WO2007128968A1 - Telomerase inhibitors - Google Patents

Abstract

The present invention relates to metal complexes of formulae (I), (IIa) or (IIb) which interaction with quadruplex DNA and act as inhibitors of telomerase activity, the synthesis of these complexes and their use in the treatment of cancer.

Description

TELOMERASE INHIBITORS

The present invention relates to novel compounds and their use as telomerase inhibitors. The invention provides the compounds for use in the treatment of cancer.

Eukaryotic chromosomes comprise sequences of coding DNA (i.e. genes) and sequences of non-coding DNA. The non-coding DNA includes telomeres which are long sequences of DNA at the end of the chromosomes. Telomeres consist of many tandem repeats of a short sequence 5'TTAGGG 3' which protect the end of the chromosome. Repeated cycles of replication of the DNA however results in a shortening of the telomere (estimated at lOObp per mitosis), ultimately resulting in senescence or apoptosis of the cell.

Telomerase is a reverse transcriptase enzyme which adds telomere repeat sequences to the 3 'end of DNA strands. Telomerase has an elevated activity in 85-90% of human cancer cells in comparison to normal somatic cells. Telomerase inhibition has thus been identified as an attractive target for cancer chemotherapy with the potential for selective toxicity for cancer cells over normal ones.

Telomerase maintains telomeric DNA integrity and prevents critical shortening of the telomere so that cells cannot reach the crisis points of senescence and apoptosis. Human telomeric DNA is typically 3-6 KB in length in cancer cells. The 3' terminal 100-200 bases are single-stranded . Crystallographic and NMR studies have shown that repeats of this sequence can fold into guanine-rich quadruplex structures. Since the substrate of telomerase is the 3 '-single- stranded overhang of telomeric DNA, the stabilization of these quadruplex structures by small molecules can lead to the inhibition of telomerase thereby selectively interfering with telomere maintenance in tumor cells.

The development of new and effective methods of treating of cancer is a well established desire in the art.

Metal-salen complexes have previously been shown to interact with duplex DNA. However, in spite of the unique electronic, structural and optical properties of metal complexes, such as metal-salen complexes, their abilities as quadruplex DNA stabilizers have not previously been explored. The present invention provides novel metal complexes which show good interaction with quadruplex DNA and inhibition of telomerase activity. Consequently these compounds can be used in the treatment of cancer.

The first aspect of the invention therefore provides a compound of formula (I)

(D wherein A is an C₅-C₆ aryl or heteroaryl group or is absent, B and C are independently an C₅-C₆ aryl or heteroaryl group, M is a metal ion or M=O;

R¹ and R² are independently hydrogen, C_1-2O alkyl, C_3-12carbocyclic, halo, C_{1- 20}haloalkyl, OR¹³, CN, NO₂, NR¹³R¹³, COR¹³, CO₂R¹³, O-(CH₂)_n-N(R¹⁴)₃ or (CH₂)_n-R¹⁵, CONR¹³R¹³, C_3-12heterocyclic, C_1-20alkylC₃,₁₂carbocyclic, C_1- i₂alkylC_3-12heterocyclic, or wherein R¹ and R² together form a carbocyclic or heterocyclic group having 5 to 18 members, fused to ring A, optionally substituted with one or more of group R¹³, or R¹ and R² are independently

R³, R⁴, R⁵ and R⁶ are independently hydrogen, halides, C_1-20 alkyl, OR¹³ or CN, or R³ and R⁵ and/or R⁴ and R⁶ together form a 5-6 membered carbocyclic or heterocyclic ring,

or R¹ and R³ and/or R² and R⁴ together form a carbocyclic or heterocyclic group having 5 to 18 members fused to ring A, optionally substituted with one or more of group R ,

X¹ and X² are independently O, S or NR¹³,

R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹² are independently hydrogen, halide, OR¹⁴, 0-(CH₂V N(R¹⁴)₃ or O-(CH₂)_n-R¹⁵, or where one or more of R⁷ and R⁸ or R¹⁰ and R¹¹ together form a carbocyclic or heterocyclic ring having 5 to 12 members,

G¹ and G² are independently OR¹⁴, O-(CH₂)_n-N(R¹⁴)₃ or O-(CH₂)_n-R¹⁵ wherein R¹⁴ is hydrogen or C_1-20 alkyl and R¹⁵ is HN-C(=NH)-NH₂, [-NH-C(- SR¹³)-NH-]⁺ a 5 or 6 membered carbocyclic or heterocyclic ring;

or G¹ and G² can together form

wherein n is 1 to 6, and M is a metal ion, M=O or is absent;

R¹³ is independently hydrogen, C_1-12 alkyl, C_3-12 carbocyclic, C_3-12 heterocyclic, C_1-6 alkylC_3-12 carbocyclic, C_1-6 alkylC_3-12 heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂; and R³⁰, R³¹, R³², R³³ and R³⁴ are independently C_1-12 alkyl, C_3-12 carbocyclic, C_3-12 heterocyclic, C_1-6 alkylC_3-12 carbocyclic, C_1-6 alkylC_3-i₂ heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂.

It will be appreciated that when R¹ and R² are both

a metal ion M can be co-ordinated between the two N atoms and the two X¹ atoms of the groups R¹ and R². Furthermore, any of groups R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹² can combine with any of groups R³⁰, R³¹, R³² or R³³ to form a bridged structure.

For the purposes of this invention, the groups A, B or C are independently preferably a six membered aryl or heteroaryl group, more preferably selected from phenyl or pyridine. Preferably, the groups A, B or C independently are a six membered aryl or heteroaryl group fused to an aromatic ring, for example naphthyl.

M is preferably selected from Ni, Co, Cu, Mn, Cr, V, Pt, Rh, Ir, Au, Pd, Zn or V=O.

The substituents R¹ and R² are preferably selected from hydrogen, F, Cl, CO₂H, O-(CH₂)_q-N(CH₃)₃, O-(CH₂)_q-C_5-6-carbocyclic, O-(CH₂)_q-C_5-6-heterocyclic, CONH-(CH₂)_m-carbocyclic or CONH(CH₂)_m-heterocyclic, wherein m is 1 to 6. wherein q is 1 to 6 preferably 2, 3, 4 or 5.

When R¹ and R², or R¹ and R³ or R² and R⁴ together form a carbocyclic or heterocyclic group, said carbocyclic or heterocyclic group preferably has 5 to 14 members fused to ring A, more preferably 5 to 10 members fused to ring A.

G₁ or G₂ are preferably selected from

wherein p is 0 or 1 and n is 1 to 6, preferably 2, 3, 4 or 5.

The first aspect of the invention preferably relates to a compound of formula (Ia)

(Ia) wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², X¹, X², M, G¹ and G² are as defined above.

Preferably, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are hydrogen and R¹, X¹, X², M, G¹ and G² are as defined above.

In an alternative feature of the first aspect of the invention, ring A is absent from the compound of formula (I). The resulting compound has the formula (Ib) or (Ic) as set out below:

(Ib)

(Ic) wherein B, C, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², X¹, X², M, G¹ and G² are as defined above.

In a further alternative feature of the invention, R³ and R⁵ together form a 5-6 membered carbocyclic or heterocyclic ring and R⁴ and R⁶ together form a 5-6 membered carbocyclic or heterocyclic ring, ring B is absent and ring C is replaced with

wherein Y is a group -CH₂-, CR²⁶ or CO, X is NH, N, or O, D is a carbocycyl or heterocycyl group having 5 to 10 members, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are hydrogen, C_1-6 alkyl, C_3-12 carbocyclic, C_3-12 heterocyclic, C_1-6 alkylC_3-i₂ carbocyclic, C_1-6 alkylC_3-12 heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂ and

G³ is H, OH, NH₂, OR²⁰, O-(CH₂)_n-N(R²⁰)₃ or O-(CH₂)_n-R²¹ wherein R²⁰ is C_1-12 alkyl, R²¹ is a 5 or 6 membered carbocyclic or heterocyclic ring and R²⁶ is hydrogen or C_1-12 alkyl.

Thus, the invention provides a compound of formula (Ha) or (lib):

(Ha) (lib)

Preferably the compound is a compound of formula (Ha) as illustrated below:

(Ha)

wherein E and F are independently a 5-6 membered carbocyclic or heterocyclic ring and R >22 , T R-.23 , R r»2^z4^q and J R r>2^/5^D are hydrogen, halides, OH, OR 20 , O-(CH₂)_n- N(R²⁰)₃, O-(CH₂)_n-R²¹, or CN,

wherein P is OR¹³, CO₂R¹³, CN, NO₂, CN, halide, SCN, H₂O, NO₃, OH, CH₃CN or OCN, and

wherein R .1¹, r R>2^z, T R_Ϊ 1ⁱ3^J, T R, 1¹6⁰, τ R> 17 , n Rl¹8⁸, r R.1ⁱ9^y, r G^^J> and M are as defined above.

Preferably, G³ is OR²⁰, O-(CH₂)_n-N(R²⁰)₃ or O-(CH₂)_n-R²¹.

Alternatively, ring C is replaced with

and G³ is H or NH₂. The present invention particularly relates to the following preferred compounds of the invention:

It should be appreciated that, although M is shown as present in the preferred compounds above, the invention encompasses corresponding compounds, useful for example as intermediates in the synthesis of the above complexes, in which M is absent.

Previously reported crystallographic and NMR studies, and computer modeling have provided a detailed picture of the guanine-rich quadruplex structures of the telomere. This has allowed the identification of features of a compound useful for stabilizing the quadruplex structure. The compounds of the present invention provide 1) a π-delocalized system that is able to stack on the face of a guanine quartet (i.e. rings A, B and C); 2) a partial positive charge that is able to lie in the centre of the quartet increasing stabilization by substituting the cationic charge of the potassium or sodium that would normally occupy that site (the metal M); and

3) positively charged substituents to interact with the grooves and loops of the quadruplex and the negatively charged backbone phosphates (groups G¹ and

G²).

The interaction of the compounds of the invention with the guanine-rich quadruplex structures of the telomere allows stabilization of the quadruplex, preventing the interaction of the telomerase enzyme with the telomeres and ultimately resulting in senescence or apoptosis of the cells.

As previously discussed, metal salen and metal salphen complexes have previously been shown to interact with duplex DNA. It will be appreciated that the interaction of a compound with duplex DNA, by for example intercalation does not provide compounds which can be used therapeutically, as such compounds interact with and damage the DNA of non-cancerous cells. The compounds of the present invention contain bulky groups (for example G¹ and G²) which it is postulated fall outside the plane of the planar ring systems A, B and C and interact with the grooves and loops of the quadruplex.. The presence of G¹ and G² prevent the compounds of the present invention from intercalating with duplex DNA and therefore minimize the interaction of the compounds of the invention with duplex DNA (and therefore with non-cancerous cells). The compounds of the present invention do not therefore significantly intercalate duplex DNA in particular compared to the interaction of the compounds with quadruplex DNA. The compounds of the present invention therefore show selectivity for cells with high telomerase activity, such as cancerous cells unlike the metal-salen and metal-salphen compounds known in the art. For the purposes of this invention, alkyl relates to both straight chain and branched alkyl radicals of 1 to 20 carbon atoms, preferably 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, preferably 1 to 8 carbon atoms, more preferably 1 to 6 carbon atoms and most preferably 1 to 4 carbon atoms including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, sec- butyl, isobutyl, tert-butyl n-pentyl, n-hexyl, n-heptyl, n-octyl. The alkyl radical can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19 or 20 carbon atoms.

"Haloalkyl" means an alkyl radical as defined above preferably having 1 to 20 carbon atoms, substituted with one or more halide atoms for example CH₂CH₂Br, CF₃ or CCl₃.

"Carbocyclic" means a cyclic 3 to 10 membered hydrocarbon, preferably a 4, 5, 6, 7, or 8 membered ring system. The carbocyclic ring can be unsaturated, partially saturated or fully saturated. The term carbocyclic encompasses both cycloalkyl groups and aryl groups.

"Cycloalkyl" means cycloalkyl radicals of 3 to 12 carbon atoms, preferably 4 to 8 carbon atoms, and most preferably 5 to 6 carbon atoms including but not limited to cyclopropyl, cyclobutyl, CH₂-cyclopropyl, CH₂-cyclobutyl, cyclopentyl or cyclohexyl. Cycloalkyl groups may be optionally substituted or fused to one or more carbocyclyl or heterocyclyl group. The cycloalkyl radical can have 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon atoms.

"Aryl" means an aromatic 3 to 10 membered hydrocarbon preferably a 6 to 10 membered ring system containing one ring or being fused to one or more saturated or unsaturated rings including but not limited to phenyl, napthyl, anthracenyl or phenanthracenyl. "Heterocyclic" means a cyclic 3 to 10 membered ring system, preferably a 4, 5, 6, 7, or 8 membered ring system containing one or more heteroatoms selected from N, O or S. The heterocyclic ring can be unsaturated, partially saturated or fully saturated. The heterocyclyl system can contain one ring or may be fused to one or more saturated or unsaturated rings. The term carbocyclic encompasses heteroaryl groups.

"Heteroaryl" means an aromatic 3 to 10 membered aryl preferably a 6 to 10 membered ring system containing one or more heteroatoms selected from N, O or S and containing one ring or being fused to one or more saturated or unsaturated rings.

Examples of carbocyclyl or heterocyclyl groups include but are not limited to cyclohexyl, phenyl, acridine, benzimidazole, benzofuran, benzothiophene, benzoxazole, benzothiazole, carbazole, cinnoline, dioxin, dioxane, dioxolane, ditbiane, dithiazine, dithiazole, dithiolane, furan, imidazole, imidazoline, imidazolidine, indole, indoline, indolizine, indazole, isoindole, isoquinoline, isoxazole, isothiazole, morpholine, napthyridine, oxazole, oxadiazole, oxathiazole, oxathiazolidine, oxazine, oxadiazine, phenazine, phenothiazine, phenoxazine, phthalazine, piperazine, piperidine, pteridine, purine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, pyrroline, quinoline, quinoxaline, quinazoline, quinolizine, tetrahydrofuran, tetrazine, tetrazole, thiophene, thiadiazine, thiadiazole, thiatriazole, thiazine, thiazole, thiomorpholine, thianaphthalene, thiopyran, triazine, triazole, and trithiane.

"Halogen" or "halide" means F, Cl, Br or I, preferably F, or Cl. The compounds of the first aspect may be provided as a salt, preferably as a pharmaceutically acceptable salt of a compound of formula (I), (Ha) or (lib). Examples of pharmaceutically acceptable salts of these compounds include those derived from organic acids such as acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic acid, phenylacetic acid, mandelic acid, methanesulfonic acid, benzenesulfonic acid and p-toluenesulfonic acid, mineral acids such as hydrochloric and sulfuric acid and the like, giving methanesulfonate, benzenesulfonate, p-toluenesulfonate, hydrochloride and sulphate, and the like, respectively or those derived from bases such as organic and inorganic bases. Examples of suitable inorganic bases for the formation of salts of compounds for this invention include the hydroxides, carbonates, and bicarbonates of ammonia, lithium, sodium, calcium, potassium, aluminium, iron, magnesium, zinc and the like. Salts can also be formed with suitable organic bases. Such bases suitable for the formation of pharmaceutically acceptable base addition salts with compounds of the present invention include organic bases, which are nontoxic and strong enough to form salts. Such organic bases are already well known in the art and may include amino acids such as arginine and lysine, mono-, di-, or trihydroxyalkylamines such as mono-, di-, and triethanolamine, choline, mono-, di-, and trialkylamines, such as methylamine, dimethylamine, and trimethylamine, guanidine; N-methylglucosamine; N-methylpiperazine; morpholine; ethylenediamine; N-benzylphenethylamine; tris(hydroxymethyl) aminomethane; and the like.

Salts may be prepared in a conventional manner using methods well known in the art. Acid addition salts of said basic compounds may be prepared by dissolving the free base compounds according to the first aspect of the invention in aqueous or aqueous alcohol solution or other suitable solvents containing the required acid. Where a compound of the invention contains an acidic function, a base salt of said compound may be prepared by reacting said compound with a suitable base. The acid or base salt may separate directly or can be obtained by concentrating the solution e.g. by evaporation. The compounds of this invention may also exist in solvated or hydrated forms.

The invention also extends to a prodrug of the aforementioned compounds such as an ester or amide thereof. A prodrug is any compound that may be converted under physiological conditions or by solvolysis to any of the compounds of the invention or to a pharmaceutically acceptable salt of the compounds of the invention. A prodrug may be inactive when administered to a subject but is converted in vivo to an active compound of the invention.

The compounds of the invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. The compounds of the invention may exist in trans or cis form. The first aspect of the invention covers all of these compounds.

The compound of the invention may exist in one or more crystalline forms. The invention therefore relates to a single crystal form of a compound of the invention or a mixture of one or more forms.

The second aspect of the invention provides a process for the preparation of a compound of the first aspect of the invention. The compounds of the first aspect of the invention may be prepared by methods known to those skilled in the art for analogous compounds, as illustrated by the general schemes and procedures below and with reference to the examples. In particular, the compound of the first aspect may be provided by the simultaneous separate or sequential addition of a group L-G¹ and a group L-G² to a compound of formula (III),

cm) wherein L is a leaving group such as a halide and R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸ R⁹, R¹⁰, R¹¹, R¹², G¹, G², X¹, X² and M are as defined in the first aspect of the invention.

It will be appreciated that where G and G are identical, the compound of

Λ O formula (III) can be incubated with an excess of L-G or L-G to form a compound of formula (I). However, where G¹ and G² are different, the groups L-G¹ and L-G² are added separately. For example, a compound of formula (III) can be incubated with a group L-G¹ to form a compound of formula (IV),

(IV) followed by incubation with a group L-G² to form a compound of formula (I). In this case, the hydroxyl groups in the compound of formula (III) can be protected with different protecting groups such as allylic protecting groups, p- methoxybenzyl chloromethyl ether, silyl ether and other standard alcohol protecting groups known to a person skilled in the art to allow the sequential addition of the groups L-G¹ and L-G².

A compound of formula (III) can be prepared by the reaction of a compound of formula (VI) with a metal, a compound of formula (VII) and a compound of formula (VIII)

(Vl) (VII) (VIII)

(III)

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², G¹, G², X¹, X² and M are as defined in the first aspect of the invention.

Again, when the compound of formulas (VII) and (VIII) are identical, the compound of formula (VI) can be incubated with an excess of a compound of formula (VII) or an excess of a compound of formula (VIII). However, where the compounds of formula (VI) and (VII) are different, the compound of formula (VII) and the compound of formula (VIII) are added separately. For example, a compound of formula (VI) can be incubated with a compound of formula (VII) to form a compound of formula (V),

followed by incubation with a compound of formula (VIII) to form a compound of formula (III). In this case, the amino groups in the compound of formula (Vl) can be protected with different protecting groups such as Fmoc, benzyl or BOC or any other standard amino protecting group known to a person skilled in the art. Alternatively, the addition of a compound of formula (VII) to a compound of formula (VI) can be carried out under reaction conditions, such as in an excess of the compound of formula (VI) wherein the compound of formula (VII) is added slowly and under high dilution conditions, such that the monosubstituted compound of formula (V) forms without the need for the use of amino protection to allow the sequential addition of the compound of formula (VII) and the compound of formula (VIII). For the purposes of this invention, the term "incubating" encompasses reacting the intermediate compounds of the invention.

The third aspect of the invention provides a composition comprising a compound according to the first aspect of the invention in combination with a pharmaceutically acceptable carrier, diluent or excipient.

The composition may also comprise one or more additional active agent, such as a chemotherapeutic agent and/or an antiproliferative agent. In particular, the composition of the present invention can comprise one or more of an alkylating agent (such as cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine, Busulfan or Thiotepa), an antimetabolite (such as Cytarabine, Gemcitabine, 6-mercaptopurine, 6- thioguanine, Fludarabine and Cladribine), an anthracycline (such as Idarubicin, Epirubicin), an antibiotic (such as Bleomycin), a camptothecin, a etoposide, a vinca alkaloid, a taxane (such as taxol, paclitaxel and docetaxel), and/or a platinium (such as cisplatin or oxaliplatin). The composition of the present invention may contain one or more compounds or agents which inhibit cell cycle regulation, inhibit DNA damage repair, inhibit cancer related genes or inhibit any other cancer selective process.

The composition may contain from 0.1% to 99% (w/w) preferably from 0.1- 60% (w/w), more preferably 0.2-12% by weight and most preferably 0.25 to 8% (w/w) of a compound of the first aspect depending on the method of administration.

Suitable carriers and/or diluents are well known in the art and include pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, (or other sugar), magnesium carbonate, gelatin, oil, alcohol, detergents, emulsifiers or water (preferably sterile). The composition may be a mixed preparation of a composition or may be a combined preparation for simultaneous, separate or sequential use (including administration).

The composition according to the invention for use in the aforementioned indications may be administered by any convenient method, for example by oral (including by inhalation), parenteral, mucosal (e.g. buccal, sublingual, nasal), rectal or transdermal administration and the compositions adapted accordingly. For oral administration, the composition can be formulated as liquids or solids, for example solutions, syrups, suspensions or emulsions, tablets, capsules and lozenges.

A liquid formulation will generally consist of a suspension or solution of the compound or physiologically acceptable salt in a suitable aqueous or nonaqueous liquid carrier(s) for example water, ethanol, glycerine, polyethylene glycol or oil. The formulation may also contain a suspending agent, preservative, flavouring or colouring agent.

A composition in the form of a tablet can be prepared using any suitable pharmaceutical carrier(s) routinely used for preparing solid formulations. Examples of such carriers include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.

A composition in the form of a capsule can be prepared using routine encapsulation procedures. For example, powders, granules or pellets containing the active ingredient can be prepared using standard carriers and then filled into a hard gelatine capsule; alternatively, a dispersion or suspension can be prepared using any suitable pharmaceutical carrier(s), for example aqueous gums, celluloses, silicates or oils and the dispersion or suspension then filled into a soft gelatine capsule.

Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the alimentary tract, for example by an outer coating of the formulation on a tablet or capsule. Typical parenteral compositions consist of a solution or suspension of the compound or physiologically acceptable salt in a sterile aqueous or nonaqueous carrier or parenterally acceptable oil, for example polyethylene glycol, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil. Alternatively, the solution can be lyophilised and then reconstituted with a suitable solvent just prior to administration.

Compositions for nasal or oral administration may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve, which is intended for disposal once the contents of the container have been exhausted. Where the dosage form comprises an aerosol dispenser, it will contain a pharmaceutically acceptable propellant. The aerosol dosage forms can also take the form of a pump-atomiser.

Compositions suitable for buccal or sublingual administration include tablets, lozenges and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

Compositions for rectal or vaginal administration are conveniently in the form of suppositories (containing a conventional suppository base such as cocoa butter), pessaries, vaginal tabs, foams or enemas. Compositions suitable for transdermal administration include ointments, gels, patches and injections including powder injections.

Conveniently the composition is in unit dose form such as a tablet, capsule or ampoule.

The fourth aspect of the invention provides a process for the manufacture of a composition according to the third aspect of the invention. The manufacture can be carried out by standard techniques well known in the art and comprises combining a compound according to the first aspect of the invention and the pharmaceutically acceptable carrier or diluent and optionally one or more additional active agents. The composition may be in any form including a tablet, a liquid, a capsule, and a powder or in the form of a food product, e.g. a functional food. In the latter case the food product itself may act as the pharmaceutically acceptable carrier.

The fifth aspect of the present invention relates to a compound of the first aspect, or a composition of the third aspect, for use in medicine. The fifth aspect of the invention particularly provides a compound of the first aspect or a composition of the third aspect for use in the treatment of cancer.

For the purposes of this invention, the compound of the first aspect or the composition of the third aspect can be provided for the treatment of adrenal cancer, AIDS-related lymphoma, anal cancer, ataxia-telangiectasia, bladder cancer, brain tumours, breast cancer, carcinoma, cervical cancer, chronic lymphocytic leukaemia, chronic myelogenous leukaemia, colorectal cancer, crainiopharyngioma, cutaneous T-cell lymphoma/mycosis fungoides, endometrial and uterine cancer, espophageal cancer, Ewing's sarcoma, fallopian tube cancer, gallbladder cancer, gastric cancer, getational trophoblastic disease and choriocarcinoma, hairy cell leukaemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leukaemia, LiFraumeni syndrome, liver cancer, lung cancer, lymphomas, medulloblastoma, melanoma, mesothelioma, metastates, myelomas, myeloproliferative disorder, neuroblastoma, Non-Hodgkin's disease, non-small cell lung cancer, oropharyngeal cancers, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer_* pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, sarcoma, small intestinal cancer, small cell lung cancer, testicular cancer, thymoma, urethral cancer, vaginal cancer, vulvar cancer or WiIm' s tumour.

The compounds of the present invention interact with and stabilize the guanine- rich quadraplex structures of the telomere. The present invention therefore provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for the stabilization of the guanine-rich quadruplex structure of a telomere. The present invention further provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for use in the inhibition of telomerase. The present invention further provides a compound of the first aspect of the invention or a composition of the third aspect of the invention for use in promoting senescence or apoptosis of a cancer cell.

The compounds of the invention are provided for the treatment of cancer. For the purposes of this invention, the term "treatment" means any amelioration, reduction in severity or reduction in the progress of the condition or a reduction in the symptoms of the condition. It will be appreciated that in some cases, the degree of the disease will be such that it is not possible to cure the patient. In this case, the term "treatment" means preventing the condition from deteriorating or getting worse for example by halting the progress of the disease without necessary ameliorating the condition or slowing the progress of the disease such that the life span and/or quality of life of the patient is improved.

A compound of the present invention may be administered simultaneously, subsequently or sequentially with one or more other active agent, such as a chemotherapeutic agent or an antiproliferative agent. In particular, the compound of the present invention can be administered with one or more of an alkylating agent (such as cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine, Busulfan or Thiotepa), an antimetabolite (such as Cytarabine, Gemcitabine, 6- mercaptopurine, 6-thioguanine, Fludarabine and Cladribine), an anthracycline (such as Idarubicin, Epirubicin), an antibiotic (such as Bleomycin), a camptothecin, a etoposide, a vinca alkaloid, a taxane (such as taxol, paclitaxel and docetaxel),and/or a platinium (such as cisplatin or oxaliplatin). The compound or composition of the invention may be administered in combination with one or more active agents for use in the treatment of side effects of cancer treatment such as antiemetics, antibiotics etc.

The compounds of the invention will normally be administered in a daily dosage regimen (for an adult patient) of, for example, an oral dose of between 1 mg and 2000 mg, preferably between 30 mg and 1000 mg, e.g. between 10 and 250 mg or an intravenous, subcutaneous, or intramuscular dose of between 0.1 mg and 100 mg, preferably between 0.1 mg and 50 mg, e.g. between 1 and 25 mg of the compound of the formula (I) or (II) or a physiologically acceptable salt thereof calculated as the free base, the compound being administered 1 to 4 times per day. Suitably the compounds will be administered for a period of continuous therapy, for example for a week or more. The sixth aspect of the invention relates to the use of a compound of the first aspect of the invention in the manufacture of a medicament for the treatment of cancer.

The medicament of the sixth aspect of the invention may further comprise one or more other active agent, such as a chemotherapeutic agent or an antiproliferative agent. In particular, the medicament of the sixth aspect may comprise one or more of an alkylating agent (such as cyclophosphamide, Ifosphamide, Melphalan, Chlorambucil, BCNU, CCNU, Decarbazine, Procarbazine, Busulfan or Thiotepa), an antimetabolite (such as Cytarabine, Gemcitabine, 6-mercaptopurine, 6-thioguanine, Fludarabine and Cladribine), an anthracycline (such as Idarubicin, Epirubicin), an antibiotic (such as Bleomycin), a camptothecin, a etoposide, a vinca alkaloid, a taxane (such as taxol, paclitaxel and docetaxel),and/or a platinium (such as cisplatin or oxaliplatin).

The medicament may contain from 0.1% to 99% (w/w) preferably from 0.1- 60% (w/w), more preferably 0.2-12% by weight and most preferably 0.25 to 8% (w/w) of a compound of the first aspect, depending on the method of administration.

The seventh aspect of the invention relates to a method of treating cancer comprising administering to a person in need therefore a compound as defined in the first aspect of the invention or a composition of the third aspect of the invention.

The compound of the first aspect of the invention is preferably provided in a therapeutically effective amount. The amount of the compound of the first aspect of the invention effective to treat a disorder as set out above depends on the nature and severity of the disorder being treated and the weight of the patient in need thereof. However, a single unit dose for a 70kg adult will normally contain. 0.01 to lOOmg, for example 0.1 to 50mg, preferably 0.5 to 6mg of the compound of the invention per day. Unit doses may be administered once or more than once a day, for example, 2, 3 or 4 times a day, usually 1 to 3 times a day, more preferably 1 or 2 times per day. The total daily dosage can be in range of approximately 0.0001 to 0.2mg per kg per day, more usually 0.001 to O.lmg per kg per day, preferably 0.01 to O.lmg per kg per day. The unit dose is preferably provided in the form of a capsule or a tablet.

It will be appreciated that the compound of the first aspect or composition of the third aspect may be provided prior to, in combination with and/or subsequent to a different cancer treatment. The compound and composition of the invention therefore may be administered prior to, in combination with and/or subsequent to chemotherapy, radiotherapy, surgery etc. The compound or composition of the invention may be administered in combination with one or more active agents for use in the treatment of side effects of cancer treatment such as antiemetics, antibiotics etc.

The compound or composition of the invention may be provided as a single course of treatment or repeated courses of treatment over a period of time to be determined by a physician.

All preferred features of each of the aspects of the invention apply to all other

aspects mutatis mutandis. The invention may be put into practice in various ways and a number of specific embodiments will be described by way of example to illustrate the invention with reference to the accompanying drawings, in which:

Figure 1 shows the docking of the nickel(II) complex 3 with the human parallel intramolecular quadruplex formed from four repeats of telomeric DNA. The model shows very good stacking between the rings of the metal complex and three of the guanine rings of the quadruplex DNA;

Figure 2 shows the TRAP gel for compound 3 showing the characteristic ladders produced by PCR amplification of the oligonucleotides generated by the activity of telomerase on a TS primer;

Figure 3 show the observed changes in quadruplex and duplex melting temperature (ΔTm) with changes in ligand concentrations;

Figure 4 shows two views (a) and (b) of the docking of the nickel(II) complex 3 with the human parallel intramolecular quadruplex formed from four repeats of telomeric DNA. The model shows very good stacking of the metal complex with the quadruplex DNA and also a good interaction between the side chains and the grooves of DNA; and

Figure 5 shows the effects of compound 3 on MACl 5 A tumours.

The present invention will now be illustrated by reference to one or more of the following non- limiting examples.

EXAMPLES Synthesis of iV,Λr'-Bis-4-(hydroxysalicylidene)phenylenediainine-Nickel(II)

(1).

1,2-phenylenediamine (0.2800 g, 2.53 mmol) and 2,4-dihydroxybenzaldehyde (0.6901 g, 4.89 πunol) were dissolved in methanol (50 mL) and heated for 30 min at reflux (70-75⁰C). Ni(OAc)₂-4H₂O (1.2621 g, 4.96 mmol) was then added to this yellow mixture. A red solid precipitated immediately. The reaction was refluxed for a further 3h (70-75⁰C). After this time, the reaction mixture was cooled to room temperature. The red precipitate was filtered and washed with methanol (100 mL), diethyl ether (50 mL) and water (50 mL) to afford 1 as a red solid. Yield: 0.5630 g, 53 %. IR (cm^"1): 3410, 2910, 1601, 1541, 1488, 1442, 1369, 1287, 1266, 1236, 1197, 1126, 1025, 954, 860, 840, 786, 737, 657. ¹H NMR (400 MHz, DMSO-d₆): δ 6.21 (s, 2H, ArH); 6.23 (d, J =2.2 Hz, 2H, ArH); 7.23-7.20 (dd, J = 3.1, 5.9 Hz, 2H, ArH); 7.40 (d, J = 8.5 Hz, 2H, ArH); 8.00-7.98 (dd, J= 3.5, 6.2 Hz, 2H, ArH); 8.55 (s, 2H, -CH=N-); 10.19 (s, -OH). ¹³C DEPT45 NMR (400 MHz, DMSO-d₆) δ 102.8, 107.0, 114.7, 125.7, 135.1, 153.5. ESI-MS Calcd for C₂₀H₅₄N₂NiO₄ (M⁺): 405.03. Found: 405.21. Anal. Calcd for C₂₀H₅₄N₂NiO₄-2H₂O: C, 54.46; H, 4.11; N, 6.35. Found: C, 54.02; H, 4.12; N, 6.16.

Synthesis of N,N'-Bis-4-[hydroxysalicylidene]-4-fluoro-l,2- phenylenediamine-Νickel (II) (2).

4-Fluoro-l,2-phenylenediamine (0.2988 g, 2.29 mmol) and 2,4- dihydroxybenzaldehyde (0.6528 g, 4.63 mmol) were dissolved in MeOH (50 mL) and heated for 30 min at reflux (70-75⁰C). Ni(OAc)₂-4H₂O (1.1792 g, 4.64 mmol) was then added to this black mixture. The reaction was boiled at reflux for a further 3h (70-75⁰C). After this time, the reaction mixture was cooled down to room temperature. The black precipitate was filtered and washed with methanol (50 mL), diethyl ether (50 mL) and water (50 mL) to afford 2 as a black solid. Yield: 0.5200 g, 49 %. IR (cm^"1): 3063, 1601, 1550, 1490, 1439, 1370, 1278, 1220, 1167, 1118, 979, 838, 809, 786, 730, 650, 533, 506. ¹H NMR (500 MHz, DMSO-d₆): δ 6.21 (s, 2H, ArH), 6.24-6.23 (t br, IH, ArH), 6.25 (d, J = 2.2, IH, ArH), 7.14-7.10 (td, J = 2.4, 8.5, IH, ArH), 7.39-7.36 (dd, J = 3.5, 8.5, 2H, ArH), 7.93-7.91 (dd, J = 2.5, 11.6, IH, ArH), 8.03-8.00 (dd, J = 5.3, 9.2 ,1H, ArH), 8.47 (s, IH, -CH=N-), 8.51 (s, IH, -CH=N-), 10.24 (s, - OH). ¹³C NMR (400 MHz, DMSO-d₆): δ 102.5, 103.4, 107.7, 108.1, 112.8 (²J_CF = 23 Hz), 114.4 (⁴J_CF = 8 Hz), 115.9 (³J_CF = 9 Hz), 135.8 (²J_CF = 18 Hz), 138.8, 143.7 (³J_CF = 9 Hz), 154.1, 154.8, 159.5, 161.9, 164.2, 166.2 (¹Jc_P = 256 Hz), 166.8. ¹⁹F NMR (500 MHz, DMSO-d₆, 390K): δ -116.83 (m br). ESI-MS Calcd for C₂₀Hi₃FN₂NiO₄ (M⁺): 423.02 a.m.u. Found: 423.01 a.m.u. Anal. Calcd for C₂₀H₁₃FN₂NiO₄-2H₂0: C, 52.33; H, 3.73; N, 6.10. Found: C, 51.65; H, 3.73; N, 6.13.

Synthesis N,N'-Bis[4-[[l-(2-ethyl)ρiperidine]oxy]salicylidene] phenylenediamine-Nickel (II) (3).

A suspension of 1 (0.1310 g, 0.30 mmol), l-(2-chloroethyl)piperidine hydrochloride (0.2199 g, 1.17 mmol) and K₂CO₃ (0.2869 g, 2.05 mmol) in dry DMF (25 mL) was stirred for 72 h under N₂. After this time an orange precipitate formed which was filtered and washed with DMF (5 mL) and diethyl ether (5 mL). The resulting solid was dissolved in CH₂Cl₂ and washed with water. The organic layer was evaporated under reduced pressure to give 3 as an orange solid. Yield: 0.0886 g, 47%. IR (cm^"1): 3258, 2930, 2790, 1605, 1577, 1514, 1500, 1473, 1433, 1385, 1368, 1319, 1268, 1248, 1200, 1153, 1122, 1090, 1032, 997, 941, 915, 853, 834, 818, 777, 767, 651, 621, 584, 554, 536, 516. ¹R NMR (500 MHz, DMSO-de): δ 1.38-1.37 (br m, 4H, piperidine H-4), 1.52-1.47 (m, 8H, piperidine H-3, H-5), 2.42 (br, 8H, piperidine H-2, H- 6), 2.65-2.63 (t, J = 5.8 Hz, 4H, CH₂N-), 7.09-4.07 (t, J = 5.8 Hz, 4H, CH₂O-), 6.33-6.30 (dd, J = 2.2, 8.9 Hz, 2H, ArH), 6.35 (d, J = 1.8 Hz, 2H, ArH), 7.26- 7.24 (dd, J = 3.2, 6.2 Hz, 2H, ArH), 7.47 (d, J = 8.9 Hz, 2H, ArH), 8.05-8.03 (dd, J = 3.3, 6.2 Hz, 2H, ArH), 8.66 (s, 2H, -CH=N-). ¹³C DEPT45 NMR (400 MHz, CDCl₃-d₆) δ 25.0, 26.7, 55.8, 58.5, 66.7, 104.0, 109.9, 115.3, 127.4, 135.1, 153.2. ESI-MS Calcd for C₃₄H₄₀N₄NiO₄ (M⁺): 627.4 a.m.u. Found: 627.3 a.m.u. mp: 211.0-213.7.Anal. Calcd for C₃₄H₄₀N₄NiO₄: C, 65.09; H, 6.43; N, 8.93; Found: C, 65.29; H, 6.40; N, 8.54. Synthesis of N,N'-Bis[4-[[l-(2-ethyl)piperidine]oxy]salicylidene]-4-fluoro- 1,2-phenylenediamine-Nickel (II) (4).

A suspension of 2 (0.1310 g, 0.31 mmol), l-(2-chloroethyl)piperidine hydrochloride (0.2335 g, 1.24 mmol) and K₂CO₃ (0.3036 g, 2.17 mmol) in dry DMF (30 mL) was stirred for 72 h under N₂. After this time an orange precipitate formed which was filtered and washed with DMF (5 mL) and diethyl ether (5 mL). The resulting solid was dissolved in CH₂Cl₂ and washed with water. The organic layer was evaporated under reduced pressure to give 5 as an orange solid. Yield: 0.11 g, 55 %. IR (cm ¹): 2933, 2781, 1662, 1605, 1581, 1504, 1470, 1432, 1370, 1318, 1278, 1246, 1214, 1177, 1120, 1025, 982, 964, 945, 834, 788, 769, 656, 541, 518. ¹H NMR (500 MHz, DMSO-d₆): δ 1.39-1.38 (m, 4H, piperidine H-4), 1.51-1.49 (m, 8H, piperidine H-3, H-5), 2.42 (br, 8H_; piperidine H-2, H-6), 2.66-2.64 (t, J = 5.6 Hz, 4H, -CH₂N-), 4.10- 4.07 (m, Hz, 4H, -CH₂O-), 6.33-6.31 (br dd, 2H, ArH), 6.35 (br, 2H, ArH), 7.17-7.14 (br td, J = 8.3 Hz, IH, ArH), 7.45-7.42 (br td, J = 7.18, 2H, ArH), 7.96-7.94 (br, IH, ArH), 8.06-8.03 (dd, J = 9.1, 5.3 Hz, IH, ArH), 8.61 (s, IH, -CH=N-), 8.57 (s, IH, -CH=N-). ¹³C DEPT45 NMR (400 MHz, CDCl₃-d₆) δ 24.1, 25.8, 54.9, 57.6, 57.6, 65.9, 101.5, 102.9, 109.8, 113.1 (²7_CF = 24 Hz), 114.6 (³J_CF = 10 Hz), 134.2 (²J_CF = 16 Hz), 151.9, 152.4. ¹⁹F NMR (500 MHz, CDCl₃-d₆): δ -115.53 (m br). ESI-MS Calcd for C₃₄H₃₉FN₄NiO₄ (M⁺): 645.4 a.m.u. Found: 645.2 a.m.u. mp: 204.9-206.5. Anal. Calcd for C₃₄H₃₉FN₄NiO₄: C, 63.27; H, 6.09; N, 8.68; Found: C, 62.99; H, 6.12; N, 8.72.

Synthesis of 2-hydroxy-4-(2-(piperidin-l-yl)ethoxy)benzaldehyde (5) 5 was prepared by modification of a synthetic method reported in Barbera et at, Liquid Cryst, 2003, 30 (6), 651-661.

A suspension of 2,4-dihydroxybenzaldehyde (3.00 g, 21.29 mmol), l-(2- Chloroethyl)piperidine hydrochloride (3.9969 g, 21.28 mmol) and NaHCO₃ (3.5764, 42.57 mmol) in dry acetone (150 mL) was refluxed (60-65⁰C) for 68h. After that period of time the excess of salt was removed by filtration and the acetone was evaporated to dryness and then purified by column chromatography over silica gel using Ethyl acetate. Upon concentration of the eluted fraction 5 was obtained as a yellow solid (1.6501 g. Yield: 29 %). ¹H NMR (400 MHz, CDCl₃): δ 1.34-1.40 (m, 2H, -0-CH₂-CH₂-CH₂-N-), 1.51- 1.56 (q, 4H, piperidine H-3, H-5), 2.42-2.45 (br, 4H, piperidine H-2, H-6), 2.69-2.72 (t, 2H, -CH₂N-, ³J_11H = 6.0 Hz), 4.06-4.09 (t, 2H, -CH₂O-, ³J_HH = 6.0 Hz), 6.34-6.35 (d, IH, ArH, ³J_HH = 2.4 Hz), 6.44-6.47 (dd, IH, ArH, ³J_HH = 2.4 Hz, ⁴J_HH = 8.8 Hz), 7.32-7.35 (d, IH, ArH, ³J_H11 = 8.8 Hz), 9.63 (s, IH, - CH=O). ¹³C NMR (400 MHz, CDCl₃): 23.0, 24.8, 54.0, 56.4, 65.4, 100.2, 107.7, 114.1, 134.1, 163.4, 165.0, 193.3.

Synthesis of N,N'-Bis-4-(hydroxysalicylidene)phenylenediamine- oxovanadiura(IV) (6)

6 was prepared by modification of a synthetic method reported in Plitt et al, J. Inorg. Biochem 2005, 99, 1230-1237. 1,2-phenylenediamine (0.3931 g, 3.63 mmol) in methanol (10 mL) was added to a methanolic solution (10 mL) of 2,4-dihydroxybenzaldehyde (1.0033 g, 7.26 mmol) and refluxed for Ih. The yellow solution was cooled at room temperature and then VOSO₄H₂O (0.5914 g, 3.63 mmol) in water (20 mL) and after that Et₃N (1 mL) was added to the methanolic solution and refluxed for a further 3h. The green precipitated formed was filtered and washed with methanol (50 mL), water (25 mL) and ether (50 mL) to afford 6 as a green solid. Yield: 1.2703 g, 84 %. IR (KBr, cm^"1): 1600 (C=N), 1542 (Ar), 1493 (Ar), 984 (V=O), 849 (Ar), 803 (Ar), 748 (Ar). FAB⁺-MS Calcd for C₂₀H₁₈N₂O₇V (M⁺): 413.23. Found: 414 a.m.u. UV/Vis (DMSO) λ (nm) (log ε, M-^cnϊ¹): 336 (4.23), 398 (4.36). Anal. Calcd for C₂₀Hi₈N₂O₇V -2H₂O: C, 53.46; H, 4.04; N, 6.23. Found: C, 53.83; H, 3.79; N, 5.90.

Synthesis of N,N'-Bis[4-[(2-ethyl)morpholine]oxy]salicylidene]-l,2- phenylenediamine-nickel (II) (7)

A suspension of 1 (0.3610 g, 0.89 mmol), 4-(2-chloroethyl)morpholine hydrochloride (0.9781 g, 5.26 mmol) and K₂CO₃ (1.0125 g, 7.33 mmol) in DMF (35 mL) was heated at 90°C overnight. After completion of the reaction the salts were removed by filtration. The DMF was evaporated under vacuum. The resulting solid was re-crystallized using a CH₂Cl₂:n-pentane mixture to give 7 as an red solid. Yield: 0.4865 g, 86%. IR (KBr, cm^"1): 2801 (CH₂), 1615 (C=N), 1577 (Ar), 1515 (Ar), 1117 (C-O), 854 (Ar), 753 (Ar). ¹H NMR (400 MHz, DMSO-d₆): 2.48 (br s, 8H, -CIj₂-O-CH₂-), 2.51-2.52 (t, 4H, -CH₂N-, ³J_HH= 1.8 Hz), 3.58-3.60 (t, 8H, -CH₄-N-CH₅-, ³J_HH= 5.3 Hz), 4.10-4.13 (t, 4H, CH₂O-, ³J_HH = 4.4 Hz), 6.31-6.34 (dd, 2H, ArH, ³J_11H = 8.8 Hz, ⁴Jm = 6.4 Hz), 6.36 (br d, 2H, ArH), 7.24-7.26 (dd, 2H, ArH, ³J_11H = 6.3 Hz, ⁴J_HH = 3.3 Hz), 7.46-7.48 (d, 2H, ArH, ³J_HH = 8.8 Hz), 8.02-8.04 (dd, 2H, ArH, ³J_11n = 6.9 Hz, ⁴J_HH = 3.6 Hz), 8.64 (s, 2H, -CH=N-). ¹³C NMR (400 MHz, DMSO-d₆): δ 54.0, 57.4, 66.3, 102.0, 108.2, 115.8, 127.2, 143.1, 155.1, 165.1, 167.8. ESI-MS Calcd for C₃₂H₃₆N₄NiO₆ (M⁺): 631.3 a.m.u. Found: 631 a.m.u. Anal. Calcd. For C₃₂H₃₆N₄NiO₆-IH₂O: C, 59.2; H, 5.90; N, 8.63. Found: C, 59.36; H, 5.47; N, 8.55.

Synthesis of N,N'-Bis[4-[(2-ethyl)morpholine]oxy]salicylidene]-4-fluoro-

1,2-phenylenediamine-nickel (II) (8)

A suspension of 2 (0.2986 g, 0.68 mmol), 4-(2-chloroethyl)morpholine hydrochloride (0.8073 g, 4.34 mmol) and K₂CO₃ (0.9170 g, 6.63 mmol) in DMF (50 rnL) was heated at 90⁰C for 20h. After completion of the reaction the K₂CO₃ was removed by filtration. The DMF was evaporated under vacuum. The resulting solid was re-crystallized twice using a CH₂Cl₂:pentane mixture to give 8 as an red solid. Yield: 0.3651 g, 83%. IR (KBr, cm^"1): 2954 (CH₂), 1610 (C=N), 1584 (Ar), 1505 (Ar), 1116 (C-O), 832 (Ar), 788 (Ar). ¹H NMR (400 MHz, CDCl₃): 2.57-2.59 (t, 8H, -CH^-O-CH₂-, ³J_11H= 4.4 Hz), 2.79-2.82 (t, 4H, -CH₂N-, ³JR_H = 5.6 Hz), 3.74-3.76 (t, 8H, -CEb-N-CH₂-, ³JH_H = 4.6 Hz), 4.07- 4.10 (t, 4H, CH₂O-, ³JH_H = 5.6 Hz), 6.23-6.26 (dd, IH, ArH, ³J_HH = 8.8 Hz, ⁴J_11H = 2.3 Hz), 6.27-6.29 (dd, IH, ArH, ³J_HH = 8.8 Hz, ⁴J_11H = 2.3 Hz), 6.49-6.50 (br, 2H, ArH), 6.80-6.84 (td, IH, ArH, ³J_nH = 9.4 Hz, ³J_HF= 2.2 Hz), 7.05-7.24 (d, 2H, ArH, ³J_HH = 25.3 Hz, ⁴J_nH = 8.9 Hz), 7.21-7.24 (dd, IH, ArH, ³J_HP = 9.6 Hz, ⁴J_HH = 2.2 Hz), 7.56-7.60 (dd, IH, ArH, ³J_HH = 9.1 Hz, V = 4.9 Hz), 7.80 (s, IH, -CH=N-), 7.86 (s, IH, -CH=N-). ¹³C NMR (400 MHz, CDCl₃): δ 54.1, 57.3, 56.8, 66.9, 101.5, 101.7, 102.8, 108.8, 109.3, 113.3 (²J_CF = 24 Hz), 114.6 (⁴Jc_F = 15 Hz), 114.7 (³Jc_F = 10.3 Hz), 134.3 (²J_CF = 17 Hz), 139.0, 143.8 (³J_CF = 9 Hz), 152.0, 152.4, 160, 162.5, 164.7, 166.4 (¹Jc_F = 253 Hz), 168.6. ¹⁹F NMR (500 MHz, CDCl₃): δ -114.2 (br). FAB⁺-MS Calcd for C₃₂H₃₅FN₄NiO₆ (M⁺): 649.3 a.m.u. Found: 649 a.m.u. Anal. Calcd. For C₃₂H₃₅FN₄NiO₆^H₂O: C, 56.08; H, 5.74; N, 8.17. Found: C, 55.95; H, 5.68; N, 8.09.

Synthesis of N,N'-Bis[4-[[l-(2ethyl) piperidine)oxy]salicylidene]phenylenediamine-zinc (II) (9)

1,2-phenylenediamine (1.0363 g, 1.04 mmol) and 5 (0.4605 g, 1.85 mmol) were dissolved in ethanol (25 mL) and heated to reflux for 2h. The solution become to a yellow colour immediately. The ethanol was evaporated at reduced pressure and the yellow solid formed was then dissolved in methanol (15 mL) and Zn(OAc)₂-2H₂0 (0.2277g, 1.04 mmol) was added to the yellow solution at once. A yellow precipitated was formed immediately. It was refluxed for 3h. After cooled to room temperature the yellow precipitated was filtered and washed with methanol (50 mL) to obtain a yellow bright solid. Yield: 0.3908 g, 59 %. IR (KBr, cm^"1): 2934 (CH₂), 2784 (CH-O), 1608 (C=N), 1581 (Ar), 1522 (Ar), 1190 (C-O), 1124 (Ar), 1038 (Ar), 746 (Ar). ¹H NMR (400 MHz, DMSO-d₆): δ 1.38-1.39 (br, 4H, piperidine H-4), 1.48-1.53 (m, 8H, piperidine H-3, H-5), 2.43 (br, 8H, piperidine H-2, H-6), 2.64-2.66 (t, 4H, -CH₂N-, ³J_HH = 5.7 Hz), 4.05-4.08 (t, 4H, -CH₂O-, ³J_nH = 5.7 Hz), 6.14-6.17 (dd, 2H, ArH, ³J_nH = 9.0, ⁴J_HH = 2.2 Hz), 6.19 (br d, 2H, ArH), 7.28-7.30 (br, 4H, ArH), 7.78-7.81 (dd, 2H₅ ³J_HH = 6.0, ⁴J_nH = 3.0 Hz), 8.9 (s, 2H, -CH=N-). ¹³C DEPT45 NMR (400 MHz, DMSO-d₆) δ 24.4, 26.0, 54.8, 57.7, 65.8, 104.6, 105.2, 114.7, 116.3, 126.8, 137.9, 139.8, 161.4, 164.6, 174.7. FAB⁺-MS Calcd for C₃₄H₄₀N₄ZnO₄ (M⁺): 634.1 a.m.u. Found: 633 a.m.u. Anal. Calcd for C₃₄H₄₀N₄ZnO₄: C, 64.40; H, 6.36; N, 8.84; Found: C, 64.55; H, 6.18; N, 9.05.

Synthesis of N,N'-Bis[4-[[l-(2- ethyl)piperidine]oxy]salicylidene]phenylenediamine-oxovanadium(IV) (10)

A suspension of 6 (0.3233 g, 0.78 mmol), l-(2-chloroethyl)piperidine hydrochloride (0.5753 g, 3.12 mmol) and K₂CO₃ (0.7700 g, 5.57 mmol) in DMF (35 mL) was stirred for 72 h at room temperature. After this time the excess salts were removed by filtration and the DMF evaporated at reduced pressure. This product was re-crystallized using a mixture of CH₂Cl₂ and pentane to give 10 as a green solid. Yield: 0.3021 g, 61%). IR (KBr, cm^"1): 1613 (C=N), 1521 (Ar), 1422 (Ar), 1125 (C-O), 985 (V=O), 839 (Ar), 798 (Ar), 747 (Ar). FAB⁺-MS Calcd for C₃₄H₄₀N₄O₅V (M⁺): 635.65. Found: 636 a.m.u. UV/Vis (DMSO) λ (nm) (log ε, M^"1 ^m^"1): 334 (4.29), 398 (4.42). Anal. Calcd for C₃₄H₄₀N₄O₅V: C, 64.24; H, 6.34; N, 8.81. Found: C, 64.10; H, 6.45; N, 8.68.

Synthesis of 4-hydroxy-phenyl-l,10-phenanthroline-2-carboxamide (11).

l,10-phenanthroline-2-carbonyl chloride (Ig, 4.4 mmol) was suspended in dry and degassed DCM (40 mL) and freshly distilled triethylamine (1.78 mL, 13.5 mmol) was added to this solution. Trimethylsilyl protected 4-aminophenol (1.6g, 8.9 mmol) was dissolved in dry and degassed DCM (10 mL) and added dropwise to the phenanthroline solution via canular transfer under nitrogen. This solution was heated to reflux for 12 hours to give a brown solution with some pale precipitate. The reaction mixture was filtered and the resulting dark brown solution was concentrated under vacuum. This was then columned using silica and 1% ethanol in 99% ethyl acetate to give PhenL4 11 as yellow solid (yield = 0.98g, 70%). ¹H NMR (DMSO-d₆): 6 = 10.9 (s, H_h, IH), 9.37 (s, H_k, IH) 9.21-9.19 (dd, H_a, J_ab = 4.4 Hz, J_ac = 1.6 Hz, IH), 8.76-8.74 (d, H_g, J_gf = 8.4 Hz, IH), 8.60-8.56 (dd, H_c, J_cb = 8.0, Hz, J_ca = 1.6 Hz, IH), 8.48-8.46 (d, H_f, J_fg = 8.4 Hz, IH), 8.13 (m, H_d, H_e, 2H), 7.89-7.86 (dd, H_b, J_ba = 4.4, J_bc = 8.0 Hz, IH), 7.73-7.71 (d, H₁, H_m, Jy = J_lm = 8.8 Hz, 2H), 6.83-6.81 (d, H_j, H₁, Jy = J_lm = 8.8 Hz, 2H) ppm. ¹³C NMR (DMSO-d₆): δ = 162.34 (C=O), 154.43, 150.38 (C- H₈), 149.29, 138.86 (C-HΛ 137.80 (C-H_c), 131.43, 130.85, 130.43, 129.52 (C- Hd), 128.75, 127.15 (C-H₆), 124.69 (C-H_b), 122.34 (C-H_i/m), 121.69 (C-H_f), 115.70 (C-H_jZ1) ppm. IR (KBr): v = 3425, 3232, 1648 (C=O), 1595, 1536, 1514, 1439, 1265, 1226, 1134, 1083, 1037, 865, 825, 717. ES MS: m/z [PhenL4 + H+] = 316. Elemental analysis for C₁₉H₁₃N₃O₂-C₂H₅O: Expected C, 69.79%; H, 5.30%; N, 11.63%. Found C, 69.85%, H, 4.99%, N, 11.74%.

Synthesis of ljlO-phenanthroline-Z-carboxamide-phenyl^-ethoxy- piperidine (12).

Compound 11 (150mg, 0.47 mmol) was

suspended in dry and degassed DMF (20 mL). NaH (60% disperssion in mineral oil) (85 mg, 2.3 mmol) was added to this suspension and heated to HO⁰C. Chloroethylpiperidine (175 mg, 0.95 mmol) was dissolved in DMF (40 mL) and added dropwise over a period of 2 hours during which time the solution turned from deep red to transparent yellow-orange. After the addition was complete the reaction mixture was allowed to cool and left to stir at room temperature for 3 days. The reaction mixture was then filtered to give a yellow solution that was concentrated under vacuum, with heating to 35°C, to give a yellow solid. HCl(aq) was added to a solution of the yellow solid dissolved in methanol and Et3N was added to neutralise the solution. The resulting solution was concentrated under vacuum and columned on silica using a gradient of ethanol (0-10%) in ethyl acetate with 0.5% Et₃N. On reduction the combined fractions gave PhenL5 12 as a yellow solid (yield = 175 mg, 85%). ¹H NMR (DMSO-d₆): δ = 10.8 (s, H_h, IH), 9.23-9.21 (dd, H_a, J_ab = 4.8 Hz, J_ac = 1.2 Hz, IH), 8.77-8.75 (d, H_g, J_≠ = 6.8 Hz, IH), 8.60-8.58 (dd, H_c, J_cb = 6.8, Hz, J_ca = 1.2 Hz, IH), 8.48-8.46 (d, H_f, J_fg = 6.8 Hz, IH), 8.14 (m, H_d, H_e, 2H), 7.84-7.83 (dd, H_b, J_ba = 4.4, J_bc = 8.0 Hz, IH), 7.84-7.83 (d, H₁, Jy = 7.2 Hz, 2H), 7.03- 7.00 (d, H_j, Jy = 7.2 Hz, 2H), 4.10-4.08 (t, H_k, J_a = 4.8 Hz, 2H), 2.68-2.66 (t, H₁, J_lk = 4.8 Hz, 2H), 2.45 (m, H_m, 4H), 1.50 (m, H_n, 4H), 1.39 (m, H₀, 2H) ppm. ¹³C NMR (DMSO-d₆): δ = 162.66 (C=O), 155.66, 150.81 (C-H₃), 145.60, 144.35, 138.96 (C-H_g), 137.28 (C-H_c), 132.01, 130.48, 129.65, 128.99 (C-H₁O, 126.92 (C-H₆), 124.42 (C-H_b), 122.12 (C-HO, 121.56 (C-H_f), 115.29 (C-H_j), 66.41 (C-H_k), 58.32 (C-H₁), 54.92 (C-HJ, 26.42 (C-H_n), 24.58 (C-H₀) ppm. IR (KBr): v = 3356, 2923, 1664 (C=O), 1540, 1509, 1491, 1265, 1242, 1229, 1137, 1043, 1034, 950, 853, 833, 821, 719. ES MS: m/z [PhenL5 + H+] = 427. Elemental analysis for C₂₆H₂₆N₄O₂-H₂O: Expected C, 70.25%; H, 6.35%; N, 12.60%. Found C, 70.07%, H, 5.99%, N, 12.45%.

l,10-phenanthroIine-2-carboxamide-phenyl-4-ethoxy-piperidine platinum (II) chloride (13).

Compound 12 (40 mg, 0.09 mmol) was dissolved in ethanol (15 mL) to give a pale yellow solution. To this an aqueous solution of K₂PtCl₄ (30 mg, 0.09 mmol) was added and the resulting mixture heated to reflux for 6 hours to give a red-brown coloured solution. This was left to stir at room temperature overnight. The reaction mixture was filtered to give a red solution which was concentrated under vacuum to give a bright red solid. This was washed twice with small quantites of methanol and once with ether to give PhenL5Pt 13 as a bright orange solid (yield = 25 mg, 44%). ¹H NMR (DMSO-d₆): δ = 9.23-9.21 (dd, H_a, IH), 9.01-8.99 (m, H_c, H_g, 2H), 8.29 (m, H_d, H_e, 2H), 8.13-8.11 (dd, H_b, IH), 8.08-8.07 (d, H_f, 2H), 7.22 (d, H₁, 2H), 6.89 (d, H_J5 2H), 4.10-4.08 (t, H_k, J_ki = 4.8 Hz, 2H), 2.68-2.66 (t, H₁, J_lk = 4.8 Hz, 2H), 2.45 (m, H_m, 4H), 1.50 (m, H_n, 4H), 1.39 (m, H₀, 2H) ppm. ¹³C NMR (DMSO-d₆): δ = 150.88, 139.80, 127.40, 130.30, 128.98, 127.67, 124.24, 113.95 ppm. IR (KBr): v (cm^"1) = 3424, 1626 (C=O), 1603, 1504, 1235. ES MS: m/z [PhenL5PtCl + H+] = 657. Elemental analysis for C₂₆H₂₅N₄O₂PtCl-SKCl: Expected C, 35.50%; H, 2.86%; N, 6.36%. Found C, 35.10%, H, 2.94%, N, 6.11%.

l,10-phenanthroline-2-carboxamide-phenyl-4-ethoxy-piperidine palladium (II) chloride (14).

Compound 12 (50 mg, 0.12 mmol) and tetrabutylammonium acetate (93 mg, .29 mmol) were dissolved in dry and degassed DMF (15 ml) to give a pale yellow solution. Palladium (II) cyclooctadiene dichloride (33 mg, 0.12 mmol) dissolved in DMF (5 mL) was added via canular transfer to the stirring ligand solution. After 30 minutes the reaction had turned bright red-orange in colour; this was left to stir at room temperature over night. The reaction mixture was filtered and the bright red orange solution concentrated under vacuum with heating to 35C. The resulting orange solid was washed thrice with small quantities of methanol and once with ether to give PhenL5Pd 14 (yield = 12 mg, 20%). ¹H NMR (DMSO-d₆): δ = 8.93-8.90 (m, H_a, H_c, H_g, 3H), 8.31 (m, H_d, H_e, 2H), 8.12-8.10 (m, H_b, H_f, 2H), 7.21 (d, H_i5 Jy = 7.2 Hz, 2H), 6.85 (d, H_j, Jij = 7.2 Hz, 2H), 4.10-4.08 (t, H_k, J_H = 4.8 Hz, 2H), 2.68-2.66 (t, H₁, J_lk = 4.8 Hz, 2H), 2.45 (m, H_m, 4H), 1.50 (m, H_n, 4H), 1.39 (m, H₀, 2H) ppm. ¹³C NMR (DMSO-d₆): δ = 140.59, 151.67, 129.78, 128.72, 127.14, 127.93, 124.24, 113.68 ppm. IR (KBr): v (cm^"1) = 3433, 1622 (C=O), 1600, 1235, 863, 700. ES MS: m/z [PhenL5PdCl + H+] = 567.

Stock Solution Preparation.

3 and 4 were dissolved in a mixture of DMSO (75% by volume), H₂O (20%) and ImM HCl (5%) to give an 8mM stock solution. Complexes were dissolved in DMSO or methanol to give a 10, 5 or 2.5mM stock solution. All solutions were stored at -80 ⁰C and defrosted and diluted immediately before use.

FRET measurements.

All oligonucleotides and their fluorescent conjugates were purchased from Eurogentec (UK) or Invitrogen (UK). DNA was dissolved as a stock 20 μM solution. All dilutions were carried out with 50 mM potassium cacodylate buffer (pH 7.4) The ability of the compounds to stabilise G-quadraplex DNA was investigated using a fluorescence resonance energy transfer (FRET) assay modified to be used as a high-throughput screen in a 96-well format. The labelled oligonucleotide F21T (5-FAM-dGGG(TTAGGG)₃-rAMRA-3; donor fluorophore FAM: 6-carboxyfluorescein; acceptor fluorophore TAMRA: 6- carboxy-tetramethylrhodamine) used as the FRET probe was diluted from stock to the correct concentration (400 nM) in a 50 mM potassium cacodylate buffer (pH 7.4) and then annealed by heating to 92°C for 5 min (FRET protocol A) or by heating to 85°C for 10 mins (FRET protocol B), followed by cooling to room temperature in the heating block. Compounds were prepared from stock concentrations (described above) on the day of use. Final solutions were prepared using DMSO in the initial 1:10 dilution, after which 50 mM potassium cacodylate buffer (pH 7.4) was used in all subsequent steps. The maximum HCl concentration in the reaction volume (at a ligand concentration of 20 μM) is thus 200 μM, well within the range of the buffer used. 96-well plates (MJ Research, Waltham, MA) were prepared using a dilution robot resulting in 200 nM DNA concentration in each well and the appropriate drug concentration. Measurements were made on a DNA Engine Opticon (MJ Research) with excitation at 450-495 nm and detection at 515-545 nm. Fluorescence readings were taken at intervals of 0.5⁰C over the range 30- 100°C, with a constant temperature being maintained for 30 seconds prior to each reading to ensure a stable value. Final analysis of the data was carried out using a script written in the program Origin 7.0 (OriginLab Corp., Northampton, MA). The advanced curve-fitting function in Origin7.0 was also used to obtain the relevant curves for the calculation of [conc]Δ7m = 20 values.

TRAP (telomere repeat amplification protocol) assay

Two TRAP assay protocols were used to assess telomerase activity in the presence of compounds of the invention. TRAP assay protocols A and B are both modified versions of standard published TRAP protocols.

TRAP Assay Protocol A

Telomerase activity in the presence of the compounds was assessed using a modified version of standard published TRAP protocols, with cell extract from exponentially growing A2780 human ovarian carcinoma cells used as the enzyme source. The TRAP assay was carried out in two steps with an initial primer-elongation step and subsequent PCR amplification of the telomerase products to enable detection. In part 1, a master reaction mix (40 μl) was prepared containing the TS forward primer (0.1 μg; 5- AATCCGTCGAGCAGAGTT-3), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 0.05% v/v Tween- 20), BSA (0.05 μg), and dNTPs (125 μM each). Protein (1 μg) was then incubated with the reaction mixture with or without drug (made up in solution as the HCl salt) for 10 min at 30 ⁰C. Following heat inactivation of telomerase at 94 ⁰C for 4 min and cooling to 20⁰C, 10 μl of a PCR reaction mix containing ACX primer (0.1 μg; 5-GTG[CCCTTA]3CCCTAA-3) and 2U Taq polymerase (RedHot, Surrey, UK) was added to each tube to start the PCR protocol for part 2, with thermal cycling being carried out in 3 parts following an initial 5 min denaturing period at 94 ⁰C (30 cycles of 94 ⁰C for 30 s, 65 ⁰C for 60 s, 72 ⁰C for 60 s). PCR- amplified reaction products were then run out on a 10% w/v non-denaturing PAGE gel and visualised by staining with SYBR Green I (Sigma). ¹⁶¹EC₅₀ values were subsequently calculated by quantitating the TRAP product using a gel scanner and GeneTools software (Syngene, Cambridge, UK), Measurements were made with respect to a negative control run using the equivalent TRAP-PCR conditions but omitting the protein extract, thus ensuring that the ladders observed were not due to artefacts of the PCR reaction.

TRAP Assay Protocol B

Telomerase activity in the presence of the compounds was assessed using a modified version of previously published TRAP protocols, with cell extract from exponentially growing A2780 human ovarian carcinoma cells used as the enzyme source. The TRAP assay was carried out in two main steps with an initial primer-elongation step and subsequent PCR amplification of the telomerase products to enable detection. Step 1: a master reaction mixture (40 μl) was prepared containing lhe TS forward primer (0.1 μg; 5'- AATCCGTCGAGCAGAGTT-3'), TRAP buffer (20 niM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 μg), and dNTPs (125 μM each). Protein (1 μg) was then incubated with the reaction mixture with or without the compound to be tested (made up in solution as the HCl salt) for 10 min at 30 ⁰C. Step 2: Following heat inactivation of tclomerase at 94 ⁰C for 4 min and cooling to 20⁰C, purification of the telomerase products was performed using QIAquick nucleotide removal spintubes, following the protocol described for their use with the exception that the elution stage was performed with 40 μL PCR-grade water. Samples were then dried using a SpeedVac centrifuge. Step 3: A master mix (50μL) of (TS forward primer (0.1 μg; 5'-AATCCGTCGAGCAGAGTT-S'), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl₂, 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 μg), and dNTPs (125 μM each) ACX primer (0.1 μg; 5'-GTG[CCCTTA]₃CCCTAA-3') and 2μM Taq polymerase (RedHot, AB gene, Surrey, UK) were added to each tube to start the PCR protocol. Thermal cycling was carried out in three parts following an initial 5 min denaturing period at 94 ⁰C (30 cycles of 94 ⁰C for 30 s, 61 ⁰C for 60 s, 72 ⁰C for 60 s). PCR-amplified reaction products were then run out on a 10% w/v non- denaturing PAGE gel and visualised by staining with SYBR Green I (Sigma). ^tclEC₅o values were calculated by quantitating the TRAP product using a gel scanner and GeneTools software (Syngene, Cambridge, UK), Measurements were made with respect to a negative control run using the equivalent TRAP- PCR conditions but omitting the protein extract, thus ensuring that the ladders observed were not due to artefacts of the PCR reaction.

Computer modelling figures.

A qualitative computer model to investigate the stacking of the nickel-salphen complexes and DNA was carried out. This was done using Maestro, Jaguar. The metal complex was optimized using DFT with a LAV3P basis set and a HF initial guess LFT + dd methods. The optimized metal complex was used to do the docking shown in figure 5. The structure of quadraplex DNA used for the qualitative modeling corresponds to the Human Telomere Repeat Sequence 5'- DCApGpGpGpTpTpApGpGpGpTpTpApGpGpGpTpTpAp GpGpG)-3 (PDB code: IKFl). These initial molecular modeling studies, as yet, have not been carried out quantitatively, which is a significant task given the problem of parameterizing the nickel atom in the context of a quadruplex. Even though the modeling is qualitative at this stage, the nature of the Ni complex with its side chains forces it into the position shown (see figure 5).

Sulforhodamine B (SRB) Growth Inhibition Assay

Growth inhibition was measured using the sulforhodamine B assay. Cells are exposed to a single dose of drug and the percentage of cell viability is measured after four days exposure. This is performed over a wide range of drug concentrations in order to establish the IC₅₀ value (the concentration where 50% of cell growth is inhibited). 4,000 cells were seeded into the wells of 96- well microtiter plates and allowed to attach overnight. Compounds dissolved as the HCl or KOH salt (see stock solution preparation above) were dissolved to 1 mM solutions in sterilised water before being dissolved in media to the final concentrations of 0.05, 0.25, 1, 5, and 25 μM, which was added to wells containing cells, in 8-fold replication. Following an incubation period of 96 hours, remaining cells were fixed with ice-cold 10% (w/v) trichloroacetic acid (30 minutes) and stained with 0.4% sulforhodamine B in 1% (v/v) acetic acid (15 minutes). Mean absorbance at 540 nm for each drug concentration was expressed as a percentage of the control untreated well absorbance and an IC50 value (the concentration required to inhibit cell growth by 50%) was determined for the compound. RESULTS

X = H, (3); F, (4)

Scheme 1. Schematic representation of the synthesis of compounds 3 and 4 under study

Compounds 1 and 2 were functionalized with piperidine (as shown in Scheme 1) to yield the substituted derivatives 3 and 4. Under slightly acidic conditions (pH range 5-6) complexes 3 and 4 showed increased water solubility enabling their ability to stabilize quadruplex DNA and inhibit telomerase to be investigated.

In order to study whether changes in the electronic distribution of the phenyl ring on the backbone of the ligand would modify the ability of the nickel(II) complex to stack onto the G-quadruplex, a fluorine substituent was introduced in 4 (see Scheme 1). The electron withdrawing effect of the fluorine lowers the electron density in the π system, which should favor a stronger interaction with the electron-rich π system of the guanine quartet. Furthermore, the presence of fluorine in the complex provides an extra spectroscopic handle (e.g. ¹⁹F NMR spectroscopy) to study the interaction of this species with DNA.

The ability of 3 and 4 to stabilize G-quadruplex DNA (sequence: 5 ' -FAM- d(GGG[TTAGGG]3)-rAMRA-3') was first investigated by a FRET (Fluorescence Resonance Energy Transfer) melting assay (using FRET protocol A). This study showed that both the nickel(II) complexes induce a very high degree of stabilization for quadruplex DNA (T_m = 59°C in absence of complex; ΔT_m = 20⁰C at ~ 0.1 μM concentration - see Table 1), whilst high DNA concentrations are required for even a very low level of duplex DNA stabilization (sequence: 5'-FΛM-dTATAGCTATA-HEG-TATAGCTATA- TAMRA-y; Tm = 60⁰C in absence of complex), suggesting a selectivity of >50-fold. Data for the trisubstituted acridine compound BRACO- 19 is shown for comparison.

Table 1. Stabilization temperatures determined by FRET

Further investigations of the ability of metal-salphen compounds of the invention to stabilize quadruplex and duplex DNA were carried out using FRET protocol B. Data for these compounds is set out in Table 2. Table 2. Stabilization temperatures (determined by FRET) of quadruplex and duplex DNA in the presence of different metal-salphen complexes.

Notes

In table 2, when no complex concentration is given, it means that up to a lOμM complex concentration the ΔTm was less than 2O⁰C. However, the complex concentration could be determined for: ¹ATm = 2⁰C giving a concentration of 4. lμM, ²ATm = 10⁰C giving a concentration of 6.4μM, ³ΔTm = 2⁰C giving a concentration of 5.3μM, ⁴ΔTm = 10⁰C giving a concentration of 6.5μM, ⁵ΔTm = 10⁰C giving a concentration of 5.8μM, and ⁶ΔTm = 2°C giving a concentration of 3.9μM

Compounds 3 and 4 were further investigated using the two-step TRAP assay protocol A. Figure 2 illustrates a decrease in the intensity of the ladder produced by PCR amplification of the oligonucleotides generated by the activity of telomerase on a TS primer with increasing concentration of 3 (i.e. increase in telomerase inhibition). The negative control was run under identical conditions but omitting the protein extract to ensure absence of PCR artefacts. The intensity of the ladders was normalised with respect to the positive and negative controls and a dose-response curve fitted to calculate the concentration for 50% enzyme inhibition (EC₅₀ value). This assay has been widely used to provide qualitative and quantitative estimates of telomerase inhibition. Both complexes showed high activity with ^telEC₅₀ values in the region of 0.1 μM (^telEC₅₀ = 0.14±0.01 and 0.12±0.01 μM for 3 and 4 respectively). This is comparable to results from the lead 3,6,9-substituted acridine compound (BRACO-19).

A separate Taq inhibition assay was performed to measure non-specific inhibition of Taq polymerase. Results showed inhibition of the Taq assay occurred at ca 50-fold greater concentration (5.69±0.15 and 4.78±1.08 mM for 3 and 4) than that needed for TRAP inhibition.

The results presented here show that the planar nickel(II) complexes 3 and 4 are excellent G-quadruplex DNA stabilizers. Computer modelling (Figure 1) predicts that the Ni²⁺ lies directly above the central ion channel of the quadruplex. It is therefore postulated that the Ni²⁺ ion is playing an important role in replacing one of the metal ions that is normally coordinated in and at the ends of the channel. The investigated compounds showed surprisingly high selectivity for quadruplex vs duplex DNA. Previous reports on metal-salphen complexes have shown binding to duplex DNA, but in no case has a salphen complex been reported with the specific pendant side chains as in 3 and 4. We therefore speculate that these are the origin of the quadruplex selectivity shown by 3 and 4.

All compounds were tested for their toxicity towards a variety of cancerous cell lines and the normal cell line, IMR90. The Sulforhodiamine B (SRB) growth inhibition assay is used to establish the acute cytotoxicity of a drug towards a particular cell line. The results for 3 and 4 are shown in Table 3 below:

Table 3 Cytotoxicity of compounds 3 and 4 against cell lines

IC₅₀ (μM) for different cell lines

Compound MCF7 DU 145 A2780 A549 IMR90

3 0.5 0.5 0.5 0.6 2

4 0.5 0.5 0.5 0.8 2

Preliminary in vivo studies were carried out with complex 3 to investigate the chemotherapy of MAC 15 A tumours. A MAC 15A murine colon adenocarcinoma tumour was implanted subcutaneously in NMRI strain mice. The mice were treated as follows: Group 1 - Untreated controls

Group 2 - Complex 3, 20mg/Kg, days 0-3, i.p.

The results are set out below in table 4

Table 4 Results of chemotherapy of MAC15A tumours with compound 3.

1 mouse was sacrificed in the treated group 2 on day 7 and exhibited >15% body weight loss. The results are shown in figure 5 wherein AA2 is compound 3.

Studies were also carried out on phenanthroline ligands of the invention. The synthetic route use to generate these compounds is represented schematically by scheme 2:

Scheme 2. Schematic representation of the synthesis of phenanthroline ligands.

Palladium(II) and platinum(II) complexes were formed with ligands iΛL⁴ using palladium (II) cyclooctadiene dichloride and K₂PtCl₄ respectively. The Pt(II)/Pd(II) complexes of L¹ and L², in solution, are in equilibrium with the dimeric form as represented for the Pt(II) complex in Scheme 3.

Scheme 3. Equilibrium of L IVnL 2 Pt(II) complex in solution. The results of studies using FRET protocol A, for compounds 13 and 14 are summarised in table 5:

Table 5. Stabilization temperatures determined by FRET

In addition, the ability of two phenanthroline ligands (L³ and L⁴ , which are more soluble as free ligands than L and L ) and the platinum(II) complexes of L¹, L² and L⁴ (compounds 15, 16 and 14, respectively) to stabilize G- quadruplex DNA (sequence: 5'-FAM-d(GGG[TTAGGG]3)-TAMRA-3') was investigated by a FRET (Fluorescence Resonance Energy Transfer) melting assay (protocol B) and the results are set out in Table 6:

Table 6. Stabilization temperatures (determined by FRET) of quadruplex and duplex DNA in the presence of different phenanthrolines and platinum(II) complexes.

These compounds were further investigated to determine if they would also show telomerase inhibition in the two-step TRAP assay (using TRAP protocol B).

The results of TRAP protocol B for these compounds are shown in Table 7. The phenanthroline complexes 15, 16 and 14 all show 50% telomerase inhibition (J⁶¹EC₅₀ values) in the range 20 to 60 μM.

Table 7. Modified TRAP assay data for selected phenanthrolines and platinum(II) complexes.

¹TlIe '⁶EC₅₀ error was calculated from plotting a dose-response logistics curve using Origin 6.0

Claims

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof

(I)

wherein A is an Cs-C₆ aryl or heteroaryl group or is absent, B and C are independently an C₅-C₆ aryl or heteroaryl group, M is a metal ion or M=O;

R¹ and R² are independently hydrogen, C_1-20 alkyl, C_3-12carbocyclic, halo, C_{1- 20}haloalkyl, OR¹³, CN, NO₂, NR¹³R¹³, COR¹³, CO₂R¹³, O-(CH₂)_n-N(R¹⁴)₃ or (CH₂)_n-R¹⁵, CONR¹³R¹³, C₃,₁₂heterocyclic, Ci_i₂alkylC₃_i₂carbocyclic, C_{1- 12}alkylC_3-12heterocyclic, or wherein R¹ and R² together form a carbocyclic or heterocyclic group having 5 to 18 members, fused to ring A, optionally substituted with one or more of group R¹³,

1 1 or R and R are independently

R³, R , R⁵ and R⁶ are independently hydrogen, halides, C_1-12 alkyl, OR¹³ or CN, or R³ and R⁵ and/or R⁴ and R⁶ together form a 5-6 membered carbocyclic or heterocyclic ring,

or R¹ and R³ and/or R² and R⁴ together form a carbocyclic or heterocyclic group having 5 to 18 members, fused to ring A, optionally substituted with one or more of group R¹³

X¹ and X² are independently O, S or NR¹³,

R⁷, R⁸, R⁹, R¹⁰, R¹¹ or R¹² are independently hydrogen, halide, OR¹⁴, O-(CH₂)_n- N(R¹⁴)₃ or O-(CH₂)_n-R¹⁵, or where one or more of R⁷ and R⁸ or R¹⁰ and R¹¹ together form a carbocyclic or heterocyclic ring having 5 to 12 members;

G¹ and G² are independently OR¹⁴, O-(CH₂)_n-N(R¹⁴)₃ or O-(CH₂)_n-R¹⁵ wherein R¹⁴ is hydrogen or C_1-12 alkyl and R¹⁵ is HN-C(=NH)-NH₂ a 5 or 6 membered carbocyclic or heterocyclic ring;

or G¹ and G² can together form

wherein n is 1 to 6 and M is a metal ion, M=O or is absent,

R¹³ is independently hydrogen, C_1-6 alkyl, C_3-12 carbocyclic, C_3-12 heterocyclic, Ci_-6 alkyl C_3-12 carbocyclic, C_1-6 alkyl C_3-12 heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂, and R³⁰, R³¹, R³², R³³ and R³⁴ are independently C_W2 alkyl, C_3-I2 carbocyclic, C_3-12 heterocyclic, Ci_-6 alkylC_3-12 carbocyclic, C_1-6 alkylC_3-12 heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂.

2. A compound as claimed in claim 1 wherein the groups A, B or C are independently a six membered aryl or heteroaryl group.

3. A compound as claimed in claim 1 or claim 2 wherein M is selected from Ni, Pt Co, Cu, Mn, Cr, V, Pt, Rh, Ir, Au, Pd, Zn or V=O.

4. A compound as claimed in any one of claims 1 to 3 wherein R¹ and R² are selected from hydrogen, F, Cl, CO₂H, O-(CH₂)_q-N(CH₃)₃, O-(CH₂)_q-C_{5- 6}-carbocyclic, O-(CH₂)_q-C_5-6-heterocyclic, CONH-(CH₂)_m-carbocyclic or CONH(CH₂)_m-heterocyclic, wherein m is 1 to 6. wherein q is 1 to 6 preferably 2, 3, 4 or 5.

5. A compound as claimed in any one of claims 1 to 4 wherein G₁ or G₂ are selected from

wherein p is 0 or 1 and n is 1 to 6.

6. A compound as claimed in any one of claims 1 to 5 of formula (Ia)

(Ia) wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², X¹, X², M, G¹ and G² claimed in any one of claims 1 to 5.

7. A compound as claimed in any one of claims 1 to 5 of formula (Ib) or (Ic)

(Ib)

(Ic) wherein B, C, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², X¹, X², M, G¹ and G² are as claimed in any one of claims 1 to 5.

8. A compound of formula (Ha) or (lib)

(Ha) (lib) wherein Y is a group -CH₂-, CR²⁶ or CO, X is NH, N, or O, D is a carbocycyl or heterocycyl group having 5 to 10 members, R¹⁶, R¹⁷, R¹⁸ and R¹⁹ are hydrogen, C_1-6 alkyl, C_3-12 carbocyclic, C_3-12 heterocyclic, Ci_-6 alkylC₃_i₂ carbocyclic, C_1-6 alkylC_3-12 heterocyclic, halo, CO₂H, OH, NH₂ or CONH₂ and G³ is H, OH, NH₂, OR²⁰, O-(CH₂)_n-N(R²⁰)₃ or O-(CH₂)_n-R²¹ wherein R²⁰ is C_1-I2 alkyl, R²¹ is a 5 or 6 membered carbocyclic or heterocyclic ring and R is hydrogen or C_1-I2 alkyl.

9. A compound according to claim 8 of formula (Ha)

(Ha) wherein Y is a group -CH₂- CR _>26 o, r CO, X is NH or O, D is a carbocycyl or heterocycyl group having 5 to 10 members, R¹⁶, R¹⁷, R¹⁸, R¹⁹, R²², R²³, R²⁴ and

R²⁵ are hydrogen, a halide, OH, OR²⁰, 0-(CHa)_n-, N(R²⁰)₃, O-(CH₂)_n-R²¹ or CN and G³ is OR²⁰, O-(CH₂)_n-N(R²⁰)₃ or O-(CH₂)_n-R²¹ wherein R²⁰ is C_1-12 alkyl, R²¹ is a 5 or 6 membered carbocyclic or heterocyclic ring and E and F are independently a 5-6 membered carbocyclic or heterocyclic ring and R²⁶ is hydrogen or C_1-12 alkyl and wherein P is OR¹³, CO₂R¹³, CN, NO₂, CN, halide, SCN, H₂O, NO₃, OH,

CH₃CN or OCN.

10. A compound as claimed in any one of claims 1 to 5 selected from

11. A compound as claimed in claim 8 or 9 selected from

12. A process for the production of a compound of formula (I) as claimed in any of claims 1 to 7 and 10 comprising the simultaneous separate or sequential addition of a group L-G¹ and a group L-G² to a compound of formula (III),

(HI) wherein L is a leaving group such as a halide and R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², G¹, G², X and M are as defined in any one of claims 1 to 5.

13. A process as claimed in claim 12 wherein where G¹ and G² are identical, the compound of formula (III) can be incubated with an excess of L-G¹ or L-G² to form a compound of formula (I).

14. A process as claimed in claim 12 wherein a compound of formula (III) is incubated with a group L-G¹ to form a compound of formula (IV),

(IV) followed by incubation with a group L-G to form a compound of formula (I).

15. A process for the formation of a compound of formula (III) by reaction of a compound of formula (VI) with a metal, a compound of formula (VII) and a compound of formula (VIII)

(Vl) (VIl) (VIIi)

(III)

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², G¹, G², X and M are as defined in any one of claims 1 to 5.

16. A pharmaceutical composition comprising a compound as claimed in any one of claims 1 to 11 in combination with a pharmaceutically acceptable carrier, diluent or excipient.

17 A pharmaceutical composition as claimed in claim 16 further comprising one or more additional active agents.

18. A process for the manufacture of a composition as claimed in claim 16 or claim 17 comprising combining a compound as claimed in any one of claims 1 to 11 with the pharmaceutically acceptable carrier or diluent and optionally one or more additional active agents.

19. A compound as claimed in any one of claims 1 to 11 or a composition as claimed in claim 16 or claim 17 for use in medicine.

20. A compound as claimed in claim 19 for use in the inhibition of a telomerase enzyme.

21. A compound as claimed in claim 19 for use in the treatment of cancer.

22. The use of a compound as claimed in any one of claims 1 to 11 in the manufacture of a medicament for the treatment of cancer.

23. The use as claimed in claim 22 wherein the medicament may further comprise one or more additional active agent.

24. The use as claimed in claim 22 wherein the compound as claimed in any one of claims 1 to 11 and the additional active agent are administered separately, sequentially or simultaneously.

25. A method of treating cancer comprising administering to a person in need therefore a compound as claimed in any one of claims 1 to 11 or a composition as claimed in claim 16 or claim 17.

26. The method as claimed in claim 25 wherein the compound or composition are administered prior to, in combination with and/or subsequent to chemotherapy, radiotherapy, and/or surgery.

27. A compound as substantially described herein with reference to one or more of the examples and/or drawings.

28. A process as substantially described herein with reference to one or more of the examples and/or drawings.

29. A composition as substantially described herein with reference to one or more of the examples and/or drawings.

30. A use as substantially described herein with reference to one or more of the examples and/or drawings.

31. A method as substantially described herein with reference to one or more of the examples and/or drawings.