WO2010109230A1 - Novel prodrugs - Google Patents

Abstract

The present invention relates to novel prodrug compounds, or pharmaceutically acceptable salts thereof, which convert in vivo to the active species 4-(1-benzenesulfonyl-6-fluoro-1H-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone. The prodrug compounds of the invention have dramatically improved solubility relative to 4-(1-benzenesulfonyl-6-fluoro-1H- indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone and, accordingly, can be easily formulated for administration to a patient. The prodrugs of the invention are therefore useful for the treatment cellular proliferation disorders, such as cancer.

Description

NOVEL PRODRUGS

FIELD OF THE INVENTION

The present invention relates to novel prodrug compounds, to processes for the preparation of these compounds, pharmaceutical compositions comprising them and their use as therapeutic agents. In particular, the prodrug compounds of the present invention are potentially useful therapeutic agents for the treatment and/or prevention of proliferative diseases, such as cancer.

BACKGROUND OF THE INVENTION WO2004/056361 discloses a series of 4-(l-(arylsulfonyl)-lH-indol-2-yl)-4-(hydroxy)- cyclohexa-2,5-dienone compounds and analogues thereof, which possess thioredoxin/thioredoxin reductase inhibitory activity. The compounds defined in WO2004/056361 are potentially useful therapeutic agents for the treatment of proliferative conditions, such as cancer.

Particular 4-(l-(arylsulfonyl)-lH-indol-2-yl)-4-(hydroxy)-cyclohexa-2,5-dienone compounds and their antitumour activity are also described in a paper by Berry et al. (J. Med. Chem. 2005; 48(2); 639-644). One particular compound described by Berry et al. is 4-(l- benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone, the structure of which is shown below.

4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone

One problem associated with 4-(l-(arylsulfonyl)-lH-indol-2-yl)-4-(hydroxy)-cyclohexa- 2,5-dienone compounds, such as 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone, is poor solubility. Poorly soluble therapeutic agents are typically more challenging to formulate for administration to a patient, and, if administered orally, they generally have poor bioavailability.

Accordingly, there is a desire to find more soluble 4-(l-(arylsulfonyl)-lH-indol-2-yl)-4- (hydroxy)-cyclohexa-2,5-dienone derivatives which possess improved pharmaceutical properties. In particular, it is an object of the present invention to provide more soluble derivatives of 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone.

SUMMARY OF THE INVENTION In accordance with the present invention, the applicants have hereby discovered novel prodrug compounds, or pharmaceutically acceptable salts thereof, as defined herein which convert in vivo to 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5- dienone, which is the active species. The prodrug compounds of the invention have dramatically improved solubility relative to 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone and, accordingly, can be easily formulated for administration to a patient.

The administration of the prodrugs of the present invention therefore provides a convenient means by which therapeutically effective levels of 4-(l-benzenesulfonyl-6-fluoro- lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone may be achieved in a warm-blooded animal, such as man. The anti-proliferation properties of 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)- 4-hydroxy-cyclohexa-2,5-dienone and related compounds has been described previously in WO2004/056361 and Berry et al. (J. Med. Chem. 2005; 48(2); 639-644). Accordingly, the prodrug compounds of the present invention are expected to be useful therapeutic agents which, following administration to a warm-blooded animal, such as man, will produce anti-proliferative effects by the formation of 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa- 2,5-dienone. The prodrugs of the invention are therefore useful for the treatment cellular proliferation disorders, such as cancer.

Accordingly, the invention pertains to novel prodrug compounds as described herein. The present invention also provides processes for the manufacture of said prodrug compounds, or pharmaceutically acceptable salts thereof, to pharmaceutical compositions containing them and to their use in the manufacture of medicaments for use in the production of anti-proliferation activity in a warm-blooded animal such as man.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless otherwise stated, the following terms used in the specification and claims have the following meanings:

The term "prodrug" refers to a compound which is converted in vivo to an active species/metabolite/metabonate which is pharmacologically active and exerts a pharmacological effect. The term "pharmaceutically acceptable cation" refers to a cation which is suitable for use in pharmaceutical compositions or formulations and is safe for administration to the human or animal body.

It is to be appreciated that references to "treating" or "treatment" include prophylaxis as well as the alleviation of established symptoms of a condition. "Treating" or "treatment" of a state, disorder or condition includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in a human that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition, (2) inhibiting the state, disorder or condition, i.e. , arresting, reducing or delaying the development of the disease or a relapse thereof (in case of maintenance treatment) or at least one clinical or subclinical symptom thereof, or (3) relieving or attenuating the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.

A "therapeutically effective amount" means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated.

Compounds

According to a first aspect of the present invention there is provided a compound of formula I:

I wherein: X⁺ is a pharmaceutically acceptable cation; or a pharmaceutically acceptable salt thereof. In a second aspect the present invention provides a compound of formula II:

II wherein: X⁺ is as defined above; or a pharmaceutically acceptable salt thereof.

A suitable pharmaceutically acceptable salt of a compound of the formula I or II above is, for example, an alkali metal salt, for example a sodium or potassium salt, an alkaline earth metal salt, for example a calcium or magnesium salt, an ammonium salt or a salt with an organic base which affords a physiologically-acceptable cation, for example a salt with methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine.

The compounds of formula I comprise two sulfonate salt groups and the compounds of formula II comprise one sulfonate salt group. Consequently, in a particular embodiment of the invention, the compounds of formula I or formula II above are present as such and are not further derivatised as a further pharmaceutically acceptable salt. Therefore, in a further aspect, the present invention provides a compound of formula I:

wherein:

X⁺ is as defined above.

In another aspect the present invention provides a compound of formula II:

II wherein:

X⁺ is as defined above.

In a particular group of compounds of formula I, X⁺ is a monovalent cation.

In a further group of compounds of formula I, X⁺ is Na⁺. In a particular group of compounds of formula II, X⁺ is a monovalent cation.

In a further group of compounds of formula II, X⁺ is Na⁺.

A particular compound of formula I is disodium 2,2'-(2-(6-fluoro-l-(phenylsulfonyl)-lH- indol-2-yl)-2-hydroxy-5-oxocyclohexane- 1 ,3-diyl)bis(sulfanediyl)diethanesulfonate, which has the structural formula III shown below:

III A particular compound of formula II is sodium 2-(2-(6-fluoro-l-(phenylsulfonyl)-lH-indol-2- yl)-2-hydroxy-5-oxocyclohex-3-enylthio)ethanesulfonate, which has the structural formula IV shown below:

IV

The compounds of this invention possess two asymmetric centres; each of which can be present as the (R)- or (S)-stereoisomer, or as mixtures thereof. Unless indicated otherwise, the structure or naming of a particular compound in the specification and claims is intended to include all individual enantiomers and mixtures, racemic or otherwise, thereof. The methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (see discussion in Chapter 4 of "Advanced Organic Chemistry", 4th edition J. March, John Wiley and Sons, New York, 2001), for example by synthesis from optically active starting materials or by resolution of a racemic form.

It is to be understood that the present invention encompasses all optical, diastereoisomers and geometric isomers and mixtures thereof that possess anti-proliferative activity.

It is also to be understood that the compounds of the invention may exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms that possess anti-proliferative activity.

It is also to be understood that certain compounds of the invention may exhibit polymorphism, and that the invention encompasses all such polymorphic forms that possess antiproliferative activity.

Synthesis

The compounds of the present invention can be prepared in accordance with the general process described below and by the processes described in the accompanying examples.

In the description of the synthetic methods described below and in the referenced synthetic methods that are used to prepare the staring materials, it is to be understood that all proposed reaction conditions, including choice of solvent, reaction atmosphere, reaction temperature, duration of the experiment and workup procedures, can be selected by a person skilled in the art.

It is understood by one skilled in the art of organic synthesis that the functionality present on various portions of the molecule must be compatible with the reagents and reaction conditions utilised.

It will be appreciated that during the synthesis of the compounds of the invention in the processes defined below, or during the synthesis of certain starting materials, it may be desirable to protect certain substituent groups to prevent their undesired reaction. The skilled chemist will appreciate when such protection is required, and how such protecting groups may be put in place, and later removed.

For examples of protecting groups see one of the many general texts on the subject, for example, 'Protective Groups in Organic Synthesis' by Theodora Green (publisher: John Wiley & Sons). Protecting groups may be removed by any convenient method described in the literature or known to the skilled chemist as appropriate for the removal of the protecting group in question, such methods being chosen so as to effect removal of the protecting group with the minimum disturbance of groups elsewhere in the molecule.

Thus, if reactants include, for example, groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein. By way of example, a suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or f-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed by, for example, hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a ferz-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulfuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine. A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium, sodium hydroxide or ammonia. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.

A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a f-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon. Resins may also be used as a protecting group.

Process A

In another aspect, the present invention provides a process for the production of a compound of the formula I, or a pharmaceutically acceptable salt thereof, which comprises: the reaction of a compound of formula A

A with two or more molar equivalents of a compound of formula B:

B in the presence of a suitable solvent; wherein X⁺ is as defined above in relation to formula I; and thereafter: optionally forming a pharmaceutically acceptable salt thereof.

Process B

In another aspect, the present invention provides a process for the production of a compound of the formula II, or a pharmaceutically acceptable salt thereof, which comprises: the reaction of a compound of formula A

A with between 0.01 and 1 molar equivalents of a compound of formula B:

,SO₃-X⁺

B in the presence of a suitable solvent; wherein X⁺ is as defined above; and thereafter: optionally forming a pharmaceutically acceptable salt thereof.

Process C In another aspect, the present invention provides a process for the production of a compound of the formula III, or a pharmaceutically acceptable salt thereof, which comprises: the reaction of a compound of formula A

A with two or more molar equivalents of compound of formula C: ,SO₃Na

C in the presence of a suitable solvent; and thereafter: optionally forming a pharmaceutically acceptable salt thereof.

Process D

In another aspect, the present invention provides a process for the production of a compound of the formula IV, or a pharmaceutically acceptable salt thereof, which comprises: the reaction of a compound of formula A

A with between 0.01 to 1 molar equivalents of a compound of formula C:

,SO₃Na

C in the presence of a suitable solvent; and thereafter: optionally forming a pharmaceutically acceptable salt thereof.

The compound of formula A can be prepared by processes known in the art. For example, a compound of formula A can be prepared in accordance with the processes described in WO2004/056361 (Method C) or Berry et al. (J. Med.Chem. 2005; 48(2); 639-644), the entire contents of which are incorporated herein by reference. Alternatively, the compound of formula A can be prepared by the alternative process outlined in the Example section below.

The compound of formula C is generally known as "Mesna" and is commercially available. Compounds of formula B are commercially available when X⁺ is Na⁺ (i.e. when the compound is Mesna), or can be prepared by techniques known in the art when X⁺ is a cation other than Na⁺.

The reaction between compounds A and B defined in processes A and B above, and compounds A and C defined in processes C and D above, are carried out in the presence of a suitable solvent. Any suitable solvent or solvent mixture may be used for these reactions. A person skilled in the art will know how to select suitable solvents or solvent mixtures for use in these reactions. Particular examples of suitable solvents include acetone, an acetone/water mixture, methanol, isopropanol, methyl ethylketone, ethanol or an ethanol/chloroform mixture.

In an embodiment of process A or process B, compound A is dissolved in a first solvent and compound B is dissolved in a second solvent and the resultant solutions are then mixed to facilitate the reaction. In a particular embodiment of process A or process B, compound A is dissolved in acetone and compound B is dissolved in water and the two resultant solutions are then mixed together to facilitate the reaction.

In an embodiment of process C or process D above, compound A is dissolved in a first solvent and compound C is dissolved in a second solvent and the resultant solutions are then mixed to facilitate the reaction. In a particular embodiment, compound A is dissolved in acetone and compound C is dissolved in water and the two resultant solutions are then mixed together to facilitate the reaction.

Suitably the reactions defined in processes A to D above are performed in the presence of a suitable base. Any suitable base may be used. A person skilled in the art will know how to select a suitable base for use in these reasons. Particular examples of suitable bases include triethylamine and diisopropylethylamine. In one embodiment, 0.01 to 0.5 molar equivalents (relative to compound A) of a suitable base is present in the reaction mixture. In a further embodiment, 0.01 to 0.2 molar equivalents (relative to compound A) of a suitable base is present in the reaction mixture. In a particular embodiment, 0.05 to 0.15 molar equivalents (relative to compound A) of a suitable base is present in the reaction mixture.

Suitably, the reactions defined in processes A to D above are carried out under reflux at an elevated temperature. In a particular embodiment, temperatures within the range of 30 to 90°C, particularly 40 to 70°C, and more particularly 50 to 65°C may be used. In addition, reflux times of two or more hours are typically used.

In process A defined above, an excess of two or more molar equivalents of compound B (relative to compound A) is provided. In a particular embodiment of process A, an excess of 2.2 or more molar equivalents of compound B (relative to compound A) is provided. In process B defined above, 0.01 to 1 molar equivalents of compound B (relative to compound A) are present. In a particular embodiment, 0.5 to 0.95 molar equivalents of compound B (relative to compound A) are present. In a further embodiment, 0.8 to 0.95 molar equivalents of compound B (relative to compound A) are present. In a particular embodiment, 0.8 to 0.9 molar equivalents of compound B (relative to compound A) are present.

In process C defined above, an excess of two or more molar equivalents of compound C (relative to compound A) is provided. In a particular embodiment of process A, an excess of 2.2 or more molar equivalents of compound C (relative to compound A) is provided.

In process D defined above, 0.01 to 1 molar equivalents of compound C (relative to compound A) are present. In a particular embodiment, 0.5 to 0.95 molar equivalents of compound C (relative to compound A) are present. In a further embodiment, 0.8 to 0.95 molar equivalents of compound C (relative to compound A) are present. In a particular embodiment, 0.8 to 0.9 molar equivalents of compound C (relative to compound A) are present.

Processes A to D may further comprise a step of isolating the desired end product. In the case of processes B and D, this may involve isolating the compound of formula II or IV respectively from and compound of formula I or

In a further aspect of the invention, there is provided a compound of formula I obtainable by process A defined above.

In a further aspect of the invention, there is provided a compound of formula I obtained by process A defined above.

In a further aspect of the invention, there is provided a compound of formula II obtainable by process B defined above.

In a further aspect of the invention, there is provided a compound of formula II obtained by process B defined above. In a further aspect of the invention, there is provided a compound of formula III obtainable by process C defined above.

In a further aspect of the invention, there is provided a compound of formula III obtained by process C defined above.

In a further aspect of the invention, there is provided a compound of formula IV obtainable by process D defined above.

In a further aspect of the invention, there is provided a compound of formula IV obtained by process D defined above.

In a further aspect of the invention, there is provided a compound of formula III obtainable by process as defined in either Example 1 or 2. In a further aspect of the invention, there is provided a compound of formula III obtained by process as defined in either Example 1 or 2.

In a further aspect of the invention, there is provided a compound of formula IV obtainable by process as defined in Example 3. In a further aspect of the invention, there is provided a compound of formula IV obtained by process as defined in Example 3.

Biological Activity

The following in vitro assay can be used to measure the pharmacological effects of the compounds of the present invention.

In vitro MTT cellular proliferation assay (i) Assay principle

MTT cellular assays are a well established means for quantifying viable cells. The non- radioactive, colorimetric assay system using MTT was first described by Mosmann, T. et al. (J. Immunol. Methods (1983) 65, 55-63) and improved in subsequent years by several other investigators (Tada, H. et al, J. Immunol. Methods (1986) 93, 157-165; Denizot, F. & Lang, R., J. Immunol. Methods (1986) 89, 271-277; Gerlier, D. & Thomasset, N., J. Immunol. Methods (1986) 94, 57-63; Hansen, M. B.,et al., J. Immunol. Methods (1989) 119, 203-210; and Vistica, D. T. et al. (1991) Cancer Res. 51, 2515-2520).

The assay is designed for the spectrophotometric quantification of cell growth and viability without the use of radioactive isotopes and can be used for quantification of the cytotoxicity effects of growth inhibiting agents, such as potential antitumour agents.

(U) Assay protocol

HCTl 16, HT29, MCF7, MDA468, and A549 cells were grown in RPMI1640 media (Sigma R8758) supplemented with 10% heat inactivated Foetal Bovine Serum (Sigma F7524) at 37°C in 95% air/5% CO₂. A sub-confluent (-70%) 25 cm² flask (Costar) was harvested with IX trypsin/EDTA solution (diluted from Sigma T4174 with phosphate buffered saline (PBS)). Cells were re-suspended in fresh complete medium, gently passed through a 23g needle and counted using a neubauer haemocytometer. A cell suspension was prepared with 2500-5000 cells/180 μl complete medium and 180 ml pipetted into 96 well plates (Nunc). Plates were incubated overnight at 37°C in 95%air/5% CO₂ to allow the cells to attach. Serial dilutions of a 1OmM stock (in DMSO) of each compound were prepared in complete medium at 10 times the required final concentration and 20 μl added to triplicate wells. Controls were treated with media alone or DMSO diluted appropriately.

An extra plate was set up so that an MTT assay was carried out at the same time as cell treatment with test compounds in order to calculate the initial optical density (OD).

Following 72 hour incubation at 37°C in 95% air/5% CO_2, 50 ml of a 2mg/ml in PBS solution of Thiazolyl Blue (MTT, Sigma M2128) was added to each well and the plates incubated for a further 2 hours.

The media/MTT is then aspirated and 150 μl DMSO (Sigma D5879) was added to solubilise the formazan product. The optical density (OD) at 550nm for each well was analysed using an Anthos Labtec 2001 plate reader and means of triplicates were used to plot graphs from which GI₅₀ values were calculated by interpolation using OD at GI₅₀ = [(control OD - initial OD)/2] + initial OD.

The compounds of the present invention were found to be active in the above screen. By way of example, the GI₅₀ activity of the compound of Examples 1 and 3 (Formula III) in the above screen is shown below in Table 1 below. For comparative purposes, the activity of the active species, 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone, is also shown in Table 1. The activity of the compound of Examples 1 and 2 (Formula III) demonstrates cytotoxic activity which is comparable to that of the active species, 4-(l- benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone.

Table 1

Unless otherwise indicated in the table above each compound was tested at least in triplicate in the assay (i.e. n > 1) and the GI₅₀ value quoted is the mean of the measured GI₅₀ values. Therefore, as will be understood, the GI₅₀ values quoted above are not absolute and further measurements of the GI₅₀ value for a compound may result in a different mean GI₅₀ value. Pharmaceutical Compositions

According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier.

The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular, intraperitoneal or intramuscular dosing or as a suppository for rectal dosing).

The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.

An effective amount of a compound of the present invention for use in therapy of infection is an amount sufficient to symptomatically relieve in a warm-blooded animal, particularly a human the symptoms of infection, to slow the progression of infection, or to reduce in patients with symptoms of infection the risk of getting worse.

The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 0.5 g of active agent (more suitably from 0.5 to 100 mg, for example from 1 to 30 mg) compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition.

The size of the dose for therapeutic or prophylactic purposes of a compound of the formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine.

In using a compound of the invention for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.1 mg/kg to 75 mg/kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous or intraperitoneal administration, a dose in the range, for example, 0.1 mg/kg to 30 mg/kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.05 mg/kg to 25 mg/kg body weight will be used. Oral administration may also be suitable, particularly in tablet form. Typically, unit dosage forms will contain about 0.5 mg to 0.5 g of a compound of this invention.

Therapeutic Uses and Applications As previously stated, the compounds of the present invention are prodrugs that are converted to the active species 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone, which has been shown to possess anti-proliferative activity in various in vitro assays (see Berry et al J. Med.Chem. 2005; 48(2); 639-644).

The present invention therefore provides, in another aspect, a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, for use as a medicament.

In another aspect the present invention provides the use of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament. In another aspect the present invention provides a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, for use in the treatment of a cell proliferation disorder, such as cancer.

In another aspect the present invention provides the use of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of a cell proliferation disorder such as cancer.

In another aspect, the present invention provides a method of treating a cell proliferation disorder, such as cancer, said method comprising administering to a human or animal in need of such treatment, a therapeutically effective amount of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof.

In another aspect the present invention provides a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, for use in the treatment of cancer. In another aspect the present invention provides the use of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of cancer.

In another aspect, the present invention provides a method of treating cancer, said method comprising administering to a human or animal in need of such treatment, a therapeutically effective amount of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof.

In another embodiment the present invention provides a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, for use in the treatment of a solid tumour.

In another aspect the present invention provides the use of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment of a cell proliferation disorder such as cancer. In another aspect, the present invention provides a method of treating a solid tumour, said method comprising administering to a human or animal in need of such treatment, a therapeutically effective amount of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof.

In another embodiment the present invention provides a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof for use in the treatment of neoplastic disease such as carcinoma of the breast, ovary, lung (including small cell lung cancer, non-small cell lung cancer and bronchioalveolar cancer), colon, rectum, prostate, bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, pancreas, skin, testes, thyroid, uterus, cervix, vulva or other tissues, as well as leukemias and lymphomas including CLL and CML, tumours of the central and peripheral nervous system, and other tumour types such as melanoma, multiple myeloma, fibrosarcoma and osteosarcoma, and malignant brain tumours.

In another embodiment the present invention provides the use of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the treatment or prophylaxis of neoplastic disease such as carcinoma of the breast, ovary, lung (including small cell lung cancer, non-small cell lung cancer and bronchioalveolar cancer), colon, rectum, prostate, bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, esophagus, pancreas, skin, testes, thyroid, uterus, cervix, vulva or other tissues, as well as leukemias and lymphomas including CLL and CML, tumours of the central and peripheral nervous system, and other tumour types such as melanoma, multiple myeloma, fibrosarcoma and osteosarcoma, and malignant brain tumours.

In another embodiment the present invention provides a method of prophylaxis or treatment of a human or animal suffering from a neoplastic disease such as carcinoma of the breast, ovary, lung (including small cell lung cancer, non-small cell lung cancer and bronchioalveolar cancer), colon, rectum, prostate, bile duct, bone, bladder, head and neck, kidney, liver, gastrointestinal tissue, oesophagus, pancreas, skin, testes, thyroid, uterus, cervix, vulva or other tissues, as well as leukemias and lymphomas including CLL and CML, tumours of the central and peripheral nervous system, and other tumour types such as melanoma, multiple myeloma, fibrosarcoma and osteosarcoma, and malignant brain tumours, comprising administering to said human or animal a therapeutically effective amount of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof.

In a particular embodiment, the neoplastic disease is pancreatic cancer.

In a particular embodiment, the neoplastic disease is colon cancer or renal cancer. In a particular embodiment, the neoplastic disease is colon cancer.

In a particular embodiment, the neoplastic disease is renal cancer.

In a particular embodiment, the neoplastic disease is prostate cancer.

In a particular embodiment, the neoplastic disease is melanoma.

In a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of the formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof, in association with a pharmaceutically acceptable diluent or carrier for use in the production of an anti-cancer effect in a warm-blooded animal such as man.

Another aspect of the invention pertains to a method of (a) inhibiting cell proliferation; (b) inhibiting cell cycle progression; (c) promoting apoptosis; or (d) a combination of one or more of these, in vitro or in vivo, comprising contacting a cell with an effective amount of a compound of formula I, II, III or IV as defined hereinbefore, or a pharmaceutically acceptable salt thereof. Combination Therapies

The anti-cancer treatment defined hereinbefore may be applied as a sole therapy or may involve, in addition to the compound of the invention, conventional surgery or radiotherapy or chemotherapy. Such chemotherapy may include one or more of the following categories of anti- tumour agents :-

(i) other antiproliferative/antineoplastic drugs and combinations thereof, as used in medical oncology, such as alkylating agents (for example cis-platin, oxaliplatin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, busulphan, temozolamide and nitrosoureas); antimetabolites (for example gemcitabine and antifolates such as fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed, methotrexate, cytosine arabinoside, and hydroxyurea); antitumour antibiotics (for example anthracyclines like adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin and mithramycin); antimitotic agents (for example vinca alkaloids like vincristine, vinblastine, vindesine and vinorelbine and taxoids like taxol and taxotere and polokinase inhibitors); and topoisomerase inhibitors (for example epipodophyllotoxins like etoposide and teniposide, amsacrine, topotecan and camptothecin);

(ii) cytostatic agents such as antioestrogens (for example tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), antiandrogens (for example bicalutamide, flutamide, nilutamide and cyproterone acetate), LHRH antagonists or LHRH agonists (for example goserelin, leuprorelin and buserelin), progestogens (for example megestrol acetate), aromatase inhibitors (for example as anastrozole, letrozole, vorazole and exemestane) and inhibitors of 5α- reductase such as finasteride;

(iii) anti-invasion agents [for example c-Src kinase family inhibitors like 4-(6-chloro-2,3- methylenedioxyanilino)-7-[2-(4-methylpiperazin-l-yl)ethoxy]-5-tetrahydropyran-4- yloxyquinazoline (AZD0530; International Patent Application WO 01/94341), N-(2-chloro-6- methylphenyl)-2- { 6- [4-(2-hydroxyethyl)piperazin- 1 -yl] -2-methylpyrimidin-4-ylamino } thiazole- 5-carboxamide (dasatinib, BMS-354825; J. Med. Chem.. 2004, 47, 6658-6661) and bosutinib (SKI-606), and metalloproteinase inhibitors like marimastat, inhibitors of urokinase plasminogen activator receptor function or antibodies to Heparanase] ; (iv) inhibitors of growth factor function: for example such inhibitors include growth factor antibodies and growth factor receptor antibodies (for example the anti-erbB2 antibody trastuzumab [Herceptin™] , the anti-EGFR antibody panitumumab, the anti-erbB 1 antibody cetuximab [Erbitux, C225] and any growth factor or growth factor receptor antibodies disclosed by Stern et al. Critical reviews in oncology/haematology, 2005, Vol. 54, ppl 1-29); such inhibitors also include tyrosine kinase inhibitors, for example inhibitors of the epidermal growth factor family (for example EGFR family tyrosine kinase inhibitors such as N-(3-chloro-4- fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine (gefitinib, ZD1839), N- (3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (erlotinib, OSI-774) and 6- acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazolin-4-amine (CI 1033), erbB2 tyrosine kinase inhibitors such as lapatinib); inhibitors of the hepatocyte growth factor family; inhibitors of the insulin growth factor family; inhibitors of the platelet-derived growth factor family such as imatinib and/or nilotinib (AMΝ107); inhibitors of serine/threonine kinases (for example Ras/Raf signalling inhibitors such as farnesyl transferase inhibitors, for example sorafenib (BAY 43-9006), tipifarnib (Rl 15777) and lonafarnib (SCH66336)), inhibitors of cell signalling through MEK and/or AKT kinases, c-kit inhibitors, abl kinase inhibitors, PI3 kinase inhibitors, Plt3 kinase inhibitors, CSF-IR kinase inhibitors, IGF receptor (insulin- like growth factor) kinase inhibitors; aurora kinase inhibitors (for example AZDl 152, PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 AND AX39459) and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4 inhibitors;

(v) antiangiogenic agents such as those which inhibit the effects of vascular endothelial growth factor, [for example the anti-vascular endothelial cell growth factor antibody bevacizumab (Avastin™) and for example, a VEGF receptor tyrosine kinase inhibitor such as vandetanib (ZD6474), vatalanib (PTK787), sunitinib (SUl 1248), axitinib (AG-013736), pazopanib (GW 786034) and 4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-l- ylpropoxy)quinazoline (AZD2171 ; Example 240 within WO 00/47212), compounds such as those disclosed in International Patent Applications WO97/22596, WO 97/30035, WO 97/32856 and WO 98/13354 and compounds that work by other mechanisms (for example linomide, inhibitors of integrin αvβ3 function and angiostatin)]; (vi) vascular damaging agents such as Combretastatin A4 and compounds disclosed in International Patent Applications WO 99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO 02/08213;

(vii) an endothelin receptor antagonist, for example zibotentan (ZD4054) or atrasentan; (viii) antisense therapies, for example those which are directed to the targets listed above, such as ISIS 2503, an anti-ras antisense;

(ix) gene therapy approaches, including for example approaches to replace aberrant genes such as aberrant p53 or aberrant BRCAl or BRCA2, GDEPT (gene-directed enzyme pro-drug therapy) approaches such as those using cytosine deaminase, thymidine kinase or a bacterial nitroreductase enzyme and approaches to increase patient tolerance to chemotherapy or radiotherapy such as multi-drug resistance gene therapy; and

(x) immunotherapy approaches, including for example ex-vivo and in-vivo approaches to increase the immunogenicity of patient tumour cells, such as transfection with cytokines such as interleukin 2, interleukin 4 or granulocyte-macrophage colony stimulating factor, approaches to decrease T-cell anergy, approaches using transfected immune cells such as cytokine-transfected dendritic cells, approaches using cytokine-transfected tumour cell lines and approaches using anti-idiotypic antibodies.

Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate dosing of the individual components of the treatment. Such combination products employ the compounds of this invention within the dosage range described hereinbefore and the other pharmaceutically-active agent within its approved dosage range.

According to this aspect of the invention there is provided a combination suitable for use in the treatment of a cancer (for example a cancer involving a solid tumour) comprising a compound of formula I, II, III or IV, as defined hereinbefore, or a pharmaceutically acceptable salt thereof, and another anti-tumour agent.

According to this aspect of the invention there is provided a combination suitable for use in the treatment of a cancer (for example a cancer involving a solid tumour) comprising a compound of formula I, II, III or IV, as defined hereinbefore, or a pharmaceutically acceptable salt thereof, and any one of the anti-tumour agents listed under (i) - (ix) above.

In a further aspect of the invention there is provided a compound of formula I, II, III or IV, or a pharmaceutically acceptable salt thereof, in combination with an anti-tumour agent selected from one listed under (i) - (ix) herein above.

Herein, where the term "combination" is used it is to be understood that this refers to simultaneous, separate or sequential administration. In one aspect of the invention "combination" refers to simultaneous administration. In another aspect of the invention "combination" refers to separate administration. In a further aspect of the invention "combination" refers to sequential administration. Where the administration is sequential or separate, the delay in administering the second component should not be such as to lose the beneficial effect of the combination. According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a compound of formula I, II, III or IV, or a pharmaceutically acceptable salt thereof in combination with an anti-tumour agent selected from one listed under (i) - (ix) herein above, in association with a pharmaceutically acceptable diluent or carrier. Examples

The invention will now be illustrated in the following Examples in which, generally: (i) Operations were carried out at ambient temperature, i.e. in the range 17 to 25°C and optionally under an atmosphere of an inert gas such as nitrogen or argon. (ii) In general, the course of the reactions described herein were followed by liquid chromatography mass spectrometry (LCMS). The reaction times that are given are not necessarily the minimum attainable.

(iii) When necessary, organic solutions were dried over anhydrous magnesium sulfate, work-up procedures were carried out using traditional layer separating techniques, evaporations were carried out by rotary evaporation in vacuo.

(iv) Yields, where present, are not necessarily the maximum attainable, and when necessary, reactions were repeated if a larger amount of the reaction product was required. (v) In general, the structures of the end-products were confirmed by nuclear magnetic resonance (NMR) and/or mass spectral techniques. Electrospray mass spectral data were obtained using Agilent 1200 series High Performance Liquid Chromatography unit coupled to an Agilent 6110 series, single quadrupole mass spectrometer acquiring both positive and negative ion data. Generally, only ions relating to the parent structure are reported. Proton NMR chemical shift values were measured on the delta scale at 300 or 400 MHz using a Bruker AV400. The following abbreviations have been used: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br, broad;

(vi) Unless stated otherwise, compounds containing an asymmetric carbon atom were not resolved.

(vii) Intermediates were not necessarily fully purified but their structures and purity were assessed by TLC, analytical HPLC, infra-red (IR) and/or NMR analysis; (viii) Unless otherwise stated, column chromatography (by the flash procedure) and medium pressure liquid chromatography (MPLC) were performed on Merck Kieselgel silica (Art. 9385). (ix) The following analytical HPLC methods were used:

- in general, reversed-phase silica was used with a flow rate of about 1 ml per minute and detection was by Electrospray Mass Spectrometry and by UV absorbance at a wavelength of 254 nm.

- Method 1 (for use with 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone (formula A)): reverse-phase HPLC using a LiChrospher 100 RPl 8 column ( 5μm silica, 125 mm length, 40mm diameter) running with increasing percentage of acetonitrile against water (both containing 0.1% acetic acid). The gradient increases from 5% acetonitrile to 95% over 6 minutes, then plateaus at 95% for 2 minutes, then decreases back to 5% over a further 0.1 minutes. The flow rate is l.Oml/min with the column running at room temperature. The total run time is 13 minutes with UV and MS detection. - Method 2 (for use with Mesna (formula C)): reverse-phase HPLC using a Gemini NX

C18 column (3μm silica, 30 mm length, 2mm diameter) running with increasing percentage of acetonitrile against water (both containing 0.1% trifluoroacetic acid). The gradient increases from 2% acetonitrile to 20% over the first 2 minutes, then over the next 2 it rises to 95%. Then there is a sharp fall back down to 5% acetonitrile over 0.1 minutes. The flow rate is 1.5ml/min with the column running at 30 Degrees Centigrade.

The total run time is 6 minutes with UV and MS detection.

(xi) Compounds were purified by flash silica chromatography using Merck Kieselgel silica

(Art. 9385).

Preparation of starting Materials

Preparation of 4-(l -benzenesulfonyl-6-fluoro- lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone As previously stated, 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone (Formula A) was prepared by reacting 1 -benzenesulfonyl-6-fluoro- IH- indole with 4,4-dimethoxy-cyclohexa-2,5-dienone according to Method C of WO2004/056361 or by the process described in Berry et al. (J. Med.Chem. 2005; 48(2); 639-644).

In an alternative process 4-(l -benzenesulfonyl-6-fluoro- IH- indol-2-yl)-4-hydroxy- cyclohexa-2,5-dienone can be prepared by the process shown in Scheme I below.

cheme I

Formula A

Stage 1

4-Methoxyphenol (50 g; 1 equivalent) was charged to a reaction vessel followed by methanol (500 ml) and the resultant mixture stirred under nitrogen at 15 - 25 ⁰C. (Diacetoxyiodo)benzene (136.3 g, 1.03 equivalents) was charged portion-wise to the reaction mixture which was then maintained at 15 - 25 ⁰C for 60-90 minutes. The completed reaction was quenched with saturated sodium hydrogen carbonate solution (500 ml) and additional charges of solid sodium hydrogen carbonate (180 g) were also added. Water (200 ml) was charged to the resulting suspension and the methanol was removed by distillation. Dichloromethane (500 ml) was then added to the residue. The solids were removed by filtration under vacuum and the organic phase separated from the filtrate. The remaining aqueous phase was extracted with dichloromethane (2 x 500 ml). The combined organic phases were washed with water (300 ml), dried (MgSO₄), filtered and concentrated in vacuo to give a crude residue which was purified by dry flash chromatography (Siθ₂) using heptanes: ethyl acetate (4:1) to give the desired 4,4- dimethoxycyclohexa-2,5-dienone as a yellow oil (53.0 g, 85.4% yield)

Characterising data:

¹H-NMR (400MHz, CDCl₃): 3.38 (6H, s), 6.27 (2H, d, J = 10.4 Hz), 6.84 (2H, d, J = 10.4 Hz).

Stage 2

6-Fluoro- IH- indole (5.0 g; 1 equivalent) was charged to a 3 necked flask under nitrogen with stirring followed by tetrahydrofuran (34 ml, 6.8 vol). The flask contents were cooled to -70 to -78°C and n-butylithium (2.5 M solution in hexanes, 15.2 ml, 1.05 equivalents) charged over 15 to 20 minutes maintaining the reaction below -60 ⁰C. The reaction was warmed to -5 to 0 ⁰C over 50 to 60 minutes, re-cooled to -70 to -78 ⁰C, benzenesulphonyl chloride (5.2 ml, 1.1 equivalents) added with the temperature maintained in the range -60 to -78 ⁰C and then allowed to warm to 15 to 25⁰C and stir for 16 to 18 hours. The reaction was quenched into 2% aqueous sodium hydrogen carbonate solution (100 ml), the organic phase separated and the aqueous phase extracted with ethylacetate (3 x 50ml). The combined organic extracts were washed with 2% aqueous sodium hydrogen carbonate solution (100 ml, 20 vol), water (2 x 50 ml, 2 x 10 vol) and saturated brine solution (50 ml), dried over magnesium sulphate (1Og), filtered under vacuum and the filter-cake washed with ethyl acetate (10 ml). The combined filtrates were concentrated under vacuum to afford crude residue (9.2g) The crude product was re-crystallised from ethanol at reflux, the solution allowed to cool to 15 to 25 ⁰C over 1.5 hours, further cooled to and aged at 0 to 5 ⁰C for 26 minutes. The precipitated solid was then collected by filtration, washed with pre-cooled ethanol (0 to 5 ⁰C, 10 ml, 1.1 vol) and dried under vacuum to afford 1— benzenesulfonyl-6-fluoro-lH-indole as an off-white solid (8.4g, 82.5%).

Characterising data:

¹H-NMR (400MHz, CDCl₃): 6.63 (IH, dd, J = 3.7, 0.7 Hz), 6.98 (IH, td, J = 8.8, 2.3 Hz), 7.43-

7.48 (3H, m), 7.54-7.58 (2H, m), 7.73 (IH, dd, J = 9.7, 2.3 Hz), 7.88-7.92 (2H, m).

Stage 3 l-Benzenesulfonyl-6-fluoro- IH- indole (17.Og, 1 equ) was charged to a 3- necked flask under nitrogen with stirring followed by tetrahydrofuran (170ml). The resulting solution was cooled to -70 to -78°C and n-butyllithium (2.5M solution in hexanes, 32ml, 1.3 equ) added dropwise whilst maintaining the temperature below -60⁰C. The reaction was cooled to between -70 and - 78⁰C and stirred for 90 to 100 minutes (reaction becomes opaque during this time), 4,4- dimethoxycyclohexa-2,5-dienone (9.5g, 61.5mmol, 1 equ) as a solution in tetrahydrofuran (85ml) was then added dropwise at < 60⁰C and the reaction warmed to and maintained at 15 to 20⁰C for 12 to 18 hours. The reaction was then quenched by the addition of 10% aqueous ammonium chloride solution and the pH noted (205ml, pHIO). The pH was adjusted to pH9 by the addition of aqueous hydrochloric acid solution (I M, 40ml) and then adjusted to pH3 by the addition of concentrated hydrochloric acid (2 to 3 ml). The tetrahydrofuran and methanol removed under vacuum at 35 to 40⁰C and the aqueous phase extracted with ethyl acetate (3x 170ml). The combined organic extracts were washed with saturated sodium chloride solution (170ml), dried over magnesium sulphate (5Ig), filtered and the filter-cake washed with ethyl acetate (2x 34ml). The combined organic filtrates were concentrated under vacuum at 35 to 40°C to afford the crude product as a brown solid (22.Ig). The solid was stirred in ethyl acetate:heptanes (2:1, 51 ml) for 10 to 15 minutes at 15 to 25⁰C. The solids were collected under vacuum, washed with ethyl acetate :heptanes (2:1, 2x 0.5 vol) and pulled dry under vacuum to afford 4-(l-,benzenesulfonyl-6-fluoro-l H- indol-2-yl)-4-hydroxycyclohexa-2,5-dienone as a light brown solid (15 g, 64% yield).

Characterising data:

¹H-NMR (400MHz, D₆ DMSO): δ_H= 8.01 (2H, d, J=5.2 Hz), 7.95 (2H, dd, J=2.4, 11.2 Hz), 7.74 (H, t, J=7.6 Hz), 7.60-7.68 (3H, m), 7.52 (2H, d, J= 10.4 Hz), 7.23 (IH, dt, J=2.4, 9.2 Hz), 6.28 (2H, d, J=10.4 Hz)

Example 1 - preparation of a compound of Formula III (disodium 2,2'-(2-(6-fluoro-l- (phenylsulfonvD-lH-indol^-vD^-hydroxy-S-oxocyclohexane-l^- diyl)bis(sulfanediyl)diethanesulfonate)

Formula C (2.2 equivalents, 188 mg) was dissolved in ΗPLC grade water (1.0 ml) in a 25 ml glass reaction vessel. Triethylamine (0.1 equivalents, 7.3 μl) was added and the mixture heated to 60 ⁰C with stirring. 4-(l-Benzenesulfonyl-6-fluoro-lΗ-indol-2-yl)-4-hydroxy-cyclohexa-2,5- dienone (Formula A, 1 equivalent, 200 mg) was dissolved in HPLC acetone (12 ml) and added drop- wise to the Mesna/TEA solution over 10-13 minutes with stirring, whilst maintaining the reaction temperature at approximately 60 ⁰C. The solution was stirred at reflux for 6 hours and then left to cool down to 28-30 ⁰C over 40 minutes. The resultant white solids were filtered off, washed with 7 ml of acetone and dried under vacuum overnight to give the desired product in pure form. Characterising data:

¹H-NMR (300MHz, D₆ DMSO): δ_H= 7.89-7.92 (2H, m), 7.78 (IH, dd, J=2, 11.2 Hz), 7.69-7.57 (4H, m), 7.15 (IH, dt, J=9.2, 2.4 Hz), 7.07 (IH, s), 4.21 (IH, t, J=4 Hz), 3.95 (IH, m), 3.43 (IH, dd, J=4.4, 15.2 Hz), 2.94.(1H, dd, J=I 1.2, 14.8 Hz), 2.88-2.84 (2H, m), 2.72-2.42 (6H, m) m/z (LC-MS, ESP), RT= 3.8 min, (M^2~+Na)= 332.7

Example 2 - Alternative preparation of a compound of Formula III (disodium 2,2'-(2-(6- fluoro-l-fphenylsulfonvD-lH-indol^-vD^-hydroxy-S-oxocvclohexane-l^- diyl)bis(sulfanediyl)diethanesulfonate)

Formula C (2.2 equivalents; 2.35g) was dissolved in ΗPLC grade water (10 ml) in a 250 ml 3 neck round-bottom flask fitted with a thermometer, condenser and dropping funnel. Triethylamine (TEA) (0.1 equivalents; 91 μl) was added and the mixture was heated to 60⁰C with stirring. 4-(l-Benzenesulfonyl-6-fluoro-lΗ-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone (Formula A; 1 equivalent; 2.5 g) was dissolved in HPLC grade acetone (120 ml) and transferred into the 150 ml dropping funnel. This solution was added drop-wise to the Mesna/TEA solution (reactor temperature 59°C) over 30 minutes with stirring. The reaction was stirred at reflux for 6 hours. The reaction was then slowly cooled over 40 minutes to 30⁰C. The resultant white solids were removed by filtration before being transferred into a beaker and washed with acetone (30 ml), filtered once more and washed with acetone (45ml). The resultant solids were dried to constant weight at room temperature over 5 days under vacuum in a desiccator with CaCl₂ to yield the desired product. Yield: 4.37g of a white solids were recovered (94% crude yield), 98.5%. Characterising data:

¹H-NMR (400MHz, D₆ DMSO): δ_H= 7.89-7.92 (2H, m), 7.78 (IH, dd, J=2, 11.2 Hz), 7.69-7.57 (4H, m), 7.15 (IH, dt, J=9.2, 2.4 Hz), 7.07 (IH, s), 4.21 (IH, t, J=4 Hz), 3.95 (IH, m), 3.43 (IH, dd, J=4.4, 15.2 Hz), 2.94.(1H, dd, J=I 1.2, 14.8 Hz), 2.88-2.84 (2H, m), 2.72-2.42 (6H, m) m/z (LC-MS, ESP), RT= 3.8 min, (M^2~+Na)= 332.7

Example 3 - Preparation of a compound of Formula IV (sodium 2-(2-(6-fluoro-l- (phenylsulfonvD-lH-indol^-vP^-h^ydroxy-S-oxocvclohex-S-en^ylthiolethanesulfonate)

IV

To 4-(l-benzenesulfonyl-6-fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone (Formula A; 1 g; 2.61 mmol) and triethylamine (0.05 g) in acetone (50 mL) at 50 ⁰C was added Mesna (sodium-2-mercaptoethanesulfonate or Formula C; 0.387 g; 2.357 mmol; 0.9 eq.) in water (3.5 mL) drop-wise over 5 minutes. Heating continued for 1 hour, then the mixture was allowed to cool. The mixture was then filtered and the precipitate washed with acetone to give disodium 2,2'-(2-(6-fluoro-l-(phenylsulfonyl)-lH-indol-2-yl)-2-hydroxy-5-oxocyclohexane-l,3- diyl)bis(sulfanediyl)diethanesulfonate (Formula III; nmr: ¹H-NMR (400MHz, D₆ DMSO): δ_H= 7.89- 7.92 (2H, m), 7.78 (IH, dd, J=2, 11.2 Hz), 7.69-7.57 (4H, m), 7.15 (IH, dt, J=9.2, 2.4 Hz), 7.07 (IH, s), 4.21 (IH, t, J=4 Hz), 3.95 (IH, m), 3.43 (IH, dd, J=4.4, 15.2 Hz), 2.94.(1H, dd, J=I 1.2, 14.8 Hz), 2.88- 2.84 (2H, m), 2.72-2.42 (6H, m) as a white powder (0.329 g). The filtrate was concentrated to an oil, to which ether was added and the mixture stirred, and the solvent decanted. This was repeated twice. The ether layer was concentrated to yield 0.291 g of 4-(l-benzenesulfonyl-6- fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone/Formula A (after recrystallisation from iso-propyl alcohol). After the traces of ether had evaporated from the oil residue, iso- propylalcohol was added and, with swirling and sonication, a white powder formed. This was filtered to give the title compound (Formula IV; sodium 2-(2-(6-fluoro-l-(phenylsulfonyl)-lH- indol-2-yl)-2-hydroxy-5-oxocyclohex-3-enylthio)ethanesulfonate). Characterising data:

¹H-NMR (400MHz, D₆ DMSO): δ_H= 7.84 (2H, d, J=8.0 Hz), 7.78 (IH, d, J=9.2 Hz), 7.71-7.62 (3H, m), 7.58 (IH, t, J=8.8Hz), 7.75 (2H, dt, J=2.0, 8.8 Hz), 7.07, (IH, s), 7.00 (IH, d, J=IO Hz), 6.40 (IH, Broad s), 6.06 (IH, d, J=I 1.2, 10 Hz), 4.26-4.46 (2H, m), 2.91-2.54 (4H, m) m/z (LC-MS, ESP), RT= 4.2 min, (M )= 333.9

Example 4 - Kinetic solubility

The relative solubility of the compound of formula III and 4-(l-benzenesulfonyl-6- fluoro-lH-indol-2-yl)-4-hydroxy-cyclohexa-2,5-dienone in water were assessed as follows:

A stock solution of lmg/ml of the compound was generated in DMSO. A 2 μl sample of this stock solution was injected and analysed by LC-MS and acted as a standard solution of a known concentration so that the correlation between peak area (as viewed by a UV detector tuned to an absorption frequency of 254 nm, versus time) of the saturated aqueous analyte solution and that of the DMSO analyte standard solution could be compared and a time limited kinetic solubility thus determined.

Further to this, 100 μl of the stock solution was added every 30 seconds to a vial containing 1 ml water. As soon as the mixture became turbid, the sample was filtered (single use syringe filter, PTFE-membrane, PP-housing, poresize 45 μM). 2 μl of the filtered solution was then injected directly onto the LCMS for analysis.

The comparison of peak areas between standard and analyte solutions then allowed the direct determination of aqueous solubility. The results are shown in Table 2 below:

Table 2

Claims

1. A compound of formula I:

I wherein:

X⁺ is a pharmaceutically acceptable cation; or a pharmaceutically acceptable salt thereof.

2. A compound of formula II:

II wherein:

X⁺ is a pharmaceutically acceptable cation; or a pharmaceutically acceptable salt thereof.

3. A compound according to claim 1 or claim 2, wherein X⁺ is a monovalent cation.

4. A compound according to any one of the preceding claims, wherein X⁺ is Na⁺.

5. A compound according to claim 1, which has the structural formula III shown below:

III

6. A compound according to claim 2, which has the structural formula IV shown below:

7. A pharmaceutical composition which comprises a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable thereof, in association with a pharmaceutically- acceptable diluent or carrier.

8. A method of treatment of a cancer comprising administering to a human or animal in need of such treatment a therapeutically effective amount of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.

9. A compound according to any one of claims 1 to 6, for use as a medicament.

10. A compound according to any one of claims 1 to 6, for use in the treatment of cancer.