POLYKETIDE XANTHONES AND USES THEREOF

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. For example, 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) and simaomicins are known to have antibacterial and antiparasitic activity and may sensitise cancer cells to anticancer agents (Arai et al. , 2004).

However, new treatments for cancer and bacterial infections are urgently required. The present invention is predicated in part on the determination that the newly discovered kibdelone polyketide xanthones have potent antitumour activity and antibacterial activity.

In one aspect of the invention, there is provided a compound of formula (I):

( I )

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ ; 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; and

A is

in which 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 preferred compounds of formulae (I) at least one of the following apply

R ¹ is H, alkyl, acyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ , especially H or alkyl, more especially C _1-4 alkyl, most especially methyl; 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:

OR OR

(l) R = Hkibdelone A (2) R = H kibdelone B (5) R = rha kibdelone A rhamnoside (6) R = rha kibdelone B rhamnoside

especially Compound (1), (kibdelone A), Compound (2) (kibdelone B) and Compound (3), (kibdelone C). The structural formulae provided above for Compounds (1) to (16) depict relative stereochemistry only and are not intended to infer absolute stereochemistry of kibdelone compounds.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

As used herein, the term "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. Examples of straight chain and branched alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, rø-pentyl and branched isomers thereof, rc-hexyl and branched isomers thereof, n-heptyl and branched isomers thereof, rø-octyl and branched isomers thereof, «-nonyl and branched isomers thereof, and rø-decyl and branched isomers thereof. Examples of cyclic alkyl include mono- or polycyclic alkyl residues such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl and the like.

The term "alkenyl" as used herein 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 ). Examples of alkenyl 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

1 ,3,5,7-cyclooctatetraenyl.

As used herein the term "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-proρynyl, 2-propynyl, and butynyl isomers, and pentynyl isomers.

The term "acyl" denotes a residue containing the moiety C=O (and not being a carboxylic acid, ester or amide). 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. Examples of acyl 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, toluoyl and naphthoyl; aralkanoyl such as phenylalkanoyl (e.g. phenylacetyl, phenylpropanoyl, phenylbutanoyl, phenylisobutylyl, phenylpentanoyl and phenylhexanoyl) and naphthylalkanoyl (e.g. naphthylacetyl, naphthylpropanoyl and naphthylbutanoyl]; aralkenoyl such as phenylalkenoyl (e.g. phenylpropenoyl, phenylbutenoyl, phenylpentenoyl and phenylhexenoyl and naphthylalkenoyl (e.g. naphthylpropenoyl, naphthylbutenoyl and naphthylpentenoyl); aryloxyalkanoyl such as phenoxyacetyl and phenoxypropionyl; heterocycliccarbonyl; heterocyclicalkanoyl such as thienylacetyl, thienylpropanoyl, thienylbutanoyl, thienylpentanoyl, thienylhexanoyl, thiazolylacetyl, thiadiazolylacetyl and tetrazolylacetyl; and heterocyclicalkenoyl such as heterocyclicpropenoyl, heterocyclicbutenoyl, heterocyclicpentenoyl and heterocyclichexenoyl.

As used herein, the term "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.

The term "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. Suitable examples of 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, benzoxazolyl, benzothiazolyl and the like.

As used herein, the term "halo" refers to fluoro, chloro, bromo and iodo substituents.

As used herein, the term "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.

The term "sugar moiety" as used herein 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.

The compounds of the invention may be in the form of complexes with metal ions. Suitable metal ions include divalent and trivalent metal ions. For example, 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 ⁺ ^.

It will also be recognised that many compounds of the invention possess asymmetric centres and are therefore capable of existing in more than one stereoisomeric form. 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. Alternatively, 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. For example, and without wishing to be bound by theory, it is proposed that, under suitable conditions, 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).

Other compounds of formula (I) may be prepared by partial synthesis from compounds isolated during fermentation using synthetic procedures known in the art. For example, 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. For example, 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 hydrosulfϊte (Na ₂ S ₂ O ₄ ), to yield compounds such as Compounds (4) or (8).

The 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).

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. For example, 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. For example 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. In particular, 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. In particular, 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. For example, 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.

In another aspect of the invention there is provided a process for preparing compounds of formula (I) comprising culturing a microorganism that produces kibdelone compounds.

In preferred embodiments, the microorganism that produces kibdelone compounds is an actinomycete microorganism from the genus Kibdelosporangium. In particular, 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.

As used herein, the term "mutant" refers to a microorganism having similar genetic identity and which has retained the ability to produce kibdelone compounds. For example,

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.

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.

(MST-108465).

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.

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. Examples of simple nitrogen compounds include, but are not limited to, amino acids and mixtures thereof. Examples of complex nitrogen compounds include, but are not limited to, peptides and proteins. Examples of 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.

In order to extract a liquid fermentation, 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).

In another aspect of the invention, there is provided an isolated microorganism which is a strain of Kibdelosporangium designated MST-108465 or a mutant thereof.

While it is possible that, for use in therapy, 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.

Thus, in a further aspect of the invention there is provided a pharmaceutical composition comprising a compound of formula (I):

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=CCa^yI)R ¹ °;

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; and

A is

in which 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.

Pharmaceutical formulations 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. The compounds of the invention, together with a conventional adjuvant, carrier, or diluent, 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.

For preparing pharmaceutical compositions from the compounds of the present 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.

In powders, the carrier is a finely divided solid which is in a mixture with the finely divided active component.

In tablets, 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. Similarly, cachets and lozenges are included. Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid forms suitable for oral administration.

For preparing suppositories, a low melting wax, such as a mixture of fatty acid glycerides or cocoa butter, is first melted and 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. For example, 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

15 or in multi-dose containers with an added preservative. The 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. Alternatively, 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,

20 pyrogen-free water, before use.

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.

30 Also included are solid form preparations which are intended to be converted, shortly before use, to liquid form preparations for oral administration. Such 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.

For topical administration to the epidermis 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. To improve nasal delivery and retention 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. The aerosol may conveniently also contain a surfactant such as lecithin. The dose of drug may be controlled by provision of a metered valve.

Altematively 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).

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.

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.

When desired, formulations adapted to give sustained release of the active ingredient may be employed.

The pharmaceutical preparations are preferably in unit dosage forms. In such form, 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. Also, 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. Thus in another aspect of the present invention, there is provided a method of treatment or prophylaxis of cancer in a mammal comprising administering an effective amount of a compound of formula (I):

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ ; 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; and

A is

in which 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.

The term "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). Preferably, 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, for example, 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 lμg 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 μg to 1 mg per kg of body weight per dosage. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided 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. Alternatively, the dosage may be administered continuously of a given time period, such as minutes, hours or days.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "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. For example, the compounds of the present invention may be administered in combination with other chemotherapeutic drugs, or with other treatments such as radiotherapy. Suitable chemotherapeutic drugs include, but are not limited to, cisplatin, cyclophosphamide, doxorubicin, etoposide phosphate, paclitaxel and vincristine.

As used herein, the term "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. There are several main types of cancer. 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. In some embodiments the cancer treated is breast cancer, prostate cancer, melanoma, ovarian cancer, lung cancer, colon cancer, CNS cancer, kidney cancer or leukemia.

The compounds of the present invention have been found to have an antibacterial effect. Thus in another aspect of the present invention, there is provided a method of treatment or prophylaxis of a bacterial infection in a mammal comprising administering an effective amount of a compound of formula (I):

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ ; 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; and

A is

in which 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. haemolyticus, S. saprophytics), 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. avium), Aerococcus, Gemella, Corynebacterium, Listeria, Kurthia, Lactobacillus, Erysipelothrix, Arachnia, Actinomyces, Propionibacterium, Rothia, Bifidobacterium, Clostridium, Eubacterium, Nocardia, Mycobacterium, Neisseria, Haemophilus, Bordetella, Francisella, Brucella, Yersinia, Vibrio, Campylobacter, Helicobacter, Pseudomonas, Burkholderia, Legionella, Chlamydia, Mycoplasma and Rickettsia.

In yet another aspect of the present invention, there is provided a use of a compound of formula (I):

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ ; 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; and

A is

in which 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.

In yet a further aspect of the invention there is provided a use of a compound of formula (I):

wherein R ¹ is H, alkyl, alkenyl, alkynyl, acyl, aryl, arylalkyl, heterocyclyl, heterocyclylalkyl, NH ₂ , NH(alkyl), N(alkyl) ₂ or N=C(alkyl)R ¹⁰ ; 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; and

A is

in which 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.

The invention will now be described with reference to the following Examples that illustrate some preferred aspects of the present invention. However, it is to be understood that the particularity of the following description of the invention is not to supersede the generality of the preceding description of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 graphically depicts the effect of compound 3 on Colo205 xenografts in nude mice compared to vehicle-treated and control mice.

EXAMPLES

Example 1: Fermentation of Kibdelosporangium (MST-108465)

Mixed media fermentation of MST-108465

A spore suspension of MST-108465, stored at -20 ⁰ C was thawed and used to inoculate a selection of solid and liquid media to explore the culture's metabolic diversity. 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:

(1) 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.

(2) The solid phases were excised from the Petri plate and transferred to a flask or vial and extracted with MeOH. The amounts of MeOH varied with the nature of the media. Generally, a solid medium was extracted 1 mL per 1 g with MeOH by gentle mixing and shaking overnight. However, for grains, a larger volume (2 mL per g) was necessary to ensure coverage of the grain during extraction.

The addition of nutrients during the fermentation resulted in an increase in production, as did an increase in medium volume.

Table 1: Media ingredients and preparation

Table 2: Production of extracts of MST-108465

*Additional extraction of the grains to generate a second and third extract was undertaken to provide supplementary extracts for the isolation of the active metabolites.

**In some cases, 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.

Fermentation of MST-108465 on barley grain Barley grain gave the same distribution of kibdelone-like metabolites but in higher yields than obtained on ISP2 agar. To isolate the remaining minor analogues, not isolated in the individual extracts of different fermentation conditions, a series of solid phase fermentations using barley was undertaken. Solid fermentations (7 x 500 g barley, incubated for 18 days at 28 °C) were extracted with MeOH (7 x 1.5 L). The extracts were

concentrated in vacuo and pooled to give an aqueous residue (2.6 L) and extracted with ethyl acetate (EtOAc) (2.6 L).

Example 2: Isolation of compounds from the fermentation extracts

Isolation of compounds of formula (I) from pooled media culture

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). 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 μm 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.

The most polar of the fractions (18.2 mg) were pooled and fractionated by HPLC (10 mL/min isocratic 35% H ₂ OZMeCN over 20 min through a Phenomenex LUNA C ₁₈ 5μm (2) 250 x 10 mm column), to yield Compound (1) (kibdelone A) (3.2 mg, 0.37 % yield) and Compound (3) (kibdelone C) (1.7 mg, 0.19 % yield).

Isolation of compounds of formula (I) from barley grain fermentation The MeOH extracts from the barley grain fermentation were concentrated in vacuo and pooled to give an aqueous residue (2.6 L) which was extracted with EtOAc (2.6 L). The EtOAc was evaporated in vacuo to yield 8 g of an enriched residue. The residue was dissolved in MeOH and diluted with water to approximately 10% and passed through four parallel C ₁₈ solid phase extraction (SPE) cartridges (4 x 10 g, Varian HF C ₁₈ ). The bound metabolites were eluted with 80% MeOH (4 x 80 mL each), followed by elution with 100% MeOH (4 x 80 mL each) and then 100% EtOAc (4 x 80 mL each). HPLC and

bioassay confirmed the presence of the activity and non-polar metabolites in the 80% MeOH fraction (4.9 g).

Residual amounts of compounds remaining in the aqueous phase after EtOAc extraction were recovered by adsorption onto two parallel C ₁₈ solid phase extraction (SPE) cartridges (2 x 10 g, Varian HF C ₁₈ ) and eluted with 100% MeOH (2 x 80 mL each), followed by 100% EtOAc (2 x 80 mL each). HPLC and bioassay confirmed the presence of compounds of formula (I) which were pooled to yield 6.4 g of enriched residues.

Duplicate batches of the enriched residues (2 x 2 g) were dissolved in a mixture of DMSO and MeOH and subjected to preparative HPLC (60 mL/min with gradient elution of 25% to 55% H ₂ OMeCN over 20 minutes followed by MeCN for 10 minutes, through a 5 μm 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.

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 5μm (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.

Spectroscopic characterisation of compounds of formula (I) are set out below:

Compound 1 (kibdelone A)

Orange amorphous solid [α] _D +72°(c 0.01, CHCl ₃ ); UV (EtOH) λ _max /nm (ε/dm ³ mol ^"1 cm- ¹ ) 447(sh), 420 (11000), 311 (20000), 254 (24900), 214 (24500) nm; ¹ H NMR and ¹³ C NMR tø-DMSO, 600 MHz and 150 MHz respectively) Table 3; ESI(±)MS (10OkV) m/z 582.1/584.2 [M+H] ⁺ , 580.0/582.1 [M-H] ^" ; HRESI(+)MS 604.0996 ([M+Na] ⁺ , C ₂₉ H ₂₄ ³⁵ ClNO ₁₀ Na requires 604.0986).

Table 3: NMR tø-DMSO, 600MHz) assignments for kibdelone A (1)

Assignments are made in comparison with literature data and co-metabolites.

Compound 2 (kibdelone B)

Orange amorphous solid [α] _D +157°(c 0.01, CHCl ₃ ); UV (EtOH) λ _max /nm (ε/dm ³ mol ^"1 cm ^"1 ) 444 (sh), 402 (9670), 308 (sh), 258 (23800), 208 (22000) nm; ¹ H NMR and ¹³ C NMR tø-DMSO, 600 MHz and 150 MHz respectively) Table 4; ESI(±)MS (10OkV) m/z 584.4/586.2 [M+H] ⁺ , 582.1/584.0 [M-H] ^" ; HRESI(+)MS 606.1128 ([M+Naf, C ₂₉ H ₂₆ ³⁵ ClNO ₁₀ Na requires 606.1143).

¹ 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.

Compound 6 (kibdelone B rhamnoside)

Orange amorphous solid [α] _D +150°(c 0.015, CHCl ₃ ); UV (EtOH) λ _max /nm (ε/dm ³ mol ^'1 cm ^'1 ) 435(5700), 308 (sh), 258 (1630O) ₅ 205 (18600) nm; ¹ H NMR and ¹³ C NMR (d ₆ - DMSO, 600 MHz and 150 MHz respectively) Table 5; ESI(±)MS (10OkV) m/z 730.2/732.2 [M+H] ⁺ 728.0/730.0 [M-H] ^' ; HRESI(+)MS 752.1740 ([M+Na] ⁺ , C ₃₅ H ₃₆ ³⁵ ClNO ₁₄ Na requires 752.1722).

Table 5: NMR (ck-DMSO, 600MHz) assignments for kibdelone B rhamnoside (6)

¹ Assignments may be interchanged. Assignments are made in comparison with literature data for reported compounds and biosynthetic analogues from same culture.

Compound 3 (kibdelone C)

Yellow amorphous solid [α] _D +49°(c 0.02, CHCl ₃ ); UV (EtOH) λ _max /nm (ε/dm ³ mol ^"1 cm- ¹ ) 396 (20100), 339 (sh), 308 (sh), 272 (sh), 258 (27500), 217 (22800) nm; ¹ H NMR and ¹³ C NMR tø-DMSO, 600 MHz and 150 MHz respectively) Table 6; ESI(±)MS (10OkV) m/z 586.2/588.2 [M+H] ⁺ 584/586 [M-H] ^' ; HRESI(+)MS 608.1259 ([M+Na] ⁺ , C ₂₉ H ₂₈ ³⁵ ClNO ₁₀ Na requires 608.1299).

The assignments for the chelated phenolic OH and the associated carbon assig ;:nments may interchange, Assignments are made in comparison with literature data for reported compounds and biosynthetic analogues from same culture.

Example 3: Interconversion of Compounds 1, 2 and 3

Samples of Compound 1 (kibdelone A), Compound 2 (kibdelone B) and Compound 3

(kibdelone C) were heated at 40°C in MeOH for 2 hours then analysed by LC/MS. HPLC conditions used were standard analytical conditions: 1 mL/minute gradient elution from 90% H ₂ OMeCN (0.05% HCOOH) to MeCN (0.05% HCOOH) over 15 minutes, followed

by a 5 minute flush with MeCN on a 5 μm Zorbax Stable Bond C ₈ 15O x 4.6 mm column. LC/MS analysis (DAD 254 nm, ESI (±) MS) confirmed that Compound (3) (kibdelone C) converts to Compound (2) (kibdelone B), and that Compound (2) (kibdelone B) converts to both Compound (3) (kibdelone C) and Compound (1) (kibdelone A), and that Compound (1) (kibdelone A) does not undergo conversion to either of Compounds (3) or (2).

Example 4: Reduction of Compound 1 (kibdelone A) to give (Compound 4) (dihydrokibdelone A)

Compound 1 (0.5 mg) was dissolved in J ₆ -DMSO (300 μL) in an NMR tube and a small amount of Na ₂ S ₂ O ₄ was added. ¹ H NMR (600 MHz) experiments were carried out immediately and after 24 hours of standing. A colour change was observed from orange to greenish yellow, which disappeared with vigorous shaking, suggesting a possible equilibrium between quinone and hydroquinone. ¹ H NMR and ¹³ C NMR (Jg-DMSO, 600 MHz and 150 MHz respectively) are shown in Table 7.

Table 7: NMR tø-DMSO, 600MHz) assignments for dihydrokibdelone A (6)

Assignments are based on gHMBC correlations. C NMR assignments were not possible as direct carbon spectrum was not obtained.

Example 5: Preparation of Compounds (9-11) (bromokibdelones A-C)

Mixed media fermentation of MST-108465

A spore suspension of MST-108465, stored at -20 ⁰ C was thawed and used to inoculate ISP2 agar containing 0.5% NaBr, and the agar incubated at 28 °C for 7 days. The agar was then extracted overnight with MeOH (1 mL per g agar) to prepare a crude extract. The crude extract was analysed by LC/D AD/MS to reveal the production of kibdelones in which the chloro substituent had been replaced by a bromo substituent. Comparative retention times and MS data for kibdelones A-C (1-3) and bromokibdelones A-C (9-11) are documented in Table 8.

Table 8: LCMS (DAD @ 254 nm and ESI(±)MS) data comparison of kibdelones A-C (1-3) and bromokibdelones A-C (9-11)

* The Cl: Cl isotope ratio was 3:1 ; ** The B Br isotope ratio was 1:1

Example 6: Biological activity

The anticancer activities of Compounds 1 to 3 (kibdelones A-C) 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:

Cells of each cell line cells were seeded (5,000/well) into individual 96-well tissue culture plates (CSL Biosciences, Australia) and were grown for 24 hours before treatment. Treatment compounds were dissolved in 100% DMSO and were diluted in complete medium; the DMSO concentration in the medium did not exceed

0.01%. Controls included no treatment, and treatment with the equivalent of 0.01% DMSO. Treatments were analysed in triplicate, and repeated as per study numbers (2-4). Three days after treatment, the cells were washed with phosphate-buffered saline (PBS) and fixed with methylated spirits, and total protein was determined using sulforhodamine B (SRB) as described previously (Skehan et al, 1990).

Briefly, at the end of the required treatment time, 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 μL/well of SRB solution (0.4% SRB (w/v) in 1% (v/v) acetic acid) over a period of at least 30 minutes. The 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 μL 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.

Table 9: IC ₅₀ (ng/mL) against human cancer cell lines

Metabolite NFF MCF7 DU145 MM96L MM418c5 C180-13s K562 kibdelone A 14 16 3.7 1.6 4 5 6.5 kibdelone B 7 9 9 4 20 7 22 kibdelone C 11 11 11 2 14 11 35

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 cell lines used, the type of cancer each cell line represents, the doubling time of the cell line and the inoculation density is given in Table 10.

Table 10: NCI cell-line panel*

http://dtp.nci.nih.gov/docs/misc/common files/cell list.html (17 February 2007)

Methodology of the In Vitro Cancer Screen

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. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in

100 μL 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.

After 24 hours, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/mL gentamicin. Additional four, 10-fold or ¹ A log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 μL of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μL of medium, resulting in the required final drug concentrations.

Following drug addition, the plates are incubated for an additional 48 hours at 37 ⁰ C, 5% CO ₂ , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μL 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 μL) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is

removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μL of 80%

TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as:

[(Ti-Tz)/(C-Tz)] x 100 for concentrations for which Ti > Tz [Ti-Tz/Tz] x 100 for concentrations for which Ti < Tz

Three dose response parameters are calculated for each experimental agent. Growth inhibition of 50% (GI ₅₀ ) is calculated from [(Ti-Tz)/(C-Tz)] x 100 = 50, which is the drug concentration resulting in ¹ a 50% reduction in the net protein increase (as measured by SRJB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. 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) indicating a net loss of cells following treatment is calculated from [(Ti- Tz)/Tz] x 100 = -50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the effect is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.

The above methodology is provided at httpV/dtp.nci.nih.gov/branches/btb/ivclsp.html (17 February 2007).

Results

The results of the screening panel in terms of the GI _5O , TGI and LC ₅₀ for kibdelones A-C (Compounds 1-3) and kibdelone B rhamnoside (Compound 6) are given in Tables 11-14 below.

On analysis of the results in Tables 11-14, it is evident that Compounds 1-3 are potent anti-tumour compounds. The rhamnoside, Compound 6, was less active than the corresponding aglycone, compound (2).

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.

Example 8: Anti-cancer efficacy studies for Compound 3 (kibdelone C) in mice

The human tumour cell line Colo205 was implanted onto nude mice and treatments were carried out i.p. with 20 μg of Compound 3 daily. A control vehicle group and a no-vehicle control group were included. Test Compound 3 was pre-weighed in 80 μg aliquots in screw cap microfuge tubes and stored at -20 ⁰ C. Compound 3 (80 μg) was dissolved in 10 μL of DMSO then diluted with 390 μL of 25% propylene glycol in saline to give 200 μg/mL of Compound 3. Injection of 100 μL then gave a dose of 20 μg. 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.

Queensland Institute of Medical Research (QIMR) AEC Ethics approval P 865. Nude (BALB/c derived) male mice (4-5 weeks, supplied by the Australian Research Centre, Perth and allowed to acclimatize at QIMR for 1 week minimum) were injected sc with tumour cells as described above (in 50 μL culture medium per site) on 4 sites on the flanks. Treatment with 100 μL (20 μg) of Compound 3 by i.p. administration was commenced next day and continued daily for 2 days. Tumour size was monitored and recorded. The mice were monitored daily for a week, then twice weekly. Euthanasia was carried out according to the score sheet, Compound 3 primarily, when the total tumour volume/mouse reaches 1 cubic cm.

Monitoring of drug-treated and tumour-bearing mice

The 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.

Clinical scoring

Scores for each parameter are summed to give a possible total of 8. Mild: <3

Moderate: 3 to 6

Severe: >6

Additional criterion for euthanasing tumour-bearing mice

2 or 4 sites/mouse: total tumour volume/mouse 1000 cubic mm (1 mL).

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:

Volume = A x b x b x 0.5, where A is the length and b is the measured breadth of the tumour lump (skin included, thus still an overestimate). (Teicher et al, ibid., p92). Mouse weight, area of local inflammation and tumour volume was measured every second day for

2 weeks from initial treatment, then twice weekly for the last 2 weeks.

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.

Monitoring occurred 6 hours after drug delivery, then daily for seven days followed by twice weekly for 12 weeks if required.

There were 3 mice in a non-injected control group, 3 mice in the control vehicle group, and

3 in the drug group.

Table 15: Protocol supplied Anticancer efficacy studies for Compound 3 using mouse models (Nude mice). ^a

^a A prior QIMR pilot study carried out on Compound 3 determined the maximum dosage tolerance = 40 μg/mouse. The dosage regime proposed for this study is based on this figure. Two sets of controls - control A with tumours only and control B with tumours

and the vehicle (DMSO) - applied at same frequency as the drug Compound 3. ^c Four xenograft sites / mouse. IP application; proposed 2 daily 20 μg doses then 10 μg at day 5.

Study Results

Treatment ofColo205 colon tumours. The results with this fast growing model shown in Table 16 and Figure 1 indicate that Compound 3 may have slowed the growth of the tumours, compared with control vehicle treatment and no vehicle.

Table 16: Treatment of Colo 205 colon tumours with Compound 3

Example 9: Antibacterial activity as assessed using ProTOX assay

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.

Briefly, in ProTOX, the bacteria (24 hour broth) were applied to the surface of an agar matrix containing the test sample and allowed to grow for 48 hours. The assay was monitored at 24 and 48 hours and the active wells noted. Known antibiotics yield consistent colour transitions which are concentration and time dependent. These patterns provided an important guide to the early recognition of interesting characteristics. Generally bactericidal actives give no colour change at both 24 and 48 hours while bacteriostatic actives are active at 24 hours but less potent or inactive at 48 hours.

In this experiment, combined extracts from Kibdelosporangium (MST- 108465) fermentations were pre-fractionated by using solvent partitioning and preparative C ₁₈ solid-phase extraction (SPE) returned to a Methanol fraction enriched in bioactive metabolites. This fraction was then subjected to C ₁₈ and C ₈ HPLC fractionation to yield

100 fractions. Bioassay with ProTOX provided antibacterial activity for fractions containing kibdelones with LD ₉₉ between 1 and 11 μg 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.

Table 17: Antibacterial Activity

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