WO2008101186A1 - Inhibitors for hdac8 - Google Patents

Abstract

This invention discloses a new HDAC inhibitor scaffold designed to exploit a unique sub-pocket of the HDAC8 active site. These compounds are based on inspection of HDAC8 crystal structures bound to various inhibitors, which showed that the HDAC8 active site is surprisingly malleable and can accommodate inhibitor structures that are distinct from the canonical 'zinc-binding group-linker-cap group' structures of SAHA, TSA and similar HDAC inhibitors. Some of the new inhibitors based on this new scaffold are >100 fold selective for HDAC8 over other class I and class II HDACs with IC50 values < 1µM against HDAC8. The present invention provides a new type of 'linkerless' HDAC8 inhibitors and methods of treating a pathological condition using the same. Treatment of human cells with the new inhibitors of the present invention show a unique pattern of hyperacetylated proteins compared with the broad spectrum HDAC inhibitor TSA.

Description

INHIBITORS FOR HDAC8

RELATED PATENT APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application number 60/890,119, filed February 15, 2007 entitled "INHIBITORS FOR HDAC8," the disclosure of which is incorporated by reference herein in its entirety.

BACKGROUND OF THE INVENTION

[0002] Posttranslational ε-acetylation of lysine residues on histone tails is a central mechanism of transcriptional control in eukaryotes (Kornberg and Lorcj, 1999, Cell 98:285- 294; Jenuwein and AlHs, 2001, Science 293: 1074-1080; Peterson and Laniel, 2004, Curr Biol 14:R546-551). Lysine acetylation was first identified as a post-translational modification of histones, with hypoacetylation correlated with transcriptional repression and hypoacetylation with transcriptional activity (Struhl, 1998, Genes Dev 12:599-606). Patterns of lysine acetylation along regions of nucleosomes have distinct biological meanings, through recruitment of specific proteins as well as structural changes to the chromatin fiber (Hecht et al, 1995, Cell 89:583-592; Shogren-Knaak et al., 2006, Science 311 :844-847). Histone lysine acetylation patterns affect diverse cellular processes including differentiation, cellular response to stimuli and tumorigenesis. More recently, however, it has been shown that nonhistone proteins such as tubulin, and p53 are also targets of reversible lysine acetylation, suggesting that acetylation may play a much broader role in controlling cellular events akin to protein phosphorylation (North et al, 2003, MoI Cell 11 :437-444; Vaziri et al, 2001, Cell 107:149-159).

[0003] The lysine acetylation state of cellular proteins is determined by the action of histone acetyl transferase (HATs) and histone deacetylases (HDACs). At least 18 HDAC subtypes exist and they are subdivided into three families of HDACs: class I (HDACs 1, 2, 3, and 8) and class II (HDACs 4, 5, 6, 7, 9, and 10) HDACs are zinc-dependent amidohydrolases with a conserved catalytic core but differing in size, domain structure, tissue expression pattern and cellular localization (Johnstone, 2002, Nat Rev Drug Discov 1 :287- 289; Gao et al, 2002, J Biol Chem 277:25748-25755). Another HDAC, HDACI l, lies at the boundary between the two classes. Class III HDACs (Sirtuins 1-7) are NAD⁺-dependent and unrelated in sequence to classes I and II (Holbert and Marmorstein 2005, Curr Opin Struct Biol 15:673-680). HDACs catalyze the removal of acetyl groups from lysine residues near the N-termini of hi stones.

[0004] Zinc-dependent HDACs have received much attention as anticancer drug targets. Inhibitors of these enzymes show a remarkable ability to induce terminal differentiation of transformed cells, presumably by altering patterns of gene expression through influencing the acetylation state of selected histone lysine residues (Marks et al, 2004, Adv Cancer Res 91 : 137-168). HDAC inhibitors are also exceedingly useful as tools to study the biology of histone deacetylases. Indeed, determining whether a cellular process involves HDACs is readily ascertained by using potent, cell-permeable molecules available.

[0005] Most HDAC inhibitors, such as trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA) act as competitive inhibitors; they closely mimic the aliphatic acetyl-lysine substrate and deliver a hydroxamic acid or other zinc-binding group to the catalytic zinc at the bottom of a narrow active site pocket as seen in the co-crystal structures of inhibited HDLP (HDAC -like protein; Finnin et al, 1999, Nature 401 : 188-193), HDAH (HDAC-like aminohydrolase; Nielsen et al, 2005, J Mo/5/o/ 354: 107-120), and human HDAC 8 (Somoza et α/., 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-9). These inhibitors have similar structures. All have a zinc-binding group (also referred to as a "metal chelator") linked via a straight chain alkyl, vinyl or aryl linker to a polar "cap group" (also referred to as "surface recognition group") (Figure 1).

[0006] TSA, SAHA, CBHA, 5-(4-methyl-benzoylamino)-biphenyl-3,4'-dicarboxylic acid 3-dimethylamide 4'-hydroxyamide (CRA-A), and 4-dimethylamino-N-(6- hydroxycarbamoyethyl)benzamide-N-hydroxy-7-(4- dimethylaminobenzoyl)aminoheptanamide (MS-344) are nonselective or poorly selective, i.e., they active against class I and class II HDACs and induce multiple effects within cells, including cell differentiation, induction of cell cycle arrest, and suppression of tumor growth (Jung et α/., 1999, J Med Chem 42:4669-4679; Almenara et α/., 2002, Leukemia 16: 1331- 1343; Richon et α/., 1998, Proc Natl Acad Sci USA 9:3003-3007; Yoshida et α/., 1990, J Biol Chem 265: 17174-17179; Yoshida et al, 1990, J Antibiot 43: 11101-1106).

[0007] Attempts to generate isozyme-specific HDAC inhibitors focused on varying the cap group to exploit variability in the HDAC surface surrounding the active site (Sternson et al, 2001, OrgLett 3:4239-4242). Despite much effort, truly selective compounds remain difficult to find. Many are modestly selective for individual HDAC family members or a subgroups of HDACs among the ten human zinc-binding HDACs. Due to the central role of HDACs in basic biological processes and their importance as novel anticancer targets, chemical tools that help elucidate then function of HDAC isozymes are of great importance. A set of inhibitors specific for each individual HDAC family member would enable the cellular role of each to be determined as well as help uncover the mechanism of the anticancer properties of HDAC inhibitors. Further, identifying selective HDAC inhibitors may open the way for developing pharmaceutical compositions with enhanced efficacy and/or tolerability.

[0008] The invention described herein provides for the rational design of HDAC inhibitors based on a new scaffold. The newly designed HDAC inhibitors show remarkable selectivity towards human HDAC8 by targeting an active site pocket unique to this HDAC family member.

BRIEF SUMMARY OF THE INVENTION [0009] In its most general aspect, this invention discloses a new HDAC inhibitor scaffold designed to exploit a unique sub-pocket of the HDAC8 active site. These compounds are based on inspection of HDAC8 crystal structures bound to various inhibitors, which showed that the HDAC8 active site is surprisingly malleable and can accommodate inhibitor structures that are distinct from the canonical "zinc-binding group-linker-cap group" structures of SAHA, TSA and similar HDAC inhibitors. Some of the new inhibitors based on this new scaffold are >100 fold selective for HDAC8 over other class I and class II HDACs with IC50 values < 1 μM against HDAC8. The present invention provides a new type of "linkerless" HDAC8 inhibitors and methods of treating a pathological condition using the same.

[0010] In one aspect, this invention provides a compound having the formula

[0011] In one aspect, this invention provides a compound having the formula

in which Z is -NH, O or S,

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl.

[0012] In yet another aspect, this invention provides a compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, NO₂, lower alkyl carboxylate, lower alkoxy, or — CN.

[0013] In another aspect, the invention provides a compound of the formula

[0014] In a further aspect, the invention provides a compound of the formula

[0015] In another aspect, the invention provides a compound of the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN.

[0016] In a further aspect, the present invention provides a compound having the formula

[0017] In yet another aspect, the present invention provides a compound having the formula

wherein X is oxygen or sulphur.

[0018] The present invention also provides pharmaceutical compositions comprising a compound of the invention and one or more pharmaceutically acceptable excipients. In a preferred embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

and (ii) a pharmaceutically acceptable excipient or carrier.

[0019] In another embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and (ii) a pharmaceutically acceptable excipient or carrier.

[0020] In yet another embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN and (ii) a pharmaceutically acceptable excipient or carrier.

[0021] In another embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

and (ii) a pharmaceutically acceptable excipient or carrier.

[0022] In a further embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

and (ii) a pharmaceutically acceptable excipient or carrier.

[0023] In another preferred embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN and (ii) a pharmaceutically acceptable excipient or carrier. [0024] In a further preferred embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

and (ii) a pharmaceutically acceptable excipient or carrier.

[0025] In yet another preferred embodiment of the present invention, the pharmaceutical composition comprises (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

wherein X is oxygen or sulphur and (ii) a pharmaceutically acceptable excipient or carrier.

[0026] The present invention also provides a method for selectively inhibiting an activity of an HDAC8 polypeptide. In a preferred embodiment, the method comprises the step of contacting an HDAC8 polypeptide with an effective inhibiting amount of a compound having the formula:

W - X - Y

in which

W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and

Y represents a zinc-binding moiety,

or

R,

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which substituents are as defined for moiety W.

[0027] The zinc binding moiety may be a hydroxamic acid group or a hydroxamic acid derivative. [0028] In a preferred embodiment of this invention, the compound is selected from the group consisting of compounds of the formula

which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl,

R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN,

R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN,

[0029] A preferred activity of the HDAC8 polypeptide is a histone deacetylase activity or a tubulin deacetylase activity.

[0030] A preferred compound of the present invention inhibits the HDAC8 polypeptide with an efficiency of greater than 50 fold over HDACl or HDAC6

[0031] A preferred compound of the present invention inhibits the HDAC8 polypeptide with an IC50 of less than 1 μM.

[0032] The method for selectively inhibiting an activity of an HD AC8 polypeptide may be practiced in vitro or in vivo.

[0033] In a preferred embodiment of the present invention, the compound is provided as a prodrug.

[0034] In another aspect, the present invention provides a method for regulating smooth muscle cell contraction in an animal. In a preferred embodiment, this method comprises the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula

W - X - Y

in which

W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and

Y represents a zinc-binding moiety,

or

in which Z is -NH, O or S; R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which substituents are as defined for moiety W.

[0035] In yet another aspect, the present invention provides a method for treating a pathological condition characterized by an aberrant genetic repression of gene expression. In a preferred embodiment, this method comprises the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula

W - X - Y

in which

W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and

Y represents a zinc-binding moiety,

or

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are as defined for moiety W.

[0036] In a preferred embodiment of the present invention, the animal is a human.

[0037] Preferred pathological conditions are cancer and acute myeloid leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 depicts common structural features of HDAC inhibitors SAHA and TSA, such as cap group, linker and zinc-binding group.

[0039] Figure 2 depicts an HDAC8:TSA co-crystal structure showing the conserved hydrophobic amino acid residues (F 152, F208, and M274) which form the narrow active site channel, at the bottom of which is the catalytic zinc ion. Also shown is a TSA molecule entering the narrow active site channel.

[0040] Figure 3 A depicts the structure of HDAC8 bound to SAHA, an alkyl-linker HDAC inhibitor. Amino acid residues M274 and F 152 (indicated by arrows) pack against each other to form the wall of the active site pocket. [0041] Figure 3B depicts the structure of HDAC8 bound to CRA-A, an aryl-linker HDAC inhibitor. In this case amino acid residue F 152 rotates away from amino acid residue M274, exposing a large sub-pocket.

[0042] Figure 4 depicts the structures of Compounds 1 to 6, linkerless, sterically demanding hydroxamates designed to bind the sub-pocket of the F1DAC8 active site.

[0043] Figure 5 depicts a scheme for the synthesis of hydroxamates shown in Figure 4. Details are described in Examples 2 and 3.

[0044] Figure 6 depicts selective inhibition of Compounds 2, 5, and 6 for HDAC8. A. Inhibition plot of Compound 2 against HDACs 1, 6, and 8. B. Inhibition plot of Compound 5 against HDACs 1, 6, and 8. C. Inhibition plot of Compound 6 against HDACs 1, 6, and 8. Details are described in Examples 6 and 7.

[0045] Figure 7 depicts that cellular proteins in human cells are acetylated in response to treatment with TSA, Compound 2 or Compound 5. A. and B. Anti-acetyl lysine Western blot of HeLa and HEK293 cell lysates pretreated with TSA (a relatively non-specific HDAC inhibitor), no inhibitor ("0") and increasing concentrations of Compound 2 (panel A) and

Compound 5 (panel B). C. and D. Western blotting of the same lysates used in panels A and B, respectively, with antibodies specific for lysine acetylated histone H4 (α-Ac-Histone H4) lysine acetylated tubulin (α-Ac-Tubulin) and tubulin (α- Tubulin). Details are described in Example 8.

[0046] Figure 8 depicts an HDAC8: CRA-A co-crystal structure showing the right-angle orientation of the aryl linker relative to the induced sub-pocket. The aryl group of CRA-A bearing the hydroxamic acid does not bind the sub-pocket but rather is positioned well above it, as can be seen in Fig. 3B.

[0047] Figures 9A and 9B show reaction schemes for the making of a phenyl -substituted pyrrol (e.g., Compound 7). See also Example 4 for details.

[0048] Figure 1OA shows a reaction scheme for making naphthyl-substituted pyrroles, furans and thiophenes (e.g., Compounds 12-15, wherein S is changed to O or N) using a naphthyl boronic acid. Figure 1OB shows a reaction scheme for making compounds 10 and 11 as described in Example 5 DETAILED DESCRIPTION OF THE INVENTION

I. DEFINITIONS

[0049] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them unless specified otherwise.

[0050] As used herein, "acetylation status" refers to the presence or absence of an acetyl group on a polypeptide, preferably a histone polypeptide.

[0051] As used herein, "activity of HDAC8" or "activity of an HDAC8 polypeptide" refers to (i) binding of an HDAC8 polypeptide to a polypeptide or peptide, preferably a histone or tubulin polypeptide or a cellular polypeptide, (ii) interaction of an HDAC8 polypeptide with a polypeptide or peptide, preferably a histone or tubulin polypeptide or a cellular polypeptide, (iii) assembly of an HDAC8 polypeptide into a multiprotein complex, preferably a multiprotein complex comprising an inversion(lό) protein product or a cellular polypeptide, (iv) localization of an HDAC8 polypeptide in the cytoplasm of a eukaryotic cell, or (v) enzymatic deacetylating of an acetylated polypeptide or peptide, preferably a histone or tubulin polypeptide.

[0052] As used herein, the term "active site of HDAC8" or grammatical equivalents thereof refer to a region of an HDAC8 polypeptide surface within 50 angstroms of the zinc ion.

[0053] As used herein, an "agent" or "candidate agent" can be any chemical compound, for example, a macromolecule or a small molecule. The candidate agent can have a formula weight of less than about 10,000 grams per mole, less than 5,000 grams per mole, less than 1,000 grams per mole, or less than about 500 grams per mole. The candidate agent can be naturally occurring (e.g., a herb or a nature product), synthetic, or both. Examples of macromolecules are proteins, protein complexes, and glycoproteins, nucleic acids, e.g., DNA, RNA and PNA (peptide nucleic acid). Examples of small molecules are peptides, peptidomimetics (e.g., peptoids), amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds e.g., heteroorganic or organometallic compounds. A candidate agent can be the only substance assayed by the method described herein. Alternatively, a collection of candidate agents can be assayed either consecutively or concurrently by the methods described herein.

[0054] Candidate agents encompass numerous chemical classes, typically synthetic, semisynthetic, or naturally-occurring inorganic or organic molecules. Candidate agents may be small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents may comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and may include at least an amine, carbonyl, hydroxyl or carboxyl group, and may contain at least two of the functional chemical groups. The candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0055] As used herein, the term "alkyl" refers to a straight or branched chain saturated hydrocarbon moiety, and can include di- and multivalent groups, having the number of carbon atoms designated (i.e. C₁-C₁O means one to ten carbons). Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, and the various pentyl, hexyl, heptyl, octyl, etc. groups. The term "lower alkyl" refers to alkyl groups having from 1 to 4 carbon atoms. The term "haloalkyl" refers to alkyl groups substituted by one or more halogen atoms, which may be the same or different. The terms "alkoxy" and "alkylthio" refer to oxygen and sulfur atoms, respectively, substituted by alkyl groups of the indicated number of carbon atoms; "haloalkoxy" and "haloalkylthio" refer to such groups substituted by one or more halogens, which may be the same or different. The terms

"alklysulfonyl" and "haloalkylsulfonyl" refer to sulfonyl (-SO₂-) moieties substituted by an alkyl or haloalkyl group, respectively. The term "alkylene" refers to a divalent group derived from an alkyl group and includes, e.g., methylene, -CH₂-, ethylene, -CH₂CH₂ -, propylene, - CH₂CH₂CH₂- and the like.

[0056] As used herein, the term "alkenyl" refers to a straight or branched chain unsaturated hydrocarbyl moiety having one or more double bonds. Examples of alkenyl groups include vinyl, allyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl and 3 -(1,4- pentadienyl). The term "lower alkenyl" refers to alkenyl groups having from 2 to 4 carbon atoms. The term "alkenylene" refers to a divalent group derived from an alkenyl group and includes, for example, ethenylene, -CH=CH-, propenylene, -CH=C=CH-, and the like.

[0057] The term "alkylene" by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified by -CH₂CH₂CH₂CH₂-, and further includes those groups described below as "heteroalkylene." Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred in the present invention. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0058] As used herein, the term "alkynyl" refers to an unsaturated alkyl group one having 2-4 carbon atoms and a triple bond. Examples of alkynyl groups include ethynyl (acetylenyl), 1-propynyl, 1- and 2-butynyl.

[0059] As used herein, "antagonist" means a chemical substance that diminishes, abolishes or interferes with the physiological action of a polypeptide. The antagonist may be, for example, a chemical antagonist, a pharmacokinetic antagonist, a non-competitive antagonist, or a physiological antagonist, such as a biomolecule, e.g., a polypeptide. A preferred antagonist diminishes, abolishes or interferes with the physiological action of an HDAC8 polypeptide.

[0060] Specifically, an antagonist may act at the level of the interaction between a first polypeptide, e.g., an HDAC8 polypeptide and a second polypeptide, for example, a binding partner, such as a histone polypeptide, an inversion(lό) protein product or a cellular polypeptide. The antagonist, for example, may by competitively or non-competitively (e.g., allosterically) inhibit binding of the first polypeptide to the second polypeptide. A "pharmacokinetic antagonist" effectively reduces the concentration of the active drug at its site of action, e.g., by increasing the rate of metabolic degradation of the first polypeptide. A "competitive antagonist" is a molecule which binds directly to the first polypeptide in a manner that sterically interferes with the interaction of the first polypeptide with the second polypeptide. Non-competitive antagonism describes a situation where the antagonist does not compete directly with the binding, but instead blocks a point in the signal transduction pathway subsequent to the binding of the first polypeptide to the second polypeptide.

Physiological antagonism loosely describes the interaction of two substances whose opposing actions in the body tend to cancel each other out. An antagonist can also be a substance that diminishes or abolishes expression of a first polypeptide. Thus, an HDAC8 antagonist can be, for example, a substance that diminishes or abolishes: (i) the expression of the gene encoding HDAC8, (ii) the translation of HDCA8 RNA, (iii) the post-translational modification of HDAC8, such as phosphorylation, or (iv) the interaction of an HDAC8 polypeptide with other polypeptides in the formation of a multi-protein complex.

[0061] The term "aryl" means, unless otherwise stated, a polyunsaturated, typically aromatic, hydrocarbon substituent which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2- quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

[0062] For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(l- naphthyloxy)propyl, and the like).

[0063] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0064] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be a variety of groups selected from: -OR', =0, =NR', =N-0R\ -NR'R", -SR', -halogen, -SiR'R"R"\ -OC(O)R', -C(O)R', - CO₂R', -CONR'R", -0C(0)NR'R", -NR"C(O)R', -NR'-C(0)NR"R"', -NR"C(0)₂R', -NH- C(NH₂)=NH, -NR'C(NH₂)=NH, -NH-C(NH₂)=NR', -S(O)R', -S(O)₂R', -S(0)₂NR'R", -CN and -NO₂ in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R', R" and R'" each independently refer to hydrogen, unsubstituted (Ci-C8)alkyl and heteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens, unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(Ci-C₄)alkyl groups. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups such as haloalkyl (e.g., -CF₃ and - CH₂CF₃) and acyl (e.g., -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH₃, and the like).

[0065] Similarly, substituents for the aryl and heteroaryl groups are varied and are selected from: -halogen, -OR', -OC(O)R', -NR'R", -SR', -R', -CN, -NO₂, -CO₂R', -CONR'R", -C(O)R', -0C(0)NR'R", -NR"C(0)R', -NR"C(0)₂R', ,-NR'-C(0)NR"R'", -NH-C(NH₂)=NH, -NR'C(NH₂)=NH, -NH-C(NH₂)=NR', -S(O)R', -S(O)₂R', -S(0)₂NR'R", - N₃, -CH(Ph)₂, perfluoro(Ci-C₄)alkoxy, and perfluoro(Ci-C₄)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R" and R'" are independently selected from hydrogen, (Ci-C₈)alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstituted aryl)oxy-(Cr C₄)alkyl.

[0066] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(0)-(CH₂)_q-U-, wherein T and U are independently -NH-, -0-, -CH₂- or a single bond, and q is an integer of from O to 2. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently -CH₂-, -0-, -NH-, -S-, -S(O)-, -S(O)₂-, -S(O)₂NR'- or a single bond, and r is an integer of from 1 to 3. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CH₂)_S- X-(CH₂)_r, where s and t are independently integers of from O to 3, and X is -0-, -NR'-, -S-, - S(O)-, -S(O)₂-, or -S(O)₂NR'-. The substituent R' in -NR'- and -S(O)₂NR'- is selected from hydrogen or unsubstituted (Ci-C₆)alkyl.

[0067] As used herein, "associated with HDAC8" means contact, interact, or bind to HDAC8.

[0068] As used herein, the term "biologically active" when referring to an agent or compound is art-recognized and refers to a form of an agent or compound that allows for it, or a portion of the amount of agent or compound administered, to be absorbed by, incorporated to, or otherwise physiologically available to a subject or patient to whom it is administered.

[0069] As used herein, "biological sample" means a sample of biological tissue or fluid that contains nucleic acids or polypeptides. Such samples are typically from humans, but include tissues isolated from non-human primates, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histological purposes, cerebral spinal fluid, blood, plasma, serum, sputum, stool, tears, mucus, hair, skin, etc. Biological samples also include explants and primary and/or transformed cell cultures derived from patient tissues. A "biological sample" also refers to a cell or population of cells or a quantity of tissue or fluid from an animal. Most often, the biological sample has been removed from an animal, but the term "biological sample" can also refer to cells or tissue analyzed in vivo, i.e., without removal from the animal. Typically, a "biological sample" will contain cells from the animal, but the term can also refer to noncellular biological material, such as noncellular fractions of, blood, serum, saliva, cerebral spinal fluid or urine, that can be used to measure expression level of a polynucleotide or polypeptide. Numerous types of biological samples can be used in the present invention, including, but not limited to, a tissue biopsy or a blood sample. As used herein, a "tissue biopsy" refers to an amount of tissue removed from an animal, preferably a human, for diagnostic analysis. "Tissue biopsy" can refer to any type of biopsy, such as needle biopsy, fine needle biopsy, surgical biopsy, etc.

[0070] "Providing a biological sample" means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells {e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome history, will be particularly useful.

[0071] "Cancer," "cancer cells," "transformed" cells or "transformation" in tissue culture, refers to spontaneous or induced phenotypic changes that do not necessarily involve the uptake of new genetic material. Although transformation can arise from infection with a transforming virus and incorporation of new genomic DNA, or uptake of exogenous DNA, it can also arise spontaneously or following exposure to a carcinogen, thereby mutating an endogenous gene. Transformation is associated with phenotypic changes, such as immortalization of cells, aberrant growth control, nonmorphological changes, and/or malignancy {see, Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)).

[0072] The phrase "changes in cell growth" refers to any change in cell growth and proliferation characteristics in vitro or in vivo, such as formation of foci, anchorage independence, semi-solid or soft agar growth, changes in contact inhibition and density limitation of growth, loss of growth factor or serum requirements, changes in cell morphology, gaining or losing immortalization, gaining or losing tumor specific markers, ability to form or suppress tumors when injected into suitable animal hosts, and/or immortalization of the cell. See, e.g., Freshney, Culture of Animal Cells a Manual of Basic Technique pp. 231-241 (3^rd ed. 1994).

[0073] As used herein, a "combinatorial chemical library" refers to a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

[0074] As used herein, the term "cycloalkyl" refers to a saturated cyclic hydrocarbon having 3 to 8 carbon atoms, and 1 to 3 rings that can be fused or linked covalently. Cycloalkyl groups useful in the present invention include, but are not limited to, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. Bicycloalkyl groups useful in the present invention include, but are not limited to, [3.3.0]bicyclooctanyl, [2.2.2]bicyclooctanyl, [4.3.0]bicyclononane, [4.4.0]bicyclodecane (decalin), spiro[3.4]octanyl, spiro[2.5]octanyl, and so forth.

[0075] As used herein, the term "cycloalkenyl" refers to an unsaturated cyclic hydrocarbon having 3 to 15 carbons, and 1 to 3 rings that can be fused or linked covalently. Cycloalkenyl groups useful in the present invention include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Bicycloalkenyl groups are also useful in the present invention.

[0076] As used herein, the term "decreased expression" refers to the level of a gene expression product that is lower and/or the activity of the gene expression product is lowered. Preferably, the decrease is at least 20%, more preferably, the decrease is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% and most preferably, the decrease is at least 100%, relative to a control.

[0077] Synonyms of the term, "determining" are contemplated within the scope of the present invention and include, but are not limited to, detecting, measuring, assaying, testing or determining, the presence, absence, amount or concentration of a molecule, such as a an HDAC8, a label, or a compound of the invention and the like. The term refers to both qualitative and quantitative determinations.

[0078] As used herein, "determining the effect" or "determining the functional effect" means assaying for an agent or compound that increases or decreases a parameter that is indirectly or directly under the influence of the agent or compound, e.g., functional, enzymatic, physical and chemical effects. Such effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of a gene, such as HDAC8; measuring binding activity, e.g., binding of an HDAC8 polypeptide to another polypeptide; assaying for deacetylation activity of HDAC8; determining the acetylation status of an HDAC8 substrate, such as a histone polypeptide, tubulin polypeptide or another cellular polypeptide; measuring cellular proliferation, measuring apoptosis, measuring subcellular localization of a polypeptide, such as HDAC8; or the like. Determination of the functional effect of an agent or compound on a disease, disorder, cancer or other pathology can also be performed using assays known to those of skill in the art such as an in vitro assays, e.g., cellular proliferation; growth factor or serum dependence; mRNA and protein expression in cells, and other characteristics of cells. The effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, measurement of changes in RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, β-gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays, apoptosis assays, measuring the production of acetyl-CoA and AMP, and the like. "Functional effects" include in vitro, in vivo, and ex vivo activities.

[0079] As used herein, "disorder", "disease" or "pathological condition" are used inclusively and refer to any deviation from the normal structure or function of any part, organ or system of the body (or any combination thereof). A specific disease is manifested by characteristic symptoms and signs, including biological, chemical and physical changes, and is often associated with a variety of other factors including, but not limited to, demographic, environmental, employment, genetic and medically historical factors. Certain characteristic signs, symptoms, and related factors can be quantitated through a variety of methods to yield important diagnostic information.

[0080] As used herein, "effective amount", "effective dose", "sufficient amount", "amount effective to", "therapeutically effective amount" or grammatical equivalents thereof mean a dosage sufficient to produce a desired result, to ameliorate, or in some manner, reduce a symptom or stop or reverse progression of a condition. In some embodiments, the desired result is an increase in cytoplasmic localization of an F1DAC8 polypeptide. In other embodiments, the desired result is an increase in nuclear localization of an HDAC8 polypeptide. In yet other embodiments, the desired result is an increase in the deacetylation activity of an HDAC8 polypeptide. In another embodiment, the desired result is an increase or decrease in the acetylation status of an HDAC8 substrate, such as a histone polypeptide or tubulin polypeptide. Amelioration of a symptom of a particular condition by administration of a pharmaceutical composition described herein refers to any lessening, whether permanent or temporary, lasting or transit that can be associated with the administration of the pharmaceutical composition. An "effective amount" can be administered in vivo and in vitro.

[0081] A "full length" polypeptide or nucleic acid refers to a polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, polynucleotide or polypeptide sequences. The "full length" may be prior to, or after, various stages of post-translation processing or splicing, including alternative splicing and signal peptide cleavage. Thus, in some embodiments the HDAC8 polypeptide is a "full length" HDAC8 polypeptide referring to a polypeptide that has at least the length of a naturally occurring HDAC8 polypeptide. A "full-length" HDAC8 polypeptide or a fragment thereof can also include other sequences, e.g., a purification tag (such as FLAG or HA), or other attached compounds (such as an attached fluorophore, a label, or cofactor).

[0082] As used herein, the terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (br), or iodine (I) atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C₄)alkyl" is mean to include trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

[0083] "FIDAC" means histone deacetylase.

[0084] As used herein, the term "F1DAC8" refers to nucleic acids, polypeptides and polymorphic variants, alleles, mutants, and interspecies homologues thereof and as further described herein, that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acid sequence identity, preferably over a region of at least about 25, 50, 75, 100, 150, 200, 250, 300, 350, or 377 amino acids, to an F1DAC8 sequence as deposited under GenBank Accession Nos., e.g., CAB90213, AAF73428, NP_060956, Q9BY41, AAF73076, AAH50433, AAH61257, AAL47569,

Q8VH37, NP_081658, AAH55541, or NP_998596; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence as deposited under GenBank Accession Nos. e.g., CAB90213, AAF73428, NP_060956, Q9BY41, AAF73076, AAH50433, AAH61257, AAL47569, Q8VH37, NP_081658, AAH55541, or NP 998596, or conservatively modified variants thereof or a fragment thereof; (3) associate with a histone polypeptide or inversion(lό) protein product; (4) modulate at least partially and indirectly the production of acetyl-CoA and AMP; (5) specifically hybridize under stringent hybridization conditions to a nucleic acid sequence as deposited under GenBank Accession Nos. BC061257, BC055541, BC050433, NM_018486, NM_027382, NM_213431, AJ277724, AY556472, AY066003, AF245664, and AF230097, or conservatively modified variants thereof; (6) have a nucleic acid sequence that has greater than about 90%, preferably greater than about 96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferably over a region of at least about 30, 50, 100, 200, 500, 1000, 1,500 or more nucleotides, to a nucleic acid sequences as deposited under GenBank Accession Nos. BC061257, BC055541, BC050433, NM_018486, NM_027382, NM_213431, AJ277724, AY556472, AY066003, AF245664, and AF230097; (7) have at least 25, often 50, 75, 100, 150, 200, 250, 300, 350, or 377 contiguous amino acid residues of a polypeptide the sequence of which is deposited under GenBank Accession Nos. CAB90213, AAF73428, NP_060956, Q9BY41, AAF73076, AAH50433, AAH61257, AAL47569, Q8VH37, NP_081658, AAH55541, or NP_998596; and/or at least 25, often 50, 75, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1,000, 1,200, 1,500, or more contiguous nucleotides of a nucleic acid sequences as deposited under GenBank Accession Nos. BC061257, BC055541, BC050433, NM_018486,

NM_027382, NM_213431, AJ277724, AY556472, AY066003, AF245664, and AF230097. HDAC8 was cloned and characterized by several independent research groups (e.g., Van den Wyngaert et al, 2000, FEBS Lett 478(l-2):77-83; Buggy et al., 2000, Biochem J350 PtI : 199-205; Hu et al., 2000, J Biol Chem 275(20): 15254-64; incorporated herein by reference in their entireties).

[0085] An HDAC8 polynucleotide or polypeptide sequence is typically from a human, but may be from other mammals, but not limited to, a non-human primate, a rodent, e.g., a rat, mouse, or hamster; a cow, a pig, a horse, a sheep, or other mammal. In certain embodiments, it is desirable to use an HDAC8 or HDAC8-like polynucleotide or polypeptide from yeast, Drosophila, trypanosome, chicken, C. elegans, or Xenopus. An "HDAC8" polypeptide and polynucleotide includes both naturally occurring or recombinant forms. Therefore, in some embodiments, an HDAC8 polypeptide and an HDAC8 subdomain polypeptide as described herein can comprise a sequence that corresponds to a human HDAC8 polypeptide sequence. Thus, exemplary HDAC8 polypeptide sequences and are known in the art. For example, GenBank accession numbers for human HDAC8 polypeptides are AAF73428, NP 060956, Q9BY41, CAB90213, AAH50433, and AAF73076. GenBank accession numbers for mouse HDAC8 polypeptides are, for example, AAH61257, Q8VH37, NP_081658, and AAL47569; for zebrafish HDAC8, NP-998596 and AAH55541. Exemplary HDAC8 polynucleotide sequences are known in the art. Exemplary, GenBank accession numbers for human HDAC8 nucleic acids are BC050433, NM_018486, AJ277724, AF245664, and AF230097. GenBank accession numbers for mouse HDAC8 include BC061257, NT_039706, NM_027382, and AY066003; pig HDAC8, AY556472; and for zebrafish HDAC8, BC055541 and NM 213431. [0086] An "HDAC8 fusion protein" as used herein refers to a polypeptide that comprises (i) an amino acid sequence of an HDAC8, an HDAC8 fragment, an HDAC8 subdomain polypeptide, an HDAC8 related polypeptide or a fragment of an HDAC8 related polypeptide and (ii) an amino acid sequence of a heterologous polypeptide (i.e., a non-HDAC8, non- HDAC8 fragment or non-HDAC8 related polypeptide).

[0087] An "HDAC8 homolog" refers to a polypeptide that comprises an amino acid sequence similar to that of HDAC8, but does not necessarily possess a similar or identical function as HD AC8.

[0088] An "HDAC8 isoform" refers to a variant of HDAC8 that is encoded by the same gene, but differs in its pi or MW, or both. Such isoforms can differ in their amino acid composition (e.g., as a result of alternative splicing or limited proteolysis) and in addition, or in the alternative, may arise from differential post-translational modification (e.g., glycosylation, acetylation or phosphorylation).

[0089] An "HDAC8 ortholog" as used herein refers to a non-human polypeptide that (i) comprises an amino acid sequence similar to that of human HDAC8 and (ii) possess a similar or identical function to that of human HDAC8.

[0090] As used herein, an "HDAC8 substrate" refers to a polypeptide with which an HDAC8 polypeptide interacts or interacts and/or a polypeptide which is deacetylated by an HDAC polypeptide. A preferred HDAC8 substrate is a histone polypeptide or tubulin polypeptide.

[0091] As used herein, "HDAC8 structure" or "structure of HDAC8" refers to the crystal structure of HDAC8 as determined by Somoza et ah. (Somoza et ah., 2004, Structure 12: 1325- 1334; incorporated herewith by reference in its entirety) and/or Vannini et ah, (Vannini et ah., 2004, Proc Nath Acad Sci USA 101(42): 15064-15069; incorporated herewith by reference in its entirety). Additional structural information is deposited with GenBank under accession numbers IVKGB (Chain B, crystal structure of human HDAC8 complexed with CRA- 19156), IVKGA (Chain A, crystal structure of inhibited human HDAC8 complexed with CRA-19156), 1W22B (Chain B, crystal structure of human HDAC8), 1W22A (Chain A, crystal structure of human HDAC8), 1T69A (Chain A, crystal structure of inhibited human HDAC8 complexed with SAHA), 1T67A (Chain A, crystal structure of inhibited human HDAC8 complexed with CMS-344), 1T64B (Chain B, crystal structure of inhibited human HDAC8 complexed with TSA), and 1T64A (Chain A, crystal structure of inhibited human HDAC8 complexed with TSA).

[0092] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), Boron (B), phosphorous (P) and sulfur (S).

[0093] As used herein, the term "heteroaryl" refers to a polyunsaturated aromatic hydrocarbon substituent having 5-12 ring members, which can be a single ring or multiple rings (up to three rings) which are fused together or linked covalently, and which has at least one heteroatom in the ring, such as N, O, or S. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, A- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3- quinolyl, and 6-quinolyl. Additional heteroaryl groups useful in the present invention include pyridyl N-oxide, tetrazolyl, benzofuranyl, benzothienyl, indazolyl, or any of the radicals substituted, especially mono- or di-substituted.

[0094] As used herein, the term "heteroatom" or "ring heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0095] As used herein, the term "heterocycloalkyl" refers to a saturated cyclic hydrocarbon having 3 to 15 ring members, and 1 to 3 rings that can be fused or linked covalently, and which has at least one heteroatom in the ring, such as N, O, or S. Additionally, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of heterocycloalkyl include 1 -(1,2,5, 6-tetrahydropyridyl), 1-piperidinyl, 2- piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2-piperazinyl, and the like.

[0096] A "host cell" is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector. Host cells may be cultured cells, explants, cells in vivo, and the like. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect cells, amphibian cells, or mammalian cells such as CHO, 293, 3T3, HeLa, and the like {see, e.g., the American Type Culture Collection catalog).

[0097] The terms "hydroxamic acid group" or "hydroxamic acid" refer to an N- hydroxylated amide and its derivatives include any type of chemical functionality attached to the carbonyl of the hydroxamic acid.

[0098] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, felines, canines, equines, bovines, mammalian farm animals, mammalian sport animals, and mammalian pets and humans. Preferred is a human. In certain embodiments, the terms also include Xenopus, zebrafish, trypanosome, C. elegans, Drosophila, and yeast.

[0099] As used herein, "inhibitor" refers to an agent or compound that, e.g., represses or inactivates the expression of a polypeptide of the invention or binds to, decreases, closes, inactivates, impedes, or reduces activation, desensitizes or down regulates the activity of a polypeptide of the invention. Inhibitors include nucleic acids such as siRNA, antisense RNA, and ribozymes that interfere with the expression of e.g., HDAC8 as well as naturally occurring and synthetic compounds and agents, small chemical molecules and the like. Preferred HDAC8 inhibitors are the compounds described herein. Assays for inhibitors are described herein. Samples or assays comprising e.g., an HDAC8 polypeptide that are treated with a potential inhibitor are compared to control samples without the inhibitor to examine the extent of the effect. Control samples (untreated with candidate agents or compounds) are assigned a relative activity value of 100%. Inhibition of the HDAC8 polypeptide is achieved when the level or activity value relative to a control is reduced by 10%, optionally 20%, optionally 30%, optionally 40%, optionally 50%, 60%, 70%, 80%, or 90-100%.

[0100] As used herein, "in vitro" means outside the body of the organism from which a cell or cells is obtained or from which a cell line is isolated.

[0101] As used herein, "in vivo" means within the body of the organism from which a cell or cells is obtained or from which a cell line is isolated.

[0102] As used herein, the term "isomers" refers to compounds of the present invention that possess asymmetric carbon atoms (optical or chiral centers) or double bonds. The enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)- or as (D)- or (L)- for amino acids, and individual isomers are all intended to be encompassed within the scope of the present invention.

[0103] A "label" or a "detectable moiety" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. For example, useful labels include ¹H, ³H, ¹²⁵1, ³²P, ¹³C, and ¹⁴C, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins or other entities which can be made detectable, e.g., by incorporating a radiolabel into an HDAC8 polypeptide, a substrate of an HDAC8 polypeptide, or a compound. A preferred label is ¹⁴C or ³H, preferably in an acetyl group.

[0104] As used herein, "level of a mRNA" in a biological sample refers to the amount of mRNA transcribed from a gene that is present in a cell or a biological sample. The mRNA generally encodes a functional protein, although mutations may be present that alter or eliminate the function of the encoded protein. A "level of mRNA" need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample. A preferred mRNA is an HDAC8 mRNA.

[0105] As used herein, "level of a polypeptide" in a biological sample refers to the amount of polypeptide translated from a mRNA that is present in a cell or biological sample. The polypeptide may or may not have protein activity. A "level of a polypeptide" need not be quantified, but can simply be detected, e.g., a subjective, visual detection by a human, with or without comparison to a level from a control sample or a level expected of a control sample. A preferred polypeptide is an HDAC8 polypeptide.

[0106] As used herein, "mammal" or "mammalian" means or relates to the class mammalia including, but not limited to the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and primates (e.g., humans, chimpanzees, and monkeys).

[0107] As used herein, a "multi-protein complex" refers to the binding and non-covalent attachment of two or more polypeptides to each other.

[0108] As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature. For example a naturally occurring nucleic acid molecule can encode a natural protein. [0109] As used herein, a "naturally-occurring" polypeptide refers to a polypeptide molecule having an amino acid sequence that occurs in nature.

[0110] As used herein, "pharmaceutically acceptable" refers to compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction when administered to a subject, preferably a human subject. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of a Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0111] As used herein, the term "pocket" within an HDAC8 polypeptide refers to any surface site of HDAC8 that is a binding site for a small molecule. A "subpocket" within an HDAC8 polypeptide refers to the pocket adjacent to the active site between amino acid residues M274 and F 152.

[0112] As used herein, "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers, those containing modified residues, and non-naturally occurring amino acid polymer. Preferred polypeptides are HDACs, in particular HDAC8.

[0113] A "purified" or "isolated" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. "Substantially free" means that the protein of interest in the preparation is at least 10% pure. In an embodiment, the preparation of the protein has less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of a contaminating component (e.g., a protein not of interest, chemical precursors, and so forth). When the protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0114] The term "recombinant" when used with reference to, e.g., a cell, nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, e.g., recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under-expressed or not expressed at all. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid, e.g., using polymerases and endonucleases, in a form not normally found in nature. In this manner, operably linkage of different sequences is achieved. Thus an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention. It is understood that once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e., using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention. Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e., through the expression of a recombinant nucleic acid as depicted above.

[0115] As used herein, the term "salt" refers to salt of an active compound or agent of the present invention, such as an HDAC8 inhibitor, which is prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds and agents of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds and agents of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like {see, e.g., Berge et al., 1977, J Pharm Science 66: 1-19). Certain specific agents of the present invention contain both basic and acidic functionalities that allow the agents to be converted into either base or acid addition salts.

[0116] The neutral forms of the compounds of the present invention may be regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound and agent for the purposes of the present invention.

[0117] As used herein, the phrases "selective inhibition of HDAC8" or "selectively inhibiting HDAC8" or grammatical equivalents thereof refer to the inhibition of an HDAC8 polypeptide by a compound of the present invention which occurs at an IC₅₀ concentration of a the compound which is at least 2 fold less, at least 5 fold less, at least 10 fold less, at least 20 fold less, at least 50 fold less, or at least 100 fold less than the IC50 concentration of the same compound for HDACl or HDAC6.

[0118] The terms "subdomain", "domain ", "functional domain", or grammatical equivalents thereof in the context of a protein, such as an HDAC8 polypeptide refer to a fragment of that protein, such as a fragment of HDAC8 polypeptide, which participates in an interaction, e.g., an intramolecular or an inter-molecular interaction, e.g., a binding or catalytic interaction. An inter-molecular interaction can be a specific binding interaction or an enzymatic interaction (e.g., the interaction can be transient and a covalent bond is formed or broken). An inter-molecular interaction can be between the protein and another protein, between the protein and another compound, or between a first molecule and a second molecule of the protein (e.g., a dimerization interaction). Biologically active portions/functional domains of a protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the protein which include fewer amino acids than the full length, natural protein, and exhibit at least one activity of the natural protein. Biological active portions/functional domains can be identified by a variety of techniques including truncation analysis, site-directed mutagenesis, and proteolysis. Mutants or proteolytic fragments can be assayed for activity by an appropriate biochemical or biological (e.g., genetic) assay. In some embodiments, a functional domain is independently folded. Typically, biologically active fragments comprise a domain or motif with at least one activity of the protein, e.g., an HDAC8 core catalytic domain. A biologically active portion/functional domain of a protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions/functional domain of a protein , such as an HDAC8 polypeptide (i) can bind to a histone polypeptide or inversion(lό) protein product, (ii) have deacetylation activity, or (iii) assemble into a multiprotein complex, comprising e.g., a histone polypeptide or inversion(lό) protein product, or (iv) are useful for treatment of a pathological condition as described herein.

[0119] A " related" polypeptide as used herein, refers to a homolog, an isoform, an ortholog, a fusion protein or fragments of a polypeptide or any combination thereof.

[0120] As used herein, the term "solvate" refers to compounds and agents of the present invention that are complexed to a solvent. Solvents that can form solvates with the compounds and agents of the present invention include common organic solvents such as alcohols (methanol, ethanol, etc.), ethers, acetone, ethyl acetate, halogenated solvents (methylene chloride, chloroform, etc.), hexane and pentane. Additional solvents include water. When water is the complexing solvent, the complex is termed a "hydrate."

[0121] As used herein, "subject" or "patient" to be treated for a pathological condition, disorder, or disease by a subject method means either a human or non-human animal in need of treatment for a pathological condition, disorder, or disease. The term "non-human animal" includes all vertebrates, e.g., mammals, such as non-human primates (particularly higher primates), sheep, dog, rodent (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbits, cow, and non-mammals, such as chickens, amphibians, reptiles, etc. In a preferred embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or animal suitable as a disease model.

[0122] As used herein, the term "tautomer" refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another. It will be apparent to one of ordinary skill in the art that certain compounds of the present invention may exist in tautomeric form. All such tautomeric forms of the compounds are within the scope of this invention.

[0123] As used herein, the terms "treat", "treating", and "treatment" include: (1) preventing a pathological condition, disorder, or disease, i.e. causing the clinical symptoms of the pathological condition, disorder, or disease not to develop in a subject that may be predisposed to the pathological condition, disorder, or disease but does not yet experience any symptoms of the pathological condition, disorder, or disease; (2) inhibiting the pathological condition, disorder, or disease, i.e. arresting or reducing the development of the pathological condition, disorder, or disease or its clinical symptoms; or (3) relieving the pathological condition, disorder, or disease, i.e. causing regression of the pathological condition, disorder, or disease or its clinical symptoms. These terms encompass also prophylaxis, therapy and cure. Treatment means any manner in which the symptoms of a pathological condition, disorder, or disease are ameliorated or otherwise beneficially altered. Preferably, the subject in need of such treatment is a mammal, more preferable a human.

[0124] As used herein, the term "zinc-binding domain" or "zing-binding group" within a compound of the present invention refers to any chemical functionality that is a ligand for transition metals (see, e.g., Cohen et al, 2004, JAm Chem Soc 126(27):8388-8389).

II. INHIBITORS FOR HDAC8 A. Selection Of A Target Site For Selective HDAC8 Inhibitors

[0125] Somoza et al. described the crystal structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors and deposited the complexes in the Protein Data Bank, e.g., HDAC8 with MS344 (ID code 1T67), HDAC8 with SAHA (ID code 1T69), HDAC8 with TSA (ID code 1T64) and GHDAC8 with CRA-A (ID code IVKG) (Somoza et al, 2004, Structure 12: 1325-1334; incorporated herewith by reference in its entirety). An additional structure, wherein HDAC8 was complexed with the hydroxamic acid inhibitor, N- hydroxy-4-(methyl[(5-pyridin-2-yl-2-thienyl)sulfonyl]amino)benzamide, was reported by Vannini et al, (Vannini et al, 2004, Pr oc Natl Acad Sci USA 101(42): 15064-15069; incorporated herewith by reference in its entirety).

[0126] The rational design of specific HDAC8 inhibitors is based upon a comparative analysis of the HDAC8, HDAH, and HDLP structures with bound hydroxamate inhibitors (Somoza et al., 2004, Structure 12: 1325-1334). The amino acid sequences and overall active site topology of the enzymes are similar. In HDAC8, the catalytic machinery, comprising the zinc ion that facilitates amide hydrolysis is found at the bottom of a long, narrow pocket (about 12 A deep; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069); just above which are the conserved and catalytically important amino acid residues Y306 and H180 (Somoza et al, 2004, Structure 12: 1325-1334). The zinc ion is bound to carboxylate oxygens of D 178 and D267, and to the Nδl atom of Hl 80 (Somoza et al, 2004, Structure 12: 1325-1334). The rim of the pocket is formed by three conserved hydrophobic amino acid residues, F 152, F208, and M274 (Figure 2). These form the tunnel that the acetyl-lysine substrate and straight-chain hydroxamate inhibitors penetrate to access the catalytic machinery. Similar architecture is found in FIDLP and FIDAH..

[0127] Vannini et al. noted that immediately below the active site is a tube-like internal cavity filled by several water molecules that could as a shuttle for the reaction product acetate (Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069). There is also a second metal binding site buried in the interior of the HDAC8 protein in the vicinity of the active site, approximately 7 A from the zinc, which is occupied by a sodium ion (Somoza et al., 2004, Structure 12: 1325-1334) or a potassium ion (Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069).

[0128] The HDAC8 structure was solved with four different hydroxamate inhibitors bound. The structure of HDAC8 was used to guide the design of HDAC 8 -selective inhibitors. The active site topology of HDAC8 showed large structural differences depending on which inhibitor was bound to the active site. It is surprisingly malleable. For example in the SAHA:HDAC8 co-crystal structure, the active site is deep and narrow similar to the HDLP and HDAH structures. (Somoza et al, 2004, Structure 12: 1325-1334). However, when a hydroxamate inhibitor with an aryl linker (CRA-A) is bound to the HDAC8, a large subpocket forms in the side of the active site, adjacent to amino acid residue M274. This pocket is created by movement of amino acid residue F 152 away from its normal position packed against amino acid residue M274 to form the lip of the active site tunnel (Figure 3). This shift may be a consequence of the more sterically demanding aryl hydroxamate CRA-A versus the aliphatic hydroxamate SAHA binding the active site.

[0129] The inventors of the present invention reasoned that this sub-pocket can be targeted by a new HDAC8 inhibitor scaffold. If this pocket is accessible only to HDAC8, inhibitors that bind to it should be selective for HDAC8. To test this idea, a panel of simple, bulky aryl hydroxamic acids that are unlike the canonical "zinc-binding group/linker/cap group" structure of most HDAC inhibitors were chemically synthesized, purified, characterized and tested for HDAC inhibition (Figure 4). B. Compounds Of The Present Invention

[0130] In general, the compounds of the present invention possess a zinc-binding group linked to a moiety or group of moieties that are positioned to a hydrophilic subpocket between HDAC8 residues methionine 274 (M274) and phenylalanine 152 (F 152) in proximity to the active site zinc ion of the HDAC8 polypeptide.

[0131] More particularly, the compounds of this invention can be divided into two groups, one group of which comprises primarily compounds that are known for other applications, but that have not been described as HDAC inhibitors (although this group includes at least one novel compound active for the purpose of this invention), and a second group that comprises novel compounds and that have this property.

[0132] The compounds of the first type have the general formula

W - X - Y

in which:

W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkyl carbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and

Y represents a zinc-binding moiety.

[0133] Preferably Y represents a hydroxamic acid group or a hydroxamic acid derivative, i.e. a group having the formula

in which Ri represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defied above in connection with moiety W.

[0134] Compounds of this group include a number of compounds described by Summers et al. and by Boldt et al. (Summers et al. 1987, J Med Chem 30:574-580; Summers et al. 1990, JMed Chem 33:992-998; BoXάX et al, 2006, Organic Letters 8: 1729-1732; incorporated by reference in their entirety).

[0135] Specific compounds of this type that have been found useful and satisfactory in carrying out this invention are indicated as Compounds 1-6 below and are represented by the following formulas

Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

[0136] Compound 5 above is novel and, per se, constitutes an aspect of this invention.

[0137] The second type of compounds of this invention have the formula

in which Z is -NH, O, or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl.

[0138] One group of preferred compounds of this type includes those having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN.

[0139] A particularly preferred compound of this group has the above formula in which R₃ and R₄ are both hydrogen, namely

Compound 7

[0140] Another group of preferred compounds of this type is that in which R₂ is 1- or 2- naphthyl, which have the formula

Compound 8

and

Compound 9

[0141] Another preferred group of compounds of this type has the formula

in which X is oxygen or sulfur and R₃ and R₄ are as defined above. Preferred compounds of this type include those in which X is sulfur or oxygen, respectively and R₃ and R₄ are both hydrogen, namely

Compound 10 and

Compound 11

[0142] Other preferred compounds of this type are those in which R₂ is a 1- or 2- naphthyl group, namely

Compounds 12 (X=S) and 13 (X=O)

and

Compounds 14 (X=S) and 15 (X=O)

[0143] Compounds of the present invention wherein the zinc-binding moity is a hydroxamic acid group or a hydroxamic acid derivative are also referred to as "linker-less" hydroxamic acids to distinguish them from most of the known, nonselective HDAC inhibitors, such as TSA, SAHA, which as shown in Figure 1 comprise a "linker" domain.

[0144] Compounds 1-6 synthesized, purified, characterized and tested for HDAC inhibition as described herein, are depicted in Figure 4. Figure 5 depicts a scheme for the synthesis of HDAC8 inhibitors of the present invention. These inhibitors are aryl hydroxamates like CRA-A, however, lacking the linker domain of CRA-A, and were expected to induce formation of the sub-pocket. Because the typical linker domain is missing in compounds 1-6, the aryl groups are only a short distance from the hydroxamic acid group, the zinc-binding group. Such molecules should be excluded from HDACs that lack the sub-pocket of HDAC8, yet be able to bind and chelate zinc (Zn²⁺) in HDAC8 due to its enlarged pocket. [0145] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0146] Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention.

[0147] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0148] The compounds of the present invention may exist as salts. The present invention includes such salts.

[0149] In addition to salt forms, the present invention provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the compounds of the present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent.

[0150] Certain compounds of the present invention can exist in solvated forms, including hydrated forms, as well as unsolvated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. C. Chemical Synthesis Of Compounds

[0151] Compounds 1-6 were synthesized in two steps from the corresponding carboxylic acids by conversion to acid chlorides followed by treatment with hydroxylamine in water/THF under basic conditions, followed by recrystallization from ethanol/water (Summers et al, 1987, J Med Chem 30:574-580). For example, for Compound 6, first a

Suzuki coupling between 1-naphtyl boronic acid and p-bromobenzoic acid was performed to make the naphtyl-benzoic acid, which was then converted to its corresponding hydroxamate. Details are described in Examples 2 and 3 and Figure 5.

[0152] Phenyl-substituted pyrroles (e.g., Compound 7) can be made by reaction of methyl cinnamate or another cinnamate with tosylmethyl isocyanate, followed by hydrolysis of the resulting pyrrole ester and reaction with thionyl chloride (Figure 9A).

[0153] An example of the preparation of such a compound (Compounds 7, 8 and 9) is given below as Example 4.

[0154] Phenyl-substituted pyrroles, furans and thiophenes (e.g., Compounds 10 and 11) can be made by the reaction of bromothiophenecarboxylic acid or bromofurancarboxylic acid with an aryl boronic acid. Example 5 below depicts such a synthesis.

[0155] Naphthyl- substituted pyrroles, furans and thiophenes (e.g., Compounds 12-15, wherein S is changed to O or N) can be made similarly, using a naphthyl boronic acid (Figure 10B):

III. PURIFICATION AND CHARACTERIZATION OF INHIBITORS FOR HDAC8

[0156] The compounds of the present invention inhibiting an HDAC8 activity were purified using standard laboratory methods and characterized.

[0157] All hydroxamates have ¹H-NMR and ¹³C-NMR spectra consistent with the structures shown herein. Details are provided in Examples 2-5. Details for performing ¹H- NMR and ¹³C-NMR spectra analyses are described in Examples IF through IH. IV. IDENTIFICATION AND TESTING OF INHIBITORS FOR HDAC8

[0158] HDAC Inhibitors are identified using methods known in the art and described herein. A number of different screening protocols can be used to identify compounds that inhibit the expression or activity of an HDAC8.

[0159] Assays for identifying selective HDAC8 inhibitors versus general inhibitors may be conducted in a cell based or cell free format. For example, an assay may comprise incubating or contacting a subject polypeptide or subject nucleic acid, with a test agent or compound of the present invention under conditions in which a level or an activity of the subject polypeptide or subject nucleic acid can be inhibited, and monitoring or determining the level of inhibition in the presence of the test agent or compound relative to the absence of the test agent or compound.

A. Detection Of HDAC Polypeptides

[0160] The methods of the present invention described above, may optionally comprise the step of determining or detecting a polypeptide, such as an HDAC8 polypeptide. Other polypeptides described herein, such as a histone polypeptide or a tubulin polypeptide or other HDACs can also be determined using the following methods.

[0161] Determining or detecting a polypeptide, such as an HDAC8 and others may be done in a variety of ways, including, but not limited to detecting the respective polypeptides in a biological sample, a cell, an organ, or in an animal, including human and non-human animals.

[0162] The expression level of a polypeptide may be determined by a variety of methods, including, but not limited to, affinity capture, mass spectrometry, traditional immunoassays and immunoprecipitation assays, PAGE, Western Blotting, RIA, or HPLC as described herein, or as known by one of skill in the art.

[0163] Detection paradigms that can be employed to this end include optical methods, electrochemical methods (voltametry and amperometry techniques), atomic force microscopy, and radio frequency methods, e.g., multipolar resonance spectroscopy. Illustrative of optical methods, in addition to microscopy, both confocal and non-confocal, are detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, and birefringence or refractive index (e.g., surface plasm on resonance, ellipsometry, a resonant mirror method, a grating coupler waveguide method or interferometry). [0164] Using, e.g., antibodies, such as the anti-HDAC8 antibodies raised against HDAC8 peptides and described herein, the level of an HDAC8 polypeptide in the absence or presence of a compound, can be assessed. Other antibodies specifically detecting a phosphorylated HDAC8 polypeptide are useful for detecting differences in the phosphorylation status of HDAC8 in the absence or presence of a compound.

[0165] Polypeptides acetylated at lysine residues, e.g., in response to treatment of a cell or sample with a compound of the present invention, may be detected using the anti-lysine antibodies described herein. Details are described in Example 8 and Figure 7.

B. Purification Of HDAC Polypeptides [0166] HDAC polypeptides for use in the testing a property (e.g., selective inhibition of an HDAC8 polypeptide) of compounds of the present invention and a property of compounds that can be prepared by one of skill in the art using the teachings described herein include naturally occurring HDAC polypeptides and recombinantly expressed HDAC polypeptides. Thus, for example, an HDAC8 polypeptide can be a naturally occurring HDAC8 polypeptide or a recombinant HDAC8 polypeptide. A naturally occurring HDAC8 polypeptide can be purified, e.g., from human or mouse tissue or e.g., from human or mouse cells. Recombinant HDAC8 polypeptide can be purified from any suitable expression system as known in the art, e.g., purification of recombinant proteins form a host cell, preferably a mammalian host cell.

[0167] HDAC8 polypeptides can be purified to substantial purity by standard techniques, e.g., including, but not limited to column chromatography, immunopurification methods, selective precipitation using ammonium sulfate, and others. An HDAC8 polypeptide may be expressed and purified as described, e.g., see Somoza et ah, (Somoza et ah, 2004, Structure 12:1325-1334) who purified an HDAC8 polypeptide from Spodoptera frugiperda SF9 cells or Trichoplusia ni Hi5 cells using a baculovirus expression system. Alternatively, an HDAC8 polypeptide is purified from E.coli as described (Hu et ah, 2000, J Biol Chem 275(20): 15254-64).

[0168] An HDAC8 polypeptide may be expressed and purified with or without an affinity tag, such as an N-terminal poly-histidine affinity tag, FLAG-epitope tag, HA-epitope tag, and the like. [0169] HDAC polypeptides useful to practice the present invention include HDAC fusion proteins, HDAC homologs, HDAC isoforms, HDAC orthologs, and in particular HDAC8 fusion proteins, HDAC8 homologs, HDAC8 isoforms, and HDAC8 orthologs.

C. Detection Of HDAC8 Enzymatic Activity [0170] Various assays have been described to detect HDAC8 deacetylase activity. For example, useful assays are those described by North et al. and Verdin et al, who reported assays for detecting deacetylase activity using, e.g., histones as a substrate (North et al, 2005, Methods 36(4):338-45; Verdin et al, 2004, Methods Enzymol 377: 180-96; incorporated by reference in their entirety).

[0171] HDAC8 deacetylating activity of a histone substrate can be monitored by, e.g., immunoblotting using an anti histone antibody detecting both acetylated and deacetylated histone and an antibody which is specific for acetylated histone.

[0172] An additional assay is described in the present invention in Example 8 and Figure 7. This assay involves immunoblotting using anti-tubulin antibodies and anti-acetylated lysine antibodies to detect the acetylation status of tubulin, one of the HDAC8 substrate. The acetylation status of an HDAC8 substrate cane be determined in the absence or presence of a compound of the present invention. A compound of the present invention that inhibits HDAC8 deacetylase activity will result in an increase of the acetylation status of the HDAC8 substrate (see Example 8, Figure 7).

[0173] Preferred compounds of the invention have an IC50 (inhibition potency or, by definition, the concentration of inhibitor which reduces HDAC8 activity by 50%) of less than about 500 μM, preferably less than about 100 μM, more preferably less than about 25 uM, even more preferably less than about 10 μM and most preferably less than about 1 μM. Exemplary compounds of the invention are listed in Tables 1 and 2. Table shows inhibition ofHDAC8.

[0174] Preferably, a compound of the present invention is a selective inhibitor of HDAC8 when compared

D. Detection of Interaction Between HDAC8 And A Compound Of The Present Invention [0175] The interaction between two molecules, such as HDAC8 and a compound of the present invention or a derivative compound thereof can also be detected, e.g., using a fluorescence assay in which at least one molecule is fluorescently labeled. One example of such an assay includes fluorescence energy transfer (FET or FRET for fluorescence resonance energy transfer) (see, for example, Lakowicz et al, U.S. Pat. No. 5,631,169; Stavrianopoulos et al., U.S. Pat. No. 4,868,103). A fluorophore label on the first, 'donor' molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second, 'acceptor' molecule, which in turn is able to fluoresce due to the absorbed energy. Alternately, the 'donor' protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the 'acceptor' molecule label may be differentiated from that of the 'donor.' Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the 'acceptor' molecule label in the assay should be maximal. A FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e.g., using a fluorimeter.

[0176] Another example of a fluorescence assay is fluorescence polarization (FP). For FP, only one component needs to be labeled. A binding interaction is detected by a change in molecular size of the labeled component. The size change alters the tumbling rate of the component in solution and is detected as a change in FP (see, e.g., Nasir et ah, 1999, Comb Chem HTS 2: 177-190; Jameson et ah, 1995, Methods Enzymol 246:283; Seethala et ah, 1998, Anal Biochem 255:257. Fluorescence polarization can be monitored in multiwell plates, e.g., using the Tecan Polarion.TM. reader (see, e.g., Parker et ah, 2000, J Biomol Screen 5:77-88; and Shoeman, et ah, 1999, Biochemistry 38: 16802-16809).

[0177] In another embodiment, determining the ability of a protein to bind to a target molecule or the ability of a compound to bind to a subject polypeptide can be accomplished using real-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander and Urbaniczky, 1991, Anal C hem 63:2338-2345; Szabo et ah, 1995, Curr Opin Struct Biol 5:699-705). "Surface plasmon resonance" or "BIA" detects biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the mass at the binding surface (indicative of a binding event) result in alterations of the refractive index of light near the surface (the optical phenomenon of surface plasmon resonance (SPR)), resulting in a detectable signal which can be used as an indication of real-time reactions between biological molecules. [0178] Binding of HDAC8 inhibitors described herein to an HDAC8 polypeptide can be determined by a variety of methods. One method involves crystallization of the HDAC8 polypeptide in the presence of an HDAC8 inhibitor of the invention as described by Somoza et al. and Vannini et al. (Somoza et al, 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069).

E. Selectivity Of HDAC8 Inhibitors

[0179] Comparing HDCA8 inhibition to inhibition of other HDACs by the compounds of the present invention can also be performed. Example 6 and Figure 6 present exemplary assays and results for determining the selectivity of inhibition of a compound of the present invention.

[0180] A compound of the present invention inhibits an HDAC8 polypeptide with an efficiency of greater than 5 fold, preferably greater than 10 fold, more preferably greater than 25 fold, even more preferably greater than 50 fold, and most preferably greater than 100 fold over another HDAC polypeptide, such as HDACl or HDAC6.

F. In vitro Assays Using A Purified HDAC8 Polypeptide

[0181] Identification and testing of compounds for inhibiting a level or activity of an HDAC8 and compounds for modulating a level, acetylation status, or activity of a cellular substrate of HDAC8 can be performed using in vitro assays. Exemplary in vitro assays are described herein, and specifically in Example IE.

G. Cell-Based Assays

[0182] Identification and testing of compounds for inhibiting a level or activity of an HDAC8 and compounds for modulating a level, acetylation status, or activity of a cellular substrate of HDAC8 can also be performed using cell-based assays. An exemplary cell-based assay is described in Examples II and 8 and shown in Figure 7.

[0183] For cell-based assays, typically, eukaryotic cells, such as mammalian cells are used. In certain embodiments, yeast cells my be used. The cell can be a primary cell isolated from a donor biological sample. Alternatively, the cell can be an established cell line as made available by the American Type Culture Collection.

[0184] A cell can also be a cell that is transiently or stably transfected with an expression construct, such as an HDAC8 expression construct. An HDAC8 expression construct comprises an HDAC8 encoding nucleic acid in a vector suitable for expression of HDAC8 in a prokaryotic or eukaryotic cell. The HDAC8 encoding nucleic acid can either encode a full- length HDAC8 polypeptide or a fragment thereof as described herein. Expression control elements, such as promoter and enhancer elements are operably linked to the HDAC8 encoding nucleic acid. The making and using of expression construct is within the skill of one of skilled in the art.

H. In vivo Assays

[0185] Identification and testing of a compound for inhibiting a level or activity of an HDAC8 polypeptide can also be performed in vivo. In this method, a compound is administered to an animal, preferably a mouse, and blood samples or tissue samples are taken from the animal at various times after administration of the compound and tested for the presence or activity of the HDAC8 polypeptide, a level, acetylation status, or activity of a cellular substrate of HDAC8, such as a histone polypeptide or a tubulin polypeptide.

I. Toxicity Of HDAC8 Inhibitors

[0186] Cell viability assays and in vivo assays can be performed to determine the toxicity of a compound of the present invention. The LD50 (abbreviation for "Lethal Dose, 50%") or median lethal dose of a toxic substance or radiation is the dose required to kill half the members of a tested population, e.g., a mammalian cell or in an animal. LD₅₀ figures are frequently used as a general indicator of a substance's toxicity.

[0187] The LD50 is usually expressed as the mass of substance, e.g., a compound of the present invention, administered per unit mass of test subject, such as grams of substance per kilogram of body mass. Stating it this way allows the relative toxicity of different compounds to be compared, and normalizes for the variation in the size of the animals exposed. Typically, the LD₅₀ of a substance is given in milligrams per kilogram of body weight. Lethal dosage often varies depending on the method of administration; for instance, many substances are less toxic when taken by mouth than when intravenously addministered. For this reason, LD₅₀ figures are often qualified with the mode of administration, e.g. "LD₅₀ i.v.

J. Computer-Based Assays

[0188] As demonstrated herein, it is also possible to use structure-activity relationships (SAR) and structure-based design principles to identify compounds inhibiting a level or activity of an HDAC8 polypeptide. SARs provide information about the activity of related compounds or agents in at least one relevant assay. Correlations are made between structural features of an agent of interest and an activity. For example, it may be possible by evaluating SARs for a family of agents that interact with an HDAC8 polypeptide to identify one or more structural features required for activity. A library of agents can then be produced that vary these features, and then the library is screened. Structure-based design can include determining a structural model of the physical interaction of the agent and its target, such as an HDAC8 polypeptide. The structural model can indicate how an antagonist of the target can be engineered. Such antagonist may be useful in altering lifespan regulation.

[0189] Both the SAR and the structure-based design approach can be used to identify a pharmacophore. Pharmacophores are a highly valuable and useful concept in drug discovery and drug-lead optimization. A pharmacophore is defined as a distinct three dimensional (3D) arrangement of chemical groups essential for biological activity. Since a pharmaceutically active molecule must interact with one or more molecular structures within the body of the subject in order to be effective, and the desired functional properties of the molecule are derived from these interactions, each active compound must contain a distinct arrangement of chemical groups which enable this interaction to occur. The chemical groups, commonly termed descriptor centers, can be represented by (a) an atom or group of atoms; (b) pseudo- atoms, for example a center of a ring, or the center of mass of a molecule; (c) vectors, for example atomic pairs, electron lone pair directions, or the normal to a plane. Once formulated a pharmacophore can be used to search a database of chemical compound, e.g., for those having a structure compatible with the pharmacophore (see, for example, U.S. Pat. No. 6,343,257; Martin, 1992, JMedChem 35, 2145-54). Database search queries are based not only on chemical property information but also on precise geometric information.

[0190] Once a compound is identified that matches the pharmacophore, it can be tested for activity, e.g., for binding to a polypeptide and/or for modulating a biological activity of a polypeptide, e.g., decreasing the enzymatic activity of an HDAC8 polypeptide.

[0191] Thus, in one aspect of the present invention, a compound is identified that is designed to interact with an HDAC8 polypeptide or binds to an HDAC8 polypeptide by employing a structure of the HDAC8 polypeptide. Preferably the HDAC8 is a human HDAC8 (Somoza et al, 2004, Structure 12: 1325-1334; Vannini et al, 2004, Proc Natl Acad Sci USA 101(42): 15064-15069). However, structures of other mammalian HD AC8 polypeptides may also be used. Once the structure has been generated, potential binding regions for compounds are identified by the computer system and/or are selected by one of skill in the art. Three-dimensional structures for potential compounds are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described herein. The three-dimensional structure of the potential compound is then compared to that of HDAC8 to identify binding sites HDAC8. Binding affinity between the HDAC8 polypeptide and compounds is determined using energy terms to determine which compounds have an enhanced probability of binding to the HDAC8 polypeptide.

K. Detection Of HDAC8 mRNA

[0192] Methods for testing and assaying compounds, agents or antagonists identified by methods described herein, are provided herein and involve a variety of accepted tests to determine whether a given candidate agent, or compound is useful to practice a method of the present invention. Methods of the present invention may optionally comprise the step of detecting a nucleic acid, such as a mRNA or a polypeptide. In one embodiment, such a method comprises determining or detecting a mRNA, preferably an HDAC8 mRNA or an mRNA encoding a cellular substrate of HDAC8. Other mRNAs encoding polypeptides described herein, such as histone polypeptide or tubulin polypeptide can also be determined using the following methods. Methods of evaluating mRNA expression of a particular gene are well known to those of skill in the art, and include, inter alia, hybridization and amplification based assays.

1. Direct Hybridization-based Assays

[0193] Methods of detecting and/or quantifying the level of a gene transcript (mRNA or cDNA made therefrom) using nucleic acid hybridization techniques are known to those of skill in the art. For example, one method for evaluating the presence, absence, or quantity of a polynucleotide involves a Northern blot. Gene expression levels can also be analyzed by techniques known in the art, e.g., dot blotting, in situ hybridization, RNase protection, probing DNA microchip arrays, and the like (e.g., see Sambrook, J., Fritsch, E. F., and Maniatis, "Molecular Cloning A Laboratory Manual" by T. published by Cold Spring Harbor Laboratory Press, 2nd edition, 1989).

2. Amplification-based Assays [0194] In another embodiment, amplification-based assays are used to measure the expression level of a gene. In such an assay, the nucleic acid sequences act as a template in an amplification reaction {e.g., Polymerase Chain Reaction, or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the level of an mRNA in the sample. Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).

[0195] In one embodiment, a TaqMan based assay is used to quantify a polynucleotide. TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the 5' nuclease activity of the polymerase, e.g., AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification {see, for example, Heid et al., 1996, Genome Res 6(10):986-94; Morris et al., 1996, J Clin Microbiol 34(12):2933-6).

[0196] Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see, Wu and Wallace, 1989, Genomics 4:560; Landegren et al., 1988, Science 241 : 1077; and Barringer et al., 1990, Gene 89: 117), transcription amplification (Kwoh et al., 1989, Proc Natl Acad Sci USA 86: 1173), self-sustained sequence replication (Guatelli et al. , 1990, Proc Nat Acad Sci USA 87: 1874), dot PCR, and linker adapter PCR, etc.

L. High Throughput Assays

[0197] In one preferred embodiment of the present invention, high throughput screening methods are employed for identifying additional compounds inhibiting a level or activity of an HDAC8 polypeptide. High throughput assays involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds. Such "combinatorial chemical libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity, such as inhibiting a level or activity of an HDCA8 polypeptide. The compounds already identified herein serve as conventional "lead compounds" or can themselves be used as therapeutics. [0198] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, 1991, Int J Pept Prot Res 37:487- 493 (1991) and Houghton et al, 1991, Nature 354:84-88). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al., 1993, Proc Natl Acad Sci USA 90:6909-6913), vinylogous polypeptides (Hagihara et al, 1992, J Amer Chem Soc 114:6568), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., 1992, J Amer Chem Soc 114:9217-9218), analogous organic syntheses of small compound libraries (Chen et al., 1994, J Amer Chem Soc 116:2661), oligocarbamates (Cho et al., 1993, Science 261 : 1303), and/or peptidyl phosphonates (Campbell et al., 1994, J Org Chem 59:658), nucleic acid libraries (see Ausubel, Berger and Sambrook), peptide nucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibody libraries (see, e.g., Vaughn et al., 1996, Nature Biotechnology 14(3):309-314 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, 1996, Science, 274: 1520-1522 and U.S. Pat. No. 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, January 18, 1993, page 33; isoprenoids, U.S. Pat. No. 5,569,588; thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974; pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino compounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No. 5,288,514, and the like). Additional examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, 1993, Proc Natl Acad Sci USA 90:6909; Erb et al, 1994, Proc Natl Acad Sci USA 91 : 11422; Zuckemmann et al, 1994, J Med Chem 37:2678; Cho et al, 1993, Science 261 : 1303; Carrell et al, 1994, Angew Chem Int Ed Engl 33:2059; Carell et al, 1994, Angew Chem Int Ed Engl. 33:2061; and Gallop et al, 1994, JMed.Chem 37: 1233.

[0199] High throughput assays are often used in screening for modulators, i.e., identifying inhibitors and activators. Thus, in high throughput assays for identifying inhibitors for HDAC8 polypeptide, it is possible to screen up to several thousand different candidate agents or ligands in a single day. In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential agent, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single agent. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) agents. If 1536 well plates are used, then a single plate can easily assay from about 100 to about 1500 different agents. It is possible to assay several different plates per day; assay screens for up to about 6,000-20,000 different agents are possible using the integrated systems of the invention. More recently, microfluidic approaches to reagent manipulation have been developed.

[0200] The molecule of interest, such as an HDAC8 polypeptide, can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage, e.g., via a tag. The tag can be any of a variety of components. In general, a molecule that binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.

[0201] A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders (see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis Mo).

[0202] Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody-antigen interactions, receptor-ligand interactions are also appropriate as tag and tag-binder pairs, such as agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand interactions such as transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I (1993)). Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g., which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

[0203] Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

[0204] Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to those of skill in the art. For example, poly(ethylene glycol) linkers are available from Shearwater Polymers, Inc., Huntsville, Ala. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

[0205] Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature (see, e.g., Merrifield, 1965, Endeavour 24:3-7; Merrifield, 1964, Biochemistry 3: 1385-90; Merrifield and Stewart, 1965, Nature 207(996):522-3; Merrifield, 1965, Science 150(693): 178-85 (describing solid phase synthesis of, e.g., peptides); Geysen et al., 1987, J ImmunMeth 102:259-274 (describing synthesis of solid phase components on pins); Frank and Doring, 1988, Tetrahedron 44:6031-6040 (describing synthesis of various peptide sequences on cellulose disks); Fodor et al, 1991, Science 251 :767-777; Sheldon et al, 1993, Clinical Chemistry 39(4):718-719; and Kozal et al, 1996, Nature Medicine 2(7):753-759 (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

[0206] The invention provides in vitro assays for identifying, in a high throughput format, agents that can decrease a level or activity of and F1DAC8 polypeptide. Control reactions that measure a level or activity of HDAC8 in a reaction that does not include a potential inhibitor are optional, as the assays are highly uniform. Such optional control reactions are appropriate and increase the reliability of the assay. Accordingly, in some embodiments, the methods of the invention include such a control reaction. For each of the assay formats described, "no inhibitor" control reactions which do not include an inhibitor of HDAC8 provide a background level of binding activity.

V. METHODS FOR USING INHIBITORS FOR HDAC8

[0207] The compounds identified herein find use in a variety of methods, for example compounds can be used for (i) inhibiting a level or activity of an HDAC8 polypeptide, (ii) modulating a level, acetylation status, or activity of an HDAC8 substrate polypeptide, (iii), or (iii) treatment of a pathological condition, disorder or disease. These methods can be practiced in vitro and in vivo. Preferably, patients treated by any one of the methods are humans and non-human animals.

[0208] Some compounds within the scope of this invention are known compounds as such, but have not heretofore been shown to possess HDAC8 inhibitory function, or the pharmaceutical or pharmacological properties described herein. Yet other compounds of this invention are novel compounds that have been designed as described above and which have been found to inhibit a level or activity of an HDAC8 polypeptide.

[0209] According to the present invention, it has been ascertained that, in addition to the above-mentioned known functions for compounds 1, 2, 3, 4, and 6, these compounds also inhibit an activity of an HDAC8 polypeptide. Accordingly, in one aspect this invention relates to methods of inhibiting an HDAC8 polypeptide using compounds 1, 2, 3, 4, or 6.

A. Inhibiting Enzymatic Activity of HDAC8

[0210] In one aspect, the present invention provides a method for inhibiting the enzymatic activity of an HDAC8 polypeptide, i.e., the catalytic activity of histone deacetylation. In a preferred embodiment inhibiting the enzymatic activity of an HDAC8 polypeptide is selective.

[0211] Using novel compounds identified herein, such as Compounds 5, and 7-15 or compounds 1, 2, 3, 4, or 6 a level or activity of an HDAC8 polypeptide can be inhibited, i.e., decreased or repressed, in vitro or in vivo. Thus, in one aspect of the present invention, a method for inhibiting a level or an activity of an HDAC8 polypeptide is provided. In a preferred embodiment of the present invention, this method comprises the step of contacting an HDAC8 polypeptide with a compound described herein, wherein the level or activity of the HDAC8 polypeptide is inhibited. Agents identifiable by a screening method of the present invention can also be used to inhibit a level or activity of an HDAC8 polypeptide.

[0212] The HDAC8 polypeptide may be in a cell, preferably a mammalian cell and more preferred in a human cell. A preferred activity of HDAC8 polypeptide is the deacetylase activity of HDAC8, preferably the deacetylation of a histone.

[0213] The effect of the compounds in vitro or in vivo can be assayed as described herein.

[0214] The method of inhibiting a level or an activity of an HDAC8 polypeptide may include the step of determining the level or activity of the HDAC8 polypeptide prior to contacting the HDAC8 polypeptide with a compound of the present invention.

[0215] In another embodiment of the method of inhibiting a level or an activity of an HDAC8 polypeptide may include the step of determining the effect of the compound of the present invention on the level or activity of the HDAC8 polypeptide.

B. Increasing The Acetylation Status of Histone Polypeptide And Tubulin

Polypeptide

[0216] As shown herein, compounds of the present invention, as exemplified by Compounds 2 and 5, inhibit HDAC8 deacetylation of histone polypeptide and tubulin polypeptides (Figure 7). As a consequence of this inhibition, tubulin polypeptides and histone polypeptides become more acetylated in the presence of an compound of the present invention. Thus, in another aspect, the present method provides a method for increasing the acetylation status of a histone polypeptide or a tubulin polypeptide. In a preferred embodiment of the present invention, this method comprises the step of contacting an HDAC8 polypeptide with a compound described herein, wherein the contacting of said compound to the HDAC8 polypeptide in a cell comprising a histone polypeptide or a tubulin polypeptide results in an increase of the acetylation status of the histone polype4ptide or tubulin polypeptide. Assays for determining the acetylation status of an HDAC8 target polypeptide are described herein.

C. Inhibiting The Interaction Between HDAC8 And A Polypeptide [0217] Class I HDACs, such as HDAC8, play an important role in gene silencing as they are recruited to key locations in nucleosomes through their interactions with transcription complexes. (Ng and Bird, 2000, Trends Biochem Sci 25: 121-128). The interaction of HDAC8 with a transcription complex is indicative of a multiprotein complex forming in a mammalian cell.

[0218] Thus, in another aspect of the present invention, a method is provided for using a compound of the present invention in the inhibition of a complex formation between an HDAC8 polypeptide and a transcription factor. In a preferred embodiment of the present invention, this method comprises the step of contacting a cell expressing an HDAC8 polypeptide and a transcription factor interacting with an HDAC8 polypeptide with a compound of the present invention, wherein the interaction of HADC8 and a transcription factor is inhibited.

[0219] In yet another aspect, the present invention provides a method for modulating a level, acetylation status, or activity of an HDAC8 substrate. A preferred HDAC8 substrate is a histone polypeptide or a tubulin polypeptide. In a preferred embodiment of the present invention, this method comprises the step of contacting a sample comprising an HDAC8 polypeptide and an HDAC8 substrate with a compound described herein, wherein the level, acetylation status, or activity of the HDAC8 substrate is modulated.

[0220] The HDAC8 polypeptide and the HDAC8 substrate may be in a cell, preferably a mammalian cell and more preferred in a human cell. A preferred activity of an HDAC8 substrate is modulation of gene expression, i.e., increasing or decreasing either alone or in combination with a transcription factor the expression level of a gene of interest.

[0221] The effect of the agents in vitro or in vivo can be assayed as described herein.

D. Increasing Cellular Expression of Repressed HDAC8 Target Genes

[0222] In yet another aspect, the present invention provides a method for increasing the cellular expression of a repressed HDAC8 target gene. In a preferred embodiment of the present invention, this method comprises the step of contacting a cell comprising an HDAC8 polypeptide and a repressed HDAC8 target gene with a compound described herein, wherein the cellular expression of the repressed HDAC8 target gene is increased. E. Inhibiting Unwanted Growth, Proliferation Or Survival Of A Cell Using Compounds Of The Present Invention

[0223] Vannini et al, reported that short interfering RNAs (siRNAs), targeting HDAC8 inhibited cell proliferation in three tumor cell lines tested, A549, HeLa, and HCTl 16 (Vannini et al. , 2004, Proc Natl Acad Sci USA 101 (42): 15064- 15069).

[0224] Thus, in yet another aspect, the present invention provides a method for inhibiting unwanted growth, proliferation or survival of a cell. A preferred cell is a cancer cell. In a preferred embodiment of the present invention, this method comprises the step of contacting a cell comprising an HDAC8 polypeptide with a compound described herein, wherein the unwanted growth, proliferation or survival of the cell is inhibited.

F. Use of Compounds To Regulate Smooth Muscle Cell Contraction

[0225] HDAC 8 polypeptide is an unusual HDAC family member. Recent data suggest that HDAC8 polypeptide is constitutively localized to the cytoplasm and its expression in primary cells is restricted to smooth muscle (Waltregny et al, 2004, Am J Pathol 165:553- 564). Cell fractionation assays performed with primary human smooth muscle cells (HSMCs) showed that HDAC8, in contrast to HDACl and HDAC3, was enriched in cytoskeleton-bound protein fractions and insoluble cell pellets, suggesting an association of HDAC8 with the cytoskeleton. Coimmunoprecipitation experiments using HSMCs, NIH- 3T3 cells, and human prostate tissue lysates further demonstrated that HDAC8 associates with smooth muscle α-actin (α-SMA), but not with β-actin (Waltregny et al., 2005, Faseb J 19(8):966-8). Interestingly, RNAi ablation of HDAC8 in these cells by siRNA interference results in a contraction-deficient phenotype, i.e., the capacity of HSMCs to contract collagen lattices was strongly reduced (Waltregny et al, 2005, Faseb J 19:966-968). As such, it may be that HDAC8 deacetylates non-histone protein(s) reminiscent of the role of HDAC6 plays in deacetylating tubulin (Hubbert et al, 2002, Nature 417(6887):455-458). Identifying the proteins that are targets of HDAC8 may expand the range of targets and functions of the HDAC family yet further.

[0226] Thus, in another aspect, the present invention provides a method for regulating smooth muscle cell contraction. In a preferred embodiment of the present invention, this method comprises the step of contacting a smooth muscle cell comprising an HDAC8 polypeptide with a compound described herein, wherein the contraction of the smooth muscle cell is regulated. [0227] Using the compounds of the present invention to inhibit an HDAC8 polypeptide activity can lead to a relaxation of smooth muscle cell contraction. This has important implications in the treatment of diseases that involve smooth muscle cells, such as asthma, hypertension, and accelerated intestinal transit.

[0228] Preferably, the method of regulating smooth muscle cell contraction is performed in an animal, a human or a non-human animal.

G. Treatment Of A Pathological Condition

[0229] The compounds of the present invention are also useful in methods for the treatment of a pathological condition, disorder, or disease.

1. Acute Myeloid Leukemia

[0230] Recent evidence suggests that HDAC8 may play a role in one of the most frequent types of acute myeloid leukemia (AML). A common form of acute myeloid leukemia (AML) results from a chromosomal translocation, Inversion(lό), creating an abnormal fusion protein, Invl (Durst et al, 2003, MoI Cell Biol 23 :607-619). The inversion(lό) protein product (Invl) associates with HDAC8 into a protein complex and is associated with aberrant, constitutive genetic repression that is thought to cause the disease. The repression that is mediated by this complex is sensitive to HDAC inhibitors (Durst et al, 2003, MoI Cell Biol 23:607-619). As such, the specific HDAC8 inhibitors provided herein are useful for the treatment of AML.

[0231] Thus, a preferred pathological condition, disorder or disease that can be treated according to the present invention is acute myeloid leukemia. In addition, disorders related to, associated with or caused (directly or indirectly) by acute myeloid leukemia, are also amenable to treatment using a method according to the present invention.

[0232] Thus, in one aspect, the present invention provides a method for the treatment of an individual having acute myeloid leukemia. In a preferred embodiment, this method comprises the step of administering to an individual having acute myeloid leukemia a therapeutically effective amount of a compound that inhibits a level or activity of an HD AC8 polypeptide, wherein the individual having acute myeloid leukemia is treated.

[0233] In another preferred embodiment, the method for the treatment of an individual having acute myeloid leukemia comprises the step of administering a pharmaceutical composition to the individual; wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier wherein the individual having acute myeloid leukemia is treated.

2. Cancer

[0234] The removal of acetyl groups from histone promotes the condensation of chromatin, leading to the repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a use for HDAC inhibitors in oncology. Treatment with HDAC inhibitors causes tumor cells to cease growth and to either differentiate or become apoptotic. Several small molecules, including SAHA, are currently in clinical trials for oncology indications (Johnstone, 2002, Nat Rev Drug Di scov 1 :287-289).

[0235] Thus, another preferred pathological condition, disorder or disease that can be treated according to the present invention is cancer. In addition, disorders related to, associated with or caused (directly or indirectly) by cancer, are also amenable to treatment using a method according to the present invention.

[0236] Thus, in one aspect, the present invention provides a method for the treatment of an individual having cancer. In a preferred embodiment, this method comprises the step of administering to an individual having cancer a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having cancer is treated.

[0237] In another preferred embodiment, the method for the treatment of an individual having cancer comprises the step of administering a pharmaceutical composition to the individual; wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having cancer is treated.

3. Neurodegenerative Disorders [0238] Huntington's disease (HD) is an inherited, progressive neurological disorder that is caused by a CAG/polyglutamine repeat expansion and for which there is no effective therapy. Recent evidence indicated that transcriptional dysregulation may contribute to the molecular pathogenesis of this disease. Hockly et al. have conducted preclinical trials with SAHA in the R6/2 HD mouse model and reported that SAHA increased histone acetylation in the brain (Hockly et al., 2003, Proc Natl Acad Sci USA, 100:2041-2046). Further, SAHA dramatically improved the motor impairment in R6/2 mice, clearly validating the pursuit of this class of compounds as HD therapeutics (Hockly et al, 2003, Proc Natl Acad Sci USA, 100:2041- 2046).

[0239] Thus, another preferred pathological condition, disorder or disease that can be treated according to the present invention is a neurodegenerative disorder. In addition, disorders related to, associated with or caused (directly or indirectly) by a neurodegenerative disorder, are also amenable to treatment using a method according to the present invention.

[0240] A preferred neurodegenerative disorder is Huntington's disease, and amyotrophic lateral sclerosis (Lou Gehrig's).

[0241] Thus, in one aspect, the present invention provides a method for the treatment of an individual having a neurodegenerative disorder. In a preferred embodiment, this method comprises the step of administering to an individual having a neurodegenerative disorder a therapeutically effective amount of a compound that inhibits a level or activity of an HD AC8 polypeptide, wherein the individual having the neurodegenerative disorder is treated.

[0242] In another preferred embodiment, the method for the treatment of an individual having a neurodegenerative disorder comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the neurodegenerative disorder is treated.

4. Genetic Disorders [0243] Severe β-thalassemia (thalassemia major, Cooley anemia) is characterized by insufficient production of adult β-globin chains with subsequent excess of α-globin chains leading to ineffective erythropoiesis, intramedullar degradation of erythroid cells, and lifelong transfusion requirement of affected patients. One molecular treatment strategy of this disease comprises the reactivation of fetal γ-globin production to substitute for the lack of β-globin chains. Witt et al. have investigated the role of HDAC inhibitors for use in stimulating fetal hemoglobin expression in human K562 erythroleukemia cells (Witt et al, 2003, Blood 101 :2001-2007). They reported that apicidin (cyclo-[L-(2-amino-8- oxodecanoy^-L-fN-methoxytryptopha^-L-isoleucyl-D-pipecolinyl) was the most potent compound compared with other HDAC inhibitors, such as TSA, MS-275, HC-toxin, SAHA and previously tested compounds such as butyrate, phenylbutyrate, isobutyramide, hydroxyurea, 5-aza-cytidine (Witt et al, 2003, Blood 101 :2001-2007). Hyperacetylation of histones correlated with the ability of HDAC inhibitors to stimulate fetal hemoglobin expression (Witt et al, 2003, Blood 101 :2001-2007).

[0244] Thus, another preferred pathological condition, disorder or disease that can be treated according to the present invention is a genetic disorder. In addition, disorders related to, associated with or caused (directly or indirectly) by a genetic disorder, are also amenable to treatment using a method according to the present invention.

[0245] A preferred genetic disorder which can be treated using the compounds of the present invention to inhibit HDAC8, is β-thalassemia.

[0246] Thus, in one aspect, the present invention provides a method for the treatment of an individual having a genetic disorder. In a preferred embodiment, this method comprises the step of administering to an individual having a genetic disorder a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having the genetic disorder is treated.

[0247] In another preferred embodiment, the method for the treatment of an individual having a genetic disorder comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the genetic disorder is treated.

5. Antiviral Response [0248] RNA interference experiments indicated that several HDACs, including HDACl, HDAC8, and HDAC6 influence beta interferon (IFN-beta) gene expression in response to virus infection (Nusinzon and Horvath, 2006, MoI Cell Biol 26(8):3106-13). While HDACl and HDAC8 repressed IFN-beta expression, HDAC6 acted as a coactivator.

[0249] Thus, another preferred pathological condition, disorder or disease that can be treated according to the present invention is a viral infection. In addition, disorders related to, associated with or caused (directly or indirectly) by a viral infection, are also amenable to treatment using a method according to the present invention.

[0250] A viral infection that can be treated using the subject method is a viral infection caused by, e.g., Sendai virus and vesicular stomatitis virus (VSV). [0251] The HDAC8 inhibitors of the present invention may also find use in the reactivation of latent viruses such as varicella-roster virus (VZV), herpes, and human immunodeficiency virus (HIV).

[0252] Thus, in one aspect, the present invention provides a method for the treatment of an individual having a viral infection. In a preferred embodiment, this method comprises the step of administering to an individual having a viral infection a therapeutically effective amount of a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the individual having the viral infection is treated.

[0253] In another preferred embodiment, the method for the treatment of an individual having a viral infection comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the individual having the viral infection is treated.

[0254] Treatment of the viral infection by the subject method can me monitored by detecting, e.g., mRNA levels for beta interferon or beta interferon polypeptides. An increase of mRNA levels for beta interferon or beta interferon polypeptides indicates treatment.

[0255] The HDAC8 inhibitors of the present invention may also find use in the reactivation of latent viruses such as varicella-roster virus (VZV), herpes, and human immunodeficiency virus (HIV).

[0256] Thus, in one aspect, the present invention provides a method for the reactivation of a latent virus in a cell. In a preferred embodiment, this method comprises the step of contacting a cell with a compound that inhibits a level or activity of an HDAC8 polypeptide, wherein the latent virus in the cell is reactivated.

[0257] In another preferred embodiment, the method for the reactivation of a latent virus is performed in an individual and comprises the step of administering a pharmaceutical composition to the individual, wherein the pharmaceutical composition comprises a biologically active compound as described herein and a pharmaceutically acceptable carrier, wherein the latent virus in the individual is reactivated.

[0258] Reactivation of a latent virus by the subject method can me monitored by detecting, e.g., mRNA levels for a viral RNA or viral polypeptides. An increase of viral mRNA levels or viral polypeptides indicates reactivation of the latent virus. [0259] In another aspect of the present invention, a compound of the present invention is combined with a methylation inhibitor, such as 5-aza-2'deoxycytidine. Co administration of an HDAC8 inhibitor of the present invention and a methylation inhibitor may lead to a synergistic effect between the HDAC8 inhibitor and the methylation inhibitor (Cameron et al, 1999, Nat Genet 21(1): 103-7).

VL PHARMACEUTICAL COMPOSITIONS

A. Use Of Compounds In The Manufacture Of A Pharmaceutical Composition Or A Medicament

[0260] Compounds of the present invention are useful in the manufacture of a pharmaceutical composition or a medicament. A pharmaceutical composition or medicament can be administered to a subject for the treatment of, for example, a pathological condition or disease as described herein.

[0261] In one aspect, the present invention provides a pharmaceutical composition or a medicament comprising at least a compound as described herein that inhibits the level or activity of an HDAC8 polypeptide and a pharmaceutically acceptable carrier.

B. Formulation And Administration

[0262] Compounds and agents of the present invention and compounds and agents identified by a method of the present invention, are useful in the manufacture of a pharmaceutical composition or a medicament comprising an effective amount thereof in conjunction or mixture with excipients or carriers suitable for either enteral or parenteral application.

[0263] A preferred pharmaceutical composition for inhibiting a level or activity of an HDAC8 polypeptide comprises (i) a compound as described herein or a compound obtained or obtainable according to a subject screening method described herein, and (ii) a pharmaceutical acceptable carrier. The compound may be provided in a therapeutically effective dose for use in a method for treatment as described herein.

[0264] Pharmaceutical compositions or medicaments for use in the present invention can be formulated by standard techniques using one or more physiologically acceptable carriers or excipients. Suitable pharmaceutical carriers are described herein and in "Remington's Pharmaceutical Sciences" by E.W. Martin. Compounds and agents of the present invention and their physiologically acceptable salts and solvates can be formulated for administration by any suitable route, including via inhalation, topically, nasally, orally, parenterally, or rectally. Thus, the administration of the pharmaceutical composition may be made by intradermal, subdermal, intravenous, intramuscular, intranasal, intracerebral, intratracheal, intraarterial, intraperitoneal, intravesical, intrapleural, intracoronary or intratumoral injection, with a syringe or other devices. Transdermal administration is also contemplated, as are inhalation or aerosol administration. Tablets and capsules can be administered orally, rectally or vaginally.

[0265] For oral administration, a pharmaceutical composition or a medicament can take the form of, for example, a tablet or a capsule prepared by conventional means with a pharmaceutically acceptable excipient. Preferred are tablets and gelatin capsules comprising the active ingredient, i.e., a compound of the present invention, together with (a) diluents or fillers, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose (e.g., ethyl cellulose, microcrystalline cellulose), glycine, pectin, polyacrylates and/or calcium hydrogen phosphate, calcium sulfate, (b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt, metallic stearates, colloidal silicon dioxide, hydrogenated vegetable oil, corn starch, sodium benzoate, sodium acetate and/or polyethyleneglycol; for tablets also (c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone and/or hydroxypropyl methylcellulose; if desired (d) disintegrants, e.g., starches (e.g., potato starch or sodium starch), glycolate, agar, alginic acid or its sodium salt, or effervescent mixtures; (e) wetting agents, e.g., sodium lauryl sulphate, and/or (f) absorbents, colorants, flavors and sweeteners.

[0266] Tablets may be either film coated or enteric coated according to methods known in the art. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives, for example, suspending agents, for example, sorbitol syrup, cellulose derivatives, or hydrogenated edible fats; emulsifying agents, for example, lecithin or acacia; non-aqueous vehicles, for example, almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils; and preservatives, for example, methyl or propyl-p-hydroxybenzoates or sorbic acid. The preparations can also contain buffer salts, flavoring, coloring, and/or sweetening agents as appropriate. If desired, preparations for oral administration can be suitably formulated to give controlled release of the active compound. [0267] Compounds and agents of the present invention can be formulated for parenteral administration by injection, for example by bolus injection or continuous infusion. Formulations for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. Injectable compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are preferably 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. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use. In addition, they may also contain other therapeutically valuable substances. The compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.

[0268] For administration by inhalation, the compounds and agents, may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base, for example, lactose or starch.

[0269] Suitable formulations for transdermal application include an effective amount of a compound or agent of the present invention with carrier. Preferred carriers include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used.

[0270] Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives. [0271] The compounds and agents can also be formulated in rectal compositions, for example, suppositories or retention enemas, for example, containing conventional suppository bases, for example, cocoa butter or other glycerides.

[0272] Furthermore, the compounds and agents can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

[0273] The compositions can, if desired, be presented in a pack or dispenser device that can contain one or more unit dosage forms containing the active ingredient. The pack can, for example, comprise metal or plastic foil, for example, a blister pack. The pack or dispenser device can be accompanied by instructions for administration.

[0274] In one embodiment of the present invention, a pharmaceutical composition or medicament comprises (i) an effective amount of a compound as described herein that inhibits a level or activity of an HDAC8 polypeptide and (ii) another therapeutic agent. When used with a compound or an agent of the present invention, such therapeutic agent may be used individually, sequentially, or in combination with one or more other such therapeutic agents (e.g., a first therapeutic agent, a second therapeutic agent, and a compound of the present invention). Administration may be by the same or different route of administration or together in the same pharmaceutical formulation.

[0275] In one preferred embodiment, the therapeutic agent is a methylation inhibitor, such as 5-aza-2'deoxycytidine.

C. Therapeutic Effective Amount And Dosing [0276] In one embodiment of the present invention, a pharmaceutical composition or medicament is administered to a subject, preferably a human or a non-human animal, at a therapeutically effective dose to prevent, treat, or control a pathological condition or disease as described herein. The pharmaceutical composition or medicament is administered to a subject in an amount sufficient to elicit an effective therapeutic response in the subject. An effective therapeutic response is a response that at least partially arrests or slows the symptoms or complications of the pathological condition, disorder, or disease. An amount adequate to accomplish this is defined as "therapeutically effective dose" also referred to as "therapeutically effective amount."

[0277] The dosage of active agents administered is dependent on the species of warmblooded animal (mammal), the body weight, age, individual condition, surface area or volume of the area to be treated and on the form of administration. The size of the dose also will be determined by the existence, nature, and extent of any adverse effects that accompany the administration of a particular compound in a particular subject. A unit dosage for oral administration to a mammal of about 50 to 70 kg may contain between about 5 and 500 mg of the active ingredient. Typically, a dosage of the active compounds of the present invention, is a dosage that is sufficient to achieve the desired effect. Optimal dosing schedules can be calculated from measurements of agent accumulation in the body of a subject. In general, dosage may be given once or more daily, weekly, or monthly. Persons of ordinary skill in the art can easily determine optimum dosages, dosing methodologies and repetition rates.

[0278] The dosage of active agents administered is also dependent on the nature of the agent. For example, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks.

[0279] Exemplary doses of the compounds described herein, include milligram or microgram amounts of the compound per kilogram of subject or sample weight (e.g., about 1 microgram per-kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a compound depend upon the potency of the compound with respect to the expression or activity to be modulated. When one or more of these compounds is to be administered to an animal (e.g., a human) in order to inhibit, e.g., a level or activity of an HDAC8 polypeptide, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0280] In one embodiment of the present invention, a pharmaceutical composition or medicament comprising compounds or agents of the present invention is administered in a daily dose in the range from about 1 mg of each compound per kg of subject weight (1 mg/kg) to about lg/kg for multiple days. In another embodiment, the daily dose is a dose in the range of about 5 mg/kg to about 500 mg/kg. In yet another embodiment, the daily dose is about 10 mg/kg to about 250 mg/kg. In another embodiment, the daily dose is about 25 mg/kg to about 150 mg/kg. A preferred dose is about 10 mg/kg. The daily dose can be administered once per day or divided into subdoses and administered in multiple doses, e.g., twice, three times, or four times per day. However, as will be appreciated by a skilled artisan, compounds or agents identified by methods of the present invention may be administered in different amounts and at different times. The skilled artisan will also appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a compound can include a single treatment or, preferably, can include a series of treatments.

[0281] To achieve the desired therapeutic effect, compounds or agents may be administered for multiple days at the therapeutically effective daily dose. Thus, therapeutically effective administration of compounds to treat a pathological condition or disease described herein in a subject requires periodic (e.g., daily) administration that continues for a period ranging from three days to two weeks or longer. Typically, agents will be administered for at least three consecutive days, often for at least five consecutive days, more often for at least ten, and sometimes for 20, 30, 40 or more consecutive days. While consecutive daily doses are a preferred route to achieve a therapeutically effective dose, a therapeutically beneficial effect can be achieved even if the agents are not administered daily, so long as the administration is repeated frequently enough to maintain a therapeutically effective concentration of the agents in the subject. For example, one can administer the agents every other day, every third day, or, if higher dose ranges are employed and tolerated by the subject, once a week. [0282] Optimum dosages, toxicity, and therapeutic efficacy of such compounds or agents may vary depending on the relative potency of individual compounds or agents and can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, by determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio, LD₅₀ZED₅₀. Agents that exhibit large therapeutic indices are preferred. While agents that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such agents to the site of affected tissue to minimize potential damage to normal cells and, thereby, reduce side effects.

[0283] The data obtained from, for example, cell culture assays and animal studies can be used to formulate a dosage range for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any agents used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (the concentration of the agent that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography (HPLC). In general, the dose equivalent of agents is from about 1 ng/kg to 100 mg/kg for a typical subject.

[0284] Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the condition or disease treated.

D. Food, Drink, And Feed

[0285] Furthermore, the present invention relates to food, drink or feed with an activity to inhibit a level or activity of an HDAC8 polypeptide. Such food drink or feed can be produced by a general method for producing foods and drinks or feeds, including, adding an active compound as described herein, e.g., a compound that inhibits a level or activity of an HDAC8 polypeptide, to a raw or cooked material of the food, drink or feed. The food, drink or feed in accordance with the present invention can be molded and granulated in the same manner as generally used for foods, drinks or feeds. [0286] The concentration of the active compound is preferably 0.001 to 10 % by weight, more preferably 0.01 to 10 % by weight and most preferably 0.1 to 10 % by weight of the food, drink or feed comprising such active agent.

[0287] Specific foods or drinks, to which the active agent is added, include, for example, juices, refreshing drinks, soups, teas, sour milk beverages, dairy products such as fermented milks, ices, butter, cheese, yogurt, processed milk and skim milk, meat products such as ham, sausage, and hamburger, fish meat, cereal, bran, cake products, egg products such as seasoned egg rolls and egg curd, confectioneries such as cookie, jelly, snacks, and chewing gum, breads, noodles, pickles, smoked products, dried fishes, soy sauce-seasoned boiled foods and seasonings.

[0288] For example, SAHA could be administered orally in drinking water when complexed with cyclodextrins (Hockly et al, 2003, Proc Natl Acad Sci USA, 100:2041- 2046). This, in a preferred embodiment, a compound of the present invention is complexed with a cyclodextrin. "Cyclodextrin" includes α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin. Included within "cyclodextrin" are derivatives of cyclodextrin, e.g., ether, ester and amide derivatives and modified cyclodextrins as described in U.S. Patent Nos. 5,134,127 and 6,407,079.

[0289] Food, drinks and feed with an activity to inhibit a level or activity of an F1DAC8 polypeptide may be further supplemented with a nutritious composition (protein, lipid, saccharide, vitamins and/or mineral).

VII. KITS

[0290] For use in diagnostic, research, and therapeutic applications described above, kits are also provided by the present invention. In the diagnostic and research applications such kits may include any or all of the following: assay reagents, buffers, a compound, agent or compound of the present invention, an HDAC8 polypeptide, a histone polypeptide or any other polypeptide described herein, an HDAC8 nucleic acid, a histone nucleic acid or any other nucleic acid described herein, an anti-HDAC8 antibody, an anti-histone antibody, an anti-acetyl-lysine antibody, or any other antibody described herein, hybridization probes and/or primers detecting an HDAC8 nucleic acid, a histone nucleic acid or any other nucleic acid described herein, an HDACC8 expression construct, a histone expression construct or an expression construct for any other polypeptide described herein, or any other compound or composition described herein. A therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.

[0291] Reference to particular buffers, media, reagents, cells, culture conditions and the like, or to some subclass of the same, is not intended to be limiting, but should be read to include all such related materials that one of ordinary skill in the art would recognize as being of interest or value in the particular context in which they are presented. For example, it is often possible to substitute one buffer system or culture medium for another, such that a different but known way is used to achieve the same goals as those to which the use of a suggested method, material or composition is directed.

[0292] Typically, the components of a kit are provided in a container. In a preferred embodiment of the present invention, a kit for inhibiting a level or activity of an HDAC8 polypeptide comprises a container containing a compound as described herein or a compound obtained or obtainable according to a subject screening method.

[0293] In addition, a kit may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention. The instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit.

While the instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention. Such media include, but are not limited to electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,

CD ROM), and the like. Such media may include addresses to internet sites that provide such instructional materials.

[0294] In a preferred embodiment of the present invention, the kit comprises an instruction for contacting the compound to a mammalian cell for inhibiting a level or activity of an HDAC8 polypeptide. In a preferred embodiment, a compound inhibits HDAC8 deacetylase activity.

[0295] Optionally, the instruction comprises warnings of possible side effects and drug- drug or drug-food interactions.

[0296] A wide variety of kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. [0297] In a preferred embodiment of the present invention, the kit is a pharmaceutical kit and comprises a pharmaceutical composition comprising (i) a compound as described herein that inhibits a level or activity of an HDAC8 polypeptide, and (ii) a pharmaceutical acceptable carrier. Pharmaceutical kits optionally comprise an instruction stating that the pharmaceutical composition can or should be used for treating a pathological condition, disorder or disease or any other subject method described herein.

[0298] Additional kit embodiments of the present invention include optional functional components that would allow one of ordinary skill in the art to perform any of the method variations described herein.

[0299] Although the forgoing invention has been described in some detail by way of illustration and example for clarity and understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain variations, changes, modifications and substitution of equivalents may be made thereto without necessarily departing from the spirit and scope of this invention. As a result, the embodiments described herein are subject to various modifications, changes and the like, with the scope of this invention being determined solely by reference to the claims appended hereto. Those of skill in the art will readily recognize a variety of non-critical parameters that could be changed, altered or modified to yield essentially similar results.

[0300] While each of the elements of the present invention is described herein as containing multiple embodiments, it should be understood that, unless indicated otherwise, each of the embodiments of a given element of the present invention is capable of being used with each of the embodiments of the other elements of the present invention and each such use is intended to form a distinct embodiment of the present invention.

[0301] The referenced patents, patent applications, and scientific literature, including accession numbers to GenBank database sequences, referred to herein are hereby incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Likewise, any conflict between an art-understood definition of a word or phrase and a definition of the word or phrase as specifically taught in this specification shall be resolved in favor of the latter. [0302] As can be appreciated from the disclosure above, the present invention has a wide variety of applications. The invention is further illustrated by the following examples, which are only illustrative and are not intended to limit the definition and scope of the invention in any way.

VIII. EXAMPLES

Example 1: General Methods

A. Cell Culture

[0303] HEK293 and HeLa cells were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 LVmL penicillin, and 100 μg/mL streptomycin and grown in 5% CO₂ at 37⁰C.

B. Purification of HDAC Polypeptides; Preparation Of Protein Lysates, Cell Fractionation And Coimmunoprecipitations

[0304] Purification methods of HDAC8 polypeptides, preparation of HDAC8 polypeptides lystaes, cell fractionation and co-immunoprecipitations are described in North et al, Verdin et al, and Waltregny et al. (North et al, 2005, Methods, 36(4):338-45; Verdin et al, 2004, Methods Enzymol 377: 180-96; Waltregny et al, 2005, Faseb J, 19(6):966-8; incorporated by reference in their entireties).

C. SDS-PAGE, Western Blotting, And Antibodies

[0305] SDS-PAGE and western blotting was performed using standard laboratory methods. Western blots were developed with the ECL detection kit (Amersham Pharmacia Biotech, Piscataway, NJ) or West Supersignal reagent (Pierce). Membranes were either nitrocellulose or polyvinylidene fluoride (PVDF) (Immuno-Blot; Bio-Rad Laboratories). The following antisera were employed: antisera specific for acetyl-lysine (#9441, Cell Signalling Technology, Waltham, MA), for acetyl-tubulin (6-1 IB-I, Abeam, Cambridge, MA), tubulin (B-5-1-2. Abeam, Cambridge, MA), acetyl -Hi stone H4 (06-866, Upstate/Millipore).

D. HDAC8 Enzymatic Assays

[0306] In vitro deacetylation assay may be performed. Equimolar amounts of purified or recombinant HDAC8 and purified recombinant histone or tubulin are incubated in SDAC deacetylation buffer (50 mM Tris-HCl (pH 9.0), 4 mM MgCl₂, 50 mM NaCl, 0.5 mM DTT) in the presence or absence of NAD (1 mM), in the presence or absence of nicotinamide (10 mM), in the presence of trichostatin A (500 nM), or a Compound of the present invention for 3 h at 32⁰C. For time course deacetylation experiments, aliquots of the deacetylation reaction are removed at various time points, mixed with 10 mM nicotinamide and incubated on ice until further analysis. Reactions were analyzed by SDS-PAGE and immunoblotting.

1. Tritiated Histone Peptide Assay [0307] Compounds of the present invention were tested as inhibitors of recombinant

HDAC8 and immunoprecipitated HDACs 1 and 6 using a triatiated histone peptide assay as described (Verdin et al, 2004, Methods Enzymol 377: 180-196). Exemplary data are shown in Table 1.

E. Acetylated Protein Assay For Characterization Of HDAC8 Inhibitors [0308] HeIa cells or HEK293 cells were grown in RPMI medium containing 100 units/mL penicillin G sodium and 100 μg/mL streptomycin sulfate supplemented with 10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere. To test compounds for their ability to induce protein acetylation, log-phase cells were used. Serial dilutions of compounds were prepared and added to cells cultured in 6-well plates. If compounds were dissolved in DMSO, control wells contained the same amount of solvent (0.1% final concentration). Cells were treated with TSA, Compound 2 or Compound 5 for 17 hours. Cells were harvested by scraping, transferred to microfuge tubes and centrifuged. Pelleted cells were resuspended in Laemmli loading buffer, boiled for 5 minutes and loaded on 15% polyacrylamide gels. Gels were transferred to nitrocellulose membranes and analyzed by western blotting using antibodies described herein and in Figure 7.

Example 2: Chemical Synthesis And Characterization Of Compounds 1-5

[0309] Compounds 1-5 were synthesized from the corresponding carboxylic acids, which were purchased from Aldrich. The carboxylic acids (1.0 mmol) were combined with neat thionyl chloride (5.0 mmol) and heated to reflux for 1 hour with vigorous evolution of gas. Excess thionyl chloride was removed under reduced pressure, the crude material taken up in toluene (50 mL) and the toluene was removed by rotary evaporation (3x). The crude acid chloride was treated with a solution of hydroxylamine hydrochloride (4 mmol) and triethylamine (6 mmol) in THF/H₂O (5:1, 10 mL) and stirred at room temperature for 1 hour. The mixture was then acidified with 10% HCl and extracted with CH₂Cl₂ (3x 50 mL). The combined organic layers were dried with magnesium sulfate, filtered and the solvent removed by rotary evaporation. The crude hydroxamic acids were purified by recrystallization from ethanol/water (4:1). Typical yields from the starting carboxylic acids were >80%. [0310] Compound 1 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 7.34 (m, IH), 7.51 (m, 3H), 7.69 (m, 4H), 7.99 (s, IH), 9.07 (s, IH), 11.30 (s, IH). ¹³C NMR (DMSO- J₆, 100 MHz) δ = 164.21, 140.76, 140.01, 134.51, 129.90, 129.68, 129.56, 128.35, 127.36, 126.59, 125.53 ppm.

[0311] Compound 2 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 7.51 (m, 4H), 7.96 (m, 2H), 8.14 (m, IH), 9.24 (s IH), 11.09 (s, IH). ¹³C NMR (DMSO- J₆, 75 MHz) δ = 166.18, 133.65, 132.77, 130.56, 128.79, 127.33, 126.87, 126.02, 125.71, 125.54.

[0312] Compound 3 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 7.00 (m, 2H), 7.11 (m, 2H), 7.42 (m, 5H), 9.04 (s IH), 11.23 (s, IH). ¹³C NMR (DMSO- J₆, 75 MHz) δ = 117.14, 119.56, 119.87, 121.65, 122.19, 124.44, 124.58, 124.70, 130.72, 130.80, 163.88.

[0313] Compound 4 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 4.67 (s, IH), 7.21 (m, 4H), 7.29 (m, 6H), 8.94 (s, IH), 10.90 (s, IH). ¹³C NMR (DMSO- J₆, 100 MHz) δ = 168.55, 140.43, 128.97, 128.77, 127.28, 53.82 ppm.

[0314] Compound 5 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 6.52 (d, IH, J=28 Hz), 7.54 (m, 3H), 7.75 (d, IH, J= 7.1 Hz), 7.94 (m, 2H), 8.14 (m, IH), 8.17 (d, IH, J=28 Hz), 9.10 (s lH), 10.86 (s, IH). ¹³C NMR (DMSO- J₆, 100 MHz) δ = 163.11, 135.31, 133.85, 132.45, 131.26, 130.12, 129.21, 127.47, 126.79, 126.30, 124.94, 123.69, 122.75 ppm.

Example 3: Chemical Synthesis And Characterization Of Compound 6

[0315] To prepare Compound 6, a Suzuki coupling between 1-naphthyl boronic acid andp- bromobenzoic acid to make the/>-(l-naphthyl)benzoic acid is first performed which was then converted to its corresponding hydroxamate in the same manner (Figure 5). Briefly, the carboxylic acid precursor to Compound 6 (carboxylic acid A) was prepared by combining p- bromobenzoic acid (5.0 mmol) with 1-naphthyl boronic acid (5.0 mmol) and sodium carbonate (15 mmol) in methanol: water (3: 1, 50 mL). This solution was degassed with N₂ for 30 min, then treated with Pd(PPh₃)₄ (0.5 mmol) and heated to reflux under N₂ for 6 hr. The methanol was then removed by rotary evaporation and the remaining solution acidified with 10% HCl and extracted with CH₂Cl₂ (3x, 100 mL). The combined organic layers with extracted with aqueous sodium bicarbonate (3x, 50 mL) and the combined aqueous layers acidified with 10 % HCl, resulting in a thick white precipitate. The carboxylic acid precipitate was filtered and used in the next reaction (described above) without further purification.

[0316] Compound 6 was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 7.51 (m, 4H), 7.96 (m, 2H), 8.14 (m, IH), 9.24 (s IH), 11.09 (s, IH). ¹³C NMR (DMSO- d₆, 75 MHz) δ = 166.18, 133.65, 132.77, 130.56, 128.79, 127.33, 126.87, 126.02, 125.71, 125.54.

Example 4: Chemical Synthesis And Characterization Of Compounds 7, 8, And 9

[0317] Compound 7 is chemically synthesized following the scheme shown in Figure 9B.

[0318] Specifically, a solution of 5 mmol methyl cinnamate 1 (or related substrate) in 25 mL Et₂OZDMSO (2: 1) is added dropwise to a stirred suspension of 6 mmol NaH in Et₂O (10 mL). After 10 min, the reaction is diluted with water (100 mL) and extracted with Et₂O (3x 100 mL). The combined organic layers are dried with magnesium sulfate and the solvent is removed under reduced pressure. The aryl pyrrole ester 2 is then recrystallized from benzene. Hydrolysis of the ester is carried out by stirring 2 overnight in basic methanol/water solution, followed by acidification and extraction.

[0319] The acid is converted to the corresponding hydroxamate by treatment with 2 molar equivalents SOCl₂ under reflux for 2 hours. Excess thionyl chloride is removed under reduced pressure, and the crude acid chloride is treated with a solution of hydroxylamine hydrochloride (4 equivalents), triethylamine (6 equivalents) in THF/water (5: 1) and stirred at room temperature for 2 hours. The reaction is then poured into 2M HCl and extracted with CH₂Cl₂. The organic phase is dried with magnesium sulfate and evaporated under reduced pressure. The residue is recrystallized from 95% ethanol to yield the hydroxamic acid.

Example 5: Chemical Synthesis And Characterization Of Compounds 10 And 11 [0320] Compounds 10 and 11 are chemically synthesized following the scheme shown in Figure 1OB.

[0321] Specifically, bromothiophenecarboxylic acid (or bromofurancarboxylic acid) 1 is mixed with 1 molar equivalent of aryl boronic acid in ethanol with 2 equivalents of saturated aqueous potassium carbonate under nitrogen. Pd[P(Ph₃)J₄ is added (0.1 equivalents) and the mixture heated to reflux for 4 hours. Ethanol is then removed under reduced pressure and the residue acidified with 10 % HCl and extracted with EtOAc (3x). The combined organic layers is extracted with saturated aqueous bicarbonate. The bicarbonate is then acidified with 10% HCl, producing a precipitate, which is filtered.

[0322] The carboxylic acid 2 (Figure 10B) is refluxed in 2 molar equivalents SOCl₂ for 2 hours. Excess thionyl chloride is removed under reduced pressure, and the crude acid chloride is treated with a solution of hydroxy 1 amine hydrochloride (4 equivalents), triethylamine (6 equivalents) in THF/water (5: 1) and stirred at room temperature for 2 hours. The reaction was then poured into 2M HCl and extracted with CH₂Cl₂. The organic phase was dried with magnesium sulfate and evaporated under reduced pressure. The residue was recrystallized from 95% ethanol to yield the hydroxamic acid.

[0323] Similarly, naphthyl-substituted compounds were synthesized and characterized. 5- naphthalen- 1 -yl-thiophene-2-carboxylic acid

was characterized as follows: ¹U NMR (DMSO-J₆, 400 MHz) δ = 7.37 (d, IH, J= 3.68), 7.58 (m, 5H), 7.81 (d, IH, .7=3.68), 8.00 (m, IH), 8.11 (m, IH). ¹³C NMR (DMSO- J₆, 100 MHz) δ = 163.31, 148.05, 135.00, 134.16, 134.00, 131.17, 131.07, 129.89, 129.30, 129.13, 128.81, 127.80, 127.02, 126.07, 125.20 ppm.

[0324] 5-naphthalen-l-yl-thiophene-2-carboxylic acid hydroxyamide

was characterized as follows: ¹H NMR (DMSO-J₆, 400 MHz) δ = 7.34 (m, 4H), 7.56 (m, 3H), 7.99 (m, IH), 8.11 (m, IH), 9.20 (s, IH), 11.32 (s, IH). ¹³C NMR (DMSO- J₆, 100 MHz) δ = 156.69, 137.54, 134.01, 129.60, 129.38, 129.18, 129.09, 128.90, 128.67, 128.37, 128.31, 127.67, 126.95, 126.07, 125.30 ppm.

Example 6: Compounds 1-6 Inhibit HDAC8 Enzymatic Activity [0325] Compounds 1-6 were tested as inhibitors of recombinant HDAC8 using the tritiated histone peptide assay as described herein. The data, represented as IC₅₀ values in Table 1, show that these linkerless, sterically demanding aryl hydroxamates inhibit HDAC8 polypeptide. While some of the compounds are moderately potent in this assay, Compounds 5 and 6 have IC50 values of below 1 μM.

Table 1 : Linkerless Compounds 1-6 inhibit HD AC8.

Example 7: Compounds 1, 2, 5 And 6 Are Selective for HDACC8 OverHDACl And HDAC6 [0326] To examine the selectivity of the compounds of the present invention towards

HDAC8 some were also tested as inhibitors against other HDAC family members. For this analysis, HDACs from class I (HDACl) and class II (HD AC6) were chosen as representatives of the larger family. In particular, the inhibitory activity of Compounds 1, 2, 5, and 6 against HDACl and HDAC6 was tested to determine their selectivity towards HDAC8. Because HDACl and HDAC6 are difficult to express as recombinant proteins, immunoprecipitated HDACs 1 and 6 were used in the tritiated histone peptide assay as described herein. The data, represented as IC50 values in Table 2, show that all hydroxamates with the linkerless scaffold, as represented by Compounds 1, 2, 5 and 6 are > 100-fold selective for HDAC8 over HDACl. Further, the linkerless compounds 1 and 2 are >100-fold selective for HDAC8 over HDAC6 and the linkerless compounds 5 and 6 are 82 and 55 fold selective for HDAC8 over HDAC6, respectively. The less potent compounds may be even more selective, since they present steric bulk in closer proximity to the zinc binding group, potentially clashing with the narrow site seen in HDAH and HDLP.

Table 2: Linkerless Compounds 1, 2, 5 and 6 are selective for HDAC8

Example 8: Treatment Of Human Cells With Compounds 2 And 5 Results In

Different Acetylated Cellular Proteins

[0327] In order to determine the effect of the compounds of the present invention on the levels of lysine acetylated proteins in cells, HeLa and HEK293 cells were treated with Compounds 2, 5, and the broad spectrum HDAC inhibitor TSA. The cell lysate was then analyzed by Western blotting using ant-acetyllysine antibodies (Figures 7A, 7B). Treating either cell type with TSA increased the lysine acetylation level of three proteins. The same three proteins became hyperacetylated upon treatment with Compound 5. (Figures 7 A, 7B) However, when either cell type was treated with Compound 2, only the high molecular weight protein became hyperacetylated on lysine.

[0328] The high molecular weight protein was determined to be tubulin when the experiment was repeated with antibodies specific against acetyl tubulin and acetyl histone (Figures 7C, 7D). These data indicate that Compound 2 inhibits a restricted subset of HDACs (possibly only HDAC8) in cells and its target(s) do not deacetylates histones. Rather, the target(s) of Compound 2 deacetylate tubulin and possibly other non-histone proteins.

Example 9: Discussion

[0329] The level of selectivity for the HDAC8 inhibitors described herein is adequate for these compounds to be useful as HDAC8-specific inhibitor compounds to examine HDAC8's biological roles, identify its acetylation targets as well as determine the role of HDAC8 in the pathological conditions described herein, including cancer, AML, neurodegenerative dieses, genetic diseases and its role in antiviral responses.

[0330] Compounds 1-6 were designed based on a simple blocking effect, where the malleability of the HDAC8 active site with the inducible subpocket allows the bulky hydroxamates to access the catalytic zinc while HDACs with more rigid, narrow actie sites prohibit zinc chelation. Without being bound by theory, the relative potency of compounds 5 and 6 suggests that their selectivity is not due to a simple "blocking" effect. Rather, specific interactions between the aryl groups and the sub-pocket may be involved. Inspection of the CRA-A:HDAC8 co-crystal structure reveals that the primary pocket between amino acid residues F 152 and F208 where the aryl group bearing the hydroxamate binds is at a right angle to the induced sub-pocket (Figure 9). Interestingly, the more potent Compounds 5 and 6 have "T-shape" conformations accessible to them. The ability to potently bind the subpocket may require inhibitors to adopt this specific conformation. As such, the conformationally rigid Compounds 1-4 are less potent inhibitors of HDAC8 than Compounds 5 and 6.

[0331] The compounds of the present invention represent rationally designed, structure- based inhibitors which are useful for specific inhibition for an individual HDAC family member and a step towards the goal of a series of isoform-specific inhibitors for every HDAC member. The relatively simple structures of the compounds described herein may exploit the active-site malleability of HDAC8 and its unique subpocket, resulting in selective inhibition. As more HDAC family members are structurally characterized, it will be seen if HDACs that are insensitive to these compounds also lack the HDAC sub-pocket shown in Figure 3, which may emerge as a structural feature that can be exploited to generate additional selective inhibitors. Chemical tools have played a prominent role in HDAC biology since their discover (Taunton et al, 1996, Science 272:408-411).

[0332] The compounds described herein are useful tools to study the biological function of HDAC8 in smooth muscle cell contraction, identify its protein targets, as well as serve as lead compounds for the treatment of cancer, in particular AML, neurodegenerative diseases, genetic diseases and in regulating the response to viral infections.

Claims

WHAT IS CLAIMED IS:

1. A compound having the formula

2. A compound having the formula:

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkyl sulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl.

3. A compound having the formula:

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, - CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN.

4. A compound selected from the group consisting of

5. A compound having the formula

6. A compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN.

7. A compound having the formula

or

A compound having the formula

or

wherein X is oxygen or sulphur.

9. A pharmaceutical composition comprising:

(i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

(ii) a pharmaceutically acceptable excipient or carrier.

10. A pharmaceutical composition comprising:

(i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkyl sulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroarylsulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; and (ii) a pharmaceutically acceptable excipient or carrier.

11. A pharmaceutical composition comprising: (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN; and (ii) a pharmaceutically acceptable excipient or carrier.

12. A pharmaceutical composition comprising: (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

(ii) a pharmaceutically acceptable excipient or carrier.

13. A pharmaceutical composition comprising: (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

(ii) a pharmaceutically acceptable excipient or carrier.

14. A pharmaceutical composition comprising:

(i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN; and

(ii) a pharmaceutically acceptable excipient or carrier.

15. A pharmaceutical composition comprising:

(i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

or

(ii) a pharmaceutically acceptable excipient or carrier.

16. A pharmaceutical composition comprising: (i) a pharmaceutically effective amount to inhibit an HDAC8 polypeptide function of a compound having the formula

or

wherein X is oxygen or sulphur; and (ii) a pharmaceutically acceptable excipient or carrier.

17. A method for selectively inhibiting an activity of an HDAC8 polypeptide, the method comprising the step of contacting an HDAC8 polypeptide with an effective inhibiting amount of a compound having the formula: W - X - Y in which W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkyl carbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, and lower haloalkylthio; and

Y represents a zinc-binding moiety, or

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are as defined for moiety W.

18. The method of claim 17, wherein the zinc binding moiety is a hydroxamic acid group or a hydroxamic acid derivative.

19. The method of claim 17, wherein the compound is selected from the group consisting of compounds of the formula

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are as defined for moiety W;

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN,

wherein R₃ and R₄ are independently hydrogen, lower haloalkyl, lower alkyl, — CO₂H, — NO₂, lower alkyl carboxylate, lower alkoxy, or — CN,

20. The method of claim 17, wherein the activity of the HDAC8 polypeptide is a histone deacetylase activity or a tubulin deacetylase activity.

21. The method of claim 17, wherein the compound inhibits the HDAC8 polypeptide with an efficiency of greater than 50 fold over HDACl or HDAC6.

22. The method of claim 17, wherein the compound inhibits the HDAC8 polypeptide with an IC₅₀ of less than 1 μM.

23. The method of claim 17, which is practiced in vitro.

24. The method of claim 17, which is practiced in vivo.

25. The method of claim 24, wherein the compound is provided as a prodrug.

26. A method for regulating smooth muscle cell contraction in an animal, the method comprising the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula W - X - Y in which W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio; and

Y represents a zinc-binding moiety, or

Ri

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are the optional substituents are as defined for moiety W.

27. A method for treating a pathological condition characterized by an aberrant genetic repression of gene expression, the method comprising the step of administering to an animal an effective amount of a compound or a pharmaceutically acceptable salt thereof, the compound having the formula: W - X - Y in which W represents a phenyl, naphthyl, cycloalkyl, heteroaryl, or heterocycloalkyl group optionally substituted by one or more groups selected from optionally substituted phenyl, optionally substituted phenoxy, optionally substituted heteroaryloxy, optionally substituted naphthyl, optionally substituted heteroaryl, optionally substituted heterocycloalkyl, optionally substituted phenyl, carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted phenethyl, optionally substituted cycloalkyl, optionally substituted cycloalkyl-methyl, optionally substituted cycloalkyl-ethyl, optionally substituted heterocycloalkyl-methyl, optionally substituted heterocycloalkyl-ethyl, halogen, hydroxyl, carboxyl, lower alkyl carboxylate, sulfhydryl, cyano, isocyano, thiocycano, isothiocycano, nitro, amino, mono-and di- lower alkylamino, mono- and di- lower haloalkylamino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, optionally substituted phenyl sulfonyl, optionally substituted heteroaryl sulfonyl, methylenedioxy, lower alkylcarbonyl, lower alkylcarbamoyl, lower alkenyl, lower alkynyl, wherein the substituents for said optionally substituted phenyl, optionally substituted phenoxy, optionally substituted phenyl carbonyl, optionally substituted benzyl, optionally substituted benzyloxy, optionally substituted heteroaryl, optionally substituted heterocyloalkyl and optionally substituted naphthyl are selected from one or more of halogen, hydroxyl, sulfhydryl, cyano, isocyano, thiocycano, isothiocyano, nitro, amino, amido, lower alkyl, lower haloalkyl, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio, lower alkylsulfonyl, aminosulfonyl, lower alkylcarbamoyl, methylenedioxy, lower alkylcarbonyl, lower alkenyl, and lower alkynyl; provided that W does not represent a naphthyl group substituted by an optionally substituted naphthyl group;

X represents a bond, an optionally substituted C₁-C₃ alkylene group, an optionally substituted C₂-C₃ alkenylene group or -C≡C-, in which optional substituents are selected from one or more of halogen, hydroxy, lower alkoxy, lower alkylthio, lower haloalkoxy, lower haloalkylthio; and

Y represents a zinc-binding moiety, or

in which Z is -NH, O or S;

R₁ represents hydrogen or an optionally substituted lower alkyl group in which the optional substituents are as defined for moiety W; and

R₂ is an optionally substituted phenyl or naphthyl group in which the substituents are the optional substituents are as defined for moiety W.

28. The method of claim 27, wherein the animal is a human.

29. The method of claim 27, wherein the pathological condition is a cancer.

30. The method of claim 27, wherein the pathological condition is acute myeloid leukemia.