Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
  • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
  • C07D295/08—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms
  • C07D295/084—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
  • C07D295/088—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by singly bound oxygen or sulfur atoms with the ring nitrogen atoms and the oxygen or sulfur atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
  • C07C251/00—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton
  • C07C251/02—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups
  • C07C251/24—Compounds containing nitrogen atoms doubly-bound to a carbon skeleton containing imino groups having carbon atoms of imino groups bound to carbon atoms of six-membered aromatic rings

Definitions

• 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.
• 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)
• R 1 and R 2 are independently hydrogen, C 1-2O alkyl, C 3-12 carbocyclic, halo, C 1- 20 haloalkyl, OR 13 , CN, NO 2 , NR 13 R 13 , COR 13 , CO 2 R 13 , O-(CH 2 ) n -N(R 14 ) 3 or (CH 2 ) n -R 15 , CONR 13 R 13 , C 3-12 heterocyclic, C 1-20 alkylC 3 , 12 carbocyclic, C 1- i 2 alkylC 3-12 heterocyclic, or wherein R 1 and R 2 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 13 , or R 1 and R 2 are independently
• R 3 , R 4 , R 5 and R 6 are independently hydrogen, halides, C 1-20 alkyl, OR 13 or CN, or R 3 and R 5 and/or R 4 and R 6 together form a 5-6 membered carbocyclic or heterocyclic ring,
• R 1 and R 3 and/or R 2 and R 4 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 ,
• X 1 and X 2 are independently O, S or NR 13 ,
• R 7 , R 8 , R 9 , R 10 , R 11 or R 12 are independently hydrogen, halide, OR 14 , 0-(CH 2 V N(R 14 ) 3 or O-(CH 2 ) n -R 15 , or where one or more of R 7 and R 8 or R 10 and R 11 together form a carbocyclic or heterocyclic ring having 5 to 12 members,
• G 1 and G 2 can together form
• n 1 to 6
• R 13 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, CO 2 H, OH, NH 2 or CONH 2 ; and R 30 , R 31 , R 32 , R 33 and R 34 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- i 2 heterocyclic, halo, CO 2 H, OH, NH 2 or CONH 2 .
• a metal ion M can be co-ordinated between the two N atoms and the two X 1 atoms of the groups R 1 and R 2 .
• any of groups R 7 , R 8 , R 9 , R 10 , R 11 or R 12 can combine with any of groups R 30 , R 31 , R 32 or R 33 to form a bridged structure.
• the groups A, B or C are independently preferably a six membered aryl or heteroaryl group, more preferably selected from phenyl or pyridine.
• the groups A, B or C independently are a six membered aryl or heteroaryl group fused to an aromatic ring, for example naphthyl.
• R 1 and R 2 are preferably selected from hydrogen, F, Cl, CO 2 H, O-(CH 2 ) q -N(CH 3 ) 3 , O-(CH 2 ) q -C 5-6 -carbocyclic, O-(CH 2 ) q -C 5-6 -heterocyclic, CONH-(CH 2 ) m -carbocyclic or CONH(CH 2 ) m -heterocyclic, wherein m is 1 to 6. wherein q is 1 to 6 preferably 2, 3, 4 or 5.
• R 1 and R 2 , or R 1 and R 3 or R 2 and R 4 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.
• G 1 or G 2 are preferably selected from
• 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)
• R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 and R 12 are hydrogen and R 1 , X 1 , X 2 , M, G 1 and G 2 are as defined above.
• ring A is absent from the compound of formula (I).
• the resulting compound has the formula (Ib) or (Ic) as set out below:
• R 3 and R 5 together form a 5-6 membered carbocyclic or heterocyclic ring and R 4 and R 6 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 -CH 2 -, CR 26 or CO, X is NH, N, or O, D is a carbocycyl or heterocycyl group having 5 to 10 members
• R 16 , R 17 , R 18 and R 19 are hydrogen, C 1-6 alkyl, C 3-12 carbocyclic, C 3-12 heterocyclic, C 1-6 alkylC 3- i 2 carbocyclic, C 1-6 alkylC 3-12 heterocyclic, halo, CO 2 H, OH, NH 2 or CONH 2 and
• G 3 is H, OH, NH 2 , OR 20 , O-(CH 2 ) n -N(R 20 ) 3 or O-(CH 2 ) n -R 21 wherein R 20 is C 1-12 alkyl, R 21 is a 5 or 6 membered carbocyclic or heterocyclic ring and R 26 is hydrogen or C 1-12 alkyl.
• the compound is a compound of formula (Ha) as illustrated below:
• E and F are independently a 5-6 membered carbocyclic or heterocyclic ring and R >22 , T R-.23 , R r»2 z 4 q and J R r>2 / 5 D are hydrogen, halides, OH, OR 20 , O-(CH 2 ) n - N(R 20 ) 3 , O-(CH 2 ) n -R 21 , or CN,
• P is OR 13 , CO 2 R 13 , CN, NO 2 , CN, halide, SCN, H 2 O, NO 3 , OH, CH 3 CN or OCN, and
• R .1 1 , r R>2 z , T R ⁇ 1 i 3 J , T R, 1 1 6 0 , ⁇ R> 17 , n Rl 1 8 8 , r R.1 i 9 y , r G ⁇ J > and M are as defined above.
• G 3 is OR 20 , O-(CH 2 ) n -N(R 20 ) 3 or O-(CH 2 ) n -R 21 .
• G 3 is H or NH 2 .
• the present invention particularly relates to the following preferred compounds of the invention:
• the compounds of the present invention provide 1) a ⁇ -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
• 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.
• the compounds of the present invention contain bulky groups (for example G 1 and G 2 ) 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.
• G 1 and G 2 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.
• 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 CH 2 CH 2 Br, CF 3 or CCl 3 .
• 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, CH 2 -cyclopropyl, CH 2 -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.
• 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.
• 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, phenoxa
• 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.
• 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
• 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.
• 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.
• 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-benzy
• 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.
• the compound of the first aspect may be provided by the simultaneous separate or sequential addition of a group L-G 1 and a group L-G 2 to a compound of formula (III),
• L is a leaving group such as a halide and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 R 9 , R 10 , R 11 , R 12 , G 1 , G 2 , X 1 , X 2 and M are as defined in the first aspect of the invention.
• ⁇ O formula (III) can be incubated with an excess of L-G or L-G to form a compound of formula (I). However, where G 1 and G 2 are different, the groups L-G 1 and L-G 2 are added separately. For example, a compound of formula (III) can be incubated with a group L-G 1 to form a compound of formula (IV),
• 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)
• R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , G 1 , G 2 , X 1 , X 2 and M are as defined in the first aspect of the invention.
• 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).
• the compound of formula (VII) and the compound of formula (VIII) are added separately.
• a compound of formula (VI) can be incubated with a compound of formula (VII) to form a compound of formula (V),
• 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.
• 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).
• 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.
• compositions may also comprise one or more additional active agent, such as a chemotherapeutic agent and/or an antiproliferative agent.
• 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 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).
• 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.
• 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.
• 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.
• suitable pharmaceutical carrier(s) include magnesium stearate, starch, lactose, sucrose and microcrystalline cellulose.
• a composition in the form of a capsule can be prepared using routine encapsulation procedures.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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,
• 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.
• 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.
• 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
• 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.
• 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.
• 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 o
• 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.
• 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.
• 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.
• 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 ( ⁇ Tm) 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;
• Figure 5 shows the effects of compound 3 on MACl 5 A tumours.
• 1,2-phenylenediamine (0.2800 g, 2.53 mmol) and 2,4-dihydroxybenzaldehyde (0.6901 g, 4.89 ⁇ unol) were dissolved in methanol (50 mL) and heated for 30 min at reflux (70-75 0 C).
• Ni(OAc) 2 -4H 2 O (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 (70-75 0 C). After this time, the reaction mixture was cooled to room temperature.
• 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 -2H 2 0 (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.
• 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.
• oligonucleotides and their fluorescent conjugates were purchased from Eurogentec (UK) or Invitrogen (UK). DNA was dissolved as a stock 20 ⁇ M 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.
• FRET fluorescence resonance energy transfer
• 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.
• Fluorescence readings were taken at intervals of 0.5 0 C over the range 30- 100°C, with a constant temperature being maintained for 30 seconds prior to each reading to ensure a stable value.
• TRAP telomere repeat amplification protocol
• TRAP assay protocols A and B are both modified versions of standard published TRAP protocols.
• 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.
• a master reaction mix (40 ⁇ l) was prepared containing the TS forward primer (0.1 ⁇ g; 5- AATCCGTCGAGCAGAGTT-3), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween- 20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each). Protein (1 ⁇ g) was then incubated with the reaction mixture with or without drug (made up in solution as the HCl salt) for 10 min at 30 0 C.
• telomerase Following heat inactivation of telomerase at 94 0 C for 4 min and cooling to 20 0 C, 10 ⁇ l of a PCR reaction mix containing ACX primer (0.1 ⁇ g; 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 0 C (30 cycles of 94 0 C for 30 s, 65 0 C for 60 s, 72 0 C 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). 161 EC 50 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.
• 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 ⁇ l) was prepared containing lhe TS forward primer (0.1 ⁇ g; 5'- AATCCGTCGAGCAGAGTT-3'), TRAP buffer (20 niM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each). Protein (1 ⁇ g) 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 0 C.
• TRAP buffer 20 niM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20
• BSA 0.05 ⁇ g
• dNTPs 125 ⁇ M each
• Step 2 Following heat inactivation of tclomerase at 94 0 C for 4 min and cooling to 20 0 C, 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 ⁇ L PCR-grade water. Samples were then dried using a SpeedVac centrifuge.
• Step 3 A master mix (50 ⁇ L) of (TS forward primer (0.1 ⁇ g; 5'-AATCCGTCGAGCAGAGTT-S'), TRAP buffer (20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20), BSA (0.05 ⁇ g), and dNTPs (125 ⁇ M each) ACX primer (0.1 ⁇ g; 5'-GTG[CCCTTA] 3 CCCTAA-3') and 2 ⁇ M Taq polymerase (RedHot, AB gene, Surrey, UK) were added to each tube to start the PCR protocol.
• TRAP buffer 20 mM Tris-HCl [pH 8.3], 68 mM KCl, 1.5 mM MgCl 2 , 1 mM EGTA, 0.05% v/v Tween-20
• BSA 0.05 ⁇ g
• Thermal cycling was carried out in three parts following an initial 5 min denaturing period at 94 0 C (30 cycles of 94 0 C for 30 s, 61 0 C for 60 s, 72 0 C 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).
• tcl EC 5 o 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.
• 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'- DCApGpGpGpTpTpApGpGpGpTpTpApGpGpGpTpTpApGpGpGpTpTpAp GpGpG)-3 (PDB code: IKFl).
• sulforhodamine B assay 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 IC 50 value (the concentration where 50% of cell growth is inhibited).
• IC 50 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 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 ⁇ M, which was added to wells containing cells, in 8-fold replication.
• FIG. 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 (EC 50 value). This assay has been widely used to provide qualitative and quantitative estimates of telomerase inhibition.
• tel EC 50 0.14 ⁇ 0.01 and 0.12 ⁇ 0.01 ⁇ M for 3 and 4 respectively. This is comparable to results from the lead 3,6,9-substituted acridine compound (BRACO-19).
• 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.
• SRB Sulforhodiamine B
• Group 2 - Complex 3 20mg/Kg, days 0-3, i.p.
• Palladium(II) and platinum(II) complexes were formed with ligands i ⁇ L 4 using palladium (II) cyclooctadiene dichloride and K 2 PtCl 4 respectively.
• the Pt(II)/Pd(II) complexes of L 1 and L 2 are in equilibrium with the dimeric form as represented for the Pt(II) complex in Scheme 3.
• TRAP protocol B The results of TRAP protocol B for these compounds are shown in Table 7.

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