a1H-Benzo[c][1,2]thiadiazine-2,2-dioxides with heterocyclic linkers as selective histone deacetylase 6 inhibitors

Abstract

Histone deacetylase inhibitors (HDACbls) and compositions comprising the same are disclosed. Methods for inhibiting HDAC6 to treat diseases and conditions such as cancer, neurodegenerative disorders, neurological disorders including peripheral neuropathy such as Charcot-Marie-Tooth disease, traumatic brain injury, stroke, malaria, autoimmune diseases, autism, and inflammation are also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to Provisional Patent Application No. 63 / 307,932, filed February 8, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Government Statement of Interest This invention was made with Government support under Grant Nos. 5R01NS079183, R41AG058283, and R43HD093464 awarded by the National Institutes of Health. The U.S. Government has certain rights in this invention.

[0003] The present invention relates to histone deacetylase inhibitors (HDAC6Is) selective for histone deacetylase 6, pharmaceutical compositions comprising one or more of the HDAC6Is, methods of enhancing the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy comprising contacting the cells with one or more of the HDAC6Is, and therapeutic methods of treating conditions and diseases in which inhibition of HDAC6 is beneficial (e.g., cancer, inflammation, neurological diseases, neurodegenerative disorders, stroke, traumatic brain injury, allograft rejection, autoimmune diseases, and malaria) comprising administering a therapeutically effective amount of the HDAC6I of the present invention to an individual in need thereof. Preferably, the compounds of the present invention may be useful in the treatment of peripheral neuropathy, e.g., Charcot-Marie-Tooth disease, and various other neurodegenerative disorders, including Alzheimer's dementia. [Background technology]

[0004] The histone deacetylase HDAC protein family consists of currently 18 enzymes that are classified into four subgroups according to their homology with the yeast family. HDACs 1, 2, 3, and 8, classified as class I HDACs according to their homology with yeast Rpd3, are characterized by ubiquitous expression and nuclear localization. Class II HDACs exhibit tissue-specific expression and shuttling between the nucleus and cytoplasm. These enzymes, which are homologous to yeast Hda1, are divided into class IIa (HDACs 4, 5, 7, and 9) and class IIb (HDACs 6 and 10). HDAC11, the only member of the class IV subfamily, shows similarity to the catalytic domains of both class I and class II enzymes. Class I, II, and IV HDACs use Zn as a cofactor for their deacetylation activity. 2+ Sirtuins 1 to 7, whose activity depends on nicotinamide adenine dinucleotide, form class III of HDACs.

[0005] HDAC inhibitors offer immense potential in the treatment of a variety of diseases. Within the HDAC field, there is a large amount of HDAC inhibitor (HDACI) compounds that can block deacetylase enzymes. The majority of HDACI compounds inhibit across multiple classes of HDAC enzymes and are therefore classified as pan-inhibitors. It has proven difficult to find HDACI compounds that are highly selective for a given subtype, such as HDAC6. To date, only non-selective pan-HDACI compounds, such as Vorinostat® and Panbinostat®, are commercially available. However, pan-HDACIs are associated with dose-limiting side effects, limiting their use in cancer treatment. Among the various HDAC isoforms that appear to be promising therapeutic targets for treating human diseases other than cancer, HDAC6 has emerged as a particularly attractive target (Simoes-Pires, C. et al., Mol. Neurodegener. 2013;8: 7). For example, HDAC6Is have shown potential to treat peripheral neuropathies such as Charcot-Marie-Tooth (CMT) disease (d'Udewalle, C. et al., Nat. Med. 2011, 17(8), 968-74), major inherited neuropathies, and tauopathies including Alzheimer's disease (Selenica, ML et al., Alzheimers Res. Ther. 2014 6(1):12). Furthermore, HDAC6Is are expected to be safe for the treatment of non-cancer diseases with long-term administration, as HDAC6 knockout animals are viable. HDAC6 is primarily involved in regulating the acetylation status of cytosolic proteins, e.g., α-tubulin, HSP-90, colactin, HSF-1, and other protein targets. This enzyme also plays a role in the recognition and clearance of polyubiquitinated misfolded proteins from cells via aggresome formation. The development of HDAC6-selective compounds has been recently reviewed (Kalin, JH et al., J. Med. Chem. 2013, 56, 6297-6313).In general, HDACIs are composed of three main motifs: a zinc-binding group (ZBG), a capping group, and a linker bridging the previous two (Figure 1). A properly optimized capping group can improve both potency and selectivity, presumably due to its ability to enter into appropriate contacts with residues on the enzyme surface.

[0006] Many HDACIs, such as trichostatin A (TSA) and SAHA, contain hydroxamic acid functions as ZBGs (Figure 1). Many of the hydroxamic acid HDAC inhibitors, including the marketed drug Vorinostat®, have been found to be genotoxic. Genotoxicity is an undesirable side effect that limits the scope of drugs to diseases that may be considered life-threatening, such as cancer. Indeed, non-genotoxic HDACI compounds would be essential for use in non-cancer diseases that require chronic and longer-term administration. Thus, there is a great need for the discovery of potent and selective HDACIs, especially HDACIs that do not exhibit genotoxic activity.

[0007] There is extensive evidence supporting the therapeutic use of HDAC6I in the treatment of various conditions and diseases, including neurodegeneration. However, despite showing overall beneficial effects, such as beneficial neuroprotective effects, HDAC6Is known to date, for example, have little specificity in terms of HDAC inhibition, and therefore may inhibit more than one zinc-dependent histone deacetylase. It is still unclear which is the prominent HDAC(s) that can confer neuroprotection. Emerging evidence suggests that at least some of the HDAC isozymes, for example HDAC1, are absolutely necessary for neuronal maintenance and survival. In addition, the problem of adverse side effects from non-specific HDAC inhibition has been raised. Thus, the clinical effectiveness of current non-specific HDAC6Is against stroke, neurodegenerative disorders, neurological diseases, and other diseases and conditions may ultimately be limited. It is therefore important to design, synthesize, and test compounds that can function as potent, isozyme-selective HDAC6Is that can ameliorate the effects of neurological, neurodegenerative, and peripheral neuropathy disorders, including chemotherapy-induced neuropathy, traumatic brain injury, cancer, inflammation, malaria, autoimmune diseases, and other conditions and diseases mediated by HDAC6.

[0008] An important advance in the art is the discovery of HDAC6I, particularly selective HDAC6I, which is useful for treating diseases that benefit from HDAC6 inhibition, such as cancer, neurological disease, traumatic brain injury, neurodegenerative disorders and other peripheral neuropathy, stroke, hypertension, malaria, allograft rejection, rheumatoid arthritis, and various inflammatory conditions.Therefore, there is a great need in the art for effective compounds, compositions, and methods that are useful for treating such diseases, either alone or in combination with other treatments that are used to treat these diseases and conditions.The present invention is directed to meeting this need. Summary of the Invention

[0009] The present invention relates to histone deacetylase 6 inhibitors (HDAC6I), pharmaceutical compositions comprising HDAC6I, and methods for treating diseases and disorders that benefit from HDAC6 inhibition, such as cancer, neurological disorders, psychiatric disorders, neurodegenerative disorders, peripheral neuropathy, stroke, hypertension, inflammation, traumatic brain injury, rheumatoid arthritis, allograft rejection, sepsis, and autoimmune diseases, comprising administering a therapeutically effective amount of HDAC6I to an individual in need thereof. The present invention also relates to methods for increasing the sensitivity of cancer cells to radiation therapy and / or chemotherapy. The present invention also allows the use of these HDAC6I in combination with other drugs and / or therapeutic approaches. In some embodiments, the HDAC6I of the present invention exhibits selectivity, particularly over certain other HDAC isozymes, such as HDAC1. In particular, the present invention relates to the discovery of compounds that comprise a tetrahydroquinoline moiety or an analog thereof as a capping residue.

[0010] More specifically, the present invention relates to compounds of general formula I: [ka] or a pharma- ceutically acceptable salt and / or solvate thereof, [ka] represents a single bond or a double bond, and in the case of a 5-membered ring moiety, there are two double bonds and three single bonds, which, when taken together, give rise to a 5-membered ring heteroaryl group; X and Y are independently carbon or nitrogen and Z is N or C(F); m=0, 1, or 2; with the exception that when R1 is deuterium, m is an integer from 0 to 4; When at least one of X and Y is carbon, n=0, 1, or 2; or when both X and Y are nitrogen, n=0 or 1; The sum of o and p is 0 or 1; R1 and R2 are independently hydrogen, deuterium, halogen, hydroxyl, -CH2OH, -CH2CH2OH, cyano, -NR a R b , -CH2NR a R b , -C(O)NR a R b , -S(O)NR a R b , acetyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 1-C6 alkoxy, C1-C6 haloalkyl, F3C-S-, C3-C6 cycloalkyl, (C3-C5 cycloalkyl)-(C1-C3 alkyl)-, (C1-C3 alkyl)-(C3-C5 cycloalkyl)-, (C3-C5 cycloalkyl)-O-, aryl, aryl-O-, aryl-(C1-C3 alkyl)-, aryl-(C1-C3 alkoxy) )-, heteroaryl, heteroaryl-O-, heteroaryl-(C1-C3 alkyl)-, heteroaryl-(C1-C3 alkoxy)-, 4- to 6-membered heterocyclyl, or (4- to 6-membered heterocyclyl)-(C1-C3 alkyl)-; or two of R1 and / or two of R2 are bonded to adjacent carbon atoms of their respective aromatic rings and linked to form a 5- or 6-membered carbocyclic or heterocyclic ring; R a and R b is independently selected from the group consisting of hydrogen and C1-C6 alkyl or C3-C6 cycloalkyl, or these groups may be combined to form a 3-7 membered heterocyclyl; a is [ka] If represents a single bond, CR c R d , C=O, NR e , O, or S; or [ka] If represents a double bond, CR c or N; b is [ka] If represents a single bond, CR c R d or [ka] If represents a double bond, CR c or b is [ka] represents a double bond, and a represents CR c If , then N; R c and R d are independently hydrogen, deuterium, fluorine, chlorine, C1-C6 alkyl, or C3-C6 cycloalkyl, or are joined together to form a 3- to 6-membered cycloalkyl; R e is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or 4-6 membered heterocyclyl; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano). [Brief description of the drawings]

[0011] [Figure 1] The structures of SAHA and TSA, as well as the general structure of HDAC inhibitors, are shown. [Figure 2A] 1 shows the biological activity of exemplary compounds disclosed herein. [Figure 2B] 1 shows the biological activity of exemplary compounds disclosed herein. [Figure 3A] 1 shows the biological activity of exemplary compounds disclosed herein. [Figure 3B] 1 shows the biological activity of exemplary compounds disclosed herein. [Figure 4] 1 shows the biological activity of exemplary compounds disclosed herein. [Diagram 5] An exemplary method for synthesizing compound 51 is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] In particular, the present invention relates to HDAC6Is, compositions comprising the HDAC6Is of the invention, and compounds of general formula I: [ka] or a pharma- ceutically acceptable salt and / or solvate thereof, [ka] represents a single bond or a double bond, and in the case of a 5-membered ring moiety, there are two double bonds and three single bonds, which, when taken together, give rise to a 5-membered ring heteroaryl group; X and Y are independently carbon or nitrogen and Z is N or C(F); m=0, 1, or 2; with the exception that when R1 is deuterium, m is an integer from 0 to 4; When at least one of X and Y is carbon, n=0, 1, or 2; or when both X and Y are nitrogen, n=0 or 1; The sum of o and p is 0 or 1; R1 and R2 are independently hydrogen, deuterium, halogen, hydroxyl, -CH2OH, -CH2CH2OH, cyano, -NR a R b , -CH2NR a R b , -C(O)NR a R b , -S(O)NR a R b, acetyl, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, 1-C6 alkoxy, C1-C6 haloalkyl, F3C-S-, C3-C6 cycloalkyl, (C3-C5 cycloalkyl)-(C1-C3 alkyl)-, (C1-C3 alkyl)-(C3-C5 cycloalkyl)-, (C3-C5 cycloalkyl)-O-, aryl, aryl-O-, aryl-(C1-C3 alkyl)-, aryl-(C1-C3 alkoxy) )-, heteroaryl, heteroaryl-O-, heteroaryl-(C1-C3 alkyl)-, heteroaryl-(C1-C3 alkoxy)-, 4- to 6-membered heterocyclyl, or (4- to 6-membered heterocyclyl)-(C1-C3 alkyl)-; or two of R1 and / or two of R2 are bonded to adjacent carbon atoms of their respective aromatic rings and linked to form a 5- or 6-membered carbocyclic or heterocyclic ring; R a and R b is independently selected from the group consisting of hydrogen and C1-C6 alkyl or C3-C6 cycloalkyl, or these groups may be combined to form a 3-7 membered heterocyclyl; a is [ka] If represents a single bond, CR c R d , C=O, NR e , O, or S; or [ka] If represents a double bond, CR c or N; b is [ka] If represents a single bond, CR c R d or [ka] If represents a double bond, CRc or b is [ka] represents a double bond, and a represents CR c If , then N; R c and R d are independently hydrogen, deuterium, fluorine, chlorine, C1-C6 alkyl, or C3-C6 cycloalkyl, or are joined together to form a 3- to 6-membered cycloalkyl; R e is selected from the group consisting of hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl, aryl, heteroaryl, or 4-6 membered heterocyclyl; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano).

[0013] In one embodiment, the present invention comprises formula Ib [ka] where R1 and R2 are defined as in formula I.

[0014] In another embodiment, the invention comprises an HDAC6I of formula Ib, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0015] Another embodiment includes an HDAC6I of formula Ic [ka] where R1 and R2 are defined as in formula I.

[0016] In another embodiment, the invention comprises an HDAC6I of formula Ic, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0017] In another embodiment, the present invention comprises formula Id [ka] where R1 and R2 are defined as in formula I.

[0018] In another embodiment, the invention comprises an HDAC6I of formula Id, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0019] In another embodiment, the present invention includes formula Ie [ka] where R1 and R2 are defined as in formula I.

[0020] In another embodiment, the invention comprises an HDAC6I of formula Ie, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0021] In another embodiment, the present invention provides a compound comprising the formula If [ka] where R1 and R2 are defined as in formula I.

[0022] In another embodiment, the invention comprises an HDAC6I of formula If, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0023] In another embodiment, the present invention includes formula Ig [ka] where R1 and R2 are defined as in formula I.

[0024] In another embodiment, the invention comprises HDAC6I of formula Ig, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0025] In another embodiment, the present invention includes a compound comprising formula Ih [ka] where R1 and R2 are defined as in formula I.

[0026] In another embodiment, the invention comprises HDAC6I of formula Ih, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0027] In another embodiment, the present invention includes formula Ii [ka] where R1 and R2 are defined as in formula I.

[0028] In another embodiment, the invention comprises an HDAC6I of formula Ii, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0029] In another embodiment, the present invention includes formula Ij [ka] where R1 and R2 are defined as in formula I.

[0030] In another embodiment, the invention comprises HDAC6I of formula Ij, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0031] In another embodiment, the present invention includes formula Ik [ka] where R1 and R2 are defined as in formula I.

[0032] In another embodiment, the invention encompasses HDAC6I of formula Ik, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0033] In another embodiment, the present invention provides a compound comprising the formula IL [ka] where R1 and R2 are defined as in formula I.

[0034] In another embodiment, the invention comprises an HDAC6I of formula IL, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0035] In another embodiment, the present invention includes the formula Im [ka] where R1 and R2 are defined as in formula I.

[0036] In another embodiment, the invention comprises an HDAC6I of formula Im, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0037] In another embodiment, the present invention provides a compound comprising the formula In [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0038] In another embodiment, the invention encompasses HDAC6I of formula In, where R1 and R2 are independently selected from H, D, Cl, and F.

[0039] In another embodiment, the present invention includes a compound comprising formula Io [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0040] In another embodiment, the invention comprises an HDAC6I of formula Io, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0041] In another embodiment, the present invention comprises the formula Ip [ka] where R1 and R2 are defined as in formula I.

[0042] In another embodiment, the invention comprises an HDAC6I of formula Ip, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0043] In another embodiment, the present invention includes formula Iq [ka] where R1 and R2 are defined as in formula I.

[0044] In another embodiment, the invention comprises HDAC6I of formula Iq, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0045] In another embodiment, the present invention provides a compound comprising formula Ir [ka] where R1 and R2 are defined as in formula I.

[0046] In another embodiment, the invention comprises an HDAC6I of formula Ir, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0047] In another embodiment, the present invention includes formula Is [ka] where R1 and R2 are defined as in formula I.

[0048] In another embodiment, the invention comprises an HDAC6I of formula Is, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0049] In another embodiment, the present invention relates to a compound comprising the formula It [ka] where R1 and R2 are defined as in formula I.

[0050] In another embodiment, the invention comprises an HDAC6I of formula It, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0051] In another embodiment, the present invention includes formula Iu [ka] where R1 and R2 are defined as in formula I.

[0052] In another embodiment, the invention comprises an HDAC6I of formula Iu, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0053] In another embodiment, the present invention includes a compound comprising formula Iv [ka] where R1 and R2 are defined as in formula I.

[0054] In another embodiment, the invention comprises an HDAC6I of formula Iv, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0055] In another embodiment, the present invention includes a compound comprising formula Iw [ka] where R1 and R2 are defined as in formula I.

[0056] In another embodiment, the invention comprises HDAC6I of formula Iw, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0057] In another embodiment, the present invention includes a compound comprising formula Ix [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0058] In another embodiment, the invention encompasses HDAC6I of formula Ix, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0059] In another embodiment, the present invention includes formula Iy [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0060] In another embodiment, the invention encompasses HDAC6I of formula Iy, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0061] In another embodiment, the present invention includes formula Iz [ka] where R1 and R2 are defined as in formula I.

[0062] In another embodiment, the invention encompasses HDAC6I of formula Iz, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0063] In another embodiment, the present invention includes formula Iaa [ka] where R1 and R2 are defined as in formula I.

[0064] In another embodiment, the invention comprises an HDAC6I of formula Iaa, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0065] In another embodiment, the present invention includes the compound of formula Ibb [ka] where R1 and R2 are defined as in formula I.

[0066] In another embodiment, the invention comprises an HDAC6I of formula Ibb, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0067] In another embodiment, the present invention includes the formula Icc [ka] where R1 and R2 are defined as in formula I.

[0068] In another embodiment, the invention comprises an HDAC6I of formula Icc, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0069] In another embodiment, the present invention provides a compound comprising the formula Idd [ka] where R1 and R2 are defined as in formula I.

[0070] In another embodiment, the invention comprises HDAC6I of formula Idd, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0071] In another embodiment, the present invention includes the compound of formula Iee [ka] where R1 and R2 are defined as in formula I.

[0072] In another embodiment, the invention encompasses HDAC6I of formula Iee, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0073] In another embodiment, the present invention provides a compound comprising the formula Iff [ka] where R1 and R2 are defined as in formula I.

[0074] In another embodiment, the invention comprises an HDAC6I of formula Iff, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0075] In another embodiment, the present invention provides a compound comprising the formula Igg [ka] where R1 and R2 are defined as in formula I.

[0076] In another embodiment, the invention comprises an HDAC6I of formula Igg, where R1 and R2 are independently selected from H, D, Cl, and F.

[0077] In another embodiment, the present invention includes the formula Ihh [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0078] In another embodiment, the invention encompasses HDAC6I of formula Ihh, where R1 and R2 are independently selected from H, D, Cl, and F.

[0079] In another embodiment, the present invention comprises formula Iii [ka] (wherein R1 and R2 are defined as in claim 1, and both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2).

[0080] In another embodiment, the invention comprises an HDAC6I of formula Iii, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0081] In another embodiment, the present invention includes the formula Ijj [ka] where R1 and R2 are defined as in formula I.

[0082] In another embodiment, the invention comprises HDAC6I of formula Ijj, where R1 and R2 are independently selected from H, D, Cl, and F.

[0083] In another embodiment, the present invention includes the formula Ikk [ka] where R1 and R2 are defined as in formula I.

[0084] In another embodiment, the invention comprises an HDAC6I of formula Ikk, wherein R1 and R2 are independently selected from H, D, Cl, and F.

[0085] In another embodiment, the present invention comprises the formula ILL [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0086] In another embodiment, the invention comprises an HDAC6I of formula ILL, wherein R1 and R2 are independently selected from H, D, Cl, and F, c and d are nitrogen, and e is oxygen.

[0087] In another embodiment, the present invention includes the formula Imm [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0088] In another embodiment, the invention comprises an HDAC6I of formula Imm, wherein R1 and R2 are independently selected from H, D, Cl, and F, c and d are nitrogen, and e is oxygen.

[0089] In another embodiment, the present invention provides a compound comprising the formula Inn [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R fis hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0090] In another embodiment, the invention includes HDAC6I of formula Inn, wherein R1 and R2 are independently selected from H, D, Cl, and F, c and d are nitrogen, and e is oxygen.

[0091] In another embodiment, the present invention includes a compound comprising the formula Ioo [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0092] In another embodiment, the invention includes HDAC6I of formula Ioo, wherein R1 and R2 are independently selected from H, D, Cl, and F, c and d are nitrogen, and e is oxygen.

[0093] In another embodiment, the present invention includes the formula Ipp [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0094] In another embodiment, the invention includes HDAC6I of formula Ipp, wherein R1 and R2 are independently selected from H, D, Cl, and F, c and d are nitrogen, and e is oxygen.

[0095] In another embodiment, the present invention includes the compound of formula Iqq [ka] (In the formula, [ka] represents a single or double bond, and of the five bonds so marked, two are double bonds and three are single bonds, which, when arranged together, give rise to a five-membered heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f ), wherein one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; R1 and R2 are defined as in formula I).

[0096] In another embodiment, the invention comprises one of the compounds listed in Table 1.

[0097] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0098] In addition, the salts, prodrugs, hydrates, isotopically labeled and fluorescently labeled derivatives of the HDAC6I of the present invention, as well as any other therapeutically or diagnostically relevant derivatives, are also included in the present invention and can be used in the methods disclosed herein. The present invention further includes all possible stereoisomers and geometric isomers of the compounds of the present invention. The present invention includes both racemic and optically active isomers. If the HDAC6I of the present invention is desired as a single enantiomer, it can be obtained by resolution of the final product or by stereospecific synthesis from either isomerically pure starting materials or the use of chiral auxiliary reagents, see, for example, Ma, Z. et al., Tetrahedron: Asymmetry, 1997, 8, 883-888. Resolution of the final product, intermediate, or starting material can be achieved by any suitable method known in the art. Furthermore, in the situation where tautomers of the compounds of the present invention are possible, the present invention is intended to include all tautomeric forms of the compounds.

[0099] The deuterium labeled compounds of the invention are of interest due to the finding that C-D bonds are broken less readily than C-H bonds and therefore metabolic degradation involving attack on these bonds is slowed down upon replacement of H with D, increasing the half-life of the drug substance (see, e.g., Halford, B. Chem. Eng. News 2016, 94(27), 32-36). Preferred C-H bonds replaced with C-D bonds are aliphatic C-H bonds adjacent to amine nitrogen atoms; aliphatic C-H bonds adjacent to the oxygen atom of an alkoxy substituent, and aromatic C-H bonds can be replaced fully or partially with C-D bonds.

[0100] Lactams and amides can be reduced to deuterated amines containing the moiety CD2 adjacent to their amine nitrogen atom using commercially available deuterated versions of reducing agents commonly used for reduction to amines, such as LiAlD4 and BD3-THF. Alternatively, alkylation of the nitrogen atom with a deuterated alkyl group can be achieved by treatment with the corresponding deuterated alkyl halide in the presence of base, or by reductive alkylation with the corresponding deuterated aldehyde [containing the moiety C(D)O] (e.g., using NaBD(OAc)3 in AcOD as the reducing agent). When a methoxy substituent is present on one of the aromatic rings of the compounds of the invention, their trideuteriomethoxy analogs can be obtained by alkylation of the corresponding phenol with CD3I or (CD3)2SO4 in the presence of a suitable base, such as K2CO3 or NaH. Similarly, alkylation of the same phenol with RCD2Br or RCD2I and base produces 1,1-dideuterioalkyl ethers. Compounds containing deuterium as a substituent on one or more aromatic rings can be obtained by carrying out the synthesis from perdeuterated starting materials or by selectively introducing deuterium atoms by reduction of aryl bromides or aryl iodides with tri-n-butyltin deuteride in the presence of a transition metal catalyst such as a palladium compound.

[0101] The prodrug of the compound of the present invention is also included in the present invention.It is well established that the prodrug approach, which derivatizes the compound into a form suitable for formulation and / or administration and then releases it as a drug in vivo, has been used successfully to temporarily (e.g., bioreversibly) change the physicochemical properties of the compound (see H. Bundgaard, Ed., "Design of Prodrugs," Elsevier, Amsterdam, (1985); RB Silverman, "The Organic Chemistry of Drug Design and Drug Action," Academic Press, San Diego, chapter 8, (1992); KM Hillgren et al., Med. Res. Rev., 15, 83 (1995)).

[0102] The compounds of the present invention can exist as salts. The pharma- ceutically acceptable salts of the HDAC6Is of the present invention are often preferred in the methods of the present invention. As used herein, the term "pharma- ceutically acceptable salts" refers to salts or zwitterionic forms of the compounds of the present invention. The salts of the compounds of the present invention can be prepared during the final isolation and purification of the compounds, or separately by reacting the compounds with an acid having a suitable anion. The pharma- ceutically acceptable salts of the compounds of the present invention can be acid addition salts formed with pharma- ceutically acceptable acids. Examples of acids that can be used to form pharma- ceutically 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. Examples of salts of the compounds of the present invention include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, 2-hydroxyethanesulfonate, phosphate, hydrogen phosphate, acetate, adipate, alginate, aspartate, benzoate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerol phosphate, hemisulfate, heptanoate, hexanoate, formate, succinate, fumarate, maleate, ascorbate, isethionate, salicylate, methanesulfonate, methion ... The salts of the amino groups that can be used include, but are not limited to, styrenesulfonate, 1-naphthalenesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, trichloroacetate, trifluoroacetate, phosphate, glutamate, bicarbonate, undecanoate, lactate, citrate, tartrate, gluconate, ethanedisulfonate, benzenesulfonate, and p-toluenesulfonate. In addition, the available amino groups present in the compounds of the present invention can be quaternized with methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides; dimethyl sulfate, diethyl sulfate, dibutyl sulfate, and diamyl sulfate; decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides; and benzyl and phenethyl bromides.In view of the foregoing, any reference to a compound of the invention appearing herein is intended to include the compound of the invention, as well as its pharma- ceutically acceptable salts, hydrates, or prodrugs.

[0103] The compounds of the present invention can also be conjugated or linked to auxiliary moieties that promote the beneficial properties of the compounds in therapeutic use. Such conjugates can improve the delivery of the compounds to specific anatomical sites or regions of interest (e.g., tumors), allow sustained therapeutic concentrations of the compounds in target cells, modify the pharmacokinetic and pharmacodynamic properties of the compounds, and / or improve the therapeutic index or safety profile of the compounds. Suitable auxiliary moieties include, for example, amino acids, oligopeptides, or polypeptides, for example, antibodies, such as monoclonal antibodies and other engineered antibodies, as well as natural or synthetic ligands for receptors in target cells or tissues. Other suitable auxiliary agents include fatty acids or lipid moieties that promote biodistribution and / or uptake of the compounds by target cells (see, for example, Bradley et al., Clin. Cancer Res. (2001) 7:3229).

[0104] The compounds of the present invention inhibit HDAC6 and are useful for treating various diseases and conditions.In particular, the HDAC6I of the present invention is used in a method for treating diseases or conditions that benefit from the inhibition of HDAC6, such as cancer, neurological diseases, neurodegenerative conditions, peripheral neuropathy, autoimmune diseases, inflammatory diseases and conditions, stroke, hypertension, traumatic brain injury, autism, and malaria.The method comprises administering a therapeutically effective amount of the HDAC6I of the present invention to an individual in need thereof.

[0105] The methods of the invention also include administering to the individual a second therapeutic agent in addition to the HDAC6I of the invention, the second therapeutic agent being selected from agents such as drugs and adjuvants known to be useful in treating the disease or condition afflicting the individual, e.g., chemotherapeutic agents and / or radiation known to be useful in treating certain cancers.

[0106] The compounds of the present invention have been evaluated for their activity at and selectivity for HDAC6 compared to other HDACs. It has previously been shown that selective HDAC6 inhibitors may be applied to a variety of disease states, including but not limited to arthritis, autoimmune disorders, inflammatory disorders, cancer, neurological diseases such as Rett syndrome, peripheral neuropathy such as CMT, stroke, hypertension, and diseases in which oxidative stress is a causative factor or a consequence. Selective HDAC6 inhibitors have also been shown to extend the lifespan of mice bearing kidney xenografts when administered in combination with rapamycin. This model was used to evaluate the immunosuppressive properties of the compounds of the present invention and served as a model of graft rejection. In addition, it has previously been shown that selective HDAC6 inhibitors confer neuroprotection in a rat primary cortical neuron model of oxidative stress. In these studies, selective HDAC6 inhibitors were identified as non-toxic neuroprotective agents. The compounds of the present invention behave similarly, as they are also selective HDAC6 agents. The compounds of the present invention demonstrate ligand efficiency, which makes them more drug-like in their physiochemical properties. Thus, the compounds of the present invention are drug candidates and research tools for identifying specific functions of HDAC6.

[0107] Thus, in one embodiment, the present invention relates to a method for treating an individual suffering from a disease or condition in which inhibition of HDAC6 would be beneficial, comprising administering to the individual in need thereof a therapeutically effective amount of a claimed HDAC6I compound.

[0108] The method of the present invention can be achieved by administering one of the HDAC6Is of the present invention as a raw compound or as a pharmaceutical composition.The administration of the pharmaceutical composition of the present invention or the raw HDAC6I can be carried out during or after the onset of the disease or condition of interest.Typically, the pharmaceutical composition is sterile and does not contain toxic, carcinogenic or mutagenic compounds that cause adverse reactions when administered.

[0109] In some embodiments, the HDAC6I of the present invention can be administered in combination with a second therapeutic agent useful for treating a disease or condition that benefits from the inhibition of HDAC6. The second therapeutic agent is different from the HDA6CI of the present invention. The HDAC6I of the present invention and the second therapeutic agent can be administered simultaneously or sequentially. In addition, the HDAC6I of the present invention and the second therapeutic agent can be administered from a single composition or two separate compositions. The HDAC6I of the present invention and the second therapeutic agent can be administered simultaneously or sequentially to achieve the desired effect.

[0110] The second therapeutic agent is administered in an amount that will produce its desired therapeutic effect. Effective dosage ranges for each second therapeutic agent are known in the art, and the second therapeutic agent is administered to an individual in need thereof within such established ranges.

[0111] Therefore, the present invention relates to compositions and methods of using such compounds in the treatment of diseases or conditions that benefit from the inhibition of HDAC6.The present invention also relates to pharmaceutical compositions comprising the HDAC6I of the present invention and any second therapeutic agent useful for the treatment of diseases and conditions that benefit from the inhibition of HDAC6.Furthermore, a kit is provided that comprises the HDAC6I of the present invention, packaged separately or together, and optionally a second therapeutic agent useful for the treatment of diseases and conditions that benefit from the inhibition of HDAC6, and a package insert with instructions for use of these active agents.

[0112] The HDAC6I of the present invention and the second therapeutic agent can be administered together as a single unit dose or separately as multiple unit doses, and the HDAC6I of the present invention is administered before the second therapeutic agent, or vice versa.One or more doses of the HDAC6I of the present invention and / or one or more doses of the second therapeutic agent can be administered.Thus, the HDAC6I of the present invention can be used in combination with one or more second therapeutic agents, for example, but not limited to, anticancer agents.

[0113] Within the meaning of the present invention, the term "disease" or "condition" refers to a disorder and / or abnormality that is generally considered to be a pathological condition or function and may manifest itself in the form of certain signs, symptoms, and / or dysfunction. As shown below, the HDAC6I of the present invention is a potent inhibitor of HDAC6 and can be used in the treatment of diseases and conditions in which inhibition of HDAC6 is beneficial, such as cancer, neurological diseases, neurodegenerative conditions, traumatic brain injury, stroke, inflammation, autoimmune diseases, and autism.

[0114] In one embodiment, the present invention provides a method for treating cancer, including but not limited to killing cancer cells or tumor cells; inhibiting the growth of cancer cells or tumor cells; inhibiting the replication of cancer cells or tumor cells; or improving symptoms thereof, comprising administering to a subject in need thereof an amount of the HDAC6I of the present invention or its pharma- ceutically acceptable salt sufficient to treat cancer.In addition, it should be noted that selective HDAC6I may promote the killing of cancer cells through the reactivation of immune system by a mechanism related to PDI receptor.The HDAC6I of the present invention can be used as a single anti-cancer agent or in combination with another anti-cancer treatment, such as radiation therapy, chemotherapy, and surgery.

[0115] In another embodiment, the present invention provides a method for increasing the sensitivity of cancer cells to the cytotoxic effects of radiotherapy and / or chemotherapy, comprising contacting the cells with the HDAC6I of the present invention or a pharma- ceutically acceptable salt thereof in an amount sufficient to increase the sensitivity of the HDAC6I, thereby increasing the sensitivity of the cells to the cytotoxic effects of radiotherapy and / or chemotherapy.Hence, the HDAC6is of the present invention can be combined with antibodies against PD-1 and / or PD-L1 to achieve higher efficacy.

[0116] In a further embodiment, the present invention provides a method for treating cancer, comprising: (a) administering to an individual in need thereof an amount of the HDAC6I compound of the present invention; and (b) administering to the individual an amount of radiation therapy, chemotherapy, or both. Each of the administered amounts is effective to treat cancer. In another embodiment, the amounts together are effective to treat cancer.

[0117] Therefore, this combination therapy of the present invention can be used in various settings for the treatment of various cancers.In certain embodiments, the individual in need of treatment has previously undergone treatment for cancer.Such previous treatment includes, but is not limited to, previous chemotherapy, radiation therapy, surgery, or immunotherapy such as cancer vaccine.

[0118] In another embodiment, the cancer to be treated is a cancer that is known to be sensitive or responsive to radiation and / or chemotherapy, including, but not limited to, non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, laryngeal cancer, cervical cancer, nasopharyngeal cancer, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophageal cancer, rectal cancer, small cell lung cancer, non-small cell lung cancer, brain tumor, or other CNS neoplasm.

[0119] In yet another embodiment, the cancer to be treated is known to be resistant to radiation therapy and / or chemotherapy or refractory to radiation therapy and / or chemotherapy. A cancer is refractory to a treatment when at least some significant portion of cancer cells are not killed or their cell division is not stopped in response to the treatment. Such a determination can be made by any method known in the art for assaying the effectiveness of a treatment on cancer cells, either in vivo or in vitro, using the art-accepted meaning of "refractory" in such context. In certain embodiments, a cancer is refractory when the number of cancer cells is not significantly reduced or is increased.

[0120] Other cancers that may be treated using the compounds and methods of the present invention include, but are not limited to, cancers and metastases such as brain cancer (glioblastoma) and melanoma, as well as other common tumors.

[0121] In certain embodiments, leukoplakia, a benign-appearing hyperplastic or dysplastic lesion of the epithelium, and Bowen's disease, a carcinoma in situ, are preneoplastic lesions indicating the desirability of preventive intervention.

[0122] In another embodiment, fibrocystic diseases (cystic hyperplasia, breast dysplasia, especially adenosis (benign epithelial thickening)) indicate the desirability of preventive intervention.

[0123] The prophylactic use of the compounds and methods of the invention has also been indicated for several viral infections that may result in cancer. For example, human papillomavirus can cause cervical cancer (see, e.g., Hernandez-Avila et al., Archives of Medical Research (1997) 28:265-271), Epstein-Barr virus (EBV) can cause lymphoma (see, e.g., Herrmann et al., J. Pathol. (2003) 199(2):140-5), hepatitis B virus or hepatitis C virus can cause liver cancer (see, e.g., El-Serag, J. Clin. Gastroenterol. (2002) 35(5 Suppl 2):S72-8), and human T-cell leukemia virus (HTLV)-I can cause T-cell leukemia (see, e.g., Mortreux et al., Leukemia (2003) 35(5 Suppl 2):S72-8). 17(1):26-38), human herpesvirus-8 infection can cause Kaposi's sarcoma (see, e.g., Kadow et al., Curr. Opin. Investig. Drugs (2002) 3(11):1574-9), and human immunodeficiency virus (HIV) infection contributes to the development of cancer as a result of immune deficiency (see, e.g., Dal Maso et al., Lancet Oncol. (2003) 4(2):110-9).

[0124] In other embodiments, subjects exhibiting one or more of the following malignant predispositions may be treated by administration of an HDAC6I of the invention and methods of the invention: chromosomal translocations associated with malignancies (e.g., Philadelphia chromosome for chronic myeloid leukemia, t(14;18) associated with follicular lymphoma, etc.), familial polyposis or Gardner's syndrome (possibly a precursor to colon cancer), benign monoclonal gammopathy (possibly a precursor to multiple myeloma), cancers or precancers exhibiting Mendelian inheritance patterns. First-degree blood relationship to an affected individual (e.g., familial adenomatous polyposis coli, Gardner's syndrome, hereditary osteomatosis, polyendocrine neoplasia, medullary thyroid carcinoma with amyloid production and pheochromocytoma, Peutz-Jeghers syndrome, von Recklinghausen neurofibromatosis, retinoblastoma, carotid body tumor, cutaneous melanoma, intraocular melanoma, xeroderma pigmentosum, ataxia-telangiectasia, Chediak-Higashi syndrome, albinism, Fanconi aplastic anemia, and Bloom's syndrome (see, e.g., Robbins and Angell, 1976, Basic Pathology, 2nd Ed., WB Saunders Co., Philadelphia, pp. 112-113) and exposure to carcinogens (e.g., smoking and inhalation or contact with certain chemicals).

[0125] In another specific embodiment, the HDAC6I and methods of the invention are administered to a human subject to prevent the progression of breast cancer, colon cancer, ovarian cancer, or cervical cancer.

[0126] In one embodiment, the present invention provides a method for treating cancer, comprising: (a) administering to an individual in need thereof an amount of the HDAC6I of the present invention; and (b) administering to the individual one or more additional anti-cancer treatment modalities, including but not limited to radiation therapy, chemotherapy involving specific antibodies, e.g., antibodies against PD-1 and PD-L1, surgery, or immunotherapy, such as cancer vaccines. In one embodiment, the administration of step (a) is prior to the administration of step (b). In another embodiment, the administration of step (a) is subsequent to the administration of step (b). In yet another embodiment, the administration of step (a) is simultaneous with the administration of step (b).

[0127] In one embodiment, the additional anti-cancer treatment modality is radiation therapy and / or chemotherapy, hi another embodiment, the additional anti-cancer treatment modality is surgery.

[0128] In yet another embodiment, the additional anti-cancer treatment modality is an immunotherapy, such as a cancer vaccine.

[0129] In one embodiment, the HDAC6I of the present invention, or a pharma- ceutically acceptable salt thereof, is administered adjunctively with an additional anti-cancer treatment modality.

[0130] In a preferred embodiment, the additional anti-cancer treatment modality is radiation therapy. Any radiation treatment protocol can be used in the methods of the present invention depending on the type of cancer being treated. Embodiments of the present invention include the use of electromagnetic radiation of various wavelengths, i.e. gamma radiation (10 -20 ~10 -13 m), X-ray (10 -12 ~10 -9 m), ultraviolet light (10nm to 400nm), visible light (400nm to 700nm), infrared radiation (700nm to 1mm), and microwave radiation (1mm to 30cm).

[0131] For example, without limitation, X-ray radiation can be administered, particularly high energy megavoltage (radiation with energy above 1 MeV) can be used for deep tumors, and electron beam and orthovoltage X-ray radiation can be used for skin cancer. Radioisotopes that emit gamma rays, such as radioisotopes of radium, cobalt, and other elements, can also be administered. Exemplary radiation therapy protocols useful in the present invention include, but are not limited to, routine methods in which multiple low-dose radiation sources are simultaneously focused into a tissue volume from multiple angles; "internal radiation therapy" such as brachytherapy, interstitial irradiation, and intracavitary irradiation, which involves placing radioactive implants directly into a tumor or other target tissue; intraoperative irradiation, which directs large amounts of external radiation to target tissue exposed during surgery; and particle beam radiation therapy, which uses fast-moving subatomic particles to treat localized cancers.

[0132] Many cancer treatment protocols currently use radiosensitizers that are activated by electromagnetic radiation, such as X-rays. Examples of X-ray activated radiosensitizers include, but are not limited to, metronidazole, misonidazole, desmethylmisonidazole, pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233, EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR), 5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine (FUdR), hydroxyurea, cisplatin, and their therapeutically active analogs and derivatives.

[0133] Photodynamic therapy (PDT) of cancer uses visible light as a radioactivator of photosensitizers. Examples of photosensitizers include, but are not limited to, hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives, NPe6, tin etioporphyrin (SnET2), feboborbide-a, bacteriochlorophyll-a, naphthalocyanines, phthalocyanines, zinc phthalocyanines, and their therapeutically active analogs and derivatives.

[0134] In addition to the HDAC6I of the present invention, the radiosensitizer can be administered in combination with one or more therapeutically effective compounds, including, but not limited to, compounds that promote the uptake of the radiosensitizer into target cells, compounds that control the flow of therapeutic agents, nutrients, and / or oxygen to target cells, chemotherapeutic agents that act on tumors with or without additional radiation, or other therapeutically effective compounds for treating cancer or other diseases. Examples of additional therapeutic agents that can be used in combination with the radiosensitizer include, but are not limited to, 5-fluorouracil (5-FU), leucovorin, oxygen, carbogen, red blood cell transfusions, perfluorocarbons (e.g., FLUOSOLW®-DA), 2,3-DPG, BW12C, calcium channel blockers, pentoxifylline, antiangiogenic compounds, hydrazine, and L-BSO.

[0135] In one embodiment, the HDAC6I of the present invention, or a pharma- ceutically acceptable salt thereof, is administered prior to the administration of radiation therapy and / or chemotherapy.

[0136] In another embodiment, the HDAC6I of the present invention, or a pharma- ceutically acceptable salt thereof, is administered adjunctively with radiation therapy and / or chemotherapy.

[0137] The HDAC6I of the present invention and the additional therapeutic modality may act additively or synergistically (i.e., the combination of the HDAC6I of the present invention or its pharma- ceutically acceptable salt with the additional anti-cancer therapeutic modality is more effective than their additive effects when each is administered alone). A synergistic combination allows the use of lower doses of the HDAC6I of the present invention and / or the additional therapeutic modality of the present invention and / or the administration of the HDAC6I of the present invention and / or the additional therapeutic modality less frequently to a subject with cancer. The ability to utilize lower doses of the HDAC6I of the present invention and / or the additional therapeutic modality of the present invention and / or the ability to administer the compound of the present invention and the additional therapeutic modality less frequently can reduce the toxicity associated with administration without reducing the efficacy of the HDAC6I of the present invention and / or the additional therapeutic modality in treating cancer. Furthermore, the synergistic effect can result in improved efficacy of the treatment of cancer and / or reduced adverse or undesirable side effects associated with the administration of the HDAC6I of the present invention and / or the additional anti-cancer therapeutic modality as a monotherapy.

[0138] In one embodiment, the HDAC6I of the present invention can act synergistically with radiation therapy when administered at a dose typically used when such HDAC6I is used alone to treat cancer.In another embodiment, the HDAC6I of the present invention can act synergistically with radiation therapy when administered at a dose lower than the dose typically used when such HDAC6I is used as a monotherapy to treat cancer.

[0139] In one embodiment, radiation therapy may act synergistically with the HDAC6I of the present invention when administered at doses typically used when radiation therapy is used as a monotherapy for the treatment of cancer. In another embodiment, radiation therapy may act synergistically with the compounds of the present invention when administered at doses lower than those typically used when radiation therapy is used as a monotherapy for the treatment of cancer.

[0140] The efficacy of HDAC6I as an HDAC6 inhibitor for sensitizing cancer cells to the effects of radiation therapy can be determined by determining survival after treatment in vitro and / or in vivo using techniques known in the art. In one embodiment, in vitro determinations can involve exposing exponentially growing cells to a known dose of radiation and monitoring cell survival. Irradiated cells are seeded and cultured for about 14 to about 21 days, and colonies are stained. Viability is the number of colonies divided by the plating efficiency of unirradiated cells. Survival curves are generated by plotting survival on a logarithmic scale against absorbed dose on a linear scale. Survival curves generally show an exponential decrease in the percentage of surviving cells at higher radiation doses after an initial shoulder region where the dose was sublethal. Similar protocols can be used for chemical agents when used in the combination therapy of the present invention.

[0141] The inherent radiosensitivity of tumor cells and environmental influences, such as hypoxia and host immunity, can be further evaluated by in vivo studies. Growth delay assays are commonly used. These assays measure the time interval required for a tumor exposed to radiation to regrow to a specific volume. The dose required to control approximately 50% of the tumors is calculated using the TCD 50 Determined by assay.

[0142] In vivo assay systems typically use transplantable solid tumor systems in experimental subjects. Normal tissue and tumor radiation survival parameters can be assayed using in vivo methods known in the art.

[0143] The present invention provides a method for treating cancer, comprising administering an effective amount of the HDAC6I of the present invention in combination with recognized surgical, radiotherapy, and chemotherapy methods (e.g., chemical-based mimics of radiotherapy) to achieve synergistic enhancement of the effectiveness of recognized treatments.The effectiveness of treatment can be measured in clinical trials or in model systems such as tumor models in mice or cell culture sensitivity assays.

[0144] The present invention provides combination therapies that result in improved efficacy and / or reduced toxicity.Thus, in one aspect, the present invention relates to the use of HDAC6I of the present invention as a radiosensitizer in combination with radiotherapy.

[0145] When the combination therapy of the present invention comprises administering the HDAC6I of the present invention together with one or more additional anti-cancer drugs, the HDAC6I of the present invention and the additional anti-cancer drugs can be administered simultaneously or sequentially to an individual.The drugs can also be administered cyclically.Cyclic therapy comprises administering one or more anti-cancer drugs for a period of time, followed by administering one or more different anti-cancer drugs for a period of time, and repeating this sequential administration, i.e., this cycle, to reduce the development of resistance to one or more of the administered anti-cancer drugs, to avoid or reduce the side effects of one or more of the administered anti-cancer drugs, and / or to improve the efficacy of treatment.

[0146] Additional anti-cancer drugs may be administered over a series of sessions. Any one or combination of the additional anti-cancer drugs listed below may be administered.

[0147] The present invention includes a method for treating cancer, comprising administering to an individual in need thereof HDAC6I of the present invention and one or more additional anti-cancer drugs or pharma- ceutically acceptable salts thereof.The HDAC6I of the present invention and the additional anti-cancer drugs may act additively or synergistically.Suitable anti-cancer drugs include, but are not limited to, gemcitabine, capecitabine, methotrexate, taxol, taxotere, etc.

[0148] Furthermore, the present invention provides a method for treating cancer using the HDAC6I of the present invention as an alternative to chemotherapy alone or radiation therapy alone, where chemotherapy or radiation therapy has proven or may prove too toxic, e.g., causing unacceptable or intolerable side effects, for the subject being treated. The individual being treated may optionally be treated with another anti-cancer treatment modality, e.g., chemotherapy, surgery, or immunotherapy, depending on which treatment is found to be acceptable or tolerable.

[0149] The HDAC6I of the present invention can also be used in in vitro or ex vivo methods, such as the treatment of certain cancers, including but not limited to leukemia and lymphoma, treatment involving autologous stem cell transplantation. This can include a multi-step process of harvesting the subject's autologous hematopoietic stem cells and removing all cancer cells, the subject is then administered an effective amount of the HDAC6I of the present invention to eradicate the subject's remaining bone marrow cell population, and the stem cell transplant is then infused back into the subject. Bone marrow function is then restored, and supportive care is provided while the subject recovers.

[0150] The method of the present invention for treating cancer may further comprise administering the HDAC6I of the present invention and an additional therapeutic agent or its pharma- ceutically acceptable salt or hydrate.In one embodiment, the composition comprising the HDAC6I of the present invention is administered simultaneously with the administration of one or more additional therapeutic agent(s), and the additional therapeutic agent(s) may be part of the same composition or may be in a different composition from that comprising the HDAC6I of the present invention.In another embodiment, the HDAC6I of the present invention is administered before or after the administration of another therapeutic agent(s).

[0151] In the method of the present invention for treating cancer, the other therapeutic agent can be an antiemetic.Suitable antiemetics include, but are not limited to, metoclopramide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acetylleucine monoethanolamine, alizapride, azasetron, benzamide, bietanautine, bromopride, buclizine, clebopride, cyclozine, dimenhydroninate, diphenidol, dolasetron, meclizine, methanol, metopimazine, nabilone, oxyperundyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinol, thiethylperazine, thioproperazine, and tropisetron.

[0152] In one embodiment, the antiemetic agent is granisetron or ondansetron. In another embodiment, the other therapeutic agent can be a hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramotim, molgramotim, and epoetin alfa.

[0153] In yet another embodiment, the other therapeutic agent may be an opioid or non-opioid analgesic.Suitable opioid analgesics include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprozine, diphenoxylate, fentanyl, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclozocine, methadone, isomethadone, and propoxyphene. Suitable non-opioid analgesics include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofenac, diflucinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam, and sulindac.

[0154] In yet another embodiment, the other therapeutic agent may be an anti-anxiety agent. Suitable anti-anxiety agents include, but are not limited to, buprene, and benzodiazepines such as diazepam, lorazepam, oxapam, clorazep, clonazepam, chlordiazepoxide, and alprazolam.

[0155] In addition to treating cancer and sensitizing cancer cells to the cytotoxic effects of radiation therapy and chemotherapy, the HDAC6I of the present invention is used in methods for treating diseases, conditions, and injuries of the central nervous system, such as neurological diseases, neurodegenerative disorders, and traumatic brain injury (TBI). In a preferred embodiment, the HDAC6I of the present invention can cross the blood-brain barrier and inhibit HDAC in the brain of an individual.

[0156] The HDAC6I compounds of the present invention also provide therapeutic effects in models of peripheral neuropathy, such as CMT.HDAC6 inhibitors have been shown to cross the blood-nerve barrier and rescue the phenotype observed in transgenic mice that exhibit symptoms of distal hereditary motor neuropathy.Administering HDAC6 inhibitors to symptomatic mice increases acetylated α-tubulin levels, restores proper mitochondrial motility and axonal transport, and increases muscle renervation.Other peripheral neuropathy includes, but is not limited to, giant axonal neuropathy and various forms of mononeuropathy, polyneuropathy, autonomic neuropathy, and neuritis.

[0157] The HDAC6I compound of the present invention also improves the associative memory loss after the elevation of Aβ or tau protein.In this test, Aβ42 is injected into mice via a cannula implanted in the dorsal hippocampus 15 minutes before training.Test compound is administered intraperitoneally (25mg / kg) 2 hours before training.Fear learning is evaluated 24 hours later.

[0158] Contextual fear conditioning performed 24 hours after training shows a reduction in freezing in Aβ-injected mice compared to vehicle-injected mice. Treatment with the compounds of the present invention improves the lack of freezing response in Aβ-injected mice and has no effect on vehicle-injected mice. Test compounds alone do not affect the memory performance of mice. Furthermore, treatment does not affect the motor, sensory, or motivational performance assessed using the visible platform test in which the compounds are injected twice a day for two days. No obvious signs of toxicity are observed during these experiments, including changes in food and liquid intake, weight loss, or changes in locomotor and exploratory behavior.

[0159] These results demonstrate that HDAC6Is of the present invention are beneficial against the impairment of associative memory following the elevation of certain proteins, including Aβ and tau.

[0160] Therefore, the HDAC6I of the present invention is useful for treating neurological diseases by administering an effective amount of the HDAC6I of the present invention to treat the neurological disease, or by administering an effective amount of a pharmaceutical composition containing the HDAC6I of the present invention to treat the neurological disease. Treatable neurological diseases include Huntington's disease, lupus, schizophrenia, multiple sclerosis, muscular dystrophy, dentate red cell atrophy (DRRLA), spinal and 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 small intestine, and cerebrovascular disease. These conditions include, but are not limited to, somatic dementia, corticobasal degeneration, dystonia, myoclonus, Tourette's syndrome, tremor, chorea, restless legs syndrome, Parkinson's disease, Parkinsonian syndrome, anxiety, depression, psychosis, manic depression, Friedreich's ataxia, Fragile X syndrome, spinal muscular dystrophy, Rett syndrome, Rubinstein-Taybi syndrome, Wilson's disease, multiform infarct state, CMT, GAN, and other peripheral neuropathies.

[0161] In one embodiment, the neurological disease being treated is Huntington's disease, Parkinson's disease, Alzheimer's disease, Down's syndrome, spinal muscular atrophy, lupus, or schizophrenia.

[0162] Charcot-Marie-Tooth disease (CMT) is one of the most common inherited neuropathies affecting approximately 1 in 2,500 people in the United States. CMT affects both motor and sensory nerves, which can result in a high-stepping gait with a dropped foot and a tendency to stumble and fall. Mutations in small heat shock protein 27 (HSPB1) cause axonal CMT or distal hereditary motor neuropathy (distal HMN). Expression of mutant HSPB1 reduced acetylated α-tubulin levels and induced severe axonal transport defects. Pharmacological inhibition of histone deacetylase 6 (HDAC6)-induced α-tubulin deacetylation caused by the HDAC6I Tubastatin A corrects the axonal transport defects induced by HSPB1 mutations and rescues the CMT phenotype in symptomatic mutant HSPB1 mice. The pathogenic role of α-tubulin deacetylation has been demonstrated in mutant HSPB1-induced neuropathy, providing valuable insights into HDAC6 inhibitors as a therapeutic strategy for inherited axonal disorders. The compounds of the present invention show potent HDAC6 isoform inhibition, high HDAC6 selectivity, and surprising α-tubulin acetylation in various cell lines.

[0163] Thus, in another embodiment, the neurological disease is Charcot-Marie-Tooth disease. Current studies using CMT2A mutant animals show that some of the HDAC6Is of the present invention can correct both motor and sensory problems in these animals, restoring their abilities to levels similar to those displayed by wild-type animals.

[0164] The HDAC6I of the present invention can also be used in methods of treating CNS conditions, diseases, and injuries with a second therapeutic agent, such as a drug known in the art to treat a particular 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.

[0165] The HDAC6I of the present invention is also useful for treating TBI. Traumatic brain injury (TBI) is a severe and complex injury that occurs in approximately 1.4 million people per 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.

[0166] TBI produces many lesions, including axonal injury, cell death, contusion, and inflammation. The inflammatory cascade is characterized by proinflammatory cytokines and microglial activation, which may exacerbate other pathologies. Although the role of inflammation in TBI is well established, there are currently no effective anti-inflammatory therapies available for the treatment of TBI.

[0167] Several known HDAC inhibitors have been found to be preventative in different cell and animal models of acute and chronic neurodegenerative injuries and diseases, such as Alzheimer's disease, ischemic stroke, multiple sclerosis (MS), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Recent studies in experimental pediatric TBI reported a decrease in hippocampal CA3 histone H3 acetylation that lasted from hours to days after injury. These changes were attributed to upstream excitotoxicity and stress cascades documented in association with TBI. HDAC6I has also been reported to have anti-inflammatory effects acting through the acetylation of non-histone proteins. It was found that the HDAC6 selective inhibitor 4-dimethylamino-N-[5-(2-mercaptoacetylamino)pentyl]benzamide (DMA-PB) could increase histone H3 acetylation and reduce microglial inflammatory responses after traumatic brain injury in rats, indicating the utility of HDAC6Is as therapeutic agents to inhibit neuroinflammation associated with TBI.

[0168] Therefore, the HDAC6I of the present invention is also useful for treating inflammation and stroke, and for treating autism and autism spectrum disorders.The HDAC6I of the present invention can also be used to treat parasitic infections (e.g., malaria, toxoplasmosis, trypanosomiasis, helminths, and protozoan infections) (see Andrews et al. Int. J. Parasitol. 2000, 30(6), 761-768).

[0169] The HDAC6I of the present invention can also be used as an imaging agent. In particular, by providing radioactively, isotopically, or fluorescently labeled HDAC6I, the labeled compound can image HDAC, tissues expressing HDAC, and tumors. The labeled HDAC6I of the present invention can also image patients suffering from cancer, or other HDAC-mediated diseases, such as stroke, by administering an effective amount of the labeled compound or a composition containing the labeled compound. In a preferred embodiment, the labeled HDAC6I can emit positron radiation and is suitable for use in positron emission tomography (PET). Typically, the labeled HDAC6I of the present invention is used to identify tissues or target regions expressing high concentrations of HDAC. The extent of accumulation of the labeled HDAC6I can be quantified using known methods for quantifying radioactive emissions. In addition, the labeled HDAC6I can include a fluorophore or similar reporter that can track the movement of specific HDAC isoforms or organelles in vitro.

[0170] The HDAC6I of the present invention useful in imaging methods includes one or more radioisotopes capable of emitting one or more forms of radiation suitable for detection by any standard radiological instrument, such as PET, SPECT, gamma cameras, MRI, and similar devices. Preferred isotopes include tritium ( 3 H) and carbon ( 11 C). The substituted HDAC6I of the present invention may also be used in combination with fluorine ( 18 F) and iodine ( 123Typically, the labeled HDAC6I of the present invention can include an isotope of 11 C-labeled alkyl or aryl groups, i.e. 11 C-methyl group, or 18 F, 123 I, 125 I, 131 I, or a combination thereof.

[0171] Fluorescently labeled HDAC6I of the invention can also be used in the imaging methods of the invention, such compounds having FITC, carbocyanine moieties, or other fluorophores to allow visualization of HDAC proteins in vitro.

[0172] The labeled HDAC6I and methods of use can be used in vivo, particularly in humans, as well as in vitro applications, such as diagnostic and research applications using body fluids and cell samples. Imaging methods using the labeled HDAC6I of the present invention are discussed in WO03 / 060523, designated US, and incorporated herein in its entirety. Typically, the method involves contacting cells or tissues with a radiolabeled, isotopically labeled, fluorescently labeled, or tagged (such as biotin tagged) compound of the present invention, and creating a radioactive, fluorescent, or similar type image, depending on the visualization method employed (i.e., for radiographic images, an amount sufficient to provide about 1 to about 30 mCi of the radiolabeled compound).

[0173] Preferred imaging methods include the use of a labeled HDAC6I of the present invention capable of generating a target to background ratio of radiation intensity of at least 2:1, or more preferably a target to background ratio of radiation intensity of about 5:1, about 10:1, or about 15:1.

[0174] In a preferred method, the labeled HDAC6I of the present invention is rapidly excreted from tissues in the body to prevent long-term exposure to radiation of the radiolabeled compound administered to an individual. Typically, the labeled HDAC6I of the present invention is excreted from the body in less than about 24 hours. More preferably, the labeled HDAC6I is 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, the preferred labeled HDAC6I is eliminated in about 60 to about 120 minutes.

[0175] In addition to isotopically and fluorescently labeled derivatives, the present invention also embodies the use of derivatives that contain tags (such as biotin) to identify biomolecules associated with HDAC isoforms of interest for diagnostic, therapeutic, or research purposes.

[0176] The HDAC6Is of the present invention are also useful in the treatment of autoimmune diseases and inflammation.The compounds of the present invention are particularly useful in overcoming transplant and graft rejection, and in treating forms of arthritis.

[0177] Despite the success of modern transplant programs, the incidence of post-transplant malignancies and graft loss due to chronic rejection, in addition to nephrotoxicity, cardiovascular disease, diabetes, and hyperlipidemia associated with current treatment regimens, drive efforts to achieve long-term graft function with minimal immunosuppression. Similarly, the incidence of inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is increasing. Animal studies have shown that regulatory T cells (Tregs), which express Foxp3, a member of the forkhead transcription family, are key to limiting autoreactive and alloreactive immunity. Moreover, following their induction by costimulatory blockade, immunosuppression, or other strategies, Tregs can be adoptively transferred into naive hosts to achieve beneficial therapeutic effects. However, attempts to develop sufficient Tregs to maintain their suppressive function after transplantation have failed in clinical trials. Mouse studies have shown that HDAC6I limits immune responses, at least in important part, by increasing Treg suppressive function (R. Tao et al., Nat. Med, 13, 1299-1307 (2007)), and selective targeting of HDAC6 is particularly effective in this regard.

[0178] In the case of organ transplants, rejection begins to occur in the first few days after transplantation, so prevention rather than treatment of rejection is the primary consideration. The opposite is true for autoimmunity, where patients present with disease that is already causing problems. Therefore, it will be 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, which is a long-term ongoing significant loss of graft function in clinical transplant patient populations. Tolerance will be evaluated by testing whether mice with long-term surviving allografts reject subsequent third-party heart transplants and accept additional donor allografts without the immunosuppression that can occur using nonselective HDAC6I plus RPM. These in vivo tests involve the evaluation of ELISPOT and MLR activity using recipient lymphocytes challenged with donor cells. Protection against chronic rejection is assessed by analysis of the host anti-donor humoral response, as well as graft arteriosclerosis and interstitial fibrosis in long-term surviving allograft recipients.

[0179] The importance of HDAC6 targeting will be evaluated in additional transplant models that seek a readout of biochemical significance as observed clinically. Thus, we will evaluate the effect of HDAC6 targeting in kidney transplant recipients (monitoring BUN, proteinuria) and in islet allografts (monitoring blood glucose levels). Since kidney transplantation is the most common organ transplant performed and the kidney performs multiple functions, e.g., regulating acid / base metabolism, blood pressure, and red blood cell production, efficacy in this model will demonstrate the utility of HDAC6 targeting. Similarly, islet transplantation is a major unmet need, considering that clinical islet allografts are typically lost in the first 1-2 years after transplantation. Having a safe and non-toxic means of extending islet survival without maintenance CNI therapy would be an important advancement. Transplantation studies will also be enhanced by using mice with floxed HDAC6. Existing Foxp3-Cre mice will be used to test, for example, the impact of HDAC6 deletion in Tregs. This approach can be extended to target HDAC6 in, for example, T cells (CD4-Cre) and dendritic cells (CD11c-Cre).Using tamoxifen-regulated Cre, the importance of HDAC6 in graft induction versus maintenance (including the impact on short-term versus maintenance HDAC6I therapy) will be assessed by administering tamoxifen at various times post-transplant to induce HDAC6 deletion.

[0180] Autoimmunity trials will also be performed. In this case, interruption of existing disease is particularly important and HDAC6 targeting may be effective without the need for additional therapy (as opposed to the need for brief low-dose RPM in highly invasive and completely MHC-mismatched transplant models). Trials in colitis mice showed that HDAC6- / - Tregs were more effective than WT Tregs in modulating disease and that Tubacin was able to rescue mice if treatment was started after the onset of colitis. These trials were extended to evaluate whether deletion of HDAC6 in Tregs (Foxp3 / Cre) vs. T cells (CD4=Cre) vs. DCs (CD11c-Cre) differentially affected the onset and severity of colitis. Similarly, control of colitis will be assessed by inducing deletion of HDAC6 at various intervals after onset of colitis using tamoxifen-regulated Cre.

[0181] The compounds of the present invention are expected to show anti-arthritic effects in a collagen-induced arthritis model in DBA1 / J mice. In this study, DBA1 / J mice (male, 7-8 weeks old) are used with 8 animals per group. Systemic arthritis is induced with bovine type II collagen and CFA plus an IFA booster injection on day 21. HDAC6I of the present invention is administered at 50 mg / kg and 100 mg / kg on day 28 for 2 consecutive weeks, and the effect was determined from the data of the mean arthritis score versus the number of days of treatment.

[0182] Despite efforts to avoid graft rejection through host-donor tissue type matching, in the majority of transplant procedures immunosuppressive therapy is critical for the survival of the donor organ in the host. A variety of immunosuppressive agents are used in transplantation therapy, including azathioprine, methotrexate, cyclophosphamide, FK-506, rapamycin, and corticosteroids.

[0183] The HDAC6I of the present invention is a potent immunosuppressant that suppresses humoral and cellular immune responses, such as allograft rejection, delayed hypersensitivity, experimental allergic encephalomyelitis, Freund's adjuvant arthritis, and graft-versus-host disease. The HDAC6I of the present invention 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.

[0184] The therapeutically effective amount of HDAC6I of the present invention can be used for immunosuppression, including, for example, preventing organ rejection or graft-versus-host disease, and treating diseases and conditions, particularly autoimmune and inflammatory diseases and conditions. Examples of autoimmune and inflammatory diseases include thyroid disease, pernicious anemia, Addison's disease, psoriasis, diabetes, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogrenick syndrome, multiple sclerosis, myasthenia gravis, Reiner's syndrome, arthritis (rheumatoid arthritis, chronic progressive arthritis, and osteomanic arthritis) and rheumatic diseases, autoimmune blood disorders (hemolytic anemia, aplastic anemia, pure red blood cell anemia, and idiopathic thrombocytopenia), multiple cartilage disorders, and inflammatory disorders. These include, but are not limited to, inflammatory bowel disease, scleroderma, Wegener's granulomatosis, chronic active hepatitis, Steven-Johnson syndrome, idiopathic sprue, autoimmune inflammatory bowel disease (ulcerative colitis and Crohn's disease), endocrine ophthalmopathy, Graves' disease, sarcoidosis, primary biliary cirrhosis, juvenile diabetes mellitus (type I diabetes), uveitis (frontal and posterior), keratoconjunctivitis sicca and cervical conjunctivitis, interstitial pulmonary fibroalicia, psoriatic arthritis, and glomerulonephritis.

[0185] The HDAC6Is of the present invention can be used alone or in combination with second therapeutic agents known to be useful in the treatment of autoimmune diseases, inflammation, transplantation, and grafts, such as cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those of skill in the art.

[0186] Additional diseases and conditions mediated by HDACs, particularly HDAC6, include, but are not limited to, asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, neuritis in general, and neuropathy in general, which can also be treated by the methods of the invention.

[0187] In the method of the present invention, a therapeutically effective amount of one or more HDAC6Is of the present invention, typically formulated according to pharmaceutical practice, is administered to a person in need thereof.Whether such treatment is indicated depends on the individual case and is subject to a medical evaluation (diagnosis) that takes into account the signs, symptoms, and / or dysfunctions present, the risk of developing the particular signs, symptoms, and / or dysfunctions, and other factors.

[0188] The HDAC6I of the present invention can be administered by any suitable route, for example, oral, buccal, inhalation, sublingual, rectal, vaginal, intracisternal or intrathecal via lumbar puncture, transurethral, ​​nasal, percutaneous, i.e., transdermal, or parenteral (intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a specific site) administration. Parenteral administration can be achieved, for example, by using a needle and syringe or by using high pressure techniques.

[0189] The pharmaceutical composition includes the HDAC6I of the present invention present in an amount sufficient to be administered in an effective amount to achieve its intended purpose.The exact formulation, route of administration and dosage are determined by individual physicians in consideration of the diagnosed condition or disease.Dosage amount and interval can be individually adjusted to provide the level of the HDAC6I of the present invention that is sufficient to maintain therapeutic effect.

[0190] Toxicity and therapeutic efficacy of the HDAC6I compounds of the invention can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., LD 50(50% lethal dose in the population), and maximum tolerated dose (MTD), and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 and ED 50 Therapeutic indices are expressed as a ratio between the ED and the dose with little or no toxicity. Compounds that exhibit large therapeutic indices are preferred. Data obtained from such procedures can be used in formulating a dosage range for use in humans. The dosage is determined by the ED with little or no toxicity. 50 It is preferred that the therapeutically effective amount of the compound be within the range of circulating concentrations that include the above range. Dosages can vary within this range depending on the dosage form employed and / or the route of administration used. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0191] The therapeutically effective amount of the HDAC6I of the present invention required for use in treatment varies according to the nature of the condition to be treated, the length of time that activity is desired, the age and condition of the patient, and is ultimately determined by the attending physician.Dosage amount and interval can be adjusted individually to provide a plasma level of HDAC6I sufficient to maintain the desired therapeutic effect.The desired dose can be conveniently administered in a single dose or as multiple doses administered at appropriate intervals, for example, one, two, three, four or more partial doses per day.Often, multiple doses are desired or required. For example, the HDAC6I may be administered at a frequency of 4 doses delivered as one dose per day spaced 4 days apart (q4d×4), 4 doses delivered as one dose per day spaced 3 days apart (q3d×4), 1 dose delivered per day spaced 5 days apart (qd×5), 1 dose delivered per week spaced 3 weeks apart (qwk3), 5 doses once per day, 2 days off, and 5 more doses once per day (5 / 2 / 5), or any dose regimen determined to be appropriate for the situation.

[0192] The dosage of the composition comprising or containing HDAC6I of the present invention may be about 1 ng / kg body weight to about 200 mg / kg body weight, about 1 μg / kg body weight to about 100 mg / kg body weight, or about 1 mg / kg body weight to about 50 mg / kg body weight. The dosages above are exemplary of the average case, but there may be individual cases in which higher or lower dosages are beneficial, and these are within the scope of the present invention. In practice, the physician will determine the actual dosage regimen that is most suitable for an individual subject, which may vary with the age, weight, and response of the particular subject.

[0193] The HDAC6I of the present invention used in the method of the present invention is usually administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the HDAC6I of the present invention can 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 dose (including all doses from 0.005 to 500 milligrams).

[0194] The HDAC6I of the present invention is typically administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the HDAC6I of the present invention.

[0195] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with the HDAC6I of the present invention. Such pharmaceutical carriers can be liquids, such as water, oils, including those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants can be used. The pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the HDAC6I is administered intravenously. Saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions of the present invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0196] These pharmaceutical compositions may be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Appropriate formulations depend on the route of administration selected. When a therapeutically effective amount of the HDAC6I of the present invention is administered orally, the composition is typically in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition may further comprise a solid carrier, such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 0.01% to about 95%, preferably about 1% to about 50%, of the HDAC6I of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, or oil of animal or vegetable origin may be added. The composition in liquid form may further comprise saline, dextrose or other sugar solution, or glycol. When administered in liquid form, the composition contains about 0.1% to about 90% by weight, preferably about 1% to about 50% by weight of the compound of the present invention.

[0197] When a therapeutically effective amount of the HDAC6I of the present invention is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, taking into consideration pH, isotonicity, stability, etc., is within the skill of the art. A composition preferred for intravenous, cutaneous, or subcutaneous injection typically includes an isotonic vehicle. The HDAC6I of the present invention can be infused with other fluids for a period of 10 to 30 minutes or for several hours.

[0198] The HDAC6I of the present invention can be easily combined with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the active agent to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding the HDAC6I of the present invention to a solid excipient, optionally grinding the resulting mixture, processing the mixture, and adding suitable auxiliary agents as necessary, followed by obtaining tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added as desired.

[0199] The HDAC6I of the present invention can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers with added preservatives.The composition can take the form of a suspension, solution, or emulsion in oily or aqueous vehicle, and can contain compounding agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0200] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active agent in water-soluble form. In addition, the suspension of the HDAC6I of the present invention can be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension. Optionally, such suspensions can also contain suitable stabilizers or agents that increase the solubility of the pharmaceutical agent, allowing for the preparation of highly concentrated solutions. Alternatively, the compositions of the present invention can be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.

[0201] The HDAC6I of the present invention can also be formulated into rectal compositions, such as suppositories or retention enemas that contain conventional suppository bases.In addition to the formulations described above, the HDAC6I of the present invention can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, the HDAC6I of the present invention can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins.

[0202] In particular, the HDAC6I of the present invention can be administered bucally or sublingually in the form of tablets containing excipients such as starch or lactose, or in the form of capsules or ovules, either alone or mixed with excipients, or in the form of elixirs or suspensions containing flavorings or colorings.Such liquid preparations can be prepared with pharma- ceutically acceptable additives, such as suspensions.The HDAC6I of the present invention can also be parenterally injected, for example, intravenously, intramuscularly, subcutaneously, or into a coronary artery.For parenteral administration, the HDAC6I of the present invention is most often used in the form of a sterile aqueous solution, which may contain other substances, such as salts or simple sugars, such as mannitol or glucose, to make the solution isotonic with blood.

[0203] As an additional embodiment, the present invention includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use to practice the method of the present invention. In one simple embodiment, the kit comprises a compound or composition described herein as useful for practicing the method (e.g., a composition comprising the HDAC6I of the present invention and an optional second therapeutic agent), packaged in a container such as a sealed bottle or container, and a label that describes the use of the compound or composition to practice the method of the present invention is attached to the container or included in the kit. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device suitable for administering the composition according to the intended route of administration, such as a syringe, drip bag, or patch. In another embodiment, the compound of the present invention is a lyophilizate. In this case, the kit may further comprise an additional container that contains a solution useful for reconstituting the lyophilizate.

[0204] Previous HDAC6Is had properties that prevented their development as therapeutic agents. In accordance with an important feature of the present invention, the HDAC6Is of the present invention were synthesized and evaluated as inhibitors of HDACs. The compounds of the present invention show increased HDAC6 potency and selectivity compared to HDAC1 and HDAC8. The improved properties of the compounds of the present invention indicate that the compounds of the present invention are useful for applications such as, but not limited to, immunosuppressants and neuroprotectants. For example, the compounds of the present invention typically have inhibitory potencies (IC) against HDAC6 of 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. 50 ).

[0205] Use of HDAC6 inhibitors.

[0206] The HDAC6Is of the present invention can be used alone or in combination with second therapeutic agents known to be useful in the treatment of a variety of diseases, including autoimmune diseases, inflammation, or transplant and graft rejection, such as cyclosporine, rapamycin, methotrexate, cyclophosphamide, azathioprine, corticosteroids, and similar agents known to those of skill in the art.

[0207] Additional diseases and conditions mediated by HDACs, particularly HDAC6, include, but are not limited to, asthma, cardiac hypertrophy, giant axonal neuropathy, mononeuropathy, mononeuritis, polyneuropathy, autonomic neuropathy, neuritis in general, and neuropathy in general, which can also be treated by the methods of the invention.

[0208] In the method of the present invention, a therapeutically effective amount of one or more HDAC6Is of the present invention, typically formulated according to pharmaceutical practice, is administered to a person in need thereof.Whether such treatment is indicated depends on the individual case and is subject to a medical evaluation (diagnosis) that takes into account the signs, symptoms, and / or dysfunctions present, the risk of developing the particular signs, symptoms, and / or dysfunctions, and other factors.

[0209] The HDAC6I of the present invention can be administered by any suitable route, for example, oral, buccal, inhalation, sublingual, rectal, vaginal, intracisternal or intrathecal via lumbar puncture, transurethral, ​​nasal, percutaneous, i.e., transdermal, or parenteral (intravenous, intramuscular, subcutaneous, intracoronary, intradermal, intramammary, intraperitoneal, intraarticular, intrathecal, retrobulbar, intrapulmonary injection and / or surgical implantation at a specific site) administration. Parenteral administration can be achieved, for example, by using a needle and syringe or by using high pressure techniques.

[0210] The pharmaceutical composition includes the HDAC6I of the present invention present in an amount sufficient to be administered in an effective amount to achieve its intended purpose.The exact formulation, route of administration and dosage are determined by individual physicians in consideration of the diagnosed condition or disease.Dosage amount and interval can be individually adjusted to provide the level of the HDAC6I of the present invention that is sufficient to maintain therapeutic effect.

[0211] Toxicity and therapeutic efficacy of the HDAC6I compounds of the invention can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, e.g., LD 50 (50% lethal dose in the population), and maximum tolerated dose (MTD), and ED 50 The dose ratio between toxic and therapeutic effects is the therapeutic index, which is the LD 50 and ED 50 Therapeutic indices are expressed as a ratio between the ED and the dose with little or no toxicity. Compounds that exhibit large therapeutic indices are preferred. Data obtained from such procedures can be used in formulating a dosage range for use in humans. The dosage is determined by the ED with little or no toxicity. 50 It is preferred that the therapeutically effective amount of the compound be within the range of circulating concentrations that include the above range. Dosages can vary within this range depending on the dosage form employed and / or the route of administration used. Determination of a therapeutically effective amount is within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0212] The therapeutically effective amount of the HDAC6I of the present invention required for use in treatment varies according to the nature of the condition to be treated, the length of time that activity is desired, the age and condition of the patient, and is ultimately determined by the attending physician.Dosage amount and interval can be adjusted individually to provide a plasma level of HDAC6I sufficient to maintain the desired therapeutic effect.The desired dose can be conveniently administered in a single dose or as multiple doses administered at appropriate intervals, for example, one, two, three, four or more partial doses per day.Often, multiple doses are desired or required. For example, the HDAC6I may be administered at a frequency of 4 doses delivered as one dose per day spaced 4 days apart (q4d×4), 4 doses delivered as one dose per day spaced 3 days apart (q3d×4), 1 dose delivered per day spaced 5 days apart (qd×5), 1 dose delivered per week spaced 3 weeks apart (qwk3), 5 doses once per day, 2 days off, and 5 more doses once per day (5 / 2 / 5), or any dose regimen determined to be appropriate for the situation.

[0213] The dosage of the composition comprising or containing HDAC6I of the present invention may be about 1 ng / kg body weight to about 200 mg / kg body weight, about 1 μg / kg body weight to about 100 mg / kg body weight, or about 1 mg / kg body weight to about 50 mg / kg body weight. The dosage of the composition may be any dosage, including but not limited to about 1 μg / kg, 10 μg / kg to 200 mg / kg. The dosages above are exemplary of the average case, but there may be individual instances in which higher or lower dosages are beneficial, and these are within the scope of the present invention. In practice, the physician will determine the actual dosing regimen that is most suitable for an individual subject, which may vary with the age, weight, and response of the particular subject.

[0214] The HDAC6I of the present invention used in the method of the present invention is usually administered in an amount of about 0.005 to about 500 milligrams per dose, about 0.05 to about 250 milligrams per dose, or about 0.5 to about 100 milligrams per dose. For example, the HDAC6I of the present invention can 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 dose (including all doses from 0.005 to 500 milligrams).

[0215] The HDAC6I of the present invention is typically administered in admixture with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions for use in accordance with the present invention are formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants that facilitate processing of the HDAC6I of the present invention.

[0216] The term "carrier" refers to a diluent, adjuvant, or excipient that is administered with the HDAC6I of the present invention. Such pharmaceutical carriers can be liquids, such as water, oils, including those of animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The carriers can be saline, gum arabic, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. In addition, auxiliary agents, stabilizers, thickeners, lubricants, and colorants can be used. The pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the HDAC6I is administered intravenously. Saline and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, etc. The compositions of the present invention, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[0217] These pharmaceutical compositions may be prepared, for example, by conventional mixing, dissolving, granulating, dragee-making, emulsifying, encapsulating, encapsulating, or lyophilizing processes. Appropriate formulations depend on the route of administration selected. When a therapeutically effective amount of the HDAC6I of the present invention is administered orally, the composition is typically in the form of a tablet, capsule, powder, solution, or elixir. When administered in tablet form, the composition may further comprise a solid carrier, such as gelatin or an adjuvant. Tablets, capsules, and powders contain about 0.01% to about 95%, preferably about 1% to about 50%, of the HDAC6I of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, or oil of animal or vegetable origin may be added. The composition in liquid form may further comprise saline, dextrose or other sugar solution, or glycol. When administered in liquid form, the composition contains about 0.1% to about 90% by weight, preferably about 1% to about 50% by weight of the compound of the present invention.

[0218] When a therapeutically effective amount of the HDAC6I of the present invention is administered by intravenous, cutaneous, or subcutaneous injection, the composition is in the form of a pyrogen-free parenterally acceptable aqueous solution. The preparation of such parenterally acceptable solutions, taking into consideration pH, isotonicity, stability, etc., is within the skill of the art. A composition preferred for intravenous, cutaneous, or subcutaneous injection typically includes an isotonic vehicle. The HDAC6I of the present invention can be infused with other fluids for a period of 10 to 30 minutes or for several hours.

[0219] The HDAC6I of the present invention can be easily combined with pharma- ceutically acceptable carriers well known in the art. Such carriers allow the active agent to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc., for oral ingestion by the patient to be treated. Pharmaceutical preparations for oral use can be obtained by adding the HDAC6I of the present invention to a solid excipient, optionally grinding the resulting mixture, processing the mixture, and adding suitable auxiliary agents as necessary, followed by obtaining tablets or dragee cores. Suitable excipients include, for example, fillers and cellulose preparations. Disintegrants can be added as desired.

[0220] The HDAC6I of the present invention can be formulated for parenteral administration by injection, for example, bolus injection or continuous infusion.The preparation for injection can be provided in unit dosage form, for example, in ampoules or in multi-dose containers with added preservatives.The composition can take the form of a suspension, solution, or emulsion in oily or aqueous vehicle, and can contain compounding agents such as suspending agents, stabilizing agents, and / or dispersing agents.

[0221] Pharmaceutical compositions for parenteral administration include aqueous solutions of active agents in water-soluble form.In addition, the suspension of HDAC6I of the present invention can be prepared as a suitable oily injection suspension.Suitable lipophilic solvents or vehicles include fatty oils or synthetic fatty acid esters.Aqueous injection suspensions can contain substances that increase the viscosity of the suspension.

[0222] Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the pharmaceutical agents to allow for the preparation of highly concentrated solutions. Alternatively, the compositions of the invention may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

[0223] The HDAC6I of the present invention can also be formulated into rectal compositions, such as suppositories or retention enemas that contain conventional suppository bases.In addition to the formulations described above, the HDAC6I of the present invention can also be formulated as depot preparations.Such long-acting preparations can be administered by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection.Thus, for example, the HDAC6I of the present invention can be formulated with suitable polymers or hydrophobic materials (e.g., as emulsions in acceptable oils) or ion exchange resins.

[0224] In particular, the HDAC6I of the present invention can be administered bucally or sublingually in the form of tablets containing excipients such as starch or lactose, or in the form of capsules or ovules, either alone or mixed with excipients, or in the form of elixirs or suspensions containing flavorings or colorings.Such liquid preparations can be prepared with pharma- ceutically acceptable additives, such as suspensions.The HDAC6I of the present invention can also be parenterally injected, for example, intravenously, intramuscularly, subcutaneously, or into a coronary artery.For parenteral administration, the HDAC6I of the present invention is most often used in the form of a sterile aqueous solution, which may contain other substances, such as salts or simple sugars, such as mannitol or glucose, to make the solution isotonic with blood.

[0225] As an additional embodiment, the present invention includes a kit comprising one or more compounds or compositions packaged in a manner that facilitates their use to practice the method of the present invention. In one simple embodiment, the kit comprises a compound or composition described herein as useful for practicing the method (e.g., a composition comprising the HDAC6I of the present invention and an optional second therapeutic agent), packaged in a container such as a sealed bottle or container, and a label that describes the use of the compound or composition to practice the method of the present invention is attached to the container or included in the kit. Preferably, the compound or composition is packaged in a unit dosage form. The kit may further comprise a device 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 lyophilizate. In this case, the kit may further comprise an additional container that contains a solution useful for reconstituting the lyophilizate.

[0226] Some conventional HDAC6Is often exhibit activity against several known HDACs, properties that may prevent their development as therapeutic agents for diseases other than cancer. Thus, an important feature of the present invention relates to the fact that the compounds of the present invention exhibit isoform selectivity. The compounds of the present invention exhibit increased inhibitory potency and selectivity for HDAC6 compared to other HDACs, particularly greater selectivity for class II over class I. The improved properties of the compounds of the present invention indicate that these compounds should be useful for applications that are not limited to immunosuppressants and neuroprotectants, but also include, but are not limited to, Alzheimer's disease, depression, Rett syndrome, Charcot-Marie-Tooth disease, brain cancer, and the like. For example, the compounds of the present invention typically exhibit binding affinities (IC) for HDAC6 of less than 1 μM, and in some cases less than 10 nM. 50 ).

[0227] definition The following terms and expressions used herein have the meanings given above.

[0228] The terms used herein may be preceded and / or followed by a single dash, "-", or a double dash "=" to indicate the bond order of the bonds between the designated 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, and further, the substituents are intended to be read "left to right" unless the dash indicates otherwise. For example, C1-C6 alkoxycarbonyloxy and -OC(O)OC1 -C6 Alkyl refers to the same functional group; similarly, arylalkyl and -alkylaryl refer to the same functional group.

[0229] "Acetyl" means a group of the formula -C(O)CH3.

[0230] "Alkenyl" means, unless otherwise specified, a straight or branched chain hydrocarbon containing 2 to 10 carbons and containing at least one carbon-carbon double bond. Representative examples of alkenyl include, but are not limited to, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl, 4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, 3-decenyl, and 3,7-dimethylocta-2,6-dinyl.

[0231] As used herein, "alkynyl" refers to a straight or branched chain hydrocarbon group containing 2 to 10 carbon atoms and containing at least one carbon-carbon triple bond. Representative examples of alkynyl include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 3-butynyl, and 2-pentynyl.

[0232] "Alkoxy," as defined herein, means an alkyl group appended to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, and hexyloxy.

[0233] "Alkyl" means a straight or branched chain hydrocarbon containing 1 to 10 carbon atoms, unless otherwise specified. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, isobutyl, 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 can also be straight or branched, examples include, but are not limited to, -CH2-, -CH2CH2-, -CH2CH2C(CH3)2-, and -CH2CH(CH2CH3)CH2-.

[0234] "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 system. The bicyclic aryl can be azulenyl, naphthyl, and the like. The aryl is attached to the parent molecular moiety through any carbon atom contained within the aryl ring system. One or two hydrogen atoms that are part of the aryl group may be replaced with a substituent selected from the group of F, Cl, CH3, CHF2, CF3, OCH3, OCHF2, OCF3, SCF3, and CN, or two adjacent hydrogen atoms may be replaced with the moiety -OCHO-. In certain embodiments, the aryl group is phenyl or naphthyl. In certain other embodiments, the aryl group is phenyl.

[0235] "Cyano" and "nitrile" refer to the group --CN.

[0236] "Cycloalkyl" refers to a 3- to 6-membered monocyclic ring. Cycloalkyls can be saturated or unsaturated, but are not aromatic. Representative cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, and cyclohexenyl.

[0237] "Halo" or "halogen" means -Cl, -Br, -I, or -F.

[0238] "Haloalkyl" means at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl groups include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, and 2-chloro-3-fluoropentyl.

[0239] "Heteroaryl" refers to a monocyclic heteroaryl or a bicyclic ring system containing at least one aromatic heterocycle. The monocyclic heteroaryl can be a 5- or 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, or the nitrogen atom can be absent if the oxygen or sulfur atom is present. The 6-membered ring consists of three double bonds and one, two, three, or four nitrogen atoms. The 5- or 6-membered heteroaryl is attached to the parent molecular moiety through any carbon atom or any nitrogen atom contained within the heteroaryl. Representative examples of monocyclic heteroaryl include, but are not limited to, furyl, imidazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, oxazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrazolyl, pyrrolyl, tetrazolyl, thiadiazolyl, thiazolyl, thienyl, triazolyl, and triazinyl. Bicyclic heteroaryl consists of a monocyclic heteroaryl, a monocyclic cycloalkyl, a monocyclic cycloalkenyl, a monocyclic heterocyclyl, or a monocyclic heteroaryl fused to a phenyl. The fused cycloalkyl or heterocyclyl portion of the bicyclic heteroaryl group is optionally substituted with one or two groups that are independently oxo or thioxo. When the bicyclic heteroaryl contains a fused cycloalkyl, cycloalkenyl, or heterocyclyl ring, the bicyclic heteroaryl group is attached to the parent molecular moiety through any carbon or nitrogen atom contained within the monocyclic heteroaryl portion of the bicyclic ring system. When the bicyclic heteroaryl is a monocyclic heteroaryl fused to a phenyl ring, the bicyclic heteroaryl group is attached to the parent molecular moiety through any carbon atom or nitrogen atom within the bicyclic ring system.Representative examples of bicyclic heteroaryl include, but are not limited to, benzimidazolyl, benzofuranyl, benzothienyl, benzoxadiazolyl, benzothiadiazolyl, benzothiazolyl, cinnolinyl, 5,6-dihydroquinolin-2-yl, 5,6-dihydroisoquinolin-1-yl, fluopyridinyl, 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, and 4,5,6,7-tetrahydrobenzo[c][1,2,5]oxadiazolyl. In certain embodiments, the fused bicyclic heteroaryl is a 5- or 6-membered monocyclic heteroaryl ring fused to either a phenyl ring, a 5- or 6-membered monocyclic cycloalkyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic cycloalkenyl, a 5- or 6-membered monocyclic heterocyclyl, or a 5- or 6-membered monocyclic heteroaryl, where the fused cycloalkyl, cycloalkenyl, and heterocyclyl groups are optionally substituted with one or two groups that are independently oxo or thioxo. 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, and the like. One or two hydrogen atoms that are part of a heteroaryl group may be replaced with a substituent selected from the group of F, Cl, CH3, CHF2, CF3, OCH3, OCHF2, OCF3, SCF3, and CN.

[0240] "Heterocyclyl" refers to a monocyclic 4-7 membered heterocycle containing one or more heteroatoms independently selected from O, N, and S, where the ring is saturated or unsaturated, but not aromatic. Representative examples of monocyclic heterocycles include, but are not limited to, azetidinyl, hexahydroazepinyl, imidazolinyl, imidazolidinyl, isothiazolinyl, isothiazolidinyl, morpholinyl, oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl, oxetanyl, piperazinyl, piperidinyl, pyrazolinyl, pyrazolidinyl, pyrrolinyl, pyrrolidinyl, and thiopyranyl. In certain embodiments, the heterocyclyl is imidazolinyl, pyrrolidinyl, piperidinyl, or piperazinyl.

[0241] "Deuterated" means containing one or more carbon-deuterium bonds replacing a carbon-hydrogen bond, up to a maximum of all carbon-hydrogen bonds in the group under consideration.

[0242] The present invention relates to novel HDAC6Is of formula I and Ib-Iqq and their use in the therapeutic treatment of, for example, cancer, inflammation, traumatic brain injury, neurodegenerative disorders, neurological diseases, peripheral neuropathy, stroke, hypertension, autoimmune diseases, inflammatory diseases, and malaria. The HDAC6Is of the present invention also enhance the sensitivity of cancer cells to the cytotoxic effects of radiation therapy and / or chemotherapy. In some embodiments, the HDAC6Is of the present invention selectively inhibit HDAC6 over other HDAC isozymes.

[0243] The present invention will be described in connection with the preferred embodiment. However, it should be recognized that the present invention is not limited to the disclosed embodiment. In consideration of the description of the embodiment of the present invention herein, it is understood that various modifications can be made by those skilled in the art. Such modifications are encompassed by the following claims.

[0244] The term "disease or condition benefiting from inhibition of HDAC" relates to a condition in which HDAC and / or the action of HDAC is important or necessary, for example, for the onset, progression, or manifestation of the disease or condition, or a disease or condition known to be treated with an HDAC inhibitor (e.g., TSA, pivaloyloxymethylbutane (AN-9, Pivanex), FK-228 (Depsipetide), PXD-101, NVP-LAQ824, SAHA, MS-275, and / or MGCD0103). Examples of such conditions include cancer, psoriasis, fibroproliferative disorders (e.g., hepatic fibrosis), smooth muscle proliferative disorders (e.g., atherosclerosis, restenosis), neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington's chorea, amyotrophic diseases, lateral sclerosis, spinocerebellar degeneration, Rett syndrome), peripheral neuropathies (Charcot-Marie-Tooth disease, giant axonal neuropathy (GAN)), inflammatory diseases (e.g., osteoarthritis, rheumatoid arthritis, colitis), diseases involving angiogenesis (e.g., cancer, rheumatoid arthritis, psoriasis, diabetic retinopathy), hematopoietic disorders (e.g., anemia, sickle cell anemia, thalassemia), fungal infections, parasites. Those skilled in the art can readily determine whether a compound treats a disease or condition mediated by HDAC for any particular cell type, for example, by assays that can be conveniently used to assess the activity of a particular compound, including, but not limited to, infectious diseases (e.g., malaria, trypanosomiasis, helminthiasis, protozoan infections), bacterial infections, viral infections, and conditions treatable by immunomodulation (e.g., multiple sclerosis, autoimmune diabetes, lupus, atopic dermatitis, allergies, asthma, allergic rhinitis, inflammatory bowel disease; and to improve graft engraftment).

[0245] The term "second therapeutic agent" refers to a therapeutic agent that is different from the HDAC6I of the present invention and is known to treat the disease or condition of interest.For example, when the disease or condition of interest is cancer, the second therapeutic agent can be a known chemotherapeutic agent, such as taxol, or radiation.

[0246] The term "HDAC" refers to a family of enzymes that remove acetyl groups from proteins, such as the ε-amino group of lysine residues at the N-terminus of histones. HDACs can be human HDACs, including HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11. HDACs can also be from protozoan or fungal sources.

[0247] As used herein, the terms "treat", "treating", "treatment", and the like refer to the elimination, relief, alleviation, recovery, and / or amelioration of a disease or condition and / or symptoms associated therewith. Although not precluded, treating a disease or condition does not require that the disease, condition, or symptoms associated therewith be completely eliminated, including the treatment of acute or chronic signs, symptoms, and / or dysfunctions. As used herein, the terms "treat", "treating", "treatment", and the like may include "prophylactic treatment", which refers to reducing the likelihood of developing or re-developing a disease or condition, or the recurrence of a previously controlled disease or condition, in a subject who has not developed or re-developed a disease or condition, or is at risk of or susceptible to a recurrence of the disease or condition, or who has not developed or re-developed a disease or condition. Thus, "treatment" also includes recurrence prevention or stepwise prevention. The term "treat" and synonyms contemplate administering a therapeutically effective amount of a compound of the present invention to an individual in need of such treatment. Treatment may be symptom-directed, for example, to suppress symptoms. Treatment may be short-term, directed over the medium term, or may be long-term treatment, for example within the scope of maintenance therapy.

[0248] As used herein, the term "therapeutically effective amount" or "effective dose" refers to an amount of active ingredient(s) sufficient to effectively deliver the active ingredient(s) to an individual in need thereof for the treatment of a condition or disease of interest when administered. In the case of cancer or other proliferative disorders, a therapeutically effective amount of an agent can reduce (i.e., slow to some extent, preferably stop) unwanted cell proliferation, reduce the number of cancer cells, reduce tumor size, inhibit (i.e., slow to some extent, preferably stop) cancer cell invasion into peripheral organs, inhibit (i.e., slow to some extent, preferably stop) tumor metastasis, inhibit tumor growth to some extent, and / or alleviate to some extent one or more of the symptoms associated with cancer. To the extent that the administered compound or composition prevents and / or kills existing cancer cells, it can be cytostatic and / or cytotoxic.

[0249] "Co-administration", "administered in combination", "co-administration" and similar expressions mean that two or more agents are administered to a subject to be treated at the same time. "Co-administration" means that each agent is administered either at the same time or sequentially in any order at different times. However, if not administered at the same time, it means that they are administered to an individual successively close enough in time to provide the desired therapeutic effect and can act in concert. For example, the HDAC6I of the present invention can be administered at the same time as the second therapeutic agent or sequentially in any order at different times. The HDAC6I of the present invention and the second therapeutic agent can be administered separately in any suitable form and by any suitable route. If the HDAC6I of the present invention and the second therapeutic agent are not administered at the same time, it is understood that they can be administered to a subject in need thereof in any order. For example, the HDAC6I of the present invention may be administered to an individual in need thereof prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks prior to) administration of a second therapeutic treatment modality (e.g., radiation therapy), or may be administered simultaneously with or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) administration of a second therapeutic treatment modality. In various embodiments, the HDAC6I of the invention and the second therapeutic agent are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1-2 hours apart, 2-3 hours apart, 3-4 hours apart, 4-5 hours apart, 5-6 hours apart, 6-7 hours apart, 7-8 hours apart, 8-9 hours apart, 9-10 hours apart, 10-11 hours apart, 11-12 hours apart, 24 hours or less apart, or 48 hours or less apart. In one embodiment, the components of the combination therapy are administered 1 minute to 24 hours apart.

[0250] The use of the terms "a", "an", "the" and similar referents in the context of describing the present invention (particularly in the context of the claims) should be construed to encompass both the singular and the plural unless otherwise indicated. Reference to ranges of values ​​herein merely serves as a shorthand method of referring individually to each individual value falling within the range, unless otherwise indicated herein, and each individual value and subrange is incorporated herein as if it were individually referred to herein. The use of any and all examples or exemplary language (e.g., "such as" and "like") provided within this specification is intended merely to better illuminate the invention and does not pose limitations on the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention. EXAMPLES

[0251] Having now generally described the invention, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the invention and are not intended to be limiting of the invention.

[0252] Example 1: Synthesis of exemplary compounds of the present disclosure Synthesis Methods and Procedures The central (hetero-)aromatic linker moiety is commercially available in a variety of structural modifications, such as: [ka] Carboxylic acids, their methyl esters, and their nitriles can further be interconverted by standard synthetic methods, or the nitriles may be obtained from (hetero-)aryl halides by transition metal catalyzed cyanation. Brominated building blocks are useful for the modification of these linker moieties at later stages of the synthesis via organometallic reactions, in particular transition metal catalyzed coupling reactions. For simplicity, the building blocks are depicted below without the substituent R2. Similar reactions are used for intermediates in which these substituents are present.

[0253] To prepare 1,3,4-oxadiazoles, hydrazides are obtained from carboxylic acid esters by hydrazinolysis or from free carboxylic acids by coupling with hydrazines protected, for example, under the action of amide / peptide coupling reagents of the carbodiimide type, preferably by the addition of an activating agent such as 4-(dimethylamino)pyridine (DMAP). Protecting groups for hydrazines include, for example, tert-butoxycarbonyl (Boc), which is then removed by treatment with mild acid, for example by warming in hexafluoroisopropanol as solvent, or benzyloxycarbonyl (Cbz), which can be removed under various conditions, including catalytic hydrogenolysis with a Pd catalyst. The NH2 group of the hydrazide is then acylated with di- or trifluoroacetic anhydride in the presence of a mild base. The resulting mixed 1,2-diacylhydrazines can be isolated and dehydrated in a separate step (e.g., with trifluoroanhydride and base or with CBr4 / PPh3) to the desired 1,3,4-oxadiazoles, or more conveniently dehydrated with an excess of the acylation mixture under conditions of their formation.

[0254] [ka] To prepare 1,2,4-oxadiazoles with a fluoroalkyl group at the 5-position, the nitrile is converted to an amidoxime with hydroxylamine. The amidoxime is N-acylated with di- or trifluoroacetic anhydride in the presence of a weak base. If an excess of the acylation mixture is used, the acylated amidoxime is dehydrated simultaneously with its formation to give the 3-(hetero-)aryl-5-(di- or trifluoromethyl)-1,2,4-oxadiazole.

[0255] [ka] The reverse substitution pattern in the 1,2,4-oxadiazole series, i.e., fluorinated alkyl at position 3 and (hetero-)aryl at position 5, is accessible from N-acylated di- and trifluoroacetamidoximes. Trifluoroacetamidoximes are commercially available and can be N-acylated with (hetero-)aroyl halides in the presence of a weak base such as pyridine, or with carboxylic acids in the presence of amide / peptide coupling reagents. Cyclization of the resulting intermediates, if not spontaneous, can be achieved using dehydrating agents such as trifluoroacetic anhydride / base.

[0256] [ka] To prepare analogous difluoromethylated 1,2,4-oxadiazoles, it may be advantageous to proceed via 3-carboxaldehyde as intermediate, which is then defluorinated with reagents commonly used for this purpose, such as sulfur tetrafluoride, diethylaminosulfur trifluoride (DAST), bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluo®), or N,N-diethyl-S,S-difluorosulfiminium tetrafluoroborate (XtalFluor-E®). Starting from 2,2-diethoxyacetamidoximes, the 3-carboxaldehydes are obtained from their diethylacetals in a similar sequence to that described above. This building block is then formed by adding hydroxylamine to diethoxyacetonitrile, a commercially available liquid that is more convenient to handle than the gaseous difluoroacetonitrile required for the preparation of difluoroacetamidoximes.

[0257] [ka] Oxazoles are commonly encountered in heterocyclic chemistry and can be prepared by a wide variety of methods. The classical Robinson-Gabriel approach consisting of dehydration of 2-(acylamino)ketones can be used to prepare certain compounds of the invention in which a (hetero-)aryl group is attached to the 5-position of the oxazole ring. Many dehydrating agents, such as those shown below, effect this transformation. Acylaminoketones are prepared by N-acylation of aminoketones in the form of stable salts, for example hydrochlorides. Aminoketone salts can be prepared from nitriles containing the required linker (hetero-)aromatic ring by a two-step procedure consisting of the addition of a Grignard reagent to form an imine, followed by a Neber rearrangement (Baumgarten, HE et al., J. Org. Chem. 1963, 28, 2369).

[0258] [ka] 2-(Acylamino)ketone intermediates may also be obtained from diazoketones by Rh-catalyzed formation of carbenoids and insertion of a primary carboxamide (in this case di- or trifluoroacetamide) into the NH bond (Davies, JR et al. Tetrahedron 2004, 60, 3967). Diazoketones can be synthesized from diazoalkanes and their derivatives by acylation with acyl halides in the presence of a weak base.

[0259] The oxazole is suitable for bromination at the 4-position with NBS in acetone (Khan, AH; Chen, JS Org. Lett. 2015, 17, 3718). The 4-bromide is a useful derivative for the introduction of various substituents via transition metal catalyzed substitution and coupling reactions, e.g., trifluoromethylation via Cu catalyzed reaction with methyl 2,2-difluoro-2-(fluorosulfonyl)acetate (Chen, QY; Wu, SWJ Chem. Soc., Chem. Commun. 1989, 705; Clarke, SL; McGlacken, GP Chem. Eur. J. 2017, 23, 1219). Methylation can be achieved by Stille coupling with Me4Sn; cyanation by various protocols using cyanide anion or various sources thereof, typically Cu or Pd catalyzed (e.g. Ren, Y. et al. Tetrahedron Lett. 2009, 50, 4595); fluorination by Pd catalyzed Br-F exchange (Lee, HG et al. J. Am. Chem. Soc. 2014, 136, 3792); and chlorination by Cu catalyzed Br-Cl exchange (Feng, X. et al. Chem. Commun. 2012, 48, 9468). The 4-carbonitrile resulting from cyanation can be reduced to the aldehyde, which is the precursor of 4-(difluoromethyl)oxazole, via deoxofluorination (e.g. DAST), for example, with diisobutylaluminum hydride.

[0260] [ka] A series of isomeric oxazoles, i.e. oxazoles with a (hetero-)aryl group attached to the 4-position of the ring, are available, for example, via a recently reported oxidative procedure from (hetero-)aryl methyl ketones (Xiao, F. Org. Lett. 2019, 21, 8533). These starting materials can be prepared from carboxylic acids by reaction of their derived Weinreb amides with methylmagnesium halides or (not shown) by the action of the same reagents on nitriles followed by imine hydrolysis; some (e.g. 5-acetyl-2-methylpyridine) are commercially available. In the protocol, an arylthio substituent is placed at the 5-position, which is removed by standard desulfurization with Raney nickel or nickel boride.

[0261] [ka] With the linker / zinc-binding group substructure assembled, the methyl group attached to the six-membered ring needs to be converted to an alkylating agent. This can be achieved via free-radical bromination. The brominating agent is usually N-bromosuccinimide (NBS) or 1,3-dibromo-5,5-dimethylhydantoin, and the radical initiator is an azo compound such as azobis(isobutyronitrile) (AIBN), or a peroxide such as dibenzoyl peroxide. These initiators are widely used in reactions carried out at the boiling temperature (77 °C) of carbon tetrachloride, the traditionally preferred solvent, at which temperature they have favorable decomposition rates. This environmentally harmful solvent has been replaced in the current body of work by fluorobenzene, a non-polar solvent of low reactivity and similar boiling point (85 °C). Photochemical initiation is also possible. In addition to the desired monobrominated product, unreacted starting material and dibrominated products are also encountered. They are typically easily separable by normal phase column chromatography. The dibrominated products can be recycled to the starting materials by free radical (e.g., Bu3SnH or H3PO2 / AIBN) or catalytic (e.g., H2, Pd / C) hydrodebromination.

[0262] [ka] The sultam building blocks are linked together by reaction with the alkylating agents described above in the presence of a weak base in a dipolar aprotic solvent such as DMF. Methods for preparing the required sultams are disclosed in the following paragraphs.

[0263] 3,4-Dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide and certain derivatives are accessible by literature procedures starting from 2-arylethanesulfonyl chlorides (Moroda, A.; Togo, H. Synthesis 2008, 1257). Acceptable substituents include halo and alkyl. Unsubstituted sulfochlorides are commercially available, but certain derivatives need to be synthesized, for example through the sequences shown. Final reductive cleavage of the NO bond can be achieved in a variety of ways, such as reduction with SmI2, catalytic hydrogenolysis, dissolving metal reduction protocols, and electrochemical methods. Accidental cleavage during the performance of certain other transformations has also been observed.

[0264] [ka] Ortho and para substituents in 2-arylethanesulfonyl chlorides clearly lead to 5- and 7-substituted 3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxides, respectively, whereas meta substituents can lead to a mixture of 6- and 8-substituted compounds that need to be separated. The preferred entry into the 6-substituted series is electrophilic substitution on intermediates lacking the 6-substituent, where the nitrogen atom exerts a predominantly or entirely para-directing influence. An example is the 6-bromination with N-bromosuccinimide (NBS) of either the N-methoxy precursors of the parent series or the free sultams (all R1=H).

[0265] [ka] The same general sequence is applicable to compounds in which the aliphatic carbon atoms bear substituents. The required substituted phenethyl alcohols are accessible, for example, by reduction of substituted phenylacetic acids or their esters. The latter can be synthesized, for example, by alkylation of enolic acid esters or by their arylation under transition metal catalysis. An example is shown below, where the enolic acid ester is a zinc enolate formed by the Reformatsky reaction of the corresponding α-bromo ester with Zn metal, and the arylation step is a Negishi coupling reaction (Sakuma, D. et al. Chem. Lett. 2015, 44, 818).

[0266] [ka] 3,4-Dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxides bearing halogen substituents such as Cl and Br on the aromatic ring are suitable starting materials for the introduction of other substituents by transition metal catalyzed exchange and coupling reactions. This approach is illustrated by the example of the Suzuki coupling reaction between 3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide and arylboronic acids. The main product is not a simple coupling product, but instead the product of coupling and the concomitant reductive cleavage of the NO bond. Other possibilities include, but are not limited to, halogen-halogen exchange, alkoxylation, amination, amidation, trifluoromethylation, trifluoromethylthiolation, cyanation, and carbonylation reactions, as well as Heck, Kumada, Negishi, Sonogashira, and Stille coupling reactions. Such reactions are of great importance in modern synthetic methodology research and are well known to those skilled in the art.

[0267] 3,4-Dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxides bearing halogen substituents such as Cl and Br on the aromatic ring are suitable starting materials for the introduction of other substituents by transition metal catalyzed exchange and coupling reactions. This approach is illustrated by the example of the Suzuki coupling reaction between 3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide and arylboronic acids. The main product is not a simple coupling product, but instead the product of coupling and the concomitant reductive cleavage of the NO bond. Other possibilities include, but are not limited to, halogen-halogen exchange, alkoxylation, amination, amidation, trifluoromethylation, trifluoromethylthiolation, cyanation, and carbonylation reactions, as well as Heck, Kumada, Negishi, Sonogashira, and Stille coupling reactions. Such reactions are of great importance in modern synthetic methodology research and are well known to those skilled in the art.

[0268] [ka] For example, if neither 1-methoxysultam nor the corresponding free sultam is suitable for the intended reaction, e.g. due to premature loss of the 1-methoxyl group or due to acidity or limited solubility of the NH of the free sultam, a different N-protecting group can be introduced and later removed when no longer needed. An example is the methoxymethyl group, which can be introduced under mild alkylation conditions and removed by treatment with acid. Other suitable protecting groups include, but are not limited to, benzyloxymethyl, which provides an additional option of hydrogenolytic cleavage; silyl groups such as tert-butyldimethylsilyl, tert-butyldiphenylsilyl, and triisopropylsilyl, which are removed by treatment with acid, protic base, or fluoride anion; benzyl and benzyloxycarbonyl, which are removed by hydrogenolysis; tert-butoxycarbonyl, which is removed with acid; and 4-methoxybenzyl and 2,4-dimethoxybenzyl, which are removed by oxidation or treatment with strong acid.

[0269] [ka] The 4-keto derivatives, i.e., 1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxides, can be synthesized following the scheme described below in Shafiq, M. et al. J. Chil. Chem. Soc. 2011, 56, 527, similar to the above work, in which N-Boc replaces N-Me, allowing access to N-unsubstituted sultams.

[0270] [ka] Another method for preparing 1H-benzo[c][1,2]thiazin-4(3H)-one 2,2-dioxides consists in the N-sulfonylation of anilines with methoxycarbonylmethanesulfochloride. After ester hydrolysis, the carboxylic acid is cyclized in an intramolecular Friedel-Crafts acylation reaction (Pasteris, RJ US4867781, 9 / 19 / 1989; Nie, H.; Widdowson, KL US 6436927, 8 / 20 / 2002).

[0271] [ka] 1H-Benzo[c][1,2]thiazine-4(3H)-one 2,2-dioxide reacts with tosylhydrazine to form tosylhydrazone, the reduction of which with catecholborane constitutes an alternative synthesis of 3,4-dihydro-1H-benzo[c][1,2]thiazine 2,2-dioxide (Pasteris, RJ US4867781, 9 / 19 / 1989). Upon treatment with base, the tosylhydrazone undergoes a Bamford-Stevens reaction to give 1H-benzo[c][1,2]thiazine 2,2-dioxide (Nie, H.;Widdowson, KL US 6436927, 8 / 20 / 2002).

[0272] [ka] Sultam building blocks containing a heteroatom at position 4 are accessible from o-phenylenediamines, o-aminophenols, and o-mercaptoanilines by reaction with chloromethanesulfonyl chloride in the presence of base. In the case of X=NH, oxidation with MnO2 delivers the corresponding unsaturated heterocycles (Nie, H.; Widdowson, KL US 6436927, 8 / 20 / 2002).

[0273] [ka] 3,4-Dihydro-1H-benzo[c][1,2,6]thiadiazine 2,2-dioxides are formed upon heating of 2-aminobenzylamine with sulfamide in pyridine (CN1909909B, 2010 / 12 / 15). One method of synthesizing the required diamine intermediate is by oxime formation from a 2-nitrophenyl alkyl ketone followed by reduction, for example, by catalytic hydrogenation.

[0274] [ka] Up to this paragraph, the compounds of the present invention are synthesized by first constructing a zinc-linked fluoroalkylated oxadiazole or oxazole moiety on a linker ring, and then connecting this assembly to a sultam capping group. It is also possible to first connect a capping group to a linker, and then construct a fluoroalkylated oxadiazole or oxazole moiety on the assembly using the same type of reaction as the approach first disclosed. Possible functional group incompatibility can be corrected by using appropriate protecting groups, or in a later step, by generating interfering functional groups from less reactive precursors, such as aromatic bromides, by reactions such as substitution or coupling reactions.

[0275] [ka] The following synthetic schemes are representative of the reactions used to synthesize the HDAC6Is of the present invention. Modifications and alternative schemes for preparing the HDAC6Is of the present invention are readily within the capabilities of one of ordinary skill in the art.

[0276] All starting materials and solvents were purchased from commercial suppliers of reagent purity and used as obtained without further purification. Reactions were monitored by visualization at 254 nm and / or by thin-layer chromatography on silica gel-coated glass plates using appropriate stains. 1 H NMR spectra were recorded on Bruker Avance-400 and Avance-500 spectrometers at 400 or 500 MHz, respectively. Chemical shifts (δ scale) are reported in parts per million (ppm) relative to TMS (in CDCl3) or to the solvent signal (in DMSO-d6; the solvent signal is set to δ 2.50). Signals are characterized as s (singlet), d (doublet), t (triplet), m (multiplet), and br (broad).

[0277] Compound 1. 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide N-(6-methylnicotinoyl)hydrazine [ka] In a 500 mL round bottom flask equipped with a stir bar, heating mantle, reflux condenser, and Ar balloon, a mixture of methyl 6-methylnicotinate (7.39 g, 48.9 mmol), 1-propanol (60 mL), and hydrazine monohydrate (9.5 mL, 195 mmol, 4.0 equiv.) was heated to reflux for 22 h. TLC analysis (SiO2, MeOH / CHCl3) indicated R fApproximately 0.25 showed hydrazide as the only significant component. The mixture was evaporated and the residue was taken up in MeOH and adsorbed onto SiO2 (50 g). The residue was chromatographed on SiO2 (15 x 6 cm) starting with 1:9 MeOH / CH2Cl2 and then changing to 15:85 MeOH / CH2Cl2. Upon appearance of hydrazide in the eluent, the mixture ratio was further changed to 1:4. The eluent containing the product was concentrated to a small volume and toluene (50 mL) was added to completely remove MeOH. Evaporation was completed and the residue was dried under vacuum to give 6.83 g (92%) of a colorless solid. 1 H NMR (400 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.84 (narrow m, 1H), 8.04 (dd, 1H, J = 8.1, 2.4 Hz), 7.33 (d, 1H, J = 8.1 Hz), 4.52 (s, 2H); CH3 is masked by DMSO-d5 signals. 1 H NMR (400 MHz, D2O) δ 8.69 (narrow m, 1H), 8.02 (dd, 1H, J = 8.2, 2.4 Hz), 7.41 (d, 1H, J = 8.2 Hz), 2.56 (s, 3H). 5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyridine [ka] A 500 mL round bottom flask equipped with a stir bar, dropping funnel, and balloon was charged with N-(6-methylnicotinoyl)hydrazine (6.83 g, 45.2 mmol), anhydrous CHCl (80 mL), and triethylamine (31.5 mL, 226 mmol, 5.0 equiv). The suspension was cooled in an ice bath and difluoroacetic anhydride (16.0 mL, 136 mL, 3.0 equiv) was added dropwise over 45 min. The starting material dissolved immediately. The solution was stirred at ambient temperature for 22.5 h, at which point it turned very dark. TLC (SiO, 1:1 EtOAc / Hexanes) indicated R fApproximately 0.35 of product was shown, with a diffuse polar tail and a brown baseline spot. Water (10 mL) was added dropwise over 20 min with cooling, followed by a solution of citric acid (17.3 g, 90 mmol, 2.0 equiv) in water (40 mL). The pH of the aqueous phase was approximately 3.5. The phases were separated and the aqueous phase was extracted successively with CH2Cl2 and EtOAc (50 mL each). The combined organic phases were washed with brine (50 mL, organic phase = bottom) and evaporated to leave a dark brown oil, which was chromatographed on SiO2 (20 x 5 cm, EtOAc / Hexane 1:1). Most of the precursor (not visible on bulk TLC) was removed, but some of the polar tail remained in the product. Evaporation left 6.9 g of a brown oil, which solidified on standing. This crude material was bulb-to-bulb distilled twice (105-125 °C / oil pump vacuum) to give 6.17 g of a mixture of colorless crystals and a yellow oil. The distillate was taken up in MeOH (20 mL) and the solution was placed in the freezer. No crystallization occurred initially. Crystals formed when the solution was added dropwise and evaporated. These were used to seed the remaining solution from which the product rapidly crystallized. After a further 3 h in the freezer, the product was isolated by suction filtration, washed with cold MeOH (10 mL) and dried under vacuum to give 3.77 g (40%) of colorless crystals, but ( 1 H NMR showed that the mixture was contaminated with approximately 3 mol % difluoroacetic acid. 1 H NMR (CDCl3, 400 MHz) δ 9.22 (narrow m, 1H), 8.29 (dd, 1H, J = 8.2, 2.3 Hz), 7.37 (d, 1H, J = 8.2 Hz), 6.95 (t, 1H, J H-F = 51.7 Hz), 2.69 (s, 3H);δ 5.97 (t, 1H, J H-F = 54.7 Hz) 13 C NMR (CDCl3, 100 MHz) δ 164.44, 163.27, 158.32 (t, J C-F = 29.2 Hz), 147.77, 134.85, 123.61, 116.48, 105.68 (t, J C-F= 241.2 Hz), 24.79.

[0278] The mother liquor contained a large amount of difluoroacetic acid (by NMR). It was evaporated and taken up in fresh MeOH (30 mL). Amberlite IRA-67 (weakly basic anion exchange resin, free base; Sigma no. A9960; CAS no. 65899-87-7; 4 g) was added. The mixture was swirled occasionally for 50 min. The resin was removed by suction filtration and washed with MeOH (3 x 5 mL). An aliquot of the filtrate was 1 Analysis by H NMR showed that low concentrations of difluoroacetic acid persisted. Treatment with anion exchange resin (2 g) was repeated for 5.7 h. After filtration from the resin, the filtrate was adsorbed onto SiO2 (8 g). The residue was analyzed by SiO2 (19 × 3.8 cm, t-BuOMe; TLC, R f Chromatography at 300 rpm (approximately 0.5, slight streaking) was performed to remove some of the polar material. The eluent was evaporated and dried under vacuum to give an additional 1.24 g (13%) of the oxadiazole as yellowish crystals.

[0279] 2-(Bromomethyl)-5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]pyridine [ka] A 250 mL 3-neck flask equipped with a stir bar, heating mantle, stopper, reflux condenser (connected to an Ar balloon), and a dropping funnel with a septum was charged with 5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyridine (3.75 g, 17.8 mmol), 1,3-dibromo-5.5-dimethylhydantoin (2.78 g, 9.8 mmol, 0.55 equiv), and fluorobenzene (50 mL). The atmosphere was exchanged with Ar. The dropping funnel was charged with a solution of azobis(isobutyronitrile) (AIBN; 0.44 g, 2.7 mmol, 0.15 equiv) in fluorobenzene (5 mL) and approximately 20% was added to the reaction mixture. The mixture was heated to reflux and the remainder of the AIBN solution was added in portions over 1 h. At this point, most of the color of the Br2 that had formed temporarily had faded. Reflux was continued for 10 min and the mixture was cooled to ambient temperature. TLC (SiO2, EtOAc / Hexane 2:3) showed R f Three major components were shown, approximately 0.75, 0.5, and 0.3, corresponding to the dibrominated by-product, the desired monobrominated product, and the starting material, respectively. The dark solution was suction filtered over Celite, the precipitated hydantoin was filtered off, and the fluorobenzene was recovered by rotary evaporation. The hydantoin was washed with the recovered fluorobenzene, and the combined liquids were evaporated again. The residue was chromatographed on SiO2 (29 x 5 cm, 1:3 EtOAc / Hexane for the dibromide, 1:2 for the monobromide, and 3:2 for the starting material). Evaporation of the appropriate fractions gave 1.34 g (semi-solid, 20%) of the dibromide, 2.55 g (off-white solid, 49%) of the monobromide, and 1.07 g (light brown solid, 29%) of the starting material. Monobromide: 1 H NMR (400 MHz, CDCl3) δ 9.29 (narrow m, 1H), 8.42 (dd, 1H, J = 8.2, 2.2 Hz), 7.66 (dd, 1H, J = 8.2, 0.4 Hz), 6.95 (t, 1H, J H-F = 51.6 Hz), 4.62 (s, 2H).

[0280] N-Methoxy-2-phenylethanesulfonamide [ka] In a 250 mL round bottom flask equipped with a stir bar and a dropping funnel, O-methylhydroxylamine hydrochloride (5.2 g, 62 mmol, 2.2 equiv.) was suspended in CH3CN (40 mL). The mixture was cooled in an ice bath and a solution of NaOH (2.5 g, 62 mmol, 2.2 equiv.) in water (15 mL) was added dropwise in 10 min. After another 25 min at 0 °C, the dropping funnel was replaced with a dry one and a solution of 2-phenylethanesulfonyl chloride (5.75 g, 28.1 mmol) in anhydrous CH3CN (20 mL) was added dropwise over 15 min. Stirring was continued in the ice bath for 20 min and then at ambient temperature for 22 h. After partial evaporation to remove the organic solvent (it's foaming!), water (80 mL) was added and the product was extracted into EtOAc (3 x 50 mL). The combined organic phase was evaporated over Na2SO4 (20 g) and the residue was dried under vacuum to give 5.93 g (98%) of the sulfonamide as a colorless solid. 1 H NMR (CDCl3, 400 MHz) δ 7.37-7.32 (m, 1H), 7.31-7.23 (m, 3H), 6.63 (br s, 1H), 3.77 (s, 3H), 3.51, 3.13 (AA'XX' multiplets, slightly broadened at high field, 4H, J AX + J AX’ = 15.6 Hz).

[0281] 3,4-Dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] In a 500 mL round bottom flask equipped with a stir bar, O-methoxy-2-phenylethanesulfonamide (5.98 g, 27.8 mmol) was dissolved in 2,2,2-trifluoroethanol (83 mL) with gentle warming. Iodobenzene (0.31 mL, 2.8 mmol, 0.10 equiv) was added. m-Chloroperoxybenzoic acid (75%, remaining m-chlorobenzoic acid and water; 7.05 g, 30.6 mmol, 1.1 equiv) was added in portions over 10 min resulting in a mild exotherm. The flask was loosely stoppered and the mixture was stirred without temperature control for 4 h. Early on, the peroxyacid went into solution. A pale yellow, then tan suspension formed. Just prior to quenching, TLC analysis (SiO2, 1:9 EtOAc / toluene) indicated R f Approximately 0.5 of product, iodobenzene near the solvent front, baseline material, and polar streaks were observed. The mixture was stirred with a solution of Na2SO3 (2.5 g, 20 mmol) in water (50 mL) for 25 min at room temperature. Evaporation of the organic solvent (bumping!) left a mostly solid residue which was taken up in EtOAc (50 mL) and brine (30 mL). The phases were separated (rather slowly but completely) and the aqueous phase was extracted with EtOAc (2 x 50 mL). The pale yellow combined organic phases were dried over Na2SO4 (20 g) and evaporated to near dryness. The residual oil was taken up in MeOH (50 mL) where rapid crystallization ensued. After standing overnight in the freezer the precipitate was isolated by suction filtration, washed with cold MeOH (2 x 10 mL) and dried under vacuum to give 4.25 g of colorless crystals. According to TLC, the mother liquor still contained the product besides polar and non-polar impurities, which was adsorbed onto SiO2 (5 g). After evaporation and drying under vacuum, by chromatography of the residue on SiO2 (20 x 4 cm, EtOAc / toluene 1:19 then 1:9), a further 0.90 g of the cyclized product was obtained in the form of a colorless solid. Total yield: 5.15 g (87%). 1 H NMR (CDCl3, 400 MHz) δ 7.40-7.36 (m, 1H), 7.36-7.29 (m, 2H), 7.23-7.19 (m, 1H), 4.08 (s, 3H), 3.50, 3.42 (AA'BB' multiplets, slightly broadened high field region, 4H).

[0282] 3,4-Dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] A 250 mL round bottom flask equipped with a stir bar was charged with 3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide (2.05 g, 9.61 mmol), p-toluenesulfonic acid monohydrate (1.83 g, 9.6 mmol), 20% Pd(OH)2 / C (50% H2O; Alfa Aesar No. 42578; 0.22 g), THF (20 mL), and EtOH (undenatured, 40 mL). A H2 balloon was placed on the flask via a three-way valve, and the flask was evacuated and flushed with H2 (3 times). Hydrogenation was allowed to proceed at room temperature for 6.5 h, then the H2 atmosphere was replaced with Ar. TLC analysis (SiO2, EtOAc / Hexane 1:1) indicated the product R f A single spot of approximately 0.6) was observed, which was the starting material (R f The mixture was adsorbed onto SiO2 (12 g) and the residue was filtered through SiO2 (12 x 3 cm, 43:57 EtOAc / Hexanes). The eluate containing the product was contaminated with some baseline material and on evaporation gave a pinkish semi-solid. Evaporation of this material with MeOH gave a crystalline solid which was taken up in tert-butyl methyl ether. Filtration with suction, repeated washing with tert-butyl methyl ether and drying under vacuum afforded 1.27 g (72%) of product in suitable purity ( 1 H NMR) as an off-white powder. 1H NMR (DMSO-d6, 400 MHz) δ 10.09 (s, 1H), 7.19 (d, 1H, J = 7.4 Hz), 7.16 (t, 1H, J = 7.5 Hz with unresolved long-range couplings), 6.96 (dt, 1H, J = 1.1 Hz (d), 7.5 Hz (t)), 6.76 (dd, 1H, J = 8.0, 0.8 Hz), 3.36-3.27 (AA'BB' multiplet, 4H). 1 H NMR (CDCl3, 400 MHz) δ 7.23-7.16 (m, 2H), 7.05 (dt, 1H, J = 0.7 Hz (d), 7.5 Hz (t)), 6.75 (d, 1H, J = 8.0 Hz), 6.37 (br s, 1H), 3.49, 3.32 (AA'XX' multiplet, slightly broadened in the low field, 4H, J AX + J AX’ = 13.5 Hz).

[0283] 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] A 100 mL round bottom flask equipped with a stir bar was charged with 3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide (615 mg, 3.36 mmol), 2-[6-(bromomethyl)pyridin-3-yl]-5-(difluoromethyl)-1,3,4-oxadiazole (1.022 g, 3.52 mmol, 1.05 equiv), finely ground K2CO3 (0.56 g, 4.05 mmol, 1.2 equiv), KI (0.11 g, 0.67 mmol, 0.2 equiv), and anhydrous DMF (10 mL). The flask was wrapped in Al foil for light protection, and the mixture was stirred at ambient temperature for 19 h. Water (200 mL) and EtOAc / Hexanes 1:1 (100 mL, then 2×50 mL) were added and the phases were separated. The combined organic layers were washed with water (2×60 ml) and brine (100 ml). TLC (SiO2, EtOAc / Hexane 2:3) showed R f The solution was adsorbed onto SiO2 (10 g) and the material was chromatographed on SiO2 (9 x 5 cm, 2:3 EtOAc / Hexanes). Evaporation of the product-containing fractions, evaporation of the residue with CH3CN, and drying under vacuum afforded the product (1.28 g) containing 2.4 wt% CH3CN ( 1 Corrected yield by 1 H NMR: 1.25 g, 95%) as a colorless glass. 1 H NMR (CDCl3, 400 MHz) δ 9.30 (narrow m, 1H), 8.37 (dd, 1H, J = 8.3, 2.2 Hz), 7.75 (d, 1H, J = 8.3 Hz), 7.19 (d, 1H, J = 7.2 Hz), 7.13 (dt, 1H, J = 1.2 Hz (d), 7.8 Hz (t)), 6.94 (t, 1H, J H-F = 51.7 Hz), 6.79 (d, 1H, J = 8.3 Hz), 5.23 (s, 2H), 3.56, 3.52 (AA'BB' multiplet, 4H).

[0284] Compound 9. 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl]methyl]-6-(4-fluorophenyl)-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide 6-Bromo-3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] To a solution of 3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide (0.95 g, 4.45 mmol) in DMF (8 mL) was added N-bromosuccinimide (NBS; 0.79 g, 4.45 mmol) all at once. The mixture was stirred at room temperature for 21 h. TLC (trace aqueous workup: water / EtOAc; SiO2, EtOAc / hexane 1:4) indicated no starting material (R f Approximately 0.25) and the product (R f 0.3) was observed. Another amount of NBS (0.40 g, 2.25 mmol, 0.5 equiv) was added and the bromination was allowed to proceed for another 44 h. TLC at this point indicated complete conversion. A solution of Na2SO3 (1 g) in water (20 mL) was added and the mixture was stirred at room temperature for 20 min. More water (80 mL) and 1:1 EtOAc / Hexanes (50 mL) were added. The phases were separated and the aqueous phase was extracted with 1:1 EtOAc / Hexanes (20 mL). The combined organic phase was washed with water (2 x 100 mL), dried over Na2SO4 (5 g) and evaporated to near dryness. MeOH (5 mL) was added and the solution was placed in the freezer. After scratching, crystallization occurred slowly and was allowed to proceed for 2 days. The mother liquor was removed by pipette and the solid was washed with cold MeOH (5 mL). Drying under vacuum gave 96% purity (OMe 1 H NMR signal integration) to give 0.84 g (65%) of a colorless material. Evaporation of the mother liquor left another 0.33 g (25%) of a pale yellow glass, contaminated by traces of starting material (by TLC) but suitable for use in Suzuki coupling reactions. 1H NMR (CDCl3, 400 MHz) δ 7.44 (dd, 1H, J = 8.5, 2.1 Hz), 7.37 (narrow m, 1H), 7.24 (d, 1H, J = 8.6 Hz), 4.05 (s, 3H), 3.47, 3.37 (AA'XX' multiplet, 4H, J AX + J AX’ = 13.0 Hz).

[0285] 6-(4-Fluorophenyl)-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] A sealed tube equipped with a stir bar was charged with 6-bromo-3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide (332 mg, 1.14 mmol), 4-fluorophenylboronic acid (175 mg, 1.25 mmol, 1.1 equiv), bis(dibenzylideneacetone)dipalladium(0) (14.5 mg, 16 μmol, 1.4%), tricyclohexylphosphonium tetrafluoroborate (10.5 mg, 28 μmol, 2.5%), K3PO4 (1.27 M in water; 1.5 mL, 1.9 mmol, 1.7 equiv), and dioxane (3.0 mL). The mixture was purged with Ar, the vial was capped, and heated to 100 °C (oil bath temperature) for 24 h. TLC (SiO2, EtOAc / toluene 1:9) indicated R f The main product is about 0.2, and R f It showed small spots at approximately 0.25 and 0.45. The phases were separated and the aqueous phase was extracted with EtOAc (2 x 3 mL). The mixture was adsorbed onto SiO2 (3 g). The residue was chromatographed on SiO2 (15 x 3 cm, EtOAc / toluene 1:9 to 1:6). The non-polar fraction (16 mg after evaporation) consisted mainly of the initially expected product, 6-(4-fluorophenyl)-3,4-dihydro-1-methoxy-1H-benzo[c][1,2]thiazine-2,2-dioxide, in low purity. 1H NMR (CDCl3, 400 MHz) δ 7.49, 7.12 (AA'XX' multiplet with additional HF coupling, 4H), 7.44 (dd, 1H, J = 8.5, 2.1 Hz), 7.34 (d, 1H, J = 1.9 Hz), 7.03 (d, 1H, J = 8.5 Hz), 3.52, 3.38 (AA'XX' multiplet, 4H, J AX + J AX’ = 13.8 Hz), 3.34 (s, 3H).

[0286] 1 According to the H NMR spectrum, the main fraction consisted of two N-demethoxylated compounds, one from the coupling product and one from the hydrodehalogenation product. This material gave 98 mg of crystals upon recrystallization from ethanol (ambient temperature to freezer reflux), improving the ratio of the components to 8:1. Another recrystallization from ethanol (same temperature interval) finally gave 78 mg (25%) of the title compound in yellowish compact crystalline form. 1 H NMR (DMSO-d6, 400 MHz) δ 10.23 (br s, 1H), 7.65, 7.26 (AA'XX' multiplet with additional HF couplings, 4H), 7.51 (d, 1H, J = 2.0 Hz), 7.46 (dd, 1H, J = 8.3, 2.1 Hz), 6.83 (d, 1H, J = 8.3 Hz), 3.38 (s, 4H).

[0287] 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl]methyl]-6-(4-fluorophenyl)-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] In a 20 mL vial equipped with a stir bar were placed 6-(4-fluorophenyl)-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide (76.5 mg, 276 μmol), 2-[6-(bromomethyl)pyridin-3-yl]-5-(difluoromethyl)-1,3,4-oxadiazole (88 mg, 303 μmol, 1.1 equiv), finely ground K2CO3 (48 mg, 0.35 mmol, 1.25 equiv), KI (9 mg, 54 μmol, 0.2 equiv), and anhydrous DMF (0.8 mL). The vial was wrapped in Al foil for light protection, and the mixture was stirred at ambient temperature for 16.5 h. Water (25 mL) and EtOAc / Hexane 1:1 (20 mL) were added, and the phases were separated. The aqueous phase was extracted with 1:1 EtOAc / hexane (10 mL) and the combined organic phases were washed with brine (10 mL). f Approximately 0.2% of product was shown. The solution was adsorbed onto SiO2 (1 g) and the material was chromatographed on SiO2 (17 x 3 cm, EtOAc / Hexanes 2:3 then 1:1). Evaporation of the product-containing fractions and drying under vacuum gave the product (125 mg, 93%) as an off-white solid. 1 H NMR (CDCl3, 400 MHz) δ 9.32 (narrow m, 1H), 8.39 (dd, 1H, J = 8.2, 2.2 Hz), 7.76 (d, 1H, J = 8.2 Hz), 7.44, 7.10 (AA'XX' multiplet with additional HF coupling, 4H), 7.36 (narrow m, 1H), 7.30 (dd, 1H, J = 8.5, 1.9 Hz), 6.93 (t, 1H, J H-F = 51.6 Hz), 6.87 (d, 1H, J = 8.5 Hz), 5.26 (s, 2H), 3.63, 3.56 (AA'BB' multiplet, 4H).

[0288] Compound 15. 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyrimidin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide 1-(tert-butoxycarbonyl)-2-(2-methylpyrimidine-5-carbonyl)hydrazine [ka] 2-Methylpyrimidine-5-carboxylic acid (1.93 g, 14.0 mmol) and tert-butylcarbazate (2.22 g, 16.8 mmol) were dissolved in CHCl (50 mL). DMAP (2.22 g, 18.2 mmol, 1.3 equiv) and EDCI·HCl (3.09 g, 16.1 g, 1.15 equiv) were added. The flask was fitted with a balloon to remove moisture and the brown solution was magnetically stirred at room temperature for 70.5 h. TLC (SiO, EtOAc / CH3 3:1) showed a single mobile UV-absorbing product (R f The reaction mixture was adsorbed onto SiO2 (25 g) and the residue was filtered onto SiO2 (9 cm L x 6 cm D, EtOAc / CH3CN 3:1; Note 1). The eluent was evaporated and the residue was dried under vacuum to give a colourless solid (3.15 g, 89%). 1 H NMR (CDCl3, 400 MHz) δ 9.02 (s, 2H), 8.42 (br, 1H), 6.77 (br, 1H), 2.81 (s, 3H), 1.50 (s, 9H). A prominent impurity showed a tert-butyl singlet at δ 1.46.

[0289] Notes 1. Due to the relatively high viscosity of the solvent, a column with good permeability must be used to achieve practical flow rates. In this case, the use of a repurposed dropping funnel resulted in elution lasting several days. A coarser grade of silica gel than the usual flash chromatography grade may be advantageous.

[0290] Note 2. When the methyl ester was reacted with hydrazine hydrate in the same manner as described for the pyridine series, a mixture of products was obtained.

[0291] N-(2-methylpyrimidine-5-carbonyl)hydrazine [ka] A solution of 1-(tert-butoxycarbonyl)-2-(2-methylpyrimidine-5-carbonyl)hydrazine (3.15 g, 12.5 mmol) in 1,1,1,3,3,3-hexafluoro-2-propanol (34 mL) was heated to reflux for 38.5 h in a 250 mL round bottom flask equipped with a stir bar, heating mantle, reflux condenser, and balloon (to exclude water). Most of the solvent was distilled off at atmospheric pressure. The residue began to solidify upon cooling. EtOAc (30 mL) was added and the mixture was stirred to break up clumps. A tan powder (1.26 g) was obtained by suction filtration, washing with EtOAc (2×5 mL), and drying under vacuum. The mother liquor was adsorbed onto SiO2 (10 g) and the residue was chromatographed on SiO2 (14×3 cm, MeOH / CH2Cl2 1:5). The eluent was evaporated with the addition of toluene (10 mL, to completely remove MeOH) to give an additional 0.34 g of the hydrazide as an off-white solid. Total: 1.60 g (84%). 1 H NMR (DMSO-d6, 400 MHz) δ 10.03 (br s, 1H), 9.02 (s, 2H), 4.59 (br s, 2H), 2.66 (s, 3H).

[0292] 5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyrimidine [ka] This compound is prepared from N-(2-methylpyrimidine-5-carbonyl)hydrazine in the same manner as described for 5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyridine.

[0293] 2-(Bromomethyl)-5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]pyridine [ka] This compound is prepared from 5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyrimidine in the same manner as described for 2-(bromomethyl)-5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-2-methylpyrimidine.

[0294] 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyrimidin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] This compound is prepared from 3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide and 2-(bromomethyl)-5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]pyrimidine in the same manner as described for 1-[[5-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)pyridin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide.

[0295] Compound 31. 1-[[5-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]pyridin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide 2-Methyl-5-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]pyridine [ka] A 150 mL round bottom flask equipped with a stir bar and reflux condenser (open to air) was charged with 6-methylnicotinonitrile (2.23 g, 18.9 mmol), EtOH (undenatured, 20 mL), and 50% aqueous hydroxylamine (15.1 M, 1.50 mL, 22.7 mmol, 1.2 equiv). The mixture was heated to reflux for 23 h. After cooling, TLC (SiO2, MeOH / CHCl3 1:9) showed Rf One major spot of approximately 0.25 was shown. Toluene (20 mL) was added, the mixture was evaporated to dryness, and the residue was dried under vacuum. The crude amidoxime formed an off-white powder and was carried on without purification.

[0296] The amidoxime was suspended in CH2Cl2 (180 mL) and transferred to a 500 mL round bottom flask equipped with a stir bar, dropping funnel, and a balloon to remove moisture. With ice cooling, trifluoroacetic anhydride (11.9 mL, 84.3 mmol, 4.45 equiv) was added dropwise over 20 min to give a clear solution. The cold bath was removed and the mixture was stirred for 2 h. Triethylamine (19.5 mL, 140 mmol, 7.4 equiv) was added dropwise over 10 min at ambient temperature, resulting in a mild exotherm. The reaction mixture was stirred at ambient temperature for 3 h. Just before this time, TLC (SiO2, 1:4 EtOAc / Hexanes) indicated R f The desired product, R f The residue on SiO2 (22 x 5 cm, 1:5 EtOAc / hexanes) gave a dark orange precursor followed by a still impure product-containing fraction which was evaporated to leave 3.3 g of a dark orange-red oil along with some solids. TLC analysis of this material (SiO2, 1:4 EtOAc / toluene) showed several non-polar impurities and some baseline R f Approximately 0.25 showed the desired product, notably one of these by-products closely preceding the oxadiazole. The oil was applied to a SiO2 column (30 x 5 cm) which was eluted with EtOAc / toluene 1:6. The product-containing fractions (still contaminated by some of the previous spot) were evaporated to leave 2.04 g of an oil. This material was finally purified by bulb-to-bulb distillation (approximately 60-65 °C / oil pump) to give 1.62 g (37%) of the oxadiazole as a yellowish mobile oil (a substantial amount of amber crystalline residue was observed). 1H NMR (400 MHz, CDCl3) δ 9.23 (narrow m, 1H), 8.27 (dd, 1H, J = 8.1, 2.3 Hz), 7.34 (d, 1H, J = 8.1 Hz), 2.67 (s, 3H). 13 C NMR (100 MHz, CDCl3) δ 167.45, 166.05 (q, J C-F = 44.6 Hz), 162.76, 148.21, 135.09, 123.48, 118.51, 115.88 (q, J C-F = 273.9 Hz), 24.72.

[0297] 2-(Bromomethyl)-5-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]pyridine [ka] A 100 mL 3-neck flask equipped with a stir bar, heating mantle, stopper, reflux condenser (connected to an Ar balloon), and septum-equipped dropping funnel was charged with 2-methyl-5-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]pyridine (1.60 g, 6.98 mmol), 1,3-dibromo-5.5-dimethylhydantoin (1.10 g, 3.84 mmol, 0.55 equiv), and fluorobenzene (12 mL). The atmosphere was exchanged with Ar. The dropping funnel was charged with a solution of azobis(isobutyronitrile) (AIBN; 0.17 g, 1.05 mmol, 0.15 equiv) in fluorobenzene (2 mL) and approximately 10% was added to the reaction mixture. The mixture was heated to reflux and the remainder of the AIBN solution was added in portions over 1.5 h. At this point, most of the transiently formed Br2 color had faded. Reflux was continued for 10 min and the mixture was cooled to ambient temperature. cTLC (SiO2, EtOAc / Hexane 1:4 or 1:9) showed baseline material plus R f Approximately 0.8, 0.6, and 0.35 or R fThree major components of approximately 0.6, 0.35, and 0.2 were observed, corresponding to the dibrominated by-product, the desired monobrominated product, and the starting material, respectively. The dark solution was adsorbed onto SiO2 (8 g) and the residue was chromatographed on SiO2 (27 x 4 cm, 1:12 EtOAc / Hexanes). Evaporation of the monobromide-containing fractions afforded 0.83 g (38%) of a colorless crystalline solid of good purity. 1 H NMR (400 MHz, CDCl3) δ 9.30 (narrow m, 1H), 8.41 (dd, 1H, J = 8.1, 2.1 Hz), 7.64 (d, 1H, J = 8.2 Hz), 4.62 (s, 2H).

[0298] 1-[[5-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]pyridin-2-yl]methyl]-3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide [ka] A 100 mL round bottom flask equipped with a stir bar was charged with 3,4-dihydro-1H-benzo[c][1,2]thiazine-2,2-dioxide (181 mg, 988 μmol), 3-[6-(bromomethyl)pyridin-3-yl]-5-(trifluoromethyl)-1,2,4-oxadiazole (320 mg, 1.04 mmol, 1.05 equiv), finely ground K2CO3 (165 mg, 1.20 mmol, 1.2 equiv), KI (33 mg, 0.20 mmol, 0.20 equiv), and anhydrous DMF (10 mL). The flask was wrapped in Al foil for light protection, and the mixture was stirred at ambient temperature for 25.5 h. Water (50 mL) and EtOAc / Hexane 1:1 (50 mL) were added and the phases were separated. The organic phase was washed with water (50 mL). TLC (SiO2, EtOAc / hexane 3:7) showed that R f Approximately 0.3 of the product was shown, which was indistinguishable from the starting sultam, but no alkylating agent was present (reference spot, R f 9.30 The product was purified by elution with 1:9 EtOAc / CHCl3. fThe starting sultam was detected at approximately 0.5 but was absent (reference spot, R f 0.4). The solution was adsorbed onto SiO2 (4 g) and the material was chromatographed on SiO2 (16 x 3 cm, EtOAc / Hexanes 3:7, tailings). Evaporation of the product-containing fractions and drying under vacuum afforded the product (392 mg, 97%) as a very pale yellowish glass that gradually solidified on standing. 1 H NMR (CDCl3, 500 MHz) δ 9.29 (narrow m, 1H), 8.36 (dd, 1H, J = 8.2, 2.1 Hz), 7.71 (d, 1H, J = 8.2 Hz), 7.19 (d, 1H, J = 7.5 Hz), 7.13 (t, 1H, J = 7.7 Hz), 7.02 (t, 1H, J = 7.6 Hz), 6.82 (d, 1H, J = 8.2 Hz), 5.22 (s, 2H), 3.56, 3.50 (AA'BB' multiplet, 4H); 13 C NMR (CDCl3, 125 MHz) δ 167.13, 166.21 (q, J C-F = 44.8 Hz), 161.36, 148.39, 139.85, 136.13, 129.72, 127.90, 123.38, 122.28, 121.73, 120.17, 117.78, 115.82 (q, J C-F = 273.9 Hz), 51.93, 45.78, 28.22.

[0299] Example 2: Exemplary in vitro activity of compounds disclosed herein Hdac enzyme activity inhibition assay The efficacy or potency of the HDAC6Is of the present invention in inhibiting the activity of HDACs is determined by IC 50 It is measured by the quantitative IC 50 The IC value indicates the concentration of a particular compound required to inhibit 50% of the activity of an enzyme in vitro. In other words, the IC 50 The IC value is the half-maximal (50%) inhibitory concentration of the compound tested with a particular enzyme of interest, e.g., HDAC.50 The smaller the value, the lower the concentration of the compound required to inhibit enzyme activity by 50%, and therefore the stronger the inhibitory effect of the compound.

[0300] In preferred embodiments, the HDAC6I of the present invention inhibits HDAC enzyme activity by at least about 50%, preferably at least about 75%, at least 90%, at least 95%, or at least 99%.

[0301] The compounds of the present invention were tested against both HDAC6 and HDACL 50 In some embodiments, compounds of the invention were also tested against HDACs 1, 2, 3, 4, 5, 8, 10, and 11. Test compounds were also tested against HDAC6 and IC 50 The IC values ​​ranged from approximately 1 nM to more than 30 μM, indicating that HDAC1 50 Values ​​range from about 91 nM to more than 30 μM. Thus, in some embodiments, the HDAC6I of the present invention is a selective HDAC6 inhibitor and has a low affinity for other HDAC isozymes, such as HDAC1, and thus produces fewer side effects than compounds that are non-selective HDAC inhibitors.

[0302] In some embodiments, the HDACI of the present invention interacts with and reduces the activity of all histone deacetylases in a cell. In some preferred embodiments, the HDACI of the present invention interacts with and reduces the activity of fewer than all histone deacetylases in a cell. In certain preferred embodiments, the HDACI of the present invention interacts with and reduces the activity of one histone deacetylase (e.g., HDAC6), but does not substantially interact with or substantially reduce the activity of other histone deacetylases (e.g., HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC7, HDAC8, HDAC9, HDAC10, and HDAC11). Thus, the present invention provides HDACIs for the treatment of various diseases and conditions in which inhibition of HDACI has beneficial effects. Preferably, the HDAC6I of the present invention is selective for HDAC6 over other HDAC isozymes by a factor of at least 2, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1000, at least 2000, at least 3000, preferably up to about 4000. For example, in various embodiments, the HDAC6I of the present invention has an IC 50 IC for HDAC6 is about 350-fold or about 1000-fold lower than 50 , i.e., a selectivity ratio (HDAC1 IC 50 / HDAC6 IC 50 ) is shown.

[0303] Other assays also demonstrated the selectivity of the HDAC6I of the present invention for HDAC6 over approximately 1000 HDACs 1, 2, 3, 4, 5, 8, 10, and 11.

[0304] IC of the compound of structural formula (I) for HDAC1 and HDAC6 50 The values ​​were determined as follows: Assays for HDAC1, 2, 4, 5, 6, 7, 8, 9, 10, and 11 used isolated recombinant human proteins. HDAC3 / NcoR2 complex was used for HDAC3 assay. The substrate for HDAC1, 2, 3, 6, 10, and 11 assays is a fluorogenic peptide from p53 residues 379-382 (RHKKAc). The substrate for HDAC8 is a fluorogenic diacylated peptide based on residues 379-382 of p53 (RHKAcKAc). Acetyl-Lys(trifluoroacetyl)-AMC substrate was used for assays for HDAC4, 5, 7, and 9. Compounds were dissolved in DMSO and assayed at IC of 10 doses with 3-fold serial dilutions starting at 30 μM. 50 The control compound trichostatin A (TSA) was tested at 10 doses with IC20 starting at 5 μM in 3-fold serial dilutions. 50 The dose / response slope was curve-fitted to determine the IC 50 The assays were performed in duplicate and IC values ​​were extracted. 50 Values ​​are the average of data from both experiments.

[0305] material Human HDAC1 (GenBank accession number NM_004964): full length with C-terminal GST tag, MW=79.9 kDa, expressed by baculovirus expression system in Sf9 cells. The enzyme is in 50 mM Tris-HCl (pH 8.0), 138 mM NaCl, 20 mM glutathione, and 10% glycerol and is stable at -80°C for >6 months. Purity by SDS-PAGE is >10%. Specific activity is 20 U / μg, where 1 U=25 mM Tris / Cl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 0.1 mg / mL BSA, 100 μM HDAC substrate, and 13.2 ng / μL HDAC1, incubated at 1 pmol / min for 30 min at 30°C under the following assay conditions. Human HDAC6 (GenBank Accession No. BC069243): Full length with N-terminal GST tag, MW=159 kDa, expressed by baculovirus expression system in Sf9 cells. The enzyme is in 50 mM Tris-HCl (pH 8.0), 138 mM NaCl, 20 mM glutathione, and 10% glycerol and is stable at -80°C for >6 months. Purity by SDS-PAGE is >90%. Specific activity is 50 U / μg, where 1 U=25 mM Tris / Cl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, and 0.1 mg / mL BSA, 30 μM HDAC substrate, and 5 ng / μL HDAC6, incubated at 1 pmol / min for 60 min at 30°C.

[0306] Substrates for HDAC1 and HDAC6: Acetylated peptide substrates for HDACs based on residues 379-382 (Arg-His-Lys-Lys(Ac)) of p53, a regulatory acetylation site by p300 and CBP acetyltransferases (lysines 381, 382)1-6, are optimal for HDACs among a panel of substrates patterned on acetylation sites in p53, histone H3, and histone H47.

[0307] References: Gu, W. et al., Cell 1997, 90, 595;Sakaguchi, K. et al., Genes Dev. 1998, 12, 2831;Liu, L. et al., Mal. Cell. Biol. 1999, 19, 1202;Ito, A. et al., EMBO J., 2001, 20, 1331;Barlev, NA et al., Mal. Cell 2001, 8, 1243;Ito, A. et al., EMBO J. 2002, 21, 6236. Reaction buffer: 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mg / mL BSA.

[0308] Assay conditions HDAC1: 75 nM HDAC1 and 50 μM HDAC substrate in reaction buffer and 1% DMSO final. Incubate at 30° C. for 2 hours.

[0309] HDAC6: 12.6 nM HDAC6 and 50 μM HDAC substrate in reaction buffer and 1% DMSO final. Incubate at 30° C. for 2 hours.

[0310] I C 50 Value Calculation All ICs 50 Values ​​are automatically calculated using GraphPad Prism version 5 and the equation for Sigmoidal dose-response (variable slope): Y=Bottom+(Top-Bottom) / (1+10”((LogEC 50 -X)*HillSlope)) where X is the log of concentration, Y is the response, and Y starts at Bbottom and goes to Top with a sigmoidal shape. In most cases, "Bottom" is set to 0 and "Top" is set to "Less than 120%". This is the same as the "4-parameter logistic equation". IC 50 The curves were also drawn using GraphPad Prism, and the IC 50 The values ​​and hill slopes are provided.

[0311] HDAC activity assay: HDAC assays are performed using fluorescently labeled acetyl substrates that contain acetylated lysine side chains. After incubation with HDAC, deacetylation of the substrate increases the sensitivity of the substrate, which then generates a fluorophore in a second step upon treatment with a detection enzyme. HDAC1 and 6 were expressed as full-length fusion proteins. Purified proteins were incubated with 50 μM fluorescently labeled acetylated peptide substrate and test compounds in HDAC assay buffer containing 50 mM Tris-HCl (pH 8.0), 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl12, 1% DMSO, and 1% BSA for 2 h at room temperature.

[0312] The reaction was terminated after 2 hours by adding developer and the development of a fluorescent signal, relative to the amount of deacetylated peptide, was monitored by a time course measurement on an EnVision (PerkinElmer). HDAC activity was estimated from the slope of the time course measurement of fluorescence intensity. The slope of the no enzyme control (substrate alone) was used as background and the % enzyme activity was calculated using the background subtracted slope of the no inhibitor control (DMSO) as 100% activity.

[0313] To date, HDACIs have shown relatively non-specific inhibition of various HDAC isozymes. Most HDACIs identified to date primarily inhibit HDAC1, 2, 3, and 8, generating anti-proliferative phenotypes useful for oncology applications, but not many of the non-oncology applications of HDACIs. (KB Glaser et al., Biochem. Biophys. Res. Commun. 2003, 310, 529-536). Potential toxicity associated with inhibition of specific HDAC isozymes may pose additional challenges to the clinical development of pan-HDAC, i.e., non-selective HDAC inhibitors. Because the network of cellular effects mediated by acetylation is so extensive that inhibition of some HDAC isozymes may result in undesirable side effects, HDACI isozyme-selective inhibitors hold greater therapeutic promise than their non-selective counterparts.

[0314] Several panselective compounds have been approved by the FDA for use in cutaneous T-cell lymphoma and multiple myeloma (Kelly, WK et al., Nat. Clin. Pract. Oncol. 2005, 2, 150-157). Avoiding cytotoxicity through isozyme selectivity may ultimately prove advantageous and open the door to a variety of other therapeutic areas.

[0315] cell The human melanoma cell line WM164 was obtained from Smalley's Lab at the Moffitt Cancer Center. Cells were cultured in RPMI1640 medium supplemented with 10% FBS, penicillin / streptomycin (50 U / mL), L-glutamine (2 mM), and 2-mercaptoethanol (50 mM) (complete medium) and grown under humidified conditions at 37°C and 5% CO2.

[0316] Assay conditions wm164 melanoma cells were seeded at 105 cells / well in 12-well plates and allowed to adhere overnight. 50 mM stocks of compounds were then added by serial dilution in complete medium to the indicated concentrations. Cells were incubated for 24 hours under humidified conditions (37°C, 5% CO2). Wells were then washed with cold PBS and cells were lysed in a buffer containing 10 mM Tris-HCl (pH 8.0), 10% SDS, 4 mM urea, 100 mM DTT, and 1x protease inhibitors (Roche). Cells were lysed on ice for 30 minutes and then sonicated for 8 minutes (8 cycles of 30 seconds on / 30 seconds rest). Cells were then boiled for 10 minutes in 6x gel loading buffer and run on a 4-15% gradient gel, which was then transferred onto a nitrocellulose membrane. Membranes were blocked with 5% milk in PBS-T and specific antigens were detected using antibodies against acetyl-H3 and H3 (Cell Signaling), and acetyl-α-tubulin and α-tubulin (Sigma). Bands were detected by scanning the blots with a LI-COR Odyssey imaging system using both the 700 and 800 channels.

[0317] Hyperacetylation of α-tubulin without increasing the levels of acetylated histones is a hallmark of HDAC6 inhibition. HDAC6 contains two catalytic domains. Its C-terminal domain is the functional domain for both synthetic and physiological substrates, while the N-terminal domain lacks enzymatic activity (Zou, H. et al., Biochem. Biophys. Res. Commun., 2006, 341, 45-50). To evaluate the activity of compounds acting in cells, the ability of some of the HDAC inhibitors to induce increased levels of tubulin acetylation was evaluated. Western blots are shown in Figure 1. Low micromolar treatment of example compounds on WM 164 melanoma cells resulted in a dose-dependent increase in acetyl α-tubulin levels without a concomitant increase in histone H3 acetylation (Figure 1), indicating binding to the second enzymatically active catalytic domain. No observable increase in histone H3 acetylation was found until concentrations of 1 μM and 10 μM were used. Biochemical IC of example compounds against class 1 HDACs responsible for histone acetylation 50 This is not surprising since HDAC6 activity is in the micromolar range, and there are clear preferences for activity in the cellular environment that corresponds to selective HDAC6 inhibition.

[0318] Cytotoxicity against HDAC inhibitors cell The B16-F10-luc mouse melanoma cell line was obtained from ATCC and cultured in RPMI 1640 supplemented with 10% FBS, 100 IU / mL penicillin, and 100 mg / mL streptomycin. The SM1 cell line was obtained from Dr. Antoni Ribas's lab at the University of California, Los Angeles. The human melanoma cell line WM164 was obtained from Smalley's Lab at the Moffitt Cancer Center. Cells were cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin / streptomycin (50 U / mL), L-glutamine (2 mM), and 2-mercaptoethanol (50 mM) (complete medium) and grown under humidified conditions at 37°C and 5% CO2.

[0319] Assay conditions Mouse melanoma cells were plated in 96-well flat-bottom plates at 5 × 10 3 Cells were seeded at 100 / well. The next day, medium was changed to contain various concentrations of HDAC6I or matching DMSO vehicle concentrations diluted in complete medium and performed in triplicate. Cells were incubated at 37°C and 5% CO2 for 48 hours. The density of viable and metabolically active cells was quantified using a standard MTS assay (CellTiter 96 AQueous One, Promega, Madison, WI) according to the manufacturer's instructions. Briefly, 20 μL of reagent was added per well and incubated at 37°C for 3 hours. Absorbance at 490 nM was measured spectrophotometrically with background subtraction at 690 nM. All values ​​were then normalized and expressed as a percentage (100%) relative to medium control.

[0320] Analysis of compound 1 Compound 1, when run by DRC, exhibits a potency of 29 nM at hHDAC6 (dual) and exhibits complete selectivity over HDAC1 (DRC), but also over other HDAC subtypes (% inhibition at 10 μM: less than 20%, see below and raw data) (Figure 3A).

[0321] Compound 1 has high kinetic solubility, high permeability, and low excretion. It is characterized by low clearance in human microsomes but high clearance in mouse microsomes. It has high free fraction in both mouse and human plasma. The compound is chemically stable at pH 7.4 and 2. Although Clint is higher than the Schedule B set standard of 50ul / min / mg protein, we agree to proceed with profiling in mouse PK studies to clarify the iv-IV clearance relationship (Figure 3B).

[0322] Mouse iv PK results for compound 1 indicate that a clearance of 44.9 mL / min / kg was observed, which is approximately half of the hepatic blood flow in mice. This moderate clearance is consistent with the observed microsomal stability. Oral PK studies of compound 1 indicate moderate clearance, a moderate volume of distribution, a half-life of 2.2 hours, a mean residence time of 3.14 hours, and low oral bioavailability (14.7%). A mean Cmax value of 456 ng / mL was observed relatively soon after oral gavage (Tmax = 15 minutes). The compound has been detected in moderate concentrations in the brain and sciatic nerve, with a brain / plasma ratio of 0.8 (1 h) to 1.2 (4 h) and a sciatic nerve / plasma ratio of 0.4 at 1 hour.

[0323] The PK / BBB properties of compound 1 combined with the reported functional activity (0.76 uM) may not yet be sufficiently good to further evaluate this compound in vivo (at least in mice).

[0324] Compound 1 may be a promising starting point for compound optimization towards more potent, selective and brain-penetrant HDAC6 inhibitors for CNS applications. Further characterization of compound 1 in other species remains to be done, as well as optimization of the functional bioactivity and ADME-T / PK properties of future molecules, followed by appropriate efficacy studies required to identify preclinical candidates for CNS applications.

[0325] Incorporation by Reference All publications and patents mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0326] Equivalent While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon consideration of this specification and the claims that follow. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. Compounds of general formula I, 【Chemistry 1】 or a pharmaceutically acceptable salt and / or solvate thereof (wherein, 【Chemistry 2】 This represents a single or double bond; in the case of a five-membered ring portion, there are two double bonds and three single bonds, which are arranged together to form a five-membered ring heteroaryl group; X and Y are independently carbon or nitrogen, and Z is N or C(F); m = 0, 1, or 2; however, R １ If it is deuterium, then m is an integer between 0 and 4; If at least one of X and Y is carbon, then n = 0, 1, or 2; or if both X and Y are nitrogen, then n = 0 or 1; The sum of o and p is 0 or 1; R １ and R ２ are independently hydrogen, deuterium, halogen, hydroxyl, -CH ２ OH, -CH ２ CH ２ OH, cyano, -NR ａ R ｂ , -CH ２ NR ａ R ｂ , -C(O)NR ａ R ｂ , -S(O) ２ NR ａ R ｂ , acetyl, C １ -C ６ alkyl, C １ -C ６ alkenyl, C １ -C ６ alkynyl, C １ -C ６ alkoxy, C １ -C ６ haloalkyl, F ３ C-S-, C ３ -C ６ cycloalkyl, (C ３ -C ５ [[ID=6e]]cycloalkyl)-(C １ -C ３ alkyl)-, (C １ -C s ３ alkyl)-(C ３ -C ５ s cycloalkyl)-, (C ３ s -C ５ s cycloalkyl)-O-, aryl, aryl-O-, aryl-(C １ -C ３ alkyl)-, aryl-(C １ -C ３ alkoxy)-, heteroaryl, heteroaryl-O-, heteroaryl-(C １ -C ３ alkyl)-, heteroaryl-(C １ -C ３ alkoxy)-, 4- to 6-membered heterocyclyl, or (4- to 6-membered heterocyclyl)-(C １ -C ３ s alkyl)-; or R １ two of the following and / or R ２ Two of these atoms bond to adjacent carbon atoms on their respective aromatic rings, linking together to form a five-membered or six-membered carbon ring or heterocycle; R ａ and R ｂ These are, independently, hydrogen and C １ -C ６ Alkyl or C ３ -C ６ They may be selected from the group consisting of cycloalkyl groups, or these groups may be bonded together to form a 3- to 7-membered heterocycline; a is, 【Transformation 3】 When represents a single bond, CR ｃ R ｄ , C=O, NR ｅ , O, or S, or 【Chemistry 4】 When represents a double bond, CR ｃ or N; b is, 【Transformation 5】 When represents a single bond, CR ｃ R ｄ And, or 【Transformation 6】 When represents a double bond, CR ｃ And, or b is, 【Transformation 7】 The symbol represents a double bond, and a is a CR bond. ｃ If that is the case, then N is; R ｃ and R ｄ These are independently hydrogen, deuterium, fluorine, chlorine, and C １ -C ６ Alkyl, or C ３ -C ６ They are cycloalkyl or, when combined, form a 3- to 6-membered cycloalkyl group; R ｅ is hydrogen, C １ -C ６ Alkyl, C ３ -C ６ Selected from the group consisting of cycloalkyl, aryl, heteroaryl, or 4- to 6-membered heterocyclines; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R ｆ ) and one of d and e is oxygen and the other is nitrogen; R ｆ (These are hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyanomethyl.)

2. Compounds having formula Ib 【Transformation 8】 (In the formula, R １ and R ２ are each independently hydrogen, deuterium, halogen, hydroxyl, -CH ２ OH, -CH ２ CH ２ OH, cyano, -NR ａ R ｂ 、-CH ２ NR ａ R ｂ 、-C(O)NR ａ R ｂ 、-S(O) ２ NR ａ R ｂ 、acetyl, C １ -C ６ alkyl, C １ -C ６ alkenyl, C １ -C ６ alkynyl, C １ -C ６ alkoxy, C １ -C ６ haloalkyl, F ３ C-S-, C<q ３ -C ６ cycloalkyl, (C ３ -C ５ cycloalkyl)-(C １ -C ３ alkyl)-, (C １ -C ３ alkyl)-(C ３ -C ５ cycloalkyl)-, (C ３ -C ５ cycloalkyl)-O-, aryl, aryl-O-, aryl-(C １ -C ３ alkyl)-, aryl-(C １ -C ３ alkoxy)-, heteroaryl, heteroaryl-O-, heteroaryl-(C １ -C ３ alkyl)-, heteroaryl-(C １ -C ３ [[ID=..]]alkoxy)-, 4- to 6-membered heterocyclyl, or (4- to 6-membered heterocyclyl)-(C １ -C ３ alkyl)- selected from the group consisting of; R ａ and R ｂ These are, independently, hydrogen and C １ -C ６ Alkyl or C ３ -C ６ They may be selected from the group consisting of cycloalkyl groups, or these groups may be bonded together to form a 3- to 7-membered heterocycline.

3. Formula Ic 【Chemistry 9】 Formula Id 【Chemistry 10】 Formula Ie 【Chemistry 11】 Formula If 【Chemistry 12】 Formula Ig 【Chemistry 13】 Formula Ih 【Chemistry 14】 Formula II 【Chemistry 15】 Formula Ij 【Chemistry 16】 Formula Ik 【Chemistry 17】 Formula Im [Chemistry 18] Formula Ip 【Chemistry 19】 Formula Iq 【Chemistry 20】 Formula Ir 【Chemistry 21】 Formula Is 【Chemistry 22】 Formula It 【Chemistry 23】 Formula Iu 【Chemistry 24】 Formula Iw 【Chemistry 25】 Formula Iz 【Chemistry 26】 Formula Iaa 【Chemistry 27】 Formula Ibb 【Chemistry 28】 Formula Icc 【Chemistry 29】 Formula Idd 【Transformation 30】 Formula IEEE 【Chemistry 31】 Formula If 【Chemistry 32】 Formula Igg 【Transformation 33】 Formula Ijj 【Transformation 34】 and Formula Ikk 【Chemistry 35】 (wherein R1 and R2 are defined in claim 1) The compound according to claim 1, having a formula selected from the following.

4. Formula IL 【Transformation 36】 Formula IV 【Chemistry 37】 and Formula If 【Transformation 38】 (wherein R1 and R2 are defined in claim 1) The compound according to claim 1, having a formula selected from the following.

5. Formula In 【Chemistry 39】 Formula I 【Chemistry 40】 Formula Ix 【Chemistry 41】 Formula Iy 【Chemistry 42】 Formula Ihh 【Chemistry 43】 and Formula III 【Chemistry 44】 (wherein R1 and R2 are defined as in claim 1, either both R3 are methyl, or one of R3 is methyl and the other is H, or both R3 together are CH2CH2 or CH2CH2CH2) The compound according to claim 1, having a formula selected from the following.

6. The compound according to claim 2, wherein R1 and R2 are independently selected from H, D, Cl, and F.

7. The compound according to claim 3, wherein R1 and R2 are independently selected from H, D, Cl, and F.

8. The compound according to claim 4, wherein R1 and R2 are independently selected from H, D, Cl, and F.

9. The compound according to claim 5, wherein R1 and R2 are independently selected from H, D, Cl, and F.

10. Formula ILL 【Chemistry 45】 Formula Inn 【Chemistry 46】 Formula Ioo 【Chemistry 47】 Formula Ipp 【Chemistry 48】 and Formula Iqq 【Chemistry 49】 (In the formula, [Transformation 50] However, each represents either a single or double bond, and of the five bonds thus marked, two are double bonds and three are single bonds, which together form a five-membered ring heteroaryl group; One of c, d, and e is oxygen and the rest are nitrogen; or c is C(R f), one of d and e is oxygen and the other is nitrogen; R f is hydrogen, deuterium, fluorine, chlorine, methyl, difluoromethyl, trifluoromethyl, or cyano; (R1 and R2 are defined in claim 1) The compound according to claim 1, having a formula selected from the following.

11. The compound according to claim 7, wherein R1 and R2 are independently selected from H, D, Cl, and F, where c and d are nitrogen and e is oxygen.

12. A compound according to claim 1, selected from the group consisting of the following compounds, or a pharmaceutically acceptable salt thereof. 【Chemistry 51】 【change】 【change】 【change】 【change】 【change】 【change】

13. (a) A compound according to any one of claims 1 to 12, (b) A second therapeutic agent useful for treating a disease or condition in which inhibition of HDAC is beneficial, (c) an excipient and / or a pharmaceutically acceptable carrier, at the discretion of the party. A composition containing the following:

14. The composition according to claim 13, wherein the second therapeutic agent comprises a chemotherapeutic agent useful for the treatment of cancer.

15. A pharmaceutical composition comprising a compound according to any one of claims 1 to 12 and a pharmaceutically acceptable carrier or vehicle.

16. A composition for use in a method of treating a disease or condition in which inhibition of an HDAC is beneficial, comprising a compound according to any one of claims 1 to 12, wherein the treatment comprises administering a therapeutically effective amount of the compound to an individual in need thereof.

17. The composition according to claim 16, further comprising administering a therapeutically effective amount of a second therapeutic agent useful for treating a disease or condition.

18. The composition according to claim 16, wherein the disease or condition is selected from cancer, neurological disease, neurodegenerative disorder, peripheral neuropathy, traumatic brain injury, stroke, inflammation, and autoimmune disease.

19. The composition according to claim 17, wherein the disease is cancer, and the second therapeutic agent is one or more of chemotherapy drugs, radiation, and immunotherapy.

20. A composition for use in a method for increasing the sensitivity of cancer cells to the cytotoxic effects of radiotherapy and / or chemotherapy, comprising a compound according to any one of claims 1 to 12, wherein the method comprises contacting the cells with an amount of the compound sufficient to increase the sensitivity of the cells to the radiotherapy and / or chemotherapy.

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