WO2015166041A1 - Flavones as cyp1a1 inhibitors for the treatment of cancer - Google Patents

Abstract

The present application relates to a compound of formula (I) for use in the prevention and/or treatment of cancer, wherein R¹ to R⁹ are independently selected from hydrogen, aliphatic, alkoxy, thioalkyi, alkylamino, halogen, hydroxy, cyano, nitro, hydroxyalkyl, alkylcarbonyloxy, alkoxycarbonyl, alkylcarbonyl, haloalkyl, alkylsulfonylamino NH₂, NO₂, SO₂R^x, SOR^x and COOR^x, where R^x is hydrogen, aliphatic or aryl. The compounds are particularly provided for the prevention and/or treatment of cancer of the lung, colon, pancreas, liver and/or kidney.

Description

FLAVONES AS CYP1A1 INHIBITORS FOR THE 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 human cytochrome P450 1 (CYP1 ) family consists of three members; namely CYP1A1 , CYP1A2 and CYP1 B1. While the mRNAs of CYP1 A1 and CYP1 B1 genes are known to be expressed in extra-hepatic tissues such as lung, ovary, prostate, kidney and mammary gland and the mRNA of CYP1 A2 gene is found in the liver amongst other tissues, protein expression of these genes is rarely seen in human tissues, under normal circumstances. Expression of CYP1A1 , CYP1 B1 and CYP1A2 are however implicated in cancer. In particular, CYP1A1 expression is induced by poly-aromatic hydrocarbons (PAHs) which are found mainly in cigarette smoke, high-boiling fraction of crude oil, charred meat and vegetables. PAHs like 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene, 7,12-dimethylbenz(a) anthracene (DMBA) can bind to the aromatic hydrocarbon receptor as ligands. The activated AhR performs the role of a transcription factor that is responsible for the induction of CYP1A1 genes.

Induction leads to increased levels of CYP1A1 enzymes. The PAHs also act as ideal substrates for CYP1A1 enzymes which efficiently hydroxylate the PAHs leading to the formation of carcinogenic entities from pro-carcinogenic molecules. The PAHs appear to play a dominant role in the CYP1A1 mediated positive feedback mechanism that underlies the formation of carcinogenic substances capable of intercalating DNA. Hydroxylated PAHs are carcinogenic since they have great propensity to intercalate with double- stranded DNA and cause breaks in the double-stranded DNA. Hence all PAHs in general have tumour promoting properties.

Besides PAHs and its derivatives, CYP1A1 metabolises other xenobiotic compounds such as nitrogenous heterocyclics, caffeine, aromatic amines and an assortment of other compounds. Metabolism (biotransformation) of these compounds (i.e. pro-carcinogens) by CYP1A1 enzymes leads to the formation of carcinogenic substances.

Induction of CYP1A1 therefore results in the biotransformation (metabolism) of PAHs which are potential pro-carcinogens to carcinogenic substances that can eventually lead to cancer. CYP1A1 has been suggested to have a role in many cancers and appears to have a major role in the genesis of lung cancer.

Cigarette smoke which contains polyaromatic hydrocarbons (PAHs) and aromatic amine pro-carcinogenic compounds is particularly associated with the induction of CYP1A1 gene and the resultant metabolism of the PAHs in cigarette smoke is thought to be one of the primary causes of lung cancer.

CYP1A1 gene induction occurs at the transcriptional level which, in turn, leads to the induction of CYP1A1 enzyme activity. CYP1A1 enzyme is also known as aromatic hydrocarbon hydroxylase (AHH) since it metabolises PAHs through hydroxylation to form carcinogens. For induction of CYP1A1 gene transcription a transcription factor composed of two proteins is required. Ligand (PAH)-bound aromatic (aryl) hydrocarbon receptor (AhR) forms a complex with the aryl hydrocarbon receptor nuclear translocator (Arnt) to form the active transcription factor.

Recent animal and human data suggested that AhR is involved in various signalling pathways critical to cells' normal homeostasis, which includes physiological processes such as cell proliferation and differentiation, gene regulation, cell motility and migration, inflammation and others. Dysregulation of these processes is known to contribute to events such as tumour initiation, promotion, and progression.

While natural products such as flavonoids, chalcones and stilbenes have a general ability to modulate the activity of cytochrome P450 (CYP) enzymes, until now no potent CYP 1A1 -specific inhibitor has been identified. There is therefore a need in the art for compounds that inhibit CYP1A1 and which have the potential to prevent conversion of pro-carcinogenic substances to carcinogens and thereby act as chemo-preventive agents in the treatment of cancer. The first aspect of the invent d of formula (I)

for use in the prevention and/or treatment of cancer, wherein R¹ to R⁹ are independently selected from hydrogen, 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.

In particular, R¹ to R⁹ 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.

Preferably, R¹, R⁵ and R⁹ are hydrogen and one or more of R², R³, R⁴, R⁶, R⁷ and R⁸ are selected from 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, with the remaining groups being hydrogen. In particular, one or more of R², R³, R⁴, R⁶, R⁷ and R⁸ are independently selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy with the remaining groups being hydrogen. The phenyl ring is preferably mono- or di-substituted, that is one or two of R², R³ and R⁴ are preferably selected from 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 one or two of R², R³ and R⁴ are independently aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy and the remainder are hydrogen. In a particularly preferred feature of the first aspect, R³ is selected from 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 R³ is selected from aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy; and R² and R⁴ are independently both hydrogen. Alternatively, R² and R³ are independently selected from 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 R² and R³ are independently aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy; and R⁴ is hydrogen. Alternatively, R² is hydrogen and R³ and R⁴ are independently selected from 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 R³ and R⁴ are independently aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy.

The fused phenyl ring is preferably di- or tri-substituted, that is two or three of R⁶, R⁷ and R⁸ are independently selected from 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 two or three of R⁶, R⁷ and R⁸ are independently aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy with the remainder of R⁶, R⁷, R⁸ being hydrogen. Preferably all three of R⁶, R⁷ and R⁸ are independently selected from 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 R⁶, R⁷ and R⁸ are independently aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy, more preferably hydroxyl or alkoxy.

Where one or more of R¹ to R⁹ are alkoxy, the one or more 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, for example a methoxy or ethoxy group, most particularly methoxy.

In a particular feature of the first aspect of the invention, R¹ to R⁹ are independently selected from hydrogen, hydroxy or alkoxy.

Preferably, R¹, R⁵ and R⁹ are hydrogen.

The phenyl ring is preferably mono- or di-substituted, that is one or two of R², R³ and R⁴ are preferably selected from alkoxy or hydroxy, and the remainder are hydrogen. In a particularly preferred feature of the first aspect, R³ is alkoxy or hydroxyl, preferably alkoxy and R² and R⁴ are selected from hydrogen, alkoxy or hydroxy, particularly R² and R⁴ are independently both hydrogen or R² is alkoxy or hydroxyl and R⁴ is hydrogen or R² is hydrogen and R⁴ is alkoxy or hydroxyl.

In a particular feature of the first aspect of the invention, R¹ to R⁹ are independently selected from hydrogen, hydroxy or methoxy.

Preferably, R¹, R⁵ and R⁹ are hydrogen.

The phenyl ring is preferably mono- or di-substituted, that is one or two of R², R³ and R⁴ are preferably selected from methoxy or hydroxy, and the remainder are hydrogen. In a particularly preferred feature of the first aspect, R³ is methoxy or hydroxyl, preferably methoxy and R² and R⁴ are selected from hydrogen, methoxy or hydroxy, particularly R² and R⁴ are independently both hydrogen or R² is methoxy or hydroxyl and R⁴ is hydrogen or R² is hydrogen and R⁴ is methoxy or hydroxyl.

The fused phenyl ring is preferably di- or tri-substituted, that is two or three of R⁶, R⁷ and R⁸ are alkoxy or hydroxyl with the remainder of R⁶, R⁷, R⁸ being hydrogen. Preferably two or three of R⁶, R⁷ and R⁸ are methoxy or hydroxyl with the remainder of R⁶, R⁷ or R⁸ being hydrogen. In a particularly preferred feature of the first aspect R⁶, R⁷ and R⁸ are each hydroxyl or alkoxy. Alternatively R⁶, R⁷ and R⁸ are each hydroxyl or alkoxy. Preferably all three groups are methoxy as illustrated below.

where R¹ to R⁵ are as disclosed above.

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, naphthyl 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 "Ci_-4 alkoxy group". The alkoxy group particularly includes 1 , 2, 3 or 4 carbon atoms. Particularly preferably alkoxy groups include methoxy, ethoxy, propyloxy and butyloxy. Halogen means F, CI, Br or I, preferably F.

In a particularly preferred aspect, the compounds of formula (I) are one or more of

(Tetramethyl luteolin ether)

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.

Methods of obtaining or synthesising flavonoids are known in the art and such methods can be adapted or modified by the skilled person to provide the compounds of the first aspect of the invention.

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), (IA) or (IB) 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), (IA) or (IB) 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. In particular, the compounds are provided for the prevention and/or treatment of cancer of the lung, colon, pancreas, liver and/or kidney. 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 a 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 compounds of the first aspect of the invention are provided as inhibitors of CYP1 A1 which has been implicated in cancer of the lung, colon, pancreas, liver and/or kidney. Inhibition of CYP1A1 by the claimed compounds will allow the prevention and/or treatment of cancer.

It is therefore a particular desire of the invention to provide compounds which selectively inhibit CYP1A1 when compared with other CYPs. The present invention therefore particular provides a compound of formula (IB)

(IB)

wherein R¹, R⁵ and R⁹ are hydrogen, R³, R⁶ and R⁸ are alkoxy and R², R⁴ and R⁷ are hydrogen, hydroxyl or alkoxy, preferably hydrogen or alkoxy.

In particular, wherein R¹, R⁵ and R⁹ are hydrogen, R³, R⁶ and R⁸ are methoxy and R², and R⁷ are hydrogen, hydroxyl or methoxy, preferably hydrogen or methoxy.

Particularly preferred compounds of formula (IB) include

Compound Chemical structure

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 non- aqueous 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 aspect 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 first 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 or treating cancer comprising administering a compound of the first aspect of the invention to a patient in need thereof.

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 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 compound of the first aspect 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 second aspect of the invention.

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.

Brief description of the figures Figures 1 and 2 show the restoration of yeast cell growth by DMU 1 10;

Figure 3 shows the restoration of HEK-293 cell growth by DMU 1 10;

Figure 4 shows a plasmid map of pSYE263/BamHI-Xbal/h_CYP1A1 (with restriction sites that cut the plasmid only once); Figure 5 shows a plasmid map of pcDNA3.1/BamHI-Xbal/h_CYP1A1 (with restriction sites that cut the plasmid only once).

The invention will now be described with reference to the following non-limiting examples. Examples

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 provide evidence of 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.

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 below:

Table 1 : The constitution of the regenerating system used per reaction in each single well for different CYPs was as follows.

CYP1A1 5 μΐ 1 μΐ 5 μΐ 39 μ1 0.2 Μ -

CYP1B1 5 μΐ 1 μΐ 5 μΐ 39 μ1 0.2 Μ -

CYP1A2 5 μΐ 1 μΐ 5 μΐ 20 μΐ 0.5 Μ 19 μΐ

CYP2D6 5 μΐ 1 μΐ 5 μΐ 25 μΐ 0.2 Μ 14 μΐ

CYP3A4 5 μΐ 1 μΐ 5 μΐ 25 μΐ 0.2 Μ

14 μΙ

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.

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. Comparing the IC50 values of flavonoids in CYP1A1 , CYP1 B1 and CYP1A2 enzyme assays (using CYP1A1 , CYP1 B1 and

CYP1A2 yeast microsomes) obtained from CYP Design Ltd

In the compound library that was screened initially for CYP1 A1 inhibition, a number of compounds were flavonoids. Out of these two compounds were found to inhibit CYP1 A1 potently when compared with CYP1 B1 and CYP1A2 enzyme inhibition. These compounds were DMU 1 14 and DMU 1 10 which were shown to have IC50 values of 2 μΜ and 397 nM as shown in Table 3. These flavonoids (natural products) had been synthesised previously within the Chemistry group at the Leicester School of Pharmacy (De Montfort University) and are also commercially available.

Table: 3: IC₅₀ values obtained by screening flavonoids on CYP1A1 , CYP1 B1 and

CYP1A2 bearing microsomes

Example 3. IC50 values of DMU 110 in CYP1A1 , CYP1 B1 , CYP1A2, CYP2D6 and CYP3A4 enzyme assays (using CYP1A1 , CYP1 B1 , CYP1A2, CYP2D6 and CYP3A4 yeast microsomes) from CYP Design Ltd

Table:4: IC₅₀ values obtained by screening DMU 1 10 on CYP1A1 , CYP1 B1 , CYP1A2, CYP2D6 and CYP3A4 bearing microsomes

The results indicate that DMU 1 10 is a relatively potent CYP1-specific inhibitor

Table 5: Chemical structure of commercial flavonoids (analogues of DMU 1 10;

Tetramethyl luteolin ether) and the IC₅₀ values obtained by screening them on CYP1A1 and CYP1 B1 enzymes from CYP Design Ltd.

Diosmetin 447nM 152nM

OMe

Example 4. 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 OD₆oo 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 μ1 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 6).

Table 6: Outline of kinetic assay parameters used for analysing cytochrome P450 enzymes using live cells and the Bio-Tek Synergy HT fluorescent plate reader.

Table 7 Compounds screened with yeast strain that expresses the CYP1A1 enzyme via live cell based inhibition assays. Comparison of CYP1A1 IC₅₀ values of selected compounds obtained from microsomal and live cell assays.

The above results show that besides screening potential inhibitors of a CYP isozyme in microsomal assays, compounds could also be screened conventionally in live cells that express a CYP isoyme and that there is a strict correlation between the two forms of assays. Example 5. Restoration of yeast cell growth by DMU 110 after recombinant yeast cells were treated with benzo(a)pyrene and TCDD

Yeast transformed with ADH2p-CYP1A1 plasmid (figure 4) was treated with

benzo(a)pyrene [BaP; 1 and 5μΜ] and 2,3,7,8-Tetrachlorodibenzo-p-dioxin [TCDD; 1 and 5μΜ] for 16 hours. Cells were blocked in growth as determined by measurement of optical density at 600nm (OD600). Treatment with DMU 1 10 (4μΜ) restored growth (confirmed through measurement of OD600, Figure 1 & Figure 2). This indicates that the CYP1A1 specific inhibitor, DMU 1 10, can prevent CYP1A1 -mediated biotransformation of BaP and TCDD into toxic substances in yeast cells.

Example 6. Restoration of HEK-293 cell growth by DMU 110 after recombinant human cells were treated with benzo(a)pyrene and TCDD

Human embryonic kidney HEK-293 cells transformed with pcDNA3.1/h_CYP1A1 plasmid (figure 5) were treated with benzo(a)pyrene [BaP; 0.5μΜ] and 2,3,7,8-Tetrachlorodibenzo- p-dioxin [TCDD; 50nM]. Cells were blocked in growth. Treatment with DMU 1 10 (4μΜ) restored growth. This indicates that the CYP1A1 specific inhibitor, DMU 1 10, can prevent CYP1A1-mediated biotransformation of BaP and TCDD into toxic substances in human cells.

Claims

A compound of formula (I)

for use in the prevention and/or treatment of cancer, wherein R¹ to R⁹ are independently selected from hydrogen, 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.

2. The compound as claimed in claim 1 wherein R¹ to R⁹ are independently selected from hydrogen, aliphatic, halogen, hydroxy, alkoxy, thioalkyi, alkylamino or cyano, preferably hydroxyl, halogen or alkoxy.

3. The compound as claimed in claim 1 or claim 2 wherein R¹, R⁵ and R⁹ are hydrogen.

4. The compound as claimed in any one of claims 1 to 3 wherein one or two of R², R³ and R⁴ are selected from 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 with the remainder or R², R³ and R⁴ being hydrogen.

5. The compound as claimed in any preceding claim wherein two or three of R⁶, R⁷ and R⁸ are independently selected from 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 with the remainder of R⁶, R⁷, R⁸ being hydrogen.

6. The compound as claimed in claim 1 wherein R¹ to R⁹ are independently selected from hydrogen, hydroxy or methoxy.

7. The compound of claim 6 wherein R¹, R⁵ and R^M are hydrogen.

8. The compound of claim 6 or 7 wherein one or two of R², R³ and R⁴ are selected from methoxy or hydroxyl.

9. The compound as claimed in any one of claims 6 to 8 wherein two or three of R⁶, R⁷ and R⁸ are methoxy or hydroxyl.

10. The compound as claimed in any one of claims 6 to 9 having the structure

where R¹ to R⁵ are as claimed in any one of claims 6 to 9.

1 1 . The compound of any preceding claim, selected from

12. The compound of any preceding claim for the prevention and/or treatment of cancer of the lung, colon, pancreas, liver and/or kidney.

13. The compound of any preceding claim having the formula (IB)

(IB)

wherein R¹, R⁵ and R⁹ are hydrogen, R³, R⁶ and R⁸ are methoxy and R², R⁴ and R⁷ are hydrogen, hydroxyl or methoxy

14. The compounds of formula (IB), as claimed in claim 13 selected from

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

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

17. A method of preventing and/or treating cancer comprising administering a compound as claimed in any one of claims 1 to 14 to a patient in need thereof.

18. The use of a compound as claimed in any one of claims 1 to 14 in the manufacture of a medicament for the prevention and/or treatment of cancer.