EP2097393A1 - Ligands g-quadruplex thérapeutiques - Google Patents

Ligands g-quadruplex thérapeutiques

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Publication number
EP2097393A1
EP2097393A1 EP07824923A EP07824923A EP2097393A1 EP 2097393 A1 EP2097393 A1 EP 2097393A1 EP 07824923 A EP07824923 A EP 07824923A EP 07824923 A EP07824923 A EP 07824923A EP 2097393 A1 EP2097393 A1 EP 2097393A1
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EP
European Patent Office
Prior art keywords
compound according
optionally substituted
aryl
formula
alkyl
Prior art date
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EP07824923A
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German (de)
English (en)
Inventor
Stephen Neidle
John Edward Moses
Adam Donald Moorhouse
Michael Moore
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University College London
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University College London
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of compounds which bind G- quadruplexes that can be formed in human telomeres, and more specifically, to those compounds which stabilise G-quadruplex structures and thereby inhibit the action of the enzyme telomerase.
  • the present invention also relates to pharmaceutical compositions comprising such compounds and their use in the treatment of proliferative conditions, such as cancer.
  • telomere a specialised DNA polymerase, telomerase, appears and utilises its associated RNA template to synthesise the telomeric sequences which have become critically shortened in these cells. This prevents further shortening of telomeres, and the resulting stabilisation of their length contributes to immortilisation.
  • Telomerase is not usually active in normal mammalian somatic cells. However, telomerase activity has been detected in up to 80-90% of all human cancers examined. Thus there has been much active research to discover telomerase inhibitors which selectively target tumour cells and cause tumour cell death well before damage to regenerative tissues occurs, thereby minimising undesirable side-effects.
  • polycyclic compounds including polycyclic acridines, anthraquinones, and fluorenones have been shown to inhibit telomerase and/or have anti-tumour effects in vitro. These are described in Bostock-Smith et al [2] and Gimenez-Arrau et al [3], amongst other publications. Improved therapeutic acridone and acridine compounds are described in WO02/08193 [1].
  • G-quadruplexes may be formed in human telomeres, at regions of single stranded G-rich DNA found at the ends of chromosomes [4].
  • the G-quadruplex is a tertiary structure which can be formed by guanine rich DNA.
  • Four guanine bases can congregate to form a tetrad structure called a G-quartet, which is held together with Hoogsteen hydrogen bonding.
  • Guanine rich DNA sequences can form many tetrads, which can associate and form cylindrical structures through ⁇ -stacking. It is these cylindrical structures formed from 1 ,2 or 4 strands of DNA that are called G- quadruplexes.
  • a range of G-quadruplex structures have been reported.
  • Quadruplex binding ligands need to be highly selective for quadruplex structures, as opposed to other tertiary formations of DNA (for example double helix forming DNA) to avoid toxic side-effects when they are administered to patients as therapeutic agents.
  • DNA for example double helix forming DNA
  • X and Y are each independently a group of formula
  • Z is absent, a group of formula II, optionally substituted Ci -7 alkyl, optionally substituted C 3-20 heterocyclyl, optionally substituted C 5-20 aryl, halo, amino, hydroxy, ether, thio, thioether, carboxy or cyano;
  • L 1 and L 2 are each independently selected from NR 3 , C 2 H 2 , CH 2 , -O-, -S- and a bond;
  • Ar 2 and Ar 3 are independently optionally substituted C 5 or C 6 aryl or heteroaryl; n is an integer from 1 to 5;
  • R 1 and R 2 are independently hydrogen, Ci -7 alkyl, C 3-20 heterocyclyl, or C 5-20 aryl, or R 1 and R 2 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms;
  • R 3 is H or Ci -7 alkyl; provided that at least one of Ar 1 , Ar 2 and Ar 3 is oxazole, triazole or tetrazole.
  • the planar triazole or tetrazole units in these compounds form part of a planar pharmacophore that is capable of ⁇ -stacking interactions with the G- quadruplex structures in telomeres.
  • the group of formula Il may advantageously bind the quadruplex grooves.
  • the compounds are highly selective for quadruplex structures and stabilise the DNA such that telomerase cannot access the DNA bases to extend the length of the telomeres.
  • the compounds are thus highly potent and selective telomerase inhibitors, having cytotoxic action only on those cells which are actively expressing telomerase (usually cancer cells), and do not bind normal duplex DNA.
  • the present invention may avoid the toxicity problems of the anti-cancer compounds of the prior art.
  • a third aspect of the invention relates to a method of inhibiting telomerase in vitro or in vivo, comprising contacting a cell with an effective amount of compound according to the first aspect of the invention.
  • a fourth aspect of the invention relates to a method of regulating cell proliferation in vitro or in vivo, comprising contacting a cell with an effective amount of compound according to the first aspect of the invention.
  • a fifth aspect of the invention relates to a method for the treatment of a proliferative condition comprising administering to a subject suffering from said proliferative condition a therapeutically effective amount of a compound according to the first aspect of the invention.
  • a final aspect of the invention is a method of manufacturing a compound of formula V in which a compound of formula III is reacted with a compound of formula IV;
  • Ar 4 is a monocyclic aryl or heteroaryl; each Ar 5 is independently optionally substituted C 5 or C ⁇ aryl or heteroaryl; Z is absent, optionally substituted Ci -7 alkyl, optionally substituted C 3-2O heterocyclyl, optionally substituted C 5-20 aryl, halo, amino, hydroxy, ether, thio, thioether, carboxy or cyano; p is an integer from 1 to 5; each L 3 and L 4 is independently selected from NR 3 , C 2 H 2 , CH 2 , -O-, -S-, and a bond;
  • R 3 is H or Ci. 7 alkyl
  • R 4 and R 5 are independently hydrogen, Ci -7 alkyl, C 3-20 heterocyclyl, or C 5-2 O aryl, or R 4 and R 5 , taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms.
  • the compounds according to the first aspect of this invention are ideally manufactured using "click chemistry".
  • click chemistry originally conceived by Barry K. Sharpless [12] makes use of "near perfect” reactions in order to bring about reliable transformations and provide rapid access to large areas of chemical space.
  • the Cu(I) catalysed Huisgen cycloaddition in particular has proven to be a very useful ligation reaction in fragment based drug discovery [13].
  • the clicked triazole acts as a reliable sturdy linkage which can be formed selectively between a complimentary azide and alkyne pair, in good yield and without the need for purification. This approach offers a reliable and efficient method for manufacturing the novel compounds of the invention.
  • a planar core with a ⁇ -delocalised system enables stacking on the face of a guanine quartet.
  • Side chain groups which may be protonated are preferred, since these provide stabilising interactions with the sugar-phosphate loops of the G-quadruplex, facilitating stacking of the compound with the quadruplex.
  • Amine side chain groups typically have suitable pK B values for protonation at physiological pH. The pK B value is typically in the range 6.5-8.5.
  • Ar 1 is phenylene. Multicyclic ring systems are known to bind to duplex DNA. It is thought that the monocyclic aryl ring Ar 1 ensures that the compounds of the invention preferentially bind to G-quadruplex structures rather than duplex DNA.
  • X and Y are meta substituents on the phenyl ring and Z is absent.
  • X and Y may be meta substituents on the phenyl ring and Z is absent.
  • Z may be different, they are preferably the same.
  • Z is present, it is preferably a group of formula II, optionally substituted phenyl, or C 5 or C 6 optionally substituted heteroaryl. However, Z may be non- aromatic.
  • Ar 1 is phenyl
  • X, Y and Z are preferably each meta substituents on the phenyl ring.
  • Ar 1 is a triazole.
  • Z is absent and X and Y are 1 ,4-substituents on the ring Ar 1 , giving a compound of formula
  • X and Y may alternatively be 1 ,5 substituents on the triazole ring.
  • Ar 1 may be pyrazine, pyrimidine or pyridazine.
  • Preferred compounds wherein Ar 1 is pyrazine, pyrimidine or pyridazine include
  • X, Y and Z are preferably the same.
  • heteroaryl groups for Ar 1 , Ar 2 and Ar 3 include, but are not limited to, those derived from pyrrole, pyridine, furan, thiophene, oxazole, isoxazole, isoxazine, oxadiazole, oxatriazole, thiazole, isothiazole, imidazole, pyrazole, pyridazine, pyrimidine, pyrazine, triazole, triazine and tetrazole.
  • each Ar 2 is triazole.
  • Ar 1 and each Ar 3 is phenyl.
  • Further preferred compounds are those of formula I wherein one or both of L 1 and L 2 is a bond.
  • R 1 and R 2 are each independently Ci -7 alkyl, which is optionally substituted.
  • each -NR 1 R 2 is independently selected from -N(Me) 2 , -N(Et) 2 , -N(nPr) 2 , -N(JPr) 2 , -N(nBu) 2 and -N(tBu) 2 .
  • R 1 and R 2 together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms, which heterocyclic ring may saturated, partially unsaturated, or fully unsaturated, and is optionally substituted.
  • R 1 and R 2 taken together with the nitrogen atom to which they are attached, form a saturated heterocyclic ring having from 3 to 8 ring atoms, wherein only one of said ring atoms is nitrogen, and all others are carbon, and which heterocyclic ring is optionally substituted.
  • R 1 and R 2 taken together with the nitrogen atom to which they are attached may form a cyclic amino group of the following formula, wherein q is an integer from 2 to 7, and wherein said group is optionally substituted:
  • Suitable, and particularly preferred terminal amino groups include the following cyclic amino groups, which may be optionally substituted:
  • This cyclic amino group may be substituted with one or more substituents selected from C 1 - 7 alkyl, C 3-20 aryl-Ci- 7 alkyl, C 3-20 aryl, Ci- 7 alkyl-C 3 - 2 o aryl, hydroxy, and Ci. 7 hydroxyalkyl.
  • each -NR 1 R 2 may be independently selected from
  • R 1 and R 2 taken together with the nitrogen atom to which they are attached, form a saturated heterocyclic ring having from 3 to 8 ring atoms, wherein said ring has at least two heteroatoms selected from nitrogen, oxygen, and sulfur, which heterocyclic ring is optionally substituted.
  • the terminal amino group, -NR 1 R 2 is one of the following cyclic amino groups, and is optionally substituted:
  • R is hydrogen, C 1-7 alkyl, C 3 . 20 heterocyclyl, or C 5 . 2 oaryl.
  • n is preferably 1 or 2, most preferably 2.
  • R 6 and R 7 are each C 1 - 2 alkyl, C 4 - 5 heterocyclyl or C 3 - 5 heteroaryl, or R 6 and
  • R 7 taken together with the nitrogen to which they are attached, form a heterocyclic ring having 5-7 ring atoms.
  • the terminal amino acid group is preferably one of the following amino groups, and is optionally substituted:
  • hetero refers to compounds and/or groups which have at least one heteroatom, for example boron, silicon, nitrogen, phosphorus, oxygen and sulphur (multivalent heteroatoms), and fluorine, chlorine, bromine and iodine (monovalent heteroatoms).
  • monocyclic aryl or heteroaryl refers to cyclic compounds which have one aromatic ring, which may contain one or more multivalent heteroatoms in the case of monocyclic heteroaryl.
  • substituted refers to a parent group which may be substituted or unsubstituted.
  • substituted refers to a parent group which bears one or more substituents.
  • substituted is used herein in the conventional sense and refers to a chemical moiety which is covalently attached to, appended to, or if appropriate, fused to, a parent group.
  • substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.
  • the substituent(s) are independently selected from: halo; hydroxy; ether (e.g., C 1 -7 alkoxy); formyl; acyl (e.g., C 1 .7 alkylacyl, C5- 2 0 arylacyl); acylhalide; carboxy; ester; acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., C 1 -7 alkylthio); sulfonic acid; sulfonate; sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulf
  • C 1 .7 alkyl including, e.g. Ci -7 haloalkyl, Ci -7 hydroxyalkyl, C 1 .7 carboxyalkyl, Ci -7 aminoalkyl, C 5-2 O aryl-Ci -7 alkyl); C 3-2 O heterocyclyl; or C 5-20 aryl (including, e.g., C 5-20 aryl, C 5-20 heteroaryl, Ci -7 alkyl-C 5-20 aryl and C 5-20 haloaryl)). More specifically, the substituents may be selected from:
  • -CF 3 -CHF 2 , -CH 2 F, -CCI 3 , -CBr 3 , -CH 2 CH 2 F, -CH 2 CHF 2 , and -CH 2 CF 3 ; -OCF 3 , -OCHF 2 , -OCH 2 F, -OCCI 3 , -OCBr 3 , -OCH 2 CH 2 F, -OCH 2 CHF 2 , and -
  • Compounds of the invention may be chiral. They may be in the form of a single enantiomer or diastereomer, or a racemate.
  • Chiral compounds of the invention may be prepared in racemic form, or prepared in individual enantiomeric form by specific synthesis or resolution as will be appreciated by the person skilled in the art.
  • the compounds may, for example, be resolved into their enantiomers by Standard techniques, such as the formation of diastereomeric pairs by salt formation with an optically active acid followed by fractional crystallisation and regeneration of the free base.
  • the enantiomers of the novel compounds may be separated by HPLC using a chiral column.
  • a compound of the invention may be in a protected amino, protected hydroxy or protected carboxy form.
  • protected amino refers to amino, hydroxy and carboxy groups which are protected in a manner familiar to those skilled in the art.
  • an amino group can be protected by a benzyloxycarbonyl, tert- butoxycarbonyl, acetyl or like group, or in the form of a phthalimido or like group.
  • a carboxyl group can be protected in the form of a readily cleavable ester such as the methyl, ethyl, benzyl or tert-butyl ester.
  • a hydroxy group can be protected by an alkyl or like group.
  • Compounds of the invention may be in the form of pharmaceutically acceptable salts, for example, addition salts of inorganic or organic acids.
  • inorganic acid addition salts include, for example, salts of hydrobromic acid, hydrochloric acid, nitric acid, phosphoric acid and sulphuric acid.
  • Organic acid addition salts include, for example, salts of acetic acid, benzenesulphonic acid, benzoic acid, camphorsulphonic acid, citric acid, 2-(4-chlorophenoxy)-2- methylpropionic acid, 1 ,2-ethanedisulphonic acid, ethanesulphonic acid, ethylenediaminetetraacetic acid (EDTA), fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, N-glycolylarsanilic acid, 4-hexylresorcinol, hippuric acid, 2-(4- hydroxybenzoyl)benzoic acid, 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid, 2-hydroxyethanesulphonic acid, lactobionic acid, n-dodecyl sulphuric acid, maleic acid, malic acid, mandelic acid, methanesulphonic acid, methyl sulphuric
  • Salts may also be formed with inorganic bases.
  • inorganic base salts include, for example, salts of aluminium, bismuth, calcium, lithium, magnesium, potassium, sodium, zinc and the like.
  • Organic base salts include, for example, salts of N, N'-dibenzylethylenediamine, choline (as a counterion), diethanolamine, ethanolamine, ethylenediamine, N,N'-bis(dehydroabietyl)ethylenediamine, N- methylglucamine, procaine, tris(hydroxymethyl)aminomethane (“TRIS”) and the like.
  • TIS tris(hydroxymethyl)aminomethane
  • Such salts may be prepared by reacting the compound with a suitable acid or base in a conventional manner.
  • the compounds of the invention may be used in the treatment of numerous conditions, the cause of which is linked to telomerase activity and unregulated cell division.
  • the present invention provides compounds which are antiproliferative agents.
  • antiproliferative agent as used herein is a compound which is useful in the treatment of a proliferative condition.
  • cell proliferation as used herein is a compound which is useful in the treatment of a proliferative condition.
  • proliferative condition a proliferative condition
  • proliferative disorder proliferative disorder
  • proliferative disease are used interchangeably herein and refer to an unwanted or uncontrolled cellular proliferation of excessive or abnormal cells which is undesired, such as, neoplastic or hyperplastic growth, whether in vitro or in vivo.
  • proliferative conditions include, but are not limited to, benign, pre- malignant, and malignant cellular proliferation, including but not limited to, neoplasms and tumours (e.g., histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. ovarian carcinoma, breast carcinoma, bowel cancer, colon cancer, renal cancer, lung cancer, small cell lung cancer, testicular cancer, prostate cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis.
  • neoplasms and tumours e.g., histocytoma, glioma, astrocyoma, osteoma
  • cancers e.g. ovarian carcinoma, breast carcinoma, bowel cancer, colon cancer, renal cancer, lung cancer, small cell lung cancer
  • anticancer agent refers to a compound which treats a cancer (i.e., a compound which is useful in the treatment of a cancer).
  • the anti-cancer effect may arise through one or more mechanisms, including but not limited to, the regulation of cell proliferation, the inhibition of angiogenesis (the formation of new blood vessels), the inhibition of metastasis (the spread of a tumour from its origin), the inhibition of invasion (the spread of tumour cells into neighbouring normal structures), or the promotion of apoptosis (programmed cell death).
  • the invention further provides active compounds for use in a method of treatment of the human or animal body, for example, in the treatment of a proliferative condition, for example cancer. Such a method may comprise administering to such a subject a therapeutically-effective amount of an active compound, preferably in the form of a pharmaceutical composition.
  • treatment refers generally to treatment and therapy, whether of a human or an animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and cure of the condition.
  • Treatment as a prophylactic measure i.e., prophylaxis is also included.
  • terapéuticaally-effective amount refers to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
  • treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
  • treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g. drugs, antibodies (e.g., as in immunotherapy), prodrugs (e.g. as in photodynamic therapy, GDEPT, ADEPT, etc).; surgery; radiation therapy; and gene therapy.
  • the active compound may be administered orally, intravenously, rectally, parenterally, by inhalation (pulmonary delivery), topically, ocularly, nasally, or to the buccal cavity.
  • Oral administration is preferred.
  • a composition of the present invention may take the form of any of the known pharmaceutical compositions for such methods of administration.
  • the compositions may be formulated in a manner known to those skilled in the art so as to give a controlled release, for example rapid release or sustained release, of the compounds of the present invention.
  • Pharmaceutically acceptable carriers suitable for use in such compositions are well known in the art.
  • the compositions of the invention may contain 0.1-99% by weight of active compound.
  • the compositions of the invention are generally prepared in unit dosage form. Preferably, a unit dose comprises the active ingredient in an amount of 1-500 mg.
  • the excipients used in the preparation of these compositions are the excipients known in the art.
  • compositions for oral administration include known pharmaceutical forms for such administration, for example tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups and elixirs.
  • Compositions intended for oral use may be prepared according to any method known to the art.
  • compositions may contain one or more agents such as sweetening agents, flavouring agents, colouring agents and preserving agents, in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.
  • excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch or alginic acid; binding agents, for example starch gelatin, acacia, microcrystalline cellulose or polyvinyl pyrrolidone; and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.
  • Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • an inert solid diluent for example calcium carbonate, calcium phosphate or kaolin
  • water or an oil medium for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids, for example polyoxyethylene sorbitan monooleat ⁇ .
  • suspending agents for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl pyrrolidone, gum tragacanth and gum
  • the aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more colouring agents, one or more flavouring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents, such as those set forth above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable sweetening, flavouring and colouring agents may also be present.
  • the pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions.
  • the oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin, or mixtures of these.
  • Suitable emulsifying agents may be naturally occurring gums, for example gum acacia or gum tragacanth, naturally occurring phosphatides, for example soya bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions may also contain sweetening and flavouring agents.
  • Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative and flavouring and colouring agents.
  • the pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above.
  • the sterile injectable preparation may also be in a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1 ,3-butanediol.
  • Suitable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono- or diglycerides.
  • fatty acids such as oleic acid, find use in the preparation of injectables.
  • the compounds of the invention may also be administered in the form of suppositories for rectal administration of the drug.
  • These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug.
  • Such materials are cocoa butter and polyethylene glycols.
  • compositions for topical administration may also be suitable for use in the present invention.
  • the active compound may be dispersed in a pharmaceutically acceptable cream, ointment or gel.
  • a suitable cream may be prepared by incorporating the active compound in a topical vehicle such as light liquid paraffin, dispersed in a aqueous medium using surfactants.
  • An ointment may be prepared by mixing the active compound with a topical vehicle such as a mineral oil or wax.
  • a gel may be prepared by mixing the active compound with a topical vehicle comprising a gelling agent.
  • Topically administrable compositions may also comprise a matrix in which the pharmaceutically active compounds of the present invention are dispersed so that the compounds are held in contact with the skin in order to administer the compounds transdermally.
  • a compound of the invention may be prepared by any suitable method known in the art.
  • a method according to the final aspect of this invention is used.
  • Ar 4 and Ar 5 are each optionally substituted phenyl.
  • the preferred features of the compounds of this invention are equally applicable to a compound of formula V manufactured according to the final aspect of this invention.
  • the substituents ⁇ -L 4 are arranged 1 ,3 on aryl ring Ar 4 .
  • L 3 and L 4 are each a bond, Z is absent, and compounds III and IV have formulae
  • Triazole units are suitably synthesised using the Cu(I) catalysed Huisgen cycloaddition as detailed in the examples. It has been found that reaction yield is maximised when an alcohol and water are added to the reaction mixture.
  • Particularly preferable conditions are a 1 :1 mixture of t-BuOH:H 2 O with CuSO 4 .5H 2 O and sodium ascorbate. Stirring at room temperature may be sufficient for the reaction to proceed for some starting compounds. Alternatively, microwave radiation may be needed to drive the reaction to completion.
  • Tetrazole units may be synthesised using a nitrile as shown in the following reaction scheme:
  • cyanide instead of a cyanide, alternative reagents may be used such as R 8 C ⁇ S or an isonitrile.
  • FIG. 2 shows the FRET melting curves (with error bars) obtained for compound 46 on four oligomer sequences tested, F2IT, C-kit7, C-kit2 and FIOT (ds) DNA;
  • Figure 3 displays the results of a competition FRET experiment. The melting temperatures ( ⁇ T 1/2 ) of F2IT in the presence of 1 ⁇ M of ligand with various concentrations of calf thymus DNA are shown;
  • Figure 4 shows the TRAP gel for (a) telomestatin, (b) compound 42 and (c) compound 44;
  • Figure 5 shows the effect of combination and single agent treatments in MCF7 cells up to 5 weeks.
  • Figure 6 shows induction of senescence following treatment of MCF7 cells with compound 46.
  • the following examples illustrate the invention.
  • Preparative reversed-phase HPLC was carried out on a Gilson Chromatograph with a Gilson 215 Liquid Handler, and a Gilson 845Z injection module coupled to a Gilson UV ⁇ /IS 155 detector using a YMC C18 5 ⁇ (100 x 20 mm) column and gradients from 0.1 % aqueous TFA and MeOH containing 0.1% TFA (flow rate: 10 mL min '1 ).
  • Analytical HPLC were carried out on a Gilson Chromatograph with a YMC C18 5 ⁇ (100 x 4.6 mm) column and an Aglient 1100 series Photo Diode array detector.
  • the chloro- compounds, 14-15 were synthesized from p-nitro aniline by treatment with both chloroacetyl chloride (14) and 3-chloropropionyl chloride (15).
  • the tertiary amines 16-23 were obtained by treatment of the appropriate chloride with a range of cyclic and straight chain secondary amines, in MeOH. Following stirring at room temperature for 12 hours, the products 16-23 were obtained by desolvation by addition of water, and subsequent filtration. Conversion to the aniline derivatives was achieved by palladium catalysed reduction with ammonium formate. Products 24-31 were obtained in sufficient purity to be used in the diazotization immediately.
  • the diazotization reaction of 24-31 was carried out by using tert-butyl nitrite followed by substitution with NaN 3 .
  • the azide products 32-39 were purified by column chromatography before the final Cu(I) catalysed Huisgen 'click' reaction with 1 ,3-diethynyl benzene.
  • the final bis-triazole compounds 40-47 were obtained by filtration of the reaction mixture and purification was achieved by preparative HPLC.
  • FRET Fluorescence Resonance Energy Transfer
  • F10T is a hairpin double helix forming labelled oligomer, (with an internal hexaethyleneglycol (HEG) linker to make the hairpin loop).
  • C-Kit1 and C- Kit2 were tested as representative examples of non-telomeric regions of G-rich DNA in the human genome which are inherently capable of forming stable G-quadruplex structures. Putative quadruplexes have been identified in a number of nontelomeric genes and genomic sequences [11] including that of the oncogene c-myc.
  • C-kit1 and C-kit2 are sections from the promoter region of the oncogene c-myc.
  • C- kit1 and C-kit2 are of no consequence with regards to the inhibition of telomerase, but were tested currently to assess whether the compounds interacted with other quadruplex DNA, but they may also potentially be of therapeutic interest due to the oncogenic nature of the C-kit gene.
  • FIG. 1 Figure 2 illustrates the results for compound 47.
  • Table 1 shows the labelled oligomers used in the FRET experiments.
  • Table 2 shows the substituent structure, some chemical and physical properties, and increase in stabilisation temperatures (ATy 2 ) at 1 ⁇ m or 5 ⁇ m concentration determined by FRET:
  • the most active compound 47 (melting curves for 47 are shown in figure 2), displayed a high stabilisation, comparable to the best ligands known to date, for example the acridone based ligand BRACO-19, has ATy 2 of 27.5 0 C at 1 ⁇ M concentration on the F21T quadruplex [14], whereas the bis triazole compound 47 has a ATy 2 of 18.7 0 C at 1 ⁇ M concentration on the F21T quadruplex.
  • a comparison of greater importance is that of the ATy 2 on F10T (ds) DNA, for BRACO-19, the value is 14.5 0 C at 1 ⁇ M, but for 47 the value was -0.5 0 C at 1 ⁇ M, i.e. no stabilisation.
  • the ligands are highly selective for Quadruplex DNA.
  • Example 4 In this Example we investigated whether the claimed compounds would also show telomerase inhibition in the two-step TRAP assay ( Figure 4). This assay has been widely used to provide qualitative and quantitative estimates of telomerase inhibition.
  • Figure 4 shows the TRAP gel for (a) telomestatin, (b) 42 and (c) 44, showing characteristic ladders produced by PCR amplification of the oligonucleotides generated by the activity of telomerase on a TS primer.
  • telomere inhibition With increasing concentration of each compound, a decrease in the intensity of the ladder is observed (i.e. increase in telomerase inhibition).
  • the negative control was run under identical conditions but omitting the protein extract to ensure absence of PCR artifacts.
  • the intensity of the ladders was normalized with respect to the positive and negative controls, and a dose-response curve was fitted to calculate the concentration for 50% enzyme inhibition (EC value).
  • tel compounds 42, 43 and 44 showed high activity with EC 50 values of 13.25, 17.10 tel and 23.5 ⁇ M respectively.
  • ultra-potent telomestatin displayed a EC 50 of
  • MCF7 and A549 cells were purchased from American Type Cell Culture (ATCC). Both MCF7 and A549 cells were maintained in Dulbecco's Modified Eagles Media containing 10% foetal bovine serum (Invitrogen, UK), 0.5 mg/ml hydrocortisone (Acros Chemicals, Loughborough, UK), 2 mM L-glutamine (Invitrogen, Netherlands), and non-essential amino acids 1X (Invitrogen, Netherlands), and incubated at 37°C, 5% CO2. WI38 cell line was maintained in Minimal Essential Medium. MCF7 and A549 cell lines were routinely passaged at 1 :6 and WI38 at 1 :3. Sulphorhodamine B short-term cytotoxicity assay
  • Short-term growth inhibition was measured using the SRB assay as described previously [15]. Briefly, cells were seeded (4000 cells/wells) into the wells of 96 well-plates in DMEM and incubated overnight as before to allow the cells to attach. Subsequently cells were exposed to freshly-made solutions of BRACO-19 at increasing concentrations of 0, 0.1 , 1 , 5 and 25 mM in quadruplicate and incubated for a further 96 h. Following this the cells were fixed with ice cold trichloaceticacid (TCA) (10%, w/v) for 30 min and stained with 0.4% SRB dissolved in 1% acetic acid for 15 min. All incubations were carried out at room temperature. The IC50 value, concentration required to inhibit cell growth by 50%, was determined from the mean absorbance at 540 nm for each drug concentration expressed as a percentage of the control untreated well absorbance. Short term combination studies
  • Combination studies were carried out again in short-term assay with varying ratios of cispaltinum and quadruplex ligand to elucidate the optimum ratios of the two drugs.
  • Cells were exposed to varying concentration ratios of quadruplex ligand and cisplatinum for 96hrs as described under short term cytotoxicity assay in a 96 well plate. Plates were stained and data was obtained as before.
  • Data from combination studies were analysed using Calcusyn software to derive Combination Index (Cl) values.
  • the programme is based on the multiple drug-effect equation of the enzyme kinetic models originally derived by Chou-Talalay [16, 17].
  • 1 X 10 5 cells were seeded in 75 cm 2 tissue culture flasks and exposed to appropriate concentrations of drugs in single agents and in combination. The concentrations were chosen according to individual Cl values and IC 50 values as determined in the Cl analysis and SRB assay.
  • Cells were grown in a final volume of 10ml DMEM and incubated as described previously. Cells were exposed to ligands twice a week by replacing with fresh media containing drug on day 3. On day 7 media was removed and cells were washed with PBS once and trypsinised using 3ml of trypsin. Cells were then pelleted and resuspended in 10 ml of DMEM and viability was determined with a haemocytometer. From this 1 X 10 5 cells were reseeded and experiment was continued as described before for four weeks. Staining for Senescence-Associated ⁇ -Galactosidase Activity.
  • ⁇ -Galactosidase activity was carried out according to the instructions of the supplier (Cell Signaling Technology, Inc., Beverly, MA). In brief, cells from long-term exposure studies were retrieved at the end of each week and seeded in 35 mm 6-well plates (Nunc A/S) at a density of 1 X 10 5 cells in 2 ml_ media and incubated overnight under standard conditions together with the appropriate concentration of the compound under investigation.
  • the growth medium was removed, and the cells were washed, fixed, and stained using the supplied staining solution [400 mM citric acid/sodium phosphate (pH 6.0), 1.5 M NaCI, 20 mM MgCI 2 , 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg of X-gal (5-bromo- 4-chloro-3-indolyl- ⁇ -D-galactopyranoside)], followed by incubation overnight at 37 0 C (5% CO 2 ). Cells were examined by light microscope (mag. 200-800X) and counted the next day for the characteristic senescence-associated development of blue coloration. Results
  • Figure 5 shows the effect of combination and single agent treatments in MCF7 cells up to 5 weeks (C46 is compound 46).
  • VC stands for Vehicle Control.
  • CisPt (CP) was kept at 0.25 ⁇ M and the concentration of compound 46 was increased periodically.
  • Figure 5 shows that with compound 46 at 3 ⁇ M combined with cis-Pt at 0.25 ⁇ M, potent sub-cytotoxic inhibition of long-term cell growth occurs, whereas with either agent alone at these concentrations, no such inhibition occurs. The behaviour is typical for a telomerase inhibitor.
  • Assays for senescence following each week's treatment show induction of senescence using compound 46 as a single agent treatment and in combination (see Figure 6).

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Abstract

L'invention concerne des composés de formule (I) : dans laquelle Ar1 est un aryle ou hétéroaryle monocyclique ; X et Y sont chacun indépendamment un groupe de formule (II) : Z est absent, un groupe de formule (II), un alkyle en C1-7 facultativement substitué, un hétérocyclyle en C3-20 facultativement substitué, un aryle en C5-20 facultativement substitué, halo, amino, hydroxy, éther, thio, thioéther, carboxy ou cyano ; L1 et L2 sont chacun indépendamment choisis parmi NR3, C2H2, CH2, -O-, -S- et une liaison ; Ar2 et Ar3 sont indépendamment aryle ou hétéroaryle en C5 ou C6 facultativement substitué ; n est un entier de 1 à 5 ; R1 et R2 sont indépendamment hydrogène, alkyle en C1-7, hétérocyclyle en C3-20, ou aryle en C5-20, ou R1 et R2, pris conjointement avec l'atome d'azote auquel ils sont attachés, forme un noyau hétérocyclique ayant 3 à 8 atomes de cycle ; R3 est H ou alkyle en C1-7 ; et à la condition qu'au moins l'un de Ar1, Ar2 et Ar3 est oxazole, triazole ou tétrazole. Ces composés sont estimés se lier aux G -quadruplex formés dans des télomères humains et sont donc utiles en thérapie anti-cancer. L'invention propose également des compositions pharmaceutiques comprenant les nouveaux composés, et des procédés pour leur fabrication.
EP07824923A 2006-11-24 2007-11-26 Ligands g-quadruplex thérapeutiques Withdrawn EP2097393A1 (fr)

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