Pyrimidine hydroxy amide compounds as histone deacetylase inhibitors

Provided herein are compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds to treat or prevent diseases or disorders associated with HDAC activity, particularly diseases or disorders that involve activity of HDAC1, HDAC2, and/or HDAC6. Also provided herein are methods for inhibiting migration of a neuroblastoma cell, inducing maturation of a neuroblastoma cell, and altering cell cycle progression of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof.

BACKGROUND

A biological target of recent interest is histone deacetylase (HDAC) (see, for example, a discussion of the use of inhibitors of histone deacetylases for the treatment of cancer: Marks et al.Nature Reviews Cancer2001, 7, 194; Johnstone et al.Nature Reviews Drug Discovery2002, 287). Post-translational modification of proteins through acetylation and deacetylation of lysine residues plays a critical role in regulating their cellular functions. HDACs are zinc hydrolases that modulate gene expression through deacetylation of the N-acetyl-lysine residues of histone proteins and other transcriptional regulators (Hassig et al.Curr. Opin. Chem. Biol.1997, 1, 300-308). HDACs participate in cellular pathways that control cell shape and differentiation, and an HDAC inhibitor has been shown to be effective in treating an otherwise recalcitrant cancer (Warrell et al.J. Natl. Cancer Inst.1998, 90, 1621-1625).

There remains a need for preparing structurally diverse HDAC inhibitors, particularly ones that are potent and/or selective inhibitors of particular classes of HDACs and individual HDACs.

SUMMARY OF THE INVENTION

Provided herein are compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds to treat or prevent diseases or disorders associated with HDAC activity, particularly diseases or disorders that involve any type of HDAC1, HDAC2, and/or HDAC6 expression. Diseases that involve HDAC1, HDAC2 and/or HDAC6 expression include, but are not limited to, various types of cancer, neurodegenerative diseases, and hemoglobinopathies, such as sickle-cell anemia and beta-thalassemia.

Thus, in one aspect, provided herein is a compound of Formula I:

or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the invention, provided herein is a compound of Formula II:

or a pharmaceutically acceptable salt thereof.

In a particular embodiment of the invention, provided herein is a compound of Formula III:

In another aspect, provided herein is a pharmaceutical composition comprising a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier.

In another aspect, provided herein is a method of inhibiting the activity of HDAC1, HDAC2, and/or HDAC6 in a subject comprising administering to the subject a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of selectively inhibiting the activity of each of HDAC1, HDAC2, and/or HDAC6 over other HDACs in a subject comprising administering to the subject a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the compound has a selectivity for each of HDAC1, HDAC2, and/or HDAC6 that is 5 to 1000 fold greater than for other HDACs. In other embodiments, the compound has a selectivity for each of HDAC1, HDAC2, and/or HDAC6 when tested in a HDAC enzyme assay, of about 5 to 1000 fold greater than for other HDACs.

In another aspect, provided herein is a method of treating a disease mediated by one or more HDACs in a subject comprising administering to the subject in need thereof a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or pharmaceutically acceptable salts thereof. In some embodiments, the disease is mediated by HDAC1 and/or HDAC2. In other embodiments, the disease is mediated by HDAC6. In other embodiments, the disease is mediated by HDAC1 and/or HDAC2 and/or HDAC6.

In another aspect, provided herein is a method of treating a disease in a subject comprising administering to the subject a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or a pharmaceutically acceptable salt thereof. In an embodiment, the disease is a hemoglobinopathy. In another embodiment, the disease is sickle-cell disease. In yet another embodiment, the disease is beta-thalassemia.

In a further embodiment, the disease is a neurodegenerative disease. The neurodegenerative disease can be selected from a group consisting of Alzheimer's disease, frontotemporal lobe dementia, progressive supranuclear palsy, corticobasal dementia, Parkinson's disease, Huntington's disease, amytrophic lateral sclerosis, Charcot-Marie-Tooth disease and peripheral neuropathy.

In a further embodiment, the disease is a cancer or a proliferation disease. The cancer can be selected from a group consisting of lung cancer, colon and rectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, glioma, glioblastoma, neuroblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia, lymphomas, myelomas, retinoblastoma, cervical cancer, melanoma and/or skin cancer, bladder cancer, uterine cancer, testicular cancer, esophageal cancer, and solid tumors. In another embodiment, the cancer is lung cancer, colon cancer, breast cancer, neuroblastoma, leukemia, or lymphomas. In still another embodiment, the cancer is non-small cell lung cancer (NSCLC) or small cell lung cancer. In another embodiment, the cancer is a hematologic cancer. In a further embodiment, the hematologic cancer is a leukemia or lymphoma. The lymphoma can be Hodgkin's or Non Hodgkin's lymphoma.

Provided in some embodiments are methods for inhibiting migration of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing maturation of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for altering cell cycle progression of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for decreasing viability and survival of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing differentiation of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for enhancing low-concentration ATRA treatment of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing cell cycle arrest of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for treating neuroblastoma in a subject comprising administering to the subject a therapeutically effective amount of Compound 001, Compound X, or Compound Y.

In a further embodiment of the methods of treatment described herein, the subject to be treated is a human.

Other objects, features, and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention.

DETAILED DESCRIPTION

The instant application is directed, generally, to compounds, pharmaceutical compositions comprising such compounds, and methods of using such compounds to treat or prevent diseases or disorders associated with HDAC activity, particularly diseases or disorders that involve any type of HDAC1, HDAC2, or HDAC6 expression. Such diseases include, but are not limited to, cancer, neurodegenerative disease, sickle-cell anemia, and beta-thalassemia.

Inhibition of HDAC1 and HDAC2 has been shown to derepress fetal globin. Fetal hemoglobin (HbF) derepression, or induction, is an established therapeutic strategy in sickle cell disease, and could also be effective in treating beta-thalassemia. Hydroxyurea is currently the only drug with proven efficacy in sickle cell disease (SCD). This therapy is cytotoxic, poorly tolerated, and only reduces the frequency and severity of sickle cell crises in a subset of patients. There are no approved drugs for the treatment of beta-thalassemia. Fetal (γ) globin expression is silenced in adults partly through the action of a complex containing BCL11A and HDACs 1 and 2. Genetic ablation and chemical inhibition of HDAC1 or HDAC2 results in the derepression of γ globin in adult bone marrow derived erythroid cells (Bradner,Proc Natl Acad Sci2010). While a variety of non-specific HDAC inhibitors have been used successfully to induce HbF, further clinical development has been limited by their variable efficacy and concerns over off target side-effects observed in small clinical trials. Therefore, development of selective and potent HDAC1 and HDAC2 inhibitors leading to HbF reactivation represents a refined and more targeted therapeutic approach for the treatment of SCD and beta-thalassemia.

It has also been shown that HDAC2 expression and activity are elevated in neurodegenerative diseases (Guan, 2009; Morris, 2013). Increasing the expression of HDAC2 impairs cognitive function in mice. Inhibition of HDAC2 by gene disruption restores cognitive function in mouse models of Alzheimer's disease (Guan, 2009; Morris, 2013; Graff, 2014). In addition, the activity of HDAC6 is implicated in neurodegenerative diseases (Xiong, 2013; Simoes-Peres, 2013; Kim, 2012). Combined inhibition of HDAC2 and HDAC6 could have a more profound effect on the development of neurodegenerative diseases than inhibition of either enzyme alone.

It has also been shown that deregulated HDAC1 expression is particularly common in advanced cancers of the gastrointestinal system, such as, for example, pancreatic, colorectal, and liver (hepatocellular) carcinomas, as well as in prostate and breast cancer. HDAC2 and HDAC3 expression are also associated with advanced stage disease and poor prognosis in gastric, prostate and colorectal cancers. HDAC2 is also over expressed in cervical cancer. Clinical trials for the treatment of patients with advanced solid tumors, lymphomas, and leukemias utilizing class I selective HDAC inhibitors such as MS275, depsipeptide, and MGCD0103 have been published (O. Witt et al., Cancer Letters, 2009, 277, 8-21 and H-J. Kim and S.-C. Bae, Am. J. Transl. Res. 2011; 3(2): 166-179). HDACs have also been found to repress HIV-1 (Human Immunodeficiency Virus) transcription through deacetylation events, particularly in latently infected resting CD4+ T cells.

As such, it is known that HDAC inhibitors can induce the transcriptional activation of the HIV-1 promoter, or re-activate latent HIV-1 from the patient viral reservoir. It is generally accepted that the use of HDAC inhibitors in the treatment of HIV infection can be valuable in purging the latently infected reservoirs in patients, particularly patients undergoing Highly Active Antiretroviral Therapy (HAAT).

The compounds described herein have HDAC1 IC50values ranging from 1 to 2000 nM and HDAC2 IC50values ranging from 10 to 3000 nM, demonstrating approximately 2- to 100-fold selectivity over HDAC3, respectively.

The compounds described herein have HDAC6 IC50values ranging from 1 to 20 nM, demonstrating approximately 5- to 1000-fold selectivity than for other HDACs.

DEFINITIONS

The number of carbon atoms in an alkyl substituent can be indicated by the prefix “Cx-y,” where x is the minimum and y is the maximum number of carbon atoms in the substituent. Likewise, a Cxchain means an alkyl chain containing x carbon atoms.

The term “about” generally indicates a possible variation of no more than 10%, 5%, or 1% of a value. For example, “about 25 mg/kg” will generally indicate, in its broadest sense, a value of 22.5-27.5 mg/kg, i.e., 25±2.5 mg/kg.

The term “alkenyl” denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to eight carbon atoms having at least one carbon-carbon double bond. The double bond may or may not be the point of attachment to another group. Alkenyl groups include, but are not limited to, for example, ethenyl, propenyl, butenyl, 1-methyl-2-buten-1-yl, heptenyl, octenyl and the like.

The term “alkynyl” denotes a monovalent group derived from a hydrocarbon moiety containing, in certain embodiments, from two to six, or two to eight carbon atoms having at least one carbon-carbon triple bond. The alkynyl group may or may not be the point of attachment to another group. Representative alkynyl groups include, but are not limited to, for example, ethynyl, 1-propynyl, 1-butynyl, heptynyl, octynyl and the like.

The term “alkoxy” refers to an —O-alkyl moiety.

The term “aryl” refers to a mono- or poly-cyclic carbocyclic ring system having one or more aromatic rings, fused or non-fused, including, but not limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, idenyl, and the like. In some embodiments, aryl groups have 6 carbon atoms. In some embodiments, aryl groups have from six to ten carbon atoms. In some embodiments, aryl groups have from six to sixteen carbon atoms.

The term “cycloalkyl” denotes a monovalent group derived from a monocyclic or polycyclic saturated or partially unsaturated carbocyclic ring compound. Examples of C3-8-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentyl and cyclooctyl; and examples of C3-12-cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl. Also contemplated are monovalent groups derived from a monocyclic or polycyclic carbocyclic ring compound having at least one carbon-carbon double bond by the removal of a single hydrogen atom. Examples of such groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like.

The term “heteroaryl” refers to a mono- or poly-cyclic (e.g., bi-, or tri-cyclic or more) fused or non-fused moiety or ring system having at least one aromatic ring, where one or more of the ring-forming atoms is a heteroatom such as oxygen, sulfur, or nitrogen. In some embodiments, the heteroaryl group has from one to six carbon atoms, and in further embodiments from one to fifteen carbon atoms. In some embodiments, the heteroaryl group contains five to sixteen ring atoms of which one ring atom is selected from oxygen, sulfur, and nitrogen; zero, one, two, or three ring atoms are additional heteroatoms independently selected from oxygen, sulfur, and nitrogen; and the remaining ring atoms are carbon. Heteroaryl includes, but is not limited to, pyridinyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiazolyl, thiadiazolyl, oxadiazolyl, thiophenyl, furanyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzooxazolyl, quinoxalinyl, acridinyl, and the like.

The term “heterocycloalkyl” refers to a non-aromatic 3-, 4-, 5-, 6- or 7-membered ring or a bi- or tri-cyclic group fused of non-fused system, where (i) each ring contains between one and three heteroatoms independently selected from oxygen, sulfur, and nitrogen, (ii) each 5-membered ring has 0 to 1 double bonds and each 6-membered ring has 0 to 2 double bonds, (iii) the nitrogen and sulfur heteroatoms may optionally be oxidized, (iv) the nitrogen heteroatom may optionally be quaternized, and (iv) any of the above rings may be fused to a benzene ring. Representative heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

In accordance with the invention, any of the aryls, substituted aryls, heteroaryls and substituted heteroaryls described herein, can be any aromatic group.

The terms “halo” and “halogen” refer to an atom selected from fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” refers to alkyl radicals wherein any one or more of the alkyl carbon atoms is substituted with halo as defined above. Haloalkyl also embraces monohaloalkyl, dihaloalkyl, and polyhaloalkyl radicals. Examples of haloalkyl include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl, and pentafluoroethyl.

The term “HDAC” refers to histone deacetylases, which are enzymes that remove the acetyl groups from the lysine residues in core histones, thus leading to the formation of a condensed and transcriptionally silenced chromatin. There are currently 18 known histone deacetylases, which are classified into four groups. Class I HDACs, which include HDAC1, HDAC2, HDAC3, and HDAC8, are related to the yeast RPD3 gene. Class II HDACs, which include HDAC4, HDAC5, HDAC6, HDAC7, HDAC9, and HDAC10, are related to the yeast Hda1 gene. Class III HDACs, which are also known as the sirtuins are related to the Sir2 gene and include SIRT1-7. Class IV HDACs, which contains only HDAC11, has features of both Class I and II HDACs. The term “HDAC” refers to any one or more of the 18 known histone deacetylases, unless otherwise specified.

The term “inhibitor” is synonymous with the term antagonist.

The terms “isolated,” “purified,” or “biologically pure” refer to material that is substantially or essentially free from components that normally accompany it as found in its native state. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Particularly, in embodiments the compound is at least 85% pure, more preferably at least 90% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

The term “subject” refers to a mammal. A subject therefore refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, and the like. Preferably the subject is a human. When the subject is a human, the subject may be referred to herein as a patient.

The terms “treat”, “treating” and “treatment” refer to a method of alleviating or abating a disease and/or its attendant symptoms.

Compounds of the Invention

or a pharmaceutically acceptable salt thereof,
wherein,

Rxis selected from the group consisting of C1-6-alkyl, C1-6-alkoxy, halo, —OH, —C(O)R1, —CO2R1, —C(O)N(R1)2, aryl, —C(S)N(R1)2, and S(O)2R1, wherein aryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl;

Rzis selected from the group consisting of C1-6-alkyl, C1-6-alkenyl, C1-6-alkynyl, C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, and heteroaryl, each of which may be optionally substituted by C1-6-alkyl, C1-6-alkoxy, halo, or —OH; and

In an embodiment of the compound of Formula I or a pharmaceutically acceptable salt thereof,

Rzis selected from the group consisting of C1-6-alkyl, C1-6-alkenyl, C1-6-alkynyl, C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, and heteroaryl, each of which may be optionally substituted by C1-6-alkyl, C1-6-alkoxy, halo, or —OH; and

In one embodiment of the compound of Formula I, provided herein is a compound of Formula II:

or a pharmaceutically acceptable salt thereof,

Rxis independently selected from the group consisting of aryl, —C(O)R1, —CO2R1, —C(O)N(R1)2, —C(S)N(R1)2, and S(O)2R1;

Ryis selected from the group consisting of H, C1-6-alkyl, and halo; and

Rzis selected from the group consisting of C1-6-alkyl, C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, and heteroaryl.

In one embodiment of the compound of Formula II, or a pharmaceutically acceptable salt thereof, Rxis independently selected from the group consisting of —C(O)R1, —CO2R1, and —C(O)N(R1)2; and Rzis selected from the group consisting of C1-6-alkyl, C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, and heteroaryl.

In an embodiment of the compounds of Formula I or II, Rzis C1-6-alkyl or aryl. In preferred embodiments of the compounds of Formula I or II, Rzis isopropyl or phenyl. In another embodiment of the compounds of Formula I or II, Rzis methyl.

In another embodiment of the compounds of Formula I or II, Rxis —C(O)N(R1)2or —C(O)NHR1. In yet another embodiment of the compounds of Formula I or II, Rxis —C(O)R1or —CO2R1. In yet another embodiment of the compounds of Formula I or II, Rxis —C(S)N(R1)2, —C(S)NHR1, or S(O)2R1.

In an embodiment of the compounds of Formula I or II, at least one of R1is selected from the group consisting of C1-6-alkyl, aryl, C1-6-alkyl-aryl and C1-6-alkyl-heteroaryl, wherein aryl, C1-6-alkyl-aryl and C1-6-alkyl-heteroaryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl. In a further embodiment, R1is —CH3, —CH2CH3, phenyl, —CH2-phenyl, or —CH2-indolyl, wherein phenyl, —CH2-phenyl, or —CH2-indolyl may be optionally substituted by one or more groups selected from C1-6-alkyl and halo.

In another embodiment of the compounds of Formula I or II, at least one of R1is, independently for each occurrence, selected from the group consisting of C1-6-alkyl, aryl, and C1-6-alkyl-aryl. In a further embodiment, at least one of R1may be —CH3, —CH2CH3, —CH2-phenyl, or phenyl.

In another embodiment of the compounds of Formulas I or II, at least one of R1is phenyl, wherein phenyl is optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, halo, and haloalkyl. In preferred embodiments, at least one of R1is phenyl, wherein phenyl is optionally substituted by one or more groups selected from CH3, —OCH3, fluoro, chloro, and CF3.

In yet another preferred embodiment of the compounds of Formula I or II, Ryis H.

In another embodiment of the compounds of Formula I or II, Rxis —C(O)R1; and

R1is C1-6-alkyl, C1-6-alkyl-aryl or C1-6-alkyl-heteroaryl, wherein C1-6-alkyl-aryl or C1-6-alkyl-heteroaryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl. In a preferred embodiment, R1is CH2-phenyl or CH2-indolyl, wherein CH2-phenyl or CH2-indolyl may be optionally substituted by one or more groups selected from C1-6-alkyl and halo.

In another embodiment of the compound of Formula I, provided herein is a compound of Formula III:

or a pharmaceutically acceptable salt thereof,

Rxis selected from the group consisting of aryl, —C(O)R1, —CO2R1, —C(O)N(R1)2, —C(S)N(R1)2, and S(O)2R1, wherein aryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl; and

In an embodiment of the compounds of Formula III, Rxis —C(O)NHR1, —C(S)NHR1, or S(O)2R1; and

R1is, independently for each occurrence, selected from the group consisting of C1-6-alkyl, C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, heteroaryl, C1-6-alkyl-cycloalkyl, C1-6-alkyl-heterocycloalkyl, C1-6-alkyl-aryl, and C1-6-alkyl-heteroaryl, wherein C3-8-cycloalkyl, C3-7-heterocycloalkyl, aryl, and heteroaryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl.

In another embodiment of the compounds of Formula III, at least one of R1is selected from the group consisting of C1-6-alkyl, aryl, heteroaryl, C1-6-alkyl-aryl, and C1-6-alkyl-heteroaryl, wherein aryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl.

In another embodiment of the compounds of Formula III, at least one of R1is aryl, wherein aryl is optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, halo, and haloalkyl.

In a preferred embodiment of the compounds of Formula III, at least one of R1is phenyl, wherein phenyl is optionally substituted by one or more groups selected from CH3, —OCH3, fluoro, chloro, and CF3.

In another embodiment of the compounds of Formula III, Rxis —C(O)R1; and

R1is C1-6-alkyl, C1-6-alkyl-aryl or C1-6-alkyl-heteroaryl, wherein C1-6-alkyl-aryl or C1-6-alkyl-heteroaryl may be optionally substituted by one or more groups selected from C1-6-alkyl, C1-6-alkoxy, —OH, halo, and haloalkyl. In a preferred embodiment, R1is CH2-phenyl or CH2-indole, wherein CH2-phenyl or CH2-indole may be optionally substituted by one or more groups selected from C1-6-alkyl or halo.

Representative compounds of Formulas I, II, and III include, but are not limited to the following compounds of Table 1:

In preferred embodiments, the compounds of the instant invention have one or more of the following properties: the compound is capable of inhibiting at least one histone deacetylase (HDAC); the compound is capable of inhibiting HDAC1, HDAC2, and/or HDAC6; the compound selectively inhibits HDAC1, HDAC2 and/or HDAC6 over other HDACs.

Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) in the manufacture of a medicament for use in the treatment of a disorder or disease herein. Another object of the present invention is the use of a compound as described herein (e.g., of any formulae herein) for use in the treatment of a disorder or disease herein.

In another aspect, the invention provides a method of synthesizing a compound of Formula I, Formula II, or any of the compounds presented in Table 1. The synthesis of the compounds of the invention can be found in the Examples below.

Another embodiment is a method of making a compound of any of the formulae herein using any one, or combination of, reactions delineated herein. The method can include the use of one or more intermediates or chemical reagents delineated herein.

Another aspect is an isotopically labeled compound of any of the formulae delineated herein. Such compounds have one or more isotope atoms which may or may not be radioactive (e.g.,3H,2H,14C,13C,35S,32P,125I, and131I) introduced into the compound. Such compounds are useful for drug metabolism studies and diagnostics, as well as therapeutic applications.

A compound of the invention 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. Alternatively, a pharmaceutically acceptable base addition salt of a compound of the invention can be prepared by reacting the free acid form of the compound with a pharmaceutically acceptable inorganic or organic base.

Alternatively, the salt forms of the compounds of the invention can be prepared using salts of the starting materials or intermediates.

The free acid or free base forms of the compounds of the invention can be prepared from the corresponding base addition salt or acid addition salt from, respectively. For example a compound of the invention in an acid addition salt form can be converted to the corresponding free base by treating with a suitable base (e.g., ammonium hydroxide solution, sodium hydroxide, and the like). A compound of the invention in a base addition salt form can be converted to the corresponding free acid by treating with a suitable acid (e.g., hydrochloric acid, etc.).

Protected derivatives of the compounds of the invention can be made by means known to those of ordinary skill in the art. A detailed description of techniques applicable to the creation of protecting groups and their removal can be found in T. W. Greene, “Protecting Groups in Organic Chemistry,” 3rd edition, John Wiley and Sons, Inc., 1999, and subsequent editions thereof.

Compounds of the present invention can be conveniently prepared, or formed during the process of the invention, as solvates (e.g., hydrates). Hydrates of compounds of the present invention can be conveniently prepared by recrystallization from an aqueous/organic solvent mixture, using organic solvents such as dioxan, tetrahydrofuran or methanol.

In addition, some of the compounds of this invention have one or more double bonds, or one or more asymmetric centers. Such compounds can occur as racemates, racemic mixtures, single enantiomers, individual diastereomers, diastereomeric mixtures, and cis- or trans- or E- or Z-double isomeric forms, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids. All such isomeric forms of these compounds are expressly included in the present invention. Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques which are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., “Enantiomers, Racemates, and Resolutions” (John Wiley & Sons, 1981). The compounds of this invention may also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion. All such isomeric forms of such compounds are expressly included in the present invention. All crystal forms of the compounds described herein are expressly included in the present invention.

The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. As can be appreciated by the skilled artisan, further methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. In addition, the solvents, temperatures, reaction durations, etc. delineated herein are for purposes of illustration only and one of ordinary skill in the art will recognize that variation of the reaction conditions can produce the desired compounds of the present invention. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

The compounds of the invention are defined herein by their chemical structures and/or chemical names. Where a compound is referred to by both a chemical structure and a chemical name, and the chemical structure and chemical name conflict, the chemical structure is determinative of the compound's identity.

Pharmaceutical Compositions

The invention also provides for a pharmaceutical composition comprising a compound of instant invention, or a pharmaceutically acceptable salt thereof, together with a pharmaceutically acceptable carrier.

In another aspect, the invention provides a pharmaceutical composition comprising any of the compounds of the instant invention (Formula I, Formula II, Formula III, or any of the compounds presented in Table 1) or pharmaceutically acceptable salts thereof, together with a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound of the present invention formulated together with one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier” means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of this invention can be administered to humans and other animals orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, or drops), buccally, or as an oral or nasal spray.

Compounds of the invention can be administered as pharmaceutical compositions by any conventional route, in particular enterally, for example, orally, e.g., in the form of tablets or capsules, or parenterally, e.g., in the form of injectable solutions or suspensions, topically, e.g., in the form of lotions, gels, ointments or creams, or in a nasal or suppository form. Pharmaceutical compositions comprising a compound of the present invention in free form or in a pharmaceutically acceptable salt form in association with at least one pharmaceutically acceptable carrier or diluent can be manufactured in a conventional manner by mixing, granulating or coating methods. For example, oral compositions can be tablets or gelatin capsules comprising the active ingredient together with a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine; b) lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol; for tablets also c) binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and or polyvinylpyrrolidone; if desired d) disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and/or e) absorbents, colorants, flavors and sweeteners. Injectable compositions can be aqueous isotonic solutions or suspensions, and suppositories can be prepared from fatty emulsions or suspensions. The compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances. Suitable formulations for transdermal applications include an effective amount of a compound of the present invention with a carrier. A carrier can include absorbable pharmacologically acceptable solvents to assist passage through the skin of the host. For example, transdermal devices are in the form of a bandage comprising a backing member, a reservoir containing the compound optionally with carriers, optionally a rate controlling barrier to deliver the compound to the skin of the host at a controlled and predetermined rate over a prolonged period of time, and means to secure the device to the skin. Matrix transdermal formulations may also be used. Suitable formulations for topical application, e.g., to the skin and eyes, are preferably aqueous solutions, ointments, creams or gels well-known in the art. Such may contain solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives.

Methods of the Invention

According to the methods of treatment of the present invention, disorders are treated or prevented in a subject, such as a human or other animal, by administering to the subject a therapeutically effective amount of a compound of the invention, in such amounts and for such time as is necessary to achieve the desired result. The term “therapeutically effective amount” of a compound of the invention means a sufficient amount of the compound so as to decrease the symptoms of a disorder in a subject. As is well understood in the medical arts, a therapeutically effective amount of a compound of this invention will be at a reasonable benefit/risk ratio applicable to any medical treatment.

In general, compounds of the invention will be administered in therapeutically effective amounts via any of the usual and acceptable modes known in the art, either singly or in combination with one or more therapeutic agents. A therapeutically effective amount may vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the compound used and other factors. In general, satisfactory results are indicated to be obtained systemically at daily dosages of from about 0.03 to 2.5 mg/kg per body weight (0.05 to 4.5 mg/m2). An indicated daily dosage in the larger mammal, e.g. humans, is in the range from about 0.5 mg to about 100 mg, conveniently administered, e.g. in divided doses up to four times a day or in retard form. Suitable unit dosage forms for oral administration comprise from ca. 1 to 50 mg active ingredient.

In certain embodiments, a therapeutic amount or dose of the compounds of the present invention may range from about 0.1 mg/kg to about 500 mg/kg (about 0.18 mg/m2to about 900 mg/m2), alternatively from about 1 to about 50 mg/kg (about 1.8 to about 90 mg/m2). In general, treatment regimens according to the present invention comprise administration to a patient in need of such treatment from about 10 mg to about 1000 mg of the compound(s) of this invention per day in single or multiple doses. Therapeutic amounts or doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents.

Upon improvement of a subject's condition, a maintenance dose of a compound, composition or combination of this invention may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. The subject may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific inhibitory dose for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

In one aspect, the invention provides a method of selectively inhibiting the activity of each of HDAC1, HDAC2, and/or HDAC6 over other HDACs in a subject, comprising administering a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or pharmaceutically acceptable salts thereof.

In one embodiment, the compound has a selectivity for each of HDAC1, HDAC2 and HDAC6 of about 2 to 1000 fold greater than for other HDACs. In another embodiment, the compound has a selectivity for each of HDAC1, HDAC2, and or HDAC6 when tested in a HDAC enzyme assay of about 2 to 1000 fold greater than for other HDACs.

In another aspect, the invention provides a method of treating a disease mediated by an HDAC, specifically HDAC1, HDAC2, or HDAC6 in a subject comprising administering to the subject a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides a method of treating a disease mediated by one or more HDACs in a subject comprising administering to the subject in need thereof a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1, or pharmaceutically acceptable salts thereof.

Inhibition of HDAC1 and HDAC2 is sufficient to derepress fetal globin. In cultured human CD34+ bone marrow cells undergoing erythroid differentiation, these compounds induced a dose dependent increase in fetal hemoglobin expression, with a 2-fold induction observed at 1 μM and 5-fold induction observed at 10 μM. Cytotoxicity of these compounds was minimal, showing IC50values ranging from 1 to 5 μM. The selective HDAC1 and HDAC2 inhibitors of the present invention have favorable pharmacokinetic profiles. Thus, the compounds are capable of derepressing fetal globin through HDAC inhibition. In a preferred embodiment, the compounds are able to treat sickle-cell disease or beta-thalessemia. Further, the compounds are able to treat a subject suffering from or susceptible to a hemoglobinopathy.

Inhibition of HDAC, including inhibition of HDAC1 and HDAC2 by selective compounds, can induce the expression of genes associated with synapse formation and memory in cultured neurons. In addition, inhibition of HDAC2 by gene disruption can lead to the formation of new synapses and increase cognitive performance in mice. Inhibition of HDAC6 by selective molecules can reverse the effects of neurodegenerative transgenes in mice, including amyloid precursor protein and presenelin 1. The selective inhibitors of HDAC1, HDAC2 and HDAC6 of the present invention would be capable of enhancing synapse formation and reversing the effects of amyloid protein, thus lessening the symptoms of neurodegenerative diseases such as Alzheimer's disease by two complimentary mechanisms.

In another aspect, the invention provides a method of activating latent HIV in a subject comprising administering to the subject a compound of Formula I, Formula II, Formula III, or any of the compounds presented in Table 1. The same compounds can be used treat HIV infections. In another embodiment, the compounds can be used in combination with one or more anti-retroviral agents for the treatment of HIV infections. In an embodiment, the HIV infection is HIV-1.

Anti-retroviral agents that can be used in combination with the HDAC inhibitors of the instant invention include nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors, virus uptake/adsorption inhibitors, virus receptor antagonists, viral fusion inhibitors, viral integrase inhibitors, entry inhibitor, co-receptor antagonist, cyclin dependent kinase inhibitor, and transcription inhibitors or other anti-retroviral agents used in treatment of HIV infection. Preferred anti-retroviral agents include efavirenz, indinavir sulfate, and raltegravir potassium

As discussed above, the present invention provides compounds useful for the treatment of various diseases. In certain embodiments, the compounds of the present invention are useful as inhibitors of histone deacetylases (HDACs) and thus are useful as anti-cancer agents, and thus may be useful in the treatment of cancer, by effecting tumor cell death or inhibiting the growth of tumor cells. The compounds of the invention are capable of inducing apoptosis in cancer cells thereby able to treat a disease such as a cancer or proliferation disease.

In further embodiments, the cancer is a hematologic cancer, such as a leukemia or a lymphoma. In a certain embodiment, the lymphoma is Hodgkins lymphoma or Non Hodgkin's lymphoma. In certain embodiments, the inventive compounds are effective anticancer agents, which are active against leukemia cells and thus are useful for the treatment of leukemias, e.g., myeloid, lymphocytic, myelocytic and lymphoblastic leukemias.

In another aspect, the present invention provides for a method of treating a subject suffering from or susceptible to Hodgkins lymphoma or Non Hodgkin's lymphoma comprising administering to a subject in need thereof a therapeutically effective amount of a compound of the instant invention to thereby treat the subject suffering from or susceptible to Hodgkins lymphoma or Non Hodgkin's lymphoma.

In another embodiment, the chemotherapeutic agent is an aromatase inhibitor.

In an embodiment, the biological agent is rituximab, ipilimumab, bevacizumab, cetuximab, panitumumab, trastuzumab, or other monoclonal antibodies used for the treatment of cancer.

Methods delineated herein include those wherein the subject is identified as in need of a particular stated treatment. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

Also, as discussed above, the compounds of the invention are selective inhibitors of HDAC1, HDAC2, and/or HDAC6 and, as such, are useful in the treatment of disorders modulated by these histone deacetylases (HDACs). For example, compounds of the invention may be useful in the treatment of cancer (e.g., lung cancer, colon cancer, breast cancer, neuroblastoma, leukemia, or lymphomas, etc.). Accordingly, in yet another aspect, according to the methods of treatment of the present invention, tumor cells are killed, or their growth is inhibited by contacting said tumor cells with an inventive compound or composition, as described herein.

Thus, in another aspect of the invention, methods for the treatment of cancer are provided comprising administering a therapeutically effective amount of an inventive compound (i.e., of any of the formulae herein), as described herein, to a subject in need thereof. In certain embodiments, the subject is identified as in need of such treatment. In certain embodiments, a method for the treatment of cancer is provided comprising administering a therapeutically effective amount of an inventive compound, or a pharmaceutical composition comprising an inventive compound to a subject in need thereof, in such amounts and for such time as is necessary to achieve the desired result. In certain embodiments of the present invention a “therapeutically effective amount” of the inventive compound or pharmaceutical composition is that amount effective for killing or inhibiting the growth of tumor cells. The compounds and compositions, according to the method of the present invention, may be administered using any amount and any route of administration effective for killing or inhibiting the growth of tumor cells. Thus, the expression “amount effective to kill or inhibit the growth of tumor cells,” as used herein, refers to a sufficient amount of agent to kill or inhibit the growth of tumor cells. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the infection, the particular anticancer agent, its mode of administration, and the like.

In certain embodiments, the method involves the administration of a therapeutically effective amount of the compound or a pharmaceutically acceptable derivative thereof to a subject (including, but not limited to a human or animal) in need of it. In certain embodiments, the inventive compounds as useful for the treatment of cancer and other proliferative disorders including, but not limited to lung cancer (e.g. non-small cell lung cancer), colon and rectal cancer, breast cancer, prostate cancer, liver cancer, pancreatic cancer, brain cancer, kidney cancer, ovarian cancer, stomach cancer, skin cancer, bone cancer, gastric cancer, breast cancer, glioma, glioblastoma, neuroblastoma, hepatocellular carcinoma, papillary renal carcinoma, head and neck squamous cell carcinoma, leukemia (e.g., CML, AML, CLL, ALL), lymphomas (non-Hodgkin's and Hodgkin's), myelomas, retinoblastoma, cervical cancer, melanoma and/or skin cancer, bladder cancer, uterine cancer, testicular cancer, esophageal cancer, and solid tumors.

Provided in some embodiments are methods for inhibiting migration of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing maturation of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for altering cell cycle progression of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for decreasing viability and survival of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing differentiation of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for enhancing low-concentration ATRA treatment of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for inducing cell cycle arrest of a neuroblastoma cell comprising administering to the cell a therapeutically effective amount of a HDAC1, HDAC2, and/or HDAC6 selective inhibitor or a pharmaceutically acceptable salt thereof. The HDAC1, HDAC2, and/or HDAC6 selective inhibitor can be any compound selected from the group consisting of a compound of Formula I, Formula II, Formula III, any of the compounds presented in Table 1, Compound X, and Compound Y.

Provided in some embodiments are methods for treating neuroblastoma in a subject comprising administering to the subject a therapeutically effective amount of Compound 001, Compound X, or Compound Y.

In certain embodiments, the invention provides a method of treatment of any of the disorders described herein, wherein the subject is a human.

In accordance with the foregoing, the present invention further provides a method for preventing or treating any of the diseases or disorders described above in a subject in need of such treatment, which method comprises administering to said subject a therapeutically effective amount of a compound of the invention or a pharmaceutically acceptable salt thereof. For any of the above uses, the required dosage will vary depending on the mode of administration, the particular condition to be treated and the effect desired.

EXAMPLES

Examples have been set forth below for the purpose of illustration and to describe certain specific embodiments of the invention. However, the scope of the claims is not to be in any way limited by the examples set forth herein. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substitutents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the appended claims. Definitions of the variables in the structures in the schemes herein are commensurate with those of corresponding positions in the formulae presented herein.

To a solution of 1 (10.4 g, 56.5 mmol) and TEA (11.4 g, 113 mmol) in DCM (60 mL) was added dropwise CbzCl (10 g, 56.5 mmol) over 30 mins at 0° C. Then the mixture was stirred at room temperature (r.t.) for 6 hrs. H2O (50 ml) was added, the organic layer was washed with aqueous NaCl, dried by anhydrous Na2SO4, concentrated in vacuo and the residue was purified by silica gel chromatography (PE/EA=20:1) to afford compound 2 as a white solid (11.6 g, yield: 70%).

To a flask containing compound 3 (1.52 g, 13.1 mmol) and compound 2 (3 g, 10.9 mol) in DMF (25 ml) was added NaH (1.09 g, 27.2 mmol) at 0° C. It was stirred at 60° C. for 3 hrs. H2O was added, the resulting mixture was extracted with ethyl acetate (EA). The combined EA layers were concentrated in vacuo and the residue was purified by silica gel chromatography (PE/EA=2:1) to afford compound 4 as a yellow solid (1.9 g, yield: 54%).

To a mixture of compound 4 (1.89 g, 5.91 mmol) in DMSO (15 mL) was added K2CO3(2.4 g, 17.7 mmol), the mixture was stirred at 60° C. Then to the reaction 30% H2O2(17 ml, 177 mmol) was added dropwise. After the reaction was complete, H2O was added, and the reaction mixture was filtered. The resulting white solid was dried to afford compound 5 1.99 g, yield: 70%).

HBr/AcOH (6.0 mL) was added to a flask containing compound 7 (3.0 g, 6.52 mmol) at r.t. for 3 hrs. Then 12 ml Et2O was added, the reaction mixture filtered, the solid was dried to give compound 8 (1.85 g, yield: 70%) as a yellow solid.

To a solution of compound 8 (100 mg, 0.31 mmol) in DCM (4 mL) was added Ac2O (47 mg, 0.46 mmol), and Et3N (0.5 ml) at r.t. The reaction was stirred for 2 hrs and the reaction mixture was concentrated in vacuo to give compound 9 (120 g, yield: 100%).

To a solution of compound 8 (106 mg, 1.0 mmol), ethyl chloroformate (400 mg, 1.0 mmol) in 5 ml THF was added DIPEA (252 mg, 2.0 mmol). The mixture was stirred at r.t. for 4 hrs. LCMS monitored the reaction to completion. Upon completion, the reaction mixture was concentrated and the residue was purified by flash chromatography with PE/EA from 6:1 to 5:1 to give the target compound, compound 9 (320 mg, 82%).

To a solution of compound 1 (3 g, 14.28 mmol) in a 3-neck-flask flushed with N2was added lithium bis(trimethylsilyl)amide (LiHDMS) (1M, 21.4 ml) at −78° C. The reaction was stirred for 3 h at which time 2-iodopropane (3.6 g, 21.43 mmol) was added slowly. The reaction solution was stirred at −78° C., and then warmed to r.t. overnight. The mixture was quenched with H2O (2 ml), concentrated, dissolved in EA (200 ml), and washed with water (100 ml×2) and saturated NaCl (aq, 100 ml). The organic layer was concentrated to afford compound 2 as a brown solid (4 g, 100%).

To a solution of compound 2 (1 g, 3.97 mmol) in DMSO (30 ml) was added K2CO3(1.6 g, 11.9 mmol) stirred at 60° C. Over a period of 2 hrs, H2O2(30% aq., 5 ml) was added dropwise. TLC was used to monitor completion of the reaction. EA (100 ml) was added to the reaction mixture and subsequently washed with water (50 ml×2) and saturated NaCl (aq, 50 ml). The combined organic solutions were dried with anhydrous Na2SO4. The solvent was removed in vacuo to obtain compound 3 as a white solid (1 g, 90%).

To a solution of compound 3 (2.7 g, 10 mmol) in acetonitrile (AN) (50 ml) was added KOH (4N, aq., 50 ml) and 1,3-dibromo-5,5-dimethylhydantoin (DBDMH) (2.81 g, 5 mmol) at 0° C. The reaction was stirred at r.t. overnight. The mixture was concentrated and 1N HCl was added to adjust the pH to ˜6. The resulting mixture was extracted with EA (50 ml). The aqueous phase was then adjusted to pH˜9 by addition of KOH, and was subsequently extracted with EA (50 ml×3). The EA phase was dried with anhydrous Na2SO4and the solvent was concentrated to obtain compound 4 as a colorless liquid (1 g, 40%).

A solution of compound 4 (500 mg, 2.06 mmol) and ethyl 2-chloropyrimidine-5-carboxylate (384 mg, 2.06 mmol) in N-methyl-2-pyrrolidone (NMP) (10 ml) flushed with N2was stirred at 140° C. for 1 hour. EA (100 ml) was added to the reaction and resulting mixture was washed with water (50 ml×2) and saturated NaCl (aq, 50 ml). The resulting organic solution was concentrated and purified by silica gel chromatography column (PE/EA=5/1) to obtain compound 5 as a white solid (120 mg 15%).

To a solution of compound 5 (200 mg, 0.51 mmol) in DCM (5 ml) was added TFA (2 ml). The reaction was stirred at r.t. for 30 min. The mixture was concentrated to obtain compound 6 as a brown liquid (200 mg, 90%).

To a solution of compound 6 (200 mg, 0.709 mmol) in DCM was added Et3N (214 mg, 2.13 mmol) and acetyl chloride (56 mg, 0.709 mmol) at 0° C. The reaction stirred for 1 hour at which time the reaction mixture was concentrated to obtain the compound 7 as a brown liquid (220 mg, 95%)

To a solution of compound 6 (200 mg, 0.709 mmol) in DCM was added Et3N (214 mg, 2.13 mmol) and ethyl carbonochloridate (77 mg, 0.709 mmol) at 0° C. for 1 hour. The reaction mixture was concentrated to obtain compound 9 as a brown liquid (250 mg, 95%).

To a solution of compound 1 (10 g, 56.5 mmol) in DCM (50 mL) was added TEA (11.4 g, 113 mmol), followed by CbzCl (10.4 g, 56.5 mmol) while the system was in a water bath. The mixture was stirred for 3 hrs at r.t. Water (50 ml) was added to the reaction mixture and extracted with EA (150 ml×2). The organic phase was washed with saturated salt and dried over Na2SO4. Concentration and purification by silica gel column with EA/PE=1/20 yielded compound 2 (3 g, 18%) as an oil.

To a solution of compound 2 (100 g, 0.36 mol) and benzyl cyanide (59 g, 0.43 mol) in DMF (400 ml) was added NaH (37 g, 0.94 mol) at 0° C. After increasing the temperature to 60° C., the mixture was stirred at 60° C. overnight. TLC was used to monitor the reaction to completion. After cooling, water was added into the mixture resulting in a green solid. The target compound was purified by flash chromatography with PE/EA from 30:1 to 2:1 to yield compound 3 (38 g, 79%) as a white solid.

To a solution of compound 3 (38 g, 112 mmol) in 300 ml DMSO was added 30% H2O2(190 ml, 2248 mmol) slowly at 0° C. followed by stirring for 30 mins. Then the temperature was slowly increased to 40° C. and stirred for an additional 30 mins. After increasing the temperature to 60° C., the mixture was stirred at 60° C. overnight. TLC was used to monitor the reaction to completion. After cooling, water was added into the mixture to give a white solid, which was isolated by filtration (38 g, ˜95%).

To a solution of compound 4 (38 g, 106 mmol) in 400 ml BuOH was slowly added NaClO (64.2 ml, 149 mmol) followed by 3N NaOH (99 ml, 298 mmol) at 0° C. Then the mixture was stirred at r.t. overnight. TLC was used to monitor the reaction to completion. The mixture was concentrated and extracted with EtOAc. The organic layer was separated, washed and dried. Then the mixture was dissolved in Et2O, and the pH was adjusted to 2 using HCl/Dioxane. The precipitate was collected, yielding the target compound 5 (38 g 100%).

To a solution of compound 5 (9.6 g, 26 mmol), 2-Cl-pyrimidine (4.9 g, 26 mmol) in 150 ml 1,4-Dioxane was added DIPEA (7.7 g, 60 mmol). The mixture was stirred at 110° C. overnight. LCMS was used to monitor the reaction to completion. Water (50 ml) was added and the mixture was extracted with EtOAc. The combined organic extracts were washed and dried. The target compound 6 (11 g, 90%) was purified by flash chromatography with PE/EA from 30:1 to 2:1.

To a solution of compound 6 (1 g, 2.17 mmol) in MeOH (15 mL) was added Pd/C (0.1 g, 10% wq) under N2. The reaction was stirred under an H2atmosphere overnight, after which it was filtered through celite and washed with MeOH. Concentration yielded compound 7 (690 mg, 98%) as a light yellow solid.

To a mixture of compound 1 (50 mg, 0.15 mmol) and 1-fluoro-2-isocyanatobenzene (21 mg, 0.15 mmol) in THF (4 ml) was added DIPEA (39 mg, 0.30 mmol) at r.t. followed by stirring for 1 hour. The reaction mixture was concentrated and purified by gel chromatography (PE:EA=1:1) to afford compound 2 (60 mg, 86%) as a white solid.

To a mixture of compound 1 (60 mg, 0.18 mmol) and 1,2-dichloro-4-isocyanatobenzene (34 mg, 0.18 mmol) in THF (4 ml) was added DIPEA (46 mg, 0.36 mmol) at r.t. followed by stirring for 1 hour. The reaction mixture was concentrated and purified by gel chromatography (PE:EA=1:1) to afford compound 2 (60 mg, 70%) as a white solid.

To a mixture of compound 1 (81 mg, 0.2 mmol) and methylcarbamic chloride (19 mg, 0.2 mmol) in THF (4 ml) was added DIPEA (46 mg, 0.36 mmol) at r.t. followed by stirring for 1 hr. The reaction was concentrated and purified by gel chromatography (DCM:MeOH=10:1) to afford compound 2 (50 mg, 61%) as a white solid.

Step 1: A procedure analogous to step 2 in Example 9 afforded compound 2 (40 mg, 58%).

To a solution of compound 1 (55 mg, 0.14 mmol), 3-fluorobenzene sulfochloride (27 mg, 0.14 mmol) in 5 ml THF was added DIPEA (44 mg, 0.34 mmol). The mixture was stirred at r.t. for 2 h. LCMS was used to monitor the reaction to completion. The target compound (30 mg, 46%) was purified by flash chromatography with PE/EA (3:1).

To a solution of compound 1 (70 mg, 0.17 mmol) and 4-chlorobenzene sulfochloride (36 mg, 0.17 mmol) in 5 ml THF was added DIPEA (44 mg, 0.34 mmol). The mixture was stirred at r.t. for 2 h. LCMS was used to monitor the reaction to completion. The target, compound 2, (56 mg, 65%) was purified by flash chromatography with PE/EA (3:1).

To a solution of compound 1 (55 mg, 0.14 mmol) and 2-methylbenzene sulfochloride (26 mg, 0.14 mmol) in 5 ml THF was added DIPEA (44 mg, 0.34 mmol). The mixture was stirred at r.t. for 2 h. LCMS was used to monitor the reaction to completion. The target compound (40 mg, 62.5%) was purified by flash chromatography with PE/EA (3:1).

To a solution of compound 1 (55 mg, 0.14 mmol) and 4-methylbenzene sulfochloride (26 mg, 0.14 mmol) in 5 ml THF was added DIPEA (44 mg, 0.34 mmol). The mixture was stirred at r.t for 2 h. LCMS was used to monitor the reaction to completion. The target compound (48 mg, 75%) was purified by flash chromatography with PE/EA (3:1).

To a mixture of compound 1 (55 mg, 0.13 mmol) and 4-(trifluoromethyl)benzene-1-sulfonyl chloride (33 mg, 0.13 mmol) in THF (4 ml) was added DIPEA (31 mg, 0.24 mmol) at r.t. followed by stirring for 20 min. The reaction mixture was concentrated and purified by gel chromatography (PE:EA=2:1) to afford compound 2 (55 mg, 79%) as a yellow solid.

To a solution of compound 1 (80 mg, 0.25 mmol) and benzene sulfochloride (44 mg, 0.25 mmol) in 5 ml THF was added DIPEA (80 mg, 0.63 mmol). The mixture was stirred at r.t. for 3 h. LCMS was used to monitor the reaction to completion. The target compound (60 mg, 51%) was purified by flash chromatography with PE/EA (2:1).

To a mixture of compound 1 (60 mg, 0.18 mmol) and 1,3-difluoro-2-isocyanatobenzene (28 mg, 0.18 mmol) in THF (4 ml) was added DIPEA (46 mg, 0.36 mmol) at r.t. followed by stirring for 20 mins. The reaction was concentrated and purified by gel chromatography (PE:EA=2:1) to afford compound 2 (70 mg, 81%) as a yellow solid.

To a solution of compound 7 (200 mg, 0.50 mmol) and 3-methyl-2-phenylbutanoic acid (90 mg, 0.50 mmol) in 5 ml DMF was added HOAT (68 mg, 0.50 mmol), EDCI (78 mg, 050 mmol) and DIPEA (129 mg, 1 mmol). The mixture was stirred at 60° C. overnight and LCMS was used to monitor the reaction to completion. The racemic compound 8 (200 mg, 83%) was purified by filtration through silica gel after extraction by EA. Chiral-HPLC afforded R and S targets separately.

Lithium bis(trimethylsilyl)amide (1.0 M solution in THF, 240 mL, 240 mmol) was slowly added to a round-bottomed flask with compound 1 (25 g, 120 mmol) at −76° C. under N2. The reaction was stirred for 4 h at −76° C. Then iodomethane (15 ml, 240 mmol) was added into the system. The reaction mixture was stirred at −76° C. for 30 min and then warmed to room temperature overnight. The resulting mixture was quenched with 150 ml saturated aqueous NH4Cl, diluted with water, and extracted with EtOAc. The organic layers were washed with water and brine then dried over sodium sulfate, filtered and concentrated to afford the target compound 2 (25 g, 93%) as a white solid.

K2CO3(31 g, 223 mmol) was added to the solution of the compound 2 (25 g, 111 mmol) in DMSO (120 mL). Then H2O2(100 mL) was slowly added to the reaction dropwise at 60° C. The reaction was stirred overnight at 60° C. After completion, cold water was added and the mixture was extracted with EA. The organic layers were washed with water and brine, and dried over sodium sulfate, filtered and concentrated to afford the target, compound 3, (26 g, 96%) as a white solid.

Compound 3 (26 g, 107 mmol) was dissolved with CH3CN (200 mL) and 2N KOH (100 mL). Then 1,3-dibromo-5,5-dimethylimidazolidine-2,4-dione (15 g, 54 mmol) was added to the reaction and stirred overnight. Then the reaction pH was adjusted to 5 with 2N HCl and extracted with EA to remove the impurity. The aqueous phase was adjusted to a pH of 10. The precipitate was collected to afford the desired product as a white solid (16 g, 69%).

To a solution of compound 5 (150 mg, 0.41 mmol) in DCM (3 ml) was added TFA (3 ml) at r.t. The reaction was stirred for 30 min. and the resulting mixture was concentrated to afford compound 6 without further purification (108 mg, 100%).

To a solution of compound 6 (108 mg, 0.41 mmol) and (R)-2-phenylpropanoic acid (61.5 mg, 0.41 mmol) in 5 ml DCM was added 2 ml TEA. The mixture was stirred at r.t. for 2 h and LCMS was used to monitor the reaction to completion. The target compound 7 (100 mg, 62%) was purified by filtration through silica gel.

To a solution of compound 7 (100 mg, 0.25 mmol) and 2-(1H-indol-3-yl)acetic acid (44 mg, 0.25 mmol) in 5 ml DMF was added HOAT (68 mg, 0.50 mmol), EDCI (78 mg, 050 mmol) and DIPEA (129 mg, 1 mmol). The mixture was stirred at 60° C. for overnight. LCMS was used to monitor the reaction to completion. The target compound 8 (90 mg, 75%) as a yellow solid was obtained by filtration through silica gel after extraction by EA.

To a solution of 2-phenylacetic acid (250 mg, 1.84 mmol) in THF (3 mL) was added LHDMS (4 mL, 4 mmol) at 0° C. under N2. The reaction was stirred for 15 min, then 1-iodo-2-methylpropane (0.23 mL, 2.02 mmol) was added into the solution and stirred at r.t. overnight. After completion, water was added and the mixture was extracted with EA. The target compound 2 (335 mg, 95%) was obtained as a white solid following purification by a silica gel column.

To a solution of compound 7 (100 mg, 0.25 mmol) and 2-(1H-indol-2-yl)acetic acid (44 mg, 0.25 mmol) in 5 ml DMF was added HOAT (68 mg, 0.50 mmol), EDCI (78 mg, 0.50 mmol) and DIPEA (129 mg, 1 mmol). The mixture was stirred at 60° C. overnight, LCMS monitored the reaction to completion. The target compound 8 (80 mg, 766%) was obtained as a yellow solid following extraction with EA and filtration through silica gel.

HDAC Enzyme Assays

Compounds for testing were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 μM TCEP) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate and trypsin at 0.05 μM final concentration were diluted in assay buffer at 6 fold their final concentration. The final enzyme concentrations used in these assays were 3.3 ng/ml (HDAC1), 0.2 ng/ml (HDAC2), 0.08 ng/ml (HDAC3) and 2 ng/ml (HDAC6). The final substrate concentrations used were 16 μM (HDAC1), 10 μM (HDAC2), 17 μM (HDAC3) and 14 μM (HDAC6).

Five μl of compounds and 20 μl of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five μ1 of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated. The IC50was determined using Graph Pad Prism by a four parameter curve fit. The IC50values obtained for several of the compounds of this invention are found in Table 1.

Pharmacological Inhibition of Histone Deacetylase (HDAC) 1, 2 or 3 have Distinct Effects on Cellular Viability, Erythroid Differentiation, and Fetal Globin (HbG) Induction

In this example, the effects of selective inhibitors of HDAC1, 2, or 3, on cytoxicity, erythroid differentiation, and HbG induction in cultured human CD34+ bone marrow cells was investigated.

A prior compound, Compound A, is a class I HDAC inhibitor with IC50values of 5, 5, and 8 nM against HDAC1, 2, and 3, respectively (i.e., it is a non-selective HDAC inhibitor). Compound 001 is 30-fold selective for HDAC1 and 2, with IC50values of 38, 34, and 1010 nM against HDAC1, 2, and 3, respectively. Treatment of cells for 4 days with Compound A (1 μM) resulted in a 20-fold decrease in cells viability, while treatment with Compound 001 (1 μM) resulted in a minimal reduction in viability (1.2-fold) and a 2-fold increase in the percentage of HbG relative to other beta-like globin transcripts (seeFIG. 1). This result suggests that pharmacological inhibition of HDAC3 is cytotoxic and is consistent with the therapeutic rationale for the design of selective inhibitors of HDAC1 and 2.

Evaluation of Test Compounds on Human Erythroid, Myeloid and Megakaryocyte Hematopoietic Progenitor Proliferation in Media Formulations Containing Various Cytokines

This study evaluated the potential effect of test compounds on human erythroid, myeloid and megakaryocyte hematopoietic progenitor proliferation in media formulations containing various cytokines. Normal human bone marrow light density cells derived from a normal bone marrow donor (Lonza, Md.) were used for these studies. Clonogenic progenitors of human erythroid (CFU-E, BFU-E) and granulocyte-monocyte (CFU-GM) lineages were assessed in a semi-solid methylcellulose-based media formulation containing rhIL-3 (10 ng/mL), rhGM-SCF (10 ng/mL), rhSCF (50 ng/mL) and Epo (3 U/mL). Clonogenic progenitors of human megakaryocyte lineage were assessed in a semi-solid collagen based matrix containing rhIL-3 (10 ng/mL), rhIL-6 (10 ng/mL) and rhTpo (50 ng/mL).

Compounds were added to the medium to give the final desired concentrations. Solvent control cultures (containing no compound but 0.1% DMSO) as well as standard controls (containing no compound and no DMSO) were also initiated for both media formulations. Human myeloid and erythroid progenitor assays were initiated at 2.5×104cells per culture and human megakaryocyte progenitor assays were initiated with 1×105cells per culture. Following 14-16 days in culture, myeloid and erythroid colonies were assessed microscopically and scored by trained personnel. The colonies were divided into the following categories, based on size and morphology; CFU-E, BFU-E, and CFU-GM. For the human megakaryocyte assay, the cultures were transferred from the 35 mm dishes to labeled glass slides, were fixed (methanol/acetone) and then stained using an anti-human CD41 antibody and an alkaline phosphate detection system according to manufactures' instructions. The colonies were assessed and scored by trained personnel and divided into the following categories based on size; CFU-Mk (3-20), CFU-Mk (21-49) and CFU-Mk (≧50).

The mean±1 standard deviation of three replicate cultures was calculated for progenitors in both media formulations. To calculate the concentration of 50% inhibition of colony growth (IC50) for each compound, a dose response curve was generated plotting the log of the compound concentration versus the percentage of control colony growth using Origin® 8. A sigmoidal curve was then fit to the graph and from this curve the inhibitory concentration (μM) was then calculated using the Boltzman equation

y=[A1-A21+ⅇ(x-x0dx)]+A2
where A1=the initial value (baseline response), A2=0 (maximum response), xo=center (drug concentration that provokes a response halfway between A1and A2) and dx=slope of the curve at midpoint as determined by Origin® 8. Results are shown inFIGS. 2A-F.

This example demonstrates that Compound-001, an HDAC1,2-selective compound, has significantly less cytotoxicity against erythroid, myeloid and megakaryocytes than does MS-275, an HDAC1,2,3-selective compound. These results suggest that selective inhibition of HDAC1 and 2 using Compound-001 may result in significantly less in vivo cytotoxicity in the hematopoietic compartment than pan-HDAC inhibitors.

In Vitro Cell Proliferation

H929 human myeloma cells were seeded in 96-well plates and grown in the presence of increasing levels of Compound 001 for a period up to 7 days. Cellular viability was assessed using Aqueous One MTS reagent at Days 0 (immediately after seeding), 3, 5, and 7.FIG. 3Ashows dose-response curves for Compound 001 at Day 0, Day 3, Day 5, and Day 7, with the half-maximal dose (IC50) at each day indicated by a dashed line.FIG. 3Bshows the relative growth of H929 cells over time in the absence of drug as well as in the presence of increasing doses of Compound 001. The dashed line indicates the level of viability at Day 0, thus doses over 3 uM resulted in a net decrease in the viability of H929 cells.

Experimental Procedure
Step 1:

To a solution of compound 1 in DCE was added POBr3and imidazole. The reaction was stirred at 80° C. overnight. Water and DCM were added to the reaction, and the organic layer was separated, washed with brine, and dried under reduced pressure to give compound 2.

To a solution of compound 2 in DMSO was added compound a and KOH. The resulting reaction mixture was stirred at 45° C. for 4 h, quenched with H2O, and extracted with EA. The combined organic layers were purified by gel chromatography to yield the desired product, compound 3.

A mixture of compound 3, cyclopropyl boronic acid, Pd(OAc)2, tricyclohexylphosphine, and K3PO4in toluene and water was stirred at 100° C. under N2atmosphere overnight. The mixture was cooled, filtered, and concentrated to obtain a residue, which was purified by Prep-TLC to get compound 4.

A mixture of compound 4 and NaOH in EtOH and THF was stirred at 60° C. for 5 h. The mixture was concentrated to obtain a residue, to which was added aq. sat. citric acid and extracted with EA. The organic layers were separated, dried, filtered and concentrated to obtain compound 5.

A mixture of compound 5, tert-butyl 2-amino-4-(thiophen-2-yl)phenylcarbamate, HOAT, EDCI, and DIPEA in DMF was stirred at 55° C. for overnight. Water was added to the mixture, and extracted with EA. The organic layers were separated, dried, filtered, and concentrated to get a residue, which was purified by Prep-TLC to afford compound 6.

Table 2 below shows the IC50(nM) of Compound X for HDACs 1, 2, and 3.

HDAC1/2/6 Selective Inhibitor Blocks Neuroblastoma Migration

Many cultured cancer cells are able to migrate across a membrane and this activity indicates the metastatic potential of the cancer cells. The migration of neuroblastoma cell line SK-N-SH was compared in the presence or absence of Compound 001, a HDAC1/2/6 inhibitor. The cancer cells were seeded and grown on a membrane surface, and the cell numbers on the other side of the membrane were counted under a microscope after 12 hours. A decreased number of migrated cells by the HDAC1/2/6 inhibitor suggests a migration suppression activity of HDAC1/2/6 inhibitor. In the study, an HDAC inhibitor was added to the cells either 2 hours before or when the migration was measured. The effect of HDAC1/2/6 inhibitor on Epidermal Growth Factor (EGF) stimulated cancer cell migration using 40 ng/ml of EGF in the assay was investigated.

The protocol for the migration assay was as follows. The compounds were prepared in DMSO at 400× stock of the final required concentrations. See Table 3.

The results of these experiments are shown inFIGS. 4A-C.

The following is a quick summary of the HDAC neuroblastoma maturation experiments. Adherent cells were plated into 12- or 24-well plates at 106cells/well and allowed to adhere for 2-4 hours. Compounds were dispensed in DMSO at the indicated concentrations using an automated liquid handling system (Tecan D300) and incubated for the indicated times at 37° C. with 5% CO2. Cells were harvested and RNA extracted using the Qiagen RNeasy Mini Kit according to manufacture protocols. The RNA was quantified and relative expression levels were assessed using the Applied Biosystems TaqMan RNA-to-Ct Kit according to manufacture protocols using the indicated TaqMan probes.

The protocol for the maturation experiments was as follows. The day before the experiment, the cells were fed in the flask by doubling the media. The cells were then harvested by adding non-enzymatic cell dissociation media and incubating at 37° C. for 15 minutes. The cells were transferred to a 50 ml tube and pipet to create a single-cell suspension. The cells were washed with PBS buffer. The cells were then resuspended in complete media at 5×105cells per ml. Next, 2 ml of cells were transferred to 24- or 12-well plates. The treatment compounds were then dispensed into each well using the D300 liquid handler. The cells were then incubated for the indicated time at 37° C. The cells were harvested by scraping the cells and transferring to a 2 ml tube. The cells were spun to form a pellet. Next, RNA was extracted using the Qiagen RNeasy Mini kit according to manufacture protocols. The RNA concentration was recorded. The relative RNA levels were assessed using the Applied Biosystems TaqMan RNA-to-Ct kit according to manufacture protocols using the indicated Taqman probes.

In one set of experiments, BE(2)-C neuroblastoma cells were treated for 4 days with Compound X at 0.5 μM (FIG. 5A), 1 μM (FIG. 5B), and 3 μM (FIG. 5C). Each of the experiments measured the fold change of various genes associated with maturation, such as TGM2, KCTD13, EGR1, JARID2, MAFF, p21, DUSP6, DDAH2, CRABP2, SLC29A1, KCTD12, ASCL1, and GATA3.FIG. 5Dshows the results of a positive control experiment in which BE(2)-C cells were treated for 4 days with 1 μM ATRA (all trans retinoic acid).FIG. 5Eshows the results of a negative control experiment in which BE(2)-C cells were treated with 1 μM of a HDAC6 selective inhibitor. The results of these experiments show that Compound X, a HDAC1/2 selective inhibitor, alters genes associated with maturation. The strongest effects were seen at 3 μM of compound.

In another set of experiments, SH-SY5Y neuroblastoma cells were treated for 72 hours with 1 μM ATRA (all trans retinoic acid) (FIG. 6A), a HDAC6 selective inhibitor (FIG. 6B), Compound X (FIG. 6C), and another HDAC6 selective inhibitor (FIG. 6D). Each of the experiments measured the fold change of various genes associated with maturation, such as HOXD4, ADD3, p21, DDAH2, IGBFP5, PPIF, GATA3, CHGA, and ASCL1. Table 4 below shows the IC50s in nM of the various compounds.

TABLE 4HDAC1HDAC2HDAC3HDAC6HDAC6i21232570112237another HDAC6i3354615Compound X636445—
The results of these experiments show that Compound X, a HDAC1/2 selective inhibitor, alters genes associated with maturation.

In yet another set of experiments, BE(2)-C neuroblastoma cells were treated for 72 hours with 1 μM ATRA (all trans retinoic acid) (FIG. 7A), a HDAC6 selective inhibitor (FIG. 7B), Compound X (FIG. 7C), and another HDAC6 selective inhibitor (FIG. 7D). Each of the experiments measured the fold change of various genes associated with maturation, such as HOXD4, ADD3, p21, DDAH2, IGBFP5, PPIF, GATA3, CHGA, and ASCL1. Table 5 below shows the IC50s in nM of the various compounds.

TABLE 5HDAC1HDAC2HDAC3HDAC6HDAC6i21232570112237another HDAC6i3354615Compound X636445—
The results of these experiments show that Compound X, a HDAC1/2 selective inhibitor, alters genes associated with maturation.

In another set of experiments, the fold change of genes associated with maturation were assessed. In one experiment, BE(2)-C neuroblastoma cells were treated for 2 days with 3 μM Compound 001 (FIG. 8A). In a second experiment, SH-SY5Y neuroblastoma cells were treated for 2 days with 3 μM Compound 001 (FIG. 8B). In a third experiment, BE(2)-C neuroblastoma cells were treated for 2 days with 3 μM Compound X (FIG. 8C). Each of the experiments measured the fold change of various genes associated with maturation, such as p21, CRABP2, JARID2, KCTD13, TGM2, ASCL1, and GATA3. The results of these experiments show that Compound 001, a HDAC1/2/6 selective inhibitor, induces gene expression changes that are consistent with maturation.

In a set of experiments, BE(2)-C neuroblastoma cells were treated for 48 hours with Compound 001 at 0.5 μM (FIG. 9A), 2 μM (FIG. 9B), and 4 μM (FIG. 9C).FIG. 9Dshows the results of a positive control experiment in which BE(2)-C cells were treated for 48 hours with 1 μM ATRA (all trans retinoic acid). Each of the experiments measured the fold change of various genes associated with maturation, such as VGF, TGM2, SYT11, RBP1, MAFF, JARID2, PTK2, HOXD4, EGR1, DUSP6, DDAH2, CRABP2, ADD3, SLC29A1, PPIF, KCTD12, IGFBP5, GATA3, CHGA, and ASCL1. The results of these experiments show that Compound 001 induces gene expression changes consistent with maturation at 4 μM, but not at 2 μM or less. HDAC2glo assay data suggest maximal HDAC2 inhibition was reached at 3-4 μM.

A set of experiments shows that a HDAC3 selective inhibitor fails to modulate genes associated with maturation. BE(2)-C neuroblastoma cells were treated for 4 days with a HDAC3 selective inhibitor at 1 μM (FIG. 10A), 0.5 μM (FIG. 10B), and 3 μM (FIG. 10C). Each of the experiments measured the fold change of various genes associated with maturation, such as EGR1, CRABP2, DUSP6, p21, DDAH2, ASCL1, GATA3, IGFBP5, KCTD12, and SLC29A1.FIG. 10Dshows the results of a positive control experiment in which BE(2)-C neuroblastoma cells were treated for 4 days with 1 μM ATRA (all trans retinoic acid).FIG. 10Eshows the results of a negative control experiment in which BE(2)-C neuroblastoma cells were treated for 4 days with 1 μM of a HDAC6 selective inhibitor. The results of these experiments show that a HDAC3 selective inhibitor did not alter gene expression in a manner consistent with neuroblastoma maturation. In addition, the dose response was modest, if present at all.

A set of experiments shows that a HDAC6 selective inhibitor fails to modulate genes associated with maturation. BE(2)-C neuroblastoma cells were treated for 48 hours with HDAC6 selective inhibitor at 0.5 μM (FIG. 11A), 2 μM (FIG. 11B), and 4 μM (FIG. 11C).FIG. 11Dshows the results of a positive control experiment in which BE(2)-C neuroblastoma cells were treated for 48 hours with 1 μM ATRA (all trans retinoic acid). Each of the experiments measured the fold change of various genes associated with maturation, such as VGF, TGM2, SYT11, RBP1, MAFF, JARID2, PTK2, HOXD4, EGR1, DUSP6, DDAH2, CRABP2, ADD3, SLC29A1, PPIF, KCTD12, IGFBP5, GATA3, CHGA, and ASCL1. The results of these experiments show that a HDAC6 selective inhibitor failed to robustly induce gene changes consistent with maturation, even at 4 μM of exposure. These results are consistent with a previous experiment where maturation was not evident after 1 μM of treatment.

HDAC1/2 Inhibition Induces Increased Sub-G1 Cell Populations at a Concentration where Maturation is Occurring

The following is a quick summary of the neuroblastoma cell cycle experiments. Adherent cells were plated into 12-well plates at 106cells/well and allowed to adhere for 2-4 hours. Compounds were dispensed in DMSO at the indicated concentrations using an automated liquid handling system (Tecan D300) and incubated for the indicated times at 37° C. with 5% CO2. Cells were harvested with enzyme-free cell disassociation solution and washed with buffered saline. Cells were fixed overnight with 100% ethanol. Cell cycle was assessed by flow cytometry using the Molecular Probes FxCycle PI/RNase Staining Solution kit according to manufacture protocols.

The protocol for the cell cycle experiments was as follows. The day before the experiment, the cells were fed in the flask by doubling the media. Then, the cells were harvested by adding non-enzymatic cell dissociation media and incubating at 37° C. for 15 minutes. Next, the cells were transferred to a 50 ml tube and pipet to create a single-cell suspension. The cells were then washed with PBS buffer. Then, the cells were resuspended in complete media at 5×105cells per ml. Next, 2 ml of cells were transferred to 12-well plates. Then, the treatment compounds were dispensed into each well using the D300 liquid handler. The cells were incubated for the indicated time at 37° C. Then, the cells were harvested by adding 500 μl enzyme free cell dissociation solution and incubating at 37° C. for 15 minutes. Next, the cells were spun into a pellet and then washed with PBS. Then, 500 μl 100% EtOH was added and incubated at 4° C. overnight. Then, the cells were washed 3× with PBS. The cells were then resuspended in 500 ml FxCycle PI/RNase solution, and incubated for 2-4 hours at room temperature. Finally, the cells were assayed by flow cytometry.

This set of experiments shows that selective HDAC inhibition alters cell cycle progression in neuroblastoma cells. In a first experiment, SH-SY5Y neuroblastoma cells were treated for 72 hours with 0, 0.5, 2, and 5 μM of a HDAC6 selective inhibitor (FIG. 12A). In a second experiment, SH-SY5Y neuroblastoma cells were treated for 72 hours with 0, 0.5, 2, and 5 μM Compound X (FIG. 12B). In a third experiment, SH-SY5Y neuroblastoma cells were treated for 72 hours with 0, 0.5, 2, and 5 μM Compound 001 (FIG. 12C). In a control experiment, SH-SY5Y neuroblastoma cells were treated for 72 hours with 0 and 1 μM ATRA (all trans retinoic acid) (FIG. 12D). Each of the experiments looked at the percent of the cell population in the G2 phase, S phase, G1 phase, and Sub G1 phase. The results of this experiment show that Compound 001 induced a reduction in G1/G2 and increase in sub-G1 at concentrations where maturation was observed. Also, Compound X induced similar cell cycle changes in all treatment groups, even at low doses associated with sub-optimal maturation. In addition, a HDAC6 selective inhibitor induced a dose-dependent decrease in G1/G2 with a corresponding increase in sub-G1. Finally, ATRA had little impact on cell cycle at concentrations associated with robust maturation at this time point.

HDAC Inhibition Decreases Neuroblastoma Viability and Survival

SK-N-BE(2) or SH-SY5Y neuroblastoma cells were treated with varying concentrations of either Compound X or Compound Y. Viability and the Caspase 3/7 Signal were measured at 48 hours. The percentage of the population of the cells at various stages of the cell cycle were measured at 96 hours. SeeFIGS. 13A-DandFIGS. 14A-D. The results of these experiments show that low levels of apoptosis and cell death were detected at 48 hours after HDACi, the time when gene expression changes associated with differentiation were observed. An increase in the sub-G1 population became evident at 96 hours after treatment, indicating cell death at later times.

HDAC Inhibition Drives Neuroblastoma Differentiation

SK-N-BE(2) or SH-SY5Y neuroblastoma cells were treated with varying concentrations of either Compound X or Y, and/or ATRA (all trans retinoic acid). The differentiation index was measured. SeeFIGS. 15A-D. The results of these experiments show that both Compound X and Compound Y induced an increase in the differentiation index, and the effect was markedly enhanced when an HDACi was combined with retinoic acid.

SK-N-BE(2) or SH-SY5Y neuroblastoma cells were treated with varying concentrations of either Compound X or Y, and/or ATRA (all trans retinoic acid). The differentiation index was measured. As a control, SK-N-BE(2) or SH-SY5Y neuroblastoma cells were treated with varying concentrations of ATRA. SeeFIGS. 16A-C. The results of these experiments show that ATRA differentiation was sub-optimal at 0.25 μM, and both Compound X and Compound Y potentiated 0.25 μM ATRA.

HDAC Inhibition Induce Cell Cycle Arrest in Neuroblastoma Cells

SK-N-BE(2) neuroblastoma cells were treated with varying concentrations of either Compound X or Y, and/or ATRA (all trans retinoic acid). The percentage of the population of the cells at various stages of the cell cycle were measured after 4 days. In addition, the fold change of p21 was also calculated. SeeFIGS. 17A-D. Both Compound X and Compound Y induced cell cycle arrest, with Compound Y being the more potent agent. The HDACi/ATRA combination effects were modest, with little difference compared to single agents.

HDAC Inhibition Induce Cell Cycle Arrest in Neuroblastoma Cells

SH-SY5Y neuroblastoma cells were treated with varying concentrations of either Compound X or Y, and/or ATRA (all trans retinoic acid). The percentage of the population of the cells at various stages of the cell cycle were measured after 4 days. In addition, the fold change of p21 was also calculated. SeeFIGS. 18A-D. Both Compound X and Compound Y induced cell cycle arrest, with Compound Y being the more potent agent. The HDACi/ATRA combination effects were modest, with little difference compared to single agents.

Synthesis of Intermediate 2:

A mixture of aniline (3.7 g, 40 mmol), compound 1 (7.5 g, 40 mmol), and K2CO3(11 g, 80 mmol) in DMF (100 ml) was degassed and stirred at 120° C. under N2overnight. The reaction mixture was cooled to r.t. and diluted with EtOAc (200 ml), then washed with saturated brine (200 ml×3). The organic layers were separated and dried over Na2SO4, evaporated to dryness and purified by silica gel chromatography (petroleum ethers/EtOAc=10/1) to give the desired product as a white solid (6.2 g, 64%).

Synthesis of Intermediate 3:

Synthesis of Intermediate 4:

2N NaOH (200 ml) was added to a solution of compound 3 (3.0 g, 9.4 mmol) in EtOH (200 ml). The mixture was stirred at 60° C. for 30 min. After evaporation of the solvent, the solution was neutralized with 2N HCl to give a white precipitate. The suspension was extracted with EtOAc (2×200 ml), and the organic layers were separated, washed with water (2×100 ml), brine (2×100 ml), and dried over Na2SO4. Removal of the solvent gave a brown solid (2.5 g, 92%).

Synthesis of Intermediate 6:

A mixture of compound 4 (2.5 g, 8.58 mmol), compound 5 (2.52 g, 12.87 mmol), HATU (3.91 g, 10.30 mmol), and DIPEA (4.43 g, 34.32 mmol) was stirred at r.t. overnight. After the reaction mixture was filtered, the filtrate was evaporated to dryness and the residue was purified by silica gel chromatography (petroleum ethers/EtOAc=2/1) to give a brown solid (2 g, 54%).

A mixture of the compound 6 (2.0 g, 4.6 mmol), sodium hydroxide (2N, 20 mL) in MeOH (50 ml) and DCM (25 ml) was stirred at 0° C. for 10 min. Hydroxylamine (50%) (10 ml) was cooled to 0° C. and added to the mixture. The resulting mixture was stirred at r.t. for 20 min. After removal of the solvent, the mixture was neutralized with 1M HCl to give a white precipitate. The crude product was filtered and purified by pre-HPLC to give a white solid (950 mg, 48%).

Synthesis of Intermediate 2:

See synthesis of intermediate 2 in Example 45.

Synthesis of Intermediate 3:

Synthesis of Intermediate 4:

See synthesis of intermediate 4 in Example 45.

Synthesis of Intermediate 6: See synthesis of intermediate 6 in Example 45.

See synthesis of Compound A in Example 45.

INCORPORATION BY REFERENCE

The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

EQUIVALENTS