FIELD OF THE INVENTION

The invention relates to compounds of formula (I) and salts and solvates thereof, and pharmaceutical compositions containing them, and the use of such compounds as inhibitors of tankyrase and in the treatment of disorders related to signalling by Tankyrase 1 and tankyrase 2, and to treat diseases or conditions modulated by tankyrase 1 and/ or 2 for example proliferative diseases and autoimmune diseases such as cancer and diabetes respectively.

BACKGROUND TO THE INVENTION

Tankyrases (TNKSs) consist of two human isoforms (TNKS1 and TNKS2) and are members of the poly(ADP-ribose)polymerase (PARP) family of enzymes that use NAD ⁺ as a substrate to transfer ADP-ribose units to target proteins, a post-translational modification process known as poly(ADP)-ribosylation. ² Target proteins include telomere repeating binding factor- 1 (TRF1), ^{3 4} nuclear mitotic apparatus protein (NuMA), ⁵ (which is essential for the resolution of chromatids during mitosis) and axin, the concentration-limiting component of the Wnt signalling axis. ⁶ Increased levels of Wnt signalling occur in many human cancer / tumour types and TNKSs are

overexpressed in several human cancers. ^{7 10}

Inhibition of TNKSs is reported to lead to stabilization of axin and decreased nuclear β- catenin-driven proliferation of cancer cells. ¹¹ The axin-TNKS-KIF3 A complex is required for insulin-stimulated GLUT4 translocation. ¹² Additionally, insulin-regulated amino-peptidase (IRAP) is also a binding partner of TNKSs. ¹³ Together, IRAP and TNKSs can enhance insulin-stimulated exocytosis of GLUT4 which could result in increased uptake of glucose. ¹³ TNKS-knockout mice display increased sensitivity to insulin and reduced adiposity and pan-PARP inhibitors have been used to investigate the role of TNKSs in studies on the translocation of GLUT4. ¹⁴

XAV939 1 (shown below) has been extensively used as an inhibitor of TNKSs and of Wnt signalling. ⁶ Like 1, compounds that bind to TNKSs include flavones, ¹⁵ 2- arylquinazolin-4-ones 2 , ^{1 21} isoquinolin- l-ones ¹⁷ and aryltetrahydronaphthyridinones which maintain a classic binding mode in the nicotinamide-binding site. ¹⁸ IWR- 1 3 (shown below) is an inhibitor of the Wnt signalling cascade through inhibiting TNKSs and binds exclusively to the adenosine-binding site. ¹⁹ The norbornane of 3 forces a conformational change of Tyr ¹⁰⁵⁰ (TNKS2 numbering), allowing the quinoline of 3 to π- stack with His ¹⁰⁴⁸ within the adenosine-binding site and so the norbornyl is required for potency. ²⁰

Previou sly, novel inhibitors including 2-aryl-8 -methylquinazolin-4-ones with 4 '-large or electron-withdrawing substituents (e.g. 2 ) and 3 -arylisoquinoline- l-ones have been reported to provide potent and selective inhibition of TNKS and Wnt signalling. ^{16 21} Some compounds have been evalu ated as TNKS inhibitors that bind to both the nicotinamide-binding site and the adenosine-binding domain , ^{22 23} although the increase in potency over 1 were modest. In addition , such compounds showed a similar selectivity for TNKS- 1 over PARP2 as 3 .

It has previously been shown that a 2-aryl group (or equivalent) is required for potent inhibition of TNKSs ; this reiterates the requirement for the 2-aryl unit when designing inhibitors of TNKSs that bind only to the nicotinamide-binding site. ^{16 18 21}

A number of TNKSs inhibitors have been reported such as WO 20 14/ 023390 and WO 20 14/ 036022.

Despite such disclosures, there is a need to find compounds having TNKS l and TNKS2 inhibitory activity. There is a further need to find compounds having selective inhibition of TNKS 1 and TNKS2 over other PARPs.

The present invention seeks to overcome problem(s) associated with the prior art.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a a compound of formula (I) :

(I)

and salts, solvates, tautomers and stereoisomers thereof, wherein :

X ¹ is selected from C-R or N;

R ¹ , R ² and R ³ are each independently selected from -H, -Ci-Ce alkyl, -Ci-Ce haloalkyl, - C(0)NR ^b R ^c , -C0 ₂ R ^b , -CN, -OR ^b , -NR ^b R ^c and -halo;

L is selected from -C1-C5 alkylene-, -S-(Ci-C4 alkylene) _z - and phenylene; wherein the phenylene is optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and -halo;

z is 0 or 1;

Yi is selected from - C(0)NH -, -NHC(O)- and -C(O)- ;

Ar is selected from a heteroarylene with 5 or 6 ring members; and phenylene wherein the heteroarylene and the phenylene are optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl and -halo;

Y ₂ is selected from -C(0)NH-, -NHC(O)- and -C(O)-;

Ar ² is a 9- or 10 -membered heteroaryl group or a napthalyl group optionally substituted with up to seven optional substituents independently selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl, -OR ^d , -NR ^d R ^e and -halo; and

R , R ^b , R ^c , R ^d and R ^e are each independently selected from -H and -Ci-Ce alkyl.

In a further aspect, the present invention provides a compound of formula (I), wherein the compound is a com

(Π)

and salts, solvates, tautomers and stereoisomers thereof, wherein :

X ¹ is selected from C-R or N;

R ¹ , R ² and R ³ are each independently selected from -H , -Ci-Ce alkyl, -Ci-Ce haloalkyl, C(0)NR ^b R ^c , -C0 ₂ R ^b , -CN, -OR ^b , -NR ^b R ^c and -halo; L is selected from -C1-C5 alkylene- , -S-(Ci-C4 alkylene) _z - and phenylene; wherein the phenylene is optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and -halo;

z is 0 or 1;

Yi is selected from -C(0)NH-, -NHC(O)- and -C(O)-;

Ar is selected from a heteroarylene with 5 or 6 ring members ; and phenylene wherein the heteroarylene and the phenylene are optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl , -Ci-Ce haloalkyl and -halo;

Y ₂ is selected from - C(0)NH-, -NHC(O)- and -C(O)- ;

X ² is selected from N and C-R ⁴ ;

X ³ is selected from N and C-R ⁵ ;

X ⁴ is selected from N and C-R ⁶ ;

X ⁵ is selected from N and C-R ⁷ ;

X ⁶ is selected from N and C-R ⁸ ;

X ⁷ is selected from N and C-R ⁹ ;

X ⁸ is selected from N and C-R ¹⁰ ;

R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently selected from -H , -G-C ₆ alkyl, -G- C ₆ haloalkyl, -OR ^d , -NR ^d R ^e and -halo; and

R , R ^b , R ^c , R ^d and R ^e are each independently selected from -H and -Ci-Ce alkyl.

In a further aspect, the present invention provides a pharmaceutical composition comprising a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof, and a pharmaceutically acceptable excipient, carrier, or diluent. In a further aspect, the present invention provides a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof, for u se in therapy.

In a further aspect, the present invention provides a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof, for u se as a medicament.

In a further aspect, the present invention provides a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof, for u se in the treatment of a disease or condition selected from a proliferative disease (such as cancer) , diabetes and fibrosis. In a further aspect, the present invention provides the u se of a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof, in the manufacture of a medicament for treating a disease or condition selected from a proliferative disease (such as cancer), diabetes and fibrosis.

In a further aspect, the present invention provides a method of treating a disease or condition selected from a proliferative disease, diabetes and fibrosis in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof or a pharmaceutical composition comprising a compound of formula (I) and salts, solvates, tautomers and stereoisomers thereof.

The compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, may be used for the isolation and investigation of the activity or expression of TNKSs. In addition, they are suitable for use in diagnostic methods for diseases in connection with TNKSs activity, in particular, diseases in connection with unregulated or disturbed TNKSs activity.

In a further aspect, the present invention provides the use of the compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, as molecular probes; in particular, as molecular probes for a disease or conditions selected from a proliferative disease (such as cancer), diabetes and fibrosis.

Compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, have been found to be highly potent and specific inhibitors of TNKS, and they can effectively inhibit Wnt signalling, increase glucose uptake into adipocytes and reduce colon cancer growth in vitro using functional cellular assays.

In a further aspect, and salts, solvates, tautomers and stereoisomers thereof, may be administered alone or in combination with other treatments, either simultaneously or sequentially depending upon the condition to be treated. The pharmaceutical composition of the present invention may further comprise one or more (e.g. two, three or four) further active agents (such as further tankyrase inhibitors and/ or further active agents for the treatment of a proliferative disease (such as cancer) and/ or diabetes and/ or fibrosis). Definitions

The following abbreviations are used throughout the specification: Ac acetyl; Ar argon ; DCM dichloromethane; DMF dimethylformamide; Et ethyl; EtOAc ethyl acetate; Me methyl; MeOH methanol; PBS phosphate-buffered saline; Ph phenyl; and THF tetrahydrofuran.

"Ci-Ce alkyl" refers to straight chain and branched saturated hydrocarbon groups, generally having from 1 to 6 carbon atoms; more suitably C ₁ - C5 alkyl; more suitably Ci- C4 alkyl, more suitably C ₁ - C3 alkyl. Examples of alkyl groups include methyl, ethyl, n- propyl, i-propyl, n-butyl, s-butyl, i-butyl, t-butyl, pent- l-yl, pent-2-yl, pent-3-yl, 3- methylbut- l-yl, 3-methylbut-2-yl, 2-methylbut-2-yl, 2,2,2-trimethyleth- l-yl, n-hexyl, and the like.

"Alkylene" refers to a divalent radical derived from an alkane which may be a straight chain or branched, as exemplified by -Ct -, - CH2CH2-, -CH(CH3)CH2- and - "Anticancer agent" refers to any agent which is administered to a patient with cancer for the purposes of treating the cancer.

"Drug", "drug substance", "active pharmaceutical ingredient", and the like, refer to a compound (e.g., compounds of Formula 1 and compounds specifically named above) that may be used for treating a subject in need of treatment.

"Excipient" refers to any substance that may influence the bioavailability of a drug, but is otherwise pharmacologically inactive. "Halo" refers to -F, -CI, -Br, and -I.

"Haloalkyl" refers to an alkyl as defined herein, which is substituted by one or more halo groups as defined herein. The haloalkyl can be monohaloaikyl, dihaloalkyl or polyhaloalkyi including perhaloalkyl. A monohaloaikyl can have one iodo, bromo, chloro or fiuoro within the alkyl group. Dihaloalkyl and polyhaloalkyi groups can have two or more of the same halo atoms or a combination of different halo groups within the alkyl. Typically the polyhaloalkyi contains up to 12, or 10 , or 8 , or 6, or 4, or 3, or 2 halo groups. Non-limiting examples of haloalkyl include fluoromethyl, difiuoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, pentafluoroethyl, heptafluoropropyl, difluorochloromethyl, dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl and dichloropropyl. A perhalo-alkyl refers to an alkyl having all hydrogen atoms replaced with halo atoms. "Heteroaryl with 5 or 6 ring members": refers to unsaturated monocyclic aromatic groups comprising from 5 or 6 ring atoms, whether carbon or heteroatoms, of which from 1 to 4 are ring heteroatoms. Suitably, each ring has from 1, 2 or 3 ring

heteroatoms. Suitably each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. The heteroaryl group may be attached to a parent group or to a substrate at any ring atom and may include one or more non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound.

Examples of monocyclic heteroaryl groups include, but are not limited to, those derived from:

Ni: pyrrole, pyridine;

Oi: furan ;

Si: thiophene;

NiOi: oxazole, isoxazole, isoxazine;

N ₂ O ₁ : oxadiazole (e.g. l-oxa-2,3-diazolyl, l-oxa-2,4-diazolyl, l-oxa-2,5-diazolyl, 1-oxa- 3,4-diazolyl) ;

N3O1: oxatriazole;

N ₁ S ₁ : thiazole, isothiazole;

N ₂ : imidazole, pyrazole, pyridazine, pyrimidine (e.g., cytosine, thymine, uracil), pyrazine;

N3 : triazole, triazine; and,

N4 : tetrazole.

"A 9- or 10-membered heteroaryl group": refers to unsaturated bicyclic aromatic groups comprising from 9 to 10 ring atoms, whether carbon or heteroatoms, of which from 1 to 10 are ring heteroatoms. Suitably, each ring has from 5 to 6 ring atoms and from 0 to 4 ring heteroatoms. Suitably each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. In one embodiment, the heteroaryl group is attached to the parent group at a hetero ring atom. In an alternative embodiment, the heteroaryl group is attached to a parent group or to a substrate at a carbon ring atom. Heteroaryl groups may include one or more optional non-hydrogen substituents unless such attachment or substitution would violate valence requirements or result in a chemically unstable compound. Suitable optional substituents includes one or more optional substituents independently selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl, -OR ^d , - NR ^d R ^e and -halo. Examples of heteroaryl groups which comprise two fused rings, include, but are not limited to, those derived from :

Oi: benzofuran, isobenzofuran ;

Ni: indole, isoindole, quinoline, isoquinoline;

Si: benzothiofuran ;

NiOi: benzoxazole, benzisoxazole;

NiSi: benzothiazole;

N ₂ : benzimidazole, indazole, quinoxaline, quinazoline, cinnoline, phthalazine, naphthyridine;

N2O1: benzofurazan ;

N ₂ S ₁ : benzothiadiazole;

N3 : benzotriazole; and

N4 : purine (e.g., adenine, guanine), pteridine.

"Heteroarylene" refers to a divalent radical derived from a heteroaryl group, as exemplified by pyridinylene - (C5H3N)-.

"Independently selected" is used in the context of statement that, for example, "R and R ^b are independently selected from -H and -Ci-6 alkyl, etc." and means that each instance of the functional group R or R ^b is selected from the listed options

independently of any other instance of R or R ^b in the compound. Hence, for example, methyl may be selected as an example of a Ci-6 alkyl for the first instance of R in the compound and -H may be selected for the next instance of R in the compound.

"Optionally substituted" refers to a parent group which may be unsubstituted or which may be substituted with one or more substituents. Suitably when optional substituents are present, the optional substituted parent group comprises from one to three optional substituents, i.e. one, two, or three optional substituents.

"Pharmaceutically acceptable" substances refers to those substances which are within the scope of sound medical judgment suitable for use in contact with the tissues of subjects without undue toxicity, irritation, allergic response, and the like,

commensurate with a reasonable benefit-to-risk ratio, and effective for their intended use. "Pharmaceutical composition" refers to the combination of one or more drug substances and one or more excipient, carrier or diluent.

"Phenylene" refers to a divalent radical derived from a phenyl group, i.e. - (CeEU)- .

The term "subject" as used herein refers to a human or non-human mammal.

Examples of non -human mammals include livestock animals such as sheep, horses, cows, pigs, goats, rabbits and deer; and companion animals such as cats, dogs, rodents, and horses.

"Substituted", when used in connection with a chemical substituent or moiety (e.g., an alkyl group), means that one or more hydrogen atoms of the substituent or moiety have been replaced with one or more non-hydrogen atoms or groups, provided that valence requirements are met and that a chemically stable compound results from the substitution.

"Therapeutically effective amount" of a drug refers to the quantity of the drug or composition that is effective in treating a subject and thus producing the desired therapeutic, ameliorative, inhibitory or preventative effect. The therapeutically effective amount may depend on the weight and age of the subject and the route of administration, among other things.

"Treating" refers to reversing, alleviating, inhibiting the progress of, or preventing a disorder, disease or condition to which such term applies, or to reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of such disorder, disease or condition.

"Treatment" refers to the act of "treating", as defined immediately above. "Tankyrase inhibitor" means a compound or that can inhibit tankyrase activity such as to reduce and/ or eliminate and/ or prevent the detrimental action of tankyrase. In some embodiments, tankyrase is selectively inhibited, that is tankyrase is preferentially inhibited compared to another enzyme. The inhibition of tankyrase can be assessed using the assay provided in the Examples.

As used herein the term "comprising" means "including at least in part of and is meant to be inclusive or open ended. When interpreting each statement in this specification that includes the term "comprising", features, elements and/ or steps other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner. R ¹ . R ² and R ³

For compound of formula (I)

(I)

R ² and R ³ above are drawn without specifying the location on the ring to which they are attached by having a bond going in to the middle of the ring. In one aspect, the compound of formula (I) is a compound of formula (IV) :

(IV)

and salts, solvates, tautomers and stereoisomers thereof.

In another aspect, the compound of formula (I) is a compound of formula (V):

(V)

and salts, solvates, tautomers and stereoisomers thereof.

In another aspect, the compound of formula (I) is a compound of formula (VI):

(VI)

and salts, solvates, tautomers and stereoisomers thereof. Suitably, R ¹ , R ² and R ³ are each independently selected from -H, -methyl, -ethyl, - propyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, - dichloromethyl, -trichloromethyl, -pentafluoroethyl, -difluoroethyl, -dichloroethyl, - C(0)NH ₂ , -C(0)NH(CH ₃ ), -C(0)NH(CH ₂ CH ₃ ), -C(0)N(CH ₃ ) ₂ , -C(0)N(CH ₂ CH ₃ ) ₂ , - C0 ₂ H, -C0 ₂ CH ₃ , -C0 ₂ CH ₂ CH ₃ , -C0 ₂ CH ₂ CH ₂ CH ₃ , -CN, -OH, -0-CH ₃ , -0-CH ₂ CH ₃ , -NH ₂ , -NH(CH ₃ ), -NH(CH ₂ CH ₃ ), -N(CH ₃ ) ₂ , -N(CH ₂ CH ₃ ) ₂ , -F, -CI, -Br and -I.

Suitably, R ¹ , R ² and R ³ are each independently selected from -H, -methyl, -ethyl, - fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, -dichloromethyl, - trichloromethyl, , -C(0)NH ₂ , -C(0)NH(CH ₃ ), -C(0)N(CH ₃ ) ₂ , -C0 ₂ H, -C0 ₂ CH ₃ , -CN, - OH, -0-CH ₃ , -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -F, -CI, -Br and -I.

Suitably, R ¹ , R ² and R ³ are each independently selected from -H, -methyl, -ethyl, - fluoromethyl, -difluoromethyl, -trifluoromethyl, -OH , -0-CH ₃ , -NH ₂ , -NH(CH ₃ ), -F and -CI.

Suitably, at least one of R ¹ , R ² and R ³ is -H. Suitably, at least two of R ¹ , R ² and R ³ are

L

Suitably, L is selected from -CH ₂ -, -CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ -, -CH ₂ CH(CH ₃ )-, - CH ₂ CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ -, -CH ₂ CH ₂ CH(CH ₃ )-, - CH ₂ CH ₂ CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ CH ₂ CH ₂ -, -CH ₂ CH(CH ₃ )CH ₂ CH ₂ -, - CH ₂ CH ₂ CH(CH ₃ )CH ₂ -, -CH ₂ CH ₂ CH ₂ CH(CH ₃ )-, -CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -, -S-, - S-CH ₂ -, - S-CH ₂ CH ₂ -, -S-CH(CH ₃ )CH ₂ -, -S-CH ₂ CH(CH ₃ )-, -S-CH ₂ CH ₂ CH ₂ -, -S- CH(CH ₃ )CH ₂ CH ₂ -, -S-CH ₂ CH(CH ₃ )CH ₂ -, -S-CH ₂ CH ₂ CH(CH ₃ )-, -S-CH ₂ CH ₂ CH ₂ CH ₂ - and phenylene; wherein the phenylene is optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and -halo. Suitably, L is selected from -CH ₂ -, - CH ₂ CH ₂ - ,- CH ₂ CH ₂ CH ₂ - , -CH ₂ CH ₂ CH ₂ CH ₂ -, - CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -, -S-,— S-CH ₂ -,— S-CH ₂ CH ₂ -,— S-CH ₂ CH ₂ CH ₂ -, -S- CH ₂ CH ₂ CH ₂ CH ₂ - and phenylene; wherein the phenylene is optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and - halo.

In one aspect, suitably L is selected from - CH ₂ -, -CH ₂ CH ₂ -, -CH(CH ₃ )CH ₂ - and - CH ₂ CH ₂ CH 2- . In a further aspect, suitably L is phenylene optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and -halo. Suitably, L is phenylene optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -propyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, -dichloromethyl, -trichloromethyl, -pentafluoroethyl, -difluoroethyl, - dichloroethyl, -F, -CI, -Br and -I.

Suitably, L is phenylene optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -fluoromethyl, -difluoromethyl, - trifluoromethyl, -chloromethyl, -dichloromethyl, -trichloromethyl, -F, -CI, -Br and -I

More suitably, in this further aspect, L is phenylene optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -fluoromethyl, - difluoromethyl, -trifluoromethyl, -F and -CI. In a further aspect, suitably L is-S-, - S-CH ₂ -, - S-CH ₂ CH ₂ -, - S-CH ₂ CH ₂ CH ₂ - and -S- CH ₂ CH ₂ CH ₂ CH 2- .

Yi is selected from - C(0)NH -, -NHC(O)- and -C(O)-. When Yi is -C(0)NH - it is attached to the adjacent L and Ar as -L-C(0)-NH-Ar-. Similarly, when Yi is - NHC(O)- it is attached to the adjacent L and Ar as -L-NH -C(0)-Ar-. Hence, Yi may be an amide group attached to the adjacent L and Ar groups in either direction .

Suitably, Yi is selected from - C(0)NH - and - NHC(O)- .

More suitably, Yi is - C(0)NH- . Ar

Suitably, Ar is selected from a heteroarylene with 5 or 6 ring members and 1 Or 2 heteroatoms ; and phenylene wherein the heteroarylene and the phenylene are optionally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and -halo

Suitably, Ar is selected from pyrrolylene, pyridinylene, furanylene, thiophenylene, oxazolylene, imidazolylene and phenylene; wherein these groups are option ally substituted with one to three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and-halo.

Suitably, Ar is selected from pyrrolylene, pyridinylene, imidazolylene and phenylene; wherein these groups are optionally substituted with one, two or three optional substituents selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl and-halo.

Suitably, the Ar group is optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -propyl, -fluoromethyl, -difluoromethyl, - trifluoromethyl, -chloromethyl, -dichloromethyl, -trichloromethyl, -pentafluoroethyl, - difluoroethyl, -dichloroethyl, -F, -CI, -Br and -I.

Suitably, the Ar group is optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -fluoromethyl, -difluoromethyl, - trifluoromethyl, -chloromethyl, -dichloromethyl, -trichloromethyl, -F, -CI, -Br and -I More suitably, the Ar group is optionally substituted with one, two or three optional substituents selected from -methyl, -ethyl, -fluoromethyl, -difluoromethyl, - trifluoromethyl, -F and - CI.

Yi

Y ₂ is selected from - C(0)NH-, -NHC(O)- and -C(O)-. When Y ₂ is - C(0)NH - it is attached to the adjacent Ar and bicyclic ring groups as - Ar-C(0)-NH-bicyclic ring. Similarly, when Y ₂ is - NHC(O)- it is attached to the adjacent Ar and bicyclic ring groups as -Ar-NH -C(0)-bicyclic ring. Hence, Y ₂ may be an amide group attached to the adjacent Ar and bicyclic ring groups in either direction .

Suitably, Y ₂ is selected from - C(0)NH- and -NHC(O)-. More suitably, Y ₂ is -C(0)NH-

Ar ²

Suitably, Ar ² is a 9- or 10 -membered heteroaryl group or a napthalyl group selected from benzofuranyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiofuranyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzimidazolyl, indazolyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, benzofurazanyl,

benzothiadiazolyl, benzotriazolyl, purinyl, pteridinyl and napthalyl optionally substituted with up to seven optional substituents independently selected from -Ci-Ce alkyl, -G-C ₆ haloalkyl, -OR ^d , -NR ^d R ^e and -halo.

In a further aspect, suitably Ar ² is a 9- or 10 -membered nitrogen -containing heteroaryl group or a napthalyl group optionally substituted with up to seven optional substituents independently selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl, -OR ^d , -NR ^d R ^e and -halo.

Suitably, Ar ² is a 9- or 10 -membered heteroaryl group or a napthalyl group selected from indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzimidazolyl, indazolyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl, purinyl, pteridinyl and napthalyl optionally substituted with up to seven optional substituents

independently selected from -Ci-Ce alkyl, -Ci-Ce haloalkyl, -OR ^d , -NR ^d R ^e and -halo.

Suitably, Ar ² is a 9- or 10 -membered heteroaryl group or a napthalyl group optionally substituted with 0 , 1, 2, 3 , 4, 5 , 6 or 7 optional substituents independently selected from -Ci-Ce alkyl, -G-C ₆ haloalkyl, -OR ^d , -NR ^d R ^e and -halo.

Suitably, ² is selected from groups (VII), (VIII) and (IX) :

(VII) (VIII) and (IX) wherein :

X ⁴ is selected from N and C-R ⁶ ;

X ^s is selected from N and C-R ⁷ ;

X ⁶ is selected from N and C-R ⁸ ; X ⁷ is selected from N and C-R ⁹ ;

X ⁸ is selected from N and C-R ¹⁰ ;

X ⁹ is N-R ^d ; and

one of X ¹⁰ and X ⁿ is N-R ^d ; whilst the other is selected from C-R ⁷ and N.

For each of groups (VII), (VIII) and (IX) both rings are aromatic.

In this specification a zig-zag line indicates the point of attachment of the shown group (e.g. the options for A ² shown for groups (VII), (VIII) and (IX) above) to the rest of the compound of formula (I).

Suitably ² is selected from groups (X), (XI) and (XII) :

(X), (XI) and (XII) wherein :

X ² is selected from N and C-R ⁴ ;

X ³ is selected from N and C-R ⁵ ;

X ⁴ is selected from N and C-R ⁶ ;

X ⁵ is selected from N and C-R ⁷ ;

X ⁶ is selected from N and C-R ⁸ ;

X ⁷ is selected from N and C-R ⁹ ;

X ⁸ is selected from N and C-R ¹⁰ ;

X ⁹ is N-R ^d ; and

one of X ¹⁰ and X ¹¹ is N-R ^d ; whilst the other of X ¹⁰ and X ¹¹ is selected from C-R ⁷ and N.

More suitably, Ar ² is a group (X).

More suitably, Ar ² is:

Xi

In one aspect, suitably X ² is N.

In a further aspect, suitably X ² is C-R ⁴ .

In one aspect, suitably X ³ is N.

In a further aspect, suitably X ³ is C-R ⁵ .

X

In one aspect, suitably X ⁴ is N.

In a further aspect, suitably X ⁴ is C-R ⁶ .

X!

In one aspect, suitably X ^s is N.

In a further aspect, suitably X ^s is C-R ⁷ .

X£

In one aspect, suitably X ⁶ is N.

In a further aspect, suitably X ⁶ is C-R ⁸ .

XI

In one aspect, suitably X ⁷ is N.

In a further aspect, suitably X ⁷ is C-R ⁹ . x_.

In one aspect, suitably X ⁸ is C-R ¹⁰ .

In a further aspect, more suitably X ⁸ is N.

XI

Suitably, X ⁹ is selected from N-H, N-CH ₃ and N-CH ₂ CH ₃ . X ¹⁰ and X"

Suitably, one X ¹⁰ and X" is selected from N-H, N-CH ₃ and N-CH ₂ CH ₃ ; whilst the other of X ¹⁰ and X» is selected from C-R ⁷ and N.

Combinations of X ² . X ³ . X ⁴ . X ^s . X ⁶ . X ⁷ and X ⁸

Suitably at least one of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ is N. Suitably at least X ⁸ is N.

Suitably at least two of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ are N. Suitably at least three of X ² , X ³ , X ⁴ , X ⁵ , X ⁶ , X ⁷ and X ⁸ are N.

In one embodiment, suitably, X ^s , X ⁶ and X ⁷ are N R ⁴ . R ^s . R ⁶ . R ⁷ . R ⁸ . R ⁹ and R ¹⁰

Suitably, R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently selected from -H , - methyl, -ethyl, -propyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, - chloromethyl, -dichloromethyl, -trichloromethyl, -pentafluoroethyl, -difluoroethyl, - dichloroethyl, -OH , -0-CH ₃ , -0-CH ₂ CH ₃ , -Nth, -NH(CH ₃ ), -NH(CH ₂ CH ₃ ), -N(CH ₃ ) ₂ , - N(CH ₂ CH ₃ ) ₂ , -F, -CI, -Br, and -I. Suitably, R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently selected from -H , - methyl, -ethyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, - dichloromethyl, -trichloromethyl, -OH, -0-CH ₃ , -NH ₂ , -NH(CH ₃ ), -N(CH ₃ ) ₂ , -F, -CI, - Br, and -I. Suitably, R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are each independently selected from -H , - methyl, -ethyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, -OH, -0-CH ₃ , -NH ₂ , - NH(CH ₃ ), -F and -CI.

Suitably, at least one of R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ is -H. Suitably, at least two of R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are -H. Suitably, at least three, at least four, at least five, at least six of R ⁴ , R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ and R ¹⁰ are -H.

R ^a . R ^b . R ^c . R ^d and R ^e

Suitably, R , R ^b , R ^c , R ^d and R ^e are each independently selected from -H, -methyl, -ethyl, -n-propyl, -i-propyl, -n-butyl, -s-butyl, -i-butyl and -t-butyl. Suitably, R , R ^b , R ^c , R ^d and R ^e are each independently selected from -H , -methyl, -ethyl, -n-propyl and -i-propyl.

More suitably, R , R ^b , R ^c , R ^d and R ^e are each independently selected from -H and - methyl,

Suitable Structures

Suitably, the compoun (III) :

and salts, solvates, tautomers and stereoisomers thereof, wherein :

R ⁿ , R ¹² and R ¹³ are independently selected from -H , -Ci-Ce alkyl, -Ci-Ce haloalkyl and - halo.

Suitably, the compoun (XIII)

(xiii)

and salts, solvates, tautomers and stereoisomers thereof, wherein :

R ⁿ , R ¹² and R ¹³ are independently selected from -H , -Ci-Ce alkyl, -Ci-Ce haloalkyl and - halo.

Suitably, the compound of formula (I), is a compound is selected from :

(14)

and salts, solvates, tautomers and stereoisomers thereof. R". R ¹² and R ¹³

Suitably, R ⁿ , R ¹² and R ¹³ are each independently selected from -H, -methyl, -ethyl, - propyl, -fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, - dichloromethyl, -trichloromethyl, -pentafluoroethyl, -difluoroethyl, -dichloroethyl, -F, - CI, -Br and -I.

Suitably, R ⁿ , R ¹² and R ¹³ are each independently selected from -H, -methyl, -ethyl, - fluoromethyl, -difluoromethyl, -trifluoromethyl, -chloromethyl, -dichloromethyl, - trichloromethyl, -F, -CI, -Br and -I

Suitably, R ⁿ , R ¹² and R ¹³ are each independently selected from -H, -methyl, -ethyl, - fluoromethyl, -difluoromethyl, -trifluoromethyl, -F and -CI.

Applications

The invention finds application in the treatment of diseases or conditions selected from proliferative diseases, diabetes and fibrosis.

The term "proliferative disease" refers 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. lung cancer, small cell lung cancer, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, bowel cancer, colon cancer, hepatoma, breast cancer, glioblastoma, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, liver cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, head and neck cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma), leukaemias, psoriasis, bone diseases, fibroproliferative disorders (e.g. of connective tissues), and atherosclerosis. Cancers of particular interest include, but are not limited to, breast cancer (both ER positive and ER negative), pancreatic cancer, lung cancer and leukaemia.

Compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, are useful for treating proliferative diseases in any type of cell, including but not limited to, the head, neck, eye, mouth, throat, oesophagus, bronchus, larynx, pharynx, chest, bone, lung, bowel, colon, rectum, stomach, prostate, urinary bladder, uterine, cervix, breast, ovaries, testicles or other reproductive organs, skin, thyroid, blood, lymph nodes, kidney, liver, pancreas, brain, central nervous system, solid tumours and blood-borne tumours.

Suitably, compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, are used in the treatment of a proliferative disease selected from bladder cancer, bone cancer, bowel cancer, brain cancer, breast cancer, cervical cancer, colon cancer, gastric cancer, head and neck cancer, leukaemia, liver cancer, lung cancer, lymphoma, melanoma, oesophageal cancer, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, renal cancer, retinoblastoma, sarcoma, skin cancer, testicular cancer, thyroid cancer and uterine cancer.

Suitably, compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, are useful in the treatment of a proliferative disease wherein the proliferative disease is a diabetes-related cancer. Suitable diabetes-related cancers include colon cancer, gastric cancer, prostate cancer and rectal cancer.

More suitably, compounds of formula (I) and salts, solvates, tautomers and

stereoisomers thereof, are useful in the treatment of a proliferative disease wherein the proliferative disease is colon cancer.

In one aspect, more suitably the disease is prostate cancer. Suitably, the prostate cancer is androgen-independent prostate cancer.

A skilled person is readily able to determine whether or not a candidate compound treats a proliferative condition for any particular cell type. In some aspects, compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, are used in the treatment of diabetes; in particular, in the treatment of type II diabetes. In some aspects, compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, are used in the treatment of fibrosis.

Suitably subjects are human, livestock animals and companion animals. The compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, may be used for the isolation and investigation of the activity or expression of TNKSs. In addition, they are suitable for use in diagnostic methods for diseases in connection with TNKSs activity, in particular, diseases in connection with unregulated or disturbed TNKSs activity.

The compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, may be used as molecular probes; in particular, as molecular probes for disease or conditions selected from a proliferative disease (such as cancer), diabetes and fibrosis. Compounds of formula (I) and salts, solvates, tautomers and stereoisomers thereof, have been found to be highly potent and specific TNKS inhibitors, and they can effectively inhibit Wnt signalling, increase glucose uptake into adipocytes and reduce colon cancer growth in vitro using functional cellular assays. Administration & Dose

Compounds of formula (I) may be administered alone or in combination with one or another or with one or more pharmacologically active compounds which are different from the compounds of formula (I). Suitably, the compounds of formula (I) may be administered simultaneously, sequentially or in alternation with administration of at least one or more pharmacologically active compounds. Suitably the one or more pharmacologically active compounds are anticancer agents.

Compounds of the invention may suitably be combined with various components to produce compositions of the invention. Suitably the compositions are combined with a pharmaceutically acceptable carrier or diluent to produce a pharmaceutical

composition (which may be for human or animal use). Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Useful pharmaceutical compositions and methods for their preparation may be found in standard pharmaceutical texts. See, for example, Handbook for Pharm aceutical Additives, 3rd Edition (eds. M. Ash and I. Ash), 2007 (Synapse Information Resources, Inc., Endicott, New York, USA) and Rem ington: The Science and Practice of

Pharm acy , 21st Edition (ed. D. B. Troy) 2006 (Lippincott, Williams and Wilkins, Philadelphia, USA) which are incorporated herein by reference.

The compounds of the invention may be administered by any suitable route. Suitably the compounds of the invention will normally be administered orally or by any parenteral route, in the form of pharmaceutical preparations comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form.

The compounds of the invention, their pharmaceutically acceptable salts, and pharmaceutically acceptable solvates of either entity can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. For example, the compounds of the invention or and salts, solvates, tautomers and stereoisomers thereof, can be administered orally, buccally or sublingually in the form of tablets, capsules (including soft gel capsules), ovules, elixirs, solutions or

suspensions, which may contain flavouring or colouring agents, for immediate-, delayed-, modified-, sustained-, controlled-release or pulsatile delivery applications. The compounds of the invention may also be administered via fast dispersing or fast dissolving dosages forms.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate,

croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/ or elixirs, the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/ or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and

combinations thereof.

Modified release and pulsatile release dosage forms may contain excipients such as those detailed for immediate release dosage forms together with additional excipients that act as release rate modifiers, these being coated on and/ or included in the body of the device. Release rate modifiers include, but are not exclusively limited to, hydroxypropylmethyl cellulose, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, Xanthan gum, Carbomer, ammonio methacrylate copolymer, hydrogenated castor oil, carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymer and mixtures thereof. Modified release and pulsatile release dosage forms may contain one or a combination of release rate modifying excipients. Release rate modifying excipients maybe present both within the dosage form i.e. within the matrix, and/ or on the dosage form i.e. upon the surface or coating. Fast dispersing or dissolving dosage formulations (FDDFs) may contain the following ingredients: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, gelatin,

hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint flavouring, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol.

The compounds of the invention can also be administered parenterally, for example, intravenously, intra-arterially, or they may be administered by infusion techniques. For such parenteral administration they are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Suitably formulation of the invention is optimised for the route of administration e.g. oral, intravenously, etc. Administration may be in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) during the course of treatment. Methods of determining the most effective means and dosage are well known to a skilled person and will vary with the formulation used for therapy, the purpose of the therapy, the target cell(s) being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and the dose regimen being selected by the treating physician, veterinarian, or clinician. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses. For example, a typical dosage for an adult human may be 100 ng to 25 mg (suitably about 1 micro g to about 10 mg) per kg body weight of the subject per day. Suitably guidance may be taken from studies in test animals when estimating an initial dose for human subjects. For example when a particular dose is identified for mice, suitably an initial test dose for humans may be approx. 0.5x to 2x the mg/ Kg value given to mice. Other Forms

Unless otherwise specified, included in the above are the well known ionic, salt, solvate, and protected forms of these substituents. For example, a reference to carboxylic acid (-COOH) also includes the anionic (carboxylate) form (-COO ), a salt or solvate thereof, as well as conventional protected forms. Similarly, a reference to an amino group includes the protonated form (-N ⁺ HR'R ² ), a salt or solvate of the amino group, for example, a hydrochloride salt, as well as conventional protected forms of an amino group. Similarly, a reference to a hydroxyl group also includes the anionic form (-0 ), a salt or solvate thereof, as well as conventional protected forms. Isomers, Salts and Solvates

Certain compounds may exist in one or more particular geometric, optical,

enantiomeric, diasteriomeric, epimeric, atropic, stereoisomeric, tautomeric, conformational, or anomeric forms, including but not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, and r- forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d- and 1- forms; (+) and (-) forms; keto-, enol-, and enolate-forms; syn- and anti-forms; synclinal- and anticlinal-forms; alpha- and beta-forms; axial and equatorial forms; boat-, chair-, twist-, envelope-, and halfchair-forms; and combinations thereof, hereinafter collectively referred to as "isomers" (or "isomeric forms").

Note that, except as discussed below for tautomeric forms, specifically excluded from the term "isomers", as used herein, are structural (or constitutional) isomers (i.e.

isomers which differ in the connections between atoms rather than merely by the position of atoms in space). For example, a reference to a methoxy group, -OCH ₃ , is not to be construed as a reference to its structural isomer, a hydroxymethyl group, - CH ₂ OH .

A reference to a class of structures may well include structurally isomeric forms falling within that class (e.g. C ₁ -7 alkyl includes n-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl; methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl). The above exclusion does not apply to tautomeric forms, for example, keto-, enol-, and enolate-forms, as in, for example, the following tautomeric pairs: keto/ enol, imine/ enamine, amide/ imino alcohol, amidine/ amidine, nitroso/ oxime,

thioketone/ enethiol, N-nitroso/hydroxyazo, and nitro/ aci-nitro. Note that specifically included in the term "isomer" are compounds with one or more isotopic substitutions. For example, H may be in any isotopic form, including Ή , ² H (D), and ³ H (T) ; C may be in any isotopic form, including ¹² C, ¹³ C, and ¹⁴ C; O may be in any isotopic form, including ¹ 0 and ¹⁸ 0; and the like. Unless otherwise specified, a reference to a particular compound includes all such isomeric forms, including (wholly or partially) racemic and other mixtures thereof.

Methods for the preparation (e.g. asymmetric synthesis) and separation (e.g. fractional crystallisation and chromatographic means) of such isomeric forms are either known in the art or are readily obtained by adapting the methods taught herein, or known methods, in a known manner.

Unless otherwise specified, a reference to a particular compound also includes ionic, salt, solvate, and protected forms of thereof, for example, as discussed below. Compounds of formula (I), which include compounds specifically named above, may form pharmaceutically acceptable complexes, salts, solvates and hydrates. These salts include nontoxic acid addition salts (including di-acids) and base salts. If the compound is cationic, or has a functional group which may be cationic (e.g. -NH ₂ may be -Nth ^"1" ), then an acid addition salt may be formed with a suitable anion.

Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids hydrochloric acid, nitric acid, nitrous acid, phosphoric acid, sulfuric acid, sulphurous acid, hydrobromic acid, hydroiodic acid, hydrofluoric acid, phosphoric acid and phosphorous acids. Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetyoxybenzoic, acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric, edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic, gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalene carboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic, methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic, phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic, succinic, sulfanilic, tartaric, toluenesulfonic, and valeric. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose. Such salts include acetate, adipate, aspartate, benzoate, besylate, bicarbonate, carbonate, bisulfate, sulfate, borate, camsylate, citrate, cyclamate, edisylate, esylate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hibenzate,

hydrochloride/ chloride, hydrobromide/ bromide, hydroiodide/ iodide, isethionate, lactate, malate, maleate, malonate, mesylate, methylsulfonate, naphthylate, 2- napsylate, nicotinate, nitrate, orotate, oxalate, palmitate, pamoate, phosphate, hydrogen phosphate, dihydrogen phosphate, pyroglutamate, saccharate, stearate, succinate, tannate, tartrate, tosylate, trifluoroacetate and xinofoate salts.

For example, if the compound is anionic, or has a functional group which may be anionic (e.g. -COOH may be -COO ), then a base salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, metal cations, such as an alkali or alkaline earth metal cation, ammonium and substituted ammonium cations, as well as amines. Examples of suitable metal cations include sodium (Na ⁺ ) potassium (K ⁺ ), magnesium (Mg ²⁺ ), calcium (Ca ²⁺ ), zinc (Zn ²⁺ ), and aluminum (Al ³⁺ ). Examples of suitable organic cations include, but are not limited to, ammonium ion (i.e. NH4 ⁺ ) and substituted ammonium ions (e.g. Nt R*, NH2 2 ⁺ , NHR ₃ ⁺ , NR ₄ ⁺ ). Examples of some suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine,

phenylbenzylamine, choline, meglumine, and tromethamine, as well as amino acids, such as lysine and arginine. An example of a common quaternary ammonium ion is Ν(ΟΪ3)4 ⁺ . Examples of suitable amines include arginine, Ν,Ν'- dibenzylethylenediamine, chloroprocaine, choline, diethylamine, diethanolamine, dicyclohexylamine, ethylenediamine, glycine, lysine, N-methylglucamine, olamine, 2- amino-2-hydroxymethyl-propane- l,3-diol, and procaine. For a discussion of useful acid addition and base salts, see S. M. Berge et al., /. Pharm . Sci. ( 1977) 66 : 1- 19 ; see also Stahl and Wermuth, Handbook of Pharm aceutical Salts: Properties, Selection, and Use (2011)

Pharmaceutically acceptable salts may be prepared using various methods. For example, one may react a compound of formula (I) with an appropriate acid or base to give the desired salt. One may also react a precursor of the compound of formula (I) with an acid or base to remove an acid- or base-labile protecting group or to open a lactone or lactam group of the precursor. Additionally, one may convert a salt of the compound of formula (I) to another salt through treatment with an appropriate acid or base or through contact with an ion exchange resin. Following reaction, one may then isolate the salt by filtration if it precipitates from solution, or by evaporation to recover the salt. The degree of ionization of the salt may vary from completely ionized to almost non-ionized.

It may be convenient or desirable to prepare, purify, and/ or handle a corresponding solvate of the active compound. The term "solvate" describes a molecular complex comprising the compound and one or more pharmaceutically acceptable solvent molecules (e.g., EtOH). The term "hydrate" is a solvate in which the solvent is water. Pharmaceutically acceptable solvates include those in which the solvent may be isotopically substituted (e.g., D ₂ O, acetone-d6, DMSO-d6).

A currently accepted classification system for solvates and hydrates of organic compounds is one that distinguishes between isolated site, channel, and metal-ion coordinated solvates and hydrates. See, e.g., K. R. Morris (H . G. Brittain ed.)

Polymorphism in Pharmaceutical Solids ( 1995). Isolated site solvates and hydrates are ones in which the solvent (e.g., water) molecules are isolated from direct contact with each other by intervening molecules of the organic compound. In channel solvates, the solvent molecules lie in lattice channels where they are next to other solvent molecules. In metal-ion coordinated solvates, the solvent molecules are bonded to the metal ion.

When the solvent or water is tightly bound, the complex will have a well-defined stoichiometry independent of humidity. When, however, the solvent or water is weakly bound, as in channel solvates and in hygroscopic compounds, the water or solvent content will depend on humidity and drying conditions. In such cases, non- stoichiometry will typically be observed. These compounds may be isolated in solid form, for example, by lyophilisation.

Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate, and in combinations other than those explicitly set out in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described further, with reference to the accompanying drawings, in which :

Figure 1 shows a model of docking of compound ( 13 ) into the structure of TNKS- 1. The protein is shown in pink and ( 13) in cyan. Key hydrogen bonds shown as gray dashed lines.

Figure 2 show an example image of DLD- 1 colony forming assay (1000 cells/ well) with Ο μΜ (control with 1% DMSO v/ v only), ΙμΜ and ΙΟΟηΜ of nicotinamide-site binder 1, adenosine-site binder 3 , compound ( 13) and (14 ) .

Figure 3 shows a graphical representation of colony forming assay with compounds 1, 3 , ( 13) and ( 14 ) (n = 3).

Figure 4 shows the effect of 1, ( 13) and ( 14 ) on percentage insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Basel levels (B) with or without compound contain no insulin (I). I (with and without compound) is administered at a final concentration of ΙΟΟηΜ. Results are mean and SEM relative to I only (n = 3 for 1 and n = 5 for ( 13) and ( 14 )).

Figure 5 shows IC5 ₀ graphs obtained from the Tankyrase-2 assay for compounds 1, 3 , (6 ) , ( 11) , ( 12) , ( 13 ) and ( 14 ) . In the graphs the x-axis shows the % inhibition and the y-axis shows the amount of the inhibitor compound (μΜ). Figure 6 shows IC5 ₀ graphs obtained from the Takyrase-1 assay for compounds 1, 3, (6), (11), (12), (13) and (14). In the graphs the x-axis shows the % inhibition and the y-axis shows the amount of the inhibitor compound (μπι).

Figure 7 shows IC5 ₀ graphs obtained from the PARP-1 assay for compounds 1, 3, (6), (11), (12), (13) and (14). In the graphs the x-axis shows the % inhibition and the y- axis shows the amount of the inhibitor compound (μπι).

Figure 8 shows IC5 ₀ graphs obtained from the PARP-2 assay for compounds 1, 3, (11), (13) and (14). In the graphs the x-axis shows the % inhibition and the y-axis shows the amount of the inhibitor compound (μπι).

Figure 9 shows Wnt signalling graphs obtained from the Wnt signalling cellular assay for compounds (13), (14) and 1.

Figure 10 shows exemplary images of prostate cancer PC3 cell colony forming assays for control (C) and for samples treated with XAV939 (1), IWR-1 (3), compound (13) and compound (14).

Figure 11 shows camera images using an inverted light microscope of colonies for control (C) and for samples treated with XAV939 (1), IWR-1 (3), compound (13) and compound (14).

Figure 12 shows a histogram of data from the colony forming assay (n=3) for control (C) and for samples treated with XAV939 (1), IWR-1 (3), compound (13) and compound (14).

EXAMPLES

General methods

Chemical reagents were purchased from Sigma, Aldrich, Fluka, Acros, Lancaster and Novabiochem. Anhydrous CH ₂ CI ₂ was obtained by distillation over calcium hydride, anhydrous THF was obtained by distillation over sodium / benzophenone. All other solvents were purchased from Fisher Scientific. Analytical TLC was performed using silica gel 60 F254 pre-coated on aluminium sheets (0.25 mm thickness). Column chromatography was performed on silica gel 60 (35-70 micron) from Fisher Scientific. Melting points were recorded on a Reichert-Jung Kofler block apparatus and are uncorrected. Ή and ¹³ C NMR were recorded using a Bruker Advance DPX 500 MHz and 400 MHz (Ή) instruments. High resolution mass spectra were determined using the electrospray ionization or electron impact techniques and were calibrated with sodium formate using a Bruker Daltonics MicroTOF instrument. The brine was saturated. Experiments were conducted at ambient temperature, unless otherwise stated. Solutions in organic solvents were dried with anhydrous MgSCu. Solvents were evaporated under reduced pressure. Molecular m odelling m eth od

Modelling was performed with the SYBYL software suite with UCSF Chimera for visualisation. Ligands were constructed and charged within SYBYL using the TNKS-2 structure 4I9I. ²³ The original ligand was removed from the 4i9i structure. Once docking experiments in SYBYL was complete using the surflex™ docking suite, the complete ligand-receptor complex was minimized in SYBYL (without restraints) to give the final structures. Modelling. Structural alignment of the co-crystal structures of 2 (PDB 4UFU) and 3 (PDB 3UA9) with TNKS2 ^{20 21} provided initial insights towards the design of 13 and 14. The quinazolinone of 2 binds into the nicotinamide-binding site, making hydrogen bonds with Ser ¹⁰⁶⁸ and Gly ¹⁰³² and π-stacks with Tyr ¹⁰⁷¹ (TNKS2 numbering). The quinolone of 3 is located in the adenosine-binding site, making a π-stack with His ¹⁰⁴⁸ (TNKS2 numbering). Features binding at each site were linked to create chimeric compounds 13 and 14 combining important hydrogen bonds and stacking interactions at both binding sites. Modelling of 13 and 14 into the active site of TNKS1 (PDB 4I9I) ²³ predicted that the designed compounds could bind to the pocket in the intended way and that the linker length was appropriate. The quinazolin-4-one core could bind to the nicotinamide-binding site to create conserved H-bond and π-stacking interactions. Compounds 1 and 2 contain a 2-aryl ring, which is shown to occupy a hydrophobic pocket. Modelling of 13 into TNKS1 suggests that the chosen linker could allow Tyr ¹²⁰³ to move towards the nicotinamide-binding site and decrease the size of the

hydrophobic pocket. However, this shrinkage of the pocket could still allow the propanamide linker to thread through. The modelling study reveals that 13 could interact further with TNKS1: The C=0 of the propanamide linker is located to H-bond with the backbone NH of Tyr ¹²¹³ ; the central phenyl ring and the quinoline of 13 are predicted to cause movement of His ¹²⁰¹ of the adenosine-binding site to locate its imidazole for ^-stacking with His ¹²⁰¹ . Modelling of 14 shows similar interactions with TNKS1.

Rea

(4 ) (6 )

= H (9) R = H (11) R= H

(13) R = H

(14) R= Me

Scheme 1. Synthesis of (13) and (14). Reagents and conditions: i) 4-nitrobenzoyl chloride, pyridine, THF, 16 h, Ar; ii) ⁺ NH ₄ HC0 ₂ , 10% Pd/C, DMF/MeOH (2:1), 2 h, Ar; iii) Et0 ₂ CCH ₂ CH ₂ COCl, pyridine, THF, 16 h; iv) aq. NaOH (0.5 M), 60°C; v) 6, DMF, Pr' ₂ NEt, carbonyldiimidazole, 72 h, Ar.

The synthesis of target compounds, shown in Reaction Scheme 1, began by acylation of 8-aminoquinoline (4) with 4-nitrobenzoyl chloride to give (5), followed by transfer hydrogenation to provide amine (6). The synthetic strategy employed involved acylation of anthranilamides (7) and (8) ²⁴ with ethyl 4-chloro-4-oxobutyrate to provide (9) and (10). One-pot cyclisation and saponification under basic aqueous conditions gave the quinazolinonepropanoic acids (11) and (12). Optimization of the coupling conditions enabled (11) / (12) to be joined with (6). Simultaneous addition of both the activating agent (CDI) and quinazolinones (11) / (12) to (6) provided the dual-binding TNKS inhibitors (13) and (14).

(5)

A solution of 8-aminoquinoline (4) (4.00 g, 27.7 mmol) in anhydrous THF (500 mL) was treated with pyridine (2.85 g, 36.1 mmol) and 4-nitrobenzoyl chloride (5.66 g, 30.5 mmol). The mixture was stirred under argon for 16 h. Evaporation and

chromatography (1:9 EtOAc / DCM) gave (5) (5.56 g, 68%) as a yellow solid: mp 175- 177°C; Ή NMR ((CD ₃ ) ₂ SO) δ 7.67-7.71 (2 H, m), 7.80 (lH,dd,/= 8.5, 1.5 Hz), 8.28 (2 H, dt, / = 8.5, 2.5 Hz), 8.44 (2 H, dt, 8.5, 2.5 Hz), 8.69 (1 H, dd, / = 7.5, 1.0 Hz), 8.99 (1 H, dd, / = 4.5, 1.5 Hz), 10.78 (1 H, s); ¹³ C NMR ((CD ₃ ) ₂ SO) δ 117.65, 122.44, 123.17, 124.09, 126.98, 127.92, 128.83, 133.73, 136.81, 138.62, 140.06, 149.37, 149.43, 163.19; MS (ES) m/z 294.0812 (M + H) ⁺ (C16H12N3O3 requires 294.0800).

(6)

A solution of 4-nitro-N-(quinolin-8-yl)-benzamide (5) (500 mg, 1.70 mmol) in anhydrous DMF/MeOH (2:1, 35 mL) was treated with ammonium formate (1.07 g, 17.0 mmol) and 10% palladium on charcoal (100 mg). The mixture was stirred for 2 h under Ar at room temperature. The reaction mixture was filtered through Celite and the solvents were evaporated to give (6) (330 mg, 82%) as a white solid: mp 169-173°C; Ή NMR ((CD ₃ ) ₂ SO) δ 5.92 (br, s) 5.92 (2 H, brs), 6.69 (1 H, dt, / = 9.5, 3.0 Hz), 7.62 (1 H, t, / = 8.5 Hz), 7.65-7.68 (2 H, m), 7.73 (2 H, dt, / = 9.5, 2.5 Hz), 8.44 (1 H, dd, / = 8.0, 2.0 Hz), 8.73 (1 H, dd, /= 7.5, 1.0 Hz), 8.96 (1 H, dd, / = 4.5, 1.5 Hz), 10.45 (1 H, brs); ¹³ CNMR((CD ₃ ) ₂ SO)5113.06, 115.63, 120.44, 121.26, 122.26, 127.16, 127.82, 128.71, 134.59, 136.76, 138.04, 148.96, 125.71, 164.32; MS (ES) m/z 264.1014 (M + H) ⁺ (Ci6Hi ₄ N ₃ 0requires 264.1059).

Example 3: 2-Amino-3-methylbenzamide (8)

(8)

2-Amino-3-methylbenzoic acid (2.93 g, 19.8 mmol) in dry DMF (78 mL) was treated with 1,1-carbonyl-diimidazole (3.14 g, 19.4 mmol) at 70°C under Ar for lh, after which aq. NH ₃ (35%, 49 mL) was added dropwise and the mixture was stirred for 16 h. The mixture was allowed to cool to 20°C and was diluted with EtOAc (100 mL). The mixture was washed with water (2 x 40 mL) and brine (2 x 40 mL). The organic solution was dried and the solvent was evaporated to give (8) (2.14 g, 98%) as a white solid: mp 150-152°C (lit. ²⁴ mp 150-152°C); Ή NMR ((CD ₃ ) ₂ SO) δ 2.05 (3 H, s,), 6.35 (2 H, br), 6.41 (1 H, brt, / = 7.6 Hz,), 7.00 (1 H, brs), 7.04 (1 H, d, / = 6.8 Hz), 7.34 (1 H, dd, / = 8.0, 0.8 Hz), 7.67 (1 H, brs); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 17.56, 113.59, 114.17, 122.99, 126.61, 132.67, 148.21, 171.73.

Example 4: 2-(4-Ethoxy-4-oxobutanamido) benzamide (9)

(9)

A solution of 2-amino benzamide (7) (200 mg, 1.47 mmol) in anhydrous THF (20mL) was treated with pyridine (151 mg, 1.91 mmol) and 4-ethoxy-4-oxobutanoyl chloride (266 mg, 1.62 mmol). The mixture was stirred for 16 h. Evaporation and

chromatography (EtOAc) gave (9) (358 mg, 92%) as a white solid: mp 96-100°C; Ή NMR ((CD ₃ ) ₂ SO) δ 1.16 (3 H, t, / = 7.5 Hz), 2.59-2.61 (4 H, m), 4.05 (2 H, q, / = 7.0 Hz), 7.09-7.12 (1 H, m), 7.45-7.49 (1 H, m), 7.74 (1 H, brs), 7.79 (2 H, dd, / = 8.0, 1.5 Hz), 8.27 (1 H, brs), 8.42 (1 H, d, / = 8.0 Hz); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 14.09, 28.68, 31.94, 59.97, 119.46, 120.05, 122.29, 128.59, 132.23, 139.62, 169.73, 170.77, 172.18; MS (ES) m/z 265.1154 (M + H) ⁺ (G ₃ Hi ₇ N ₂ 0 ₄ requires 265.1110).

Example 5: 2-(4-Ethoxy-4-oxobutanamido)-3-methyl benzamide (10)

(10)

A solution of 2-amino-3-methyl benzamide (8) (200 mg, 1.33 mmol) in anhydrous THF (20mL) was treated with pyridine (136 mg, 1.73 mmol) and 4-ethoxy-4- oxobutanoyl chloride (240 mg, 1.46 mmol). The mixture was stirred for 16 h.

Evaporation and chromatography (EtOAc) gave (10) (342 mg, 92%) as a white solid: mp 181-183°C; Ή NMR ((CD ₃ ) ₂ SO) δ 1.17 (3 H, t, / = 7.0 Hz), 2.14 (3 H, s), 2.56 (4 H, m), 4.05 (2 H, q, / = 7.0 Hz), 7.09 (1 H, t, / = 8.0 Hz), 7.30-7.34 (3 H, m), 7.52 (1 H, brs), 9.53 (1 H, s); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 14.13, 18.17, 29.02, 30.29, 59.92, 125.74, 125.85, 131.81, 133.61, 133.91, 135.87, 169.48, 169.89, 172.26; MS (ES) m/z 279.1261 (M + H) ⁺ (Ci ₄ Hi ₉ N ₂ 0 ₄ requires 279.1267). Example 6: 3-(4-Oxoquinazolin-2-yl)propanoic acid (11)

(11)

Asuspension of 2-(4-ethoxy-4-oxobutanamido)benzamide (9) (2.20 g, 9.31 mmol) was stirred in aq. NaOH (0.5 M, 600 mL) at 60°C for 3 h. The mixture was acidified by addition of aq. HQ (12 M) to pH 2. The precipitate was collected by filtration, washed with water and dried to give (11) (1.29 g, 63%) as a white solid: mp 239-242°C; Ή NMR ((CD ₃ ) ₂ SO) δ 2.75 (2 H, t, / = 7.0 Hz), 2.85 (2 H, t, / = 6.5 Hz), 7.45 (1 H, t, / = 7.0 Hz), 7.56 (1 H, d, / = 8.0 Hz), 7.76 (1 H, td, / = 8.5, 1.5 Hz), 8.07 (1 H, dd, / = 7.5, 1.0 Hz), 12.22 (1 H, s); ¹³ C NMR ((CD ₃ ) ₂ SO) δ 29.09, 29.81, 120.91, 125.75, 126.05, 126.85, 134.34, 148.71, 156.29, 161.67, 173.61; MS (ES) m/z 217.0685 (M - H)

(C ₁₁ H ₉ N ₂ O ₃ requires 217.0691).

Example 7: 3-(8-Methyl-4-oxoquinazolin-2-yl)propanoic acid (12) (12)

A suspension of 2-(4-ethoxy-4-oxobutanamido)-3-methylbenzamide (10 ) (1.42 g, 5.1 mmol) was stirred in aq. NaOH (0.5 M, 600 mL) at 60°C for 3 h. The mixture was acidified by addition of aq. HQ (12M) to pH 2. The precipitate was collected by filtration, washed with water and dried to give (12) (1.10 g, 95%) as a white solid: mp 235-237°C; Ή NMR ((CD ₃ ) ₂ SO) δ 2.76 (2 H, t, / = 7.0 Hz), 2.87 (2 H, t, / = 6.5 Hz),

7.34 (1 H, t, / = 8.0 Hz), 7.63 (1 H, d, / = 7.0 Hz), 7.91 (1 H, t, / = 7.0 Hz), 12.17 (1 H, s), 12.21 (lH,s); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 16.94, 29.01, 29.62, 120.69, 123.29, 125.41, 134.57, 134.81, 146.96, 154.91, 161.88, 173.68; MS (ES) m/z 231.0852 (M - H) (G2H11N2O3 requires 231.0848).

Example 8 : 2-(3-Oxo-3-(4-((quinolin-8-yl)aminocarbonyl)phenylamino- propyl)quinazolin-4-o (13)

(13) Asolution of 4-amino-N-(quinolin-8-yl)-benzamide (6) (548 mg, 2.08 mmol) in anhydrous DMF (50 mL) was treated with diisopropylethylamine (2.96 g, 22.9 mmol) then carbonyldiimidazole (371 mg, 2.29 mmol) followed by addition of 3-(4- oxoquinazolin-2-yl)propanoic acid (11) (500 mg, 2.29 mmol). The mixture was stirred for 72 h under Ar. The reaction was evaporated and the resulting residue was dissolved in EtOAc/ MeoH (1:2, 70 mL). The organic solvent was washed with water (3 x 30 mL) and brine (3 x 30 mL) then dried. Evaporation and chromatography (1:9 EtOAc / DCM → 1:9 MeOH / EtOAc) gave a mixture containing (13). Further chromatography (EtOAc) gave (13) (74 mg, 8%) as an off-white solid: mp 265-268°C; Ή NMR

((CD ₃ ) ₂ SO) δ 2.95-2.98 (4 H, m), 7.47 (1 H, t, / = 7.0 Hz), 7.63 (1 H, d, / = 8.0 Hz), 7.64-7.69 (2 H, m), 7.72-7.83 (2 H, m), 7.82 (2 H, d, / = 8.5 Hz), 8.00 (2 H, d, / = 8.5 Hz), 8.08 (1H, d,J= 8.0 Hz), 8.46 (lH,dd,/= 8.0, 1.5 Hz), 8.72 (lH,d,/= 7.5 Hz), 8.97(lH,dd,/= 4.0, 1.5 Hz), 10.45 (lH,s), 10.59 (lH,s), 12.27 (lH,s); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 29.09, 32.34, 116.44, 118.70, 120.93, 122.13, 122.38, 125.77, 126.04, 126.76, 127.11, 127.86, 128.12, 128.44, 134.16, 134.35, 136.81, 138.26, 142.76, 148.74, 149.18, 156.52, 161.64, 163.96, 170.73; MS (ES) m/z 464.1722 (M + H) ⁺ (C27H22N5O3 requires 464.1723).

Exam le 9 : 8-Methyl-2-(3-oxo-3-(4-((quinolin-8-yl)aminocarbonyl)phenyl- amino ro yl)quinazolin-4-one (14)

(14)

Asolution of 4-amino-N-(quinolin-8-yl)-benzamide (6) (514 mg, 1.95 mmol) in anhydrous DMF (50 mL) was treated with diisopropylethylamine (2.78 g, 21.5 mmol) then carbonyldiimidazole (348 mg, 2.15 mmol) followed by addition of 3-(8-methyl-4- oxoquinazolin-2-yl)propanoic acid (12) (500 mg, 2.15 mmol). The mixture was stirred for 72 h under Ar. The reaction was evaporated and the resulting residue was dissolved in EtOAc/ MeoH (1:2, 70 mL). The organic solvent was washed with water (3 x 30 mL) and brine (3 x 30 mL) then dried. Evaporation and chromatography (1:9 EtOAc / DCM → EtOAc) gave (14) (119 mg, 21%) as a beige solid: mp 280-282°C; Ή NMR

((CD ₃ ) ₂ SO) δ 2.44 (3 H, s), 2.93 (2 H, t, / = 6.0 Hz), 3.01 (2 H, t, / = 7.0 Hz), 7.31 (1 H, t, / = 7.5 Hz), 7.58 (1 H, m), 7.63-7.69 (2 H, m), 7.70 (1 H, dd, / = 8.5, 1.0 Hz), 7.83 (2 H,d,/= 8.5 Hz), 7.91(lH,dd,/= 8.0, 1.0 Hz), 8.00 (2 H, d,J= 8.5 Hz), 8.46 (lH,dd, /= 8.5,2.0 Hz), 8.72 (1H, dd,/= 7.5, 1.5 Hz), 8.97 (1 H, dd, /= 4.5, 2.0 Hz), 10.76 (1 H, s), 10.59 (1H, s), 12.25 (1 H, s); ¹³ CNMR ((CD ₃ ) ₂ SO) δ 17.06, 28.99, 32.08, 116.45, 118.67, 120.76, 122.11, 122.38, 123.36, 125.44, 127.11, 127.86, 128.09, 128.34, 134.17, 134.59, 134.84, 136.81, 138.26, 142.91, 147.07, 149.17, 155.12, 161.94, 163.98, 170.98; MS (ES) m/z 478.1876 (M + H) ⁺ (C28H24N5O3 requires 478.1879).

Example 10 : Tankyrase-2 assay experimental

As suspension of tankyrase protein (7.5ng, BPS Bioscience and AMS Bio Europe Ltd. Catalogue # 80515) in buffer solution (25 μΕ consisting of 50mM TRIS-HClpH 8.0, 5mM MgCl ₂ , 20μΜ ZnCl ₂ ) was loaded into ELISA quality, half- volume, high binding 96 well plates (greiner bio-one) and stored at 4 °C for 16h. The wells were then washed with phosphate buffer saline containing 0.05% tween 20 (v/ v) (PBS-T) (Aldrich), (4 x -250 μί) and treated with 100 μΕ skimmed milk power (marvel) suspended in buffer solution (5% w/v) and left at room temperature for 1 h. The wells were then washed with PBS-T (4 x -250 μΕ) and treated with buffer (15μΕ), varying concentrations of inhibitor containing a final concentration of 1% DMSO (5μΕ) and 5μΜ (5μΕ) 1:1 mixture of biotinylated (BioLog life science institute) and cold NAD ⁺ (Enzo life sciences). The reaction was incubated for 2 h at 30 °C. The wells were then washed with PBS-T (4 x -250 μν> and treated with 100 μΕ HRP/ Strep solution (R&D systems) for 2 h at room temperature. The wells were then washed with PBS-T (4 x -250 μΕ) and treated with a 1:1 mixture of substrate solution A and B (ΙΟΟμΕ, R&D systems) for 30 minutes at room temperature and then quenched with 2M H ₂ SO ₄ . The plate was read immediately at 450 nm. The determination of IC5 ₀ data was calculated using a four parameter logistic curve and SigmaPlot 12.0 software. IC5 ₀ graphs obtained for compounds 1, 3, (6), (11), (12), (13) and (14) from this assay are shown in Figure 5.

Example 11: Tankyrase-1 assay experimental

Tankyrase- 1 assays were performed using a commercial kit (Amsbio Europe Ltd.

Catalogue # 4700-096-K) using pre-coated histone well plates. A solution of 20X 1- PAR assay buffer (catalogue # 4684-096-07) was diluted to IX (1:20) with dH ₂ 0. The IX 1-PAR assay buffer solution (was used to rehydrate the histone-coated wells (50 μL· per well at room temperature for 30 min.). The lX-PAR assay buffer was removed by aspiration. A suspension of Tankyrase-1 protein was prepared (5 mU in 25 μL· IX 1-PAR assay buffer solution) and added to appropriate wells (25 μΚ) containing the appropriate volume of IX 1-PAR assay buffer solution. The addition inhibitor containing a final concentration of 1% DMSO (5 μί) prepared in IX 1-PAR assay buffer solution was followed by the addition of assay substrate (15 μL· catalogue # 4700-096- 02). Background wells were treated with IX 1-PAR assay buffer solution (50 μί). Maximum enzyme activity was established using wells containing enzyme only + 1% DMSO. The PARsylating reaction was left at room temperature for 30 min. The wells were washed with 2 x PBS-T and 2 x PBS. A solution of anti-PAR monoclonal antibody was prepared by dilution of 5X antibody diluent (catalogue # 4684-096-03) to IX ( 1:5) using dt O. The anti-PAR monoclonal antibody (catalogue # 4684-096-04) was diluted 1000 fold with IX antibody diluent and 50 μΐ. was used per well. The reaction was left at room temperature for 30 min. The wells were washed with 2 x PBS-T and 2 x PBS. A solution of goat anti-mouse IgG-HRP congugate was prepared by dilution of 5X antibody diluent (catalogue # 4684-096-03) to IX ( 1:5) using dH ₂ 0. The goat anti- mouse IgG-HRP conjugate (catalogue # 4684-096-05) was diluted 1000 fold with IX antibody diluent and 50 μΐ. was used per well. The reaction was left at room

temperature for 30 min. The wells were washed with 2 x PBS-T and 2 x PBS. Pre- warmed TACS-Sapphire™ (50 μΚ) was added per well and left at room temperature in the dark. The reaction was quenched by the addition of 0.2M HQ (50 μΚ) and the absorbance recorded immediately at 450 nm. The determination of IC5 ₀ data was calculated using a four parameter logistic curve and SigmaPlot 12.0 software. IC5 ₀ graphs obtained for compounds 1, 3 , (6 ) , ( 11) , ( 12) , ( 13) and ( 14 ) from this assay are shown in Figure 6. Exam ple 12 : PARP-1 assay e xperim ental

PARP- 1 assays were performed using a commercial kit (Amsbio Europe Ltd. Catalogue # 4676-096-K) using pre-coated histone well plates. A solution of 20X PARP assay buffer (catalogue # 4671-096-02) was diluted to IX (1:20) with dH ₂ 0. The IX PARP assay buffer solution was used to rehydrate the histone-coated wells (50 μL· per well) at room temperature for 30 min. The IX PARP assay buffer was removed by aspiration. A suspension of PARP- 1 protein was prepared (0.5 mU in 25 μL· IX PARP assay buffer solution) and added to appropriate wells (25 μΐ.) containing the appropriate volume of IX PARP assay buffer solution. Varying concentrations of inhibitor containing a final concentration of 0.1% DMSO were prepared in IX PARP assay buffer solution and added to appropriate wells (5 μ¾. A solution of substrate consisting of 10X PARP cocktail (2.5 μΐ. per well, catalogue # 4671-096-03), 10X activated DNA (2.5 μΐ. per well, catalogue # 4671-096-06) and IX PARP assay buffer ( 15 μL· per well) was added to appropriate wells (20 μΙ, ρεΓ well). The reaction was left at room temperature for 1 hour. The wells were washed with 2 x PBS-T and 2 x PBS. A solution of Strep-HRP (catalogue # 4800-30-06) was diluted 500 fold with IX strep-HRP diluent (catalogue # 4671-096-04) and added to each well (50 μΚ) and left for lhour at room temperature. The wells were washed with 2 x PBS-T and 2 x PBS. Pre-warmed TACS-Sapphire™ (50 μν> was added per well and left at room temperature in the dark. The reaction was quenched by the addition of 0.2M HQ (50 μί) and the absorbance recorded immediately at 450 nm. The determination of IC5 ₀ data was calculated using a four parameter logistic curve and SigmaPlot 12.0 software. IC5 ₀ graphs obtained for compounds 1, 3 , (6 ) , ( 11) , ( 12) , ( 13) and ( 14 ) from this assay are shown in Figure 7.

Exam ple 13 : PARP-2 assay expe rim e ntal

PARP- 1 assays were performed using a commercial kit (Amsbio Europe Ltd. Catalogue # 80552). A solution of 5X histone mixture solution (catalogue # 52029) was diluted to IX (1:5) with PBS. Each well was treated with histone solution (50 μΕ per well) at 4°C for 16 hours. The wells were washed with 3 x PBS-T (200 μΕ per well). The blocking buffer solution provided was added to the wells (200 μΕ per well) and the plate was incubated at room temperature for 90 minutes. The wells were washed with 3 x PBS-T (200 μΕ per well). A solution of 10X PARP assay buffer (catalogue # 80602) was diluted to IX (1: 10) with dF O. Varying concentrations of inhibitor containing a final concentration of 0.1% DMSO were prepared in IX PARP assay buffer solution and added to appropriate wells (5 μΕ). Substrate solution mixture for the ribosylation reaction was prepared by preparing a solution of 10X PARP assay buffer (2.5 μΕ per well), 10X PARP assay mixture (2.5 μΕ per well, catalogue # 40305), Activated DNA (5.0 μΕ ρεΓ well, catalogue # 80605) and dt O (15 μΕ ρεΓ well). The substrate solution was added to all wells (25 μΕ ρεΓ well). PARP-2 enzyme solution (catalogue # 80502) was diluted with IX PARP assay buffer to 2.0 ng/ mL final concentration. The diluted PARP-2 solution was added to appropriate wells (20 μΕ per well). The reaction was left at room temperature for 1 hour. The wells were washed with 3 x PBS-T (200 μΕ per well). A solution of Strep-HRP (catalogue # 130708) was diluted 50 fold with blocking buffer and added to each well (50 μΕ) and left for 30 minutes at room temperature. The wells were washed with 3 x PBS-T (200 μΕ per well). The wells were treated with a 1: 1 mixture of HRP chemiluminescent solution A and HRP chemiluminescent solution B ( 100 μΕ ρεΓ well, catalogue # 140613) and the luminescence was recorded immediately using a chemiluminescence plate reader. The determination of IC5 ₀ data was calculated using a four parameter logistic curve and SigmaPlot 12.0 software. IC5 ₀ graphs obtained for compounds 1, 3 , ( 11) , ( 12) , ( 13) and ( 14 ) from this assay are shown in Figure 8. Exam ple 14 : Wnt signalling cellular assay experim ental

The Wnt Signalling Pathway TCF/ LEF Reporter-HEK293 Cell Line (rHEK293) containing stably integrated Wnt reporter assay was bought from BPS Bioscience, catalog #60501. rHEK293 cells were grown at 37° C with 5% CO ₂ using Dulbecco's Modified Eagle's Medium (DMEM, Sigma), supplemented with 10 % of fetal bovine serum (Lonza), 1% non-essential amino acid (Sigma), IX of penicillin and streptomycin (Sigma), and 400 μg/ ml of Geneticin (Roche). Wnt-3a and L cells (ATCC) were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % fetal bovine serum, penicillin and streptomycin at 37° C in a 5% CO ₂ atmosphere. Wnt3a and control conditioned medium were prepared as described previously. ³¹

For Wnt reporter assay, 30 000 rHEK293 cells/ well in 40μ1 of serum-free DMEM were plated on 96 well plate (white wells, clear bottom, Perkin Elmer). 10 μΐ of 50 mM LiCl in serum-free DMEM with 5x concentration of tankyrase inhibitors was added and cells were incubated for 16 hours. Next, 25μ1 of Wnt3a or control conditioned medium supplemented with DMSO or tankyrase inhibitors was added on cells and cells were incubated for 20 hours. After the second incubation, the plates were allowed to settle in room temperature for 30 minutes before luciferase measurements. Wnt3a induced luciferase activity was determined using ONE-Glo Luciferase Assay System (Promega) according to manufacturer's protocol. Wnt signalling graphs obtained from the Wnt signalling cellular assay for compounds ( 13) , ( 14 ) and 1 are shown in Figure 9. Inhibition of Wnt signalling in TCF/ LEF reporter-HEK293 cells

A TCF/ LEF Reporter-HEK293 Cell line (BPS Bioscience, catalog #60501) was utilized to evaluate the cellular potency of the compounds. ²⁷ Upon extracellular Wnt signals, β- catenin is stabilized and associates with TCF/ LEF transcription factors activating transcription. ^{6 27} Tankyrases control the stability of the β-catenin destruction complex and TNKS inhibition is expected to stabilize the destruction complex and subsequently lower the levels of β-catenin. ⁶ The TCF/ LEF Reporter-HEK293 cell line is used to measure this interference with this pathway. Both ( 13) (cellular assay IC5 ₀ = 29 nM) and ( 14 ) (cellular assay IC5 ₀ = 37 nM) were extremely effective and potent inhibitors of the Wnt signalling axis compared to 1 (220nM), demonstrating their usefulness in a cellular context (Table 1). Cell viability was monitored with a light microscope and ( 13) and ( 14 ) did not display any cytotoxicity in TCF/ LEF Reporter-HEK293 assay.

Biochemical evaluation

Known compounds 1 and 3 were used as control inhibitors against TNKS1, TNKS2, PARPl and PARP2 to compare their potency and isoform-selectivity in comparison to the new dual-binding inhibitors ( 13 ) and ( 14 ) (see Table 1). Inhibitor compounds ( 13) and ( 14 ) were also tested against other PARPs (see Table 1). The nicotinamide-site binder XAV939 1 showed good potency and 25- and 4-fold selectivity comparing TNKS1 with PARPl and PARP2, respectively, and 131- and 22-fold selectivity for inhibition of TNKS2 vs. PARPl and PARP2, respectively. IWR- 1 3 , which binds to the adenosine-binding site, had moderate potency against the TNKS1 and TNKS2 (IC5 ₀ = 343 and 31 nM, respectively) and did not inhibit PARPl and PARP2 up to 10 μΜ. IWR- 1 3 has previously been shown to be a selective inhibitor of TNKSs (TNKS1 IC5 ₀ = 131 nM; TNKS2 IC ₅₀ = 56 nM), compared with PARPl and PARP2 (ICso > 18.7 μΜ). ⁶ Narwal et al. confirmed the selectivity of 3 with ICso = ca. 100 μΜ (PARPl) and ICso = ca. 35 μΜ (PARP2). ²⁰ Thus binding at the adenosine site has potential for greater selectivity for TNKSs vs. PARPl and PARP2. Intermediate (6 ) was evaluated to investigate its ability to bind to the adenosine-binding site in the absence of the anchoring quinazolinone but failed to inhibit any of the isoforms (see Table 2). From this observation, it is clear that the norbornene of 3 is essential, in that it interacts with Ty _r i203 _to modify the geometry of the adenosine-binding site to accept the

aminobenzoylaminoquinoline.

Compounds ( 11) and ( 12) both contain a propanoic acid at the 2-position of the quinazolinone scaffold and were used to explore the contribution of the quinazolinone- CH2CH2CO unit towards potency and selectivity; both showed no inhibition of TNKSs. It has previously been shown that a 2-aryl group (or equivalent) is required for potent inhibition of TNKSs; this reiterates the requirement for the 2-aryl unit when designing inhibitors of TNKSs that bind only to the nicotinamide-binding site. ^{16 18 21} However, ( 11) and ( 12) do inhibit PARPl and PARP2 (see Table 2). Previously, we showed that polar groups at the 4'-position of 2-arylquinazolin-4-ones decreased selectivity for TNKSs in that inhibition of PARPl was enhanced; ^{16 17 21} therefore, the 2-propanoic acid of ( 11) and ( 12) is well suited to interact with the corresponding region of protein of PARPl that contains hydrophilic residues. Furthermore, compounds ( 11) and ( 12) displayed moderately potent and selective inhibition of PARP2 (53- and 6-fold, respectively, vs. PARPl), results which place ( 11) amongst the most PARP2-selective agents known. ²⁵

The designed compounds ( 13) and ( 14) are extremely potent and selective inhibitors of TNKSs, in comparison with the nicotinamide mimic and lead inhibitor 1 and the adenosine-site-binding 3. Both inhibited TNKS2 in the pM range and TNKS1 in the low nM range. Interestingly, ( 13 ) and ( 14 ) have similar activities against the TNKSs, showing that the 8 -Me does not contribute significantly to binding, unlike in the less potent simple 2-arylquinazolin-4-ones. ¹ > ²¹ Remarkably, this increase in potency of inhibition of the TNKSs for the dual-site-binding ( 13) and ( 14 ) was not accompanied by an increase in activity against PARPl and PARP2, leading to exquisite selectivity. Inhibitor ( 13) is 9 x 10 ⁴ -fold selective for TNKS2 vs. PARPl and 6.9 x 10 ⁴ -fold selective for TNKS2 vs. PARP2. Similarly, ( 14 ) is 7 x 10 ⁴ -fold selective for TNKS2 vs. PARPl and 1.2 x 10 ⁵ -fold selective for TNKS2 vs. PARP2. The somewhat lesser potency of these agents in TNKSl corresponds to selectivities for TNKSl vs. PARPl and PARP2 in the range 1-2.3 x 10 ³ -fold.

Table 1. IC ₅₀ (pIC ₅ o ± standard error) values (n=3) for inhibition of TNKS l, TNKS2, and PARP isoforms in biochemical assa s and for inhibition of Wnt-si nallin in a cellular

'Limited by solubility. N.D = not determined , determined

Table 1 - IC ₅₀ values (nM) for inhibition of TNKSl, TNKS2, PARPl, PARP2, PARP3, PARP4, PARPIO , PARP 12, PARP 14, PARP 15 and PARP 16 in biochemical assays and inhibition of Wnt signalling in a cellular assay.

Not determined ^b Limited by solubility

Table 2 - IC ₅₀ values (nM) for inhibition of TNKSl, TNKS2, PARPl and PARP2 biochemical assays and for inhibition of Wnt signalling in a cellular assay. Exam ple 15 : Colony form ation cell assay experim ental ⁶

DLD-1 cells were seeded in DMEM medium ( lx catalogue # 41966-029 ; gibco life technologies) supplemented with 10 % FBS. Plates contained 1000 cells per well and incubated at 37°C in 5% CO ₂ for 16 hr after which, 2 mL of tankyrase inhibitors (2 x final concentration) was added to the appropriate wells containing 2 mL of media to give the final concentration of inhibitor in each well (4mL total volume) and a final DMSO concentration of 1% (v/ v). Media was replenished every 72 h until colony formation in the inhibitor treated wells was observed (18 days). Cells were viewed using a Moti™ AE 2000 light microscope equipped with Moticam™5.0 Mega Pixel camera and a computer monitor for visualisation. Colonies contained a minimum of 50 cells. After colony formation, media was removed and washed with lx PBS solution (2 mL). Cells were fixed using 70 % EtOH and water solution (2 mL) for 5 minutes at room temperature. The fixing solution was removed and the cells were stained with a solution Trypan Blue ( 1 mL; 0.4% v/ v; Sigma Aldrich catalogue # T8 154) at room temperature for 15 minutes. The stain was removed and cells were washed gently with PBS (lx) solution. Colonies were visually counted using a Moti™ AE 2000 light microscope equipped with Moticam™5.0 Mega Pixel camera.

Anti-proliferative activity towards DLD -1 hum an colon carcinom a ce lls Aberrant Wnt signalling is found in over 90 % of colorectal cancers, due to mutation in the adenomatous polyposis coli (APC) protein which is a component of the β-catenin destruction complex. ⁶ Compounds ( 13 ) and ( 14 ) were evaluated for anti-proliferative activity against DLD- 1 human colon cancer cells in a colony-forming assay under normal serum conditions as opposed to serum deprived conditions used in previous studies. ⁶ Both ( 13 ) and ( 14 ) significantly inhibited colony formation at 100 nM and 1.0 μΜ, compared to wells containing control inhibitors 1 and 3 and wells containing 1% DMSO (v/ v) only (Figure 3).

Exam ple 16 : Insulin -stim ulated glucose uptake

3T3-L1 fibroblasts were obtained from the American Type Culture Collection, cultured in DMEM and differentiated to adipocytes by treatment with insulin, dexamethasone and isobutylmethylxanthine, as described previously. ²⁸ On the day of the experiment, 10- 12 d post-differentiation, the cells were incubated with serum-free DMEM for 2 h at 37°C. Cells in the treatment group were treated with increasing concentrations of ( 13 ) or ( 14 ) for 1 h. At the end of the incubation period, the cells were washed three times with Krebs-Ringer-HEPES (KRH) buffer ( 140 mM NaCl, 4.7 mM KC1, 2.5 mM CaCl ₂ , 1.25 mM MgS0 ₄ , 2.5 mM NaH ₂ P0 ₄ , 10 mM HEPES, (pH 7.4)) and incubated for 30 min in the presence of ( 13) or ( 14 ) and in either the absence or presence of insulin (100 nM) at 37°C. After the 30 min incubation period, 2-deoxy-D-[2,6- ³ H]glucose (final concentration 50 μΜ, 0.1 μΟ/ \νε11) was added for 5 min and the cells were washed four times with ice-cold KRH buffer. Nonspecific uptake of 2-deoxy-D-glucose was measured in the presence of 10 μΜ cytochalasin B. The cells were lysed in aq.

NaOH (0.1 M) and radioactivity was counted in a TriCarb Packard scintillation counter (Perkin-Elmer). The concentrations of proteins were measured using BCA protein assay kit (Thermo Fisher Scientific). Results are expressed in nmol of 2-deoxy-D- glucose min ¹ (mg protein)

Insulin-stimulated glucose uptake

The axin-TNKS-KIF3A complex is stabilized through inhibition of TNKS. ¹² Ablation of expression of TNKSs has been reported to upregulate GLUT4 at the post- transcriptional level, potentially increasing uptake of glucose into adipocytes. ¹⁴ To address this pharmacologically, our highly potent and selective TNKS inhibitors were examined for their ability of insulin -stimulated uptake of glucose. Adipocyte cells, derived from 3T3-L1 fibroblasts, were treated with ( 13 ) and ( 14 ) in the presence of insulin (100 nM). ²⁸ XAV939 1 was used for comparison, as a standard but weaker and less selective inhibitor of TNKSs. The uptake of radiolabeled 2-deoxy-D-glucose into the cells was measured. In comparison to insulin only, 1 did not increase insulin- stimulated glucose uptake, even at 1.0 μΜ. In comparison to insulin only and 1, both ( 13 ) and ( 14 ) significantly increased insulin-stimulated glucose uptake. Glucose uptake increased by ca. 40 % and 20 % using 100 nM and 1.0 μΜ of ( 13) , respectively. Insulin-stimulated uptake of glucose increased by ca. 20 % using ( 14 ) at 10 nM, 100 nM and 1.0 μΜ in comparison to insulin only (Figure 4). These observations again confirm potent intracellular inhibition of TNKSs by ( 13) and ( 14 ) .

Exam ple 17: Prostate Cancer Cells

Metastatic androgen-independent prostate cancer PC3 cells (obtained from ATCC) were seeded at 200 cells/ well in 6-well plates and treated with a final concentration of ΙΟΟηΜ inhibitor (final DMSO concentration = 1% v/ v) or control (1% DMSO v/ v only). Inhibitor, control and media were replenished every 72 hours until colonies in the treated wells were observed (after 13 days). Colonies that contained >50 cells were scored as a colony. Cells were fixed with formaldehyde and stained with crystal violet. Images of each well were captured using a USB camera. Compound ( 13 ) and compound ( 14 ) formed significantly fewer colonies than cells treated with control ( C) , XAV939 ( 1) or IWR-1 (3) . Example images of PC3 colony forming assays for each of these five samples are shown in Figure 10. This novel data suggests potent and isoform-selective tankyrase inhibition, such as that achieved by using compounds ( 13 ) and ( 14 ) , antagonises colony formation of PC3 metastatic androgen-independent cells. Colonies were imaged using an inverted light microscope which was attached to a Moticam 5.0MP camera. In most cases, compound ( 13 ) and compound ( 14 ) gave fewer and smaller colonies compared to DMSO (as control 1% DMSO v/ v only) ( C) , XAV939 ( 1) or IWR- 1 (3 ) as shown Figure 11. Hence, the potent and isoform-selective tankyrase inhibitors, such as compound ( 13 ) and compound ( 14 ) , reduce the density and size of PC3 cell colonies.

Colonies were counted and those that were difficult to visualise by eye were viewed under an inverted light microscope under magnification to verify colony formation and colony density. Compounds ( 13) and ( 14 ) reduce colony formation compared to the commercial inhibitors XAV939 ( 1) or IWR-1 (3) as shown in Figure 12 a histogram of data from the colony forming assay (n=3). Summary of results (n=3) in histogram Figure 12 for the effect of TNKS/ Wnt inhibitors XAV939 ( 1) , IWR- 1 (3) , TA-91 ( 13 ) , and TA-92 ( 14 ) on anti-proliferative activity of PC3 androgen-independent PCa cells. Positive control (c) = cells treated with vehicle; 1% DMSO (v/ v). Results are the mean and standard error (n = 3 for all inhibitors). Statistical data and significance was derived using GraphPad Prism 6.0 in comparison to positive control, p-values were obtained using ordinary one-way AN OVA Dunett analysis. Control vs XAV939 = not significant (n.s.); Control vs IWR- 1 = n.s.; control vs TA-91 p = <0.001; control vs TA- 92 p = <0.0001.

Hence, potent and isoform-selective tankyrase inhibitors, such as compounds ( 13) and ( 14 ) , reduce colony survival of prostate cancer cells, such as androgen-independent prostate cancer cells. Conclusion.

The compounds were designed by molecular modelling so that the quinazolin-4-one moiety occupied the nicotinamide-binding site, setting up the linker so that the quinoline moiety interacts with the adenosine-binding region. This dual-site design has led to some of the most potent and isoform-selective inhibitors reported to date, with ICso = 100 pM for inhibition of TNKS2 by ( 13) and ( 14 ) and 1.2 x 10 ^s -fold selectivity for inhibition of TNKS2 vs. the major PARP isoform, PARPl. Cellular uptake of these agents was demonstrated by their potent inhibition of Wnt / β-catenin signalling in the low nM range. Significant anti-proliferative activity was demonstrated in DLD- 1 human colon carcinoma cells even in the presence of ΙΟΟηΜ inhibitor.

Moreover, for the first time, these potent and selective TNKS inhibitors have been used to effectively increase insulin -stimulated glucose uptake, whereas previous studies have used non-selective pan-PARP inhibitors for this application ; ¹³ this provides further evidence of a role of TNKs in insulin-stimulated glucose transport, without wishing to be bound by theory this role is thought to be through the axin-TNKS-KIF3A complex. ¹²

Compounds of the present invention, such as inhibitors ( 13 ) and ( 14 ) , are efficient molecular tools as Wnt antagonists and in relation to diabetes-related cancers. These results also indicate that potent and selective inhibition of TNKSs can increase the uptake of glucose in response to insulin ; high doses of insulin can cause dysregulation of various signalling cascades (PI3K / Akt / mTOR). ²⁹ Such compounds can be used for investigating the activity and/ or expression of TNKSs and are useful in diagnostic methods for diseases connected with TNKSs activity. The compounds may be used to treat diseases or conditions modulated by TNKSs such as cancer.

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All publications mentioned in the above specification are herein incorporated by reference. Although illustrative embodiments of the invention have been disclosed in detail herein, with reference to the accompanying drawings, it is understood that the invention is not limited to the precise embodiment and that various changes and modifications can be effected therein by one skilled in the art without departing from the scope of the invention as defined by the appended claims and their equivalents.