LACTONE DERIVATIVES

Field of the Invention

The invention relates to bioactive molecules. More particularly, the invention relates to lactone derivatives useful as pharmaceutical, agricultural or pesticidal agents.

Background of the Invention

Bio-discovery is a growing field, which investigates and screens for bioactive natural products from natural environments, including plants, microorganisms, coral and other marine life. In the search for bioactive natural products, biological material is screened for molecules having properties that may be of therapeutic benefit for potential use in a range of treatments, for example treatments for cancer, antiprotozoal treatments, antiparasitic treatments, antifungal treatments, antibiotic treatments and anti-inflammatory treatments, or for pesticidal activity.

Summary of the Invention

The present invention is predicated in part on the discovery that newly isolated lactone derivatives have cytotoxic, antiprotozoal, antiparasitic, antibiotic and/or insecticidal activity. Such compounds have potential for therapeutic, veterinary or agricultural use.

One aspect of the present invention provides a compound of formula (I):

(I)

wherein

X is selected from -O-, -S-, -NH-, -N(C ₁ -QaIlCyI)- and -CH ₂ -;

R ¹ and R ² are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R, -OR, -SR, -N(R) ₂ , -NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ , -C(Y)R and -YC(Y)R; or R ¹ and R ² together form a carbonyl group; R ³ to R ⁶ are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R, -OR, -SR, -N(R) ₂ , -NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ , -C(Y)R and -YC(Y)R; or R ³ and R ⁵ together form a double bond or -0-, or R ³ and R ⁴ or R ⁵ and R ⁶ form a carbonyl group;

R ⁷ and R ⁸ are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R, -OR, -SR, -N(R) ₂ , -NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ , -C(Y)R and -YC(Y)R; or R ⁷ and R ⁸ together form a carbonyl group;

Each R ⁹ to R ¹² are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R, -OR, -SR, -N(R) ₂ , -NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ , -C(Y)R and -YC(Y)R; or one or more R ¹⁰ and R ¹² together form a double bond or -O- or one or more R ⁹ and R ¹⁰ or one or more R ¹¹ and R ¹² together form a carbonyl group; R ¹³ is selected from hydrogen, -cycloalkyl, -aryl, -heterocyclyl and -heteroaryl;

R is selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl, -aryl, -heteroaryl, -heterocyclyl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl and haloalkyl; m is an integer from 1 to 15; n is an integer from 1 to 10;

Y is selected from O, S, NH or N(Ci-C ₆ alkyl); wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl is optionally substituted; or a pharmaceutically, agriculturally or pesticidally acceptable salt thereof.

In some embodiments of formula (I), one or more of the following applies:

X is selected from -O- or -S-;

R ¹ is hydrogen and R ² is selected from -OH, -OCi _-10 alkyl, -OC _2-10 alkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OCi _-10 alkylcycloalkyl, -OCj.ioalkylaryl,

-Od-ioalkylheterocyclyl, -OCi-iQalkylheteroaryl and -OC(O)R or R ¹ and R ² taken together form a carbonyl group;

R ³ to R ⁶ are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl, -cycloalkyl,

-aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl, -alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R, -OR, -SR, -N(R) ₂ ,

-NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(O)(R) ₃ , -OSi(R) ₃ ,

-OB(R) ₂ , -C(Y)R and -YC(Y)R; or R ³ and R ⁵ form a double bond or -O- (epoxide);

R ⁷ is hydrogen;

R is selected from hydrogen, -OH, -OCi-ioalkyl, -OC _2-10 alkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OCi-njalkylcycloalkyl, -OC _1-10 alkylaryl,

-Od-ioalkylheterocyclyl, -OC _MO alkylheteroaryl and -OC(O)R;

Each R ⁹ is hydrogen;

Each R ¹⁰ is hydrogen;

Each R ¹¹ is hydrogen; Each R ¹² is independently selected from -OH, -OCi.i ₀ alkyl, -OC ₂ . _! oalkenyl, -Ocycloalkyl,

-Oaryl, -Oheterocyclyl, -Oheteroaryl, -OCi.joalkylcycloalkyl, -OC _1-10 alkylaryl,

-OCi-ioalkylheterocyclyl, -OC _1-10 alkylheteroaryl and -OC(O)R; or one or more R ¹⁰ and R ¹² together form a double bond or -O-, especially where one R ¹⁰ and R ¹² together form a double bond; or one or more R ' and R ¹² together from a carbonyl group, especially one

R ¹¹ and R ¹² together form a carbonyl group;

R ¹³ is selected from -cycloalkyl, -aryl, -heterocyclyl and -heteroaryl;

R is selected from -Ci.i _O alkyl, -C _2-10 alkenyl, -C _2-10 alkynyl, -cycloalkyl, -aryl, -heterocyclyl,

-heteroaryl, -C _1-6 alkylcycloalkyl, -C _1-6 alkylaryl, -Ci- _ό alkylheterocyclyl,

-Ci- ₆ alkylheteroaryl and haloalkyl-; m is an integer from 1 to 12; and n is an integer from 2 to 7; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl is optionally substituted; or a pharmaceutically, agriculturally or pesticidally acceptable salt thereof.

In some embodiments, the compound of formula (I) is a compound of formula (II):

wherein R ¹ is hydrogen,

R ² is selected from -OH, -OCj.ioalkyl, -OC ₂ .i ₀ alkenyl, -Ocycloalkyl, -Oaryl,

-Oheterocyclyl, -Oheteroaryl, -OC _t -ioalkylcycloalkyl, -OCj.ioalkylaryl,

-OC _M oalkylheterocyclyl, -OC _1-10 alkylheteroaryl and -OC(O)R; or

R ¹ and R ² taken together form a carbonyl group; R ³ and R ⁵ are independently selected from hydrogen, -alkyl, -alkenyl, -alkynyl,

-cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -alkylcycloalkyl, -alkylaryl,

-alkylheterocyclyl, -alkylheteroaryl, haloalkyl-, -alkoxyalkyl, -halo, -CN, -NO ₂ , -CO ₂ R,

-OR, -SR, -N(R) ₂ , -NROR, -ON(R) ₂ , -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ ,

-P(O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ , -C(Y)R and -YC(Y)R; or R ³ and R ⁵ together form a double bond or -0-; R ⁸ is selected from hydrogen, -OH, -OC]. ₁₀ alkyl, -OC _2- ioalkenyl, -Ocycloalkyl, -Oaryl,

-Oheterocyclyl, -Oheteroaryl, -OCuioalkylcycloalkyl, -OCi _-10 alkylaryl,

-OCi.Kjalkylheterocyclyl, -OC _1-10 alkylheteroaryl and -OC(O)R;

Each R ¹⁰ is hydrogen;

Each R ¹² is selected from -OH, -OCi.i ₀ alkyl, -OC _2-1 oalkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OC _M oalkylcycloalkyl, -OCi _-10 alkylaryl,

-OCi-ioalkylheterocyclyl, -Od-ioalkylheteroaryl and -OC(O)R; or one or more R ¹⁰ and R ¹² together from a double bond or -O- ;

R ¹³ is selected from -cycloalkyl, -aryl, -heterocyclyl and -heteroaryl;

R is selected from -C _1-10 alkyl, -C ₂ -i ₀ alkenyl, -C _2-10 alkynyl, -cycloalkyl, -aryl, -heterocyclyl, -heteroaryl, -Ci- _ό alkylcycloalkyl, -Ci-βalkylaryl, -Cj-όalkylheterocyclyl,

-Cj-ealkylheteroaryl and haloalkyl-; m is an integer from 1 to 12; n is an integer from 2 to 7; wherein each alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heterocyclyl and heteroaryl group is optionally substituted; or a pharmaceutically, agriculturally or pesticidally acceptable salt thereof.

In some embodiments of the compounds of formula (II), one or more of the following applies:

R ¹ is hydrogen;

R ² is selected from -OH, -OCi _-6 alkyl, -OC _2-6 alkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OC(O)C _1-6 alkyl, -OC(O)C ₂ . ₆ alkenyl and -OC(O)aryl or R ¹ and R ² taken together form a carbonyl group; especially where R ¹ and R ² taken together form a carbonyl group;

R ³ and R ⁵ are each independently selected from hydrogen, -C _1-6 alkyl, haloalkyl-, -halo, -OH, -OCi _-6 alkyl, -CO ₂ H, -CO ₂ C _1-6 alkyl, -SH, -SC^alkyl, -NH ₂ , -NH(Ci _-6 alkyl), -N(Ci _-6 alkyl) ₂ and -OC(O)C _! . ₆ alkyl or R ³ and R ⁵ taken together form a double bond or -O-; especially where R ³ and R ⁵ taken together form a double bond or -O-; more especially where R ³ and R ⁵ taken together form a double bond;

R ⁸ is selected from hydrogen, -OH, -OC^alkyl, -OC ₂ . ₆ alkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OC(O)Ci _-6 alkyl, -OC(O)C _2-6 alkenyl and -OC(O)aryl; especially hydrogen, -OH, -OCi _-6 alkyl and -OC(O)C _1-6 alkyl; more especially hydrogen and -OH; Each R ¹⁰ is hydrogen;

Each R ¹² is selected from -OH, -OCi _-6 alkyl, -OC _2-6 alkenyl, -Ocycloalkyl, -Oaryl, -Oheterocyclyl, -Oheteroaryl, -OC(O)C i _-6 alkyl, -OC(O)C ₂ . ₆ alkenyl and -OC(O)aryl; or each R ¹⁰ and R ¹² taken together form a double bond or -0-; especially where one or more R ¹⁰ is hydrogen and one or more R ¹² is -OH or -OC(O)Ci _-6 alkyl or where one or more R ¹⁰ and R ¹² taken together form a double bond; more especially where one or more R ¹⁰ is hydrogen and R ¹² is -OH or -OC(O)CH ₃ or where one or more R ¹⁰ and R ¹² taken together form a double bond, especially where one R ¹⁰ and R ¹² taken together form a double bond; R ¹³ is -aryl or -heteroaryl; especially -aryl; more especially optionally substituted phenyl; most especially unsubstituted phenyl; m is an integer from 2 to 12, especially an even number selected from 2, 4, 6, 8, 10 and 12; n is an integer from 3 to 5;

Each alkyl, alkenyl, cycloalkyl, aryl, heterocyclyl and heteroaryl are optionally substituted; or a pharmaceutically, agriculturally or pesticidally acceptable salt thereof.

In some embodiments, the compound of the invention is selected from:

also referred to as EBI-15;

also referred to as EBI- 16; also referred to as EBI- 17; also referred to as EBI-11 ; also referred to as EBI-19; also referred to as EBI-32; and

also referred to as EBI-33.

The term "alkyl" refers to optionally substituted linear and branched hydrocarbon groups having 1 to 20 carbon atoms. Where appropriate, the alkyl group may have a specified number of carbon atoms, for example, -C ₁ -C ₆ alkyl which includes alkyl groups having 1,

2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements. Non-limiting examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s- and t-butyl, pentyl,

2-methylbutyl, 3-methylbutyl, hexyl, heptyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl.

The term "alkenyl" refers to optionally substituted unsaturated linear or branched hydrocarbon groups, having 2 to 20 carbon atoms and having at least one double bond. Where appropriate, the alkenyl group may have a specified number of carbon atoms, for example, C ₂ -C ₆ alkenyl which includes alkenyl groups having 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements. Non-limiting examples of alkenyl groups include, ethenyl, propenyl, isopropenyl, butenyl, s- and t-butenyl, pentenyl, hexenyl, hept-1,3- diene, hex-l,3-diene, non-l,3,5-triene and the like.

The term "alkynyl" refers to optionally substituted unsaturated linear or branched hydrocarbon groups, having 2 to 20 carbon atoms and having at least one triple bond. Where appropriate, the alkynyl group may have a specified number of carbon atoms, for example, C ₂ -C ₆ alkynyl groups have 2, 3, 4, 5 or 6 carbon atoms in linear or branched arrangements. Non-limiting examples of alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl and the like.

The term "cycloalkyl" refers to optionally substituted saturated or unsaturated mono-cyclic, bicyclic or tricyclic carbon groups. Where appropriate, the cycloalkyl group may have a specified number of carbon atoms, for example, C ₃ -C ₆ cycloalkyl is a carbocyclic group having 3, 4,; 5 or 6 carbon atoms. Non-limiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl and the like.

"Aryl" means a C ₆ -C ₁₄ membered monocyclic, bicyclic or tricyclic carbocyclic ring system having up to 7 atoms in each ring, wherein at least one ring is aromatic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl and biphenyl. The aryl may comprise 1-3 benzene rings. If two or more aromatic rings are present, then the rings may be fused together, so that adjacent rings share a common bond.

"Heterocyclic" or "heterocyclyl" refers to a non-aromatic ring having 3 to 8 atoms in the ring and of those atoms 1 to 4 are heteroatoms, said ring being isolated or fused to a second ring selected from 3- to 7-membered alicyclic ring containing 0 to 4 heteroatoms, wherein said heteroatoms are independently selected from O, N and S. Heterocyclic includes partially and fully saturated heterocyclic groups. Heterocyclic systems may be attached to another moiety via any number of carbon atoms or heteroatoms of the radical and may be both saturated and unsaturated, which includes all forms of carbohydrate moieties. Non-limiting examples of heterocyclic include pyrrolidinyl, pyrrolinyl, pyranyl, piperidinyl, piperazinyl, morpholinyl, tetrahydrofuranyl, tetrahydrothiophenyl, pyrazolinyl, dithiolyl, oxathiolyl, dioxanyl, dioxinyl, oxazinyl, azepinyl, diazepinyl, thiazepinyl, oxepinyl and thiapinyl, imidazolinyl, thiomorpholinyl, and the like.

The term "heteroaryl" as used herein means a stable monocyclic or bicyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and at least one ring contains from 1-4 heteroatoms, selected from sulfur, oxygen and nitrogen. Heteroaryl includes, but is not limited to, oxazolyl, thiazolyl, thienyl, furyl, 1-isobenzofuranyl, pyrrolyl, imidazolyl, pyrazolyl, isothiazolyl, isobxazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl, indolyl, purinyl, phthalazinyl, 1,2,3-triazolyl, 1 ,2,4-triazolyl,

1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,

1,2,3,4-oxatriazolyl, 1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl, 1,2,3-triazinyl, benzofuranyl, isobenzofuranyl, thionaphthenyl, isothionaphthenyl, indoleninyl, 2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl, isoindazolyl, indoxazinyl, benzoxazolyl, quinolinyl, isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl, pyrido[3,4-b]pyridinyl, pyrjdo[3,2-b]pyridinyl, pyrido[4,3-b]pyridinyl, acridinyl, carbazolyl, quinaoxalinyl, pyrazolyl, benzotriazolyl, thiophenyl, isoquinolinyl, pyridinyl, tetrahydroquinolinyl, benzazepinyl, benzodioxanyl, benzoxepinyl, benzodiazepinyl, benzothiazepinyl and benzothiepinyl and the like.

The alkyl, alkenyl, alkynyl, cycloalkyl, aryl, heteroaryl and heterocyclyl groups may be substituted with one or more substituent independently selected from -F, -Cl, -Br, -I, -CO ₂ R, -CN, -OR, -SR, -N(R) ₂ , -NO ₂ , -NROR, -ON(R) ₂ , -SOR, -SO ₂ R, -SO ₃ R, -SON(R) ₂ , -SO ₂ N(R) ₂ , -SO ₃ N(R) ₂ , -P(R) ₃ , -P(=O)(R) ₃ , -OSi(R) ₃ , -OB(R) ₂ wherein R is as defined above. Each cycloalkyl, aryl, heterocyclyl and heteroaryl may also be substituted with one or more -Q- _ό alkyl or -C _2-6 alkenyl either alone or with other optional substituents listed above.

As used herein, the terms "halo" or "halogen" refers to fluorine (fluoro), chlorine (chloro), bromine (bromo) and iodine (iodo).

Yet another aspect of the invention provides a pharmaceutically, agriculturally or pesticidally acceptable salt of a compound of formula (I) or formula (II).

The terms "pharmaceutically acceptable salts", "agriculturally acceptable salts" or "pesticidally acceptable salts" as used herein refer to salts which are toxicologically safe for systemic or localised administration or suitable for application to a plant or an agricultural, industrial or household environment. The pharmaceutically, agriculturally or pesticidally acceptable salts may be selected from the group including alkali and alkali earth, ammonium, aluminium, iron, amine, glucosamine, chloride, sulphate, sulphonate, bisulphate, nitrate, citrate, tartrate, bitarate, phosphate, carbonate, bicarbonate, malate,

maleate, napsylate, fumarate, succinate, acetate, benzoate, terephthalate, palmoate, pectinate and s-methyl methionine salts, piperazine and the like.

It will also be recognised that compounds of the invention may 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 e.g., greater than about 90% ee, such as about 95% or 97% ee or greater than 99% ee, as well as mixtures, including racemic mixtures, thereof. Such isomers may be obtained by isolation from natural sources, by asymmetric synthesis, for example using chiral intermediates, or by chiral resolution. The compounds of the invention may exist as geometrical isomers. The invention also relates to compounds in substantially pure cis (Z) or trans (E) forms or mixtures thereof.

The compounds of the present invention may be obtained by isolation from a plant or plant part, or by derivatisation of an isolated compound or by synthetic methods from known starting materials.

In another aspect of the invention there is provided a method of isolating one or more compounds of formula (I) or formula (II), the method including the step of extracting one or more compounds of formula (I) or formula (II) from a plant or plant part.

Preferably, the plant is of the family Lauraceae.

Preferably, the genus is Cryptocarya, Litsea, Cinnamomum, Beilschmiedia, Endiandra, Neolitsea and Lindera.

Preferably the species is Cryptocarya spp. such as C. alba, C. angulata, C. aristata, C. ashersoniana, C. chinensis, C. cinnamomifolia, C. corrugata, C. crassinervia, C. cunninghamiana, C. densiflora, C. ferrea, C. foetida, C. gigantocarpa, C. glaucescens, C. grandis, C. hypospodia, C. invasorium, C. laevigata, C. leptospermoides, C. mackinnoniana, C. massoia, C. meissneri, C. membranaceae, C. multipaniculata,

C. murrayi, C. nigra, C. nitens, C. oblata, C. odorata, C. palawanensis, C. pleurosperma, C. pluricostata, C. rigida, C. scortechinii, C. transversa, C. tomentosa, C. triplinervis, C. vulgaris, C. angulata, C. bamagana, C. bellendenkerana, C. bidwillii, C. brassii, C. burckiana, C. clarksoniana, C. claudiana, C. cocosoides, C. cunninghamii, C. endiandrifolia, C. erythoxylon, C. exfoliata, C. floydii, C. foveolata, C. glaucocarpa, C. leucophylla, C. lividula, C. macdonaldii, C. meisneriana, C. melanocarpa, C. microneura, C. obovata, C. onoprienkoana, C. putida, C. rhodosperma, C. saccharata, C. sclerophylla, C. smaragdina, C. sp Boonjee, C. sp Gadgarra, C. triplinervis var. riparia; especially C. oblata, C. leucophylla, C. angulata, C. bamagana, C. bellendenkerana, C. bidwillii, C. brassii, C. clarksoniana, C. cocosoides, C. corrugata, C. cunninghamii, C. exfoliata, C. glaucescens, C. grandis, C. hypospodia, C. laevigata, C. lividula, C. macdonaldii, C. mackinnoniana, C. melanocarpa, C. microneura, C. murrayi, C. onoprienkoana, C. pleurosperma, C. putida, C. rhodosperma, C. triplinervis var. riparia, C. vulgaris; or Litsea spp. such as Litsea sebifera, Litsea polyantha, Litsea cassiaefolia, Litsea elliptica, Litsea ferruginea, Litsea flrma, Litsea garciae, Litsea oppositifolia, Litsea australis, Litsea bennettii, Litsea bindoniana, Litsea breviumbellata, Litsea connorsii, Litsea fawcettiana, Litsea glutinosa, Litsea granitica, Litsea leefeana, Litsea macrophylla, Litsea reticulata; especially Litsea breviumbellata, Litsea connorsii and Litsea leefeana; Cinnamomum spp. such as Cinnamomum acuminatifolium, Cinnamomum acuminatissimum, Cinnamomum acutatum, Cinnamomum africanum, Cinnamomum aggregatum, Cinnamomum alainii, Cinnamomum alatum, Cinnamomum albiflorum, Cinnamomum alcinii, Cinnamomum alexei, Cinnamomum alibertii, Cinnamomum alternifolium, Cinnamomum altissimum, Cinnamomum ammannii, Cinnamomum amoenum, Cinnamomum amplexicaule, Cinnamomum amplifolium, Cinnamomum anacardium, Cinnamomum andersonii, Cinnamomum angustifolium, Cinnamomum angustitepalum, Cinnamomum antillarum, Cinnamomum appelianum, Cinnamomum arbusculum, Cinnamomum archboldianum, Cinnamomum areolatocostae, Cinnamomum areolatum, Cinnamomum areolatum, Cinnamomum arfakense, Cinnamomum argenteum, Cinnamomum aromaticum, Cinnamomum arsenei, Cinnamomum asa-grayi, Cinnamomum assamicum, Cinnamomum aubletii, Cinnamomum aureo-fulvum, Cinnamomum australe, Cinnamomum austro-sinense, Cinnamomum

austro-yunnanense, Cinnamomum bahianum, Cinnamomum bahiense, Cinnamomum baileyanum, Cinnamomum baillonii, Cinnamomum balansae, Cinnamomum bamoense, Cinnamomum barbato-axillatum, Cinnamomum barbeyanum, Cinnamomum barlowii, Cinnamomum bartheifolium, Cinnamomum barthii, Cinnamomum bazania, Cinnamomum beccarii, Cinnamomum bejolghota, Cinnamomum bengalense, Cinnamomum biafranum, Cinnamomum bintulense, Cinnamomum birmanicum, Cinnamomum blumei, Cinnamomum bodinieri, Cinnamomum bonii, Cinnamomum bonplandii, Cinnamomum borneense, Cinnamomum bourgeauvianum, Cinnamomum boutonii, Cinnamomum brachythyrsum, Cinnamomum bractefoliaceum, Cinnamomum burmannii, Cinnamomum camphora, Cinnamomum cassia (syn. C. aromaticum), Cinnamomum caudiferum, Cinnamomum chartophyllum, Cinnamomum citriodorum, Cinnamomum contractum, Cinnamomum filipes, Cinnamomum glanduliferum, Cinnamomum glaucescens, Cinnamomum ilicioides, Cinnamomum impressinervium, Cinnamomum iners, Cinnamomum japonicum, Cinnamomum javanicum, Cinnamomum jensenianum, Cinnamomum kotoense, Cinnamomum kwangtungense, Cinnamomum liangii, Cinnamomum longepaniculatum, Cinnamomum longipetiolatum, Cinnamomum loureiroi, Cinnamomum mairei, Cinnamomum micranthum, Cinnamomum migao, Cinnamomum mollifolium, Cinnamomum oliveri, Cinnamomum osmophloeum, Cinnamomum parthenoxylon, Cinnamomum pauciflorum, Cinnamomum philippinense, Cinnamomum pingbienense, Cinnamomum pittosporoides, Cinnamomum platyphyllum, Cinnamomum porphyrium, Cinnamomum propinquum, Cinnamomum reticulatum, Cinnamomum rigidissimum, Cinnamomum saxatile, Cinnamomum septentrionale, Cinnamomum subavenium, Cinnamomum tamala, Cinnamomum tenuipilum, Cinnamomum tonkinense, Cinnamomum triplinerve, Cinnamomum tsangii, Cinnamomum tsoi, Cinnamomum validinerve, Cinnamomum verum, Cinnamomum virens, Cinnamomum wilsonii and Cinnamomum laubatii especially Cinnamomum laubatii, Cinnamomum oliveri, Cinnamomum virens and Cinnamomum camphora; or Beilschmiedia bancroftii, Beilschmiedia brunnea, Beilschmiedia castrisinensis, Beilschmiedia collina, Beilschmiedia elliptica, Beilschmiedia obtusifolia, Beillschmiedia oligandra, Beilschmiedia peninsularis, Beilschmiedia recurva, Beilschmiedia tooram, Beilschmiedia volckii; especially Beilschmiedia bancroftii, Beilschmiedia castrisinensis, Beilschmiedia peninsularis, Beilschmiedia recurva,

Beilschmiedia tooram, Beilschmiedia volckii; Endiandra acuminata, Endiandra anthropophagorum, Endiandra bellendenkerana, Endiandra bessaphila, Endiandra collinsii, Endiandra compressa, Endiandra cooperana, Endiandra cowleyana, Endiandra crassiflora, Endiandra dichrophylla, Endiandra dielsiana, Endiandra discolor, Endiandra floydii, Endiandra glauca, Endiandra globosa, Endiandra grayi, Endiandra hayesii, Endiandra hypotephra, Endiandra impressicosta, Endiandra insignis, Endiandra introrsa, Endiandra jonesii, Endiandra leptodendron, Endiandra limnophila, Endiandra longipedicellata, Endiandra microneura, Endiandra monothyra subsp monothyra, Endiandra monothyra subsp trichophylla, Endiandra montana, Endiandra muelleri, Endiandra palmerstonii, Endiandra phaeocarpa, Endiandra sankeyana, Endiandra sideroxylon, Endiandra sieberi, Endiandra virens, Endiandra wolfei, Endiandra xanthocarpa; especially Endiandra bessaphila, Endiandra compressa, Endiandra globosa, Endiandra insignis, Endiandra jonesii, Endiandra microneura, Endiandra monothyra subsp monothyra, Endiandra montana, Endiandra palmerstonii, Endiandra sankeyana; Neolitsea australiensis, Neolitsea brassii, Neolitsea dealbata; especially Neolitsea dealbata; and Lindera queenslandica.

The parts of the plant may include fruit, seed, bark, leaf, flower, roots and wood.

Preferably the extract is obtained from the epicarp or mesocarp.

For example, the biomass obtained from fruit, seed, bark, leaf, flower, roots and wood of the plant is subject to initial solvent extraction, for example with a polar solvent such as methanol. The initial extraction is then concentrated and diluted with water and subject to extraction with a second solvent, for example, ethyl acetate. The solvent samples from the second extraction are pooled and subject to separation by preparative HPLC fractionation. The fractions are analysed by analytical HPLC and pooled according to the retention time of compounds found in the samples. The pooled fractions are weighed, bioassayed and analysed by analytical HPLC. Further fractionation using one or more preparative HPLC is performed to isolate specific compounds. Each compound is bioassayed and its structure identified by UV, NMR and mass spectrometry techniques.

Other compounds of the invention may be obtained by derivatising compounds isolated from plants or parts of plants, especially from the genus Cryptocarya, Litsea and Cinnamomum.

Derivatives of the natural compounds can be obtained by techniques known in the art. For example, hydroxy groups may be oxidised, to ketones, aldehydes or carboxylic acids by exposure to oxidising agents such as chromic acid, Jones' reagent, KMnO ₄ , peracids such as mCPBA (metachloroperbenzoic acid) or dioxiranes such as dimethyldioxirane (DMDO) and methyl(trifluoromethyl)dioxirane (TFDO). Oxidising agents may be chosen such that other functional groups in the molecule are or are not also oxidised. For example, a primary alcohol may be selectively oxidised to an aldehyde or carboxylic acid in the presence of secondary alcohois using reagents such as RuCl ₂ (PPh ₃ ) ₃ -benzene. Secondary alcohols may be selectively oxidised to ketones in the presence of a primary alcohol using Cl ₂ -pyridine or NaBrO ₃ -ceric-ammonium nitrate. Alcohols may be oxidised in the presence of double and triple bonds and without epimerisation at adjacent stereocentres using Jone's reagent. Alternatively, reagents chosen may be less selective resulting in oxidation at more than one functional group.

Hydroxy groups may also be derivatised by etherification or acylation. For example, ethers may be prepared by formation of an alkoxide ion in the presence of base and reacting the alkoxide with an appropriate alkylhalide, alkenylhalide, alkynylhalide or arylhalide. Similarly acylation may be achieved by formation of an alkoxide ion and reaction with an appropriate carboxylic acid or activated carboxylic acid (such as an anhydride).

Acyl groups may be hydrolysed to provide alcohols by acid or base hydrolysis as known in the art.

Silyl groups may be introduced onto hydroxy groups to provide silyl ethers using mild base and a silyl chloride reagent, for example Me ₃ SiCl and triethylamine in THF or agents such as MeSiNHCO ₂ SiMe ₃ in THF.

Sulfonates may be readily introduced onto hydroxy groups by reaction with a suitable sulfonate group. For example, methanesulfonates may be introduced by treatment of a hydroxy group with MsCl and triethylamine in dichloromethane. Tosylate groups may be introduced by reacting a hydroxy group with TsCl and pyridine. Allylsulfonates may be introduced by reacting a hydroxy group with allylsulfonyl chloride and pyridine in dichloromethane .

Ketones may be reduced to secondary alcohols by reducing agents such as lithium aluminium hydride and other metal hydrides without reducing double bonds, including α-unsaturated ketones.

Double bonds and triple bonds may be reduced to single bonds using catalytic reduction, for example, H ₂ /Pd. Double bonds may also be oxidised to epoxides using oxidising agents such as per acids, for example mCPBA or dioxiranes, such as DMDO and TFDO. Double bonds may also be subject to addition reactions to introduce substituents such as halo groups, hydroxy or alkoxy groups and amines.

A person skilled in the art would be able to determine suitable conditions for obtaining derivatives of isolated compounds, for example, by reference to texts relating to synthetic methodology, examples of which are Smith M.B. and March J., March's Advanced Organic Chemistry, Fifth Edition, John Wiley & Sons Inc., 2001 and Larock R.C., Comprehensive Organic Transformations, VCH Publishers Ltd., 1989. Furthermore, selective manipulations of functional groups may require protection of other functional groups. Suitable protecting groups to prevent unwanted side reactions are provided in Green and Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons Inc., 3 ^rd Edition, 1999.

The compounds of the invention may also be synthesised from commercially available starting materials.

For example, compounds of formula (I) may be prepared as set out in the following schemes.

2. R ₃ BHNa protection

MeO^ _^ OMe

( 1 )

Scheme 1

An important intermediate (1) may be prepared by reacting epoxymethylchloride under Grignard conditions with an appropriate alkyl-, arylalkyl-, heterocyclylalkyl- or heteroarylalkyl-magnesium bromide compound to provide the hydrophobic portion of the compound. The resulting alkyl epoxy compound may be reacted further with reagents that provide disguised or protected alkylhydroxy groups in the form of dithianes or epoxides. An example of such a reaction is described by Smith et al, J. Am. Chem. Soc, 2003,

14435. Deprotection to expose γ-hydroxy substituents and then reprotection provides intermediate (1). Suitable protecting agents, methods for their introduction and removal are provided in Green and Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons, 3 ^rd Edition, 1999.

The polyhydroxy chain may be extended by further reactions known in the art to provide the required number of hydroxy groups. For example, a hydroxyalkyl extension is shown in Scheme 2.

3. mCPBA ( D ( 2 )

Scheme 2

One means of preparing compounds having a polyhydroxyalkyl chain directly attached to the lactone group is to use intramolecular cyclisation by ring closing metathesis to form the lactone ring, as shown in Scheme 3 :

Scheme 3

Deprotection of all protecting groups can provide a polyhydroxy compound. Selective deprotection to expose one hydroxy group followed by reaction with acetic anhydride, can be used to provide selective acetylation of hydroxy groups as shown in Scheme 4.

Scheme 4

Alternatively, compounds having a polyhydroxyalkenyllactone moiety may be prepared by initially preparing an alkynyl substituted lactone and reacting this compound with a suitable polyhydroxy epoxide as shown in Scheme 5:

OR ₃

Scheme 5

The initial preparation of the cyclic ether may be prepared by the method of Jacobsen (Jacobsen, Angew. Chem., 2001, 3667). The resulting cyclic ether may be reacted with a suitable epoxide by the method of Wipf (Wipf, J. Org. Chem., 1993, 825) to provide the cyclic ether.

Deprotection of all protecting groups and selective oxidation of an allylic hydroxy group can provide the required lactone. Alternatively, selective deprotection, oxidation and/or acetylation can provide the compounds of the invention as shown in Scheme 6.

Scheme 6

Furthermore, the compounds prepared by the methods above may be further manipulated during or after preparation by methods known in the art. For example, reduction of double bonds may be achieved by catalytic hydrogenation, eg Pd/H ₂ . Substituents may be included by addition across a double bond or by nucleophilic substitution as known in the art. Suitable conditions for manipulating compounds of the invention during or after

synthesis are known to those in the art, for example, those described in Smith and March, March's Advanced Organic Chemistry, J. Wiley & Sons, 5 ^th Edition, 2001; Larock, Comprehensive Organic Transformations, VCH, 1989 and Li, Name Reactions, Springer, 2 ^nd Edition, 2003.

A further aspect of the invention provides a pharmaceutical composition for treatment or prophylaxis of a disease or condition comprising an effective amount of one or more compounds of formula (I) or formula (II), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, diluent and/or excipient.

Dosage form and rates for pharmaceutical use and compositions are readily determinable by a person of skill in the art.

Dosage forms include tablets, dispersions, suspensions, injections, solutions, syrups, troches, capsules, suppositories, aerosols, transdermal patches and the like. These dosage forms may also include injecting or implanting devices designed specifically for, or modified to, controlled release of the pharmaceutical composition. Controlled release of the therapeutic agent may be effected by coating the same, for example, with hydrophobic polymers including acrylic resins, waxes, higher aliphatic alcohols, polylactic and polyglycolic acids and certain cellulose derivates such as hydroxypropylmethyl cellulose.

In addition, the controlled release may be affected by using other polymer matrices, liposomes and/or microspheres.

Pharmaceutically acceptable carriers and acceptable carriers for systemic administration may also be incorporated into the compositions of this invention.

Suitably, the pharmaceutical composition comprises a pharmaceutically acceptable excipient or an acceptable excipient. By "pharmaceutically acceptable excipient" is meant a solid or liquid filler, diluent or encapsulating substance that may be safely used in systemic administration. Depending upon the particular route of administration, a variety of carriers, well known in the art may be used. These carriers or excipients may be selected

from a group including sugars, starches, cellulose and its derivates, malt, gelatine, talc, calcium sulphate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffered solutions, emulsifiers, isotonic saline, and pyrogen-free water.

Any suitable route of administration may be employed for providing a human or non- human with the pharmaceutical composition of the invention. For example, oral, rectal, parenteral, sublingual, buccal, intravenous, intraarticular, intra-muscular, intra-dermal, subcutaneous, inhalational, intraocular, intraperitoneal, intracerebroventricular, transdermal and the like may be employed.

Pharmaceutical compositions of the present invention suitable for administration may be presented in discrete units such as vials, capsules, sachets or tablets each containing a predetermined amount of one or more pharmaceutically active compounds of the invention, as a powder or granules or as a solution or a suspension in an aqueous liquid, a non-aqueous liquid, an oil-in- water emulsion or a water-in-oil emulsion. Such compositions may be prepared by any of the method of pharmacy but all methods include the step of bringing into association one or more pharmaceutically active compounds of the invention with the carrier which constitutes one or more necessary ingredients, hi general, the compositions are prepared by uniformly and intimately admixing the agents of the invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product in to the desired presentation.

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 admixture 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, 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 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 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 m powder form, obtained by aseptic isolation of sterile solid or by lyophilisation from solution, for constitution with a suitable vehicle, e.g. sterile, 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, stabilizing and thickening agents, as desired.

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.

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, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing 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.

Alternatively 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).

Conveniently the powder carrier will form a gel in the nasal cavity. 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.

The active compounds of the invention and of the composition of this invention are present in an amount sufficient to prevent, inhibit or ameliorate one or more diseases or conditions selected from the group consisting of: a bacterial infection, a protozoal infection, a parasitic infestation, a cell proliferative disorder, an inflammatory disorder or a pest infestation. Suitable dosages of the compounds of the invention and the pharmaceutical compositions containing such may be readily determined by those skilled in the art.

In a further aspect of the invention, there is provided a method of treating or preventing of a disease or condition comprising administering to a subject in need of such treatment an effective amount of one or more compounds according to the invention, or a pharmaceutically acceptable salt thereof.

In yet another aspect of the invention, there is provided the use of one or more of the compounds according to the invention, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment or prophylaxis of a disease or condition.

In non-limiting embodiments compounds of the invention have one or more activities selected from antiparasitic activity (e.g. against an endoparasite and/or an ectoparasite, such as, Haemonchus contortus), antibiotic activity (e.g. against Bacillus subtilis), antiprotozoal activity (e.g. against Giardia sp. Portland) cytotoxic activity (e.g. against a basal cell carcinoma and/or a squamous cell carcinoma and/or a melanoma and/or a fibrosarcoma and/or a murine myeloma, and/or antitumour activity (e.g. against a leukemia, a melanoma, a prostate cancer, a breast cancer, an ovarian cancer and/or other solid tumour cancers), anti-fuήgal and/or pesticidal activity.

In one aspect of the invention, there is provided a method of treating or preventing a bacterial infection comprising administering to a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the compound of formula (I) and formula (II) is one of EBI- 16, and EBI- 17.

The bacterial infection may be caused by a Gram positive or Gram negative bacteria, especially Gram positive bacteria including bacteria of the Genus Bacillus (e.g. B. subtilis, B. anthracis, B. cereus, B. flrmis, B. licheniformis, B. megaterium, B. pumilus, B. coagulans, B. pantothenticus, B. alvei, B. brevis, B. circulans, B. laterosporus, B. macerans, B. polymyxd, ¹ ' stearothermophilus, B. thuringiensis, sphaericus),

Staphylococcus (e.g. S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus), 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.

In another aspect of the invention there is provided a method of treating or preventing a parasitic infection comprising administering to a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In preferred embodiments, the parasite is a helminth (worm), especially nematodes, trematodes and cestodes, especially Haemonchus contortus, Trichinella spiralis, H. placei, Bursaphelenchus xylophilus, Ostertagia circumcincta, O. ostertagi, Mecistocirrus digitatus, Trychostrongylus axei, Trichuris trichiura, T. vulpis, T. campanula, T. suis, T. ovis, Bunostomum trigonocephalum, B. phleboyomum, Oesophagostomum columbianum, O. radiatum, Cooperia curticei, C. punctata, C. oncophora, C. pectinata, Strongyloides papillosus, Chabertia ovina, Ancylostoma duodenale, A. braziliense. A. tubaeforme, A. caninum, Ascaris lumbricoides, Enterobius vermicularis, E. gregorii, Ascaris lumbricoides, Paragonimus Westermani, Clonorchis sinensis, Fasciola hepatica, Taenia solium, T. saginata, Capillaria aerophila, Necator americanus, species of the genus Trichuris, Baylisascaris, Aphelenchoides, Meliodogyne, Heterodera, Globodera, Nacobbus, Pratylenchus, Ditylenchus, Xiphinema, Longidorus, Trichodorus, Nematodirus.

In this embodiment, preferred compounds include EBI- 16, EBI- 17, EBI- 19 and EBI-33.

In yet another aspect of the invention, there is provided a method of treating or preventing a cell proliferative disorder comprising administering to a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the cell proliferative disorder is a cancer, especially where the cancer is selected from leukaemia, melanoma, prostate cancer, breast cancer, ovarian cancer, basal cell carcinoma, squamous cell carcinoma, fibrosarcoma, colon cancer, lung cancer, a neoplasm and other solid tumour cancers.

In this embodiment, preferred compounds include EBI-I l, EBI-15, EBI-16, EBI-17, EBI-19, EBI-32 and EBI-33.

The present invention further contemplates a combination of therapies, such as the administration of the compounds of the invention or pharmaceutically acceptable salts thereof together with the subjection of the subject to other agents or procedures which are useful in the treatment of cell proliferative disorders such as tumours. 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, cyclophosphamide, doxorubicine, etoposide phosphate, paclitaxel, topotecan, camptothecins, 5-fluorouracil, tamoxifen, staurosporine, avastin, erbitux, imatinib and vincristine. The compounds of the invention may be administered simultaneously, separately or sequentially with the chemotherapeutic drug.

In yet another embodiment of the present invention, there is provided a method of treating or preventing a protozoan infection comprising administering to a subject a compound of formula (I) or a pharmaceutically acceptable salt thereof.

In a preferred embodiment, the protozoan infection is selected from Giardia spp., Trichomonas spp., African trypanosomiasis, amoebic dysentery, babesiosis, balantidial dysentery, Chaga's disease, coccidiosis, malaria and toxoplasmosis, especially Giardia spp. and Trichomonas spp. infections.

In this embodiment, a preferred compound is EBI-17.

In yet another aspect of the invention, there is provided a method of treating or prophylaxis of a fungal infection in a subject comprising administering to a subject, an effective amount of one or more compounds of formula (I), or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of this aspect, the fungal infection is caused by Absidia corymbifera, Acremonium sp., Agaricus bisporus, Agaricus campestris, Alternaria alternate, Aphanoascus flavescens, Apophysomyces elegans, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aureobasidium pullulans, Basidiobolus ranarum, Beauveria bassiana, Bipolar is (Drechslera) australiensis, Candida albicans, Candida sp., Chaetomium globosum, Cryptococcus neoformans, Chrysosporium sp., Cladophialophora bantiana, Cladophialophora carrionii, Cladosporium cladosporioides, Coccidioides immitis, Conidiobolus coronatus, Cunninghamella bertholletiae, Curvularia lunata, Epicoccum nigrum, Epidermophyton floccosum, Exophiala jeanselmei, Exophiala spinifera, Exophiala werneckii, Exserohilum sp., Fonsecaea pedrosoi, Fusarium solani, Gelasinospora sp., Geotrichum candidum, Gliocladium sp., Gymnoascus sp., Hendersonula toruloidea, Histoplasma capsulatum, Lasiodiplodia theobromae, Leptosphaerulina sp., Madurella mycetomatis, Malassezia furfur, Microsporum audouinii, Microsporum canis, Microsporum ferrugineum, Microsporum gypseum, Microsporum nanum, Mortierella wolfii, Mucor sp., Paecilomyces variotii, Paracoccidioides brasiliensis, Penicillium sp., Penicillium marneffei, Phialophora sp., Phialophora verrucosa, Pithomyces chartarum, Pseudallescheria boydii, Pseudoarachniotus sp., Rhizomucor pusillus, Rhizopus sp., Rhizopus homothallicus, Rhizopus oryzae, Rhizopus sexualis, Rhizopus stolonifer, Saksenaea vasiformis, Scedosporium apiospermum, Scedosporium prolificans, Scopulariopsis brevicaulis, Scytalidium sp., Sepedonium sp., Sordaria βmicola, Sporothrix schenckii, Stemphylium sp., Syncephalastrum sp., Trichoderma harzianum, Trichophyton concentricum, Trichophyton equinum, Trichophyton mentagrophytes var. inter digitale, Trichophyton mentagrophytes var. mentagrophytes, Trichophyton mentagrophytes var. quinckeanum, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton soudanense,

Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichophyton roseum, Ulocladium sp., Veronaea botryose, Verticillium sp. or Wangiella dermatitidis.

In some embodiments of this aspect, the compound of formula (I) is EBI-17.

In yet another aspect of the present invention, there is provided a use of a compound of formula (I) in the manufacture of a medicament for treating or preventing a bacterial infection, a parasitic infection, a protozoan infection, a fungal infection or a cell proliferative disorder.

The term "subject" 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), birds (eg. chickens, ducks, geese, parrots, cockatoos, pigeons, finches, raptors, ratites, quail, canaries), captive wild animals (eg. foxes, kangaroos, deer) and reptiles (eg. lizards and snakes). Preferably, the subject is human, a companion animal, a livestock animal or a laboratory test animal. Even more preferably, the subject is a human, a companion animal or livestock animal.

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.

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.

In another aspect of the invention, the compounds of the invention are suitable for use as a pesticide. The invention therefore further provides a pesticidal composition comprising a compound of the invention or a pharmaceutically, an agriculturally or pesticidally acceptable salt thereof and an agriculturally or pesticidally acceptable carrier.

The pesticidal composition may be in the form of an emulsifiable concentrate, a flowable, a wettable powder, a soluble powder, a solution, an aerosol, a dust, a granule or a bait. A person skilled in the formulation of pesticidal compositions would be able to prepare such formulations.

Suitable carriers for pesticidal compositions include, but are not limited to, oils, especially petroleum oils, emulsifiers, solvents such as water or hydrocarbons, surfactants, aerosol spray components such as CFCs, talc or clay.

In yet another aspect of the invention, there is provided a method of controlling pests comprising applying an effective amount of a compound of formula (I) or an agriculturally or pesticidally acceptable salt thereof to a subject and/or an agricultural or other environment infested with the pest.

In some embodiments, the pest is an insect, especially flies, beetles, grasshoppers, locusts, butterflies and moths and their larvae or nymphs, especially the flies (Diptera) such as true flies, fleas, lice, ticks, mosquitoes, gnats and midges. In some embodiments, the pest is a bacterial or fungal pathogen, particularly of plants.

In some embodiments, the pest infests plants. Examples of such pests include, but are not limited to, Acyrthosiphon kondoi (blue-green aphid), Acyrthosiphon pisum (pea aphid), Agrotis spp. (cutworm), Agrypnus variabilis (sugarcane wireworm), Anoplognathus spp. (christmas beetles), Aphodius tα57wαrmαe(blackheaded pasture cockchafer), Austroasca alfalfae (lucerne leaf hopper), Bathytricha truncate (sugarcane and maize stemborer), Bemisia tabaci fwhitefly), Brachycaudus helichrysi (Zeaf curl plum aphid), Brevicoryne brassicae (cabbage aphid), Bruchophagus roddi (lucerne seed wasp), Bruchus pisorum (pea weevil), Bryobia spp. (bryobia mite), Ciampa arietaria (brown pasture looper), Chortoicetes terminifera (Australian plague locust), Chrysodeitis angentifena (tobacco looper), Chrysodeitis eriosoma (green looper), Contarinia sorghicola (sorghum midge), Deroceras spp. (slugs), Diachrysia oricalcea (soybean looper), Etiella behrii (lucerne seed-web moth), Frankliniella schultzei (tomato thrips), Graphognathus leucoloma (white fringed weevil), Halotydeus destructor (ϊedlegged earth mite), Hednota pedionoma (pasture webworm), Helicoverpa armigera (corn earworm), Helicoverpa punctigera (native budworm), Helix spp. (snails), Heteronychus arator (African black beetle), Leucania convecta (common armyworm), Lipaphis erysimi (turnip aphid), Listroderes difficilis (vegetable weevil), Melanacanthus scutellaris (brown bean bug), Merophyas divulsana (lucerne leaf roller), Myzus persicae (green peach aphid), NaIa lividipes (black field earwig), Mythimna convector (common armyworm), Nezara viridula (green vegetable bug), Nysius vinitor (rutherglen bug), Nysius clevelandensis (grey cluster bug), Oncopera rufobrunnea (underground grass grub), Orondina spp. (false wireworm),

Othnonius batesi (black soil scarabs), Penthaleus major (blue oat mite), Persectania ewingii (southern armyworm), Petrobia lateens φrown wheat mite), Pieris rapae (cabbage white butterfly), Piezodorus hybneri fredbanded shield bug), Plutella xylostella (cabbage moth/diamondback moth), Rhopalosiphum maidis (corn aphid), Sericesthis spp. (small brownish cockchafers), Sitona discoideus fsitona weevil), Sminthurus viridis (lucerne flea), Spodoptera exigua (lesser armyworm), Spodoptera letura (cluster caterpillar Spodoptera mauritia (lawn armyworm), Stomopteryx simplexella (soybean moth), Tetranychus ludeni (bean spider mite), Tetranychus urticae (two spotted mite), Therioaphis trifoliif. maculata (spotted alfalfa aphid), Thrips tabaci (onion thrips), Thrips imaginis ("plague thrips), Zizina labradus (grass blue butterfly), Zygrita diva (lucerne crown borer). Pests that cause fungal infections in plants include species from the genera Alternaria, Aspergillus, Fusarium, Penicillium, Rhizopus, Verticillium, Thielaviopsis, Magnaporthe, Rhizoctonia, Phakospora, Puccinia, Pythium and Phytophthora. Pests that cause bacterial infections in plants include species from the genera Agrobacterium, Clavibacter, Erwinia, Pseudomonas, Xanthomonas and Xyella.

In other embodiments, the pests infest subject and/or environments other than plants. Examples of such pests include, but are not limited to, lice, ants including Camponotus spp., Lasius alienus, Acanthomyops interjectus, Monomorium pharaonis, Solenopsis molesta, Tetramorium caepitum, Monomorium minimum, Prenolepis impairs, Formica exsectoides, Iridomyrmex pruinosus, Cremastogaster lineolata, Tapinoma sessile, Paratrechina longicornis, cockroachs, mosquitos, bed bugs including Leptoglassus occidentalis, Acrosternum hiare, Chlorochroa sayi, Podius maculiventris, Murgantia histrionica, Oncopeltus fasciatus, Nabis alternatus, Leptopterna dolabrata, Lygus lineolaris, Adelpocoris rapidus, Poecilocapsus lineatus, Orius insidiosus, Corythucha ciliata, bees, wasps, black widow spider, booklice, boxelder bug, brown recluse spider, clothes moths including Tineola spp., Tinea spp., Trichophaga spp., carpet beetles, centipedes, clover mites, cluster and face flies, cigarette and drugstore beetles, crickets including Acheta spp., Gryllus spp., Gryllus spp., Nemobius spp., Oecanthus spp., Ceuthophilus spp., Neocurtilla spp., daddy-long-legs, domestic flies, drain flies, earwigs, European hornet, fleas including Ctenocephalides felis, Ctenocephalides canis,

Ctenocephalides spp., Nosopsyllus fasciatus, Nosopsyllus spp., Xenopsylla cheopis, Xenopsylla spp., Cediopsylla simplex, Cediopsylla spp., fungus gnats, ground beetles, hide and larder beetles, horse/cattle/deer/pig flies, house dust mites including Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides spp., mites including Ornithonyssus sylviarum, Dermanyssus gallinae, Ornithonyssus bacoti, Liponyssoides sanuineus, Demodex folliculorum, Sarcoptes scabiei hominis, Pyemotes tritici, Acarus siro, Tyrophagus putrescentiae, Dermatophagoides sp., human lice, humbacked flies, Indian meal moth, millipedes, mud daubers, multicolored asian lady beetle, house borer, midges and crane flies, periodical and "dog-day" cicadas, powderpost beetles, roundheaded and flatheaded borers, pseudoscorpions, psyllids or jumping plant lice, spider beetles, sac spiders, sap beetles, termites, silverfish and firebrats, sowbugs and pillbugs, springtails, stinging hair caterpillars, tarantulas, vinegar flies, wasps and hornets, wharf borer, woods cockroach, yellowjacket wasps, fungus beetles, seed weevils, sawtoothed and merchant grain beetles, confused and red flour beetles, granery and rice weevils, indian meal moth, mealworms, drain flies, ticks including Dermacentar spp., Ixodes spp., Rhipicenphalus' spp., carpenter bees, fleas, assassin bugs, human lice, chiggers, mystery bugs, european hornet, stinging hair caterpillars, black-legged tick, mayflies, black flies, horsehair worms, crickets, gypsy moths, grasshoppers, gnats, midges, locusts, mosquitoes including Aedes albopictus, Aedes Canadensis Aedes triseriatus, Aedes tivittatus, Aedes vexans, Aedes spp., Anopheles quadrimaculatus, Anopheles spp., Coquillettidia perturbans, Coquillettidia spp., Culex pipiens, Culex spp.

An agriculturally effective amount may be determined by those skilled in the art using known methods and would typically range from 5 g to 500 g per hectare.

The environment that is infested with a pest may be an agricultural environment, a household environment or an industrial environment.

As used herein, the term "agricultural environment" refers to an environment in which agriculture is carried out, for example, the growing of crops, trees, and other plants of commercial importance. The agricultural environment includes not only the plant itself,

but also the soil and area around the plants as they grow and also areas where parts of plants, for example, seeds, grains, leaves or fruit, may be stored.

A "household environment" includes environments that are inhabited by humans or animals and may include indoor environments such as carpets, curtains, cupboards, bedding and the air inside a house. An "industrial environment" includes environments which are used for industrial purposes such as manufacture, storage or vending of products. Industrial environments include warehouses, manufacturing plants, shops, storage facilities and the like.

In this aspect, preferred compounds of the invention include EBI-15, EBI-19 and EBI-33.

The invention further provides use of a compound of the invention as an agrochemical.

Accordingly, the compound of the invention may be formulated in an appropriate manner for delivery to crops, pastures, forests and other agricultural environments, preferably for the alleviation and/or eradication of one or more insect pests.

Detailed Description of the Invention

Activity Screening

Solvent extraction samples from Cryptocarya oblata and Cryptocarya leucophylla containing compounds of formula (I) were tested to determine therapeutic activity by screening in a range of Microbial Screening Technologies bioassays, notably NemaTOX, ProTOX, MycoTOX, CyTOX, DipteraTOX and TriTOX. For ease of description these bioassays will be described briefly prior to the extraction and chemical structure elucidation methodologies.

NemaTOX (alternatively referred to herein as Ne) is an anthelmintic bioassay, applicable to all parasitic nematodes with free-living life cycle stages, and can be used as a screen to

detect activity and define the species spectrum of compounds against parasitic nematodes and examine the impact of pre-existing resistance to other anthelmintic classes on potency. Haemonchus contortus was utilised for this assay.

The effect on larval development is determined in this assay by the method described by Gill et al. (1995) Int. J. Parasitol. 25: 463-470. Briefly, in this assay nematode eggs were applied to the surface of an agar matrix containing the test sample and allowed to develop through to the L3, infective stage (6 days). At this time the stage of larval development reached and any unusual features (deformity, paralysis, toxicity) were noted by microscopic examination.

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 ¹ 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 h. 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.

MycoTOX (alternatively referred to herein as Tr) is a non-chromogenic bioassay used to detect activity against filamentous fungal pathogens of plants and animals. The bioassay

features a solid phase agar base into which the test compound has been incorporated. As the growth patterns of filamentous fungi are readily apparent on the agar surface the extent of mycelial growth, sporulation (if relevant to the species under investigation) and colour changes with maturation are measured. Compounds with potent antifungal activity inhibit germination of fungal spores and provide a stark contrast to wells containing inactive compounds with the excessive fungal growth. Lower concentrations of such compounds, or compounds exhibiting a more fungistatic mode of action, show reductions in mycelial growth, extent of sporulation or reductions in other characteristic patterns of colony maturation.

MycoTOX, involves a fungus (spore suspension or mycelial fragments) applied to the surface of an agar matrix containing the test chemical and allowed to grow for a period of up to a week (depending on species). The assay was monitored at two discrete times to identify key development phases in the life cycle (for example mycelial growth and extent of sporulation) and the active wells noted. The monitoring times were dependent on the fungal species under investigation.

The MycoTOX assays were carried out using Trichophyton rubrum.

CyTOX (alternatively referred to herein as Cy) is a microtitre plate bioassay use to identify potential antitumour actives. CyTOX is a chromogenic bioassay with broad application to a wide range of tumour and non-tumour cell lines. The colour transitions in CyTOX are proportional to cell metabolism and turnover and hence offer useful recognition patterns to support the diagnostic classification of actives within a framework of known cytotoxic and antitumour actives.

CyTOX features a liquid media into which the test compound has been incorporated together with a novel chromogen. As the cells grow and divide the chromogen is metabolised from purple in a single step process to a colourless metabolite. CyTOX is routinely undertaken using NSl murine myeloma cell line as a guide to mammalian cell toxicity.

Briefly, in CyTOX the cells were applied to the media containing the test chemical and allowed to grow for 72 hours. The assay was monitored at 24, 48 and 72 hours and the active wells identified.

DipteraTOX,

DipteraTOX is referred to herein as DipG, DipP and DipH. DipG represents no grazing of larva. DipP represents no pupae formation and Dip H represents no hatching of flies. A value of A in DipG, Dip P or Dip H represents very active and a value of P represents active. In DipteraTox the fly eggs are applied to the surface of an agar matrix containing 250 μg per mL of the test chemical and allowed to hatch, develop and pupate for a period up of two weeks. The assay was monitored at two discrete times to determine the extent of grazing of the agar matrix at Week 1 and the presence of adult flies at Week 2. Activity was scored qualitatively as active or inactive at Days 7 and 14 to denote failure to feed and failure to development to the adult stage, respectively. Drosophila melanogaster was utilised for this assay.

TriTOX (alternatively referred to herein as Gi) is a microtitre plate based chromogenic bioassay for the screening of anti-protozoan activity of pathogenic, anaerobic/microaerophilic protozoans for example Giardia spp. and Trichomonas spp. The bioassays are run under anaerobic conditions and features species specific chromogens. The minimum inhibitory concentrations (approximate LD99) are determined by the following method: stock solutions of the unknowns are serially diluted 1/2 to give 12 concentrations over a 2,048-fold range. Aliquots of each concentration(s) are applied to the wells of 96-well microtitre plates and diluted with media. Test substances are scored as active or inactive based on the chromogen colour change. The lowest concentration at which the compound is active is noted as the minimum inhibitory concentration (MIC). Additionally, microscopic inspection is carried out to identify any patterns of morphological change that may be consistent with a type of toxicity and therefore mode of action. Giardia spp. was utilized for this assay.

In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non- limiting examples.

EXAMPLE 1

METHODS

Extraction

Biomass samples, including epicarp and mesocarp from Cryptocarya leucophylla and Cryptocarya oblata were collected and subject to the following extraction process. Two samples and their subsequent fractions are referred in the below examples as EB 84 (C. leucophylla) and EB85 (C. oblata).

Phase 1 - Extraction

The biomass was generously covered with methanol and shaken (~2 L, overnight) followed by filtration to give the first extract. This process was repeated a second time (~2 L, ~5 hours) to generate the second extract. Each extract was examined by analytical HPLC and bioassayed. The sequential methanol extracts are combined and the solvent removed by rotary evaporation to afford an aqueous concentrate.

Phase 2 - Solvent Partition

The aqueous concentrate from the extraction was diluted with water to 400 mL. The diluted sample (code 'Cr') was subsampled for HPLC and bioassay, then shaken with an equal volume of ethyl acetate (EtOAc) in a separatory funnel and the individual layers, EtOAcI and H2O1, collected. Note, occasionally a precipitate would form that was insoluble in either layer. This precipitate was collected by filtration and dissolved in methanol (code 'Me'). The lower aqueous layer (H2O1) was twice more extracted with ethyl acetate to give EtOAc2 and EtOAc3 along with the remaining H2O3 layer. Subsamples of all layers were examined by analytical HPLC and bioassay.

The sequential ethyl acetate extracts were pooled and the solvent removed by rotary evaporation to afford a residue that is weighed. On occasions, analytical HPLC indicated the EtOAc extract contained considerable amounts of extremely lipophilic (RT >9 minutes) material. To remove this material a 10:9:l-hexane:methanol:water partition was performed.

Phase 3 - Preparative HPLC Fractionation

The residue from the solvent partition was investigated by analytical HPLC to find optimum chromatographic conditions for separation of the metabolites present. Using these optimum conditions the residue (~2 g) was fractionated by preparative reverse phase HPLC (C 18, single injection) into 100 fractions. Subsamples of all 100 fractions are examined by analytical HPLC. After analysis of the HPLC traces, the 100 fractions were consolidated into 20 to 30 pooled fractions (pools), some of which may be >80% pure. These pooled fractions were weighed, bioassayed and examined by analytical HPLC.

Solvent Partition Summary for EB84 and EB85

Biomass samples of Cryptocarya leucophylla and Cryptocarya oblata under went extraction and solvent partitioning, using phase 1 and 2 described above to give extracts EB84 and EB85 respectively. Table 1 summarises the amounts of extractable material obtained after solvent partitioning with ethyl acetate.

Table 1: Weights after Ethyl Acetate Partition of Extracts

'WeighfcTotal sample weight in grams of plant material supplied and used for the study. ² EtOAc: Ethyl acetate extractables. ³ %Ext: Ethyl acetate extractables expressed as a percentage of the total sample weight.

Preparative HPLC

The preparative HPLC was carried out on a system consisting of two Shimadzu LC-8A Preparative Liquid Chromatographs with static mixer, Shimadzu SPD-MlOAVP Diode Array Detector and Shimadzu SCL-IOAVP System Controller. The column used was 50 x 100 mm (diameter x length) packed with C 18 Platinum EPS (Alltech).

Approximately 2 grams of ethyl acetate extracted material was dissolved in dimethyl sulphoxide (4 mL) and subjected to preparative HPLC with typically conditions being 60 mL/minute with gradient elution of 30% to 100% acetonitrile/water over 20 minutes followed by acetonitrile for 10 minutes. One hundred fractions (20 mL) were collected, evaporated under nitrogen, and then combined on the basis of HPLC analysis.

UV Analysis

UV spectra were acquired during HPLC with the Shimadzu SPD-MlOAVP Diode Array Detector as mentioned above.

NMR Analysis

All NMR spectra were acquired in d6-dimethyl sulphoxide and referenced to the residual dimethyl sulphoxide signals or deuterated chloroform (CDCl ₃ ) and referenced to residual chloroform signals. ID NMR spectra, ¹ H and ¹³ C [APT], were acquired at 300 and 75 MHz respectively on a Varian Gemini 300BB (Palo Alto CA. USA) spectrometer. 2D NMR spectra, HSQC, HMBC, COSY and TOCSY, and a ID NMR ¹ H spectrum were acquired on a Bruker DRX600 (600 MHz) NMR spectrometer.

Analysis of NMR data was performed using ACD/SpecManager and ACD/Structure Elucidator, both version 6.0 from Advanced Chemistry Development, Inc. (Toronto, ON, Canada).

Electrospray Mass Sepctrometry Analysis (ES-MS) All positive electrospray mass spectra were performed on a Finnigan/Mat TSQ7000 LCMS/MS (San Jose CA. USA).

EXAMPLE 2

EB84 : Extraction and Solvent Partition Each of the extraction and solvent partition layers were tested for bioactivity using the above bioassays. The results are shown in Table 2.

TABLE 2: Activity of Extracts and Solvent Partitions.

LD ₉₉ in μg/mL.

The successive aqueous concentrated extracts were subjected to HPLC. The column used was 50 x 100 mm (diameter x length) packed with Cl 8 Platinum EPS (Alltech). Approximately 2 grams of extracted material was dissolved in dimethyl sulphoxide (4 mL) and subjected to preparative HPLC with typical conditions being 60 mL/minute with gradient elution of 30% to 100% acetonitrile/water over 20 minutes followed by acetonitrile for 10 minutes.

For comparison purposes the first ethyl acetate partition and the third water layers were analysed by HPLC. There was little or no compounds of interest remaining in the third water layer of the third water/ethyl acetate solvent partition.

EB84 : Preparative HPLC Fractionation

In a manner similar to that described in Phase 3 above the EB84 ethyl acetate solvent partition samples were pooled and further worked up using preparative HPLC chromatograph.

The preparative HPLC was used to produce 100 fractions. These fractions were pooled depending on the relative concentration of compounds indicated in the preparative HPLC chromatograph.

The bioactivity of each fraction or pooled fraction resulting from the preparative HPLC was determined using the above bioassay method. The results are summarised below in Table 3.

TABLE 3: Activity of Preparative HPLC Pools.

⁴ LD99 in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point not attained. 5 Weight in mg.

EB85 : Extraction and Solvent Partition

Each of the extraction and solvent partition layers of EB85 were tested for bioactivity using the above bioassays. The results are given in Table 4.

TABLE 4: Activity of Extracts and Solvent Partitions.

4LD ₉₉ in μg/mL.

The successive aqueous concentrated extracts were subjected to HPLC. The column used was 50 x 100 mm (diameter x length) packed with Cl 8 Platinum EPS (Alltech). Approximately 2 grams of extracted material was dissolved in dimethyl sulphoxide (4 mL) and subjected to preparative HPLC with typical conditions being 60 mL/minute with gradient elution of 30% to 100% acetonitrile/water over 20 minutes followed by acetonitrile for 10 minutes.

For comparison purposes the first ethyl acetate partition and the third water layers were analysed by HPLC. There were little or no compounds of interest remaining in the third water layer of the third water/ethyl acetate solvent partition.

EB85 : Preparative HPLC Fractionation

In a manner similar to that described in Phase 3 above the EB85 ethyl acetate solvent partition samples were pooled and further worked up using preparative HPLC chromatograph.

The preparative HPLC was used to produce 100 fractions. These fractions were pooled depending on the relative concentration of compounds indicated in the preparative HPLC chromatograph.

The bioactivity of each fraction or pooled fraction resulting from the preparative HPLC was determined using the above bioassay method. The results are summarised below in Table 5.

TABLE 5: Activity of Preparative HPLC Pools.

I EB85.LA2.132-91/100 | I 41 I I 8 I I o I I o I 2048 I

⁴ LD99 in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point not attained.

⁵ Weight in mg.

EXAMPLE 3

Chemical Structural Elucidation EBI-15

Fractions 51 to 53 of EB85, 204 mg, from the gradient preparative HPLC run were pooled and dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 20 x 100 mm column).

Fractions 30 and 31 of the above preparative HPLC were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 6). From spectral data, it was determined that these fractions contained the following compound, referred to herein as EBI-15.

Molecular weight calculated to be 578.78. Mass spectral analysis: (m/z) 579 [M+H] ⁺ , 601 [M+Na] ⁺ and 1179 [2M+Na ⁺ ].

TABLE 6: NMR Data for EBI-15 in DMSO-d6 at 75/600 MHz

The bioassay results in Table 7 demonstrate that EBI- 15 has efficacy as a cytotoxic agent and as an insecticide:

TABLE 7: Bioassay Results for EBI- 15

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained. P: Active.

EBI-16

Fractions 51 to 53 of EB85, 204 mg, from the gradient preparative HPLC run were pooled and dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 20 x 100 mm column).

Fractions 51 to 59 of the above preparative HPLC were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and

NMR analysis (Table 8). From the spectral data, it was determined that these fractions contained the following compound referred to herein as EBI- 16.

Molecular weight calculated at 620.81. Mass spectral analysis: (m/z) 643 [M+Na] ⁺ and 1263 [2M+Na ⁺ ].

TABLE 8: NMR Data for EBI- 16 in DMSO-d6 at 75/600 MHz.

The bioassay results in Table 9 demonstrate that EBI- 16 has efficacy as a cytotoxic agent, an anthelmintic or antiparasitic agent and an antibacterial agent.

TABLE 9: Bioassay Results for EBI- 16.

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained.

In summary EBI- 16 has anthelmintic activity at 13 μg/mL against H. contortus, cytotoxic activity against murine myeloma NS-I at less than or equal to 0.78 μg/mL and antibacterial action at less than or equal to 13 μg/mL.

EBI-17

Fractions 51 to 53 of EB85, 204 mg, from the gradient preparative ηPLC run were pooled and dissolved in methanol and subjected to preparative ηPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 20 x 100 mm column).

Fractions 61 to 69 of the above preparative ηPLC were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 10). From the spectral data, it was determined that these fractions contained the following compound referred to herein as EBI-17.

Molecular weight calculated as 620.81. Mass spectral analysis: 643 [M+Na] ⁺ and 1263 [2M+Na ⁺ ].

TABLE 10: NMR Data for EBI- 17 in DMSO-d6 at 75/600 MHz.

The bioassay results in Table 11 demonstrate that EBI- 17 has efficacy as a cytotoxic agent, an anthelmintic or antiparasitic agent, an antibacterial agent, an antifungal agent and an antiprotazoal agent.

TABLE 11:

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained.

EBI-Il

Fraction 22 of EB84 (702 mg) from the gradient preparative HPLC run was dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 2O x 100 mm column).

Fractions 9 and 10 of the above preparative HPLC were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 12). From the spectral data, it was determined that these fractions contained the following compound, referred to herein as EBI-11.

Molecular weight calculated as 360.44. Mass spectral analysis: (m/z) 361 [M+H] ⁺ , 383 [M+Na] ⁺ , 721 [2M+H] ⁺ and 743 [2M+Na ⁺ ] .

TABLE 12: NMR Data for EBI-I l in DMSO-d6 at 75/600 MHz.

No. ¹³ C ¹ H Multiplicity (J in Hz)

2 163.6

3 120.3 5.94 m

4 146.6 7.01 ddd (9.7, 5.7, 2.8)

5 29.2 2.46, 2.37 m

6 77.7 4.90 m

7 128.8 5.60 dd (15.5, 6.6)

8 131.7 5.83 m

9 40.7 2.12 t (6.5)

10 66.5 3.72 m

11 45.1 1.33 m

12 63.8 3.90 m

13 45.6 1.37 m

14 66.2 3.66 m

15 39.9 1.60 m

The bioassay results in Table 13 demonstrate that EBI-11 has cytotoxic activity.

TABLE 13:

4 LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained.

EBI-19

Fractions 31 and 32 (62 mg) of the gradient preparative HPLC run of EB84 were combined, dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 20 x 100 mm column).

Fraction 8 of this HPLC run was concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 14). From the spectral data, it was determined that this fraction contained the following compound referred to herein as EBI-19.

Molecular weight calculated as 344.44. Mass spectral analysis: (m/z) 345 [M+H] ⁺ , 362 [M+NH ₄ ] ⁺ , 367 [M+Na] ⁺ , 689 [2M+H] ⁺ and 711 [2M+Na ⁺ ].

TABLE 14: NMR Data for EBI- 19 in DMSO-d6 at 75/600 MHz.

The bioassay results in Table 15 demonstrate that EBI- 19 has efficacy as a cytotoxic agent, an anthelmintic agent or antiparasitic agent and as an insecticide.

TABLE 15:

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained.

⁶ Very active - no grazing of larva.

⁷ Very active - no pupae formed.

⁸ Very active - no hatching of flies.

EBI-32

Fractions 28 and 29 (313 mg) of the EB 84 gradient preparative HPLC run were combined, dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna C 18(2) 20 x 100 mm column).

Fractions 4 to 6 of this HPLC run were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 16). From the spectral data, it was determined that these fractions contained the following compound referred to herein as EBI-32.

Molecular weight calculated as 402.48. Mass spectral analysis: (m/z) 425 [M+Na] ⁺ and 827 [2M+Na ⁺ ].

TABLE 16: NMR Pata for EBI-32 in DMSO-d6 at 75/600 MHz.

No. "C ¹ H Multiplicity (J in Hz)

2 163.5

3 120.3 5.94 m

4 146.6 7.01 ddd (9.7, 5.6, 2.8)

5 29.1 2.44, 2.38 m

6 77.3 4.90 m

7 130.2 5.63 dd (15.5, 6.4)

8 129.3 5.71 m

9 37.1 2.34, 2.25 m

10 70.1 5.00 m

11 41.7 1.52 m

12 63.3 3.67 m

13 45.6 1.36 m

14 65.9 3.64 m

15 39.9 1.59 m

16 31.5 2.67, 2.56 m

17 142.5

18,22 128.3 7.18 d (7.0)

19,21 128.2 7.25 t (7.4)

20 125.5 7.14 t (7.2)

23 169.9

24 21.0 1.97 S

12-OH n.d

14-OH n.d

The bioassay results in Table 17 demonstrate that EBI-32 has efficacy as a cytotoxic agent.

TABLE 17:

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained.

EBI-33

Fractions 28 and 29 (313 mg) of the EB 84 gradient preparative HPLC run were combined, dissolved in methanol and subjected to preparative HPLC (10 mL/min with isocratic elution with 55% water/acetonitrile over 30 minutes through a 5 μm Phenomenex Luna Cl 8(2) 20 x 100 mm column).

Fractions 10 to 13 of this HPLC run were combined, concentrated under vacuum, freeze dried and the resulting product analysed by UV spectroscopy, ES-MS and NMR analysis (Table 18). From the spectral data, it was determined that these fractions contained the following compound referred to herein as EBI-33.

Molecular weight calculated as 402.48. Mass spectral analysis: (m/z) 425 [M+Na] ⁺ and 827 [2M+Na ⁺ ].

TABLE 18: NMR Data for EBI-33 in DMSO-d6 at 75/600 MHz.

No. ^1J C ¹ H Multiplicity (J in Hz)

2 163.6

3 120.3 5.94 m

4 146.6 7.01 ddd (9.7, 5.7, 2.8)

5 29.2 2.46, 2.37 m

6 77.6 4.90 m

7 128.9 5.60 dd (15.5, 6.6)

8 131.5 5.82 m

9 40.7 2.12 m

10 66.2 3.71 m

11 45.0 1.35, 1.28 m

12 63.3 3.67 m

13 42.2 1.58 m

14 71.0 5.00 m

15 36.1 1.81 m

16 30.8 2.56 m

17 141.6

18,22 128.2 7.18 m

19,21 128.2 7.25 m

20 125.7 7.15 m

23 170.1

24 21.0 1.97 S

10-OH n.d

12-OH n.d

The bioassay results shown in Table 19 demonstrate that EBI-33 has efficacy as a cytotoxic agent, an anthelmintic agent or antiparasitic agent and an insecticide.

TABLE 19:

⁴ LD ₉₉ in μg/mL calculated as weight of chemical in last well with activity, however the real value may be lower as end point was not attained. 6 Very active - no grazing of larva. 7 Very active - no pupae formation. Very active - no hatching of flies.

EXAMPLE 4

Inhibition of tumour cell line growth in vitro

Method

The SRB (sulfurhodamine) assay was used to quantitate inhibition of cell growth by the compounds. Reagent was added the day after seeding of cells into 96-well plates (3000-5000 cells/well) and allowed to remain on the cells for the duration of the assay. After 5-6 days, cells were fixed and stained with SRB, essentially a comparison of cell number with untreated controls. The assay was similar to that used by the NCI, with the important exception that continuing the cultures for 5-6 days maximizes the clonogenic nature of the assay. This avoids false negatives, arising with some agents where cell growth requires several rounds of replication before it is inhibited.

The human cell lines screened were:

NFF normal cell line (neonatal foreskin fibroblasts)

MM96L

DU 145 prostate cancer

CI80-13S ovarian cancer (cisplatin-resistant)

MM418c5 melanoma

• MCF7 breast cancer

• K562, HL60 leukemia (MTS assay)

• Colo205, colon cancer

The results of the inhibition assay are shown in Table 20.

TABLE 20: Bioactivity of chemicals (IC50 in μg/ml)