SMALL MOLECULE INHIBITORS OF HISTONE DEACTEYLASES

FIELD

Embodiments of the disclosure relate to the fields of cell biology, stem cells, cell differentiation, cancer, and therapeutics. More specifically, embodiments of the disclosure relate small molecules and methods, compositions and kits comprising the small molecules for reprogramming cells, cell-based therapeutics, and treatment of diseases.

BACKGROUND

[0001] Regenerative medicine holds great promise as a therapy for many human ailments, but also entails some difficult technical challenges, which include low cloning efficiency, a short supply of potentially pluripotent tissues, and a generalized lack of knowledge as to how to control cell differentiation and what types of embryonic stem cells can be used for selected therapies. While ES cells have tremendous plasticity,

undifferentiated ES cells can form teratomas (benign tumors) containing a mixture of tissue types. In addition, transplantation of ES cells from one source to another likely would require the administration of drugs to prevent rejection of the new cells.

[0002] Attempts have been made to identify new avenues for generating stem cells from tissues that are not of fetal origin. One approach involves the manipulation of autologous adult stem cells. The advantage of using autologous adult stem cells for regenerative medicine lies in the fact that they are derived from and returned to the same patient, and are therefore not subject to immune-mediated rejection. A drawback is that these cells lack the plasticity and pluripotency of ES cells and thus their potential is uncertain. Another approach is aimed at reprogramming somatic cells from adult tissues to create pluripotent ES-like cells. However, this approach has been difficult as each cell type within a multi-cellular organism has a unique epigenetic signature that is thought to become fixed once cells differentiate or exit from the cell cycle.

[0003] Cellular DNA generally exists in the form of chromatin, a complex

comprising of nucleic acid and protein. Indeed, most cellular RNA molecules also exist in the form of nucleoprotein complexes. The nucleoprotein structure of chromatin has been the subject of extensive research, as is known to those of skill in the art. In general,

chromosomal DNA is packaged into nucleosomes. A nucleosome comprises a core and a linker. The nucleosome core comprises an octamer of core histones (two each of H2A, H2B, H3 and H4) around which is wrapped approximately 150 base pairs of chromosomal DNA. In addition, a linker DNA segment of approximately 50 base pairs is associated with linker histone HI . Nucleosomes are organized into a higher-order chromatin fiber and chromatin fibers are organized into chromosomes. See, for example, Wolffe "Chromatin: Structure and Function" 3.sup.rd Ed., Academic Press, San Diego, 1998.

[0004] Chromatin structure is not static, but is subject to modification by processes collectively known as chromatin remodeling. Chromatin remodeling can serve, for example, to remove nucleosomes from a region of DNA; to move nucleosomes from one region of DNA to another; to change the spacing between nucleosomes; or to add nucleosomes to a region of DNA in the chromosome. Chromatin remodeling can also result in changes in higher order structure, thereby influencing the balance between transcriptionally active chromatin (open chromatin or euchromatin) and transcriptionally inactive chromatin (closed chromatin or heterochromatin).

[0005] Chromosomal proteins are subject to numerous types of chemical

modification. One mechanism for the posttranslational modification of these core histones is the reversible acetylation of the epsilon-amino groups of conserved highly basic N-terminal lysine residues. The steady state of histone acetylation is established by the dynamic equilibrium between competing histone acetyltransferase(s) and histone deacetylase(s) herein referred to as HDAC.

[0006] Histone acetylation and deacetylation has long been linked to transcriptional control. The reversible acetylation of histones can result in chromatin remodeling and as such can act as a control mechanism for gene transcription. In general, hyperacetylation of histones facilitates gene expression, whereas histone deacetylation is correlated with transcriptional repression. Histone acetyltransferases were shown to act as transcriptional coactivators, whereas deacetylases were found to belong to transcriptional repression pathways. [0007] The dynamic equilibrium between histone acetylation and deacetylation is essential for normal cell growth. Inhibition of histone deacetylation results in cell cycle arrest, cellular differentiation, apoptosis and reversal of the transformed phenotype.

[0008] The development of pluripotent or totipotent cells into a differentiated, specialized phenotype is determined by the particular set of genes expressed during development. Gene expression is mediated directly by sequence-specific binding of gene regulatory proteins that can effect either positive or negative regulation. However, the ability of any of these regulatory proteins to directly mediate gene expression depends, at least in part, on the accessibility of their binding site within the cellular DNA. As discussed above, accessibility of sequences in cellular DNA often depends on the structure of cellular chromatin within which cellular DNA is packaged.

[0009] Therefore, it would be useful to identify methods, compositions and kits that can induce the expression of genes required for pluripotency, including methods,

compositions, and kits that can inhibit the activity of HDACs involved in repressing transcription.

BRIEF SUMMARY

[0010] The disclosure relates to small molecules that inhibit a histone deacetylase

(HDAC). In another embodiment, the disclosure relates to compositions, articles of manufacture and kits comprising these small molecules, as well as methods for inhibiting a HDAC and treatment methods using small molecules according to the disclosure. In, still another embodiment, the disclosure relates to methods of making compositions comprising these small molecules, as well as intermediates useful in such methods.

[0011] In one embodiment, the disclosure relates to methods, compositions and kits comprising small molecules disclosed herein for reprogramming a cell. Embodiments of the disclosure relate to methods comprising using a small molecule disclosed herein to induce the expression of a pluripotent or multipotent gene. In yet another embodiment, the disclosure further relates to producing a reprogrammed cell using small molecules disclosed herein.

[0012] In one embodiment, the disclosure relates to a compound of the formula selected from the group consisting of:

 (4) (5) (6)

[0014] In still yet another embodiment, the disclosure relates to a method comprising inhibiting the activity of a protein that is involved in transcriptional repression. In yet another embodiment, the disclosure relates to a method for reprogramming a cell comprising exposing a cell to a small molecule disclosed herein and altering the activity, expression or activity and expression of a regulatory protein. The method further comprises using a small molecule disclosed herein to induce the expression of a pluripotent or multipotent gene, and reprogramming the cell.

[0015] In one embodiment, the compounds, compositions, kits and articles of manufacture comprising the small molecules disclosed herein are used to inhibit the activity of a HDAC. In another embodiment, the compounds, compositions, kits and articles of manufacture are used to treat a disease state for which a HDAC possess activity that contributes to the pathology and/or symptomology of the disease state. [0016] In one embodiment, a pharmaceutical composition is provided that comprises a small molecule according to the disclosure as an active ingredient. Pharmaceutical compositions according to the disclosure may optionally comprise from 0.001% to 100% of one or more small molecules of this disclosure. These pharmaceutical compositions may be administered or co-administered by a wide variety of routes, including but not limited to orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadiposally, intraarticularly, or intrathecally. The compositions may also be administered or co-administered in slow release dosage forms.

[0017] In yet another embodiment, the disclosure is also directed to kits and other articles of manufacture for treating disease states associated with histone deacteylases.

[0018] In another embodiment, an article of manufacture is provided that comprises a composition comprising at least one small molecule of the disclosure in combination with packaging materials. The packaging material may comprise a container for housing the composition. The container may optionally comprise a label indicating the disease state for which the composition is to be administered, storage information, dosing information and/or instructions regarding how to administer the composition. The kit may also optionally comprise additional components, such as syringes for administration of the composition. The kit may comprise the composition in single or multiple dose forms.

[0019] Also provided are methods for preparing compounds, compositions and kits according to the disclosure. Also provided are methods for using compounds, compositions, kits and articles of manufacture according to the disclosure.

[0020] In another embodiment, a small molecule according to the disclosure is administered to a subject wherein HDAC activity within the subject is altered. In another embodiment, a prodrug of a small molecule according to the disclosure is administered to a subject that is converted to the small molecule in vivo where it inhibits a HDAC.

[0021] In another embodiment, a method of inhibiting a HDAC is provided that comprises contacting a HDAC with a small molecule according to the disclosure. [0022] In another embodiment, a method of inhibiting a HDAC is provided that comprises causing a small molecule according to the disclosure to be present in a subject in order to inhibit a HDAC in vivo.

[0023] In another embodiment, a method of inhibiting a HDAC is provided that comprises administering a first compound to a subject that is converted in vivo to a second compound wherein the second compound inhibits HDAC in vivo. It is noted that the small molecule disclosed herein may be the first or second compounds.

[0024] In another embodiment, a therapeutic method is provided that comprises administering a small molecule or compositions comprising a small molecule disclosed herein.

[0025] In another embodiment, a method is provided for treating a condition in a patient that is known to be mediated by a HDAC, or which is known to be treated by a HDAC inhibitor, the method comprising administering to the patient a therapeutically effective amount of a small molecule or composition comprising a small molecule disclosed herein.

[0026] In another embodiment, a method is provided for treating a disease state for which a HDAC possess activity that contributes to the pathology and/or symptomology of the disease state, the method comprising: causing a small molecule disclosed herein to be present in a subject in a therapeutically effective amount for the disease state.

[0027] In another embodiment, a method is provided for treating a disease state for which HDAC contributes to the pathology and/or symptomology of the disease state, the method comprising: administering a first compound to a subject that is converted in vivo to a second compound such that the second compound is present in the subject in a

therapeutically effective amount for the disease state. It is noted that the small molecules disclosed herein may be the first or second compounds.

[0028] In another embodiment, a method is provided for treating a disease state for which a HDAC contributes to the pathology and/or symptomology of the disease state, the method comprising: administering a small molecule disclosed herein to a subject such that the small molecule is present in the subject in a therapeutically effective amount for the disease state. [0029] In another embodiment, a method is provided for using a small molecule according to the disclosure in order to manufacture a medicament for use in the treatment of a disease state that is known to be mediated by a HDAC, or that is known to be treated by a HDAC inhibitors.

[0030] It is noted in regard to all of the above embodiments that the disclosure is intended to encompass all pharmaceutically acceptable ionized forms (e.g., salts) and solvates (e.g., hydrates) of the compounds, regardless of whether such ionized forms and solvates are specified since it is well known in the art to administer pharmaceutical agents in an ionized or solvated form. It is also noted that unless a particular stereochemistry is specified, recitation of a small molecule is intended to encompass all possible stereoisomers (e.g., enantiomers or diastereomers depending on the number of chiral centers), independent of whether the compound is present as an individual isomer or a mixture of isomers. Further, unless otherwise specified, recitation of a compound is intended to encompass all possible resonance forms and tautomers. With regard to the claims, the language "compound comprising the formula," "compound having the formula" and "compound of the formula" is intended to encompass the compound and all pharmaceutically acceptable ionized forms and solvates, all possible stereoisomers, and all possible resonance forms and tautomers unless otherwise specifically specified in the particular claim.

[0031] It is further noted that prodrugs may also be administered, which are altered in vivo and become a small molecule or compound comprising a small molecule disclosed herein. The various methods of using the small molecules of the disclosure are intended, regardless of whether prodrug delivery is specified, to encompass the administration of a prodrug that is converted in vivo to a small molecule according to the disclosure. It is also noted that certain small molecules of the disclosure may be altered in vivo to inhibit a histone deacetylase and thus may themselves be prodrugs for another compound. Such prodrugs of another compound may or may not themselves independently have histone deacetylase inhibitory activity.

[0032] Embodiments of the disclosure also relate to kits for preparing the methods and compositions of the disclosure. The kit can be used for, among other things,

reprogramming a cell and generating ES-like and stem cell-like cells. [0033] In one embodiment, a kit is provided that comprises a composition comprising at least one small molecule of the disclosure n in combination with instructions. The instructions may indicate the disease state for which the composition is to be administered, storage information, dosing information and/or instructions regarding how to administer the composition. The kit may also comprise packaging materials. The packaging material may comprise a container for housing the composition. The kit may also optionally comprise additional components, such as syringes for administration of the composition. The kit may comprise the composition in single or multiple dose forms.

[0034]

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 A- 1L are bar graphs depicting the activity of HDAC inhibitors screened against a triple-negative breast-cancer cell line. The cells were treated at the concentration of 10 μΜ for 48 (open bars) and 72 hours (hatched bars) followed by

Sulforhodamine B based in vitro toxicology assay. Results are representative of three independent experiments performed in quadruplicate. Results are shown as percent of control.

[0036] Figure 1 A shows the results of HDAC inhibitors N0007, NO 16, N027, N029,

N031 , N032, N034, and N036.

[0037] Figure I B shows the results of HDAC inhibitors N052, N054, N055, N057,

N060, N061, N062, and N063.

[0038] Figure 1C shows the results of HDAC inhibitors N084, N085, N087, N090,

N091 , N093, N098, and N104.

[0039] Figure ID shows the results of HDAC inhibitors N066, N067, N068, N069,

N071 , N077, N079, and N081.

[0040] Figure 1 E shows the results of HDAC inhibitors N084, N085, N087, N090,

N091 , N093, N098 and N104.

[0041] Figure IF shows the results of HDAC inhibitors N105, N107, N108, Nl 1 1 ,

N1 14, N116, N117, and N1 18.

[0042] Figure 1G shows the results of HDAC inhibitors N121 , N134, N135, N137,

N140, N141 , N149 and N150. [0043] Figure 1 H shows the results of HDAC inhibitors N152, N155, N156, N159,

N160, N161 , N162, and N163.

[0044] Figure II shows the results of HDAC inhibitors N165, N166, N167, N169,

N170, N173, N175 and N176.

[0045] Figure 1J shows the results of HDAC inhibitors N177, N182, N185, N186,

N187, N191 , N219 and N235.

[0046] Figure IK shows the results of HDAC inhibitors N258, N260, N263, N308,

N314, N322, N323, and N336.

[0047] Figure 1 L shows the results of HDAC inhibitors N337, N341 , N347, N350, and N377.

[0048] Figure 2A-2N are line graphs depicting the activity of HDAC inhibitors screened against triple-negative breast cancer cell line. The cells were treated at the concentration of 100, 50, 20, 10, 5, 2 and 1 μΜ for 48 and 72 hours followed by

Sulforhodamine B based in vitro toxicology assay. IC50 inhibitory concentrations were determined by constructing a dose response curve.

[0049] Figures 2 A and 2B show the inhibitory activity of NO 16 for cells treated for

48 hours (Figure 2A) and 72 hours (Figure 2B).

[0050] Figures 2C and 2D show the inhibitory activity of N121 for cells treated for

48 hours (Figure 2C) and 72 hours (Figure 2D).

[0051] Figures 2E and 2F show the inhibitory activity of N137 for cells treated for 48 hours (Figure 2E) and 72 hours (Figure 2F).

[0052] Figures 2G and 2H show the inhibitory activity of N 140 for cells treated for

48 hours (Figure 2G) and 72 hours (Figure 2H).

[0053] Figures 21 and 2J show the inhibitory activity of N152 for cells treated for 48 hours (Figure 21) and 72 hours (Figure 23).

[0054] Figures 2K and 2L show the inhibitory activity of N155 for cells treated for 48 hours (Figure 2K) and 72 hours (Figure 2L).

[0055] Figures 2M and 2N show the inhibitory activity of N160 for cells treated for

48 hours (Figure 2M) and 72 hours (Figure 2N).

[0056] Figure 3 A-3N are cytograms of selected HDAC inhibitors in triple-negative cancer cell line. Annexin V/Propidium Iodine cytograms following 48 h and 72 hour treatment (X axis, Annexin V; y axis, PI staining). Annexin V-positive, PI -negative cells reflect cells in the early stages of apoptosis (lower right quadrant), whereas Annexin V- positive, Pl-postivie cells reflect dead cells or cells at the late stages of apoptosis (upper rig quadrant).

[0057] Figure 3A is a cytogram depicting the autofluorescence.

[0058] Figure 3B is a cytogram depicting the staining of the control.

[0059] Figures 3C (48 h) and 3D (72 h) are cytograms depicting the staining of tripl negative cancer cell line with NO 16.

[0060] Figures 3E (48 h) and 3F (72 h) are cytograms depicting the staining of triple negative cancer cell line with N121.

[0061] Figures 3G (48 h) and 3H (72 h) are cytograms depicting the staining of triple negative cancer cell line with N137.

[0062] Figures 31 (48 h) and 3J (72 h) are cytograms depicting the staining of triple- negative cancer cell line with N140.

[0063] Figures 3K (48 h) and 3L (72 h) are cytograms depicting the staining of triple negative cancer cell line with N152.

[0064] Figures 3M (48 h) and 3N (72 h) are cytograms depicting the staining of triple-negative cancer cell line with N155.

[0065] Figure 4A-4K are bar graphs depicting the activity of HDAC inhibitors on human BJ fibroblasts. Cells were treated for six days at concentrations of 10 μΜ and 1 μΜ. Graphs represent the fold difference of Oct-4 expression normalized to a control. Data are expressed as meant ± SEM.

[0066] Figure 4 A shows the results of HDAC inhibitors N0007, NO 16, N027, N029,

N031 , N032, and N034.

[0067] Figure 4B shows the results of HDAC inhibitors N036, N38, N040, N041 ,

N043, N044, and N045.

[0068] Figure 4C shows the results of HDAC inhibitors N049, N052, N054, N055,

N057, N060, and N061.

[0069] Figure 4D shows the results of HDAC inhibitors N062, N063, N066, N067,

N068, N069, and N071. [0070] Figure 4E shows the results of HDAC inhibitors N077, N079, N081 , N084,

N085, N087, and N090.

[0071] Figure 4F shows the results of HDAC inhibitors N091, N093, N098, N104,

N105, N107, and N108.

[0072] Figure 4G shows the results of HDAC inhibitors Nl 1 1 , Nl 14, N159, N160,

N161, N162 and N163.

[0073] Figure 4H shows the results of HDAC inhibitors N165, N166, N167, N169,

N170, N173, and N175.

[0074] Figure 41 shows the results of HDAC inhibitors N176, N177, N182, N185,

N186, N187, and N191.

[0075] Figure 4J shows the results of HDAC inhibitors N219, N235, N258, N260,

N263, N308, and N314.

[0076] Figure 4K shows the results of HDAC inhibitors N323, N336, N337, N341 ,

N347, N350, and N377.

[0077] Figures 5A and 5B depict the relative increase of HDAC inhibitor potency between 48 and 72 hours in a triple negative cancer cell line. Figure 5A depicts the relative increase in order of highest potency at 48 h time point. Figure 5B depicts the relative increase in order of value.

DETAILED DESCRIPTION

[0078] Definitions

[0079] It is noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Further, definitions of standard chemistry terms may be found in reference works, including Carey and Sundberg "ADVANCED ORGANIC CHEMISTRY 4.sup.TH ED" Vols. A (2000) and B (2001), Plenum Press, New York. Also, unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art are employed.

[0080] The numerical ranges in this disclosure are approximate, and thus may include values outside of the range unless otherwise indicated. Numerical ranges include all values from and including the lower and the upper values, in increments of one unit, provided that there is a separation of at least two units between any lower value and any higher value. As an example, if a compositional, physical or other property, such as, for example, molecular weight, melt index, temperature etc., is from 100 to 1 ,000, it is intended that all individual values, such as 100, 101 , 102, etc., and sub ranges, such as 100 to 144, 155 to 170, 197 to 200, etc., are expressly enumerated. For ranges containing values which are less than one or containing fractional numbers greater than one (e.g., 1.1 , 1.5, etc.), one unit is considered to be 0.0001 , 0.001 , 0.01 or 0.1 , as appropriate. For ranges containing single digit numbers less than ten {e.g., 1 to 5), one unit is typically considered to be 0.1. These are only examples of what is specifically intended, and all possible combinations of numerical values between the lowest value and the highest value enumerated, are to be considered to be expressly stated in this disclosure. Numerical ranges are provided within this disclosure for, among other things, relative amounts of components in a mixture, and various temperature and other parameter ranges recited in the methods.

[0081] As used herein, "C _m to C _n " in which "m" and "n" are integers refers to the number of carbon atoms in an alkyl, alkenyl or alkynyl group or the number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, the alkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of the cycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of the heteroaryl or ring of the heteroalicyclyl can contain from "m" to "n", inclusive, carbon atoms. Thus, for example, a "Q to C ₄ alkyl" group refers to all alkyl groups having from 1 to 4 carbons, that is, CH. ₃ ~, CH3CH2-, CH3CH ₂ CH ₂ -, (CH ₃ ) ₂ CH~, CH3CH ₂ CH2CH2--, CH ₃ CH ₂ CH(CH ₃ )~ and (CH ₃ ) ₃ C-. If no "m" and "n" are designated with regard to an alkyl, alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group, the broadest range described in these definitions is to be assumed.

[0082] As used herein, "alkyl" refers to a straight or branched hydrocarbon chain fully saturated (no double or triple bonds) hydrocarbon group. The alkyl group may have 1 to 50 carbon atoms (whenever it appears herein, a numerical range such as " 1 to 50" refers to each integer in the given range; e.g., "1 to 50 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 50 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated). The alkyl group may also be a medium size alkyl having 1 to 30 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 5 carbon atoms. The alkyl group of the compounds may be designated as "C1 -C4 alkyl" or similar designations. By way of example only, "C1-C4 alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.

[0083] The alkyl group may be substituted or unsubstituted. When substituted, the substituent group(s) is(are) one or more group(s) individually and independently selected from alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,

heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N- sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl (mono-, di- and tri- substituted haloalkyl), haloalkoxy (mono-, di- and tri-substituted haloalkoxy),

trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di- substituted amino groups, and the protected derivatives thereof.

[0084] As used herein, "alkenyl" refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more double bonds. An alkenyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution unless otherwise indicated.

[0085] As used herein, the term "analogs" refers to compounds which are

substantially the same as another compound but which may have been modified by, for example, adding side groups, oxidation or reduction of the parent structure. Analogs of the small molecules disclosed herein can be readily prepared using commonly known standard reactions. These standard reactions include, but are not limited to, hydrogenation, alkylation, acetylation, and acidification reactions. Chemical modifications can be accomplished by those skilled in the art by protecting all functional groups present in the molecule and deprotecting them after carrying out the desired reactions using standard procedures known in the scientific literature (Greene, T. W. and Wuts, P. G. M. "Protective Groups in Organic Synthesis" John Wiley & Sons, Inc. New York. 3rd Ed. pg. 819, 1999; Honda, T. et al.

Bioorg. Med. Chem. Lett., 1997, 7: 1623-1628; Honda, T. et al. Bioorg. Med. Chem. Lett., 1998, 8:271 1-2714; Konoike, T. et al. J Org. Chem., 1997, 62:960-966; Honda, T. et al. J. Med. Chem., 2000, 43:4233-4246; each of which are hereby incorporated herein by reference in their entirety). Analogs exhibiting the desired biological activity (such as inhibition of an HDAC, potential to differentiate cells, etc.) can be identified or confirmed using cellular assays or other in vitro or in vivo assays.

[0086] As used herein, "alkynyl" refers to an alkyl group that contains in the straight or branched hydrocarbon chain one or more triple bonds. An alkynyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be selected from the same groups disclosed above with regard to alkyl group substitution unless otherwise indicated.

[0087] As used herein, "aryl" refers to a carbocyclic (all carbon) monocyclic or multicyclic aromatic ring system that has a fully delocalized pi-electron system. Examples of aryl groups include, but are not limited to, benzene, naphthalene and azulene. The ring of the aryl group may have 5 to 50 carbon atoms. The aryl group may be substituted or

unsubstituted. When substituted, hydrogen atoms are replaced by substituent group(s) that is(are) one or more group(s) independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,

(heteroalicyclyl)alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, ester, mercapto, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N- thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C- carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl (mono-, di- and tri-substituted haloalkyl), haloalkoxy (mono-, di- and tri- substituted haloalkoxy), trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof, unless the substituent groups are otherwise indicated.

[0088] As used herein, "cell" or "cells," unless specifically limited to the contrary, includes any somatic cell, embryonic stem (ES) cell, adult stem cell, an organ specific stem cell, nuclear transfer (NT) units, and stem-like cells. The cell or cells can be obtained from any organ or tissue. The cell or cells can be human or other animal. For example, a cell can be mouse, guinea pig, rat, cattle, horses, pigs, sheep, goats, etc. A cell also can be from non- human primates.

[0089] As used herein, "culture medium" or "growth medium" means a suitable medium capable of supporting growth of cells.

[0090] As used herein, "cycloalkenyl" refers to a cycloalkyl group that contains one or more double bonds in the ring although, if there is more than one, the double bonds cannot form a fully delocalized pi-electron system in the ring (otherwise the group would be "aryl," as defined herein). When composed of two or more rings, the rings may be connected together in a fused, bridged or spiro-connected fashion. A cycloalkenyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be an alkyl or selected from the substituents disclosed above with respect to alkyl group substitution unless otherwise indicated.

[0091] As used herein, "cycloalkynyl" refers to a cycloalkyl group that contains one or more triple bonds in the ring. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro-connected fashion. A cycloalkynyl group may be unsubstituted or substituted. When substituted, the substituent(s) may be an alkyl or selected from the substituents disclosed above with respect to alkyl group substitution unless otherwise indicated.

[0092] As used herein, "cycloalkyl" refers to a completely saturated (no double bonds) mono- or multi-cyclic hydrocarbon ring system. When composed of two or more rings, the rings may be joined together in a fused, bridged or spiro-connected fashion.

Cycloalkyl groups may range from C ₃ to C] ₀ , in other embodiments it may range from C ₃ to C ₈ . A cycloalkyl group may be unsubstituted or substituted. Typical cycloalkyl groups include, but are in no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. If substituted, the substituent(s) may be an alkyl or selected from those substituents indicated above with respect to substitution of an alkyl group unless otherwise indicated.

[0093] As used herein, "differentiation" means the process by which cells become structurally and functionally specialized during embryonic development.

[0094] As used herein, "epigenetics" refers to the state of DNA with respect to heritable changes in function without a change in the nucleotide sequence. Epigenetic changes can be caused by modification of the DNA, such as by methylation and demethylation, without any change in the nucleotide sequence of the DNA.

[0095] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C4)alkyl" includes trifluoromethyl, 2,2,2-trifluoroethyl, 4- chlorobutyl, 3-bromopropyl, and the like.

[0096] As used herein, "heterocyclyl" and "heteroalicyclyl" refer to a stable 3- to 18 membered ring that consists of carbon atoms and from one to five heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. The "heterocyclyl" or "heteroalicyclyl" may be monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may be joined together in a fused, bridged or spiro-connected fashion; and the nitrogen, carbon and sulfur atoms in the "heterocyclyl" or "heteroalicyclyl" may be optionally oxidized; the nitrogen may be optionally quaternized; and the rings may also contain one or more double bonds provided that they do not form a fully delocalized pi-electron system throughout all the rings.

Heterocyclyl and heteroalicyclyl groups may be unsubstituted or substituted. When substituted, the substituent(s) may be one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, ester, mercapto, alkylthio, arylthio, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C- amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O- carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, haloalkyl (mono-, di- and tri- substituted haloalkyl), haloalkoxy (mono-, di- and tri-substituted haloalkoxy),

trihalomethanesulfonyl, trihalomethanesulfonamido, and amino, including mono- and di- substituted amino groups, and the protected derivatives thereof. Examples of such

"heteroalicyclic" or "heteroalicyclyl" include but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, 1 ,3-dioxin, 1 ,3-dioxane, 1 ,4-dioxane, 1 ,2-dioxolanyl, 1,3-dioxolanyl, 1,4-dioxolanyl, 1,3 -oxathiane, 1 ,4-oxathiin, 1 ,3-oxathiolane, 1 ,3-dithiole, 1 ,3-dithiolane, 1 ,4- oxathiane, tetrahydro-l,4-thiazine, 2H-l ,2-oxazine, maleimide, succinimide, barbituric acid, thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil, trioxane, hexahydro-1 ,3,5- triazine, imidazolinyl, imidazolidine, isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone, thiazoline, thiazolidine, morpholinyl, oxiranyl, piperidinyl N-Oxide, piperidinyl, piperazinyl, pyrrolidinyl, pyrrolidone, pyrrolidione, 4-piperidonyl, pyrazoline, pyrazolidinyl, 2-oxopyrrolidinyl, tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholinyl, thiamorpholinyl sulfoxide, thiamorpholinyl sulfone, and their benzo-fused analogs (e.g., benzimidazolidinone, tetrahydroquinoline, 3,4-methylenedioxyphenyl).

[0097] As used herein, "heteroalkyl" refers to an alkyl group as described herein in which one or more of the carbons atoms in the backbone of alkyl group has been replaced by a heteroatom such as nitrogen, sulfur and/or oxygen.

[0098] As used herein, "heteroalkenyl" refers to an alkenyl group as described herein in which one or more of the carbons atoms in the backbone of alkenyl group has been replaced by a heteroatom, for example, nitrogen, sulfur and/or oxygen.

[0099] As used herein, "heteroalkynyl" refers to an alkynyl group as described herein in which one or more of the carbons atoms in the backbone of alkynyl group has been replaced by a heteroatom such as nitrogen, sulfur and/or oxygen.

[00100] As used herein, "heteroaryl" refers to a monocyclic or multicyclic aromatic ring system (a ring system with fully delocalized pi-electron system) that contain(s) one or more heteroatoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur. The ring of the heteroaryl group may have 5 to 50 atoms. The heteroaryl group may be substituted or unsubstituted. Examples of heteroaryl rings include, but are not limited to, furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole, benzoxazole, 1,2,3-oxadiazole, 1 ,2,4-oxadiazole, thiazole, 1 ,2,3-thiadiazole, 1 ,2,4- thiadiazole, benzothiazole, imidazole, benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine, pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline, and triazine. A heteroaryl group may be substituted or unsubstituted. When substituted, hydrogen atoms are replaced by substituent group(s) that is(are) one or more group(s) independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxy, alkoxy, aryloxy, acyl, ester, mercapto, cyano, halogen, carbonyl, thiocarbonyl, O-carbamyl, N-carbamyl, O- thiocarbamyl, N-thiocarbamyl, C-amido, N-aniido, S-sulfonamido, N-sulfonamido, C- carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl (mono-, di- and tri-substituted haloalkyl), haloalkoxy (mono-, di- and tri-substituted haloalkoxy), trihalomethanesulfonyl,

trihalomethanesulfonamido, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof.

[00101] As used herein, "histone deacetylase inhibitor" and "inhibitor of histone deacetylase" refer to a compound that is capable of interacting with a histone deacetylase and inhibiting its enzymatic activity. "Inhibiting histone deacetylase activity" means reducing the ability of a histone deacetylase to remove an acetyl group from a suitable substrate, such as a histone, or other protein. In some embodiments, such reduction of histone deacetylase activity is at least about 50%, in other embodiments at least about 75%, and still in other embodiments at least about 90%. In still yet other embodiments, histone deacetylase activity is reduced by at least 95% and in other embodiments by at least 99%.

[00102] As used herein, "histone" refers to a class of protein molecules found in chromosomes responsible for compacting DNA enough so that it will fit within a nucleus.

[00103] As used herein, "knock down" means to suppress the expression of a gene in a gene-specific fashion. A cell that has one or more genes "knocked down," is referred to as a knock-down organism or simply a "knock-down."

[00104] As used herein, "pluripotent" means capable of differentiating into cell types of the 3 germ layers or primary tissue types.

[00105] "Pluripotent gene" means a gene that contributes to a cell being pluripotent.

[00106] "Pluripotent cell cultures" are said to be "substantially undifferentiated" when they display morphology that clearly distinguishes them from differentiated cells of embryo or adult origin. Pluripotent cells typically have high nuclear/cytoplasmic ratios, prominent nucleoli, and compact colony formation with poorly discernable cell junctions, and are easily recognized by those skilled in the art. It is recognized that colonies of undifferentiated cells can be surrounded by neighboring cells that are differentiated. Nevertheless, the

substantially undifferentiated colony will persist when cultured under appropriate conditions, and undifferentiated cells constitute a prominent proportion of cells growing upon splitting of the cultured cells. Useful cell populations described in this disclosure contain any proportion of substantially undifferentiated pluripotent cells having these criteria. Substantially undifferentiated cell cultures may contain at least about 20%, 40%, 60%, or even 80% undifferentiated pluripotent cells (in percentage of total cells in the population).

[00107] "Regulatory protein" means any protein that regulates a biological process, including regulation in a positive and negative direction. The regulatory protein can have direct or indirect effects on the biological process, and can either exert affects directly or through participation in a complex.

[00108] "Reprogramming" means removing epigenetic marks in the nucleus, followed by establishment of a different set of epigenetic marks. During development of multicellular organisms, different cells and tissues acquire different programs of gene expression. These distinct gene expression patterns appear to be substantially regulated by epigenetic modifications such as DNA methylation, histone modifications and other chromatin binding proteins. Thus each cell type within a multicellular organism has a unique epigenetic signature that is conventionally thought to become "fixed" and immutable once the cells differentiate or exit the cell cycle. However, some cells undergo major epigenetic

"reprogramming" during normal development or certain disease situations.

[00109] As used herein, "small molecule" refers to a non-peptidic, non-oligomeric organic compound either synthesized in the laboratory or found in nature. Small molecules, as used herein, can refer to compounds that are "natural product-like," however, the term "small molecule" is not limited to "natural product-like" compounds. Rather, a small molecule is typically characterized in that it possesses one or more of the following characteristics including having several carbon-carbon bonds, having multiple stereocenters, having multiple functional groups, having at least two different types of functional groups, and having a molecular weight of less than 1500, although this characterization is not intended to be limiting for the purposes of the disclosure.

[00110] As used herein, "substituent convertible to hydrogen in vivo" means any group that is convertible to a hydrogen atom by enzymological or chemical means including, but not limited to, hydrolysis and hydrogenolysis. Examples include hydrolyzable groups, such as acyl groups, groups having an oxycarbonyl group, amino acid residues, peptide residues, o-nitrophenylsulfenyl, trimethylsilyl, tetrahydro-pyranyl, diphenylphosphinyl, and the like. Examples of acyl groups include formyl, acetyl, trifluoroacetyl, and the like. Examples of groups having an oxycarbonyl group include ethoxycarbonyl, t-butoxycarbonyl [(CH ₃ ) ₃ C-OCO~], benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, vinyloxycarbonyl-β- (p-toluenesulfonyl)ethoxycarbonyl, and the like. Examples of suitable amino acid residues include amino acid residues per se and amino acid residues that are protected with a protecting group. Suitable amino acid residues include, but are not limited to, residues of Gly (glycine), Ala (alanine); Arg (arginine), Asn (asparagine), Asp (aspartic acid), Cys (cysteine), Glu (glutamic acid), His (histidine), He (isoleucine), Leu (leucine), Lys (lysine), Met (methionine), Phe (phenylalanine), Pro (proline), Ser (serine), Thr (threonine), Trp (tryptophan), Tyr (tyrosine), Val (valine), Nva (norvaline), Hse (homoserine), 4-Hyp (4- hydroxyproline), 5-Hyl (5-hydroxylysine), Orn (ornithine) and β-Ala. Examples of suitable protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethyloxycarbonyl groups (such as

benzyloxycarbonyl and p-nitrobenzyloxycarbonyl), t-butoxycarbonyl groups [(CH ₃ ) ₃ C-- OCO-], and the like.

[00111] Suitable peptide residues include peptide residues comprising two to five, and optionally two to three, of the aforesaid amino acid residues. Examples of such peptide residues include, but are not limited to, residues of such peptides as Ala-Ala

[CH.sub.3CH(NH.sub.2)CO-NHCH(CH.sub.3)CO-], Gly-Phe, Nva-Nva, Ala-Phe, Gly- Gly, Gly-Gly-Gly, Ala-Met, Met-Met, Leu-Met and Ala-Leu. The residues of these amino acids or peptides can be present in stereochemical configurations of the D-form, the L-form or mixtures thereof. In addition, the amino acid or peptide residue may have an asymmetric carbon atom. Examples of suitable amino acid residues having an asymmetric carbon atom include residues of Ala, Leu, Phe, Trp, Nva, Val, Met, Ser, Lys, Thr and Tyr. Peptide residues having an asymmetric carbon atom include peptide residues having one or more constituent amino acid residues having an asymmetric carbon atom.

[00112] Examples of suitable amino acid protecting groups include those typically employed in peptide synthesis, including acyl groups (such as formyl and acetyl), arylmethyloxycarbonyl groups (such as benzyloxycarbonyl and p-nitrobenzyloxycarbonyl), t- butoxycarbonyl groups [(CH ₃ ) ₃ C~OCO— ], and the like. Other examples of substituents "convertible to hydrogen in vivo" include reductively eliminable hydrogenolyzable groups. Examples of suitable reductively eliminable hydrogenolyzable groups include, but are not limited to, arylsulfonyl groups (such as o-toluenesulfonyl); methyl groups substituted with phenyl or benzyloxy (such as benzyl, trityl and benzyloxymethyl); arylmethoxycarbonyl groups (such as benzyloxycarbonyl and o-methoxy-benzyloxycarbonyl); and

halogenoethoxycarbonyl groups.

[00113] "Substituted or unsubstituted" means that a given moiety may consist of only hydrogen substituents through available valencies (unsubstituted) or may further comprise one or more non-hydrogen substituents through available valencies (substituted) that are not otherwise specified by the name of the given moiety. For example, isopropyl is an example of an ethylene moiety that is substituted by --CH ₃ . In general, a non-hydrogen substituent may be any substituent that may be bound to an atom of the given moiety that is specified to be substituted. Examples of substituents include, but are not limited to, aldehyde, alicyclic, aliphatic, (Ci-io)alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aromatic, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo, heterobicycloalkyl, heterocycloalkylene, heteroaryl,

heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted.

[00114] In one particular embodiment, examples of substituents include, but are not limited to, hydrogen, halo, nitro, cyano, thio, oxy, hydroxy, carbonyloxy, (Ci.io)alkoxy, (C _4- i ₂ )aryloxy, hetero(Ci-io)aryloxy, carbonyl, oxycarbonyl, aminocarbonyl, amino, (Ci.

io)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (Ci-io)alkyl, halo(Ci-io)alkyl, hydroxy(Ci.io)alkyl, carbonyl(ci-io)alkyl, thiocarbonyl(Ci_io)alkyl, sulfonyl(C]_io)alkyl, sulfinyl(Ci-io)alkyl, (Ci-io)azaalkyl, imino(Ci_io)alkyl, (C3-i2)cycloalkyl(C _{1 -5)} alkyl, hetero(C ₃ . i2)cycloalkyl(C _{M 0} )alkyl, aryl( _{cl -1} o)alkyl, hetero(C _1-1 o)aryl(C _{1 -5} )alkyl, (C ₉ -i ₂ )bicycloaryl(Ci. ₅ )alkyl, hetero(C8.i ₂ )bicycloaryl(Ci.5)alkyl, (C _3- i ₂ )cycloalkyl, hetero(C _3- i ₂ )cycloalkyl, (C _9- i ₂ )bicycloalkyl, hetero(C _3- i ₂ )bicycloalkyl, (C _4- i ₂ )aryl, hetero(Ci.io)aryl, (C _9- i ₂ )bicycloaryl and hetero(C _4- i ₂ )bicycloaryl.

[00115] In addition, the substituent is itself optionally substituted by a further substituent. In one embodiment, examples of the further substituent include, but are not limited to, hydrogen, halo, nitro, cyano, thio, oxy, hydroxy, carbonyloxy, (Ci _-10 )alkoxy, (C _4- i ₂ )aryloxy, hetero(Ci.io)aryloxy, carbonyl, oxycarbonyl, aminocarbonyl, amino, (ci - io)alkylamino, sulfonamido, imino, sulfonyl, sulfinyl, (Ci-io)alkyl, halo(C]-io)alkyl, hydroxy(Ci-io)alkyl, carbonyl(Ci_io)alkyl, thiocarbonyl(Ci-io)alkyl, sulfonyl(Ci-io)alkyl, sulfinyl(Ci-io)alkyl, (Ci-io)azaalkyl, imino(Ci.io)alkyl, (C3. i 2)cycloalkyl(Ci _-5 )alkyl, hetero(C ₃ . i ₂ )cycloalkyl(C _1-10 )alkyl, aryl(C i _i ₀ )alkyl, hetero(C , _{A 0} )aryl(C j _-5 )alkyl, ( _C 9-i ₂ )bicycloaryl(C i . ₅ )alkyl, hetero(C _8- i ₂ )bicycloaryl(Ci_5)alkyl, (C _3- i ₂ )cycloalkyl, hetero(C _3- i ₂ )cycloalkyl, (C _9- i ₂ )bicycloalkyl, hetero(C ₃ -] ₂ )bicycloalkyl, (C _4- i 2)aryl, hetero(C _{] -} io)aryl, (C9-i ₂ )bicycloaryl and hetero(C _4- ] ₂ )bicycloary 1.

[00116] "Treatment" or "treating" means any administration of a compound of the disclosure and includes (1) preventing the disease from occurring in an animal which may be predisposed to the disease but does not yet experience or display the pathology or

symptomatology of the disease; (2) inhibiting the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., arresting further development of the pathology and/or symptomatology); or (3) ameliorating the disease in an animal that is experiencing or displaying the pathology or symptomatology of the diseased (i.e., reversing the pathology and/or symptomatology).

[00117] As used herein, the term "therapeutically effective amount" is intended to encompass any amount that will achieve the desired therapeutic or biological effect. The therapeutic effect is dependent upon the disease or disorder being treated or the biological effect desired. As such, the therapeutic effect can be a decrease in the severity of symptoms associated with the disease or disorder and/or inhibition (partial or complete) of progression of the disease. The amount needed to elicit the therapeutic response can be determined based on the age, health, size and sex of the subject. Optimal amounts can also be determined based on monitoring of the subject's response to treatment.

[00118] "Totipotent" means capable of developing into a complete embryo or organism.

[00119] It is noted in regard to all of the definitions provided herein that the definitions should be interpreted as being open ended in the sense that further substituents beyond those specified may be included. Hence, a C _\ alkyl indicates that there is one carbon atom but does not indicate what are the substituents on the carbon atom. Hence, a (Ci)alkyl comprises methyl {i.e., ~CH ₃ ) as well as— CRR'R" where R, R', and R" may each independently be hydrogen or a further substituent where the atom attached to the carbon is a heteroatom or cyano. Hence, CF ₃ , CH ₂ OH and CH ₂ CN, for example, are all (Ci)alkyls. Similarly, terms such as alkylamino and the like comprise dialkylamino and the like.

[00120] The disclosure relates to small molecules that may be used to inhibit HDACs. The disclosure also relates to pharmaceutical compositions, kits and articles of manufacture comprising such small molecules. In addition, the disclosure relates to methods and intermediates useful for making the compositions. Further, the disclosure relates to methods of using said small molecules. It is noted that the small molecules of the disclosure may also possess activity for other members of the same protein family and thus may be used to address disease states associated with these other family members.

[00121] Embodiments of the disclosure relate to methods comprising using a small molecule disclosed herein to induce expression of at least one gene that contributes to a cell being pluripotent or multipotent. In some embodiments, the methods induce expression of at least one gene that contributes to a cell being pluripotent or multipotent and producing reprogrammed cells that are capable of directed differentiation into at least one lineage.

[00122] Embodiments of the disclosure also relate to a method comprising using a small molecule disclosed herein to modify chromatin structure, and reprogramming a cell to be pluripotent or multipotent.

[00123] In yet another embodiment, the disclosure relates to a method for

reprogramming a cell comprising: exposing said cell to a small molecule disclosed herein, inducing the expression of a pluripotent or multipotent gene, and selecting a cell, wherein differentiation potential has been restored to said cell. The pluripotent or multipotent gene may be induced by any fold increase in expression including but not limited to 0.25-0.5, 0.5- 1 , 1.0-2.5, 2.5-5, 5-10, 10-15, 15-20, 20-40, 40-50, 50-100, 100-200, 200-500, and greater than 500. In another embodiment, the method comprises plating differentiated cells, overexpressing a transcription factor in said plated cells; exposing said plated cells to a small molecule disclosed herein, culturing said cells, and identifying a cell that has been reprogrammed.

[00124] The activity or expression of a regulatory protein can be increased or decreased by exposure to a small molecule disclosed herein by any amount including but not limited to 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80- 90%, 90-95%, and 95-99%, 99-200%, 200-300%, 300-400%, 400-500% and greater than 500%..

[00125] In yet another embodiment, the method further comprises selecting a cell using an antibody directed to a protein or a fragment of a protein coded for by a pluripotent or multipotent gene or an antibody directed to a pluripotent or multipotent marker. Any type of antibody can be used including but not limited to a monoclonal, a polyclonal, a fragment of an antibody, a peptide mimetic, an antibody to the active region, and an antibody to the conserved region of a protein In still another embodiment, the method comprises selecting a cell and expanding or culturing said cell to a pluripotent cell culture.

[00126] In still another embodiment, the method further comprises selecting a cell using a reporter driven by a pluripotent or mulitpotent gene or a pluripotent or mulitpotent surface marker. Any type of reporter can be used including but not limited to a fluorescent protein, green fluorescent protein, cyan fluorescent protein (CFP), a yellow fluorescent protein (YFP), bacterial luciferase, jellyfish aequorin, enhanced green fluorescent protein, chloramphenicol acetyltransferase (CAT), dsRED, β-galactosidase, and alkaline phosphatase.

[00127] In still another embodiment, the method further comprises selecting a cell using resistance as a selectable marker including but not limited to resistance to an antibiotic, a fungicide, puromycin, hygromycin, dihydrofolate reductase, thymidine kinase, neomycin resistance (neo), G418 resistance, mycophenolic acid resistance (gpt), zeocin resistance protein and streptomycin.

[00128] In still another embodiment, the method further comprises comparing the chromatin structure of a pluripotent or multipotent gene of a cell that exists before exposing the cell to a small molecule disclosed herein to the chromatin structure of a pluripotent or multipotent gene obtained after exposure to the small molecule. Any aspect of chromatin structure can be compared including but not limited to euchromatin, heterochromatin, histone acetylation, histone methylation, the presence and absence of histone or histone components, the location of histones, the arrangement of histones, and the presence or absence of regulatory proteins associated with chromatin. The chromatin structure of any region of a gene may be compared including but not limited to an enhancer element, an activator element, a promoter, the TATA box, regions upstream of the start site of transcription, regions downstream of the start site of transcription, exons and introns. I. Small Molecules

[00129] The present disclosure encompasses small molecules shown in Tables I or any embodiment thereof and analogs, stereoisomers, tautomers, pharmaceutically acceptable salts, solvates, clathrates, hydrates, polymorph, prodrugs and pharmaceutical compositions thereof. In one aspect, the disclosure provides compounds as set forth below in Table I:

[00130] Table I. Small molecules.

[00131] The small molecules recited above in Table I may be referred to by the assigned number. For purposes of clarity, the molecule with ID NSPTOOl 18 may be referred to as molecule 1 18, or as molecule Nl 18. All designations refer to the same molecule.

[00132] Small molecules of the disclosure include but are not limited to: 2-(9H- Fluoren-9-yl)-N-hydroxy-acetamide, Biphenyl-4-carboxylic acid hydroxyamide,

Naphthalene-2-carboxylic acid hydroxyamide, N-Hydroxy-2,2-diphenyl-acetamide, 3- Hydroxy-naphthalene-2-carboxylic acid hydroxyamide, 4-Fluoro-naphthalene-l -carboxylic acid hydroxyamide, (E)-3-Furan-2-yl-N-hydroxy-acrylamide, 5-Methoxy-2-phenyl- benzofuran-3-carboxylic acid hydroxyamide, 3-Benzoylamino-thiophene-2-carboxylic acid hydroxyamide, Benzo[b]thiophene-2-carboxylic acid hydroxyamide, 5-Methyl-3-phenyl- isoxazole-4-carboxylic acid hydroxyamide, (E)-3-(3-Fluoro-phenyl)-N-hydroxy-acrylamide, 2,5-Dimethyl-furan-3-carboxylic acid hydroxyamide, 3-Methyl-benzofuran-2-carboxylic acid hydroxyamide, 5-Phenyl-furan-2-carboxylic acid hydroxyamide, Cyclohex-1 -enecarboxylic acid hydroxyamide, (E)-N-Hydroxy-3-(2-hydroxy-phenyl)-acrylamide, (E)-N-Hydroxy-3- (3-hydroxy-phenyl)-acrylamide, (E)-3-(2-Fluoro-phenyl)-N-hydroxy-acrylamide, 2- Methoxy-naphthalene-l -carboxylic acid hydroxyamide, (E)-3-(3,5-Dimethoxy-phenyl)-N- hydroxy-acrylamide, (E)-N-Hydroxy-3-(4-hydroxy-3,5-dimethoxy-phenyl)-acrylamide, (E)-3-Benzo[l ,3]dioxol-5-yl-N-hydroxy-acrylamide, 2-Hydroxy-biphenyl-3-carboxylic acid hydroxyamide, (E)-3-(3,4-Dimethoxy-phenyl)-N-hydroxy-acrylamide, (E)-N- Hydroxy-3-(4-hydroxy-3-methoxy-phenyl)-acrylamide, (E)-N-Hydroxy-3-(3,4,5- trimethoxy-phenyl)-acrylamide, 2-Hydroxy-naphthalene-l -carboxylic acid hydroxyamide, (E)-3-(4-Fluoro-phenyl)-N-hydroxy-acrylamide, (E)-3-(2-Chloro-4-fluoro-phenyl)-N- hydroxy-acrylamide, (E)-3-(3-Bromo-phenyl)-N-hydroxy-acrylamide, (E)-3-(5-Bromo-2- fluoro-phenyl)-N-hydroxy-acrylamide, 2',4'-Difluoro-4-hydroxy-biphenyl-3-carboxylic acid hydroxyamide, 2-Hydroxy-5-nitro-benzo[de]isoquinoline-l ,3-dione, 2,5-Dihydroxy- benzo[de]isoquinoline-l ,3-dione, N-Hydroxy-4-methoxy-benzamide, N-Hydroxy-3- trifluoromethyl-benzamide, N-Hydroxy-4-trifluoromethyl-benzamide, N-Hydroxy-4- isopropyl-benzamide, N-Hydroxy-4-methyl-benzamide, 2-Amino-N-hydroxy-terephthalamic acid methyl ester, N-Hydroxy-4-propyl-benzamide, 4-Chloro-N-hydroxy-3-methoxy- benzamide, N-Hydroxy-4-thiophen-2-yl-benzamide, 4-tert-Butyl-N-hydroxy-benzamide, 2- Chloro-N-hydroxy-5-trifluoromethyl-benzamide, 4-Cyclohexyl-N-hydroxy-benzamide, 4- Chloro-N-hydroxy-benzamide, 3-Chloro-N-hydroxy-4-methoxy-benzamide, 3-Fluoro-N- hydroxy-4-methoxy-benzamide, 4-Methyl-3,4-dihydro-2H-benzo[l ,4]oxazine-7-carboxylic acid hydroxyamide, Benzofuran-5-carboxylic acid hydroxyamide, Benzothiazole-6- carboxylic acid hydroxyamide, N-Hydroxy-3-methoxy-benzamide, Benzo[b]thiophene-5- carboxylic acid hydroxyamide, 2,3-Dihydro-benzofuran-7-carboxylic acid hydroxyamide, 2- Acetyl-N-hydroxy-benzamide, N-Hydroxy-4-(4-methyl-piperazin-l -yl)-benzamide, N- Hydroxy-4-pyn l-l -yl-benzamide, 3-Difluoromethoxy-N-hydroxy-benzamide, N- Hydroxy-3,5-bis-trifluoromethyl-benzamide, N-HydiOxy-3-trifluoromethoxy-benzamide, N-Hydroxy-4-(2,4,6-trimethyl-benzoyl)-benzamide, 4-Acetylamino-N-hydroxy- benzamide, N-Hydroxy-4-morpholin-4-yl-benzamide, N-Hydroxy-3-phenoxy-benzamide, 6-Hydroxy-naphthalene-2-carboxylic acid hydroxyamide, 4-Benzoyl-N-hydroxy-benzamide, N-Hydroxy-4-[l ,2,3]thiadiazol-4-yl-benzamide, 1 H-Indole-6-carboxylic acid hydroxyamide, 3-Acetyl-N-hydroxy-benzamide, N-Hydroxy-3-methoxy-4-methyl-benzamide, Benzo[l ,3]dioxole-5-carboxylic acid hydroxyamide, N-Hydroxy-3,5-dimethoxy-benzamide, N- Hydroxy-4-pentyl-benzamide, 4-Butyl-N-hydroxy-benzamide, 4-Ethyl-N-hydroxy- benzamide, 6-Methoxy-naphthalene-2-carboxylic acid hydroxyamide, 4-Ethoxy-N-hydroxy- benzamide, 2,N-Dihydroxy-5-methyl-benzamide, 3,5-Dihydroxy-naphthalene-2-carboxylic acid hydroxyamide, 3,7-Dihydroxy-naphthalene-2-carboxylic acid hydroxyamide, 2,N- Dihydroxy-3-methyl-benzamide, 4'-Hydroxy-biphenyl-4-carboxylic acid hydroxyamide, 4'-Trifluoromethyl-biphenyl-2-carboxylic acid hydroxyamide, N-Hydroxy-4- trifluoromethoxy-benzamide, N-Hydroxy-4-isopropoxy-benzamide, 4-Chloro-N-hydroxy-2- methoxy-benzamide, 5-Acetyl-2,N-dihydroxy-benzamide, N-Hydroxy-4-isobutyl- benzamide, 2-Benzyloxy-N-hydroxy-benzamide, N-Hydroxy-2-phenethyl-benzamide, 3- Benzoyl-N-hydroxy-benzamide, N-Hydroxy-4-propoxy-benzamide, 3,N-Dihydroxy-4- methyl-benzamide, 2,N-Dihydroxy-6-methoxy-benzamide, 2,N-Dihydroxy-4-methoxy- benzamide, 4-Butylamino-N-hydroxy-benzamide, N-Hydroxy-4-pentyloxy-benzamide, N- Hydroxy-4-methoxy-3-methyl-benzamide, 4-Benzyl-N-hydroxy-benzamide, 9-Oxo-9H- fluorene-4-carboxylic acid hydroxyamide, Adamantane-1 ,3-dicarboxylic acid bis-m- tolylamide, 5,8-Dichloro-2,3,4,9-tetrahydro-beta-carbolin-l -one, 2-Chloro-N-(2-methoxy- phenyl)-nicotinamide, 3,4,5,6-Tetrahydro-2H-[l ,2']bipyridinyl-3'-carboxylic acid (3- trifluoromethyl-phenyl)-amide, 2-(4-Methyl-piperazin- 1 -yl)-N-(3 -trifluoromethyl -phenyl- nicotinamide, 2-(4-Acetyl-phenyl)-4-nitro-isoindole-l ,3-dione, (2E,4E)-5-(l H-Indol-3-yl)-3- methyl-penta-2,4-dienoic acid, 1 -(3,4-Dimethoxy-phenyl)-2,3,4,9-tetrahydro-l H-beta- carboline-3-carboxylic acid, 6-Bromo-2,3, 7,9,1 0,1 1 -hexahydro-1 ,4-dioxa-7,9- diaza-benzo[c]fluoren-8-one, 5-Phenyl-2-[4-((E)-styryl)-phenyl]-oxazole, 2-Phenyl-5-[4-((E)- styryl)-phenyl]-[l ,3,4]oxadiazole, 2-{4-[(E)-2-(4-Chloro-phenyl)-vinyl]-phenyl}-5-phenyl- oxazole, N-[2-(2 -Methoxy -phenyl)- 1 ,3-dioxo-2,3-dihydro-l H-isoindol-4-yl]-acetamide, N-[2- (4-Methoxy-phenyl)-l ,3-dioxo-2,3-dihydro-l H-isoindol-4-yl]-acetamide, 2-Dibenzofuran-3- yl-4-nitro-isoindole-l ,3-dione, N-p-Tolyl-nicotinamide, 5-[l -(1 H-Indol-3-yl)-meth-(E)- ylidene]-imidazolidine-2,4-dione, N-(3-Fluoro-phenyl)-nicotinamide, N-(3-Chloro-phenyl)- nicotinamide, N-Naphthalen-2-yl-nicotinamide, N-(l -Phenyl-ethyl)-nicotinamide, N-(2,4- Dimethyl-phenyl)-nicotinamide, N-Pyridin-2-yl-nicotinamide, N-(6-Methoxy-benzothiazol- 2-yl)-nicotinamide, N-Pyridin-4-yl-nicotinamide, N-(4,6-Dimethyl-pyrimidin-2-yl)- nicotinamide, 3-Benzo[l ,3]dioxol-5-yl-7-hydroxy-chromen-2-one, 3-Benzo[l ,3]dioxol-5-yl- 6-chloro-chromen-2-one, 4- [(Pyridine-3-carbonyl)-amino] -benzoic acid methyl ester, 4-(l H- Indol-3-yl)-l -morpholin-4-yl-butan- 1 -one, 7-Methoxy-3-(4-nitro-phenyl)-chromen-2-one, Benzotriazol-1 -yl-phenyl-methanone, Benzotriazol-1 -yl-m-tolyl-methanone, Acetic acid 2- oxo-3-phenyl-2H-chromen-7-yl ester, 3-Methyl-N-[4-(2-oxo-2H-chromen-3-yl)-phenyl]- benzamide,4-[(Pyridine-3-carbonyl)-amino]-butyric acid methyl ester, N-(4-Acetyl-phenyl)- 5-bromo-nicotinamide, 6-Bromo-9-methyl-2,3,4,9-tetrahydro-carbazol-l -one, 4-[(2-Chloro- pyridine-3-carbonyl)-amino] -butyric acid, 5-Acetoxy-2-phenyl-naphtho[l ,2-b]furan-3- carboxylic acid ethyl ester, 5-Pentanoyloxy-2-phenyl-naphtho[l ,2-b]furan-3-carboxylic acid ethyl ester, 5-(2-Bromo-benzoyloxy)-2-phenyl-naphtho[l ,2-b]furan-3 -carboxylic acid ethyl ester, 3-Methyl-N-[3-(2-oxo-2H-chromen-3-yl)-phenyl]-benzamide, Naphthalene- 1 - carboxylic acid[4-(2-oxo-2H-chromen-3-yl)-phenyl]-amide, 2-Methoxy-N-[4-(2-oxo-2H- chromen-3-yl)-phenyl]-benzamide, 3-Methyl-l -phenyl-3H-naphtho[l ,2,3-de]quinoline- 2,7-dione, 3-Ethyl-l H-quinazoline-2,4-dione, 3-[l -(3,4-Dimethoxy~phenyl)-meth-(Z)- ylidene]-2,3-dihydro-l H-pyrrolo[2,l -b]quinazolin-9-one, 2-Fluoro-N-[4-(2-oxo-2H- chromen-3-yl)-phenyl]-benzamide, 2-Fluoro-N-[3-(2-oxo-2H-chromen-3-yl)-phenyl]- benzamide, 3-Bromo-N-[4-(2-oxo-2H-chromen-3-yl)-phenyl]-benzamide, 6-Isobutoxy- 2,3,4,9-tetrahydro-carbazol-l -one, Adamantane-1 ,3-dicarboxylic acid bis-isopropylamide, 2- Chloro-5-iodo-N-[4-(2-oxo-2H-chromen-3-yl)-phenyl]-benzamide , N-[2-(4-Methoxy- phenylcarbamoyl)-phenyl]-nicotinamide, 4-Chloro-2,6-bis-(4-methyl-piperazin- 1 -yl)- pyrimidine, 2-Chloro-N-(2,4-dimethoxy-phenyl)-nicotinamide, 2-Chloro-N-(4-isopropyl- phenyl)-nicotinamide, 3-Methoxy-naphthalene-2-carboxylic acid[4-(2-oxo-2H-chromen-3-yl)- phenyl] -amide, 9-Methyl-9H-purine-8-thiol, 3-Allyl-2-{N'-[2-oxo-l ,2-dihydro-indol-(3E)- ylidene]-hydrazino}-3H-quinazolin-4-one, 3 -Hydroxy-benzoic acid hydrazide, 3,4- Dihydroxy-benzoic acid ethyl ester, 1 ,4-Dihydroxy-naphthalene-2-carboxylic acid, 1 -(2,3,4- Trihydroxy-phenyl)-ethanone, 2-Amino-4-ethanesulfonyl-phenol, 1 H-Indol-4-ol, 2- Benzooxazol-2-yl -phenol, 4-([l ,2,3,4]Thiatriazol-5-ylamino)-phenol, 3-Hydroxy-N-phenyl- benzamide, 1 -[4-(4-Hydroxy-phenyl)-piperazin-l -yl]-ethanone, 2-[l ,8]Naphfhyridin-2-yl- phenol, 2-Benzothiazol-2-yl-phenol, (3,5-Dihydroxy-phenyl)-acetic acid methyl ester, 3-(4- Hydroxy-phenyl)-propionic acid, (E)-3-(4-Hydroxy-3,5-dimethoxy-phenyl)-acrylic acid, (E)-3-(4-Hydroxy-3-methoxy-phenyl)-acrylic acid, 4-Chloro-2-(l H-pyrazol-3-yl)-phenol, 8-Hydroxy-2,3,6,7-tetrahydro-l H,5H-pyrido[3,2,l-ij]quinoline-9-carbaldehyde, 3,4,5- Trihydroxy-benzoic acid hydrazide, N-Hydroxy-isonicotinamidine, 2-Amino-3-hydroxy- anthraquinone, 3-Hydroxy-naphthalene-2-carboxylic acid(2-methoxy-phenyl)-amide, 3- Chloro-N-hydroxy-benzamidine, 2,6-Dichloro-N-hydroxy-benzamidine, N-Hydroxy- cyclohexanecarboxamidine, 4-Imidazol-l -yl-phenol, Quinolin-8-ol, (E)-l -(4-Hydroxy- phenyl)-3 -phenyl -propenone, 6-Hydroxy-3H-benzooxazol-2-one, 5-(3-Hydroxy-phenyl)- furan-2-carboxylic acid methyl ester, 4'-Hydroxy-biphenyl-4-carboxylic acid, 2-Hydroxy-N- phenyl-benzamide, 4-Bromo-2-isoxazol-5-yl-phenol, 4-Hydroxy-3-(3-oxo-l -phenyl-butyl)- chromen-2-one, 1 -Benzyl-2-ethyl-5 -hydroxy- 1 H-indole-3-carboxylic acid methyl ester, 4- Amino-2-(5-chloro-benzooxazol-2-yl)-phenol, 3-Hydroxy-naphthalene-2-carboxylic acid(2- chloro-phenyl)-amide, 2-Hydroxy-l ,2-diphenyl-ethanone oxime, 6-Hydroxy-3,4-dihydro-l H- quinolin-2-one, Octanoic acid hydroxyamide, 4-[l ,3,4]Oxadiazol-2-yl-phenol.

[00133] In another embodiment, the small molecules recited in Table I may be substituted or unsubstituted. Examples of substituents for the small molecules in Table I include, but are not limited to, aldehyde, alicyclic, aliphatic, (Ci_io)alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aromatic, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo,

heterobicycloalkyl, heterocycloalkylene, heteroaryl, heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted

[00134] In another embodiment, small molecules disclosed herein include a tautomeric form of the small molecules recited in Table I. In another embodiment, small molecules disclosed herein include a stereoisomer of the small molecules recited in Table I. In yet another embodiment, small molecules disclosed herein include a pharmaceutically acceptable salt of the small molecules recited in Table I. In still another embodiment, small molecules disclosed herein include compositions of the small molecules recited in Table I. [00135] In yet another embodiment, small molecules disclosed herein include substituted molecules of the molecules recited in Table I. In still another embodiment, small molecules disclosed herein include unsubstituted molecules of the molecules recited in Table I.

[00136] In another embodiment, small molecules disclosed herein include

synthetically prepared compounds recited in Table I. In yet another embodiment, small molecules disclosed herein include compounds recited in Table I that are produced in vivo after administration of a different or starting compound.

[00137] In still another embodiment, small molecules disclosed herein include molecules with the following formula A:

[00138]

HN

OH Formula A

where R1 -R5 can be aldehyde, alicyclic, aliphatic, (Cj _-10 )alkyl, alkylene, alkylidene, amide, amino, aminoalkyl, aromatic, aryl, bicycloalkyl, bicycloaryl, carbamoyl, carbocyclyl, carboxyl, carbonyl group, cycloalkyl, cycloalkylene, ester, halo, heterobicycloalkyl, heterocycloalkylene, heteroaryl, heterobicycloaryl, heterocycloalkyl, oxo, hydroxy, iminoketone, ketone, nitro, oxaalkyl, and oxoalkyl moieties, each of which may optionally also be substituted or unsubstituted.

[00139] In another embodiment, any one of R1-R5 can be a halogen. In still another embodiment, Ri and R ₄ can be a halogen. In yet another embodiment, Ri and R ₃ can be a halogen. In still another embodiment, Ri and R ₅ can be a halogen. In yet another embodiment, Ri and R ₂ can be a halogen. In another embodiment, the following values of Formula I are contemplated: [00140] R ₂ and R ₃ are halogen; R ₂ and R4 are halogen, R ₂ and R ₅ are halogen;\

[00141] R ₃ and R ₄ are halogen; R ₃ and R ₅ are halogen;

[00142] R ₄ and R ₅ are halogen;

[00143] Ri , R ₂ and R ₃ are halogen; R _{l s} R ₂ and R4 are halogen; R _{l s} R ₂ and R ₅ are halogen;

[00144] Ri, R ₃ and R ₄ are halogen; Ri , R ₃ and R ₅ are halogen;

[00145] Ri, R4 and R5 are halogen;

[00146] R ₂ , R ₃ and R4 are halogen; R ₂ , R ₃ and R ₅ are halogen;

[00147] R ₂ , R4, and R ₅ are halogen;

[00148] R ₃ , R and R ₅ are halogen;

[00149] Ri, R ₂ , R ₃ , and R4 are halogen; R _{l s} R ₃ , R4, and R ₅ are halogen;

[00150] R ₂ , R ₃ , R4, and R ₅ are halogen; and

[00151] Ri, R ₂ , R ₃ , R4 and R ₅ are halogen.

[00152] In another embodiment, the small molecules of the disclosure also encompass molecules with substitutions of similar nature. For example, if a small molecule containing a halogen such as bromide is disclosed, it is contemplated that a small molecule containing another halogen such as fluoride may be useful.

[00153] It is noted that the small molecules of the disclosure may be in the form of a pharmaceutically acceptable salt, biohydrolyzable ester, biohydrolyzable amide,

biohydrolyzable carbamate, solvate, hydrate or prodrug thereof. For example, the small molecule optionally comprises a substituent that is convertible in vivo to a different substituent such as hydrogen.

[00154] It is further noted that the small molecule may be present as a mixture of stereoisomers, or the compound may be present as a single stereoisomer.

[00155] A. Stereochemistry

[00156] Many organic compounds exist in optically active forms having the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane -polarized light by the compound, with (-) or meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are non-superimposable mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture. Many of the compounds described herein can have one or more chiral centers and therefore can exist in different enantiomeric forms. If desired, a chiral carbon can be designated with an asterisk (*). When bonds to the chiral carbon are depicted as straight lines in the Formulas of the disclosure , it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within the Formula. As is used in the art, when it is desired to specify the absolute configuration about a chiral carbon, one of the bonds to the chiral carbon can be depicted as a wedge (bonds to atoms above the plane) and the other can be depicted as a series or wedge of short parallel lines is (bonds to atoms below the plane). The Cahn-Inglod-Prelog system can be used to assign the (R) or (S) configuration to a chiral carbon.

[00157] When small molecules disclosed herein contain one chiral center, the compounds exist in two enantiomeric forms and the disclosure includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixtures. The enantiomers can be resolved by methods known to those skilled in the art, such as formation of diastereoisomeric salts, which may be separated, for example, by crystallization (see, CRC Handbook of Optical Resolutions via Diastereomeric Salt Formation by David Kozma (CRC Press, 2001)); formation of diastereoisomeric derivatives or complexes that may be separated, for example, by crystallization, gas-liquid or liquid chromatography;

selective reaction of one enantiomer with an enantiomer-specific reagent, for example enzymatic esterification; or gas-liquid or liquid chromatography in a chiral environment, for example on a chiral support for example silica with a bound chiral ligand or in the presence of a chiral solvent. It will be appreciated that where the desired enantiomer is converted into another chemical entity by one of the separation procedures described above, a further step is required to liberate the desired enantiomeric form. Alternatively, specific enantiomers may be synthesized by asymmetric synthesis using optically active reagents, substrates, catalysts or solvents, or by converting one enantiomer into the other by asymmetric transformation. [00158] Designation of a specific absolute configuration at a chiral carbon of the small molecules disclosed herein is understood to mean that the designated enantiomeric form of the compounds is in enantiomeric excess (ee) or in other words is substantially free from the other enantiomer. For example, the "R" forms of the small molecules are substantially free from the "S" forms of the small molecules and are, thus, in enantiomeric excess of the "S" forms. Conversely, "S" forms of the small molecules are substantially free of "R" forms of the small molecules and are, thus, in enantiomeric excess of the "R" forms. Enantiomeric excess, as used herein, is the presence of a particular enantiomer at greater than 50%. In an embodiment when a specific absolute configuration is designated, the enantiomeric excess of depicted compounds is at least about 90%.

[00159] When a small molecule disclosed herein has two or more chiral carbons, it can have more than two optical isomers and can exist in diastereoisomeric forms. For example, when there are two chiral carbons, the compound can have up to 4 optical isomers and 2 pairs of enantiomers ((S,S)/(R,R) and (R,S)/(S,R)). The pairs of enantiomers (e.g., (S,S)/(R,R)) are mirror image stereoisomers of one another. The stereoisomers that are not mirror-images (e.g., (S,S) and (R,S)) are diastereomers. The diastereoisomeric pairs may be separated by methods known to those skilled in the art, for example chromatography or crystallization and the individual enantiomers within each pair may be separated as described above. The disclosure includes each diastereoisomer of such compounds and mixtures thereof.

B. HDACs and HDAC Inhibitors

[00160] HDACs are classified in at least four classes depending on sequence identity and domain organization: Class I: HDAC1 , HDAC2, HDAC3, HDAC8; Class II: HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC10; Class III: sirtuins in mammals (SIRT1 , SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7); and Class IV: HDAC1 1. Class I HDACs are those that most closely resemble the yeast transcriptional regulator RPD3. Class II HDACs are those that most closely resemble the yeast HDA1 enzyme.

[00161] A small molecule of the methods, compositions and kits of the disclosure may interact with one or more HDAC. The small molecules disclosed herein (see Table I and substitutions of the molecules in Table I) may interact with an HDAC of class I, class II, class III, or class IV. A small molecule may interact with one specific class of HDACs, all classes of HDACS, or with multiple classes of HDACs. The small molecules disclosed herein may also interact with HDACs that do not fall into one of the known classes.

[00162] A small molecule may have an irreversible mechanism of action or a reversible mechanism of action. A small molecule can have any binding affinity including but not limited to millimolar (mM), micromolar (μΜ), nanomolar (nM), picomolar (pM), and fentamolar (fM).

[00163] In one embodiment, one or more substituted molecules of the molecules recited in Table I, one or more tautomeric forms of molecules recited in Table I, one or more stereoisomer of molecules recited in Table I, one or more pharmaceutically acceptable salts of molecules recited in Table I or composition thereof inhibit the activity of an HDAC.

[00164] In one embodiment, one or more substituted molecules of the molecules recited in Table I, one or more tautomeric forms of molecules recited in Table I, one or more stereoisomer of molecules recited in Table I, one or more pharmaceutically acceptable salts of molecules recited in Table I or composition thereof inhibit the activity of HDAC 1 , HDAC2, HDAC3, HDAC8; HDAC4, HDAC5, HDAC6, HDAC7A, HDAC9, HDAC 10; SIRT1 , SIRT2, SIRT3, SIRT4, SIRT5, SIRT6, SIRT7); HDAC1 1. One or more than one HDAC can be inhibited by the small molecules disclosed herein.

[00165] Preferably, such inhibition is specific, i.e., the small molecule reduces the ability of a histone deacetylase to remove an acetyl group from a histone at a concentration that is lower than the concentration of the inhibitor that is required to produce another, unrelated biological effect. Preferably, the concentration of the small molecule required for histone deacetylase inhibitory activity is at least 2-fold lower, more preferably at least 5-fold lower, even more preferably at least 10-fold lower, and most preferably at least 20-fold lower than the concentration required to produce an unrelated biological effect.

[00166] In one embodiment, the disclosure relates to a method of inhibiting an HDAC in a cell comprising treating the cell with one or more small molecules recited in Table I, one or more substituted molecules of the molecules recited in Table I, one or more tautomeric forms of molecules recited in Table I, one or more stereoisomer of molecules recited in Table I, one or more pharmaceutically acceptable salts of molecules recited in Table I or composition thereof. [00167] In one embodiment, the disclosure relates to a method of treating a disease caused by an HDAC in an animal comprising treating the animal with one or more small molecules recited in Table I, one or more substituted molecules of the molecules recited in Table I, one or more tautomeric forms of molecules recited in Table I, one or more stereoisomer of molecules recited in Table I, one or more pharmaceutically acceptable salts of molecules recited in Table I or composition thereof.

[00168] In another embodiment, one or more small molecules recited in Table IA, one or more substituted molecules of the molecules recited in Table IA, one or more tautomeric forms of molecules recited in Table IA, one or more stereoisomer of molecules recited in Table IA, one or more pharmaceutically acceptable salts of molecules recited in Table IA or composition thereof inhibit the activity of HDAC6, HDAC8, or HDAC6 and HDAC8.

[00169] Table IA. Small molecules that inhibit HDACs including but not

[00170] In another embodiment, the small molecule may act by binding to the zinc containing catalytic domain of the HDACs. HDAC inhibitors with this mechanism of action fall into several groupings: (i) hyroxamic acids, such as Trichostatin A; (ii) cyclic tetrapeptides; (iii) benzamides; (iv) electrophilic ketones; and (v) the aliphatic acid group of compounds such as phenylbutyrate and valproic acid.

[00171] In yet another embodiment, the small molecule can be directed toward the sirtuin Class III HDACs, which are NAD+ dependent and include but are not limited to nicotinamide, derivatives of NAD, dihydrocoumarin, naphthopyranone, and 2- hydroxynaphaldehydes.

[00172] In yet another embodiment, the small molecule can alter the degree of acetylation of nonhistone effector molecules and thereby increase the transcription of genes. Small molecules of the methods, compositions, and kits of the disclosure should not be considered to act solely as enzyme inhibitors of HDACs. A large variety of nonhistone transcription factors and transcriptional co-regulators are known to be modified by acetylation, including but not limited to ACTR, cMyb, p300, CBP, E2F1 , EKLF, FEN 1, GATA, HNF-4, HSP90, Ku70, NFKB, PCNA, p53, RB, Runx, SFl Sp3, STAT, TFIIE, TCF, and YY1. The activity of any transcription factor or protein involved in activating transcription, which is acetylated, could be increased with the methods of the disclosure .

[00173] In one embodiment, a single small molecule disclosed herein can be used to inhibit HDAC activity. In yet another embodiment, more than one small molecule disclosed herein can be used to inhibit HDAC activity.

[00174] In yet another embodiment, any number, any combination and any

concentration of small molecules disclosed herein can be used, including but not limited to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 -15, 16-20, and 21-25, 26-35, 36-50, 51-100, 101 -150, 151-200, and greater than 200 small molecules. One or more than one family of inhibitory proteins may be inhibited. One or more than one mechanism of inhibition may be used including but not limited to small molecule inhibitors, HDAC inhibitors, shRNA, RNA interference, and small interfering RNA.

[00175] In still another embodiment, one or more small molecules disclosed herein can be used in combination with other HDAC inhibitors. Table II provides a representative list of known compounds that can function as an HDAC inhibitor. The reference to "Isotype" in Table II is meant to merely provide insight as to whether the compound has a preference for a particular class of HDAC. Listing a specific isotype or class of HDAC should not be construed to mean that the compound only has affinity for that isotype or class. HDAC inhibitors of the disclosure include derivatives and analogues of any HDAC inhibitor herein mentioned.

[00176] Butyric acid, or butyrate, was the first HDAC inhibitor to be identified.

However, in millimolar concentrations, butyrate may not be specific for HDAC, it also may inhibit phosphorylation and methylation of nucleoproteins as well as DNA methylation. The analogue, phenylbutyrate, acts in a similar manner. More specific are trichostatin A (TSA) and trapoxin (TPX). TPX and TSA have emerged as inhibitors of histone deacetylases. TSA reversibly inhibits, whereas TPX irreversibly binds to and inactivates HDAC enzymes.

Unlike butyrate, nonspecific inhibition of other enzyme systems has not yet been reported for TSA or TPX.

[00177] Valproic acid also inhibits histone deacetylase activity. VPA is a known drug with multiple biological activities that depend on different molecular mechanisms of action. VPA is an antiepileptic drug. VPA is teratogenic. When used as antiepileptic drug during pregnancy, VPA may induce birth defects (neural tube closure defects and other

malformations) in a few percent of born children. In mice, VPA is teratogenic in the majority of mouse embryos when properly dosed. VPA activates a nuclear hormone receptor (PPAR-delta.).

[00178] Table II. A representative list of compounds that can function as an

HDAC inhibitor.

Belinostat class I/II nM Hydroxamate

[00179] A variety of HDAC inhibitors also are available from Sigma Aldrich (St. Louis, MO) including but not limited to APHA Compound; Apicidin; Depudecin; Scriptaid; Sirtinol; and Trichostatin A. Further, additional HDAC inhibitors are available from Vinci- Biochem (Italy) including but not limited to 5-Aza-2'-deoxycytidine; CAY10398;

CAY10433; 6-Chloro-2,3,4,9-tetrahydro-lH-carbazole-l-carboxamide; HC Toxin; ITSA1 ; M344; MC 1293; MS-275; Oxamflatin; PXD101 ; SAHA; Scriptaid; Sirtinol; Splitomicin. Dexamethasone may also be used in combination with any HDAC inhibitor. For example, a composition comprising dexamethasone and to 5-Aza-2'-deoxycytidine can be used.

[00180] C. HDAC Assays

[00181] In one embodiment, assays to determine the activity of a small molecule can be performed with a CRO microfluidics platform using fluorescently labeled peptide substrates. Small molecules can be prediluted in DMSO, and various concentrations can be tested including but not limited to 30 μΜ and 10 μΜ.

[00182] In another embodiment, the activity of a small molecule can be determined in vitro using, for example, enzymatic assays, which show inhibition of at least one histone deacetylase. Further, determination of the accumulation of acetylated histones in cells treated with a particular small molecule or composition can be determinative of the HDAC inhibitory activity of a small molecule.

[00183] Assays for the accumulation of acetylated histones have been reported. See, for example, Marks, P. A. et al, J. Natl. Cancer Inst., 92: 1210-1215, 2000, and Butler, L. M. et al., Cancer Res. 60:5165-5170 (2000).

[00184] For example, an enzymatic assay to determine the inhibitory activity of a small molecule can be conducted as follows. Briefly, the effect of a small molecule on affinity purified human epitope-tagged (Flag) HDAC 1 can be assayed by incubating the enzyme preparation in the absence of substrate on ice for about 20 minutes with the indicated amount of inhibitor compound. Substrate ([ HJacetyl-labeled murine erythroleukemia cell- derived histone) can be added and the sample can be incubated for 20 minutes at 37°C in a total volume of 30 μί. The reaction can then be stopped and released acetate can be extracted and the amount of radioactivity release determined by scintillation counting. An alternative assay useful for determining the HDAC inhibitory activity of a small molecule is the "HDAC Fluorescent Activity Assay; Drug Discovery Kit-AK-500" available from BIOMOL Research Laboratories, Inc., Plymouth Meeting, Pa.

[00185] In another embodiment, animals, for example, mice, can be injected intraperitoneally with a small molecule of the present disclosure. Selected tissues, for example, brain, spleen, liver etc, can be isolated at predetermined times, post administration. Histones can be isolated from tissues essentially as described by Yoshida et ah, J. Biol. Chem. 265: 17174-17179, 1990. Equal amounts of histones (about 1 μg) can be

electrophoresed on 15% SDS-polyacrylamide gels and can be transferred to Hybond-P filters (available from Amersham). Filters can be blocked with 3% milk and can be probed with a rabbit purified polyclonal anti-acetylated histone H4 antibody (a-Ac-H4) and anti-acetylated histone H3 antibody (a-Ac-H3) (Upstate Biotechnology, Inc.). Levels of acetylated histone can be visualized using a horseradish peroxidase-conjugated goat anti-rabbit antibody (1 :5000) and the SuperSignal chemiluminescent substrate (Pierce). As a loading control for the histone protein, parallel gels can be run and stained with Coomassie Blue (CB).

II. Salts, Hydrates, and Prodrugs of Small Molecules

[00186] It should be recognized that the small molecules disclosed herein may be present and optionally administered in the form of salts, hydrates and prodrugs that are converted in vivo into the small molecules disclosed herein. For example, it is within the scope of the disclosure to convert the small molecules of the disclosure into and use them in the form of their pharmaceutically acceptable salts derived from various organic and inorganic acids and bases in accordance with procedures well known in the art.

[00187] When small molecules of the disclosure possess a free base form, the small molecules can be prepared as a pharmaceutically acceptable acid addition salt by reacting the free base form of the compound with a pharmaceutically acceptable inorganic or organic acid, e.g., hydrohalides such as hydrochloride, hydrobromide, hydroiodide; other mineral acids and their corresponding salts such as sulfate, nitrate, phosphate, etc.; and alkyl and monoarylsulfonates such as ethanesulfonate, toluenesulfonate and benzenesulfonate; and other organic acids and their corresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate and ascorbate. Further acid addition salts include, but are not limited to: adipate, alginate, arginate, aspartate, bisulfate, bisulfite, bromide, butyrate, camphorate, camphorsulfonate, caprylate, chloride, chlorobenzoate, cyclopentanepropionate, digluconate, dihydrogenphosphate, dinitrobenzoate, dodecylsulfate, fumarate, galacterate (from mucic acid), galacturonate, glucoheptonate, gluconate, glutamate, glycerophosphate, hemisuccinate, hemisulfate, heptanoate, hexanoate, hippurate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, iodide, isethionate, iso-butyrate, lactate,

lactobionate, malate, malonate, mandelate, metaphosphate, methanesulfonate,

methylbenzoate, monohydrogenphosphate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate, oleate, pamoate, pectinate, persulfate, phenylacetate, 3-phenylpropionate, phosphate, phosphonate and phthalate. It should be recognized that the free base forms will typically differ from their respective salt forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free base forms for the purposes of the disclosure .

[00188] When the small molecules disclosed herein possess a free acid form, a pharmaceutically acceptable base addition salt can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base. Examples of such bases are alkali metal hydroxides including potassium, sodium and lithium hydroxides; alkaline earth metal hydroxides such as barium and calcium hydroxides; alkali metal alkoxides, e.g. , potassium ethanolate and sodium propanolate; and various organic bases such as ammonium hydroxide, piperidine, diethanolamine and N-methylglutamine. Also included are the aluminum salts of the small molecules disclosed herein. Further base salts include, but are not limited to: copper, ferric, ferrous, lithium, magnesium, manganic, manganous, potassium, sodium and zinc salts. Organic base salts include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, e.g., arginine, betaine, caffeine, chloroprocaine, choline, Ν,Ν'-dibenzylethylenediamine (benzathine),

dicyclohexylamine, diethanolamine, 2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, iso-propylamine, lidocaine, lysine, meglumine, N- methyl-D-glucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethanolamine, triethylamine, trimethylamine, tripropylamine and tris-(hydroxymethyl)-methylamine (tromethamine). It should be recognized that the free acid forms will typically differ from their respective salt forms somewhat in physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid forms for the purposes of the disclosure .

[00189] Small molecules disclosed herein that comprise basic nitrogen-containing groups may be quaternized with such agents as (C ) alkyl halides, e.g., methyl, ethyl, iso- propyl and tert-butyl chlorides, bromides and iodides; di (C _{1 -4} ) alkyl sulfates, e.g., dimethyl, diethyl and diamyl sulfates; (C ₁₀ -is) alkyl halides, e.g., decyl, dodecyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; and aryl (C _{] -4} ) alkyl halides, e.g., benzyl chloride and phenethyl bromide. Such salts permit the preparation of both water-soluble and oil- soluble compounds of the disclosure .

[00190] N-oxides of small molecules disclosed herein can be prepared by methods known to those of ordinary skill in the art. For example, N-oxides can be prepared by treating an unoxidized form of the small molecule with an oxidizing agent (e.g.,

trifluoroperacetic acid, permaleic acid, perbenzoic acid, peracetic acid, meta- chloroperoxybenzoic acid, or the like) in a suitable inert organic solvent (e.g., a halogenated hydrocarbon such as dichloromethane) at approximately 0°C. Alternatively, the N-oxides of the compounds can be prepared from the N-oxide of an appropriate starting material.

[00191] Prodrug derivatives of small molecules can be prepared by modifying substituents of small molecules disclosed herein that are then converted in vivo to a different substituent. It is noted that in many instances, the prodrugs themselves also fall within the scope of the range of compounds according to the disclosure. For example, prodrugs can be prepared by reacting a compound with a carbamylating agent (e.g., 1 ,1 - acyloxyalkylcarbonochloridate, para-nitrophenyl carbonate, or the like) or an acylating agent. Further examples of methods of making prodrugs are described in Saulnier et al. (1994), Bioorganic and Medicinal Chemistry Letters, Vol. 4, p. 1985.

[00192] Protected derivatives of small molecules of the disclosure can also be made. Examples of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, Protecting Groups in Organic Synthesis, 3.sup.rd edition, John Wiley & Sons, Inc. 1999. [00193] Small molecules of the disclosure may also be conveniently prepared, or formed during the process of the disclosure , as solvates (e.g., hydrates). Hydrates of small molecules of the disclosure may be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxin, tetrahydrofuran or methanol.

[00194] A "pharmaceutically acceptable salt", as used herein, is intended to encompass any small molecule according to the disclosure that is utilized in the form of a salt thereof, especially where the salt confers on the compound improved pharmacokinetic properties as compared to the free form of compound or a different salt form of the compound. A pharmaceutically acceptable salt, as used herein, includes salts present in vivo.

[00195] The pharmaceutically acceptable salt form may also initially confer desirable pharmacokinetic properties on the compound that it did not previously possess, and may even positively affect the pharmacodynamics of the compound with respect to its therapeutic activity in the body. An example of a pharmacokinetic property that may be favorably affected is the manner in which the compound is transported across cell membranes, which in turn may directly and positively affect the absorption, distribution, biotransformation and excretion of the compound. While the route of administration of the pharmaceutical composition is important, and various anatomical, physiological and pathological factors can critically affect bioavailability, the solubility of the compound is usually dependent upon the character of the particular salt form thereof, which it utilized. One of skill in the art will appreciate that an aqueous solution of the compound will provide the most rapid absorption of the compound into the body of a subject being treated, while lipid solutions and suspensions, as well as solid dosage forms, will result in less rapid absorption of the compound.

III. Compositions Comprising Small Molecules

[00196] A wide variety of compositions and administration methods may be used in conjunction with small molecules of the disclosure. Such compositions may include, in addition to the small molecules of the disclosure, conventional pharmaceutical excipients, and other conventional, pharmaceutically inactive agents. Additionally, the compositions may include active agents in addition to the small molecules of the disclosure. These additional active agents may include additional compounds according to the disclosure , and/or one or more other pharmaceutically active agents.

[00197] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[00198] The small molecules of the disclosure may be administered for the purpose of preventing disease progression or stabilizing tumor growth.

[00199] The preparation of pharmaceutical compositions that contain an active component is well understood in the art, for example, by mixing, granulating, or tablet- forming processes. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. For oral

administration, the active agents are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions and the like as detailed above.

[00200] The amount of the compound administered to the patient is less than an amount that would cause unmanageable toxicity in the patient. In the certain embodiments, the amount of the compound that is administered to the patient is less than the amount that causes a concentration of the compound in the patient's plasma to equal or exceed the toxic level of the compound. In one embodiment, the concentration of the compound in the patient's plasma is maintained at about 10 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 25 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 50 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 100 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 1000 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 2500 nM. In another embodiment, the concentration of the compound in the patient's plasma is maintained at about 5000 nM. The optimal amount of the compound that should be administered to the patient in the practice of the disclosure will depend on the particular compound used and the type of cancer being treated. [00201] The compositions may be in gaseous, liquid, semi-liquid or solid form, formulated in a manner suitable for the route of administration to be used. For oral administration, capsules and tablets are typically used. For parenteral administration, reconstitution of a lyophilized powder, prepared as described herein, is typically used.

[00202] Compositions comprising small molecules of the disclosure may be administered or co-administered orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadipo sally, intraarticularly, or intrathecally. The compounds and/or compositions according to the disclosure may also be administered or coadministered in slow release dosage forms. The small molecules of the disclosure may be administered intravenously on the first day of treatment, with oral administration on the second day and all consecutive days thereafter.

[00203] The small molecules and compositions comprising them may be administered or coadministered in any conventional dosage form. Co-administration in the context of this disclosure is intended to mean the administration of more than one therapeutic agent, one of which includes a small molecule, in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time.

[00204] Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application may optionally include one or more of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; agents for the adjustment of tonicity such as sodium chloride or dextrose, and agents for adjusting the acidity or alkalinity of the composition, such as alkaline or acidifying agents or buffers like carbonates, bicarbonates, phosphates, hydrochloric acid, and organic acids like acetic and citric acid. Parenteral preparations may optionally be enclosed in ampules, disposable syringes or single or multiple dose vials made of glass, plastic or other suitable material. [00205] When compounds according to the disclosure exhibit insufficient solubility, methods for solubilizing the compounds may be used. Such methods are known to those of skill in this art, and include, but are not limited to, using cosolvents, such as

dimethylsulfoxide (DMSO), using surfactants, such as TWEEN, or dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as prodrugs of the compounds may also be used in formulating effective pharmaceutical compositions.

[00206] Upon mixing or adding small molecules of the disclosure to a composition, a solution, suspension, emulsion or the like may be formed. The form of the resulting composition will depend upon a number of factors, including the intended mode of administration, and the solubility of the compound in the selected carrier or vehicle. The effective concentration needed to ameliorate the disease being treated may be empirically determined.

[00207] Compositions are optionally provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, dry powders for inhalers, granules, solutions, sterile parenteral solutions or suspensions, and oral solutions or suspensions, syrup, aerosol, suspensions, and oil-water emulsions containing suitable quantities of the compounds, particularly the pharmaceutically acceptable salts, preferably the sodium salts, thereof. The pharmaceutically therapeutically active compounds and derivatives thereof are typically formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms, as used herein, refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the therapeutically active compound sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes individually packaged tablet or capsule. Unit-dose forms may be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unit-dosage forms packaged in a single container to be administered in segregated unit-dose form.

Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pint or gallons. Hence, multiple dose form is a multiple of unit-doses that are not segregated in packaging. [00208] In addition to one or more small molecules of the disclosure, the composition may comprise: a diluent such as lactose, sucrose, dicalcium phosphate, or

carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acaciagelatin, glucose, molasses, polyvinylpyrrolidine, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art. Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, or otherwise mixing an active compound as defined above and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, glycols, ethanol, and the like, to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of auxiliary substances such as wetting agents, emulsifying agents, or solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrine derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. Actual methods of preparing such dosage forms are known in the art, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15th Edition, 1975. The composition or formulation to be administered will, in any event, contain a sufficient quantity of an inhibitor of the disclosure to reduce HDAC activity in vivo, thereby treating the disease state of the subject.

[00209] Dosage forms or compositions may optionally comprise one or more small molecules of the disclosure in the range of 0.005% to 100% (weight/weight) with the balance comprising additional substances such as those described herein.

[00210] In one embodiment, the amount of one or more small molecules disclosed herein in a pharmaceutical composition is selected from the group consisting of: less than 80%) by weight, less than 70% by weight, less than 60%> by weight, less than 50% by weight, less than 40% by weight, less than 30% by weight, less than 20% by weight, and less than 10%) by weight.

[00211] For oral administration, a pharmaceutically acceptable composition may optionally comprise any one or more commonly employed excipients, such as, for example pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, sodium crosscarmellose, glucose, sucrose, magnesium carbonate, sodium saccharin, talcum. Such compositions include solutions, suspensions, tablets, capsules, powders, dry powders for inhalers and sustained release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others. Methods for preparing these formulations are known to those skilled in the art. The compositions may optionally contain 0.01%-100% (weight/weight) of one or more small molecules, optionally 0.1 -95%, and optionally 1-95%.

[00212] Salts, preferably sodium salts, of the inhibitors may be prepared with carriers that protect the compound against rapid elimination from the body, such as time release formulations or coatings. The formulations may further include other active compounds to obtain desired combinations of properties.

A. Formulations for Oral Administration

[00213] Oral pharmaceutical dosage forms may be as a solid, gel or liquid. Examples of solid dosage forms include, but are not limited to tablets, capsules, granules, and bulk powders. More specific examples of oral tablets include compressed, chewable lozenges and tablets that may be enteric-coated, sugar-coated or film-coated. Examples of capsules include hard or soft gelatin capsules. Granules and powders may be provided in non- effervescent or effervescent forms. Each may be combined with other ingredients known to those skilled in the art.

[00214] In certain embodiments, compounds according to the disclosure are provided as solid dosage forms, preferably capsules or tablets. The tablets, pills, capsules, troches and the like may optionally contain one or more of the following ingredients, or compounds of a similar nature: a binder; a diluent; a disintegrating agent; a lubricant; a glidant; a sweetening agent; and a flavoring agent.

[00215] Examples of binders that may be used include, but are not limited to, microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, sucrose, and starch paste.

[00216] Examples of lubricants that may be used include, but are not limited to, talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. [00217] Examples of diluents that may be used include, but are not limited to, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate.

[00218] Examples of glidants that may be used include, but are not limited to, colloidal silicon dioxide.

[00219] Examples of disintegrating agents that may be used include, but are not limited to, crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose.

[00220] Examples of coloring agents that may be used include, but are not limited to, any of the approved certified water-soluble FD and C dyes, mixtures thereof, and water insoluble FD and C dyes suspended on alumina hydrate.

[00221] Examples of sweetening agents that may be used include, but are not limited to, sucrose, lactose, mannitol and artificial sweetening agents such as sodium cyclamate and saccharin, and any number of spray-dried flavors.

[00222] Examples of flavoring agents that may be used include, but are not limited to, natural flavors extracted from plants such as fruits and synthetic blends of compounds that produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate.

[00223] Examples of wetting agents that may be used include, but are not limited to, propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether.

[00224] Examples of anti-emetic coatings that may be used include, but are not limited to, fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates.

[00225] Examples of film coatings that may be used include, but are not limited to, hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

[00226] If oral administration is desired, the salt of the compound may optionally be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient.

[00227] When the dosage unit form is a capsule, it may optionally additionally comprise a liquid carrier such as a fatty oil. In addition, dosage unit forms may optionally additionally comprise various other materials that modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents.

[00228] Small molecules according to the disclosure may also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may optionally comprise, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

[00229] The small molecules of the present disclosure may also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. For example, if a compound is used for treating asthma or hypertension, it may be used with other bronchodilators and antihypertensive agents, respectively.

[00230] Examples of pharmaceutically acceptable carriers that may be included in tablets comprising compounds of the present disclosure include, but are not limited to binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents, and wetting agents. Enteric-coated tablets, because of the enteric-coating, resist the action of stomach acid and dissolve or disintegrate in the neutral or alkaline intestines. Sugar-coated tablets may be compressed tablets to which different layers of pharmaceutically acceptable substances are applied. Film-coated tablets may be compressed tablets that have been coated with polymers or other suitable coating. Multiple compressed tablets may be compressed tablets made by more than one compression cycle utilizing the pharmaceutically acceptable substances previously mentioned. Coloring agents may also be used in tablets. Flavoring and sweetening agents may be used in tablets, and are especially useful in the formation of chewable tablets and lozenges.

[00231] Examples of liquid oral dosage forms that may be used include, but are not limited to, aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.

[00232] Examples of aqueous solutions that may be used include, but are not limited to, elixirs and syrups. As used herein, elixirs refer to clear, sweetened, hydroalcoholic preparations. Examples of pharmaceutically acceptable carriers that may be used in elixirs include, but are not limited to solvents. Particular examples of solvents that may be used include glycerin, sorbitol, ethyl alcohol and syrup. As used herein, syrups refer to concentrated aqueous solutions of a sugar, for example, sucrose. Syrups may optionally further comprise a preservative.

[00233] Emulsions refer to two-phase systems in which one liquid is dispersed in the form of small globules throughout another liquid. Emulsions may optionally be oil-in-water or water-in-oil emulsions. Examples of pharmaceutically acceptable carriers that may be used in emulsions include, but are not limited to non-aqueous liquids, emulsifying agents and preservatives.

[00234] Examples of pharmaceutically acceptable substances that may be used in non- effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents.

[00235] Examples of pharmaceutically acceptable substances that may be used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide.

[00236] Coloring and flavoring agents may optionally be used in all of the above dosage forms.

[00237] Particular examples of preservatives that may be used include glycerin, methyl and propylparaben, benzoic add, sodium benzoate and alcohol.

[00238] Particular examples of non-aqueous liquids that may be used in emulsions include mineral oil and cottonseed oil.

[00239] Particular examples of emulsifying agents that may be used include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate.

[00240] Particular examples of suspending agents that may be used include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Diluents include lactose and sucrose. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as sodium cyclamate and saccharin.

[00241] Particular examples of wetting agents that may be used include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and

polyoxyethylene lauryl ether.

[00242] Particular examples of organic acids that may be used include citric and tartaric acid. [00243] Sources of carbon dioxide that may be used in effervescent compositions include sodium bicarbonate and sodium carbonate. Coloring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof.

[00244] Particular examples of flavoring agents that may be used include natural flavors extracted from plants such fruits, and synthetic blends of compounds that produce a pleasant taste sensation.

[00245] For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, is preferably encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, may be diluted with a sufficient quantity of a

pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for

administration.

[00246] Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Pat. Nos. Re 28,819 and 4,358,603.

[00247] B. Injectables, Solutions, and Emulsions

[00248] The present disclosure also is directed to compositions designed to administer the small molecules by parenteral administration, generally characterized by subcutaneous, intramuscular or intravenous injection. Injectables may be prepared in any conventional form, for example as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.

[00249] Examples of excipients that may be used in conjunction with injectables according to the present disclosure include, but are not limited to water, saline, dextrose, glycerol or ethanol. The injectable compositions may also optionally comprise minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. The percentage of active compound contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

[00250] Parenteral administration of the formulations includes intravenous, subcutaneous and intramuscular administrations. Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as the lyophilized powders described herein, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

[00251] When administered intravenously, examples of suitable carriers include, but are not limited to physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

[00252] Examples of pharmaceutically acceptable carriers that may optionally be used in parenteral preparations include, but are not limited to aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

[00253] Examples of aqueous vehicles that may optionally be used include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection.

[00254] Examples of nonaqueous parenteral vehicles that may optionally be used include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil.

[00255] Antimicrobial agents in bacteriostatic or fungistatic concentrations may be added to parenteral preparations, particularly when the preparations are packaged in multiple- dose containers and thus designed to be stored and multiple aliquots to be removed.

Examples of antimicrobial agents that may be used include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride.

[00256] Examples of isotonic agents that may be used include sodium chloride and dextrose. Examples of buffers that may be used include phosphate and citrate. Examples of antioxidants that may be used include sodium bisulfate. Examples of local anesthetics that may be used include procaine hydrochloride. Examples of suspending and dispersing agents that may be used include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Examples of emulsifying agents that may be used include Polysorbate 80 (TWEEN 80). A sequestering or chelating agent of metal ions includes EDTA.

[00257] Pharmaceutical carriers may also optionally include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[00258] The concentration of an inhibitor in the parenteral formulation may be adjusted so that an injection administers a pharmaceutically effective amount sufficient to produce the desired pharmacological effect. The exact concentration of an inhibitor and/or dosage to be used will ultimately depend on the age, weight and condition of the patient or animal as is known in the art.

[00259] Unit-dose parenteral preparations may be packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration should be sterile, as is known and practiced in the art.

[00260] Injectables may be designed for local and systemic administration. Typically a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, preferably more than 1% w/w of the small molecule to the treated tissue(s). The small molecule may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment will be a function of the location of where the composition is parenterally administered, the carrier and other variables that may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the age of the individual treated. It is to be further understood that for any particular subject, specific dosage regimens may need to be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the formulations. Hence, the concentration ranges set forth herein are intended to be exemplary and are not intended to limit the scope or practice of the claimed formulations.

[00261] The small molecule may optionally be suspended in micronized or other suitable form or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the disease state and may be empirically determined.

[00262] C. Lyophilized Powders

[00263] The small molecules of the present disclosure may also be prepared as lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. The lyophilized powders may also be formulated as solids or gels.

[00264] Sterile, lyophilized powder may be prepared by dissolving the compound in a sodium phosphate buffer solution containing dextrose or other suitable excipient. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. Briefly, the lyophilized powder may optionally be prepared by dissolving dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent, about 1-20%, preferably about 5 to 15%, in a suitable buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, typically, about neutral pH. Then, a small molecule is added to the resulting mixture, preferably above room temperature, more preferably at about 30-35C, and stirred until it dissolves. The resulting mixture is diluted by adding more buffer to a desired concentration. The resulting mixture is sterile filtered or treated to remove particulates and to insure sterility, and apportioned into vials for lyophilization. Each vial may contain a single dosage or multiple dosages of the inhibitor. [00265] D. Topical Administration

[00266] The small molecules of the present disclosure may also be administered as topical mixtures. Topical mixtures may be used for local and systemic administration. The resulting mixture may be a solution, suspension, emulsions or the like and are formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration.

[00267] The small molecules may be formulated as aerosols for topical application, such as by inhalation (see, U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflation, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will typically have diameters of less than 50 microns, preferably less than 10 microns.

[00268] The small molecules may also be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the small molecule alone or in combination with other pharmaceutically acceptable excipients can also be administered.

[00269] E. Formulations for Other Routes of Administration

[00270] Depending upon the disease state being treated, other routes of administration, such as topical application, transdermal patches, and rectal administration, may also be used. For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets for systemic effect. Rectal suppositories are used herein mean solid bodies for insertion into the rectum that melt or soften at body temperature releasing one or more pharmacologically or therapeutically active ingredients. Pharmaceutically acceptable substances utilized in rectal suppositories are bases or vehicles and agents to raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax, (polyoxyethylene glycol) and appropriate mixtures of mono-, di- and triglycerides of fatty acids. Combinations of the various bases may be used. Agents to raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared either by the compressed method or by molding. The typical weight of a rectal suppository is about 2 to 3 gm. Tablets and capsules for rectal administration may be manufactured using the same pharmaceutically acceptable substance and by the same methods as for formulations for oral administration.

IV. Combination Therapy

[00271] The small molecules of the present disclosure can be administered alone or in combination with other therapies suitable for the disease or disorder being treated. Where separate dosage formulations are used, the small molecules and the other therapeutic agent can be administered at essentially the same time (concurrently) or at separately staggered times (sequentially). The pharmaceutical combination is understood to include all these regimens. Administration in these various ways are suitable for the present disclosure as long as the beneficial therapeutic effect of the small molecule and the other therapeutic agent are realized by the patient at substantially the same time. In an embodiment, such beneficial effect is achieved when the target blood level concentrations of each active drug are maintained at substantially the same time.

[00272] The small molecules of the present disclosure are also useful in combination with known therapeutic agents and anti-cancer agents. For example, instant compounds are useful in combination with known anti-cancer agents. Combinations of the presently disclosed small molecules with other anti-cancer or chemotherapeutic agents are within the scope of the disclosure . Examples of such agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6.sup.th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. A person of ordinary skill in the art would be able to discern the combinations of agents that would be useful based on the particular characteristics of the drugs and the cancer involved.

[00273] Such anti-cancer agents include, but are not limited to, the following: estrogen receptor modulators, androgen receptor modulators, retinoid receptor modulators, cytotoxic/cyto static agents, antiproliferative agents, reverse transcriptase inhibitors, HIV protease inhibitors, angiogenesis inhibitors, prenyl-protein transferase inhibitors, HMG-CoA reductase inhibitors and other angiogenesis inhibitors, inhibitors of cell proliferation and survival signaling, apoptosis inducing agents, agents that interfere with cell cycle

checkpoints, agents that interfere with receptor tyrosine kinases (RTKs) and cancer vaccines. The instant compounds are particularly useful when co-administered with radiation therapy.

[00274] "Cytotoxic/cytostatic agents" refer to compounds which cause cell death or inhibit cell proliferation primarily by interfering directly with the cell's functioning or inhibit or interfere with cell mytosis, including alkylating agents, tumor necrosis factors, intercalators, hypoxia activatable compounds, microtubule inhibitors/microtubule-stabilizing agents, inhibitors of mitotic kinesins, inhibitors of histone deacetylase, inhibitors of kinases involved in mitotic progression, antimetabolites; biological response modifiers;

hormonal/anti -hormonal therapeutic agents, haematopoietic growth factors, monoclonal antibody targeted therapeutic agents, topoisomerase inhibitors, proteasome inhibitors and ubiquitin ligase inhibitors.

[00275] Examples of cytotoxic agents include, but are not limited to, sertenef, cachectin, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil mustard, thiotepa, busulfan, fluorouracil, cytarbine, vinblastine, paclitaxel, doxorubicin, bleomycin, mitomycin, prednisone, tamoxifen, flutamide, asparaginase, rituximab, trastuzumab, imatinib, retinoic acid, colony-stimulating factor, amifostine, lenalidonmide, HDCA inhibitor, CDK inhibitor, camptothecin, topotecan, carmustine, lomustine, streptozocin, tasonermin, lonidamine, carboplatin, altretamine, dacarbazine, procarbazine, prednimustine, dibromodulcitol, ranimustine, fotemustine, methotrexate, mercaptopurine, nedaplatin, oxaliplatin, temozolomide, heptaplatin, estramustine, improsulfan tosilate, trofosfamide, nimustine, dibrospidium chloride, pumitepa, lobaplatin, satraplatin,

thioguanine, profiromycin, cisplatin, irofuilven, dexifosfamide, cis-aminedichloro(2-methyl- pyridine)platinum, benzylguanine, glufosfamide, GPX100, (trans, trans, trans)-bis-mu- (hexane-l ,6-diamine)-mu-[diamine-platinum(II)]bis[diamine(c- hloro)platinum

(II)]tetrachloride, diarizidinylspermine, arsenic trioxide, 1-(1 l -dodecylamino-10- hydroxyundecyl)-3,7-dimethylxanthine, zorubicin, doxorubicin, daunorubicin, idarubicin, anthracenedione, bleomycin, mitomycin C, dactinomycin, plicatomycin, bisantrene, mitoxantrone, pirarubicin, pinafide, valrubicin, amrubicin, antineoplaston, 3'-deamino-3'- morpholino-13-deoxo-lO-hydroxycanninomycin, annamycin, galarubicin, elinafide, MEN10755, and 4-demethoxy-3-deamino-3-aziridinyl-4-methylsulphonyl-daunoru bicin (see WO 00/50032).

V. Dosages and Dosing Schedules

[00276] The dosage regimen utilizing small molecules of the present disclosure can be selected in accordance with a variety of factors including type, species, age, weight, sex and the type of cancer being treated; the severity (i.e., stage) of the disease to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to treat, for example, to prevent, inhibit (fully or partially) or arrest the progress of the disease.

[00277] For oral administration, suitable daily dosages are for example between about 2-4000 mg administered orally once-daily, twice-daily or three times-daily, continuous (every day) or intermittently (e.g., 3-5 days a week). For example, when used to treat the desired disease, the dose of the phosphorus compound can range between about 2 mg to about 2000 mg per day.

[00278] The small molecule or pharmaceutical compositions comprising a small molecule is administered once daily (QD), or divided into multiple daily doses such as twice daily (BID), and three times daily (TID). For administration once a day, a suitably prepared medicament would therefore contain all of the needed daily dose. For administration twice a day, a suitably prepared medicament would therefore contain half of the needed daily dose. For administration three times a day, a suitably prepared medicament would therefore contain one third of the needed daily dose.

[00279] In addition, the administration can be continuous, i.e., every day, or intermittently. The terms "intermittent" or "intermittently" as used herein means stopping and starting at either regular or irregular intervals. For example, intermittent administration of a small molecule may be administration one to six days per week or it may mean

administration in cycles (e.g., daily administration for two to eight consecutive weeks, then a rest period with no administration for up to one week) or it may mean administration on alternate days.

[00280] The compounds can also be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art. To be administered in the form of a transdermal delivery system, the dosage administration will, or course, be continuous rather than intermittent throughout the dosage regime.

[00281] It should be apparent to a person skilled in the art that the various modes of administration, dosages and dosing schedules described herein merely set forth specific embodiments and should not be construed as limiting the broad scope of the disclosure . Any permutations, variations and combinations of the dosages and dosing schedules are included within the scope of the disclosure.

VI. Methods for Reprogramming a Cell

[00282] In one embodiment, the disclosure relates to a method for reprogramming a cell comprising: exposing said cell to a small molecule disclosed herein, inducing the expression of a pluripotent or multipotent gene, and selecting a cell, wherein differentiation potential has been restored to said cell.

[00283] In another embodiment, the disclosure provides a reprogrammed cell that is obtained in the absence of eggs, embryos, embryonic stem cells, or somatic cell nuclear transfer (SCNT). A reprogrammed cell produced by the methods of the disclosure may be pluripotent or multipotent. A reprogrammed cell produced by the methods of the disclosure can have a variety of different properties including embryonic stem cell like properties. For example, a reprogrammed cell may be capable of proliferating for at least 10, 15, 20, 30, or more passages in an undifferentiated state. In other forms, a reprogrammed cell can proliferate for more than a year without differentiating. Reprogrammed cells can also maintain a normal karyotype while proliferating and/or differentiating. Some reprogrammed cells also can be cells capable of indefinite proliferation in vitro in an undifferentiated state. Some reprogrammed cells also can maintain a normal karyotype through prolonged culture. Some reprogrammed cells can maintain the potential to differentiate to derivatives of all three embryonic germ layers (endoderm, mesoderm, and ectoderm) even after prolonged culture. Some reprogrammed cells can form any cell type in the organism. Some reprogrammed cells can form embryoid bodies under certain conditions, such as growth on media that do not maintain undifferentiated growth. Some reprogrammed cells can form chimeras through fusion with a blastocyst, for example.

[00284] Reprogrammed cells can be defined by a variety of markers. For example, some reprogrammed cells express alkaline phosphatase. Some reprogrammed cells express SSEA-1 , SSEA-3, SSEA-4, TRA-1 -60, and/or TRA-1-81. Some reprogrammed cells express Oct 4, Sox2, and Nanog. It is understood that some reprogrammed cells will express these at the mRNA level, and still others will also express them at the protein level, on for example, the cell surface or within the cell.

[00285] A reprogrammed cell can have any combination of any reprogrammed cell property or category or categories and properties discussed herein. For example, a reprogrammed cell can express alkaline phosphatase, not express SSEA- 1 , proliferate for at least 20 passages, and be capable of differentiating into any cell type. Another

reprogrammed cell, for example, can express SSEA-1 on the cell surface, and be capable of forming endoderm, mesoderm, and ectoderm tissue and be cultured for over a year without differentiation.

[00286] A reprogrammed cell can be alkaline phosphatase (AP) positive, SSEA-1 positive, and SSEA-4 negative. A reprogrammed cell also can be Nanog positive, Sox2 positive, and Oct-4 positive. A reprogrammed cell also can be Tell positive, and Tbx3 positive. A reprogrammed cell can also be Cripto positive, Stellar positive and Dazl positive. A reprogrammed cell can express cell surface antigens that bind with antibodies having the binding specificity of monoclonal antibodies TRA-1-60 (ATCC HB-4783) and TRA-1-81 (ATCC HB-4784). Further, as disclosed herein, a reprogrammed cell can be maintained without a feeder layer for at least 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 passages or for over a year.

[00287] A reprogrammed cell may have the potential to differentiate into a wide variety of cell types of different lineages including fibroblasts, osteoblasts, chondrocytes, adipocytes, skeletal muscle, endothelium, stroma, smooth muscle, cardiac muscle, neural cells, hemiopoetic cells, pancreatic islet, or virtually any cell of the body. A reprogrammed cell may have the potential to differentiate into all cell lineages. A reprogrammed cell may have the potential to differentiate into any number of lineages including 1 , 2, 3, 4, 5, 6-10, 1 1-20, 21-30, and greater than 30 lineages.

[00288] Embodiments of the disclosure also include methods for treating a variety of diseases using a reprogrammed cell produced according to the novel methods disclosed elsewhere herein. The skilled artisan would appreciate, based upon the disclosure provided herein, the value and potential of regenerative medicine in treating a plethora of diseases including, but not limited to, heart disease, diabetes, skin diseases and skin grafts, spinal cord injuries, Parkinson's disease, multiple sclerosis, Alzheimer's disease, and the like. The disclosure encompasses methods for administering reprogrammed cells to an animal, including humans, in order to treat diseases where the introduction of new, undamaged cells will provide some form of therapeutic relief.

[00289] Embodiments of the disclosure relate to the use of reprogrammed cells and reprogrammed cell lines produced using the methods disclosed herein for drug screening and toxicity assays. In another embodiment, the disclosure relates to the use of a cell culture of reprogrammed cells for drug screening and toxicity assays.

[00290] In another embodiment, the disclosure relates to reagents and methods for in vitro screening of pharmaceutical and non-pharmaceutical chemicals using induced pluripotent or multipotent stem cells or iPS-derived lineage-specific cells, such as neural stem cells, muscle cells, and pancreatic cells. In yet another embodiment, the disclosure relates to methods for in vitro screening of toxicity and teratogenicity of chemical compounds using induced pluripotent or multipotent stem cells or iPS-derived lineage- specific cells.

[00291] In another embodiment, the disclosure provides human-specific in vitro methods for reliably determining toxicity of pharmaceuticals and other chemical compounds using induced pluripotent or multipotent stem cells or iPS-derived lineage-specific cells, thus overcoming the limitations associated with interspecies animal models. The methods can be used to test compounds that can produce positive, negative or a combination of positive and negative affects on the cell. Compounds can be tested on lineage specific cells, including but not limited to neuronal cells, muscle cell, cardiac cells, and pancreatic cells. [00292] In another embodiment, the disclosure provides methods for using

undifferentiated induced pluripotent stem cells or iPS-derived lineage-specific cells for in vitro evaluation. In one embodiment, undifferentiated induced pluripotent or multipotent stem cells or iPS-derived lineage-specific cells, are exposed to test compounds at any concentration including but not limited to concentrations reflective of in vivo levels.

[00293] Further embodiments of this aspect of the disclosure provide for determination of the capacity of the test compound to induce differentiation of induced pluripotent or multipotent stem cells into particular cell types.

[00294] In another embodiment, the disclosure relates to methods for using

pluripotent, non-lineage restricted cells to screen compounds. The benefit of utilizing pluripotent stem cells is they permit analysis of global toxic response(s) and are isolated from the physiological target of developmental toxicity. In addition, because these cells have not differentiated into a specific lineage, the potential for false negatives is reduced.

[00295] In another embodiment, the disclosure relates to methods for identifying predictive biomarkers of toxic responses to chemical compounds, particularly pharmaceutical and non-pharmaceutical chemicals, and particularly to known teratogens. In embodiments of this aspect, a dynamic set representative of a plurality of cellular metabolites, preferably secreted or excreted by hES cells is determined and correlated with health and disease or toxic insult state. Cellular metabolites according to this aspect of the disclosure generally range from about 10 to about 1500 Daltons, more particularly from about 100 to about 1000 Daltons, and include but are not limited to compounds such as sugars, organic acids, amino acids, fatty acids and signaling low-molecular weight compounds. Said biomarker profiles are diagnostic for toxicity of chemical compounds, particularly pharmaceutical and non- pharmaceutical chemicals, that participate in and reveal functional mechanisms of cellular response to pathological or toxic chemical insult, thus serving as biomarkers of disease or toxic response that can be detected in biological fluids. In particularly preferred embodiments of this aspect of the disclosure, these biomarkers are useful for identifying active (or activated) metabolic pathways following molecular changes predicted, inter alia, by other methods (such as transcriptomics and proteomics).

[00296] Embodiments of the disclosure also relate to a method for monitoring or identifying the early stages or the initiation of reprogramming. In one embodiment, cells overexpressing a transcription factor in conjunction with a reporter construct can be used to monitor the early stages of reprogramming.

[00297] The skilled artisan will readily understand that reprogrammed cells can be administered to an animal as a re-differentiated cell, for example, a neuron, and will be useful in replacing diseased or damaged neurons in the animal. Additionally, a reprogrammed cell can be administered to the animal and upon receiving signals and cues from the surrounding milieu, can re-differentiate into a desired cell type dictated by the neighboring cellular milieu. Alternatively, the cell can be re-differentiated in vitro and the differentiated cell can be administered to a mammal in need thereof.

[00298] The reprogrammed cells can be prepared for grafting to ensure long term survival in the in vivo environment. For example, cells can be propagated in a suitable culture medium, such as progenitor medium, for growth and maintenance of the cells and allowed to grow to confluence. The cells are loosened from the culture substrate using, for example, a buffered solution such as phosphate buffered saline (PBS) containing 0.05% trypsin supplemented with 1 mg/ml of glucose; 0.1 mg/ml of MgCl.sub.2, 0.1 mg/ml

CaCl.sub.2 (complete PBS) plus 5% serum to inactivate trypsin. The cells can be washed with PBS using centrifugation and are then resuspended in the complete PBS without trypsin and at a selected density for injection.

[00299] The reprogrammed cells of this disclosure can be transplanted "naked" into patients using techniques known in the art such as those described in U.S. Pat. Nos.

5,082,670 and 5,618,531 , each incorporated herein by reference, or into any other suitable site in the body.

[00300] The reprogrammed cells can be transplanted as a mixture/solution comprising of single cells or a solution comprising a suspension of a cell aggregate. Such aggregate can be approximately 10-500 micrometers in diameter, and, more preferably, about 40-50 micrometers in diameter. A reprogrammed cell aggregate can comprise about 5-100, more preferably, about 5-20, cells per sphere. The density of transplanted cells can range from about 10,000 to 1,000,000 cells per microliter, more preferably, from about 25,000 to

500,000 cells per microliter.

[00301] Transplantation of the reprogrammed cell of the disclosure can be

accomplished using techniques well known in the art as well those developed in the future. The disclosure comprises a method for transplanting, grafting, infusing, or otherwise introducing reprogrammed cells into an animal, preferably, a human.

[00302] The reprogrammed cells also may be encapsulated and used to deliver biologically active molecules, according to known encapsulation technologies, including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883; 4,353,888; and 5,084,350, herein incorporated by reference), or macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761 ;

5,158,881 ; 4,976,859; and 4,968,733; and International Publication Nos. WO 92/19195; WO 95/05452, all of which are incorporated herein by reference). For macroencapsulation, cell

* 3 9 number in the devices can be varied; preferably, each device contains between 10 -10 cells, most preferably, about 10 ⁵ to 10 ⁷ cells. Several macroencapsulation devices may be implanted in the patient. Methods for the macroencapsulation and implantation of cells are well known in the art and are described in, for example, U.S. Pat. No. 6,498,018.

[00303] Reprogrammed cells of the disclosure also can be used to express a foreign protein or molecule for a therapeutic purpose or for a method of tracking their integration and differentiation in a patient's tissue. Thus, the disclosure encompasses expression vectors and methods for the introduction of exogenous DNA into reprogrammed cells with concomitant expression of the exogenous DNA in the reprogrammed cells such as those described, for example, in Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[00304] In yet another embodiment, the disclosure relates to an enriched population of reprogrammed cells produced according to a method comprising: exposing said cell to a small molecule disclosed herein; inducing expression of a pluripotent or multipotent gene, selecting a cell, wherein differentiation potential has been restored to said cell, and culturing said selected cell to produce population of cells. In still another embodiment, the

reprogrammed cell expresses a cell surface marker selected from the group consisting of: SSEA3, SSEA4, Tra-1 -60, and Tra-1-81 , Oct-4, Nanog, and Sox-2. In yet another embodiment, the reprogrammed cells account for at least 1-5%, 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 96-98%; or at least 99% of the enriched population of cells. [00305] Embodiments of the disclosure also relate to a composition comprising a cell that has been produced by the methods of the disclosure. In another embodiment, the disclosure relates to a composition comprising cell that has been reprogrammed by overexpressing a transcription factor and exposing said cell to an agent that inhibits the activity, expression or activity and expression of a gene, which codes for a protein, or a protein involved in transcriptional respression, and inducing somatic cell reprogramming factors through endogenous auto- and reciprocal transcriptional regulation. In yet another embodiment, the disclosure relates to a composition comprising a cell that has been reprogrammed by overexpressing a single transcription factor.

VII. Methods for treating a disease

[00306] In yet another embodiment, the small molecules of the present disclosure are useful for the treatment of a proliferative disorder. In one embodiment, the disclosure relates to a method of treating a proliferative disorder in a subject comprising administering to said subject a therapeutically effective amount of a small molecule described herein.

[00307] As used herein, a "proliferative disorder" or a "hyperproliferative disorder," and other equivalent terms, means a disease or medical condition involving pathological growth of cells. Proliferative disorders include cancer, smooth muscle cell proliferation, systemic sclerosis, cirrhosis of the liver, adult respiratory distress syndrome, idiopathic cardiomyopathy, lupus erythematosus, retinopathy, e.g., diabetic retinopathy or other retinopathies, cardiac hyperplasia, reproductive system associated disorders such as benign prostatic hyperplasia and ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, harmatomas, lymphangiomatosis, sarcoidosis, desmoid tumors.

[00308] In one embodiment, the disclosure relates to a method of inhibiting or treating a disease arising from abnormal cell proliferation and/or differentiation in an animal, comprising administering to said animal a therapeutically effective amount of one or more small molecules disclosed herein, including but not limited to the small molecules with the chemical structures in Table 1 and Table 1 A. In one embodiment, the animal is human. In another embodiment, the disease is mediated by a histone deacetylase. In another embodiment, the disease is selected from the group consisting of a cell proliferative disease, autosomal dominant disorder, genetic related metabolic disorder, fibrosis, autoimmune disease, diabetes, neurological disease, and Alzheimer's disease. In yet another embodiment, the disease is cancer selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, liver cancer, lung cancer, small cell lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer stomach cancer, and thyroid cancer.

[00309] The in vivo effects of small molecules disclosed herein may not be exerted by those compounds as such, but by one or more degradation products.

[00310] Smooth muscle cell proliferation includes hyperproliferation of cells in the vasculature, for example, intimal smooth muscle cell hyperplasia, restenosis and vascular occlusion, particularly stenosis following biologically- or mechanically-mediated vascular injury, e.g., vascular injury associated with angioplasty. Moreover, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle other than the vasculature, e.g., bile duct blockage, bronchial airways of the lung in patients with asthma, in the kidneys of patients with renal interstitial fibrosis, and the like.

[00311] Non-cancerous proliferative disorders also include hyperproliferation of cells in the skin such as psoriasis and its varied clinical forms, Reiter's syndrome, pityriasis rubra pilaris, and hyperproliferative variants of disorders of keratinization (e.g., actinic keratosis, senile keratosis), scleroderma, and the like.

[00312] In another embodiment, the proliferative disorder is cancer. Cancers that can be treated or prevented by the small molecules, compositions and methods of the present disclosure include, but are not limited to human sarcomas and carcinomas, e.g.,

fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute myelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and non- Hodgkin's disease), multiple myeloma, Waldenstrobm's macroglobulinemia, and heavy chain disease.

[00313] Other examples of leukemias include acute and/or chronic leukemias, e.g., lymphocytic leukemia {e.g., as exemplified by the p388 (murine) cell line), large granular lymphocytic leukemia, and lymphoblastic leukemia; T-cell leukemias, e.g., T-cell leukemia (e.g., as exemplified by the CEM, Jurkat, and HSB-2 (acute), YAC-1 (murine) cell lines), T- lymphocytic leukemia, and T-lymphoblastic leukemia; B cell leukemia (e.g., as exemplified by the SB (acute) cell line), and B-lymphocytic leukemia; mixed cell leukemias, e.g., B and T cell leukemia and B and T lymphocytic leukemia; myeloid leukemias, e.g., granulocytic leukemia, myelocytic leukemia {e.g., as exemplified by the HL-60 (promyelocyte) cell line), and myelogenous leukemia (e.g. , as exemplified by the K562(chronic)cell line); neutrophilic leukemia; eosinophilic leukemia; monocytic leukemia (e.g., as exemplified by the THP- 1 (acute) cell line); myelomonocytic leukemia; Naegeli-type myeloid leukemia; and nonlymphocytic leukemia. Other examples of leukemias are described in Chapter 60 of The Chemotherapy Sourcebook, Michael C. Perry Ed., Williams & Williams (1992) and Section 36 of Holland Frie Cancer Medicine 5th Ed., Bast et al. Eds., B. C. Decker Inc. (2000). The entire teachings of the preceding references are incorporated herein by reference.

[00314] In yet another embodiment, the small molecules and methods disclosed herein can be used to treat a subject with non-solid tumors such as multiple myeloma. In another embodiment, the small molecules and methods disclosed herein can be used to treat T- leukemia (e.g., as exemplified by Jurkat and CEM cell lines); B-leukemia (e.g., as exemplified by the SB cell line); promyelocytes (e.g., as exemplified by the HL-60 cell line); uterine sarcoma (e.g., as exemplified by the MES-SA cell line); monocytic leukemia (e.g., as exemplified by the THP-l(acute) cell line); and lymphoma (e.g., as exemplified by the U937 cell line). [00315] In still yet another embodiment, the small molecules and methods disclosed herein can be used to treat subjects whose cancer has become "multi-drug resistant." A cancer that initially responded to an anti-cancer drug becomes resistant to the anti-cancer drug when the anti-cancer drug is no longer effective in treating the subject with the cancer. For example, many tumors will initially respond to treatment with an anti-cancer drug, such as a tyrosine kinase inhibitor, by decreasing in size or even going into remission, only to develop resistance to the drug. Drug resistant tumors are characterized by a resumption of their growth and/or reappearance after having seemingly gone into remission, despite the administration of increased dosages of the anti-cancer drug. Cancers that have developed resistance to two or more anti-cancer drugs are said to be "multi-drug resistant." For example, it is common for cancers to become resistant to three or more anti-cancer agents, often five or more anti-cancer agents and at times ten or more anti-cancer agents.

[00316] The compounds of the disclosure may also be used as immunosuppressants or immunomodulators and can accordingly be used in the treatment or prevention of immune response or immune-mediated responses and diseases such as systemic lupus erythematosus (SLE) and acute or chronic transplant rejection in a recipient of an organ, tissue or cell transplant.

[00317] Examples of autoimmune diseases for which the small molecules of the present disclosure may be employed include autoimmune hematological disorders (including hemolytic anemia, aplastic anemia, pure red cell anemia and idiopathic thrombocytopenia), systemic lupus erythematosus, thyroiditis, Hashimoto's thyroiditis, polychondritis, scleredoma, Wegener granulomatosis, dermatomyositis, chronic active hepatitis, myasthenia gravis, psoriasis, atopic dermatitis, vasculitis, Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (including ulcerative colitis and Crohn's disease) endocrine ophthalmopathy, Graves disease, sarcoidosis, multiple sclerosis, primary biliary cirrhosis, juvenile diabetes (diabetes mellitus type I), diabetes type II and the disorders associated therewith, uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal keratoconjunctivitis, interstitial lung fibrosis, psoriatic arthritis, glomerulonephritis (with and without nephrotic syndrome, including idiopathic nephrotic syndrome or minimal change nephropathy), juvenile dermatomyositismfectious, auto-antibody mediated diseases, aplastic anemia, Evan's syndrome, autoimmune hemolytic anemia, infectious diseases causing aberrant immune response and/or activation, such as traumatic or pathogen induced immune disregulation, including for example, that which are caused by hepatitis B and C infections, staphylococcus aureus infection, viral encephalitis, sepsis, parasitic diseases wherein damage is induced by inflammatory response (e.g. leprosy); and circulatory diseases, such as arteriosclerosis, atherosclerosis, polyarteritis nodosa and myocarditis.

VIII. Methods for treating an inflammatory disease

[00318] In yet another embodiment, small molecules of the present disclosure are useful for the treatment of an inflammatory disorder. In one embodiment, the disclosure relates to a method of treating an inflammatory disorder in a subject comprising

administering to said subject a therapeutically effective amount of a small molecule described herein.

[00319] In still another embodiment, the small molecules of the present disclosure can also be used for treating or preventing pain associated with inflammation or with an inflammatory disease in an animal. Such pain can arise where there is an inflammation of the body tissue which can be a local inflammatory response and/or a systemic inflammation. For example, the small molecules of the present disclosure can be used to treat or prevent an inflammatory disease including, but not limited to: organ transplant rejection; reoxygenation injury resulting from organ transplantation (see Grupp et al., J. Mol, Cell Cardiol. 31 :297-303 (1999)) including, but not limited to, transplantation of the heart, lung, liver, or kidney; chronic inflammatory diseases of the joints, including arthritis, rheumatoid arthritis, osteoarthritis and bone diseases associated with increased bone resorption; inflammatory bowel diseases, such as ileitis, ulcerative colitis, Barrett's syndrome, and Crohn's disease; inflammatory lung diseases, such as asthma, adult respiratory distress syndrome, and chronic obstructive airway disease; inflammatory diseases of the eye, including corneal dystrophy, trachoma, onchocerciasis; uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory disease of the gum, including gingivitis and periodontitis; tuberculosis;

leprosy; inflammatory diseases of the kidney, including uremic complications,

glomerulonephritis and nephrosis; inflammatory disease of the skin, including

sclerodermatitis, psoriasis and eczema; inflammatory diseases of the central nervous system, including chronic demyelinating diseases of the nervous system, multiple sclerosis, AIDS- related neurodegeneration and Alzheimer's disease, infectious meningitis, encephalomyelitis, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis and viral or autoimmune encephalitis; autoimmune diseases, including Type I and Type II diabetes mellitus; diabetic complications, including, but not limited to, diabetic cataract, glaucoma, retinopathy, nephropathy (such as microaluminuria and progressive diabetic nephropathy), polyneuropathy, mononeuropathies, autonomic neuropathy, gangrene of the feet,

atherosclerotic coronary arterial disease, peripheral arterial disease, nonketotic

hyperglycemic-hyperosmolar coma, foot ulcers, joint problems, and a skin or mucous membrane complication (such as an infection, a shin spot, a candidal infection or necrobiosis lipoidica diabeticorum); immune-complex vasculitis, and systemic lupus erythematosus (SLE); inflammatory disease of the heart, such as cardiomyopathy, ischemic heart disease hypercholesterolemia, and artherosclerosis; as well as various other diseases that can have significant inflammatory components, including preeclampsia, chronic liver failure, brain and spinal cord trauma, and cancer. The small molecules of the present disclosure can also be used for inhibiting, treating, or preventing pain associated with inflammatory disease that can, for example, be a systemic inflammation of the body, exemplified by gram-positive or gram negative shock, hemorrhagic or anaphylactic shock, or shock induced by cancer chemotherapy in response to pro-inflammatory cytokines, e.g., shock associated with proinflammatory cytokines. Such shock can be induced, e.g., by a chemotherapeutic agent that is administered as a treatment for cancer.

IX. Kits

[00320] Embodiments of the disclosure also relate to kits for preparing the methods and compositions of the disclosure. The kit can be used for, among other things, producing a reprogrammed cell and generating ES-like and stem cell-like cells,

[00321] In another embodiment, the kit can be used to reprogram a cell. The kit can contain the reagents necessary to practice the following method: exposing a cell to a small molecule, inducing the expression of a gene that contributes to a cell being pluripotent or multipotent; and selecting a reprogrammed cell. The kit may comprise at least one agent for overexpression of a transcription factor. The kit may comprise multiple agents for overexpression of transcription factors. The agents for overexpression of a transcription factor can be provided in a single container or in multiple containers.

[00322] The kit may also comprise reagents necessary to determine if the cell has been reprogrammed including but not limited to reagents to test for the induction of a gene that contributes to a cell being pluripotent or multipotent, reagents to test for inhibition of a DNMT, regents to test for demethylating of CpG dinucleotides, and reagents to test for remodeling of the chromatin structure.

[00323] The kit may also comprise regents that can be used to differentiate the reprogrammed cell into a particular lineage or multiple lineages including but not limited to a neuron, an osteoblast, a muscle cell, an epithelial cell, and hepatic cell.

[00324] The kit may also contain an instructional material, which describes the use of the components provide in the kit. As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of the methods of the disclosure in the kit for, among other things, effecting the reprogramming of a differentiated cell. Optionally, or alternately, the instructional material may describe one or more methods of re- and/or trans-differentiating the cells of the disclosure. The instructional material of the kit of the disclosure may, for example, be affixed to a container that contains a small molecule inhibitor. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and a small molecule inhibitor, or component thereof, be used cooperatively by the recipient.

[00325] The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations that become evident as a result of the teaching provided herein. All references including but not limited to U.S. patents, allowed U.S. patent applications, or published U.S. patent applications are incorporated within this specification by reference in their entirety.

[00326] The disclosure in now further described in reference to the following number paragraphs: [00327] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00328] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00329] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceuticall acceptable salt or composition thereof:

[00330] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00331] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00332] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00333] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00334] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00335] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00336] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00337] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00338] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00339] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00340] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[003 1] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00342] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00343] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00344] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00345] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00346] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00347] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00348] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00349] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00350] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00351] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00352] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00353] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00354] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00355] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00356] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00357] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00358] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00359] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00360] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00361] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or com osition thereof:

[00362] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof: [00363] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00364] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00365] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00366] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00367] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00368] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00369] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00370] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00371] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00372] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00373] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00374] A compound of the structure below, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof:

[00375] A pharmaceutical composition comprising a compound according to any one of paragraphs [00302] through [00349], as an active ingredient, along with a

pharmaceutically acceptable carrier, diluent, or excipient.

[00376] The pharmaceutical composition according to paragraph [00350], wherein the pharmaceutical composition is in a form of a tablet, capsule, powder, syrup, solution, aerosol or suspension.

[00377] The pharmaceutical composition according to any one of paragraphs [00350] or [00351], wherein the amount of the compound of fomiula (I) in the composition is less than 70% by weight.

[00378] The pharmaceutical composition according to paragraphs [00350] through

[00352] further comprising one or more anti-cancer agents.

[00379] The pharmaceutical composition according to any one of paragraphs [00350] through [00353], wherein the one or more anti-cancer agents are selected from the group consisting of cyclophosphamide, dacarbazine, cisplatin, methotrexate, mercaptopurine, thioguanine, fluorouracil, cytarabine, vinblastine, paclitaxel, doxorubicin, bleomycin, mitomycin, prednisone, tamoxifen, flutamide, asparaginase, rituximab, trastuzumab, imatinib, retinoic acid, colony-stimulating factor, amifostine, lenalidomide, HDAC inhibitor, CDK inhibitor, camptothecin and topotecan.

[00380] A method of inhibiting or treating a disease arising from abnormal cell proliferation and/or differentiation in an animal, comprising administering to said animal a therapeutically effective amount of one or more compounds according to paragraphs [00302] through [00350].

[00381] The method of paragraph [00355], wherein the animal is human.

[00382] The method of any one of paragraphs [00355] or [00356], wherein the disease is mediated by a histone deacetylase.

[00383] The method of any one of paragraphs [00355] through [00357], wherein the disease is selected from the group consisting of a cell proliferative disease, autosomal dominant disorder, genetic related metabolic disorder, fibrosis, autoimmune disease, diabetes, neurological disease, and Alzheimer's disease.

[00384] The method of any one of paragraphs [00355] through [00358], wherein the disease is cancer selected from the group consisting of bladder cancer, breast cancer, colon cancer, rectal cancer, endometrial cancer, kidney cancer, leukemia, promylocytic leukemia, chronic myelocytic leukemia, acute lymphocytic leukemia, liver cancer, lung cancer, small cell lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer stomach cancer, and thyroid cancer.

[00385] A method of inhibiting an HDAC in a cell comprising treating the cell with the compound according to any one of paragraphs [00302] through [00349].

[00386] EXAMPLES

[00387] The following examples are illustrative only and are not intended to limit the scope of the disclosure as defined by the claims.

[00388] Example 1:

[00389] Table III provides a list of small molecules and their mean inhibition of Class I, Class Ila, Class lib, and Class III HDACs. Table I provides the chemical structure of the compound in Table III. The small molecules of Table III, or a tautomeric form, a

stereoisomer, pharmaceutically acceptable salt or composition thereof can be used in combination with other small molecules. Multiple small molecules may provide synergistic effects. In addition, the small molecules of Table III can be used with other cytotoxic agents.

[00390] The small molecules of Table III, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof can inhibit or treat a disease arising fi-om abnormal cell proliferation and/or differentiation. The small molecules of Table III can be substituted or unsubstituted. In addition, the small molecules of Table III can inhibit the activity of one or more HDACs in a cell.

[00391] Table III. HD AC inhibitory activity of novel small molecules.

Mean Enz me Inhibition (%)

30 10 3 6 22 7 8 2 3 37 3 0 1 8

NSPT00046 10 -7 0 -3 0 -3 -3 2 -1 12 0 3 0 -1

30 -3 -1 2 -1 1 -1 1 1 17 1 1 0 -1

NSPT00047 10 -1 3 5 10 4 1 5 3 6 -1 1 -1 -1

30 5 3 2 26 3 3 1 3 13 0 -1 -1

NSPT00072 10 1 1 -1 5 4 -31 0 0 2 0 1 -1 0

30 1 5 1 6 -2 5 3 3 1 1 0 10

NSPT00074 10 7 7 2 3 3 -2 3 2 13 5 -4 1

30 6 5 2 7 2 2 2 4 23 1 1 -7 0

NSPT00082 10 8 6 4 9 2 10 3 0 9 3 1 -3 1

30 6 8 1 24 2 5 2 2 16 1 -3 5

NSPT00094 10 10 5 4 2 2 -3 1 -2 12 -1 1 -2 2

30 8 0 2 2 8 -1 2 1 22 -1 -1 0

NSPT00106 10 9 3 8 4 1 -4 0 -3 13 3 1 1 -5

30 8 6 8 3 2 6 5 1 28 3 -1 0 1

NSPT001 13 10 3 4 3 1 6 4 3 6 16 0 1 0 1

30 3 3 8 7 3 3 2 1 36 0 -1 0

NSPT001 18 10 5 0 1 -1 -2 -1 0 -2 2 0 - -4 -1

30 1 3 0 -1 -2 1 1 1 2 -4 1 -3 6

NSPT00135 10 -11 -9 -9 -1 -7 -5 -7 -5 -1 -10 -5 -4 -5

30 -6 -2 -1 21 -1 0 3 1 16 -3 0 -1 0

NSPT00137 10 -5 2 -7 6 2 -5 3 0 9 -1 -3 20 -1

30 0 2 -2 13 4 5 6 2 24 0 3 38 1

NSPT00138 10 2 1 4 2 2 -1 1 2 21 0 0 -2 0

30 6 0 7 9 2 -1 2 2 36 1 -4 -5 0

NSPT00162 10 10 3 3 4 7 1 2 1 3 3 4 0 1

30 17 6 12 2 4 3 1 3 2 5 6 1 1

NSPT00166 10 3 -1 1 16 5 12 13 2 5 -4 -12 1 0

30 -4 -1 0 10 -2 -1 1 -1 6 1 0 1 -7

NSPT00171 10 7 6 7 3 5 -1 2 0 3 5 3 1 -1

30 6 4 5 4 7 6 6 2 9 5 3 1 9

NSPT00173 10 4 1 5 2 0 -4 1 2 3 0 3 0 -7

30 -1 6 2 1 2 3 1 1 2 1 4 0 1

NSPT00182 10 1 1 -2 0 3 1 3 0 9 0 -1 0 0

30 -1 -1 2 2 2 0 5 1 21 0 4 0 -1

NSPT00185 10 -3 -8 -4 -5 -3 -7 -2 -3 -2 -3 -2 -1 -3

30 4 0 -1 -2 3 0 2 2 1 1 0 2 0 NSPT00186 10 4 3 4 0 3 2 1 1 3 3 2 2 0

30 4 0 0 -1 1 -1 0 1 0 2 6 2 3

NSPT00187 10 0 1 2 -1 -4 4 -2 0 3 -1 1 1 1

30 1 2 -7 -1 -2 -1 17 0 3 -6 -4 1 2

NSPT00191 10 -6 -6 -4 -4 -3 -7 -2 -4 3 -4 -4 -1 -5

30 -4 -2 -3 -2 1 0 3 0 7 -1 0 25 -3

NSPT00193 10 -2 -2 1 0 -1 -1 1 0 1 -2 5 2 0

30 2 2 0 -4 2 1 2 1 2 1 4 3 1

NSPT00200 10 -2 -5 -2 1 0 3 -2 -2 1 -1 0 2 0

30 1 0 -4 0 1 -1 -1 1 5 0 -5 1 -2

NSPT00205 10 2 2 -1 -1 3 0 2 0 8 -1 -3 3 1

30 -4 -4 2 -1 1 1 3 -1 11 0 4 7 -1

NSPT00215 10 7 -3 3 1 4 0 4 0 11 -1 3 1 -2

30 -1 2 0 1 0 -1 1 2 31 1 1 5 4

NSPT00216 10 -9 -6 -2 -1 -3 -2 -2 -3 0 -3 0 -1 -5

30 -4 -8 1 -2 -4 -4 -2 -2 5 -3 1 1 -11

NSPT00217 10 13 2 0 1 6 -4 0 -1 -1 0 2 4 3

NSPT00217 30 4 -2 -2 3 1 1 2 -1 2 0 0 4 14

NSPT00218 10 1 5 0 1 2 2 1 -1 16 2 2 2 1

30 1 4 1 2 2 2 3 3 38 1 4 2 2

NSPT00219 10 6 2 4 0 1 0 0 2 4 2 4 1 -6

30 6 13 3 -1 2 -2 5 -3 5 4 4 5 2

NSPT00221 10 3 2 2 0 -4 -1 3 2 -2 7 3 2 1

30 8 1 6 -1 -2 0 0 -1 1 1 -2 3 -5

NSPT00225 10 2 0 -4 1 -4 2 1 1 0 1 8 2 1

30 -2 -3 0 3 0 3 1 -3 2 2 1 4 1

NSPT00226 10 -3 -1 1 2 -1 -1 0 -2 7 1 1 0 -3

30 2 -2 2 1 -1 -2 -2 -2 14 0 1 0 -8

NSPT00231 10 5 1 2 0 15 2 2 5 0 1 5 2 2

30 5 4 -37 -2 5 2 0 1 2 4 0 4 6

NSPT00232 10 5 3 1 -1 3 3 3 2 3 0 3 3 11

30 18 2 4 0 7 3 5 2 4 1 5 3 11

NSPT00233 10 -1 -2 -4 -5 1 -4 -1 -3 -2 0 0 -3 -2

30 6 3 10 0 3 -3 13 0 6 3 1 1

NSPT00234 10 7 4 4 -2 10 2 0 2 5 4 5 2 4

30 5 1 3 0 4 2 0 1 6 2 5 3 -2

NSPT00235 10 0 -3 1 -5 -1 -8 -4 -3 0 -1 4 1 3

30 3 1 4 5 0 5 3 2 4 0 3 1 1 11

NSPT00237 10 5 1 2 3 3 5 1 0 3 3 4 3 2

30 4 0 -2 0 5 4 2 0 6 2 7 3 1

NSPT00238 10 7 0 4 6 6 0 7 2 9 2 5 1 2

30 11 10 11 10 -1 0 24 4 21 4 4 -2 12

NSPT00246 10 2 5 -2 3 4 2 3 1 3 5 -2 2 2

30 2 0 0 0 8 0 6 1 7 2 -2 3 -1

NSPT00249 10 2 2 -2 7 0 0 2 1 0 3 -4 3 5

30 4 2 0 -1 -1 -1 8 -1 2 0 0 3 8 NSPT00258 10 3 -1 0 0 -2 5 4 1 0 1 -1 1 1

30 0 -4 -1 -1 3 0 8 -2 -7 -2 -3 0 7

NSPT00260 10 -4 -4 -3 -6 -4 -10 -4 -6 -3 -2 -4 -3 -3

30 -3 4 3 1 0 -1 -1 0 6 0 0 2 1

NSPT00263 10 0 1 1 -3 -1 1 1 -2 1 2 0 1 0

30 12 10 1 1 0 3 -3 0 -2 2 4 -10 0 -1

NSPT00264 10 2 0 -1 0 -2 0 2 1 5 1 -3 2 1

30 0 -2 -2 -3 2 -4 7 4 8 -3 4 3 2

NSPT00271 10 4 1 -2 3 3 -2 2 0 1 0 2 5 5

30 6 4 1 0 2 0 6 2 7 1 -2 4 3

NSPT00273 10 7 2 3 2 4 -5 4 2 14 1 -4 3 3

30 1 -3 -5 1 1 4 6 0 2 32 1 -8 1 4

NSPT00275 10 2 0 3 1 -1 0 2 2 5 -2 -9 3 1

30 -1 1 -2 2 2 1 4 2 -1 -1 -3 4 6

NSPT00276 10 -7 -9 1 -2 1 -4 -5 -3 1 -4 -6 -1 -4

30 -1 3 4 1 6 0 2 1 7 0 -1 4 0

NSPT00282 10 3 -2 -7 2 13 -4 18 0 0 -1 -2 4 3

30 -4 -3 -6 8 30 2 33 0 -2 -2 -7 12 8

NSPT00283 10 13 -2 6 -2 0 0 -6 -1 0 1 -1 6 10

30 0 3 -1 0 2 1 -2 -2 1 -1 -3 -1 1

NSPT00285 10 1 1 -6 3 4 -1 -7 -3 -2 2 2 -10 8 -3

30 6 -5 2 7 1 -1 1 -1 5 0 -8 9 5

NSPT00286 10 -1 -5 0 0 0 1 -3 0 1 4 2 1 5

30 0 -2 0 0 -4 -2 . 2 -1 5 6 -6 13 -1

NSPT00287 10 -5 -6 2 -1 -2 -1 -1 0 3 0 1 0 -8

30 -1 -3 5 -1 -3 -6 -1 ^' -3 7 -2 1 -5 -10

NSPT00291 10 8 8 12 0 0 -1 0 -2 0 0 2 2 -1

30 3 5 1 2 -2 -3 1 1 1 1 8 1 4 1

NSPT00292 10 2 3 0 -2 0 0 7 5 0 2 -3 14 1

30 0 3 1 1 0 0 9 0 0 4 2 3 1

NSPT00293 10 -1 1 1 -2 -1 -1 -1 -1 1 0 -1 9 9

30 -2 4 -1 4 6 -2 -2 0 3 0 -1 3 1

NSPT00297 10 8 -3 17 0 2 -7 2 1 -1 6 -5 1 5

30 6 2 6 -1 2 1 2 -1 8 2 2 5 0

NSPT00301 10 6 -2 1 1 0 0 3 13 8 -2 4 -2 14 3

30 12 1 20 1 1 1 -3 -1 4 6 1 2 4

NSPT00305 10 -2 0 8 1 -1 0 2 -1 5 8 -2 5 0

30 5 -5 0 -2 -3 3 -1 0 0 1 1 -3 5 -2

NSPT00308 10 -13 0 2 -7 -6 -5 5 7 -2 -1 -6 -6 0

30 2 0 4 3 -1 -1 5 -2 1 6 29 3 5

NSPT00310 10 5 4 7 1 4 0 2 2 -1 1 2 1 9

30 2 4 8 2 -2 10 4 2 0 2 4 10

NSPT00315 10 3 2 4 1 5 -1 4 1 4 12 2 2 2

30 1 5 3 -1 -5 5 12 1 1 13 0 -6 4

NSPT00317 10 2 0 0 0 4 -1 6 1 -1 4 3 -1 1

30 8 3 -6 2 1 5 34 2 4 3 1 1 3

NSPT00318 10 3 2 5 4 2 0 3 0 3 1 -4 5 1 30 3 -4 6 -6 -1 -1 0 2 1 2 5 21 3

NSPT00323 10 -1 -2 -3 5 17 2 24 2 -1 -4 1 4 2

30 0 -3 0 7 24 -1 33 2 -1 -3 0 3 -1

NSPT00334 10 4 2 0 -2 0 -1 0 -1 1 2 -2 3 -4

30 2 3 1 -1 -2 0 3 -1 -6 2 3 12 -1

NSPT00335 10 13 0 -1 -1 1 -5 6 2 2 2 0 7 5

30 -5 2 0 -3 -2 -2 2 -1 4 0 0 12 2

NSPT00336 10 7 3 3 -1 -1 -3 2 -1 1 0 6 -13

30 1 0 2 -3 1 3 1 3 3 1 3 -1 1

NSPT00343 10 -2 -3 -3 2 1 -1 23 -1 -1 3 -12 1 1

30 -9 -5 -8 2 3 -2 26 -2 1 0 -6 3 -2

NSPT00345 10 -1 -7 -6 -1 -5 -6 2 -4 3 -8 -2 -7 -2

30 0 -2 0 2 -5 -5 10 2 11 -4 2 -1 2

NSPT00346 10 0 -2 -3 -1 3 -5 5 0 5 -4 3 1 0

30 -3 0 -7 -1 -4 4 12 3 7 -10 1 -4 4

NSPT00347 10 -5 -4 -2 -5 -4 -4 -5 -3 -8 -5 -1 -3 2

30 5 -1 5 -1 1 4 3 1 3 5 8 6

NSPT00350 10 -3 1 2 0 1 0 1 2 3 -3 -4 1 3

30 13 6 4 1 1 1 5 0 7 13 6 -7 9

NSPT00351 10 9 2 8 -1 0 -2 0 -2 2 0 -6 6 -1

30 6 6 -1 2 0 0 -2 3 3 3 -4 1 0

NSPT00356 10 4 4 4 1 2 2 1 2 3 2 7 1 2

30 5 4 4 2 1 0 -1 3 3 1 4 5 1

NSPT00357 10 6 2 7 -1 2 2 0 2 2 2 6 1 1 1

30 4 0 1 1 4 2 3 1 1 1 -2 10 3

NSPT00359 10 2 1 3 -2 -5 0 -3 3 2 2 2 6 2

30 2 1 4 -3 0 -2 -5 2 1 8 -3 2 4

NSPT00360 10 5 3 3 -2 2 2 -2 1 1 1 5 2 -1

30 5 4 1 -1 2 1 0 1 1 4 3 -2 3

NSPT00361 10 5 2 4 2 3 2 2 1 3 7 1 -1 2

30 5 1 4 2 1 1 0 2 6 0 -7 0 2

NSPT00362 10 5 4 13 1 0 1 4 2 1 1 -1 4 2

30 4 3 13 3 3 5 12 2 -2 1 1 4 4

NSPT00363 10 3 2 3 15 1 3 3 1 0 4 2 0 1

30 7 5 2 2 1 2 5 3 -1 1 0 0 4

NSPT00365 10 1 2 6 2 2 0 -1 1 1 2 2 -1 2

30 1 2 2 2 -3 2 -2 0 1 27 1 -2 2

NSPT00366 10 5 4 5 4 2 0 1 1 2 4 5 18 2

30 9 9 5 4 2 1 1 1 4 4 4 0 3

NSPT00371 10 0 1 7 7 3 2 -1 1 3 2 2 5 2

30 6 4 5 7 -1 -3 1 0 9 0 3 7 4

[00392] Example 2:

[00393] Table IV provides a list of small molecules and their mean inhibition of Class

I, Class lla, Class lib, and Class III HDACs. Table I provides the chemical structure of the compounds recited in Table IV. Small molecules in Table IV have substantial HDAC inhibitory activity.

[00394] The small molecules of Table IV can be used in combination with other small molecules. Multiple small molecules may provide synergistic effects. In addition, the small molecules of Table IV, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof can be used with other cytotoxic agents. The small molecules of Table IV can be substituted or unsubstituted.

[00395] The small molecules of Table IV, or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof can be used to treat or inhibit a disease arising from abnormal cell proliferation and/or differentiation. In addition, the small molecules in Table IV , or a tautomeric form, a stereoisomer, pharmaceutically acceptable salt or composition thereof can inhibit one or more HDAC in a cell.

[00396] Table IV. HDAC inhibitory activity of novel small molecules (% enzyme inhibition)

30 90 79 109 94 43 62 43 24 99 87 -1 -1 -7

NSPT00029 10 26 8 58 76 15 15 25 9 97 22 -5 -3 -4

30 57 34 84 89 34 35 53 31 94 49 0 2 2

NSPT00031 10 63 35 81 91 19 23 25 17 93 31 1 1 1 1

30 83 59 97 96 44 48 48 39 95 57 -2 -1 -6

NSPT00032 10 1 1 -2 15 61 2 1 5 12 90 -3 -2 -4 1

30 23 10 35 83 28 25 35 36 90 14 2 0 1

NSPT00034 10 53 27 72 92 34 39 37 20 97 24 1 2 7

30 77 48 93 95 60 68 64 48 94 46 0 0 1 1

NSPT00036 10 54 24 84 85 16 12 32 17 101 61 6 2 8

30 74 47 101 94 36 38 57 38 98 72 4 5 -4

NSPT00038 10 56 30 86 87 18 19 26 19 104 34 8 2 4

30 82 53 104 94 39 50 50 38 109 58 0 1 4

NSPT00039 10 57 28 83 92 41 42 47 31 108 34 3 1 -1

30 66 44 87 100 70 71 75 55 104 50 2 1 8

NSPT00040 10 88 69 102 100 72 86 73 50 106 85 2 2 12

30 95 83 74 97 81 91 84 72 83 86 0 1 4

NSPT00041 10 12 7 15 101 17 16 30 16 101 7 -3 1 4

30 17 6 16 99 28 37 49 35 105 50 -5 1 3

NSPT00043 10 88 65 106 99 32 62 37 17 100 79 1 3 3

30 97 82 77 97 49 78 60 47 82 85 2 3 1 1

NSPT00044 10 32 21 76 94 24 23 48 27 106 30 7 5 -1

30 65 37 99 98 47 43 71 54 109 54 -1 1 -5

NSPT00045 10 7 3 23 86 5 2 1 4 94 5 -1 0 -4

30 14 5 39 94 0 5 7 7 105 1 1 -2 1 -4

NSPT00049 10 39 20 62 99 35 35 44 33 104 19 2 0 0

30 63 32 60 96 53 58 64 57 80 35 -2 -1 1

NSPT00052 10 15 4 31 74 -2 0 8 3 92 20 0 0 -4

30 32 19 58 87 8 7 24 15 94 23 -1 1 6

NSPT00053 10 -5 -6 -10 19 -9 -1 1 -10 -8 23 -6 -5 -4 4

30 8 5 5 53 -1 5 9 2 50 2 0 2 2

NSPT00054 10 42 23 69 83 17 1 1 26 19 102 26 1 1 -1

30 59 40 88 95 35 38 52 33 94 40 1 0 3

NSPT00055 10 14 5 19 92 8 1 1 27 1 1 90 7 -4 0 6

30 18 12 41 96 27 31 54 34 93 9 2 1 5

NSPT00057 10 56 27 85 85 16 14 25 14 102 34 -3 0 10

30 71 48 93 96 34 37 49 32 94 52 1 0 10

NSPT00060 10 80 51 97 93 52 51 58 43 102 50 1 0 3

30 87 76 106 99 78 80 82 70 100 75 1 0 1 1

NSPT00061 10 70 42 89 94 49 41 73 49 98 54 1 0 3

30 91 64 104 98 74 67 88 76 99 79 0 0 0

NSPT00062 10 48 28 87 82 56 47 78 48 98 38 -7 -3 -4

30 79 53 97 92 78 74 91 78 97 66 -6 1 -2

NSPT00063 10 3 0 9 94 1 1 1 1 28 18 96 3 0 1 0

30 8 0 21 98 30 37 53 42 97 3 -1 0 -10

NSPT00065 10 14 -23 -7 10 -8 -6 6 -6 32 -10 -2 -5 -4 30 31 -16 -8 34 6 19 23 8 52 -2 0 1 2

NSPT00066 10 59 32 89 82 14 16 24 13 103 34 2 -2 0

30 73 51 95 94 36 47 50 31 94 54 1 2 2

NSPT00067 10 16 8 26 90 10 1 1 25 7 94 6 2 0 7

30 32 12 56 98 25 34 49 32 96 14 -1 -1 4

NSPT00068 10 31 12 66 83 4 3 6 -1 60 12 0 -5 -1

30 61 35 88 92 21 25 31 15 80 38 -5 1 2

NSPT00069 10 56 28 79 93 30 26 55 32 103 32 -1 -1 6

30 68 45 90 99 55 55 78 61 95 50 -1 0 7

NSPT00071 10 66 40 94 87 32 33 22 14 97 42 -5 -4 -3

30 86 68 100 94 59 70 57 44 95 71 3 1 3

NSPT00077 10 81 53 102 96 27 40 28 20 103 74 1 -1 0

30 96 77 111 97 52 68 55 39 106 90 1 -1 4

NSPT00079 10 67 37 92 96 44 40 54 33 105 52 4 0 0

30 88 61 72 94 63 61 75 59 75 69 -1 1 -2

NSPT00081 10 -1 5 -3 9 -2 -2 0 0 42 0 2 -1 -2

30 6 17 10 18 -3 -2 -1 0 73 5 0 -1 -7

NSPT00084 10 35 21 70 80 29 35 38 19 104 24 1 -1 10

30 52 32 85 95 57 64 62 42 101 44 -1 1 5

NSPT00085 10 23 11 27 100 34 32 47 24 101 6 1 3 3

30 40 15 37 96 55 56 69 49 78 13 3 9 4

NSPT00087 10 -4 -23 5 24 4 6 13 4 69 1 1 -1 3

30 13 -18 4 51 9 27 16 10 87 2 3 -1 6

NSPT00090 10 5 0 1 1 95 8 9 20 8 99 3 1 -1 0

30 10 3 23 99 18 19 37 20 106 9 7 -1 -6

NSPT00091 10 24 12 37 86 8 14 17 7 93 11 6 -4 2

30 48 22 63 94 24 33 36 25 93 23 1 -3 0

NSPT00093 10 90 65 98 94 76 77 77 66 101 58 0 -2 1

30 82 78 101 101 89 93 90 85 93 74 -3 -1 4

NSPT00097 10 6 5 9 7 2 14 6 -1 26 4 0 0 3

30 4 1 7 13 1 21 5 0 44 0 2 3

NSPT00098 10 60 29 85 86 49 50 56 36 97 38 -8 -3 -5

30 82 59 97 93 72 76 81 67 94 66 3 3 0

NSPT00104 10 63 33 94 83 19 24 29 16 96 49 1 -2 -2

30 85 64 100 93 45 54 66 49 94 77 2 -1 5

NSPT00105 10 8 8 6 59 1 -2 4 4 25 4 1 0 2

30 12 12 11 82 6 13 8 4 49 6 2 0 0

NSPT00107 10 52 32 86 89 41 47 64 34 97 36 -3 -2 10

30 82 58 99 95 66 72 86 67 97 67 2 3 0

NSPT00108 10 55 30 89 88 24 17 44 22 104 44 -3 -1 4

30 75 52 104 95 45 48 62 48 100 67 -5 0 -9

NSPT0011 1 10 95 74 106 91 35 63 29 15 104 88 1 0 -2

30 87 81 100 100 63 91 47 34 102 92 0 2 1

NSPT001 14 10 24 14 50 95 19 16 44 22 101 14 2 -1 -2

30 43 22 75 102 44 39 66 45 104 31 1 0 1

NSPT00116 10 19 7 25 91 13 16 15 15 99 9 3 -1 5 30 32 18 52 95 22 27 32 26 106 14 -1 0 -4

NSPT001 17 10 5 1 5 9 3 -1 -2 1 52 1 1 -1 0

30 17 3 8 23 -1 -4 5 1 84 4 -3 -2 -5

NSPT00118 10 10 4 8 97 8 8 24 1 1 99 2 -1 -3 3

30 22 7 18 96 23 27 45 31 81 6 0 -3 0

NSPT001 19 10 4 4 -2 22 2 3 2 2 18 1 0 -2 1

30 10 5 6 42 3 5 8 4 39 3 1 -1 -1

NSPT00121 10 78 55 97 100 75 91 48 40 105 58 0 -1 0

30 92 75 73 96 83 92 71 65 79 74 1 -2 -1

NSPT00125 10 20 -14 54 85 2 7 7 0 86 1 -4 -4 -6

30 50 2 80 93 13 40 34 23 92 34 0 0 -1

NSPT00126 10 5 3 1 3 1 -7 -2 1 28 0 2 -4 2

30 2 3 -1 9 2 2 4 1 47 -1 1 -2 1

NSPT00131 10 -5 -4 -5 6 -6 -7 -7 -9 18 -7 -5 -5 -4

30 3 6 8 31 1 3 1 1 46 0 1 -1 -1

NSPT00134 10 18 7 43 91 27 31 51 28 97 8 -4 -3 -4

30 52 25 76 96 55 62 78 63 97 34 0 -3 -4

NSPT00135 10 80 45 92 93 49 43 74 56 104 59 -1 -1 -6

30 87 71 109 97 71 71 88 78 100 77 -3 -1 -9

NSPT00137 10 93 86 102 91 65 88 62 49 97 84 -7 -4 -5

30 96 94 103 96 80 91 84 78 95 95 0 0 1

NSPT00138 10 2 5 2 11 0 -4 5 2 32 0 0 -7 -1

30 3 8 4 31 2 4 3 3 55 -3 -1 -15 1

NSPT00140 10 94 86 104 92 59 78 67 43 98 85 -2 -4 -4

30 98 95 105 96 77 88 87 74 97 96 -1 1 0

NSPT00141 10 92 65 105 93 35 36 46 33 104 67 -2 0 1

30 85 79 103 100 61 69 72 58 95 77 -3 -1 3

NSPT00147 10 4 3 0 2 4 0 5 0 12 0 5 6 6

30 5 1 6 8 8 2 7 6 55 0 6 14 9

NSPT00149 10 67 44 88 96 80 94 98 87 102 38 2 2 1

30 87 67 102 97 95 100 99 96 105 60 1 6 4

NSPT00150 10 18 10 37 88 10 3 19 10 101 11 3 1 10

30 38 16 60 97 21 20 45 24 103 23 1 0 9

NSPT00151 10 13 4 9 51 7 9 8 6 71 2 4 1 9

30 17 4 1 1 72 10 16 13 16 65 5 -4 1 -1

NSPT00152 10 93 74 103 97 57 76 56 36 102 89 1 1 0

30 99 89 110 98 82 93 78 62 103 96 1 0 -1

NSPT00153 10 6 -1 6 14 -1 0 0 -1 27 1 2 0 -3

30 2 -4 1 1 25 -2 -3 1 -2 54 2 1 -1 -10

NSPT00154 10 5 2 1 39 1 1 4 2 17 1 7 -2 1

30 7 1 4 63 4 1 3 5 33 0 3 -1 1

NSPT00155 10 33 -25 -1 82 30 74 49 16 72 2 4 -10 3

30 42 -2 0 99 56 94 64 35 86 1 4 -5 1

NSPT00156 10 38 13 66 99 13 8 14 6 57 22 2 -2 12

30 53 21 80 103 24 20 30 15 78 37 2 -2 12

NSPT00157 10 5 3 3 30 4 11 7 5 12 2 2 -2 2

30 9 0 2 54 8 26 13 10 25 2 1 -2 5

NSPT00159 10 72 42 99 97 26 16 30 13 102 73 3 -1 15

30 86 65 104 102 52 40 56 32 104 93 4 0 4

NSPT00160 10 91 74 107 98 55 61 61 51 103 89 7 1 2 NSPT00160 30 97 88 76 95 73 77 79 72 76 87 0 0 4

NSPT00161 10 77 42 99 87 7 18 13 7 100 68 2 2 0

30 81 -1 108 95 3 40 18 10 104 87 35 4 -3

NSPT00162 10 10 2 33 26 3 1 3 1 63 14 4 0 -1

30 5 -1 14 29 3 0 5 3 77 7 3 -1 -7

NSPT00163 10 75 51 90 94 42 49 49 34 95 61 3 -1 -1

30 94 76 102 98 66 75 73 61 97 88 4 0 5

NSPT00165 10 98 76 108 93 46 61 45 26 103 81 4 -2 2

30 91 85 103 98 67 83 71 51 99 88 4 -1 10

NSPT00166 10 24 13 42 96 39 40 69 44 95 10 2 0 3

30 48 22 69 98 63 65 86 72 95 29 3 0 4

NSPT00167 10 3 1 2 68 1 -4 2 -2 36 0 0 -3 -3

30 11 6 11 84 9 10 17 6 61 3 5 0 3

NSPT00169 10 81 57 95 98 15 24 23 11 97 71 -1 1 7

30 97 79 104 98 30 48 44 26 96 95 2 0 3

NSPT00170 10 5 -2 3 62 88 94 94 85 26 -9 2 -4 0

30 7 5 5 82 90 91 94 92 49 0 2 2 1

NSPT00173 10 50 21 77 94 35 29 55 39 102 32 -4 -5 -10

30 75 43 91 94 60 59 76 70 98 57 3 -2 -3

NSPT00175 10 7 1 4 22 2 10 12 3 64 3 2 -2 1

30 18 3 5 48 10 28 23 12 86 5 2 -3 1

NSPT00176 10 40 18 72 81 15 9 12 6 99 27 -4 -5 -8

30 68 40 90 90 29 32 36 22 96 53 3 0 3

NSPT00177 10 5 4 7 76 5 9 5 3 46 4 -7 -1 2

30 12 7 17 90 10 28 14 6 74 11 2 -2 -1

NSPT00182 10 0 10 1 1 11 4 1 1 2 44 3 2 0 3

30 9 6 42 24 5 6 8 7 68 6 3 -1 1

NSPT00185 10 1 3 6 6 6 -1 4 3 33 2 9 1 5

30 4 4 9 6 5 -1 5 3 56 2 -5 0 2

NSPT00186 10 25 9 8 24 7 3 5 0 65 2 1 2 4

30 34 12 9 44 8 12 16 6 87 2 4 7 16

NSPT00187 10 6 6 7 63 3 3 2 6 57 4 4 1 2

30 8 7 8 79 5 -1 7 3 58 3 -1 2 6

NSPT00191 10 64 31 86 94 63 54 82 67 98 46 0 -2 1

30 85 56 102 96 86 81 92 86 102 74 2 -2 0

NSPT00219 10 0 -1 4 4 0 -4 1 -1 26 -1 2 1 5

30 2 0 -2 12 3 -1 3 0 52 -1 -1 3 8

NSPT00221 10 7 -3 3 1 4 0 4 0 11 -1 3 1 -2

30 -1 2 0 1 0 -1 1 2 31 1 1 5 4

NSPT00235 10 12 7 6 14 10 12 13 2 64 3 3 5 17

30 16 10 18 37 27 16 29 15 83 3 6 8 6

NSPT00258 10 20 3 12 56 17 23 24 12 85 6 4 0 5

30 34 2 19 81 30 53 46 23 98 7 7 1 3

NSPT00260 10 1 1 -4 8 -1 1 4 2 43 1 3 4 0

30 6 2 7 16 3 1 1 2 71 1 2 3 0

NSPT00263 10 85 60 98 97 71 74 83 76 100 83 -1 1 -1

30 95 84 106 97 87 91 93 91 103 94 -3 0 -2

NSPT00308 10 2 -2 4 5 -1 -6 -3 -4 39 1 -6 0 -6

30 5 4 16 32 4 1 5 2 65 6 -1 3 6 NSPT00314 10 26 6 45 89 16 21 31 11 99 18 -4 0 3

30 51 19 73 93 39 47 59 34 98 40 -1 8 5

NSPT00322 10 12 0 10 20 8 -1 12 5 73 7 0 -3 A

30 24 -3 9 47 1 1 10 17 11 91 5 5 -17 1

NSPT00323 10 3 -3 -1 33 -2 -7 0 1 39 1 -3 -2 -7

30 5 2 1 1 67 1 3 6 1 69 4 1 5 5

NSPT00334 10 4 2 -1 14 0 1 2 -1 37 0 1 0 -4

30 4 0 6 30 2 2 4 0 47 4 4 0 -3

NSPT00335 10 -1 -2 -3 5 17 2 24 2 -1 -4 1 4 2

30 0 -3 0 7 24 -1 33 2 -1 -3 0 3 -1

NSPT00336 10 2 2 2 7 43 2 41 -1 -7 -2 -4 3 4

30 -3 -3 5 6 62 12 59 2 2 -5 -9 6 20

NSPT00337 10 17 17 66 83 12 17 25 11 99 20 1 3 0

30 57 27 64 88 27 32 46 25 72 35 4 0 0

NSPT00341 10 -1 -2 -5 3 -6 -7 -8 -4 -2 -2 -4 58 -10

30 7 -3 2 16 3 4 6 2 9 3 0 76 -5

NSPT00347 10 2 -1 -3 7 -4 -4 -4 -5 55 0 -3 -9 -2

30 9 5 8 28 3 2 6 1 78 4 1 0 -2

NSPT00350 10 -5 -3 -4 9 -3 0 1 -1 63 -2 -3 1 -4

30 5 3 2 28 7 3 13 13 83 3 -1 3 -9

NSPT00377 10 77 52 97 78 14 34 5 3 100 74 -1 1 -1

30 94 76 107 93 27 65 14 10 105 89 0 2 0

[00397] Example 3:

[00398] HDAC inhibitors cytotoxicity in triple-negative breast cancer cell line

[00399] The triple negative (ERT, PR ^~ Her2/neif ) breast cancer cell line (MDA-MB- 231 ; ATCC #HTB-26) was grown in Leibovitz's L-15 medium containing 10% FBS. Cells (passage 18-19) were inoculated into 96-well plates in 100 at plating densities 20,000 or 10,000 cells/well. After cell inoculation, the plates were incubated at 37°C, 0% C0 ₂ , 100% air and 100 % relative humidity for 24 h prior to addition of experimental small molecule inhibitors. After 24 h, cells were fixed in situ with TCA to represent a measurement of the cell population at the time of small molecule addition (Tz).

[00400] Experimental small molecules were solubilized in DMSO and stored frozen prior to use. At the time of treatment, small molecules were diluted to 20 μΜ concentration with complete medium containing 50 μg/ml gentamicin. Aliquots of 100 μΐ of drug dilutions were added to the wells already containing 100 μΐ of medium, resulting in the final 10 μΜ concentration.

[00401] Following small molecule addition, the plates were incubated for an additional 48 hours (h) as above. The assay was terminated by the addition of cold 50% (w/v) TCA followed by incubation for 60 minutes at 4°C. The supernatant was discarded, and the plates were washed five times with tap water and air dried. Sulforhodamine B solution at 0.4 % (w/v) in 1 % acetic acid was added to each well, and plates were incubated for 20 minutes at room temperature.

[00402] After staining, unbound dye was removed by washing five times with 1% acetic acid and the plates were air dried. Bound stain was subsequently solubilized with 10 mM trizma base, and the absorbance was read on a plate reader at a wavelength of 515 nm. The background absorbance of multiwell plates was measured at 690 nm and subtracted from the measurement at 515 nm.

[00403] Using the absorbance measurements [time zero (Tz), control growth (C), and test growth in the presence of drug (Ti)], the percentage growth was calculated for each of the drug as:

[00404] [(Ti-Tz)/(C-Tz)] χ 100 for concentrations for which Ti > Tz

[00405] [(Ti-Tz)/Tz] ^χ 100 for concentrations for which Ti < Tz.

[00406] Out of 93 compounds identified, 7 HDAC inhibitors were identified with the most potent cell growth inhibition at the end of the 48h and 72h treatments as compared to that at the beginning (Day 0). The results are summarized in Figure 1 A-1 L.

[00407] Next, selected compounds were evaluated at seven concentration levels (100,

50, 20, 10, 5, 2, and 1 μΜ). Four response parameters: (1) Inhibitory 50% Concentration

(IC50); (2) Growth Inhibition of 50% (GI50); (3) Total Growth Inhibition (TGI); and (4)

50% Lethal Concentration (LC50) were calculated for each experimental compound (Figure

2 A through Figure 2N and Table V).

[00408] Table V. Response parameters of tested compounds N121 18.7 13.2 38.5 24.2 nd nd

N137 10.6 11.8 20.4 21.4 51.0 42.1

N140 9.0 7.9 16.9 19.6 nd 74.1

N152 16.2 16.5 43.8 30.1 nd 64.0

N155 35.0 30.5 54.8 55.1 nd nd

N160 44.5 43.6 91.6 82.7 143.8 122.3

[00409] Cell cycle analysis

[00410] The effects of HDAC inhibitors on cell cycle progression were analyzed by

Propidium Iodine (PI) flow cytometry at 48 and 72 hours. Both floating and trypsinized adherent cells were collected and combined for analysis. Cells were fixed in ice cold ethanol and stored at -20°C until stained for 30 min at 37°C with 3 μΜ propidium iodide in staining buffer containing 10 μg/mL ribonuclease A. As shown in Table VI, selected compounds induced G2/M cell cycle arrest, as evidenced by accumulation of cells in G2/M in the tested cell line.

[00411] Table VI. Triple-negative breast cancer cell line was treated with selected HDAC inhibitors at the concentration of 20 or 10 μΜ for 48 and 72 hours followed by Propidium Iodine staining. Cell cycle analysis was performed by flow cytometry for each experimental compound.

[00412] Measurement of apoptosis

[00413] Dual staining with Annexin V and Propidium Iodine allows discrimination between unaffected cells (Annexin V-negative and Pi-negative), early apoptotic cells (Annexin V-positive and Pi-negative), and late apoptotic cells (Annexin V-positive and PI- positive). Both floating and trypsinized adherent cells were stained with FITC Annexin V and PI for 15 min at room temperature. Flow cytometry analysis revealed that selected HDAC inhibitors decreased the viability of the cells and increased the populations of early and late apoptotic cells. The data are summarized in Table VII and Figure 3A through Figure 3N.

[00414] Table VII. Triple-negative breast cancer cell line was treated with selected HDAC inhibitors at the concentration of 20 or 10 μΜ for 48 and 72 hours followed by Annexin V/Propidium Iodine staining. Percentages of viable cells and cells undergoing early and late apoptosis were analyzed by flow cytometry for each experimental compound.

[00415] Example 4:

[00416] Several studies show that small molecules that inhibit epigenetic enzymes, including histone deacetylase inhibitors (HDACi), improve somatic cell reprogramming. Several HDACi representing pan-inhibitors, class I and class II specific, and highly selective inhibitors were screened to identify potential epi-drugs that could substitute reprogramming transcription factors and enhance reprogramming process. Human BJ fibroblasts were treated with HDACi for 6 days at the concentrations of 10 μΜ and 1 μΜ. The Oct4 induction was analyzed at mRNA by quantitative RT-PCR. Multiple compounds caused 2-3 -fold induction of Oct4 in the cells (Figure 4A through Figure 4K). These data demonstrate that the small molecules disclosed herein can increase expression of pluripotency genes. The small molecules disclosed herein can be used to reprogram an adult cell.

[00417] Example 5:

[00418] Histone deacetylase inhibitors affect gene expression, which results in reduction of cell growth and even cytotoxicity through alteration of apoptosis, cell cycle or intracellular protein degradation. Hematological malignancies have been treated using HDAC inhibitors in the clinic. HDAC inhibitors also have been shown to exert synergistic effects with Paclitaxel and potentiate the efficacy of standard chemotherapy.

[00419] The MDA-MB-231 breast cancer cell line is negative for Her2 receptors, estrogen and progesterone receptors (triple negative) and responded to known HDAC inhibitors such as SAHA (suberoylanilide hydroxamic acid, Vorinostat) and Trichostatin A (TSA). Vorinostat (Zolinza®) has been tested in Phase II clinical trials in patients with recurrent or persistent epithelial ovarian or primary peritoneal carcinoma (Modesitt S 2008) and received FDA approval in 2006 for CTCL (cutaneous T cell lymphoma).

[00420] Newly designed HDAC inhibitors were screened in a triple negative breast cancer cell line MDA-MB-231 in vitro.

[00421] Methods:

[00422] MDA-MB-231 cells (ATCC, HTB-26, passage # 13) were seeded at a density of 1000 cells per well in opaque plates. After 2 days, cells were replenished with fresh media and incubated with HDAC inhibitors (5 specific and 5 pan) at final concentrations of 0.0001 , 0.001, 0.01 , 0.1 , 1 , 5, 10 and 100 μΜ. HDAC inhibitors as 0.3 μΜ stock solutions (SAHA, 007, 040, 041, 043, 044, 049, 1 1 1 , 137, 162, 169) were prepared in complete cell culture media from a 10 mM stock solution in DMSO. TSA (Sigma T1952) was supplied as 5 mM stock in DMSO.

[00423] Controls for 100 % cell viability (0.3 % DMSO - culture media) and 100 % cell death (0.1 % Triton X 100) were incubated under the same conditions. Control HDAC inhibitors were SAHA (Vorinostat) and TSA prepared under the same conditions as the test HDACs. Cell viability was determined after 48 and 72 hours using a luminescent assay kit (Promega, Madison, WI, Cell Titer Glo, G 7572, lot 31511202). Data were processed and analyzed to obtain IC ₅ o values using Graph Pad Prizm version 5.00 for Windows, GraphPad Software, San Diego California USA, www.graphpad.com (Graph Pad 5 Prizm, Inc).

[00424] Statistical analysis for significance was determined by a two-tailed Student's

T-test. Each test was conducted using double plates of 3 wells each to achieve an N of 6.

[00425] Results:

[00426] All HDAC inhibitors showed increased cytotoxicity from 48 h to 72 h incubations (Table VIII). TSA reached IC ₅₀ values of 0.16±0.1 μΜ after 48 h and

0.02±0.001 μΜ after 72 h and represented the most potent HDACi at both time points. SAHA was significantly (pO.OOl) less potent than TSA with IC ₅ o values of 5.7±0.4 μΜ after 48 h and 2.3±0.4 μΜ after 72 h.

[00427] Table VIII: In vitro activities of HDACi for MDA-MB-231 cells after 48 and

72 h.

NSPT00169 15.2±3.1 P<0.001 1.35±0.2 Ns

NSPT00041 14.4±3.4 P<0.001 0.56±0.09 P<0.05

[00428] After 48 h HDAC inhibitors had cytotoxicity as measured by IC ₅ 0 (small numbers represent higher potency) in the following order:

[00429] TSA>40>7>SAHA> 162> 137> 1 1 1 >49=43>44> 169=41

[00430] After 72 h the order was as follows:

[00431 ] TS A> 162> 1 1 1 =41 =49=41 >44=43 =40>7= 137> 169>S AH A.

[00432] Test compound 40 the was most toxic HDAC inhibitor in the screening assay with 1.8±0.2 μΜ after 48 h and 0.88±0.2 μΜ after 72 h (p<0.05 compared to SAHA).

[00433] Test compound 162 was most toxic after 72 h with IC50 values of 0.33±0.1 μΜ (pO.01 vs 169; p<0.02 vs 7, p<0.006 vs 137, p<0.02 vs 43 and p<0.04 vs 41).

[00434] The increase of toxicity between 48 and 72 h was plotted for each HDACi.

(Figure 5A and Figure 5B). TSA had an 8 fold increase and SAHA a 2.5 fold increase. HDACi 40 had similar increase as SAHA with 2.3. Increases of cytotoxicity similar to TSA were measured for 137. Increases of > 20 fold in activity were found for three of the HDACi (1 1 1 , 49, and 25).

[00435] HDACi that were screened were generally more potent than SAHA in killing MDA-MB-231 breast cancer cells. Cytotoxicity changed between 48 and 72 hour incubations and increased up to 25 fold. HDACi are potent in killing MDA-MB-231 triple negative cancer cells in vitro with different activity profile.

[00436] Although specific embodiments have been illustrated and described herein, it will be appreciated by those of ordinary skill in the art that any arrangement that is calculated to achieve the same purpose may be substituted for the specific embodiments shown. This application is intended to cover any adaptations or variations that operate according to the principles of the disclosure as described. Therefore, it is intended that this disclosure be limited only by the claims and the equivalents thereof. The disclosures of patents, references and publications cited in the application are incorporated by reference herein.

I l l