An HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor and its preparation method and application

Abstract

This invention belongs to the field of biopharmaceuticals and relates to an HDAC6 / JNK3 dual-targeting anti-Alzheimer's disease inhibitor, its preparation method, and its application. The chemical structure is shown in Formula I: #imgabs0#, where R is H or a halogen; and X is a linker. This invention utilizes an indazole core as the cap and hydroxamic acid as the ZBG, using linkers of varying lengths and types to synthesize a series of compounds that can act as HDAC6 / JNK3 dual-targeting inhibitors, effectively treating Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology and relates to an HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor and its preparation method and application. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Although there are currently approved drugs for alleviating Alzheimer's disease (AD) symptoms, these drugs cannot reverse the pathological progression. In the past two years, aducanumab and lencanerumab, which have been developed to target Aβ oligomers, have been shown to effectively reduce Aβ levels. However, it is important to note that these drugs may cause adverse reactions such as amyloid-related imaging abnormalities and mental confusion. Therefore, it is necessary to continue exploring new, safer and more effective treatments with fewer side effects.

[0004] Single-target drugs are not very effective in treating AD, but by combining the key pharmacophores of inhibitors of different targets, the drugs can inhibit multiple targets simultaneously, thereby producing a synergistic effect. Compared with combination therapy, they have better patient compliance and pharmacokinetic parameters, so more and more medicinal chemists are beginning to pay attention to multi-target drugs. However, multi-target drugs are single-component drugs that selectively act on two or more molecular targets at the same time. The technical difficulty of developing a multi-target drug that has multiple target activities and good drugability is much higher than that of combination therapy. The difficulty includes: the selection of target combinations, the selection of pharmacophores, and the splicing and fusion of pharmacophores. Summary of the Invention

[0005] To address the deficiencies of the prior art, the present invention provides a dual-target HDAC6 / JNK3 anti-Alzheimer's disease inhibitor, its preparation method, and its application. This invention utilizes an indazole core as a surface recognition group (Cap) and a hydroxamic acid as a zinc ion chelating group (ZBG). By varying the length and type of linker, a series of compounds were synthesized. These compounds can be used as dual-target HDAC6 / JNK3 inhibitors, effectively treating Alzheimer's disease.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In the first aspect, an HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor, the chemical structure of which is shown in Formula I:

[0008]

[0009] Wherein, R is H or halogen; X is a connecting fragment selected from:

[0010] HDAC6 is overexpressed in the brain tissue of Alzheimer's patients, causing histone deacetylation, which compacts chromosome structure and prevents normal transcription. Furthermore, deacetylation of α-tubulin impairs normal mitochondrial transport, inducing the production of oxygen free radicals and the hyperphosphorylation of Tau protein, which aggregates into neurofibrillary tangles and causes neurotoxicity. JNK3 is primarily expressed in the human brain and is closely associated with cognitive impairment. Studies have shown that activation of HDAC6 and JNK3 can lead to abnormal fragmentation of amyloid proteins and hyperphosphorylation of Tau protein. Furthermore, HDAC6 and JNK3 can also cause neuronal apoptosis by causing tubulin deacetylation and c-Jun phosphorylation, respectively. Furthermore, HDAC6 inhibitors can inhibit the transcription of the JNK upstream kinase MKK7 and significantly reduce the phosphorylation levels of JNK and c-Jun. Therefore, the present invention dually targets HDAC6 and JNK3.

[0011] Based on the HDAC6 / JNK3 dual-target, the present invention adopts the HDAC pharmacophore model, retains the indazole core of the JNK3 inhibitor 25c as the Cap, uses hydroxamic acid as the ZBG, and synthesizes a series of compounds by connecting fragments. These compounds can be used as HDAC6 / JNK3 dual-target inhibitors, thereby effectively treating Alzheimer's disease.

[0012] In some embodiments, R is H, F, or Cl.

[0013] Studies have shown that the type of linker fragment is related to the HDAC inhibitory activity of the compound. In some embodiments, the linker fragment is selected from Studies have shown that when this linker fragment is selected, it has better HDAC mixed enzyme inhibitory activity. More specifically, the chemical structures are shown as follows:

[0014]

[0015] Specifically, selected from the compounds shown in the following table:

[0016]

[0017]

[0018] In a second aspect, a method for preparing the HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor according to the first aspect of the present invention comprises the steps of using raw materials 1 and 2 as starting materials to obtain the compound of formula I according to the following reaction scheme;

[0019]

[0020] In some embodiments, raw material 1 and raw material 2 undergo a nucleophilic substitution reaction to generate intermediate 3, intermediate 3 is deprotected to obtain intermediate 4, intermediate 4 is reacted with tert-butyl piperazine-1-carboxylate to generate intermediate 5 by amide condensation, intermediate 5 is deprotected to obtain intermediate 6, intermediate 6 is reacted with a carboxylic acid derivative by amide condensation to generate intermediate 7, and intermediate 7 is reacted in a methanol solution of potassium hydroxylamine to generate hydroxamic acid to obtain the compound of formula I;

[0021] Among them, the chemical structural formula of the carboxylic acid derivative is Wherein, X is as described above.

[0022] Specifically, raw material 1 and raw material 2 undergo amide condensation reaction under the action of potassium iodide and N,N-diisopropylethylamine (DIPEA) to generate intermediate 3.

[0023] Specifically, the reaction of removing the protecting group is carried out under the action of trifluoroacetic acid.

[0024] Specifically, the amide condensation reaction of intermediate 4 and tert-butyl piperazine-1-carboxylate is carried out under the conditions of HOBT, EDC.HCl and NMM.

[0025] Specifically, the amide condensation reaction of intermediate 6 and a carboxylic acid derivative is carried out under the action of HOBT, EDC.HCl and NMM.

[0026] In a third aspect, a composition comprises the HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor described in the first aspect of the present invention or a pharmaceutically acceptable salt thereof.

[0027] As used herein, a "pharmaceutically acceptable salt" refers to a salt of a compound that, within the scope of reliable pharmaceutical evaluations, is suitable for contact with human or lower animal tissues without undue toxicity, irritation, or allergic reaction, presents a reasonable benefit-risk ratio, is generally water- or oil-soluble or dispersible, and is effective for its intended use. This includes pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts that are compatible with the chemical properties of the compound of Formula I and are suitable for their intended use. For a list of suitable salts, see SM Birge et al., J. Pharm. Sci., 1977, 66, pp. 1-19.

[0028] The pharmaceutical composition of the present invention can be administered in any of the following ways: oral, spray inhalation, rectal, nasal, vaginal, topical, parenteral, such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal or intracranial injection or infusion, or by means of an explanted reservoir, wherein oral, intramuscular, intraperitoneal or intravenous administration is preferred.

[0029] In a fourth aspect, a pharmaceutical preparation comprises the HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor described in the first aspect of the present invention or the composition described in the third aspect of the present invention, and at least one pharmaceutically acceptable carrier or excipient.

[0030] The pharmaceutical compositions of the present invention are generally safe, non-toxic and biologically desirable. The pharmaceutically acceptable carriers or excipients of the present invention are non-toxic and safe, and their combination with the compounds of the present invention is also non-toxic and safe. The pharmaceutically acceptable carriers and excipients of the present invention are generally well known to those skilled in the art, or can be determined by those skilled in the art based on actual conditions. Examples of suitable carriers and excipients include glucose, water, glycerol, ethanol, propylene glycol, corn starch, gelatin, lactose, sucrose, alginic acid, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, cross-linked sodium carboxymethyl cellulose and sodium starch glycolate, polysorbate 80, polyethylene glycol 300, polyethylene glycol 400, cyclodextrin or its derivatives, such as (2-hydroxypropyl)-cyclodextrin) and (2-hydroxyethyl)-cyclodextrin, which is also known as HPCD, pegylated castor oil, poloxamer (such as poloxamer 407 or 188); hydrophilic carriers, hydrophobic carriers, or combinations thereof. Hydrophobic carriers include, for example, fat emulsions, lipids, pegylated phospholipids, biocompatible polymers, lipid spheres, liposomes, vesicles, polymer matrices, particles, etc. In addition, it is understood by those skilled in the art that diluents are included within the scope of the terms carrier and excipient.

[0031] The content of the carrier or excipient in the pharmaceutical composition can be 1wt%-98wt%, usually accounting for about 80wt%.For convenience, local anesthetics, preservatives, buffers, etc. can be directly dissolved in the carrier.

[0032] Pharmaceutical dosage forms can be liquid or solid. Liquid dosage forms can be true solutions, colloids, microparticles, emulsions, or suspensions. Other dosage forms include tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, suppositories, lyophilized powder injections, inclusion compounds, implants, patches, and liniments.

[0033] In a fifth aspect, a use of the HDAC6 / JNK3 dual-target anti-Alzheimer's disease inhibitor described in the first aspect of the present invention, the composition described in the third aspect of the present invention, or the pharmaceutical preparation described in the fourth aspect of the present invention in the preparation of a drug for treating Alzheimer's disease.

[0034] The beneficial effects of the present invention are:

[0035] The compounds provided by the present invention can effectively inhibit the activity of histone deacetylase 6 (HDAC6) and c-Jun amino-terminal kinase (JNK3). At the same time, some of the compounds provided by the present invention can also effectively inhibit Cu 2+ Induced Aβ 1-42 Aggregation, and the protection of PC12 neural cells is obvious. DETAILED DESCRIPTION

[0036] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0037] Example 1: Synthesis of compounds N1-N30 of formula I and lead compound 25c:

[0038] (1) Synthesis steps of intermediate compounds 6a, 6b, and 6c:

[0039]

[0040] Reagents and conditions: (i) KI, DIPEA, dichloromethane, 80°C; (ii) trifluoroacetic acid, dichloromethane, room temperature; (iii) tert-butyl piperazine-1-carboxylate, HOBT, EDC.HCl, NMM, DMF, 0°C to room temperature; (iv) trifluoroacetic acid, dichloromethane, room temperature.

[0041] Step 1: Synthesis of 3a. 4-(Chloromethyl)benzoic acid tert-butyl ester (1.87 g, 8.26 mmol) was added to 30 mL of DMF containing 1H-indazol-3-amine (1 g, 7.51 mmol), KI (124.7 mg, 0.751 mmol), and N,N-diisopropylethylamine (DIPEA, 2.91 g, 22.53 mmol). The mixture was heated to 80°C and stirred overnight under nitrogen. The reaction was monitored by TLC. Ethyl acetate (125 mL) and water (100 mL) were added to the mixture for extraction, and the organic layer was separated. The aqueous layer was washed with ethyl acetate (125 mL x 3). The organic layers were combined, washed with water (100 mL x 2) and brine (100 mL), dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was purified by column chromatography (petroleum ether:ethyl acetate = 5:1 to 4:1) to give a white solid 3a (1.25 g, yield 51.4%).

[0042] Step 2: Synthesis of 4a. CF3COOH (0.7 g, 6.19 mmol) was added dropwise to 10 mL of dichloromethane containing intermediate 3a (0.4 g, 1.24 mmol) and stirred at room temperature for 3-4 hours. After completion of the reaction, the reaction solvent was removed in vacuo to obtain the crude product, which was purified by column chromatography (DCM:MeOH = 30:1) to afford 4a as a white solid (0.3 g, 90.9% yield).

[0043] Step 3: Synthesis of 5a. tert-Butyl piperazine-1-carboxylate (69.8 mg, 0.38 mmol) was added to 10 mL of DMF containing intermediate 4a (50 mg, 0.19 mmol), HOBT (32.9 mg, 0.24 mmol), EDC.HCl (46.6 mg, 0.24 mmol) and NMM (83.3 mg, 0.82 mmol). The mixture was stirred at room temperature for 4 hours. The reaction was then monitored by TLC, and ethyl acetate (30 mL) and water (60 mL) were added. The organic layer was separated, and the aqueous layer was further extracted with ethyl acetate (30 mL×3); the organic layers were then combined, washed with saturated sodium chloride solution (30 mL), dried over anhydrous Na2SO4, and concentrated in vacuo to obtain the crude product 5a.

[0044] Step 4: Synthesis of 6a: Intermediate 5a was dissolved in DCM (30 mL) without purification, and TFA (68.1 mg, 0.59 mmol) was added dropwise and stirred at room temperature for 2 h. The solvent was removed in vacuo, and an appropriate amount of saturated aqueous NaHCO₃ was added to adjust the pH to 5. The reaction solution was then extracted with DCM (120 mL x 3). The organic layers were combined, washed with brine, dried over Na₂SO₄, concentrated in vacuo, and purified by column chromatography (DCM / MeOH = 20:1) to obtain a white solid 6a (40 mg, 62.9% yield). The synthesis of 6b and 6c was similar to the above synthetic route.

[0045] (2) Synthesis of compounds N1-N30 of formula I and lead compound 25c:

[0046]

[0047] Reagents and conditions: (i) HOBT, EDC.HCl, NMM, DMF, 0°C to room temperature; (ii) NH2OK, MeOH, 0°C to room temperature.

[0048] 8-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-N-hydroxy-8-oxooctamide (N1)

[0049] Under ice-bath conditions, intermediate 6a (0.15 g, 0.82 mmol), HOBT (0.144 g, 1.07 mmol), EDC.HCl (0.20 g, 1.07 mmol) and NMM (0.47 g, 4.70 mmol) were added to 12 mL of DMF and stirred for activation for half an hour. Then, monomethyl suberate (0.33 g, 0.99 mmol) was added to the above solution and reacted at room temperature for 2 h. After the reaction was completed as monitored by TLC, ethyl acetate (20 mL) and water (50 mL) were added. The organic layer was separated, and the aqueous layer was further extracted with ethyl acetate (20 mL × 3); then, the organic layers were combined, washed with saturated brine (30 mL × 3), dried over anhydrous Na2SO4, and concentrated in vacuo to obtain crude product 7a-X1, ESI-MS m / z: 506.31 [M+H] + , 504.57[MH] - .

[0050] Preparation of NH2OK solution: First, prepare Solution A by grinding 14.18 g (0.25 mol) of potassium hydroxide into a powder, then dissolving it in 35 mL of methanol by ultrasonic oscillation in an ice bath. Next, prepare Solution B by dissolving 11.68 g (0.16 mol) of NH2OH·HCl in 60 mL of methanol. Finally, slowly add Solution A to Solution B in an ice bath, stir at room temperature for 15 min, filter the resulting potassium hydroxylamine solution, and store in a refrigerator at 4°C.

[0051] 8 mL of NH2OK solution was added to 7a-X1, which was then purified using HPLC (MeOH:H2O=3:2) and dried by rotary evaporation to give 30 mg of white solid N1 with a yield of 41% and a melting point of 88-90°C. ESI-MS m / z: 507.21 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ11.39(s,1H),10.32(s,1H),8.65(s,1H),7.75(d,J=8.2Hz,1H),7.50–7.41(m,2H),7.40–7.33(m,2H),7.22( d,J=2.8Hz,2H),6.90(s,1H),6.61(s,1H),4.55–4.48(m,2H),3.47(s,8H),2.29(s,2H),2.01–1.88(m,2H),1.46(s,4H),1.24(s,4H). 13C NMR(150MHz,DMSO-d6)δ171.36,169.78,169.59,150.15,143.39,142.28,134.27,127.80,1 27.45,126.80,120.50,117.90,114.09,109.83,46.85,32.70,28.92,28.89,25.49,25.07.

[0052] 3-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclopentane-1-carboxamide (N2)

[0053] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 3-(ethoxycarbonyl)cyclopentane-1-carboxylic acid as starting materials to give 95 mg of a white solid product with a yield of 50.10% and a melting point of 194-196°C. ESI-MS m / z: 491.28 [M+H] + , 488.35[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.33(s,1H),10.36(s,1H),8.65(s,1H),7.69(d,J=8.1H z,1H),7.41(d,J=7.9Hz,2H),7.30(d,J=7.8Hz,2H),7.17(d,J=3.7Hz,2H),6.89– 6.80(m,1H),6.56(t,J=6.1Hz,1H),4.45(d,J=6.1Hz,2H),3.44(s,8H),2.95(s,1 H), 2.31 (d, J = 37.4Hz, 1H), 1.80 (t, J = 10.4Hz, 2H), 1.64 (dd, J = 19.7, 11.9Hz, 4H). 13 CNMR(100MHz,DMSO-d6)δ173.24,171.71,170.80,169.75,150.13,143.38,142.26,134.25,127. 79,127.45,126.78,120.49,117.88,114.08,109.82,46.84,42.21,40.87,34.16,29.84,29.36.

[0054] 4-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclohexane-1-carboxamide (N3)

[0055] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 4-(methoxycarbonyl)cyclohexane-1-carboxylic acid as starting materials to give 85 mg of a white solid product with a yield of 55.40% and a melting point of 188-190°C. ESI-MS m / z: 505.36 [M+H] + , 503.42[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.40(s,1H),10.35(d,J=17.1Hz,1H),8.64(d,J=23.4Hz,1H ),7.79–7.72(m,1H),7.51–7.44(m,2H),7.37(dd,J=8.1,2.3Hz,2H),7.24(t,J=2.9Hz ,2H),6.91(d,J=8.2Hz,1H),6.63(s,1H),4.52(d,J=6.6Hz,2H),3.47(s,8H),2.68(s ,1H),1.90(d,J=30.8Hz,1H),1.67(t,J=13.5Hz,3H),1.47(s,3H),1.40–1.19(m,2H). 13 C NMR(100MHz,DMSO-d6)δ173.85,172.42,169.71,150.13,143.40,142.26,134.23,127.7 8,127.45,126.78,120.49,117.88,114.08,109.82,46.83,28.68,28.56,26.42,26.28.

[0056] 4-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N4)

[0057] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 4-(methoxycarbonyl)benzoic acid as starting materials to give 60 mg of a white solid product with a yield of 55.40% and a melting point of 178-180°C. ESI-MS m / z: 499.34 [M+H] + , 497.43[MH] -.1H NMR (600MHz, DMSO-d6) δ11.37(s,1H),11.30(s,1H),9.11(s,1H),7.80(d,J=7.8Hz,2H),7.75(d,J=8.0Hz,1H),7.48(t,J=8.2Hz,4H),7.37(d, J=7.7Hz,2H),7.26–7.20(m,2H),6.90(ddd,J=7.9,4.7,2.9Hz,1H),6.60(t,J=6.1Hz,1H),4.51(d,J=6.1Hz,2H),3.65(s,4H),3.36(s,4H).13C NMR(100MHz,DMSO-d6)δ169.81,169.00,150.12,143.48,142.24,138.57, 134.09,127.78,127.51,126.81,120.50,117.90,114.05,109.83,46.80.

[0058] 3-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N5)

[0059] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 3-(methoxycarbonyl)benzoic acid as starting materials to give 50 mg of a white solid product with a yield of 52.30% and a melting point of 170-172°C. ESI-MS m / z: 499.34 [M+H] + , 497.42[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.38(s,1H),11.20(s,1H),9.13(s,1H),7.82(d,J=7.2Hz,1H),7.75(d,J=8.4Hz,2H),7.53(d,J=8.1Hz,2H),7.47(d,J= 7.8Hz,2H),7.37(d,J=7.6Hz,2H),7.23(d,J=3.9Hz,2H),6.90(dt,J=8.0,3.9Hz,1H),6.61(t,J=6.1Hz,1H),4.51(d,J=6.1Hz,2H),3.64(s,8H). 13C NMR(100MHz,DMSO-d6)δ169.82,169.08,163.83,150.13,143.46,142.26,136.20,134.13,133.59, 130.02,129.13,128.51,127.79,127.49,126.78,125.88,120.49,117.88,114.08,109.82,46.82.

[0060] 4-(2-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)-N-hydroxybenzamide (N6)

[0061] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 2-(4-(methoxycarbonyl)phenyl)acetic acid as starting materials to give 15 mg of a white solid product with a yield of 22.10% and a melting point of 142-144°C. ESI-MS m / z: 513.35 [M+H] + , 511.34[MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.39(s,1H),11.15(s,1H),9.01(s,1H),7.75(d,J =8.1Hz,1H),7.68(d,J=7.9Hz,2H),7.47(d,J=7.8Hz,2H),7.35(d,J=7.9Hz, 2H),7.28(d,J=7.9Hz,2H),7.25–7.21(m,2H),6.91(ddd,J=7.8,4.5,3.2Hz, 1H), 6.61 (t, J = 6.0Hz, 1H), 4.51 (d, J = 6.1Hz, 2H), 3.78 (s, 2H), 3.52 (s, 8H). 13 C NMR(100MHz,DMSO-d6)δ169.80,169.23,150.13,143.39,142.26,139.45,134.20,131 .48,129.60,127.79,127.44,127.32,126.79,120.50,117.89,114.08,109.82,46.83.

[0062] 2-(4-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacetamide (N7)

[0063] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 4-(2-methoxy-2-oxoethyl)benzoic acid as starting materials to give 15 mg of a white solid product with a yield of 20.10% and a melting point of 150-152°C. ESI-MS m / z: 513.25 [M+H] + . 1 H NMR (400MHz, DMSO-d6) δ11.39(s,1H),10.68(s,1H),8.87(s,1H),7.76(d,J=8.0Hz,1H),7.48(d,J=7.8Hz,2H),7.37(d,J =8.9Hz,4H),7.32(s,2H),7.24(s,2H),6.91(s,1H),6.61(s,1H),4.52(d,J=6.0Hz,2H),3.51(d,8H),3.24–3.18(m,2H). 13 C NMR(150MHz,DMSO-d6)δ169.80,169.66,167.06,150.14,143.45,142.27,138.25,134.22, 134.14,129.43,127.78,127.58,127.50,126.79,120.49,117.89,114.08,109.83,46.83.

[0064] 6-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxynicotinamide (N8)

[0065] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and 5-(methoxycarbonyl)picolinic acid as starting materials to give 35 mg of a white solid product with a yield of 30.20% and a melting point of 128-130°C. ESI-MS m / z: 500.30 [M+H] + ,498.52[MH] - . 1H NMR (400MHz, DMSO-d6) δ11.38(s,1H),8.89(s,1H),8.22(d,J=7.6Hz,1H),7.7 5(d,J=8.1Hz,1H),7.69(d,J=8.1Hz,1H),7.46(t,J=7.8Hz,2H),7.38(d,J=7. 8Hz,2H),7.31(d,J=7.8Hz,1H),7.23(d,J=3.8Hz,2H),6.91(dt,J=7.9,3.8Hz ,1H),6.60(q,J=6.6Hz,1H),4.51(t,J=5.3Hz,2H),3.70(s,4H),3.45(s,4H). 13 C NMR(150MHz,DMSO-d6)δ169.82,166.80,162.24,155.99,150.14,147.29,143.46,142.27,136 .41,134.12,127.78,127.51,127.26,126.79,123.50,120.49,117.89,114.08,109.83,46.86.

[0066] (E)-3-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N9)

[0067] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and (E)-4-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 20 mg of a white solid product with a yield of 50.30% and a melting point of 172-174°C. ESI-MS m / z: 525.37 [M+H] + ,523.30[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.38(s,1H),10.82(s,1H),9.11(s,1H),7.75(d,J=8.2Hz,1H),7.63(d,J=7.9Hz,2H),7.48(q,J=7.0Hz,5H),7.3 8(d,J=7.8Hz,2H),7.23(t,J=3.0Hz,2H),6.91(s,1H),6.62(d,J=6.5Hz,1H),6.52(d,J=15.8Hz,1H),4.52(d,J=5.9Hz,2H),3.61(s,8H). 13C NMR(100MHz,DMSO-d6)δ169.80,169.20,150.13,143.47,142.26,136.57,134.13,128 .21,127.93,127.78,127.50,126.78,120.86,120.49,117.88,114.08,109.82,46.82.

[0068] (E)-3-(3-(4-(4-(((1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N10)

[0069] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6a and (E)-3-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 20 mg of a white solid product with a yield of 51.20% and a melting point of 168-170°C. ESI-MS m / z: 525.33 [M+H] + ,523.86[MH] - . 1 H NMR (400MHz, DMSO) δ11.38(s,1H),10.81(s,1H),9.12(s,1H),7.75(d,J=8. 1Hz,1H),7.63(d,J=7.9Hz,2H),7.49(d,J=6.6Hz,1H),7.46(s,2H),7.44(s, 2H),7.37(d,J=7.8Hz,2H),7.23(d,J=3.8Hz,2H),6.95–6.86(m,1H),6.62( t,J=6.1Hz,1H),6.52(d,J=15.8Hz,1H),4.52(d,J=6.1Hz,2H),3.62(s,8H). 13 C NMR(100MHz,DMSO-d6)δ169.79,169.21,162.84,150.13,143.47,142.26,137.70,136.62,134 .13,128.20,127.91,127.78,127.50,126.78,120.93,120.49,117.88,114.08,109.82,46.82.

[0070] 8-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-N-hydroxy-8-oxooctamide (N11)

[0071] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and monomethyl suberate as starting materials to obtain 13 mg of a white solid product with a yield of 15.20% and a melting point of 80-82°C. ESI-MS m / z: 541.56 [M+H] + ,539.38[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),10.32(s,1H),8.66(s,1H),7.47(d,J=7.7Hz,2H),7.36(d,J=9.4Hz,2H),7.20(d,J=4.6Hz,2H),6.92(d ,J=5.5Hz,1H),6.07(s,1H),4.54(d,J=6.1Hz,2H),3.47(s,8H),2.29(s,2H),1.92(t,J=7.4Hz,2H),1.47(d,J=8.8Hz,4H),1.27–1.13(m,4H). 13 C NMR(150MHz,DMSO-d6)δ171.34,169.77,169.59,149.06,143.55,143.25,134.22,127.93,1 27.64,127.46,125.75,118.33,110.88,109.27,46.92,32.70,28.92,28.89,25.49,25.07.

[0072] 3-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclopentane-1-carboxamide (N12)

[0073] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 3-(ethoxycarbonyl)cyclopentane-1-carboxylic acid as starting materials to give 75 mg of a white solid product with a yield of 65.20% and a melting point of 138-140°C. ESI-MS m / z: 525.26 [M+H] + ,523.41[MH] - . 1H NMR (400MHz, DMSO-d6) δ11.84(s,1H),10.41(s,1H),8.69(s,1H),7.47(d,J=7.4Hz,2H),7.36(dd,J=8.4,2.1Hz,2H),7.20(dd,J=5.1,2.2Hz,2H),6.92 (dd,J=5.4,2.7Hz,1H),6.06(t,J=6.1Hz,1H),4.54(d,J=6.0Hz,2H),3.50( s,8H),3.02(s,1H),2.42(s,1H),1.91–1.82(m,2H),1.73(d,J=21.0Hz,4H). 13 C NMR(150MHz,DMSO-d6)δ173.26,171.73,169.77,149.06,143.55,143.25,134.21,127.93,1 27.65,127.46,125.76,118.33,110.88,109.28,46.93,42.22,40.89,34.16,29.84,29.37.

[0074] 4-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclohexane-1-carboxamide (N13)

[0075] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 4-(methoxycarbonyl)cyclohexane-1-carboxylic acid as starting materials to give 95 mg of a white solid product with a yield of 55.60% and a melting point of 160-162°C. ESI-MS m / z: 539.28 [M+H] + ,536.36[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),10.37(s,1H),8.66(s,1H),7.51–7. 45(m,2H),7.37(s,1H),7.35(d,J=2.3Hz,2H),7.22–7.20(m,1H),6.93(dd ,J=5.3,2.8Hz,1H),6.08(t,J=6.1Hz,1H),4.54(d,J=6.1Hz,2H),3.51(s, 8H),1.87(s,1H),1.67(t,J=13.2Hz,4H),1.47(s,3H),1.39–1.30(m,2H). 13C NMR(150MHz,DMSO-d6)δ173.86,172.44,169.73,149.05,143.55,143.26,134.20,127.9 2,127.64,127.47,125.75,118.33,110.88,109.27,46.93,28.68,28.57,26.43,26.29.

[0076] 4-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N14)

[0077] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 4-(methoxycarbonyl)benzoic acid as starting materials to give 20 mg of a white solid product with a yield of 17.20% and a melting point of 174-176°C. ESI-MS m / z: 533.25 [M+H] + ,530.32[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),11.33(s,1H),9.16(s,1H),7.81(d,J=7.7Hz,2H),7.48(t,J=8.1Hz,4H),7.3 7(d,J=8.0Hz,2H),7.24–7.16(m,2H),6.92(dd,J=5.5,2.7Hz,1H),6.08(s,1H),4.54(d,J=6.1Hz,2H),3.64(s,8H). 13 C NMR(150MHz,DMSO-d6)δ169.82,169.02,149.05,143.55,143.33,138.58, 134.08,127.92,127.65,127.52,125.75,118.33,110.87,109.28,46.93.

[0078] 3-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N15)

[0079] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 3-(methoxycarbonyl)benzoic acid as starting materials to give 15 mg of a white solid product with a yield of 45.20% and a melting point of 162-164°C. ESI-MS m / z: 533.21 [M+H] + ,531.32[MH] - . 1H NMR (400MHz, DMSO-d6) δ11.84(s,1H),11.40(s,1H),9.15(s,1H),7.89–7.80(m,1H),7.76(s,1H),7.53(d,J=8.6Hz,2H),7.47(d,J=7.6Hz,2H ),7.37(d,J=8.0Hz,2H),7.21(t,J=2.9Hz,2H),6.92(dt,J=5.1,2.8Hz,1H),6.05(d,J=7.3Hz,1H),4.57–4.50(m,2H),3.54(d,J=76.6Hz,8H). 13 C NMR(150MHz,DMSO-d6)δ169.83,169.10,149.05,143.55,143.32,136.20,134.10, 129.13,127.92,127.65,127.51,125.86,125.75,118.33,110.87,109.28,46.93.

[0080] 4-(2-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)-N-hydroxybenzamide (N16)

[0081] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 2-(4-(methoxycarbonyl)phenyl)acetic acid as starting materials to give 25 mg of a white solid product with a yield of 35.20% and a melting point of 138-140°C. ESI-MS m / z: 547.17 [M+H] + ,545.30[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),11.28(s,1H),9.02(s,1H),7.68(d,J=7.8Hz,2H),7.46(d,J=7.4Hz,2H),7.35(dd,J=8.3,2.2Hz,2H),7 .28(d,J=7.8Hz,2H),7.24–7.18(m,2H),6.92(dt,J=5.1,2.3Hz,1H),6.06(t,J=6.3Hz,1H),4.53(d,J=6.1Hz,2H),3.78(s,2H),3.51(s,8H). 13C NMR(150MHz,DMSO-d6)δ169.81,169.24,149.05,143.54,143.27,139.47,134.15,131 .46,129.62,127.94,127.64,127.46,127.32,125.75,118.33,110.86,109.28,46.91.

[0082] 2-(4-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacetamide (N17)

[0083] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 4-(2-methoxy-2-oxoethyl)benzoic acid as starting materials to give 45 mg of a white solid product with a yield of 55.12% and a melting point of 150-152°C. ESI-MS m / z: 547.18 [M+H] + ,545.38[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),10.71(s,1H),8.87(s,1H),7.47(d,J=7.8Hz,2H),7.37(s,2H),7.36–7.28(m,4H) ,7.25–7.15(m,2H),6.92(dd,J=5.3,2.8Hz,1H),6.05(t,J=6.3Hz,1H),4.54(d,J=6.1Hz,2H),3.67(s,2H),3.57(s,8H). 13 C NMR(150MHz,DMSO-d6)δ169.81,169.66,167.06,149.06,143.56,143.30,138.26,134.23, 134.11,129.43,127.92,127.64,127.59,127.52,125.75,118.33,110.88,109.28,46.94.

[0084] 6-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxynicotinamide (N18)

[0085] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and 5-(methoxycarbonyl)picolinic acid as starting materials to give 45 mg of a white solid product with a yield of 15.20% and a melting point of 180-182°C. ESI-MS m / z: 534.43 [M+H] +,532.35[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),11.45(s,1H),9.29(s,1H),8.89(s,1H),8.21(d,J=8.1Hz,1H),7.68(d,J=8.1Hz,1H),7.47(d,J=7.6H z,2H),7.37(d,J=7.8Hz,2H),7.20(dd,J=5.2,2.4Hz,2H),6.91(d,J=5.4Hz,1H),6.05(s,1H),4.57–4.50(m,2H),3.70(s,3H),3.45(s,5H). 13 C NMR(150MHz,DMSO-d6)δ169.82,166.80,155.99,149.05,147.29,143.55,143.30,136 .40,134.09,127.92,127.64,127.52,125.75,123.49,118.33,110.88,109.27,46.93.

[0086] (E)-3-(4-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N19)

[0087] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and (E)-4-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 31 mg of a white solid product with a yield of 35.20% and a melting point of 190-192°C. ESI-MS m / z: 559.32 [M+H] + ,557.44[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.84(s,1H),7.62(d,J=7.8Hz,2H),7.46(t,J=8.6Hz,5H),7.37(d,J=7.9Hz,2H),7.25–7 .18(m,2H),6.92(dd,J=5.3,2.7Hz,1H),6.52(d,J=15.8Hz,1H),6.06(s,1H),4.53(d,J=6.1Hz,2H),3.61(s,8H). 13CNMR(150MHz,DMSO-d6)δ169.81,169.22,149.05,143.55,143.31,136.63,134 .10,128.20,127.91,127.65,127.51,125.75,118.32,110.87,109.28,46.92.

[0088] (E)-3-(3-(4-(4-(((4-chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N20)

[0089] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6b and (E)-3-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 75 mg of a white solid product with a yield of 65.30% and a melting point of 188-190°C. ESI-MS m / z: 559.20 [M+H] + ,557.39[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.85(s,1H),10.78(s,1H),9.26(d,J=94.5Hz,1H),7.65(d,J=7.6Hz,1H),7.60(s,1H),7.56–7.44(m,4H),7.39(dd,J= 15.1,7.6Hz,3H),7.21(d,J=4.4Hz,2H),6.97–6.87(m,1H),6.52(d,J=15.8Hz,1H),6.08(s,1H),4.54(d,J=6.1Hz,2H),3.56(d,J=67.7Hz,8H). 13 C NMR(150MHz,DMSO-d6)δ169.83,169.23,162.91,149.06,143.56,143.32,137.93,136.77,135.63,134.09, 129.58,128.98,128.29,127.92,127.66,127.52,126.45,125.76,120.71,118.33,110.88,109.28,46.93.

[0090] 8-(4-(4-(((4-Fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-N-hydroxy-8-oxooctamide (N21)

[0091] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and monomethyl suberate as starting materials to obtain 40 mg of a white solid product with a yield of 45.10% and a melting point of 78-80°C. ESI-MS m / z: 525.39 [M+H] + , 523.49[MH] - .1HNMR(400MHz,DMSO-d6)δ11.75(s,1H),10.32(s,1H),8.65(s,1H),7.47(d ,J=7.8Hz,2H),7.36(d,J=7.8Hz,2H),7.26–7.16(m,1H),7.05(d,J=8.3Hz,1H ),6.64(dd,J=10.7,7.8Hz,1H),6.31–6.24(m,1H),4.49(d,J=6.1Hz,2H),3.4 8(s,8H),2.30(s,2H),1.93(t,J=7.5Hz,2H),1.51–1.43(m,4H),1.25(s,4H). 13 C NMR (150MHz, DMSO-d6) δ171.35,169.79,169.59,157.36,155.72,148.01,144.98,143.27,134.18,128. 16,128.11,127.73,127.41,106.42,103.14,102.58,102.46,46.89,32.70,28.91,28.88,25.48,25.07.

[0092] 3-(4-(4-(((4-Fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclopentane-1-carboxamide (N22)

[0093] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 3-(ethoxycarbonyl)cyclopentane-1-carboxylic acid as starting materials to give 45 mg of a white solid product with a yield of 30.10% and a melting point of 118-120°C. ESI-MS m / z: 509.20 [M+H] + , 506.41[MH] - . 1H NMR(400MHz,DMSO-d6)δ11.75(s,1H),10.41(s,1H),8.70(s,1H),7.51–7.44(m, 2H),7.36(dd,J=8.1,2.1Hz,2H),7.21(q,J=7.6Hz,1H),7.05(dd,J=8.4,2.2Hz, 1H),6.64(ddd,J=10.2,7.6,2.2Hz,1H),6.32–6.24(m,1H),4.52–4.46(m,2H),3 .51(s,8H),3.02(s,1H),2.43(s,1H),1.87(t,J=10.1Hz,2H),1.78–1.65(m,4H). 13 C NMR(150MHz,DMSO-d6)δ173.25,171.73,169.79,157.37,155.73,145.05,144.99,143.28,134.19,128.16,1 28.10,127.74,127.41,106.41,103.30,103.16,102.58,102.46,46.91,42.23,40.89,34.16,29.84,29.37.

[0094] 4-(4-(4-(((4-Fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxycyclohexane-1-carboxamide (N23)

[0095] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 4-(methoxycarbonyl)cyclohexane-1-carboxylic acid as starting materials to give 25 mg of a white solid product with a yield of 25.41% and a melting point of 130-132°C. ESI-MS m / z: 523.17 [M+H] + , 520.41[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.75(s,1H),10.35(s,1H),8.65(s,1H),7.47(d,J=7.7Hz,2H),7.36(d,J=7.6Hz,2H),7.21(d,J=7.0Hz,1H),7.05(d,J=8. 3Hz,1H),6.64(s,1H),6.28(s,1H),4.49(d,J=6.1Hz,2H),3.48(s,8H),3 .22–3.17(m,3H),1.73–1.62(m,3H),1.46(s,3H),1.35(t,J=12.4Hz,1H). 13C NMR(150MHz,DMSO-d6)δ173.88,172.45,169.76,157.37,155.73,148.02,145.06,143.29,134.1 7,128.11,127.73,127.41,106.42,103.16,102.58,102.46,46.90,28.68,28.56,26.43,26.29.

[0096] 4-(4-(4-(((4-Fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N24)

[0097] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 4-(methoxycarbonyl)benzoic acid as starting materials to give 55 mg of a white solid product with a yield of 55.40% and a melting point of 168-170°C. ESI-MS m / z: 517.22 [M+H] + , 515.32[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.75(s,1H),11.32(s,1H),9.15(s,1H),7.81(d,J=7.8Hz,2H),7.53–7.44(m,4H),7.36(d,J=7.8Hz,2H),7.21 (td,J=8.0,5.1Hz,1H),7.05(d,J=8.3Hz,1H),6.64(dd,J=10.7,7.6Hz,1H),6.29(t,J=5.8Hz,1H),4.49(d,J=6.1Hz,2H),3.65(s,8H). 13 C NMR(150MHz,DMSO-d6)δ169.85,169.03,157.37,155.73,148.02,145.03,143.36,138.59, 134.05,128.16,127.74,127.52,127.46,106.42,103.29,103.16,102.59,102.47,46.90.

[0098] 3-(4-(4-(((4-Fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxybenzamide (N25)

[0099] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 3-(methoxycarbonyl)benzoic acid as starting materials to give 35 mg of a white solid product with a yield of 50.30% and a melting point of 148-150°C. ESI-MS m / z: 517.22 [M+H] + , 515.47[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.75(s,1H),11.30(s,1H),9.13(s,1H),7.83(s,1H),7.77(s,1H),7.56(s,2H),7.48(d,J=7.9Hz,2H),7.37( d,J=7.6Hz,2H),7.21(d,J=7.8Hz,1H),7.05(d,J=8.1Hz,1H),6.64(t,J=9.2Hz,1H),6.27(s,1H),4.49(d,J=6.1Hz,2H),3.44(s,8H). 13 C NMR(150MHz,DMSO-d6)δ169.86,169.07,157.37,155.72,148.02,145.05,143.35,136.23,134.07,133 .47,130.11,129.16,128.55,128.16,127.75,127.45,125.90,106.42,103.15,102.58,102.46,46.90.

[0100] 4-(2-(4-(4-(((4-fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)-2-oxoethyl)-N-hydroxybenzamide (N26)

[0101] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 2-(4-(methoxycarbonyl)phenyl)acetic acid as starting materials to give 50 mg of a white solid product with a yield of 52.30% and a melting point of 144-146°C. ESI-MS m / z: 531.18 [M+H] + , 529.42[MH] - . 1H NMR(400MHz,DMSO-d6)δ11.75(s,1H),11.16(s,1H),9.00(s,1H),7.68(d, J=7.8Hz,2H),7.47(d,J=7.6Hz,2H),7.40–7.32(m,2H),7.29(d,J=7.8Hz,2 H),7.21(d,J=6.9Hz,1H),7.05(d,J=8.1Hz,1H),6.64(t,J=8.9Hz,1H),6. 27(t,J=6.3Hz,1H),4.49(d,J=6.1Hz,2H),3.79(s,2H),3.69–3.38(m,8H). 13 C NMR(150MHz,DMSO-d6)δ169.83,169.24,157.37,155.72,148.02,145.00,143.29,139.49,134.14,131 .44,129.62,128.16,128.11,127.74,127.40,127.34,106.40,103.29,103.16,102.59,102.47,46.91.

[0102] 2-(4-(4-(4-(((4-fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacetamide (N27)

[0103] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 4-(2-methoxy-2-oxoethyl)benzoic acid as starting materials to give 35 mg of a white solid product with a yield of 40.05% and a melting point of 138-140°C. ESI-MS m / z: 531.28 [M+H] + , 529.41[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.75(s,1H),10.70(s,1H),8.88(s,1H),7.47(d,J=7.7Hz,2H),7.40–7.29(m,6H),7.21(td,J=8.0,5.0Hz,1H) ,7.05(d,J=8.3Hz,1H),6.64(dd,J=10.8,7.6Hz,1H),6.29(t,J=6.1Hz,1H),4.49(d,J=6.2Hz,2H),3.64(d,J=27.4Hz,8H),3.33(s,2H). 13C NMR(150MHz,DMSO-d6)δ169.84,169.67,167.10,157.37,155.73,148.02(d,J=2.4Hz),145.06,143.33,138.24,1 34.23,134.08,129.43,128.16,128.11,127.74,127.59,127.46,106.43,103.29,103.16,102.59,102.47,46.91.

[0104] 6-(4-(4-(((4-fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)-N-hydroxynicotinamide (N28)

[0105] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and 5-(methoxycarbonyl)picolinic acid as starting materials to give 15 mg of a white solid product with a yield of 20.10% and a melting point of 190-192°C. ESI-MS m / z: 518.13 [M+H] + ,516.36[MH] - . 1 H NMR(400MHz,DMSO-d6)δ11.75(s,1H),11.46(s,1H),9.31(s,1H),8.89(s,1H) ,8.22(d,J=8.1Hz,1H),7.69(d,J=8.1Hz,1H),7.47(d,J=7.7Hz,2H),7.38(d, J=7.5Hz,2H),7.21(q,J=7.2Hz,1H),7.05(d,J=8.3Hz,1H),6.64(t,J=9.4Hz, 1H), 6.27 (s, 1H), 4.49 (d, J = 6.0Hz, 2H), 3.70 (s, 2H), 3.48 (d, J = 20.9Hz, 6H). 13 C NMR(150MHz,DMSO-d6)δ169.83,166.80,157.36,155.98,155.72,148.01,147.29,143.34,136 .40,134.06,128.10,127.73,127.46,123.49,106.42,103.28,103.15,102.58,102.46,46.90.

[0106] (E)-3-(4-(4-(4-(4-fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N29)

[0107] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and (E)-4-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 65 mg of a white solid product with a yield of 60.03% and a melting point of 182-184°C. ESI-MS m / z: 543.17 [M+H] + ,541.37[MH] - . 1 H NMR (400MHz, DMSO-d6) δ11.74(s,1H),10.82(s,1H),9.11(s,1H),7.63(d,J=7.7Hz,2H),7.50(s,1H),7.47(d,J=7.3Hz,4H),7.37(d,J=7.7Hz,2H) ,7.26–7.15(m,1H),7.05(d,J=8.3Hz,1H),6.64(dd,J=10.6,7.8Hz,1H), 6.52(d,J=15.8Hz,1H),6.27(s,1H),4.49(d,J=6.1Hz,2H),3.61(s,8H). 13 C NMR(150MHz,DMSO-d6)δ169.83,169.22,157.37,155.72,148.01,143.35,136.59,134.07, 128.20,127.92,127.74,127.46,120.88,106.42,103.29,103.15,102.58,102.46,46.90.

[0108] (E)-3-(3-(4-(4-(((4-fluoro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazine-1-carbonyl)phenyl)-N-hydroxyacrylamide (N30)

[0109] The synthesis of the title compound was similar to that of target compound N1, using intermediate 6c and (E)-3-(3-methoxy-3-oxoprop-1-en-1-yl)benzoic acid as starting materials to give 35 mg of a white solid product with a yield of 55.80% and a melting point of 168-170°C. ESI-MS m / z: 543.18 [M+H] + ,541.48[MH] - . 1H NMR (400MHz, DMSO-d6) δ11.74(s,1H),10.77(s,1H),9.10(s,1H),7.65(d,J=7.8 Hz,1H),7.59(s,1H),7.55–7.44(m,4H),7.41(d,J=7.6Hz,1H),7.37(d,J=7.6Hz ,2H),7.24–7.16(m,1H),7.05(d,J=8.2Hz,1H),6.64(dd,J=10.8,7.9Hz,1H),6. 52(d,J=15.8Hz,1H),6.28(d,J=6.6Hz,1H),4.49(d,J=6.1Hz,2H),3.63(s,8H). 13 CNMR(150MHz,DMSO-d6)δ169.85,169.22,162.99,157.36,155.72,148.00,145.05,144.99,143.35,138.02,136.76,135.60,134 .05,129.59,128.99,128.30,128.16,128.11,127.74,127.46,120.67,106.41,106.40,103.28,103.14,102.58,102.46,46.89.

[0110] (4-(4-(((4-Chloro-1H-indazol-3-yl)amino)methyl)benzoyl)piperazin-1-yl)(2,4-dichlorophenyl)methanone (25c)

[0111] Intermediate 6b (0.086 g, 0.45 mmol), HOBT (0.079 g, 0.59 mmol), EDC-HCl (0.11 g, 0.59 mmol), and NMM (0.20 g, 1.99 mmol) were added to 12 mL of DMF and stirred for half an hour. 2,4-Dichlorobenzoic acid (0.086 g, 0.99 mmol) was added to the solution and allowed to react at room temperature for 2 h. The reaction was monitored by TLC, and ethyl acetate (20 mL) and water (50 mL) were added. The organic layer was separated, and the aqueous layer was further extracted with ethyl acetate (20 mL x 3). The organic layers were then combined, washed with brine (30 mL x 3), dried over anhydrous Na2SO4, and concentrated in vacuo to afford the crude product. This was then purified by column chromatography to afford the product 25c as a white solid with a purity of 97% and a melting point of 188-190°C. ESI-MS m / z: 543.17 [M+H] + , 541.36[MH] - . 1H NMR (400MHz, DMSO-d6) δ11.83(s,1H),7.73(s,1H),7.52(d,J=8.3Hz,1H),7.46(d,J=8.1Hz,3H),7.36(d,J=7.6Hz,2H), 7.23–7.14(m,2H),6.91(dt,J=5.2,2.6Hz,1H),6.05(s,1H),4.56–4.50(m,2H),3.68(s,3H),3.45(s,3H),3.20(s,2H). 13 C NMR(100MHz,DMSO-d6)δ169.83,165.41,149.03,143.52,143.36,134.88,134.82,133.97,130 .85,129.88,129.57,128.41,127.93,127.63,127.53,125.74,118.32,110.84,109.26,46.89.

[0112] Example 2: In vitro HDACs inhibitory activity test of the compound represented by general formula (I)

[0113] Using HeLa cell nuclear extract (mainly containing HDAC1 and HDAC2) as the enzyme source, all target compounds were preliminarily screened for HDACs enzyme inhibitory activity. The HDAC fluorescence analysis method (two-step method) is a rapid and convenient method for detecting HDAC activity with simple operation. In the first step, the HDAC fluorescent substrate (containing an acetylated lysine side chain - Boc-Lys(acetyl)-AMC) is incubated with a sample containing HDAC activity to deacetylate and activate the substrate. In the second step, Boc-Lys-AMC is hydrolyzed with trypsin to produce the fluorescent group (or chromophore) AMC, and the fluorescence intensity is measured at the excitation wavelength / emission wavelength (390nm / 460nm).

[0114] A blank group, a 100% control group, a compound group, and a positive drug group were set up, with three replicates for each group:

[0115] Blank group: 60 μL of blank HDACs buffer was first incubated at 37°C for 5 min. After adding 40 μL of substrate, the reaction was carried out at 37°C for 30 min. Then, 100 μL of Trypsin solution was added and incubated at 37°C for another 20 min. The fluorescence intensity was measured at 390 nm / 460 nm.

[0116] 100% control group: 50 μL of blank HDACs buffer was mixed with 10 μL of HDACs enzyme solution and pre-incubated at 37°C for 5 min. After adding 40 μL of substrate, the reaction was incubated at 37°C for 30 min. Then, 100 μL of Trypsin solution was added to terminate the reaction and the reaction was incubated at 37°C for 20 min. The fluorescence intensity was measured at 390 nm / 460 nm.

[0117] Compound group: 50 μL of HDACs buffer containing different concentrations of compounds was mixed with 10 μL of HDACs enzyme solution and incubated at 37°C for 5 min. After adding 40 μL of substrate, the reaction was incubated at 37°C for 30 min. Then, 100 μL of Trypsin solution was added to terminate the reaction and the reaction was incubated at 37°C for 20 min. The fluorescence intensity was measured at 390 nm / 460 nm.

[0118] Positive drug group: SAHA and PXD101 were selected as positive control drugs, and the method was the same as that of the compound group.

[0119] Inhibition rate (%) = (A1-A2) / (A1-A0) × 100%

[0120] A0: fluorescence intensity of blank control; A1: 100% fluorescence intensity; A2: fluorescence intensity of compound.

[0121] The inhibition rate was calculated according to the formula, and nonlinear curve fitting was performed using Graphpad Prism 8 to further obtain the IC of the test compound. 50 The results are shown in Table 1.

[0122] Table 1 In vitro HDACs inhibitory activity of target compounds and positive drugs

[0123]

[0124]

[0125] a Numerical values are expressed as mean ± SD of three experiments.

[0126] b Inhibition rate of compound at 5 μM.

[0127] Experimental conclusion analysis:

[0128] The experimental results show that the HDACs enzyme activity of the 30 designed compounds is related to the linker type. Compounds with n-hexyl and styryl linkers (N1, N9, N10, N11, N19, N20, N21, N29, N30) IC50 It reaches the sub-micromolar level and has good HDAC enzyme inhibitory activity. Among them, N19, N20, and N30 have the best activity, IC 50 They are 311.1nM, 146.6nM and 182.6nM respectively.

[0129] Example 3: In vitro HDAC6 inhibitory activity assay of N19, N20, and N30

[0130] Using HDAC6 as the enzyme source, some compounds were tested for their inhibitory activity. The HDAC fluorescence analysis method (two-step method) was used to quickly and conveniently detect HDAC activity with ease. In the first step, the HDAC6 fluorescent substrate (containing an acetylated lysine side chain - Boc-Lys(acetyl)-AMC) was incubated with a sample containing HDAC6 activity to deacetylate and activate the substrate. In the second step, Boc-Lys-AMC was hydrolyzed with trypsin to produce the fluorescent group (or chromophore) AMC, and the fluorescence intensity was measured at the excitation / emission wavelengths (360nm / 460nm).

[0131] A blank group, a 100% control group, a compound group, and a positive drug group were set up, with three replicates for each group:

[0132] Blank group: 40 μL of blank HDAC6 buffer was first incubated at 30°C for 1 h, 10 μL of 20 μM substrate was added, and the reaction was carried out at 30°C for 2 h. Then, 10 μL of a solution containing 35 mg / mL trypsin and 5 μM trichostatin A (TSA) was added, and the cells were incubated at 30°C for another 30 min. The fluorescence intensity was measured at 360 nm / 460 nm.

[0133] 100% control group: 20 μL of blank HDAC6 buffer was mixed with 20 μL of HDAC6 enzyme solution, pre-incubated at 30°C for 5 min, 10 μL of 20 μM substrate was added, and the reaction was carried out at 30°C for 30 min. Then, 10 μL of a solution containing 35 mg / mL trypsin and 5 μM trichostatin A (TSA) was added to terminate the reaction, and the reaction was carried out at 30°C for 30 min. The fluorescence intensity was measured at 360 nm / 460 nm.

[0134] Compound group: 20 μL of HDACs buffer containing different concentrations of compounds was mixed with 20 μL of HDAC6 enzyme solution, pre-incubated at 30°C for 1 h, 10 μL of 20 μM substrate was added, and the reaction was carried out at 30°C for 2 h. The reaction was terminated by adding 10 μL of a solution containing 35 mg / mL trypsin and 5 μM trichostatin A (TSA), and the reaction was carried out at 30°C for 30 min. The fluorescence intensity was measured at 360 nm / 460 nm.

[0135] Positive drug group: ACY-1215 was selected as the positive control drug, and the method was the same as that of the compound group.

[0136] Inhibition rate (%) = [(A 1 -A 0 )-(A 2 -A 3 )] / (A 1 -A 0 )×100%

[0137] A 0 :100% fluorescence intensity. A 1 :100% fluorescence intensity. A 2 : Fluorescence intensity of the compound. A 3 : Blank fluorescence intensity.

[0138] The inhibition rate was calculated according to the formula, and nonlinear curve fitting was performed using Graphpad Prism 8 to further obtain the IC of the test compound. 50 The results are shown in Table 2.

[0139] Table 2 HDAC6 IC of some compounds 50

[0140]

[0141] Experimental conclusion analysis:

[0142] From the experimental results, we can see that the HDAC6 subtype IC of N19, N20 and N30 50 The values were all in the nanomolar range, and the IC 50 111.2, 144.6 and 66.76 nM respectively.

[0143] Example 4: In vitro JNK3 inhibitory activity assay of N19, N20, and N30

[0144] Experimental principle:

[0145] Radioactive enzyme assays are compatible with a variety of substrate types, including peptides, proteins, lipids, and small molecules. 33 The in vitro enzyme activity data were obtained by transferring P-labeled ATP to the substrate.

[0146] Experimental methods:

[0147] Compound inhibition of JNK3 kinase was assayed using the Lance Ultra method, with ATP concentration as the Km and staurosporine (antibiotic AM-2282 or STS) as the reference compound. Triplicate wells were used. First, the compound was diluted in 100% DMSO to 100 times the maximum inhibitor concentration required in the reaction as a stock solution. Then, 100 μL of 100% DMSO was added to six empty wells of the same 96-well plate for blank and standard controls, designated as the source plate. 40 μL of compound was transferred from the source plate to a new 384-well Echo plate, serving as the intermediate plate. A 2x 1.2 nM kinase mix solution was prepared by adding JNK3 to 1x kinase buffer. 10 μL of the kinase mix solution was transferred to the standard control wells, followed by 10 μL of 1x kinase buffer to the blank control wells. A 2x substrate mix solution was prepared by adding the ULight-4E-BP1 peptide and 4.2 μM ATP to 1x kinase reaction buffer. Next, 10 μL of substrate solution was added to each well of the assay plate and incubated at room temperature for 30 minutes. A 2x detection solution of Eu-anti-phospho-4E-BP1 antibody was prepared, and 20 μL of the detection solution was added to the assay plate and incubated at room temperature for 60 minutes. Data were processed on Envision, and the results are shown in Table 3.

[0148] Table 3 JNK3 IC of some compounds 50

[0149]

[0150] Experimental conclusion analysis:

[0151] In this example, three compounds with good inhibitory activity against HDACs and HDAC6 were selected for JNK3 inhibitory activity testing. As shown in Table 3, the inhibitory activity of selected compounds N19 and N20 against JNK3 was comparable to that of the positive drug 25c. However, N30, which had the best HDAC6 inhibitory activity, had a sharp decrease in JNK3 inhibitory activity. This may be because the polarizability and atomic radius of fluorine atoms are smaller than those of chlorine atoms, making them unable to form effective halogen bonds with amino acid residues in the hydrophobic pocket I of JNK3.

[0152] Example 5: Some compounds represented by general formula (I) inhibit Cu 2+ Induced Aβ 1-42 Aggregation experiments

[0153] Experimental principle:

[0154] The experiment used thioflavin T (ThT) as a fluorescent colorimeter, which can react with Aβ 1-42The compound binds to the β-folded structure in the aggregated state. The complex can be detected at excitation wavelength / emission wavelength = 450 / 490nm, and the fluorescence intensity is proportional to the concentration of the complex. Therefore, the fluorescence value can be used to evaluate the effect of the compound on Aβ 1-42 The ability to inhibit self-aggregation.

[0155] Experimental methods:

[0156] Add the test compound, positive drug and Aβ to different groups in the 96-well fluorescence plate. 1-42 Solution, keep the total volume of each well at 80 μL, the specific addition volume and reaction time are as follows: Positive drug group: positive drug clioquinol (40 μL 40 μM), Cu 2+ (20 μL 40 μM) and Aβ 1-42 solution (20 μL 40 μM); experimental group: test compound (40 μL 40 μM), Cu 2+ (20 μL 40 μM) and Aβ 1-42 Solution (20 μL 40 μM); Negative group: Cu 2+ (20 μL 40 μM), Aβ 1-42 Solution (20 μL 40 μM) and 40 μL 1× PBS buffer; blank control: 80 μL 1× PBS. Set up at least three replicate wells for each test concentration. After addition, seal the 96-well plate with sealing film and shake in a 37°C incubator for 24 hours. After shaking, add 120 μL of Thioflavin T in glycine-sodium hydroxide buffer to each well, shake for 10 minutes, and measure fluorescence at excitation / emission wavelengths of 450 / 490 nm.

[0157] Calculation formula for fluorescence intensity and inhibition rate:

[0158] Inhibition rate = (fluorescence value of negative group - fluorescence value of experimental group) / (fluorescence value of negative group - fluorescence value of blank group) × 100%.

[0159] Table 4 Effects of some compounds on Cu 2+ Induced Aβ 1-42 Aggregation inhibition rate

[0160]

[0161]

[0162] Experimental conclusion analysis:

[0163] Among the selected compounds, N11, N19, N20, N29 and N30 had better inhibitory effects than the positive drug clioquinol, with inhibition rates of 63.62%, 76.90%, 77.23%, 74.93% and 64.23%, respectively.

[0164] Example 6: Neuroprotective Experiment on PC12 Cells Injured by H2O2 by Some Compounds of Formula (I)

[0165] Experimental principle:

[0166] Adding a certain concentration of H2O2 during cell culture damages PC12 cells and causes them to die. In the presence of 1-methoxy-5-methylphenazine methylsulfate, WST-8 can be reduced to water-soluble formazan by succinate dehydrogenase in the mitochondria of living cells, which appears orange in the culture medium, while dead cells cannot be stained.

[0167] Experimental methods:

[0168] PC12 cells were diluted to 1 × 10 5 / mL, seeded into 96-well plates, 100 μL per well. 100 μL of complete medium containing cells was added to the experimental, H2O2-injured, and negative groups, while 100 μL of complete medium without cells was added to the blank group. The cells were incubated in a cell culture incubator for 24 hours. After incubation, the medium was aspirated. 100 μL of 50 μM compound was added to the experimental group, and six replicates were set up. 100 μL of complete medium was added to the H2O2-injured, negative, and blank groups. After addition, the cells were incubated in a cell culture incubator for 1 hour. 100 μL of 1600 μM H2O2 solution was added to the experimental and H2O2-injured groups, while 100 μL of complete medium was added to the negative and blank groups. After addition, the cells were incubated in a cell culture incubator at 37°C, 5% CO2 for 12 hours. The culture medium was removed using a 100 μL pipette, and 100 μL of basal culture medium containing 10% CCK-8 was added to each well in the dark. The cells were incubated in a cell culture incubator for 2 h, and the absorbance was measured at 450 nm.

[0169] Protection rate (%) = (absorbance value of experimental group - absorbance value of H2O2 damaged group) / (absorbance value of negative group - absorbance value of H2O2 damaged group) × 100%.

[0170] Table 5 Neuroprotective rate of some compounds against H2O2-injured PC12 cells

[0171]

[0172] Experimental conclusion analysis:

[0173] Compound N30 showed comparable neuroprotective activity to the positive drug Trolox, with a protection rate of 38.96%. In addition, N20 also had some protective activity, with a protection rate of 32.72%.

[0174]

[0175] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. An anti-Alzheimer's disease inhibitor, characterized in that: Its chemical structure is shown in Formula I: Wherein, R is H or halogen; X is a connecting fragment, and the connecting fragment is selected from 、 、 , thus forming the following structures: 、 、 。 2. The anti-Alzheimer's disease inhibitor according to claim 1, characterized in that: R is H, F or Cl.

3. The anti-Alzheimer's disease inhibitor according to claim 1, characterized in that: Selected from the compounds shown in the following table: 。 4. A method for preparing the anti-Alzheimer's disease inhibitor according to claim 1, characterized in that: The method comprises the steps of using raw material 1 and raw material 2 as starting materials to obtain the compound represented by formula I according to the following reaction scheme: ， Wherein, R and X are as shown in claim 1.

5. The method for preparing the anti-Alzheimer's disease inhibitor according to claim 4, characterized in that: Raw material 1 and raw material 2 undergo a nucleophilic substitution reaction to generate intermediate 3, intermediate 3 is deprotected to obtain intermediate 4, intermediate 4 is reacted with tert-butyl piperazine-1-carboxylate to generate intermediate 5, intermediate 5 is deprotected to obtain intermediate 6, intermediate 6 is reacted with a carboxylic acid derivative to generate intermediate 7, and intermediate 7 is reacted in a methanol solution of potassium hydroxylamine to generate hydroxamic acid to obtain the compound of formula I; Among them, the chemical structural formula of the carboxylic acid derivative is , wherein X is as described in claim 1.

6. A composition characterized in that: Comprising the anti-Alzheimer's disease inhibitor according to claim 1 or a pharmaceutically acceptable salt thereof.

7. A pharmaceutical preparation, characterized in that: The invention comprises the anti-Alzheimer's disease inhibitor according to claim 1 or the composition according to claim 6, and at least one pharmaceutically acceptable carrier or excipient.

8. Use of the anti-Alzheimer's disease inhibitor according to any one of claims 1 to 3, the composition according to claim 6, or the pharmaceutical preparation according to claim 7 in the preparation of a drug for treating Alzheimer's disease.

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