Difluoroacetylhydrazides and trifluoroacetylhydrazides as selective HDAC6 inhibitors

Abstract

The present invention relates to acyl hydrazides obtained in situ by enzymatic hydrolysis of the parent prodrugs 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole on histone deacetylase 6 (HDAC6).

Description

[Technical Field]

[0001] The present invention relates to acyl hydrazides obtained in situ by enzymatic hydrolysis of the parent prodrugs 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole on histone deacetylase 6 (HDAC6). [Background technology]

[0002] Zn-dependent histone deacetylases (HDACs) are a family of 11 evolutionarily related hydrolases that catalyze the removal of acetyl or myristyl residues from histones, nonhistone proteins, and polyamines (Biochemistry. 57, 3105-3114; Mol. Cell. Proteomics. 21, 100193; J. Genet. Genomics. 44, 243-250). Given the involvement of HDACs in many diseases, the pharmaceutical industry has been developing HDAC inhibitors (HDACis) for nearly two decades, leading to the approval of five molecules for the treatment of cancer (Br. J. Clin. Pharmacol. 87, 4577-4597). Unfortunately, the therapeutic efficacy of HDACis has been limited by side effects due to the poor selectivity of these first-generation molecules, which inhibit several to all members of the Zn-dependent HDAC family, potentially affecting important physiological functions.

[0003] Among the 11 human zinc-dependent HDAC isoforms, HDAC6 stands out as the only one with two homologous tandem catalytic domains (CD1 and CD2) and a zinc finger-ubiquitin binding domain. Furthermore, HDAC6 is primarily localized in the cytoplasm, and its primary substrates are not histones but various nonhistone proteins, such as α-tubulin, Foxp3, Hsp90, β-catenin, cortactin, and peroxiredoxin. Interestingly, HDAC6 knockout mice are viable and fertile and show no obvious physiological dysfunction (Mol. Cell. Biol. 28, 1688-1701). Furthermore, selective HDAC6 inhibition has been shown to be well tolerated in preclinical and human clinical trials (Oncologist. 26, 184-e366).

[0004] Because HDAC6 plays a role in both the ubiquitin-proteasome and aggresome pathways, the regulation of immune responses, the development of neurological disorders, and Alzheimer's disease, there has been much interest in identifying highly specific HDAC6 inhibitors (J. Med. Chem. 64, 1362-1391).

[0005] The classical HDACi pharmacophore consists of a zinc-binding group (ZBG) that interacts with the active site Zn ion, a cap that interacts with the outer region of the enzyme, and a linker that connects the ZBG and the cap. Most HDACi have a hydroxamic acid moiety as the ZBG, a chemical group that has inherent stability and safety issues. The hydroxamate group is a metabolic hotspot associated with suboptimal pharmacokinetics and potential genotoxicity.

[0006] WO 2022 / 029041, WO 2022 / 013728, WO 2021 / 127643, WO 2020 / 212479, and WO 2022 / 049496 disclose oxadiazole-based HDAC6 inhibitors that are potential alternatives to hydroxamates, although their mechanism of action is unknown. Summary of the Invention [Means for solving the problem]

[0007] We have identified difluoroacetylhydrazides and trifluoroacetylhydrazides as tight-binding inhibitors of HDAC6 and demonstrated that they form long-lived complexes with the enzyme when formed in situ from appropriately designed prodrugs.

[0008] Surprisingly, we found that difluoromethyl-1,3,4-oxadiazole (DFMO) and trifluoromethyl-1,3,4-oxadiazole (TFMO) are substrates of HDAC6, which can hydrolyze them to the corresponding difluoroacetylhydrazides and trifluoroacetylhydrazides. Therefore, we selected DFMO and TFMO as prodrugs for the in situ generation of acylhydrazides.

[0009] Using sub-steady-state kinetics and rapid chromatography and mass spectrometry, we investigated the inhibitory mechanisms of DFMO- and TFMO-containing compounds in detail and showed that HDAC6-catalyzed ring hydration / opening of DFMO and TFMO leads to the formation of tight, long-lived complexes between the enzyme and the corresponding acyl hydrazides. Once dissociated from the enzyme, the hydrated inhibitors eventually rebind to HDAC6 and can undergo further hydrolysis to generate low-affinity hydrazide complexes.

[0010] We were also able to confirm this mechanism by solving the X-ray crystal structure of HDAC6-CD2 bound to the DFMO compound. Surprisingly, we found that the electron density in the active site did not fit the structure of the parent prodrug, but was a perfect fit for the structure of the hydrazide (i.e., the final hydrolysis product).

[0011] The acylhydrazide-containing compounds that are the subject of this invention are inactive when administered directly to enzymes or cells, but are surprisingly potent and selective when generated in situ from DFMO or TFMO prodrugs.

[0012] Focusing on prodrugs (DFMO- and TFMO-containing HDAC6 inhibitors), we were able to identify molecules with >10,000-fold selectivity for HDAC6 over all other HDACs, as well as excellent drug-like properties, safety profiles, and pharmacological activity in vitro and in vivo. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows a hypothetical mechanism for DFMO ring opening and associated acylhydrazide and hydrazide formation. [Figure 2] FIG. 1 shows the isolation of long-lived / tight complexes (Example 10). DETAILED DESCRIPTION OF THE INVENTION

[0014] (definition) Unless otherwise defined, all technical terms, notations and other scientific terms used herein are intended to have the meaning that is generally understood by those skilled in the art to which this disclosure pertains.In some cases, for the sake of clarity and / or quick reference, this specification defines terms that have the meaning that is generally understood.Therefore, the inclusion of such definitions herein should not be interpreted as representing a substantial difference from what is generally understood in the art.

[0015] As used herein, the term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).

[0016] As used herein, the term "C1-C6 alkyl" refers to a branched or linear hydrocarbon containing 1 to 6 carbon atoms. Examples of C1-C6 alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, and n-hexyl.

[0017] As used herein, the term "aryl" refers to monocyclic and polycyclic aromatic ring systems (i), wherein the individual carbon rings in the polycyclic ring system may be fused or may be joined to each other by a single bond. Suitable aryl groups include, but are not limited to, phenyl, naphthyl, and biphenyl.

[0018] As used herein, the term "aryloxy" refers to the group O-aryl, where "aryl" is as defined above.

[0019] As used herein, the term "alkoxy" refers to an O-alkyl group, where "alkyl" is as defined above.

[0020] As used herein, the term "thioalkoxy" refers to an S-alkyl group, where "alkyl" is as defined above. Preferred thioalkoxy groups are thioethoxy (-SEt) or thiomethoxy (-SMe), more preferably thiomethoxy. In different embodiments, the thioalkoxy group refers to an alkyl group in which one of the non-terminal hydrocarbon units of the alkyl chain has been replaced with a sulfur atom.

[0021] As used herein, the term "halogenated" refers to halogen substitution. In other words, any of the aforementioned alkyl, alkoxy, and thioalkoxy groups may be fully or partially substituted with halogen atoms. Preferably, the halogen atoms are F or Cl, more preferably F.

[0022] As used herein, the term "cycloalkyl" refers to a saturated or unsaturated hydrocarbon ring, preferably having 3 to 10 carbon atoms. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl.

[0023] As used herein, the term "arylalkyl" refers to an aryl radical, as defined herein, attached to an alkyl radical, as defined herein. An example of an arylalkyl is benzyl.

[0024] As used herein, the term "deuterated" refers to deuterium substitution. In other words, some or all of the hydrogen atoms can be replaced with deuterium.

[0025] As used herein, the term "heterocycle" refers to a 4-, 5-, 6-, 7-, or 8-membered monocyclic ring, saturated or unsaturated, composed of carbon atoms and one or more heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur heteroatoms are optionally oxidized, and the nitrogen heteroatom is optionally quaternized. The heterocycle can be attached to any heteroatom or carbon atom, provided that such attachment results in the creation of a stable structure. The term also includes bicyclic ring systems in which any of the aforementioned heterocycles is fused to an aryl or another heterocycle. If the heterocycle is an aromatic heterocycle, it can be defined as a "heteroaromatic ring."

[0026] As used herein, the term "unsaturated ring" refers to a partially or fully unsaturated ring. For example, unsaturated C6 monocyclic rings refer to cyclohexene, cyclohexadiene, and benzene.

[0027] As used herein, the term "substituted" means mono- or polysubstitution with defined (or undefined) substituents, provided that mono- or polysubstitution is chemically permissible.

[0028] As used herein, the term "physiologically acceptable excipient" refers to a substance that has no pharmacological effect itself and does not cause adverse reactions when administered to a mammal, preferably a human. Physiologically acceptable excipients are well known in the art and are disclosed, for example, in the Handbook of Pharmaceutical Excipients, sixth edition 2009, which is incorporated herein by reference.

[0029] As used herein, the term "pharmaceutically acceptable salt or derivative thereof" refers to a salt or derivative that has the biological effectiveness and properties of the salted or derivatized compound and does not produce adverse reactions when administered to a mammal, preferably a human. A pharmaceutically acceptable salt may be an inorganic salt or an organic salt. Examples of pharmaceutically acceptable salts include, but are not limited to, carbonate, hydrochloride, hydrobromide, sulfate, hydrogen sulfate, citrate, maleate, fumarate, trifluoroacetate, 2-naphthalenesulfonate, paratoluenesulfonate, and the like. Further information regarding pharmaceutically acceptable salts can be found in "Handbook of Pharmaceutical Salts," P. Stahl, C. Wermuth, WILEY-VCH, 127-133, 2008, which is incorporated herein by reference. Pharmaceutically acceptable derivatives include esters, ethers, and N-oxides.

[0030] The terms "comprising," "having," "including," and "containing" are understood to be open-ended terms (meaning "including, but not limited to") and are also considered to support terms such as "essentially consist of," "consist of," and "consisting of."

[0031] The terms "essentially consist of" and "essentially consisting of" are understood as semi-closed-ended terms and mean that no other ingredients that affect the novel properties of the invention are included (and therefore, optional excipients may be included).

[0032] The terms "consist of" and "consisting of" are to be understood as closed-ended terms.

[0033] The term "isomer" refers to stereoisomers (or spatial isomers), i.e. diastereoisomers and enantiomers.

[0034] Description of the Invention Difluoromethyl-1,3,4-oxadiazole (DFMO) and trifluoromethyl-1,3,4-oxadiazole (TFMO) were found to be substrates for HDAC6, which can hydrolyze them to the corresponding difluoroacetylhydrazides or trifluoroacetylhydrazides.

[0035] Kinetic studies were performed by incubating several DFMO and TFMO inhibitors with HDAC6 and analyzing the culture medium by LC-MS. Indeed, the disappearance of the DFMO compounds and the formation of the corresponding acyl hydrazides (used as standards in mass spectrometry studies) were observed. Hydrazide formation was also observed when extreme non-physiological conditions for inhibitor and protein concentrations were used. In the absence of enzymes, no degradation events were observed when the same DFMO and TFMO inhibitors were dissolved in the same buffer for the same time period. These experiments confirmed that DFMO and TFMO are potential substrates for HDAC6.

[0036] Intrigued by the slow binding / slow release kinetics associated with DFMO and TFMO compounds, we used spin column chromatography coupled with LC-HRMS to identify tight-binding species that form long-lived inhibitor-HDAC6 complexes (see Example 10). Upon incubation with DFMO or TFMO compounds, the predominant species coeluting with zHDAC6-CD2 was the corresponding acyl hydrazide. This hydrate form and the corresponding hydrazide were also detected in fractions containing the free compound unbound to the enzyme. Interestingly, the parent compounds of DFMO and TFMO did not coelute with the enzyme.

[0037] Interestingly, direct incubation of the enzyme with difluoromethyl acyl hydrazide did not induce enzyme inhibition, suggesting that the high-affinity species is the acyl hydrazide formed in situ.

[0038] Crystallographic data combined with molecular modeling supported the hypothesized mechanism. A hydrazide was found when co-crystallizing DFMO-containing compounds. The hypothesized hydrazide could be formed as a result of two subsequent reactions. After the inhibitor entered the catalytic pocket, the active site Zn cation was directly bound to the CHF2 group of the DFMO moiety via sp 2 This enhances the electrophilic behavior of the carbon atom, allowing for nucleophilic attack by a water molecule, whose presence in the coordination sphere of the metal cation is supported by modeling studies. The hydrated intermediate can undergo further ring-opening to give the acyl hydrazide (Figure 1, Step 1). To explain the hydrazide detected in the crystal structure, we must assume an additional hydrolysis reaction, which may be enzyme-catalyzed or may occur in solution after the release of the acyl hydrazide from the enzyme active site. If the second hydrolysis reaction is indeed enzyme-catalyzed, the zinc coordination sphere must be restored by the entry of a second water molecule. The metal cation then activates the acyl carbonyl, allowing for routine deacetylation and subsequent release of the hydrazide and difluoroacetic acid (Figure 1, Step 2).

[0039] Surprisingly, the present inventors have found that DFMO inhibitors are hydrolyzed in the presence of HDAC6 to give the corresponding difluoromethyl acyl hydrazides, which are true active and selective HDAC6 inhibitors.

[0040] According to a first aspect, the present invention provides compounds of formula (I), their pharmaceutically acceptable salts and isomers:

[0041] [ka]

[0042] During the ceremony, W is H or F; G is a 5-membered heteroaromatic ring consisting of carbon atoms and 1 to 4 heteroatoms selected from N, O, S and Se, optionally substituted with C1-C3 alkyl, alkoxy, thioalkoxy, halogenated derivatives thereof, or halogen, hydroxy; or G is of formula (Ia):

[0043] [ka]

[0044] wherein X, X', Y and Y' are independently selected from CH, N, CF or CCl. and; Z is -CD2-, -CF2-, -CHR 2 -, -NH-, -S-; R 2 is H, halogen, C1-C6 alkyl or C3-C6 cycloalkyl, each of which is unsubstituted or selected from the following groups: hydroxy, carbonyl, C1-C3 alkoxy, aryloxy or thioalkoxy, or their halogenated derivatives; · halogens; primary, secondary or tertiary amines substituted with C1-C6 alkyl, C3-C6 cycloalkyl or their halogenated derivatives; phenyl, pyridyl, thiophenyl, furan or pyrrole, which may be unsubstituted or substituted with C1-C3 alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen; or The following substructures:

[0045] [ka]

[0046] or its halogenated derivatives, H, halogen, C1-C6 alkyl or C3-C6 cycloalkyl; L is absent, C1-C6 alkyl, alkoxy or thioalkoxy, -(CH2) m -CHR 4 -(CH2) o -, -(CH2) m -CH(NHR 4 )-(CH2) o -, -(CH2) m -NR 4 -(CH2) o - or halogenated derivatives thereof, wherein m and o are each independently 0, 1 or 2; L is the following substructures (IIa) to (IIf) and their halogenated derivatives:

[0047] [ka]

[0048] wherein a, b, c, and d are independently 0, 1, 2, or 3, and a and b are not simultaneously 0; Q is CH2, NR 4 or O)

[0049] [ka]

[0050] wherein n is 0, 1, or 2; Y' is absent, C1-C2 alkenyl, or the following substructure:

[0051] [ka]

[0052] wherein a, b and Q are as defined above) and halogenated derivatives thereof Selected from among; R 4 is H, unsubstituted or: Halogen Phenyl, pyridyl, thiophenyl, furan or pyrrole, unsubstituted or substituted with C1-C3 alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen. C1-C4 alkyl substituted with; P is an unsubstituted or substituted, aromatic or non-aromatic 5-10 membered heterocycle, the ring consisting of carbon atoms and one or more heteroatoms selected from N, O and S; R 1 is absent, halogen, unsubstituted or substituted -C(=O)C1-C4 alkyl, C1-C4 alkyl, C4-C6 cycloalkenyl, C6-C 12 aryl or a 5- to 9-membered heteroaryl containing at least one heteroatom selected from N, O, and S; The following compounds: 3-[2-[[4-[[(2,2-difluoroacetyl)amino]carbamoyl]phenyl]methyl]tetrazol-5-yl]benzoic acid, 2-((5-(6-aminopyridin-3-yl)-2H-tetrazol-2-yl)methyl)-N'-(2,2-difluoroacetyl)pyrimidine-5-carbohydrazide, tert-butyl (5-(1-((6-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridazin-3-yl)methyl)-1H-1,2,3-triazol-4-yl)pyridin-2-yl)carbamate, N'-(2,2-difluoroacetyl)-4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolidin-1-yl)methyl)-3-fluorobenzohydrazide, 4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolidin-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide, N'-(2,2-difluoroacetyl)-4-((2,5-dioxo-3-phenylimidazolidin-1-yl)methyl)-3-fluorobenzohydrazide, N'-(2,2-difluoroacetyl)-6-((1-(3-fluorophenyl)-8-(oxetan-3-yl)-2,4-dioxo-1,3,8-triazaspiro[4.5]decan-3-yl)methyl)nicotinohydrazide, 4-((1H-imidazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide, N'-(2,2-difluoroacetyl)-4-(pyrimidin-2-ylamino)benzohydrazide, benzyl 4-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyrimidin-2-yl)amino)-4-phenylpiperidine-1-carboxylate, 4-((1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)-N'-(2,2,2-trifluoroacetyl)benzohydrazide and pharmaceutically acceptable salts and isomers thereof, provided that:

[0053] Another class of preferred compounds includes compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 2 is H, or C1-C3 alkyl, either unsubstituted or selected from the following groups: C1-C3 alkyl or C3-C6 cycloalkyl hydroxy, carbonyl, C1-C2 alkoxy, aryloxy or thioalkoxy, or their halogenated derivatives; · halogens; · Primary, secondary or tertiary amines substituted with C1-C3 alkyl, C3-C6 cycloalkyl or their halogenated derivatives; phenyl, pyridyl, thiophenyl, furan or pyrrole, each of which is unsubstituted or substituted with C1-C3 alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen; or The following substructures:

[0054] [ka]

[0055] or its halogenated derivatives is H, or C1-C3 alkyl, optionally substituted with

[0056] Another class of preferred compounds includes compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein P has the following structure:

[0057] [ka]

[0058] and A is C, N, O, S; B is C, N; D is C, N, O; E is C, N, O; M is C,N; R 5 and R 6 are independently -H, halogen, ═O, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or halogenated derivatives thereof, optionally substituted with carbonyl or carboxy; or R 5 and R 6 may independently be selected from the following substructures:

[0059] [ka]

[0060] Selected from among; R 3 is absent, —H, C1-C6 alkyl optionally substituted with —OH or —N(C1-C5 alkyl), —LR 1 or R 3 is the following substructure:

[0061] [ka]

[0062] Selected from among; R 3 Ga-LR 1 then there is no substitution on M; Ra and Rb are independently H, halogen, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or halogenated derivatives thereof.

[0063] Another class of preferred compounds includes compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein P is the following substructure:

[0064] [ka]

[0065] [ka]

[0066] Selected from: Ra and Rb are as defined in claim 3, or -LR 1 and; Rc is H, halogen, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or halogenated derivatives thereof, or -NH2.

[0067] Another class of preferred compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 is the following substructure:

[0068] [ka]

[0069] [ka]

[0070] [ka]

[0071] Selected from among; R 6 and R 7 are independently: -H, -D, -OH, C1-C4 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl or halogenated derivatives thereof, halogen, -(CH2) a NR'R”, -NHR 8 , -C(=O)OR', -C(=O)R 9 , -C(=NH)R 9 , -NO2, -CN, -Ph, -SO2-NR'R”, =O, =NR 8 , —SO2—C1-C4 alkyl; or R 6 and R 7 may independently be selected from the following substructures:

[0072] [ka]

[0073] [ka]

[0074] Selected from among; R 8 is -H, -D, -OH, C1-C6 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof, -(CH2) a NR'R", -C(=O)OR', -C(=O)R 9 , -C(=NH)R 9 , -(CH2) a Ph, -(CH2) a Py, -SO2-C1-C4 alkyl, or R 8 is the following substructure:

[0075] [ka]

[0076] Selected from among; R 9 is -NR'R", C1-C4 alkyl, or a halogenated derivative thereof, or is the following substructure:

[0077] [ka]

[0078] Selected from among; R 10 and R 11 are independently selected from -H, C1-C4 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof, -OR', -C(=O)OR', -C(=O)R', or halogen; Q 1 are CH2, O, S, NR 8 and; Q 2 and Q 3 are independently CR'R", CF2, O, S, NR 8 and; R' and R" are independently -H, C1-C4 alkyl, C3-C6 cycloalkyl, or halogenated derivatives thereof; a, b, c and R 8 is as defined above.

[0079] Another class of preferred compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 is the following substructure:

[0080] [ka]

[0081] [ka]

[0082] [ka]

[0083] Selected from among; R 6 and R 7 are independently -H, -D, -OH, C1-C4 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl or halogenated derivatives thereof, halogen, -(CH2) a NR'R”, -NHR 8 , -C(=O)R 9 , -NO2, -Ph, -SO2-NR'R”, =O, =NR 8 , —SO—C1-C4 alkyl, or independently selected from the group consisting of the following substructures:

[0084] [ka]

[0085] and; R 8 is -H, -D, -OH, C1-C6 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof, -(CH2) a NR'R", -C(=O)OR', -C(=O)R 9 , -C(=NH)R 9, -SO2-C1-C4 alkyl, or R 8 is the following substructure:

[0086] [ka]

[0087] Selected from among; R 9 is -NR'R", C1-C4 alkyl, or a halogenated derivative thereof, or is the following substructure:

[0088] [ka]

[0089] Selected from among; R 10 and R 11 are independently selected from -H, C1-C4 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof, -OR', -C(=O)OR', -C(=O)R', or halogen; Q 1 are CH2, O, S, NR 8 and; Q 2 and Q 3 are independently CR'R", CF2, O, S, NR 8 and; R' and R" are independently -H, C1-C4 alkyl, C3-C6 cycloalkyl, or halogenated derivatives thereof; a, b, c and R 8 is as defined above.

[0090] Another class of preferred compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 is the following substructure:

[0091] [ka]

[0092] Selected from among; a, b are independently 0, 1, 2, or 3, and a and b cannot simultaneously be 0; Z1 is CH2, NH, or O; R 1 wherein at least one H may optionally be substituted with halogen, —(CH)(n)-Q-Q-R; n is 0, 1 or 2; Q4 is absent, -SO2-, -NH-, -N(C1-C5 alkyl)-, -NHC(=O)-, -N(C1-C5 alkyl)C(=O)-, or -C(=O)-; Q5 is absent, C1-C5 alkylene, -NH-, -(C1-C5 alkylene)-NH-C(=O)-, or -N(C1-C5 alkyl); Rd is -OH, C1-C5 alkyl, C1-C5 haloalkyl, -NR'R", C1-C5 alkoxy, 5- or 6-membered heteroaryl containing 1 to 3 N, or the following substructure:

[0093] [ka]

[0094] and; e and f are independently 1 or 2; M1 is CH2, O, NH or SO2; M2 is CH or N; At least one H in Rd is optionally selected from OH, halogen, C1-C5 alkyl, C1-C5 haloalkyl, —C(═O)—(C1-C5 alkyl), —C(═O)O(C1-C5 alkyl); —NH—C(═O)—O(C1-C5 alkyl), —NR′R″, the following substructures:

[0095] [ka]

[0096] may be substituted with; g and h are independently 0 or 1, but cannot both be 0; M4 is CH2, O, NH, and at least one H of M4 may be optionally substituted with halogen, C1-C5 alkyl, C3-C6 cycloalkyl, or -C(=O)-O(C1-C5 alkyl); R' and R" are independently -H or C1-C4 alkyl.

[0097] The following compounds of formula (I) are preferred: N'-(2,2-difluoroacetyl)-4-((4-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-1H-1,2,3-triazol-1-yl)methyl)benzohydrazide (compound 1), 4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 2), N'-(2,2-difluoroacetyl)-6-((4-(p-tolyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 8), N'-(2,2-difluoroacetyl)-6-((4-(m-tolyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 9), N-(3-(1-(4-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)-2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)phenyl)acetamide (compound 12), N'-(2,2-difluoroacetyl)-6-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 13), N'-(2,2-difluoroacetyl)-6-((4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 14), N'-(2,2-difluoroacetyl)-5-fluoro-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 15), N'-(2,2-difluoroacetyl)-6-((5-phenyl-2H-tetrazol-2-yl)methyl)nicotinohydrazide (compound 16), N'-(2,2-difluoroacetyl)-6-((5-(thiophen-2-yl)-2H-tetrazol-2-yl)methyl)nicotinohydrazide (compound 17), N'-(2,2-difluoroacetyl)-6-((4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 18), N'-(2,2-difluoroacetyl)-6-((4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 19), N'-(2,2-difluoroacetyl)-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinohydrazide (compound 20), 6-((4-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 27), 4-(1-(4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)ethyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 30), 4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-2-chloro-N'-(2,2-difluoroacetyl)benzohydrazide (compound 31), 4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (compound 32), 6-((4-(1H-indazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 34), 4-((4-(1H-indol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 36), 4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-3-chloro-N'-(2,2-difluoroacetyl)benzohydrazide (compound 37), 6-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 38), 6-(1-(4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 39), 6-((4-(1H-indazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-5-fluoronicotinohydrazide (compound 40), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-3,5-difluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 42), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 43), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-2-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 44), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (compound 45), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3,5-difluorobenzohydrazide (compound 46), 4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 47), 5-[[4-(2-amino-1,3-benzothiazol-6-yl)triazol-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (compound 48), 5-[[4-(6-aminopyridin-3-yl)triazol-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (compound 49).

[0098] [Table 1]

[0099] [Table 2]

[0100] The compounds of the present invention may contain one or more chiral centers (asymmetric carbon atoms) and therefore may exist in the form of enantiomers and / or diastereoisomers.

[0101] All possible optical isomers, either alone or in mixture with one another, fall within the scope of the present invention.

[0102] A second object of the present invention is the compounds of formula (I) above for use as pharmaceuticals.

[0103] A third object of the present invention is a compound as described above for use in the prevention and / or treatment of diseases or disorders modulated by HDAC6.

[0104] The compounds of the present invention are useful for treating peripheral neuropathies of genetic origin, such as Charcot-Marie-Tooth disease, drug (chemotherapy or antibiotics such as metronidazole and fluoroquinolones)-induced peripheral neuropathies, peripheral neuropathies caused by systemic diseases such as diabetes or leprosy, or peripheral neuropathies generally associated with severe axonal transport defects. The compounds of the present invention are also useful for treating chemotherapy-related cognitive impairment (CRCI).

[0105] The compounds of the present invention are preferably useful for the treatment of transplant rejection, GVHD, myositis, diseases associated with lymphocyte dysfunction, multiple myeloma, non-Hodgkin's lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer and neurodegenerative diseases, ophthalmic diseases (such as uveitis).

[0106] (General synthesis route) The compounds described in this invention can be prepared using methods known to those skilled in the art. All starting materials, reactants, acids, bases, solvents and catalysts used in the synthesis of the compounds described are commercially available. The progress of the reaction was monitored by TLC, HPLC, UPLC or HPLC-MS analysis.

[0107] The acylhydrazides are obtained in situ by enzymatic hydrolysis of the parent prodrugs, 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole, in histone deacetylase 6 (HDAC6). Similarly, although less efficiently, 2-(difluoromethyl)-1,3,4-oxadiazole and 2-(trifluoromethyl)-1,3,4-oxadiazole can be hydrolyzed in aqueous acid (TFA) or basic (LiOH or methanolic ammonia) solutions to produce difluoroacetylhydrazide and trifluoroacetylhydrazide, respectively.

[0108] The 2-(difluoromethyl)-1,3,4-oxadiazole and 2-(trifluoromethyl)-1,3,4-oxadiazole moieties are described in the following literature: International Patent Application Publication No. 2022 / 029041, Marchini, M. et al., 2021, "2-(4-((5-(benzo[b]thiophen-3-yl)-1H-tetrazol-1-yl)methyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazole derivatives and similar compounds as selective inhibitors of histone deacetylase 6 (HDAC6) for use in treating, e.g., peripheral neuropathy"; International Patent Application Publication No. 2022 / 013728, Lee, JK et al, "Novel compounds as histone deacetylase 6 inhibitors, and pharmaceutical compositions comprising the same"; International Patent Application Publication No. 2020 / 212479, Vara Salazar, YI et al, 2020, "1,3,4-oxadiazole derivatives as histone deacetylase inhibitors"; International Patent Application Publication No. 2018 / 165520, "Yates, C, 2018, Metalloenzyme inhibitor compounds" In most cases, the corresponding hydrazide was treated with an excess of difluoroacetic acid or trifluoroacetic anhydride, which acted as both an acylating agent and a dehydrating agent (WO 2017 / 018805; Lee, J. et al., 2017, "1,3,4-Oxadiazole sulfonamide derivatives as histone deacetylase 6 inhibitors and their pharmaceutical composition and preparation"). In some cases, starting from the corresponding tetrazole, the 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole moiety was prepared and converted to 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole in the presence of difluoroacetic anhydride or trifluoroacetic anhydride (Vereshchagin et al Rus.J. Org. Chem. 2007, 43(11), 1710-1714). Furthermore, acylhydrazides can be converted to oxadiazoles in the presence of other dehydrating reagents such as Burgess reagent (WO 2017 / 23133, Lee, J. et al., "1,3,4-Oxadiazole derivative compounds as histone deacetylase 6 inhibitors, and the pharmaceutical composition consisting of the same") or tosyl chloride (WO 2019 / 027054, Ito M. et al., 2019, "Heterocylic compound").

[0109] Other methods for preparing acyl hydrazides include acylation of hydrazides in the presence of approximately stoichiometric amounts of difluoroacetic acid or trifluoroacetic anhydride (WO 2017 / 018805, Lee, J. et al., 2017; "1,3,4-Oxadiazole sulfonamide derivatives as histone deacetylase 6 inhibitors and their pharmaceutical composition and preparation"), or functionalization of carboxylic acids with difluoroacetyl hydrazides or trifluoroacetyl hydrazides using common amide coupling procedures and reagents (i.e., via activated esters using HATU or HOBt / EDC hydrochloride) (WO 2019 / 027054, Ito M. et al., 2019, "Heterocylic compounds"), or via acryloyl chloride (Shchekotikhin et al., Rus. J. Org. Chem. 2007, 43(11), All synthetic routes are summarized in Scheme 1.

[0110] Scheme 1: Synthesis of difluoroacetylhydrazide or trifluoroacetylhydrazide and the prodrug 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole moiety a )

[0111] [ka]

[0112] aReactants and conditions: (a) DFAA or TFAA; (b) DFAA or TFAA; (c) TsCl or Burgess reagent; (d) TFA, water; (e) NH3 (7 M solution in MeOH), water or LiOH, THF / water; (f) HDAC6; (g) HATU or HOBt, EDC; (h) SOCl2; (i) difluoroacetylhydrazide or trifluoroacetylhydrazide.

[0113] The acylhydrazide targets of the present invention were obtained by decomposition of the corresponding 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole. Unless otherwise stated, the synthesis of the parent compounds was carried out as described in the literature.

[0114] Unless otherwise specified, 1,2,3-triazole compounds rely on the common intermediate 2-(4-(bromomethyl)aryl)-5-(difluoromethyl)-1,3,4-oxadiazole or 2-(4-(bromomethyl)aryl)-5-(trifluoromethyl)-1,3,4-oxadiazole, the preparation of which has been described (WO 2022 / 029041; Marchini, M. et al., 2021, "2-(4-((5-(benzo[b]thiophen-3-yl)-1H-tetrazol-1-yl)methyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazole derivatives and similar compounds as selective inhibitors of histone deacetylase 6 (HDAC6) for treating e.g., peripheral neuropathy"). Treatment of the methyl or ethyl esters with hydrazine gave the corresponding hydrazides, which were converted to the difluoromethyl-1,3,4-oxadiazole and trifluoromethyl-1,3,4-oxadiazole moieties as previously described. Subsequent benzylic bromination using N-bromosuccinimide and azobisisobutyronitrile (AIBN) or dibenzoyl peroxide (BPO) as catalysts afforded the bromomethyl intermediates (Scheme 2).

[0115] Scheme 2: Synthesis of 2-(4-(bromomethyl)aryl)-5-(difluoromethyl)-1,3,4-oxadiazole or 2-(4-(bromomethyl)aryl)-5-(trifluoromethyl)-1,3,4-oxadiazole common intermediate a )

[0116] [ka]

[0117] aReactants and conditions: (a) N2H4·H2O, MeOH, reflux; (b) DFAA or TFAA, DMF, room temperature; (c) NBS, AIBN or BPO, CCl4, 80 °C.

[0118] Conversion of the bromide to the azide in the presence of sodium azide and a one-pot CuAAC click reaction with the appropriate alkyne afforded the 1,2,3-triazole-containing products bearing 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole.

[0119] Scheme 3: Synthesis of 1,2,3-triazole-incorporated compounds a )

[0120] [ka]

[0121] a Reagents and conditions: (a) NaN3, DMF, 1 h, room temperature; (b) CuSO4-5H2O, sodium ascorbate, DMF:H2O (1:1), 16 h, 40 °C; (c) Pd(dppf)Cl2, CuI, Et3N, DMF; (d) TBAF, DMF or K2CO3, MeOH; (e) K2CO3, MeOH, followed by Ohira-Bestmann reagent.

[0122] Commercially unavailable aryl alkynes were prepared by the Sonogashira coupling of appropriate aryl halides with ethynyl(trimethyl)silane in the presence of triethylamine using [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) (Pd(dppf)Cl2) and copper(I) iodide as catalysts (AG Sams et al., Bioorg. Med. Chem. Lett. 2011, 21(11), 3407-3410), followed by cleavage of the TMS protection with tetrabutylammonium fluoride (TBAF) or potassium carbonate in methanol. Aliphatic alkynes were synthesized starting from the corresponding aldehydes under Ohira-Westmann conditions using potassium carbonate in methanol (Honig, M., Carreira, EM Angew. Chem. Int. Ed. 2020, 59(3), 1192-1196).

[0123] Compounds based on a tetrazole core were synthesized by nucleophilic substitution. The common intermediate, 2-(4-(bromomethyl)aryl)-5-(difluoromethyl)-1,3,4-oxadiazole, was reacted with the appropriate substituted tetrazole in DMF using potassium carbonate as the base at room temperature overnight (see Scheme 4). This reaction afforded a mixture of regioisomers, which could be efficiently separated by chromatographic methods. In general, the 2,5-disubstituted tetrazole is the most abundant of the two regioisomers. The common intermediate methyl bromide derivatives were synthesized in a similar manner to the compounds with a 1,2,3-triazole core (Scheme 2).

[0124] Scheme 4: Synthesis of compounds with tetrazole as the central skeleton a )

[0125] [ka]

[0126] aReactants and conditions: (a) K2CO3, DMF, 16 h, room temperature; (b) NaN3, NH4Cl.

[0127] Most of the substituted tetrazoles used are commercially available. Non-commercially available building blocks were synthesized from the corresponding carbonitriles by reaction with excess sodium azide in the presence of ammonium chloride.

[0128] The following examples are intended to further illustrate, but not limit, the present invention. [Example]

[0129] Example 1. Synthesis of 2-(4-(bromomethyl)-3-chlorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (Intermediate C) (Process 1)

[0130] [ka]

[0131] Methyl 3-chloro-4-methylbenzoate (10 g, 54.1 mmol, 1 equiv.) was dissolved in MeOH and hydrazine hydrate (5 equiv.) was added. Following conversion to the hydrazide by UPLC, the mixture was refluxed under stirring for 5 hours. The desired intermediate precipitated out. It was then collected by filtration and washed with fresh MeOH. The crude residue was dissolved in DMF and the mixture was cooled to 0 °C. DFAA (2.5 equiv.) was added dropwise. The resulting mixture was stirred at room temperature overnight, and complete conversion was observed. The reaction mixture was diluted with saturated aqueous NaHCO3. The product precipitated out as a solid and was collected by filtration, washed with water, and dried under vacuum (8.47 g, 34.6 mmol, 64% yield).

[0132] (Process 2)

[0133] [ka]

[0134] A mixture of 2-(3-chloro-4-methylphenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (3.44 g, 14 mmol, 1 equiv.) and N-bromosuccinimide (1.1 equiv.) in 70 mL of carbon tetrachloride was stirred under argon until complete dissolution. Then, AIBN (0.015 equiv.) was added to the reaction mixture, which was then stirred at 70 °C overnight. The conversion was monitored by LCMS. The mixture was allowed to reach room temperature, diluted with DCM, and washed successively with saturated aqueous NaHCO3, water, and brine. The organic phase was separated, dried over MgSO4, filtered, and concentrated under reduced pressure. The residue was purified by flash chromatography (silica gel, hexane / ethyl acetate, 0-15%) to give the desired product as a white solid (2.9 g, 8.9 mmol, 63% yield). The following compounds were prepared using similar procedures:

[0135] [Table 3]

[0136] The synthesis of the following analogous intermediates has been described elsewhere (WO 2022 / 029041, Marchini, M. et al, 2022, Example 1):

[0137] [Table 4]

[0138] Example 2. General Procedure A for the Conversion of 2-(Difluoromethyl)-1,3,4-oxadiazole or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoroacetylhydrazide or Trifluoroacetylhydrazide

[0139] [ka]

[0140] To a DMSO solution of 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole-containing parent compound (0.16 mmol, 1 equivalent, 0.2 M), ammonia (7 M solution in MeOH, 10 equivalents) was added. The resulting mixture was then added with water (excess, 1.5-3 mL / mmol) and heated to 30-70 °C with stirring for 2 days to achieve complete conversion. The ammonia and water were removed under vacuum, and the residue was purified by preparative HPLC.

[0141] The following compounds were prepared according to general procedure A. The synthesis of the parent compounds is described elsewhere.

[0142] [Table 5]

[0143] Example 3. General Procedure B for the Conversion of 2-(Difluoromethyl)-1,3,4-oxadiazole or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoroacetylhydrazide or Trifluoroacetylhydrazide

[0144] [ka]

[0145] 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole-containing compound (1 equivalent, 0.07M) is dissolved in DMSO / water 95:5. TFA (60 equivalents) is added to the mixture and stirred at room temperature for 3 days to achieve complete conversion. The reaction mixture is concentrated, and the residue is purified by preparative HPLC.

[0146] The following compounds were prepared according to general procedure B. The synthesis of the parent compounds is described elsewhere.

[0147] [Table 6]

[0148] Example 4. General Procedure C for CuAAC Click Reaction and One-Pot Conversion of 2-(Difluoromethyl)-1,3,4-oxadiazole or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoroacetylhydrazide or Trifluoroacetylhydrazide

[0149] [ka]

[0150] Reagent A (Intermediate AJ, Example 1, 0.26 mmol, 1 equiv.) was dissolved in 1 mL of DMSO. Sodium azide (0.26 mmol, 1 equiv.) was added, and the reaction mixture was stirred at room temperature for 20 minutes. Alkyne (0.26 mmol, 1 equiv.), copper(II) sulfate solution (1 M aqueous solution, 0.2 equiv.), and sodium (+)-L-ascorbate solution (0.5 M aqueous solution, 0.4 equiv.) were added sequentially, and the reaction mixture was stirred at room temperature until complete consumption of the starting reagent (2-12 hours) was confirmed by UPLC or / and TLC monitoring. 0.5 mL of 7 M ammonia solution in MeOH and 0.5 mL of water were added, and the reaction mixture was stirred at 50°C overnight. The reaction mixture was concentrated to remove volatile solvents, and the residue was purified by preparative HPLC. In some cases, prodrugs could also be isolated.

[0151] The following compounds were prepared according to general procedure C. As mentioned above, the synthesis of alkyne building blocks was described separately when not commercially available.

[0152] [Table 7]

[0153] [Table 8]

[0154] Example 5. General Procedure D for Tetrazole Nucleophilic Substitution and One-Pot Conversion of 2-(Difluoromethyl)-1,3,4-oxadiazole or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoroacetylhydrazide or Trifluoroacetylhydrazide

[0155] [ka]

[0156] Tetrazole (0.34 mmol, 1 eq.) was dissolved in 2 mL of DMF. The appropriate reagent (Intermediate AJ, Example 1, 1 eq.) and potassium carbonate (2 eq.) were added. The reaction mixture was stirred for 3 hours (or until complete conversion), after which methanolic ammonia (7 M, 5 eq.) and excess water were added. The resulting mixture was stirred at 50° C. overnight, concentrated, and subjected to preparative HPLC.

[0157] The following compounds were prepared according to general procedure D. As mentioned above, the synthesis of prodrugs was also described separately:

[0158] [Table 9]

[0159] Example 6 Synthesis of 4-(1-(4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)ethyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (Compound 30) and 6-(1-(1-(4-(5-(difluoromethyl)-1,3,4-oxadiazol-2-yl)-2-fluorophenyl)ethyl)-1H-1,2,3-triazol-4-yl)benzo[d]thiazol-2-amine (Compound 30-A) (Process A)

[0160] [ka]

[0161] A solution of methyl 3-fluoro-4-formylbenzoate (1 g, 5.49 mmol, 1 equiv.) in THF (20 mL) was cooled to -70 °C. Methylmagnesium bromide (1 equiv.) was added dropwise, and the resulting mixture was stirred at -70 °C for 20 min. The reaction was quenched with aqueous NH4Cl and extracted with MTBE. The organic phases were combined, dried over Na2SO4, filtered, and concentrated. The crude material was purified by flash chromatography (silica gel, hexane / ethyl acetate, 0-30%) to give the desired product (1.09 g, 5.49 mmol, 100% yield).

[0162] (Process B)

[0163] [ka]

[0164] Methyl 3-fluoro-4-(1-hydroxyethyl)benzoate (1.09 g, 5.49 mmol, 1 eq.) was dissolved in 20 mL of DCM. Triethylamine (2 eq.) and methanesulfonyl chloride (1.2 eq.) were added, and the mixture was stirred at room temperature overnight. Complete conversion was observed. The reaction mixture was diluted with ethyl acetate, washed with brine, dried over Na2SO4, filtered, and concentrated.

[0165] The crude intermediate mesylate salt was dissolved in 10 mL of DMSO, and sodium azide (1 equivalent) was added. The resulting mixture was stirred at room temperature for 45 minutes, diluted with MTBE, and washed with brine. The organic phase was dried over Na2SO4, filtered, and concentrated. The resulting product was used in the next step without further purification (1.2 g, 5.38 mmol, 98% yield).

[0166] (Process C)

[0167] [ka]

[0168] To a solution of methyl 4-(1-azidoethyl)-3-fluorobenzoate (1.2 g, 5.38 mmol, 1 equiv.) in 10 mL of MeOH was added hydrazide monohydrate (6 equiv.) The reaction mixture was refluxed under stirring for 3 h and evaporated to dryness.

[0169] The intermediate hydrazide was dissolved in 5 mL of DMF and difluoroacetic anhydride (2.5 equiv.) was added. The reaction mixture was stirred overnight at room temperature, diluted with saturated aqueous NaHCO3, and extracted with MTBE. The organic phase was dried over Na2SO4, filtered, and concentrated. The crude material was purified by flash chromatography (silica gel, hexane / ethyl acetate, 0-15%) to give the desired product (860 mg, 3.04 mmol, 56% yield).

[0170] (Process D)

[0171] [ka]

[0172] 6-Bromo-1,3-benzothiazol-2-amine (8 g, 34.9 mmol, 1 equiv.) was dissolved in 75 mL of dioxane. Triethylamine (2 equiv.) was added, and the mixture was degassed with Ar. Copper iodide (0.1 equiv.) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II) DCM complex (0.1 equiv.) were added, and the mixture was degassed again. Ethynyl(trimethyl)silane (3 equiv.) was added, and the mixture was stirred at 95 °C overnight. After the reaction mixture reached room temperature, it was diluted with ethyl acetate and filtered through Celite. The filtrate was washed with 5% aqueous NH3, followed by saturated aqueous NaHCO3 and brine. The organic phase was dried over Na2SO4, filtered, and concentrated to dryness. The crude material was purified by flash chromatography (silica gel, 20-50% hexane / ethyl acetate) to give 7.38 g of the desired intermediate (29.9 mmol, 86% yield).

[0173] (Process E)

[0174] [ka]

[0175] 6-((trimethylsilyl)ethynyl)benzo[d]thiazol-2-amine (7.38 g, 29.9 mmol, 1 equiv.) was suspended in 75 mL of MeOH and potassium carbonate (1.5 equiv.) was added. The resulting mixture was stirred at room temperature overnight to obtain complete conversion. The crude material was purified by flash chromatography (silica gel, dry load, 0-4% MeOH / DCM) to give 4.2 g of the desired intermediate (24.1 mmol, 80% yield).

[0176] (Process F-1)

[0177] [ka]

[0178] 2-[4-(1-azidoethyl)-3-fluorophenyl]-5-(difluoromethyl)-1,3,4-oxadiazole (60 mg, 0.34 mmol, 1 equiv.) and 6-ethynyl-1,3-benzothiazol-2-amine (60 mg, 0.34 mmol, 1 equiv.) were dissolved in 2 mL of DMSO. Copper sulfate pentahydrate (0.3 equiv., 0.5 M aqueous solution) and sodium (+)-L-ascorbate (0.5 equiv., 1 M aqueous solution) were added, and the mixture was stirred at room temperature overnight. UPLC showed complete conversion to the desired click product.

[0179] The reaction mixture was heated to 50° C. A mixture of water (0.5 mL) and methanolic ammonia (0.5 mL, 7 M solution) was added to the reaction mixture and stirred at 50° C. overnight. Complete conversion to the desired product was detected by UPLC. The mixture was concentrated by rotary evaporation, filtered, and subjected to purification. RP-flash chromatography and subsequent preparative HPLC afforded 12.3 mg of the desired product as the free base (0.02 mmol, 7% yield). Compound 30: [M+H] + Actual value 476.35; 1 H NMR (400 MHz, DMSO-d6) δ 10.82 (br s, 2H), 8.69 (s, 1H), 8.15 (d, J=1.7Hz, 1H), 7.80-7.69 (m, 3H), 7.57 (s, 2H), 7.52 (t, J=7.8Hz, 1H), 7.38 (d, J=8.3Hz, 1H), 6.45 (t, J=52.9Hz, 1H), 6.26 (q, J=7.0Hz, 1H), 1.97 (d, J=7.0Hz, 3H).

[0180] (Process F-2)

[0181] [ka]

[0182] 2-[4-(1-azidoethyl)-3-fluorophenyl]-5-(difluoromethyl)-1,3,4-oxadiazole (400 mg, 1.4 mmol, 1 equiv.) and 6-ethynyl-1,3-benzothiazol-2-amine (246 mg, 1.4 mmol, 1 equiv.) were dissolved in 5 mL of DMSO. Copper sulfate pentahydrate (0.1 equiv., 0.5 M aqueous solution) and sodium (+)-L-ascorbate (0.2 equiv., 1 M aqueous solution) were added, and the mixture was stirred at room temperature for 1 h. UPLC showed complete conversion to the desired product. The mixture was directly subjected to RP-flash chromatography (water / ACN 40%, 0.1% FA) to give the pure product (566 mg, 1.24 mmol, 88% yield). Compound 30-A: [M+H] + Actual value 458.17; 1 H NMR (400 MHz, DMSO-d6) δ 8.73 (s, 1H), 8.32 (s, 1H), 8.15 (d, J=1.7Hz, 1H), 7.97-7.91 (m, 2H), 7.75-7.68 (m, 1H), 7.63-7.55 (m, 4H), 7.38 (d, J=8.3Hz, 1H), 6.30 (q, J=7.0Hz, 1H), 1.99 (d, J=7.1Hz, 3H).

[0183] Example 7. Synthesis of 5-[[4-(2-amino-1,3-benzothiazol-6-yl)triazol-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (Compound 48) (Process 1)

[0184] [ka]

[0185] Methyl 5-(bromomethyl)thiophene-2-carboxylate (1 g, 4.2 mmol, 1 equiv.) was dissolved in 8 mL of DMSO, and sodium azide (1.05 equiv.) was added. After 1 h, complete conversion was observed by LCMS. Water was added, and the reaction mixture was extracted with ethyl acetate and concentrated under reduced pressure. The crude product was used in the next step without purification.

[0186] (Process 2)

[0187] [ka]

[0188] Methyl 5-(azidomethyl)thiophene-2-carboxylate (402 mg, 2 mmol, 1 equivalent) was dissolved in 10 mL of ethanol, and hydrazine (5 equivalents) was added. The reaction mixture was refluxed overnight under stirring and concentrated under reduced pressure. The crude residue obtained was used in the next step without purification.

[0189] (Step 3)

[0190] [ka]

[0191] To a solution of 5-(azidomethyl)thiophene-2-carbohydrazide (200 mg, 1.0 mmol, 1 equiv.) in 5 mL of DMF was added dropwise difluoroacetic anhydride (1 equiv.). After 1 h, water was added, and the reaction mixture was extracted with ethyl acetate, dried over Na2SO4, and concentrated under reduced pressure. The resulting product (210 mg) was used in the next step without further purification.

[0192] (Step 4)

[0193] [ka]

[0194] 5-(azidomethyl)-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (210 mg, 0.51 mmol, 1 equiv.) and 6-ethynyl-1,3-benzothiazol-2-amine (1 equiv.) were dissolved in 3 mL of DMSO. Copper sulfate pentahydrate (0.2 equiv.) and sodium ascorbate (0.4 equiv.) were added as aqueous solutions. After 2 h, the reaction mixture was filtered and purified by preparative HPLC to give the final product (120 mg, 0.26 mmol, 52% yield). [M+H] + Actual value 450.23; 1 H NMR (400 MHz, DMSO-d6) δ 10.98 (s, 1H), 10.70 (s, 1H), 8.56 (s, 1H), 8.15 (d, J=1.7Hz, 1H), 7.75 (d, J=3.8Hz, 1H), 7.71 (dd, J=8.3Hz, 1.8Hz, 1H), 7.57 (s, 2H), 7.38 (d, J=8.3Hz, 1H), 7.27 (d, J=3.8Hz, 1H), 6.44 (t, J=52.9Hz, 1H), 5.91 (s, 2H).

[0195] Example 8. General Procedure E for the Bioconversion of 2-(difluoromethyl)-1,3,4-oxadiazole or 2-(trifluoromethyl)-1,3,4-oxadiazole to Difluoroacetylhydrazide or Trifluoroacetylhydrazide HDAC6 (1 μM) was incubated with 5 μM of prodrug compound in assay buffer at 25° C. At distinct time points, aliquots (40 μL) were transferred to test tubes containing acetonitrile (240 μL) to stop the reaction. Samples were stored frozen at −80° C. until LC-HRMS analysis.

[0196] LC-HRMS analysis was performed using a Vanquish Flex UHPLC (Thermo Fisher Scientific) and a high-resolution mass spectrometer, Orbitrap QExactive Focus (Thermo Fisher Scientific), equipped with heated electrospray ionization, operating in positive ion mode. Full scan analysis was performed over the m / z range of 50–500 amu. An XSelect HSS T3 50 × 2.1 mm, 2.5 μm chromatography column (Waters) was used. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was set to 0.5 mL / min, and a gradient program of 3–20% mobile phase B over 3 min was used.

[0197] When incubated with HDAC6 enzyme, the disappearance of the prodrug as a function of time was observed. HR-LCMS analysis revealed that the molecular weight of the acyl hydrazide was MW = [プロドラッグ] The production of +18 new compounds was demonstrated. Using general procedure E, the following compounds were obtained and identified:

[0198] [Table 10]

[0199] Example 9. LC-HRMS analysis for enzyme reaction monitoring HDAC6 (1 μM) was incubated with 5 μM DFMO compounds in assay buffer at 25°C. At distinct time points, aliquots (40 μL) were transferred to test tubes containing acetonitrile (240 μL) to terminate the reaction. Samples were stored frozen at -80°C until LC-HRMS analysis. Quantitation of test substances in samples was performed by reference to calibration curves obtained by varying compound concentrations. Calculated compound concentrations are shown in Table 1 as a percentage of the compound concentration at each time point.

[0200] LC-HRMS analysis was performed using a Vanquish Flex UHPLC (Thermo Fisher Scientific) and a high-resolution mass spectrometer, Orbitrap QExactive Focus (Thermo Fisher Scientific), equipped with heated electrospray ionization, operating in positive ion mode. Full scan analysis was performed over the m / z range of 50–500 amu. An XSelect HSS T3 50 × 2.1 mm, 2.5 μm chromatography column (Waters) was used. Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The flow rate was set to 0.5 mL / min, and a gradient program of 3–20% mobile phase B over 3 min was used.

[0201] [Table 11]

[0202] All compounds were found to disappear upon incubation with HDAC6 enzyme. HR-LCMS analysis also confirmed the formation of novel compounds with molecular weights of 18, corresponding to hydrate forms. For compounds 2-A, 31-A, and 37-A, the retention times of HPLC analysis confirmed that the products were the corresponding compounds 2, 31, and 37.

[0203] All compounds were incubated in the same buffer for the same time in the absence of enzyme, and all were stable (>80% by 8 hours, except for 42-A, which remained at 60% after 8 hours).

[0204] Example 10. Isolation of long-lived / tight complexes High-performance spin column chromatography coupled with LC-HRMS was used to attempt to isolate long-lived / tight complexes between HDAC6 and Examples 1 and 1-A. zHDAC6-CD2 wt (1 μM) was incubated with 1 (5 μM) or 1-A (also 5 μM) for 6 h as described above. To separate the HDAC6-inhibitor complex from the free compound, aliquots (60 μL) were loaded onto a Bio-Spin P-6 Gel Column (Bio-Rad) equilibrated with assay buffer. After centrifugation to elute the first fraction containing the majority of the enzyme, ten 200 μL aliquots were added and eluted by centrifugation to recover the low molecular weight (free) compound. Aliquots of the fractions were subjected to LC-HRMS analysis (as described above) to identify and quantify 1 and its derivatives.

[0205] The graph in Figure 2 reveals that the only compound coeluting with the enzyme (fraction 1) is compound 1, suggesting that it is a true inhibitor, while compound 1-A behaves as a prodrug (note that at this extreme concentration, the hydrolysis product of compound 1, the hydrazide, is also formed and binds to the enzyme, albeit to a lesser extent).

[0206] Example 11. Enzyme Screening For each test compound, eight doses of a 100x concentrated DMSO solution were prepared and diluted with assay buffer (25 mM Tris-HCl, pH 8, 130 mM NaCl, 0.05% Tween-20, 10% glycerol) to obtain 5x concentrated solutions relative to the final concentration (typical final concentration range: 6.4-200,000 nM or 0.18-50,000 nM, final DMSO content: 1%). Next, 10 μL of each test compound concentration was placed in triplicate in a 96-well plate, and 15 μL of a 3.33x concentrated enzyme solution in assay buffer containing 3.33x concentrated BSA (final BSA concentration: 1 mg / mL) and, in the case of HDAC6, 3.33x concentrated TCEP (final TCEP concentration: 200 μM) was added to each well. After preincubation at 25°C for a set time (30 min for HDAC6 and 120 min for HDAC1), 25 μL of a solution containing substrate was added: FLUOR DE LYS® Deacetylase Substrate (Enzo Life Sciences, cat: BML-KI104, FdL), FLUOR DE LYS®-Green Substrate (Enzo Life Sciences, cat: BML-KI572, FdL_G), or a 2x concentrated solution of Boc-Lys(Tfa)-AMC (Bachem, cat: 4060676.005, Tfal) in assay buffer. After a reaction time (30 min at 25°C), 50 μL of developer solution was added, consisting of concentrated FLUOR DE LYS® Developer I (Enzo Life Sciences, ca: BML-KI105) diluted 200-fold in buffer (50 mM Tris-HCl, pH=8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl) supplemented with 2 μM TSA, and after 25 min in the dark and at room temperature, fluorescence measurements were performed using a Victor 1420 Multilabel Counter Perkin Elmer Wallac instrument (excitation / emission 485 / 535 nm for Fluor de Lys Green and 355 / 460 nm for Tfal and Fluor de Lys).

[0207] Each synthetic compound was evaluated for its enzymatic activity against recombinant human HDAC6 and HDAC1 (Table 2).

[0208] [Table 12]

[0209] All tested compounds were virtually inactive against HDAC1 (IC50>100 μM) but showed adequate activity against HDAC6 (IC50<600 nM), confirming the selectivity of this class of compounds.

[0210] Example 12. In vitro acetylation of α-tubulin in the 697 cell line Human B-cell precursor leukemia 697 was evaluated for in vitro α-tubulin acetylation. 697 cells were maintained in RPMI medium 1640 (Gibco, cat: 21875-034) supplemented with 10 mM HEPES (Gibco, cat: 15630-080), Pen-Strep (100 U / ml penicillin, 100 μg / ml streptomycin, Gibco, cat: 15140-122), and 10% fetal bovine serum (Gibco, cat: 10270-106). Cells, 5.5 x 10 5 Cells were plated in 12-well plates (Costar, cat: 3512) at a density of 1000 cells / ml.

[0211] Serial dilutions of test compounds in DMSO were prepared using a 20 mM stock solution, with eight doses 200-fold concentrated relative to the final dose (2.7-100,000 nM). The DMSO solution was then diluted 10-fold with culture medium to obtain a 20-fold concentrated solution used for cell treatment (125 μl of medium solution was added to 2.375 ml of cell suspension). The final DMSO content was 0.5%. Plates were incubated at 37°C, 5% CO2 for 16 hours.

[0212] After the incubation period, the cells were harvested, centrifuged at 200 × g for 5 minutes, and washed with 0.9% NaCl at 4 °C. The resulting pellet was treated with complete Lysis-M buffer containing protease inhibitors (Complete Lysis-M Roche + Complete Tablets, Mini Easypack, cat: 4719956001) and phosphatase inhibitor cocktail (PhosStop Easypack, Roche, cat: 4906837001) for 30 minutes at 4 °C, and then centrifuged at 18,213 × g for 10 minutes. The protein concentration in each supernatant was measured using a BCA protein assay kit (Pierce, cat: 23227). Samples were diluted to a concentration of 2 μg / ml with 1 × PBS and applied to a MaxiSorp 96-well plate (Nunc, cat: 442404). The plate was incubated overnight at room temperature.

[0213] Plates were washed twice with wash buffer (1x PBS + 0.005% Tween 20) and saturated with 300 μL of 1x PBS containing 10% FBS for 1 hour at room temperature. After washing twice with wash buffer, plates were incubated for 2 hours at room temperature in the presence of 100 μL / well of either anti-acetylated-α-tubulin antibody (monoclonal anti-tubulin, acetylated antibody produced in mouse, Sigma-Aldrich, cat:T6793) or total anti-α-tubulin antibody (anti-α-tubulin produced in mouse, Sigma-Aldrich, cat:T6074) diluted 1:1000 in 1x PBS containing 10% FBS. After five washing cycles with wash buffer, enzyme-HRP-linked secondary antibody (goat anti-mouse IgG, IgM, IgA (H+L), stock concentration 0.5 mg / ml, Thermo Fisher Scientific, cat: A10668) diluted 1:1000 in 1x PBS + 10% FBS was added in a volume of 100 μl / well. After 2 hours of incubation at room temperature, the plate was washed four times with wash buffer, followed by the addition of 100 μl / well of TMB substrate (TMB substrate kit, Thermo Fisher Scientific, cat: 34021) for 10 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl of 2 M H2SO4. The plate was read at a wavelength of 450 nm using a BioTek Synergy H1 multimode microplate reader.

[0214] The measured absorbance was corrected by subtracting the mean value of the blank values (samples without primary antibody). The absorbance ratio of acetyl-tubulin to total tubulin was calculated and normalized to a four-parameter logistic curve of a reference compound (positive control) (0% is the fitted bottom of the curve, 100% is the fitted top of the curve). The results were expressed as relative EC 50 As shown.

[0215] [Table 13]

[0216] All the tested compounds showed very high activity in inducing tubulin acetylation in the 697 cell line.

[0217] Example 13. In vitro acetylation of α-tubulin in the N2a cell line The mouse neuroblastoma N2a cell line was evaluated for in vitro α-tubulin acetylation. Cells were maintained in Eagle's minimum essential medium (ATCC, cat: 30-2003) supplemented with 10% fetal bovine serum-FBS (Gibco, cat: 10270-106). 6 x 10 each 4 Cells were plated in 12-well plates (Costar, cat: 3512) at a density of 1000 cells / ml. Test compounds were prepared as 20-fold concentrated media solutions relative to the final concentration. Cells were treated the following day. Compounds were tested at three doses: 10 μM, 1 μM, and 0.1 μM. The final DMSO content was 0.5%. Cells were incubated with compounds at 37°C for 16 hours.

[0218] After the incubation period, cells were harvested, centrifuged at 200 × g for 5 minutes, and washed with 0.9% NaCl at 4°C. The resulting pellets were treated with complete Lysis-M buffer containing protease inhibitors (Complete Lysis-M Roche + Complete Tablets, Mini Easypack, cat: 4719956001) and phosphatase inhibitor cocktail (PhosStop Easypack, Roche, cat: 4906837001), and then centrifuged at 18,213 × g for 10 minutes. Protein concentrations in the supernatants were measured using a BCA protein assay kit (Pierce, cat: 23227). Samples were diluted with 1 × PBS to a concentration of 2 μg / ml and applied to a MaxiSorp 96-well plate (Nunc, cat: 442404). After overnight incubation at room temperature, the plates were washed twice with wash buffer (1x PBS + 0.005% Tween-20) and saturated with 300 μL of 1x PBS containing 10% FBS for 1 hour at room temperature. After washing twice with wash buffer, the plates were incubated for 2 hours at room temperature in the presence of 100 μL / well of either anti-acetylated-α-tubulin antibody (monoclonal anti-tubulin, acetylated antibody produced in mouse, Sigma-Aldrich, cat:T6793) or total anti-α-tubulin antibody (anti-α-tubulin produced in mouse, Sigma-Aldrich, cat:T6074) diluted 1:1000 in 1x PBS containing 10% FBS. After five washing cycles with washing buffer, enzyme-HRP-linked secondary antibody (goat anti-mouse IgG, IgM, IgA (H+L), stock concentration 0.5 mg / ml, Thermo Fisher Scientific, cat: A10668) diluted 1:1000 in 1x PBS + 10% FBS was added in a volume of 100 μl / well.After 2 hours of incubation at room temperature, the plate was washed four times with wash buffer, and then 100 μl / well of TMB substrate (TMB substrate kit, Thermo Fisher Scientific, cat: 34021) was added for 15 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl of 2 M H2SO4. The plate was read at a wavelength of 450 nm using a BioTek Synergy H1 multimode microplate reader.

[0219] The absorbance was corrected by subtracting the mean value of the blank (samples without primary antibody). The absorbance ratio of acetyl-tubulin to total tubulin was calculated and normalized to a four-parameter logistic curve of a reference compound (positive control) (100% is the fitted top of the curve, 0% is the DMSO control, a protein extract obtained from untreated cells). The results are expressed as the fold increase compared to the control (DMSO).

[0220] [Table 14]

[0221] All the tested compounds showed very high activity in inducing tubulin acetylation in the N2a cell line.

[0222] Example 14. In vitro acetylation of α-tubulin in undifferentiated SH-SY5Y cell line SH-SY5Y cells (ATCC, cod. CRL-2266) are plated at 5000 cells / well in 100 μl / well of growth medium (DMEM / F12 (1:1) + 10 mM HEPES + 100 units / mL penicillin + 100 μg / mL streptomycin + 10% inactivated fetal calf serum (FCS, Hyclone)) in optically optimized 96-well black plates (Perkin Elmer, cod. 6055302). 24 hours after seeding, cells were incubated overnight with 0.1-1-10 μM of the selected molecule. At the same dose, ACY1083 and Tubastatin A were tested as positive controls for α-tubulin acetylation, while untreated cells were incubated with 0.01% DMSO and labeled "control DMSO."

[0223] At the end of the incubation period, 100 μL / well of 8% formaldehyde in PBS (final formaldehyde concentration is 4% in 200 μL / well) is added directly to 100 μL / well of medium to fix the cells for 30 minutes at room temperature. The fixative is carefully removed, and the wells are washed twice with PBS for 10 minutes. Fixed cells are stored in PBS at 4°C until staining.

[0224] On the day of the staining experiment, the fixed cells are incubated with blocking buffer (PBS with 5% FCS + 0.3% Triton® X-100) for 60 minutes. During blocking, primary antibodies are prepared by diluting α-tubulin Alexa Fluor 488 conjugate (Cell Signaling, cod. 5063) antibody at 1:200 and acetyl α-tubulin Alexa Fluor 647 conjugate (Cell Signaling, cod. 81502) antibody at 1:50 in antibody dilution buffer (PBS with 1% BSA + 0.3% Triton® X-100). After aspirating the blocking solution, the diluted primary antibodies are applied and incubated overnight at 4°C. The next day, the cells are washed twice with PBS (10 minutes each), incubated with 300 nM DAPI in PBS for 5 minutes, and washed twice with PBS (10 minutes each). Triplicate wells are stained for each treatment.

[0225] Images of stained cells are acquired using an IN Cell Analyzer 2500 HS instrument using the far-red channel for acetyl-α-tubulin staining (0.02 s exposure), the green channel for α-tubulin staining (0.02 s exposure), and the blue channel for DAPI (nuclear) staining. Ten images are acquired for each well.

[0226] Images of stained cells were analyzed using InCarta software (Molecular Devices) to calculate the fluorescence intensity for the entire cell. For each treatment, the average cell concentration of both stains, Bckg (cells), was calculated for each FOV (field of view) using the raw InCarta data. Results are expressed as the fold increase in the ratio of acetylated tubulin to total tubulin relative to the control (DMSO control).

[0227] [Table 15]

[0228] All the tested compounds showed very high activity in inducing tubulin acetylation in the SH-SY5Y cell line.

Claims

1. Compounds of formula (I), their pharmaceutically acceptable salts and isomers: 【Chemistry 1】 During the ceremony, W is either H or F; G is a five-membered heteroaromatic ring consisting of a carbon atom and 1 to 4 heteroatoms selected from N, O, S, and Se, and optionally C １ ~C ３ Alkyl, alkoxy, thioalkoxy, halogenated derivatives thereof, or a five-membered heteroaromatic ring which may be optionally substituted with a halogen or hydroxyl, or G is equation (Ia): 【Chemistry 2】 (In the formula, X, X', Y, and Y' are independently selected from CH, N, CF, or CCl.) And; Z is -CD ２ -, -CF ２ -----CHR ２ -, -NH-, -S-; R ２ is H, halogen, C １ ~C ６ alkyl or C ３ ~C ６ cycloalkyl, each being unsubstituted or the following group: ・ Hydroxy, carbonyl, C １ ~C ３ Alkoxy, aryloxy, or thioalkoxy compounds, or their halogenated derivatives; Halogen; C １ ~C ６ Alkyl, C ３ ~C ６ Primary, secondary, or tertiary amines substituted with cycloalkyl groups or their halogenated derivatives; - Non-substitution or C １ ~C ３ Alkyl, alkoxy, thioalkoxy or halogenated derivatives thereof, or phenyl, pyridyl, thiophenyl, furan or pyrrole, which may be substituted with halogens; or The following substructure: 【Transformation 3】 or it may be substituted with a halogenated derivative thereof. H, halogen, C １ ~C ６ Alkyl or C ３ ~C ６ It is cycloalkyl; L is absent, C １ ~C ６ Alkyl, alkoxy or thioalkoxy, -(CH ２ ) ｍ - CHR ４ - (CH ２ ) ｏ -, - (CH ２ ) ｍ -CH (NHR) ４ )-(CH ２ ) ｏ -, - (CH ２ ) ｍ -NR ４ - (CH ２ ) ｏ -. Or halogenated derivatives thereof, where m and o are independently 0, 1 or 2, or L is the following substructures (IIa) to (IIf) and their halogenated derivatives: 【Chemistry 4】 (In the formula, a, b, c, and d are independently 0, 1, 2, or 3, and a and b cannot be 0 at the same time; Q is CH ２ , NR ４ (or O) 【Transformation 5】 (wherein n is 0, 1, or 2; Y' is nonexistent, C １ -C ２ It is either an alkenyl or has the following substructure: 【Transformation 6】 (wherein a, b, and Q are as defined above) and selected from among its halogenated derivatives. Selected from among; R ４ H, unsubstituted or: Halogen - Non-substitution, or C １ -C ３ Alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or phenyl, pyridyl, thiophenyl, furan, or pyrrole substituted with halogens. C is replaced by １ ~C ４ It is alkyl; P is an unsubstituted or substituted, aromatic or non-aromatic 5- to 10-membered heterocycle, where the ring is composed of a carbon atom and one or more heteroatoms selected from N, O, and S; R １ -C(=O)C is absent, halogenated, unsubstituted, or substituted. １ ~C ４ Alkyl, C １ ~C ４ Alkyl, C ４ ~C ６ Cycloalkenyl, C ６ ~C １２ An aryl or a 5- to 9-membered heteroaryl containing at least one heteroatom selected from N, O, and S; The following compounds: 3-[2-[[4-[[(2,2-difluoroacetyl)amino]carbamoyl]phenyl]methyl]tetrazole-5-yl]benzoic acid, 2-((5-(6-aminopyridine-3-yl)-2H-tetrazole-2-yl)methyl)-N'-(2,2-difluoroacetyl)pyrimidine-5-carbohydrazide, tert-butyl (5-(1-((6-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyridazin-3-yl)methyl)-1H-1,2,3-triazole-4-yl)pyridine-2-yl)carbamate, N'-(2,2-difluoroacetyl)-4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolidined-1-yl)methyl)-3-fluorobenzohydrazide, 4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolidined-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide, N'-(2,2-difluoroacetyl)-4-((2,5-dioxo-3-phenylimidazolidined-1-yl)methyl)-3-fluorobenzohydrazide, N'-(2,2-difluoroacetyl)-6-((1-(3-fluorophenyl)-8-(oxetan-3-yl)-2,4-dioxo-1,3,8-triazaspiro[4.5]decane-3-yl)methyl)nicotinohydrazide, 4-((1H-imidazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide, N'-(2,2-difluoroacetyl)-4-(pyrimidine-2-ylamino)benzohydrazide, Benzyl 4-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyrimidine-2-yl)amino)-4-phenylpiperidine-1-carboxylate, 4-((1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyrazole-4-yl)methyl)-N'-(2,2,2-trifluoroacetyl)benzohydrazide Subject to the exclusion of Compounds of formula (I), their pharmaceutically acceptable salts and isomers.

2. R ２ is H, or C １ ~C ３ Alkyl groups, all of which are unsubstituted or have the following groups: C １ ~C ３ Alkyl or C ３ ~C ６ Cycloalkyl ・ Hydroxy, carbonyl, C １ ~C ２ Alkoxy, aryloxy, or thioalkoxy compounds, or their halogenated derivatives; Halogen; C １ ~C ３ Alkyl, C ３ ~C ６ Primary, secondary, or tertiary amines substituted with cycloalkyl groups or their halogenated derivatives; - Both are non-substituted or C １ ~C ３ Alkyl, alkoxy, thioalkoxy or halogenated derivatives thereof, or halogen-substituted phenyl, pyridyl, thiophenyl, furan or pyrrole; or The following substructure: 【Transformation 7】 or its halogenated derivative H or C may be substituted with １ ~C ３ The compound according to claim 1, wherein it is alkyl.

3. P has the following structure: 【Transformation 8】 And, A is C, N, O, S; B is C, N; D is C, N, O; E is C, N, O; M is C, N; R ５ and R ６ These may be independently and optionally substituted with carbonyl or carboxyl molecules: -H, halogen, =O, C １ ~C ６ Alkyl, alkoxy, or thioalkoxy, C ３ ~C ６ Cycloalkyls, or halogenated derivatives thereof, R ５ and R ６ It has the following substructure independently: 【Chemistry 9】 Selected from among; R ３ is absent, -H, optionally -OH or -N(C) １ ~C ５ Alkyl) ２ C may be optionally replaced with １ ~C ６ Alkyl, -LR １ is it, or, R ３ is the following partial structure: 【Chemistry 10】 Selected from among; R ３ ga-LR １ In this case, no substitutions exist on M; Ra and Rb are independently H, halogen, and C １ ~C ６ Alkyl, alkoxy, or thioalkoxy, C ３ ~C ６ Cycloalkyls, or halogenated derivatives thereof, The compound according to claim 1.

4. P has the following substructure: 【Chemistry 11】 【Chemistry 12】 Selected from: Ra and Rb are independently H, halogen, C １ ~C ６ alkyl, alkoxy or thioalkoxy, C ３ ~C ６ cycloalkyl, or a halogenated derivative thereof, or -L-R １ where; Rc stands for H, halogen, C １ ~C ６ Alkyl, alkoxy, or thioalkoxy, C ３ ~C ６ Cycloalkyls, or halogenated derivatives thereof, or -NH ２ That is, The compound according to claim 1.

5. R １ It has the following substructure: 【Chemistry 13】 【Chemistry 14】 【Chemistry 15】 Selected from among; R ６ and R ７ These are independently: -H, -D, -OH, C １ ~C ４ Alkyl, alkoxy, or thioalkoxy, C ３ ~C ６ Cycloalkyl or halogenated derivatives thereof, halogen, -(CH ２ ) ａ NR'R", -NHR ８ , -C(=O)OR', -C(=O)R ９ , -C(=NH)R ９ , -NO ２ , -CN, -Ph, -SO ２ -NR'R", =O, =NR ８ , -SO ２ -C １ ~C ４ Selected from the group consisting of alkyl, or R ６ and R ７ It has the following substructure independently: 【Chemistry 16】 【Chemistry 17】 Selected from among; R ８ -H, -D, -OH, C １ ~C ６ Alkyl, C ３ ~C ６ Cycloalkyl or halogenated derivatives thereof, -(CH ２ ) ａ NR'R", -C(=O)OR', -C(=O)R ９ , -C(=NH)R ９ ,-(CH ２ ) ａ Ph, -(CH ２ ) ａ Py, -SO ２ -C １ ~C ４ Is it alkyl, or R ８ It has the following substructure: [Chemistry 18] Selected from among; R ９ is -NR'R”, C １ ~C ４ Alkyl, or halogenated derivatives thereof, or the following substructures: 【Chemistry 19】 Selected from among; R １０ and R １１ These are independently -H, C １ ~C ４ Alkyl, C ３ ~C ６ Selected from cycloalkyls or their halogenated derivatives, -OR', -C(=O)OR', -C(=O)R', or halogens; Q １ CH ２ O, S, NR ８ And; Q ２ and Q ３ Independently, CR'R", CF ２ O, S, NR ８ And; R' and R'' are independently -H, C １ ~C ４ Alkyl, C ３ ~C ６ They are cycloalkyl or halogenated derivatives thereof; a, b, c and R ８ This is as defined above. The compound according to claim 1.

6. R １ It has the following substructure: 【Chemistry 20】 【Chemistry 21】 【Chemistry 22】 Selected from among; R ６ and R ７ These are independently: -H, -D, -OH, C １ ~C ４ Alkyl, alkoxy, or thioalkoxy, C ３ ~C ６ Cycloalkyl or halogenated derivatives thereof, halogen, -(CH ２ ) ａ NR'R", -NHR ８ , -C(=O)R ９ , -NO ２ -Ph, -SO ２ -NR'R", =O, =NR ８ , -SO ２ -C １ ~C ４ Selected from the group including alkyl groups, or independently, the following substructures: 【Chemistry 23】 And; R ８ -H, -D, -OH, C １ ~C ６ Alkyl, C ３ ~C ６ Cycloalkyl or halogenated derivatives thereof, -(CH ２ ) ａ NR'R", -C(=O)OR', -C(=O)R ９ , -C(=NH)R ９ , -SO ２ -C １ ~C ４ Is it alkyl, or R ８ The following substructure: 【Chemistry 24】 Selected from among; R ９ is -NR'R”, C １ ~C ４ Alkyl, or halogenated derivatives thereof, or the following substructures: 【Chemistry 25】 Selected from among; R １０ and R １１ These are independently -H, C １ ~C ４ Alkyl, C ３ ~C ６ Selected from cycloalkyls or their halogenated derivatives, -OR', -C(=O)OR', -C(=O)R', or halogens; Q １ CH ２ O, S, NR ８ And; Q ２ and Q ３ Independently, CR'R", CF ２ O, S, NR ８ And; R' and R'' are independently -H, C １ ~C ４ Alkyl, C ３ ~C ６ They are cycloalkyl or halogenated derivatives thereof; a, b, c and R ８ This is as defined above. The compound according to claim 1.

7. R １ It has the following substructure: 【Chemistry 26】 Selected from among; a and b are independently 0, 1, 2, or 3, and a and b cannot be 0 at the same time; Z １ CH ２ , NH, or O; R １ At least one H is optionally a halogen, -(CH ２ )(n)-Q4-Q5-Rd may also be substituted; n is 0, 1, or 2; Q4 is absent, -SO ２ -, -NH-, -N(C １ ~C ５ Alkyl)-, -NHC(=O)-, -N(C １ ~C ５ Alkyl)C(=O)- or -C(=O)-; Q5 is absent, C １ ~C ５ Alkylene, -NH-, -(C １ ~C ５ Alkylene)-NH-C(=O)-, or -N(C １ -C ５ It is alkyl; Rd is -OH, C １ ~C ５ Alkyl, C １ ~C ５ Haloalkyl, -NR'R'', C １ ~C ５ Alkoxy, 5-membered or 6-membered heteroaryl containing 1-3 N, the following substructures: 【Chemistry 27】 And; e and f are independently 1 or 2; M １ CH ２ , O, NH or SO ２ And, M ２ It is CH or N, At least one H in Rd is optionally OH, halogen, or C １ ~C ５ Alkyl, C １ ~C ５ Haloalkyl, -C(=O)-(C １ ~C ５ Alkyl), -C(=O)O(C １ ~C ５ Alkyl); -NH-C(=O)-O(C １ ~C ５ Alkyl), -NR'R'', the following substructure: 【Chemistry 28】 It is also fine if it is replaced with; g and h are independently either 0 or 1, but cannot be 0 at the same time; M ４ CH ２ ,O,NH, and at least one H of M4 is optionally halogen, C １ ~C ５ Alkyl, C ３ ~C ６ Cycloalkyl or -C(=O)-O(C １ ~C ５ It may also be substituted with alkyl; R' and R'' are independently -H or C １ ~C ４ It is alkyl The compound according to claim 1.

8. • N'-(2,2-difluoroacetyl)-4-((4-(4-((4,5-dihydro-1H-imidazole-2-yl)amino)phenyl)-1H-1,2,3-triazole-1-yl)methyl)benzohydrazide (compound 1), 4-((4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 2), • N'-(2,2-difluoroacetyl)-6-((4-(p-tolyl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 8), • N'-(2,2-difluoroacetyl)-6-((4-(m-tolyl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 9), • N-(3-(1-(4-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)-2-fluorobenzyl)-1H-1,2,3-triazole-4-yl)phenyl)acetamide (compound 12), • N'-(2,2-difluoroacetyl)-6-((4-phenyl-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 13), • N'-(2,2-difluoroacetyl)-6-((4-(2-fluorophenyl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 14), • N'-(2,2-difluoroacetyl)-5-fluoro-6-((4-(pyridine-2-yl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 15), • N'-(2,2-difluoroacetyl)-6-((5-phenyl-2H-tetrazole-2-yl)methyl)nicotinohydrazide (compound 16), • N'-(2,2-difluoroacetyl)-6-((5-(thiophen-2-yl)-2H-tetrazole-2-yl)methyl)nicotinohydrazide (compound 17), • N'-(2,2-difluoroacetyl)-6-((4-(3-fluorophenyl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 18), • N'-(2,2-difluoroacetyl)-6-((4-(thiophen-2-yl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 19), • N'-(2,2-difluoroacetyl)-6-((4-(pyridine-2-yl)-1H-1,2,3-triazole-1-yl)methyl)nicotinohydrazide (compound 20), 6-((4-(1H-pyrrolo[2,3-b]pyridine-5-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 27), 4-(1-(4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)ethyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 30), 4-((4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)methyl)-2-chloro-N'-(2,2-difluoroacetyl)benzohydrazide (compound 31), 4-((4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (compound 32), 6-((4-(1H-indazole-4-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 34), 4-((4-(1H-indole-4-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 36), 4-((4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)methyl)-3-chloro-N'-(2,2-difluoroacetyl)benzohydrazide (compound 37), 6-((4-(2-aminobenzo[d]thiazole-6-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 38), 6-(1-(4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)ethyl)-N'-(2,2-difluoroacetyl)nicotinohydrazide (compound 39), 6-((4-(1H-indazole-4-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)-5-fluoronicotinohydrazide (compound 40), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-3,5-difluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 42), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 43), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-2-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (compound 44), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (compound 45), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3,5-difluorobenzohydrazide (compound 46), 4-((4-(6-aminopyridine-3-yl)-1H-1,2,3-triazole-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (compound 47), 5-[[4-(2-amino-1,3-benzothiazole-6-yl)triazole-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (compound 48), 5-[[4-(6-aminopyridine-3-yl)triazole-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbozide (Compound 49) A compound according to claim 1, selected from the following.

9. The compound according to claim 1, for use as a pharmaceutical product.

10. The compound for use according to claim 9 in the treatment of one or more HDAC6-mediated diseases selected from the group consisting of chemotherapy-related cognitive impairment (CRCI), graft rejection, GVHD, myositis, lymphocyte dysfunction-related diseases, multiple myeloma, non-Hodgkin lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer and neurodegenerative diseases, and ocular diseases.

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