ES2368033T3 - HISTONE DEACETILASE INHIBITORS. - Google Patents

Abstract

A histone deacetylase inhibitor represented by the formula in which Cy-L 2 -Ar-Y 2 - (O) NH-Z Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted with one and four substituents selected from halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonamido, alkanesulfonidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamidoamide , alkylcarbonyl, acyloxy, cyano and ureido, and wherein said aryl, heterocyclyl and heteroaryl may also be optionally substituted with one or more groups selected from (C1-C8) alkyl, di (C1-C6) amino, alkane alkane (C1-C6) acylamino and arene (C6-C10) acylamino, and wherein said cycloalkyl and heterocyclyl may also be optionally substituted with oxo, and wherein said heterocyclyl may be ta Also optionally independently substituted on nitrogen with a group selected from alkylsulfonyl, arylcarbonyl, arylsulfonyl and aralkoxycarbonyl, with the proviso that Cy is not a (spirocycloalkyl) heterocyclyl; L 2 is saturated C1-C8 alkylene or C2-C8 alkenylene, the alkylene or alkenylene may be optionally substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino , acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido on the condition that it is not in the condition of wherein one of the carbon atoms of the alkylene may optionally be replaced by a heteroatomic moiety selected from the group consisting of S; or S (O): o L 2 is S (O) 2- (CH2) 1-4: Ar is arylene, which arylene may optionally be fused with an aryl or heteroaryl ring, or with a saturated cycloalkyl or heterocyclic ring or unsaturated; and Y 2 is a saturated straight or branched chain alkylene; and Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, being M H or a pharmaceutically acceptable cation, thiadiazolyl may be optionally substituted with a substituent selected from the group consisting of thiol, trifluoromethyl, amino and sulfonamido; with the proviso that when the carbon atom to which Cy is attached is substituted with oxo, then Cy and Z are not both pyridyl.

Description

Background of the invention

Field of the Invention

The present invention relates to the inhibition of histone deacetylase. More particularly, the invention relates to compounds and methods for inhibiting the enzymatic activity of histone deacetylase.

Summary of related technique

In eukaryotic cells, nuclear DNA associates with histones to form a compact complex called chromatin. Histones constitute a family of basic proteins that are generally very conserved at

10 through eukaryotic species. The central histones, called H2A, H2B, H3, and H4, associate to form a protein nucleus. The DNA is wrapped around this protein nucleus, interacting the basic amino acids of the histones with the negatively charged phosphate groups of the DNA. Approximately 146 base pairs of DNA are wrapped around a histone nucleus to constitute a nucleosome particle, the structural motif of chromatin repeat.

15 Csordas, Biochem. J., 286: 23-38 (1990) teaches that histones are subjected to post-translational acetylation of the ε-amino groups of N-terminal lysine residues, a reaction that is catalyzed by histone acetyl transferase (HAT1). Acetylation neutralizes the positive charge of the lysine side chain, and is believed to affect the structure of chromatin. In fact, Taunton et al., Science, 272: 408-411 (1996), teach that access to transcription factors to chromatin templates is enhanced by histone hyperacetylation. Taunton et al.

20 further teach that enrichment has been found in subacetylated histone H4 in transcriptionally mute regions of the genome.

Histone acetylation is a reversible modification, with deacetylation catalyzed by a family of enzymes called histone deacetylases (HDAC). Grozinger et al., Proc. Natl Acad. Sci. USA, 96: 4868-4873 (1999), teaches that HDACs can be divided into two classes, the first represented by proteins similar to Rpd3 yeast, and the second represented by proteins similar to Hda1 yeast. Grozinger et al. It also teaches that human HDAC1, HDAC2, and HDAC3 proteins are members of the first class of HDAC, and unveils new proteins, called HDAC4, HDAC5, and HDAC6, which are members of the second class of HDAC. Kao et al., Genes & Dev., 14: 55-66 (2000), reveals HDAC7, a new member of the second class of HDAC. Van den Wyngaert, FEBS, 478: 77-83 (2000) reveals HDAC8, a new member of the first class of

30 HDAC

Richon et al., Proc. Natl Acad. Sci. USA, 95: 3003-3007 (1998), discloses that HDAC activity is inhibited by tricostatin A (TSA), a natural product isolated from Streptomyces hygroscopicus, and by a synthetic compound, hydroxamic suberylanilide acid (SAHA ). Yoshida and Beppu, Exper. Cell Res., 177: 122-131 (1988), teaches that TSA causes the arrest of rat fibroblasts in the G1 and G2 phases of the cell cycle, which implies HDAC in the

35 cell cycle regulation. In fact, Finnin et al., Nature, 401: 188-193 (1999), teaches that TSA and SAHA inhibit cell growth, induce terminal differentiation, and prevent tumor formation in mice.

These findings suggest that the inhibition of HDAC activity represents a new procedure to intervene in the regulation of the cell cycle and that HDAC inhibitors have great therapeutic potential in the treatment of cell proliferative diseases or conditions. To date, only a few 40 histone deacetylase inhibitors are known in the art. Thus, there is a need to identify additional HDAC inhibitors and to identify the structural characteristics required for a potent HDAC inhibitor activity. The relevant prior art includes documents JP-11302173; J Med Chem 42 (22), 199, 4669-79; Beilstein Database, BRN 3436503, 4265970, 2813528, 7299261, m2890108, 2793974; Beilstein Database BRN 5634742, 6816677, 2852507, 2855506, 3454395: Beilstein Database BRN 6864971, 4446205; Beilstein Database

45 BRN 7655985, 3313150, 3313231; Beilstein Database BRN 3137042; Chem Abstr 91: 107818; Chem Abstr 131: 97120; WO 98/55449; Chem Abstr 131: 317417; EP 827742; WO 02/22577; WO 95/06554: WO 00/69819; WO 98/38859; WO 00/56704: JP 56051441; US 4013768; US 4173577; WO 93/12075; WO 02/30879; GB0023986D; and JP10182583.

Brief summary of the invention

In accordance with the present invention a histone deacetylase inhibitor represented by the formula is provided

Cy-L2-Ar-Y2-C (O) NH-Z

in which

Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted with between one and four substituents selected from halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino , alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenosulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido, and in which one or more may be substituted or heteroaryl and may be optionally substituted groups selected from alkyl (C1-C6) amino, di-alkyl (C1-C8) amino, alkane (C1-C8) acylamino and arene (C6-C10) acylamino, and wherein said cycloalkyl and heterocyclyl can also be optionally substituted with oxo, and wherein said heterocyclyl is also optionally independently substituted in nitrogen with a group selected from alkyl lsulfonyl, arylcarbonyl, arylsulfonyl and aralkoxycarbonyl, with the proviso that Cy is not a (spirocycloalkyl) heterocyclyl;

L2 is saturated C1-C8 alkylene or C2-C8 alkenylene, in which the alkylene or alkenylene may be optionally substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy , amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido L, with the condition C-2 and wherein one of the alkylene carbon atoms may optionally be replaced by a heteroatomic moiety selected from the group consisting of S; bear); or L2 is -S (O) 2- (CH2) 1-4;

Ar is arylene, said arylene may optionally be fused with an aryl or heteroaryl ring, or with a saturated or unsaturated cycloalkyl or heterocyclic ring; Y

Y2 is a saturated straight or branched chain alkylene; Y

Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, with M H or a pharmaceutically acceptable cation, said thiadiazolyl being optionally substituted with a substituent selected from the group consisting of thiol, trifluoromethyl, amino and sulfonamido;

with the proviso that when the carbon atom to which Cy is attached is substituted with oxo, then Cy and Z are not both pyridyl.

Preferably, L2 is saturated C1-C8 alkylene or C2-C8 alkenylene, in which the alkylene or alkenylene may be optionally substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy , aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenosulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido -, and in which one of the alkylene carbon atoms may optionally be replaced by a heteroatomic moiety selected from the group consisting of S; bear).

Preferably, Y2 is C1-C6 alkylene. Preferably, Y2 is C1-C3 alkylene. Preferably, Y2 is C1-C2 alkylene.

Preferably, one or two carbons saturated in L2 are substituted with a substituent independently selected from the group consisting of C1-C6 alkyl, C6-C10 aryl, amino, oxo, hydroxy, C1-C4 alkoxy, and C6-C10 aryloxy.

Preferably, no carbon atom of the L2 alkylene is replaced by a heteroatomic moiety. Alternatively, preferably an L2 alkylene carbon atom is replaced by a heteroatomic moiety selected from the group consisting of S; bear).

Preferably, L2 is selected from the group consisting of -S- (CH2) n, -S (O) - (CH2) n, and - S (O) 2- (CH2) n-, where n is 1, 2, 3 or 4.

Preferably, L2 is saturated C1-C6 alkylene. Preferably, L2 is saturated C1-C4 alkylene.

Preferably, L2 is saturated C1-C6 alkylene, substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido, with the proviso that L2 is not C (O) -.

Preferably, L2 is saturated C1-C4 alkylene, substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido, with the proviso that L2 is not C (O) -.

Preferably, Z is selected from the group consisting of 2-anilinyl, 2-pyridyl, 1,3,4-thiadiazol-2-yl, and -O-M, with M H or a pharmaceutically acceptable cation. Preferably, Z is -O-M, with M being hydrogen or any pharmaceutically acceptable cation. Preferably, Z is -O-M, with M being hydrogen.

Preferably, Ar is unsubstituted phenylene, which may be optionally condensed with an aryl or heteroaryl ring, or with a saturated or partially unsaturated cycloalkyl or heterocyclic ring. Preferably, the phenylene is 4-phenylene.

Preferably, Cy is selected from the group consisting of phenyl, naphthyl, thienyl, benzothienyl, and quinolyl, any of which may be optionally substituted.

Preferably, the phenyl, naphthyl, thienyl, benzothienyl, or quinolyl is not substituted or is substituted by one or two substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl, C6-C10 aryl, ar (C6- C10) (C1-C8) alkyl, halo, nitro, hydroxy, C1-C8 alkoxy, C1-C8 alkoxy carbonyl, carboxy, and amino.

Preferably, Cy is unsubstituted phenyl.

Preferably, the inhibitor is selected from the group consisting of

image 1

Y

fifteen

Preferably, the inhibitor is selected from the group consisting of

image 1

image 1

Preferably, the inhibitor has the structure

twenty

image 1

Preferably, the inhibitor has the structure

image 1

Preferably, the inhibitor has the structure

image 1

Preferably, the inhibitor has the structure

image 1

Preferably, the inhibitor has the structure

image 1

Also provided according to the present invention is a pharmaceutical composition comprising a histone deacetylase inhibitor according to any of the preceding claims, and a pharmaceutically acceptable carrier, excipient, or diluent.

A histone deacetylase inhibitor according to the present invention is also provided in accordance with the present invention for use in the inhibition of histone deacetylase in a cell.

Preferably, the cell is a neoplastic cell. Preferably, the neoplastic cell is in an animal. Preferably, the neoplastic cell is in a neoplastic growth.

Preferably, the inhibitor is for use with an antisense oligonucleotide that inhibits the expression of a histone deacetylase.

Preferably, the inhibitor is for use in inhibiting cell proliferation. Preferably, the inhibitor is for use in the treatment of a fungal disease or infection.

The use of an inhibitor according to the present invention in the preparation of a medicament for inhibiting cell proliferation is also provided in accordance with the present invention.

The use of an inhibitor according to the present invention in the preparation of a medicament for treating a fungal disease or infection is also provided in accordance with the present invention.

Detailed description of the preferred embodiments

The invention provides compounds and methods for inhibiting histone deacetylase enzyme activity. The invention also provides compositions and methods for treating diseases and conditions.

25 cell proliferatives. The patent and scientific literature mentioned in this document establish the knowledge that is available to those skilled in the art. In case of inconsistencies, the present disclosure will prevail.

For the purposes of the present invention, the following definitions will be used:

As used herein, the terms "histone deacetylase" and "HDAC" are intended to refer to

Any of a family of enzymes that remove the acetyl groups from the ε-amino groups of the lysine residues at the N-terminus of a histone. Unless otherwise indicated by the context, the term "histone" is intended to refer to any histone-like protein, including H1, H2A, H2B, H3, H4, and H5, of any species. Preferred histone deacetylases include class I and class II enzymes. Preferably, histone deacetylase is a human HDAC, including; although without limitation, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC, HDAC-7, and

35 HDAC-8. In some other preferred embodiments, histone deacetylase comes from a protozoan or fungal source.

The term "histone deacetylase inhibitor" is used to identify a compound that has a structure as defined herein, which is capable of interacting with a histone deacetylase and inhibiting its enzymatic activity. Inhibiting the enzymatic activity of histone deacetylase means reducing the ability of a histone deacetylase to remove an acetyl group from a histone. In some preferred embodiments, said reduction in histone deacetylase activity is at least about 50%, more preferably at least about 75%, and even more preferably at least about 90%. In other preferred embodiments, the activity of histone deacetylase is reduced by at least 95% and, more preferably, at least 99%.

Preferably, said inhibition is specific, that is, the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone at a concentration that is less than the concentration of the inhibitor that is required to produce another biological effect. related. Preferably, the concentration of the inhibitor required to inhibit histone deacetylase activity is at least 2 times less, more preferably at least 5 times less, even more preferably at least 10 times less, and most preferably at least 20 times less than the concentration. required to produce an unrelated biological effect.

The term "alkyl," as used herein, refers to straight or branched chain aliphatic groups having 1 to 12 carbon atoms, preferably 1-8 carbon atoms, and more preferably, 1-6 atoms. carbon, which may be optionally substituted with one, two or three substituents. Unless, from the context, something else is evident, the term "alkyl" is intended to include saturated, unsaturated and partially unsaturated aliphatic groups. When unsaturated groups are particularly required, the terms "alkenyl" or "alkynyl" will be used. When only saturated groups are required, the term "saturated alkyl" will be used. Preferred saturated alkyl groups include, without limitation, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, and hexyl .

An "alkylene" group is an alkyl group, as defined hereinbefore, which is located between and which serves to connect two other chemical groups. Preferred alkylene groups include, without limitation, methylene, ethylene, propylene, and butylene.

The term "cycloalkyl", as used herein, includes cyclic, saturated and partially unsaturated hydrocarbon groups, having 3 to 12 carbons, preferably 3 to 8 carbons and, more preferably, 3 to 6 carbons, in that the cycloalkyl group may be additionally optionally substituted.The preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

An "aryl" group is a C6-C14 aromatic moiety comprising one to three aromatic rings, which may be optionally substituted. Preferably, the aryl group is a C6-C10 aryl group. Preferred aryl groups include, without limitation, phenyl, naphthyl, anthracenyl, and fluorenyl. An "aralkyl" or "arylalkyl" group comprises an aryl group covalently linked to an alkyl group, which may be independently optionally substituted or unsubstituted. Preferably, the aralkyl group is (C 1 -C 6) alkyl (C 6 -C 10) aryl, including, without limitation, benzyl, phenethyl, and naphthylmethyl. An "alkaryl" or "alkylaryl" group is an aryl group having one or more alkyl substituents. Examples of alkaryl groups include, without limitation, tolyl, xylyl, mesityl, ethylphenyl, tert-butylphenyl, and methylnaphthyl.

An "arylene" group is an aryl group, as defined hereinbefore, which is located between, and which serves to connect two other chemical groups. Preferred arylene groups include, without limitation, phenylene and naphthylene. The term "arylene" is also intended to include heteroaryl bridge linkage groups, including, but not limited to, benzothienyl, benzofuryl, quinolyl, isoquinolyl, and indolyl.

A "heterocyclyl" or "heterocyclic" group is a ring structure having from about 3 to about 8 atoms, in which one or more atoms are selected from the group consisting of N, O, and S. The heterocyclic group may be optionally substituted on carbon in one or more positions. The heterocyclic group may also be independently substituted on nitrogen with alkyl, aryl, aralkyl, alkylcarbonyl, alkylsulfonyl, arylcarbonyl, arylsulfonyl, alkoxycarbonyl, aralkoxycarbonyl, or on sulfur with oxo or lower alkyl. Preferred heterocyclic groups include, without limitation, epoxy, aziridinyl, tetrahydrofuranoyl, pyrrolidinyl, piperidinyl, piperazinyl, thiazolidinyl, oxazolidinyl, oxazolidinonyl, and morpholino. In certain preferred embodiments, the heterocyclic group is fused to an aryl or heteroaryl group. Examples of such condensed heterocycles include, without limitation, tetrahydroquinoline and dihydrobenzofuran.

As used herein, the term "heteroaryl" refers to groups that have 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms; that have 6, 10, or 14  electrons shared in a cyclic arrangement; and having, in addition to carbon atoms, between one and about three heteroatoms selected from the group consisting of N, O, and S. Preferred heteroaryl groups include, without limitation, thienyl, benzothienyl, furyl, benzofuryl, dibenzofuryl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, indolyl, quinolyl, isoquinolyl, quinoxalinyl, tetrazolyl, oxazolyl, thiazolyl, and isoxazolyl.

As used herein, a "substituted" alkyl, cycloalkyl, aryl, heteroaryl, or heterocyclic group is one having between one and about four, preferably between one and about three, more preferably one or two, substituents other than hydrogen . Suitable substituents include, without limitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy groups; amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfanantido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido.

The term "halogen" or "halo", as used herein, refers to chlorine, bromine, fluorine, or iodine.

As used herein, the term "acyl" refers to an alkylcarbonyl or arylcarbonyl substituent.

The term "acylamino" refers to an amide group attached to the nitrogen atom. The term "carbamoyl" refers to an amide group attached to the carbonyl carbon atom. The nitrogen atom of an acylamino or carbamoyl substituent may be additionally substituted. The term "sulfonamido" refers to a sulfonamide substituent fixed through any of the sulfur or nitrogen atom. The term "amino" is intended to include NH2 groups,

10 alkylamino, arylamino, and cyclic amino. The term "ureido", as used herein, refers to a urea moiety, substituted or unsubstituted.

Specific examples of synthetic compounds and routes are given below. However, compounds that are not included within the general formula detailed in claim 1 do not form part of the present invention.

15 Synthesis

Scheme 3

image 1

Compounds of the formula Cy-L2-Ar-Y2- (O) -NH-O-M are preferably prepared according to the synthetic routes described in Schemes 3-5. Accordingly, in certain preferred embodiments, the compounds of

Formulas XIX and XXI (L2 = -C (O) -CH = CH- or -C (O) -CH2CH2-) are preferably prepared according to the route described in Scheme 3. Therefore, an aryl Substituted acetophenone (XV) is treated with an aryl aldehyde (XVI) in a protic solvent, such as methanol, in the presence of a base, such as sodium methoxide, giving enone XVII.

The acid substituent of XVII (R = H) is coupled with an O-protected hydroxylamine, such as Otetrahydropyranylhydroxylamine (R1 = tetrahydropyranyl) to give the O-protected N-hydroxybenzamide XVIII. The coupling reaction is preferably carried out by treating the acid and hydroxylamine with dicyclohexylcarbodiimide in a solvent, such as methylene chloride or with 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide in the presence of N-hydroxybenzotriazole in a solvent, such as dimethylformamide Other coupling reagents are known in the art, and can also be used in this reaction. O-deprotection is achieved by treatment of XVIII with a

acid, such as camphorsulfonic acid, in a solvent, such as methanol, giving hydroxamic acid XIX (L2 = C (O) -CH = CH-).

Saturated compounds of formula XXI (L2 = -C (O) -CH2CH2-) are preferably prepared by hydrogenation of XVII (R = Me) on a palladium catalyst, such as 10% Pd / C, in a solvent , such as methanol tetrahydrofuran. Basic hydrolysis of the resulting product XIX with lithium hydroxide, followed by formation of N-hydroxy amide and acid hydrolysis, as described above, then gives hydroxamic acid XXI.

Scheme 4

image 1

Compounds of formula XXVI (L2 = - (CH2) or + 2-) are preferably prepared by general procedures

10 described in Schemes 4 and 5. Therefore, in some embodiments, a terminal olefin (XXII) is coupled with an aryl halide (XXIII) in the presence of a catalytic amount of a source of palladium, such as palladium acetate or tris (dibenzylideneacetone) dipaladium (0), a phosphine, such as triphenylphosphine, and a base, such as triethylamine, in a solvent, such as acetonitrile, to give the coupled product XXIV. Hydrogenation, followed by Nhydroxyamide formation and acid hydrolysis, as described above, gives hydroxamic acid XXVI.

15 Scheme 5

image 1

Alternatively, in some other embodiments, a phosphonium salt of formula XXVII is treated with an aryl aldehyde of formula XXVIII, in the presence of base, such as lithium hexamethyldisilazide, in a solvent, such as tetrahydrofuran, to produce compound XXIV. Hydrogenation, followed by formation of N-hydroxyamide and

20 acid hydrolysis, then gives compounds XXVI.

Scheme 6

image 1

Compounds of the formula Cy-L-Ar-YC (O) -NH-Z, in which L is L2, Y is Y2, and Z is anilinyl or pyridyl, are preferably prepared according to the synthetic routes described in Scheme 6. An acid of formula Cy-L-Ar-Y5 C (O) -OH (XXIX), prepared by one of the procedures shown in Schemes 3-5, is converted into the corresponding addition chloride XXX, of according to conventional procedures, for example, by treatment with sodium hydride and oxalyl chloride. Treatment of XXX with 2-aminopyridine and a tertiary base, such as N-methylmorpholine, preferably in dichloromethane at reduced temperature, then gives pyridyl amide XXXL. Similarly, the acid chloride XXX can be treated with 1,2-phenylenediamine giving the anilinyl

10 amide XXXII. Alternatively, the acid chloride XXX can be treated with a mono-protected 1,2-phenylenediamine, such as 2- (t-BOC-amino) aniline, followed by deprotection, giving XXXII.

In another alternative procedure, XXIX acid can be activated by treatment with carbonyldiimidazole (CDI), followed by treatment with 1,2-phenylenediamine and trifluoroacetic acid, giving the anilinyl amide XXXII.

Scheme 7

image 1

Compounds of formula XXXVIII (L2 = -C (O) -alkylene-) are preferably prepared according to the general procedure described in Scheme 7. Therefore, the aldol condensation of ketone XXXIII (R1 = H or alkyl) with aldehyde XXXIV gives adduct XXXV. The adduct XXXV can be converted, directly, into the corresponding hydroxamic acid XXXVI, or it can first undergo hydrogenation, giving the saturated compound XXVII and then becoming the hydroxamic acid XXXVIII.

Scheme 8

image 1

The compounds of formula (2) in which one of the carbon atoms in L2 is replaced with S, S (O), or S (O) 2 are preferably prepared in accordance with the general procedure described in Scheme 8 Therefore, thiol XXXIX is added to olefin XL to produce XLI. The reaction is preferably carried out in the presence of a radical initiator, such as 2,2'-azobisisobutyronitrile (AIBN) or 1,1'-azobis (cyclohexanecarbonitrile) (VAZO ™). Oxidation of the sulfide, preferably by treatment with m-chloroperbenzoic acid (mCPBA), gives the corresponding sulfone, which is conveniently isolated after conversion into the methyl ester by treatment with diazomethane. Hydrolysis of the ester then gives XLII acid, which is converted to XLIII hydroxamic acid according to any of the procedures described above. The XLI sulfide can also be converted directly into the corresponding XLIV hydroxamic acid, which can then be selectively oxidized to the XLV sulfoxide, for example, by treatment with hydrogen peroxide and tellurium dioxide.

Pharmaceutical compositions

The pharmaceutical compositions of the present invention comprise a histone deacetylase inhibitor as defined in the appended claims, and a pharmaceutically acceptable carrier, excipient, or diluent. The compounds of the invention may be formulated by any method well known in the art, and may be prepared for administration by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain preferred embodiments, the compounds of the invention are administered intravenously in a hospital setting. In certain other preferred embodiments, administration may preferably be orally.

The characteristics of the vehicle will depend on the route of administration. As used herein, the term "pharmaceutically acceptable" means a non-toxic material, which is compatible with a biological system, such as a cell, a cell culture, tissue, or organism, and which does not interfere with the efficacy of the biological activity of the active ingredient or ingredients. Therefore, the compositions according to the invention may contain, in addition to the inhibitor, diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The preparation of pharmaceutically acceptable formulations is described, for example, in Remington's Pharmaceutical Sciences, 18th Edition, ed A. Gennaro, Mack Publishing Co., Easton, PA, 1990.

Histone Deacetylase Inhibition

Measurement of the enzymatic activity of a histone deacetylase can be achieved using known methodologies. For example, Yoshida et al., J. Biol. Chem., 265: 17174-17179 (1990), describes the evaluation of the enzymatic activity of histone deacetylase by detecting acetylated histones in cells treated with tricostatin A. Taunton et al. ., Science, 272: 408-411 (1996), analogously describes procedures for measuring the enzymatic activity of histone deacetylase using endogenous and recombinant HDAC-1.

In some preferred embodiments, the histone deacetylase inhibitor interacts with, and reduces the activity of, all histone deacetylases in the cell. In some other preferred embodiments according to this aspect of the invention, the histone deacetylase inhibitor interacts with, and reduces the activity of, less than all histone deacetylases in the cell. In certain preferred embodiments, the inhibitor interacts with, and reduces the activity of, a histone deacetylase (for example, HDAC-1), but does not interact with or reduces the activities of other histone deacetylase (for example, HDAC-2, HDAC- 3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, and HDAC-8). As discussed below, certain particularly preferred histone deacetylase inhibitors are those that interact with and reduce the enzymatic activity of a histone deacetylase that is involved in tumorigenesis. Certain other preferred histone deacetylase inhibitors interact with and reduce the enzymatic activity of a fungal histone deacetylase.

Preferably, inhibition of histone deacetylase in a cell causes an inhibition of cell proliferation of contact cells. The term "inhibition of cell proliferation" is used to denote a capacity of a histone deacetylase inhibitor to retard the growth of cells in contact with the inhibitor, compared to cells with which there has been no contact. An evaluation of cell proliferation can be done by counting the cells that have been contacted and those that have not been contacted, using a Coulter cell counter (Coulter, Miami, FL) or a hema-cytometer. When the cells are in a solid growth (for example, a solid tumor or an organ), said evaluation of cell proliferation can be done by measuring the growth with calipers and comparing the growth size of the cells with which contact has been made with that of the cells with which no contact has been made.

Preferably, the growth of cells that have come into contact with the inhibitor is retarded by at least 50%, compared to the growth of cells that have not come into contact. More preferably, cell proliferation is 100% inhibited (i.e., the cells with which contact has been made do not increase in number). More preferably, the expression "inhibition of cell proliferation" includes a reduction in the number or size of cells with which contact has been made, as compared to cells with which no contact has been made. Therefore, a histone deacetylase inhibitor according to the invention that inhibits cell proliferation in a cell with which contact has been made can induce that the cell with which contact has been made experience growth retardation, experience a stop of growth, experience a programmed cell death (i.e., apoptosis), or experience a necrotic cell death.

The ability to inhibit cell proliferation of histone deacetylase inhibitors according to the invention allows synchronization of a population of cells that grow in a non-synchronized manner. For example, histone deacetylase inhibitors of the invention can be used to stop a population of non-neoplastic cells that grows in vitro, in the G1 or G2 phase of the cell cycle. Such synchronization allows, for example, the identification of the gene and / or gene products expressed during the G1 or G2 phase of the cell cycle. Such synchronization of cultured cells could also be useful for testing the efficacy of a new transfection protocol, in which the transfection efficiency varies and depends on the particular phase of the cell cycle of the cell to be transfected. The use of histone deacetylase inhibitors of the invention allows the synchronization of a population of cells, thus aiding in the detection of enhanced transfection efficiency.

In some preferred embodiments, the cell with which it is contacted is a neoplastic cell. The term "neoplastic cell" is used to denote a cell that shows aberrant cell growth. Preferably, aberrant cell growth of a neoplastic cell increases cell growth. A neoplastic cell can be a hyperplastic cell, a cell that shows an absence of contact growth inhibition, in vitro, a benign tumor cell that is incapable of metastasis in vivo, or a cancer cell that is capable of metastasis in vivo , and that may reappear after the withdrawal attempt. The term "tumorigenesis" is used to denote the induction of cell proliferation that leads to the development of neoplastic growth. In some embodiments, the histone deacetylase inhibitor induces cell differentiation in the cell with which it has come into contact. Therefore, a neoplastic cell, when it comes into contact with a histone deacetylase inhibitor, can be induced to differentiate, resulting in the production of a daughter cell that is phylogenetically more advanced than the cell with which it has been placed. Contact.

In some preferred embodiments, the cell with which it comes in contact is in an animal. Therefore, the invention provides the use of an inhibitor according to the present invention in the preparation of a medicament for inhibiting cell proliferation. Preferably, the animal is a mammal, more preferably a domesticated mammal. More preferably, the animal is a human being.

The term "cell proliferative disease or condition" is intended to refer to any condition characterized by aberrant cell growth, preferably abnormally increasing cell proliferation. Examples of such cell proliferative diseases or conditions include, but are not limited to, cancer, restenosis, and psoriasis. In particularly preferred embodiments, the invention provides a method for inhibiting neoplastic cell proliferation in an animal comprising administering to an animal, which has at least one neoplastic cell present in its body, a therapeutically effective amount of a histone deacetylase inhibitor of the invention.

Thus, the invention provides the use of an inhibitor according to the present invention in the preparation of a medicament for treating a fungal disease or infection. Preferably, the animal is a mammal, more preferably a human being. Preferably, the histone deacetylase inhibitor used in accordance with this embodiment of the invention inhibits a fungal histone deacetylase to a greater extent that inhibits mammalian histone deacetylases, particularly human histone deacetylases.

The term "therapeutically effective amount" is intended to denote a dosage sufficient to cause inhibition of histone deacetylase activity in the subject's cells, or a dosage sufficient to inhibit cell proliferation or induce cell differentiation in the subject. Administration may be by any route, including, without limitation, parenteral, oral, sublingual, transdermal, topical, intranasal, intratracheal, or intrarectal. In certain particularly preferred embodiments, the compounds of the invention are administered intravenously in a hospital setting. In certain other preferred embodiments, administration may preferably be orally.

When administered systemically, the histone deacetylase inhibitor is preferably administered at a dosage sufficient to achieve a blood level of the inhibitor of about 0.01 M to about 100 M, more preferably about 0.05 M to about 50 M, even more preferably from about 0.1 M to about 25 M, and most preferably from about 0.5 M to about 25 M. For localized administration, concentrations much lower than these can be effective, and concentrations much can be tolerated greater. One skilled in the art will appreciate that the dosage of histone deacetylase inhibitor necessary to produce a therapeutic effect can vary considerably depending on the tissue, organ, or the particular animal or patient to be treated.

In certain preferred embodiments of the present invention, the inhibitor is for use with an antisense oligonucleotide that inhibits the expression of a histone deacetylase. The combined use of a nucleic acid level inhibitor (i.e. antisense oligonucleotide) and a protein level inhibitor (i.e. histone deacetylase enzyme activity inhibitor) results in an improved inhibitory effect, thereby reducing the amounts of the required inhibitors, obtaining a given inhibitory effect, as a comparison with the necessary amounts when any of them is used individually. The antisense oligonucleotides according to this aspect of the invention are complementary to the double stranded RNA or DNA regions encoding HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC -7, and / or HDAC-8.

For the purposes of the invention, the term "oligonucleotide" includes polymers of two or more deoxyribonucleosides, ribonucleosides, or 2'-O-substituted ribonucleoside moieties, or any combination thereof. Preferably, said oligo-nucleotides have from about 6 to about 100 nucleoside residues, more preferably from about 8 to about 50 nucleoside residues, and even more preferably from about 12 to about 30 nucleoside residues. The nucleoside moieties can be coupled to each other by any of the numerous known internucleoside junctions. Such internucleoside linkages include, without limitation, phosphorothioate, phosphorodithioate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboxymethyl ester, acetamidate, carbamate, thioether, bridge linked phosphoramidate, interlinked phosphonium linked by phosphonate of sulfone. In certain preferred embodiments, these internucleoside linkages may be phosphodiester, phosphotriester, phosphorothioate, or phosphoramidate linkages, or combinations thereof. The term "oligonucleotide" also includes polymers that have chemically modified bases or sugars and / or that have additional substituents including, without limitation, lipophilic groups, intercalating agents, diamines and adamantane. For the purposes of the invention the term "2'-O-substituted" means replacement of the 2'-position of the pentose moiety with a lower -O-alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with a group -O-aryl or allyl having 2-6 carbon atoms, wherein said alkyl, aryl or allyl group may be unsubstituted or may be substituted, for example, with halo, hydroxy, trifluoromethyl, cyano, nitro, acyl groups , acyloxy, alkoxy, carboxyl, carbalkoxy, or amino; or said 2 'substitution may be with a hydroxy group (to produce a ribonucleoside), an amino or halo group, but not with a 2' group

H. The term "oligonucleotide" also includes bound nucleic acid and peptide nucleic acid.

Particularly preferred antisense oligonucleotides used in this aspect of the invention include chimeric oligonucleotides and hybrid oligonucleotides.

For the purposes of the invention, a "chimeric oligonucleotide" refers to an oligonucleotide having more than one type of internucleoside binding. A preferred example of said chimeric oligonucleotide is a chimeric oligonucleotide comprising a region of phosphorothioate, phosphodiester or phosphorodithioate, which preferably comprises from about 2 to about 12 nucleotides, and an alkyl phosphonate or alkylphosphonothioate region (see, for example, Pederson et al. Patents of the United States No. 5,635,377 and 5,366,878). Preferably, said chimeric oligonucleotides contain at least three consecutive internucleoside linkages, selected from phosphodiester and phosphorothioate linkages, or combinations thereof.

For the purposes of the invention, a "hybrid oligonucleotide" refers to an oligonucleotide having more than one type of nucleoside. A preferred example of said hybrid oligonucleotide comprises a ribonucleotide or region of

2'-O-substituted ribonucleotide, which preferably comprises from about 2 to about 12 2'-O-substituted nucleotides, and a deoxyribonucleotide region. Preferably, said hybrid oligonucleotide will contain at least three consecutive deoxyribonucleosides and will also contain ribonucleosides, 2'-O-substituted ribonucleosides, or combinations thereof (see, for example, Metelev and Agrawal, U.S. Patent No. 5,652,355).

The exact nucleotide sequence and chemical structure of an antisense oligonucleotide used in the invention may vary, as long as the oligonucleotide maintains its ability to inhibit the expression of the gene of interest. This is easily determined by testing whether the particular antisense oligonucleotide is active, quantifying the mRNA encoding a gene product, or in a Western blot analysis assay for the gene product,

or in an activity assay for an enzymatically active gene product, or in a growth assay of

Soft agar, or in a reporter gene construction assay, or an in vivo tumor growth assay, all of which are described in detail herein, or in Ramchandani et al. (1997) Proc. Natl Acad. Sci. USA 94: 684-689.

The antisense oligonucleotides used in the invention can conveniently be synthesized on a suitable solid support, using well known chemical approaches, including H-phosphonate chemistry, chemistry of

20 phosphoramidite, or a combination of H-phosphonate chemistry and phosphoramidite chemistry (ie, Hphosphonate chemistry for some cycles and phosphoramidite chemistry for the other cycles). Suitable solid supports include any of the conventional solid supports used for solid phase oligonucleotide synthesis, such as controlled pore size glass (CPG) (see, for example, Pon, RT (1993) Methods in Molec. Biol. 20 : 465-496).

Particularly, preferred oligonucleotides have nucleotide sequences of about 13 to about 35 nucleotides, which include the nucleotide sequences shown in Tables 1-3. Other particularly preferred additional oligonucleotides have nucleotide sequences of about 15 to about 26 nucleotides of the nucleotide sequences shown in Tables 1-3.

Table 1 Table 2

SEQ ID NO.

SEQUENCE DIANA (**)

one

5'-GAG ACA GCA GCA CCA GCG GG -3 ' 17-36

2

5'-ATG ACC GAG TGG GAG ACA GC-3 ' 21-49

3

5'-GGA TGA CCG AGT GGG AGA CA-3 ' 31-50

4

5'-CAG GAT GAC CGA GTG GGA GA -3 ' 33-52

5

5'-TGT GTT CTC AGG ATG ACC GA-3 ' 41-60

6

5'-GAG TGA CAG AGA CGC TCA GG-3 ' 62-81

7

5'-TTC TGG CTT CTC CTC CTT GG-3 ' 1504-1523

8

5'-CTT GAC CTC CTC CTT GAC CC-3 ' 1531-1550

9

5'-GGA AGC CAG AGC TGG AGA GG-3 ' 1565-1584

10

5'-GAA ACG TGA GGG ACT CAG CA-3 ' 1585-1604

eleven

5'-CCG TCG TAG TAG TAA CAG ACT TT-3 ' 138-160

12

5'-TGT CCA TAA TAG TAA TTT CCA A-3 ' 166-187

13

5'-CAGCAAATTATGAGTCATGCGGATTC-3 ' 211-236

(**) The reference number of the target is in accordance with HDAC-1, GenBank Reference Number U50079.

SEQ ID NO.

SEQUENCE DIANA (***)

14

5'-CTC CTT GAC TGT ACG CCA TG-3 ' 1-20

fifteen

5'-TGC TGC TGC TGC TGC TGC CG-3 ' 121-141

16

5'-CCT CCT GCT GCT GCT GCT GC-3 ' 132-152

17

5'-CCG TCG TAG TAG TAG CAG ACT TT-3 ' 138-160

18

5'-TGT CCA TAA TAA TAA TTT CCA A-3 ' 166-187

19

5'-CAG CAA GTTATG GGT CAT GCG GAT TC-3 ' 211-236

twenty

5'-GGT TCC TTT GGT ATC TGT TT-3 ' 1605-1625

(***) The reference number of the target is in accordance with HDAC-2, GenBank Reference Number U31814.

Table 3

SEQ ID NO.

SEQUENCE DIANA (***)

twenty-one

5'-GCTGCCTGCCGTGCCCACCC-3 ' 514-533

(***) the reference number of the target is in accordance with HDAC-4

The following examples are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.

Example 21:

N-Hydroxy-4- (3-oxo-3-phenylpropenyl) -benzamide (45) (a compound not claimed)

image 1

10 Stage 1: 4- (3-oxo-3-phenylpropenyl) -benzoic acid (43)

Sodium methoxide (1.8 g, 33.3 mmol) was added to a stirred suspension of 4-carboxybenzaldehyde (2.5 g, 16.6 mmol) and acetophenone (20 g ul, 16.6 mmol) in methanol (50 ml) at room temperature. The mixture was stirred at room temperature for 16 hours, and half the volume of methanol was removed under reduced pressure. The mixture was poured into 1M HCl (50 ml) (until pH = 2) and ethyl acetate was added. The separated aqueous phase was extracted with ethyl acetate.

15 (3 x 30 ml), dried (MgSO, anh.), Filtered and evaporated. The residue was triturated with dichloromethane-hexanes (1: 1) to give 3 g of 43 (72% yield).

1H NMR (300 MHz, CDCl,); ,50 7.50-7.87 (m, 7 H), 8.04 (d, 2 H, J = 8 Hz), 8.16 (d, 2 H, J = 8 Hz)

Stage 2: 4- (3-Oxo-3-phenylpropenyl) -N- (O-tetrahydropyranyl) -benzamide (44)

The carboxylic acid 43 (260 mg, 1.0 mmol) was dissolved in anhydrous CH2Cl2 (10 ml) and DCC (256 mg, 1.2 mmol) was added followed by NH2OTHP (145 mg, 1.2 mmol). The mixture was allowed to stir at room temperature for 2

h. Sat NH4Cl was added, and extracted with EtOAc. The organic phase was dried over MgSO4, filtered and the solvent evaporated in vacuo. (Purification by column chromatography using 1% MeOH / CH2Cl2 gave the title compound, which was used directly in the next step.

Stage 3: N-Hydroxy-4- (3-oxo-3-phenylpropenyl) -benzamide (45)

Protected hydroxamic acid 44 (234 mg, 0.67 mmol) was dissolved in MeOH (7 ml) and then CSA (31 mg, 0.13 mmol) was added. The mixture was allowed to stir at reflux for 2 hours or until the reaction was completed by TLC. 1N HCl was added, extracted with EtOAc, the organic phase was dried over anhydrous MgSO4 and filtered. The solvent was evaporated in vacuo. Purification by column chromatography using 5% MeOH / CH2Cl2, gave the title compound. 1 H NMR (300 MHz, DMSO-d6),  7.53-8.20 (m, 11 H); 9.12 (s a, 1 H); 11.35 (s at, 1 H)

Example 22:

N-Hydroxy-4- (3-oxo-3-phenylpropyl) -benzamide (50) (a compound not claimed)

image 1

10 Stage 1: 4- (3-Oxo-phenylpropenyl) -methylbenzoate (46)

To 4-carbomethoxybenzaldehyde (79 mg, 0.48 mmol) and acetophenone (56 ml, 0.48 mmol) in anhydrous methanol (1.6 ml), pure sodium methoxide (26 mg, 0.48 mmol) was added. The mixture was stirred at room temperature overnight and then heated at reflux for 1 hour, cooled to room temperature and 1N HCl and EtOAc were added. The phases were separated and the organic phase was dried over anhydrous MgSO4 and filtered. The solvent was evaporated in vacuo

15 giving a yellow solid, which was recrystallized from acetonitrile / water to give a pale yellow crystalline solid.

1H NMR (300 MHz, CDCl3); ,95 3.95 (s, 3 H), 7.50-8.12 (m, 11 H)

Stage 2: 4- (3-Oxo-3-phenylpropyl) -methylbenzoate (47)

The aromatic enone 46 (321 mg, 1.20 mmol) was dissolved in anhydrous THF (6 ml) and anhydrous MeOH (6 ml). They were added

20 2 teaspoons of 10% Pd on activated C, placed under a hydrogen atmosphere and allowed to stir for 2 hours at room temperature. It was purged with nitrogen, filtered through Celite and the solvent removed by evaporation in vacuo. The benzyl alcohol is reoxidized to the ketone by the following procedure. The crude product was taken up again in anhydrous CH2Cl2 (10 ml), with 3 Å molecular sieves, TPAP (1 teaspoon) was added followed by NMO (212 mg, 1.8 mmol). It was stirred at room temperature for 30 minutes and filtered through a

25 bed of silica gel. The solvent was evaporated in vacuo and purified by column chromatography using 10% EtOAc / Hexane.

1H NMR (300 MHz, CDCl3); ,1 3.14 (t, 2 H), 3.34 (t, 2 H), 3.90 (s, 3 H), 7.30-7.60 (m, 6 H), 7.92-7 , 99 (m, 4 H).

Stage 3: 4- (3-Oxo-3-phenylpropyl) -benzoic acid (48)

To a solution of methyl ester 47 (195 mg, 0.73 mmol) in water / THF (1: 1, 0.07 M) was added LiOH (46 mg, 1.1

30 mmol). The resulting solution was stirred overnight at room temperature or until no starting material was detected by TLC. 1N HCl was added and the solution was extracted with EtOAc and the organic phase was dried over anhydrous MgSO4. Filtration and evaporation of the solvent in vacuo followed by purification by column chromatography using 10% MeOH / CH2Cl2 gave the title compound. 1H NMR (310 MHz, CDCl4); ,1 3.16 (t, 2 H), 3.36 (t, 2 H), 7.33-7.60 (m, 5 H), 7.93-806 (m, 4 H).

Step 4: N-hydroxyl-4- (3-oxo-3-phenylpropyl) -benzamide (50)

Following the procedure described in Example 21, Steps 2-3, but substituting carboxylic acid 4 for compound 48, the title compound was obtained.

1H NMR (300 MHz, DMSO-d6);  2.97 (t, 2 H), 3.38 (t, 2 H), 7.34 (d, 2 H, J = 8 Hz), 7.45-7.70 (m, 5 H), 7.96 (dd, 2 H, J = 8 Hz, 1 Hz), 11.14 (sa, 1 H)

image2

Stage 1: 4-Carboxy-N- (O-tetrahydropyranyl) -benzamide (51)

Hydroxylamine-O-THP (3.9 g, 33.2 mmol) was added to a suspension of 4-formylbenzoic acid (4.2 g, 27.7 mmol) and DCC (6.8 g, 33.2 mmol) in dichloromethane (200 ml). The mixture was stirred at room temperature overnight and quenched with saturated ammonium chloride. The separated aqueous phase was extracted with ethyl acetate (3 x 100 ml) and

The combined organic phases were washed with brine, dried (MgSO4 anh.), Filtered and evaporated. Flash chromatography of the residue (10% methanol in CH2Cl2), gave (51).

1H NMR (300 MHz, CDCl2);  ppm. 10.04 (s, 1 H), 8.95 (s, 1 H), 7.99 (d, 2 H, J = 7.0 Hz), 7.93 (d, 2 H, J = 7, 0 Hz), 5.1 (s, 1 H), 3.60 (m, 2 H), 1.60 (m, 6 H)

Stage 2: 4- (3-Oxo-3-phenyl-1-hydroxypropyl) -N- (O-tetrahydropyranyl) -benzamide (52)

N-BuLi (1.4 M / hexane, 1.6 ml, 2.2 mmol) was added to a solution at 0 ° C of diisopropylamine (337 ml, 2.4 mmol) in anhydrous THF (15 ml). It was stirred at 0 ° C 10 minutes, then cooled to -78 ° C. Acetophenone was added, then stirred 30 minutes at -78 ° C. It was cannulated in a solution at -78 ° C of aldehyde 9 (50 mg, 2.0 mmol) in anhydrous THF (10 ml). It was stirred 3 hours at -78 ° C and then NH4Cl was added. It was heated to room temperature, extracted with EtOAc, dried over MgSO4, filtered and the solvent evaporated in vacuo. HPLC purification

CH4CN: H2O: 0.1% TFA; 10-95% gave the title compound 52.

Stage 3: N-Hydroxy-4- (3-oxo-3-phenyl-1-hydroxypropyl) -benzamide (53)

Following the same procedure as described in Example 21, Step 3, but substituting compound 44 for compound 52, the title compound was obtained.

1H NMR (300 MHz, DMSO-d6); ,20 3.20 (dd, 1 H, J = 4 Hz, J = 16 Hz), 3.42 (dd, 1 H, J = 16 Hz, 8 Hz), 5.20 (m, 1 25 H), 7.44-8.18 (m, 9 H), 11.15 (sa, 1 H), 11.32 (sa, 1 H)

Example 24:

N-Hydroxy-4- (3-phenylpropyl) -benzamide (56) (a compound not claimed) Stage 1: 4- (3-phenylpropenyl) -benzoic acid / 4- (3-phenyl-2-propenyl) -benzoic acid (54)

image 1

Allylbenzene (255 ml, 1.9 mmol), 4-bromobenzoic acid (523 mg, 2.6 mmol), Et3N (0.91 ml, 6.5 mmol), palladium (II) acetate (16 mg, 0.052 mmol ), triphenylphosphine (60 mg, 0.21 mmol) and acetonitrile (5 ml) were stirred at reflux overnight in a round bottom flask. 1N HCl was added, extracted with EtOAc, the organic phase was dried over

Anhydrous MgSO4 was filtered, the solvent was evaporated in vacuo. Purification by column chromatography using 10% MeOH / CH2Cl2 afforded 90 mg (14%) of a mixture of two 54 regioisomers. The mixture was then subjected to hydrogenation without further characterization.

Stage 2: 4- (3-Phenylpropyl) -benzoic acid (55)

A mixture of regioisomeric olefins 54 (100 mg, 0.42 mmol) and 10% Pd on C (10 mg) in methanol (4 ml) is

10 stirred vigorously under H2 atmosphere (96.5 kPa). The mixture was stirred for 2 hours at room temperature, filtered through Celite and evaporated to give 55 as an oil. Flash chromatography of the residue gave 55 (88 mg, 88%).

1H NMR (300 MHz, CDCl3);  ppm 8.10 (d, 2 H, J = 8.0 Hz), 7.35 (m, 7 H), 2.73 (m, 4 H), 2.00 (m, 2 H)

Stage 3: N-Hydroxy-4- (3-phenylpropyl) -benzamide (56)

Following the same procedure as described in Example 21, Steps 2-3, but substituting compound 43 for compound 55, the title compound was obtained as a beige solid. (24 mg, 26% yield)

1H NMR (300 MHz, CD3OD);  (ppm) 7.63 (d, 2 H, J = 8.0 Hz); 7-38-7.05 (m, 7 H), 2.63 (m, 4 H), 1.91 (m, 2 H)

Example 25:

N-Hydroxy-4- (4-phenylbutyl) -benzamide (61) (a compound not claimed)

image 1

Stage 1: 4- (1-Butenyl-4-phenyl) -benzoic acid / 4- (2-butenyl-4-phenyl) -benzoic acid (57/58)

Under a nitrogen atmosphere, in a 25 ml round bottom flask, 4-phenyl-1-butene (568 ml, 3.8 mmol), 4-bromobenzoic acid (634 mg, 3.2 mmol), tris were mixed (dibenzylideneacetone) dipaladium (0) (87 mg, 0.1 mmol), tri-o25 tolylphosphine (58 mg 0.2 mmol), triethylamine (1.1 ml, 7.9 mmol) in N, N-dimethylformamide (7 ml, 0.5 M solution). The mixture was stirred for 22 hours at 100 ° C. Then, the resulting suspension was cooled to room temperature, filtered through Celite and rinsed with ethyl acetate. The filtrate was acidified with 1 N HCl, the phases were separated and the aqueous phase was extracted with ethyl acetate. The combined organic phases were washed with water and brine, dried over MgSO4, filtered and concentrated. The resulting solid was triturated with hexane: dichloromethane (9: 1)

30 giving 367 mg (46%) of a beige solid 57/58. 1H NMR (300 MHz, (CD3) 2CO):  (ppm) 2.50-2.60 (m, 2 H), 2.80 (t, 2 H, J = 9.0 Hz), 6, 40-6.50 (m, 2 H), 7.12-7.35 (m, 5 H), 7.41 (d, 2 H, J = 9.0 Hz), 7.92 (d, 2 H, J = 9.0 Hz).

Stage 2: 4- (4-phenylbutyl) -benzoic acid (59)

Following the procedure described in Example 24, Step 2, but substituting compound 54 for compounds 57/58, the title compound was obtained as a white solid in a yield of 92%.

1H NMR (300 MHz, CD3OD);  (ppm) 1.60-1.75 (m, 4 H), 2.65 (t, 2 H, J = 9.0 Hz), 2.72 (t, 2 H, J = 9.0 Hz ), 7,127.30 (m, 5 H), 7.33 (d, 2 H, J = 9.0 Hz), 7.96 (d, 2 H, J = 9.0 Hz)

Stage 3: 4- (4-Phenylbutyl) -N- (O-tetrahydropyranyl) -benzamide (60)

Under a nitrogen atmosphere, in a 25 ml round bottom flask, to 4- (4-phenylbutyl) benzoic acid 59 (341 mg, 1.3 40 mmol) in 5 ml of N, N-dimethylformamide (0.3 M solution) 1- (3-Dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (308 mg, 1.6 mmol) and 1-hydroxybenzotriazole hydrate (272 mg, 2.0 mmol) were added at room temperature. The mixture was stirred for 30 minutes and then 2- (tetrahydropyranyl) hydroxylamine (235 mg, 2.0 mmol) was added and the mixture was stirred for 4 days. The N, N-dimethylformamide was removed in vacuo, the resulting oil was dissolved in ethyl acetate, washed with water and brine, dried over MgSO4, filtered and concentrated giving a yield.

5 of 95% of the crude title compound 60. 1H NMR (300 MHz, CD3OD);  (ppm) 1.50-1.75 (m, 10 H), 2.65 (t, 2 H, J = 9.0 Hz), 2.72 (t, 2 H, J = 9.0 Hz ), 3.51 (d, 1H, J = 15 Hz), 4.05 (t, 1H, J = 15 Hz), 5.05 (s, 1 H), 7.10-7.35 (m, 7 H), 7.75 (d, 2 H, J = 9.0 Hz), 10.60 (s, 1 H)

Stage 4: N-Hydroxy-4- (4-phenylbutyl) -benzamide (61)

Under nitrogen, to the crude oil in a 25 ml round bottom flask, 5 ml of

10 methyl alcohol (0.3 M solution) and camphorsulfonic acid (333 mg, L4 mmol). The mixture was stirred for 2 hours at room temperature. The methyl alcohol was removed in vacuo without heating and the resulting oil was purified by flash chromatography eluting with methyl alcohol and dichloromethane (1:19). The solid was eluted with hexane: dichloroamethane (9: 1) giving 212 mg (59%) of a beige solid 61.

1H NMR (300 MHz, (CD3) 2CO):  1.66 (m, 4 H), 2.65 (t, 2 H, J = 7.2 Hz), 2.70 (t, 2 H, J = 7.1 Hz), 7.15-7.31 (m, 7 15 H), 7.75 (d, 2 H, J = 7.8 Hz), 8.18 (wide s, 1 H) , 10.68 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  31.6 (t), 31.8 (t), 36.1 (t), 36.2 (t), 2 X 126.4 ( d), 127.8 (d), 2 X 129.1 (d), 2 X 129.2 (d), 2 X 129.3 (d), 130.6 (s), 143.3 (s) , 147.3 (s), 165.9 (s).

Example 26:

N-Hydroxy-3- (3-phenylpropyl) -benzamide (64) (a compound not claimed)

20 Stage 1: 3- (3-phenylpropenyl) -benzoic acid (62)

Following the same procedure as described in Example 24, step 1, but substituting 3-bromobenzoic acid for 4-bromobenzoic acid, the title compound was obtained as a mixture of olefins. The mixture was taken to the next stage without purification.

1H NMR (300 MHz, CDCl2);  (ppm); 3.6 (dd, 2 H, CH2); 6.4 (dd, 2 vinyl H.); 7.0-7.5 (m, 8 H, CHAr); 8.0 (s, 1H, 25 CHAr)

Stage 2: 3- (3-Phenylpropyl) -benzoic acid (63)

Following the same procedure as described in Example 24, Step 2, but substituting compound 54 for compound 62, the compound was obtained in a yield of 52% and was taken to the next stage without further purification.

1 H NMR (300 MHz, CDCl3);  (ppm); 2.0 (m, 2 H, CH2); 2.7 (m, 4 H, 2CH2); 7.0-7.4 (m, 8 H, CHAr); 8.0 (s, 1 H, CHAr)

Stage 3: N-Hydroxy-3- (3-phenylpropyl) -benzamide (64)

Following the procedure described in Example 25, Step 3-4, but substituting compound 59 for compound 63, the title compound was obtained. Purification by flash chromatography using

CH2Cl2: MeOH (9.5: 0.5) gave compound 64 in 20% yield.

1H NMR (300 MHz, DMSO-d6);  1.8 (m, 2 H, CH2); 2-8 (m, 4 H, CH2); 7.0-7.4 (m, 7 H, CHAr); 7.6 (s, CHAr); 9.0 (s, NH); 11.2 (s, OH)

Example 27:

N-Hydroxy-3- (2-phenylethyl) -benzamide (68) (a compound not claimed) Stage 1: 3- (2-phenyletenyl) -benzoic acid (65/66)

image 1

A 1.0 M solution of lithium bis (trimethylsilyl) amide (3.3 ml, 3.3 mmol) in THF was added to a stirred suspension of benzyltriphenylphosphonium bromide (1.44 g, 3.6 mmol) in THF (35 ml) at 0 ° C. The resulting orange solution was added by cannula to a mixture of 3-carboxybenzaldehyde (500 mg, 3-3 mmol) and lithium bis (trimethylsilyl) amide (3.3 ml, 3.3 mmol) in THF (10 ml) The mixture was stirred overnight at room temperature. Be

5 added a 1 N solution of HCl (75 ml) and ethyl acetate (75 ml) and the separated aqueous phase was extracted with ethyl acetate (3 x 50 ml), dried (MgSO, anh.), Filtered and It vanished. The residue was purified by HPLC (CH3CN: H2O 10:95, 0.1% TFA) to give 130 mg of the title compound (17%)

1H NMR (300 MHz, CDCl3);  (ppm) mixture E: Z (1: 1) 8.22 (s, 1 H), 7.98 (s, 1 H), 7.90-7.10 (m, 16 H), 6.70 (d, 1 H, J = 15.0 Hz), 6.62 (d, 1H, J = 15.0 Hz)

10 Stage 2: 3- (2-Phenylethyl) -benzoic acid (67)

Following the same procedure as described in Example 24, Step 2, but substituting compound 54 for compounds 65/66, the title compound was obtained quantitatively.

1H NMR (300 MHz, CDCl3);  (ppm) 2.98 (m, 4 H); 7.30 (m, 7 H); 7.99 (m, 2 H)

Stage 3: N-Hydroxy-3- (2-phenyletin-benzamide (68)

Following the same procedure as described in Example 25, Step 3 and 4, but substituting compound 59 for compound 67, the title compound was obtained in 22% yield.

1H NMR (300 MHz, DMSO-d6);  (ppm) 2.82 (s, 4 H); 7.03-7.08 (m, 8 H); 7.62 (s, 1 H); 8.98 (s at, 1 H); 11.15 (s a, 1 H)

Example 28:

20 N-Hydroxy-4- (2-thiophenyl) -ethyl benzamide (70) (a compound not claimed)

image 1

Stage 1: 4- (2-Thiophenyl) -ethyl benzoic acid (69)

According to the published procedure (Gareau et al., Tet. Lett., 1994, 1837), under a nitrogen atmosphere in a 50 ml round bottom flask, containing 4-vinyl-benzoic acid (1.0 g, 6.75 mmol), in 10 ml of benzene

25 (0.7 M) benzenethiol (797 ml, 7.76 mmol) was added followed by VAZO ™ (Aldrich Chemical Company, 495 mg, 2.02 mmol). The mixture was stirred for 12 hours at reflux. The resulting solution was cooled to room temperature and the solvent evaporated in vacuo. The solid was purified by trituration using hexane and dichloromethane to give 1.94 g (85%) of white solid.

1H NMR (300 MHz, CDCl3):  3.01 (t, 2 H, J = 8.4 Hz), 3.28 (dd, 2 H, J = 7.2, 7.8 Hz), 7 , 21 (tt, 1 H, J = 1.2, 7.2 30 Hz), 7.34 (t, 2 H, J = 8.1 Hz), 7.38-7.43 (m, 1 H ), 7.41 (d, 2 H, J = 8.4 Hz), 7.97 (d, 2 H, J = 8.1 Hz).

Stage 2: N-Hydroxy-4- (2-thiophenyl) -ethyl benzamide (70)

Under a nitrogen atmosphere, in a 50 ml round bottom flask containing 4- (2-thiophenyl) -ethyl benzoic acid (600 mg, 2.32 mmol) in 12 ml of N, N-dimethylformamide (0.2 M ) 1- (3-Dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride (579 mg, 3.02 mmol) and 1-hydroxybenzotriazole hydrate (377 mg, 2.79 mmol) were added at room temperature. The mixture was stirred 30 minutes, then hydroxylamine hydrochloride (242 mg, 3.48 mmol) and tri-ethylamine (971 ml, 6.97 mmol) were added and the mixture was stirred for 12 hours at 50 ° C. The N, N-dimethylformamide was removed in vacuo and the resulting oil was dissolved in ethyl acetate, washed with water, saturated sodium hydrogen carbonate solution, water and brine. The organic phase was dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The crude solid was purified by trituration using hexane and dichloromethane to give 450 mg (71%) of a beige solid. RP-HPLC (Hewlett-Packard 1100, column C18 HP 4.6 x 250 mm, flow 1 ml / min, CH3CN 10-95% / H2O in 42 min with 0.1% TFA); Purity: 95.8% (220 nm), 93.2% (254 nm).

1H NMR (300.072 MHz, (CD3) 2CO):  2.98 (t, 2 H, J = 7.2 Hz), 3.26 (dd, 2 H, J = 6.6, 8.4 Hz ), 7.21 (tt, 1 H, J = 1.5, 6.9 Hz), 7.31-7.42 (m, 6 H), 7.77 (d, 2 H, J = 9, 3 Hz), 8.08 (wide s, 1 H), 10.69 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  34.8 (t), 35.9 (t), 126.7 (d), 127.9 (d), 2 X 129.6 ( d), 2 X 129.7 (d), 2 X 129.9 (d), 131.3 (s), 137.3 (s), 145.0 (s).

Elemental Analysis; Calc. For C15H15O2NS x 0.1 H2O:% C = 75.31,% H = 7.14,% N = 5.17. Found:% C = 75.2  0.1,% H = 7.41  0.07,% N = 5.17  0.01.

N-Hydroxy-4- (2-benzenesulfonyl) -ethyl benzamide (73)

Stage 1: 4- (2-Benzenesulfonyl) -ethyl benzoic acid (72)

Under a nitrogen atmosphere in a 100 ml round bottom flask containing 4- (2-thiophenyl) -ethyl benzoic acid

(69) (600 mg, 2.32 mmol) in 20 ml of dichloromethane (0.1 M) at 0 ° C was added portionwise 3-chloroperbenzoic acid (Aldrich Chemical Co., 57-86% pure solid, 2 g, 6.97 mmol), as described by Nicolaou et al., J. Am. Chem. Soc., 114: 8897 (1992). The mixture was allowed to reach room temperature and stirred for 1 hour. Dimethyl sulfide (5 ml) was added, the mixture was diluted in dichloromethane and washed 3 times with water. The organic phase was dried over anhydrous magnesium sulfate, filtered and the solvent evaporated in vacuo to give 3 g of white solid. This mixture of 3-chlorobenzoic acid and the desired 4- (2-benzenesulfonyl) -ethyl benzoic acid was placed in a 125 ml Erlenmeyer flask, dissolved in 30 ml of dichloromethane and treated with an excess of freshly prepared diazomethane solution in diethyl ether (0.35 M). Nitrogen was bubbled to remove excess diazomethane and the solvents evaporated in vacuo. The resulting solid was purified by flash chromatography, eluting with 20% ethyl acetate: 80% hexane to give 341.6 mg (48%) of the corresponding ester. Saponification of this ester was performed using the same procedure as described in Example 1, step 2, giving 312.4 mg (96%) of 4- (2-benzenesulfonyl) -ethyl benzoic acid (72) 1 H NMR ( 300 MHz, CDCl3):  3.06-3.11 (m, 2 H), 3.56-3.61 (m, 2 H), 7.37 (d, 2 H, J = 8.4 Hz ), 7.67 (tt, 2 H, J = 1.5, 7.2 Hz), 7.76 (tt, 1H, J = 1.2, 7.5 Hz), 7.93 (d, 2 H, J = 8.7 Hz), 7.97 (dd, 2 H, J = 1.8, 6.9 Hz).

Stage 2: N-Hydroxy-4- (2-benzenesulfonyl) -ethyl benzamide (73)

Following the procedure described for N-hydroxy-4- (2-thiophenyl) -ethyl benzamide, but substituting 4- (2-benzenesulfonyl) -ethyl benzoic acid for 4- (2-thiophenyl) -ethyl benzoic acid, the compound of the title in the form of a beige solid. RP-HPLC (Hewlett-Packard 1100, column C18 HP 4.6 x 250 mm, flow 1 ml / min, CH3CN 10-95% / H2O in 42 min with 0.1% TFA); Purity: 98.8% (220 nm), 97.6% (254 nm).

1H NMR (300.072 MHz, (CD3) 2CO):  2.98 (t, 2 H, J = 7.2 Hz), 3.26 (dd, 2 H, J = 6.6, 8.4 Hz ), 7.21 (tt, 1 H, J = 1.5, 6.9 Hz), 7.31-7.42 (m, 6 H), 7.77 (d, 2 H, J = 9, 3 Hz), 8.08 (wide s, 1 H), 10.69 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  25.2 (t), 34.3 (t), 55.6 (t), 128.0 (d), 2 X 128.8 ( d), 129.4 (d), 2 X 130.2 (d), 131.1 (s), 134.5 (d), 140.7 (s), 145.5 (s), 165.8 (s)

N-Hydroxy-4- (2-benzenesulfoxide) -ethyl benzamide (71)

According to the procedure described by Van Der Borght et al., J. Org. Chem., 65: 288 (2000), in a nitrogen atmosphere in a 10 ml round bottom flask containing N-hydroxy-4- (2-thiophenyl) -ethyl benzamide (70) (50 mg, 0.18 mmol) in 2 ml of methanol (0.1 M) tellurium dioxide (3 mg, 0.018 mmol) was added followed by a 35% solution in hydrogen peroxide water (32 ml, 0.36 mmol). The mixture was stirred for five days and then brine was added. The aqueous phase was extracted 3 times with ethyl acetate and the combined organic phases were dried over anhydrous magnesium sulfate, filtered and the solvent evaporated in vacuo. The resulting solid (43.3 mg) was purified by trituration using acetonitrile, giving 10 mg (20%) of a beige solid.

RP-HPLC (Hewlett-Packard 1100, column C18 HP 4.6 x 250 mm, flow 1 ml / min, CH3CN 10-95% / H2O in 42 min with 0.1% TFA); Purity: 98.8% (220 nm), 97.9% (254 nm).

1H NMR (300.072 MHz, (CD3) 2CO):  2.76-2.91 (m, 1 H), 3.00-3.29 (m, 3 H), 7.34 (d, 2 H , J = 8.4 Hz), 7.55-7.62 (m, 3 H), 7.70 (dd, 2 H, J = 1.5, 8.1 Hz), 7.76 (d, 2 H, J = 8.1 Hz), 8.08 (wide s, 1 H), 10.70 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  28.3 (t), 57.8 (t), 2 X 124.8 (d), 128.0 (d), 2 X 129, 6 (d), 2 X 130.0 (d), 131.5 (d), 144.1 (s), 145.7 (s).

image3

Stage 1: 3- (4-Bromophenyl) -propanoic acid (74)

Under nitrogen atmosphere, in a 250 ml round bottom flask, containing 4-bromocynamic acid (5.0 g,

10 22 mmol) in 45 ml of N, N-dimethylformamide (0.5 M) benzenesulfonylhydrazide (7.6 g, 44 mmol) was added. The mixture was stirred at reflux for 12 hours. The solution was cooled to room temperature, saturated aqueous ammonium chloride solution was added and the aqueous phase was extracted with ethyl acetate 3 times. The combined organic phases were washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated in vacuo. The resulting solid was purified by flash chromatography eluting with 5% methanol: 95%

Dichloromethane giving 3.66 g (73%) of a beige solid.

1H NMR (300 MHz, CDCl3):  2.66 (t, 2 H, J = 7.5 Hz), 2.91 (d, 2 H, J = 7.5 Hz), 7.08 (d , 2 H, J = 8.4 Hz), 7.41 (d, 2 H, J = 8.4 Hz).

Stage 2: N-Hydroxy-3- (4-bromophenyl) -propanamide (75)

Following a procedure analogous to that described for the preparation of 70, 1.54 g (39%) of the title compound were obtained.

1H NMR (300 MHz, CDCl3):  2.39 (t, 2 H, J = 7.8 Hz), 2.89 (d, 2 H, J = 7.2 Hz), 7.18 (d , 2 H, J = 8.1 Hz), 7.42 (d, 2 H, J = 8.7 Hz), 8.18 (wide s, 1 H), 9.98 (wide s, 1 H) .

Stage 3: N-Hydroxy-3- [4- (3-phenyl-1-propenyl) -phenyl] -propanamide and N-Hydroxy-3- [4- (3-phenyl-2-propenyl) -phenyl] -propanamide

(76)

Following a procedure analogous to that described in Example 25, step 1, but replacing 4-bromobenzoic acid with N-hydroxy-3- (4-bromophenyl) -propanamide (75) (250 mg, 1.02 mmol) and 4- phenyl-1-butene (163 ml, 1.2 mmol) per allyl benzene, yielding 155.4 mg (54%) of the title compounds mixed.

1H NMR (300 MHz, CDCl3):  2.39 (m, 2 H), 2.88 (t, 2 H, J = 8.4 Hz), 3.51 (t, 2 H, J = 8 , 1 Hz), 6.32-6.53 (m, 2 H), 7.14-7.44 (m, 9 H), 8.60 (wide s, 1 H), 10.04 (wide s , 1 HOUR).

30 Stage 4: N-Hydroxy-3 [4- (3-phenylpropyl) -phenyl] -propanamide (77)

Following a procedure analogous to that described in Example 24, step 2, but replacing olefins 54 (155 mg, 0.55 mmol) with the mixture of N-hydroxy-3- [4- (3-phenyl-1-propenyl) -phenyl] -propanamide and N-hydroxy-3- [4- (3-phenyl-2-propenyl) -phenyl] -propanamide, 155.4 mg (99%) of the title compound were obtained.

RP-HPLC: (Hewlett-Packard 1100, column C18 HP 4.6 x 250 mm, flow 1 ml / min, CH3CN 10-95% / H2O in 42 min 35 with 0.1% TFA); Purity: 99.9% (220 nm) (2 peaks but the same compound, tested by LCMS, 99.9% (254 nm).

1H NMR (300.072 MHz, (CD3) 2CO):  1.91 (quintuple, 2 H, J = 8.1 Hz), 2.38 (t, 2 H, J = 7.8 Hz), 2, 61 (c, 4 H, J = 9.6 Hz), 2.87 (t, 2 H, J = 7.2 Hz), 7.12-7.29 (m, 9 H), 8.42 ( s wide, 1 H), 10.01 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  26.3 (t), 28.7 (t), 29.8 (t), 30.3 (t), 30.7 (t) , 121.1 (d), 3 X 123.7 (d), 3 X 123.8 (d), 133.9 (s), 133.4 (s), 137.8 (s), 164.9 (s)

Elemental Analysis; Calc. For C18H21O2N x 0.1 H2O:% C = 75.81,% H = 7.49,% N = 4.91. Found:% C = 75.7  0.3,% H = 7.54  0.02,% N = 4.85  0.03.

Example 36:

The following additional compounds were prepared by procedures analogous to those described in the previous Examples:

image 1

1H NMR (300.072 MHz, (CD3) 2CO):  1.63 (m, 4 H, J = 45 Hz), 2.37 (t, 2 H, J = 7.8 Hz), 257-266 ( m, 4 H), 2.86 (t, 2 H, J = 7.5 Hz), 7.10-7.28 (m, 9 H), 8.01 (wide s, 1 M, 9.98 (s wide, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  31.0 (t), 2 X 3L9 (t), 35.1 (t), 35.8 (t), 36.2 (t) , 126-4 (d), 2 X 129.0 (d), 2 X 129.1 (d), 2 X 129.1 (d), 129.2 (d), 138.8 (s), 141 , 2 (s), 143.4 (s), 164.1 (s).

image 1

1H NMR (300.072 MHz, (CD3) 2CO):  1.83-1.98 (m, 4 H), 2.08-2.14 (m, 2 H), 2.56-2.67 ( m, 6 H), 7.12-7.30 (m, 9 H), 9.98 (wide s, 1 H).

image 1

1H NMR (300.072 MHz, (CD3) 2CO):  1.60-1.68 (m, 4 H), 1.87 (quintuple, 2 H, J = 7.5 Hz), 2.03-2 , 14 (m, 2 H), 2.55-2.67 (m, 6 H), 7.09-7.28 (m, 9 H).

image 1

1H NMR (300.072 MHz, (CD3) 2CO):  2.37 (t, 2M, J = 7.2 Hz), 2.78-2.89 (m, 6 H), 7.13-7, 29 (m, 9 H), 7.84 (wide s, 1 H), 9.90 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  31.6 (t), 35.1 (t), 38.2 (t), 38.6 (t), 2 X 126.6 ( d), 2 X 129.1 (d), 2 X 129.2 (d), 2 X 129.3 (d), 139.4 (s), 140.4 (s), 142.8 (s) , 170.1 (s).

image 1

1H NMR (300.072 MH2, (CD3) 2CO):  1.96 (quintuple, 2 H, J = 6.0 Hz), 2.69 (t, 2 H, J = 8.0 Hz), 3, 19 (dd, 2 H, J = 6.0, 9.0 Hz), 3.38 (s, 2 H), 7.09 (d, 2 H, J = 7.5 Hz), 7.21 ( d, 2 H, J = 7.5 Hz), 7.66 (t, 2 H, J = 8.1 Hz), 7.747 (t, 1 H,

J = 6.9 Hz), 7.90 (d, 2 H, J 6.6 Hz), 10.08 (wide s, 1 H).

13C NMR (75.46 MHz, (CD3) 2CO):  25.5 (t), 34.1 (t). 39.9 (t), 55.7 (t), 2 X 128.8 (d), 130.0 (d), 2 X 130.2 (d), 134.4 (s), 139.9 ( s), 140.7 (s), 168.5 (s).

Example 40:

5 Inhibition of histone deacetylase enzyme activity

HDAC inhibitors were selected against histone deacetylase enzyme in nuclear extracts prepared from the H446 human lung microcytic cancer cell line (ATTC HTB-171) and against a cloned, expressed and purified recombinant human HDAC-1 enzyme. from a Baculovirus insect cell expression system.

10 For deacetylase assays, 20,000 cpm of the metabolically labeled acetylated histone substrate was incubated with 3H (M. Yoshida et al., J. Biol. Chem. 265 (28): 17174-17179 (1990)) with 30 mg of extract nuclear H446 or an equivalent amount of recombinant HDAC-1 cloned for 10 minutes at 37 ° C. The reaction was stopped by adding acetic acid (0.04 M, final concentration) and HCl (250 mM, final concentration). The mixture was extracted with ethyl acetate and the [3 H] -acetic acid released was quantified by scintillation counting. For studies of

In inhibition, the enzyme was pre-incubated with compounds at 4 ° C for 30 minutes before starting the enzymatic assay. IC50 values for HDAC enzyme inhibitors were determined by performing dose-response curves with individual compounds, and determining the concentration of inhibitor that produces fifty percent of the maximum inhibition.

Representative data are presented in Table 4. The first column shows the IC50 values.

20 determined against histone deacetylase in nuclear extracts of H446 cells (combined HDAC). In the second column the IC50 values determined against the recombinant human HDAC-1 enzyme (HDAC-1r) are presented. For less active compounds, the data is expressed as the percentage of inhibition at the specified concentration.

Table 4: Histone Deacetylase Inhibition

Example

 Compound Structure Combined HDAC IC50 (M) HD50-1r IC50 (M)

Ex 29

77 3 0.65

Ex 36

154 2 0.9

Ex 36

155 39% at 20 M

Ex 36

156 5 0.73

Ex 36

157 6 2-4

Ex 36

158 image 1 > 209

 174

> 20

175

> 20

Example 41:

Inhibition of Histone Deacetylase in Complete Cells

one.

Histone H4 acetylation in whole cells by immunoblotting

T24 human bladder cancer cells, which were grown in a culture, were incubated with HDAC inhibitors for 16 hours. Histones were extracted from the cells after the culture period, as described by M. Yoshida et al. (J. Biol. Chem. 265 (28): 17174-17179 (1990)). 20 mg of total histone protein was loaded into SDS / PAGE and transferred to nitrocellulose membranes. The membranes were probed with polyclonal antibodies specific for acetylated histone H-4 (Bitotech Inc. above), followed by conjugated secondary antibodies of horseradish peroxidase (Sigma). Enhanced chemiluminescence (ECL) (Amersham) detection was performed using Kodak films (Eastman Kodak). The acetylated H-4 signal was quantified by densitometry.

2.

Urea Triton Acid (AUT) gel analysis of histone acetylation.

Human cancer cells (T24, 293T or Jurkat cells), which were grown in a culture, were incubated with HDAC inhibitors for 24 h. Histones are extracted from cells as described by M. Yoshida et al. (J. Biol. Chem. 265 (28): 17174-17179 (1990)). Urea triton acid gel (AUT) electrophoresis is used for the detection of acetylated histone molecules. Histones (150 mg of total protein) are electrophoresed at 80 V for 16 h, at room temperature, as described by M. Yoshida et al., Supra. The gels are stained with Coomassie bright blue to visualize histones, dried and scanned by densitometry to quantify histone acetylation.

Example 42:

Antineoplastic Effect of Histone Deacetylase Inhibitors on In Vivo Tumor Cells

Female BALB / c mice, athymic, eight to ten weeks old (Taconic Labs, Great Barrington, NY) are injected subcutaneously in the side area with 2 x 106 preconditioned human lung carcinoma A549 cells. The preconditioning of these cells is performed with a minimum of three consecutive tumor transplants in the same strain of hairless mice. Subsequently, tumor fragments of approximately 30 mg are excised and implanted subcutaneously in mice, in the area of the left side, under Forene anesthesia (Abbott Labs, Geneve, Switzerland). When the tumors reached a volume of 100 mm3, the mice were treated intravenously, subcutaneously, or intraperitoneally by daily injection, with a solution of the histone deacetylase inhibitor in an appropriate vehicle, such as PBS, DMSO / water , or Tween 80 / water, at an initial dose of 10 mg / kg. The optimal dose of the HDAC inhibitor is established by dose response experiments, in accordance with conventional protocols. Tumor volume is calculated every second day after infusion, according to conventional procedures (for example, Meyer et al., Int. J. Cancer 43: 851-856 (1989)). Treatment with HDAC inhibitors according to the invention causes a significant reduction in tumor weight and volume compared to controls treated only with saline solution (ie, without HDAC inhibitor). In addition, histone deacetylase activity, when measured, is expected to be significantly reduced compared to controls treated with saline.

Example 43:

Synergistic Antineoplastic Effect of Histone Deacetylase Inhibitors and Histone Deacetylase Antisense Oligonucleotides on In Vivo Tumor Cells

The purpose of this example is to illustrate the ability of the histone deacetylase inhibitor of the invention and a histone deacetylase antisense oligonucleotide to synergistically inhibit tumor growth in a mammal. Preferably, the antisense oligonucleotide and the HDAC inhibitor inhibit the expression and activity of histone deacetylase itself.

As described in Example 10, mice carrying implanted A549 tumors (average volume 100 mm3) are treated daily with saline preparations containing from about 0.1 mg to about 30 mg per kg body weight of histone antisense oligonucleotide deacetylase A second group of mice is treated daily with pharmaceutically acceptable preparations containing from about 0.01 mg to about 5 mg per kg body weight of HDAC inhibitor.

Some mice received both the antisense oligonucleotide and the HDAC inhibitor. Of these mice, a group can receive the antisense oligonucleotide and HDAC inhibitor simultaneously intravenously through the tail vein. Another group may receive the antisense oligonucleotide through the tail vein, and the HDAC inhibitor subcutaneously. Another group can receive both the antisense oligonucleotide and the HDAC inhibitor subcutaneously. Groups of control mice are established analogously, which do not receive treatment (e.g., saline only), a single antisense oligonucleotide, a control compound that does not inhibit the activity of histone deacetylase, and a missing antisense oligonucleotide with a compound. of control.

Tumor volume is measured with calipers. Treatment with the antisense oligonucleotide plus the histone deacetylase protein inhibitor according to the invention causes a significant reduction in the weight and volume of the tumor with respect to the controls.

Claims (35)

1. A histone deacetylase inhibitor represented by the formula   Cy-L2-Ar-Y2- (O) NH-Z in which Cy is cycloalkyl, aryl, heteroaryl, or heterocyclyl, any of which may be optionally substituted. 5 with between one and four substituents selected from halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, arenesulfonyl, arenesulfonyl, arenesulfonyl , arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido, and wherein said aryl, heterocyclyl and heteroaryl can also be optionally substituted with one or more groups selected from (C1-C8) amino, di (C1-C6) alkyl ) Not me, (C 1 -C 6) alkylamino and (C 6 -C 10) acylamino arene, and wherein said cycloalkyl and heterocyclyl may also be optionally substituted with oxo, and wherein said heterocyclyl may also be optionally independently substituted on the nitrogen with a group selected from alkylsulfonyl, arylcarbonyl, arylsulfonyl and aralkoxycarbonyl, with the proviso that Cy is not a (spirocycloalkyl) heterocyclyl; L2 is saturated C1-C8 alkylene or C2-C8 alkenylene, the alkylene or alkenylene may be optionally 15 substituted by a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido , arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido with the proviso that L2 is not -C (O) -, and in which one of the carbon atoms of the alkylene can be optionally replaced by a heteroatomic moiety selected from the 20 group consisting of S; o S (O): o L2 is S (O) 2- (CH2) 1-4: Ar is arylene, said arylene may optionally be fused with an aryl or heteroaryl ring, or with a saturated or unsaturated cycloalkyl or heterocyclic ring; Y Y2 is a saturated straight or branched chain alkylene; Y Z is selected from the group consisting of anilinyl, pyridyl, thiadiazolyl, and -O-M, where M H or a cation Pharmaceutically acceptable, thiadiazolyl may be optionally substituted with a substituent selected from the group consisting of thiol, trifluoromethyl, amino and sulfonamido; with the proviso that when the carbon atom to which Cy is attached is substituted with oxo, then Cy and Z are not both pyridyl. 2. The inhibitor of claim 1, wherein L2 is saturated C1-C8 alkylene or C2-C8 alkenylene, wherein the Alkylene or alkenylene may be optionally substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl , alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfanamido, alkylcarbonyl, acyloxy, cyano and ureido with the proviso that L2 is not C (O) -, and in which one of the alkylene carbon atoms may be optionally replaced by rest Heteroatomic selected from the group consisting of S; bear).

3.

 The inhibitor of claim 1 or 2, wherein Y2 is C1-C6 alkylene.

Four.

 The inhibitor of claim 1 or 2, wherein Y2 is C1-C3 alkylene.

5.

 The inhibitor of claim 1 or 2, wherein Y2 is C1-C2 alkylene.

6.

 The inhibitor of any one of claims 1 to 5, wherein one or two carbons saturated in L2 are

40 substituted with a substituent independently selected from the group consisting of C1-C6 alkyl, C6-C10 aryl, amino, oxo, hydroxy, C1-C4 alkoxy, and C6-C10 aryloxy. 7. The inhibitors of any one of claims 1 to 5, wherein no carbon atom of the L2 alkylene is replaced by a heteroatomic moiety. 8. The inhibitor of any one of claims 1 to 5, wherein a carbon atom of the L2 alkylene is replaced by a heteroatomic moiety selected from the group consisting of S; bear).

9.

 The inhibitor of claim 1, wherein L2 is selected from the group consisting of -S- (CH2)., -S (O) - (CH2) n, and -S (O) 2- (CH2) n- , in which n is 1, 2, 3 or 4.

10.

 The inhibitor of any one of claims 1 to 5, wherein L2 is saturated C1-C6 alkylene.

eleven.

 The inhibitor of any one of claims 1 to 5, wherein L2 is saturated C1-C4 alkylene.

12.

 The inhibitor of any one of claims 1 to 5, wherein L2 is saturated C1-C6 alkylene substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino , acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido,

5 with the proviso that L2 is not -C (O) -. 13. The inhibitor of any one of claims 1 to 5, wherein L2 is saturated C1-C4 alkylene substituted with a group selected from oxo, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy , amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano and ureido, 10 with the proviso that L2 is not -C (O) -.

14.

 The inhibitor of any one of claims 1 to 13, wherein Z is selected from the group consisting of 2-anilinyl, 2-pyridyl, 1,3,4-thiadiazol-2-yl, and -OM, being MH or a pharmaceutically acceptable cation.

fifteen.

 The inhibitor of any one of claims 1 to 14, wherein Z is -O-M, M being hydrogen or any pharmaceutically acceptable cation.

16. The inhibitor of any one of claims 1 to 14, wherein Z is -O-M, where M is hydrogen. 17. The inhibitor of any one of claims 1 to 16, wherein Ar is unsubstituted phenylene, which may be optionally fused with an aryl or heteroaryl ring, or with a saturated cycloalkyl or heterocyclic ring or partially unsaturated. 18. The inhibitor of claim 17, wherein the phenylene is 4-phenylene. The inhibitor of any one of claims 1 to 18, wherein Cy is selected from the group consisting of phenyl, naphthyl, thienyl, benzothienyl, and quinolyl, any of which may be optionally substituted. 20. The inhibitor of claim 19, wherein the phenyl, naphthyl, thienyl, benzothienyl, or quinolyl is not substituted or substituted with one or two substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 haloalkyl , C6-C10 aryl, ar (C6-C10) (C1-C6) alkyl, halo, nitro, hydroxy, C1-C6 alkoxy, C1-C6 alkoxycarbonyl, 25 carboxy, and amino.

twenty-one.

 The inhibitor of claim 19, wherein Cy is unsubstituted phenyl.

22

 The inhibitor according to claim 1 or 2 selected from the group consisting of

2. 3.

 The inhibitor according to claim 1 selected from the group consisting of

image 1 image2 image2 24. The inhibitor according to claim 22 having the structure image2

25.

 The inhibitor according to claim 22 having the structure

26.

 The inhibitor according to claim 22 having the structure

27.

 The inhibitor according to claim 22 having the structure

image2 image2 image2 The inhibitor according to claim 22 having the structure image2 29. A pharmaceutical composition comprising a histone deacetylase inhibitor according to any of the preceding claims, and a pharmaceutically acceptable carrier, excipient, or diluent.

30

 A histone deacetylase inhibitor according to any one of claims 1 to 28 for use in the inhibition of histone deacetylase in a cell.

31.

 The inhibitor of claim 30, wherein the cell is a neoplastic cell.

32

 The inhibitor of claim 31, wherein the neoplastic cell is in an animal.

33.

 The inhibitor of claim 32, wherein the neoplastic cell is in a neoplastic growth.

3. 4.

The inhibitor of any one of claims 30 to 33, for use with an antisense oligonucleotide that inhibits the expression of a histone deacetylase.

35

 The inhibitor of any one of claims 30 to 34, for use in inhibiting cell proliferation.

36.

 The inhibitor according to any one of claims 1 to 28 for use in the treatment of a fungal disease or infection.

37. Use of an inhibitor according to any of claims 1 to 28 in the preparation of a medicament for inhibiting cell proliferation. 38. Use of an inhibitor according to any of claims 1 to 28 in the preparation of a medicament for treating a fungal disease or infection.