A histone deacetylase 6 inhibitor and application thereof

Abstract

The application discloses a histone deacetylase 6 inhibitor and application thereof, and belongs to the technical field of chemical medicines. The inhibitor is the following compound or pharmaceutically acceptable salt thereof: which can be used for the treatment of lung cancer, melanoma, liver cancer, kidney cancer, leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, thyroid follicular cancer, gastrointestinal cancer, tumors of the central or peripheral nervous system, mesothelioma, type II or non-insulin-dependent diabetes, and autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of chemical drug technology, and in particular relates to a histone deacetylase 6 inhibitor and its application. Background Technology

[0002] Orderly transcriptional regulation of genes is a prerequisite for the normal functioning of cells. Disruptions in gene transcriptional regulation can lead to cell carcinogenesis. The acetylation and deacetylation of core histones are closely related to gene regulation, and the enzymes responsible for this process are a pair of antagonistic proteases—histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs are a group of enzymes that regulate a range of biological effects at the chromatin level by inducing histone deacetylation, including chromatin reorganization, transcriptional activation or inhibition, cell cycle, cell differentiation, and apoptosis, particularly related to the regulation of gene transcriptional expression after cell activation. Histone deacetylase inhibitors, by inhibiting HDAC activity, induce apoptosis, differentiation, and inhibit proliferation, and are considered promising targets for anticancer drugs. Current research on HDAC inhibitors covers numerous oncology fields, such as hematologic malignancies, melanoma, breast cancer, ovarian cancer, prostate cancer, lung cancer, and colon cancer.

[0003] HDACs are a class of promising anti-tumor targets. There is still an unmet clinical need for novel and effective HDAC inhibitors. It is hoped that new and alternative HDAC inhibitors can be developed to prevent and / or treat diseases related to uncontrolled histone deacetylase activity, especially cancer. Summary of the Invention

[0004] This invention provides an effective inhibitor targeting HDACs, which is compound (I) or a pharmaceutically acceptable salt thereof:

[0005] (I) in: R 1 R 2 Each can independently represent hydrogen, alkyl, alkenyl, alkynyl, aryl, aralkyl, or Het; R 3 It can be hydrogen, alkyl, haloalkyl, cyano, hydroxyl, halogen, mercapto, alkoxy, alkylthio, alkoxyalkyl, aryl, aralkyl, or Het; Q 1 It is selected from aryl, aralkyl, or Het; Q 2 It is selected from aryl or Het; The alkyl group is a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms connected to a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms. The alkoxy group is a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms connected to a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; wherein each carbon atom may optionally be substituted with oxygen. The alkylamino group is a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms; or a cyclic saturated hydrocarbon group having 3-6 carbon atoms connected to a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; wherein each carbon atom may optionally be replaced by an NH atom group; Alkoxyalkyl refers to an alkoxy group connected to an alkyl group as defined above; Alkenyl and alkynyl are straight-chain or branched unsaturated hydrocarbon groups containing double or triple bonds and having 1-6 carbon atoms; The aryl group is a carbon ring selected from phenyl, naphthyl, acenaphthel or tetrahydronaphthyl, each of which is optionally substituted by 1, 2 or 3 substituents, each substituent being independently selected from hydrogen, alkyl, cyano, halogen, haloalkyl, hydroxyl, mercapto, alkoxy, alkylthio, alkoxyalkyl, aralkyl, diarylalkyl, aryl or Het. Arylalkyl and diarylalkyl are aryl groups linked to alkyl groups as defined above; Het is a monocyclic heterocyclic compound selected from pyridinyl, pyrroloyl, pyrazolyl, imidazolyl, thiophene, furanyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrimidinyl, pyrazinyl, or pyridazinyl; or selected from quinolinyl, quinoxolinyl, indolyl, benzimidazolyl, benzoxazolyl, benzoisoxazolyl, benzothiazolyl, benzoisothiazolyl, benzothiophene, benzofuranyl, 2,3-dihydrobenzo[1,4] A bicyclic heterocycle of dioxanehexenyl or benzo[1,3]dioxanepentenyl; or a monocyclic saturated hydrocarbon group of 3-6 carbon atoms or a bicyclic saturated hydrocarbon group of 6-12 carbon atoms, wherein the carbon atoms on the ring are optionally substituted by 1-4 O, S, N or NH atoms; each monocyclic or bicyclic ring is optionally substituted by 1, 2 or 3 substituents, each substituent being optionally selected from halogen, haloalkyl, hydroxyl, alkyl or alkoxy groups; Halogens are substituents selected from fluorine, chlorine, bromine or iodine; The haloalkyl group is a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms, or a cyclic saturated hydrocarbon group having 3-6 carbon atoms, or a cyclic saturated hydrocarbon group having 3-6 carbon atoms connected to a straight-chain or branched saturated hydrocarbon group having 1-6 carbon atoms; wherein one or more carbon atoms are replaced by one or more halogen atoms.

[0006] Preferably, where R 1 R 2 Each can independently represent hydrogen, alkyl, aryl, or Het; R 3 It can be hydrogen, alkyl, hydroxyl, halogen, cyano, haloalkyl, mercapto, alkoxy, alkylthio, or alkoxyalkyl; Q 1 It is selected from aryl, aralkyl, or Het; Q 2 It is selected from aryl or Het; More preferably, wherein: R 1 R 2 Each can independently represent hydrogen, methyl, and ethyl; R 3 It can be hydrogen, methyl, ethyl, 1-2 halogens, or trifluoromethyl; Q 1 It is an aromatic ring or a substituted aromatic ring selected from the following: phenyl, naphthyl, pyrrole, furanyl, thiophene, pyridyl, pyrazinyl, pyrimidinyl, and the substituent can be methyl, ethyl, 1 to 2 halogens or trifluoromethyl; Q 2 It is selected from phenyl.

[0007] More preferably, wherein: R 1 R 2 Each represents hydrogen independently; R 3 It can be hydrogen, methyl, or 1 to 2 halogens; Q 1 It is selected from the following aromatic rings or substituted aromatic rings: phenyl, and the substituents can be methyl or methoxy; Q 2 It is selected from phenyl.

[0008] Preferably, the compound is at least one of the following compounds: N -hydroxy-4-(((7-phenylpyrazolo[1,5- a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-1); N -hydroxy-4-(((7-(p-tolylpyrazolo[1,5-) a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-2); N -hydroxy-4-(((7-(4-chlorobenzyl)pyrazolo[1,5- a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-3); N- Hydroxy-4-(((7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide (I-4); N- Hydroxy-4-(((7-(4-methoxyphenyl)pyrazolo[1,5-) a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-5); N- Hydroxy-4-(((7-(3-methoxyphenyl)pyrazolo[1,5-) a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-6); N -hydroxy-4-(((7-(3,4-dichlorophenyl)pyrazolo[1,5- a ]Pyrimidin-5-yl)amino)methyl)-benzamide (I-7); N- Hydroxy-4-(((7-(3-(methanesulfonyl)phenyl)pyrazolo[1,5- a ]Pyrimidin-5-yl)amino)methyl)benzamide (I-8); N -hydroxy-4-(((7-(4-(methoxymethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide (I-9); N-hydroxy-4-(((7-(thiophen-2-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide (I-10); N-hydroxy-7-((7-(4-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino)heptamide (I-11); N-hydroxy-7-((7-(3-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino) heptamethamide (I-12); N-hydroxy-7-((7-(3,4-dichlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)heptamide (I-13); The preparation method of the above-mentioned compound of the present invention is as follows: .

[0009] All compounds of the present invention can be prepared by the above or similar preparation methods. The appropriate raw materials can be selected according to the different substituents and their positions.

[0010] Pharmacological testing results indicate that compounds of formula (I) and their pharmaceutically acceptable salts exhibit excellent inhibitory activity against HDAC6. Therefore, compounds of formula (I) and their pharmaceutically acceptable salts can be used to treat clinical conditions related to the aforementioned targets. Diseases related to HDAC6 can include, but are not limited to: lung cancer, melanoma, liver cancer, kidney cancer, leukemia, non-small cell lung cancer, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, follicular thyroid carcinoma, gastrointestinal cancer, tumors of the central or peripheral nervous system (e.g., astrocytoma, neuroblastoma, glioma, or schwannoma), mesothelioma, type II or non-insulin-dependent diabetes mellitus, and autoimmune diseases.

[0011] Compared with the prior art, the present invention has the following beneficial effects: This invention provides new drugs for the treatment of various diseases. Attached Figure Description

[0012] Figure 1 In Example I-1 N -hydroxy-4-(((7-phenylpyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0013] Figure 2 The NMR spectrum of N-hydroxy-4-(((7-(p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide in Example I-2.

[0014] Figure 3 In Example I-3 N -hydroxy-4-(((7-(4-chlorobenzyl)pyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0015] Figure 4 In Example I-4 N- Hydroxy-4-(((7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0016] Figure 5 In Examples I-5 N- Hydroxy-4-(((7-(4-methoxyphenyl)pyrazolo[1,5-) a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0017] Figure 6The NMR spectrum of N-hydroxy-4-(((7-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide in Examples I-6.

[0018] Figure 7 In Examples I-7 N -hydroxy-4-(((7-(3,4-dichlorophenyl)pyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)-benzamide.

[0019] Figure 8 In Examples I-8 N- Hydroxy-4-(((7-(3-(methanesulfonyl)phenyl)pyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0020] Figure 9 In Examples I-9 N Nuclear magnetic resonance image of 4-hydroxy-(((7-(4-(methoxymethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide.

[0021] Figure 10 In Example I-10 N -hydroxy-4-(((7-(thiophen-2-yl)pyrazolo[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)methyl)benzamide.

[0022] Figure 11 In Example I-11 N -hydroxy-7-((7-(4-methoxyphenyl)pyrazole[1,5- a Nuclear magnetic resonance image of pyrimidin-5-yl)amino)heptamethamide.

[0023] Figure 12 In Example I-12 N Nuclear magnetic resonance image of 7-hydroxy-7-((7-(3-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino)heptamide.

[0024] Figure 13 In Example I-13 N Nuclear magnetic resonance image of 7-hydroxy-((7-(3,4-dichlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)heptamide.

[0025] Figure 14The image shows a Western blot diagram to verify the selective inhibition of HDAC6 by compound I-1 in Experiment Example 1. In the diagram, TA and SA are abbreviations for the positive control drugs tubastatin A and SAHA, respectively, and 0.1, 1, and 10 are the concentrations of compound I-1 in micromoles.

[0026] Figure 15 This is a diagram showing how compound I-1 in Experiment 1 inhibits melanoma growth in a mouse xenograft model. Detailed Implementation

[0027] Example I-1 N -hydroxy-4-(((7-phenylpyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide

[0028] The method for synthesizing the above compounds is as follows: Step a: 5-Chloro-7-phenylpyrazolo[1,5- a Preparation of pyrimidine 5,7-Dichloropyrazole [1,5- a Pyrimidine (188 mg, 1 mmol), potassium carbonate (345.5 mg, 2.5 mmol), and 1,1'-bis(diphenylphosphine)ferrocene palladium dichloride (36.6 mg, 0.05 mmol) were added sequentially to a 100 mL round-bottom flask and dissolved in 1,4-dioxane (40 mL). The reaction was monitored by TLC for 8 hours until complete, followed by silica gel column chromatography (PE:EA = 2:1 eluent). The pure product was collected in 90.2% yield. White solid, yield 62.1%. ¹H NMR (400 MHz, DMSO-) d 6) δ 8.14 (s, 1H), 8.06 (dd, J = 7.6, 1.9 Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.49 –7.45 (m, 1H), 6.60 (s, 1H), 6.50 (d, J = 2.2 Hz, 1H).

[0029] Step b: 4-(((7-phenylpyrazole[1, 5- a Preparation of methyl pyrimidin-5-yl)amino)methyl)benzoate The above-mentioned intermediates (229.7 mg and 1 mmol) were added to 100 mL eggplant-shaped flasks respectively. N ,N -Diisopropylethylamine (0.7 mL, 4 mmol), methyl 4-aminomethylbenzoate (165.2 mg, 1 mmol) in n-butanol (100 mL) solution, 120 The mixture was heated under reflux for 8 h, and the reaction was monitored by TLC until complete. Silica gel column chromatography (evolving solvent PE:EA = 5:1) yielded the pure product as a white solid, with a yield of 72.3%. 1 H NMR (400 MHz, DMSO- d 6) δ 8.74 (t, J = 6.6 Hz, 1H), 8.14 (s, 1H), 8.05 (dd, J = 7.6, 1.9 Hz, 2H), 7.94 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H),7.49 – 7.42 (m, 3H), 6.61 (s, 1H), 6.50 (d, J = 2.2 Hz, 1H), 4.86 (d, J = 6.6 Hz, 2H), 3.81 (s, 3H).

[0030] Step c: N -hydroxy-4-(((7-phenylpyrazolo[1,5- a Preparation of pyrimidin-5-yl)amino)methyl)benzamide The above intermediate (1.91 g, 5.34 mmol) and 50 mL of 50% hydroxylamine methanol solution were added to a 100 mL three-necked flask. The mixture was reacted overnight at room temperature. The pH was adjusted to neutral with 1 N hydrochloric acid. After concentration, 50 mL of ethyl acetate was added, and then 50 mL of water was used to wash away the hydroxylamine. The organic phase was dried and concentrated to dryness. 20 mL of ethanol / water mixture (ethanol to water ratio of 2:1) was added to crystallize. The product was filtered to obtain a white solid with a yield of 54.1%; mp: 123.4~125.0 °C. 1 H NMR (400 MHz, DMSO- d 6) δ : 11.13(s, 1H), 8.98 (s, 1H), 8.71 (t, J = 6.6 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 8.07(dd, J = 7.8, 1.8 Hz, 2H), 7.71 (d,J = 8.3 Hz, 2H), 7.55 – 7.42 (m, 5H), 6.63(s, 1H), 6.49 (d, J = 2.2 Hz, 1H), 4.81 (d, J = 6.6 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ : 163.95, 156.04, 151.30, 151.16, 140.76, 137.62, 132.16, 130.91,130.27, 128.94, 128.68, 127.35, 127.32, 114.86, 100.21, 47.11. Calcd. for360.1455. (C 20 H 18 N5O2 + [M+H] + Found 360.1452, see atlas. Figure 1 .

[0031] Example I-2 N -hydroxy-4-(((7-(p-tolylpyrazolo[1,5-) a ]pyrimidin-5-yl)amino)methyl)benzamide

[0032] Yield 54.1%; white solid; mp: 123.4~125.0 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.18 (s, 1H), 9.08 (s, 1H), 8.70 (t, J = 6.6 Hz, 1H), 8.12 (d, 1H), 7.98 (d, J =8.1 Hz, 2H), 7.72 (d, J = 8.1 Hz, 2H), 7.53 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.0Hz, 2H), 6.61 (s, 1H), 6.47 (d, J= 2.1 Hz, 1H), 4.81 (d, 2H), 2.34 (s, 3H). 13 CNMR (126 MHz, DMSO- d 6) δ : 164.09, 155.73, 148.85, 146.99, 143.91, 141.72,139.37, 135.36, 131.72, 129.17, 127.11, 127.04, 126.89, 94.87, 81.82, 44.14,20.86. Calcd. for 374.1612. (C 21 H 20 N5O2 + [M+H] + ). Found 374.1610, see atlas Figure 2 .

[0033] Example I-3 N -hydroxy-4-(((7-(4-chlorobenzyl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide

[0034] Yield 58.2%; white solid; mp: 124.5~125.7 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.16 (s, 1H), 9.01 (s, 1H), 8.77 (t, J = 6.3 Hz, 1H), 8.17 – 8.09 (m, 3H), 7.71 (d, J = 7.8 Hz, 2H), 7.52 (t, J = 6.9 Hz, 4H), 6.68 (s, 1H), 6.50 (s, 1H), 4.82 (d, J = 6.3 Hz, 2H). 13 C NMR (126 MHz, DMSO- d 6) δ: 164.07, 154.45, 148.72,147.15, 144.11, 141.64, 136.92, 134.53, 131.73, 128.76, 128.59, 127.11,127.05, 95.17, 81.89, 44.11. Calcd. for 394.1065. (C 20 H 17 Cl 35 N5O2 + [M+H] + ). Found394.1061, see atlas Figure 3 .

[0035] Example I-4 N- Hydroxy-4-(((7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide

[0036] Yield 66.0%; white solid; mp: 133.0~131.8 °C. 1 H NMR (400 MHz, DMSO- d 6) δ : 11.17 (s, 1H), 9.03 (s, 1H), 8.83 (t, J = 6.6 Hz, 1H), 8.41 (t, 2H), 8.17(d, J = 2.2 Hz, 1H), 7.82 (d, J = 7.8 Hz, 1H), 7.73 (dd, J = 7.8, 3.5 Hz, 3H), 7.54(d, J = 8.2 Hz, 2H), 6.82 (s, 1H), 6.57 (d, J = 2.2 Hz, 1H), 4.86 (d, J = 6.6 Hz, 2H). 13 C NMR (126 MHz, DMSO- d 6) δ: 172.03, 164.07, 153.97, 148.73, 147.32,144.24, 141.67, 139.13, 131.76, 130.92, 129.81, 126.13, 123.35, 95.43, 82.12,44.16, 21.05.Calcd. for 428.1329. (C 21 H 17 F3N5O2 + [M+H] + Found 428.1322, see atlas. Figure 4 .

[0037] Example I-5 N- Hydroxy-4-(((7-(4-methoxyphenyl)pyrazolo[1,5-) a ]pyrimidin-5-yl)amino)methyl)benzamide

[0038] Yield 59.0%; white solid; mp: 126.2~127.8 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.16 (s, 1H), 8.12 (d, J = 2.2 Hz, 1H), 8.03 (d, J = 8.9 Hz, 3H), 7.71 (d, J = 8.3Hz, 2H), 7.52 (d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.9 Hz, 3H), 6.59 (s, 1H), 6.45(d, J = 2.2 Hz, 1H), 4.81 (d, J = 6.5 Hz, 2H), 3.81 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ: 164.10, 160.83, 155.23, 147.13, 144.05, 141.68, 131.75, 128.59,127.14, 127.10, 114.01, 94.52, 81.68, 55.34, 44.17. Calcd. for 390.1561.(C 21 H 20 N5O3 + [M+H] + Found 390.1563, see atlas. Figure 5 .

[0039] Example I-6 N- Hydroxy-4-(((7-(3-methoxyphenyl)pyrazolo[1,5-) a ]pyrimidin-5-yl)amino)methyl)benzamide

[0040] Yield 64.3%; white solid; mp: 127.8~128.8 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.14 (s, 1H), 8.99 (s, 1H), 8.72 (t, J = 6.6 Hz, 1H), 8.14 (d, J = 2.2 Hz, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.64 – 7.58 (m, 2H), 7.52 (d, J = 8.3 Hz, 2H), 7.38 (t, J = 7.9 Hz, 1H), 7.02 (m, 1H), 6.61 (s, 1H), 6.50 (d, J = 2.3 Hz, 1H), 4.81 (d, J = 6.6 Hz, 2H), 3.82 (s, 3H). 13 C NMR (126 MHz, DMSO- d 6) δ: 164.08, 159.51,155.55, 148.74, 147.01, 144.03, 141.71, 139.65, 131.74, 129.68, 127.09,119.37, 115.43, 112.18, 95.09, 82.35, 55.21, 44.14, 21.06. Calcd. for390.1561. (C 21 H 20 N5O3 + [M+H] + Found 390.1560, see atlas. Figure 6 .

[0041] Example I-7 N -hydroxy-4-(((7-(3,4-dichlorophenyl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)-benzamide

[0042] Yield 69.0%; white solid; mp: 128.0~129.5 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.15 (s, 1H), 9.01 (s, 1H), 8.83 (s, 1H), 8.37 (d, J = 2.1 Hz, 1H), 8.17 (d, J =2.3 Hz, 1H), 8.12 (dd, J = 8.5, 2.1 Hz, 1H), 7.75 (d, J = 8.5 Hz, 1H), 7.70 (d, J =8.3 Hz, 2H), 7.51 (d, J = 8.2 Hz, 2H), 6.80 (s, 1H), 6.53 (d, J = 2.3 Hz, 1H), 4.84 (d, J = 6.6 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ: 164.07, 153.09, 148.62,147.30, 144.28, 141.69, 138.70, 132.35, 131.72, 131.59, 130.81, 128.70,127.11, 127.09, 95.43, 81.94, 44.07. Calcd. for 428.0676. (C 20 H 16 Cl 35 2N5O2 + [M+H] + Found 428.0671, see atlas. Figure 7 .

[0043] Example I-8 N- Hydroxy-4-(((7-(3-(methanesulfonyl)phenyl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide

[0044] Yield 55.2%; white solid; mp: 132.4~133.7 °C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.48 (s, 1H), 8.31 (d, J = 7.6 Hz, 1H), 8.12 (d, J = 8.1 Hz, 1H), 8.01 (s, 1H),7.88 – 7.83 (m, 2H), 7.72 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 7.9 Hz, 2H), 6.57 (s,1H), 6.11 (d, J = 2.1 Hz, 1H), 4.63 (d, J = 5.0 Hz, 2H), 3.30 (s, 3H). 13 C NMR (101MHz, DMSO- d 6) δ: 163.96, 149.49, 143.71, 141.08, 134.20, 134.13, 132.47,129.71, 128.77, 127.59, 127.17, 126.96, 113.79, 43.47. Calcd. for 438.1231.(C 21 H 20 N5O4S + [M+H] + Found 438.1227, see atlas. Figure 8 .

[0045] Example I-9 N -hydroxy-4-(((7-(4-(methoxymethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide

[0046] Yield 59.6%; white solid; mp: 134.8~136.0 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :11.17 (s, 1H), 9.02 (s, 1H), 7.92 (d, J = 8.3 Hz, 3H), 7.81 (d, J = 2.1 Hz, 1H), 7.72 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H), 6.45(s, 1H), 6.07 (d, J = 2.1 Hz, 1H), 4.63 (d, J = 5.6 Hz, 2H), 4.51 (s, 2H), 3.34(s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ: 158.35, 155.55, 149.58, 144.74, 143.36,130.87, 128.66, 127.12, 126.87, 125.16, 114.67, 113.76, 91.80, 64.77, 64.44,43.42. Calcd. for 404.1717. (C 22 H 22 N5O3 + [M+H] + Found 404.1711, see atlas. Figure 9 .

[0047] Example I-10 N -hydroxy-4-(((7-(thiophen-2-yl)pyrazolo[1,5- a ]pyrimidin-5-yl)amino)methyl)benzamide

[0048] 62.8% yield; white solid;mp: 121.1~122.8 °C. 1 H NMR (400 MHz, DMSO- d 6) δ : 11.14 (s, 1H), 8.98 (s, 1H), 8.69 (t, J = 6.7 Hz, 1H), 8.10 (d, J = 2.2 Hz, 1H), 7.83 (d, J = 3.0 Hz, 1H), 7.74 – 7.65 (m, 3H), 7.53 (d, J = 8.2 Hz, 2H),7.17 – 7.14 (m, 1H), 6.64 (s, 1H), 6.43 (d, J = 2.2 Hz, 1H), 4.77 (d, J = 6.6 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6) δ: 164.10, 151.30, 148.43, 146.85, 144.16,143.99, 141.71, 131.75, 129.51, 128.25, 127.18, 127.12, 127.04, 94.71, 80.73,44.14. Calcd. for 366.1019. (C 18 H 16 N5O2S + [M+H] + Found 366.1017, see atlas. Figure 10 .

[0049] Example I-11 N -hydroxy-7-((7-(4-methoxyphenyl)pyrazole[1,5- a Pyrimidin-5-yl)amino)heptadecamide

[0050] Yield 54.6%; white solid; mp: 105.6~107.2 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :10.34 (s, 1H), 8.67 (d, 1H), 8.14 (d, J = 8.9 Hz, 2H), 8.05 (d, J = 2.2 Hz, 1H), 7.90 (t, J = 6.1 Hz, 1H), 7.04 (d, J = 8.9 Hz, 2H), 6.60 (s, 1H), 6.40 (d, J = 2.2Hz, 1H), 3.83 (s, 3H), 1.92 (d, J = 14.0 Hz, 4H), 1.66 (p, J = 7.3 Hz, 2H), 1.50(p, J = 7.4 Hz, 2H), 1.38 – 1.27 (m, 4H). 13 C NMR (101 MHz, DMSO- d 6) δ: 169.18,160.63, 155.62, 148.87, 147.00, 143.64, 130.72, 128.61, 113.90, 94.51, 80.84,55.31, 41.27, 32.28, 28.38, 26.12, 25.13, 21.09. Calcd. for 384.2030.(C 20 H 26 N5O3 + [M+H] + Found 384.2030, see atlas. Figure 11 .

[0051] Example I-12 N -Hydroxy-7-((7-(3-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino)heptanamide

[0052] Yield 57.2%; white solid; mp: 110.2~111.3 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :10.33 (s, 1H), 8.65 (s, 1H), 8.08 (m, 1H), 7.95 (t, J = 6.1 Hz, 1H), 7.75 –7.69 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H), 7.04 (dd, J = 8.1, 2.0 Hz, 1H), 6.65 (s,1H), 6.47 (d, J = 2.2 Hz, 1H), 3.85 (s, 3H), 3.51 (d, J = 6.8 Hz, 2H), 1.93 (dd, J = 13.8, 6.4 Hz, 2H), 1.67 (p, J = 7.3 Hz, 2H), 1.50 (p, J = 7.4 Hz, 2H), 1.33 (m, 4H). 13 C NMR (101 MHz, DMSO- d 6) δ: 170.36, 160.05, 156.53, 149.10, 147.63,144.57, 140.23, 130.43, 120.14, 116.02, 112.77, 95.50, 82.39, 55.77, 41.80,32.74, 28.80, 26.50, 25.59. Calcd. for 384.2030. (C 20 H 26 N5O3 + [M+H] + ). Found384.2033, see atlas Figure 12 .

[0053] Example I-13 N -Hydroxy-7-((7-(3,4-dichlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)heptamide (I-13)

[0054] Yield 58.0%; white solid; mp: 114.7~115.9 °C. 1 H NMR (400 MHz, DMSO- d 6) δ :10.34 (s, 1H), 8.67 (s, 1H), 8.47 – 8.42 (m, 1H), 8.23 ​​– 8.18 (m, 1H), 8.13 –8.07 (m, 2H), 7.76 (d, J = 8.5 Hz, 1H), 6.78 (s, 1H), 6.50 (s, 1H), 1.94 (t, J =7.3 Hz, 2H), 1.71 – 1.62 (m, 2H), 1.54 – 1.46 (m, 2H), 1.39 – 1.20 (m, 6H). 13 CNMR (101 MHz, DMSO- d 6) δ : 169.19, 153.15, 148.63, 147.26, 144.02, 138.91,132.25, 131.56, 130.74, 128.78, 127.24, 95.22, 81.51, 41.34, 32.29, 28.44,26.12, 25.13. Calcd. for 422.1145. (C19 H 22 Cl 35 2N5O2 + [M+H] + Found 422.1143, see atlas. Figure 13 .

[0055] Experimental Example 1 (1) Determination and results of the inhibitory activity of the target compound against HDAC6 The inhibitory activity of the synthesized compounds against HDAC6 was determined by fluorescence resonance energy transfer (FRET), and compared with positive control drugs to screen for compounds with better activity. HDAC was obtained by purification or by purchasing kits directly.

[0056] Specific method: Add enzyme to reaction wells, add reaction buffer to control wells, add sample dissolved in DMSO to reaction wells, incubate using a non-contact nano-level ultrasonic pipetting system, add the corresponding fluorescent substrate to each reaction well, rotate and incubate at 30°C for 1-2 hours, add chromogenic reagent containing TMP26 to stop the reaction, fluorescence is generated, and the fluorescence intensity (excitation light: 490 nM, emission light: 520 nM) is detected using an EnVision multi-label microplate detector (Perkin Elmer). After the color development reaches stability, read the endpoint values, and calculate the percentage (relative to the DMSO control group) and half-maximal inhibition rate using GraphPad Prism 4 software.

[0057] Table 1. Results of the inhibitory activity of the compounds against HDAC6

[0058] (2) Determination of the in vitro antitumor activity of the target compound In this experiment, the CTG method was used to determine the inhibitory effect of the compound on leukemia cell line HL60 and melanoma cell line SK-MEL-2.

[0059] Experimental Methods: Cells in the exponential growth phase were collected for viable cell counting. The cell suspension concentration was adjusted using the corresponding culture medium for each cell type. 90 µL of cell suspension was added to each well in a 96-well cell culture plate. Each test compound was dissolved in DMSO to prepare a 10 mM or 5 mM stock solution, which was then diluted 10-fold with culture medium. Two replicates were performed, with 10 µL of the corresponding 10-fold solution added to each well for each cell line. The final drug concentration was 10 µM or 25 µM, and the final DMSO concentration was 0.1%–0.5% (see Compound Preparation Method and Sample Addition Design: Experimental Plate Sample Addition Design). The plates were incubated at 37 ºC in a 5% CO2 incubator for 72 h. After 72 h of drug treatment, 50 µL (1 / 2 culture volume) of pre-melted and equilibrated CTG solution was added to each well. The mixture was shaken for 2 min using a microplate shaker, and after standing at room temperature for 10 min, the fluorescence signal was measured using an Envision 2104 plate reader. The cell inhibition rate was calculated using the formula: (1-V) sample / V vehicle control Calculated by (×100%), where V sample V represents the mean value of the drug-treated groups. vehicle control This represents the average value of the solvent control group.

[0060] Table 2. Results of antiproliferative activity of some compounds against two tumor cell lines (IC50). 50 (μM)

[0061] (3) Western blot experiment: After step (1) is completed, the loading amount is determined according to the protein concentration. 10 μL of each sample containing 15 μg of protein is taken. Then, the protein is separated by SDS-PAGE gel electrophoresis (conditions: 80 V electrophoresis for 30 min, followed by 120 V electrophoresis for 60 min). The protein is then transferred to a polyvinylidene fluoride (PVDF) membrane (0.45 / 0.22 μm in diameter) (conditions: constant current of 300 mA for transfer, and the transfer time is adjusted according to the molecular weight, approximately 1 minute per 1 kDa). The PVDF membrane is blocked at room temperature with TBST blocking buffer containing 5% skim milk for 2 hours. Then, the membrane is placed in diluted primary antibody (1:2000 dilution ratio) and incubated on a shaker at 4 ℃ for 12 hours. After incubation, the membrane is washed three times with TBST for 5 minutes each time, and then incubated with the corresponding secondary antibody (antibody diluted with 5% skim milk at a ratio of 1:2000) for 2 hours. Finally, the reaction signal on the membrane was detected using a chemiluminescent gel imaging system. ImageJ 2.0 software was used for quantitative analysis of the protein blot images, and GraphPadPrism 8.0 software was used for data analysis and plotting. The results are shown in [reference needed]. Figure 14 .

[0062] (4) Determination of the in vivo antitumor activity of the target compound This experiment used a cell-line-derived xenograft (CDX) mouse model to determine the inhibitory effect of the compound on melanoma.

[0063] Experimental methods: Female NOD / SCID mice (6-8 weeks old) were purchased and housed at the Experimental Animal Center of Xi'an Jiaotong University, fed a standard diet. Before the start of the experiment, the mice were acclimatized for one week in a 25°C, 12-hour light and dark cycle. All animal experiments were approved by the Ethics Committee of the School of Medicine, Xi'an Jiaotong University, and conducted strictly in accordance with relevant standards.

[0064] By containing 2×10 6A melanoma CDX model was successfully established by injecting 200 μL of SK-MEL-2 cell suspension into the back of mice. Mice were then randomly divided into two groups: the treatment group received intraperitoneal injections of 5 mg / kg of compound I-1 (100 μL) every two days for 14 days (a total of 7 injections); the control group received an equal volume of physiological saline every two days. Throughout the treatment period, mouse weight was measured and recorded every two days, tumor size was measured using calipers, and tumor volume was calculated using the formula: V = 0.5(a × b) 2 (), where a is the long axis of the tumor and b is the short axis of the tumor. See the results. Figure 15 .

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A compound having formula (I) or a pharmaceutically acceptable salt thereof: in: R 1 R 2 Each can independently represent hydrogen, methyl, and ethyl; R 3 hydroxyl group; Q 1 It is selected from the following aromatic rings or substituted aromatic rings: phenyl, with substituents being methyl or methoxy; Q 2 Selected from phenyl.

2. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The compound is at least one of the following compounds: (1) N-hydroxy-4-(((7-phenylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (2) N-hydroxy-4-(((7-(p-tolylpyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (3) N-hydroxy-4-(((7-(4-chlorobenzyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (4) N-hydroxy-4-(((7-(3-(trifluoromethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (5) N-hydroxy-4-(((7-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (6) N-hydroxy-4-(((7-(3-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (7) N-hydroxy-4-(((7-(3,4-dichlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (8) N-hydroxy-4-(((7-(3-(methanesulfonyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (9) N-hydroxy-4-(((7-(4-(methoxymethyl)phenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (10) N-hydroxy-4-(((7-(thiophen-2-yl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)methyl)benzamide; (11) N-hydroxy-7-((7-(4-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino)heptamide; (12) N-hydroxy-7-((7-(3-methoxyphenyl)pyrazol[1,5-a]pyrimidin-5-yl)amino)heptamide; (13) N-hydroxy-7-((7-(3,4-dichlorophenyl)pyrazolo[1,5-a]pyrimidin-5-yl)amino)heptamide.

3. The compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein the compound is prepared by the following method: 。 4. The use of the compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof in the preparation of a drug for inhibiting HDAC6.

5. The application according to claim 4, wherein the drug further comprises a medically acceptable carrier.

6. In the application according to claim 5, the compound of any one of claims 1-2 or its pharmaceutically acceptable salt is the sole active ingredient in the medicament.

7. The use of the compound of any one of claims 1-2 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating lung cancer, melanoma, liver cancer, kidney cancer, leukemia, prostate cancer, thyroid cancer, skin cancer, pancreatic cancer, ovarian cancer, testicular cancer, breast cancer, bladder cancer, gallbladder cancer, myelodysplastic syndrome, lymphoma, esophageal cancer, gastrointestinal cancer, tumors of the central or peripheral nervous system, mesothelioma, type II or non-insulin-dependent diabetes mellitus, or autoimmune diseases.

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