US20060189674A1

US20060189674A1 - Deacetylase inhibitors

Info

Publication number: US20060189674A1
Application number: US11/408,129
Authority: US
Inventors: Stacy Remiszewski; Kenneth Bair; Richard Versace; Lawrence Perez; Michael Green; Lidia Sambucetti; Sushil Sharma
Original assignee: Individual
Current assignee: Secura Bio Inc
Priority date: 2000-09-01
Filing date: 2006-04-20
Publication date: 2006-08-24
Also published as: CY2015053I2; ECSP034492A; AU2001282129B2; US6552065B2; NZ524365A; LU92890I2; PL361408A1; NO20030867L; PL221738B1; KR100585484B1; FR15C0086I1; ES2292610T3; TWI286544B; EP1318980B1; IL154574A; JP4012819B2; SK2422003A3; EP1318980A2; RU2302408C2; MY136892A

Abstract

The present invention provides hydroxamate compounds which are deacetylase inhibitors. The compounds are suitable for pharmaceutical compositions having anti-proliferative properties.

Description

The present invention relates to hydroxamate compounds which are inhibitors of histone deacetylase. The inventive compounds are useful as pharmaceuticals for the treatment of proliferative diseases.

BACKGROUND

Reversible acetylation of histones is a major regulator of gene expression that acts by altering accessibility of transcription factors to DNA. In normal cells, histone deacetylase (HDA) and histone acetyltrasferase together control the level of acetylation of histones to maintain a balance. Inhibition of HDA results in the accumulation of hyperacetylated histones, which results in a variety of cellular responses.
Inhibitors of HDA have been studied for their therapeutic effects on cancer cells. For example, butyric acid and its derivatives, including sodium phenylbutyrate, have been reported to induce apoptosis in vitro in human colon carcinoma, leukemia and retinoblastoma cell lines. However, butyric acid and its derivatives are not useful pharmacological agents because they tend to be metabolized, rapidly and have a very short half-life in vivo. Other inhibitors of HDA that have Been widely studied for their anti-cancer activities are trichostatin A and trapoxin. Trichostatin A is an antifungal and antibiotic and is a reversible inhibitor of mammalian HDA. Trapoxin is a cyclic tetrapeptide, which is an irreversible inhibitor of mammalian HDA. Although trichostatin and trapoxin have been studied for their anti-cancer activities, the in vivo instability of the compounds makes them less suitable as anti-cancer drugs. There remains a need for an active compound that is suitable for treating tumors, including cancerous tumors, that is highly efficacious and stable.

SUMMARY

The present invention provides efficacious deacetylase inhibitor compounds that are useful as pharmaceutical agents having the formula (I):

wherein

- R₁is H, halo, or a straight chain C₁-C₆alkyl (especially methyl, ethyl or n-propyl, which methyl, ethyl and n-propyl substituents are unsubstituted or substituted by one or more substituents described below for alkyl substituents);
- R₂is selected from H, C₁-C₁₀alkyl, (e.g. methyl, ethyl or —CH₂CH₂—OH), C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g. benzyl), heteroarylalkyl (e.g. pyridylmethyl), —(CH₂)_nC(O)R₆, —(CH₂)—OC(O)R₆, amino acyl, HON—C(O)CH═C(R₁)-aryl-alkyl- and —(CH₂)_nR₇;
- R₃and R₄are the same or different and independently H, C₁-C₆alkyl, acyl or acylamino, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR₈, or R₂together with the nitrogen to which it is bound and R₃together with the carbon to which it is bound can form a C₄-C₉heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;
- R₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl (e.g. benzyl), heteroarylalkyl (e.g. pyridylmethyl), aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheterocycles; n, n₁, n₂and n₃are the same or different and independently selected from 0-6, when n, is 1-6, each carbon atom can be optionally and independently substituted with R₃and/or R₄;
- X and Y are the same or different and independently selected from H, halo, C₁-C₄alkyl, such as CH₃and CF₃, NO₂, C(O)R₁, OR₉, SR₉, CN, and NR₁₀R₁₁;
- R₆is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g., benzyl, 2-phenylethenyl), heteroarylalkyl (e.g., pyridylmethyl), OR₁₂, and NR₁₃R₁₄;
- R₇is selected from OR₁₅, SR₁₅, S(O)R₁₆, SO₂R₁₇, NR₁₃R₁₄, and NR₁₂SO₂R₆;
- R₈is selected from H, OR₁₅, NR₁₃R₁₄, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl);
- R₉is selected from C₁-C₄alkyl, for example, CH₃and CF₃, C(O)-alkyl, for example O(O)CH₃, and C(O)CF₃;
- R₁₀and R₁₁are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)-alkyl;
- R₁₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl);
- R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), amino acyl, or R₁₃and R₁₄together with the nitrogen to which they are bound are C₄-C₉heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and non-aryl polyheterocycle;
- R₁₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
- R₁₆is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
- R₁₇is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, aromatic polycycles, heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and NR₁₃R₁₄;
- m is an integer selected from 0 to 6; and
- Z is selected from O, NR₁₃, S and S(O),
  or a pharmaceutically acceptable salt thereof.

The compounds of the present invention are suitable as active agents in pharmaceutical compositions that are efficacious particularly for treating cellular proliferative ailments. The pharmaceutical composition has a pharmaceutically effective amount of the present active agent along with other pharmaceutically acceptable exicipients, carriers, fillers, diluents and the like. The term pharmacuetically effective amount as used herein indicates an amount necessary to administer to'a host to achieve a therapeutic result, especially an anti-tumor effect, e.g., inhibition of proliferation of malignant cancer cells, benign tumor cells or other proliferative cells.

DETAILED DESCRIPTION

The present invention provides hydroxamate compounds, e.g., hydroxamic acids, that are inhibitors of deacetylases, preferably inhibitors of histone deacetylases. The hydroxamate compounds are highly suitable for treating tumors, including cancerous tumors. The hydroxamate compounds of the present invention have the following structure (I):

wherein

- R₁is H, halo, or a straight chain C₁-C₆alkyl (especially methyl, ethyl or n-propyl, which methyl, ethyl and n-propyl substituents are unsubstituted or substituted by one or more substituents described below for alkyl substituents);

R₂is selected from H, C₁-C₁₀alkyl, (preferably C₁-C₆alkyl, e.g. methyl, ethyl or —CH₂CH₂—OH), C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g. benzyl), heteroarylalkyl (e.g. pyridylmethyl), —(CH₂)_nC(O)R₆, —(CH₂)_nOC(O)R₆, amino acyl, HON—C(O)—CH═C(R₁)-aryl-alkyl- and —(CH₂)_nR₇;
R₃and R₄are the same or different and independently H, C₁-C₆alkyl, acyl or acylamino, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR₈, or R₂together with the nitrogen to which it is bound and R₃together with the carbon to which it is bound can form a C₄-C₉heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;

- R₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl (e.g. benzyl), heteroarylalkyl (e.g. pyridylmethyl), aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryl, non-aromatic polyheterocycles, and mixed aryl and non-aryl polyheterocycles;
- n, n₁, n₂and n₃are the same or different and independently selected from 0-6, when n₁is 1-6, each carbon atom can be optionally and independently substituted with R₃and/or R₄;
- X and Y are the same or different and independently selected from H, halo, C₁-C₄alkyl, such as CH₃and CF₃, NO₂, C(O)R₁, OR₉, SR₉, CN, and NR₁₀R₁₁;
- R₆is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g., benzyl, 2-phenylethenyl), heteroarylalkyl (e.g., pyridylmethyl), OR₁₂, and NR₁₃R₁₄;
- R₇is selected from OR₁₅, SR₁₅, S(O)R₁₆, SO₂R₁₇, NR₁₃R₁₄, and NR₁₂SO₂R₆;
- R₈is selected from H, OR₁₅, NR₁₃R₁₄, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl);
- R₉is selected from C₁-C₄alkyl, for example, CH₃and CF₃, C(O)-alkyl, for example C(O)CH₃, and C(O)CF₃;
- R₁₀and R₁₁are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)-alkyl;
- R₁₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl);
- R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), amino acyl, or R₁₃and R₁₄together with the nitrogen to which they are bound are C₄-C₉heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and non-aryl polyheterocycle;
- R₁₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
- R₁₆is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
- R₁₇is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, aromatic polycycles, heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and NR₁₃R₁₄;
- m is an integer selected from 0 to 6; and
- Z is selected from O, NR₁₃, S and S(O),
  or a pharmaceutically acceptable salt thereof.

As appropriate, unsubstituted means that there is no substituent or that the only substituents are hydrogen.
Halo substituents are selected from fluoro, chloro, bromo and iodo, preferably fluoro or chloro.
Alkyl substituents include straight and branched C₁-C₆alkyl, unless otherwise noted. Examples of suitable straight and branched C₁-C₆alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, and the like. Unless otherwise noted, the alkyl substituents include both unsubstituted alkyl groups and alkyl groups that are substituted by one or more suitable substituents, including unsaturation (i.e. there are one or more double or triple C—C bonds), acyl, cycloalkyl, halo, oxyalkyl, alkylamino, aminoalkyl, acylamino and OR₁₅, for example, alkoxy. Preferred substituents for alkyl groups include halo, hydroxy, alkoxy, oxyalkyl, alkylamino, and aminoalkyl.
Cycloalkyl substituents include C₃-C₉cycloalkyl groups, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. Unless otherwise noted, cycloalkyl substituents include both unsubstituted cycloalkyl groups and cycloalkyl groups that are substituted by one or more suitable substituents, including C₁-C₆alkyl, halo, hydroxy, aminoalkyl, oxyalkyl, alkylamino, and OR₁₅, such as alkoxy. Preferred substituents for cycloalkyl groups include halo, hydroxy, alkoxy, oxyalkyl, alkylamino and aminoalkyl.
The above discussion of alkyl and cycloalkyl substituents also applies to the alkyl portions of other substituents, such as without limitation, alkoxy, alkyl amines, alkyl ketones, arylalkyl, heteroarylalkyl, alkylsulfonyl and alkyl ester substituents and the like.
Heterocycloalkyl substituents include 3 to 9 membered aliphatic rings, such as 4 to 7 membered aliphatic rings, containing from one to three heteroatoms selected from nitrogen, sulfur, oxygen. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1,3-diazapane, 1,4-diazapane, 1,4-oxazepane, and 1,4-oxathiapane. Unless otherwise noted, the rings are unsubstituted or substuted on the carbon atoms by one or more suitable substituents, including C₁-C₆alkyl, C₄-C₉cycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl), halo, amino, alkyl amino and OR₁₅, for example alkoxy. Unless otherwise noted, nitrogen heteroatoms are unsubstituted or substituted by H, C₁-C₄alkyl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl), acyl, aminoacyl, alkylsulfonyl, and arylsulfonyl.
Cycloalkylalkyl substituents include compounds of the formula —(CH₂)_n5-cycloalkyl wherein n5 is a number from 1-6. Suitable alkylcycloalkyl substituents include cyclopentylmethyl-, cyclopentylethyl, cyclohexylmethyl and the like. Such substituents are unsubstituted or substituted in the alkyl portion or in the cycloalkyl portion by a suitable substituent, including those listed above for alkyl and cycloalkyl.
Aryl substituents include unsubstituted phenyl and phenyl substituted by one or more suitable substituents, including C₁-C₆alkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), O(CO)alkyl, oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl ketones, nitrile, carboxyalkyl, alkylsulfonyl, aminosulfonyl, arylsulfonyl, and OR₁₅, such as alkoxy. Preferred substituents include including C₁-C₆alkyl, cycloalkyl (e.g., cyclopropylmethyl), alkoxy, oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl ketones, nitrile, carboxyalkyl, alkylsulfonyl, arylsulfonyl, and aminosulfonyl. Examples of suitable aryl groups include C₁-C₄alkylphenyl, C₁-C₄alkoxyphenyl, trifluoromethylphenyl, methoxyphenyl, hydroxyethylphenyl, dimethylaminophenyl, aminopropylphenyl, carbethoxyphenyl, methanesulfonylphenyl and tolylsulfonylphenyl.
Aromatic polycycles include naphthyl, and naphthyl substituted by one or more suitable substituents, including C₁-C₆alkyl, alkylcycloalkyl (e.g., cyclopropylmethyl), oxyalkyl, halo, nitro, amino, alkylamino, aminoalkyl, alkyl ketones, nitrile, carboxyalkyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl and OR₁₅, such as alkoxy.
Heteroaryl substituents include compounds with a 5 to 7 member aromatic ring containing one or more heteroatoms, for example from 0.1 to 4 heteroatoms, selected from N, O and S. Typical heteroaryl substituents include furyl, thienyl, pyrrole, pyrazole, triazole, thiazole, oxazole, pyridine, pyrimidine, isoxazolyl, pyrazine and the like. Unless otherwise noted, heteroaryl substituents are unsubstituted or substituted on a carbon atom by one or more suitable substituents, including alkyl, the alkyl substituents identified above, and another heteroaryl substituent. Nitrogen atoms are unsubstituted or substituted, for example by R₁₃; especially useful N substituents include H, C₁-C₄alkyl, acyl, aminoacyl, and sulfonyl.
Arylalkyl substituents include groups of the formula —(CH₂)_n5-aryl, —(CH₂)_n5-1-(CHaryl)-(CH₂)_n5-aryl or —(CH₂)_n5-1CH(aryl)(aryl) wherein aryl and n5 are defined above. Such arylalkyl substituents include benzyl, 2-phenylethyl, 1-phenylethyl, tolyl-3-propyl, 2-phenylpropyl, diphenylmethyl, 2-diphenylethyl, 5,5-dimethyl-3-phenylpentyl and the like. Arylalkyl substituents are unsubstituted or substituted in the alkyl moiety or the aryl moiety or both as described above for alkyl and aryl substituents.
Heteroarylalkyl substituents include groups of the formula —(CH₂)_n5-heteroaryl wherein heteroaryl and n5 are defined above and the bridging group is linked to a carbon or a nitrogen of the heteroaryl portion, such as 2-, 3- or 4-pyridylmethyl, imidazolylmethyl, quinolylethyl, and pyrrolylbutyl. Heteroaryl substituents are unsubstituted or substituted as discussed above for heteroaryl and alkyl substituents.
Amino acyl substituents include groups of the formula —C(O)—(CH₂)_n—C(H)(NR₁₃R₁₄)—(CH₂)_n—R₅wherein n, R₁₃, R₁₄and R₅are described above. Suitable aminoacyl substituents include natural and non-natural amino acids such as glycinyl, D-tryptophanyl, L-lysinyl, D- or L-homoserinyl, 4-aminobutryic acyl, ±-3-amin-4-hexenoyl.
Non-aromatic polycycle substituents include bicyclic and tricyclic fused ring systems where each ring can be 4-9 membered and each ring can contain zero, 1 or more double and/or triple bonds. Suitable examples of non-aromatic polycycles include decalin, octahydroindene, perhydrobenzocycloheptene, perhydrobenzo-[1-azulene. Such substituents are unsubstituted or substituted as described above for cycloalkyl groups.
Mixed aryl and non-aryl polycycle substituents include bicyclic and tricyclic fused ring systems where each ring can be 4-9 membered and at least one ring is aromatic. Suitable examples of mixed aryl and non-aryl polycycles include methylenedioxyphenyl, bis-methylenedioxyphenyl, 1,2,3,4-tetrahydronaphthalene, dibenzosuberane, dihdydroanthracene, 9H-fluorene. Such substituents are unsubstituted or substituted by nitro or as described above for cycloalkyl groups.
Polyheteroaryl substituents include bicyclic and tricyclic fused ring systems where each ring can independently be 5 or 6 membered and contain one or more heteroatom, for example, 1, 2, 3, or 4 heteroatoms, chosen from O, N or S such that the fused ring system is aromatic. Suitable examples of polyheteroaryl ring systems include quinoline, isoquinoline, pyridopyrazine, pyrrolopyridine, furopyridine, indole, benzofuran, benzothiofuran, benzindole, benzoxazole, pyrroloquinoline, and the like. Unless otherwise noted, polyheteroaryl substituents are unsubstituted or substituted on a carbon atom by one or more suitable substituents, including alkyl, the alkyl substituents identified above and a substituent of the formula —O—(CH₂CH═CH(CH₃)(CH₂))_1-3H. Nitrogen atoms are unsubstituted or substituted, for example by R₁₃; especially useful N substituents include H, C₁-C₄alkyl, acyl, aminoacyl, and sulfonyl.
Non-aromatic polyheterocyclic substituents include bicyclic and tricyclic fused ring systems where each ring can be 4-9 membered, contain one or more heteroatom, for example, 1, 2, 3, or 4 heteroatoms, chosen from O, N or S and contain zero or one or more C—C double or triple bonds. Suitable examples of non-aromatic polyheterocycles include hexitol, cis-perhydro-cyclohepta[b]pyridinyl, decahydro-benzo[f][1,4]oxazepinyl, 2,8-dioxabicyclo[3.3.0]octane, hexahydro-thieno[3,2-b]thiophene, perhydropyrrolo[3,2-b]pyrrole, perhydronaphthyridine, perhydro-1H-dicyclopenta[b,e]pyran. Unless otherwise noted, non-aromatic polyheterocyclic substituents are unsubstituted or substituted on a carbon atom by one or more substituents, including alkyl and the alkyl substituents identified above. Nitrogen atoms are unsubstituted or substituted, for example, by R₁₃; especially useful N substituents include H, C₁-C₄alkyl, acyl, aminoacyl, and sulfonyl.
Mixed aryl and non-aryl polyheterocycles substituents include bicyclic and tricyclic fused ring systems where each ring can be 4-9 membered, contain one or more heteroatom chosen from O, N or S, and at least one of the rings must be aromatic. Suitable examples of mixed aryl and non-aryl polyheterocycles include 2,3-dihydroindole, 1,2,3,4-tetrahydroquinoline, 5,11-dihydro-10H-dibenz[b,e][1,4]diazepine, 5H-dibenzo[b,e][1,4]diazepine, 1,2-dihydropyrrolo[3,4-b][1,5]benzodiazepine, 1,5-dihydro-pyrido[2,3-b][1,4]diazepin-4-one, 1,2,3,4,6,11-hexahydro-benzo[b]pyrido[2,3-e][1,4]diazepin-5-one. Unless otherwise noted, mixed aryl and non-aryl polyheterocyclic substituents are unsubstituted or substituted on a carbon atom by one or more suitable substituents, including, —N—OH, ═N—OH, alkyl and the alkyl substituents identified above. Nitrogen atoms are unsubstituted or substituted, for example, by R₁₃; especially useful N substituents include H, C₁-C₄alkyl, acyl, aminoacyl, and sulfonyl.
Amino substituents include primary, secondary and tertiary amines and in salt form, quaternary amines. Examples of amino substituents include mono- and di-alkylamino, mono- and di-aryl amino, mono- and di-aryalkyl amino, aryl-arylalkylamino, alkyl-arylamino, alkyl-arylalkylamino and the like.
Sulfonyl substituents include alkylsulfonyl and arylsulfonyl, for example methane sulfonyl, benzene sulfonyl, tosyl and the like.
Acyl substituents include groups of formula —C(O)—W, —OC(O)—W, —C(O)—O—W or —C(O)NR₁₃R₁₄, where W is R₁₆, H or cycloalkylalkyl.
Acylamino substituents include substituents of the formula —N(R₁₂)C(O)—W, —N(R₂)C(O)—O—W, and —N(R₁₂)C(O)—NHOH and R₁₂and W are defined above.
The R₂substituent HON—C(OYCH═C(R₁)-aryl-alkyl- is a group of the formula
Preferences for each of the substituents include the following:

- R₁is H, halo, or a straight chain C₁-C₄alkyl;
- R₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, amino acyl, and —(CH₂)_nR₇;
- R₃and R₄are the same or different and independently selected from H, and C₁-C₆alkyl, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR₈;
- R₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, a aromatic polycycle, a non-aromatic polycycle, a mixed aryl and non-aryl polycycle, polyheteroaryl, a non-aromatic polyheterocycle, and a mixed aryl and non-aryl polyheterocycle;
- n, n₁, n₂and n₃are the same or different and independently selected from 0-6, when n, is 1-6, each carbon atom is unsubstituted or independently substituted with R₃and/or R₄;
- X and Y are the same or different and independently selected from H, halo, C₁-C₄alkyl, CF₃, NO₂, C(O)R₁, OR₉, SR₉, CN, and NR₁₀R₁₁;
- R₆is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, OR₁₂, and NR₁₃R₁₄;
- R₇is selected from OR₁₅, SR₁₅, S(O)R₁₆, SO₂R₁₇, NR₁₃R₁₄, and NR₁₂SO₂R₆;
- R₈is selected from H, OR₁₅, NR₁₃R₁₄, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
- R₉is selected from C₁-C₄alkyl and C(O)-alkyl;
- R₁₀and R₁₁are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)-alkyl;
- R₁₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;
- R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and amino acyl;
- R₁₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
- R₁₆is selected from C₁-C₈alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;
R₁₇is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and NR₁₃R₁₄;
- m is an integer selected from 0 to 6; and
- Z is selected from O, NR₁₃, S, S(O),
  or a pharmaceutically acceptable salt thereof.

Useful compounds of the formula (I) include those wherein each of R₁, X, Y, R₃, and R₄is H, including those wherein one of n₂and n₃is zero and the other is 1, especially those wherein R₂is H or —CH₂—CH₂—OH.
One suitable genus of hydroxamate compounds are those of formula Ia:

wherein

- n₄is 0-3,
- R₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, amino acyl and —(CH₂)_nR₇;
- R₅′ is heteroaryl, heteroarylalkyl (e.g., pyridylmethyl), aromatic polycycles, non-aromatic polycycles, mixed aryl and non-aryl polycycles, polyheteroaryl, or mixed aryl and non-aryl polyheterocycles,
  or a pharmaceutically acceptable salt thereof.

Another suitable genus of hydroxamate compounds are those of formula Ia:

wherein

- n₄is 0-3,
- R₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, amino acyl and —(CH₂)_nR₇;
- R₅′ is aryl, arylalkyl, aromatic polycycles, non-aromatic polycycles, and mixed aryl and non-aryl polycycles; especially aryl, such as p-fluorophenyl, p-chlorophenyl, p-O—C₁-C₄-alkylphenyl, such as p-methoxyphenyl, and p-C₁-C₄-alkylphenyl; and arylalkyl, such as benzyl, ortho, meta or para-fluorobenzyl, ortho, meta or para-chlorobenzyl, ortho, meta or para-mono, di or tri-O—C₁-C₄-alkylbenzyl, such as ortho, meta or para-methoxybenzyl, m,p-diethoxybenzyl, o,m,p-triimethoxybenzyl, and ortho, meta or para- mono, di or tri C₁-C₄-alkylphenyl, such as p-methyl, m,m-diethylphenyl,
  or a pharmaceutically acceptable salt thereof.

Another interesting genus are the compounds of formula Ib:

wherein

- R₂′ is selected from H, C₁-C₆alkyl, C₄-C₆cycloalkyl, cycloalkylalkyl (e.g., cyclopropylmethyl), (CH₂)_2-4OR₂₁where R₂₁is H, methyl, ethyl, propyl, and i-propyl, and
- R₅″ is unsubstituted 1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl, or substituted 1H-indol-3-yl, such as 5-fluoro-1H-indol-3-yl or 5-methoxy-1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl,
  or a pharmaceutically acceptable salt thereof.

Another interesting genus of hydroxamate compounds are the compounds of formula (Ic)

wherein

- the ring containing Z, is aromatic or non-aromatic, which non-aromatic rings are saturated or unsaturated,
- Z₁is O, S or N—R₂₀,
- R18 is H, halo, C₁-C₆alkyl (methyl, ethyl, t-butyl), C₃-C₇cycloalkyl, aryl, for example unsubstituted phenyl or phenyl substituted by 4-OCH₃or 4-CF₃, or heteroaryl, such as 2-furanyl, 2-thiophenyl or 2-, 3- or 4-pyridyl;
- R₂₀is H, C₁-C₆alkyl, C₁-C₆alkyl-C₃-C₉cycloalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), acyl (acetyl, propionyl, benzoyl) or sulfonyl (methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfonyl)
- A₁is 1, 2 or 3 substituents which are independently H, C₁-C₆alkyl, —OR₁₉, halo, alkylamino, aminoalkyl, halo, or heteroarylalkyl (e.g., pyridylmethyl),
- R₁₉is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl) and —(CH₂CH═CH(CH₃)(CH₂))_1-3H;

R₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, alkylcycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, amino acyl and —(CH₂)_nR₇;

- v is 0, 1 or 2,
- p is 0-3, and
- q is 1-5 and r is 0 or
- q is 0 and r is 1-5,
  or a pharmaceutically acceptable salt thereof. The other variable substituents are as defined above.

Especially useful compounds of formula (Ic) are those wherein R₂is H, or —(CH₂)_pCH₂OH, wherein p is 1-3, especially those wherein R₁is H; such as those wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3, especially those wherein Z₁is N—R₂₀. Among these compounds R₂is preferably H or —CH₂—CH₂—OH and the sum of q and r is preferably 1.
Another interesting genus of hydroxamate compounds are the compounds of formula (Id)

wherein
Z₁is O, S or N—R₂₀,
R₁₈is H, halo, C₁-C₈alkyl (methyl, ethyl, t-butyl), C₃-C₇cycloalkyl, aryl, for example, unsubstituted phenyl or phenyl substituted by 4-OCH₃or 4-CF₃, or heteroaryl,
R₂₀is H, C₁-C₆alkyl, C₁-C₆alkyl-C₃-C₉cycloalkyl (e.g., cyclopropylmethyl), aryl, heteroaryl, arylalkyl (e.g., benzyl), heteroarylalkyl (e.g., pyridylmethyl), acyl (acetyl, propionyl, benzoyl) or sulfonyl (methanesulfonyl, ethanesulfonyl, benzenesulfonyl, toluenesulfonyl),
A₁is 1, 2 or 3 substituents which are independently H, C₁-C-₆alkyl, —OR₁₉, or halo,
R₁₉is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl (e.g., benzyl), and heteroarylalkyl (e.g., pyridylmethyl);.
p is 0-3, and
q is 1-5 and r is 0 or
q is 0 and r is 1-5,
or a pharmaceutically acceptable salt thereof. The other variable substituents are as defined above.
Especially useful compounds of formula (Id) are those wherein R₂is H, or —(CH₂)_pCH₂OH, wherein p is 1-3, especially those wherein R₁is H; such as those wherein R₁is H and X and Y are each H, and wherein 4 is 1-3 and r is 0 or wherein q is 0 and r is 1-3. Among these compounds R₂is preferably H or —CH₂—CH₂—OH and the sum of q and r is preferably 1.
The present invention further relates to compounds of the formula (Ie)

or a pharmaceutically acceptable salt thereof. The variable substituents are as defined above.
Especially useful compounds of formula (Ie) are those wherein R₁₈is H, fluoro, chloro, bromo, a C₁-C₄alkyl group, a substituted C₁-C₄alkyl group, a C₃-C₇cycloalkyl group, unsubstituted phenyl, phenyl substituted in the para position, or a heteroaryl (e.g., pyridyl) ring.
Another group of useful compounds of formula (Ie) are those wherein R₂is H, or —(CH₂)_pCH₂OH, wherein p is 1-3, especially those wherein R₁is H; such as those wherein R₁is H and of and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3. Among these compounds R₂is preferably H or —CH₂—CH₂—OH and the sum of q and r is preferably 1.
Another group of useful compounds of formula (Ie) are those wherein R₁₈is H, methyl, ethyl, t-butyl, trifluoromethyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thiophenyl, or 2-, 3- or 4-pyridyl wherein the 2-furanyl, 2-thiophenyl and 2-, 3- or 4-pyridyl substituents are unsubstituted or substituted as described above for heteroaryl rings; R₂is H, or —(CH₂)_pCH₂OH, wherein p is 1-3; especially those wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3. Among these compounds R₂is preferably H or —CH₂—CH₂—OH and the sum of q and r is preferably 1.
Those compounds of formula Ie wherein R₂₀is H, or C₁-C₆alkyl, especially H, are important members of each of the subgenuses of compounds of formula Ie described above.
N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, are important compounds of formula (Ie).
The present invention further relates to the compounds of the formula (If):

or a pharmaceutically acceptable salt thereof. The variable substituents are as defined above.
Useful compounds of formula (If) are include those wherein R₂is H, or —(CH₂)_pCH₂OH, wherein p is 1-3, especially those wherein R₁is H; such as those wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3. Among these compounds R₂is preferably H or —CH—CH₂—OH and the sum of q and r is preferably 1.
N-hydroxy-3-[4-[[[2-(benzofur-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof, is an important compound of formula (If).
The compounds described above are often used in the form of a pharmaceutically acceptable salt. Pharmaceutically acceptable salts include, when appropriate, pharmaceutically acceptable base addition salts and acid addition salts, for example, metal salts, such as alkali and alkaline earth metal salts, ammonium salts, organic amine addition salts, and amino acid addition salts, and sulfonate salts. Acid addition salts include inorganic acid addition salts such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as alkyl sulfonate, arylsulfonate, acetate, maleate, fumarate, tartrate, citrate and lactate. Examples of metal salts are alkali metal salts, such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt, and zinc salt. Examples of ammonium salts are ammonium salt and tetramethylammonium salt. Examples of organic amine addition salts are salts with morpholine and piperidine. Examples of amino acid addition salts are salts with glycine, phenylalanine, glutamic acid and lysine. Sulfonate salts include mesylate, tosylate and benzene sulfonic acid salts.
As is evident to those skilled in the art, the many of the deacetylase inhibitor compounds of the present invention contain asymmetric carbon atoms. It should be understood, therefore, that the individual stereoisomers are contemplated as being included within the scope of this invention.
The hydroxamate compounds of the present invention can be produced by known organic synthesis methods. For example, the hydroxamate compounds can be produced by reacting methyl 4-formyl cinnamate with tryptamine and then converting the reactant to the hydroxamate compounds. As an example, methyl 4-formyl cinnamate 2, is prepared by acid catalyzed esterification of 4-formylcinnamic acid 3 (Bull. Chem. Soc. Jpn. 1995; 68:2355-2362). An alternate preparation of methyl 4-formyl cinnamate 2 is by a Pd-catalyzed coupling of methyl acrylate 4 with 4-bromobenzaldehyde 5.

Additional starting materials can be prepared from 4-carboxybenzaldehyde 6, and an exemplary method is illustrated for the preparation of aldehyde 9, shown below. The carboxylic acid in 4-carboxybenzaldehyde 6 can be protected as a silyl ester (e.g., the t-butyidimethylsilyl ester) by treatment with a silyl chloride (e.g., t-butyldimethylsilyl chloride) and a base (e.g. triethylamine) in an appropriate solvent (e.g., dichloromethane). The resulting silyl ester 7 can undergo an olefination reaction (e.g., a Horner-Emmons olefination) with a phosphonate ester (e.g., triethyl 2-phosphonopropionate) in the presence of a base (e.g., sodium hydride) in an appropriate solvent (e.g., tetrahydrofuran (THF)). Treatment of the resulting diester with acid (e.g., aqueous hydrochloric acid) results in the hydrolysis of the silyl ester providing acid 8. Selective reduction of the carboxylic acid of 8 using, for example, borane-dimethylsuflide complex in a solvent (e.g., THF) provides an intermediate alcohol. This intermediate alcohol could be oxidized to aldehyde 9 by a number of known methods, including, but not limited to, Swern oxidation, Dess-Martin periodinane oxidation, Moffatt oxidation and the like.

The aldehyde starting materials 2 or 9 can be reductively aminated to provide secondary or tertiary amines. This is illustrated by the reaction of methyl 4-formyl cinnamate 2 with tryptamine 10 using sodium triacetoxyborohydride (NaBH(OAc)₃) as the reducing agent in dichloroethane (DCE) as solvent to provide amine 11. Other reducing agents can be used, e.g., sodium borohydride (NaBH₄) and sodium cyanoborohydride (NaBH₃CN), in other solvents or solvent mixtures in the presence or absence of acid catatylysts (e.g., acetic acid and trifluoroacetic acid). Amine 11 can be converted directly to hydroxamic acid 12 by treatment with 50% aqueous hydroxylamine in a suitable solvent (e.g., THF in the presence of a base, e.g., NaOH). Other methods of hydroxamate formation are known and include reaction of an ester with hydroxylamine hydrochloride and a base (e.g., sodium hydroxide or sodium methoxide) in a suitable solvent or solvent mixture (e.g., methanol, ethanol or methanol/THF).

Aldehyde 2 can be reductively aminated with a variety of amines, exemplified by, but not limited to, those illustrated in Table 1. The resulting esters can be converted to target hydroxamates by the methods listed.

TABLE 1

	Reducing	Hydroxamate
Amine	Conditions	Conditions	R


	NaBH(OAc)₃HOAc, DCE	2 M HONH₂in MeOH

	″	″

	″	″

	″	″

	″	″

	″	″

	″	″

	″	″

	″	″

Ph(CH₂)₃NH₂	NaBH₃CN/MeOH/HOAc		Ph(CH₂)₃

An alternate synthesis of the compounds of this invention starts by reductive amination of 4-formyl cinnamic acid 3, illustrated below with 3-phenylpropylamine 13, using, for example, NaBH₃CN as the reducing agent in MeOH and HOAc as a catalyst. The basic nitrogen of the resulting amino acid 14 can be protected, for example, as t-butoxycarbamate (BOC) by reaction with di-t-butyldicarbonate to give 15.

The carboxylic acid can be coupled with a protected hydroxylamine (e.g., O-trityl hydroxylamine) using a dehydrating agent (e.g., 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCl)) and a catalyst (e.g., 1-hydroxybenzotriazole hydrate (HOBT)) in a suitable solvent (e.g., DMF) to produce 16. Treatment of 16 with a strong acid (e.g., trifluoroacetic acid (TFA)) provides a hydroxamic acid 17 of the present invention. Additional examples of compounds that can be prepared by this method are:

Tertiary amine compounds can be prepared by a number of methods. Reductive amination of 30 with nicotinaldehyde 32 using NaBH₃CN as the reducing agent in dichloroethane and HOAc as a catalyst provides ester 34. Other reducing agents can be used (e.g., NaBH₄and NaBH(OAc)₃) in other solvents or solvent mixtures in the presence or absence of acid catalysts (e.g., acetic acid, trifluoroacetic acid and the like). Reaction of ester 34 with HONH₂.HCl, NaOH in MeOH provides hydroxamate 36.

Tertiary amine compounds prepared by this methodology are exemplified, but not limited to, those listed in Table 2.

TABLE 2





or

		Hydroxamate
Reducing Conditions	Conditions


	NaBH(OAc)₃HOAc, DCE	HONH₂.HCl/NaOMe/MeOH

	NaBH(OAc)₃HOAc, DCE	HONH₂.HCl/NaOMe/MeOH

	NaBH(OAc)₃HOAc, DCE	2 M HONH₂in MeOH

	NaBH₃CN/MeOH/HOAc	2 M HONH₂in MeOH

	NaBH(OAc)₃HOAc, DCE	2 M HONH₂in MeOH

An alternate method for preparing tertiary amines is by reacting a secondary amine with an alkylating agent in a suitable solvent in the presence of a base. For example, heating a dimethylsulfoxide (DMSO) solution of amine 11 and bromide 40 in the presence of (i-Pr)₂NEt yielded tertiary amine 42. Reaction of the tertiary amine 42 with HONH₂.HCl, NaOH in MeOH provides hydroxamate 43. The silyl group can be removed by any method known to those skilled in the art. For example, the hydroxamate 43 can be treated with an acid, e.g., trifluoroacetic acid, or fluoride to produce hydroxyethyl compound 44.
The hydroxamate compound, or salt thereof, is suitable for preparing pharmaceutical compositions, especially pharmaceutical compositions having deacetylase, especially histone deacetylase, inhibiting properties. Studies with athymic mice demonstrate that the hydroxamate compound causes HDA inhibition and increased histone acetylation in vivo, which triggers changes in gene expression that correlate with tumor growth inhibition.
The present invention further includes pharmaceutical compositions comprising a pharmaceutically effective amount of one or more of the above-described compounds as active ingredient. Pharmaceutical compositions according to the invention are suitable for enteral, such as oral or rectal, and parenteral administration to mammals, including man, for the treatment of tumors, alone or in combination with one or more pharmaceutically acceptable carriers.
The hydroxamate compound is useful in the manufacture of pharmaceutical compositions having an effective amount the compound in conjunction or admixture with excipients or carriers suitable for either enteral or parenteral application. Preferred are tablets and gelatin capsules comprising the active ingredient together with (a) diluents; (b) lubricants, (c) binders (tablets); if desired, (d) disintegrants; and/or (e) absorbents, colorants, flavors and sweeteners. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, the compositions may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain preferably about 1 to 50% of the active ingredient.
Suitable formulations also include formulations for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
As discussed above, the compounds of the present invention are useful for treating proliferative diseases. A proliferative disease is mainly a tumor disease (or cancer) (and/or any metastases). The inventive compounds are particularly useful for treating a tumor which is a breast cancer, genitourinary cancer, lung cancer, gastrointestinal cancer, epidermoid cancer, melanoma, ovarian cancer, pancreas cancer, neuroblastoma, head and/or neck cancer or bladder cancer, or in a broader sense renal, brain or gastric cancer; in particular (i) a breast tumor; an epidermoid tumor, such as an epidermoid head and/or neck tumor or a mouth tumor; a lung tumor, for example a small cell or non-small cell lung tumor; a gastrointestinal tumor, for example, a colorectal tumor; or a genitourinary tumor, for example, a prostate tumor (especially a hormone-refractory prostate tumor); or (ii) a proliferative disease that is refractory to the treatment with other chemotherapeutics; or (iii) a tumor that is refractory to treatment with other chemotherapeutics due to multidrug resistance.
In a broader sense of the invention, a proliferative disease may furthermore be a hyperproliferative condition such as leukemias, hyperplasias, fibrosis (especially pulmonary, but also other types of fibrosis, such as renal fibrosis), angiogenesis, psoriasis, atherosclerosis and smooth muscle proliferation in the blood vessels, such as stenosis or restenosis following angioplasty.
Where a tumor, a tumor disease, a carcinoma or a cancer are mentioned, also metastasis in the original organ or tissue and/or in any other location are implied alternatively or in addition, whatever the location of the tumor and/or metastasis.
The compound is selectively toxic or more toxic to rapidly propiferating cells than to normal cells, particularly in human cancer cells, e.g., cancerous tumors, the compound has significant antiproliferative effects and promotes differentiation, e.g., cell cycle arrest and apoptosis. In addition, the hydroxamate compound induces p21, cyclin-CDK interacting protein, which induces either apoptosis or G1 arrest in a variety of cell lines.
The following examples are intended to illustrate the invention and are not to be construed as being limitations thereto.

EXAMPLE P1

Preparation of N-Hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide

4-formylcinnamic acid methylester is produced by adding 4-formylcinnamic acid (25 g, 0.143 mol) in MeOH and HCl (6.7 g, 0.18 mol). The resulting suspension is heated to reflux for 3 hours, cooled and evaporated to dryness. The resulting yellow solid is dissolved in EtOAc, the solution washed with saturated NaHCO₃, dried (MgSO₄) and evaporated to give a pale yellow solid which is used without further purification (25.0 g, 92%). To a solution of tryptamine (16.3 g, 100 mmol) and 4-formylcinnamic acid methylester (19 g, 100 mmol) in dichloroethane, NaBH(OAc)₃(21 g, 1.00 mmol) is added. After 4 hours the mixture is diluted with 10% K₂CO₃solution, the organic phase separated and the aqueous solution extracted with CH₂Cl₂. The combined organic extracts are dried (Na₂SO₄), evaporated and the residue purified by flash chromatography to produce 3-(4-{[2-(1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic acid methyl ester (29 g). A solution of KOH (12.9 g 87%, 0.2 mol) in MeOH (100 mL) is added to a solution of HONH₂.HCl (13.9 g, 0.2 mol) in MeOH (200 mL) and a precipitate results. After 15 minutes the mixture is filtered, the filter cake washed with MeOH and the filtrate evaporated under vacuum to approximately 75 mL. The mixture is filtered and the volume adjusted to 100 mL with MeOH. The resulting solution 2M HONH₂is stored under N₂at −20° C. for up to 2 weeks. Then 3-(4-{[2-(1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic acid methyl ester (2.20 g, 6.50 mmol) is added to 2 M HONH₂in MeOH (30 mL, 60 mmol) followed by a solution of KOH (420 mg, 6.5 mmol) in MeOH (5 mL). After 2 hours dry ice is added to the reaction and the mixture is evaporated to dryness. The residue is dissolved in hot MeOH (20 mL), cooled and stored at −20° C. overnight. The resulting suspension is filtered, the solids washed with ice cold MeOH and dried under vacuum, producing N-Hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (m/z 336 [MH⁺]).

EXAMPLE P2

Preparation of N-Hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide

A solution of 3-(4-{[2-(1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic acid methyl ester (12.6 g, 37.7 mmol), (2-bromoethoxy)-tert-butyldimethylsilane (12.8 g, 53.6 mmol), (i-Pr)₂NEt, (7.42 g, 57.4 mmol) in DMSO (100 mL) is heated to 50° C. After 8 hours the mixture is partitioned with CH₂Cl₂/H₂O. The organic layer is dried (Na₂SO₄) and evaporated. The residue is chromatographed on silica gel to produce 3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-yl)-ethyl]-amino}-methyl)-phenyl]-(2E)-2-propenoic acid methyl ester (13.1 g). Following the procedure described for the preparation of the hydroxamate compound in Example. P1, 3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-yl)-ethyl]-amino}-methyl)-phenyl]-(2E)-2-propenoic acid methyl ester (5.4 g, 11 mmol) is converted to N-hydroxy-3-[4-({[2-(tert-butyldimethylsilanyloxy)-ethyl]-[2-(1H-indol-3-yl)-ethyl]-amino}-methyl)-phenyl]-(2E)-2-propenamide (5.1 g,) and used without further purification. The hydroxamic acid (5.0 g, 13.3 mmol) is then dissolved in 95% TFA/H₂O (59 mL) and heated to 40-50° C. for 4 hours. The mixture is evaporated and the residue purified by reverse phase HPLC to produce N-Hydroxy-3-[4-4[(2-hydroxyethyl)[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide as the trifluoroacetate salt (m/z 380 [MH⁺]).

EXAMPLE P3

Preparation of N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide

A suspension of LiAlH₄(17 g, 445 mmol) in dry THF (1000 mL) is cooled to 0° C. and 2-methylindole-3-glyoxylamide (30 g, 148 mmol) is added in portions over 30 min. The mixture is stirred at room temperature for 30 min. and then maintained at reflux for 3 h. The reaction is cooled to 0° C. and treated with H₂O (17 ml), 15% NaOH (aq., 17 ml) and H₂O (51 ml). The mixture is treated with MgSO₄, filtered and the filtrate evaporated to give 2-methyltryptamine which is dissolved in MeOH. Methyl 4-formylcinnamate (16.9 g, 88.8 mmol) is added to the solution, followed by NaBH₃CN (8.4 g) and AcOH (1 equiv.). After 1 h the reaction is diluted with NaHCO₃(aq.) and extracted with EtOAc. The organic extracts are dried (MgSO₄), filtered and evaporated. The residue is purified by chromatography to give 3-(4-[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl)phenyl)-(2E)-2-propenoic acid methyl ester. The ester is dissolved in MeOH, 1.0 M HCl/dioxane (1-1.5 eqiv.) is added followed by Et₂O. The resulting precipitate is filtered and the solid washed with Et₂O and dried thoroughly to give 3-(4-{[2-(2-methyl-1H-indol-3-yl)-ethylamino]-methyl}-phenyl)-(2E)-2-propenoic acid methyl ester hydrochloride. 1.0 M NaOH (aq., 85 mL) is added to an ice cold solution of the methyl ester hydrochloride (14.9 g, 38.6 mmol) and HONH₂(50% aq. solution, 24.0 mL, ca. 391.2 mmol). After 6 h, the ice cold solution is diluted with H₂O and NH₄Cl (aq., 0.86 M, 100 mL). The resulting precipitate is filtered, washed with H₂O and dried to afford N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (m/z 350 [MH^+]).

EXAMPLES 1-265

The following compounds are prepared by methods analogous to those disclosed in Examples P1, P2 and P3:



Example	STRUCTURE	m/z (MH⁺)


1		426

2

3

4		325

5

6

7

8		465

9

10

11

12		420

13		420

14

15		465

16		385

17		550

18		432

19		366

20		350

21

22		442

23		338

24		464

25		541

26

27

28		417

29

30

31		380

32		436

33

34		493

35		477

36		586

37		513

38		378

39		408

40		449

41		438

42		452

43		507

44		565

45

46

47

48

49

50

51		470

52

53		548

54		623

55		456

56		478

57		394

58		422

59		479

60		603

61		477

62		539

63		523

64

65

66

67

68		539

69		495

70

71		379

72		478

73		462

74		378

75

76		493

77		503

78		350

79		549

80		471

81		350

82		418

83		486

84		524

85		424

86		364

87		440

88		420

89		390

90

91

92		484

93		498

94		490

95

96		475

97		525

98		422

99		528

100		448

101		437

102		451

103		505

104		519

105		514

106		507

107		626

108		499

109

110

111		429

112		464

113		432

114		422

115		390

116		501

117		484

118

119		587

120		602

121		539

122

123		528

124		487

125

126		556

127

128

129		552

130		519

131		450

132		464

133		558

134		533

135

136		527

137		381

138		364

139

140		448

141		558

142

143		427

144

145		432

146		384

147		354

148

149

150

151

152

153

154		350

155		366

156		408

157		322

158		364

159		364

160		378

161		350

162		463

163

164		381

165		463

166		476

167

168

169

170		368

171		493

172		527

173		515

174		323

175		540

176		441

177		276

178

179		455

180

181		336

182		347

183		447

184

185		420

186		424

187		422

188

189		398

190		418

191		350

192

193		352

194		499

195		408

196		394

197		499

198

199

200		350

201

202

203

204		365

205		465

206

207		410

208		410

209

210		366

211		352

212

213		368

214		338

215		356

216		408

217		368

218		396

219

220		342

221		392

222		412

223		337

224		337

225		456

226		364

227		481

228		355

229		312

230		424

231

232		351

233		392

234

235

236		322

237

238		366

239

240		368

241

242		406

243		398

244		442

245		350

246		364

247		402

248		418

249		364

250

251		408

252

253

254		413

255		405

256

257		394

258		390

259		434

260		386

261		368

262		412

263		406

264

265		378

The compounds of Examples 1-265 show an HDA enzyme IC₅₀in the range from about 0.005 to about 0.5 μM.

EXAMPLE B1

Cell lines H1299 (human lung carcinoma cell) and HCT116 (colon tumor cell) are obtained from the American Type Culture Collection, Rockville, Md. The cell lines are free of Mycoplasma contamination (Rapid Detection System by Gen-Probe, Inc., San Diego, Calif.) and viral contamination (MAP testing by MA BioServices, Inc., Rockville, Md.). The cell lines are propagated and expanded in RPMI 1640 medium containing 10% heat-inactivated FBS (Life Technologies, Grand Island, N.Y.). Cell expansions for implantation are performed in cell factories (NUNC, purchased from Fisher Scientific, Springfield, N.J.). Cells are harvested at 50-90% confluency, washed once with HBSS (Hank's Balanced Salt Solution) containing 10% FBS, and suspended in 100% HBSS.
Cell proliferation is measured with a commercial MTS kit (Promega, Madision, Wis.) assay using an adaptation of published procedures, for example, that disclosed in Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay, Alley M C, et al., Cancer Res. 1988; 48:589-601. Cells are plated in 96-well tissue culture dishes, with top and bottom rows left empty. H1299 and HCT116 cells are suspended in complete media at a density of 5.3×10³and 3.6×10³cell/mL, respectively, and 190 μl are added per well. Each cell line is added to one half of the plate. Complete medium (200 μL) is added to the top and bottom rows. Twenty-four hours later, 10 μl of MTS solution is added to one of the plates to determine the activity at the time of compound addition (T₀). The plate is incubated at 37° C. for 4 hours and the OD₄₉₀is measured on a Molecular Devices Thermomax at 490 nm using the Softmax program. The To plate serves as a reference for initial activity at the beginning of the experiment.
Five serial dilutions (1:4) of each compound are made in a 96-deep well plate with the highest concentrations on the edge of plate. Two cell lines are tested with two compounds per plate.0 Ten microliters of each of the five dilutions are added in triplicate and complete medium alone is added to columns six and seven. The plates are incubated at 37° C. for 72 hours. The MTS solution is added (as for the T₀plate) and read four hours later.
In order to analyze the data, the average background value (media alone) is subtracted from each experimental well; the triplicate values are averaged for each compound dilution. The following formulas are used to calculate percent growth.
If X>T₀, % Growth=((X−T ₀)/(GC−T ₀))×100
If X<T₀, % Growth=(X−T ₀)/T ₀)×100
in which T₀=(average value of cell viability at time 0)−background
GC=average value of untreated cells (in triplicate)−background
X=average value of compound treated cells (in triplicate)−background
The “% Growth” is plotted against compound concentration and used to calculate IC₅₀s employing the linear regression techniques between data points to predict the concentration of compounds at 50% inhibition.
Lactate salts of N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (CMD1), N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (CMD2), N-hydroxy-3-[4-[[[2-(5-methoxy-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (CMD3), N-hydroxy-3-[4-[[[2-(5-fluoro-1H-indol-3-yl) ethyl]-amino]methyl]phenyl]-2E-2-propenamide (CMD4), N-hydroxy-3-[4-[[[2-(benzofur-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide (CMD5) having a purity of higher than 95% are dissolved in pure dimethylsulfoxide (DMSO) to create a stock solution. The stock solution is diluted with 5% dextrose injection, USP, just prior to dosing. In addition, N-(2-aminophenyl[N-pyridin-3-yl)methoxycarbonylaminomethyl]benzamide is synthesized in accordance with Example 48 of EP 0 847 992 and used as a control compound (CMDC). Inhibition of cell growth in monolayer for 72 hours of compound treatment is measured in triplicate-experiments and used to derive the IC₅₀by MTS assay. The results are shown in Table B1.

TABLE B1

Active Monolayer Growth IC₅₀(μM)

Compound H1299 HCT116

CMD1 0.40 0.03

CMD2 0.15 0.01

CMD3 0.58 0.03

CMD4 0.28 0.03

CMD5 0.18 0.03

CMDC 6.8 0.67

The results show that the hydroxamate compounds of the present invention are highly active in inhibition of tumor cell growth. In addition to the above results, it has been observed that the compounds selectively inhibited tumor cells while showing minimal inhibition activities in non-tumorous cells.
The cells treated with the hydroxamate compounds are also tested for the induction of p21 promoter, which is a key mediator of G1 arrest and differentiation. The hydroxamate compounds activate the p21 promoter to a readily detectable level at a concentration within two-fold of their respective IC₅₀for monolayer cell growth inhibition in H1299. Without being bound by amy particular theory, the correlation appears to demonstrate that HDA inhibition leads to transcriptional activation of genes that inhibit tumor cell proliferation.

EXAMPLE B2

HDA is partially purified from H1299, human non-small cell lung carcinoma cells (obtained from American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA). Cells are grown to 70-80% confluence in RPMI media in the presence of 10% FCS, harvested and lysed by sonication. The lysate is centrifuged at 23,420 g for 10-15 min, the supernatant is applied to a Hiload 26/10 High performance Q-sepharose column (Amersham Pharmacia Biotech), and equilibrated with a buffer containing 20 mM Tris pH8.0, 1 mM EDTA, 10 mM NH₄Cl₂, 1 mM W Mercaptoethanol, 5% glycerol, 2 μg/mL aprotinin, 1 μg/mL leupeptin, and 400 mM PMSF. Proteins are eluted in 4 mL aliquots with a linear gradient from 0-500 mM NaCl in the above buffer at a flow rate of 2.5 mL/min. Each preparation of partially purified HDA enzyme is titrated to determine the optimal amount needed to obtain a signal to noise ratio of at least 5 to 1. Generally, 20-30 μl of partially purified HDA (5-10 mg protein/mL) is mixed with 2 μL of compound solution in DMSO in a deep well titer plate (Beckman). The compounds are serially diluted in DMSO to generate stocks at 20-fold of the assay concentrations. Final concentrations of compounds in the assay are 10 μM, 2 μM, 400 nM, 80 nM, and 16 nM with the final percentage of DMSO in each enzyme reaction equaling 0.1%. Each concentration of compound is assayed in duplicate. The substrate used in the reaction is a peptide of amino acid sequence, SGRGKGGKGLGKGGAKRHRKVLRD, corresponding to the twenty-four N-terminal amino acids of human histone H4, biotinylated at the N-terminus and penta-acetylated, at each lysine residue with ³H-acetate. To initiate the reaction, the substrate is diluted in 10 μL of Buffer A (100 mM Tris pH 8.0, 2 mM EDTA), added to the enzyme mixture and collected at the bottom of the deep well plate by centrifugation for 5 minutes at 1500 rpm. Following centrifugation, the mixture is incubated at 37° C. for 1.5 hr. The reaction is stopped by the addition of 20 μL of the Stop Buffer (0.5N HCl, 0.08M Acetic Acid). At this point, the assay proceeds to the robotic extraction phase or is frozen for several days at −0° C.
The extraction of enzymatically cleaved ³H-acetate groups from the reaction mixture is achieved with the solvent TBME (t-butyl methyl ether) using the Tomtec Quadra 96 workstation. A program is written to add 200 μL of TBME to a 96 “deep well” plate. The workstation is programmed to aspirate 50 μL of air followed by 200 μL of TBME and finally another 25 μL of air, which is dispensed into the each well of the plate. The contents of the deep well, were mixed thoroughly by pipetting 160 μL up and down 10 times. Before addition of TBME to the reaction mixture, it is necessary to “pre-wet” the pipette tips with TBME to prevent the solvent from dripping during the transfer to the deep well plate. The organic and aqueous phases in the deep well are separated by centrifugation at 1500 rpm for 5 min. Opti-Phase Supermix liquid scintillation cocktail (200 μL) (Wallac) is added to each well of the 96-well Trilux plate (Wallac). The deep well and Trilux plates are placed back on the workstation programmed to aspirate 25 μL of air into the pipette tips followed by 100 μL of the upper TBME phase and transfer it into the Trilux plate. The solutions are mixed by pipetting and expelling 50 μL, five times, within the same well. The Trilux plate is covered with clear film and read on a 1450 MicroBeta Trilux liquid scintillation and luminescence counter (Wallac) with a color/chemical quench and dpm correction.
In order to determine the IC₅₀values, the data are analyzed on a spreadsheet. The analysis requires a correction for the background luminescence that is accomplished by subtracting the dpm values of wells without ³H substrate from the experimental wells. The corrected dpm values along with the concentrations of the compounds are used to calculate IC₅₀using the user-defined spline function. This function utilizes linear regression techniques between data points to calculate the concentration of compounds that produced 50% inhibition. The results are shown in Table B2.

TABLE B2

Compound HDA Enzyme Activity IC₅₀(μM)

CMD1 0.032

CMD2 0.063

CMD3 0.014

CMD4 0.014

CMD5 0.016

CMDC >10

EXAMPLE B3

The A549 non-small cell lung human tumor cell line is purchased from the American Type Culture Collection, Rockville, Md. The cell line is free of Mycoplasma contamination (Rapid Detection System by Gen-Probe, Inc., San Diego, Calif.) and viral contamination (MAP testing by MA BioServices, Inc., Rockville, Md.). The cell line is propagated and expanded in RPMI 1640 medium containing 10% heat-inactivated FBS (Life Technologies, Grand Island, N.Y.). Cell expansions for implantation are performed in cell factories (NUNC, purchased from Fisher Scientific, Springfield, N.J.). Cells are harvested at 50-90% confluency, washed once with HBSS containing 10% FBS, and suspended in 100% HBSS.
Outbred athymic (nu/nu) female mice (“Hsd:Athymic Nude-nu” from Harlan Sprague Dawley, Indianapolis, Ind.) are anesthetized with Metofane (Mallinckrodt Veterinary, Inc., Mundelein, Ill.), and 100 μL of the cell suspension containing 1×10⁷cells is injected subcutaneously into the right axillary (lateral) region of each animal. Tumors are allowed to grow for about 20 days until a volume of 10-100 mm is achieved. At this point, mice bearing tumors with acceptable morphology and size are sorted into groups of eight for the study. The sorting process produces groups balanced with respect to mean and range of tumor size. Antitumor activity is expressed as % T/C, comparing differences in tumor volumes for treatment group (T) to vehicle control group (C). Regressions are calculated using the formula: (1−T/T₀)×100%, where T is the tumor volume for the treatment group at the end of the experiment, and T₀is the tumor volume at the beginning of the experiment.

CMD1 is administered intravenously, once daily 5×/week for three weeks, at doses of 10, 25, 50, or 100 mg/kg. The final DMSO concentration is 10%. Each test group has eight mice. Tumors are measured, and individual animal body weights recorded. Table B3 shows the results on the 41^stday.

TABLE B3


		Δ MEAN		Δ %
		TUMOR		BODY
	DOSE	VOLUME*¹	%	WEIGHT*²
COMPOUND	(mg/kg)	(mm³± SEM*³)	T/C	(% ± SEM*³)

10% DMSO/D5W*⁴	—	376 ± 55	—	+11.9 ± 0.2
CMD1	10	121 ± 27	32	+1.3 ± 0.3
CMD1	25	77 ± 32	20	−0.9 ± 0.3
CMD1	50	57 ± 10	15	−0.4 ± 0.3
CMD1	100	28 ± 25	7	+0.4 ± 0.3

Note:
*¹Difference in mean tumor volume for a group of animals at the end of the experiment minus mean tumor volume at the beginning.
*²Difference in body weight for a group of animals at the end of the experiment minus mean tumor volume at the beginning.
*³Standard error of the mean
*⁴5% dextrose injection, USP.

EXAMPLE B4

Example B3 repeated except CMD2 is used. Table B4 shows the results.

TABLE B4


		Δ MEAN		Δ %
		TUMOR		BODY
	DOSE	VOLUME		WEIGHT
COMPOUND	(mg/kg)	(mm³± SEM)	% T/C	(% ± SEM)

10% DMSO/D5W	—	135 ± 43	—	+6.7 ± 1.1
CMD2	25	37 ± 16	27	−4.2 ± 2.5
CMD2	50	29 ± 15	21	−2.9 ± 1.5

EXAMPLE B5

Example B3 is repeated except the HCT116 colon tumor cell line is used in place of the A549 cell line. The HCT116 cell line is also obtained from American Type Culture Collection, Rockville, Md., and the cell line is free of Mycoplasma contamination and viral contamination. The results are recorded on the 34^thday and are shown in Table B5.

TABLE B5


		Δ MEAN
		TUMOR		Δ %
	DOSE	VOLUME	%	BODY WEIGHT
COMPOUND	(mg/kg)	(mm³± SEM)	T/C	(% ± SEM)

10% DMSO/D5W	—	759 ± 108	—	−0.4 ± 0.4
CMD1	50*¹⁰	186 ± 40	25	−7.4 ± 0.8
CMD1	100	140 ± 38	18	−3.2 ± 0.4

Note:
*¹⁰Seven mice are tested in this group.

EXAMPLE B6

Example B4 is repeated except the HCT116 colon tumor cell line is used in place of the A549 cell line. The HCT116 is also obtained from American Type Culture Collection, Rockville, Md., and the cell line is free of Mycoplasma contamination and viral contamination. The results are recorded on the 34^thday and are shown in Table B6.

TABLE B6


		Δ MEAN
		TUMOR		Δ %
	DOSE	VOLUME	%	BODY WEIGHT
COMPOUND	(mg/kg)	(mm³± SEM)	T/C	(% ± SEM)

10% DMSO/D5W	—	759 ± 108	—	−0.4 ± 0.4
CMD2	10	422 ± 75	56	−10.2 ± 0.5
CMD2	25	305 ± 47	40	−7.0 ± 0.2
CMD2	50	97 ± 30	13	−7.3 ± 0.3
CMD2	100	132 ± 30	17	−9.4 ± 0.4

EXAMPLE B7

Annexin V binding was used as a marker for the early stages of apoptosis. A549, HCT116 and Normal Dermal Human Fibroblasts (NDHF) cells are treated separately with four compounds (CMD1, CMD2, CMD3 and CMD4) for 24 or 48 hours, stained with annexin V and compared to cells treated similarly with vehicle (DMSO). Cells are examined by fluorescence microscopy. Those undergoing apoptosis exhibit green fluorescent membrane staining. Viability is assessed by the counterstain, propidium iodide. Cells detected by red fluorescence are not viable. A small percentage of A549 and the majority of HCT116 cells exhibit cell surface staining with annexin V after 24 hour exposure to each of the four compounds. After 48 hour treatment, the majority of the A549 and HCT116 stain, with annexin V and/or propidium iodide indicating that the compounds induce apoptotic cell death. In contrast, NDHF cells do not show noticeable annexin V staining after 24 hour exposure and limited annexin V staining with CMD3 after 48 hour. These data show that NDHF cells predominantly underwent non-lethal growth arrest upon compound treatment, consistent with the cell cycle profile.
The staining results demonstrate that the hydroxamate compounds of the present invention cause tumor cells to die by apoptosis, while causing normal fibroblast to predominantly undergo cell cycle arrest, clearly demonstrating the selective efficacy of the present compounds.

Claims

1. A compound of the formula (I)

wherein

R₁is H, halo, or a straight chain C₁-C₆alkyl;

R₂is selected from H, C₁-C₁₀alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, —(CH₂)_nOC(O)R₆, amino acyl, HON—C(O)—CH═C(R₁)-aryl-alkyl- and —(CH₂)_nR₇;

R₃and R₄are the same or different and independently H, C₁-C₆alkyl, acyl or acylamino, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR₈, or R₂together with the nitrogen to which it is bound and R₃together with the carbon to which it is bound can form a C₄-C₉heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;

R₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, acyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, aromatic polycycle, non-aromatic polycycle, mixed aryl and non-aryl polycycle, polyheteroaryl, non-aromatic polyheterocycle, and mixed aryl and non-aryl polyheterocycle;

n, n₁, n₂and n₃are the same or different and independently selected from 0-6, when n, is 1-6, each carbon atom can be optionally and independently substituted with R₃and/or R₄;

X and Y are the same or different and independently selected from H, halo, C₁-C₄alkyl, NO₂, C(O)R₁, OR₉, SR₉, CN, and NR₁₀R₁₁;

R₆is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, OR₁₂, and NR₁₃R₁₄;

R₇is selected from OR₁₅, SR₁₅, S(O)R₁₆, SO₂R₁₇, NR₁₃R₁₄, and NR₁₂SO₂R₆;

R₈is selected from H, OR₁₅, NR₁₃R₁₄, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

R₉is selected from C₁-C₄alkyl and C(O)-alkyl;

R₁₀and R₁₁are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)-alkyl;

R₁₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₆heterocycloalkylalkyl, aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl, and heteroarylalkyl;

R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, amino-acyl, or R₁₃and R₁₄together with the nitrogen to which they are bound are C₄-C₉heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and non-aryl polyheterocycle;

R₁₅is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;

R₁₆is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, polyheteroaryl, arylalkyl, heteroarylalkyl and (CH₂)_mZR₁₂;

R₁₇is selected from C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, aromatic polycycle, heteroaryl, arylalkyl, heteroarylalkyl, polyheteroaryl and NR₁₃R₁₄;

m is an integer selected from 0 to 6; and

Z is selected from O, NR₁₃, S and S(O);

or a pharmaceutically acceptable salt thereof.

2. A compound of claim 1 wherein each of R₁, X, Y, R₃, and R₄is H.

3. A compound of claim 2 one of n₂and n₃is zero and the other is 1.

4. A compound of claim 3 wherein R₂is H or —CH₂—CH₂—OH.

5. A compound of claim 1 of the formula (Ia)

wherein

n₄is 0-3,

R₅′ is heteroaryl, heteroarylalkyl, an aromatic polycycle, a non-aromatic polycycle, a mixed aryl and non-aryl polycycle, polyheteroaryl, or a mixed aryl and non-aryl polyheterocycle

or a pharmaceutically acceptable salt thereof.

6. A compound of claim 1 of the formula (Ia)

wherein

n₄is 0-3,

R₅′ is aryl, arylalkyl, an aromatic polycycle, a non-aromatic polycycle or a mixed aryl and non-aryl polycycle,

or a pharmaceutically acceptable salt thereof.

7. A compound of claim 6 wherein R₅′ is aryl or arylalkyl.

8. A corm pound of claim 7 wherein R₅′ is p-fluorophenyl, p-chlorophenyl, p-O—C₁-C₄-alkylphenyl, p-C₁-C₄-alkylphenyl, benzyl, ortho, meta or para-fluorobenzyl, ortho, meta or para-chlorobenzyl, or ortho, meta or para- mono, di or tri-O—C₁-C₄-alkylbenzyl.

9. A compound of claim 1 of formula Ib:

wherein

R₂′ is selected from H, C₁-C₆alkyl, C₄-C₆cycloalkyl, alkylcycloalkyl, and —(CH₂)_2-4OR₂₁where R₂₁is H, methyl, ethyl, propyl, or isopropyl, and

R₅″ is unsubstituted or substituted 1H-indol-3-yl, benzofuran-3-yl or quinolin-3-yl

or a pharmaceutically acceptable salt thereof.

10. A compound of claim 9 wherein R₅″ is substituted 1H-indol-3-yl or substituted benzofuran-3-yl.

11. A compound of claim 1 of formula (Ic)

wherein

the ring containing Z₁is aromatic or non-aromatic which non-aromatic rings are saturated or unsaturated,

Z₁is O, S or N—R₂₀;

R₁₈is H, halo, C₁-C₆alkyl, C₃-C₇cycloalkyl; aryl, or heteroaryl;

R₂₀is H, C₁-C₆alkyl, C₁-C₆alkyl-C₃-C₉cycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, acyl or sulfonyl;

A₁is 1, 2 or 3 substituents which are independently H, C₁-C-₆alkyl, —OR₁₉, halo, alkylamino, aminoalkyl, halo, or heteroarylalkyl,

R₁₉is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

v is 0, 1 or 2,

p is 0-3, and

q is 1-5 and r is 0 or

q is 0 and r is 1-5

or a pharmaceutically acceptable salt thereof.

12. A compound of claim 11 wherein Z₁is N—R₂₀.

13. A compound of claim 11 wherein R₂is H or —CH₂—CH₂—OH and the sum of q and r is 1.

14. A compound of claim 1 of the formula (Id)

wherein

Z₁is O, S or N—R₂₀,

R18 is H, halo, C₁-C₆alkyl, C₃-C₇cycloalkyl, unsubstituted phenyl, substituted phenyl, or heteroaryl,

A₁is 1, 2 or 3 substituents which are independently H, C_1-C- ₆alkyl, —OR₁₉, or halo,

R₁₉is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl and —(CH₂CH═CH(CH₃)(CH₂))_1-3H;

p is 0-3, and

q is 1-5 and r is 0 or

q is 0 and r is 1-5,

or a pharmaceutically acceptable salt thereof.

15. A compound of claim 14 wherein R₂is H or —CH₂—CH₂—OH and the sum of q and r is 1.

16. A compound of claim 11 of formula (Ie)

or a pharmaceutically acceptable salt thereof.

17. A compound of claim 16 wherein R₁₈is H, fluoro, chloro, bromo, a C₁-C₄alkyl group, a C₃-C₇cycloalkyl group, phenyl or a heteroaryl ring.

18. A compound of claim 16 wherein R₂is H, or —(CH₂)_sCH₂OH and wherein s is 1-3.

19. A compound of claim 18 wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3.

20. A compound of claim 16 wherein R₁₈is H, methyl, ethyl, t-butyl, trifluoromethyl, cyclohexyl, phenyl, 4-methoxyphenyl, 4-trifluoromethylphenyl, 2-furanyl, 2-thiophenyl, or 2-, 3- or 4-pyridyl.

21. A compound of claim 20 wherein R₂is H, or —(CH₂)_sCH₂OH and wherein s is 1-3.

22. A compound of claim 21 wherein p is 1-3

23. A compound of claim 22; wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3.

24. A compound of claim 23 wherein R₂is H or —CH₂—CH₂—OH and the sum of q and r is 1.

25. A compound of claim 16 wherein R₂₀is H or C₁-C₆alkyl.

26. A compound of claim 16 selected from the group consisting of N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.

27. A compound of claim 1 of the formula (If)

or a pharmaceutically acceptable salt thereof.

28. A compound of claim 27 wherein R₂is H or —(CH₂)_pCH₂OH and p is 1-3.

29. A compound of claim 28 wherein R₁is H and X and Y are each H, and wherein q is 1-3 and r is 0 or wherein q is 0 and r is 1-3.

30. A compound of claim 29 wherein R₂is H or —CH₂—CH₂—OH and the sum of q and r is 1.

31. A compound of claim 27 which is N-hydroxy-3-[4-[[[2-(benzofur-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.

32. A pharmaceutical composition comprising a pharmaceutically effective amount of a compound of formula (I)

wherein

R₁is H, halo, or a straight chain C₁-C₆alkyl;

R₂is selected from H, C₁-C₁₀alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉-heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, —(CH₂)_nOC(O)R₆, amino acyl, HON—C(O)—CH═C(R₁)-aryl-alkyl- and —(CH₂)_nR₇;

R₃and R₄are the same or different and independently H, C₁-C₆alkyl, acyl or acylamino, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR₈, or, R₂together with the nitrogen to which it is bound and R₃together with the carbon to which it is bound can form a C₄-C₉heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;

R₉is selected from C₁-C₄alkyl and C(O)-alkyl;

R₁₀and R₁₁are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)alkyl;

R₁₂is selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, aryl, mixed aryl and non-aryl polycycle, heteroaryl, arylalkyl, and heteroarylalkyl;

R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, amino acyl, or R₁₃and R₁₄together with the nitrogen to which they are bound are C₄-C₉heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and non-aryl polyheterocycle;

m is an integer selected from 0 to 6; and

Z is selected from O, NR₁₃, S and S(O);

or a pharmaceutically acceptable salt thereof.

33. A pharmaceutical composition of claim 32 wherein the compound of formula (I) is selected from the group consisting of selected N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.

34. A pharmaceutical composition of claim 32 which is N-hydroxy-3-[4-[[[2-(benzofur-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.

35. A method for treating a proliferative disorder in a mammal which comprises administering to said mammal a compound of the formula (I)

wherein

R₁is H, halo, or a straight chain C₁-C₆alkyl;

R₃and R₄are the same or different and independently H, C₁-C₆alkyl, acyl or acylamino, or R₃and R₄together with the carbon to which they are bound represent C═O, C═S, or C═NR, or R₂together with the nitrogen to which it is bound and R₃together with the carbon to which it is bound can form a C₄-C₉heterocycloalkyl, a heteroaryl, a polyheteroaryl, a non-aromatic polyheterocycle, or a mixed aryl and non-aryl polyheterocycle ring;

n, n₁, n₂and n₃are the same or different and independently selected from 0-6, when n₁is 1-6, each carbon atom can be optionally and independently substituted with R₃and/or R₄;

R₉is selected from C₁-C₄alkyl and C(O)-alkyl;

R₁₀and R₁₁, are the same or different and independently selected from H, C₁-C₄alkyl, and —C(O)-alkyl;

m is an integer selected from 0 to 6; and

Z is selected from O, NR₁₃, S and S(O);

or a pharmaceutically acceptable salt thereof.

36. A method of claim 35 wherein the compound of formula (I) is selected from the group consisting of N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.

37. A method for regulating p21 promoter which comprises introducing a compound of the formula (I)

wherein

R₁is H, halo, or a straight chain C₁-C₆alkyl;

R₂is selected from H, C₁-C₁₀alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, C₄-C₉heterocycloalkylalkyl, cycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, —(CH₂)_nC(O)R₆, —(CH₂)_nOC(O)RN, amino acyl, HON—C(O)—CH═C(R₁)-aryl-alkyl- and —(CH₂)_nR₇;

R₉is selected from C₁-C₄alkyl and C(O)-alkyl;

R₁₃and R₁₄are the same or different and independently selected from H, C₁-C₆, alkyl, C₄-C₉cycloalkyl, C₄-C₉heterocycloalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, amino acyl, or R₁₃and R₁₄together with the nitrogen to which they are bound are C₄-C₉heterocycloalkyl, heteroaryl, polyheteroaryl, non-aromatic polyheterocycle or mixed aryl and non-aryl polyheterocycle;

m is an integer selected from 0 to 6; and

Z is selected from O, NR₁₃, S and S(O);

or a pharmaceutically acceptable salt thereof,

into the environment of a mammalian cell.

38. A method of claim 37 wherein the compound of formula (I) is selected from the group consisting of N-hydroxy-3-[4-[[(2-hydroxyethyl)[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide, N-hydroxy-3-[4-r[[[2-(1H-indol-3-yl)ethyl]-amino]methyl]phenyl]-2E-2-propenamide and N-hydroxy-3-[4-[[[2-(2-methyl-1H-indol-3-yl)-ethyl]-amino]methyl]phenyl]-2E-2-propenamide, or a pharmaceutically acceptable salt thereof.