WO2015166043A1 - Compounds - Google Patents

Abstract

The invention relates to a compound of formula (I) for use in the prevention and/or treatment of cancer, wherein rings A and B are independently an optionally substituted aryl or an optionally substituted heteroaryl. The compounds are particularly provided for the prevention and/or treatment of hormone- induced cancers, such as breast, ovarian, uterine, endometrial and prostate cancer.

Description

Compounds

The present invention relates to compounds for use in the prevention and/or treatment of cancer. Cytochrome P450 (CYP) enzymes belong to a large family of detoxification enzymes that are present in different organs of the human body. The CYP isoform CYP1 B1 has been found to be expressed in all cancers, regardless of oncogenic origin, while being absent from healthy tissue. It is understood that CYP1 B1 may have a dominant role in the genesis of breast cancer.

Activated estrogen receptor (ER) is responsible for breast cell division (proliferation). ER is activated by its ligand, estradiol (a steroidal hormone). In pre-menopausal women, estradiol is primarily produced in the ovaries by the pituitary via a cascade of biochemical reactions initiated by the LH-RH receptor whereas, in post-menopausal women, estradiol is synthesised solely in the adrenal glands from testosterone. Hyper-activated ER, through constant overproduction of estradiol, is the cause of ER-positive breast cancers and 80% of breast cancers are ER-positive. It is thought that preventing the synthesis of the ER ligand, estradiol, could lead to an ideal treatment of ER-positive breast cancers. However, estradiol plays an essential role in most healthy tissues.

Estradiol is synthesised through the aromatisation of the 'A' ring of testosterone with the help of cytochrome P450 19 (CYP19) enzyme which is also known as aromatase. The CYP19 enzyme (i.e. aromatase) catalyses the conversion of testosterone to estradiol. Inhibitors of aromatase have been hugely successful for the treatment of estrogen receptor (ER)-positive post-menopausal breast cancers. Unfortunately the use of aromatase inhibitors is restricted to the treatment of post-menopausal women suffering from breast cancer. The inhibitors avert the formation of estradiol from testosterone thereby inhibiting cell division by preventing activation of ER. However, aromatase inhibitors have profound adverse effects on pre-menopausal ER-positive breast cancer patients, as a result of a biochemical feedback loop that affects the pituitary.

In particular, the use of aromatase inhibitors in premenopausal women, results in a decrease in estrogen, which activates the hypothalamus and pituitaries to increase gonadotropin secretion, which in turn stimulates the ovary to increase testosterone production. The heightened gonadotropin levels also up-regulate the aromatase promoter, increasing aromatase production in a setting where the substrate, testosterone, levels have increased. In premenopausal women the effect of an aromatase inhibitor results in the increase of total estrogen rather than the intended decrease in its levels. Hence, aromatase inhibitors can only be used for the treatment of post-menopausal women.

Regio-specific conversion of the overproduced estradiol to its carcinogenic (cancer- causing) form by CYP1 B1 , present in high amounts only in pre-cancerous and cancerous cells, is responsible for the onset of breast cancer.

The present invention provides compounds for selectively inhibiting CYP1 B1 for the prevention and treatment of ER-positive breast cancers in both pre- and post-menopausal women. The inventors have recognised that preventing the 'conversion' of overproduced estradiol to the cancer-causing 4-hydroxy estradiol is an attractive way of preventing the onset and progression of majority of breast cancers. Hence, the identification of CYP1 B1 - specific inhibitors provides a novel way of treating the majority (i.e. >80%) of breast cancers. Moreover, proteomic analysis has revealed that CYP1 B1 is over-produced in tumours which have become resistant to chemotherapy with cisplatin. It has been suggested that CYP1 B1 inhibitors would be able to re-sensitise cancer cells to currently available cancer therapies that involve platinum compounds, taxanes and also nucleoside analogues. The present invention therefore provides CYP1 B1 inhibitors for use in the treatment of drug-resistant cancer cells, as alternatives to currently used aromatase inhibitors and in gynaecological cancers and prostate cancer.

The present invention therefore provides inhibitors which inhibit CYP1 B1 and more preferably provide some selectivity towards the inhibition of CYP1 B1.

The first aspect of the invention therefore provides a compound of formula (I)

for use in the prevention and/or treatment of cancer, wherein rings A and B are independently an optionally substituted aryl or an optionally substituted heteroaryl.

For the purposes of this invention, the optionally substituted aryl or optionally substituted heteroaryl can be substituted with one or more substituents selected from the group consisting of aryl, aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl. Preferred substituents include aryl, aliphatic, halogen, hydroxy, alkoxy, thioalkyl, alkylamino or cyano, preferably aryl, hydroxyl, halogen or alkoxy.

The invention particularly relates to compounds of formula II,

wherein ring A is an optionally substituted aryl and ring B is an optionally substituted heteroaryl.

For such compounds of formula II, an aryl group can comprise an optionally substituted phenyl or a polycyclic aryl group such as a naphthalene or a phenanthroline group and a heteroaryl group can comprise an optionally substituted monocyclic heteroaryl having 5 or 6 ring atoms, at least one ring atom being a heteroatom selected from O, N or S, preferably N. More preferably, one ring atom of the heteroaryl is a heteroatom, selected from O, N or S, preferably N. In particular, the heteroaryl group is pyridine. The optionally substituted aryl or optionally substituted heteroaryl can be substituted with one or more substituents selected from the group consisting of aliphatic, alkoxy, thioalkyi, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl.

Preferred substituents include aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy.

In particular, Ring A is an aryl group optionally substituted with one or more subsitutuents selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy. Where the aryl group is substituted with one or more alkoxy group, the alkoxy group is particularly one or more Ci_-6 alkoxy group, more particularly one or more Ci_-4 alkoxy group, more particularly a methoxy, ethoxy, propoxy or butoxy group, more particularly a methoxy or ethoxy group, most particularly methoxy.

Ring A can be a phenyl group optionally substituted with one or more subsitutuents selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy. Where the phenyl group is substituted with one or more alkoxy group, the alkoxy group is particularly one or more Ci_-6 alkoxy group, more particularly one or more Ci_-4 alkoxy group, more particularly a methoxy, ethoxy, propoxy or butoxy group, more particularly a methoxy or ethoxy group, most particularly methoxy. The aryl group and more particularly the phenyl group is preferably substituted with one, two or three substituents.

The compound of formula (II) will therefore preferably have the structure;

wherein R¹, R², R³, R⁴ or R⁵ are independently hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydrogen, hydroxyl, halogen or alkoxy.

Preferably wherein one, two or three of R¹, R², R³, R⁴ or R⁵ are independently, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy.

Preferably the R⁵ position is unsubstituted (i.e. R⁵ is hydrogen)

Ring B is preferably a heteroaryl group optionally substituted with one or more subsitutuents selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy. Preferably Ring B is a pyridine group. The pyridine group is preferably attached to the ketone functionality at the 3 or 4 position. The pyridine group is optionally substituted with one or more of aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably alkoxy, such as methoxy or ethoxyl, more preferably methoxy. The compound of formula (II) will therefore preferably have the structure:

wherein R¹, R², R³, R⁴ or R⁵ are independently hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydrogen, hydroxyl, halogen or alkoxy.

Preferably wherein one, two or three of R¹, R², R³, R⁴ or R⁵ are independently, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy.

Preferably the R⁵ position is unsubstituted (i.e. R⁵ is hydrogen) In a preferred feature of the first aspect of the invention, the invention provides a compound of formula (IIC) for use in the treatment of cancer,

(IIC) wherein R¹ is hydrogen or alkoxy,

R² is hydrogen, alkoxy, halogen or hydroxyl,

R³ is hydrogen, alkoxy or halogen;

R⁴ is hydrogen or alkoxy; and

R⁵ is hydrogen

wherein R² and R³ are together -0-CH₂-0- or wherein R³ and R⁴ are -CH=CH-CH=CH- to form a napthyl group with the phenyl ring.

Where R¹, R², R³ or R⁴ are alkoxy, the alkoxy group is particularly one or more C_1-6 alkoxy group, more particularly one or more C_1-4 alkoxy group, more particularly a methoxy, ethoxy, propoxy or butoxy group, more particularly a methoxy or ethoxy group, most particularly methoxy.

In a preferred feature of the first aspect, there is provided a compound of formula (IIC) wherein R¹ is hydrogen or methoxy,

R² is hydrogen, methoxy, chloride, fluoride or hydroxyl,

R³ is hydrogen, methoxy, fluoride or chloride;

R⁴ is hydrogen or methoxy; and

R⁵ is hydrogen

wherein R² and R³ are together -0-CH₂-0- or wherein R³ and R⁴ are -CH=CH-CH=CH- to form a napthyl group with the phenyl ring. Most preferably, the groups R³ and R⁴ together form a napthyl with the phenyl ring.

Preferably the phenyl ring is mono, di or tri substituted. In particular, the ring is preferably mono-substituted with hydroxy or methoxy more preferably at R¹ or R². The ring is preferably di-substituted with hydroxyl, methoxy, chloride, or fluoride, preferably at R² and R³, more preferably R² and R³ are the same. The ring is preferably tri-substituted with methoxy, preferably at R¹, R² and R³. In particular, the compound is one or more selected from

The invention further provides a compound of formula (III)

wherein ring A is an optionally substituted aryl and ring B is an optionally substituted heteroaryl.

In particular for the compounds of formula (III), an aryl group can comprise an optionally substituted phenyl or a polycyclic aryl group such as a naphthalene or a phenanthroline group. A heteroaryl group can comprise an optionally substituted monocyclic heteroaryl having 5 or 6 ring atoms, at least one ring atom being a heteroatom selected from O, N or S, preferably N. More preferably, one ring atom of the heteroaryl is a heteroatom, selected from O, N or S, preferably N. In particular, the heteroaryl group is pyridine or imidazole.

The optionally substituted aryl or optionally substituted heteroaryl can be substituted with one or more subsitutuents selected from the group consisting of aryl, aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂ ^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl. Preferred substituents include aryl, aliphatic, halogen, hydroxy, alkoxy, thioalkyl, alkylamino or cyano, preferably aryl, hydroxyl, halogen or alkoxy.

In particular, Ring A is an aryl group optionally substituted with one or more subsitutuents selected from aryl, aliphatic, halogen, hydroxy, alkoxy, thioalkyl, alkylamino or cyano, preferably aryl, halogen or alkoxy. Where the aryl group is substituted with one or more alkoxy group, the alkyl group is particularly one or more Ci_-6 alkoxy group, more particularly one or more Ci_-4 alkoxy group, more particularly a methoxy, ethoxy, propoxy or butoxy group, more particularly a methoxy or ethoxy group, most particularly methoxy. Where the aryl group is substituted with an aryl group, the aryl group is preferably phenyl. Ring A can be a phenyl group optionally substituted with one or more subsitutuents selected from aryl, aliphatic, halogen, hydroxy, alkoxy, thioalkyl, alkylamino or cyano, preferably aryl, halogen or alkoxy. Where the phenyl group is substituted with one or more alkoxy group, the alkoxy group is particularly one or more Ci_-6 alkoxy group, more particularly one or more Ci_-4 alkoxy group, more particularly a methoxy, ethoxy, propoxy or butoxy group, more particularly a methoxy or ethoxy group, most particularly methoxy. Wherein the phenyl group is substituted with one or more aryl group, the aryl group is preferably phenyl.

The aryl group and more particularly the phenyl group is preferably substituted with one, two or three substituents, more preferably one or two substituents. The invention therefore relates to a compound of formula (III) wherein Ring A is an aryl ring optionally substituted with one or more of methoxy, chloride or phenyl and Ring B is a five or six membered heteroaryl group containing one or two nitrogen atoms, preferably pyridine, pyrrole, imidazole, pyridazine, pyrimidine or pyrazine, more preferably pyridine or imidazole.

More, particularly, the first aspect of the invention relates to a compound of formula (Ilia)

ia) wherein Ring A is an aryl ring optionally substituted with one or more of methoxy, chloride or phenyl

The aryl group of formula (III) or (Ilia) is preferably phenyl or a fused aryl group comprising two, three or four fused phenyl groups such as napthyl or phenanthroline.

Particularly the fused aryl group can be selected from one or more of

When the fused aryl group is napthyl, it is preferably substituted with methoxy. Where the aryl group is a phenyl group, it can have the structure

where R¹, R², R³, R⁴ and R⁵ are preferably hydrogen, methoxy, chloride or phenyl. The phenyl ring can be mono, di or tri substituted. Where the phenyl ring is mono-substituted, it is preferably substituted with methoxy, chloride or phenyl preferably at the R² or R³ position. When the phenyl ring is di-substituted, it is preferably substituted with methoxy, preferably at the R¹ and R³, R² and R³ or R² and R⁴ positions. When the phenyl ring is tri- substituted, it is preferably substituted with methoxy at the R¹, R² and R³ positions.

In particular, the compound is one or more selected from

For the purposes of the present invention and the compounds of formula (I), (II) and (III) as described herein, the term aryl includes for example optionally substituted unsaturated monocyclic, bicyclic or tricyclic rings of up to 14 carbon atoms, such as phenyl, naphthy and phenanthroline. Alternatively, the term aryl may include partially saturated bicyclic rings such as tetrahydro-naphthyl. Preferably, the aryl group is phenyl, naphthy or phenanthroline.

A heteroaryl group or moiety may be for example an optionally substituted 5- or 6- membered heterocyclic aromatic ring which may contain from 1 to 4 heteroatoms selected from O, N or S. The heterocyclic ring may optionally be fused to a phenyl ring. Examples of heteroaryl groups thus include furyl, thienyl, pyrrolyl, oxazolyl, oxazinyl, thiazolyl, imidazolyl, oxadiazolyl, thiadiazolyl, pyridyl, triazolyl, triazinyl, pyridazyl, pyrimidinyl, pyrazolyl, indolyl, indazolyl, isoxazolyl, benzofuranyl, benzothienyl, benzimidazolyl, benzoxazolyl, benzoxazinyl, quinoxalinyl, quinolinyl, quinazolinyl, cinnolinyl, benzothiazolyl and pyridopyrrolyl. The heteroaryl group or moiety may be fully or partially reduced. For the purposes of this invention the terms 'reduced' or 'reduction' relate to the addition of one or more electrons to an atom or the addition of hydrogen to a moiety. Examples of such reduced heteroaryl groups or moieties include any fully or partially saturated derivative of the aforementioned heteroaryl groups and include pyrrolidinyl, tetrahydrofuranyl, tetrahydrothienyl and piperidinyl groups.

For the compounds of formula (II), the heteroaryl group is preferably pyridyl, thienyl, furyl or pyrrolyl. For the compounds of formula (III), the heteroaryl group is preferably imidazoyl, pyridyl, thienyl, furyl or pyrrolyl.

The term "aliphatic" as used herein refers to a straight or branched chain hydrocarbon which is completely saturated or contains one or more units of unsaturation. Thus, aliphatic may be alkyl, alkenyl or alkynyl, preferably having 1 to 12 carbon atoms, up to 6 carbon atoms or up to 4 carbon atoms. For the purposes of this invention, alkyl relates to both straight chain and branched alkyl radicals of 1 to 12 carbon atoms, preferably 1 to 8 carbon atoms and most preferably 1 to 4 carbon atoms including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl, tert-butyl n-pentyl, n-hexyl, n-heptyl, n-octyl. The term alkyl therefore relates to radicals comprising 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms. The term alkyl also encompasses cycloalkyl radicals of 3 to 12 carbon atoms, preferably 4 to 8 carbon atoms, and most preferably 5 to 6 carbon atoms including but not limited to cyclopropyl, cyclobutyl, CH₂-cyclopropyl, CH₂-cyclobutyl, cyclopentyl or cyclohexyl. Cycloalkyl groups may be optionally substituted or fused to one or more carbocyclyl or heterocyclyl group. Haloalkyl relates to an alkyl radical preferably having 1 to 8 carbon atoms, preferably 1 to 4 carbon atoms substituted with one or more halide atoms for example CH₂CH₂Br, CF₃ or CCI₃.

The term "alkenyl" means a straight chain or branched alkylenyl radical of 2 to 12 carbon atoms, preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms, and containing one or more carbon-carbon double bonds and includes but is not limited to ethylene, n-propyl-1-ene, n-propyl-2-ene, isopropylene, etc.. The term "alkynyl" means a straight chain or branched alkynyl radical of 2 to 12 carbon atoms, preferably 2 to 6 carbon atoms and most preferably 2 to 4 carbon atoms, and containing one or more carbon-carbon triple bonds and includes but is not limited to ethynyl, 2-methylethynyl etc. The term alkenyl and alkynyl therefore encompass radicals comprising 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 or 12 carbon atoms.

The term "alkoxy" as used herein refers to an oxy group that is bonded to an alkyl group as defined herein. An alkoxy is preferably a "Ci_-8 alkoxy group", even more preferably a "Ci-6 alkoxy group" and more preferably a "Ci_-4 alkoxy group". The alkoxy group particularly includes 1 , 2, 3 or 4 carbon atoms. Particularly preferably alkoxy groups include methoxy and ethoxy.

Halogen means F, CI, Br or I, preferably F.

Preferred compounds of the invention listed above extend to the tautomers thereof, as well as (but not limited to) pharmaceutically acceptable salts, esters, amides, carbamates, carbonates, ureides or prodrugs thereof or a derivative optionally with one or more lipid groups (natural or synthetic) attached. The invention extends to prodrugs of the aforementioned compounds. A prodrug is any compound that may be converted under physiological conditions or by solvolysis to any of the compounds of the invention or to a pharmaceutically acceptable salt of the compounds of the invention. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound of the invention.

The compounds of the invention may contain one or more stereogenic (asymmetric) carbon atoms and may exist in racemic and optically active forms (enantiomers or diastereoisomers). The first aspect of the invention includes all such enantiomers or diastereoisomers and mixtures thereof, including racemic mixtures. Examples of pharmaceutically acceptable salts of the compounds of formulae (I), (II) and (III) include those derived from organic acids such as methanesulphonic acid, benzenesulphonic acid and p-toluenesulphonic acid, mineral acids such as hydrochloric and sulphuric acid and the like, giving methanesulphonate, benzenesulphonate, p- toluenesulphonate, hydrochloride and sulphate, and the like, respectively or those derived from bases such as organic and inorganic bases. Examples of suitable inorganic bases for the formation of salts of compounds for this invention include the hydroxides, carbonates, and bicarbonates of ammonia, lithium, sodium, calcium, potassium, aluminium, iron, magnesium, zinc and the like. Salts can also be formed with suitable organic bases. Such bases suitable for the formation of pharmaceutically acceptable base addition salts with compounds of the present invention include organic bases which are nontoxic and strong enough to form salts. Such organic bases are already well known in the art and may include amino acids such as arginine and lysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine, choline, mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and trimethylamine, guanidine; N- methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine; N- benzylphenethylamine; tris(hydroxymethyl) aminomethane; and the like.

Salts may be prepared in a conventional manner using methods well known in the art. Acid addition salts of said basic compounds may be prepared by dissolving the free base compounds according to the first or second aspects of the invention in aqueous or aqueous alcohol solution or other suitable solvents containing the required acid. Where a compound of formula (I), (II) or (III) contain an acidic function a base salt of said compound may be prepared by reacting said compound with a suitable base. The acid or base salt may separate directly or can be obtained by concentrating the solution eg. by evaporation. The compounds of this invention may also exist in solvated or hydrated forms. The compounds of the invention are provided for the prevention and/or treatment of cancer. As discussed above, conventional aromatase inhibitors cannot be used in premenopausal women as they result in an increase in total estrogen rather than the intended decrease. The provision of inhibitors which selectively inhibit CYP1 B1 allows the treatment of hormone-induced cancers (such as breast, ovarian, uterine, endometrial and prostate cancer). In particular, the claimed invention provides a compound for use in the prevention and/or treatment of hormone induced cancers in both pre- and postmenopausal women.

The compounds of formula (I), (II) and/or (III) as described above are therefore provided for the prevention and/or treatment of hormone-induced cancers, preferably breast, ovarian, uterine, endometrial and prostate cancer. The compounds of formula (I), (II) and/or (III) are further provided for the prevention and/or treatment of hormone-induced cancers, preferably breast, ovarian, uterine, endometrial and prostate cancer in pre- and/or post-menopausal women, preferably pre-menopausal women.

Throughout this text, the prevention and/or treatment of cancer means any effect which mitigates any damage, to any extent. The term "treatment" means any amelioration of disorder, disease, syndrome, condition, pain or a combination of two or more thereof. The term prevention means to prevent the condition from occurring, lessening the severity of the condition or to prevent from deteriorating or getting worse for example by halting the progress of the disease without necessary ameliorating the condition.

The present invention particularly relates to the treatment of cancer. A second aspect of the invention provides a composition comprising a compound, in particular a novel compound according to the first aspect of the invention, in combination with a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may comprise a pharmaceutically acceptable carrier and/or pharmaceutically acceptable diluent. Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).

A pharmaceutical composition may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

The compounds according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration and the compositions adapted accordingly. Such compositions may be prepared by any method known in the art of pharmacy, for example by admixing the active ingredient with a carrier(s) or excipient(s) under sterile conditions.

For oral administration, the compounds can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges. Pharmaceutical compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as powders or granules; as solutions, syrups or suspensions (in aqueous or non-aqueous liquids; or as edible foams or whips; or as emulsions). Suitable excipients for tablets or hard gelatine capsules include lactose, starch including maize starch or derivatives thereof, stearic acid or salts thereof, such as magnesium stearate, sucrose or microcrystalline cellulose. A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatin capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatin capsule. Suitable excipients for use with soft gelatine capsules include for example vegetable oils, waxes, fats, semi-solid, or liquid polyols etc. Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule.

A liquid formulation, such as a solution or a syrup will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or nonaqueous liquid carrier(s) for example water, ethanol, glycerine, sugars, polyethylene glycol or an oil. For the preparation of suspensions oils (e.g. vegetable oils) may be used to provide oil-in-water or water in oil suspensions. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent.

Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or nonaqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump- atomiser.

Pharmaceutical compositions adapted for nasal administration wherein the carrier is a solid include a coarse powder having a particle size for example in the range 20 to 500 microns which is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable compositions wherein the carrier is a liquid, for administration as a nasal spray or as nasal drops, include aqueous or oil solutions of the active ingredient.

Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. For application to the eye or other external tissues, for example mouth and skin, the compositions are preferably applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil- in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles and mouth washes. Pharmaceutical compositions adapted for rectal administration may be presented as suppositories or enemas. Pharmaceutical compositions adapted for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations. Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin. Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.

Pharmaceutical compositions adapted for parenteral administration include aqueous and non-aqueous sterile injection solution which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. Excipients which may be used for injectable solutions include water, alcohols, polyols, glycerine and vegetable oils, for example. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

The pharmaceutical compositions may contain preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colourants, odourants, salts, buffers, coating agents or antioxidants. They may also contain an adjuvant and/or therapeutically active agents in addition to the substance of the present invention.

Dosages of the substance of the present invention can vary between wide limits, depending upon a variety of factors including the disease or disorder to be treated, the age, weight and condition of the individual to be treated, the route of administration etc. and a physician will ultimately determine appropriate dosages to be used. Typically, however, the dosage adopted for each route of administration when a compound of the invention is administered to adult humans is 0.001 to 500 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily by bolus infusion, infusion over several hours and/or repeated administration. The compositions may be administered in conjunction with one or more other therapeutically active agents, especially those effective for treating cancers (i.e. a chemotherapeutic agent). Another chemotherapeutic agent may be, for example, mitoxantrone, Vinca alkaloids, such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents such as chlorambucil and melphalan, taxanes such as paclitaxel, anti-folates such as methotrexate and tomudex, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN-38 and DNA methylation inhibitors. The other active compound(s) may be incorporated in the same composition as the compounds of the present invention or they may be administered alongside the compounds of the present invention, e.g. simultaneously or sequentially. Thus, the invention provides a kit of parts comprising a compound of the invention and another chemotherapeutic agent, optionally with instructions for use.

Alternatively, the compound of the first aspect of the invention may be administered by their addition to a food or drink. In an alternative feature of the second aspect of the invention, the compounds of the first aspect of the invention are formulated into a powder or liquid for addition to food or drink and administration by these means. In this feature of the second aspect, the compounds of the first or second aspects will be formulated with an excipient or diluent but such excipient or diluent does not need to be pharmaceutically acceptable but instead should be acceptable for consumption. A third aspect of the invention provides a process for the manufacture of a composition according to the second aspect of the invention. The manufacture can be carried out by standard techniques well known in the art and involves combining a compound according to the second aspect of the invention and a pharmaceutically acceptable carrier or diluent. The composition may be in any form including a tablet, a liquid, a capsule, and a powder or in the form of a food product, e.g. a functional food. In the latter case the food product itself may act as the pharmaceutically acceptable carrier.

The fourth aspect of the invention provides a method of preventing and/or treating cancer comprising administering a compound of the first aspect of the invention to a patient in need thereof.

The method is particularly provided for the prevention and/or treatment of hormone- induced cancers, preferably breast, ovarian, uterine, endometrial and prostate cancer. The compounds of formula (I), (II) and/or (III) are further provided for the prevention and/or treatment of hormone-induced cancers, preferably breast, ovarian, uterine, endometrial and prostate cancer in pre- and/or post-menopausal women, preferably premenopausal women. As discussed above, the compounds of the present invention inhibit the conversion of pre- carcinogens into carcinogenic compounds thereby reducing or removing the risk of cancer. The patient in need thereof does not therefore need to be suffering from cancer but can instead wish to reduce his or her risk of cancer. Thus the fourth aspect of the invention provides a method of reducing the risk of developing cancer, comprising administering a compound of the first aspect of the invention. A person wishing to reduce this or her risk of cancer may be a person who is genetically predisposed to cancer or who is at risk of cancer due to environmental factors (i.e. smoking, pollution, exposure to toxins etc.). The compound of the first aspect of the invention can be provided in combination with one or more other therapeutic agents, especially those effective for treating cancers (i.e. a chemotherapeutic agent) as described in the second aspect of the invention. The fifth aspect of the invention relates to the use of the compounds of the first and/or second aspect of the invention in the manufacture of a medicament for the prevention and/or treatment of cancer. As discussed above, the compounds of the present invention inhibit the conversion of pre- carcinogens into carcinogenic compounds thereby reducing or removing the risk of cancer. The medicament can therefore be provided to patient who is not suffering from cancer but instead wishes to reduce his or her risk of cancer.

The sixth aspect of the invention relates to a composition comprising a compound of formula (I), (II) or (III) as defined in the first aspect of the invention and a drug for treating cancer.

The drug for treating cancer can be for example, mitoxantrone, Vinca alkaloids, such as vincristine and vinblastine, anthracycline antibiotics such as daunorubicin and doxorubicin, alkylating agents such as chlorambucil and melphalan, taxanes such as paclitaxel, anti- folates such as methotrexate and tomudex, epipodophyllotoxins such as etoposide, camptothecins such as irinotecan and its active metabolite SN-38 and DNA methylation inhibitors. Preferably the drug for treating cancer is one or more of a platinum compound, such as cisplatin, a taxane or a nucleoside analogue.

The compound of formula (I), (II) or (III) acts to re-sensitise cancer cells which are resistant to currently available cancer therapies. The seventh aspect of the invention therefore relates to a composition comprising a compound of formula (I), (II) or (III) as defined in the first aspect of the invention and a drug for treating cancer for use in treating cancer, wherein the cancer is resistant to the cancer treating drug.

For the purposes of this invention, the term "resistant" indicates that the cancer therapy either has a decreased effect or no effect on the cancer cells. All preferred features of the first to seventh aspects of the invention relate to all other aspects of the invention mutandis mutandi. In particular, cancer according to the fifth and sixth aspects of the invention is as defined in the first aspect of the invention. Brief Description of the Figures

Figure 1 illustrates the plasmid map of pcDNA3.1/n_CYP1 B1 ;

Figure 2 illustrates the confirmation of the presence of CYP1 B1 protein in HEK293 and CHO-K1 cells transferred with pcDNA3.1/n_CYP1 B1 by Western blotting; and

Figure 3 illustrates confirmation of the presence of CYP1 B1 protein in A2780 and A2780 cis cells by western blotting. 1.74μg of protein for A2780 and O^g of protein for A2780 cis cells were fractionated by 10% SDS-PAGE followed by immunoblotting.

Examples

General method of chalcone synthesis

There is set out below, a general method of chalcone synthesis

Chalcones were synthesised using the following solvent free method: The benzaldehyde (10.1 mmol) and 4-acetylpyridine (1.12ml, 10.1 mmol) were mixed for 5 minutes using a mortar and pestle. Powdered sodium hydroxide (0.81 g, 20.2 mmol) was added and the resulting orange mixture ground until no further colour change was observed (approx. 25 minutes). The resulting mixture was quenched with water (50ml) and extracted with ethyl acetate (3x30ml). The combined organic layers were washed with saturated brine, dried with anhydrous magnesium sulphate and concentrated in vacuo. The crude product was purified by column chromatography using hexane (15%): ethyl acetate (80%): triethylamine (5%) as eluent.

Example 1. Determination of IC50 values

This method is used to measure the IC50 values (the concentration at which 50 % of the enzyme activity is inhibited) of the compounds. IC50 values, which effectively reflect the inhibitory potential of a compound, also hint at the possible effectiveness of a compound in a biological process.

An IC50 assay includes microsomes which either contain cytochrome P450 enzymes, a chosen chemical compound in six serial dilutions, DMSO, 96-well flat-bottomed microtitre plate, substrates such as ER or CEC or EOMCC or DBF (which form fluorescent compounds upon CYP metabolism) and a fluorescent plate reader which ultimately determines IC50 values via endpoint fluorescence assays. CYP1 B1 End Point Assay

Regenerating System consists of:

5 μΙ Solution A (183 mg of NADP⁺ + 183 mg of glucose-6-phosphate + 654 μΙ of 1.0 M magnesium chloride solution + 9.15 ml of sterile ultra-pure water) + 1 μΙ Solution B (250 Units of glucose-6-phosphate dehydrogenase + 6.25 ml of 5 mM sodium citrate, mixed in a tube and made up to 10 ml with sterile ultra-pure water) + 39 μΙ 0.2 M Kpi (0.6 ml of 1.0M K₂HP0₄ + 9.4 ml of 1.0M KH₂P0₄ were mixed and made up to 50 ml with sterile ultra-pure water) + 5 μΙ potential inhibitory compound.

Enzyme System consists of:

0.5 μΙ CYP1 B1 (0.5 pmoles; CYP Design Ltd) + 1 .7 μΙ control protein (denatured proteins from yeast cells that do not contain recombinant CYP450 proteins) + 5 μΙ 0.1 mM 7-ER (7- ethoxyresorufin substrate) + 42.8 μΙ 0.1 M Kpi (0.3 ml of 1.0 M K₂HP0₄ + 4.7 ml of 1.0 M KH2PO4 were mixed and made up to 50 ml with sterile ultra-pure water). The Assay is performed using (a) Sensitivity (Gain): 65/70/75 of the Biotek Synergy plate reader (this would differ from one instrument to the other) and (b) Filters: 530/590 nm that monitors fluorescence excitation/ emission.

Procedure for IC₅o determination

The computer was switched on and the KC4 software (on the BioTek plate reader) was opened to select the assay parameters and plate layout. The plate reader machine was warmed at 37°C. Compounds were serially diluted to six different concentrations with 10% DMSO in a Sero-Wel white microplate. Serial dilutions were made with a dilution factor of 1 :20. 45 μΙ of regenerating system was prepared and pre-warmed at 37°C. Table 1 : The constitution of the regenerating system used per reaction in each single well for different CYPs was as follows.

Meanwhile, 50 μΙ of enzyme substrate mix reaction was prepared and kept for incubation at 37°C for 10 minutes (Table 2).

Table 2: Enzyme-Substrate mixtures per reaction in each well were as follows.

5 μΙ 0.1 mM

CYP1 B1 0.5 μΙ 1 .7 μΙ 42.8 μΙ 0.1 Μ - E.R

5 μΙ 320 μΜ

CYP1A2 1 μΙ 1 .6 μΙ 42.4 μΙ 0.1 Μ - CEC

0.5 μΙ 2 mM

CYP2D6 2.5 μΙ 0.4 μΙ 25 μΙ 0.2 Μ 21 .6 μΙ

EOMCC

CYP3A4 1.1 μΙ 0.102 μΙ 0.1 μΙ 2 mM 25 μΙ 0.2 Μ 23.96 μΙ

In wells of a 96-well flat-bottomed microplate, 45 μΙ of regenerating system, 5 μΙ serial dilutions of inhibitor were added from the dilution plate and 50 μΙ of enzyme/substrate was added except in control well (positive control); for this well, instead of inhibitor 5 μΙ of 10% DMSO was added. In the background well (negative control), only 45 μΙ regenerating system and 5 μΙ 10% DMSO were added but no enzyme and then microplate was vortexed for few seconds. The microplate was incubated for 10 minutes. After 10 minutes, 75 μΙ of Tris-acetonitrile was added to all wells using an 8-channel multi-pipette to stop the reaction; after that 50 μΙ of enzyme/substrate reaction was added into the background well. The plate was left to shake for 10 seconds and an endpoint assay was run using appropriate settings.

Calculation of IC₅o values

• Step 1 : Calculated percentage inhibition of the samples

· Step 2: Subtracted the value below 50% by 50 = A

• Step 3: Chose the value above 50% and below 50% and subtract the values = B

• Step 4: Divided A by B = C

• Step 5: Multiplied C by ( high concentration obtained - minus lower concentration obtained) = D

· Step 6: Added lower concentration than D which probably will lead to IC50 value • Step 7: IC50 = (50- low percentage below 50%) x (higher concentration - lower concentration) + lower concentration.

Example 2. The IC50s of potent inhibitors of CYP1 B1 based on their percentage inhibition

The IC50s were determined using the CYP1 B1 endpoint protocol. The IC50 graphs were produced using GraphPad Prism 6 and the structures of compounds were drawn using Symyx Draw. The compounds are grouped together on the basis of their structural similarities. In the graphs shown in Table 3, effects of an inhibitor on EROD activity catalysed by CYP1 B1 are shown. All assays included the substrate 7-ER in the presence of indicated concentrations of the inhibitor. Each point represents the mean of triplicate readings; bars denote ± SD. The X-axis represents the logarithmic values of the concentration of inhibitor in μΜ.

Table 3: Structures and IC50 graphs of the compounds that inhibit in the CYP1 B1 assay.

Heterocyclic chalcones with 3-N-Pyrido "A" ring

B1

- Log values - Log values - Log values B1

- Log values

B1

- Log values - Log values B1

- Log values - Log values

Heterocyclic Chalcones with Pyrido "B" ring and bi-aromatic "A" ring

B1

- Log values B1

Log values B1

- Log values B1

- Log values

Heterocylic Chalcones with Pyrido "B" ring and poly-aromatic "A" ring B1

- Log values B1

- Log values

Example 3. Cell-based enzyme inhibition assays

The assays provide a rapid and inexpensive method of determining the inhibitory potential of compounds. The assays could also be used to determine the expression levels of a particular CYP from different clones. The cells can be grown and expressed at various time points and the metabolism of a fluorescence substrate can be analysed to determine the relative amounts of a CYP that is produced from different clones.

The cell-based enzyme inhibition assays were carried out to find if the earlier results obtained from the in vitro enzyme assays (using isolated microsomes) have any bearing in the cellular context. This can be achieved by comparing results from the in vitro assays with those obtained from cellular assays. As observed with microsomes, P450 activity is inhibited by certain compounds. However, it is important to consider if live cells expressing CYP1A1 , CYP1 B1 and CYP1A2 enzymes have the potential to take up the compounds of interest through the yeast cell wall.

Recombinant yeast cells that harbour the CYP1A1, CYP1B1 and CYP1A2 genes and are activated by a modified human P450 reductase, AhRDM, were grown. Assays were carried out using selected compounds which had already shown specificity towards microsomal CYP1A1 , CYP1 B1 and CYP1A2 enzymes during in vitro IC₅₀ determinations of these compounds.

The live cell procedures include the use of 96-well flat-bottomed microplates, the substrates and a multi-mode filter plate reader to obtain fluorescence outputs that help in determining IC50 values. Procedure for live cell assays

From frozen glycerol stocks yeast strains were streaked out for growth on SD-minimal medium agar plates that contained the required nutrients and 2% glucose. The plates were then incubated at 30°C for 3 days. A loop-full of cells, from one of the many colonies that grew on the SD-minimal medium agar plate, were taken and were inoculated in 10 ml of autoclaved minimal medium broth that contained 0.02% casamino acids (SW6 broth) in a sterile conical flask. The broth was incubated in a shaking incubator at 30°C at 220 rpm for 16 hours. The culture was then diluted 1 :10 and optical density was measured at 600 nm. Once the optical density of SW6 broth culture reached OD₆₀₀ between 5 and 6, 10 ml of SW6 pre-culture was transferred to a sterile 10 ml tube and centrifuged at 3000 g for 10 minutes, the pellet obtained was transferred to a sterile conical flask containing 10 ml of SE medium which contained yeast nitrogen base, 2% ethanol, 12.5 ml/litre L-adenine, 8.3 ml/litre of L-histidine, L-leucine, and L-tryptophan. 10 ml of cell culture was incubated at 30°C at 220 rpm for 4 hours. After 4 hours, approximately 0.4 X 10⁷ cells per ml were aliquoted into sterile Eppendorf tubes and centrifuged at 13,000 rpm. The supernatant was poured off and cell pellet was washed with TE buffer 3 times. Finally, the cell pellet was re-suspended in 450 μΙ of TE buffer (0.5M Tris-HCI, pH7.4 and 0.1 M EDTA). Meanwhile, serial dilutions of selected compounds were made to yield different concentrations of compound. In each well of black microplate, 50 μΙ of cell suspension (containing a specific CYP), 5 μΙ of potential inhibitor (selected chemical compounds) and 50 μΙ of substrate mixture (containing CYP-specific substrates) were added. In the control wells, 45μΙ of cell suspension (contain a specific CYP) was mixed with 5 μΙ 10% DMSO and 50μΙ of CYP-specific substrate. After preparation of all wells, the microplate was vortexed for 10 seconds so that contents were mixed well in each well and kinetic assay was carried out for 30 minutes at 30°C using appropriate settings (see Table 4).

Table 4 Outline of kinetic assay parameters used for analysing cytochrome P450 enzymes using live cells and the Bio-Tek Synergy HT fluorescent plate reader.

Bandwidth of filter Final

Dilution Substrate

Enzyme Substrate Product Excitation Emission Sensitivity of

Cone, per

Substrate reaction

7-Ethoxy

CYP1A1 Resorufin 530 nm 590 nm 72 3 μΜ TE buffer resorufin

7-Ethoxy

CYP1 B1 Resorufin 530 nm 590 nm 72 3 μΜ TE buffer resorufin

CYP1A2 CEC CHC 400 nm 460 nm 72 16 μΜ TE buffer

Example s IC50 values in CYP1 B1 , CYP1A1 , CYP1A2, CYP2D6 and

CYP3A4 enzyme assays (using CYP1A1 , CYP1 B1 and CYP1A2 yeast microsomes obtained from CYP Design Ltd

5 The results below show that besides screening potential inhibitors of a CYP isozyme in microsomal assays, compounds could also be screened conveniently in live cells that express a CYP isozyme.

Table 5. Comparative data for a set of compounds.

DMU

29 nM — 1.5 μΜ — 1.5 μΜ — — 769

2105 analogue

DMU

32 nM — 10.5 μΜ — 752 nM 1.28 μΜ 1 μΜ 789

2105 analogue

DMU 200 nM -

13 nM — 4.7 μΜ 220 nM — — 790 800 nM

DMU

1 12 nM — 2.6 μΜ — 1.7 μΜ — — 2103

DMU

19 nM 40 nM 1.4 μΜ — 258 nM 53 μΜ 1 μΜ 774

DMU

349 nM — 288 nM — 515 nM — — 21 14

DMU

31 nM — 297 nM 200 nM 168 nM — — 21 17

DMU

96 nM — 2 μΜ — 624 nM — — 778

DMU

120 nM — 1 1.76 μΜ — 860 nM — — 2101

DMU

4 nM — 742 nM 800 nM 1.2 μΜ 18 μΜ 10 μΜ 2105

DMU

77 nM — 31.8 μΜ — 4.7 μΜ — — 21 18

IC50 IC50 IC50

IC50

CYP1 B1 CYP1 B1 CYP1A1 IC50 IC50 IC50

Name Structure CYP1A1

(a) in- (b) live- (b) live- CYP1A2 CYP3A4 CYP2D6

(a) in-vitro

vitro cell cell

DMU

189 nM — 17 μΜ — 2 3 μΜ — — 2134

DMU

187 nM — 907 nM — 300 nM — — 780

Heterocyclic

(Pyrido-"B" Ring;

poly-aromatic "A"

ring) Chalcones

DMU

48 nM — 122 nM — 1 μΜ — — 2136

DMU

133 nM 58 nM 669 nM — — 2137

DMU

162 nM 2.2 μΜ 2.1 μΜ — — 2140

Example 5. Re-sensitization of recombinant HEK-293 cells carrying the

CYP1 B1 gene to cisplatin and paclitaxel

CYP1 B1 is expressed in high amounts in tissues which overproduce oestrogen, tissues 5 like the breast, uterus and ovaries. Overproduction of oestrogen causes the perpetual activation of the oestrogen receptor ultimately leading to tumour formation.

Experimental studies have also suggested that CYP1 B1 may offer a mechanism of anticancer drug resistance. Hence inhibition of CYP1 B1 by CYP1 B1 -specific inhibitors may offer a novel mechanism for overcoming drug resistance in some form of cancers.

10 A cell line which overproduces CYP1 B1 was created to confirm that CYP1 B1 overproduction indeed induces resistance to cisplatin and paclitaxel, two widely used anticancer agents. The CYP1 B1 overproducing cell line was used to explore if a potent CYP1 B1 specific inhibitor would be able to overcome cisplatin resistance.

15 Transfection

The plasmid pcDNA3.1/h_CYP1 B1 (Figure 1 ) was used for the transfection of human embryonic kidney HEK-293 cells. The plasmid pcDNA3.1/h_CYP1 B1 was introduced into HEK-293 and CHO cells (1 * 10⁶ cells) via an electroporation device (Nucleofector I, Amaxa GmbH, Cologne, Germany). The Nucleofector I is especially designed to facilitate high efficiency transfections. A specific Nucleofector solution kit that has been developed by Amaxa for HEK-293 and CHO cells was used for transfections. After transfection, cells were expanded in T75 flasks in the presence of 1000 μg/μl of G418 antibiotic. All cell lines, before and after transfection, were maintained in the required medium. After transfection the cell lines were grown for several days and then cell lysates were made. Protein concentrations were determined by Bradford assays and this information was used to perform western blotting so as to confirm the presence of the CYP1 B1 protein. Western blotting

For Western blotting, cell lysates (12 μg/lane for HEK293 cells and 3 g/lane for CHO-K1 cells) were separated on 10 % SDS-polyacrylamide gels. The proteins were electro- transferred to Immobilon-P-membranes (Millipore) by the semi-dry transfer method. The membranes were blocked with 5 % non-fat dry milk in PBS. The blots were probed first with primary antibody for CYP1 B1 (AbCam, Cat No Ab32649) and the secondary antibody (AbCam, Cat No Ab6721 ; goat polyclonal secondary antibody to rabbit IqG-HyL conjugated to HKP). Chemiluminescence was detected using the Gel Doc (Bio Rad) system using an ECL kit (Abeam, Cat. No. Ab65623). Western blotting confirmed the presence of the CYP1 B1 protein in HEK-293 and CHO-K1 cells transfected with pcDNA3.1/h_CYP1 B1 plasmid. Proteins from CYP1 B1 Saccharosomes (CYP Design Ltd) were used as positive control. Non-transfected cells were used as negative control. The results are shown in Figure 2. Based on the results of western blotting it was decided that HEK293 cells transfected with pcDNA3.1/h_CYP1 B1 would be used to carry out further studies.

A2780 and A2780cis are epithelial human ovarian cancer cell lines; A2780 is the parent cell line, whereas A2780cis is a cisplatin-resistant cell line. It was developed by chronic exposure of the parent cisplatin-sensitive A2780 cells to increasing concentrations of cisplatin. It has been reported that cisplatin resistant lines overproduce CYP1 B1. Figure 3 confirms that this is true. Table 6 depicts the EC50s obtained from MTT assays performed with different cisplatin concentrations (0.05-1 ΟΟμΜ; each concentration in triplicate) to determine the cytotoxicity of cisplatin. The results show that EC50 for cell survival in the presence of cisplatin for a normal or non-transfected HEK-293 cell line is 12μΜ, which is very close to the EC50 obtained in HEK293 cells transfected with the empty pcDNA3.1 plasmid (that contains no CYP1B1 gene). When the HEK293 cell line is transfected with pcDNA3.1/h_CYP1 B1 , the EC₅₀ of cisplatin increased to 61 μΜ (i.e. increase seen in the presence of CYP1 B1 protein). Hence, it can be said that the cytotoxicity of cisplatin decreased in the presence of CYP1 B1 indicating the cells confer resistance to cisplatin in the presence of CYP1 B1 .

Table. 6. EC50 data for HEK293 cells, HEK293 cells transfected with pcDNA3.1 and pcDNA3.1/h_CYP1 B1 , and treated with cisplatin. Cell growth was monitored via the MTT assay. EC50 is the concentration of cisplatin that gives half-maximal response to inhibition of cell growth.

Table 7 depicts that when CYP1 B1 -specific inhibitors (using IC50 concentrations obtained in in vitro enzyme assays) are co-administered with cisplatin in HEK-293 cells lines transfected with pcDNA3.1/h_CYP1 B1 , the EC50s are reversed completely suggesting that inhibition of CYP1 B1 effectively overcomes cisplatin resistance.

In the presence of DMU 2105 the EC₅₀ goes down to 1 μΜ from 61 μΜ (seen in the presence of CYP1 B1 ). In the presence of CYP1 B1 inhibitor DMU 2139 the EC50 is back to 8.3 μΜ from 61 μΜ (seen in the presence of CYP1 B1 ) which is close to 8.7 μΜ (seen with the cell line transfected with the control plasmid). a-NF (a-napthoflavone) which is a known inhibitor of CYP1 enzymes only reduced the toxicity to 40 μΜ from 61 μΜ (seen in the presence of CYP1 B1 ). This is much higher than 8.7 μΜ (seen with the cell line transfected with the control plasmid) indicating that a-NF is ineffective in overcoming resistance to cisplatin that is conferred by the presence of active CYP1 B1 enzyme.

Table. 7. EC50 data for inhibition studies performed on HEK-293 cells transfected with pcDNA3.1/h_CYP1 B1 using the CYP1 B1 -specific inhibitors DMU 2105, DMU 2139 and the known inhibitor a-naphthoflavone (a-NF).

The CYP1 B1 -specific inhibitors DMU 2105 and DMU2139 have also been used to treat A2780 and A2780cis (cisplatin resistant cells). The EC50 of both DMU 2105 and DMY2139 in A2780 cells is at least 100-fold higher than in A2780cis cells indicating that cisplatin resistant cells are extremely sensitive to the CYP1 B1 -specific inhibitors.

Similar results have been obtained with paclitaxel using HEK-293 pcDNA3.1/ and HEK- 293 pcDNA3.1/nh CYP1 B1 cells

HEK-293_pcDNA3.1/h_CYP1 B1 cells are resistant to paclitaxel; EC50 increases at least 10-fold from the parent line HEK-293_pcDNA3.1 .

Resistance is overcome in the presence of the inhibitors DMU 2105 and DMU 2139. Example 6. CYP1 B1 inhibitors prevent conversion of estradiol to 4- hydroxyestradiol

Yeast produced CYP1 B1 was 5μΜ concentration of estradiol and incubated for 45 minutes at 37°C. The formation of 4-hydroxyestradiol was monitored via LC-MS (Agilent) and 500 MHz NMR (Bruker).

Table. 8. CYP1 B1 inhibitors prevent formation of 4-hydroxyestradiol. The results below were obtained using LC-MS. Similar results can be obtained using NMR.

The results indicate that CYP1 B1 inhibitors have potential application in hormone- sensitive cancers.

Claims

for use in the prevention and/or treatment of cancer, wherein rings A and independently an optionally substituted aryl or an optionally substituted heteroaryl.

2. The compound according to claim 1 having the formula

wherein ring A is an optionally substituted aryl and ring B is an optionally substituted heteroaryl, wherein the optional substitutents are one or more selected from the group consisting of aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl. 3. The compound as claimed in claim 1 or 2 having the structure

wherein R¹, R², R³, R⁴ or R⁵ are independently hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydrogen, hydroxyl, halogen or alkoxy.

4. The compound as claimed in any one of claims 1 to 3 having the structure:

wherein R¹, R², R³, R⁴ or R⁵ are independently hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydrogen, hydroxyl, halogen or alkoxy.

5. The compound as claimed in claim 4

wherein R¹ is hydrogen or alkoxy,

R² is hydrogen, alkoxy, halogen or hydroxyl,

R³ is hydrogen, alkoxy or halogen;

R⁴ is hydrogen or alkoxy; and

R⁵ is hydrogen

wherein R² and R³ are together -0-CH₂-0- or wherein R³ and R⁴ are -CH=CH-CH=CH- to form a napthyl group with the phenyl ring.

6. A compound of claim 4 or claim 5 wherein R¹ is hydrogen or methoxy,

R² is hydrogen, methoxy, chloride, fluoride or hydroxyl,

R³ is hydrogen, methoxy, fluoride or chloride, R⁴ is hydrogen or methoxy; and

R⁵ is hydrogen

wherein R² and R³ are together -0-CH₂-0- or wherein R³ and R⁴ are -CH=CH-CH=CH- to form a napthyl group with the phenyl ring.

7. A compound as claimed in any of claims 4 to 6 wherein the phenyl ring is mono, di or tri substituted.

8. The compound of any one of claims 5, 6 or 7 wherein the groups R³ and R⁴ together form a napthyl group with the phenyl ring.

9. The compound of any preceding claim selected from

10. The compound according to claim 1 having the formula (III)

wherein ring A is an optionally substituted aryl and ring B is an optionally substituted heteroaryl; wherein the optional substituents are one or more of aryl, aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl. 1 1 . The compound as claimed in claim 10 wherein

wherein Ring A is an aryl ring optionally substituted with one or more of alkoxy, halogen or aryl and Ring B is a five or six membered heteroaryl group containing one or two nitrogen atoms selected from pyridine, pyrrole, imidazole, pyridazine, pyrimidine or pyrazine. The compound as claimed in claim 10 or claim 11 having the formula

wherein Ring A is an aryl ring optionally substituted with one or more of methoxy, chlorid or phenyl.

The compound as claimed in any one of claims 10 to 12 wherein the aryl group or a fused aryl group comprising two, three or four fused phenyl groups. 14. The compound as claimed in claim 13 wherein the fused aryl group can be selected from one or more of

15. The compound as claimed in any one of claims 10 to 14 wherein the aryl group has the structure

where R¹, R², R³, R⁴ and R³ are preferably hydrogen, methoxy, chloride or phenyl.

16. The compound as claimed in any one of claims 10 to 15 selected from

17. The compound of any preceding claim wherein the cancer is a hormone-induced cancer. 18. The compound of claim 17 wherein the cancer is one or more of breast, ovarian, uterine, endometrial and prostate cancer.

19. The compound as claimed in any preceding claim wherein the compound is provided for the prevention and/or treatment of cancer in pre-menopausal women.

20. A composition comprising a compound as claimed in any one of claims 1 to 16, in combination with a pharmaceutically acceptable carrier or diluent.

21 . A process for the manufacture of a composition as claimed in claim 20 comprising combining a compound as claimed in any one of claims 1 to 16, and a pharmaceutically acceptable carrier or diluent.

22. A method of preventing and/or treating cancer comprising administering a compound as claimed in any one of claims 1 to 16, to a patient in need thereof.

23. The method as claimed in claim 22 for the prevention and/or treatment of hormone-induced cancers.

24. The method as claimed in claim 23 wherein the cancer is one or more of breast, ovarian, uterine, endometrial and prostate cancer.

25. The method as claimed in any one of claims 22 to 24 for the prevention and/or treatment of cancer in pre-menopausal women. 26. The use of the compounds as claimed in any one of claims 1 to 16, in the manufacture of a medicament for the prevention and/or treatment of cancer.

27. A composition comprising a compound as claimed in any one of claims 1 to 16, and a drug for use in treating cancer.

28. A composition as claimed in claim 27 wherein the drug for treating cancer is one or more of a platinum compound, such as cisplatin, a taxane or a nucleoside analogue.

29. A composition for use in treating cancer comprising a compound of formula (I) as claimed in any one of claims 1 to 16, and a drug for treating cancer, wherein the cancer is resistant to the cancer treating drug.