HDAC6 inhibitor, pharmaceutical composition and application

Abstract

The invention discloses an HDAC6 inhibitor, a medicinal composition and application, relates to the technical field of medicines, and solves the technical problems that the existing HDAC6 is poor in selectivity and poor in brain permeability and metabolic stability. The HDAC6 inhibitor disclosed by the invention is selected from any one or more of compounds with structural formulas as shown in formulas 1-13, meanwhile, the invention provides a preparation method of the compounds with the formulas 1-13, and on the basis, the invention further provides a medicinal composition comprising the inhibitor and a pharmaceutically acceptable carrier or auxiliary material thereof. According to the HDAC6 inhibitor provided by the invention, the inhibition activity is effectively improved, the subtype selectivity, fat solubility and metabolic stability to molecules are improved, and the HDAC6 inhibitor has high brain penetration.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an HDAC6 inhibitor, a pharmaceutical composition and applications. Background Art

[0002] Histone modification in the epigenetic regulatory mechanism is one of the important pathways involved in regulating neuroinflammation. Histone deacetylases (HDACs) play a vital role in regulating the dynamic balance of acetylation and deacetylation of histone and non-histone lysine residues. Their abnormal expression is associated with some diseases such as tumors, central nervous system diseases, inflammation, and metabolic abnormalities. Therefore, HDACs have become potential drug targets for the treatment of these diseases, and the development of inhibitors targeting HDACs is one of the effective strategies for the treatment of these diseases. 2+ Among the dependent HDAC family, HDAC6 is the largest member (consisting of 1215 amino acids) and the only subtype in the HDAC family with two tandem catalytic domains. Its unique biological functions and broad therapeutic potential make it one of the most attractive targets in the HDAC family. Recent studies have shown that HDAC6 is involved in deacetylation of non-histone substrates such as tubulin and heat shock proteins, and plays an important role in cellular homeostasis, stress response, protein degradation and other processes. Abnormal expression of HDAC6 is associated with a variety of diseases, including cancer, neurodegenerative diseases and immune diseases. By regulating the activity of HDAC6, new therapeutic strategies are expected to be developed.

[0003] Selective HDAC6 inhibitors are expected to improve drug effectiveness and reduce adverse reactions through precise targeted therapy, thereby inducing tumor cell apoptosis and inhibiting its metastatic ability in cancer treatment. In addition, these inhibitors also have potential in neurodegenerative diseases, slowing neuronal loss by regulating protein processing pathways. At the same time, HDAC6 inhibitors can also regulate immune responses and provide new immunotherapy strategies. However, most of the existing HDAC6 inhibitors have problems such as low target selectivity and difficulty in penetrating the blood-brain barrier. Therefore, further development of HDAC6 inhibitors with high target affinity, good selectivity, and good blood-brain barrier permeability will help achieve better efficacy in the treatment of tumors, nerves and other diseases. Summary of the invention

[0004] The present invention aims to solve the technical problems that the existing HDAC6 inhibitors have poor selectivity, insufficient brain permeability and metabolic stability, and aims to provide an HDAC6 inhibitor, a pharmaceutical composition and an application, which effectively improve the inhibitory activity of the HDAC6 inhibitor, improve the subtype selectivity of the molecule, lipid solubility and metabolic stability, and have high brain permeability.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide an HDAC6 inhibitor, the structural formula of the inhibitor is as follows:

[0007]

[0008] Wherein, X, Y, M, N=CH or nitrogen;

[0009] R1 = any one of halogen, alkyl, alkoxy, and long-chain aromatic ring;

[0010] R2=H, halogen or OMe,

[0011] R3 is a zinc ion chelating group;

[0012] A and B form aliphatic cyclic amines;

[0013] C is a methylene group or a carbonyl group.

[0014] Furthermore, the R1=alkyl group, and the alkyl group is selected from any one of Me, Et, and n-Pr.

[0015] Furthermore, the R1=alkoxy group, and the alkoxy group is selected from any one of MeO, EtO, and n-PrO.

[0016] Furthermore, the R1=long-chain aromatic ring, the long-chain aromatic ring structure is (-CH2-) n -Ar, wherein n=1 to 3, Ar=any one of a benzene ring, a pyridine ring, a pyrimidine ring, an indole, an azaindole, and an aromatic fused bicyclic ring.

[0017] Further, the R3 comprises Any one of .

[0018] Furthermore, the aliphatic cyclic amine substance composed of A and B is selected from any one of the following structures:

[0019]

[0020] Further, the compound includes the structural formula shown in Formula 1-13:

[0021]

[0022]

[0023] In Formula 1 to Formula 7, Cap is an azabicyclic amine substance, and R is selected from any one of H, 2'-F, 2'-OMe, and 3'-OMe;

[0024] In Formulae 8 to 13, R is selected from H or 2'-F, and X is selected from C or N.

[0025] Further, in Formula 1-Formula 7, Cap is selected from any one of the following structures:

[0026]

[0027] The second object of the present invention is to provide a pharmaceutical composition comprising the aforementioned inhibitor and a pharmaceutically acceptable carrier or excipient thereof.

[0028] The third object of the present invention is to provide an application of an HDAC6 inhibitor or a pharmaceutical composition in the preparation of a drug for preventing or treating neuroinflammation.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] The present invention designs and synthesizes novel brain-permeable HDAC6 compounds, and adopts a variety of HDAC6 inhibitor structure modification strategies, including ring expansion of the Cap part, introduction of halogen modification and polar groups, symmetrical double F substitution of the linker part and use of a semi-flexible styryl linker, and replacement of the oxime acid ZGB with non-oxime acids such as mercaptoacetamide, hydrazide and trifluorooxazole. These structure optimization strategies are not limited to a single enhancement of the HDAC6 inhibitory activity of the molecule, but also have an improvement effect on the subtype selectivity, lipid solubility and metabolic stability of the molecule, and have high brain permeability. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:

[0032] Figure 1 The PET imaging study results of compound No. 21 synthesized by the present invention are shown in Figure 1, wherein A represents a representative whole-body PET image of mice, and B represents the heart, liver, spleen, lung and kidney of chronic unpredictable mild stress mouse model (CUMS) and healthy control (HC) mice. 18 F]21 radioactive uptake; C represents [ 18 F] Representative PET images of the mouse brain at 21°C (30–60 min; coronal, sagittal, and axial planes; D indicates [ 18F] Time-activity curve of 21 in the whole brain of mice (n=4); E represents [ 18 Mean radioactivity uptake of [F]21 in brain regions of HC and CUMS mice; data are expressed as SD±mean, n=4; two-way ANOVA: p value<0.05; **p value<0.01; ***p value<0.001. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the technical solutions in the embodiments of the present invention are clearly and completely described below in conjunction with embodiments. Obviously, the schematic implementation modes of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

[0034] The experimental instruments and reagents involved in the present invention are described as follows:

[0035] 1 H NMR and 13 C NMR was measured on a Bruker Avance 400MHz NMR spectrometer. Unless otherwise specified, tetramethylsilane (TMS) was used as the internal standard and the test temperature was 25°C. Mass spectrometry was high-resolution mass spectrometry (HRMS), MicroTOF QII (Bruker, Germany) Q-TOF. Constant temperature water bath (Shanghai Shenke Technology Co., Ltd.) and circulating vacuum pump (Shanghai Yarong Technology Co., Ltd.); electronic balance (HXK-FA210, Fuzhou Huazhi Scientific Instrument Co., Ltd.); three-purpose UV analyzer (Beijing Haifuda Technology Co., Ltd.).

[0036] The experiments involved were all carried out in conventional reaction devices. The reagents used in the reactions were all domestic brands (Bi De Pharmaceuticals, Exploration Platform, Anaiji Chemical, McLean Reagent, etc.). The reaction solvents were all commercially available analytical grade, and the drying solvents were commercial ultra-dry solvents (Anaiji Chemical). The eluents used in column chromatography, ethyl acetate, petroleum ether, dichloromethane, and methanol were all domestic analytical grade solvents (Chengdu Jinshan and Chengdu Kelong), and were used directly without further treatment. The organic reaction was tracked using thin layer silica gel plates (TLC) and detected by ultraviolet light (254nm). The products were separated using TLC silica gel plates (Yantai Jiangyou) or column chromatography (silica gel powder 200-300 mesh and 300-400 mesh, produced by Qingdao Ocean Chemical Plant).

[0037] The following is the synthetic route required for the examples.

[0038] Synthetic route A: connection between Cap and Linker

[0039] At -78°C, add 1.2-1.3 eq of NaHMDS (2M) solution to a tetrahydrofuran solution containing a substrate (aza-fused bicyclic amine), stir at low temperature for 30 min, then slowly drop a solution of substituted benzyl bromide dissolved in tetrahydrofuran, and continue the reaction at -78°C for 30-50 min after the addition is completed. Then transfer to room temperature and stir. After the reaction is completed, remove the solvent by reduced evaporation, extract by dilution with ethyl acetate / water, dry with saturated brine and sodium sulfate, remove the solvent under reduced pressure again, and obtain the target product by column chromatography.

[0040] Synthetic route B: Construction of oxime acid ZGB

[0041] Add 30eq hydroxylamine hydrochloride and an appropriate amount of methanol (5-7mL) to a 25mL flask, add 23eq LiOH at 0°C, then stir at room temperature for 30min to prepare a hydroxylamine methanol solution, add a methanol solution containing the substrate after the solution is clarified, continue stirring for 10-30min, quench with 4M hydrochloric acid after the reaction is completed, adjust the pH to neutral, remove the solvent under reduced pressure, dilute and extract with ethyl acetate / water, use saturated brine and sodium sulfate to dry the organic phase, and purify the system by pulping or column chromatography with ethyl acetate / petroleum ether = 10:1 after deevaporation.

[0042] Synthetic route C: connection between Cap and Linker.

[0043] The substrate (aza-condensed bicyclic amine) was dissolved in DMF, 1.2 eq of NaH was added at 0°C, and the mixture was stirred for 30 min under nitrogen protection at room temperature. A solution of substituted benzyl bromide dissolved in DMF was added dropwise at 0°C, and the mixture was stirred for 30-120 min at room temperature. After the reaction was completed, saturated ammonium chloride was used to quench the reaction, the solvent was removed by evaporation, and the mixture was diluted and extracted with ethyl acetate / water. The solvent was removed by evaporation again, and the mixture was purified by column chromatography.

[0044] Synthesis route D: The de-Boc or Trt protected substrate is placed in a 25 mL round-bottom flask, and about 12 mL of trifluoroacetic acid is added at room temperature for stirring. After reacting for 10 min under nitrogen protection, the trifluoroacetic acid is removed under reduced pressure. The concentrate is diluted and dissolved with an appropriate amount of ethyl acetate and washed with saturated sodium carbonate solution until the aqueous phase has a pH of 9-10. The organic phase is collected, concentrated under reduced pressure, and purified by column chromatography.

[0045] Synthetic route E: Construction of amide bond.

[0046] The substrate amine (1.1 eq), carboxylic acid (1 eq), and nitrogen methyl imidazole (NMI, 2.2 eq) were dissolved in an appropriate amount of acetonitrile or DMF, stirred evenly, and 2-bromo-1-ethylpyridin-1-ium tetrafluoroborate (BEP) or N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (TCFH) 1.1 eq was added at 0°C, and then the mixture was transferred to room temperature and stirred overnight. After the reaction was completed, the solvent was removed by evaporation, and the mixture was diluted and extracted with ethyl acetate / water. The organic phase was dried with saturated brine and sodium sulfate, and the ethyl acetate was removed by evaporation and then purified by column chromatography.

[0047] Example 1

[0048] Synthesis of Compound 01-84

[0049] Cap is selected from any of the following structures:

[0050]

[0051] The synthetic route is:

[0052]

[0053] Reaction conditions: i) a.2-1a~m, NaHMDS, THF, -78℃, 30min; b.2-1n~t, NaH, DMF, 0~RT; ii) NH2OH·HCl, LiOH, MeOH, 10~30min, RT; iii) LiOH, MeOH / THF / H2O, 65℃; (iv) tert-butyl 1-ethylhydrazinecarboxylate, BEP, NMI, DMF, 30℃; v) TFA, DCM, RT.

[0054] In this embodiment, the synthetic route of compounds 01-42 is generally divided into two steps. The first step is to use azabicyclic amines as the Cap starting material, and connect the Cap and the aromatic Linker part by nucleophilic substitution to obtain intermediates 2-2a-y, 2-3a-n containing ester groups. When the nitrogen hydrogen acidity of the azabicyclic amine is strong, such as pyrrole nitrogen hydrogen (2-1n), lactam nitrogen hydrogen (2-1o-s) and 1,8-naphthyridine-2-amine (2-1t), the reaction adopts the reaction conditions of NaH / DMF / 0-RT; when the nitrogen hydrogen is a relatively weakly acidic tetrahydronaphthyridine (2-1f-m) nitrogen hydrogen, the reaction adopts the reaction conditions of NaHMDS / THF / -78°C. The second step is to construct the oxime acid ZGB, and the obtained intermediates 2-2a-x, 2-3a-v are subjected to a one-step amine ester exchange in an excess hydroxylamine environment to construct the oxime acid ZGB to obtain the final product.

[0055] The synthesis of compound 43-84 uses intermediates 2-2a~y, 2-3a~n as starting materials, and obtains the corresponding carboxylic acid compounds 3-2a~y, 3-3a-n through alkaline hydrolysis, and then connects 3-2a~y, 3-3a-n with tert-butyl 1-ethylhydrazinecarboxylate through condensation reaction to obtain intermediates 4-2a~y, 4-3a-n, and finally uses trifluoroacetic acid to remove Boc protection to obtain the corresponding hydrazide chelating group.

[0056] The specific synthesis steps are as follows:

[0057] (1) Synthesis of intermediates 2-2a~y, 2-3a~n: see general synthetic route A;

[0058] (2) Synthesis of intermediates 2-2a~y, 2-3a~n: see general synthetic route C;

[0059] (3) Synthesis of compounds 01-42: See general synthetic route B;

[0060] (4) Synthesis of intermediates 3-2a~y, 3-3a~n: The substrates 3-2a~y, 3-3a~n were dissolved in appropriate solvents (THF / MeOH / H2O=3 / 1 / 1), 3 eq of LiOH was added at 0°C, and then stirred at room temperature for 5 h. After the reaction was completed, 4 M hydrochloric acid was used to adjust the pH to weak acidity, and the solvent was removed under reduced pressure. The organic phase was extracted with ethyl acetate / water, and saturated brine and sodium sulfate were used to dry the organic phase. After degassing, the system was purified by column chromatography using ethyl acetate / petroleum ether.

[0061] (5) Synthesis of intermediates 4-2a~y, 4-3a~n: see general synthetic route E;

[0062] (6) Synthesis of compounds 43-84: Experimental step v, see general synthetic route D.

[0063] Example 2

[0064] Synthesis of Compounds 85-124

[0065] The synthetic route is:

[0066]

[0067] Reaction conditions: (i) a. 2-1a~m, NaHMDS, THF, -78℃, 30min; b. 2-1n~t, NaH, DMF, 0~RT; (ii) a. TMS-CF3, CsF, THF, RT; b. 4N hydrochloric acid.

[0068] The synthesis process of this example uses Cap and 1-[4-(bromomethyl)phenyl]-2,2,2-trifluoroethanone as raw materials to connect Cap, Linker and ZGB through nucleophilic substitution reaction. 115-136 convert the ester group into trifluoroethanone through a one-step reaction under TMS-CF3 / CsF conditions.

[0069] The specific synthesis steps are as follows:

[0070] (1) Synthesis of Compounds 85-104 and Intermediate 6: See steps (1) and (2) in Example 1.

[0071] (2) Step ii of the synthesis of compounds 104-124: Substrate 6 was dissolved in an appropriate amount of THF, TMS-CF3 (2.5 eq) and CsF (3 eq) were added, and the mixture was reacted at room temperature for 2 h under nitrogen protection. After the reaction was completed, the reaction system was quenched with 4 M hydrochloric acid and stirred for 1 h. The solvent was removed by evaporation, and the mixture was diluted and extracted with ethyl acetate / water. The organic phase was dried with saturated brine and sodium sulfate, and the ethyl acetate was removed by evaporation. The system was purified by thin layer chromatography.

[0072] Example 3

[0073] Synthesis of Compounds 125-164

[0074] The synthetic route is:

[0075]

[0076] Reaction conditions: (i) a.2-1a~m, NaHMDS, THF, -78℃, 30min; b.2-1n~t, NaH, DMF, 0~RT; (ii) 10% Pd / C, NH4COOH, MeOH / THF, reflux; (iii) TCFH, NMI, CH3CN, RT; (iv) TFA / DCM, RT.

[0077] This embodiment is divided into four steps: the first step is to use Cap as the starting material, and Linker is 3-nitrobenzyl bromide or 1-(bromomethyl)-2-fluoro-4-nitrobenzene, and connect Cap and the aromatic Linker part by nucleophilic substitution to obtain intermediate 8, and then reduce the nitro group of the intermediate under the condition of Pd / C / NH4COOH to obtain intermediate 9, and then continue to condense with 2-(tritylthio)acetic acid to obtain intermediate 10, and finally remove the protection under the condition of trifluoroacetic acid to obtain the target product.

[0078] The specific synthesis steps are as follows:

[0079] (1) Synthesis of intermediate 8: See synthesis steps (1) and (2) in Example 1.

[0080] (2) Synthesis of intermediate 9: The nitro substrate 13 was dissolved in a THF / MeOH = 1:3 (volume ratio) mixed solvent, and 20 eq of formic acid amine and 10% of the substrate mass of Pd / C (10% wt) were added. The system was heated to reflux. The reaction was completed in about 10 minutes. The palladium carbon was removed by diatomaceous earth filtration. The filtrate was evaporated and diluted with ethyl acetate / water for extraction. The product was dried with saturated brine and sodium sulfate, and the ethyl acetate was evaporated to obtain the target product without further purification.

[0081] (3) Synthesis of intermediate 10: refer to synthetic route E.

[0082] (4) Synthesis steps of compounds 125-164: refer to synthesis route D.

[0083] Example 4

[0084] Synthesis of Compounds 165-184

[0085] The synthetic route is as follows:

[0086]

[0087] Reaction conditions: i) BEP, NMI, CH3CN, RT; ii) LiAlH 4, THF, 0℃~RT.

[0088] This embodiment is divided into two steps, and the specific synthesis steps are as follows:

[0089] (1) Intermediate 11 is obtained by a one-step condensation reaction of Cap and 4-[5-(trifluoromethyl)-1,2,4-oxadiazol-3-yl]benzoic acid.

[0090] (2) Synthesis of compounds 165-184: Under nitrogen atmosphere, intermediate 11 was dissolved in anhydrous tetrahydrofuran (300 ml), LiAlH4 (5 eq) was slowly added at 0°C, and the mixture was then warmed to room temperature and reacted for 2 hours. After the reaction was completed, water was slowly added to the reaction solution to quench the reaction, and the temperature of the reaction solution was maintained at 0-10°C. The mixture was then diluted and extracted with ethyl acetate / water, and the organic phase was successively dried with saturated brine and sodium sulfate. After degassing, the system was purified by column chromatography using ethyl acetate / petroleum ether system.

[0091] Example 5

[0092] Synthesis of Compounds 185-192

[0093] The synthetic route is as follows:

[0094]

[0095] Reaction conditions: i) dimethyl acetonylphosphonate, K2CO3, THF, 30℃, 10h; ii) 2-amino-3-pyridinecarboxaldehyde, LiOH·H2O, H2O, 80℃, 2h; iii) Pd / C, H2, 8h; iv) NaHMDS, THF, -78℃, 30min; v) NH2OH·HCl, LiOH, MeOH, 10~30min, RT.

[0096] The synthetic routes of compounds 185-188 are consistent, all of which use aryl methylene acetone as the starting material, and obtain intermediates 14-2a~b through Friedlander quinoline synthesis, and further use palladium carbon to reduce the double bond and pyridine ring to obtain Cap part 15-2a~b. Then, Cap and Linker are connected by nucleophilic substitution to obtain intermediates 16-2a~d, and finally the oxime acid ZGB is constructed, which is a one-step amine ester exchange reaction.

[0097] The synthesis of compounds 189-192 uses intermediates 16-2a~d as starting materials, and obtains the corresponding carboxylic acid compounds 17-2a~d through alkaline hydrolysis, and then connects 17-2a~d with tert-butyl 1-ethylhydrazinecarboxylate through condensation reaction to obtain intermediates 18-2a~d, and finally uses trifluoroacetic acid to remove Boc protection to obtain the corresponding hydrazide chelating group.

[0098] The specific synthesis steps are as follows:

[0099] (1) Synthesis of intermediates 13-2a~b: 4-pyridinecarboxaldehyde (1 eq) and potassium carbonate (2.5 eq) were added to a round-bottom flask in sequence, and an appropriate amount of THF (substrate concentration 0.4 mmol / mL) was added for dissolution and stirring. Subsequently, dimethyl acetonephosphonate (1.1 eq) was added and reacted at room temperature for 20 h. The potassium carbonate was filtered off, and the filtrate was concentrated under reduced pressure. The concentrate was diluted and extracted twice with ethyl acetate and water. The organic phases were combined, dried over anhydrous sodium sulfate, and the crude product was obtained under reduced pressure. It was purified by column chromatography using acetone:petroleum ether = 1:10.

[0100] (2) Synthesis of intermediates 14-2a~b: 2-Amino-3-pyridinecarboxaldehyde (1.0 eq) and substrate 12-2a or 12-2b (1.1 eq) were dissolved in an appropriate amount of water and stirred, followed by the addition of 1.0 eq of LiOH·H2O and the reaction was carried out at 80°C for 2 h. After the reaction was completed, the mixture was diluted and extracted twice with dichloromethane to synthesize the organic phase, which was purified by column chromatography.

[0101] (3) Synthesis of intermediates 15-2a~b: The substrate 13-2a or 13-2b was dissolved in an appropriate amount of tetrahydrofuran, and Pd / C (10%) was added at 30% by weight of the substrate. After hydrogen substitution, the mixture was reacted at room temperature overnight. After the reaction was completed, the palladium carbon was filtered out through crude silica gel (100-200 mesh), and the filtrate was concentrated and purified by column chromatography.

[0102] (4) For the synthesis of intermediates 16-2a~d, see Synthesis Scheme A.

[0103] (5) Synthesis of compounds 185-188, refer to Synthesis Scheme B.

[0104] (6) Synthesis of intermediates 17-2a~d: Substrates 16-2a~d were dissolved in appropriate solvents (THF / MeOH / H2O=3 / 1 / 1), 3 eq of LiOH was added at 0°C, and then stirred at room temperature for 5 h. After the reaction was completed, 4 M hydrochloric acid was used to adjust the pH to weak acidity, and the solvent was removed under reduced pressure. The organic phase was extracted with ethyl acetate / water, and saturated brine and sodium sulfate were used in turn. After degassing, the system was purified by column chromatography using ethyl acetate / petroleum ether system.

[0105] (7) Synthesis of intermediates 18-2a~d: See general synthetic route E.

[0106] (8) Synthesis of Compounds 189-192: For synthetic step v, see General Synthetic Route D.

[0107] Example 6

[0108] Synthesis of Compounds 193-196

[0109] The synthetic route is:

[0110]

[0111] The synthesis method of compounds 193-196 refers to Example 2.

[0112] Example 7

[0113] Synthesis of Compounds 197-200

[0114] The synthetic route is:

[0115]

[0116] The synthesis method of compounds 197-200 refers to Example 3.

[0117] Example 8

[0118] Synthesis of Compounds 201-202

[0119] The synthetic route is:

[0120]

[0121] The synthesis method of compounds 201 and 202 refers to Example 4.

[0122] Example 9

[0123] Determination of the inhibitory activity of compounds on HDAC6

[0124] The HDAC6 enzyme inhibitory activity of the target compound was determined at Nanosyn (Santa Clara CA, Santa Clara, California, USA). The target compound to be determined was diluted 3 times with 100% DMSO, and the final compound concentration range was 10 μM to 0.056 nM, and the compound was tested in a single well, and the final concentration of DMSO was kept at 1%. The IC50 value was obtained by curve fitting of the 12 points of the determination. The positive control of Quinostat (JNJ-26481585) was also determined in the same way; the inhibitory activity of the compound on HDAC6 is shown in Table 1 below.

[0125] Table 1. Inhibitory activity of compounds on HDAC6

[0126]

[0127]

[0128]

[0129]

[0130] Note: Compound No. 203 is Quinostat (JNJ-26481585) as a positive control.

[0131] It can be seen from the data in Table 1 that the compounds prepared in the present invention have a high inhibitory activity against HDAC6.

[0132] Example 10

[0133] In vivo brain-to-blood ratio test of compounds

[0134] The brain-to-blood ratio of the compound was investigated by monitoring the content of representative compounds in mouse brain and plasma. The experimental steps are as follows:

[0135] (1) Weigh compound No. 4, add a small amount of DMSO, and then add sodium chloride solution for injection to make 2 mg / mL -1 PB131 compound solution for administration to mice.

[0136] (2) 9 C57BL / 6J mice (male), 20 mg / kg -1The drug was administered by intraperitoneal injection. Whole blood and whole brain were collected at 0.5h, 1h, and 3h after administration (n=3). Whole blood was centrifuged for 15min (3500rpm) and the supernatant plasma was collected. The weight of the centrifuge tube was weighed as M1, the weight of the centrifuge tube containing the whole brain was weighed as M2, the weight of the centrifuge tube after adding water homogenate was weighed as M3, and the weight of the centrifuge tube after taking out 20μL of homogenate was weighed as M4. 20μL of plasma and 20μL of brain homogenate were taken into a centrifuge tube, and 80μL of 20ng·ml -1 The internal standard SAHA was precipitated with acetonitrile, vortexed for 30 seconds, centrifuged at 13000 rpm for 15 minutes, and the supernatant was taken and placed in a sample injection bottle for testing.

[0137] (3) Brain homogenate standard curve range: 0.1~100ng·ml -1 .

[0138] (4) Plasma standard curve range: 1~1000ng·ml -1 .

[0139] (5) Drug content in brain = measured value × 0.02 × (M3-M1) / [(M2-M1) × (M3-M4)].

[0140] (6)LC-MS / MS conditions:

[0141] This example uses Waters ACQUITY ultra-high performance liquid chromatography and AB SCIEX The 5500 mass spectrometer system was used for quantitative detection of the analyte. A BEH C18 (2.1 mm × 100 mm, 1.7 μm) chromatographic column was used, and the column temperature was 35°C. The injection volume was 1 μL, the mobile phase A was an aqueous solution containing 0.1% formic acid, the mobile phase B was acetonitrile, and a gradient elution was used, and the gradient was shown in the table. The needle washing solution was water-methanol (v:v=10:90), and the needle washing method was internal and external washing.

[0142] The mass spectrometer scanning mode was multiple reaction ion monitoring (MRM) mode; the electrospray ion source was in positive ion mode (ESI+); the nebulizer gas pressure was 55 psi; the auxiliary gas pressure was 55 psi; the curtain gas pressure was 40 psi; the ion spray voltage was 5500 V; and the ion source temperature was 450°C.

[0143] From the data in Table 2, it can be seen that compound No. 4 has a high inhibitory activity against HDAC6 and a high brain permeability.

[0144]

[0145] Brain / blood distribution dynamics studies

[0146]

[0147] Note: In vivo PK study was conducted by Huiyuan Biotechnology (Shanghai) Co., Ltd. The data are presented as mean ± SEM of three repeated experiments.

[0148] Embodiment 11

[0149] Blood-brain barrier permeability evaluation

[0150] Here, we investigated the expression changes of HDAC6 in the pathological process of major depressive disorder (MDD) by radiolabeling in a noninvasive manner. We established a mild stress mouse model (CUMS) and used an HDAC6-specific probe [ 18 F]21 conducted a PET imaging experiment. The specific method is as follows:

[0151] Compound 21 was labeled with radioactive fluorine-18 and injected into male C57 / BL mice via the tail vein. Dynamic positron emission tomography (PET) scanning was performed for 60 minutes and CT scanning was performed for 3 minutes. The dynamic PET data were reconstructed and analyzed. 18 F]T21 compound uptake and distribution in the mouse brain to investigate the expression changes of HDAC6 in MDD.

[0152] PET imaging results showed that compared with healthy mice, CUMS mice had increased 18 F]21 in the lungs showed no significant difference ( Figure 1 AB). In addition, we investigated the changes of HDAC6 in the brain under MDD pathology. Figure 1 As shown in Figure 5, the uptake of radiotracer in the brain of CUMS mice was significantly increased (SUV 30-60 =0.7, for SUV 30-60 =1.0), indicating that HDAC6 expression was upregulated in the model mouse brain. Further analysis of CUMS mice compared with healthy controls showed that [ 18 F]21 uptake showed increased radioactivity uptake, especially in the striatum, hippocampus, and hypothalamus ( Figure 1 E).

[0153] Finally, it should be noted that the above specific embodiments are only used to explain in detail the purpose, technical solutions and beneficial effects of the present invention. It should be understood that the above description is only a specific implementation method of the present invention and is not used to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the above specific embodiments, ordinary technicians in this field should understand that it is still possible to modify the technical solutions recorded in the above embodiments, or to replace or improve some or all of the technical features therein. These modifications, equivalent replacements and improvements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and description of the present invention.

Claims

1. An HDAC6 inhibitor, characterized in that The structural formula of the inhibitor is as follows: Wherein, X, Y, M, N=CH or nitrogen; R1 = any one of halogen, alkyl, alkoxy, and long-chain aromatic ring; R2=H, halogen or OMe, R3 is a zinc ion chelating group; A and B form aliphatic cyclic amines; C is a methylene group or a carbonyl group.

2. An HDAC6 inhibitor according to claim 1, characterized in that The R1=alkyl group, and the alkyl group is selected from any one of Me, Et, and n-Pr.

3. An HDAC6 inhibitor according to claim 1, characterized in that The R1=alkoxy group, and the alkoxy group is selected from any one of MeO, EtO, and n-PrO.

4. The HDAC6 inhibitor according to claim 1, characterized in that The R1=long chain aromatic ring, the long chain aromatic ring structure is (-CH2-) n -Ar, wherein n=1 to 3, Ar=any one of a benzene ring, a pyridine ring, a pyrimidine ring, an indole, an azaindole, and an aromatic fused bicyclic ring.

5. The HDAC6 inhibitor according to claim 1, characterized in that The R3 is selected from Any one of .

6. The HDAC6 inhibitor according to claim 1, characterized in that The aliphatic cyclic amine substance composed of A and B is selected from any one of the following structures:

7. The HDAC6 inhibitor according to claim 1, characterized in that Including compounds with structural formulas as shown in Formula 1-13: In Formula 1 to Formula 7, Cap is an azabicyclic amine substance, and R is selected from any one of H, 2'-F, 2'-OMe, and 3'-OMe; In Formulae 8 to 13, R is selected from H or 2'-F, and X is selected from C or N.

8. The HDAC6 inhibitor according to claim 7, characterized in that In Formula 1-Formula 7, Cap is selected from any one of the following structures:

9. A pharmaceutical composition, characterized in that The invention comprises the inhibitor according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier or excipient thereof.

10. Use of the HDAC6 inhibitor according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 9 in the preparation of a drug for preventing or treating neuroinflammation.

Images