-, etc) will create an amine. For example, replacement of a methylene group in 1,3-propylene with 25 -NH- will create a methaminomethyl linking group as shown below: H and replacement of a methylene group in 1,3-propylene with -NMe- will create a methamino(methyl)methyl linking group as shown below: 30 Likewise it will be understood that the replacement of a methylene group in an alkylene C:NRPonbKDCCDAR\4061895_1 DOC-21/12/2011 -23 group with -0- will create an ether. For example, replacement of a methylene group in 1,3 propylene with -0- will create a methoxymethyl linking group as shown below: 5 In some embodiments, more than one methylene group in the alkylene linking group, Q, will be independently replaced with -NH-, -N(alkyl)- or -0-. Whilst mixed ether-amine linking groups are contemplated, typically the linking group will be either an alkylene, a (poly)aminoalkylene or a (poly)oxyalkylene. 10 Examples of polyaminoalkylene linking groups are provided below: H H N N \ H H N - N N \ H H H N , N N N H H Examples of (poly)oxyalkylene linking groups are provided below: 15 It will be understood that the linking group may be used to alter the lipophilicity, flexibility and size of the ruthenium complexes of the present invention. For example, the skilled person will recognise that under certain conditions the polyaminoalkylene linking groups shown above will become protonated. In some embodiments this protonation may be preferable to aid in water solubility, or in other embodiments it may be deleterious if 20 increased lipophilicity is desired. The skilled worker is therefore provided with a useful handle to alter the lipophilicity of the complexes of the present invention. One such way in which the lipophilicity of the complexes of the present invention may be altered is by C:\NRPonblDCCDARM'A61895_1 DOC-21/12/2012 - 24 making a pharmaceutically acceptable salt of an amine group in Q. As used herein the term "counteranion" refers to any negatively charged group, such as organic or inorganic anionic groups, which renders the overall charge of the compounds of 5 the invention neutral. In particular, the entity [Z]b~ refers to one or more counteranions providing an overall negative charge "b~" which is sufficient to render the charge of the compounds of the invention neutral. Counteranions may be atomic, such as halide including fluoride, chloride, bromide and iodide counteranions. Counteranions can also be molecular, such as acetate, succinate, maleate, trifluoromethanesulfonate (triflate) and 10 hexafluorophosphate. Counteranions can have a charge greater than 1, such as 2 or more. By way of example, in the compound represented by the following structure: 3+ NMeMe N N -PN N N Ru(II) Ru(Ii) N N N N N CI N .3 Cl I N(CH 2 )n the skilled worker shall appreciate that the entity [Z] b- referred to herein corresponds to the three chloride counteranions such that b is equal to 3. In preferred embodiments the 15 counteranion is pharmaceutically acceptable. The compounds and methods of the present invention may be used in the treatment and/or prevention of a range of microbial infections. As used herein, treatment may include alleviating or ameliorating the symptoms, diseases or conditions associated with the 20 microbial infection being treated, including reducing the severity and/or frequency of the microbial infection. As used herein, prevention may include preventing or delaying the onset of, inhibiting the progression of, or halting or reversing altogether the onset or progression of the particular symptoms, disease or condition associated with a microbial infection. 25 CANRPorlCC\DARW061895_1 DOC-21/12/20II -25 The terms "microbial", "microorganism", etc includes any microscopic organism or taxonomically related macroscopic organism within the categories algae, bacteria, fungi, yeast and protozoa or the like. 5 The bacterial infection may be caused by one or more species selected from one or more of the Gram-negative bacterial genera: Acinetobacter; Actinobacillus; Bartonella; Bordetella; Brucella; Burkholderia; Campylobacter; Cyanobacteria; Enterobacter; Erwinia; Escherichia; Francisella; Helicobacter; Hemophilus; Klebsiella; Legionella; Moraxella; Morganella; Neisseria; Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella; 10 Serratia; Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia. Specific examples include, but are not limited to, infections caused by Bacteroides, Bordetella pertussis, Brucella, Campylobacter infections, enterohaemorrhagic Escherichia coli (EHEC) enteroinvasive Escherichia coli (EIEC), enterotoxigenic Escherichia coli (ETEC), Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella spp., 15 Moraxella catarrhalis, Neisseria gonnorrhoeae, Neisseria meningitidis, Proteus spp., Pseudomonas aeruginosa, Salmonella spp., Shigella spp., Vibrio cholera and Yersinia; acid fast bacteria including Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Myobacterium johnei, Mycobacterium leprae, atypical bacteria, Chlamydia, Mycoplasma, Rickettsia, Spirochetes, Treponema pallidum, Borrelia recurrentis, Borrelia 20 burgdorfii and Leptospira icterohemorrhagiae and other miscellaneous bacteria, including Actinomyces and Nocardia. The bacterial infection may be caused by one or more species selected from one or more of the Gram-positive bacterial genera: Actinobacteria; Bacillus; Clostridium; 25 Corynebacterium; Enterococcus; Listeria; Nocardia; Staphylococcus; and Streptococcus. Specific examples include, but are not limited to, infections caused by Bacillus cereus, Bacillus anthracis, Clostridium botulinum, Clostridium difficile, Clostridium tetani, Clostridium perfringens, Corynebacteria diphtheriae, Enterococcus (Streptococcus D), Listeria monocytogenes, Pneumoccoccal infections (Streptococcus pneumoniae), 30 Staphylococcal infections and Streptococcal infections.

C:\NRP.nbPDCCDAR\4O61895-IDOC21112101 I -26 Fungal infections include, but are not limited to, infections caused by Alternaria alternata, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus versicolor, Blastomyces dermatiditis, Candida albicans, Candida dubliensis, Candida krusei, Candida parapsilosis, Candida tropicalis, Candida glabrala, 5 Coccidioides immitis, Cryptococcus neoformans, Epidermophyton floccosum, Histoplasma capsulatum, Malassezia furfur, Microsporum canis, Mu cor spp., Paracoccidioides brasiliensis, Penicillium marneffei, Pityrosporum ovale, Pneumocystis carinii, Sporothrix schenkii, Trichophyton rubrum, Trichophyton interdigitale, Trichosporon beigelii and Rhodotorula spp.. 10 Yeast infections include, but are not limited to, infections caused by Brettanomyces clausenii, Brettanomyces custerii, Brettanomyces anomalous, Brettanomyces naardenensis, Candida himilis, Candida intermedia, Candida saki, Candida solani, Candida tropicalis, Candida versatilis, Candida bechii, Candida famata, Candida 15 lipolytica, Candida stellata, Candida vini, Debaromyces hansenii, Dekkera intermedia, Dekkera bruxellensis, Geotrichium sandidum, Hansenula fabiani, Hanseniaspora uvarum, Hansenula anomala, Hanseniaspora guillermondii, Hanseniaspora vinae, Kluyveromyces lactis, Kloekera apiculata, Kluveromyces marxianus, Kluyveromyces fragilis, Metschikowia pulcherrima, Pichia guilliermodii, Pichia orientalis, Pichia fermentans, 20 Pichia memranefaciens, Rhodotorula Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces dairiensis, Saccharomyces exigus, Saccharomyces uinsporus, Saccharomyces uvarum, Saccharomyces oleaginosus, Saccharomyces boulardii, Saccharomycodies ludwigii, Schizosaccharomyces pombe, Torulaspora delbruekii, Torulopsis stellata, Zygoaccharomyces bailli and Zygosaccharomyces rouxii. 25 Protozoal infections include, but are not limited to, infections caused by Leishmania, Toxoplasma, Plasmodia (which are understood to be the causative agent(s) of malarial infection), Theileria, Anaplasma, Giardia, Trichomonas, Trypanosoma, Coccidia, and Babesia. Specific examples include Trypanosoma cruzi, Eimeria tenella, Plasmodium 30 falciparum, Plasmodium vivax, Plasmodium malaria, Plasmodium knowlesi or Plasmodium ovale.

C:\NRPonb\DCC\DAR\461895_ .DOC-21/12201 - 27 Preferably, the microbial infection is caused by either a Gram-positive or a Gram-negative bacterium, for example, Staphylococcus aureus (including MRSA), Enterococcus fecalis, Escherichia coli, Klebsiella pneumonia, Salmonella typhimurium or pseudotuberculosis, 5 Acinetobacter, Pseudomonas aeruginosa, Clostridium perfringens, Clostridium difficile, Campylobacter jejuni or Bacteroides fragilis; a fungal or yeast infection, for example, Trichophyton interdigitale; Aspergillus fumigatus or Candida albicans; or a protozoal infection, for example Plasmodiumfalciparum. 10 Examples of microbial infections include bacterial or fungal wound infections, mucosal infections, enteric infections, septic conditions, pneumonia, trachoma, ornithosis, trichomoniasis, fungal infections and salmonellosis, such as in veterinary practice. The compounds of the invention may also be used for the treatment of resistant microbial species or in various fields where antiseptic treatment or disinfection of materials is 15 required, for example, surface disinfection. The term "subject" as used herein refers to any animal having a disease or condition which requires treatment with a pharmaceutically-active agent. The subject may be a mammal, preferably a human, or may be a domestic or companion animal. While it is particularly 20 contemplated that the compounds of the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment of animals. The compounds of the invention may be in crystalline form or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. The term 25 "solvate" is a complex of variable stoichiometry formed by a solute (in this invention, a compound of the invention) and a solvent. Such solvents should preferably not interfere with the biological activity of the solute. Solvents may be, by way of example, water, acetone, ethanol or acetic acid. Methods of solvation are generally known within the art. 30 The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound as hereinbefore defined, or a C:\NRoRtnb\DCC\DARW4061895_L.DOC-21/12/2011 -28 pharmaceutically acceptable salt thereof, together with at least one pharmaceutically acceptable carrier or diluent. Pharmaceutically acceptable acid addition salts may be prepared from inorganic and 5 organic acids. Examples of inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like. Examples of organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic 10 acid, salicylic acid and the like. For example, where the linking group represented by Q contains one or more amino groups, the amino groups may undergo reaction with an acid to form the acid addition salt. Pharmaceutically acceptable base addition salts may be prepared from inorganic and 15 organic bases. Corresponding counterions derived from inorganic bases include the sodium, potassium, lithium, ammonium, calcium and magnesium salts. Organic bases include primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, and cyclic amines, including isopropylamine, trimethyl amine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2 20 dimethylaminoethanol, tromethamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, N-alkylglucamines, theobromine, purines, piperazine, piperidine, and N-ethylpiperidine. Acid/base addition salts tend to be more soluble in aqueous solvents than the 25 corresponding free acid/base forms. The term "composition" is intended to include the formulation of an active ingredient with encapsulating material as carrier, to give a capsule in which the active ingredient (with or without other carrier) is surrounded by carriers. 30 C-\NRPortbI\DCC\DARU61895_ DOC.21/12/2011 -29 While the compound as hereinbefore described, or pharmaceutically acceptable salt thereof, may be the sole active ingredient administered to the subject, the administration of other active ingredient(s) with the compound is within the scope of the invention. For example, the compound could be administered with one or more therapeutic agents in 5 combination. The combination may allow for separate, sequential or simultaneous administration of the compound as hereinbefore described with the other active ingredient(s). The combination may be provided in the form of a pharmaceutical composition. 10 As will be readily appreciated by those skilled in the art, the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the condition and the mammal to be treated. It is believed that the choice of a particular carrier or delivery system, and route of administration could be readily determined by a person skilled in the art. In the preparation of any formulation containing the compound 15 care should be taken to ensure that the activity of the compound is not destroyed in the process and that the compound is able to reach its site of action without being destroyed. In some circumstances it may be necessary to protect the compound by means known in the art, such as, for example, micro encapsulation. Similarly the route of administration chosen should be such that the compound reaches its site of action. 20 Those skilled in the art may readily determine appropriate formulations for the compounds of the present invention using conventional approaches. Identification of preferred pH ranges and suitable excipients, for example antioxidants, is routine in the art. Buffer systems are routinely used to provide pH values of a desired range and include carboxylic 25 acid buffers for example acetate, citrate, lactate and succinate. A variety of antioxidants are available for such formulations including phenolic compounds such as BHT or vitamin E, reducing agents such as methionine or'sulphite, and metal chelators such as EDTA. The compounds as hereinbefore described, or pharmaceutically acceptable salt thereof, 30 may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery. The pharmaceutical forms suitable for C\NRPonb\DCC\DARWM61895_ I DOC.21/12/l01 - 30 injectable use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions. They should be stable under the conditions of manufacture and storage and may be preserved against reduction or oxidation and the contaminating action of microorganisms such as bacteria or fungi. 5 The solvent or dispersion medium for the injectable solution or dispersion may contain any of the conventional solvent or carrier systems for the compound, and may contain, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The 10 proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about where necessary by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it 15 will be preferable to include agents to adjust osmolarity, for example, sugars or sodium chloride. Preferably, the formulation for injection will be isotonic with blood. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Pharmaceutical forms suitable for injectable use may be delivered by any 20 appropriate route including intravenous, intramuscular, intracerebral, intrathecal, epidural injection or infusion. Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients such as 25 those enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum 30 drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.

C:XRPortbhDCC\DARW061K95_ LDOC-21/12/2011 -31 Other pharmaceutical forms include oral and enteral formulations of the present invention, in which the active compound may be formulated with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it 5 may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal or sublingual tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage will 10 be obtained. The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the 15 like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For 20 instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active 25 compound may be incorporated into sustained-release preparations and formulations, including those that allow specific delivery of the active compound to specific regions of the gut. Liquid formulations may also be administered enterally via a stomach or oesophageal tube. 30 C:WRPortbrDCC\DAR406189)5I.DOC.21/1201 1 -32 Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases. It is also possible, but not necessary, for the compounds of the present invention to be administered topically, intranasally, intravaginally, intraocularly and the like. 5 The present invention also extends to any other forms suitable for administration, for example topical application such as creams, lotions and gels, or compositions suitable for inhalation or intranasal delivery, for example solutions, dry powders, suspensions or emulsions. 10 The compounds of the present invention may be administered by inhalation in the form of an aerosol spray from a pressurised dispenser or container, which contains a propellant such as carbon dioxide gas, dichlorodifluoromethane, nitrogen, propane or other suitable gas or combination of gases. The compounds may also be administered using a nebuliser. 15 Pharmaceutically acceptable vehicles and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is 20 incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions. It is especially advantageous to formulate the compositions in dosage unit form for ease of 25 administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically acceptable vehicle. The specification for the novel dosage unit forms of the invention are 30 dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the C:WRPotnbDCC\DARMW61895_I.DOC.21/12/2011 - 33 art of compounding active materials for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail. As mentioned above the principal active ingredient may be compounded for convenient 5 and effective administration in therapeutically effective amounts with a suitable pharmaceutically acceptable vehicle in dosage unit form. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.25 pg to about 200 mg. Expressed in proportions, the active compound may be present in from about 0.25 pg to about 200 mg/mL of carrier. In the case of compositions containing 10 supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients. The terms "therapeutically effective amount" and "effective amount" refer to that amount which is sufficient to effect treatment, as defined below, when administered to an animal, 15 preferably a mammal, more preferably a human in need of such treatment. The therapeutically effective amount or effective amount will vary depending on the subject and nature of bacterial infection being treated, the severity of the infection and the manner of administration, and may be determined routinely by one of ordinary skill in the art. 20 The terms "treatment" and "treating" as used herein cover any treatment of a condition or disease in an animal, preferably a mammal, more preferably a human, and includes: (i) inhibiting the microbial infection, eg arresting its proliferation; (ii) relieving the infection, eg causing a reduction in the severity of the infection; or (iii) relieving the conditions caused by the infection, eg symptoms of the infection. The terms "prevention" and 25 preventing" as used herein cover the prevention or prophylaxis of a condition or disease in an animal, preferably a mammal, more preferably a human and includes preventing the microbial infection from occurring in a subject which may be predisposed to infection but has not yet been diagnosed as being infected. 30 Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will C :NRorbT\DCC\DARWO61895_I.DOC-21/12/2011 - 34 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The invention will now be described with reference to some specific examples and 5 drawings. However, it is to be understood that the particularity of the following description is not to supercede the generality of the invention as hereinbefore described. EXAMPLES 10 Physical measurements 'H NMR data were 'H NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer at room temperature in CD 2

CI

2 (>99.8%, Aldrich). Materials and methods 15 Ethylene glycol, 1,10-phenanthroline (phen), potassium hexafluorophosphate (KPF 6 ), ammonium hexafluorophosphate (NH 4

PF

6 ), tetraethylammonium chloride were purchased from Aldrich and used as supplied. SP-Sephadex* C-25 cation-exchanger and Sephadex* LH-20 were obtained from Amersham Pharmacia Biotech. 3,4,7,8-Tetramethyl-1,10 phenanthroline (Me 4 phen) was obtained from GFS chemicals. 20 Synthesis of Ligands Syntheses of bridging ligzands "bb" The synthesis of ligands bb, (J.L. Morgan, C.B. Spillane, J.A. Smith, D.P. Buck, J. G. 25 Collins and F.R. Keene, Dalton Trans. 2007, 4333-4342), A-[Ru(phen) 2 (py) 2

]{(-)

AsOtart} 2 (X. Hua and A. von Zelewsky, Inorg. Chem., 1995, 34, 5791-5797), [Ru(terpy)C1 3 ] (P. A. Adcock, F.R. Keene, R.S. Smythe, M.R. Snow, Inorg. Chem. 1984, 23, 2336-2343) and [Ru(Me 4 phen) 2

C

2 ] (T. Tagano, Inorg. Chim. Acta, 1992, 195, 221 225) were performed according to literature methods. 30 The ligands bb 2 (n=2), bb 5 (n=5), bb7 (n=7), bbio (n=10), bb1 2 (n=12), bb 14 (n=14) and bb1 6 C:\NRPonb\DCC\DAR4061895_ I DOC-21/12/0 11 - 35 (n= 16) are shown below: N N_

(CH

2 )n N N By way of example, to prepare mono-lithiated Me 2 bpy, Me 2 bpy (1.6 g, 8.7 mmol) was 5 dissolved in dry THF (50 mL) under an inert atmosphere (Ar) at room temperature and the mixture cooled to -78 *C. A solution of lithium diisopropylamide (9.6 mmol) in THF (10 mL) was added dropwise over the course of 30 min, and the mixture stirred for a further 1.5 h at -78 *C, during which time the colour turned from white to dark brown-red. This mixture was brought to -10 *C over the course of 30 min. 10 For bb1 2 , 1,10-dibromodecane (1,12-dibromododecane for bbi4, 1,14-dibromotetradecane for bbi 6 ; 4.3 mmol) was injected into the suspension of mono-lithiated Me 2 bpy. The reaction was brought to room temperature and left to stir under an inert atmosphere (Ar). A colour change from dark red to dark green to grey-green then to cream was typically 15 observed within the first 2 h. After a further 24 h the reaction was quenched with water (10 mL), and the product extracted into diethyl ether (3 x 80 mL) and DCM (1 x 80 mL). The organic layers were combined, washed with water (1 x 50 mL), dried over anhydrous Na 2

SO

4 , filtered, and then evaporated to dryness in vacuo to yield a fluffy white powder in each case. The crude product was dissolved in a minimal volume of DCM and then loaded 20 onto a silica gel column (230-400 mesh, 3 cm diam. x 10 cm). The unreacted dibromopropane (pale yellow band) and Me 2 bpy (yellow band) were eluted using DCM. bba 2 and bb1 4 (yellow band) and side products (yellow-brown band, suspected to be due to the di-lithiation of Me 2 bpy in the first step) were gradient-eluted using 1-10% (v/v) methanol in DCM. The purity and contents of each fraction were determined by TLC using 25 15% (v/v) methanol in DCM as a mobile phase. The purest fractions were combined and the solvent was removed in vacuo to yield fluffy white solids. bbi 6 , was recrystallised from boiling DCM after extraction. Yields: bb1 2 (30%); bb, 4 (33%); bbi 6 (44%). Characterisation was achieved using 'H NMR in CDCl 3 . In some cases there was a C :WRPortbl\DCC\DAR\4061895_I.DOC.2 /12/2011 -36 presence of a small impurity of unreacted Me 2 bpy, which was difficult to completely eliminate by silica gel chromatography. The ligands were used for complex synthesis without further purification. bb1 2 - 'H NMR (300 MHz, CDCl 3 ): d 8.55 (4H, dd, J = 4.5, 3.0 Hz, bipy6); 8.24 (4H, s, 5 bipy3); 7.14 (4H, dd, J= 5.0, 1.0 Hz, bipy5); 2.70 (4H, t, 2 x CH2bipy); 2.45 (6H, s, 2 x CH3bipy); 1.28 - 1.70 (20H, m, 10 x CH2). bb1 4 - IH NMR (300 MHz, CDCl 3 ): d 8.56 (4H, dd, J= 4.5, 3.0 Hz, bipy6); 8.24 (4H, s, bipy3); 7.15 (4H, d, J= 4.5, 1.0Hz, bipy5); 2.70 (4H, t, 2 x CH2bipy); 2.45 (6H, s, 2 x CH3bipy); 1.26 - 1.72 (24H, m, 12 x CH2). 10 bb1 6 - 'H NMR (300 MHz, CDC 3 ): d 8.57 (4H, dd, J= 4.5, 3.0 Hz, bipy6). 8.24 (4H, s, bipy3); 7.15 (4H, dd, J= 5.0, 1.0 Hz, bipy5); 2.70 (4H, t, 2 x CH 2 bipy); 2.45 (6H, s, 2 x

CH

3 bipy); 1.26 -1.66 (28H, m, 14 x CH 2 ). Synthesis of bridging ligands bbN, 15 Analogous bridging ligands containing flexible polyamine chains were also synthesised. Using similar methodology to Sasaki and co-workers (I. Sasaki, M. Imberdis, A. Gaudemer, B. Drahi, D. Azhari and E. Amouyal, New J. Chem., 1994, 18, 759-764.) which provides polyamine-linked ligands via a condensation between 4-methyl-2,2' bipyridine-4'-carboxaldehyde and appropriate primary amines, followed by the reduction 20 of the resulting Schiff base products, in the present work 4-methyl-2,2'-bipyridine-4' carboxaldehyde was reacted with 1,3-diaminopropane, diethylenetriamine (dien) and triethylenetetramine (trien) to afford polyamine-linked bis[4(4'-methyl-2,2'-bipyridine)] ligating groups (bbNn), as shown below: H dio n H H H NN \ NN / 2. NBH '-- bbN12 25 The ligands bbN 7 (n=7), bbN 9 (n=9) and bbN1 2 (n=12) are shown below: C:NRPortblDCC\DAR\406J893_ .DOC-21/12/2011 - 37 -~ ~ - N-~N H H bbN 7 \/ \// NN N N H H H bbN N N N H H bbN 12 N - N \ N N H H A mixture of 4-methyl-2,2'-bipyridine-4'-carboxaldehyde (4.04 mmol) and the appropriate polyamine compound (1,3-diaminopropane, diethylenetriamine or triethylenetetramine, 2.02 mmol) was stirred in methanol (30 ml) at room temperature for 4 h. Sodium 5 borohydride (4.04 mmol) was then added to the reaction with further stirring for I h. The solvent was removed and the crude residue redissolved in a minimum amount of water. The organic component was extracted three times with ethyl acetate, then washed with water and brine. After removing the solvent, the crude residue was chromatographed using silica gel with MeOH/ammonia (9:1) eluent to afford bbN, in 20-50% yield. 10 bbN 7 - 'H NMR (300 MHz, CDC 3 ): d 8.58 (2H, d, J= 6.0, bipy6'); 8.51 (2H, d, J = 5.0 Hz, bipy6); 8.21 (2H, s, bipy3); 8.31 (2H, s, bipy3'); 7.55 (2H, d, J= 5.3, 1.0Hz, bipy5'); 7.12 (2H, dd, J = 5.0, 1.0Hz, bipy5); 3.86 (4H, 2 x CH 2 bipy); 2.43 (6H, s, 2xCH 3 bipy); 1.80 (2H, 2x-NH); 1.60, 2.61 (6H, m, 3 x -CH 2 ). bbN 9 - 'H NMR (300 MHz, CDCl 3 ): d 8.58 (2H, d, J = 6.0, bipy6'); 8.51 (2H, d, J = 5.0 15 Hz, bipy6); 8.31 (2H, s, bipy3'); 8.21 (2H, s, bipy3); 7.55 (2H, d, J= 5.3, 1.0 Hz, bipy5'); 7.12 (2H, dd, J= 5.0, 1.0 Hz, bipy5); 3.86 (4H, s, 2 x CH 2 bipy); 2.80 (8H, s, 4 x -CH2); 2.43 (6H, s, 2 x CH 3 bipy); 1.80 (3H, 3 x -NH). bbN1 2 - 'H NMR (300 MHz, CDCl 3 ): d 8.58 (2H, d, J= 6.0, bipy6'); 8.51 (2H, d, J= 5.0 Hz, bipy6); 8.31 (2H, s, bipy3'); 8.21 (2H, s, bipy3); 7.55 (2H, d, J= 5.3, 1.0 Hz, bipy5'); 20 7.12 (2H, dd, J= 5.0, 1.0 Hz, bipy5); 3.86 (4H, s, 2 x CH 2 bipy); 3.04 (4H, s, 4 x -NH); 2.78 (12H, s, 6 x -CH 2 ); 2.43 (6H, s, 2 x CH 3 bipy).

C:WRNonbl\DCC\DAR\4061895I DOC-21112/201 I - 38 Synthesis of ligands bbO, Analogous bridging ligands containing (poly)ether groups (bbOn) were synthesised from the dibromopolyethoxy precursors (obtained from appropriate polyethylene glycols as reported in G. Bdrubd, D. Rabouin, V. Perron, B. N'Zemba, R.-C. Gaudreault, S. Parent 5 and E. Asselin, Steroids, 2006, 71, 911-921) as shown below: Mesyl chloride HO O OH LI~r' Br Or 1. L A In N O- N THIF N / 2. Br O O ,Br bbO The ligands bb0 7 (n=7), bbO 10 (n=10), bbO 13 (n=13) and bbO 1 6 (n=16) are shown below: bb0 7 0 / \/ N N N N N bbO 1 0 / O N N bb0 1 3 O O O bbO 16 O_ O O O ~ N N Lithium diisopropylamide (2 M solution in heptane/THF, 4.34 mmol) was added dropwise 10 to a stirred solution of 4,4'-dimethyl-2,2'-bipyridine (8.68 mmol) in dry THF while maintaining the temperature at -78 *C (acetone/dry ice) under Ar. The dark-coloured mixture was further stirred for 2 h while the temperature was raised slowly to -10 0 C. An appropriate dibromopolyether compound (1,5-dibromo-3-oxapentane, 1,8-dibromo-3,6 dioxaoctane, 1,11 -dibromo-3,6,9-trioxadecane or 1,1 4-dibromo-3,6,9,12 15 tetraoxatetradecane; 4.34 mmol) was then added and stirring continued for further 20 h. Water (30 ml) was added to the mixture, followed by extraction with diethyl ether and DCM. The organic phases were combined, washed with water and brine and dried over C:\NRPtnbT\DCCDARMU6I895_ IDOC-21112/2011 -39 anhydrous Na 2

SO

4 followed by filtration. Evaporation of the filtrate to dryness gave crude oil of bbOn, which was purified using a silica gel column with DCM-methanol (9:1) as the eluent. The pure bbOn was obtained in the middle band, while the first and the last were assigned as the 4,4'-dimethyl-2,2'-bipyridine and di-lithiation product impurities, 5 respectively. bb0 7 - 'H NMR (300 MHz, CDC1 3 ): d 8.55 (4H, m, bipy6); 8.25 (4H, s, bipy3); 7.16 (4H, m, bipy5); 3.48 (4H, m, 2 x CH 2 0); 2.81 (4H, t, 2 x CH 2 bipy); 2.45 (6H, s, 2 x CH 3 bipy); 2.00 (4H, m, 2 x CH 2 ). bbOo - 'H NMR (300 MHz, CDCl 3 ): d 8.55 (4H, m, bipy6); 8.25 (4H, s, bipy3); 7.16 10 (4H,m, bipy5); 3.51 - 3.62 (8H,m, 4 x CH 2 0); 2.81 (4H, t, CH 2 bipy); 2.45 (6H, s, 2 x

CH

3 bipy); 2.00 (4H, m, 2 x CH 2 ). bbO, 3 - 'H NMR (300 MHz, CDCl 3 ): d 8.55 (4H, m, bipy6); 8.25 (4H, s, bipy3); 7.16 (4H, m, bipy5); 3.51 - 3.62 (12H, m, 6 x CH 2 0); 2.81 (4H, t, CH 2 bipy); 2.45 (6H, s, 2 x

CH

3 bipy); 2.00 (4H, m, 2 x CH 2 ). 15 bb01 6 - 'H NMR (300 MHz, CDCl 3 : d 8.55 (4H, m, bipy6); 8.25 (4H,s, bipy3); 7.16 (4H,m, bipy5); 3.51 - 3.65 (16H, m, 8 x CH 2 0); 2.81 (4H, t, CH 2 bipy); 2.45 (6H, s, 2 x

CH

3 bipy); 2.00 (4H, m, 2 x CH 2 ). Synthesis of Complexes 20 Synthesis of dinuclear complexes AA- and AA-({Ru(phen)ju-bb2)1(PF) 4 (n = 2, 5, 7,10, 12, 14 and 16) As used herein these complexes are also referred to by shorthand notation. For example 25 AA-[{Ru(phen)212(p-bb 2 )1 is referred to as AA-Rubb1 2 . The syntheses of AA- and AA-[{Ru(phen) 2

}

2 (p-bb.)]C 4 (n = 12, 14, and 16) complexes were adapted from J. L. Morgan, C. B. Spillane, J. A. Smith, D. P. Buck, J. G. Collins and F. R. Keene, Dalton Trans., 2007, 4333-4342. 30 A typical procedure was as follows: A-[Ru(phen) 2 (py) 2 1{(-)-AsOtart} 2 (180 mg, 0.169 C \NR.PonblDCC\DAR\406O195_ LDOC-21/12/0 1I -40 mmol) and bb 1 2 (43 mg, 0.085 mmol) were dissolved in ethylene glycol (4 mL) containing 10% (v/v) water, and stirred at 110 'C in the dark under an inert atmosphere (Ar) for 6 h. The completed reaction was cooled to room temperature, followed by the addition of water (10 mL) and ethanol (10 mL). 5 The dark red-orange solution was loaded onto an SP-Sephadex C-25 column (2 cm diam. x 25 cm), and rinsed with water. Mononuclear impurities were eluted as a bright red-orange band using aqueous 0.3M NaCl solution, and the desired dinuclear species eluted as a bright red-orange band using 0.6 M NaCl solution. The dinuclear product was isolated 10 from the eluate as its PF 6 ~ salt by a slow addition of saturated aqueous KPF 6 , and then extraction into DCM. The organic layer was washed with water, dried over anhydrous Na 2

SO

4 , and evaporated to dryness in vacuo to yield a bright red-orange precipitate, AA-[{Ru(phen) 2

}

2 (p 15 bb 12

)](PF

6

)

4 . The product was further purified by dissolving it in a minimal volume of acetone (AR), loading onto a silica gel column (1 cm diam. x 5 cm), rinsed with acetone (AR) and then eluting with 5% (w/v) NH 4

PF

6 in acetone (AR). An equal volume of water was added to the red-orange eluate and the acetone removed in vacuo. The resulting bright orange precipitate was collected by filtration and washed with cold water (- 50 mL). 20 The precipitate was then converted into its chloride salt by stirring it in an aqueous solution using AmberliteR* IRA-400 (chloride form) anion-exchange resin. The resin was removed by filtration, and the dark red filtrate was freeze dried to obtain a fluffy bright orange powder. Typical yield - 20%. The corresponding AA complexes were synthesised 25 as described above, by using A-[Ru(phen) 2 (py)2]]{(+)-AsOtart} 2 as the mononuclear precursor. In the cases of [{Ru(phen) 2

}

2 (p-bb 14 )]Cl 4 and [{Ru(phen) 2

}

2 (t-bbi 6 )]Cl 4 , the eluents were required to include 10% acetone to keep the complexes solubilised during SP Sephadex C-25 purification. 30 Circular dichroism spectra and ID 'H NMR were consistent with those reported in J. L. Morgan, C. B. Spillane, J. A. Smith, D. P. Buck, J. G. Collins and F. R. Keene, Dalton C:RPonbl\DCC\DAR\461895_.DOC-2112nO2 11 -41 Trans., 2007, 4333-4342 for bb 2 , bb 5 , bb 7 , and bbio bridged species. 4+ \N // (SI N4+ ~ N \ Diastereoisomeric forms of [{Ru(phen) 2

}

2 (p-bb 2

)]

4 *: (a) rac {AA (- AA)}; (b) meso. 5 Hydrogen atoms are omitted for clarity; the notation shown is used in the assignment of the 'H NMR spectra in D 2 0. The hexafluorophosphate salts were able to be metathesised to the water-soluble bromide salts by dissolution in a minimum volume of acetone and the addition of [(n-C 4

H

9

)

4 N]Br 10 until complete precipitation had occurred. The products were filtered and washed with cold acetone. In some cases the hexafluorophosphate salts were converted to the water soluble chloride salts by stirring with Dowex ion-exchange resin in water. After filtering, the water solution was freeze-dried to obtain a fluffy orange powder. 15 [{Ru(phen) 2

}

2 (p-bb 2

)](PF

6

)

4 -3H20. Found: C, 45.0; H, 3.41; N 8.3%. Calc. for

C

72

H

54 N1 2 Ru 2

P

4

F

24 -3H 2 0: C, 45.0; H, 3.14; N, 8.7%. UV/Vis (MeCN) - Xma/nm (s/M-1cm'): 451 (0.28 x 105) 285 sh (0.71 x 105), 264 (1.37 x 105). Meso-[{Ru(phen) 2

}

2 (pi-bb 2

)](PF

6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.65 (4H, dd, J= 8.1, 1.0 Hz, H7); 8.54 (4H, dd, J= 8.1, 1.0 Hz, H4); 8.51 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 20 8.45 (2H, dd, J= 1.2, -0.5 Hz, bpy3'); 8.28-8.21(8H, m, H5,6); 8.20 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, dd, J = 5.0, 1.0 Hz, H2); 7.76 (4H, ddd, J = 8.1, 5.0, 1.0 Hz, H8); 7.59 7.55 (6H, m, H3, bpy6); 7.50 (2H, d, J= 5.7 Hz, bpy6'); 7.21 (2H, dd, J= 5.7, 1.0 Hz, bpy5); 7.14 (2H, dd, J= 5.7, 1.0 Hz, bpy5), 3,13, (4H, s, 2 x CH2), 2.52 (6H, s, 2 x Me). Rac-[{Ru(phen) 2

}

2 (p-bb 2

)](PF

6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.66 (4H, dd, J= 8.1, C:\R nb\CC\DAR\61895_ .DOC-21/12/201I - 42 1.0 Hz, H7); 8.57 (4H, dd, J = 8.1, 1.0 Hz, H4); 8.53 (2H, dd, J = 1.2, -0.5 Hz, bpy3); 8.46 (2H, dd, J= 1.2, -0.5 Hz, bpy3'); 8.29-8.21(8H, m, H5,6); 8.21 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, dd, J = 5.0, 1.0 Hz, H2); 7.77 (4H, ddd, J = 8.1, 5.0, 1.0 Hz, H8); 7.59-7.55 (6H, m, H3, bpy6); 7.50 (2H, d, J= 5.7 Hz, bpy6'); 7.21 (2H, dd, J= 5.7, 1.0 Hz, bpy5); 5 7.14 (2H, dd, J = 5.7, 1.0Hz, bpy5), 3,14, (4H, s, 2x CH2), 2.52 (6H, s, 2 x Me). CD {X/nm (Ae/cm-'M-') CH 3 CN} - AA: 468 (-24), 420 (19), 283 (-267), 269 (-296), 258 (367). AA: 468 (23), 420 (-25), 283 (246), 269 (281), 258 (-354). [{Ru(phen) 2

}

2 (p-bbs)](PF 6

)

4 -7H 2 0. Found: C, 44.1; H, 3.43; N, 8.2%. Calc. for

C

75

H

60 Nl 2 Ru 2

P

4

F

24 -7H 2 0: C, 44.2; H, 3.66; N, 8.2%. 10 Meso-[{Ru(phen) 2

}

2 (p-bbs)](PF 6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.66 (4H, dd, J = 8.1, 1.0 Hz, H7); 8.54 (4H, dd, J = 8.1, 1.0 Hz, H4); 8.42 (2H, dd, J = 1.2, -0.5 Hz, bpy3); 8.39 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.28-8.21 (8H, m, H5,6); 8.22 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, dd, J= 5.0, 1.0 Hz, H3); 7.79 (4H, ddd, J= 8.1, 5.1, 1.0 Hz, H8); 7.57 7.47 (8H, m, H3, bpy6); 7.15-7.11 (4H, m, bpy5); 2.79, (4H, bt, J = 5.1 Hz, 2 x CH2-bpy); 15 2.52 (6H, s, 2 x Me); 1.77-1.65 (6H, m, J = Hz, 3 x CH2). Rac-[{Ru(phen) 2

}

2 (p-bb)](PF 6

)

4 : 'H NMR (300 MHz, CD3CN): d 8.65 (4H, dd, J = 8.1, 1.0 Hz, H7); 8.55 (4H, dd, J= 8.1, 1.0 Hz, H4); 8.42 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.38 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.28-8.22 (8H, m, H5,6); 8.21 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, dd, J = 4.5, 1.0 Hz, H3); 7.80 (4H, ddd, J = 8.1, 5.1, 1.0 Hz, H8); 7.56 (4H, 20 dd, J= 8.2, 5.1 H3); 7.50 (4H, dd, J = 5.4, 5.1, bpy6) 7.18-7.08 (4H, m, bpy5); 2.79, (4H, bt, J= -7 Hz, 2 x CH2-bpy); 2.53 (6H, 2 x Me); 1.78-1.66 (6H, m, J = Hz, 3 x CH2). CD (k/nm (De/cm-IM-1) CH3CN) - AA: 466 (-24), 418 (22), 285 (-233), 268 (-312), 260 (334). AA: 466 (26), 418 (-20), 285 (239), 268 (318), 258 (-345). [{Ru(phen) 2

}

2 (p-bb 7

)](PF

6

)

4 -2(acetone)-6H 2 0: Found C, 45.8; H, 3.83; N, 7.5%. Calc. 25 for C 77 H64N 2 Ru 2

P

4

F

24 -2C 3

H

6 0-6H 2 0: C, 46.1; H, 4.10;N, 7.8%. Rac-[{Ru(phen) 2

}

2 (p-bb 7

)](PF

6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.64 (4H, ddd, J = 8.1, 4.8, 1.0 Hz, H8); 8.54 (2H, dd, J = 8.1, 1.0Hz, H4); 8.42 (2H, dd, J = 1.2, -0.5Hz, C:WRPofnb\DCCDAR061395_1 DOC-21112/2011 - 43 bpy3); 8.37 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.28-8.22 (8H, m, H5,6); 8.21 (4H, dd, J= 5.0, 1.0 Hz, H7); 7.89 (4H, dd, J= 4.5, 1.0 Hz, H2); 7.80 (4H, ddd, J= 8.1, 5.1, 1.0 Hz, H9); 7.57 (4H, dd, J= 8.2, 5.1 H3); 7.50 (4H, dd, J= 5.4, 5.1, bpy6); 7.16-7.08 (4H, m, bpy5); 2.77, (4H, bt, J= -7 Hz, 2 x CH2-bpy); 2.53 (6H, s, 2 x Me); 1.74-1.58 (6H, m, J 5 = Hz, 3 x CH2). CD {X/nm (Ae/cm~'M~') CH 3 CN} - AA: 465 (-23), 417 (20), 284 (-221), 268 (-296), 260 (313). AA: 465 (24), 417 (-20), 284 (231), 268 (293), 260 (-336). [{Ru(phen) 2

}

2 (p-bbio)](PF 6

)

4 -10H 2 0. Found C, 44.5; H, 3.96; N, 7.4%. Calc. for

C

8 oH 7 0 N1 2 Ru 2

P

4

F

2 4 -10H 2 0: C, 44.3; H, 4.20; N, 7.8%. Meso-[{Ru(phen) 2

}

2 (p-bbio)](PF 6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.66 (4H, dd, J= 10 8.1, 1.0 Hz, H7); 8.55 (4H, dd, J = 8.1, 1.0 Hz, H4); 8.42 (2H, dd, J = 1.2, -0.5 Hz, bpy3); 8.37 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.28-8.24 (8H, m, H5,6); 8.21 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, ddd, J= 5.0, 1.0, -0.3 Hz, H2); 7.79 (4H, ddd, J= 8.1, 5.1, 1.0 Hz, H8); 7.56 (4H, ddd, J= 8.1, 5.7, -0.3 Hz, H3); 7.50 (4H, dd, J= 5.3, 3.6 Hz, bpy6); 7.15 7.08 (4H, m, bpy5); 2.77, (4H, bt, J= 5.1 Hz, 2 x CH2-bpy); 2.53 (6H, s, 2 x Me); 1.73 15 1.60 (6H, m, J= Hz, 2 x CH2); 1.40-1.24 (12H, m, 6 x CH2). Rac-[{Ru(phen) 2

}

2 (p-bbio)J(PF 6

)

4 : 'H NMR (300 MHz, CD 3 CN): d 8.66 (4H, dd, J= 8.1, 1.0 Hz, H7); 8.55 (4H, dd, J= 8.1, 1.0 Hz, H4); 8.42 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.37 (2H, dd, J= 1.2, -0.5 Hz, bpy3); 8.28-8.24 (8H, m, H5,6); 8.21 (4H, dd, J= 5.0, 1.0 Hz, H9); 7.89 (4H, ddd, J= 5.0, 1.0, -0.3 Hz, H2); 7.79 (4H, ddd, J = 8.1, 5.1, 1.0 Hz, H8); 20 7.56 (4H, ddd, J=8.1, 5.7,-0.3Hz, H3); 7.50 (4H, dd, J=5.3, 3.6Hz, bpy6); 7.15-7.08 (4H, m, bpy5); 2.77, (4H, bt, J= 5.1 Hz, 2 x CH2-bpy); 2.53 (6H, s, 2 x Me); 1.73-1.60 (6H, m, J = Hz, 2 x CH2); 1.40-1.24 (12H, m, 6 x CH2). CD (k/nm (De/cm-1M-1) CH3CN} AA: 465 (-21), 418 (17), 284 (-203), 268 (-254), 259 (289). AA: 465 (22), 418 (-18), 284 (202), 268 (251), 259 (-290). 25 AA-[{Ru(phen)2} 2 (p-bbl2)](PF 6

)

4

-H

2 0. Anal. Found C, 48.8; H, 3.82: N, 8.5%. Calcd. for

C

82

H

76

N,

2

F

24

OP

4 Ru 2 : C, 48.6; H, 3.78; N, 8.3%. 'H NMR (300 MHz, CD 3 CN) : d 8.69 C WRPorblDCCDARU061895_1 DOC-2112/20 11 - 44 (4H, d, J = 8.5 Hz, H2,H9); 8.58 (4H, d, J = 9.0 Hz, H2,H9); 8.42 (4H, d, J = 14.0 Hz, bipy6); 8.27 (8H, dd, J = 3.0, 1.0, H4,H7); 8.24 (4H, m, bipy3); 7.92 (4H, m, H5, H6); 7.82 (4H, m, H5,H6); 7.58 (4H, dd, J = 8.0, 5.3 Hz, H3,H8); 7.52 (4H, dd, J = 5.8, 4.0 Hz, H3, H8); 7.15 (4H, m, bipy5); 2.80 (4H, t, 2 x CH 2 bipy); 2.55 (6H, s, CH 3 bipy); 1.29 5 1.69 (20H, m, 10 x CH 2 ). CD {k/nm(AE/cm~1M~'), Cl- salt in H 2 0}: AA: 473.5(-28.6); 419(28.5); 287.5(-277); 269(-227); 259.5(292); 218(52.9). AA: 471(29.0); 415.5(-26.3); 287.5(282); 268.5(242); 260(-315); 218.5(-57.0). AA-[{Ru(phen) 2

)

2 (p-bbi 4

)](PF

6

)

4

-H

2 0. Anal. Found C, 48.9; H, 3.75: N, 8.6%. Calcd. for

C

84 HsoN, 2

F

24

OP

4 Ru 2 : C, 49.1; H, 3.92; N, 8.2%. 'H NMR (300 MHz, CD 3 CN) : d 8.68 10 (4H, d, J = 8.3 Hz, H2,H9); 8.58 (4H, d, J = 8.2 Hz, H2,H9); 8.42 (4H, d, J = 14.0 Hz, bipy6); 8.28 (8H, dd, J= 3.5, 1.0, H4,H7); 8.24 (4H, m, bipy3); 7.91 (4H, m, H5, H6); 7.83 (4H, m, H5,H6); 7.59 (4H, dd, J= 8.5, 5.0 Hz, H3,H8); 7.52 (4H, dd, J= 6.0, 4.0 Hz, H3,H8); 7.15 (4H, m, bipy5); 2.80 (4H, t, 2 x CH 2 bipy); 2.55 (6H, s, CH 3 bipy); 1.29 1.69 (24H, m, 12 x CH 2 ). CD {k/nm(AE/cm~'M~'), Cl- salt in H 2 0}: AA: 472.5(-31.9); 15 417.5(24.2); 286(-279); 269(-296); 260(353); 216(62.1). AA: 468.5(32.5); 415(-22.8); 286(276); 269(293); 260(-352); 216.5(-63.5. AA-I{Ru(phen) 2

}

2 (p-bbl 6

)](PF

6

)

4 '3H 2 0-NH 4

PF

6 . Anal. Found C, 44.5; H, 3.38: N, 7.8%. Calcd. for C 86

H

9 2

N,

3

F

30 0 3

P

5 Ru 2 : C, 45.2; H, 4.07; N, 8.0%. 'H NMR(300 MHz,

CD

3 CN) : d 8.71 (4H, d, J= 8.3Hz, H2,H9); 8.59 (4H, d, J= 8.2 Hz, H2,H9); 8.52 (4H, d, 20 J= 14.0 Hz, bipy6); 8.29 (8H, d, J= 3.0 Hz, H4,H7); 8.24 (4H, m, bipy3); 7.92 (4H, m, H5, H6); 7.84 (4H, m, H5,H6); 7.60 (4H, dd, J= 8.5, 5.0 Hz, H3,H8); 7.52 (4H, t, H3,H8); 7.15 (4H, m, bipy5); 2.80 (4H, t, 2 x CH 2 bipy); 2.55 (6H, s, CH 3 bipy); 1.70 - 1.27 (28H, m, 14 x CH 2 ). CD {/nm(AE/cm~1M~'), Cl~ salt in H 2 0}: AA: 468.5(-35.6); 418(22.7); 285.5(-292); 270(-306); 260(357); 218.5(72.1). AA: 468(36.0); 417.5(-20.5); 285.5(285); 25 269.5(306); 260(-343); 219.5(-70.4). Representative synthesis of{{Ru(Me 4 phen)(A-bb)7Cl 4 (n = 7, 12, 16) C RW onbf\DCCDAR\46195_1 DOC-21/12201 I - 45 Me Me /, N~ Me Me ~ Me Me Me Me Me N N N N Me Ru(II) Ru(lI) Me N IN N N I2-'N Me Me N N Me Me Me Me Me [Ru(Me 4 phen) 2 Cl 2 ] (0.15 mmol) and bb, (0.075 mmol) were refluxed in EtOH/water (1:1, 20 ml) for four hours. After cooling, the solvent was evaporated under reduced pressure until half of the original volume. The mixture was then loaded onto a Sephadex C-25 5 cation exchange column, eluted with water, 0.3 M NaCl then with 1.0 M NaCl to remove the impurities. Elution with 1.0 M NaCl containing 5% acetone gave the pure [{ Ru(Me4phen) 2 } 2 (p-bb 7 )]2+. For [{Ru(Me 4 phen) 2

}

2 (p-bb2)]2+, the complex was obtained by elution with 1.0 M NaCl containing 10% acetone. For [{Ru(Me 4 phen) 2

}

2 (pI-bbI 6 )]2+, the complex was obtained by elution with 1.0 M NaCl containing 20% acetone. After acetone 10 removal, excess KPF 6 was added causing the precipitation of the PF 6 ~ salt of the complex which was then extracted into dichloromethane followed by evaporation to dryness to give the corresponding [{Ru(Me 4 phen) 2

}

2 (p-bb,)](PF 6

)

2 . The PF6' salt was converted to the chloride by dissolving the solid in the minimum amount of acetone followed by drop wise addition of the saturated solution of tetraethylammonium chloride in acetone while stirring 15 for half an hour. The resulting solid was filtered and washed with acetone and dried under reduced pressure to afford [{Ru(Me 4 phen) 2

}

2 (p-bb,)](Cl) 4 . Typical yield: 30-50%. [{Ru(Me 4 phen) 2

)

2 (A-bb 7 )j(PF 6 )2 'H NMR (300 MHz, CD 2 Cl 2 ) 8 8.30 (m); 8.90 (s), 8.85 (s); 8.55 (d), 8.51 (t); 7.20 (m); 2.90-2.85 (m), 2.51 (s), 2.49 (d), 2.30 (s), 1.80 (s), 1.33 (s). 20 Synthesis of dinuclear complexes AA- and AA-({Ru(phen) ju-bbX) *b+ (X = N, 0) Using similar synthetic techniques to those described above to produce the alkylene bridged complexes stereoselectively, dinuclear complexes AA-[{Ru(phen) 2

}

2 (pi-bbXn)] 4 * (X = N, 0) were produced. These complexes were separated from mononuclear species and other impurities using an SP-Sephadex C-25 cation exchange column with a gradient C:\NRPonbl\DCCOARWO61895_I.DOC.2 P/12/2011 -46 concentration of aqueous sodium chloride solution as eluent. In contrast to the chromatographic purification of AA-[{Ru(phen) 2

}

2 (i-bbn)]4* and AA-[{Ru(phen) 2 }2(t bbOn)]4 *, difficulties were encountered with AA-[{Ru(phen) 2

}

2 (pt-bbN )] 4 + species as there was a significant broadening of the bands, presumably due to the protonation of the free 5 amine moieties. Compared with the alkylene- and (poly)ether-bridged analogues, the purification of AA-[{Ru(phen) 2

}

2 (i-bbN )] 4 + species generally required much slower flow rates and higher concentration of electrolyte in the eluent. The protonation of the free secondary amine groups during the separation processes is not unexpected at the neutral pH values of these procedures, and was exemplified by the microanalytical data of the 10 isolated dinuclear species which were consistent with bbN 12 being protonated to give the AA-[{Ru(phen) 2

}

2 (pt-bbH 4

N

1 2)]8+ form rather than non-protonated AA-[{Ru(phen) 2 }2(p bbNi 2

)]

4 : {Ruphn(pyW3 'NN- 2 15 Synthesis of Dinuclear Complexes AA-{{Ru(phen)J)(u-bbX,) 1(PF) 4 (RubbX,; X = N, 0) As used herein these complexes are also referred to by shorthand notation. For example AA-[{Ru(phen)} (4-bbN 7 )1 is referred to as AA-RubbN 7 . 20 A-[Ru(phen) 2 (py) 2 ]{(-)-AsOtart} 2 (0.18 g, 0.169 mmol) and bbXn (0.085 mmol) were dissolved in ethylene glycol (4 mL) containing 10% (v/v) water, and stirred at 110 0 C in the dark under Ar for 6 h. The completed reaction was cooled to room temperature, followed by the addition of water (10 mL). The dark red-orange solution was loaded onto 25 a SP-Sephadex C-25 column and washed with water. Elution with 0.3 M NaCl solution removed the mononuclear impurities. The desired dinuclear species were eluted with 0.6 M NaCl solution, except for the one case of AA-[{Ru(phen) 2

}

2 (t-bbN 2 )]Cl 4 , which was C:\NRPonb\DCC\DAR\4O61895_ LDOC-2II2/2011 - 47 obtained by using 1.0 M NaCl solution. Solid KPF 6 was added to the eluates, followed by extraction into DCM. The organic layer was washed with water, dried over anhydrous Na 2

SO

4 , and evaporated to dryness in vacuo to yield a bright red-orange precipitate, AA [{Ru(phen) 2

}

2 (p-bbX,)](PF 6

)

4 . The complexes were further purified by dissolving them in 5 minimum amount of acetone, loading onto Sephadex LH-20 and then eluting with acetone. The solvent was removed to dryness and the resultant bright orange complexes then converted to their chloride salts by stirring them to in aqueous solution using Amberlite* IRA-400 (chloride form) anion-exchange resin. The resin was removed by filtration, and the filtrate was freeze-dried to afford a fluffy bright orange AA-[{Ru(phen) 2

}

2 (pi-bbX")]Cl 4 10 in 20 - 25% yield. 'H NMR (aromatic regions) and CD spectral data of these complexes are consistent with previously reported AA-[{Ru(phen) 2

}

2 (p-bbn)]complexes. AA-[{Ru(phen) 2

}

2 (p-bbH 2

N

7

)](PF

6

)

6 -4H 2 0-acetone. Anal. Found C, 39.8; H, 3.65: N, 8.5%. Calcd. for C 7 8

H

78

N

14

F

3 6 0 5

P

6 Ru 2 : C, 39.6; H, 3.33; N, 8.3%. 'H NMR (300 MHz,

CD

3 CN) : d 8.73 - 8.55 (12H, m, H2, H9, bipy6); 8.28 (8H, d, J = 3.0 Hz, H4, H7); 8.24 15 (4H, m, bipy3); 7.92 (4H, m, H5,H6) 7.69 - 7.53 (8H, m, H3,H8); 7.30 (2H, d, J = 4.3 Hz, bipy5'); 7.18 (2H, d, J = 6.0 Hz, bipy5); 4.14 (4H, s, 2 x CH 2 bipy); 3.02 (4H, s, 2 x

CH

2 N); 2.55 (6H, s, 2 x CH 3 bipy); 1.88 (2H, m, CH 2 ). CD {2/nm (A/cm-'M-), Cl~ salt in

H

2 0} 468 (-23), 420 (21), 286.5 (-188), 268.5 (-246), 259.5 (294), 216.5 (58). AA-[{Ru(phen) 2

}

2 (p-bbH 4

NI

2

)](PF

6

)

8 -2H 2 0. Anal. Found C, 35.8; H, 2.85: N, 7.8%. 20 Calcd. for C 7 8

H

7 8

N

16

F

4 8

O

2

P

8 Ru 2 : C, 35.6; H, 2.99; N, 8.5%. 'H NMR (300 MHz, CD 3 CN): d 8.70 - 8.56 (12H, m, H2, H9, bipy6); 8.28 (8H, d, J = 3.0, H4, H7); 7.92 (4H, m, H5,H6); 7.83 (4H, m, H5,H6); 7.65 - 7.52 (8H, m, H3,H8); 7.31 (2H, d, J = 5.0 Hz, bipy6'); 7.15 (2H, d, J = 5.0 Hz, bipy6); 4.07 (4H, s, 2 x CH 2 bipy). 3.10 (12H, s, 6 x

CH

2 N); 2.53 (6H, s, 2 x CH 3 bipy). CD {/nm (AE/cm~'M'), Cl' salt in H 2 0} 467.5 (-23), 25 417.5 (21), 288 (-194), 268.5 (-253), 259.5 (292), 219.5 (55). AA-[{Ru(phen)2}2(p-bbO 7

)](PF

6

)

4 -2H 2 0. Anal. Found C, 46.1; H, 3.11: N, 8.1%. Calc. for C 76

H

66 N1 2

F

2 4 0 3

P

4 Ru 2 : C, 46.2; H, 3.36; N, 8.5%. 'H NMR (300 MHz, CD 3 CN) : d 8.68 (4H, dd, J = 8.0, 1.0 Hz, H2, H9); 8.58 (4H, dt, J = 9.0 Hz, H2, H9); 8.43 (4H, d, J = 8.0 Hz, bipy6); 8.28 (8H, m, H4, H7); 8.23 (4H, m, bipy3); 7.92 (4H, m, H5,H6); 7.83 30 (4H, m, H5,H6); 7.61 - 7.51 (8H, m, H3,H8); 7.15 (4H, d, J= 6.0 Hz, bipy5); 3.48 (4H, t, 2 x CH 2 0); 2.86 (4H, t, 2 x CH 2 bipy); 2.53 (6H, s, 2 x CH 3 bipy); 1.96 (4H, m, 2 x CH 2

).

C:\P orb CC \AR\461895_1.DOC-21/12/2011 -48 CD {X/nm (AE/cm~'M-1), Cl~ salt in H 2 0} 469.5 (-20), 420 (19), 288 (-212), 268.5 (-21 1), 259.5 (262), 216.5 (53). AA-[{Ru(phen) 2

}

2 (p-bbOlo)](PF 6

)

4

H

2 0. Anal. Found C, 46.7; H, 3.22: N, 8.0%. Calcd. for C 78

H

6 8

N,

2

F

24 0 3

P

4 Ru 2 : C, 46.8; H, 3.42; N, 8.4%. 'H NMR (300 MHz, CD 3 CN): d 8.70 5 (4H, d, J = 7.0 Hz, H2, H9); 8.58 (4H, d, J = 9.0 Hz, H2, H9); 8.44 (4H, d, J = 10.3 Hz, bipy6); 8.29 (8H, m, H4, H7); 8.24 (4H, m, bipy3); 7.92 (4H, m, H5,H6); 7.83 (4H, m, H5,H6); 7.61 - 7.51 (8H, m, H3,H8); 7.15 (4H, d, J= 5.0 Hz, bipy5); 3.53 - 3.50 (8H, m, 4 x CH 2 0); 2.87 (4H, t, CH 2 bipy); 2.55 (6H, s, 2 x CH 3 bipy); 1.97 (4H, m, 2 x CH 2 ). CD {X/nm (A/cm-'M-1), Cl~ salt in H 2 0} 468.5 (-29), 416 (27), 286.5 (-286), 268.5 (-297), 10 259.5 (300), 217 (71). AA-[{Ru(phen) 2 }2(p-bbOi 3

)](PF

6

)

4 g2H 2 0. Anal. Found C, 46.7; H, 3.51: N, 7.8%. Calcd. for C 8 oH 74 N1 2

F

24 0 5

P

4 Ru 2 : C, 46.5; H, 3.61; N, 8.1% 'H NMR (300 MHz, CD 3 CN) : d 8.61 (4H, d, J= 8.0 Hz, H2, H9); 8.58 (4H, d, J= 8.0 Hz, H2, H9); 8.44 (4H, d, J= 10.3 Hz, bipy6); 8.29 (8H, m, H4, H7); 8.24 (4H, m, bipy3); 7.92 (4H, m, H5,H6); 7.83 (4H, m, 15 H5,H6); 7.61 - 7.51 (8H, m, H3,H8); 7.15 (4H, d, J= 5.0 Hz, bipy5); 3.53 - 3.48 (12H, s, 6 x CH 2 0); 2.85 (4H, t, CH 2 bipy); 2.54 (6H, s, 2 x CH 3 bipy); 1.96 (4H, m, 2 x CH 2 ); CD {X/nm (AE/cm~'M-'), Cl~ salt in H 2 0} 469 (-26), 420 (23), 286.5 (-254), 269 (-276), 259.5 (300), 217 (67). AA-[{Ru(phen) 2

}

2 (g-bbOI 6

)](PF

6

)

4

'H

2 0. Anal. Found C, 47.2; H, 3.48: N, 7.6%. Calcd. 20 for C 82

H

76 N1 2

F

24 0 5

P

4 Ru 2 : C, 47.1; H, 3.66; N, 8.0% 'H NMR (300 MHz, CD 3 CN) : d 8.68 (4H, d, J = 8.0 Hz, H2, H9); 8.58 (4H, d, J = 8.0 Hz, H2, H9); 8.44 (4H, d, J = 10.3 Hz, bipy6); 8.29 (8H, m, H4, H7); 8.24 (4H, m, bipy3); 7.92 (4H, m, H5,H6); 7.83 (4H, m, H5,H6); 7.61 - 7.51 (8H, m, H3,H8); 7.15 (4H, d, J= 5.0 Hz, bipy5); 3.53 - 3.48 (16H, m, 8 x CH 2 O); 2.87 (4H, t, CH 2 bipy); 2.55 (6H, s, 2 x CH 3 bipy); 1.98 (4H, m, 2 x CH 2 ); CD 25 {/nm (AE/cm~'M-1), Cl~ salt in H 2 0} 470.5 (-27), 415.5 (25), 28.5 (-273), 269 (-294), 260.5 (300), 216.5 (67). The synthesis of the above dinuclear (poly)oxy and polyamino complexes generally produced mononuclear species as side products. In the cation exchange chromatography 30 purification procedure, the mononuclear A-[Ru(phen) 2 (bbN 7

)]

2 + species was well separated from the dinuclear analogue AA-[{Ru(phen) 2

}

2 (p-bbH 2

N

7

)]

6 +, although the microanalysis CANRPortb\DCC\DAR\4061895_ .DOC-21/12/2011 - 49 of the isolated mononuclear product suggested that it also underwent protonation to form a higher charged complex A-[Ru(phen) 2 (bbH 4

N

7

)]

6 : N N 'N ftARu(phen(py,) 2 . RubbHNymon RubbH2N, 5 Synthesis of mononuclear complexes The mononuclear complex A-[Ru(phen) 2 (bbH 4

N

7

)]

6 + was obtained as the side product from the purification of dinuclear AA-[{Ru(phen) 2

}

2 (pt-bbH 2

N

7 )]6* using the above procedure. The syntheses of mononuclear A-[{Ru(phen) 2 (Me 2 bipy)] 2 + and A [Ru(phen) 2 (bb 7

)]

2 + were also carried out according to the above procedure using excess 10 4,4'-dimethyl-2,2'-bipyridine and bb 7 , while the synthesis of mononuclear A [Ru(phen) 2 (bbi 6 )]C1 2 was modified as follows. A solution of A-[Ru(phen) 2 (py) 2

]{(-)

AsOtart} 2 (180 mg, 0.169 mmol) in ethylene glycol/water (9 : 1; 20 ml) was added dropwise to a hot solution of bb 16 (0.676 mmol) in 2-methoxyethanol (50 ml) over a period of 4 h in the dark under Ar at 115 *C. Stirring was continued for further 2 h, after which 15 the solution was cooled to room temperature and the unreacted bb 16 was removed by filtration. Water (10 ml) was added to the filtrate and loaded onto an SP-Sephadex C-25 cation exchange column. Washing with water and elution with 0.6 M NaCl solution removed the impurities. 20 The bright orange product was eluted with a 1 M NaCl solution containing 40% acetone. Solid KPF 6 was added to the eluate and the complex extracted into DCM. The organic layer was washed with water, dried over anhydrous Na 2

SO

4 , and evaporated to dryness in vacuo to yield a bright red-orange mixture of mononuclear and dinuclear species. The mixture was dissolved in minimal acetone and a saturated solution of tetraethylammonium 25 chloride in acetone was added dropwise until no more precipitation occurred. The dinuclear species precipitated out as its chloride salt, while the mononuclear complex C:\NPob\DCCDAR"6I895_LDOC-21/I2/2h1 - 50 remained in solution. Both the precipitate and the solution were loaded onto a Sephadex LH-20 column and washed with acetone, resulting in the elution of the PF 6 ~ form of the mononuclear species, 5 while the chloride salt of the dinuclear complex was retained in the column. The dinuclear complex was finally eluted with methanol. Due to low solubility, the conversion of the PF6~ salt of the mononuclear A-[Ru(phen) 2 (bbi 6

)](PF

6

)

2 into A-[Ru(phen) 2 (bb6)] Cl 2 using Amberlite IRA-400 anion-exchange resin required high dilution and prolonged stirring. A-IRu(phen) 2 (Me 2 bipy)](PF 6

)

2

-H

2 0. Anal.Found C, 45.4; H, 2.70: N, 8.5%. Calcd. for 10 C 36

H

3 0

N

6 Fl 2

OP

2 Ru: C, 45.3; H, 3.17; N, 8.8% 'H NMR (300 MHz, CD 3 CN) : d 8.68 (2H, d, J= 8.0 Hz, H2,H9); 8.57 (2H, d, J= 8.0 Hz, H2, H9); 8.42 (2H, s, bipy6); 8.28 (4H, d, J = 4.0 Hz, H4,H7); 8.25 (2H, m, bipy3); 7.93 - 7.81 (4H, m, H5, H6); 7.61 - 7.50 (4H, m, H3, H8); 7.15 (2H, d = 5.0 Hz, bipy5); 2.55 (6H, s, 2 x CH 3 bipy). CD {X/nm (AE/cm~'M''), Cl- salt in H 2 0} 468 (-14), 418 (14), 285 (-147), 268.5 (-187), 260 (208), 218 (27). 15 A-[Ru(phen)2(bbH 4

N

7

)](PF

6

)

6 -3CH 2

C

2 . Anal. Found C, 32.1; H, 2.51: N, 7.1%. Calc. for

C

54

H

56 NioC1 6

F

36

P

6 Ru: C, 32.0; H, 2.78; N, 6.9% 'H NMR, (300 MHz, CD 3 CN) d 8.69 (2H, dd, J= 8.5, 1.0Hz, H2,H9); 8.58 (2H, dd, J= 8.0, 1.0Hz, H2,H9); 8.47 (4H, s, bipy6); 8.28 (4H, d, J= 4.0Hz, H4,H7); 8.23 (4H,m, bipy3); 7.94 - 7.81 (4H, m, H5,H6); 7.62 7.52 (4H, m, H3, H8); 7.23 (2H, dd, J= 5.5, 1.0Hz, bipy5'); 7.16 (2H, d, J= 5.5, bipy5); 20 3.94 (4H, s, 2 x CH 2 bipy); 3.16 (2H, t,NCH 2 ); 2.85 (2H, t,NCH 2 ); 2.57 (6H, s, 2 x

CH

3 bipy);1.80 (2H, m,CH 2 ). CD{X/nm (AS/cm~'M~'), Cl~ salt in H 2 0} 470 (-22), 421.5 (24), 286.5 (-208), 268 (-273), 260 (330), 217 (57). A-[Ru(phen) 2 (bb 7

)](PF

6

)

2

-H

2 0. Anal. Found C, 52.6; H, 4.01: N, 8.9%. Calc. for

C

53

H

5 oN 8 Fi 2

OP

2 Ru: C, 52.8; H, 4.18; N, 9.3% 'H NMR, (300 MHz, CD 3 CN) d 8.66 (2H, 25 d, J = 8.0 Hz, H2,H9); 8.58 - 8.48 (2H, m, H2,H9); 8.42 (2H, s, bipy6'); 8.38 (2H, s, bipy6), 8.27 (4H, m, H4,H7); 8.22 (4H, m, bipy3), 7.92 - 7.77 (4H, m, HS,H6); 7.60 - 7.49 (4H, m, H3,H8); 7.24 (2H, s, bipy5); 7.14 (2H, dd, J= 6.0, 1.0 Hz, bipy5); 2.81 (2H, t,

CH

2 bipy); 2.73 (2H, t, CH 2 bipy); 2.55 (3H, s, CH 3 bipy); 2.46 (3H, s, CH 3 bipy); 1.69 1.38 (10H, m, 5 x CH 2 ). CD {l/nm (AE/cm~'M- 1 ), Cl~ salt in H 2 0} 469.5 (-17), 421 (15), 30 285.5 (-152), 269 (-177), 260 (195), 218 (46). A-[Ru(phen) 2 (bbid)J(PFd) 2 and A-[Ru(phen) 2 (bbd)j(PF,2 C:\NRPortbl\DCC\DAR\406]895_.DOC-2 1/12/2011 -51 Solid bb1 2 (0.26 mmol) was heated at 120 *C in 2-methoxyethanol (30 ml) on a two neck round bottom flask under Argon. A solution of A-[Ru(phen) 2 (py) 2 ](-)AsOTart (0.09 mmol) in ethylene glycol/water (9:1, 20 ml) was added dropwise for five hours and the mixture was further heated for two hours. After cooling, water (10 ml) was added and the 5 mixture was loaded onto Sephadex C-25 cation exchange column, eluted with water and then 0.3 M NaCl to remove the impurities. Elution with 1.0 M NaCl containing 5% acetone gave the pure A-[Ru(phen) 2 (bb1 2

)]

2 +. In the case of A-[Ru(phen) 2 (bbi 6

)]

2 +, the complex was obtained using 1.0 M NaCl containing 10% acetone. After acetone removal, excess KPF 6 was added causing the precipitation of the PF 6 ~ salt of the complex which was 10 then extracted into dichloromethane followed by evaporation to dryness to give the corresponding A-[Ru(phen) 2 (bb,)](PF 6

)

2 . Typical yield: 61 - 64%. A-[Ru(phen) 2 (bbj2)](PF 6

)

2 'H NMR, (300 MHz, CD 2 Cl 2 ) 8 8.61-8.50 (m); 8.31-8.18 (m); 7.92-7.88 (m); 7.65 (t), 7.51 (t); 7.30 (d), 7.18 (d); 2.85 - 2.79 (m); 2.59 (s); 2.53 (s); 1.71 1.67 (m); 1.39-1.24 (m). A-[Ru(phen) 2 (bbl 6

)](PF

6

)

2 -27H 2 0-acetone. Anal. Found C, 42.1; 15 H, 6.51: N, 5.7%. Calc. for C 65

H,

26

N

8 FI20 28

P

2 Ru: C, 42.0; H, 6.83; N, 6.0%. 'H NMR (300 MHz, CD 3 CN) d 8.66 (2H, d, J = 8.4 Hz, H2,H9); 8.56 (2H, dd, J = 8.0 Hz, H2, H9); 8.43 (2H, s, bipy6'); 8.39 (2H, s, bipy6); 8.26 (4H, dd, J= 3.0, 1.0 Hz, H4,H7); 8.23 (4H, m, bipy3); 7.91 (2H, m, H5,H6); 7.82 (2H, m, H5,H6); 7.60 - 7.50 (4H, m, H3,H8); 7.24 (2H, s, bipy5); 7.14 (2H, d, J= 5.0 Hz); 2.80 (2H, t, CH 2 bipy); 2.74 (2H, t, CH 2 bipy); 2.55 20 (3H, s, CH 3 bipy); 2.44 (3H, s, CH 3 bipy); 1.69 - 1.22 (28H, m, 24 x CH 2 ). CD{X/nm (A/cmIM-1), Cl- salt in H 2 0} 471 (-4), 421.5 (4), 286 (-36), 269.5 (-40), 259.5 (45), 215.5 (6). The successful isolation of the mononuclear complexes A-[Ru(phen) 2 (bbn)] 2 + (referred to 25 as "Rubbmono") enabled the synthesis of higher nuclearity complexes due to the availability of the free 2,2'-bipyridine coordination site that can undergo a further reaction with other species in a complex-as-ligand strategy. For example, its reaction with [Ru(phen)C1 4 ]~ afforded the important precursor A-[Ru*(phen) 2 (pt-bb 7 )Ru(phen)C 2

]

2 + {referred to as "Rubb 7 dichloro"; only the Ru centre (asterisked) originating f rom the 30 mononuclear complex containing bb 7 is chiral}, which reacted with another A [Ru(phen) 2 (bb 7

)]

2 + to produce the trinuclear complex C:\NRPonbl\DCC\DARND61893_I DOC-211V2011 - 52 [{Ru*(phen) 2 }(p.-bb 7 ){Ru(phen)} (p.-bb 7 ) {Ru*(phen) 2 } ]6 (referred to as "Rubb 7 trinuclear"; only the two terminal Ru centres are chiral). In addition to microanalytical and NMR characterisation of the trinuclear species, a high-resolution electrospray ionisation mass spectrum of Ru 3 [Ci1sHio 4 Ns](PF 6

)

6 gave +2, +3, +4 and +5 5 ions corresponding to five successive losses of PF6- ions. Reaction of this same precursor with bb 7 in 2:1 ratio gave the tetranuclear complex [{Ru*(phen) 2 }(pt-bb 7 ){Ru(phen)}-(p-bb 7 ){Ru(phen)}( t-bb 7 ){Ru*(phen) 2 } ]8+ (referred to as "Rubb 7 tetranuclear"; only the two terminal Ru centres are chiral) as shown below: 2+ Rubbmono [Ru(phen)CI) - C 1 2+ N/ 'N Ru Ru.N - Rubb rchioro Rubb ono b. (2:1 ratio) 1 ~ ~ N .

1 - .

..N.N , N 10 Rubbrlnuclear Rubb,ttiranuclear The replacement of the chloro ligands by the 2,2'-bpy moieties was carried out in ethanol water solution under reflux, as reported in T. Tagano, Inorg. Chim. A cta, 1992, 195, 221 225. Characterisation was achieved by microanalysis and NMR methods: despite 15 extensive attempts, the ESI-FTMS determination was unsuccessful for the parent tetranuclear ion, consistent with a sensitivity observed even for the trinuclear species. Circular dichroism spectra of the trinuclear and tetranuclear complexes were consistent with those for the mononuclear and dinuclear complexes with D chiral metal centres, 20 suggesting that the stereochemistry in both terminal "Ru(phen) 2 (bpy)" moieties is maintained. It should be noted that the central "Ru(phen)(bpy) 2 " entities may adopt more C:\NRPTb\DCCDAR\461895 I.DOC-21112/2011 - 53 than one geometric isomeric form, due to the relative orientations of the two Me-bpy and x-CH 2 -bpy sites. These stereochemical differences can be observed from the 'H NMR spectra of both complexes, which show the presence of the multiplet methylene bpy-CH 2 protons (2.82 ppm), in contrast to a typical triplet observed in the (symmetrical) dinuclear 5 complexes. In addition, the presence of the three closely-positioned bpy-CH 3 protons at 2.50, 2.55 and 2.60 ppm further attests to the isomeric complexity. 10 Synthesis of dichloro "precursor complex" Rubbzdichloro, ^*-{{Ru*(phen)(u bb7)[Ru(phen)ClW7(PFsh (only the Ru centre designated by an asterisk is resolved (A)) The synthesis of the complex was adapted from K. Hara, H. Sugihara, L. P. Singh, A. Islam, R. Katoh, M. Yanagida, K. Sayama, S. Murata and H. Arakawa, J. Photochem. Photobiol., A, 2001, 145, 117-122. A typical procedure was as follows. A mixture of A 15 [Ru(phen) 2 (bb 7

)](PF

6

)

2 (80 mg, 0.05 mmol), (phenH+)[Ru(phen)Cl 4 ] (40 mg, 0.05 mmol) and lithium chloride (50 mg) in dry DMF (5 ml) was heated to reflux at 150 'C for 8 h in the dark under Ar. Acetone (20 ml) was added to the resulting dark brown solution, causing the precipitation of a dark brown material, which was kept with the mother liquor in the fridge for 12 h. The precipitate was then filtered and washed with acetone and 20 redissolved in ethanol. Solid NH 4

PF

6 (50 mg) was added to the solution, resulting in the precipitation of the PF6~ salt of the complex, which was then filtered and washed with ethanol and diethyl ether to afford a dark brown solid. Purification of this product was performed on Sephadex LH-20 using acetone as the eluent. The major first brown band containing A-[Ru 2 (phen) 3 (p.-bb 7 )Cl 2

](PF

6

)

2 was collected, and the product isolated in 20 25 30% yield. A separation of any possible isomeric products was not attempted. A-[{Ru*(phen) 2 (p-bb 7 ){Ru(phen)CI 2

)](PF

6

)

2 -3H 2 0. Anal. Found C, 49.0; H, 3.75: N, 8.5%. Calc. for C 6 5

H

6 2 NIOFi20 3

P

2 Ru 2 : C, 49.0; H, 3.92; N, 8.8%. 'H NMR (300 MHz,

CD

2 Cl 2 ) d 8.65 - 8.58 (3H, m, H2,H9); 8.50 (6H, dd, J= 8.0, 1.0, H2, H9); 8.36 (4H, m, bipy6); 8.25 - 8.17 (10H, m, H4,H7,bipy3); 7.90 - 7.86 (6H, m, H5,H6); 7.64 (3H, m, 30 H3,H8); 7.49 (3H, m, H3,H8); 7.16 (4H, m, bipy5); 2.73 (4H, m, CH 2 bipy); 2.58 (6H, s, 2 x CH 3 bipy); 1.68 - 1.40 (10H, m, 5 x CH 2 ). CD{X/nm (AE/cm-'M~), Cl~ salt in H 2 0} 468 C:\NRPorbl\DCCDAR\46I95_1 DOC-21/12/20lI - 54 (-11), 420 (12), 286.5 (-112), 268.5 (-136), 259 (160), 216.5 (31). Synthesis of trinuclear complex Rubb 7 trinuclear, A *A *-Ru *(phen) (p bb 7 )Ru(phen)(u-bbz)(Ru*(phen))(PF6)6 [only the Ru centres designated by an asterisk 5 are resolved (A) A mixture of A-[Ru 2 (phen) 3 (pt-bb 7

)C

2

](PF

6

)

2 (40 mg, 0.025 mmol) and A [Ru(phen) 2 (bb 7

)](PF

6

)

2 (40 mg, 0,025 mmol) in ethanol-water (1 : 1, 20 ml) was refluxed for three hours in the dark under an Ar atmosphere. The solution slowly turned from brown to dark red during the course of the reaction. The resulting solution was cooled to 10 room temperature and the solvent was evaporated under reduced pressure to obtain a dark orange solid, which was redissolved in a minimum amount of acetone and converted to chloride salt in water using Amberlite IRA-400 anion-exchange resin. After filtration, the solution was loaded onto an SP Sephadex C-25 cation exchange column, washed with water and eluted with 0.3 M NaCl solution to remove mononuclear impurities. The 15 desired trinuclear species was eluted with 10% acetone in 1 M NaCl solution. After removing the acetone, solid KPF 6 was added followed by extraction into dichloromethane. The organic layer was washed with water, dried over anhydrous Na 2

SO

4 , and evaporated to dryness to yield a bright red-orange precipitate, AA -[(Ru 3 (phen) 5 (I-bb) 2

](PF

6

)

6 . The complex was further purified on Sephadex LH-20 using acetone as the eluent. Typical 20 yield 40-50%. A separation of any possible isomeric products was not attempted. A*A*-[{Ru*(phen) 2 }(p-bb 7 ){Ru(phen)}(p-bb 7 ){Ru*(phen) 2 1-(PF 6

)

6 -2H 2 0-2acetone. Anal: Found C, 48.0; H, 3.75: N, 8.0%. Calcd. for C1 24

HI

2 aNI 8

F

36 0 4

P

6 Ru 3 : C, 48.0; H, 3.90; N, 8.1%. 'H NMR (300 MHz, CD 3 CN) d 8.69 - 8.56 (1OH, m, H2,H9); 8.44 - 8.31 (8H, m, bipy6); 8.27 (10H, m, H4,H7); 8.23 (8H, m, bipy3); 7.92 (5H, m, H5,H6); 7.86 25 7.68 (5H,m,H5,H6); 7.61 - 7.51 (10H, m, H3,H8); 7.34 (4H, m, bipy5'); 7.12 (4H, m, bipy5); 2.82 (8H, m, CH 2 bipy; 2.60 (3H, s, CH 3 bipy); 2.55 (6H, s, 2 x CH 3 bipy); 2.50 (3H, s, CH 3 bipy); 1.72 - 1.38 (20H, m, 10 x CH 2 ). CD{)/nm (As/cm~'M~ 1 ), Cl~ salt in H 2 0} 470 (-22), 419.5 (21), 286.5 (-215), 270 (-235), 258.5 (280), 217.5 (69). FTMS (+ESI): most abundant ion m/z 837.4985 ([M-3PF 6 13*) calc. for Ru 3

[C

1 8 Hi 04

NI

8

](PF

6 )33*, 837.4929; 30 most abundant ion m/z 1328.7241 ([M-2PF 6

]

2 +) calc. for Ru 3 [CIisHio 4 Ni 8

](PF

6

)

4 2+' 1328.7218.

C:NRPonbflCC\DARw061895_I.DOC-21/12nOI1 -55 Synthesis of tetranuclear complex Rubb7tetranuclear, A*A*-({Ru*(phen)2j(p bb 7 ){Ru(phen)) (p-bb 7 ){Ru(phen)|(py-bb 7 )-[Ru*(phen)2 |(PF 2 8 [only the Ru centres designated by an asterisk are resolved (A)) 5 A mixture of A-[Ru 2 (phen) 3 (pi-bb 7 )(Cl) 2

](PF

6

)

2 (40 mg, 0.025 mmol) and bb 7 (5 mg, 0.013 mmol) was heated to reflux in ethanol-water (1 : 1, 20 ml) for 3 h in the dark under Ar atmosphere. The solution slowly turned from brown to dark red during the course of the reaction. The resulting solution was cooled to room temperature and the solvent was evaporated under reduced pressure to obtain a dark orange solid, which was redissolved in 10 acetone (2 ml) followed by a dropwise addition of a saturated solution of tetraethylammonium chloride, causing the precipitation of its chloride salt. After the acetone removal, the solid was dissolved in water (5 ml) and loaded onto an SP-Sephadex C-25 cation exchange column, washed with water and eluted with 0.3 M NaCl solution to remove the impurities. The desired tetranuclear species was eluted with a 1 M NaCl 15 solution containing 10% acetone. After removing the acetone, solid KPF 6 was added followed by extraction into DCM. The organic layer was washed with water, dried over anhydrous Na 2

SO

4 , and evaporated to dryness to yield a bright red-orange precipitate, AA [(Ru 4 (phen) 6 (t-bb 7

)

3

](PF

6 )s. The complex was further purified using Sephadex LH-20 with acetone as the eluent. Typical yield 60%. A separation of any possible isomeric 20 products was not attempted. A*A*-[{Ru*(phen)2}(p-bb 7 ){Ru(phen) 2 }(i-bb 7 ){Ru(phen) 2 }(pi-bb 7 ) {Ru*(phen) 2

}J(PF

6 ).8H 2 0 Anal. Found C, 48.1; H, 3.63: N, 8.3%. Calcd. for Ci 5 9

HI

4 6

N

24

F

4 8

OP

8 Ru 4 : C, 48.1; H, 3.70; N, 8.5%. 'H NMR (300 MHz, CD 3 CN) d 8.70 8.57 (12H, m, H2,H9); 8.45 - 8.35 (12H, m, bipy6); 8.27 (12H, m, H4,H7); 8.25 (12H, m, 25 bipy3); 7.92 (6H, m, H5,H6); 7.85 - 7.64 (6H, m, H5,H6); 7.60 - 7.52 (12H, m, H3,H8); 7.34 (6H, m, bipy5'); 7.12 (6H, m, bipy5); 2.82 (8H,m, CH 2 bipy); 2.60 (3H, s,CH 3 bipy); 2.56 (12H, s, CH 3 bipy); 2.51 (3H, s, CH 3 bipy); 1.70 - 1.41 (30H, m, 15 x CH 2 ). CD{/nm (As/cm-'M'), C~ salt in H 2 0} 470.5 (-26), 417.5 (25), 289 (-255), 268 (-266), 258 (293), 218 (58). 30 Representative synthesis of ({Ru(terpy)Clb(p-bb)/' (n=7, 10, 12 and 14) C:\NRPotnb\CCDAR\4061K95_ DOC-21/12/2011 -56 2+ Me Me N N I N N N _N Ru(II) Ru(I1) N N N 1-1111N; CI CI .2 Cl

(CH

2 )n As used herein these complexes are also referred to by shorthand notation. For example [{Ru(terpy)Clh(g-bb7)1 is referred to as Rubb 7 -Cl. 5 Solid [Ru(terpy)C1 3 ] (0.20 g, 0.45 mmol) and bb, (0.23 mmol) were refluxed in EtOH/H 2 0 (4:1; 40 ml) for 4 h. After cooling, the solvent mixture was evaporated to approximately half of the original volume and excess NH 4

PF

6 was added causing the precipitation of the dark brown-purple material, which was filtered and washed with cold ethanol followed by 10 diethyl ether. The crude product was dissolved in acetone and loaded onto a Sephadex LH 20 exclusion column, and separated using acetone as the eluent. The pure dinuclear [{Ru(terpy)(Cl)} 2 (p-bb.)](PF 6

)

2 complexes were isolated as dark purple materials. The chloride salts were obtained by stirring the solid in an aqueous solution using Amberlite* IRA-400 (chloride form) anion-exchange resin. The resin was removed by filtration, and 15 the dark purple solution was freeze-dried to obtain a fluffy dark red purple powder of [{Ru(terpy)(Cl)} 2 (t-bb.)]Cl 2 . Typical yields after conversion: 30-60%. Separation of any possible geometric isomers of [{Ru(terpy)(Cl)} 2 (p-bbn)] 2 + was not attempted. [{Ru(terpy)(Cl)} 2 (p-bb 7 )](PF)2.3H20: Anal. Found C, 46.8; H, 3.63: N, 8.7%. Calcd. for Cs 9

H

6 oNioFI 2

O

3

P

2 Ru 2 : C, 46.6; H, 3.98; N, 9.2% 'H NMR (300 MHz, CD 3 CN) d 10.04 (s); 20 8.61 (s); 8.49 (m); 8.40 (d, J = 7.2 Hz); 8.31 (d, J = 7.2 Hz); 8.20 (d, J = 11.7 Hz); 8.10 (t); 7.90 (t); 7.81 (s); 7.70 (s); 7.55 (s); 7.30 (s); 7.12 (t); 6.81 (d, 5.1 Hz); 3.04 (t); 2.84 (t); 2.78 (s); 2.77-2.55 (m); 1.76 (s); 1.53 (s); 1.37-1.22 (m). [{Ru(terpy)(Cl)} 2 (p-bblo)](PF 6

)

2 .3acetone: Anal. Found C, 50.7; H, 4.49: N, 8.4%. Calcd. for C 7 1

H

7 8 NioF 2

O

3

P

2 Ru 2 : C, 50.7; H, 4.67; N, 8.3% 'H NMR(300 MHz,CD 3 CN) d 25 10.04 (s); 8.64 (s); 8.51 (d, J = 8.1 Hz); 8.40 (d, J = 7.8 Hz); 8.33 (m); 8.22 (m); 8.10 (t); 7.91 (t); 7.81 (s); 7.71 (s); 7.60 (s); 7.30 (s); 7.12 (t); 6.80 (s); 3.04 (t); 2.85 (t); 2.78 (s); C:\NRPotbl\DCC\DARW61895_I DOC-21/12/2011 -57 2.60 (s); 1.75 (s); 1.50 (s); 1.41-1.22 (m). [{Ru(terpy)(Cl)} 2 (p-bbl 2

)](PF

6

)

2 .5acetone: Anal. Found C, 52.5; H, 4.69: N, 8.5%. Calcd. For C 79

H

94 NoFI20 5

P

2 Ru 2 : C, 52.0; H, 5.19; N, 7.8% 'H NMR (300 MHz, CD 3 CN) d 10.04 (s); 8.65 (s); 8.50 (m); 8.47-8.34 (m); 8.20 (d, J = 12.0 Hz); 8.09 (t); 7.90 (t); 7.82 5 (s); 7.71 (s); 7.30 (s); 7.12 (t); 6.81 (d, 5.7 Hz); 3.04 (t); 2.89 (t); 2.78 (s); 2.63-2.55 (m); 1.76 (s); 1.53 (s); 1.37-1.22 (m). [{Ru(terpy)(C)}2(p-bb,4)](PF 6 )2.3acetone: Anal. Found C,52.1; H, 4.88: N, 8.5%. Calcd. for C 7 5

H

8 6 NiaF 1 2 0 3

P

2 Ru 2 : C, 51.8; H, 4.99; N, 8.1% 'H NMR(300 MHz, CD 3 CN) d 10.04 (s); 8.61 (s); 8.49 (m); 8.40 (d, J = 7.2 Hz); 8.31 (d, J = 7.2 Hz); 8.20 (d, J = 11.7 Hz); 8.10 10 (t); 7.90 (t); 7.81 (s); 7.70 (s); 7.55 (s); 7.30 (s); 7.12 (t); 6.81 (d, 5.1 Hz); 3.04 (t); 2.84 (t); 2.78 (s); 2.77-2.55 (m); 1.76 (s); 1.53 (s); 1.37-1.22 (m). Representative synthesis of (Ru(phen)4(p-bb,)Ru(terpv)Cl7Cl3 3+ N M/ Me Me - NN N N N Ru(II) Ru(I1) N N N N I IIC N CI .3 Cl

(CH

2 )n 15 As used herein these complexes are also referred to by shorthand notation. For example [Ru(phen) (p-bb 7 )Ru(terpy)Cl] is referred to as Ru(w-bb)-RuCl. Solid [Ru(terpy)C1 3 ] (0.032 mmol) and A-[Ru(phen) 2 (bbn)](PF 6

)

2 (0.032 mmol) were 20 refluxed in ethanol/water (4:1, 10 ml) for three hours. After cooling, excess amount of

NH

4

PF

6 was added causing the precipitation of a dark brown material which was filtered and washed with ethanol. The brown crude was then loaded onto Sephadex LH20 exclusion chromatography using acetone as the eluent. The [Ru(phen) 2

(P

bbr)Ru(terpy)Cl](PF 6

)

3 fraction was obtained as the major dark brown band which was 25 isolated and evaporated to dryness. The PF6- salt was converted to the chloride by C:\NRorb\DCCDAR\46895_.DOC-21/12/01 I -58 dissolving the solid in the minimum amount of acetone followed by addition of the saturated solution of tetraethylammonium chloride in acetone drop wise while stirring for half an hour. The resulting fluffy precipitates were centrifuged, decanted, washed several times with cold acetone and dried under reduced pressure to afford [Ru(phen) 2

(A

5 bb,)Ru(terpy)Cl](Cl) 3 in 70-80% yield. Separation of any possible geometric isomers was not attempted. [Ru(phen)2(p-bb)Ru(terpy)CII(PF) 3 'H NMR (300 MHz, CD 2 Cl 2 ) 6 10.13 (s); 8.63 (d); 8.53(d); 8.43-8.35 (m); 8.27-8.11 (m), 8.01-7.62 (m); 7.55-7.45 (m), 7.26-7.15 (m); 7.00 (t); 6.78 (t); 3.01 -2.79 (m); 2.64-2.59 (m); 2.38 (s); 1.69-1.26 (m). 10 [Ru(phen)2(pi-bbi 2 )Ru(terpy)CII(PF) 3 'H NMR (300 MHz, CD 2 Cl 2 ) 6 10.22 (s); 8.64 (d); 8.53(d); 8.43-8.35 (m); 8.34-8.09 (m), 8.91-7.87 (m); 7.76-7.65 (m), 7.53-7.49 (m); 7.35 (s); 7.18 (s); 6.89 (s); 6.81 (s); 3.24 (t); 3.08 (t); 2.84 (s); 2.76-2.70 (m); 2.59 (s); 2.55 (s), 1.74-1.30 (m). [Ru(phen)2(p.-bbi 6 )Ru(terpy)C](PF) 3 'H NMR (300 MHz, CD 2 Cl 2 ) 6 10.25 (s); 8.63 15 (d); 8.52 (d); 8.37-8.17 (m); 8.07 (d), 7.91-7.66 (m); 7.51-7.47 (m), 7.32-7.19 (m); 6.99 (d); 6.81 (d); 3.07 (t); 2.86 (t); 2.66-2.59 (m); 2.43 (s); 1.60-1.20 (m). Minimum inhibitory concentration (MIC) determination Note: The bacterial strains used in this study are classified as risk group 2 according to 20 the Australian/New Zealand Standard (AS/NZS 2243.4:2010) and must be manipulated in a PC2 class laboratory. Four strains of bacteria were chosen for the susceptibility tests: S. aureus ATCC 25923; MRSA (a wild clinical strain from the JCU culture collection); E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The bacteria were grown on Mueller Hinton agar (OXOID, cat. 25 No. CM0337) and suspended in growth medium cation-adjusted Mueller Hinton broth (CAMHB, OXOID, cat. No. CM0405). The MIC of each complex was determined in duplicate by standard micro-dilution methodology (British Society for Antimicrobial Chemotherapy Working Party, J. Antimicrob. Chemother., 1991, 27(suppl. D), 22) using gentamicin as the positive control. The ruthenium complexes were dissolved in CAMHB 30 to a stock solution of 512 mg ml-1 and two-fold diluted in the broth in 96-well plates in a final volume of 100 ml in each well. The same volume (100 ml) of bacterial suspension C:NPonbl\DCC\ AR\46195_t DOC-21112201 1 -59 was added in each well making a concentration range of 0.25 mg ml-' to 128 mg ml-' for each complex. The plates were incubated at 37 'C for 24 h. The results are summarised in Table 1 below, using the following abbreviations: Rubpm = AA-[{Ru(phen) 2 }2(pt-2,2'-bipyrimidine)]Cl 4 5 Rudppm = AA-[{Ru(phen) 2

}

2 (p-4,6-bis(2-pyridyl)pyrimidine)]Cl 4 Rubb 7 trinuclear = A*A*-[{Ru*(phen) 2 }(p-bb 7 ){Ru(phen)}(p-bb 7 ){Ru*(phen) 2 }]C1 6 (only the two terminal metal centres are chiral, the two central metal centres are racemic) Rubb 7 tetranuclear = A*A*-[{Ru*(phen) 2 }(p-bb 7 ){Ru(phen)}(p-bb 7 ){Ru(phen)}(p-bb 7

)

{Ru*(phen) 2 }]Cls (only the two terminal metal centres are chiral, the two central 10 metal centres are racemic) The minimum inhibitory concentration (MIC) results for the ruthenium complexes against four bacterial strains shown in Table I demonstrate that some of the ruthenium(II) complexes are highly active against both classes of bacteria, although Gram-positive 15 bacteria appeared to be more susceptible than the Gram-negative counterparts. Of particular note are the low MIC values of the AA/AA enantiomers of Rubbi 2 , Rubb 14 and Rubbi 6 against both S. aureus and the drug-resistant strain MRSA (an MIC of 1 Ig mL' is equivalent to 0.5 pM). Only slight differences in activity were observed between the AA and AA-enantiomers of Rubb 7 , Rubb, 2 , Rubb 4 and Rubbi 6 , suggesting that chiral receptors 20 - such as proteins or nucleic acids - may not be the main intracellular target for these metal complexes. Interestingly, the dinuclear complexes with a short linking chain (bb 2 and bbs), a rigid polycyclic aromatic linking ligand (bpm and dppm) or those containing an ether or amine in the linking ligand, showed very little or no observable activity against any of the bacterial strains. The trinuclear and tetranuclear analogues of AA-Rubb 7 showed 25 slightly better activity than the dinuclear complex. The highly lipophilic mononuclear complexes (as determined by octanol-water partition coefficient - logP values) containing the bb 7 or bbi 6 ligand exhibited intermediate MIC values, although Rubb 7 mono was more active (on a mole basis) against S. aureus than its dinuclear counterpart. 30 The mononuclear complex [Ru(phen) 3 ] 2 exhibited no activity; however, the incorporation of methyl groups on the phenanthroline ligands significantly increased the activity against C -NRPoJthDCC\DAR\40618195_.DOC.21/12/201 - 60 all strains. [Ru(Me 4 phen) 3

]

2 + showed equal activity (on a mole basis) to AA/AA Rubb 12 , Rubb 14 and Rubb 16 against S. aureus, but was less active against the drug-resistant strain MRSA. This indicates that MRSA is resistant to the mononuclear complexes but not the dinuclear complexes. It is possible that the dinuclear complexes could just pass through 5 the cell wall and/or the cytoplasmic membrane more easily, or alternatively the mechanisms of resistance are not effective against the dinuclear complexes.

C:NRPotnb\DCC\DAR\4061155l.DOC-21/12/2011 -61 Table 1: In vitro antimicrobial activity MIC (minimum inhibitory concentration) of ruthenium complexes against four bacterial strains M IC (pg.ml-') Gram-positive Gram-negative Group Compound S aureus MRSA E. coli P.aeruginosa Rigid link Rubpm >128 >128 >128 >128 dinuclear Rudppm >128 >128 >128 >128 Flexible link AA-Rubb 2 >128 >128 >128 dinuclear AA-Rubb 2 >128 >128 >128 >128 AA-Rubb 5 128 128 64 128 AA-Rubb 5 128 128 128 >128 AA-Rubb 7 64 32 16 64 AA-Rubb 7 16 32 16 128 AA-Rubbio 8 8 4 32 AA-Rubbio 4 4 4 64 AA-Rubb 1 2 2 2 2 16 AA-Rubb1 2 1 1 2 16 AA-Rubb 1 4 1 1 2 8 AA-Rubb 14 1 1 4 8 AA-Rubb 16 I 1 4 8 AA-Rubb 16 1 1 4 8 Flexible link AA-RubbN 7 >128 >128 >128 >128 dinuclear with AA-RubbO 7 128 128 128 >128 N, 0 in the AA-RubbN 1 2 128 128 128 >128 chain Mononuclear [Ru(phen) 2 (Me 2 bpy)]* 128 >128 >128 >128 with bb, Rubb 7 mono 4 16 16 32 ligands Rubb 16 mono 16 16 64 64 Mononuclear [Ru(phen) 3

]

2 * >128 >128 >128 >128 with phen or [Ru(Me 2 phen) 3

]

2 + 8 128 128 >128 Menphen [Ru(Me 4 phen) 3

]

2 + 0.5 4 8 32 ligands Tri- and tetra- Rubb 7 trinuclear 4 4 16 2 nuclear Rubb 7 tetranuclear 8 8 32 8 Control Gentamicin 50.0625 16 0.5 0.125 C:\NRPortb1DCCDAR4U061895_ 1.DOC-21/12/201I - 62 It has been shown that some Gram-positive bacteria, such as S. aureus, develop resistance to cationic antibiotics by altering the structure of the teichoic acids in the cell wall in order to decrease the negative charge on the cell surface (A. Peschel, M. Otto, R. W. Jack, H. 5 Kalbacher, G. Jung and F. Gotz, J. Biol. Chem., 1999, 274, 8405). MRSA strains with reduced negatively-charged teichoic acids will exhibit lower electrostatic attraction to cationic antimicrobial molecules on the cellwall and membrane. Consistent with this proposal, the positive control drug used in this study - gentamicin (an aminoglycoside) - is positively charged at neutral pH and it was considerably less active against MRSA than the 10 susceptible ATCC 25923 S. aureus. Similarly, mononuclear ruthenium complexes with a +2 charge may be less effective against MRSA than the susceptible strain. By contrast, the dinuclear Rubbe complexes, which have a higher charge (+4) that is also spread over two metal centres, exhibited equal activity against both strains. It was also noted that the tri and tetranuclear complexes exhibited equal activity against S. aureus and MRSA. The 15 results also suggest that a trinuclear complex based upon either a bb 12 , bb 1 4 or bbi 6 ligand could be even more effective than their dinuclear counterparts. The primary difference between the members of the Rubbn family of complexes is the number of methylene groups in the flexible linking chain. The observed differences in the 20 MIC values of the complexes therefore suggest that the antibacterial activity is correlated with the lipophilicity of the metal complex. Lipophilicity is a significant factor affecting the biological activity of most therapeutic compounds, as it relates to the capacity of the drug to penetrate through the cell membrane. The standard octanol-water partition coefficient (logP) was determined for the Rubbn series (F. Lombardo, B. Faller, M. 25 Shalaeva, I. Tetko and S. Tilton, Methods and Principles in Medicinal Chemistry, 2008, 37, 407) parent model mononuclear species ([Ru(phen) 2 (Me 2 bpy)] 2 +) and the trinuclear and tetranuclear complexes. The partition coefficients (logP) were measured using the shake flask technique: each 30 ruthenium complex (at both 0.05 and 0.1 mM) was dissolved in the water phase (0.2 M Na 2

HPO

4 -NaH 2

PO

4 buffer, pH = 7.4) and an equivalent volume of n-octanol was added.

C:\NRPotb\DCCDAR 16895_I.DOC.2112/2OI 1 - 63 The two phases were mutually saturated by shaking overnight at ambient temperature and then were allowed to separate on standing. The concentration of the metal complex in each phase was determined spectrophotometrically at 1 = 450 nm. The results are summarised in Table 2 below: 5 Table 2: The octanol-water partition coefficient (logP) values Metal complex Charge logP Rubb 2 +4 -3.6 Rubb 5 +4 -3.5 Rubb 7 +4 -3.4 Rubbio +4 -3.3 Rubb 12 +4 -2.7 Rubb 14 +4 -2.3 Rubb 16 +4 -1.9 Rubb 7 trinuclear +6 -1.2 Rubb 7 tetranuclear +8 -1.9 [Ru(phen) 2 (Me 2 bpy)] 2 + +2 -2.9 Rubb 7 monoa +2 -0.7 Rubbi 6 monoa +2 +2.1 a As the Rubb 7 mono and Rubb 1 6 mono complexes contain ionisable bpy-N groups, logD should be used (F. Lombardo, B. Faller, M. Shalaeva, I. Tetko and S. Tilton, Methods and Principles in Medicinal Chemistry, 2008, 37, 407). However the experimental pH 7.4 10 > pKa (5.45), (B. R. James and R. J. P. Williams, J. Chem. Soc., 1961, 2007) and therefore, logD = logP.(B. R. James and R. J. P. Williams, J. Chem. Soc., 1961, 2007) For the dinuclear Rubbn complexes, it is apparent that the activity is related to the lipophilicity as measured by logP. However, it is also noted that the highly lipophilic 15 mononuclear complex Rubb] 6 mono was considerably less active than AA/AA Rubb 14 and Rubb 16 . This observation indicates that the overall complex charge is important as well as the lipophilicity, and further highlights the greater antibacterial potential of the oligonuclear species compared to the mononuclear complexes. The logP values do not C:NPotbDCC AR4061895_1 DOC-21/12/2011 -64 change in a consistent linear manner along the series from Rubb 2 to Rubbi 6 , with AlogP/CH 2 greater for Rubbi 0 -Rubb 16 than for Rubb 2 -Rubbio. Analysis of the dinuclear complexes by NMR spectroscopy indicates that the ruthenium metal centres can fold back upon themselves (intramolecular folding), with the maximum folding occurring for 5 Rubbio. Consequently, from Rubb 2 to Rubbio the increasing octanol solubility arising from the increasing number of methylene groups is partially offset by the intramolecular folding. On the other hand, for Rubb 12 to Rubb 16 the lipophilicity increases with the number of methylene groups and the unfolding of the linking chain. 10 Further MIC assays were conducted against five different bacteria with Rubb" (n = 7, 10, 12, 16), two rigid dinuclear species (bridges bpm and dppm) and Gentamicin - Whole spectrum bacteria MIC test results (Broth: CAMHB). The results of the further MIC assays are shown in Table 3 below: 15 Table 3: MIC assays against five different bacteria Gram-positive Gram-negative S. epidermidis Efaecalis VRE S. typhimurium A. baumanii AA-Rubb 7 1 32 16 >128 64 AA-Rubbio 0.5 8 4 32 64 AA-Rubb 12 0.5 4 2 8 8 AA-Rubb 16 1 2 2 8 8 Rubpm >128 >128 >128 >128 >128 Rudppm >128 >128 >128 >128 >128 Gentamicin <0.125 32 16 2 16 The results shown in table 3 indicate that the dinuclear complexes of the present invention containing flexible linking groups (Q) were as active, or significantly more active, against a number of the bacterial strains tested than the control gentamicin. 20 C \NRPorbl\DCC\DARAU061995_ .DOC-21/12/2 I -65 Further MIC assays and Minimum Bactericidal Concentration (MBC) assays were conducted against Gram-positive and Gram-negative bacterial strains using symmetric and non-symmetric, inert and labile complexes. The results are summarised in Table 4 below: 5 Table 4: MICs of three groups of dinuclear ruthenium complexes (all AA- enantiomers) Broth Gram-positive Gram-negative (CAMHB) S. aureus MRSA E. coli P.aeruginosa MIC MBC MIC MBC MIC MBC MIC MBC Symmetric Rubb 7 16 32 16 32 16 16 64 128 Inert Rubb 12 1 2 1 2 2 2 16 32 dinuclear Rubb 16 1 1 1 2 2 2 8 8 Rubb 7 -Cl 8 8 8 16 8 8 32 64 Symmetric Rubb 12 -Cl 1 1 1 1 2 2 8 16 labile dinuclear Rubb 14 -Cl 1 1 1 2 2 2 32 64 Rubb 16 -Cl 8 8 8 8 8 8 >128 >128 Non- Ru(p-bb 7 )Ru-Cl 8 16 8 32 1 1 16 32 symmetric Ru(p-bbi 2 )Ru-Cl 1 1 1 2 1 1 16 32 dinuclear (one side inert, one Ru(p-bbi)Ru-Cl 2 2 2 2 4 4 64 64 side labile) The results appear to suggest that both the MIC and MBC activities increased against Gram-positive and Gram-negative strains up to an alkylene linker group length of approximately C 1 2 for both the symmetric inert dinuclear ruthenium complexes and the 10 symmetric labile dinuclear ruthenium chloro complexes. The activity trend appeared to be matched for the non-symmetric dinuclear ruthenium chloro complexes against Gram positive strains whereas the activity of the non-symmetric dinuclear ruthenium chloro complexes against Gram-negative strains remained approximately the same for alkylene linker group lengths of C 7

-

12 and became worse as the size increased beyond C 12 . 15 C.NRonbrt\DCC\DAR\4061895_1.DOC-21/12/201I - 66 The results appear to suggest that for Gram-positive strains all the dinuclear complexes were approximately equally effective against the susceptible and the resistant strain. When n=12 the complexes of the three groups showed similar activity, and the complexes with the labile ligands showed better activity than their inert counterparts when n<12 while 5 became less effective when n>12. Without wishing to be bound by theory, this observation might be due to the labile ligand affecting the uptake rate and amount of the complexes and/or the DNA attraction inside the cells weakened the interaction of the main target - the cell membrane. 10 For Gram-negative strains, the three groups of complexes showed quite different patterns but the complexes with n=12 were the most active in every group. Ru(p-bb 7 )Ru-Cl and Ru( t-bb] 2 )Ru-Cl were the most active complexes against E.coli. All the complexes tested are considered bactericidal, having MBC/MIC 5 2. The 15 Ru(t-bbi 2 )Ru-Cl complex showed the best activity against the four bacteria strains among all the compounds tested. The variations of MIC values are possibly related to the lipophilicity of these complexes. Rubb 16 is considerably more lipophilic than Rubb 1 2 (almost one log 10 unit), and the non 20 symmetric complexes are more lipophilic than the inert dinuclear. The symmetric labile complexes are the most lipophilic. It would appear that for Gram-positive, the best degree of lipophilicity occurs with the Rubb 12 symmetric labile complex. Rubb 14 symmetric labile complex appears to be 25 slightly worse than the equivalent Rubb 12 symmetric labile complex. Haemolytic assay While excellent antibacterial activity is typically essential for a potential new drug, the compound should ideally also exhibit low toxicity towards human or animal eukaryotic 30 cells. As the ruthenium complexes are water-soluble and could potentially be distributed throughout the body in the blood, the haemolytic activity was determined for the dinuclear C:WRPortbPDCDAR\4O61895_ IDOC-21/12201 1 -67 complexes that showed the best activity (AA-Rubb 7 , AA-Rubbia, AA-Rubb 12 and AA Rubb 16 ) in the antibacterial assays. Freshly-drawn human blood was collected using lithium heparin vacuum blood collection 5 tubes (Vacutainer, BD Australia). Erythrocytes were separated by centrifugation for 10 min at IOOOg at 10 *C and washed six times with phosphate-buffered saline (PBS) solution and diluted with PBS to a 10% solution. The concentration of erythrocytes was determined by haemocrit centrifugation (Biofuge Haemo, Heraeus instruments). A 1% Triton-X solution was prepared as the positive control. The complexes to be tested were 10 dissolved in PBS to a stock solution of 2048 mg ml' and two-fold diluted in PBS in round bottomed 96-well plates in a final volume of 100 ml in each well. 100 ml red blood cell suspension samples were added to the wells making a concentration range from I mg ml- 1 to 512 pg ml~' for Rubb, (n = 10, 12, 16) and 2 ptg ml~' to 1024 ptg ml~' for Rubb7, and the plates with each complex were respectively incubated for 1, 2, 4, 8 and 24 h at 37 OC. 15 After incubation, the plates were centrifuged at 1 OOg for 4 min. 100 ml of supernatant in each well was transferred to a corresponding well in a new 96-well microtitre plate. The absorbance of each well was measured at 540 nm using a plate reader (Mutiskan Ascent, Thermo). Each complex was analysed in duplicate. The solutions of the metal complex at the same tested concentrations without erythrocytes were measured as a negative control. 20 The extent of haemolysis induced by incubation of the ruthenium complexes for 24 h with freshly-collected human red blood cells is shown in Fig. I and summarised in Table 5 below: 25 30 C:\NRPotb\DCC\ARW61895_1 DOC-21/122011 -68 Table 5: HC 50 values of four Rubb, complexes (AA-Rubb 7 , AA-Rubbio, AA-Rubb 2 and AA-Rubbi 6 ) after 24 h incubation with red blood cells Complex HC 5 o (pM) HC 50 (mg ml-) First indication (>2%) of haemolysis (pg.ml-') AA-Rubb 7 >682 >1024 64 AA-Rubbio 265.6 410 32 AA-Rubb, 2 101.8 160 16 AA-Rubb 16 13.5 22 4

HC

5 o: concentration needed to induce 50% haemolysis. The experimental error is ± 5%. 5 Incubation with AA-Rubb 16 caused the most severe haemolysis and AA-Rubb 7 the least; however, the HC 50 values for all the complexes were significantly higher than the corresponding MIC values. The concentration of the ruthenium complex at the first indication of haemolysis (>2%) was also higher than the MIC values in all cases. Of the complexes tested, AA-Rubb 12 gave the most encouraging results, with excellent 10 antimicrobial activity but low toxicity to human red blood cells, and hence showed the greatest potential for further investigations as an antimicrobial agent. The dose-response curves of AA-Rubb 1 2 and AA-Rubb 16 for different incubation times are shown in Fig. 2. The haemolytic activity of AA-Rubb 16 at 10 ptM was low for 8 h, but noticeably increased 15 over 24 h. By contrast, AA-Rubb 12 at 10 gM essentially exhibited no haemolysis over a 24 h period. Significant haemolysis was only observed after 4 h at high concentrations (>256 jigml~'), while no significant haemolytic activity was observed at all the tested concentrations AA-Rubb 7 and AA-Rubbio over 24 h. In summary, for the haemolytic activity, the Rubba complexes were more concentration-dependent than time-dependent. 20 Cytotoxicity assay The haemolytic assay indicated that the Rubb, dinuclear complexes (especially AA Rubb 12 ) were highly active against bacteria but exhibited low toxicity to human red blood cells. However, red blood cells are a special type of eukaryotic cell that do not contain a C :NRPonbl\CC\DAR\4061895_l.DOC.21/12/2011 -69 cell nucleus and the cell membrane has a unique structure of three layers. Given the demonstrated DNA binding ability of several ruthenium complexes (C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32, 215; B. M. Zeglis, V. C. Pierre and J. K. Barton, Chem. Commun., 2007, 4565; and F. R. Keene, J. A. Smith and J. G. Collins, Coord. 5 Chem. Rev., 2009, 253, 2021) it was believed to be important to investigate the toxicity of the complexes against a nucleated eukaryotic cells. Consequently, the toxicity (IC 50 ) against THP-1 cells (a human acute monocytic leukemia cell line and a good model for nucleated eukaryotic cells) was examined (J. Auwerx, Experientia, 1991, 47, 22). 10 Human monocytic THP-1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in RPMI media (Gibco Labs, Grand Island, NY, USA) and supplemented with 10% fetal bovine serum (FBS) (Gibco Labs, Grand Island, NY, USA), with 20 mM L-glutamine (Sigma Aldrich, Sydney, NSW, Australia), and 12.5 mM HEPES buffer at 37 *C in a humidified atmosphere of 5% CO 2 . 15 Cells were seeded at a density of 5 x 105 per well in a sterile flat bottom 96-well plate, and incubated with the desired drug concentration in duplicate, to a total volume of 200 ml. The concentration range for all the complexes was between 2 mg ml-' and 1024 mg ml"'. The plates containing each complex were respectively incubated for 24 and 48 h at 37 *C with 5% CO 2 . After incubation, the concentration of cells in each well was counted with a 20 haemacytometer and IC 50 values were calculated and the results are summarise in Table 6 below: Table 6: The cytotoxicity of AA-Rubb 7 , AA-Rubbio, AA-Rubb1 2 and AA-Rubbi 6 against the THP-1 cell line 24 h 48 h Ruthenium IC 5 0 (fig mL') IC 5 0 [ptM] ICSO (ptg mL~') IC 50 [IM] complex AA-Rubb 7 400 266 260 173 AA-Rubbio 300 194 160 103 AA-Rubb1 2 135 86 90 57 AA-Rubb 16 78 48 40 25 C:\NPortbl\DCCDAR\406L895_1 DOC-21/12/2011 - 70 The cytotoxicity of the dinuclear complexes against THP-1 cells increased as the length of the linking chain increased, but again all the complexes exhibited IC 50 values greater than their corresponding MICs against susceptible S. aureus and MRSA, as shown in Fig. 3. 5 AA-Rubb 12 displayed the highest HC 50 /MIC and IC 5 0 /MIC ratios, suggesting that it is the most selectively toxic of the Rubb, complexes, and consequently has the greatest potential as an antimicrobial agent. The apparent selectivity of the Rubbn complexes for bacterial cells over human eukaryotic 10 cells might be due to a number of reasons. Firstly, bacterial membranes have a higher proportion of negatively-charged phospholipids, whereas, the phospholipids in eukaryotic membranes are predominantly zwitterionic and uncharged. Furthermore, the cell wall of bacteria commonly contain teichoic acids and lipopolysaccharides which are negatively charged. Consequently, due to favourable electrostatic interactions, cationic drugs are 15 preferentially bound to the outer surface of bacterial cells compared to eukaryotic cells. Alternatively, due to the higher reproduction rate of bacteria compared to human cells, it is possible that the observed selectivity is due to inhibition of the synthesis of important macromolecules, such as DNA, RNA or the cell wall. 20 In vitro antimalarial activity Continuous in vitro cultivation of Plasmodium falciparum strains The P. falciparum laboratory adapted strains utilised in this project (Table 7) were cultured in vitro and routinely maintained in RPMI-1640-LPLF complete medium, which contained low concentrations of para-amino benzoic acid (0.0005 mg/L) and folic acid (0.01 mg/L). 25 Table 7. Plasmodiumfalciparum strains used in this project. Strain Origin Drug Resistance D6 Sierra-Leone, Africa Sensitive to chloroquine and pyrimethamine W2 Indochina Resistant to chloroquine and pyrimethamine The low concentration of folic acid in RPMI-1640-LPLF prevents inhibition of the compound if its activity targets the parasite's folate metabolic pathway. Parasites were C:\NRonbl\DCC\DARW6I895_ LDOC-21/2/2011 -71 cultured in human red blood cells (RBCs) in vitro at 37 *C in special gas mixture (5% 02, 5% CO 2 and 90% N 2 ) as described in Trager and Jensen (1979) Science 193: 673-675. Preparation of cultivation medium 5 Base cultivation medium consisted of 10.4 g/L RPMI-1640-LPLF powder (Gibco BRL), 5.97 g/L HEPES buffer (MP Biomedicals, USA), 2.0 g/L D-glucose (BDH chemicals, Australia), 0.05 g/L hypoxanthine (Sigma, USA) and 40 mg/L gentamycin (Pfizer, Australia). The pH of the medium was adjusted to 6.9 and the solution was filtered using 0.2 pm pore size (AcroCap, Gelman Science, USA). Complete medium was prepared by 10 adding sodium bicarbonate solution (final concentration, 0.21%) and drug-free heat inactivated human plasma obtained from the Australian Red Cross Blood Service (Brisbane) (final concentration, 10%) to the base RPMI-1640-LPLF. RPMI-1640-LPLF complete medium which lacked [3H]-hypoxanthine ([3H]-RPMI-1640-LPLF) was used during the [3H]-hypoxanthine inhibition growth assay to prevent uptake of hypoxanthine 15 by parasites, as radioactive hypoxanthine uptake is measured as a surrogate marker of growth. All complete medium was used within three days of preparation. Preparation of red blood cells Red blood cells (RBC) were required for P. falciparum parasites to proliferate in vitro. 0 20 (Rh+) type blood was obtained from the Australian Red Cross Blood Service. The RBC were washed twice in phosphate-buffered saline (PBS) and once in [ 3 H]-RPMI-1640-LPLF complete medium by centrifugation at 1,500 x g for 5 minutes. Following the final wash, the haematocrit was measured as the percent of RBC to total culture volume. The haematocrit was adjusted to 50% by removing or adding [3H]-RPMI-LPLF complete 25 medium. Continuous cultivation ofparasites All P. falciparum strains were grown in RPMI-1640-LPLF complete medium at 4% haematocrit and 1% to 8% parasitaemia at 37 *C in sealed flasks in a gas mixture of 5% 30 02, 5% CO 2 and 90% N 2 (BOC Gases, Brisbane, Australia). For drug susceptibility assays, cultures were routinely synchronised when the majority of parasites (>85%) were C:NRPrb\DCCDAR\461895_l.DOC.21/12/2011 -72 at ring stage. Synchronisation involved removing the more mature erythrocytic parasite stages by lysis, resulting in the retention only of early trophozoite stages. Synchronisation was performed by resuspending the infected red blood cell (iRBC) pellet in 5 to 10 times its volume of 5% D-sorbitol (Bacto Laboratories Pty. Ltd., Australia) for 5 minutes 5 (Lambros and Vanderberg, 1979 J Parasitol 65: 418-420). The mixture was centrifuged (1,500 rpm for 5 min) and the supernatant removed. The iRBC were washed twice using PBS and once using [ 3 H]-RPMI-LPLF plain medium. Following synchronisation, a new culture was prepared with an initial parasitaemia of 1% in RPMI-LPLF complete medium. 10 Evaluation of in vitro antimalarial activity of the dinuclear ruthenium(II) complexes The in vitro antimalarial activities of two of the dinuclear ruthenium(II) complexes and chloroquine were assessed by exposing P. falciparum strains to ten serially diluted two fold concentrations of each compound. Parasite growth was measured by uptake of tritiated [ 3 H]-hypoxanthine into newly synthesised parasitic DNA. 15

[

3 H]-Hypoxanthine growth inhibition assay The [ 3 H]-hypoxanthine growth inhibition assay (Desjardins et al., 1979 Antimicrobial Agents Chemother 16: 710-718) was used to evaluate the in vitro antimalarial activity of the compounds. Briefly, synchronised parasite cultures (>90% rings, 6 to 8 h post 20 invasion) in [ 3 H]-RPMI-LPLF complete medium with 0.5 % parasitaemia and 2% haematocrit were exposed to the compounds at ten two-fold concentrations, ranging from 10,000 to 20 nM in 96-well microtitre plates. Chloroquine was used as a reference drug. Uninfected RBCs at 2% haematocrit were used as background controls. For the 96 h exposure period, the plates were incubated in the gas mixture at 37 *C for approximately 25 48 h, followed by the addition of 0.2 pCi of 3 H-hypoxanthine (GE Healthcare, Amersham) in [ 3 H]-RPMI-1640-LPLF to each well and a further 48 h of incubation and then frozen at 20 *C. Plates were thawed and harvested using Tomtech Harvester 96 Mach III and radioactive counts were obtained using Wallac TriLux 1450 Microbeta Liquid Scintillation Counter (Perkin Elmer, USA). All assays were performed in triplicate for each strain and 30 at least on two separate occasions.

C:\RPorbl\DCC\DAR406I895_ LDOC-2 1/12/2011 - 73 In vitro inhibition concentrations of the dinuclear ruthenium(II) complexes Tritiated hypoxanthine uptake data were analysed in Graphpad Prism V5.0 software (GraphPad Software Inc. USA). The concentrations of the dinuclear ruthenium(Il) complexes and chloroquine were transformed into logarithmic values. After subtracting 5 the background values, the data from drug-treated wells were normalised against drug-free control wells. Non-linear regression analysis was carried out of the compound's concentration versus parasitic hypoxanthine incorporation. The in vitro antimalarial activity of the compound is defined as inhibitory concentrations (IC 50 ) and (IC 90 ) that cause 50% and 90% inhibition of parasite growth as determined by measuring [ 3

H]

10 hypoxanthine incorporation. The averaged results for two replicate experiments are presented in Table 8. Table 8: In vitro antimalarial susceptibility of two dinucelar ruthenium(II) complexes Compound mg MWt D6 W2

IC

5 o (nM) IC 9 0 (nM) IC 50 (nM) IC 90 (nM) Rubb 12 2.54 1572 733 (28) 1250(71) 739(118) 1400 (0.000) [mean (SD)] Rubb 16 6.31 1628 226 (13) 413 (8) 289 (28) 444 (29) [mean (SD)] chloroquine 9.8 (0.5) 12.5 (2.2) 176.1 (1.5) 382.9 (152.5) 15 The results suggest that the complexes are equally potent against chloroquine sensitive and chloroquine resistant lines. Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will 20 be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or C:WNRPonblDCC\DAR\406IX95_ .DOC-21/12/20 1 - 74 admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Claims (24)

1. Compound of the following formula: b+ L L L L L 1L Ru(Il) Ru(ll) [Z]b L L- -Q-- L- -H L L -a a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; wherein when the compound does not contain a labile ligand and a = I then Q contains at least one -NH-, -N(alkyl)- or -0- group.

2. Compound according to claim I wherein each L may be the same or different and is independently selected from optionally substituted bipyridine, optionally substituted terpyridine, optionally substituted phenanthroline and labile ligand.

3. Compound according to claim 2 wherein, where present, the or each optional substituent is independently selected from alkyl.

4. Compound according to any one of claims 1 to 3 wherein, where present, the or each labile ligand is independently selected from halide or water. C:\RPnbl\DCC\DAR\4061895_I DOC-21/12/2D I - 76

5. Compound according to claim 4 wherein halide is chloride.

6. Compound according to any one of claims 1 to 5 wherein Q is selected from C 2 - 1 6 alkylene wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-.

7. Compound according to any one of claims 1 to 6 wherein Q is selected from C 2 -1 6 alkylene.

8. Compound according to any one of claims 1 to 7 wherein at least one Ru(II) atom coordinates one labile ligand.

9. Compound according to claim 8 wherein at least one Ru(II) atom does not coordinate one labile ligand.

10. The compound according to any one of claims 1 to 9 wherein the two terminal ruthenium centres have the same absolute configuration.

11. The compound according to claim 10 wherein the two terminal ruthenium centres have the A-absolute configuration.

12. The compound according to any one of claims I to 11 of the following formula: b+ L 1 L 1 Ll,1 1 L1 L1,, L1 Ru(Il) Ru(ll) [Z]b L 2 L 1 a wherein: a is an integer from 1 to 3, wherein when a is greater than 1 each Q may be the same or different; C.NRPorbl\DCC\DARG6JS95. 1.DOC-21/12/2011 - 77 b is an integer from 2 to 8; Z represents one or more counteranions; each L, may be the same or different and is independently selected from pyridyl ligand such that each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; L 2 is a labile ligand; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-.

13. Method of preventing or treating a microbial infection comprising administering to a subject in need thereof an effective amount of a compound of the following formula: b+ L L L I L L I L Ru(II) Ru(II) [Z]b Q L- -- Q- L HL--H L L a wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-.

14. Method according to claim 13 wherein at least one Ru(II) atom coordinates one labile ligand. C:\RPortb\DCC\DAR\4061895_I.DOC.21/12/O211 - 78

15. Method according to claim 14 wherein at least one Ru(II) atom does not coordinate one labile ligand.

16. Method according to any one of claims 13 to 15 wherein the microbial infection is a bacterial infection.

17. Method according to claim 16 wherein the bacterial infection is a Gram-negative bacterial infection.

18. A compound of the following formula: b+ L L L L L L Ru(II) Ru(II) [Z]b L L- -Q- L L- -H L L -a wherein: a is an integer from 1 to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; for use in preventing or treating a microbial infection.

19. Compound according to claim 18 wherein at least one Ru(II) atom coordinates one labile ligand. C RPonblDCCDAR\4061895_1 DOC-21/12/2011 - 79

20. Use of a compound of the following formula: b+ L L L I L L I L Ru(II) Ru(II) [Z]b Q L- Q-L L- - H L L a wherein: a is an integer from I to 3, wherein when a is greater than 1 each Q may be the same or different; b is an integer from 2 to 8; Z represents one or more counteranions; each L may be the same or different and is independently selected from pyridyl ligand or labile ligand such that each Ru(II) atom coordinates no more than one labile ligand and each pyridyl ligand forms a polydentate ligand together with one or more other pyridyl ligands on the same Ru(II) atom; and Q is an alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-; in the manufacture of a medicament for preventing or treating a microbial infection.

21. Use according to claim 20 wherein at least one Ru(II) atom coordinates one labile ligand.

22. Pharmaceutical composition comprising a compound according to any one of claims 1 to -12 or a pharmaceutically acceptable salt thereof together with at least one pharmaceutically acceptable carrier or diluent.

23. Compound according to claim 1 or claim 18 substantially as hereinbefore described with reference to any one of the examples.

24. Method according to claim 13 substantially as hereinbefore described with reference to any one of the examples.