Amino-derivatives as novel inhibitors of histone deacetylase

Abstract

This invention comprises the novel compounds of formula (I) ##STR00001##
wherein n, m, t, R.sup.1, R.sup.2, L, 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), ##STR00088## the N-oxide form, pharmaceutically acceptable addition salt, or stereo-chemically isomeric form thereof, wherein n is 1 and m is 2; t is 0 or 1 and when t is 0 then a direct bond is intended; Q is C; X is N or C; Y is N; Z is CH.sub.2; R.sup.1 is C(O)NR.sup.3R.sup.4, 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.3 and R.sup.4 are each independently hydrogen, hydroxy, C.sub.1-6alkyl, hydroxyC.sub.1-6alkyl, aminoC.sub.1-6alkyl or aminoaryl; R.sup.7 is hydrogen, C.sub.1-6alkyl, C.sub.1-6alkylcarbonyl, arylC.sub.1-6alkyl, C.sub.1-6alkylpyrazinyl, pyridinone, pyrrolidinone, or methylimidazolyl; and R.sup.8 is hydrogen or C.sub.1-6alkyl; R.sup.2 is hydrogen, hydroxy, amino, hydroxyC.sub.1-6alkyl, C.sub.1-6alkyl, C.sub.1-6alkyloxy, arylC.sub.1-6alkyl, aminocarbonyl, hydroxycarbonyl, aminoC.sub.1-6alkyl, aminocarbonylC.sub.1-6alkyl, hydroxycarbonylC.sub.1-6alkyl, hydroxyaminocarbonyl, C.sub.1-6alkyloxycarbonyl, C.sub.1-6alkylaminoC.sub.1-6alkyl, or di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; -L- is a bivalent radical that is C.sub.1-6alkanediyl, carbonyl, sulfonyl, or C.sub.1-6alkanediyl substituted with phenyl; ##STR00089## is a radical selected from the group consisting of ##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094## wherein each s is independently 0, 1, 2, 3, 4 or 5, as allowed; each R.sup.5 and R.sup.6 is independently selected from the group consisting of hydrogen; halo; hydroxy; amino; nitro; trihaloC.sub.1-6alkyl; trihaloC.sub.1-6alkyloxy; C.sub.1-6alkyl; C.sub.1-6alkyl substituted with aryl and C.sub.3-10cycloalkyl; C.sub.1-6alkyloxy; C.sub.1-6alkyloxyC.sub.1-6alkyloxy; C.sub.1-6alkylcarbonyl; C.sub.1-6alkyloxycarbonyl; C.sub.1-6alkylsulfonyl; cyanoC.sub.1-6alkyl; hydroxyC.sub.1-6alkyl; hydroxyC.sub.1-6alkyloxy; hydroxyC.sub.1-6alkylamino; aminoC.sub.1-6alkyloxy; di(C.sub.1-6alkyl)aminocarbonyl; di(hydroxyC.sub.1-6alkyl)amino; (aryl)(C.sub.1-6alkyl)amino; di(C.sub.1-6alkyl)aminoC.sub.1-6alkyloxy; di(C.sub.1-6alkyl)aminoC.sub.1-6alkylamino; di(C.sub.1-6alkyl)aminoC.sub.1-6alkylaminoC.sub.1-6alkyl; aryl sulfonyl; arylsulfonylamino; aryloxy; aryloxyC.sub.1-6alkyl; arylC.sub.2-6alkenediyl; di(C.sub.1-6alkyl)amino; di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; di(C.sub.1-6alkyl)amino(C.sub.1-6alkyl)amino; di(C.sub.1-6alkyl)amino(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl(C.sub.1-6alkyl)amino; di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; aminosulfonylamino(C.sub.1-6alkyl)amino; aminosulfonylamino(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; di(C.sub.1-6alkyl)aminosulfonylamino(C.sub.1-6alkyl)amino; di(C.sub.1-6alkyl)aminosulfonylamino(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; cyano; thiophenyl; thiophenyl substituted with di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl(C.sub.1-6alkyl)aminoC.sub.1-6alkyl, di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl, C.sub.1-6alkylpiperazinylC.sub.1-6alkyl, hydroxyC.sub.1-6alkylpiperazinylC.sub.1-6alkyl, hydroxyC.sub.1-6alkyloxyC.sub.1-6alkylpiperazinylC.sub.1-6alkyl, di(C.sub.1-6alkyl)aminosulfonylpiperazinylC.sub.1-6alkyl, C.sub.1-6alkyloxypiperidinyl, C.sub.1-6alkyloxypiperidinylC.sub.1-6alkyl, morpholinylC.sub.1-6alkyl, hydroxyC.sub.1-6 alkyl(C.sub.1-6alkyl)aminoC.sub.1-6alkyl, or di(hydroxyC.sub.1-6alkyl)aminoC.sub.1-6alkyl; furanyl; furanyl substituted with hydroxyC.sub.1-6alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolyl substituted with aryl and C.sub.1-6alkyl; C.sub.1-6alkyltriazolyl; tetrazolyl; pyrrolidinyl; pyrrolyl; piperidinylC.sub.1-6alkyloxy; morpholinyl; C.sub.1-6alkylmorpholinyl; morpholinylC.sub.1-6alkyloxy; morpholinylC.sub.1-6alkyl; morpholinylC.sub.1-6alkylamino; morpholinylC.sub.1-6alkylaminoC.sub.1-6alkyl; piperazinyl; C.sub.1-6alkylpiperazinyl; C.sub.1-6alkylpiperazinylC.sub.1-6alkyloxy; piperazinylC.sub.1-6alkyl; naphtalenylsulfonylpiperazinyl; naphtalenylsulfonylpiperidinyl; naphtalenylsulfonyl: C.sub.1-6alkylpiperazinylC.sub.1-6alkyl; C.sub.1-6alkylpiperazinylC.sub.1-6alkylamino; C.sub.1-6alkylpiperazinylC.sub.1-6alkylaminoC.sub.1-6alkyl; C.sub.1-6alkylpiperazinylsulfonyl; aminosulfonylpiperazinylC.sub.1-6alkyloxy; aminosulfonylpiperazinyl; aminosulfonylpiperazinylC.sub.1-6alkyl; di(C.sub.1-6alkyl)aminosulfonylpiperazinyl; di(C.sub.1-6alkyl)aminosulfonylpiperazinylC.sub.1-6alkyl; hydroxyC.sub.1-6alkylpiperazinyl; hydroxyC.sub.1-6alkylpiperazinylC.sub.1-6alkyl; C.sub.1-6alkyloxypiperidinyl; C.sub.1-6alkyloxypiperidinylC.sub.1-6alkyl; piperidinylaminoC.sub.1-6alkylamino; piperidinylaminoC.sub.1-6alkylaminoC.sub.1-6alkyl; (C.sub.1-6alkylpiperidinyl)(hydroxyC.sub.1-6alkyl)aminoC.sub.1-6alkylamino; (C.sub.1-6alkylpiperidinyl)(hydroxyC.sub.1-6alkyl)aminoC.sub.1-6alkylaminoC.sub.1-6alkyl; hydroxyC.sub.1-6alkyloxyC.sub.1-6alkylpiperazinyl; hydroxyC.sub.1-6alkyloxyC.sub.1-6alkylpiperazinylC.sub.1-6alkyl; (hydroxyC.sub.1-6alkyl)(C.sub.1-6alkyl)amino; (hydroxyC.sub.1-6alkyl)(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; hydroxyC.sub.1-6alkylaminoC.sub.1-6alkyl; di(hydroxyC.sub.1-6alkyl)aminoC.sub.1-6alkyl; pyrrolidinylC.sub.1-6alkyl; pyrrolidinylC.sub.1-6alkyloxy; pyrazolyl; thiopyrazolyl; pyrazolyl substituted with two substituents selected from the group consisting of C.sub.1-6alkyl and trihaloC.sub.1-6alkyl; pyridinyl; pyridinyl substituted with C.sub.1-6alkyloxy, aryloxy or aryl; pyrimidinyl; tetrahydropyrimidinylpiperazinyl; tetrahydropyrimidinylpiperazinylC.sub.1-6alkyl; quinolinyl; indolyl; phenyl; phenyl substituted with one, two, or three substituents independently selected from the group consisting of halo, amino, nitro, C.sub.1-6alkyl, C.sub.1-6alkyloxy, hydroxyC.sub.1-4alkyl, trifluoromethyl, trifluoromethyloxy, hydroxyC.sub.1-4alkyloxy, C.sub.1-4alkylsulfonyl, C.sub.1-4alkyloxyC.sub.1-4alkyloxy, C.sub.1-4alkyloxycarbonyl, aminoC.sub.1-4alkyloxy, di(C.sub.1-4alkyl)aminoC.sub.1-4alkyloxy, di(C.sub.1-4alkyl)amino, di(C.sub.1-4alkyl)aminocarbonyl, di(C.sub.1-4alkyl)aminoC.sub.1-4alkyl, di(C.sub.1-4alkyl)aminoC.sub.1-4alkylaminoC.sub.1-4alkyl, di(C.sub.1-4alkyl)amino(C.sub.1-4alkyl)amino, di(C.sub.1-4alkyl)amino(C.sub.1-4alkyl)aminoC.sub.1-4alkyl, di(C.sub.1-4alkyl)aminoC.sub.1-4alkyl(C.sub.1-4alkyl)amino, di(C.sub.1-4alkyl)aminoC.sub.1-4alkyl(C.sub.1-4alkyl)aminoC.sub.1-4alkyl, aminosulfonylamino(C.sub.1-4alkyl)amino, aminosulfonylamino(C.sub.1-4alkyl)aminoC.sub.1-4alkyl, di(C.sub.1-4alkyl)aminosulfonylamino(C.sub.1-4alkyl)amino, di(C.sub.1-4alkyl)aminosulfonylamino(C.sub.1-4alkyl)aminoC.sub.1-6alkyl, cyano, piperidinylC.sub.1-4alkyloxy, pyrrolidinylC.sub.1-4alkyloxy, aminosulfonylpiperazinyl, aminosulfonylpiperazinylC.sub.1-4alkyl, di(C.sub.1-4alkyl)aminosulfonylpiperazinyl, di(C.sub.1-4alkyl)aminosulfonylpiperazinylC.sub.1-4alkyl, hydroxyC.sub.1-4alkylpiperazinyl, hydroxyC.sub.1-4alkylpiperazinylC.sub.1-4alkyl, C.sub.1-4alkyloxypiperidinyl, C.sub.1-4alkyloxypiperidinylC.sub.1-4alkyl, hydroxyC.sub.1-4alkyloxyC.sub.1-4alkylpiperazinyl, hydroxyC.sub.1-4alkyloxyC.sub.1-4alkylpiperazinylC.sub.1-4alkyl, (hydroxyC.sub.1-4alkyl)(C.sub.1-4alkyl)amino, (hydroxyC.sub.1-4alkyl)(C.sub.1-4alkyl)aminoC.sub.1-4alkyl, di(hydroxyC.sub.1-4alkyl)amino, di(hydroxyC.sub.1-4alkyl)aminoC.sub.1-4alkyl, furanyl, furanyl substituted with CHCHCHCH, pyrrolidinylC.sub.1-4alkyl, pyrrolidinylC.sub.1-4alkyloxy, morpholinyl, morpholinylC.sub.1-4alkyloxy, morpholinylC.sub.1-4alkyl, morpholinylC.sub.1-4alkylamino, morpholinylC.sub.1-4alkylaminoC.sub.1-4alkyl, piperazinyl, C.sub.1-4alkylpiperazinyl, C.sub.1-4alkylpiperazinylC.sub.1-4alkyloxy, piperazinylC.sub.1-4alkyl, C.sub.1-4alkylpiperazinylC.sub.1-4alkyl, C.sub.1-4alkylpiperazinylC.sub.1-4alkylamino, C.sub.1-4alkylpiperazinylC.sub.1-4alkylaminoC.sub.1-6alkyl, tetrahydropyrimidinylpiperazinyl, tetrahydropyrimidinylpiperazinylC.sub.1-4alkyl, piperidinylaminoC.sub.1-4alkylamino, piperidinylaminoC.sub.1-4alkylaminoC.sub.1-4alkyl, (C.sub.1-4alkylpiperidinyl)(hydroxyC.sub.1-4alkyl)aminoC.sub.1-4alkylamino, (C.sub.1-4alkylpiperidinyl)(hydroxyC.sub.1-4alkyl)aminoC.sub.1-4alkylaminoC.sub.1-4alkyl, pyridinylC.sub.1-4alkyloxy, hydroxyC.sub.1-4alkylamino, hydroxyC.sub.1-4alkylaminoC.sub.1-4alkyl, di(C.sub.1-4alkyl)aminoC.sub.1-4alkylamino, aminothiadiazolyl, aminosulfonylpiperazinylC.sub.1-4alkyloxy, and thiophenylC.sub.1-4alkylamino; each R.sup.5 and R.sup.6 can be placed on the nitrogen in replacement of the hydrogen; wherein aryl in the above is phenyl, or phenyl substituted with one or more substituents that is each independently halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, cyano, or hydroxycarbonyl.

2. A compound of formula (I), ##STR00095## the N-oxide form, pharmaceutically acceptable addition salt, or stereo-chemically isomeric form thereof, wherein n is 1; m is 2; t is 0; Q is C; X is N or C; Y is N; Z is CH.sub.2; R.sup.1 is C(O)NR.sup.3R.sup.4, 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.3 and R.sup.4 are each independently selected from the group consisting of hydrogen, hydroxy, hydroxyC.sub.1-6alkyl, and aminoC.sub.1-6alkyl; R.sup.2 is hydrogen, hydroxy, amino, hydroxyC.sub.1-6alkyl, C.sub.1-6alkyl, C.sub.1-6alkyloxy, arylC.sub.1-6alkyl, aminocarbonyl, aminoC.sub.1-6alkyl, C.sub.1-6alkylaminoC.sub.1-6alkyl, or di(C.sub.1-6alkyl)aminoC.sub.1-6alkyl; -L- is a bivalent radical selected from the group consisting of C.sub.1-6alkanediyl, carbonyl and sulfonyl; ##STR00096## is a radical selected from the group consisting of ##STR00097## ##STR00098## ##STR00099## ##STR00100## ##STR00101## s is 0, 1, 2, 3 or 4, as allowed; R.sup.5 is selected from the group consisting of hydrogen; halo; hydroxy; amino; nitro; trihaloC.sub.1-6alkyl; trihaloC.sub.1-6alkyloxy; C.sub.1-6alkyl; C.sub.1-6alkyloxy; C.sub.1-6alkylcarbonyl; C.sub.1-6alkyloxycarbonyl; C.sub.1-6alkylsulfonyl; hydroxyC.sub.1-6alkyl; aryloxy; di(C.sub.1-6alkyl)amino; cyano; thiophenyl; furanyl; furanyl substituted with hydroxyC.sub.1-6alkyl; benzofuranyl; imidazolyl; oxazolyl; oxazolyl substituted with aryl and C.sub.1-6alkyl; C.sub.1-6alkyltriazolyl; tetrazolyl; pyrrolidinyl; pyrrolyl; morpholinyl; C.sub.1-6alkylmorpholinyl; piperazinyl; C.sub.1-6alkylpiperazinyl; hydroxyC.sub.1-6alkylpiperazinyl; C.sub.1-6 alkyloxypiperidinyl; pyrazolyl; pyrazolyl substituted with one or two substituents selected from the group consisting of C.sub.1-6alkyl and trihaloC.sub.1-6alkyl; pyridinyl; pyridinyl substituted with C.sub.1-6alkyloxy, aryloxy, or aryl; pyrimidinyl; quinolinyl; indolyl; phenyl; and phenyl substituted with one or two substituents independently selected from the group consisting of halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, and trifluoromethyl; and R.sup.6 is selected from the group consisting of hydrogen; halo; hydroxy; amino; nitro; trihaloC.sub.1-6alkyl; trihaloC.sub.1-6alkyloxy; C.sub.1-6alkyl; C.sub.1-6alkyloxy; C.sub.1-6alkylcarbonyl; C.sub.1-6alkyloxycarbonyl; C.sub.1-6alkylsulfonyl; hydroxyC.sub.1-6alkyl; aryloxy; di(C.sub.1-6alkyl)amino; cyano; pyridinyl; phenyl; and phenyl substituted with one or two substituents independently selected from the group consisting of halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, and trifluoromethyl; wherein aryl in the above is phenyl, or phenyl substituted with one or more substituents that is each independently halo, C.sub.1-6alkyl, C.sub.1-6alkyloxy, trifluoromethyl, cyano, or hydroxycarbonyl.

3. The compound as claimed in claim 1, wherein R.sup.1 is C(O)NH(OH); R.sup.2 is hydrogen; -L- is a bivalent radical that is carbonyl, sulfonyl, or C.sub.1-6alkanediyl substituted with phenyl; ##STR00102## is a radical that is (a-1), (a-20), or (a-43); s is 0 or 1; and each R.sup.5 is hydrogen or phenyl.

4. The compound as claimed in claim 1, wherein R.sup.1 is C(O)NH(OH); R.sup.2 is hydrogen; -L- is a bivalent radical that is carbonyl or sulfonyl; ##STR00103## is a radical that is (a-1) or (a-20); each s is independently 0 or 1; and each R.sup.5 is independently selected from the group consisting of hydrogen and phenyl.

5. The compound according to claim 1 that is ##STR00104##

6. A pharmaceutical composition comprising a pharmaceutically acceptable carrier 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 carrier and the compound.

8. A process for preparing a compound as claimed in claim 1, comprising reacting an intermediate of formula (II) ##STR00105## with an appropriate acid, yielding a hydroxamic acid of formula (I-a) ##STR00106##

9. The method of claim 8 wherein the acid is trifluoroacetic acid.

10. 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 labelled compound as defined in claim 1 and a HDAC.

11. A composition comprising a compound of claim 1 and an additional anti-cancer agent that is cisplatin, carboplatin, oxalyplatin, paclitaxel, docetaxel, irinotecan, topotecan, etoposide, teniposide, vinblastine, vincristine, vinorelbine, 5-fluorouracil, gemcitabine, capecitabine, cyclophosphamide, chlorambucil, carmustine, lomustine, daunorubicin, doxorubicin, idarubicin, mitoxantrone, trastuzumab, tamoxifen, toremifene, droloxifene, faslodex, raloxifene, exemestane, anastrozole, letrazole, vorozole, vitamin D, accutane, azacytidine, flavoperidol, imatinib mesylate, gefitinib, butyrate, 4-phenylbutyrate or valproic acid, suberoylanilide hydroxamic acid (SAHA), biaryl hydroxamate A-161906, bicyclic aryl-N-hydroxycarboxamides, pyroxamide, CG-1521, PXD-101, sulfonamide hydroxamic acid, LAQ-824, trichostatin A (TSA), oxamflatin, scriptaid, m-carboxy cinnamic acid bishydroxamic acid, trapoxin-hydroxamic acid analogue, trapoxin, apidicin, depsipeptide, MS-275, CI-994, or depudecin.

Description

EXPERIMENTAL PART

(1) The following examples are provided for purposes of illustration.

(2) Hereinafter DCM means dichloromethane, DMA means dimethylacetamide, DMF means dimethylformamide, EtOAc means ethyl acetate, iPrOH means isopropyl, MeOH means methanol, EtOAc means ethyl acetate, EtOH means ethanol, TEA means triethylamine, TFA means trifluoroacetic acid and THF means tetrahydrofuran.

A. Preparation of the Intermediates

Example A1

a) Preparation of

(3) ##STR00035##

(4) A solution of 2-naphthalenesulfonyl chloride (0.0094 mol) in DCM (10 ml) was added at 5 C. to a mixture of 2-(4-piperidinyl)-1H-isoindole-1,3(2H)-dione (0.0078 mol) and TEA (0.0109 mol) in DCM (20 ml) under N.sub.2 flow. The mixture was kept 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, yielding 3.3 g (100%) of intermediate 1.

b) Preparation of

(5) ##STR00036##

(6) A mixture of intermediate 1 (0.0078 mol) in hydrazine monohydrate (3.5 ml) and EtOH (35 ml) was stirred and refluxed for 1 hour. Satured sodium chloride 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, yielding 2 g (92%) of intermediate 2, melting point 135 C.

c) Preparation of

(7) ##STR00037##

(8) A mixture of intermediate 2 (0.0036 mol), 6-chloro-3-pyridinecarboxylic acid, ethyl ester (0.0043 mol) and sodium carbonate (0.0054 ml) in DMA (10 ml) was stirred at 130 C. for 18 hours, poured out into water and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (2.5 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH 99.5/0.5;). The pure fractions were collected and the solvent was evaporated. The residue (0.68 g, 43%) was crystallized from diethyl ether. The precipitate was filtered off and dried, yielding 0.477 g (30%) of intermediate 3, melting point 215 C.

Example A2

a) Preparation of

(9) ##STR00038##

(10) TEA (0.0112 mol) then a solution of [1,1-biphenyl]-4-sulfonyl chloride (0.0112 mol) in DCM (5 ml) were added at 5 C. to a mixture of 1,4-dioxa-8-azaspiro[4.6]undecane (0.01 mol) in DCM (10 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, yielding 5.1 g (>100%) of intermediate 4.

b) Preparation of

(11) ##STR00039##

(12) A mixture of intermediate 4 (0.01 mol) in HCl 3N (40 ml) and MeOH (20 ml) was stirred and refluxed, then cooled, poured out on ice, basified with NH.sub.4OH conc. and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness, yielding 3.4 g (100%) of intermediate 5.

c) Preparation of

(13) ##STR00040##

(14) Sodium hydroborate (0.011 mol) was added portionwise at 5 C. to a mixture of intermediate 5 (0.01 mol) in MeOH (35 ml) under N.sub.2 flow. The mixture was kept at room temperature for 1 hour, poured out into water and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness, yielding 3.2 g (97%) of intermediate 6.

d) Preparation of

(15) ##STR00041##

(16) Diazenedicarboxylic acid, bis(1-methylethyl) ester (0.0126 mol) was added dropwise to a mixture of intermediate 6 (0.0096 mol), 1H-isoindole-1,3(2H)-dione (0.0126 mol) and triphenyl-phosphine (0.0126 mol) in THF (35 ml) under N.sub.2 flow. The mixture was kept 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 (13.1 g) was purified by column chromatography over silica gel (15-40 nm) (eluent: DCM/EtOAc 99/1). The pure fractions were collected and the solvent was evaporated, yielding 2.9 g (66%) of intermediate 7.

e) Preparation of

(17) ##STR00042##

(18) A mixture of intermediate 7 (0.0063 mol) in hydrazine monohydrobromide (2.9 ml) and EtOH (30 ml) was stirred and refluxed for 1 hour, then cooled. Satured NaCl 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 (2.5 g) was crystallized from acetonitrile/diethyl ether. The precipitate was filtered off and dried, yielding 0.89 g (89%) of intermediate 8, melting point 141 C.

Preparation of

(19) ##STR00043##

(20) Intermediate 10 was handled analogously as described in example A2 (c, d, e) to give 0.157 g (52%) of intermediate 19, melting point 123 C.

Example A3

a) Preparation of

(21) ##STR00044##

(22) TEA (0.0112 mol) then a solution of 2-naphthalenesulfonyl chloride (0.0112 mol) in DCM (5 ml) were added at 5 C. to a mixture of 1,4-dioxa-8-azaspiro[4.6]undecane (0.01 mol) in DCM (10 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, yielding 3.9 g (>100%) of intermediate 9.

b) Preparation of

(23) ##STR00045##

(24) A mixture of intermediate 9 (0.01 mol) in hydrochloric acid (35 ml) and MeOH (35 ml) was stirred and refluxed for 30 minutes, poured out on ice, basified with NH.sub.4OH and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness, yielding 3.4 g (>100%) of intermediate 10.

c) Preparation of

(25) ##STR00046##

(26) Tetraisopropanolatotitanium (0.012 mol) was added at room temperature to a mixture of intermediate 10 (0.01 mol) and benzenemethanamine (0.011 mol) in EtOH (40 ml). The mixture was stirred for 18 hours. Sodium hydroborate (0.011 mol) was added portionwise. The mixture was stirred at room temperature for 1 hour and 30 minutes. Potassium carbonate 10% was added. The mixture was extracted with DCM. The salts were filtered and washed with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness, yielding 2.7 g (70%) of intermediate 11.

d) Preparation of

(27) ##STR00047##

(28) A mixture of intermediate 11 (0.0068 mol) and Pd/C 10% (0.5 g) in acetic acid (3 ml) and MeOH (30 ml) was hydrogenated at 50 C. for 48 hours under a 3 bar pressure, then filtered and washed with DCM. The filtrate was evaporated, yielding: 2.55 g (>100%) of intermediate 12.

Example A4

a) Preparation of

(29) ##STR00048##

(30) Sodium hydride 60% (0.008 mol) was added portionwise at 0 C. to a mixture of 4-(aminomethyl)-1-piperidinecarboxylic acid, 1,1-dimethylethyl ester (0.004 mol) in THF (20 ml) under N.sub.2 flow. The mixture was stirred at 0 C. for 1 hour. A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0052 mol) in THF (10 ml) was added dropwise at 0 C. The mixture was brought to room temperature, stirred for 2 hours, poured out into ice water and extracted with EtOAc. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue (1.5 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH/NH.sub.4OH 98/2/0.1). The pure fractions were collected and the solvent was evaporated. The residue (0.5 g, 35%) was crystallized from DIPE. The precipitate was filtered off and dried, yielding 0.28 g of intermediate 13, melting point 110 C.

b) Preparation of

(31) ##STR00049##

(32) A mixture of intermediate 13 (0.016 mol) in hydrochloric acid 3N (60 ml) and THF (15 ml) was stirred at 80 C. for 6 hours, poured out into ice water, basified with NH.sub.4OH and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated, yielding 1.4 g (33%) of intermediate 14.

Example A5

a) Preparation of

(33) ##STR00050##

(34) A solution of 1-hydroxy-1H-benzotriazole (0.011 mol) in DCM (5 ml) was added at 5 C. to a mixture of (3-piperidinylmethyl)-carbamic acid, 1,1-dimethylethyl ester (0.01 mol) and TEA (0.014 mol) in DCM (5 ml) under N.sub.2 flow. The mixture was stirred at room temperature for 18 hours. Water 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, yielding 4 g (100%) of intermediate 15.

b) Preparation of

(35) ##STR00051##

(36) A mixture of intermediate 15 (0.01 mol) in HCl/2-propanol (50 ml) was stirred at 50 C. for 15 minutes, basified with NH.sub.4OH concentrated and the solvent was evaporated till dryness. The residue was taken up in DCM and filtered. The filtrate was dried (MgSO.sub.4), filtered and the solvent was evaporated till dryness, yielding 0.53 g (18%) of intermediate 16.

Example A6

a) Preparation of

(37) ##STR00052##

(38) Sodium hydride 60% (0.014 mol) was added portionwise at 0 C. to a mixture of (2-morpholinylmethyl)-carbamic acid, 1,1-dimethylethyl ester (0.0092 mol) in THF (30 ml) under N.sub.2 flow. The mixture was stirred at 0 C. for 1 hour. A solution of 2-naphthalenesulfonyl chloride (0.0111 mol) in THF (30 ml) was added. The mixture was brought to room temperature overnight, poured out into ice water and extracted with EtOAc. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue (4.9 g) was purified by column chromatography over silica gel (15-40 m) (eluent: cyclohexane/EtOAc 70/30). The pure fractions were collected and the solvent was evaporated, yielding 2.15 g (58%) of intermediate 17.

b) Preparation of

(39) ##STR00053##

(40) A mixture of intermediate 17 (0.0052 mol) in HCl 6N (25 ml) was stirred at 80 C. for 12 hours, poured out on ice, basified with sodium hydroxide and extracted with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated, yielding 1 g (63%) of intermediate 18.

B. Preparation of the Final Compounds

Example B1

a) Preparation of

(41) ##STR00054##

(42) A mixture of intermediate 3 (0.0014 mol) and sodium hydroxide (0.0028 mol) in EtOH (10 ml) was stirred and refluxed for 2 hours, then cooled. The precipitate was filtered, washed with EtOH, then with diethyl ether and dried, yielding 0.5 g (82%) of intermediate 20.

b) Preparation of

(43) ##STR00055##

(44) N-(ethylcarbonimidoyl)-N,N-dimethyl-1,3-propanediamine, monohydrochloride (0.0013 mol) was added to a mixture of intermediate 20 (0.0011 mol), O-(tetrahydro-2H-pyran-2-yl)-hydroxylamine (0.0013 mol) and 1-hydroxy-1H-benzotriazole (0.0013 mol) in DCM/THF (10 ml) under N.sub.2 flow. The mixture was stirred at room temperature overnight. 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 (0.95 g) was purified by column chromatography over silica gel (15-40 m)(eluent: DCM/MeOH/NH.sub.4OH 97/3/0.2). The pure fractions were collected and the solvent was evaporated. The residue (0.48 g, 82%) was crystallized from acetonitrile/diethyl ether. The precipitate was filtered off and dried, yielding 0.455 g (77%) of intermediate 21, melting point 129 C.

c) Preparation of

(45) ##STR00056##

(46) Trifluoroacetic acid (0.5 ml) was added to a mixture of intermediate 21 (0.0007 mol) in MeOH (5 ml). The mixture was stirred at room temperature for 18 hours. Trifluoroacetic acid (0.5 ml) was added. The mixture was stirred at room temperature for 18 hours. The solvent was evaporated till dryness. The residue was crystallized from MeOH/DCM/diethyl ether. The precipitate was filtered off and dried, yielding 0.195 g (48%) of compound 1. 0.79C.sub.2HF.sub.3O.sub.2, melting point 160 C.

Example B2

Preparation of

(47) ##STR00057##

(48) A mixture of intermediate 8 (0.0031 mol), 6-chloro-3-pyridinecarboxylic acid, ethyl ester (0.0037 mol) and sodium carbonate (0.0046 mol) in DMA (10 ml) was stirred at 130 C. for 18 hours. Water was added. The mixture was extracted several times with EtOAc. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (2.1 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH 99/1). The pure fractions were collected and the solvent was evaporated. The residue (0.414 g, 28%) was crystallized from diethyl ether. The precipitate was filtered off and dried, yielding 0.294 g (20%) of intermediate 22, melting point 176 C.

(49) Intermediate 22 was handled analogously as described in example [B1] to give 0.084 g (61%) of compound 2. 1.2H.sub.2O. 0.71C.sub.2HF.sub.3O.sub.2 (1:1), melting point 115 C.

(50) ##STR00058##

Example B3

Preparation of

(51) ##STR00059##

(52) Sodium hydride 60% in oil (0.0045 mol) was added at 5 C. to a mixture of intermediate 19 (0.003 mol) in THF (20 ml) under N.sub.2 flow. The mixture was kept for 1 hour. A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0039 mol) in THF (10 ml) was added. The mixture was stirred at 5 C. for 2 hours. Water was added. The mixture was extracted twice with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (1.5 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH 99/1). The pure fractions were collected and the solvent was evaporated. The residue (0.47 g, 34%) was crystallized from diethyl ether. The precipitate was filtered off and dried, yielding 0.224 g (16%) of intermediate 23, melting point 186 C.

(53) Intermediate 23 was handled analogously as described in example [B1] to give 0.032 g (26%) of compound 3. 0.22C.sub.2HF.sub.3O.sub.2, melting point 140 C.

(54) ##STR00060##

Example B4

Preparation of

(55) ##STR00061##

(56) Sodium hydride (0.0038 mol) was added at 5 C. to a mixture of intermediate 8 (0.0025 mol) in THF (20 ml) under N.sub.2 flow. The mixture was kept for 1 hour. A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0033 mol) in THF (10 ml) was added. The mixture was stirred at 5 C. for 18 hours. Water 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 (1.1 g) was purified by column chromatography over silica gel (15-35 m) (eluent: cyclohexane/EtOAc 70/30). The pure fractions were collected and the solvent was evaporated. The residue (0.18 g, 15%) was crystallized from diethyl ether. The precipitate was filtered off and dried, yielding 0.141 g (12%) of intermediate 24, melting point 151 C.

(57) Intermediate 24 was handled analogously as described in example [B1] to give 0.04 g (57%) of compound 4. C.sub.2HF.sub.3O.sub.2, melting point 227 C.

(58) ##STR00062##

Example B5

Preparation of

(59) ##STR00063##

(60) Sodium hydride 60% in oil (0.0045 mol) was added at 5 C. to a mixture of intermediate 2 (0.003 mol) in THF (15 ml) under N.sub.2 flow. The mixture was kept for 1 hour. A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0039 mol) in THF (10 ml) was added. The mixture was stirred at 5 C. for 2 hours. Water was added. The mixture was extracted twice with DCM. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (1.4 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH 99/1). The pure fractions were collected and the solvent was evaporated. The residue (0.49 g, 37%) was crystallized from acetonitrile. The precipitate was filtered, washed with diethyl ether and dried, yielding 0.172 g (13%) of intermediate 25, melting point 226 C.

(61) Intermediate 25 was handled analogously as described in example [B1] to give 0.064 g (49%) of compound 5. 0.17C.sub.2HF.sub.3O.sub.2, melting point 256 C.

(62) ##STR00064##

Example B6

Preparation of

(63) ##STR00065##

(64) A mixture of intermediate 12 (0.0052 mol), 6-chloro-3-pyridinecarboxylic acid, ethyl ester (0.0062 mol) and sodium carbonate (0.0078 mol) in DMA (20 ml) was stirred at 130 C. for 18 hours. Water was added. The mixture was extracted with EtOAc. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated till dryness. The residue (2.6 g) was purified by column chromatography over silica gel 915-40 m) (eluent: DCM/MeOH 99/1;). The pure fractions were collected and the solvent was evaporated, yielding 0.64 g (27%) of intermediate 26, melting point 146 C. Intermediate 26 was handled analogously as described in example [B1] to give 0.263 g (67%) of compound 6 H.sub.2O (1:1) 0.0.73C.sub.2HF.sub.3O.sub.2, melting point 115 C.

(65) ##STR00066##

Example B7

b) Preparation of

(66) ##STR00067##

(67) A solution of 2-naphthalenesulfonyl chloride (0.0006 mol) in DCM (2 ml) was added dropwise at 0 C. to a mixture of intermediate 14 (0.0005 mol) and TEA (0.0008 mol) in DCM (3 ml). The mixture was stirred at room temperature for 12 hours, poured out into water and extracted with EtOAc. The organic layer was separated, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue (0.33 g) was purified by column chromatography over silica gel (10 m) (eluent: DCM 100). The pure fractions were collected and the solvent was evaporated, yielding 0.21 g (80%) of intermediate 27. Intermediate 27 was handled analogously as described in example [B1] to give 0.043 g (33%) of compound 7, melting point 240 C.

(68) ##STR00068##

Example B8

Preparation of

(69) ##STR00069##

(70) Sodium hydride 60% in oil (0.0023 mol) was added portionwise at 5 C. to a mixture of intermediate 16 (0.0017 mol) in THF (5 ml) under N.sub.2 flow. The mixture was stirred for 30 minutes. A solution of 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0021 mol) in THF (2 ml) was added. 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 (0.72 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/MeOH 99/1). The pure fractions were collected and the solvent was evaporated, yielding 0.3 g (39%) of intermediate 28. Intermediate 28 was handled analogously as described in example [B1] to give 0.119 g (75%) of compound 8, melting point 137 C.

(71) ##STR00070##

Example B9

Preparation of

(72) ##STR00071##

(73) A mixture of intermediate 18 (0.0019 mol), 2-(methylsulfonyl)-5-pyrimidinecarboxylic acid, ethyl ester (0.0025 mol) and potassium carbonate (0.0039 mol) in acetonitrile (15 ml) was stirred at room temperature for 12 hours, poured out into ice water and extracted with EtOAc. The organic layer was washed with water, dried (MgSO.sub.4), filtered, and the solvent was evaporated. The residue (1 g) was purified by column chromatography over silica gel (15-40 m) (eluent: DCM/EtOAc 80/20). The pure fractions were collected and the solvent was evaporated. The residue (0.48 g, 89%) was crystallized from CH.sub.3CN/diethyl ether. The precipitate was filtered off and dried, yielding 0.2 g of intermediate 29, melting point 168 C.

(74) Intermediate 29 was handled analogously as described in example [B1] to give 0.08 g (73%) of compound 9, melting point 226 C.

(75) ##STR00072##

(76) Table F-1 lists the compounds that were prepared according to one of the above Examples. The following abbreviations were used in the tables: .C.sub.2HF.sub.3O.sub.2 stands for the trifluoroacetate salt.

(77) TABLE-US-00001 TABLE F-1 0

C. Pharmacological Example

(78) 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).

(79) 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).

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

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

(82) 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.4). 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.

(83) 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 p21 WAF 1 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.

(84) The capacity of the compounds to induce p21 WAF 1 was measured with the p21 WAF 1 enzyme linked immunosorbent assay (WAF 1 ELISA of Oncogene). The p21 WAF 1 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 p21 WAF 1 (provided lyophilised)(see example C.5).

Example C.1

In Vitro Assay for Inhibition of Histone Deacetylase

(85) 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.

(86) 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 subtracted 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 14 compounds had a pIC.sub.50 5 (see table F-2).

Example C.2

Determination of Antiproliferative Activity on A2780 Cells

(87) 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).

(88) 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: BioMerieux).

(89) 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). 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 14 compounds had a pIC.sub.50 5 (see table F-2).

Example C.3

Kinetic Solubility in Aqueous Media

(90) 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 14 compounds was measured. From these compounds 6 showed a score of 3, 2 had a score of 2 and 6 demonstrated a score of 1 (see table F-2).

Example C.4

Metabolic Stability

(91) 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.

(92) 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.

(93) 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.

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

(95) 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 mM at 95 degrees Celsius) microsomes. Recovery of the compounds in the control samples was always 100%.

(96) The mixtures were preincubated for 5 mM 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 C18 (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.

(97) 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.

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

(99) $\left(E\left(\mathrm{act}\right)\right)(\%\mathrm{metabolism}=100\%-\left(\left(\frac{\mathrm{Total}\mathrm{Ion}\mathrm{Current}\left(\mathrm{TIC}\right)\mathrm{of}E\left(\mathrm{act}\right)\mathrm{at}t=15}{\mathrm{TIC}\mathrm{of}E\left(\mathrm{act}\right)\mathrm{at}t=0}\right)100\right).$
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.

(100) Three compounds were tested. One compound had a percentage metabolism less than 20% and two compounds had a percentage metabolism between 20 and 70%.

Example C.5

p21 Induction Capacity

(101) 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.

(102) 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 p21WAF standard was reconstituted with distilled H.sub.2O and further diluted with sample diluent (provided in the kit).

(103) The samples were prepared by diluting them 1:4 in sample diluent. The samples (100 l) and the p21WAF1 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 p21WAF1 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.

(104) 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 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 and showed significant induction.

(105) 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 Cellular activity activity Solubility Co. No. pIC50 pIC50 Score 15 <5 <5 3 10 6.81 5.396 3 1 7.676 5.61 3 3 7.306 5.506 2 11 6.594 5.6 1 5 7.525 5.473 3 4 7.314 5.838 1 14 7.077 5.201 1 6 6.853 5.345 2 12 7.592 5.718 3 2 6.871 5.804 1 13 6.934 5.452 7 7.408 5.109 1 8 7.23 5.721 1 9 7.161 5.667 3

D. Composition Example

Film-Coated Tablets

(106) Preparation of Tablet Core

(107) 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).

(108) Coating

(109) 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.