2,6-Diarylpyridine HDAC / Tubulin dual-function inhibitors and preparation methods and uses thereof

Abstract

This invention belongs to the field of synthetic medicinal chemistry technology, and relates to novel 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitors with significant antitumor activity and their application in antitumor drug development. This invention also includes the application of the aforementioned compounds, their pharmaceutical salts, and their compound formulations in the preparation of drugs for the prevention or treatment of tumor-related diseases. The compounds of this invention, or their pharmaceutically acceptable salts, can inhibit the regulatory mechanisms of tumor cell proliferation by inhibiting histone deacetylases and microtubule aggregation, effectively inhibiting tumor cell growth in vitro and in vivo, and can be further used to prepare drugs for the prevention or treatment of tumor-related diseases, including benign and malignant tumors, as well as other diseases caused by tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of synthetic pharmaceutical chemistry, and relates to 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitors, preparation methods and uses thereof, and specifically to 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitors with significant anti-tumor activity, preparation methods thereof, in vivo and in vitro anti-tumor activities thereof, and use of such compounds and acceptable pharmaceutical salts thereof or a compound medicine with the same as one of its components in the preparation of medicines for preventing and treating tumor-related diseases. Background Art

[0002] The World Health Organization's International Agency for Research on Cancer (IARC) published its global cancer data report online in December 2020. The latest statistics show that the global incidence of cancer is becoming increasingly severe, with 19.29 million new cancer cases in 2020 and 9.96 million deaths in the same period. Among them, there were 4.57 million new cancer cases and 3 million cancer deaths in my country. Due to factors such as an aging population, environmental degradation, smoking, unhealthy diet, and obesity, it is predicted that by 2040, the global cancer burden will increase by 50%, and the number of new cases per year will reach nearly 30 million. This shows that we need to take immediate and effective medical measures to address the threat of cancer to human health.

[0003] Microtubules are the main components of the cytoskeleton and are essential for cell mitosis. They also play an indispensable role in maintaining cell morphology, cell movement, cell division and proliferation. Malignant tumor cells undergo extremely vigorous mitosis and metastasis. Therefore, targeting microtubules and breaking the dynamic balance of tubulin polymerization and depolymerization can selectively inhibit the division and proliferation of tumor cells (Science 2013, 339, 587-590). Although some significant progress has been made in the research on tubulin aggregation inhibitors acting on colchicine, especially the research on the structural modification of Combretastatin A-4, such as the disodium phosphate salt of Combretastatin (CA-4P) and the disodium phosphate salt of BNC105 (BNC105P), which have entered Phase I and Phase II clinical trials respectively, the results show that these drugs have poor clinical effects and have certain toxic and side effects, such as nausea, vomiting, visual impairment and headache. In addition, there are still deficiencies in prolonging the survival of patients. So far, no such drugs have been approved for marketing (J. Med. Chem. 2016, 59, 8685-8711). Therefore, the development of tubulin aggregation inhibitors with better anti-tumor activity, higher selectivity and less toxic and side effects is of concern to those skilled in the art and is the main direction of future research and development of such drugs.

[0004] Studies have shown that histone deacetylases (HDACs), as key enzymes for regulating cell epigenetics, are highly expressed in multiple tumor cell lines. They are highly correlated with the growth, proliferation and invasion of tumor cells and are considered to be one of the most promising targets for tumor treatment (Nat. Rev. Drug Discovery 2012, 11, 384-400). Pan-HDAC inhibitors are mainly used for the treatment of hematological tumors, but have poor therapeutic effects on solid tumors. Therefore, HDAC inhibition is often used in combination with other anti-tumor drugs. Although the combination therapy model can produce synergistic anti-tumor effects, it can cause complex and unpredictable pharmacokinetics, poor patient compliance, and even drug-drug interactions. The "one drug, multiple targets" drug model can effectively overcome these shortcomings and is an ideal alternative treatment model for traditional "single target, single drug" and combination therapy (J. Med. Chem. 2005, 48, 6523-6543). A large number of research reports have proved that the design of HDAC dual-target drugs is reasonable and feasible, and is also effective in improving the efficacy of tumor treatment.

[0005] Preclinical studies have shown that tubulin aggregation inhibitors (vincristine) and HDAC inhibitors SAHA can produce synergistic anti-tumor effects when combined to treat leukemia. In the MOLT-4 nude mouse transplant tumor model, the tumor inhibition rate of the vincristine and SAHA combined administration group was significantly higher than that of the two single-drug groups, and the combined administration group prolonged the survival of mice (J. Hematol. Oncol. 2017, 8, 82). In addition, there are also mechanistic studies showing that tubulin and HDAC are interrelated and synergistic in the occurrence and development of tumors. In early studies, HDAC mainly catalyzed the deacetylation of histone lysine residues on chromosomes in the cell nucleus, but in fact, HDAC's catalytic substrates are also widely present in the cytoplasm, such as α-tubulin and HSP90. Overexpressed HDAC in tumor cells can cause excessive deacetylation of α-tubulin in the cytoplasm, thereby accelerating the aggregation and depolymerization of microtubules and promoting the migration and invasion of tumor cells. Microtubules themselves also have the dynamic characteristics of aggregation and depolymerization. This synergistic interaction between microtubules and HDAC is very obvious in malignant tumor cells with vigorous division (J.Med.Chem.2020,63,23-39). These mechanistic studies and data from preclinical studies provide a reasonable basis for the design of HDAC / Tubulin dual-target inhibitors.

[0006] Based on the foundation and current status of the prior art, the inventors of the present application intend to provide 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitors, preparation methods and pharmaceutical uses. Summary of the invention

[0007] The purpose of the present invention is to provide a novel 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitor and a preparation method thereof, and the use of the compound and its pharmaceutical salt or a compound drug with the compound as a component in the preparation of drugs for preventing and treating tumor-related diseases based on the basis and status of the prior art.

[0008] Based on the structural characteristics of Tubulin aggregation inhibitors and HDAC inhibitors, the present invention adopts fusion pharmacophore drug design to construct a 2,6-diarylpyridine HDAC / Tubulin dual-target molecular compound library. Through anti-tumor activity tests at the molecular and cellular levels, lead compounds with good activity are screened and finally candidate compounds with novel structures and potential drug development prospects are obtained.

[0009] Specifically, the present invention provides a 2,6-diarylpyridine HDAC / Tubulin dual-function inhibitor or a pharmaceutical salt thereof of the following general structure:

[0010]

[0011] Among them, R 1 With R 2 is selected from hydrogen, alkyl, substituted alkyl, alkoxy, halogen, amino, hydroxy, acyloxy, methoxycarbonyl, allyloxy, propargyloxy, sulfonyloxy, alkylamino, acylamino, sulfonylamino or a combination of 2-3 of the same or different groups; X is selected from carbon, nitrogen, oxygen, ester or amide; L is selected from Where n is selected from 0, 1, 2, 3, 4, 5, 6, 7; L can also be selected from

[0012] Alicyclic and aromatic heterocyclic rings; R 3 Taken from

[0013] The preferred compounds of the present invention are:

[0014]

[0015]

[0016] The "pharmaceutically acceptable salt" mentioned in the present invention specifically includes salts formed with organic acids such as malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, and oxalic acid, and inorganic acids such as phosphoric acid, hydrohalic acid, sulfuric acid, and nitric acid.

[0017] Another object of the present invention is to provide the use of the above-mentioned compound or pharmaceutically acceptable salts of these compounds and compositions comprising the compound or its salt for preparing drugs for preventing or treating tumor-related diseases.

[0018] The tumor-related diseases can be specifically listed as thyroid cancer, lymphoma, prostate cancer, kidney cancer, bladder cancer, glioma, nasopharyngeal carcinoma, neuroendocrine cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, stromal sarcoma, choriocarcinoma, malignant hydatidiform mole, malignant teratoma, gastric cancer, lung cancer, liver cancer, melanoma, undifferentiated cancer and benign tumors, but are not limited thereto.

[0019] The present invention provides and demonstrates a 2,6-diarylpyridine HDAC / Tubulin bifunctional inhibitor or a pharmaceutically acceptable salt thereof with significant anti-tumor activity, a regulatory mechanism for inhibiting tumor cell growth by inhibiting HDAC and tubulin aggregation, and significant proliferation inhibition and angiogenesis inhibition effects on tumor cells in in vitro and in vivo anti-tumor experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 . In vitro inhibition of microtubule self-assembly by compound 7a—absorbance-time curve.

[0021] Figure 2 .Effect of compound 7a on the expression of HDAC-related proteins.

[0022] Figure 3 .Effect of compound 7a on tumor cell cycle.

[0023] Figure 4 .Effect of compound 7a on the expression of tumor cell cycle-related proteins.

[0024] Figure 5 .Compound 7a induced cell apoptosis test.

[0025] Figure 6 .Effect of compound 7a on the expression of apoptosis-related proteins.

[0026] Figure 7 .Inhibition of tumor cell colony formation by compound 7a. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with examples. These examples are only used to further illustrate the present invention and do not change the scope of protection of the present invention. The preparation method of the target compound of the present invention can be further embodied in the following representative compound preparation process:

[0028] Example 1 Synthesis of intermediate (1a-1b)

[0029]

[0030] The synthesis method is reported in the literature. The specific operation is as follows: arylboronic acid (5.0mmol), 2,6-dibromopyridine (1.30g, 5.5mmol), [1,1-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (36mg, 0.05mmol) and sodium carbonate (1.59g, 15mmol) are added to a 50mL Schlenk tube in sequence. After nitrogen is replaced, toluene (8mL) and ethanol (2mL) are injected, and the mixture is heated under reflux for 12 hours under nitrogen protection. After the reaction is complete, the mixture is cooled, water is added, and the mixture is extracted three times with ethyl acetate. After the organic phases are combined, the mixture is washed once with saturated brine and dried over anhydrous sodium sulfate. After rotary evaporation and concentration, the mixture is separated by silica gel column chromatography (PE / EA8:1) and dried to obtain the corresponding intermediate.

[0031] 1. Synthesis of 16-(3,4,5-trimethoxyphenyl)-2-bromopyridine (1a)

[0032] White solid (1a), yield 66%. 1 H NMR (400MHz, CDCl3): δ8.60(d,J=2.5Hz,1H),7.85(dd,J=2.5,8.4Hz,1H),7.58( d,J=8.4Hz,1H),7.11(s,2H),3.95(s,6H),3.90(s,3H).ESI-MS(m / z):325.3(M+H + ).

[0033] 1. Synthesis of 2-(2,4-dimethoxyphenyl)-2-bromopyridine (1b)

[0034] Pale yellow solid (1b), yield 68%. 1 H NMR (400MHz, CDCl3): δ8.11(m,1H),7.76-7.72(m,3H),6.63(dd,J=2.4,8.7Hz, 1H),6.52(d,J=2.4Hz,1H),3.85(s,3H),3.82(s,3H).ESI-MS(m / z):295.3(M+H + ).

[0035] Example 2 Synthesis of intermediates (2a-2b)

[0036]

[0037] 3-Hydroxy-4-methoxyphenylboronic acid pinacol ester (0.75g, 2.4mmol), tetrakis(triphenylphosphine)palladium (0.21g, 0.18mmol), potassium carbonate (0.51g, 3.7mmol) and raw material 1 (1.85mmol) were added to a 50mL Schlenk tube in sequence. After nitrogen was replaced, tetrahydrofuran (10mL) and water (5mL) were injected, and the mixture was heated under reflux for 12 hours under nitrogen protection. After the reaction was complete, the mixture was cooled, water was added, and the mixture was extracted three times with ethyl acetate. After the organic phase was combined, it was washed once with saturated brine and dried over anhydrous sodium sulfate. After rotary evaporation and concentration, the mixture was separated by silica gel column chromatography (PE / EA 3:1) and dried to obtain the corresponding intermediate.

[0038] 2.1 Synthesis of 2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)phenol (2a)

[0039] Yellow solid (2a), yield 68%. 1 H NMR (400MHz, CDCl3): δ7.86(m,2H),7.76(m,1H),7.71(m,1H),7.60(d,J=8.4Hz,1H),7.48(s,2H),7 .03(d,J=8.4Hz,1H),5.16(s,1H),3.92(s,6H),3.82(s,3H),3.74(s,3H).ESI-MS(m / z):368.5(M+H + ).

[0040] 2.1 Synthesis of 2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)phenol (2b)

[0041] Light yellow solid (2b), yield 58%. 1 H NMR (400MHz, CDCl3): δ8.04-8.02(m,1H),7.77(d,J=7.9Hz,1H),7.71(m,2H),7.64(d,J=8.4Hz,1H),7.54(d,J=8.5Hz,1H), 6.94(d,J=8.5Hz,1H),6.66(d,J=8.5Hz,1H),6.57(s,1H),5.67(s,1H),3.95(s,3H),3.88(s,6H).ESI-MS(m / z):338.5(M+H + ).

[0042] Example 3 Synthesis of intermediate compounds (3a-3h)

[0043]

[0044] The synthesis method is reported in the literature. The specific operation is as follows: Add raw material 2 (0.27mmol), potassium carbonate (75mg, 0.54mmol), bromoalkyl ester (0.54mmol) and acetonitrile (2mL) into a 25mL eggplant-shaped bottle, and react overnight under heating reflux. After the reaction is completed, cool, add water, extract with ethyl acetate three times, combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. After rotary evaporation and concentration, separate by silica gel column chromatography (PE / EA 3:1), and spin dry to obtain the corresponding intermediate product.

[0045] 3.1 Synthesis of ethyl 2-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxyacetate (3a)

[0046] Yellow solid (3a), yield 76%. mp 130-122℃. 1 H NMR (400MHz, CDCl3): δ7.82(s,1H),7.77-7.72(m,2H),7.62-7.59(m,2H),7.38(s,2H),7.02(d,J=6.1Hz ,1H),4.79(s,2H),4.16(q,J=7.1Hz,2H),4.00(s,6H),3.96(s,3H),3.92(s,3H),1.27(t,J=7.1Hz,3H). 13 CNMR (150MHz, CDCl3): δ168.9,156.3,155.8,153.4,150.6,147.4,139.1,137.5,135.1,132.2,121 .0,118.0,117.9,113.1,111.8,104.2,66.6,61.3,60.9,56.3,56.0,14.2.ESI-MS(m / z):454.2(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 28 NO7[M+H + ],454.1860;found,454.1865.

[0047] 3. Synthesis of ethyl 2-4-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxybutyrate (3b)

[0048] Yellow solid (3b), yield 77%. mp 86-88℃. 1H NMR (400MHz, CDCl3): δ7.82(s,1H),7.79-7.76(m,1H),7.67(d,J=8.9Hz,1H),7.64-7.58(m,2H),7.39(s,2H),6.99(d,J=8.5Hz,1H),4 .21(t,J=6.9Hz,2H),4.14(q,J=6.9Hz,2H),3.99(s,6H),3.93(s,6H),2.57(t,J=7.4Hz,2H),2.25-2.20(m,2H),1.25(t,J=6.9Hz,3H). 13 C NMR (150MHz, CDCl3): δ173.2,156.2,153.4,150.6,148.4,139.1,137.4,135.2,132.3,119.8,117 .9,112.1,111.6,104.2,67.9,60.9,60.4,56.2,56.0,30.8,24.6,14.2.ESI-MS(m / z):482.2(M+H + ).ESI-HRMS(m / z):calcd for C 27 H 32 NO7[M+H + ],482.2173;found,482.2181.

[0049] 3. Synthesis of ethyl 5-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxyvalerate (3c)

[0050] Yellow solid (3c), yield 80%. mp 61-63℃. 1 H NMR (400MHz, CDCl3): δ7.81(s,1H),7.77(d,J=7.6Hz,1H),7.63-7.67(m,2H),7.60(d,J=6.7Hz,1H),7.40(s,2H),6.99(d,J=6.7Hz,1H ),4.17-4.10(m,4H),3.98(s,6H),3.92(s,6H),2.33(m,2H),1.92(m,2H),1.77-1.71(m,2H),1.56-1.51(m,2H),1.26(t,J=6.8Hz,3H). 13C NMR (150MHz, CDCl3): δ173.6,156.3,153.4,150.5,148.6,139.1,137.4,135.2,132.2,119.6,117.9 ,111.7,111.5,104.2,68.7,60.9,60.3,56.2,34.2,28.9,25.6,24.8,14.2.ESI-MS(m / z):496.2(M+H + ).ESI-HRMS(m / z):calcd for C 28 H 34 NO7[M+H + ],496.2330;found,496.2330.

[0051] 3.4 Synthesis of 6-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxyhexanoic acid ethyl ester (3d)

[0052] Yellow solid (3d), yield 76%. mp 84-86℃. 1 H NMR (400MHz, CDCl3): δ7.81(s,1H),7.79-7.76(m,1H),7.69-7.67(m,2H),7.61(d,J=8.5Hz,1H),7.40(s,2H),6.99(d,J=8.8Hz ,1H),4.20-4.13(m,4H),3.99(s,6H),3.93(s,6H),2.45-2.39(m,2H),1.98-1.94(m,2H),1.88-1.82(m,2H),1.28-1.25(m,5H). 13 C NMR (150MHz, CDCl3): δ173.4,156.3,153.4,150.5,148.5,139.1,137.5,135.2,132.2,128.6,119.7,11 7.9,111.8,111.5,104.2,68.5,60.9,60.3,56.2,56.1,33.9,28.7,21.6,14.3.ESI-MS(m / z):510.2(M+H + ).ESI-HRMS(m / z):calcd for C 29 H 36 NO7[M+H + ],510.2486;found,510.2490.

[0053] 3.5 Synthesis of ethyl 2-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)phenoxyacetate (3e)

[0054] Yellow solid (3e), yield 72%. mp 101-103℃. 1 H NMR (400MHz, CDCl3): δ7.99(d,J=8.5Hz,1H),7.78(d,J=7.9Hz,1H),7.73-7.67(m,3H),7.53(d,J=7.7Hz,1H), 6.99(d,J=8.3Hz,1H),6.67(d,J=9.5Hz,1H),6.58(s,1H),4.81(s,2H),3.94(s,3H),3.88(s,6H),3.82(s,3H). 13 C NMR (150MHz, CDCl3): δ169.5,161.3,158.4,155.7,155.0,150.3,147.3,136.3,132.9,132.2,122.6,1 22.2,121.0,117.1,113.0,111.8,105.2,98.9,66.6,56.0,55.6,55.5,52.3.ESI-MS(m / z):410.2(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 24 NO6[M+H + ],410.1598;found,410.1605.

[0055] 3. Synthesis of ethyl 6-(4-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)phenoxybutyrate (3f)

[0056] Pale yellow solid (3f), yield 70%. mp 68-70℃. 1 H NMR (400MHz, CDCl3): δ7.99(d,J=9.2Hz,1H),7.77-7.70(m,3H),7.63(d,J=8.3Hz,1H),7.55(d,J=7.6Hz,1H),6.96(d,J=8.3Hz ,1H),6.67(d,J=8.6Hz,1H),6.58(s,1H),4.17(m,4H),3.91(s,3H),3.88(s,6H),2.57(m,2H),2.22-2.19(m,2H),1.25(m,3H). 13C NMR (150MHz, CDCl3): δ173.3,161.3,158.4,156.2,155.0,150.3,148.4,136.2,132.9,132.3,122.5,122.3, 119.8,117.2,112.3,111.6,105.1,98.9,68.0,60.4,56.0,55.5,30.9,24.7,14.2.ESI-MS(m / z):452.5(M+H + ).ESI-HRMS(m / z):calcd for C 26 H 30 NO6[M+H + ],452.2068;found,452.2069.

[0057] 3.7 Synthesis of ethyl 5-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)phenoxyvalerate (3 g)

[0058] White solid (3 g), yield 67%. mp 56-58°C. 1 H NMR (400MHz, CDCl3): δ7.99(d,J=9.2Hz,1H),7.77-7.70(m,3H),7.63(d,J=8.3Hz,1H),7.55(d,J=7.6Hz,1H),6.96(d,J=8.3Hz ,1H),6.67(d,J=8.6Hz,1H),6.58(s,1H),4.17(m,4H),3.91(s,3H),3.88(s,6H),2.57(m,2H),2.22-2.19(m,2H),1.25(m,3H). 13 C NMR (150MHz, CDCl3): δ173.7,161.3,158.4,156.3,155.0,150.2,148.6,136.2,132.9,132.2,122.5,122.3,119.6,1 17.2,111.9,111.5,105.1,98.9,68.8,60.2,56.1,55.6,55.5,34.3,29.0,25.7,24.8,14.3.ESI-MS(m / z):466.5(M+H + ).ESI-HRMS(m / z):calcd for C 27 H 32 NO6[M+H + ],466.2224;found,466.2229.

[0059] 3.8 Synthesis of 6-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)phenoxyhexanoic acid ethyl ester (3h)

[0060] Yellow solid (3h), yield 56%. mp 61-63℃. 1 H NMR (400MHz, CDCl3): δ8.00(d,J=8.5Hz,1H),7.78-7.68(m,3H),7.62(d,J=8.2Hz,1H),7.55(d,J=7.6Hz,1H),6.96(d,J=8.3Hz,1H),6.68(d,J =8.5Hz,1H),6.58(s,1H),4.46-4.41(m,4H),3.92(s,3H),3.88(s,6H), 2.41(t,J=6.6Hz,2H),1.93(m,2H),1.91(m,2H),1.25(t,J=7.1Hz,3H). 13 C NMR (150MHz, CDCl3): δ173.5,161.3,158.4,156.2,155.0,150.2,148.5,136.2,132.9,132.2,122.5,119.6,11 7.2,112.0,111.5,105.1,98.9,68.6,60.3,56.1,55.6,55.5,34.0,28.7,21.6,14.3.ESI-MS(m / z):480.5(M+H + ).ESI-HRMS(m / z):calcd for C 28 H 34 NO6[M+H + ],480.2381; found,480.2388.

[0061] Example 4 Synthesis of target compounds (4a-4h)

[0062]

[0063] Add raw material 3 (0.1mmol), sodium hydroxide (40mg, 1.0mmol), and hydroxylamine aqueous solution (0.15mL) to a 25mL eggplant-shaped bottle, and dissolve with dichloromethane (0.5mL) and methanol (1.0mL). React under ice bath conditions. After the raw material disappears completely, add water, adjust the pH to 6 with acetic acid, extract three times with ethyl acetate, combine the organic phases, wash once with saturated brine, and dry over anhydrous sodium sulfate. Filter, concentrate by rotary evaporation, and separate by silica gel column chromatography (CH2Cl2 / MeOH 30:1).

[0064] 4.1 Synthesis of 2-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxyacetamide (4a)

[0065] White solid (4a), yield 53%. mp 68-70℃. 1 H NMR (400MHz, CDCl3): δ7.76-7.74(m,3H),7.57(d,J=7.5Hz,2H),7.32(s,2H) ,7.00(d,J=7.6Hz,1H),4.73(s,2H),3.97(s,6H),3.94(s,3H),3.91(s,3H). 13 C NMR (150MHz, CDCl3): δ165.7,156.5,155.5,153.5,150.6,147.2,139.2,137.5,135.0,13 2.9,122.0,118.3,117.9,114.6,112.0,104.3,61.0,56.3,56.0.ESI-MS(m / z):441.2(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 25 N2O7[M+H + ],441.1656;found,441.1667.

[0066] 4. Synthesis of 2-4-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxybutyramide (4b)

[0067] Red solid (4b), yield 33%. mp 96-98℃. 1 H NMR (400MHz, CDCl3): δ7.78-7.71(m,2H),7.65(d,J=7.5Hz,1H),7.57(m,2H),7.33(s,2H),6 .95(d,J=7.0Hz,1H),4.15-4.11(m,2H),3.95(s,6H),3.90(s,6H),2.44(m,2H),2.15(m,2H). 13C NMR (151MHz, CDCl3): δ170.8,156.3,156.0,153.4,150.1,147.9,139.1,137.5,135.2,132.5,1 20.1,118.0,118.0,111.7,111.4,104.3,68.1,61.0,56.2,56.0,24.8.ESI-MS(m / z):469.2(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 29 N2O7[M+H + ],469.1969;found,469.1975.

[0068] 4. Synthesis of 3-(5-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxypentanamide (4c)

[0069] Red solid (4c), yield 37%. mp 90-92℃. 1 H NMR (400MHz, CDCl3): δ7.78-7.75(m,2H),7.66-7.61(m,3H),7.37(s,2H),7.00(d,J=6.6H z,1H),4.21-4.16(m,2H),3.98(s,9H),3.92(s,3H),2.49-2.38(m,2H),1.98-1.89(m,4H). 13 C NMR (150MHz, CDCl3): δ169.9,155.7,155.5,152.8,148.8,147.6,138.6,136.8,134.6,132.1,1 18.9,117.3,110.4,109.8,103.8,69.0,60.3,55.6,55.3,25.9,24.1.ESI-MS(m / z):483.2(M+H + ).ESI-HRMS(m / z):calcd for C 26 H 31 N2O7[M+H + ],483.2126;found,483.2133.

[0070] 4.4 Synthesis of 6-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxyhexanamide (4d)

[0071] Yellow solid (4d), yield 40%. mp 76-78℃.1 H NMR (400MHz, CDCl3): δ7.77-7.70(m,2H),7.61(d,J=8.6Hz,1H),7.59-7.53(m,2H),7.35(s,2H),6.94(d,J=8.2Hz,1H),4 .12-4.04(m,2H),3.95(s,6H),3.90(s,6H),2.13-2.12(m,2H),1.86-1.82(m,2H),1.68-1.65(m,2H),1.47-1.45(m,2H). 13 C NMR (150MHz, CDCl3): δ171.1,156.2,153.4,150.2,148.5,139.0,137.4,135.2,132.3,119.6, 117.9,111.5,104.3,68.7,60.9,56.2,56.0,32.6,28.7,25.6,25.1.ESI-MS(m / z):497.2(M+H + ).ESI-HRMS(m / z):calcd for C 27 H 33 N2O7[M+H + ],497.2282;found,497.2288.

[0072] 4.5 Synthesis of 2-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxyacetamide (4e)

[0073] Yellow solid (4e), yield 45%. mp 98-100℃. 1 H NMR (400MHz, CDCl3): δ9.80(s,1H),7.92(d,J=6.1Hz,1H),7.71(m,3H),7.64(d,J=7.1Hz,1H),7.50(s,1 H),6.94(d,J=7.2Hz,1H),6.68(m,1H),6.55(s,1H),5.29(s,1H),4.69(s,1H),3.88(s,3H),3.85(s,6H). 13C NMR (150MHz, CDCl3): δ166.0,161.4,158.3,155.4,155.3,150.3,147.2,136.5,133.2,132.2,122. 9,121.9,121.8,117.3,114.7,111.8,105.2,98.9,69.0,55.9,55.6,55.5.ESI-MS(m / z):411.2(M+H + ).ESI-HRMS(m / z):calcd for C 22 H 23 N2O6[M+H + ],411.1551;found,411.1554.

[0074] 4.6 Synthesis of 4-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxybutyramide (4f)

[0075] Red solid (4f), yield 35%. mp 86-88℃. 1 H NMR (400MHz, CDCl3): δ7.94 (dd, J=6.0, 2.4Hz, 1H), 7.73-7.69 (m, 3H), 7.59-7.52 (m, 2H), 6.94 (d, J=7. 4Hz,1H),6.66(d,J=8.8Hz,1H),6.57(s,1H),4.20-4.11(m,2H),3.94(s,3H),3.86(s,6H),2.13(m,4H). 13 C NMR (150MHz, CDCl3): δ170.9,161.4,158.3,156.1,155.2,149.8,147.9,136.4,133.0,132.2,122.8,1 22.1,120.1,117.4,111.9,111.3,105.1,98.9,68.2,56.1,55.6,29.3,24.8.ESI-MS(m / z):439.2(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 27 N2O6[M+H + ],439.1864;found,439.1869.

[0076] 4.7 Synthesis of 5-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxypentanamide (4 g)

[0077] White solid (4 g), yield 37%. mp 96-98°C. 1 H NMR (400MHz, CDCl3): δ7.95(d,J=6.8Hz,1H),7.74-7.68(m,3H),7.58-7.56(m,2H),6.94(d,J=5.8Hz,1H), 6.66(d,J=6.8Hz,1H),6.57(s,1H),4.16(s,2H),3.97(s,3H),3.86(s,6H),2.44-2.34(m,2H),1.90(m,4H). 13 C NMR (150MHz, CDCl3): δ161.4,158.4,156.1,155.2,149.1,148.1,136.3,132.2,122.7,122.6,119.8,119 .4,117.3,112.2,111.6,105.2,98.9,68.7,56.1,55.6,55.5,29.7,29.3,28.5.ESI-MS(m / z):453.2(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 29 N2O6[M+H + ],453.2020;found,453.2025.

[0078] 4.8 Synthesis of 6-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenoxy-N-hydroxyhexanamide (4h)

[0079] Red solid (4h), yield 32%. mp 68-71℃. 1 H NMR (400MHz, CDCl3): δ7.93(m,1H),7.1(m,3H),7.55(d,J=6.8Hz,2H),6.93(m,1H),6.65(d,J=7.8Hz,1H),6.56 (s,1H),4.08(s,2H),3.85(s,9H),2.09-2.02(m,2H),1.83-1.77(m,2H),1.65-1.56(m,2H),1.47-1.40(m,2H). 13C NMR (150MHz, CDCl3): δ171.4,161.3,158.3,156.3,155.2,149.9,148.5,136.4,132.9,132.2,122.7,122.1,11 9.6,117.5,111.8,111.5,105.1,98.9,68.7,56.0,55.6,55.5,29.7,28.6,25.5,25.0.ESI-MS(m / z):467.2(M+H + ).ESI-HRMS(m / z):calcd forC 26 H 31 N2O6[M+H + ],467.2177; found,467.2185.

[0080] Example 5 Synthesis of intermediate compounds (5a-5d)

[0081]

[0082] The synthesis method is similar to that of intermediates (2a-2b).

[0083] 5.1 Synthesis of 2-(3-bromo-4-methoxyphenyl)-6-(3,4,5-trimethoxyphenyl)pyridine (5a)

[0084] White solid (5a), yield 72%. mp 140-142℃. 1 H NMR (400MHz, CDCl3): δ8.36(s,1H),8.07(dd,J=8.6,2.2Hz,1H),7.79(t,J=8.8Hz,1H),7.61 (d,J=7.9Hz,2H),7.36(s,2H),7.02(d,J=8.7Hz,1H),4.00(s,6H),3.97(s,3H),3.92(s,3H). 13 C NMR (150MHz, CDCl3): δ156.6,154.9,153.5,139.1,137.6,135.1,133.3,131.9,12 7.0,118.4,117.9,112.1,111.8,104.4,60.9,56.4,56.3.ESI-MS(m / z):430.2(M+H + ).ESI-HRMS(m / z):calcd forC 21 H 21 BrNO4[M+H + ],430.0648;found,430.0655.

[0085] 5.2 Synthesis of 2-(3-bromo-4-methoxyphenyl)-6-(2,4-dimethoxyphenyl)pyridine (5b)

[0086] Yellow solid (5b), yield 76%. mp 63-65℃. 1 H NMR (400MHz, CDCl3): δ8.34(s,1H),8.01(t,J=9.8Hz,2H),7.79(d,J=7.9Hz,1H),7.71(t,J=7.6Hz,1H),7. 54(d,J=7.7Hz,1H),6.98(d,J=8.6Hz,1H),6.68(d,J=10.4Hz,1H),6.58(s,1H),3.95(s,3H),3.88(s,6H). 13 CNMR (150MHz, CDCl3): δ161.4,158.4,156.3,155.3,154.8,136.4,132.3,131.8,1 26.9,122.9,117.0,111.7,105.2,98.9,56.4,55.6,55.5.ESI-MS(m / z):400.5(M+H + ).ESI-HRMS(m / z):calcd for C 20 H 19 BrNO3[M+H + ],400.0543;found,400.0546.

[0087] 5.3 Synthesis of 2-(3-bromophenyl)-6-(3,4,5-trimethoxyphenyl)pyridine (5c)

[0088] Yellow oily liquid (5c), yield 86%. 1 H NMR (400MHz, CDCl3): δ8.30 (s, 1H), 8.04 (d, J = 7.8Hz, 1H), 7.85-7.80 (m, 1 H),7.67(m,2H),7.57-7.55(m,1H),7.37(s,3H),4.00(s,6H),3.93(s,3H). 13 C NMR (150MHz, CDCl3): δ156.1,154.5,152.9,140.8,138.6,137.1,134.3,131.3,129 .6,129.5,124.8,122.4,118.5,118.0,103.7,60.4,55.7.ESI-MS(m / z):401.3(M+H +).ESI-HRMS(m / z):calcd for C 20 H 19 BrNO3[M+H + ],401.0312;found,401.0315.

[0089] 5.3 Synthesis of 2-(4-bromophenyl)-6-(3,4,5-trimethoxyphenyl)pyridine (5d)

[0090] Yellow oily liquid (5d), yield 76%. 1 H NMR (400MHz, CDCl3): δ8.01-7.98(m,2H),7.80(t,J=7.0Hz,1H),7.64-7.60(m,4H),7.35(s,2H),3.97(s,6H),3.90(s,3H). 13 C NMR (150MHz, CDCl3): δ156.09,154.97,152.87,138.60,137.65,137.01,134.37,131 .22,127.89,122.85,118.26,117.72,103.69,60.36,55.66.ESI-MS(m / z):401.2(M+H + ).ESI-HRMS(m / z):calcd for C 20 H 19 BrNO3[M+H + ],401.0316; found,401.0319.

[0091] Example 6 Synthesis of intermediate compounds (6a-6f)

[0092]

[0093] The synthesis method is reported in the literature. The specific operation is as follows: add the raw material (1.5mmol), ethyl acrylate or 1-butenoic acid methyl ester (11.7mmol), triethylamine (2.0mL, 14.6mmol) and N-methylpyrrolidone (8mL) into a 15mL stoppered threaded tube, add palladium acetate (49mg, 0.22mmol) and tri(o-methylphenyl)phosphine (133mg, 0.44mmol) after nitrogen is passed through to fully deoxygenate, and heat at 128°C for 12 hours after strict sealing. After the reaction is completed, cool, add ethyl acetate, wash three times with water, wash the organic phase once with saturated brine, and dry it with anhydrous sodium sulfate. After rotary evaporation and concentration, separate by silica gel column chromatography (PE / EA 5:1), and spin dry to obtain the corresponding intermediate.

[0094] 6.1 Synthesis of ethyl (E)-3-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenylacrylate (6a)

[0095] White solid (6a), yield 42%. mp 118-120℃. 1 H NMR (400MHz, CDCl3): δ8.36(s,1H),8.14(d,J=6.6Hz,1H),8.07(d,J=16.1Hz,1H),7.80(t,J=8.1Hz,1H),7.63(t,J=8.5Hz,2H),7.39(s, 2H),7.05(d,J=6.8Hz,1H),6.65(d,J=16.1Hz,1H),4.28(q,J=6.9Hz,2H),4.00(s,6H),3.97(s,3H),3.92(s,3H),1.35(t,J=7.0Hz,3H). 13 C NMR (150MHz, CDCl3): δ167.4,159.1,156.4,155.6,153.5,139.9,139.2,137.5,135.1,131.9,129.8,1 27.5,123.5,119.2,118.1,117.7,111.3,104.3,60.9,60.4,56.3,55.7,14.4.ESI-MS(m / z):450.5(M+H + ).ESI-HRMS(m / z):calcd for C 26 H 28 NO6[M+H + ],450.1911;found,450.1920.

[0096] 6.2 Synthesis of methyl (E)-4-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenyl-3-butenoate (6b)

[0097] Yellow oily liquid (6b), yield 52%. 1H NMR (400MHz, CDCl3): δ8.27(s,1H),8.03(d,J=7.2Hz,1H),7.78(d,J=6.8Hz,1H),7.66(d,J=8.1Hz,1H),7.60(d,J=7.6Hz,1H),7.40(s ,2H),6.99(d,J=8.8Hz,1H),6.89(d,J=16.0Hz,1H),6.48-6.41(m,1H),4.00(s,6H),3.92(s,6H),3.73(s,3H),3.32(d,J=6.8Hz,2H). 13 C NMR (150MHz, CDCl3): δ172.1,157.4,156.2,153.4,139.0,137.4,135.3,131.8,128.2,127.3,1 25.9,125.5,122.7,117.8,110.9,104.2,60.9,56.3,55.6,51.9,38.7.ESI-MS(m / z):450.3(M+H + ).ESI-HRMS(m / z):calcd for C 26 H 28 NO6[M+H + ],450.1911;found,450.1920.

[0098] 6.3 Synthesis of ethyl (E)-3-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenylacrylate (6c)

[0099] Pale yellow solid (6c), yield 46%. mp 86-88℃. 1 H NMR (400MHz, CDCl3): δ8.25(s,1H),8.12-8.07(m,1H),8.03-7.98(m,1H),7.78(d,J=8.1Hz,1H),7.72(t,J=7.8Hz,1H),7.56(d,J=8.1Hz ,1H),7.01(d,J=8.8Hz,1H),6.65(d,J=16.3Hz,1H),6.58(s,1H),4.28(q,J=6.8Hz,2H),3.95(s,3H),3.88(s,6H),1.35(t,J=6.8Hz,3H). 13C NMR (150MHz, CDCl3): δ166.9,160.7,158.3,157.8,154.9,154.6,139.6,135.7,131.9,131.7,129.4,127. 1,122.8,122.1,121.6,118.6,116.4,110.6,104.6,98.3,59.8,55.1,54.9,13.8.ESI-MS(m / z):420.3(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 26 NO5[M+H + ],420.1805;found,420.1813.

[0100] 6. Synthesis of methyl 4-(E)-3-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenyl-3-butenoate (6d)

[0101] Yellow oily liquid (6d), yield 56%. 1 H NMR (400MHz, CDCl3): δ8.13(s,1H),8.02-7.97(m,2H),7.77-7.71(m,3H),7.57(d,J=7.9Hz,1H),6.95(d,J=8.3Hz,1H),6.85- 6.82(m,1H),6.67(d,J=7.9Hz,1H),6.57(d,J=8.2Hz,1H),6.46-6.41(m,1H),3.88(s,9H),3.73(s,3H),3.31(d,J=7.2Hz,2H). 13 C NMR (150MHz, CDCl3): δ171.6,160.7,157.8,156.6,155.6,154.5,135.6,131.8,131.7,127.8,126.8,125. 2,124.9,121.9,121.9,121.7,116.6,110.2,104.5,98.3,54.9,54.9,51.3,38.2.ESI-MS(m / z):420.7(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 26 NO5[M+H + ],420.1805;found,420.1810.

[0102] 6.5 Synthesis of ethyl (E)-3-[3-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenylacrylate (6e)

[0103] Yellow oily liquid (6e), yield 78%. 1 H NMR (400MHz, CDCl3): δ8.34(s,1H),8.14(s,1H),7.81(d,J=16.1Hz,2H),7.68(m,2H),7.60(m,1H),7 .53(m,1H),7.40(s,2H),6.56(d,J=15.8Hz,1H),4.28(m,2H),4.01(s,6H),3.93(s,3H),1.36(m,3H). 13 C NMR (150MHz, CDCl3): δ166.4,156.1,155.2,152.9,143.9,139.4,138.6,137.0,134.4,134.3,128.7,12 8.1,127.9,126.0,121.8,118.3,118.1,117.9,103.7,60.4,59.9,55.7,13.7.ESI-MS(m / z):420.3(M+H + ).ESI-HRMS(m / z):calcd for C 25 H 26 NO5[M+H + ],420.3005;found,420.3010.

[0104] 6. Synthesis of ethyl 6(E)-3-[4-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenylacrylate (6f)

[0105] Yellow oily liquid (6f), yield 62%. 1 H NMR (400MHz, CDCl3): δ8.16(m,2H),7.83-7.75(m,2H),7.72-7.66(m,4H),7.38(s ,2H),6.52(d,J=16.3Hz,1H),4.29(m,2H),4.00(s,6H),3.92(s,3H),1.36(m,3H). 13C NMR (150MHz, CDCl3): δ166.4,156.1,155.1,152.9,143.4,140.4,138.6,136.9,134.4 ,127.8,126.7,118.4,118.0,103.7,60.4,59.9,55.7,13.7.ESI-MS(m / z):420.4(M+H + ).ESI-HRMS(m / z):calcd C 25 H 26 NO5[M+H + ],420.4106; found,420.4108.

[0106] Example 7 Synthesis of target compounds (7a-7f)

[0107]

[0108] The synthesis method is similar to that of 4a-d.

[0109] 7.1 Synthesis of (E)-3-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxyacrylamide (7a)

[0110] White solid (7a), yield 52%. mp 140-142℃. 1 H NMR (400MHz, DMSO-d6): δ10.82(s,1H),9.09(s,1H),8.39(s,1H),8.22(m,1H),7.93(s,3H),7.7 6(d,J=16.2Hz,1H),7.50(s,2H),7.23(m,1H),6.66(d,J=16.3Hz,1H),3.92(s,9H),3.73(s,3H). 13 C NMR (150MHz, DMSO-d6): δ158.1,155.1,154.6,152.9,138.4,137.9,134.2,131.0,128. 8,125.8,123.3,118.3,117.8,111.9,103.8,59.9,55.8,55.7.ESI-MS(m / z):437.7(M+H + ).ESI-HRMS(m / z):calcdfor C 24 H 25 N2O6[M+H + ],437.1707;found,437.1716.

[0111] 7.2 Synthesis of (E)-4-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxy-3-butenamide (7b)

[0112] Red solid (7b), yield 42%. mp 108-110℃. 1 H NMR (400MHz, DMSO-d6): δ10.55(s,1H),8.83(s,1H),8.37(s,1H),8.11(dd,J=8.3,2.0Hz,1H),7.92(s,3H),7.52(s,2H), 7.15(d,J=7.2Hz,1H),6.79(d,J=16.2Hz,1H),6.46(m,1H),3.92(s,6H),3.89(s,3H),3.73(s,3H),2.98(d,J=5.5Hz,2H). 13 C NMR (150MHz, DMSO-d6): δ166.8,156.8,154.8,154.7,152.9,138.3,137.9,134.1,130.7,126.7,126. 4,125.2,124.8,124.1,117.9,117.6,111.4,103.7,59.9,55.8,55.5,37.2.ESI-MS(m / z):451.7(M+H + ).ESI-HRMS(m / z):calcdfor C 25 H 27 N2O6[M+H + ],451.1864;found,451.1873.

[0113] 7.3 Synthesis of (E)-3-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxyacrylamide (7c)

[0114] Red solid (7c), yield 66%. mp 144-146℃. 1 H NMR (400MHz, DMSO-d6): δ10.78(s,1H),9.06(s,1H),8.30(s,1H),8.13(s,1H),7.89(d,J=7.7Hz,1H),7.81(s,1H),7.74 (d,J=8.0Hz,2H),7.19(d,J=6.9Hz,1H),6.70(s,2H),6.65(s,1H),5.75(s,1H),3.92(s,3H),3.85(s,3H),3.83(s,3H). 13C NMR (150MHz, DMSO-d6): δ160.9,158.0,154.6,154.4,136.7,131.5,131.4,128.8,125.8,123. 2,122.3,120.9,119.7,116.9,111.8,105.6,98.6,55.7,55.2,54.7.ESI-MS(m / z):407.7(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 23 N2O5[M+H + ],407.1601;found,407.1612.

[0115] 7. Synthesis of 4-(E)-4-[2-methoxy-5-(6-(2,4-dimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxy-3-butenamide (7d)

[0116] Yellow solid (7d), yield 56%. mp 124-126℃. 1 H NMR (400MHz, DMSO-d6): δ8.20(s,1H),8.04(d,J=8.2Hz,1H),7.89(d,J=8.4Hz,1H),7.82-7.79(m,2H),7.71-7.74(m,1H),7.12(d,J=8.4 Hz,1H),6.76(d,J=16.3Hz,1H),6.71(m,2H),6.43(dt,J=16.3,7.8Hz,1H),3.86(s,3H),3.84(s,3H),3.82(s,3H),3.22(d,J=7.8Hz,2H). 13 C NMR (150MHz, DMSO-d6): δ172.6,160.9,157.9,156.7,154.9,154.3,136.7,131.4,131.3,126.8,125 .2,124.4,122.1,121.1,116.9,111.3,105.5,98.6,55.6,55.5,55.2,38.3.ESI-MS(m / z):421.7(M+H + ).ESI-HRMS(m / z):calcd for C 24 H 25 N2O5[M+H + ],421.1758;found,421.1762.

[0117] 7.5 Synthesis of (E)-3-[3-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxyacrylamide (7e)

[0118] Red solid (7e), yield 56%. mp 101-103℃. 1 H NMR (400MHz, DMSO-d6): δ8.44(s,1H),8.26(d,J=9.1Hz,1H),8.06-7.94(m,2H),7.74(d,J=9. 11Hz,1H),7.66-7.54(m,4H),7.52(s,2H),6.70(d,J=16.2Hz,1H),3.92(s,6H),3.73(s,3H). 13 C NMR (150MHz, CDCl3): δ155.4,154.8,153.0,139.1,138.4,138.1,135.2,134.1,129 .3,127.9,127.5,125.4,119.1,118.7,103.9,59.9,55.8.ESI-MS(m / z):407.4(M+H + ).ESI-HRMS(m / z):calcd forC 23 H 23 N2O5[M+H + ],407.4104;found,407.1308.

[0119] 7.6 Synthesis of (E)-3-[4-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenyl-N-hydroxyacrylamide (7f)

[0120] Red solid (7f), yield 46%. mp 104-106℃. 1 H NMR (400MHz, DMSO-d6): δ12.36(s,1H),8.21(d,J=8.4Hz,1H),7.93(s,2H),7.80(d,J=8.8Hz,1H),7.61(d,J=16.7H z,1H),7.45(s,3H),7.35(s,1H),7.21(s,1H),7.93-6.88(m,1H),6.57(d,J=16.8Hz,1H),3.86(s,6H),3.68(s,3H). 13C NMR (150MHz, CDCl3): δ167.4,155.4,154.4,153.0,143.2,142.2,139.9,138.5,138.1,134.7,134 .0,132.0,128.6,126.8,125.7,119.6,119.2,118.8,103.9,59.9,55.8.ESI-MS(m / z):407.4(M+H + ).ESI-HRMS(m / z):calcd for C 23 H 23 N2O5[M+H + ],407.4302; found,407.4306.

[0121] Example 8 (E) Synthesis of 3-[2-methoxy-5-(6-(3,4,5-trimethoxyphenyl)-2-pyridyl)]phenylacrylic acid (8a)

[0122]

[0123] Add the raw material (0.98mmol) to a 25mL eggplant-shaped bottle, dissolve it with methanol (3mL), and then slowly drip into a sodium hydroxide aqueous solution (1M, 1.9mL). Heat and reflux the reaction. After the raw material disappears completely, add water, adjust the pH to 6 with acetic acid, extract with ethyl acetate three times, combine the organic phases, wash once with saturated brine, and dry with anhydrous sodium sulfate. Filter, concentrate by rotary evaporation, separate by silica gel column chromatography (PE / EA 2:1), and spin dry to obtain a white solid (8a) with a yield of 67%. mp 166-168℃. 1 H NMR (400MHz, DMSO-d6): δ8.48(d,J=2.0Hz,1H),8.28(dd,J=8.8,2.0Hz,1H),8.00(m,1H),7.92(s,2H),7.79(d,J =16.0Hz,1H),7.52(s,2H),7.23(d,J=8.8Hz,1H),6.71(d,J=16.1Hz,1H),3.94(s,3H),3.92(s,6H),3.74(s,3H). 13 C NMR (150MHz, DMSO-d6): δ158.03,154.87,154.45,152.97,138.35,137.92,134.13,130.92,128.9 0,125.64,123.38,118.08,117.86,111.82,103.76,59.94,55.76,55.67.ESI-MS(m / z):422.4(M+H+ ).ESI-HRMS(m / z):calcd for C 24 H 23 NO6[M+H + ],422.1598; found,422.1602.

[0124] Example 9 In vitro tumor cell proliferation inhibition activity detection experiment

[0125] Take the tumor cells in the logarithmic growth phase, inoculate them in a 96-well plate after treatment, and culture them for 24 hours at 37°C and 5% CO2. Add 6 gradient concentrations of the test compound, and use CA-4 as a positive control. Continue to culture for 48 hours under the same conditions, then add MTT. After 4 hours of culture, discard the supernatant containing MTT, add DMSO to each well, shake to dissolve the purple crystals, and then measure the OD value at 490nm or 540nm on the microplate reader, and calculate the inhibition rate. The half inhibitory concentration (IC 50 The inhibition rate of 6 concentrations was calculated. Three replicate wells were set for each concentration gradient and the test was repeated three times. The activity results are shown in Tables 1 and 2.

[0126] Table 1 Inhibitory activity of compounds on tumor cell proliferation (IC 50 / μM)

[0127]

[0128]

[0129]

[0130] Notes: a The antitumor activity was determined by the MTT method, and the data are the average of three measurements; b BE-(2)-C is a human neuroblastoma cell line; c A549 is a human lung cancer cell line; d U87MG is a human malignant glioblastoma cell line; e HCT-116 is a human colon cancer cell line.

[0131] Table 2 Inhibitory activity of compound 7a on tumor cell proliferation (IC 50 / μM)

[0132]

[0133] Notes: a The antitumor activity was determined by the MTT method, and the data are the average of three measurements; b HeLa is a human cervical cancer cell line; cA2780 is a human ovarian cancer cell line; d MDA-MB-231 is a human breast cancer cell line; e HUH-7 is a human hepatoma cell line; f SKOV3 is a human ovarian cancer cell line; g MCF-7 is a human breast cancer cell line; h MDA-MB-468 is a human breast cancer cell line.

[0134] Example 10 In vitro tubulin self-assembly experiment

[0135] The inhibitory effect of the preferred compound 7a on microtubule aggregation in vitro was tested by turbidimetry, and the detection kit was purchased from Cytoskeleton, USA. The operation steps are as follows: the microtubule aggregation system (0.1M PIPES, 10mM MgCl2, 1mM GTP, 1mM EGTA and 3.4M glycerol) with pH = 6.6 was pre-incubated on ice, and different concentrations of the test compound 7a were added. The Colchicine treatment group was set as a positive control and the DMSO (4%, v / v) treatment group was set as a negative control. After adding tubulin (10mM) to the above system, it was immediately transferred and placed at 37°C for aggregation reaction. The temperature was kept constant, and the absorbance was measured at 340nm with a spectrophotometer (Synergy H4 Hybrid) every 1 minute. The test was continued for a total of 30 minutes, and then the absorbance curve was plotted (as shown in FIG. 2 ). Figure 1 The results showed that compound 7a could significantly inhibit microtubule aggregation, and its IC 50 It is 2.4 μM (as shown in Table 3).

[0136] Table 3. In vitro inhibition of microtubule self-assembly by compound 7a

[0137]

[0138] Example 11 In vitro HDAC inhibitory activity test

[0139] The inhibitory activity of the compounds on HDACs was tested by enzyme-linked immunofluorescence assay, and all enzymatic reactions were carried out at 37°C for 30 minutes. The compounds were diluted with 5% DMSO to different concentration ranges, and 5 μl of the compound solution was added to 50 μl of pH = 8.0 and containing 25 mM Tris, 1 mM MgCl2, 0.1 mg / ml BSA, 137 mM NaCl, 2.7 mM KCl, HDAC and its substrate reaction mixture. After the enzymatic reaction was completed, the fluorescence intensity was detected on a SpectraMax M5 microtiter plate reader with an excitation wavelength of 350-360 nm and an emission wavelength of 450-460 nm. Finally, Prism GraphPad software was used to calculate the IC of the test compound by fitting after normalization and performing nonlinear regression. 50 The test results are shown in Table 4. Both 7a and 7c can significantly inhibit the activity of HDAC8. Their IC 50 The values ​​were 0.117 μM and 0.482 μM, respectively. At the same time, the inhibitory activity of both on HDAC1 and HDAC6 was not obvious. Among them, the preferred compound 7a had a selectivity of 50 times and 42 times for HDAC1 and HDAC6, respectively, indicating that 7a is a selective HDAC8 inhibitor.

[0140] Table 4 Inhibitory activity of compounds on HDAC1 / 6 / 8 (IC 50 / μM)

[0141]

[0142] Notes: a IC 50 All are the average of three measurements; b SAHA is a non-selective HDACs inhibitor; c PCI-34051 is a selective HDAC8 inhibitor; NT=not tested.

[0143] Example 12 Detection of HDAC-related protein expression

[0144] BE-(2)-C cells in the logarithmic growth phase were treated and then cultured at 2×10 5 The number of cells / well was inoculated in a 6-well plate and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours. After adding gradient concentrations of compound 7a to treat tumor cells (DMSO treatment group was set as negative control), the cells were collected and lysed with lysis buffer after 24 hours. After heat denaturation, the protein sample was loaded on polyacrylamide gel, separated by SDS-PAGE electrophoresis, wet transferred to the membrane, blocked, and reacted with primary antibody and secondary antibody in sequence, and then exposed to color. The results are shown in Figure 2As shown, compound 7a can significantly promote the acetylation of HDAC8 substrate protein SMC3, but has no obvious effect on the acetylation of HDAC6 substrate protein α-tubulin and HDAC1 substrate protein histone H3, indicating that the preferred compound 7a can selectively inhibit HDAC8.

[0145] Example 13 In vitro cell cycle arrest experiment

[0146] BE-(2)-C cells in the logarithmic growth phase were treated and then cultured at 2×10 5 The number of cells / well was inoculated in a 6-well plate and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 12 hours. After the cells adhered to the wall, fresh culture medium was replaced and the cells were treated with different concentrations of compound 7a for 24 hours. At the same time, a DMSO treatment group was set as a negative control. The supernatant was discarded, the adherent cells were collected, rinsed twice with PBS, and then fixed with 75% ethanol, fixed overnight at -20°C, and tested by flow cytometry after staining with PI. The results showed that compound 7a could significantly block the tumor cell cycle at the G2 / M phase (such as Figure 3 shown).

[0147] Example 14 In vitro cell cycle related protein detection

[0148] BE-(2)-C cells in the logarithmic growth phase were treated and then cultured at 2×10 5 The number of cells / well was inoculated in a 6-well plate and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours. After adding gradient concentrations of compound 7a to treat tumor cells (DMSO treatment group was set as negative control), the cells were collected and lysed with lysis buffer after 24 hours. After heat denaturation, the protein sample was loaded on polyacrylamide gel, separated by SDS-PAGE electrophoresis, wet transferred to the membrane, blocked, and reacted with primary antibody and secondary antibody in sequence, and then exposed to color. The results are shown in Figure 4 As shown, compound 7a can significantly promote the expression of mitotic checkpoint protein Bubr-1, phosphorylated histone P-Histone 3, and cyclin B1.

[0149] Example 15 In vitro cell apoptosis induction experiment

[0150] BE-(2)-C cells in the logarithmic growth phase were treated and then cultured at 2×10 5 The number of cells / well was inoculated in a 6-well plate and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours. Gradient concentrations of compound 7a were added, and a DMSO treatment group was set as a negative control. After 48 hours of continuous culture, the supernatant cells and adherent cells were collected, double-stained with PI and Annexin V, and detected by flow cytometry. The results are shown in Figure 5As shown, compound 7a can induce cell apoptosis in a concentration-dependent manner.

[0151] Example 16 In vitro apoptosis-related protein detection experiment

[0152] BE-(2)-C cells in the logarithmic growth phase were treated and then cultured at 2×10 5 The number of cells / well was inoculated in a 6-well plate and cultured in an incubator at 37°C, 5% CO2 and saturated humidity for 24 hours. Gradient concentrations of compound 7a were added, and the DMSO treatment group was set as a negative control. After 48 hours, the cells were collected and lysed with lysis buffer. The samples were loaded on polyacrylamide gel, separated by SDS-PAGE electrophoresis, wet transferred to the membrane, blocked, and reacted with primary and secondary antibodies in sequence, and then exposed to color. The results are shown in Figure 6 As shown, compound 7a can significantly promote the expression of tumor suppressor gene p53, pro-apoptotic protein Bax and cleaved DNA repair enzyme PAPR-1.

[0153] Example 17 Colony formation inhibition experiment

[0154] BE-(2)-C cells in the logarithmic growth phase were taken and inoculated in a 6-well plate after treatment, with 1500 cells in each well, and cultured for 24 hours at 37°C, 5% CO2 and saturated humidity. After the cells adhered to the wall, different concentrations of compound 7a were added to treat the cells for 48 hours, and the DMSO treatment group was set as a negative control. Fresh culture medium was replaced and culture was continued for two weeks, after which the supernatant was discarded and the cells were rinsed twice with PBS. The cells were fixed with anhydrous methanol for 30 minutes and stained with crystal violet dye for 1 hour. The staining solution was washed off, and after sufficient drying, the number of colonies formed by more than 50 cells was counted under a microscope. The results showed that compound 7a could significantly inhibit the formation of HeLa cell colonies (such as Figure 7 shown).

Claims

1. 2,6-Diarylpyridine compounds, characterized in that: Having the structure of general formula I, Among them, R 1 With R 2 is selected from hydrogen atom, methoxy group, halogen atom, amino group, hydroxyl group or a combination of 2-3 of the same or different groups mentioned above; L is selected from Where n is selected from 0, 1, 2, 3, 4, 5, 6, 7, m is selected from 1, 2; R 3 for 2. The 2,6-diarylpyridine compound according to claim 1, wherein the preferred compound is:

3. Use of the compound according to any one of claims 1 to 2 and its pharmaceutically acceptable salt in the preparation of a medicament for preventing and treating tumor-related diseases, wherein the diseases are neuroblastoma, stromal sarcoma, choriocarcinoma, malignant hydatidiform mole, thyroid cancer, head and neck squamous cell carcinoma, cervical cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, gastric cancer, lung cancer, liver cancer, melanoma, lymphoma, glioma, nasopharyngeal carcinoma, neuroendocrine carcinoma, undifferentiated carcinoma, malignant teratoma and benign tumors, wherein the "pharmaceutically acceptable salt" is a salt formed with malic acid, lactic acid, camphorsulfonic acid, citric acid, fumaric acid, oxalic acid, as well as phosphoric acid, hydrohalic acid, sulfuric acid and nitric acid.

4. A compound medicine for preventing and treating tumor-related diseases, characterized in that It contains the compound described in any one of claims 1-2, and the tumor-related diseases are neuroblastoma, thyroid cancer, lung cancer, liver cancer, melanoma, lymphoma, prostate cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, breast cancer, colorectal cancer, pancreatic cancer, esophageal cancer, osteosarcoma, renal cancer, undifferentiated cancer, stromal sarcoma, choriocarcinoma, gastric cancer, bladder cancer, brain glioma, nasopharyngeal carcinoma, neuroendocrine cancer, malignant hydatidiform mole, malignant teratoma and benign tumor.

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