Preparation method of isoflavone compound and use thereof in neuroprotection

Abstract

This invention relates to a novel isoflavone compound represented by formula (I), a method for preparing this compound, and its use in the preparation of neuroprotective small molecule drugs. The general chemical formula of this compound is shown in formula (I). The compound in formula (I) is prepared by reacting a polyphenolic compound with a substituted phenylacetonitrile, or by reacting a polyphenolic compound with a substituted phenylacetic acid. A series of neuroprotective activity experiments have demonstrated that the compound described in this invention possesses excellent neuroprotective activity, and the compound with the best activity also exhibits acetylcholinesterase inhibitory activity, making it suitable as a lead molecule for the development of neuroprotective and neurodegenerative disease drugs. The preparation process of this invention is simple, the raw materials are inexpensive and readily available, and the product has high purity. (I).

Description

Technical Field

[0001] This invention relates to a novel isoflavone compound and its intermediates, as well as the use of this compound in the preparation of neuroprotective drugs. It belongs to the pharmaceutical field. Background Technology

[0002] Neurons play a crucial role in activities such as learning, thinking, and memory. The gradual decline in neuronal function and viability caused by a variety of pathological factors is a major cause of neurodegenerative diseases. These multifactorial neurodegenerative diseases affect millions of people worldwide. Protein misfolding, mitochondrial damage, and oxidative stress are all present 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 a major cause of dementia, a progressive decline in at least two cognitive domains that leads to an inability to perform normal social and occupational activities. AD typically affects older adults, but it is not a normal aging defect. AD is characterized by amyloid-β peptide (Aβ) aggregation, accumulation of hyperphosphorylated tau protein (p-tau), production of inflammatory mediators, oxidative stress, cholinergic dysfunction, and deficits in synaptic and cognitive function. Genetic factors, environmental factors, and typical lifestyle are part of the etiological factors associated with AD. Currently, Alzheimer's disease (AD) affects more than 50 million people, and this number is projected to reach 150 million by 2050. However, treating AD remains a challenge because its pathogenesis is not fully understood. Many phase III clinical trials targeting Aβ production and accumulation have failed. Recently, clinical trials of Crenezumab (NCT03491150), lanabecestat (NCT02972658), and solanezumab (NCT01900665) were terminated due to a lack of significant efficacy. This systemic failure in the search for new AD drugs has prompted scientists to explore new strategies to combat the disease.

[0003] Alzheimer's disease (AD) is characterized by a variety of pathological features, such as massive DNA oxidation, severe mitochondrial damage, widespread lipid peroxidation, high levels of neurotoxic trace metals, and elevated Aβ levels. All of these factors increase the formation of reactive oxygen species (ROS), leading to oxidative stress in the brains of AD patients. Under physiological conditions, ROS homeostasis is strictly regulated by the ROS-generating system and the cellular antioxidant network. The antioxidant defense system includes various enzymes and small molecules, such as heme oxygenase-1 (HO-1), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), thioredoxin (Trx), thioredoxin reductase (TrxR), the glutathione peroxidase (GPX) family, and NAD(P)H:quinone oxidoreductase 1 (NQO1). Studies of human AD have found that the levels of nuclear transcription factors Nrf2, SOD1, CAT, and GPX in the brains of AD patients are significantly lower than in healthy individuals. Due to the lower levels of Nrf2 and these important antioxidant enzymes, the brains of AD patients are more susceptible to ROS attack. In this situation, ROS causes progressive and irreversible damage to the brain. Furthermore, substantial research evidence shows a significant decrease in the level of the neurotransmitter acetylcholine (ACh) in AD patients, possibly due to reduced ACh production or increased acetylcholinesterase (AChE) activity. Moreover, increased AChE activity further leads to increased and accumulated Aβ levels.

[0004] Direct use of 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 early stages. Activating Nrf2 to increase the level of antioxidant proteins is considered a better approach to achieving neuroprotection. Nrf2 can induce the expression of approximately 500 genes encoding various cytoprotective enzymes and detoxification proteins. High levels of Nrf2 can improve damage caused by ROS and / or mitochondrial dysfunction. Simultaneously, inhibiting AChE activity can not only increase neurotransmitter levels in the human brain but also reduce the production and accumulation of Aβ, thereby reducing ROS production. Currently, dimethyl fumarate (trade name Tecfidera) and oleanolic acid derivatives (trade name Skyclarys) are used as Nrf2 activators to treat multiple sclerosis and Friedrich's ataxia, respectively. Donepezil, rivastigmine, and galantamine are AChE inhibitors approved by the FDA for clinical treatment of AD.

[0005] Natural products are a vital source of innovation for new drug discovery. Naturally derived isoflavones, due to their polyphenolic structures, often possess strong antioxidant properties and other multiple physiological functions. Based on the structural characteristics of Nrf2 activators and our previous analysis of the structural characteristics of AChE inhibitors and the AChE protein structure, we designed and synthesized 40 structurally diverse isoflavone compounds and their intermediates (see attached product manual). Figure 6 and 7 (as shown in the figure), and tested the free radical scavenging ability of these compounds, their cytotoxic and protective activities against neuron-like PC12 cells, their activation effect on Nrf2, and their inhibitory activity against AChE. Summary of the Invention

[0006] The purpose of this invention is to provide an isoflavone compound or an intermediate thereof capable of activating Nrf2 and inhibiting AChE. Another objective of this invention is to provide the use of such a compound as a candidate molecule for neuroprotective drugs in neurodegenerative diseases.

[0007] The series of isoflavone compounds and their intermediates described in this invention are included in the specification. Figure 6 and 7 The chemical structure shown is:

[0008] Structural Specifications Figure 6 In the following: Substituent R1 is selected from phenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-nitrophenyl, 4-aminophenyl, 3,4-dimethoxyphenyl, 3,4-dihydroxyphenyl, 4-bromophenyl, 4-chlorophenyl; Substituent R2 is selected from hydroxyl and hydrogen; Substituent R3 is selected from methoxy and hydrogen; Substituent R4 is selected from methoxy and hydroxyl; Substituent R5 is selected from hydroxyl and hydrogen.

[0009] Structural Specifications Figure 7 In the middle: substituent R1 is selected from phenyl, 4-hydroxyphenyl, 4-methoxyphenyl, 4-nitrophenyl, 3,4-dimethoxyphenyl, 4-bromophenyl, 4-chlorophenyl; substituents R2 and R3 are both selected from hydroxyl and hydrogen.

[0010] The preparation method of this invention is as per the appendix to the instruction manual. Figure 1 , 2 The chemical reaction equations shown in 3, 4 and 5 proceed.

[0011] The isoflavone compounds and their intermediates described in this invention (see attached specification) Figure 6 and 7 The optimal preparation method for compounds is carried out through the following steps, which can be generally 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.

[0012] Preparation of compounds 1-11: Phloroglucinol (3.6 g, 28.6 mmol), various substituted phenylacetonitrs (32.5 mmol), and anhydrous zinc chloride (1.9 g, 13.9 mmol) were dissolved in 40 mL of 1,2-dimethoxyethane and stirred vigorously at room temperature. Hydrogen chloride gas was then slowly and continuously bubbled into the reaction solution until the solution became clear, and the bubbling continued for 30 minutes. The resulting reaction mixture was further stirred at room temperature for 48 hours. After stirring, the reaction mixture was cooled to below 10 °C, and then 1.4 g of water was slowly added. The reaction continued for 2.5 hours below 10 °C. The solid intermediate ketimine hydrochloride was then obtained by filtration and washed with 1,2-dimethoxyethane (2.6 g) and water (4.4 g).

[0013] The resulting solid was then dissolved in a mixture comprising water (35 g), concentrated hydrochloric acid (3 g), and methanol (1.5 g). After thorough stirring, the mixture was heated to reflux at 110 °C and reacted for 15 hours. The reaction mixture was then cooled to below 10 °C and stirred for another 2 hours. The resulting mixture was filtered under vacuum to obtain a solid cake, which was washed with (5 g) water. The solid was dried in a fluidized bed dryer at 65 °C for 5 hours to obtain intermediate ketone 1, namely compounds 2, 5, 8, and 9, which could be used directly in the next reaction without further purification.

[0014] Under stirring, a solution of boron trifluoride diethyl ether (6 mmol) was added dropwise to 8 mL of DMF containing intermediate 1 (1 mmol). 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 proceed for 7 hours, with the reaction progress monitored by thin-layer chromatography (TLC). After the reaction was complete, the mixture was washed successively 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 crude products were purified by column chromatography (elution system: dichloromethane:methanol) to give the corresponding pure compounds, namely compounds 1, 3, 4, 6, 7, 10, and 11.

[0015] Preparation method of compound 12-40:

[0016] ① Preparation of intermediate compounds: Under dry conditions, 25 mL of boron trifluoride diethyl ether 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, with the reaction progress monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was cooled to room temperature, and the reaction mixture was washed with saturated sodium acetate and sodium bicarbonate solution, followed by extraction with ethyl acetate. The organic layers were combined, concentrated under reduced pressure, and the residue was purified by column chromatography to obtain the corresponding pure target compounds, namely intermediate compounds 13, 18, 19, 21, 23, and 26.

[0017] ② Preparation of isoflavones: Under dry conditions, boron trifluoride diethyl ether (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, with the reaction progress monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was cooled to room temperature and then further cooled to below 0 °C in an ice bath. At this point, DMF (15 mL) was slowly added dropwise to the reaction solution. 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 slowly 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 to be complete by TLC. After the reaction was complete 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 isoflavones 12, 14-17, 20, 22, 24, 25 and 27-40.

[0018] Preparation of the polyphenolic reactant, 4-methoxyresorcinol: Hydrogen peroxide aqueous 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 in an ice bath (0 °C). Then, 3-hydroxy-4-methoxybenzaldehyde (6.0 g, 39.4 mmol) was added in portions to the reaction mixture. After all the aldehydes were added, the ice bath was removed, and the reaction was stirred overnight at room temperature. The reaction progress was confirmed 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% sodium bisulfite aqueous solution and saturated sodium chloride solution, and concentrated again under reduced pressure. The resulting residue was dissolved in 20 mL of methanol and stirred vigorously with 200 mL of saturated sodium carbonate solution at room temperature for approximately 2 hours, with the reaction progress monitored by TLC. After the reaction was complete, 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 give a brown solid (5.2 g, 84% yield) of the target product. TLC confirmed that the product could be used directly in subsequent reactions without further purification.

[0019] Preliminary in vitro antioxidant assays and screening revealed that the product listed in the instructions... Figure 6 and 7 Some of the compounds shown exhibit strong scavenging ability against DPPH and ABTS free radicals (see attached instruction manual). Figure 49 and 50 Compounds 2, 5, 8, 9, 10, and 23-29 (a total of 12 compounds) with the strongest in vitro free radical scavenging ability were selected for cytotoxic activity assays and screening. It was found that all 12 compounds were non-toxic to PC12 cells (rat adrenal pheochromocytoma cells) at a concentration of 5 mM, while at concentrations up to 20 mM, only compound 26-28 showed weak toxicity (see product information appendix). Figure 51 These 12 compounds were further screened using an in vitro neurodegenerative disease cell model (PC12 cells were damaged by a certain concentration of hydrogen peroxide). The results showed that these 12 compounds had significant protective effects on PC12 cells in a concentration-dependent manner (see product manual attached). Figure 52 Compound 29, which exhibited the best protective activity, was selected. Its activation effect on the nuclear transcription factor Nrf2 was determined using a luciferase reporter gene assay. The results showed that its activity was significantly stronger than that of the classic Nrf2 activator tert-butylhydroquinone (THC) at the same concentration. t -BHQ (Instruction manual included) Figure 53Compound 29 also possesses AChE inhibitory activity and is a non-competitive AChE inhibitor (see product manual). Figure 54 Therefore, the isoflavone compounds and their intermediates of the present invention can serve as effective Nrf2 activators and AChE inhibitors, and as lead molecules for the development of neuroprotective and neurodegenerative disease drugs. The isoflavone compounds and their intermediates of the present invention have a simple synthetic process, high product purity, and exhibit strong protective effects on nerve cells, showing excellent application prospects.

[0020] The following detailed description of specific embodiments further illustrates the above-mentioned aspects of the present invention. However, this should not be construed as a limitation of the present invention.

[0021] Detailed implementation method.

[0022] Example 1: Target compound 1 (attached to the specification) Figure 8 Preparation of ).

[0023] Compound 2 (attached to instruction manual) Figure 9 The preparation method of the intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethyl ketone: 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) were dissolved in 40 mL of 1,2-dimethoxyethane and stirred vigorously at room temperature. Subsequently, hydrogen chloride gas was slowly and continuously bubbled into the reaction solution until the solution became clear, and then the bubbling of hydrogen chloride gas was continued for 30 minutes. The resulting reaction mixture was further stirred at room temperature for 48 hours. After stirring, the reaction mixture was cooled to below 10 °C, and then 1.4 g of water was slowly added. The reaction continued for 2.5 hours below 10 °C. The intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethylimine hydrochloride solid was then obtained by filtration. This solid was washed with 1,2-dimethoxyethane (2.6 g) and water (4.4 g) and used directly in the next step of the reaction without drying.

[0024] The resulting solid was then dissolved in a mixture comprising water (35 g), concentrated hydrochloric acid (3 g), and methanol (1.5 g). After thorough stirring, the mixture was heated to reflux at 110 °C and reacted for 15 hours. The reaction mixture was then cooled to below 10 °C and stirred for another 2 hours. The resulting mixture was filtered under vacuum to obtain a solid cake, which was washed with (5 g) water. The solid was dried in a fluidized bed dryer at 65 °C for 5 hours to obtain compound 2, namely intermediate 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethyl ketone 6.4 g, which can be used directly in the next reaction without further purification.

[0025] Preparation of Compound 1: A solution of boron trifluoride diethyl ether (6 mmol) was added dropwise to 8 mL of DMF containing 2-(4-methoxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethyl ketone (274 mg, 1 mmol) under stirring. 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 proceed for 7 hours, with the reaction progress monitored by thin-layer chromatography (TLC). After the reaction was complete, the mixture was washed successively with saturated sodium acetate and sodium bicarbonate solutions, followed by extraction with ethyl acetate. The combined organic phases were concentrated under reduced pressure, and the crude product was purified by column chromatography (elution system: dichloromethane:methanol) to give the corresponding pure compound 1, namely 236 mg of 5,7-dihydroxy-3-(4-methoxyphenyl)-4H-chroman-4-one.

[0026] The reaction is shown in reaction formula 1 (attached to the instruction manual). Figure 10 ).

[0027] 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] - .

[0028] 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 [MH] - .

[0029] Example 2: Target compound 3 (attached to the instruction manual) Figure 11 Preparation of ).

[0030] Similar to Example 1, except that phenylacetonitrile was used instead of p-methoxyphenylacetonitrile as the starting material. The reaction product detection data 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 [MH] - .

[0031] Example 3: Target compound 4 (attached to the instruction manual) Figure 12 Preparation of ).

[0032] Compound 5 (with instruction manual) Figure 13 The preparation method of the intermediate 2-(4-hydroxyphenyl)-1-(2,4,6-trihydroxyphenyl)ethyl ketone is the same as in Example 1, except that p-hydroxyphenylacetonitrile is used instead of p-methoxyphenylacetonitrile as the starting material. The detection 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 [MH] - .

[0033] Preparation method of compound 4: Same as in Example 1, except that p-hydroxyphenylacetonitrile was used instead of p-methoxyphenylacetonitrile as the starting material. The detection 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 [MH] - .

[0034] Example 4: Target compound 6 (attached to the instruction manual) Figure 14 Preparation of ).

[0035] Compound 8 (with instruction manual) Figure 15 The preparation method of the intermediate 2-(4-nitrophenyl)-1-(2,4,6-trihydroxyphenyl)ethyl ketone is the same as in Example 1, except that p-nitrophenylacetonitrile is used instead of p-methoxyphenylacetonitrile 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 [MH] - .

[0036] Compound 7 (with instruction manual) Figure 16 Preparation method of compound 7: Same as in Example 1, except that p-nitrophenylacetonitrile was used instead of p-methoxyphenylacetonitrile 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 [MH] - .

[0037] Preparation of Compound 6: Compound 7 (299 mg, 1 mmol) was added to a round-bottom flask containing stannous chloride (950 mg, 5 mmol), concentrated hydrochloric acid (5 mL), and water (2 mL). The mixture was refluxed for 12 hours, after which the reaction was stopped. After cooling, the pH of the reaction solution was adjusted to 7 with 5% sodium hydroxide aqueous solution, resulting in the formation of a yellow solid precipitate. The precipitate was filtered, and the filtrate was extracted three times with ethyl acetate. The filter cake and the ethyl acetate solution obtained from the extraction were combined and purified by column chromatography to yield 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 [MH] - .

[0038] Example 5: Target compound 9 (attached to the instruction manual) Figure 17 Preparation of ).

[0039] Similar to Example 1, except that 3,4-dimethoxyphenylacetonitrile was used instead of p-methoxyphenylacetonitrile as the starting material. The product analysis data 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 [MH] - .

[0040] Example 6: Target compound 10 (attached to the instruction manual) Figure 18 Preparation of ).

[0041] Compound 11 (attached to instruction manual) Figure 19 Preparation method of compound 11: Same as in Example 1, except that 3,4-dimethoxyphenylacetonitrile was used instead of p-methoxyphenylacetonitrile as the starting material. The detection 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 = 2.4 Hz, 1H), 7.11 (dd, J = 2.4, 8.4 Hz, 1H), 7.00 (d, J = 8.4 Hz, 1H), 6.39 (d, J = 2.4 Hz, 1H), 6.23 (d, 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 [MH] - .

[0042] Compound 10 (with instruction manual) Figure 18 Preparation method of compound 11: Compound 11 (314 mg, 1 mmol) and pyridine hydrochloride (1.15 g, 10 mmol) were mixed in a round-bottom flask. The reaction system was then heated to 190 °C, at which point the system was in a molten state. The reaction was continued at this temperature for 12 hours, after which the reaction was stopped. After the reaction was cooled to room temperature, 50 mL of 2M HCl aqueous solution was added, and the mixture was stirred for 15 minutes. The solution was extracted three times with ethyl acetate. The organic phases were combined under reduced pressure to obtain the crude product, which was purified by column chromatography to give 236 mg of pure compound 10. The detection data of the product obtained from the reaction 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 = 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 [MH] - .

[0043] Example 7: Target compound 12 (attached to the instruction manual) Figure 20 Preparation of ).

[0044] Preparation of Compound 12: Under dry conditions, 25 mL of boron trifluoride diethyl ether 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, with the reaction progress monitored by TLC. After the reaction was complete, it was cooled to room temperature and then placed in an ice bath to below 0 °C, at which point 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 to be complete by TLC. After the reaction was completed and cooled, the reaction mixture was washed with saturated sodium acetate and sodium bicarbonate solution and then extracted with ethyl acetate. The organic layers were combined and concentrated under reduced pressure. The resulting residue was purified by column chromatography to give 2.16 g of pure target compound 12. Compound 12, yield: 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] + .

[0045] Example 8: Target compound 13 (attached to the instruction manual) Figure 21 Preparation of ).

[0046] Preparation of Compound 13: Under dry conditions, 25 mL of boron trifluoride diethyl ether 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, with the reaction progress monitored by TLC. After the reaction was completed, the mixture was cooled to room temperature, washed with saturated sodium acetate and sodium bicarbonate solution, and then extracted with ethyl acetate. The organic layers were combined, concentrated under reduced pressure, and the residue was purified by column chromatography to give 1.93 g of pure target compound 13. The detection data of the product obtained from the reaction 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 CNMR (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 [MH] - .

[0047] Example 9: Target compound 14 (attached to the specification) Figure 22 Preparation of ).

[0048] Preparation method of compound 14: Same as in Example 7, except that 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The reaction product detection data 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] + .

[0049] Example 10: Target compound 15 (attached to the instruction manual) Figure 23 Preparation of ).

[0050] Preparation method of compound 15: Same as in Example 7, except that phenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The reaction product detection data 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] + .

[0051] Example 11: Target compound 16 (attached to the specification) Figure 24 Preparation of ).

[0052] Preparation method of compound 16: Same as in Example 7, except that p-bromophenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The reaction product detection data 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 = 8.4Hz, 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 [MH] - .

[0053] Example 12: Target compound 17 (attached to the specification) Figure 25 Preparation of ).

[0054] Preparation method of compound 17: Same as in Example 7, except that p-hydroxyphenylacetic acid was used instead of p-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 [MH] - .

[0055] Example 13: Target compound 18 (attached to the specification) Figure 26 Preparation of ).

[0056] Preparation method of compound 18: Same as in Example 8, except that p-hydroxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The detection data of the reaction product 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] - .

[0057] Example 14: Target compound 19 (attached to the specification) Figure 27 Preparation of ).

[0058] Preparation method of compound 19: Same as in Example 8, except that p-nitrophenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The reaction product detection 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-ESIm / z: 272.05 [MH] - .

[0059] Example 15: Target compound 20 (attached to the instruction manual) Figure 28 Preparation of ).

[0060] Preparation method of compound 20: Same as in Example 7, except that p-nitrophenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting material. The detection 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 [MH] - .

[0061] Example 16: Target compound 21 (attached to the specification) Figure 29 Preparation of ).

[0062] Preparation method of compound 21: Same as in Example 8, except that p-chlorophenylacetic acid was used instead of p-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-ESIm / z: 261.00 [MH] - .

[0063] Example 17: Target compound 22 (attached to the instruction manual) Figure 30 Preparation of ).

[0064] Preparation method of compound 22: Same as in Example 7, except that p-chlorophenylacetic acid was used instead of p-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 [MH] - .

[0065] Example 18: Target compound 23 (attached to the instruction manual) Figure 31 Preparation of ).

[0066] Preparation method of compound 23: Same as in Example 8, except that pyrogallol was 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 [MH] - .

[0067] Example 19: Target compound 24 (attached to the instruction manual) Figure 32 Preparation of ).

[0068] Preparation method of compound 24: Same as in Example 7, except that pyrogallol was used instead of resorcinol as the starting material. The detection 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 [MH] - .

[0069] Example 20: Target compound 25 (attached to the instruction manual) Figure 33 Preparation of ).

[0070] Preparation method of compound 25: Same as in Example 7, except that pyrogallol 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 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 [MH] - .

[0071] Example 21: Target compound 26 (attached to the instruction manual) Figure 34 Preparation of ).

[0072] Preparation method of compound 26: Same as in Example 8, except that pyrogallol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as starting materials. The reaction product detection data 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 [MH] - .

[0073] Example 22: Target compound 27 (attached to the instruction manual) Figure 35 Preparation of ).

[0074] Preparation method of compound 27: Same as in Example 7, except that pyrogallol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as 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 [MH] - .

[0075] Example 23: Target compound 28 (attached to the instruction manual) Figure 36 Preparation of ).

[0076] Preparation method of compound 28: Same as in Example 7, except that pyrogallol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as 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] - .

[0077] Example 24: Target compound 29 (attached to the instruction manual) Figure 37 Preparation of ).

[0078] Preparation method of compound 29: Same as in Example 7, except that pyrogallol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as starting materials. The reaction product detection 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] - .

[0079] Example 25: Target compound 30 (attached to the instruction manual) Figure 38 Preparation of ).

[0080] Preparation method of compound 30: Same as in Example 7, except that 4-methoxyresorcinol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as starting materials. The reaction product detection data 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 [MH] - .

[0081] Example 26: Target compound 31 (attached to the specification) Figure 39 Preparation of ).

[0082] Preparation method of compound 31: Same as in Example 7, except that 4-methoxyresorcinol was used instead of resorcinol as the starting material. The detection 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] + .

[0083] Example 27: Target compound 32 (attached to instruction manual) Figure 40 Preparation of ).

[0084] Preparation method of compound 32: Same as in Example 7, except that 4-methoxyresorcinol was used instead of resorcinol and p-bromophenylacetic acid was used instead of p-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 ) 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] + .

[0085] Example 28: Target compound 33 (attached to the instruction manual) Figure 41 Preparation of ).

[0086] Preparation method of compound 33: Same as in Example 7, except that 4-methoxyresorcinol was used instead of resorcinol and p-chlorophenylacetic acid was used instead of p-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 [MH] - .

[0087] Example 29: Target compound 34 (attached to specification) Figure 42 Preparation of ).

[0088] Preparation method of compound 34: Same as in Example 7, except that 4-methoxyresorcinol 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 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 [MH] - .

[0089] Example 30: Target compound 35 (attached to the instruction manual) Figure 43 Preparation of ).

[0090] Preparation method of compound 35: Same as in Example 7, except that 4-methoxyresorcinol was used instead of resorcinol and p-hydroxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting materials. The detection 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] + .

[0091] Example 31: Target compound 36 (attached to the instruction manual) Figure 44 Preparation of ).

[0092] Preparation method of compound 36: Same as in Example 7, except that 3-methoxyphenol was used instead of resorcinol and p-bromophenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting materials. The detection 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] + .

[0093] Example 32: Target compound 37 (attached to the instruction manual) Figure 45 Preparation of ).

[0094] Preparation method of compound 37: Same as in Example 7, except that 3-methoxyphenol was used instead of resorcinol and 3,4-dimethoxyphenylacetic acid was used instead of p-methoxyphenylacetic acid as starting materials. The reaction product detection data 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 = 7.8Hz, 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] + .

[0095] Example 33: Target compound 38 (attached to the instruction manual) Figure 46 Preparation of ).

[0096] Preparation method of compound 38: Same as in Example 7, except that 3-methoxyphenol was used instead of resorcinol and p-chlorophenylacetic acid was used instead of p-methoxyphenylacetic acid as the starting materials. The detection data of the reaction product are as follows: Compound 38. Yield: 77%; 1H NMR (600 MHz, DMSO- d 6 ) δ 8.53 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.63 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 9.0 Hz, 2H), 7.20 (d, J = 2.4 Hz, 1H), 7.63(dd, J = 2.4, 9.0 Hz, 2H), 3.91 (s, 3H); 13 C 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] + .

[0097] Example 34: Target compound 39 (attached to specification) Figure 47 Preparation of ).

[0098] Preparation method of compound 39: 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 starting materials. The detection data of the reaction product are as follows: Compound 39. Yield: 90%; 1 HNMR (600 MHz, DMSO- d 6 ) δ : 8.47 (s, 1H), 8.04 (d, J = 9.0 Hz, 1H), 7.58 (d, J = 7.2 Hz, 2H), 7.43 (t, J = 7.2 Hz, 2H), 7.38 (t, J = 7.2 Hz, 1H), 7.17 (d, J = 2.4 Hz, 1H), 7.09 (dd, J = 2.4, 9.0 Hz, 1H), 3.91 (s, 3H); 13C 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] + .

[0099] Example 35: Target compound 40 (attached to the instruction manual) Figure 48 Preparation of ).

[0100] Preparation method of compound 40: Same as in Example 7, except that 3-methoxyphenol was used instead of resorcinol as the starting material. The detection data of the reaction product are as follows: Compound 40. Yield: 76%; 1 H NMR (600 MHz, DMSO- d 6 ) δ 8.43 (s, 1H), 8.04 (d, J = 8.4 Hz, 1H), 7.54 – 7.52 (m, 2H), 7.17 (d, J = 2.4Hz, 1H), 7.09 (dd, J = 2.4, 9.0 Hz, 1H), 7.01 – 6.98 (m, 2H), 3.91 (s, 3H), 3.79 (s, 3H); 13 C 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] + .

[0101] The experimental methods and results for determining the free radical scavenging activity, cytotoxic activity, neuroprotective activity, luciferase reporter gene activity, and AChE activity of compounds 1-40.

[0102] (1) In vitro free radical scavenging experiment: 20 mM of the target compound was mixed with 50 mM DPPH solution, 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 published methods ( Free Radical Biol. Med. 1999, 26, 1231- 1237. The ABTS solution was prepared by oxidizing ABTS (7 mM) with potassium persulfate (2.5 mM) at room temperature in the dark for 12–16 hours, and then diluting it to an absorbance of 0.700 ± 0.020 at 734 nm. 2 mL of the ABTS solution was mixed with 100 mL of the target compound solution, and the absorbance at 734 nm was recorded after 30 minutes. Free radical scavenging results are shown in the appendix to the instruction manual. Figure 49 and 50 .

[0103] (2) Cell Culture: PC12 cells are rat adrenal pheochromocytoma cells, derived from the Lanzhou Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. To ensure their growth in vitro is similar to their in vivo growth, the in vitro environment must simulate in vivo conditions. This involves providing the necessary conditions for cell survival, such as culturing in DMEM medium (containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM glutamine) in a 5% CO2, 37 ℃, or 95% humidity CO2 cell culture incubator.

[0104] (3) MTT assay: Digested PC12 cells were subjected to a 1×10⁻⁶ t / t assay. 4 Cells were seeded in 96-well plates and cultured for 24 h. Then, cells were treated with different concentrations of compounds and incubated for 24 h. Next, 0.5 mg / mL MTT was added, and the 96-well plates were placed in a CO2 cell culture incubator for 4 h, resulting in the formation of blue-purple formazan. Finally, 100 μL of a triplet solution (10% SDS, 5% isobutanol, and 0.1% hydrochloric acid) was added to dissolve the cells overnight. Absorbance was measured using a microplate reader at 570 nm. Cytotoxic activity results are shown in the instruction manual appendix. Figure 51 .

[0105] (4) Experiment on oxidative damage of hydrogen peroxide (H2O2) to PC12 cells: PC12 cells were subjected to 1×10 4 Cells were seeded in 96-well plates and incubated with different concentrations of the compound for 24 h. The culture medium containing the compound was then discarded, and the cells were incubated with medium containing hydrogen peroxide (500 μM) for another 24 h. Cell viability was then determined using an MTT assay. Neuroprotective activity is detailed in the product manual. Figure 52 .

[0106] (5) Luciferase reporter gene experiment: pARE-luc plasmid was transformed and amplified by E. coli, and extracted using a high-purity medium-volume kit for later use. The cells to be transfected were seeded in 6-well plates. When the cells grew to about 50%, the pARE-luc plasmid was transfected into PC12 cells using transfection reagent. After incubation with the drug for 24 h, the cells were collected and then lysis buffer (1% Triton X-100, 25 mM Glycylglycine, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT) was added. The cells were then pulverized using an ultrasonic cell disruptor. The protein quantification was 2 mg / mL. Add 200 μL of detection solution (100 μM luciferin, 25 mM M lycylglycine, 15 mM K3PO4, 15 mM MgSO4, 4 mM EGTA, 2 mM ATP, 1 mM DTT) and 10 μL of protein supernatant to each well. Finally, measure the biofluorescence using a fluorescence spectrometer. Luciferase reporter gene activity is shown in the instruction manual appendix. Figure 53 .

[0107] (6) AChE Activity and Kinetic Assay: AChE inhibitory activity was tested according to the method of Ellman et al. (Biochem. Pharmacol., 1961, 7, 2, 88-90.). Each compound (10 mL) was dispensed into 96-well microplates in triplicate and mixed with 190 mL of Ellman's reagent, which included 20 mL of AChE, 140 mL of phosphate buffer at pH = 8 (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 inhibition type of the most active inhibitor, Ellman's method was performed at multiple substrate and inhibitor concentrations. Data were analyzed using Graph Pad Prism version 9.0, and Ki values ​​were calculated using the standard method of double reciprocal plotting (Lineweaver-Burk). AChE activity and kinetic results are attached to the instruction manual. Figure 54 .

[0108] In vitro free radical scavenging experiments demonstrated that some compounds possess strong free radical scavenging capabilities (see instruction manual). Figure 49 and 50These highly active compounds (compounds 2, 5, 8, 9, 10, and 23-29) were selected for further cytotoxic activity screening experiments. It was found that most compounds showed no toxicity at the experimental concentration of 20 mM (compounds 26-28 showed slight toxicity). (See attached product manual) Figure 51 Neuroprotective activity experiments demonstrated that these compounds exhibited varying degrees of protection against hydrogen peroxide-damaged PC12 cells (see instruction manual). Figure 52 Compound 29 exhibited the highest protective activity among the compounds. Luciferase reporter gene assays showed that compound 29, at the same concentration, was more effective than... t -BHQ (the classic Nrf2 activator) has a much higher Nrf2 activation ability (instructions included). Figure 53 This demonstrates that one of the mechanisms by which compound 29 protects nerve cells is by activating Nrf2. Simultaneously, compound 29 can also non-competitively inhibit AChE (see product manual). Figure 54 This is another mechanism by which it protects nerve cells. In summary, compound 29, as an Nrf2 activator and AChE inhibitor, has great potential for development of neuroprotective drugs or drugs for neurodegenerative diseases.

[0109] Attached image description.

[0110] Figure 1 Synthetic routes for target compounds 1-11.

[0111] Figure 2 Synthetic routes for target compounds 12-22.

[0112] Figure 3 Synthetic routes for target compounds 23-29.

[0113] Figure 4 Synthetic routes for target compounds 30-35.

[0114] Figure 5 Synthetic routes for target compounds 36-40.

[0115] Figure 6 This is the general chemical structural formula for the target isoflavone compounds.

[0116] Figure 7 This is the general chemical structural formula of the target compound, which is an isoflavone intermediate.

[0117] Figure 8 The specific chemical structural formula of target compound 1 is given.

[0118] Figure 9 The specific chemical structural formula of target compound 2 is given.

[0119] Figure 10This refers to reaction formula 1, which describes the specific chemical reaction process for synthesizing target compounds 1 and 2.

[0120] Figure 11 The specific chemical structural formula of target compound 3 is given.

[0121] Figure 12 The specific chemical structural formula of target compound 4 is given.

[0122] Figure 13 The specific chemical structural formula of target compound 5 is given.

[0123] Figure 14 The specific chemical structural formula of target compound 6 is given.

[0124] Figure 15 The specific chemical structural formula of target compound 8.

[0125] Figure 16 The specific chemical structural formula of target compound 7 is given.

[0126] Figure 17 The specific chemical structural formula of target compound 9 is given.

[0127] Figure 18 The specific chemical structural formula of target compound 10 is given.

[0128] Figure 19 The specific chemical structural formula of target compound 11 is given.

[0129] Figure 20 The specific chemical structural formula of target compound 12 is given.

[0130] Figure 21 The specific chemical structural formula of target compound 13 is given.

[0131] Figure 22 The specific chemical structural formula of target compound 14 is given.

[0132] Figure 23 The specific chemical structural formula of target compound 15 is given.

[0133] Figure 24 The specific chemical structural formula of target compound 16 is given.

[0134] Figure 25 The specific chemical structural formula of target compound 17 is given.

[0135] Figure 26 The specific chemical structural formula of target compound 18.

[0136] Figure 27 The specific chemical structural formula of target compound 19.

[0137] Figure 28 The specific chemical structural formula of target compound 20 is given.

[0138] Figure 29 The specific chemical structural formula of target compound 21 is given.

[0139] Figure 30 The specific chemical structural formula of target compound 22 is given.

[0140] Figure 31 The specific chemical structural formula of target compound 23 is given.

[0141] Figure 32 The specific chemical structural formula of target compound 24 is given.

[0142] Figure 33 The specific chemical structural formula of target compound 25.

[0143] Figure 34 The specific chemical structural formula of target compound 26.

[0144] Figure 35 The specific chemical structural formula of target compound 27.

[0145] Figure 36 The specific chemical structural formula of target compound 28.

[0146] Figure 37 The specific chemical structural formula of target compound 29.

[0147] Figure 38 The specific chemical structural formula of target compound 30 is given.

[0148] Figure 39 The specific chemical structural formula of target compound 31 is given.

[0149] Figure 40 The specific chemical structural formula of target compound 32 is given.

[0150] Figure 41 The specific chemical structural formula of target compound 33.

[0151] Figure 42 The specific chemical structural formula of target compound 34.

[0152] Figure 43 The specific chemical structural formula of target compound 35 is given.

[0153] Figure 44 The specific chemical structural formula of target compound 36.

[0154] Figure 45 The specific chemical structural formula of target compound 37 is given.

[0155] Figure 46 The specific chemical structural formula of target compound 38.

[0156] Figure 47 The specific chemical structural formula of target compound 39.

[0157] Figure 48 The specific chemical structural formula of target compound 40.

[0158] Figure 49 The in vitro scavenging activity of all forty target compounds against the free radical DPPH was determined.

[0159] Figure 50 The in vitro scavenging activity of all forty target compounds against the free radical ABTS was determined.

[0160] Figure 51 The cytotoxic activity of twelve target compounds obtained through preliminary selection against PC12 cells was measured.

[0161] Figure 52 The study investigated the neuroprotective activity of twelve target compounds obtained through preliminary selection against PC12 cells at different concentrations.

[0162] Figure 53 For target compound 29 and classic Nrf2 activator t -BHQ luciferase reporter gene activity.

[0163] Figure 54 The results show the inhibitory activity and kinetics of target compound 29 against AChE.

Claims

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

2. The method for preparing an isoflavone compound according to claim 1, characterized in that: Under dry conditions, 25 mL of boron trifluoride diethyl ether 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, with the reaction progress monitored by thin-layer chromatography (TLC). After the reaction was complete, the reaction solution was cooled to room temperature and then further cooled to below 0 °C in an ice bath. At this point, N,N-dimethylformamide (DMF, 15 mL) was slowly added dropwise to the reaction solution. 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 slowly added dropwise at this temperature. After the addition was complete, the mixture was maintained at this temperature for another 15 minutes and then heated to 100 °C. The mixture was heated to ℃ 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 crude product was purified by column chromatography with dichloromethane / methanol as the elution system to obtain the purified target compound.

3. The isoflavone compound according to claim 1, characterized in that: It is used in the preparation of drugs for the prevention or treatment of the neurodegenerative disease Alzheimer's disease.

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