WO2004058252A2 - Anti-cancer agents comprising alkb inhibitors and alkylating agents - Google Patents

Abstract

A pharmaceutical combination which comprises: (a) a compound of one of the formulae: formula (I) wherein each of RI and RII, which max be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CRIRII, NRIII or O wherein RIII is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NRIII, OH or SH, or Y and Z together represents =O or =S, or formula (II) where R1 to R11 may independently be H, a branched or straight C1 to C6 alkyl chain, OH, O-alkyl having a branched or straight C1 to C6 alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C1 to C6 alkyl ester (alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and formula (A) represents formula (B) or formula (C) or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent. This combination is useful in the treatment of cancer.

Description

ANTI-CANCER AGENTS

This invention relates to anti-cancer agents and, in particular agents which have an effect on AlkB.

Summary

The integrity of the genome is maintained by a set of DNA repair enzymes, including those that repair alkylation damage (1). DNA can be alkylated by a variety of agents that occur both exogenously and endogenously (2,3). Alkylating agents are used in some chemotherapy treatments and alkylated DNA bases have been detected in the urine of smokers (4). AlkB is one of four proteins involved in the adaptive response to DNA alkylation damage in Escherichia coli and is highly conserved from bacteria to humans. Recent analyses have verified the prediction that AlkB is a member of the iron (II) and 2-oxoglutarate (2OG) dependent oxygenase family of enzymes. AlkB mediates repair of methylated DNA by direct demethylation of 1-methyladenine and 3-methylcytidine lesions. It has been demonstrated that AlkB repairs the cyto toxic 1- methyladenine and 3-methylcytidine lesions in both single and double stranded DNA by an oxidative process (Fig. 1). The reaction most probably proceeds by hydroxylation of the methyl group, leading to its extrusion as formaldehyde. Such a direct repair mechanism is likely to be efficient, as unlike the nucleotide excision repair and base excision repair pathways, it is not reliant on a complementary strand for maintenance of genetic information. Other members of the iron (II) and 2OG dependent oxygenase family, including those involved in the hypoxic response, are targets for therapeutic intervention.

We have found that the nucleosides 1-methyladenosine, l-methyl-2'- deoxyadenosine, 3-methyl-2'-deoxyclytidine and 3-methylcytidine stimulate 2OG turnover by AlkB, but are not demethylated indicating an uncoupling of 2OG and prime substrate oxidation; thus oligomeric DNA is required for hydroxylation and subsequent demethylation to occur. Nucleosides such as 1-methyladenosine, 1- methyl-2'-deoxyadenosine, 3-methyl-2'-deoxycytidine and 3-methylcytidine may be used in assays for AlkB directed towards the discovery of AlkB inhibitors. Note the advantages of using these compounds compared with oligomers are cost, ease of preparation and that they are chemically defined (cf methylated oligomers which are mixtures) thereby enabling reproducible assay results. In contrast adenosine and cytidine do not stimulate 2OG turnover indicating that the presence of a methyl group in the substrate is important in initiating oxidation of 2OG. The stimulation of 2OG turnover by 1-methyladenosine was shown to be highly dependent on the presence of a reducing agent, ascorbate or dithiothreitol. Following the observation that AlkB is inhibited by high concentrations of 2OG, structural analogues of 2OG, such as 2- mercaptoglutarate, were also found to inhibit AlkB. The flavonoid quercetin inhibits both AlkB and the 2OG oxygenase factor inhibiting hypoxia-inducible factor (FIH) in vitro. FIH inhibition occurs in the presence of excess iron indicating a specific interaction, while the inhibition of AlkB is, predominantly, due to non-specific iron chelation.

We have found, according to the present invention, certain analogues of 2OG which inhibit AlkB. The ability in this way to selectively inhibit AlkB can allow a reduction in the amount of cytotoxic alkylating agents used in cancer chemotherapy. Accordingly, the present invention provides a pharmaceutical combination which comprises (a) a compound which is an analogue of 2OG as defined below, or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent. The present invention also provides a product comprising (a) a said analogue of 2OG or a pharmaceutically acceptable salt thereof; and (b) an anti-cancer alkylating agent; for separate, simultaneous or sequential use in the treatment of cancer. The invention further provides the use, in the manufacture of a medicament for the treatment of cancer, of (a) a compound which is an OG analogue as defined below, or a pharmaceutically acceptable salt thereof; and (b) an anti-cancer alkylating agent. Yet further, the invention provides a method for treating cancer in a patient, which method comprises administering to the patient, separately, simultaneously or sequentially, a said analogue of 2OG, or a pharmaceutically acceptable salt thereof, and an anti-cancer alkylating agent.

The OG analogues used in the present invention are defined by one of the following formulae:

wherein each of R¹ and R^π, which may be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CR ¹¹, NR^πι or O wherein R¹¹¹ is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NR^m, OH or SH or Y and Z together represents =O or =S. Preferably R¹ and R^π are not alkyl and especially are both hydrogen. It is also preferred that Y and Z together form =O or =S or Y is -SH and Z is H. The alkyl groups include methyl, ethyl and propyl and isopropyl; methyl is preferred. Specific compounds of formula (I) include N-oxalylglycine, -thiooxalylglycine and 1-mercaptoglutarate.

wherein R¹ to R^u may independently be H, a branched or straight C to C₆ alkyl chain, OH, O-alkyl having a branched or straight C_j to C₆ alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C_x to C₆ alkyl ester (alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and

represents

Specific compounds of formula II include:

Rl

isomers)

R2

H

R3 R4

(racemate or single stereo isomers)

(R² is quercetin.) Thus typically R, R³, R⁷, R⁸ and R^π are hydrogen and R², R⁴, R⁹ and R¹⁰ are

OH.

Where appropriate the compounds can be in the form of pharmaceutically acceptable salts. Pharmaceutically acceptable salts of compounds with acid groups include salts formed with alkali metal cations, alkaline earth metal cations or ammonium cations. Such salts are prepared by treating the free compound of formula (I) or (II) with the corresponding metal base or ammonia. Examples of these salts include salts of sodium, potassium, calcium, magnesium and ammonium. Compounds which possess a basic centre, such as compounds of formula (I) in which X is NH, may also form acid addition salts. Pharmaceutically acceptable acid addition salts include salts of inorganic acids such as hydrochloric acid, hydrobromic acid and sulphuric acid and salts of organic acids such as acetic acid, oxalic acid, malic acid, methanesulphonic acid, trifluoroacetic acid, benzoic acid, citric acid and tartaric acid.

Pharmaceutical Compositions

In one embodiment of the pharmaceutical combination of the invention components (a) and (b) are formulated as a single pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or diluent. In an alternative embodiment components (a) and (b) are formulated as separate pharmaceutical compositions, each of which further comprises a pharmaceutically acceptable carrier or diluent. A pharmaceutical composition is prepared by admixing the or each active component with a pharmaceutically acceptable carrier or diluent, and optionally other ingredients.

The anti-cancer alkylating agent is typically a methylating agent. Suitable alkylating agents include nitrogen mustards such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil, azirizidines and epoxides such as thiotepa, mitomycin C and diaziquone, alkyl sulphonates such as alkylalkane sulphonates, more particularly busulfan, nitrosoureas including carmustine, lowmustine, 4-methyl lowmustine and semustine, triazenes and hydrazines including 5-(3,3-dimethyl-l-triazeno) imidazole-4-carboxamide

(dacarbazine or DTIC) and 8-carbamoyl-3-methylimidazo[5,l-d]-l,2,3,5-tetrazin-4- (3H)-one (temozolomide, TMZ) which are pro-drugs for the active methylating species, 5-(3-methyltriazen-l-yl) imidazole-4-carboxamide (MTIC) that generates the methyldiazonium cation. It has been reported TMZ generates different methyl adducts including N7-methyl-guanine and N3-methyladeine, as well as O⁶- methylguanine which has been viewed as providing the anti-tumour activity. Tumour cell susceptibility to TMZ is said ⁽⁴⁰⁾ to be strongly affected by the functional status of DNA repair systems involved either in the removal of methyl adducts from O⁶G or in the apoptotic signalling triggered by O⁶-methylG:T mispairs. The compound of formula (I) or (II) and the anti-cancer alkylating agent are administered separately, simultaneously or sequentially.

Administration of the compound of formula (I) or (II), or a pharmaceutically acceptable salt thereof, and the anti-cancer alkylating agent, is preferably in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy). This means that the dosage is sufficient to show benefit to the individual. The actual amount of each agent administered, and rate and time-course of administration, will depend on a variety of factors including the age, weight and condition of the patient, the nature and severity of what is being treated, and the route of administration. Prescription of treatment including decisions on dosage is within the responsibility of general practitioners and other medical doctors.

Typically, the dosage adopted for each route of administration when a compound of formula (I) or (II) or a salt thereof is administered to an adult human is from 0.001 to 500 mg/kg body weight per day, most commonly in the range of 0.01 to 100 mg/kg, for instance 0.01 to 50 mg/kg or 0.01 to 20 mg/kg. Such a dosage may be given, for example, from 1 to 5 times daily. Administration may be orally or parenterally, for instance intravenously such as by bolus infusion, infusion over several hours and/or repeated administration.

Dosage regimens for anti-cancer alkylating agents are well established and can be determined by medical practitioners by reference to standard resources such as drug directories and pharmacopoeias. The recommended dose for an adult human is critically dependent on the identity of the alkylating agent and the route of administration. For instance, chlorambucil is typically given in tablet form at a dose of 0.1 - 0.2 mg/kg per day; mitomycin is typically given intravenously, in a single dose of 10-20 mg/m²by infusion; ifosfamide is typically given intravenously at a dose of 1.2g/m²per day for 5 days; cyclophosphamide may be given either intravenously, at a dose of 40-50 mg/kg in a divided dose over 2-5 days, or in tablet form at a dose of from 1 - 5 mg/kg; and thiotepa is typically administered in a total dose of from 2 - 15 mg per day. These examples are provided for illustration purposes only; different dosages and regimens will be recommended for other anti- cancer alkylating agents.

Pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to one or more active ingredients, a pharmaceutically acceptable carrier or diluent, buffer, stabiliser or other materials well known to those skilled in the art. In particular they may include a pharmaceutically acceptable excipient. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous. Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Liposomes, particularly cationic liposomes, may be used in carrier formulations. Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The substance or composition may be administered in a localised manner to a particular site or may be delivered in a manner in which it targets particular cells or tissues, for example using intra-arterial stent based delivery.

Targeting therapies may be used to deliver the active substances more specifically to certain types of cell, by the use of targeting systems such as antibody or cell specific ligands. Targeting may be desirable for a variety of reasons, for example if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

The combination of the invention may be used in the oncolytic treatment of primary or secondary cancer, either with or without additional pro-drug therapy or stimulation of an immune response. It may be used in the therapeutic treatment of any solid tumour in a mammal, preferably in a human. For example a compound of formula (I) or (II) or a pharmaceutically acceptable salt thereof, and an anti-cancer alkylating agent, may be administered separately, simultaneously or sequentially to a subject with pancreatic, tongue, prostate, breast, lung, liver, endometrial, bladder, colon, ovarian or cervical carcinoma; adenocarcinoma; melanoma; lymphoma; glioma; or sarcomas such as soft tissue and bone sarcomas as well as haematological malignancies such as leukaemia. The condition of the patient may thereby be ameliorated.

The following Examples further illustrate the present invention.

Example 1

Materials and methods

Materials - All chemicals were purchased from Sigma-Aldrich except: oligonucleotides (Sigma-Genosys), restriction enzymes (New England Biolabs), Pfu Turbo, ligation kit and competent cells (Stratagene), pET-24a(+) vector (Novagen), Gentra systems kit for purification of E.coli DNA, l-[¹ C]-2-oxoglutarate (Perkin Elmer Life Sciences). Hyamine hydroxide (ICN radiochemicals) and Opti-phase 'SAFE' scintillant (Fisher).

Cloning - AlkB was cloned from genomic E. coli DNA by PCR using the primers; forward 5ø-GGTGGTCATATGTTGGATCTGTTTGCC-3ø and reverse 5ø- GGTGGTGGATCCTTATTCTTTTTTACCTGC-30. The PCR product was digested with Nde I and BamB. I and cloned into pET-24a(+).

Cell growth - The pET-24a(+)-AlkB plasmid was transformed into E. coli BL21(DE3) Gold competent cells and growth conditions were optimised. Initially cultures were grown at 37°C until the OD₆₀₀ reached 0.6, where isopropyl-β-D- thiogalactoside was added to a final concentration of 0.2mM and the temperature was dropped to 28°C. Cells were harvested after 4 hours by centrifugation. AlkB was produced as ca. 10% of the total soluble protein (by SDS-PAGE analysis). Cell lysis and protein purification - Cells (15g) were resuspended on ice in 50mL of lOOmM MES, pH5.8, lmM dithiothreitol, 40μg mL^"1 lysozyme, 4μg mL^"1 DNasel, then sonicated (Ultrasonics Inc, W-380) with 4x30s pulses and centrifuged (Beclαnann JA25-50) for 20mins at 19000rpm. The supernatant was filtered through a 0.2μm membrane and loaded on to a (50mL) S-Sepharose column (Pharmacia), run at lOmL min ^! in lOOmM MES, ρH5.8. AlkB was eluted with a gradient of 0-1M NaCl over 400mL. AlkB containing fractions were pooled and diluted 3 fold prior to loading on a (6mL) Resource S column (Pharmacia) run at 8mL min^" \ again AlkB was eluted with a gradient of 0-1 M NaCl over 180mL. AlkB containing fractions were concentrated and loaded onto a (720mL, Superdex-75) gel filtration column at 3mL min^"1 in 150mM TRIS, pH7.5. This yielded approximately 30mg of AlkB >95% purity by SDS-PAGE analysis. Methylation of oligonucleotides with MMS - Methylated DNA oligonucleotides were prepared using a modified version of literature procedures (21 ,22).

Polydeoxyadenine (poly(dA)) and polydeoxycytosine (poly(dC)) were both 15mers. The 27mer and 33mer were of random sequence and contained all four DNA bases. Prior to reaction with MMS oligomers were dissolved at 0.05mM in lOmM TRIS, pH7.5. MMS (240mM in EtOH) was added to a final concentration of 24mM. Reactions were carried out at room temperature for 24 hours. The DNA was precipitated by addition of 3 volumes of ice cold EtOH (0.3M acetate, pH5.2) and placing at -80°C for 30mins. The sample was then centrifuged and the supernatant removed, the precipitated DNA was washed 6 times with EtOH. DNA concentration was measured by UN spectroscopy. Methylation of 2 ø-deoxyadenosine - l-Methyl-2ø-deoxyadenosine was prepared using the procedure of Singer et al (23). l-Methyl-2ø-deoxyadenosine was the major product, produced in approximately 20% yield (~75% of starting material remained). Yield was estimated by integration of the HPLC trace at 260nm. 1- Methyl-2ø-deoxyadenosine was purified by HPLC using a Phenomenex LUΝA C-18 column (250' 10mm, 5μm). All solvents were filtered through 0.2μm filters and sparged with He (g) at lOOmL min^"1 for 20mins before use. The column was run at 3.2mL min^"1 in 50mM ammonium acetate pH6.4, 5% acetonitrile for 25mins, then a gradient was run to 30% acetonitrile over 5mins and held there for lO ins. Isolated l-methyl-2ø-deoxyadenosine m/z(ESl+) 266; UN: λ_ttax(pH7) 259nm, λ_max(ρHl) 259nm, λ_max(pH13) 260nm, λ_inflection(pH13) 279nm. As expected these UN profiles were the same as those obtained for the commercial 1-methyladenosine. Methylation of 2 ø-deoxycytidine - 3-Methyl-2ø-deoxycytidine was prepared using the same procedure as for methylation of 2ø-deoxyadenosine. Isolated 3-methyl-2ø- deoxycytidine m/z(ESI+) 242; UN: λ_a pΕLT) 278nrn, λ_max(pHl) 278nm, λ_max(pHl 3) 267nm. As expected these UN profiles were the same as those obtained for the commercial 3-methylcytidine. l-[¹⁴C],2-Oxoglutarate assays - This assay is based on the method used to measure ¹⁴CO₂ release by α-ketoisocaproate oxygenase (24). Standard assay conditions comprised of a total volume of 1 OOμL, 50mM TRIS, pH7.5, 4mM ascorbate, 160μM 2OG (2.5% 1-[¹⁴C]), 80μM (ΝH₄)₂SO₄.FeSO₄.6H₂O, 0.48mg mL^"1 catalase, 12.5μM AlkB. Generally two stocks were made, one total volume 25 μL contained AlkB and Fe¹¹, the second total volume 75 μL contained all other reagents. Assays were started by the addition of the Fe¹¹, AlkB stock to the other stock. A tube containing 200μL hyamine hydroxide was added and the vial sealed. The assays were incubated at 37°C for 5 mins and quenched with 20% (v/v) trifluoroacetic acid (300μL). They were then left on ice for 30 mins to collect ¹⁴CO₂ gas, before the hyamine hydroxide was removed and treated with scintillant for counting (Beclαnann, LS6500). Assay points were performed in triplicate unless otherwise stated, values quoted are an average with standard deviation given as the error. DNA oligomers were incubated at a final concentration of lOμM. Nucleoside and base substrates were dissolved at 20mM in DMSO, these were added to a final concentration of 400μM, AlkB activity was not affected by the presence of 2% (v/v) DMSO. For the inhibitor, replacement of ascorbate and variation of 2OG concentration assays, 800μM 1-methyladenosine was present. Inhibitors were assayed at 5 concentrations around the approximate IC₅₀ value, the maximum concentration of inhibitor tested was 4mM. Initial rate was plotted against inhibitor concentration in an Excel worksheet and a curve was fitted to the data, the equation for the curve was solved for half the rate in the absence of inhibitor to give an approximate IC₅₀ value. For assays in which Fe¹¹ concentration was varied with inhibitor concentration held at IC₅₀, Fe¹¹ was added to a final concentration of 40, 80, 160, 250 and 500μM. For the assays using alternative reducing agent a lOOmM stock of reducing agent was made and added to a final concentration of 4mM. For variation of ascorbate concentration assays, ascorbate was added to a final concentration of 0, 0.25, 0.5, 1 and 4mM. For variation of 2OG concentration assays, 2OG was added to a final concentration 50, 75, 100, 125, 150,

175, 200, 250, 300, 400, 500 and 700μM, these assays were single points and carried out at both 80 and 250μM Fe^π. Graphs showing variation of ascorbate and 2OG concentration were plotted in SigmaPlot. HPLC/LCMS assays - HPLC was carried out using a Synergi C-18 Hydro column, (250'4.6mm 4μm) from Phenomenex, the elutant was analysed using a Photodiode Array Detector. All solvents were filtered through 0.2μm filters and sparged with He (g) at lOOmL min^"1 for 20mins before use. Prior to injection 1% (v/v) acetic acid was added to samples and they were centrifuged at 13000rpm for lOmins. The column was run at 0.8mL min^"1 in 50mM ammonium acetate pH6.4, 2% acetonitrile for 25mins, then a gradient was run to 30% acetonitrile over 5mins and held there for lOmins. AlkB assays for HPLC were carried out using the same concentrations of reagents as above, except that no radiolabelled 2OG was present and they were quenched with 1 volume of MeOH after 40 minutes and placed on ice to precipitate the protein. HPLC runs for 1-methyladenosine, l-methyl-2ø-deoxyadenosine, 3- methylcytidine and 3-methyl-2ø-deoxycytidine assays were also analysed with some demethylated nucleoside added to confirm that product would be detected if demethylation had occurred. The same conditions were used for the LCMS assays with products being analysed with a Micromass ZMD mass spectrometer ESI(+/-). Factor inhibiting HIF assays - Factor inliibiting HIF (FIH) was purified and assayed as described previously with the C- terminal fragment of HIFlα(775-826) as a GST fusion protein used as a substrate (20).

Synthesis of inhibitors - N-Oxalylglycine, N-oxalyl-4S-alanine, N-oxalyl-4R-alanine (25) and 2-hydroxyglutarate (26) were prepared according to literature procedures. N- Tl iono-oxalylglycine was prepared as follows: The dimethyl ester of thiono- oxalylglycine was prepared as reported (27), and subsequently deprotected to give the desired product (25) (1.7mmol, 85%) as a yellow solid, mp 105-106 °C; v_max (NaCl, MeOHycnf¹ 1730, 1697 (C=O); 4(200 MHz; OMSO-d₆) 4.27 (2 H, d, ³J_W 6.0, CH₂), 10.9 (1 H, br t, NH); <?_c(50 MHz; ΩMSO-d₆) 47.9 (CH₂), 163.4, 169.4, 189.5 (CO, C=S); m/z(AP-) 162 (M - H⁺, 30%), 118 (M - H⁺ - CO₂, 100%); HRMS 161.9861 calculated for M - it (C₄H₄NO₄S), 161.9861 found.

(±)-2-Mercaptoglutarate was prepared according to the literature procedure (28)

Results and discussion

Expression and purification - The AlkB gene was cloned from E.coli and inserted into the pET-24a(+) vector. A three column protocol was developed for purification of AlkB based on cation exchange and gel filtration chromatography, giving the desired protein at >95% purity by SDS-PAGE analysis.

Substrate assays and structural recognition - AlkB activity was analysed by incubating AlkB with l-[¹⁴C]-2OG and measuring the release of ¹⁴CO₂ upon formation of succinate (23). The results were consistent with the conclusions of Trewick et al and Fames et al that AlkB is a 2OG dependent Fe¹¹ dioxygenase (16,17). Several oligonucleotides including polyadenine and polycytosine were reacted with the S_N2 methylating agent MMS. When assayed with AlkB, the unmethylated oligonucleotides did not increase 2OG turnover significantly above the uncoupled rate (see Table 1 - the prefix Me indicates the oligomer had been treated with MMS.) However, the methylated oligonucleotides gave rise to a significant increase in decarboxylation of 2OG by AlkB. The methylated oligomers used to ascertain the identity of the substrates of AlkB (17) are not precisely defined in a chemical sense as they are made by non-specific methylation of an oligomeric substrate. Consequently, commercially available monomeric, potential substrates were assayed to probe the structural features of DNA that are required for AlkB catalysis.

Various methylated bases, nucleotides and nucleosides were assayed as potential substrates for AlkB, including those with the 1-methyladenine and 3- methylcytosine bases that are substrates for AlkB when incorporated into oligomeric DNA (see Table 2) (16,17). Incubation with 1-methyladenosine or l-methyl-2ø- deoxyadenosine was found to induce up to a seven-fold increase in 2OG turnover. Whilst 3-methylcytidine and 3-methyl-2ø-deoxycytidine only brought about a two and three fold increase respectively. This suggests that 1-methyladenine rather than 3-methylcytosine lesions may be preferred substrates for AlkB. An HPLC based assay was developed to investigate the production of nucleosides from their methylated counterparts by AlkB. Neither analysis by photodiode array HPLC or by ESI-LCMS led to the detection of any demethylated nucleoside for the incubation of either 1-methyladenosine, l-methyl-2ø-deoxyadenosine, 3-methylcytidine or 3- methyl-2ø-deoxycytidine with AlkB. Thus, unlike the situation with oligomeric DNA the turnover of 2OG and hydroxylation of the prime substrate appear to be predominantly uncoupled in the presence of nucleoside substrate analogues. This is of interest with regard to substrate recognition by AlkB. Crystallographic, spectroscopic and solution studies of other 2OG oxygenases have indicated that binding of the prime substrate to the enzyme complex is required to initiate dioxygen binding and subsequent reaction of 2OG (30-32). It appears that 1-methyladenosine and 3-methylcytidine nucleosides are recognised by the AlkB active site and can stimulate reaction of 2OG with dioxygen, but the reactive oxidising species, believed to be [Fe^IV=O « Fe^m-O], does not react with the methyl group. It may be that when the requisite connections are not made to the DNA oligomer the reactive oxidised species is not positioned correctly to effect hydroxylation. To effect further 2OG catalysis the original Fe^π center must be regenerated by an alternative pathway possibly involving ascorbate (see below). To further investigate the structural motifs recognised by AlkB, assays with 1-methyladenine and various non-methylated bases and nucleosides were carried out (see Table 2). The rate of 2OG turnover remained consistent at the level observed in the absence of substrate when AlkB was incubated with adenosine, 2ø-deoxyadenosine or adenine, i.e. in the absence of the 1 -methyl group. For both 1-methyladenine, l-methyl-2ø-deoxyadenosine and 1- methyladenosine there was an increase in the rate of 2OG turnover, indicating that the 1 -methyl group is an absolute requirement for stimulation of the reaction between 2OG and O₂. 2OG conversion was lower for 1-methyladenine than for 1- methyladenosine and l-methyl-2ø-deoxyadenosine, indicating that the presence of a sugar moiety improves substrate recognition by AlkB, possibly by direct contacts between the enzyme and the sugar giving rise to tighter enzyme/ substrate analogue binding. Similarly, cytidine, 2ø-deoxycytidine and cytosine all failed to bring about an increase in the rate of 2OG conversion, indicating that AlkB activity is stimulated by binding the methyl group of 3-methylcytidine nucleosides.

Stimulation of 2OG oxidation without coupling to hydroxylation of the prime substrate has previously been observed for procollagen prolyl-4-hydroxylase with substrate analogues (33). Further, mutagenesis studies on deacetoxycephalosporin C synthase (DAOCS) have resulted in an uncoupling of 2OG turnover and prime substrate hydroxylation (34). For procollagen prolyl-4-hydroxylase the uncoupled oxidation of 2OG has been linked to the requirement for ascorbate as well as Fe¹¹ and 2OG. It has been proposed that an ascorbate molecule regenerates the Fe¹¹ center in the event of uncoupled turnover of 2OG to the ferryl intermediate and succinate (35). Role of ascorbate - Uncoupled 2OG turnover by AlkB was found to be highly dependent on ascorbate both in the presence and absence of 1-methyladenosine, but more so in the former case (see Fig. 2 - filled circles = no substrate; open circles = with methyladenosine). However, the amount of ascorbate required for optimal uncoupled 2OG turnover was far in excess of stoichiometry to 2OG. The role of ascorbate was investigated further by attempting to replace it in the assay with alternative reducing agents Table 3 gives the results. Activity was maintained with .D-isoascorbate and dithiothreitol, the latter giving ca. 90% of the activity withE- ascorbate. All other agents tested resulted in a significant loss of activity, though, when compared to the rate of reaction in the absence of reducing agent, β- mercaptoethanol gave a moderate increase in activity, whilst addition of dithionite and 4-nitrocatechol brought about a reduction in rate.

For prolyl-4-hydroxylase, in the presence of poly(L-proline), (a substrate that stimulates uncoupled 2OG turnover but is not hydroxylated) a stoichiometric reaction of 2OG with disappearance of ascorbate has been reported (35). Recent work on anthocyanidin synthase (ANS), a 2OG dependent oxygenase involved in the biosynthesis of flavonoids in plants, has suggested that an ascorbate molecule may bind at the active site in the presence of the prime substrate and be involved in the catalytic cycle (36). Elucidation of the exact role of ascorbate in AlkB catalysis will require further work. However, the observation that it can be replaced by dithiothreitol suggests that its role may not be entirely specific. Inhibition of AlkB - It has been found that 2OG concentrations greater than 200μM caused a reduction in the initial rate of 2OG turnover (see Fig. 3). This inhibition was unaffected by an increase in Fe¹¹ concentration and the same pattern was observed at both 80μM and 250μM Fe¹¹. Therefore, AlkB inhibition at high 2OG concentrations is unlikely to be a result of Fe¹¹ chelation by 2OG. The 2OG dependent dioxygenases DAOCS and thymidine-7-hydroxylase have also been observed to be inhibited by high concentrations of 2OG (32,37). Enzyme inhibition by high substrate concentrations is often thought to be a result of two molecules of substrate binding to the enzyme to produce an inactive complex (38). It is possible, though unlikely, that inhibition of AlkB by 2OG has physiological significance. The inhibition of AlkB by high concentrations of 2OG prompted us to investigate the inhibition of AlkB by a variety of structural analogues of 2OG. Various compounds have therefore been assayed as inhibitors of AlkB. The results are given in Table 4. N-Oxalylglycine differs from 2OG by the replacement of the 3-CH₂ with an NH and has previously been reported to be a competitive inhibitor of both the procollagen (25) and the HIF hydroxylases (19,20). N- Oxalylglycine was found to be a moderate inhibitor of AlkB with an IC₅₀ of 0.70mM. The 2-thione derivative of N-oxalylglycine gave a similar IC₅₀ of 0.8 lmM. Both R- and S- enantiomers of N-oxalylalanine were then tested as inhibitors. Neither stereochemistry was found to be a good inhibitor, with IC₅₀ values of 2.4 and 3.3mM for the S- and R- enantiomers respectively, both significantly higher than that for N- oxalylglycine. This observation implies that space within the 2OG binding pocket of AlkB may be limited compared to some other 2OG oxygenases. N-Oxalyl-4S- alanine was also found to be a better inhibitor than the 4R of procollagen prolyl-4- hydroxylases (25). Inhibitors in which the 2-keto of 2OG was replaced with a thiol or an alcohol were then tested. As these compounds lack the carbonyl group that reacts with dioxygen it is theoretically impossible for them to undergo nucleophilic attack by an activated dioxygen molecule. The C-2 thiol, (+)-2-mercaptoglutarate had the lowest IC₅₀ (0.12mM) of the 2OG analogues tested; in contrast the C-2 alcohol showed no inhibition up to a concentration of 4mM. The significant difference between inhibition by the alcohol and the thiol is interesting and is possibly a result of the high affinity of the thiol for Fe¹¹. Changing the concentration of Fe¹¹ had no effect on inhibition by (±)-2-mercaptoglutarate, indicating that simple solution based Fe¹¹ chelation was not responsible for inhibition. It is also noteworthy that, during catalysis by isopenicillin N synthase, an oxidase with a close structural relationship to the 2OG oxygenases, ligation of its thiol substrate (L)-δ-(α- aminoadipyl)-(L)-cysteinyl-(D)-valine to the Fe¹¹ center is a key step in catalysis (39). Accordingly, it is not unreasonable to conclude that related thiols of formula I will behave similarly.

Recently, it has been proposed that the naturally occurring flavonoid quercetin, (a compound used in dietary supplements) can regulate the hypoxic response by inhibition of the HIF hydroxylases involved in the degradation of the HIF-lα protein (40). HIF-lα is hydroxylated by members of the Fe¹¹, 2OG dependent dioxygenase family at an asparagine residue by FIH and two proline residues by the prolyl hydroxylase (PHD) enzymes. Quercetin is a good iron chelator and its regulatory role could in part be due to its effect on iron concentrations and consequently the activity of the Fe¹¹ dioxygenases. Under the standard AlkB assay conditions, quercetin was found to have an IC₅₀ of 0.08mM for inhibition of 2OG turnover by AlkB. However, AlkB activity could be returned to normal levels by addition of excess Fe^π (160μM) (data not shown). This indicates that there is unlikely to be any specific and tight binding interaction between quercetin and AlkB. References

I. Lindahl, T., and Wood, R. D. (1999) Science 286, 1897-1905 2. Lindahl, T. (1993) Nature 362, 709-715

3. Sedgwick, B., and Naughan, P. (1991) Mutat. Res. 250, 211-221

4. Kopplin, A., Eberleadamkiewicz, G., Glusenkamp, K. H., Νehls, P., and Kirstein, U. (1995) Carcinogenesis 16, 2637-2641

5. Lindahl, T., Sedgwick, B., Sekiguchi, M., andΝakabeppu, Y. (1988) Annu. Rev. Biochem. 57, 133-157

6. Pegg, A. E. (2000) Mutat. Res.-Rev. Mutat. Res. 462, 83-100

7. Myers, L. C, Terranova, M. P., Ferentz, A. E., Wagner, G., and Verdine, G. L. (1993) Science 261, 1164-1167

8. Landini, P., and Volkert, M. R. (2000) J. Bacteriol 182, 6543-6549 9. Wyatt, M. D., Allan, j. M., Lau, A. Y., Ellenberger, T. E., and Samson, L. D. (1999) Bioessays 21, 668-676 10. Hollis, T., Ichikawa, Y., and Ellenberger, T. (2000) Embo J. 19, 758-766

I I . Wei, Y. F., Carter, K. C, Wang, R. P., and Shell, B. K. (1996) Nucleic Acids Res. 24, 931-937 12. Kataoka, H., Yamamoto, Y., and Sekiguchi, M. (1983) J. Bacteriol. 153, 1301- 1307

13. Chen, B. R. j., Carroll, P., and Samson, L. (1994) J. Bacteriol. 176, 6255-6261

14. Dinglay, S., Trewick, S. C, Lindahl, T., and Sedgwick, B. (2000) Genes Dev. 14, 2097-2105 15. Aravind, L., and Koonin, E. V. (2001) Genome Biol 2, research/0007.0001- 0007.0008

16. Falnes, P. O., Johansen, R. F., and Seeberg, E. (2002) Nature 419, 178-182

17. Trewick, S. C, Henshaw, T. F., Hausinger, R. P., Lindahl, T., and Sedgwick, B. (2002) Nature 419, 174-178 18. Prescott, A. G., and Lloyd, M. D. (2000) Nat. Prod. Rep. 17, 367-383 19. Jaakkola, P., Mole, D. R., Tian, Y. M., Wilson, M. I., Gielbert, J., Gaskell, S. J., vonKriegsheim, A., Hebestreit, H. F., Mukherji, M., Schofield, C. J., Maxwell, P. H., Pugh, C. W., and Ratcliffe, P. J. (2001) Science 292, 468-472

20. Hewitson, K. S., McNeill, L. A., Riordan, M. V., Tian, Y. M., Bullock, A. N., Welford, R. W., Elkins, J. M., Oldham, N. J., Bhattacharya, S., Gleadle, J. M.,

Ratcliffe, P. J., Pugh, C. W., and Schofield, C. J. (2002) J. Biol Chem. 277, 26351-26355

21. Bodell, W. J., and Singer, B. (1979) Biochemistry 18, 2860-2863

22. Encell, L., Shuker, D. E. G., Foiles, P. G., and Gold, B. (1996) Chem. Res. Toxicol 9, 563-567

23. Sabourin, P. J., and Bieber, L. L. (1982) J. Biol. Chem. 257, 7460-7467

24. Cunliffe, C. J., Franklin, T. J., Hales, N. J., and Hill, G. B. (1992) J. Med. Chem. 35, 2652-2658

25. Battersby, A. R., Cardwell, K. S., and Leeper, F. J. (1986) J. Chem. Soc.-Perkin Trans. 1, 1565-1580

26. Harris, P. A. (1989) Sulphur letters 10, 117-122

27. Danielli, J. F., Danielli, M., Fraser, J. B., Mitchell, P. D., and Owen, L. N. (1947) Biochem. J. 41, 325-333

28. Garbiras, B. J., and Marburg, S. (1999) Synthesis, 270-274 29. Zhang, Z. H., Ren, J. S., Harlos, K., McKinnon, C. H., Clifton, I. J., and

Schofield, C. J. (2002) FEBSLett. 517, 7-12 30. Solomon, E. I., Brunold, T. C, Davis, M. I., Kemsley, J. N., Lee, S. K., Lehnert,

N., Neese, F., Skulan, A. J., Yang, Y. S., and Zhou, J. (2000) Chem. Rev. 100,

235-349 31. Holme, E. (1975) Biochemistry 14, 4999-5003

32. Venkateswara Rao, N., and E, A. (1978) J. Biol. Chem. 253, 6327-6330

33. Lipscomb, S. J., Lee, H. J., Mukherji, M., Baldwin, J. E., Schofield, C. J., and Lloyd, M. D. (2002) Eur. J. Biochem. 269, 2735-2739

34. Myllyla, R., Majamaa, K., Gunzler, V., Hanauskeabel, H. M., and Kivirikko, K. I. (1984) J. Biol. Chem. 259, 5403-5405 35. Wi nouth, R. C, Turnbull, J. J., Welford, R. W. D., Clifton, I. J., Prescott, A. G., and Schofield, C. J. (2002) Structure 10, 93-103

36. Dubus, A., Lloyd, M. D., Lee, H. J., Schofield, C. J., Baldwin, J. E., and Frere, J. M. (2001) Cell. Mol Life Sci. 58, 835-843 37. Cornish-Bowden, A. (2001) Fundamentals of Enzyme Kinetics, Portland Press

38. Roach, P. L., Clifton, I. J., Hensgens, C. M. H., Shibata, N., Schofield, C. J., Hajdu, J., and Baldwin, J. E. (1997) Nature 387, 827-830

39. Wilson, W. J., and Poellinger, L. (2002) Biochem. Biophys. Res. Commun. 293, 446-450 40. Tentori, L and and Graziani, G (2002) Current Medicinal Chemistry, 9, 1285- 1301.

Table 1

Table 2

Table 3: The effect on rate of 2OG turnover by AlkB brought about by replacing ascorbate in the assay with alternative reducing agents

Table 4: IC,5_n0 values and structures of the inhibitors tested with AlkB.

Example 2: Tablet composition

Tablets, each weighing 0.15 g and containing 25 mg of a compound of the invention were manufactured as follows:

Composition for 10,000 tablets

Compound of the invention (250 g) Lactose (800 g) Corn starch (415g) Talc powder (30 g) Magnesium stearate (5 g)

The compound of the invention, lactose and half of the corn starch were mixed. The mixture was then forced through a sieve 0.5 mm mesh size. Corn starch (10 g) is suspended in warm water (90 ml) . The resulting paste was used to granulate the powder. The granulate was dried and broken up into small fragments on a sieve of 1.4 mm mesh size. The remaining quantity of starch, talc and magnesium was added, carefully mixed and processed into tablets.

Example 3: Iniectable Formulation

Compound of the invention 200mg Hydrochloric Acid Solution 0.1M or

Sodium Hydroxide Solution 0.1M q.s. to pH 4.0 to 7.0

Sterile water q.s. to 10 ml

The compound of the invention was dissolved in most of the water (35° - 40° C) and the pH adjusted to between 4.0 and 7.0 with the hydrochloric acid or the sodium hydroxide as appropriate. The batch was then made up to volume with water and filtered through a sterile micropore filter into a sterile 10 ml amber glass vial (type 1) and sealed with sterile closures and overseals. Example 4: Intramuscular Injection

Compound of the invention 200 mg Benzyl Alcohol 0.10 g

Glycofurol 75 1.45 g

Water for inj ection q. s to 3.00 ml

The compound of the invention was dissolved in the glycofurol. The benzyl alcohol was then added and dissolved, and water added to 3 ml. The mixture was then filtered through a sterile micropore filter and sealed in sterile 3 ml glass vials (type 1).

Example 5: Syrup Formulation

Compound of invention 250 mg Sorbitol Solution 1.50 g

Glycerol 2.00 g

Sodium benzoate 0.005 g

Flavour 0.0125 ml

Purified Water q.s. to 5.00 ml

The compound of the invention was dissolved in a mixture of the glycerol and most of the purified water. An aqueous solution of the sodium benzoate was then added to the solution, followed by addition of the sorbital solution and finally the flavour. The volume was made up with purified water and mixed well.

Claims

1. A pharmaceutical combination which comprises : (a) a compound of one of the formulae:

wherein each of R¹ and R^π, which may be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CR ¹¹- NR^m or O wherein R^m is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NR^m, OH or SH, or Y and Z together represents =O or =S, or

where R¹ to R¹¹ may independently be H, a branched or straight to C₆ alkyl chain, OH, O-alkyl having a branched or straight to C₆ alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C_l to C₆ alkyl ester (alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and

represents

or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent.

2. A combination according to claim 1 wherein components (a) and (b) are formulated as a single pharmaceutical composition, further comprising a pharmaceutically acceptable carrier or diluent.

3. A combination according to claim 1 wherein components (a) and (b) are formulated as separate pharmaceutical compositions, each of which further comprises a pharmaceutically acceptable carrier or diluent.

4. A combination according to any one of the preceding claims wherein component (a) is a compound of formula (I) in which R¹ and R^π are not alkyl.

5. A combination according to any one of the preceding claims wherein component (a) is a compound of formula (I) in which R¹ and R^π are both hydrogen.

6. A combination according to any one of the preceding claims wherein component (a) is a compound of formula (I) in which Y and Z together form =O or =S or Y is SH and Z is hydrogen.

7. A combination according to any one of the preceding claims wherein component (a) is N-oxalylglycine, N-thiooxalylglycine, 2-mercaptoglutarate, or a pharmaceutically acceptable salt thereof.

8. A combination according to any one of the preceding claims wherein the alkylating agent is a methylating agent.

9. A combination according to claim 8 wherein the alkylating agent is a nitrogen mustard, aziridine or epoxide, alkyl sulfonate, nitrosourea or triazine or hydrazine.

10. A combination according to claim 9 wherein the alkylating agent is dacarbazine, temozolomide or 5-(3-methyltriazen-l-yl) imidazole-4-carboxamide.

11. A product comprising:

(a) a compound of one of the formulae

wherein each of R¹ and R^π, which may be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CRΪt.^π, NR^m or O wherein R^m is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NR^m, OH or SH, or Y and Z together represents =O or =S, or

where R¹ to R¹¹ may independently be H, a branched or straight to C₆ alkyl chain, OH, O-alkyl having a branched or straight C_j to C₆ alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C_x to C₆ alkyl ester

(alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and

represents

or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent; for separate, simultaneous or sequential use in the treatment of cancer.

12. Use, in the manufacture of a medicament for the treatment of cancer, of (a) a compound of one of the formulae:

wherein each of R¹ and R^π, which may be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CRR¹¹, NR^m or O wherein R^m is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NR^m, OH or SH, or Y and Z together represents =O or =S, or

where R¹ to R¹¹ may independently be H, a branched or straight C_j to C₆ alkyl chain, OH, O-alkyl having a branched or straight to C₆ alkyl chain optionally containing

1 or 2 N, O or S atoms, COOH, a branched or straight Cj to C₆ alkyl ester

(alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N,

S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and

represents

or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent.

13. A method of treating cancer, which method comprises administering to a patient in need thereof, separately, simultaneously or sequentially: (a) a compound of one of the formulae

wherein each of R¹ and R^π, which may be the same or different, represents hydrogen, fluorine or alkyl of 1 to 6 carbon atoms, X represents CR ¹¹, NR or O wherein R^m is hydrogen or alkyl of 1 to 6 carbon atoms, each of Y and Z, which may be the same or different, represents hydrogen, NR^m, OH or SH, or Y and Z together represents =O or =S, or

where R¹ to R^u may independently be H, a branched or straight to C₆ alkyl chain, OH, O-alkyl having a branched or straight C_t to C₆ alkyl chain optionally containing 1 or 2 N, O or S atoms, COOH, a branched or straight C_x to C₆ alkyl ester (alkoxycarbonyl), a 4 to 7 membered heterocyclic ring optionally containing 1 or 2 N, S, O or P atoms or a 5 or 6 membered aromatic ring, optionally containing 1 or more N, O or S atoms, which can be fused to another ring, or a said alkyl chain substituted by a said aromatic ring, and

represents

or a pharmaceutically acceptable salt of a said compound; and (b) an anti-cancer alkylating agent.