An isoquercetin ester dimer and its preparation method and use

Abstract

The present invention discloses an isoquercetin ester dimer and a preparation method and use thereof, wherein the structure of the isoquercetin ester dimer is shown in the following formula (I): The isoquercetin ester dimer of the present invention not only retains the anti-inflammatory activity of isoquercetin itself, but also strengthens the therapeutic effect on neuroinflammation due to the introduction of anti-inflammatory drugs such as ibuprofen, aspirin, naproxen, etc., and can improve the symptoms of a series of neurodegenerative diseases, such as Parkinson's movement disorder, Alzheimer's and vascular dementia learning and memory impairment, etc., and broadens the use of isoquercetin in the treatment of neuroinflammation-related diseases.

Description

Technical Field

[0001] The invention relates to a preparation method and application of an isoquercetin ester dimer, which can effectively alleviate neuroinflammation, improve Parkinson's movement disorder, and improve learning and memory impairment in Alzheimer's and vascular dementia.

[0002] Background Art

[0003] Neuroinflammation is an inflammatory response to neuronal damage in the central nervous system (CNS). It is known that glial cells are closely related to the occurrence of a series of neurodegenerative diseases, mainly including Alzheimer's disease, Parkinson's disease, etc. At present, the research progress on therapeutic drugs for neurodegenerative diseases is relatively slow. According to research reports, after activation, glial cells can produce substances related to neuroinflammation. These inflammatory substances can cause severe ischemic brain damage to the human body, thereby threatening human life and health. Therefore, from the perspective of regulating neuroinflammation, a strategy can be provided for the prevention and treatment of neurodegenerative diseases.

[0004] As a natural flavonol glycoside compound, isoquercitrin has good antioxidant, anti-inflammatory, antibacterial and antiviral effects. Isoquercitrin exerts neuroprotective effects by regulating RhoGTPase, which is closely related to human nervous system disease damage (PLoS One, 2012, 7(11): e49979); isoquercitrin has a protective effect on 6-OHDA-induced neurotoxicity and is expected to be used to treat Parkinson's disease-related neurodegenerative diseases (BMCRes Notes, 2014, 7(1): 49-57); Xie et al. showed that isoquercitrin inhibits oxidative stress, nitrosative stress and inflammatory response and antagonizes hepatotoxicity by regulating the activity of sulfotransferases (SULTs) and cytochrome P4502E1 enzyme (CYP2E1) (LifeScience, 2016, 152: 2016, 152: 180-189). In addition, isoquercetin exerts an anti-inflammatory effect on lipopolysaccharide-induced RAW264.7 cell inflammatory factors by inhibiting the production of TNF-α, NO, iNOS, and COX-2 (Medical Guide, 2017, 36: 601-605). The above studies suggest that isoquercetin is expected to be used in the prevention and treatment of neuroinflammatory diseases. However, the content of natural isoquercetin in nature is low, and the extraction process is relatively cumbersome. On the other hand, isoquercetin has low solubility and poor bioavailability, so it is necessary to structurally modify isoquercetin to improve its pharmaceutical properties and enhance its anti-inflammatory effect.

[0005] The ester compounds obtained by selective acylation of isoquercetin improve the physicochemical properties of isoquercetin (Journal of Molecular Catalysis B: Enzymatic, 2003, 23(2): 411-417), and its antioxidant and antibacterial activities are also enhanced (Food Chemistry, 2010, 123(1): 45-50; Journal of Biotechnology, 2005, 116(3): 295-304).

[0006] Some commonly used non-steroidal anti-inflammatory drugs (NSAIDs) in clinical practice, such as aspirin, ibuprofen, naproxen, indomethacin, etc., have a relieving or inhibitory effect on Alzheimer's disease or neuroinflammation (Acta Pharmaceutica Sinica, 2014, 49 (9): 1211-1217). It is also reported that lipoic acid has a protective effect on Alzheimer's neurological damage (Journal of Alzheimers Disease, 2014, 38 (1): 111-120), can also improve the motor symptoms of Parkinson's rats, and can reduce the aggregation of inflammatory factors and reduce dopaminergic neuron death by inhibiting the activation of microglia (Metabolish Brain Diseases, 2015, 30 (5): 1-10). Accordingly, the present invention mainly uses carboxylic acid anti-inflammatory drug structural units to acylate isoquercetin to obtain isoquercetin ester dimer. Summary of the invention

[0007] The present invention aims to provide an ester dimer with structural units of isoquercetin and carboxylic acid anti-inflammatory drugs as the mother core, and a preparation method and use thereof. The technical problem to be solved is to select a suitable molecular structure so that it has better activity in antagonizing neuroinflammation, can improve Parkinson's movement disorders, alleviate symptoms such as Alzheimer's and vascular dementia memory impairment, and has good blood-brain barrier penetration and relatively low toxicity.

[0008] The present invention solves the technical problem by adopting the following technical solutions:

[0009] The isoquercetin ester dimer of the present invention uses isoquercetin and carboxylic acid anti-inflammatory drug structural units as the parent core, and its structure is shown in the following formula (I):

[0010]

[0011] The R group includes but is not limited to the following structures:

[0012]

[0013] The preparation method of the isoquercetin ester dimer of the present invention comprises the following steps:

[0014] Step 1: Preparation of vinyl carboxylate

[0015] PdCl2, LiOAc and CuBr2 are added to a solution of vinyl acetate and tetrahydrofuran, and the carboxylic acid anti-inflammatory drug RCOOH is added under slow stirring, and the mixture is stirred at 70-75°C for 20-24 hours. After the reaction is completed, the mixture is filtered and washed with ethyl acetate, and concentrated under reduced pressure. The crude product is separated and purified by silica gel column chromatography to obtain a vinyl carboxylate intermediate, whose structural formula is:

[0016]

[0017] Step 2: Esterification reaction of isoquercetin with vinyl carboxylate

[0018] The isoquercetin prepared by the rutin degrading enzyme and the vinyl carboxylate obtained in step 1 are added to an anhydrous reaction solvent having a water content of less than 1‰, and the immobilized lipase is added, dried, and the mixture is stirred for 2 hours. The reaction was carried out under inert gas with closed stirring at a speed of 150-250 rpm. After 3-8 days of reaction, the immobilized enzyme and molecular sieve were removed by filtration, and the filtrate was concentrated under reduced pressure to obtain a crude product, which was separated and purified by silica gel column chromatography to obtain the target product (I).

[0019] In step 1, the molar ratio of PdCl2, LiOAc, CuBr2, vinyl acetate and carboxylic acid anti-inflammatory drug is 0.004-0.006:0.4-0.6:0.01-0.02:8-12:1.

[0020] In step 2, the molar ratio of isoquercetin to the vinyl carboxylate obtained in step 1 is 1:4-6.

[0021] In step 2, the amount of immobilized lipase added is 50-120% of the mass of isoquercetin, and the dried The added amount of the molecular sieve is 40-60% of the mass of isoquercetin.

[0022] In step 2, when the carboxylic acid anti-inflammatory drug is ibuprofen, lipoic acid, aspirin or naproxen, the anhydrous reaction solvent is anhydrous acetone, the immobilized lipase is Novozym 435, and the reaction temperature is 40-45°C.

[0023] In step 2, when the carboxylic acid anti-inflammatory drug is indomethacin, diclofenac, mefenamic acid or sulindac, the anhydrous reaction solvent is anhydrous acetonitrile, the immobilized lipase is Lipozyme RM IM, and the reaction temperature is 65-70°C.

[0024] The reaction route is as follows:

[0025]

[0026] The purpose of the isoquercetin ester dimer of the present invention is to prepare a pharmaceutical preparation with the isoquercetin ester dimer. The pharmaceutical preparation can effectively alleviate neuroinflammation and can be used for the prevention and treatment of neurodegenerative diseases.

[0027] The neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, vascular dementia and the like.

[0028] Furthermore, the pharmaceutical preparation can improve Parkinson's movement disorder, and the learning and memory impairment of Alzheimer's and vascular dementia.

[0029] The isoquercetin ester dimer of the present invention is a single ester compound synthesized by introducing structural units of carboxylic acid drugs such as aspirin, ibuprofen, diclofenac, lipoic acid, etc., which have clinical anti-inflammatory effects, into isoquercetin. The compound has the effect of inhibiting neuroinflammation, and can improve symptoms such as Parkinson's movement disorders, Alzheimer's and vascular dementia learning and memory impairment, and can be used as an improved isoquercetin ester derivative for the prevention and treatment of neurodegenerative diseases (such as Parkinson's, Alzheimer's, and vascular dementia).

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] Compared with the existing active molecule isoquercetin, the isoquercetin ester dimer of the present invention has increased blood-brain barrier permeability and is superior to isoquercetin in anti-neuroinflammatory effect; compared with the existing clinical carboxylic acid anti-inflammatory drug molecules (such as ibuprofen), the isoquercetin ester dimer of the present invention not only retains the antagonistic neuroinflammatory activity of ibuprofen, but also enhances the protective effect against neuroinflammatory damage due to the anti-inflammatory effect of isoquercetin itself and the ester structure, which helps to improve Parkinson's movement disorders, Alzheimer's and vascular dementia learning and memory impairment, and broadens the use of flavonol glycoside molecules (taking isoquercetin as an example) in the prevention and treatment of neurodegenerative diseases. DETAILED DESCRIPTION

[0032] The following content is a description of specific embodiments of the present invention, which is intended to further describe the technical solution of the present invention, but the present invention includes but is not limited to the following embodiments.

[0033] Example 1: Preparation of Ibuprofen Isoquercetin

[0034] 1a. Preparation of Ibuprofen Vinyl Ester

[0035]

[0036] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr2178 mg (0.8 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, slowly add ibuprofen 8.2 g (40 mmol, M = 206) under stirring, react at 70 ° C for 24 h, filter the mixture, and wash with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1. The components are collected and concentrated to obtain light yellow ibuprofen vinyl ester with an isolation yield of 74%.

[0037] 1b. Preparation of Ibuprofen Isoquercetin

[0038]

[0039] Weigh 0.3 g of isoquercetin (5 mmol, M = 464) into a reaction bottle, add 5.8 g of ibuprofen vinyl ester (25 mmol, M = 232), add 15 mL of anhydrous acetone as a reaction solvent, add 0.3 g of Novozym 435, and dry. Molecular sieve 0.15g, evacuate and pass nitrogen, react with stirring at 45℃ for 3d, filter to remove enzyme to terminate the reaction, concentrate the filtrate under reduced pressure to obtain the crude product, perform silica gel column chromatography, eluent is chloroform:methanol=30:1, collect the sample eluate with Rf=0.3, concentrate under reduced pressure to obtain the pure product, with a yield of 32%. 1 HNMR (DMSO, 600MHz) δ: 12.73 (s, 1H), 10.94 (s, 1H), 9.73 (s, 1H), 9.32 (s, 1 H),7.04-7.20(m,5H),6.80-6.83(m,1H),6.52(s,1H),5.95-6.00(m,2H),5 .60(d,1H,J=7.5Hz),4.20-4.32(m,2H),4.00-4.05(m,2H),3.60-3.70(m,3 H),2.43(m,2H),1.82(m,1H),1.51(d,J=2.0Hz,3H),0.87(d,J=2.0Hz,6H).

[0040] Example 2: Preparation of isoquercetin lipoate

[0041] 2a. Preparation of lipoic acid vinyl ester

[0042]

[0043] In a 100 mL round-bottom flask, PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.32 g (20 mmol, M = 66), CuBr2155 mg (0.7 mmol, M = 223) were added to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, and thioctic acid 8.2 g (40 mmol, M = 206) was slowly added under stirring. The mixture was reacted at 70° C. for 20 h, and the mixture was filtered and washed with ethyl acetate. The filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was subjected to silica gel column chromatography with an eluent of petroleum ether: ethyl acetate = 15:1. The corresponding components were collected and concentrated to obtain the product thioctic acid vinyl ester with a yield of 45%.

[0044] 2b. Preparation of isoquercetin lipoate

[0045]

[0046] Weigh 0.3 g of isoquercetin (5 mmol, M = 464) into a reaction bottle, add 5.8 g of lipoic acid vinyl ester (25 mmol, M = 232), add 15 mL of anhydrous acetone as the reaction solvent, add 0.3 g of Novozym 435, and dry. Molecular sieve 0.15g, closed reaction at 40℃ for 3d, filtered to remove enzyme to terminate the reaction, concentrated the filtrate under reduced pressure to obtain the crude product, silica gel column chromatography, eluent chloroform: methanol = 30:1, collect the eluate corresponding to the product point of Rf = 0.3, and concentrate under reduced pressure to obtain the product with a yield of 43%. 1 H NMR (DMSO, 600MHz) δ: 12.71 (s, 1H), 10.94 (s, 1H), 9.65 (s, 1H), 9.42 (s, 1H) ),7.04-7.20(m,1H),6.80-6.84(m,1H),6.52(s,1H),5.94-6.01(m,2H),5 .60(d,1H,J=7.5Hz),4.20-4.32(m,2H),4.00-4.05(m,2H),3.60-3.70(m, 2H),2.33-2.59(m,5H),1.85-1.90(m,2H),1.50-1.63(m,4H),1.25(m,2H).

[0047] Example 3: Preparation of isoquercetin aspirin ester

[0048] 3a. Preparation of vinyl aspirin

[0049]

[0050] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), and CuBr20.2 g (1.0 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, and slowly add aspirin 7.2 g (40 mmol, M = 180) under stirring. The mixture is reacted at 70°C for 24 h, and the mixture is filtered and washed with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with an eluent of petroleum ether:ethyl acetate = 20:1. The components are collected and concentrated to obtain the product aspirin vinyl ester with a yield of 62%.

[0051] 3b. Preparation of isoquercetin aspirin ester

[0052]

[0053] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) in a reaction bottle, add 5.2 g of aspirin vinyl ester (25 mmol, M = 206), add 15 mL of anhydrous acetone as the reaction solvent, add 0.3 g of Novozym 435, 0.15 g of dry The reaction was carried out at 45 °C in a sealed state for 4 days, and the enzyme was filtered to terminate the reaction. The filtrate was concentrated under reduced pressure to obtain a crude product, and the crude product was subjected to silica gel column chromatography with the eluent being chloroform:methanol=30:1. The product was obtained by concentration with a yield of 35%. 1 H NMR(DMSO,600MHz)δ:12.80(s,1H),10.84(s,1H),9.63(s,1H),9.42(s,1H),7.80-8.05(m,4H),7.04-7.10(m,1H),6.82-6.85(m, 1H),6.52(s,1H),5.95-6.00(m,2H),5.61(d,1H,J=7.5Hz),4.20-4.38(m,3H),4.00-4.05(m,1H),3.60-3.70(m,2H),2.39(s,3H).

[0054] Example 4: Preparation of Naproxen Ester of Isoquercetin

[0055] 4a. Preparation of naproxen vinyl ester

[0056]

[0057] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr2178 mg (0.8 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, slowly add naproxen 9.2 g (40 mmol, M = 230) under stirring, react at 70 ° C for 24 h, filter the mixture, and wash with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with petroleum ether: ethyl acetate = 20:1 as the eluent. The components are collected and concentrated to obtain the product naproxen vinyl ester.

[0058] 4b. Preparation of isoquercetin naproxen ester

[0059]

[0060] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) into a reaction bottle, add 5.2 g of naproxen vinyl ester (25 mmol, M = 206), add 15 mL of anhydrous acetone as the reaction solvent, add 0.3 g of Novozym 435, and dry. The mixture was added with 0.15 g molecular sieve and sealed at 45°C for 5 days. The reaction was terminated by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography with the eluent being chloroform:methanol=30:1. The product was concentrated to obtain a product with a yield of 41%. 1 HNMR (DMSO, 600MHz) δ: 12.67 (s, 1H), 10.95 (s, 1H), 9.71 (s, 1H), 9.35 (s, 1H), 7.43-7.54 (m, 3H), 7.00-7.15 (m, 3H), 6.50-6.63 ( m,3H),5.95-6.00(m,2H),5.65(d,1H,J=7.5Hz),4.25-4.32(m,2H),4.05-4.10(m,1H),3.50-3.70(m,7H),1.58(d,J=2.0Hz,3H).

[0061] Example 5: Synthesis of Isoquercetin Indomethacin Ester

[0062] 5a. Preparation of indomethacin vinyl ester

[0063] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr2180 mg (0.8 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, and slowly add indomethacin 14.3 g (40 mmol, M = 358) under stirring. The mixture is reacted at 70 ° C for 24 h, and the mixture is filtered and washed with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with petroleum ether: ethyl acetate = 20:1 as the eluent. The components are collected and concentrated to obtain the product indomethacin vinyl ester.

[0064]

[0065] 5b. Preparation of isoquercetin indomethacin ester

[0066]

[0067] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) in a reaction bottle, add 9.6 g of indomethacin vinyl ester (25 mmol, M = 384), add 15 mL of anhydrous acetonitrile as the reaction solvent, add 0.3 g of Lipozyme RM IM, and dry. The mixture was added with 0.15 g molecular sieve and sealed at 65°C for 5 days. The reaction was terminated by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography with the eluent being chloroform:methanol=30:1. The product was concentrated to obtain a yield of 37%. 1 HNMR (DMSO, 600MHz) δ: 12.65 (s, 1H), 10.93 (s, 1H), 9.70 (s, 1H), 9.39 (s, 1H), 7.70-7.76 (m, 2H), 7.45-7.50 (m, 2H), 6.50-6.73 (m, 5H) ),6.20-6.23(m,1H),5.95-6.00(m,2H),5.68(d,1H,J=7.5Hz),4.25-4.32(m,2H),4.05-4.10(m,1H),3.50-3.70(m,8H),2.30(s,3H).

[0068] Example 6: Synthesis of Isoquercetin Diclofenac

[0069] 6a. Preparation of diclofenac vinyl ester

[0070] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr2178 mg (0.8 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, slowly add diclofenac 11.8 g (40 mmol, M = 296) under stirring, react at 70 ° C for 24 h, filter the mixture, and wash with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with petroleum ether: ethyl acetate = 20:1 as the eluent. The components are collected and concentrated to obtain the product diclofenac vinyl ester.

[0071]

[0072] 6b. Preparation of isoquercetin diclofenac

[0073]

[0074] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) in a reaction bottle, add 9.6 g of diclofenac vinyl ester (25 mmol, M = 322), add 15 mL of anhydrous acetonitrile as the reaction solvent, add 0.3 g of Lipozyme RM IM, and dry. The mixture was added with 0.15 g molecular sieve and sealed at 70°C for 6 days. The reaction was terminated by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography with an eluent of chloroform:methanol=30:1 and concentrated to obtain the product. The yield of isoquercetin diclofenac was 29%. 1 H NMR (DMSO, 600MHz) δ: 12.69 (s, 1H), 10.95 (s, 1H), 9.73 (s, 1H), 9.43 (s, 1H), 6.75-6.90 (m, 5H), 6.30-6.53 (m,5H),5.95-6.00(m,2H),5.68(d,1H,J=7.5Hz),4.25-4.32(m,2H),4.05-4.10(m,1H),3.50-3.70(m,5H).

[0075] Example 7: Synthesis of Isoquercetin Mefenamate

[0076] 7a. Preparation of vinyl mefenamic acid ester

[0077]

[0078] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr2180 mg (0.8 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, and slowly add mefenamic acid 10.2 g (40 mmol, M = 255) under stirring. React at 70 ° C for 24 h, filter the mixture, and wash with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product, and the product is subjected to silica gel column chromatography with an eluent of petroleum ether: ethyl acetate = 20:1. The components are collected and concentrated to obtain vinyl mefenamic acid ester.

[0079] 7b. Preparation of Isoquercetin Mefenamate

[0080]

[0081] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) in a reaction bottle, add 7.0 g of mefenamic acid vinyl ester (25 mmol, M = 281), add 15 mL of anhydrous acetonitrile as the reaction solvent, add 0.3 g of Lipozyme RM IM, dry the molecular sieves, 0.15 g, sealed at 65 ° C for 7 days, filtered to remove the enzyme to terminate the reaction, the filtrate was concentrated under reduced pressure to obtain a crude product, silica gel column chromatography, the eluent was chloroform: methanol = 30:1, concentrated to obtain the product, the yield was 32%. 1 H NMR (DMSO, 600MHz) δ: 12.73 (s, 1H), 10.90 (s, 1H), 9.73 (s, 1H), 9.42 (s, 1H), 6.70-6.85 (m, 4H), 6.50-6.60 (m, 3H), 6.15-6. 30(m,3H),5.95-6.00(m,2H),5.68(d,1H,J=7.5Hz),4.27-4.32(m,2H),4.05-4.10(m,1H),3.50-3.70(m,5H),2.35(s,6H).

[0082] Example 8: Synthesis of isoquercetin sulindole ester

[0083] 8a. Preparation of sulindac vinyl ester

[0084]

[0085] In a 100 mL round-bottom flask, add PdCl20.035 g (0.2 mmol, M = 177), LiOAc 1.3 g (19.5 mmol, M = 66), CuBr20.1 g (0.5 mmol, M = 223) to vinyl acetate 34.4 g (400 mmol, M = 86) and 20 mL THF, and slowly add sulindac 14.2 g (40 mmol, M = 356) under stirring. The mixture is reacted at 70 ° C for 24 h. The mixture is filtered and washed with ethyl acetate. The filtrate is concentrated under reduced pressure to obtain a crude product. The product is subjected to silica gel column chromatography with petroleum ether: ethyl acetate = 20:1 as the eluent. The components are collected and concentrated to obtain the product vinyl mefenamic acid ester.

[0086] 8b. Preparation of isoquercetin sulindole ester

[0087]

[0088] Weigh 0.3 g of isoquercetin (5 mmol, M = 192) in a reaction bottle, add 9.6 g of sulindac vinyl ester (25 mmol, M = 382), add 15 mL of anhydrous acetonitrile as the reaction solvent, add 0.3 g of Lipozyme RM IM, and dry. The mixture was added with 0.15 g molecular sieve and sealed at 65°C for 8 days. The reaction was terminated by filtration to remove the enzyme. The filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was subjected to silica gel column chromatography with the eluent being chloroform:methanol=30:1. The product was concentrated to obtain a product with a yield of 30%. 1 H NMR (DMSO, 600MHz) δ: 12.79 (s, 1H), 10.95 (s, 1H), 9.75 (s, 1H), 9.40 (s, 1H), 7 .65-7.70(m,4H),7.35-7.38(m,1H),7.01-7.04(m,1H),6.70-6.85(m,3H),6. 50-6.60(m,2H),5.95-6.00(m,2H),5.60(d,1H,J=7.5Hz),4.27-4.32(m,2H), 4.05-4.10(m,1H),3.45-3.70(m,3H),2.92(s,2H),2.64(s,2H),1.71(s,3H).

[0089] Example 9: Effects of Compounds 1-8 on the Survival Rate of LPS-Induced BV-2 Neuroinflammatory Cells

[0090] BV-2 microglia cells in the logarithmic growth phase were taken at 1×10 6Cells / well were inoculated into 96-well plates and incubated overnight in a 37°C, 5% CO2 incubator. Different concentrations of compounds 1-5 (1, 10, 20 μmmol) were added, with 3 replicates per group. After pre-incubation for 4 hours, the supernatant was discarded, and LPS (1 μg / mL) was added to continue incubation for 12 hours. After the reaction was completed, the old culture medium was discarded, sterile PBS was added for washing once, 10 μL CCK-8 solution was added, and after incubation for 4 hours, OD was measured using a multifunctional microplate reader. 450nm , and calculate the survival rate of BV2 microglia: cell survival rate / % = (OD 药物处理组 / OD 对照组 )×100.

[0091] Table 1 Effects of compounds (1-8) on the survival rate of BV-2 cells

[0092]

[0093]

[0094] * P<0.05, **P<0.01 vs LPS model group

[0095] The above experimental results show that the synthesized isoquercetin ester compounds 1-8 have a high activity at a concentration of 10 μmol·L -1 , 50 μmol·L -1 The survival rate of BV-2 cells induced by LPS was effectively increased at 50 μmol·L -1 Judging from the cell survival rate, compounds 1, 2, and 3 may have stronger ability to inhibit neuroinflammation.

[0096] Example 10: Antagonism of Anti-neuroinflammatory Activity by Compounds 1-8

[0097] A neuroinflammatory cell model was established by incubating BV2 microglia with LPS, and the effects of the target compounds on the production and release of nitric oxide (NO) in this cell model were studied.

[0098] BV2 cells in the logarithmic growth phase were cultured at 3 × 10 5 The density of each well was inoculated into a 96-well culture plate, 100 μL per well, and the culture plate was placed in an incubator at 37° C. and 5% CO 2 .

[0099] Table 2 Effects of compounds (1-8) on LPS-induced NO production in BV-2 cells

[0100]

[0101] * P<0.05, **P<0.01 vs LPS model group

[0102] The experimental groups were divided into normal control group, model group and sample group. After 24h of cell culture, 10μL of corresponding solvent was added to the normal control group, and 10μL of sample was added to the sample group. Each sample had 3 concentrations, namely 0.1, 1, and 10μmol·L -1 After culturing in an incubator for 24 h, the culture medium was discarded and 90 μL of fresh culture medium was added to each well. 10 μL of corresponding solvent was added to the normal control group, and LPS (1 μg mL -1 ) were incubated for 24 h, and the supernatant was collected. Nitrite, the stable metabolite of NO, was used as an indicator for NO detection and was determined using a Griess kit.

[0103] The experimental results in Table 2 show that the synthesized isoquercetin ester compounds 1-8 have a high activity at a concentration of 50 μmol·L -1 All of them can effectively reduce LPS-induced excessive microglia production and the release of inflammatory mediator NO, and have an inhibitory effect on activated microglia. The results suggest that compounds 1, 2, 3, 6, and 8 may have better potential to inhibit neuroinflammation.

[0104] Example 11: Study on the blood-brain barrier permeability of compounds (1, 2, 3)

[0105] The experimental process is as follows: 72 Kunming mice, half male and half female, with a body weight range of 20±2g, were randomly divided into isoquercetin and ibuprofen control groups, compound (1) administration groups, compound (2) administration groups, and compound (3) administration groups. The mice were respectively injected into the tail vein at a dose of 0.032mmol / kg (equimolar doses for the control group and the experimental group). After administration, one group was taken from each group at 10min, 30min, 90min, 180min, and 480min, and plasma and brain tissue were obtained. The brain tissue was weighed, and 3 times the weight of physiological saline was added to make a homogenate, which was frozen at -80℃ for later use.

[0106] Accurately aspirate 200 μL of plasma or brain homogenate, add 20 μL of internal standard solution, vortex mix, add 1 mL of methanol and vortex mix for 5 min for extraction, centrifuge at 4000 rpm for 10 min, take 1 μL of supernatant and inject it into HPLC-MS, record the chromatogram and the peak areas of compound 1 (2, 3), isoquercetin (As) and internal standard (Ai), and calculate the drug content in plasma and brain homogenate according to the linear equation.

[0107] The blood-brain barrier permeability of a drug is expressed as logBB. The definition of logBB is: the logarithmic value of the ratio of the drug concentration in the brain (ng / g) to the blood concentration (ng / mL) when the drug distribution reaches a steady state. The mathematical expression is logBB = log(C brain / C blood ).

[0108] The research results of compound 1 are shown in Table 3 below.

[0109] Table 3 Blood-brain barrier permeability study of compounds (1-3)

[0110]

[0111] After mice were injected with isoquercetin via the tail vein, the drug concentration detected in the brain homogenate was low, presumably because isoquercetin had difficulty passing through the blood-brain barrier. Within 30-180 minutes after administration, its logBB was in a steady state, with an average value of -4.00; while for compounds 1, 2, and 3, within 30-180 minutes or 30-480 minutes after administration, the logBB was in a relatively steady state, with average values ​​of -1.20, -1.11, and -1.50, respectively.

[0112] From the results in Table 3, it can be seen that after isoquercetin was transformed into ester derivatives, its ability to penetrate the blood-brain barrier was greatly improved, and the steady-state logBB of compounds 1, 2, and 3 was also improved to a certain extent compared with ibuprofen. Compounds 1, 2, and 3 were rapidly transported from the blood to the brain, and the brain drug concentration could be maintained for a long time (the BB value of 180min or 480min was relatively stable), which is speculated to have good blood-brain barrier penetration ability.

[0113] Example 12: Effects on movement disorders, dopamine content and related inflammatory factors in Parkinson's rats

[0114] After 7 days of adaptive feeding, C57BL / 6 mice were subjected to Parkinson's neuroinflammation modeling and pre-protection experiments with compounds 1 and 2, i.e., 50 mg / kg of 1-methyl-4-phenyl-1,2,3.6-tetrahydropyridine (MPTP) was intraperitoneally injected for 7 days (0-7 days), and MPTP injection was simultaneously administered by gavage for two weeks (0-14 days), once a day; behavioral tests were performed on the 7th and 14th days after administration, respectively, and mice were killed after the last administration and fasting for 12 hours, and blood and brain samples were collected.

[0115] The medication of each group is as follows:

[0116] (1) Blank group: injected with normal saline for 7 days and gavaged with CMC-Na aqueous solution for 7 days;

[0117] (2) Model group: MPTP injection for 7 days, followed by intragastric administration of CMC-Na aqueous solution for 7 days;

[0118] (3) Positive group: MPTP injection for 7 days, followed by oral administration of levodopa solution (3 mg / kg) dissolved in CMC-Na for 14 days;

[0119] (4) Compound (1) administration group: MPTP injection model was established for 7 days, and compound 1 (15 mg / kg) was gavaged for 14 days.

[0120] (5) Compound (2) administration group: MPTP injection model was established for 7 days, and compound 2 (15 mg / kg) was gavaged for 14 days.

[0121] There are three behavioral test items:

[0122] The pole climbing test was used to evaluate the slow movement state of mice caused by MPTP. A stopwatch was used to time the time (s) for the mice to climb to the bottom of the pole. Three parallel experiments were performed on each mouse, with a 10-min interval between each experiment to allow the mice to fully recover their strength.

[0123] The hanging test can detect the grip strength, coordination of movements and the effect of MPTP on the motor function of mice. The reaction of mice after hanging is recorded and scored according to their performance. The scoring criteria are as follows: 3 points for stable hanging and moving to the platform at either end within 1 minute, 2 points for a long stable hanging time but unable to move to the platform, 1 point for a short hanging time and unable to crawl on the wire, and 0 points for neither hanging nor moving. Three mice were selected from each group for testing, and three parallel experiments were conducted on each mouse, with a 10-minute interval to allow the mice to fully recover their strength.

[0124] The swimming test is used to evaluate the coordination of the mice's limbs. The scoring criteria are: 3.0 points for continuous swimming for 60 seconds, 2.5 points for swimming between 30-60 seconds, 2.0 points for swimming between 10-30 seconds, 1.5 points for occasional swimming but with limb movement, 1.0 points for occasional swimming but with a tendency to move the hind limbs and always floating, and 0 points for floating on the water surface without limb movement. Three mice were selected from each group for testing, and three parallel experiments were performed on each mouse.

[0125] Table 4 Effects of drug administration on behavioral indicators of Parkinson's neuroinflammation mice

[0126]

[0127] *P<0.05, **P<0.01 vs. LPS model group

[0128] The mice are in a state of dynamic equilibrium. The recovery status of PD mice can be judged by detecting the dopamine (DA) content in the serum. The content of inflammatory factors such as inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6) in the mouse serum can be measured by Elisa kits to infer the effect of oral administration on PD neuroinflammation.

[0129] Table 4 lists the effects of compound 1 and 2 administration on behavioral indicators of PD neuroinflammatory mice; Table 5 lists the effects of compound 1 and 2 administration on dopamine and inflammatory factor contents in PD neuroinflammatory mice.

[0130] Table 5 Effects of drug administration on dopamine and inflammatory factor indicators in PD neuroinflammatory mice

[0131]

[0132] *P<0.05, **P<0.01 vs. model group

[0133] As shown in Tables 4 and 5, the behavior of mice in each group was significantly improved after administration, the levels of four inflammatory factors, iNOS, COX-2, TNF-α, and IL-6 in their serum were significantly reduced, and the dopamine content was greatly increased. The improvement effect was similar to or slightly better than that of the positive drug dopamine.

[0134] Example 13: Effects on learning and memory ability and inflammatory factors in Alzheimer's (AD) neuroinflammatory mice

[0135] The AD neuroinflammation mouse model was established by intraperitoneal injection of lipopolysaccharide (LPS, 250 μg / kg), and the therapeutic effect of compound 2 (isoquercetin lipoate) on the impairment of learning and memory function in AD mice was studied.

[0136] Male ICR mice trained in the eight-arm maze for 7 days were randomly divided into 6 groups, namely, a blank group, a model group (250 μg / kg LPS), a positive drug donepezil group (2 mg·kg-1·d-1); low-, medium-, and high-dose groups of compound (2) were given drugs at doses of 2 mg·kg-1·d-1, 5 mg·kg-1·d-1, and 10 mg·kg-1·d-1, respectively; the blank group was only given an equal amount of solvent. The mice were intraperitoneally injected once a day for 14 consecutive days.

[0137] According to the pre-training method, the eight-arm maze test was started at a fixed time before administration on the 14th to 21st day. The eight-arm maze test was started on the 14th day, with training for the first 5 days and testing for the last 2 days. The working memory error (WME) of the rat re-entering the baited arm was recorded in each experiment, and entering the unbaited arm was called the referent memory error (RME). The WME and RME of each rat in each experiment were recorded within 10 minutes. After the eight-arm maze test, the mice were dislocated and killed, and the brain tissue was dissected and taken. The hippocampus of one side of the brain was prepared into a 10% homogenate, and the supernatant was taken after centrifugation at 4°C. ELISA was used to detect the content of TNF-α, IL-1β and ROS in the hippocampus of the brain.

[0138] Table 6 lists the behavioral test results of AD mice, and Table 7 lists the experimental results of inflammatory factor levels in mouse hippocampal tissue.

[0139] Table 6 Results of behavioral test of compound 2 on rats with Alzheimer's disease

[0140]

[0141] *P<0.05vs model group

[0142] Table 7 Effects of compound 2 on ACh content and AChE activity in the cerebral cortex of AD mice

[0143]

[0144] *P<0.05vs model group

[0145] The results in Tables 6 and 7 show that compared with the AD model group, compound 2 can effectively reduce the number of behavioral errors in mice and inhibit the release of inflammatory factors to a certain extent.

[0146] Example 14: Acute toxicity test of compounds (1, 2)

[0147] Experimental animals: Kunming mice, weighing 18-22 g, half male and half female, were used for preliminary and formal experiments after 3 days of adaptive feeding.

[0148] Drug preparation: Compounds (1, 2) are dissolved in a mixed solvent, and the maximum solubility reaches 100-120 mg / mL.

[0149] Experimental method: Compounds (1, 2) (500, 350, 245, 171.5, 120.1, 84.1, 58.9 mg / kg) were injected into the tail vein of mice respectively. The injection volume was calculated as 0.1 mL / 20 g body weight. The drugs were administered by single tail vein injection of equal volume and different concentrations. The appearance, mental state, behavior, food intake and other toxic reactions and death distribution of the animals were observed and recorded every day for 14 consecutive days.

[0150] Experimental results: The median lethal dose (LD) was calculated using the Bliss method. 50 ) and its 95% confidence limit, LD of compound 1 injected into the tail vein of mice 50 The LD of compound 2 was 221.6 mg / kg, with a 95% confidence interval of (166.5-315.22) mg / kg. 50 It is 208.94 mg / kg, and the 95% confidence interval is (147.43-324.12) mg / kg, which indicates that the toxicity of compounds 1 and 2 is relatively small and their clinical use is relatively safe.

Claims

1. An isoquercetin ester dimer, characterized in that Its structure is shown in the following formula (I): ； The R group is selected from the following structures: 。 2. A method for preparing the isoquercetin ester dimer according to claim 1, characterized in that The steps include: Step 1: Preparation of vinyl carboxylate PdCl2, LiOAc and CuBr2 are added to a solution of vinyl acetate and tetrahydrofuran, and a carboxylic acid anti-inflammatory drug RCOOH is added under stirring, and the mixture is stirred at 70-75°C for 20-24 hours. After the reaction is completed, the mixture is filtered and washed with ethyl acetate, and concentrated under reduced pressure. The crude product is separated and purified by silica gel column chromatography to obtain a vinyl carboxylate intermediate, whose structural formula is: ； Step 2: Esterification reaction of isoquercetin with vinyl carboxylate Isoquercetin and the vinyl carboxylate obtained in step 1 are added to an anhydrous reaction solvent, and immobilized lipase and dry 4Å molecular sieves are added, and the reaction is carried out under inert gas sealed stirring. After the reaction is continued for 3-8 days, the immobilized enzyme and molecular sieves are removed by filtration, and the filtrate is concentrated under reduced pressure to obtain a crude product, which is separated and purified by silica gel column chromatography to obtain the target product (I).

3. The preparation method according to claim 2, characterized in that: In step 1, the molar ratio of PdCl2, LiOAc, CuBr2, vinyl acetate and carboxylic acid anti-inflammatory drug is 0.004-0.006:0.4-0.6:0.01-0.02:8-12:

1.

4. The preparation method according to claim 2, characterized in that: In step 2, the molar ratio of isoquercetin to the vinyl carboxylate obtained in step 1 is 1:4-6.

5. The preparation method according to claim 2, characterized in that: In step 2, the amount of immobilized lipase added is 50-120% of the mass of isoquercetin, and the amount of dry 4Å molecular sieve added is 40-60% of the mass of isoquercetin.

6. The preparation method according to claim 5, characterized in that: In step 1, the carboxylic acid anti-inflammatory drug is ibuprofen, lipoic acid, aspirin or naproxen; in step 2, the anhydrous reaction solvent is anhydrous acetone, the immobilized lipase is Novozym 435, and the reaction temperature is 40-45°C.

7. The preparation method according to claim 5, characterized in that: In step 1, the carboxylic acid anti-inflammatory drug is indomethacin, diclofenac, mefenamic acid or sulindac; in step 2, the anhydrous reaction solvent is anhydrous acetonitrile, the immobilized lipase is Lipozyme RM IM, and the reaction temperature is 65-70°C.

8. A use of the isoquercetin ester dimer according to claim 1, characterized in that: The isoquercetin dimer is used to prepare a pharmaceutical preparation, which can effectively alleviate neuroinflammation and can be used for the prevention and treatment of neurodegenerative diseases.

9. The use according to claim 8, characterized in that: The neurodegenerative disease is Parkinson's disease, Alzheimer's disease or vascular dementia.

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