RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No. 60/679,923, filed May 11, 2005, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds that may be used to inhibit histone deacetylases (HDACs), as well as compositions of matter and kits comprising these compounds. The invention also relates to methods for inhibiting HDACs and treatment methods using compounds according to the present invention. In particular, the present invention relates to compounds, compositions of matter, kits and methods used to inhibit Class I HDACs, such as HDAC1, HDAC2, HDAC6 and HDAC8.

BACKGROUND OF THE INVENTION

DNA in eukaryotic cells is tightly complexed with proteins (histones) to form chromatin. Histones are small, positively charged proteins that are rich in basic amino acids (positively charged at physiological pH), which contact the phosphate groups (negatively charged at physiological pH) of DNA. There are five main classes of histones H1, H2A, H2B, H3, and H4. The amino acid sequences of H2A, H2B, H3, and H4 show remarkable conservation between species, wherein H1 varies somewhat and in some cases is replaced by another histone, e.g., H5. Four pairs of each of H2A, H2B, H3 and H4 together form a disk-shaped octomeric protein core, around which DNA (about 140 base pairs) is wound to form a nucleosome. Individual nucleosomes are connected by short stretches of linker DNA associated with another histone molecule to form a structure resembling a beaded string, which is itself arranged in a helical stack, known as a solenoid.

The majority of histones are synthesized during the S phase of the cell cycle, and newly synthesized histones quickly enter the nucleus to become associated with DNA. Within minutes of its synthesis, new DNA becomes associated with histones in nucleosomal structures.

A small fraction of histones, more specifically, the amino acid side chains thereof, are enzymatically modified by post-translational addition of methyl, acetyl, or phosphate groups, neutralizing the positive charge of the side chain, or converting it to a negative charge. For example, lysine and arginine groups may be methylated, lysine groups may be acetylated, and serine groups may be phosphorylated. For lysine, the â€”(CH₂)₄â€”NH₂sidechain may be acetylated, for example by an acetyltransferase enzyme to give the amide â€”(CH₂)₄â€”NHC(â• O)CH₃. Methylation, acetylation, and phosphorylation of amino termini of histones that extend from the nucleosomal core affect chromatin structure and gene expression. Spencer and Davie 1999. Gene 240:1 1-12.

Acetylation and deacetylation of histones is associated with transcriptional events leading to cell proliferation and/or differentiation. Regulation of the function of transcriptional factors is also mediated through acetylation. Recent reviews on histone deacetylation include Kouzarides et al., 1999, Curr. Opin. Genet. Dev. 9:1, 40-48 and Pazin et al., 1997, 89:3 325-328.

The correlation between acetylation status of histones and the transcription of genes has been known for quite some time. Certain enzymes, specifically acetylases (e.g., histone acetyltransferases (HAT) and deacetylases (histone deacetylases or HDACs), which regulate the acetylation state of histones have been identified in many organisms and have been implicated in the regulation of numerous genes, confirming a link between acetylation and transcription. In general, histone acetylation is believed to correlate with transcriptional activation, whereas histone deacetylation is believed to be associated with gene repression.

A growing number of histone deacetylases (HDACs) have been identified. HDACs function as part of large multiprotein complexes, which are tethered to the promoter and repress transcription. Well characterized transcriptional repressors such as MAD, nuclear receptors and YY1 associate with HDAC complexes to exert their repressor function.

Studies of HDAC inhibitors have shown that these enzymes play an important role in cell proliferation and differentiation. HDACs are believed to be associated with a variety of different disease states including, but not limited to cell proliferative diseases and conditions (Marks, P. A., Richon, V. M., Breslow, R. and Rifkind, R. A., J. Natl. Cancer Inst. (Bethesda) 92, 1210-1215, 2000) such as leukemia (Lin et al., 1998. Nature 391: 811-814; Grignani et al. 1998. Nature 391: 815-818; Warrell et al., 1998, J. Natl. Cancer Inst. 90:1621-1625; Gelmetti et al., 1998, Mol. Cell Biol. 18:7185-7191; Wang et al., 1998, PNAS 951 0860-10865), melanomas/squamous cell carcinomas (Gillenwater et al., 1998, Int. J. Cancer 75217-224; Saunders et al., 1999, Cancer Res. 59:399-404), breast cancer, prostrate cancer, bladder cancer (Gelmetti et al., 1998, Mol. Cell Biol. 18:7185-7191; Wang et al., 1998, PNAS 951 0860-10865), lung cancer, ovarian cancer, colon cancer (Hassig et al., 1997, Chem. Biol. 4:783-789; Archer et al., 1998, PNAS, 956791-6796; Swendeman et al., 1999, Proc. Amer. Assoc. Cancer Res. 40, Abstract #3836), and hyperproliferative skin disease such as cancerous and precancerous skin lesions, as well as inflammatory cutaneous disorders.

Histone deacetylase inhibitors are potent inducers of growth arrest, differentiation, or apoptotic cell death in a variety of transformed cells in culture and in tumor bearing animals (Histone deacetylase inhibitors as new cancer drugs, Marks, P. A., Richon, V. M., Breslow, R. and Rifkind, R. A., Current Opinions in Oncology, 2001, Nov. 13 (6): 477-83; Histone deacetylases and cancer: causes and therapies, Marks, P., Rifkind, R. A., Richon, V. M., Breslow, R., Miller, T. and Kelly, W. K., Nat. Rev. Cancer 2001 Dec. 1 (3):194-202). In addition, HDAC inhibitors are useful in the treatment or prevention of protozoal diseases (U.S. Pat. No. 5,922,837) and psoriasis (PCT Publication No. WO 02/26696).

Accordingly, despite the various HDAC inhibitors that have been reported to date, a need continues to exist for new and more effective inhibitors of HDACs.

SUMMARY OF THE INVENTION

The present invention relates to compounds that have activity for inhibiting histone deacetylases (HDACs). The present invention also provides compositions, articles of manufacture and kits comprising these compounds.

In one embodiment, a pharmaceutical composition is provided that comprises an HDAC inhibitor according to the present invention as an active ingredient. Pharmaceutical compositions according to the invention may optionally comprise 0.001%-100% of one or more HDAC inhibitors of this invention. These pharmaceutical compositions may be administered or coadministered by a wide variety of routes, including for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally. The compositions may also be administered or coadministered in slow release dosage forms.

The invention is also directed to kits and other articles of manufacture for treating disease states associated with one or more HDAC.

In one embodiment, a kit is provided that comprises a composition comprising at least one HDAC inhibitor of the present invention in combination with instructions. The instructions may indicate the disease state for which the composition is to be administered, storage information, dosing information and/or instructions regarding how to administer the composition. The kit may also comprise packaging materials. The packaging material may comprise a container for housing the composition. The kit may also optionally comprise additional components, such as syringes for administration of the composition. The kit may comprise the composition in single or multiple dose forms.

In another embodiment, an article of manufacture is provided that comprises a composition comprising at least one HDAC inhibitor of the present invention in combination with packaging materials. The packaging material may comprise a container for housing the composition. The container may optionally comprise a label indicating the disease state for which the composition is to be administered, storage information, dosing information and/or instructions regarding how to administer the composition. The kit may also optionally comprise additional components, such as syringes for administration of the composition. The kit may comprise the composition in single or multiple dose forms.

Also provided are methods for preparing compounds, compositions and kits according to the present invention. For example, several synthetic schemes are provided herein for synthesizing compounds according to the present invention.

Also provided are methods for using compounds, compositions, kits and articles of manufacture according to the present invention.

In one embodiment, the compounds, compositions, kits and articles of manufacture are used to inhibit one or more HDAC.

In another embodiment, the compounds, compositions, kits and articles of manufacture are used to treat a disease state for which one or more HDAC possesses activity that contributes to the pathology and/or symptomology of the disease state.

In another embodiment, a compound is administered to a subject wherein HDAC activity within the subject is altered, preferably reduced.

In another embodiment, a prodrug of a compound is administered to a subject that is converted to the compound in vivo where it inhibits one or more HDAC.

In another embodiment, a method of inhibiting one or more HDAC is provided that comprises contacting an HDAC with a compound according to the present invention.

In another embodiment, a method of inhibiting one or more HDAC is provided that comprises causing a compound according to the present invention to be present in a subject in order to inhibit the HDAC in vivo.

In another embodiment, a method of inhibiting an HDAC is provided that comprises administering a first compound to a subject that is converted in vivo to a second compound wherein the second compound inhibits the HDAC in vivo. It is noted that the compounds of the present invention may be the first or second compounds.

In another embodiment, a therapeutic method is provided that comprises administering a compound according to the present invention.

In another embodiment, a method of treating a condition in a patient which is known to be mediated by one or more HDAC, or which is known to be treated by HDAC inhibitors, comprising administering to the patient a therapeutically effective amount of a compound according to the present invention.

In another embodiment, a method is provided for treating a disease state for which one or more HDAC possesses activity that contributes to the pathology and/or symptomology of the disease state, the method comprising: causing a compound according to the present invention to be present in a subject in a therapeutically effective amount for the disease state.

In another embodiment, a method is provided for treating a disease state for which one or more HDAC possesses activity that contributes to the pathology and/or symptomology of the disease state, the method comprising: administering a first compound to a subject that is converted in vivo to a second compound such that the second compound is present in the subject in a therapeutically effective amount for the disease state. It is noted that the compounds of the present invention may be the first or second compounds.

In another embodiment, a method is provided for treating a disease state for which one or more HDAC possesses activity that contributes to the pathology and/or symptomology of the disease state, the method comprising: administering a compound according to the present invention to a subject such that the compound is present in the subject in a therapeutically effective amount for the disease state.

In another embodiment, a method is provided for using a compound according to the present invention in order to manufacture a medicament for use in the treatment of a disease state that is known to be mediated by one or more HDAC, or that is known to be treated by HDAC inhibitors.

It is noted in regard to all of the above embodiments that the present invention is intended to encompass all pharmaceutically acceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) of the compounds, regardless of whether such ionized forms and solvates are specified since it is well know in the art to administer pharmaceutical agents in an ionized or solvated form. It is also noted that unless a particular stereochemistry is specified, recitation of a compound is intended to encompass all possible stereoisomers (e.g., enantiomers or diastereomers depending on the number of chiral centers), independent of whether the compound is present as an individual isomer or a mixture of isomers. Further, unless otherwise specified, recitation of a compound is intended to encompass all possible resonance forms and tautomers. With regard to the claims, the language â€œcompound comprising the formulaâ€ is intended to encompass the compound and all pharmaceutically acceptable ionized forms and solvates, all possible stereoisomers, and all possible resonance forms and tautomers unless otherwise specifically specified in the particular claim.

It is further noted that prodrugs may also be administered which are altered in vivo and become a compound according to the present invention. The various methods of using the compounds of the present invention are intended, regardless of whether prodrug delivery is specified, to encompass the administration of a prodrug that is converted in vivo to a compound according to the present invention. It is also noted that certain compounds of the present invention may be altered in vivo prior to inhibiting HDAC and thus may themselves be prodrugs for another compound. Such prodrugs of another compound may or may not themselves independently have HDAC inhibitory activity.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

illustrates residues 1-482 of HDAC1 and a Flag tag at both the N- and C-terminus (SEQ. I.D. No. 1).

FIG. 2

illustrates the DNA sequence (SEQ. I.D. No. 2) that was used to encode SEQ. I.D. No. 1.

FIG. 3

illustrates residues 1-488 of HDAC2 and a 6-histidine tag at the C-terminus (SEQ. I.D. No. 3).

FIG. 4

illustrates the DNA sequence (SEQ. I.D. No. 4) that was used to encode SEQ. I.D. No. 3.

FIG. 5

illustrates residues 73-845 of HDAC6 and a 6-histidine tag at the C-terminus (SEQ. I.D. No. 5).

FIG. 6

illustrates the DNA sequence (SEQ. I.D. No. 6) that was used to encode SEQ. I.D. No. 5.

FIG. 7

illustrates residues 1-377 of HDAC8 and a 6-histidine tag at the N-terminus (SEQ. I.D. No. 7).

FIG. 8

illustrates the DNA sequence (SEQ. I.D. No. 8) that was used to encode SEQ. I.D. No. 7.

DEFINITIONS

Unless otherwise stated, the following terms used in the specification and claims shall have the following meanings for the purposes of this Application.

â€œAlicyclicâ€ means a moiety comprising a non-aromatic ring structure. Alicyclic moieties may be saturated or partially unsaturated with one, two or more double or triple bonds. Alicyclic moieties may also optionally comprise heteroatoms such as nitrogen, oxygen and sulfur. The nitrogen atoms can be optionally quaternerized or oxidized and the sulfur atoms can be optionally oxidized. Examples of alicyclic moieties include, but are not limited to moieties with C_3-8rings such as cyclopropyl, cyclohexane, cyclopentane, cyclopentene, cyclopentadiene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptene, cycloheptadiene, cyclooctane, cyclooctene, and cyclooctadiene.

â€œAliphaticâ€ means a moiety characterized by a straight or branched chain arrangement of constituent carbon atoms and may be saturated or partially unsaturated with one, two or more double or triple bonds.

â€œAlkoxyâ€ means an oxygen moiety having a further alkyl substituent. The alkoxy groups of the present invention can be optionally substituted.

â€œAlkylâ€ represented by itself means a straight or branched, saturated or unsaturated, aliphatic radical having a chain of carbon atoms, optionally with oxygen (see â€œoxaalkylâ€ ) or nitrogen atoms (see â€œaminoalkylâ€ ) between the carbon atoms. C_Xalkyl and C_X-Yalkyl are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C_1-6alkyl includes alkyls that have a chain of between 1 and 6 carbons (e.g., methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-methylallyl, ethynyl, 1-propynyl, 2-propynyl, and the like). Alkyl represented along with another radical (e.g., as in arylalkyl, heteroarylalkyl) means a straight or branched, saturated or unsaturated aliphatic divalent radical having the number of atoms indicated or when no atoms are indicated means a bond (e.g., (C_6-10)aryl(C_1-3)alkyl includes, benzyl, phenethyl, 1-phenylethyl, 3-phenylpropyl, 2-thienylmethyl, 2-pyridinylmethyl and the like).

â€œAlkenylâ€ means a straight or branched, carbon chain that contains at least one carbon-carbon double bond. Examples of alkenyl include vinyl, allyl, isopropenyl, pentenyl, hexenyl, heptenyl, 1-propenyl, 2-butenyl, 2-methyl-2-butenyl, and the like.

â€œAlkynylâ€ means a straight or branched, carbon chain that contains at least one carbon-carbon triple bond. Examples of alkynyl include ethynyl, propargyl, 3-methyl-1-pentynyl, 2-heptynyl and the like.

â€œAlkyleneâ€ , unless indicated otherwise, means a straight or branched, saturated or unsaturated, aliphatic, divalent radical. C_Xalkylene and C_X-Yalkylene are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C_1-6alkylene includes methylene (â€”CH₂â€”), ethylene (â€”CH₂CH₂â€”), trimethylene (â€”CH₂CH₂CH₂â€”), tetramethylene (â€”CH₂CH₂CH₂CH₂â€”)2-butenylene (â€”CH₂CHâ• CHCH₂â€”), 2-methyltetramethylene (â€”CH₂CH(CH₃)CH₂CH₂â€”), pentamethylene (â€”CH₂CH₂CH₂CH₂CH₂â€”) and the like.

â€œAlkenyleneâ€ means a straight or branched, divalent carbon chain having one or more carbon-carbon double bonds. Examples of alkenylene include ethene-1,2-diyl, propene-1,3-diyl, methylene-1,1-diyl, and the like.

â€œAlkynyleneâ€ means a straight or branched, divalent carbon chain having one or more carbon-carbon triple bonds. Examples of alkynylene include ethyne-1,2-diyl, propyne-1,3-diyl, and the like.

â€œAlkylideneâ€ means a straight or branched saturated or unsaturated, aliphatic radical connected to the parent molecule by a double bond. C_Xalkylidene and C_X-Yalkylidene are typically used where X and Y indicate the number of carbon atoms in the chain. For example, C_1-6alkylidene includes methylene (â• CH₂), ethylidene (â• CHCH₃), isopropylidene (â• C(CH₃)₂), propylidene (â• CHCH₂CH₃), allylidene (â• CHâ€”CHâ• CH₂), and the like).

â€œAminoâ€ means a nitrogen moiety having two further substituents where, for example, a hydrogen or carbon atom is attached to the nitrogen. For example, representative amino groups include â€”NH₂, â€”NHCH₃, â€”N(CH₃)₂, â€”NHC_1-10-alkyl, â€”N(C_1-10-alkyl)₂, â€”NHaryl, â€”NHheteroaryl, â€”N(aryl)₂, â€”N(heteroaryl)₂, and the like. Optionally, the two substituents together with the nitrogen may also form a ring. Unless indicated otherwise, the compounds of the invention containing amino moieties may include protected derivatives thereof. Suitable protecting groups for amino moieties include acetyl, tert-butoxycarbonyl, benzyloxycarbonyl, and the like.

â€œAminoalkylâ€ means an alkyl, as defined above, except where one or more substituted or unsubstituted nitrogen atoms (â€”Nâ€”) are positioned between carbon atoms of the alkyl. For example, an (C_2-6)aminoalkyl refers to a chain comprising between 2 and 6 carbons and one or more nitrogen atoms positioned between the carbon atoms.

â€œAnimalâ€ includes humans, non-human mammals (e.g., dogs, cats, rabbits, cattle, horses, sheep, goats, swine, deer, and the like) and non-mammals (e.g., birds, and the like).

â€œAromaticâ€ means a moiety wherein the constituent atoms make up an unsaturated ring system, all atoms in the ring system are sp²hybridized and the total number of pi electrons is equal to 4n+2. An aromatic ring may be such that the ring atoms are only carbon atoms or may include carbon and non-carbon atoms (see Heteroaryl).

â€œArylâ€ means a monocyclic or polycyclic ring assembly wherein each ring is aromatic or when fused with one or more rings forms an aromatic ring assembly. If one or more ring atoms is not carbon (e.g., N, S), the aryl is a heteroaryl. C_Xaryl and C_X-Yaryl are typically used where X and Y indicate the number of atoms in the ring.

â€œBicycloalkylâ€ means a saturated or partially unsaturated fused bicyclic or bridged polycyclic ring assembly.

â€œBicycloarylâ€ means a bicyclic ring assembly wherein the rings are linked by a single bond or fused and at least one of the rings comprising the assembly is aromatic. C_Xbicycloaryl and C_X-Ybicycloaryl are typically used where X and Y indicate the number of carbon atoms in the bicyclic ring assembly and directly attached to the ring.

â€œBridging ringâ€ as used herein refers to a ring that is bonded to another ring to form a compound having a bicyclic structure where two ring atoms that are common to both rings are not directly bound to each other. Non-exclusive examples of common compounds having a bridging ring include borneol, norbornane, 7-oxabicyclo[2.2.1]heptane, and the like. One or both rings of the bicyclic system may also comprise heteroatoms.

â€œCarbamoylâ€ means the radical â€”OC(O)NR_aR_bwhere R_aand R_bare each independently two further substituents where a hydrogen or carbon atom is attached to the nitrogen.

â€œCarbocycleâ€ means a ring consisting of carbon atoms.

â€œCarbocyclic ketone derivativeâ€ means a carbocyclic derivative wherein the ring contains a â€”COâ€” moiety.

â€œCarbonylâ€ means the radical â€”COâ€”. It is noted that the carbonyl radical may be further substituted with a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

â€œCarboxyâ€ means the radical â€”CO₂â€”. It is noted that compounds of the invention containing carboxy moieties may include protected derivatives thereof, i.e., where the oxygen is substituted with a protecting group. Suitable protecting groups for carboxy moieties include benzyl, tert-butyl, and the like.

â€œCyanoâ€ means the radical â€”CN.

â€œCycloalkylâ€ means a non-aromatic, saturated or partially unsaturated, monocyclic, fused bicyclic or bridged polycyclic ring assembly. C_Xcycloalkyl and C_X-Ycycloalkyl are typically used where X and Y indicate the number of carbon atoms in the ring assembly. For example, C_3-10cycloalkyl includes cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,5-cyclohexadienyl, bicyclo[2.2.2]octyl, adamantan-1-yl, decahydronaphthyl, oxocyclohexyl, dioxocyclohexyl, thiocyclohexyl, 2-oxobicyclo[2.2.1]hept-1-yl, and the like.

â€œCycloalkyleneâ€ means a divalent saturated or partially unsaturated, monocyclic or polycyclic ring assembly. C_Xcycloalkylene and C_X-Ycycloalkylene are typically used where X and Y indicate the number of carbon atoms in the ring assembly.

â€œDiseaseâ€ specifically includes any unhealthy condition of an animal or part thereof and includes an unhealthy condition that may be caused by, or incident to, medical or veterinary therapy applied to that animal, i.e., the â€œside effectsâ€ of such therapy.

â€œFused ringâ€ as used herein refers to a ring that is bonded to another ring to form a compound having a bicyclic structure when the ring atoms that are common to both rings are directly bound to each other. Non-exclusive examples of common fused rings include decalin, naphthalene, anthracene, phenanthrene, indole, furan, benzofuran, quinoline, and the like. Compounds having fused ring systems may be saturated, partially saturated, carbocyclics, heterocyclics, aromatics, heteroaromatics, and the like.

â€œHaloâ€ means fluoro, chloro, bromo or iodo.

â€œHalo-substituted alkylâ€ , as an isolated group or part of a larger group, means â€œalkylâ€ substituted by one or more â€œhaloâ€ atoms, as such terms are defined in this Application. Halo-substituted alkyl includes haloalkyl, dihaloalkyl, trihaloalkyl, perhaloalkyl and the like (e.g., halo-substituted (C1-3)alkyl includes chloromethyl, dichloromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, perfluoroethyl, 2,2,2-trifluoro-1,1-dichloroethyl, and the like).

â€œHeteroatomâ€ refers to an atom that is not a carbon atom. Particular examples of heteroatoms include, but are not limited to nitrogen, oxygen, and sulfur.

â€œHeteroatom moietyâ€ includes a moiety where the atom by which the moiety is attached is not a carbon. Examples of heteroatom moieties include â€”Nâ• , â€”NRcâ€”, â€”N+(Oâ€”)â• , â€”Oâ€”, â€”Sâ€” or â€”S(O)2-, wherein Rc is further substituent.

â€œHeterobicycloalkylâ€ means bicycloalkyl, as defined in this Application, provided that one or more of the atoms within the ring is a heteroatom. For example hetero(C9-12)bicycloalkyl as used in this application includes, but is not limited to, 3-aza-bicyclo[4.1.0]hept-3-yl, 2-aza-bicyclo[3.1.0]hex-2-yl, 3-aza-bicyclo[3.1.0]hex-3-yl, and the like.

â€œHeterocycloalkyleneâ€ means cycloalkylene, as defined in this Application, provided that one or more of the ring member carbon atoms is replaced by a heteroatom.

â€œHeteroarylâ€ means a cyclic aromatic group having three to fourteen ring atoms, wherein at least one ring atom is a heteroatom and the remaining ring atoms are carbon. The nitrogen atoms can be optionally quaternerized and the sulfur atoms can be optionally oxidized. Heteroaryl groups of this invention include, but are not limited to, those derived from furan, imidazole, isothiazole, isoxazole, oxadiazole, oxazole, 1,2,3-oxadiazole, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrroline, thiazole, 1,3,4-thiadiazole, triazole and tetrazole. â€œHeteroarylâ€ also includes, but is not limited to, bicyclic or tricyclic rings, wherein the heteroaryl ring is fused to one or two rings independently selected from the group consisting of an aryl ring, a cycloalkyl ring, a cycloalkenyl ring, and another monocyclic heteroaryl or heterocycloalkyl ring. These bicyclic or tricyclic heteroaryls include, but are not limited to, those derived from benzo[b]furan, benzo[b]thiophene, benzimidazole, imidazo[4,5-c]pyridine, quinazoline, thieno[2,3-c]pyridine, thieno[3,2-b]pyridine, thieno[2,3-b]pyridine, indolizine, imidazo[1,2a]pyridine, quinoline, isoquinoline, phthalazine, quinoxaline, naphthyridine, quinolizine, indole, isoindole, indazole, indoline, benzoxazole, benzopyrazole, benzothiazole, imidazo[1,5-a]pyridine, pyrazolo[1,5-a]pyridine, imidazo[1,2-a]pyrimidine, imidazo[1,2-c]pyrimidine, imidazo[1,5-a]pyrimidine, imidazo[1,5-c]pyrimidine, pyrrolo[2,3-b]pyridine, pyrrolo[2,3-c]pyridine, pyrrolo[3,2-c]pyridine, pyrrolo[3,2-b]pyridine, pyrrolo[2,3-d]pyrimidine, pyrrolo[3,2-d]pyrimidine, pyrrolo[2,3-b]pyrazine, pyrazolo[1,5-a]pyridine, pyrrolo[1,2-b]pyridazine, pyrrolo[1,2-c]pyrimidine, pyrrolo[1,2-a]pyrimidine, pyrrolo[1,2-a]pyrazine, triazo[1,5-a]pyridine, pteridine, purine, carbazole, acridine, phenazine, phenothiazene, phenoxazine, 1,2-dihydropyrrolo[3,2,1-hi]indole, indolizine, pyrido[1,2-a]indole and 2(1H)-pyridinone. The bicyclic or tricyclic heteroaryl rings can be attached to the parent molecule through either the heteroaryl group itself or the aryl, cycloalkyl, cycloalkenyl or heterocycloalkyl group to which it is fused. The heteroaryl groups of this invention can be substituted or unsubstituted.

â€œHeterobicycloarylâ€ means bicycloaryl, as defined in this Application, provided that one or more of the atoms within the ring is a heteroatom. For example, hetero(C4-12)bicycloaryl as used in this Application includes, but is not limited to, 2-amino-4-oxo-3,4-dihydropteridin-6-yl, tetrahydroisoquinolinyl, and the like.

â€œHeterocycloalkylâ€ means cycloalkyl, as defined in this Application, provided that one or more of the atoms forming the ring is a heteroatom selected, independently from N, O, or S. Non-exclusive examples of heterocycloalkyl include piperidyl, 4-morpholyl, 4-piperazinyl, pyrrolidinyl, perhydropyrrolizinyl, 1,4-diazaperhydroepinyl, 1,3-dioxanyl, 1,4-dioxanyl and the like.

â€œHydroxyâ€ means the radical â€”OH.

â€œIC₅₀â€ means the molar concentration of an inhibitor that produces 50% inhibition of the target enzyme.

â€œIminoketone derivativeâ€ means a derivative comprising the moiety â€”C(NR)â€”, wherein R comprises a hydrogen or carbon atom attached to the nitrogen.

â€œIsomersâ€ mean any compound having an identical molecular formulae but differing in the nature or sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed â€œstereoisomers.â€ Stereoisomers that are not mirror images of one another are termed â€œdiastereomersâ€ and stereoisomers that are nonsuperimposable mirror images are termed â€œenantiomersâ€ or sometimes â€œoptical isomers.â€ A carbon atom bonded to four nonidentical substituents is termed a â€œchiral center.â€ A compound with one chiral center has two enantiomeric forms of opposite chirality. A mixture of the two enantiomeric forms is termed a â€œracemic mixture.â€ A compound that has more than one chiral center has 2nâˆ’1 enantiomeric pairs, where n is the number of chiral centers. Compounds with more than one chiral center may exist as ether an individual diastereomer or as a mixture of diastereomers, termed a â€œdiastereomeric mixture.â€ When one chiral center is present a stereoisomer may be characterized by the absolute configuration of that chiral center. Absolute configuration refers to the arrangement in space of the substituents attached to the chiral center. Enantiomers are characterized by the absolute configuration of their chiral centers and described by the R- and S-sequencing rules of Cahn, Ingold and Prelog. Conventions for stereochemical nomenclature, methods for the determination of stereochemistry and the separation of stereoisomers are well known in the art (e.g., see â€œAdvanced Organic Chemistryâ€ , 4th edition, March, Jerry, John Wiley & Sons, New York, 1992).

â€œNitroâ€ means the radical â€”NO2.

â€œOxaalkylâ€ means an alkyl, as defined above, except where one or more oxygen atoms (â€”Oâ€”) are positioned between carbon atoms of the alkyl. For example, an (C2-6)oxaalkyl refers to a chain comprising between 2 and 6 carbons and one or more oxygen atoms positioned between the carbon atoms.

â€œOxoalkylâ€ means an alkyl, further substituted with a carbonyl group. The carbonyl group may be an aldehyde, ketone, ester, amide, acid or acid chloride.

â€œPharmaceutically acceptableâ€ means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic and neither biologically nor otherwise undesirable and includes that which is acceptable for veterinary use as well as human pharmaceutical use.

â€œPharmaceutically acceptable saltsâ€ means salts of compounds of the present invention which are pharmaceutically acceptable, as defined above, and which possess the desired pharmacological activity. Such salts include acid addition salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; or with organic acids such as acetic acid, propionic acid, hexanoic acid, heptanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, o-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, p-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, p-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4â€²-methylenebis(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid and the like.

Pharmaceutically acceptable salts also include base addition salts which may be formed when acidic protons present are capable of reacting with inorganic or organic bases. Acceptable inorganic bases include sodium hydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide and calcium hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like.

â€œProdrugâ€ means a compound that is convertible in vivo metabolically into an inhibitor according to the present invention. The prodrug itself may or may not also have HDAC inhibitory activity. For example, an inhibitor comprising a hydroxy group may be administered as an ester that is converted by hydrolysis in vivo to the hydroxy compound. Suitable esters that may be converted in vivo into hydroxy compounds include acetates, citrates, lactates, tartrates, malonates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-b-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates, quinates, esters of amino acids, and the like. Similarly, an inhibitor comprising an amine group may be administered as an amide that is converted by hydrolysis in vivo to the amine compound.

â€œProtected derivativesâ€ means derivatives of inhibitors in which a reactive site or sites are blocked with protecting groups. Protected derivatives are useful in the preparation of inhibitors or in themselves may be active as inhibitors. A comprehensive list of suitable protecting groups can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

â€œRingâ€ means a carbocyclic or a heterocyclic system.

â€œSubstituted or unsubstitutedâ€ means that a given moiety may consist of only hydrogen substituents through available valencies (unsubstituted) or may further comprise one or more non-hydrogen substituents through available valencies (substituted) that are not otherwise specified by the name of the given moiety. For example, isopropyl is an example of an ethylene moiety that is substituted by â€”CH₃. In general, a non-hydrogen substituent may be any substituent that may be bound to an atom of the given moiety that is specified to be substituted. Examples of substituents include, but are not limited to, aldehyde, alicyclic, aliphatic, (C_1-10)alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aromatic, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo, heterobicycloalkyl, heterocycloalkylene, heteroaryl, heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted.

â€œSulfinylâ€ means the radical â€”SOâ€”. It is noted that the sulfinyl radical may be further substituted with a variety of substituents to form different sulfinyl groups including sulfinic acids, sulfinamides, sulfinyl esters, and sulfoxides.

â€œSulfonylâ€ means the radical â€”SO₂â€”. It is noted that the sulfonyl radical may be further substituted with a variety of substituents to form different sulfonyl groups including sulfonic acids, sulfonamides, sulfonate esters, and sulfones.

â€œTherapeutically effective amountâ€ means that amount which, when administered to an animal for treating a disease, is sufficient to effect such treatment for the disease.

â€œThiocarbonylâ€ means the radical â€”C(S)â€”. It is noted that the thiocarbonyl radical may be further substituted with a variety of substituents to form different thiocarbonyl groups including thioacids, thioamides, thioesters, and thioketones.

â€œTreatmentâ€ or â€œtreatingâ€ means any administration of a compound of the present invention and includes:

(1) preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display the pathology or symptomatology of the disease,

(2) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology), or

(3) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).

It is noted in regard to all of the definitions provided herein that the definitions should be interpreted as being open ended in the sense that further substituents beyond those specified may be included. Hence, a C₁alkyl indicates that there is one carbon atom but does not indicate what are the substituents on the carbon atom. Hence, a C₁alkyl comprises methyl (i.e., â€”CH₃) as well as â€”CR_aR_bR_cwhere R_a, R_b, and R_cmay each independently be hydrogen or any other substituent where the atom attached to the carbon is a heteroatom or cyano. Hence, CF₃, CH₂OH and CH₂CN, for example, are all C₁alkyls.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds, compositions, kits and articles of manufacture that may be used to inhibit histone deacetylases (HDACs) and, in particular, Class I HDACs such as HDAC1, HDAC2, HDAC6 and HDAC8.

At least seventeen human genes that encode proven or putative HDACs have been identified to date, some of which are described in Johnstone, R. W., â€œHistone-Deacetylase Inhibitors: Novel Drugs for the Treatment of Cancerâ€ , Nature Reviews, Volume I, pp. 287-299, (2002) and PCT Publication Nos. 00/10583, 01/18045, 01/42437 and 02/08273.

HDACs have been categorized into three distinct classes based on their relative size and sequence homology. The different HDACs (Homo sapiens), HDAC classes, sequences and references describing the different HDACs are provided in Tables 1-3.

TABLE 1CLASS I HDACs

GenBankHDACAccession NumberReference

1NP_004955Histone deacetylase: a regulator of transcription,Wolffe, A. P., Science 272 (5260), 371-372 (1996)2NP_001518Isolation and mapping of a human gene (RPD3L1) thatis homologous to RPD3, a transcription factor inSaccharomyces cerevisiae; Furukawa, Y.,Kawakami, T., Sudo, K., Inazawa, J., Matsumine, A.,Akiyama, T. and Nakamura, Y., Cytogenet. Cell Genet.73 (1-2), 130-133 (1996)3NP_003874Isolation and characterization of cDNAs correspondingto an additional member of the human histonedeacetylase gene family, Yang, W. M., Yao, Y. L.,Sun, J. M., Davie, J. R. and Seto, E., J. Biol. Chem. 272(44), 28001-28007 (1997)8NP_060956Buggy, J. J., Sideris, M. L., Mak, P., Lorimer, D. D.,McIntosh, B. and Clark, J. M.Biochem. J. 350 Pt 1, 199-205 (2000)11NP_079103Cloning and Functional Characterization of HDAC11,a Novel Member of the Human Histone DeacetylaseFamily, Gao, L., Cueto, M. A., Asselbergs, F. andAtadja, P., J. Biol. Chem. 277 (28), 25748-25755(2002)

TABLE 2CLASS II HDACs

GenBankHDACAccession NumberReference

4NP_006028Transcriptional control. Sinful repression, Wolffe, A. P.,Nature 387 (6628), 16-17 (1997)5NP_631944Prediction of the coding sequences of unidentifiedhuman genes. IX. The complete sequences of 100 newcDNA clones from brain which can code for largeproteins in vitro, Nagase, T., Ishikawa, K., Miyajima, N.,Tanaka, A., Kotani, H., Nomura, N. and Ohara, O., DNARes. 5 (1), 31-39 (1998)6NP_006035Transcriptional control. Sinful repression, Wolffe, A. P.,Nature 387 (6628), 16-17 (1997)7NP_057680Isolation of a novel histone deacetylase reveals thatclass I and class II deacetylases promote SMRT-mediated repression, Kao, H. Y., Downes, M.,Ordentlich, P. and Evans, R. M., Genes Dev. 14 (1), 55-66(2000)9NP_478056MEF-2 function is modified by a novel co-repressor,MITR, Sparrow, D. B., Miska, E. A., Langley, E.,Reynaud-Deonauth, S., Kotecha, S., Towers, N.,Spohr, G., Kouzarides, T. and Mohun, T. J., EMBO J. 18(18), 5085-5098 (1999)10NP_114408Isolation and characterization of mammalianHDAC10, a novel histone deacetylase, Kao, H. Y.,Lee, C. H., Komarov, A., Han, C. C. and Evans, R. M., J.Biol. Chem. 277 (1), 187-193 (2002)

TABLE 3CLASS III HDACs

GenBankHDACAccession NumberReferenceSirtuin 1NP_036370Characterization of five human cDNAs with homologyto the yeast SIR2 gene: Sir2-like proteins (sirtuins)metabolize NAD and may have protein ADP-ribosyltransferase activity; Frye, R. A.; Biochem.Biophys. Res. Commun. 260 (1), 273-279 (1999)Sirtuin 2NP_085096/A â€˜double adaptorâ€™ method for improved shotgunNP_036369library construction; Andersson, B., Wentland, M. A.,Ricafrente, J. Y., Liu, W. and Gibbs, R. A.; Anal.Biochem. 236 (1), 107-113 (1996)Sirtuin 3NP_036371Characterization of five human cDNAs with homologyto the yeast SIR2 gene: Sir2-like proteins (sirtuins)metabolize NAD and may have protein ADP-ribosyltransferase activity; Frye, R. A.; Biochem.Biophys. Res. Commun. 260 (1), 273-279 (1999)Sirtuin 4NP_036372Characterization of five human cDNAs with homologyto the yeast SIR2 gene: Sir2-like proteins (sirtuins)metabolize NAD and may have protein ADP-ribosyltransferase activity; Frye, R. A.; Biochem.Biophys. Res. Commun. 260 (1), 273-279 (1999)Sirtuin 5NP_112534/Characterization of five human cDNAs with homologyNP_036373to the yeast SIR2 gene: Sir2-like proteins (sirtuins)metabolize NAD and may have protein ADP-ribosyltransferase activity; Frye, R. A.; Biochem.Biophys. Res. Commun. 260 (1), 273-279 (1999)Sirtuin 6NP_057623Phylogenetic classification of prokaryotic andeukaryotic Sir2-like proteins; Frye, R. A.; Biochem.Biophys. Res. Commun. 273 (2), 793-798 (2000)Sirtuin 7NP_057622Phylogenetic classification of prokaryotic andeukaryotic Sir2-like proteins; Frye, R. A.; Biochem.Biophys. Res. Commun. 273 (2), 793-798 (2000)

Of particular note are Class I HDACs. All Class I HDACs appear to be sensitive to inhibition by trichostatin A (TSA). Of particular note HDAC2 and HDAC8, proteins whose crystal structures Applicants determined and used in conjunction with arriving at the present invention.

HDAC2 is a 488 residue, 55 kDa protein localized to the nucleus of a wide array of tissues, as well as several human tumor cell lines. The wild-type form of full length HDAC2 is described in GenBank Accession Number NM 001527, Furukawa, Y. et al., Cryogenet. Cell Genet., 73 (1-2), 130-133 (1996). Zn²⁺ is likely native to the protein and required for HDAC2 activity.

HDAC8 is a 377 residue, 42 kDa protein localized to the nucleus of a wide array of tissues, as well as several human tumor cell lines. The wild-type form of full length HDAC8 is described in GenBank Accession Number NP 060956; Buggy, J. J. et al., Biochem. J., 350 (Pt 1), 199-205 (2000). Zn²⁺ is likely native to the protein and required for HDAC8 activity.

It is noted that the compounds of the present invention may also possess inhibitory activity for other HDAC family members and thus may be used to address disease states associated with these other family members.

Crystal Structure of Histone Deacetylase

Takeda San Diego, Inc. (formerly Syrrx, Inc.) solved the crystal structure for HDAC2 (U.S. patent Ser. Nos. 10/826,134 and 10/826,170, both filed Apr. 16, 2004, each of which is hereby incorporated by reference in its entirety) and HDAC8 (U.S. patent Ser. Nos. 10/601,058 and 10/601,335, both filed Jun. 20, 2003, each of which is hereby incorporated by reference in its entirety).

HDAC2 was found to adopt an open-faced Î±/Î² structure consisting of 8 central parallel Î²-sheets sandwiched between 12 Î±-helices. The ligand binding cleft lies almost in the plane of the central Î²-sheet, and is formed primarily by loops emanating from the carboxy-terminal ends of the Î²-strands comprising the sheet. Residues which form loop regions extending between Î²-strand 1 and Î±-helix 1 and between Î±-helix 4 and Î±-helix 5, provide key surface interactions with bound ligands. Residues which form loop regions extending between Î²-strand 3 and Î±-helix 6 and between Î²-strand 4 and Î±-helix 7 and between Î²-strand 8 and Î±-helix 10 play important roles in defining the shape of the ligand binding pocket, and are involved in a number of key interactions with the bound ligands.

HDAC8 was found to have a single domain structure belonging to the open Î±/Î² class of folds. The structure consists of a central 8-stranded parallel Î²-sheet sandwiched between layers of Î±-helices. The ligand binding clefts lie almost in the plane of the central Î²-sheet, and are formed primarily by loops emanating from the carboxy-terminal ends of the Î²-strands comprising the sheet. There are two large structural extensions, which occur beyond the core of the Î±/ [Image Omitted] motif, off the second and last Î²-strands of the central Î²-sheet. Residues contained in the extension off the second Î²-strand form a globular â€œcapâ€ over the core of the protein, play an important role in defining the shape of the ligand binding pockets, and are involved in a number of key interactions with the bound ligands.

Knowledge of the crystal structures was used to guide the design of the HDAC inhibitors provided herein.

HDAC Inhibitors

In one embodiment, HDAC inhibitors of the present invention comprise:

[Image Omitted]

wherein:

- n is selected from the group consisting of 0, 1, 2, 3 and 4;
- A₁is selected from the group consisting of (C_3-12)cycloalkyl, hetero(C_3-12)cycloalkyl, (C_9-12)bicycloalkyl, hetero(C_3-12)bicycloalkyl, aryl, heteroaryl, (C_9-12)bicycloaryl, and hetero(C_4-12)bicycloaryl, each substituted or unsubstituted;
- L is a linker providing a 1-6 atom separation between A₁and the N to which L is attached;
- X is selected from the group consisting of CH₂, CS, SO and SO₂;
- Y is selected from the group consisting of oxo, sulfo and R₁₀;
- R₁and R₂are each independently selected from the group consisting of hydrogen, hydroxy, alkoxy, aryloxy, heteroaryloxy, carbonyl, amino, (C_1-10)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (C_1-10)alkyl, halo(C_1-10)alkyl, carbonyl(C_1-3)alkyl, thiocarbonyl(C_1-3)alkyl, sulfonyl(C_1-3)alkyl, sulfinyl(C_1-3)alkyl, amino(C_1-10)alkyl, imino(C_1-3)alkyl, (C_3-12)cycloalkyl(C_1-5)alkyl, hetero(C_3-12)cycloalkyl(C_1-5)alkyl, aryl(C_1-10)alkyl, heteroaryl(C_1-5)alkyl, (C_9-12)bicycloaryl(C_1-5)alkyl, hetero(C_8-12)bicycloaryl(C_1-5)alkyl, (C_3-12)cycloalkyl, hetero(C_3-12)cycloalkyl, (C_9-12)bicycloalkyl, hetero(C_3-12)bicycloalkyl, aryl, heteroaryl, (C_9-12)bicycloaryl and hetero(C_4-12)bicycloaryl, each substituted or unsubstituted, or R₁and R₂are taken together to form a ring;
- R₃is selected from the group consisting of hydrogen, hydroxy, alkoxy, aryloxy, heteroaryloxy, carbonyl, amino, (C_1-10)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (C_1-10)alkyl, halo(C_1-10)alkyl, carbonyl(C_1-3)alkyl, thiocarbonyl(C_1-3)alkyl, sulfonyl(C_1-3)alkyl, sulfinyl(C_1-3)alkyl, amino(C_1-10)alkyl, imino(C_1-3)alkyl, (C_3-12)cycloalkyl(C_1-5)alkyl, hetero(C_3-12)cycloalkyl(C_1-5)alkyl, aryl(C_1-10)alkyl, heteroaryl(C_1-5)alkyl, (C_9-12)bicycloaryl(C_1-5)alkyl, hetero(C_8-12)bicycloaryl(C_1-5)alkyl, (C_3-12)cycloalkyl, hetero(C_3-12)cycloalkyl, (C_9-12)bicycloalkyl, hetero(C_3-12)bicycloalkyl, aryl, heteroaryl, (C_9-12)bicycloaryl and hetero(C_4-12)bicycloaryl, each substituted or unsubstituted;
- each R₄is independently selected from the group consisting of hydrogen, halo, nitro, cyano, thio, hydroxy, alkoxy, aryloxy, heteroaryloxy, carbonyl, amino, (C_1-10)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (C_1-10)alkyl, halo(C_1-10)alkyl, carbonyl(C_1-3)alkyl, thiocarbonyl(C_1-3)alkyl, sulfonyl(C_1-3)alkyl, sulfinyl(C_1-3)alkyl, amino (C_1-10)alkyl, imino(C_1-3)alkyl, (C_3-12)cycloalkyl(C_1-5)alkyl, hetero(C_3-12)cycloalkyl(C_1-5)alkyl, aryl(C_1-10)alkyl, heteroaryl(C_1-5)alkyl, (C_9-12)bicycloaryl(C_1-5)alkyl, hetero(C_8-12)bicycloaryl(C_1-5)alkyl, (C_3-12)cycloalkyl, hetero(C_3-12)cycloalkyl, (C_9-12)bicycloalkyl, hetero(C_3-12)bicycloalkyl, aryl, heteroaryl, (C_9-12)bicycloaryl and hetero(C_4-12)bicycloaryl, each substituted or unsubstituted;
- R₅, R₆, R₇and R₈are each independently selected from the group consisting of hydrogen, halo, nitro, cyano, thio, hydroxy, alkoxy, aryloxy, heteroaryloxy, carbonyl, amino, (C_1-10)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (C_1-10)alkyl, halo(C_1-10)alkyl, carbonyl(C_1-3)alkyl, thiocarbonyl(C_1-3)alkyl, sulfonyl(C_1-3)alkyl, sulfinyl(C_1-3)alkyl, amino (C_1-10)alkyl, imino(C_1-3)alkyl, (C_3-12)cycloalkyl(C_1-5)alkyl, hetero(C_3-12)cycloalkyl(C_1-5)alkyl, aryl(C_1-10)alkyl, heteroaryl(C_1-5)alkyl, (C_9-12)bicycloaryl(C_1-5)alkyl, hetero(C_8-12)bicycloaryl(C_1-5)alkyl, (C_3-12)cycloalkyl, hetero(C_3-12)cycloalkyl, (C_9-12)bicycloalkyl, hetero(C_3-12)bicycloalkyl, aryl, heteroaryl, (C_9-12)bicycloaryl and hetero(C_4-12)bicycloaryl, each substituted or unsubstituted, or R₆and R₈are absent when the C to which they are bound form part of a double bond, or any two of R₅, R₆, R₇and R₈are taken together to form a ring;
- R₉is selected from the group consisting of hydrogen, hydroxy, alkoxy, aryloxy, heteroaryloxy, carbonyl, amino, (C_1-10)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (C_1-10)alkyl, halo(C_1-10)alkyl, carbonyl(C_1-3)alkyl, thiocarbonyl(C_1-3)alkyl, sulfonyl(C_1-3)alkyl, sulfinyl(C_1-3)alkyl, amino(C_1-10)alkyl, imino(C_1-3)alkyl, (C_3-12)cycloalkyl(C_1-5)alk