AU2015201006A1 - Benzopyran compounds and use thereof - Google Patents

Abstract

Benzopyran Compounds and Use Thereof Abstract The present invention relates broadly to anti-cancer agents. In particular, the present invention relates to selected benzopyran compounds, the preparation thereof, and their use in methods for treating cancer and reducing the incidence or risk of cancer recurrence.

Description

1 Benzopyran Compounds and Use Thereof Field of the Invention The present invention relates broadly to anti-cancer agents. In particular, the present invention relates to selected benzopyran compounds, the preparation thereof, and their use in methods for treating cancer and reducing the incidence or risk of cancer recurrence. Background of the Invention Cancer kills many thousands of people annually throughout the world. There have been significant breakthroughs made in the treatment and prevention of a wide variety of cancers. For example breast cancer has seen early screening programs as well as a variety of surgical techniques. However, these often prove physically and emotionally debilitating. Moreover, patients who have undergone surgery and subsequent chemotherapy often experience a recurrence. In recent years research has indicated the heterogeneous tumorigenic potential of cancer cells which has lead to the cancer stem cell (CSC) hypothesis. In brief, this hypothesis states that only a fraction of cells within a tumor have stem cell like features, including unlimited proliferative potential. Further evidence in the literature supports the concept that tumours are complex heterogeneous organ-like systems with a hierarchical cellular organization, rather than simply as collections of homogeneous single lineage tumour cells. The initiator tumour cell retains the capacity to generate diverse progeny at various levels of differentiation, from uncommitted pluripotent stem cells, to committed progenitor cells, to fully differentiated senescent descendent cells. In this way, the tumour cell population itself is heterogeneous, adding diverse architecture afforded by the immune, stromal, and vascular cells that are also present in tumours. Some of the cells within this "cancer organ" or tumour have the potential for continued proliferation. The phylogeny of these tumor cells thus suggests the existence of a cell population that retains the ability to self renew while also often possessing the capacity to generate progeny that differentiate. Hence, the cancer stem cell is defined as being a cell within a tumour that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumour. Indeed laboratory evidence confirms that injection of isolated ovarian, brain, colon, breast, prostate or pancreatic cancer stem-like cells into immunocompromised mice results in the formation of tumours that are phenotypically identical to the original tumour and contain both stem-like cells and non-stem-like cells.

2 Hence there are two distinct populations; a relatively well-differentiated subset with limited proliferative capacity forming the bulk of the tumor which phenotypically characterises the disease, and a second smaller, less differentiated subset that contains clonogenic CSCs. Importantly CSCs exhibit multiple-drug resistance, an additional property that contributes to their longevity and metastatic potential by permitting them to survive toxic insults, including many of the drugs currently used to treat cancer. There is therefore a need to develop therapies that specifically target the self-renewal capabilities of the stem cell population, thereby abrogating the source of tumour recurrence as a result of resistance to conventional therapies. Putative CSC markers that have been described for other malignancies, including acute myeloid leukemia (CD34-positive/CD38- negative), breast (CD44-positive/CD24 negative/-low/Linnegative), prostate (CD44-positive/_2_1-high/CD133-positive) and brain (CD133-positive/nestinpositive), have reflected those expressed by their normal tissue counterparts' original status. Recent evidence confirms that CD44+ ovarian cancer cells also posses the ability to form tumours in immunocompromised mice. As with other CSC phenotypes, ovarian cancer stem cells are slow growing, chemoresistant and form tumours in immunocompromised mice that are phenotypically identical to the original tumour in that there are mainly CD44-ve cells forming the bulk of the tumour with small pockets of CD44+ve cells. Many advanced cancers recur despite the use of chemotherapeutic and radiation modalities that initially lead to therapeutic responses. For example, irradiation of glioblastomas can lead to significant radiographic responses, yet these tumors invariably recur and lead to patient death. Frequently, glioblastomas recur in a nodular pattern, suggesting a clonal or polyclonal source of recurrent tumor cells that are able to withstand conventional cytotoxic therapies, including radiation therapy, to cause recurrence of disease. Furthermore, recurrent tumors also demonstrate heterogeneity within the tumor cell population with regard to the presence of both CSCs and non CSCs as well as in histologic and cytogenetic differences. This suggests that the CSCs that populated the original tumor may have withstood therapeutic intervention to repopulate the recurrent tumor even after the bulk of the tumor had been removed by resection or chemoradiation therapy, hence the concept that CSCs are the source of post-therapeutic tumour recurrence. A shift in therapeutic strategy that leads to the development of unique targeted agents that attack CSCs may enhance cancer care and prolong the survival of many patients. The present inventors have surprisingly discovered that a selection of benzopyran compounds are able to exert powerful biological effects on non-CSCs as well as CSCs.

Such compounds offer alternative chemotherapeutic strategies for treating cancer and reducing the incidence or risk of cancer recurrence. Summary of the Invention In a first aspect the present invention provides a compound selected from the group consisting of: HO O HO O HO H OH O H OH F F F OH OH HO O O HO O OMe F: F OH OH HO O HO O F F OH OH F OH OH HO O HO O HO O OH OH FOH C1 HO F OMe F OH 4 HO O HO O HOH F0 OHO HO 0 H OH HO O HO O FF OH OH F F OH OH HO O NHEt F OH or a pharmaceutically acceptable salt, hydrate, derivative, solvate or prodrug thereof. In a second aspect the present invention provides a pharmaceutical composition comprising a compound according to the first aspect together with a pharmaceutically acceptable carrier, diluent or excipient. In a third aspect the present invention provides a method for the treatment of cancer in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound according to the first aspect, or a composition of the second aspect. The cancer may be lung cancer, pancreatic cancer, melanoma, colorectal cancer, ovarian cancer, breast cancer, brain cancer or prostate cancer.

5 The cancer may be resistant to one or more chemotherapeutic agents. The cancer may be temozolomide-resistant glioblastoma. The cancer may be carboplatin-resistant and/or carboplatin refractory ovarian cancer. The cancer may be a cancer that has recurred. In a fourth aspect the present invention provides use of a compound according to the first aspect in the manufacture of a medicament for treating cancer. The medicament may further comprise, or may be administered with, another chemotherapeutic agent. In a fifth aspect the present invention provides a compound according to the first aspect for use in the treatment of cancer. In a sixth aspect the present invention provides a method for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence, the method comprising administration to the subject of an effective amount of a compound according to the first aspect, or a composition of the second aspect. The subject may be a subject who is in cancer remission. The subject may be in remission from ovarian cancer. In a seventh aspect the present invention provides use of a compound according to the first aspect in the manufacture of a medicament for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence. In an eighth aspect the present invention provides a compound according to the first aspect for use in reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence. In a ninth aspect the present invention provides a method for inducing apoptosis in, or inhibiting the proliferation of, a cancer stem cell, the method comprising contacting the cancer stem cell with an effective amount of a compound according to the first aspect. The cancer stem cell may be an ovarian cancer stem cell. In a tenth aspect the present invention provides use of a compound according to the first aspect in the manufacture of a medicament for inducing apoptosis in, or inhibiting the proliferation of, a cancer stem cell. In an eleventh aspect the present invention provides a method for treating a disease in a subject caused by cancer stem cells, the method comprising administration to the subject of a therapeutically effective amount of a compound according to the first aspect, or a composition of the second aspect.

6 The disease may be cancer. The cancer may be a metastatic cancer. The cancer stem cells may be ovarian cancer stem cells. In a twelfth aspect the present invention provides use of a compound according to the first aspect in the manufacture of a medicament for treating a disease caused by cancer stem cells. In a thirteenth aspect the present invention provides a compound according to the first aspect for use in treating a disease caused by cancer stem cells. In a fourteenth aspect the present invention provides a method for preparing a compound of the first aspect comprising the steps of: (a) reducing a compound of formula (V) to produce a compound of formula (VI): R1 R1 HO O O HO O

R

1 0 10 I1 R 1 , R 1 RI R 13

R

11 R15 13 R 14 1 R R1 (V) (VI) wherein R 1 , R 10 , R", R, 13

R

14 and R" are as defined in the following table: Compound R' R'R R I Me 4-OH H 4-F H H 2 Me 4-OH H 3-F 4-OH H 3 Me 2-F 4-OH 3-F 4-OH H 4 Me 3-OH H 3-F 4-OH H 5 Me 3-Me 4-OMe 3-F 4-OH H 6 Me 3-F 4-OH 3-F 4-OH H 7 Me 3-F 4-OH 4-OH H H 8 Me 4-OH H 3-Cl 4-OMe H 9 Me 4-OH H 3-OH 4-F H 10 Me 4-OH H 2-F 3-F 4-OH 7 11 Me 3-Me 4-OMe 4-OH H H 12 Me 4-OH H 3-OMe 4-OMe H 13 Pr 4-OH H 3-F 4-OH H 14 Pr 3-F 4-OH 3-F 4-OH H 15 Pr 4-NHEt H 3-F 4-OH H (b) hydrogenating a compound of formula (VI) to produce a compound of the first aspect. Step (a) may be carried out by reacting a compound of formula (V) with a borane reagent, for example borane dimethylsulfide complex, decborane, 9-BBN or borane tetrahydrofuran complex. Step (b) may be carried out by reacting a compound of formula (VI) with a heterogenous metal catalyst. In one embodiment the method may further comprise: (c) reacting a compound of formula (Ill) with a compound of formula (IV) to produce a compound of formula (V) R1 R1 HO O O HO OH R 3HO >R1 R10 7< 0 O R -- R 13

R

11 R 1 (I1I) (IV) wherein R 1 , R 1 ", R", R, 13

R

14 and R 15 are as defined in the above table. Step (c) may be carried out in the presence of a base. In another embodiment the method may further comprise: (d) reacting a compound of formula (I) with a compound of formula (II) to produce a compound of the formula (111) 8 HO OH

R

1 ±HO HO OH

R

1 3 OOHR1 (I) (II)O (111)0 wherein R', R 10 , R 11 , R 13 , R 14 and R" are as defined in the above table. Step (d) may be carried out by combining compounds (I) and (11) in the presence of phosphorous oxychloride and zinc chloride. In an alternative embodiment step (d) may be carried out by reacting compound (II) with thionyl chloride, followed by reaction with aluminium chloride and compound (1). Definitions The following are some definitions that may be helpful in understanding the description of the present invention. These are intended as general definitions and should in no way limit the scope of the present invention to those terms alone, but are put forth for a better understanding of the following description. Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. The terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element. In the context of this specification, the term "prodrug" means a compound which is able to be converted in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of the invention. For example, an ester prodrug of a compound of the invention containing a hydroxy group may be hydrolysed in vivo to the parent molecule. Suitable esters are, for example, acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates and maleates. In the context of this specification, the term "effective amount" includes a non-toxic but sufficient amount of an active compound to provide the stated effect. When used in reference to cancer recurrence "effective amount" means an amount of a compound of the invention that is required to reduce the incidence of, or risk of an individual 9 experiencing cancer recurrence. Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact "effective amount". However, for any given case an appropriate "effective amount" may be determined by one of ordinary skill in the art. In the context of this specification, the term "therapeutically effective amount" includes a non-toxic but sufficient amount of an active compound to provide the desired therapeutic effect. Those skilled in the art will appreciate that the exact amount of a compound required will vary based on a number of factors and thus it is not possible to specify an exact "therapeutically effective amount". However, for any given case an appropriate "therapeutically effective amount" may be determined by one of ordinary skill in the art. In the context of this specification, the terms "treating", "treatment", "preventing" and "prevention" refer to any and all uses which remedy cancer or symptoms thereof, prevent the establishment of cancer, or otherwise prevent, hinder, retard or reverse the progression of cancer or other undesirable symptoms in any way whatsoever. Thus, the terms "treating", "treatment", "preventing" and "prevention" and the like are to be considered in their broadest context. For example, treatment does not necessarily imply that a subject is treated until total recovery. In the context of this specification, the term "subject" includes human and also non human animals. As such, in addition to being useful in the treatment of cancer in humans, the compounds of the present invention also find use in the treatment of cancer in non-human animals, for example mammals such as companion animals and farm animals. Non-limiting examples of companion animals and farm animals include dogs, cats, horses, cows, sheep and pigs. Preferably, the subject is a human. In the context of this specification the term "recurrence" as it relates to cancer is understood to mean the return of cancerous cells and/or a cancerous tumour after cancerous cells and/or a cancerous tumour have been successfully treated previously. In the context of this specification the term "administering" and variations of that term including "administer" and "administration", includes contacting, applying, delivering or providing a compound or composition of the invention to an organism by any appropriate means. In the context of this specification the term "compounds of the invention" refers to compounds 1 to 15, including compounds 1a to 15a and 1b to 15b. Brief Description of the Drawings Figure 1: CS-6 exhibits anti-proliferative activity against a broad range of cancers.

10 Figure 2: The CS-6 enantiomer (CS-25) exhibits superior anti-proliferative activity against Glioma compared with the racemate. Figure 3: CS-6 demonstrates equipotent anti-proliferative activity against TMZ susceptible and TMZ-resistant GBM cell lines. A and C: CS-6 activity against matched TMZ-susceptible and resistant GBM cell lines D54 and U87 respectively; B and D: TMZ activity against matched TMZ-susceptible and resistant GBM cell lines D54 and U87 respectively. Inset tables show respective IC50 values. All experiments were assayed after 72hr drug exposure. Figure 4: Differential activity of CS-6 against two different GBM patient derived explants. A. GBM14 and B. OAD14. Each of the coloured traces within each graph represents a different experiment. Figure 5: CS-6 inhibits the proliferation of differentiated ovarian cancer cells (OCC-2) and ovarian cancer stem cells (OCSC-2). Panels A (OCC-2 - differentiated ovarian cancer cells) and B (OCSC-2 - ovarian cancer stem cells). Duplicate experiments were set up and the Incucyte real-time imaging system was used to measure proliferation over 72hr. Cells were treated with either, control, 0.0125, 0.025, 0.05, 0.125, .25, 12.5, 25 pg/ml CS-6. Results were expressed as percent cell confluence. Panels C (OCSC-2) and D (OCC-2) show representative cell morphology for each cell type after 1 exposure to CS-6 (right panels) or no treatment (left panels). Duplicate experiments were set up and the Incucyte real-time imaging system was used to monitor proliferation over 72 hrs. Figure 6: Both CS-6 and CS-25 inhibit the proliferation of undifferentiated ovarian cancer stem cells (OCSC-1). Undifferentiated ovarian cancer stem cells (OCSC-1) were treated with the indicated concentrations of CS-6 (Panel A) and CS-25 (Panel B). Duplicate experiments were set up and the Incucyte real-time imaging system was used to monitor proliferation over 72 hrs. Results were expressed as percent cell confluence. Figure 7: Pre-incubation of ovarian cancer stem cells with CS-25 inhibit their subsequent proliferation. Undifferentiated ovarian cancer stem cells OCSC-1 (panel A) and OSCS-2 (panel B) were treated with the indicated concentrations of CS-25. After 22hrs, cells were replenished with drug-free culture medium and monitored for a further 50 hr. Duplicate experiments were set up and the Incucyte real-time imaging system was used to monitor proliferation over 72 hrs. Results were expressed as percent cell confluence. Figure 8: Analysis of CS-25 induced cellular effects in ovarian cancer stem cells. Effect of CS-25 treatment on the apoptotic cascade. OCSC1 (A, B) and F2-CHERRY (CD) cells were treated with CS-25 (0.1 pg/mL) over 24 hrs. Lysates were analyzed for 11 caspase-9 (A, C) and caspase-3 (B,D) activity using the Caspase-Glo 9, 3/7 assays. The expression of XIAP in those lysates was also assessed at the stipulated timepoints using Western blot analyses (see inset figures in B, D). The effect of CS-25 (0.1 pg/ml over 24 hr) on mitochondrial membrane potential of OCSC1 was assessed using MitoTracker red methodology and compared with those cells treated with vehicle. Figure 9: Summary of CS-25 associated cellular effects in ovarian cancer stem cells. Figure 10: CS-6 exhibits superior potency and anti-proliferative activity against ovarian cancer stem cells (OCSC-2) when compared with carboplatin. Undifferentiated ovarian cancer stem cells OCSC-2 (panel A) were treated with the indicated concentrations of CS-25 (panel A) and carboplatin (panel B). Duplicate experiments were set up and the Incucyte real-time imaging system was used to monitor proliferation over 72 hrs. Results were expressed as percent cell confluence. Figure 11: : Pharmacokinetics of CS-25 delivered via the oral, intraperitoneal and intravenous routes. Intravenous (.); intraperitoneal ( ); oral (A) delivery. Inset table shows PK parameters assessed. AUCO-48h: cumulative area under the curve from 0 48h; Cmax: maximum concentration (observed); Tmax: time to maximum concentration (observed); Lambda Z (A\z): elimination rate constant; T 112 (el): terminal half-life; VdObS: volume of distribution (observed); CLObS: clearance (observed); NA: not available. Figure 12: In vivo efficacy and toxicity of CS-25 in U87MG tumour bearing nude mice .Tumour bearing nude mice (n=10 per group) were treated with daily intraperitoneal injections of either vehicle (30% Captisol) or CS-25 (150 mg/kg). Average tumour volume (A) and average body weights (B) were calculated overe the study duration. Inset in panel A shows the average terminal tumour mass calculated for both the vehicle and CS-25 treated animals/ Figure 13: CS-25 Maximum Tolerated Dose assessment in femal BALB/c mice. Mice were administered with CS-25 (30 mg/mI formulated in 30% Captisol), i.p. over 7 consecutive days with either 37.5, 75, 150 or 300 mg/kg (Treatment Phase). After 7 days, CS-25 administration was stopped and animals were monitored for clinical signs and weight gain. Red arrow indicates the first dose of 300mg/kg CS-25. Reference to "CS6" in the above descriptions and in any other part of this specification refers to Compound 2. Detailed Description of the Invention The present invention relates to selected benzopyran compounds of the general formula (I), the preparation of such compounds and their use in treating cancer and reducing the 12 incidence of cancer recurrence. The compounds disclosed herein represent a selection invention with respect to US2012/0251630, W02012/061409, W02006/032086 and W02006/032085. In one aspect the present invention provides a compound selected from the group consisting of: HO 0 HO 0 HO O F OH OH OH F F F OH OH 1 2 3 HO 0 HO 0 HO 0 OH F OMe OH F F F OH OH OH 4 5 6 HO 0 F OH OH 7 13 HO 0 HO 0 HO 0 OH OH F OH CI HO F OMe F OH 8 9 10 HO 0 HO 0 HO 0 O OH OH MeO F OH OH OH 11 12 13 HO 0 HO 0 F . OH NHEt F F OH OH 14 15 or a pharmaceutically acceptable salt, hydrate, derivative, solvate or prodrug thereof. The compounds of the invention include two chiral centres. The present invention includes all 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 persons skilled in the art. In some embodiments compounds of the invention are racemic mixtures. In other embodiments compounds of the invention are present in optically pure form. It will also be recognised by those skilled in the art that in the compounds of the invention the phenyl substituents attached to the heterocyclic ring can be either cis or trans relative to each other. Preferably, in the compounds of the invention these 14 substituents will be cis relative to each other. For example, in some embodiments compounds 1 to 15 have the following structures: HO 0 HO 0 HO O F OH OH OH F F F OH OH 1a 2a 3a HO 0 HO 0 HO 0 OH F OMe OH F F F OH OH OH 4a 5a 6a HO 0 F OH OH 7a 15 HO 0 HO 0 HO 0 OH OH F OH CI HO F OMe F OH 8a 9a 10a HO 0 HO 0 HO 0 0 OH OH MeO F OH OH OH 11a 12a 13a HO O HO 0 F 0H NHEt F F OH OH 14a 15a 16 HO HO HO F q OH F' OH F OH F OH OH lb 2b 3b HO 0H HO HO 0 1 l'/-,-OH -l Ko"- ,, OMe H F F F OH OH OH OH 4b 5b 6b HO 0 OH 7b 17 HO O HO O HO 0 z z F OH OH OH Cl HO >F OMe F OH 8b 9b 10b HO O HO O HO 0 0 -OH OH MeOP F OH OH OH 11b 12b 13b HO O HO >0 - 3 ,,, F I'a N F- OH NHEt F> F OH OH 14b 15b In some embodiments the compound may be compound 2 or one of its enantiomers 2a or 2b. Compounds of the invention are also taken to include hydrates and solvates. Solvates are complexes formed by association of molecules of a solvent with a compound of the invention. In the case of compounds of the invention that are solids, it will be understood by those skilled in the art that such compounds may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention. The compounds of the invention may be in the form of pharmaceutically acceptable salts. Such salts are well known to those skilled in the art. S. M. Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66:1 19. Pharmaceutically acceptable salts can be prepared in situ during the final isolation 18 and purification of compounds of the invention, or separately by reacting the free base compound with a suitable organic acid. Suitable pharmaceutically acceptable acid addition salts of the compounds of the present invention may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucoronic, fumaric, maleic, pyruvic, alkyl sulfonic, arylsulfonic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, ambonic, pamoic, pantothenic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, p-hydroxybutyric, galactaric, and galacturonic acids. Suitable pharmaceutically acceptable base addition salts of the compounds of the present invention include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine. Alternatively, organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N methylglucamine), procaine, ammonium salts, quaternary salts such as tetramethylammonium salt, amino acid addition salts such as salts with glycine and arginine. The compounds of the invention also extend to include all derivatives with physiologically cleavable leaving groups that can be cleaved in vivo to provide the compounds of the invention. Suitable leaving groups 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 are readily cleavable to the corresponding hydroxyl-substituted compounds. Compounds of the invention may be synthesised as described below. In the first step of the synthesis benzophenone intermediate (ll) is prepared from compound (I) and a suitably functionalized benzoic acid (II) according to Scheme 1. In this scheme, R 1 , R", R 14 and R 15 are selected depending on the compound of the invention that is desired to be prepared. For example, if it is desired to prepare compound 1, then R' would be methyl, R 14 would be 4-F and R' 3 and R" would be H.

19 HO OH RH +HO R 15 R15 R R15 R4 (I) (I1)O (Ill) Scheme 1: Preparation of a benzophenone intermediate Where appropriate or necessary, protecting groups may be employed. Standard protecting groups are known to those skilled in the art and include those described, for example, in 'Protective Groups in Organic Synthesis' by Theodora Greene and Peter Wuts (Third edition, 1999, John Wiley and Sons). Typically, in the reaction depicted in Scheme 1, the phenolic compound (I) and the benzoic acid compound (II) are reacted under acylating conditions. For example, in one method described in the Indian Journal of Chemistry, 1971, 619-62, the phenolic compound (1) and the benzoic acid compound (II) may be combined with phosphorous oxychloride and zinc chloride and the mixture heated for a period of time sufficient for the reaction to proceed substantially to completion. The precise period of time will depend on the scale of the reaction, however those skilled in the art will readily be able to determine suitable time and temperature conditions. In a typical reaction, the reagents are heated at a temperature of about 70 CC for about 1 to 3 hours. When the reaction is judged to be sufficiently complete, the reaction mixture is cooled, for example by pouring onto ice, after which the benzophenone intermediate (111) may be isolated and purified using standard techniques known to those skilled in the art. In an alternative method, the benzoic acid compound (II) may be stirred in refluxing thionyl chloride for about 2 to 6 hours, followed by addition of catalytic NN dimethylformamide in a suitable organic solvent (for example dichloromethane), for about 20 mins to 1 hour. After removing residual thionyl chloride the mixture is typically cooled (for example in an ice bath) then aluminium chloride and the phenolic compound (I) are added and the mixture stirred for a suitable period of time, typically about 18 to 36 hours, while slowly warming to room temperature, then heated at reflux for about 2 to 8 hours. The reaction may be conducted under an inert atmosphere. The benzophenone intermediate (ll) may be purified using standard techniques known to those skilled in the art. For example, the benzophenone intermediate (111) may be collected by filtration, washed (for example with water), then recrystallised from a suitable solvent system. Examples of recrystallisation solvents include methanol, 20 ethanol, water and mixtures thereof. Alternatively, the benzophenone intermediate (Ill) may be purified by column chromatography. The next step of the synthesis involves reaction of the benzophenone intermediate (111) with a suitably functionalized phenylcarboxylic acid (IV) to provide functionalized benzopyranone (V) (see Scheme 2). In this scheme, Rio and R" are selected depending on the compound of the invention that is desired to be prepared. For example, if it is desired to prepare compound 1, then Rio would be 4-OH and R" would be H. Where appropriate or necessary, protecting groups may be employed. R1 R1 HO O O HO OH R 13 HO 0 R 15 R 1 4 O R R 1 -R 13 R11 R K (Ill)(IV) (V Scheme 2: Preparation of a functionalized benzopyranone Typically, in the condensation reaction depicted in Scheme 2, the benzophenone intermediate (Ill) is reacted with the suitably functionalized phenylacetic acid (IV) in the presence of a base and acetic anhydride. Typically, the base is a non-nucleophilic base, such as N,N-diisopropylethyl amine (DIEA), N-methylmorpholine or triethylamine. During this reaction any hydroxy substituents present on the phenyl rings may be converted to the corresponding acetate. The reaction is typically carried out with heating at a temperature and for a period of time until the reaction is judged to be substantially complete (for example by TLC or GC analysis). Those skilled in the art will know that suitable time periods will depend on the scale of the reaction and the particular reagents employed. Typically, the reagents may be warmed at a temperature of about 40-600C (for example about 500C) for about 20 to 30 minutes to ensure that all of the reagents are in solution, then heated at a higher temperature, such as about 130 150'C (for example about 135"C), for about 6 to 48 hours (for example about 18 hours). The functionlized benzopyranone (V) may be isolated by conventional means, such as solvent extraction (for example, using an organic solvent such as ethyl acetate, chloroform, or the like), and washing with aqueous alkaline solution (for example sodium carbonate, or sodium hydrogen carbonate solution), followed by standard purification using techniques known to those skilled in the art, such as column chromatography, 21 recrystallisation from a suitable solvent (for example ethanol or an ethanol/water mixture), or trituation with a suitable solvent (for example methanol, ethanol or mixtures thereof). The next step of the synthesis involves reduction of the lactone of the functionalized benzopyranone (V) to provide functionalized chromene compound (VI) (see Scheme 3). R1 R1 HO O O HO O R10 10 R R 13

R

11 15 R 13

R

11 R 14 (V) (VI) Scheme 3. Preparation of a functionalized chromene Typically, the reduction reaction is carried out by treating the benzopyranone (V) with a suitable reducing agent capable of reducing the ketone moiety of the pyranone ring. Preferably, the reducing reagent selectively reduces the ketone moiety of the pyranone ring but does not reduce the 3,4 double bond. The reduction may also deprotect any acylated hydroxy groups present on the phenyl rings. Suitable reducing agents will be known to those skilled in the art and include borane reagents, such as, for example borane dimethylsulfide complex, decborane, 9-BBN and borane tetrahydrofuran complex. In some embodiments the reducing agent is borane dimethylsulfide. The reduction may be facilitated by the use of a chiral auxiliary. For example, borane dimethylsulfide is amenable to asymmetric ketone reduction using a chiral oxazaborolidine catalyst (Corey, E.J.; Helal, C. J. Angew.Chem. Int. Ed. 1998, 1986). The reaction may be carried out in an organic solvent, such as tetrahydrofuran, toluene or chloroform. The reaction may be performed under an inert atmosphere at a temperature below room temperature, typically at a temperature from about -10'C to about 10 C, or at about -50C to about 00C, or at about 00C, for about 15 minutes to about 4 hours, typically for about 30 minutes to about 2 hours. When the reduction reaction is judged to be complete (or substantially complete) the product may be isolated by acidic work up using standard methods known to the skilled person, then purified using conventional techniques such as column chromatography. With globally deprotected chromene compound (VI) in hand, the final step of the 22 synthesis involves catalytic cisoid reduction of the olefin of chromene compound (VI) to give compounds of the invention (see Scheme 4). R1 R1 HO O HO O K K' R5 S -- R13 R11 R - j R13 R11 R R4 (VI) compounds of the invention Scheme 4. Catalytic reduction to provide compounds of the invention The reduction of the double bond may be performed by hydrogenation using reagents and conditions that are well known to those skilled in the art. Suitable reagents include heterogenous metal catalysts, such as palladium and platinum catalysts in the presence of a hydrogen atmosphere. Presently preferred catalysts include, but are not limited to, Pd/C, Pd(OH) 2 /C, Pt/C, Raney Nickel, Rh catalysts, including chiral Rh catalysts, such as Rh DIPAMP and Wilkinsons catalysts. Examples of suitable solvents include methanol and ethanol. The reaction may be performed at room temperature or the reaction mixture may be heated (for example to about 50 to 60 0 C). Alternatively, the hydrogenation reaction may be performed under pressure. Those skilled in the art will readily be able to determine when the reaction is complete (or substantially complete) using standard techniques (for example TLC, GC-MS). The product may bc purified using standard techniques (for example chromatography). After purification, compounds of the invention may be substantially pure. For example, the compounds of the invention may be isolated in a form which is at least about 80%, 85%, 90%, 95%, 98%, 99%, 99.5% or 99.9% pure. Compounds of the invention may be obtained as racemic mixtures. Enantiomers may be isolated using techniques known to those skilled in the art, including chiral resolution, supercritical fluid chromatography and enantioselective syntheses. Individual enantiomers may be isolated in a substantially pure form or in an enantiomeric excess (ee). For example, in preferred embodiments an enantiomer may be isolated in an enantiomeric excess of about 70%, 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or greater than 99%.

23 The present inventors have discovered that compounds of the invention are able to exert surprisingly powerful biological effects on differentiated cancer cells as well as on undifferentiated cancer cells, which are variously referred to as "cancer stem cells" or "cancer progenitor cells". The biological effects may include inhibition of cell proliferation, induction of cell death, induction of cellular differentiation and reversal of aberrant behavior. The compounds of the invention may therefore find use in the treatment of cancer. In particular, the compounds of formula (I) may be used in the treatment of cancer where it is desirable to target both undifferentiated and differentiated cancer cells, and where the effect on both cancer cell types might be different or even opposite. For example, compounds of the invention may induce cell death in differentiated cancer cells and induce cellular differentiation in undifferentiated cancer cells. In other embodiments compounds of the invention may inhibit the proliferation of cancer stem cells and differentiated cancer stem cells, such as somatic cancer cells. The compounds of the invention may be used in conjunction with, or alternatively in the absence of, other chemotherapeutic agents. The compounds of the invention may be used in the treatment of cancer that is resistant to one or more chemotherapeutic agents. In one embodiment the compounds of the invention may be used in the treatment of temozolomide-resistant glioblastoma. In another embodiment the compounds of the invention may be used in the treatment of carboplatin resistant ovarian cancer and/or carboplatin refractory ovarian cancer. By virtue of their biological effects on undifferentiated cancer cells the compounds of the invention find particular use in treating cancer that has recurred in a subject and in reducing the incidence of, or the risk of, reccurence of cancer in a subject deemed to be at risk of cancer recurrence, for example a subject who is in cancer remission. The subject may be in remission from a solid tumour as defined herein. The compounds of the invention may also find use in inducing apoptosis in, or inhibiting the proliferation of, cancer stem cells. The compounds of the invention may also find use in treating diseases caused by cancer stem cells, such as cancer. The cancer may be a metastatic cancer. Furthermore, compounds of the invention may possess superior pharmaceutical properties, such as improved resistance to conjugation via glucuronyl transferases and other water-solubilising transferases such as sulfases, which may be over-expressed on proliferative cells, such as cancer cells. This may advantageously confer superior pharmaceutical properties, such as an enhanced pharmacokinetic profile through 24 reduced conjugation and elimination. In all aspects of the invention the cancer may be a solid tumour, such as for example, breast cancer, lung cancer (NSCLC and SCLC), prostate cancer, ovarian cancer, uterine cancer, brain cancer (including for example glioma, medulloblastoma and glioblastoma), skin cancer, colon cancer, bladder cancer, colorectal cancer, gastric cancer, liver cancer, pancreatic cancer, head and neck cancer, melanoma or mesothelioma. In particular embodiments the cancer is ovarian cancer. In other embodiments the cancer is glioma, colorectal, melanoma, pancreatic, prostate, breast, lung or liver cancer. In one embodiment the invention provides a method for inhibiting the proliferation of ovarian cancer stem cells, the method comprising contacting the ovarian cancer stem cells with an effective amount of compound 2 or its enantiomers 2a or 2b. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). In another embodiment the invention provides a method for the treatment of ovarian cancer in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of compound 2 or its enantiomers 2a or 2b. The cancer may be a cancer that has recurred. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). In another embodiment the invention provides a method for the treatment of carboplatin resistant ovarian cancer and/or carboplatin refractory ovarian cancer in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of compound 2 or its enantiomers 2a or 2b. The cancer may be a cancer that has recurred. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). In another embodiment the invention provides a method for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence, the method comprising administration to the subject of an effective amount of compound 2 or its enantiomers 2a or 2b. The subject deemed to be at risk of cancer recurrence may be a subject in remission from ovarian cancer. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). In a further embodiment the invention provides a method for treating a disease in a subject caused by ovarian cancer stem cells, the method comprising administration to the subject of a therapeutically effective amount of compound 2 or its enantiomers 2a or 2b. The disease may be cancer. The cancer may be ovarian cancer or some other 25 cancer, for example a metastatic cancer. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). In a further embodiment the invention provides a method for the treatment of glioblastoma in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of compound 2 or its enantiomers 2a or 2b. The glioblastoma may be temozolomide-resistant glioblastoma. The compounds may be used in the absence of other chemotherapeutic agents (i.e. as a monotherapy). Those skilled in the art will recognise that compounds and pharmaceutical compositions of the present invention may be administered via any route which delivers an effective amount of the compounds to the tissue or site to be treated. In general, the compounds and compositions may be administered by the parenteral (for example intravenous, intraspinal, subcutaneous or intramuscular), oral or topical route. Administration may be systemic, regional or local. The particular route of administration to be used in any given circumstance will depend on a number of factors, including the nature of the cancer to be treated, the severity and extent of the cancer, the required dosage of the particular compound to be delivered and the potential side-effects of the compound. In general, suitable compositions may be prepared according to methods which are known to those of ordinary skill in the art and may include pharmaceutically acceptable carriers, diluents and/or excipients. The carriers, diluents and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralised or distilled water; saline solution; vegetable based oils such as peanut oil, safflower oil, olive oil, cottonseed oil, maize oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethylcellulose, sodium carboxymethylcellulose or hydroxypropylmethylcellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1,3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinylpyrridone; agar; carrageenan; gum tragacanth or gum acacia and petroleum jelly. Typically, the carrier or carriers will form from 10% to 99.9% by weight of the compositions.

26 Pharmaceutical compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, caplets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an eye drop, in an aerosol form suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in a form suitable for parenteral administration, that is, subcutaneous, intramuscular or intravenous injection. For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or carriers can include cyclodextrins (for example Captisol@) Cremaphor, Ringer's solution, isotonic saline, phosphate buffered saline, ethanol and 1,2 propylene glycol. Some examples of suitable carriers, diluents, excipients and adjuvants for oral use include cyclodextrins, Captisol, Cremaphor, peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, gum acacia, gum tragacanth, dextrose, sucrose, sorbitol, mannitol, gelatine and lecithin. In addition these oral formulations may contain suitable flavouring and colourings agents. When used in capsule form the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which delay disintegration. Adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and buffering agents. Solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavourings, coating agents, preservatives, lubricants and/or time delay agents. Suitable binders include gum acacia, gelatine, corn starch, gum tragacanth, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharine. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavouring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavouring. Suitable coating agents include polymers or copolymers of acrylic acid and/or methacrylic acid and/or their esters, waxes, fatty alcohols, zein, shellac or gluten. Suitable preservatives include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl paraben, propyl paraben or sodium bisulphite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate.

27 Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol, isopropanol, glycerol, fatty alcohols, triglycerides or mixtures thereof. Suspensions for oral administration may further comprise dispersing agents and/or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, polyoxyethylene sorbitol mono- or di-oleate, -stearate or -laurate, polyoxyethylene sorbitan mono- or di-oleate, -stearate or -laurate and the like. Emulsions for oral administration may further comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, gum acacia or gum tragacanth. Methods for preparing parenterally administrable compositions are apparent to those skilled in the art, and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pa., hereby incorporated by reference herein. Topical formulations may comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where treatment is required, such as liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. Drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These may be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and/or fungicidal agent and/or any other suitable preservative, and optionally including a surface active agent. The resulting solution may then be clarified by filtration, transferred to a suitable container and sterilised. Sterilisation may be achieved by autoclaving or maintaining at 90 0 C to 100 0C for half an hour, or by filtration, followed by transfer to a container by an aseptic technique. Examples of bactericidal and fungicidal agents suitable for inclusion in the drops are phenylmercuric nitrate or acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate (0.01%). Suitable solvents for the preparation of an oily solution 28 include glycerol, diluted alcohol and propylene glycol. Lotions according to the present invention include those suitable for application to the skin or eye. An eye lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared by methods similar to those described above in relation to the preparation of drops. Lotions or liniments for application to the skin may also include an agent to hasten drying and to cool the skin, such as an alcohol or acetone, and/or a moisiteriser such as glycerol, or oil such as olive oil. Creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. They may be made by mixing the active ingredient in finely divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy basis. The basis may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metallic soap; a mucilage; an oil of natural origin such as almond, corn, arachis, castor or olive oil; wool fat or its derivatives, or a fatty acid such as stearic or oleic acid together with an alcohol, such as propylene glycol or macrogols. The composition may incorporate any suitable surfactant such as an anionic, cationic or non-ionic surfactant, such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inoraganic materials such as silicaceous silicas, and other ingredients such a lanolin, may also be included. The compositions may also be administered or delivered to target cells in the form of liposomes. Liposomes are generally derived from phospholipids or other lipid substances and are formed by mono- or multi-lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Specific examples of liposomes used in administering or delivering a composition to target cells are synthetic cholesterol (Sigma), the phospholipid 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); Avanti Polar Lipids), the PEG lipid 3-N-[(-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2 dimyrestyloxy-propylamine (PEG-cDMA), and the cationic lipid 1,2-di-o-octadecenyl-3 (N,N-dimethyl)aminopropane (DODMA) or 1,2-dilinoleyloxy-3-(N,N dimethyl)aminopropane (DLinDMA) in the molar ratios 55:20:10:15 or 48:20:2:30, respectively, PEG-cDMA, DODMA and DLinDMA. Any non-toxic, physiologically acceptable and metabolisable lipid capable of forming liposomes can be used. The compositions in liposome form may contain stablisers, preservatives, excipients and the like. The preferred lipids are the phospholipids and the phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art, and in relation to this, specific reference is made to: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq., the contents of 29 which is incorporated herein by reference. The compositions may also be administered in the form of microparticles or nanoparticles. Biodegradable microparticles formed from polyactide (PLA), polylactide co-glycolide (PLGA), and epsilon-caprolactone (t-caprlactone) have been extensively used as drug carriers to increase plasma half life and thereby prolong efficacy (R. Kumar, M., 2000, J. Pharm. Pharmaceut. Sci. 3(2) 234-258). Microparticles have been formulated for the delivery of a range of drug candidates including vaccines, antibiotics, and DNA. Moreover, these formulations have been developed for various delivery routes including parenteral subcutaneous injection, intravenous injection and inhalation. The compositions may incorporate a controlled release matrix that is composed of sucrose acetate isobutyrate (SAIB) and an organic solvent or organic solvents mixture. Polymer additives may be added to the vehicle as a release modifier to further increase the viscosity and slow down the release rate. SAIB is a well known food additive. It is a very hydrophobic, fully esterified sucrose derivative, at a nominal ratio of six isobutyrate to two acetate groups. As a mixed ester, SAIB does not crystallise but exists as a clear viscous liquid. Mixing SAIB with a pharmaceutically acceptable organic solvent, such as ethanol or benzyl alcohol decreases the viscosity of the mixture sufficiently to allow for injection. An active pharmaceutical ingredient may be added to the SAIB delivery vehicle to form SAIB solution or suspension formulations. When the formulation is injected subcutaneously, the solvent differs from the matrix allowing the SAIB-drug or SAIB-drug-polymer mixtures to set up as an in situ forming depot. For the purposes of the present invention compounds and compositions may be administered to subjects either therapeutically or preventively. In a therapeutic application compositions are administered to a patient already suffering from cancer in an amount sufficient to cure or at least partially arrest thecancer and its complications. The composition should provide a quantity of the compound or agent sufficient to effectively treat the subject. The therapeutically effective amount for any particular subject will depend upon a variety of factors including: the cancer being treated and the severity thereof; the activity of the compound administered; the composition in which the compound is present; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of sequestration of the compound; the duration of the treatment; drugs used in combination or coincidental with the compound, together with other related factors well known in medicine. One skilled in the art would be able, by routine experimentation, to determine an 30 effective, non-toxic amount of a compound which would be required to treat or prevent a particular cancer. Generally, an effective dosage is expected to be in the range of about 0.0001 mg to about 1000 mg per kg body weight per 24 hours; typically, about 0.001 mg to about 750 mg per kg body weight per 24 hours; about 0.01 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 500 mg per kg body weight per 24 hours; about 0.1 mg to about 250 mg per kg body weight per 24 hours; about 1.0 mg to about 250 mg per kg body weight per 24 hours. More typically, an effective dose range is expected to be in the range about 1.0 mg to about 200 mg per kg body weight per 24 hours; about 1.0 mg to about 100 mg per kg body weight per 24 hours; about 1.0 mg to about 50 mg per kg body weight per 24 hours; about 1.0 mg to about 25 mg per kg body weight per 24 hours; about 5.0 mg to about 50 mg per kg body weight per 24 hours; about 5.0 mg to about 20 mg per kg body weight per 24 hours; about 5.0 mg to about 15 mg per kg body weight per 24 hours. Alternatively, an effective dosage may be up to about 500 mg/m 2 . Generally, an effective dosage is expected to be in the range of about 25 to about 500 mg/m 2 , preferably about 25 to about 350 mg/m 2 , more preferably about 25 to about 300 mg/m 2 , still more preferably about 25 to about 250 mg/m 2 , even more preferably about 50 to about 250 mg/m 2 , and still even more preferably about 75 to about 150 mg/m 2 Typically, in therapeutic applications, the treatment would be for the duration of the disease state. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the cancer being treated, the form, route and site of administration, and the nature of the particular individual being treated. Also, such optimum conditions can be determined by conventional techniques. The compounds of the invention may be used alone in the treatment of cancer, or alternatively in combination with radiotherapy and/or surgery and/or other therapeutic agents, for example chemotherapeutic agents and immunostimulatory agents, as part of a combination therapy. The compounds of the invention may sensitise undifferentiated cancer cells to other chemotherapeutic agents and/or radiotherapy. The terms "combination therapy" and "adjunct therapy" are intended to embrace administration of multiple therapeutic agents in a sequential manner in a regimen that will provide beneficial effects and is intended to embrace administration of these agents in either a single formulation or in separate formulations.

31 Combination therapy may involve the active agents being administered together, sequentially, or spaced apart as appropriate in each case. Combinations of active agents including compounds of the invention may be synergistic. The co-administration of compounds of the invention with other therapeutic agent(s) may be effected by a compound of the invention being in the same unit dose form as the other therapeutic agent(s), or the compound of the invention and the other therapeutic agent(s) may be present in individual and discrete unit dosage forms that are administered sequentially, at the same, or at a similar time. Sequential administration may be in any order as required, and may require an ongoing physiological effect of the first or initial agent to be current when the second or later agent is administered, especially where a cumulative or synergistic effect is desired. When administered separately, it may be preferred for the compound of the invention and the other agent to be administered by the same route of administration, although it is not necessary for this to be so. In accordance with various embodiments of the present invention one or more compounds of the invention may be included in combination therapy with surgery and/or radiotherapy and/or one or more chemotherapeutic agents. There are large numbers of chemotherapeutic agents that are currently in use, in clinical evaluation and in pre-clinical development, which could be selected for treatment of cancers in combination with compounds of the invention. Such agents fall into several major categories, namely, antibiotic-type agents, alkylating agents, anti-metabolite agents, hormonal agents, immunological agents, interferon-type agents and a category of miscellaneous agents. Alternatively, other chemotherapeutic agents, such as metallomatrix proteases (MMP) inhibitors may be used. Suitable agents which may be used in combination therapies include those listed, for example, in the Merck Index, An Encyclopaedia of Chemicals, Drugs and Biologicals, 12th Ed., 1996, the entire contents of which are incorporated herein by reference. When used in the treatment of solid tumours compounds of the invention may be administered with one or more of the following chemotherapeutic agents: adriamycin, taxol, docetaxel, fluorouracil, melphalan, cisplatin, alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD (methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP (prednisone, methotrexate (w/leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine, vincristine, prednisone and procarbazine), vincristine, vinblastine, angioinhibins, TNP 470, pentosan polysulfate, platelet factor 4, angiostatin, LM 609, SU 101, CM 101, Techgalan, 32 thalidomide, SP-PG and the like. Those skilled in the art will appreciate that the aspects and embodiments described herein are susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The citation of any reference herein should not be construed as an admission that such reference is available as prior art to the present application. Further, the reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgement or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endevour to which this specification relates. The present invention is further described below by reference to the following non limiting examples. Examples Example 1 - Synthesis of 4-(4-fluorophenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-o (Compound 1) Step 1: HO OH H POC1s ZnC2 HO OH F HO POI 3 ZCI I F 0 (2,4-Dihydroxy-3-methylphenyl)(4-fluorophenyl)methanone A 250 mL round bottom flask fitted with a septum and outlet needle was charged with 4-fluorobenzoic acid (5.15 g, 0.03678 mol), 2-methylresorcinol (4.1g, 0.0330 mol), zinc chloride (9.89 g, 0.0725 mol) and phosphorus oxychloride (45 mL). The white suspension was heated at 70 0C for 2 h and turned dark brown in colour. The mixture was cooled to room temperature and then poured slowly onto crushed ice. A red suspension resulted which was kept in the ice bath for 1.5 h and then collected under vacuum filtration. The filter cake was twice re-suspended in water (50 mL) and filtered, then the residue was re-crystallised from 1:1 ethanol:water (30 mL). The collected solid was washed (5 mL, 1:1 ethanol:water) and dried to give an orange powder (3.66 g, 44%). 1 H NMR (400 MHz, DMSO-d 6 ) & 12.05 (s, 1H), 9.76 (s, 1H), 7.78 (d, J = 9.04Hz, 33 2H), 7.34 (d, J = 9.00 Hz, 2H), 7.25 (d, J = 8.41 Hz, 1 H), 6.95 (d, J = 9.00 Hz, 1 H), 2.16 (s, 3H). Step 2: HO OH AcO 0 0 0 HO Ac 2 O DiPEA OH OAc F F 4-(7-Acetoxy-4-(4-fluorophenyl)-8-methyl-2-oxo-2H-chromen-3-yl)pheny acetate (2,4-Dihydroxy-3-methylphenyl)(4-fluorophenyl)methanone from Step 1 (0. 95g, 3.86 mmol), 4-hydroxyphenylacetic acid (0.587 g, 3.86 mmol), N,N-diisopropylethylamine (DiPEA) (2.2 mL, 12.6 mmol) and acetic anhydride (2 mL) were combined and heated at 50 0C for 20 mins giving an orange solution which was subsequently heated at 135 0C for 18 h. On cooling to room temperature, water (25 mL) was added and the contents stood overnight resulting in the precipitation of a gummy yellow solid. The mixture was partitioned between ethyl acetate (100 mL) and a saturated solution of sodium hydrogen carbonate (100 mL). The aqueous layer (-pH 8) was separated and further extracted with ethyl acetate (50 mL). The organic phases were combined, washed with brine (50 mL), dried (magnesium sulfate), filtered and concentrated under reduced pressure. Purification by column chromatography (eluent 1:1 ethyl acetate:hexanes) gave (1.3 g, 77%). 1 H NMR (400 MHz, Chloroform-d) 6 7.72 (d, J = 8.3 Hz, 2H), 7.62 (d, J = 9.01 Hz, 1H), 7.48 (d, J = 8.5 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24 (d, J = 8.5 Hz, 2H), 7.04 (d, J = 9 Hz, 1H), 2.36 (s, 3H), 2.35 (s, 3H), 2.18 (s, 3H). Step 3: AcO O 0 HO 0

BH

3 .SMe 2 , THF OAc OH F F 34 4-(4-Fluorophenyl)-3-(4-hydroxyphenyl)-8-methyl-2H-chromen-7-oI Under an atmosphere of nitrogen, a reaction vessel was charged with 7-acetyloxy-3-(4 acetyloxyphenyl)-4-(4-fluorophenyl)-8-methyl-2H-chromen-2-one from Step 2 (100 mg, 0.2 mmol) and anhydrous tetrahydrofuran (10 mL) and the resulting solution stirred in an ice bath for 15-30 mins. Borane dimethylsulfide complex (2 M solution in THF, 2.5 mL, 5 mmol) was added and the mixture stirred for 2 h in the ice bath, allowed to come to room temperature then heated at 35 0C overnight. On cooling to room temperature, the reaction vessel was placed in an ice bath and 2 M HCI was added dropwise with vigorous gas evolution. Addition was continued until no more gas evolution was observed then an extra 5 mL of 2 M HCI was added. The mixture was allowed to warm to room temperature, transferred to a separating funnel containing water (50 mL) and extracted with ethyl acetate (75 mL). The organics were washed with brine (50 mL), dried (magnesium sulfate), filtered and concentrated. Purification by column chromatography (eluent 1:1 ethyl acetate:hexanes) gave product (33 mg, 37%). 'H NMR (400 MHz, Acetone-d 6 ) 6 7.48 (d, J = 9.00 Hz, 2H), 7.33 (d, J = 9.06 Hz, 2H), 7.24 (d, J = 9.12 Hz, 2H), 7.01 (d, J = 9.6 Hz, 1H), 6.82 (d, J = 9.7 Hz, 1H), 6.66 (d, J = 8.6 Hz, 2H) 4.66 (s, 2H), 2.16 (s, 3H). Step 4: HO 0 HO 0

H

2 Pd/C OH OH F F 4-(4-Fluorophenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-of (Compound 1) A 50 mL round bottom flask was charged with 4-(4-fluorophenyl)-3-(4-hydroxyphenyl)-8 methyl-2H-chromen-7-ol from Step 3 (25 mg, 0.069 mmol) and absolute ethanol (10 mL). Palladium hydroxide on carbon (10 mg, 20% by wt) was added and a rubber septum was fitted. Using a balloon, H 2 gas was flushed through the reaction flask, then a fresh balloon of H 2 gas was attached and the reaction mixture stirred at 60 OC for 72 hours. The mixture was cooled to room temperature, filtered through celite, the residue washed with ethanol (2 x 10 mL) and the filtrates combined and concentrated in vacuo to give product, a yellow solid (15 mg, 60%). 1 H NMR (400 MHz, Acetone-d 6 ) 6 8.38 (br s, 1H), 8.11 (br s, 1H), 8.05 (br s, 1H), 7.33 (d, J = 9.00 Hz, 2H), 7.27 (d, J = 35 8.68 Hz, 2H), 7.19 (d, J = 8.65 Hz, 2H), 6.82 (d, J = 8.6 Hz, 1H), 6.67 (d, J = 8.1 Hz, 2H), 6.34 (d, J = 8.6Hz, 1H) 4.30 - 4.41 (m, 1H), 4.21 - 4.22 (m, 1H), 4.10 (d, J = 5.48 Hz, 1H), 3.91 (m, 1H), 2.14 (s, 3H). Example 2 - Synthesis of additional compounds of the invention 0T~ R13 0 R 14

R

13 HOO STE I HO HO O HO OH HO R12 STP OR12 H i HO( 'H14- OH 0 R, R1 4-la RI = Me, R12= F, R13 = OH, R14 = H 4-1d R1 - Me, R12= CI, R13 =OMe, R14 H 4-1bR1=Me,R12=Me, R13=OH,R14=H 4-le RI=Me, R12=OH,R13=F, R14=H 4-1cR=Me, R12=H,R13=OH,R14=H 4-IfRI=Pr,R12=F, R13 ,R14=H 4-Ig RI = Me, R12 = OH, R13 = F, R14 = F 4-lh RI - Me, R12 = OH R13 = OMe R/R R12 R 12 R3R 13 I!x R 0

R

1 STEP 2 1R4 STEP 3 R 14 STEP 4 1 R

R

11 RI -Rii AcO 0 0 HO 0 HO 0 R1R R, 4-2 4-3 4-4 a R1 = Me, RI0 = OH, R11 = H, R12 F, R13 = OH, R14 = H g R1 = Me, RIO = OH, R11 = F, R12= H, R13 =OH, R14 H bR1=Me,R1O=OH,R1I -F,R12=F,R13=OHR14=H hRI=Me,R1O=OH,R11=H,R12=CI,R13=OMe,R14=H c R1 - Me, RIO = H, RI = OH, R12 F, R13 OH, R14 = H i RI = Me, RIO = OH, RIl = H, R12 = OH, R13 = F, R14 = H d RI - Me, RI0 = OMe, R1I = Me. R12 = F, R13 = OH, R14 - H j R1 = Pr, R1O OH, R11 H, R12 F,R13 = OH, R14 = H e RI = Me, RIO = OMe, R11= Me, R12= H, R13 = OH, R14 = H k RI Pr, R1O OH, R11 F, R12 F,R13 = OH, R14 = H fR1 = Me, RIO = OH, RI = F, R12= F, R13 = OH, R14 = H I R1 Pr, R1O - NHEt, R11 = H R12 =F, R13 - OH, R14 = H m R1= Me, R10 = OH, R1I =H, R12 = Oh, R13 = F, R14 = F n R1= Me, R10 = OH, R11 = H, R12 = OH, R13 - OMe, R14 = H Step 1. ZnCl 2 , POC 3 , 70 C, 2 h; Step 2. DiPEA, Ac 2 0, 135 *C, 18 h. Step 3. THF,

BH

3 .Me 2 S in THF, 35 0C, 18 h; Step 4. H 2 , Pd/C, EtOH, 39 psi, RT, 18 h Step 1. (2,4-Dihydroxy-3-methylphenyl)(3-fluoro-4-hydroxyphenyl)methanone (4-1a) Procedure A 1 : A 250 mL round bottom flask fitted with a septum and outlet needle was charged with 3-fluoro-4-hydroxybenzoic acid (5.05 g, 0.0324 mol), 2-methylresorcinol (4.02 g, 0.0324 mol), zinc chloride (9.71 g, 0.0712 mol) and phosphorus oxychloride (45 mL). The white suspension was heated at 70 0C for 2 h and turned dark brown in colour. The mixture was cooled to room temperature and then poured slowly onto crushed ice. A red suspension resulted which was kept in the ice bath for 1.5 h and then collected under vacuum filtration. The filter cake was twice re-suspended in water (50 mL) and filtered, then the residue was re-crystallised from 1:1 ethanol:water (33 mL). The collected solid was washed (5 mL, 1:1 ethanol:water) and dried to give (4-1a) as an orange powder (3.52 g, 41%). 'H NMR (400 MHz, DMSO-d 6 ). 5 12.72 (s, 1H), 10.76 (s, 36 1 H), 10.65 (s, 1 H), 7.43 (dd, J = 1.96, 11.74 Hz, 1 H), 7.33 (d, J = 9.00 Hz, 2H), 7.06 (t, J = 8.41 Hz, 1 H), 6.45 (d, J = 9.00 Hz, 1 H), 1.99 (s, 3H). (1 Indian Journal of Chemistry, 1971, 619-62) Other analogues prepared via this method: (2,4-Dihydroxy-3-methylphenyl)(4-hydroxy-3-methylphenyl)methanone (4-1 b) (71 %) (2,4-Dihydroxy-3-methylphenyl)(4-hydroxyphenyl)methanone (4-1c) (77%) (3-Chloro-4-methoxyphenyl)(2,4-dihydroxy-3-methylphenyl)methanone (4-1d) (19%) (chromatography) (2,4-Dihydroxy-3-propylphenyl)(3-fluoro-4-hydroxyphenyl)methanone (4-If) (80%) (2,4-Dihydroxy-3-methylphenyl)(2,3-difluoro-4-hydroxyphenyl)methanone (4-1g) 50% (2,4-Dihydroxy-3methylphenyl)(3-methoxy-4-hydroxyphenyl)methanone (4-1 h) 70% Procedure B: A 250 mL round bottom flask fitted with a septum and outlet needle was charged with 3-fluoro-4-hydroxybenzoic acid (10.0 g, 0.064 mol), 2-methylresorcinol (7.95 g, 0.064 mol), zinc chloride (19.2 g, 0.141 mol) and phosphorus oxychloride (90 mL). The suspension was heated at 70 0C for 2 h and turned dark brown in colour. The mixture was cooled to room temperature and then poured slowly onto crushed ice. A red suspension resulted which was stirred in the ice bath for 5 h then left to sit overnight. The solid was then collected under vacuum filtration. The filter cake was washed with water (200 mL) and air dried. The residue (27.4 g) was dissolved in a mixture of dichloromethane/methanol/triethylamine (92.5/7.5/0.1) and filtered through a plug of silica eluting with the same solvent mixture. After concentration of the solvent, the resulting residue (16.4 g) was dissolved in -0.6 M sodium hydroxide solution (350 mL), stirred for a short time and then acidified to pH<2 with concentrated hydrochloric acid solution. The mixture was stirred for 2 days and the resulting solid was collected by vacuum filtration, washed with water (2 x 50 mL). The wet solid was concentrated from methanol and air dried to give (4-1a) as an orange solid (12.0 g, 72%) which was used without further purification. 1 H NMR (400 MHz, DMSO-d 6 ) 6 12.70 (s, 1H), 10.74 (s, 1H), 10.63 (s, 1H), 7.42 (dd, J = 1.96, 11.74 Hz, 1H), 7.32 (d, J = 9.00 Hz, 2H), 7.07 (t, J = 8.41 Hz, 1 H), 6.45 (d, J = 9.00 Hz, 1 H), 1.99 (s, 3H). Other analogues prepared via this method after plug column only (i.e., without dissolution into base then re-acidification): (2,4-Dihydroxy-3-methylphenyl)(4-fluoro-3-hydroxyphenyl)methanone (4-1 e) (61 %) 37 Step 2. 4-(7-Acetoxy-3-(4-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)-2 fluorophenyl acetate (4-2a) Compound 4-1a (11.8 g, 0.045 mol), 4-hydroxyphenylacetic acid (6.85 g, 0.045 mol), N,N-diisopropylethylamine (25.9 mL, 0.149 mol) and acetic anhydride (23.8 mL) were combined in a 250 mL round bottom flask and stirred at 130-135 0C for 18 h. On cooling to room temperature, the mixture was partitioned between ethyl acetate (250 mL) and a saturated solution of sodium hydrogen carbonate (150 mL). The organic phase was separated and washed with a saturated solution of sodium hydrogen carbonate (100 mL), water (50 mL), brine (50 mL), dried (magnesium sulfate), filtered and concentrated under reduced pressure. The dark gummy residue (22.5 g) was stirred in ethanol (150 mL) for 1-2 h and the resulting powder was collected by vacuum filtration, washed with ethanol (25 mL) and air dried to give an off-white residue (4-2a) (13.6 g). The residue was recrystallised from ethanol to give (4-2a) (9.33 g, 41%). 1 H NMR (400 MHz, Chloroform-d) 5 E7.06 - 7.16 (m, 4H), 6.98 (d, J = 8.61 Hz, 2H), 6.92 - 6.96 (m, 2H), 6.87 (d, J= 8.22 Hz, 1H), 2.38 (s, 3H), 2.36 (s, 3H), 2.33 (s, 3H), 2.25 (s, 3H). Other analogues prepared via this method using the same procedure through workup to concentrated organic extract. (The specific purification method is as indicated): 3-(4-Acetoxy-2-fluorophenyl)-4-(4-acetoxy-3-fluorophenyl)-8-methyl-2-oxo-2H-chromen 7-yl acetate (4-2b) (45%) (ethanol trituration) 4-(7-Acetoxy-3-(3-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)-2-fluoropheny acetate (4-2c) (45%) (ethanol trituration) 4-(7-Acetoxy-3-(4-methoxy-3-methylphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)-2 fluorophenyl acetate (4-2d) (53%) (methanol trituration) 4-(7-Acetoxy-3-(4-methoxy-3-methylphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)phenyl acetate (4-2e) (46%) (methanol trituration) (7-Acetoxy-8-methyl-2-oxo-2H-chromene-3,4-diyl)bis(2-fluoro-4,1-phenylene) diacetate (4-2f) (19%) (chromatography) 4-(7-Acetoxy-3-(4-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)-2-fluoropheny acetate (4-2g) (39%) (chromatography) 4-(7-Acetoxy-4-(3-chloro-4-methoxyphenyl)-8-methyl-2-oxo-2H-chromen-3-yl)pheny acetate (4-2h) (29%) (chromatography) 5-(7-Acetoxy-3-(4-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-4-yl)-2-fluorophenyl acetate (4-2i) (23%) (chromatography) 38 4-(7-Acetoxy-3-(4-acetoxyphenyl)-2-oxo-8-propyl-2H-chromen-4-yl)-2-fluoropheny acetate (4-2j) (33%) (chromatography) (7-Acetoxy-2-oxo-8-propyl-2H-chromene-3,4-diyl)bis(2-fluoro-4, 1 -phenylene) diacetate (4-2k) (50%) (chromatography) 4-(7-Acetoxy-3-(4-(ethylaminophenyl)-2-oxo-8-propyl-2H-chromen-4-yl)-2-fluoropheny acetate (4-21) (50%) (chromatography) 4-(7-Acetoxy-4-(4-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-3-yl)-2,3-diflourophenyl acetate (4-2m) (30%) (chromatography) 4-(7-Acetoxy-4-(4-acetoxyphenyl)-8-methyl-2-oxo-2H-chromen-3-yl)3-methoxyphenyl acetate (4-2n) (50%) (chromatography) Step 3. 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-methyl-2H-chromen-7-ol (4-3a). Under an atmosphere of nitrogen, a 1 L reaction vessel was charged with 4-2a (9.33 g, 0.018 mol) and anhydrous tetrahydrofuran (200 mL) and the resulting solution stirred in an ice bath for 15-20 mins. Borane dimethylsulfide complex (2 M solution in THF, 231 mL, 0.462 mol) was added slowly over 1.5-2 h. The mixture was then heated at 35 OC overnight. On cooling to room temperature, the reaction mixture was slowly poured into 2 L of cold 2 M hydrochloric acid solution (the temperature was maintained below 15 0C). The mixture was allowed to warm to room temperature, transferred to a separating funnel and extracted with ethyl acetate (2 x 250 mL). The organics were combined and washed with water (2 x 250 mL), brine (100 mL), dried (magnesium sulfate), filtered and concentrated. Purification by column chromatography (eluent ethyl acetate:hexanes 2:3 increasing to 1:1) gave 4-3a (3.82 g, 57%). 1 H NMR (400 MHz, Acetone-d 6 ) 18.30 (bs, 2H), 6.90 - 6.95 (m, 3H), 6.81 (dd, J = 2.00, 12.40 Hz, 1H), 6.75 (dd, J = 0.80, 8.00 Hz, 1H), 6.65 (dd, J = 2.00, 8.40 Hz, 2H), 6.48 (d, J = 8.40 Hz,1H), 6.38 (d, J= 8.00 Hz, 1H), 5.01 (s, 2H), 2.10 (s, 3H). Other analogues prepared via this method: 3-(2-Fluoro-4-hydroxyphenyl)-4-(3-fluoro-4-hydroxyphenyl)-8-methy-2H-chromen-7-o (4-3b) (52%) 4-(3-Fluoro-4-hydroxyphenyl)-3-(3-hydroxyphenyl)-8-methyl-2H-chromen-7-o (4-3c) (22%) 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-methoxy-3-methylphenyl)-8-methyl-2H-chromen-7-o (4-3d) (60%) 39 4-(4-Hydroxyphenyl)-3-(4-methoxy-3-methylphenyl)-8-methyl-2H-chromen-7-oI (4-3e) (67%) 3,4-bis(3-Fluoro-4-hydroxyphenyl)-8-methyl-2H-chromen-7-o (4-3f) (65%) 3-(3-Fluoro-4-hydroxyphenyl)-4-(4-hydroxyphenyl)-8-methyl-2H-chromen-7-o (4-3g) (24%) 4-(3-Chloro-4-methoxyphenyl)-3-(4-hydroxyphenyl)-8-methyl-2H-chromen-7-o (4-3h) (66%) 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-propyl-2H-chromen-7-o (4-3j) (50%) 4,4'-(7-Hydroxy-8-propyl-2H-chromene-3,4-diyl)bis(2-fluorophenol) (4-3k) (50%) 3-(4-(Ethylamino)phenyl)-4-(3-fluoro-4-hydroxyphenyl)-8-propyl-2H-chromen-7-o (4-31) (50%) 4-(2,3-Difluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-propyl-2H-chromen-7-o (4-3m) (40%) 4-(3-methoxy-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-propyl-2H-chromen-7-o (4-3n) (50%) Step 4. 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-oI (Compound 2) (4-4a) Procedure A: A 50 mL round bottom flask was charged with 4-3a (25 mg, 0.069 mmol) and abs. ethanol (10 mL). Palladium hydroxide on carbon (10 mg, 20% by wt) was added and a rubber septum was fitted. Using a balloon, H 2 gas was flushed through the reaction flask, then a fresh balloon of H 2 gas was attached and the reaction mixture stirred at 60 'C for 72 hours. The mixture was cooled to room temperature, filtered through celite, the residue washed with ethanol (2 x 10 mL) and the filtrates combined and concentrated. Purification by Flashmaster 11 chromatography (eluent 1:1 ethyl acetate:hexanes) gave 4-4a as an amber gel (11 mg, 44%). 'H NMR (400 MHz, acetone-d6) 5 ppm 2.14 (s,3H), 3.40-3.50 (m, 1H),4.19 (d, J = 5.48 Hz, 1H), 4.22 4.29 (m, 1 H), 4.33 - 4.42 (m, 1 H),6.25 - 6.34 (m, 2 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.57 (d, J = 8.22 Hz, 1 H) 6.61 - 6.73 (m, 5 H), 8.05 (br. s., 1H), 8.17 (br. s., 1H), 8.30 (br. s., 1 H). Procedure B: A 500 mL Parr hydrogenation bottle was charged with palladium hydroxide (20% by weight on carbon, 562 mg). A solution of 4-3a (1.40 g, 3.84 mmol) in 40 ethanol (120 mL) was added. The mixture was hydrogenated at 39-34 psi (2.70 2.35 bar) over an 80 min period. The reaction mixture was filtered on celite and the residue washed further with ethanol (3 x 20 mL). The combined filtrates were concentrated to dryness and the residue purified by chromatography (eluent ethyl acetate:hexanes 2:3 increasing to 1:1) to give 4-4a (0.94 g, 67%). 'H NMR (400 MHz, Acetone-d 6 ) 6E] ppm 2.14 (s, 3 H) 3.41 - 3.51 (m, 1 H) 4.15 - 4.29 (m, 2 H) 4.33 - 4.43 (m, 1 H) 6.23 - 6.34 (m, 2 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.57 (d, J = 8.22 Hz, 1 H) 6.65 (d, J= 3.52 Hz, 5 H) 8.05 (s, 1 H) 8.17 (br s, 1 H) 8.35 (br s, 1 H) Other compounds of the invention prepared via this method (final products purified by preparative HPLC): 3-(2-Fluoro-4-hydroxyphenyl)-4-(3-fluoro-4-hydroxyphenyl)-8-methylchroman-7-o (Compound 3) (4-4b) (106%). 1 H NMR (400 MHz, Acetone-d 6 ) 6E ppm 2.14 (s, 3 H) 3.65 - 3.75 (m, 1 H) 4.21 - 4.32 (m, 2 H) 4.37 - 4.49 (m, 1 H) 6.24 - 6.47 (m, 5 H) 6.58 (d, J = 8.61 Hz, 2 H) 6.71 (s, 1 H) 4-(3-Fluoro-4-hydroxyphenyl)-3-(3-hydroxyphenyl)-8-methylchroman-7-o (Compound 4) (4-4c) (111%). 'H NMR (400 MHz, Acetone-d 6 ) 6K ppm 2.14 (s, 3 H) 3.41 - 3.52 (m, 1 H) 4.24 (d, J = 4.70 Hz, 1 H) 4.26 - 4.33 (m, 1 H) 4.34 - 4.44 (m, 1 H) 6.24 - 6.37 (m, 4 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.57 (d, J = 8.22 Hz, 1 H) 6.67 (t, J = 8.41 Hz, 2 H) 7.00 (s, 1 H) 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-methoxy-3-methylphenyl)-8-methylchroman-7-o (Compound 5) (4-4d) (99%). 'H NMR (400 MHz, Acetone-d 6 ) 6E ppm 2.14 (s, 3 H) 3.40 - 3.49 (m, 1 H) 3.77 (s, 3 H) 4.15 - 4.28 (m, 2 H) 4.33 - 4.43 (m, 1 H) 6.23 - 6.33 (m, 2 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.49 - 6.63 (m, 3 H) 6.66 - 6.75 (m, 2 H) 4-(4-Hydroxyphenyl)-3-(4-methoxy-3-methylphenyl)-8-methylchroman-7-o (Compound 11) (4-4e) (83%). 1 H NMR (400 MHz, Acetone-d 6 ) 6E ppm 2.13 (s, 3 H) 3.42 (d, J = 11.74 Hz, 1 H) 3.77 (s, 3 H) 4.11 - 4.24 (m, 2 H) 4.33 - 4.43 (m, 1 H) 6.39 (d, J = 8.22 Hz, 1 H) 6.44 (d, J = 8.22 Hz, 2 H) 6.50 (br. s., 1 H) 6.54 (d, J = 4.30 Hz, 4 H) 6.68 (d, J = 8.61 Hz, 1 H) 3,4-bis(3-Fluoro-4-hydroxyphenyl)-8-methylchroman-7-o (Compound 6) (4-4f) (96%) (50% HPLC purity). 1 H NMR (400 MHz, Acetone-d 6 ) 6E ppm 2.00 (s, 3 H) 3.30 - 3.40 (m, 1 H) 4.07 - 4.18 (m, 2 H) 4.21 - 4.30 (m, 1 H) 6.14 - 6.25 (m, 2 H) 6.28 (d, J = 8.36 Hz, 1 H) 6.34 - 6.46 (m, 3 H) 6.58 (d, J = 8.80 Hz, 1 H) 6.69 (s, 1 H) 3-(3-Fluoro-4-hydroxyphenyl)-4-(4-hydroxyphenyl)-8-methylchroman-7-ol (Compound 7) (4-4g) (89%). 'H NMR (400 MHz, Acetone-d 6 ) K6 ppm 2.00 (s, 3 H) 3.28 - 3.36 (m, 1 H) 4.06 (d, J = 5.28 Hz, I H) 4.08 - 4.14 (m, 1 H) 4.20 - 4.29 (m, 1 H) 6.26 (d, J = 8.22 Hz, 41 1 H) 6.30 - 6.38 (m, 4 H) 6.39 - 6.47 (m, 3 H) 6.63 - 6.70 (m, 1 H) 4-(3-Chloro-4-methoxyphenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-ol (Compound 8) (4-4h) (96%). 1 H NMR (400 MHz, Acetone-d 6 ) 6D ppm 2.14 (s, 3 H) 3.48 (dt, J = 11.54, 4.40 Hz, 1 H) 3.80 (s, 3 H) 4.19 - 4.30 (m, 2 H) 4.32 - 4.41 (m, 1 H) 6.42 (d, J = 8.61 Hz, 1 H) 6.49 - 6.70 (m, 7 H) 6.81 (s, 1 H) 4-(4-Fluoro-3-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-o (Compound 9) (4-4i) (82%). 'H NMR (400 MHz, Acetone-d 6 ) 6 ppm 2.13 (s, 3 H) 3.46 (dt, J = 11.54, 4.40 Hz, 1 H) 4.17 (d, J = 5.48 Hz, 1 H) 4.23 (dd, J = 10.56, 2.35 Hz, 1 H) 4.34 - 4.45 (m, 1 H) 6.07 (ddd, J = 6.16, 4.40, 1.96 Hz, 1 H) 6.29 (dd, J = 8.61, 1.96 Hz, 1 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.56 (d, J = 8.22 Hz, 1 H) 6.59 - 6.69 (m, 5 H) 6.72 - 6.81 (m, 1 H) 4-(3-Fluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-propylchroman-7-o (compound 13) (4-4j) (96%) 1 H NMR (400 MHz, Acetone-d 6 ) 6 ppm 0.82 (t, J= 7.6 Hz, 3 H) 1.45 (m, 2H), 2.4(t, J= 7.6 Hz 2H), 3.56 (dt, J = 11.54, 4.40 Hz, 1 H) 4.07 (d, J = 5.3 Hz, 1 H) 4.13 (dd, J = 10.2, 2.35 Hz, 1 H) 4.24 (m, 1 H) 6.17 (ddd, J = 6.16, 4.40, 1.96 Hz, 1 H) 6.27 (dd, J = 8.61, 1.96 Hz, 1 H) 6.31 (d, J = 8.22 Hz, 1 H) 6.56 (d, J = 8.22 Hz, 1 H) 6.58 6.61 (m, 5 H) 6.68 - 6.78 (m, 1 H) 4,4'-(7-Hydroxy-8-propylchromane-3,4-diyl)bis(2-fluorophenol) (4-4k) (compound 14) (98%) 'H NMR (400 MHz, Acetone-d 6 ) 50 ppm 0.82 (t, J= 7.6 Hz, 3 H) 1.43 (m, 2H), 2.45(t, J= 7.6 Hz 2H) ,3.33 - 3.42 (m, 1 H) 4.17 - 4.23 (m, 2 H) 4.25 - 4.30 (m, 1 H) 6.14 - 6.26 (m, 2 H) 6.32 (d, J = 8.36 Hz, 1 H) 6.38 - 6.46 (m, 3 H) 6.65 (d, J = 8.80 Hz, 1 H) 6.75 (s, 1 H) 3-(4-(Ethylamino)phenyl)-4-(3-fluoro-4-hydroxyphenyl)-8-propylchroman-7-o (4-41) (75%) (compound 15) 6 ppm 0.82 (t, J= 7.6 Hz, 3 H) 1.15 (t, J = 6.7 Hz, 3H), 1.45 (m, 2H), 2.4 (t, J= 7.6 Hz 2H), 2.89 (q, J = 6.9 Hz, 2H) , 3.2 (br, 1H), 3.46 (dt, J = 10.5, 4.20 Hz, 1 H) 4.17 (d, J = 5.01 Hz, 1 H) 4.28 (dd, J = 10.03, 2.45 Hz, 1 H) 4.34 (m, 1 H) 6.27 (ddd, J = 6.6, 4.20, 2.03 Hz, 1 H) 6.27 (dd, J = 9.2, 2.05 Hz, 1 H) 6.31 (d, J = 7.82 Hz, 1 H) 6.56 (d, J = 7.92 Hz, 1 H) 6.58 -6.65 (m, 5 H) 6.72 - 6.79 (m, 1 H) 4-(2,3-difluoro-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-o (4-4m) (76% Compound 10) 1 H NMR (400 MHz, Acetone-d 6 ) 6 ppm 2.10 (s, 3 H) 3.42 (dt, J = 11.54, 4.40 Hz, 1 H) 4.27 (d, J = 5.48 Hz, 1 H) 4.39 (dd, J = 10.56, 2.35 Hz, 1 H) 4.41 4.45 (m, 1 H) 6.08 (brdd, J = 7.16, 4.40 Hz, 1 H) 6.41 (d, J = 8.22 Hz, 1 H) 6.56 (d, J = 8.22 Hz, 1 H) 6.57 - 6.70 (m, 5 H) 6.76 - 6.80 (m, 1 H). 4-(3-methoxy-4-hydroxyphenyl)-3-(4-hydroxyphenyl)-8-methylchroman-7-o (4-4n) (Compound 12) 42 1 H NMR (400 MHz, Acetone-d 6 ) 60 ppm 2.11 (s, 3 H) 3.43 (dt, J = 11.11, 4.43 Hz, 1 H) 3.82 (s, 3 H) 4.18 - 4.35 (m, 2 H) 4.38 - 4.46 (m, 1 H) 6.45 (d, J = 8.21 Hz, 1 H) 6.49 6.90 (m, 7 H) 6.95 (s, 1 H) Example 3 - Chiral resolution Chiral resolution of compound 4 was performed by the following methods. Analytical separation method. Instrument: Thar analytical SFC. Column: ChiralCel OD 3, 150 x 4.6mm. Mobile phase: A for C02 and B for Ethanol (0.05%DEA). Gradient: B 40%. Flow rate: 2.4mL/min. Back pressure: 100bar. Column temperature: 350C. Wavelength: 220nm. Preparative separation method. Instrument: MGII preparative SFC. Column: ChiralCel OD-H, 250x30mmI.D. Mobile phase: A for C02 and B for Ethanol (0.1%NHH 3

-H

2 0). Gradient: B 40%. Flow rate: 50mL /min. Back pressure: 100bar. Column temperature: 38'C. Wavelength: 254nm. Cycletime: -1.6min. Sample preparation: Compound was dissolved in ethanol to -0.3mg/ml. Injection: 1.5ml per injection. Work up: After separation, the fractions were dried off via rotary evaporator at bath temperature 40 0C to get the desired isomers. Fastest eluting enantiomer (98.4% e.e., referred to as CS-25 herein) and had a specific optical rotation of [a]D25.5 = + 3.30 (c 0.85, MeOH) . Slowest eluting enantiomer (99.2% e.e., referred to as CS-26 herein) and had a specific optical rotation of [ca]D 25 - 3.50 (c 1.21, MeOH). Example 4 - In vitro testing Activity of compounds 1-9 and 11 against various cancer cell lines Selected compounds were screened for oncology activity against a number of cancer cell lines using an MTT-based assay from which IC50 values were ascribed. The MTT assay was run using the following general procedure: Short well 96 assay. Each condition was performed in triplicate or more according to the following general procedure: Day One: Trypsinise one T-25 flask and add 5 ml of complete media to trypsinized cells. Centrifuge in a sterile 15 ml falcon tube at 500 rpm in the swinging bucked rotor (-400 x g) for 5 min.

43 Remove media and resuspend cells to 1.0 ml with complete media. Count and record cells per ml. The cells should be removed aseptically when counting. Dilute the cells (cv=cv) to 75,000 cells per ml. Use complete media to dilute cells. Add 100 pl of cells (7500 total cells) into each well and incubate overnight. Day Two: Treat cells on day two with agonist, inhibitor or drug. (If removing media, do very carefully to minimise variation in data. Final volume should be 100 pl per well). Day Three: Add 20 pl of 5 mg/ml MTT to each well. Include one set of wells with MTT but no cells (control). All should be done aseptically. Incubate for 3.5 hours at 370C in culture hood. Carefully remove media. Do not disturb cells and do not rinse with PBS. Add 150 pl MTT solvent. Cover with tinfoil and agitate cells on orbital shaker for 15 min. Read absorbance at 590 nm with a reference filter of 620 nm. The IC 50 values (micromolar) for Compounds 1-9 and 11 were determined for the human cancer lines indicated in Tables 1-5. Table 1. IC 50 (pM) Compounds tested against selected brain cancer cell lines Compound No U87-MG T98G A172 SNB-19 DAOY 1 2.23 0.44 2.47 NT NT 2 0.19 0.07 0.41 0.13 0.13 3 0.61 NT NT 0.43 0.43 4 3.18 NT NT 1.34 1.34 5 1.57 NT NT 0.57 0.57 11 0.67 NT NT 0.21 0.21 6 0.56 NT NT 0.22 0.22 7 0.55 NT NT 0.24 0.24 8 1.17 NT NT 0.51 0.51 9 1.28 NT NT 0.42 0.42 NT = Not tested Table 2. IC 50 (pM) Compounds tested against colon cancer and pancreatic cancer cell lines Compound No HT-29 HCT-15 HCT-116 SW480 MiaPaCa-2 1 3.40 0.89 1.10 6.11 NT 2 5.70 0.30 0.30 0.27 NT 44 3 64.76 NT 0.65 NT 0.33 4 72.81 NT 3.59 NT 2.21 5 NT NT 1.77 NT 0.57 11 8.37 NT 1.27 NT 1.28 6 NT NT 1.19 NT 0.23 7 NT NT 0.71 NT 0.21 8 NT NT 2.07 NT >50 9 NT NT 3.20 NT 26.77 NT = Not tested Table 3. IC50 (pM) Compounds tested against pancreatic cancer, prostate cancer and melanoma cancer cell lines Compound No PANC-1 A375 G361 A431 PC3 1 NT 0.96 1.01 4.94 1.95 2 NT 0.22 0.28 0.41 0.29 3 0.56 0.45 0.55 NT 0.49 4 19.12 1.89 2.38 NT 1.93 5 >50 0.49 0.98 NT 1.14 11 >50 0.23 0.48 NT 0.54 6 >50 0.58 0.51 NT 0.51 7 >50 0.54 0.52 NT 0.50 8 >50 0.32 1.71 NT 1.08 9 >50 0.45 0.87 NT 1.30 NT = Not tested Table 4. IC5o (pM) Compounds tested against lung cancer, prostate cancer and breast cancer cell lines

MDA-MB

Compound No LNCaP DU145 MCF-7 231 NCI-H460 1 3.20 2.52 27.71 2.03 0.70 2 0.37 0.26 1.17 0.50 0.28 3 0.47 NT NT NT NT 4 2.69 NT NT NT NT 5 1.32 NT NT NT NT 11 0.47 NT NT NT NT 6 0.52 NT NT NT NT 7 0.49 NT NT NT NT 8 0.85 NT NT NT NT 9 0.93 NT NT NT NT NT = Not tested Table 5. IC50 (pM) Compounds against lung cancer cell lines Compound No A549 NCI-H69 1 0.80 NT 2 0.32 NT 3 25.85 1.27 45 4 38.56 3.47 5 2.35 29.26 11 1.13 0.58 6 2.71 0.58 7 2.15 0.54 8 1.05 >50 9 0.94 >50 NT = Not tested These results demonstrate the broad activity of compounds of the present invention against various cancer cell lines. In particular, these studies indicate that Compound 2 is active against cancer cell lines representative of a broad range of cancer types at concentrations some 1000 times lower than anti-proliferative activity seen in NV-128 and triphendiol (which are representative compounds of W02006/032085 and W02006/032086). Furthermore these results indicate that placement of certain functional groups on the phenyl ring attached to the 3-position of the chromen-7-ol system increase the propensity of compounds to be conjugated by the glucuronyl transferase enzymes over expressed by the cell line HT-29. This allows for the specific design of compounds with lower rates of conjugation. This may confer the advantages of superior pharmaceutical properties, in particular an enhanced pharmacokinetic profile through reduced conjugation and elimination. When assessed in vitro, Compound 2 (racemate) demonstrated potent anti-proliferative activity against a broad range of human cancer cells, including glioblastoma multliforme (T-98G), melanoma (G-361), lung (NCI-H460), colorectal (HCT116), breast (MDA-MB 231) and prostate cancer (DU-145) (see Figure 1). IC 50 values ranged from 70 nm for T 98G (Glioma) to 500 nm for the triple negative breast cancer cell line MDA-MB-231 (Breast cancer) (see Figure 1). Activity of Compound 2 and its enantiomers against 3 glioblastoma multiforme cell lines The activity of Compound 2 (racemate) was compared with that of Compound 2 enantiomers CS-25 and CS-26 (-ve ORD enantiomer) against 3 glioblastoma multiforme cell lines (SNB19, DAOY and U87MG). The IC 50 value for CS-25 was calculated at -100nM for each of the cell lines assessed. The Compound 2 racemate was slightly less active when compared with CS-25 with IC 50 values ranging from 130 nM for SNB19 to 250 nm for U87MG. The other enantiomer CS-26 had activity that was >100-fold less active than Compound 2 and CS-25 against all three glioma cell lines (see Figure 2). Activity of Compound 2 against Temozolomide-resistant glioblastoma multiforme cell lines 46 The anti-proliferative activity of Compound 2 (racemate) was assessed in matched cell lines that are susceptible (D54-S and U87-S) or resistant (D54-R and U87-R) to temozolomide (TMZ). Briefly, a sulforhodamine B assay (Sigma-Aldrich) was performed to assess cell survival in the absence or presence of serial concentrations of TMZ or Compound 2. Seeded cells (5000 cells/well) were cultured in 96-well plates for 72 h. Cellular protein content was used to represent surviving cell density, as measured by absorbance of optical density at 490 nm. The percentages of surviving cells were calculated after normalization to the controls (cells without prior drug treatment). The half maximal IC 50 value was calculated by derivation of the best-fit line, using 3 independent experiments performed in triplicate. TMZ-resistant sub-clones were established by exposing parental TMZ-sensitive cells (D54-S and U87-S) to TMZ until stable TMZ-resistant subclones (D54-R and U87-R) were established. A resistant phenotype was confirmed by inhibitory concentration (IC 5 0 ) testing with resistant cub-clones exhibiting >2-fold greater resistance to TMZ compared with the parental lines (Figure 3B and D). The resistant subclones were maintained in a low dose (100 pM) of TMZ. Compound 2 demonstrated equipotent anti-proliferative activity against both the D54 and U87 GBM cell lines regardless of their TMZ resistance status with IC 50 values observed at between 200 and 300 nM compared with IC50's of -600 pM in the TMZ susceptible lines and -2000 pM against the TMZ-resistant lines (Figure 3A and C). These data demonstrate that Compound 2 exhibits superior anti-proliferative activity against TMZ-susceptible and resistant GBM cell lines. Compound 2 activity in patient-derived explants of glioblastoma multiforme The anti-cancer activity of Compound 2 (racemate) was assessed by XenTech in two glioblastome multiforme patient derived explants established from tumour biopsies following the methodology detailed. Briefly, primary cell culture was obtained from from explanted and dissociated ODA14-RAV and GBM14-CHA xenografts. Cells were thawed quickly in a 37 0 C water bath. One vial of cells (~10 millions cells) was diluted into 10 mL of complete growth medium (F12/DMEM supplemented with 8% foetal bovine serum, 100 pg/ml penicillin G sodium, 100 pg/ml streptomycin sulphate). After centrifugation at 150 g, 5 minutes, the cell pellet was resuspended in complete growth medium and plated at a density of at least 140 000 cells/cm 2 in 75 cm 2 cell culture Flasks. Cells were maintained at 37 0 C in a humidified atmosphere with 5% C02 for at least one week. Then, cells were harvested and seeded in 96-wells plates at a density of 2.5x103 cells/well for cytotoxicity assays. Cells were incubated 48h at 37 0 C prior to 47 addition of the test molecules. Test drugs were added at desired final concentrations and further incubated for 72 hr. Cell viability is assessed before drugs' addition (TO) and 72 hr after drugs' addition by measuring ATP cell content using CellTiter-Glo@ Luminescent Cell Viability Assay (Promega) according to the manufacturer's instructions. ODA14 was designated as grade IlIl (determined by histopathology), susceptible to TMZ when assessed in xenograft studies, p53 mutant, pTEN wildtype and had amplified EGFR expression. GBM14 was designated as TMZ resistant, p53 wt, pTEN mutant, EGFR wt. The GBM grade is not known. After 72 hr exposure to Compound 2, an IC 50 of 0.16 pM against GBM14 and 36.5 pM against ODA14 was observed (see Figure 4). These data suggest that GBM cells that have an EGFR amplification and mutant p53 will exhibit resistance to Compound 2. However, upon reviewing EGFR, p53 and pTEN status in the 5 commercially available GBM cell lines, it was apparent that compound 2 was equally active in cells with a molecular profile similar to that of ODA14 (i.e. DAOY, SNB-19 and T98G) when compared with Compound 2 activity in U87MG and A172 (not shown). These data indicate that compound 2 is active against GBM cells regardless of the identified molecular traits. Furthermore, the grade of tumour does not appear to correlate with compound 2 sensitivity although only two cell lines have been assessed (compare SNB19; grade IV and ODA14; grade Ill). Example 5 - Anti-proliferative activity of Compound 2 against ovarian cancer The ability of Compound 2 to inhibit the proliferation of both differentiated and undifferentiated ovarian cancer cells was established from patient derived explants. Cell proliferation was assessed using the Incucyte Kinetic Imaging System. Briefly, monolayer cells were trypsinized and plated in 96-well plates. After 24h, once the cells have attached, treatment was dispensed in RPMI with 10% FBS. Doses used were: 0.0001, 0.001, 0.01, 0.1, 1, and 10 ug/mL. Culture plates were immediately placed in the Incucyte system and imaged every 2h. Growth curves were calculated as a measure of cell confluence using an integrated confluence algorithm as a surrogate for cell number to determine proliferation rate. In duplicate experiments it was found that Compound 2 was able to retard the proliferation of both undifferentiated ovarian cancer stem cells (OCSC-2 which are CD44+ve) and their differentiated progeny of OCSC-2 (termed OCC-2 which are CD44-ve) (Figure 5A and B). Using the Incucyte real-time imaging system, all concentrations of CS-6 used down to 0.0125 pg/ml completely inhibited both differentiated ovarian cancer cell and ovarian cancer stem cell proliferation. Microscopic evaluation of both ovarian cancer cells and ovarian cancer stem cells treated with 48 0.0125 pg/ml revealed a change in morphology from dendritic at t=0 (Figure 5C - left tile), to rounded with granular nuclei at 1 hr (Figure 5C, right tile). All treated cells had similar appearance 5 and 10 hr post treatment (data not shown). Earlier studies in GBM cell lines indicated that CS-25 (the +ve ORD enantiomer of compound CS-6) exhibited marginally superior anti-cancer activity compared with Compound 2 against GBM (see Figure 2). Similarly, using Incucyte methodology, CS 25 (1 and 0.1pM) retarded the proliferation rate of the highly chemoresistant ovarian cancer stem cell (OCSC-1) with cells only reaching -30% confluence at 42hrs post treatment which was marginally superior to that of Compound 2 (0.1pM) (40% confluence achieved at 48hrs) (Figure 6). Given the concept that residual cancer progenitor cells within the tumour post treatment are responsible for tumour relapse, a critical therapeutic strategy to prolong survival is eradicate those tumour progenitor cells driving relapse. Therefore, studies were conducted to determine whether CS-25 was able to inhibit OCSC proliferation once drug pressure was removed. OCSC-1 and OCSC-2 cells were treated with 0.1 and 1 pg/ml CS-25 for 22hrs, washed with culture medium and allowed to recover for a further 50 hrs under standard incubation conditions. While proliferation of the highly resistant ovarian cancer stem cell OCSC-1 was initially inhibited when cultured in the presence of both 0.1 and 1 pg/ml (over 22 hrs) when compared to control, once drug pressure was removed those cells initially cultured in 0.1 pg/ml initiated proliferation two hrs after culture medium replenishment. Those cells cultured in 1 pg/ml CS-25 took longer to begin proliferating (-25hr) once drug pressure was removed (Figure 7A). In contrast to the OCSC-1 result, OCSC-2 failed to enter logarithmic cell proliferation at both CS-25 concentrations tested after 72hrs (Figure 7B). Like OCSC2, F1 cells also failed to enter logarithmic growth phase at all concentrations used except for 0.1 pg/ml and (Figure 7C). Indeed, concentrations of 1 pg/ml and above appeared to reduce non-proliferating cell numbers which suggests that Compound 2 is able to induce apoptosis in resting cells. The data presented in Figure 7 indicate that CS-25 induced a transient cytostatic effect on OCSC-1 which was time-dependently abrogated after CS-25 removal. CS-25 at low concentrations (0.1 pM), elicited a more durable anti-proliferative effect on OCSC-2 cells, still apparent at 50hrs. Studies were conducted using ovarian cancer stem cells to determine the effects CS-25 had on cellular machinery. Briefly, ovarian cancer stem cells, OCSC2 and F2 cells that had been transfected with CHERRY reporter vector construct, were treated with CS-25 over 24 hr. Cell lysates from each timepoint were assayed for caspase 9 and caspase 3 activity using Caspase-Glo 3/7 or 9 reagents (Promega). After incubating cell lysate and 49 the respective caspase reagent at room temperature for 1 hour, luminescence was measured using TD 20/20 Luminometer (Turner Designs, Sunnyvale, CA). Blank values were subtracted and fold-increase in activity was calculated based on activity measured from untreated cells. Each sample was measured in triplicate. To determine whether CS-25 had any effect on the status of X-linked inhibitor of apoptosis (XIAP) protein levels, lysates collected from the above cell treatment regimen were subjected to SDS PAGE and blotted on to PVDF membrane. Mouse anti-XIAP (BD Biosciences,1:1,000) was then use to visualize XIAP labeled protein using enhanced chemiluminescence (Pierce, Rockford, IL). Mitochondrial membrane potential was determined using Mitocapture apoptosis detection kit based on JC1 dye staining (Biovision Inc.) and Mitotracker Red CMXRos (Molecular Probes). The treatment of both ovarian cancer stem cell lines (OSCS1 and F2-CHERRY) with low levels of CS-25 (0.1 pg/ml) resulted in reduced levels of cellular XIAP thereby explaining the induction of caspase-9 and executioner caspase-3 observed some 4-8hr post CS-25 treatment (Figure 8A-D). Previous studies have demonstrated that XIAP is a target for proteasomal degradation however, conversely, phosphorylated XIAP, cataylsed by AKT, is protected from proteasomal degradation. It is plausible the CS-25 inhibits AKT mediated phosphorylation of XIAP thereby enabling its degradation by the proteasome. CS-25 was also shown to induce mitochondrial depolarization in OCSC1 as demonstrated by a reduction in the number cells with normal mitochondria in control cells (89.6%) versus those cells that have been treated with CS-25 (43.2%) (Figure 8E,F). The subsequent release of cytochrome c from compromised mitochondria is thought to result in caspase-9 activation however further studies are required to confirm the involvement of cytochrome c. The cellular effects of CS-25 treated ovarian cancer stem cells are summarised in Figure 9. The ability of CS-25 to induce apoptosis is in direct contrast to its other structural analogue NV-128 which induces autophagy only. Compound 2 has been shown to inhibit plasma membrane-associated NADH oxidase activity. Carboplatin is regarded as a standard of care for the treatment of ovarian cancer. Disease recurrence post the end of first-line therapy can occur from 6 months with those patients whose disease showed initial response to platinum and recurred having >6 months progression free Interval defined as platinum sensitive. Patients whose disease showed response to treatment or stable disease prior to platinum treatment and who recurred within 6 months of final treatment are defined as platinum resistant. Patients, whose disease progressed during platinum treatment and have less than 3-month progression free interval, are defined as having refractory disease, which means that these patients have very little chance to respond to a platinum-based therapy. OCSC-1 50 and OSCC-2 ovarian cancer stem cells were established from patients that had platinum-refractory disease. The efficacy of Compound 2 assessed at 0.1 and 1 pg/ml was compared with carboplatin at 0.001, 0.01, 0.1, 1, 10 and 100 pg/ml against OCSC-1. As observed previously, 0.1 pg/ml Compound 2 completely inhibited OCSC-1 proliferation (0.01 pg/ml CS-6 had no effect on proliferation) (Figure 10A). In contrast, carboplatin when assessed at 0.1 pg/ml had no effect on OCSC-1 proliferation with cells proliferating at the same rate as vehicle control. Indeed, carboplatin concentrations up to 10 pg/ml carboplatin (100-fold greater than CS-6) had very little effect on OCSC-1 proliferation with 100 pg/ml carboplatin strongly inhibiting OCSC-1 proliferation (Figure 10B). Those OCSC-1 cells treated with 0.1 pg/ml CS-6 failed to proliferate out to 70hrs post treatment whereas OCSC-1 treated with 100 pg/ml carboplatin began to slowly proliferate by 50 hrs post treatment. These data indicate that when assessed in vitro, Compound 2 has superior anti proliferative activity on carboplatin-resistant ovarian cancer stem cells when compared with carboplatin. Example 6 - Pre-clinical overview - In vivo efficacy CS-25 Formulation in Captisol The utility of Captisol as a vehicle to formulate Compound 2 was assessed. A High Pressure Liquid Chromatography (HPLC)-based method for determination of the concentration of Compound 2 in aqueous solutions was developed and subject to partial validation to confirm accuracy, precision and linearity. This method was used to determine the concentration of CS-25 (Compound 2 enantiomer) when formulated as an aqueous solution in the presence of varying concentrations of Captisol. Briefly, CS-25 and Captisol were dispensed into a mortar and ground to a fine powder. The combined powders were then transferred to volumetric flasks and reconstituted to a 1 mL final volume by the gradual stepwise addition of water. The samples were vortexed/agitated with each addition of solvent. The samples were then centrifuged at 8000xg for 10 min to pellet insoluble material. An aliquot was then taken for dose analysis (0 hrs) and samples were incubated at room temperature with constant agitation shielded from light. After 6, 24 and 48 hours, additional aliquots were removed from each formulation following additional centrifugation steps. All samples were then diluted into 70% aqueous methanol and analyzed by HPLC.

51 Results demonstrate that the maximum concentration of soluble CS-25 achieved in this study was approximately 30 mg/mL in 30% Captisol. The formulations were generally stable for at least 48 hours at ambient temperature protected from the light. CS-25 Pharmacokinetics The pharmacokinetics of CS-25 were assessed. Briefly, 36 male Balb/c mice were allocated to three groups of twelve mice each. Each group of mice was administered a single dose of CS-25 (150 mg/kg) via either intravenous (i.v.), intraperitoneal (i.p.) injection or oral gavage (p.o.). Interim bleeds via the submandibular route were collected from three mice per time-point at 15 and 30 min, 1 and 2 hours post-initial treatment. Terminal blood samples were collected at 4, 8, 24 and 48 hours post-initial treatment via cardiac puncture. Plasma samples prepared from non-coagulated whole blood samples were analysed for Test Article concentration using a liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method. Briefly, free and total concentration of CS-6 was determined in mouse plasma using a protein precipitation for sample preparation followed by the determination of free or total Compound 2 concentration by LC-MS/MS using NV-196 as an internal standard. Calibration standards containing nominal amounts of Compound 2 in blank mouse plasma will be used to generate calibration curves. Prior to protein precipitation, those samples to be analysed for free CS-25 was treated with glucuronidase to liberate the test article from glucuronides. LC-MS/MS data collection was performed using Analyst 1.4.2 (API 4000 QTRAP LC-MS/MS software, Applied Biosystems). The LLOQ will be defined as the lowest concentration standard in the calibration curve that gives rise to a response 2 3 times the corresponding response in the blank sample, while meeting the criteria for precision of 20%CV and accuracy of 80-120%. PK parameters were determined using non-compartmental modeling methods. Like its predecessor drugs, CS-25 was subject to glucuronidation by t-glucuronyl transferases which are a defence mechanism responsible for detoxifying foreign molecules by catalyzing the addition of sugar moieties to the drug (via glucuronidation) resulting glucuronide conjugates that are more readily excreted. Intravenous delivery of CS-25 formulated in Captisol yielded high plasma levels of conjugated CS-25 (Total CS 25) (CMax = 204 pg/ml). Importantly, i.v. delivery also yielded high levels of the active (free) form of CS-25 (CMax = 37.6 pg/ml or 44 pM) which is some 140 times higher than the amount of drug required to inhibit the proliferation of glioblastoma cells in the test tube (IC 50 = ~300 nM for GBM cell lines, see Figure 2) (Figure 11). Free CS-25 however, was rapidly eliminated as indicated by the high observed clearance rate of 6.5L/hr thus explaining the rapid elimination of free CS-25 in plasma.

52 Indeed, by 1 hr post injection CS-25 was largely eliminated and by 2yrs the level of CS 25 in plasma is below the amount of drug required to inhibit the proliferation of cancer cells by 50% (IC 50 ). Intraperitoneal delivery yielded a lower Cmax level free CS-25 in plasma (16.4 pg/ml or 44 pM) and total CS-25 (Figure 11). While this plasma concentration is some 50 fold higher than the IC 50 concentrations observed against glioblastoma cancer cells, the clearance rate via this mode of delivery, while being some 1.3 fold slower than observed clearance rate for i.v. delivery, was still high (4.9 L/hr). This slower rate of elimination can be observed in Figure 11 with therapeutic levels of free CS-25 remaining in plasma for up to 3hr post administration. CS-25 efficacy as a monotherapy in a murine xenograft model of GBM This study was undertaken to determine whether the maximum tolerated dose of CS-25 using daily delivery at 15 mg/kg exerted an anti-cancer effect in nude mice bearing U87 MG xenografts. Briefly, Twenty-five mice were inoculated while under isoflurane-induced anaesthesia a 100 pL cell suspension consisting of 2 x 106 cells. Twenty of the inoculated mice were selected for the study, with mean tumour volume of approximately 67 mm 3 17 days post-inoculation (Day 0). The mice were randomised, based on tumour size, into two groups of ten mice. The Vehicle for CS-25, 30% w/v Captisol in water, was prepared weekly and stored at room temperature protected from light. CS-25 stock (30 mg/mL) was prepared and dosed within 48 of preparation. Vehicle Control (30% w/v Captisol in sterile water; Group 1) and CS-25 (150 mg/kg; Group 2) were administered daily via intraperitoneal injection (i.p.). Treatment began on Day 0 and continued for 19 days. The Vehicle Control and Test Article were administered in a dosing volume of 5 mL/kg. The volume of dosing solution administered to each animal was calculated and adjusted based on individual body weight measured immediately prior to dosing. Body weights and tumour dimensions (length and diameter) were measured for all animals on the first treatment day (Day 0) and then three times per week, including the termination day of the study (Day 22). Tumours were excised from each animal at termination and weighed. Tumour volume was calculated using the equation: V(mm.) = length x diameter. x Tr /6. Comparisons of tumour size and weight measurements at termination of the study were made between the CS-25 treatment group (Group 2) and the Vehicle Control (Group 1) using an unpaired t-test. A p value less than 0.05 was considered significant. There was significant inhibition of tumour growth by CS-25 (Group 2) compared with Vehicle Control (Group 1) (AT/AC% = 71.8%) (p 5 0.05) (Figure 12A). Tumour weights 53 and volumes at study termination were significantly lower (p 5 0.05) in mice treated with CS-25 (Group 2) compared with Vehicle Control-treated mice (Group 1) (See inset in Figure 12A). Clinically, Seven mice treated with Vehicle Control and six mice treated with CS-25 showed a loss in body condition on Day 22. Two mice with Vehicle Control and seven mice treated with CS-25 exhibited mild dehydration on Day 22. All mice treated with CS-25 showed mild diarrhoea beginning on Day 14 that resolved on Day 17. In five of these mice mild diarrhoea recurred on Day 22 following the resumption of dosing. Mice receiving Vehicle Control and CS-25 showed mild gain in body weight from Days 0-11 (mean 4.4% and 5.4% of initial weight, respectively, before a mild decrease in body weight was observed from Days 12-22 (Figure 12B). Mean body weight gain over the study duration (Days 0-22) (0.9% of initial weight) for the Vehicle Control group was not significant. Mice treated with CS-25 had a mean body weight loss of 1.1% of initial weight, which was also not significant these mice. Mean kidney weights were similar in mice treated with Vehicle Control or CS-25 (0.331 g and 0.341 g, Groups 1 and 2, respectively). There was also no considerable difference between the mean liver weights of Vehicle Control and CS-25 mice (data not shown). Spleens appeared enlarged in some animals treated with CS-25. Example 7 - Pre-clinical overview - Toxicology A study was undertaken to determine the maximum tolerated dose of CS-25 following once daily dose administration to female Balb/c mice for seven days. Balb/c mice are commonly used to assess the acute toxicity of test compounds but were used in this instance to identify a MTD dose to use in subsequent xenograft efficacy studies. Briefly, twelve female Balb/c mice were administered one of three doses of CS-25 (n=4 mice per dose) via intraperitoneal (i.p.) injection daily for seven consecutive days followed by a seven-day recovery phase. The three doses chosen were 75 mg/kg (low dose), 150 mg/kg (middle dose) and 300 mg/kg (High dose). The dosing plan: starting on Day 0 of the study, one mouse in the group receiving the lowest dose concentration will be dosed with 75 mg/kg CS-25 via i.p. injection in a dosing volume of 10 mL/kg. This mouse will be monitored for 2 hours post-dose and if no adverse clinical signs are observed, the remaining mice in the group will be dosed. After 24 hours, if no adverse toxicity is observed, one mouse in the next highest dose group (150mg/kg) will be dosed. If no adverse clinical signs are observed following a 2-hour monitoring period, the remaining mice in this group will be dosed. This staggered dosing procedure will be 54 continued until all scheduled doses are administered. All mice will be weighed prior to treatment and actual dosing volume adjusted accordingly. The Body weight progression and observation of clinical signs were used to assess the effects of CS-25 on the ability to thrive. This study was conducted under the principles outlined in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes, 7th Edition, 2004 (National Health and Medical Research Council). The study will be carried out according to the approval conditions of the RMIT University Animal Ethics Committee Project No. 1335. After the second dose at 75 mg/kg (>48 hrs post administration) 1 of 4 animals was found moribund and had to be euthanased. While Clinically, the remaining animals appeared OK apart from some minor piloerection, dosing was suspended and animals observed for 24hrs, after which dosing was recommenced. In terms of body weight gain all animals continued to gain weight over the treatment period (the mean weight gain over the treatment period compared with day 0 was 8%) (Figure 13). Apart from some minor piloerection, animals in the 150 mg/kg group had no apparent clinical symptomology 48hrs post the initial administration, and were subject to continued daily dosing for 7 days. Over the treatment period animals failed to thrive and the average weight gain was 0.4%. Once treatment with CS-25 was removed, the mean average weight gain was 5.2% at the end of the 7-day recovery period. Indicating that any toxicity being elicited by CS-25 was reversible (Figure 13) Due to the adverse event observed in the low dose group, the protocol was amended such that animals in the 300 mg/kg group were initially dosed at 35 mg/kg rather than 300 mg/kg for the first 4 days. After 4 doses the animals were then switched to 300 mg/kg due to essentially normal clinical signs. Over the first 4 days of treatment with CS 25, animals gained -4% of their initial starting weight. When switched to 300 mg/kg, animals lost the weight gained finishing with an average weight gain of 1.9% at the end of the treatment period. During the 7-day recovery phase, all animals started to thrive and by the end of this period had gained 4.1% of their starting weight (Figure 13). These data indicate that female BALB/c mice can tolerate i.p. administered CS-25 at doses up to 150 mg/kg without losing any of their initial starting weight. Hence, 150 mg/kg has been assigned as our Maximum Tolerated Dose (MTD) and will be used in subsequent xenograft studies. These data also demonstrate that the observed CS-25 toxicity (piloerection and failure to thrive) was transient with all animals gaining weight once removed from drug pressure.

55 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, features, compositions and compounds.

Claims (20)

1. 2. The compound of claim 1, which is compound 2.
2. 3. A pharmaceutical composition comprising a compound according to claim 1 or claim 2 together with a pharmaceutically acceptable carrier, diluent or excipient.
3. 4. A method for the treatment of cancer in a subject in need thereof, the method comprising administration to the subject of a therapeutically effective amount of a compound according to claim 1 or claim 2, or a composition according to claim 3.
4. 5. The method of claim 4, wherein the cancer is selected from the group consisting of: lung cancer, pancreatic cancer, melanoma, colorectal cancer, ovarian cancer, breast cancer, brain cancer or prostate cancer. 58
5. 6. The method of claim 4 or claim 5, wherein the cancer is resistant to one or more chemotherapeutic agents.
6. 7. The method of claim 6, wherein the cancer is temozolomide-resistant glioblastoma.
7. 8. The method of claim 6, wherein the cancer is carboplatin-resistant or carboplatin refractory ovarian cancer.
8. 9. The method of any one of claims 4 to 8, wherein the cancer is a cancer that has recurred.
9. 10. A method for reducing incidences of, or risk of, cancer recurrence in a subject deemed to be at risk of cancer recurrence, the method comprising administration to the subject of an effective amount of a compound according to claim 1 or claim 2, or a composition of claim 3.
10. 11. The method of claim 10, wherein the subject is a subject who is in cancer remission.
11. 12. The method of claim 11, wherein the subject is in remission from ovarian cancer.
12. 13. A method for inducing apoptosis in, or inhibiting the proliferation of, a cancer stem cell, the method comprising contacting the cancer stem cell with an effective amount of a compound according to claim 1 or claim 2.
13. 14. The method of claim 13, wherein the cancer stem cell is an ovarian cancer stem cell.
14. 15. A method for treating a disease in a subject caused by cancer stem cells, the method comprising administration to the subject of a therapeutically effective amount of a compound according to claim 1 or claim 2, or a composition according to claim 3.
15. 16. The method of claim 15, wherein the disease is cancer.
16. 17. The method of claim 16, wherein the cancer is a metastatic cancer.
17. 18. The method of any one of claims 15 to 17, wherein the cancer stem cells are ovarian cancer stem cells.
18. 19. A method for preparing a compound of claim 1 comprising the steps of: (a) reducing a compound of formula (V) to produce a compound of formula (VI): 59 R1 R1 HO O O HO O R10 10 13 R11 R13 (V) (VI) wherein R 1 , R' 0 , R", R", R' 4 and R 1 are as defined in the following table: Compound R R 10 R R 13 R R 15 1 Me 4-OH H 4-F H H 2 Me 4-OH H 3-F 4-OH H 3 Me 2-F 4-OH 3-F 4-OH H 4 Me 3-OH H 3-F 4-OH H 5 Me 3-Me 4-OMe 3-F 4-OH H 6 Me 3-F 4-OH 3-F 4-OH H 7 Me 3-F 4-OH 4-OH H H 8 Me 4-OH H 3-Cl 4-OMe H 9 Me 4-OH H 3-OH 4-F H io Me 4-OH H 2-F 3-F 4-OH 11 Me 3-Me -4-OMe 4-OH H H 12 Me 4-OH H 3-OMe 4-OMe H 13 Pr 4-OH H 3-F 4-OH H 14 Pr 3-F 4-OH 3-F 4-OH H 15 Pr 4-NHEt H 3-F 4-OH H (b) hydrogenating a compound of formula (VI) to produce a compound of claim 60
19. 20. The method of claim 19, wherein step (a) is carried out by reacting a compound of formula (V) with a borane reagent.
20. 21. The method of claim 19 or claim 20, wherein step (b) is carried out by reacting a compound of formula (VI) with a heterogenous metal catalyst.