Difluoro and trifluoroacetyl hydrazide as selective HDAC6 inhibitors

Abstract

The present invention relates to an acyl hydrazide obtained in situ by enzymatic hydrolysis of the parent prodrug 2-(difluoromethyl)-or 2-(trifluoromethyl)-1, 3, 4-oxadiazole in the histone deacetylase 6 (HDAC6). # imgabs0 #

Description

Technical Field

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

[0002] Zinc-dependent histone deacetylases (HDACs) are a family of 11 evolutionarily related hydrolases that catalyze the removal of acetyl or myristoyl residues from histones, non-histone proteins, and polyamines (Biochemistry. 57, 3105–3114; Mol. Cell. Proteomics. 21, 100193; J. Genet. Genomics. 44, 243–250). Given the involvement of HDACs in a variety of diseases, the pharmaceutical industry has been developing HDAC inhibitors (HDACis) for nearly two decades, an effort that has resulted in five molecules approved for the treatment of cancer (Br. J. Clin. Pharmacol. 87, 4577–4597). Unfortunately, the therapeutic benefits of HDACs have been limited by side effects due to the poor selectivity of these first-generation molecules, which inhibit several to all zinc-dependent HDAC family members, thereby also affecting key physiological functions.

[0003] HDAC6 stands out among the 11 human isoforms of zinc-dependent HDACs and is the only isoform with two homologous tandem catalytic domains (CD1 and CD2) and a zinc finger ubiquitin binding domain. In addition, HDAC6 is mainly located in the cytoplasm, and its main substrates are not histones, but various non-histone proteins, such as α-tubulin, Foxp3, Hsp90, β-catenin, cortactin, and peroxidase. Interestingly, HDAC6 KO mice are viable and fertile and do not show obvious physiological dysfunction (Mol. Cell. Biol. 28, 1688–1701). In addition, selective HDAC6 inhibition has been shown to be well tolerated in preclinical species and human clinical trials (Oncologist. 26, 184-e366).

[0004] Since HDAC6 plays an important role in the ubiquitin-proteasome and aggresome pathways, regulating immune responses, the development of neuropathy and Alzheimer's disease, there is great interest in identifying highly specific HDAC6 inhibitors (J. Med. Chem. 64, 1362–1391).

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

[0006] WO2022 / 029041, WO2022 / 013728, WO2021 / 127643, WO2020 / 212479 and WO2022 / 049496 disclose oxadiazole-based HDAC6 inhibitors as potential alternatives to hydroxamates, but their mechanism of action is still unclear. Summary of the invention

[0007] The present inventors identified difluoro and trifluoroacetyl hydrazides as tight binding inhibitors of HDAC6 that, when formed in situ by appropriately designed prodrugs, form durable complexes with the enzyme.

[0008] The inventors surprisingly found that difluoromethyl-1,3,4-oxadiazole (DFMO) and trifluoromethyl-1,3,4-oxadiazole (TFMO) are substrates of HDAC6, which can hydrolyze them into the corresponding difluoro or trifluoroacetyl hydrazides. Therefore, DFMO and TFMO are selected as prodrugs for in situ generation of acyl hydrazides.

[0009] The inventors used pre-steady state kinetics, flash chromatography and mass spectrometry to dissect the inhibitory mechanism of DFMO and TFMO containing compounds, indicating that HDAC6-catalyzed DFMO and TFMO ring hydration / opening leads to the enzyme forming a tight and durable complex with the corresponding acyl hydrazide. Once separated from the enzyme, the hydrated inhibitor may eventually re-bind with HDAC6, thereby further hydrolyzing to produce a hydrazide complex with lower affinity.

[0010] The inventors were also able to confirm this mechanism by solving the X-ray crystal structure of HDAC6-CD2 bound to the DFMO compound. The inventors surprisingly found that the electron density in the active site was incompatible with the structure of the parent prodrug, but could fully adapt to the structure of the hydrazide (i.e., the final hydrolysis product).

[0011] The acyl hydrazide-containing compounds that are the subject of the present invention are inactive if directly applied to enzymes or cells, but when formed in situ from the DFMO and TFMO prodrugs, they possess impressive potency and selectivity.

[0012] Focusing on prodrugs (HDAC6 inhibitors containing DFMO and TFMO), the inventors have been able to identify molecules that are more than 10,000-fold selective for HDAC6 over all other HDACs, and have excellent drug-like properties, safety profiles, and pharmacological activity in vitro and in vivo.

[0013] definition

[0014] Unless otherwise defined, all technical terms, symbols and other scientific terms used herein are intended to have the meanings commonly understood by those skilled in the art to which the present disclosure belongs. In some cases, for the sake of clarity and / or ease of reference, terms with commonly understood meanings are defined herein; therefore, the inclusion of such definitions herein should not be construed as representing a substantial difference from the general understanding of the art.

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

[0016] In this article, the term “C 1 -C 6 "Alkyl" refers to a branched or straight chain hydrocarbon containing 1 to 6 carbon atoms. 1 -C 6 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl.

[0017] In this context, the term "aryl" refers to monocarbocyclic and polycarbocyclic aromatic ring systems (i), wherein the individual carbocyclic rings in the polycarbocyclic system may be fused or connected to each other via single bonds. Suitable aryl groups include, but are not limited to, phenyl, naphthyl and biphenyl.

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

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

[0020] As used herein, the term "thioalkoxy" refers to an S-alkyl group, wherein "alkyl" is as defined above. Preferred thioalkoxy groups are thioethoxy (-SEt) or thiomethoxy (-SMe), even more preferably thiomethoxy. In another embodiment, thioalkoxy refers to an alkyl group in which one of the non-terminal hydrocarbon units of the alkyl chain is substituted by a sulfur atom.

[0021] In this document, the term "halogen" refers to halogen substitution, that is, any of the above alkyl, alkoxy, thioalkoxy groups can be completely or partially substituted by a halogen atom. Preferably, the halogen atom is 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 "aralkyl" refers to an aryl group, as defined herein, attached to an alkyl group, as defined herein. An example of an aralkyl group is benzyl.

[0024] In this context, the term "deuterated" refers to deuterium substitution, in other words, hydrogen atoms may be partially or completely replaced by deuterium.

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

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

[0027] In the present context, the term "substituted" refers to mono- or poly-substitution with defined (or undefined) substituents, provided that such mono- or poly-substitution is chemically permitted.

[0028] In this article, the term "physiologically acceptable excipient" refers to a substance that does not have any pharmacological effect itself and does not produce adverse reactions when administered to mammals, preferably humans. Physiologically acceptable excipients are well known in the art, for example, disclosed in the Handbook of Pharmaceutical Excipients, 2009, the sixth edition, which is incorporated herein by reference.

[0029] As used herein, the term "pharmaceutically acceptable salt or derivative thereof" refers to salts or derivatives that have the biological effectiveness and properties of the salt-forming or derived compound and do not produce adverse reactions when administered to a mammal (preferably a human). Pharmaceutically acceptable salts may be inorganic or organic salts; Examples of pharmaceutically acceptable salts include, but are not limited to, carbonates, hydrochlorides, hydrobromides, sulfates, bisulfates, citrates, maleates, fumarates, trifluoroacetates, 2-naphthalenesulfonates, and p-toluenesulfonates. More information about 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” should be construed as open terms (meaning “including, but not limited to,”) and should be considered support for terms such as “essentially consist of,” “essentially consisting of,” “consist of,” or “consisting of.”

[0031] The terms "essentially consist of" and "essentially consisting of" should be understood as semi-closed terms, meaning that other ingredients that affect the novel characteristics of the invention are not included (and therefore may include optional excipients).

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

[0033] The term "isomer" refers to stereoisomers (or spatial isomers), ie, diastereomers and enantiomers. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 A hypothesized mechanism showing DFMO ring opening and the formation of related acyl hydrazides and acyl hydrazides.

[0035] Figure 2 Isolation of a persistent / tight complex (Example 10) was shown. DETAILED DESCRIPTION

[0036] Difluoromethyl-1,3,4-oxadiazole (DFMO) and trifluoromethyl-1,3,4-oxadiazole (TFMO) were found to be substrates of HDAC6, which was able to hydrolyze them into the corresponding difluoro- or trifluoroacetyl hydrazides.

[0037] Kinetic studies were performed, in which some DFMO and TFMO inhibitors were incubated with HDAC6 and the medium was analyzed by LC-MS. In fact, the disappearance of the DFMO compound and the formation of the corresponding acyl hydrazide (used as a standard for mass spectrometry studies) were observed. The formation of hydrazide was also observed using extreme non-physiological conditions in terms of inhibitor and protein concentrations. The same DFMO and TFMO inhibitors dissolved in the same buffer medium for the same time in the absence of enzyme did not show any degradation events. These experiments confirm that DFMO and TFMO can be considered as substrates of HDAC6.

[0038] Due to the interest in the slow binding / slow release kinetics associated with DFMO and TFMO compounds, spin column chromatography associated with LC-HRMS was used to identify tightly bound species that form persistent inhibitor-HDAC6 complexes (see Example 10). The major species that co-eluted with zHDAC6-CD2 when incubated with DFMO or TFMO compounds was the corresponding acyl hydrazide. This hydrate form and the corresponding hydrazide were also detected in fractions containing free compound that was not bound to the enzyme. Interestingly, the DFMO and TFMO parent compounds did not co-elute with the enzyme.

[0039] Interestingly, direct incubation of the difluoromethyl acyl hydrazide with the enzyme did not induce enzyme inhibition, suggesting that this species is active only when formed in situ. The present data instead suggest that the high affinity species is the acyl hydrazide formed in situ.

[0040] Crystallographic data combined with molecular modeling support the hypothesized mechanism. Hydrazide was found upon co-crystallization of the DFMO-containing compound. The hypothesized hydrazide could be formed by two subsequent reactions. After the inhibitor enters the catalytic pocket, the active site Zn cation could enhance the CHF binding directly to the DFMO moiety. 2 Group-bound sp 2 The electrophilic behavior of the carbon atom allows nucleophilic attack by a water molecule, and its presence in the coordination sphere of the metal cation is supported by model studies. The hydrated intermediate can further react in a ring-opening reaction to produce the acyl hydrazide ( Figure 1, step 1). To explain the hydrazide detected in the crystal structure, one must assume that upon release of the acyl hydrazide from the enzyme active site, an additional hydrolysis reaction occurs, which may be enzyme-catalyzed or may occur in solution. If the second hydrolysis reaction is indeed enzyme-catalyzed, the entry of a second water molecule is required to restore the zinc coordination sphere. The metal cation can then activate the acyl carbonyl group, allowing a typical deacetylation followed by release of the hydrazide and difluoroacetic acid ( Figure 1 , step 2).

[0041] The present inventors surprisingly found that DFMO inhibitors are hydrolyzed in the presence of HDAC6 to produce the corresponding difluoromethyl acyl hydrazide, which is a truly active and selective HDAC6 inhibitor.

[0042] According to a first aspect, the present invention relates to compounds of formula (I), pharmaceutically acceptable salts and isomers thereof:

[0043]

[0044] in:

[0045] W=H or F

[0046] G is a 5-membered heteroaromatic ring consisting of carbon atoms and 1 to 4 heteroatoms selected from N, O, S and Se, which is optionally replaced by C 1 -C 3 Alkyl, alkoxy or thioalkoxy, halogenated derivatives thereof, or halogen, or hydroxyl substitution;

[0047] Alternatively, G is of formula (Ia):

[0048]

[0049] wherein X, X', Y and Y' are independently selected from CH, N, CF or CCl;

[0050] Z=-CD 2 -、-CF 2 -、-CHR 2 -, -NH-, -S-;

[0051] R 2 =H, halogen, C 1 -C 6 Alkyl or C 3 -C 6 Cycloalkyl, which is unsubstituted or substituted with:

[0052] Hydroxyl, carbonyl, C 1 -C 3 Alkoxy, aryloxy or thioalkoxy, or halogenated derivatives thereof;

[0053] ·halogen;

[0054] Primary, secondary or tertiary amines, which are C 1 -C 6 Alkyl, C 3 -C 6 Substituted with cycloalkyl or its halogenated derivatives;

[0055] · phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or replaced by C 1 -C 3 Alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen substitution; or

[0056] The following substructures or their halogenated derivatives:

[0057]

[0058] L does not exist, 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 its halogenated derivatives;

[0059] wherein m and o are each independently 0, 1 or 2; or

[0060] L is selected from the following substructures (IIa)-(IIf) and their halogenated derivatives:

[0061]

[0062] Wherein a, b, c and d are independently 0, 1, 2, or 3, and a and b cannot be 0 at the same time;

[0063] Q is CH 2 NR 4 or O;

[0064]

[0065] Where n is 0, 1, or 2;

[0066] Y" does not exist, C1 -C 2 Alkylene, or selected from the following substructures and halogenated derivatives thereof:

[0067]

[0068] wherein a, b and Q are as defined above;

[0069] R 4 =H、C 1 -C 4 Alkyl, the C 1 -C 4 Alkyl is unsubstituted or substituted with:

[0070] ·halogen

[0071] · phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or replaced by C 1 -C 3 Alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen substitution;

[0072] P is an unsubstituted or substituted, aromatic or non-aromatic 5- to 10-membered heterocyclic ring, wherein the ring consists of carbon atoms and one or more heteroatoms selected from N, O and S;

[0073] R 1 is absent, halogen, unsubstituted or substituted -C(=O)C 1 -C 4 Alkyl, C 1 -C 4 Alkyl, C 4 -C 6 Cycloalkenyl, C 6 -C 12 aryl, or a 5- to 9-membered heteroaryl group containing at least one heteroatom selected from N, O and S;

[0074] Provided that the following compounds are not included:

[0075] -3-[2-[[4-[[(2,2-difluoroacetyl)amino]carbamoyl]phenyl]methyl]tetrazol-5-yl]benzoic acid

[0076] -2-((5-(6-aminopyridin-3-yl)-2H-tetrazol-2-yl)methyl)-N'-(2,2-difluoroacetyl)pyrimidine-5-carbohydrazide

[0077] 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

[0078] -N'-(2,2-difluoroacetyl)-4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolin-1-yl)methyl)-3-fluorobenzohydrazide

[0079] -4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolin-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide

[0080] -N'-(2,2-difluoroacetyl)-4-((2,5-dioxo-3-phenylimidazolin-1-yl)methyl)-3-fluorobenzohydrazide

[0081] -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)nicotinylhydrazide

[0082] -4-((1H-imidazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide

[0083] -N'-(2,2-difluoroacetyl)-4-(pyrimidin-2-ylamino)benzohydrazide

[0084] -4-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyrimidin-2-yl)amino)-4-phenylpiperidine-1-carboxylic acid benzyl ester

[0085] -4-((1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)-N'-(2,2,2-trifluoroacetyl)benzohydrazide.

[0086] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts or isomers thereof, wherein R 2 H or C 1 -C 3 Alkyl, the C 1 -C 3 Alkyl is unsubstituted or substituted with:

[0087] ·C 1 -C 3 Alkyl or C 3 -C 6 Cycloalkyl

[0088] Hydroxyl, carbonyl, C 1 -C 2 Alkoxy, aryloxy or thioalkoxy, or halogenated derivatives thereof;

[0089] ·halogen;

[0090] Primary, secondary or tertiary amines, which are C 1 -C 3 Alkyl, C 3 -C 6 Substituted with cycloalkyl or its halogenated derivatives;

[0091] · phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or replaced by C 1 -C 3 Alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen substitution; or

[0092] The following substructures or their halogenated derivatives:

[0093]

[0094] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts or isomers thereof, wherein P is

[0095]

[0096] in:

[0097] A=C, N, O, S;

[0098] B=C、N;

[0099] D=C, N, O;

[0100] E=C, N, O;

[0101] M = C, N;

[0102] R 5 and R 6 are independently -H, halogen, =O, C 1 -C 6 Alkyl, alkoxy or thioalkoxy, C 3 -C 6 Cycloalkyl, or a halogenated derivative thereof, which is optionally substituted by a carbonyl or carboxyl group, or R 5 and R 6 Independently selected from the following substructures:

[0103]

[0104] R 3 is absent, -H, optionally replaced by -OH or -N(C 1 -C 5 alkyl) 2 Substituted C 1 -C 6 Alkyl, -LR 1Or from the following substructures:

[0105]

[0106] When R 3 =-LR 1 When , substitution on M does not exist;

[0107] Ra and Rb are independently H, halogen, C 1 -C 6 Alkyl, alkoxy or thioalkoxy, C 3 -C 6 Cycloalkyl or its halogenated derivatives.

[0108] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein P is selected from the following substructures:

[0109]

[0110]

[0111] wherein Ra and Rb are as defined in claim 3, or are -LR 1 ;

[0112] Rc is H, halogen, C 1 -C 6 Alkyl, alkoxy or thioalkoxy, C 3 -C 6 Cycloalkyl, or its halogenated derivative, or -NH 2 .

[0113] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 Select from the following substructures:

[0114]

[0115]

[0116] Where R 6 and R 7 Independently selected from: -H, -D, -OH, C 1 -C 4 Alkyl, alkoxy or thioalkoxy, C 3 -C 6 Cycloalkyl or its halogenated derivatives, halogen, -(CH 2 ) a NR'R", -NHR 8 , -C(=O)OR', -C(=O)R 9 、-C(=NH)R9 、-NO 2 、-CN、-Ph、-SO 2 -NR'R", =O, =NR 8 、-SO 2 -C 1 -C 4 Alkyl, or

[0117] R 6 and R 7 Independently selected from the following substructures:

[0118]

[0119]

[0120] R 8 =-H, -D, -OH, C 1 -C 6 Alkyl, C 3 -C 6 Cycloalkyl or its halogenated derivatives, -(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 Alkyl, or R 8 Select from the following substructures:

[0121]

[0122] R 9 =-NR'R", C 1 -C 4 Alkyl, or a halogenated derivative thereof, or selected from the following substructures:

[0123]

[0124] R 10 and R 11 Independently selected from -H, C 1 -C 4 Alkyl, C 3 -C 6 Cycloalkyl or its halogenated derivatives, -OR', -C(=O)OR', -C(=O)R', or halogen;

[0125] Q1 CH 2 , O, S, NR 8 ;

[0126] Q 2 and Q 3 Independently CR'R", CF 2 , O, S, NR 8 ;

[0127] R' and R" are independently -H, C 1 -C 4 Alkyl, C 3 -C 6 Cycloalkyl or its halogenated derivatives;

[0128] a, b, c and R 8 As defined above.

[0129] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 Select from the following substructures:

[0130]

[0131]

[0132] Where R 6 and R 7 Independently selected from: -H, -D, -OH, C 1 -C 4 Alkyl, alkoxy or thioalkoxy, C 3 -C 6 Cycloalkyl or its halogenated derivatives, 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 Alkyl, or independently the following substructures:

[0133]

[0134]

[0135] R 8 =-H, -D, -OH, C 1 -C 6 Alkyl, C3 -C 6 Cycloalkyl or its halogenated derivatives, -(CH 2 ) a NR'R", -C(=O)OR', -C(=O)R 9 、-C(=NH)R 9 、-SO 2 -C 1 -C 4 Alkyl, or R 8 Select from the following substructures:

[0136]

[0137] R 9 =-NR'R", C 1 -C 4 Alkyl, or a halogenated derivative thereof, or selected from the following substructures:

[0138]

[0139] R 10 and R 11 Independently selected from -H, C 1 -C 4 Alkyl, C 3 -C 6 Cycloalkyl or its halogenated derivatives, -OR', -C(=O)OR', -C(=O)R', or halogen;

[0140] Q 1 CH 2 , O, S, NR 8 ;

[0141] Q 2 and Q 3 Independently CR'R", CF 2 , O, S, NR 8 ;

[0142] R' and R" are independently -H, C 1 -C 4 Alkyl, C 3 -C 6 Cycloalkyl or its halogenated derivatives;

[0143] a, b, c and R 8 As defined above.

[0144] Another preferred class of compounds comprises compounds of formula (I), pharmaceutically acceptable salts and isomers thereof, wherein R 1 Select from the following substructures:

[0145]

[0146] Where a and b are independently 0, 1, 2, or 3, and a and b cannot be 0 at the same time;

[0147] Z 1 CH 2 , NH, or O;

[0148] Where R 1 At least one H of 2 ) n -Q4-Q5-Rd substitution;

[0149] n = 0, 1 or 2;

[0150] Q4=does not exist, -SO 2 -、-NH-、-N(C 1 -C 5 alkyl)-, -NHC(=O)-, -N(C 1 -C 5 alkyl)C(═O)- or –C(═O)-;

[0151] Q5=does not exist, C 1 -C 5 Alkylene, -NH-, -(C 1 -C 5 Alkylene)-NH-C(=O)- or –N(C 1 -C 5 alkyl);

[0152] Rd=-OH、C 1 -C 5 Alkyl, C 1 -C 5 Haloalkyl, -NR'R", C 1 -C 5 alkoxy, 5- or 6-membered heteroaryl containing 1 to 3 N,

[0153]

[0154] wherein e and f are independently 1 or 2;

[0155] M1 is CH 2 , O, NH or SO 2 , M 2 is CH or N, and wherein at least one H of Rd is optionally replaced by OH, halogen, C 1 -C 5 Alkyl, C 1 -C 5 Haloalkyl, -C(=O)-(C 1 -C 5alkyl), -C(=O)O(C 1 -C 5 alkyl), -NH-C(=O)-O(C 1 -C 5 Alkyl), -NR'R" substituted,

[0156]

[0157] Where g and h are independently 0 or 1, but cannot be 0 at the same time;

[0158] M 4 CH 2 , O, NH, and at least one H of M4 is optionally replaced by halogen, C 1 -C 5 Alkyl, C 3 -C 6 Cycloalkyl or -C(=O)-O(C 1 -C 5 Alkyl) substituted;

[0159] R' and R" are independently -H or C 1 -C 4 alkyl.

[0160] The following compounds of formula (I) are preferred:

[0161] -N'-(2,2-difluoroacetyl)-4-((4-(4-((4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)-1H-

[0162] 1,2,3-Triazol-1-yl)methyl)benzoylhydrazide (Compound 1)

[0163] -4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (Compound 2)

[0164] -N'-(2,2-difluoroacetyl)-6-((4-(p-methylphenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0165] (Compound 8)

[0166] -N'-(2,2-difluoroacetyl)-6-((4-(m-tolyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0167] (Compound 9)

[0168] -N-(3-(1-(4-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)-2-fluorobenzyl)-1H-1,2,3-triazol-4-yl)

[0169] Phenyl)acetamide (Compound 12)

[0170] -N'-(2,2-difluoroacetyl)-6-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide (Compound 13)

[0171] -N'-(2,2-difluoroacetyl)-6-((4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0172] (Compound 14)

[0173] -N'-(2,2-difluoroacetyl)-5-fluoro-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 15)

[0174] -N'-(2,2-difluoroacetyl)-6-((5-phenyl-2H-tetrazol-2-yl)methyl)nicotinylhydrazide (Compound 16)

[0175] -N'-(2,2-difluoroacetyl)-6-((5-(thiophen-2-yl)-2H-tetrazolyl-2-yl)methyl)nicotinylhydrazide (Compound 17)

[0176] -N'-(2,2-difluoroacetyl)-6-((4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0177] (Compound 18)

[0178] -N'-(2,2-difluoroacetyl)-6-((4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0179] (Compound 19)

[0180] -N'-(2,2-difluoroacetyl)-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide

[0181] (Compound 20)

[0182] -6-((4-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-di

[0183] (Fluoroacetyl)nicotinic acid hydrazide (Compound 27)

[0184] -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)

[0185] -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)

[0186] -4-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (Compound 32)

[0187] -6-((4-(1H-indazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinic acid hydrazide (Compound 34)

[0188] -4-((4-(1H-indol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (Compound 36)

[0189] -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)

[0190] -6-((4-(2-aminobenzo[d]thiazol-6-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinic acid hydrazide (Compound 38)

[0191] -6-(1-(4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethyl)-N'-(2,2-difluoroacetyl)nicotinic acid hydrazide (Compound 39)

[0192] -6-((4-(1H-indazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-5-fluoronicotinic acid hydrazide (Compound 40)

[0193] -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)

[0194] -4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (Compound 43)

[0195] -4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-2-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide (Compound 44)

[0196] -4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide (Compound 45)

[0197] -4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3,5-difluorobenzohydrazide (Compound 46)

[0198] -4-((4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-3-fluorobenzohydrazide (Compound 47)

[0199] -5-[[4-(2-amino-1,3-benzothiazol-6-yl)triazol-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (Compound 48)

[0200] -5-[[4-(6-aminopyridin-3-yl)triazol-1-yl]methyl]-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (Compound 49).

[0201]

[0202]

[0203] The compounds of the invention may contain one or more chiral centers (asymmetric carbon atoms) and they may therefore exist in the form of enantiomers and / or diastereoisomers.

[0204] All possible optical isomers, whether existing alone or mixed with each other, belong to the scope of the present invention.

[0205] A second object of the present invention is the use of the above-mentioned compound of formula (I) as a medicine.

[0206] The third object of the present invention is the use of the above compounds for preventing and / or treating diseases or disorders regulated by HDAC6.

[0207] The compounds of the present invention are preferably useful for treating hereditary peripheral neuropathy, for example, but not limited to Charcot-Marie-Tooth disease induced by drugs (chemotherapy or antibiotics, such as metronidazole and fluoroquinolones) and peripheral neuropathy caused by systemic diseases such as diabetes or leprosy, or generally for treating peripheral neuropathy associated with severe axonal transport defects. The compounds of the present invention can also be used for treating chemotherapy-related cognitive impairment (CRCI).

[0208] The compounds of the invention are preferably used to treat transplant rejection, GVHD, myositis, diseases associated with abnormal lymphocyte function, multiple myeloma, non-Hodgkin's lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer and neurodegenerative diseases, eye diseases (e.g. uveitis).

[0209] General synthetic route

[0210] The compounds described in this invention can be prepared by methods known to those skilled in the art.

[0211] All starting materials, reagents, acids, bases, solvents and catalysts used in the synthesis of the described compounds are commercially available.

[0212] The reaction progress was monitored by TLC, HPLC, UPLC or HPLC-MS analysis.

[0213] Acyl hydrazides are obtained in situ by enzymatic hydrolysis of the parent prodrug 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole in the presence of histone deacetylase 6 (HDAC6).

[0214] Similarly, although less efficiently, 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazoles can be hydrolyzed under acidic aqueous (TFA) or basic aqueous (LiOH or methanolic ammonia) conditions to yield difluoro- and trifluoroacetyl hydrazides, respectively.

[0215] As described in the literature (Marchini, M. et al., 2021, “2-(4-((5-(benzo[b]thiophen-3-yl)-1H-tetrazol-1-yl)methyl)phenyl)-5-(difluoromethyl)-1,3,4-oxadiazoledelivativesand similar compounds as selective inhibitors of histone deacetylase 6(HDAC6) for use in treating egperipheral neuropathy", WO2022029041; Lee, JK et al., "Novel compounds as histone deacetylase 6inhibitor, and pharmaceutical composition comprising the same", WO2022013728; Vara Salazar, YI et al., 2020, "1,3,4-oxadiazoledelivatives as histone deacetylase inhibitors",WO2020212479; Yates, C., 2018, "Metalloenzyme inhibitor compounds" WO2018165520), 2-(difluoromethyl)- and 2-(trifluoromethyl)-1,3,4-oxadiazole moieties were synthesized. In most cases, the corresponding hydrazides were treated with excess difluoroacetic anhydride or trifluoroacetic anhydride, which acted as both an acylating agent and a dehydrating agent (Lee, J. et al., 2017; "1,3,4-Oxadiazole sulfonamide derivatives as histone deacetylase 6 inhibitors and their pharmaceutical composition and preparation"; WO2017018805). In some cases, the 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole moiety is prepared starting from the corresponding tetrazole, which is converted to 2-(difluoromethyl)- 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).In addition, acyl hydrazides can be converted to oxadiazoles in the presence of other dehydrating reagents such as Burgess reagent (Lee, J. et al., "1,3,4-Oxadiazole derivative compounds as histone deacetylase 6 inhibitor, and the pharmaceutical composition comprising the same", WO201723133) or p-toluenesulfonyl chloride (Ito M. et al., 2019, "Heterocylic compound", WO2019027054).

[0216] Other methods for preparing acyl hydrazides include acylation of hydrazides in the presence of nearly stoichiometric amounts of difluoroacetic anhydride or trifluoroacetic anhydride (Lee, J. et al., 2017; "1,3,4-Oxadiazole sulfonamide derivatives ashistone deacetylase 6 inhibitors and their pharmaceutical composition and preparation"; WO2017018805), or using common amide coupling procedures and reagents, i.e., by using activated esters of HATU or HOBt / EDC hydrochloride (Ito M. et al., 2019, "Heterocylic compound", WO2019027054), or by acyl chlorides (Shchekotikhin et al. Rus. J. Org. Chem. 2007, 43 (11), 1686–1695), functionalizing carboxylic acids with difluoroacetyl hydrazides or trifluoroacetyl hydrazides. All synthetic routes are summarized in Scheme 1.

[0217] Scheme 1 – Difluoro- or trifluoro-acetyl hydrazide and prodrug 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4- Synthesis of the oxadiazole moiety a

[0218]

[0219] a Reagents and conditions: (a) DFAA or TFAA; (b) DFAA or TFAA; (c) TsCl or Burgess reagent; (d) TFA, water; (e) NH 3 (7M MeOH solution), water or LiOH, THF / water; (f) HDAC6; (g) HATU or HOBt, EDC; (h) SOCl 2 ; (i) difluoroacetyl hydrazide or trifluoroacetyl hydrazide.

[0220] The acyl hydrazide target of the present invention is obtained by degradation of the corresponding 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole. Unless otherwise stated, the synthesis of the parent compound is as described in the literature.

[0221] 1,2,3-triazole compounds not described elsewhere rely on common intermediates of 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 is described as follows (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 delivatives and similar compounds as selective inhibitors of histone deacetylase 6 (HDAC6) for use in treating egperipheral neuropathy”, WO2022029041). Treatment of the methyl or ethyl ester with hydrazine affords the corresponding hydrazide, which is converted to the difluoromethyl- and trifluoromethyl-1,3,4-oxadiazole moieties as described previously. Bromination at the benzylic position using N-bromosuccinimide and azobisisobutyronitrile (AIBN) or dibenzoyl peroxide (BPO) as catalysts then affords the bromomethyl intermediate (Scheme 2).

[0222] Scheme 2 – 2-(4-(bromomethyl)aryl)-5-(difluoromethyl)-1,3,4-oxadiazole or 2-(4-(bromomethyl)aryl) Synthesis of conventional intermediates of 5-(trifluoromethyl)-1,3,4-oxadiazole a

[0223]

[0224] a Reagents and conditions: (a) N 2 H 4 ·H 2 O, MeOH, reflux; (b) DFAA or TFAA, DMF, rt; (c) NBS, AIBN or BPO, CCl 4 , 80℃.

[0225] In the presence of sodium azide, the bromide was converted to the azide, followed by a one-pot CuAAC click reaction with an appropriate alkyne to afford 1,2,3-triazole-containing products, either 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole.

[0226] Scheme 3 – Synthesis of 1,2,3-triazole compounds a

[0227]

[0228] a Reagents and conditions: (a) NaN 3 , DMF, 1h, rt; (b) CuSO 4 ·5H 2 O, sodium ascorbate, DMF:H 2 O(1:1), 16h, 40C; (c) Pd(dppf)Cl 2 , CuI, Et 3 N, DMF; (d) TBAF, DMF or K 2 CO 3 , MeOH; (e) K 2 CO 3 , MeOH, and then Ohira-Bestmann reagent.

[0229] Non-commercial aryl alkynes are prepared by Sonogashira coupling using [1,1'-bis(diphenylphosphino)ferrocene]palladium(II) chloride (Pd(dppf)Cl2) and copper(I) iodide as catalysts to react appropriate aryl halides with ethynyl(trimethyl)silane in the presence of triethylamine (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 ( M.,Carreira,EMangew.Chem.Int.Ed.2020,59(3),1192–1196).

[0230] Compounds with a tetrazole core were synthesized by nucleophilic substitution reactions, using potassium carbonate as a base, to react the conventional intermediate 2-(4-(bromomethyl)aryl)-5-(difluoromethyl)-1,3,4-oxadiazole with the appropriate substituted tetrazole in DMF at room temperature overnight (see Scheme 4). The reaction provided a mixture of regioisomeric products that could be efficiently separated by chromatography. The 2,5-disubstituted tetrazole was generally the most abundant of the two regioisomers. The conventional intermediate methyl bromide derivative was synthesized as described for compounds with a 1,2,3-triazole core (Scheme 2).

[0231] Scheme 4 – Synthesis of compounds with tetrazole as the central skeleton a

[0232]

[0233] a Reagents and conditions: (a) K 2 CO 3 , DMF, 16h, rt; (b) NaN 3 , NH 4 Cl.

[0234] Most of the substituted tetrazoles used are commercially available. Non-commercially available building blocks were synthesized from the corresponding nitriles by reaction with excess sodium azide in the presence of ammonium chloride.

[0235] The following examples are intended to further illustrate the present invention, but the present invention is not limited thereto.

[0236] Example 1. Synthesis of 2-(4-(bromomethyl)-3-chlorophenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (Intermediate C)

[0237] Step 1

[0238]

[0239] Methyl 3-chloro-4-methylbenzoate (10 g, 54.1 mmol, 1 eq.) was dissolved in MeOH and hydrazine hydrate (5 eq.) was added. After conversion to hydrazide by UPLC, the mixture was refluxed under stirring for 5 hours. The desired intermediate precipitated. 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 eq.) was added dropwise. The resulting mixture was stirred at room temperature overnight and complete conversion was observed. The reaction mixture was heated to 4 ° C with saturated NaHCO 3 The reaction mixture was diluted with aqueous solution. The product precipitated as a solid which was collected by filtration, rinsed with water and dried under vacuum (8.47 g, 34.6 mmol, 64% yield).

[0240] Step 2

[0241]

[0242] Under argon, a mixture of 2-(3-chloro-4-methylphenyl)-5-(difluoromethyl)-1,3,4-oxadiazole (3.44 g, 14 mmol, 1 eq.) and N-bromosuccinimide (1.1 eq.) was stirred in 70 mL of carbon tetrachloride until completely dissolved. AIBN (0.015 eq.) was then added to the reaction mixture and stirred at 70 °C overnight. LCMS was performed after conversion. The mixture was allowed to reach room temperature, diluted with DCM, and washed with saturated NaHCO successively. 3The organic layer was separated and washed with MgSO 4 Dry, filter and concentrate under reduced pressure.The residue was purified by flash chromatography (silica gel, hexanes / EtOAc, 0-15%) to give the desired product as a white solid (2.9 g, 8.9 mmol, 63% yield).

[0243] The following compounds were prepared according to the same procedure:

[0244]

[0245] The synthesis of the following similar intermediates has been described elsewhere (Marchini, M. et al., 2022, WO2022029041, Example 1):

[0246]

[0247] Example 2. General Procedure A for the Conversion of 2-(Difluoromethyl)- or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoro- or Trifluoroacetylhydrazide

[0248]

[0249] Ammonia (7M in MeOH, 10 eq) was added to a DMSO solution containing the parent 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole compound (0.16 mmol, 1 eq, 0.2 M). Water (excess, 1.5-3 mL / mmol) was then added to the resulting mixture, which was heated to 30-70° C. and stirred for 2 days to achieve complete conversion. Ammonia and water were removed under vacuum and the residue was purified by preparative HPLC.

[0250] The following compounds were prepared following General Procedure A. The syntheses of the parent compounds were described elsewhere:

[0251]

[0252] Example 3. General Procedure B for the Conversion of 2-(Difluoromethyl)- or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoro- or Trifluoroacetylhydrazide

[0253]

[0254] The compound containing 2-(difluoromethyl)- or 2-(trifluoromethyl)-1,3,4-oxadiazole (1 eq., 0.07 M) was dissolved in DMSO / water 95:5. TFA (60 eq.) was added to the mixture and stirred at room temperature for 3 days to achieve complete conversion. The reaction mixture was concentrated and the residue was purified by preparative HPLC.

[0255] The following compounds were prepared following General Procedure B. The syntheses of the parent compounds were described elsewhere:

[0256]

[0257] Example 4. General Procedure for the CuAAC Click Reaction and One-Pot Method for the Conversion of 2-(Difluoromethyl)- or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoro- or Trifluoroacetyl Hydrazide C

[0258]

[0259] Reagent A (Intermediate AJ Example 1, 0.26mmol, 1 equivalent) was dissolved in 1mL DMSO. Sodium azide (0.26mmol, 1 equivalent) was added and the reaction mixture was stirred at room temperature for 20 minutes. Alkynes (0.26mmol, 1 equivalent) and copper sulfate (II) (1M aqueous solution, 0.2 equivalent) and (+)-L-sodium ascorbate (0.5M aqueous solution, 0.4 equivalent) solutions were added successively, and the reaction mixture was stirred at room temperature to achieve complete consumption of the starting reagent, as monitored by UPLC or / and TLC (2-12 hours). 0.5mL 7M ammonia in MeOH solution and 0.5mL 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 may also be separated.

[0260] The following compounds were prepared following General Procedure C. When not commercially available, the synthesis of their alkyne building blocks was described elsewhere:

[0261]

[0262]

[0263] Example 5. General Procedure D for the Tetrazolium Nucleophilic Substitution Reaction and One-Pot Method for the Conversion of 2-(Difluoromethyl)- or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoro- or Trifluoroacetyl Hydrazide

[0264]

[0265] Tetrazole (0.34mmol, 1 equivalent) was dissolved in 2mL DMF. Suitable reagent (Intermediate AJ, Example 1, 1 equivalent) and potassium carbonate (2 equivalents) were added. After stirring the reaction mixture for 3 hours (or until complete conversion), methanolic ammonia (7M, 5 equivalents) and excess water were added. The resulting mixture was stirred at 50°C overnight, concentrated and submitted to preparative HPLC.

[0266] The following compounds were prepared according to General Procedure D. The synthesis of prodrugs was also described elsewhere:

[0267]

[0268] 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)

[0269] Step A

[0270]

[0271] A solution of methyl 3-fluoro-4-formylbenzoate (1 g, 5.49 mmol, 1 eq) in THF (20 mL) was cooled to -70°C. Methylmagnesium bromide (1 eq) was added dropwise and the resulting mixture was stirred at -70°C for 20 minutes. 4 The reaction was quenched with aqueous Cl solution and extracted with MTBE. The organic layers were combined and washed with Na 2 SO 4 Dry, filter and concentrate.The crude product was purified by flash chromatography (silica gel, hexanes / EtOAc 0-30%) to give the desired product (1.09 g; 5.49 mmol, 100% yield).

[0272] Step B

[0273]

[0274] Methyl 3-fluoro-4-(1-hydroxyethyl)benzoate (1.09 g; 5.49 mmol, 1 eq) was dissolved in 20 mL of DCM. Trifluoroethylamine (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 EtOAc, washed with brine, and washed with Na 2 SO 4 Dry, filter and concentrate.

[0275] The crude intermediate mesylate was dissolved in 10 mL DMSO and sodium azide (1 eq.) was added. The resulting mixture was stirred at room temperature for 45 minutes, then diluted with MTBE and washed with brine. The organic layer was washed with Na 2 SO 4Dry, filter and concentrate. The product thus obtained was used in the next step without further purification (1.2 g, 5.38 mmol, 98% yield).

[0276] Step C

[0277]

[0278] Hydrazine monohydrate (6 eq) was added to a solution of methyl 4-(1-azidoethyl)-3-fluorobenzoate (1.2 g, 5.38 mmol, 1 eq) in 10 mL of MeOH. The reaction mixture was refluxed with stirring for 3 h and then evaporated to dryness.

[0279] The intermediate hydrazide was dissolved in 5 mL of DMF and difluoroacetic anhydride (2.5 eq.) was added. The reaction mixture was stirred at room temperature overnight and washed with saturated NaHCO 3 The aqueous solution was diluted and extracted with MTBE. The organic layer was washed with Na 2 SO 4 Dry, filter and concentrate.The crude product was purified by flash chromatography (silica gel, hexanes / EtOAc 0-15%) to give the desired product (860 mg, 3.04 mmol, 56% yield).

[0280] Step D

[0281]

[0282] 6-Bromo-1,3-benzothiazol-2-amine (8 g, 34.9 mmol, 1 eq) was dissolved in 75 mL of dioxane. Trifluoroethylamine (2 eq) was added and the mixture was degassed with Ar. Copper iodide (0.1 eq) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II)DCM complex (0.1 eq) were added and the mixture was degassed again. Ethylene(trimethyl)silane (3 eq) was added and the mixture was stirred at 95 °C overnight. The reaction mixture was allowed to reach room temperature and then diluted with EtOAc and filtered through celite. The filtrate solution was washed with 5% NH 3 The solution was washed with saturated NaHCO 3 The mixture was washed with aqueous solution and brine. 2 SO 4 The organic phase was dried, filtered and concentrated to dryness.The crude product was purified by flash chromatography (silica gel, 20-50% Hex / EtOAc) to afford 7.38 g of the desired intermediate (29.9 mmol, 86% yield).

[0283] Step E

[0284]

[0285] 6-((Trifluorosilyl)benzo[d]thiazol-2-amine (7.38 g, 29.9 mmol, 1 eq) was suspended in 75 mL MeOH and potassium carbonate (1.5 eq) was added. The resulting mixture was stirred at room temperature overnight to obtain complete conversion. The crude product was purified by flash chromatography (silica gel, dry loaded, 0-4% MeOH / DCM) to give 4.2 g of the desired intermediate (24.1 mmol, 80% yield).

[0286] Step F-1

[0287]

[0288] 2-[4-(1-Azidoethyl)-3-fluorophenyl]-5-(difluoromethyl)-1,3,4-oxadiazole (60 mg, 0.34 mmol, 1 eq) and 6-ethynyl-1,3-benzothiazol-2-amine (60 mg, 0.34 mmol, 1 eq) were dissolved in 2 mL of DMSO. Copper sulfate pentahydrate (0.3 eq, 0.5 M aqueous solution) and (+)-sodium L-ascorbate (0.5 eq, 1 M aqueous solution) were added and the mixture was stirred at room temperature overnight. UPLC showed complete conversion to the desired click reaction product.

[0289] 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 overnight at 50°C. UPLC detected complete conversion to the desired product. The mixture was concentrated by rotary evaporation, filtered, and then purified. RP-flash chromatography and continuous preparative HPLC gave 12.3 mg of the desired product in the form of the free base (0.02 mmol, 7% yield).

[0290] Compound 30: [M+H] + Measured value: 476.35; 1 H NMR(400MHz,DMSO-d6)δ10.82(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,1 H), 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).

[0291] Step F-2

[0292]

[0293] 2-[4-(1-azidoethyl)-3-fluorophenyl]-5-(difluoromethyl)-1,3,4-oxadiazole (400 mg, 1.4 mmol, 1 eq.) and 6-ethynyl-1,3-benzothiazol-2-amine (246 mg, 1.4 mol, 1 eq.) were dissolved in 5 mL DMSO. Copper sulfate pentahydrate (0.1 eq., 0.5 M aqueous solution) and (+)-sodium L-ascorbate (0.2 eq., 1 M aqueous solution) were added and the mixture was stirred at room temperature for 1 hour. UPLC showed complete conversion to the desired product. The mixture was directly subjected to RP-flash chromatography (water / ACN 40%, 0.1% FA) to obtain the pure product (566 mg, 1.24 mmol, 88% yield).

[0294] Compound 30-A: [M+H] + Measured value: 458.17; 1 H NMR(400MHz, DMSO-d6)δ8.73(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).

[0295] 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)

[0296] Step 1

[0297]

[0298] 5-(Bromomethyl)thiophene-2-carboxylic acid methyl ester (1 g, 4.2 mmol, 1 eq) was dissolved in 8 mL DMSO and sodium azide (1.05 eq) was added. After 1 hour, LCMS observed complete conversion. Water was added and the reaction mixture was extracted with EtOAc and concentrated under reduced pressure. The crude product was used in the next step without purification.

[0299] Step 2

[0300]

[0301] 5-(Azidomethyl)thiophene-2-carboxylic acid methyl ester (402 mg, 2 mmol, 1 eq) was dissolved in 10 mL of ethanol and hydrazine (5 eq) was added. The reaction mixture was refluxed overnight with stirring and then concentrated under reduced pressure. The crude product thus obtained was used in the next step without purification.

[0302] Step 3

[0303]

[0304] Difluoroacetic anhydride (1 eq) was added dropwise to a solution of 5-(azidomethyl)thiophene-2-carbohydrazide (200 mg, 1.0 mmol, 1 eq) in 5 mL of DMF. After 1 hour, water was added and the reaction mixture was extracted with EtOAc and washed with Na 2 SO 4 The product (210 mg) was used in the next step without further purification.

[0305] Step 4

[0306]

[0307] 5-(Azidomethyl)-N'-(2,2-difluoroacetyl)thiophene-2-carbohydrazide (210 mg, 0.51 mmol, 1 eq) and 6-ethynyl-1,3-benzothiazol-2-amine (1 eq) were dissolved in 3 mL DMSO. Copper sulfate pentahydrate (0.2 eq) and sodium ascorbate (0.4 eq) were added as aqueous solutions. After 2 hours, the reaction mixture was filtered and purified by preparative HPLC to give the final product (120 mg, 0.26 mmol, 52% yield). [M+H] + Measured value: 450.23; 1 H NMR (400MHz, DMSO) δ10.98(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=8.3,1.8Hz,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).

[0308] Example 8. General Procedure E for the Bioconversion of 2-(Difluoromethyl)- or 2-(Trifluoromethyl)-1,3,4-oxadiazole to Difluoro- or Trifluoroacetyl Hydrazide

[0309] HDAC6 (1 μM) was incubated with 5 μM prodrug compounds in assay buffer at 25°C. At different times, aliquots (40 μL) were transferred to test tubes containing acetonitrile (240 μL) to quench the reaction. Samples were stored frozen at -80°C until LC-HRMS analysis.

[0310] 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), which was equipped with a heated electrospray ionization device and operated in positive mode. Full scan analysis was set within the m / z range of 50-500amu. XSelect HSS T3 50x2.1 mm, 2.5μm chromatographic column (Waters) was used. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The flow rate was set to 0.5mL / min, and the gradient program was from 3% B to 20% B in 3 minutes.

[0311] When incubated with HDAC6 enzyme, the prodrug was observed to disappear over time. HR-LCMS analysis showed the formation of a molecular weight = MW [前药] The new compound with +18 corresponds to the acyl hydrazide.

[0312] The following compounds were obtained and confirmed using General Procedure E:

[0313]

[0314] Example 9. LC-HRMS analysis for monitoring enzyme reactions

[0315] At 25°C, HDAC6 (1 μM) was incubated with 5 μM DFMO-compounds in assay buffer. At different times, aliquots (40 μL) were transferred to test tubes containing acetonitrile (240 μL) to quench the reaction. The samples were frozen at -80°C until LC-HRMS analysis. The test items in the samples were quantified according to the calibration curves obtained for the different concentrations of the compounds. The calculated compound concentrations are plotted in Table 1 as a percentage of the compound concentration at each time point.

[0316] 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), which was equipped with a heated electrospray ionization device and operated in positive mode. Full scan analysis was set within the m / z range of 50-500amu. XSelect HSS T3 50x2.1 mm, 2.5μm chromatographic column (Waters) was used. Mobile phase A consisted of 0.1% formic acid in water, and mobile phase B consisted of 0.1% formic acid in acetonitrile. The flow rate was set to 0.5mL / min, and the gradient program was from 3% B to 20% B in 3 minutes.

[0317] Table 1. Kinetics of prodrug disappearance when incubated with HDAC6

[0318]

[0319] For all tested compounds, its disappearance could be recorded when incubated with the HDAC6 enzyme. HR-LCMS analysis also showed the formation of a new compound with a molecular weight of MW+18, corresponding to the hydrate form. For compounds 2-A, 31-A and 37-A, the retention times in the HPLC analysis confirmed that the products formed were the corresponding compounds 2, 31 and 37.

[0320] All compounds were also incubated in the same buffer without enzyme for the same time, and they were all stable (>80% within 8 hours, except for Example 42-A, where 60% remained after 8 hours).

[0321] Example 10. Isolation of persistent / tight complexes

[0322] Use flash chromatography on a spin column coupled to LC-HRMS to attempt to separate the persistent / tight complex between HDAC6 and Examples 1 and 1-A. As previously described, zHDAC6-CD2 wt (1 μM) was incubated with 1 (5 μM) or 1-A (also 5 μM) for 6 hours. An aliquot (60 μL) was loaded onto a Bio-Spin P-6 gel column (Bio-Rad) equilibrated with assay buffer to separate the HDAC6 inhibitor complex from the free compound. After eluting the initial fraction containing most of the enzyme by centrifugation, 10 aliquots of 200 μL were taken and eluted by centrifugation to collect low molecular weight (free) compounds. LC-HRMS analysis (as described above) was performed on aliquots of each fraction to identify and quantify 1 and its derivatives.

[0323] from Figure 2It can be clearly seen in the graph that the only compound that co-eluted with the enzyme (fraction 1) was compound 1, suggesting that this is the true inhibitor, while compound 1-A behaves as a prodrug (note that at this extreme concentration, the hydrolysis product of compound 1, hydrazide, is also formed, which also binds the enzyme, even if to a small extent).

[0324] Example 11. Enzyme screening

[0325] For each test compound, 8 doses of 100× concentrated DMSO solution were prepared and then diluted in assay buffer (25mM Tris-HCl, pH 8, 130mM NaCl, 0.05% Tween-20, 10% glycerol) to obtain a 5× concentrated solution related to the final concentration (typical final concentration range is 6.4-2000000nM or 0.18-50000nM, and the final DMSO content is 1%). Then 10 μL of each test compound concentration was placed in a 96-well plate in triplicate, and 15 μL of 3.33X concentrated enzyme solution was placed in an assay buffer containing 3.33X concentrated BSA (final BSA concentration of 1mg / mL), and if it is HDAC6, 3.33X concentrated TCEP was added to each well (final TCEP concentration of 200μM). After a period of preincubation at 25°C (30 minutes for HDAC6 and 120 minutes for HDAC1), 25 μL of a solution containing a substrate was added. As substrate, FLUORDE Deacetylase substrate (Enzo Life Sciences, cat: BML-KI104, FdL), FLUOR DE -Green substrate (Enso Life Sciences, cat: BML-KI572, FdL_G) or Boc-LYS(Tfa)-AMC (Bachem, cat: 4060676.005, Tfal)-2X concentrated assay buffer. After the reaction period (25°C, 30 minutes), 50 μL of concentrated FLUOR DE The developing solution consisted of developer I (Enzo Life Sciences, ca: BML-KI105) in a buffer (50 mM Tris-HCl, pH 8, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2) and diluted 200-fold in 5% paraformaldehyde, 2 μM TSA was added, and after 25 minutes at room temperature in the dark, fluorescence reading was performed using a Victor 1420 Multilabel Counter Perkin-Elmer Wallac instrument (excitation / emission: 485 / 535 nM-FLUOR de Lys Green, 355 / 460 nM-Tfal, Fluor deLys).

[0326] The enzymatic activity of each synthetic compound against recombinant human HDAC6 and HDAC1 was evaluated (Table 2).

[0327] Table 2. Enzyme inhibition activity assay (IC) of HDAC6 and HDAC1 50 , unit is nM)

[0328]

[0329]

[0330] All tested compounds had little activity against HDAC1 (IC 50 >100 μM), while showing relevant activity against HDAC6 (IC 50 <600 nM). This confirms the selectivity associated with this class of compounds.

[0331] Example 12. Acetylation of α-tubulin in vitro in 697 cell line

[0332] Human B-cell precursor leukemia 697 was evaluated for in vitro α-tubulin acetylation.

[0333] 697 cells were maintained in RPMI medium 1640 (Gibco, cat: 21875-034) supplemented with 10 mM HEPES (Gibco, cat: 15630-080), Pen-Strep (penicillin 100 U / ml, streptomycin 100 μg / ml, Gibco, cat: 15140-122) and 10% fetal bovine serum (Gibco, cat: 10270-106).

[0334] The cells were cultured at 5.5 x 10 5 The cells were plated at a density of 10 cells / ml in 12-well plates (Costar, cat: 3512).

[0335] Serial dilutions of the test compounds in DMSO were prepared using the 20 mM stock solution to obtain 8 doses, 200× concentrated relative to the final dose (2.7-100000 nM). The DMSO solution was then diluted 10× in culture medium to obtain a 20× concentrated solution for cell treatment (125 μl of culture medium solution was added to 2.375 ml of cell suspension). The final DMSO content was set to 0.5%. The well plate was incubated at 37°C, 5% CO 2 Incubate for 16 hours.

[0336] At the end of the incubation period, the cells were harvested, centrifuged at 200 x g for 5 minutes, and washed with 0.9% NaCl at 4°C. The resulting particles were treated with 100 μl of Complete Lysis-M buffer containing protease inhibitors (Complete Lysis-M Roche + Complete Tablets, MiniEasypack, cat: 4719956001) and a phosphatase inhibitor cocktail (PhosStopEasypack, Roche, cat: 4906837001) at 4°C for 30 minutes, and then centrifuged at 18213 x g for 10 minutes. The protein concentration in each supernatant was determined using a BCA protein assay kit (Pierce, cat: 23227). The samples were diluted in PBS 1x to obtain a concentration of 2 μg / ml and coated in a MaxiSorp 96-well plate (Nunc, cat: 442404). The well plate was incubated overnight at room temperature.

[0337] The plate was washed twice with washing buffer (PBS 1x + 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 washing buffer solution, the plate was incubated at room temperature for 2 hours in the presence of 100 μL / well of anti-acetylated-α-tubulin antibody (monoclonal anti-tubulin, acetylated antibody produced in mouse, Sigma-Aldrich, cat: T6793) or in the presence of total anti-α-tubulin antibody (monoclonal anti-α-tubulin produced in mouse, Sigma-Aldrich, cat: T6074) diluted 1:1000 in 1x PBS containing 10% FBS. After 5 cycles of washing with wash buffer, a secondary antibody conjugated to the enzyme HRP (goat anti-mouse IgG, IgM, IgA (H+L), stock concentration 0.5 mg / ml, Thermo Fisher Scientific, cat: A10668) was added at a volume of 100 μL / well, which was diluted 1:1000 in 1x PBS+10% FBS. After incubation for two hours at room temperature, the plate was washed 4 times with wash buffer and then 100 μl / well of TMB substrate (TMB substrate kit, Thermo Fisher Scientific, cat: 34021) was added for 10 minutes at room temperature in the dark. The reaction was stopped by adding 50 μl 2M H2SO4. The plate was read at a wavelength of 450 nm in a BioTek Synergy H1 multi-mode microplate reader.

[0338] The measured absorbance was corrected by subtracting the mean of the blank values ​​(samples without primary antibody). The absorbance ratio of the acetyl to total tubulin assay was calculated and normalized to the reference compound (positive control) 4-parameter logistic curve, where 0% is the fitted bottom of the curve and 100% is the fitted top of the curve. The results are expressed as relative EC 50 .

[0339] Table 3. Tubulin acetylation (EC 50 , unit is nM)

[0340]

[0341]

[0342] In the 697 cell line, all tested compounds showed very high activity in inducing tubulin acetylation.

[0343] Example 13. Acetylation of α-tubulin in vitro in N2a cell line

[0344] In vitro α-tubulin acetylation was assessed on the mouse neuroblastoma N2a cell line.

[0345] Cells were maintained in Eagle's Minimum Essential Medium (ATCC, cat: 30-2003) supplemented with 10% fetal bovine serum-FBS (Gibco, cat: 10270-106).

[0346] The cells were plated at 6 x 10 4 cells / cm 2 The density of 1 μM was plated in a 12-well plate (Costar, cat: 3512). The test compound was prepared as a 20X concentrated medium solution relative to the final concentration. The cells were treated the next day. At 3 doses: 10 μM, 1 μM and 0.1 μM test compound. The final DMSO content was set to 0.5%. The cells were incubated with the compound at 37°C for 16 hours.

[0347] At the end of the incubation period, the cells were harvested, centrifuged at 200 x g for 5 minutes, and washed with 0.9% NaCl at 4°C. The resulting particles were treated with 100 μl of Complete Lysis-M buffer containing protease inhibitors (Complete Lysis-M Roche + Complete Tablets, MiniEasypack, cat: 4719956001) and phosphatase inhibitor cocktail (PhosStopEasypack, Roche, cat: 4906837001) at 4°C for 30 minutes, and then centrifuged at 18213 x g for 10 minutes. The protein concentration in each supernatant was determined using a BCA protein assay kit (Pierce, cat: 23227). The samples were diluted in PBS 1x to obtain a concentration of 2 μg / ml and coated in a MaxiSorp 96-well plate (Nunc, cat: 442404). The plate was incubated overnight at room temperature, then washed twice with washing buffer (PBS 1x + 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 washing buffer solution, the plate was incubated at room temperature for 2 hours in the presence of 100 μL / well of anti-acetylated-α-tubulin antibody (monoclonal anti-tubulin, acetylated antibody produced in mouse, Sigma-Aldrich, cat: T6793) or in the presence of total anti-α-tubulin antibody (monoclonal anti-α-tubulin produced in mouse, Sigma-Aldrich, cat: T6074) diluted 1:1000 in 1x PBS containing 10% FBS. After five cycles of washing with wash buffer, a secondary antibody conjugated to the enzyme HRP (goat anti-mouse IgG, IgM, IgA (H+L), stock concentration 0.5 mg / ml, Thermo Fisher Scientific, cat: A10668) was added at a volume of 100 μL / well, which was diluted 1:1000 in 1xPBS+10% FBS. After incubation for two hours 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 plate was incubated for 24 hours at room temperature for 15 minutes ... 2 SO 4 The reaction was stopped and the plate was read at a wavelength of 450 nm in a BioTek Synergy H1 Multi-Mode Microplate Reader.

[0348] The measured absorbance was corrected by subtracting the mean of the blank values ​​(samples without the primary antibody). The absorbance ratio of the acetyl to total tubulin assay was calculated and normalized to the reference compound (positive control) 4-parameter logistic curve, where 100% was the fitted top of the curve and 0% was the DMSO control (protein extract obtained from untreated cells). The results are expressed as the fold increase compared to the control (DMSO).

[0349] Table 4. Tubulin acetylation of selected compounds in N2a cell line (fold increase compared to control group)

[0350] Example Increase multiple 13-A 6 14-A 7 20-A 15 32-A 20 37-A 31 38-A 22 48-A 45 49-A 26

[0351] In the N2a cell line, all tested compounds showed very high activity in inducing tubulin acetylation.

[0352] Example 14. Acetylation of α-tubulin in undifferentiated SH-SY5Y cell line in vitro

[0353] SH-SY5Y cells (ATCC, cod. CRL-2266) were 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 (PerkinElmer, cod. 6055302).

[0354] After 24 h of plating, cells were incubated overnight with 0.1-1-10 μM of the selected molecules. The same doses of ACY1083 and Tubastatin A were tested as positive controls for α-tubulin acetylation, while untreated cells were incubated with 0.01% DMSO, labeled as CTRL-DMSO.

[0355] At the end of the incubation, fix the cells by adding 100 μL / well of 8% formaldehyde in PBS (final formaldehyde concentration of 4% in 200 μL / well) directly to the culture medium and keep at room temperature for 30 minutes. Carefully remove the fixation solution and wash the wells twice with PBS for 10 minutes each. Store the fixed cells in PBS at 4°C until staining.

[0356] On the day of the staining experiment, fixed cells were mixed with blocking buffer (containing 5% FCS + 0.3% Triton TM Incubate with the cells in PBS (containing 1% BSA + 0.3% Triton X-100) for 60 minutes. TMPrepare the primary antibodies by diluting 1:200 α-tubulin Alexa Fluor 488 conjugate (Cell Signaling, cod.5063) antibody and 1:50 acetyl-α-tubulin Alexa Fluor 647 conjugate (Cell Signaling, cod.81502) antibody in PBS (X-100). Once the blocking solution is aspirated, apply the diluted primary antibody and incubate overnight at 4°C. The next day, rinse the cells twice with PBS (10 minutes each), incubate with 300nM DAPI in PBS for 5 minutes, and then rinse twice with PBS (10 minutes each). For each treatment, three wells were stained.

[0357] Images of stained cells were collected by IN Cell Analyzer 2500HS instrument, using the far red channel (exposure 0.02 seconds) for acetyl-α-tubulin staining, the green channel (exposure 0.02 seconds) for α-tubulin staining, and the blue channel for DAPI (nucleus) staining. 10 images were collected per well.

[0358] The images of the stained cells were analyzed with InCarta software (Molecular Devices) to obtain the fluorescence intensity considering the whole cell. For each treatment, the InCarta raw data of FOV (Field of View) was used to obtain the cell intensity mean value of the two stainings - Bckg (cell). The results are expressed as the increase fold of the ratio of acetylated tubulin and total tubulin to the control (CTRL-DMSO).

[0359] Table 5. Tubulin acetylation in the undifferentiated SH-SY5Y cell line (fold increase in acetylated tubulin and total tubulin compared to control at 1 μM).

[0360]

[0361] In the SH-SY5Y cell line, all tested compounds showed very high activity in inducing tubulin acetylation.

Claims

1. A compound of formula (I), its pharmaceutically acceptable salts and isomers in: W=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, which is optionally substituted by C1-C3 alkyl, alkoxy or thioalkoxy, halogenated derivatives thereof, or halogen, or hydroxy; Alternatively, G is of formula (Ia): wherein X, X', Y and Y' are independently selected from CH, N, CF or CCl; Z=-CD2-、-CF2-、-CHR 2 -、-NH-、-S-; R 2 =H, halogen, C1-C6 alkyl or C3-C6 cycloalkyl, which is unsubstituted or substituted by: Hydroxyl, carbonyl, C1-C3 alkoxy, aryloxy or thioalkoxy, or halogenated derivatives thereof; ·halogen; A primary amine, a secondary amine, or a tertiary amine substituted with a C1-C6 alkyl group, a C3-C6 cycloalkyl group, or a halogenated derivative thereof; phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or substituted by C1-C3 alkyl, alkoxy, thioalkoxy or a halogenated derivative thereof, or halogen; or The following substructures or their halogenated derivatives: 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 its halogenated derivatives; wherein m and o are each independently 0, 1 or 2; or L is selected from the following substructures (IIa)-(IIf) and their halogenated derivatives: Wherein 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 CH2, NR 4 or O; Where n is 0, 1, or 2; Y" is absent, C1-C2 alkylene, or selected from the following substructures or halogenated derivatives thereof: wherein a, b and Q are as defined above; R 4 =H, C1-C4 alkyl, the C1-C4 alkyl is unsubstituted or substituted by the following groups: ·halogen phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or substituted by C1-C3 alkyl, alkoxy, thioalkoxy or their halogenated derivatives, or halogen; P is an unsubstituted or substituted, aromatic or non-aromatic 5- to 10-membered heterocyclic ring, wherein the ring consists 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 group containing at least one heteroatom selected from N, O and S; Provided that the following compounds are not included: -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-phenylimidazolin-1-yl)methyl)-3-fluorobenzohydrazide -4-((5,5-dimethyl-2,4-dioxo-3-phenylimidazolin-1-yl)methyl)-3-fluoro-N'-(2,2,2-trifluoroacetyl)benzohydrazide -N'-(2,2-difluoroacetyl)-4-((2,5-dioxo-3-phenylimidazolin-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)nicotinylhydrazide -4-((1H-imidazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)benzohydrazide -N'-(2,2-difluoroacetyl)-4-(pyrimidin-2-ylamino)benzohydrazide -4-((5-(2-(2,2-difluoroacetyl)hydrazine-1-carbonyl)pyrimidin-2-yl)amino)-4-phenylpiperidine-1-carboxylic acid benzyl ester -4-((1-(3-chloro-4-cyanophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)methyl)-N'-(2,2,2-trifluoroacetyl)benzohydrazide.

2. The compound according to claim 1, wherein R 2 is H, or C1-C3 alkyl, wherein the C1-C3 alkyl is unsubstituted or substituted by: ·C1-C3 alkyl or C3-C6 cycloalkyl Hydroxyl, carbonyl, C1-C2 alkoxy, aryloxy or thioalkoxy, or halogenated derivatives thereof; ·halogen; Primary, secondary or tertiary amines, which are substituted by C1-C3 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof; phenyl, pyridyl, thiophenyl, furan or pyrrole, which is unsubstituted or substituted by C1-C3 alkyl, alkoxy, thioalkoxy or a halogenated derivative thereof, or halogen; or The following substructures or their halogenated derivatives:

3. The compound according to claim 1 or 2, wherein P is in: A=C, N, O, S; B=C、N; D=C, N, O; E=C, N, O; M = C, N; R 5 and R 6 is independently -H, halogen, =O, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or a halogenated derivative thereof, which is optionally substituted with a carbonyl or carboxyl group, or R 5 and R 6 Independently selected from the following substructures: R 3 is absent, -H, C1-C6 alkyl optionally substituted with -OH or -N(C1-C5 alkyl)2, -LR 1 Or from the following substructures: When R 3 =-LR 1 When , substitution on M does not exist; Ra and Rb are independently H, halogen, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or halogenated derivatives thereof.

4. The compound according to claim 1 or 2, wherein P is selected from the following substructures: wherein Ra and Rb are as defined in claim 3, or are -LR 1 ; Rc is H, halogen, C1-C6 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl, or a halogenated derivative thereof, or -NH2.

5. A compound according to any one of the preceding claims, wherein R 1 Select from the following substructures: Where R 6 and R 7 Independently selected from: -H, -D, -OH, C1-C4 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl or its halogenated derivatives, 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 Independently selected from the following substructures: R 8 =-H, -D, -OH, C1-C6 alkyl, C3-C6 cycloalkyl or its halogenated derivatives, -(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 Select from the following substructures: R 9 =-NR'R", C1-C4 alkyl, or a halogenated derivative thereof, or selected from the following substructures: R 10 and R 11 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 CH2, O, S, NR 8 ; Q 2 and Q 3 are independently CR'R", CF2, O, S, NR 8 ; R' and R" are independently -H, C1-C4 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof; a, b, c, and R 8 As defined above.

6. A compound according to any one of the preceding claims, wherein R 1 Select from the following substructures: Where R 6 and R 7 Independently selected from: -H, -D, -OH, C1-C4 alkyl, alkoxy or thioalkoxy, C3-C6 cycloalkyl or its halogenated derivatives, halogen, -(CH2) a NR'R", -NHR 8 、-C(=O)R 9 , -NO2, -Ph, -SO2-NR'R”, =O, =NR 8 , -SO2-C1-C4 alkyl, or independently selected from the following substructures: R 8 =-H, -D, -OH, C1-C6 alkyl, C3-C6 cycloalkyl or its halogenated derivatives, -(CH2) a NR'R", -C(=O)OR', -C(=O)R 9 、-C(=NH)R 9 , -SO2-C1-C4 alkyl, or R 8 Select from the following substructures: R 9 =-NR'R", C1-C4 alkyl, or a halogenated derivative thereof, or selected from the following substructures: R 10 and R 11 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 CH2, O, S, NR 8 ; Q 2 and Q 3 are independently CR'R", CF2, O, S, NR 8 ; R' and R" are independently -H, C1-C4 alkyl, C3-C6 cycloalkyl or halogenated derivatives thereof; a, b, c, and R 8 As defined above.

7. A compound according to any one of claims 1 to 4, wherein R 1 Select from the following substructures: Where a and b are independently 0, 1, 2, or 3, and a and b cannot be 0 at the same time; Z1 is CH2, NH, or O; Where R 1 At least one H is optionally replaced by halogen, -(CH2) n -Q4-Q5-Rd substitution; n = 0, 1 or 2; Q4 = absent, -SO2-, -NH-, -N(C1-C5 alkyl)-, -NHC(=O)-, -N(C1-C5 alkyl)C(=O)-, or -C(=O)-; Q5 = absent, C1-C5 alkylene, -NH-, -(C1-C5 alkylene)-NH-C(=O)-, or -N(C1-C5 alkyl); Rd=-OH, C1-C5 alkyl, C1-C5 haloalkyl, -NR'R", C1-C5 alkoxy, 5- or 6-membered heteroaryl containing 1 to 3 N, wherein e and f are independently 1 or 2; M1 is CH2, O, NH or SO2, M2 is CH or N, and at least one H of Rd is optionally substituted by 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", Where g and h are independently 0 or 1, but cannot be 0 at the same time; M4 is CH2, O, NH, and at least one H of M4 is optionally substituted by halogen, C1-C5 alkyl, C3-C6 cycloalkyl or -C(=O)-O(C1-C5 alkyl); R' and R" are independently -H or C1-C4 alkyl.

8. The compound according to claim 1, which is selected from: -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-methylphenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide (Compound 8) -N'-(2,2-difluoroacetyl)-6-((4-(m-methylphenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinic acid hydrazide (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)nicotinylhydrazide (Compound 13) -N'-(2,2-difluoroacetyl)-6-((4-(2-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 14) -N'-(2,2-difluoroacetyl)-5-fluoro-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 15) -N'-(2,2-difluoroacetyl)-6-((5-phenyl-2H-tetrazol-2-yl)methyl)nicotinic acid hydrazide (Compound 16) -N'-(2,2-difluoroacetyl)-6-((5-(thiophen-2-yl)-2H-tetrazolyl-2-yl)methyl)nicotinylhydrazide (Compound 17) -N'-(2,2-difluoroacetyl)-6-((4-(3-fluorophenyl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 18) -N'-(2,2-difluoroacetyl)-6-((4-(thiophen-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 19) -N'-(2,2-difluoroacetyl)-6-((4-(pyridin-2-yl)-1H-1,2,3-triazol-1-yl)methyl)nicotinylhydrazide (Compound 20) -6-((4-(1H-pyrrolo[2,3-b]pyridin-5-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)nicotinic acid hydrazide (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)nicotinic acid hydrazide (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)nicotinic acid hydrazide (Compound 38) -6-(1-(4-(6-aminopyridin-3-yl)-1H-1,2,3-triazol-1-yl)ethyl)-N'-(2,2-difluoroacetyl)nicotinic acid hydrazide (Compound 39) -6-((4-(1H-indazol-4-yl)-1H-1,2,3-triazol-1-yl)methyl)-N'-(2,2-difluoroacetyl)-5-fluoronicotinic acid hydrazide (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).

9. A compound according to any one of the preceding claims for use as a medicament.

10. A compound for use according to claim 9, for treating one or more diseases regulated by HDAC6, the disease being selected from chemotherapy-related cognitive impairment (CRCI), transplant rejection, GVHD, myositis, diseases associated with abnormal lymphocyte function, multiple myeloma, non-Hodgkin's lymphoma, peripheral neuropathy, autoimmune diseases, inflammatory diseases, cancer and neurodegenerative diseases, eye diseases.

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