Title of Invention	A SUBSTITUTED PIPERIDINES AS MDM2-p53 INHIBITORS
Abstract	The present invention provides compounds having formula (II) or formula (III) and the pharmaceutically acceptable salts thereof; wherein X, n, and R1-R12 are defined herein. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutically acceptable carrier or excipient. The compounds are useful for treating cancer.

Title of Invention

A SUBSTITUTED PIPERIDINES AS MDM2-p53 INHIBITORS

Abstract

The present invention provides compounds having formula (II) or formula (III) and the pharmaceutically acceptable salts thereof; wherein X, n, and R1-R12 are defined herein. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutically acceptable carrier or excipient. The compounds are useful for treating cancer.

Full Text	Substituted piperidines as novel MDM2-p53 inhibitors The present invention provides a novel series of piperidines with ris-3,4-dialkoxy substitutions represented by formula (I): and the pharmaceutically acceptable salts thereof; wherein n is an integer independently selected from 1 or 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbon)7!, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy, R12 is selected from the group consisting of alkyl or alkenyl having from 1 to about 5 carbon atoms, and wherein R to R are independently selected from the group consisting of hydrogen, halogen, -CN, -N02, CF3, -OCH3, -COOCH3, and -C6H5.; R2 to R6 are independently selected from the group consisting of hydrogen, halogen, . lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; and in case R12 is alkyl or alkenyl having from 1 to about 5 carbon atoms, R2 to R6 are hydrogen. The compounds of formula I are small molecule inhibitors of MDM2-p53 interaction. These compounds are useful in the treatment or control of cancer. p53 is a tumor suppressor protein that plays a central role in protection against the development of cancer. p53 guards cellular integrity and prevents the propagation of permanently damaged clones of cells by the induction of growth arrest or apoptosis. At the molecular level, p53 is a transcription factor that can activate a panel of genes implicated in the regulation of cell cycle and apoptosis. p53 is a potent cell cycle inhibitor, which is tightly regulated by MDM2 at the cellular level. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 and inhibit its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin-dependent degradation of p53. p53 can activate the expression of the MDM2 gene, thus raising the cellular level of MDM2 protein. This feedback control loop insures that both MDM2 and p53 are kept at a low level in normal proliferating cells. MDM2 is also a cofactor for E2F, which plays a central role in cell cycle regulation. The ratio of MDM2 to p53 (E2F) is deregulated in many cancers. Frequently occurring molecular defects in the pl6INK4/pl9ARF locus, for instance, have been shown to affect MDM2 protein degradation. Inhibition of MDM2-p53 interaction in tumor cells with wild-type p53 should lead to accumulation of p53, cell cycle arrest and/or apoptosis. MDM2 antagonists, therefore, can offer a novel approach to cancer therapy as single agents or in combination with a broad spectrum of other antitumor therapies. The feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of MDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides). MDM2 also binds E2F through a conserved binding region as p53 and activates E2F-dependent transcription of cyclin A, suggesting that MDM2 antagonists might have effects in p53 mutant cells. The present invention provides a compound represented by formula (II): and the pharmaceutical acceptable salts thereof; wherein n is an integer independently selected from 1 or 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyi, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyi, or hydroxy, R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; and R7 to R11 are independendy selected from the group consisting of hydrogen, halogen, -CN, -N02, -CF3, -OCH3, -COOCH3, and -C6H5. The present invention also provides a compound represented by formula (III): and the pharmaceutical^ acceptable salts thereof; wherein n is an integer from 1 to 2; X is halogen; R is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy; and R is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutically acceptable carrier or excipient The invention further provides a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound represented by formula (II) or (III). Furthermore, the invention provides the use of a compound represented by formula (II) or (III) for the preparation of medicaments for treating cancer. The present invention provides a novel series of small molecule inhibitors of MDM2-p53 interaction, piperidines with a5-3,4-dialkoxy substitutions, which have been tested in ELISA assays. The most potent compounds in this series have been shown to inhibit interaction of MDM2 protein with a p53-like peptide with a potency that is approximately the same as a p53-derived peptide. Binding of these compounds to the p53-binding pocket of MDM2 protein has been confirmed by NMR studies of selected compounds in this series as well as fragments of these compounds. These compounds have been shown to bind the same binding pockets as ris-imidazolines, which have demonstrated clear mechanistic activity in cell-based assays as well as antiproliferative activity against wild-type-p53 containing tumor cells both in vitro and in vivo. Therefore these compounds are useful as anticancer agents. As used herein, the following terms have the given meanings: "AlkyP or 'lower alkyl group" means a straight-chained or branched saturated aliphatic hydrocarbon having from 1 to 10, preferably 1 to 6 carbon atoms and more preferably 1 to 4 carbon atoms. Typical lower alkyl groups include methyl, ethyl, propyl, and isopropyl. "AlkenyP means a straight-chain or branched aliphatic hydrocarbon having from 2 to 10, preferably 2 to 6 carbon atoms, and at least one carbon-carbon double bond, for example, vinyl, 2-butenyl, and 3-methyl-2-butenyl. "Carbonyl" means a divalent -CO- radical. "Effective amount" means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. "Halogen" means fluorine, chlorine, bromine, or iodine. "Hydroxy" means a monovalent -OH group. "Substituted/' as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options. "SulfonyF means the bivalent radical -SO2-. "IC50" means a concentration of a particular compound required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described subsequently. "Pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyiammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. aL, Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457. "Pharmaceutically acceptable," such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered. "Therapeutically effective amount" means an amount of at least one compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof, which significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines. Compounds of the present invention as exemplified advantageously show IC50 values from about 3 μ M to about 100 μ M. The compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular, oncological disorders. These compounds and formulations containing the compounds maybe useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors. A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art. The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and maybe determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10>000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it maybe given as continuous infusion. In accordance with the present invention, a compound is provided represented by formula (II): and the pharmaceutically acceptable salts thereof; wherein n is an integer independently selected from 1 or 2; X is halogen; R is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy; r to R° are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; and R' to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -NO* CF3, -OCH3, -COOCH3, and -QH5. In a preferred embodiment of the invention, n is 1. In another preferred embodiment of the invention, X is chloro or fluoro; more preferably chloro. In another preferred embodiment of the invention, X is a para substituenL In another preferred embodiment of the invention, Ri is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3; more_preferably Ri is hydrogen. In another preferred embodiment of the invention, R2 and R$ are independently selected from the group consisting of hydrogen and halogen; more preferably hydrogen, fluoro, chloro, andbromo. In another preferred embodiment of the invention, R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CH3l -OCH3i -CN, and -N02; more preferably R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, and bromo. In another preferred embodiment of the invention, R4 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -C(CH3)3, -CH3> -CN, and -CF3; more preferably R4 is selected from the group consisting of hydrogen, fluoro, chloro, and bromo. In another preferred embodiment of the invention, R7 and Rn are independently selected from the group consisting of hydrogen, halogen, -CN, -N02> and -QH5; more preferably R7 and Rn are independently selected from the group consisting of hydrogen, fluoro, chloro, andbromo. In another preferred embodiment of the invention, Rs and R10 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CN, -N02, and -OCH3; more preferably Rs and Rio are independently selected from the group.consisting of hydrogen, fluoro, chloro, and bromo. In another preferred embodiment of the invention, R9 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -OCH3, -CN, -CF3, and -COOCH3; more preferably R9 is selected from the group consisting of hydrogen, fluoro, chloro, and bromo. as-[rac]-3-[4-Benzylo^ 1-yl] -propane- 1,2-diol; and ds-[rac]-3-[4-Benzy!ox^^ piperidin-1-yl] -methane sulfonyl. Further in accordance with the present invention, a compound is provided having formula (HI): and the pharmaceutical^ acceptable salts thereof, wherein n is an integer from 1 to 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy; and R12 is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms. In a preferred embodiment of the invention, n is 1. In another preferred embodiment of the invention, X is chloro or fluoro; more preferably chloro. In another preferred embodiment of the invention, X is a para substituent In another preferred embodiment of the invention, Ri is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3) -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3; more preferably Ri is hydrogen. In another preferred embodiment of the invention, Ri2 is selected from the group consisting of -(CH2)3CH3, -(CH2)4CH3> -CH2CH=CHCH3, and -CH2CH=CH CH2CH3. The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutical^ acceptable carrier or excipient The invention further provides a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound represented by formula (II) or (HI). Preferably, the cancer is breast or colon cancer. Furthermore, the invention provides a process for the preparation of a compound of formula I of claim 1, which process comprises a) coupling a compound of the formula IV wherein R2 to R6, X and n have the significances as defined hereinbefore, , with a suitable linker to a solid phase to obtain a compound of formula V wherein R1 is H, and if desired, d) reacting the compound of formula la with an acyl halide, alkyl halide or sulfonyl chloride to obtain a compound of formula I, wherein R1 is carbonyl, sulfonyl, lower alkyl or lower alkyl substituted by carbonyl, sulfonyl or hydroxy. The compounds of the present invention, and intermediates thereof, can be prepared according to the schemes set out below. Starting materials are made using known procedures or as illustrated. The abbreviations used in the descriptions of the schemes, preparations, and the examples are set out below. ACE-C1 = 1-chloroethyl chloroformate (Aldrich) BEMP = 2-tert-butyhmino-2-diethylamino-l,3-dimethyl-perhydro-l,3>2-diazaphosphorine (Huka) Boc = tert-butyloxycarbonyl f-BuOK = potassium tert-butoxide CH2CI2 = dichloromethane DCE = 1,2-dichloroethane DMF = N,N-dimethylformamide Et20 = diethyl ether EtOH = ethanol H2SO4 = sulfuric acid HC1 = hydrogen chloride H2O = water MeOH = methanol NaH = sodium hydride NMO = N-methylmorpholine-N-oxide NMP = l-methyl-2-pyrrolidinone OSO4 = osmium tetroxide Polymer (supported) BEMP = 2-tert-butylimino-2-diethylamino-l,3-dimethyl-perhydro-1,3,2- diazaphosphorine supported by polystyrene resin (Fluka) i-Pr2NEt = N, N-diisopropylethylamine TEA = triethylamine TFA = trifluoroacetic acid THF = tetrahydrofuran Schemes 1-3 outline a general method for preparing compounds of the invention having formula (II) and (III) using a novel solid phase synthesis. Scheme 4 outlines a general method for preparing the compounds in solution phase. Scheme 5 illustrates various methods for modifying the Rj group using a novel polymer supported reagent In Scheme 1, the protection of a secondary amine of a commercially available 4-aryl-l,2,3,6-tetrahydropyridine is outlined. The preferred protecting group is a tert-butyloxycarbonyl, which can be synthesized from known procedures (Greene, T. W. and Wuts, P. G. M, 2nd Edition, John Wiley & Sons, N. Y. 1991). For liquid phase synthesis, the resulting tert-butyi ester can be treated with OsCVNMO to form the dihydroxy intermediate A. Selective o-alkylation of intermediate A at C3 can be carried out using NaH and 4-methoxybenzyl chloride. The resulting intermediate can be benzylated according to known methods. Deprotection of both 4-methoxylbenzyl and tert-butyloxycarbonyl groups using 50% TFA/CH2CI2 yields 4-aryl-4-benzyloxy-3-hydroxypiperidine B. For solid phase synthesis, the resulting piperidine B can be coupled to a suitable linker, such as a chloromethyl derivative ArgoPore-Cl linker (Argonaut). Alkylation on solid phase can be accomplished using f-BuOK to generate the anion followed by coupling with various alkyl halides (RX). The final reaction product can be cleaved from the N-benzyl linked tertiary amine linker using a/p/za-chloroethyl chloroformat (ACE-Cl)/MeOH to give the piperidine compounds of the invention (Leysen, D. et al. Tetrahedron Lett. 1997,2915-2918). coupling of the resulting 4-aryl-3,4-dihydro:^iperidine with a ArgoPore-Cl linker. Dialkylation using f-BuOK and alkyl halides (RX) followed by cleavage of the polymer support according to the described conditions affords the piperidine compounds of the invention. In Scheme 4, a general synthetic method is outlined for preparing compounds of formula (II) and (IE) in solution phase. Sequential alkylation of intermediate A using NaH and alkyl halides followed by acidic deprotection of tert-butyloxycarbonyl provides the piperidine compounds of the invention. In Scheme 5, a general method is outlined for the post-modification of intermediate C. The secondary amine of intermediate C can be treated with a variety of agents such as acyl chlorides, alkyl halides, and sulfonyl chlorides to form the corresponding amide, tert-amine, or sulfonamide. The intermediate C can also be allylated using polymer support BEMP and allyl iodide to form the N-allyl substituted intermediate, which can be further treated with OSO4/NMO to form 3-propane-1,2-diol-substituted piperidines of the invention. The compounds of the present invention can be prepared according to the examples below. The examples are presented for purposes of demonstrating, but not limiting, the preparation of the compounds and compositions of this invention. Examples The inhibitory activity (IC5o) of the compounds prepared in the examples below, and represented by formula (II) or (III), is in the range of 3 pM to 100 yM. Example 1 4-(4-Chlorophenyl)-l,2,3>6-tetrahydxopyridine hydrochloride (Lancaster, 1.00 g, 4.35 mmol), triethylamine (0.92 g, 9.13 mmol), and di-tert-butyl carbonate (Aldrich, 1.04 g, 4.78 mmol) were combined in dichloromethane (100 mL) and stirred at room temperature for 14 hours. The mixture was washed with saturated aqueous ammonium chloride solution (30 mL), water (30 mL), saturated sodium chloride solution (30 mL) and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give 4-(4-chlorophenyl)-3,6-dihydro-2H«pyridine-l-carboxylic acid tert-butyl ester (1.41 g, 100%) of as a viscous yellow oil, which was used without further purification. 4-(4-Chlorophenyl)-3,6-dihydro-2H-pyridine-l-carboxylic acid tert-butyl ester (334 mg, 1.14 mmoL) was dissolved in acetone/water (1:1,10 mL), and N- methylmorpholine-N-oxide (Aldrich, 462 mg, 3.42 mmoL) and a catalytic amount of osmium tetroxide (Aldrich) were added. The mixture was stirred at room temperature for 3 hours. A saturated aqueous solution of sodium sulfite (2 mL) was added, and the mixture was stirred for 30 minutes at room temperature. The mixture was diluted with ethyl acetate (10 mL) and water (5 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 X10 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give os-[rac]-4-(4-chIorophenyl)-3,4-dihydroxypiperidine-1-carboxylic acid tert-butyl ester (364 mg, 1.11 mmol, 97%) as a yellow oil. Example 2 To an ice-cold suspension of sodium hydride (62 mg, 2.6 mmoL) in a mixture of telrahydrofiiran/NjN-dimethylformainide (4/1, 4 mL) was added dropwise a solution of c^-[rac]-4-(4-chlorophenyl)"3,4-dihydroxypiperidine-l-carboxylic acid tert-butyl ester (Example 1,340 mg, L04 mmol) in tetrahydrofuran/N,N-dimethylformamide (1 mL, 4/1). After the addition was complete, the mixture was stirred with ice cooling for 15 minutes. Then 3,4-dichloroben2yl chloride (Aldrich, 239 mg, 1.14 mmol) was added, and the mixture was warmed to room temperature and stirred for 20 hours. The mixture was poured into a saturated aqueous solution of ammonium chloride (30 mL), and the mixture was extracted with ethyl acetate (3 X15 mL). The combined organic layers were washed with water (2 X 20 mL), saturated aqueous sodium chloride solution (1X 20 mL), and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give a viscous oil (500 mg), which was purified by flash silica gel chromatography (Merck silica gel 60,230 to 400 mesh) to afford ris-[rac]-4~(4-chlorophenyl)-3-(3,4-dichlorobenzyta^ butyl ester (310 mg, 61%) as a colorless oil. Example 3 ds-[rac]-4-(Ben2ylo:^)^ carboxylic acid tert-butyi ester To an ice cold solution of d5-[rac]-4-(4-chlorophenyl)-3-(3,4-dichloro-benz)4ox7)-4-hydrox)^iperidine-l-carboxylic acid tert-butyi ester (Example 2,224 mg, 0.46 mmol) in tetrahydrofuran (1.5 mL) was added sodium hydride (22 mg, 0.92 mmol) portionwise. After stirring with ice cooling for ten minutes, benzyl bromide (86 mg, 0.51 mmol) was added followed by N,N-dimethylformamide (0.5 mL). The cooling bath was removed, and the mixture was warmed to room temperature and stirred for 3 hours. Several drops of methanol were added, and the mixture was stirred and room temperature for 1 hour. The mixture was'partitioned between ethyl acetate (30 mL) and saturated aqueous ammonium chloride solution (15 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 X 15 mL). The combined organic layers were washed with water (3 X 30 mL), saturated aqueous sodium chloride solution (15 mL) and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give ri5-[rac]-4-(benzyioxy)-4-(4-chlorophenyl)«3-(3,4-dichloroben2yloxy)-piperidine-l-carboxylic acid tert-butyi ester (240 mg), which was used without further purification. Example 4 Crude ds-[racH-(benzylox^ piperidine -1-carboxyiic acid tert-butyl ester (Example 3,240 mg) was dissolved in anhydrous diethyl ether (10 mL). The mixture was cooled in an ice bath, and hydrogen chloride gas was passed through the solution for 10 minutes. The reaction vessel was tightly stoppered and stored at 0°C for 24 hours. The precipitated solid was collected and washed with diethyl ether to afford a5-[rac]-4-(benzyloxyr)-4-(4-chlorophenyl)'3-(354-dichlorobenzyloxy)-piperidine hydrochloride as a white solid (130 mg, 55%). HR-FAB m/e calcd for C24H24NO2CI: [M+Hf 476.0952, found 476.0931. Example 5 cis- [rac] -4-Benzyloxy-4- (4-chloro-phenyl)-3- (4-methoxy-benzyloxy) -piperidine-1 -carboxylic acid tert-butyl ester ds-[rac]-4-Ben2ylox7-4-(4-cMoro-phenyl)-3-(4-methoxy-benzyloxy-)-piperidine--1-carboxylic acid tert-butyl ester was prepared from ds-[rac]-4-(4-chlorophenyl)-3,4-dlhydroxypiperidine-1-carboxylic acid tert-butyl ester (Example 1) and 4-methoxybenzyl chloride (Aldrich) following the procedures used in Examples 2 and 3, which was used without purification. Example 6 cis- [rac] -4-Benzyloxy-4-(4-cUoro-phenyI)-3-(4-me&^^ 1-carboxylic acid tert-butyl ester (Example 5,550 mg, 1.02 mmol) was dissolved in 50% trifluoroacetic acid/dichloromethane (10 mL). After stirring 1.5 hours, the solution was evaporated in vacuo. The residue was triturated in 1:1 ethyl acetate/hexane to provide as-\ [rac]-4,4-[benzyIoxy-(4-chlorophenyl)]-3-hydroxypiperidine (150 mg) as a white powder. Mass spectrum (ES) [M+CH3CN]+ = 359. Example 7 A mixture of as-[rac]-4,4-[benzyloxy-(4«chlorophenyl)]-3-hydroxypiperidine (Example 6, 7.3 g, 23 mmol), ArgoPore-Cl resin (16 g, 15.3 mmol, loading capacity: 0.96 mmol from Argonaut Inc.), and N>N-diisopropylethylamine (13 mL, 76.7 mmol) in N-methylpyrrolinone (12 mL) was heated at 60°C overnight The resin was filtered and washed successively with CH2C12, and MeOH. The resin was dried at 40°C/high vacuum overnight to provide resin-bound 4,4-[benzyloxy-(4-chlorophenyl)]-3-hydroxypiperidine. Example 8 ris-[rac]-4-Benzylox}r-4-(4-chloro-phenyl)-3-substituted piperidine library: General procedure To a suspension of resin-bound as-[rac]-4,4-[benzylo37-(4-chlorophenyi)]-3-hydroxypiperidine (Example 7,200 mg> 0.192 mmol, loading capacity: 0.96 mmol/g) and alkyl halide (RX, 0.58 mmol) in 4:1 THF:DMF solution (2 mL) was added potassium t-butoxide (0.77 M in THF; 1 mL, 0.77 mmol). After stirring at room temperature overnight, the resin was filtered and washed successively with N,N-dimethyiformamide, tetrahydrofuran, methanol, dichloromethane, and diethyl ether. The resin was dried at 40°C/high vacuum overnight. 1-Chloroethyl chloroformate (0.2 mL, 1.9 mmol) was added to the resin in 1,2-dichloroethane (2 mL). After shaking 4 hours at room temperature, the resin was filtered off and washed with 1,2-dichloroethane (3x2 mL). The filtrate was evaporated in vacuo. Dry methanol (2 mL) was added to the residue and the solution was heated at 60°C for 3 hours. The solution was evaporated in vacuo to provide the desired product. -^ 4-(4-Chloro-phenyl)»3-(3,4-dichloro-benzyioxy)-4-substituted piperidine was prepared from resin-bound 3-(3>4-dichlorobenzyloxyr)-4,4-[(4-chlorophenyl)-hydroxy] piperidine (Example 12) and alkyl bromide following the procedure used in Example 8. In the manner described above> the following compounds were prepared. This resin-bound compound was prepared from cis- [rac]-4-(4-chlorophenyl)-3,4-dihydroxypiperidine-1-carboxylic acid tert-butyi ester (Example 1) following the procedure used in Example 6 and 7. Example 15 To a suspension of resin-bound 4-(4-Chloro-phenyl)-piperidine-3,4-diol (Example 14, 200 mg, 0.192 mmol, loading capacity: 0.96 mmol/g) and 2,4-difluorobenzyi bromide (Lancaster, 1.54 mmol) in 4:1 THF:DMF solution (2 mL) was added potassium t-butoxide (0.77 M in THF; 1 mL, 0.77 mmol). After stirring at room temperature overnight, the resin was filtered and washed successively with N,N-dimethylformamide, tetrahydrofuran, methanol, dichloromethane, and diethyl ether. The resin was dried at 40°C/high vacuum overnight 1-Chloroethyl chloroformate (0.2 mL, 1.9 mmol) was added to the resin in 1,2-dichloroethane (2 mL). After shaking 4 hours at room temperature, the resin was filtered off and washed with 1,2-dichloroethane (3x2 mL). The filtrate was evaporated in vacuo. Dry methanol (2 mL) was added to the residue and the solution was heated at 60°C for 3 hours. The solution was evaporated in vacuo to provide the desired product Mass spectrum (ES) M+ = 480. Example 16 cis- [rac] -4-(4-CUoro-phenyl)-3,4-bis-(3-xiitro-benzylox7)-piperidine was prepared from resin-bound 4-(4-Chloro-phenyl)-piperidine-354-diol (Example 14) and 3-nitrobenzyl bromide (Lancaster) following the procedure used in Example 15. Mass spectrum (ES) M+ = 498. Example 17 cis- [rac] -1- [4-Benzyloxy-4~ (4-chloro-phenyl)-3-substituted«piperidin- 1-yl] -ethanone library: General procedure A mixture of 4,4-[benzyloxy-(4-chlorophenyl)]-3-aryloxypiperidine (Example 8, 0.012 mmol), polymer-supported BEMP (10 mg, 0.023 mmol, loading capacity. 2.3 mmol/g from Fluka Inc.), and acetyl chloride (0.01 mL, 0.14 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature overnight The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo to proAdde the desired product Example 17K has an IC50 of 5.5 ^M in the assay described in Example 28. Example 18 cts-[rac]-l-[4-Benzyloxy-4-(4-chlo^ yl] -propan-1 -one cw-[rac]-l-[4-Benzyiox^ piperidin-l-yl]-propan-l-one was prepared from 4-Ben2yloxy-4-(4-chloro-phenyl)-3-(2>3>4-trifluoro-benzyioxy)-piperidine (Example 8Ad) and propionyl chloride (Aldrich) following the procedure used in Example 17. Mass spectrum (ES) M+ = 518. Example 19 c^rac] - l-[4-Ben2yloxy^ yl]-propan-l-one ris-fracj-l-t^Benzyloxy^-C^cUoro-pheny^-S-CS^-dicUoro-benzyloxy)-piperidin-l-yl]-propan-l-one was prepared from cis- [rac]-4-(benzyloxy)-4-(4-chlorophenyl)-3-(3,4»dichloroben2yloxy)-piperidine (Example 4) following the procedure used in Example 18. Mass spectrum (ES) M+ = 533. Example 20 Resin-boimdds-[rac]-4-ben2yloxy"4-(4-fluoro-phenyl)-piperidin-3-ol This resin-bound compound was prepared from 4-(4-fluorophenyi)-l,2?3,6-tetrahydropyridine hydrochloride (Acros) following the procedure used in Examples 1,5, 6, and 7. as-[rac]-4-Benzyloxy-3-(4-bromo-bei^ as-[rac]-4-Benzyloxy-3-(^ prepared from resin-bound cis-[rac]-4-benzyloxy-4-(4-fluoro-phen7l)-piperidin-3-ol (Example 20) and 4-bromobenzyl bromide (Aldrich) following the procedure used in Example 8. Mass spectrum (ES) Mtf" = 471. Example 22 as-[rac]-4-Benzyloxy^-4«(4«fluorO"phenyl)-3-pentylox7-piperidine as-[rac]- 4-Benzylox7-4-(4«fluoro-phenyl)-3-pentyloxy-piperidine was prepared from resin-bound cis-[rac]-4-benzyloxy-4-(4-fluoro-phenyl)-piperidin-3-ol (Example 20) and 1-iodopentane (Aldrich) following the procedure used in Example 8. Mass spectrum (ES) MH+ = 372. IC5o = 56 \iM (in the assay described in Example 28). Example 23 as-[rac]-4-BenzyIoxy^ piperidine A mixture of 4-benzyioxyp-4-(4-cMoro-phenyl)-3-(3,4-dichloro-benz7loxyr)-piperidine (Example 4,0.01 mmol), polymer-supported BEMP (6.5 mg, 0.015 mmol, loading capacity: 2.3 mmol/g from Fluka Inc.), iodoethane (Aldrich, 0.015 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature overnight. The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) MH* = 506. IC50 = 4.9 \xM (in the assay described in Example 28). Example 24 a5-[rac]-4-Ben27loxy-4-(4-cUoro-phenyl)-3-(3,4-dicUoro-ben^4oxy)-l-propyl-piperidine d5-[rac]-4"Benzyloxy-4-(4-cUoro-phenyl)-3-(3,4-dichloro-benzyloxy)-l-propyl-piperidine was prepared from 4-benzyloxy-4-(4-chloro-phenyl) -3- (3,4-dichloro-benzyloxy)-piperidine (Example 4) and 1-iodopropane (Aldrich) following the procedure used in Example 23. Mass spectrum (ES) M* = 519. l A mixture of 4-benz7loxy^-4-(4-chloro-phenyl)-3-(2,3,4-trifluoro-benzylox7)-piperidine (Example 8Ac, 6 mg, 0.013 mmol)> polymer-supported BEMP (10 mg, 0.023 mmol, loading capacity: 2.3 mmol/g from Fluka Inc.), allyl iodide (Aldrich, 3 uL, 0.003 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature for 2 hours. The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo. Without purification, the residue was dissolved in 9:1 acetone/water solution (1 mL). To the solution was added osmium tetroxide (Aldrich, 10 uL, 0.008 mmol) and 4-methylmorpholine N-oxide (Aldrich, 12 mg, 0.1 mmol). The mixture was stirred overnight and Na2SC>3 was added. After stirring for 2 hours, the mixture was filtered through a silica gel pad. The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) M~"= 536. Example 26 as-[rac]-3-[4-Benzyloxy^3-(4-bromo-ben propane-1,2-diol This compound was prepared from 4~benzyloxy-3-(4-bromo-benzyloxy)-4-(4-chloro-phenyl)-piperidine (Example 8P) followed the procedure used in Example 25. Mass spectrum (ES) MH = 562. IC5o = 25.8 ^M (in the assay described in Example 28). Example 27 cis- [rac] -3- [4-Benzyloxy-4- (4-chloro-phenyl)-3-(2,3>4-trifluoro-benzyloxy)-piperidin-1-yl]-methane sulfonyl To a solution of 4-benzylox)^4-(4-chloro-phenyl)-3-(2,3>4-trifluoro-benzylox}0-piperidine (Example 8Ac, 4 mg, 0.008 mmol) and triethylamine (6 uL, 0.08 mmol) in dichloromethane (0.5 mL) at 0°C was added dropwise methanesulfonyl chloride (Aldrich, 2.5 uL, 0.015 mmol). After stirring for 2 hours, the mixture was filtered through an amino silica gel pad (Silicycle). The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) MH+= 541. Example 28 In Vitro Activity Assay The ability of the substituted piperidine compounds of the invention to inhibit the interaction between p53 and MDM2 proteins was measured by an ELISA (Enzyme-Linked Immuno Sorbent Assay) in which recombinant GST-tagged MDM2 binds to a peptide that resembles the MDM2-interacting region of p53 (Bottger et al., J. Mol. Bio. 1997, Vol. 269, pgs. 744-756). This peptide is immobilized to the surface of a 96 well plate via N-terminal biotin, which binds to streptavidin-coated wells. MDM2 is added to each well in the presence of anti-MDM2 mouse monoclonal antibody (SMP-14, Santa Cruz Biotech). After removal of the unbound MDM2 protein, a peroxydase-linked secondary antibody (anti-mouse IgG, Roche Molecular Biochemicals) and the amount of peptide-bound MDM2 is determined colorimetrically by the addition of a peroxydase substrate (MTB Microwell Peroxydase Substrate System, Kirkegaard 8c Perry Labs). Test plates were prepared by coating with streptavidin (5 mg/ml in PBS) for 2 hours followed by a PBS (phosphate-buffered saline) wash and overnight blocking with 150 \|jl of blocking buffer containing 2 mg/ml bovine serum albumin (Sigma) and 0.05% Tween 20 (Sigma) in PBS at 4°C. Biotinylated peptide (1 \M) is added to each well in 50 \xl of blocking buffer and washed extensively after a 1-hour incubation. Test compounds were diluted in a separate 96 well plate and added in triplicate to a compound incubation plate containing a mix of the MDM2 protein and anti-MDM2 antibody. After 20 minutes incubation, the content of the plate is transferred to the test plate and incubated for an additional 1 hour. The secondary anti-mouse IgG antibody is added to the test plate preceded and followed by a triple wash with 0.05% Tween 20 in PBS. Finally, peroxydase substrate is added to each well and the absorption was read using a plate reader (MR7000, Dynatech) at 450nm. The inhibitory activity of the test compounds was measured as a percentage of the bound MDM2 in treated vs. untreated wells and IC5o was calculated. The inhibitory activity (IC50) of the compounds prepared in the examples above, and represented by formula (I) or (II), is in the range of 3 joM to 100 pM, While a number of embodiments of this invention have been represented, it is apparent that the basic construction can be altered to provide other embodiments that utilize the invention without departing from the spirit and scope of the invention. All such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims rather than the specific embodiments that have been presented by way of example. and the pharmaceutically acceptable salts thereof, wherein n is an integer independently selected from 1 or 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy, R12 is selected from the group consisting of alkyl or alkenyi having from 1 to about 5 carbon atoms, and wherein R7 to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -N02, CF3> -OCH3, -COOCH3, and -C6H5.; R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; Ti 7 fit and in case R is alkyl or alkenyi having from 1 to about 5 carbon atoms, R to R are hydrogen. 2. Compounds of formula I according to claim 1 represented by formula (II): and the pharmaceutical^" acceptable salts thereof, wherein n is an integer independently selected from 1 or 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyi, and lower alkyi substituted by carbonyl, sulfonyl, or hydroxy, R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyi, -C(CH3)3, CF3, -OCH3, -NO* and -CN; and R' to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -N02> CF3, -OCH3, -COOCH3, and -C6H5. 3. Compounds of formula II according to claim 2, wherein n is 1. 4. Compounds of formula II according to claim 2, wherein X is chloro. 5. Compounds of formula II according to claim 2, wherein X is a, para substituent 6. Compounds of formula II according to claim 2, wherein R1 is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3. 7. Compounds of formula II according to claim 2, wherein R and R are independently selected from the group consisting of hydrogen, fluoro, chloro, and bromo. 8. Compounds of formula II according to claim 2, wherein R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CH3, -OCH3> -CN, and -N02- 9. Compounds of formula II according to claim 2, wherein R4 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -C(CH3)3, -CH3> -CN, and -CF3. 10. Compounds of formula II according to claim 2, wherein R7 and R11 are independently selected from the group consisting of hydrogen, fluoro, chloro, -CN, -N02, and -C6H5. 11. Compounds of formula II according to claim 2, wherein R8 and R10 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CN, -N02, and -OCH3. 12. Compounds of formula II according to claim 2, wherein R9 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -OCH3, -CN, -CF3, and -COOCH3. 13. Compounds of formula II according to claim 2, wherein the compound is selected from the group consisting of: os-[rac]-4-benzyloxy-4-(4-chloro-phenyl)-3-(3,4-difluoro-benzyloxy)-piperidine; ds-[rac]-4-benzylox7-4-(4-cUoro-phenyl)-3-(2,6-difluoro-benzyloxy)-piperidine; ds-[rac]-4-benz)doxj^3-(biphenyl-2-ylmethox50"4-(4-cMoro-phenyl)-piperidine; cis- [rac] -4- [4-ben2rj4oxy-4-(4-cUoro-phenyi)-piperidin-3-yloxymethyl] -benzonitrile; ds-[rac]-4-benzyloxy-4-(4-cUoro-phenyl)-3-(3-ni1ro-benzyloxy)-piperidine; ds-[rac]-4-benzyloxy-3-(3-bromo-benzyloxyp)-4-(4-chloro-phenyl)-piperidine; ds-[rac]-4-benzyloxy-4-(4-cMoro-phenyl)-3-(2,4-difluoro-benzylox}0-piperidine; aV[rac]-4-benz7loxy-3-(2-cUoro-4-fluoro-benzyloxy)-4-(4-chloro-phenyl)-piperidine; cis- [rac] -4- [4-benzyloxy-4- (4-chloro-phenyl) -piperidin-3-yloxymethyl] -benzoic acid methyl ester; d5-[rac]-4-benz7lox7-4-(4-chloro-phenyl)-3-(3,5-difluoro-benzyloxy)-piperidine; ds-[rac]-4-ben2yloxy-3-(3-cUoro-2-fluoro-benzyloxy)-4-(4-cUoro-phenyl)-piperidine; and the pharmaceutical^ acceptable salts thereof, wherein n is an integer from 1 to 2; X is halogen; R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy, and R12 is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms. 19. Compounds of formula III according to claim 18, wherein n is 1. 20. Compounds of formula III according to claim 18, wherein X is chloro. 21. Compounds of formula III according to claim 18, wherein X is a para substituent 22. Compounds of formula III according to claim 18, wherein R1 is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -SO2CH3. 23. Compounds of formula III according to claim 18> wherein R is selected from the group consisting of-(CH2)3CH3> -(CH2)4CH3, -CH2CH=CHCH3> and -CH2CH=CH-CH2CH3. 24. Compounds of formula III according to claim 18, wherein the compound is selected from the group consisting of: cis- [rac] -4-ben2yloxy-4-(4-fluoro-phenyl)-3-butyloxy-piperidine; ds«[rac]"4-benzyloxy-4-(4«fluoro-phenyl)-3-pentyloxy-piperidine; cis- [rac] -4-benzyloxy-4-(4-fluoro-phenyl)-3-(but-2-en)yloxy-piperidine; a>[rac]"4-benzyloxyr-4-(4-fluoro-phenyl)--3-(pent-2-enyl)oxy-piperidine; cis- [rac] -4-benzyloxy-4-(4-chloro-phenyl)-3-butyloxy-piperidine; ds- [rac] -4-benz)doxy-4-(4-chloro-phenyl)-3-pentyloxy-piperidine; as-[rac]-4-benzylox7-4-(4--chloro-phenyl)-3-(but-2-en)yloxy-piperidine;and cis- [rac] -4-benzyloxy-4- (4-chloro-phenyl)-3- (pent-2-enyl)oxy-piperidine. 25. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I according to claim 1 and a pharmaceutically acceptable carrier or excipient 26. The pharmaceutical composition according to claim 25 comprising a therapeutically effective amount of a compound of formula II according to claim 2 and a pharmaceutically acceptable carrier or excipient 27. The pharmaceutical composition according to claim 25 comprising a therapeutically effective amount of a compound of formula III according to claim 18 and a pharmaceutically acceptable carrier or excipient 28. The pharmaceutical composition according to claim 25, wherein the compound is suitable for administration to a patient having cancer. 29. Compounds of any one of claims 1 to 24 for use as medicaments. 30. A process for the preparation of a compound of formula I of claim 1, which process comprises a) coupling a compound of the formula IV

Full Text

Substituted piperidines as novel MDM2-p53 inhibitors
The present invention provides a novel series of piperidines with ris-3,4-dialkoxy substitutions represented by formula (I):

and the pharmaceutically acceptable salts thereof; wherein
n is an integer independently selected from 1 or 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbon)7!, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy,
R12 is selected from the group consisting of alkyl or alkenyl having from 1 to about 5 carbon atoms, and
wherein R to R are independently selected from the group consisting of hydrogen, halogen, -CN, -N02, CF3, -OCH3, -COOCH3, and -C6H5.;

R2 to R6 are independently selected from the group consisting of hydrogen, halogen, . lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN;
and in case R12 is alkyl or alkenyl having from 1 to about 5 carbon atoms, R2 to R6 are hydrogen.
The compounds of formula I are small molecule inhibitors of MDM2-p53 interaction. These compounds are useful in the treatment or control of cancer.
p53 is a tumor suppressor protein that plays a central role in protection against the development of cancer. p53 guards cellular integrity and prevents the propagation of permanently damaged clones of cells by the induction of growth arrest or apoptosis. At the molecular level, p53 is a transcription factor that can activate a panel of genes implicated in the regulation of cell cycle and apoptosis. p53 is a potent cell cycle inhibitor, which is tightly regulated by MDM2 at the cellular level. MDM2 and p53 form a feedback control loop. MDM2 can bind p53 and inhibit its ability to transactivate p53-regulated genes. In addition, MDM2 mediates the ubiquitin-dependent degradation of p53. p53 can activate the expression of the MDM2 gene, thus raising the cellular level of MDM2 protein. This feedback control loop insures that both MDM2 and p53 are kept at a low level in normal proliferating cells. MDM2 is also a cofactor for E2F, which plays a central role in cell cycle regulation.
The ratio of MDM2 to p53 (E2F) is deregulated in many cancers. Frequently occurring molecular defects in the pl6INK4/pl9ARF locus, for instance, have been shown to affect MDM2 protein degradation. Inhibition of MDM2-p53 interaction in tumor cells with wild-type p53 should lead to accumulation of p53, cell cycle arrest and/or apoptosis. MDM2 antagonists, therefore, can offer a novel approach to cancer therapy as single agents or in combination with a broad spectrum of other antitumor therapies. The feasibility of this strategy has been shown by the use of different macromolecular tools for inhibition of MDM2-p53 interaction (e.g. antibodies, antisense oligonucleotides, peptides). MDM2 also binds E2F through a conserved binding region as p53 and activates E2F-dependent transcription of cyclin A, suggesting that MDM2 antagonists might have effects in p53 mutant cells.
The present invention provides a compound represented by formula (II):

and the pharmaceutical acceptable salts thereof; wherein
n is an integer independently selected from 1 or 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyi, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyi, or hydroxy,
R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; and
R7 to R11 are independendy selected from the group consisting of hydrogen, halogen, -CN, -N02, -CF3, -OCH3, -COOCH3, and -C6H5.
The present invention also provides a compound represented by formula (III):

and the pharmaceutical^ acceptable salts thereof; wherein n is an integer from 1 to 2;

X is halogen;
R is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy; and
R is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutically acceptable carrier or excipient The invention further provides a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound represented by formula (II) or (III). Furthermore, the invention provides the use of a compound represented by formula (II) or (III) for the preparation of medicaments for treating cancer.
The present invention provides a novel series of small molecule inhibitors of MDM2-p53 interaction, piperidines with a5-3,4-dialkoxy substitutions, which have been tested in ELISA assays. The most potent compounds in this series have been shown to inhibit interaction of MDM2 protein with a p53-like peptide with a potency that is approximately the same as a p53-derived peptide. Binding of these compounds to the p53-binding pocket of MDM2 protein has been confirmed by NMR studies of selected compounds in this series as well as fragments of these compounds. These compounds have been shown to bind the same binding pockets as ris-imidazolines, which have demonstrated clear mechanistic activity in cell-based assays as well as antiproliferative activity against wild-type-p53 containing tumor cells both in vitro and in vivo. Therefore these compounds are useful as anticancer agents.
As used herein, the following terms have the given meanings:
"AlkyP or 'lower alkyl group" means a straight-chained or branched saturated aliphatic hydrocarbon having from 1 to 10, preferably 1 to 6 carbon atoms and more preferably 1 to 4 carbon atoms. Typical lower alkyl groups include methyl, ethyl, propyl, and isopropyl.
"AlkenyP means a straight-chain or branched aliphatic hydrocarbon having from 2 to 10, preferably 2 to 6 carbon atoms, and at least one carbon-carbon double bond, for example, vinyl, 2-butenyl, and 3-methyl-2-butenyl.
"Carbonyl" means a divalent -CO- radical.

"Effective amount" means an amount that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated.
"Halogen" means fluorine, chlorine, bromine, or iodine.
"Hydroxy" means a monovalent -OH group.
"Substituted/' as in substituted alkyl, means that the substitution can occur at one or more positions and, unless otherwise indicated, that the substituents at each substitution site are independently selected from the specified options.
"SulfonyF means the bivalent radical -SO2-.
"IC50" means a concentration of a particular compound required to inhibit 50% of a specific measured activity. IC50 can be measured, inter alia, as is described subsequently.
"Pharmaceutically acceptable salt" refers to conventional acid-addition salts or base-addition salts that retain the biological effectiveness and properties of the compounds of the present invention and are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases. Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like. Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as for example, tetramethyiammonium hydroxide. Chemical modification of a pharmaceutical compound (i.e. drug) into a salt is a technique well known to pharmaceutical chemists to obtain improved physical and chemical stability, hygroscopicity, flowability and solubility of compounds. See, e.g., H. Ansel et. aL, Pharmaceutical Dosage Forms and Drug Delivery Systems (6th Ed. 1995) at pp. 196 and 1456-1457.
"Pharmaceutically acceptable," such as pharmaceutically acceptable carrier, excipient, etc., means pharmacologically acceptable and substantially non-toxic to the subject to which the particular compound is administered.
"Therapeutically effective amount" means an amount of at least one compound of formula (II) or (III), or a pharmaceutically acceptable salt thereof, which significantly inhibits proliferation and/or prevents differentiation of a human tumor cell, including human tumor cell lines.

Compounds of the present invention as exemplified advantageously show IC50 values from about 3 μ M to about 100 μ M.
The compounds of the present invention are useful in the treatment or control of cell proliferative disorders, in particular, oncological disorders. These compounds and formulations containing the compounds maybe useful in the treatment or control of solid tumors, such as, for example, breast, colon, lung and prostate tumors.
A therapeutically effective amount of a compound in accordance with this invention means an amount of compound that is effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is within the skill in the art.
The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and maybe determined in a manner known in the art. Such dosage will be adjusted to the individual requirements in each particular case including the specific compound(s) being administered, the route of administration, the condition being treated, as well as the patient being treated. In general, in the case of oral or parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 10 mg to about 10>000 mg, preferably from about 200 mg to about 1,000 mg, should be appropriate, although the upper limit may be exceeded when indicated. The daily dosage can be administered as a single dose or in divided doses, or for parenteral administration, it maybe given as continuous infusion.
In accordance with the present invention, a compound is provided represented by formula (II):

and the pharmaceutically acceptable salts thereof; wherein

n is an integer independently selected from 1 or 2; X is halogen;
R is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy;
r to R° are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN; and
R' to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -NO* CF3, -OCH3, -COOCH3, and -QH5.
In a preferred embodiment of the invention, n is 1.
In another preferred embodiment of the invention, X is chloro or fluoro; more preferably chloro. In another preferred embodiment of the invention, X is a para substituenL
In another preferred embodiment of the invention, Ri is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3; more_preferably Ri is hydrogen.
In another preferred embodiment of the invention, R2 and R$ are independently selected from the group consisting of hydrogen and halogen; more preferably hydrogen, fluoro, chloro, andbromo.
In another preferred embodiment of the invention, R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CH3l -OCH3i -CN, and -N02; more preferably R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, and bromo.
In another preferred embodiment of the invention, R4 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -C(CH3)3, -CH3> -CN, and -CF3; more preferably R4 is selected from the group consisting of hydrogen, fluoro, chloro, and bromo.
In another preferred embodiment of the invention, R7 and Rn are independently selected from the group consisting of hydrogen, halogen, -CN, -N02> and -QH5; more preferably R7 and Rn are independently selected from the group consisting of hydrogen, fluoro, chloro, andbromo.

In another preferred embodiment of the invention, Rs and R10 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CN, -N02, and -OCH3; more preferably Rs and Rio are independently selected from the group.consisting of hydrogen, fluoro, chloro, and bromo.
In another preferred embodiment of the invention, R9 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -OCH3, -CN, -CF3, and -COOCH3; more preferably R9 is selected from the group consisting of hydrogen, fluoro, chloro, and bromo.

as-[rac]-3-[4-Benzylo^ 1-yl] -propane- 1,2-diol; and
ds-[rac]-3-[4-Benzy!ox^^
piperidin-1-yl] -methane sulfonyl.
Further in accordance with the present invention, a compound is provided having formula (HI):
and the pharmaceutical^ acceptable salts thereof, wherein
n is an integer from 1 to 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy; and
R12 is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms.
In a preferred embodiment of the invention, n is 1.
In another preferred embodiment of the invention, X is chloro or fluoro; more preferably chloro. In another preferred embodiment of the invention, X is a para substituent
In another preferred embodiment of the invention, Ri is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3) -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3; more preferably Ri is hydrogen.
In another preferred embodiment of the invention, Ri2 is selected from the group consisting of -(CH2)3CH3, -(CH2)4CH3> -CH2CH=CHCH3, and -CH2CH=CH CH2CH3.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of a compound represented by formula (II) or (III) and a pharmaceutical^ acceptable carrier or excipient The invention further provides a method for treating cancer comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound represented by formula (II) or (HI). Preferably, the cancer is breast or colon cancer.
Furthermore, the invention provides a process for the preparation of a compound of formula I of claim 1, which process comprises
a) coupling a compound of the formula IV

wherein R2 to R6, X and n have the significances as defined hereinbefore, , with a suitable linker to a solid phase to obtain a compound of formula V

wherein R1 is H, and if desired,
d) reacting the compound of formula la with an acyl halide, alkyl halide or sulfonyl chloride to obtain a compound of formula I, wherein R1 is carbonyl, sulfonyl, lower alkyl or lower alkyl substituted by carbonyl, sulfonyl or hydroxy.
The compounds of the present invention, and intermediates thereof, can be prepared according to the schemes set out below. Starting materials are made using known procedures or as illustrated. The abbreviations used in the descriptions of the schemes, preparations, and the examples are set out below.
ACE-C1 = 1-chloroethyl chloroformate (Aldrich)
BEMP = 2-tert-butyhmino-2-diethylamino-l,3-dimethyl-perhydro-l,3>2-diazaphosphorine (Huka)
Boc = tert-butyloxycarbonyl
f-BuOK = potassium tert-butoxide
CH2CI2 = dichloromethane
DCE = 1,2-dichloroethane
DMF = N,N-dimethylformamide
Et20 = diethyl ether
EtOH = ethanol
H2SO4 = sulfuric acid
HC1 = hydrogen chloride
H2O = water
MeOH = methanol
NaH = sodium hydride
NMO = N-methylmorpholine-N-oxide
NMP = l-methyl-2-pyrrolidinone
OSO4 = osmium tetroxide

Polymer (supported) BEMP = 2-tert-butylimino-2-diethylamino-l,3-dimethyl-perhydro-1,3,2- diazaphosphorine supported by polystyrene resin (Fluka)
i-Pr2NEt = N, N-diisopropylethylamine
TEA = triethylamine
TFA = trifluoroacetic acid
THF = tetrahydrofuran
Schemes 1-3 outline a general method for preparing compounds of the invention having formula (II) and (III) using a novel solid phase synthesis. Scheme 4 outlines a general method for preparing the compounds in solution phase. Scheme 5 illustrates various methods for modifying the Rj group using a novel polymer supported reagent
In Scheme 1, the protection of a secondary amine of a commercially available 4-aryl-l,2,3,6-tetrahydropyridine is outlined. The preferred protecting group is a tert-butyloxycarbonyl, which can be synthesized from known procedures (Greene, T. W. and Wuts, P. G. M, 2nd Edition, John Wiley & Sons, N. Y. 1991). For liquid phase synthesis, the resulting tert-butyi ester can be treated with OsCVNMO to form the dihydroxy intermediate A. Selective o-alkylation of intermediate A at C3 can be carried out using NaH and 4-methoxybenzyl chloride. The resulting intermediate can be benzylated according to known methods. Deprotection of both 4-methoxylbenzyl and tert-butyloxycarbonyl groups using 50% TFA/CH2CI2 yields 4-aryl-4-benzyloxy-3-hydroxypiperidine B. For solid phase synthesis, the resulting piperidine B can be coupled to a suitable linker, such as a chloromethyl derivative ArgoPore-Cl linker (Argonaut). Alkylation on solid phase can be accomplished using f-BuOK to generate the anion followed by coupling with various alkyl halides (RX). The final reaction product can be cleaved from the N-benzyl linked tertiary amine linker using a/p/za-chloroethyl chloroformat (ACE-Cl)/MeOH to give the piperidine compounds of the invention (Leysen, D. et al. Tetrahedron Lett. 1997,2915-2918).

coupling of the resulting 4-aryl-3,4-dihydro:^iperidine with a ArgoPore-Cl linker. Dialkylation using f-BuOK and alkyl halides (RX) followed by cleavage of the polymer support according to the described conditions affords the piperidine compounds of the invention.

In Scheme 4, a general synthetic method is outlined for preparing compounds of formula (II) and (IE) in solution phase. Sequential alkylation of intermediate A using NaH and alkyl halides followed by acidic deprotection of tert-butyloxycarbonyl provides the piperidine compounds of the invention.

In Scheme 5, a general method is outlined for the post-modification of intermediate C. The secondary amine of intermediate C can be treated with a variety of agents such as acyl chlorides, alkyl halides, and sulfonyl chlorides to form the corresponding amide, tert-amine, or sulfonamide. The intermediate C can also be allylated using polymer support BEMP and allyl iodide to form the N-allyl substituted intermediate, which can be further treated with OSO4/NMO to form 3-propane-1,2-diol-substituted piperidines of the invention.

The compounds of the present invention can be prepared according to the examples below. The examples are presented for purposes of demonstrating, but not limiting, the preparation of the compounds and compositions of this invention.
Examples
The inhibitory activity (IC5o) of the compounds prepared in the examples below, and represented by formula (II) or (III), is in the range of 3 pM to 100 yM.
Example 1

4-(4-Chlorophenyl)-l,2,3>6-tetrahydxopyridine hydrochloride (Lancaster, 1.00 g, 4.35 mmol), triethylamine (0.92 g, 9.13 mmol), and di-tert-butyl carbonate (Aldrich, 1.04 g, 4.78 mmol) were combined in dichloromethane (100 mL) and stirred at room temperature for 14 hours. The mixture was washed with saturated aqueous ammonium chloride solution (30 mL), water (30 mL), saturated sodium chloride solution (30 mL) and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give 4-(4-chlorophenyl)-3,6-dihydro-2H«pyridine-l-carboxylic acid tert-butyl ester (1.41 g, 100%) of as a viscous yellow oil, which was used without further purification. 4-(4-Chlorophenyl)-3,6-dihydro-2H-pyridine-l-carboxylic acid tert-butyl ester (334 mg, 1.14 mmoL) was dissolved in acetone/water (1:1,10 mL), and N-

methylmorpholine-N-oxide (Aldrich, 462 mg, 3.42 mmoL) and a catalytic amount of osmium tetroxide (Aldrich) were added. The mixture was stirred at room temperature for 3 hours. A saturated aqueous solution of sodium sulfite (2 mL) was added, and the mixture was stirred for 30 minutes at room temperature. The mixture was diluted with ethyl acetate (10 mL) and water (5 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (2 X10 mL). The combined organic layers were washed with brine and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give os-[rac]-4-(4-chIorophenyl)-3,4-dihydroxypiperidine-1-carboxylic acid tert-butyl ester (364 mg, 1.11 mmol, 97%) as a yellow oil.
Example 2

To an ice-cold suspension of sodium hydride (62 mg, 2.6 mmoL) in a mixture of telrahydrofiiran/NjN-dimethylformainide (4/1, 4 mL) was added dropwise a solution of c^-[rac]-4-(4-chlorophenyl)"3,4-dihydroxypiperidine-l-carboxylic acid tert-butyl ester (Example 1,340 mg, L04 mmol) in tetrahydrofuran/N,N-dimethylformamide (1 mL, 4/1). After the addition was complete, the mixture was stirred with ice cooling for 15 minutes. Then 3,4-dichloroben2yl chloride (Aldrich, 239 mg, 1.14 mmol) was added, and the mixture was warmed to room temperature and stirred for 20 hours. The mixture was poured into a saturated aqueous solution of ammonium chloride (30 mL), and the mixture was extracted with ethyl acetate (3 X15 mL). The combined organic layers were washed with water (2 X 20 mL), saturated aqueous sodium chloride solution (1X 20 mL), and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give a viscous oil (500 mg), which was purified by flash silica gel chromatography (Merck silica gel 60,230 to 400 mesh) to afford ris-[rac]-4~(4-chlorophenyl)-3-(3,4-dichlorobenzyta^ butyl ester (310 mg, 61%) as a colorless oil.

Example 3
ds-[rac]-4-(Ben2ylo:^)^ carboxylic acid tert-butyi ester

To an ice cold solution of d5-[rac]-4-(4-chlorophenyl)-3-(3,4-dichloro-benz)4ox7)-4-hydrox)^iperidine-l-carboxylic acid tert-butyi ester (Example 2,224 mg, 0.46 mmol) in tetrahydrofuran (1.5 mL) was added sodium hydride (22 mg, 0.92 mmol) portionwise. After stirring with ice cooling for ten minutes, benzyl bromide (86 mg, 0.51 mmol) was added followed by N,N-dimethylformamide (0.5 mL). The cooling bath was removed, and the mixture was warmed to room temperature and stirred for 3 hours. Several drops of methanol were added, and the mixture was stirred and room temperature for 1 hour. The mixture was'partitioned between ethyl acetate (30 mL) and saturated aqueous ammonium chloride solution (15 mL). The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (3 X 15 mL). The combined organic layers were washed with water (3 X 30 mL), saturated aqueous sodium chloride solution (15 mL) and dried over anhydrous magnesium sulfate. The mixture was filtered and concentrated in vacuo to give ri5-[rac]-4-(benzyioxy)-4-(4-chlorophenyl)«3-(3,4-dichloroben2yloxy)-piperidine-l-carboxylic acid tert-butyi ester (240 mg), which was used without further purification.
Example 4

Crude ds-[racH-(benzylox^ piperidine -1-carboxyiic acid tert-butyl ester (Example 3,240 mg) was dissolved in anhydrous diethyl ether (10 mL). The mixture was cooled in an ice bath, and hydrogen chloride gas was passed through the solution for 10 minutes. The reaction vessel was tightly stoppered and stored at 0°C for 24 hours. The precipitated solid was collected and washed with diethyl ether to afford a5-[rac]-4-(benzyloxyr)-4-(4-chlorophenyl)'3-(354-dichlorobenzyloxy)-piperidine hydrochloride as a white solid (130 mg, 55%).
HR-FAB m/e calcd for C24H24NO2CI: [M+Hf 476.0952, found 476.0931.
Example 5
cis- [rac] -4-Benzyloxy-4- (4-chloro-phenyl)-3- (4-methoxy-benzyloxy) -piperidine-1 -carboxylic acid tert-butyl ester

ds-[rac]-4-Ben2ylox7-4-(4-cMoro-phenyl)-3-(4-methoxy-benzyloxy-)-piperidine--1-carboxylic acid tert-butyl ester was prepared from ds-[rac]-4-(4-chlorophenyl)-3,4-dlhydroxypiperidine-1-carboxylic acid tert-butyl ester (Example 1) and 4-methoxybenzyl chloride (Aldrich) following the procedures used in Examples 2 and 3, which was used without purification.
Example 6

cis- [rac] -4-Benzyloxy-4-(4-cUoro-phenyI)-3-(4-me&^^ 1-carboxylic acid tert-butyl ester (Example 5,550 mg, 1.02 mmol) was dissolved in 50% trifluoroacetic acid/dichloromethane (10 mL). After stirring 1.5 hours, the solution was evaporated in vacuo. The residue was triturated in 1:1 ethyl acetate/hexane to provide as-\ [rac]-4,4-[benzyIoxy-(4-chlorophenyl)]-3-hydroxypiperidine (150 mg) as a white powder. Mass spectrum (ES) [M+CH3CN]+ = 359.
Example 7

A mixture of as-[rac]-4,4-[benzyloxy-(4«chlorophenyl)]-3-hydroxypiperidine (Example 6, 7.3 g, 23 mmol), ArgoPore-Cl resin (16 g, 15.3 mmol, loading capacity: 0.96 mmol from Argonaut Inc.), and N>N-diisopropylethylamine (13 mL, 76.7 mmol) in N-methylpyrrolinone (12 mL) was heated at 60°C overnight The resin was filtered and washed successively with CH2C12, and MeOH. The resin was dried at 40°C/high vacuum overnight to provide resin-bound 4,4-[benzyloxy-(4-chlorophenyl)]-3-hydroxypiperidine.
Example 8
ris-[rac]-4-Benzylox}r-4-(4-chloro-phenyl)-3-substituted piperidine library:
General procedure

To a suspension of resin-bound as-[rac]-4,4-[benzylo37-(4-chlorophenyi)]-3-hydroxypiperidine (Example 7,200 mg> 0.192 mmol, loading capacity: 0.96 mmol/g) and alkyl halide (RX, 0.58 mmol) in 4:1 THF:DMF solution (2 mL) was added potassium t-butoxide (0.77 M in THF; 1 mL, 0.77 mmol). After stirring at room temperature overnight, the resin was filtered and washed successively with N,N-dimethyiformamide, tetrahydrofuran, methanol, dichloromethane, and diethyl ether. The resin was dried at 40°C/high vacuum overnight. 1-Chloroethyl chloroformate (0.2 mL, 1.9 mmol) was added to the resin in 1,2-dichloroethane (2 mL). After shaking 4 hours at room temperature, the resin was filtered off and washed with 1,2-dichloroethane (3x2 mL). The filtrate was evaporated in vacuo. Dry methanol (2 mL) was added to the residue and the solution was heated at 60°C for 3 hours. The solution was evaporated in vacuo to provide the desired product.

-^

4-(4-Chloro-phenyl)»3-(3,4-dichloro-benzyioxy)-4-substituted piperidine was prepared from resin-bound 3-(3>4-dichlorobenzyloxyr)-4,4-[(4-chlorophenyl)-hydroxy] piperidine (Example 12) and alkyl bromide following the procedure used in Example 8.
In the manner described above> the following compounds were prepared.

This resin-bound compound was prepared from cis- [rac]-4-(4-chlorophenyl)-3,4-dihydroxypiperidine-1-carboxylic acid tert-butyi ester (Example 1) following the procedure used in Example 6 and 7.
Example 15

To a suspension of resin-bound 4-(4-Chloro-phenyl)-piperidine-3,4-diol (Example 14, 200 mg, 0.192 mmol, loading capacity: 0.96 mmol/g) and 2,4-difluorobenzyi bromide (Lancaster, 1.54 mmol) in 4:1 THF:DMF solution (2 mL) was added potassium t-butoxide (0.77 M in THF; 1 mL, 0.77 mmol). After stirring at room temperature overnight, the resin was filtered and washed successively with N,N-dimethylformamide, tetrahydrofuran, methanol, dichloromethane, and diethyl ether. The resin was dried at 40°C/high vacuum overnight 1-Chloroethyl chloroformate (0.2 mL, 1.9 mmol) was added to the resin in 1,2-dichloroethane (2 mL). After shaking 4 hours at room temperature, the resin was filtered off and washed with 1,2-dichloroethane (3x2 mL). The filtrate was evaporated in vacuo. Dry methanol (2 mL) was added to the residue and the solution was heated at 60°C for 3 hours. The solution was evaporated in vacuo to provide the desired product Mass spectrum (ES) M+ = 480.
Example 16

cis- [rac] -4-(4-CUoro-phenyl)-3,4-bis-(3-xiitro-benzylox7)-piperidine was prepared from resin-bound 4-(4-Chloro-phenyl)-piperidine-354-diol (Example 14) and 3-nitrobenzyl bromide (Lancaster) following the procedure used in Example 15. Mass spectrum (ES) M+ = 498.
Example 17
cis- [rac] -1- [4-Benzyloxy-4~ (4-chloro-phenyl)-3-substituted«piperidin- 1-yl] -ethanone library:
General procedure

A mixture of 4,4-[benzyloxy-(4-chlorophenyl)]-3-aryloxypiperidine (Example 8, 0.012 mmol), polymer-supported BEMP (10 mg, 0.023 mmol, loading capacity. 2.3 mmol/g from Fluka Inc.), and acetyl chloride (0.01 mL, 0.14 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature overnight The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo to proAdde the desired product

Example 17K has an IC50 of 5.5 ^M in the assay described in Example 28.
Example 18
cts-[rac]-l-[4-Benzyloxy-4-(4-chlo^ yl] -propan-1 -one
cw-[rac]-l-[4-Benzyiox^
piperidin-l-yl]-propan-l-one was prepared from 4-Ben2yloxy-4-(4-chloro-phenyl)-3-(2>3>4-trifluoro-benzyioxy)-piperidine (Example 8Ad) and propionyl chloride (Aldrich) following the procedure used in Example 17. Mass spectrum (ES) M+ = 518.

Example 19
c^rac] - l-[4-Ben2yloxy^ yl]-propan-l-one
ris-fracj-l-t^Benzyloxy^-C^cUoro-pheny^-S-CS^-dicUoro-benzyloxy)-piperidin-l-yl]-propan-l-one was prepared from cis- [rac]-4-(benzyloxy)-4-(4-chlorophenyl)-3-(3,4»dichloroben2yloxy)-piperidine (Example 4) following the procedure used in Example 18. Mass spectrum (ES) M+ = 533.
Example 20
Resin-boimdds-[rac]-4-ben2yloxy"4-(4-fluoro-phenyl)-piperidin-3-ol

This resin-bound compound was prepared from 4-(4-fluorophenyi)-l,2?3,6-tetrahydropyridine hydrochloride (Acros) following the procedure used in Examples 1,5, 6, and 7.

as-[rac]-4-Benzyloxy-3-(4-bromo-bei^

as-[rac]-4-Benzyloxy-3-(^ prepared from resin-bound cis-[rac]-4-benzyloxy-4-(4-fluoro-phen7l)-piperidin-3-ol (Example 20) and 4-bromobenzyl bromide (Aldrich) following the procedure used in Example 8. Mass spectrum (ES) Mtf" = 471.
Example 22 as-[rac]-4-Benzyloxy^-4«(4«fluorO"phenyl)-3-pentylox7-piperidine

as-[rac]- 4-Benzylox7-4-(4«fluoro-phenyl)-3-pentyloxy-piperidine was prepared from resin-bound cis-[rac]-4-benzyloxy-4-(4-fluoro-phenyl)-piperidin-3-ol (Example 20) and 1-iodopentane (Aldrich) following the procedure used in Example 8. Mass spectrum (ES) MH+ = 372. IC5o = 56 \iM (in the assay described in Example 28).

Example 23
as-[rac]-4-BenzyIoxy^ piperidine

A mixture of 4-benzyioxyp-4-(4-cMoro-phenyl)-3-(3,4-dichloro-benz7loxyr)-piperidine (Example 4,0.01 mmol), polymer-supported BEMP (6.5 mg, 0.015 mmol, loading capacity: 2.3 mmol/g from Fluka Inc.), iodoethane (Aldrich, 0.015 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature overnight. The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) MH* = 506. IC50 = 4.9 \xM (in the assay described in Example 28).
Example 24
a5-[rac]-4-Ben27loxy-4-(4-cUoro-phenyl)-3-(3,4-dicUoro-ben^4oxy)-l-propyl-piperidine
d5-[rac]-4"Benzyloxy-4-(4-cUoro-phenyl)-3-(3,4-dichloro-benzyloxy)-l-propyl-piperidine was prepared from 4-benzyloxy-4-(4-chloro-phenyl) -3- (3,4-dichloro-benzyloxy)-piperidine (Example 4) and 1-iodopropane (Aldrich) following the procedure used in Example 23. Mass spectrum (ES) M* = 519.

l

A mixture of 4-benz7loxy^-4-(4-chloro-phenyl)-3-(2,3,4-trifluoro-benzylox7)-piperidine (Example 8Ac, 6 mg, 0.013 mmol)> polymer-supported BEMP (10 mg, 0.023 mmol, loading capacity: 2.3 mmol/g from Fluka Inc.), allyl iodide (Aldrich, 3 uL, 0.003 mmol) in tetrahydrofuran (1 mL) was shaken at room temperature for 2 hours. The resin was filtered and washed successively with CH2CI2, and MeOH. The filtrate was evaporated in vacuo. Without purification, the residue was dissolved in 9:1 acetone/water solution (1 mL). To the solution was added osmium tetroxide (Aldrich, 10 uL, 0.008 mmol) and 4-methylmorpholine N-oxide (Aldrich, 12 mg, 0.1 mmol). The mixture was stirred overnight and Na2SC>3 was added. After stirring for 2 hours, the mixture was filtered through a silica gel pad. The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) M~*"= 536.
Example 26
as-[rac]-3-[4-Benzyloxy^3-(4-bromo-ben propane-1,2-diol

This compound was prepared from 4~benzyloxy-3-(4-bromo-benzyloxy)-4-(4-chloro-phenyl)-piperidine (Example 8P) followed the procedure used in Example 25. Mass spectrum (ES) MH* = 562. IC5o = 25.8 ^M (in the assay described in Example 28).
Example 27
cis- [rac] -3- [4-Benzyloxy-4- (4-chloro-phenyl)-3-(2,3>4-trifluoro-benzyloxy)-piperidin-1-yl]-methane sulfonyl

To a solution of 4-benzylox)^4-(4-chloro-phenyl)-3-(2,3>4-trifluoro-benzylox}0-piperidine (Example 8Ac, 4 mg, 0.008 mmol) and triethylamine (6 uL, 0.08 mmol) in dichloromethane (0.5 mL) at 0°C was added dropwise methanesulfonyl chloride (Aldrich, 2.5 uL, 0.015 mmol). After stirring for 2 hours, the mixture was filtered through an amino silica gel pad (Silicycle). The filtrate was evaporated in vacuo to provide the desired product. Mass spectrum (ES) MH+= 541.
Example 28
In Vitro Activity Assay
The ability of the substituted piperidine compounds of the invention to inhibit the interaction between p53 and MDM2 proteins was measured by an ELISA (Enzyme-Linked Immuno Sorbent Assay) in which recombinant GST-tagged MDM2 binds to a peptide that resembles the MDM2-interacting region of p53 (Bottger et al., J. Mol. Bio. 1997, Vol. 269, pgs. 744-756). This peptide is immobilized to the surface of a 96 well plate via N-terminal biotin, which binds to streptavidin-coated wells. MDM2 is added to each well in the presence of anti-MDM2 mouse monoclonal antibody (SMP-14, Santa Cruz Biotech). After removal of the unbound MDM2 protein, a peroxydase-linked secondary antibody (anti-mouse IgG, Roche Molecular Biochemicals) and the amount of peptide-bound MDM2 is determined colorimetrically by the addition of a peroxydase substrate (MTB Microwell Peroxydase Substrate System, Kirkegaard 8c Perry Labs).

Test plates were prepared by coating with streptavidin (5 mg/ml in PBS) for 2 hours followed by a PBS (phosphate-buffered saline) wash and overnight blocking with 150 |jl of blocking buffer containing 2 mg/ml bovine serum albumin (Sigma) and 0.05% Tween 20 (Sigma) in PBS at 4°C. Biotinylated peptide (1 \M) is added to each well in 50 \xl of blocking buffer and washed extensively after a 1-hour incubation. Test compounds were diluted in a separate 96 well plate and added in triplicate to a compound incubation plate containing a mix of the MDM2 protein and anti-MDM2 antibody. After 20 minutes incubation, the content of the plate is transferred to the test plate and incubated for an additional 1 hour. The secondary anti-mouse IgG antibody is added to the test plate preceded and followed by a triple wash with 0.05% Tween 20 in PBS. Finally, peroxydase substrate is added to each well and the absorption was read using a plate reader (MR7000, Dynatech) at 450nm. The inhibitory activity of the test compounds was measured as a percentage of the bound MDM2 in treated vs. untreated wells and IC5o was calculated.
The inhibitory activity (IC50) of the compounds prepared in the examples above, and represented by formula (I) or (II), is in the range of 3 joM to 100 pM,
While a number of embodiments of this invention have been represented, it is apparent that the basic construction can be altered to provide other embodiments that utilize the invention without departing from the spirit and scope of the invention. All such modifications and variations are intended to be included within the scope of the invention as defined in the appended claims rather than the specific embodiments that have been presented by way of example.

and the pharmaceutically acceptable salts thereof, wherein
n is an integer independently selected from 1 or 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy,
R12 is selected from the group consisting of alkyl or alkenyi having from 1 to about 5 carbon atoms, and
wherein R7 to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -N02, CF3> -OCH3, -COOCH3, and -C6H5.;
R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyl, -C(CH3)3, CF3, -OCH3, -N02, and -CN;
Ti 7 fit
and in case R is alkyl or alkenyi having from 1 to about 5 carbon atoms, R to R are hydrogen.

2. Compounds of formula I according to claim 1 represented by formula (II):

and the pharmaceutical^" acceptable salts thereof, wherein
n is an integer independently selected from 1 or 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyi, and lower alkyi substituted by carbonyl, sulfonyl, or hydroxy,
R2toR6 are independently selected from the group consisting of hydrogen, halogen, lower alkyi, -C(CH3)3, CF3, -OCH3, -NO* and -CN; and
R' to R11 are independently selected from the group consisting of hydrogen, halogen, -CN, -N02> CF3, -OCH3, -COOCH3, and -C6H5.
3. Compounds of formula II according to claim 2, wherein n is 1.
4. Compounds of formula II according to claim 2, wherein X is chloro.
5. Compounds of formula II according to claim 2, wherein X is a, para substituent
6. Compounds of formula II according to claim 2, wherein R1 is selected from the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3, -CH2CHOHCH2OH, and -S02CH3.
7. Compounds of formula II according to claim 2, wherein R and R are independently selected from the group consisting of hydrogen, fluoro, chloro, and bromo.

8. Compounds of formula II according to claim 2, wherein R3 and R5 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CH3, -OCH3> -CN, and -N02-
9. Compounds of formula II according to claim 2, wherein R4 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -C(CH3)3, -CH3> -CN, and -CF3.

10. Compounds of formula II according to claim 2, wherein R7 and R11 are independently selected from the group consisting of hydrogen, fluoro, chloro, -CN, -N02, and -C6H5.
11. Compounds of formula II according to claim 2, wherein R8 and R10 are independently selected from the group consisting of hydrogen, fluoro, chloro, bromo, -CN, -N02, and -OCH3.
12. Compounds of formula II according to claim 2, wherein R9 is selected from the group consisting of hydrogen, fluoro, chloro, bromo, -OCH3, -CN, -CF3, and -COOCH3.
13. Compounds of formula II according to claim 2, wherein the compound is selected from the group consisting of:
os-[rac]-4-benzyloxy-4-(4-chloro-phenyl)-3-(3,4-difluoro-benzyloxy)-piperidine;
ds-[rac]-4-benzylox7-4-(4-cUoro-phenyl)-3-(2,6-difluoro-benzyloxy)-piperidine;
ds-[rac]-4-benz)doxj^3-(biphenyl-2-ylmethox50"4-(4-cMoro-phenyl)-piperidine;
cis- [rac] -4- [4-ben2rj4oxy-4-(4-cUoro-phenyi)-piperidin-3-yloxymethyl] -benzonitrile;
ds-[rac]-4-benzyloxy-4-(4-cUoro-phenyl)-3-(3-ni1ro-benzyloxy)-piperidine;
ds-[rac]-4-benzyloxy-3-(3-bromo-benzyloxyp)-4-(4-chloro-phenyl)-piperidine;
ds-[rac]-4-benzyloxy-4-(4-cMoro-phenyl)-3-(2,4-difluoro-benzylox}0-piperidine;
aV[rac]-4-benz7loxy-3-(2-cUoro-4-fluoro-benzyloxy)-4-(4-chloro-phenyl)-piperidine;
cis- [rac] -4- [4-benzyloxy-4- (4-chloro-phenyl) -piperidin-3-yloxymethyl] -benzoic acid methyl ester;
d5-[rac]-4-benz7lox7-4-(4-chloro-phenyl)-3-(3,5-difluoro-benzyloxy)-piperidine;
ds-[rac]-4-ben2yloxy-3-(3-cUoro-2-fluoro-benzyloxy)-4-(4-cUoro-phenyl)-piperidine;

and the pharmaceutical^ acceptable salts thereof, wherein
n is an integer from 1 to 2;
X is halogen;
R1 is selected from the group consisting of hydrogen, carbonyl, sulfonyl, lower alkyl, and lower alkyl substituted by carbonyl, sulfonyl, or hydroxy, and
R12 is selected from the group consisting of alkyl and alkenyl having from 1 to about 5 carbon atoms.
19. Compounds of formula III according to claim 18, wherein n is 1.
20. Compounds of formula III according to claim 18, wherein X is chloro.
21. Compounds of formula III according to claim 18, wherein X is a para
substituent
22. Compounds of formula III according to claim 18, wherein R1 is selected from
the group consisting of hydrogen, -CH2CH3, -CH2CH2CH3, -COCH3, -COCH2CH3,
-CH2CHOHCH2OH, and -SO2CH3.

23. Compounds of formula III according to claim 18> wherein R is selected from the group consisting of-(CH2)3CH3> -(CH2)4CH3, -CH2CH=CHCH3> and -CH2CH=CH-CH2CH3.
24. Compounds of formula III according to claim 18, wherein the compound is selected from the group consisting of:
cis- [rac] -4-ben2yloxy-4-(4-fluoro-phenyl)-3-butyloxy-piperidine;
ds«[rac]"4-benzyloxy-4-(4«fluoro-phenyl)-3-pentyloxy-piperidine;
cis- [rac] -4-benzyloxy-4-(4-fluoro-phenyl)-3-(but-2-en)yloxy-piperidine;
a>[rac]"4-benzyloxyr-4-(4-fluoro-phenyl)--3-(pent-2-enyl)oxy-piperidine;
cis- [rac] -4-benzyloxy-4-(4-chloro-phenyl)-3-butyloxy-piperidine;
ds- [rac] -4-benz)doxy-4-(4-chloro-phenyl)-3-pentyloxy-piperidine;
as-[rac]-4-benzylox7-4-(4--chloro-phenyl)-3-(but-2-en)yloxy-piperidine;and
cis- [rac] -4-benzyloxy-4- (4-chloro-phenyl)-3- (pent-2-enyl)oxy-piperidine.
25. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula I according to claim 1 and a pharmaceutically acceptable carrier or excipient
26. The pharmaceutical composition according to claim 25 comprising a therapeutically effective amount of a compound of formula II according to claim 2 and a pharmaceutically acceptable carrier or excipient
27. The pharmaceutical composition according to claim 25 comprising a therapeutically effective amount of a compound of formula III according to claim 18 and a pharmaceutically acceptable carrier or excipient
28. The pharmaceutical composition according to claim 25, wherein the compound is suitable for administration to a patient having cancer.
29. Compounds of any one of claims 1 to 24 for use as medicaments.
30. A process for the preparation of a compound of formula I of claim 1, which process comprises
a) coupling a compound of the formula IV

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Patent Number

232301

Indian Patent Application Number

2226/CHENP/2005

PG Journal Number

13/2009

Publication Date

27-Mar-2009

Grant Date

16-Mar-2009

Date of Filing

12-Sep-2005

Name of Patentee

F. HOFFMANN-LA ROCHE AG

Applicant Address

124 GRENZACHERSTRASSE, CH-4070 BASEL,

Inventors:

#	Inventor's Name	Inventor's Address
1	KIM, KYUNGJIN	48 ASHWOOD DRIVE, LIVINGSTON, NEW JERSEY 07039,
2	LIU, EMILY, AIJUN	3133 FRONTERA WAY, UNIT 221, BURLINGAME, CALIFORNIA 94010,
3	MISCHKE, STEVEN, GREGORY	118 BEECHWOOD ROAD, FLORHAM PARK, NEW JERSEY 07932,

PCT International Classification Number

A61K31/451

PCT International Application Number

PCT/EP04/02339

PCT International Filing date

2004-03-08

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/454,387	2003-03-13	U.S.A.