NZ553835A - 7-hydroxy-3,4-diphenyl-chroman derivatives - Google Patents

Abstract

Disclosed are 7-hydroxy-3,4-diphenyl-chroman derivatives as represented by formula (I-a), wherein R1 is hydrogen; R2 and R3 are independently hydrogen, hydroxy, alkoxy, halo or OC(O)R7, with the exception that R2 and R3 are both not hydrogen; R4, R5 and R6 are hydrogen, hydroxy, alkoxy, alkyl, acyl, OC(O)R7 or amino; R7 is hydrogen, alkyl, aryl, aryl-alkyl or amino; or a pharmaceutically acceptable salt thereof. Also disclosed is the use of a compound of formula (I-a) as defined above for the manufacture of a medicament for the treatment of cancer or a tumour mass or as a radiosensitising or chemosensitising agent (wherein the medicament can be used to chemosensitise an anti-cancer agent such as cisplatin, dehydroequol, paclitaxel, gemcitabine, dcxorubicin, topotecan or camptothecin).

Description

C:\NRPonbrDCC\RMH\2718459_l DOC-10/02/2010 RECEIVED at IPONZ on 22 February 2010 553835 CHROMAN DERIVATIVES, MEDICAMENTS AND USE IN THERAPY Field of the Invention The present invention relates to certain novel chroman derivatives and their salts and derivatives, compositions containing same, methods for their preparation and uses thereof as therapeutic agents particularly as anti-cancer and chemotherapeutic selective agents.

Background of the Invention Over 700 different naturally occurring isoflavones are known some of which have biological properties with potential therapeutic benefit.

US 5,726,202 generically discloses certain isoflavan compounds, particularly 3,4-diarylchroman and centchroman for the treatment of benign prostatic hypertrophy.

WO 01/17986 also discloses certain isoflavan compounds.

Summary of the Invention Surprisingly, the present inventors have found a novel group of compounds of the general formula (I) and sub-formulae thereof which exhibit important therapeutic activities including strong anti-cancer activity, chemotherapeutic selectivity and radiosensitisation of cancers.

Thus according to an aspect of the invention as claimed there is provided a compound of formula (I-a): RECEIVED at IPONZ on 22 February 2010 C:\NRPortbl\DCC\RMH\2718459J.DOC-10/02/2010 553835 - 1A- r-io (I-a) r3 r2 wherein Ri is hydrogen, R2 and R3 are independently hydrogen, hydroxy, Cj-6-alkoxy, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, R», R5 and R<5 are independently hydrogen, hydroxy, Ci-6-alkoxy, Ci-6-alkyl, Ci-6-acyl, 0C(O)R7, amino, and R7 is hydrogen, Ci-g-alkyl, aryl, aryl-Ci-6-alkyl or amino, or a pharmaceutical^ acceptable salt thereof.

These and other aspects and embodiments of the present invention are described below and in the claims that follow.

According to another aspect of the present invention there is provided a compound of the general formula (I): r-|0 "r3 r2 (I) wherein 553835 PCT/AIJ2005/001436 Ri is hydrogen, alkyl, cycloalkyl or C(0)R7, R2 and R3 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, R4, Rs and R6 are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, Cj-4-alkylamino or di(Cj_4-alkyl)amino, 0C(0)R7 or ORg.. R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and Rs is aryl such as phenyl or arylalkyl such as benzyl, and R9 is hydrogen hydroxy, alkyl, alkoxy, cycloalkyl or halo, or a pharmaceutically acceptable salt or derivative thereof.

In a preferred embodiment of the present invention R9 is hydrogen. Accordingly, in another aspect of the invention there is provided a compound of the formula (I-a): wherein Ri is hydrogen, alkyl, cycloalkyl or C(0)R7, R2 and R3 are independently hydrogen, hydroxy, alkoxy, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, R4, R5 and Rfi are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, 0C(0)R7, amino, and R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.

According to another aspect of the present invention there is provided a process for the preparation of a compound of formula (I) comprising the step of reacting the keto group of a compound of the formula (II): 553835 RlO 0 (II) o ■Rs r2 or the analogue thereof including a substituent which corresponds to Rg in compounds of 5 formula (I) wherein Ri, is alkyl or a protecting group such as Si(Rio)3, R2 and R3 are independently hydrogen, alkoxy or OSi(Rio)3, with the exception that R2 and R3 are not both hydrogen, and Rio is independently alkyl or aryl, with an arylating agent W"M+, wherein W" is an optionally substituted aryl radical, and M+ is one or more counter ions, preferably [MgBr]+, to form the intermediate tertiary alcohol of formula (III): r-,0 (III) r3 r2 or protected derivative thereof or a salt thereof (or an analogue thereof including a 20 substituent which corresponds to R9 in compounds of formula (I)) and which is dehydrated to form a compound of formula (IV): 553835 _4_ (or an analogue thereof including a substituent which corresponds to R9 in compounds of formula (I)) the double bond of which is subsequently reduced, for example, by hydrogenation and optionally deprotected to form a compound of formula (I) .

According to another aspect of the present invention there is provided a compound of the general formula (III), compositions containing same and uses thereof.

In another aspect, there is provided a compound of the general formula (IV), compositions 10 containing same and uses thereof.

Thus, according to another aspect of the present invention there is provided the use of a compound of formula (I) in therapy, particularly chemotherapy and/or as a radiosensitising or chemosensitising agent.

According to another aspect of the present invention there is provided a method for the treatment, prevention or amelioration of a disease or disorder, which comprises administering to a subject one or more compounds of the formula (I) or a pharmaceutically acceptable salt or derivative thereof optionally in association with a carrier and/or 20 excipient.

According to another aspect of the present invention there is provided the use of one or more compounds of formula (I) or a pharmaceutically acceptable salt or derivative thereof in the manufacture of a medicament for the treatment of a disease or disorder.

According to another aspect of the present invention there is provided an agent for the treatment, prophylaxis or amelioration of a disease or disorder which agent comprises one or more compounds of formula (I) or a pharmaceutically acceptable salt or derivative thereof. 553835 According to another aspect of the present invention there is provided a pharmaceutical composition which comprises one or more compounds of formula (I) or a pharmaceutically acceptable salt or derivative thereof in association with one or more pharmaceutical 5 carriers, excipients, auxiliaries and/or diluents.

According to another aspect of the present invention there is provided a drink or food-stuff, which contains one or more compounds of formula (I) or a pharmaceutically acceptable salt or derivative thereof.

These and other aspects of the invention will become evident from the description and claims which follow, together with the accompanying drawings.

Brief Description of the Figures 15 Fig. 1 represents a comparison of dehydroequol (DHE graph A), 3-(4~hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (HMC compound 1 according to the invention graph B) and cisplatin (graph C) toxicity in neonatal foreskin fibroblasts.

Fig. 2 represents HMC efficacy in melanoma cells in comparison with cisplatin, Fig. 3 represents a pharmacokinetic profile of free and total forms of HMC (A) and DHE (B) after p.o (peri oral) administration to BALB/c mice (50 mg/kg).

Fig. 4 represents a comparison of the pharmacokinetic profile the HMC concentration in 25 serum after i.v (intravenously) and i.p (intraperitoneally) administration of HMC formulated in 20% hydroxypropyl-beta-cyclodextrin at a dose of 50 mg/kg.

Fig. 5 represents comparative mean tumour volume data taken from nude mice bearing HPAC pancreatic cancer tumours treated with either i.p dosed 20% HPBCD (vehicle 30 control, qdxl5) or HMC (100 mg/kg, qdxl5). Data represented as mean ± SEM*, student's T-test, p < 0.01. 553835 PCT/AIJ2005/001436 Fig. 6 represents comparative mean terminal tumour mass data taken from nude mice bearing HP AC pancreatic cancer tumours treated with either i.p dosed 20% HPBCD (vehicle control, qdx 15) or HMC (100 mg/kg, qdx 15). Data represented as mean ± SEM*, student's T-test,_p <0.01.

Fig. 7 represents comparative mean terminal tumour mass data taken from nude mice bearing HP AC pancreatic-cancer tumours treated with either i.p dosed 20% HPBCD (vehicle control, qdx 15) or HMC (100 mg/kg, qdx 15). Data represented as mean ± SEM*, student's T-test, p <0.01.

Fig. 8 represents a summary of apoptosis incidence in DHE and HMC treated melanoma cells over a 24 and 48 hour period.

Fig. 9 represents selective initiation of programmed cell death in HMC and DHE treated 15 malignant melanoma cells (Mel-RM and Me4405). The same concentration of DHE and HMC and exposure times do not induce apoptosis in normal fibroblasts (MRC-5).

Fig. 10 represents a 3D analysis of HMC-cisplatin synergy cytotoxicity data in the MM200 melanoma cell line. HMC-cisplatin combinations were assessed using a 5-day 20 combination protocol (Fig 10 A), or a 24 hr HMC-> anti-cancer sequence (Fig 10 B). For each combination experiment HMC was assessed at 10, 5, 2 and 1 pM. See Table 8 for raw data.

Fig. 11 represents the percentage inhibition of TNFa in murine macrophages by 25 compounds 6 and 7 of the invention.

Fig. 12 represents the 1II n.m.r. spectrum of 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyI)chroman-7-ol. 553835 Detailed Description of the Invention The present inventors have found that a class of isoflavan derivatives of the general formula (I) show surprising and unexpected biological and pharmaceutical properties.

The compounds of formula (I) of the invention are believed to have favourable toxicity profiles with normal cells and good bioavailability. Surprisingly the compounds of the invention-exhibit anti-cancer activity, significantly better than or at least comparable to known cancer treatments.

The compounds of formula (I) are cytostatic and cytotoxic against a broad range of cancer cells of human and animal origin. By cancer cells, it is meant cells that display malignant characteristics and which are distinguished from non-cancer cells by unregulated growth and behaviour which usually ultimately is life-threatening unless successfully treated.

The cancer cells that have been found to be responsive to compounds of formula (I) are of epithelial origin (for example, prostate, ovarian, cervical, breast, gall-bladder, pancreatic, colorectal, renal, and non-small lung cancer cells), of mesenchymal origin (for example, melanoma, mesothelioma and sarcoma cancer cells), and of neural origin (for example glioma cancer cells). It is highly unusual and surprising to fmd a related group of 20 compounds that display such potent cytotoxicity against cancer cells, but with low toxicity against non-cancer cells such as keratinocytes derived from human foreskin. Such cancer cell selectivity is highly unusual and unexpected.

Advantageously the compounds of formula (I) show cytotoxicity against cancer cells that 25 are well recognised for being poorly sensitive to standard anti-cancer drugs. It is highly unusual and unexpected to fmd such potent activity against cancers, for example, cholangiocarcinoma, pancreatic adenocarcinoma and melanoma.

Advantageously the compounds of formula (I) also unexpectedly display an ability to 30 radio-sensitise cancer cells, by which it is meant that these compounds either lower the amount of gamma-irradiation that is required to kill the cells, or they convert cancer cells from a state of radio-resistance to a radio-sensitive state. 553835 Additionally the compounds of formula (I) are thought to possess chemo-sensitising activity, that is they increase the cytotoxicity of chemotherapeutic agents, especially to cancer cells, and/or convert cancerous cells from a state of chemo-resistance to a chemo-5 sensitive state.

Compounds of the invention may also provide chemo and/or radio-protective properties for non-cancerous cells. This has significant therapeutic implications because the traumatic side-effects of chemotherapy and radiotherapy are caused by the toxicity of the traditional 10 treatments to non-cancerous cells.

The properties described above offer significant clinical advantages.

The radio and/or chemo-protective properties of the compounds of the invention may be 1.5 employed to protect healthly individuals from the effects of radiation and/or chemical toxins, or lessen the effects of the same.

Thus, the invention also provides the use of compounds of formula (I) to treat patients with cancer by either reducing the rate of growth of such tumours or by reducing the size of 20 such tumours through therapy with said compounds alone, and/or in combination with each other, and/or in combination with other anti-cancer agents, and/or in combination with radiotherapy.

The use of compounds of the present invention either alone or in combination therapy as 25 described above may reduce the adverse side-effects often experienced by patients when treated with standard anti-cancer treatments. The use of compounds of the invention may mean that lower doses can be employed in such therapy which represents an important advance for cancer sufferers.

Preferably R3 in compounds of formula (I) is in the 3-position.

In another aspect of the invention R9 is Ci-4-alkyl, such as methyl. 553835 Preferably in compounds of formula (I-a): Ri is hydrogen, C M-alkyl or C(0)R7, R2 and R3 are independently hydrogen, hydroxy, C-M-alkoxy, halo or 0C(0)R7, 5 provided that R2 and R3 are not both hydrogen, R4, R5 and Rg are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, 0C(0)R7, and R7 is Ci-4-alkyl, phenyl or benzyl, or a pharmaceutically acceptable salt or derivative thereof.

More preferably in compounds of formula (I-a): Ri is hydrogen, methyl, ethyl, propyl, isopropyl or acetyl, R2 and R3 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, bromo, chloro, fluoro or acetyloxy, with the exception that R2 and R3 15 are not both hydrogen, R4 is hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy or acetyloxy, and R5 and R6 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, acetyl, or acetyloxy, or a pharmaceutically acceptable salt or derivative thereof.

Particular preferred compounds of formula (I-a) have the following substituents where: Ri is hydrogen, methyl or acetyl, R2 and R3 are independently hydrogen, hydroxy, methoxy, bromo or acetyloxy, with the exception that R2 and R3 are not both hydrogen, R4 and R6 are independently hydrogen, hydroxy, methoxy or acetyloxy, and R5 is hydrogen, or a pharmaceutically acceptable salt or derivative thereof.

The invention also extends to compounds of formula (I-b): WO 2006/032086 553835 PCT/AU2005/001436 rio.

(I-b) wherein: Ri represents hydrogen or Ci_(,-alkyl, more preferably hydrogen or methyl, especially hydrogen.

R2 represents hydrogen, hydroxy or Ci-6-alkoxy such as methoxy, ethoxy, propoxy, more preferably hydroxy or methoxy, especially hydroxy.

R3 represents hydrogen, hydroxy, Ci-6-alkoxy such as methoxy, ethoxy, propoxy, more preferably hydrogen or methoxy, especially hydrogen, with the proviso that R2 and R3 do not both represent hydrogen, 10 R4 represents hydrogen, hydroxy, Ci-6-alkoxy such as methoxy, ethoxy, propoxy, Ci„6-alkjd such as methyl, ethyl, propyl, isopropyl, especially hydrogen, hydroxy, methoxy or methyl particularly methoxy or hydroxy, R5 represents hydrogen, Ci-6-alkoxy, Ci.^-alkyl, especially hydrogen, methoxy, hydroxy, particularly hydrogen, or a pharmaceutically acceptable salt or derivative thereof.

Preferred compounds of the invention include those of the general formula (I-c): (I-c) wherein: 553835 PCT/AIJ2005/001436 Ri is hydrogen or Ci-Cg alkyl such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, R2 is hydroxy or Ci.6-alkoxy such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, secbutoxy, tertiary butoxy, and R4 is hydroxy or Ci_6-alkoxy such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, secbutoxy, tertiary butoxy or a pharmaceutically acceptable salt or a derivative thereof.

More preferably in compounds of formula (I-c) Ri is hydrogen or methyl, especially hydrogen.

More preferably in compounds of formula (I-c) R2 is hydroxy or methoxy, especially hydroxy.

More preferably in compounds of formula (I-c) R4 is hydroxy or methoxy, especially methoxy.

In an alternative aspect the invention provides compounds of formula (I-d): wherein: Ri is hydrogen, alkyl, cycloalkyl or C(0)R7, and R3 is hydroxy, alkoxy, alkyl, cycloalkyl, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, R4 is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, C]-4-alkylamino or di(C]-4-alkyl)amino or 0C(0)R7, and (I-d) 553835 WO 2006/032086 PCT/AU2005/001436 R.7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino.

In a further alternative aspect the invention provides compounds of formula (I-e); wherein Ri is hydrogen, alkyl, cycloalkyl or C(0)R7, and R2 and Ri are independently hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, Preferably in compounds of formula (I-e) R] represents hydrogen or methyl, especially hydrogen.

Preferably in compounds of formula (I-e) R2 represents hydroxy or Ci-Cg alkoxy such as 15 methoxy.

In compounds of formula (I-e) preferably R3 represents hydrogen, hydroxy or methoxy, especially hydrogen.

In a further alternative aspect the invention provides compounds of formula (I-f): 553835 PCT/AIJ2005/001436 (I-f) wherein Rj is hydrogen, alkyl, cycloalkyl or C(0)R7, and R3 is hydroxy, alkoxy, alkyl, cycloalkyl, halo or 0C(0)R7, with the exception that R2 R4 is hydrogen, hydroxy, alkoxy, alkyl, cycloalkyl, acyl, amino, Ci_4-alkylamino or di(Ci.4-alkyl)amino, 0C(0)Ry or ORs, and R7 is hydrogen, alkyl, cycloalkyl, aryl, arylalkyl or amino, and Rg is aryl such as phenyl or arylalkyl such as benzyl.

Preferably in compounds of formula (I-f) Ri represents hydrogen or methyl, especially hydrogen.

Preferably in compounds of formula (I-f) R2 represents hydroxy or C] ^-alkoxy such as 15 methoxy, especially hydroxy.

Preferably in compounds of formula (I-f) R3 represents hydrogen or Ci-6-alkoxy such as methoxy, especially hydrogen.

Preferably in compounds of formula (I-f) R3 is in the 3-position.

Preferably in compounds of formula (I-f) Rja represents amino, Cm-alkylamino or di(Ci_4-alkyl)amino, especially amino. and R3 are not both hydrogen, Especially preferred compounds of formula (I) include: 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (HMC; Cpd. 1); 3-(4-hydroxyphenyl)-4-phenylcliroman-7-ol (Cpd. 2); 553835 PCT/AIJ2005/001436 3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 3); 3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 4); 3-(4-hydroxyphenyl)-4-(4-methylphenyl)chroman-7-ol (Cpd. 5); 3-(4-methoxyphenyl)-4~(4-methoxyphenyI)-7-methoxychroman (Cpd. 6); 5 3-(4-hydroxyphenyl)-4-(2,6-dimethoxy-4-hydroxyphenyl)chroman-7-ol (Cpd. 7); 3-(4-hydroxyphenyl)-4-(2-hydroxyphenyl)chroman-7-ol (Cpd. 8); 3-(4-hydroxyphenyl)-4-(3-acyl-2-hydroxy-4-methoxyphenyl)chroman-7-ol (Cpd. 9); 3-(3-hydroxypheny])-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 10); 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chxoman-7-ol (HHC; Cpd. 11); 10 3-(4-bromophenyl)-4-(4-methoxyphenyl)chroman-7-ol (Cpd. 12); 3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol (Cpd. 13); 3-(4-hydroxyphenyl)-4-(3-aminophenyl)chroman-7-ol (Cpd. 14); 3-(4-hydroxyphenyl)-4-(4-phenoxyphenyl)chroman-7-ol (Cpd 15); 3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyl)-8-methylchroman-7-ol (Cpd 16).

HO OMe HO OMe (4) OMe OMe 553835 553835 HO (16) OMe OMe or a pharmaceutically acceptable salt thereof.

The compounds of formula (I) according to the invention include two chiral centres. The 5 present invention includes all the enantiomers and diastereoisomers as well as mixtures thereof in any proportions. The invention also extends to isolated enantiomers or pairs of enantiomers. Methods of separating enantiomers and diastereoisomers are well known to person skilled in the art.

It will be clear to persons skilled in the art that the in compounds of formula (I) the aryl substituents on the heterocyclic ring can be cis or trans relative to each other. Preferably in the compounds of formula (I) these substituents will be cis.

A particularly preferred compound of the present invention is the c/s-isomer of compound No. (1), HMC: HO OH OMe or a pharmaceutically acceptable salt thereof.

Likewise, particularly preferred compounds are compound Nos. (2) to (16) in the cis-conformation. 553835 PCT/AIJ2005/001436 The compounds of formulae (III) and (IV) are intermediates as set out herein. Each corresponding isoflavan-4-oI and isoilavan-3-ene intermediate of compound Nos. (1) to (16) are also preferred compounds of the present invention.

W in compounds of formula (III) and (IV) may, for example, represent the following radicals: or a protected derivative thereof wherein R4, R5 and R& are as defined above for compounds of formula (I).

The term "isoflavone" as used herein is to be taken broadly to include as isoflavones, isoflavenes, isoflavans, isoflavanones, isoflavanols and the like.

The term "alkyl" is taken to include straight chain and branched chain saturated alkyl groups of 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, secbutyl, tertiary butyl, pentyl and the like. The alkyl group more preferably contains preferably from 1 to 4 carbon atoms, especially methyl, ethyl, propyl or isopropyl.

Cycloalkyl includes C3.6 cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.

(V-1) 553835 The alkyl group or cycloalkyl group may optionally be substituted by one or more of fluorine, chlorine, bromine, iodine, carboxyl, Ci-C4-alkoxycarbonyl, Ci-C4-alkyIamino-carbonyl, di-(Ci-C4-alkyl)-amino-carbonyl, hydroxyl, Ci-C4-alkoxy5 formyloxy, C1-C4-5 alkyl-carbonyloxy, Ci-C4-alkylthio, Cs-Q-cycloalkyl or phenyl.

Preferably the alkyl group does not bear any substituents.

The term "aryl" is taken to include phenyl, benzyl, biphenyl and naphthyl and may be 10 optionally substituted by one or more C]-C4-alkyl, hydroxy, Ci-C4-alkoxy, carbonyl, C|-C4-alkoxycarbonyl, C1 -C4-alkylcarbonyloxy, nitro or halo.

The term "halo" is taken to include fluoro, chloro, bromo and iodo, preferably fluoro and chloro, more preferably fluoro, Reference to for example "haloalkyl" will include 15 monohalogenated, dihalogenated and up to perhalogenated alkyl groups. Preferred haloalkyl groups are trifluoromethyl and pentafluoroethyl.

The compounds of the invention include all salts, such as acid addition salts, anionic salts and zwitterionic salts, and in particular include pharmaceutically acceptable salts as would 20 be known to those skilled in the art. The term "pharmaceutically acceptable salt" refers to an organic or inorganic moiety that carries a charge and that can be administered in association with a pharmaceutical agent, for example, as a counter-cation or counter-anion in a salt, Pharmaceutically acceptable cations are known to those of skilled in the art, and include but are not limited to sodium, potassium, calcium, zinc and quaternary amine. 25 Pharmaceutically acceptable anions are known to those of skill in the art, and include but are not limited to chloride, acetate, tosylate, citrate, bicarbonate and carbonate.

Pharmaceutically acceptable salts include those formed from: acetic, ascorbic, aspartic, benzoic, benzenesulphonic, citric, cinnamic, ethanesulphonic, fumaric, glutamic, glutaric, 30 gluconic, hydrochloric, hydrobromic, lactic, maleic, malic, methanesulphonic, naphthoic, hydroxynaphthoic, naphthalenesulphonic, naphthalenedisulphonic, naphthaleneacrylic, oleic, oxalic, oxaloacetic, phosphoric, pyruvic, j?-toluenesulphonic, tartaric, trifluoroacetic, 553835 PCT/AIJ2005/001436 triphenylacetic, tricarballylic, salicylic, sulphuric, sulphamic, sulphanilic and succinic acid.

The term "pharmaceutically acceptable derivative" or "prodrug" refers to a derivative of 5 the active compound that upon administration to the recipient is capable of providing directly or indirectly, the parent compound or metabolite, or that exhibits activity itself and includes for example phosphate derivatives and sulphonate derivatives. Thus, derivatives include solvates, pharmaceutically active esters, prodrugs or the like. This also includes derivatives with physiologically cleavable leaving groups that can be cleaved in 10 vivo to provide the compounds of the invention or their active moiety. The leaving groups may include acyl, phosphate, sulfate, sulfonate, and preferably are mono-, di- and per-acyl oxy-substituted compounds, where one or more of the pendant hydroxy groups are protected by an acyl group, preferably an acetyl group. Typically acyloxy substituted compounds of the invention are readily cleavable to the corresponding hydroxy substituted 15 compounds.

Chemical functional group protection, deprotection, synthons and other techniques known to those skilled in the art may be used where appropriate to aid in the synthesis of the compounds of the present invention, and their starting materials.

The protection of functional groups on the compounds and derivatives of the present invention can be carried out by well established methods in the art, for example as described in T. W. Greene, Protective Groups in Organic Synthesis, John Wiley & Sons, New York, 1981.

Hydroxyl protecting groups include but are not limited to carboxylic acid esters, eg acetate esters, aryl esters such as benzoate, acetals/ketals such as acetonide and benzylidene, ethers such as o-benzyl and j?-methoxy benzyl ether, tetrahydropyranyl ether and silyl ethers such as f-butyldimethyl silyl ether.

Protecting groups can be removed by, for example, acid or base catalysed hydrolysis or reduction, for example, hydrogenation. Silyl ethers may require hydrogen fluoride or WO 2006/032086 553835 PCT/AU2005/001436 tetrabutylammonium fluoride to be cleaved.

It will be clear to persons skilled in the art of medicinal chemistry that compounds of formula (I) may be converted into other compounds of formula (I), for example, where a 5 compound of formula (I) bears one or more hydroxyl substituents then one or more of these substituents can be converted in to a halo substituent such as bromo, chloro or iodo by treating the alcohol with a halogenating agent. Halogenating agents include compounds like NBS, hydrobromic acid, chlorine gas etc. It may be necessary during processes such as halogenation to use protecting groups to protect other functionality in the molecule.

Phenolic type hydroxyls may not be readily convertible to the corresponding halogen compound by treatment with a halogenating agent. However, the desired halogen compound may be prepared by, for example, treating an appropriate aryl amine starting material with NaNC>2 in the presence of HCI under reduced temperature conditions such as 15 0°C, to form the corresponding azide salt. Subsequent treatment with CuCl, CuBr, KI or HBF4 may be used to convert the azide into the required halo-compound.

A general process for preparing compounds of formula (I) comprises the step of treating a compound of formula (IV): (IV) wherein Ri, R2, R3 and W are as defined above in relation to compounds of formula (II) with a reducing agent to provide a compounds of formula (I) or a protected derivative 25 thereof.

Reducing agents are well known to persons skilled in the art and can include hydride sources like borohydrides and alkali metal borohydrides, but would include hydrogen in 553835 catalytic hydrogenation where a suitable catalyst such as palladium on carbon may be used. Other suitable hydride sources include sodium triacetoxyborohydride tetrabutyl ammonium triacetoxyborohydride and sodium cyanoborohydride.

Preferably the double bond in compounds of formula (IV) is reduced by hydrogenation.

Compounds of formula (IV) are prepared by dehydrating a compound of formula (III): wherein Ri, R2, R3 and W are as defined above, in relation to compounds of formula (II) or a protected derivative thereof, Dehydration can, for example, be catalysed by acid, by base or facilitated by conversion of 15 the tertiary alcohol into a better leaving group as would be known to those skilled in the Preferably compounds of formula (III) are dehydrated, for example, by treatment with para-toluene sulphonic acid.

(HI) art.

Compounds of formula (III) may be prepared by treating compounds of formula (II): (II) wherein Rj, R2, R3 are as defined above for compounds of formula (II) or a protected 553835 PCT/AIJ2005/001436 derivative thereof with an arylating agent, for example, a compound of formula W"M+ wherein W' is an optionally substituted aryl radical and M+ is one ore more counter ions, preferably [MgBr]+.

The arylating agent \ArM+ may be prepared by Grignard chemistry where the haloaryl compound (V): or a protected derivative thereof wherein 10 R4, R5 and R& are independently hydrogen, alkoxy, alkyl, acyl, 0C(0)R7, a protected hydroxy such as OSi(Rio)3 or protected amino such as trimethyisilylamino phenyl halide, and Rio is independently alkyl or aryl, and X is halo, preferably bromo, is reacted with a metal such as magnesium to form the arylating agent.

Preferably the haloaryl compound (V) is selected from: X X X X wherein R4, R5, R6 and X are as defined above for compounds of formula (V). 553835 PCT/AIJ2005/001436 Reaction of the arylating agent with the ketone of formula (II) provides access to the corresponding isoflavan-4-ols (iii), isoflav-3-enes (IV) and isoflavans (I) of the present invention.

Alternatively compounds of formula (iii) may be prepared by reacting compounds of formula (ii) with a compound analogous to compounds of formula (V) wherein X represents any appropriate leaving group L which is lost in the formation of the product by nucleophilic addition of the aryl moiety to a ketone by reactions well known by those skilled in the art.

Preferably any free alcohols, esters or other such reactive groups in the keto compounds of formula (ii) will be protected, for example, as /-butyldimethylsilyl ethers during the nucleophilic addition reaction.

Compounds of formula (II) can be prepared by reducing the eneone double bond in compounds of formula (VI): or a protected derivative thereof, wherein Ri, R2 and R3 are as defined above, for 20 compounds of formula (ii).

Suitable reducing agents are described above. Preferably reduction of the carbon-carbon double bond can be effected, for example, by hydrogenation.

Access to compounds of general formula (VI) is available by general synthetic methods as set out in Scheme 1 below and as described in published International application No. WOOl/17986, the disclosure of which is incorporated herein by reference. A typical synthesis is depicted in Scheme 1.

RiO (VI) ■r3 r2 553835 R<\0 OH + HO BF3/Et20 70°C r-jo (vi) MeS02CI ,Rs DMF r2 Scheme 1 Access to the variatious 3-phenyl substituted chromans is available by varying the substitution pattern on the phenylacetic acid derived group.

Access to the 4-phenyl substituted chromans is available by varying the substitution pattern of the arylating agent (V).

Analogues of compounds employed in the processes may be used which include a 10 substituent which corresponds to Rg as defined for compounds of formula (I).

As used herein, the terms "treatment", "prophylaxis" or "prevention", "amelioration" and the like are to be considered in their broadest context. In particular, the term "treatment" does not necessarily imply that an animal is treated until total recovery, Accordingly, 15 "treatment" includes amelioration of the symptoms or severity of a particular condition or preventing or otherwise reducing the risk of developing a particular condition.

The amount of one or more compounds of formula (I) which is required in a therapeutic treatment according to the invention will depend upon a number of factors, which include 20 the specific application, the nature of the particular compound used, the condition being treated, the mode of administration and the condition of the patient. 553835 PCT/AIJ2005/001436 Compounds of formula (I) may be administered in a manner and amount as is conventionally practised. See, for example, Goodman and Gilman, "The pharmacological basis of therapeutics", 7th Edition, (1985). The specific dosage utilised will depend upon the condition being treated, the state of the subject, the route of administration and other 5 well known factors as indicated above. In general, a daily dose per patient may be in the range of 0.1 mg to 5 g; typically from 0.5 mg to 1 g; preferably from 50 mg to 200 mg. The length of dosing may range from a single dose given once every day or two, to twice or thrice daily doses given over the course of from a week to many months to many years as required, depending on the severity of the condition to be treated or alleviated.

It will be further understood that for any particular subject, specific dosage regimens should be adjust over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.

Relatively short-term treatments with the active compounds can be used to cause stabilisation or shrinkage or remission of cancers. Longer-term treatments can be employed to prevent the development of cancers in high-risk patients.

The production of pharmaceutical compositions for the treatment of the therapeutic 20 indications herein described are typically prepared by admixture of the compounds of the invention (for convenience hereafter referred to as the "active compounds") with one or more pharmaceutically or veterinary acceptable carriers and/or excipients as are well known in the art.

The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the subject. The carrier or excipient may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose, for example, a tablet, which may contain up to 100% by weight of the active compound, preferably from 0.5% to 59% by weight of the active compound, One or more active compounds may be incorporated in the formulations of the invention, which may be prepared by any of the well known techniques of pharmacy consisting WO 2006/032086 PCT/AU2005/001436 essentially of admixing the components, optionally including one or more accessory ingredients. The preferred concentration of active compound in the drug composition will depend on absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those of skill in the art.

The formulations of the invention include those suitable for oral, rectal, ocular, buccal (for example, sublingual), parenteral (for example, subcutaneous, intramuscular, intradermal, or intravenous), transdermal administration including mucosal administration via the nose, mouth, vagina or rectum, and as inhalants, although the most suitable route in any given 10 case will depend on the nature and severity of the condition being treated and on the nature of the particular active compound which is being used.

Formulation suitable for oral administration may be presented in discrete units, such as capsules, sachets, lozenges, or tablets, each containing a predetermined amount of the 15 active compound; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which includes the step of bringing into association the active compound and a suitable carrier (which may contain one or more accessory ingredients as noted above).

In general, the formulations of the invention are prepared by uniformly and intimately admixing the active compound with a liquid or finely divided solid carrier, or both, and then, if necessary, shaping the resulting mixture such as to form a unit dosage. For example, a tablet may be prepared by compressing or moulding a powder or granules 25 containing the active compound, optionally with one or more other ingredients.

Compressed tablets may be prepared by compressing, in a suitable machine, the compound of the free-flowing, such as a powder or granules optionally mixed with a binder, lubricant, inert diluent, and/or surface active/dispersing agent(s). Moulded tablets may be made by 30 moulding, in a suitable machine, the powdered compound moistened with an inert liquid binder. 553835 PCT/AIJ2005/001436 Formulations suitable for buccal (sublingual) administration include lozenges comprising the active compound in a flavoured base, usually sucrose and acacia or tragacanth; and pastilles comprising the compound in an inert base such as gelatine and glycerin or sucrose and acacia.

Formulations suitable for ocular administration include liquids, gels and creams comprising the active compound in an ocularly acceptable carrier or diluent.

Compositions of the present invention suitable for parenteral administration conveniently 10 comprise sterile aqueous preparations of the active compounds, which preparations are preferably isotonic with the blood of the intended recipient. These preparations are preferably administered intravenously, although administration may also be effected by means of subcutaneous, intramuscular, or intradermal injection. Such preparations may conveniently be prepared by admixing the compound with water or a glycine buffer and 15 rendering the resulting solution sterile and isotonic with the blood. Injectable formulations according to the invention generally contain from 0.1% to 60% w/v of active compound and can be administered at a rate of 0.1 ml/minute/kg.

Formulations for infusion, for example, may be prepared employing saline as the carrier 20 and a solubilising agent such as a cyclodextrin or derivative thereof. Suitable cyclodextrins include a-cyclodextrin, P-cyclodextrin. y-cyclodextrin, dimethyl-(3-cyclodextrin, 2-hydroxyethyl-p-cyclodextrin, 2-hydroxypropyl-cyclodextrin, 3-hydroxypropyl-P-cyclodextrin and tri-methyl-p-cyclodextrin. More preferably the cyclodextrin is hydroxypropyl-P-cyclodextrin. Suitable derivatives of cyclodextrins include Captisol® a sulfobutyl ether derivative of cyclodextrin and analogues thereof as described in US 5,134,127.

Formulations suitable for rectal administration are preferably presented as unit dose suppositories. Formulations suitable for vaginal administration are preferably presented as unit dose pessaries. These may be prepared by admixing the active compound with one or more conventional solid carriers, for example, cocoa butter, and then shaping the resulting mixture.

WO 2006/032086 PCT/AU2005/001436 Formulations or compositions suitable for topical administration to the skin preferably take the form of an ointment, cream, lotion, paste, gel, spray, aerosol, or oil. Carriers which may be used include Vasoline, lanoline, polyethylene glycols, alcohols, and combination of two or more thereof. The active compound is generally present at a concentration of 5 from 0.1% to 5% w/w, more particularly from 0.5% to 2% w/w. Examples of such compositions include cosmetic skin creams.

Formulations suitable for transdermal administration may be presented as discrete patches adapted to remain in intimate contact with the epidermis of the recipient for a prolonged 10 period of time. Such patches suitably contain the active compound as an optionally buffered aqueous solution of, for example, 0.1 M to 0.2 M concentration with respect to the said active compound. See for example Brown, L., et al. (1998).

Formulations suitable for transdermal administration may also be delivered by 15 iontophoresis (see, for example, Panchagnula R, et al., 2000) and typically take the form of an optionally buffered aqueous solution of the active compound. Suitable formulations comprise citrate or Bis/Tris buffer (pH 6) or ethanol/water and contain from 0.1 M to 0.2 M active ingredient.

Formulations suitable for inhalation may be delivered as a spray composition in the form of a solution, suspension or emulsion. The inhalation spray composition may further comprise a pharmaceutically acceptable propellant such as carbon dioxide or nitrous oxide or a hydrogen containing fluorocarbon such as 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or mixtures thereof.

The active compounds may be provided in the form of food stuffs, such as being added to, admixed into, coated, combined or otherwise added to a food stuff. The term food stuff is used in its widest possible sense and includes liquid formulations such as drinks including dairy products and other foods, such as health bars, desserts, etc. Food formulations 30 containing compounds of the invention can be readily prepared according to standard practices. 553835 PCT/AIJ2005/001436 Therapeutic methods, uses and compositions may be for administration to humans or other animals, including mammals such as companion and domestic animals (such as dogs and cats) and livestock animals (such as cattle, sheep, pigs and goats), birds (such as chickens, turkeys, ducks), marine animals including those in the aquaculture setting (such as fish, 5 crustaceans and shell fish) and the like.

The active compound or pharmaceutically acceptable derivatives prodrugs or salts thereof can also be co-administered with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antibiotics, 10 antifungals, antiinflammatories, or antiviral compounds. The active agent can comprise two or more isoflavones or derivatives thereof in combination or synergistic mixture. The active compounds can also be administered with lipid lowering agents such as probucol and nicotinic acid; platelet aggregation inhibitors such as aspirin; antithrombotic agents such as Coumadin; calcium channel blockers such as verapamil, diltiazem, and nifedipine; 15 angiotensin converting enzyme (ACE) inhibitors such as captopril and enalapril, and (5-blockers such as propanolol, terbutalol, and labetalol. The compounds can also be administered in combination with nonsteriodal antiinflammatories such as ibuprofen, indomethacin, aspirin, fenoprofen, mefenamic acid, flufenamic acid and sulindac. The compounds can also be administered with corticosteroids or an anti-emetic such as 20 zofran®.

Compounds of formula (I) seem to be particularly suitable for co-administration with one or more anti-cancer drugs such as cisplatin, dehydroequol (DHE), taxol (paclitaxel), gemcitabine, doxorubicin, topotecan and/or camptothecin, especially cisplatin, 25 dehydroequol (DHE), taxol. This may result in improved effects in the treatment, for example in the form of synergistic effects, in comparison to when only one of the medicaments is employed. Particularly the compounds of the presently claimed invention, especially HMC (ie compound 1) seem to be chemosensitisers and increase the cytotoxicity of the one or more anticancer drug co-administered therewith. This seems to 30 be the case even though said anticancer drugs work through a variety of different mechanisms, for example cisplatin is thought to work by interacting with nuclear DNA, taxol is thought to work by blocking cells in the G2/M phase of the cell cycle and prevent 553835 them forming normal mitotic apparatus, gemcitabine is thought to work by incorporating itself into the DNA of the cell, ultimately preventing mitosis, doxorubicin is though to be a topoisomerase II inhibitor thereby preventing DNA replication and transcription and topotecan is thought to be a topoisomerase I inhibitor.

Interestingly, in some situations this increased cytotoxicity to cancerous cells is not associated with a corresponding increase in toxicity to non-cancerous cells.

Whilst this observation has important implications for the treatment of many cancers, it is especially important to the treatment of cancers such as melanoma, which are extremely difficult to treat.

The co-administration may be simultaneous or sequential. Simultaneous administration may be effected by the compounds being in the same unit dose, or in individual and discrete unit doses administered at the same or similar time. Sequential administration may be in any order as required and typically will require an ongoing physiological effect of the first or initial active agent to be current when the second or later active agent is administered, especially where a cumulative or synergistic effect is desired.

The invention also extends to a pack comprising the combination therapy.

Compounds for use in the preferred synthetic methods of the present invention may be derived from any number of sources readily identifiable to a person skilled in the art. For example, daidzein is readily available or can be synthesised by standard methods known in the art. Suitable methods may be found in, for example, published international patent applications WO 98/08503 and WO 00/49009, and references cited therein, which are incorporated herein in their entirety by reference.

Compounds of the general formulae (II), (III) and (IV) described above are intermediates in the production of the active isoflavan compounds of formula (I). These intermediates also represent further aspects of the present invention. 553835 PCT/AIJ2005/001436 Whilst not wishing to be bound by theory the compounds of the present invention are thought to regulate a wide variety of signal transduction processes within animal cells and that these signal transduction processes are involved in a wide range of functions that are vital to the survival and function of all animal cells. Therefore, these compounds have 5 broad-ranging and important health benefits in animals including humans, and in particular have the potential to prevent and treat important and common human diseases, disorders and functions, which represents a substantial unexpected benefit.

Thus it seems that the compounds of the present invention have activity as TNFoc 10 inhibitors. It Is hypothesised that TNFa is part of a tightly regulated cytokine network, activating multiple signal transduction pathways and inducing or suppressing a wide variety of genes. TNFa can provide a survival signal for cancer cells and hence it has been referred to as a tumour promoting factor. As a central mediator of inflammation, TNFa provides a molecular link between chronic inflammatory stimuli and the subsequent 15 development of malignant disease. Consequently its inhibition by the compounds of the invention may provide one mechanism by which they exert anti-cancer and/or antiinflammatory activity. Alternatively, these compounds may be used as chemopreventative agents.

The particular benefits of this invention lie in (a) the large range of signal transduction processes targeted by the compounds, (b) the fact that regulation of these various processes includes both up-regulation of some processes and down-regulation of others, and (c) that such a broad and varied effect on signal transduction processes also is accompanied by an independent effect on a range of important enzymes that are fundamental to metabolism 25 and steroidogenesis.

The isoflavan compounds of the present invention exhibit good in vitro toxicity profiles against normal cells. The isoflavans have broad activity, markedly better than or at least comparable with dehydroequol. The isoflavans are highly active against cancer cells 30 representative of leukaemia, glioma, prostate, ovarian, breast and lung cancer. The isoflavan compounds show potent activity against melanoma and cholangiocarcinoma (gall bladder cancer) cell lines. Good activity was observed against colorectal cancer 553835 PCT/AIJ2005/001436 cells.

Radio-sensitisation in vivo may be tested for example employing human epidermoid vulval carcinoma A431 tumours established on the upper leg and subjected to several doses of 5 local radiation (to the tumour bearing leg only). A radiation treatment regimen of 2.5Gy/day for 4 days will delay tumour growth, and the effect of the radiation dose in combination with the test compound could be assessed by monitoring tumour growth delay. Tumour growth delay of ~6 days can be expected using radiation alone. Tumour growth delay using orally dosed test compound can be determined separately. Evidence of 10 test compound mediated radio-sensitisation of A431 tumours is then determined by measuring tumour growth delay using a regimen of orally dosed test compound pre-treated animals followed by the standard radiation therapy regimen described above. A mean growth delay of up to 30 days using the combination treatment compared to up to 10 days using either radiation or test compound monotherapy regimens is evidence of the radio-15 sensitisation properties of the compounds of the invention.

Radio-sensitisation in vitro may be tested, for example, employing clonogenic assays using human the human epidermoid vulva carcinoma A431 cell line to measure response to radiation alone or in combination with test compounds. A drug dose causing 10% toxicity 20 to the cells may be used in combination with graded doses of radiation. The appropriate dose of compound would be determined by clonogenic assay. Evidence of test compound mediated radio-sensitisation is shown by, for example, a >20% toxicity to cells using chemoradiation therapy compared to 10% toxicity using the corresponding monotherapy regimens.

The compounds of the invention are useful in the treatment, prevention or amelioration of diseases associated with aberrant cell survival, aberrant cell proliferation, abnormal cellular migration, abnormal angiogenesis, abnormal estrogen/androgen balance, dysfunctional or abnormal steroid genesis, degeneration including degenerative changes within blood vessel 30 walls, inflammation, and immunological imbalance.

The invention is further illustrated by the following non-limiting Examples and 553835 accompanying drawings.

Examples In the Examples and accompanying drawings which follow the abbreviation "DHE" is used 5 for dehydroequol, "HMC" is used for compound No. 1, being 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-chroman-7-ol and "HHC" is used for compound No. 11, being 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)-chroman-7~ol. 1.0. Synthesis 10 Example 1: 4',7-Diacetoxydaidzein A mixture of daidzein (2.0 g), acetic anhydride (10 ml) and pyridine (2 ml) was heated at 15 105-110 C for lh. After cooling the mixture to room temperature, it was stirred for a further 30 min during which time the diacetate crystallised from solution. The product was filtered, washed thoroughly with water and recrystallised from methanol to yield 4',7-diacetoxydaidzein as colourless prisms (2.4 g, 90%).

Example 2: 7-Acetoxy-3-(4-acetoxyphenyl)chroman-4-one Palladium-on-charcoal (5%, 0.02g) was added to a solution of 4',7-diacetoxydaidzein 25 (0.50g, 1.5 mmol) in ethyl acetate (80 ml) and the mixture was stirred at room temperature under a hydrogen atmosphere for 72h. The catalyst was removed by filtration through Celite and the resulting filtrate was evaporated in vacuo. The residue was recrystallised 553835 from ethanolto yield 7-acetoxy-3-(4-acetoxyphenyl)chroman-4-one (0,40g, 80%) as colourless plates.

Example 3: 7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one AcO Imidazole HO 'OAc OH Imidazole (0.63g) was added to a suspension of 4',7-diacetoxydihydrodaidzein (0.26g, 0.08 mmol) in absolute ethanol (5.0 ml) and the mixture was refluxed for 45 min under argon. The solution was concentrated under reduced pressure and distilled water (10 ml) was added to the residue. The mixture was left overnight under refrigeration and the resulting precipitate was filtered. The crude product was recrystallised from ethyl acetate/dichloromethane to yield 7-hydroxy-3-(4-hydroxyphenyl)chroman-4-one (0.14g, 71%) as a white powder.

Example 4: 7-(toV-ButyldimethlysilyIoxy)-3-(4-(tert-butyIdimethIysiIyloxy) phenyI)chroman-4-one 7-Hydroxy-3-(4-hydroxyphenyl)chroman-4-one 42g, imidazole 130g, tert-20 butyldimethylsilyl chloride 127g, and N,N-dimethylformamide (500ml) were combined in a 2L round bottom flask and stirred under nitrogen at room temperature for 16 hours. The reaction was quenched with the addition of chilled H2O (200ml) with the reaction mix cooled in an ice bath. The resultant white solid was filtered and rinsed with water. Recrystallisation from ethanol afforded the product as white fluffy crystals (35.7g). 553835 PCT/AIJ2005/001436 Example 5: 7-(te^-ButyldimcthylsiIyIoxy)-3-(4-(te^-butyIdimethyIsiIyIoxy)phenyI)-4-(4-methoxyphenyl)chroman-4-ol 7-(ferr-Butyldimethylsilyloxy)-3-(4-(fer/-butyldimcthylsilyloxy)phenyl)-4-(4-methoxypenyl)chroman-4-ol 25g was weighed in a 2-neck round bottom flask, and flushed under nitrogen. Anhydrous THF 80ml was added to the reaction vessel to give a clear slightly yellow solution. A condenser was attached and the reaction vessel placed in an ice bath. Commercial 4-methoxyphenylmagnesium bromide (0.5M solution in THF) 225ml was added to the reaction mix dropwise over 10 minutes. The reaction was quenched by the dropwise addition of wet ether (50:50 H2O: diethyl ether) while still under nitrogen, with a white precipitate forming as increasing amounts of H2O was added. A further amount of HjO was added to the reaction mix before extraction with diethyl ether.

The organic layers were combined and washed with water, brine, dried over anhydrous MgSC>4 and solvent removed on rotovap to give clear yellow oil which solidified overnight to give an off-white solid. The crude product was used in the next step without purification.

MgBr MeO OH II J.

OtBDMS 553835 PCT/AIJ2005/001436 Example 6: 3-(4-Hydroxyphenyl)-4-(4-methoxyphenyl)-2//-chromen-7-ol tBDMSO pTsOH HO OtBDMS "OH MeO OMe 7-(/CT'/-Butyldimethylsilyloxy)-3-(4-(/er/-butyldimethylsily]oxy)phenyl)-4-(4- methoxyphenyl)chroman-4-ol (42g), pTsOH (43 5g), boiling chips and 2.5L of ethanol were combined in a 2-neck 5L round bottom flask with condenser attached. The reaction was heated at reflux for 3 hours. The solvent was concentrated in vacuo to ~ 100ml before being poured into chilled, stirred water (700ml). The mixture was then extracted with 10 ethyl acetate, the combined organic layers washed with water (3 x 2L), brine (1 x 500ml), dried over anhydrous magnesium sulphate and filtered and solvent removed in vacuo to give red/ brown oil. The oil was dissolved in methanol (~ 100ml) and put in freezer overnight.

A white precipitate had formed overnight, which was filtered off and rinsed with methanol. The filtrate was concentrated in vacuo to give a brown oil.

Example 7: 3-(4-Hydroxyphenyl)-4-(4rmethoxyphenyl)-chroman-7-oI HO HO H2 / Pd OH ethanol OH OMe OMe 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-2H-chromen-7-ol 25,5g (70mmoles), 10% PCI/AI2O3 3.95g and 200ml of ethanol were combined in a 2-neck 500ml round bottom 553835 WO 2006/032086 PCT/AU2005/001436 flask. The reaction was hydrogenated at low pressure using standard conditions for 3 hours. The reaction was filtered through Celite to remove the catalyst, rinsed through with ethanol (300ml). The filtrate was concentrated to ~50ml before being poured into chilled, stirred water (1.4L). A pale orange precipitate formed which then formed a brown oil.

The mixture was then extracted with diethyl ether, the combined organic layers washed with water (3 x 1L), brine (1 x 500ml), dried over anhydrous magnesium sulphate and filtered; The solvent was removed in vacuo to give red/ brown oil. The product was recrystallised from diethyl ether (~ 100ml), to give brown solid which was rinsed with chilled diethyl ether to give off-white crystals 11,3g. The 1H NMR spectrum and 10 numbering scheme being shown below.

OMe H ppm Peak Shape J Hz Integrates Comments C2equatorial 4.14 Dd .98 1 C2axial 4.35 Dd 1 Dd is overlapping C3 3.47 Ddd 1 C4 4.20 Dd .12 1 C5 6.71 D 8.05 1 C6 6.36 Dd 2.56, 8.42 1 C8 6.41 D 2.20 1 C9 3.71 S - 3 C2\ C6' 6,61 D - 2 Doublets overlapping for C2',C3',C2" C3" Total integration is 8 C3', C5' 6.61 D . 2 C2", C6" 6.61 D - 2 C3", C5" 6.61 D - 2 In the above general methods, the structures may be optionally substituted or protected 15 with appropriate substituents, or synthons or derivatives thereof. The compounds may be present as, for example, their salts, acetates, benzyl or silyloxy derivatives as can be determined by a skilled synthetic chemist. Hydroxy groups can be readily alkylated 553835 (Mel/base), acylated (AC2O/PV) or silylated (Cl-SiR^/base) and likewise deprotected by standard methods known in the art.

Example 8: 3-(4~Hydroxyphenyl)-4-(4-hydroxyphenyl)-chroman-7-ol 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol (3.17g) was transferred to a round bottom flask and the flask was purged with nitrogen. 33 wt,% Hydrogen bromide in acetic 10 acid (13ml) was added drop-wise to the flask and the contents were heated to reflux at 130°C for 7 hours, The reaction mixture was placed in an ice bath and adjusted to pH 6 using sodium hydroxide solution (40%w/v). The mixture was extracted with ethyl acetate and the ethyl acetate layer was further washed with water and brine prior to drying over magnesium sulphate. The mixture was filtered and the solvent was removed in vacuo to 15 yield a brown solid (2.89g). The solid was dissolved in minimal ethyl acetate and purified by column chromatography (Silica 60H, 200-400mesh using ethyl acetatexhloroform (40:60) eluant). 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol was obtained in ~80% purity and was further purified by semi-preparative high performance liquid chromatography (HPLC). The *H n.m.r, is shown in Fig. 12. 2.0. Materials and Methods 2.1. Tissue culture The human pancreatic cancer cell line, HP AC (CRL-2119) was routinely cultured in 1:1 mixture DMEM (Dulbecco's Modified Eagle Medium Sigma) plus Ham's F12 (Sigma) 25 medium containing HEPES (15 mM), insulin (0.002 mg/ml), transferrin (0.005 mg/ml), hydrocortisone, (40 ng/ml), epidermal growth factor (10 ng/ml). The ovarian cancer cell lines; CP70 was obtained as a gift from Dr. Gil Mor (Yale University) and cultured in a 1:1 mixture DMEM plus Ham's F12 medium, and SKOV-3 (ovarian cancer cell line) was 553835 purchased from ATCC and cultured in McCoys 5a medium. The breast cancer cell line MDA-MB-468 cultured in Leibovitz's L-15 medium. The melanoma cell line MM200 was obtained as a gift from Peter Hersey (University of Newcastle) and A2058 was obtained as a gift from Dr Peter Parsons (QIMR). Both were cultured in DMEM medium.

All cultures were supplemented with 10% FCS (fetal calf serum CSL, Australia), penicillin (lOOU/ml), streptomycin (lOOmg/ml), L-glutamine (2mM) and sodium bicarbonate (1.2 g/L), and cultured at 37°C in a humidified atmosphere of 5% CO2. All cell lines were purchased from ATCC (Maryland, USA) except where noted.

The normal cell line NFF (neonatal foreskin fibroblasts) was a gift from Dr. Peter Parsons (Queensland Institute of Medical Research), RK (rabbit kidney) cells were obtained from Miller Whalley (Macquarie University). Both cell lines were cultured in RPMI supplemented with 10% FCS (CSL, Australia), penicillin (lOOU/ml), streptomycin (lOOmg/ml), L-glutamine (2mM) and sodium bicarbonate (1.2 g/L), and cultured at 37°C in a humidified atmosphere of 5% CO2. 2.2. Proliferation assays IC50 values were determined for each cell line. Cells were seeded in 96-well plates at an appropriate cell density as determined from growth kinetics analysis and cultured for 5 days in the absence and presence of the test compounds. Cell proliferation was assessed after the addition of 20 |al of 3-4,5 dimethylthiazol-2,5-diphenyl tetrazolium bromide (MTT, 2.5mg/ml in PBS, Sigma) for 3-4hrs at 37°C according to manufacturer's instructions. IC50 values were calculated from semi-log plots of % of control proliferation on the y-axis against log dose on the x-axis. 2.3. DHE and HMC Pharmacokinetics - Oral HMC and DHE were prepared as homogenous suspensions in 1% CMC (m:v, water). Both formulations were delivered orally by gavage to female BALB/c mice at a dosage of 50 mg/kg. Three animals were allocated to each timepoint (15 min, 30 min, lhr, 4 hr and 24 hr). At each respective timepoint, animals were euthanased by cervical dislocation and blood collected. Free HMC was analysed by mass spectroscopy. 553835 2.4. HMC Pharmacokinetics - i.v. and i.p.

HMC was prepared as a solution in 20% hydroxypropyl-beta-cyclodextrin (m:v, water). The formulation was delivered either orally by gavage or by i.p. injection to female 5 BALB/c mice at a dosage of 50 mg/kg. Three animals were allocated to each timepoint (15 min, 30 min, lhr, 4 hr and 24 hr). At each respective timepoint, animals were euthanased by cervical dislocation and blood collected. Urine was also collected and analysed for HMC. Free HMC was analysed by mass spectroscopy, 2.5 Pilot in vivo efficacy study - HP AC tumour bearing mice Subconfluent (80%) flasks of HP AC cells were trypsinised, washed in Hanks balanced salt solution (Sigma), resuspended in dubellco's minimal essential medium (Sigma) and an equal volume of matrigel™ (Becton Dickson) at a density of 3,7 x 107 cells per ml. Athymic nu/nu BALB/c mice were s.c. inoculated with 3.7xl06 HPAC cells bi-laterally 15 midway along the dorsal surface. For the HMC (n=3 per dosage regimen) and control groups (n=2), treatment commenced five days post inoculation to allow tumour formation. HMC was formulated 20% HPBCD and delivered i.p. daily for 15 days. The control group received equivalent (weight:weight) i.p. doses of 20% HPBCD. Tumour measurements commenced on day 5 post inoculation (10x10 mm2) and were measured in 2 dimensions, 20 length (a) and width (b), using calipers. Tumor weight (W) was calculated by the formula W=ab2 /2, where a, is the longer of the 2 measurements (Odwyer et al., 1994). Tumour proliferation curves were analyzed with respect to maximal tumour inhibition (treated/control, T/C). On sacrifice, liver, kidney, femur, stomach and colon tissue were fixed in buffered formalin, embedded in paraffin, sections cut and stained with H&E. 25 Stained sections were then submitted to Rothwell consulting for histopathology analysis. Serum biochemistry was conducted on bloods taken from control, vehicle control and HMC treatment groups. Serum analysis was conducted by Veterinary Clinical pathology (U. Syd). 553835 2.6 Three-D model analysis of synergy 3-D model analysis of the cytotoxic interaction between drug A and drug B enables the representation of predicted inhibitory effect of two drugs in combination in 3 dimensions to reveal actual regions of synergy or antagonism. The 3D synergy plots are based on a 5 theory of "Theretical Additivity" (TA) as outlined by Kanzawa et al (Int. J. Cancer 71, 311-319(1997)). Theoretical Additivity was calculated from the cytotoxicities of drug A and drug B as monotherapies using the following formula which assumes the drugs are mutually exclusive inhibitors: (fa)A + (Qb " 2(fa)A(fa)B TA/ 0) 1 " (fa)A(fa)B Where: (Qa = fraction of cells affected by drug A (fa)e = fraction of cells affected by drug B The TA is calculated for each combination of drug concentrations and subtracted from the observed experimental effect for each combination to give a measurement of synergistic action. A positive difference indicates that more cells are affected by the drug combination than would be expected in theory if the two drugs were administered together - hence synergism. A negative difference indicates that less cells were affected than theoretically 20 expected - hence antagonism. 3.0. Results 3.1. Normal cell toxicity Dehydroequol (DHE) was less toxic to both NFF and rabbit kidney cells with IC50 values 25 above 150 |iM when compared to HMC (86 and 61 pM respectively) (Table 1 and Figure 1). In a separate study, HHC was found to be non-toxic to both NFF and RK cells (see again Table 1). When compared to cisplatin, a benchmark chemotherapeutic agent, the degree of toxicity exhibited by HMC and HHC is mild. 553835 Table 1. Relative toxicity of DHE, HMC, HHC and cisplatin against Neonatal foreskin fibroblasts (NFF) and rabbit kidney cells, Tissuc/cell Type Designation Analogue (IC50 uM) Antineoplastic (IC50 uM) DHE HMC HHC Cisplatin Fibroblast Neonatal Foreskin Fibroblasts (Human, NFF) >150 86.12 ± 7.6 60.8 9.85 ±5 - Kidney Rabbit Kidney >150 61 ± 4.3 >150 Not tested 3.2. In vitro efficacy against cancer cells When compared to DHE IC50 values, HMC demonstrated markedly superior activity (~5-10 fold greater) against the multi-drug resistant, p53 mt ovarian cancer cell line (SKOV-3), the AR negative, p53 Mt prostate cancer cell line (PC3), both ER positive (p53 wt) and negative (p53 mt) breast cancer cell lines (MCF-7 and MDA-MB-468 respectively), p53 Mt Glioma (HTB-138), p53 Mt pancreatic cancer (HPAC) and p53 Mt large cell lung 10 cancer (Table 2), HMC exhibited anti-cancer activity comparable to that of DHE against all other cell lines tested (Table 1). Particular efficacy of HMC was noted against melanoma cells. (Table 2.1 and Fig 2). This represents a substantial advantage over the prior art.

HMC was differentially active against 2 separate colorectal cell lines, with marked activity observed against HT-29 cells and somewhat less activity against HCT-15. It is noted that HT-29 and HCT-15 are COX-2 positive and deficient respectively. When examined microscopically and compared to cells treated with vehicle only, HMC treated SKOV-3 cells exhibited morphological changes consistent with cells undergoing apoptosis (cell 20 enlargement, granular appearance in cytosol and blebbling of plasma membrane). In contrast SKOV-3 cells exposed to 100 fiM Dehydroequol after 18 hr retained a relatively normal morphology, comparable with that of vehicle only treated cells. 553835 Table 2.1. Comparison of Dehydroequol and HMC cytoxicity against cell lines representative of different malignancies.

Indication Designation Analogue (IC50 uM) Antineoplastic (IC50 uM) DHE HMC Cisplatin Ovarian A2780 1,7 ± 0.61 1.58 ±0.59 2.10 CP70 1,69 ±0.62 1.21 ±0.29 .30 SKOV-3 21.83 ±4.65 2.26 .40 Prostate PC3 9.09 ±8.12 . 2.9 ±0.92 2.11 LNCaP 4.8 ±3.8 4.52 >10 DU145 .95 ±1.5 3.78 2.07 Breast MCF-7 21.5 ± 13.2 7.15 ±7 3.69 MDA-MB-468 7.9 ±3.5 1.1 ±0.35 0.58 Glioma HTB-138 7.35 ±0.89 1.9 ±0.27 42.30 Pancreatic CRL-2119 56.62 ± 16.8 14.1 ± 1.16 9.36 Leukemic RPMI-8226 3.72 ±0.08 NT NT CCRP-CEM 1.7 ± 0.68 1.90 NT Lung NCI-H23 8.75 ±7.2 3.75 ±2.5 NT NCI-H460 .6 ±3.8 2.23 ±0.15 22.29 Colorectal HT-29 50.45 ±21.9 3.7 ± 1.4 22.7 ±35 HCT-15 24.4 ± 12.57 37.8 ±33 129.9 ±39 Melanoma MM200 2.90 0.7 ± .03 8.3 ±0.7 A2058 NT 1.2 ±0.65 .73 ±2.3 IGR-3 NT 0.53 ±0.02 NT In further studies, the cytotoxicity of various compounds described herein against various cell lines was determined. Compound 14-ene is the 3-ene chromene precursor to the correspondingly reduced chroman, compound 14. It was observed that compounds 1, 2 and 11 show the best efficacy against most all cancer cell lines. Compound 14 shows slightly better efficacy in general compared to its corresponding 14-ene and to compound 6 10 (Table 2.2). 553835 Table 2.2. Chroman compounds 1,2, 6,11 and 14 and chrom-3-ene compound 14-ene cytoxicity against cell lines representative of different malignancies.

Indication Cell line Compound (IC50 uM) HMC 1 2 6 HHC 11 14-ene 14 Ovarian CP70 2.1 3± 1.2 >100 1.1 ±0.9 >100 >100 Prostate PC3 2.5 ± 1 4.2 ± 0.02 116± 57 0.88 ±0.4 46.2 ± 7 32.5 ±2,1 Breast MDA-MB-468 2,8 ± 4.2 1.9 28.7 ± 3.6 1 ±0.1 NT 56.6 Glioma HTB-138 1.9 ±0.3 3.77 >100 0.52 ±0.1 82 ± 10 53.5 ±14 Pancreatic HP AC 2.8 ± 0.9 24.4 ± 12 >100 31.6 ± 27 81.5 ±59 79 ±56 Leukaemia CCRF-CEM 2 ±0.97 4.02 92 ± 81.6 0.6 ±0.01 >100 73 ±51.6 NSC Lung NCI-H460 2.8 ±2 .4±2.1 NT 0,5 ±0.1 >100 65 Colorectal HT-29 .4 ± 1.8 59.4 >100 2.5 ± 1 97.5 ±30 45 ± 4.7 Melanoma MM200 1.07 ±0.5 7.34 >100 0.6 ±0,3 58 ±2.7 93 ± 2.9 3.3.1. HMC Pharmacokinetics - oral When compared with the pharmacokinetic profile of orally dosed DHE, HMC administered via the same route and dosage (50 mg/kg), HMC exhibited a Cmax of 141 |aM (achieved after 1 hr) compared to 511 |iM for DHE (achieved after 15 min) (Table 3 and Figure 3). Like DHE, HMC is also subject to conjugation with low plasma 10 concentrations of the free form of the molecule observed (1.3 |iM after 30 min) (Table 3 and Figure 3). This is less than half the maximum concentration of free dehydroequol achieved using the same dosage regimen (3.3 juM after 15 min) (Figure 3). The ratio of free:total is greater for HMC when compared to DHE (0.92 vs 0.64 respectively).

Table 3.1. Comparison of free and total plasma concentrations achieved in mice dosed with 50 mg/kg of either HMC or DHE p.o.

Time HMC (uM) DHE (uM) Total Free Total Free 0.25 72 ±4.4 0.38 ±0.04 511.5 ± 99 3.3 ±0.13 0.5 122 ±18.4 1.3 ±0.2 357 ±82 2.9 ± 0.05 1 141 ±45.8 0.95 ±0.4 387 ±22.8 1.5 ±0.11 4 33.9 ±12.7 0.19 ±0.08 117.6 ±42 1.3 ±0.07 24 0 0 0.13 ±0.1 0.15 ±0.04 3.3.2. HMC and HHC Pharmacokinetics - oral Human patients were orally dosed with 200 mg of either HMC or HHC. For each 20 challenged patient, blood was taken over a 6 hour period and the results averaged to 553835 characteri.se the plasma pharmacokinetics. The initial results give an oral half life of 3.99 hours for HMC and 3.26 hours for HHC (Table 3.2).

Table 3.2. Comparison of plasma half life concentrations achieved in humans dosed with 5 200 mg of either HMC or HHC Compound Cmax (ng/mL) T max (h) ti/2 (h) HMC (1) 513 2.17 3.99 HHC (11) 341 2.67 3.26 3.4. HMC Pharmacokinetics - i.v. and i.p.

When formulated in HPBCD and delivered i.v., extremely high levels of HMC were 10 observed in the blood equating to 1 mM of drug, 15 min post administration (Figure 4). Elimination kinetics of i.v. delivered HMC were biphasic with HMC being rapidly excreted from blood at a rate of ~1000 uM/hr in the first hr post administration. Assuming linear excretion, this rate slowed to 0.97 uM/hr in hours 1-4 hr post administration. When the same formulation was administered i.p., approximately 1 log less HMC was observed 15 in plasma (131 jaM by i.p. administration vs 1069 piM by i.v. administration) up to 1 hour post administration (Fig 4). Elimination kinetics by i.p. administration however, was much slower during this period (112 uM/hr) thus resulting in a serum concentration some 4.5 fold higher at 1 hr post administration (18.7 by i.p. vs 3.98 by i.v.). Conversely, in hours 1-4 post administration, elimination kinetics was faster after i.p. administration when 20 compared to i.v. (4.6 |j,M/hr by i.p. vs 0.97 [jM/hr by i.v.). These data confirm that HMC is highly bioavailable in its free state when administered by i.v. or i.p. routes. In conjunction with oral PK data, these data also suggest that HMC is susceptible to rapid removal by GI detoxification enzymes. Large concentrations of free HMC were observed in urine over 0.5, 1 and 4hr where collected (3.3 mM, 3.9 mM and 0.093 mM).

WO 2006/032086 PCT/AU2005/001436 ( Table 4. Comparison of the pharmacokinetic profile of HMC in serum after i.v and i.p administration of HMC formulated in 20% hydroxypropyl beta cyclodextrin at a dose of 50 mg/kg). Inset shows HMC concentrations in serum.

Time (hr) Serum HMC (uM) iv ip 0.25 1069.75 131.37 0.50 198.66 31.78 1 3.98 18.74 4 0.07 0.15 24 0.05 0.17 3.5. Pilot in vivo efficacy study - HP AC tumour bearing mice HMC when dosed daily, i.p. at 100 mg/kg significantly retarded the proliferation of HP AC tumours over the treatment period when compared to vehicle control (Figure5). When the mean terminal tumour mass was assessed a significant reduction in final tumour burden (%T/C = 62) was also noted (Figure 6). Importantly, no signs of toxicity were noted in 10 animals dosed with HMC at 100 mg/kg daily for 15 days as determined by weight loss. Indeed animals treated with HMC appeared to thrive when compared to control (Figure 7). Organs (liver, kidney, spleen, femur, stomach and colon) were collected and submitted for histopathological assessment by Rothwell consulting. A limited serum biochemistry analysis was also conducted. These data confirm that HMC demonstrates antitumorigenic 15 activity against HP AC tumours in vivo. 3.5.1. Histpatbological examination of HMC treated groups Histopathological examination of haematoxylin and eosin-stained sections cut from formalin-fixed tissues from two series of experimental mice was made. The liver, kidney, 20 stomach and colon were examined for evidence of toxic damage, the spleen and bone marrow for evidence of myelosuppression and the tumour for degree of necrosis. A score of 0-5 was allocated to each tumour specimen for the degree of necrosis present, a 0 score representing, no necrosis and a score of 5, total necrosis. The sections were scored 'blind' on two separate occasions and the final score given in the results is the mean of these two 25 scores. 553835 Table 5. HMC Toxicology Sample Description Necrosis Score 0-5 4/04 & 1/8 Vehicle control 0.5 4/04 & 2/8 HMC 2 4/04 & 4/8 2 4/04 & 5/8 2 4/04 & 1/11 No treatment control 0.5 3.5.1.1. Overview of results No evidence of toxicity or myelosuppression was detected in sections cut from the tissues of the drug-treated mice. However, in all the drug-treated mice there were patchy mild/moderately severe chronic inflammatory changes affecting the serosa and attached mesentery, as well as reactive changes of the mesothelial cells, in some of the tissues examined. These changes are consistent with the intra-peritoneal injection of a mildly irritant material.

Significant necrosis of tumour tissue was not detected in control specimens 1/8 and 1/11. However, there was considerable necrosis in the tumour sections from the drug-treated mice. 3.5.1.2. Serum biochemistry of HMC treated mice in comparison to control Alakalines phosphatase (ALP), alanine transferase (ALT) and creatine (Cre) were assessed in HMC treated vs control animals. ALP and Cre levels were similar to control and fell within normal ranges (for rat) however, ALT levels in vehicle control and HMC treated groups were much lower than no treatment control levels. 553835 Table 6. Serum biochemistry of HMC treated mice in comparison to control Clinical marker (mice) Sample Group ALP ALT Cre Urea U/L U/L uM mM Control 1/11 116 713 7 6.92 Vechicle Control 1/S 152 441 26 9.81 HMC treated (100 mg/kg) 2/8 74 505 17 7.27 4/8 111 482 8 8.11 /8 100 494 8 7.79 Normal ranges) - 86- 246 - 84-143 -6 6.3 - 8" * for rat ALP: Alakaline phosphatase ALT: Alanine aminotransferase Cre: Creatinine 3.6. HMC induced apoptosis in melanoma cells and normal fibroblasts 3.6.1. Melanoma HMC induced apoptosis in all TRAIL-sensitive and -resistant melanoma cells at concentrations down to 2\xM (-7-10% apoptosis) over 24 and 48 hrs of exposure (Table 7 15 and Figure 8A). At the clinically significant drug concentration of 4 |iM, the incidence of apoptotic cells after 24 hr exposure to HMC rose to 25% and 39% in TRAIL sensitive (MEL-RM) and TRAIL negative (IGR3) cell lines respectively (Table 7 and Figure 8A). The incidence of HMC induced apoptosis at 4 (iM after 24 hr exposure in the other cell lines was ~9%. In comparison, the incidence apoptosis in DHE- treated cells at a 20 concentratoin of 4 |j,M after 24 hr exposure was 0-1%. Over 48 hr at the same concentration of HMC (4jaM), the incidence of apoptosis rose to 21-42 % in all cell lines examined (Table 7 and Figure 8B). DHE was the only other agent to induce moderate levels of apoptosis after 48 hr exposure at a concentration of 4iiM, but only in ME4405 (14%>) and Mel-AT (15%) cell lines (Table 7 and Figure 8B). 553835 PCT/AIJ2005/001436 Table 7. Summary of apoptosis incidence in DHE and HMC treated melanoma cells over 24 and 48 hi.

Drug Percent Apoptosis Concentration TRAIL sensitive Trail-resistant Trail +ve / (uM) Caspase 8 -ve MEL-RM ME4405 IGR3 Mel-AT DHE HMC DHE HMC DHE HMC DHE HMC Control 0 0 0 0 0 0 0 0 a 1 0 0 0 0 0 8 0 0 fc S3 2 0 8 2 8 0 11 3 6 2 n g. a> 4 0 2 9 1 39 4 9 8 2 39 6 11 3 41 8 23 38 11 19 4 32 24 Control 0 0 0 0 0 0 0 0 01 1 1 1.5 1 1.5 1 1 1.5 1 - a xt 5 2 1.5 8 3 8 1 9 7 oo 2 0) 4 3 22 14 42 2 21 8 38 40 S 38 43 59 12 18 8 38 40 3.6.2. Normal fibroblasts Studies were conducted on normal fibroblasts (MRC-5) and TRAIL-sensitive melanoma cells (ME4405 and MEL-RM) using 8|j,M of either DHE or HMC over 24 and 48 hr of exposure (Figure 9). These data demonstrate that HMC, and to a lesser extent DHE, 10 induced marked levels of apoptosis in both melanoma cell lines over 24 and 48hr.

Importantly, while promoting programmed cell death in malignant cells, normal fibroblasts were shown to both HMC and DHE induced apoptosis at 8 jiiM drug over 24 and 48 hr of exposure. These data confirm that HMC is selectively cytotoxic to cancer cells.

The isoflavan compounds of the invention exhibit a superior efficacy profile against all cancers tested when compared to DHE. While HMC is marginally more toxic than DHE in NFF and RIC cells, HMC is markedly less toxic than cisplatin. HMC delivered orally in mice is less bioavailable when compared to DHE but the ratio of free:total is greater. HPBCD-formulated HMC was markedly bioavailable in its free form when delivered i.v 20 and i.p. Significant serum concentrations of free HMC post delivery i.p. were some 18 fold above that of orally delivered HMC. It has been demonstrated that HMC, formulated in 553835 % HPBCD and delivered i.p., exerts a moderate antitumorigenic activity against HP AC tumours in vivo. HMC when delivered at 100 mg/kg to mice is not toxic to major organs as determined by histopathology however, in all the drug-treated mice there were patchy mild/moderately severe chronic inflammatory changes affecting the serosa and attached 5 mesentery, as well as reactive changes of the mesothelial cells which are consistent with the intra-peritoneal injection of a mildly irritant material.

HMC induced moderate-strong levels of apoptosis in TRAIL-resistant and TRAIL-sensitive melanoma cells after both 24 and 48 hrs of exposure. Normal fibroblast cells 10 were resistant to apoptosis after 48 hrs exposure DHE induces mild-moderate levels of apoptosis in TRAIL-resistant and TRAIL-sensitive melanoma cells after 48 hrs of exposure. Normal fibroblast cells were resistant to apoptosis after 48 hr exposure. The ability of both HMC and DHE to induce apoptosis in caspase negative cells suggest that an operational extrinsic programmed cell death pathway is not essential for HMC and DHE 15 mediated apoptosis. 3.7 In vitro HMC synergistic toxicity in cancer cells when combined with cisplatin, paclitaxel and gemcitabine, camptothecin, topotecan and doxorubicin 3.7.1. HMC combination with cisplatin against the MM200 melanoma cell line.

HMC synergy with cisplatin was assessed either in combination over 5 days exposure or in sequence (HMC -» cisplatin) against the MM200 melanoma cell line. It was difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to HMC toxicity as monotherapy (Table 8). 3D analysis of the data reveals that only additive 25 toxicity was apparent using the 5-day combination protocol (Fig. 11). Evidence of synergy using the HMC-cisplatin combination was further assessed using the HMC—» cisplatin sequence (24hr exposure to each compound in sequence) against the melanoma cell line MM200. Using change in IC50 to assess for synergy it was noted that HMC at concentrations of 2 jjM markedly chemosensitised the MM200 cells to cisplatin by >1000 30 fold (Table 8). HMC induced chemosensitisation of MM200 cells to cisplatin was confirmed using 3D analysis of the data (Fig. 1 IB). These data demonstrate that HMC is able to chemosensitise cancer cells, in this case melanoma, to cisplatin. 553835 Table 8. Comparative assessment of synergy between HMC and the cisplatin against the Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a monotherapy or in combination are shown DRUG TREATMENT (IC50 uM) Combined Change Factor 24 hr HMC first Change Factor cisplatin 8.82 — 6.32 — — HMC 0.72 — .64 -- ..

HMC lOuM 1.00E-06 HMC effect — 1.28E-05 >10000 HMC 5uM 1.00E-06 HMC effect — 2.71E-05 >10000 HMC 2uM 1.00E-06 HMC effect -- 1.00E-06 >10000 HMC luM 0.45 HMC effect — 8.29 -1.31 3.7.2 HMC combination with gemcitabine against the Mel-RM melanoma cells.

HMC synergy with gemcitabine was assessed either in combination over 5 days exposure or in sequence (HMC —> gemcitabine) against the Mel-RM melanoma cell line. It was 10 difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to HMC toxicity as monotherapy (Table 9). 3D analysis of the data reveals that 5-day combination protocol did not elicit synergisitic toxicity against the Mel-RM cell line. Evidence of synergy using the HMC-gemcitabine combination was further assessed using the HMC-» gemcitabine sequence (24hr exposure to each compound in sequence) against 15 the melanoma cell line Mel-RM. Using change in IC50 to assess for synergy it was noted that HMC at concentrations of 2 and l(j,M markedly chemosensitised the Mel-RM cells to gemcitabine by >1000 fold. HMC-induced chemosensitisation of Mel-RM cells to gemcitabine was confirmed using 3D analysis of the data. These data demonstrate that HMC is able to chemosesnsitise cancer cells to gemcitabine. 553835 Table 9. Comparative assessment of synergy between HMC and gemcitabine against the Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a monotherapy or in combination are shown.

DRUG TREATM CENT (IC50 uM) Combined Change Factor 24 hr HMC first Change Factor HMC 0.51 — 27.79 — — HMC 1 uM 1.00E-06 ?HMC effect — 1.76E-03 >1000 HMC 2 uM 1.00E-06 ?HMC effect — 1.00E-06 >1000 Gemcitabine 3.88E-03 — 4.67E-03 -- — HMC 0.51 — 27.79 — — HMC 1 uM 1.00E-06 ?HMC effect — 1.00E-06 >1000 HMC 2 uM 1.00E-06 ?HMC effect - 3.89E-05 >100 3.7.3. HMC combination with paclitaxel against the 4405 melanoma cell line.

HMC synergy with paclitaxel was assessed either in combination over 5 days exposure or in sequence (HMC -» paclitaxel) against the 4405 melanoma cell line. It was difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to HMC toxicity as monotherapy (Table 10). A 30 fold reduction in IC50 was noted in the 10 combination experiment when compared to the paclitaxel monotherapy. However, 3D analysis of the data revealed that the 5-day combination protocol did not elicit synergisitic toxicity against the 4405 cell line. Evidence of synergy using the HMC-paclitaxel combination was further assessed using the HMC—> paclitaxel sequence (24hr exposure to each compound in sequence) against the melanoma cell line 4405. Using change in IC50 15 to assess for synergy it was noted that HMC at concentrations of 2 jj.M markedly chemosensitised the 4405 cells to paclitaxel by >1000 fold (Table 10). HMC-induced chemosensitisation of MM200 cells to paclitaxel was confirmed using 3D analysis of the data. These data demonstrate that HMC is able to chemosesnsitise cancer cells to pacltaxel. 553835 Table 10. Comparative assessment of synergy between HMC and paclitaxel against the 4405 melanoma cell line. Average IC50 data for each agent when assessed as a monotherapy or in combination are shown.

DRUG TREATMENT (IC50 uM) Combined Change Factor 24 hr HMC first Change Factor HMC 1 uM 0.006 1.00 — 0.03 -4.44 HMC 2 uM 1.00E-06 5964.400 — 0.01 -1.035 Paclitaxel 1.25E-07 .84E-04 — — HMC 1.26 -- 55.50 — — HMC 1 uM 4.12E-09 .36 — .31E-05 11.00 HMC 2 uM 3.91E-10 ?HMC effect — .48E-09 106633.75 3.7.4. HMC combination with topotecan against the MM200 melanoma cell line.

HMC synergy with topotecan was assessed either in combination over 5 days exposure or in sequence (HMC -»• topotecan) against the MM200 melanoma cell line. It was difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy due to 10 HMC toxicity as monotherapy (Table 8). 3D analysis of the data confirmed that the 5-day combination protocol did not elicit synergisitic toxicity against the MM200 cell line. Evidence of synergy using the HMC-gemcitabine combination was further assessed using the HMC-» topotecan sequence (24hr exposure to each compound in sequence) against the melanoma cell line MM200. Using change in IC50 to assess for synergy it was noted that 15 HMC at a concentration of 2 juM markedly chemosensitised the MM200 cells to topotecan by >1000 fold (Table 11). HMC-induced chemosensitisation of MM200 cells to topotecan was confirmed using 3D analysis of the data. These data demonstrate that HMC is able to chemosesnsitise cancer cells to topotecan. From the 3D analysis the optimum combination of HMC and topotecan against the MM200 melanoma cell line would appear to be 2 |aM 20 HMC and between 1 and 0.1 (iM topotecan. 553835 Table 11, Comparative assessment of synergy between HMC and topotecan against the MM200 melanoma cell line. Average IC50 data for for each agent when assessed as a monotherapy or in combination are shown.

DRUG TREATMENT (IC50 uM) Combined Change Factor 24 hr HMC first Change Factor HMC 1 uM 0.115 -14.145- - « 0.009 9.304 HMC 2 uM 1.00E-06 ?HMC effect — 7.85E-05 >1000 Topotecan 0.095 ... 2.216 — -- HMC 0.702 — 17.952 -- — HMC 1 uM 0.000 ?HMC effect — 0.044 50.656 HMC 2 uM 1.00E-06 ?HCM effect — 1.00E-06 >10000 3.7.5. HMC combination with camptothecin against the Mel-RM melanoma cell line.

HMC synergy with doxorubicin was assessed either in combination over 5 days exposure or in sequence (HMC —» doxorubicin) against the Mel-RM melanoma cell line. It was difficult to assess for synergisitic toxicity using a change in IC50 as a measure of synergy 10 due to HMC toxicity as monotherapy (Table 12). 3D analysis of the data confirmed that the 5-day combination protocol did not elicit synergisitic toxicity against the Mel-RM cell line, indeed evidence of antagonism was noted. Evidence of synergy using the HMC-doxorubicin combination was further assessed using the HMC-* doxorubicin sequence protocol (24hr exposure to each compound in sequence) against the melanoma cell line 15 Mel-RM. Using change in IC50 to assess for synergy it was noted that HMC at a concentration of 2 |j,M chemosensitised the Mel-RM cells to camptothecin by -12 fold (Table 12). 3D analysis of the data, however, reveal a marked degree of synergy between HMC and doxorubicin against Mel-RM cells. These data demonstrate that HMC is able to chemosesnsitise cancer cells to pacltaxel. From the 3D analysis the optimum combination 20 of HMC and camptothecin against the MM200 melanoma cell line would appear to be 2 jjMHMC and between 1 and O.ljoM doxorubicin. 553835 Table 12. Comparative assessment of synergy between HMC and doxorubicin against the Mel-RM melanoma cell line. Average IC50 data for each agent when assessed as a monotherapy or in combination are shown.

DRUG TREATM1 ENT (IC5 ) uM) Combined Change Factor 24 hr HMC first Change Factor Doxorubicin 0.19 — 0.100 — -- HMC 0.54 — 50.515 -- -- HMC 1 uM 0.40 -2.06 — 0.062 1.62 HMC 2 uM 0.18 ?HMC effect 8.16E-03 12.22 3.8 Inhibition of TNFa in murine macrophages (RAW 264.7) by compounds 4,6 & 7 The mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with FCS, 2mM glutamine and 50U/ml penicillin/streptomycin, Subconfluent cells were e detached from the flask by gentle scraping and 24-well plates seeded at 5 x 10 cells per 10 . well and allowed to adhere for lhr. Cells were treated with either test agent (in 0.025% DMSO) or vehicle alone, lhr prior to the addition of 50ng/ml LPS. After incubation for 16 hrs, culture media was collected and stored at -80°C for TNFa measurement using an enzyme immunometric assay (Becton Dickinson).

Compound 6 and compound 7 of the present invention were tested and the results are shown in Figure 12, which indicated that the compounds tested inhibit TNFa in murine macrophages in a dose dependent manner over the concentration ranges tested.

The raw data for compounds 6, 7 and additionally compound 4 is shown in Table 13 20 below.

Table 13. Inhibition of TNFa in murine macrophages by compounds 4, 6 and 7 Concentration (pM) Compound 6 Compound 7 Compound 4 -48.7 -26.2 -6.1 1 -13.0 -25.4 -11.9 0.1 -10.4 -1.4 -6.3 0.001 1.0 19.2 -14.3 0.01 - - -11.3 553835 3.9 Assessment of HHC as a chemosensitiser HHC was screened as chemosensitiser against a panel cell lines representative of a range cancer indications using a panel of cytotoxics commonly used in the treatment of cancer. It 5 has emerged that HHC has an ability to strongly chemosensitise cancer cell lines from different pathologies to gemcitabine (ovarian, prostate, breast and pancreatic cancers, and glioma) (Table 14). Strong synergy has been noted using HHC:cisplatin against ovarian and prostate cancer, mild synergy against colorectal cancer cell lines and synergy was not observed in pancreatic cancer and glioma. Moderate synergy has been noted using the 10 HHC;paclitaxel combination against breast and colorectal cancer, and melanoma cell lines. Equivocal synergy data using the HHC:paclitaxel combination has been noted against ovarian cancer and glioma cell lines and there was no evidence of synergy against prostate and pancreatic cancer cell lines. Data has revealed that HHC is able to strongly chemosensitise the MM96L melanoma cell line to cisplatin, carboplatin and decarbazine 15 (Table 7).

Table 14. Assessment of HHC as a chemosensitiser using a panel of cancer cell lines and standard cytotoxics Drug Cancer Indication Ovarian Prostate Breast Melanoma Glioma Pancreatic CP70 PC3 MDA-468 MM96L HTB-138 HP AC Cisplatin SSSSS SSSSS - SSSSS n.o. n.o.

- SSSSS - - n.o.

- - - - - - - Gemcitabine SSSSS SSSSS SSSSS - SSSSS SSSSS - - - - SSSSS - - - - - - - Paclitaxel n.o. n.o.

MS - MS n.o.

MS - - - n.o. - - - - - n.o.

- Carboplatin - - - SSSSS - - Dacarbazine - - - SSSSS - - Key: SSSSS = Synergy MS = moderate synergy n.o. - not observed = not tested 553835 3.10. Efficacy of DHE, HMC and HHC against selected melanoma cell lines.

In comparison with DHE, both HMC and HHC showed excellent anti-cancer activity against a range of melanoma cell lines. HHC was the most efficacious agent against all melanoma cell lines tested to date having sub 1 |-iM IC50 values (Table 15).

Table 15. Comparison of DHE, HMC and HHC efficacy against melanoma monotherapy Melanoma cell Kive (I C5Q uM) Analogue MM200 A2058 IsR.3 "" RM 4405 MM96L SKMel28 DHE 4.77 ±4 NT NT NT NT 4.76 4.40 HMC 1.13 ±0.52 1.42 ±0.48 0.51 ±0.08 1.4±1.6 1.46 ±0.18 1.31 ±0.38 0.68 ±0.16 HHC 0.66 ±0.28 0.34 0.20 0.39 ±0.28 0.50 0.2 ±0.09 0.36 ±0.34 4.0 Effect on murine macrophages (RAW 264.7) stimulated with LPS The mouse macrophage cell line RAW 264.7 was cultured in DMEM supplemented with foetal calf serum (FCS), 2mM glutamine and 50U/ml penicillin/streptomycin. Subconfluent cells were detached from the flask by gentle scraping and 24-well plates seeded at 5 x 105 cells per well and allowed to adhere for lhr. Cells were then treated either test compound at a concentration of 10pM (in 0.025% DMSO) or vehicle alone, and incubated for lhr. LPS 50ng/ml (LPS -Sigma-Aldrich) was then added. After incubation for 16hrs, culture media was collected and stored at -80°C for ecosanoid measurements using enzyme immunometric assays (PGE2 and TXB2 - Cayman Chemical).

Table 16: Percentage change in eicosanoid synthesis after incubating test compound at 10p,M compared with incubation with vehicle alone. Positive values indicate enhanced synthesis; negative values indicate inhibition of synthesis and consequently suggest antiinflammatory activity.

Compound PGE2 TXB2 1 -33.8 0 2 -12.6 16 6 -37.7 -16.4 11 27.2 51.4 The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation.

WO 2006/032086 553835 PCT/AU2005/001436 -58 However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent without departing from the scope of the invention. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read 5 as limiting the scope of the present invention.

The entire disclosures of all applications, patents and publications, cited herein, if any, are hereby incorporated by reference.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" or "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.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification individually or collectively, and any and all combinations of any two or more of said steps or features.

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 the field of endeavour. 553835 Selected Reference Articles Constantinou Al, Mehta R, Husband A. 2003 Phenoxodiol, a novel isoflavone derivative, inhibits dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis in female Sprague-Dawley rats. Eur J Cancer, 39, 1012-8.

Constantinou Al, Husband A. 2002 Phenoxodiol (2H-l-benzopyran-7-0,l,3-(4-hydroxyphenyl)), a novel isoflavone derivative, inhibits DNA topoisomerase II by stabilizing the cleavable complex. Anticancer Res. 22, 2581-5.

Gamble, JR., Xia, P., Hahn, C., Drew, J., Drogemuller, C., Carter, C., Walker, C., Brown, DM., Vadas, MA. 2003 Phenoxodiol, a derivative of plant flavanoids, shows potent anti-tumour and anti-angiogenic properties, Nature Medicine. Submitted.

Hersey, P and Zhang, X. D. 2001 How melanoma cells evade Trail-induced apoptosis. Nature reviews, Cancer, 1, 142-150.

Kamsteeg, M., Rutherford, T., Sapi, E., Hanczaruk, B., Shahabi, S., Flick, M., Brown, D.M and Mor, G. 2003 Phenoxodiol-an isoflvone analogue- induces apoptosis in chemo-resistant ovarian cancer cells. Oncogene, ;22, 2611-20.

O'Dwyer PJ, Moyer JD, Suffness M, Harrison SD Jr, Cysyk R, Hamilton TC, Plowman J. 1994 Antitumor activity and biochemical effects of aphidicolin glycinate (NSC 303812) alone and hi combination with cisplatin in vivo. Cancer Res. 54, 724-9 Todorov PT, Field WN, Tisdale MJ 1999 Role of a proteolysis-inducing factor (PIF) in cachexia induced by a human melanoma (G361). Br J Cancer. 80,1734-7.

Bellisarii, F. L., S. Gallina, et al. (2001). "Tumor necrosis factor-alpha and cardiovascular diseases." Italian Heart Journal: Official Journal of the Italian Federation of Cardiology. 2(6): 408-17. 553835 PCT/AIJ2005/001436 Szlosarek, P. W. and F. R. Balkwill (2003). "Tumour necrosis factor alpha: a potential target for the therapy of solid tumours." Lancet Oncol 4(9): 565-73.

Nakata E, Hunter N, Mason K, Fan Z, Ang KK, Milas L. 2004 C225 antiepidermal growth 5 factor receptor antibody enhances the efficacy of docetaxel chemoradiotherapy. Int J Radiat Oncol Biol Phys. 59(4): 1163-73.

RECEIVED at IPONZ on 22 February 2010 C:\NRPortbl\DCC\RMH\2718431_l.DOC - NV-196 NZ claims 553835

Claims (17)

Claims

1. A compound of the formula (I-a): R^O (I-a) wherein Ri is hydrogen, R2 and R3 are independently hydrogen, hydroxy, Ci-6-alkoxy, halo or 0C(0)R7, with the exception that R2 and R3 are not both hydrogen, R4, R5 and R6 are independently hydrogen, hydroxy, Ci-6-alkoxy, Ci.6-alkyl, C1-6- acyl, 0C(0)R7, amino, and R7 is hydrogen, Ci^-alkyl, aryl, aryl-Ci-^-alkyl or amino, or a pharmaceutically acceptable salt thereof.

2, A compound of claim 1, wherein Ri is hydrogen, R2 and R3 are independently hydrogen, hydroxy, Ci-4-alkoxy, halo or 0C(0)R7, provided that R2 and R3 are not both hydrogen, R4, R5 and R6 are independently hydrogen, hydroxy, Ci-6-alkoxy, Ci-6-alkyl, C1-6- acyl, 0C(0)R7, and R7 is C^-alkyl, phenyl or benzyl, or a pharmaceutically acceptable salt thereof.

3. A compound of claim 2, wherein Ri is hydrogen, R2 and R3 are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy, C:\NRPortbl\DCC\RMH\2718431_l.DOC - NV-196 NZ claims 553335 RECEIVED at IPONZ on 22 February 2010 -62- isopropoxy, bromo, chloro, fluoro or acetyloxy, with the exception that R2 and R3 are not both hydrogen, R4 is hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy or acetyloxy, mid R5 and R(S are independently hydrogen, hydroxy, methoxy, ethoxy, propoxy, isopropoxy, acetyl, or acetyloxy, or a pharmaceutically acceptable salt thereof.

4. A compound of claim 3, wherein Ri is hydrogen, R2 and R3 are independently hydrogen, hydroxy, methoxy, bromo or acetyloxy, with the exception that R2 and R3 are not both hydrogen, R4 and R$ are independently hydrogen, hydroxy, methoxy or acetyloxy, and R5 is hydrogen, or a pharmaceutically acceptable salt thereof.

5. A compound selected from: 3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-phenylchroman-7-ol; 3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol; 3-(3,4-dimethoxyphenyl)-4-(4-methoxyphenyI)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(4-methylphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(2,6-dimethoxy-4-hydroxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(2-hydroxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(3-acyl-2-hydroxy-4-methoxyphenyl)chroman-7-ol; 3-(3-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol; 3-(4-bromophenyl)-4-(4-methoxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(3-methoxyphenyl)chroman-7-ol; 3-(4-hydroxyphenyl)-4-(3-aminophenyl)chroman-7-ol; and 3-(4-hydroxyphenyl)-4-(4-phenoxyphenyl)chroman-7-ol or a pharmaceutically acceptable salt thereof. RECEIVED at IPONZ on 22 February 2010 C:\NRPortbl\DCC\RMH\2718431_l.DOC -NV-196 NZciaims 553835 -63-

6. A compound according to claim 5 selected from: 3 -(4-hydroxyphenyl)-4-(4-methoxyphenyl)chroman-7-ol; and 3-(4-hydroxyphenyl)-4-(4-hydroxyphenyl)chroman-7-ol.

7. A process for the preparation of a compound of formula (I-a) according to claim 1 comprising the step of reacting the 4-keto group of a compound of the formula (II): wherein Ri is alkyl or Si(Rio)3, R2 and R3 are independently hydrogen, Ci-6-alkoxy, halo or OSi(Rio)3, with the exception that R2 and R3 are not both hydrogen, and Rio is independently Ci-6-alkyl or aryl, with a nucleophilic arylating agent W"M+, wherein W" is optionally substituted aryl, and M+ is one or more counter ions, to form the intermediate tertiary alcohol (III): or a salt thereof, wherein W is optionally substituted aryl, and which intermediate RECEIVED at IPONZ on 22 February 2010 C:''NRPortbl\DCORMH\2118431 _1 .DOC - N V-196 NZ claims 553835 -64- tertiary alcohol (III) is dehydrated to form a compound of formula (IV): RiO (IV) r3 ^ r2 which compound of formula (IV) is subsequently hydrogenated and optionally deprotected to form a compound of formula (la).

8. A process of claim 7, wherein M+ is [MgBr]+.

9. Use of one or more compounds as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment of cancer or a tumour mass or as a radiosensitising or chemosensitising agent.

10. Use of claim 9, wherein the cancer or tumour mass is of epithelial origin, of mesenchymal origin or of neural origin.

11. Use of claim 10, wherein the cancer or tumour mass of epithelial origin is from prostate, ovarian, cervical, breast, gall-bladder, pancreatic, colorectal, renal or non-small lung cancer cells.

12. Use of claim 10, wherein the cancer or tumour mass of mesenchymal origin is from melanoma, mesothelioma or sarcoma cancer cells.

13. Use of claim 10, wherein the cancer or tumour mass of neural origin is from glioma cancer cells.

14. Use of claim 9, where the medicament chemosensitises an anti-cancer agent selected from cisplatin, dehydroequol, paclitaxel, gemcitabine, doxorubicin, topotecan and camptothecin. RECEIVED at IPONZ on 22 February 2010 C:\NRPortblMXlC\RMH\2718431_l.DOC -NV-196 NZclaims 553335 -65-

15. A pharmaceutical composition which comprises one or more compounds as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof in association with one or more pharmaceutical carriers, excipients, auxiliaries and/or diluents.

16. A pharmaceutical composition according to claim 15 which further comprises an additional chemotherapeutic agent.

17. A drink or food-stuff, which contains one or more compounds as defined in any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.