WO2007095696A1 - Polycétide-xanthones et applications - Google Patents

Polycétide-xanthones et applications Download PDF

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Publication number
WO2007095696A1
WO2007095696A1 PCT/AU2007/000228 AU2007000228W WO2007095696A1 WO 2007095696 A1 WO2007095696 A1 WO 2007095696A1 AU 2007000228 W AU2007000228 W AU 2007000228W WO 2007095696 A1 WO2007095696 A1 WO 2007095696A1
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WIPO (PCT)
Prior art keywords
hydrogen
alkyl
methyl
cancer
compound according
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PCT/AU2007/000228
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English (en)
Inventor
Robert John Capon
Ernest Lacey
Ranjala Ratnayake
Jennifer Helen Gill
Shaun Tennant
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The University Of Queensland
Microbial Screening Technologies Pty Ltd
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Priority claimed from AU2006900980A external-priority patent/AU2006900980A0/en
Application filed by The University Of Queensland, Microbial Screening Technologies Pty Ltd filed Critical The University Of Queensland
Publication of WO2007095696A1 publication Critical patent/WO2007095696A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
    • C12P17/188—Heterocyclic compound containing in the condensed system at least one hetero ring having nitrogen atoms and oxygen atoms as the only ring heteroatoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/02—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains two hetero rings
    • C07D491/04—Ortho-condensed systems
    • C07D491/044—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring
    • C07D491/052—Ortho-condensed systems with only one oxygen atom as ring hetero atom in the oxygen-containing ring the oxygen-containing ring being six-membered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • the present invention relates generally to polyketide xanthones and their use as anticancer or antibacterial agents. More particularly the present invention relates to polyketide xanthones of the kibdelone class. The present invention also relates to methods of preparing the kibdelone compounds and their derivatives, pharmaceutical compositions containing them and their use in methods for treating cancers, particularly mammalian cancers, and bacterial infections.
  • a number of polyketide xanthones are known and have varying biological activities.
  • actinoplanones are known to have antibiotic, antifungal and cytotoxic activity (JP 63188683; Kobayashi et al, 1988a; Kobayashi et al, 1988b)
  • IB-00208 has cytotoxic activity (Rodriguez et al, 2003; Malet-Cascon et al, 2003)
  • citreamicins have antitumour activity (WO 01/87283)
  • simaomicins are known to have antibacterial and antiparasitic activity and may sensitise cancer cells to anticancer agents (Arai et al. , 2004).
  • the present invention is predicated in part on the determination that the newly discovered kibdelone polyketide xanthones have potent antitumour activity and antibacterial activity.
  • R 2 is H or alkyl,
  • R 3 is H or halo,
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R 8 and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H, C0 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R 11 is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R 12 and R 13 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof, stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers.
  • R 2 is alkyl, especially C 1-4 alkyl, more especially propyl;
  • R 3 is halo, especially chloro;
  • R 4 is H, alkyl, acyl or a sugar moiety, especially alkyl, more especially methyl;
  • R 5 is H, alkyl, acyl or a sugar moiety, especially H or alkyl, more especially H;
  • R 6 and R 7 are independently selected from H, alkyl, acyl or a sugar moiety, especially H, alkyl or a sugar moiety, more especially H;
  • R 8 is OR 11 ;
  • R 9 is H
  • R 11 is H 3 alkyl, acyl or a sugar moiety, especially H or a sugar moiety, more especially H or a sugar moiety, most especially H or rhamnose;
  • R 12 and R 13 are independently selected from H, alkyl, acyl or a sugar moiety, especially H or alkyl, more especially H.
  • Particularly preferred compounds are:
  • alkyl used either alone or in compound words, denotes saturated, straight chain, branched and cyclic hydrocarbon residues typically having from 1 to 18 carbon atoms, preferably 1 to 10 or 1 to 6 carbon atoms.
  • straight chain and branched alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, r ⁇ -pentyl and branched isomers thereof, rc-hexyl and branched isomers thereof, n-heptyl and branched isomers thereof, r ⁇ -octyl and branched isomers thereof, «-nonyl and branched isomers thereof, and r ⁇ -decyl and branched isomers thereof.
  • cyclic alkyl examples include mono- or polycyclic alkyl residues such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.
  • alkenyl denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon to carbon double bond including ethylenically mono-, di- or poly-unsaturated alkyl or cycloalkyl residues as previously defined, preferably C 2-18 alkenyl (eg C 2-10 or C 2-6 ).
  • alkenyl examples include, but are not limited to, vinyl, allyl, 1-methylvinyl, butenyl, wo-butenyl, 3-methyl-2-butenyl, 1-pentenyl, cyclopentenyl, 1-methyl-cyclopentenyl, 1-hexenyl, 3-hexenyl, cyclohexenyl, 1-heptenyl, 3-heptenyl, 1-octenyl, cyclooctenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 3-decenyl, 1,3-butadienyl, 1,4-pentadienyl, 1,3-cyclopentadienyl, 1,3-cycloheptadienyl, 1,3,5-cycloheptatrienyl and
  • alkynyl denotes groups formed from straight chain, branched or cyclic hydrocarbon residues containing at least one carbon-carbon triple bond including ethynically mono-, di- or poly- unsaturated alkyl or cycloalkyl residues as previously defined.
  • the term preferably refers to C 2-18 alkynyl. Examples include, but are not limited to, ethynyl, 1-pro ⁇ ynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers.
  • Preferred acyl residues include C(O)-R, wherein R is hydrogen or an alkyl, alkenyl, alkynyl, aryl, arylalkyl, heterocyclyl or heterocyclylalkyl residue, preferably a C 1-18 residue.
  • acyl examples include formyl; straight chain or branched alkanoyl such as, acetyl, propanoyl, butanoyl, 2-methylpropanoyl, pentanoyl, 2,2-dimethylpropanoyl, hexanoyl, heptanoyl, octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, tridecanoyl, tetradecanoyl, pentadecanoyl, hexadecanoyl, heptadecanoyl, octadecanoyl, nonadecanoyl and icosanoyl; cycloalkylcarbonyl such as cyclopropylcarbonyl cyclobutylcarbonyl, cyclopentylcarbonyl and cyclohexylcarbonyl; aroyl such as benzoyl, tolu
  • naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl
  • heterocycliccarbonyl heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl
  • heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl.
  • aryl used either alone or in compound words, denotes a C 6 -C 14 aromatic hydrocarbon residue.
  • Suitable aryl residues include phenyl, biphenyl, naphthyl, tetrahydronaphthyl, anthracenyl, dihydroanthracenyl and phenanthrenyl.
  • Preferred aryl residues include phenyl and naphthyl.
  • heterocyclyl used either alone or in compound words, denotes monocyclic, polycyclic or fused, saturated, unsaturated or aromatic hydrocarbon residues, wherein one or more carbon atoms (and where appropriate, hydrogen atoms attached thereto) are replaced by a heteroatom.
  • Suitable heteroatoms include, O, N and S. Where two or more carbon atoms are replaced, this may be by two or more of the same heteroatom or by different heteroatoms.
  • heterocyclic residues may include pyrrolidinyl, pyrrolinyl, piperidyl, piperazinyl, morpholinyl, indolinyl, imidazolidinyl, pyrazolidinyl, thiomorpholinyl, oxazolyl, dioxanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyrrolyl, pyridyl, thienyl, furyl, pyrrolyl, imidazolyl, indolyl, pyridazinyl, pyrazolyl, pyrazinyl, thiazolyl, pyrimidyl, pyrimidinyl, quinolinyl, isoquinolinyl, benzofuranyl, benzothienyl, purinyl, quinazolinyl, phenazinyl, acridinyl, coumarinyl, benzo
  • halo refers to fluoro, chloro, bromo and iodo substituents.
  • Mbdelone is used to refer to compounds of formula (I), particularly a compound of formula (I) isolated from an actinomycete microorganism from the genus Kibdelosporangium or a derivative of such a compound.
  • Examples of compounds referred to as kibdelones include Compound (1) referred to as kibdelone A, Compound (2) referred to as kibdelone B and Compound (3) referred to as kibdelone C.
  • sugar moiety includes mono- and poly-saccharides containing one to five furanose and/or pyranose sugar molecules, especially mono-, di- or tri-saccharides or saccharide derivatives. Suitable mono-saccharide units that may occur alone or as part of a poly-saccharide include, but are not limited to, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose and rhamnose.
  • Suitable mono-saccharide derivatives that may occur alone or as part of a poly-saccharide include, but are not limited to, JV-glycosamines, O-acyl derivatives, O-alkyl derivatives, O-alkenyl derivatives, O-aryl derivatives, sugar alcohols, sugar acids and deoxy sugars. Saccharides may also be present in D- or L-isomeric form.
  • the compounds of the invention may be in the form of pharmaceutically acceptable salts.
  • suitable pharmaceutically acceptable salts include, but are not limited to, salts of pharmaceutically acceptable inorganic acids such as hydrochloric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic, and hydrobromic acids, or salts of pharmaceutically acceptable organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, benzoic, succinic, oxalic, phenylacetic, methanesulphonic, toluenesulphonic, benzenesulphonic, salicyclic sulphanilic, aspartic, glutamic, edetic, stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids.
  • Base salts include, but are not limited to, those formed with pharmaceutically acceptable cations, such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium.
  • Basic nitrogen-containing groups may be quarternised with such agents as lower alkyl halide, such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • lower alkyl halide such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides
  • dialkyl sulfates like dimethyl and diethyl sulfate; and others.
  • the compounds of the invention may be in the form of complexes with metal ions.
  • Suitable metal ions include divalent and trivalent metal ions.
  • suitable metal ions for complexation include, but are not limited to Mn +"1" , Mg +"1" , Co 4+ , Fe 4+ , Ni + *, Cu +"1" , Zn 4+ and Fe + ⁇ .
  • the invention thus also relates to compounds in substantially pure isomeric form at one or more asymmetric centres eg., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, where ee refers to the enantiomeric excess of one enantiomer compared to the other enantiomer, as well as mixtures, including racemic mixtures, thereof.
  • the stereoisomeric forms may be epimers, diastereomers or enantiomers.
  • Such isomers may be prepared by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution.
  • asymmetric compounds may be prepared by fermentation with suitable microorganisms and optionally further derivatised.
  • the kibdelone compounds of formula (I) may undergo air oxidation and may exist in tautomeric form.
  • Compound (3) (kibdelone C) readily undergoes oxidation to Compound (2) (kibdelone B), and likewise Compound (2) (kibdelone B) readily undergoes keto-enol tautomerism and oxidation to Compound (1) (kibdelone A).
  • This interconversion process may allow for controlled interconversion of kibdelones A, B and C, and their analogues and derivatives.
  • the present invention encompasses mixtures of compounds that may be present as an equilibrium of tautomeric forms, or as the result of partial oxidation or reduction.
  • the present invention also encompasses mixtures of stereoisomers such as enantiomers, diastereomers and epimers.
  • a number of the compounds of formula (I) may be prepared by fermentation of an appropriate microorganism. This is particularly so of Compounds (1) to (3) and (5) to (7), (kibdelones A-C and kibdelone A-C rhamnosides). Furthermore, altering the fermentation conditions may alter the ratio of compounds produced by the fermentation. Altering the fermentation conditions may also allow manipulation of substituents. For example, addition of sodium chloride (NaCl) to the fermentation medium will produce a compound of formula (I) in which R 3 is Cl and addition of sodium bromide (NaBr) to the fermentation medium will produce a compound of formula (I) in which R 3 is Br, for example, Compounds (9) to (11) and (13) to (15).
  • NaCl sodium chloride
  • NaBr sodium bromide
  • Other compounds of formula (I) may be prepared by partial synthesis from compounds isolated during fermentation using synthetic procedures known in the art.
  • free hydroxy groups, phenol hydroxy groups and amides may be alkylated, alkenylated or alkynylated using an appropriate activated reagent such as an alkyl halide, alkenyl halide or alkynyl halide.
  • a hydroxy group may be methylated with methyl iodide and K 2 CO 3 in acetone.
  • Acylated derivatives may be prepared by treatment of hydroxy groups, including phenol hydroxy groups, and amides with suitable activated carboxylic acid derivatives such as acid anhydrides or acid chlorides in dry pyridine.
  • Alkali metal salts may be prepared by treatment of hydroxy groups, particularly phenol hydroxy groups with appropriate basic salts such as Na 2 CO 3 or K 2 CO 3 .
  • Glycosides may be isolated directly from the fermentation medium or may be prepared by methods known in the art, such as, treatment of hydroxy groups or amide groups with appropriate mono-, di-, tri- or other poly-saccharides under acidic conditions, by treatment with activated sugar reagents, or by treatment with suitable enzyme preparations.
  • Ketones present in the compounds of formula (I), particularly on the quinone moiety A, such as Compounds (1) or (5), may be reduced using suitable reducing agents such as hydride reagents, LiAlH 4 or NaBH 4 or other reducing agents such as SnCl 2 -HCl or sodium hydrosulf ⁇ te (Na 2 S 2 O 4 ), to yield compounds such as Compounds (4) or (8).
  • suitable reducing agents such as hydride reagents, LiAlH 4 or NaBH 4 or other reducing agents such as SnCl 2 -HCl or sodium hydrosulf ⁇ te (Na 2 S 2 O 4 ), to yield compounds such as Compounds (4) or (8).
  • hydroquinones such as those of Compounds (3) or (7), may be readily oxidised to provide quinones using oxidising agents such as acid dichromate, silver oxide, lead tetraacetate, HIO 4 and Fremy's salt ((KSO 3 ) 2 N-O).
  • oxidising agents such as acid dichromate, silver oxide, lead tetraacetate, HIO 4 and Fremy's salt ((KSO 3 ) 2 N-O).
  • Hydrolysis of alkyl, alkenyl, alkynyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and sugar moieties to provide hydroxy groups may be performed using procedures known in the art.
  • methoxy groups may be cleaved using BBr 3 , Me 2 SiI, MeSSiMe 3 or PhSSiMe 3 and benzyl ethers may be cleaved to provide a hydroxy group with H 2 ZPd-C in ethanol.
  • Acyl groups may also be cleaved to provide hydroxy groups by known means in the art.
  • acetyl groups may be cleaved by K 2 CO 3 in methanol/water and benzoyl groups may be cleaved using 1% NaOH or triethylamine in methanol.
  • Double bonds may be reduced to provide single bonds by reduction techniques known in the art.
  • a compound of formula (I) in which is a double bond may be reduced to provide a compound of formula (I) in which is a single bond by, for example, catalytic hydrogenation.
  • Single bonds may also be oxidised to provide a double bond.
  • a compound of formula (I) in which is a single bond may be prepared by the interconversion and air oxidation as described above. - l i ⁇
  • lt may be required that in order to prepare compounds of formula (I) from compounds isolated from fermentation, one or more functional groups must be protected.
  • Suitable protecting groups for hydroxy groups, carboxylic acids, amines, ketones and other functional groups, their introduction and cleavage, including selective introduction and cleavage, are known and can be found, for example, in Protective Groups in Organic Synthesis, T.W. Greene and P.G.M. Wuts, 3 rd Edition, John Wiley and Son, 1999.
  • Compounds of formula (I) may also be prepared using microorganisms genetically manipulated to include the genes required for their preparation using techniques known in the art.
  • the genes required for preparation of compounds of formula (I) may be isolated from the Kibdelosporangium sp. MST- 108465 and inserted in a second microorganism. The second microorganism may then be fermented to produce the kibdelone compounds, or further manipulated to provide analogues not isolated from the original Kibdelosporangium sp. It may also be possible to prepare greater quantities (overproduce) of the compounds of formula (I) than those originally produced by the Kibdelosporangium sp. Methods for the isolation of genes and their incorporation into a second microorganism are known in the art.
  • the microorganism that produces kibdelone compounds is an actinomycete microorganism from the genus Kibdelosporangium.
  • the preferred microorganism is a strain of Kibdelosporangium designated MST-108465 or a mutant thereof.
  • the microorganism MST- 108465 was deposited with National Measurement Institute under the Accession No. NM06/00003 on 3 February 2006.
  • mutant refers to a microorganism having similar genetic identity and which has retained the ability to produce kibdelone compounds.
  • the mutant may have one or more substitutions, deletions or additions in its DNA or RNA provided it retains the ability to produce kibdelone compounds.
  • MST- 108465 The microbial strain, MST- 108465 was isolated from a soil sample collected from a timber woolshed 15 km north of Port Augusta in South Australia in 1996.
  • a 16S rRNA analysis identified MST-108465 as belonging to the Pseudonocardiaceae and having 98% identity with Kibdelosporangium spp. (aff phillipinense).
  • Investigation of the metabolites produced by the known species of Kibdelosporangium showed no shared common secondary metabolites with MST-108465. Accordingly, based on rRNA and metabolite data MST-108465 is regarded tentatively as a novel species, Kibdelosporangium sp.
  • Fermentation of the microorganism that produces compounds of formula (I) may be achieved by inoculating suitable fermentation media and incubation for an appropriate time at a temperature in the range of 16 °C to 40 0 C, especially about 25 0 C to 30 0 C, more especially about 28 °C. After incubation, the fermentation medium is extracted with a polar solvent such as methanol, ethanol, acetone or ethyl acetate.
  • a polar solvent such as methanol, ethanol, acetone or ethyl acetate.
  • Suitable fermentation media include solid, liquid or mixed media.
  • a suitable solid medium is agar, especially ISP2 Agar, suitable liquid media include ISP2 liquid medium and rice flour liquid medium, and suitable mixed media include barley grain and wheat grain media.
  • Improved yields of compounds of formula (I) may be obtained by increasing the volume of the medium and addition of nutrients.
  • Suitable nutrients are known to those skilled in the art of fermentation and include carbon sources, nitrogen sources and trace elements.
  • Suitable carbon sources include, but are not limited to, carbohydrates and may be simple or complex and may be added as a single compound or a mixture of compounds. Examples of simple sugars include, but are not limited to, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose and rhamnose, or mixtures of these compounds.
  • Complex carbohydrates include, but are not limited to, starch and other polysaccharides. Examples of complex mixtures include but are not limited to molasses, syrup, grains and cane sugar.
  • Suitable nutrients that are nitrogen sources may also be simple or complex and added as single compounds or mixtures.
  • simple nitrogen compounds include, but are not limited to, amino acids and mixtures thereof.
  • complex nitrogen compounds include, but are not limited to, peptides and proteins.
  • complex mixtures suitable as a nutrient nitrogen source include, but are not limited to, peptone, soytone and meals such as cotton seed meal, fish meal and beef meal.
  • Nutrients that are trace elements are sources of metals such a Fe, Mb, Co and the like. A person skilled in the art could readily provide suitable nutrients for fermentation of a microorganism.
  • the culture is incubated for a period of between 5 to 40 days, preferably 7 to 18 days and more preferably 7 to 14 days.
  • the medium may be centrifuged to provide a pellet of mycelia.
  • the supernatant may then be decanted and the mycelial pellet extracted with solvent to obtain a mycelial extract.
  • the decanted supernatant may be concentrated in vacuo and/or freeze dried, then extracted with solvent to yield a supernatant extract.
  • Solid fermentations may be extracted by excising solid media from the Petri plate and transferring to a flask or vial before extraction of the media with solvent.
  • Extracts may then be concentrated in vacuo and purified by chromatography. At least one of solid phase extraction, high performance liquid chromatography (HPLC) and preparative HPLC may be performed to obtain semi-purified and isolated compounds of formula (I).
  • HPLC high performance liquid chromatography
  • preparative HPLC may be performed to obtain semi-purified and isolated compounds of formula (I).
  • an isolated microorganism which is a strain of Kibdelosporangium designated MST-108465 or a mutant thereof.
  • a compound of the invention may be administered as a neat chemical, it is preferable to present the claimed active compound as a pharmaceutical composition.
  • a pharmaceutical composition comprising a compound of formula (I):
  • R 2 is H or alkyl
  • R 3 is H or halo
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R 8 and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H, C0 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R 11 is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R 12 and R 13 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof, stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers, together with one or more pharmaceutically acceptable carriers.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the other components of the composition and not deleterious to the recipient thereof.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sub-lingual), vaginal or parenteral (including intramuscular, sub-cutaneous and intravenous) administration or in a form suitable for administration by inhalation or insufflation.
  • compositions and unit dosages thereof may thus be placed into the form of pharmaceutical compositions and unit dosages thereof, and in such form may be employed as solids, such as tablets or filled capsules, or liquids such as solutions, suspensions, emulsions, elixirs, or capsules filled with the same, all for oral use, in the form of suppositories for rectal administration; or in the form of sterile injectable solutions for parenteral (including subcutaneous) use.
  • Such pharmaceutical compositions and unit dosage forms thereof may comprise conventional ingredients in conventional proportions, with or without additional active compounds or principles, and such unit dosage forms may contain any suitable effective amount of the active ingredient commensurate with the intended daily dosage range to be employed.
  • Formulations containing ten (10) milligrams of active ingredient or, more broadly, 0.1 to two hundred (200) milligrams, per tablet, are accordingly suitable representative unit dosage forms.
  • the compounds of the present invention can be administered in a wide variety of oral and parenteral dosage forms. It will be obvious to those skilled in the art that the following dosage forms may comprise, as the active component, either a compound of the invention or a pharmaceutically acceptable salt or derivative of the compound of the invention.
  • pharmaceutically acceptable carriers can be either solid or liquid.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances which may also act as diluents, flavouring agents, solubilisers, lubricants, suspending agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the carrier is a finely divided solid which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding capacity in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from five or ten to about seventy percent of the active compound.
  • Suitable carriers are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating material as carrier providing a capsule in which the active component, with or without carriers, is surrounded by a carrier, which is thus in association with it.
  • cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogenous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • Formulations suitable for vaginal administration may be presented as pessaries, tampons, 5 creams, gels, pastes, foams or sprays containing in addition to the active ingredient such carriers as are known in the art to be appropriate.
  • Liquid form preparations include solutions, suspensions, and emulsions, for example, water or water-propylene glycol solutions.
  • parenteral injection liquid 10 preparations can be formulated as solutions in aqueous polyethylene glycol solution.
  • the compounds according to the present invention may thus be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusion
  • compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilising and/or dispersing agents.
  • the active ingredient may be in powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile,
  • Aqueous solutions suitable for oral use can be prepared by dissolving the active component in water and adding suitable colorants, flavours, stabilising and thickening agents, as desired. 25
  • Aqueous suspensions suitable for oral use can be made by dispersing the finely divided active component in water with viscous material, such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • viscous material such as natural or synthetic gums, resins, methylcellulose, sodium carboxymethylcellulose, or other well known suspending agents.
  • solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration.
  • liquid forms include solutions, suspensions, and emulsions.
  • These preparations may contain, in addition to the active component, colorants, flavours, stabilisers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilising agents, and the like.
  • the compounds according to the invention may be formulated as ointments, creams or lotions, or as a transdermal patch.
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents, thickening agents, or colouring agents.
  • Formulations suitable for topical administration in the mouth include lozenges comprising active agent in a flavoured base, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert base such as gelatin and glycerin or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.
  • Solutions or suspensions are applied directly to the nasal cavity by conventional means, for example with a dropper, pipette or spray.
  • the formulations may be provided in single or multidose form. In the latter case of a dropper or pipette, this may be achieved by the patient administering an appropriate, predetermined volume of the solution or suspension. In the case of a spray, this may be achieved for example by means of a metering atomising spray pump.
  • the compounds according to the invention may be encapsulated with cyclodextrins, or formulated with their agents expected to enhance delivery and retention in the nasal mucosa.
  • Administration to the respiratory tract may also be achieved by means of an aerosol formulation in which the active ingredient is provided in a pressurised pack with a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • a suitable propellant such as a chlorofluorocarbon (CFC) for example dichlorodifluoromethane, trichlorofluoromethane, or dichlorotetrafluoroethane, carbon dioxide, or other suitable gas.
  • CFC chlorofluorocarbon
  • the aerosol may conveniently also contain a surfactant such as lecithin.
  • the dose of drug may be controlled by provision of a metered valve.
  • the active ingredients may be provided in the form of a dry powder, for example a powder mix of the compound in a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • a suitable powder base such as lactose, starch, starch derivatives such as hydroxypropylmethyl cellulose and polyvinylpyrrolidone (PVP).
  • the powder composition may be presented in unit dose form for example in capsules or cartridges of, e.g., gelatin, or blister packs from which the powder may be administered by means of an inhaler.
  • the compound In formulations intended for administration to the respiratory tract, including intranasal formulations, the compound will generally have a small particle size for example of the order of 1 to 10 microns or less. Such a particle size may be obtained by means known in the art, for example by micronization.
  • formulations adapted to give sustained release of the active ingredient may be employed.
  • the pharmaceutical preparations are preferably in unit dosage forms.
  • the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • Tablets or capsules for oral administration and liquids for intravenous administration are preferred compositions.
  • the compounds of the present invention have been found to inhibit the growth of mammalian cancers.
  • a method of treatment or prophylaxis of cancer in a mammal comprising administering an effective amount of a compound of formula (I):
  • R 2 is H or alkyl,
  • R 3 is H or halo,
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R 8 and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H, CO 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R 11 is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R 12 and R 13 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof, stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers.
  • mammal as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer).
  • livestock animals eg. sheep, pigs, cattle, horses, donkeys
  • laboratory test animals eg. mice, rabbits, rats, guinea pigs
  • companion animals eg. dogs, cats
  • captive wild animals eg. foxes, kangaroos, deer.
  • the mammal is human or a laboratory test animal. Even more preferably, the mammal is a human.
  • an "effective amount” means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated.
  • the amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • An effective amount in relation to a human patient may lie in the range of about 0.1 ng per kg of body weight to 1 g per kg of body weight per dosage.
  • the dosage is preferably in the range of l ⁇ g to 1 g per kg of body weight per dosage, such as is in the range of lmg to Ig per kg of body weight per dosage. In one embodiment, the dosage is in the range of 1 mg to 500mg per kg of body weight per dosage. In another embodiment, the dosage is in the range of 1 mg to 250 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 mg to 100 mg per kg of body weight per dosage, such as up to 50 mg per kg of body weight per dosage. In yet another embodiment, the dosage is in the range of 1 ⁇ g to 1 mg per kg of body weight per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response.
  • doses may be administered daily, weekly, monthly or other suitable time intervals, or the dose may be proportionally reduced as indicated by the exigencies of the situation.
  • the dosage may be administered continuously of a given time period, such as minutes, hours or days.
  • treatment does not necessarily imply that a subject is treated until total recovery.
  • prophylaxis does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.
  • the term “prophylaxis” may be considered as reducing the severity or onset of a particular condition. “Treatment” may also reduce the severity of an existing condition.
  • the present invention further contemplates a combination of therapies, such as the administration of the compounds of the invention or pharmaceutically acceptable salts or prodrugs thereof together with the subjection of the mammal to other agents or procedures which are useful in the treatment of cancers.
  • the compounds of the present invention may be administered in combination with other chemotherapeutic drugs, or with other treatments such as radiotherapy.
  • chemotherapeutic drugs include, but are not limited to, cisplatin, cyclophosphamide, doxorubicin, etoposide phosphate, paclitaxel and vincristine.
  • cancer refers to any benign or malignant growth or tumour caused by abnormal and uncontrolled cell division and which may occur in tissues, the lymphatic system or the blood stream and may spread to other parts of the body. Cancer cells can invade nearby tissues and can spread through the bloodstream and lymphatic system to other parts of the body.
  • Carcinoma is cancer that begins in the skin or in tissues that line or cover internal organs.
  • Sarcoma is cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.
  • Blastoma is a cancer or tumour that arises from embryonic tissue.
  • Leukemia is cancer that starts in blood-forming tissue such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the bloodstream.
  • Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.
  • the cancer that may be treated by the present invention includes benign or malignant growth or tumours or abnormal and uncontrolled cell division in tissues including, but not limited to, squamous cell cancers, central nervous system tumours such as brain tumours; lungs cancer such as small lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung; ovarian cancer, endometrial or uterine cancer, cervical cancer, testicular cancer, stomach cancer, colon cancer, prostate cancer, bowel cancer, liver cancer, kidney or renal cancer, tongue cancer, throat cancer, bone cancer, blood cancers, for example leukemia, lymphatic cancer, breast cancer, skin cancers, for example melanoma, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, rectal cancer, colorectal cancer, salivary gland carcinoma, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer
  • the compounds of the present invention have been found to have an antibacterial effect.
  • a method of treatment or prophylaxis of a bacterial infection in a mammal comprising administering an effective amount of a compound of formula (I):
  • R 2 is H or alkyl,
  • R 3 is H or halo,
  • R 4 , R 5 , R 6 and R 7 are independently selected from H 3 alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R s and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H, CO 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R 11 is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R and R are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof, stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers.
  • the bacterial infection may be caused by a Gram positive or Gram negative bacterium, especially Gram positive bacteria including all bacteria belonging to the genus Bacillus (e.g. B, subtilis, B. anthracis, B. cereus, B. firmis, B. licheniformis, B. megaterium, B. pumilus, B. coagulans, B. pantothenticus, B. alvei, B. brevis, B, circulans, B. laterosporus, B. macerans, B. polymyxa, stearothermophilus, B. thuringiensis, sphaericus), Staphylococcus (e.g. S. aureus, S. epidermidis, S.
  • Bacillus e.g. B, subtilis, B. anthracis, B. cereus, B. firmis, B. licheniformis, B. megaterium, B. pumilus, B. coagulans, B. pantothenticus, B.
  • Streptococcus e.g. S. pyogenes, S. pneumoniae, S, agalactiae, S. pyogenes, S. agalactiae, S. dysgalactiae, S. equisimilis, S. equi, S. zooepidemicus, S. anginosus, S. salivarius, S. milleri, S. sanguis, S. mitior, S. mutans, S. faecalis, S.faecium, S. bovis, S. equinus, S. uberus, S.
  • Streptococcus e.g. S. pyogenes, S. pneumoniae, S, agalactiae, S. pyogenes, S. agalactiae, S. dysgalactiae, S. equisimilis, S. equi, S. zooe
  • R 2 is H or alkyl,
  • R 3 is H or halo,
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R 8 and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H 5 C0 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R 11 is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R 12 and R 13 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof and stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers in the manufacture of a medicament for the treatment or prophylaxis of cancer.
  • R 2 is H or alkyl,
  • R 3 is H or halo,
  • R 4 , R 5 , R 6 and R 7 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, one of R 8 and R 9 is H and the other is OR 11 ;
  • R 10 is selected from H, alkyl, alkenyl, alkynyl, OH, Oalkyl, CO 2 H, CO 2 alkyl, CO 2 NH 2 , CO 2 NH(alkyl) and CO 2 N(alkyl) 2 ;
  • R is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety, represents a single or double bond;
  • R 12 and R 13 are independently selected from H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl and a sugar moiety; and pharmaceutically acceptable salts thereof, stereoisomers and tautomers thereof and mixtures of stereoisomers and/or tautomers in the manufacture of a medicament for the treatment or prophylaxis of a bacterial infection.
  • Figure 1 graphically depicts the effect of compound 3 on Colo205 xenografts in nude mice compared to vehicle-treated and control mice.
  • the media selected were: (a) ISP2 liquid medium and (b) rice flour liquid medium and solid phase: (a) ISP2 agar, (b) barley grain and (c) wheat grain (Table 1).
  • Each media was incubated at 28 °C for a range of times as defined in Table 2. The incubation times were selected based on previous optimal times for other actinomycete cultures. At the conclusion of the incubation each treatment was processed as follows:
  • the liquid media were centrifuged at 10,000 rpm for 30 min to pellet the mycelia (approximately, 10 to 15 mL).
  • the supernatant was decanted as a separate fraction and the mycelia extracted with methanol (MeOH) to generate a mycelial extract.
  • the decanted supernatant was concentrated in vacuo and/or freeze dried, then extracted with solvent to yield a supernatant extract.
  • the fermentation included additional treatment.
  • “Taped” refers to sealing of the agar plate with parafilm to assess the effects of limiting air oxidation.
  • C 18 resin refers to the addition of a Cl 8 resin to provide a hydrophobic support for growth of the microorganism.
  • “Nutrients” refers to periodic addition of ISP2 media during incubation.
  • the individual extracts from the fermentation were pooled to provide 4 L of methanolic extract.
  • This extract was concentrated in vacuo to an aqueous residue (500' mL) that was diluted with H 2 O to a final volume of 2 L.
  • This cloudy solution was passed through two parallel C 18 solid phase extraction (SPE) cartridges (2 x 10 g, Varian HF C 18 ), eluting with 50% aqueous MeOH (2 x 80 mL each) followed by elution with 100% MeOH (2 x 80 mL each).
  • SPE solid phase extraction
  • the pooled MeOH fraction (160 mL) was defatted with hexane and the residue (875 mg) dissolved in a mixture of dimethylsulfoxide (DMSO) and MeOH, and fractionated by preparative HPLC (60 mL/min with gradient elution of 70% to 10% H 2 OMeCN over 20 minutes followed by acetonitrile (MeCN) for 10 min, through a 5 ⁇ m Platinum EPS C 18 50 x 100 mm column). One hundred fractions were collected, concentrated in vacuo and combined on the basis of analytical HPLC analysis.
  • DMSO dimethylsulfoxide
  • MeOH acetonitrile
  • the fractions containing the major kibdelone metabolites were pooled (248 mg) and fractionated by HPLC (10 mL/min gradient of 35% H 2 O/MeCN to 45% H 2 0/MeCN over 20 min through a Phenomenex LUNA C 18 5 ⁇ m (2) 250 x 10 mm column) to yield Compound (1) (kibdelone A) (26 mg, 0.6 % yield), Compound (2) (kibdelone B) (52 mg, 1.1% yield), Compound (3) (kibdelone C) (97 mg, 2.0 % yield) and Compound (6) (kibdelone B rhamnoside) (5.2 mg, 0.11 % yield). All yields are calculated relative to the active 80% MeOH fraction from the enriched EtOAc residue.
  • Assignments may be interchanged. Assignments are made in comparison with literature data for reported compounds and biosynthetic analogues from the same culture. c Assignments are based on HMBC correlations and the exact chemical shifts can be interchanged.
  • Assignments may be interchanged. Assignments are made in comparison with literature data for reported compounds and biosynthetic analogues from same culture.
  • Table 8 LCMS (DAD @ 254 nm and ESI( ⁇ )MS) data comparison of kibdelones A-C (1-3) and bromokibdelones A-C (9-11)
  • the anticancer activities of Compounds 1 to 3 were determined in vitro against the following human cancer cell lines; breast (MCF7), prostate (DU145), melanoma (MM96L), pigmented melanoma (MM418c5), ovarian (C 180- 13 s) and leukemia (K562).
  • AU samples were also tested against a non-cancerous human normal fibroblast (NFF) cell line.
  • the IC 50 values expressed in ng/mL are listed in Table 9.
  • the experimental and analytical methods employed were as follows:
  • the medium was removed from the plates, and they were washed twice with PBS.
  • the cells were then fixed with methylated spirits for a minimum of 15 minutes. After this time the cells were washed with water twice and the fixed cells stained with 50 ⁇ L/well of SRB solution (0.4% SRB (w/v) in 1% (v/v) acetic acid) over a period of at least 30 minutes.
  • SRB solution was then removed from the wells and the plates were then rapidly washed twice with 1% (v/v) acetic acid.
  • Protein bound dye was then solubilised by addition of 100 ⁇ L of 10 mM unbuffered Tris and incubated at room temperature for 15 minutes. Plates were then read at 564 nm on a VERSA max tuneable microplate reader (Molecular Devices, Sunnyvale, CA). Data was presented as a percentage of control cell protein.
  • Example 7 Biological Activity in NCI 60-cell line panel
  • the NCI 60-cell line panel screen utilizes 60 different human tumour cell lines representing leukaemia, melanoma, cancers of the lung, colon, brain, ovary, breast, prostate and kidney.
  • the human tumour cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.
  • RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine.
  • cells are inoculated into 96 well microtiter plates in
  • microtiter plates 100 ⁇ L at plating densities ranging from 5000 to 40000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO 2 , 95% air and 100% relative humidity for 24 hours prior to addition of experimental drugs.
  • the plates are incubated for an additional 48 hours at 37 0 C, 5% CO 2 , 95% air, and 100% relative humidity.
  • the assay is terminated by the addition of cold TCA.
  • Cells are fixed in situ by the gentle addition of 50 ⁇ L of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4 °C. The supernatant is discarded, and the plates are washed five times with tap water and air dried.
  • Sulforhodamine B (SRB) solution 100 ⁇ L) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature.
  • GI 50 Growth inhibition of 50%
  • C-Tz C-Tz
  • TGI total growth inhibition
  • the LC 50 concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning
  • Compounds 1-3 displayed similar profiles with high Pearson's correlation coefficients between all three compounds (data not shown).
  • a Pearson's correlation coefficient is a measure of the strength of association between two variables. With similar mean profiles across the 60 cell line screen, it is thought that Compounds 1-3 operate by a common mode of action.
  • the human tumour cell line Colo205 was implanted onto nude mice and treatments were carried out i.p. with 20 ⁇ g of Compound 3 daily. A control vehicle group and a no-vehicle control group were included. Test Compound 3 was pre-weighed in 80 ⁇ g aliquots in screw cap microfuge tubes and stored at -20 0 C. Compound 3 (80 ⁇ g) was dissolved in 10 ⁇ L of DMSO then diluted with 390 ⁇ L of 25% propylene glycol in saline to give 200 ⁇ g/mL of Compound 3. Injection of 100 ⁇ L then gave a dose of 20 ⁇ g. Placebo (control vehicle) was made up in the same way, but without the compound. Cultured human tumour cells (Colo 205 colon tumour cells; 2 million cells/site) were injected into the flanks of mice and growth measured over time, during treatment with the test compounds.
  • mice were monitored by the following clinical assessment criteria for distress during the period of the experiment to determine whether the treatments described in this application (i.e., tumour burden and effects of drugs) are causing distress to the mice to a degree to where they should be euthanased.
  • treatments described in this application i.e., tumour burden and effects of drugs
  • Total tumour burden was limited to 1 gm/mouse (e.g., Teicher et ah, p91).
  • the commonly- used literature method was used to calculate volume:
  • mice were euthanased if the above score reaches 3 on the above cumulative criteria, or if the total tumour burden/mouse reaches the above dimensions.
  • mice There were 3 mice in a non-injected control group, 3 mice in the control vehicle group, and
  • ProTOX (alternatively referred to herein as Bs) is an antibacterial bioassay, broadly applicable to most aerobic and anaerobic bacteria.
  • the bioassay features a solid phase agar base into which the test compound has been incorporated together with a chromogen. As the bacteria multiply in the well, the chromogen is metabolised from blue in a two-step process to a colourless compound. Compounds with potent bactericidal activity inhibit bacterial metabolism of the chromogen while bacteriostatic compounds induce limited metabolism as indicated by an intermediate pink colour.
  • ProTOX is broadly applicable to a range of gram-positive and gram-negative bacteria under aerobic and microaerophilic conditions. ProTOX assays were carried out using Bacillus subtilis.
  • Bioassay with ProTOX provided antibacterial activity for fractions containing kibdelones with LD 99 between 1 and 11 ⁇ g mL '1 .
  • Testing of isolated compounds 1-3 with Bacillus subtilis and compound 1 with Escherichia coli also provided good antibacterial results as shown in Table 17.

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Abstract

La présente invention concerne des polycétide-xanthones de la classe des kibdélones et leur application en tant qu'agents antibactériens et anticancéreux. La présente invention concerne également des méthodes de synthèse de kibdélones, des compositions pharmaceutiques les contenant et leur application dans le traitement des cancers et des infections bactériennes.
PCT/AU2007/000228 2006-02-27 2007-02-27 Polycétide-xanthones et applications WO2007095696A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009085359A2 (fr) * 2007-10-04 2009-07-09 Novobiotic Pharmaceuticals Llc Antibiotique à base de citreamicine isolé du gendre nocardia
WO2009085360A3 (fr) * 2007-10-04 2009-11-12 Novobiotic Pharmaceuticals Llc Antibiotique à base de citreamicine doté d'un résidu glucidique
WO2009085359A3 (fr) * 2007-10-04 2009-12-30 Novobiotic Pharmaceuticals Llc Antibiotique à base de citreamicine isolé du gendre nocardia
WO2009085360A2 (fr) * 2007-10-04 2009-07-09 Novobiotic Pharmaceuticals Llc Antibiotique à base de citreamicine doté d'un résidu glucidique
ES2368236A1 (es) * 2009-12-23 2011-11-15 MERCK SHARP & DOHME CORP. Agentes antibacterianos.
US11459635B2 (en) 2010-07-14 2022-10-04 Vacuumschmelze Gmbh & Co. Kg Device and method for the production of a metallic strip
CN103992333A (zh) * 2014-05-28 2014-08-20 中国人民解放军军事医学科学院毒物药物研究所 色原酮二聚体衍生物及其制备方法和用途
NO20160680A1 (en) * 2016-04-22 2017-10-23 Marbileads New antimicrobial compound, a method of producing it, pharmaceutical compositions and uses in therapy
NO341815B1 (en) * 2016-04-22 2018-01-29 Marbileads New antimicrobial compound, a method of producing it, pharmaceutical compositions and uses in therapy
CN109195971A (zh) * 2016-04-22 2019-01-11 挪威科技工业研究院Tto股份公司 新的抗微生物化合物
US10494375B2 (en) 2016-04-22 2019-12-03 Sintef Tto As Antimicrobial compound
CN109195971B (zh) * 2016-04-22 2021-08-27 挪威科技工业研究院Tto股份公司 抗微生物化合物
WO2017182632A1 (fr) * 2016-04-22 2017-10-26 Marbileads As Nouveau composé antimicrobien
CN109956951A (zh) * 2019-04-11 2019-07-02 中国医学科学院医药生物技术研究所 鹿色霉素(cervinomycin)C1、C2、C3、C4及其生产方法及应用

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