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WO2013091014A1 - Agents antimicrobiens - Google Patents

Agents antimicrobiens Download PDF

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Publication number
WO2013091014A1
WO2013091014A1 PCT/AU2012/001583 AU2012001583W WO2013091014A1 WO 2013091014 A1 WO2013091014 A1 WO 2013091014A1 AU 2012001583 W AU2012001583 W AU 2012001583W WO 2013091014 A1 WO2013091014 A1 WO 2013091014A1
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WIPO (PCT)
Prior art keywords
ligand
pyridyl
same
phen
labile
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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PCT/AU2012/001583
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English (en)
Inventor
Yanyan Mulyana
Marshall Leonard Feterl
Frank Richard Keene
Fangfei Li
John Grant Collins
Jeffrey Mitchell Warner
Kirsten Ruth Maria Heimann
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James Cook University
NewSouth Innovations Pty Ltd
Original Assignee
James Cook University
NewSouth Innovations Pty Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US13/334,741 external-priority patent/US20130172308A1/en
Priority claimed from AU2011265513A external-priority patent/AU2011265513A1/en
Application filed by James Cook University, NewSouth Innovations Pty Ltd filed Critical James Cook University
Publication of WO2013091014A1 publication Critical patent/WO2013091014A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table
    • C07F15/0006—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic Table compounds of the platinum group
    • C07F15/0046—Ruthenium compounds
    • C07F15/0053—Ruthenium compounds without a metal-carbon linkage
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • 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.
  • VRE vancomycin-resistant Enterococcus
  • the present invention provides a compound of the following formula:
  • 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-;
  • Q contains at least one -NH-, -N(alkyl)- or -O- group.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of the following formula:
  • a is an integer from 1 to 3, wherein when a is greater than 1 each Q may be the same 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(Il) 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-;
  • Q contains at least one -NH-, -N(alkyl)- or -0- group
  • the present invention provides a 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:
  • 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-.
  • the present invention provides a compound of the following formula:
  • 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-;
  • a is an integer from 1 to 3, wherein when a is greater than 1 each Q may be the same 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-;
  • a is an integer from 1 to 3, preferably a is 1 ;
  • Q is ah 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 . i 6 alkylene linking group wherein any one or more methylene moieties in alkylene is optionally independently replaced with -NH-, -N(alkyl)- or -0-, more preferably Q is a C 2 .i 6 alkylene linking group;
  • b is an integer from 2 to 8, typically when a is 1 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 1 then b is 3, typically when a - 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 ruthenium centre does not complex a labile ligand;
  • 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 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
  • phen 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.
  • halide such as iodide, bromide and chloride, preferably chloride
  • Figure 1 depicts the results of 24 h haemolysis assays for AA-Rubb 7 , ⁇ -Rubbio, ⁇ - Rubbi2 and AA-Rubbi6 with freshly-collected human red blood cells, fitted as logarithmic curves.
  • Figure 2 depicts haemolytic dose-response curves of AA-Rubb]6 (a) and AA-Rubb
  • Figure 3 depicts a comparison of the antimicrobial activity, haemolytic activity and cytotoxicity against the THP-1 cell line after 24 h incubation with ⁇ -Rub , ⁇ -Rubbio, AA-Rubbi2 and AA-Rubb
  • Figure 4 depicts time-kill curves of ruthenium complexes (Rubbi 2 and Rubbi 6 ) and two control antibiotics (Gentamicin and Ampicillin) against two Gram positive strains.
  • Figure 5 depicts time-kill curves of ruthenium complexes (Rubbi 2 and Rubbi 6 ) and two control antibiotics (Gentamicin and Ampicillin) against the two Gram negative strains. No curve of Ampicillin against P. aeruginosa is provided as it is ineffective.
  • Figure 8 depicts the results of pharmacokinetic concentration curves by different administration routes:
  • Figure 8(b) Rubbn serum concentration curves with different administration routes. The doses given to mice: 8 mg/kg s.c, 1 mg/kg i.v. and 8 rrig/kg i.m.
  • each ligand (L) that complexes the ruthenium centres of the compounds of the invention is selected from pyridyl ligand or labile ligand:
  • the compounds of the invention are symmetric with respect to the types of ligands on the ruthenium centres, such as [ ⁇ Ru(phen) 2 ⁇ 2 ⁇ - (bpy(Me)CH 2 CH 2 bpy(Me)))](PF ⁇ ;)4 where each ruthenium centre complexes the same group of pyridyl ligands, and [ ⁇ Ru(terpy)Cl ⁇ 2 ( ⁇ -(b ⁇ y(Me)CH 2 CH2bpy(Me)))]Cl 2 where each ruthenium centre complexes the same group of pyridyl and labile ligands (choride).
  • the complexes are non-symmetric with respect to the types of ligands on the ruthenium centres, such as
  • 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 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:
  • 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 Li 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;
  • L2 is a labile ligand
  • 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.
  • 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 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.
  • 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 be optionally substituted with suitable groups including alkyl groups such as methyl groups.
  • optionally substituted polypyridyl ligands according to the invention include optionally substituted bipyridine (bipy or bpy), optionally substituted “terpyridine (terpy) and optionally substituted phenanthroline (phen), such as methylbipyridine, terpyridine, phenanthroline and tetramethylphenanthroline.
  • the ruthenium centre on the left of the structure coordinates two phenanthroline ligands and one substituted bipyridine ligand
  • the ruthenium centre on the right of the structure coordinates one terpyridine ligand, one substituted bipyridine ligand and one chloride ligand.
  • 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.
  • the ruthenium atom on the right of the structure coordinates a labile chloride ligand.
  • a water molecule may substitute for the labile chloride ligand.
  • the overall charge of the cationic portion of the compound shall be increased to 4 + thereby leading to an association with four chloride counteranions as shown below:
  • 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.
  • the 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).
  • 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 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-;
  • 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.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • the present invention provides a compound of the following formula:
  • 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 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-.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • the present invention provides a compound of the following formula:
  • a is an integer from 1 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; 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, 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.
  • the present invention provides a compound of the following formula:
  • a is an integer from 1 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; 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-.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • Q is not selected from 1,7-heptylene, decylene, 1,12-dodecylene and 1,14-tetradecylene.
  • the present invention provides a compound of the following formula:
  • a is an integer from 1 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; 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, 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.
  • the present invention provides a compound of the following formula:
  • a is an integer from 1 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;
  • 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-.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • 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.
  • the compounds of the invention may exist as mixtures of ⁇ - and ⁇ -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 ⁇ - or the ⁇ - stereoisomeric form.
  • each ruthenium centre within the compound has the same absolute configuration such that for a compound bearing two ruthenium centres the stereochemical configuration is either ⁇ - or ⁇ -.
  • the non-terminal ruthenium centres may exist as racemic mixtures whereas the terminal ruthenium centres may be in either the ⁇ - or ⁇ - stereoisomeric form.
  • enantioenriched or enantiopure forms of the compounds may be produced through stereoselective synthesis and/or through the use of chromatographic or selective recrystallisation techniques. Accordingly in some embodiments the present invention provides a compound of the formula:
  • 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 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-;
  • 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.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • 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 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-.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • 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-;
  • Z represents one or more counteranions.
  • the compound may be used in the prevention or treatment of a microbial infection.
  • Q contains at least one -NH-, -N(alkyl)- or -0- group. In further embodiments Q is not selected from 1,7-heptylene.
  • a is 3 and the compound has the stereochemistry shown below:
  • 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-;
  • Z represents one or more counteranions
  • the compound may be used in the prevention or treatment of a microbial infection.
  • Q contains at least one -NH-, -N(alkyl)- or -O- group. In further embodiments Q is not selected from 1,7-heptylene.
  • alkylene is intended to denote the divalent form of “alkyl” as herein defined.
  • alkyl denotes straight chain, branched or cyclic alkyl, for example C O alkyl, or C 1.20 or C 2 .i6.
  • straight chain and branched alkyl examples include methyl, ethyl, n-propyl, isopropyl, «-butyl, sec-butyl, i-butyl, «-pentyl, 1 ,2- dimethylpropyl, 1,1-dimethyl-propyl, hexyl, 4-methylpentyl, 1 -methylpentyl, 2- 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, 1- methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2- dimethylpentyl, 1 ,3-dimethylpentyl,
  • cyclic alkyl examples include mono- or polycyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.
  • the alkylene linking group "Q" is a flexible alkylene linking group.
  • 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.
  • the alkylene linking group links through the terminal carbon atoms of the group, namely through the ⁇ , ⁇ -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
  • 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 -NH- will create a methaminomethyl linking group as shown below:
  • more than one methylene group in the alkylene linking group, Q will be independently replaced with -NH-, -N(alkyl)- or -0-.
  • the linking group will be either an alkylene, a (poly)aminoalkylene or a (poly)oxyalkylene.
  • polyaminoalkylene linking groups examples are provided below:
  • the linking group may be used to alter the lipophilicity, flexibility and size of the ruthenium complexes of the present invention.
  • 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 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 making a pharmaceutically acceptable salt of an amine group in Q.
  • counteranion refers to any negatively charged group, such as organic or inorganic anionic groups, which renders the overall charge of the compounds of the invention neutral.
  • 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 hexafluorophosphate. Counteranions can have a charge greater than 1, such as 2 or more.
  • treatment may include alleviating or ameliorating the symptoms, diseases or conditions associated with the microbial infection being treated, including reducing the severity and/or frequency of the microbial infection.
  • 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.
  • microbial includes any microscopic organism or taxonomically related macroscopic organism within the categories algae, bacteria, fungi, yeast and protozoa or the like.
  • 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;
  • Morganella Neisseria; Pasteurella; Proteus; Providencia; Pseudomonas; Salmonella; Serratia; Shigella; Stenotrophomonas; Treponema; Vibrio; and Yersinia.
  • EHEC enterohaemorrhagic Escherichia coli
  • EIEC enteroinvasive Escherichia coli
  • ETEC enterotoxigenic Escherichia coli
  • Haemophilus influenzae Helicobacter pylori
  • Klebsiella pneumoniae Legionella spp.
  • Moraxella catarrhalis Neisseria gonnorrhoeae
  • Neisseria meningitidis Proteus spp.
  • Pseudomonas aeruginosa Salmonella spp., Shigella spp., Vibrio cholera and resort Yersinia
  • acid fast bacteria including Mycobacterium tuberculosis, Mycobacterium avium- intracellular e, Myobacterium johnei, Mycobacterium leprae
  • 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; Coryne bacterium; 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), Staphylococcal infections and Streptococcal infections.
  • 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 glabrata, 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 inter digitale, Trichosporon beigelii and Rho
  • 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 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 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.
  • 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 falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium knowlesi or Plasmodium ovale.
  • 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, " 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 Plasmodium falciparum.
  • Staphylococcus aureus including MRSA
  • Enterococcus fecalis Enterococcus fecalis
  • Escherichia coli Escherichia coli
  • Klebsiella pneumonia Salmonella
  • 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 required, for example, surface disinfection.
  • subject 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 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.
  • 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.
  • the present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound as hereinbefore defined, or a 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 organic acids.
  • inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like.
  • 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 acid, salicylic acid and the like.
  • 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 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- 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 corresponding free acid/base forms.
  • 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.
  • 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.
  • the compound could be administered with one or more therapeutic agents in 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.
  • 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.
  • any formulation containing the compound 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.
  • 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 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 may be prepared in parenteral dosage forms, including those suitable for intravenous, intrathecal, and intracerebral or epidural delivery.
  • the pharmaceutical forms suitable for 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.
  • 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 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.
  • agents to adjust osmolality for example, sugars or sodium chloride.
  • 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 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 those enumerated above, as required, followed by filtered sterilization.
  • 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.
  • preferred methods of preparation are vacuum drying or freeze-drying of a previously sterile-filtered solution of the active ingredient plus any additional desired ingredients.
  • compositions 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 may be compressed into tablets, or it may be incorporated directly with the food of the diet.
  • 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 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 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.
  • 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 like
  • a lubricant such as magnesium stearate
  • a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of winter
  • 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.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active 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.
  • Enteral formulations may be prepared in the form of suppositories by mixing with appropriate bases, such as emulsifying bases or water-soluble bases.
  • bases such as emulsifying bases or water-soluble bases.
  • the compounds of the present invention may be administered topically, intranasally, intravaginally, intraocularly and the like.
  • 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.
  • 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.
  • 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.
  • 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 incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • Dosage unit form 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 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 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.
  • the principal active ingredient may be compounded for convenient 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 ⁇ g to about 200 mg. Expressed in proportions, the active compound may be present in from about 0.25 g to about 200 mg/mL of carrier.
  • 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, 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.
  • treatment covers ' 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.
  • prevention and preventing 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.
  • H NMR data were H NMR spectra were recorded on a Varian Mercury 300 MHz spectrometer at room temperature in CD 2 C1 2 (>99.8%, Aldrich). Materials and methods
  • Ethylene glycol, 1 ,10-phenanthroline (phen), potassium hexafluorophosphate (KPF 6 ), ammonium hexafluorophosphate (NH PFe), 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.
  • Me 2 bpy (1.6 g, 8.7 mmol) was 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.
  • 1,10-dibromodecane (1,12-dibromododecane for bbi4, 1 ,14-dibromotetradecane for bbie; 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 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 ⁇ 80 mL) and DCM (1 * 80 mL).
  • bb 12 and bb] 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 arid contents of each fraction were determined by TLC using 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: bb 12 (30%); bb H (33%); bb 16 (44%). Characterisation was achieved using ⁇ NMR in CDCI3. In some cases there was a 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.
  • a typical procedure was as follows: A-[Ru(phen) 2 (py) 2 ] ⁇ (-)-AsOtart ⁇ 2 (180 mg, 0.169 mmol) and bb] 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).
  • the dark red-orange solution was loaded onto an SP-Sephadex C-25 column (2 cm diam. 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 from the eluate as its PF 6 " salt by a slow addition of saturated aqueous PF6, and then extraction into DCM. The organic layer was washed with water, dried over anhydrous Na 2 S0 , and evaporated to dryness in vacuo to yield a bright red-orange precipitate,
  • Circular dichroism spectra and ID ⁇ 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 Trans. , 2007, 4333-4342 for bb 2 , bbs, bb , and bbio bridged species.
  • 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 Hg) 4 N]Br until complete precipitation had occurred.
  • the products were filtered and washed with cold acetone.
  • 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.
  • CD ⁇ /nmtAe/cm-'M- 1 CI " salt in ⁇ 2 0 ⁇ : ⁇ : 468.5(-35.6); 418(22.7); 285.5(-292); 270(-306); 260(357); 218.5(72.1). ⁇ : 468(36.0); 417.5(-20.5); 285.5(285); 269.5(306); 260(-343); 219.5(-70.4).
  • These complexes were separated from mononuclear species and other impurities using an SP-Sephadex C-25 cation exchange qolumn with a gradient concentration of aqueous sodium chloride solution as eluent.
  • the desired dinuclear species were eluted with 0.6 M NaCl solution, except for the one case of which was obtained by using 1.0 M NaCl solution.
  • Solid PF 6 was added to the eluates, followed by extraction into DCM.
  • the organic layer was washed with water, dried over anhydrous Na 2 S0 4 , and evaporated to dryness in vacuo to yield a bright red-orange precipitate, ⁇ - [ ⁇ Ru(phen) 2 ⁇ 2 ⁇ -bbX n )](PF 6 ) 4 .
  • the complexes were further purified by dissolving them in minimum amount of acetone, loading onto Sephadex LH-20 and then eluting with acetone.
  • 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(ph.en) 2 ⁇ 2 ⁇ -bbH 2 N7)] 6+ using the above procedure.
  • the syntheses of mononuclear A-[ ⁇ Ru(phen) 2 (Me 2 bipy)] 2+ and ⁇ - [Ru(phen) 2 (bb 7 )] were also carried out according to the above procedure using excess 4,4'-dimethyl-2,2'-bipyridine and bb 7 , while the synthesis of mononuclear ⁇ - [Ru(phen)2(bbi6)]Cl 2 was modified as follows.
  • 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 S0 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 tetraethylammoniurii chloride in acetone was added dropwise until no more precipitation occurred.
  • the dinuclear species precipitated out as its chloride salt, while the mononuclear complex remained in solution.
  • Solid bb] 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 mixture was loaded onto Sephadex C-25 cation exchange column, eluted with water and then 0.3 M NaCl to remove the impurities.
  • the pure dinuclear [ ⁇ Ru(terpy)(Cl) ⁇ 2 ⁇ -bb n )](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 the dark purple solution was freeze-dried to obtain a fluffy dark red purple powder of [ ⁇ Ru(terpy)(Cl) ⁇ 2 ⁇ -bb n )]Cl 2 .
  • the PF 6 " salt was converted to the chloride by 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- bb n )Ru(terpy)Cl](Cl)3 in 70-80% yield. Separation of any possible geometric isomers was not attempted.
  • the bacterial strains used in this study are classified as risk group 2 according to the Australian/New Zealand Standard (AS/NZS 2243.4:2010) and must be manipulated in a PC2 class laboratory.
  • S. aureus ATCC 25923 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. 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 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 was added in each well making a concentration range of 0.25 mg ml "1 to 128 mg ml "1 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:
  • Rudppm AA-[ ⁇ Ru(phen) 2 ⁇ 2 ⁇ -4,6-bis(2-pyridyl)pyrimidine)]Cl 4
  • Rubb 7 trinuclear ⁇ * ⁇ *-[ ⁇ Ru*(phen) 2 ⁇ ( ⁇ -bb 7 ) ⁇ Ru( hen) ⁇ ( ⁇ -bb7) ⁇ Ru*(phen) 2 ⁇ ]Cl 6 (only the two terminal metal centres are chiral, the two central metal centres are racemic)
  • Rubbytetranuclear ⁇ * ⁇ *-[ ⁇ Ru*(phen)2 ⁇ ( ⁇ -bb 7 ) ⁇ Ru(phen) ⁇ ( ⁇ -bb7) ⁇ Ru(phen) ⁇ ( ⁇ -bb 7 )* ⁇ Ru*(phen) 2 ⁇ ]Cl8 (only the two terminal metal centres are chiral, the two central metal centres are racemic)
  • the minimum inhibitory concentration (MIC) results for the ruthenium complexes against four bacterial strains shown in Table 1 demonstrate that some of the ruthenium(II) complexes are highly active against both classes of bacteria, although Gram-positive bacteria appeared to be more susceptible than the Gram-negative counterparts.
  • MIC minimum inhibitory concentration
  • the trinuclear and tetranuclear analogues of AA-Rubb 7 showed slightly better activity than the dinuclear complex.
  • the highly lipophilic mononuclear complexes (as determined by octanol-water partition coefficient - log? values) containing the bb or bb 16 ligand exhibited intermediate MIC values, although Rubb 7 mono was more active (on a mole basis) against S. aureus than its dinuclear counterpart.
  • 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 all strains.
  • [Ru(Me 4 phen) 3 ] 2+ showed equal activity (on a mole basis) to ⁇ / ⁇ Rubbi 2 , Rubb
  • 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 susceptible ATCC 25923 S. aureus.
  • mononuclear ruthenium complexes with a +2 charge may be less effective against MRSA than the susceptible strain.
  • the dinuclear Rubbrush complexes which have a higher charge (+4) that is also spread over two metal centres, exhibited equal activity against both strains.
  • the tri- and tetranuclear complexes exhibited equal activity against S. aureus and MRSA.
  • the results also suggest that a tnnuclear complex based upon either a bbi 2 , bb ⁇ or bbi 6 ligand could be even more effective than their dinuclear counterparts.
  • MIC values are possibly related to the lipophilicity of these complexes.
  • Rubbi6 is considerably more lipophilic than Rubbi 2 (almost one log 10 unit), and the non- symmetric complexes are more lipophilic than the inert dinuclear.
  • the symmetric labile complexes are the most lipophilic.
  • the compound should ideally also exhibit low toxicity towards human or animal eukaryotic cells.
  • the haemolytic activity was determined for the dinuclear complexes that showed the best activity ( ⁇ -Rub , ⁇ -Rubbio, ⁇ -Rubbu and ⁇ - Rubbie) in the antibacterial assays.
  • the complexes to be tested were dissolved in PBS to a stock solution of 2048 mg ml '1 and two-fold diluted in PBS in round- bottomed 96-well plates in a final volume of 100 ml in each well.
  • HC5 0 concentration needed to induce 50% haemolysis.
  • the experimental error is ⁇ 5%.
  • Incubation with ⁇ -Rubbie caused the most severe haemolysis and ⁇ -Rubb? the least; however, the HC50 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 was also higher than the MIC values in all cases.
  • ⁇ -Rubbu gave the most encouraging results, with excellent 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 ⁇ -Rubb ⁇ and AA-Rubbi 6 for different incubation times are shown in Fig. 2.
  • the haemolytic activity of AA-Rubbi 6 at 10 ⁇ was low for 8 h, but noticeably increased over 24 h.
  • AA-Rubbi2 at 10 ⁇ essentially exhibited no haemolysis over a 24 h period.
  • Significant haemolysis was only observed after 4 h at high concentrations (>256 ⁇ gm ⁇ 1 ), while no significant haemolytic activity was observed at all the tested concentrations ⁇ -Rubb? and ⁇ -Rubbjo over 24 h.
  • the Rubb n complexes were more concentration-dependent than time-dependent.
  • the haemolytic assay indicated that the Rubb n dinuclear complexes (especially ⁇ - Rubb
  • red blood cells are a special type of eukaryotic cell that do not contain a cell nucleus and the cell membrane has a unique structure of three layers.
  • ruthenium complexes C. Metcalfe and J. A. Thomas, Chem. Soc. Rev., 2003, 32, 215; B. M. Zeglis, V. C. Pierre and J. . Barton, Chem. Commun., 2007, 4565; and F. R. eene, J. A. Smith and J. G.
  • THP-1 cells a human acute monocytic leukemia cell line and a good model for nucleated eukaryotic cells
  • 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% C0 2 .
  • FBS fetal bovine serum
  • L-glutamine Sigma Aldrich, Sydney, NSW, Australia
  • HEPES buffer 12.5 mM HEPES buffer at 37 °C in a humidified atmosphere of 5% C0 2 .
  • Cells were seeded at a density of 5 * 10 5 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 '1 and 1024 mg ml
  • Table 8 The cytotoxicity of AA-Rubb 7 , ⁇ -Rubbio, AA-Rubb 12 and AA-Rubbi 6 against the THP-1 cell line
  • AA-Rubbi6 78 48 40 25 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 IC50 values greater than their corresponding MICs against susceptible S. aureus and MRS A, as shown in Fig. 3. AA-Rubbi2 displayed the highest HC50/MIC and IC50 MIC ratios, suggesting that it is the most selectively toxic of the Rubbrush complexes, and consequently has the greatest potential as an antimicrobial agent.
  • the apparent selectivity of the Rubb n complexes for bacterial cells over human eukaryotic 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 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. In vitro antimalarial activity
  • the P. falciparum laboratory adapted strains utilised in this project 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).
  • Table 10 Plasmodium falciparum strains used in this project.
  • folic acid in RPMI-1640-LPLF prevents inhibition of the compound if its activity targets the parasite's folate metabolic pathway.
  • Parasites were cultured in human red blood cells (RBCs) in vitro at 37 °C in special gas mixture (5% 0 2 , 5% C0 2 and 90% N 2 ) as described in Trager and Jensen (1979) Science 193: 673-675. Preparation of cultivation medium
  • 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 ⁇ pore size (AcroCap, Gelman Science, USA).
  • RPMI-1640-LPLF complete medium which lacked [3 H] -hypoxanthine ([3H]-RPMI-1640-LPLF) was used during the [3H]-hypoxanthine inhibition growth assay to prevent uptake of hypoxanthine 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 were required for P. falciparum parasites to proliferate in vitro.
  • O (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 medium. Continuous cultivation of parasites
  • 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 (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.
  • the [ 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 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.
  • the plates were incubated in the gas mixture at 37 °C for approximately 48 h, followed by the addition of 0.2 ⁇ 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 at least on two separate occasions. 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(II) complexes and chloroquine were transformed into logarithmic values. After subtracting 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 (IC50) and (IC90) that cause 50% and 90% inhibition of parasite growth as determined by measuring [ 3 H]- hypoxanthine incorporation. The averaged results for two replicate experiments are presented in Table 1 1. Table 11: In vitro antimalarial susceptibility of two dinuclear ruthenium(II) compl
  • Q[10] is a macrocyclic host called cucurbituril composed of 10 monomerie units, see Pisani, M.J. Zhao, Y., Wallace, L., Woodward, C.E., Keene, F.R., Day, A.I. and Collins, J.G. Cucurbit[10]uril binding of dinuclear platinum(II) and ruthenium(II) complexes: association/dissociation rates from seconds to hours. Dalton Trans., 2010, 39, 2078-2086.
  • liver function failure Studies of the histopathology of the iri vivo models showed no significant lesions of main organs (i.e. liver, lung, kidney and heart). There was no obvious evidence for the cause of death from the evaluation of these tissues. As the animals died on the second day of starting the experiment, such an acute toxicity did not appear to lead to physical lesions to the damaged organs.
  • ALP alkaline phosphatase
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase

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Abstract

La présente invention porte sur des complexes de ruthénium et en particulier sur des complexes de ruthénium dinucléaires et multinucléaires qui peuvent être utilisés comme agents antimicrobiens. L'invention porte également sur des compositions pharmaceutiques comprenant de tels complexes et sur leurs procédés d'utilisation dans le traitement ou la prévention d'infections microbiennes.
PCT/AU2012/001583 2011-12-22 2012-12-21 Agents antimicrobiens Ceased WO2013091014A1 (fr)

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CN105601672A (zh) * 2014-11-25 2016-05-25 中国科学院大连化学物理研究所 一种双核钌nnn配合物及其制备方法
CN112638922A (zh) * 2018-05-03 2021-04-09 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法
CN115925752A (zh) * 2022-12-30 2023-04-07 中山大学·深圳 一种近红外光释放双核钌配合物及其制备和抗非小细胞肺癌耐药应用
US12404291B2 (en) 2019-04-04 2025-09-02 The University Of Sheffield Antimicrobial agent

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105601672A (zh) * 2014-11-25 2016-05-25 中国科学院大连化学物理研究所 一种双核钌nnn配合物及其制备方法
CN105601672B (zh) * 2014-11-25 2018-05-15 中国科学院大连化学物理研究所 一种双核钌nnn配合物及其制备方法
CN112638922A (zh) * 2018-05-03 2021-04-09 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法
CN112638922B (zh) * 2018-05-03 2024-04-05 阿尔梅里亚大学 金(iii)配合物、其缀合物、包含其的药物组合物以及用途和制备其的方法
US12404291B2 (en) 2019-04-04 2025-09-02 The University Of Sheffield Antimicrobial agent
CN115925752A (zh) * 2022-12-30 2023-04-07 中山大学·深圳 一种近红外光释放双核钌配合物及其制备和抗非小细胞肺癌耐药应用
CN115925752B (zh) * 2022-12-30 2025-09-26 中山大学·深圳 一种近红外光释放双核钌配合物及其制备和抗非小细胞肺癌耐药应用

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