Inhibitors of histone deacetylase

Abstract

This invention comprises the novel compounds of formula (I) ##STR00001##
wherein n, R.sup.1, R.sup.2, R.sup.3, R.sup.4, Q, X, Y, Z and ##STR00002##
have defined meanings, having histone deacetylase inhibiting enzymatic activity; their preparation, compositions containing them and their use as a medicine.

Claims

1. A compound of formula (I), ##STR00061## the N-oxide forms, the pharmaceutically acceptable addition salts and the stereo-chemically isomeric forms thereof, wherein n is 1, 2 or 3; Q is ##STR00062## X is ##STR00063## Y is ##STR00064## Z is ##STR00065## R.sup.1 is C(O)NR.sup.5R.sup.6, N(H)C(O)R.sup.7, C(O)C.sub.1-6alkanediylSR.sup.7, NR.sup.8C(O)N(OH)R.sup.7, NR.sup.8C(O)C.sub.1-6alkanediylSR.sup.7, or NR.sup.8C(O)CN(OH)R.sup.7 wherein R.sup.5 and R.sup.6 are each independently selected from hydrogen, hydroxy, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl, aminoC.sub.1-6alkyl or aminoaryl; R.sup.7 is independently selected from hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylcarbonyl, arylC.sub.1-6alkyl, C.sub.1-6alkylpyrazinyl, pyridinone, pyrrolidinone or methylimidazolyl; R.sup.8 is independently selected from hydrogen or C.sub.1-6alkyl; R.sup.2 is hydrogen, halo, hydroxy, amino, nitro, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, di(C.sub.1-6alkyl)amino, hydroxyamino or naphtalenylsulfonylpyrazinyl; R.sup.3 and R.sup.4 are present on adjacent carbon atoms, and R.sup.3 and R.sup.4 together form a bivalent radical of formula
CHCHCHCH(b-1); aryl in the above is phenyl, or phenyl substituted with one or more substituents each independently selected from halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, cyano or hydroxycarbonyl.

2. The compound as claimed in claim 1 wherein R.sup.1 is C(O)NR.sup.5R.sup.6, C(O)C.sub.1-6alkanediylSR.sup.7, NR.sup.8C(O)N(OH)R.sup.7, NR.sup.8C(O)C.sub.1-6alkanediylSR.sup.7, or NR.sup.8C(O)CN(OH)R.sup.7 wherein R.sup.5 and R.sup.6 are each independently selected from hydrogen, hydroxy, hydroxyC.sub.1-6alkyl or aminoC.sub.1-6alkyl; and R.sup.2 is hydrogen, halo, hydroxy, amino, nitro, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl or di(C.sub.1-6alkyl)amino.

3. The compound as claimed in claim 1 wherein n is 1; each R.sup.1 is C(O)NH(OH) or NHC(O)C.sub.1-6alkanediylSH; and R.sup.2 is hydrogen or nitro.

4. The compound as claimed in claim 1 wherein n is 1; R.sup.1 is C(O)NH(OH); and R.sup.2 is nitro.

5. The compound according to claim 1 that is ##STR00066##

6. A pharmaceutical composition comprising pharmaceutically acceptable carriers and as an active ingredient, a therapeutically effective amount of a compound as claimed in claim 1.

7. A process of preparing a pharmaceutical composition as claimed in claim 6 comprising intimately mixing the pharmaceutically acceptable carriers and the compound.

8. A method of treating ovarian carcinoma in a patient comprising administering to the patient, an effective amount of a compound of claim 1.

9. A process for preparing a compound as claimed in claim 1, comprising reacting an intermediate of formula (II) with an acid, yielding a hydroxamic acid of formula (I-a) ##STR00067## wherein n is 1, 2 or 3; Q is ##STR00068## X is ##STR00069## Y is ##STR00070## Z is ##STR00071## R.sup.2 is hydrogen, halo, hydroxy, amino, nitro, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, di(C.sub.1-6alkyl)amino, hydroxyamino or naphtalenylsulfonylpyrazinyl; R.sup.3 and R.sup.4 are present on adjacent carbon atoms, and R.sup.3 and R.sup.4 together form a bivalent radical of formula
CHCHCHCH(b-1); aryl in the above is phenyl, or phenyl substituted with one or more substituents each independently selected from halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, cyano or hydroxycarbonyl.

10. The process of claim 9, wherein the acid is trifluoro acetic acid.

11. A method of detecting or identifying a histone deactylase (HDAC) in a biological sample comprising detecting or measuring the formation of a complex between a labeled compound as defined in claim 1 and a HDAC.

12. A combination of anti-cancer agents and a compound of claim 1.

Description

Experimental Part

(1) The following examples are provided for purposes of illustration. BSA means bovine serum albumine, DCM means dichloromethane, DIEA means diisopropylethylamine, DMF means dimethylformamide, DMSO means dimethylsulfoxide, EtOAc means ethyl acetate, Fmoc means fluorenylmethoxycarbonyl, Hepes means 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid, HOBT means 1-hydroxy-1H-benzotriazole, MeOH means methanol, PyBop means benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate, PyBrOP means bromo-tris-pyrrolidino-phosphonium hexafluorophosphate, TEA means triethylamine, TFA means trifluoroacetic acid THF means tetrahydrofuran, Extrelut is a product of Merck KgaA, Darmstadt, Germany, and is a short column comprising diatomaceous earth.

A. Preparation of the Intermediates

Example A1

(2) a) Preparation of

(3) ##STR00016##

(4) A solution of 1-(phenylmethyl)-piperazine (0.068 mol) in acetonitrile p.a. (135 ml) was added gradually to a solution of potassium carbonate (0.18 mol) and 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.082 mol) in acetonitrile p.a. (135 ml) and the reaction mixture was stirred for 45 min at room temperature. Then, the reaction mixture was stood overnight. DCM (400 ml) was added. Water (300 ml) was added and the organic layer was separated, dried (MgSO.sub.4), filtered and the solvent was evaporated. The residue (28 g) was purified by column chromatography over silica gel (eluent: DCM/MeOH 95/5). The pure fractions were collected and the solvent was evaporated. The residue was crystallized from acetonitrile, filtered off and dried in vacuo, yielding 15.1 g of intermediate 1.

(5) b) Preparation of

(6) ##STR00017##

(7) A mixture of intermediate 1 (0.03 mol) in EtOH (250 ml) was hydrogenated at 50 C. with Pd/C 10% (2 g) as a catalyst. After uptake of H.sub.2 (1 equiv), the catalyst was filtered off and the filtrate was evaporated. The residue was purified by column chromatography over silica gel (eluent: DCM/(MeOH/NH.sub.3) 90/10). The product fractions were collected and the solvent was evaporated, yielding 6.8 g (>96%) of intermediate 2.

(8) c) Preparation of

(9) ##STR00018##

(10) A solution of dimethyl-sulfamoyl chloride (0.0015 mol) in DCM (1 ml) was added at 5 C. to a mixture of intermediate 2 (0.0012 mol) and TEA (0.0017 mol) in DCM (1 ml) under N.sub.2 flow. The mixture was stirred at room temperature for 18 hours. Potassium carbonate 10% was added. The mixture was extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (0.69 g) was taken up in diethyl ether. The precipitate was filtered off and dried, yielding 0.64 g (73%) of intermediate 3, melting point 193 C.

Example A2

(11) Preparation of

(12) ##STR00019##

(13) A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0434 mol) in acetonitrile (100 ml) was added dropwise at 10 C. to a solution of 4-piperidinemethanamine (0.0868 mol) and potassium carbonate (0.0434 mol) in acetonitrile (200 ml) under N.sub.2 flow. The mixture was stirred at room temperature for 2 hours, poured out into ice water and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue (14.18 g) was purified by column chromatography over silica gel (20-45 m) (eluent: DCM/MeOH/NH.sub.4OH 90/10/1 to 80/20/2). The pure fractions were collected and the solvent was evaporated, yielding 3.7 g (32%) of intermediate 4.

Example A3

(14) a) Preparation of

(15) ##STR00020##

(16) A mixture of intermediate 2 (0.0002 mol), <-phenyl-benzeneacetyl chloride (0.0003 mol) and morpholinomethyl-PS-scavenger (Supplier Novabiochem cat No 01-64-0171: Morpholinomethyl polystyrene HL (200-400 mesh), 2% divinylbenzene) (0.150 g) in DCM (5 ml) was stirred at room temperature for 20 hours, then tris(2-aminoethyl)amine-PS-scavenger (Supplier Novabiochem cat No 01-64-0170: Tris-(2-aminomethyl)-amine polystyrene HL(200-400 mesh), 1% divinylbenzene) (0.150 g) was added and the reaction mixture was stirred for another 4 hours. The scavengers were filtered off, washed with DCM and the solvent was evaporated, yielding intermediate 5.

(17) b) Preparation of

(18) ##STR00021##

(19) A mixture of intermediate 5 (0.0003 mol) in sodium hydroxide 1N (1.5 ml), THF (4 ml) and MeOH (1 ml) was stirred at room temperature for 3 days, then the reaction mixture was neutralised with HCl (1.5 ml, 1N). The mixture was filtered through Extrelut NT (supplier: Merck) and dried under N.sub.2-flow, yielding intermediate 6.

(20) c) Preparation of

(21) ##STR00022##

(22) A mixture of intermediate 6 (0.0003 mol), HOBT-6-carboxamidomethyl-PS-scavenger (0.200 g; Novabiochem Cat. No. 01-64-0425) and N,N-dimethyl-4-pyridinamine (0.00015 mol) in DCM/DMF (5 ml) was stirred at room temperature for 15 min., then N,N-methanetetraylbis-2-propanamine (0.070 ml) was added and the reaction mixture was shaken for 4 hours. The resin was washed 3 times with DCM, 3 times with DMF and again 3 times with DCM and 3 times with DMF, finally 6 times with DCM. A solution of O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.00026 mol) in DCM (5 ml) was added and the reaction mixture was shaken for 20 hours, then PS linked methylisocyanate (Supplier Novabiochem cat No 01-64-0289: Methylisothiocyanate polystyrene HL(200-400 mesh), 2% divinylbenzene) (0.150 g) was added and the mixture was shaken for 4 hours. The scavengers were filtered off, washed 2 times with DCM and the filtrate was used, yielding intermediate 7.

B. Preparation of the Final Compounds

Example B1

(23) N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) was deprotected by 50% piperidine in DMF (RT, 24 hr). The resin was washed several times with DCM and DMF and swelled in DMF. Two equivalents of acid.sup.1, PyBrOP and 4 equivalents of DIEA were added as one portion. The mixture was shaken for 24 hr, liquid was drained and the resin was washed several times by DCM and DMF. The resin was swelled in DMF containing 2 equivalents of amine, was shaken 24 hr at RT, the liquid was drained and the resin was washed by DCM and DMF. An arylsulfonyl chloride (2 eq.) was added as one portion to the resin swelled in DMF with 4 equivalents of TEA. Reaction was stirred overnight, drained and the resin was washed by DCM and DMF. The final product was cleaved by 5% TFA in DCM, analyzed by HPLC and MS and evaporated in the pre-weighted test-tubes. .sup.1. Based on the loading of the resin.

(24) For illustrative purposes the scheme hereunder is included.

(25) ##STR00023##

Example B2

(26) N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) was deprotected by 50% piperidine in DMF (RT, 24 hr).sup.1. The resin was washed.sup.2 several times with DCM and DMF and swelled in DMF. Two equivalents of acid.sup.3, PyBrOP.sup.4 and 4 equivalents of DIEA were added as one portion. The mixture was shaken for 24 hr, liquid was drained and the resin was washed several times by DCM and DMF. The resin was swelled in DMF containing 2 equivalents of amine, was shaken 24 hr at RT, the liquid was drained and the resin was washed by DCM and DMF. The final product was cleaved by 5% TFA in DCM, analyzed by HPLC and MS and evaporated in the pre-weighted test-tubes. .sup.1. In one example compound 1 carboxymethanethiol 4-methoxytrityl resin (Novabiochem, 01-64-0238) was used. .sup.2. In one case also MeOH was used in the different washing procedures compound 1. .sup.3. Based on the loading of the resin. .sup.4. In one case PyBrOP was replaced by PyBOP compound 1.

Example B3

(27) N-Fmoc-hydroxylamine 2-chlorotrityl resin (Novabiochem, 01-64-0165) was deprotected by 50% piperidine in DMF (RT, 24 hr).sup.1. The resin was washed.sup.2 several times with DCM and DMF and swelled in DMF. Two equivalents of acid.sup.3, PyBrOP.sup.4 and 4 equivalents of DIEA were added as one portion. The mixture was shaken for 24 hr, liquid was drained and the resin was washed several times by DCM and DMF. The resin was swelled in DMF containing 2 equivalents of amine, was shaken 24 hr at RT, the liquid was drained and the resin was washed by DCM and DMF. Three equivalents of acid, DIC and DIEA were shaken with resin overnight at RT. The resin was drained and washed by DCM and DMF. The final product was cleaved by 5% TFA in DCM, analyzed by HPLC and MS and evaporated in the pre-weighted test-tubes.

Example B4

(28) a) Preparation of

(29) ##STR00024##

(30) A mixture of intermediate 3 (0.0016 mol) and sodium hydroxide (0.0033 mol) in EtOH (6 ml) was stirred and refluxed for 2 hours, then cooled to room temperature. The precipitate was filtered, washed with EtOH and dried, yielding 0.59 g (>100%) of intermediate 8.Na.

(31) b) Preparation of

(32) ##STR00025##

(33) N-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine, monohydrochloride (0.0021 mol) was added portionwise to a mixture of intermediate 8.Na (0.0016 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0021 mol) and 1-hydroxy-1H-benzotriazole (0.0021 mol) in DCM/THF (10 ml) under N.sub.2 flow. The mixture was stirred at room temperature for a week end. Potassium carbonate 10% was added. The mixture was extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (0.94 g) was purified by column chromatography over kromasil (eluent: DCM/MeOH/NH.sub.4OH 97/3/0.1; 15-40 m). The pure fractions were collected and the solvent was evaporated. The residue (0.45 g, 65%) was taken up in diethyl ether. The precipitate was filtered off and dried, yielding 0.422 g (61%) of intermediate 9, melting point 183 C.

(34) c) Preparation of

(35) ##STR00026##

(36) Trifluoroacetic acid (0.5 ml) was added to a mixture of intermediate 9 (0.0009 mol) in MeOH (10 ml). The mixture was stirred at room temperature for 18 hours. The precipitate was filtered, washed with DCM and dried., yielding 0.176 g (59%) of compound 2, melting point >260 C.

Example B5

(37) Preparation of

(38) ##STR00027##

(39) A mixture of intermediate 2 (0.0019 mol) and sulfamide (0.0021 mol) in 1,2-dimethoxy-ethane (5 ml) was stirred and refluxed for 4 days. Water was added. The mixture was filtered off and dried, yielding 0.51 g (83%) of intermediate 10, melting point 192 C.

(40) Intermediate 10 was handled analogously as described in example [B4] to give 0.034 g (13%) of compound 3, melting point 212 C.

(41) ##STR00028##

Example B6

(42) Preparation of

(43) ##STR00029##

(44) A solution of dimethyl-sulfamoyl chloride (0.007 mol) in DCM (5 ml) was added at 10 C. to a solution of intermediate 4 (0.0057 mol) and TEA (0.0085 mol) in DCM (5 ml) under N.sub.2 flow. The mixture was stirred overnight, poured out into ice water and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue was crystallized from CH.sub.3CN/diethyl ether. The precipitate was filtered off and dried, yielding 0.492 g (24%) of intermediate 11, melting point 142 C.

(45) Intermediate 11 was handled analogously as described in example [B4] to give 0.7 g (85%) of compound 4, melting point 182 C.

(46) ##STR00030##

Example B7

(47) Preparation of

(48) ##STR00031##

(49) A mixture of intermediate 7 (0.0003 mol) in acetic acid, trifluoro-acetic acid (5 ml, 5% in MeOH) was stirred at room temperature for 20 hours, then the reaction mixture was blown dry, yielding compound 5.

Example B8

(50) Preparation of

(51) ##STR00032##

(52) A mixture of intermediate 2 (0.0025 mol), 2-naphthalenecarbonyl chloride (0.003 mol) and potassium carbonate (0.005 mol) in acetonitrile (20 ml) was stirred and refluxed overnight, then cooled to room temperature, poured out into ice water and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, yielding 0.97 g (100%) of intermediate 12, melting point 140 C. Intermediate 12 was handled analogously as described in example [B4] to give 0.338 g (86%) of compound 6, melting point 130 C.

(53) ##STR00033##

(54) Table F-1 lists the compounds that were prepared according to one of the above Examples. The following abbreviations were used in the tables. Co.No. stands for Compound Number, Ex. [Bn] referred to the same method as described in the Bn examples, C.sub.2HF.sub.3O.sub.2 stands for the trifluoroacetate salt. Some compounds have been characterized via melting point (mp.), other compounds were characterized via Mass Spectral data [MH.sup.+] (ms.).

(55) TABLE-US-00001 TABLE F-1 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 1; Ex. [B2] Co. No. 2; Ex. [B4]; mp >260 C. Co. No. 3; Ex. [B5]; mp 212 C. Co. No. 4; Ex. [B6]; mp 182 C. C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 7; Ex. [B2]; ms. 412 C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 8; Ex. [B2]; ms. 383 0 C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 9; Ex. [B2]; ms. 383 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 10; Ex. [B2]; ms. 361 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 11; Ex. [B2]; ms. 314 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 12; Ex. [B3]; ms. 366 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 13; Ex. [B2]; ms. 314 C.sub.2HF.sub.3O.sub.2 (1:1), Co. No. 14; Ex. [B2]; ms. 412 C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 15; Ex. [B2]; ms. 281 C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 16; Ex. [B2]; ms. 365 C.sub.2HF.sub.3O.sub.2 (1:2), Co. No. 17; Ex. [B2]; ms. 295 Co. No. 18; Ex. [B1]; ms. 398 0 Co. No. 19; Ex. [B1]; ms. 328 Co. No. 20; Ex. [B1]; ms. 300 Co. No. 21; Ex. [B1]; ms. 329 Co. No. 22; Ex. [B1]; ms. 354 Co. No. 5; Ex. [B7]; ms. 418 Co. No. 6; Ex. [B8]; mp. 130 C. Co. No. 23; Ex. [B7]; ms. 348 Co. No. 24; Ex. [B7]; ms. 378 Co. No. 25; Ex. [B7]; ms. 329 Co. No. 26; Ex. [B7]; ms. 402 0 Co. No. 27; Ex. [B7]; ms. 329

C. Pharmacological Example

(56) The in vitro assay for inhibition of histone deacetylase (see example C.1) measures the inhibition of HDAC enzymatic activity obtained with the compounds of formula (I).

(57) Cellular activity of the compounds of formula (I) was determined on A2780 tumour cells using a colorimetric assay for cell toxicity or survival (Mosmann Tim, Journal of Immunological Methods 65: 55-63, 1983)(see example C.2).

(58) Kinetic solubility in aqueous media measures the ability of a compound to stay in aqueous solution upon dilution (see example C.3).

(59) DMSO-stock solutions are diluted with a single aqueous buffer solvent in 3 consecutive steps. For every dilution turbidity is measured with a nephelometer.

(60) A drug's permeability expresses its ability to move from one medium into or through another. Specifically its ability to move through the intestinal membrane into the blood stream and/or from the blood stream into the target. Permeability (see example C.4) can be measured through the formation of a filter-immobilized artificial membrane phospholipid bilayer. In the filter-immobilized artificial membrane assay, a sandwich is formed with a 96-well microtitre plate and a 96-well filter plate, such that each composite well is divided into two chambers with a donor solution at the bottom and an acceptor solution at the top, separated by a 125 m micro-filter disc (0.45 m pores), coated with 2% (wt/v) dodecane solution of dioleoylphosphatidyl-choline, under conditions that multi-lamellar bilayers form inside the filter channels when the system contacts an aqueous buffer solution. The permeability of compounds through this artificial membrane is measured in cm/s. The purpose is to look for the permeation of the drugs through a parallel artificial membrane at 2 different pH's: 4.0 and 7.4. Compound detection is done with UV-spectrometry at optimal wavelength between 250 and 500 nm.

(61) Metabolism of drugs means that a lipid-soluble xenobiotic or endobiotic compound is enzymatically transformed into (a) polar, water-soluble, and excretable metabolite(s). The major organ for drug metabolism is the liver. The metabolic products are often less active than the parent drug or inactive. However, some metabolites may have enhanced activity or toxic effects. Thus drug metabolism may include both detoxication and toxication processes. One of the major enzyme systems that determine the organism's capability of dealing with drugs and chemicals is represented by the cytochrome P450 monooxygenases, which are NADPH dependent enzymes. Metabolic stability of compounds can be determined in vitro with the use of subcellular human tissue (see example C.5). Here metabolic stability of the compounds is expressed as % of drug metabolised after 15 minutes incubation of these compounds with microsomes. Quantitation of the compounds was determined by LC-MS analysis.

(62) The tumour suppressor p53 transcriptionally activates a number of genes including the WAF1/CIP1 gene in response to DNA damage. The 21 kDa product of the WAF1 gene is found in a complex involving cyclins, cyclin dependent kinases (CDKs), and proliferating cell nuclear antigen (PCNA) in normal cells but not transformed cells and appears to be a universal inhibitor of CDK activity. One consequence of p21WAF1 binding to and inhibiting CDKs is to prevent CDK-dependent phosphorylation and subsequent inactivation of the Rb protein, which is essential for cell cycle progression. Induction of p21WAF1 in response to cellular contact with a HDAC inhibitor is therefore a potent and specific indicator of inhibition of cell cycle progression at both the G1 and G2 checkpoints.

(63) The capacity of the compounds to induce p21WAF1 was measured with the p21WAF1 enzyme linked immunosorbent assay (WAF1 ELISA of Oncogene). The p21WAF1 assay is a sandwich enzyme immunoassay employing both mouse monoclonal and rabbit polyclonal antibodies. A rabbit polyclonal antibody, specific for the human WAF1 protein, has been immobilized onto the surface of the plastic wells provided in the kit. Any p21WAF present in the sample to be assayed will bind to the capture antibody. The biotinylated detector monoclonal antibody also recognizes human p21WAF1 protein, and will bind to any p21WAF1, which has been retained by the capture antibody. The detector antibody, in turn, is bond by horseradish peroxidas-conjugated streptavidin. The horseradish peroxidase catalyses the conversion of the chromogenic substrate tetra-methylbenzidine from a colorless solution to a blue solution (or yellow after the addition of stopping reagent), the intensity of which is proportional to the amount of p21WAF1 protein bond to the plate. The colored reaction product is quantified using a spectrophotometer. Quantitation is achieved by the construction of a standard curve using known concentrations of p21WAF1 (provided lyophilised) (see example C.6).

Example C.1

In Vitro Assay for Inhibition of Histone Deacetylase

(64) HeLa nuclear extracts (supplier: Biomol) were incubated at 60 g/ml with 210.sup.8 M of radiolabeled peptide substrate. As a substrate for measuring HDAC activity a synthetic peptide, i.e. the amino acids 14-21 of histone H4, was used. The substrate is biotinylated at the NH.sub.2-terminal part with a 6-aminohexanoic acid spacer, and is protected at the COOH-terminal part by an amide group and specifically [.sup.3H]acetylated at lysine 16. The substrate, biotin-(6-aminohexanoic)Gly-Ala-([.sup.3H]-acetyl-Lys-Arg-His-Arg-Lys-Val-NH.sub.2), was added in a buffer containing 25 mM Hepes, 1 M sucrose, 0.1 mg/ml BSA and 0.01% Triton X-100 at pH 7.4. After 30 min the deacetylation reaction was terminated by the addition of HCl and acetic acid. (final concentration 0.035 mM and 3.8 mM respectively). After stopping the reaction, the free .sup.3H-acetate was extracted with ethylacetate. After mixing and centrifugation, the radioactivity in an aliquot of the upper (organic) phase was counted in a -counter.

(65) For each experiment, controls (containing HeLa nuclear extract and DMSO without compound), a blank incubation (containing DMSO but no HeLa nuclear extract or compound) and samples (containing compound dissolved in DMSO and HeLa nuclear extract) were run in parallel. In first instance, compounds were tested at a concentration of 10.sup.5M. When the compounds showed activity at 10.sup.5M, a concentration-response curve was made wherein the compounds were tested at concentrations between 10.sup.5M and 10.sup.12M. In each test the blank value was substracted from both the control and the sample values. The control sample represented 100% of substrate deactylation. For each sample the radioactivity was expressed as a percentage of the mean value of the controls. When appropriate IC.sub.50-values (concentration of the drug, needed to reduce the amount of metabolites to 50% of the control) were computed using probit analysis for graded data. Herein the effects of test compounds are expressed as pIC.sub.50 (the negative log value of the IC.sub.50-value). All tested compounds showed enzymatic activity at a test concentration of 10.sup.5M and 21 compounds had a pIC.sub.505 (see table F-2).

Example C.2

Determination of Antiproliferative Activity on A2780 Cells

(66) All compounds tested were dissolved in DMSO and further dilutions were made in culture medium. Final DMSO concentrations never exceeded 0.1% (v/v) in cell proliferation assays. Controls contained A2780 cells and DMSO without compound and blanks contained DMSO but no cells. MTT was dissolved at 5 mg/ml in PBS. A glycine buffer comprised of 0.1 M glycine and 0.1 M NaCl buffered to pH 10.5 with NaOH (1 N) was prepared (all reagents were from Merck).

(67) The human A2780 ovarian carcinoma cells (a kind gift from Dr. T. C. Hamilton [Fox Chase Cancer Centre, Pennsylvania, USA]) were cultured in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 g/ml gentamicin and 10% fetal calf serum. Cells were routinely kept as monolayer cultures at 37 C. in a humidified 5% CO.sub.2 atmosphere. Cells were passaged once a week using a trypsin/EDTA solution at a split ratio of 1:40. All media and supplements were obtained from Life Technologies. Cells were free of mycoplasma contamination as determined using the Gen-Probe Mycoplasma Tissue Culture kit (supplier: BioMrieux).

(68) Cells were seeded in NUNC 96-well culture plates (Supplier: Life Technologies) and allowed to adhere to the plastic overnight. Densities used for plating were 1500 cells per well in a total volume of 200 l medium. After cell adhesion to the plates, medium was changed and drugs and/or solvents were added to a final volume of 200 l. Following four days of incubation, medium was replaced by 200 l fresh medium and cell density and viability was assessed using an MTT-based assay. To each well, 25 l MTT solution was added and the cells were further incubated for 2 hours at 37 C. The medium was then carefully aspirated and the blue MTT-formazan product was solubilized by addition of 25 l glycine buffer followed by 100 l of DMSO. The microtest plates were shaken for 10 min on a microplate shaker and the absorbance at 540 nm was measured using an Emax 96-well spectrophotometer (Supplier: Sopachem).

(69) Within an experiment, the results for each experimental condition are the mean of 3 replicate wells. For initial screening purposes, compounds were tested at a single fixed concentration of 10.sup.6 M. For active compounds, the experiments were repeated to establish full concentration-response curves. For each experiment, controls (containing no drug) and a blank incubation (containing no cells or drugs) were run in parallel. The blank value was subtracted from all control and sample values. For each sample, the mean value for cell growth (in absorbance units) was expressed as a percentage of the mean value for cell growth of the control. When appropriate, IC.sub.50-values (concentration of the drug, needed to reduce cell growth to 50% of the control) were computed using probit analysis for graded data (Finney, D. J., Probit Analyses, 2.sup.nd Ed. Chapter 10, Graded Responses, Cambridge University Press, Cambridge 1962). Herein the effects of test compounds are expressed as pIC.sub.50 (the negative log value of the IC.sub.50-value). Most of the tested compounds showed cellular activity at a test concentration of 10.sup.6 M and 9 compounds had a pIC.sub.505 (see table F-2)

Example C.3

Kinetic Solubility in Aqueous Media

(70) In the first dilution step, 10 l of a concentrated stock-solution of the active compound, solubilized in DMSO (5 mM), was added to 100 l phosphate citrate buffer pH 7.4 and mixed. In the second dilution step, an aliquot (20 l) of the first dilution step was further dispensed in 100 l phosphate citrate buffer pH 7.4 and mixed. Finally, in the third dilution step, a sample (20 l) of the second dilution step was further diluted in 100 l phosphate citrate buffer pH 7.4 and mixed. All dilutions were performed in 96-well plates. Immediately after the last dilution step the turbidity of the three consecutive dilution steps were measured with a nephelometer. Dilution was done in triplicate for each compound to exclude occasional errors. Based on the turbidity measurements a ranking is performed into 3 classes. Compounds with high solubility obtained a score of 3 and for this compounds the first dilution is clear. Compounds with medium solubility obtained a score of 2. For these compounds the first dilution is unclear and the second dilution is clear. Compounds with low solubility obtained a score of 1 and for these compounds both the first and the second dilution are unclear. The solubility of 9 compounds was measured. From these compounds 7 showed a score of 3, and 2 demonstrated a score of 1 (see table F-2).

Example C.4

Parallel Artificial Membrane Permeability Analysis

(71) The stock samples (aliquots of 10 l of a stock solution of 5 mM in 100% DMSO) were diluted in a deep-well or Pre-mix plate containing 2 ml of an aqueous buffer system pH 4 or pH 7.4 (PSR4 System Solution Concentrate (pION)).

(72) Before samples were added to the reference plate, 150 l of buffer was added to wells and a blank UV-measurement was performed. Thereafter the buffer was discarded and the plate was used as reference plate. All measurements were done in UV-resistant plates (supplier: Costar or Greiner).

(73) After the blank measurement of the reference plate, 150 l of the diluted samples was added to the reference plate and 200 l of the diluted samples was added to donorplate 1. An acceptor filter plate 1 (supplier: Millipore, type:MAIP N45) was coated with 4 l of the artificial membrane-forming solution (1,2-Dioleoyl-sn-Glycer-3-Phosphocholine in Dodecane containing 0.1% 2,6-Di-tert-butyl-4-methylphenol and placed on top of donor plate 1 to form a sandwich. Buffer (200 l) was dispensed into the acceptor wells on the top. The sandwich was covered with a lid and stored for 18 h at room temperature in the dark.

(74) A blank measurement of acceptor plate 2 was performed through the addition of 150 l of buffer to the wells, followed by an UV-measurement. After the blank measurement of acceptor plate 2 the buffer was discarded and 150 l of acceptor solution was transferred from the acceptor filter plate 1 to the acceptor plate 2. Then the acceptor filter plate 1 was removed form the sandwich. After the blank measurement of donor plate 2 (see above), 150 l of the donor solution was transferred from donor plate 1 to donor plate 2. The UV spectra of the donor plate 2, acceptor plate 2 and reference plate wells were scanned (with a SpectraMAX 190). All the spectra were processed to calculate permeability with the PSR4p Command Software. All compounds were measured in triplo. Carbamazepine, griseofulvin, acycloguanisine, atenolol, furosemide, and chlorothiazide were used as standards in each experiment. Compounds were ranked in 3 categories as having a low permeability (mean effect<0.510.sup.6 cm/s; score 1), a medium permeability (110.sup.6 cm/s>mean effect0.510.sup.6 cm/s; score 2) or a high permeability (0.510.sup.6 cm/s; score 3). Two compounds showed a score of 1 at one of the pH's measured.

Example C.5

Metabolic Stability

(75) Sub-cellular tissue preparations were made according to Gorrod et al. (Xenobiotica 5: 453-462, 1975) by centrifugal separation after mechanical homogenization of tissue. Liver tissue was rinsed in ice-cold 0.1 M Tris-HCl (pH 7.4) buffer to wash excess blood. Tissue was then blotted dry, weighed and chopped coarsely using surgical scissors. The tissue pieces were homogenized in 3 volumes of ice-cold 0.1 M phosphate buffer (pH 7.4) using either a Potter-S (Braun, Italy) equipped with a Teflon pestle or a Sorvall Omni-Mix homogeniser, for 710 sec. In both cases, the vessel was kept in/on ice during the homogenization process.

(76) Tissue homogenates were centrifuged at 9000g for 20 minutes at 4 C. using a Sorvall centrifuge or Beckman Ultracentrifuge. The resulting supernatant was stored at 80 C. and is designated S9.

(77) The S9 fraction can be further centrifuged at 100.000g for 60 minutes (4 C.) using a Beckman ultracentrifuge. The resulting supernatant was carefully aspirated, aliquoted and designated cytosol. The pellet was re-suspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1 ml per 0.5 g original tissue weight and designated microsomes.

(78) All sub-cellular fractions were aliquoted, immediately frozen in liquid nitrogen and stored at 80 C. until use.

(79) For the samples to be tested, the incubation mixture contained PBS (0.1M), compound (5 M), microsomes (1 mg/ml) and a NADPH-generating system (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8 Units of glucose-6-phosphate dehydrogenase). Control samples contained the same material but the microsomes were replaced by heat inactivated (10 min at 95 degrees Celsius) microsomes. Recovery of the compounds in the control samples was always 100%.

(80) The mixtures were preincubated for 5 min at 37 degrees Celsius. The reaction was started at timepoint zero (t=0) by addition of 0.8 mM NADP and the samples were incubated for 15 min (t=15). The reaction was terminated by the addition of 2 volumes of DMSO. Then the samples were centrifuged for 10 min at 900g and the supernatants were stored at room temperature for no longer as 24 h before analysis. All incubations were performed in duplo. Analysis of the supernatants was performed with LC-MS analysis. Elution of the samples was performed on a Xterra MS C 18 (504.6 mm, 5 m, Waters, US). An Alliance 2790 (Supplier: Waters, US) HPLC system was used. Elution was with buffer A (25 mM ammoniumacetate (pH 5.2) in H.sub.2O/acetonitrile (95/5)), solvent B being acetonitrile and solvent C methanol at a flow rate of 2.4 ml/min. The gradient employed was increasing the organic phase concentration from 0% over 50% B and 50% C in 5 min up to 100% B in 1 min in a linear fashion and organic phase concentration was kept stationary for an additional 1.5 min. Total injection volume of the samples was 25 l.

(81) A Quattro (supplier: Micromass, Manchester, UK) triple quadrupole mass spectrometer fitted with and ESI source was used as detector. The source and the desolvation temperature were set at 120 and 350 C. respectively and nitrogen was used as nebuliser and drying gas. Data were acquired in positive scan mode (single ion reaction). Cone voltage was set at 10 V and the dwell time was 1 sec.

(82) Metabolic stability was expressed as % metabolism of the compound after 15 min of incubation in the presence of active microsomes

(83) $\left(E\left(\mathrm{act}\right)\right)(\%\mathrm{metabolism}=100\%-\left(\left(\frac{\mathrm{Total}\mathrm{Ion}\mathrm{Current}\left(\mathrm{TIC}\right)\mathrm{of}E\left(\mathrm{act}\right)\mathrm{at}t=15}{\mathrm{TIC}\mathrm{of}E\left(\mathrm{act}\right)\mathrm{at}t=0}\right)100\right).$
Compounds that had a percentage metabolism less than 20% were defined as highly metabolic stable. Compound that had a metabolism between 20 and 70% were defined as intermediately stable and compounds that showed a percentage metabolism higher than 70 were defined as low metabolic stable. Three reference compounds were always included whenever a metabolic stability screening was performed. Verapamil was included as a compound with low metabolic stability (% metabolism=73%). Cisapride was included as a compound with medium metabolic stability (% metabolism 45%) and propanol was included as a compound with intermediate to high metabolic stability (25% metabolism). These reference compounds were used to validate the metabolic stability assay.

(84) One compound was tested and showed a percentage metabolism less than 20%.

Example C.6

p21 Induction Capacity 20

(85) The following protocol has been applied to determine the p21 protein expression level in human A2780 ovarian carcinoma cells. The A2780 cells (20000 cells /180 l) were seeded in 96 microwell plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 g/ml gentamicin and 10% fetal calf serum. 24 hours before the lysis of the cells, compounds were added at final concentrations of 10.sup.5, 10.sup.6, 10.sup.7 and 10.sup.8 M. All compounds tested were dissolved in DMSO and further dilutions were made in culture medium. 24 hours after the addition of the compound, the supernatants were removed from the cells. Cells were washed with 200 l ice-cold PBS. The wells were aspirated and 30 l of lysisbuffer (50 mM Tris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40 and 10% glycerol) was added. The plates were incubated overnight at 70 C.

(86) The appropriate number of microtiter wells were removed from the foil pouch and placed into an empty well holder. A working solution (1) of the Wash Buffer (20 plate wash concentrate: 100 ml 20-fold concentrated solution of PBS and surfactant. Contains 2% chloroacetamide) was prepared. The lyophilised p21 WAF standard was reconstituted with distilled H.sub.2O and further diluted with sample diluent (provided in the kit)

(87) The samples were prepared by diluting them 1:4 in sample diluent. The samples (100 l) and the p21 WAF1 standards (100 l) were pipetted into the appropriate wells and incubated at room temperature for 2 hours. The wells were washed 3 times with 1 wash buffer and then 100 l of detector antibody reagent (a solution of biotinylated monoclonal p21 WAF1 antibody) was pipetted into each well. The wells were incubated at room temperature for 1 hour and then washed three times with 1 wash buffer. The 400 conjugate (peroxidase streptavidine conjugate: 400-fold concentrated solution) was diluted and 100 l of the 1 solution was added to the wells. The wells were incubated at room temperature for 30 min and then washed 3 times with 1 wash buffer and 1 time with distilled H.sub.2O. Substrate solution (chromogenic substrate)(100 l) was added to the wells and the wells were incubated for 30 minutes in the dark at room temperature. Stop solution was added to each well in the same order as the previously added substrate solution. The absorbance in each well was measured using a spectrophotometric plate reader at dual wavelengths of 450/595 nm.

(88) For each experiment, controls (containing no drug) and a blank incubation (containing no cells or drugs) were run in parallel. The blank value was substracted from all control and sample values. For each sample, the value for p21WAF1 induction (in absorbance units) was expressed as the percentage of the value for p21WAF1 present in the control. Percentage induction higher than 130% was defined as significant induction. Three compounds were tested in this assay. Two showed significant induction.

(89) TABLE-US-00002 TABLE F-2 Table F-2 lists the results of the compounds that were tested according to example C.1, C.2, and C.3. Enzyme activity Cellular activity Solubility Co. No. pIC50 pIC50 Score 8 >5 7 >5 9 >5 10 >5 11 >5 12 >5 1 <5 <5 1 18 6.173 6.166 1 5 7.096 6.181 23 6.932 5.796 24 7.073 6.084 25 6.29 <5 26 6.984 5.378 27 6.433 <5 6 7.104 5.828 3 19 5.536 <5 3 20 5.451 <5 21 5.679 <5 3 22 5.599 5.297 3 2 6.615 5.534 3 3 6.881 <5 3 4 7.27 5.528 3

D. Composition Example:

Film-Coated Tablets

(90) Preparation of Tablet Core

(91) A mixture of 100 g of a compound of formula (I), 570 g lactose and 200 g starch is mixed well and thereafter humidified with a solution of 5 g sodium dodecyl sulphate and 10 g polyvinyl-pyrrolidone in about 200 ml of water. The wet powder mixture is sieved, dried and sieved again. Then there is added 100 g microcrystalline cellulose and 15 g hydrogenated vegetable oil. The whole is mixed well and compressed into tablets, giving 10.000 tablets, each comprising 10 mg of a compound of formula (I).

(92) Coating

(93) To a solution of 10 g methyl cellulose in 75 ml of denaturated ethanol there is added a solution of 5 g of ethyl cellulose in 150 ml of dichloromethane. Then there are added 75 ml of dichloromethane and 2.5 ml 1,2,3-propanetriol 10 g of polyethylene glycol is molten and dissolved in 75 ml of dichloromethane. The latter solution is added to the former and then there are added 2.5 g of magnesium octadecanoate, 5 g of polyvinyl-pyrrolidone and 30 ml of concentrated colour suspension and the whole is homogenated. The tablet cores are coated with the thus obtained mixture in a coating apparatus.