This application claims the benefit of the priority date established by UK patent application number 1317166.5 filed on 27 September 2013, the contents of which are incorporated herein by reference.

Field of the Invention

This invention relates to dinucleotide compounds. More particularly, the invention relates to dinucleotide derivatives of gemcitabine and cytarabine. Also provided are pharmaceutical compositions containing the compounds and the therapeutic uses of the compounds.

Background of the Invention

Gemcitabine and cytarabine (also known as Ara-C) are antimetabolite drugs that are useful in the treatment of cancers. Although beneficial for some patients, new agents with improved properties are needed to overcome issues of resistance, poor pharmacokinetics, toxicity and inter-individual variability. One example of an enzyme whose expression and activity are linked with poor efficacy is cytidine deaminase (CDA). Others include deoxycytidine kinase (DCK), transporters such as human equilibrative nucleoside transporter 1 (ENT1) and ribonucleotide reductase (RR). The poor pharmacokinetics of intravenous gemcitabine and cytarabine dosing are demonstrated by the short mean residence time (MRT).

A number of derivatives and prodrugs based on cytarabine and gemcitabine have been explored but, to date, none have progressed to market. Examples of these include elacytarabine (Clavis Pharma ASA), which is an elaidic acid derivative of cytarabine. This molecule was resistant to metabolism by CDA and was designed to improve the pharmacokinetics of cytarabine. It reached a phase 3 study but failed to show advantage versus a clinician's choice of treatment in acute myeloid leukaemia (AML) patients.

The elaidic acid derivative of gemcitabine (CP-4126, Clovis Oncology Inc and Clavis Pharma ASA) has also reached phase 2 clinical trials. It was hoped that this molecule would be more ac ^t ive in patients with transporter deficiencies (ENT1 ) as the prodrug was shown to work in transporter deficient (ENT1 ) cell lines. However the clinical trial failed to show any improvement in overall survival with the prodrug compared to the gemcitabine standard of care. The Invention

The invention provides dinucleotide derivatives of gemcitabine and cytarabine.

Accordingly, in a first aspect, the present invention provides a compound of the formula (1 ):

or a salt or tautomer thereof, wherein either (i) R ¹ is hydroxy and R ² is hydrogen; or (ii) R ¹ and R ² are both fluorine; and R ³ is hydroxy or a group convertible under physiological conditions to hydroxy.

The group R ³ can be hydroxy or a group that can be transformed under physiological conditions to give a hydroxy group. Compounds containing such groups therefore act as prodrugs of compounds wherein R ³ is hydroxy. The transformation takes place in vivo when the compound of formula (1 ) is administered to a subject and can, for example, involve simple hydrolysis or an enzymatic conversion.

In one embodiment, R ³ is hydroxy.

In another embodiment, R ³ is a group which is convertible under physiological conditions to hydroxy.

Examples of groups R ³ that can be transformed into a hydroxy group include those containing an organic moiety and an oxygen, nitrogen or sulphur atom which is linked to the phosphorus atom of the phosphoryl group.

Examples of groups R ³ wherein R ³ is an O-linked group include esters such as: acyloxyalkyl esters (for example pivaloyloxymethyl 'POM' ester prod

S-acylthioethyl (SATE) esters (for example, methyl or tert-butyl SATE esters);

• aryl esters (for example, ortho-ethoxyphenyl ester);

benzyl esters (for example, para-acetoxybenzyl ester); and

• lipid esters (for example, hexadecyloxypropyl ester).

Examples of groups R ³ wherein R ³ is an N-linked group include phosphoramidates and include amino acid ester phosphoramidates such as methyl alanyl phosphoramidate.

Examples of groups R ³ wherein R ³ is an S-linked group are phosphorothioates such as S-(isopropoxycarbonyl)oxymethyl phosphorothioate. Thus, in another embodiment, (i) R is hydroxy and R ² is hydrogen; or (ii) R and R ² are both fluorine; and R ³ is a group that is convertible under physiological conditions to hydroxy and is selected from acyloxyalkoxy; S-acylthioethoxy; aryloxy; arylalkoxy; alkoxyalkoxy; an N-linked amino acid ester residue; and an S-linked substituted alkyl residue. In another embodiment, R ¹ is hydroxy, R ² is hydrogen and R ³ is hydroxy and hence the compound ha? the formula (2):

OH

(2)

or a salt or tautomeric form thereof. In a further embodiment, R ¹ and R ² are both fluorine and R ³ is hydroxy and hence the compound has the formula (3):

OH

(3)

or a salt or tautomeric form thereof. Definitions

All references to compounds of the formula (1), (2) or (3) herein include salts, tautomers, isotopic variants and solvates thereof, as defined below, unless the context indicates otherwise.

As used herein, the term "combination", as applied to two or more compounds and/or agents (also referred to herein as the components), is intended to define material in which the two or more compounds/agents are associated. The terms "combined" and "combining" in this context are to be interpreted accordingly.

The association of the two or more compounds/agents in a combination may be physical or non-physical. Examples of physically associated combined

compounds/agents include:

• compositions (e.g. unitary formulations) comprising the two or more

compounds/agents in admixture (for example within the same unit dose);

• compositions comprising material in which the two or more compounds/agents are chemically/physicochemically linked (for example by crosslinking, molecular agglomeration or binding to a common vehicle moiety); • compositions comprising material in which the two or more compounds/agents are chemically/physicochemically co-packaged (for example, disposed on or within lipid vesicles, particles (e.g. micro- or nanoparticles) or emulsion droplets);

· pharmaceutical kits, pharmaceutical packs or patient packs in which the two or more compounds/agents are co-packaged or co-presented (e.g. as part of an array of unit doses);

Examples of non-physically associated combined compounds/agents include:

• material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds/agents together with instructions for the extemporaneous association of the at least one compound to form a physical association of the two or more compounds/agents;

• material (e.g. a non-unitary formulation) comprising at least one of the two or more compounds/agents together with instructions for combination therapy with the two or more compounds/agents;

• material comprising at least one of the two or more compounds/agents together with instructions for administration to a patient population in which the other(s) of the two or more compounds/agents have been (or are being) administered;

• material comprising at least one of the two or more compounds/agents in an amount or in a form which is specifically adapted for use in combination with the other(s) of the two or more compounds/agents.

As used herein, the term "combination therapy" is intended to define therapies which comprise the use of a combination of two or more compounds/agents (as defined above). Thus, references to "combination therapy", "combinations" and the use of compounds/agents "in combination" in this application may refer to compounds/agents that are administered as part of the same overall treatment regimen. As such, the posology of each of the two or more compounds/agents may differ: each may be administered at the same time or at different times. It will therefore be appreciated that the compounds/agents of the combination may be administered sequentially (e.g. before or after) or simultaneously, either in the same pharmaceutical formulation (i.e. together), or in different pharmaceutical formulations (i.e. separately). Simultaneously in the same formulation is as a unitary formulation whereas simultaneously in different pharmaceutical formulations is non-unitary. The posoiogies of each of the two or more compounds/agents in a combination therapy may also differ with respect to the route of administration.

As used herein, the term "pharmaceutical kit" defines an array of one or more unit doses of a pharmaceutical composition together with dosing means (e.g. measuring device) and/or delivery means (e.g. inhaler or syringe), optionally all contained within common outer packaging. In pharmaceutical kits comprising a combination of two or more compounds/agents, the individual compounds/agents may unitary or non-unitary formulations. The unit dose(s) may be contained within a blister pack. The

pharmaceutical kit may optionally further comprise instructions for use.

As used herein, the term "pharmaceutical pack" defines an array of one or more unit doses of a pharmaceutical composition, optionally contained within common outer packaging. In pharmaceutical packs comprising a combination of two or more compounds/agents, the individual compounds/agents may unitary or non-unitary formulations. The unit dose(s) may be contained within a blister pack. The pharmaceutical pack may optionally further comprise instructions for use.

As used herein, the term "patient pack" defines a package, prescribed to a patient, which contains pharmaceutical compositions for the whole course of treatment.

Patient packs usually contain one or more blister pack(s). Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

The combinations of the invention may produce a therapeutically efficacious effect relative to the therapeutic effect of the individual compounds/agents when

administered separately.

Salts

Compounds of the formulae (1 ), (2) and (3) can form salts with acids or bases.

Acid addition salts can be synthesized from the parent compound by conventional chemical methods such as methods described in Pharmaceutical Salts: Properties, Selection, and Use, P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3- 90639-026-8, Hardcover, 388 pages, August 2002. Generally, such salts can be prepared by reacting the free base form of the compound with the acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are used.

Acid addition salts may be formed with a wide variety of acids, both inorganic and organic. Examples of acid addition salts include salts formed with an acid selected from the group consisting of acetic, 2,2-dichloroacetic, adipic, alginic, ascorbic (e.g. L- ascorbic), L-aspartic, benzenesulphonic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, D-gluconic, glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a- oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, (+)-L- lactic, (±)-DL-lactic, lactobionic, maleic, malic, (-)-L-malic, malonic, (±)-DL-mandelic, methanesulphonic, naphthalene-2-sulphonic, naphthalene-1 ,5-disulphonic, 1-hydroxy- 2-naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, (+)-L-tartaric, thiocyanic, p-toluenesulphonic, undecyienic and valeric acids, as well as acylated amino acids and cation exchange resins.

Particular acid addition salts are hydrochloric acid salts.

Metal salts can arise from the addition of an inorganic base to a compound described herein. The inorganic base consists of a metal cation paired with a basic counterion, such as, for example, hydroxide, carbonate, bicarbonate, or phosphate. The metal can be an alkali metal, alkaline earth metal, transition metal, or main group metal. Non-limiting examples of suitable metals include lithium, sodium, potassium, caesium, cerium, magnesium, manganese, iron, calcium, strontium, cobalt, titanium, aluminium, copper, cadmium, and zinc.

Non-limiting examples of suitable metal salts include a lithium salt, a sodium salt, a potassium salt, a caesium salt, a cerium salt, a magnesium salt, a manganese salt, an iron salt, a calcium salt, a strontium salt, a cobalt salt, a titanium salt, a aluminium salt, a copper salt, a cadmium salt, and a zinc salt.

Ammonium salts can arise from the addition of ammonia or an organic amine to a compound described herein. Non-limiting examples of suitable organic amines include triethyl amine, diisopropyl amine, ethanol amine, diethanol amine, triethanol amine, morpholine, N-methylmorpholine, piperidine, N-methylpiperidine, N-ethylpiperidine, dibenzyl amine, piperazine, pyridine, pyrrazole, pipyrrazole, imidazole, pyrazine, pipyrazine, ethylenediamine, Ν,Ν'-dibenzylethylene diamine, procaine, chloroprocaine, choline, dicyclohexyl amine, and N-methylglucamine.

Non-limiting examples of suitable ammonium salts include a triethyl amine salt, a diisopropyl amine salt, an ethanol amine salt, a diethanol amine salt, a triethanol amine salt, a morpholine salt, an N-methylmorpholine salt, a piperidine salt, an N- methylpiperidine salt, an N-ethylpiperidine salt, a dibenzyl amine salt, a piperazine salt, a pyridine salt, a pyrrazole salt, a pipyrrazole salt, an imidazole salt, a pyrazine salt, a pipyrazine salt, an ethylene diamine salt, an Ν,Ν'-dibenzylethylene diamine salt, a procaine salt, a chloroprocaine salt, a choline salt, a dicyclohexyl amine salt, and a N- methylglucamine salt.

In one embodiment, the salts are selected from sodium, ammonium and substituted ammonium salts. More particularly, the salts can be selected from ammonium (NH ₄ ⁺ ) and sodium salts.

The salt forms of the compounds of the invention are typically pharmaceutically acceptable salts, and examples of pharmaceutically acceptable salts are discussed in Berge er a/., 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1 - 19. However, salts that are not pharmaceutically acceptable may also be prepared as intermediate forms which may then be converted into pharmaceutically acceptable salts. Such non-pharmaceutically acceptable salts forms, which may be useful, for example, in the purification or separation of the compounds of the invention, also form part of the invention.

Tautomers

The compounds of the invention may in principle exist in a number of different tautomeric forms and references to the compounds of formulae (1), (2) and (3) include all such forms. For the avoidance of doubt, where a compound can exist in one of several tautomeric forms and only one is specifically described or shown, all others are nevertheless embraced by formulae (1 ), (2) or (3).

Isotopic variants

The compounds of formulae (1 ), (2) and (3) may contain one or more isotopic substitutions, and a reference to a particular element includes within its scope all isotopes of the element. For example, a reference to hydrogen includes within its scope ¹ H, ² H (D), and ³ H (T). Similarly, references to carbon and oxygen include within their scope respectively ² C, ³ C and ¹⁴ C and ^δ Ο and ¹⁸ 0. The isotopes may be radioactive or non-radioactive. In one embodiment of the invention, the compounds contain no radioactive isotopes. Such compounds are preferred for therapeutic use. In another embodiment, however, the compound may contain one or more radioisotopes. Compounds containing such radioisotopes may be useful in a diagnostic context.

Solvates

Compounds of the formulae (1 ), (2) and (3) may form solvates.

Preferred solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates.

Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3.

Biological Activity

The compounds of the formulae (1 ), (2) and (3) inhibit the growth of cancer cells and are useful as anticancer agents.

The activity of the compounds against cancer cell lines can be determined using the protocol described in Example A below.

Accordingly, in further embodiments, the invention provides:

· A compound of the formula (1 ), (2) or (3) or a salt or tautomer thereof for use in medicine or therapy; • A compound of the formula (1 ), (2) or (3) or a salt or tautomer thereof for use in the treatment of a proliferative disease such as cancer;

• The use of a compound of the formula (1), (2) or (3) or a salt or tautomer

thereof for the manufacture of a medicament for the treatment of a proliferative disease such as cancer;

• A method for the prophylaxis or treatment of a proliferative disease such as cancer, which method comprises administering to a patient, optionally in combination with radiotherapy or another chemotherapeutic agent a compound of the formula (1 ), (2) or (3) or a salt or tautomer thereof;

· A method for the prophylaxis or treatment of a proliferative disease such as cancer, which method comprises administering to a patient a compound of the formula (1). (2) or (3) or a salt or tautomer thereof.

In each of the foregoing embodiments, the cancer may be selected from carcinomas, for example carcinomas of the bladder, breast, colon, kidney, epidermis, liver, lung, oesophagus, gall bladder, ovary, pancreas, stomach, cervix, thyroid, prostate, gastrointestinal system, or skin, hematopoieitic tumours such as leukaemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma, or Burkett's lymphoma; hematopoieitic tumours of myeloid lineage, for example acute and chronic myelogenous leukaemias, myelodysplasia syndrome, or promyelocytic leukaemia; thyroid follicular cancer; tumours of mesenchymal origin, for example fibrosarcoma or habdomyosarcoma; tumours of the central or peripheral nervous system, for example astrocytoma, neuroblastoma, glioma or schwannoma; melanoma; seminoma; teratocarcinoma; osteosarcoma; xeroderma pigmentosum; keratoctanthoma; thyroid follicular cancer; or Kaposi's sarcoma.

In one embodiment, the cancer is selected from non-small cell lung cancer, pancreatic cancer, bladder cancer, breast cancer, acute myeloid leukemia (AML), acute lymphocytic leukaemia (ALL) and lymphomas such as non-Hodgkin lymphoma.

In another embodiment, a compound of formula (1 ) wherein R ¹ is hydroxy and R ² is hydrogen is used for the treatment of hematopoieitic tumours. For example, the compound can be used for the treatment of acute myeloid leukemia (AML), acute lymphocytic leukaemia (ALL) and in lymphomas such as non-Hodgkin lymphoma.

In another embodiment, the compound of formula (1 ) wherein R is hydroxy and R ² is hydrogen is used for the treatment of a cancer selected from acute myeloid leukaemia (AML), acute non-lymphoblastic leukaemias, acute lymphoblastic leukaemias, acute lymphocytic leukaemia (ALL), erythroleukaemia, blast crises of chronic myeloid leukaemia, diffuse histiocytic lymphomas (non-Hodgkin's lymphomas of high malignancy), meningeal leukaemia and meningeal neoplasms.

In another embodiment, a compound of formula (1 ) wherein R and R ² are both fluorine can be used for the treatment of a cancer selected from ovarian cancer; breast cancer; non-small cell lung cancer; pancreatic cancer; and bladder cancer. For example, the compound can be used for the treatment of a cancer selected from locally advanced or metastatic bladder cancer; locally advanced or metastatic adenocarcinoma of the pancreas; locally advanced or metastatic non-small cell lung cancer (NSCLC); locally advanced or metastatic epithelial ovarian carcinoma; and unresectable, locally recurrent or metastatic breast cancer.

Pharmaceutical Formulations

While it is possible for the active compound to be administered alone, it is preferable to present it as a pharmaceutical composition (e.g. formulation).

Accordingly, in another embodiment of the invention, there is provided a

pharmaceutical composition comprising at least one compound of the formula (1 ), (2) or (3) or a salt or tautomer as defined herein together with a pharmaceutically acceptable excipient.

The pharmaceutically acceptable excipient can be, for example, a carrier (e.g. a solid, liquid or semi-solid carrier), a diluent or bulking agent, a granulating agent, coating agent, binding agent, disintegrant, lubricating agent, preservative, antioxidant, buffering agent, suspending agent, thickening agent, flavouring agent, sweetener, taste masking agent or any other excipient conventionally used in pharmaceutical compositions. Examples of excipients for various types of pharmaceutical

compositions are set out in more detail below.

The pharmaceutical compositions can be in any form suitable for oral, parenteral, topical, intranasal, ophthalmic, otic, rectal, intra-vaginal, or transdermal administration. Typically, however, they are formulated for parenteral administration, for example for administration by intravenous, intramuscular, intraperitoneal, subcutaneous administration or for direct delivery into a target organ or tissue by injection, infusion or other means of delivery. The delivery can be by bolus injection, short term infusion or longer term infusion and can be via passive delivery or through the utilisation of a suitable infusion pump. In one particular embodiment, the compounds are administered sub-cutaneously.

Pharmaceutical formulations adapted for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats, co-solvents, organic solvent mixtures, cyclodextrin complexation agents, emulsifying agents (for forming and stabilizing emulsion formulations), liposome components for forming liposomes, gellable polymers for forming polymeric gels, lyophilisation protectants and combinations of agents for, inter alia, stabilising the active ingredient in a soluble form and rendering the formulation isotonic with the blood of the intended recipient. Pharmaceutical formulations for parenteral administration may also take the form of aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents (R. G. Strickiy, Solubilizing Excipients in oral and injectable formulations, Pharmaceutical Research, Vol 21 (2) 2004, p 201 -230).

A drug molecule that is ionizable can be solubilized to the desired concentration by pH adjustment if the drug's pK _a is sufficiently away from the formulation pH value. The acceptable range is pH 2-12 for intravenous and intramuscular administration, but subcutaneously the range is pH 2.7-9.0. The solution pH is controlled by either the salt form of the drug, strong acids/bases such as hydrochloric acid or sodium hydroxide, or by solutions of buffers which include but are not limited to buffering solutions formed from glycine, citrate, acetate, maleate, succinate, histidine, phosphate, tris(hydroxymethyl)- aminomethane (TRIS), or carbonate.

The combination of an aqueous solution and a water-soluble organic solvent/surfactant (i.e., a cosolvent) is often used in injectable formulations. The water-soluble organic solvents and surfactants used in injectable formulations include but are not limited to propylene glycol, ethanol, polyethylene glycol 300, polyethylene glycol 400, glycerin, dimethylacetamide (DMA), N-methyl-2-pyrrolidone (NMP; Pharmasolve),

dimethylsulphoxide (DMSO), Solutol HS 15, Cremophor EL, Cremophor RH 60, and polysorbate 80. Such formulations can usually be, but are not always, diluted prior to injection.

Propylene glycol, PEG 300, ethanol, Cremophor EL, Cremophor RH 60, and polysorbate 80 are the entirely organic water-miscible solvents and surfactants used in commercially available injectable formulations and can be used in combinations with each other. The resulting organic formulations are usually diluted at least 2-fold prior to IV bolus or IV infusion. Alternatively increased water solubility can be achieved through molecular complexation with cyclodextrins.

The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

The pharmaceutical formulation can be prepared by lyophilising a compound of Formula (1 ), (2) or (3) or a salt or tautomer thereof. Lyophilisation refers to the procedure of freeze-drying a composition. Freeze-drying and lyophilisation are therefore used herein as synonyms. A typical process is to solubilise the compound and the resulting formulation is clarified, sterile filtered and aseptically transferred to containers appropriate for lyophilisation (e.g. vials). In the case of vials, they are partially stoppered with lyo-stoppers. The formulation can be cooled to freezing and subjected to lyophilisation under standard conditions and then hermetically capped forming a stable, dry lyophile formulation. The composition will typically have a low residual water content, e.g. less than 5% e.g. less than 1 % by weight based on weight of the lyophile.

The lyophilisation formulation may contain other excipients for example, thickening agents, dispersing agents, buffers, antioxidants, preservatives, and tonicity adjusters. Typical buffers include phosphate, acetate, citrate and glycine. Examples of antioxidants include ascorbic acid, sodium bisulphite, sodium metabisulphite, monothioglycerol, thiourea, butylated hydroxytoluene, butylated hydroxyl anisole, and ethylenediaminetetraacetic acid salts. Preservatives may include benzoic acid and its salts, sorbic acid and its salts, alkyl esters of para-hydroxybenzoic acid, phenol, chlorobutanol, benzyl alcohol, thimerosal, benzalkonium chloride and cetylpyridinium chloride. The buffers mentioned previously, as well as dextrose and sodium chloride, can be used for tonicity adjustment if necessary.

Bulking agents are generally used in lyophilisation technology for facilitating the process and/or providing bulk and/or mechanical integrity to the lyophilized cake. Bulking agent means a freely water soluble, solid particulate diluent that when co- lyophilised with the compound or salt thereof, provides a physically stable lyophilized cake, a more optimal freeze-drying process and rapid and complete reconstitution. The bulking agent may also be utilised to make the solution isotonic.

The water-soluble bulking agent can be any of the pharmaceutically acceptable inert solid materials typically used for lyophilisation. Such bulking agents include, for example, sugars such as glucose, maltose, sucrose, and lactose; polyalcohols such as sorbitol or mannitol; amino acids such as glycine; polymers such as

polyvinylpyrrolidine; and polysaccharides such as dextran.

The ratio of the weight of the bulking agent to the weight of active compound is typically within the range from about 1 to about 5, for example of about 1 to about 3, e.g. in the range of about 1 to 2.

Alternatively they can be provided in a solution form which may be concentrated and sealed in a suitable vial. Sterilisation of dosage forms may be via filtration or by autoclaving of the vials and their contents at appropriate stages of the formulation process. The supplied formulation may require further dilution or preparation before delivery for example dilution into suitable sterile infusion packs.

Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In one preferred embodiment of the invention, the pharmaceutical composition is in a form suitable for i.v. administration, for example by injection or infusion.

In another preferred embodiment, the pharmaceutical composition is in a form suitable for sub-cutaneous (s.c.) administration.

Particular examples of formulations for parenteral (e.g. sub-cutaneous) administration include compositions in which the compound of formula (1 ), (2) or (3) or a

pharmaceutically-ucceptable salt thereof is dissolved in a substantially anhydrous solvent comprising about 45% to about 85% propylene glycol; about 5% to about 45% glycerin; and 0% to about 30% ethanol.

In one embodiment, the solvent comprises about 65% to about 70% propylene glycol; about 25% to about 30% glycerin, and 0% to about 10% ethanol.

Ethanol can be incorporated as a thinning agent or can be eliminated while retaining suitable handling/reconstitution characteristics.

In some embodiments, the solvent comprises about 65% propylene glycol; about 25% glycerin; and about 10% ethanol, for example being 65% propylene glycol; 25% glycerin; and 10% ethanol.

In other embodiments, the solvent comprises 65% to 70% propylene glycol and 25% to 30% glycerin, any balance being ethanol. In other embodiments, the solvent comprises about 70% propylene glycol and about 30% glycerin, ethanol being absent.

In further embodiments, the solvent comprises: 45% to 85% propylene glycol; 5% to 45% glycerin; and 0% to 30% ethanol; or 65% to 70% propylene glycol; 25% to 30% glycerin, and 0% to 10% ethanol.

The compound of formula (1), (2) or (3) can be present at a concentration of about 80 mg/mL to about 1 10 mg/mL, for example about 100 mg/mL

The sub-cutaneous formulations may further comprise dimethyl sulfoxide (DMSO), optionally at a DMSOxompound ratio of about 2: about 1 ; about V. about 1 ; about 0.5: about 1 ; about 0.3: about 1 ; or about 0.2 - about 0.3: about 1.

In another aspect, the invention provides a kit comprising: (a) a first vessel containing a compound of the formula (1 ), (2) or (3) or pharmaceutically-acceptable salt thereof as described herein; and (b) a second vessel containing a substantially anhydrous solvent comprising about 45% to about 85% propylene glycol; about 5% to about 45% glycerin; and 0% to about 30% ethanol.

The compound in the kit of the invention can be present in the form of a substantially anhydrous powder, for example a lyophilized powder. The compound can be present in the first vessel in an amount of about 80 mg to about 1 10 mg, for example about 100 mg. In some embodiments, the kit further comprises instructions for

administration by subcutaneous injection.

When the compounds are orally active, pharmaceutical dosage forms suitable for oral administration include tablets, capsules, caplets, pills, lozenges, syrups, solutions, powders, granules, elixirs and suspensions. The preparation of oral dosage forms can be carried out in accordance with methods well known to the skilled person, , see for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA, USA.

The compound of formula (1 ), (2) or (3) or their salts or tautomers as defined herein, may be formulated with a carrier and administered in the form of nanoparticles.

Nanoparticles offer the possibility of direct penetration into the cell. Nanoparticle drug delivery systems are described in "Nanoparticle Technology for Drug Delivery", edited by Ram B Gupta and Uday B. Kompella, Informa Healthcare, ISBN 9781574448573, published 13 ^th March 2006. Nanoparticles for drug delivery are also described in J. Control. Release, 2003, 91 (1-2), 167-172, and in Sinha et al., Mol. Cancer Ther. August 1 , (2006) 5, 1909. The pharmaceutical formulations may be presented to a patient in "patient packs" containing an entire course of treatment in a single package, usually a blister pack. Patient packs have an advantage over traditional prescriptions, where a pharmacist divides a patient's supply of a pharmaceutical from a bulk supply, in that the patient always has access to the package insert contained in the patient pack, normally missing in patient prescriptions. The inclusion of a package insert has been shown to improve patient compliance with the physician's instructions.

Compositions for parenteral administration are typically presented as sterile aqueous or oily solutions or fine suspensions, or may be provided in finely divided sterile powder form for making up extemporaneously with sterile water for injection.

Examples of formulations for rectal or intra-vaginal administration include pessaries and suppositories which may be, for example, formed from a shaped moldable or waxy material containing the active compound.

The compounds of the formula (1 ) will generally be presented in unit dosage form and, as such, will typically contain sufficient compound to provide a desired level of biological activity. For example, a formulation may contain from 1 nanogram to 2 grams of active ingredient, e.g. from 1 nanogram to 2 milligrams of active ingredient. Within this range, particular sub-ranges of compound are 0.1 milligrams to 2 grams of active ingredient (more usually from 10 milligrams to 1 gram, e.g. 50 milligrams to 500 milligrams), or 1 microgram to 20 milligrams (for example 1 microgram to 10 milligrams, e.g. 0.1 milligrams to 2 milligrams of active ingredient).

For oral compositions, a unit dosage form may contain from 1 milligram to 2 grams, more typically 10 milligrams to 1 gram, for example 50 milligrams to 1 gram, e.g. 100 miligrams to 1 gram, of active compound.

The active compound will be administered to a patient in need thereof (for example a human or animal patient) in an amount sufficient to achieve the desired therapeutic effect.

Methods of Treatment and Combination Therapy

It is envisaged that the compounds of the formula (1 ), (2) or (3) or their salts or tautomers as defined herein will be useful either alone or in combination therapy with other chemotherapeutic agents or radiation therapy in the prophylaxis or treatment of a range of proliferative disease states or conditions. Examples of such disease states and conditions are set out above. The compounds of formula (1 ), (2) or (3) or their salts or tautomers whether administered alone, or in combination with anti-cancer agents and therapies such as radiotherapy, are generally administered to a subject in need of such administration, for example a hurr.an or animal patient, preferably a human.

Examples of chemotherapeutic agents that may be co-administered with the compounds of formula (1), (2) or (3) or their salts or tautomers as defined herein include:

Examples of other therapeutic agents or treatments that may be administered together (whether concurrently or at different time intervals) with the compounds of the formula (1 ) include but are not limited to:

topoisomerase I inhibitors;

other antimetabolites;

tubulin targeting agents;

DNA binder and topoisomerase II inhibitors;

· alkylating Agents;

monoclonal Antibodies;

anti-hormones;

signal transduction inhibitors;

proteasome inhibitors;

· DNA methyl transferase inhibitors;

cytokines and retinoids;

chromatin targeted therapies, e.g. HDAC or HAT modulators;

T-cell activating agents, including immunomodulating antibodies;

cancer vaccines;

· radiotherapy; and

other therapeutic or prophylactic agents; for example agents that reduce or alleviate some of the side effects associated with chemotherapy; for example anti-emetic agents and agents that prevent or decrease the duration of chemotherapy-associated neutropenia and prevent complications that arise from reduced levels of red blood cells or white blood cells, such as

erythropoietin (EPO), granulocyte macrophage-colony stimulating factor (GM- CSF), and granulocyte-colony stimulating factor (G-CSF).

In one embodiment, an alkylating agent is used in combination with the compound of formula (1), (2) or (3). Examples of alkylating agents include bischloroethylamines (nitrogen mustards, e.g. chlorambucil, cyclophosphamide, ifosfamide,

mechlorethamine, melphaian, uracil mustard), aziridines (e.g. thiotepa), alkyl alkone sulfonates (e.g. busulfan), nitrosoureas (e.g. carmustine, lomustine, streptozocin), nonclassic alkylating agents (altretamine, dacarbazine, and procarbazine), platinum compounds (carboplastin and cisplatin).

In another embodiment the compound of formula (1 ), (2) or (3) is used in combination with a platinum compound such as cisplatin or carboplatin.

In another embodiment, the compound of formula (1 ), (2) or (3) is used in combination with a member of the retinoids superfamily such as all-trans-retinol, all-trans-retinoic acid (tretinoin), 13-cis retinoic acid (isotretinoin) and 9-cis-retinoic acid.

In a further embodiment, the compound of formula (1 ), (2) or (3) is used in combination with a hormonal agent such as a synthetic oestrogen (e.g. diethylstibestrol), antiestrogen (e.g. tamoxifen, toremifene, fluoxymesterol and raloxifene), antiandrogen (bicalutamide, nilutamide, flutamide), aromatase inhibitor (e.g., aminoglutethimide, anastrozole and tetrazole), ketoconazole, goserelin acetate, leuprolide, megestrol acetate and mifepristone.

In yet another embodiment, the compound of formula (1 ), (2) or (3) is used in combination with a plant-derived agent such as a vinca alkaloid (e.g., vincristine, vinblastine, vindesine, vinzolidine and vinorelbine), camptothecin (20(S)-camptothecin, 9-nitro-20(S)-camptothecin, and 9-amino-20(S)-camptothecin), a podophyllotoxin (e.g., etoposide (VP-16) and teniposide (VM-26)), and taxane (e.g., paclitaxel and docetaxel).

In a particular embodiment, the compound of formula (1 ), (2) or (3) is used in combination with a taxane such as paclitaxel and docetaxel, an in particular paclitaxel. In another embodiment, a compound of formula (1 ), and in particular a compound of formula (1) wherein R ¹ is hydroxy and R ² is hydrogen, can be used in combination with an anthracycline such as daunorubicin or idarubicin.

In a further embodiment, a compound of the formula (1 ) wherein R ¹ is hydroxy and R ² is hydrogen can be used in combination with a compound wherein R ¹ and R ² are both fluorine, and optionally a further therapeutic agent as defined herein.

In a further embodiment, the compound of formula (1 ), (2) or (3) is used in combination with a biological agent such as an immuno-modulating protein (e.g. a cytokine), a monoclonal antibody against a tumour antigen, a tumour suppressor gene or a cancer vaccine.

Examples of interleukins that may be used in combination with the compound of formula (1), (2) or (3) include, but are not limited to, interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin 12 (IL-12). Examples of interferons that may be used in conjunction with the compound of formula (1 ), (2) or (3) include, but are not limited to, interferon [alpha], interferon [beta broblast interferon) and interferon [gamma] (fibroblast interferon). Examples of such cytokines include, but are not limited to erythropoietin (epoietin), granulocyte-CSF (filgrastim), and granulocyte, macrophage-CSF

(sargramostim). Immuno-modulating agents other than cytokines include, but are not limited to bacillus Calmette-Guerin, levamisole, and octreotide.

Examples of monoclonal antibodies against tumour antigens that can be used in conjunction with the inventive formulations include, but are not limited to,

HERCEPTIN(R) (Trastruzumab), RITUXAN(R) (Rituximab), MYLOTARG(R) (anti- CD33), and CAMPATH(R) (anti-CD52).

In a further embodiment, the compound of formula (1 ), (2) or (3) can be used in combination with a cancer vaccine, for example a cancer vaccine selected from a CTA cancer vaccine, such as a vaccine based on a CTA antigen selected from: NY-ESO-1 , LAGE-1 , MAGE-A1 , -A2, -A3, -A4, -A6, -A10, -A12, CT7, CT10, GAGE1 -6, GAGE 1 -2, BAGE, SSX1 -5, SSX 2, HAGE, PRAME, RAGE-1 , XAGE-1 , MUC2, MUC5B and

HMW-MAA. Non-limiting examples of CTA vaccines include those based on MAGE-A3 (for example recMAGE-A3), NY-ESO-1 and PRAME.

In another embodiment, the compound of formula (1 ), (2) or (3) can be used in combination with a T-cell activating agent, for example a T-cell activating agent which is an antibody (optionally a mAb), for example selected from: (a) a CD137 agonist; (b) a CD40 agonist; (c) an OX40 agonist; (d) a PD-1 mAb; (e) a PD-L1 mAb; (f) a CTLA-4 mAb; and (g) combinations of (a)-(f). In some embodiments, the ancillary therapeutic component is Tremelimumab or Ipilimumab.

The compound of formula (1 ), (2) or (3) and any other therapeutic agents may be presented separately or presented together in a pharmaceutical package, kit or patient pack.

The compounds of the invention and combinations with other therapeutic agents or radiation therapies as described above may be administered over a prolonged term to maintain beneficial therapeutic effects or may be administered for a short period only. Alternatively they may be administered in a pulsatile or continuous manner.

The compounds of the invention will be administered in an effective amount, i.e. an amount which is effective to bring about the desired therapeutic effect either alone (in monotherapy) or in combination with one or more chemotherapeutic agents or radiation therapy. For example, the "effective amount" can be a quantity of compound which, when administered to a subject suffering from cancer, slows tumour growth, ameliorates the symptoms of the disease and/or increases longevity.

The amount of compound of formula (1), (2) or (3) or their salts or tautomers administered to the subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. The skilled person will be able to determine appropriate dosages depending on these and other factors. Effective dosages for commonly used anti-cancer drugs and radiation therapy are well known to the skilled person.

A typical daily dose of the compounds of formula (1 ), (2) or (3) or their salts or tautomers, whether administered on their own in monotherapy or administered in combination with another therapeutic agent, can be in the range from 100 picograms to 100 milligrams per kilogram of body weight, more typically 5 nanograms to 25 milligrams per kilogram of bodyweight, and more usually 10 nanograms to 15 milligrams per kilogram (e.g. 10 nanograms to 10 milligrams, and more typically 1 microgram per kilogram to 20 milligrams per kilogram, for example 1 microgram to 10 milligrams per kilogram) per kilogram of bodyweight although higher or lower doses may be administered where required. The compound can be administered on a daily basis or on a repeat basis every 2, or 3, or 4, or 5, or 6, or 7, or 10 or 14, or 21 , or 28 days for example.

Dosages may also be expressed as the amount of drug administered relative to the body surface area of the patient (mg/m ² ). In one embodiment, the compounds of the invention can be administered in amounts from 3mg/m ² to 125mg/m ² daily. Treatment can be by continuous daily dosing or more usually consist of multiple cycles of treatment separated by treatment breaks. One example of a single treatment cycle is 5 consecutive daily doses followed by 3 weeks without treatment.

A compound of formula (1 ) wherein R ¹ and R ² are both fluorine can be used, for example, in the following indications: • for the treatment of locally advanced or metastatic bladder cancer in combination with cisplatin.

• for the treatment of patients with locally advanced or metastatic

adenocarcinoma of the pancreas. · in combination with cisplatin, for the treatment of patients with locally advanced or metastatic non-small cell lung cancer (NSCLC).

• for the treatment of patients with locally advanced or metastatic epithelial ovarian carcinoma, in combination with carboplatin, in patients with relapsed disease following a recurrence-free interval of at least 6 months after platinum- based, first-line therapy.

• with paclitaxel, for the treatment of patients with unresectable, locally recurrent or metastatic breast cancer who have relapsed following adjuvant/neoadjuvant chemotherapy. Prior chemotherapy should have included an anthracycline unless clinically contraindicated.

In one embodiment, a compound of the formula (1 ) wherein R ¹ and R ² are both fluorine can be administered in combination with carboplatin for the treatment of ovarian cancer. For example, a treatment regimen can comprise 21 -day cycles in which the carboplatin and the compound of formula (3) are coadministered over 30 minutes on days 1 & 8 of each 21 day cycle. The individual dosages of the compound of the invention can be, for example, stoichiometrically equivalent to 1000mg/m ² of gemcitabine.

In another embodiment, a compound of formula (1 ) wherein R ¹ and R ² are both fluorine can be administered in combination with paclitaxel for the treatment of breast cancer. For example, a treatment regimen can comprise 21 -day cycles in which the paclitaxel and the compound of formula (1 ) wherein R ¹ and R ² are both fluorine are coadministered over 30 minutes on days 1 & 8 of each 21 day cycle. The individual dosages of the compound of the invention can be, for example, stoichiometrically equivalent to 1250mg/m ² gemcitabine.

In another embodiment, a compound of formula (1 ) wherein R ¹ and R ² are both fluorine can be administered in combination with cisplatin for the treatment of non- small cell lung cancer. For example, a treatment regimen can comprise 28-day cycles in which the cisplatin and the compound of formula (1 ) wherein R ¹ and R ² are both fluorine are coadministered over 30 minutes on days 1 , 8 & 15 of each 28 day cycle. The individual dosages of the compound of the invention can be, for example, stoichiometrically equivalent to 1000mg/m ² gemcitabine. Alternatively, the compound of the invention can be administered in an amount stoichiometrically equivalent to 1250mg/m ² gemcitabine over 30 minutes on days 1 & 8 of a 21 day cycle.

In a further embodiment, a compound of formula (1 ) wherein R ¹ and R ² are both fluorine is administered as a single agent for the treatment of pancreatic cancer. For example, a compound of formula (3) can be administered over 30 minutes once weekly for 7 weeks then for 3 out of every 4 weeks. The individual dosages of the compound of the invention can be, for example, stoichiometrically equivalent to 1000mg/m ² gemcitabine.

In a further embodiment, a compound of formula (1 ) wherein R ¹ is hydroxy and R ² is hydrogen can be used in combination with another therapeutic agent selected from daunorubicin, idarubicin, fludarabine and clofarabine for the treatment of AML.

Ultimately, however, the quantity of compound administered and the type of composition used will be commensurate with the nature of the disease or physiological condition being treated and will be at the discretion of the physician.

EXAMPLES

The invention will now be illustrated, but not limited, by reference to the specific embodiments described in the following examples.

In the examples, the following abbreviations are used.

Tac = 4-terf-butylphenoxyacetyl

Pac = phenoxyacotyl

Ac = acetyl

DMT = 4,4'-dimethoxytrityl

Ph = phenyl

dG = 2'-deoxyguanosine

Example 1

1 -(3-D-Arabinofuranosvl)cvtosinyl-(3'→5')-2'-deoxvguanosine (Compound (2))

1 A. Synthesis of 2'-deoxy-N2,3'-0-bis-f[4-ferf-butylphenoxy1acetyl1-guanosine , Intermediate 1-2

Step 1 - DMT protection

To a solution of N2-Tac-dG, (100 g, 0.218 mol, commercially available from Prime Organics, or synthesised from 2'-deoxyguanosine via known methods) and pyridine (2.5 L) was added 4-dimethylaminopyridine (5.34 g, 0.043 mol). 4,4'-Dimethoxytrityl chloride (92 g, 0.272 mol) was added in four equal portions after a 5 minute interval, maintaining the reaction temperature between 0 to 5°C. The reaction mixture was stirred overnight at room temperature and the resulting mixture was diluted with dichloromethane (3.0 L), washed with sodium bicarbonate solution (10% w/v, 2.0 L) followed by saturated brine solution (2 X 2L). The organic layer was then dried over anhydrous sodium sulphate (100 g) and concentrated under reduced pressure to yield 5'-ODMT-N2-Tac-dG (160 g, quantitative, HPLC purity: 78.03% contains 13.5% pyridine) as thick oil which was taken ino the next step without further purification.

Step 2 - Tac protection

Crude 5'-ODMT-N2-Tac-dG (160 g, 0.21 1 mol) from Step 1 was dissolved in pyridine (3.2 L) and 4-dimethylaminopyridine (5.15 g, 0.042 mol) was added. The reaction mixture was cooled to 0-5 °C and 4-teri-butylphenoxyacetyl chloride (1 19.68 g, 0.528 mol) was added slowly, dropwise through an addition funnel, maintaining the temperature of the reaction mixture between 0 °C and 5 °C. The reaction mixture was then stirred at room temperature for 4-5 hrs. After completion, the reaction mixture was quenched with water (100 ml) and the resulting mass was diluted with dichloro- methane (3 L) and washed with saturated sodium bicarbonate (2.0 L), followed by saturated brine solution (2 X 2 L). The organic layer was then separated and dried over anhydrous sodium sulphate (100 g). The solvents were removed under reduced pressure to give 5'-ODMT-N2,3'-0-Bis-Tac-dG (155 g, quantitative, HPLC Purity:

64.17% contains 15% pyridine) as a foam, which was used in the next step without further purification.

Step 3 - DMT removal 5'-ODMT-N2,3'-0-Bis-Tac-dG (150 g, 0.157 mol) from Step 2 was dissolved in dichloromethane (3 L) and, to the resulting solution, a 10% solution of TFA (36 g, 0.31 mol) in dichloromethane (3 L) was added in two equal portions within a 10 minute interval. The reaction mixture was stirred at room temperature for 30 minutes, methanol (0.3 L) was added to the reaction mixture and the mixture was then stirred for a further 10 minutes. The reaction mixture was neutralized with sodium bicarbonate (10 %, w/v) and diluted with dichloromethane (2.5 L), and the organic layer was separated, washed with brine (2 x 2 L) and dried over anhydrous sodium sulphate (100g). The solvents were removed under reduced pressure to give the crude product (HPLC purity 65.5%) which was then purified by flash silica gel column

chromatography (230-400 mesh, 20 vol) using ethyl acetate: hexane (25: 75 to 75: 25). Fractions containing pure products were pooled together and concentrated under reduced pressure to afford pure 2'-deoxy-N2,3'-0-bis-[[4-terf-butylphenoxy]-acetyl]- guanosine (30 g, HPLC Purity: 98.9%).

1 H NMR (400 MHz, DMSO-d6): 1.25-1.24 (d, 18 H), 2.54 (m, 1 H), 2.84 (m, 1 H), 3.62 (bd, 2H), 4.1 1 (m, 1 H), 4.84-4.83 (d, 4H), 5.15 (m, 1 H, exchangeable), 5.48-5.47 (d, 1 H), 6.24-6.21 (q, 1 H), 6.9-6.8 (m,4H), 7.32-7.30 (m, 4H), 8.3 (s, 1 H), 1 1.82 (bs, 2H, exchangeable).

1 B. Synthesis of 1 -(3-D-Arabinofuranosvl)cvtosinvl-(3'→5')-2'-deoxyquanosine

(Compound (21)

Step 1 - Synthesis of 1-(5-0-DMT-p-D-arabinofuranosyl)cytosinyl-(3'→5')-2'-deoxy - guanosine (I-5) from phosphoramidite (1-1 ) To a solution of protected arabinocytidinyl phosphoramidite (Intermediate 1-1 , which can be synthesised according to WO01/85751 Example 6, 0.318mmol) in

dichloromethane (1 ml) were added 4 angstrom molecular sieves (160mg) and 4,5- dicyanoimidazole (38mg, 0.318mmol). After stirring at room temperature for 3 minutes, 2'-deoxy-N2,3'-0-bis-[[4-fert-butylphenoxy]acetyl]-guanosine (I-2, 206mg, 0.318mmol) in dichloromethane (1 ml) was added and the resulting mixture was stirred at room temperature for 16 hours to yield intermediate I-3 which was not isolated. Teri-butyl hydroperoxide in decane (5-6M solution, 10Oul) was added and the reaction was further stirred for 3 hours before the mixture was filtered and concentrated to give intermediate 1-4 which was also not isolated. The residue was dissolved in methanolic ammonia (2M, 5ml) and stirred for 16 hours and the resulting mixture was then concentrated and purified by preparative HPLC (100x19mm Waters XBridge BEH C18 5μηι column; 25ml/min overall flow rate; solvent A water, solvent B acetonitrile at 24ml/min; 250mM aqueous ammonium bicarbonate modified to pH9.2 with ammonia added at 1 ml/min; 0.4min at 5%B, gradient to 40%B at 7 min; detection by UV and MS). The product, intermediate I-5, was obtained as a white solid after freeze drying.

MS [M+H] ⁺ 874

Step 2 - Synthesis of 1-(P-D-Arabinofuranosyl)cytosinyl-(3'→5')-2'-deoxyguanosin e (2)

To 1 -(5-0-DMT- -D-arabinofuranosyl)cytosinyl-(3'→5')-2'-deoxy-guanosine (I-5) (30mg, 0.034mmol) was added a solution of glacial acetic acid (1.6g) in water (2ml). The mixture was left at room temperature for 5 minutes. The solvent was then evaporated, water was added and the mixture was washed with diethyl ether (x2). The aqueous fraction was concentrated and re-concentrated from water (x2) to form a colourless solid. This crude material was purified by preparative HPLC (100x19mm Waters XBridge BEH C18 5μιη column; 25ml/min flow rate; solvent A water, solvent B acetonitrile; 0.4min at 0%B, gradient to 30%B at 7 min; detection by UV and MS). The fractions were freeze dried to furnish the desired product 1 -(β-D-arabinofuranosyl)- cytosinyl-(3'→5')-2'-deoxyguanosine (Compound 2) as a partial ammonium salt (ca. 15mol% ammonium).

MS [M+H] ⁺ 573

H NMR (400 MHz, DMSO-c/ ₆ ) δ 10.69 (s, 1 H), 8.79 (s, 1 H), 8.46 (s, H), 7.92 (s, 1 H), 7.86 (d, J = 7.7 Hz, 1 H), 6.53 (s, 2H), 6.14 (dd, J = 8.0, 6.1 Hz, 1 H), 6.04 (d, J = 4.4 Hz, 1 H), 5.97 (d, J = 7.7 Hz, 1 H), 5.40 (s, 1 H), 4.45 - 4.25 (m, 3H), 4.07 - 3.80 (m, 4H), 3.62 (d, J = 5.1 Hz, 2H), 2.57 (ddd, J = 12.7, 7.8, 5.3 Hz, 1 H), 2.20 (ddd, J = 13.1 , 6.0, 2.7 Hz, 1 H). Note: 3 x OH resonances not determined.

³¹ P NMR (162 MHz, DMSO-d ₆ ) δ -1.20. Example 2

Synthesis of 2',2'-Difluoro-2'-deoxycvtidinyl-(3'→5')-2'-deoxyguanosine (Compound (3)1

Step 1 - Pac protection of 2',2'-difluoro-2'-deoxycytidine (gemcitabine)

A solution of gemcitabine (5.0g, 19mmol) and phenoxyacetic anhydride (6.0g, 20.9mmol) in anhydrous Ν,Ν-dimethylformamide (200ml) was stirred at room temperature. After 5 hours, the mixture was concentrated in vacuo and triturated with ethyl acetate. The solids were isolated by filtration and dried in a vacuum oven for 30 minutes furnishing N-phenoxyacetyl-gemcitabine (6.54g) as a white solid.

1 H NMR (400 MHz, DMSO-d6): 1 1.15 (1 H, s), 8.29 (1 H, d), 7.31 (2H, t), 7.20 (1 H, d), 7.06-6.88 (3H, m), 6.32 (1 H, d), 6.19 (1 H, t), 5.36-5.25 (1 H, m), 4.85 (2H, s), 4.28-4.12 (1 H, m), 3.97-3.87 (1 H, m), 3.81 (1 H, d), 3.74-3.60 (1 H, m).

Step 2 - DMT protection of N-pac gemcitabine

A solution of N-pac gemcitabine (7.72g, 19.4mmol) and 4,4'-dimethoxytrityl chloride (7.25g, 21.4mmol) in pyridine (125ml) was stirred at room temperature. After 4 hours an additional 7.6g of 4,4'-dimethoxytrityl chloride was added. After 2 hours, the reaction mixture was concentrated and re-concentrated from toluene (x2).

Dichloromethane (200ml) was added to the residue and this was washed with water (x2) and saturated brine solution before concentration. Partial purification of the crude product was achieved by chromatography on silica using 0-15% methanol/ dichloromethane furnishing 14g of a yellow foam. Further purification of 7.4g of this foam was achieved using a second silica column eluting with 30-75% ethyl acetate/ petroleum ether furnishing 5'-0-DMT-N-pac-gemcitabine (intermediate I-6) (4.8g) as a pale yellow foam/ solid.

MS [M+H] ⁺ 700

Step 3 - Synthesis of 2',2'-difluoro-2'-deoxy-5 ^, -0-DMT-cytidinyl-(3'→5')-2'- deoxyguanosine (intermediate 1-10) from 5'-0-DMT-N-pac-gemcitabine (intermediate I-6)

To a solution of 2-cyanoethyl Λ/,Λ/,Λ/',Λ/'-tetraisopropylphosphordiamidite (431 mg, 1.4mmol) in dichloromethane (10ml) at room temperature was added 4 angstrom molecular sieves (700mg) and 4,5-dicyanoimidazole (169 mg, 1.4 mmol). 5'-0-DMT-N- pac-gemcitabine (intermediate I-6) (1 g, 1.4mmol) in dichloromethane (10ml) was then added dropwise over 20 minutes. After stirring at room temperature for 4 hours, during which time intermediate I-7 was formed (but not isolated), 4,5-dicyanoimidazole (169mg, 1.4mmol) was added, followed by dropwise addition of 2'-deoxy-N2-3'-0-bis- [[4-fe/f-butyl-phenoxy]-acetyl]-guanosine (intermediate 1-2, 926mg, 1.4mmol) in dichloromethane (10ml). The mixture was stirred for 3 days to give intermediate !-8 which was also not isolated. t-Butyl hydroperoxide solution in decane (4-5M, 0.5ml) was then added and the reaction mixture was stirred for 16 hours, after which time the reaction mixture was filtered and concentrated in vacuo to give intermediate I-9 which was used without further purification. The crude intermediate 1-9 was dissolved in methanolic ammonia (2M, 15ml) and stirred for 6 hours to give intermediate 1-10. The crude DMT protected product (intermediate 1-10) was purified by preparative HPLC (100x19mm Waters XBridge BEH C18 5μιτι column; 25ml/min overall flow rate; solvent A water, solvent B acetonitrile at 24ml/min; 250mM aqueous ammonium bicarbonate modified to pH9.2 with ammonia added at 1 ml/min; 0.4min at 5%B, gradient to 40%B at 7 min; detection by UV and MS). The isolated fractions were freeze dried to furnish a white solid

MS [M+H] ⁺ 895

Step 4 - DMT removal

2',2'-Difluoro-2'-deoxy-5'-0-DMT-cytidinyl-(3'→5')-2'-d eoxyguanosine (intermediate 1-10, 105mg, 0.1 1 mmol) was dissolved in 10ml 80% glacial acetic acid in water. The solution was stirred for 3 minutes and was then concentrated in vacuo. Water was added and the mixture was washed with diethyl ether (x2). The aqueous solution was concentrated in vacuo and re-concentrated again from water to furnish a white solid which was purified by preparative HPLC (100x19mm Waters XBridge BEH C18 5μιη column; 25ml/min flow rate; solvent A water, solvent B acetonitrile; 0.4min at 0%B, gradient to 30%B at 7 min; detection by UV and MS). The desired product 2', 2'- difluoro-2'-deoxycytidinyl-(3'→5')-2'-deoxyguanosine (Compound 3) was isolated as a partial ammonium salt (ca. 50mol% ammonium, 51 mg) as a white solid after freeze drying.

MS [M+H] ⁺ 593

¹ H NMR (400 MHz, DMSO-cfe) δ 10.63 (s, 1 H), 7.92 (s, 1 H), 7.78 (s, 1 H), 7.69 (d, J = 7.6 Hz, 1 H), 7.64 (s, 1 H), 6.51 (s, 2H), 6.17 - 6.07 (m, 2H), 5.87 (d, J = 7.6 Hz, 1 H), 4.68 - 4.52 (m, 1 H), 5.35 (s, 1 H), 4.38 (dt, J = 5.2, 2.5 Hz, 1 H), 3.96 - 3.80 (m, 4H), 3.78 - 3.65 (m, 2H), 2.60 - 2.49 (m, 1 H), 2.18 (ddd, J = 13.1 , 6.1 , 2.8 Hz, 1 H). Note: 3 x OH resonances not determined.

¹⁹ F NMR (376 MHz, DMSO-cfe) δ -1 15.58

³ P NMR (162 MHz, DMSO-d ₆ ) δ -1.35

Preparation of compounds of the formula (1 ) wherein R ³ is a group convertible under physiological conditions to hydroxy

Compounds of the formula (1 ) wherein R ³ is a group convertible under physiological conditions to hydroxy can be prepared from the compounds of the formulae (1 ), (2) or (3) or the intermediates described herein. For example, phosphorothiolates can be prepared by sulfurization of intermediates 1-3 or 1-8. Alternatively substitution of Ν,Ν,Ν',Ν'-tetraisopropylphosphorodiamidite in the synthetic routes described with a suitable alternative that contains the desired N, O or S- substituent will provide the desired analogues. BIOLOGICAL ACTIVITY

EXAMPLE A

Proliferation Assays

Inhibition of cell growth was measured using the Alamar Blue assay (Nociari, M. M, Shalev, A., Benias, P., Russo, C. Journal of Immunological Methods 1998, 213, 157- 167). The method is based on the ability of viable cells to reduce resazurin to its fluorescent product resorufin. Cells were seeded into 96-well plates and allowed to recover for 16 hours prior to the addition of inhibitor compounds (in 0.1 % DMSO v/v) for a further 144 hours. At the end of the incubation period, 10% (v/v) Alamar Blue (Biosource International) was added and incubated for a further 6 hours, prior to determination of fluorescent product at 535nM ex / 590nM em.

The above protocol was used to determine the activity of compounds of the invention against the HL60 {Human causian promyelocytic leukaemia - ECACC cat. No 98070106) cell line. The results are shown in the table below.

EXAMPLE B

Pharmacokinetic Properties

The pharmacokinetics of compounds of the invention were assessed in male Balb C mice using the protocol described below. Gemcitabine hydrochloride or Ara-C were administered intravenously at a dose level of 2.5 mg freebase/kg. Compounds were formulated in 0.9 % w/v sodium chloride (aq). 2',2'-Difluoro-2'-deoxycytidinyl-(3'→5')- 2'-deoxyguanosine or 1 -( -D-arabinofuranosyl)cytosinyl-(3'→5')-2'-deoxyguanosine were administered subcutaneously as partial ammonium salts at dose levels which were stoichiometrically equivalent to 2.5mg freebase/kg of the 2',2'-difluoro-2'- deoxycytidinyl or -( -D-arabinofuranosyl)cytosinyl moieties. Compounds were formulated in 65% polypropylene glycol/10% ethanol/25% glycerin. Blood samples were collected at timed intervals following dosing and centrifuged to separate the plasma. Compounds were then extracted by the addition of acetonitrile containing an internal standard. All extracts are analysed for gemcitabine or Ara-C and the internal standard using a compound specific LCMS-MS assay. Mean plasma compound concentration-time profiles were plotted and pharmacokinetic parameters obtained using Phoenix WinNonLin® non-compartmental analysis software.

Results

The results demonstrate that 2',2'-difluoro-2'-deoxy-cytidinyl-(3'→5')-2'-deoxy- guanosine (Compound (3)) functions as a pro-drug for gemcitabine whereas 1-(β-ϋ- arabinofuranosyl)-cytosinyl-(3'→5')-2'-deoxy-guanosine (Compound (2)) functions as a pro-drug for cytarabine (Ara-C). An advantage of both Compound (2) and Compound (3) is that they can be administered subcutaneously which is a less invasive route of administration than intravenous administration. In addition, the Mean Residence Time (MRT) of the active moiety was markedly increased when dosed as the pro-drug compared with either gemcitabine or cytarabine administered intravenously.

Advantages associated with an increase in MRT include a prolonged therapeutic effect, reduced toxicity, and the possibility of using lower dosages.

PHARMACEUTICAL FORMULATIONS (i) Injectable Formulation I

A parenteral composition for administration by injection can be prepared by dissolving a compound of the formula (1 ) (e.g. in a salt form) in water containing 10% propylene glycol to give a concentration of active compound of 1.5 % by weight. The solution is then sterilised by filtration, filled into an ampoule and sealed.

(ii) Injectable Formulation II

A parenteral composition for injection is prepared by dissolving in water a compound of the formula (1) (e.g. in salt form) (2 mg/ml) and mannitol (50 mg/ml), sterile filtering the solution and filling into sealable 1 ml vials or ampoules.

(iii) Injectable formulation III

A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (1 ) (e.g. in a salt form) in water at 20 mg/ml. The vial is then sealed and sterilised by autoclaving.

(iv) Injectable formulation IV

A formulation for i.v. delivery by injection or infusion can be prepared by dissolving the compound of formula (1 ) (e.g. in a salt form) in water containing a buffer (e.g. 0.2 M acetate pH 4.6) at 20mg/ml. The vial is then sealed and sterilised by autoclaving.

(v) Subcutaneous Injection Formulation

A composition for sub-cutaneous administration is prepared by mixing a compound of the formula (1 ) with pharmaceutical grade corn oil to give a concentration of 5 mg/ml. The composition is sterilised and filled into a suitable container.

vi) Lvophilised formulation

Aliquots of formulated compound of formula (1 ) are put into 50 ml vials and lyophilized. During lyophilisation, the compositions are frozen using a one-step freezing protocol at (-45 °C). The temperature is raised to -10 °C for annealing, then lowered to freezing at -45 °C, followed by primary drying at +25 °C for approximately 3400 minutes, followed by a secondary drying with increased steps if temperature to 50 °C. The pressure during primary and secondary drying is set at 80 millitor.

Equivalents

The foregoing examples are presented for the purpose of illustrating the invention and should not be construed as imposing any limitation on the scope of the invention. It will readily be appjrent that numerous modifications and alterations may be made to the specific embodiments of the invention described above and illustrated in the examples without departing from the principles underlying the invention. All such modifications and alterations are intended to be embraced by this application.