Selective histone deacetylase 6 inhibitors

Abstract

The present disclosure provides compounds of formula I: TIFF2023532047000033.tif53165 or a pharmaceutically acceptable salt thereof, wherein R 1 , R 2 , L 1 , L 2 , m, n, p, X, Y, and Z are as defined herein, including methods of increasing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy in a subject.

Description

[Technical field]

[0001] Statement of Government Interest This invention was made with Government support under Grant No. 5R01 NS079183 awarded by the National Institutes of Health. The United States Government has certain rights in this invention.

[0002] The present disclosure relates to histone deacetylase (HDAC) inhibitors, e.g., selective HDAC6 inhibitors, pharmaceutical compositions comprising one or more of said HDAC inhibitors, methods of increasing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy comprising contacting said cells with one or more of said HDAC inhibitors, and methods of treating conditions and diseases benefiting from inhibition of HDAC, e.g., cancer, inflammation, neurological diseases, neurodegenerative disorders, stroke, traumatic brain injury, allograft rejection, autoimmune diseases, and malaria, comprising administering a therapeutically effective amount of the HDAC inhibitor to an individual in need thereof. [Background technology]

[0003] Reversible acetylation of lysine side chains on the surface of enzymes and other proteins is controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Protein lysine acetylation or deacetylation serves as an important regulatory pathway for a variety of cellular processes, such as transcription, cell cycle, and cell metabolism (see, for example, Zhao et al., Science 327:1000-1004 (2010); Wang et al., Science 327:1004-1007 (2010); or Choudhary et al., Nat. Rev. Mol. Cell Biol. 15:536-550, (2014)). HDACs are considered effective targets for therapeutic intervention in cancer treatment, neurological diseases, and immune disorders (see, for example, Li et al., Cold Spring Harb. Perspect. Med. 6 (2016); Eckschlager et al., Int. J. Mol. Sci. 18: E1414 (2017); or Falkenberg et al., Nat. Rev. Drug Discov. 13: 673-691 (2014)). 2+ )-dependent HDACs (classes I, II, and IV) and seven nicotinamide adenine dinucleotide (NAD +)-dependent sirtuins (SIRTs) (class III) have been identified. Unlike other members, HDAC6 of the class IIb subgroup has the unique ability to deacetylate a variety of non-histone proteins as preferred substrates, such as α-tubulin, cortactin, HSP-90, and HSF-1 (see, e.g., Matthias et al., Cell Cycle 7:7-10 (2008), or Imai et al., Cancer Sci. 107:1543-1549 (2016)). Three HDAC6 inhibitors (HDAC6i), Ricolinostat (ACY-1215), Citarinostat (ACY-241), and KA2507, are being studied in clinical trials in a variety of cancer types, either as monotherapy or in combination (see, e.g., Shen et al., Expert Opin. Ther. Pat. 30:121-136 (2020)), and HDAC6i are being developed and emerging as a promising approach for cancer treatment. The immunomodulatory properties of HDAC6 have led to them being considered as novel therapeutic agents for cancer immunotherapy. HDAC6 has been reported to interact with the transcription factor STAT3, a key regulator of immune responses in the tumor microenvironment (see, e.g., Rebe et al., Cancers (Basel) 11:1280 (2019)), and to regulate STAT3-mediated gene expression (see, e.g., Cheng et al., J. Immunol. 193:2850-2862 (2014)). In antigen-presenting cells (APCs), such as macrophages and dendritic cells, selective inhibition of HDAC6 reduces the production of the immunosuppressive cytokine IL-10, thereby maintaining the proinflammatory state of APCs (see, e.g., Cheng et al., J. Immunol. 193:2850-2862 (2014)). In melanoma tumor cells, inhibition of HDAC6 leads to a decrease in the expression of the immunosuppressive molecule PD-L1 by affecting the recruitment and activation of STAT3 (see, e.g., Lienlaf et al., Mol. Oncol. 10:735-750 (2016)). Furthermore, using a syngeneic murine melanoma mouse model, it was demonstrated that combination therapy of selective HDAC6i with PD-1 antibody resulted in significantly improved effects on tumor growth compared to monotherapy (see, e.g., Knox et al., Sci. Rep. 9:6136 (2019)), thereby highlighting the immunomodulatory capabilities of selective HDAC6 inhibitors. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Zhao et al.,Science 327:1000-1004(2010) [Non-Patent Document 2] Wang et al.,Science 327:1004-1007(2010) [Non-Patent Document 3] Choudhary et al.,Nat.Rev.Mol.Cell Biol.15:536-550,(2014)

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Summary of the Invention

Means for Solving the Problems

[0005] The present disclosure relates to histone deacetylase inhibitors (HDACIs), pharmaceutical compositions comprising the HDACIs, and methods of treating diseases and conditions that benefit from inhibition of HDACs, such as cancer, neurological disorders, psychiatric disorders, neurodegenerative disorders, peripheral neuropathy, stroke, hypertension, inflammation, traumatic brain injury, rheumatoid arthritis, allograft rejection, sepsis, and autoimmune diseases, comprising administering a therapeutically effective amount of the HDACI to an individual in need of treatment. The present disclosure also relates to methods of increasing the sensitivity of cancer cells to radiation therapy and / or chemotherapy. The present disclosure also allows for the use of these HDAC inhibitors in combination with other drugs and / or therapies. In some embodiments, the present HDACIs exhibit selectivity for HDAC isozymes, such as HDAC6, over other HDAC isozymes. In certain embodiments, the present disclosure relates to phenylhydroxamic acids that selectively inhibit HDAC6. In other embodiments, the present disclosure relates to the use of the inhibitors in combination with anti-PD1 immunotherapy for the treatment of cancer.

[0006] In some embodiments, the present disclosure relates to histone deacetylase inhibitors (HDACIs) having structural formula I: [ka] In the formula, R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R 2 are linked to form a 3- to 7-membered heterocyclyl, L 1 is CO2H, C(O)NH2, C(O)NHOH, or B(OH)2, and L 2 is H or OR 3 and R 3is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl, each X is independently hydrogen or halogen, p is 0, 1, 2, or 3, Y and Z are independently selected from the group consisting of carbon and nitrogen, m is 1, 2, 3, or 4, and n is 0, 1, or 2. In another embodiment, R 1 , R 2 and R 3 is independently a C1-C6 branched alkyl. [Brief description of the drawings]

[0007] [Figure 1A] Immunoblot analysis of WM164 human melanoma cells treated overnight with increasing concentrations of Suprastat (6a) from 0.1 μM to 10 μM. Analysis was performed for Ac-α-tubulin and Ac-H3. Total α-tubulin and total H3 are loading controls. Immunoblots were repeated at least twice for data robustness and the best representation is shown.

[0008] [Figure 1B] Immunoblot analysis of WM164 human melanoma cells treated overnight with increasing concentrations of compound 6b from 0.1 μM to 10 μM. Analysis was performed for Ac-α-tubulin and Ac-H3. Total α-tubulin and total H3 are loading controls. Immunoblots were repeated at least twice for data robustness and the best representation is shown.

[0009] [Figure 1C] Immunoblot analysis of WM164 human melanoma cells treated overnight with increasing concentrations of compound 6c from 0.1 μM to 10 μM. Analysis was performed for Ac-α-tubulin and Ac-H3. Total α-tubulin and total H3 are loading controls. Immunoblots were repeated at least twice for data robustness and the best representation is shown.

[0010] [Figure 1D] Immunoblot analysis of WM164 human melanoma cells treated overnight with increasing concentrations of NextA from 0.1 μM to 10 μM. Analysis was performed for Ac-α-tubulin and Ac-H3. Total α-tubulin and total H3 are loading controls. Immunoblots were repeated at least twice for data robustness and the best representation is shown.

[0011] [Figure 1E] FIG. 1 is a bar graph showing densitometric analysis of Ac-α-tubulin bands with increasing concentrations of Suprastat (6a), compound 6b, compound 6c, and NextA from 0.1 μM to 10 μM.

[0012] [Figure 1F] FIG. 1 is a bar graph showing densitometric analysis of the Ac-H3 band with increasing concentrations of Suprastat (6a), compound 6b, compound 6c, and NextA from 0.1 μM to 10 μM.

[0013] [Diagram 2] FIG. 1 is a line graph showing a cytotoxicity assay with increasing concentrations of Suprastat (6a), compound 6b, compound 6c, and NextA.

[0014] [Figure 3A] FIG. 1 is a box plot graph showing gene expression of IL10 measured by quantitative PCR in bone marrow-derived macrophages exposed to interferon-gamma (20 ng / mL) and LPS (100 ng / mL) after treatment with Suprastat.

[0015] [Figure 3B] FIG. 1 shows immunoblot analysis of WM164 mouse melanoma cells pretreated with 5 μM Suprastat or NextA followed by treatment with IL-6 (30 ng / mL) for 20 minutes.

[0016] [Figure 4A] Immunoblot analysis of RAW264.7 macrophages treated with increasing concentrations of Suprastat. Band intensities quantified and expressed as fold change relative to vehicle show a dose-dependent effect of Suprastat on α-tubulin acetylation.

[0017] [Figure 4B] 1 is a bar graph of RAW264.7 macrophages treated with increasing concentrations of Suprastat.

[0018] [Figure 5A] FIG. 1 is a line graph showing cumulative tumor growth rates in C57BL / 6 mice (n=10) following treatment with Suprastat, anti-PD1 therapy, or a combination of both. Tumor growth rates of treatment groups were compared to a PBS-treated control group.

[0019] [Figure 5B] 4 is a set of four spider graphs showing tumor growth rates for individual mice following treatment with Suprastat, anti-PD1 therapy, or a combination of both.

[0020] [Figure 6A] 13 is a box plot graph showing Macrophage-1 cells stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0021] [Figure 6B] 1 is a box plot graph showing Macrophage 2 cells stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0022] [Figure 6C]1 is a box plot graph showing Macrophage 1 / Macrophage 2 cells stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0023] [Figure 6D] Box plot graphs showing T cells (CD8+, CD8 CM, and CD8 EM) stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0024] [Figure 6E] Box plot graphs showing T cells (CD4+, CD4 CM, and CD4 EM) stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0025] [Figure 6F] Graph showing T cells (Tregs) stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0026] [Figure 6G] 1 is a box plot graph showing NK cells stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both.

[0027] [Figure 6H] 1 is a box plot graph showing NKT cells stained with cell surface markers and analyzed by multicolor flow cytometry after treatment with Suprastat or anti-PD1 therapy or a combination of both. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure relates to novel HDAC inhibitors (HDACIs) and their use in the therapeutic treatment of, for example, cancer, inflammation, traumatic brain injury, neurodegenerative disorders, neurological diseases, peripheral neuropathy, stroke, hypertension, autoimmune diseases, inflammatory diseases, and malaria.The HDACIs also increase the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy.In some embodiments, the HDACIs selectively inhibit HDAC6 over other HDAC isozymes.

[0029] The present disclosure is described in relation to preferred embodiments. However, it should be understood that the present disclosure is not limited to the embodiments of the present disclosure. It should be understood that various modifications may be made by those skilled in the art based on the description of the embodiments of the present disclosure herein. Such modifications are encompassed within the scope of the following claims.

[0030] definition As used herein, the following terms and expressions have the meanings indicated.

[0031] Terms used herein may be preceded and / or followed by a single macron "-" or a double macron "=" to indicate the bond order of the bond between the designated substituent and its parent moiety. A single macron indicates a single bond and a double macron indicates a double bond. In the absence of a single or double macron, it is understood that a single bond is formed between the substituent and its parent moiety. Additionally, substituents are intended to be read "left to right" unless otherwise indicated by the macron. For example, C1-C6 alkoxycarbonyloxy and -OC(O)C1-C6 alkyl indicate the same functional group, and similarly, arylalkyl and -alkylaryl indicate the same functional group.

[0032] "Acetyl" means a group of the formula CH3C(O)-.

[0033] "Alkoxy" means an alkyl group, as defined herein, appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0034] "Alkyl" means a straight or branched chain hydrocarbon containing 1 to 10 carbon atoms, unless otherwise specified. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl" group is a linking group between two other moieties, it may be straight or branched. Examples include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2CHC(CH3)-, and -CH2CH(CH2CH3)CH2-.

[0035] "Aryl" refers to phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring, or an aromatic bicyclic ring system containing only carbon atoms. The bicyclic aryl can be azulenyl, naphthyl, etc. The aryl is bonded to the parent molecular moiety through any carbon atom contained within the aryl ring system. In certain embodiments, the aryl group is phenyl or naphthyl. In certain embodiments, the aryl group is phenyl.

[0036] As used herein, "cycloalkyl" refers to a monocyclic cycloalkyl ring system. A monocyclic ring system is a cyclic hydrocarbon group containing 3 to 6 carbon atoms, which may be saturated or unsaturated, but is not aromatic. In certain embodiments, a cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl.

[0037] "Halo" or "halogen" means -Cl, -Br, -I or -F.

[0038] "Haloalkyl" means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.

[0039] "Heteroaryl" refers to a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring. The monocyclic heteroaryl may be a 5-membered or 6-membered ring. The 5-membered ring consists of two double bonds and 1, 2, 3 or 4 nitrogen atoms, and optionally an oxygen or sulfur atom. The 6-membered ring consists of three double bonds and 1, 2, 3 or 4 nitrogen atoms. The 5-membered or 6-membered heteroaryl is connected to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. Representative examples of monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. The bicyclic heteroaryl is composed of a monocyclic heteroaryl fused to a phenyl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl. The fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups, which are independently oxo or thia. When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring, the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system. When the bicyclic heteroaryl is a monocyclic heteroaryl fused to a phenyl ring, the bicyclic heteroaryl group is connected to the parent molecular moiety through any carbon or nitrogen atom contained within the bicyclic ring system.Representative examples of bicyclic heteroaryls include benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzoxathiadiazolyl, benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, furopyridinyl, indazolyl, indolyl, isoquinolinyl, naphthyridinyl, quinolinyl, purinyl, 5,6,7,8-tetrahydroquinolinyl, quinolinyl, purinyl, quinolin ... quinolin-2-yl, 5,6,7,8-tetrahydroquinolin-3-yl, 5,6,7,8-tetrahydroquinolin-4-yl, 5,6,7,8-tetrahydroisoquinolin-1-yl, thienopyridinyl, 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl, and 6,7-dihydrobenzo[c][1,2,5]oxadiazol-4(5H)-onyl. In certain embodiments, the fused bicyclic heteroaryl is a 5- or 6-membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, and the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups that are independently oxo or thia. In certain embodiments of the present disclosure, the heteroaryl group is furyl, imidazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, thiazolyl, thienyl, triazolyl, benzimidazolyl, benzofuranyl, indazolyl, indolyl, quinolinyl, etc.

[0040] "Heterocyclyl" refers to a monocyclic 5- or 6-membered heterocyclic ring containing at least one N atom and optionally one or more additional heteroatoms independently selected from the group consisting of O, N, and S, where the ring is saturated or unsaturated, but not aromatic.Representative examples of monocyclic heterocycles include, but are not limited to, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, and thiopyranyl.In certain embodiments, the heterocyclyl is imidazolinyl, pyrrolidinyl, piperidinyl, or piperazinyl.

[0041] The present disclosure relates to novel HDACIs of formula I, Ib, and Ic, and their use in the therapeutic treatment of, for example, cancer, inflammation, traumatic brain injury, neurodegenerative disorders, neurological diseases, peripheral neuropathy, stroke, hypertension, autoimmune diseases, inflammatory diseases, and malaria.The HDACIs also increase the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy.In some embodiments, the HDACIs selectively inhibit HDAC6 over other HDAC isozymes.

[0042] The term "disease or condition benefiting from inhibition of HDAC" relates to a condition in which HDAC and / or the action of HDAC is important or necessary, for example, for the onset, progression, or manifestation of the disease or condition, or a disease or condition known to be treated with an HDAC inhibitor (e.g., TSA, pivalolyloxymethylbutane (AN-9, Pivanex), FK-228 (depsipeptide), PXD-101, NVP-LAQ824, SAHA, MS-275, and / or MGCD0103, etc.). Examples of such conditions include cancer, psoriasis, fibroproliferative disorders (e.g., hepatic fibrosis), smooth muscle proliferative disorders (e.g., atherosclerosis, restenosis), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic lateral sclerosis, spinocerebellar degeneration, Rett syndrome), peripheral neuropathies (Charcot-Marie-Tooth disease, giant axonal neuropathy (GAN)), inflammatory diseases (e.g., osteoarthritis, rheumatoid arthritis, colitis), diseases involving angiogenesis (e.g., osteoporosis, osteoarthritis, osteoporosis ... , cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia, thalassemia), fungal infections, parasitic infections (e.g., malaria, trypanosomiasis, helminthiasis, protozoan infections), bacterial infections, viral infections, and conditions treatable by immunomodulation (e.g., multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergies, asthma, allergic rhinitis, inflammatory bowel disease, and to improve graft survival). Those skilled in the art can readily determine whether a compound treats a disease or condition mediated by HDAC for any cell type, for example, by assays that can be conveniently used to evaluate the activity of the compound.

[0043] "The second treatment factor " refers to a treatment different from this HDACI. factor and a therapeutic agent known to treat the disease or condition of interest. factor For example, when cancer is the disease or condition of interest, the second treatment factor can be, for example, a known chemotherapy drug such as taxol, or radiation.

[0044] "HDAC" refers to a family of enzymes that remove acetyl groups from the ε-amino groups of lysine residues at the N-terminus of proteins, such as histones. The HDACs can be human HDACs, including HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11. The HDACs can also be obtained from protozoan or fungal sources.

[0045] "Treat", "treating", "treatment" and the like refer to eliminating, reducing, alleviating, reversing and / or ameliorating a disease or condition and / or its associated symptoms. Although not excluded, treating a disease or condition does not require that the disease, condition or its associated symptoms be completely eliminated, including treatment of acute or chronic signs, symptoms and / or disorders. As used herein, the terms "treat", "treating", "treatment" and the like may include "prophylactic treatment", which refers to reducing the likelihood of reoccurrence of a disease or condition, or the likelihood of a previously controlled disease or condition reoccurring, in a subject who does not have the disease or condition or has not reoccurred with the disease or condition but is at risk or susceptible to reoccurrence. Thus, "treatment" also includes recurrence prevention or stage prevention. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the present disclosure to an individual in need of such treatment. Treatment may also be performed symptomatically, for example to suppress symptoms. This may be in the short term, medium term or long term, for example as part of maintenance therapy.

[0046] The term "therapeutically effective amount" or "effective dose" refers to the amount of active ingredient(s) that is sufficient when administered to effectively deliver the active ingredient(s) to an individual in need of treatment for a condition or disease of interest. In the case of cancer or other proliferative disorders, the factorA therapeutically effective amount of may reduce (i.e., slow to some extent, preferably stop) unwanted cell proliferation, reduce the number of cancer cells, reduce the size of a tumor, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit (i.e., slow to some extent, preferably stop) tumor growth, reduce HDAC signaling in target cells, and / or alleviate to some extent one or more symptoms associated with the cancer. To the extent that the administered compound or composition prevents the proliferation and / or kills existing cancer cells, it may be cytostatic and / or cytotoxic.

[0047] "Co-administration," "administered in combination," "co-administration," and similar expressions refer to the administration of two or more factor "Simultaneously" means that each factor It means that the HDACIs are administered to an individual at the same time or at different times, sequentially in any order. However, if not administered simultaneously, it means that they are administered to an individual in a sequence close enough in time that they can act in concert to provide the desired therapeutic effect. For example, the present HDACIs may be administered to an individual in combination with a second therapeutic agent. factor The HDACI and the second therapy may be administered simultaneously or sequentially at different times and in any order. factor The HDACI and the second therapy may also be administered separately, in any suitable form, and by any suitable route. factor It is understood that when the are not administered simultaneously, they may be administered in any order to a subject in need thereof. For example, the subject HDACI may be administered in combination with a second treatment. factorThe HDACI and the second therapy may be administered to an individual in need thereof prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to), concurrently therewith, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of the first therapy (e.g., radiation therapy). factor are administered at intervals of 1 minute, 10 minutes, 30 minutes, less than 1 hour, 1 hour, 1-2 hours, 2-3 hours, 3-4 hours, 4-5 hours, 5-6 hours, 6-7 hours, 7-8 hours, 8-9 hours, 9-10 hours, 10-11 hours, 11-12 hours, 24 hours or less, or 48 hours or less. In one embodiment, the components of the combination therapy are administered at intervals of 1 minute to 24 hours.

[0048] In the context of describing this disclosure (particularly in the context of the claims), the use of the terms "a", "an", "the" and similar referents are to be construed to cover both the singular and the plural unless otherwise indicated. The recitation of ranges of values ​​herein merely serves as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value and subrange is incorporated herein as if individually recited herein. The use of any and all examples or exemplary language (e.g., "such as" and "such as") provided herein is intended to better illustrate the disclosure and does not limit the scope of the disclosure unless otherwise asserted. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0049] In some embodiments, the disclosure relates to HDACIs of Formula I, compositions comprising the HDACIs, and therapeutic uses of the HDACIs: [ka] In the formula, R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R 2 are linked to form a 3- to 7-membered heterocyclyl, L 1 is CO2H, C(O)NH2, C(O)NHOH, or B(OH)2, and L 2 is H or OR 3 and R 3 is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl, each X is independently hydrogen or halogen, p is 0, 1, 2, or 3, Y and Z are independently selected from the group consisting of carbon and nitrogen, m is 1, 2, 3, or 4, and n is 0, 1, or 2. In another embodiment, R 1 , R 2 and R 3 is independently a C1-C6 branched alkyl.

[0050] In certain embodiments, L 1 is C(O)NHOH.

[0051] In other embodiments, the disclosure relates to HDACIs of formula Ib, compositions comprising the HDACIs, and therapeutic uses of the HDACIs: [ka] In the formula, R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R 2 are linked to form a 3- to 7-membered heterocyclyl, L 2 is H or OR 3 and R 3is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl, each X is independently hydrogen or halogen, p is 0, 1, 2, or 3, Y and Z are selected from the group consisting of carbon and nitrogen, m is 1, 2, 3, or 4, and n is 0, 1, or 2. In another embodiment, R 1 , R 2 and R 3 is independently a C1-C6 branched alkyl.

[0052] In other embodiments, the disclosure relates to HDACIs of formula Ic, compositions comprising the HDACIs, and therapeutic uses of the HDACIs: [ka] In the formula, R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R 2 are linked to form a 3- to 7-membered heterocyclyl, L 2 is H or OR 3 and R 3 is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl, each X is independently hydrogen or halogen, p is 0, 1, 2, or 3, m is 1, 2, 3, or 4, and n is 0, 1, or 2. In another embodiment, R 1 , R 2 and R 3 is independently a C1-C6 branched alkyl.

[0053] In other embodiments, the disclosure relates to HDACIs of formula Id, compositions comprising the HDACIs, and therapeutic uses of the HDACIs: [ka] wherein each X is independently hydrogen or halogen; m is 1, 2, 3, or 4; p is 0, 1, 2, or 3; m is 1, 2, 3, or 4; and n is 0, 1, or 2.

[0054] In other embodiments, the disclosure relates to HDACIs of formula Ie, compositions comprising the HDACIs, and therapeutic uses of the HDACIs: [ka] wherein each X is independently hydrogen or halogen; p is 0, 1, 2, or 3; m is 1, 2, 3, or 4; and n is 0, 1, or 2.

[0055] In other embodiments, the present disclosure provides the following HDACIs: [ka] Compositions comprising these HDACIs and therapeutic uses of these HDACIs are also disclosed.

[0056] Additionally, salts, prodrugs, hydrates, isotopically labeled, fluorescently labeled, and any other therapeutically or diagnostically relevant derivatives of the present HDACI are also included in the present disclosure and may be used in the methods disclosed herein. The present disclosure further includes all possible stereoisomers and geometric isomers of the present compounds. The present disclosure includes both racemic and optically active isomers. When the present HDACI is desired as a single enantiomer, it may be obtained by resolution of the final product or by stereospecific synthesis from isomerically pure starting materials or by use of chiral auxiliary reagents. See, for example, Ma et al., Tetrahedron:Asymmetry 8:883-888 (1997). Resolution of the final product, intermediate, or starting material may be accomplished by any suitable method known in the art. Additionally, in situations where tautomers of the present compounds are possible, the present disclosure is intended to include all tautomeric forms of the compounds.

[0057] Prodrugs of the compounds are also included in the present disclosure. Prodrug methods, in which a compound is derivatized into a form suitable for formulation and / or administration and then released as a drug in vivo, are well established for the temporary (e.g., bioreversible) modification of the physicochemical properties of the compound (see, for example, Bundgaard, Ed., "Design of Prodrugs," Elsevier, Amsterdam, (1985); Silverman, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992); or Hillgren et al., "Prodrugs: A Guide to Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992)). (See, e.g., et al., Med. Res. Rev. 15:83 (1995)). Certain prodrugs of HDACIs are described in WO 2008 / 055068, which is incorporated herein by reference in its entirety.

[0058] The compounds of the present disclosure can exist as salts. The pharma- ceutically acceptable salts of the present HDACIs are often preferred for the method of the present disclosure. As used herein, the term "pharma- ceutically acceptable salts" refers to the salts or zwitterionic forms of the present compounds. The salts of the present compounds may be prepared during the final isolation and purification of the compounds, or may be prepared separately by reacting the compounds with an acid having a suitable cation. The pharma- ceutically acceptable salts of the present compounds may be acid addition salts formed by pharma- ceutical acceptable acids. Examples of acids that can be used to form pharma- ceutical acceptable salts include inorganic acids, such as nitric acid, boric acid, hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and organic acids, such as oxalic acid, maleic acid, succinic acid, tartaric acid, and citric acid. Non-limiting examples of salts of the compounds of the present disclosure include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethanesulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylene sulfate, and the like. The amino acid salts include, but are not limited to, phonates, naphthylenesulfonates, nicotinates, 2-naphthalenesulfonates, oxalates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, trichloroacetates, trifluoroacetates, phosphates, glutamates, bicarbonates, paratoluenesulfonates, undecanoates, lactates, citrates, tartrates, gluconates, methanesulfonates, ethanedisulfonates, benzenesulfonates, and p-toluenesulfonates. In addition, the available amino groups present in the compounds of the present disclosure may be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides, dimethyl, diethyl, dibutyl, and diamyl sulfates, decyl chlorides, bromides, and iodides, lauryl, myristyl, and stearyl, and benzyl and phenethyl bromides.In view of the above, any reference to a compound of the present disclosure appearing herein is intended to include the compound and its pharma- ceutically acceptable salts, hydrates, or prodrugs.

[0059] The compounds may also be conjugated or linked to auxiliary moieties that promote the beneficial properties of the compounds in the method of therapeutic use. Such conjugates may facilitate the delivery of the compounds to the anatomical site or region of interest (e.g., tumors), allow sustained therapeutic concentrations of the compounds in target cells, modify the pharmacokinetic and pharmacodynamic properties of the compounds, and / or improve the therapeutic index or safety profile of the compounds. Suitable auxiliary moieties include, for example, amino acids, oligopeptides, or polypeptides, for example, antibodies, such as monoclonal antibodies and other engineered antibodies, and natural or synthetic ligands for receptors of target cells or tissues. Other suitable auxiliary moieties include fatty acid or lipid moieties that promote the biodistribution and / or uptake of the compounds by target cells (see, for example, Bradley et al., Clin. Cancer Res. 7:3229 (2001)).

[0060] The compounds of the present disclosure inhibit HDAC and are useful for treating various diseases and conditions.In some embodiments, the HDACI is used in a method for treating diseases or conditions that benefit from the inhibition of HDAC, such as cancer, neurological diseases, neurodegenerative conditions, peripheral neuropathy, autoimmune diseases, inflammatory diseases and conditions, stroke, hypertension, traumatic brain injury, autism, and malaria.The method comprises administering a therapeutically effective amount of the HDACI to an individual in need thereof.

[0061] The compounds have been evaluated for their activity at HDAC6 and their selectivity for HDAC6 compared to HDAC1. Selective HDAC6 inhibitors have been shown to be involved in a variety of disease states, including but not limited to arthritis, autoimmune diseases, inflammatory diseases, cancer, neurological diseases such as Rett syndrome, peripheral neuropathies such as CMT, stroke, hypertension, and diseases in which oxidative stress is a causative factor or consequence. It has also been shown that administration of selective HDAC6 inhibitors in combination with rapamycin extends the life span of mice bearing kidney xenografts. This model was used to evaluate the immunosuppressive properties of the compounds and serve as a model of graft rejection. In addition, selective HDAC6 inhibitors have been shown to confer neuroprotection in a rat primary cortical neuron model of oxidative stress. These studies have confirmed that selective HDAC6 inhibitors are non-toxic neuroprotective agents. As the compounds are also selective HDAC6 agents, they behave similarly. The compounds exhibit ligand efficiency that makes their physiochemical properties more drug-like. Furthermore, the compounds maintain the potency and selectivity observed with previous HDACIs. Thus, the compounds are drug candidates and research tools for identifying the specific functions of HDAC6.

[0062] Accordingly, in one embodiment, the present disclosure relates to a method of treating an individual suffering from a disease or condition for which inhibition of HDAC is beneficial, the method comprising administering to an individual in need thereof a therapeutically effective amount of a claimed HDACI compound.

[0063] The method of the present disclosure can be achieved by administering one of the HDACIs of the present disclosure as a raw compound or as a pharmaceutical composition.The pharmaceutical composition of the present disclosure or the raw HDACI can be administered during or after the onset of the disease or condition of interest.Usually, the pharmaceutical composition is sterile and does not contain any toxic, carcinogenic or mutagenic compounds that cause adverse reactions when administered.

[0064] In some embodiments, the HDACI is a second therapeutic agent useful for treating a disease or condition benefiting from inhibition of HDAC. factor The second treatment may be administered in combination with factor is different from the present HDACI. factor is known to be useful in treating a disease or condition from which the individual suffers. factor The HDACI and the second treatment are selected from, for example, drugs and adjuvants, such as chemotherapeutic agents and / or radiation, known to be useful in the treatment of cancer. factor may be administered together as a single unit dose or separately as multiple unit doses, and the HDACI is factor The one or more administrations of the HDACI and / or the second treatment may be administered simultaneously with, prior to, or vice versa. factor may be administered.

[0065] The second treatment factor Each second treatment is administered in an amount that provides its desired therapeutic effect. factor The effective dosage ranges of the second treatment are known in the art. factor is administered within such established ranges to an individual in need thereof.

[0066] Accordingly, the present disclosure relates to compositions and methods of using such compounds in the treatment of diseases or conditions that benefit from inhibition of HDACs. The present disclosure also relates to the use of the present HDACIs and optional second therapeutic agents useful in the treatment of diseases or conditions that benefit from inhibition of HDACs. factor Also provided is a pharmaceutical composition comprising the HDACI and, optionally, a second therapeutic agent, packaged separately or together, useful in the treatment of diseases and conditions benefiting from inhibition of HDAC. factor , and their activities factor The kit includes an insert having instructions for use of the composition.

[0067] Within the meaning of this disclosure, the term "disease" or "condition" generally refers to a derangement and / or abnormality that is considered to be a pathological condition or function and may be manifested in the form of signs, symptoms, and / or disorders. As shown below, the present HDACIs are potent inhibitors of HDACs and may be used to treat diseases and conditions in which inhibition of HDACs is beneficial, such as cancer, neurological diseases, neurodegenerative conditions, traumatic brain injury, stroke, inflammation, autoimmune diseases, and autism.

[0068] In one embodiment, the present disclosure provides a method for treating cancer, including but not limited to killing cancer or neoplastic cells, inhibiting the growth of cancer or neoplastic cells, inhibiting the replication of cancer or neoplastic cells, or improving symptoms thereof, comprising administering to a subject in need thereof an amount of the present HDACI or its pharma- ceutically acceptable salt sufficient to treat the cancer.In addition, it is known that the selective HDACI may be able to promote the killing of cancer cells through the reactivation of immune system by the mechanism related to PDI receptor.The present HDACI may be used as the only anticancer agent, or may be used in combination with other anticancer treatments, such as radiation, chemotherapy, and surgery.

[0069] In another embodiment, the disclosure provides a method of increasing the sensitivity of a cancer cell to the cytotoxic effects of radiation therapy and / or chemotherapy, the method comprising contacting the cell with the HDACI or a pharma- ceutical acceptable salt thereof in an amount sufficient to increase the sensitivity of the cell to the cytotoxic effects of radiation therapy and / or chemotherapy.

[0070] In a further embodiment, the present disclosure provides a method for treating cancer, the method comprising: (a) administering to an individual in need thereof an amount of a HDACI compound of the present disclosure; and (b) administering to the individual an amount of radiation therapy, chemotherapy, or both. Each of the administered amounts is effective for treating cancer. In another embodiment, the amounts are jointly effective for treating cancer.

[0071] The combination therapy of the present disclosure can be used in various situations according to the treatment of various cancers.In certain embodiments, the individual in need of treatment has been previously treated for cancer.Such previous treatment includes, but is not limited to, previous chemotherapy, radiation therapy, surgery, or immunotherapy, such as cancer vaccine.

[0072] In another embodiment, the cancer to be treated is a cancer with demonstrated sensitivity to radiation therapy and / or chemotherapy or a cancer known to respond to radiation therapy and / or chemotherapy, including but not limited to non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, laryngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small cell lung cancer, non-small cell lung cancer, brain tumor, or other CNS neoplasm.

[0073] In yet another embodiment, the cancer to be treated is a cancer that has been proven resistant to radiation therapy and / or chemotherapy, or is a cancer that is known to be ineffective against radiation therapy and / or chemotherapy.A cancer is ineffective when at least a significant portion of the cancer cells are not killed in response to the treatment, or when their cell division is not stopped.This determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of a treatment on cancer cells, using the art-standard meaning of "ineffective" in this context.In certain embodiments, a cancer is ineffective when the number of cancer cells is not significantly reduced or is increasing.

[0074] Other cancers that may be treated with the compounds and methods of the present disclosure include, but are not limited to, cancers and metastases such as brain cancer (glioblastoma) and melanoma, as well as other common tumors.

[0075] In certain embodiments, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, and Bowen's disease, a carcinoma in situ, are precancerous lesions indicating the desirability of preventive intervention.

[0076] In another embodiment, fibrocystic diseases (cystic hyperplasia, mammary dysplasia, and adenosis (benign epithelial hyperplasia)) indicate the desirability of preventative intervention.

[0077] Prophylactic use of the disclosed compounds and methods is also indicated for some viral infections that can lead to cancer. For example, human papillomaviruses can cause cervical cancer (see, e.g., Hernandez-Avila et al., Archives of Medical Research 28:265-271 (1997)), Epstein-Barr virus (EBV) can cause lymphoma (see, e.g., Herrmann et al., J Pathol 199(2):140-5 (2003)), hepatitis B or C viruses can cause liver cancer (see, e.g., El-Serag, J Clin Gastroenterol 35(5 Suppl 2):S72-8 (2002)), human T-cell leukemia virus (HTLV)-I can cause T-cell leukemia (see, e.g., Mortreux et al., Leukemia 17(1):26-38 (2003)), and human herpesvirus-8 infection can cause Kaposi's sarcoma (see, e.g., Kadow et al., J Clin Gastroenterol 35(5 Suppl 2):S72-8 (2002)). al., Curr Opin Investig Drugs 3(11):1574-9 (2002)), and human immunodeficiency virus (HIV) infection contributes to the development of cancer as a result of immune deficiency (see, e.g., Dal Maso et al., Lancet Oncol 4(2):110-9 (2003)).

[0078] In other embodiments, subjects exhibiting one or more of the following predispositions to malignancies may be treated by administration of the HDACIs and methods of the disclosure: chromosomal translocations associated with malignancies (e.g., Philadelphia chromosome in chronic myeloid leukemia, t(14;18) in follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possibly a precursor to colon cancer), benign monoclonal gammopathy (possibly a precursor to multiple myeloma), cancers exhibiting Mendelian (inherited) inheritance patterns or predisposition to malignancies. First-degree relatives with cancerous diseases (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostoses, polyendocrine neoplasia, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, von Recklinghausen neurofibromatosis, retinoblastoma, carotid bulb tumor, cutaneous malignant melanoma, intraocular melanoma, xeroderma pigmentosum, ataxia-telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi aplastic anemia, and Bloom's syndrome; see Robbins and Angell, Basic Pathology, 2d Ed., WB Saunders Co., Philadelphia, pp. 112-113 (1976)), and exposure to carcinogens (e.g., smoking, and inhalation or contact with certain chemicals).

[0079] In another specific embodiment, the HDACIs and methods of the disclosure are administered to a human subject to prevent the progression of breast cancer, colon cancer, ovarian cancer, or cervical cancer.

[0080] In one embodiment, the present disclosure provides a method for treating cancer, comprising (a) administering to an individual in need thereof an amount of the present HDACI, and (b) administering to the individual one or more additional anti-cancer therapies, including but not limited to radiation therapy, chemotherapy, surgery, or immunotherapy, such as cancer vaccines. In one embodiment, the administration of step (a) is prior to the administration of step (b). In another embodiment, the administration of step (a) is subsequent to the administration of step (b). In yet another embodiment, the administration of step (a) is simultaneous with the administration of step (b).

[0081] In one embodiment, the additional anti-cancer therapy is radiation therapy and / or chemotherapy, hi another embodiment, the additional anti-cancer therapy is surgery.

[0082] In yet another embodiment, the additional anti-cancer therapy is an immunotherapy, e.g., a cancer vaccine.

[0083] In one embodiment, the immunotherapy comprises anti-PD1 immunotherapy. In another embodiment, the anti-PD1 immunotherapy comprises administration of a PD-1 antibody. In another embodiment, the PD-1 antibody is nivolumab, pembrolizumab, STI-A1014, or pidilizumab.

[0084] In one embodiment, the HDACI or a pharma- ceutically acceptable salt thereof is administered in combination with the additional anti-cancer therapy.

[0085] In another embodiment, the additional anti-cancer treatment is radiation therapy. In the methods of the present disclosure, any radiation therapy protocol may be used depending on the type of cancer being treated. An embodiment of the present disclosure is radiation therapy using gamma radiation (10 -20 ~10 -13 m), X-ray radiation (10 -12 ~10 -9 m), ultraviolet light (10nm-400nm), visible light (400nm-700nm), infrared (700nm-1mm), and microwave radiation (1mm-30cm).

[0086] For example, but not limited to, X-ray radiation can be administered, and in some embodiments, high energy megavoltage (radiation with energies greater than 1 MeV) can be used for deep tumors, and electron beam and orthovoltage X-ray radiation can be used for skin cancer. Gamma ray emitting radioisotopes, such as radioisotopes of radium, cobalt and other elements, can also be administered. Exemplary radiation therapy protocols useful in the present disclosure include, but are not limited to, stereotactic techniques, in which multiple low dose radiation sources are simultaneously focused into a tissue volume from multiple angles, "internal radiation therapy," which involves placing radioactive implants directly into a tumor or other target tissue, such as brachytherapy, interstitial irradiation, and intracavitary irradiation, intraoperative irradiation, in which large amounts of external radiation are directed at exposed target tissue during surgery, and particle beam therapy, which involves the use of high speed subatomic particles to treat localized cancers.

[0087] Many cancer treatment protocols currently use radiosensitizers that are activated by electromagnetic radiation, such as X-rays. Examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU1069, SR4233, EO9, RB6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cis-platin, and their therapeutically active analogs and derivatives.

[0088] Photodynamic therapy (PDT) of cancer uses visible light as a radioactivator of sensitizers. Examples of photodynamic radiosensitizers include, but are not limited to, hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), pheophorbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and their therapeutically active analogs and derivatives.

[0089] The radiosensitizer may be administered with a therapeutically effective amount of one or more compounds in addition to the HDACI. Such compounds include, but are not limited to, compounds that promote the uptake of the radiosensitizer into target cells, compounds that control the flow of therapeutic agents, nutrients, and / or oxygen to the target cells, chemotherapeutic agents that act on tumors with or without additional radiation, or other therapeutically effective compounds for treating cancer or other diseases. Examples of additional therapeutic agents that may be used with the radiosensitizer include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red blood cell transfusions, perfluorocarbons (e.g., FLUOSOLW®-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, angiogenesis inhibitor compounds, hydralazine, and L-BSO.

[0090] In one embodiment, the HDACI or a pharma- ceutically acceptable salt thereof is administered prior to the administration of radiation therapy and / or chemotherapy.

[0091] In another embodiment, the HDACI or a pharma- ceutically acceptable salt thereof is administered in combination with radiation therapy and / or chemotherapy.

[0092] The HDACI and the additional therapy may act additively or synergistically (i.e., the combination of the HDACI or a pharma- ceutically acceptable salt thereof with the additional anti-cancer therapy is more effective than the additive effects of each administered alone). A synergistic combination allows for the use of lower doses of the HDACI and / or the additional therapy and / or less frequent administration of the HDACI and / or the additional therapy to a subject with cancer. The use of lower doses of the HDACI and / or the additional therapy and / or less frequent administration of the compounds of the present disclosure and the additional therapy may reduce toxicity associated with administration without reducing the efficacy of the HDACI and / or the additional therapy in treating cancer. Furthermore, synergistic effects may improve the efficacy of cancer treatment and / or reduce adverse or undesirable side effects associated with administration of the HDACI and / or the additional anti-cancer therapy as monotherapy.

[0093] In one embodiment, the HDACI may act synergistically with radiation therapy when administered at a dose typically used when such HDACI is used alone to treat cancer, hi another embodiment, the HDACI may act synergistically with radiation therapy when administered at a dose lower than that typically used when such HDACI is used as a monotherapy to treat cancer.

[0094] In one embodiment, radiation therapy may act synergistically with the present HDACIs when administered at doses typically used when radiation therapy is used as a monotherapy for the treatment of cancer. In another embodiment, radiation therapy may act synergistically with the compounds of the present disclosure when administered at doses lower than those typically used when radiation therapy is used as a monotherapy for the treatment of cancer.

[0095] The effectiveness of the HDACI as an HDAC inhibitor for sensitizing cancer cells to the effects of radiation therapy can be determined by measuring survival after treatment in vitro and / or in vivo using techniques known in the art. In one embodiment, for in vitro measurements, cells in logarithmic growth phase can be exposed to a known dose of radiation and cell survival observed. Irradiated cells are seeded and cultured for about 14 to about 21 days, and colonies are stained. Viability is the number of colonies divided by the plating efficiency of unirradiated cells. Survival curves are generated by plotting the survival rate on a logarithmic scale against the absorbed dose on a uniform scale. Survival curves generally show an exponential decrease in the percentage of surviving cells at higher radiation doses after an initial shoulder region where the dose is sublethal. Similar protocols can be used for chemicals when used in combination therapies of the present disclosure.

[0096] The inherent radiosensitivity of tumor cells and environmental influences, such as hypoxia and host immunity, can be further evaluated by in vivo studies. Growth delay assays are commonly used. They measure the time interval required for a tumor exposed to radiation to regrow to a specific volume. The dose required to control approximately 50% of the tumors is estimated as the TCD 50 Identified by assay.

[0097] In vivo assay systems typically use solid tumor systems that are implantable into experimental subjects. Radiation survival parameters of normal tissues and tumors can be assayed using in vivo techniques known in the art.

[0098] The present disclosure provides a method for treating cancer comprising administering an effective amount of the present HDACI in conjunction with accepted methods of surgery, radiation therapy, and chemotherapy, including, for example, mimicking radiation therapy with chemical systems to achieve a synergistic increase in the efficacy of accepted therapies. The efficacy of the treatment can be measured in clinical studies or model systems, such as mouse tumor models, or cell culture susceptibility assays.

[0099] The present disclosure provides combination therapies that improve efficacy and / or reduce toxicity. Thus, in one aspect, the present disclosure relates to the use of the present HDACIs as radiosensitizers in combination with radiation therapy.

[0100] When the combination therapy of the present disclosure comprises administering the HDACI with one or more additional anti-cancer drugs, the HDACI and the additional anti-cancer drugs can be administered simultaneously or sequentially to an individual.The drugs can also be administered cyclically.Cycling therapy includes the administration of one or more anti-cancer drugs for a certain period of time, followed by the administration of one or more different anti-cancer drugs for a certain period of time and repeating this sequential administration, i.e., cycle, to reduce the development of resistance to one or more of the administered anti-cancer drugs, avoid or reduce the side effects of one or more of the administered anti-cancer drugs, and / or improve the efficacy of the treatment.

[0101] Additional anti-cancer drugs may be administered over a course of time. Any or a combination of additional anti-cancer drugs described below may be administered.

[0102] The present disclosure includes a method for treating cancer, comprising administering to an individual in need of cancer treatment the present HDACI and one or more additional anti-cancer drugs or pharma- ceutically acceptable salts thereof. The present HDACI and the additional anti-cancer drugs may act additively or synergistically. Suitable anti-cancer drugs include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, and the like.

[0103] Additionally, the present disclosure provides methods of treating cancer using the present HDACIs as an alternative to chemotherapy alone or radiation therapy alone when chemotherapy or radiation therapy has proven or may prove to be too toxic to the subject being treated, e.g., when it results in unacceptable or intolerable side effects. The individual being treated may optionally be treated with another anti-cancer therapy, e.g., chemotherapy, surgery, or immunotherapy, depending on which treatment is found to be tolerated or tolerable.

[0104] The HDACI can also be used in an in vitro or ex vivo manner to treat certain cancers, including but not limited to leukemia and lymphoma, and such treatments include autologous stem cell transplantation. This can include a multi-step process in which the subject's autologous hematopoietic stem cells are harvested and all cancer cells are removed, the subject is then administered an effective amount of the HDACI to eradicate the subject's remaining bone marrow cell population, and the stem cell transplant is then infused back into the subject. Supportive care is then administered until bone marrow function is restored and the subject is cured.

[0105] The present methods for treating cancer further include administering to a subject a subject a subject a subject a further therapy. factor or a pharma- ceutical acceptable salt or hydrate thereof. In one embodiment, the composition comprising the HDACI is administered with one or more additional therapeutic agents, which may be part of the same composition as the composition comprising the HDACI or in a different composition. factor In another embodiment, the HDACI is administered simultaneously with the administration of another treatment. factor It is administered before or after administration of (several).

[0106] In this method for treating cancer, the other treatment factormay be an antiemetic. Suitable antiemetics include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methalathal, metopimazine, nabilone, oxyperundyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine, and tropisetron. In one embodiment, the antiemetic is granisetron or ondansetron. In another embodiment, the other treatment factor can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim, and epoetin alfa.

[0107] In yet another embodiment, the other treatment factor The opioid analgesic may be an opioid or a non-opioid analgesic. Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, loperamide, anileridine, ethoheptazine, piminidine, betaprozine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone, and propoxyphene. Suitable non-opioid analgesics include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflunisal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamic acid, mefanamic acid, nabumetone, naproxen, piroxicam, and sulindac.

[0108] In yet another embodiment, the other treatment factor may be an anti-anxiety drug. Suitable anti-anxiety drugs include, but are not limited to, buspirene, and benzodiazepines such as diazepam, lorazepam, oxazepam, clorazepate, clonazepam, chlordiazepoxide, and alprazolam.

[0109] In addition to treating cancer and sensitizing cancer cells to the cytotoxic effects of radiation and chemotherapy, the present HDACIs find use in methods of treating diseases, conditions, and injuries to the central nervous system, such as neurological diseases, neurodegenerative disorders, and traumatic brain injury (TBI). In preferred embodiments, the present HDACIs are capable of crossing the blood-brain barrier and inhibiting HDACs in the brain of an individual.

[0110] The HDACI compounds also provide therapeutic effects in models of peripheral neuropathy, such as CMT. HDAC6 inhibitors have been shown to cross the blood-nerve barrier and rescue the phenotype observed in transgenic mice that exhibit symptoms of distal hereditary motor neuropathy. Administration of HDAC6 inhibitors to symptomatic mice increases the level of acetylated α-tubulin, restores proper mitochondrial motility and axonal transport, and increases muscle reinnervation. Other peripheral neuropathies include, but are not limited to, giant axonal neuropathy and various forms of mononeuropathy, polyneuropathy, autonomic neuropathy, and neuritis.

[0111] The HDACI compounds also improve associative memory loss following Aβ elevation. In this study, mice were infused with Aβ42 via a cannula implanted in the dorsal hippocampus 15 min before training. Test compounds were administered ip (25 mg / kg) 2 h before training. Fear learning was assessed 24 h later.

[0112] Contextual fear conditioning performed 24 hours after training shows reduced freezing in Aβ-injected mice compared to vehicle-injected mice. Treatment with the compound improves the freezing defect in Aβ-injected mice and has no effect on vehicle-injected mice. The test compound alone has no effect on the memory performance of the mice. Furthermore, the compound was injected twice daily for two days and there was no effect of the treatment on motor, sensory, or motivational skills assessed using the visible platform test. No obvious signs of toxicity were observed during these experiments, including changes in food and water intake, weight loss, or changes in locomotion and exploration behavior.

[0113] These results indicate that HDACIs of the present disclosure are beneficial against impairment of associative memory following Aβ elevation.

[0114] Thus, the HDACIs are useful for treating neurological disorders by administering an effective amount of the HDACI to treat the neurological disorder, or by administering an effective amount of a pharmaceutical composition comprising the HDACI to treat the neurological disorder. Neurological disorders that may be treated include Huntington's disease, lupus, schizophrenia, multiple sclerosis, muscular dystrophies, dentatorubral-pallidoluysian atrophy (DRRLA), spinal-bulbar muscular atrophy (SBMA), and spinocerebellar ataxias (SCA1, SCA2, SCA3 / MJD (Machado-Joseph disease), SCA6, and SCA7), drug-induced movement disorders, Creutzfeldt-Jakob disease, amyotrophic lateral sclerosis, Pick's disease, Alzheimer's disease, Lewy body syndrome, and others. These conditions include, but are not limited to, dementia, corticobasal degeneration, dystonia, myoclonus, Tourette's syndrome, tremor, chorea, restless legs syndrome, Parkinson's disease, Parkinsonian syndrome, anxiety, depression, psychosis, manic depression, Friedreich's ataxia, Fragile X syndrome, spinal muscular dystrophy, Rett syndrome, Rubinstein-Taybi syndrome, Wilson's disease, multi-infarct states, CMT, GAN as well as other peripheral neuropathies.

[0115] In one embodiment, the neurological disease being treated is Huntington's disease, Parkinson's disease, Alzheimer's disease, spinal muscular atrophy, lupus, or schizophrenia.

[0116] Charcot-Marie-Tooth disease (CMT) is one of the most common genetic neurological disorders affecting approximately 1 in 2,500 people in the United States. CMT affects both motor and sensory nerves and can lead to foot drop and elevated gait with frequent stumbling and falling. Mutations in small heat shock protein 27 (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). Expression of mutant HSPB1 reduced acetylated α-tubulin levels and induced severe axonal transport defects. Pharmacological inhibition of histone deacetylase 6 (HDAC6)-induced α-tubulin deacetylation caused by the HDAC6i Tubastatin A corrects the axonal transport defects induced by HSPB1 mutations and rescues the CMT phenotype in symptomatic mutant HSPB1 mice. The pathogenic role of α-tubulin deacetylation has been demonstrated in mutant HSPB1-induced neuropathy, providing valuable insight into HDAC6 inhibitors as a therapeutic strategy for inherited axonal degeneration. Compounds of the present disclosure show potent HDAC6 isoform inhibition, high HDAC6 selectivity, and excellent α-tubulin acetylation in various cell lines.

[0117] Thus, in another embodiment, the neurological disorder is Charcot-Marie-Tooth disease.

[0118] The present HDACIs may also be used as second therapeutic agents in methods for treating conditions, diseases, and injuries to the CNS. factor Such a second treatment may be used in combination with factor are drugs known in the art for treating a condition, disease, or injury, such as, but not limited to, lithium in the treatment of mood disorders, estradiol benzoate, and nicotinamide in the treatment of Huntington's disease.

[0119] The HDACIs are also useful in the treatment of traumatic brain injury (TBI), a severe and complex injury that affects approximately 1.4 million people in the United States each year. TBI is associated with a wide range of symptoms and disorders, including risk factors for developing neurodegenerative disorders such as Alzheimer's disease.

[0120] TBI induces many pathologies, including axonal injury, cell death, contusion, and inflammation. The inflammatory cascade is characterized by inflammatory cytokines and microglial activation that may exacerbate other pathologies. Although the role of inflammation in TBI is well established, there are currently no effective anti-inflammatory therapies for the treatment of TBI.

[0121] Several known HDAC inhibitors have been found to be protective in various cell and animal models of acute and chronic neurodegenerative injuries and diseases, such as Alzheimer's disease, ischemic stroke, multiple sclerosis (MS), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal-bulbar muscular atrophy (SBMA). A recent study on experimental pediatric TBI reported that decreased hippocampal CA3 histone H3 acetylation lasted from hours to days after injury. These changes were attributed to identified upstream excitotoxicity and stress cascades associated with TBI. HDACs have also been reported to have anti-inflammatory effects acting through acetylation of non-histone proteins. It was found that the HDAC6 selective inhibitor, 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB), could increase histone H3 acetylation and reduce microglial inflammatory responses after traumatic brain injury in rats. This demonstrates the utility of HDACIs as therapeutic agents to inhibit neuroinflammation associated with TBI.

[0122] Thus, the present HDACIs are also useful for treating inflammation and stroke, as well as for treating autism and autism spectrum disorders. The present HDACIs may further be used to treat parasitic infections (e.g., malaria, toxoplasmosis, trypanosomiasis, helminthiasis, protozoan infections (see Andrews et al., Int. J. Parasitol. 30(6):761-768 (2000)).

[0123] The HDACI can also be used as an imaging agent. In some embodiments, by providing a radioactively, isotopically, or fluorescently labeled HDACI, the labeled compound can image HDACs, tissues expressing HDACIs, and tumors. The labeled HDACI of the present disclosure can also image patients suffering from cancer or other HDACI-mediated diseases, such as stroke, by administering an effective amount of the labeled compound or a composition comprising the labeled compound. In a preferred embodiment, the labeled HDACI is capable of emitting positron radiation and is suitable for use in positron emission tomography (PET). Typically, the labeled HDACI of the present disclosure is used to identify tissues or target regions expressing high concentrations of HDACIs. The extent of accumulation of the labeled HDACI can be quantified using known methods for quantifying radioactive emissions. Additionally, the labeled HDACI can include a fluorophore or similar reporter capable of tracking the movement of HDACI isoforms or organelles in vitro.

[0124] The present HDACIs useful for imaging procedures include one or more radioisotopes capable of emitting one or more forms of radiation suitable for detection by any standard radiological instrument, such as PET, SPECT, gamma cameras, MRI, and similar devices. A preferred isotope is tritium ( 3 H) and carbon ( 11 The substituted HDACIs of the present disclosure also include isotopes of fluorine ( 18 F) and iodine isotopes ( 123 Typically, the labeled HDACI of the present disclosure can include: 11C-labeled alkyl groups, i.e. 11 C-methyl group, or 18 F, 123 I, 125 I, 131 I, or combinations thereof.

[0125] Fluorescently labeled HDACIs of the present disclosure can also be used in the imaging methods of the present disclosure, such compounds bearing FITC, carbocyanine moieties or other fluorophores that allow visualization of HDAC proteins in vitro.

[0126] The labeled HDACI and method of use can be used in vivo and in humans, and can also be used in vitro, for example, for diagnostic and research applications using body fluids and cell samples. Imaging methods using the labeled HDACI of the present disclosure are discussed in WO03 / 060523, which is designated U.S. and is incorporated herein in its entirety. Typically, the method includes contacting cells or tissue with a radiolabeled, isotopically labeled, fluorescently labeled, or tagged (such as biotin tagged) compound of the present disclosure, and producing an X-ray, fluorescent, or similar type of image, depending on the visualization method used. That is, for X-ray images, the amount is sufficient to provide about 1 to about 30 mCi of the radiolabeled compound.

[0127] Preferred imaging methods include the use of labeled HDACIs of the present disclosure capable of generating a target to background emission intensity ratio of 2:1, or more preferably, a target to background emission intensity ratio of about 5:1, about 10:1, or about 15:1.

[0128] In preferred methods, the labeled HDACIs of the present disclosure are rapidly excreted from tissues in the body, preventing long-term exposure to radiation of the radiolabeled compound administered to an individual. Typically, the labeled HDACIs of the present disclosure are cleared from the body in less than about 24 hours. More preferably, the labeled HDACIs are cleared from the body in less than about 16 hours, 12 hours, 8 hours, 6 hours, 4 hours, 2 hours, 90 minutes, or 60 minutes. Typically, preferred labeled HDACIs are cleared from the body in about 60 minutes to about 120 minutes.

[0129] In addition to isotopically and fluorescently labeled derivatives, the present disclosure also embodies the use of derivatives that contain tags (such as biotin) to identify biomolecules associated with HDAC isoforms of interest for diagnostic, therapeutic or research purposes.

[0130] The present HDACIs are also useful for treating autoimmune diseases and inflammation. The compounds of the present disclosure are useful in overcoming transplant and transplant rejection and in treating arthritis.

[0131] Despite the success of modern transplantation programs, the incidence of post-transplant malignancies and graft loss due to chronic rejection, in addition to nephrotoxicity, cardiovascular disease, diabetes, and hyperlipidemia associated with current therapies, has driven efforts to achieve long-term allograft function with minimal immunosuppression. Similarly, the incidence of inflammatory bowel diseases (IBD), such as Crohn's disease and ulcerative colitis, is increasing. Animal studies have shown that regulatory T cells (Tregs), which express Foxp3, a member of the forkhead transcription family, are key to limiting autoreactive and alloreactive immunity. Furthermore, after induction by costimulatory blockade, immunosuppression, or other strategies, Tregs can be adoptively transferred into naive hosts to achieve beneficial therapeutic effects. However, in clinical trials, attempts to develop sufficient Tregs to maintain suppressive function after transfer have been unsuccessful. Studies in mice have shown that HDACIs enhance the suppressive function of Tregs. It has been shown that selective targeting of HDAC6 limits the immune response, at least in part, by inhibiting IL-1 and IL-2 (Tao et al., Nat Med 13:1299-1307 (2007)), and selective targeting of HDAC6 is particularly effective in this regard.

[0132] In organ transplantation, the prevention of rejection is a priority consideration rather than its treatment, since rejection begins soon after transplantation. The opposite is true for autoimmunity, where the patient already presents with a disease that is causing problems. Accordingly, HDAC6- / - mice treated with low-dose RPM (rapamycin) for 14 days will be evaluated to see whether they show signs of tolerance induction and resistance to the development of chronic rejection, i.e., a continued major loss of long-term graft function in clinical transplant populations. Tolerance will be assessed by examining whether mice with long-term surviving allografts subsequently reject a third-party heart graft and accept a further donor allograft without immunosuppression, as occurs when nonselective HDACI and RPM are used. These in vivo tests will be achieved by evaluation of ELISPOT and MLR activity using lymphocytes from recipients administered donor cells. Protection against chronic rejection will be evaluated by analysis of host antidonor humoral responses, as well as graft arteriosclerosis and interstitial fibrosis in long-term surviving allograft recipients.

[0133] The importance of HDAC6 targeting will be evaluated in further transplantation models that seek a readout of biochemical significance as observed clinically. Thus, the effect of HDAC6 targeting in kidney transplant recipients (monitoring BUN, proteinuria) and in pancreatic islet allografts (monitoring blood glucose) will be evaluated. Since kidney transplantation is the most commonly performed organ transplant and the kidney performs multiple functions, e.g., regulating acid / base metabolism, blood pressure, and red blood cell production, efficacy in this model will indicate the utility of HDAC6 targeting. Similarly, islet transplantation is a major unmet need given that clinical islet allografts are usually lost after the first year or two after transplantation. Having a safe and non-toxic means to extend islet survival without maintenance CNI therapy would be an important development. Transplantation studies will also be enhanced by the use of mice with floxed HDAC6. For example, the effect of deleting HDAC6 only in Tregs will be tested using existing Foxp3-Cre mice. This approach can be extended to targeting HDAC6 in, for example, T cells (CD4-Cre) and dendritic cells (CD11c-Cre).Using tamoxifen-controlled Cre, the importance of HDAC6 in engraftment and maintenance (impact on short-term and maintenance HDAC6I therapy) can be assessed by administering tamoxifen and inducing HDAC6 deletion at various times after engraftment.

[0134] Autoimmunity is also being studied. In this case, blocking existing disease is particularly important, and targeting HDAC6 may be effective without the need for further treatment (in contrast to the need for short, low-dose RPM in the highly aggressive, fully MHC-mismatched transplant model). Studies in mice with colitis have shown that HDAC6- / - Tregs are more effective at controlling disease than WT Tregs, and that Tubacin can rescue mice if treatment is initiated after colitis has developed. These studies will be extended by assessing whether deletion of HDAC6 in Tregs (Foxp3 / Cre), T cells (CD4=Cre), or DCs (CD11c-Cre) differentially affects the onset and severity of colitis. Similarly, control of colitis will be assessed by inducing HDAC6 deletion with tamoxifen-controlled Cre at various intervals after colitis onset.

[0135] The compound is expected to show anti-arthritic efficacy in a collagen-induced arthritis model in DBA1 / J mice. In this study, DBA1 / J mice (male, 7-8 weeks old) are used, 8 per group. Systemic arthritis is induced with bovine type II collagen and CFA plus an IFA booster injection on day 21. The HDACI is administered at 50 mg / kg on day 28. and 100 mg / kg for 2 weeks, and the effects are judged from data on mean arthritis scores versus days of treatment.

[0136] Despite efforts to match host and donor tissue types to avoid graft rejection, in most transplant procedures immunosuppressive therapy is critical for the survival of the donor organ in the host. A variety of immunosuppressive agents are employed in transplant procedures, including azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids.

[0137] The present HDACIs are potent immunosuppressants that suppress humoral and cellular immune responses, such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis, and graft-versus-host disease. The HDACIs of the present disclosure are useful for preventing organ rejection after organ transplantation, treating rheumatoid arthritis, treating psoriasis, and treating other autoimmune diseases, such as type I diabetes, Crohn's disease, and lupus.

[0138] Therapeutically effective amounts of the subject HDACIs may be used for immunosuppression, including, for example, prevention of organ rejection or graft-versus-host disease, and treatment of diseases and conditions such as autoimmune and inflammatory diseases and conditions. Examples of autoimmune and inflammatory diseases include Hashimoto's thyroiditis, pernicious anemia, Addison's disease, psoriasis, diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, dermatomyositis, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, arthritis (rheumatoid arthritis, chronic progressive arthritis, osteoarthritis) and rheumatic diseases, autoimmune blood diseases (hemolytic anemia, aplastic anemia, true erythrocytic anemia, and idiopathic thrombocytopenia), systemic lupus erythematosus, and the like. These include, but are not limited to, glaucoma, polychondritis, scleroderma, Wegener's granulomatosis, dermatomyositis, chronic active hepatitis, psoriasis, Stevens-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (ulcerative colitis, Crohn's disease), endocrine eye disorders, Graves' disease, sarcoidosis, primary biliary cirrhosis, juvenile diabetes mellitus (type I diabetes), uveitis (anterior and posterior), keratoconjunctivitis sicca and vernal conjunctivitis, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis.

[0139] The present HDACIs may be used alone or as a second therapy known to be useful in the treatment of autoimmune diseases, inflammation, transplantation, and grafts. factor For example, they may be used in combination with cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those skilled in the art.

[0140] Additional diseases and conditions mediated by HDACs, such as HDAC6, include, but are not limited to, asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, general neuritis, and general neuropathy, which may also be treated by the methods of the present disclosure.

[0141] In the methods, a therapeutically effective amount of one or more HDACIs of the present disclosure are typically formulated and administered in accordance with pharmaceutical practice to a human in need thereof. Whether such treatment is indicated will depend on the individual case and will be subject to a medical evaluation (diagnosis) taking into account the signs, symptoms, and / or disorders present, the risk of exhibiting the signs, symptoms, and / or disorders, and other factors.

[0142] The HDACIs may be administered by any suitable route, for example, orally, bucally, by inhalation, topically, sublingually, rectally, intravaginally, intracisternally or intrathecally via lumbar puncture, transurethrally, intranasally, percutaneously, i.e., transdermal, or parenterally (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at the site) administration. Parenteral administration may be accomplished using a needle and syringe or using high pressure techniques.

[0143] A pharmaceutical composition includes one in which the HDACI is present in an amount sufficient to be administered in an effective amount to achieve its intended purpose. The exact formulation, route of administration, and dosage will be determined by the individual physician in light of the diagnosed condition or disease. Dosage amount and interval can be individually adjusted to provide a level of the HDACI sufficient to maintain therapeutic effect.

[0144] The toxicity and therapeutic efficacy of the present HDACI compounds can be evaluated in cell cultures or experimental animals, e.g., by measuring the toxicity of HDACI compounds in vitro and in vivo. 50 (the dose at which 50% of the population is lethal) and ED 50The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be determined by standard pharmaceutical procedures to measure the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 and ED 50 Therapeutic indices are expressed as a ratio between ED and ED. Compounds that exhibit high therapeutic indices are preferred. Data obtained from such procedures can be used in formulating a range of dosages for use in humans. The dosages are preferably within the range of ED with little or no toxicity. 50 The therapeutically effective amount is within the range of blood compound concentrations that include the above range. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0145] The therapeutically effective amount of the HDACI required for use in treatment varies according to the nature of the condition to be treated, the length of time activity is desired, and the age and condition of the patient, and is ultimately determined by the attending physician.Dosage amount and interval can be individually adjusted to provide a plasma level of the HDACI sufficient to maintain the desired therapeutic effect.The desired dose can be conveniently administered in a single dose or as multiple doses administered at appropriate intervals, for example, once, twice, three times, four times or more as partial doses per day.In many cases, multiple doses are desirable or necessary. For example, the HDACIs may be administered once daily, 4 days apart for 4 doses (q4d×4), once daily, 3 days apart for 4 doses (q3d×4), once daily, 5 days apart (qd×5), once weekly for 3 weeks (qwk3), 5 consecutive days on, 2 days off, and 5 additional consecutive days on (5 / 2 / 5), or any dosing schedule deemed appropriate for the situation.

[0146] The dosage of the composition comprising or containing the HDACI may be from about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg of body weight. The dosages above are exemplary of the average case, but there may be individual cases that merit higher or lower dosages and are within the scope of this disclosure. In practice, the physician will determine the actual dosing regimen that is most appropriate for the individual patient and may vary with the patient's age, weight, and response.

[0147] The HDACIs used in the methods of the present disclosure are typically administered in amounts of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the HDACIs can be administered in amounts of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams per dose, including all doses between 0.005 and 500 milligrams.

[0148] The HDACIs of the present disclosure are typically administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or more physiologically acceptable carriers that contain excipients and adjuvants that facilitate processing of the HDACI.

[0149] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with the HDACI. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary substances, stabilizers, thickeners, lubricants, and colorants can be used. The pharma-ceutically acceptable carriers are sterile. When the HDACI is administered intravenously, water is a preferred carrier. For example, saline solution and aqueous dextrose and glycerol solutions can also be employed as liquid carriers for injections. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0150] These pharmaceutical compositions may be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Appropriate formulations depend on the route of administration selected. When a therapeutically effective amount of the present HDACI is administered orally, the composition is usually in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition may further comprise a solid carrier, such as gelatin or an adjuvant. The tablets, capsules, and powders contain about 0.01% to about 95%, preferably about 1% to about 50%, of the present HDACI. When administered in liquid form, a liquid carrier, such as water, petroleum, or oil of animal or vegetable origin, may be added. The liquid form of the composition may further comprise saline, dextrose or other saccharide solution, or glycol. When administered in liquid form, the composition contains about 0.1% to about 90% by weight, preferably about 1% to about 50% by weight of the present compound.

[0151] When a therapeutically effective amount of the HDACI is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. It is within the skill of the artisan to prepare such a parenterally acceptable solution, taking into due consideration pH, isotonicity, stability, etc. Preferred compositions for intravenous, cutaneous, or subcutaneous injection usually include an isotonic medium. The HDACI may be infused with other liquids over 10-30 minutes or over several hours.

[0152] The HDACI can be easily mixed with pharma- ceutically acceptable carriers known in the art. Such carriers allow the formulation of the active agent as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc., for oral ingestion by the patient to be treated. A pharmaceutical preparation for oral use can be obtained by adding the HDACI to a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries as necessary, and then processing the granules of the mixture to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added as needed.

[0153] The HDACI can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of preservatives.The composition can take the form of suspension, solution, or emulsion in oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0154] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form.Furthermore, the suspension of the HDACI can be prepared as a suitable oily injection suspension.Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension.Optionally, the suspension can also contain suitable stabilizers or agents that increase the solubility of the compound and allow the preparation of highly concentrated solutions.Alternatively, the composition can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0155] The HDACI can also be formulated into rectal compositions, such as suppositories containing conventional suppository bases or retention enemas.In addition to the formulations described above, the HDACI can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, the HDACI can be formulated with suitable polymers or hydrophobic materials (e.g., as an emulsion in acceptable oil) or ion exchange resins.

[0156] In some embodiments, the HDACI may be administered orally, bucally, or sublingually in the form of a tablet containing excipients such as starch or lactose, or in a capsule or suppository, alone or in a mixture with excipients, or in the form of an elixir or suspension containing flavoring or coloring agents. Such liquid preparations may be prepared with pharma- ceutically acceptable additives, such as suspending agents. The HDACI may also be parenterally injected, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the HDACI is most suitable for use in the form of a sterile aqueous solution, which may contain other substances, such as salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.

[0157] In further embodiments, the present disclosure includes kits containing one or more compounds or compositions packaged in a manner that facilitates their use in practicing the methods of the present disclosure. In one simple embodiment, the kit includes a compound or composition described herein as useful for practicing the methods (e.g., the present HDACI and optional second treatments) packaged in a container, such as a sealed bottle or container. factor The composition comprises a compound or composition that is a soluble ...

[0158] Previously, HDACIs were factor In accordance with an important feature of the present disclosure, the present HDACIs were synthesized and evaluated as inhibitors of HDACs. The present compounds exhibit increased HDAC6 potency and selectivity for HDAC1 and HDAC8, and have improved BEI, compared to previous compounds. The improved properties of the present compounds, such as increased BEI and reduced potency at HDAC8, indicate that the present compounds are useful for applications such as, but not limited to, immunosuppressants and neuroprotectants. For example, the compounds of the present disclosure generally exhibit improved binding affinity (IC) for HDAC6. 50 ) is less than 100 μM, less than 25 μM, less than 10 μM, less than 1 μM, less than 0.5 μM, and less than 0.2 μM.

[0159] Synthesis Methods and Procedures All starting materials and solvents were purchased from commercial suppliers at reagent purity and used as is without further purification unless otherwise noted. Dry solvents used as media in moisture sensitive reactions were purchased in anhydrous grade from Sigma-Aldrich and operated under argon. All reactions were carried out dry under an inert (argon) atmosphere. Microwave reactions were carried out in a Biotage Initiator microwave reactor. Reactions were run on silica gel coated glass plates (TLC LuxPlate Silica gel 60 F 254 Reactions were monitored by thin layer chromatography on a HPLC column (Merck) visualized at 254 nm and / or using appropriate dyes. Where indicated, synthetic intermediates were purified by 230-400 mesh silica gel flash chromatography on a CombiFlash® system using appropriate solvent mixtures. Final products were purified by preparative HPLC using a Shimadzu preparative liquid chromatograph [ACE 5AQ (150×21.2 mm) 5 μm particle size. Method 1: 25-100% MeOH / H2O, 30 min, 100% MeOH, 5 min, 100-25% MeOH / H2O, 4 min. Method 2: 8-100% MeOH / H2O, 30 min, 100% MeOH, 5 min, 100-8% MeOH / H2O, 4 min. Method 3: 0% MeOH, 5 min, 0-100% MeOH / H2O, 25 min, 100% MeOH, 5 min, 100-0% MeOH / H2O, 4 min. Flow rate = 17 mL / min], monitored at 254 and 280 nm. 0.05% TFA was added to both solvents. 1 H and 13 C NMR spectra were recorded on a Bruker DPX-400 or AVANCE-400 spectrometer at 400 MHz and 100.6 MHz, respectively. Chemical shifts (δ scale) are reported in parts per million (ppm) relative to TMS. 1H NMR spectra are reported in the following order: multiplicity and number of protons, signals are s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), bs (broad signal). HRMS spectra were recorded using an ESI equipped LCMS-IT-TOF (Shimadzu). The purity of all final compounds was determined by analytical HPLC [ACE 3AQ C18 column (150 × 4.6 mm, particle size 3 μM), gradient elution system of H2O with 0.05% TFA / MeOH with 0.05% TFA, flow rate = 1.0 mL / min]. All compounds were tested to be >95% pure as determined by HPLC analysis.

[0160] The synthetic route of Suprastat, a representative HDAC6 inhibitor of the present disclosure, is shown in Scheme 1. According to Scheme 1, the general carbamate intermediate 2 was prepared from phenyl chloroformate and aniline 1 under K2CO3 / acetone conditions. Methyl 4-formylbenzoate 3a was rapidly reductively aminated with 4-amino-1-butanol to give intermediate 4a. The combined reaction between 2 and 4a under TEA / THF conditions then gave the key urea precursor 5a, which was further converted to the final hydroxamic acid product 6a (Suprastat) using aqueous hydroxylamine under basic conditions and TFA / THF conditions to deprotect the Boc group. The same synthetic route as Suprastat was also used to prepare analog 6b with the original butyl chain attached to the aminomethyl group and the proximal urea nitrogen for evaluation alongside Suprastat in subsequent biological experiments. The synthesis of 6c (Table 1), which contains a hydroxyl butyl side chain, has been reported previously (see, for example, Bergman et al., J. Med. Chem. 55:9891-9899 (2012)). In addition, non-hydroxamic acid analogs 6d-f, which contain the same cap as Supratstat, were prepared and evaluated to investigate whether additional hydrogen-bonding interactions could retain activity without the hydroxamic acid ZBG, originating from ketone / amide-based class I HDACIs (see, for example, Koya et al., Cancer Res. 72:3928-3937 (2012) or O'Donnell et al., Cancer Treat Rev. 52:71-81 (2017)). The carboxylic acid analog 6d was obtained directly from the urea ester 5a by hydrolysis and Boc deprotection under basic conditions. To synthesize the amide analog 6e, 4-formylbenzonitrile 3b underwent a two-step reductive amination followed by reaction with carbamate 2 to generate intermediate urea 5c. The nitrile group of 5c was further converted to an amide group by treatment with aqueous hydrogen peroxide under basic conditions, and final product 6e was obtained by Boc deprotection as described above. The synthetic route to boronic acid analog 6f began with reductive amination of 4-bromobenzaldehyde 3c followed by urea formation using the same procedure as described above to give intermediate urea 5d.The precursor 5d was coupled with bis(pinacolato)diboron under KOAc / Pd(dppf)Cl conditions. Finally, the desired boronic acid product 6f was obtained by pinacol deprotection and Boc deprotection under NaIO / NHOAc and TFA / THF, respectively. [ka] Scheme 1. Synthetic route to 6a-b. Reagents and conditions: (a) phenyl chloroformate, K2CO3, acetone, rt, 2 h, (b) i) 4-amino-1-butanol for 4a, 4c, and 4d, n-butylamine, EtOH, reflux, 2 h for 4b, ii) NaBH4, MeOH, 0 °C to rt, 2 h, (c) 2, TEA, THF, reflux, 2 h, (d) for 6a and 6b: i) aq. NH2OH (50%), NaOH, THF / MeOH, 0 °C, 15 min, ii) TFA, THF, rt, 0.5 h, (e) for 6d: i) 1N NaOH, THF / MeOH, rt, overnight, ii) TFA, THF, rt, 0.5 h, (f) for 6e: i) H2O2 (30%), K2CO3, DMSO, rt, 5 h, ii) TFA, THF, rt, 0.5 h, (g) for 6f: i) B2pin2, KOAc, Pd(dppf)Cl2, DMF, 80 °C, overnight, ii) NaIO4, NH4OAc, acetone-H2O, rt, overnight, iii) TFA, THF, rt, 0.5 h.

[0161] Use of HDAC inhibitors The HDACIs of the present disclosure may be used alone or in combination with a second therapy known to be useful in the treatment of a variety of diseases, including autoimmune diseases, inflammation, transplantation, and grafts. factor For example, they may be used in combination with cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those skilled in the art.

[0162] Additional diseases and conditions mediated by HDACs, and for example, HDAC6, include, but are not limited to, asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, general neuritis, and general neuropathy, which may also be treated by the methods of the present disclosure.

[0163] In the methods, a therapeutically effective amount of one or more HDACIs of the present disclosure are typically formulated and administered in accordance with pharmaceutical practice to a human in need thereof. Whether such treatment is indicated will depend on the individual case and will be subject to a medical evaluation (diagnosis) taking into account the signs, symptoms, and / or disorders present, the risk of exhibiting the signs, symptoms, and / or disorders, and other factors.

[0164] The HDACIs may be administered by any suitable route, for example, orally, bucally, by inhalation, topically, sublingually, rectally, intravaginally, intracisternally or intrathecally via lumbar puncture, transurethrally, intranasally, percutaneously, i.e., transdermal, or parenterally (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at the site) administration. Parenteral administration may be accomplished using a needle and syringe or using high pressure techniques.

[0165] A pharmaceutical composition includes one in which the HDACI is present in an amount sufficient to be administered in an effective amount to achieve its intended purpose. The exact formulation, route of administration, and dosage will be determined by the individual physician in light of the diagnosed condition or disease. Dosage amount and interval can be individually adjusted to provide a level of the HDACI sufficient to maintain therapeutic effect.

[0166] The toxicity and therapeutic efficacy of the HDACI compounds can be determined in cell cultures or experimental animals, e.g., by measuring the LDso (the dose that is lethal to 50% of the population) and ED 50The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be determined by standard pharmaceutical procedures to measure the dose that is therapeutically effective in 50% of the population. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 and ED 50 The therapeutic index is expressed as a ratio between ED50 and ED50. Compounds that exhibit high therapeutic indices are preferred. Data obtained from such procedures can be used in formulating a dosage range for use in humans. The dosage is preferably within a range of blood compound concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0167] The therapeutically effective amount of the HDACI required for use in treatment varies according to the nature of the condition to be treated, the length of time activity is desired, and the age and condition of the patient, and is ultimately determined by the attending physician.Dosage amount and interval can be individually adjusted to provide a plasma level of the HDACI sufficient to maintain the desired therapeutic effect.The desired dose can be conveniently administered in a single dose or as multiple doses administered at appropriate intervals, for example, once, twice, three times, four times or more as partial doses per day.In many cases, multiple doses are desirable or necessary. For example, the HDACIs may be administered once daily, 4 days apart for 4 doses (q4d×4), once daily, 3 days apart for 4 doses (q3d×4), once daily, 5 days apart (qd×5), once weekly for 3 weeks (qwk3), 5 consecutive days on, 2 days off, and 5 additional consecutive days on (5 / 2 / 5), or any dosing schedule deemed appropriate for the situation.

[0168] The dosage of the composition comprising the HDACI or the composition comprising the same may be about 1 ng / kg to about 200 mg / kg, about 1 μg / kg to about 100 mg / kg, or about 1 mg / kg to about 50 mg / kg of body weight. The dosage of the composition may be any dosage, including but not limited to about 1 μg / kg, 10 μg / kg to 200 mg / kg. The dosages above are exemplary of the average case, but there may be individual cases that merit higher or lower dosages, which are within the scope of this disclosure. In practice, the physician will determine the actual dosage regimen that is most appropriate for the individual patient, which may vary with the age, weight, and response of the patient.

[0169] The HDACIs used in the methods of the present disclosure are typically administered in amounts of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the HDACIs can be administered in amounts of about 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 milligrams per dose, including all doses between 0.005 and 500 milligrams.

[0170] The HDACIs of the present disclosure are typically administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present disclosure are formulated in a conventional manner using one or more physiologically acceptable carriers that contain excipients and adjuvants that facilitate processing of the HDACI.

[0171] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with the HDACI. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary substances, stabilizers, thickeners, lubricants, and colorants can be used. The pharma-ceutically acceptable carriers are sterile. When the HDACI is administered intravenously, water is a preferred carrier. For example, saline solution and aqueous dextrose and glycerol solutions can also be employed as liquid carriers for injections. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0172] These pharmaceutical compositions may be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Appropriate formulations depend on the route of administration selected. When a therapeutically effective amount of the present HDACI is administered orally, the composition is usually in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition may further comprise a solid carrier, such as gelatin or an adjuvant. The tablets, capsules, and powders contain about 0.01% to about 95%, preferably about 1% to about 50%, of the present HDACI. When administered in liquid form, a liquid carrier, such as water, petroleum, or oil of animal or vegetable origin, may be added. The liquid form of the composition may further comprise saline, dextrose or other saccharide solution, or glycol. When administered in liquid form, the composition contains about 0.1% to about 90% by weight, preferably about 1% to about 50% by weight of the present compound.

[0173] When a therapeutically effective amount of the HDACI is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. It is within the skill of the artisan to prepare such a parenterally acceptable solution, taking into due consideration pH, isotonicity, stability, etc. Preferred compositions for intravenous, cutaneous, or subcutaneous injection usually include an isotonic medium. The HDACI may be infused with other liquids over 10-30 minutes or over several hours.

[0174] The HDACI can be easily mixed with pharma- ceutically acceptable carriers known in the art. Such carriers allow the formulation of the active agent as a tablet, pill, dragee, capsule, liquid, gel, syrup, slurry, suspension, etc., for oral ingestion by the patient to be treated. A pharmaceutical preparation for oral use can be obtained by adding the HDACI to a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries as necessary, and then processing the granules of the mixture to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added as needed.

[0175] The HDACI can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.The preparation for injection can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with the addition of preservatives.The composition can take the form of suspension, solution, or emulsion in oily or aqueous medium, and can contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0176] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form.Furthermore, the suspension of the HDACI can be prepared as a suitable oily injection suspension.Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension.

[0177] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds and allow for the preparation of highly concentrated solutions. Alternatively, the composition may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0178] The HDACI can also be formulated into rectal compositions, such as suppositories containing conventional suppository bases or retention enemas.In addition to the formulations described above, the HDACI can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, the HDACI can be formulated with suitable polymers or hydrophobic materials (e.g., as an emulsion in acceptable oil) or ion exchange resins.

[0179] In some embodiments, the HDACI may be administered orally, bucally, or sublingually in the form of a tablet containing excipients such as starch or lactose, or in a capsule or suppository, alone or in a mixture with excipients, or in the form of an elixir or suspension containing flavoring or coloring agents. Such liquid preparations may be prepared with pharma- ceutically acceptable additives, such as suspending agents. The HDACI may also be parenterally injected, for example, intravenously, intramuscularly, subcutaneously, or intracoronarily. For parenteral administration, the HDACI is most suitable for use in the form of a sterile aqueous solution, which may contain other substances, such as salts or monosaccharides, such as mannitol or glucose, to make the solution isotonic with blood.

[0180] In further embodiments, the present disclosure includes kits containing one or more compounds or compositions packaged in a manner that facilitates their use in practicing the methods of the present disclosure. In one simple embodiment, the kit includes a compound or composition described herein as useful for practicing the methods (e.g., the present HDACI and optional second treatments) packaged in a container, such as a sealed bottle or container. factorThe composition comprises a compound or composition that is a soluble ...

[0181] Due to the fact that some conventional HDACIs often exhibit activity against several known HDACs, they are not suitable for the treatment of diseases other than cancer. factor Thus, an important feature of the present disclosure relates to the fact that the compounds of the present disclosure exhibit isoform selectivity. The compounds exhibit increased inhibitory potency and selectivity for HDAC6 compared to other HDACs, e.g., greater selectivity for class II over class I. The improved properties of the compounds indicate that they should be useful as immunosuppressants and neuroprotectants, as well as for applications including, but not limited to, Alzheimer's disease, depression, Rett syndrome, Charcot-Marie-Tooth disease, brain cancer, and the like. For example, the compounds of the present disclosure typically exhibit a higher binding affinity (IC) for HDAC6. 50 ) is less than 1 μM, and in some cases, less than 10 nM.

[0182] In some aspects, the present disclosure provides the following specific embodiments.

[0183] Embodiment 1. A compound of formula I above, or a pharma- ceutical acceptable salt thereof, wherein R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R 2 are linked to form a 3- to 7-membered heterocyclyl, L 1is CO2H, C(O)NH2, C(O)NHOH, or B(OH)2, and L 2 is H or OR 3 and R 3 is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl; each X is independently hydrogen or halogen; p is 0, 1, 2, or 3; Y and Z are independently selected from the group consisting of carbon and nitrogen; m is 1, 2, 3, or 4; and n is 0, 1, or 2; or a pharma- ceutically acceptable salt thereof.

[0184] Embodiment 2.R 1 , R 2 and R 3 is independently C1-C6 branched alkyl, or a pharma- ceutically acceptable salt thereof.

[0185] Embodiment 3.L 1 is C(O)NHOH, or a pharma- ceutically acceptable salt thereof.

[0186] Embodiment 4. The compound of embodiment 1, wherein said compound is of formula Ib, or a pharma- ceutically acceptable salt thereof.

[0187] Embodiment 5. The compound of embodiment 1, wherein said compound is of formula Ic, or a pharma- ceutically acceptable salt thereof.

[0188] Embodiment 6. The compound of embodiment 1, wherein the compound is of formula Id, or a pharma- ceutically acceptable salt thereof.

[0189] Embodiment 7. The compound of embodiment 1, wherein said compound is of formula Ie, or a pharma- ceutically acceptable salt thereof.

[0190] Embodiment 8. The compound of embodiment 1, or a pharma- ceutically acceptable salt thereof, wherein the compound is compound 6a, 6b, 6d, 6e, or 6f.

[0191] Embodiment 9. (a) a compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f; and (b) a second therapy useful for treating a disease or condition benefiting from inhibition of HDAC. factor and (c) optionally an excipient and / or a pharma- ceutically acceptable carrier.

[0192] Embodiment 10. The second treatment factor 10. The composition of embodiment 9, wherein said composition comprises a chemotherapeutic agent useful for the treatment of cancer.

[0193] Embodiment 11. A pharmaceutical composition comprising a compound of any one of formulas I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and a pharma- ceutically acceptable carrier or vehicle.

[0194] Embodiment 12. Use of a compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f for the manufacture of a medicament for the treatment of a disease or condition in an individual benefiting from inhibition of HDAC.

[0195] Embodiment 13. The use according to embodiment 12, wherein said HDAC is HDAC6.

[0196] Embodiment 14. Additionally, a second therapy useful in treating said disease or condition. factor The use of embodiment 12, comprising administering a therapeutically effective amount of

[0197] Embodiment 15. A compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and said second treatment factor The use of embodiment 12, wherein said

[0198] Embodiment 16. A compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and said second treatment factor The use of embodiment 12, wherein said administering agent is administered separately.

[0199] Embodiment 17. The use according to any one of embodiments 12 to 16, wherein the disease or condition is cancer.

[0200] Embodiment 18. The disease is cancer and the second treatment factor The use according to any one of embodiments 12 to 17, wherein the therapeutic agent is one or more of a chemotherapeutic agent, radiation, and immunotherapy.

[0201] Embodiment 19. The use of embodiment 18, wherein the immunotherapy comprises anti-PD1 immunotherapy.

[0202] Embodiment 20. The use of embodiment 19, wherein the anti-PD1 immunotherapy comprises administration of a PD-1 antibody.

[0203] Embodiment 21. The use of embodiment 20, wherein the PD-1 antibody is nivolumab, pembrolizumab, STI-A1014, or pidilizumab.

[0204] Embodiment 22. The second treatment factor comprises radiation, said radiation optionally comprising a radiosensitizer and / or a treatment factor The use according to any one of embodiments 14 to 21, administered in combination with

[0205] Embodiment 23. The use of any one of embodiments 12 to 22, wherein the disease or condition is a neurological disease, a neurodegenerative disorder, a peripheral neuropathy, or a traumatic brain injury.

[0206] Embodiment 24. The use according to any one of embodiments 12 to 23, wherein the disease or condition is stroke.

[0207] Embodiment 25. The use of any one of embodiments 12 to 24, wherein the disease or condition is an inflammatory or autoimmune disease.

[0208] Embodiment 26. Additionally, a second therapy useful for treating said autoimmune disease or said inflammation. factor The use of embodiment 25, comprising administering a therapeutically effective amount of

[0209] Embodiment 27. Use of a compound of any one of formulas I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, for increasing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy.

[0210] Embodiment 28. A kit comprising a compound of any one of formulas I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and instructions for administering said compound, or a pharma- ceutically acceptable salt thereof, to a subject in need thereof.

[0211] Embodiment 29. The kit of embodiment 28, wherein the subject has cancer.

[0212] Embodiment 30. The kit of embodiment 29, further comprising an anti-PD1 antibody.

[0213] Embodiment 31. A compound of any one of formulas I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, for use in treating a disease or condition in an individual that would benefit from inhibition of HDAC.

[0214] Embodiment 32. A compound for use according to embodiment 31, wherein said HDAC is HDAC6.

[0215] Embodiment 33. Additionally, a second therapy useful in treating the disease or condition. factor 32. The compound for use according to embodiment 31, comprising administering a therapeutically effective amount of

[0216] Embodiment 34. A compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and the second treatment factor The compound for use according to embodiment 31, wherein said compound is administered simultaneously.

[0217] Embodiment 35. A compound of any one of formula I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, and the second treatment factor The compound for use according to embodiment 31, wherein said compound is administered separately.

[0218] Embodiment 36. A compound for use according to any one of embodiments 31 to 35, wherein the disease or condition is cancer.

[0219] Embodiment 37. The disease is cancer and the second treatment factor The compound for use according to any one of embodiments 31 to 36, wherein is one or more of a chemotherapeutic agent, radiation, and immunotherapy.

[0220] Embodiment 38. The compound for use according to embodiment 37, wherein said immunotherapy comprises anti-PD1 immunotherapy.

[0221] Embodiment 39. The compound for use according to embodiment 38, wherein said anti-PD1 immunotherapy comprises administration of a PD-1 antibody.

[0222] Embodiment 40. The compound for use according to embodiment 39, wherein the PD-1 antibody is nivolumab, pembrolizumab, STI-A1014, or pidilizumab.

[0223] Embodiment 41. The second treatment factor comprises radiation, said radiation optionally comprising a radiosensitizer and / or a treatment factor The compound for use according to any one of embodiments 33 to 40, administered in combination with

[0224] Embodiment 42. A compound for use according to any one of embodiments 31 to 41, wherein the disease or condition is a neurological disease, a neurodegenerative disorder, a peripheral neuropathy, or a traumatic brain injury.

[0225] Embodiment 43. A compound for use according to any one of embodiments 31 to 42, wherein the disease or condition is stroke.

[0226] Embodiment 44. A compound for use according to any one of embodiments 31 to 43, wherein the disease or condition is an inflammatory or autoimmune disease.

[0227] Embodiment 45. Additionally, a second therapy useful for treating said autoimmune disease or said inflammation. factor 45. The compound for use according to embodiment 44, comprising administering a therapeutically effective amount of

[0228] Embodiment 46. A compound of any one of formulas I, Ib, Ic, Id, or Ie, or compounds 6a, 6b, 6d, 6e, or 6f, for use in increasing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy.

[0229] The foregoing may be better understood with reference to the following examples, which are presented for illustrative purposes and are not intended to limit the scope of the disclosure. EXAMPLES

[0230] General information 1 H and 13C NMR spectra were obtained on Bruker spectrometers at 400 / 101 and 500 / 126 MHz using residual peaks of the solvent as internal standard (chemical shifts: CDCl3, δ 7.26 / 77.16 and DMSO-d6, 2.50 / 39.52) unless otherwise stated. The following abbreviations were used for multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br s = broad singlet. TLC plates (Merck silica gel 60 F 254 The progress of the reaction was monitored using a column chromatography (50×2.1 mm, id), and the spots were visualized under UV (254 nm). High-resolution mass spectrometry (HRMS) was performed on a Shimadzu IT-TOF instrument under the following conditions: column, ACE 3AQ (50×2.1 mm, id), mobile phase, 5-100% acetonitrile / water with 0.1% formic acid, flow rate 0.5 mL / min for 4 min. Flash chromatography was performed on a Combi-Flash® Rf system (Teledyne ISCO) equipped with a silica gel cartridge. Preparative HPLC was used for the purification of all final compounds, and a Shimadzu preparative LC was used under the following conditions: column, ACE 5AQ (150×21.2 mm, id), mobile phase: 5-100% acetonitrile / water with 0.05% TFA, flow rate 17 mL / min for 30 min, UV detection at 254 and 280 nm. Analytical HPLC was performed on an Agilent 1260 series instrument with the following conditions: column, ACE 3 (150 x 4.6 mm, id), mobile phase, 5-100% acetonitrile / water with 0.05% TFA, flow rate 1.0 mL / min for 25 min, UV detection at 254 nm. Purity of all test compounds for in vitro biological testing was >95%. Purity of Suprastat for crystallographic studies and in vivo testing was >98%.

[0231] [ka] Phenyl (4-(((tert-butoxycarbonyl)amino)methyl)phenyl)carbamate (2). To a stirred solution of tert-butyl (4-aminobenzyl)carbamate (1, 500 mg, 2.25 mmol) and K2CO3 (373 mg, 2.70 mmol) in acetone (15 mL) was added phenyl chloroformate (352 mg, 2.25 mmol) over 10 min. After stirring at room temperature for 2 h, excess solids were filtered off. The filtrate was collected and concentrated in vacuo. The crude product was purified by flash chromatography (0-50% EtOAc / Hexanes) to give 2 as a pale yellow solid (700 mg, 91% yield). 1 H NMR(400MHz, CDCl3)δ 7.41-7.37(m,4H),7.25-7.16(m,6H),4.86(s,1H),4.27(d,J=4.7Hz,2H),1.46(s,9H). 13 C NMR(101MHz, CDCl3)δ 156.1,151.8,150.7,136.8,134.6,129.5(2C),128.4(2C),125.8,121.8(2C),119.1(2C),79.7,44.3,28.5(3C).

[0232] [ka] Methyl 4-(((4-hydroxybutyl)amino)methyl)benzoate (4a). (i) A solution of 4-amino-1-butanol (0.28 mL, 6.10 mmol) and methyl-4-formylbenzoate (3a, 500 mg, 3.05 mmol) in EtOH (25 mL) was heated to reflux for 2 h. After cooling to room temperature, the mixture was concentrated and the crude product was used directly in the next step. (ii) To a stirred solution of this crude product in MeOH (50 mL) was added sodium borohydride (116 mg, 3.10 mmol) at 0° C. over 10 min. The mixture was allowed to warm to room temperature and stirred at room temperature for 2 h. The reaction was then quenched with water and extracted with EtOAc (15 mL×3). The combined organic layers were washed with brine, dried over Na2SO4 and concentrated in vacuo to give 4a as a colorless oil. This product was used directly in the next step without further purification (630 mg, yield: 87% for two steps). 1 H NMR(400MHz,CDCl3)δ 7.98(d,J=8.1Hz,2H),7.36(d,J=8.0Hz,2H),3.88(s,3H),3.82(s,2H),3.58(t,J=5.0Hz,2H),3.07(br s,2H,NH+OH),2.67(t,J=5.4Hz,2H),1.71-1.53(m,4H). 13 C NMR(101MHz, CDCl3)δ 167.0,144.6,130.0(2C),129.2,128.3(2C),62.7,53.6,52.2,49.4,32.2,28.4.

[0233] [ka] Methyl 4-((butylamino)methyl)benzoate (4b) was synthesized from n-butylamine (0.31 mL, 6.10 mmol) and methyl-4-formylbenzoate (3a, 500 mg, 3.05 mmol) using a procedure similar to that described for the synthesis of 4a and obtained as a colorless oil (500 mg, yield: 74% for two steps). 1H NMR(400MHz,CDCl3)δ 7.99(d,J=8.3Hz,2H),7.39(d,J=8.3Hz,2H),3.90(s,3H),3.84(s,2H),2.7 5-2.51(m,2H),1.53-1.45(m,2H),1.39-1.30(m,2H),0.91(t,J=7.3Hz,3H). 13 C NMR(101MHz, CDCl3)δ 166.7,145.9,129.5(2C),128.5,127.7(2C),53.5,51.7,49.0,32.1,20.3,13.8.

[0234] [ka] 4-(((4-Hydroxybutyl)amino)methyl)benzonitrile (4c) was synthesized from 4-amino-1-butanol (0.10 mL, 1.0 mmol) and 4-formylbenzonitrile (3b, 131 mg, 1.0 mmol) using a similar procedure as described for the synthesis of 4a and obtained as a colorless oil (120 mg, yield: 59% for two steps).

[0235] [ka] 4-((4-Bromobenzyl)amino)butan-1-ol (4d) was synthesized from 4-amino-1-butanol (0.276 mL, 3 mmol) and 4-bromobenzaldehyde (3c, 550 mg, 3 mmol) using a similar procedure as described for the synthesis of 4a and obtained as a colorless oil (0.53 g, yield: 68% for two steps).

[0236] [ka] Methyl 4-((3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)-1-(4-hydroxybutyl)-ureido)methyl)-benzoate (5a). To a stirred solution of 4a (284 mg, 1.2 mmol) and 2 (350 mg, 1.0 mmol) in THF (10 mL) was added TEA (0.28 mL, 2.0 mmol). The resulting mixture was heated to reflux for 2 h. The reaction was then cooled to room temperature, quenched with water (10 mL), and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue was purified by flash chromatography (0-50% EtOAc / Hexane) to give 5a as a colorless oil (420 mg, yield: 87%). 1 H NMR(400MHz,CDCl3)δ 7.94(d,J=8.1Hz,2H),7.65(br s,1H),7.29(d,J=8.1Hz,4H),7.05(d,J=8.1Hz,2H),5.10(br s,1H),4.56(s,2H),4.14(s,2H),3.87(s,3H),3.65(d,J=4.9Hz,2H),3.53(br s,1H,-OH),3.31(d,J=6.2Hz,2H),1.64(s,2H),1.53-1.45(m,2H),1.40(s,9H). 13 C NMR(101MHz,CDCl3)δ 167.0,156.2,156.1,143.7,138.7,133.2,130.0(2C),129.2,127.8(2C),127 .3(2C),120.3(2C),79.5,62.5,52.2,49.9,46.9,44.1,28.4(3C),27.6,25.4.

[0237] [ka] Methyl 4-((3-(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)-1-butylureido)-methyl)benzoate (5b) was synthesized from 4b (265 mg, 1.2 mmol) and 2 (350 mg, 1.0 mmol) using a similar procedure as described for the synthesis of 5a and obtained as a colorless oil (400 mg, yield: 85%). 1H NMR (400MHz, CDCl3) δ 7.98(d,J=8.2Hz,2H),7.33(d,J=8.1Hz,2H),7.24(d,J=8.4Hz,2H),7.12(d,J= 8.0Hz,2H),6.52(d,J=6.6Hz,1H),4.92(s,1H),4.60(s,2H),4.19(d,J=5.1Hz,2 H),3.89(s,3H),3.40-3.23(m,2H,-OH),1.99(t,J=10.2Hz,1H),1.59(dd,J=14. 7,7.2Hz,2H),1.42(s,9H),1.32(dd,J=14.9,7.4Hz,2H),0.91(t,J=7.3Hz,3H). 13 C NMR(101MHz,CDCl3)δ 166.9,156.0,155.5,143.3,138.2,133.7,130.2(2C),129.4,128.1(2C),1 27.1,120.3(2C),79.4,52.2,50.4,47.7,44.2,30.5,28.5(3C),20.2,13.9.

[0238] [ka] Methyl tert-butyl (4-(3-(4-cyanobenzyl)-3-(4-hydroxybutyl)ureido)benzyl)carbamate (5c) was synthesized from 4c (120 mg, 0.58 mmol) and 2 (200 mg, 0.58 mmol) using a similar procedure as described for the synthesis of 5a and obtained as a colorless oil (80 mg, yield: 30%). 1 H NMR(400MHz,CDCl3)δ 7.80(s,1H),7.57(d,J=7.9Hz,2H),7.43-7.29(m,4H),7.07(d,J=7.9Hz,2H),5.01( s,1H),4.58(s,2H),4.16(d,J=4.5Hz,2H),3.72(s,2H),3.35-3.32(m,2H),3.27(br s, 1H), 1.69 (s, 2H), 1.54 (s, 2H), 1.42 (s, 9H). 13C NMR(101MHz,CDCl3)δ 156.0,155.9,144.1,138.6,133.1,132.3(2C),128.0(2C),127.7(2C),120 .1(2C),118.7,110.9,79.4,62.6,49.8,46.8,44.1,28.3(3C),27.0,25.5.

[0239] [ka] tert-Butyl (4-(3-(4-bromobenzyl)-3-(4-hydroxybutyl)ureido)benzyl)carbamate (5d) was synthesized from 4d (520 mg, 2 mmol) and 2 (680 mg, 2 mmol) using a procedure similar to that described for the synthesis of 5a and obtained as a colorless oil (960 mg, 95%). 1 H NMR(400MHz,DMSO-d6)δ 8.35(s,1H),7.52(d,J=8.3Hz,2H),7.37(d,J=8.1Hz,2H),7.29(br.t,1H),7.22(d,J=8.3Hz,2H),7.08(d,J=8.1Hz,2H),4.52(s,1H),4.48(br t,2H),4.03(d,J=5.6Hz,2H),3.39-3.37(m,2H),3.29-3.27(m,2H),1.58-1.46(m,2H),1.45-1.35(m,2H),1.36(s,9H).

[0240] [ka] 4-((3-(4-(aminomethyl)phenyl)-1-(4-hydroxybutyl)ureido)methyl)-N-hydroxybenz-amide (6a, Suprastat). (i) In a round bottom flask, NaOH (68 mg, 1.69 mmol) was dissolved in 50% aqueous NH2OH (0.7 mL, ca. 50 equiv.) at 0 °C. A solution of 5a (200 mg, 0.43 mmol) in 1:1 THF / MeOH (2 / 2 mL) was added dropwise and stirring was continued for 30 min while warming to room temperature. The solution was neutralized with 2N HCl and extracted with EtOAc (10 mL x 3). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The crude product was used directly in the next step without further purification. (ii) The crude product was dissolved in THF (2 mL) followed by the addition of TFA (3 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h and then the excess solvent was removed in vacuo. The crude product was purified by preparative HPLC and lyophilized to give 6a as a white powder (62 mg, TFA salt, purity: 95%, yield: 29% for two steps). 1 H NMR(500MHz,DMSO-d6)δ 11.17(br s,1H),8.51(s,1H),8.07(br s,3H,NH3 + ),7.72(d,J=8.7Hz,2H),7.50(d,J=8.7Hz,2H),7.32(d,J=6.6Hz,2H),7.30(d,J=7.0Hz,2H),4.61(s,2H),3.94(q,J=5 .8Hz,3H),3.39(t,J=6.4Hz,2H),3.33(t,J=7.4Hz,2H),1.54(ddd,J=9.2,6.4,2.5Hz,2H),1.41(dt,J=8.7,6.5Hz,2H). 13 C NMR(126MHz,DMSO-d6)δ 164.0,155.1,142.2,140.8,131.4,129.1(2C),127.01(2C),126.97(2C),126.9,119.7(2C),60.6,49.0,46.3,42.0,29.4,24.6. ESI HRMS C 20 H 25 Calculated value for N4O4: [MH] - , m / z 385.1881; measured value: 385.1871.

[0241] [ka] 4-((3-(4-(aminomethyl)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide (6b) was synthesized from 5b (420 mg, 0.87 mmol) using a procedure similar to that described for the synthesis of 6a and obtained as a white solid (160 mg, TFA salt, purity: >99%, yield: 77% for two steps). 1 H NMR(400MHz,DMSO-d6)δ 11.17(br s,1H),9.00(br s,1H),8.48(s,1H),8.01(br s,3H,NH3 + ),7.72(d,J=8.2Hz,2H),7.50(d,J=8.6Hz,2H),7.32(d,J=6.4Hz,2H),7.30(d,J=6.8Hz,2H),4.61(s,2H),3.9 3(t,J=5.6Hz,2H),3.30-3.26(m,2H,overlapping with water peak),1.53-1.40(m,2H),1.31-1.16(m,2H),0.86(t,J=7.3Hz,3H). 13 C NMR(126MHz,DMSO-d6)δ 164.0,155.1,142.2,140.8,131.4,129.1(2C),127.0,126.9(4C),119.8(2C),49.0,46.2,42.0,29.9,19.4,13.8. ESI HRMS C 20 H 25 Calculated value for N4O3: [MH] - , m / z 369.1932; measured value: 369.1923.

[0242] [ka] 4-((3-(4-(aminomethyl)phenyl)-1-(4-hydroxybutyl)ureido)methyl)benzoic acid (6d). To a stirred solution of 5a (62 mg, 0.13 mmol) in THF / MeOH (1 / 1 mL) was added 1N NaOH solution (1 mL). The resulting mixture was then stirred at room temperature overnight. After completion of the reaction was confirmed by HPLC, the reaction was acidified with 2N HCl and extracted with THF (15 mL×3). The combined organic extracts were washed with H2O and brine, dried over Na2SO4, and concentrated in vacuo. The crude product was dissolved in THF (2 mL) followed by addition of TFA (3 mL) at room temperature. The resulting mixture was stirred at room temperature for an additional 0.5 h, then excess solvent was removed in vacuo. The crude product was purified by preparative HPLC and lyophilized to give 6d as a white powder (40 mg, TFA salt, purity: 95%, yield: 63% for two steps). 1 H NMR(500MHz,DMSO-d6)δ 12.92(br s,1H),8.53(s,1H),8.07(br s,3H,NH3 + ),7.91(d,J=8.3Hz,2H),7.51(d,J=8.6Hz,2H),7.37(d,J=8.2Hz,2H),7.30(d,J=8.7Hz,2H),4.65(s,2H),3.94 (q,J=5.6Hz,2H),3.39(t,J=6.4Hz,2H),3.34(t,J=7.5Hz,2H),1.54(tt,J=8.2,6.3Hz,2H),1.45-1.32(m,2H). 13 C NMR(126MHz,DMSO-d6)δ 167.2,155.2,144.2,140.8,129.5(2C),129.4,129.1(2C),127.1(2C),127.0,119.8(2C),60.6,49.1,46.4,42.0,29.4,24.6. ESI HRMS C 20 H 24 Calculated value for N3O4: [MH] - , m / z 370.1772; measured value: 370.1769.

[0243] [ka] 4-((3-(4-(aminomethyl)phenyl)-1-(4-hydroxybutyl)ureido)methyl)benzamide (6e). To a stirred solution of 5c (80 mg, 0.18 mmol) in DMSO (2 mL) was added K2CO3 (2.4 mg, 0.018 mmol) and H2O2 (30%, 0.2 mL) at room temperature. The resulting mixture was stirred for 5 h. After completion of the reaction, the solution was quenched with water (5 mL) and extracted with EtOAc (10 mL x 3). The organic layer was separated, washed with brine, dried over Na2SO4 and concentrated under vacuum. The crude product was dissolved in THF (2 mL) followed by addition of TFA (3 mL) at room temperature. The resulting mixture was stirred at room temperature overnight and then the excess solvent was removed under vacuum. The crude product was purified by preparative HPLC and lyophilized to give 6e as a white powder (30 mg, TFA salt, purity: 98%, yield: 35% for two steps). 1 H NMR(500MHz,DMSO-d6)δ 8.51(s,1H),8.06(br s,3H,NH3 + ), 7.92 (br s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.6 Hz, 2H), 7.32-7.29 (m, 5H, one proton of CONH2 overlaps with the signal of the phenyl ring), 4.62 (s, 2H), 3.94 (q, J = 5.8 Hz, 3H), 3.39 (t, J = 6.4 Hz, 2H), 3.33 (t, J = 7.5 Hz, 2H), 1.54 (ddd, J = 12.1, 8.8, 6.4 Hz, 2H), 1.45-1.34 (m, 2H). 13 C NMR(126MHz,DMSO-d6)δ 167.6,155.1,142.3,140.9,133.0,129.1(2C),129.0,127.6(2C),126.8(2C),126.7,119.7(2C),60.6,49.0,42.0,29.4,24.6. ESI HRMS C 20 H 27 Calculated value for N4O3: [M+H] + , m / z 371.2078; measured value: 371.2087.

[0244] [ka] (4-((3-(4-(aminomethyl)phenyl)-1-(4-hydroxybutyl)ureido)methyl)phenyl)boronic acid (6f). (i) To a stirred solution of 5d (510 mg, 1.0 mmol), bis(pinacolato)diboron (280 mg, 1.1 mmol), and potassium acetate (290 mg, 3 mmol) in DMF (5 mL) was added Pd(dppf)Cl2 at room temperature under argon atmosphere. After stirring at 80 °C overnight, the reaction was cooled to room temperature and excess solids were filtered off. The filtrate was collected, quenched with saturated aqueous NaHCO3 (20 mL), and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with 10% aqueous LiCl, washed with brine (30 mL), dried over Na2SO4, and concentrated in vacuo. The crude product was purified by flash chromatography (0-50% EtOAc / Hexane) to give the pinacol ester intermediate as a brown solid (0.25 g, yield: 44%). (ii) To a solution of the pinacol ester intermediate (240 mg, 0.44 mmol) in acetone / water (2:1, 9 mL) was added NaIO4 (280 mg, 1.32 mmol) and NHOAc (100 mg, 1.32 mmol) at room temperature. After stirring at room temperature overnight, the reaction mixture was quenched with water and extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine (30 mL), dried over Na2SO4, and concentrated in vacuum. The crude product was used directly in the next step without further purification. (iii) The crude product was dissolved in THF (1 mL) followed by the addition of TFA (1 mL) at room temperature. The resulting mixture was stirred at room temperature for 0.5 h, and then the excess solvent was removed under vacuum. The crude product was purified by preparative HPLC and lyophilized to give 6f as a white powder (190 mg, TFA salt, purity: 99%, yield: 39% over three steps). 1 H NMR(400MHz,DMSO-d6)δ 8.49(s,1H),8.06(br s,3H,NH3 +),7.74(d,J=7.5Hz,2H),7.50(d,J=8.2Hz,2H),7.30(d,J=8.2Hz,2H),7.21(d,J=7.6Hz,2H),4.57(s, 2H),3.95(s,2H),3.48(t,J=5.7Hz,2H),3.31(t,J=6.7Hz,2H),1.61-1.57(m,2H),1.46-1.43(m,2H). ESI HRMS C 19 H 27 Calculated value for BN3O4 [M+H] + : m / z 372.2089, actual value: 372.2098.

[0245] Expression and purification of HDAC1-9 and 11 Large-scale expression of human HDACs was performed in HEK293 / T17 cells essentially as previously described (e.g., Falkenberg et al., Nat. Rev. Drug Discov. 13, 673-691 (2014) or Matthias et al. (see, e.g., et al., Cell Cycle 7:7-10 (2008)). Briefly, transiently transfected cells were harvested 3 days after transfection and the cell pellet was resuspended in lysis buffer (50 mM Tris, 150 mM NaCl, 10 mM KCl, 2 mM MgCl2, 10% glycerol, 0.2% NP-40, 2 units / mL benzonase, pH 8) supplemented with a cocktail of protease inhibitors (Roche, Basel, Switzerland). Cells were lysed by sonication (30 W, 3 x 20 sec) on ice and the cell lysate was clarified by centrifugation at 40000 x g for 30 min at 4 °C. Recombinant fusion HDAC proteins were purified by Strep-Tactin affinity chromatography (IBA, Goettingen, Germany) in an elution buffer containing 50 mM HEPES, 100 mM NaCl, 50 mM KCl, 10% glycerol, and 3 mM desthiobiotin, pH 7.5. Purified proteins were concentrated to 1 mg / mL, aliquoted, flash frozen in liquid nitrogen, and stored at -80°C until further use.

[0246] Measurement of inhibitory activity against HDAC1-9 and 11 Table 1 IC 50 Values ​​were measured using a fluorescence-based assay (e.g., Immunohistochemistry) with 10 μM Ac-GAK(Ac)-AMC (HDACs 1, 2, 3, 6) or 10 μM Boc-Lys(TFA)-AMC (HDACs 4, 5, 7, 8, 9, 11) as substrate (e.g., Immunohistochemistry). (See, et al., Cancer Sci. 107:1543-1549 (2016)). Briefly, individual HDACs were preincubated with a dilution series of test inhibitors (0–100 μM) in a 384-well plate in a total volume of 40 μL for 10 min at 37°C in a reaction buffer containing 50 mM HEPES, 140 mM NaCl, 10 mM KCl, 1 mM TCEP, 0.1% BSA, pH 7.4. The deacetylation reaction was initiated by adding 10 μL of 10 μM substrate to the HDAC / inhibitor mixture. After 30 min of incubation at 37°C, the reaction was terminated by adding 25 μL of trypsin solution (4 mg / mL). Trypsin-induced fluorescence development was performed for 15 and 60 min at 37°C for Ac-GAK(Ac)-AMC and Boc-Lys(TFA)-AMC substrates, respectively. The released aminomethylcoumarin was quantified using a CLARIOstar fluorometer with excitation and emission wavelengths set at 365 and 440 nm, respectively. Nonlinear regression analysis was employed to determine the IC using GraphPad Prism software. 50 IC values ​​were calculated using a 3-fold dilution series of inhibitors, with 14 IC 50 Curves were generated using the following inhibitor concentration ranges: 100 μM to 0.063 pM for HDACs 1-5, 7-9, 11, and 3 μM to 1.88 pM for HDAC 6. Reactions without enzyme or inhibitor were used to define 0% and 100% HDAC activity, respectively.

[0247] Cell culture and antibodies SM1 mouse melanoma cells were obtained from Dr. A. Ribas, University of California, Los Angeles. WM164 human melanoma cells were obtained from ATCC. These cells were cultured in RPMI1640, 1% penicillin-streptomycin, and 10% fetal bovine serum in an incubator at 37°C and 5% CO2. HDAC inhibitors including 6a-c and NextA were added at concentrations of 0.1 μM, 0.5 μM, 1 μM, 2.5 μM, 5 μM, and 10 μM and incubated overnight. For STAT3 phosphorylation assay, cells were pretreated with or without HDAC6i (5 μM) overnight and then treated with recombinant human IL-6 (Biolegend) for 20 min. PBS was added as a control. RAW264.7 macrophages were purchased from ATCC and cultured in DMEM medium supplemented with 10% FBS, 1% non-essential amino acids, and 2-mercaptoethanol (50 μM). RAW macrophages were treated with Suprastat at 1 μM, 5 μM, and 10 μM overnight, after which lysates were collected for immunoblot assays. Cytotoxicity assays were performed using CellTox Green (Promega, Cat#G8731) according to the manufacturer's instructions, and fluorescence measurements were obtained on a Spectramax i3 (Molecular Devices) multimode plate reader at wavelengths EX 485 nm and EM 520 nm. SM1 cells were treated with various concentrations of compounds for 24 hours to measure cytotoxicity.

[0248] Immunoblot analysis Cells were harvested and lysed by sonication in a Bioruptor (Diagenode) in RIPA buffer (ThermoScientific, 89900) containing protease and phosphatase inhibitors (ThermoScientific, 78440) for 8 cycles of 30 s on and 30 s off at high setting. To assess protein expression, total protein samples were heat denatured in SDS sample loading buffer and 15–20 μg of protein was analyzed on 4–20% SDS-PAGE gels (Bio-Rad, 456-1093). Proteins were transferred to low-fluorescence PVDF membranes (Bio-Rad, 1704274) using a Trans-Blot Turbo transfer system (Bio-Rad). These membranes were blocked for 1 h with Odyssey blocking buffer (Licor, 927-40000) and then incubated with primary antibodies (1:1000 dilution) at 4 °C. The membranes were washed three times with TBST buffer and then incubated with near-infrared fluorophore-conjugated secondary antibodies (1:10000 dilution) for 1 hour at room temperature. The membranes were scanned with an Azure Biosystems C600 imager at near-infrared wavelengths. Images were analyzed and processed with Image Studio™ Lite software. The antibodies used were HDAC6 (Assay biotech, C0266), α-tubulin (Cell Signaling, 3873), acetyl-α-tubulin (Cell Signaling, 3971), histone 3 (Cell Signaling, 3638S), and acetyl-histone 3 (Cell Signaling, 9649S).

[0249] Quantitative analysis of gene expression Total RNA was isolated from cells according to the manufacturer's instructions with QIAzol (Qiagen, 79306). RNA quantification was performed using a NanoDrop One spectrophotometer (NanoDrop Technologies). Samples with an absorbance ratio of 260 / 280 greater than 1.9 were used for cDNA synthesis with the iScript cDNA synthesis kit (Bio-Rad, 1708891). cDNA synthesized from 1 μg of total RNA was diluted 1:10 in nuclease-free water. Quantitative PCR analysis was performed on a CFX96 real-time system (Bio-Rad) using iQ SYBR Green Supermix (Bio-Rad, 1708882). Gene expression analysis was performed on a 2-well plate using a 100% PBS buffer. -ΔΔCt PCR was performed using the RT-PCR method and target mRNA levels were normalized to GAPDH expression. Cycling conditions were used as per the manufacturer's instructions. Amplification of a single PCR product was confirmed by melting curve analysis in all experiments performed. The sequences of the primers used in the analysis are as follows:

[0250] mouse Animal studies involving mice were performed according to a protocol (#A354) approved by The George Washington University's Institutional Care and Use Committee (IACUC). Forty C57BL / 6 female mice were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA). In vivo studies were performed with tumor cells that had been passaged a minimum of five times in vivo from mouse to mouse prior to tumor implantation. Mice were inoculated with 1.0 × 10 6In vivo passaged melanoma cells were subcutaneously injected. The pretreatment group was started when the tumors were palpable, approximately 5 days after tumor implantation. Cages were randomly assigned to different treatment groups and mice were treated with Suprastat, anti-PD1 antibody or vehicle control. Control mice were intraperitoneally injected with 100 μL PBS as vehicle control, anti-PD1 antibody (BioXcell, clone RMP1-14) at a dose of 15 mg / kg, and Suprastat at 25 mg / kg. Mice were treated 5 days a week until the tumors in the control group reached their maximum size according to our IACUC protocol. Tumor volumes were measured every other day using caliper measurements and calculated using the formula L × W 2 The mean mean basal t-test was calculated using the RT-PCR method. All animal studies were performed with consideration for toxicity and were monitored regularly for early signs of toxicity. Emphasis was placed on mortality, body weight, and food consumption. Postmortem evaluation of endpoints was performed for each condition, including gross visual inspection of organs such as the liver for hepatotoxicity, splenomegaly, and pulmonary metastatic nodules.

[0251] In vitro evaluation of 6a-c To assess the effect of each additional functional group on potency and isoform selectivity, the potencies of 6a-c against human HDACs 1-9 and 11 were examined with NextA under optimized conditions in vitro (see, e.g., Osko et al., J. Med. Chem. 63:295-308 (2020)). The results in Table 1 suggest that 6a and 6c are more potent and selective HDAC6i (IC 50 =0.4 and 0.5 nM), with at least 290-fold selectivity over the class I isoforms HDAC1-3 and 8, and 1000-fold selectivity over the class IIa isoforms HDAC4, 5, 7, and 9. Furthermore, 6a-c showed no activity against the class IV isoform HDAC11 below 50 μM. For HDAC6, experimental IC 50 Note that the values ​​are at the limit of our assay, which uses a concentration of the full-length enzyme of approximately 0.6 nM (measured by absorbance measurement at 280 nm). Thus, the IC values ​​for Suprastat and 6c are 50The values ​​are at the limit of our assay, and theoretically these two inhibitors may have even higher potency than reported here. Overall, these inhibition data suggest that the hydroxyl butyl chain is more important for improving the enzymatic potency and selectivity of HDAC6 compared to the aminomethyl group. Furthermore, the incorporation of polar aminomethyl and hydroxyl butyl groups into inhibitors 6a-c increases the number of heavy atoms, but their clogP (calculated by SwissADME) decreases compared to NextA (Table 3), which leads to a significant increase in the lipophilic ligand efficiency (LipE), especially for 6a (LipE=7.57 (6a) vs. 6.19 (NexA)), while the ligand efficiency of 6a and 6b is slightly lower. In summary, compared with NextA and related analogs, Suprastat (6a), which has both hydroxybutyl and aminomethyl moieties, exhibits improved potency against HDAC6 and superior selectivity against HDAC isoforms, while also exhibiting the highest LipE value due to its significantly decreased clogP. Meanwhile, the inhibitory potency against HDAC6 of non-hydroxamic acid analogs 6d-f, which contain carboxylic acids, amides, and boronic acids as alternative ZBGs, is greatly compromised (Table 2), with significant hydroxamic acid-Zn 2+ Without coordination, the additional hydrogen bonding interactions between this cap and the pocket of HDAC6 may not prove strong enough to maintain nanomolar potency. [Table 1] [Table 2]

[0252] As part of the initial ADME profiling, the stability of the test compounds was measured in PBS, simulated gastric fluid (SGF), human plasma, and liver microsomes, and protein binding was also measured in human plasma (Table 3). Overall, the stability of Suprastat (6a) is very good, ranging from more than 24 hours in PBS to a half-life of 173 minutes in rat liver microsomes. Consistent with the expected physicochemical properties, the plasma binding of Suprastat is very low (plasma protein bound fraction 1.9%), beneficial for increasing the free drug concentration in vivo compared to the more lipophilic parent compound NextA (89%), while the plasma bound fraction of the singly modified derivatives 6b and 6c is approximately 50%. [Table 3]

[0253] In vitro characterization of 6a-c in melanoma cells To evaluate the potency and isoform selectivity of 6a-c in cells, in vitro analysis was performed using the WM164 human melanoma cell line. WM164 cells are mutated for BRAF V600E, a mutation frequently found in melanoma patients (see, e.g., Daina et al., Sci. Rep. 7:42717 (2017)). WM164 cells were treated with 6a-c and NextA at concentrations ranging from 0.1 to 10 μM, respectively. Their ability to increase levels of acetylated α-tubulin (Ac-α-tubulin) was measured by immunoblot analysis and compared against controls. Figure 1A-D shows an increase in Ac-α-tubulin with increasing concentrations of HDAC6i, but to varying degrees (Figure 1E). However, the increase in Ac-α-tubulin was also associated with a slight increase in the levels of acetylated histone H3 (Ac-H3) at higher concentrations (Figure 1F). Treatment with Suprastat resulted in the highest Ac-α-tubulin levels at 0.1-10 μM, with the largest increase at 10 μM compared to other HDAC6i. At higher concentrations, a slight increase in the levels of Ac-H3 was observed with Suprastat, which was much lower than other HDAC6i. Thus, this data indicates that Suprastat is a highly selective and potent HDAC6 inhibitor. Based on the concentration range of these α-tubulin / histone acetylation experiments, further cytotoxicity assays were performed in SM1 mouse melanoma cells. The results shown in Figure 2 demonstrated that NextA and 6b began to induce cytotoxicity at a concentration of 10 μM, whereas Suprastat and 6c were not cytotoxic up to 25 μM.

[0254] Functional characterization of Suprastat Immune cells such as macrophages are major cellular components of the tumor microenvironment. Tumor-associated macrophages often promote tumors by secreting anti-inflammatory cytokines such as TGFβ and IL-10. It has already been demonstrated that HDAC6 forms a complex with STAT3 and that either pharmacological inhibition or shRNA-mediated knockdown of HDAC6 reduces STAT3 recruitment at the IL10 promoter region in antigen-presenting cells (see, e.g., Cheng et al., J. Immunol. 193:2850-2862 (2014)). Consistent with these experiments, as shown in Figure 3A, treatment of mouse bone marrow-derived macrophages with 5 μM Suprastat reduced the expression of the IL10 gene compared to vehicle-treated macrophages, as judged by mRNA quantification. Gene expression of IL10 is normalized to β-actin (ACTB) as a reference gene. A dose-dependent increase in Ac-α-tubulin levels in RAW264.7 macrophages (Figure 4) was observed upon treatment with increasing concentrations of Suprastat (1, 5, and 10 μM), indicating that Suprastat affects macrophages and melanoma cells in a similar manner. Furthermore, as shown in Figure 3B, immunoblot analysis of lysates obtained from WM164 melanoma cells pretreated with either Suprastat or NextA and then exposed to IL-6 cytokine (30 ng / mL) for 20 min resulted in decreased phosphorylation of Y705 of STAT3 compared to IL-6 alone. This result indicates that Suprastat, like NextA, mediates its immunomodulatory effects by affecting the interaction of HDAC6 and STAT3 transcription factors.

[0255] In vivo combination testing with immunotherapy Immunotherapy has emerged as a primary treatment for solid tumors, but patients often develop resistance, and currently combination therapies are needed to both enhance the efficacy of immunotherapy and overcome resistance (see, for example, O'Donnell et al., Cancer Treat Rev. 52:71-81 (2017)). Using a syngeneic SM1 mouse melanoma model, we previously demonstrated that pretreatment with NextA significantly reduced tumor size in C57BL / 6 mice. These mice have an effective immune system, allowing for the testing of immune checkpoint inhibitors such as anti-PD1 therapy. The combination of NextA and anti-PD1 therapy significantly controlled tumor growth compared to monotherapy, suggesting that inhibition of HDAC6 plays an important role in enhancing anti-tumor immunity. Following a similar approach, C57BL / 6 mice bearing SM1 melanoma tumors were administered 25 mg / kg Suprastat intraperitoneally (IP) before initiating anti-PD1 immune checkpoint blockade therapy (15 mg / kg, IP). This pretreatment method was performed to precondition the tumor microenvironment (TME) in a manner conducive to the induction of antitumor immune responses. As shown in Figure 5A, compared to the control (PBS) group, monotherapy with Suprastat similarly reduced tumor burden, as indicated by tumor volume. However, no significant differences were observed between Suprastat and anti-PD1 therapy. On the contrary, the combination of Suprastat and anti-PD1 therapy showed a significant reduction in tumor burden compared to the control and monotherapy groups, suggesting that pretreatment with Suprastat enhances the antitumor immune response resulting from anti-PD1 therapy. Figure 5B shows the tumor growth of each mouse in each treatment group. The data thus far indicate that Suprastat has an immunomodulatory effect in vivo and, when combined with anti-PD1 therapy, significantly enhances the antitumor immune response. It was noted that the group combined with Suprastat and anti-PD1 therapy showed an enhanced inhibitory effect on tumor growth by day 14, compared to the other groups.In contrast, in our previous study, the combination of NextA and anti-PD1 therapy began to show clear antitumor effects from day 20 onwards compared to the monotherapy group (see, for example, Knox et al., Sci. Rep. 9:6136 (2019)), suggesting that Suprastat can promote immunotherapy at an earlier stage.

[0256] Immunomodulatory Properties of Suprastat To understand the immune-modulatory properties of Suprastat, we performed comprehensive immune cell phenotyping by flow cytometry. The number of F4 / 80+CD80+H2+ anti-tumor M1 macrophages as a percentage of CD45+ cells did not change significantly in all treatment groups (Figure 6A). However, Suprastat significantly reduced F4 / 80+CD206+ tumor-induced M2 macrophages (Figure 6B), thus shifting the balance towards anti-tumor immune responses as indicated by a significantly higher M1 / M2 ratio in the Suprastat and combination groups compared to the control and anti-PD1 groups (Figure 6C). Interestingly, the increase in the M1 / M2 ratio in the combination group was not observed in our previous study with NextA (see, e.g., Knox et al., Sci. Rep. 9:6136 (2019)). Analysis of lymphoid cells showed a significant increase in CD8+ effector T cells and effector memory cells in all treatment groups compared to the control group (Figure 6D), and the fold increase was much greater in all groups than in previous studies with NextA. However, the only increase in the percentage of CD8+ central memory (CM) cells was observed in the Suprastat-treated group, suggesting that it enhances the effector memory function of CD8 T cells and enables long-term antitumor immune responses. Analysis of CD4+ T cells showed no significant changes in central memory (CM) or effector memory (EM) function (Figure 6E). No significant changes were observed in immunosuppressive T-regs (Figure 6F). Further analysis of natural killer (NK) cells showed an increase in the anti-PD1 and combination groups, but with a favorable trend in the Suprastat group (Figure 6G), which was not observed in previous combination studies using NexA (see, e.g., Knox et al., Sci. Rep. 9:6136 (2019)). NK T cells were significantly decreased in all treatment groups compared to the control group (Figure 6H), which can be inferred to be a significant increase in CD8+ effector T cells.Overall, this immune cell phenotyping demonstrated that Suprastat has improved immunomodulatory properties compared to the parent compound NextA by reducing tumor-induced M2 macrophages and increasing the infiltration of anti-tumor CD8+ effector T cells and memory cells, which may contribute to enhanced immunomodulatory effects in vivo and improved anti-tumor immune responses in combination with anti-PD1 therapy.

[0257] conclusion Compared to other related analogues, Suprastat exhibits potent HDAC6 activity, isoform selectivity, and the ability to selectively increase the levels of acetylated tubulin without any apparent effect on histone acetylation. The added polar functional group of Suprastat reduces its lipophilicity, thereby increasing ligand efficacy and plasma protein binding while maintaining metabolic stability in different media. In vivo combination studies with PD-1 antibodies have revealed that Suprastat significantly improves therapeutic outcomes due to its immunomodulatory properties. References 1. S. Zhao, W. KLGuan,Science,2010,327,1000-1004. 2. Q. Wang, Y. Zhang, C. Yang, H. Xiong, Y. Lin, J. Yao, H. Li, L. 3.C.Choudhary,BTWeinert,Y.Nishida,E.Verdin and M.Mann,Nat.Rev.Mol.Cell Biol.,2014,15,536-550. 4. Y. Li and E. Seto, Cold Spring Harb. Perspective. Med.,2016.6. https: / / doi.org / 10.1103 / PhysRevLett.18.1414 , Google Scholar Crossref , CAS 5. T. Eckschlager, J. Plch, M. 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(Item 1) Compounds of Formula I: [ka] or a pharma- ceutical acceptable salt thereof, wherein R 1 and R 2 is independently selected from the group consisting of hydrogen and C1-C6 alkyl, or R 1 and R2 are linked to form a 3- to 7-membered heterocyclyl, L 1 is CO2H, C(O)NH2, C(O)NHOH, or B(OH)2, L 2 is H or OR 3 and R 3 is selected from the group consisting of hydrogen, acetyl, C1-C6 alkyl, C1-C6 haloalkyl, C3-C6 cycloalkyl, aryl, heteroaryl, and C5-C6 heterocyclyl; each X is independently hydrogen or halogen; p is 0, 1, 2, or 3; Y and Z are independently selected from the group consisting of carbon and nitrogen; m is 1, 2, 3 or 4; The compound, or a pharma- ceutically acceptable salt thereof, wherein n is 0, 1 or 2. (Item 2) R 1 , R 2 and R 3 is independently C1-C6 branched alkyl, or a pharma- ceutically acceptable salt thereof. (Item 3) L 1 is C(O)NHOH, or a pharma- ceutically acceptable salt thereof. (Item 4) Compounds according to item 1 of formula Ib: [ka] or a pharma- ceutically acceptable salt thereof. (Item 5) The compound according to item 1 of formula Ic: [ka] or a pharma- ceutically acceptable salt thereof. (Item 6) Compounds according to item 1 of formula Id: [ka] or a pharma- ceutically acceptable salt thereof. (Item 7) Compounds according to item 1 of formula Ie: [ka] or a pharma- ceutically acceptable salt thereof. (Item 8) The compound according to item 1, selected from the group consisting of: [ka] or a pharma- ceutically acceptable salt thereof. (Item 9) (a) a compound according to any one of items 1 to 8; and (b) a second therapeutic agent useful in the treatment of a disease or condition benefiting from inhibition of HDAC. factor and (c) optionally an excipient and / or a pharma- ceutically acceptable carrier. (Item 10) The second treatment factor The composition according to item 9, further comprising a chemotherapeutic agent useful for the treatment of cancer. A pharmaceutical composition comprising a compound according to any one of items 1 to 8 and a pharma- ceutically acceptable carrier or vehicle. (Item 12) A method for treating a disease or condition in which inhibition of HDAC is beneficial, comprising administering to an individual in need thereof a therapeutically effective amount of a compound according to any one of items 1 to 8. (Item 13) 13. The method of claim 12, wherein the HDAC is HDAC6. (Item 14) Additionally, a second therapeutic agent useful in treating said disease or condition. factor 13. The method of claim 12, comprising administering a therapeutically effective amount of (Item 15) the compound and the second treatment factor The method of claim 14, wherein said (Item 16) the compound and the second treatment factor 15. The method of claim 14, wherein said first and second agonists are administered separately. (Item 17) 17. The method according to any one of items 12 to 16, wherein the disease or condition is cancer. (Item 18) the disease is cancer, and factor 18. The method according to any one of items 14 to 17, wherein the therapeutic agent is one or more of a chemotherapy agent, radiation, and / or immunotherapy. (Item 19) 20. The method of claim 18, wherein the immunotherapy comprises anti-PD1 immunotherapy. (Item 20) 20. The method of claim 19, wherein the anti-PD1 immunotherapy comprises administration of a PD-1 antibody. (Item 21) 21. The method of claim 20, wherein the PD-1 antibody is nivolumab, pembrolizumab, STI-A1014, or pidilizumab. (Item 22) The second treatment factor comprises radiation, said radiation optionally comprising a radiosensitizer and / or a treatment factor 22. The method according to any one of items 14 to 21, wherein the method is administered together with (Item 23) 23. The method according to any one of items 12 to 22, wherein the disease or condition is a neurological disease, a neurodegenerative disorder, a peripheral neuropathy, or a traumatic brain injury. (Item 24) 24. The method according to any one of items 12 to 23, wherein the disease or condition is stroke. (Item 25) 25. The method according to any one of items 12 to 24, wherein the disease or condition is an inflammatory or autoimmune disease. (Item 26) Additionally, a second therapy useful for treating said autoimmune disease or said inflammation. factor 26. The method of claim 25, comprising administering a therapeutically effective amount of (Item 27) 9. A method for increasing the sensitivity of a cancer cell to the cytotoxic effects of radiotherapy and / or chemotherapy, comprising contacting said cell with a compound according to any one of items 1 to 8 in an amount sufficient to increase the sensitivity of said cell to said radiotherapy and / or chemotherapy. (Item 28) A kit comprising the compound according to any one of items 1 to 8, or a pharma- ceutically acceptable salt thereof, and instructions for administering the compound or a pharma-ceutically acceptable salt thereof to a subject in need thereof. (Item 29) 29. The kit of claim 28, wherein the subject has cancer. (Item 30) 29. The kit according to item 28, further comprising an anti-PD1 antibody.

Claims

1. Compounds of Formula I: 【Chemistry 23】 or a pharma- ceutical acceptable salt thereof, wherein R １ and R ２ are independently hydrogen and C １ -C ６ alkyl, or R １ and R ２ are linked to form a 3- to 7-membered heterocyclyl, L １ is C(O)NHOH, L ２ is H or OR ３ and R ３ is hydrogen, acetyl, C １ -C ６ Alkyl, C １ -C ６ Haloalkyl, C ３ -C ６ Cycloalkyl, aryl, heteroaryl, and C ５ -C ６ heterocyclyl; Each X is independently a halogen; p is 0, 1, 2, or 3; Y and Z are independently selected from the group consisting of carbon and nitrogen; m is 1, 2, 3 or 4; The compound, or a pharma- ceutically acceptable salt thereof, wherein n is 0, 1 or 2.

2. R １ , R ２ and R ３ But independently, C １ -C ６ 2. The compound of claim 1, or a pharma- ceutically acceptable salt thereof, which is branched alkyl.

3. L １ is C(O)NHOH, or a pharma- ceutically acceptable salt thereof.

4. Compound according to claim 1 of formula Ib: 【Chemistry 24】 or a pharma- ceutically acceptable salt thereof.

5. The compound according to claim 1 of formula Ic: 【Chemistry 25】 or a pharma- ceutically acceptable salt thereof.

6. Compound according to claim 1 of formula Id: 【Chemistry 26】 or a pharma- ceutically acceptable salt thereof.

7. Compound according to claim 1 of formula Ie: 【Chemistry 27】 or a pharma- ceutically acceptable salt thereof.

8. The compound of claim 1 selected from the group consisting of: 【Chemistry 28】 or a pharma- ceutically acceptable salt thereof.

9. A composition comprising (a) a compound according to any one of claims 1 to 8, (b) a second therapeutic agent useful in the treatment of a disease or condition benefiting from inhibition of HDAC, and (c) optionally an excipient and / or a pharma- ceutically acceptable carrier.

10. The composition of claim 9 , wherein the second therapeutic agent comprises a chemotherapeutic agent useful in the treatment of cancer.

11. A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier or vehicle.

12. A composition for treating a disease or condition in which inhibition of HDAC is beneficial, said composition comprising a compound according to any one of claims 1 to 8.

13. The composition of claim 12, wherein the HDAC is HDAC6.

14. 13. The composition of claim 12, wherein the composition is administered in combination with a second therapeutic agent useful in the treatment of the disease or condition.

15. The composition of claim 14 , wherein the composition and the second therapeutic agent are administered simultaneously.

16. The composition of claim 14 , wherein the composition and the second therapeutic agent are administered separately.

17. The composition of any one of claims 12 to 16, wherein the disease or condition is cancer.

18. The composition of any one of claims 14 to 17, wherein the disease is cancer and the second therapeutic agent is one or more of a chemotherapeutic agent, radiation, and / or immunotherapy.

19. 20. The composition of claim 18, wherein the immunotherapy comprises an anti-PD1 immunotherapy.

20. The composition of claim 19, wherein the anti-PD1 immunotherapy comprises administration of a PD-1 antibody.

21. 21. The composition of claim 20, wherein the PD-1 antibody is nivolumab, pembrolizumab, STI-A1014, or pidilizumab.

22. The composition of any one of claims 14 to 21, wherein the second therapeutic agent comprises radiation, optionally administered together with a radiosensitizer and / or a chemotherapeutic agent.

23. The composition of any one of claims 12 to 22, wherein the disease or condition is a neurological disease, a neurodegenerative disorder, a peripheral neuropathy, or a traumatic brain injury.

24. The composition of any one of claims 12 to 23, wherein the disease or condition is stroke.

25. The composition of any one of claims 12 to 24, wherein the disease or condition is an inflammatory or autoimmune disease.

26. 26. The composition of claim 25, wherein the composition is administered in combination with a second therapeutic agent useful in treating the autoimmune disease or the inflammation.

27. 10. A composition for use in a method of increasing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy, said composition comprising a compound according to any one of claims 1 to 8, said method comprising contacting said cells with said compound in an amount sufficient to increase the sensitivity of said cells to said radiation therapy and / or chemotherapy.

28. 9. A kit comprising a compound according to any one of claims 1 to 8, or a pharma- ceutically acceptable salt thereof, and instructions for administering said compound, or a pharma- ceutically acceptable salt thereof, to a subject in need thereof.

29. 30. The kit of claim 28, wherein the subject has cancer.

30. 30. The kit of claim 28, further comprising an anti-PD1 antibody.

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