A substituted benzisoxazole compound and its application in preparing anti-tumor drugs

Abstract

The present invention relates to a substituted benzisoxazole compound, which has strong inhibitory activity on HDAC1, also shows specificity and selectivity on tumor cells and normal cells, has low potential cardiac toxicity, and has less toxic and side effects. The present invention also discloses the use of the compound as an anti-tumor drug.

Description

Technical Field

[0001] The invention relates to a substituted benzisoxazole compound, a preparation method thereof and application of the compound in preventing and treating tumors and other related diseases by inhibiting HDAC activity. Background Art

[0002] The occurrence of cancer is associated with changes in the genome and epigenetic modifications, such as DNA methylation and histone modification. Histone modification mainly includes histone acetylation, phosphorylation and methylation, among which histone acetylation is an important epigenetic modification in cancer.

[0003] Histone deacetylases (HDACs) are a class of enzymes that remove acetyl groups from histone ε-N-acetylysine and play an important role in gene regulation, transcription, cell proliferation, metastasis and angiogenesis. In the cell nucleus, histone acetylation and deacetylation are in a dynamic equilibrium, which is jointly regulated by histone acetyltransferases (HATs) and histone deacetylases. HATs transfer the acetyl group of acetyl-CoA to the lysine residue at the end of histones, making the structure of chromosomes loose, which is conducive to the specific binding of various transcription factors and co-transcription factors to DNA binding sites and activating gene transcription. HDACs deacetylate histones, and histones bind tightly to negatively charged DNA, hindering the binding of DNA polymerase to DNA and inhibiting gene transcription. Under pathological conditions, HDACs are overexpressed, causing dynamic imbalance, reducing the expression of certain anti-cancer genes and tumor suppressor genes, and ultimately leading to tumorigenesis.

[0004] According to the catalytic mechanism, location in the cell, homology with yeast sequences, and peptide chain folding, HDACs can be divided into two families and four categories: Class I: HDAC1, 2, 3, 8; Class II: HDAC4, 5, 6, 7, 9; Class III: SIRT1-7; Class IV: HDAC11. Among them, Class I, II, and IV belong to Zn 2+ The family of histone deacetylation catalyzed by the 2+ ; Class III belongs to the nicotinamide adenine dinucleotide-dependent family, and its catalytic histone deacetylation requires nicotinamide adenine dinucleotide.

[0005] HDAC inhibitors inhibit the activity of HDAC, enhance the acetylation of histones, promote the binding of transcription factors to DNA chains, and activate the expression of specific genes such as tumor suppressor genes, thereby inhibiting the proliferation of tumor cells and promoting tumor cell apoptosis. The structure of HDAC inhibitors is generally composed of Cap group, Linker and Zn 2+The Cap group is generally a hydrophobic and aromatic group that can interact with the surface recognition area of ​​the HDAC active pocket; ZBG interacts with the Zn at the bottom of the HDAC active pocket. 2+ Chelation forms a complex, which is the key pharmacophore; Linker is composed of a linear or cyclic hydrophobic structure connecting the Cap group and ZBG. Currently, there are 5 HDAC inhibitors on the market, of which Vorinostat, Romidepsin, Belinostat, and Panobinostat are approved by the US FDA for the treatment of cutaneous T-cell lymphoma, multiple myeloma, and peripheral T-cell lymphoma. Chidamide is approved in China for the treatment of relapsed or refractory peripheral T-cell lymphoma.

[0006]

[0007] Currently, HDAC inhibitor drugs still have the following shortcomings: (1) they have significant clinical toxic side effects, such as nausea, vomiting, and bone marrow suppression; (2) they are only effective in treating hematological tumors, but not in treating solid tumors; and (3) they are prone to drug resistance during use.

[0008] Based on the above shortcomings, it is urgent to develop new HDAC inhibitors with high efficiency and low toxicity for the research of new anti-tumor drugs. Therefore, seeking new anti-tumor active molecules with more development value than existing clinical drugs is an important research content in this field and is also urgently needed in clinical practice. Summary of the invention

[0009] The purpose of the present invention is to provide a substituted benzisoxazole compound and its use in the preparation of anti-tumor drugs, so as to discover HDAC inhibitor anti-tumor compounds with new structural types to meet the needs of clinical application.

[0010] The present invention adopts the following technical solution:

[0011] A substituted benzisoxazole compound, the general formula of which is:

[0012]

[0013] R 1 Selected from H, halogen, aryl, C 1-3 alkyl, saturated heterocyclic group, substituted saturated heterocyclic group, alkoxy acyl group, N-alkylcarbamoyl group;

[0014] R 2 Selected from H, halogen groups, C 1-3 Alkyl, C 1-3 Alkoxy, nitro.

[0015] As a preference, R1 is selected from H, F, methyl, phenyl, pyridyl, piperidinyl, N-methylpiperazinyl, N-acetylpiperazinyl, N-isopropylpiperazinyl, N-cyclopropylpiperazinyl, morpholinyl;

[0016] R 2 Selected from the following groups: H, F, Cl, methyl, ethyl, methoxy, ethoxy, nitro.

[0017] More preferably, R 1 Selected from the following groups:

[0018] R 2 Selected from H, F, Cl, methyl, methoxy, nitro.

[0019] More preferably, the following specific compounds are selected from the compounds of formula (I): I-1: N-hydroxy-3-phenylbenzo[c]isoxazole-5-carboxamide;

[0020] I-2: N-hydroxy-3-(p-toluene)benzo[c]isoxazole-5-carboxamide;

[0021] I-3: 3-([1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide;

[0022] I-4: N-hydroxy-3-(4-(pyridin-4-yl)phenyl)benzo[c]isoxazole-5-carboxamide;

[0023] I-5: N-hydroxy-3-(4-(piperidin-4-yl)phenyl)benzo[c]isoxazole-5-carboxamide; I-6: N-hydroxy-3-(4-(4-methylpiperazin-1-yl)phenyl)benzo[c]isoxazole-5-carboxamide; I-7: 3-(4-(4-acetylpiperazin-1-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide; I-8: N-hydroxy-3-(4-(4-isopropylpiperazin-1-yl)phenyl)benzo[c]isoxazole-5-carboxamide; I-9: 3-(4-(4-cyclopropylpiperazin-1-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide; I-10: N-hydroxy-3-(4-morpholinophenyl)benzo[c]isoxazole-5-carboxamide;

[0024] I-11: 3-(2-fluoro-[1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide; I-12: N-hydroxy-3-(2-methoxy-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide;

[0025] I-13: 3-(2-chloro-[1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide; I-14: N-hydroxy-3-(2-methyl-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide;

[0026] I-15: N-hydroxy-3-(2-isopropyl-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide;

[0027] I-16: N-hydroxy-3-(2-nitro-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide;

[0028] I-17: 3-(3-fluorophenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide;

[0029] I-18: 3-(3-Fluoro-4-(piperidin-4-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide. The structural formula of the above compound is shown in Table 1.

[0030] Table 1 Preferred compound numbers and corresponding structural formulas

[0031]

[0032]

[0033] A method for preparing the above-mentioned substituted benzisoxazole compounds, the synthesis route is as follows:

[0034]

[0035] The specific steps are:

[0036] 1) dissolving compound II in a protic solvent, adding a base catalyst, and reacting with 2-(4-nitrophenyl)-1,3-dioxolane to obtain intermediate III;

[0037] 2) intermediate III is deprotected in the presence of a catalyst to obtain intermediate IV;

[0038] 3) Intermediate IV is subjected to oxidation reaction to obtain intermediate V;

[0039] 4) intermediate V is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate VI;

[0040] 5) The intermediate VI is deprotected to obtain the target compound I.

[0041] Furthermore, in step 1), the protic solvent is methanol, ethanol or isopropanol; the base catalyst is sodium hydroxide, potassium hydroxide or potassium tert-butoxide; and the reaction temperature is 10-40°C.

[0042] Furthermore, in step 2), the catalyst is trifluoroacetic acid, hydrochloric acid or sulfuric acid; the reaction solvent is acetonitrile / water, tetrahydrofuran / water, 1,4-dioxane / water; and the reaction temperature is 10-40°C.

[0043] Further, in step 3), the oxidant is KHSO 5 1 / 2KHSO 4 1 / 2K 2 SO 4 ; The reaction solvent is DMF or DMA.

[0044] Furthermore, in step 4), the condensation agent is HBTU, HATU, HOBt or TFCH; the base catalyst is triethylamine, diisopropylethylamine or pyridine; the reaction solvent is DMF or DMA; and the reaction temperature is 10-40°C.

[0045] Furthermore, in step 5), the catalyst is trifluoroacetic acid, hydrochloric acid or sulfuric acid; the reaction solvent is acetonitrile / water, tetrahydrofuran / water, 1,4-dioxane / water; and the reaction temperature is 10-40°C.

[0046] A use of the substituted benzisoxazole compound in the preparation of histone deacetylase inhibitors or anti-tumor drugs.

[0047] Furthermore, the histone deacetylase inhibitor is an HDAC1 inhibitor.

[0048] Furthermore, the indications of the anti-tumor drug include one or more of T-cell lymphoma, multiple myeloma, colorectal cancer, liver cancer and prostate cancer.

[0049] The compounds of the present invention can be used in combination for mammals (including humans) that need tumor treatment by oral administration, injection, etc., wherein oral administration is the best, and the dosage is 0.0001 mg / kg to 200 mg / kg body weight per day. The best dosage depends on the individual, usually starting with a small dosage and then gradually increasing the dosage.

[0050] The pharmaceutical composition comprises a therapeutically effective dose of the compound represented by formula (I) and a pharmaceutically acceptable carrier.

[0051] The carrier refers to a common carrier in the pharmaceutical field, for example: a diluent, an excipient such as water, etc.; a binder such as cellulose derivatives, gelatin, polyvinyl pyrrolidone, etc.; a filler such as starch, etc.; a disintegrant such as calcium carbonate, sodium bicarbonate; in addition, other auxiliary agents such as flavoring agents and sweeteners may also be added to the composition.

[0052] The composition of the present invention can be prepared into conventional solid preparations, such as tablets, capsules, etc., for oral administration, or can be prepared into injections for injection.

[0053] Various dosage forms of the composition of the present invention can be prepared by conventional methods in the pharmaceutical field, wherein the content of the active ingredient I is 0.1% to 99.5% (by weight) of the weight of the compound.

[0054] The beneficial effects of the present invention are:

[0055] 1) The compounds of the present invention have novel structures, good HDAC enzyme inhibitory activity, and good inhibitory effects on a variety of human tumor cells, with broad-spectrum and high-efficiency characteristics. They may also have good therapeutic effects on diseases caused by abnormal gene expression, such as endocrine disorders, immune system diseases, genetic diseases and nervous system diseases.

[0056] 2) The compounds of the present invention have a weak inhibitory effect on normal cells while effectively inhibiting tumor cells, showing good selective inhibitory activity and having a good prospect for clinical application in tumors.

[0057] 3) The compounds of the present invention have weak inhibitory effects on hERG potassium channels, have low potential cardiac toxicity, have good safety, and are worth further study.

[0058] In summary, the present invention structurally transforms the binding site acting on the HDAC enzyme to replace the benzisoxazole structure as the Cap region, retains the ZBG domain, and designs and synthesizes a series of substituted benzisoxazole compounds. In the structure of such compounds, the substituted benzisoxazole structure can theoretically form a π-π interaction with the receptor, making it more suitable for binding to the HDAC enzyme. In addition, the new structural type compound of the present invention has strong enzyme inhibitory activity while also showing specificity and selectivity for tumor cells and normal cells. At the same time, the new structural compound has higher safety, less toxic and side effects, and is easier to use as an anti-tumor drug. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0060] Example 1 Synthesis of Intermediate 5-(1,3-dioxolan-2-yl)-3-phenylbenzo[c]isoxazole

[0061]

[0062] NaOH (6.15 g, 153.70 mmol) was dissolved in isopropanol (200 mL), ultrasonically dissolved, 2-(4-nitrophenyl)-1,3-dioxolane (3.00 g, 15.37 mmol) and 2-phenylacetonitrile (2.70 g, 23.06 mmol) were added, and the reaction was stirred at room temperature for 23 h. After the reaction was completed, the reaction solution was filtered, the filter cake was added to 350 mL of water for slurrying, filtered, and the filter cake was dried. The filter cake was recrystallized from isopropanol at 70 ° C for 1 h, filtered, and the filter cake was dried to obtain 3.46 g of yellow solid with a yield of 84.2%. HRMS: m / zC 16 H 14 NO 3 [M+H] + 268.0895,Found 268.0976.

[0063] Example 2 Synthesis of Intermediate 3-phenylbenzo[c]isoxazole-5-aldehyde

[0064]

[0065] The intermediate 5-(1,3-dioxolan-2-yl)-3-phenylbenzo[c]isoxazole (1.56 g, 5.83 mmol) was dissolved in MeCN (52 mL) / H 2 O (13 mL), TFA (3 mL) was added, and the reaction was stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was filtered and the filter cake was dried. The filtrate was concentrated and slowly dripped into 200 mL of water and diluted with 2M Na 2 CO 3 Adjust pH to about 7.0, extract with dichloromethane (25 mL × 3), combine the organic phases, dry over anhydrous magnesium sulfate, filter, and concentrate to obtain 1.28 g of yellow solid with a yield of 98.5%. The next step can be directly carried out. HRMS: m / z C 14 H 10 NO 2 [M+H] + 224.0633,Found 224.0892.

[0066] Example 3 Synthesis of Intermediate 3-phenylbenzo[c]isoxazole-5-carboxylic acid

[0067]

[0068] The intermediate 3-phenylbenzo[c]isoxazole-5-aldehyde (0.89 g, 4.01 mmol) was dissolved in DMF (10 mL), potassium persulfate (1.23 g, 4.01 mmol) was added, and the reaction was stirred at room temperature for 18 h. After the reaction was completed, the reaction was quenched with 2M HCl, and the reaction solution was slowly dripped into 100 mL of water. Solids precipitated, filtered, and the filter cake was dried. The filter cake was recrystallized with methanol at 60 ° C for 1 h, cooled and filtered, and the filter cake was dried. The filter cake was slurried with petroleum ether: ethyl acetate = 2:1, filtered, and the filter cake was dried. The filter cake was recrystallized with methanol at 60 ° C for 1 h, cooled and filtered, and the filter cake was dried to obtain 608.7 mg of a yellow solid with a yield of 63.5%. HRMS: m / z C 14 H 10 NO 3 [M+H] + 240.0582,Found 240.1052.

[0069] Example 4 Synthesis of Intermediate 3-phenyl-N-((tetrahydro-2H-pyran-2-yl)oxy)benzo[c]isoxazole-5-carboxamide

[0070]

[0071] The intermediate 3-phenylbenzo[c]isoxazole-5-carboxylic acid (227.11 mg, 0.95 mmol), O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (123.01 mg, 1.05 mmol), HATU (433.46 mg, 1.14 mmol), triethylamine (480.65 mg, 4.75 mmol) were dissolved in DMF (4 mL) and N 2 After the reaction was completed, the reaction solution was slowly dripped into 100 mL of ice water. Solids were precipitated and filtered. The filter cake was dried. The filter cake was slurried with dichloromethane and dried to obtain 180.1 mg of yellow solid with a yield of 56.1%. HRMS: m / z C 19 H 19 N 2 O 4 [M+H] + 339.1267, Found 339.2413.

[0072] Example 5 Synthesis of N-hydroxy-3-phenylbenzo[c]isoxazole-5-carboxamide (target compound I-1)

[0073]

[0074] The intermediate 3-phenyl-N-((tetrahydro-2H-pyran-2-yl)oxy)benzo[c]isoxazole-5-carboxamide (81.15 mg, 0.24 mmol) was dissolved in MeCN (24 mL) / H 2 O (6 mL), TFA (0.5 mL) was added, and the reaction was stirred at room temperature for 3 h. After the reaction was completed, the reaction solution was filtered and the filter cake was dried. The filter cake was slurried with dichloromethane and methanol and dried to obtain 20.7 mg of a yellow solid with a yield of 33.9%. HRMS: m / z C 14 H 11 N 2 O 3 [M+H] + 255.0691,Found 255.0842.

[0075] 1 H NMR (500 MHz, DMSO-d 6 )δ10.48(s,1H),9.49(s,1H),8.24(s,1H),7.98(s,2H),7.78(s,3H),7.53(s,1H),7.46(s,1H).

[0076] Example 6 Synthesis of N-hydroxy-3-(p-toluene)benzo[c]isoxazole-5-carboxamide (target compound I-2)

[0077]

[0078] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-2 can be prepared with a yield of 50.6%. HRMS: m / z C 15 H 13 N 2 O 3 [M+H] + 269.0848,Found 269.1253.

[0079] 1 H NMR (500 MHz, DMSO-d 6 )δ10.50(s,1H),9.34(s,1H),8.29(s,2H),7.75(s,3H),7.55(s,1H),7.50(s,1H),2.34(s,3H).

[0080] Example 7 Synthesis of 3-([1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-3)

[0081]

[0082] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-3 can be prepared with a yield of 35.6%. HRMS: m / z C 20 H 15 N 2 O 3 [M+H] + 331.1004,Found 331.1765.

[0083] 1 H NMR (500 MHz, DMSO-d 6 )δ11.48(s,1H),9.23(s,1H),8.53(s,1H),8.26(s,2H),7.98(s,2H),7.80(s,3H),7.76(s,1H),7.54(s,2H),7.46(s,1H).

[0084] Example 8 Synthesis of N-hydroxy-3-(4-(pyridin-4-yl)phenyl)benzo[c]isoxazole-5-carboxamide (target compound I-4)

[0085]

[0086] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-4 can be prepared with a yield of 57.1%. HRMS: m / z C 19 H 14 N 3 O 3 [M+H] + 332.0957,Found 332.0993.

[0087] 1 H NMR (500 MHz, DMSO-d 6 )δ11.50(s,1H),9.35(s,1H),8.29(s,2H),7.93(s,2H),7.78(s,3H),7.65(s,1H),7.60(s,2H),7.50(s,1H).

[0088] Example 9 Synthesis of N-hydroxy-3-(4-(piperidin-4-yl)phenyl)benzo[c]isoxazole-5-carboxamide (target compound I-5)

[0089]

[0090] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-5 can be prepared with a yield of 48.0%. HRMS: m / z C19 H 20 N 3 O 3 [M+H] + 338.1426, Found 338.1297.

[0091] 1 H NMR (500 MHz, DMSO-d 6 )δ10.95(s,1H),9.37(s,1H),8.76(s,1H),8.69(s,2H),7.89(s,2H),7.73(s,1H),6.87(s,1H ), 2.82 (s, 1H), 2.80-2.68 (m, 4H), 2.00 (s, 1H), 1.79 (t, J = 2.4Hz, 2H), 1.49 (t, J = 2.7Hz, 2H).

[0092] Example 10 Synthesis of N-hydroxy-3-(4-(4-methylpiperazin-1-yl)phenyl)benzo[c]isoxazole-5-carboxamide (target compound I-6)

[0093]

[0094] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-6 can be prepared with a yield of 30.2%. HRMS: m / z C 19 H 21 N 4 O 3 [M+H] + 353.1535, Found 353.1678.

[0095] 1 H NMR (500 MHz, DMSO-d 6 )δ11.60(s,1H),9.49(s,1H),8.36(s,2H),7.90(s,1H),7.87(s,1H),7.50(s ,2H),7.48(s,1H),3.42(t,J=3.5Hz,4H),2.23(t,J=2.8Hz,4H),2.10(s,3H).

[0096] Example 11 Synthesis of 3-(4-(4-acetylpiperazin-1-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-7)

[0097]

[0098] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-7 can be prepared with a yield of 40.9%. HRMS: m / z C 20 H 21 N 4 O 4 [M+H] + 381.1485, Found 381.1672.

[0099] 1 H NMR (500 MHz, DMSO-d 6 )δ11.60(s,1H),9.49(s,1H),8.36(s,2H),7.90(s,1H),7.87(s,1H),7.50(s ,2H),7.48(s,1H),3.42(t,J=3.5Hz,4H),2.23(t,J=2.8Hz,4H),2.10(s,3H).

[0100] Example 12 Synthesis of N-hydroxy-3-(4-(4-isopropylpiperazin-1-yl)phenyl)benzo[c]isoxazole-5-carboxamide (target compound I-8)

[0101]

[0102] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-8 can be prepared with a yield of 39.4%. HRMS: m / z C 21 H 25 N 4 O 3 [M+H] + 381.1848,Found 381.1994.

[0103] 1 H NMR (500 MHz, DMSO-d 6 )δ11.08(s,1H),9.76(s,1H),8.21(s,2H),7.88(s,1H),7.64(s,1H),7.52(s,2H),7.46(s ,1H),3.40(t,J=3.4Hz,4H),2.25(t,J=2.7Hz,4H),2.69(s,1H),1.20(s,3H),1.00(s,3H).

[0104] Example 13 Synthesis of 3-(4-(4-cyclopropylpiperazin-1-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-9)

[0105]

[0106] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-9 can be prepared with a yield of 50.6%. HRMS: m / z C 21 H 23 N 4 O 3 [M+H] + 379.1692,Found 379.1962.

[0107] 1 H NMR (500 MHz, DMSO-d 6 )δ10.50(s,1H),9.23(s,1H),8.26(s,2H),7.83(s,1H),7.65(s,1H),7.54(s,2H),7.37( s, 1H), 3.44 (t, J = 5.2Hz, 4H), 2.57 (t, J = 2.7Hz, 4H), 2.25 (s, 1H), 0.82 (d, J = 7.6Hz, 4H).

[0108] Example 14 Synthesis of N-hydroxy-3-(4-morpholinophenyl)benzo[c]isoxazole-5-carboxamide (target compound I-10)

[0109]

[0110] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-10 can be prepared with a yield of 59.1%. HRMS: m / z C 18 H 18 N 3 O 4 [M+H] + 340.1219, Found 340.1129.

[0111] 1 H NMR (500 MHz, DMSO-d 6 )δ10.76(s,1H),8.79(s,1H),8.26(s,2H),7.91(s,1H),7.67(s,1H),7.50(s,2H),6.98(s,1H),3.53(t,J=3.4Hz,4H),3.23(t,J=2.7Hz,4H).

[0112] Example 15 Synthesis of 3-(2-fluoro-[1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-11)

[0113]

[0114] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-11 can be prepared with a yield of 49.9%. HRMS: m / z C 20 H 14 FN 2 O 3 [M+H] + 349.0910,Found 349.1098.

[0115] 1 H NMR (500 MHz, DMSO-d 6 )δ11.40(s,1H),9.28(s,1H),8.56(s,1H),8.39(s,2H),7.90(s,2H),7.77(s,3H),7.68(s,1H),7.60(s,2H).

[0116] Example 16 Synthesis of N-hydroxy-3-(2-methoxy-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide (target compound I-12)

[0117]

[0118] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-12 can be prepared with a yield of 46.3%. HRMS: m / z C 21 H 17 N 2 O 4 [M+H] + 361.1110, Found 361.1256.

[0119] 1 H NMR (500 MHz, DMSO-d 6 )δ11.20(s,1H),9.46(s,1H),8.46(s,1H),8.30(s,2H),8.22(s,1H),7.80(s,2H),7.76(s,2H),7.64(s,1H),7.62(s,2H),3.79(s,3H).

[0120] Example 17 Synthesis of 3-(2-chloro-[1,1'-biphenyl]-4-yl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-13)

[0121]

[0122] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-13 can be prepared with a yield of 50.9%. HRMS: m / z C 20 H 14 C1N 2 O 3 [M+H] + 365.0615,Found 365.0764.

[0123] 1 H NMR (500 MHz, DMSO-d 6 )δ10.95(s,1H),8.76(s,1H),8.69(s,2H),7.89(s,2H),7.73(s,3H),7.59(s,1H),7.40(s,2H),6.87(s,1H).

[0124] Example 18 Synthesis of N-hydroxy-3-(2-methyl-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide (target compound I-14)

[0125]

[0126] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-14 can be prepared with a yield of 30.8%. HRMS: m / z C 21 H 17 N 2 O 3 [M+H] + 345.1161, Found 345.1698.

[0127] 1 H NMR (500 MHz, DMSO-d 6 )δ11.09(s,1H),8.58(s,1H),8.42(s,2H),7.86(s,2H),7.74(s,3H),7.60(s,1H),7.42(s,2H),6.86(s,1H),2.57(s,3H).

[0128] Example 19 Synthesis of N-hydroxy-3-(2-isopropyl-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide (target compound I-15)

[0129]

[0130] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-15 can be prepared with a yield of 45.0%. HRMS: m / z C 23 H 21 N 2 O 3 [M+H] + 373.1474,Found 373.1751.

[0131] 1 H NMR (500 MHz, DMSO-d 6 )δ11.02(s,1H),8.68(s,1H),8.40(s,2H),7.96(s,2H),7.68(s,3H),7. 58(s,1H),7.43(s,2H),6.68(s,1H),2.88(m,1H),1.18(d,J=2.5Hz,6H).

[0132] Example 20 Synthesis of N-hydroxy-3-(2-nitro-[1,1'-biphenyl]-4-yl)benzo[c]isoxazole-5-carboxamide (target compound I-16)

[0133]

[0134] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-16 can be prepared with a yield of 61.0%. HRMS: m / z C 20 H 14 N 3 O 5 [M+H] + 376.0855, Found 376.0978.

[0135] 1 H NMR (500 MHz, DMSO-d 6 )δ10.82(s,1H),8.86(s,1H),8.42(s,2H),8.02(s,2H),7.70(s,3H),7.56(s,1H),7.28(s,2H),6.64(s,1H).

[0136] Example 21 Synthesis of 3-(3-fluorophenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-17)

[0137]

[0138] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-17 can be prepared with a yield of 38.8%. HRMS: m / z C 14 H 10 FN 2 O 3 [M+H] + 273.0597,Found 273.0813.

[0139] 1 H NMR (500 MHz, DMSO-d 6 )δ10.84(s,1H),8.80(s,1H),8.44(s,1H),8.06(s,1H),7.76(s,3H),7.65(s,1H),7.28(s,1H).

[0140] Example 22 Synthesis of 3-(3-fluoro-4-(piperidin-4-yl)phenyl)-N-hydroxybenzo[c]isoxazole-5-carboxamide (target compound I-18)

[0141]

[0142] According to the preparation method of the target compound I-1 in Examples 1 to 5, the target compound I-18 can be prepared with a yield of 50.0%. HRMS: m / z C 19 H 19 FN 3 O 3 [M+H] + 356.1332, Found 356.1653.

[0143] 1 H NMR (500 MHz, DMSO-d 6 )δ10.90(s,1H),9.37(s,1H),8.86(s,1H),8.69(s,2H),7.79(s,2H),6.87(s,1H),2.8 4(s,1H),2.82-2.74(m,4H),2.00(s,1H),1.78(t,J=2.5Hz,2H),1.50(t,J=2.7Hz,2H).

[0144] Example 23 In vitro HDAC1 inhibitory activity test of the compounds of the present invention

[0145] The inhibitory activity of the test compound on HDAC1 was tested using a kit, and the test operation was carried out according to the instructions of the kit. 50 Test. Vorinostat was selected as the control drug, and the test operation was as follows:

[0146] (1) Compound preparation

[0147] The compound was prepared into a DMSO solution with a stock concentration of 10 mM and stored in the dark for future use.

[0148] (2) HDAC1 reaction process

[0149] a. Prepare 1× reaction solution.

[0150] b. Compound preparation: The final concentration of the compound test is 10uM, 3-fold dilution, 8 concentrations, and single-well detection. The final concentration of the positive control compound Vorinostat is 3μM, 3-fold dilution, 10 concentrations, and multiple-well detection. Gradually dilute to the corresponding 100-fold final concentration solution in the 384-well Source plate, and then use Echo550 to transfer 250nL to the 384-well reaction plate for testing. Transfer 250nL of 100% DMSO to both the Max well and the Min well.

[0151] c. Use 1× reaction solution to prepare 1.67× enzyme solution.

[0152] d. Add 15 μL of 1.67× enzyme solution to each well and 15 μL of 1.6× reaction solution to the Min well. Incubate at room temperature for 15 minutes.

[0153] e. Use 1× reaction solution to prepare 2.5× substrate mixed solution.

[0154] f. Add 10 μL of 2.5× substrate mixed solution to each well of the reaction plate to start the reaction.

[0155] g. Use Synergy to continuously read the fluorescence signal.

[0156] (3) Data analysis

[0157] The linear response segment was selected to obtain the slope and the percentage inhibition rate was calculated using the following formula:

[0158]

[0159] Where: Mean(Max) is the mean of the slope values ​​of the Max wells (containing DMSO and enzyme); Mean(Min) is the mean of the slope values ​​of the Min wells (wells without enzyme); Sample Signal is the slope value of the compound well.

[0160] Fitting the dose-effect curve: The log value of the compound concentration is used as the X-axis, and the corresponding percentage inhibition rate is used as the Y-axis. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism 5 is used to fit the dose-effect curve, so as to obtain the IC value of each compound for inhibiting enzyme activity. 50 The test results are shown in Table 2.

[0161] Table 2 Results of in vitro inhibitory activity test of compounds on HDAC1

[0162]

[0163] As can be seen from Table 2 above, the compounds of the present invention have strong inhibitory activity against HDAC1, such as compounds I-3, I-4, I-7, I-8, I-10 and I-13, whose inhibitory activity against HDAC1 is comparable to or better than that of the positive control drug Vorinostat. Among them, compound I-4 has an activity 2.4 times that of the positive control drug Vorinostat.

[0164] Example 24 In vitro tumor cell inhibition activity test of the compounds of the present invention

[0165] Compounds with good inhibitory activity on HDAC1 were selected and tested for their activity on human liver cancer cell line HepG2, human prostate cancer cell line PC-3, human colorectal cancer cell line HCT116 and non-small cell lung cancer A549. 50 The value was measured by CCK-8 method, Vorinostat was selected as the control drug, and the experimental operation was as follows:

[0166] (1) Cell inoculation: Take cells in the logarithmic growth phase and inoculate them into a 96-well plate, with 10,000 cells per well. Culture for 12 hours to allow the cells to adhere normally.

[0167] (2) Drug preparation: Weigh the drug and dissolve it in the corresponding solvent to prepare a stock solution with a concentration of 1 mM. Perform a 2-fold gradient dilution in complete culture medium during the test.

[0168] (3) Drug treatment: The culture medium in the 96-well plate was aspirated and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to the wells, and 3 replicate wells were set for each measurement; at the same time, DMSO control wells and blank culture medium control wells were set. The drug was allowed to act for 12 hours.

[0169] (4) CCK8 detection: 10 μL CCK8 was added to each well, and the cells were cultured for 1-2 hours. The detection wavelength was 450 nm and the reference wavelength was 600-650 nm.

[0170] (5) Result statistics: Cell survival rate: [(As-Ab) / (Ac-Ab)]×100%

[0171] Wherein: As: absorbance of the experimental well (culture medium containing cells, CCK8, and the drug to be tested); Ab: absorbance of the blank well (culture medium without cells and the drug to be tested, CCK8); Ac: absorbance of the control well (culture medium containing cells, CCK8, and no drug to be tested).

[0172] (6) Graphpad Prism 9.5.1 software was used to calculate the IC for the above three different cell types. 50 The test results are shown in Table 3 (unit: μM).

[0173] Table 3 In vitro inhibitory activity of compounds on tumor cells

[0174]

[0175] As can be seen from Table 3 above, compared with the positive control drug Vorinostat, the compounds of the present invention exhibited good in vitro anti-tumor cell proliferation activity against a variety of tumor cells, and some compounds had better anti-tumor cell proliferation activity than the positive control drug Vorinostat. Among them, I-4, I-8 and I-10 have high inhibitory activity against human liver cancer cell line HepG2; compounds I-3 and I-8 have high inhibitory activity against human prostate cancer cell line PC-3; compound I-3 has high inhibitory activity against human colorectal cancer cell line HCT116, and compounds I-7 and I-13 have high inhibitory activity against non-small cell lung cancer cell line A549, with the characteristics of broad spectrum and high efficiency.

[0176] Example 25 In vitro inhibitory activity test of the compounds of the present invention on normal cells

[0177] The activity of the compounds of the present invention on MRC-5 human embryonic lung fibroblasts was determined, and the IC 50 The value was measured by CCK-8 method. Vorinostat was selected as the control drug to measure the IC inhibitory activity of normal cell lines in vitro. 50 Test, the test operation is as follows:

[0178] (1) Cell inoculation: Take cells in the logarithmic growth phase and inoculate them into a 96-well plate, with 10,000 cells per well. Culture for 12 hours to allow the cells to adhere normally.

[0179] (2) Drug preparation: Weigh the drug and dissolve it in the corresponding solvent to prepare a stock solution with a concentration of 1 mM. Perform a 2-fold gradient dilution in complete culture medium during the test.

[0180] (3) Drug treatment: The culture medium in the 96-well plate was aspirated and the cells were washed once with PBS. 100 μL of culture medium containing different drug concentrations was added to the wells, and 3 replicate wells were set for each measurement; at the same time, DMSO control wells and blank culture medium control wells were set. The drug was allowed to act for 12 hours.

[0181] (4) CCK8 detection: 10 μL CCK8 was added to each well, and the cells were cultured for 1-2 hours. The detection wavelength was 450 nm and the reference wavelength was 600-650 nm.

[0182] (5) Result statistics: Cell survival rate: [(As-Ab) / (Ac-Ab)]×100%

[0183] Wherein: As: absorbance of the experimental well (culture medium containing cells, CCK8, and the drug to be tested); Ab: absorbance of the blank well (culture medium without cells and the drug to be tested, CCK8); Ac: absorbance of the control well (culture medium containing cells, CCK8, and no drug to be tested).

[0184] (6) Graphpad Prism 9.5.1 software was used to calculate the IC values ​​of different drugs on MRC-5 human embryonic lung fibroblasts 50 The test results are shown in Table 4 (unit: μM).

[0185] Table 4 In vitro inhibitory activity of compounds and control drugs on normal cells

[0186]

[0187] As can be seen from Table 4 above, the tested compounds of the present invention have weaker inhibitory activity on normal cells and lower toxic side effects compared to the control drug Vorinostat, which reveals that the hydroxamic acid HDAC1 inhibitor of the present invention has better selectivity in inhibiting the proliferation of tumor cells and normal cells, indicating that it will have lower toxic side effects when used as an anti-tumor drug.

[0188] Example 26 hERG potassium channel effect test of the compounds of the present invention

[0189] The hERG potassium channel inhibition test was used to preliminarily investigate the potential cardiac toxicity and side effects of the compounds of the present invention in vitro. The test procedure is as follows:

[0190] (1) Cell preparation: CHO-hERG cells were cultured at 175 cm 2In the culture flask, when the cell density grows to 60-80%, remove the culture medium, wash once with 7mL PBS (Phosphate Buffered Saline), and then add 3mL Detachin for digestion. After digestion is complete, add 7mL culture medium for neutralization, then centrifuge, aspirate the supernatant, and add 5mL culture medium for resuspending to ensure that the cell density is 2-5×10 6 / mL.

[0191] (2) Electrophysiological recording process: The single-cell high-impedance sealing and whole-cell pattern formation process were all automatically completed by the Qpatch instrument. After obtaining the whole-cell recording mode, the cell was clamped at -80 mV. Before a 5-second +40 mV depolarizing stimulus was given, a 50-millisecond -50 mV pre-voltage was given, and then repolarized to -50 mV for 5 seconds, and then returned to -80 mV. This voltage stimulus was applied every 15 seconds. After recording for 2 minutes, extracellular solution was given for 5 minutes of recording, and then the drug administration process began. The compound concentration started from the lowest test concentration, and each test concentration was given for 2.5 minutes. After all concentrations were given continuously, the positive control compound 3 μM Cisapride was given. At least 3 cells (n≥3) were tested for each concentration.

[0192] (3) Compound preparation: Dilute the compound stock solution with DMSO, take 10 μL of the compound stock solution and add it to 20 μL of DMSO solution, and dilute it 3 times to 6 DMSO concentrations. Take 4 μL of the compound with 6 DMSO concentrations respectively, add it to 396 μL of extracellular fluid, dilute it 100 times to 6 intermediate concentrations, then take 80 μL of the 6 intermediate concentration compounds respectively, add it to 320 μL of extracellular fluid, and dilute it 5 times to the final concentration to be tested. The highest test concentration is 40.00 μM, and there are 6 concentrations of 40.00, 13.33, 4.44, 1.48, 0.49, and 0.16 μM respectively. The DMSO content in the final test concentration does not exceed 0.2%, and this concentration of DMSO has no effect on hERG potassium channels. The compound preparation is completed by the Bravo instrument throughout the dilution process.

[0193] (4) Data analysis: The experimental data were analyzed using GraphPad Prism 5.0 software.

[0194] Table 5 hERG potassium channel effect test results of compounds (IC 50 / μM)

[0195]

[0196] The hERG test results show that the inhibitory activity of the compounds of the present invention on the hERG potassium ion channel is greater than 30 μM, indicating that the potential cardiac toxicity of the compounds of the present invention is relatively low.

[0197] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitution combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the scope of the present invention.

Claims

1. A substituted benzisoxazole compound, characterized in that: The general formula is: R 1 Selected from the following groups: R 2 Selected from H, F, Cl, methyl, methoxy, nitro.

2. A method for preparing the substituted benzisoxazole compound according to claim 1, characterized in that: The synthetic route is as follows: The steps are: 1) dissolving compound II in a protic solvent, adding a base catalyst, and reacting with 2-(4-nitrophenyl)-1,3-dioxolane to obtain intermediate III; 2) intermediate III is deprotected in the presence of a catalyst to obtain intermediate IV; 3) Intermediate IV is subjected to oxidation reaction to obtain intermediate V; 4) intermediate V is reacted with O-(tetrahydro-2H-pyran-2-yl)hydroxylamine in the presence of a base catalyst to obtain intermediate VI; 5) The intermediate VI is deprotected to obtain the target compound I.

3. The method according to claim 2, characterized in that In step 1), the protic solvent is methanol, ethanol or isopropanol; the base catalyst is sodium hydroxide, potassium hydroxide or potassium tert-butoxide.

4. The method according to claim 2, characterized in that: In step 2), the catalyst is trifluoroacetic acid, hydrochloric acid or sulfuric acid.

5. The method according to claim 2, characterized in that: In step 3), the oxidant is KHSO5·1 / 2KHSO4·1 / 2K2SO4; In step 4), the condensation agent is HBTU, HATU, HOBt or TFCH; and the base catalyst is triethylamine, diisopropylethylamine or pyridine.

6. Use of the substituted benzisoxazole compound according to claim 1 in the preparation of a histone deacetylase inhibitor or an anti-tumor drug.

7. The use according to claim 6, characterized in that: The histone deacetylase inhibitor is an HDAC1 inhibitor.

8. The use according to claim 6, characterized in that: The anti-tumor drug is indicated for one or more of T-cell lymphoma, multiple myeloma, colorectal cancer, liver cancer and prostate cancer.

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