Preparation method of isoflavone compound and application of isoflavone compound in neuroprotection

Abstract

The invention relates to a novel isoflavone compound as shown in a formula (I), a preparation method of the compound and application of the compound in preparation of neuroprotective small molecule drugs. The chemical general formula of the compound is as shown in formula (I). The compound in the formula (I) is prepared by reacting a polyphenol compound with substituted phenylacetonitrile or by reacting the polyphenol compound with substituted phenylacetic acid. A series of neuroprotective activity experiments prove that the compounds have good neuroprotective activity, and the compounds with the best activity also have acetylcholin esterase inhibitory activity and can be used as lead molecules for development of neuroprotective and neurodegenerative disease drugs. The preparation process is simple, raw materials are cheap and easy to obtain, and the product purity is high. # imgabs0 # (I).

Description

Technical Field

[0001] The present invention relates to a novel isoflavone compound and its intermediate, and the use of such a compound in the preparation of a neuroprotective drug. It belongs to the field of medicine. Background Art

[0002] Neurons play a crucial role in activities such as learning, thinking, and memory. The progressive decline in neuronal function and viability caused by various pathological factors is the main cause of neurodegenerative diseases. These multi-factorial neurodegenerative diseases affect millions of people worldwide. Protein misfolding, mitochondrial damage, and oxidative stress are all manifested in neurodegenerative diseases such as Alzheimer's Disease (AD). AD is an irreversible, progressive brain disorder characterized by memory loss and a decline in thinking and behavioral abilities. AD is considered the main cause of dementia, which is a progressive decline in at least two cognitive domains, leading to an inability to perform normal social and occupational activities. AD usually affects the elderly, but it is not a normal aging defect. The characteristics of AD include amyloid-β peptide (Aβ) aggregation, hyperphosphorylated tau protein (p-tau) accumulation, production of inflammatory mediators, oxidative stress, cholinergic dysfunction, and defects in synaptic and cognitive functions. Genetic factors, environmental factors, and common lifestyle are part of the etiological factors associated with AD. Currently, AD affects more than 50 million people, and this number is expected to reach 150 million by 2050. However, the treatment of AD remains a challenge because its pathogenesis has not been fully understood. Many Phase III clinical trials targeting Aβ generation and aggregation have failed. Recently, clinical trials for Crenezumab (NCT03491150), lanabecestat (NCT02972658), and solanezumab (NCT01900665) were terminated due to lack of significant efficacy. This systematic failure in finding new drugs for AD has prompted scientists to explore new strategies to combat this disease.

[0003] AD has multiple pathological features, such as a large amount of oxidized DNA, severe mitochondrial damage, extensive lipid peroxidation, high levels of neurotoxic trace metal elements, and elevated Aβ levels. All these factors increase the formation of reactive oxygen species (ROS), leading to the brain of AD patients being affected by oxidative stress. Under physiological conditions, ROS homeostasis is strictly regulated by the ROS generation system and the cellular antioxidant network. The antioxidant defense system includes a variety of enzymes and small molecules, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), thioredoxin (Trx), thioredoxin reductase (TrxR), glutathione peroxidase (Gpx) family, and NAD(P)H: quinone oxidoreductase 1 (NQO1), etc. During the study of human AD, it was found that the levels of nuclear transcription factor Nrf2, SOD1, CAT, and Gpx in the brains of patients were severely decreased compared with those of normal people. Due to the low levels of Nrf2 and these important antioxidant enzymes, the brains of AD patients are more vulnerable to ROS attack. In this case, ROS will cause progressive and irreversible damage to the brain. In addition, a large amount of research evidence also shows that the level of the neurotransmitter acetylcholine (ACh) is significantly decreased in AD patients, and this phenomenon may be caused by the reduction of ACh production or the enhancement of acetylcholinesterase (AChE) activity. Moreover, the enhancement of AChE activity will further lead to the increase and aggregation of Aβ levels.

[0004] Direct treatment with antioxidants such as vitamin E and vitamin C may have a positive effect on AD, but the efficacy of these antioxidants in clinical patients is still in its infancy. Activating Nrf2 to increase the level of antioxidant proteins is considered a better way to achieve neuroprotection. Nrf2 can induce about 500 genes, encoding the expression of different cytoprotective enzymes and detoxifying proteins. High levels of Nrf2 can improve the damage caused by ROS and / or mitochondrial dysfunction. At the same time, inhibiting the activity of AChE can not only increase the level of neurotransmitters in the human brain but also reduce the production and aggregation of Aβ levels, thereby reducing the generation of ROS. Currently, dimethyl fumarate (trade name Tecfidera) and oleanolic acid derivatives (trade name Skyclarys) are used as Nrf2 activators to treat multiple sclerosis and Friedreich's ataxia, respectively. And the AChE inhibitors approved by the FDA for clinical treatment of AD are donepezil, rivastigmine, and galantamine.

[0005] Natural products are an innovative source for new drug discovery. Natural isoflavonoid compounds often have strong antioxidant effects due to their polyphenolic structures and also possess other multiple physiological functions. Based on the structural characteristics of current Nrf2 activators and our previous analysis and research on the structural characteristics of AChE inhibitors and the AChE protein structure, we designed and synthesized 40 structurally diverse isoflavonoid compounds and their intermediates (attached in the specification Figure 6 and 7 as shown), and tested the free radical scavenging ability, cytotoxic and protective activities against neuronal-like cells PC12, activation effect on Nrf2, and inhibitory activity against AChE of these compounds. Summary of the Invention

[0006] The object of the present invention is to provide an isoflavonoid compound or its intermediate that can activate Nrf2 and inhibit AChE. At the same time, another object of the present invention is to provide the use of such a compound as a candidate molecule for neuroprotective drugs in neurodegenerative diseases.

[0007] A series of isoflavonoid compounds and their intermediates described in the present invention have the chemical structures attached in the specification Figure 6 and 7 as shown: Structural formula attached in the specification Figure 6 In the formula: the substituent R1 is selected from phenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-nitrophenyl, 4-aminophenyl, 3,4-dimethoxyphenyl, 3,4-dihydroxyphenyl, 4-bromophenyl, 4-chlorophenyl; the substituent R2 is selected from hydroxyl and hydrogen; the substituent R3 is selected from methoxy and hydrogen; the substituent R4 is selected from methoxy and hydroxyl; the substituent R5 is selected from hydroxyl and hydrogen.

[0008] Structural formula attached in the specification Figure 7 In the formula: the substituent R1 is selected from phenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-nitrophenyl, 3,4-dimethoxyphenyl, 4-bromophenyl, 4-chlorophenyl; both the substituents R2 and R3 are selected from hydroxyl and hydrogen.

[0009] The preparation method of the present invention is carried out according to the chemical reaction formulas attached in the specification Figure 1 、 2 、3, 4 and 5 as shown.

[0010] The best preparation method of such isoflavonoid compounds and their intermediates (compounds attached in the specification Figure 6 and 7 ) of the present invention is completed through the following steps and can generally be divided into two types: the preparation of compounds 1-11 is the first method, and the preparation of compounds 12-40 is the second method.

[0011] Preparation method of compounds 1-11: Dissolve phloroglucinol (3.6 g, 28.6 mmol), various substituted phenylacetonitriles (32.5 mmol) and anhydrous zinc chloride (1.9 g, 13.9 mmol) in 40 mL of 1,2-dimethoxyethane, and stir vigorously at room temperature. Subsequently, slowly and continuously introduce hydrogen chloride gas into the reaction solution until the solution becomes clear, and then continue to introduce hydrogen chloride gas for 30 minutes. The resulting reaction mixture is further stirred at room temperature for 48 hours. After the stirring is completed, cool the reaction mixture to below 10 °C, and then slowly add 1.4 g of water. The reaction continues for 2.5 hours under the condition of below 10 °C. Then filter to obtain the solid of intermediate ketimine hydrochloride, and wash the solid with 1,2-dimethoxyethane (2.6 g) and water (4.4 g).

[0012] Subsequently, dissolve the obtained solid in a mixed solution including water (35 g), concentrated hydrochloric acid (3 g) and methanol (1.5 g). After sufficient stirring, heat the mixture to reflux at 110 °C for 15 hours. Then cool the reaction solution to below 10 °C and stir for another 2 hours. Filter the obtained mixture under vacuum to obtain a solid cake, and wash it with (5 g) water. Dry the solid in a fluidized dryer at 65 °C for 5 hours to obtain intermediate ketone 1, namely compounds 2, 5, 8 and 9, which can be directly used for the next reaction without further purification.

[0013] While stirring, dropwise add a solution of boron trifluoride diethyl etherate (6 mmol) to an 8 mL DMF solution containing intermediate 1 (1 mmol). Heat the reaction mixture to 53 °C, and at this time slowly add a mixture of DMF (2 mL) and methanesulfonyl chloride (5 mmol). After the addition is completed, keep the mixture at this temperature for 15 minutes. Subsequently, heat the reaction mixture to 100 °C and let the reaction continue for 7 hours while monitoring the reaction process by thin layer chromatography (TLC). After the reaction is completed, wash the mixture successively with saturated sodium acetate solution and sodium bicarbonate solution, and then extract with ethyl acetate. Concentrate the combined organic phases under reduced pressure, and purify the obtained crude product by column chromatography (elution system: dichloromethane: methanol) to obtain the corresponding pure compounds, namely compounds 1, 3, 4, 6, 7, 10 and 11.

[0014] Preparation method of compounds 12-40: ① Preparation of intermediate compounds: Under dry conditions, boron trifluoride etherate (25 mL) was added to a round-bottom flask containing the corresponding starting material polyphenol compound (10 mmol) and substituted phenylacetic acid (10 mmol). The mixture was heated to 90 °C and reacted for 6 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the reaction solution was cooled to room temperature, and the reaction mixture was washed with saturated sodium acetate and sodium bicarbonate solutions, and then extracted with ethyl acetate. The organic layers were combined and concentrated under reduced pressure, and the resulting residue was purified by column chromatography to obtain the corresponding pure target compounds, namely intermediate compounds 13, 18, 19, 21, 23 and 26.

[0015] ② Preparation of isoflavone compounds: Under dry conditions, boron trifluoride etherate (25 mL) was added to a round-bottom flask containing the corresponding starting material polyphenol compound (10 mmol) and substituted phenylacetic acid (10 mmol). The mixture was heated to 90 °C and reacted for 6 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the reaction solution was cooled to room temperature and then further cooled to below 0 °C in an ice bath. At this time, DMF (15 mL) was slowly added dropwise to the reaction solution. After the addition was completed, the mixture was reheated to 53 °C and stirred for 15 minutes. Subsequently, a mixture of methanesulfonyl chloride (50 mmol) and DMF (8 mL) was slowly added dropwise at this temperature. After the addition was completed, the mixture was kept at this temperature for another 15 minutes, then heated to 100 °C and reacted for 8 hours until the reaction was confirmed to be complete by TLC. After the reaction was completed and cooled, the mixture was washed successively with saturated sodium acetate solution and saturated sodium bicarbonate solution, and then extracted with ethyl acetate. The combined organic layers were concentrated under reduced pressure, and the resulting residue was purified by column chromatography (elution system: dichloromethane: methanol) to obtain the purified target compounds, namely isoflavone compounds 12, 14 - 17, 20, 22, 24, 25 and 27 - 40.

[0016] Preparation method of polyphenol reaction raw material, namely compound 4-methoxyresorcinol: Aqueous hydrogen peroxide solution (30%, 8.9 mL, 86.7 mmol) and selenium dioxide (SeO2, 350 mg, 3.1 mmol) were added to 100 mL of dichloromethane, and stirred thoroughly under an ice bath (0 °C). Subsequently, 3-hydroxy-4-methoxybenzaldehyde (6.0 g, 39.4 mmol) was added to the reaction mixture in batches. After all the aldehyde substances were added, the ice bath was removed, and the reaction was continued to stir overnight at room temperature. After the reaction process was confirmed to be completed by thin-layer chromatography (TLC), the reaction mixture was filtered, and the filter cake was washed with water (100 mL). The filtrate was extracted three times with dichloromethane, and the combined organic layers were concentrated under reduced pressure. Subsequently, the organic phase was washed successively with 10% aqueous sodium bisulfite solution and saturated sodium chloride solution, and concentrated again under reduced pressure. The obtained residue was dissolved in 20 mL of methanol, and vigorously stirred with 200 mL of saturated sodium carbonate solution at room temperature for about 2 hours, and the reaction process was monitored by TLC. After the reaction was completed, the reaction mixture was adjusted to pH 4-5 with 1 M hydrochloric acid, and extracted three times with ethyl acetate. The combined organic phases were concentrated under reduced pressure to obtain a brown solid of the target product (5.2 g, yield 84%). Confirmed by TLC, this product can be directly used for subsequent reactions without further purification.

[0017] After preliminary in vitro antioxidant assays and screening, it was found that some of the compounds shown in the attached Figure 6 and 7 have strong scavenging abilities for two free radicals, DPPH and ABTS (attached to the specification Figure 49 and 50 ). Twelve compounds with the strongest in vitro free radical scavenging abilities, namely compounds 2, 5, 8, 9, 10, and 23-29, were selected for cytotoxicity assays and screening. It was found that these 12 compounds were non-toxic to PC12 cells (rat adrenal pheochromocytoma cells) at a concentration of 5 mM, while at a concentration as high as 20 mM, only compounds 26-28 had weak toxicity (attached to the specification Figure 51 ). These 12 compounds were further screened using an in vitro cell model of neurodegenerative diseases (PC12 cells were damaged with a certain concentration of hydrogen peroxide), and it was found that these 12 compounds had a significant protective effect on PC12 cells and showed a concentration-dependent form (attached to the specification Figure 52 ). Compound 29 with the best protective activity was selected, and its activation effect on the nuclear transcription factor Nrf2 was determined using a luciferase reporter gene assay. It was found that its activity was much stronger than that of the classical Nrf2 activator tert-butylhydroquinone ( t -BHQ) at the same concentration (attached to the specification Figure 53). Meanwhile, Compound 29 also has AChE inhibitory activity and is a non-competitive AChE inhibitor (description attached Figure 54 ). Therefore, the isoflavone compounds and their intermediates of the present invention can be used as effective Nrf2 activators and AChE inhibitors, and as lead molecules for the research and development of drugs for neuroprotection and neurodegenerative diseases. The synthesis process of the isoflavone compounds and their intermediates described in the present invention is simple, the product purity is high, and they show strong protective effects on nerve cells, having excellent application prospects.

[0018] The following further details the above content of the present invention through specific embodiments. However, this should not be construed as a limitation of the present invention.

[0019] Specific embodiments.

[0020] Example 1: Preparation of target compound 1 (description attached Figure 8 ).

[0021] Preparation method of compound 2 (description attached Figure 9 ), that is, intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethanone: Dissolve phloroglucinol (3.6 g, 28.6 mmol), p-methoxyphenylacetonitrile (4.8 g, 32.5 mmol) and anhydrous zinc chloride (1.9 g, 13.9 mmol) in 40 mL of 1,2-dimethoxyethane, and stir vigorously at room temperature. Subsequently, hydrogen chloride gas is slowly and continuously introduced into the reaction solution until the solution becomes clear, and then hydrogen chloride gas is continued to be introduced for 30 minutes. The obtained reaction mixture is further stirred at room temperature for 48 hours. After the stirring is completed, the reaction mixture is cooled to below 10 °C, and then 1.4 g of water is slowly added. Under the condition of below 10 °C, the reaction continues for 2.5 hours. Then, filter to obtain the solid of intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethanimine hydrochloride, and this solid is washed with 1,2-dimethoxyethane (2.6 g) and water (4.4 g), and directly used for the next step of reaction without drying.

[0022] Subsequently, dissolve the obtained solid in a mixed solution including water (35 g), concentrated hydrochloric acid (3 g) and methanol (1.5 g). After sufficient stirring, heat the mixture to reflux at 110 °C for 15 hours. Then cool the reaction solution to below 10 °C and stir for another 2 hours. Filter the obtained mixture under vacuum to obtain a solid cake, and wash it with (5 g) water. Dry the solid in a fluidized dryer at 65 °C for 5 hours to obtain 6.4 g of compound 2, that is, intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethanone, which can be directly used for the next step of reaction without further purification.

[0023] Preparation of Compound 1: Boron trifluoride in ether (6 mmol) was added dropwise to a stirred solution of 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethanone (274 mg, 1 mmol) in 8 mL of DMF. The reaction mixture was heated to 53°C, at which point a mixture of DMF (2 mL) and methanesulfonyl chloride (5 mmol) was slowly added. After the addition was complete, the mixture was maintained at this temperature for 15 minutes. The reaction mixture was then heated to 100°C and allowed to react for 7 hours, while monitoring the reaction progress by thin-layer chromatography (TLC). After completion of the reaction, the mixture was washed sequentially with saturated sodium acetate and sodium bicarbonate solutions, and then extracted with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the resulting crude product was purified by column chromatography (eluent: dichloromethane:methanol) to yield 236 mg of the corresponding pure compound 1, 5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chroman-4-one.

[0024] The reaction is shown in reaction formula 1 (attached to the specification). Figure 10 ).

[0025] Compound 1. Yield: 94%; 1 H NMR (400 MHz, DMSO- d 6 ) δ : 12.92 (s, 1H), 10.89 (s, 1H), 8.25 (s, 1H), 7.46 (d, 2H, J = 8.4 Hz), 6.95 (d, 2H, J = 8.4 Hz), 6.34 (s, 1H), 6.21 (s, 1H), 3.76 (s, 3H); 13 C NMR (100 MHz, DMSO- d 6 ) δ : 180.1,164.4, 162.1, 159.2, 157.6, 154.1, 130.2, 123.0, 122.0, 113.7, 104.5, 99.1,93.8, 55.2; MS-ESI m / z: 283.05 [MH] - ..

[0026] Compound 2. Yield: 86%; 1 H NMR (600 MHz, DMSO- d 6 )δ : 12.24 (s, 1H), 10.39(s, 1H), 7.13 (d, 2H, J = 9.0 Hz), 6.85 (d, 2H, J = 9.0 Hz), 5.82 (s, 2H),4.27 (s, 2H), 3.72 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 202.8, 164.9, 164.3,157.9, 130.7, 127.8, 113.6, 103.6, 94.7, 55.0, 48.1; MS-ESI m / z: 273.05 [M-H] - .。

[0027] Example 2: Preparation of the target compound 3 (attached to the specification Figure 11 ).

[0028] Same as Example 1, except that phenylacetonitrile was used instead of p-methoxyphenylacetonitrile as the starting material. The detection data of the reaction product are as follows: Compound 3. Yield: 96%; 1 H NMR (400 MHz, DMSO- d 6 ) δ : 12.89 (s, 1H), 10.96(s, 1H), 8.42 (s, 1H), 7.57 – 7.55 (m, 2H), 7.46 – 7.37 (m, 3H), 6.41 (d, J =2.0 Hz, 1H), 6.24 (d, J = 2.0 Hz, 1H); 13 C NMR (100 MHz, DMSO- d 6 ) δ : 179.9,164.4, 162.0, 157.6, 154.9, 130.9, 129.0, 128.2, 128.0, 122.3, 104.5, 99.1,93.8; MS-ESI m / z: 253.00 [M-H] - .。

[0029] Example 3: Preparation of the target compound 4 (attached to the specification Figure 12 ).

[0030] Compound 5 (description attached Figure 13 ), namely the preparation method of intermediate 2-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethanone: same as Example 1, only using 4-hydroxybenzonitrile instead of 4-methoxybenzonitrile as the starting material. The test data of the reaction product are as follows: Compound 5. Yield: 86%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.26 (s,2H), 10.39 (s, 1H), 9.21 (s, 1H), 7.02 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.4Hz, 2H), 5.83 (s, 2H), 4.22 (s, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 203.1,164.9, 164.4, 155.9, 130.6, 126.0, 115.0, 103.7, 94.8, 48.1; MS-ESI m / z:259.05 [M-H] - .。

[0031] The preparation method of Compound 4: same as Example 1, only using 4-hydroxybenzonitrile instead of 4-methoxybenzonitrile as the starting material. The test data of the reaction product are as follows: Compound 4. Yield: 81%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.95 (s, 1H), 10.87 (br, 1H), 9.58 (br, 1H), 8.31 (s, 1H), 7.37 (d, J =8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz, 2H), 6.38 (d, J = 1.8 Hz, 1H), 6.22 (d, J =1.8 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ: 180.2, 164.3, 162.0, 157.6, 157.4, 154.0, 130.2, 122.3, 121.2, 115.1, 104.5, 99.0, 93.7; MS-ESI m / z: 269.05 [M-H] - .。

[0032] Example 4: Preparation of the target compound 6 (attached to the specification) Figure 14 ).

[0033] Compound 8 (attached to the specification) Figure 15 ), that is, the preparation method of the intermediate 2-(4-nitrophenyl)-1-(2,4,6-trihydroxyphenyl)ethanone: The same as in Example 1, only using p-nitrobenzonitrile instead of p-methoxybenzonitrile as the starting material. The detection data of the reaction product are as follows: Compound 8. Yield: 81%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.18 (s, 2H), 10.47 (s, 1H), 8.16 (d, J = 9.0 Hz, 2H), 7.50 (d, J = 9.0 Hz, 2H), 5.85(s, 2H), 4.52 (s, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 200.9, 165.2, 164.3, 146.2, 144.3, 131.2, 123.1, 103.8, 94.8, 48.9; MS-ESI m / z: 288.00 [M-H] - .。

[0034] Preparation method of compound 7 (attached to the specification) Figure 16 ): The same as in Example 1, only using p-nitrobenzonitrile instead of p-methoxybenzonitrile as the starting material. The detection data of the reaction product are as follows: Compound 7. Yield: 74%; 1 H NMR (400MHz, DMSO- d 6 ) δ : 12.71 (s, 1H), 11.06 (s, 1H), 8.63 (s, 1H), 8.31 (d, J = 8.8Hz, 2H), 7.89 (d,J = 8.8 Hz, 2H), 6.45 (d, J = 2.0 Hz, 1H), 6.27 (d, J = 2.0Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 179.7, 165.2, 162.4, 158.0, 156.9, 147.4, 138.5, 130.5, 123.7, 120.9, 104.8, 99.9, 94.5; MS-ESI m / z: 298.00 [M-H] - .。

[0035] Preparation method of Compound 6: Add Compound 7 (299 mg, 1 mmol) to a round-bottom flask containing stannous chloride (950 mg, 5 mmol), concentrated hydrochloric acid (5 mL) and water (2 mL), reflux the reaction for 12 hours and then stop the reaction. After the reaction solution cools, adjust the pH of the reaction solution to 7 with 5% aqueous sodium hydroxide solution, and a yellow solid precipitate is formed. Filter, extract the filtrate with ethyl acetate three times, combine the filter cake with the ethyl acetate solution obtained by extraction, and purify by column chromatography to obtain 164 mg of pure Compound 6. The detection data of the reaction product are as follows: Compound 6. Yield: 61%; 1 H NMR (600 MHz, DMSO- d 6 ) δ :13.04 (s, 1H), 10.84 (s, 1H), 8.26 (s, 1H), 7.23 (d, J = 8.4 Hz, 2H), 6.60(d, J = 8.4 Hz, 2H), 6.37 (d, J = 1.8 Hz, 1H), 6.21 (d, J = 1.8 Hz, 1H), 5.24(s, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ: 180.5, 164.1, 162.0, 157.6, 153.3, 148.8, 129.6, 122.7, 117.6, 113.4, 104.5, 98.9, 93.6; MS-ESI m / z: 268.05 [M-H] - .。

[0036] Example 5: Preparation of target compound 9 (description attached Figure 17 ).

[0037] Same as Example 1, except that 3,4-dimethoxybenzyl cyanide was used instead of p-methoxybenzyl cyanide as the starting material. The test data of the reaction product are as follows: Compound 9. Yield: 94%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.25 (s, 2H), 10.40 (s, 1H), 6.85 (d, J = 8.4 Hz, 2H), 6.73 (dd, J = 1.8, 8.4 Hz, 1H), 5.83 (s, 2H), 4.27 (s, 2H), 3.71 (s, 6H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 202.7, 164.9, 164.3, 148.5, 147.5, 128.3, 121.7, 113.8, 111.7, 103.7, 94.8, 55.5(55.538), 55.5 (55.451), 48.5; MS-ESI m / z: 303.05 [M-H] - .。

[0038] Example 6: Preparation of target compound 10 (description attached Figure 18 ).

[0039] Preparation method of compound 11 (description attached Figure 19 ): Same as Example 1, except that 3,4-dimethoxybenzyl cyanide was used instead of p-methoxybenzyl cyanide as the starting material. The test data of the reaction product are as follows: Compound 11. Yield: 89%; 1 HNMR (600 MHz, DMSO- d 6 ) δ: 12.94 (s, 1H), 10.89 (s, 1H), 8.38 (s, 1H), 7.17(d, J J = 2.4 Hz, 1H), 7.11 (dd, J J = 2.4, 8.4 Hz, 1H), 7.00 (d, J J = 8.4 Hz,1H), 6.39 (d, J J = 2.4 Hz, 1H), 6.23 (d, J J = 1.8 Hz, 1H), 3.78 (s, 6H); 13 C NMR(150 MHz, DMSO- d 6 ) δ : 180.1, 164.3, 162.0, 157.5, 154.5, 148.8, 148.4, 123.2,122.1, 121.4, 112.8, 111.6, 104.5, 99.0, 93.7, 55.6 (55.575), 55.6 (55.551);MS-ESI m / z: 313.05 [M-H] - .。

[0040] Preparation method of Compound 10 (description attached Figure 18 ): Mix Compound 11 (314 mg, 1 mmol) and pyridine hydrochloride (1.15 g, 10 mmol) in a round-bottom flask, then heat this reaction system to 190 °C. At this time, the reaction system is in a molten state. Stop the reaction after continuously reacting at this temperature for 12 hours. After the reaction cools to room temperature, add 50 mL of 2M HCl aqueous solution, stir for 15 minutes, and then extract this solution three times with ethyl acetate. Combine the organic phases under reduced pressure to obtain the crude product, and purify it by column chromatography to obtain 236 mg of pure Compound 10. The test data of the reaction product are as follows: Compound 10. Yield: 82%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 13.00 (s, 1H), 10.86 (s, 1H), 9.06 (s, 1H), 8.99(s, 1H), 8.29 (s, 1H), 7.00 (d, J J = 1.8 Hz, 1H), 6.81 – 6.77 (m, 2H), 6.38(d, J= 1.8 Hz, 1H), 6.22 (d, J = 2.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 180.2, 164.2, 162.0, 157.5, 154.0, 145.5, 144.9, 122.4, 121.6, 119.9, 116.5, 115.4, 104.5, 98.9, 93.6; MS-ESI m / z: 285.05 [M-H] - .。

[0041] Example 7: Preparation of target compound 12 (attached to the specification Figure 20 ).

[0042] Preparation method of compound 12: Under dry conditions, boron trifluoride diethyl ether (25 mL) was added to a round-bottom flask containing resorcinol (10 mmol) and p-methoxyphenylacetic acid (10 mmol). The mixture was heated to 90 °C and reacted for 6 hours, and the reaction progress was monitored by TLC. After the reaction was complete, it was cooled to room temperature and then cooled to below 0 °C in an ice bath. At this time, DMF (15 mL) was added dropwise. After the addition was complete, the mixture was reheated to 53 °C and stirred for 15 minutes. Subsequently, a mixture of methanesulfonyl chloride (50 mmol) and DMF (8 mL) was added dropwise at this temperature. After the addition was complete, the mixture was maintained at this temperature for another 15 minutes, then heated to 100 °C and reacted for 8 hours until the reaction was confirmed complete by TLC. After the reaction was completed and cooled, the reaction mixture was washed with saturated sodium acetate and sodium bicarbonate solutions, and then extracted with ethyl acetate. The organic layers were combined, concentrated under reduced pressure, and the resulting residue was purified by column chromatography to obtain 2.16 g of pure target compound 12. Yield of compound 12: 80%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.80 (s, 1H), 8.33 (s, 1H), 7.97 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 9.0 Hz, 2H), 6.98 (d, J = 9.0 Hz, 2H), 6.94(dd, J = 2.4, 9.0 Hz, 1H), 6.87 (d, J= 2.4 Hz, 1H), 3.78 (s, 3H); 13 C NMR(150 MHz, DMSO- d 6 ) δ : 174.6, 162.6, 159.0, 157.4, 153.1, 130.1, 127.3, 124.2,123.2, 116.6, 115.2, 113.6, 102.1, 55.1; MS-ESI m / z: 269.05 [M+H] + .。

[0043] Example 8: Preparation of the target compound 13 (attached to the specification Figure 21 ).

[0044] Preparation method of compound 13: Under dry conditions, boron trifluoride diethyl ether (25 mL) was added to a round-bottom flask containing resorcinol (10 mmol) and p-methoxyphenylacetic acid (10 mmol). The mixture was heated to 90 °C and reacted for 6 hours, and the reaction progress was monitored by TLC. After the reaction was completed, it was cooled to room temperature, and the reaction mixture was washed with saturated sodium acetate and sodium bicarbonate solutions, and then extracted with ethyl acetate. The organic layers were combined, concentrated under reduced pressure, and the resulting residue was purified by column chromatography to obtain 1.93 g of the pure target compound 13. The test data of the reaction product are as follows: Compound 13. Yield: 75%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.56 (s, 1H), 10.66 (s, 1H), 7.94 (d, J =9.0 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.39 (dd, J= 2.4, 9.0 Hz, 1H), 6.25 (d, J = 2.4 Hz, 1H), 4.20 (s, 2H), 3.72 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ: 202.5, 164.9, 164.7, 158.0, 133.6, 130.5, 127.0, 113.8, 112.1, 108.3, 102.5, 55.0, 43.2; MS-ESI m / z: 257.05 [M-H] - .。

[0045] Example 9: Preparation of target compound 14 (attached to the specification) Figure 22 ).

[0046] Preparation method of compound 14: The same as Example 7, only using 3,4-dimethoxyphenylacetic acid instead of p-methoxyphenylacetic acid as the starting material. The detection data of the reaction product are as follows: Compound 14. Yield: 85%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.66 (s, 1H), 8.37 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.19 (d, J = 2.4 Hz, 1H), 7.12 (dd, J = 2.4, 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.94 (dd, J = 2.4, 8.4 Hz, 1H), 6.87 (d, J = 1.8 Hz, 1H), 3.78 (s, 6H); 13 C NMR(150 MHz, DMSO- d 6 ) δ : 174.6, 162.6, 157.4, 153.3, 148.6, 148.3, 127.3, 124.5, 123.2, 121.2, 116.6, 115.2, 112.8, 111.5, 102.1, 55.6, 55.5; MS-ESI m / z: 299.05 [M+H] + .。

[0047] Example 10: Preparation of target compound 15 (attached to the specification) Figure 23 ).

[0048] Preparation method of Compound 15: The same as in Example 7, except that phenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting material. The test data of the reaction product are as follows: Compound 15. Yield: 87%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.84 (s, 1H), 8.38 (s, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.56 (d, J = 7.2 Hz, 2H), 7.42 (t, J = 7.8 Hz, 2H), 7.40 (t, J = 7.8 Hz, 1H), 6.95 (dd, J = 2.4, 9.0 Hz, 1H), 6.89 (d, J = 1.8 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.4, 162.7, 157.5, 153.8, 132.1, 129.0, 128.1, 127.7, 127.3, 123.6, 116.6, 115.3, 102.2; MS-ESI m / z: 239.05 [M+H] + .。

[0049] Example 11: Preparation of the target compound 16 (attached to the specification Figure 24 ).

[0050] Preparation method of Compound 16: The same as in Example 7, except that p-bromophenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting material. The test data of the reaction product are as follows: Compound 16. Yield: 71%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 11.17 (s, 1H), 8.44 (s, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.00 (dd, J= 1.8, 8.4 Hz, 1H), 6.97 (d, J = 2.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.2, 163.1, 157.4, 154.1, 131.4, 131.0, 131.0, 127.2, 122.3, 121.0, 116.4, 115.5, 102.2; MS-ESI m / z: 314.95 [M-H] - .。

[0051] Example 12: Preparation of target compound 17 (attached to the specification Figure 25 ).

[0052] Preparation method of compound 17: The same as Example 7, only using 4-hydroxyphenylacetic acid instead of 4-methoxyphenylacetic acid as the starting material. The detection data of the reaction product are as follows: Compound 17. Yield: 70%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.77 (s, 1H), 9.52 (s, 1H), 8.28 (s, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.38 (d, J = 7.8 Hz, 2H), 6.93 (dd, J = 2.4, 8.4 Hz, 1H), 6.86 (d, J = 2.4 Hz, 1H), 6.81 (d, J = 8.4 Hz, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.7, 162.5, 157.4, 157.2, 152.8, 130.1, 127.3, 123.5, 122.6, 116.6, 115.1, 115.0, 102.1; MS-ESI m / z: 253.05 [M-H] - .。

[0053] Example 13: Preparation of target compound 18 (attached to the specification Figure 26 ).

[0054] Compound 18 was prepared as in Example 8, except that p-hydroxyphenylacetic acid was used as the starting material instead of p-methoxyphenylacetic acid. The product analysis data are as follows: Compound 18. Yield: 92%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.60 (s, 1H), 10.68 (br, 1H), 9.31 (br, 1H), 7.92 (d, J = 9.0 Hz, 1H),7.07 (d, J = 8.4 Hz, 2H), 6.70 (d, J = 8.4 Hz, 2H), 6.39 (dd, J = 2.4, 9.0Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 4.13 (s, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ :202.7, 165.0, 164.8, 156.1, 133.6, 130.4, 125.2, 115.2, 112.0, 108.3, 102.5,43.3; MS-ESI m / z: 243.05 [MH] - ..

[0055] Example 14: Target compound 19 (attached to the specification Figure 27 ) preparation.

[0056] Compound 19 was prepared as in Example 8, except that p-nitrophenylacetic acid was used as the starting material instead of p-methoxyphenylacetic acid. The product analysis data are as follows: Compound 19. Yield: 61%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.25 (s, 1H), 10.71 (s, 1H), 8.19 (d, J = 8.4 Hz, 2H), 7.94 (d, J = 9.0Hz, 1H), 7.56 (d, J = 8.4 Hz, 2H), 6.42 (dd, J= 2.4, 9.0 Hz, 1H), 6.28 (d, J = 2.4 Hz, 1H), 4.54 (s, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 200.5, 165.1,164.3, 146.4, 143.4, 133.4, 131.2, 123.3, 112.4, 108.4, 102.5, 43.9; MS-ESI m / z: 272.05 [M-H] - .。

[0057] Example 15: Preparation of target compound 20 (attached to the specification Figure 28 ).

[0058] Preparation method of compound 20: The same as that in Example 7, only using 4-nitrophenylacetic acid instead of 4-methoxyphenylacetic acid as the starting material. The test data of the reaction product are as follows: Compound 20. Yield: 53%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.92 (s, 1H), 8.60 (s, 1H), 8.28 (d, J = 9.0 Hz, 2H), 8.00 (d, J = 8.4Hz, 1H), 7.90 (d, J = 9.0 Hz, 2H), 6.98 (dd, J = 2.4, 9.0 Hz, 1H), 6.92 (d, J = 2.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 173.9, 163.0, 157.4, 155.4,146.7, 139.3, 129.9, 127.4, 123.2, 121.6, 116.4, 115.6, 102.3; MS-ESI m / z:282.00 [M-H] - .。

[0059] Example 16: Preparation of target compound 21 (attached to the specification Figure 29 ).

[0060] Preparation method of Compound 21: Identical to Example 8, except that 4-chlorophenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting material. The detection data of the reaction product are as follows: Compound 21. Yield: 96%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.41 (s, 1H), 10.69 (s, 1H), 7.93 (d, J = 9.0 Hz, 1H), 7.37 (d, J = 8.4Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.40 (dd, J = 2.4, 9.0 Hz, 1H), 6.27 (d, J = 2.4 Hz, 1H), 4.32 (s, 2H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 201.5, 165.0,164.5, 134.2, 133.4, 131.6, 131.3, 128.2, 112.3, 108.3, 102.5, 43.4; MS-ESI m / z: 261.00 [M-H] - .。

[0061] Example 17: Preparation of the target compound 22 (attached to the specification Figure 30 ).

[0062] Preparation method of Compound 22: Identical to Example 7, except that 4-chlorophenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting material. The detection data of the reaction product are as follows: Compound 22. Yield: 87%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.73 (s, 1H), 8.43 (s, 1H), 7.98 (d, J = 8.4 Hz, 1H), 7.61 (d, J = 8.4Hz, 2H), 7.48 (d, J = 8.4 Hz, 2H), 6.96 (dd, J= 2.4, 8.4 Hz, 1H), 6.90 (d, J = 2.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.2, 162.8, 157.5, 154.1,132.5, 131.0, 130.7, 128.1, 127.3, 122.3, 116.5, 115.4, 102.2; MS-ESI m / z:271.00 [M-H] - .。

[0063] Example 18: Preparation of target compound 23 (description attached Figure 31 ).

[0064] Preparation method of compound 23: The same as in Example 8, except that pyrogallol is used instead of resorcinol as the starting material. The detection data of the reaction product are as follows: Compound 23. Yield: 76%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.55(s, 1H), 10.11 (s, 1H), 8.62 (s, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.20 (d, J =7.8 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.42 (d, J = 8.4 Hz, 1H), 4.20 (s,2H), 3.72 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 203.2, 158.0, 152.6(152.600), 152.6 (152.583), 132.4, 130.5, 127.1, 123.0, 113.8, 112.4, 107.8,55.0, 43.1; MS-ESI m / z: 273.05 [M-H] - .。

[0065] Example 19: Preparation of target compound 24 (description attached Figure 32 ).

[0066] Preparation method of Compound 24: The same as Example 7, except that pyrogallol is used instead of resorcinol as the starting material. The test data of the reaction product are as follows: Compound 24. Yield: 69%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.33(s, 1H), 9.45 (s, 1H), 8.38 (s, 1H), 7.51 (d, J = 8.4 Hz, 2H), 7.48 (d, J =9.0 Hz, 1H), 6.99 – 6.96 (m, 3H), 3.78 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ :175.1, 158.9, 153.0, 150.0, 146.7, 132.9, 130.2, 124.4, 122.6, 117.5, 115.7,114.2, 113.6, 55.2; MS-ESI m / z: 283.05 [M-H] - .。

[0067] Example 20: Preparation of the target compound 25 (attached to the specification Figure 33 ).

[0068] Preparation method of Compound 25: The same as Example 7, except that pyrogallol is used instead of resorcinol and phenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting materials. The test data of the reaction product are as follows: Compound 25. Yield: 87%; 1 H NMR(600 MHz, DMSO- d 6 ) δ : 9.99 (s, 3H), 8.43 (s, 1H), 7.58 (d, J = 7.2 Hz, 2H),7.50 (d, J = 8.4 Hz, 1H), 7.42 (t, J = 7.2 Hz, 2H), 7.37 – 7.35 (m, 1H), 7.01(d, J = 8.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d6 ) δ : 174.9, 153.7, 150.2, 146.8, 133.0, 132.3, 129.1, 128.1, 127.7, 123.0, 117.5, 115.7, 114.4; MS-ESI m / z: 253.00 [M-H] - .。

[0069] Example 21: Preparation of the target compound 26 (attached to the specification) Figure 34 )

[0070] Preparation method of compound 26: The same as Example 8, only using pyrogallol instead of resorcinol and 3,4-dimethoxyphenylacetic acid instead of p-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 26. Yield: 72%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 12.59 (s, 1H), 10.14 (s, 1H), 8.66 (s, 1H), 7.51 (d, J = 9.0 Hz, 1H), 6.91 (d, J = 1.8 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.80 – 6.79 (m, 1H), 6.44 (d, J = 9.0 Hz, 1H), 4.19 (s, 2H), 3.72 (s, 3H), 3.71 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 203.2, 152.7 (152.693), 152.7(152.665), 148.7, 147.7, 132.5, 127.6, 123.1, 121.6, 113.4, 112.5, 111.9, 107.9, 55.5 (55.535), 55.5 (55.513), 43.7; MS-ESI m / z: 303.05 [M-H] - .。

[0071] Example 22: Preparation of the target compound 27 (attached to the specification) Figure 35 )

[0072] Preparation method of Compound 27: The same as in Example 7, except that pyrogallol is used instead of resorcinol, and 3,4-dimethoxyphenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 27. Yield: 54%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.53 (s, 1H), 9.49 (s, 1H), 8.42 (s, 1H),7.47 (d, J = 8.4 Hz, 1H), 7.21 (d, J = 1.8 Hz, 1H), 7.13 (dd, J = 2.4, 8.4Hz, 1H), 7.04 (d, J = 9.0 Hz, 1H), 6.99 (d, J = 8.4 Hz, 1H), 3.78 (s, 3H),3.77 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 175.1, 153.2, 150.0, 148.5, 148.3,146.7, 133.0, 124.8, 122.7, 121.3, 117.5, 115.6, 114.5, 112.9, 111.6, 55.6(55.578), 55.6 (55.556); MS-ESI m / z: 313.05 [M-H] - .。

[0073] Example 23: Preparation of the target compound 28 (attached to the specification Figure 36 ).

[0074] Preparation method of Compound 28: The same as in Example 7, except that pyrogallol is used instead of resorcinol, and 3,4-dimethoxyphenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 28. Yield: 80%; 1 H NMR (600 MHz, DMSO- d 6 ) δ: 10.38 (s, 1H), 9.50 (s, 1H), 8.49 (s, 1H), 7.63 – 7.61 (m, 2H), 7.50 – 7.48 (m, 3H), 6.98 (d, J = 8.4 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.7, 154.0, 150.2, 146.7, 133.0, 132.4, 131.2, 130.7,128.1, 121.8, 117.3, 115.7, 114.3; MS-ESI m / z: 287.00 [MH] - ..

[0075] Example 24: Target compound 29 (attached to the specification Figure 37 ) preparation.

[0076] Preparation of Compound 29: The same method as in Example 7 was used, except that pyrogallol was substituted for resorcinol and 3,4-dimethoxyphenylacetic acid was substituted for p-methoxyphenylacetic acid as the starting materials. The product analysis data are as follows: Compound 29. Yield: 77%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.19 (s, 1H), 9.67 (s, 1H), 8.48 (s, 1H), 7.63 – 7.61 (m, 2H), 7.57 – 7.54 (m, 2H), 7.48 (d, J = 9.0 Hz, 1H), 6.98 (d, J = 9.0 Hz, 1H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.6, 153.9, 150.3, 146.7,133.0, 131.6, 131.1, 131.0, 121.8, 121.0, 117.3, 115.7, 114.4; MS-ESI m / z:330.90 [MH] - ..

[0077] Example 25: Target compound 30 (attached to the specification sheet) Figure 38 ) preparation.

[0078] Preparation method of Compound 30: The same as Example 7, except that 4-methoxyresorcinol is used instead of resorcinol, and 3,4-dimethoxyphenylacetic acid is used instead of p-methoxyphenylacetic acid as the starting materials. The test data of the reaction product are as follows: Compound 30. Yield: 75%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.61 (s, 1H), 8.35 (s, 1H), 7.45 (s,1H), 7.21 (d, J = 1.8 Hz, 1H), 7.12 (dd, J = 2.1, 8.4 Hz, 1H), 7.00 (d, J =8.4 Hz, 1H), 6.95 (s, 1H), 3.88 (s, 3H), 3.78 (s, 6H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.2, 153.0, 152.9, 151.7, 148.5, 148.3, 147.0, 124.8, 122.7, 121.1,116.2, 112.8, 111.6, 104.7, 102.8, 55.8, 55.5 (55.547), 55.5 (55.496); MS-ESIm / z: 327.05 [M-H] - .。

[0079] Example 26: Preparation of the target compound 31 (attached to the specification Figure 39 ).

[0080] Preparation method of Compound 31: The same as Example 7, except that 4-methoxyresorcinol is used instead of resorcinol as the starting material. The test data of the reaction product are as follows: Compound 31. Yield: 81%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.66 (s, 1H), 8.33 (s, 1H), 7.53 – 7.49 (m, 2H), 7.43 (s, 1H), 7.00 –6.97 (m, 2H), 6.94 (s, 1H), 3.88 (s, 3H), 3.79 (s, 3H);13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.2, 158.9, 153.1, 152.8, 151.8, 147.0, 130.1, 124.5, 122.6, 116.1, 113.6, 104.7, 102.8, 55.8, 55.1; MS-ESI m / z: 299.05 [M+H] + .。

[0081] Example 27: Preparation of the target compound 32 (attached to the specification Figure 40 ).

[0082] Preparation method of compound 32: The same as Example 7, except that 4-methoxyresorcinol is used instead of resorcinol, and 4-bromophenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 32. Yield: 88%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.64 (s, 1H), 8.41 (s, 1H), 7.63 – 7.60 (m, 2H), 7.55 – 7.53 (m, 2H), 7.43 (s, 1H), 6.95 (s, 1H), 3.88 (s, 3H); 13 C NMR(150 MHz, DMSO- d 6 ) [[ID=I09]]δ : 173.8, 153.7, 153.1, 151.8, 147.1, 131.6, 131.0, 130.9, 121.8, 121.0, 116.2, 104.7, 102.9, 55.8; MS-ESI m / z: 346.90 [M+H] + .。

[0083] Example 28: Preparation of the target compound 33 (attached to the specification Figure 41 ).

[0084] Preparation method of compound 33: The same as Example 7, except that 4-methoxyresorcinol is used instead of resorcinol, and 4-chlorophenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 33. Yield: 91%; 1 H NMR (600 MHz, DMSO- d6 ) δ : 10.65 (s, 1H), 8.42 (s, 1H), 7.63 – 7.60 (m,2H), 7.50 – 7.48 (m, 2H), 7.44 (s, 1H), 6.97 (s, 1H), 3.88 (s, 3H); 13 C NMR(150 MHz, DMSO- d 6 ) δ : 173.8, 153.7, 153.1, 151.8, 147.1, 132.4, 131.2, 130.6,128.1, 121.8, 116.2, 104.7, 102.9, 55.8; MS-ESI m / z: 301.00 [M-H] - .。

[0085] Example 29: Preparation of the target compound 34 (attached to the specification Figure 42 ).

[0086] Preparation method of compound 34: The same as in Example 7, only using 4-methoxyresorcinol instead of resorcinol and phenylacetic acid instead of p-methoxyphenylacetic acid as starting materials. The detection data of the reaction product are as follows: Compound 34. Yield: 90%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 10.64 (s, 1H), 8.37 (s, 1H), 7.58 – 7.56 (m, 2H),7.45 – 7.41 (m, 3H), 7.38 – 7.35 (m, 1H), 6.97 (s, 1H), 3.88 (s, 3H); 13 C NMR(150 MHz, DMSO- d 6 ) δ : 174.1, 153.5, 153.0, 151.8, 147.1, 132.4, 129.0, 128.1,127.7, 123.1, 116.3, 104.7, 102.9, 55.9; MS-ESI m / z: 267.05 [M-H] - .。

[0087] Example 30: Preparation of the target compound 35 (attached to the specification Figure 43 ).

[0088] Preparation method of Compound 35: The same as in Example 7, except that 4-methoxyresorcinol is used instead of resorcinol and 4-hydroxyphenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting materials. The test data of the reaction product are as follows: Compound 35. Yield: 76%; 1 H NMR (400 MHz, DMSO- d 6 ) δ : 10.96 (s, 1H), 9.71 (s, 1H), 8.27 (s, 1H),7.41 (s, 1H), 7.39 – 7.35 (m, 2H), 7.08 (s, 1H), 6.85 – 6.81 (m, 2H), 3.86(s, 3H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.8, 157.7, 153.6, 152.9, 152.2,147.5, 130.4, 123.4, 123.1, 116.5, 115.5, 105.1, 103.3, 56.2; MS-ESI m / z:285.05 [M+H] + .。

[0089] Example 31: Preparation of the target compound 36 (attached to the specification Figure 44 ).

[0090] Preparation method of Compound 36: The same as in Example 7, except that 3-methoxyphenol is used instead of resorcinol and 4-bromophenylacetic acid is used instead of 4-methoxyphenylacetic acid as the starting materials. The test data of the reaction product are as follows: Compound 36. Yield: 51%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 8.54 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.65– 7.63 (m, 2H), 7.58 – 7.56 (m, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.11 (dd, J =2.4, 9.0 Hz, 1H), 3.92 (s, 3H); 13 C NMR (150 MHz, DMSO- d 6 )δ : 174.7, 164.4, 158.0, 154.9, 131.7, 131.6, 131.4, 127.4, 123.1, 121.6, 118.0, 115.5, 101.2, 56.6; MS-ESI m / z: 331.00 [M+H] + .。

[0091] Example 32: Preparation of target compound 37 (description attached Figure 45 ).

[0092] Preparation method of compound 37: The same as Example 7, only using 3-methoxyphenol instead of resorcinol and 3,4-dimethoxyphenylacetic acid instead of p-methoxyphenylacetic acid as starting materials. The detection data of the reaction product are as follows: Compound 37. Yield: 83%; 1 H NMR (600 MHz, DMSO- d 6 ) δ : 8.45 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.21 (s, 1H), 7.16 – 7.14 (m, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.01 (d, J = 7.8 Hz, 1H), 3.91 (s, 3H), 3.79 (s, 6H); 13 C NMR (150 MHz, DMSO- d 6 ) δ : 174.6, 163.7, 157.4, 153.7, 148.7, 148.3, 127.0, 124.4, 123.5, 121.2, 117.6, 114.8, 112.7, 111.5, 100.6, 56.1, 55.5; MS-ESI m / z: 313.10 [M+H] + .。

[0093] Example 33: Preparation of target compound 38 (description attached Figure 46 ).

[0094] Preparation method of compound 38: The same as Example 7, only using 3-methoxyphenol instead of resorcinol and p-chlorophenylacetic acid instead of p-methoxyphenylacetic acid as starting materials. The detection data of the reaction product are as follows: Compound 38. Yield: 77%; 11H NMR (600 MHz, DMSO- d 6 ) δ : 8.53 (s, 1H), 8.04 (d, J J = 9.0 Hz, 1H), 7.63 (d, J J = 8.4 Hz, 2H), 7.50 (d, J J = 9.0 Hz, 2H), 7.20 (d, J J = 2.4 Hz, 1H), 7.63(dd, J J = 2.4, 9.0 Hz, 2H), 3.91 (s, 3H); 13 13C NMR (150 MHz, DMSO- d 6 ) δ : 174.3,163.9, 157.5, 154.5, 132.6, 130.9, 130.7, 128.2, 127.0, 122.6, 117.5, 115.0,100.7, 56.2; MS-ESI m / z: 287.00 [M+H] + .。

[0095] Example 34: Preparation of the target compound 39 (see attached Figure 47 ).

[0096] Preparation method of compound 39: The same as in Example 7, except that 3-methoxyphenol was used instead of resorcinol and phenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 39. Yield: 90%; 1 1HNMR (600 MHz, DMSO- d 6 ) δ : 8.47 (s, 1H), 8.04 (d, J J = 9.0 Hz, 1H), 7.58 (d, J J = 7.2 Hz, 2H), 7.43 (t, J J = 7.2 Hz, 2H), 7.38 (t, J J = 7.2 Hz, 1H), 7.17 (d, J J = 2.4 Hz, 1H), 7.09 (dd, J J = 2.4, 9.0 Hz, 1H), 3.91 (s, 3H); 1313C NMR (150 MHz, DMSO- d 6 ) δ : 174.4, 163.8, 157.5, 154.2, 132.0, 128.9, 128.1, 127.8, 127.0, 123.8, 117.6, 114.9, 100.6, 56.1; MS-ESI m / z: 253.10 [M+H] + .

[0097] Example 35: Preparation of the target compound 40 (description attached Figure 48 ).

[0098] Preparation method of compound 40: The same as Example 7, only using 3-methoxyphenol instead of resorcinol as the starting material. The test data of the reaction product are as follows: Compound 40. Yield: 76%; 1 1H NMR (600 MHz, DMSO- d 6 ) δ : 8.43 (s, 1H), 8.04 (d, J J = 8.4 Hz, 1H), 7.54 – 7.52 (m, 2H), 7.17 (d, J J = 2.4Hz, 1H), 7.09 (dd, J J = 2.4, 9.0 Hz, 1H), 7.01 – 6.98 (m, 2H), 3.91 (s, 3H), 3.79 (s, 3H); 13 13C NMR (150 MHz, DMSO- d 6 ) δ : 174.6, 163.7, 159.0, 157.5, 153.5, 130.1, 127.0, 124.1, 123.4, 117.6, 114.8, 113.6, 100.6, 56.1, 55.2; MS-ESI m / z: 283.05 [M+H] + .

[0099] Test methods and results for the determination of radical scavenging activity, cytotoxic activity, neuroprotective activity, luciferase reporter gene and AChE activity of compounds 1 - 40.

[0100] (1)In vitro free radical scavenging experiment: The target compound at 20 mM was mixed with DPPH solution (50 mM). The mixture was shaken vigorously and allowed to stand in the dark at room temperature for 30 minutes. The absorbance of the reaction solution was measured spectrophotometrically at 517 nm. The ABTS scavenging ability was determined according to the published method ( Free Radical Biol. Med. 1999, 26, 1231- 1237. ). The ABTS solution was prepared by oxidizing ABTS (7 mM) with potassium persulfate (2.5 mM) in the dark at room temperature for 12 - 16 hours, and then diluted to an absorbance of 0.700 ± 0.020 at 734 nm. 2 mL of the ABTS solution was mixed with 100 μL of the target compound solution, and the absorbance at 734 nm was recorded after 30 minutes. The results of the free radical scavenging experiment are shown in the attached instructions Figure 49 and 50 .

[0101] (2)Cell culture: PC12 cells are rat adrenal pheochromocytoma cells, which are from the Lanzhou Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. When cultured in vitro, in order to make their growth conditions similar to those in vivo, it is necessary to simulate the in - vivo growth environmental conditions in vitro. Provide the necessary conditions for cell survival, such as culturing in DMEM medium (containing 10% fetal bovine serum, 1% penicillin - streptomycin, 2 mM glutamine) in a CO2 cell incubator at 5% CO2, 37 °C and 95% humidity.

[0102] (3)MTT assay: The digested PC12 cells were seeded at 1×10 4 cells / well in a 96 - well plate. After 24 - hour culture, the cells were then treated with different concentrations of the compound and incubated for 24 hours. Then, 0.5 mg / mL MTT was added, and the 96 - well plate was placed in a CO2 cell incubator for 4 hours to generate purple - blue formazan. Finally, 100 μL of the triple solution (10% SDS, 5% isobutanol, and 0.1% hydrochloric acid) was added to dissolve overnight, and the absorbance was measured using a microplate reader at 570 nm. The results of the cytotoxic activity are shown in the attached instructions Figure 51 .

[0103] (4)Oxidative damage experiment of hydrogen peroxide (H2O2) on PC12 cells: PC12 cells were seeded at 1×10 4 cells / well in a 96 - well plate, incubated with different concentrations of the compound for 24 hours, then the medium containing the compound was discarded, and the cells were incubated with the medium containing hydrogen peroxide (500 μM) for 24 hours. Then, the cell viability was determined by MTT assay. The neuroprotective activity is shown in the attached instructions Figure 52 .

[0104] (5) Luciferase reporter gene experiment: pARE-luc plasmid was transformed and amplified by Escherichia coli, extracted by high-purity medium kit and set aside. The cells to be transfected were inoculated in 6-well plates. When the cells grew to about 50%, the pARE-luc plasmid was transferred into PC12 cells using transfection reagent. After incubation with drugs for 24 h, the cells were collected and lysis buffer (1% TritonX-100, 25 mM Glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT) was added. The cells were pulverized with an ultrasonic cell crusher, and the protein quantification was 2 mg / mL. Add 200 μL of detection solution (100 μM luciferin, 25 mM Glycylglycine, 15 mM K3PO4, 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mM DTT) and 10 μL of protein supernatant to each well, and measure the bioluminescence using a fluorimeter. Luciferase reporter gene activity is shown in the appendix of the instruction manual. Figure 53 .

[0105] (6) AChE activity and kinetics assay: AChE inhibitory activity was tested based on the method of Ellman et al. (Biochem.Pharmacol., 1961, 7, 2, 88-90). Each compound (10 mL) was dispensed into a 96-well microplate in triplicate and mixed with 190 mL of Ellman's reagent, which included 20 mL of AChE, 140 mL of pH 8 phosphate buffer (containing 10 mL of 0.5 mM 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB)), and 20 mL of ATCh. Control wells contained only ethanol. Enzyme activity was monitored at 412 nm, read every 30 seconds for 10 minutes (linear reaction). To investigate the type of inhibition of the most active inhibitor, Ellman's assay was performed at multiple substrate and inhibitor concentrations. The data were analyzed using Graph Pad Prism version 9.0, and the Ki values were calculated using the standard method of double reciprocal (Lineweaver-Burk) plotting. The results of AChE activity and kinetics assays are shown in the appendix of the manual. Figure 54 .

[0106] In vitro free radical scavenging experiments have shown that some compounds have strong free radical scavenging ability (see Appendix Figure 49 and 50), these highly active compounds (Compounds 2, 5, 8, 9, 10, and 23 - 29) were selected for further cytotoxic activity screening experiments, and it was found that most of the compounds were non-toxic at the experimental concentration of 20 mM (Compounds 26 - 28 had weak toxicity) (see attached instructions Figure 51 ). The neuroprotective activity experiments proved that these compounds had varying degrees of protective effects on hydrogen peroxide - damaged PC12 cells (see attached instructions Figure 52 ), among which Compound 29 had the highest protective activity. The luciferase reporter gene experiment showed that Compound 29 had much higher ability to activate Nrf2 than t -BHQ (a classical Nrf2 activator) at the same concentration (see attached instructions Figure 53 ), proving that one of the mechanisms by which Compound 29 protects nerve cells is to activate Nrf2. At the same time, Compound 29 can also inhibit AChE in a non - competitive manner (see attached instructions Figure 54 ), which is another mechanism for it to protect nerve cells. In summary, Compound 29 has good application potential as an Nrf2 activator and AChE inhibitor for the development of neuroprotective drugs or drugs for neurodegenerative diseases.

[0107] Description of the drawings.

[0108] Figure 1 is the synthetic route of target compounds 1 - 11.

[0109] Figure 2 is the synthetic route of target compounds 12 - 22.

[0110] Figure 3 is the synthetic route of target compounds 23 - 29.

[0111] Figure 4 is the synthetic route of target compounds 30 - 35.

[0112] Figure 5 is the synthetic route of target compounds 36 - 40.

[0113] Figure 6 is the general chemical structure formula of isoflavone - type target compounds.

[0114] Figure 7 is the general chemical structure formula of isoflavone intermediate target compounds.

[0115] Figure 8 is the specific chemical structural formula of target compound 1.

[0116] Figure 9 is the specific chemical structural formula of target compound 2.

[0117] Figure 10For Reaction Scheme 1, which is the specific chemical reaction process for synthesizing target compounds 1 and 2.

[0118] Figure 11 This is the specific chemical structural formula of target compound 3.

[0119] Figure 12 This is the specific chemical structural formula of target compound 4.

[0120] Figure 13 This is the specific chemical structural formula of target compound 5.

[0121] Figure 14 This is the specific chemical structural formula of target compound 6.

[0122] Figure 15 This is the specific chemical structural formula of target compound 8.

[0123] Figure 16 This is the specific chemical structural formula of target compound 7.

[0124] Figure 17 This is the specific chemical structural formula of target compound 9.

[0125] Figure 18 This is the specific chemical structural formula of target compound 10.

[0126] Figure 19 This is the specific chemical structural formula of target compound 11.

[0127] Figure 20 This is the specific chemical structural formula of target compound 12.

[0128] Figure 21 This is the specific chemical structural formula of target compound 13.

[0129] Figure This is the specific chemical structural formula of target compound 14.

[0130] ​ This is the specific chemical structural formula of target compound 15.

[0131] ​ This is the specific chemical structural formula of target compound 16.

[0132] ​ This is the specific chemical structural formula of target compound 17.

[0133] ​ This is the specific chemical structural formula of target compound 18.

[0134] ​ This is the specific chemical structural formula of target compound 19.

[0135] ​ This is the specific chemical structural formula of target compound 20.

[0136] ​ This is the specific chemical structural formula of target compound 21.

[0137] ​ This is the specific chemical structural formula of target compound 22.

[0138] ​ This is the specific chemical structural formula of target compound 23.

[0139] ​ This is the specific chemical structural formula of target compound 24.

[0140] ​ This is the specific chemical structural formula of target compound 25.

[0141] ​ This is the specific chemical structural formula of target compound 26.

[0142] ​ This is the specific chemical structural formula of target compound 27.

[0143] ​ This is the specific chemical structural formula of target compound 28.

[0144] ​ This is the specific chemical structural formula of target compound 29.

[0145] ​ This is the specific chemical structural formula of target compound 30.

[0146] ​ This is the specific chemical structural formula of target compound 31.

[0147] ​ This is the specific chemical structural formula of target compound 32.

[0148] ​ This is the specific chemical structural formula of target compound 33.

[0149] ​ This is the specific chemical structural formula of target compound 34.

[0150] ​ This is the specific chemical structural formula of target compound 35.

[0151] ​ This is the specific chemical structural formula of target compound 36.

[0152] ​ This is the specific chemical structural formula of target compound 37.

[0153] ​ It is the specific chemical structural formula of target compound 38.

[0154] ​ It is the specific chemical structural formula of target compound 39.

[0155] ​ It is the specific chemical structural formula of target compound 40.

[0156] ​ It is the in vitro scavenging activities of all forty target compounds against the free radical DPPH.

[0157] ​ It is the in vitro scavenging activities of all forty target compounds against the free radical ABTS.

[0158] ​ It is the cytotoxic activities of twelve target compounds obtained through preliminary optimization against PC12 cells.

[0159] ​ It is the neuroprotective activities of twelve target compounds obtained through preliminary optimization against PC12 cells at different concentrations.

[0160] ​ It is the luciferase reporter gene activities of target compound 29 and the classical Nrf2 activator t -BHQ.

[0161] ​ It is the inhibition activities of target compound 29 against AChE and the results of kinetic experiments.

Claims

1. An isoflavone compound, characterized in that The described compound has the chemical structural formula shown in Formula (I): （Ⅰ） In Structural Formula (I): the substituent R1 is selected from 4-bromophenyl, the substituent R2 is selected from hydrogen, the substituent R3 is selected from hydrogen, the substituent R4 is selected from hydroxyl, and the substituent R5 is selected from hydroxyl.

2. The preparation method of an isoflavone compound according to claim 1, characterized in that Under dry conditions, boron trifluoride diethyl ether (25 mL) was added to a round-bottom flask containing pyrogallol (10 mmol) and 4-bromophenylacetic acid (10 mmol). The mixture was heated to 90 °C and reacted for 6 hours, and the reaction progress was monitored by thin-layer chromatography (TLC). After the reaction was completed, the reaction solution was cooled to room temperature and then further cooled to below 0 °C in an ice bath. At this time, N,N-dimethylformamide (DMF, 15 mL) was slowly added dropwise to the reaction solution. After the addition was completed, the mixture was reheated to 53 °C and stirred for 15 minutes. Subsequently, a mixture of methanesulfonyl chloride (50 mmol) and DMF (8 mL) was slowly added dropwise at this temperature. After the addition was completed, the mixture was kept at this temperature for another 15 minutes, then heated to 100 °C and reacted for 8 hours until the reaction was confirmed to be completed by TLC. After the reaction was completed and cooled, the mixture was washed successively with saturated sodium acetate solution and saturated sodium bicarbonate solution, and then extracted with ethyl acetate. The combined organic layers were concentrated under reduced pressure, and the obtained crude product was purified by column chromatography (elution system: dichloromethane: methanol) to obtain the purified target compound.

3. An isoflavone compound according to claim 1, characterized in that It can be used in the preparation of drugs for preventing or treating neurodegenerative diseases, specifically Alzheimer's disease.

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