WO2015166042A1 - Stilbene derivatives and their use for the treatment of cancer - Google Patents

Abstract

The present application relates to a compound of formula (I) wherein each of R¹, R², R³, R⁴ or R⁵ are independently selected from hydrogen, aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyi, alkylsulfonylamino NH₂, NO₂, SO₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl, for use in the prevention and/or treatment of cancer. 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

STILBENE DERIVATIVES AND THEIR USE FOR THE TREATMENT OF CANCER

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 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 selective inhibition of CYP1 B1 .

The first aspect of the invention relates to a compound of formula (I) for use in the prevention and/or treatment of cancer,

wherein each of R¹, R², R³, R⁴ or R⁵ are independently selected from hydrogen, aliphatic, alkoxy, thioalkyi, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyi, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl.

In particular, the compounds of formula (I) are provided as selective inhibitors of CYP1 B1.

In particular, each of R¹, R², R³, R⁴ or R⁵ are independently selected from hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydrogen, hydroxyl, halogen or alkoxy, more preferably hydrogen, hydroxyl or alkoxy.

In a particular, the phenyl ring can be mono-, di- or tri-substituted. Thus, one, two or three of R¹, R², R³, R⁴ or R⁵ can be substitued with one or more group independently selected from aliphatic, alkoxy, thioalkyi, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyi, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl.

Preferably one, two or three of R¹, R², R³, R⁴ or R⁵ can be substituted with one or more group independently selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy.

In particular, when R¹ and R² are independently selected from aliphatic, alkoxy, thioalkyi, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyi, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino, NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl, the remaining groups R³, R⁴ or R⁵ are hydrogen.

Preferably when R¹ and R² are independently selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy, the remaining groups R³, R⁴ or R⁵ are hydrogen.

Alternatively, when R¹, R³, R⁴ and R⁵ are hydrogen, R² is 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, preferably aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy.

When R¹, R² and R³ are 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, preferably aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy, R⁴ and R⁵ are hydrogen.

Alternatively, when R¹, R², R4 and R⁵ are hydrogen, R³ is 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, preferably, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy. When R², R³ and R⁴ are 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, preferably, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, more preferably hydroxyl, halogen or alkoxy, most preferably hydroxyl or alkoxy, R¹ and R⁵ are hydrogen.

In a particular feature of the first aspect of the invention, each of R¹, R², R³, R⁴ or R⁵ are independently hydrogen, hydroxyl or alkoxy, preferably methoxy.

To this end, preferred compounds of the first aspect of the invention, are those in which the phenyl ring can be mono-, di- or tri-substituted with hydroxyl or alkoxy, preferably alkoxy. In particular, when R³, R⁴ and R⁵ are hydrogen and R¹ is alkoxy, R² can be hydrogen or alkoxy. When R² is alkoxy, R⁴ and R⁵ are hydrogen, R¹ and R³ can be hydrogen or alkoxy. When R³ is alkoxy and R¹ and R⁵ are hydrogen, R² and R⁴ can be hydrogen or alkoxy.

Preferably when R³, R⁴ and R⁵ are hydrogen, R¹ and R² are alkoxy or hydroxyl, preferably alkoxy.

To this end, preferred compounds of the first aspect of the invention, are those in which the phenyl ring can be mono-, di- or tri-substituted with hydroxyl or methoxy, preferably methoxy. In particular, when R³, R⁴ and R⁵ are hydrogen and R¹ is methoxy, R² can be hydrogen or methoxy. When R² is methoxy, R⁴ and R⁵ are hydrogen, R¹ and R³ can be hydrogen or methoxy. When R³ is methoxy and R¹ and R⁵ are hydrogen, R² and R⁴ can be hydrogen or methoxy.

Preferably when R³, R⁴ and R⁵ are hydrogen, R¹ and R² are methoxy or hydroxyl, preferably methoxy.

As discussed above, the compounds of formula (I) are provided as selective inhibitors of CYP1 B1. For the purposes of the present invention, 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. 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 "C_1-8 alkoxy group", even more preferably a "Ci_-6 alkoxy group" and more preferably a "C_1-4 alkoxy group". The alkoxy group particularly includes 1 , 2, 3 or 4 carbon atoms. Particularly preferably alkoxy groups include methoxy, ethoxy, propyloxy or butyloxy. Halogen means F, CI, Br or I, preferably F.

In particular, the compound is one or more selected from

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) 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) 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 can not 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 compound of formula (I) as described above is 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) 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. The second aspect of the invention relates to novel compounds of formula (I)

wherein each of R¹, R², R³, R⁴ or R⁵ are independently hydrogen, or methoxy.

In particular, the compounds of formula (I) are provided as selective inhibitors of CYP1 B1. To this end, preferred compounds of the first aspect of the invention are those in which the phenyl ring can be mono-, di- or tri-substituted with hydroxyl or methoxy, preferably methoxy. In particular, when R³, R⁴ and R⁵ are hydrogen and R¹ is methoxy, R² can be hydrogen or methoxy. When R² is methoxy and R⁴ and R⁵ are hydrogen, R¹ and R³ can be hydrogen or methoxy. When R³ is methoxy and R¹ and R⁵ are hydrogen, R² and R⁴ can be hydrogen or methoxy.

Preferably when R³ and R⁴ and R⁵ are hydrogen, R¹ and R² are methoxy or hydroxyl, preferably methoxy.

In particular, the compound is one or more selected from

A third aspect of the invention provides a composition comprising a compound, in particular a novel compound according to the first or second aspects 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 non- aqueous 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 or second aspects of the invention may be administered by their addition to a food or drink. In an alternative feature of the third aspect of the invention, the compounds of the first or second aspects 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 third 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 fourth aspect of the invention provides a process for the manufacture of a composition according to the third 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 first or 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 fifth aspect of the invention provides a method of preventing and/or treating cancer comprising administering a compound of the first or second aspects 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) 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.

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 fifth aspect of the invention provides a method of reducing the risk of developing cancer, comprising administering a compound of the first or second aspects 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 third aspect of the invention. The sixth aspect of the invention relates to the use of the compounds of the first or second aspects 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 compounds of the first or second aspects of the invention can be provided in combination with one or more other therapeutic agents, especially those effective from treating cancers (i.e. a chemotherapeutic agent) as described in the third aspect of the invention. The seventh aspect of the invention relates to a composition comprising a compound of formula (I) as defined in the first and second aspects of the invention and a drug for treating cancer.

The drug for treating cancer is one or more selected from 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) acts to re-sensitise cancer cells which are resistant to currently available cancer therapies. The eighth aspect of the invention therefore relates to a composition comprising a compound of formula (I) as defined in the first and second aspects 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 fifth aspects of the invention relate to all other aspects of the invention mutandis mutandi. In particular, cancer according to the fourth and fifth aspects of the invention is as defined in the first aspect of the invention.

Description of figures. Figure 1 illustrates the plasmid map of pcDNA3.1/h_CYP1 B1.

Figure 2. illustrates confirmation of the presence of CYP1 B1 protein in HEK293 and CHO- K1 cells transfected with pcDNA3.1/h_CYP1 B1 by western blotting; and Figure 3. illustrates confirmation of the presence of CYP1 B1 protein in A2780 and A2780cis cells by western blotting. 1.74 μg of protein for A2780 and 0.3 μg of protein for A2780cis cells were fractionated by 10% SDS-PAGE followed by immunoblotting.

Examples

Methods for synthesis of stilbene derivatives Synthesized Stilbene derivatives

Lithium hydroxide (21 mg, 0.5mmol) was added to solution of 3-pyridylacetonitrile (0.54ml, 5.09mmol) in ethanol (5ml). The mixture was stirred at room temperature for 10 minutes before addition of the benzaldehyde (5.09mmol). The reaction was stirred overnight resulting in the formation of a coloured precipitate. The mixture was quenched with water (50 ml) and extracted with ethyl acetate (3 x 30ml). The combined organic extracts were washed with saturated brine, dried with anhydrous magnesium sulphate and the solvent removed in vacuo. The crude product was purified by column chromatography using hexane (15%): ethyl acetate (80%): triethylamine (5%) as eluent.

Chemical structure and physical properties of synthesised stilbenes

1. DMU-SB1

Brown fluffy powder (889mg, 65%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (83-85°C); m/z [FAB⁺] 297.1236 ([M+H]⁺, 100%); Y_max (KBr)/cm^"1 1574 (C=N), δ_Η (CDCIs): 3.90 (3H, s, OCH₃), 3.98 (3H, s, OCH₃), 4.10 (3H, s, OCH₃), 6.89 (2H, s, ArH), 7.98 (1 H, d, C=CH), 7.76 (1 H, d, PyH), 8.10 (1 H, t, PyH), 8.78 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5c (CDCI₃): 55.3, 56.6, 60.1 , 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 144.3, 149.7, 151.7, 156.4 HRMS found [M+H]⁺ 297.1236, Ci₇H₁₇0₃N₂ requires [M+H]⁺ 297.1239. 2. DMU-SB2

Light brown fluffy powder (890mg, 65%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (83-85°C); m/z [FAB⁺] 297.1236 ([M+H]⁺, 100%); Y_max (KBr)/cm^"1 1591 (C=N), δ_Η (CDCI₃): 3.90 (3H, s, OCH₃)_, 3.98 (3H, s, OCH₃), 4.10 (3H, s, OCH₃), 6.89 (2H, d, ArH), 7.98 (1 H, s, C=CH), 7.76 (1 H, dd, PyH), 8.10 (1 H, d, PyH), 8.78 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5_C (CDCI₃): 55.3, 56.6, 60.1 , 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 144.3, 149.7, 151.7, 156.4 HRMS found [M+H]⁺ 297.1235, C₁₇H₁₇0₃N₂ requires [M+H]⁺ 297.1234. 3. DMU-SB3

Cream colour powder (880mg, 73%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (87-89°C); m/z [FAB⁺] 237.1023 ([M+H]⁺, 100%); Y_max (KBr)/cm^"1 1598 (C=N), δ_Η (CDCIs): 3.98 (3H, s, OCH₃), 6.92 (1 H, d, ArH), 6.98 (1 H, d, ArH), 7.09 (2H, s, ArH), 7.98 (1 H, s, C=CH), 8.10 (1 H, d, PyH), 8.78 (1 H, dd, PyH), 8.86 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5c (CDCI₃): 55.3, 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 144.3, 149.7, 151.7, 156.4, 160.2 HRMS found [M+H]⁺ 237.1023, Ci₅H₁₃0₂N₂ requires [M+H]⁺ 237.1022.

4. DMU-SB4

Pale yellow colour powder (886mg, 73%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (85-87°C); m/z [FAB⁺] 237.1018 ([M+H]⁺, 100%); Y_max (KBr)/cm^"1 1604 (C=N), δ_Η (CDCI₃): 3.98 (3H, s, OCH₃), 6.92 (2H, d, ArH), 6.98 (2H, dd, ArH), 7.98 (1 H, d, C=CH), 8.10 (1 H, dd, PyH), 8.78 (1 H, d, PyH), 8.86 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5c (CDCI₃): 55.3, 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 130.2, 144.3, 149.7, 151.7, 156.4, 160.1 HRMS found [M+H]⁺ 237.1018, Ci₅H₁₃0iN₂ requires [M+H]⁺ 237.1022.

5. DMU-SB5

Cream colour powder (884mg, 65%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (1 10-1 15°C); m/z [FAB⁺] 267.1 131 ([M+H]⁺, 100%); Y_max (KBr)/cm^"1 1592 (C=N), δ_Η (CDCI₃): 3.90 (3H, s, OCH₃), 4.10 (3H, s, OCH₃), 6.97 (2H, dd, ArH), 7.34 (1 H, d, ArH), 7.98 (1 H, s, C=CH), 8.68 (1 H, dd, PyH), 8.78 (1 H, d, PyH), 8.99 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5_C (CDCI₃): 55.3, 56.6, 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 135.2, 144.3, 149.7, 151.7, 156.4 HRMS found [M+H]⁺ 267.1 131 , C₁₆H₁₅0₂N₂ requires [M+H]⁺ 267.1 128. 6. DMU-SB6

White fluppy powder (940mg, 69%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (107-1 10°C); m/z [FAB⁺] 267.1 125 ([M+H]⁺, 100%); y_max (KBr)/cm^"1 1567 (C=N), δ_Η (CDCIs): 3.90 (3H, s, OCH₃), 4.10 (3H, s, OCH₃), 7.10 (1 H, d, ArH), 7.34-7.44 (2H, M, ArH), 7.98 (1 H, s, C=CH), 8.68 (1 H, d, PyH), 8.78 (1 H, dd, PyH), 8.99 (1 H, d, PyH), 9.10 (1 H, d, PyH), 5_C (CDCI₃): 55.3, 56.6, 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 135.1 , 140.1 , 144.3, 149.7, 151 .7, 156.4 HRMS found [M+H]⁺ 267.1 125, Ci₆H₁₅0₂N2 requires [M+H]⁺ 267.1 128.

7. DMU-SB7

Pale yellow powder (920mg, 88%), TLC: R_f 0.65 (ethyl acetate/hexane/ triethylamine 6:3.5:0.5); m.p. (107-1 10°C); m/z [FAB⁺] 206.1 105 ([M+H]⁺, 100%); v_max (KBrycrrf¹ 1566 (C=N), δ_Η (CDCIs): 7.10 (1 H, d, ArH), 7.44 (2H, M, ArH), 7.76 (1 H, d, ArH), 7.98 (1 H, d, C=CH), 8.10 (1 H, d, PyH), 8.67 (1 H, d, PyH), 8.86 (1 H, dd, PyH), 8.98 (1 H, d, PyH), 5_C (CDCIs): 109.6, 1 10.3, 1 19.9, 122.3, 124.6, 130.2, 135.3, 144.3, 149.7, 151.7, 156.4 HRMS found [M+H]⁺ 206.1 1 10, Ci₃H₁₀N₂ requires [M+H]⁺ 206.1 1 10. 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, provides evidence of 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 KH₂P0₄ 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 IC₅₀ 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 as detailed in (Table 2). Meanwhile, 50 μΙ of enzyme substrate mix reaction was prepared and kept for incubation at 37°C for 10 minutes (Table 3). 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 IC₅₀ 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 1 , 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 axis represents the logarithmic values of the concentration of inhibitor in μΜ.

Table 1 : Structures and IC50 graphs of compounds that inhibit CYP1 B1 assay. Stilbenes

Synthesised Stilbenes

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 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 2).

Table 2: Outline of kinetic assay parameters used for analysing cytochrome P450 enzymes using live cells and the Bio-Tek Synergy HT fluorescent plate reader.

Example s IC50 values in CYP1 B1 , CYP1A1 , CYP1A2, CYP2D6 and CYP3A4 enzyme (in vitro) and live cell assays (using CYP1A1 , CYP1 B1 and CYP1A2 yeast microsomes and recombinant cells)

5 Table 3.

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 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 15 may offer a novel mechanism for overcoming drug resistance in some form of cancers.

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. 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 IgG-HyL conjugates 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 4 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 4. 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.

Conclusion

We expect that the compounds listed in this application would be re-sensitize HEK-293 cells transferred with pcDNA 3.1/h CYP1 B1 , which are normally resistant to the treatment of cisplatin and paclitaxel.

Claims

1. A compound of formula (I) for use in the prevention and/or treatment of cancer,

(!) wherein each of R¹, R², R³, R⁴ or R⁵ are independently selected from hydrogen, aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyi, alkylsulfonylamino NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl.

2. The compound as claimed in claim 1 wherein each of R¹, R², R³, R⁴ or R⁵ are independently selected from hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyl, alkylamino or cyano.

3. The compound as claimed in claim 1 or claim 2 wherein one, two or three of R¹, R², R³, R⁴ or R⁵ can be substitued with one or more group independently selected from aliphatic, alkoxy, thioalkyl, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyi, alkylsulfonylamino NH₂, N0₂, S0₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl and wherein in the remaining groups are hydrogen.

4. The compound as claimed in any one of claims 1 to 3 wherein each of R¹, R², R³ or R⁴ are independently hydrogen, hydroxyl or methoxy.

5. The compound as claimed in any preceding claim wherein the phenyl mono-, di- or tri-substituted with hydroxyl or methoxy.

6. The compound as claimed in any preceding claim wherein when R³, R⁴ and R⁵ are hydrogen, R¹ and R² are methoxy or hydroxyl.

7. The compound as claimed in any preceding claim selected from

8. The compound of any preceding claim wherein the cancer is a hormone-induced cancer.

9. The compound of claim 5 wherein the cancer is one or more of breast, ovarian, uterine, endometrial and prostate cancer.

10. 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.

A compound of formula (IA)

(I)

wherein each of R¹, R², R^3, R⁴ or R⁵ are independently hydrogen, or methoxy.

12. A compound as claimed in claim 1 1 wherein when R and R and R⁵ are hydrogen, R¹ and R² are methoxy or hydroxyl.

13. A compound as claimed in claim 1 1 or claim 12 selected from

14. A composition comprising a compound as claimed in any one of claims 1 to 13, in combination with a pharmaceutically acceptable carrier or diluent.

15. A process for the manufacture of a composition as claimed in claim 14 comprising combining a compound as claimed in any one of claims 1 to 13, and a pharmaceutically acceptable carrier or diluent.

16. A method of preventing and/or treating cancer comprising administering a compound as claimed in any one of claims 1 to 13, to a patient in need thereof.

17. The method as claimed in claim 16 for the prevention and/or treatment of hormone-induced cancers.

18. The method as claimed in claim 17 wherein the cancer is one or more of breast, ovarian, uterine, endometrial and prostate cancer.

19. The method as claimed in any one of claims 16 to 18 for the prevention and/or treatment of cancer in pre-menopausal women.

20. The use of the compounds as claimed in any one of claims 1 to 13, in the manufacture of a medicament for the prevention and/or treatment of cancer.

21 . A composition comprising a compound of formula (I) as claimed in any one of claims 1 to 13, and a drug for treating cancer.

22. A composition as claimed in claim 21 wherein the drug for treating cancer is one or more of a platinum compound, such as cisplatin, a taxane or a nucleoside analogue.

23. A composition for treating cancer comprising a compound of formula (I) as claimed in any one of claims 1 to 13, and a drug for treating cancer for use in treating cancer, wherein the cancer is resistant to the cancer treating drug.