WO2008062235A1 - Therapeutic g-quadruplex ligands - Google Patents

Abstract

The invention provides compounds of formula (I): wherein Ar1 is a monocyclic aryl or heteroaryl; X and Y are each independe ntly a group of formula (II): Z is absent, a group of formula (II), optionally substituted C1-7 alkyl, optionally substituted C3-20 heterocyclyl, optionally substituted C5-20 aryl, halo, amino, hydroxy, ether, thio, thioet her, carboxy or cyano; L1 and L2 are each independently selected from NR 3, C2H2, CH2, -O-, -S- and a bond; Ar2 and Ar3 are independently optionally substituted C 5 or C6 aryl or heteroaryl; n is an integer from 1 to 5; R1 and R2 are independently hydrog en, C1-7 alkyl, C3-20 heterocyclyl, or C 5-20 aryl, or R1 and R2, taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms; R3 is H or C 1-7 alkyl; and provided that at least one of Ar 1, Ar2 and Ar3 is oxazole, triazole or tetrazole. These compounds are thought to bind G -quadruplexes formed in human telomeres and are therefore useful in anti -cancer therapy. The invention also provides pharmaceutical compositions comprising the novel compounds, and methods for their manufacture.

Description

THERAPEUTIC G-QUADRUPLEX LIGANDS

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.

The concepts behind cell cycle regulation are described in detail in WO02/08193 [1]. Briefly, when the tight controls regulating cell replication break down, uncontrolled proliferation results, resulting in tumour growth and the disease cancer. In many immortalised cells, 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.

A number of 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 π-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. Stabilisation of these G-quadruplex structures with small molecules has been shown to inhibit the action of telomerase [5]. Thousands of molecules have been screened for G-quadruplex binding in recent years [6]. These molecules can be categorised into 3 main classes [5]:- i) fused polycyclic intercalators, exemplified by BRACO-19; ii) macrocyclic compounds, including the natural product telemostatin (a potent binder which has been shown to induce telomere shortening, apoptosis, cell growth suppression and enhancement of chemosensitivity in certain cancer cell lines) [7]; and iii) polyaromatic unfused systems, for example the peptide hemi-cyanide ligand described in [8].

The understanding of the quadruplex structure is increasing and with crystallographic, NMR, and with in silico studies, there is the possibility of a rational approach to the design of G-quadruplex ligands. Recently, there are also some examples of selective ligands formed by the combination of fragments of known binders, and this was developed further by Balasubramanian et al who used the quadruplex as a template in a fragment based dynamic covalent chemistry approach [9]. Most studies, however, employ lead identification and elucidation of a structure-activity relationships (SAR) [10]. Although great advances have been made, there remains a great need for potent telomerase inhibitors and anti-tumour agents which selectively target tumour cells and have little or no cytotoxic effects on normal cells. 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. To date, none of the compounds suggested in the literature are specific enough to be free of these cytotoxic side-effects.

In accordance with a first aspect of this invention, we provide a compound of formula I:

^(l) wherein Ar¹ is a monocyclic aryl or heteroaryl;

X and Y are each independently a group of formula

O

-L¹ Ar²- -Ar³ N- Il

H -c- -(CH₂)_n- -NR¹ 1RQ2 (H)

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;

L¹ and L² are each independently selected from NR³, C₂H₂, CH₂, -O-, -S- and a bond;

Ar² and Ar³ are independently optionally substituted C₅ or C₆ aryl or heteroaryl; n is an integer from 1 to 5;

R¹ and R² are independently hydrogen, Ci_-7 alkyl, C_3-20 heterocyclyl, or C_5-20 aryl, or R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms; and

R³ is H or Ci_-7 alkyl; provided that at least one of Ar¹, Ar² and Ar³ is oxazole, triazole or tetrazole.

The planar triazole or tetrazole units in these compounds form part of a planar pharmacophore that is capable of π-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. Thus the present invention may avoid the toxicity problems of the anti-cancer compounds of the prior art.

In accordance with a second aspect of the invention we provide use of the compound according to the first aspect of the invention in the manufacture of a medicament for use in the treatment of a proliferative conduction. 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;

(HI) (IV)

(V)

to give a compound of formula V; wherein Ar⁴ is a monocyclic aryl or heteroaryl; each Ar⁵ is independently optionally substituted C₅ or Cβ 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 L³ and L⁴ is independently selected from NR³, C₂H₂, CH₂, -O-, -S-, and a bond;

R³ is H or Ci.₇alkyl; R⁴ and R⁵ are independently hydrogen, Ci_-7 alkyl, C_3-20 heterocyclyl, or C_5-2O aryl, or R⁴ and R⁵, 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". The concept of 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]. In these applications 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.

Certain compounds and combinations of substituents are preferred, in particular see the sub-claims. With regard to the structural preferences, a planar core with a π-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.

With regard to the compound of formula I, it is preferred that Ar¹ is phenylene. Multicyclic ring systems are known to bind to duplex DNA. It is thought that the monocyclic aryl ring Ar¹ ensures that the compounds of the invention preferentially bind to G-quadruplex structures rather than duplex DNA. In this embodiment, preferably X and Y are meta substituents on the phenyl ring and Z is absent. Alternatively, X and Y may be meta substituents on the phenyl ring and Z is absent. Although X and Y may be different, they are preferably the same. If Z is present, it is preferably a group of formula II, optionally substituted phenyl, or C₅ or C₆ optionally substituted heteroaryl. However, Z may be non- aromatic. In the embodiment, wherein Ar¹ is phenyl, X, Y and Z are preferably each meta substituents on the phenyl ring.

In another embodiment, Ar¹ is a triazole. Preferably, in this embodiment, Z is absent and X and Y are 1 ,4-substituents on the ring Ar¹, giving a compound of formula

X and Y may alternatively be 1 ,5 substituents on the triazole ring.

Alternatively, Ar¹ may be pyrazine, pyrimidine or pyridazine. Preferred compounds wherein Ar¹ is pyrazine, pyrimidine or pyridazine include

In any of the compounds according to the present invention, X, Y and Z (if present) are preferably the same.

Further examples of suitable heteroaryl groups for Ar¹, Ar² and Ar³ 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. Preferably, each Ar² is triazole. Preferably Ar¹ and each Ar³ is phenyl. Further preferred compounds are those of formula I wherein one or both of L¹ and L² is a bond.

In preferred embodiments, R¹ and R² are each independently Ci_-7 alkyl, which is optionally substituted. Preferably, each -NR¹R² is independently selected from -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(JPr)₂, -N(nBu)₂ and -N(tBu)₂.

Alternatively, R¹ and R², 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. In one preferred embodiment, preferably R¹ and R², 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. In these embodiments R¹ and R², 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 C₁-₇ alkyl, C_3-20 aryl-Ci-₇ alkyl, C_3-20 aryl, Ci-₇ alkyl-C₃-₂o aryl, hydroxy, and Ci.₇ hydroxyalkyl.

Alternatively, each -NR¹R² may be independently selected from

In other preferred embodiments, R¹ and R², 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. It is preferred in this embodiment that the terminal amino group, -NR¹R², is one of the following cyclic amino groups, and is optionally substituted:

wherein R is hydrogen, C_1-7alkyl, C₃.₂₀heterocyclyl, or C₅.₂oaryl. In the preferred compounds of the invention, n is preferably 1 or 2, most preferably 2.

Some individual embodiments of the present invention include compounds of formula

(Vl)

wherein m is an integer 1 or 2; and R⁶ and R⁷ are each C₁-₂ alkyl, C₄-₅ heterocyclyl or C₃-₅ heteroaryl, or R⁶ and

R⁷, taken together with the nitrogen to which they are attached, form a heterocyclic ring having 5-7 ring atoms.

In these compounds of the formula Vl, the terminal amino acid group is preferably one of the following amino groups, and is optionally substituted:

Compounds 46 and 47, as described in the Examples, are particularly preferred. The term "hetero" as used herein 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). The term "monocyclic aryl or heteroaryl" as used herein refers to cyclic compounds which have one aromatic ring, which may contain one or more multivalent heteroatoms in the case of monocyclic heteroaryl.

The phrase "optionally substituted" as used herein refers to a parent group which may be substituted or unsubstituted. Unless otherwise specified, the term "substituted", as used herein, refers to a parent group which bears one or more substituents. The term "substituent" 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. A wide variety of substituents are well known, and methods for their formation and introduction into a variety of parent groups are also well known.

Suitably, the substituent(s) are independently selected from: halo; hydroxy; ether (e.g., C₁-7 alkoxy); formyl; acyl (e.g., C₁.7 alkylacyl, C5-₂0 arylacyl); acylhalide; carboxy; ester; acyloxy; amido; acylamido; thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., C₁-7 alkylthio); sulfonic acid; sulfonate; sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulfamyl; sulfonamido;

C₁.7 alkyl (including, e.g. Ci_-7 haloalkyl, Ci_-7 hydroxyalkyl, C ₁.7 carboxyalkyl, Ci_-7 aminoalkyl, C_5-2O aryl-Ci_-7 alkyl); C_3-2O 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:

-F, Cl, -Br, and -I;

-OH;

-OMe, -OEt, -O(tBu), and -OCH₂Ph;

-SH; -SMe, -SEt, -S(tBu), and -SCH₂Ph;

-C(=O)H;

-C(=0)Me, -C(=O)Et, -C(=O)(tBu), and -C(=O)Ph;

-C(=O)OH; -C(=O)OMe, -C(=O)OEt, and -C(=O)O(tBu);

-C(=O)NH₂, -C(=O)NHMe, -C(=O)NMe_2l and -C(=O)NHEt;

-NHC(=O)Me, -NHC(=O)Et, -NHC(=O)Ph, succinimidyl, and maleimidyl;

-NH₂, -NHMe, -NHEt, -NH(iPr), -NH(nPr), -NMe₂, -NEt₂, -N(JPr)₂, -N(nPr)₂, - N(nBu)₂, and -N(tBu)₂;

-CN;

-NO₂;

-Me, -Et, -nPr, -iPr, -nBu, -tBu;

-CF₃, -CHF₂, -CH₂F, -CCI₃, -CBr₃, -CH₂CH₂F, -CH₂CHF₂, and -CH₂CF₃; -OCF₃, -OCHF₂, -OCH₂F, -OCCI₃, -OCBr₃, -OCH₂CH₂F, -OCH₂CHF₂, and -

OCH₂CF₃;

-CH₂OH, -CH₂CH₂OH, and -CH(OH)CH₂OH;

-CH₂NH₂, -CH₂CH₂NH₂, and -CH₂CH₂NME₂; and, optionally substituted phenyl. 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. Alternatively, 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. The terms "protected amino", "protected hydroxy" and "protected carboxy" as used herein refer to amino, hydroxy and carboxy groups which are protected in a manner familiar to those skilled in the art. For example, 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. Some compounds of formula I and Vl may exist in the form of solvates, for example hydrates, which also fall within the scope of the present invention.

Compounds of the invention may be in the form of pharmaceutically acceptable salts, for example, addition salts of inorganic or organic acids. Such 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 acid, mucic acid, 2-naphthalenesulphonic acid, pamoic acid, pantothenic acid, phosphanilic acid ((4-aminophenyl)phosphonic acid), picric acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, terephthalic acid, p- toluenesulphonic acid, 10-undecenoic acid and the like.

Salts may also be formed with inorganic bases. Such 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. It will be appreciated that such salts, provided that they are pharmaceutically acceptable, may be used in therapy. 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. The term "antiproliferative agent" as used herein is a compound which is useful in the treatment of a proliferative condition. The terms "cell proliferation", "proliferative condition", "proliferative disorder", and "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. Examples of 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. Any type of cell may be treated, including but not limited to, colon, kidney (renal), breast (mammary), lung, ovarian, liver (hepatic), pancreas, skin, and brain. Antiproliferative compounds of the present invention have application in the treatment of cancer, and so the present invention further provides anticancer agents. The term "anticancer agent" as used herein, 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. The term "treatment", as used herein in the context of treating a condition, 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.

The term "therapeutically-effective amount", as used herein, 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.

The term "treatment" includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously. Examples of 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.

In therapeutic use, 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. Thus, 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.

Appropriate dosage levels may be determined by any suitable method known to one skilled in the art. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health and sex of the patient, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the disease undergoing treatment. 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. The 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. These 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. For example, 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.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such 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 monooleatθ. 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. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, 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. 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. Preferably, a method according to the final aspect of this invention is used. In this method, preferably Ar⁴ and Ar⁵ 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. In a preferred method, the substituents ≡-L⁴ are arranged 1 ,3 on aryl ring Ar⁴. In a preferred embodiment of the method, L³ and L⁴ are each a bond, Z is absent, and compounds III and IV have formulae

III' IV

Preferably "click" chemistry is used to synthesise the compounds of the invention. 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:H₂O with CuSO₄.5H₂O 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:

Instead of a cyanide, alternative reagents may be used such as R⁸C≡S or an isonitrile.

The invention is illustrated further with reference to the following figures, in which Figure 1 shows the synthesis of bis-triazole compounds 40-47;

Figure 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 (ΔT_1/2) of F2IT in the presence of 1 μM 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; and

Figure 6 shows induction of senescence following treatment of MCF7 cells with compound 46. The following examples illustrate the invention.

Materials and Methods

All Chemicals were purchased from Sigma-Aldrich and Fischer Scientific, were reagent grade and used without further purification. Flash Chromatography employed Merck™ silica gel [Kieselgel 60 (0.040-0.063 mm)] and Florisil purchased from Avocado™ [Florisil 60 - 100 Mesh]. Analytical TLC was performed on 0.2 mm Silica-coated aluminium sheets from Merck [Kieselgel 60 F₂₅₄], and visualized with UV Light. Microwave reactions were carried out in Emrys™ Process v/als sealed with a Reseal™ septum, and heated in an Emrys™ Optimizer Microwave Station (Personal Chemistry™). 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 UVΛ/IS 155 detector using a YMC C18 5μ (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 5μ (100 x 4.6 mm) column and an Aglient 1100 series Photo Diode array detector. Infra Red measurements were carried out on a Perkin Elmer, FT-IR Spectrometer: Spectrum 1000 with a Specac™ golden gate. ¹H and ¹³C NMR spectra were recorded on a Bruker™ Avance400 spectrometer (400MHz for ¹H and 100MHz for ¹³Cspectra). Spectra were recorded at 295K either in CDCI₃ or DMSO-d6; chemical shifts are calibrated to the residual proton and carbon resonance of the solvent: CDCI₃ (dH 7.25, dC 77.0 ppm), DMSO-d6 (dH 2.49, dC 39.5ppm). Signal patterns are indicated as s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Melting Points were recorded on an Electrothermal digital melting point apparatus and are uncorrected, 'dec' indicates decomposition temperature. High Resolution Mass Spectroscopy was carried out on a Micromass™ Q-TOF Ultima Global Tandem Mass Spectrometer. All samples were run under Electrospray ionization mode using 50% Acetonitrile in Water and 0.1 % Formic acid as solvent. Compound names were obtained from CS ChemDraw Ultra™. Example 1

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 NaN₃. 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.

Specific reaction conditions for a selection of compounds 14-47 are given below. 2-Chloro-N-(4-nitro-phenyl)-acetamide, (14).

4-Nitroaniline (61 g, 443mmol) was dissolved in THF (3 L) and cooled to -

15°C in a salt-ice bath. To this stirred mixture was added TEA (44.7g, 443mmol), followed by the addition of Chloroacetylchloride (10Og, 885mmol) in THF (1.5 L) dropwise over 30 min. The reaction was then allowed to warm up to RT, then stirred overnight. The reaction was monitered by TLC, and when complete (approx. 14 hr), the insoluble precipitate of NH₄CI was removed by filtration. The filtrate was the basified with sat. NaHCO_3(aq) soln. (500 ml_), the solid formed was then removed by filtration. This was then successively washed with water (2 x 1 L) portions, then Acetone (2 x 500 ml_) portions. This gave the pure product, a pale yellow solid (84.43 g, 90%); R, = 0.05 (5% MeOH/ 95% DCM), m/p 187-191¹C, IR (film) 3272.90, 3225.90, 3162.90, 3103.87, 2939.90, 1682.77 cm^"1, ¹H NMR (DMSO, 400MHz) δ: 4.34 (s, 2H), 7.84 (d, 2H₁ J = 9.2 Hz), 8.25 (d, 2H, J = 9.2 Hz), 10.88 (s, 1 H). ¹³C NMR (DMSO, 100MHz) δ: 43.53, 119.10, 125.00, 142.63, 144.54, 165.56, HRMS [ES⁺] calcd for C₈H₇CIN₂O₃215.0218 [M+H] ⁺, found 215.0220. 3-Chloro-N-(4-nitro-phenyl)-propionamide, (15).

4-Nitroaniline (85.8 g, 623 mmol) was suspended in 3- Chloropropionylchloride (175 mL, 1.83 mol). The suspension was stirred and heated at 50⁹C overnight. The reaction was monitored by TLC, and when the reaction was complete (approx. 16 hr), the solid precipitate was filtered and washed with ether (3 x 150 mL) potions. This gave the product, a pale green solid (147 g, 97%); R_f = 0.2 (EtOAc: hexane 1 :4); m/p 157-159C, IR (film) 3346.74, 3132.92, 2970.93, 2911.92, 1702.60 cm^"1, ¹H NMR (DMSO, 400MHz) δ: 2.70 (t, 2H, J = 6.3 Hz), 3.69 (t, 2H, J = 6.2 Hz), 7.65 (d, 2H, J = 9.3 Hz), 8.02 (d, 2H, J = 9.3 Hz), 10.52 (s, 1 H), ¹³C NMR (DMSO, 100MHz) δ: 39.36, 40.38, 118.77, 124.98, 142.24, 145.02, 169.95; HRMS [ES⁺] calcd for C₉H₉CIN₂O₃229.0374 [M+H] ⁺, found 229.0380. 3-Dimethylamino-N-(4-nitro-phenyl)-propionamide, (23).

To a suspension of 3-Chloro-N-(4-nitro-phenyl)-propionamide (4.56 g, 20 mmol) in MeOH (20 mL) at O⁰C was added dimethylamine as a 2.0 M solution in THF (30 mL, 60 mmol), dropwise over 30 min. The mixture was allowed to warm to room temperature and stirring was continued until the reaction was complete by TLC (approx. 12hr). The mixture was then diluted with distilled water (300 mL). The diluted mixture was filtered to give the product, a bright yellow solid (3.9 g, 82%); m/p 80-82C, R_f = 0.2 (10% MeOH/ 90% CHCI₃), IR (film) 2947.21 , 2812.80, 2777.81 , 1698.76 cm^"1, ¹H NMR (CDCI₃, 400 MHz) δ: 2.44 (s, 6H), 2.56 (t, 2H, J= 5.7 Hz), 2.70 (t, 2H, J= 5.7 Hz), 7.68 (d, 2H, J= 9.2 Hz), 8.21 (d, 2H, J= 9.2 Hz), 11.69 (S₁ 1 H). ¹³C NMR (CDCI₃, 100 MHz) δ: 33.35, 44.37, 54.86, 1 19.13, 125.05, 143.06, 144.68, 171.24. HRMS [ES⁺]: calcd for CnH₁₆N₃O₃ 238.1186 [M+H]⁺, found 238.1192.

N-(4-Amino-phenyl)-3-dimethylamino-propionamide, (31 ).

To a solution of 3-Dimethylamino-N-(4-nitro-phenyl)-propionamide (3.4 g,

14.7 mmol) in DMF (60 mL) was added ammonium formate (4.5 g, 73.8 mmol), followed by 10% Pd/C (340 mg, 10 %m/m). The mixture was stirred at room temperature overnight and then monitored to completion by TLC. After the reaction was complete (approx. 16 hr) the mixture was filtered through a pad of celite, washed with DMF (50 mL) and then concentrated in vacuo. The residue was then suspended in 3% NH_3(aq) solution (50 mL), upon which the product crashed out. The mixture was then cooled to 0⁰C for 10 min then filtered and washed with water (2 x 50 mL). This yielded the product, a dark brown solid (1.62 g, 46%); R_f = 0.05 (10% MeOH/ 90% CHCI₃), m/p 90-9213, IR (film) 3178.75, 3067.79, 2974.79, 2827.80, 2796.77, 1641.40 cm^'1, ¹H NMR (CDCI₃, 400MHz) δ: 2.28 (S, 6H), 2.40 (t, 2H, J= 6.3 Hz), 2.56 (t, 2H, J= 6.3 Hz), 3.49 (S, 2H), 6.58 (d, 2H, J= 8.7 Hz), 7.23 (d, 2H, J= 8.7 Hz), 10.52 (s, 1 H). ¹³C NMR (CDCI₃, 100MHz) δ: 33.35, 44.47, 55.26, 115.44, 121.66, 130.31 , 142.67, 170.18. HRMS [ES⁺]: calcd for C₁₁H₁₈N₃O 248.1757 [M+H]⁺, found 248.1752. N-(4-Azido-phenyl)-3-dimethylamino-propionamide, (39).

To a solution of N-(4-Amino-phenyl)-2-dimethylamino-propionamide (1.262 g, 6.1 mmol) in THF (8OmL) at O⁰C was added cone. HCI_(aq) (2.5 ml_), followed by t- BuONO (1.81 nriL, 15.25 mmol). The mixture was stirred at O⁰C for 1 hr, then NaN₃ (1.19 g, 18.3 mmol) was added, followed by the careful addition of water (10 mL). The reaction mixture was allowed to warm to room temperature and left overnight. After the reaction is complete by TLC it was quenched with NaHCO₃ (30 mL). The THF is then removed in vacuo, before the remaining aqueous mixture is extracted with EtOAc (3 x 100 mL). The organics are then dried over anhydrous MgSO₄ and concentrated in vacuo. The crude product was purified by flash chromatography on florisil (1-5% MeOH/ DCM) to yield the product, a yellow solid (1.21g, 85%); R_f = 0.2 (20% MeOH/ 80% DCM), m/p 73-76C, IR (film) 2945.78, 2821.81 , 2354.90, 21 12.44, 1658.61 cm^'1, ¹H NMR (CDCI₃, 400MHz) δ: 2.39 (s, 6H), 2.52 (t, 2H, J= 6.3 Hz), 2.67 (t, 2H, J= 6.3 Hz), 6.98 (d, 2H_x J= 8.8 Hz), 7.54 (d, 2H, J= 8.8 Hz), 11.00 (S, 1 H). ¹³C NMR (CDCI₃, 100MHz) δ: 33.33, 44.44, 55.12, 1 19.39, 121.18, 134.97, 135.89, 170.62. HRMS [ES⁺]: calcd for CnH₁₆N₅O 234.1349 [M+H]⁺, found 234.1353.

3-Dimethylamino-N-{4-[4-(3-{1-[4-(3-dimethylamino propionylamino)-phenyl]- 1 H-[1 ,2,3]triazol-4-yl}-phenyl)-[1 ,2,3]triazol-1 -yl]-phenyl}-propionamide, (47).

To a solution of N-(4-Azido-phenyl)-3-dimethylamino-1-yl-propionamide (580 mg, 2.3 mmol), in 1 :1 t-BuOH: H₂O (10 mL) was added 1 ,3-diethynylbenzene (95 mg, 0.75 mmol), Sodium Ascorbate (75 mg, 0.38 mmol) and CuSO₄.5 H₂O (10 mg, 5 mol%). The mixture was stirred vigorously for 4 days at RT, reaction completion was monitored by TLC. The reaction mixture was then diluted with 20 ml. water and cooled to dC for 10 min. The reaction mixture was then filtered to yield the crude product, a dark green solid (377 mg, 80%). The crude product was purified using HPLC (Solvents: A = MeOH (0.1% TFA), B = H₂O (0.1% TFA), 0 min (20% A, 80% B), 2 min (20% A, 80% B), 40 min (80% A, 20% B), Retention Time = 21.3 min). R, = 0.05 (20% MeOH/ 80% DCM), m/p Dec. 306C; IR (film) 331 1.87, 3136.90, 2937.87, 2818.87, 2769.88, 1654.60 cm^"1, ¹H NMR (d⁶-DMSO, 400MHz) δ: 2.13 (s, 12H), 2.44 (m, 4H), 2.54 (t, 4H, J= 7.1 Hz), 7.57 (t, 1 H, J= 7.8 Hz), 7.83 (d, 4H, J = 9.0Hz), 7.92 (d, 4H, J = 9.3Hz), 7.94 (dd, 2H, J = 1.5, 7.8 Hz), 8.58 (t, 1 H, J = 1.5 Hz), 9.32 (s, 2H), 10.36 (s, 2H); ¹³C NMR (d⁶-DMSO, 100MHz) δ: 34.74, 44.89, 54.95, 119.73, 119.77, 120.63, 122.09, 125.08, 129.69, 131.04, 131.63, 139.60, 146.90, 170.50; HRMS [ES⁺]: calcd for C₃₂H₃₇N₁₀O₂ 593.3096 [M+H]⁺, found 593.3106. Example 2

The interaction of the molecules with DNA was then investigated using a high-throughput FRET (Fluorescence Resonance Energy Transfer) assay, a valuable and extensively used tool for the rapid and accurate identification and comparison of quadruplex stabilising ligands. The effect of different dosings of the final compounds 40-47 on the melting temperature (Δ7V₂) of four FRET labelled oligomers was investigated. The three different types of quadruplex forming labelled oligomers were F21T, which represents the Human telomeric sequence, and C-kit1 and C-kit2, which represent the sequences from the oncogenes C-kit1 and C-kit2. F10T (ds) 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. Thus, 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.

Figure 2 illustrates the results for compound 47.

Table 1 shows the labelled oligomers used in the FRET experiments.

Table 1 - Labelled Oligomers used in FRET experiments

Key: 'X¹ = HEG (hexaethyleneglycol [(-CH₂-CH₂-O)₆])

Note: Sequences are labelled at the 5'-end with FAM (6-carboxyfluoroscein) and at the 3'-end with TAMRA (6-carboxytetramethylrhodamine).

Table 2 shows the substituent structure, some chemical and physical properties, and increase in stabilisation temperatures (ATy₂) at 1 μm or 5μm concentration determined by FRET:

Compd Substituent n Yield F21T F10T ds C-kit1 C-kit2

(1 μM) (1 μM) (5μM) (5μM)

40 morpholinyl- 1 100 -0.1±0.1 -0.5±0.1 N/A N/A

41 pyrrolidinyl- 1 78 13.6±0.2 -0.4±0.1 5.5±0.6 8.7±0.1

42 piperidinyl- 1 100 2.4±0.1 -0.6±0.1 1.3±1.2 2.4±0.2

43 diethylamino- 1 95 4.0±0.3 -0.4±0.2 1.2+0.1 2.9±0.3

44 dimethylamino- 1 100 9.2+0.2 -0.4±0.1 2.9±0.5 6.5±0.3

45 piperidinyl- 2 100 14.4±0.3 -0.5±£>.1 15.7±0.2 17.2±0.5

46 pyrrolidinyl- 2 88 17.8+0.2 -0.5±0.2 27.0±0.2 27.7±0.8

47 dimethylamino- 2 80 18.7+0.3 -0.5±0.4 24.7±0.2 25.7±0.3

Table 2 - Substituent structure, some chemical and physical properties, and increase in stabilisation temperatures (ATy₂) determined by FRET

[a] Yields are isolated yields before HPLC, which was carried out to ensure greater than 90% purity before FRET analysis The melting temperature (T_1/2) is indicative of the stability of the DNA when bound to the compound under investigation. Compounds which increase the Tv₂ value of the DNA by a greater amount are binding more strongly to the DNA.

Moderate to very high stabilisations were obtained across all of the three Quadruplex forming labelled Strands, except in the case of compound 40, which contained a morpholinyl substituent and displayed no binding. The binding increases approximately 1.1 to 6-fold with an increase in the length of the alkyl side chains by one -CH₂- unit - this can be seen in comparison of ΔT_V2 values between 41 and 46, and 42 and 45. Binding affinity is also dependant on the nature of the amino substituent. For compounds 40 to 44 which only contain one -CH₂- unit in the side chain, the binding increases in the sequence morpholinyl-, piperidinyl-, diethylamino-, dimethylamino- then pyrrolidinyl-. That pattern emerges for compounds 45 to 47 which contain two -CH₂- units in each side chain, where the binding increases in the sequence piperidinyl-, pyrrolidinyl-, then dimethylamino-. Importantly in terms of selectivity, none of the compounds increased the melting temperature of the duplex forming strand F10T (ds) across the concentration range tested. This may be a consequence of the steric requirements of these compounds which prevent intercalative binding to duplex DNA.

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 ATy₂ of 27.5⁰C at 1 μM concentration on the F21T quadruplex [14], whereas the bis triazole compound 47 has a ATy₂ of 18.7⁰C at 1 μM concentration on the F21T quadruplex. A comparison of greater importance is that of the ATy₂ on F10T (ds) DNA, for BRACO-19, the value is 14.5⁰C at 1 μM, but for 47 the value was -0.5⁰C at 1 μM, i.e. no stabilisation. Thus the ligands are highly selective for Quadruplex DNA.

Example 3

We also performed a competition experiment of our own design to further assess the specificity of some of the compounds. This experiment involved performing the FRET assay with a fixed concentration of F21T labelled oligonucleotide, in the presence of increasing concentrations of a competitor substrate in the form of Calf Thymus DNA (The calf thymus concentrations are given as μM (Phosphate)). The effect of a duplex DNA competitor could then be deduced from the suppression of the melting temperature of the labelled F21T DNA (at 200 nM) in the presence of different quadruplex ligands (at 1 μM). The results of the experiment are shown in Figure 3. Although BRACO-19 gives rise to greater stabilization of the F21T quadruplex with no competitor, in the presence of 26μM calf thymus DNA, the melting temperature falls below that of 47 and 46 in analogous conditions. 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. 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). Using this assay, tel compounds 42, 43 and 44 showed high activity with EC₅₀ values of 13.25, 17.10 tel and 23.5 μM respectively. Although ultra-potent telomestatin displayed a EC 50 of

0.66 μM in the same assay, the results for 42-44 are significant. Example 5 Cell lines

Human cancer cell lines, breast (MCF7), lung (A549) and normal human fibroblast line (WI38) 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]. The equation, however, ascertains only the additive effect (Cl=1) and cannot determine synergism or antagonism. Nevertheless, synergism (Cl=<1 ) is considered as more than expected additive effect, and antagonism (Cl=>1) as a less than expected additive effect. A classical Isobologram, when the interaction between the two drugs are additive, can be plotted and effect levels, eg: ED₅₀, ED₇₅ and ED₉₀ (effective dose or the concentration of drug eliciting response in 50%, 75% and 90% of cell population respectively) can be derived. Long-term combination growth inhibition studies

1 X 10⁵ cells were seeded in 75 cm² 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 IC₅₀ 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 10⁵ cells were reseeded and experiment was continued as described before for four weeks. Staining for Senescence-Associated β-Galactosidase Activity.

Staining for β-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 10⁵ cells in 2 ml_ media and incubated overnight under standard conditions together with the appropriate concentration of the compound under investigation. After an approximately 24-h incubation period, 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 MgCI₂, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 1 mg of X-gal (5-bromo- 4-chloro-3-indolyl-α-D-galactopyranoside)], followed by incubation overnight at 37 ⁰C (5% CO₂). Cells were examined by light microscope (mag. 200-800X) and counted the next day for the characteristic senescence-associated development of blue coloration. Results

Short term acute cytotoxicity IC₅₀ data for four ligands in two cancer cell lines and a normal human fibroblast cell line (WI38) are shown in Table 3. This shows that all four compounds have potent short term cell kill properties, and that compound 46 in particular has selectivity for cancer cells.

Table 3 - Short term acute cytotoxicity (C₅₀ data)

Combination index studies were carried out in A549 and MCF7 with compound 46 and Cisplatinum, in a range of molar ratios, in order to find out if a particular combination could be more effective than either compound alone in inhibiting cell growth. Tables 4 and 5 show the results:

Table 5 - MCF7 cell line + compound 46 + Cis Pt

The numbers in tables 4 and 5 represent combination indexes (activity of the two drugs together) which are categorised as follows: Cl of <1 = Synergism

Cl of 1 = Additive Cl of >1 = Antagonism Both in MCF7 and A549 cell lines, ratios of 1 :1 , 1 :2 and 2:1 produce a synergistic effect. Increasing the amount/ratio of quadruplex ligand enhances the effect of the combination treatment. This is evident from the ratios of 3:1 (CisPtcompound 46, last column) where increased amounts of Cisplatinum diminishes the effect. This is true for all cell lines, except in MCF7 where it produces an additive rather than an antagonistic effect.

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.

In the combinations in Figure 5, CisPt (CP) was kept at 0.25μM and the concentration of compound 46 was increased periodically.

Figure 5 shows that with compound 46 at 3 μM combined with cis-Pt at 0.25μM, 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).

These results show that the novel compounds of this invention show short- term cell kill. The compounds show selectivity for cancer cells and show marked synergy with the established anti-cancer drug cisplatinium. References

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Claims

1. A compound of formula I:

wherein Ar¹ is a monocyclic aryl or heteroaryl;

X and Y are each independently a group of formula II:

o L¹ Ar² L² Ar³ N C (CH₂)_n NR¹ R² (II)

H

Z is absent, a group of formula II, optionally substituted C_1-7 alkyl, optionally substituted C_3-20 heterocyclyl, optionally substituted C_5-2O aryl, halo, amino, hydroxy, ether, thio, thioether, carboxy or cyano;

L¹ and L² are each independently selected from NR³, C₂H₂, CH₂, -O-, -S- and a bond;

Ar² and Ar³ are independently optionally substituted C₅ or C₆ aryl or heteroaryl; n is an integer from 1 to 5;

R¹ and R² are independently hydrogen, Ci-₇ alkyl, C_3-20 heterocyclyl, or C_5-20 aryl, or R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms;

R³ is H or Ci-7 alkyl; and provided that at least one of Ar¹, Ar² and Ar³ is oxazole, triazole or tetrazole.

2. A compound according to claim 1 , wherein Ar¹ is a phenyl ring.

3. A compound according to claim 2, wherein X and Y are meta substituents on the phenyl ring and Z is absent.

4. A compound according to claim 2, wherein X and Y are para substituents on the phenyl ring and Z is absent.

5. A compound according to claim 1 or 2 wherein Z is a group of formula II, optionally substituted phenyl, or C₅ or C₆ optionally substituted heteroaryl.

6. A compound according to claim 5 when dependent upon claim 2 wherein X, Y and Z are each meta substituents on the phenyl ring.

7. A compound according to claim 1 wherein Ar¹ is a triazole ring.

8. A compound according to claim 7, wherein Z is absent and X and Y are 1 ,4 substituents on the ring Ar¹, having the formula

9. A compound according to claim 1 wherein Ar¹ is pyrazine, pyrimidine or pyridazine.

10. A compound according to claim 9 having formula

11. A compound according to any of the preceding claims, wherein X and Y [and optionally Z (if present)] are the same.

12. A compound according to any preceding claim, wherein each Ar² is triazole.

13. A compound according to any preceding claim, wherein each Ar³ is phenyl.

14. A compound according to any preceding claim, wherein each L¹ is a bond.

15. A compound according to any preceding claim, wherein each L² is a bond.

16. A compound according to any preceding claim, wherein R¹ and R² are each independently d-₇ alkyl, which is optionally substituted.

17. A compound according to claim 16, wherein each -NR¹R² is independently selected from -N(Me)₂, -N(Et)₂, -N(nPr)₂, -N(iPr)₂, -N(nBu)₂, or - N(tBu)₂.

18. A compound according to any one of claims 1 to 15, wherein in each group NR¹R² R¹ and R², taken 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.

19. A compound according to claim 18, wherein R¹ and R², 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.

20. A compound according to claim 19, wherein R¹ and R², taken together with the nitrogen atom to which they are attached 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:

21. A compound according to claim 20, wherein each group -NR¹R², is one of the following cyclic amino groups, and is optionally substituted:

22. A compound according to claim 21 , wherein said cyclic amino group is substituted with one or more substituents selected from Ci_-7 alkyl, C_3-20 aryl-Ci_-7 alkyl, C_3-20 aryl, C_1-7alkyl-C₃.₂₀aryl, hydroxy, and Ci_-7 hydroxyalkyl.

23. A compound according to claim 21 , wherein each -NR¹R² is independently selected from:

24. A compound according to claim 18, wherein in each NR¹R² group R¹ and R², 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.

25. A compound according to claim 24, wherein the group -NR¹R², is one of the following cyclic amino groups, and is optionally substituted:

wherein R is hydrogen, Cwalkyl, Ca-ao heterocyclyl, or C₅-₂oaryl.

26. A compound according to any preceding claim, wherein n is 1 or 2.

27. A compound according to claim 1 of formula

(Vl)

wherein m is an integer 1 or 2; and R⁶ and R⁷ are each C_1-2 alkyl, C_4-5 heterocyclyl or C_3-5 heteroaryl, or R⁶ and

R⁷, taken together with the nitrogen to which they are attached, form a heterocyclic ring having 5-7 ring atoms.

28. A compound according to claim 27, wherein the terminal amino acid group is one of the following amino groups, and is optionally substituted:

29. A pharmaceutical composition comprising a compound according to any of claims 1 -28 or a pharmaceutically acceptable salt, ester, amide, solvate, hydrate or protected form thereof, and a pharmaceutically acceptable diluent or carrier.

30. A compound according to any of claims 1 -28 for use in therapy.

31. Use of a compound according to any of claims 1-28, in the manufacture of a medicament for use in the treatment of a proliferative condition.

32. A method of inhibiting telomerase in vitro or in vivo, comprising contacting a cell with an effective amount of compound according to any of claims 1-28.

33. A method of regulating cell proliferation in vitro and/or in vivo, comprising contacting a cell with an effective amount of compound according to any one of claims 1-24.

34. 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 any of claims 1-28.

35. A method of manufacturing a compound of formula (V) in which a compound of formula III is reacted with a compound of formula IV to give a compound of formula V;

(III) (IV)

(V)

wherein Ar⁴ is a monocyclic aryl or heteroaryl; each Ar⁵ is independently optionally substituted C₅ or C₆ aryl or heteroaryl; Z is absent, optionally substituted Ci_-7 alkyl, optionally substituted C_3-20 heterocyclyl, optionally substituted C₅.₂o aryl, halo, amino, hydroxy, ether, thio, thioether, carboxy or cyano; p is an integer from 4 to 5; each L³ and L⁴ is independently selected from NR³, C₂H₂, CH₂, -O-, -S-, or is a bond;

R³ is H or Ci_-7 alkyl;

R⁴ and R⁵ are independently hydrogen, Ci_-7 alkyl, C_3-2O heterocyclyl, or C_5-20 aryl, or R¹ and R², taken together with the nitrogen atom to which they are attached, form a heterocyclic ring having from 3 to 8 ring atoms.

36. A method according to claim 35 wherein the substituents ≡-L⁴ are arranged 1 ,3 on aryl ring Ar⁴.

37. A method according to claim 35 or 36 wherein Ar⁴ and Ar⁵ are each optionally substituted phenyl.

38. A method according to claim 37 wherein L³ and L⁴ are each a bond, Z is absent, and compounds III and IV have formulae

III¹ IV

39. A method according to any of claims 35-38 wherein a Cu(I) catalyst is used.

40. A method according to any of claims 35-39 wherein the reaction is carried out in a mixture of alcohol and water.