Hydroxamic acid compounds for inhibiting HDAC6 and preparation method and application thereof

Abstract

The present application provides a kind of inhibiting histone deacetylase hydroxamic acid compound and its preparation method and application, structural general formula is as shown in formula (I), or its isomer, or its pharmaceutically acceptable salt, ester or prodrug.The hydroxamic acid compound described in the present application is a new type of HDAC6 enzyme inhibitor, can selectively inhibit HDAC6 enzyme, has protective effect to nerve cell, has low potential toxicity to normal cell and small potential cardiac toxicity, and is expected to be used as neuroprotective agent for the treatment of neurodegenerative diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an isohydroxamic acid compound that inhibits HDAC6, its preparation method, and its application. Background Technology

[0002] Histone deacetylases (HDACs) catalyze the deacetylation of histones and non-histone proteins, and together with histone acetyltransferases (HATs), they regulate intracellular acetylation levels, thereby controlling gene expression. Currently, 18 subtypes of mammalian HDACs are known, divided into four classes: Class I (HDAC1, HDAC2, HDAC3, HDAC8); Class II: IIa (HDAC4, HDAC5, HDAC7, HDAC9) and IIb (HDAC6, HDAC10); Class III (Sirt1–Sirt7); and Class IV (HDAC11).

[0003] Currently, there are five marketed histone deacetylase inhibitors (HDACi): vorinostat, belinostat, panobinostat, romidepsin, and chidamide. Vorinostat and romidepsin are used to treat cutaneous T-cell lymphoma (CTCL), belinostat and chidamide are used to treat relapsed and refractory peripheral T-cell lymphoma (PTCL), and panobinostat is used in combination with bortezomib and dexamethasone to treat multiple myeloma (MM).

[0004] Although HDAC inhibitors have achieved good clinical efficacy, they still have the following drawbacks:

[0005] (1) Strong toxic side effects, such as nausea, vomiting, and bone marrow suppression;

[0006] (2) Genotoxicity;

[0007] (3) Poor pharmacokinetic characteristics, low bioavailability, short half-life, etc.

[0008] Therefore, developing novel HDAC inhibitors with high efficacy and low toxicity remains a challenge.

[0009] HDAC6 is the largest member of the HDAC family. Unlike other HDAC members, HDAC6 is the only histone deacetylase containing two catalytic domains (CD1 and CD2). It is mainly distributed in the cytoplasm rather than the nucleus and plays no significant role in post-translational modifications of histones. HDAC6 possesses strong histone deacetylase activity and can mediate the deacetylation process of non-histone proteins. Its main substrates include α-tubulin, heat shock protein 90 (HSP90), and cortactin. Due to its unique structure and substrate diversity, HDAC6 participates in various intracellular physiological processes, including cell movement, endocytosis, autophagy, apoptosis, and protein transport and degradation. Numerous studies have confirmed that overexpression of HDAC6 is closely related to various diseases, such as cancer, autoimmune diseases, and neurodegenerative diseases. Summary of the Invention

[0010] This invention provides a novel HDAC6 inhibitor. The isohydroxamic acid compound provided by this invention can selectively inhibit HDAC6 enzyme activity, has a protective effect on nerve cells, low toxicity to normal cells and low potential cardiotoxicity, and is expected to be used as a neuroprotective agent for the treatment of neurodegenerative diseases.

[0011] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides an isohydroxamic acid compound with the general structural formula shown in formula (I), or an isomer thereof, or a pharmaceutically acceptable salt, ester or prodrug thereof;

[0012]

[0013] in,

[0014] R1 and R2 are independently selected from hydrogen, deuterium, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, benzyl, heterocycloalkyl, aryl, heteroaryl, cyano, haloalkyl, acyl, sulfonyl, aminoalkyl, which may optionally be substituted;

[0015] R is selected from hydrogen, alkyl, cycloalkyl, benzyl, heterocycloalkyl, acyl, sulfonyl or -C(=O)-Y, which may optionally be substituted;

[0016] Y is selected from cycloalkyl, aryl, and heteroaryl groups, which may optionally be substituted.

[0017] In some specific embodiments of the present invention, when R is -C(=O)-Y, the general structural formula is as shown in formula (Ⅱ):

[0018]

[0019] in,

[0020] R1 and R2 are independently selected from hydrogen, deuterium, hydroxyl, halogen, alkyl, alkoxy, cycloalkyl, benzyl, heterocycloalkyl, aryl, heteroaryl, cyano, haloalkyl, acyl, sulfonyl, aminoalkyl, which may optionally be substituted;

[0021] Y is selected from cycloalkyl, aryl, heteroaryl, aminoaryl, aminoalkyl, or aminoheteraryl, which may optionally be substituted.

[0022] Preferably, the alkyl group is selected from methyl, ethyl, propyl, isopropyl, butyl, or isobutyl, and may optionally be substituted with 1-3 halogens.

[0023] Preferably, the cycloalkyl group is selected from cyclopropane, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and may optionally be substituted with 1-3 halogens;

[0024] Preferably, the heterocyclic alkyl group is selected from pyrrolyl, morpholinyl, piperidinyl, piperazine ring, tetrahydroquinolinyl, tetrahydrotriazolylpyrazine, diazacycloheptyl or piperazine;

[0025] Preferably, the aryl or heteroaryl group is selected from phenyl, naphthyl, anthracene, pyridyl, pyrimidinyl, pyrazinyl, indolyl, imidazolyl, benzoxazolyl, benzofuranyl, benzothiophene, benzothiazolyl, triazolyl, isoxazolyl, quinolinyl, pyrroleyl, pyrazolyl or 5,6,7,8-tetrahydroisoquinoline;

[0026] Preferably, the acyl group is selected from acetyl, propionyl, isobutyryl, or aryl acyl.

[0027] Preferably, the aminoalkyl group is selected from dimethylaminoalkyl, methylaminoalkyl, piperazinealkyl, and piperidinealkyl.

[0028] Preferably, the halogen is selected from fluorine, chlorine or bromine.

[0029] In some specific embodiments of the present invention, the pharmaceutically acceptable salts of the compound of formula (I) include anionic salts formed by reacting the compound of formula (I) with hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, citrate, tartrate, maleate, fumarate, methanesulfonate, malate, p-toluenesulfonate or oxalate; or cationic salts formed by reacting the compound of formula (I) with sodium ion solution or potassium ion solution.

[0030] More preferably, R1 and R2 are independently selected from hydrogen, methoxy, halogen, methyl or phenyl, which may optionally be substituted; R is selected from hydrogen or -R(=O)-Y, and Y is selected from cyclopropyl, phenyl, halophenyl or aminophenyl.

[0031] In some specific embodiments of the present invention, the isohydroxamic acid compounds include the following compounds or isomers thereof, or pharmaceutically acceptable salts, esters or prodrugs thereof:

[0032] Table 1

[0033]

[0034]

[0035]

[0036]

[0037] A second aspect of the present invention provides a method for preparing the isohydroxamic acid compounds described above, comprising the following steps:

[0038] S1, Compound (IV) and methyl 4-(formyl)benzoate were reduced with sodium borohydride to give compound (V);

[0039]

[0040]

[0041] Wherein, R1 and R2 are as defined in the above technical solution;

[0042] S2, when R is hydrogen or deuterium, the compound of formula (V) reacts with an alkaline hydroxylamine solution to give the compound of formula (III);

[0043]

[0044] When R is not hydrogen or deuterium, compound (V) undergoes an amidation reaction with compound RX to obtain compound (VI), and compound (VI) reacts with alkaline hydroxylamine solution to obtain compound (I).

[0045]

[0046] Where R is as defined in the above technical solution, and X is a halogen.

[0047] Preferably, in step S1 of this invention, the reaction temperature is -5℃ to 5℃, and / or the reaction is carried out under conditions of pH = 5 to 7; and / or the reaction solvent is dichloromethane, dichloroethane, or methanol; and / or the reducing agent is sodium borohydride or sodium triacetoxyborohydride.

[0048] In some specific embodiments of the present invention, step S1 includes: mixing compound (IV), methyl 4-(formyl)benzoate, an organic solvent, and acetic acid; slowly adding sodium borohydride; and reacting at -5°C to 5°C for 10 to 18 hours to obtain compound (V). Preferably, the molar ratio of compound (IV) to methyl 4-(formyl)benzoate is 1:1; preferably, the organic solvent is 1,2-dichloroethane; preferably, the mixture is stirred at -5°C to 5°C for 1 to 3 hours before adding sodium borohydride; preferably, the reaction temperature of step S1 is 0°C; preferably, the reaction time of step S1 is 12 hours.

[0049] Preferably, in step S2 of this invention, the alkaline hydroxylamine solution is prepared according to the following method:

[0050] Hydroxylamine hydrochloride and sodium hydroxide were dissolved in an organic solvent. After stirring, sodium chloride precipitated out. The solution was filtered to obtain an alkaline hydroxylamine solution.

[0051] More preferably, in step S2, the reaction temperature of compound (V) or compound (VI) with alkaline hydroxylamine solution is 5°C to 5°C.

[0052] In some specific embodiments of the present invention, when R is hydrogen or deuterium, step S2 includes: adding the compound of formula (V) to an alkaline hydroxylamine solution, reacting at -5°C to 5°C for 2 to 5 hours, adjusting the pH to neutral after the reaction, removing the solvent, adding ethyl acetate, and recrystallizing to obtain the isohydroxamic acid compound (compound of formula (III)) of the present invention. Preferably, the reaction temperature of step S2 is 0°C; preferably, the reaction time of step S2 is 3 hours; preferably, the pH is adjusted to neutral with acetic acid after the reaction; preferably, the solvent is removed by vacuum concentration.

[0053] In some specific embodiments of the present invention, when R is not hydrogen or deuterium, step S2 includes: mixing compound (V), compound RX, triethylamine, and an organic solvent, reacting at room temperature for 5-8 hours to obtain compound (VI); adding compound (VI) to an alkaline hydroxylamine solution, reacting at -5°C to 5°C for 2-5 hours; adjusting the pH to neutral after the reaction, removing the solvent, adding ethyl acetate, and recrystallizing to obtain the isohydroxamic acid compound of the present invention. Preferably, the organic solvent is dichloromethane; preferably, the reaction time at room temperature is 6 hours; preferably, the reaction time at -5°C to 5°C is 3 hours; preferably, the pH is adjusted to neutral with acetic acid after the reaction; preferably, the solvent is removed by vacuum concentration.

[0054] A third aspect of the present invention provides an intermediate compound for preparing the isohydroxamic acid compounds described in the foregoing technical solutions, comprising a compound of formula (IV) or an isomer thereof, a pharmaceutically acceptable salt, an ester, or a prodrug:

[0055]

[0056] And / or, compounds of formula (V) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0057]

[0058] And / or, compounds of formula (VII) or their isomers, pharmaceutically acceptable salts, esters, or prodrugs:

[0059]

[0060] R, R1, and R2 are defined as in the aforementioned technical solution.

[0061] The fourth aspect of the present invention provides the use of the isohydroxamic acid compounds described in the foregoing technical solutions, the isohydroxamic acid compounds obtained by the preparation methods described in the foregoing technical solutions, or the intermediate compounds described in the foregoing technical solutions in the preparation of histone deacetylase inhibitors or drugs for the treatment of neurodegenerative diseases.

[0062] Preferably, the histone deacetylase inhibitor of the present invention is an HDAC6 and / or HDAC1 inhibitor.

[0063] Preferably, the neurodegenerative diseases mentioned in this invention include one or more of the following: cerebral ischemia, brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and Pick's disease.

[0064] A fifth aspect of the present invention provides a pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable excipients; said active ingredient comprising isohydroxamic acid compounds as described in the foregoing technical solutions or isohydroxamic acid compounds prepared by the foregoing technical solutions.

[0065] Preferably, the pharmaceutically acceptable excipients of this invention include one or more of the following: diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbent carriers, lubricants, flavorings, and sweeteners.

[0066] The pharmaceutical composition of this invention can be formulated into various forms such as tablets, powders, granules, capsules, oral liquids, and injectable drugs. All of these dosage forms can be prepared using conventional methods in the pharmaceutical field. The active component in the pharmaceutical composition of this invention can also be combined with other effective ingredients that have therapeutic effects or enhance therapeutic effects, reduce toxic side effects, or prolong metabolic time to form a pharmaceutical composition.

[0067] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0068] α-Tubulin, a substrate of HDAC6, is a major component of microtubules. Acetylation of α-tubulin at lysine 40 is a common process in microtubules. The stability of microtubule function strongly depends on the acetylation state of α-tubulin. Impaired microtubule-based transport can disrupt mitochondrial transport between the neuronal cell body and axon / dendries, further leading to mitochondrial dysfunction and subsequent cell death. Mitochondria are prominent microtubule-based axonal transport organelles; enhancing α-tubulin acetylation by inhibiting HDAC6 can improve microtubule-based transport, thereby improving mitochondrial transport defects. HDAC6 inhibition may slow or reverse Aβ-related neuronal damage. This invention proposes a novel HDAC6 inhibitor targeting α-tubulin for the treatment of neurodegenerative diseases.

[0069] 1. Experiments show that the isohydroxamic acid compounds provided by this invention have novel structures and high inhibitory activity against HDAC6 (nM level). Some compounds show certain selective inhibitory effects against HDAC6 (compared to HDAC1).

[0070] 2. Experiments show that the hydroxamic acid compounds provided by this invention have low toxicity to human embryonic lung fibroblasts MRC-5 and exhibit good selective inhibitory effects on tumor cells;

[0071] 3. Experiments show that the hydroxamic acid compounds provided by this invention have weak anti-proliferative effects on the human neuroblastoma cell line SH-SY5Y and low toxicity to nerve cells;

[0072] 4. Experiments show that the hydroxamic acid compounds provided by this invention can enhance the viability of L-glutamate-damaged SH-SY5Y cells, demonstrating a good neuroprotective effect;

[0073] 5. Experiments show that the isohydroxamic acid compounds provided by this invention have weak inhibitory activity on hERG potassium ion channels and low potential cardiotoxicity. Detailed Implementation

[0074] In this invention, the term "isomer" includes, but is not limited to, enantiomers, diastereomers, mixtures of enantiomers and diastereomers, tautomers, mixtures of racemic mixtures and diastereomers, and their pharmaceutically acceptable salts. Unless otherwise stated, when an isomer component is not specifically specified, all possible isomers are included.

[0075] In this invention, "pharmaceutically acceptable salt" refers to a compound modified by forming an acidic or basic salt of the benzothiadiazine compound described in this invention, including but not limited to salts of inorganic acids selected from, for example, hydrochlorides, phosphates, hydrogen phosphates, hydrobromic acids, sulfates, sulfites, and nitrates; and salts of organic salts selected from, for example, malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p-toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoates such as acetates, and salts of HOOC-(CH2)n-COOH, where n can be any integer from 0 to 4. If the compound is obtained as an acid addition salt, the free base can be obtained by alkalizing a solution of the acidic salt. Conversely, if the product is a free base, the addition salt (e.g., a pharmaceutically acceptable addition salt) can be prepared by dissolving the free base in a suitable organic solvent and treating the solution with acid, consistent with the conventional process for preparing acid addition salts from basic compounds. Those skilled in the art will understand the various synthetic methods that can be used to prepare non-toxic, pharmaceutically acceptable addition salts without excessive experimentation. Similarly, "pharmaceutically acceptable ester" refers to an ester derivative formed by the formation of the small molecule inhibitors of the present invention, and "pharmaceuticalally acceptable prodrugs" include precursor compounds having the ability to form the small molecule inhibitors of the present invention in vitro and in vivo.

[0076] In this invention, "aryl" refers to a monocyclic or fused polycyclic group with 5-12 carbon atoms, possessing a fully conjugated π-electron system. Non-limiting examples of aromatic rings include benzene rings, biphenyl rings, naphthyl rings, and anthracene rings. Aromatic rings can be unsubstituted or substituted. Substituents in aromatic rings can be selected from halogens, nitro groups, amino groups, C1-C6 alkyl groups, C1-C6 alkoxy groups, halo-C1-C6 alkyl groups, halo-C1-C6 alkoxy groups, C3-C6 cycloalkyl groups, and halo-C3-C6 cycloalkyl groups.

[0077] In this invention, "heteroaryl" refers to an unsaturated carbon ring with 5-12 ring atoms, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. The heteroaryl ring can be monocyclic or bicyclic, i.e., formed by the fusion of two rings. Specific heterocyclic aryl groups can be: pyrrole, pyrazolyl, imidazolyl, furanyl, thiophene, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridinyl, pyrazinyl, pyrrole, morpholinyl, piperidinyl or piperazinyl, thiophene, benzothiophene, pyrazolyl, benzopyrazolyl, indolyl, dioxopentyl, benzo[1,3]dioxopentyl, oxazolyl, benzooxazolyl, furanyl, benzofuranyl, thiazolyl or benzothiazolyl, etc. Heterocyclic aryl groups can be unsubstituted or substituted. The substituents of the heterocyclic aryl group can be selected from halogen, nitro, amino, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, and halogenated C3-C6 cycloalkyl.

[0078] In this invention, "alkoxy" refers to an -O-alkyl group, wherein the alkyl group is as defined above. Examples of "alkoxy" as used in this invention include, but are not limited to, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and tert-butoxy, and the alkoxy group may be unsubstituted or substituted.

[0079] In this invention, "halogen" or "halogenated" means fluorine, chlorine, bromine or iodine.

[0080] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Experimental methods in the following embodiments without specific conditions are generally performed under conventional conditions or according to the manufacturer's recommendations. All raw materials without specified synthesis methods were purchased from manufacturers such as Exploration Platform, Aladdin, and Sigma-Aldrich, and were of analytical grade.

[0081] Example 1: Preparation of N-hydroxy-4-(((2-methoxyphenyl)amino)methyl)benzamide (Q-1)

[0082]

[0083] Synthesis of intermediate methyl 4-(((2-methoxyphenyl)amino)methyl)benzoate

[0084] In a 150 ml three-necked round-bottom flask, o-methoxyaniline (2 g, 16.24 mmol, 1 eq), methyl p-formylbenzoate (2.67 g, 16.24 mmol), 50 ml of 1,2-dichloroethane, and 1 ml of acetic acid were added. The mixture was stirred at 0 °C for 2 h, and then NaBH4 (1.84 g, 48.72 mmol, 3 eq) was slowly added. The mixture was then placed in an ice bath and reacted at 0 °C for 12 h. The reaction was monitored by TLC to indicate completion. Column chromatography yielded 2.7 g of a white intermediate, with a yield of 61.2%.

[0085] MS(ESI) m / z: 272.2 [M+H] + .

[0086] 1 H NMR (400MHz, DMSO-d6) δ7.93(dd,J=8.3,1.8Hz,2H),7.62–7.39(m,2H),6.84(dd,J=7.9,1.4Hz,1H),6.68(td,J=7.6,1.4Hz,1H), 6.55(td,J=7.7,1.5Hz,1H),6.35(dd,J=7.8,1.6Hz,1H),5.77(t,J=6.3Hz,1H),4.43(d,J=6.2Hz,2H),3.86(s,3H),3.84(s,3H).

[0087] Synthesis of N-hydroxy-4-(((2-methoxyphenyl)amino)methyl)benzamide (Q-1)

[0088] Hydroxylamine hydrochloride (2.00 g, 29 mmol) and potassium hydroxide (2.09 g, 32 mmol) were dissolved in methanol (50 mL) and stirred at room temperature for 2 h. The precipitated potassium chloride solid was filtered off. A white intermediate (1 g, 3.68 mmol) was added to the filtrate, and the reaction was carried out at 0 °C for 3 h. The reaction was monitored by TLC to ensure complete reaction. The pH of the reaction solution was adjusted to 7 with acetic acid, the solvent was removed under reduced pressure, ethyl acetate was added to dissolve the solid, and sonication was used to aid dissolution. The mixture was stirred at room temperature for 1 h, filtered under reduced pressure, the filter cake was discarded, the solvent was removed from the filtrate under reduced pressure, and recrystallization from ethanol yielded a white solid Q-10.57 g, yield: 57%, melting point 144.4 °C~145.5 °C. MS (ESI) m / z: 273.2 [M+H] + .

[0089] 1H NMR (400MHz, DMSO-d6) δ11.16(s,1H),9.01(s,1H),7.79–7.65(m,2H),7.42(d,J=8.0Hz,2H),6.84(dd,J=7.9,1.4Hz,1H),6.68(td,J =7.7,1.4Hz,1H),6.54(td,J=7.7,1.5Hz,1H),6.37(dd,J=7.8,1.6Hz,1H),5.70(t,J=6.3Hz,1H),4.39(d,J=6.2Hz,2H),3.84(s,3H).

[0090] Example 2: Preparation of 4-(((4-chlorophenyl)amino)methyl)-N-hydroxybenzamide (Q-2)

[0091]

[0092] The synthesis method was the same as that for Q-1, yielding 0.547 g of a white solid, with a yield of 54.7%.

[0093] Purity: 99.17%, Melting point: 156.3℃-157.7℃.

[0094] MS(ESI) m / z: 276.90 [M+H] + .

[0095] 1 H NMR(400MHz,DMSO-d6)δ11.16(s,1H),9.07(s,1H),7.80–7.70(m,2H),7.43( d,J=8.1Hz,2H),7.13–7.04(m,2H),6.63–6.51(m,3H),4.33(d,J=6.1Hz,2H).

[0096] Example 3: Preparation of N-hydroxy-4-((phenylamino)methyl)benzamide (Q-3)

[0097]

[0098] The synthesis method was the same as that for Q-1, yielding 0.437 g of a white solid, with a yield of 43.7%.

[0099] Purity: 96.69%, Melting point: 136℃-139.6℃.

[0100] MS(ESI) m / z: 243 [M+H] + .

[0101] 1H NMR (400MHz, DMSO-d6) δ7.78–7.66(m,2H),7.33(d,J=8.0Hz,2H),7.12–6.98(m,2H),6.66–6.47(m,3H),4.29(d,J=6.0Hz,2H).

[0102] Example 4: Preparation of N-hydroxy-4-((p-toluamino)methyl)benzamide (Q-4)

[0103]

[0104] The synthesis method was the same as that for Q-1, yielding 0.495 g of a white solid, with a yield of 49.5%.

[0105] Purity: 96.4%, Melting point: 152℃-156.3℃.

[0106] MS(ESI) m / z: 257 [M+H] + .

[0107] 1 H NMR (600MHz, DMSO) δ9.69(s,1H),7.67(d,J=7.8Hz,2H),7.30(d,J=7.7Hz,2H),6.84(d,J=8 .0Hz,2H),6.47(d,J=8.1Hz,2H),5.98(t,J=5.7Hz,1H),4.22(d,J=5.8Hz,2H),2.11(s,3H).

[0108] Example 5: Preparation of 4-(((2-chloro-4-fluorophenyl)amino)methyl)-N-hydroxybenzamide (Q-5)

[0109]

[0110] The synthesis method was the same as that for Q-1, yielding 0.513 g of a white solid, with a yield of 51.3%.

[0111] Purity: 96.53%, Melting point: 138.9℃-139.6℃.

[0112] MS(ESI) m / z: 295.2 [M+H] + .

[0113] 1H NMR (400MHz, DMSO-d6) δ9.12(s,1H),7.69(d,J=8.0Hz,2H),7.38(d,J=7.9Hz,2H),7.25(dd,J=8.5,3.0Hz, 1H), 6.91 (td, J=8.7, 3.0Hz, 1H), 6.48 (dd, J=9.1, 5.2Hz, 1H), 6.11 (t, J=6.2Hz, 1H), 4.42 (d, J=6.2Hz, 2H).

[0114] Example 6: Preparation of N-hydroxy-4-(((3-methoxyphenyl)amino)methyl)benzamide (Q-6)

[0115]

[0116] The synthesis method was the same as that for Q-1, yielding 0.489 g of an off-white solid, with a yield of 48.9%.

[0117] Purity: 96.48%, Melting point: 113.15℃-116℃.

[0118] MS(ESI) m / z: 273 [M+H] + .

[0119] 1 H NMR (400MHz, DMSO-d6) δ10.22(s,2H),7.74(d,J=8.0Hz,2H),7.43(d,J=8.0Hz,2H),7.05–6.90(m,1H),6 .37(t,J=6.1Hz,1H),6.20(dt,J=8.4,1.2Hz,1H),6.16–6.03(m,2H),4.32(d,J=6.0Hz,2H),3.66(s,3H).

[0120] Example 7: Preparation of N-hydroxy-4-((o-toluamino)methyl)benzamide (Q-7)

[0121]

[0122] The synthesis method was the same as that for Q-1, yielding 0.59 g of a white solid, with a yield of 59%.

[0123] Purity: 98.97%, Melting point: 123.7℃~127.9℃.

[0124] MS(ESI) m / z: 257 [M+H] + .

[0125] 1H NMR (400MHz, DMSO-d6) δ11.01(s,1H),9.22(s,1H),7.72(s,2H),7.42(d,J=7.6Hz,2H),6.99(d,J=7.1Hz,1H),6.9 0(t,J=7.6Hz,1H),6.49(t,J=7.2Hz,1H),6.34(d,J=8.0Hz,1H),5.71(s,1H),4.41(d,J=5.7Hz,2H),2.19(s,3H).

[0126] Example 8: Preparation of 4-(((3-bromo-5-fluorophenyl)amino)methyl)-N-hydroxybenzamide (Q-8)

[0127]

[0128] The synthesis method is as described in Q-1, yielding 0.463 g of a white solid, with a yield of 46.3%.

[0129] Purity: 98.89%, Melting point: 157.9℃~160.9℃.

[0130] MS(ESI) m / z: 257 [M+H] + .

[0131] 1 H NMR (400MHz, DMSO-d6) δ11.20(s,1H),9.08(s,1H),7.75(d,J=7.8Hz,2H),7.43(d,J=7.8Hz,2H),6.9 9(t,J=6.1Hz,1H),6.64(s,1H),6.57(d,J=8.3Hz,1H),6.38(d,J=12.0Hz,1H),4.36(d,J=6.0Hz,2H).

[0132] Example 9: Preparation of 4-(((3-chlorophenyl)amino)methyl)-N-hydroxybenzamide (Q-9)

[0133]

[0134] The synthesis method was the same as that for Q-1, yielding 0.654 g of a white solid, with a yield of 65.4%.

[0135] Purity: 96.34%, Melting point: 142.4℃~147.3℃.

[0136] MS(ESI) m / z: 276.9 [M+H] + .

[0137] 1H NMR (600MHz, DMSO-d6) δ11.14(s,1H),9.00(s,1H),7.76–7.62(m,2H),7.40(d,J=8.1Hz,2H),7.03(t,J=8. 0Hz,1H),6.65(t,J=6.1Hz,1H),6.55(t,J=2.2Hz,1H),6.51(dt,J=8.3,2.0Hz,2H),4.32(d,J=6.1Hz,2H).

[0138] Example 10: Preparation of 4-(([1,1'-biphenyl]-4-ylamino)methyl)-N-hydroxybenzamide (Q-10)

[0139]

[0140] The synthesis method was the same as that for Q-1, yielding 0.543 g of a white solid, with a yield of 54.3%. Purity: 91.10%, melting point: 204℃~207.1℃.

[0141] MS(ESI) m / z: 319 [M+H] + .

[0142] 1 H NMR(400MHz, DMSO-d6)δ8.60(s,1H),7.71(d,J=8.1Hz,2H),7.58–7.54(m,2H),7.40(dd,J=8.0,5.7H z,4H),7.25(dd,J=7.9,3.0Hz,3H),6.73–6.68(m,2H),6.41(t,J=5.9Hz,1H),4.30(d,J=5.9Hz,2H).

[0143] Example 11: Preparation of N-hydroxy-4-(((4-methoxyphenyl)amino)methyl)benzamide (Q-11)

[0144]

[0145] The synthesis method was the same as that for Q-1, yielding 0.478 g of a white solid, with a yield of 47.8%.

[0146] Purity: 97.24%, Melting point: 133.3℃~139.7℃.

[0147] MS(ESI) m / z: 273 [M+H] + .

[0148] 1H NMR (600MHz, DMSO) δ9.89 (s, 1H), 7.68 (d, J = 7.8Hz, 2H), 7.36 (d, J = 7.7Hz, 2H), 6.67 (d, J = 8 .6Hz,2H),6.51(d,J=8.5Hz,2H),5.84(t,J=5.6Hz,1H),4.23(d,J=5.8Hz,2H),3.60(s,3H).

[0149] Example 12: Preparation of 4-(((3-fluorophenyl)amino)methyl)-N-hydroxybenzamide (Q-12)

[0150]

[0151] The synthesis method was the same as that for Q-1, yielding 0.51 g of a white solid, with a yield of 51%.

[0152] Purity: 97.98%, Melting point: 132.4℃~135.2℃.

[0153] MS(ESI) m / z: 260.9 [M+H] + .

[0154] 1 H NMR (400MHz, DMSO-d6) δ11.18(s,1H),9.08(s,1H),7.75(d,J=8.1Hz,2H),7.45(d,J=8.1Hz,2H),7.07(q,J =7.8Hz,1H),6.69(t,J=6.1Hz,1H),6.44(dd,J=8.2,2.1Hz,1H),6.36–6.26(m,2H),4.35(d,J=6.1Hz,2H).

[0155] Example 13: Preparation of 4-(((4-fluorophenyl)amino)methyl)-N-hydroxybenzamide (Q-13)

[0156]

[0157] The synthesis method was the same as that for Q-1, yielding 0.574 g of a white solid, with a yield of 57.4%.

[0158] Purity: 94.71%, Melting point: 131.7℃~134.6℃.

[0159] MS(ESI) m / z: 260.9 [M+H] + .

[0160] 1H NMR (600MHz, DMSO) δ10.74(s,1H),9.45(s,1H),7.70(d,J=7.9Hz,2H),7.39(d,J=7.8Hz,2H),6 .87(t,J=8.8Hz,2H), 6.53(dd,J=8.6,4.4Hz,2H), 6.22(t,J=5.8Hz,1H), 4.27(d,J=5.9Hz,2H).

[0161] Example 14: Preparation of 4-(((2-fluorophenyl)amino)methyl)-N-hydroxybenzamide (Q-14)

[0162]

[0163] The synthesis method was the same as that for Q-1, yielding 0.53 g of a white solid, with a yield of 53%.

[0164] Purity: 97.29%, Melting point: 137.2℃~138.56℃.

[0165] MS(ESI) m / z: 260.9 [M+H] + .

[0166] 1 H NMR (600MHz, DMSO-d6) δ10.25(s,2H),7.70(d,J=8.1Hz,2H),7.41(d,J=8.1Hz,2H),7.01(ddd,J=12.3,8.0,1 .5Hz,1H),6.85(td,J=7.7,1.4Hz,1H),6.57–6.46(m,2H),6.26(td,J=6.3,2.2Hz,1H),4.38(d,J=6.2Hz,2H).

[0167] Example 15: Preparation of 4-(((2-chlorophenyl)amino)methyl)-N-hydroxybenzamide (Q-15)

[0168]

[0169]

[0170] The synthesis method was the same as that for Q-1, yielding 0.613 g of a white solid, with a yield of 61.3%.

[0171] Purity: 97.23%, Melting point: 57.06℃~61.4℃.

[0172] MS(ESI) m / z: 275.9 [M+H] + .

[0173] 1 H NMR (400MHz, DMSO-d6) δ10.22(s,2H),7.74(d,J=8.0Hz,2H),7.43(d,J=8.0Hz,2H),7.05–6.90(m, 1H),6.37(t,J=6.1Hz,1H),6.20(dt,J=8.4,1.2Hz,1H),6.16–6.03(m,2H),4.32(d,J=6.0Hz,2H).

[0174] Example 16: Preparation of N-hydroxy-4-((m-toluidine)methyl)benzamide (Q-16)

[0175]

[0176] The synthesis method was the same as that for Q-1, yielding 0.587 g of a white solid, with a yield of 58.7%.

[0177] Purity: 96.01%, Melting point: 120.3℃~124.5℃.

[0178] MS(ESI) m / z: 257 [M+H] + .

[0179] 1 H NMR (400MHz, DMSO-d6) δ10.12(s,1H),7.75–7.62(m,2H),7.39(d,J=8.0Hz,2H),6.90(t,J=7.7Hz,1H),6.3 9(t,J=2.0Hz,1H),6.33(dd,J=8.3,2.4Hz,2H),6.19(t,J=6.2Hz,1H),4.28(d,J=6.1Hz,2H),2.13(s,3H).

[0180] Example 17: Preparation of 4-((N-(3-fluorophenyl)cyclopropaneformamide)methyl)-N-hydroxybenzamide (Q-17)

[0181]

[0182] Preparation of methyl 4-(((3-fluorophenyl)amino)methyl)benzoate (6c)

[0183] In a 150 ml three-necked round-bottom flask, m-fluoroaniline (2 g, 18 mmol, 1 eq), methyl p-formylbenzoate (2.95 g, 18 mmol), 50 ml of 1,2-dichloroethane, and 1 ml of acetic acid were added. The mixture was stirred at 0 °C for 2 h, and then NaBH4 (2.04 g, 54 mmol, 3 eq) was slowly added. The mixture was kept in an ice bath and reacted at 0 °C for 12 h. The reaction was monitored by TLC to indicate completion. Column chromatography yielded 2.3 g of a white solid (6 c), with a yield of 49.2%.

[0184] MS(ESI) m / z: 260.2 [M+H] + .

[0185] 1H NMR (400MHz, DMSO-d6) δ8.03–7.91(m,2H),7.52(d,J=8.0Hz,2H),7.07(q,J=7.8Hz,1H),6.73(t, J=6.1Hz,1H),6.44(dd,J=8.3,2.1Hz,1H),6.40–6.27(m,2H),4.40(d,J=6.1Hz,2H),3.86(s,3H).

[0186] Preparation of methyl 4-((N-(3-fluorophenyl)cyclopropionyl)methyl)benzoate (7a)

[0187] 6c (1 g, 3.86 mmol, 1 eq), cyclopropionyl chloride (0.443 g, 4.24 mmol, 1.1 eq), triethylamine (0.78 g, 7.71 mmol, 2 eq) and 50 mL of dichloromethane were placed in a 150 mL single-necked round-bottom flask and stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate completion. Column chromatography was used to separate the product into a white solid 7a (0.7 g), yielding 55.6%.

[0188] MS(ESI) m / z: 328.40 [M+H] + .

[0189] 1H NMR(400MHz,DMSO-d6)δ7.92–7.84(m,2H),7.41(td,J=8.2,6.7Hz,1H),7.32 (d,J=8.2Hz,2H),7.21(dt,J=10.1,2.2Hz,1H),7.15(tdd,J=8.6,2.7,0.9Hz ,1H),7.08(ddd,J=7.9,2.1,0.9Hz,1H),4.99(s,2H),3.82(s,3H),1.48(tt, J=7.6,4.9Hz,1H),0.77(dd,J=4.8,3.0Hz,2H),0.70(dt,J=8.0,3.3Hz,2H).

[0190] Preparation of 4-((N-(3-fluorophenyl)cyclopropanecarboxamide)methyl)-N-hydroxybenzamide (Q-17)

[0191] Hydroxylamine hydrochloride (2.00 g, 29 mmol) and potassium hydroxide (2.09 g, 32 mmol) were dissolved in methanol (50 mL) and stirred at room temperature for 2 h. The precipitated potassium chloride solid was filtered off. 7a (0.5 g, 1.53 mmol) was added to the filtrate and reacted at 0 °C for 3 h. The reaction was monitored by TLC to ensure complete reaction. 173 mg of granular white solid was obtained by recrystallization from ethanol. Yield: 34.6%, melting point: 174.4 °C–175.7 °C.

[0192] MS(ESI) m / z: 229 [M+H] + .

[0193] 1 H NMR (400MHz, DMSO-d6) δ11.16(s,1H),9.01(s,1H),7.66(d,J=8.1Hz,2H),7.42(q,J=7.9Hz,1H),7.23(t,J=8.7Hz,3H ),7.19–7.12(m,1H),7.09(d,J=7.7Hz,1H),4.96(s,2H),1.42(s,1H),0.94–0.82(m,2H),0.70(dd,J=7.5,3.4Hz,2H).

[0194] Example 18: Preparation of N-(3-fluorophenyl)-N-(4-(hydroxycarbamoyl)benzyl)benzamide (Q-18)

[0195]

[0196] The preparation method was the same as that for Q-17, yielding 0.17 g of an off-white solid, with a yield of 34%.

[0197] Purity: 98.97%, Melting point: 197℃~208℃.

[0198] MS(ESI) m / z: 365 [M+H] + .

[0199] 1 H NMR (600MHz, DMSO-d6) δ11.17(s,1H),9.01(s,1H),7.73–7.63(m,2H),7.42–7.35(m,4H),7.35–7.29(m,1H),7.27(dd,J=8.1,6.6Hz, 2H),7.18(td,J=8.2,6.6Hz,1H),7.09(dt,J=10.3,2.3Hz,1H),6.94(td,J=8.5,2.5Hz,1H),6.87(dd,J=8.0,2.0Hz,1H),5.17(s,2H).

[0200] Example 19: Preparation of 3,4-dichloro-N-(3-fluorophenyl)-N-(4-(hydroxycarbamoyl)benzyl)benzamide (Q-19)

[0201]

[0202]

[0203] The preparation method is the same as Q-17, yielding an oily substance 4-((3,4-dichloro-N-(3-fluorophenyl)benzoyl)methyl)-N-hydroxy-benzamide, which is then salted in ethyl acetate hydrochloride solution to give 0.214 g of a white solid, with a yield of 27%.

[0204] Purity: 90.77%, Melting point: 250℃.

[0205] MS(ESI) m / z: 433.40 [M-HCl].

[0206] 1 H NMR (400MHz, DMSO-d6) δ11.19(s,1H),7.68(d,J=1.7Hz,1H),7.67(d,J=2.3Hz,2H),7.52(d,J=8.3Hz,1H),7.40–7.36(m ,2H),7.29(dd,J=8.4,2.0Hz,1H),7.24–7.16(m,2H),6.99(tdd,J=8.4,2.3,1.0Hz,1H),6.95–6.90(m,1H),5.14(s,2H).

[0207] Example 20: Preparation of 4-((1-(3-fluorophenyl)-3-phenylurea)methyl)-N-hydroxybenzamide (Q-20)

[0208]

[0209] Preparation of methyl 4-(((3-fluorophenyl)amino)methyl)benzoate (6c)

[0210] For example, in general method six, m-fluoroaniline (2 g, 18 mmol, 1 eq), methyl p-formylbenzoate (2.95 g, 18 mmol), 50 ml of 1,2-dichloroethane, and 1 ml of acetic acid were added to a 150 ml three-necked round-bottom flask. The mixture was stirred at 0 °C for 2 h, and then NaBH4 (2.04 g, 54 mmol, 3 eq) was slowly added. The mixture was then placed in an ice bath and reacted at 0 °C for 12 h. The reaction was monitored by TLC to indicate completion, and the product was separated by column chromatography to obtain a white solid 6c 2.3 g, with a yield of 49.2%.

[0211] MS(ESI) m / z: 260.2 [M+H] + .

[0212] 1H NMR (400MHz, DMSO-d6) δ8.03–7.91(m,2H),7.52(d,J=8.0Hz,2H),7.07(q,J=7.8Hz,1H),6.73(t, J=6.1Hz,1H),6.44(dd,J=8.3,2.1Hz,1H),6.40–6.27(m,2H),4.40(d,J=6.1Hz,2H),3.86(s,3H).

[0213] Preparation of methyl 4-((1-(3-fluorophenyl)-3-phenylurea)methyl)-benzoate (7c)

[0214] 6c (1 g, 3.86 mmol, 1 eq), aniline (0.36 g, 3.86 mmol, 1 eq), triphosgene (1.37 g, 4.63 mmol, 1.2 eq), triethylamine (0.78 g, 7.71 mmol, 2 eq) and 50 mL of dichloromethane were placed in a 150 mL single-necked round-bottom flask and stirred at room temperature for 6 h. The reaction was monitored by TLC to indicate completion. 0.67 g of intermediate 7c was obtained by column chromatography, with a yield of 45.9%.

[0215] MS(ESI) m / z: 379.1 [M+H] + .

[0216] 1H NMR (600MHz, DMSO-d6) δ8.52(s,1H),8.03–7.92(m,2H),7.51(td,J=6.2,3.0Hz,4H),7.43(td,J=8.2,6.8Hz,1H),7.35–7.28(m,2H),7. 25(dt,J=10.7,2.3Hz,1H),7.14(ddd,J=8.0,2.1,0.9Hz,1H),7.13–7.08(m,1H),7.04(tt,J=7.3,1.1Hz,1H),5.12(s,2H),3.89(s,3H).

[0217] Preparation of 4-((1-(3-fluorophenyl)-3-phenylurea)methyl)-N-hydroxybenzamide (Q-20)

[0218] The preparation method is as described in Q-17. After separation by column chromatography, 0.231 g of brown solid was obtained, with a yield of 23.1%.

[0219] Purity: 99.3%, Melting point: 72.5℃~76.3℃.

[0220] MS(ESI) m / z: 380 [M+H] + .

[0221] 1 H NMR (600MHz, DMSO-d6) δ11.16(s,1H),8.99(s,1H),8.47(s,1H),7.69(d,J=8.1Hz,2H),7.49–7.41(m,2H),7.37(dd,J=7.6,5.7Hz,3H),7.24(dd ,J=8.4,7.2Hz,2H),7.18(dt,J=10.7,2.3Hz,1H),7.08(dd,J=8.0,2.0Hz,1H),7.04(td,J=8.5,2.6Hz,1H),6.98(t,J=7.3Hz,1H),5.01(s,2H).

[0222] Example 21: HDAC inhibitory activity of the compound

[0223] The inhibitory effects of the compounds on HDAC6 and HDAC1 targets were detected using the established experimental platform and testing conditions, with HDAC6 inhibitors Rocilinostat and SW-100 used as positive control compounds.

[0224] 1) Reagents and consumables

[0225] Table 2

[0226]

[0227] 2) Instruments

[0228] Table 3

[0229]

[0230] 3) HDAC enzyme preparation

[0231] HDAC6 enzyme inhibition assay: Prepare a 40mM DMSO solution from the sample and store it in the dark for later use.

[0232] HDAC1 enzyme inhibition experiment: The compounds were prepared as 20 mM DMSO solutions and stored in the dark for later use.

[0233] 4) HDAC6 enzymatic reaction process

[0234] a. Prepare a 1× reaction solution.

[0235] b. Preparation of compound concentration gradients: The final concentration of the analyte was initially 80 nM, diluted 4-fold, resulting in 5 concentrations, and a single-well assay was performed. The final concentration of the positive compound was initially 3 μM, diluted 3-fold, resulting in 10 concentrations, and a replicate assay was performed. Solutions were serially diluted to the corresponding 100-fold final concentrations in a 384-well Source plate, and then 250 nL was transferred to the 384-well reaction plate using an Echo 550. 250 nL of 100% DMSO was transferred to both the Max and Min wells.

[0236] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0237] Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to each Min well. Incubate at room temperature for 15 minutes.

[0238] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0239] Add 10 μL of a 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0240] g uses Synergy to continuously read fluorescence signals.

[0241] 5) HDAC1 enzymatic reaction process

[0242] a. Prepare a 1× reaction solution.

[0243] b. Preparation of compound concentration gradients: The final concentration of the analyte was initially 2 μM, diluted 10-fold, and four concentrations were set up for single-well detection; the positive control concentration was initially 3 μM, diluted 3-fold, and ten concentrations were set up for replicate detection. Solutions were serially diluted to the corresponding 100-fold final concentrations in a 384-well Source plate, and then 250 nmol was transferred to the 384-well reaction plate using an Echo 550. 250 nmol of 100% DMSO was transferred to both the Max and Min wells.

[0244] c. Prepare a 1.67× enzyme solution using a 1× reaction solution.

[0245] Add 15 μL of 1.67× enzyme solution to each well; add 15 μL of 1.6× reaction solution to each Min well. Incubate at room temperature for 15 minutes.

[0246] e. Prepare a 2.5× substrate mixture using 1× reaction solution.

[0247] Add 10 μL of a 2.5× substrate mixture to each well of the reaction plate to initiate the reaction.

[0248] g uses Synergy to continuously read fluorescence signals.

[0249] 5) Data Analysis

[0250] The slope is obtained by selecting the linear response segment. The percentage inhibition rate is then calculated using the following formula:

[0251]

[0252] Where: Mean(Max) is the mean slope value of each Max well (containing DMSO and enzyme); Mean(Min) is the mean slope value of each Min well (without enzyme); Sample Signal is the slope value of the compound well.

[0253] Fitting dose-response curves: Using the log value of compound concentration as the X-axis and the corresponding percentage inhibition rate as the Y-axis, the dose-response curves were fitted using the log(inhibitor) vs. response-variable slope function of GraphPad Prism 5 to obtain the IC50 of each compound inhibiting enzyme activity. 50 value.

[0254] The experimental results are shown in Table 4:

[0255] Table 4

[0256]

[0257]

[0258] Experimental results show that the compound of this invention exhibits strong inhibitory activity against HDAC6 (IC50). 50 <15nM), compared with HDAC1, some compounds have stronger inhibitory activity against HDAC6, showing a certain degree of selectivity.

[0259] Example 22 Compound Anti-cell proliferation activity

[0260] Using the CCK-8 assay, with SW-100 and Ricolinostat as positive controls, a subset of the compounds of this invention were selected to test their antiproliferative activity against SH-SY5Y cells (human neuroblastoma cell line) and MRC-5 human normal embryonic lung fibroblasts at a concentration of 20 μM (replicas). The experimental data are shown in Table 5.

[0261] Table 5 Results of anti-cell proliferation assay (Inh%in 20μM)

[0262]

[0263]

[0264] Experimental results showed that at a concentration of 20 μM, the compound of the present invention generally had a weak antiproliferative effect on SH-SY5Y cells and MRC-5 human normal embryonic lung fibroblasts, and was weaker than the positive control drugs SW-100 and Ricolinostat, indicating that it had low cytotoxicity.

[0265] Example 23: Protective effect of compound on L-glutamate-induced SH-SY5Y cell damage model

[0266] Using SW-100 and Ricolinostat as positive control drugs, some compounds of this invention were selected to test their protective effect on an L-glutamate-induced SH-SY5Y cell damage model.

[0267] I. Experimental Methods

[0268] Day 0: Cell inoculation

[0269] 1. Centrifuge the suspended cells and resuspend them in growth medium, then count them using a cell counter.

[0270] 2. Dilute the cell suspension in the growth medium to the required density.

[0271] 3. Seed 100 μL of cells into 96-well plates containing growth medium according to the plate diagram. Medium is used only as a background control (Min).

[0272] Incubate overnight at 4.37℃ and 5% CO2.

[0273] Day 1: Compound Processing

[0274] 1. Prepare a 200-fold compound solution in DMSO.

[0275] 2. The compound containing the growth medium was diluted to a final concentration by adding 3 μL of a 200-fold dilution of the compound solution to 197 μL of growth medium.

[0276] 3. Add 50 μL of the diluted compound solution to the cells and incubate at 37°C and 5% CO2 for 48 h.

[0277] 4. Remove the growth medium

[0278] 5. Add 150 μL of culture medium containing 1X cpds and 16 mM L-glutamate to the cells and incubate at 37°C and 5% CO2 for 24 hours.

[0279] Day 4: Measurement

[0280] 1. Equilibrate the test plate to room temperature before measurement.

[0281] 2. Add 40 μL to each well. Reagent.

[0282] 3. Mix the contents on an orbital oscillator for 2 minutes to induce cell lysis.

[0283] 4. Incubate at room temperature for 60 minutes to stabilize the luminescence signal.

[0284] 5. Record the luminescence on Envision.

[0285] II. Data Analysis

[0286] (1) Use GraphPad Prism 5.

[0287] (2)%Inh = (maximum signal - composite signal) / (maximum signal - minimum signal) × 100.

[0288] (3) The maximum signal comes from the effect of DMSO.

[0289] (4) The minimum signal is obtained solely by the action of the medium.

[0290] The experimental results are shown in Table 6.

[0291] Table 6

[0292]

[0293]

[0294]

[0295] Results from an in vitro glutamate-induced neuronal injury model showed that:

[0296] 1) Glutamate (15 nM) can significantly reduce the viability of neurons. SW-100 can improve the viability of SH-SY5Y cells damaged by L-glutamate, reduce the cytotoxic effects of L-glutamate, and has a certain neuroprotective effect, while the neuroprotective effect of Ricolinostat is weaker and even shows some toxicity.

[0297] 2) The compounds of this invention can enhance the viability of SH-SY5Y cells and show a protective effect against L-glutamate-induced SH-SY5Y cell damage, which is superior to or equivalent to SW-100.

[0298] Example 24: Experiment on the effect of compounds on hERG potassium channels

[0299] The potential cardiotoxic side effects of some compounds of this invention were preliminarily investigated in vitro using the hERG potassium channel inhibition assay. The experimental procedure is as follows:

[0300] 1) Cell preparation

[0301] CHO-hERG cells were cultured at 175 cm⁻¹ 2 In the culture flask, when the cell density grows to 60-80%, remove the culture medium, wash once with 7 mL PBS, and then add 3 mL Detachin for digestion.

[0302] After complete digestion, add 7 mL of culture medium to neutralize, then centrifuge, aspirate the supernatant, and resuspend in 5 mL of culture medium to ensure a cell density of 2–5 × 10⁻⁶ cells / mL. 6 / mL.

[0303] 2) Electrophysiological recording process

[0304] The entire process of single-cell high-impedance sealing and whole-cell pattern formation was automated by the Qpatch instrument. After obtaining the whole-cell recording pattern, the cells were clamped at -80 mV. Before applying a 5-second +20 mV depolarization stimulus, a 50-millisecond -50 mV pre-voltage was applied, followed by repolarization to -50 mV for 5 seconds, and then back to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular fluid was applied for another 2 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was administered for 2 minutes. After all concentrations were administered, 10 μM Cisapride, a positive control compound, was administered. At least 3 cells (n≥3) were tested for each concentration.

[0305] 3) Compound preparation

[0306] The stock solution of the compound was diluted with extracellular fluid. 2 μL of the stock solution was added to 998 μL of extracellular fluid, and then the solution was serially diluted 5-fold in extracellular fluid containing 0.2% DMSO to obtain the final concentration to be tested. Experimental data were analyzed using XLFit software.

[0307] The experimental results are shown in the table below:

[0308]

[0309] hERG experimental results showed that the compounds of this invention exhibited inhibitory activity against hERG potassium ion channels greater than 20 μM, suggesting that the compounds of this invention have low potential cardiotoxicity.

[0310] Example 25 Pharmaceutical Composition 1

[0311] The compound Q-3 prepared in Example 3 was mixed with a filler, a disintegrant, and a lubricant, granulated, and tableted to obtain a pharmaceutical composition 1 with compound Q-3 as the active ingredient.

[0312] Example 26 Pharmaceutical Composition 2

[0313] The compound Q-14 prepared in Example 14 was mixed with a solvent and a stabilizer, filtered, and packaged to obtain pharmaceutical composition 2 with compound Q-14 as the active ingredient.

[0314] Example 27 Pharmaceutical Composition 3

[0315] Compound Q-3 prepared in Example 3 and compound Q-17 prepared in Example 17 were mixed with filler, disintegrant, and lubricant, granulated, and tableted to obtain pharmaceutical composition 3 with compounds Q-3 and Q-17 as active ingredients.

[0316] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hydroxamic acid compound, characterized in that, It is the following compounds or their pharmaceutically acceptable salts: 。 2. The isohydroxamic acid compound according to claim 1, characterized in that, The pharmaceutically acceptable salt is an anionic salt formed by reacting any one of compounds Q-5, Q-6, Q-12, Q-14, and Q-15 with hydrochloride, hydrobromide, sulfate, acetate, trifluoroacetate, citrate, tartrate, maleate, fumarate, methanesulfonate, malate, p-toluenesulfonate, or oxalate; or a cationic salt formed by reacting any one of compounds Q-5, Q-6, Q-12, Q-14, and Q-15 with sodium ion solution or potassium ion solution.

3. The use of the isohydroxamic acid compound according to any one of claims 1-2 in the preparation of histone deacetylase inhibitors or drugs for the treatment of neurodegenerative diseases; wherein the histone deacetylase inhibitor is an HDAC6 inhibitor.

4. The application according to claim 3, characterized in that, The neurodegenerative diseases mentioned include one or more of the following: cerebral ischemia, brain injury, epilepsy, Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, spinocerebellar ataxia, and Pick's disease.

5. A pharmaceutical composition comprising at least one active ingredient and one or more pharmaceutically acceptable excipients; said active ingredient comprising any one of the isohydroxamic acid compounds according to claims 1-2.

6. The pharmaceutical composition according to claim 5, characterized in that, Pharmaceutically acceptable excipients include one or more of the following: diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, flavorings, and sweeteners.

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