Selective histone deacetylase 6 inhibitor

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] Description Regarding Government Interests This invention was made with government support under grant number 5R01 NS079183 awarded by the National Institutes of Health. The United States government has certain rights in this invention.

[0002] This disclosure relates to histone deacetylase (HDAC) inhibitors, e.g., selective HDAC6 inhibitors, pharmaceutical compositions comprising one or more of such HDAC inhibitors, methods for increasing the sensitivity of cells to the cytotoxic effects of radiotherapy and / or chemotherapy, which comprise contacting cancer cells with one or more of such HDAC inhibitors, and to methods of treatment thereof which comprise administering to an individual in need of treatment for a condition and disease in which inhibition of HDAC is effective, e.g., cancer, inflammation, neurological diseases, neurodegenerative disorders, stroke, traumatic brain injury, allograft rejection, autoimmune diseases, and malaria, a therapeutically effective amount of such HDAC inhibitor.

Background Art

[0003] The 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 functions as an important regulatory pathway for diverse cellular processes such as transcription, the 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)). The latest 11 zinc ion (Zn 2+ )-dependent HDACs (classes I, II, and IV) and 7 nicotinamide adenine dinucleotide (NAD +)The dependency search tuin (SIRT) (class III) has been identified. Unlike other members, HDAC6 of the class IIb subgroup has the unique ability to deacetylate various non-histone proteins, such as α-tubulin, cortactin, HSP-90, and HSF-1, as preferred substrates (see, for example, 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, have been studied in clinical trials for various cancer types, either as monotherapy or in combination (see, for example, Shen et al., Expert Opin. Ther. Pat. 30:121-136 (2020)), and HDAC6i has emerged as a promising approach for cancer treatment. Due to the immunomodulatory properties of HDAC6, they are considered as novel therapeutic agents for cancer immunotherapy. HDAC6 interacts with the transcription factor STAT3, which is a major regulator of the immune response in the tumor microenvironment (see, for example, Rebe et al., Cancers (Basel) 11:1280 (2019)), and it has been reported to regulate gene expression mediated by STAT3 (see, for example, 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 pro-inflammatory state of APCs (see, for example, 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, for example, Lienlaf et al., Mol. Oncol. 10:735-750 (2016)). Furthermore, using a syngeneic mouse melanoma mouse model, it has been demonstrated that combination therapy of selective HDAC6i and anti-PD-1 antibody results in a significantly improved effect on tumor growth compared to monotherapy (see, for example, Knox et al., Sci. Rep. 9:6136 (2019)), thereby highlighting the immunomodulatory ability of selective HDAC6 inhibitors.

Prior Art Documents

Non-Patent Documents

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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 in which inhibition of HDAC results in an effect, such as cancer, neurological diseases, psychiatric diseases, neurodegenerative disorders, peripheral neuropathies, stroke, hypertension, inflammation, traumatic brain injury, rheumatoid arthritis, allograft rejection, sepsis, and autoimmune diseases, which methods comprise administering to an individual in need thereof a therapeutically effective amount of an HDACI. The present disclosure also relates to methods of increasing the sensitivity of cancer cells to radiation therapy and / or chemotherapy. The present disclosure also enables the use of these HDAC inhibitors in combination with other drugs and / or therapies. In some embodiments, the present HDACI exhibits selectivity for HDAC isoenzymes, such as HDAC6, that is higher than that for other HDAC isoenzymes. 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 inhibitor in combination with anti-PD1 immunotherapy for the treatment of cancer.

[0006] In some embodiments, the present disclosure relates to a histone deacetylase inhibitor (HDACI) having the structural formula I:

Chemical formula

Brief Description of the Drawings

[0007]

Figure 1A

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Figure 1B

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Figure 1C

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Figure 1D

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Figure 1E

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Figure 1F

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Figure 6C

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Figure 6D

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Figure 6E

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Figure 6F

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Figure 6G

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Figure 6H

Mode for Carrying Out the Invention

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

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

[0030] Definitions The following terms and expressions used herein have the indicated meanings.

[0031] The terms used herein are preceded and / or followed by a single dash “-” or a double dash “=”, indicating the order of the bond of the bond between the specified substituent and its parent moiety. A single dash indicates a single bond, and a double dash indicates a double bond. In the absence of a single or double dash, it is understood that a single bond is formed between the substituent and its parent moiety. Further, the substituent is intended to be read “from left to right” unless otherwise indicated by a dash. For example, C 1 -C 6 Alkoxycarbonyloxy and -OC(O)C 1 -C 6 Alkyl represents the same functional group, and similarly, arylalkyl and -alkylaryl represent the same functional group.

[0032] “Acetyl” means a group of the formula CH 3 C(O)-.

[0033] "Alkoxy" means an alkyl group as defined herein, attached 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 from 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, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CHC(CH 3 )-, and -CH 2 CH(CH 2 CH 3 )CH 2 -.

[0035] "Aryl" means phenyl (i.e., monocyclic aryl), or a bicyclic ring system containing at least one phenyl ring, or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring. Bicyclic aryl can be azulenyl, naphthyl, etc. The aryl is attached 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" means a monocyclic cycloalkyl ring system. A monocyclic ring system is a cyclic hydrocarbon group containing 3 to 6 carbon atoms, and such a group may be saturated or unsaturated, but is not aromatic. In certain embodiments, the 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 attached to the parent molecular moiety via 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" means a monocyclic heteroaryl or a bicyclic ring system containing at least one heteroaromatic ring. The monocyclic heteroaryl may be a 5-membered ring or a 6-membered ring. The 5-membered ring consists of two double bonds and one, two, three or four nitrogen atoms, and optionally one oxygen or sulfur atom. The 6-membered ring consists of three double bonds and one, two, three or four nitrogen atoms. The 5- or 6-membered heteroaryl is connected to the parent molecular moiety via 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 consists of phenyl, monocyclic cycloalkyl, monocyclic cycloalkenyl, monocyclic heterocyclyl, or monocyclic heteroaryl fused to a monocyclic heteroaryl. The fused cycloalkyl or heterocyclyl moiety of the bicyclic heteroaryl group is optionally substituted with one or two groups that 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 via 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 via any carbon atom or nitrogen atom contained within the bicyclic ring system.Representative examples of bicyclic heteroaryl include, but are not limited to, 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-tetrahydroquinolin-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)-on-yl. In certain embodiments, the fused bicyclic heteroaryl is a 5- or 6-membered monocyclic heteroaryl ring fused to 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 and independently substituted with one or two groups that are 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" means 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, wherein 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, 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, for example, in the treatment of cancer, inflammation, traumatic brain injury, neurodegenerative disorders, neurological diseases, peripheral neuropathy, stroke, hypertension, autoimmune diseases, inflammatory diseases, and malaria. The present HDACIs also increase the sensitivity of cancer cells to the cytotoxic effects of radiotherapy and / or chemotherapy. In some embodiments, the present HDACIs selectively inhibit HDAC6 over other HDAC isoenzymes.

[0042] The term "disease or condition in which inhibition of HDAC has an effect" relates to conditions where 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 to diseases or conditions known to be treated by HDAC inhibitors (e.g., TSA, pivaloyloxymethyl butane (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., liver fibrosis), smooth muscle proliferative disorders (e.g., atherosclerosis, restenosis), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar degeneration, Rett syndrome), peripheral neuropathy (Charcot-Marie-Tooth disease, giant axonal neuropathy (GAN)), inflammatory diseases (e.g., osteoarthritis, rheumatoid arthritis, colitis), diseases associated with angiogenesis (e.g., cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia, thalassemia), fungal infections, parasitic infections (e.g., malaria, trypanosomiasis, helminthiasis, protozoal 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 for improving graft engraftment), but are not limited thereto. 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 an assay that can be conveniently used to evaluate the activity of the compound.

[0043] "Second treatment Factor " refers to a treatment Factor different from this HDACI and known to treat the disease or condition of interest. Factor For example, if cancer is the disease or condition of interest, the second treatment Factor can be, for example, a known chemotherapeutic agent such as taxol, or radiation.

[0044] "HDAC" refers to a family of enzymes that remove an acetyl group from the ε-amino group of a lysine residue at the N-terminus of a protein, such as a histone. The HDAC can be a human HDAC including HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11. The HDAC can also be obtained from protozoa or fungi.

[0045] "Treat", "treating", "treatment", etc. refer to eliminating, reducing, alleviating, reversing, and / or improving a disease or condition and / or the symptoms associated therewith. Although not necessarily excluded, treating a disease or condition does not require complete elimination of the disease, condition, or the symptoms associated therewith, including treatment of acute or chronic signs, symptoms, and / or discomfort. As used herein, the terms "treat", "treating", "treatment", etc. may include "preventive treatment", which refers to reducing the likelihood of recurrence of a disease or condition, or the likelihood of recurrence of a previously controlled disease or condition, in a subject who does not have the disease or condition or who has not had a recurrence of the disease or condition but is at risk of recurrence or is prone to recurrence. Thus, "treatment" includes prevention of recurrence or stage prevention. The term "treat" and synonyms are intended to encompass administration of a therapeutically effective amount of a compound of the present disclosure to an individual in need of such treatment. Treatment may be carried out symptomatically, for example, to suppress symptoms. This may be carried out for a short period, or for an intermediate period, or, for example, as part of maintenance therapy for long-term treatment.

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

[0047] "Co-administration", "administer in combination", "simultaneous administration" and similar expressions mean that two or more Factor are administered simultaneously to the subject being treated. "Simultaneously" means that each Factor is administered simultaneously or sequentially in any order at different times. However, if not administered simultaneously, it means that they are administered to the individual in a sufficiently short period in sequence so that they can produce the desired therapeutic effect and act in concert. For example, the present HDACI can be administered simultaneously as a second treatment Factor or sequentially in any order at different times. The present HDACI and the second treatment Factor can also be administered separately in any suitable form and by any suitable route. It is understood that the present HDACI and the second treatment Factor can be administered to the subject in need thereof in any order if not administered simultaneously. For example, the present HDACI can be a second treatment FactorIt can be administered to an individual in need thereof before administration of a treatment method (e.g., radiotherapy) (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 before), simultaneously therewith, or after (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). In various embodiments, the present HDACI and the second treatment Factor are administered at intervals of 1 minute, 10 minutes, 30 minutes, less than 1 hour, 1 hour, 1 to 2 hours, 2 to 3 hours, 3 to 4 hours, 4 to 5 hours, 5 to 6 hours, 6 to 7 hours, 7 to 8 hours, 8 to 9 hours, 9 to 10 hours, 10 to 11 hours, 11 to 12 hours, intervals of 24 hours or less or intervals of 48 hours or less. In one embodiment, the components of the combination therapy are administered at intervals of 1 minute to 24 hours.

[0048] From the perspective of explaining the present disclosure (particularly from the perspective of the claims), the use of the terms "a", "an", "the" and similar referents is to be construed as encompassing both the singular and plural forms unless otherwise indicated. The listing of ranges of values herein serves, unless otherwise indicated herein, merely as a convenient way to individually refer to each separate value falling within the range, and each separate value and sub-range are incorporated herein as if individually recited herein. The use of any and all examples provided herein, or exemplary language (e.g., "such as" and "like") is intended to better explain the present disclosure and, unless otherwise claimed, does not limit the scope of the present disclosure. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the present disclosure.

[0049] In some embodiments, the present disclosure relates to an HDACI of formula I, a composition comprising the present HDACI, and the therapeutic use of the HDACI: [Chemical formula] In the formula, R 1 and R 2 are independently selected from the group consisting of hydrogen and C 1 -C 6 alkyl, or R 1 and R 2 are bonded to form a 3- to 7-membered heterocyclyl, L 1 is CO 2 H, C(O)NH 2 , C(O)NHOH, or B(OH) 2 , L 2 is H or OR 3 , 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 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 are independently C 1 -C 6 branched alkyl.

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

[0051] In other embodiments, the present disclosure relates to HDACIs of formula Ib, compositions comprising such HDACIs, and therapeutic uses of such HDACIs: [Chemical formula] In the formula, R 1 and R2 is independently selected from the group consisting of hydrogen and C 1 -C 6 alkyl, or R 1 and R 2 are joined to form a 3- to 7-membered heterocyclyl, L 2 is H or OR 3 wherein 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 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 are independently C 1 -C 6 branched alkyl.

[0052] In other embodiments, the present disclosure relates to HDACIs of formula Ic, compositions comprising such HDACIs, and therapeutic uses of such HDACIs:

Chemical formula

[0053] In other embodiments, the present disclosure relates to HDACIs of formula Id, compositions comprising such HDACIs, and therapeutic uses of such HDACIs:

Chemical formula

[0054] In other embodiments, the present disclosure relates to HDACIs of formula Ie, compositions comprising such HDACIs, and therapeutic uses of such HDACIs:

Chemical formula

[0055] In other embodiments, the present disclosure relates to the following HDACIs

Chemical formula

[0056] Furthermore, salts, prodrugs, hydrates, isotope labels, fluorescent labels, and any other therapeutically or diagnostically relevant derivatives of the present HDACI are also included in the present disclosure and can be used in the methods disclosed herein. The present disclosure further includes all possible stereoisomers and geometric isomers of the compounds. The present disclosure includes both racemic compounds and optically active isomers. If the present HDACI is desired as a single enantiomer, it may be obtained by resolution of the final product, or from enantiomerically pure starting materials, or by stereospecific synthesis using chiral auxiliaries. See, for example, Ma et al., Tetrahedron: Asymmetry 8:883-888 (1997). Resolution of the final product, intermediate, or starting material can be achieved by any suitable method known in the art. Further, in situations where tautomers of the 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, where the compound is derivatized into a form suitable for formulation and / or administration and then released as a drug in vivo, are well established for temporarily (e.g., bioreversibly) changing the physicochemical properties of the compound (e.g., see 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., Med. Res. Rev. 15:83 (1995)). Specific prodrugs of the HDACI are described in WO2008 / 055068, which is incorporated herein by reference in its entirety.

[0058] The compounds of the present disclosure may exist as salts. Pharmaceutically acceptable salts of the present HDACIs are often preferred for the methods of the present disclosure. As used herein, the term "pharmaceutically acceptable salts" refers to 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 compound or may be prepared separately by reacting the compound with an acid having an appropriate cation. Pharmaceutically acceptable salts of the present compounds may be acid addition salts formed by pharmaceutically acceptable acids. Examples of acids that may be used to form pharmaceutically 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, glycerophosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, mesitylenesulfonate, naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, paratoluenesulfonate, undecanoate, lactate, citrate, tartrate, gluconate, methanesulfonate, ethanedisulfonate, benzenesulfonate, and p-toluenesulfonate, but are not limited thereto. Further, 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, lauryl, myristyl, and stearyl chlorides, bromides, and iodides, and benzyl and phenethyl bromides.In light of the above, any reference to a compound of the disclosure appearing herein is intended to include the compound and its pharmaceutically acceptable salts, hydrates, or prodrugs.

[0059] The compound may also be conjugated or linked to an auxiliary moiety that promotes the beneficial properties of the compound in a method of therapeutic use. Such conjugates can promote delivery of the compound to a desired anatomical site or region (e.g., a tumor), enable persistence of a therapeutic concentration of the compound in target cells, alter the pharmacokinetic and pharmacodynamic properties of the compound, and / or improve the therapeutic index or safety profile of the compound. Suitable auxiliary moieties include, for example, amino acids, oligopeptides, or polypeptides, such as antibodies, including monoclonal antibodies and other engineered antibodies, and natural or synthetic ligands for receptors on target cells or tissues. Other suitable auxiliary moieties include fatty acid or lipid moieties that promote the biodistribution of the compound and / or its uptake by target cells (see, e.g., Bradley et al., Clin. Cancer Res. 7:3229 (2001)).

[0060] The compounds of the disclosure inhibit HDAC and are useful for the treatment of various diseases and conditions. In some embodiments, the HDACI is used in a method of treating a disease or condition in which inhibition of HDAC results in an effect, 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 includes administering a therapeutically effective amount of the HDACI to an individual in need thereof.

[0061] These compounds have been evaluated for their activities at HDAC6 and their selectivity for HDAC6 compared to HDAC1. Selective HDAC6 inhibitors have been shown to be involved in a variety of medical conditions 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 where oxidative stress is a causative factor or a consequence thereof. Administration of a selective HDAC6 inhibitor in combination with rapamycin has also been shown to extend the lifespan of mice with kidney allografts. This model was used to evaluate the immunosuppressive properties of the compounds and to function as a model of graft rejection. Furthermore, selective HDAC6 inhibitors have already 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. Since this compound is also a selective HDAC6 agent, it behaves similarly. This compound exhibits ligand efficiency that makes their physicochemical properties more drug-like. Furthermore, this compound maintains the potency and selectivity observed with the previous HDACIs. Accordingly, this compound is a pharmaceutical candidate and research tool for identifying the specific functions of HDAC6.

[0062] Accordingly, in one embodiment, the present disclosure relates to a method of treating an individual afflicted with a disease or condition in which inhibition of HDAC has an effect, the method comprising administering to the individual in need thereof a therapeutically effective amount of an HDAC1 compound according to the claims.

[0063] The methods of the present disclosure can be achieved by administering one of the HDACIs of the present disclosure as the compound as such or as a pharmaceutical composition. Administration of the pharmaceutical composition of the present disclosure or the HDACI as such can be carried out during or after the onset of the disease or condition of interest. Usually, the pharmaceutical composition is sterile and does not contain toxic, carcinogenic, or mutagenic compounds that cause adverse reactions upon administration.

[0064] In some embodiments, the present HDACI is a second treatment useful for the treatment of a disease or condition in which inhibition of HDAC results in an effect Factor and can be administered in combination. The second treatment Factor is different from the present HDACI. The second treatment Factor is known to be useful for the treatment of the disease or condition from which the individual is suffering Factor and is selected, for example, from drugs and adjuvants, for example, chemotherapeutic agents and / or radiation known to be useful for the treatment of cancer. The present HDACI and the second treatment Factor may be administered together as a single unit dose or separately as multiple unit doses, and the present HDACI may be administered simultaneously with, before, or vice versa to the second treatment Factor . One or more administrations of the present HDACI and / or one or more administrations of the second treatment Factor may be carried out.

[0065] The second treatment Factor is administered in an amount that provides its desired therapeutic effect. The effective dosage range for each second treatment Factor is known in the art, and the second treatment Factor is administered to an individual in need thereof within such established range.

[0066] Accordingly, the present disclosure relates to compositions and methods of using such compounds in the treatment of diseases or conditions in which inhibition of HDAC results in an effect. The present disclosure also relates to pharmaceutical compositions comprising the present HDACI and a second treatment of any component useful for the treatment of a disease or condition in which inhibition of HDAC results in an effect Factor . Further provided is a kit comprising the present HDACI and, optionally, a second treatment, separately or together packaged, useful for the treatment of diseases and conditions in which inhibition of HDAC results in an effect Factor , and an insert having instructions for use regarding the use of these activities Factor .

[0067] Within the scope of the present disclosure, the terms "disease" or "condition" generally refer to a pathological condition or function and the disorders and / or abnormalities that can manifest in the form of signs, symptoms, and / or malaise. As shown below, the present HDACI is a potent inhibitor of HDAC and can be used in the treatment of diseases and conditions in which inhibition of HDAC has an effect, 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 cells or neoplastic cells, inhibiting the proliferation of cancer cells or neoplastic cells, inhibiting the replication of cancer cells or neoplastic cells, or improving their symptoms, the method including administering to a subject in need thereof a sufficient amount of the present HDACI or a pharmaceutically acceptable salt thereof for treating the cancer. Further, it is known that the selective HDACI may be able to promote the killing of the cancer cells through the reactivation of the immune system by a mechanism related to the PDI receptor. The present HDACI may be used as the sole anticancer agent or in combination with another anticancer treatment, such as radiation, chemotherapy, and surgery.

[0069] In another embodiment, the present disclosure provides a method for increasing the sensitivity of cancer cells to the cytotoxic effects of radiotherapy and / or chemotherapy, the method including contacting the cells with the present HDACI or a pharmaceutically acceptable salt thereof in an amount sufficient to increase the sensitivity of the cells to the cytotoxic effects of the radiotherapy and / or chemotherapy.

[0070] In a further embodiment, the present disclosure provides a method for treating cancer, the method including (a) administering to an individual in need thereof an amount of an HDACI compound of the present disclosure, and (b) performing on the individual an amount of radiotherapy, chemotherapy, or both. The dosages are each effective for treating the cancer. In another embodiment, the amounts are effective in combination for treating the cancer.

[0071] This combination therapy of the present disclosure can be used in various situations depending on the treatment of various cancers. In certain embodiments, the individual in need of treatment has previously received cancer treatment. Such previous treatments include, but are not limited to, previous chemotherapy, radiation therapy, surgery, or immunotherapy, such as cancer vaccines.

[0072] In another embodiment, the cancer to be treated is a cancer that has been demonstrated to be sensitive to radiation therapy and / or chemotherapy, or a cancer that is known to respond to radiation therapy and / or chemotherapy. Such cancers include, but are not limited to, non-Hodgkin 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 tumors, or other CNS neoplasms.

[0073] In yet another embodiment, the cancer to be treated is a cancer that has been demonstrated to be resistant to radiation therapy and / or chemotherapy, or a cancer that is known to be ineffective to radiation therapy and / or chemotherapy. A treatment is ineffective if at least a substantial portion of the cancer cells do not respond to the treatment and are not killed, or if their cell division is not arrested. Such determination can be made either in vivo or in vitro by any method known in the art for assaying the effectiveness of a treatment against cancer cells, using the ordinary meaning of "ineffective" in the art in such context. In certain embodiments, a cancer is refractory if the number of cancer cells has not significantly decreased or has increased.

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

[0075] In certain embodiments, leukoplakia, a benign-appearing hyperplasia or dysplastic lesion of the epithelium, and Bowen's disease, an intraepithelial carcinoma, are premalignant lesions that indicate the desirability of preventive intervention.

[0076] In another embodiment, fibrocystic disease (cystic hyperplasia, mammary dysplasia, and adenosis (benign epithelial hyperplasia)) indicates the desirability of preventive intervention.

[0077] The prophylactic use of the compounds and methods of the present disclosure is also suggested for several viral infections that can lead to cancer. For example, human papillomavirus 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 virus 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)), human herpesvirus-8 infection can cause Kaposi's sarcoma (see, e.g., Kadow et 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 immunodeficiency (see, e.g., Dal Maso et al., Lancet Oncol 4(2):110-9 (2003)).

[0078] In other embodiments, a subject exhibiting one or more of the following predispositions to malignancy can be treated by administration of the present HDACI and the methods of the present disclosure: chromosomal translocations associated with malignancy (e.g., the Philadelphia chromosome in chronic myelogenous leukemia, t(14;18) in follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (potential precursors to colon cancer), benign monoclonal gammopathy (potential precursor to multiple myeloma), a first-degree relative of a patient with a cancer or pre-cancerous disease exhibiting a Mendelian (hereditary) inheritance pattern (e.g., familial polyposis of the colon, Gardner's syndrome, hereditary exostosis, multiple endocrine neoplasia, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, von Recklinghausen neurofibromatosis, retinoblastoma, carotid body tumor, cutaneous malignant melanoma, intraocular melanoma, xeroderma pigmentosum, ataxia telangiectasia, Chediak-Higashi syndrome, vitiligo, Fanconi's aplastic anemia, and Bloom's syndrome; see Robbins and Angell, Basic Pathology, 2d Ed., W.B. 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 present HDACI and the methods of the present disclosure are administered to a human subject to prevent progression of breast cancer, colon cancer, ovarian cancer, or cervical cancer.

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

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

[0082] In yet another embodiment, the further anti-cancer therapy is immunotherapy, such as a cancer vaccine.

[0083] In one embodiment, the immunotherapy includes anti-PD1 immunotherapy. In another embodiment, the anti-PD1 immunotherapy includes 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 present HDACI or a pharmaceutically acceptable salt thereof is administered in combination with the further anti-cancer therapy.

[0085] In another embodiment, the further anti-cancer therapy is radiotherapy. In the method of the present disclosure, any radiotherapy protocol can be used depending on the type of cancer being treated. Embodiments of the present disclosure employ electromagnetic radiation of gamma rays (10 -20 ~10 -13 m), X-ray radiation (10 -12 ~10 -9 m), ultraviolet light (10 nm to 400 nm), visible light (400 nm to 700 nm), infrared light (700 nm to 1 mm), and microwave radiation (1 mm to 30 cm).

[0086] For example, without limitation, X-ray radiation can be administered, and in some embodiments, high-energy super-high voltage (radiation with energy exceeding 1 MeV) can be used for deep tumors, and electron beams and conventional voltage 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 stereotactic methods in which multiple low-dose radiation sources are simultaneously focused on a tissue volume from multiple angles, "brachytherapy" including directly placing radioactive implants in tumors or other target tissues, such as low-dose-rate brachytherapy, interstitial irradiation, and intracavitary irradiation, intraoperative irradiation in which a large amount of external radiation is directed at target tissues exposed during surgery, and particle beam therapy including using high-speed subatomic particles for the treatment of localized cancer, but are not limited thereto.

[0087] Many cancer treatment protocols currently use radiation sensitizers activated by electromagnetic radiation, such as X-rays. Examples of X-ray activated radiation sensitizers 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, cisplatin, and their therapeutically effective analogs and derivatives.

[0088] In photodynamic therapy (PDT) for cancer, visible light is used as the radiation activator for the sensitizer. Examples of photodynamic radiation sensitizers include, but are not limited to: hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives, NPe6, tin etiopurpurin (SnET2), pheophorbide-a, bacteriochlorophyll-a, naphthalocyanine, phthalocyanine, zinc phthalocyanine, and their therapeutically effective analogs and derivatives.

[0089] The radiosensitizer can be administered together with one or more compounds in a therapeutically effective amount in addition to the present HDACI. Such compounds include compounds that promote the uptake of the radiosensitizer into target cells, compounds that control the flow of therapeutic agents, nutrients, and / or oxygen into 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, but are not limited thereto. Examples of additional therapeutic agents that can be used with the radiosensitizer include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red blood cell transfusion, perfluorocarbon (e.g., FLUOSOLW (registered trademark)-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, angiogenesis inhibitory compounds, hydralazine, and L-BSO.

[0090] In one embodiment, the present HDACI or a pharmaceutically acceptable salt thereof is administered prior to the administration of radiotherapy and / or chemotherapy.

[0091] In another embodiment, the present HDACI or a pharmaceutically acceptable salt thereof is administered in combination with radiotherapy and / or chemotherapy.

[0092] This HDACI and additional therapies may act additively or synergistically (i.e., the combination of this HDACI or a pharmaceutically acceptable salt thereof and an additional anti-cancer therapy is more effective than the additive effect when each is administered alone). The synergistic combination allows for the use of lower doses of this HDACI and / or additional therapies and / or less frequent administration of this HDACI and / or additional therapies to a subject having cancer. By being able to use lower doses of this HDACI and / or additional therapies and / or administer the compounds of the present disclosure and additional therapies less frequently, the toxicity associated with administration can be reduced without reducing the effectiveness of this HDACI and / or the additional therapy in the treatment of cancer. Further, the synergistic effect can improve the effectiveness of cancer treatment and / or reduce the adverse effects or undesirable side effects associated with the administration of this HDACI and / or an additional anti-cancer therapy as a monotherapy.

[0093] In one embodiment, this HDACI can act synergistically with radiation therapy when administered at a dose normally used when such HDACI is used alone for the treatment of cancer. In another embodiment, this HDACI can act synergistically with radiation therapy when administered at a dose less than the dose normally used when such HDACI is used as a monotherapy for the treatment of cancer.

[0094] In one embodiment, radiation therapy can act synergistically with this HDACI when administered at a dose normally used when radiation therapy is used alone for the treatment of cancer. In another embodiment, radiation therapy can act synergistically with the compounds of the present disclosure when administered at a dose less than the dose normally 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 radiotherapy can be determined by measuring post-treatment survival in vitro and / or in vivo using techniques known in the art. In one embodiment, for in vitro measurement, cells in the logarithmic growth phase are exposed to a known dose of radiation, and cell survival can be observed. The irradiated cells are seeded and cultured for about 14 to about 21 days, and the colonies are stained. The survival rate is the number of colonies divided by the plating efficiency of the non-irradiated cells. A survival curve is generated by graphing the survival rate on a logarithmic scale against the absorbed dose on a linear scale. The survival curve generally shows that after an initial shoulder region where the dose is sub-lethal, the proportion of surviving cells decreases exponentially at higher radiation doses. A similar protocol can be used for the chemicals when used in the combination therapies of the present disclosure.

[0096] The intrinsic radiosensitivity of tumor cells and the influence of the environment, such as hypoxia and host immunity, can be further evaluated by in vivo tests. The growth delay assay is generally used. This assay measures the time interval required for a tumor exposed to radiation to regrow to a specific volume. The dose required to control about 50% of the tumor is specified by the 50 TCD assay.

[0097] In vivo assay systems typically use solid tumor systems that can be transplanted into experimental subjects. The 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 combination with surgery, radiotherapy, and chemotherapy, which are generally recognized methods, and the generally recognized methods include, for example, mimicking in a chemical system of radiotherapy where a synergistic improvement in the effectiveness of generally recognized therapies is achieved. The effectiveness of the treatment can be measured in clinical studies or model systems, such as mouse tumor models, or sensitivity assays in cell culture.

[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 HDACI as a radiosensitizer in combination with radiation therapy.

[0100] When the combination therapy of the present disclosure involves administering the present HDACI together with one or more additional anti-cancer agents, the present HDACI and the additional anti-cancer agent(s) may be administered simultaneously or sequentially to an individual. The agents may also be administered periodically. Cycling therapy involves repeating the administration of one or more different anti-cancer agents for a defined period following a defined period of administration of one or more anti-cancer agents, i.e., including cycles, reducing the development of resistance to one or more of the administered anti-cancer agents, avoiding or reducing one or more side effects of one or more of the administered anti-cancer agents, and / or improving the efficacy of the treatment.

[0101] The additional anti-cancer agent(s) may be administered over a series of periods. Any one or combination of the additional anti-cancer agents described below may be administered.

[0102] The present disclosure includes methods of treating cancer that involve administering to an individual in need of treatment for cancer the present HDACI and one or more additional anti-cancer agents or pharmaceutically acceptable salts thereof. The present HDACI and the additional anti-cancer agent(s) may act additively or synergistically. Suitable anti-cancer agents include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, and the like.

[0103] Furthermore, the present disclosure provides methods of treating cancer using the present HDACI as an alternative to chemotherapy alone or radiation therapy alone when chemotherapy or radiation therapy has been or can be demonstrated to be overly toxic to a subject undergoing treatment, e.g., resulting in unacceptable or intolerable side effects. The individual undergoing treatment may optionally be treated with another anti-cancer therapy, e.g., chemotherapy, surgery, or immunotherapy, depending on the treatment that has been found to be acceptable or tolerable.

[0104] This HDACI can also be used for the treatment of certain cancers, including but not limited to, for example, leukemia and lymphoma, in an in vitro or ex vivo manner, and such treatment includes 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, and the subject is then administered an effective amount of this HDACI to eradicate the remaining population of bone marrow cells in the subject, and then the stem cell graft is infused back into the subject. Supportive therapy is then carried out until the bone marrow function recovers and the subject recovers.

[0105] This method for treating cancer further includes the administration of this HDACI and a further treatment Factor or a pharmaceutically acceptable salt or hydrate thereof. In one embodiment, the composition comprising this HDACI may be part of the same composition or may be included in a different composition as one or more further treatments Factor (s) administered simultaneously with the administration of this HDACI. In another embodiment, this HDACI is administered before or after the administration of another treatment Factor (s).

[0106] In this method for treating cancer, the other treatment FactorIt may be an antiemetic. Suitable antiemetics include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetyl leucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, metalatal, metopimazine, nabilone, oxypendyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thiopropperazine, and tropisetron. In one embodiment, the antiemetic is granisetron or ondansetron. In another embodiment, the other treatment Factor It may be a hematopoietic colony-stimulating factor. Suitable hematopoietic colony-stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim, and epoetin alpha.

[0107] In yet another embodiment, the other treatment Factor It may be an opioid or non-opioid analgesic. Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, methopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, loperamide, anileridine, ethoheptazine, piminidine, beta-prodine, 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 radiotherapy and chemotherapy, the present HDACI is used in methods for treating diseases, conditions, and injuries to the central nervous system such as neurological diseases, neurodegenerative disorders, and traumatic brain injury (TBI). In a preferred embodiment, the present HDACI can cross the blood - brain barrier and inhibit HDAC in the brain of an individual.

[0110] The present HDACI compounds also have a therapeutic effect on peripheral neuropathies, for example, models of CMT. HDAC6 inhibitors are known to cross the blood - nerve barrier and rescue the phenotypes observed in transgenic mice that exhibit symptoms of distal hereditary motor neuropathy. Administration of an HDAC6 inhibitor to symptomatic mice increased the levels of acetylated α - tubulin, restored proper mitochondrial motility and axonal transport, and increased 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 present HDACI compounds also improve associative memory loss after an increase in Aβ. In this study, Aβ42 was injected into the dorsal hippocampus of mice via a cannula implanted 15 minutes before training. The test compound was administered ip (25 mg / kg) 2 hours before training. Fear learning was evaluated 24 hours later.

[0112] Contextual fear conditioning conducted 24 hours after training shows a decrease in freezing in Aβ-injected mice compared to vehicle-injected mice. Treatment with this compound improves the defect in the freezing response in Aβ-injected mice and has no effect on vehicle-injected mice. The test compound alone has no effect on the memory ability of mice. Furthermore, injecting the compound twice a day for 2 days had no effect on motor, sensory, or motivational skills evaluated using the visible platform test. During these experiments, no obvious signs of toxicity were observed, including changes in food and water intake, weight loss, or changes in locomotor and exploratory behavior.

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

[0114] Therefore, the present HDACI is useful for treating neurological diseases by administering an effective amount of the present HDACI or by administering a pharmaceutical composition comprising an effective amount of the present HDACI for treating neurological diseases. Neurological diseases that can be treated include Huntington's disease, lupus, schizophrenia, multiple sclerosis, muscular dystrophy, 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 dementia, corticobasal degeneration, dystonia, myoclonus, Tourette syndrome, tremors, chorea, restless legs syndrome, Parkinson's disease, Parkinson's syndrome, anxiety, depression, psychosis, bipolar disorder, Friedreich's ataxia, fragile X syndrome, spinal muscular dystrophy, Rett syndrome, Rubinstein-Taybi syndrome, Wilson's disease, multi-infarct state, CMT, GAN, and other peripheral neuropathies, but are not limited thereto.

[0115] In one embodiment, the neurological disorder to be 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 hereditary 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 a high-stepping gait that frequently causes tripping and falling. Mutations in the low molecular weight heat shock protein 27 (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). Expression of mutant HSPB1 results in reduced levels of acetylated α-tubulin and induces severe axonal transport defects. Pharmacological inhibition of histone deacetylase 6 (HDAC6)-induced deacetylation of α-tubulin by Tubastatin A, an HDAC6i, corrects 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, giving valuable insights into HDAC6 inhibitors as a therapeutic strategy for hereditary axonal degenerative diseases. The compounds of the present disclosure exhibit potent HDAC6 isoform inhibition, high HDAC6 selectivity, and excellent α-tubulin acetylation in various cell lines.

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

[0118] This HDACI can also be used in combination with a second treatment in a method of treating conditions, diseases, and injuries to the CNS. Factor Such a second treatment Factor is a drug 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] This HDACI is also useful for the treatment of TBI. Traumatic brain injury (TBI) is a serious and complex injury that occurs in approximately 1.4 million people each year in the United States. 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 causes many pathological conditions, including axonal injury, cell death, contusion, and inflammation. The inflammatory cascade is characterized by the activation of inflammatory cytokines and microglia, which can exacerbate other pathological conditions. Although the role of inflammation in TBI is well established, there is currently no effective anti-inflammatory therapy for the treatment of TBI.

[0121] Some 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 spinobulbar muscular atrophy (SBMA). Recent studies on experimental pediatric TBI have reported that the decrease in hippocampal CA3 histone H3 acetylation persists for several hours to several days after injury. These changes were due to the confirmed upstream excitotoxicity and stress cascades associated with TBI. HDACI has also been reported to have an anti-inflammatory effect through the acetylation of non-histone proteins. The HDAC6-selective inhibitor, 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB), was found to be able to increase histone H3 acetylation and reduce the microglial inflammatory response after traumatic brain injury in rats. This demonstrates the usefulness of HDACI as a therapeutic agent for inhibiting neuroinflammation associated with TBI.

[0122] Accordingly, the present HDACI is also useful for the treatment of inflammation and stroke, as well as for the treatment of autism and autism spectrum disorders. The present HDACI can further be used for the treatment of parasitic infections (e.g., malaria, toxoplasmosis, trypanosomiasis, helminthiasis, protozoal infections (see Andrews et al., Int. J. Parasitol. 30(6):761-768(2000)).

[0123] The present HDACI can also be used as a contrast agent. In some embodiments, by providing a radiolabeled, isotope-labeled, or fluorescently labeled HDACI, the labeled compound can image HDAC, tissues expressing HDAC, and tumors. The labeled HDACI of the present disclosure can also image a patient suffering from cancer or other HDAC-mediated diseases, such as stroke, by administration of 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 positrons 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 levels of HDAC. The degree of accumulation of the labeled HDACI can be quantified using known methods for quantifying radioactivity emission. Further, the labeled HDACI can include a fluorophore or similar reporter capable of tracking the movement of HDAC isoforms or organelles in vitro.

[0124] The HDACI useful for imaging methods comprises one or more radioisotopes capable of emitting one or more forms of radiation suitable for detection by any standard radiation equipment, such as PET, SPECT, gamma camera, MRI, and similar devices. Preferred isotopes include tritium ( 3 H) and carbon ( 11 C). The substituted HDACI of the present disclosure can also include isotopes of fluorine ( 18 F) and iodine ( 123 I) for imaging methods. Typically, the labeled HDACI of the present disclosure is 11An alkyl group having a C label, i.e., 11 a C-methyl group, or 18 F, 123 I, 125 I, 131 I, or an alkyl group substituted with a combination thereof.

[0125] The fluorescently labeled HDACIs of the present disclosure can also be used in the imaging methods of the present disclosure. Such compounds have FITC, a carbocyanine moiety, or other fluorophores that enable visualization of HDAC proteins in vitro.

[0126] The labeled HDACIs and methods of use can be used in vivo and in humans, and can also be used for in vitro applications, such as diagnostic and research applications using body fluids and cell samples. The imaging method using the labeled HDACIs of the present disclosure is described in WO03 / 060523, which designates the United States and is incorporated herein by reference in its entirety. Typically, the method involves contacting cells or tissues with a radiolabeled, isotope-labeled, fluorescently labeled, or tagged (such as biotin-tagged) compound of the present disclosure, and generating an X-ray, fluorescence, or similar type of image depending on the visualization method used. That is, for X-ray imaging, an amount sufficient to provide from about 1 to about 30 mCi of the radiolabeled compound is used.

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

[0128] In a preferred method, the labeled HDACIs of the present disclosure are rapidly excreted from tissues in the body, preventing long-term exposure of an individual to the radiation of a radiolabeled compound administered to the individual. Typically, the labeled HDACIs of the present disclosure are excreted from the body in less than about 24 hours. More preferably, the labeled HDACIs are excreted 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 excreted in about 60 minutes to about 120 minutes.

[0129] In addition to isotope-labeled and fluorescent-labeled derivatives, the present disclosure also embodies the use of derivatives containing tags (such as biotin) for identifying biomolecules related to HDAC isoforms for diagnostic, therapeutic, or research purposes.

[0130] This HDACI is also useful for the treatment of autoimmune diseases and inflammation. The compounds of the present disclosure are useful in overcoming graft rejection and rejection due to transplantation and in treating arthritis.

[0131] Despite the success of modern transplantation programs, in addition to the nephrotoxicity, cardiovascular diseases, diabetes, and hyperlipidemia associated with current therapies, the incidence of post-transplant malignancies and graft loss due to chronic rejection has led to 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 also increasing. Animal experiments have shown that regulatory T cells (Tregs), which express Foxp3, a member of the forkhead transcription family, are key to restricting autoreactive and alloreactive immunity. Furthermore, after induction by co-stimulation blockade, immunosuppression, or other strategies, Tregs can be adoptively transferred to naive hosts to achieve beneficial therapeutic effects. However, clinical trials have failed in attempts to develop sufficient Tregs to maintain inhibitory function after transfer. In mouse studies, HDACIs enhance the inhibitory function of Tregs. Restricting the immune response at least in a substantial part by ablation (Tao et al., Nat Med 13:1299-1307 (2007)), and it is shown that the selective targeting of HDAC6 is particularly effective in this regard.

[0132] In organ transplantation, since rejection begins immediately after transplantation, prevention rather than treatment of rejection is the top priority issue to be considered. The opposite is true for autoimmunity in which the patient already has a disease causing problems. Accordingly, it is evaluated whether HDAC6- / - mice treated with low-dose RPM (rapamycin) for 14 days show signs of tolerance induction and resistance to the development of chronic rejection, that is, a continuous significant loss of long-term graft function in the clinical transplant population. Tolerance is evaluated by examining whether mice with long-term surviving allografts, as occurs when non-selective HDACI and RPM are used, then reject a third-party heart graft and accept additional donor allografts without immunosuppression. These in vivo tests are achieved by evaluation of ELISPOT and MLR activities using lymphocytes of recipients administered donor cells. Protection against chronic rejection is evaluated by analysis of host anti-donor humoral responses, as well as analysis of graft arteriosclerosis and interstitial fibrosis in long-term surviving allograft recipients.

[0133] The importance of HDAC6 targeting is evaluated in additional transplantation models seeking a biochemical readout, as clinically observed. Thus, the effect of HDAC6 in targeting in kidney transplant recipients (monitoring of BUN, proteinuria) and the effect of HDAC6 in targeting in islet allografts (monitoring of blood glucose levels) are evaluated. Kidney transplantation is the most commonly performed organ transplantation, and the kidney regulates multiple functions, such as acid / base metabolism, blood pressure, and erythropoiesis. Thus, the effectiveness in this model indicates the usefulness of HDAC6 targeting. Similarly, islet transplantation is a major unmet need considering that clinical islet allografts are typically lost in the first or second year after transplantation. Having a safe and non-toxic means to extend islet survival without maintenance CNI therapy would be an important advance. Transplantation research is also enhanced by the use of mice with floxed HDAC6. For example, the effect of deleting HDAC6 only in Tregs is tested using existing Foxp3-Cre mice. This approach can be extended, for example, to targeting HDAC6 in T cells (CD4-Cre) and dendritic cells (CD11c-Cre). Using tamoxifen-controlled Cre, the importance of HDAC6 in the induction and maintenance of transplantation (impact on short-term and maintenance HDAC6I therapy) is evaluated by administering tamoxifen and inducing deletion of HDAC6 at various times after transplantation.

[0134] Autoimmune research is also being conducted. In this case, blocking existing diseases is particularly important, and targeting HDAC6 may be effective without the need for further treatment (in contrast to the need for short-term, low-dose RPM in a highly aggressive, fully MHC-mismatched transplantation model). Studies in mice with colitis have shown that HDAC6− / − Tregs are more effective in disease control than WT Tregs, and that tubacin can rescue mice when treatment is initiated after the onset of colitis. These studies are extended by evaluating whether deletion of HDAC6 in Tregs (Foxp3 / Cre), T cells (CD4=Cre), or DCs (CD11c-Cre) specifically affects the onset and severity of colitis. Similarly, the control of colitis is evaluated by inducing deletion of HDAC6 at various intervals after the onset of colitis using tamoxifen-regulated Cre.

[0135] This compound is expected to exhibit anti-arthritis efficacy in the collagen-induced arthritis model of DBA1 / J mice. In this test, DBA1 / J mice (male, 7–8 weeks old) are used at 8 mice per group. Systemic arthritis is induced on day 21 by injection of bovine type II collagen and CFA, in addition to an IFA booster injection. This HDACI is administered at 50 mg / kg and 100 mg / kg for 2 weeks, and its effect is judged from data of mean arthritis score versus number of treatment days.

[0136] Despite efforts to avoid graft rejection by matching the tissue types of the host and donor, immunosuppressive therapy is important for the survival of donor organs in the host in most transplantation procedures. Various immunosuppressive agents, including azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids, are employed in transplantation.

[0137] This HDACI is a potent immunosuppressive agent that suppresses humoral and cellular immune responses, such as allograft rejection, delayed-type hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis, and graft-versus-host disease. The HDACI of the present disclosure is 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] A therapeutically effective amount of this HDACI can be used, for example, for preventing organ rejection or graft-versus-host disease and for immunosuppression, including the 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, polycythemia vera and idiopathic thrombocytopenia), systemic lupus erythematosus, 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 ophthalmopathy, Graves' disease, sarcoidosis, primary biliary cirrhosis, juvenile diabetes (type I diabetes), uveitis (anterior and posterior), dry keratoconjunctivitis and vernal catarrh, interstitial pulmonary fibrosis, psoriatic arthritis, and glomerulonephritis, but are not limited thereto.

[0139] This HDACI may be used alone or in combination with a second treatment known to be useful for the treatment of autoimmune diseases, inflammation, transplantation, and grafts, Factor for example, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those skilled in the art.

[0140] Additional diseases and conditions mediated by HDAC, 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. These diseases and conditions can also be treated by the methods of the present disclosure.

[0141] In this method, a therapeutically effective amount of one or more of the HDACIs of the present disclosure is typically formulated according to pharmaceutical practice and administered to a human in need thereof. Whether such treatment is indicated depends on the individual case and follows a medical evaluation (diagnosis) taking into account the existing signs, symptoms, and / or disorders, the risk of presenting signs, symptoms and / or disorders, and other factors.

[0142] The present HDACI can be administered by any suitable route, for example, orally, buccally, by inhalation, topically, sublingually, rectally, intravaginally, intracisternal or intrathecal via lumbar puncture, transurethrally, intranasally, percutaneously, i.e., transdermally, or parenterally (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intra-articular, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at the site). Parenteral administration can be achieved using needles and syringes or using high-pressure techniques.

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

[0144] The toxicity and therapeutic efficacy of the present HDACI compounds are determined in cell cultures or experimental animals, for example, LD 50 (the dose at which 50% of the population dies) and ED 50can be determined by standard pharmaceutical procedures for measuring the therapeutically effective dose for 50% of the population. The dose ratio between toxicity and therapeutic effect is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 . Compounds showing a high therapeutic index are preferred. Data obtained from such procedures can be used in formulating dosage ranges for use in humans. The dosage preferably lies within the range of blood compound concentrations that include little or no toxicity and an ED 50 . The dosage can vary within this range depending on the dosage form employed and the route of administration utilized. Determination of the therapeutically effective amount is within the capabilities of a fully skilled artisan, particularly in view of the detailed disclosure provided herein.

[0145] The therapeutically effective amount of the present HDACI required for use in therapy will vary depending on the nature of the condition being treated, the length of time activity is desired, as well as the age and condition of the patient, and will ultimately be determined by the attending physician. The dosage and interval can be adjusted individually to provide plasma levels of the present HDACI sufficient to maintain the desired therapeutic effect. The desired dosage can be administered as a single dose, or as multiple doses conveniently administered at appropriate intervals, for example, once, twice, three times, four times, or more times a day as fractional doses. In many cases, multiple doses are desirable or necessary. For example, the present HDACI can be administered 4 times at 4-day intervals once a day (q4d×4), 4 times at 3-day intervals once a day (q3d×4), administered once a day at 5-day intervals (qd×5), administered once a week for 3 weeks (qwk3), administered daily for 5 days, with a 2-day break, and then administered daily for another 5 days (5 / 2 / 5), or at a frequency of any dosing schedule determined to be appropriate for the situation.

[0146] The dosage of the composition comprising the present HDACI, or the composition containing 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 by body weight. The above dosages are exemplary of average cases, but there may be individual cases that merit higher or lower dosages, and they are within the scope of the present disclosure. In practice, the physician determines the actual dosing schedule that is most appropriate for an individual patient and that may vary depending on the patient's age, weight, and response.

[0147] The present HDACI used in the methods of the present disclosure is usually administered in an amount of about 0.005 to about 500 milligrams per administration, about 0.05 to about 250 milligrams per administration, or about 0.5 to about 100 milligrams per administration. For example, the present HDACI may be administered in an amount 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 administration, including all dosages between 0.005 and 500 milligrams per administration.

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

[0149] The term "carrier" refers to a diluent, adjuvant, or excipient administered together with the present 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, etc. The carrier can be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliaries, stabilizers, thickeners, lubricants, and coloring agents can be used. The pharmaceutically acceptable carrier is sterile. When the present HDACI is administered intravenously, water is a preferred carrier. For example, as a liquid carrier for injections, an aqueous solution of sodium chloride as well as aqueous solutions of dextrose and glycerol can also be employed. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The present composition can also contain, if necessary, a small amount of a wetting agent or emulsifier, or a pH buffer.

[0150] These pharmaceutical compositions can be produced, for example, by conventional mixing, dissolving, granulating, tablet-making, emulsifying, encapsulating, entrapping, or lyophilization processes. The appropriate formulation depends 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 tablets, capsules, powders, solutions, or elixirs. When administered in tablet form, the composition can further contain a solid carrier, such as gelatin or an adjuvant. The tablets, capsules, and powders contain from about 0.01% to about 95%, preferably from about 1% to about 50%, of the present HDACI. When administered in liquid form, a liquid carrier, such as water, petroleum, or an oil of animal or plant origin, can be added. The liquid form of the composition can further contain physiological saline, a dextrose or other saccharide solution, or a glycol. When administered in liquid form, the composition contains from about 0.1 wt% to about 90 wt%, preferably from about 1 wt% to about 50 wt%, of the present compound.

[0151] When a therapeutically effective amount of the present HDACI is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. Preparation of such a parenterally acceptable solution, taking full account of pH, isotonicity, stability, etc., is within the skill of those of ordinary skill in the art. Preferred compositions for intravenous, cutaneous, or subcutaneous injection usually contain an isotonic vehicle. The present HDACI can be infused with other liquids over a period of 10 - 30 minutes or several hours.

[0152] The present HDACI can be readily admixed with pharmaceutically acceptable carriers well known in the art. Such carriers enable formulation of the active agent as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by a patient undergoing treatment. Pharmaceutical preparations for oral use are obtained by adding the present HDACI to a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries if necessary, and then treating the granules of the mixture to obtain tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added if necessary.

[0153] The present HDACI can be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable formulations can be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative. The composition can take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and can contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents.

[0154] The pharmaceutical composition for parenteral administration contains an aqueous solution of the active agent in water-soluble form. Further, the suspension of the present HDACI can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. The aqueous injection suspension may contain a substance that increases the viscosity of the suspension. Optionally, the suspension can also contain a suitable stabilizer or agent that enhances the solubility of the compound and enables the preparation of a highly concentrated solution. Alternatively, the composition can be in powder form for constitution before use with a suitable vehicle, such as sterile pyrogen-free water.

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

[0156] In some embodiments, the present HDACI may be administered orally, buccally, or sublingually, in the form of tablets containing excipients such as starch or lactose, or in capsules or vaginal suppositories, either alone or as a mixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid formulations can be prepared with pharmaceutically acceptable additives, such as suspending agents. The present HDACI can also be administered parenterally, for example, by intravenous, intramuscular, subcutaneous, or intracoronary injection. In the case of parenteral administration, the present HDACI is most suitable for use in the form of a sterile aqueous solution, which can be made isotonic with blood by including other substances, such as salts or monosaccharides, such as mannitol or glucose.

[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 grade and used as received without further purification, unless otherwise noted. Dry solvents used as media in moisture-sensitive reactions were purchased from Sigma-Aldrich in anhydrous grade and handled under argon. All reactions were carried out under an inert (argon) atmosphere and in a dry state. Microwave reactions were performed in a Biotage Initiator microwave reactor. The reactions were monitored by thin-layer chromatography on silica gel-coated glass plates (TLC LuxPlate Silica gel 60 F 254 , Merck), visualized at 254 nm, and / or using appropriate dyes. When indicated, synthetic intermediates were purified by flash chromatography on 230 - 400 mesh silica gel using a CombiFlash® system with an appropriate solvent mixture. The final products were purified by preparative liquid chromatography on a Shimadzu preparative HPLC using ACE 5AQ (150×21.2 mm) particle size 5 μm. Method 1: 25 - 100% MeOH / H 2 O, 30 min, 100% MeOH, 5 min, 100 - 25% MeOH / H 2 O, 4 min. Method 2: 8 - 100% MeOH / H 2 O, 30 min, 100% MeOH, 5 min, 100 - 8% MeOH / H 2 O, 4 min. Method 3: 0% MeOH, 5 min, 0 - 100% MeOH / H 2 O, 25 min, 100% MeOH, 5 min, 100 - 0% MeOH / H 2 O, 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 at 400 MHz and 100.6 MHz, respectively, on a Bruker DPX-400 or AVANCE-400 spectrometer. Chemical shifts (δ scale) are reported in parts per million (ppm) relative to TMS. 11H NMR spectra are reported in the following order: multiplicity and number of protons, signal is s (singlet), d (doublet), dd (doublet of doublets), t (triplet), m (multiplet), bs (broad signal). HRMS spectra were recorded using ESI with LCMS-IT-TOF (Shimadzu). The purity of all final compounds was measured by analytical HPLC [ACE 3AQ C18 column (150×4.6 mm, particle size 3 μM), gradient elution system of H 2 O / MeOH containing 0.05% TFA, flow rate = 1.0 mL / min]. All compounds were tested with a purity exceeding 95% as measured 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 is K 2 CO 3From phenyl chloroformate and aniline 1 under acetone conditions, methyl 4-formylbenzoate 3a was rapidly reductively aminated with 4-aminobutanol to obtain intermediate 4a. Subsequently, under TEA / THF conditions, the important urea precursor 5a was obtained by the combinatorial reaction between 2 and 4a. This was further converted to the final hydroxamic acid product 6a (Suprastat) using an aqueous hydroxylamine solution under basic conditions and TFA / THF conditions for Boc group deprotection. For evaluation together with Suprastat in the following biological experiments, an analog 6b with the original butyl chain attached to the aminomethyl group and the proximal urea nitrogen was also prepared using the same synthetic route as Suprastat. The synthesis of 6c (Table 1) containing a hydroxylbutyl side chain has been reported previously (see, for example, Bergman et al., J. Med. Chem. 55:9891-9899 (2012)). Furthermore, non-hydroxamic acid analogs 6d-f containing the same cap as Supratstat were prepared and evaluated to investigate whether additional hydrogen bonding interactions can retain activity without a hydroxamic acid ZBG, starting from the ketone / amide-based class I HDACI (see, for example, Koya et al., Cancer Res. 72:3928-3937 (2012), or O’Donnell et al., Cancer Treat Rev. 52:71-81 (2017)). Carboxylic acid analog 6d was obtained directly from urea ester 5a by hydrolysis under basic conditions and Boc deprotection. To synthesize amide analog 6e, 4-formylbenzonitrile 3b was subjected to two-step reductive amination, followed by reaction with carbamate 2 to produce intermediate urea 5c. The nitrile group of 5c was further converted to an amide group by treatment with aqueous hydrogen peroxide solution under basic conditions, and the final product 6e was obtained by Boc deprotection as described above. The synthetic route to boronic acid analog 6f started with the reductive amination of 4-bromobenzaldehyde 3c and continued urea formation using the same procedure as above to obtain intermediate urea 5d. The precursor 5d was coupled with bis(pinacolato)diboron under the 2 conditions of 2 . Finally, the desired boronic acid product 6f was obtained by treatment with NaIO 4 / NH 4 It was obtained by deprotection of pinacol and Boc deprotection under OAc and TFA / THF.

Chemical formula

[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 for the treatment of various diseases including autoimmune diseases, inflammation, transplantation, and grafts, Factor for example, cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those skilled in the art.

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

[0163] In the method, a therapeutically effective amount of one or more of the HDACIs of the present disclosure is typically formulated according to pharmaceutical practice and administered to a human in need thereof. Whether such treatment is indicated depends on the individual case and follows a medical evaluation (diagnosis) taking into account the existing signs, symptoms, and / or disorders, the risk of presenting signs, symptoms and / or disorders, and other factors.

[0164] The present HDACI can be administered by any suitable route, for example, orally, buccally, by inhalation, topically, sublingually, rectally, vaginally, intracisternal or intrathecal via lumbar puncture, transurethrally, intranasally, percutaneously, i.e., transdermally, or parenterally (including intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intra-articular, intrathecal, retrobulbar, intrapulmonary injection, and / or surgical implantation at the site). Parenteral administration can be achieved using needles and syringes or using high pressure techniques.

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

[0166] The toxicity and therapeutic efficacy of the present HDACI compounds are determined in cell cultures or experimental animals, for example, LDso (the dose at which 50% of the population dies) and ED 50It can be determined by standard pharmaceutical procedures for measuring (the therapeutically effective dose for 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index, which is expressed as the ratio between LD 50 and ED 50 . Compounds showing a high therapeutic index are preferred. Data obtained from such procedures can be used in formulating the dosage range for use in humans. The dosage preferably lies within the range of blood compound concentrations that includes 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 the therapeutically effective amount is within the capabilities of a fully skilled artisan, particularly in view of the detailed disclosure provided herein.

[0167] The therapeutically effective amount of the present HDACI required for use in therapy varies depending on the nature of the condition being treated, the length of time for which activity is desired, and the age and condition of the patient, and will ultimately be determined by the attending physician. The dosage and interval can be adjusted individually to provide plasma levels of the present HDACI sufficient to maintain the desired therapeutic effect. The desired dosage can be administered as a single dose, or as multiple doses at appropriate intervals, for example, once, twice, three times, four times, or more times a day as fractional doses, as convenient. In many cases, multiple doses are desirable or necessary. For example, the present HDACI can be administered 4 times at 4-day intervals once a day (q4d×4), 4 times at 3-day intervals once a day (q3d×4), administered once a day at 5-day intervals (qd×5), administered once a week for 3 weeks (qwk3), administered daily for 5 days, with a 2-day break, and then administered daily for another 5 days (5 / 2 / 5), or at any dosing schedule frequency determined to be appropriate for the situation.

[0168] The dosage of the composition containing the present HDACI, or the composition containing 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 by 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 above dosages are exemplary of an average case, but there may be individual cases that merit higher or lower dosages, and they are within the scope of the present disclosure. In practice, a physician determines the actual dosage regimen that is most appropriate for an individual patient and that may vary depending on the patient's age, weight, and response.

[0169] The present HDACI used in the method of the present disclosure is usually administered in an amount of about 0.005 to about 500 milligrams per administration, about 0.05 to about 250 milligrams per administration, or about 0.5 to about 100 milligrams per administration. For example, the present HDACI may be administered in an amount 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 administration, including all dosages between 0.005 and 500 milligrams per administration.

[0170] The HDACI of the present disclosure is usually administered in admixture with a pharmaceutical carrier selected with respect to the intended route of administration and standard pharmaceutical practice. The pharmaceutical composition for use in accordance with the present disclosure is formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and adjuvants that facilitate the processing of the present HDACI.

[0171] The term "carrier" refers to a diluent, adjuvant, or excipient administered together with the present 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, etc. The carrier can be saline, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, adjuvants, stabilizers, thickeners, lubricants, and coloring agents can be used. The pharmaceutically acceptable carrier is sterile. When the present HDACI is administered intravenously, water is a preferred carrier. For example, as a liquid carrier for injections, aqueous saline solutions as well as aqueous solutions of dextrose and glycerol can also be employed. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The present composition can also contain, if necessary, a small amount of a wetting agent or emulsifier, or a pH buffering agent.

[0172] These pharmaceutical compositions can be manufactured, for example, by conventional mixing, dissolving, granulating, tablet coating, emulsifying, encapsulating, enclosing, or lyophilization processes. The appropriate formulation depends on the selected route of administration. When a therapeutically effective amount of the present HDACI is administered orally, the composition is usually in the form of tablets, capsules, powders, solutions, or elixirs. When administered in tablet form, the composition can further contain a solid carrier, such as gelatin or an adjuvant. The tablets, capsules, and powders contain from about 0.01% to about 95%, preferably from about 1% to about 50%, of the present HDACI. When administered in liquid form, a liquid carrier, such as water, petroleum, or an oil of animal or plant origin, can be added. The liquid form of the composition can further contain saline, dextrose or other saccharide solutions, or glycols. When administered in liquid form, the composition contains from about 0.1% by weight to about 90% by weight, preferably from about 1% by weight to about 50% by weight, of the present compound.

[0173] When a therapeutically effective amount of the present HDACI is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. Preparing such a parenterally acceptable solution is within the skill of those of ordinary skill in the art, taking full account of pH, isotonicity, stability, etc. Preferred compositions for intravenous, cutaneous, or subcutaneous injection usually contain an isotonic medium. The present HDACI can be infused with other liquids over 10 - 30 minutes or several hours.

[0174] The present HDACI can be readily mixed with pharmaceutically acceptable carriers well known in the art. Such carriers enable formulation of the active agent as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for oral ingestion by the patient being treated. Pharmaceuticals for oral use are obtained by adding the present HDACI to a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries if necessary, and then treating the granules of the mixture to obtain tablet or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added if necessary.

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

[0176] Pharmaceutical compositions for parenteral administration contain an aqueous solution of the active agent in water-soluble form. Additionally, suspensions of the present HDACI can be prepared as suitable oily injection suspensions. Suitable lipophilic solvents or media 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 can also contain a suitable stabilizer or agent that enhances the solubility of the compound and enables the preparation of a high-concentration solution. Alternatively, the composition can be in powder form for constitution prior to use with a suitable vehicle, such as sterile pyrogen-free water.

[0178] The present HDACI can also be formulated into rectal compositions such as suppositories or retention enemas, including, for example, conventional suppository bases. In addition to the formulations described above, the present HDACI can also be formulated as a depot formulation. Such long-acting formulations can be administered by implantation (e.g., subcutaneously or intramuscularly) or intramuscular injection. Thus, for example, the present HDACI can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin.

[0179] In some embodiments, the present HDACI may be administered orally, buccally, or sublingually, in the form of tablets containing excipients such as starch or lactose, or in capsules or vaginal suppositories, either alone or as a mixture with excipients, or in the form of elixirs or suspensions containing flavoring or coloring agents. Such liquid formulations can be prepared with pharmaceutically acceptable additives, such as suspending agents. The present HDACI can also be administered parenterally, for example, by intravenous, intramuscular, subcutaneous, or intracoronary injection. For parenteral administration, the present HDACI is most suitable for use in the form of a sterile aqueous solution, which can be made isotonic with blood by including other substances, such as salts or monosaccharides, such as mannitol or glucose.

[0180] As a further embodiment, the present disclosure includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates use for practicing the methods of the present disclosure. In one simple embodiment, the kit comprises a compound or composition described herein as being useful for practicing the method (e.g., the present HDACI and a second therapeutic of any component) packaged in a container such as a sealed bottle or vessel. FactorA composition containing the compound or composition for use in practicing the methods of the present disclosure is attached to the container or included in the kit. Preferably, the compound or composition is packaged in a unit dosage form. The kit can further include an apparatus suitable for administering the composition according to the intended route of administration, such as a syringe, drip bag, or patch. In another embodiment, the selected compound is a lyophilized product. In this example, the kit can further include an additional container containing a solution useful for reconstituting the lyophilized product.

[0181] Some conventional HDACIs often have properties that tend to impede their development as treatments for diseases other than cancer due to the fact that they are active against several known HDACs. Factor Thus, an important feature of the present disclosure relates to the fact that the compounds of the present disclosure exhibit isoform selectivity. The present compounds show an increased inhibitory potency and selectivity for HDAC6 compared to other HDACs, for example, higher selectivity for class II compared to class I. The improved properties of the present compounds indicate that these compounds should be useful for immunosuppressive agents and neuroprotective agents, as well as for uses including but not limited to Alzheimer's disease, depression, Rett syndrome, Charcot-Marie-Tooth disease, brain cancer, etc. For example, the compounds of the present disclosure typically have a binding affinity (IC 50 ) for HDAC6 of 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 pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are independently selected from the group consisting of hydrogen and C 1 -C 6 alkyl, or R 1 and R 2 are joined to form a 3- to 7-membered heterocyclyl, and L 1 is CO2 H, C(O)NH 2 , C(O)NHOH, or B(OH) 2 wherein L 2 is H or OR 3 wherein 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 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, said compound, or a pharmaceutically acceptable salt thereof.

[0184] Embodiment 2. R 1 , R 2 and R 3 are independently C 1 -C 6 branched alkyl, the compound according to Embodiment 1, or a pharmaceutically acceptable salt thereof.

[0185] Embodiment 3. L 1 is C(O)NHOH, the compound according to Embodiment 1, or a pharmaceutically acceptable salt thereof.

[0186] Embodiment 4. The compound is of formula Ib, the compound according to Embodiment 1, or a pharmaceutically acceptable salt thereof.

[0187] Embodiment 5. The compound is of formula Ic, the compound according to Embodiment 1, or a pharmaceutically acceptable salt thereof.

[0188] Embodiment 6. The compound is of formula Id, the compound according to Embodiment 1, or a pharmaceutically acceptable salt thereof.

[0189] Embodiment 7. The compound according to Embodiment 1, which is the compound of formula Ie, or a pharmaceutically acceptable salt thereof.

[0190] Embodiment 8. The compound according to Embodiment 1, which is the compound 6a, 6b, 6d, 6e, or 6f, or a pharmaceutically acceptable salt thereof.

[0191] Embodiment 9. (a) Any one compound of formula I, Ib, Ic, Id, or Ie, or the compound 6a, 6b, 6d, 6e, or 6f, (b) a second treatment useful for the treatment of a disease or condition in which inhibition of HDAC has an effect Factor , and (c) a composition optionally containing an excipient and / or a pharmaceutically acceptable carrier.

[0192] Embodiment 10. The second treatment Factor is a composition according to Embodiment 9, which contains a chemotherapeutic agent useful for the treatment of cancer.

[0193] Embodiment 11. A pharmaceutical composition comprising any one compound of formula I, Ib, Ic, Id, or Ie, or the compound 6a, 6b, 6d, 6e, or 6f, and a pharmaceutically acceptable carrier or vehicle.

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

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

[0196] Embodiment 14. Further comprising administering a therapeutically effective amount of a second treatment Factor useful for the treatment of the disease or condition, the use according to Embodiment 12.

[0197] Use according to embodiment 12, wherein any one compound of formula I, Ib, Ic, Id, or Ie, or compound 6a, 6b, 6d, 6e, or 6f, and said second treatment Factor are administered simultaneously.

[0198] Use according to embodiment 12, wherein any one compound of formula I, Ib, Ic, Id, or Ie, or compound 6a, 6b, 6d, 6e, or 6f, and said second treatment Factor are administered separately.

[0199] Use according to any one of embodiments 12 - 16, wherein said disease or condition is cancer.

[0200] Use according to any one of embodiments 12 - 17, wherein said disease is cancer and said second treatment Factor is one or more of a chemotherapeutic agent, radiation, and immunotherapy.

[0201] Use according to embodiment 18, wherein said immunotherapy comprises anti - PD1 immunotherapy.

[0202] Use according to embodiment 19, wherein said anti - PD1 immunotherapy comprises administration of a PD - 1 antibody.

[0203] Use according to embodiment 20, wherein said PD - 1 antibody is nivolumab, pembrolizumab, STI - A1014, or pidilizumab.

[0204] Use according to any one of embodiments 14 - 21, wherein said second treatment Factor comprises radiation, and said radiation is optionally administered together with a radiation sensitizer and / or a treatment Factor agent.

[0205] Use according to any one of embodiments 12 - 22, wherein said disease or condition is a neurological disease, neurodegenerative disorder, peripheral neuropathy, or traumatic brain injury.

[0206] Use according to any one of Embodiments 12 to 23, wherein the disease or condition is stroke.

[0207] Use according to any one of Embodiments 12 to 24, wherein the disease or condition is inflammation or an autoimmune disease.

[0208] Embodiment 26. Further, a second treatment useful for treating the autoimmune disease or the inflammation Factor Use according to Embodiment 25, comprising administering a therapeutically effective amount of.

[0209] Use of any one compound of Formula I, Ib, Ic, Id, or Ie, or Compounds 6a, 6b, 6d, 6e, or 6f to increase the sensitivity of cancer cells to the cytotoxic effects of radiotherapy and / or chemotherapy.

[0210] Kit comprising any one compound of Formula I, Ib, Ic, Id, or Ie, or Compounds 6a, 6b, 6d, 6e, or 6f, and instructions for use for administering the compound or a pharmaceutically acceptable salt thereof to a subject in need thereof.

[0211] Kit according to Embodiment 28, wherein the subject has cancer.

[0212] Embodiment 30. Further, a kit according to Embodiment 29, comprising an anti-PD1 antibody.

[0213] Use of any one compound of Formula I, Ib, Ic, Id, or Ie, or Compounds 6a, 6b, 6d, 6e, or 6f for treating a disease or condition in an individual in which inhibition of HDAC results in an effect.

[0214] Compound for use according to Embodiment 31, wherein the HDAC is HDAC6.

[0215] Embodiment 33. Further, a second treatment useful for treating said disease or condition Factor A compound for use according to embodiment 31, comprising administering a therapeutically effective amount of

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

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

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

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

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

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

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

[0223] Embodiment 41. Said second treatment Factorcomprises radiation, said radiation optionally comprising a radiation sensitizer and / or a therapy Factor A compound for use according to any one of embodiments 33 to 40, which is administered together 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 a stroke.

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

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

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

[0229] The above can be better understood by reference to the following examples. The examples are presented for illustrative purposes and are not intended to limit the scope of the present disclosure.

Examples

[0230] General Information 1 H and 1313C NMR spectra were obtained on Bruker spectrometers operating at 400 / 101 and 500 / 126 MHz, using the residual peak of the solvent as the internal standard, unless otherwise specified (chemical shifts: CDCl 3 , δ 7.26 / 77.16 and DMSO-d 6 , 2.50 / 39.52). The following abbreviations were used for multiplicity: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, and br s = broad singlet. The progress of the reaction was monitored using TLC plates (Merck silica gel 60 F 254 , thickness 250 μm), 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 containing 0.1% formic acid, flow rate 0.5 mL / min, for 4 min. Flash chromatography was carried out using a Combi-Flash® Rf system (Teledyne ISCO) equipped with silica gel cartridges. Preparative HPLC was used for the purification of all final compounds, and Shimadzu preparative LC was used under the following conditions: column, ACE 5AQ (150×21.2 mm, id), mobile phase: 5–100% acetonitrile / water containing 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 under the following conditions: column, ACE 3 (150×4.6 mm, id), mobile phase, 5–100% acetonitrile / water containing 0.05% TFA, flow rate 1.0 mL / min for 25 min, UV detection at 254 nm. The purity of all test compounds for in vitro biological tests was greater than 95%. The purity of Suprastat for crystallographic studies and in vivo tests was greater than 98%.

[0231] [Chemical formula] Phenyl(4-(((tert-butoxycarbonyl)amino)methyl)phenyl)carbamate (2). tert-Butyl(4-aminobenzyl)carbamate (1, 500 mg, 2.25 mmol) and K 2 CO 3 (373 mg, 2.70 mmol) in stirred acetone (15 mL) was added phenyl chloroformate (352 mg, 2.25 mmol) over 10 min. After stirring at room temperature for 2 h, the excess solid was filtered off. The filtrate was collected and concentrated under vacuum. The crude product was purified by flash chromatography (0 - 50% EtOAc / hexane) to give 2 as a pale yellow solid (700 mg, yield: 91%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.41 - 7.37 (m, 4H), 7.25 - 7.16 (m, 6H), 4.86 (s, 1H), 4.27 (d, J = 4.7 Hz, 2H), 1.46 (s, 9H). 13 C NMR (101 MHz, CDCl 3 ) 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]

Chemical Structure

[0233]

Chemical formula

[0234]

Chemical formula

[0235]

Chem.

[0236]

Chem.

[0237]

Chem.

[0238]

Chem.

[0239]

Chem.

[0240]

Chemical Structure

[0241]

Chemical Structure

[0242]

Chemical Structure

[0243]

Chemical Structure

[0244]

Chemical Structure

[0245] Expression and purification of HDAC1 - 9 and 11 Large - scale expression of human HDACs was performed in HEK293 / T17 cells basically as previously described (see, for example, Falkenberg et al., Nat. Rev. Drug Discov. 13, 673 - 691 (2014), or Matthias 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 MgCl 2 , 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, 3×20 s) on ice, and the cell lysate was clarified by centrifugation at 40000×g for 30 min at 4°C. Recombinant fusion HDAC proteins were purified by Strep - Tactin affinity chromatography (IBA, Goettingen, Germany) with an elution buffer containing 50 mM HEPES, 100 mM NaCl, 50 mM KCl, 10% glycerol, and 3 mM desthiobiotin, pH 7.5. The 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 The IC in Table 1 50 values were measured using a fluorescence-based assay with 10 μM Ac-GAK(Ac)-AMC (for HDAC1, 2, 3, 6) or 10 μM Boc-Lys(TFA)-AMC (for HDAC4, 5, 7, 8, 9, 11) as substrates (see, for example, Imai et al., Cancer Sci. 107:1543-1549 (2016)). Briefly, individual HDACs were pre-incubated with a dilution series of the test inhibitor (0 - 100 μM) in a 384-well plate at 37 °C for 10 minutes in a reaction buffer at pH 7.4 containing 50 mM HEPES, 140 mM NaCl, 10 mM KCl, 1 mM TCEP, 0.1% BSA in a total volume of 40 μL. The deacetylation reaction was initiated by adding 10 μL of 10 μM substrate to the HDAC / inhibitor mixture. After incubation at 37 °C for 30 minutes, the reaction was terminated by adding 25 μL of trypsin solution (4 mg / mL). Fluorescence generation by trypsin was carried out for 15 minutes and 60 minutes 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 nm and 440 nm, respectively. Non-linear regression analysis was employed, and the IC 50 values were calculated using GraphPad Prism software. A 3-fold dilution series of the inhibitor was used to create 14-point IC 50 curves. The inhibitor concentration ranges used: 100 μM - 0.063 pM for HDAC1-5, 7-9, 11, and 3 μM - 1.88 pM for HDAC6. 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 of the University of California, Los Angeles. WM164 human melanoma cells were obtained from ATCC. These cells were incubated in RPMI 1640, 1% penicillin-streptomycin, and 10% fetal bovine serum at 37 °C and 5% CO 2 2. The 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. In the STAT3 phosphorylation assay, cells were pretreated overnight with or without HDAC6i (5 μM) and then treated with recombinant human IL-6 (Biolegend) for 20 minutes. 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 overnight with 1 μM, 5 μM, and 10 μM of Suprastat, and then lysates for immunoblot assay were collected. The cytotoxicity assay was performed using CellTox Green (Promega, Cat#G8731) according to the manufacturer's instructions, and fluorescence measurements were obtained with 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 the compound for 24 hours to measure cytotoxicity.

[0248] Immunoblot analysis Cells were harvested and lysed in RIPA buffer (ThermoScientific, 89900) containing protease and phosphatase inhibitors (ThermoScientific, 78440) by sonication with a Bioruptor (Diagenode) at high setting for 8 cycles of 30 seconds on and 30 seconds off. To evaluate protein expression, total protein samples were heat-denatured in SDS sample loading buffer and 15 - 20 μg of protein was analyzed on a 4 - 20% SDS-PAGE gel (Bio-Rad, 456-1093). Proteins were transferred to a low-fluorescence PVDF membrane (Bio-Rad, 1704274) using a Trans-Blot Turbo transfer system (Bio-Rad). These membranes were blocked with Odyssey blocking buffer (Licor, 927-40000) for 1 hour and then incubated with primary antibodies (1:1000 dilution) at 4°C. After washing these membranes three times with TBST buffer, they were incubated with a near-infrared fluorophore-conjugated secondary antibody (1:10000 dilution) for 1 hour at room temperature. These 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 instructions of the manufacturer of QIAzol (Qiagen, 79306). Quantification of RNA was performed using a NanoDrop One spectrophotometer (NanoDrop Technologies). Samples with an absorbance ratio of 260 / 280 exceeding 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 with nuclease-free water. Quantitative PCR analysis was performed using the iQ SYBR Green Supermix (Bio-Rad, 1708882) on a CFX96 Real-Time System (Bio-Rad). Gene expression analysis was performed using 2 -ΔΔCt methods, and the 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 for the analysis were as follows:

[0250] Mouse Animal experiments involving mice were conducted in accordance with protocol #A354, approved by the Institutional Care and Use Committee (IACUC) of The George Washington University. Forty C57BL / 6 female mice were purchased from Charles River Laboratories (Wilmington, Massachusetts, USA). In vivo tests were performed using tumor cells that had been passaged in vivo from mouse to mouse at least 5 times prior to tumor transplantation. Mice were injected with 1.0 × 10 6Individual in vivo passaged melanoma cells were subcutaneously injected. When the tumor became palpable approximately 5 days after tumor transplantation, the pre-treatment group was initiated. The cages were randomly assigned to different treatment groups, and the mice were treated with Suprastat, anti-PD1 antibody, or vehicle control. Control mice were intraperitoneally injected with 100 μL of PBS as vehicle control, an anti-PD1 antibody (BioXcell, clone RMP1-14) at a dose of 15 mg / kg, and 25 mg / kg of Suprastat. The mice were treated 5 days a week until the tumors in the control group reached the maximum size according to our IACUC protocol. Tumor volume was measured every other day using caliper measurements and calculated using the formula L×W 2 / 2. All animal experiments were conducted taking toxicity into consideration, and the initial signs of toxicity were monitored regularly. Emphasis was placed on mortality, body weight, and food intake. At the postmortem evaluation of the endpoint, each condition was performed, including a general visual inspection of organs such as the liver for hepatotoxicity, splenomegaly, and pulmonary metastatic nodules.

[0251] In vitro evaluation of 6a - c To evaluate the impact of each additional functional group on potency and isoform selectivity, the potencies of 6a - c against human HDAC1 - 9 and 11 were examined in vitro under optimized conditions (see, for example, Osko et al., J. Med. Chem. 63:295 - 308 (2020)) together with NextA. The results in Table 1 suggest that 6a and 6c are more potent and selective HDAC6 inhibitors (IC 50 = 0.4 and 0.5 nM), with at least 290 - fold selectivity for class I isoforms HDAC1 - 3 and 8, and 1000 - fold selectivity for class IIa isoforms HDAC4, 5, 7, and 9. Furthermore, 6a - c did not show activity against HDAC11, a class IV isoform, at concentrations below 50 μM. Regarding HDAC6, it should be noted that the experimental IC 50 values in the range of 0.2 - 0.4 nM are at the limit of our assay using the full - length enzyme at a concentration of approximately 0.6 nM (measured by absorbance at 280 nm). Therefore, the IC 50The values have reached the limit of our assays, and theoretically these two inhibitors may have even higher potencies than those reported here. Overall, these inhibition data suggest that the hydroxylbutyl chain is more important for improving the enzymatic ability and selectivity of HDAC6 compared to the aminomethyl group. Furthermore, the incorporation of polar aminomethyl and hydroxylbutyl groups into inhibitors 6a - c increases the number of heavy atoms, but their clogP (calculated by SwissADME) decreases compared to NextA (Table 3), resulting in a significant increase in lipophilic ligand efficiency (LipE), particularly for 6a (LipE = 7.57 (6a) vs 6.19 (NextA)), while the ligand efficiencies of 6a and 6b are slightly lower. In summary, compared to NextA and related analogs, Suprastat (6a), which has both a hydroxybutyl moiety and an aminomethyl moiety, shows improved potency against HDAC6 and excellent selectivity against HDAC isoforms, and at the same time exhibits the highest LipE value due to its significantly decreased clogP. On the other hand, as alternative ZBGs, the inhibitory potencies of non-hydroxamic acid analogs 6d - f containing carboxylic acid, amide, and boronic acid against HDAC6 are severely impaired (Table 2), and significant hydroxamic acid-Zn 2+ Without coordination, it can be shown that the additional hydrogen bond interactions between this cap and the pocket of HDAC6 are not 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 measured in human plasma (Table 3). Overall, the stability of Suprastat (6a) is very good, ranging from over 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 (the plasma protein binding fraction is 1.9%), which is beneficial for increasing the free drug concentration in vivo compared to the more lipophilic parent compound NextA (89%), while the plasma binding fractions of the singly modified derivatives 6b and 6c are 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 assays were performed using the WM164 human melanoma cell line. WM164 cells are a variant of BRAF V600E, a mutation frequently found in melanoma patients (see, for example, Daina et al., Sci. Rep. 7:42717 (2017)). WM164 cells were treated with 6a - c and NextA at concentration ranges of 0.1 - 10 μM, respectively. Their ability to increase the level of acetylated α - tubulin (Ac - α - tubulin) was measured by immunoblot analysis and compared to a control. Figures 1A - D show an increase in Ac - α - tubulin with increasing concentration of HDAC6i, but the heights vary (Figure 1E). However, the increase in Ac - α - tubulin was also associated with a slight increase in the level of acetylated histone H3 (Ac - H3) at high concentrations (Figure 1F). Treatment with Suprastat resulted in the highest Ac - α - tubulin levels at 0.1 - 10 μM and the greatest increase at 10 μM compared to other HDAC6i. At high concentrations, a slight increase in the level of Ac - H3 was observed with Suprastat, but it was much lower than that of other HDAC6i. Therefore, this data indicates that Suprastat is a highly selective and potent HDAC6 inhibitor. Based on the concentration ranges of these α - tubulin / histone acetylation experiments, further cytotoxicity assays were performed on 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, while Suprastat and 6c were not cytotoxic up to 25 μM.

[0254] Functional Characterization of Suprastat Immune cells such as macrophages are the 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 decreases STAT3 recruitment at the IL10 promoter region in antigen-presenting cells (see, for example, Cheng et al., J. Immunol. 193:2850-2862 (2014)). Consistent with these experiments, as shown in FIG. 3A, when mouse bone marrow-derived macrophages were treated with 5 μM of Suprastat, the expression of the IL10 gene was decreased as judged by mRNA quantification, compared to vehicle-treated macrophages. The gene expression of IL10 was normalized to β-actin (ACTB) as a reference gene. A dose-dependent increase in the Ac-α-tubulin level of RAW264.7 macrophages (FIG. 4) was observed when treated with increasing concentrations (1, 5, and 10 μM) of Suprastat, indicating that Suprastat affects macrophages and melanoma cells in the same way. Furthermore, as shown in FIG. 3B, the results of immunoblot analysis of lysates obtained from WM164 melanoma cells exposed to IL-6 cytokine (30 ng / mL) for 20 minutes after pretreatment with either Suprastat or NextA showed a decrease in the phosphorylation of Y705 of STAT3 compared to IL-6 alone. This result indicates that Suprastat mediates an immunomodulatory effect by affecting the interaction between HDAC6 and the STAT3 transcription factor, similar to NextA.

[0255] In vivo combination test with immunotherapy Immunotherapy has emerged as a major treatment for solid tumors, but patients often develop resistance, and currently, combination therapies that enhance the effectiveness of immunotherapy while overcoming resistance are needed (see, for example, O’Donnell et al., Cancer Treat Rev. 52:71-81 (2017)). Using the syngeneic SM1 mouse melanoma model, it has previously been demonstrated that pretreatment with NextA significantly reduces tumor size in C57BL / 6 mice. Since these mice have a functional immune system, it is possible to test immune checkpoint inhibitors such as anti-PD1 therapy. The combination of NextA and anti-PD1 therapy significantly controls 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 of Suprastat intraperitoneally (IP), after which anti-PD1 immune checkpoint blockade therapy (15 mg / kg, IP) was initiated. This pretreatment method was performed to prime the tumor microenvironment (TME) in a way that leads to the induction of an anti-tumor immune response. As shown in Figure 5A, monotherapy using Suprastat similarly reduced the tumor mass as indicated by tumor volume compared to the control (PBS) group. However, no significant difference was observed between Suprastat and anti-PD1 therapy. On the contrary, the combination of Suprastat and anti-PD1 therapy showed a significant reduction in tumor mass compared to the control group and the monotherapy group, suggesting that pretreatment with Suprastat enhances the anti-tumor immune response resulting from anti-PD1 therapy. Figure 5B shows the tumor growth of each mouse in each treatment group. The data so far indicate that Suprastat has an immunomodulatory effect in vivo and, when combined with anti-PD1 therapy, significantly enhances the anti-tumor immune response. It was noted that the group combining Suprastat and anti-PD1 therapy showed an enhanced inhibitory effect on tumor growth by day 14 compared to the other groups.In contrast, the combination of NextA and anti-PD1 therapy began to show a clear anti-tumor effect after day 20 in the previous tests by the present inventors, as compared with 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 immunomodulatory properties of Suprastat, a comprehensive immunocyte phenotype examination by flow cytometry was performed. 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 decreased F4 / 80+CD206+ tumor-promoting M2 macrophages (Figure 6B), and thus the balance shifted towards an anti-tumor immune response, 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 studies with NextA (see, for example, 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 tests performed with NextA. However, an increase was only observed in the percentage of CD8+ central memory (CM) cells in the group treated with Suprastat, suggesting that it enhances the effector memory function of CD8 T cells and enables a long-term anti-tumor immune response. 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-reg (Figure 6F). Further analysis of natural killer (NK) cells showed an increase in the anti-PD1 and combination groups, but a favorable trend in the Suprastat group (Figure 6G), which was not observed in previous combination tests using NexA (see, for example, 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 presumed to be a significant increase in CD8+ effector T cells.Overall, this phenotypic examination of immune cells demonstrated that Suprastat improved immunomodulatory properties by reducing tumorigenic M2 macrophages and increasing the infiltration of anti-tumor CD8+ effector T cells and memory cells compared to its parent compound, NextA, i.e., it was shown to contribute to the promotion of in vivo immunomodulatory effects and the improvement of anti-tumor immune responses in combination with anti-PD1 therapy.

[0257] Conclusion Compared to other related analogs, what Suprastat exhibits is the ability to selectively enhance the level of acetylated tubulin rather than strong HDAC6 activity, isoform selectivity, and the obvious effect on histone acetylation. The polar functional groups added to Suprastat reduce its lipophilicity, thereby increasing ligand efficiency and plasma protein binding while maintaining metabolic stability in different media. In in vivo combination tests with PD-1 antibodies, it was revealed that Suprastat significantly improved the therapeutic outcome due to its immunomodulatory properties. 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Teng and M. J. Smyth, Cancer Treat Rev., 2017, 52, 71-81. The present invention provides, for example, the following items. (Item 1) A compound of formula I: [Chemical formula] Or a pharmaceutically acceptable salt thereof, wherein R 1 and R 2 are independently hydrogen and C 1 -C 6is selected from the group consisting of alkyl, or R 1 and R 2 are joined to form a 3- to 7-membered heterocyclyl, L 1 is CO 2 H, C(O)NH 2 , C(O)NHOH, or B(OH) 2 and 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, and is selected from the group consisting of, 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, n is 0, 1, or 2, said compound, or a pharmaceutically acceptable salt thereof. (Item 2) R 1 , R 2 and R 3 are, independently, C 1 -C 6 branched alkyl, the compound according to Item 1, or a pharmaceutically acceptable salt thereof. (Item 3) L 1 is C(O)NHOH, the compound according to Item 1, or a pharmaceutically acceptable salt thereof. (Item 4) The compound according to Item 1 of Formula Ib: [Chemical formula] or a pharmaceutically acceptable salt thereof. (Item 5) The compound according to item 1 of formula Ic:

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Claims

Claim 1 A compound of formula I: 【Chemical 23】 or a pharmaceutically acceptable salt thereof, wherein R １ and R ２ are each independently selected from the group consisting of hydrogen and C １ -C ６ -alkyl, or R １ and R ２ are joined 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 selected from the group consisting of 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, and is selected from the group consisting of: each X is independently 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, n is 0, 1 or 2, said compound, or a pharmaceutically acceptable salt thereof. Claim 2 R １ 、 R ２ and R ３ is independently C １ -C ６ branched alkyl, the compound according to claim 1, or a pharmaceutically acceptable salt thereof. Claim 3 L １ The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein L is C(O)NHOH. Claim 4 The compound according to claim 1 of formula Ib: 【Chemical 24】 or a pharmaceutically acceptable salt thereof. Claim 5 The compound according to claim 1 of formula Ic: 【Chemical 25】 or a pharmaceutically acceptable salt thereof. Claim 6 The compound according to claim 1 of formula Id: 【Chemical 26】 or a pharmaceutically acceptable salt thereof. Claim 7 The compound according to claim 1 of formula Ie: 【Chemical 27】 or a pharmaceutically acceptable salt thereof. Claim 8 The compound according to claim 1 selected from the group consisting of: 【Chemical Formula 28】 or a pharmaceutically acceptable salt thereof. Claim 9 (a) A compound according to any one of claims 1 to 8, (b) a second therapeutic agent useful for the treatment of a disease or condition in which inhibition of HDAC has an effect, and (c) optionally an excipient and / or a pharmaceutically acceptable carrier. Claim 10 The composition according to claim 9, wherein said second therapeutic agent comprises a chemotherapeutic agent useful for the treatment of cancer. Claim 11 A pharmaceutical composition comprising a compound according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier or vehicle. Claim 12 A composition for treating a disease or condition in which inhibition of HDAC has an effect, said composition comprising a compound according to any one of claims 1 to 8. Claim 13 The composition according to claim 12, wherein said HDAC is HDAC6. Claim 14 The composition according to claim 12, wherein said composition is administered in combination with a second therapeutic agent useful for the treatment of said disease or condition. Claim 15 The composition according to claim 14, wherein said composition and said second therapeutic agent are administered simultaneously. Claim 16 The composition according to claim 14, wherein said composition and said second therapeutic agent are administered separately. Claim 17 The composition according to any one of claims 12 to 16, wherein said disease or condition is cancer. Claim 18 The composition according to any one of claims 14 to 17, wherein said disease is cancer and said second therapeutic agent is one or more of a chemotherapeutic agent, radiation, and / or immunotherapy. Claim 19 The composition according to claim 18, wherein the immunotherapy comprises anti-PD1 immunotherapy.

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

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

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

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

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

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

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

28. A kit comprising a compound according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, and instructions for administration of the compound, or a pharmaceutically acceptable salt thereof, to a subject in need thereof.

29. The kit according to claim 28, wherein the subject has cancer.

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

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