WO2002096430A1 - Cephalosporin derivatives as anti-cancer agents - Google Patents

Abstract

The invention relates to a compound and/or pharmaceutically acceptable salts, esters, hydrates or solvates thereof, having the structural formula (1) or (2): (Please insert formulas 1 and 2)wherein:- X and Y are independently selected from the group comprising: H,C,N,O,S or alkyl,- W is selected from the group comprising: NH2, NH-R1, NH-CO-R1, NH-CO-Cn-O-R1, | R4 NH-CO-Cn-R1, NH-CO-O-Cn-R1, NH-CO-Cn-S-R1, NH-CO-Cn-NH- R1 | | | | R4 R4 R4 R4 wherein R4 is optional, and n = 1-12, preferably 1-6 and more preferably 1-4- V is selected from the group comprising: H, C, N, O, S or a terminal alkyl group, whereby when V is a terminal alkyl group, X and R2 are nothing,- Z is selected from the group comprising: S, S = O, O = S = O,- Q is selected from the group comprising: H, or R1, R2, R3, or R4- R1, R2, R3, or R4 are independently selected from the group comprising: H, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxycarbonyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl.

Description

CEPHALOSPORIN DERIVATIVES AS ANTI-CANCER AGENTS

The present invention concerns a class of compounds having utility as anti- cancer agents, for example melanomas, hepatoblastomas, hair follicle tumors, medulloblastomas and particularly which interact with the mechanism of colorectal cancer and other cancers, pharmaceutical composition comprising these compounds, the synthesis of these compounds, and their use in treating disease, specifically cancer.

The identification of LEF/TCF transcription factors as interaction partners of β-catenin and the identification of this active transcription complex in many tumor cells is one of the major breakthroughs in the field of signal transduction in embryonic development and tumor progression. Previously, interest had been focused on the well established function of β-catenin as a cytoplasmic mediator of cell adhesion in complex with cadherins. The more recently discovered interaction with LEF/TCF has been shown to be an integral part of the wnt signalling cascade during embryonic development and to become dysregulated, and constitutive active as an early event in tumor development. Dysregulation of the cytoplasmic pool of β-catenin is based on inhibition of degradation and translocation of the protein from the cytoplasm into the nucleus, the site where β-catenin permanently activates genetranscription in a ternary complex with LEF/TCF and DNA.

In the absence of wnt signalling under physiological conditions the cytoplasmic pool of β-Catenin is regulated by a multiprotein complex which controls β-catenin degradation. This contains β-Catenin, the serine/threonine kinase GSK3β, the tumor suppressor gene product APC, and conductin/axin. In 90% of human colon cancer, mutations in either β-catenin, conductin/axin or APC affect this complex . As a consequence cytoplasmic β-catenin accumulates and is translocated into the nucleus in complex with LEF/TCF. There target genes like c-myc or cyclin Dl then become constitutively active. In a first aspect the present application concerns compounds which

interfere with this complex formation.

According to this first aspect, the present invention concerns a compound

and/or pharmaceutically acceptable salts, esters, hydrates or solvates thereof, having the

structural formula (1) or (2):

(l)

(2)

wherein:

- X and Y are independently selected from the group comprising: H,C,N,O,S or alkyl,

- W is selected from the group comprising: NH₂, NH-Ri, NH-CO-Ri, NH-CO-Cn-O-Ri,

NH-CO-Cn-Ri, NH-CO-O-Cn-Ri, NH-CO-Cn-S-Ri, NH-CO-Cn-NH- Ri

wherein R₄ is optional, and n = 1-12, preferably 1-6 and more preferably 1-4

- V is selected from the group comprising: H, C, N, O, S or a terminal alkyl group, whereby when V is a terminal alkyl group, X and R₂ are nothing,

- Z is selected from the group comprising: S, S = O, O = S = O, - Q is selected from the group comprising: H, or Ri, R₂, R3, or R

- Ri, R₂, R3, or R₄ are independently selected from the group comprising: H, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxycarbonyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl.

The binding domains of β-catenin for all factors mentioned above were analysed by the inventors by performing an in vitro mutagenesis procedure. Point mutants of β-catenin were tested for interaction with LEF/TCF, APC, conductin/axin and E- Cadherin. Essential contact points for individual interactions were identified and termed "hot spots". Amino acid residues in the "hot spot" for LEF/TCF binding were identified which abrogate LEF/TCF binding when mutated, but do not affect binding of APC or conductin/axin. The hot spot for interaction with the LEF/TCF-transcription factors was used for a computer aided virtual drug screening for low molecular weight compounds which should fit ideally into a nearby hydrophobic pocket. These compounds were tested in an ELISA for inhibition of LEF/TCF to β-catenin binding.

Furthermore the inventors have demonstrated that the compounds according to the present invention substantially lack antibiotic activity, whereby the selectivity and activity of these compounds is enhanced and the potential for undesirable side effects is diminished.

Further preferred features of the compound according to the present invention are defined in claims 2-21.

Definitions of the various terms

Listed below definitions of various terms used to describe the compunds of the present invention.

It should be noted that any heteroatom with unsatisfied valances is assuemed to have the hydrogenatom to satisfy the valances. The term "alkyl" or "alk" refers to a monovalent alkane (hydrocarbon) derived radical containing from 1 to 12 carbon atoms unless otherwise defined.

An alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group. When substituted, the alkyl groups may be substituted, at any available point of attachement. When the alkyl group is said to be substituted with alkyl group this is used interchangeably with "branched alkyl group". Exemplary unsubstitueted such groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. Exemplary substituents may include but are not limited to one or more of the following groups: halogen (such as F, CI, Br, I), haloalkyl (such as CCb and CF₃), alkoxy, alkylthio, hydroxy, carboxy (-COOH), alkyloxycarbonyl (-C(O)R), alkylcarbonyloxy (-OCOR), amino (-NH₂), carbamoyl (-NHCOOR- or - OCONHR-), urea (-NHCONHR) or thiol (-SH).

Alkyl groups as defined may also comprise one or more carbon to carbon double bonds or one or more carbon to carbon triple bonds.

The term "alkenyl" refers to a hydrocarbon radical straight, branched or cyclic containing from 1 to 20, preferably 2 to 12 carbon atoms and at least one carbon to carbon double bond.

The term "alkynyl" refers to a hydrocarbon radical straight, branched or cyclic containing from 1 to 20, preferably 2 to 12 carbon atoms and at least one carbon to carbon triple bond.

Cycloalkyl is a specie of alkyl containing from 1 to 20, preferably 3 to 15 carbon atoms, without alterning or resonating double bonds between carbon atoms. It may contain from 1 to 4 rings. Exemplary unsubstituted such groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, etc. Exemplary substituents include one or more of the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or alkylthio. The terms "alkoxy" or "alkylthio", as used herein, denote an alkyl group as described above bonded through an oxygen linkage (-O-) or a sulfur linkage (-S-), respectively. The term "alkoxycarbonyl", as used herein, denotes an alkoxy group bonded through a carbonyl group. An alkoxycarbonyl radical is represented by the formula: - C(O)OR, where the R group is a straight or branched CM 2 alkyl group.

The term "alkylcarbonyl" refers to an alkyl group bonded through a carbonyl group.

The term "alkylcarbonyloxy", as used herein, denotes an alkylcarbonyl group which is bonded through an oxygen linkage.

The term "arylalkyl", as used herein, denotes an aromatic ring bonded to an alkyl group as described above.

The term "aryl" refers to monocyclic or bicyclic aromatic rings, e.g. phenyl, substituted phenyl and the like, as well as groups which are fused, e.g. naphtyl, phenanthrenyl and the like. An aryl group thus contains at least one ring having at least 6 atoms, with up to five such rings being present, containing up to 22 atoms therein, with alternating (resonating) double bonds between adjacent carbon atoms or suitable heteroatoms. Aryl groups may optionally be substituted with one or more groups including, but not limited to halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)_m (m = 0,1,2), or thiol.

The term "heteroaryl" refers to a monocyclic aromatic hydrocarbon group having 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing at least one heteroatom, O, S, or N, in which a carbon or nitrogen atom is the point of attachement, and in which one to three additional carbon atoms is optionally replaced by a heteroatom selected from O, N, or S, said heteroaryl group being optionally substituted as described herein. Exemplary heteroaryl groups include the following: thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl, piperidinyl, thiazolyl, pyrazinyl, pyridazinyl, pyrimidinyl, triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, oxazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, benzofuranzanyl and tetrahydropyranyl. Exemplary substituentsinclude one or more of the following: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkyl-S(O)_m (m = 0,1,2), or thiol.

The term "heterocycloalkyl" refers to a cycloalkyl group (nonaromatic) in which one to three of the carbon atoms in the ring are replaced by a heteroatom selected from O, S or N.

The term "heteroatom" means O,S or N, selected on an indepent basis. The term "halogen" refers to chlorine, bromine, fluorine or iodine. When a functional group is termed "protected", this means that the group is in modified form to preclude undesired side reactions at the protected site. Suitable protecting groups fo the compounds of the present invention will be recognized from the present application taking into account the level of skill in the art, and with reference to standard textbooks, such as Greene, T.W. et al, Protective Groups in Organic Synthesis, Wiley, N.Y. (1991).

Suitable examples of salts of the compounds according to the invention with inorganic or organic acids are hydrochloride, hydrobromide, sulfat, phosphat. Salts which are unsuitable for pharmaceutical uses but which can be employed, for example, for the isolation or purification of free compounds (1), (2) or their acceptable salts, are also included. Suitable salts of carboxylic groups of the compounds like sodium, potassium, lithium or magnesium or other pharmaceutically acceptable salts are also included.

All stereoisomers of the compounds of the instant invention are contemplated, either in a mixture or in pure or substantially pure form. The definition of the compounds according to the invention embraces all possible stereoisomers and their mixtures. It very particularly embraces the racemic forms and the isolated optical isomers having the specified activity. The racemic forms can be dissolved by physical methods, such as, for example fractional crystallisation, separation or crystallisation of diastereomeric derivatives or separation by chiral column chromatography. The individual optical isomers can be obtained from the racemates by conventional methods, such as, salt formation with an optically active acid followed by crystallisation. It should be understood that solvates (e.g. hydrates) of the compounds of formula (1), (2) are also within the scope of the present invention. Methods of solvation are generally known in the art. Accordingly, the compounds of the instant invention may be in the free or hydrate form, and may be optained by methods exemplified.

Mutations in the genes for APC, conductin/axin, and β-Catenin occur in 90% of human colorectal carcinomas, in 48% of sporadic hepatoblastomas, in melanomas, in hair-follicle tumors, medulloblastomas, and many other tumors. All these mutations result in permanent complex formation and nuclear translocation of β -Catenin complexed with LEF/TCF. Mutations in the APC gene have been demonstrated to be an early event in colorectal tumorigenesis. The compounds which interfere with complex formation have a high potential to block tumor growth.

In cancer cells harbouring mutations in the genes for APC or conductin/axin repair by expression of functional proteins results in cell death (apoptosis) or a block in cell division. This suggests that the cancer cells must be still sensitive to agents which interfere with signal transduction by LEF/TCF and β-Catenin.

In a mouse model sytem based on a mutant APC gene multiple intestinal adenomas develop. The corresponding truncated APC protein is non functional for binding to conductin/axin and as a consequence cannot regulate complex formation of β-Catenin and LEF/TCF. Reintroduction of the mutant APC protein which has been only repaired by adding the conductin/axin binding domain results in complete prevention of tumor formation. The addition only complements for initiation of β-Catenin degradation. This finding argues for the potential of drugs to prevent tumor formation in a majority of human tumors which develop through constitutive signalling of _β-Catenin and LEF/TCF.

Another aspect of the present invention concerns the synthesis of these compounds based on modifications of the lead structure which increase inhibitory activity of these to the nanomolar concentration range. The modified structures demonstrate no or only weak antibiotic activity. Furthermore the compounds efficiently inhibit intracellular reporter genes which are activated by β-Catenin and LEF/TCF. According to this aspect, there is provided a process for synthesising these compounds comprising acylating a cephalonsporanic acid of the following structural formula:

R. wherein Z, Q, V, X, R₂ and R3 have the meaning as defined in any of the claims 1-19 with an acyl halide selected from the group comprising the following members:

O

wherein Ri, X, R4, and n have the same meaning as defined in any of the claims 1-19. According to further aspects of the present invention there is provided amongst others, a medicament comprising these compounds, and an assay for determining the effectivity of these compounds.

Rar.korminri of the invention The interaction of cytoplasmic β-Catenin and LEF/TCF transcription actors is the last step in transducing a signal of the wnt signal transduction pathway to the nucleus. The pathway is named by the extracellular messenger protein wnt. Receptors on epithelial cells are activated by binding of wnt-protein. The membrane spanning receptor transduces this signal into the cell. A cascade of intracellular proteins then transmitts this signal to the nucleus, where the cellular program is modified by activation of ^β-Catenin and LEF/TCF dependent target genes. The formation of Tcf/β-catenin transcription factor complexes in the cell nucleus is normally subject to tight regulation, which serves to ensure that the target genes are only switched on at the correct time during embryonic development or for renewal of epithelia in adult organisms. However, in colon cancer for example this regulation is commonly lost through genetic alterations of key tumor suppressor genes such as APC or through mutations in β-catenin itself. These mutations result in loss of some of the β- Catenin interaction domains of APC or in loss of phosphorylation sites of β-Catenin which prevents its earmarking for degradation. As a result, Tcf/β-catenin complexes are constantly present in the cell nucleus and target genes are inappropriately activated, leading to the formation of benign tumors in the small intestine. Additional genetic alterations in other key tumor suppressor genes and oncogenes are then accumulated over time, which result in the formation of invasive colon tumors. Loss of regulation of Tcf/β-catenin complex formation is considered to be one of the earliest events in colon cancer and there is growing evidence to indicate that inhibition of Tcf/β-catenin signaling may be an effective therapy for treatment of colon carcinoma and other cancers.

In a second function β-Catenin is a key constituent of cell adhesion complexes via direct interaction with the cytoplasmic tail of cadherins. Loss of cell adhesion correlates with the maligne potential of tumors.

Experimental 1

A number of compounds were synthesised and subsequently analysed for inhibitory activity with regard to the complex formation of β-catenin and LEF/TCF.

Synthesis: General outline of compounds according to the present invention

To acylate the amino group the acids were activated by one of the following procedures if they are not commercially available as acid chlorides, active esters or in another activated form. To activate the organic acids one of the two following literature known methods was used. The carboxylic acids were activated with thionyl chloride to generate the acid chloride. M 60 °C, 6 h ° . + socι₂ »-

R OH R I scheme 1 activation with thionyl chloride

The α-hydroxy- or the β-hydroxy acids were normally activated by transformation with phosgene to the l,3-dioxolane-2,4-dione derivative.

scheme 2 activation with phosgene

In all cases, the activated acids were used without any further purification. The activated acids were then reacted with the amino compounds. This was generally done by dissolving the amino compound and the base, in almost all cases sodium hydrogen carbonate, in water/acetone at 0°C and successional adding of the activated acid, dissolved in tetrahydrofuran. If there was another purification necessary after washing this was generally done by preparative HPLC or chromatographie on silica gel.

scheme 3 exemplary acylation with an acid chloride of a cephalosporanic acid The exchange of the acetoxy group to generate a thio ether could be achieved by the transformation of an N-acyl-7-aminocephalosporanic acid with an mercapto compound. For this an /V-acyl-7-aminocephalosporanic acid and 2 equivalents sodium hydrogencarbonate were dissolved in a phosphate buffer (pH 6.4; 7.15 ml 1/15 M KH2PO₄ and 2.46 ml 1/15 M ΝaHPO₄) and 0.98 equivalents of a mercapto compound were added and the mixture was heated at 60°C for 16 h. The reaction mixture was worked up by diluting with ethylacetate and washing with hydrochloric acid. If there was purification necessary this was generally done by preparative HPLC or chrornatographie on silica gel.

scheme 4 exchange of the acetoxy group of an N-acyl-7-aminocephalosporanic acid

As the case may be one or more equivalents of dimethyldioxirane were used in the reaction with an N-acyl-7-aminocephalosporanic acid derivative you get either the two corresponding sulfoxides or the corresponding sulfon.

if x = 1 then n = 1 if x = 4-5 then n = 2 scheme 5 synthesis of the sulfon or the sulfoxide

To block the free carboxylic acid e.g. to generate the methyl ester this can be achieved with the transformation of an N-acyl-7-aminocephalosporanic acid derivative or an N-acyl-7-aminodesacetoxycephalosporanic acid derivative with dimethylsulfate.

scheme 6 generating the methyl ester with dimethylsulfate

General procedure for the activation of acids or diacids with thionyl chloride

0.5 mmol of an organic acid or diacid were dissolved in 10 eq. of thionyl chloride and heated to 60°C. After 6 -12 h the excessive thionyl chloride was removed under reduced pressure and the acid chloride could be used directly without any further purification.

General procedure for the activation of rv-hydroxy- and β-hydroxy acids with phosgene

0.5 mmol of a α-hydroxy-carboxylic acid or a β-hydroxy-carboxylic acid were dissolved in 2 ml tetrahydrofuran then 10 eq. of phosgene (2 molar solution in toluene) were added. The solution was stirred for 6 to 16 h at room temperature and the excessive phosgene and the solvent were removed under reduced pressure. The anhydride can be used directly without any further purification.

General procedure for the acylation of the amino fnnrtion of 7-aminocephalo-sporanic acid and 7-aminodesar,etoyycephalosporanic acid

To a solution of 136 mg (0.5 mmol) of 7-aminocephalosparanic acid or 7- amino-desacetoxycephalosporanic acid and 105 mg of sodium hydrogencarbonate in a mixture of 3 ml water and 1.5 ml acetone and cooled to 0°C. 0.5 mmol of an acid chloride or acid anhydride, dissolved in 1 ml tetrahydrofurane, were added. The mixture was stirred for 2-12 h at O°C.

10 ml of saturated sodium hydrogencarbonate solution were added and the alkaline water layer was extracted with 10 ml ethylacetate. The aqueous layer was separated, 10 ml ethylacetate were added and the solution was acidified with 1 N HC1 to a pH of 2. The solution was extracted 3 times with 10 ml ethylacetate. The combined organic layers were dried over magnesium sulphate and the solvent was removed under reduced pressure. Purification was performed by preparative HPLC if necessary.

General procedure for the transformation of /V-Acyl-7-aminocephalosporanic acids with mercapto-compound

To a solution of 0.077 mmol of a N-acyl-7-aminocephalosporanic acid and 0.15 mmol of sodium hydrogencarbonate in 1 ml phosphate buffer (pH 6.4; 7.15 ml 1/15 M KH₂PO₄ and 2.46 ml 1/15 M ΝaHPO₄) 0.075 mmol of a mercapto compound were added and the mixture was heated at 60°C for 16 h. After cooling to room temperature 5 ml ethylacetate were added and the solution was acidified with 1 N HC1 to a pH of 2. The aqueous layer was extracted 3 times with 10 ml ethyl acetate and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure. The raw product was purified by preparative HPLC if necessary.

General procedure for the ringopening of the β-lactamering of A -acyl-7-amino- cephalosporanic acid or /V-acyl-7-aminodesacetoxycephalosporanic acid derivatives

100 mg of a N-acyl-7-aminocephalosporanic acid or N-acyl-7- aminodesacetoxy-cephalosporanic acid derivatives were dissolved in 5 ml carbonate buffer (pH 10-11) and stirred for 16 h at 40°C. The mixture was lyphylized. Purification was performed by preparative HPLC. General procedure for the oxidation of λ -acy1-7-aminocephalosporanic acid or λ/-acy1-7- aminodesacetoxycephalosporanic acid derivatives to the corresponding sulphoxide

To a solution of 0.1 mmol of a N-acyl-7-aminocephalosporanic acid or N- acyl-7-aminodesacetoxycephalosporanic acid derivative in 2 ml acetone, cooled to 0°C, 1 equivalent dimethyldioxirane, dissolved in 1 ml acetone was added. The solution was stirred for 1 h at 0°C. The solvent was removed under reduced pressure. No further purification was necessary. (The diastereomeres can be separated by preparative HPLC if necessary.)

General procedure for the oxidation of Λ/-acyl-7-aminoceρhalosporanic acid or λ -acy1-7- aminodesacetoxycephalosporanic. acid derivatives to the corresponding snlfoxide

To a solution of 0.1 mmol of a N-acyl-7-aminocephalosporanic acid or N- acyl-7-aminodesacetoxycephalosporanic acid derivative in 2 ml acetone, cooled to 0°C, 4 equivalent dimthyldioxirane, dissolved in 1 ml acetone was added. The solution was stirred for 1 h at 0°C. The solvent was removed under reduced pressure. No further purification was necessary.

General procedure for the transformation of Λ/-acyl-7-aminocephalosporanic acid or N- acyl-7-aminodesacetoxycephalosρoranic acid derivatives to the corresponding methyl ester To a solution of 0.2 mmol of a N-acyl-7-aminocephalosporanic acid or N- acyl-7-aminodesacetoxycephalosporanic acid derivative and 0.8 mmol of sodium hydrogencarbonate in 4 ml water/acetone 1 : 1 (v/v), 0.8 mmol dimethyl sulfate were added and the reaction mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydrogencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary. Synthesis of some examples

STF 196

99 mg (0.5 mmol) of 4-biphenylcaboxylic acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 136 mg (0.5 mmol) of the 7-aminocephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and then the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 4.57 min. MS: m/e 903.9 (2M-H)⁺ Η-NMR (400 MHz, CDCb): δ = 7.83 (d, J= 10 Hz, 2 H, arom. H), 7.65 (d, J ^~~ 10 Hz, 2 H, arom. H), 7.57 (d, J= 10 Hz, 2 H, arom. H), 7.47-7.40 (m, 2 H, arom. H), 7.38-7.32 (m, 2 H, arom. H), 5.98-5.92 (m, 1 H, NH-CH), 5.10 (d, J' = 13 Hz, 1 H, CH₂-O), 5.08 (d, J" = 4.5 Hz, 1 H, CH-S-), 4.88 (d, J' = 13 Hz, 1 H, CH₂-O), 3.58 (d, J' = 16 Hz, 1 H, CH2-S), 3.35 (d, J' = 16 Hz, 1 H, CH₂-S), 2.04 (s, 3 H, CH₃).

STF?.13

83 mg (0.5 mmol) of 4-fluorophenylacetic acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 136 mg (0.5 mmol) of the 7-aminocephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and then the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 3.41 min. MS: m/e 814.9 (2M-H)⁺

STF7.1 .

86 mg (0.5 mmol) of 1-naphtoic acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 136 mg (0.5 mmol) of the 7-aminocephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and then the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 3.81 min. MS: m/e 851.9 (2M-H)⁺ Η-NMR (400 MHz, CDCb): δ = 8.49-8.32 (m, 2 H, arom. H), 7.98-7.82 (m, 3 H, arom. H), 7.72-7.42 (m, 4 H, arom. H), 6.97-6.89 (bs, 1 H, NH), 6.13-6.09 (m, 1 H, NH-CH), 5.18 (d, J = 4.5 Hz, 1 H, CH-S), 5.13 (d, J' = 13 Hz, 1 H, CH₂-O-), 4.93 (d, J' = 13 Hz, 1 H, CH2-O-), 3.63 (d, J' = 17 Hz, 1 H, CH₂-S-), 3.42 (d, J' = 17 Hz, 1 H, CH2-S-), 2.07 (s, 3 H, CH₃).

STF7.

109 mg (0.5 mmol) of 2-(+-)-cyclohexylphenylacetic acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 136 mg (0.5 mmol) of the 7-aminocephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and then the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6*125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 5.17 min. MS: m/e 943.1 (2M-H)⁺

STF9.40

82 mg (0.5 mmol) of 2-phenylbutyric acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 136 mg (0.5 mmol) of the 7-aminocephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and then the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6*125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 4.05 min. MS: m/e 834.9 (2M-H)⁺

STF287

540 mg (2 mmol) of 2-(2,4,5-trichlorophenoxy)-propionic acid were dissolved in 3.1 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 544 mg (2 mmol) of the 7- aminocephalosporanic acid and 420 mg of sodium hydrogencarbonate were dissolved in a mixture of 6 ml acetone and 12 ml water and cooled to 0°C. The acid chloride, dissolved in 8 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 20 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 40 ml ethyl acetate and the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 60 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 5.04 min. MS: m/e 1044.7, 1046.6, 1048.6 (2M-H)⁺ STF 00

92 mg (0.5 mmol) of 10-undecylenic acid were dissolved in 1.5 ml thionyl chloride and heated to 60 °C. After 6-16 h the excessive thionyl chloride was removed under reduced pressure. 107 mg (0.5 mmol) of the 7-aminodesacetoxy-cephalosporanic acid and 105 mg of sodium hydrogencarbonate were dissolved in a mixture of 1.5 ml acetone and 3 ml water and cooled to 0°C. The acid chloride, dissolved in 2 ml tetrahydrofurane was added and the mixture was stirred for 3 h at 0°C. 5 ml of a saturated sodium hydrogencarbonate solution were added and the alkaline water layer was washed twice with 10 ml ethyl acetate and the water layer was acidified with 1 N HCl to a pH of 2. The solution was extracted 3 times with 15 ml ethyl acetate. The combined organic layers were dried with magnesium sulfate and the solvent was removed under reduced pressure. No further purification was necessary.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6*125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 5.24 min. MS: m/e 760.1 (2M-H)⁺

1H-NMR (400 MHz, CDC1₃): δ = 6.35 (bs, 1 H, NH), 5.85-5.75 (m, 2 H, CH=CH₂, -NH- CH), 5.00 (s, 1 H, CH-S-), 4.98 (d, J ^~~ 11 Hz, 1 H, CH=CH ), 4.93 (d, J = 11 Hz, 1 H, CH=CH₂), 3.57 (d, J' = 17 Hz, 1 H, CH2-S-), 3.25 (d, J' = 17 Hz, 1 H, CH2-S-), 2.38-2.30 (m, 2 H, CH₂=CO-), 2.21 (s, 3 H, CH₃), 2.06-1.99 (m, 2 H, CH₂-CH=), 1.69-1.58 (m, 2 H, CH₂-CH2-CO), 1.40-1.20 (m, 10 H, 5 * CH₂).

STF 10 To a solution of 83.6 mg (0.2 mmol) of N-((+-)-3-phenylbutyryl)-7-amino- cephalosporanic acid and 68 mg (0.8 mmol) of sodium hydrogencarbonate in 2 ml water and 2 ml acetone 76 μl dimethyl sulfate were added. The mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydrogencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 10% to 90 % B gradient in 5 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 3.90 min. MS: m e 455.1 (M+ Na)⁺ 1H-NMR (400 MHz, CDCb): δ = 7.34-7.28 (m, 2 H, arom. H), 7.23-7.17 (m, 3 H, arom. H), 6.22-6.13 (m, 1 H, NH), 5.81-5.74 (m, 1 H, CO-CH-N), 5.12-5.06, 4.85-4.79 (m, m, 2 H, CH₂-O), 4.95, 4.90 (d, J = 8 Hz, 1 H, -N-CH-S-), 3.87 (s, 3H, CH₃, methylester), 3.84- 3.78, 3.57-3.50 (m, m, 2 H, CH₂-S-), 3.32-3.24 (m, 1 H, Ph-CH-), 2.65-2.45 (m,2 H, CH₂- CO), 2.08 (s, 3 H, CH₃, acetyl), 1.34-1.28 (m, 3 H, CH3).

STF 1 1

To a solution of 104.8 mg (0.2 mmol) of N-(2-(2,4,5-trichlorophenoxy)- propionyl)-7-aminocephalosporanic acid and 68 mg (0.8 mmol) of sodium hydrogencarbonate in 2 ml water and 2 ml acetone 76 μl dimethylsulfate were added. The mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydrogencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): rt = 6.21 min. MS: m e 561.0 (M+ Νa)⁺

STF 17 To a solution of 89.9 mg (0.2 mmol) of N-(6-bromohexanoyl)-7- aminocephalosporanic acid and 68 mg (0.8 mmol) of sodium hydrogencarbonate in 2 ml water and 2 ml acetone 76 μl dimethylsulfate were added. The mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydrogencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 10% to 90 % B gradient in 5 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 3.76 min. MS: m/e 487.1 (M+ Na)⁺

Η-NMR (400 MHz, CDCb): δ =6.28 (bs, IH, NH), 5.85-5.82 (m, 1 H, CO-CH-N), 5.11, 4.85 (d, d, J= 14 Hz, CH₂-O-), 4.99 (d, J = 8 Hz, 3.87 (s, 3 H, CH₃, methyl ester), 3.58 (d, J" = 18 Hz, 1 H, CH₂-S), 3.41-3.36 (m, 3 H, CH₂-Br, CH₂-S), 2.32-2.26 (m, 2 H, CH₂- CO), 2.08 (s, 3 H, CH₃, acetyl), 1.92-1.84 (m, 2 H, CH₂-CH₂Br), 1.72-1.63 (m, 2 H, CH₂- CH2-CO), 1.52-1.45 (m, 2 H, CH₂).

STF 314

To a solution of 69.7 mg (0.2 mmol) of N-(benzyloxycarbonyl)-7-amino- desacetoxy-cephalosporanic acid and 68 mg (0.8 mmol) of sodium hydrogencarbonate in 2 ml water and 2 ml acetone 76 μl dimethylsulfate were added. The mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydro-gencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6*125 mm; 10% to 90 % B gradient in 5 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 4.36 min. MS: m e 363.1 (M+ H)⁺

STF31

To a solution of 73.7 mg (0.2 mmol) of N-(hex-3-enoyl)-7-amino- cephalosporanic acid and 68 mg (0.8 mmol) of sodium hydrogencarbonate in 2 ml water and 2 ml acetone 76 μl dimethylsulfate were added. The mixture was stirred for 2 days at room temperature. The solution was extracted twice with 10 ml saturated sodium hydrogencarbonate solution, twice with 10 ml 1 Ν HCl and 10 ml brine. The organic layer was dried with magnesium sulfate and the solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): rt = 4.01 min. MS: m/e 405.1 (M+ Na)⁺

Η-NMR (400 MHz, CDCb): δ = 5.75-5.71 (m, 1 H, CO-CH-N), 5.68-5.60, 5.48-5.40 (m, 2 H, CH=CH), 5.05, 4.78 (d, J = 14 Hz, 2 H, CH₂-O), 4.94 (d, J' = 7 Hz, 1 H, N-CH-S), 3.82 (s, 3 H, CH₃, methyl ester), 3.54, 3.34 (d, d, J" = 18 Hz, 2 H, CH₂-S), 2.96-2.91 (m, 2 H, CH2-CO), 2.04-1.91 (m, 5 H, CH₂-CH=, CH3, acetyl), 0.94 (t, J"" = 9 Hz, 3 H, CH₃).

STF340

To a to 0°C cooled solution of 52.4 mg (0.1 mmol) of N-(2-(2,4,5- trichlorophenoxy)-propionyl)-7-aminocephalosporanic acid in 2 ml acetone 1 equivalent dimethyldi-oxirane, dissolved in 1 ml acetone, was added. The mixture was stirred for 1 hour in the ice bath. The solvent was removed under reduced pressure. The diastereomeres were not seperated.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 10% to 90 % B gradient in 5 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): rt = 4.06 min. MS: m/e 794.8 (2M-H )⁺ Η-ΝMR (400 MHz, CDCb): δ = 7.42 (s, 1 H, arom. H), 6.91 (s, 1 H, arom. H), 6.03-6.00 (bs, 1 H, ΝH), 5.35-5.24, 5.16-5.09 (m, m, 1 H, CO-CH-Ν), 4.93-4.88 (m, 1 H, Ν-CH-SO- ), 4.82-4.68 (m, 2 H, CH₂-O), 4.68-4.62 (m, 1 H, CH-CO), 4.02-3.89, 3.83-3.64 (m, m, 2 H, CH2-SO), 2.08-2.01 (m, 3 H, CH₃, acetyl), 1.63-1.54 (m, 3 H, CH₃).

STF353

To a to 0°C cooled solution of 52.4 mg (0.1 mmol) of N-(2-(2,4,5- trichlorophenoxy)-propionyl)-7-aminocephalosporanic acid in 2 ml acetone 5 equivalents dimethyldi-oxirane, dissolved in 5 ml acetone, were added. The mixture was stirred for 1 hour in the ice bath. The solvent was removed under reduced pressure. HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 4.87 min. MS: m/e 1106.6, 1108.6, 1112.5 (2M-H )⁺

STF354

To a to 0°C cooled solution of 44.9 mg (0.1 mmol) of N-(6- bromohexanoyl)-7-aminocephalosporanic acid in 2 ml acetone 5 equivalents dimethyldioxirane, dissolved in 5 ml acetone, were added. The mixture was stirred for 1 hour in the ice bath and the solvent was removed under reduced pressure. The diastereomeres were not separated.

HPLC (Column: Waters Spherisorb ODS II, C18, 5μm, 4.6* 125 mm; 20% to 90 % B gradient in 6 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): rt = 3.47 min. MS: m/e 958.6, 960.6, 962.7 (2M-H )⁺

Η-ΝMR (400 MHz, CDCb): δ = 5.95-5.90 (m, 1 H, CO-CH-Ν), 5.09, 4.77 (d, d, J = 13 Hz, 2 H, CH2-O-), 4.88 (d, J' = 10 Hz, 1 H, Ν-CH-SO2), 3.95, 3.68 (d, d, J" = 18 Hz, 2 H, CH2-SO2), 3.32-3.21 (m, 2 H, CH₂Br), 2.23-2.17 (m, 2 H, CH2-CO), 2.03-1.97 (m, 3 H, CH₃), 1.78-1.72 (m, 2 H, CH₂-CH₂Br), 1.56-1.50 (m, 2 H, CH2-CH2CO), 1.41-1.35 (m, 2 H, CH₂).

KPT 049

To a solution of 30 mg (0.077 mmol) of a N-phenylacetylic-7- aminocephalosporanic acid and 12.9 mg (0.15 mmol) of sodium hydrogencarbonate in 1 ml phosphate buffer (pH 6.4; 7.15 ml 1/15 M KH2PO4 nd 2.46 ml 1/15 M ΝaHPO₄), 9.7 mg

(0.075 mmol) of mercaptophenol were added and the mixture was heated at 60°C for 16 h.

After cooling to room temperature 5 ml ethylacetate were added and the solution was acidified with 1 N HCl to a pH of 2. The aqueous layer was extracted 3 times with 10 ml ethyl acetate and the combined organic layers were dried over magnesium sulfate. The solvent was removed under reduced pressure. The raw product was purified by preparative

HPLC. HPLC (Column: CC 250/4 Nucleosil 100-5 C18, 10 % to 90 % B in 10 min. Solvent A: water + 0.1 % trifluoroacetic acid, Solvent B: 100 % acetonitrile + 0.1 % trifluoroacetic acid, UV: 214 nm, 254 nm, 301 nm): r_t = 8.10 min.

Preparative HPLC: (Column: 250/21 Nucleosil 100-5 C18 PPN, 10 % to 90 % B gradient in 26 min. Solvent A: water + 1 % acetonitrile + 0.1 % trifluoroacetic acid, Solvent B: acetonitrile + 1 % water+ 0.1 % trifluoroacetic acid). MS: m/e 91 1.0 (2M-H )⁺

Figure 1 details the chemical structure of these compounds.

Figure 2 details a number of further compounds of the present invention synthesised according to the general methods.

Experimental 7.

800 different compounds were synthesized and controlled for inhibitory activity of the complex formation of β-Catenin and LEF/TCF in an ELISA. Surprisingly inhibitory compounds were found with IC50 values of 5 μM to 0.9 μM using the ELISA (Table 1).

Table 1

Table 1 shows IC₅₀ values for inhibition of β-catenin to LEF/TCF binding (ELISA) and inhibition of cellular transactivation of reporter genes (cellular). 800 compounds were tested for interference with binding to LEF/TCF data of the most active compounds in ELISA and in the cellular transactivation assay are summarized in table 1. These compounds interfere in a concentration dependent manner with complex formation. For analysis of specificity of the compounds, they were also tested in an ELISA with the β-Catenin binding domains of E-Cadherin or APC (Fig. 3). Figure 3 shows that STF 354 specifically inhibits h TCF-4 binding to

β-Catenin. Binding of human TCF-4, E-Cadherin or the 20 amino acid repeat

containing domain of APC to β-Catenin are tested in the presence of increasing

concentrations of STF 354 as an example. The percentage of complexes formed

in the absence of compounds is set to 100%.

The compounds demonstrated no inhibitory activity for binding of β- Catenin to E-cadherin or APC at concentrations to 100 μM. As the starting compound- structures contained a cephalosporin core, we tested them for antibiotic activity (Fig. 4).

Figure 4 shows that modified cephalosporines demonstrate no antibiotic activity. Suφrisingly the compounds demonstrated no antibiotic activity for the E.coli bacteria, while ampicillin blocked growth of the bacteria under the same conditions. Therefore modifications abrogated the antibiotic potential of the compounds. The compounds were also tested for interference with DNA-binding of LEF/TCF transcription factors (Fig. 5). As expected the compounds do not interfere with DNA-binding. Figure 5 shows that the compounds do not interfere with DNA-binding of

LEF/TCF.

Analysis of the synthesized compounds in cellular assays

The basis of the cell-assay

The inventors have developed synthetic reporter genes which are activated when these Tcf/β-catenin transcription complexes are present in cell nuclei. These consist of 5 optimal binding motifs for Tcf (CCTTTGATC) upstream of a Thymidine kinase promoter linked to a firefly luciferase gene (termed TKTOPFLASH; see figure 6a). These reporter genes were introduced into the genome of colon carcinoma cell-lines LS 174T and DLD-1 via standard transfection methods to generate cell-lines L4Δ3T/F1 and D7Δ15T/F5 respectively. These cell-lines were also engineered to carry an internal control reporter gene which comprises 5 copies of a non-Tcf binding motif (CCTTTGGCC) upstream of a TK promoter driving high level expression of a second luciferase gene, Renilla (termed TKFOPRENILLA; figure 6b). Unlike TKTOPFLASH, the expression of this reporter gene is not subject to regulation by Tcf/β-catenin complexes, but is a convenient read-out for changes in cell viability. Additionally, these cells harbor an inducible expression construct for a Tcf-4 protein lacking the β-catenin binding site. Induction of this ΔNTcf-4 protein via addition of Doxycycline to the culture medium results in inhibition of TKTOPFLASH activity through direct competition with endogenous Tcf/β-catenin complexes for the Tcf binding sites. This serves as a convenient positive control for inhibition of Tcf/β-catenin signaling in these colon carcinoma cell-lines. Figure 6 is a schematic overview of TKTOP-luciferase (A) and TKFOP-

Renilla (B) .

Protein expression and purification

For analysis of inhibitory activity of compounds cDNA for human β- Catenin (amino acids 1-927) is subcloned into pQE32 (Qiagen), cDNAs for human TCF-4 (amino acids 1-129) or mLEF-1 (amino acids 1-394) are subcloned into pET16b (Novagen). For analysis of specificity of compounds cDNA of E-Cadherin (amino acids 773-884, cytoplasmic domain) and APC (amino acids 1006-2069, 15 and 20 amino acid repeats) are subcloned into pET16b. Expression from these vectors results in fusion proteins with N-terminal His-tags for purification. Proteins are expressed in the E.coli strains XL-1 (β-Catenin) and BL21(DE3)pLysS (TCF-4, E-Cadherin, APC) and prepared according to the manufacturers protocol for Ni-agarose purification (Qiagen). Briefly transformed bacteria are grown in 200 ml medium each at 37° C for 2 hrs with 200 rpm shaking. Expression is induced with 1 mM IPTG at an optical density of 0.6 at 600 nm for 3 hrs. Bacteria are lysed in presence of 10 mM EDTA using sonification. Lysates are loaded in batch on 1 ml Ni-agarose after addition of MgCb to 10 mM at 4°C with gentle shaking for 1 hour. After washing the agarose with loading buffer (50 mM Tris-HCl, pH 8.0; 250 mM NaCI, 10 mM MgCb , 0.005 % NP-40, 20% glycerol) by centrifugation (2 min, 1000 rpm, in an Eppendorf centrifuge) the proteins are eluted with 10 mM, 50 mM and 150 mM imidazole (Sigma). FT IS A for testing inhibition of R-Catenin to T EF/TGF binding

Microtiterplates (96well,Greiner, Maxisoφ) are used for binding 25-50 ng LEF/TCF per well for 1 hr at room temperature in PBS. After blocking with 5% low fat dry milk powder in PBS for 2-3 hrs, the plates are used for binding assays. Compounds are diluted in 10% DMSO/PBS and added to a final concentration of 1% DMSO/PBS to the binding reaction (i.e. 5 μl compound with 10 μM concentration in 10% DMSO added to 45 μl PBS incubation volume to final concentration of 1 μM and 1% DMSO). First compounds and then β-Catenin (50-100 ng) are added to the binding reaction in PBST (0.005% Tween-20 in PBS). Binding is performed for 15 minutes at room temperature. Wells are washed three times with PBST and binding of β-Catenin is detected with monoclonal anti-β-Catenin (Transduction Lab.), peroxidase conjugated secondary antibodies (Dianova) using 0.1 mg/ml TMB (3,3,5,5-Tetramethyl-Benzidine, Sigma) in 50 mM phosphate buffer (pH 5.0) as a substrate and photometric quantification at 450 nm. Specificity of compounds for inhibition of LEF/TCF binding is analyzed by controlling β- Catenin binding to E-Cadherin (5 ng adsorbed per microtiter well) or APC (50-100 ng per well) in the presence of inhibitors.

Compounds were solubilized at 100 mM concentrations in DMSO and stored at -20C. For usage in the ELISA compounds are diluted to 10 mM in PBS (diluting DMSO to 10%). Further dilutions (500 μM, 10 μM ...) are adjusted to 10% DMSO by addition of solvent for constant DMSO concentrations in the binding reactions. IC50 values were determined by plotting concentrations of compounds (dilution series) against the percentage of complexes formed, when compared with binding reactions in the absence of compounds (100% value). The program Excel (Microsoft) was used for calculations and graphical display.

F.T .TSA for analysis of DNA-hinding of T .F.F/TC.F in presence of compounds

Streptavidin coated microtiter plates (Exiqon) are loaded with biotinylated double stranded LEF/TCF binding site containing DNA (100 ng per well, BioTez, sense strand DNA-sequence: biotin-ggt agg gca ecc ttt gaa get etc cc, anti sense strand: biotin- ggg gag age ttc aaa ggg tgc ect ac) for 30 minutes at room temperature in PBST (in a volume of 50 μL PBS per well containing 0.005% Tween-20). After two washes with PBST compounds, LEF/TCF and 50 ng sonified E. coli carrier DNA are added in 50 μl PBST for 15 minutes. After two additional washes DNA binding of LEF/TCF is quantified with anti His-tag antibodies (Sigma) conjugated with peroxidase using TMB as a substrate. Competition by unlabelled binding site containing DNA is used as a positive control.

Assay for antihiotic activity of compounds

E. coli strain XL-1 is grown in culture medium to exponential growth. Doubling of optical density from 0.1 to 0.2 and 0.4 indicates this growth phase. The bacteria are transferred to prewarmed micotiter plates and incubated for 3 hours in the presence or absence of 100, 50, 25 and 10 μg/ml ampicillin or compound. The optical density of traeted and nontreated bacteria is compared.

Outline Protocol (cell based assay)

DAY 1: The culture medium is removed from the 175cm culture flasks containing L4Δ3/T/F1 or D7Δ15T/F5 cells, the cells washed twice with sterile phosphate- buffered saline (PBS) to remove all traces of fetal calf serum (FCS) and subsequently incubated with 3ml of trypsin-EDTA for 10 minutes at 37 C to achieve separation of the cells from the flask. The flasks are examined under a microscope to ensure that a single- cell suspension is achieved. The cells are then counted using a haemocytometer and plated out at 30,000 (for L4Δ3/T/F1), or 15,000 (for D7Δ15T/F5) cells per well of a 96 well, white-walled culture plate (Corning) in a final volume of lOOul. The 96 well plates are incubated overnight at 37°C; 5% CO2. DAY 2: The 96 well culture plates contain L4Δ3/T/F1 cells at 50% confluency or D7Δ15T/F5 cells at 30% confluency. The media is removed from the plates and replaced with 75ul of the appropriate concentration of a test compound (in duplicate) or control media according to the scheme depicted in Figure 9 (below). The cells are then incubated in the presence of these compounds for 24 hours at 37 C; 5% CO2. DAY 3: The media is removed from the assay plates and the cells lysed in 50ul of passive lysis buffer on a rocking platform for 15 minutes at room temperature. The amount of Luciferase and Renilla activity in the cell lysate is then measured using the Dual Luciferase reporter assay, according to the manufacturers instructions(Promega; see Appendix B for theory of the dual luciferase assay) on a MicroLumat LB96V (Berthold Technologies) 96 well luminometer (see Appendix C for detailed method). The data is directly transferred to an Excel template, enabling graphical presentation for easy inteφretation.

Materials cell based assay

L4Δ3/T/F1 or D7Δ15T/F5 colon carcinoma cell-lines cultured in RPMI media supplemented with 10% FCS, Penicillin/Streptomycin, Zeocin, Basticydin and G418

Assay medium: RPMI supplemented with 5% FCS and Penicillin/Streptomycin

96 well flat bottom assay plates DMSO

Doxycycline

Dual Luciferase Reporter assay system lml Polypropylene storage blocks plus mat

Microlumat LB 96V WinGlow software

Microsoft Excel

-80°C Freezer

The Dual Luciferase assay Firefly and Renilla luciferases have different enzyme structures and substrate requirements. These differences make it possible to selectively discriminate between their respective luminescent reactions. Thus, using the dual luciferase assay system, the luminescence from the firefly luciferase reaction (experimental reporter) may be quenched while simultaneously activating the luminescent reaction of Renilla luciferase (control reporter).

This allowed the inventors to determine the activity of both the TKTOPFLASH and TKFOPRENILLA reporter genes in the colon carcinoma cell-lines in a single experiment.

Following injection of the Luciferase substrate the following biochemical reaction is initiated, which results in the emission of photons. These photons are "captured" by the Microlumat and a signal is generated that corresponds to the luciferase activity (TKTOPFLASH) present in the sample.

Firefly luciferase Beetle luciferin + ATP + O2 oxylluciferin + AMP + Ppi + CO2 + Light

The subsequent injection of the Renilla substrate quenches this reaction and initiates a second biochemical following reaction catalysed by the Renilla activity in the sample. Again, this reaction causes light to be generated and this is quantified as a measure of the Renilla activity (TKFOPRENILLA) in the sample.

Renilla Luciferase Coelenterazine + O2 Coelenteramide + CO2 + Light

Measurement of Luciferase and Renilla activities using the Microlumat T .R96V

Set up the injector systems pf the Microlumat with the Luciferase substrate connected to injector 1 and the Stop & Glo (Renilla substrate) connected to injector 2. Enter the WinGlow program to start measuring:

Prime the system with the substrates to ensure the tubing is full of liquid: *^♦ taskbar -» measure -» prime *♦ number of injections: 7 ** check the checkboxes for injector P and injector M

•* press start Initiate the actual measurement:

** taskbar -» measure — » integrate »> open file DLR25.IPT (C:\WinGlow)

** select all the circles (blue and red) of the wells to be measured *^♦ Specify the following parameters for injecting the substrates: Inj. P: delay 2.0 sec;

Meas. Interval 1 10.0 sec. Inj. M:delay 2.0 sec; Meas. Interval 2 10.0 sec. Injector P 25 μl; Injector M 25 μl »^♦ Press OK

** In the next window press start and the measurement will begin. • After the measurement save the raw data file (taskbar -» file -» save as..)

• Evaluate the data with Excel (taskbar -» file -» Excel evaluation)

• Import the data into the Excel window ( taskbar -» Lumi - Raw Data Spreadsheet)

• Check option 1 (All data (plate format)) and press OK • Copy the Raw data into the template by using the copy and paste function

• Name the compounds and save the excel file Repeat the last 7 steps for every plate

After measurement, unload the reagents from the system by using the soft-keys on the microlumat: • See display: Other — > other -» reagent — » other — » manual unload → Inj. P and Inj. M, several times — » exit After unloading, wash the system

• Put tubes in 80% ethanol solution

• See display: other — > other — » reagent -» other -» wash -» Inector P: on -» enter -» Injector M: on -» number of cycles 20

• Repat the washing procedure with H2O - Unload the system.

Importing the data to an Excel Template for graphical output *♦ Start Excel ** Open template 1 or template2 depending on the used dilutions, template 1 : 1 mM - 1 μM ; template2: 1 mM - 0.1 μM ** Export the data with WinGlow to Excel, select file —> Excel Evaluation. ** Import the data into Excel: Lumi —> Raw Data Spreadsheet. ** In the next box check the first option (All Data (microplate format)), press ok.

*^♦ Select the data in the new sheet (B13-M36) ** Select copy

** Open or activate the template and paste the data into the template by selecting cell B24 and using the paste command. *^♦ Fill in the codes for the compounds and save the file.

*^♦ Subsequently print the different sheets, sign and evaluate. An example of the template used is shown in figure 7.

Test compounds are dissolved in 100% DMSO to obtain lOOmM stock solutions. Serial dilutions are then prepared from this 100 mM stock: 1.0, 0.3, 0.1,

0.03, 0.01 and 0.001 mM in media supplemented with 5% FCS. The dilutions are added to the wells according to the schematic outlined below, along with the appropriate controls. Remaining dilutions and the stocks are stored at -80 C in polypropylene 1ml storage blocks. Figure 7 shows a template wherein:

A1-A6 and B1-B6 contain the controls: M: Media with 5% FCS DC: doxycycline (induction of ΔNTcf-4 1%: media with 5% FCS and 1% DMSO 0.3%: media with 5% FCS and 0.3% DMSO 0.1%: media with 5% FCS and 0.1% DMSO Values shown indicate the final concentrations (mM) of the test compounds.

Results of the cell based assays for the compounds shown in figures 1 are shown in figures 8-11, illustrating the inhibition of cellular β-catenin and LEF/TCF dependent reporter genes, whereby the compounds were added to the cell culture medium and luciferase activity for specific interference with b-Catenin dependent transactivation or renilla activity for overall transcriptional activity are determined. Concentrations of inhibitors and percentage of transcriptional activity compared to untreated cells in presence of compareable DMSO concentrations are indicated.

The invention is not restricted to the above description, the requested rights are rather determined by the following claims.

Claims

1. Compound and/or pharmaceutically acceptable salts, esters, hydrates or solvates thereof, having the structural formula (1) or (2):

0) (2)

wherein:

- X and Y are independently selected from the group comprising: H,C,N,O,S or alkyl,

- W is selected from the group comprising: NH₂, NH-Ri, NH-CO-Ri, NH-CO-Cn-O-Ri,

R₄

NH-CO-Cn-Ri, NH-CO-O-Cn-Ri, NH-CO-Cn-S-Ri, NH-CO-Cn-NH- Ri

wherein R₄ is optional, and n = 1-12, preferably 1-6, more preferably 1-4

- V is selected from the group comprising: H, C, N, O, S or a terminal alkyl group, whereby when V is a terminal alkyl group, X and R2 are nothing,

- Z is selected from the group comprising: S, S = O, O = S = O, - Q is selected from the group comprising: H, or Ri, R2, R3, or R

- Ri, R2, R3, or Rt are independently selected from the group comprising: H, alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, alkoxycarbonyl, aryl, arylalkyl, heteroaryl, heterocycloalkyl.

2. Compound according to claim 1, wherein X is O or S.

3. Compound according to claims 1 or 2, wherein Y is O.

4. Compound according to claims 1, 2 or 3, wherein Z is S.

5. Compound according to any of the preceding claims, wherein Ri, R2 , R3, and R4 are independently selected from the group comprising:

- H, - an alkyl group, being a monovalent alkane derived radical having form 1-

12 C atoms, wherein the alkyl group is an optionally substituted straight, branched or cyclic saturated hydrocarbon group, selected from the group comprising: methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, wherein the optional substituents comprise one or more members of the following groups: halogen, haloalkyl, alkoxy, alkylthio, hydroxy, carboxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl, urea or thiol,

- an alkenyl group, being a hydrocarbon radical straight, branched or cyclic group containing from 2 to 12 carbon atoms and at least one carbon to carbon double bond, optionally substituted by one or more members of the following groups: halogen, haloalkyl, alkoxy, alkylthio, hydroxy, carboxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl, urea or thiol,

- an alkynyl group, being a hydrocarbon radical straight, branched or cyclic group containing from 2 to 12 carbon atoms and at least one carbon to carbon triple bond, optionally substituted by one or more members of the following groups: halogen, haloalkyl, alkoxy, alkylthio, hydroxy, carboxy, alkyloxycarbonyl, alkylcarbonyloxy, amino, carbamoyl, urea or thiol,

- a cycloalkyl group, comprising from 3-15 C atoms without alterning or resonating double bonds between the carbon atoms, wherein the cycloalky group may contain from 1 to 4 rings, and may be substituted or usubstituted, unsubstituted cycloalky groups comprising cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, adamantyl, and the like, the cycloalky group being optionally substituted by one or more of the following groups: halogen, alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/or alkylthio,

- an alkoxy group, comprising the alkyl group bonded through an oxygen linkage (-O-), - an alkylthio group, comprising the alkyl group bonded through a sulfur linkage (-S-), respectively,

- an alkoxycarbonyl group, comprising an alkoxy group bonded through a carbonyl group,

- an alkoxycarbonyl group, being represented by the formula: -C(O)OR, where the R group is a straight or branched C1-12 alkyl group,

- an alkylcarbonyl group, being an alkyl group bonded through a carbonyl group,

- an alkylcarbonyloxy group, being an alkylcarbonyl group bonded through an oxygen linkage, - an arylalkyl group, being an aromatic ring bonded to the alkyl group,

- an aryl group comprising one or more monocyclic or bicyclic aromatic rings, including phenyl, substituted phenyl and the like, fused ring systems including naphtyl, phenanthrenyl and the like, wherein the aryl group preferably comprises at least one ring having at least 6 atoms, and at most five rings having upto 22 atoms, with alternating (resonating) double bonds between adjacent carbon atoms or heteroatoms, the aryl groups being optionally substituted with one or more members selected from the group comprising: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)_m (m = 0,1,2), or thiol,

- a heteroaryl group, being a monocyclic aromatic hydrocarbon group preferably having 5 or 6 ring atoms, or a bicyclic aromatic group preferably having 8 to 10 atoms, preferably having at least one heteroatom selected from O, S, or N, in which a carbon or nitrogen atom is the point of attachement, and in which one to three additional carbon atoms is optionally replaced by a heteroatom selected from O, N, or S, said heteroaryl group being optionally substituted, with one or more members selected from the group comprising: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)_m (m = 0,1,2), or thiol,

- a heteroaryl group comprising one or more of the following groups: thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl, piperidinyl, thiazolyl, pyrazinyl, pyridazinyl, pyrimidinal, triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl, benzofiiranzanyl and tetrahydropyranyl, optionally substituted by one or more of the following groups: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkyl-S(O)_m (m = 0,1,2), or thiol,

- a heterocycloalkyl group, being a cycloalkyl group in which preferably one to three of the carbon atoms in the ring are replaced by a heteroatom selected from the group comprising O, S or N.

6. Compound according to any of the preceding claims wherein W is NH-

7. Compound according to any of the preceding claims wherein Ri consists of a halo substituted alkyl group.

8. Compound according to any of the preceding claims wherein Ri consists of a halo substituted pentyl group.

9. Compound according to any of the preceding claims wherein Ri consists of a terminal halo substituted pentyl group.

10. Compound according to any of the preceding claims 7-9 wherein the halogen is Br.

11. Compound according to any of the preceding claims 1-6 wherein the Ri group is an alkenyl group having 2-12, preferably 5-10 C atoms, and is most preferably a penten-2,3-yl group or a decen-9,10-yl group.

12. Compound according to any of the preceding claims 6-11 wherein Ri comprises one or more, optionally substituted aryl groups, optionally substituted, with one or more members selected from the group comprising: halogen, alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)_m (m = 0,1,2), or thiol, wherein most preferably Ri is most preferably

13. Compound according to any of the preceding claims 1-5, 12 wherein W is:

Rt

and wherein:

- n is 1,

- R₄ is a methyl group,

- Ri is optionally a substituted aryl group and is preferably a tri-chloro substituted benzene ring.

14. Compound according to any of the preceding claims 1-5, 12 wherein W is:

NH-CO-Cn-O-Ri

R»

and wherein; - n is 1, - R is selected from the group consisting of a methyl, an ethyl, aryl or cyclohexyl group.

15. Compound according to any of the preceding claims 1-5, 12 wherein W is:

Rt

and wherein; - R4 is nothing, - Ri is an optionally substituted aryl group, and is preferably an unsubstituted benzene ring.

16. Compound according to any of the preceding claims wherein R2 is a carbonyl group.

17. Compound according to claim 16 wherein X is O.

18. Compound according to any of the preceding claims 1-15 wherein R2 is an, optionally substituted, aryl group halogen, wherein the optional substituents comprise one or more of the following groups: alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino, cycloalkyl, cyano, alkyl S(O)_m (m = 0,1,2), or thiol.

19. Compound according to claim 18 wherein R2 is a hydroxy substituted benzene group.

20. Compound according to any of the preceding claims wherein R3 is H or a methyl group.

21. Compound according to any of the preceding claims selected from the group comprising the following members:

STF196 STF213

STF215 STF236

STF310 STF311

STF312 STF340

STF318 STF314

STF353 STF354

KRT049

22. Process for synthesising a compound according to any of the preceding claims comprising acylating a cephalonosporanic acid of the following structural formula:

wherein Z, Q, V, X, R2, and R₃ have the meaning as defined in any of the claims 1-19, with an acyl halide selected from the group comprising the following members:

wherein Ri, X, R , and n have the same meaning as defined in any of the preceding claims 1-19.

23. Process according to claim 22 wherein the acyl halide is an acyl chloride.

24. Process according to claims 22 or 23 whereby the beta-lactone ring is opened to provide a compound of structural formula 2.

25. Compound according to any of the claims 1-21, obtainable according to the process of any of the claims 22-24.

26. Pharmaceutically acceptable salts, esters, hydrates or solvates of the compounds according to any of the claims 1- 20 and 25.

27. Medicament comprising a compound according to any of the claims 1- 20, 25 and 26, and pharmaceutically acceptable excipients.

28. Method of treating cancer comprising administering a compound according to any of the claims 1-20, 25, 26 or a medicament according to claim 27 to a patient.

29. Use of a compound according to any of the claims 1-20, 25 as a medicament.

30. Use of a compound according to any of the claims 1-20, 25 for preparing a medicament for treating cancer.

31. Compound according to any of the claims 1-20, 25, which compound substantially lacks antibiotic activity.

32. Use of a compound according to any of the claims 1-20, 25 for inhibiting the formation of one or more of the following:

- Tcf/beta catenin complexes, - beta catenin,

- LEF/TCF complexes.

33. Compound according to any of the preceding claims 1-20, 25 having an IC₅₀ value lying in the range of 0.1-20, preferably 0.5-15, more preferably 0.9-5 μM (Elisa).

34. A nucleotide sequence comprising the following sequence: CCTTTGATC and/or CCTTTGGCC or a variant thereof, which is activatable for expression when Tcf/beta catenin factors are present.

35. A nucleotide sequence according to claim 34 being a reporter gene and/or a control reporter gene.

36. The nucleotide sequence, or a variant thereof, according to claim 34 or 35, CCTTTGATC, being a reporter gene in the form of a binding motif for Tcf.

37. The nucleotide sequence, or a variant thereof, according to claim 34 or 35, CCTTTGGCC, being a control reporter gene in the form of a non-binding motif for Tcf.

38. Nucleotide sequence according to claim 36 or 37 being linked to a to promoter and/or a reporter preferably being a thymidine kinase reporter and a luciferase or renilla reporter.

39. Protein expressable by the nucleotide sequence according to any of the claims 34-38.

40. Method for analysing the presence of Tcf/beta catenin complexes comprising the step of introducing a nucleotide sequence according to any of the preceding claims 34-38 into carcinoma cell lines.