WO2018118868A1

WO2018118868A1 - Triazole derivatives as tankyrase inhibitors

Info

Publication number: WO2018118868A1
Application number: PCT/US2017/067228
Authority: WO
Inventors: Stefan Krauss; Marc Nazare; Upendra Rao ANUMALA; Lari LEHTIO; Jo Waaler; Dan Holsworth; Anita Wegert; Ruben Gerardus George Leenders
Original assignee: Forschungsverbund Berlin FVB eV; Oulu University of; Oslo Universitetssykehus hf
Current assignee: Forschungsverbund Berlin FVB eV; Oulu University of; Oslo Universitetssykehus hf
Priority date: 2016-12-19
Filing date: 2017-12-19
Publication date: 2018-06-28
Anticipated expiration: 2019-06-19
Also published as: RU2019121618A

Abstract

The present invention relates to compounds of formula (I'), tautomers, stereoisomers, and pharmaceutically acceptable salts thereof, to processes for their preparation, to pharmaceutical formulations containing such compounds and to their use in therapy (I') (wherein: Z represents an optionally substituted, 5- or 6-membered unsaturated heterocyclic group comprising at least one nitrogen atom; L represents a 4-, 5- or 6-membered cycloalkyl group, preferably a cyclobutyl group; each R1 independently represents F, CI, Br, I, C_1-3 alkyl, C1-3 haloalkyl (e.g. -CF₃), -CN, -OH or -NO2, preferably F, CI, Br or 1, e.g. CI or F; each R²independently represents F, CI, Br, I, C_1-3 alkyl, -CN, -OH or -NO₂, preferably F, CI, Br, I or -CN, e.g. F or -CN; X represents -NR³- or -0-; R³ represents H or a C_1-3 alkyl group (e.g. methyl); n is an integer from 0 to 5, preferably 0 to 3, more preferably 0, 1 or 2, e.g 1; and m is an integer from 0 to 5, preferably 0 to 3, more preferably 0, 1 or 2, e.g. 0 or 1). These compounds find particular use in the treatment and/or prevention of conditions or diseases which are affected by over-activation of signaling in the WNT pathway and increased presence of nuclear β-catenin. For example, these may be used in preventing and/or retarding proliferation of tumor cells and metastasis, for example carcinomas such as colon carcinomas.

Description

TRIAZOLE DERIVATIVES AS TANKYRASE INHIBITORS

The present invention relates to compounds, to pharmaceutical formulations containing such compounds and to their use in therapy, in particular as WNT signaling pathway inhibitors for reducing the proliferation of tumor cells and metastasis and causing an enhanced effect of immunotherapy. The invention further relates to processes for the preparation of such compounds and to intermediates formed during these processes.

The WNT family of glycoproteins control a variety of developmental processes including cell fate specification, proliferation, metabolism, migration and immune response. Consequently, the WNT pathway is instrumental in ensuring proper tissue development in embryos and tissue maintenance in adults. WNT signaling is altered in a variety of tumors including tumors emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, brain, breast and kidney. About 90%» of sporadic colon cancers show aberrant WNT signaling whereby mutations in the adenomatous polyposis coli gene (APC), β-catenin, or Axin genes lead to accumulation of nuclear β- catenin and hence an activation of the pathway.

Blocking canonical WNT activity in WNT deregulated cancers has been shown to cause cell cycle arrest in Gl , altered cellular energy metabolism and an altered differentiation status. Evidence also suggests an involvement of WNT/ -Catenin signaling in the interplay between cancer cells and the immune system.

Tankyrase 1 and 2 (TNKS 1 and TNKS2) (PARP-5a, PARP-5b) are members of the poly-ADP-ribose polymerase (PARP) family of enzymes. Tankyrase 1/2 has been identified as a positive regulator of the WN T signaling pathway via its interaction with AXIN protein. The inhibition of tankyrase 1/2 produces elevated AXIN protein levels and reduced levels of cellular β-catenin even in the absence of a functional APC protein. It has been hypothesised that the inhibition of tankyrase 1/2 may offer a novel approach to the treatment of WNT signaling-related diseases such as a variety of cancers including colon cancer and non-small cell lung cancer and fibrotic diseases.

Several groups of chemical substances (XAV939, MN-64, CMP 8, CMP 18, CMPl l, CMP30, iWR-1, CMP40, CMP4, WIKI4, JW74, JW55, G007-LK, CMP24, CMP4b, MVP- TNKS656, AZ0108, E7449 and 3-arylisoquinolin-l-one inhibitors) have been identified which stabilize the destruction complex (Chen et a!., Nat. Chem. Biol. 5 : 100-107, 2009; Huang et a!... Nature: 461 : 614-620, 2009; Haikarainen et a!. , Curr, Pharm. Des. 20(41): 6472-88, 2014; McGonigle et a!., Oncotarget 6(38): 41307-23, 2015; Paine et a!., Bioorg. Med. Chem.23(17): 5891-908, 2015; Nkizinkiko et al., Bioorg. Med. Chem.23(15): 4139- 49, 2015; and Haikarainen et al., Bioorg. Med. Chem. Lett.26(2): 328-332016, 2016). By blocking the PARP domain of Tankyrase, chemical inhibitors are thought to alter the PARsylation and ubiquitination of AXIN2 that results in its increased stability and in inhibition of canonical WNT signaling. Since elevated levels of ȕ-catenin in the nucleus are a common feature of abnormal canonical WNT signaling, down-regulation of canonical WNT activity by reducing the presence of ȕ-catenin represents a potential therapeutic strategy. Tankyrase also regulates the stability and activity of other target proteins, hence tankyrase inhibitors may also act through WNT independent mechanism.

Certain compounds which exhibit activity in blocking canonical WNT signaling are described in WO 2010/139966 and WO 2012/076898. In view of the central importance of canonical WNT signaling in a wide range of cancers, there is an ongoing desire to identify further compounds which are useful in blocking canonical WNT signaling. We have found that the compounds described herein exhibit activity in blocking canonical WNT signaling, and in particular are capable of reducing levels of activated nuclear ȕ-catenin. Furthermore, we have found that the compounds modulate the cellular energy metabolism. Such compounds are thus suitable for inhibiting tumor cells in general and, in particular, those associated with colorectal cancers, non-small cell lung cancer, breast cancer, CNS cancers, ovary cancer, liver cancer, melanoma and pancreatic adenocarcinoma.

In some embodiments, the compounds described herein demonstrate greater solubility and/or lower IC₅₀ values than other known WNT inhibitors based on a triazole core, such as those described in WO 2010/139966 and WO 2012/076898, thereby further improving their suitability for use as active pharmaceutical ingredients. Their improved solubility is advantageous for parenteral administration, e.g. intravenous injection.

In a first aspect the invention provides compounds of general formula (I’):

wherein:

Z represents an optionally substituted, 5- or 6-membered unsaturated heterocyclic group comprising at least one nitrogen atom;

L represents a 4-, 5- or 6-membered cycloalkyl group, preferably a cyclobutyl group;

each R¹ independently represents F, Cl, Br, I, C1-3 alkyl, C1-3 haloalkyl (e.g. -CF3), -CN, -OH or -NO₂, preferably F, Cl, Br or I, e.g. Cl or F;

each R² independently represents F, Cl, Br, I, C1-3 alkyl, -CN, -OH or -NO2, preferably F, Cl, Br, I or -CN, e.g. F or -CN;

X represents -NR³- or -O-;

R³ represents H or a C_1-3 alkyl group (e.g. methyl);

n is an integer from 0 to 5, preferably 0 to 3, more preferably 0, 1 or 2, e.g 1; and m is an integer from 0 to 5, preferably 0 to 3, more preferably 0, 1 or 2, e.g.0 or 1;

and the tautomers, stereoisomers, and pharmaceutically acceptable salts thereof.

In a further aspect the invention provides compounds of general formula (I):

wherein:

Z represents a 5- or 6-membered unsaturated heterocyclic group comprising at least one nitrogen atom;

each R¹ independently represents F, Cl, Br, I, C1-3 alkyl, -CN, -OH or -NO2, preferably F, Cl, Br or I, e.g. Cl;

R³ represents H or a C1-3 alkyl group (e.g. methyl);

and the tautomers, stereoisomers and pharmaceutically acceptable salts thereof.

Preferred groups Z in the compounds of formulae (I’) and (I) are 5- or 6-membered unsaturated heterocyclic groups comprising two nitrogen atoms such as pyrazolyl, imidazolyl, pyrazolinyl, imidazolinyl, pyridazinyl, pyrimidinyl, or pyrazinyl groups.

Particularly preferably Z is a pyrimidinyl group. In one embodiment, group Z in the compounds of formulae (I’) and (I) is a thiazolyl group, e.g. a 2-thiazolyl or 5-thiazolyl group.

Any of the Z groups herein described may be substituted by one or more ring substituents. Where the Z groups are substituted, it is preferred that these are substituted by one or two substituent groups, e.g. by one substituent. Suitable substituents are as herein described and include, for example, C1-3 alkyl and C1-3 alkoxy groups. In one embodiment, the Z groups are unsubstituted.

Preferred compounds in accordance with the invention are those of general formulae (II) and (III):

wherein R¹, R², R³, Z, n and m are as defined herein.

Particularly preferred compounds of formula (II) are those of formula (IIa):

Particularly preferred compounds of formula (III) are those of formula (IIIa):

In the compounds of formulae (IIa) and (IIIa), groups R¹, R², R³, n and m are as defined herein. In these compounds, n is preferably 1 and m is preferably 0 or 1.

In the compounds described herein (including compounds of formulae (I’), (I), (II), (IIa), (III) and (IIIa)) it is preferred that at least one group R¹ is present. Where one group R¹ is present this is preferably bound to the phenyl ring in the ortho position relative to the carbon atom which is bound to the triazole ring, i.e. group R¹ is located as follows:

(where * denotes the point of attachment to the triazole ring). Where two groups R¹ are present these are both preferably bound to the phenyl ring in the ortho position relative to the carbon atom which is bound to the triazole ring, i.e. the two R¹ groups are located as follows:

(where * denotes the point of attachment to the triazole ring and where each group R¹ may be the same or different). In one embodiment of the compounds described herein (including compounds of formulae (I’), (I), (II), (IIa), (III) and (IIIa)), group R¹ is absent, i.e. n = 0.

In the compounds described herein (including compounds of formulae (I’), (I), (II), (IIa), (III) and (IIIa)) it is preferred either that group R² is absent or that a single group R² is present on the benzimidazole or benzoxazole ring. In the case where a single group R² is present on the benzimidazole ring, this is preferably located as follows:

(where * denotes the point of attachment to group L).

Thus in one embodiment the compounds of the invention are compounds of formula (IV):

wherein R¹, R², R³ and Z are as defined herein, and n is 0 or 1.

Preferred compounds of formula (IV) are those of general formulae (V) and (VI):

In the compounds of formulae (Va) and (VIa), groups R¹, R² and R³ are as herein defined, and n is 0 or 1.

In any of the compounds of the invention it is preferred that n is 0 (i.e. the phenyl ring is unsubstituted) or that n is 1 and group R¹ is either Cl or F.

In any of the compounds of the invention it is preferred that m is 0 (i.e. the benzimidazole or benzoxazole ring is unsubstituted) or that m is 1 and group R² is Cl, F, or - CN.

In certain embodiments of the compounds of the invention n is 0 or 1 and R¹ is Cl or F, and m is 0 or 1 and R² is Cl, F or -CN.

In certain embodiments of the compounds of the invention n is 1, m is 1, R¹ is Cl and R² is -CN. In certain embodiments, n is 1, m is 1, R¹ is Cl and R² is F. In certain

embodiments n is 1, m is 0 and R¹ is Cl.

In preferred embodiments of compounds of general formulae (I), (II), (III), (IIa), (IIIa), (IV), (V), (VI), (Va) and (VIa), R³ is H and R¹, R², L, Z, n and m are as herein described.

In certain embodiments n is 0 or 1, m is 0 or 1, R¹ is Cl or F, R² is Cl, F or -CN, and R³ is H. In certain embodiments n is 1, m is 1, R¹ is Cl, R² is -CN, and R³ is H. In certain embodiments n is 1, m is 1, R¹ is Cl, R² is F, and R³ is H. In certain embodiments n is 1, m is 0, R¹ is Cl, and R³ is H.

In further embodiments of the compounds of general formulae (I), (II), (III), (IIa), (IIIa), (IV), (V), (VI), (Va) and (VIa), R³ is methyl and groups R¹, R², L, Z, n and m are as herein described. In certain embodiments n is 0 or 1, m is 0 or 1, R¹ is Cl or F, R² is Cl, F or -CN, and R³ is methyl. In certain embodiments n is 1, m is 1, R¹ is Cl, R² is -CN, and R³ is methyl. In certain embodiments n is 1, m is 1, R¹ is Cl, R² is F, and R³ is methyl. In certain

embodiments n is 1, m is 0, R¹ is Cl, and R³ is methyl.

As will be understood, the compounds described herein may exist in various stereoisomeric forms, including enantiomers, diastereomers, and mixtures thereof. The invention encompasses all optical isomers of the compounds described herein and mixtures of optical isomers. Hence, compounds that exist as diastereomers, racemates and/or

enantiomers are within the scope of the invention.

In particular, the invention extends to the enantiomers, diastereomers, and mixtures of diastereomers and/or enantiomers, of any of the compounds having a chiral centre in the group L.

In the compounds according to the invention, linker L is bound to both the triazole- derived moiety and to the benzimidazole or benzoxazole-derived moiety. In preferred embodiments of the invention, the bonds between the linker L and the remainder of the molecule (i.e. the bond to the triazole-derived moiety and to the benzimidazole or benzoxazole-derived moiety) are in a trans relationship. Thus, in preferred embodiments of compounds of general formula (II) and general formula (III) the compounds have the following stereochem

istr :

Compounds of general formulae (IIa), (IIIa), (V), (Va), (VI) and (VIa) having this stereochemistry form further embodiments of the invention.

Examples of preferred compounds in accordance with the invention include the following, their tautomers, stereoisomers, and pharmaceutically acceptable salts:

Compound No. (6) in accordance with the invention preferably has the following stereochemistry:

Compound No. (2) in accordance with the invention preferably has the following stereochemistry:

Compound No. (4) in accordance with the invention preferably has the following stereochemistry:

Compound No. (5) in accordance with the invention preferably has the following stereochemistry:

As used herein, the term "C_1-3 alkyl" refers to a saturated hydrocarbon group having one to three carbon atoms. Examples of such groups include methyl, ethyl, n-propyl, and iso-propyl.

As used herein, the term“C1-3 alkoxy” refers to an -O-C1-3 alkyl group. Examples of such groups include methoxy, ethoxy and propyloxy.

As used herein, the term "C1-3 haloalkyl" refers to a C1-3 alkyl group having one or more halo substituents. Examples of such groups include -CH₂F, -CHF₂, -CF₃, -CCl₃, - CHCl2,

-CH₂CF₃, etc.

As used herein, the term“cycloalkyl” refers to a saturated, cyclic hydrocarbon group. Examples of such groups which may be present in the compounds herein described include cyclobutyl, cyclopentyl, and cyclohexyl groups.

As used herein, the term "unsaturated heterocyclic group" is intended to cover any 5- or 6-membered, mono-, di or tri-unsaturated heterocyclic ring which contains at least one nitrogen atom. Additional heteroatoms selected from nitrogen, oxygen and sulphur may also be present, although it is preferred that no oxygen or sulphur atoms are present. In one embodiment the heterocyclic group may contain one or two nitrogen atoms, e.g. two nitrogen atoms. The heterocyclic ring structure may be linked to the remainder of the molecule through a carbon atom or through a nitrogen atom. Preferably it will be linked to the remainder of the molecule through a carbon atom. The unsaturated heterocyclic group may be aromatic or non-aromatic. Preferably, it will be aromatic. Unless otherwise stated, any unsaturated heterocyclic group mentioned herein may optionally be substituted by one or more groups, which may be identical or different. Examples of substituent groups include, but are not limited to, hydroxy, C_1-3 alkyl, C_1-3 alkoxy, amino, -CN, -NO₂, and halogen atoms (e.g. F, Cl or Br). Preferred substituents include C1-3 alkyl (e.g. methyl) and C1-3 alkoxy (e.g. methoxy and ethoxy) groups.

Illustrative examples of "unsaturated heterocyclic rings" are the heterocycles pyrrole, 2H-pyrrole, pyrroline, pyrazole, imidazole, oxazole, isoxazole, pyrazoline, imidazoline, thiazole, isothiazole, thiadiazole, pyridine, pyridazine, pyrimidine, pyrazine, and triazole. Of these, pyrazole, imidazole, pyrazoline, imidazoline, pyridine, pyridazine, pyrimidine and pyrazine are preferred, particularly preferably pyrimidine and pyridine.

The compounds according to the invention may be prepared from readily available starting materials using synthetic methods known in the art. Preferably, the compounds are obtained in accordance with the following method which forms part of the invention:

(a) reacting a compound of general formula (VII):

with a compound of general formula (VIII) or formula (VIII’):

wherein in formulae (VII), (VIII) and (VIII’), Z, L, R¹, R², R³, n and m are as herein defined;

(b) if desired, resolving a compound thus obtained into the stereoisomers thereof; and/or (c) if desired, converting a compound thus obtained into a salt thereof, particularly a pharmaceutically acceptable salt thereof.

The method described above may be used to prepare any compound of formula (I’) or (I) (including compounds of formula (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa)) as herein described.

The reaction of the compound of formula (VII) with the compound of formula (VIII) or (VIII’) is conveniently carried out in a solvent or mixture of solvents, such as for example a polar solvent such as acetonitirile, acetone, DMF, DMSO, toluene or dioxane or mixtures thereof. Toluene is a preferred solvent. The reaction may suitably be carried out under reflux conditions, typically for a time from 12 hours to 2.5 days (e.g.16 hours, 24 hours or 48 hours).

Where R³ is C_1-3 alkyl (e.g. methyl), this may be introduced by first reacting a compound of formula (VII) with a compound of formula (VIII) wherein R³ is hydrogen, followed by introduction of a C_1-3 alkyl group into the reaction product such that the C_1-3 alkyl R³ group replaces the initially-present R³ hydrogen atom. The C1-3 alkyl (e.g. methyl) group may be introduced using standard techniques known to those skilled in the art, such as deprotonation using a suitable base such as potassium carbonate followed by reaction with a suitable alkylating agent, e.g. methyl iodide. The step of replacing the R³ hydrogen with an R³ group which is a C1-3 alkyl (e.g. methyl) group may suitably be carried out after step (a), e.g. immediately following step (a), prior to step (b), prior to step (c), after step (b) or even after step (c) of the method as described above.

Alternatively, a compound of formula (VIII) wherein R³ is C1-3 alkyl may be prepared according to the methods described herein, prior to reaction of the compound of formula (VIII) with the compound of formula (VII).

Compounds of general formula (VII) and general formulae (VIII) and (VIII’) form a further aspect of the invention.

In the compounds of general formula (VII) and general formulae (VIII) and (VIII’), groups L, Z, R¹, R², R³, n and m can be any such group or combination of groups as hereinbefore described with reference to the compounds of general formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa).

In an embodiment, the compound of general formula (VII) may be obtained by the following method which forms part of the invention:

(aa) reacting a compound of general formula (IX) with a compound of general formula (X) to form a compound of general formula (XI):

(bb) reacting the compound of general formula (XI) with a thionylating agent to form a compound of general formula (XII):

and

(cc) methylating the compound of general formula (XII) to form a compound of general formula (VII);

wherein in formulae (IX), (X), (XI) and (XII), Z, R¹ and n are as hereinbefore defined. Step (aa) may suitably be performed under conventional amide formation conditions known to those skilled in the art. For example, the compounds of formulae (IX) and (X) may be reacted in the presence of SOCl2 at a temperature of up to 100ºC (e.g.80ºC) for a period of 1 to 5 hours (e.g.2 hours) followed by reaction with DMAP (4-dimethylaminopyridine) and TEA (triethylamine) in THF (tetrahydrofuran) at a temperature of up to 60ºC (e.g.50ºC) for a period of 12 hours or more (e.g.2 days).

Step (bb) may be performed using a conventional thionylating agent known to those skilled in the art such as Lawesson’s reagent (2,4-bis(4-methoxyphenyl)-1,3,2,4- dithiadiphosphetane-2,4-dithione) in a suitable solvent such as toluene. Suitably, about 0.5 to about 1 molar equivalent of the thionating agent may be employed. The thionation reaction may suitably be performed at a temperature of up to 100ºC (e.g.80ºC) for a period of 12 to 24 hours (e.g.16 hours).

Step (cc) may be performed using a conventional methylation reaction known to those skilled in the art. For example, the compound of general formula (XII) may suitably be reacted with at least one molar equivalent of methyl iodide in the presence of a base such as sodium hydroxide, sodium carbonate, potassium hydroxide or potassium carbonate.

In an embodiment, the compound of formula (VIII) may be obtained by the following method which forms part of the invention:

(aaa) Reacting a compound of general formula (XIII) with a compound of general formula (XIV) to form a compound of general formula (XV):

(bbb) reacting the compound of general formula (XV) with a reducing agent to form a compound of general formula (XVI):

and

(ccc) reacting the compound of general formula (XVI) with triphosgene and Hünig’s base (N,N-diisopropylethylamine (also called DIPEA or DIEA)) to form a compound of general formula (XVII):

(ddd) optionally reacting the compound of general formula (XVII) with an alkylating agent in the presence of a base to form a compound of general formula (XVIIa):

(eee) Reacting the compound of general formula (XVII) or (XVIIa) with hydrazine to form a compound of general formula (VIII);

wherein in formulae (XII), (XIII), (XIV), (XVI) and (XVII), L, R² and m are as hereinbefore defined, in formula (XVIIa) R^3’ is a C_1-3 alkyl (e.g. methyl) group, and in formula (XIII) G denotes a suitable leaving group such as F or Cl.

Step (aaa) may suitably be performed in acetonitrile as the solvent and in the presence of a mild base such as potassium carbonate or sodium carbonate. Suitably this step may be carried out at a temperature of up to 100ºC (e.g.80ºC to 90ºC or 75ºC to 85ºC) for a period of 12 to 48 hours (2 days) (e.g.16 hours or 24 hours (1 day)).

Step (bbb) may be performed using any suitable reduction reaction known to those skilled in the art. For example, reaction with SnCl2 in ethanol may be employed as the method of reduction, suitably at a temperature of up to 100ºC (e.g.80ºC to 90ºC or 75ºC to 85ºC) for a period of 0.5 to 5 hours (e.g.1 hour to 2 hours, such as 1.5 hours). Other suitable reduction reactions are known to those skilled in the art and include, for example, palladium- catalysed reduction using aqueous potassium fluoride and polymethylhydrosiloxane or triethylsilane in the presence of Pd(OAc)2; or reaction with iron and CaCl2 in an

ethanol/water solvent at 60ºC for a period of 30 minutes to 2 hours. Step (ccc) may suitably be performed in acetonitrile as the solvent. A temperature of up to 100ºC (e.g.60ºC) and a reaction time of 12 to 24 hours (e.g.16 hours) may suitably be employed. In an alternative embodiment the reaction can be performed at room temperature (e.g. between 15 and 25ºC, such as at about 20ºC) for a period of 16 to 24 hours, e.g.20 hours.

Step (ddd), when present, can be carried out using standard bases and alkylating agents known to those skilled in the art, such as potassium carbonate as base and methyl iodide as alkylating agent. Any suitable solvent may be used, such as acetonitrile or dimethylformamide (DMF).

Step (eee) may suitably be performed using hydrazine in its monohydrate or dihydrate form. Methanol or ethanol may suitably be employed as a solvent. The reaction can be performed at room temperature (e.g. between 15 and 25ºC, such as at about 20ºC) for a period of 1 to 36 hours (e.g.1.5 hours to 24 hours, such as 2 to 20 hours, e.g.16 or 20 hours). In an alternative embodiment the reaction may be performed at a temperature of up to 100ºC (e.g.85ºC) and a reaction time of 1 to 36 hours (e.g.1.5 hours to 24 hours, such as 2 to 20 hours, e.g.16 or 20 hours) may suitably be employed.

The compounds used as starting materials in the methods of preparation of compounds of formulae (VII), (VIII) and (VIII’) are either known from the literature or may be commercially available. Alternatively, these may be obtained by methods known from the literature.

In an embodiment, the method of preparing a compound of formula (I’) or (I) (including compounds of formula (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa)) as described above may include a step of preparing a compound of formula (VII) and/or a step of preparing a compound of formula (VIII) or (VIII’) prior to carrying out the reaction of the compounds of formulae (VII) and (VIII) or (VIII’). In such embodiments the preparation of the compound of formula (VII) and/or the compound of formula (VIII) or (VIII’) is performed in accordance with the methods described herein.

The compounds of general formulae (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) may be resolved into their enantiomers and/or diastereomers. For example, where these contain only one chiral centre, these may be provided in the form of a racemate or may be provided as pure enantiomers, i.e. in the R- or S-form. Any of the compounds which occur as racemates may be separated into their enantiomers by methods known in the art, such as column separation on chiral phases or by recrystallisation from an optically active solvent. Those compounds with at least two asymmetric carbon atoms may be resolved into their diastereomers on the basis of their physical-chemical differences using methods known per se, e.g. by chromatography and/or fractional crystallisation, and where these compounds are obtained in racemic form, they may subsequently be resolved into the enantiomers.

The invention further extends to tautomers of any of the compounds herein disclosed. As will be appreciated, certain compounds according to the invention may exist in tautomeric forms, i.e. in forms which readily interconvert by way of a chemical reaction which may involve the migration of a proton accompanied by a switch of a single bond and adjacent double bond. In cases where R³ is hydrogen the compounds of the invention may, in particular, undergo keto-enol tautomerism. Dependent on the conditions, the compounds may predominantly exist either in the keto or enol form and the invention is not intended to be limited to the particular form shown in any of the structural formulae given herein.

The compounds according to the invention may be converted into a salt thereof, particularly into a pharmaceutically acceptable salt thereof with an inorganic or organic acid or base. Acids which may be used for this purpose include hydrochloric acid, hydrobromic acid, sulphuric acid, sulphonic acid, methanesulphonic acid, phosphoric acid, fumaric acid, succinic acid, lactic acid, citric acid, tartaric acid, maleic acid, acetic acid, trifluoroacetic acid and ascorbic acid. Bases which may be suitable for this purpose include alkali and alkaline earth metal hydroxides, e.g. sodium hydroxide, potassium hydroxide or cesium hydroxide, ammonia and organic amines such as diethylamine, triethylamine, ethanolamine,

diethanolamine, cyclohexylamine and dicyclohexylamine. Procedures for salt formation are conventional in the art.

In a further aspect there is provided pharmaceutical formulations comprising a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers or excipients.

The compounds according to the invention and their pharmaceutically acceptable salts have valuable pharmacological properties, particularly an inhibitory effect on WNT/ß-catenin signaling through inhibition the adenosine binding site of the catalytic domain of tankyrase 1/2 and stabilization of the AXIN protein. In view of their ability to inhibit signaling in the WNT pathway, and in particular to reduce the levels of nuclear ß-catenin, the compounds according to the invention and their pharmaceutically acceptable salts are suitable for the treatment and/or prevention of any condition or disease which may be affected by over- activation of signaling in the WNT pathway, in particular those conditions or diseases which involve activation of ß-catenin. The compounds of the invention and their pharmaceutically acceptable salts also have valuable pharmacological properties through affecting other target proteins of tankyrase 1/2.

The term "WNT signaling pathway" is used to refer to the chain of events normally mediated by WNT, LRP (LDL-receptor related protein), Frizzled, AXIN and ß-catenin, among others, and resulting in changes in gene expression and other phenotypic changes typical of WNT activity.

The WNT pathway plays a central role in the pathology of a variety of cancers. The compounds of the invention are thus particularly suitable for preventing and/or retarding proliferation and metastasis of tumor cells, in particular carcinomas such as

adenocarcinomas. More specifically, the compounds are effective in treatment and/or prevention of tumors emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, cervix, thyroid, head and neck, brain, breast and kidney. Particularly preferably, the compounds herein described may be used in the treatment and/or prevention of colorectal cancer and non-small cell lung cancer.

The compounds according to the invention and their pharmaceutically acceptable salts have valuable pharmacological properties that may also be used for treatment or prevention of non-cancer indications that are influenced by the activity of tankyrase 1/2, dependent or independent of its impact on WNT signaling. These include, non-regenerative wound healing, viral infections such as Herpes Simplex Virus infections, fibrosis such as pulmonary, dermal-, renal- and liver fibrosis, myocardial fibrosis, and metabolic conditions such as aberrant systemic glucose metabolism,

As used herein, the term "proliferation" refers to cells undergoing mitosis. The term "retarding proliferation" indicates that the compounds inhibit proliferation of a cancer cell. In preferred embodiments, "retarding proliferation" indicates that DNA replication is at least 10% less than that observed in untreated cells, more preferably at least 25% less, yet more preferably at least 50% less, e.g.75%, 90% or 95% less than that observed in untreated cancer cells.

The term "carcinoma" refers to any malignant growth which arises from epithelial cells. Exemplary carcinomas include basal cell carcinoma, squamous cell carcinoma and adenocarcinoma. Adenocarcinomas are malignant tumors originating in the glandular epithelium and include colorectal, pancreatic, breast and prostate cancers. Viewed from a further aspect the invention thus provides a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, for use in therapy.

Unless otherwise specified, the term "therapy" as used herein is intended to include both treatment and prevention.

In a still further aspect the invention provides a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, for use in the treatment or prevention of a tumor emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, cervix, thyroid, head and neck, brain, breast or kidney, in the treatment of non-regenerative wound healing, or in the treatment or prevention of viral infections such as Herpes Simplex Virus infections, fibrosis such as pulmonary-, dermal-, renal- and liver fibrosis, myocardial fibrosis, or metabolic conditions such as aberrant systemic glucose metabolism.

In another aspect the invention provides the use of a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in a method of treatment or prevention of a tumor emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, cervix, thyroid, head and neck, brain, breast or kidney, in the treatment of non-regenerative wound healing, or in the treatment or prevention of viral infections such as Herpes Simplex Virus infections, fibrosis such as pulmonary-, dermal-, renal- and liver fibrosis, myocardial fibrosis, or metabolic conditions such as aberrant systemic glucose metabolism.

Also provided is a method of treatment of a human or non-human animal body to treat or prevent a tumor emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, cervix, thyroid, head and neck, brain, breast or kidney, to treat non-regenerative wound healing, or to treat or prevent viral infections such as Herpes Simplex Virus infections, fibrosis such as pulmonary-, dermal-, renal- and liver fibrosis, myocardial fibrosis, or metabolic conditions such as aberrant systemic glucose metabolism, said method comprising the step of administering to said body an effective amount of a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or

pharmaceutically acceptable salt thereof. Small molecules that selectively target the developmental pathways which control pattern formation during embryogenesis, including WNT signaling pathways, are considered to be valuable for directing differentiation of pluripotent stem cells toward many desired tissue types (see Wang et al., ACS Chemical Biology, 16 November 2010). As modulators of WNT signaling, the compounds herein described also have effects on the development of cellular differentiation. The compounds described herein therefore have valuable properties for use in regenerative medicine, for example in protocols for lineage specific in vitro differentiation of progenitor cells. By "progenitor cell" is meant a cell with the capacity to differentiate into another cell type, e.g. a stem cell.

According to this aspect, the present invention provides a method (e.g. an in vitro method) of promoting and/or directing cellular differentiation comprising contacting a progenitor cell with an effective amount of a compound of formula (I’), (I), (II), (IIa), (IIb), (III), (IIIa), (IIIb), (IV), (V), (Va), (VI), or (VIa) as herein defined, or a tautomer, stereoisomer or pharmaceutically acceptable salt thereof. In particular, the progenitor cell is contacted with said at least one compound under suitable conditions and for a sufficient time for the progenitor cell to differentiate into a new cell type. In a related aspect, the present invention provides the use of at least one compound as herein defined for promoting and/or directing cellular differentiation of a progenitor cell, especially in vitro.

Preferably, the progenitor cell is a totipotent or a pluripotent cell, especially a stem cell such as an embryonic stem cell. Preferred are mammalian progenitor cells such as mouse, rat and human cells, especially human cells. Such stem cells may be obtained from established cell cultures or may be derived directly from mammalian tissue by methods known in the art, including non tissue-destructive methods.

In a preferred embodiment, the progenitor cell is promoted and/or directed to differentiate into a new cell type which is a myocyte (e.g. a cardiomyocyte), a neuronal cell (e.g. a dopaminergic neuronal cell), an endocrine pancreatic cell or a hepatocyte or a cell type which may further differentiate into a myocyte, a neuronal cell, an endocrine pancreatic cell or a hepatocyte. Especially preferably, the progenitor cell is an embryonic stem cell and the new cell type is a cardiomyocyte, a dopaminergic neuronal cell, an endocrine pancreatic cell, a hepatocyte, or a cardiomyocyte.

The dosage required to achieve the desired activity of the compounds herein described will depend on the compound which is to be administered, the patient, the nature and severity of the condition, the method and frequency of administration and may be varied or adjusted according to choice. Typically, the dosage may be expected to be in the range from 1 to 100 mg, preferably 1 to 30 mg (when administered intravenously) and from 1 to 1000 mg, preferably from 1 to 200 mg (when administered orally).

The compounds of the invention may be formulated with one or more conventional carriers and/or excipients according to techniques well known in the art. Typically, the compositions will be adapted for oral or parenteral administration, for example by intradermal, subcutaneous, intraperitoneal or intravenous injection. Suitable pharmaceutical forms thus include plain or coated tablets, capsules, suspensions and solutions containing the active component optionally together with one or more conventional inert carriers and/or diluents, such as corn starch, lactose, sucrose, microcrystalline cellulose, magnesium stearate, polyvinylpyrrolidone, citric acid, tartaric acid, water, water/ethanol, water/glycerol, water/sorbitol, water/polyethyleneglycol, propylene glycol, stearylalcohol,

carboxymethylcellulose or fatty substances such as hard fat or suitable mixtures of any of the above.

Alternatively, the compounds of the invention may be administered topically at or near the affected site. Topical compositions include gels, creams, ointments, sprays, lotions, salves, sticks, powders, pessaries, suppositories, aerosols, drops, solutions and any of the other conventional pharmaceutical forms in the art. Topical administration to inaccessible sites may be achieved by techniques known in the art, e.g. by use of catheters or other appropriate drug delivery systems.

Due to their enhanced solubility, the compounds may suitably be formulated in a form for parenteral administration, e.g. for intravenous injection. For this purpose, sterile solutions containing the active compounds may be employed.

The pharmacological properties of the compounds of the invention can be analysed using standard assays for functional activity. Detailed protocols for testing of the compounds of the invention are provided in the Examples.

The invention will now be described in more detail in the following non-limiting Examples and Figures, in which:

Figure 1 shows solubility as a function of concentration for Compound (6); and Figure 2 shows in vivo concentrations of Compound (6) as a function of time in a mouse pharmacokinetic model when administered orally or intravenously.

Example 1 - Preparation of 1-((1R,4R)-4-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H- 1,2,4-triazol-3-yl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-one - Compound (1)

(1a) Synthesis of N-(2-chlorophenyl)pyrimidine-4-carboxamide:

To a flask charged with pyrimidine-4-carboxylic acid (1.4 g, 11.28 mmol) were added DCM (50 ml), DIPEA (5.89 ml, 33.84 mmol), HATU (4.75 g, 12.4 mmol) and 2-chloro benzenamine (1.18 ml, 11.28 mmol). The resulting mixture was stirred overnight at room temperature. Water was added to the reaction mixture and the organic layer was separated using a separating funnel. The organic layer was dried over magnesium sulfate, filtered and concentrated in vacuo, and subjected to column chromatography on silica gel using a cyclohexane and ethyl acetate gradient as eluent to afford N-(2-chlorophenyl)pyrimidine-4- carboxamide as a solid. Yield 1.79 g (68%).

MS (ESI) m/z for C₁₁H₈ClN₃O = 234.04 (calcd) 234.1 ([M + H]⁺, found)

(1b) Synthesis of N-(2-chlorophenyl)pyrimidine-4-carbothioamide:

To a solution of N-(2-chlorophenyl)pyrimidine-4-carboxamide (2.0 g, 8.55 mmol) in toluene (20 ml) Lawesson’s reagent (2.42 g, 5.99 mmol) was added and the mixture was refluxed for 7 hours. After the reaction mixture was cooled to room temperature, the solvents were evaporated. The crude product was purified by chromatography on silica gel eluting with a gradient of cyclohexane /ethyl acetate. The fractions containing the product were combined and the solvent evaporated under reduced pressure to yield N-(2- chlorophenyl)pyrimidine-4-carbothioamide. Yield 1.34 g (63%) MS (ESI) m/z for C11H8ClN3S = 249.01 (calcd) 250.1 ([M + H]⁺, found)

(1c) methyl (Z)-N-(2-chlorophenyl)pyrimidine-4-carbimidothioate:

To a flask charged with N-(2-chlorophenyl)pyrimidine-4-carbothioamide (249.7 mg, 1.0 mmol) in tetrahydrofuran (6 ml) was added potassium tert-butoxide (112.2 mg, 1.0 mmol) and stirred for 15 min. Methyl tosylate (151 ^l, 1.0 mmol) was then added dropwise and the reaction mixture was stirred at room temperature for further 18 hours. The reaction mixture was then partitioned between water and ethylacetate, the layers were separated and the organic layer was washed twice with water and brine, dried over magnesium sulfate, filtered and evaporated under reduced pressure to provide crude product. The crude mixture of methyl (Z)-N-(2-chlorophenyl)pyrimidine-4-carbimidothioate was used directly in the further step without any purification. Yield: 248 mg.94%

MS (ESI) m/z for C₁₂H₁₀ClN₃S = 263.03 (calcd) 264.1 ([M + H]⁺, found)

(1d) (1R,4R)-methyl 4-(2-nitrophenylamino)cyclohexane carboxylate:

To a solution of methyl 4-aminocyclohexane carboxylate hydrochloride (1 g, 5.16 mmol) and 1-fluoro-2-nitrobenzene (544 ^l, 5.16 mmol) in acetonitrile (50 ml) was added potassium carbonate (1.06 g, 77.4 mmol) at ambient temperature. The resulting reaction mixture was heated for 8 hours at reflux. After cooling down to room temperature, acetonitrile was removed under reduced pressure and the crude solid was dissolved in dichloromethane. The dichloromethane layer was washed with water, dried over magnesium sulfate, filtered and then concentrated under reduced pressure. The crude product was washed with cold methanol and used in the next reaction step without any further

purification. Yield 1.3 g, (91%)

MS (ESI) m/z for C14H18N2O4 = 278.13 (calcd) 279.2 ([M + H]⁺, found)

(1e) (1R,4R)-methyl 4-(2-aminophenylamino)cyclohexane carboxylate:

To a reaction flask with a mixture of (1R,4R)-methyl 4-(2-nitrophenylamino) cyclohexane carboxylate (1.85 g, 9.55 mmol) and a catalytic amount of 10% palladium on charcoal in ethanol (400 ml) was attached a hydrogen balloon and hydrogen gas bubbled into the reaction mixture at atmospheric pressure. After 2 hours, the balloon was removed and the reaction mixture purged with nitrogen, filtered through a pad of celite and washed twice with ethanol. The filtrate was concentrated under reduced pressure to afford (1R,4R)-methyl 4-(2- aminophenylamino)cyclohexane carboxylate. Yield 1.25 g. (76%)

MS (ESI) m/z for C14H20N2O2 = 248.15 (calcd) 249.2 ([M + H]⁺, found)

(1f) Synthesis of (1R,4R)-methyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclohexane carboxylate:

To a mixture of (1R,4R)-methyl 4-(2-aminophenylamino)cyclohexane carboxylate (1.00 g, 4.02 mmol) in dichloromethane (70 ml) at 0°C (ice bath) was added triphosgene (1.79 g, 6.03 mmol). The mixture was stirred at 0^oC for 1 h. Then it was allowed to warm to room temperature and stirred for 24 hours. Next the resulting mixture was refluxed for 24 hours. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, and brine, dried over magnesium sulfate filtered and evaporated to give (1R,4R)-methyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexane carboxylate. Yield 950 mg. (86%)

MS (ESI) m/z for C15H18N2O3 = 274.13 (calcd) 275.2 ([M + H]+, found)

(1g) Synthesis of (1R,4R)-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)

cyclohexanecarbohydrazide:

To a mixture of (1R,4R)-methyl 4-(1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclohexane carboxylate (500 mg, 1.82 mmol) in ethanol was added hydrazine hydrate and heated at 120^oC for 3 hours under microwave irradiation in a sealed vial (Biotage intiator⁺). Upon completion of the reaction, the solvent was removed under reduced pressure and the crude solid was washed with a mixture of dichloromethane and methanol to yield (1R,4R)-4- (1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexanecarbohydrazide. Yield 460 mg.

(92%)

MS (ESI) m/z for C₁₅H₁₈N₂O₃ = 274.14 (calcd) 275.2 ([M + H]⁺, found)

(1h) Synthesis of 1-((1R,4R)-4-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)cyclohexyl)-1H-benzo[d]imidazol-2(3H)-one:

The (1R,4R)-4-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclohexanecarbohydrazide (200 mg, 0.729 mmol) was added to a solution of N-(2-chlorophenyl)pyrimidine-4- carboiminethiomethyl (211 mg, 0.801 mmol) in N,N-dimethylacetamide (1 ml).

Trifluoroacetic acid (27.8 ^l) was added and the reaction mixture was heated at 120^oC for 14 hours. After cooling down to room temperature, the reaction mixture was filtered and the residue washed with dichloromethane and methanol. The filtrate was concentrated under reduced pressure to remove the methanol and dichloromethane. A mixture of water and dichloromethane was added and the reaction mixture portioned using a separating funnel. The organic layer was washed with water, brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude product was purified by chromatography on silica gel eluting with a gradient of dichloromethane / methanol. The fractions containing the product were combined and the solvent evaporated under reduced pressure to yield the title compound (1). Yield: 72 mg. (21%)

MS (ESI) m/z for C25H22ClN7O = 471.16 (calcd) 472.2 ([M + H]⁺, found)

Example 2 - Preparation of 1-((1R,4R)-4-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4- yl)-4H-1,2,4-triazol-3-yl)cyclohexyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5- carbonitrile– Compound (2)

(2a) Synthesis of (1R,4R)-methyl 4-(4-cyano-2-nitrophenylamino)cyclohexane carboxylate:

To a solution of methyl 4-aminocyclohexane carboxylate hydrochloride (1.5 g, 7.74 mmol) and 4-fluoro-3-nitrobenzonitrile (1.28 g, 7.74 mmol) in acetonitrile (50 ml) was added N,N-diisopropylethylamine (2.69 ml, 15.49 mmol) at ambient temperature. The resulting reaction mixture was stirred for 18 hours at room temperature. Acetonitrile was removed under reduced pressure and the crude solid was dissolved in dichloromethane and the dichloromethane layer was washed with water, dried over magnesium sulfate, filtered and then concentrated under reduced pressure. The crude solid product was washed with cold methanol and used in the next step without any further purification. Yield 1.8 g.77% MS (ESI) m/z for C₁₅H₁₇N₃O₄ = 303.12 (calcd) 304.2 ([M + H]⁺, found)

(2b) Synthesis of (1R,4R)-methyl 4-(2-amino-4-cyanophenylamino)cyclohexane carboxylate:

To a reaction flask with a mixture of (1R,4R)-methyl 4-(4-cyano-2-nitrophenylamino) cyclohexane carboxylate (2.38 g, 7.85 mmol) and a catalytic amount of 10% palladium on charcoal in ethanol (450 ml) was attached a hydrogen balloon and hydrogen gas bubbled into the reaction mixture at atmospheric pressure. After 2 hours, the balloon was removed and the reaction mixture purged with nitrogen, filtered through a pad of celite and washed twice with ethanol. Then the filtrate was concentrated under reduced pressure to afford (1R,4R)-methyl 4-(2-amino-4-cyanophenylamino)cyclohexane carboxylate. Yield 1.84 g. (86%).

MS (ESI) m/z for C₁₅H₁₉N₃O₂ = 273.15 (calcd) 274.2 ([M + H]⁺, found) (2c) Synthesis of (1R,4R-methyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3- yl) cyclohexane carboxylate:

To a mixture of (1R,4R)-methyl 4-(2-amino-4-cyanophenylamino)cyclohexane carboxylate (1.87 g, 6.84 mmol) in dichloromethane (65 ml) was added triphosgene (3.06 g, 10.31 mmol) at 0°C (ice water bath). The mixture was stirred at 0^oC for 1 h. Then it was allowed to warm to room temperature and stirred for 24 hours. Next the resulting mixture was heated to reflux for 24 hours. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, and brine, dried over magnesium sulfate and evaporated to give (1R,4R)-methyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclohexane carboxylate.1.90 g. (93%)

MS (ESI) m/z for C₁₆H₁₇N₃O₃ = 299.13 (calcd) 300.2 ([M + H]⁺, found)

(2d) Synthesis of (1R,4R)-4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclohexanecarbohydrazide:

To a mixture of (1R,4R)-methyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclohexane carboxylate (299.3 mg, 1.00 mmol) in ethanol (7 ml) was added hydrazine hydrate (7 ml) and heated to 80^oC for 3 hours under microwave irradiation in a sealed vial (Biotage intiator⁺). Upon completion of the reaction, the reaction mixture was filtered and the crude solid was washed with methanol, filtered and dried to afford (1R,3R)-3-(1,2- dihydro-2-oxobenzo[d]imidazol-3-yl)cyclobutanecarbohydrazide. Yield 269 mg (90%) MS (ESI) m/z for C15H17N5O2 = 299.14 (calcd) 300.2 ([M + H]⁺, found)

(2e) Synthesis of 1-((1R,4R)-4-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)cyclohexyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile:

The (1R,4R)-4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)

cyclohexanecarbohydrazide (142 mg, 0.47 mmol) was added to a solution of N-(2- chlorophenyl)pyrimidine-4-carboiminethiomethyl (138 mg, 0.52 mmol) in N,N- dimethylacetamide (2 ml). Trifluoroacetic acid (18.2 ^l, 0.23 mmol) was added and the reaction mixture was heated at 120^oC for 14 hours. The reaction mixture was then cooled down to room temperature and filtered. Water was added to the filtrate and the reaction mixture was extracted with dichloromethane. The combined organic layers were washed with water, brine, dried over magnesium sulfate, filtered and concentrated under reduced pressure. The crude solid was then washed with an acetonitrile/water mixture (1:1, 3 ml) followed by cold methanol resulted in compound (2). Yield 24 mg (10%)

MS (ESI) m/z for C₂₆H₂₁ClN₈O = 496.15 (calcd) 497.2 ([M + H]⁺, found);

1H NMR (600 MHz, DMSO-d6) į 11.44 (s, 1H), 9.08 (d, J = 5.3 Hz, 1H), 9.01 (d, J = 1.4 Hz, 1H), 8.34 (dd, J = 5.3, 1.4 Hz, 1H), 7.93 (dd, J = 7.8, 1.7 Hz, 1H), 7.89 (dd, J = 8.1, 1.4 Hz, 1H), 7.80 (td, J = 7.8, 1.7 Hz, 1H), 7.75 (td, J = 7.7, 1.5 Hz, 1H), 7.67 (d, J = 8.3 Hz, 1H), 7.58 (dd, J = 8.2, 1.6 Hz, 1H), 7.48 (d, J = 1.5 Hz, 1H), 4.43 (tt, J = 12.3, 3.9 Hz, 1H), 2.73– 2.66 (m, 1H), 2.31– 2.19 (m, 3H), 2.10– 1.98 (m, 3H), 1.95– 1.85 (m, 2H).

Example 3 - Preparation of 1-((1R,3R)-3-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4- yl)-4H-1,2,4-triazol-3-yl)cyclobutyl)-1H-benzo[d]imidazol-2(3H)-one– Compound (3)

(3a) Synthesis of (1R,3R)-methyl 3-(2-nitrophenylamino)cyclobutanecarboxylate:

To a solution of (1R,3R)-methyl 3-aminocyclobutanecarboxylate hydrochloride (1.0 g, 6.05 mmol) and 1-fluoro-2-nitrobenzene (1.09 g, 7.74 mmol) in acetonitrile (50 ml) was added potassium carbonate (1.60 g, 11.6 mmol) at ambient temperature. The resulting reaction mixture was heated for 16 hours at reflux. Acetonitrile was removed under reduced pressure and the crude solid was dissolved in dichloromethane and the dichloromethane layer was washed with water, dried over magnesium sulfate and then concentrated under reduced pressure. Unreacted 1-fluoro-2-nitrobenzene was removed using co-distillation with toluene. The crude product was purified by chromatography on silica gel eluting with a gradient of cyclohexane /ethyl acetate. The fractions containing the product were combined and the solvent evaporated under reduced pressure to yield 1.43 g of (1R,3R)-methyl 3-(2- nitrophenylamino) cyclobutanecarboxylate. Yield 1.435 g.95%

MS (ESI) m/z for C₁₂H₁₄N₂O₄ = 250.10 (calcd) 251.2 ([M + H]⁺, found)

(3b) Synthesis of (1R,3R)-methyl 3-(2-aminophenylamino)cyclobutanecarboxylate:

To a reaction flask with a mixture of (1R,3R)-methyl 3-(2-nitrophenylamino) cyclobutanecarboxylate (1.4 g, 5.59 mmol) and a catalytic amount of 10% palladium on charcoal in ethanol was attached a hydrogen balloon and hydrogen gas bubbled into reaction mixture at atmospheric pressure. After 2 hours, the balloon was removed and the reaction mixture purged with nitrogen, filtered through a pad of celite and washed twice with ethanol. The filtrate was concentrated under reduced pressure to afford (1R,3R)-methyl 3-(2- aminophenylamino)cyclobutanecarboxylate. The crude product was used in next step without any further purification. Yield: 936 mg. (76%)

MS (ESI) m/z for C₁₂H₁₆N₂O₂ = 220.12 (calcd) 221.2 ([M + H]⁺, found)

(3c) Synthesis of (1R,3R)-methyl 3-(2-aminophenylamino)cyclobutanecarboxylate:

To a mixture of (1R,3R)-methyl 3-(2-aminophenylamino)cyclobutanecarboxylate (1.12 g, 5.08 mmol) in dichloromethane was added triphosgene (2.26 g, 7.63 mmol) at 0°C (ice water bath). The mixture was stirred at 0^oC for 1 h. Then it was allowed to warm to room temperature and stirred for 24 hours. Next the resulting mixture was refluxed for 24 hours. The reaction mixture was diluted with dichloromethane and washed with saturated sodium bicarbonate, and brine, dried over magnesium sulfate and evaporated under reduced pressure to give (1R,3R)-methyl 3-(1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclobutanecarboxylate. Yield 1.15 gm (92%)

MS (ESI) m/z for C₁₃H₁₄N₂O₃ = 246.10 (calcd) 247.1 ([M + H]⁺, found)

(3d) Synthesis of (1R,3R)-3-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarbohydrazide:

To a mixture of (1R,3R)-methyl 3-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarboxylate (400 mg, 1.62 mmol) in ethanol (7 ml) was added hydrazine hydrate (7 ml) and heated to 100^oC for 3 hours under microwave irradiation in a sealed vial (Biotage intiator⁺). Upon completion of the reaction, the solvent was removed under reduced pressure and the crude solid was washed with methanol resulting in (1R,3R)-3-(1,2-dihydro-2- oxobenzo[d]imidazol-3-yl)cyclobutanecarbohydrazide as a solid product which was used in the next step without any further purification. Yield 360 mg. (90%).

MS (ESI) m/z for C12H14N4O2 = 246.11 (calcd) 247.1 ([M + H]⁺, found)

(3e) Synthesis of 1-((1R,3R)-3-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)cyclobutyl)-1H-benzo[d]imidazol-2(3H)-one:

The (1R,3R)-3-(1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclobutanecarbohydrazide (123 mg, 0.5 mmol) was added to a solution of N-(2-chlorophenyl)pyrimidine-4- carboiminethiomethyl (158 mg, 0.6 mmol) in N,N-dimethylacetamide (1 ml). Trifluoroacetic acid (19 ^l, 0.25 mmol) was added and the reaction mixture was heated to 120^oC for 14 hours. Water was added and the reaction mixture was extracted with dichloromethane. The organic layer was washed with water, brine, dried over magnesium sulfate, filtered and concentrated. The crude solid was purified by preparative HPLC (C18 reverse phase column, elution with a water/MeCN gradient with 0.1% TFA). The fractions containing the product were evaporated and lyophilized to yield compund (3) as a white solid. The product was obtained as its trifluoracetate salt. Yield: 46 mg. (16%).

Example 4 - Preparation of 1-((1R,3R)-3-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H- 1,2,4-triazol-3-yl)cyclobutyl)-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5-carbonitrile– Compound (6)

(6a) Synthesis of (1R,3R)-methyl 3-(4-cyano-2-nitrophenylamino)cyclobutanecarboxylate:

To a solution of (1R,3R)-methyl 3-aminocyclobutanecarboxylate hydrochloride (331.2 mg, 2.0 mmol) and 4-fluoro-3-nitrobenzonitrile (332 mg, 2.0 mmol) in acetonitrile (4 ml) was added N,N-diisopropylethylamine (1.04 ml, 6.0 mmol) at ambient temperature. The resulting reaction mixture was stirred for 24 hours at room temperature. After completion of the eaction, the acetonitrile was removed under reduced pressure and the crude solid was dissolved in dichloromethane. The dichloromethane layer was washed twice with water, brine, dried over magnesium sulfate, concentrated under reduced pressure and washed with cold methanol to yield (1R,3R)-methyl 3-(4-cyano-2-nitrophenylamino)

cyclobutanecarboxylate. Yield: 318 mg.96%

MS (ESI) m/z for C13H13N3O4 = 275.09 (calcd) 276.1 ([M + H]⁺, found)

(6b) Synthesis of (1R,3R)-methyl 3-(2-amino-4- cyanophenylamino)cyclobutanecarboxylate:

To a reaction flask with a mixture of (1R,3R)-methyl 3-(4-cyano-2-nitrophenylamino) cyclobutanecarboxylate (1.5 g, 5.45 mmol) and a catalytic amount of 10% palladium on charcoal in ethanol (400 ml) was attached a hydrogen balloon and hydrogen gas bubbled into the reaction mixture at atmospheric pressure. After 2 hours, the balloon was removed and the reaction mixture purged with nitrogen, filtered through a pad of celite and washed twice with ethanol. The filtrate was concentrated under reduced pressure to afford (1R,3R)-methyl 3-(2- amino-4-cyanophenylamino) cyclobutanecarboxylate. Yield: 962 mg. (72%)

MS (ESI) m/z for C₁₃H₁₅N₃O2 = 245.12 (calcd) 246.2 ([M + H]⁺, found)

(6c) Synthesis of (1R,3R)-methyl 3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3- yl) cyclobutanecarboxylate:

To a mixture of (1R,3R)-methyl 3-(2-amino-4- cyanophenylamino)cyclobutanecarboxylate (1 g, 4.07 mmol) in dichloromethane (60 ml) at 0°C (ice water bath) was added triphosgene (1.8 g, 6.12 mmol). The mixture was stirred at 0^oC for 1 h and was then allowed to warm to room temperature and stirred for 24 hours. Next the resulting mixture was refluxed for 24h. The reaction mixture was diluted with

dichloromethane and washed with saturated sodium bicarbonate, and brine, dried over magnesium sulfate and evaporated under reduced pressure to give (1R,3R)-methyl 3-(6- cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarboxylate. Yield: 1.05 g. (95%)

MS (ESI) m/z for C14H13N3O3 = 271.10 (calcd) 272.2 ([M + H]⁺, found)

(6d) Synthesis of (1R,3R)-3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclobutanecarboxylic acid:

To a mixture of (1R,3R)-methyl 3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3- yl)cyclobutanecarboxylate (1.0 g, 3.68 mmol) in THF/H₂O (4:1) (60 ml) was added lithium hydroxide monohydrate (386.9 mg, 9.22 mmol) and stirred for 36 hours at room temperature. The mixture was acidified with 1 N hydrochloric acid solution. The resulting precipitate was collected by filtration to afford (1R,3R)-3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarboxylic acid. Yield: 825 mg. (87%)

MS (ESI) m/z for C13H11N3O3 = 257.08 (calcd) 258.1 ([M + H]⁺, found) (6e) Synthesis of (1R,3R)-3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarbohydrazide:

A mixture of tert-butyl carbazate (409 mg, 3.1 mmol) and (1R,3R)-3-(6-cyano-1,2- dihydro-2-oxobenzo[d]imidazol-3-yl)cyclobutanecarboxylic acid (800 mg, 3.1 mmol), N,N- diisopropylethylamine (1.61 ml, 9.3 mmol) and HATU (1.3 g, 3.42 mmol) in N,N- dimethylacetamide (10 ml) was stirred at room temperature for 12 hours. Then a mixture of dichloromethane and water was added and the organic layer was separated, washed with water, brine, dried over magnesium sulfate, filtered and concentrated to afford (1R,3R)-3-(6- cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)cyclobutanecarbohydrazide as a crude solid. To the crude product a mixture of (1:5) ml of TFA: dichloromethane (24 ml) was added and stirred for 6 hours resultin in (1R,3R)-3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl) cyclobutanecarbohydrazide as a crude solid. The residue was purified by preparative HPLC (C18 reverse phase column, elution with a water/MeCN gradient with 0.1% TFA). The fractions containing the product were evaporated and lyophilized to yield a white solid. The product was obtained as its trifluoroacetate salt. Yield 81 mg.19%

MS (ESI) m/z for C13H13N5O2 = 271.11 (calcd) 272.2 ([M + H]⁺, found)

(6f) Synthesis of 1-((1R,3R)-3-((4S)-4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)cyclobutyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile:

The (1R,3R)-3-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)

cyclobutanecarbohydrazide (68.5 mg, 0.25 mmol) was added to a solution of N-(2- chlorophenyl)pyrimidine-4-carboiminethiomethyl (80 mg, 0.30 mmol) in N,N- dimethylacetamide (2 ml). Trifluoroacetic acid (9.6 ^l, 0.125 mmol) was added and the reaction mixture was heated to 120^oC for 14 hours. Water was added and the reaction mixture was extracted with ethyl acetate. The organic layer was washed with brine, dried over magnesium sulfate and concentrated under reduced pressure. The crude solid was purified by preparative HPLC (C18 reverse phase column, elution with a water/MeCN gradient with 0.1% TFA). The fractions containing the product were evaporated and lyophilized to yield compound (6) as a white solid. T he product was obtained as its trifluroacetate salt. Yield: 22.5 mg.15%

MS (ESI) m/z for C₂₄H₁₇ClN₈O = 468.12 (calcd) 469.2 ([M + H]⁺, found);

1H NMR (600 MHz, DMSO-d6) į 11.37 (s, 1H), 8.97 (d, J = 5.2 Hz, 1H), 8.90 (s, 1H), 8.25 (d, J = 5.0 Hz, 1H), 7.74 (dd, J = 8.0, 1.7 Hz, 2H), 7.63 (td, J = 7.8, 1.6 Hz, 1H), 7.57 (dt, J = 7.6, 3.5 Hz, 2H), 7.45 (dd, J = 8.3, 1.6 Hz, 1H), 7.38 (s, 1H), 5.27 (p, J = 8.9 Hz, 1H), 3.48 (dq, J = 10.0, 5.1, 3.7 Hz, 1H), 3.10 (q, J = 10.2 Hz, 2H), 2.83 (ddt, J = 12.3, 8.4, 3.7 Hz, 1H), 2.73 (ddt, J = 12.7, 8.5, 4.0 Hz, 1H).

Example 5 - Preparation of 1-((1R,3R)-3-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H- 1,2,4-triazol-3-yl)cyclobutyl)-3-methyl-2-oxo-2,3-dihydro-1H-benzo[d]imidazole-5- carbonitrile– Compound (4)

4-pyrimidinecarboxylic acid 1 and 2-chloroaniline 2 were reacted for 2 hours at 80ºC with SOCl2 and then reacted for 2 days at 50ºC with DMAP and TEA in THF, producing 1 of ure com ound 3 and 0.5 of im urities.

1.02 g of pure compound 3 were separated and then reacted with Lawesson’s reagent i toluene at 80ºC for 16 hours, yielding 0.6 g of compound 4:

_reflux

3 4

This was methylated, yielding 0.55 g of compound 5:

Separately, compound 6 was reacted with 7a in acetonitrile in the presence of K₂CO₃ for 16 hours at 80ºC ieldin 1.51 8a:

8a was reduced using SnCl2 in ethanol for 1 hour at 80 ºC yielding 1.2 g of 9a:

Reaction of this with triphosgene and DIPEA in acetonitrile for 16 hours at 60ºC yielded 0.9 g of 10a. 10a was then reacted with methyl iodide as methylating agent in the presence of K2CO3 as base and DMF as solvent to yield 0.1 g of product, from which 0.075 g of pure 11a were isolated:

11a was then reacted with hydrazine dihydrate in methanol for 20 hours at 20ºC to gve 12

Compounds 5 and 12a were reacted under reflux in toluene for 2 days giving about 50 mg of Compound (4):

Example 6 - Preparation of 1-((1R,3R)-3-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H- 1,2,4-triazol-3-yl)cyclobutyl)-5-fluoro-1,3-dihydro-2H-benzo[d]imidazol-2-one– Compound (5)

4-pyrimidinecarboxylic acid 1 and 2-chloroaniline 2 were reacted for 2 hours at 80ºC with SOCl₂ and then reacted for 2 days at 50ºC with DMAP and TEA in THF, producing 1.02 g of pure compound 3 and 0.5 g of impurities:

1.02 g of pure compound 3 were separated and then reacted with Lawesson’s reagent in toluene at 80ºC for 16 hours, yielding 0.6 g of compound 4:

This was methylated, yielding 0.55 g of compound 5:

0.5 g of 6 was reacted with 7 in acetonitrile in the presence of K2CO3 for 48 hours at 85ºC, yielding 0.88 g 8:

8 was reduced using SnCl₂ in ethanol for 1.5 hours at 85ºC, yielding a pale brown crude product which was determined by TLC to be a mixture of methyl ester 9 and the corresponding ethyl ester:

Reaction of isolated 9 with triphosgene and DIPEA in acetonitrile for 16 hours at 60ºC yielded 0.23 g of 10 which was then reacted with hydrazine dihydrate in methanol for 20 hours at 20ºC to give 0.18 g 11:

Compounds 5 and 11 were reacted under reflux in toluene for 2 days giving about 55 mg of Compound (5):

Example 7– Preparation of 1-(4-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)phenyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile– Compound (B) (Comparative)

Synthesis of ethyl 4-(2-amino-4-cyanophenylamino)benzoate:

To a solution of ethyl 4-(4-cyano-2-nitrophenylamino)benzoate (891 mg, 2.86 mmol) in EtOH(20 mL) was added SnCl₂ (5.55 g, 29.2 mmol). The orange mixture was refluxed for 45 min. It was then poured into EtOAc. The organic layer was washed with 1N HCl (50 mL), brine, and saturated NaHCO₃, and dried with Na₂SO₄. It was purified by column chromatography with 25% EtOAc in hexane to afford ethyl 4-(2-amino-4- cyanophenylamino)benzoate as a light yellow solid (743 mg, 2.64 mmol, 92%).

Synthesis of ethyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoate:

To a mixture of ethyl 4-(2-amino-4-cyanophenylamino)benzoate (320 mg, 1.21 mmol) in

CH₂Cl₂ (50 mL) under ice-water bath was added triphosgene (573 mg, 1.93 mmol). The mixture was stirred at 0^oC for 1 h. Then it was allowed to warm to room temperature and stirred overnight. The resulting mixture was refluxed overnight. It was diluted with CH2Cl2, washed with saturated NaHCO3, and brine, and evaporated to give a white solid ethyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoate (254 mg, 0.86 mmol, 72%).

Synthesis of 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoic acid:

To a mixture of ethyl 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoate (400 mg, 1.30 mmol) in THF/H2O (4:1 v/v, 15 mL) was added LiOH*H2O (133 mg, 3.16 mmol). The mixture was stirred overnight. The mixture was acidified with 1N HCl. The resulting precipitate was collected by filtration to give a white solid 4-(6-cyano-1,2-dihydro- 2-oxobenzo[d]imidazol-3-yl)benzoic acid (340 mg, 1.22 mol, 94%).

Synthesis of N''-(2-chlorophenyl)pyrimidine-4-carboximidhydrazide:

To a solution of N-(2-chlorophenyl)pyrimidine-4-carbimidoyl chloride (450 mg, 1.92 mmol) in toluene (20 ml) was added phosphorous pentachloride (1.2 g, 5.77 mmol) and phosphoryl chloride (540 μl, 5.77 mmol) and the mixture stirred at reflux for 7 hours. The solvents were then evaporated and 3 ml of hydrazine hydrate was added and stirred for 4 hours at room temperature. Solvents were evaporated and subjected to column

chromatography on silica gel using a cyclohexane and ethyl acetate gradient as eluent to afford N''-(2-chlorophenyl) pyrimidine-4-carboximidhydrazide as a solid (230 mg.48%).

Synthesis of N'-((Z)-(2-chlorophenylimino)(pyrimidin-4-yl)methyl)-4-(6-cyano-1,2- dihydro-2-oxobenzo[d]imidazol-3-yl)benzohydrazide:

To a mixture of 4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol-3-yl)benzoic acid (271 mg,

0.97 mmol) in THF (35 mL) was added compound N''-(2-chlorophenyl)pyrimidine-4- carboximidhydrazide (331 mg, 1.34 mmol), EDCI.HCl (323 mg, 1.68 mmol), HOBT (175 mg, 1.30 mmol) and Et₃N (1.5 mL, 10.8 mmol) sequentially. The mixture was stirred for 3 days. It was diluted with CH2Cl2, washed with H2O, dried with satd. NaHCO3, and chromatographed with 5% MeOH in CH₂Cl₂ to a yellow solid N'-((Z)-(2- chlorophenylimino)(pyrimidin-4-yl)methyl)-4-(6-cyano-1,2-dihydro-2-oxobenzo[d]imidazol- 3-yl)benzohydrazide (40 mg, 0.079 mmol, 8%). Synthesis of 1-(4-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3- yl)phenyl)-2,3- dih dro-2-oxo-1H-benzo d imidazole-5-carbonitrile:

A mixture of N'-((Z)-(2-chlorophenylimino)(pyrimidin-4-yl)methyl)-4-(6-cyano-1,2- dihydro-2-oxobenzo[d]imidazol-3-yl)benzohydrazide (20 mg, 0.039 mmol) in toluene (10 mL) was refluxed for 3 days. The mixture was evaporated and subjected to column chromatography with 2%MeOH in CH2Cl2 to give the crude product, which was washed with Et₂O to afford the title compound 1-(4-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4- triazol-3-yl)phenyl)-2,3-dihydro-2-oxo-1H-benzo[d]imidazole-5-carbonitrile as a pale yellow solid (6 mg, 0.01 mmol, 31%).

MS-API-ES (negative mode) calculated for

490.1, found 489.3 [M-H]- ; HPLC: 91.42%. ]⁺;

1H-NMR (300 Hz, CDCl3+CD3OD) į ppm 8.82-8.00 (m, 2H), 8.28-8.26 (m, 1H), 7.66-7.60 (m, 2H), 7.54-7.44 (m, 4H), 7.40-7.32 (m, 4H), 7.08-7.06 (m, 1H).

Example 8 - In vitro inhibition experiments

Plasmids, constructs, cell lines and conditioned media:

The L WNT3a-expressing cells were purchased from ATCC (American Type Culture Collection) and, including ST-Luc/Ren HEK293 cells (see below), maintained according to the supplier’s recommendations. A stable HEK293 cell line containing SuperTOP-Flash plasmid (ST-Luc HEK293) (7 X TCF binding sites promoter) was kindly provided by V. Korinek. To create SuperTOP-Luciferase/Renilla HEK293 cells (ST-Luc/Ren HEK293), the pRL-TK (Renilla, Promega) cassette was subcloned into pPUR (Promega) giving rise to the construct pRL-TK-puro. Linearized pRL-TK-puro was transfected (FuGENE6, Roche) into ST-Luc HEK293 before selection (2.5 μg/mL Puromycin, Sigma). WNT3a containing conditioned media (WNT3a-CM) from L WNT3a expressing cells was collected as described by ATCC. Transfection and luciferase assays:

40,000 ST-Luc/Ren HEK293 cells were seeded in 96-well plates coated with poly-L lysine. 24 hours after seeding, the cells were incubated for an additional 24 hours with various compound concentrations in 50 % WNT3a-CM. After compound exposures, the cells were lysed and the firefly luciferase and Renilla activities were measured on a GloMax® Luminometer (Promega) using Dual-Glo Luciferase Assay System (Promega).

IC50 calculations:

XLfit (idbs) was used to determine the IC50-values in inhibition experiments. The following formula was chosen to fit the data points (Langmuir Binding Isotherm):

fit = ((A+(B*x))+(((C-B)*(1-exp((-1*D)*x)))/D)), res = (y-fit)

Protein expression and purification:

The expression and purification of human TNKS were done as previously described (Narwal et al., J. Med. Chem.55(3): 1360-7, 2012). The proteins used in the assays were ARTD5/TNKS1 (residues 1030-1317), ARTD6/TNKS2 (residues 873-1162).

Biochemical inhibitor potency measurements:

The inhibitor potencies were measured with a fluorescence based activity assay (see Narwal et al., 2012 supra). The potencies of the compounds were measured using half log dilutions of the inhibitors and the reactions were done in quadruplicates with protein and inhibitor controls to exclude the effect of autofluorescence. The fluorescence intensity was measured using Tecan Infinity M1000 with excitation/emission wavelengths of 372 nm and 444 nm, respectively. Sigmoidal dose response curves were fitted with four variables using GraphPad Prism version 5.04 for Windows (GraphPad Software).

In vitro results:

Cellular IC50, TNKS1 and/or TNKS2 biochemical IC50 values were determined in accordance with the above protocols for a range of compounds according to the invention (Compounds (1) to (6) shown in Table 1 below). Compounds (A) and (B), which are not compounds according to the invention, were also tested for comparative purposes. As can be seen from Table 1, the compounds according to the invention have cellular IC50 values which are approximately 10 to 100 times lower than those of the compounds not according to the invention. The solubility results in the final column of Table 1 were determined in accordance with the methods in Example 9. The in vivo Mouse PK (pharmacokinetic) results were determined in accordance with the methods in Example 10. The in vivo Rat and Dog PK (pharmacokinetic) results were determined in accordance with the methods in Examples 11 and 12, respectively. Table 1:

Key to Table 1:

1 = Compound

2 = Structure

3 = Cellular IC50

4 = Biochemical IC₅₀ (TNKS1)

5 = Biochemical IC50 (TNKS2)

6 = Solubility / Mouse, Rat, Dog PK results

All compounds in Table 1 were determined as having >95% purity.

Example 9 - Solubility measurements

The turbidimetric (kinetic) solubility of Compound (6) was determined as follows. Serial dilutions of Compound (6) were prepared in DMSO at 100 times the final concentration. Test article solutions were diluted 100-fold into PBS buffer in a 96-well plate and mixed. After 2 hours of incubation at 37ºC, the presence of precipitate was detected by turbidity (measured by determination of absorbance at 540 nm). Precipitate is formed when maximum aqueous solubility levels are reached.

An absorbance value greater than“mean + 3 times standard deviation of the blank” (after subtracting the background) was indicative of turbidity. For brightly coloured compounds, a visual inspection of the plate was performed to verify the solubility limit determined by UV absorbance. The solubility limit was reported as the highest experimental concentration with no evidence of turbidity. Comparative experiments were performed in the same manner using reserpine, tamoxifen, and verapamil. Experimental conditions are shown in Table 2. The experimental results are shown in Table 3.

Table 2:

Table 3:

*solubility limit is highest concentration with no detectable precipitate

Example 10 - In vivo mouse pharmacokinetic model

Test Article Preparation:

The test article, Compound (6), was dissolved in 5% DMSO, 50% PEG400, 45% saline to yield a nominal concentration of 0.2 mg/mL for intravenous administration and 0.5 mg/mL for oral administration. The resulting solution (pH ~ 7) was clear and colorless solution and was stored at room temperature until picked up for dosing.

The preparation details are presented in Table 4 below. 3.5mg Compound (6) was dissolved in 0.36 ml DMSO to yield a nominal concentration of 10 mg/ml, then transferred to 0.08 ml to 0.12 ml DMSO, 1.6 ml PEG400 and 2.2 ml saline to yield a nominal

comcentration of 0.2 mg/ml for intravenous administration and transferred to 0.25 ml 10 mg/ml solution to 2.0 ml PEG400 and 2.75 ml saline to yield a nominal concentration of 0.5 mg/ml for oral administration. Animal Acquisition and Assignment to Study:

A total of 40 male experimentally ICR mice were transferred from stock colony, which were originally received from Sino-British SIPPR/BK Lab Animal Ltd, Shanghai, China, and 30 ice (body weight : 19.1-21.6 g) were placed in the study.

Dose Administration:

Dose administration information is presented in the following table (Table 4). Table 4:

* IV = intravenous

**PO = oral

Animal Final Disposition:

All animals were euthanized by carbon dioxide inhalation before sample collection. Animals received for the study but not placed in the study were used for collection of blank blood and brain after being euthanized by carbon dioxide inhalation. Euthanasia was confirmed via cervical dislocation, and the carcasses were discarded without further evaluation

Sample Collection and Bioanalysis:

Blood samples (approximately 500 μL) were collected via cardiac puncture after euthanasia by carbon dioxide inhalation post-dose (15 min, 1 h, 4 h, 8 h and 24 h). One sample was collected per animal at each time point. Blood samples were placed into tubes containing K2EDTA and centrifuged at 8000 rpm for 6 minutes at 4qC to separate plasma from the samples. Following centrifugation, the resulting plasma was transferred to clean tubes and stored frozen at -80qC pending bioanalysis.

Pharmacokinetic Analysis:

A standard set of parameters including Area Under the Curve (AUC_(0-t) and AUC_(0-^)), elimination half-life (T1/2), maximum plasma concentration (Cmax), initial concentration (C0), time to reach maximum plasma concentration (T_max), clearance (CL), and steady-state volume of distribution (Vss) were calculated using noncompartmental analysis modules in the FDA certified pharmacokinetic program WinNonlin Professional (Pharsight, USA). Furthermore, the bioavailability was estimated using the following formula:

Pharmacokinetics:

Plasma and brain concentrations from individual animals are tabulated in Table 5. The estimates of the non-compartmental pharmacokinetics parameters are summarized in Table 6. Log-linear plots of the plasma and brain concentration versus time curves are presented in Figure 2.

Intravenous administration of Compound (6), 1 mg/kg:

Following the intravenous administration of Compound (6) at a dose of 1 mg/kg, the mean values of Tmax and Cmax were 0.25 hr and 77.58 ng/mL. The mean values of AUC(0-^) were 62.93 hr*ng/mL.

Oral administration of Compound (6), 5 mg/kg:

Following the oral administration of Compound (6) at a dose of 5 mg/kg, the mean values of Tmax and Cmax were 0.25 hr and 123.48 ng/mL. The mean half-life (T½) was 1.51 hr, the mean values of AUC_(0-t) and AUC_(0-^) were 144.66 and 146.97 hr*ng/mL. The CL was 34.02 L/kg. The bioavailability was 46.71%.

Table 5: Selected pharmacokinetics parameters of Compound (6) in ICR Mice following intravenous and oral administration

Example 11– Pharmacokinetic profile of Compound (6) in Spraque-Dawley rats following intravenous and oral administration

The assay was performed by Medicilon (CN) according to their protocols. Compound (6) was dissolved in 5% DMSO, 50% PEG400, 45% saline to yield a nominal concentration of 0.7 mg/mL for intravenous administration and 1.4 mg/mL for oral administration to rats as follows:

Table 6:

Blood samples (approximately 200 μL) were collected via jugular vein at appropriate time points for determination of plasma concentrations. Blood samples were placed into tubes containing K2EDTA and centrifuged at 3500 rpm for 10 minutes at 4^oC to separate plasma from the samples. Following centrifugation, the resulting plasma was transferred to clean tubes and stored frozen at -80^oC pending bioanalysis. Standard set of parameters including Area Under the Curve (AUC(0-t) and AUC(0-^)), elimination half-life (T1/2), maximum plasma concentration (Cmax), initial concentration (C0), time to reach maximum plasma concentration (Tmax), clearance (CL), and steady-state volume of distribution (Vss) was calculated using non-compartmental analysis modules in the FDA certified

pharmacokinetic program WinNonlin Professional (Pharsight, US). Bioavailability (F) was calculated according to the same formula described in Example 9 (mouse PK).

Table 7: Selected pharmacokinetics parameters of Compound (6) in male Spraque-Rawley rats following intravenous (IV) and oral (PO) administration

t1/2: elimination half-life; Tmax: time to reach maximum plasma concentration; Cmax: maximum plasma concentration; AUC: area under concentration time curve; MRT: mean residence time; F: fraction (bioavailability)

Example 12– Pharmacokinetic profile of Compound (6) in Beagle dogs following intravenous and oral administration

The assay was performed by Medicilon (CN) according to their protocols. Compound (6) was dissolved in 5% DMSO, 50% PEG400, 45% saline to yield a nominal concentration of 1.4 mg/mL for intravenous administration and for oral administration to Beagle dogs as follows:

Table 8:

Blood samples (approximately 200 μL) were collected via jugular vein (after anesthetizia by isoflurane) at appropriate time points for determination of plasma concentrations. Blood samples were placed into tubes containing K3EDTA and centrifuged a^{t 3500 rpm for 10 minutes at 4o} _{C to separate plasma from the samples. Following} centrifugation, the resulting plasma was transferred to clean tubes and stored frozen at -80^oC pending bioanalysis. Standard set of parameters including Area Under the Curve (AUC(0-t) and AUC(0-^)), elimination half-live (T1/2), maximum plasma concentration (Cmax), initial concentration (C0), time to reach maximum plasma concentration (Tmax), clearance (CL), and steady-state volume of distribution (Vss) was calculated using non-compartmental analysis modules in the FDA certified pharmacokinetic program WinNonlin Professional (Pharsight, US). Bioavailability (F) was calculated according to the same formula described in Example 9 (mouse PK).

Table 9: Selected pharmacokinetics parameters of Compound (6) in Beagle dog following intravenous (IV) and oral (PO) administration

t_1/2: elimination half-life; T_max: time to reach maximum plasma concentration; C_max: maximum plasma concentration; AUC: area under concentration time curve; MRT: mean residence time; F: fraction (bioavailability)

Example 13– Inhibition in mouse tumor growth xenograft model

To evaluate the anti-tumor effects of Compound (6) in vivo, xenografts were established using the human colorectal cancer cell line COLO 320DM cells in male Balb/c nude mice. The tumor-bearing mice were randomized into 4 treatment groups, 2 days after inoculation: i) vehicle control (1% starch, n = 4), ii) 15 mg/kg Compound (6) (n = 5), iii) 30 mg/kg Compound (6) (n = 5) and iv) 60 mg/kg Compound (6) (n = 5). After 10 days of once daily oral drug-administration, and caliper-based tumor size measurements on day 12, 17 and 21, the experiment was terminated. Compared to vehicle control, treatment with Compound (6) resulted in 53%, 63% and 63% statistically significant tumor size reductions at 15 mg/kg, 30 mg/kg and 60 mg/kg once daily oral administration, respectively.

In a second tumor model, the syngeneic leukemic p388 mouse model was used. Immunocompetent BDF1 (DBA2×C57Bl6j) mice were implanted with p388 cells and randomized into 3 treatment groups consisting of 6 mice each after 2 days: Vehicle (1% starch, n = 6) and two treatment groups for Compound (6): 10 mg/kg (n = 5) and 30 mg/kg (n = 5). After 10 days of once daily oral administration, the tumors sizes were measured using caliper and the experiment was ended. Compared to vehicle control, treatment with Compound (6) resulted in 32% and 57% statistically significant tumor size reductions for 15 mg/kg and 30 mg/kg dosing, respectively. No animal discomforts or body weight differences were registered throughout the experiment period. Collectively, the results show that Compound (6) can significantly decrease the growth of colorectal cancer and leukemia in immunodeficient and immunocompetent models, respectively.

Statistical analyses for the animal experiments were conducted using One Way ANOVA/Holm-Sidak method, ANOVA on Ranks/Dunn's method or Students t-test.

Example 14– Synthesis of Compound (14)

(14)

Preparation of compound 14a, 3-(benzyloxy)-4-fluorobenzonitrile

A 20-ml tube was charged with 4-fluoro-3-hydroxybenzonitrile (0.206 g, 1.5 mmol) and potassium carbonate (0.207 g, 1.500 mmol). It was placed under a nitrogen atmosphere, acetonitrile (anhydrous) (3 ml) was added followed by (bromomethyl)benzene (0.196 ml, 1.650 mmol) and the white suspension was heated in a reaction block at 60°C for 3 hours. The suspension was evaporated to dryness, re-dissolved in a mixture of water and DCM and extracted three times with DCM. After drying over sodium sulfate, filtration and thorough evaporation, a batch of 0.33 g, 100% yield of a white solid was isolated and employed as such in the follow up experiment.

Preparation of compound 14b, (1R,3R)-3-(4-((S)-2-chlorophenyl)-5-(pyrimidin-4-yl)- 4H-1,2,4-triazol-3-yl)cyclobutan-1-amine hydrochloride

Step A

Methyl trans-3-amino-cyclobutanecarboxylate hydrochloride (39.94 g, 241 mmol) was suspended in dichloromethane (400 ml). The reaction mixture was cooled to 0°C.

Triethylamine (134 ml, 965 mmol) and BOC-O-BOC (63.2 g, 289 mmol) were added. The white suspension was allowed to warm to room temperature and was stirred for 20 hours. The product was extracted with DCM. The organic layer was dried with Na₂SO₄, filtered and the solvents removed in vacuo to give a white solid. The reaction mixture was filtered. Water was added to the filtrate and the layers were separated. The organic layer was washed two more times with H2O (300 mL). The combined organic layers were dried over sodium sulfate and concentrated to give a white solid (64.57 g). The product was stirred in heptane during 1 hour, filtered and the residue was dried on air during 1 hour to give the desired product (46.3 g).

Step B

Methyl (1R,3R)-3-((tert-butoxycarbonyl)amino)cyclobutane-1-carboxylate (2.5 g, 8.72

mmol) was suspended in methanol (60 ml) and hydrazine hydrate (2.54 ml, 52.3 mmol)

was added. The mixture was stirred at room temperature for 3 hours. A sample was diluted with MeCN and stirred overnight. An extra portion of hydrazine hydrate (2.54 ml, 52.3

mmol) was added and stirred at room temperature for 2 more hours. To the reaction mixture was added an extra portion of methyl (1R,3R)-3-((tertbutoxycarbonyl)

(amino)cyclobutane-1-carboxylate (2.5 g, 8.72 mmol) and this was stirred overnight. The product was filtered and the white solids were washed with H2O and Et2O twice (10 mL each). The white solid was dried on air to give the product in 4.75 g. It was used as such.

Step C

tert-Butyl ((1R,3R)-3-(hydrazinecarbonyl)cyclobutyl)carbamate (110 mg, 0.480 mmol)

together with methyl (E)-N-(2-chlorophenyl)pyrimidine-4-carbimidothioate (137 mg, 0.518 mmol) were suspended in 1-butanol (2.6 ml). The reaction container (microwave vial) was closed and the reaction mixture was stirred while heating at 120°C for 5 hours. The batch went into solution. The reaction mixture was stirred at 130°C for 18 hours and then stirred 143°C for 5 more hours. A sample was diluted with MeCN. No work up was performed, and the products were purified using MeOH in DCM as eluents with gradient 0- 6%. Fractions containing the product were pooled and evaporated to give the product as a yellow brown foam (174 mg). The compound was used as such.

Step D

tert-Butyl ((1R,3r)-3-(4-(2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3- yl)cyclobutyl)carbamate (174 mg, 0.408 mmol) was dissolved in 1,4-dioxane (5 mL) and HCl 4N in dioxane (3.66 mL, 14.64 mmol) was added. The mixture was stirred at room temperature for 60 hours. Extra addition of 4 M hydrochloric acid in 1,4- dioxane (0.305 mL, 1.22 mmol) was followed by stirring for 6 hours. The solvents were removed in vacuo to give the product as a beige solid (148 mg). The compound was used as such.

Preparation of Compound (14)

Step 1

A 0.5-2 ml microwave vial was charged 3-(benzyloxy)-4-fluorobenzonitrile (0.040 g, 0.176 mmol) and potassium carbonate (0.055 g, 0.400 mmol) followed by a solution of an impure batch of (1R,3R)-3-(4-((S)-2-chlorophenyl)-5-(pyrimidin-4-yl)-4H-1,2,4-triazol-3- yl)cyclobutan-1-amine hydrochloride (0.065 g, 0.16 mmol) in dimethyl sulfoxide (dry) (1.6 ml). The pale yellow suspension was then irradiated for 1 hour at 120°C. The mixture was then placed in a reaction block heated to 120°C for overnight (~ 15 hours). The vial was then irradiated in the microwave at 150°C for 3 hours. The heating was continued for an additional 65 hours in a reaction block at 120°C. The mixture was cooled down, diluted with water and extracted three times with DCM. After drying over sodium sulfate, filtration and solvent evaporation, circa 104 mg of a brown semi-solid of the crude was isolated. Column chromatography on a 12 g silica gel cartridge was performed, eluting with a gradient of methanol (0 to 5%) in DCM, followed by a second column with a gradient of ethyl acetate (0 to 100%) in DCM. 12 mg of a colourless glass of the desired compound was obtained. The latter fraction was directly subjected to hydrogenolysis.

Step 2

In a 10 ml tube was placed 3-(benzyloxy)-4-(((1R,3r)-3-(4-(2-chlorophenyl)-5- (pyrimidin-4-yl)-4H-1,2,4-triazol-3-yl)cyclobutyl)amino)benzonitrile (0.012 g, 0.023 mmol) in a mixture of ethanol (0.50 ml) and ethyl acetate (0.50 ml). A suspension was obtained, to which also dichloromethane (0.50 ml) was added. Palladium on carbon (4.90 mg, 4.60 ^mol) was added and the mixture was vigorously stirred under 1 bar of hydrogen. The stirring was continued overnight for additional 17 hours. An extra portion of palladium on carbon (4.90 mg, 4.60 ^mol) was added and the hydrogenolysis was continued under a fresh hydrogen atmosphere for six hours. The mixture was then filtered through celite rinsing on turns with EtOH, DCM and ethyl acetate. The whitish residue, circa 10 mg was directly employed in the final stage.

Step 3

In a 50 ml flask a ~60% pure batch of 4-(((1R,3r)-3-(4-(2-chlorophenyl)-5- (pyrimidin-4-yl)-4H-1,2,4-triazol-3-yl)cyclobutyl)amino)-3-hydroxybenzonitrile (10.36 mg, 0.014 mmol) was dissolved in dichloromethane (dried) (3.0 ml) under a nitrogen atmosphere. Acetonitrile (anhydrous) (1.0 ml) was also added to give an almost clear solution. An excess of N,N'-carbonyldiimidazole (0.011 g, 0.070 mmol) was added and the mixture was stirred for 60 hours at ambient temperature. The solvent was evaporated and submitted to preparative SFC. After freeze-drying the purified fractions from acetonitrile / water 2.8 mg of the product as a white powder was obtained.

MS (ESI) m/z for C₂₄H₁₆ClN₇O₂ = 469.89 (calcd) 470.1 ([M+H]⁺, found)

Example 15– Synthesis of Compound (15)

(15)

Preparation of 15a, methyl (E)-N-(2-chlorophenyl)thiazole-2-carbimidothioate Step A

Under nitrogen N-(2-chlorophenyl)thiazole-2-carboxamide (0.985 g, 4.13 mmol) was suspended in toluene (dry) (15 ml). Lawesson's reagent (1.669 g, 4.13 mmol) was added and the beige suspension heated to 80°C for 24 hours. The temperature was raised to 104 degrees and an extra portion of Lawesson's reagent (0.768 g, 1.898 mmol) was added and the reaction stirred overnight. The oil was suspended in 3 ml DCM and purified by column

chromatography using a 12 g column and a 0-33% EtOAc in heptane gradient over a period of 15 min to afford the product as a yellow solid (207 mg).

Step B

N-(2-chlorophenyl)thiazole-2-carbothioamide (207 mg, 0.813 mmol) was dissolved in acetone (10 ml). Iodomethane (0.061 ml, 0.975 mmol) and potassium carbonate (168 mg, 1.219 mmol) were added and the yellow suspension was stirred at room temperature for 3 hours. The temperature was raised to 34°C and the reaction mixture was stirred overnight. The solids were filtered and the filtrate was evaporated to dryness yielding the product as a yellow smelly oil (345 mg). Used as such.

Preparation of 15b, (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro-1Hbenzo[d]imidazol-1-yl) cyclobutane-1-carbohydrazide

Step 1

Methyl (1R,3R)-3-aminocyclobutane-1-carboxylate hydrochloride (10 g, 60.4 mmol) and 4-fluoro-3-nitrobenzonitrile (10.03 g, 60.4 mmol) were slurried in acetonitrile (160 ml). DIPEA (26.3 ml, 151 mmol) was added dropwise ( temperature: max 23 degrees) whereupon the yellow suspension dissolved, and the resulting reaction mixture was stirred at room temperature overnight. The mixture was concentrated and the solid yellow residue re- dissolved in DCM (100 ml). This was washed with water (2 x 100 mL), dried over Na₂SO₄ and concentrated to afford the product as a bright yellow solid (16.59 g).

Step 2:

To a degassed solution of methyl (1R,3R)-3-((4-cyano-2- nitrophenyl)amino)cyclobutane-1-carboxylate (16.59 g, 58.5 mmol) in tetrahydrofuran (dry) (200 ml) was added under a nitrogen atmosphere palladium, 10% on activated carbon (2.80 g, 2.63 mmol) in ethanol (Abs) (200 ml). The flask was evacuated and re-filled with hydrogen gas (3# cycles); then three balloons filled with hydrogen gas were connected and the reaction mixture was stirred overnight. The reaction mixture was flushed with nitrogen for 30 min, insolubles removed by filtration over a pad of Celite® while eluting with EtOH/DCM. The light yellow filtrate was evaporated to give the product as a yellow solid (14.54 g). Used as such.

Step 3:

Under nitrogen, methyl (1R,3R)-3-((2-amino-4-cyanophenyl)amino)cyclobutane-1- carboxylate (14.54 g, 57.5 mmol) was dissolved in dichloromethane (200 ml) and the mixture was cooled to 0°C. Then 1,1'-carbonyldiimidazole (11.19 g, 69.0 mmol) was added batchwise to keep the temperature below 2 degrees. The cool bath was removed and the mixture stirred while slowly warming to room temperature. Stirring was continued for 3 hours. The mixture was washed with a saturated NaHCO3 solution (2x100 mL) and water (2x100 mL). The aqueous layer was combined and extracted with DCM (1x100 mL).

Organic layers were combined and dried (Na2SO4), filtered and filtrate concentrated to afford the product as a pink solid (11.48 g). Used as such in the next experiment.

Step 4:

The experiment was performed in a 50 mL one-necked round-bottomed flask with magnetic

stirring closed with a bubble counter. To a stirred white suspension of methyl (1r,3r)- 3-(5-cyano-2-oxo-2,3-dihydro-1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carboxylate (9.06 g, 33.4 mmol) in methanol (90 ml) was added hydrazine hydrate (8.13 ml, 167 mmol) in one portion. The resulting white suspension was stirred at 50˚C. The reaction was continued for 4 hours while cooling to room temperature. The reaction mixture, a white suspension, was cooled to 0°C and stirred for 1 hour. The white suspension was filtered over a p3 glass filter and the resulting product filter cake was eluted with methanol (20 mL) and dried on the filter under suction (with an empty suction flask) and subsequently in a vacuum stove at 40°C for 60 hours affording 6.616 g of the product as a white powder.

Preparation of Compound (15)

A 2-5 ml microwave vial was charged with methyl (E)-N-(2-chlorophenyl)thiazole-2- carbimidothioate (0.054 g, 0.199 mmol) and (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (0.060 g, 0.199 mmol). Upon addition of 1-butanol (2.0 ml) a pale yellow suspension was obtained, the vial was capped and it was placed in a reaction block at 120°C for 60 hours. The temperature was elevated to 142 degrees and stirred for 17 hours. The mixture was thoroughly evaporated to dryness, stripping twice with acetonitrile. The product was purified and resulted in a white powder (23 mg).

MS m/z for C23H16ClN7OS = 473.94 (calcd) 474.1 ([M+H]⁺, found)

Example 16– Synthesis of Compound (16)

(16)

Preparation of 16a, methyl (E)-N-(2-(trifluoromethyl)phenyl)pyrimidine-4- carbimidothioate

Step A

Pyrimidine-4-carboxylic acid (1.5 g, 12.09 mmol) was slurried in N,N- dimethylformamide (dry) (15 ml), DIPEA (2.53 ml, 14.50 mmol) was added followed by HATU (5.06 g, 13.30 mmol). After 15 min, 2-(trifluoromethyl)aniline (2.142 g, 13.30 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature for 40 hours. The reaction mixture was evaporated (55°C to half volume (~7ml). This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml). The mixture was filtered, washed with cold H2O and dried on air to give a purple/brown solid. The batch was placed in a vacuum oven at 40 degrees for 24 hours. The solid was titruated in MeOH over 60 hours. The purple solids were filtered. The filtrate was evaporated under vacuum to a brown oil that solidified on standing (3.2 g). Used as such.

Step B Under nitrogen N-(2-(trifluoromethyl)phenyl)pyrimidine-4-carboxamide (1 g, 3.74 mmol)

was suspended in toluene (dry) (13 ml). Lawesson's reagent (1.514 g, 3.74 mmol) was

added and the beige suspension heated to 80°C for 4 hours. The temperature was elevated to 130°C and stirred overnight. The reaction mixture was cooled to room temperature. The solvents were removed in vacuo to give a black oil. The oil was suspended in 3 ml DCM and purified by column chromatography using a 24 g column and a 0-33% EtOAc in heptane gradient over a period of 20 min. The vials which contained the product (TLC) were collected and evaporated to give the product as an orange oil. This product was suspended in 3 ml DCM and purified by reveleris column chromatography using a 24 g column and a 0-15% EtOAc in heptane gradient over a period of 20 min. The vials which contained the product (TLC) were collected and evaporated to give the product as an orange solid (506 mg). Used as such.

Step C

N-(2-(trifluoromethyl)phenyl)pyrimidine-4-carbothioamide (506 mg, 1.786 mmol) was

dissolved in acetone (5 ml). iodomethane (0.133 ml, 2.144 mmol) and potassium carbonate (370 mg, 2.68 mmol) were added and the yellow suspension was stirred at room temperature overnight. The solids were filtered. The filtrate was evaporated to dryness to give a yellow semi solid. The solid was suspended in DCM and filtered again. The residue was washed with DCM and the filtrate was evaporated to give the product as a clear yellow oil (514 mg). Used as such.

Preparation of Compound (16)

A 2-5 ml microwave vial was charged with methyl (E)-N-(2-(trifluoromethyl)phenyl) pyrimidine-4-carbimidothioate (0.064 g, 0.215 mmol) and (1R,3R)-3-(5-cyano-2-oxo-2,3- dihydro-1H-benzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (0.060 g, 0.215 mmol). Upon addition of 1-butanol (2.0 ml) a pale yellow suspension was obtained, the vial was capped and it was placed in a reaction block at 120°C. Once at a slight reflux the mixture still stayed as a suspension with a slight colour change, and it was stirred overnight. The temperature was raised from 120°C to 150°C and stirred overnight. The reaction mixture was placed in a reaction block and stirred at 170°C for 3 days, the temperature was raised by 20°C and the reaction mixture was stirred for 3 days. The product was purified using SFC purification, and was prepared for lyophylaztion. The product was afforded as a white powder (11.4 mg).

MS (ESI) m/z for C25H17F3N8O = 502.46 (calcd) 503.2 ([M+H]⁺, found)

– Synthesis of Compound (17)

(17)

Preparation of 17a, methyl (E)-N-(2-chlorophenyl)-2-methylpyrimidine-4- carbimidothioate

Step A

2-methylpyrimidine-4-carboxylic acid (250 mg, 1.810 mmol) was slurried in

N,N,Dimethylformamide (dry) (3 ml). DIPEA (0.378 ml, 2.172 mmol) was added followed by HATU (757 mg, 1.991 mmol). After 15 mins 2-chloroaniline (0.191 ml, 1.810 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature overnight. The reaction mixture was evaporated (55°C to half volume (~4ml). This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml). The solvents were filtered and the residue was taken in DCM. The organic layer was dried with Na₂SO₄, filtered and the filtrate was evaporated to dryness to give the product as a beige solid. Used as such.

Step B

Under nitrogen N-(2-chlorophenyl)-2-methylpyrimidine-4-carboxamide (255 mg, 1.030

mmol) was suspended in toluene (dry) (4 ml). Lawesson's reagent (416 mg, 1.030 mmol) was added and the beige suspension heated to 80°C for 24 hours. The reaction mixture was cooled to room temperature. The solvents were removed in vacuo.20 ml DCM was added and the resulting solution washed with 20 ml sat. NaHCO₃ and then twice with 10 ml brine. The aqueous phases were back extracted with DCM (20 ml). The organic layers were combined, dried over Na₂SO₄ and evaporated to give the product as an orange oil. The batch was purified by reveleris using 1-30% EtOAc in heptane to afford the product as an orange solid. Used as such.

Step C N-(2-chlorophenyl)-2-methylpyrimidine-4-carbothioamide (146mg, 0.493 mmol) was dissolved in acetone (15 ml). iodomethane (0.040 ml, 0.640 mmol) and potassium carbonate (102 mg, 0.739 mmol) were added and the yellow suspension was stirred at room temperature for 60 hours. The solids were filtered, the filtrate was evaporated to dryness and dissolved in a 1:1 mixture of H2O and DCM. Separation via phase separator yielded the product as a brown smelly oil (115 mg). Used as such.

Preparation of Compound (17)

A 2-5 ml microwave vial was charged with (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (60 mg, 0.221 mmol), 1-butanol (2ml) and methyl (E)-N-(2-chlorophenyl)-2-methylpyrimidine-4-carbimidothioate (61.4 mg, 0.221 mmol). The vial was capped and it was placed in a reaction block at 130°C and stirred overnight, followed by 140°C for 24 hours. The reaction mixture was cooled and thoroughly evaporated to dryness. The product was purified using SFC purification. The product was prepared for lyophylization, and returned the product as a white solid

MS (ESI) m/z for C25H19ClN8O = 482.93 (calcd) 483.2 ([M+H]⁺, found)

– Synthesis of Compound (18)

(18) Preparation of 18a, methyl (E)-N-(2-chlorophenyl)-5-ethoxypyridine-2- carbimidothioate

Step A

5-ethoxypicolinic acid (80mg, 0.479 mmol) was slurried in N,N-Dimethylformamide (dry) (2 ml), DIPEA (0.100 ml, 0.574 mmol) was added followed by HATU (200 mg, 0.526 mmol). After 15 mins 2-chloroaniline (0.056 ml, 0.526 mmol) was added to the brown suspension and the resulting reaction mixture stirred at room temperature for 20 hours. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The filtrate was evaporated to dryness to give the product as a brown oil which solidified on standing. The product was purified further by reveleris 12 gram column using heptane-EtOAc gradient (0 to 25%) to afford a white solid. Used as such.

Step B

Under nitrogen N-(2-chlorophenyl)-5-ethoxypicolinamide (173 mg, 0.525 mmol) was suspended in toluene (dry) (4 ml). Lawesson's reagent (212 mg, 0.525 mmol) was added and the beige suspension heated to 80°C for 3 days. The reaction mixture was cooled to room temperature and stirred overnight. The solvents were removed in vacuo. The batch was purified by reveleris using 1-30% EtOAc in heptane 24 gram column. Appropriate fractions were combined and the solvents were removed in vacuo to give as a yellow solid. The product was purified further by reveleris 12 g column using heptane-EtOAc gradient (0 to 15%), to afford the product as a yellow solid (80 mg).

Step C

N-(2-chlorophenyl)-5-ethoxypyridine-2-carbothioamide (80 mg, 0.273 mmol) was dissolved in acetone (10 ml). Iodomethane (0.022 ml, 0.355 mmol) and potassium carbonate (56.6 mg, 0.410 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly solid (71 mg). Used as such.

Preparation of Compound (18)

A 2-5 ml microwave vial was charged with (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (60.2 mg, 0.222 mmol), 1-butanol (3 ml) and methyl (E)-N-(2-chlorophenyl)-5-ethoxypyridine-2-carbimidothioate (68 mg, 0.222 mmol). The vial was capped and it was placed in a reaction block at 140°C and stirred for four days. The reaction mixture was cooled and thoroughly evaporated to dryness and purified.

MS (ESI) m/z for C27H22ClN7O2 = 511.97 (calcd) 510.1 ([M-H], found)

Example 19– Synthesis of Compound (19)

(19) Preparation of 19a, methyl (E)-N-phenylpyrimidine-4-carbimidothioate

Step A

Pyrimidine-4-carboxylic acid (1 g, 8.06 mmol) was slurried in N,N- Dimethylformamide (dry) (10 ml), DIPEA (1.684 ml, 9.67 mmol) was added followed by HATU (3.37 g, 8.86 mmol). After 15 mins aniline (0.808 ml, 8.86 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature for 20 hours. The reaction mixture was evaporated to half its volume. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml), washed with cold H2O and dried on air to give a purple/brown solid. The solid was triturated in MeOH for 2 hours and the mixture was filtered. The residue was placed in a vacuum oven at 40 degrees for 24 hours. Used as such.

Step B

Under nitrogen N-phenylpyrimidine-4-carboxamide (770 mg, 3.87 mmol) was suspended in Toluene (dry) (13 ml). Lawesson's reagent (1563 mg, 3.87 mmol) was added and the beige suspension heated to 80°C for 24 hours. The reaction mixture was cooled to room temperature. The solvents were removed in vacuo, 20 ml DCM was added and the resulting solution washed with 20 ml sat. NaHCO3 and then twice with 10 ml brine. The aqueous phases were back-extracted with DCM (10ml). The organic layers were combined, dried over Na2SO4 and evaporated to give the product as a yellow oil. The batch was repurified by reveleris using 1-30% EtOAc in heptane to afford the product as an orange solid. Used as such.

Step C

N-phenylpyrimidine-4-carbothioamide (535mg, 2.485 mmol) was dissolved in acetone (10 ml). iodomethane (0.201 ml, 3.23 mmol) and potassium carbonate (515 mg, 3.73 mmol) were added and the yellow suspension was stirred at room temperature over weekend. The solids were filtered and the filtrate was evaporated to dryness and dissolved in a 1:1 mixture of H2O and DCM. Separation via phase separator yielded the product as a brown smelly oil (528 mg). Used as such.

Preparation of Compound (19)

A 2-5 ml microwave vial was charged with methyl (E)-N-phenylpyrimidine-4- carbimidothioate (0.053 g, 0.232 mmol) and (1R,3R)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (0.065 g, 0.232 mmol). Upon addition of 1-butanol (2.0 ml) a pale yellow suspension was obtained, the vial was capped and it was placed in a reaction block at 130°C and stirred for 48 hours, followed by stirring at 150°C for 24 hours. The reaction mixture was cooled and the mixture was thoroughly evaporated to dryness. The batch was purified by reveleris (12 g column) using 1-100% EtOAc in heptane. The crude was then flashed on a 12 gram silica gel cartridge eluted with a gradient of methanol (0 to 10%) in DCM, affording the product as a brown oil (35 mg). While MeOH was added for SFC purification, the batch formed crystals, which were filtered and the solids were rinsed with MeOH to give the product as a beige solid. The product was prepared for lyophylaztion. The batch returned as a beige powder (29.2 mg).

MS (ESI) m/z for C24H18N8O = 434.46 (calcd) 435.2 ([M+H]⁺, found)

– Synthesis of Compound (20)

(20) Preparation of 20a, methyl N-(2-chlorophenyl)-5-methylthiazole-2-carbimidothioate Step A

5-Methylthiazole-2-carboxylic acid (200 mg, 1.397 mmol) was slurried in

N,NDimethylformamide (dry) (2 ml), DIPEA (0.292 ml, 1.676 mmol) was added followed by HATU (584 mg, 1.537 mmol). A yellow solution was formed. After 25 min.2-chloroaniline (0.162 ml, 1.537 mmol) was added and the resulting reaction mixture stirred further at room temperature for three days. The mixture was then diluted with aqueous potassium carbonate and extracted twice with ethyl acetate. The organic extracts were rinsed with water, brine, dried over sodium sulfate, filtered and evaporated to dryness. The residue was then adsorbed on isolute and flashed on a 24 g silica gel cartridge eluted with a gradient of ethyl acetate (10 to 100) in heptane, providing 297 mg of the product as a white solid.

Step B

A 50 ml flask was charged with N-(2-chlorophenyl)-5-methylthiazole-2-carboxamide (0.297 g, 1.14 mmol) and Lawesson's reagent (0.461 g, 1.140 mmol), the content of the flask was placed under a nitrogen atmosphere and toluene (dry) (6.0 ml) was added. The suspension was first heated up to reflux for a couple of minutes in order to dissolve the major portion of the reactants. Then it was stirred at a temperature of 100°C. The mixture was stirred overnight. The mixture was cooled down, diluted with acetonitrile and evaporated to dryness. The residue was absorbed on isolute and flashed on a 12 g silica gel column eluted with a gradient ethyl acetate (5 to 50, then to 100%) in heptane. A yellow-coloured fraction was collected giving 272 mg of a yellow solid.

Step C

N-(2-chlorophenyl)-5-methylthiazole-2-carbothioamide (0.271 g, 1.00 mmol) was dissolved in acetone (10 ml), potassium carbonate (0.193 g, 1.400 mmol) was added and the yellow suspension was treated with iodomethane (0.075 ml, 1.200 mmol). The mixture was allowed to stir overnight, then it was evaporated to dryness. After re-suspending in DCM, it was filtered through celite, the yellow solution was concentrated, absorbed on isolute and flashed on a 12 g silica gel column eluted with a gradient ethyl acetate (5 to 50%) in heptane. A yellow-coloured was collected giving 268 mg of the product as a yellow oil.

Preparation of Compound (20)

A 2-5 ml microwave vial was charged with methyl N-(2-chlorophenyl)-5- methylthiazole-2-carbimidothioate (0.028 g, 0.10 mmol) and (1r,3r)-3-(5-cyano-2-oxo-2,3- dihydro-1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (0.027 g, 0.100 mmol) suspending the reactants in 1-butanol (1.0 ml). The vial was capped and the suspension was heated in a reaction block at 140°C for 20 hours. The mixture was evaporated to dryness, stripped with acetonitrile and coated on isolute. Flash column on a 12 g silica gel cartridge eluted with a gradient of ethyl acetate (10 to 100%) in heptane resulted in a white solid. This was lyophilised for 2 days from acetonitrile / water obtaining 26.2 mg of the product as a white powder.

MS (ESI) m/z for C₂₄H₁₈ClN₇OS= 487.97 (calcd) 488.1 ([M+H]⁺, found)

– Synthesis of Compound (21)

(21)

Preparation of 21a, methyl (E)-N-(2-chlorophenyl)thiazole-5-carbimidothioate Step A

Thiazole-5-carboxylic acid (104mg, 0.805 mmol) was slurried in N,N- Dimethylformamide (dry) (2 ml), DIPEA (0.168 ml, 0.966 mmol) was added followed by HATU (337 mg, 0.886 mmol). After 15 mins 2-chloroaniline (0.093 ml, 0.886 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature for 20 hours. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The filtrate was evaporated to dryness to give the product as a brown oil which solidified on standing. Used as such.

Step B

Under nitrogen N-(2-chlorophenyl)thiazole-5-carboxamide (131 mg, 0.538 mmol) was

suspended in Toluene (dry) (4 ml). Lawesson's reagent (218 mg, 0.538 mmol) was added and the beige suspension heated to 80°C for 3 days. The reaction mixture was cooled to room temperature and stirred overnight. The solvents were removed in vacuo. The batch was purified by reveleris using 1-30% EtOAc in heptane 24 g column. Appropriate fractions were combined and the solvents were removed in vacuo to give the product as a white solid. The batch was purified again by reveleris using 1-15% EtOAc in heptane 12 g column.

Appropriate fractions were combined and the solvents were removed in vacuo to give the product as a yellow solid (111 mg). Used as such.

Step C

N-(2-chlorophenyl)thiazole-5-carbothioamide (111 mg, 0.436 mmol) was dissolved in acetone (10 ml). iodomethane (0.035 ml, 0.566 mmol) and potassium carbonate (90 mg, 0.654 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly oil (106 mg). Used as such.

Preparation of Compound (21)

A 2-5 ml microwave vial was charged with (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (53.4 mg, 0.197mmol), 1-butanol (2.5 ml) and methyl (E)-N-(2-chlorophenyl)thiazole-5-carbimidothioate (53 mg, 0.197 mmol). The vial was capped and it was placed in a reaction block at 130°C and stirred overnight. The reaction mixture was stirred at 140°C for three days. The reaction mixture was cooled and thoroughly evaporated to dryness and prepared for SFC purification. Adding MeOH resulted in a solid appearing, which was filtered and the filtrate was purified, placed in the fridge with MeCN/H2O, and then freeze dried overnight. The product was purified again by SFC affording 13.1 mg product as a white solid after lyophilisation.

MS (ESI) m/z for C23H16ClN7OS = 473.94 (calcd) 474.1 ([M+H]⁺, found)

– Synthesis of Compound (22)

Preparation of 22a, methyl (E)-N-(2-chlorophenyl)-1-methyl-1H-pyrazole-4- carbimidothioate

1-methyl-1H-pyrazole-4-carboxylic acid (116mg, 0.920 mmol) was slurried in N,N Dimethylformamide (dry) (2 ml), DIPEA (0.192 ml, 1.104 mmol) was added followed by HATU (385 mg, 1.012 mmol). After 15 mins 2-chloroaniline (0.107 ml, 1.012 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature for 20 hours, and then refluxed for 4 hours. An extra portion of DIPEA (0.200 ml, 1.150 mmol) was added and reflux was continued for three days. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 15%). Appropriate fractions were combined and solvents removed to give the product as a brown solid. Used as such.

Step B

Under a nitrogen atmosphere N-(2-chlorophenyl)-1-methyl-1H-pyrazole-4- carboxamide (44 mg, 0.187 mmol) was suspended in toluene (dry) (2 ml). Lawesson's reagent (76 mg, 0.187 mmol) was added and the beige suspension heated to 80°C for 3 days. The mixture was diluted with acetonitrile and evaporated to dryness. The residue was then adsorbed on isolute and flashed on a 24 gram silica gel cartridge eluted with a gradient of ethyl acetate (5 to 50, the pure EA) in heptane. A second purification by reveleris afforded 38 mg of the product. Used as such.

Step C N-(2-chlorophenyl)-1-methyl-1H-pyrazole-4-carbothioamide (38 mg, 0.151 mmol) was

dissolved in acetone (3 ml). Iodomethane (0.012 ml, 0.196 mmol) and potassium carbonate (31.3 mg, 0.226 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H₂O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a colourless smelly oil (32 mg). Used as such.

Preparation of Compound (22)

A 2-5 ml microwave vial was charged with (1R,3R)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (32.6 mg, 0.12mmol), 1-butanol (2.5 ml) and methyl (E)-N-(2-chlorophenyl)-1-methyl-1H-pyrazole-4-carbimidothioate (32 mg, 0.120 mmol). The vial was capped and it was placed in a reaction block at 140°C and stirred for two days. The reaction mixture was cooled and thoroughly evaporated to dryness and prepared for SFC purification. Lyophilization overnight returned a white powder in an amount of 33.9 mg.

MS (ESI) m/z for

470.92 (calcd) 471.1 ([M+H]⁺, found)

– Synthesis of Compound (23)

(23)

Preparation of 23a, methyl (E)-N-(2-chlorophenyl)pyridine-2-carbimidothioate Step A

Picolinic acid (250 mg, 2.031 mmol) was slurried in N,N-dimethylformamide (dry) (2 ml), DIPEA (0.424 ml, 2.437 mmol) was added followed by HATU (849 mg, 2.234 mmol). After 15 mins 2-chloroaniline (0.236 ml, 2.234 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature for 72 hours. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 10 ml) and DCM 10 mL. The product was extracted and the layers were separated over a phase separator. The filtrate was evaporated to dryness to afford a brown solid. The product was purified by reveleris 12 g column using heptane- EtOAc gradient (0 to 25%). Appropraite fractions were combined and solvents removed to give the product a white solid (173 mg). Used as such.

Step B

Under nitrogen N-(2-chlorophenyl)picolinamide (173 mg, 0.744 mmol) was suspended in

toluene (dry) (4 ml). Lawesson's reagent (301 mg, 0.744 mmol) was added and the beige suspension heated to 80°C for 3 days. The mixture was diluted with acetonitrile and evaporated to dryness. The residue was then adsorbed on isolute and flashed on a 24 g silica gel cartridge, eluted with a gradient of ethyl acetate (5 to 30, to pure ethyl acetate) in heptane. A yellow coloured fraction was collected giving 327 mg of a yellow solid as the product.

Step C

N-(2-chlorophenyl)pyridine-2-carbothioamide (312 mg, 1.254 mmol) was dissolved in

acetone (3 ml). Iodomethane (0.102 ml, 1.631 mmol) and potassium carbonate (260 mg, 1.882 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. eparation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly oil (331 mg). Used as such.

Preparation of Compound (23)

A 2-5 ml microwave vial was charged with (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (51.5 mg, 0.19 mmol), 1-butanol (2.5 ml) and methyl (E)-N-(2-chlorophenyl)pyridine-2-carbimidothioate (50 mg, 0.190 mmol). The vial was capped and it was placed in a reaction block at 140°C and stirred overnight. The reaction mixture was cooled and the mixture was thoroughly evaporated to dryness and prepared for SFC purification. While adding MeOH a solid appeared. It was filtered and the residue was taken up into DCM/MeCN. Solvents were removed to give the product as a white solid. This was placed in fridge with MeCN/H₂O, and lyophilized overnight, returning a white powder (32.2 mg).

MS (ESI) m/z for C₂₅H₁₈ClN₇O = 467.92 (calcd) 468.1 ([M+H]⁺, found)

Example 24– Synthesis of Compound (24)

(24)

Preparation of 24a, methyl N-(2-fluorophenyl)pyrimidine-4-carbimidothioate Step A

Pyrimidine-4-carboxylic acid (500mg, 4.03 mmol) was slurried in N,N- Dimethylformamide (dry) (4 ml), DIPEA (0.842 ml, 4.83 mmol) was added followed by HATU (1685 mg, 4.43 mmol). After 15 mins 2-fluoroaniline (0.428 ml, 4.43 mmol) was added to the brown suspension and the resulting reaction mixture stirred further at room temperature during 20 hours. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 30%).

Appropriate fractions were combined and solvents removed in vacuo to give the product as a white solid. Used as such.

Step B

Under a nitrogen atmosphere N-(2-fluorophenyl)pyrimidine-4-carboxamide (381 mg, 1.754 mmol) was suspended in toluene (dry) (8 ml). Lawesson's reagent (709 mg, 1.754 mmol) was added and the beige suspension heated to 80°C for 24 hours. The batch was cooled and filtered. The filtrate was evaporated to dryness. The smelly orange-red residue was purified on a 12 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane to afford the product as an orange solid. Used as such.

Step C

N-(2-fluorophenyl)pyrimidine-4-carbothioamide (317 mg, 1.359 mmol) was dissolved in

acetone (4 ml). iodomethane (0.110 ml, 1.767 mmol) and potassium carbonate (282 mg, 2.038 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly oil (298 mg). Used as such.

Preparation of Compound (24) A 2-5 ml microwave vial was charged with (1R,3R)-3-(5-cyano-2-oxo-2,3-dihydro- 1Hbenzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (43.9 mg, 0.162 mmol), 1-butanol (2.5 ml) and methyl N-(2-fluorophenyl)pyrimidine-4-carbimidothioate (40mg, 0.162 mmol). The vial was capped and it was placed in a reaction block at 140°C and stirred overnight. The reaction mixture was cooled, thoroughly evaporated to dryness and purified. The product was a white powder (14 mg).

MS (ESI) m/z for C24H17FN8O = 452.45 (calcd) 453.1 ([M+H]⁺, found)

– Synthesis of Compound (25)

(25)

Preparation of 25a, methyl N-(2,6-dichlorophenyl)pyrimidine-4-carbimidothioate Step A

Pyrimidine-4-carboxylic acid (500mg, 4.03 mmol) was slurried in N,N- dimethylformamide (dry) (4 ml), DIPEA (0.842 ml, 4.83 mmol) was added followed by HATU (1685 mg, 4.43 mmol). After 15 mins 2,6-dichloroaniline (718 mg, 4.43 mmol) was added to the brown suspension and the resulting reaction mixture stirred for 20 hours at room temperature. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 30%). Fractions were combined and solvents removed in vacuo to give the product as a white solid. Used as such.

Step B

Under a nitrogen atmosphere N-(2,6-dichlorophenyl)pyrimidine-4-carboxamide (561 mg,

2.093 mmol) was suspended in toluene (dry) (10 ml). Lawesson's reagent (846 mg, 2.093 mmol) was added and the beige suspension heated to 80°C for 24 hours. The mixture was filtered and evaporated to dryness. The residue was then flashed on a 12 g silica gel cartridge, eluted with a gradient of ethyl acetate 0 to 20 in heptane. A yellowish semi-solid product was obtained.

Step C N-(2,6-dichlorophenyl)pyrimidine-4-carbothioamide (350 mg, 1.232 mmol) was dissolved in acetone (4 ml). Iodomethane (0.100 ml, 1.601 mmol) and potassium carbonate (255 mg, 1.848 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly oil (357 mg). Used as such.

Preparation of Compound (25)

A 2-5 microwave vial was charged with a batch of methyl N-(2,6- dichlorophenyl)pyrimidine-4-carbimidothioate (0.089 g, 0.30 mmol), purified first before use by a silica gel column, followed by (1R,3R)-3-(5-cyano-2-oxo-2,3-dihydro-1H- benzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (0.081 g, 0.300 mmol) and 1-butanol (3 ml). The suspension was then heated overnight for 14 hours in a microwave oven set at 150°C. The irradiation was continued for additional 4 hours at 200°C. The mixture was evaporated to dryness and the residue was purified by flash chromatography eluting with a gradient of methanol (0 to 5%, then to 10%). he desired compound was obtained in 71 mg as a white solid. This was freeze-dried from acetonitrile / water recovering 66.2 mg of a white solid.

MS (ESI) m/z for C24H16Cl2N8O = 503.35 (calcd) 503.1 ([M+H]⁺, found)

– Synthesis of Compound (26)

(26)

Preparation of 26a, methyl N-(2,6-difluorophenyl)pyrimidine-4-carbimidothioate Step A

Pyrimidine-4-carboxylic acid (500mg, 4.03 mmol) was slurried in N,N- dimethylformamide (dry) (4 ml), DIPEA (0.842 ml, 4.83 mmol) was added followed by HATU (1685 mg, 4.43 mmol). After 15 mins 2,6-difluoroaniline (572 mg, 4.43 mmol) was added to the brown suspension and the resulting reaction mixture stirred at room temperature for 20 hours. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 30%). Appropriate fractions were combined and solvents removed in vacuo to give the product as a white solid. Used as such.

Step B

Under a nitrogen atmosphere N-(2,6-difluorophenyl)pyrimidine-4-carboxamide (620 mg,

2.64 mmol) was suspended in toluene (dry) (10 ml). Lawesson's reagent (1066 mg, 2.64 mmol) was added and the beige suspension heated to 80°C for 24 hours, followed by stirring at 90°C for 5 more hours. The reaction mixture was cooled, filtered and the filtrate was evaporated to dryness. The smelly orange-red residue was purified on a 24 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane, affording the product as an orange solid. Used as such.

Step C

N-(2,6-difluorophenyl)pyrimidine-4-carbothioamide (310 mg, 1.234 mmol) was dissolved in acetone (4 ml). Iodomethane (0.100 ml, 1.604 mmol) and potassium carbonate (256 mg, 1.851 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo yielded the product as a yellow smelly oil (271 mg). Used as such.

Preparation of Compound (26)

A 2-5 ml microwave vial was charged with a purified by column chromatography batch of

methyl N-(2,6-difluorophenyl)pyrimidine-4-carbimidothioate (0.053 g, 0.20 mmol) followed by (1r,3r)-3-(5-cyano-2-oxo-2,3-dihydro-1H-benzo[d]imidazol-1-yl)cyclobutane-1- carbohydrazide (0.054 g, 0.200 mmol) and 1-butanol (2.0 ml). The vial was capped and the suspension was heated in a reaction block at 120°C overnight (~15 hours) staying still as a yellow suspension. The heating was continued for additional 21 hours at 150°C. The brown suspension was filtered through a P4 filter rinsing with MeCN. The filtrate was evaporated to dryness and the residue was submitted for purification by preparative SFC, affording a pale yellow residue of the desired product, 13 mg. The product was subjected to an additional purification by basic mode reversed phase column (PoraPak Rxn RP). After lyophilising from acetonitrile / water, 13.4 mg of a white solid was obtained.

MS (ESI) m/z for C24H16F2N8O = 470.44 (calcd) 471.2 ([M+H]⁺, found)

Example 27– Synthesis of Compound (27)

(27)

Preparation of 27a, methyl N-(3-fluorophenyl)pyrimidine-4-carbimidothioate Step A

Pyrimidine-4-carboxylic acid (500mg, 4.03 mmol) was slurried in N,N- dimethylformamide (dry) (4 ml), DIPEA (0.842 ml, 4.83 mmol) was added followed by HATU (1685 mg, 4.43 mmol). After 15 mins 3-fluoroaniline (0.425 ml, 4.43 mmol) was added to the brown suspension. The resulting reaction mixture was stirred for 24 hours. The reaction mixture was evaporated to dryness. To the residue was added to a cold water / sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 30%). Appropriate fractions were combined and solvents removed in vacuo to give the product as an orange solid. Used as such.

Step B

Under a nitrogen atmosphere N-(3-fluorophenyl)pyrimidine-4-carboxamide (580 mg, 2.67 mmol) was suspended in toluene (dry) (8 ml). Lawesson's reagent (1080 mg, 2.67 mmol) was added and the beige suspension heated to 80°C and stirred for 60 hours. The batch was cooled, filtered, and the filtrate was evaporated to dryness. The smelly orange-red residue was purified on a 12 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane. The orange-red residue was purified for a second time using a 24 g silica gel cartridge, eluting with a gradient of ethyl acetate 0 to 20% in heptane. The product was obtained as an orange solid (623 mg). Used as such.

Step C

N-(3-fluorophenyl)pyrimidine-4-carbothioamide (623 mg, 2.257 mmol) was dissolved in

acetone (4 ml). Iodomethane (0.183 ml, 2.93 mmol) and potassium carbonate (468 mg,

3.39 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents in vacuo was performed. The smelly orange-red residue was purified on a 12 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane to afford the product as an orange solid (386 mg). Used as such.

Preparation of Compound (27)

A 2-5 ml microwave vial was charged with methyl N-(3-fluorophenyl)pyrimidine-4- carbimidothioate (60 mg, 0.221 mmol), 1-butanol (2.5 ml) and (1R,3R)-3-(5-cyano-2-oxo- 2,3-dihydro-1H-benzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (59.9 mg, 0.221mmol). The vial was capped and the suspension was placed in a reaction block at 140°C and stirred overnight. The reaction mixture was cooled, the mixture was thoroughly evaporated to dryness and purified. The batch was placed in the fridge and was lyophilyzed overnight and returned as a white powder (33.2 mg).

MS (ESI) m/z for C24H17FN8O = 452.45 (calcd) 453.2 ([M+H]⁺, found)

Example 28– Synthesis of Compound (28)

(28)

Preparation of 28a, methyl N-(4-fluorophenyl)pyrimidine-4-carbimidothioate

Step A

Pyrimidine-4-carboxylic acid (500mg, 4.03 mmol) was slurried in N,N- Dimethylformamide (dry) (4 ml), DIPEA (0.842 ml, 4.83 mmol) was added followed by HATU (1685 mg, 4.43 mmol). After 15 mins 4-fluoroaniline (0.425 ml, 4.43 mmol) was added to the brown suspension. The resulting reaction mixture was stirred overnight. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. The product was purified by reveleris 12 g column using heptane-EtOAc gradient (0 to 30%). Appropriate fractions were combined and solvents removed in vacuo to give the product as a white solid. Used as such.

Step B Under a nitrogen atmosphere N-(4-fluorophenyl)pyrimidine-4-carboxamide (630 mg, 2.90 mmol) was suspended in toluene (dry) (8 ml). Lawesson's reagent (1173 mg, 2.90 mmol) was added and the beige suspension heated to 80°C and stirred for 60 hours. The batch was cooled and filtered. The filtrate was evaporated to dryness. The smelly orange-red residue was purified on a 12 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane. The product was obtained as an orange solid. Used as such.

Step C

N-(4-fluorophenyl)pyrimidine-4-carbothioamide (677 mg, 2.293 mmol) was dissolved in

acetone (4 ml). Iodomethane (0.186 ml, 2.98 mmol) and potassium carbonate (475 mg,

3.44 mmol) were added and the yellow suspension was stirred at room temperature overnight. An extra portion of iodomethane (0.043 ml, 0.688 mmol) was added and the reaction mixture was stirred for 24 hours. The reaction mixture was filtered and washed with acetone. The product was evaporated to dryness and the residue was dissolved in a 1:1 mixture of ice/H2O and DCM. Separation via phase separator and removal of the solvents. The smelly orange-red residue was purified on a 24 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane. The product was obtained as a yellow solid (396 mg). Used as such.

Preparation of Compound (28)

A 2-5 ml microwave vial was charged with methyl N-(4-fluorophenyl)pyrimidine-4- carbimidothioate (60 mg, 0.201 mmol), 1-butanol (2.5 ml) and (1R,3R)-3-(5-cyano-2-oxo- 2,3-dihydro-1H-benzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (54.6 mg, 0.201mmol). The vial was capped and the suspension was placed in a reaction block at 140°C and stirred overnight. The reaction mixture was cooled and thoroughly evaporated to dryness. The product was purified. The batch was placed in the fridge and lyophilized overnight to form a white powder (16.3 mg).

MS (ESI) m/z for C24H17FN8O = 452.45 (calcd) 453.1 ([M+H]⁺, found)

Example 29– Synthesis of Compound (29)

(29)

Preparation of 29a, methyl N-(2-chlorophenyl)-6-methylpyridine-2- carbimidothioate Step A

6-methylpicolinic acid (500mg, 3.65 mmol) was slurried in N,N-dimethylformamide (dry) (4 ml), DIPEA (0.762 ml, 4.38 mmol) was added followed by HATU (1525 mg, 4.01 mmol). After 15 mins 2-chloroaniline (0.422 ml, 4.01 mmol) was added to the brown suspension. The resulting reaction mixture was stirred overnight. The reaction mixture was evaporated to dryness. This mixture was added to a cold water/ sat. sodium bicarbonate mixture (2:1, 20 ml) and DCM 20 mL. The product was extracted and the layers were separated over a phase separator. T he product was purified by reveleris 24 g column using heptane-EtOAc gradient (0 to 30%). Appropriate fractions were combined and solvents removed in vacuo to give the product as a yellow solid. Used as such.

Step B

Under a nitrogen atmosphere N-(2-chlorophenyl)-6-methylpicolinamide (781 mg, 3.17

mmol) was suspended in toluene (dry) (8 ml). Lawesson's reagent (1280 mg, 3.17 mmol) was added and the beige suspension heated to 80°C overnight. The batch was then cooled to room temperature and stirred for 2 days. The filtrate was evaporated to dryness. The smelly orange-red residue was purified on a 24 g silica gel cartridge eluted with a gradient of ethyl acetate 0 to 20% in heptane to afford the product as an orange solid (542 mg). Used as such.

Step C

N-(2-chlorophenyl)-6-methylpyridine-2-carbothioamide (542 mg, 2.063 mmol) was dissolved in acetone (6 ml). Iodomethane (0.167 ml, 2.68 mmol) and potassium carbonate (428 mg, 3.09 mmol) were added and the yellow suspension was stirred at room temperature overnight. The reaction mixture was filtered and the filtrate was evaporated to dryness. The residue was dissolved in a 1:1 mixture of H₂O and DCM. Separation via phase separator and removal of the solvents in vacuo afforded the product as a green oil. Used as such.

Preparation of Compound (29)

A 2-5 ml microwave vial was charged with methyl N-(2-chlorophenyl)-6- methylpyridine-2- carbimidothioate (60 mg, 0.217 mmol), 1-butanol (2.5 ml) and (1r,3r)-3- (5-cyano-2-oxo- 2,3-dihydro-1H-benzo[d]imidazol-1-yl)cyclobutane-1-carbohydrazide (58.8 mg, 0.217 mmol). The vial was capped and the suspension was placed in a reaction block at 140°C and stirred overnight. The reaction mixture was cooled. The mixture was thoroughly evaporated to dryness and purified to afford the product.

MS (ESI) m/z for C26H20ClN7O = 481.94 (calcd) 482.1 ([M+H]⁺, found)

Example 30– IC₅₀ values

Cellular IC50 and biochemical IC50 values were determined in accordance with the protocols set out in Example 8 (see also Anumala et al., Discovery of Novel Series of Tankyrase Inhibitors by a Hybridization Approach J. Med. Chem.2017) for each of the compounds in Examples 14 to 29:

Table 10:

Claims

Claims:

1. A compound of general formula (I’):

wherein:

X represents -NR³- or -O-;

R³ represents H or a C_1-3 alkyl group (e.g. methyl);

or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

2. A compound as claimed in claim 1 of general formula (I):

wherein:

R³ represents H or a C1-3 alkyl group (e.g. methyl);

or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

3. A compound as claimed in claim 1 having the general formula (II):

wherein R¹, R², R³, Z, n and m are as defined in claim 1 or claim 2, or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

4. A compound as claimed in claim 1 having the general formula (III):

5. A compound as claimed in claim 1 having the general formula (IV):

wherein R¹, R², R³, Z and L are as defined in claim 1 or claim 2; and

n is 0 or 1;

or a tautomer, stereoisomer, or pharmaceutically acceptable salt thereof.

6. A compound as claimed in any one of claims 1 to 5 wherein Z is an optionally substituted pyrazolyl, imidazolyl, pyrazolinyl, imidazolinyl, pyridazinyl, pyridinyl, pyrimidinyl, pyrazinyl, or thiazolyl group.

7. A compound as claimed in any one of claims 1 to 5 wherein Z is a pyrimidinyl group.

8. A compound as claimed in any one of claims 1 to 7 wherein either n is 0, or n is 1 and R¹ is Cl or F.

9. A compound as claimed in any one of claims 1 to 8 wherein either m is 0, or m is 1 and R² is Cl, F, or -CN.

10. A compound as claimed in any one of claims 1 to 9 wherein R³ is H.

11. A compound as claimed in any one of claims 1 to 10 wherein the bonds to group L are disposed trans to one another.

12. A compound as claimed in claim 1 selected from the following, or a pharmaceutically acceptable salt thereof:

13. A compound as claimed in claim 1 which is

or

.

14. A compound of general formula (VII):

wherein:

Z represents an optionally substituted 5- or 6-membered unsaturated heterocyclic group comprising at least one nitrogen atom;

R¹ represents F, Cl, Br, I, C_1-3 alkyl, C_1-3 haloalkyl, -CN, -OH or -NO₂; and n is an integer from 0 to 5.

15. A compound of general formula (VIII):

wherein:

L represents a 4-, 5- or 6-membered cycloalkyl group;

R² represents F, Cl, Br, I, C_1-3 alkyl, -CN, -OH or -NO₂;

m is an integer from 0 to 5; and

R³ represents hydrogen or C_1-3 alkyl (e.g. methyl).

16. A method for the preparation of a compound of formula (I’) or formula (I) as defined in claim 1 or claim 2, said method comprising:

(a) reacting a compound of general formula (VII) as defined in claim 14 with a

compound of general formula (VIII) as defined in claim 15;

(b) optionally resolving a compound thus obtained into the stereoisomers thereof, and/or (c) optionally converting a compound thus obtained into a salt thereof, particularly a pharmaceutically acceptable salt thereof.

17. A method for the preparation of a compound of formula (VII) as defined in claim 14, said method comprising:

and

wherein in formulae (IX), (X), (XI) and (XII):

R¹ represents F, Cl, Br, I, C1-3 alkyl, C1-3 haloalkyl, -CN, -OH or–NO2; and

n is an integer from 0 to 5.

18. A method for the preparation of a compound of formula (VIII) as defined in claim 15, said method comprising:

and

wherein:

L represents a 4-, 5- or 6-membered cycloalkyl group;

R² represents F, Cl, Br, I, C1-3 alkyl, -CN, -OH or -NO2;

m is an integer from 0 to 5; R^3’ is a C1-3 alkyl (e.g. methyl) group; and

G denotes a leaving group.

19. A pharmaceutical formulation comprising a compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers or excipients.

20. A compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, for use in therapy.

21. A compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, for use in the treatment and/or prevention of any condition or disease which is affected by over-activation of signaling in the WNT pathway, e.g. in the treatment and/or prevention of such conditions or diseases which involve activation of ß-catenin, or for use in the treatment and/or prevention of any condition or disease which can be alleviated by stabilization of TNKS target proteins such as AXIN.

22. A compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, for use in preventing and/or retarding proliferation or metastasis of tumor cells, in particular carcinomas such as adenocarcinomas, or improving their immune recognition.

23. A compound for use as claimed in claim 22 in preventing and/or retarding proliferation or metastasis of a tumor emerging from colorectal tissue, uterus, pancreas, skin, liver, thyroid, prostate, ovary, stomach, lung, lymphoid, bladder, cervix, thyroid, head and neck, brain, breast or kidney, for example in the treatment and/or prevention of colorectal cancer, non-small cell lung cancer or melanoma.

24. A compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of a non-cancerous condition influenced by the activity of tankyrase 1/2.

25. A compound for use as claimed in claim 24 in the treatment of non- regenerative wound healing, in the treatment or prevention of a viral infection, in the treatment or prevention of fibrosis such as pulmonary-, dermal-, renal-, liver- or myocardial fibrosis, or in the treatment or prevention of a metabolic condition such as aberrant systemic glucose metabolism.

26. Use of a compound as claimed in any one of claims 1 to 13, or a

pharmaceutically acceptable salt thereof, in the manufacture of a medicament for use in the treatment or prevention of any condition or disease defined in any one of claims 21 to 25.

27. A method of treatment of a human or non-human animal body to combat or prevent a condition or disease defined in any one of claims 21 to 25, said method comprising the step of administering to said body an effective amount of a compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.

28. A method (e.g. an in vitro method) of promoting and/or directing cellular differentiation comprising contacting a progenitor cell with an effective amount of a compound as claimed in any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof.