APPLICATIONS

FIELD OF THE INVENTION

The present invention relates to hydroxamate compounds that are inhibitors of histone deacetylase (HDAC). More particularly, the present invention relates to benzothiophene containing compounds and methods for their preparation. These compounds may be useful as medicaments for the treatment of proliferative disorders as well as other diseases involving, relating to or associated with enzymes having histone deacetylase activities.

BACKGROUND OF THE INVENTION

Local chromatin architecture is generally recognized as an important factor in the regulation of gene expression. The architecture of chromatin, a protein-DNA complex, is strongly influenced by post-translational modifications of the histones which are the protein components. Reversible acetylation of histones is a key component in the regulation of gene expression by altering the accessibility of transcription factors to DNA. In general, increased levels of histone acetylation are associated with increased transcriptional activity, whereas decreased levels of acetylation are associated with repression of gene expression [Wade P.A. Hum. MoI. Genet. 10, 693-698 (2001 ), De Ruijter A.J. M. et al, Biochem. J., 370, 737-749 (2003)]. In normal cells, histone deacetylases (HDACs) and histone acetyltransferase together control the level of acetylation of histones to maintain a balance. Inhibition of HDACs results in the accumulation of acetylated histones, which results in a variety of cell type dependent cellular responses, such as apoptosis, necrosis, differentiation, cell survival, inhibition of proliferation and cytostasis.

Inhibitors of HDAC have been studied for their therapeutic effects on cancer cells. For example, suberoylanilide hydroxamic acid (SAHA) is a potent inducer of differentiation and/or apoptosis in murine erythroleukemia, bladder, and myeloma cell lines [Richon V.M. et al, Proc. Natl. Acad. Sci. USA, 93: 5705-5708 (1996), Richon V.M. et al, Proc. Natl. Acad. Sci. USA, 95: 3003-3007 (1998)]. SAHA has been shown to suppress the growth of prostate cancer cells in vitro and in vivo [Butler L. M. et al, Cancer Res. 60, 5165-5170 (2000)]. Other inhibitors of HDAC that have been widely studied for their anti-cancer activities are trichostatin A (TSA) and trapoxin B [Yoshida M. et al, J. Biol. Chem., 265, 17174 (1990), Kijima M. et al, J. Biol. Chem., 268, 22429 (1993)]. Trichostatin A is a reversible inhibitor of mammalian HDAC. Trapoxin B is a cyclic

tetrapeptide, which is an irreversible inhibitor of mammalian HDAC. However, due to the in vivo instability of these compounds they are less desirable as anti-cancer drugs. Recently, other small molecule HDAC inhibitors have become available for clinical evaluation [US6,552,065]. Additional HDAC inhibiting compounds have been reported in the literature [Bouchain G. et al, J. Med. Chem., 46, 820-830 (2003)] and patents [WO 03/066579A2, WO 01/38322 A1]. The in vivo activity of such inhibitors can be directly monitored by their ability to increase the amount of acetylated histones in the biological sample. HDAC inhibitors have been reported to interfere with neurodegenerative processes, for instance, HDAC inhibitors arrest polyglutamine- dependent neurodegeneration [Nature, 413(6857): 739-43, 18 October, 2001]. In addition, HDAC inhibitors have also been known to inhibit production of cytokines such as TNF, IFN, IL-1 which are known to be implicated in inflammatory diseases and/or immune system disorders. [ J. Biol. Chem. 1990; 265(18): 10230-10237; Science, 1998; 281 : 1001-1005; Dinarello CA. and Moldawer L.L. Proinflammatory and antiinflammatory cytokines in rheumatoid arthritis. A primer for clinicians. 2 ^nd Edition, Amergen Inc., 2000].

Nevertheless, there is still a need to provide further HDAC inhibitors that would be expected to have useful, improved pharmaceutical properties in the treatment of diseases such as cancer, neurodegenerative diseases, disorders involving angiogenesis and inflammatory and/or immune system disorders.

SUMMARY OF THE INVENTION

In one aspect the present invention provides compounds of the formula (I):

Formula I wherein:

Ri is selected from the group consisting of: H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy,

hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR ₅ , -COOR ₅ , -CONHR ₅ , -NHCOR ₅ , -NHSO ₂ R ₅ , -NHCOOR ₅ , -CONR ₅ R ₆ , -NR ₆ COR ₅ , -NR ₆ SO ₂ R ₅ , -NR ₆ COOR ₅ , -NR ₆ CONHR ₅ , -NHCONHR ₅ , C(=NOH)R ₅ , -alkylNR ₆ COR ₅ , alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl, SR ₆ and acyl, each of which may be optionally substituted; or R ₁ = L;

R ₂ is selected from the group consisting of H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl and acyl, each of which may be optionally substituted;

R ₃ is selected from the group consisting of: H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl and acyl, each of which may be optionally substituted;

R ₄ is independently selected from the group consisting of: H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, alkoxyalky, -COOH -C(O)OR ₅ , -COR ₅ , -SH, -SR ₆ , -OR ₆ , acyl and -NR ₇ R ₈ , each of which may be optionally substituted;

Each Y is independently selected from the group consisting of: H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkoxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, alkoxyalky, -COOH -C(O)OR ₅ , -COR ₅ , -SH, -SR ₆ , -OR ₆ , acyl and -NR ₇ R ₈ , each of which may be optionally substituted;

Each R ₅ is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl,

cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, arylheteroalkyl heteroarylalkyl, heteroarylheteroalkyl and acyl, each of which may be optionally substituted;

Each R ₆ is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylheteroalkyl, hydroxyalkyl, heteroarylheteroalkyl and acyl, each of which may be optionally substituted;

Each R ₇ and R ₈ is independently selected from the group consisting of: H, alkyl, alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylheteroalkyl, heteroarylheteroalkyl and acyl, each of which may be optionally substituted or R ₇ and R ₈ when taken together with the nitrogen atom to which they are attached from a heterocyclic group which may contain additional heteroatoms selected from O, S and N and may be optionally substituted; p is an integer selected from the group consisting of 0, 1 , 2, and 3;

L is selected from the group consisting of:

a) L=Cy-L ₁ -W- wherein

Cy is C ₁ -C ₁₅ alkyl, aminoalkyl, -NR ₇ R ₈ , heteroalkyl, heterocycloalkyl, cycloalkyl, aryl, aryloxy, heteroaryl or heteroaryloxy, each of which may be optionally substituted;.

L ₁ is selected from the group consisting of a bond, C ₁ -C ₅ alkyl and C ₂ -C ₅ alkenyl, each of which may be optionally substituted;

W is selected from the group consisting of a bond, -O-, -S-, -S(O)-, -S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(R ₉ )SO ₂ -, and

b) L=Cy-L ₁ -W-L ₂ wherein,

Cy is C ₁ -C ₁₅ alkyl, aminoalkyl, -NR ₇ R ₈ , heteroalkyl, heterocycloalkyl, cycloalkyl, aryl, aryloxy, heteroaryl, or heteroaryloxy each of which may be optionally substituted;

L ₁ and L ₂ are the same or different and are independently selected from the group consisting of Ci-C ₅ alkyl and C ₂ -C ₅ alkenyl, each of which may be optionally substituted;

W is selected from the group consisting of a bond, -O-, -S-, -S(O)-, -S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(Ri ₀ )SO ₂ -, and

c) L=Cy-(CH ₂ ) _m -W- wherein,

Cy is C ₁ -C ₁₅ alkyl, aminoalkyl, -NR ₇ R ₈ , heteroalkyl, heterocycloalkyl, cycloalkyl, aryl, aryloxy, heteroaryl or heteroaryloxy, each of which may be optionally substituted; m is 0, 1 , 2, 3, 4 or 5;

W is selected from the group consisting of a bond, -0-, -S-, -S(O)-,

-S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(R ₉ )SO ₂ -, and

d) L=L ₁ -W-L ₂

L ₁ and L ₂ are the same or different and are independently selected from the group consisting of C ₁ -C ₅ alkyl and C ₂ -C ₅ alkenyl, each of which may be optionally substituted;

W is selected from the group consisting of a bond, -0-, -S-, -S(O)-, -S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(R ₉ )SO ₂ -, and

e) L = a group of formula y ^R 13

W ₁ R ₁₂ C L ₃ - W— 4

R ₁₁ wherein,

W and Wi are each independently selected from the group consisting of a bond, -0-, -S-, -S(O)-, -S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(R ₉ )SO ₂ -, and -N(R ₉ )-C(0)-N(R _1o )-;

L ₃ is a bond or is selected from C ₁ -C ₅ alkyl or C ₂ -C ₅ alkenyl, each of which may be optionally substituted,

Ri ₁ is selected from the group consisting of H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylafkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR5, -COOR ₅ , -CONHR ₅ , -NHCOR ₅ , -NHCOOR ₅ , -HCONHR ₅ , C(=N0H)R ₅ , -alkylNCOR ₅ , alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl, SR ₆ and acyl, each of which may be optionally substituted;

R ₁₂ and R ₁₃ are independently selected from the group consisting of H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR ₅ , -COOR ₅ , -CONHR ₅ , -NHCOR ₅ , -NHCOOR ₅ , -NHCONHR ₅ , C(=NOH)R ₅ , -alkylNCOR ₅ , alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl, SR ₆ and acyl, each of which may be optionally substituted;

Each Rg and R ₁₀ are the same or different and are independently selected from H, C ₁ -C ₆ alkyl, C ₄ -C ₉ cycloalkyl, cycloalkylalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, hydroxyalkyl, heteroalkyl, heteroaryl, arylalkyl, heteroarylalkyl, heterocycloalkylalkyl, and acyl each of which may be optionally substituted;

or a pharmaceutically acceptable salt or prodrug thereof.

In one embodiment R ₂ is H and the compounds are of the formula (Ia):

³

wherein Ri, R ₃ , R ₄ , Y and p are as described above.

In another embodiment R ₃ is H and the compounds are of the formula (Ib):

Formula (Ib)

wherein R-i, R ₄ , Y and p are as described above.

In an even further embodiment R ₂ and R ₃ are H, the Ri substituent is in the 5 position and the hydroxamic acid is in the 2 position leading the compounds of Formula (Ic).

wherein Ri, R ₄ , Y and p are as described above.

As with any group of structurally related compounds which possess a particular utility, certain groups are preferred for the compounds of the Formula (I), (Ia), (Ib) and (Ic) in their end use application.

In certain embodiments Ri is a group of formula:

Cy-L ₁ -W-

wherein Cy, L ₁ and W are as described above. In this embodiment the compounds are compounds of formula (II).

Formula (II)

wherein R ₁ , R ₄ , Y, p, Cy, L ₁ and W are as described above.

There are a number of embodiments of the compounds of formula (II). In some embodiments W is -C(O)N(R ₉ )- or -SO ₂ -N(R ₉ )-. In more specific forms of these embodiment W is -CONH- or -SO ₂ -NH-. In one specific embodiment W is -CONH-. As such embodiments of the compounds of Formula (II) include compounds of formula (Na), (lib), (lie), and (lid).

Formula (Ha)

Formula (lib)

Formula (lie)

Formula (lid)

In each of these embodiments R ₄ , Y, p, Li and Cy are as defined above.

In each of these embodiments there are a number of specific values of Li and Cy. In certain embodiments

Li is a bond, methyl, ethyl, ethenyl or propyl, and

Cy is aryl, heteroaryl, aryloxy, heteroaryloxy, each of which may be optionally substituted, or Cy is a group of formula

RT

— N R ₈ , wherein R ₇ and R ₈ are as defined above. In certain embodiments R ₇ and R ₈ when taken together with the nitrogen atom to which they are attached form a heterocycloalkyl group.

In one specific embodiment Cy is selected from the group consisting of optionally substituted phenyl, optionally substituted indolinyl, optionally substituted phenoxy, optionally substituted naphthoxy, optionally substituted indolinyloxy, optionally substituted morpholinyl-4-yl, optionally substituted pyrollindin-1-yl, and optionally substituted piperazin-1-yl.

In one specific embodiment Ri is selected from the group consisting of:

10

In another embodiment Ri is a group of formula

R ₁₁ wherein W and W-i are independently selected from the group consisting of a bond, -O-, -S-, -S(O)-, -S(O) ₂ -, -N(R ₉ )-, -C(O)N(R ₉ )-, -SO ₂ N(R ₉ )-, -N(R ₉ )C(O)-, -N(R ₉ )SO ₂ -, and -N(Rg)-C(O)-N(R ₁₀ )-;

R _1I is selected from the group consisting of H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroaryialkyl, arylalkenyl, cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR ₅ , -COOR ₅ , -CONHR ₅ , -NHCOR ₅ , -NHCOOR ₅ , -NHCONHR ₅ , C(=N0H)R ₅ , -alkylNCOR ₅ , alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl,

arylsulfonyl, arylsulfinyl, aminosulfonyl, SR ₆ and acyl, each of which may be optionally substituted;

Ri ₂ and Ri ₃ are independently selected from the group consisting of H, halogen, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, heteroarylalkyl, arylalkenyl, cycloalkylheteroalkyl, arylheteroalkyl, heterocycloalkylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxyaryl, alkenyloxy, alkynyloxy, cycloalkylkoxy, heterocycloalkyloxy, aryloxy, heteroaryloxy, arylalkyloxy, phenoxy, benzyloxy, amino, alkylamino, aminoalkyl, acylamino, arylamino, sulfonylamino, sulfinylamino, -COOH, -COR ₅ , -COOR ₅ , -CONHR ₅ , -NHCOR ₅ , -NHCOOR ₅ , -NHCONHR ₅ , C(=N0H)R ₅ , -alkylNCOR ₅ , alkoxycarbonyl, alkylaminocarbonyl, sulfonyl, alkylsulfonyl, alkylsulfinyl, arylsulfonyl, arylsulfinyl, aminosulfonyl, SR ₆ and acyl, each of which may be optionally substituted.

The compounds of this embodiment are compounds of formula (III).

Formula (III)

In one form of this embodiment L ₃ is a bond.

In another form of this embodiment Rn is H.

s such in one form of these embodiments the compounds are those of formula (Ilia).

Formula (Ilia)

wherein R ₄ , Y, p, R ₁₂ , R ₁₃ , W and W ₁ are as described above for formula I.

In another form of this embodiment W and W ₁ are both a group of formula:

H |— CO — N-|

Accordingly in another embodiment of the invention Ri is a group of formula:

As such in another embodiment the compounds are compounds of formula (lllb).

Formula (lllb)

wherein R ₄ , Y, p, Ri ₂ , and R ₁₃ are as described above for formula (I).

In one form of this embodiment R ₁₂ is selected from the group consisting of cycloalkyl, cycloalkylalkyl, alkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryl

and heteroarylalkyl each of which may optionally be substituted. In one specific form Ri2 is arylalkyl. In another specific form Ri ₂ is phenylmethyl.

In another embodiment Ri ₃ is selected from the group consisting of cycloalkyl, cycloalkylalkyl, alkyl, alkenyl, heteroalkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylaikyl, heteroaryl and heteroarylalkyl each of which may optionally be substituted. In another embodiment Ri ₃ is selected from the group consisting of cycloalkyl, cycloalkylalkyl, alkyl, heterocycloalkyl, heterocycloalkylalkyl, aryl, arylalkyl, heteroaryf and heteroarylalkyl each of which may optionally be substituted.

Specific values of R« are cyclopropyl, cyclopentyl, 2-hexyl-ethyl, propyl, 2-isopropyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, 1 -hexyl-methyl, 2-piperidino-i-yl-ethyl, 4-aminoethanamide-phenyl, 3-phenylpropyl, 2-phenylethyl, 2,3-dimethoxyphenyl, thienyl-3yl, 1-thienyl-3-yl-methyl, furan-3-yl, 1-pyridin-2-ylmethyl, 1-(3,4-difluoro- phenyl)-methyl, 4-dimethylamino-phenyl and phenyl.

In another form of this embodiment W is a group of formula

H

-<3— -CO — N — £-

and Wi is a group of formula t ^→ -NH CO-NH — £-

and the Ri group is a group of the formula

wherein Ri ₂ and R ₁ 3 are as define a ove.

As such in another embodiment the compounds of the invention are compounds of formula (HIc).

Formula (NIc)

wherein R ₄ , Y, p, Ri ₂ , and Ri ₃ are as described above for formula (I).

In one form of this embodiment Ri ₂ is arylalkyl. In a specific form Ri ₂ is phenylmethyl.

In one form of this embodiment Ri ₃ is selected from the group consisting of alkyl, cycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyl, and heteroaryl alkyl, each of which may optionally be substituted.

Specific values of Ri ₃ are isopropyl-2-yl, butyl, cyclohexyl, 1-cyclohexyl-methyl, 1-tetrahydrofuran-2-yl methyl, 2-morpholin-4-yl-ethyl, 2-phenyl-ethyl, 3-phenyl-propyl, 1 -(4-methytk-phenyl)-methyl, 1 -(2,4-difluorophenyl)-methyl, 1 -(2,5-dimethoxyphenyl)- methyl, 1-then-2-yl-methyl, 1-furan-2-yl-methyl, 1-pyridin-4yl-methyl, 1-pyridin-2-yl- methyl, 3-imadaz-1-yl-methyl, 1 -phenylmethyl.

In another form of this embodiment W is a group of the formula

H

-| — CO — N— £-

and W ₁ is a group of the formula -NR ₉ -, wherein Rg is H in one specific form.

This form provides compounds of formula (llld).

Formula (IHd)

wherein R ₄ , Y, P, R9, Ri ₂ , and R ₁₃ are as described above for formula (I).

If Ri is substituted in one embodiment the substituents are selected from the group consisting of: halogen, =0, =S, -CN, -NO ₂ , alkyl, alkenyl, heteroalkyl, haloalkyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, hydroxy, hydroxyalkyl, alkoxy, alkylamino, aminoalkyl, acylamino, phenoxy, alkoxyalkyl, benzyloxy, alkylsulfonyl, arylsulfonyl, aminosulfonyl, -C(O)OR ₅ , COOH, SH, -NR ₇ R ₈ and acyl.

In one embodiment R ₁ is at the 5 or 6 position. In a specific embodiment R ₁ is at the 5 position.

Each Y may be the same or different and in one embodiment are selected from H, halogen, C ₁ -C ₄ alkyl, -CF ₃ , -NO ₂ , -C(O)R ₅ , -OR ₆ , -SR ₆ , -CN and NR ₇ R ₈ .

In one embodiment Y is H.

In one embodiment Y is at the 4 and/or 7 positions of the aromatic ring.

In one specific embodiment p is O.

In one embodiment R ₂ is H, C ₁ -C ₆ alkyl, or acyl. In another embodiment R ₂ is H or

C ₁ -C ₄ alkyl. A specific value for R ₂ is H;

In one embodiment R ₃ is H or C ₁ -C ₄ alkyl. A specific value for R ₃ is H;

In one embodiment R ₄ is H or C ₁ -C ₄ alkyl. A specific value for R ₄ is H;

In one embodiment R ₄ is located at the 3 position of the ring.

In one embodiment R ₅ is C ₁ -C ₄ alkyl, heteroalkyl, or acyl. A specific value for R ₅ is methyl;

In one embodiment R ₆ is C ₁ -C ₄ alkyl, hydroxyalkyl, heteroalkyl or acyl. A specific value for R ₆ is CrC ₄ alkyl;

In one embodiment R ₇ and R ₈ are selected from the group consisting of H, C ₁ -C ₆ alkyl,

C ₄ .C ₉ cycloalkyl, C ₄ -C ₉ heterocycloalkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl;

In one embodiment R ₉ and R ₁₀ are H, hydroxyalkyl or heteroalky.

In addition to compounds of the invention as described above the embodiments disclosed are also directed to pharmaceutically acceptable salts, pharmaceutically acceptable prodrugs, and pharmaceutically active metabolites of such compounds, and pharmaceutically acceptable salts of such metabolites. Such compounds, salts, prodrugs and metabolites are at times collectively referred to herein as "HDAC

inhibiting agents" or "HDAC inhibitors". In certain embodiments the compounds disclosed are used to modify deacetylase activity, in some cases histone deacetylase activity and in some cases HDAC 8, or HDAC 1 activity.

The embodiments disclosed also relate to pharmaceutical compositions each comprising a therapeutically effective amount of a HDAC inhibiting agent of the embodiments described with a pharmaceutically acceptable carrier or diluent for treating cellular proliferative ailments. The term "effective amount" as used herein indicates an amount necessary to administer to a host to achieve a therapeutic result, e.g., inhibition of proliferation of malignant cancer cells, benign tumor cells or other proliferative cells.

The invention also relates to pharmaceutical compositions including a compound of the invention with a pharmaceutically acceptable carrier, diluent or excipient.

In yet a further aspect the present invention provides a method of treatment of a disorder caused by, associated with or accompanied by disruptions of cell proliferation and/or angiogenesis including administration of a therapeutically effective amount of a compound of formula (I).

In one embodiment the method includes administration of a compound of formula (Ia). In another embodiment the method includes administration of a compound of formula (Ib). In one embodiment the method includes administration of a compound of formula (Ic) as described herein.

In one embodiment the disorder is selected from the group consisting of but not limited to cancer (e.g. breast cancer, colon cancer, prostate cancer, pancreatic cancer, leukemias, lymphomas, ovarian cancers, neuroblastomas, melanoma, inflammatory diseases/immune system disorders, angiofibroma, cardiovascular diseases (e.g. restenosis, arteriosclerosis), fibrotic diseases (e.g. liver fibrosis), diabetes, autoimmune diseases, chronic and acute neurodegenerative disease like disruptions of nerval tissue, Huntington's disease and infectious diseases like fungal, bacterial and viral infections. In another embodiment the disorder is a proliferative disorder. In one embodiment the proliferative disorder is cancer. The cancer can include solid tumors or hematologic malignancies.

The invention also provides agents for the treatment of a disorder caused by, associated with or accompanied by disruptions of cell proliferation and/or angiogenesis including a compound of formula (I) as disclosed herein. In one embodiment he agent is an anti-cancer agent. In another embodiment, the agent is an anti-angiogenesis agent.

In one embodiment the agent contains a compound of formula (Ia). In another embodiment the agent contains a compound of formula (Ib). in yet another embodiment the agent contains a compound of formula (Ic).

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for the treatment of a disorder caused by, associated with or accompanied by disruptions of cell proliferation and/or angiogenesis. In one embodiment the disorder is a proliferative disorder. In a specific embodiment the proliferative disorder is a cancer.

The compounds of the present invention surprisingly show low toxicity, together with a potent anti-proliferative activity.

In yet a further embodiment the invention provides a method of treatment of a disorder, disease or condition that can be treated by the inhibition of histone deacetylase including administration of a therapeutically effective amount of a compound of formula CO-

ln one embodiment the method includes administration of a compound of formula (Ia). In another embodiment the method includes administration of a compound of formula (Ib). In yet an even further embodiment the method includes administration of a compound of formula (Ic).

In one embodiment the disorder is selected from the group consisting of but not limited to Proliferative disorders (e.g. cancer); Neurodegenerative diseases including Huntington's Disease, Polyglutamine diseases, Parkinson's Disease, Alzheimer's Disease, Seizures, Striatonigral degeneration, Progressive supranuclear palsy, Torsion dystonia, Spasmodic torticollis and dyskinesis, Familial tremor, Gilles de Ia Tourette syndrome, Diffuse Lewy body disease, Pick's disease, Intracerebral haemorrhage Primary lateral sclerosis, Spinal muscular atrophy, Amyotrophic lateral sclerosis, Hypertrophic interstitial polyneuropathy, Retinitis pigmentosa, Hereditary optic atrophy,

Hereditary spastic paraplegia, Progressive ataxia and Shy-Drager syndrome; Metabolic diseases including Type 2 diabetes; Degenerative Diseases of the Eye including Glaucoma, Age-related macular degeneration, macular myopic degeneration, Rubeotic glaucoma, Interstitial keratitis, Diabetic retinopathy, Peter's anomaly retinal degeneration, Cellophane Retinopathy; Cogan's Dystrophy; Corneal Dystrophy; Iris Neovascularization (Rubeosis); Neovascularization of the Cornea; Retinopathy of Prematurity; Macular Edema; Macular Hole; Macular Pucker; Marginal Blepharitis, Myopia, nonmalignant growth of the conjunctiva; Inflammatory diseases and/or Immune system disorders including Rheumatoid Arthritis (RA), Osteoarthritis, Juvenile chronic arthritis, Graft versus Host disease, Psoriasis, Asthma, Spondyloarthropathy, Crohn's Disease, inflammatory bowel disease, Colitis Ulcerosa, Alcoholic hepatitis, Diabetes, Sjoegrens's syndrome, Multiple Sclerosis, Ankylosing spondylitis, Membranous glomerulopathy, Discogenic pain, Systemic Lupus Erythematosus, allergic contact dermatitis; Disease involving angiogenesis including cancer, psoriasis, rheumatoid arthritis; Psychological disorders including bipolar disease, schizophrenia, depression and dementia; Cardiovascular Diseases including Heart failure, restenosis, cardiac hypertrophy and arteriosclerosis; Fibrotic diseases including liver fibrosis, lung fibrosis, cystic fibrosis and angiofibroma; Infectious diseases including Fungal infections, such as Candida Albicans, Bacterial infections, Viral infections, such as Herpes Simplex, Protozoal infections, such as Malaria, Leishmania infection, Trypanosoma brucei infection, Toxoplasmosis and coccidiosis, and Haematopoietic disorders including thalassemia, anemia and sickle cell anemia.

The invention also provides agents for the treatment of a disorder, disease or condition that can be treated by the inhibition of histone deacetylase including a compound of formula (I) as disclosed herein. In one embodiment the agent is an anti-cancer agent.

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for the treatment of a disorder, disease or condition that can be treated by the inhibition of histone deacetylase.

The invention also provides a method for inhibiting cell proliferation including administration of an effective amount of a compound according to formula (I).

The invention also provides agents for inhibiting cell proliferation including a compound of formula (I) as disclosed herein.

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for inhibiting cell proliferation.

In yet an even further aspect the invention provides a method of treatment of a neurodegenerative disorder in a patient including administration of a therapeutically effective amount of a compound of formula (I). In one embodiment the method includes administration of a compound of formula (Ia). In another embodiment the method includes administration of a compound of formula (Ib). In yet an even further embodiment the method includes administration of a compound of formula (Ic) as described herein. In a specific embodiment the neurodegenerative disorder is Huntington's Disease.

The invention also provides agents for the treatment of neurodegenerative disorder including a compound of formula (I) as disclosed herein. In one embodiment the agent is an anti-Huntington's disease agent.

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for the treatment of a neurodegenerative disorder. In one embodiment the neurodegenerative disorder is Huntington's Disease.

In yet an even further aspect the invention provides a method of treatment of an inflammatory disease and/or immune system disorder in a patient including administration of a therapeutically effective amount of a compound of formula .(I). In one embodiment the method includes administration of a compound of formula (Ia). In another embodiment the method includes administration of a compound of formula (Ib). In an even further embodiment the method includes administration of a compound of formula (Ic) as described herein. In one embodiment the inflammatory disease and/or immune system disorder is rheumatoid arthritis. In another embodiment the inflammatory disease and/or immune system disorder is Systemic Lupus Erythematosus.

The invention also provides agents for the treatment of inflammatory disease and/or immune system disorder including a compound of formula (I) as disclosed herein.

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for the treatment of inflammatory disease and/or immune system disorder. In one embodiment the inflammatory disease and/or immune system disorder is

rheumatoid arthritis. In another embodiment the inflammatory disease and/or immune system disorder is Systemic Lupus Erythematosus.

In yet an even further aspect the invention provides a method of treatment of eye disease mediated by HDAC inhibition in a patient including administration of a therapeutically effective amount of a compound of formula (I). In one embodiment the method includes administration of a compound of formula (Ia). In another embodiment the method includes administration of a compound of formula (Ib). In yet an even further embodiment the method includes administration of a compound of formula (1c). In one embodiment, the eye disease is macular degeneration. In another embodiment, the eye disease is glaucoma. In another embodiment, the eye disease is retinal degeneration.

The invention also provides agents for the treatment of eye disease mediated by HDAC inhibition including a compound of formula (I). In one embodiment, the eye disease is macular degeneration. In another embodiment, the eye disease is glaucoma. In another embodiment, the eye disease is retinal degeneration.

The invention also relates to the use of compounds of formula (I) in the preparation of a medicament for the treatment of eye disease mediated by HDAC inhibition. In one embodiment the compound used is a compound of formula (Ia). In another embodiment the compound used is a compound of formula (Ib). In yet an even further embodiment the compound used is a compound of formula .(Ic). In one embodiment, the eye disease is macular degeneration. In another embodiment, the eye disease is glaucoma. In another embodiment, the eye disease is retinal degeneration.

DETAILED DESCRIPTION OF THE EMBODIMENTS

There are disclosed hydroxamate compounds, for example benzothiophene containing hydroxamic acid in one of the substituents that may be inhibitors of deacetylases, including but not limited to inhibitors of histone deacetylases. The hydroxamate compounds may be suitable for prevention or treatment of a disorder caused by, associated with or accompanied by disruptions of cell proliferation and/or angiogenesis when used either alone or together with a pharmaceutically acceptable carrier, diluent or excipient. An example of such a disorder is cancer.

As used herein the term 'cancer ¹ is a general term intended to encompass the vast number of conditions that are characterised by uncontrolled abnormal growth of cells.

It is anticipated that the compounds of the invention will be useful in treating various cancers including but not limited to bone cancers including Ewing's sarcoma, osteosarcoma, chondrosarcoma and the like, brain and CNS tumours including acoustic neuroma, neuroblastomas, glioma and other brain tumours, spinal cord tumours, breast cancers, colorectal cancers, advanced colorectal adenocarcinomas, colon cancers, endocrine cancers including adenocortical carcinoma, pancreatic cancer, pituitary cancer, thyroid cancer, parathyroid cancer, thymus cancer, multiple endocrine neoplasma, gastrointestinal cancers including stomach cancer, esophageal cancer, small intestine cancer, Liver cancer, extra hepatic bile duct cancer, gastrointestinal carcinoid tumour, gall bladder cancer, genitourinary cancers including testicular cancer, penile cancer, prostate cancer, gynaecological cancers including cervical cancer, ovarian cancer, vaginal cancer, uterus/endometrium cancer, vulva cancer, gestational trophoblastic cancer, fallopian tube cancer, uterine sarcoma, head and neck cancers including oral cavity cancer, lip cancer, salivary gland cancer, larynx cancer, hypopharynx cancer, orthopharynx cancer, nasal cancer, paranasal cancer, nasopharynx cancer, leukemias including childhood leukemia, acute lymphocytic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, acute promyelocytic leukemia, plasma cell leukemia, myelomas, haematological disorders including myelodysplastic syndromes, myeloproliferative disorders, aplastic anemia, Fanconi anemia, Waldenstroms Macroglobulinemia, lung cancers including small cell lung cancer, non-small cell lung cancer, lymphomas including Hodgkin's disease, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, peripheral T-cell lymphoma, AIDS related Lymphoma, B-cell lymphoma, Burkitt's lymphoma; eye cancers including retinoblastoma, intraocular melanoma, skin cancers including melanoma, non-melanoma skin cancer, merkel cell cancer, soft tissue sarcomas such as childhood soft tissue sarcoma, adult soft tissue sarcoma, Kaposi's sarcoma, urinary system cancers including kidney cancer, Wilms tumour, bladder cancer, urethral cancer, and transitional cell cancer.

Exemplary cancers that may be treated by the compounds of the present invention are breast cancer, lung cancer, ovarian cancer, prostate cancer, head and neck cancer, renal cancer (e.g. renal cell carcinoma), gastric cancer, colon cancer, colon cancer, colorectal cancer and brain cancer.

Exemplary cancers that may be treated by compounds of the present invention include but are not limited to B-cell lymphoma (e.g. Burkitt's lymphoma), leukemias (e.g. Acute

promyelocyte leukemia), cutaneous T-cell lymphoma (CTCL) and peripheral T-cell lymphoma.

Exemplary cancers that may be treated by compounds of the present invention include solid tumors and hematologic malignancies.

The compounds may also be used in the treatment of a disorder involving, relating to or, associated with dysregulation of histone deacetylase (HDAC).

There are a number of disorders that have been implicated by or known to be mediated at least in part by HDAC activity, where HDAC activity is known to play a role in triggering disease onset, or whose symptoms are known or have been shown to be alleviated by HDAC inhibitors. Disorders of this type that would be expected to be amenable to treatment with the compounds of the invention include the following but not limited to: Proliferative disorders (e.g. cancer); Neurodegenerative diseases including Huntington's Disease, Polyglutamine diseases, Parkinson's Disease, Alzheimer's Disease, Seizures, Striatonigral degeneration, Progressive supranuclear palsy, Torsion dystonia, Spasmodic torticollis and dyskinesis, Familial tremor, Gilles de Ia Tourette syndrome, Diffuse Lewy body disease, Pick's disease, Intracerebral haemorrhage Primary lateral sclerosis, Spinal muscular atrophy, Amyotrophic lateral sclerosis, Hypertrophic interstitial polyneuropathy, Retinitis pigmentosa, Hereditary optic atrophy, Hereditary spastic paraplegia, Progressive ataxia and Shy-Drager syndrome; Metabolic diseases including Type 2 diabetes; Degenerative Diseases of the Eye including Glaucoma, Age-related macular degeneration, macular myopic degeneration, Rubeotic glaucoma, Interstitial keratitis, Diabetic retinopathy, Peter's anomaly retinal degeneration, Cellophane Retinopathy; Cogan's Dystrophy; Corneal Dystrophy; Iris Neovascularization (Rubeosis); Neovascularization of the Cornea; Retinopathy of Prematurity; Macular Edema; Macular Hole; Macular Pucker; Marginal Blepharitis, Myopia, nonmalignant growth of the conjunctiva; Inflammatory diseases and/or Immune system disorders including Rheumatoid Arthritis (RA), Osteoarthritis, Juvenile chronic arthritis, Graft versus Host disease, Psoriasis, Asthma, Spondyloarthropathy, Crohn's Disease, inflammatory bowel disease, Colitis Ulcerosa, Alcoholic hepatitis, Diabetes, Sjoegrens's syndrome, Multiple Sclerosis, Ankylosing spondylitis, Membranous glomerulopathy, Discogenic pain, Systemic Lupus Erythematosus, allergic contact dermatitis; Disease involving angiogenesis including cancer, psoriasis, rheumatoid arthritis; Psychological disorders including bipolar disease, schizophrenia, depression and dementia; Cardiovascular Diseases including

Heart failure, restenosis, cardiac hypertrophy and arteriosclerosis; Fibrotic diseases including liver fibrosis, lung fibrosis, cystic fibrosis and angiofibroma; Infectious diseases including Fungal infections, such as Candida Albicans, Bacterial infections, Viral infections, such as Herpes Simplex, Protozoal infections, such as Malaria, Leishmania infection, Trypanosoma brucei infection, Toxoplasmosis and coccidiosis, and Haematopoietic disorders including thalassemia, anemia and sickle cell anemia.

As used herein, the term unsubstituted means that there is no substituent or that the only substituents are hydrogen.

The term "optionally substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. Preferably the substituent groups are one or more groups independently selected from the group consisting of halogen, =O, =S, -CN, -NO ₂ , -CF ₃ , -OCF ₃ , alkyl, aikenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkoxycycloalkyl, alkoxyheterocycloalkyl, alkoxyaryl, alkoxyheteroaryl, alkoxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, arylalkyl, heteroarylalkyl, cycloalkylalkyl, heterocycloalkylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -COOH, -COR ₆ , -C(O)OR ₆ , CONHR ₆ , NHCOR ₆ , NHCOOR ₆ , NHCONHR ₆ , C(=NOH)R ₆ , -SH, -SR ₆ , -OR ₆ and acyl. Substituent groups themselves may be further optionally substituted.

"Halogen" represents chlorine, fluorine, bromine or iodine.

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C ₁ -C ₁₄ alkyl, more preferably C ₁ -C ₁₀ alkyl, most preferably CrC ₆ unless otherwise noted. Examples of suitable straight and branched

CrC ₆ alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.

"Alkylamino" includes both monoalkylamino and dialkylamino, unless specified. "Monoalkylamino" means a -NH-Alkyl group, in which alkyl is as defined above. "Dialkylamino" means a -N(alkyl) ₂ group, in which each alkyl may be the same or different and are each as defined herein for alkyl. The alkyl group is preferably a C ₁ -C ₆ alkyl group.

"Arylamino" includes both mono-arylamino and di-arylamino unless specified. Mono- arylamino means a group of formula aryl NH- in which aryl is as defined herein, di-arylamino means a group of formula (aryl ₂ ) N- where each aryl may be the same or different and each are as defined herein for aryl.

"Acyl" means an alkyl-CO- group in which the alkyl group is as described herein. Examples of acyl include acetyl and benzoyl. The alkyl group is preferably a CrC ₆ alkyl group.

"Alkenyl" as group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-14 carbon atoms, more preferably 2-12 carbon atoms, most preferably 2-6 carbon atoms, in the chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. Exemplary alkenyl group include, but are not limited to, ethenyl and propenyl.

"Alkoxy" refers to an -O-alkyl group in which alkyl is defined herein. Preferably the alkoxy is a Ci-C ₆ alkoxy. Examples include, but are not limited to, methoxy and ethoxy.

"Alkenyloxy" refers to an -O- alkenyl group in which alkenyl is as defined herein. Preferred alkenyloxy groups are Ci-C ₆ alkenyloxy groups.

"Alkynyloxy" refers to an -O-alkynyl group in which alkynyl is as defined herein'. Preferred alkynyloxy groups are C ₁ -C ₆ alkynyloxy groups.

"Alkoxycarbonyl" refers to an -C(O)-O-alkyl group in which alkyl is as defined herein. The alkyl group is preferably a CrC ₆ alkyl group. Examples include, but not limited to, methoxycarbonyl and ethoxycarbonyl.

"Akylsulfinyl" means a -S(O)-alkyl group in which alkyl is as defined above. The alkyl group is preferably a C ₁ -C ₆ alkyl group. Exemplary alkylsulfinyl groups include, but not limited to, methylsulfinyl and ethylsulfinyl.

"Alkylsulfonyl" refers to a -S(O) ₂ -alkyl group in which alkyl is as defined above. The alkyl group is preferably a d-Cβ alkyl group. Examples include, but not limited to methylsulfonyl and ethylsulfonyl.

"Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-14 carbon atoms, more preferably 2-12 carbon atoms in the chain, preferably 2-6 carbon atoms in the chain. Exemplary structures include, but are not limited to, ethynyl and propynyl.

"Alkylaminocarbonyl" refers to an alkylamino-C(O)- group in which alkylamino is as defined above.

"Cycloalkyl" refers to a saturated or partially saturated, monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic system such as cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.

"Cycloalkylalkyl" means a cycloalkyl-alkyl- group in which the cycloalkyl and alkyl moieties are as previously described. Exemplary monocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl.

"Heterocycloalkyl" refers to a saturated or partially saturated monocyclic, bicyclic or polycyclic ring containing at least a heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyl substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1 ,3-diazapane, 1 ,4-diazapane, 1 ,4-oxazepane, and 1 ,4-oxathiapane.

"Heterocycloalkenyl" refers to a heterocycloalkyl as described above but containing at least one double bond.

"Heterocycloalkylalkyl" refers to a heterocycloalkyl-alkyl group in which the heterocycloalkyl and alkyl moieties are as previously described. Exemplary heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl, (2-tetrahydrothiofuranyl)methyl.

"Heteroalkyl" refers to a straight- or branched-chain alkyl group preferably having from 2 to 14 carbons, more preferably 2 to 10 atoms in the chain, one or more of which has been replaced by a heteroatom selected from S, O, and N. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, alkyl sulfides, and the like.

"Aryl" as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C _5-7 cycloalkyl or C _5-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.

"Arylalkenyl" means an aryl-alkenyl- group in which the aryl and alkenyl are as previously described. Exemplary arylalkenyl groups include phenylallyl.

"Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties are as previously described. Preferred arylalkyl groups contain a Ci _-5 alkyl moiety. Exemplary arylalkyl groups include benzyl, phenethyl and naphthelenemethyl.

"Cycloalkenyl" means an optionally substituted non-aromatic monocyclic or polycyclic ring system containing at least one carbon-carbon double bond and preferably having from 5-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl or cycloheptenyl.

The term "heteroaryl" either alone or part of another group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having 1 or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen.

Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1 H-indazo)e, purine, 4H-qu/nolidine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isoxazole, furazane, phenoxazine, 2-, 3-, or 4-pyridyl, 2-, 3-, 4-, 5-, or 8-quinolyl, 1-, 3-, A-, or 5-isoquinolyl, 1-, 2-, or 3-indolyl, 2-benzothiazolyl, 2-benzo[b]thienyl, benzo[b]furanyl, 2- or 3-thienyl, or the like. More preferred examples include 2- or 3-thienyl, 2-, 3-, or 4-pyridyl, 2- or 3-quinolyl, 1-isoquinolyl, 1- or 2-indolyl, 2-benzothiazolyl, and the like.

"Heteroarylalkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl moieties are as previously described. Preferred heteroarylalkyl groups contain a Ci to CQ alkyl moiety. Exemplary heteroarylalkyl groups include pyridylmethyl.

"Lower alkyl" as a group means unless otherwise specified, an aliphatic hydrocarbon group which may be straight or branched having 1 to 6 carbon atoms in the chain, more preferably 1 to 4 carbons such as methyl, ethyl, propyl (n-propyl or isopropyl) or butyl (n-butyl, isobutyl or tertiary-butyl).

It is understood that included in the family of compounds of Formula (I) are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers.in "E" or "Z" configurational isomer or a mixture of E and Z isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods and by those skilled in the art.

Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and /or diastereomers. All such single stereoisomers, racemates and mixtures thereof are intended to be within the scope of the subject matter described and claimed.

Additionally, Formula (I) is intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non-hydrated forms.

In addition to compounds of the Formula (I), the HDAC inhibiting agents of the various embodiments include pharmaceutically acceptable salts, prodrugs, and active metabolites of such compounds, and pharmaceutically acceptable salts of such metabolites.

The term "Pharmaceutically acceptable salts" refers to salts that retain the desired biological activity of the above-identified compounds, and include pharmaceutically acceptable acid addition salts and base addition salts. Suitable pharmaceutically acceptable acid addition salts of compounds of Formula (I) may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, sulfuric, and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, heterocyclic carboxylic and sulfonic classes of organic acids, examples of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, fumaric, maleic, alkyl sulfonic, arylsulfonic. Suitable pharmaceutically acceptable base addition salts of compounds of Formula (I) include metallic salts made from lithium, sodium, potassium, magnesium, calcium, aluminium, and zinc, and organic salts made from organic bases such as choline, diethanolamine, morpholine. Other examples of organic salts are: ammonium salts, quaternary salts such as tetramethylammonium salt; amino acid addition salts such as salts with glycine and arginine. Additional information on pharmaceutically acceptable salts can be found in Remington's Pharmaceutical Sciences, 19th Edition, Mack Publishing Co., Easton, PA 1995. In the case of agents that are solids, it is understood by those skilled in the art that the inventive compounds, agents .and salts may exist in different crystalline or polymorphic forms, all of which are intended to be within the scope of the present invention and specified formulae.

"Prodrug" means a compound which is convertible in vivo by metabolic means (e.g. by hydrolysis, reduction or oxidation) to a compound of formula (I). For example an ester prodrug of a compound of formula I containing a hydroxyl group may be convertible by hydrolysis in vivo to the parent molecule. Suitable esters of compounds of formula (I) containing a hydroxyl group, are for example acetates, citrates, lactates, tartrates, maloriates, oxalates, salicylates, propionates, succinates, fumarates, maleates, methylene-bis-β-hydroxynaphthoates, gestisates, isethionates, di-p-toluoyltartrates, methanesulphonates, ethanesulphonates, benzenesulphonates, p-toluenesulphonates, cyclohexylsulphamates and quinates. As another example an ester prodrug of a compound of formula I containing a carboxy group may be convertible by hydrolysis in

vivo to the parent molecule. (Examples of ester prodrugs are those described by F.J. Leinweber, Drug Metab. Res., 18:379, 1987).

Preferred HDAC inhibiting agents include those having an IC ₅₀ value of 10 μM or less.

Administration of compounds within Formula (I) to humans can be by any of the accepted modes for enteral administration such as oral or rectal, or by parenteral administration such as subcutaneous, intramuscular, intravenous and intradermal routes. Injection can be bolus or via constant or intermittent infusion. The active compound is typically included in a pharmaceutically acceptable carrier or diluent and in an amount sufficient to deliver to the patient a therapeutically effective dose. In various embodiments the inhibitor compound may be selectively toxic or more toxic to rapidly proliferating cells, e.g. cancerous tumors, than to normal cells.

The term "therapeutically effective amount" or "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations. An effective amount is typically sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the progression of the disease state. A therapeutically effective amount can be readily determined by a skilled practitioner by the use of conventional techniques and by observing results obtained in analogous circumstances. In determining the effective amount a number of factors are considered including the species of the patient, its size, age, general health, the specific disease involved, the degree or severity of the disease, the response of the individual patient, the particular compound administered, the mode of administration, the bioavailability of the compound, the dose regimen selected, the use of other medication and other relevant circumstances.

In using the compounds of the invention they can be administered in any form or mode which makes the compound bioavailable. One skilled in the art of preparing formulations can readily select the proper form and mode of administration depending upon the particular characteristics of the compound selected, the condition to be treated, the stage of the condition to be treated and other relevant circumstances. We refer the reader to Remingtons Pharmaceutical Sciences, 19 ^th edition, Mack Publishing Co. (1995) for further information.

The compounds of the present invention can be administered alone or in the form of a pharmaceutical composition in combination with a pharmaceutically acceptable carrier,

diluent or excipient. The compounds of the invention, while effective themselves, are typically formulated and administered in the form of their pharmaceutically acceptable salts as these forms are typically more stable, more easily crystallised and have increased solubility.

The compounds are, however, typically used in the form of pharmaceutical compositions which are formulated depending on the desired mode of administration. As such in a further embodiment the present invention provides a pharmaceutical composition including a compound of Formula (I) and a pharmaceutically acceptable carrier, diluent or excipient. The compositions are prepared in manners well known in the art.

The invention in other embodiments provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. In such a pack or kit can be found a container having a unit dosage of the agent(s). The kits can include a composition comprising an effective agent either as concentrates (including lyophilized compositions), which can be diluted further prior to use or they can be provided at the concentration of use, where the vials may include one or more dosages. Conveniently, in the kits, single dosages can be provided in sterile vials so that the physician can employ the vials directly, where the vials will have the desired amount and concentration of agent(s). Associated with such containers) can be various written materials such as instructions for use, or a notice in the form prescribed by a- governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The compounds of the invention may be used or administered in combination with one or more additional drug (s) that include chemotherapeutic drugs or HDAC inhibitor drugs and/or procedures (e.g. surgery, radiotherapy) for the treatment of the disorder/diseases mentioned. The components can be administered in the same formulation or in separate formulations. If administered in separate formulations the compounds of the invention may be administered sequentially or simultaneously with the other drug (s).

In addition to being able to be administered in combination with one or more additional drugs that include chemotherapeutic drugs or HDAC inhibitor drugs the compounds of the invention may be used in a combination therapy. When this is done the compounds

are typically administered in combination with each other. Thus one or more of the compounds of the invention may be administered either simultaneously (as a combined preparation) or sequentially in order to achieve a desired effect. This is especially desirable where the therapeutic profile of each compound is different such that the combined effect of the two drugs provides an improved therapeutic result

Pharmaceutical compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use. Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminium monostearate and gelatin.

The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium just prior to use.

Solid dosage forms for oral administration include capsules, dragees, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c)

humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes.

If desired, and for more effective distribution, the compounds can be incorporated into slow release or targeted delivery systems such as polymer matrices, liposomes, and microspheres.

The active compounds can also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water ox other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminium metahydroxide, bentonite, agar-agar, and tragacanth, and mixtures thereof.

Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non-irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at room temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.

Dosage forms for topical administration of a compound of this invention include powders, patches, sprays, ointments and inhalants. The active compound is mixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives, buffers, or propellants which may be required.

A preferred dosage will be a range from about 0.01 to 300 mg per kilogram of body weight per day. A.more preferred dosage will be in the range from 0.1 to 100 mg per kilogram of body weight per day, more preferably from 0.2 to 80 mg per kilogram of body weight per day, even more preferably 0.2 to 50 mg per kilogram of body weight per day. A suitable dose can be administered in multiple sub-doses per day.

As discussed above, the compounds of the embodiments disclosed inhibit histone deacetylases. The enzymatic activity of a histone deacetylase can be measured using known methodologies [Yoshida M. et al, J. Biol. Chem., 265, 17174 (1990), J. Taunton et al, Science 1996 272: 408]. In certain embodiments, the histone deacetylase inhibitor interabts with and/or reduces the activity of more than one known histone deacetylase in the cell, which can either be from the same class of histone deacetylase or different class of histone deacetylase. In some other embodiments, the histone deacetylase inhibitor interacts and reduces the activity of predominantly one histone deacetylase, for example HDAC-1 , HDAC-2, HDAC-3 or HDAC-8 which belongs to Class I HDAC enzymes [De Ruijter AJ. M. et al, Biochem. J., 370, 737-749 (2003)].

HDACs can also target non-histone substrates to regulate a variety of biological functions implicated in disease pathogenesis. These non-histone substrates include Hsp90, α-tubulin,p53, NFkb and HIF1α [Drummond et al., Annu Rev. Pharmacol. Toxicol. 45:495 (2004)]. Certain preferred histone deacetylase inhibitors are those that interact with, and/or reduce the activity of a histone deacetylase which is involved in tumorigenesis, and these compounds may be useful for treating proliferative diseases. Examples of such cell proliferative diseases or conditions include cancer (include any metastases), psoriasis, and smooth muscle cell proliferative disorders such as restenosis. The inventive compounds may be particularly useful for treating tumors such as breast cancer, colon cancer, lung cancer, ovarian cancer, prostate cancer, head and/or neck cancer, or renal, gastric, pancreatic cancer and brain cancer as well as hematologic malignancies such as lymphoma and leukemias. In addition, the inventive compounds may be useful for treating a proliferative disease that is refractory to the treatment with other chemotherapeutics; and for treating hyperproliferative condition such as leukemias, psoriasis and restenosis. In other embodiments, compounds of this invention can be used to treat pre-cancer conditions or hyperplasia including familial adenomatous polyposis, colonic adenomatous polyps, myeloid dysplasia, endometrial dysplasia, endometrial hyperplasia with atypia, cervical dysplasia, vaginal intraepithelial neoplasia, benign prostatic hyperplasia, papillomas of the larynx, actinic and solar keratosis, seborrheic keratosis and keratoacanthoma.

Additionally compounds of the various embodiments disclosed herein may be useful for treating neurodegenerative diseases, and inflammatory diseases and/or immune system disorders.

Examples of such disorders are selected from the group consisting of cancer, inflammatory diseases and/or immune system disorders (e.g. rheumatoid arthritis, systemic lupus erythematosus), angiofibroma, cardiovascular diseases, fibrotic diseases, diabetes, autoimmune diseases, chronic and acute neurodegenerative disease like Huntington's disease, Parkinson's disease, disruptions of nerval tissue and infectious diseases like fungal, bacterial and viral infections. In another embodiment the disorder is a proliferative disorder.

The histone deacetylase inhibitors of the invention have significant antiproliferative effects and promote differentiation, cell cycle arrest in the G1 or G2 phase, and induce apoptosis.

SYNTHESIS OF DEACETYLASE INHIBITORS

The agents of the various embodiments may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available in the art using starting materials that are readily available. The preparation of particular compounds of the embodiments is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other agents of the various embodiments. For example, the synthesis of non-exemplified compounds may be successfully performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. A list of suitable protecting groups in organic synthesis can be found in T.W. Greene and P. G. M. Wuts' Protective Groups in Organic Synthesis, 3rd Edition, Wiley-lnterScience, 1999. Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the various embodiments.

Reagents useful for synthesizing compounds may be obtained or prepared according to techniques known in the art.

In the examples described below, unless otherwise indicated, all temperatures in the following description are in degrees Celsius and all parts and percentages are by weight, unless indicated otherwise.

Various starting materials and other reagents were purchased from commercial suppliers, such as Aldrich Chemical Company or Lancaster Synthesis Ltd., and used without further purification, unless otherwise indicated. Tetrahydrofuran (THF) and N ₁ N- dimethylformamide (DMF) were purchased from Aldrich in SureSeal bottles and used as received. All solvents were purified by using standard methods in the art,, unless otherwise indicated.

The reactions set forth below were performed under a positive pressure of nitrogen, argon or with a drying tube!, at ambient temperature (unless otherwise stated), in anhydrous solvents, and the reaction flasks are fitted with rubber septa for the introduction of substrates and reagents via syringe. Glassware was oven-dried and/or heat-dried. Analytical thin-layer chromatography was performed on glass-backed silica gel 60 F 254 plates (E Merck (0.25 mm)) and eluted with the appropriate solvent ratios

(v/v). The reactions were assayed by TLC and terminated as judged by the consumption of starting material.

The TLC plates were visualized by UV absorption or with a p-anisaldehyde spray reagent or a phosphomolybdic acid reagent (Aldrich Chemical, 20wt% in ethanol) which was activated with heat, or by staining in iodine chamber. Work-ups were typically done by doubling the reaction volume with the reaction solvent or extraction solvent and then washing with the indicated aqueous solutions using 25% by volume of the extraction volume (unless otherwise indicated). Product solutions were dried over anhydrous sodium sulfate prior to filtration, and evaporation of the solvents was under reduced pressure on a rotary evaporator and noted as solvents removed in vacuo. Flash column chromatography [Still et al, J. Org. Chem., 43, 2923 (1978)] was conducted using E Merck-grade flash silica gel (47-61 mm) and a silica gehcrude material ratio of about 20:1 to 50:1 , unless otherwise stated. Hydrogenolysis was done at the pressure indicated or at ambient pressure.

1 H NMR spectra was recorded on a Bruker instrument operating at 400 MHz, and ¹³ C-NMR spectra was recorded operating at 100 MHz. NMR spectra are obtained as CDCI ₃ solutions (reported in ppm), using chloroform as the reference standard (7.25 ppm and 77.00 ppm) or CD ₃ OD (3.4 and 4.8 ppm and 49.3 ppm), or an internal tetramethylsilane standard (0.00 ppm) when appropriate. Other NMR solvents were used as needed. When peak multiplicities are reported, the following abbreviations are used: s = singlet, d = doublet, t = triplet, m = multiplet, br = broadened, dd = doublet of doublets, dt = doublet of triplets. Coupling constants, when given, are reported in Hertz.

Mass spectra were obtained using LC/MS either in ESI or APCI. All melting points are uncorrected.

All final products had greater than 80% purity (by HPLC at wavelengths of 220 nm and 254 nm).

The following examples are intended to illustrate the embodiments disclosed and are not to be construed as being limitations thereto. Additional compounds, other than those described below, may be prepared using the following described reaction scheme or appropriate variations or modifications thereof.

SYNTHESIS

A number of compounds within the scope of Formula (I) can be produced using the reaction scheme set out in Scheme I.

Scheme I

HSCH ₂ CO ₂ CH ₃ ⁰⁵ TVf SnCI ₂ , HCI

Specifically, the hydroxamate compounds Formula Vl can be synthesized by the synthetic route shown in Scheme I. The reaction of 2-Chloro-5-nitrobenzaldehyde (I) with methyl thioglyocolate in the presence of a base (e.g. K ₂ CO ₃ ) in an appropriate solvent (e.g. DMF) gave (III). The nitro group of (III) can be reduced by appropriate reducing agent (e.g. tin chloride) to afford amine IV, which then can be further modified by treating with corresponding acid chloride in the present of a base (e.g. DIEA) to give compounds V. The hydroxamate compounds (Vl) can be obtained from the compounds of formula (V) by a known synthesis method (J. Med. Chem., 2002, 45, 753-757).

Accordingly, following the generally synthetic scheme set out in Scheme I a skilled worker can produce compounds of the general formula

As will be appreciated by a skilled worker judicious selection of the starting materials I and the acid chloride used in the third step allows for the synthesis of a broad range of compounds within this general formula.

Additional compounds can be produced using the reaction scheme set out in Scheme II.

Scheme Il

IV VIl VIII

Reductive-annination, NH ₂ OH/ acylation or alkylation NaOMe

IX

Specifically, the hydroxamate compounds Formula X can be synthesized by the synthetic route shown in Scheme II. The reaction of amines (IV) with corresponding protected α-amino acid chloride in the presence of a base (e.g. DIEA) gave compounds VII. Deprotection of compounds VII gave free α-amine, which can be functionalized to give diverse products IX by using different methods (such as reductive amination, acylation, alkylation etc.) The hydroxamate compounds (X) can be obtained from the compounds of formula (IX) by a known synthesis method (J. Med. Chem., 2002, 45, 753-757).

Accordingly following the general procedure set out in Scheme Il the skilled addressee can produce compounds of the general formula

Once again modification of the starting material (IV) and the amino acid chloride used in Step 1 can be used to produce a wide variety of compounds with this general formula.

A similar methodology can be used as shown in Scheme III to provide compounds of the general formula

Scheme III

Once again, by judicious choice of the starting material (VHI) and the amine group used in step 2 a wide variety of compounds can be accessed.

A similar methodology can be used for the production of compounds of general formula

The route to these compounds are shown in Scheme IV.

Scheme IV

IV XIV

XVI

In addition compounds of the general form

can be synthesized as shown in Scheme V or Scheme Vl.

Scheme V

XIV XVII

XVIII

Scheme Vl

XIV XIX

reductive-amination or acylation

XVIII

Specifically, the hydroxamate compounds Formula XVIII can be synthesized by the synthetic route shown in Scheme Vl. The displacement of the bromide (XIV) with corresponding primary amines gave compounds XIX, which can be functionalized to give diverse products XVIl by using different methods (such as reductive amination, acylation etc.) The hydroxamate compounds XVIII can be obtained from compounds XVII using the known protocol.

As before, modification of the reagents and/or the starting materials can enable a skilled worker to make a large number of compounds within the scope of the general formula.

A number of additional compounds may be made by the general scheme outlined in scheme VII which allows entry in compounds of the general formula in which a nitrogen moiety attached to the benzothiophene ring may be disubstituted. Specifically reaction of the free amine IV with an alkylating agent such an aldehyde under reducing conditions leads to the formation of the alkylated amine XX. Reaction of the alkylated amine XX with an appropriately substituted acid chloride leads to the N-alkylated amides XXI. The hydroxamate compounds XXIV can be obtained from compounds XXI using the known protocol. Alternatively, the group R ₁ in compounds XXI can be further transformed into Ri" which may contain functional groups such as alcohol, aldehyde or amines, utilising standard chemistry to give compounds XXII. Once formed these

compounds can be converted under standard conditions to the free hydroxamates XXIII.

Scheme VII

XXI XXII

XXIV XXIII

As before, modification of the reagents and/or the starting materials can enable a skilled worker to make a large number of compounds within the scope of the general formula.

The following preparation and examples are given to enable those skilled in the art to more clearly understand and to practice the subject matter hereof. They should not be considered as limiting the scope of the disclosure, but merely as being illustrative and representative thereof.

Example 1

Preparation of δ-Phenylacetylamino-benzoFbithiophene^-carboxylic acid hvdroxyamide (1 ) Step i

To a solution of 2-Chloro-5-nitrobenzaIdehyde (3.7 g, 0.020 mol) in N, N- Dimethylformamide (40 ml_) was added methyl thioglyocolate (1.82 ml_, 0.020 mol) followed by K ₂ CO ₃ (3.3 g, 0.024 mol). The reaction mixture was stirred at rt overnight. Then, water was added to the reaction mixture and the suspension was filtered. The residue was subsequently washed with water to yield the product 6-Nitro- benzo[b]thiophene-2-carboxylic acid methyl ester as yellow solid. Yield: 95 %. Step 2

To the above product 6-Nitro-benzo[b]thiophene-2-carboxylic acid methyl ester (1 g, 0.004 mol) and SnCI ₂ -H ₂ O (4.85 g, 0.017 mol) was added concentrated HCI (20 ml_, 0.24 mol). The reaction was stirred at 80 ⁰ C for 1 hour, then cooled to low temperature. Then, the pH of the reaction mixture was raised to pH = 9 ~ 11 using 10% NaOH solution in water. The suspension was then filtered and residue was washed with H ₂ O followed by CH ₃ OH to yield the product as yellow solid quantitatively. Step 3

To a solution of above Benzothiophene amine (104 mg, 0.5 mmol) in 5 mL of anhydrous DCM was added phenylacetic acid chloride (0.13 mL, 1.0 mmol) and DIEA (di-isopropyl ethylamine, 0.18 mL, 2.0 mmol) at room temperature under N ₂ . The resulting mixture was stirred overnight. After work-up, the residue was purified on column (Hexanes:DCM : EtOAc = 3:1 :1 ) to afford compound 5-Phenylacetylamino- benzo[b]thiophene-2-carboxylic acid methyl ester. Yield: 71 %. LCMS m/z: 326 ([M+H] ⁺ ). Step 4

To a suspension of above methyl ester (45 mg, 0.14 mmol) in 0.5 mL of MeOH was added a pre-prepared 2.0 M NH ₂ OH in MeOH solution (1.5 mL, 2.0 mmol). The resulting suspension was stirred for 4 hrs. then quenched with TFA (0.1 mL, 1.3 mmol). The resulting solution was purified on preparative HPLC to give 19 mg of 5- Phenylacetylamino-benzo[b]thiophene-2-carboxylic acid hydroxamic acid. Yield: 42%. HPLC purity at 254 nm: 98.8%.t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5-95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1 % trifluoroacetic acid; UV 254): 6.23 min. LCMS m/z: 327 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 3.65 (2H, s), 7.17-7.38 (5H, m), 7.43 (1H, d, J = 8.8 Hz), 7.70 (1H, s), 7.76 (1H, d, J = 8.8 Hz), 8.20 (1H, s).

Example 2

Preparation of δ-Benzoylamino-benzoFbithiophene-Σ-carboxylic acid hvdroxyamide (2) The titled compound (2) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 96.5%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1% trifluoroacetic acid; UV 254): 6.00 min. LCMS m/z: 313 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.50-7.62 (3H, m), 7.78 (1 H, dd, J = 2.2, 8.8 Hz), 7.91 (1 H ₁ s), 7.90-8.05 (3H, m), 8.45 (1 H, d, J = 2.2 Hz), 10.40 (1 H, s), 11.46 (1H, s).

Example 3

Preparation of δ-Phenylmethanesulfonylamino-benzorblthiophene^-carboxylic acid hvdroxyamide (3)

The titled compound (3) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 82.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 10 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1% trifluoroacetic acid; UV 254): 3.71 min. LCMS m/z: 363 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 4.36 (2H, s), 7.16-7.30 (6H, m), 7.64 (1 H, d, J = 2.0 Hz), 7.69 (1H, s), 7.77 (1H, d, J = 8.8 Hz).

Example 4

Preparation of 5- (3-Phenyl-acryloylamino)-benzorblthiophene-2-carboxylic acid hvdroxyamide (4)

The titled compound (4) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1% trifluoroacetic acid; UV 254): 7.20 min. LCMS m/z: 339 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 6.87 (1 H, d, J = 20.0 Hz), 7.41-7.49 (3H ₁ m), 7.60-7.66 (4H, m), 7.90 (1 H, s), 7.97 (1 H, d, J = 8.8 Hz), 8.44 (1 H, s), 10.38 (1 H, s), 11.45 (1 H, s).

Example 5

Preparation of 5-r3-Phenyl-2-(toluene-4-sulfonylaminoVpropionylamino1- benzorbithiophene-2-carboxylic acid hydroxyamide (5)

The titled compound (5) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 98.6%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1 % trifluoroacetic acid; UV 254): 8.00 min. LCMS m/z: 510 ([IVHH] ⁺ ). ¹ H NMR (400 MHz, DMSOd ₆ ) δ 2.21 (3H, s), 2.70-2.90 (2H, m), 4.01 (1 H, m), 7.04 (2H, d, J = 8.0 Hz), 7.05-7.20 (5H, m), 7.43 (2H, d, J = 8.0 Hz), 7.76 (1 H, br), 7.80 (1 H, d, J = 8.4 Hz), 7.85 (1 H, d, J = 2.0 Hz), 8.14 (1H, d, J = 9.2 Hz), 9.95 (1 H, s), 11.36 (1 H, s).

Example 6

Preparation of 5- (4-Phenyl-butyrylamino)-benzorblthiophene-2-carboxylic acid hvdroxyamide (6)

The titled compound (6) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1 % trifluoroacetic acid; UV 254): 7.49 min. LCMS m/z: 355 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 1.85 (2H, m), 2.30 (2H, t, J = 7.6 Hz), 2.57 (2H, t, J = 7.6 Hz), 7.10-7.30 (5H, m), 7.44 (1 H, dd, J = 2.2, 8.8 Hz), 7.78 (1 H, br.), 7.83 (1 H, d, J= 8.8 Hz), 8.24 (1 H, d, J = 2.2 Hz), 9.96 (1 H, s), 11.36 (1 H, br); ¹³ C-NMR (400 MHz, DMSO) 526.7, 34.6, 35.8, 114.1 , 119.0, 122.8, 124.1, 125.8, 128.3 (4C), 134.3,136.7, 138.1 , 139.4, 141.6, 159.6, 171.1.

Example 7

Preparation of 5- (2-Pyridin-3-yl-acetylamino)-benzorblthiophene-2-carboxylic acid hvdroxyamide (71

The titled compound (7) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B: Acetonitrile with 0.1 % trifluoroacetic acid; UV 254): 2.64 min. LCMS m/z: 328 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.94 (2H, s), 7.54 (1 H, dd, J = 2.0, 8.4 Hz), 7.70-7.85

(2H ₁ m), 7.95 (1 H, d, J = 8.8 Hz), 8.20-8.30 (2H, m), 8.71 (1 H, d, J = 2.2 Hz), 8.78 (1 H ₁ s), 10.51 (1 H, s), 11.43 (1 H, br)

Example 8

Preparation of 5- [2-(5-Bromo-1 f7-indol-3-yl)-acetylamino ^' l-benzorb ^' ]thiophene-2- carboxylic acid hvdroxyamide (8)

The titled compound (8) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 96.3%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1 % trifluoroacetic acid; Solvent B: Acetonitrile with 0.1% trifluoroacetic acid; UV 254): 7.33 min. LCMS m/z: 446, 444 ([IvHH] ⁺ ). ¹ H NMR (400 MHz, DMSOd ₆ ) δ 3.68 (2H ₁ s), 7.11 (1 H, dd, J = 2.0, 8.8 Hz), 7.26 (1 H, d, J = 8.8 Hz), 7.27 (1 H, d, J = 2.0 Hz), 7.47 (1H, dd, J = 2.0, 8.8 Hz), 7.75 (1 H, d, J = 2.0 Hz), 7.77 (1 H, br.), 7.85 (1 H, d, J = 8.8 Hz), 8.23 (1 H, d, J = 2.0 Hz), 9.17 (1 H, s), 10.20 (1H, s), 11.07 (1H, br.), 11.34 (1H, br)

Example 9

Preparation of 5- (3-Phenyl-propionylamino)-benzorblthiophene-2-carboxylic acid hvdroxyamide (9)

The titled compound (9) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B:. Acetonitrile with 0.1 % trifluoroacetic acid; UV 254): 6.77 min. LCMS m/z: 341 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.67 (2H, t, J = 7.6 Hz), 2.94 (2H, t, J = 7.6 Hz), 7.16- 7.35 (5H, m), 7.50 (1H, dd, J = 2.2, 8.8 Hz) ₁ 7.86 (1 H, s), 7.91 (1H, d, J = 8.8 Hz), 8.30 (1 H, d, J = 2.2 Hz), 9.26 (1 H, br), 10.07 (1 H, s), 11.43 (1 H, br); ¹³ C-NMR (400 MHz, CDCl ₃ ) δ 30.8, 37.9, 114.1 , 119.0, 122.8, 124.1, 125.9, 128.2 (2C), 128.3 (2C), 130.4, 136.6, 138.2, 139.4, 141.1 , 159.6, 170.5.

Example 10

Preparation of 5-(2-1 H-indoJ-S-yl-acetylaminoVbenzofbithiophene^-carboxylic acid hvdroxyamide (10)

The titled compound (10) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. t _R =(LC/PDA: Phenomenex Luna C18 2.0x150mm 5μ column; 0.8 mL/min, gradient 5- 95% B over 20 min, Solvent A: H ₂ O with 0.1% trifluoroacetic acid; Solvent B:

Acetonitrile with 0.1% trifluoroacetic acid; UV 254): 6.11 min. LCMS m/z: 366 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.70 (2H, s), 6.93 (1 H, t, J = 7.6 Hz), 7.01 (1 H, t, J = 7.6 Hz), 7.21 (1 H ₁ s), 7.29 (1 H, d, J = 8.0 Hz), 7.47 (1 H ₁ d, J = 8.0 Hz) ₁ 7.55 (1 H, d, J = 8.0 Hz), 7.76 (1 H, s), 7.84 (1 H, d, J = 8.4 Hz), 8.23 (1 H, d, J = 2.0 Hz), 10.18 (1 H ₁ s), 10.84 (1H ₁ br), 11.34 (1 H ₁ br).

Example 11

Preparation of 5-r2-(3,4-Dimethoxy-phenyl)-acetylaminol-benzofb1thiophene-2 - carboxylic acid hydroxyamide (11)

The titled compound (11) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. LCMS m/z: 387 ([M+H] ⁺ ).

Example 12

Preparation of 5-(3,3-Dimethyl-butyrylam ^' inoVbenzofb1thiophene-2-carboxylic acid hydroxyamide (12)

The titled compound (12) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. LCMS m/z: 307 ([M+H] ⁺ ).

Example 13

Preparation of 5-(2-Phenoxy-acetylamino)-benzorbUhiophene-2-carboxylic acid hydroxyamide (13)

The titled compound (13) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purity at 254 nm: 99.0%. LCMS m/z: 343 ([M+H] ⁺ ).

Example 14

Preparation of 5-(2-Benzyloxy-acetylamino)-benzo[blthiophene-2-carboxylic acid hvdroxyamide (14)

The titled compound (14) was prepared according to the procedures described in Example 1 , by using appropriate starting materials. HPLC purify at 254 nm: 99.0%. LCMS m/z: 357 ([M+H] ⁺ ).

Example 15 Step i

SOCI ₂ (7.28 ml, 0.120 mol) was added to a suspension of Fmoc-Phe-OH 5 (7.74 g, 0.020 mol) in CH ₂ CI ₂ (100 ml) and N,N-Dimethylformadide (2 drops, catalytic). The

reaction mixture was warmed at 40 ⁰ C for.30 minutes. The course of the reaction was monitored by adding MeOH to a sample of the reaction mixture to detect the formation of the corresponding methyl ester. Upon completion, 2 drops of toluene was added to the reaction mixture and the solvent was removed under vacuum. The residue was used immediately for the next reaction. Step 2

The freshly prepared above acid chloride (1 equiv.) in CH ₂ CI ₂ was added to a solution of compound 6-Amino-benzo[b]thiophene-2-carboxylic acid methyl ester (1 equiv.) in CH ₂ CI ₂ and DIEA (1.1 equiv.). Reaction mixture was stirred for 3 hours at rt resulting in an orange gel-like suspension. It was then filtered and the residue was washed extensively with MeOH and dried under vacuum, yielding the product 5-[2-(9H-Fluoren- θ-ylmethoxycarbonylaminoJ-S-phenyl-propionylaminol-benzotbJ thiophene^-carboxylic acid methyl ester as white solid. Yield: 87%. Step 3

Piperidine (4.3 mL, 0.043 mol) was added to a solution of 5-[2-(9H-Fluoren-9- ylmethoxycarbonylaminoJ-S-phenyl-propionylaminol-benzoIbJthi ophene^-carboxylic acid methyl ester (5 g, 0.0086 mol) in diethyl ether (35 mL). The reaction mixture was stirred at r.t. for 5 hours. The resulting suspension was filtered and the residue was washed with ether and dried under vacuum to yield the product 5-(2-Amino-3-phenyl- propionylamino)-benzo[b]thiophene-2-carboxylic acid methyl ester as white solid quantitatively. Step 4

Parallel synthesis of analogues

The carboxylic acids (0.141 mmol) were added to above free amine compound (50 mg, 0.141 mmol), EDCI (27.1 mg, 0.141 mmol), HOBt (19.1 mg, 0.141 mmol) and DIEA (49.1 ul, 0.282 mmol) in CH ₂ CI ₂ (1-2 ml) at rt for 1 overnight. For products that precipitated out filtered and washed with CH ₂ CI ₂ . Otherwise, solvent was removed and MeOH was added to precipitate the products. For products that could not be precipitated with either CH ₂ CI ₂ or MeOH, purification was carried out via reverse phase HPLC system. Step 5

Synthesis of analogues 15a - 15t

Compounds IX of Scheme Il was individually mixed with a freshly prepared 2M cocktail of NH ₂ OH.HCI (10 equiv.), 37% NaOMe (12 equiv.) in MeOH. A few drops of N ₁ N- dimethylformamide was added to reaction with poor solubility. The reaction mixture was then allowed to stir at rt for 20 minutes and subsequently quenched with an

equivalent amount of trifluoroacetic acid under 0 ⁰ C. The crude products were then subjected to purification via reverse phase HPLC system.

Example (15i)

Preparation of 5-r3-Phenvi-2-(3-piperidin-1 -yl-propionylamino Vpropionylaminoi- benzoFblthiophene-2-carboxylic acid hvdroxyamide (150

The titled compound (15i) was prepared according to the procedures described in

Example 15, by using appropriate starting materials. LCMS m/z: 495 ([M+Hf). ¹ H NMR

(400 MHz, MeOD-d ₄ ) δ 1.64-1.87 (2H, m), 2.60 - 2.84 (2H, m), 2.93 (2H, t, J = 10 Hz),

2.96 (1 H, dd, J _gem = 14 Hz, J _vic = 9 Hz), 3.15 (1H, dd, J _gem = 14 Hz, J _vic = 6 Hz), 3.25

(2H, m), 3.40 (2H ₁ d, J = 12 Hz2H), 4.73 (1 H, dd, J = 7 Hz), 7.26 (5H, m), 7.37 (1 H, d, J

= 7 Hz), 7.71 (1H, s), 7.78 (1 H, d, J = 9 Hz), 8.12 (1H, s).

Example (15q)

Preparation of 5-r3-Phenyl-2-(2-pyridin-2-yl-acetylarnino)-propionylamino1- benzofbithiophene-2-carboxylic acid hvdroxyamide (15q)

The titled compound (15q) was prepared according to the procedures described in Example 15, by using appropriate starting materials. LCMS m/z: 475 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ): δ 2.99 (1H, dd, J _gβm = 14 Hz, J _vic = 9 Hz), 3.19 (1 H, dd, J _gθm = 14 Hz, J _vic = 8 Hz), 4.75 (3H, m), 7.22 (5H, m), 7.35 (1H, d, J = 9 Hz), 7.62 (1 H ₁ d, J = 8 Hz), 7.71 (1H ₁ 1, J = 6 Hz), 7.71 (1 H, s), 7.77 (1 H, d, J = 9 Hz), 8.11 (1 H, s), 8.23 (1H, t, J = S Hz), 8.60 (1 H, d, J = 6 Hz)

Example (15s)

Preparation of 5-[2-(4-Dimethylamino-benzoylamino)-3-Phenyl-propionylamino1 - benzofbithiophene-2-carboχylic acid hvdroxyamide (15s) The titled compound (15s) was prepared according to the procedures described in Example 15, by using appropriate starting materials. LCMS m/z: 503 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.97 (6H, s), 3.13 (2H ₁ d, J = 6.5 Hz), 6.70 (2H, d, J = 9 Hz), 7.16 - 7.42 (5H, m), 7.57 (1 H, d, J = 9 Hz), 7.74 (2H, d, J = 9 Hz), 7.87 (1 H, s), 7.95 (1 H, d, J = 9 Hz), 8.30 (1 H, s), 8.35 (1 H, d, J = 8 Hz), 10.3 (1 H, s), 11.4 (1 H, br).

Example 16

Preparation of 5-(2-Benzylamino-3-phenyl-propionylaminoVbenzorblthiophene-2 - carboxylic acid hvdroxyamide, (16)

Compound 16-1 (58 mg, 0.165 mmol) in MeOH: AcOH (4:1) (2.5 ml_) was added benzaldehyde (16 mg, 0.15 mmol). The reaction was stirred at rt for 3 days and then, NaBH ₃ CN (14 mg, 0.225 mmol) was added and reaction mixture was left for 1 day. Hydroxamic acid formation: Followed the same protocol mentioned-above. Yield = 71 %. LCMS m/z: 446 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 3.15 (1 H ₁ dd, J _gβm = 13 Hz, Jw _o = 9 Hz), 3.37 (1 H, dd, J _gem = 13 Hz, J _vic = 5 Hz), 4.14 (1 H, s), 4.21 (2H, s), 7.26 (7H ₁ m), 7.46 (5H, m), 7.88 (1 H, s), 7.95 (1 H, d, J = 9 Hz), 8.17 (1 H, s).

Example 17 Step i

To a solution of compound VHl of Scheme III (354 mg, 1mmol) in CH ₂ CI ₂ (5 ml_) was added'DIEA (150 μl_, 0.868 mmol) followed by a solution of p-nitrophenylchloroformate (250 mg, 1.24 mmol) in CH ₂ CI ₂ (5 ml_). The pale yellow solution was allowed to stir at rt for 3 hours. A white suspension was formed during the reaction. The solid was filtered, washed with CH ₂ CI ₂ and dried over vacuum to yield the desired compound Xl. Yield: 45 %.

Step 2

Parallel synthesis of analogues

To compound Xl (1 eqv.) in CH ₂ CI ₂ :THF (1 :1) (3 ml_) was added acetic acid (0.1 eqv.) followed by the amines (1.2 eqv.). Reaction was allowed to shake at rt for one overnight. Generally, products precipitated either in CH ₂ CI ₂ or MeOH and used for subsequent step without further purification.

Step 3

Synthesis of analogues 17a - 17q

Compounds XII of Scheme III were individually subjected to a freshly prepared cocktail of NH ₂ OH. HCI/ NaOMe/ MeOH. A few drops of A/,Λ/-dimethylformamide was added to reaction with poor solubility. The reaction mixture was then allowed to stir at rt for 20 minutes and subsequently quenched with an equivalent amount of trifluoroacetic acid under 0 ⁰ C. The crude products were then subjected to purification via reverse phase HPLC system.

Example 17j

Preparation of 5-{2-r3-(2,4-Difluoro-benzvh-ureido1-3-phenyl-propionylamino >- benzorblthiophene-2-carboxylic acid hvdroxyamide (17Q

The titled compound (17j) was prepared according to the procedures described in Example 17, by using appropriate starting materials. LCMS m/z: 525 ([M+H] ⁺ ). ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 2.86 (1 H, dd, J _gem = 13 Hz, J _vic = 8 Hz), 3.02 (1 H, td, J _gem = 10 Hz, J _vic = 4 Hz), 4.18 (2H, d, J = 6 Hz), 4.60 (1 H, dd, J = 8 Hz ₁ 8 Hz), 6.39 (1 H, d, J = 8 Hz), 6.58 (1 H, t, J = 6 Hz), 7.01 (1 H, td, J = 9 Hz, 2 Hz), 7.13 - 7.29 (5H, m), 7.50 (1 H, dd, J = 9 Hz, 2 Hz), 7.81 (1 H, s), 8.00 (1 H, d, J = 11 Hz), 8.24 (1 H, d, J = 2 Hz).

Example 18

Step i

To a solution of compound IV of Scheme IV (500 mg, 2.42 mmol) in CH ₂ CI ₂ (35 mL) was added slowly bromo-acetyl chloride (320 μL, 2.045 mmol) in CH ₂ CI ₂ (15 mL) at rt. Then, DIEA (422 μL, 2.42 mmol) was added dropwise. The reaction mixture was left to stir at rt for 3 hours. Water was added, and the reaction mixture was extracted with CH ₂ CI ₂ , dried and solvent evaporated in vacuo to yield 5-(2-Bromo-acetylamino)- benzo[b]thiophene-2-carboxylic acid methyl ester (XIV) quantitatively.

Step 2

Synthesis of analogues

To compound XIV (1 eqv.) in acetonitrile (7.5 mL) was added the appropriate phenols or substituted phenols (2 eqv.) followed by K ₂ CO ₃ (2eqv.) and tetrabutylammonium hydrogen sulfate (1 eqv.). The resulting mixture was stirred at room temperature for 1 day to give compounds XV.

Step 3

Synthesis of analogues 18a - 18b

Compounds XV of Scheme IV were individually subjected to a freshly prepared cocktail of NH ₂ OH-HCI/ NaOMe/ MeOH. A few drops of /V,/V-dimethylformamide was added to

reaction with poor solubility. The reaction mixture was then allowed to stir at rt for 20 minutes and subsequently quenched with an equivalent amount of trifluoroacetic acid under 0 ⁰ C. The crude products were then subjected to purification via reverse phase HPLC system.

Example 18a

Preparation of δ-^^-Methoxy-phenoxyVacetylaminol-benzofbithiophene-Σ-carb oxylic acid hvdroxyamide (18a)

The titled compound (18a) was prepared according to the procedures described in Example 18, by using appropriate starting materials. HPLC purity at 254 nm: 95.0%. LCMS m/z: 373 ([M+H] ⁺ ).

Example 18b

Preparation of 5-f2-(3.4.5-Trimethoxy-phenoxy)-acetylaminol-benzofb1thiophe ne-2- carboxylic acid hvdroxyamide (18b)

The titled compound (18b) was prepared according to the procedures described in Example 18, by using appropriate starting materials. LCMS m/z: 433 ([M+H] ⁺ ). Example 19

Step i

Synthesis of analogues

To compound XIV (1 eqv.) of Scheme V in CH ₂ CI ₂ (1 mL) was added primary or secondary amines (3.3 eqv.) in CH ₂ CI ₂ (1 mL). The resulting mixture was stirred at room temperature for 1 day to obtain compounds XVII.

Step 2

Synthesis of analogues 19a - 19d

Compounds XVII of Scheme V were individually subjected to a freshly prepared cocktail of NH ₂ OH-HCI/ NaOMe/ MeOH. A few drops of Λ/,Λ/-dimethylformamide was added to reaction with poor solubility. The reaction mixture was then allowed to stir at rt for 20 minutes and subsequently quenched with an equivalent amount of trifluoroacetic acid under 0 ⁰ C. The crude products were then subjected to purification via reverse phase HPLC system.

Example 19a

Preparation of 5-f2-(2-lsopropoxy-ethylamino)-acetylamino1-benzo[b1thiophen e-2- carboxylic acid hvdroxyamide (19a)

The titled compound (19a) was prepared according to the procedures described in Example 19, by using appropriate starting materials. HPLC purity at 254 nm: 99.9%. LCMS m/z: 352 ([M+H] ⁺ ).

Example 19b

Preparation of 5-(2-Piperidin-1-yl-acetylamino)-benzofblthiophene-2-carboxy lic acid hvdroxyamide (19b)

The titled compound (19b) was prepared according to the procedures described in Example 19, by using appropriate starting materials. LCMS m/z: 334 ([M+H] ⁺ ).

Example 19c

Preparation of 5-(2-3,6-Dihydro-2H-pyridin-1 -yl-acetylamino)-benzofb1thiophene-2- carboxylic acid hvdroxyamide (19c)

The titled compound (19c) was prepared according to the procedures described in Example 19, by using appropriate starting materials. HPLC purity at 254 nm: 99.9%.LCMS m/z: 332([M+H] ⁺ ).

Example 19d

Preparation of 5-r2-(Benzyl-methyl-amino)-acetylarnino1-benzofb1thiophene-2 - carboxylic acid hvdroxyamide (19d)

The titled compound (19d) was prepared according to the procedures described in Example 19, by using appropriate starting materials. LCMS m/z: 370 ([M+Hf).

Example 20a

Preparation of 5-rf3,3-Dimethyl-butyryl)-(2-hvdroxy-ethyl)-aminol-benzofb1t hiophene-2- carboxvlic acid hvdroxvamide (20a)

IV 20a-1

20a-3 20a

Step 1

To a solution of compound IV (194 mg, 0.9 mmol) in 10 ml_ of MeOH was added

Benzyloxy acetaldehyde (0.086 ml_, 0.6 mmol) at room temperature. The resulting mixture was stirred overnight. NaCNBH ₃ (98 mg, 1.562 mmol) and acetic acid (1 ml_) were then added and the resulting mixture was stirred at room temperature for 2 hours. After work-up, the residue was purified on column (Hexanes: EtOAc = 4:1) to afford compound 5-(2-Benzyloxy-ethylamino)-benzo[b]thiophene-2-carboxylic acid methyl ester, 20a-1. Yield: 84%. LCMS m/z: 342 ([M+H] ⁺ ).

Step 2

To a solution of compound 20a-1 (174 mg, 0.5 mmol) in 10 ml_ of anhydrous DCM was added 3,3-Dimethyl-butyryl chloride (0.14 ml_, 1.0 mmol) and DIEA (di-isopropyl ethylamine,0.18 ml_, 1.0 mmol) at room temperature under N ₂ . The resulting mixture was stirred for 1 hour. After work-up, the residue was purified on column (Hexanes:EtOAc = 4:1) to afford compound 5-[(2-Benzyloxy-ethyl)-(3,3-dimethyl- butyryl)-amino]-benzo[b]thiophene-2-carboxylic acid methyl ester 20a-2 quantitatively. LCMS m/z: 440 ([M+H] ⁺ ).

To a solution of compound 20a-2 (252 mg, 0.57 mmol) in 10 mL of anhydrous DCM was added 1.0 M BCI ₃ in DCM (0.15 mL, 1.15 mmol) at~78 ⁰ C. The resulting mixture was stirred at this temperature for 1 hour. Then the reaction mixture was gradually warmed up to -0 ⁰ C and stirred for another 3 hours. Subsequently, MeOH (1 mL) was added at ^" 78 ⁰ C and the resulting mixture was stirred for 1 hour. After work-up, the residue was purified on column (Hexanes:EtOAc = 2:3) to afford compound 5-[(3,3- Dimethyl-butyryl)-(2-hydroxy-ethyl)-amino]-benzo[b]thiophene -2-carboxylic acid methyl ester, 20a-3. Yield: 75%. LCMS m/z: 350 ([M+H] ⁺ ).

Step 4

To a suspension of compound 20a-3 (15 mg, 0.14 mmol) in 0.15 mL of MeOH was added NH ₂ OH-HCI (30 mg, 0.42 mmol) followed by 4.37 M of NaOMe (0.19 mL, 0.85 mmol). The resulting suspension was stirred for 30 minutes then quenched with 1 N HCI. The resulting solution was purified on preparative HPLC to give 4 mg of 5-[(3,3- Dimethyl-butyryl)-(2-hydroxy-ethyl)-amino]-benzo[b]thiophene -2-carboxylic acid hydroxyamide, 20a. Yield: 27%. HPLC purity at 254 nm: 99.0%. LCMS m/z: 351 ([MH-H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 0.93 (9H, s), 2.03 (2H, s), 3.69 (2H, t, J = 6.0 Hz), 3.86 (2H, t, J = 5.8 Hz), 7.37 (1 H, dd, J = 8.6, 1.6 Hz), 7.85 (1 H, s), 7.83 (1 H, s), 8.02 (1 H, d, J = 8.6 Hz).

Example 20b

Preparation of 5-f(2-Hvdroxy-ethyl)-(4-phenyl-butyryl)-aminol-benzofb1thiop hene-2- carboxylic acid hvdroxyamide (20b)

The titled compound (20b) was prepared according to the procedures described in Example 20a, by using appropriate starting materials. HPLC purity at 254 nm: 99.9%. LCMS m/z: 399 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 1.85 (2H, m), 2.07 (2H, t, J = 7.2 Hz), 2.49 (2H, t, J = 7.3 Hz), 3.67 (2H, t, J = 6.0 Hz), 3.86 (2H, t, J = 6.0 Hz), 7.11 - 6.91 (5H, m), 7.31 (1 H, dd, J = 8.6, 2.0 Hz), 7.71 (1 H, d,J = 2.9 Hz), 7.82 (1 H, s), 7.97 (1 H ₁ Cl ₁ J = 8.0 Hz). .

Example 20c

Preparation of 5-r(3,3-Dimethyl-butyrylH2-isopropylamino-ethylVamino1- benzorbithiophene-2-carboxylic acid hvdroxyamide (20c)

20c-2 20c

Step 1

To a solution of compound 20a-3 (132 mg, 0.38 mmol) in 7.5 mL of anhydrous DCM was added d ess-Martin periodinane (0.19 mL, 0.45 mmol) at 0 ⁰ C. The resulting mixture was gradually warmed up to room temperature and stirred for 30 minutes. Subsequently, saturated solution of sodium bicarbonatersodium sulfate (1 : 1) was added (15 mL). After work-up, the residue was purified on column (Hexanes:EtOAc = 9:1) to afford compound 5-[(3,3-Dfmethyl-butyryl)-(2-oxo-ethyl)-amino]- benzo[b]thiophene-2-carboxylic acid methyl ester, 20c-1. Yield: 93%. LCMS m/z: 348 ([M+H] ⁺ ).

Step 2

To a solution of compound 20c-1 (28 mg, 0.08 mmol) in 3.6 mL of MeOH was added lsopropylamine (0.069 mL, 0.8 mmol) at room temperature. The resulting mixture was stirred for 2 hours. NaCNBH ₃ (10 mg, 0.16 mmol) and acetic acid (0.3 mL) were then added and the resulting mixture was stirred at room temperature for 30 minutes. After work-up, the residue was purified on column (Hexanes.ΕtOAc = 4:1) to afford compound 5-(2-Benzyloxy-ethylamino)-benzo[b]thiophene-2-carboxylic acid methyl ester, 20c-2 quantitatively. LCMS m/z: 391 ([M+H] ⁺ ).

Step 3

To a suspension of compound 20c-2 (40 mg, 0.1 mmol) in 1.5 mL of MeOH was added NH ₂ OH-HCI (69 mg, 1 mmol) followed by 4.37 M of NaOMe (0.46 mL, 2 mmol). The resulting suspension was stirred for 30 minutes then quenched with 1N HCI. The resulting solution was purified on preparative HPLC to give 3 mg of 5-[(3,3-Dimethyl- butyryl)-(2-isopropylamino-ethyl)-amino]-benzo[b]thiophene-2 -carboxylic acid hydroxyamide, 20c. Yield: 8%. HPLC purity at 254 nm: 94.0%. LCMS m/z: 392 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ) δ 0.95 (9H, s), 1.34 (3H, s), 1.35 (3H, s), 2.11 (2H ₁ s), 3.23 (2H, t, 6.6 Hz), 3.44 (1 H, m,), 4.05 (2H, t, J = 6.6 Hz), 7.39 (1H, dd, J = 8.6, 2.1 Hz), 7.88 (2H, s), 8.01 (1 H ₁ d, J = 8.6 Hz)..

Example 2Od

Preparation of (5-{(3,3-Dimethyl-butyryl)-[2-(3-methyl-butylamino)-ethyl]-a mino}- benzo[b]thiophene-2-carboxylic acid hydroxyamide) (2Od)

The titled compound (2Od) was prepared according to the procedures described in Example 20a, by using appropriate starting materials. HPLC purity at 254 nm: 91.7%. LCMS m/z: 420 ([M+H] ⁺ ). ¹ H NMR (400 MHz, MeOD-d ₄ ): δ 0.95 (9H, s), 1.02 (3H, s), 1.04 (3H, s), 1.61 - 1.56 (2H ₁ m), 1.73 - 1.66 (1 H, m), 2.11 (2H, s), 3.07 (2H, t, J = 8.1 Hz), 3.23 (2H, t, J = 4.0 Hz), 4.06 (2H, t, 6.4 Hz), 7.38 (1 H, dd, J = 8.6, 2.1 Hz), 7.88 (1 H, s), 7.87 (1H, s), 8.08 (1 H, d, J = 8.6 Hz).

The following compounds are some representative examples prepared by methods disclosed or analogous to those disclosed in above Examples 1-2Od (Table 1 ).

By methods analogous to those disclosed above and by varying the starting materials used in the synthesis, a wide variety of compounds could be prepared according to Schemes IV - VII, including, but not limited to, those in Table 1a.

Specifically, the synthesis method of Example 1 , Example 18 and 19 can be employed to synthesize additional phenoxy ethylene, phenoxy acetyl (compounds series 18) and amino acetyl (compounds series 19) derivatives respectively. Some representative examples for these two series are shown in Table 1a (Compounds 21 - 31). Additionally, according to Scheme Vl, a series of secondary amines (XIX) can be synthesized by reacting appropriate primary amines with compound XIV, which can be functionalized to give diverse products XVII by using different methods (such as reductive amination, acylation, alkylation etc.) The hydroxamate compounds (XVIII) can be obtained from the compounds of formula (XVH) by synthesis methods known in the literature and stated before. Some representative examples are shown in Table 1a (Compounds 32 - 40).

Nonetheless, a series of compounds can be made according to Scheme VII, where the appropriate aldehydes could be reacted with compound IV to give compounds XX, which could then be reacted with the appropriate acyl chlorides or carboxylic acids to form compounds XXI, upon which could be converted to the free hydroxamates XXIV employing the similar methods mentioned above. The substituent R ₁ in intermediate XXI could be functionalized to Ri" to provide the intermediate XXII which could be converted to hydroxamate XXIII.

Table 1a

BIOLOGICAL TESTING AND ENZYME ASSAYS

Recombinant GST-HDAC1 Protein expression and purification

Human cDNA library was prepared using cultured SW620 cells. Amplification of human HDAC1 coding region from this cDNA library was cloned separately into the baculovirus expression pDEST20 vector (GATEWAY Cloning Technology, Invitrogen Pte Ltd). The pDEST20-HDAC1 construct was confirmed by DNA sequencing. Recombinant baculovirus was prepared using the Bac-To-Bac method following the manufacturer's instruction (Invitrogen Pte Ltd). Baculovirus titer was determined by plaque assay to be about 10 ⁸ PFU/ml.

Expression of GST-HDAC1 was done by infecting SF9 cells (Invitrogen Pte Ltd) with pDEST20-HDAC1 baculovirus at MOI=I for 48 h. Soluble cell lysate was incubated with pre-equilibrated Glutathione Sepharose 4B beads (Amersham) at 4 ⁰ C for 2 h. The beads were washed with PBS buffer for 3 times. The GST-HDAC1 protein was eluted by elution buffer containing 50 mM Tris, pH8.0, 15OmM NaCI, 1% Triton X-100 and 1OmM or 2OmM reduced Glutathione. The purified GST-HDAC1 protein was dialyzed with HDAC storage buffer containing 1OmM Tris, pH7.5, 10OmM NaCI and 3mM MgCI ₂ . 20% Glycerol was added to purified GST-HDAC1 protein before storage at -8O ⁰ C.

In vitro HDAC assay for determination of ICs _n values

The assay has been carried out in 96 well format and the BIOMOL fluorescent-based HDAC activity assay has been applied. The reaction composed of assay buffer, containing 25 mM Tris pH 7.5, 137 mM NaCI, 2.7 mM KCI, 1 mM MgCI ₂ , 1 mg/ml BSA, tested compounds, an appropriate concentration of HDAC1 enzyme, 500 μM Flur de lys generic substrate for HDAC1 enzyme and subsequently was incubated at room

temperature for 2 h. Flurde lys Developer was added and the reaction was incubated for 10 min. Briefly, deacetylation of the substrate sensitizes it to the developer, which then generates a fluorophore. The fluorophore is excited with 360 nm light and the emitted light (460 nm) is detected on a fluorometric plate reader (Tecan Ultra Microplate detection system, Tecan Group Ltd.).

The analytical software, Prism 3.0 (GraphPad Software Inc) has been used to generate IC ₅₀ from a series of data.

The HDAC enzyme inhibition results of representative compounds are shown in Table 2 (unit is in micromolar).

Table 2

Cell-based proliferation assay for determination of Glgn values

Human colon cancer cell lines (e.g. Colo205), human breast cancer cell lines (e.g. MDA-MB435) were obtained from ATCC. Colo205 cells were cultivated in RPMI 1640 containing 2 mM L-Glutamine, 5% FBS, 1.0 mM Na Pyruvate and MDA-MB435 cells were cultivated in DMEM containing 2 mM L-Glutamine, 5% FBS. Colo205 cells were seeded in 96-wells plate at 5000 cells per well and MDA-MB435 cells were seeded in 96-wells plate at 6000 cells per well. The plates were incubated at 37 ⁰ C, 5% CO ₂ , for 24 h. Cells were treated with compounds at various concentrations for 96 h. Cell growth was then monitored using CyQUANT ^® cell proliferation assay (lnvitrogen Pte Ltd). Dose response curves were plotted to determine Gl ₅ o values for the compounds using XL-fit.

The cellular or growth inhibition activity results of representative compounds are shown in Table 3 and Table 4 for Colo205 and MDA-MB435 respectively (unit is in micromolar). The data indicated that the compounds of this invention are active in the inhibition of tumor cell growth.

Table 3

Table 4

Histone acetylation assay

A hallmark of histone deacetylase (HDAC) inhibition is the increase in the acetylation level of histones. Histone acetylation, including histones H3, H4 and H2A can be detected by immuno-blotting (western-blot). Colo205 cells, approximately 5 x10 ⁵ cells, will be seeded in the previously described medium, cultivated for 24 h and subsequently treated with HDAC inhibitory agents and a positive control (known HDAC inhibitor) at 10 μM final concentration. After 24 h, cells will be harvested and lysed according to the instruction from Sigma Mammalian Cell Lysis Kit. The protein concentration will be quantified using BCA method (Sigma Pte Ltd). The protein lysate will be separated using 4-12% bis-tris SDS-PAGE gel (Invitrogen Pte Ltd) and will be transferred onto PVDF membrane (BioRad Pte Ltd). The membrane will be probed using primary antibody specific for acetylated histone H3 (Upstate Pte Ltd). The detection antibody, goat anti rabbit antibody conjugated with HRP will be used according to the manufacturing instruction (Pierce Pte Ltd). After removing the detection antibody from the membrane, an enhanced chemiluminescent substrate for detection of HRP (Pierce Pte Ltd) will be added onto the membrane. After removing the substrate, the membrane will be exposed to an X-ray film (Kodak) for 1 sec- 20 mins. The X-ray film will be developed using the X-ray film processor. The density of each band observed on the developed film can be qualitatively analyzed using UVP Bioimaging software (UVP, Inc, Upland, CA). The values will be then normalized against the density of actin in the corresponding samples to obtain the expression of the protein and compared to the values obtained from the positive control. It is anticipated that the compounds of this invention will demonstrate an increased acetylation level of histones such as histone H3.

Tumor xenograft model - In vivo antineoplastic (or anti-tumor) effect :

The efficacy of the compounds of the invention can then be determined using tumor xenograft studies. The tumor xenograft model is one of the most commonly used in vivo cancer models.

In these studies Female athymic nude mice (Harlan), 12-14 weeks of age will be implanted subcutaneously in the flank with 5 x 10 ⁶ cells of HCT116 human colon cancer cells, or with 5 x 10 ⁶ cells of A2780 human ovarian cancer cells, or with 5 x 106 cells of PC3 prostate cancer cells. When the tumor reaches the size 100 mm ³ , the xenograft nude mice will be paired-match into various treatment groups. The selected HDAC inhibitors will be dissolved in appropriate vehicles and administered to xenograft nude mice intraperitonelly or orally daily for 21 days. The dosing volume will be 0.01 ml/ g body weight. Paclitaxol, which can be used as positive control, will be prepared for intravenous administration in an appropriate vehicle. The dosing volume for Paclitaxol will be 0.01 ml/g body weight. Tumor volume will be calculated every second day or twice-a-week of post injection using the formula: Volume (mm ³ ) = (w ² x l)/2, where w = width and I = length in mm of an HCT116, or A2780, or PC3 tumor. Compounds of this invention that are tested will show significant reduction in tumor volume relative to controls treated with vehicle only. Acetylated histone relative to vehicle treated control group when measured shall be accumulated. The result will therefore indicate that compounds of this invention are efficacious in treating a proliferative disease such as cancer.

The details of specific embodiments described in this invention are not to be construed as limitations. Various equivalents and modifications may be made without departing from the essence and scope of this invention, and it is understood that such equivalent embodiments are part of this invention.