A coumarin derivative and its application

Abstract

The present invention discloses a coumarin derivative having a structure shown in general formula (I) and its application. The solubility of the coumarin derivative with this structure in water is significantly improved, which solves the problem of low solubility of existing coumarin compounds. Studies have shown that the coumarin derivative of the present invention exhibits strong anti-inflammatory activity in inhibiting inflammatory factors and promoting the secretion of anti-inflammatory factors, and can be used to prepare drugs for preventing and treating central nervous system inflammation or neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to the field of chemical medicine, and in particular to a coumarin derivative and application thereof. Background Art

[0002] With the worsening of population aging, environmental pollution and mental stress, the incidence of major brain diseases such as stroke, Alzheimer's disease (AD), Parkinson's disease (PD), depression and schizophrenia has continued to rise, becoming a major problem that seriously endangers human health and has received great attention. At present, there are many types of drugs used clinically to combat these diseases, but their application is limited by problems such as poor efficacy or serious adverse reactions. Therefore, it is of great significance to develop drugs that effectively treat major brain diseases.

[0003] Coumarin is a lactone compound widely found in nature. Studies have found that many coumarin derivatives (esculetin, daphnetin, osthole, etc.) have neuroprotective effects. For example, 7,8-dihydroxy-4-methylcoumarin (Dhmc) has a significant inhibitory effect on glutamate-induced PC12 cell damage and can improve cell survival. Studies at the cellular level have shown that the protective effect of osthole on Parkinson's disease is related to its ability to regulate mitochondrial permeability and inhibit oxidative stress. For example, osthole can prevent the loss of membrane potential (MMP), reduce GSH depletion, promote the activation of SOD and CAT, and also reduce the Bax / Bcl-2 ratio, reduce the release of cytochrome C, and inhibit the caspase-3 apoptosis pathway. Studies have shown that osthole can also delay the occurrence and development of Alzheimer's disease, reduce ultrastructural damage to hippocampal neurons in model rats, and improve learning and memory disorders. Osthole has a protective effect on memory impairment in AlCl3-induced Alzheimer's disease model mice. The protective mechanism may be through enhancing the activity of antioxidant enzymes glutathione peroxidase (GSH-PX) and SOD to eliminate the damage of oxygen free radicals to central nervous system neurons.

[0004] The water solubility of coumarin derivatives is generally low, which limits their wide application in the fields of biomedicine and medicine. Typical coumarin derivatives such as coumarin have a solubility of 0.17 mg / mL in water, while 7-hydroxycoumarin has a slightly higher solubility in water of about 0.8 mg / mL (25°C). Other derivatives, such as 4-methylcoumarin and 6,7-dimethoxycoumarin, have solubilities of 0.12 mg / mL and 0.05 mg / mL, respectively, indicating that methyl and dimethoxy substitutions further reduce water solubility. Coumarin-6 is almost insoluble in water, with a solubility of only about 0.002 mg / mL. Therefore, structural modification and transformation of coumarin derivatives and screening of their efficacy are of great significance for the study or treatment of major brain diseases. Summary of the invention

[0005] In order to solve the above technical problems, the object of the present invention is to provide a coumarin derivative and its use in the preparation of a drug for preventing and treating central neuritis or neurodegenerative diseases.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] In the first aspect, the present invention provides a coumarin derivative having a structure represented by the general formula (I),

[0008]

[0009] wherein R1 is selected from -OH, -OCH3 or -(CH3)2C=CH-CH2;

[0010] X is selected from -O, -S or -NH;

[0011] Y is selected from Cl, Br or I;

[0012] n represents 1-4.

[0013] Preferably, the coumarin derivatives are selected from:

[0014]

[0015] N,N,N-Trimethyl-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethylamine hydrobromide;

[0016]

[0017] N,N,N-Trimethyl-2-(2H-chromen-2-one)oxy)ethylamine hydrobromide;

[0018]

[0019] N,N,N-Trimethyl-3-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)propylamine hydrobromide;

[0020]

[0021] N,N,N-Trimethyl-3-((-2H-chromen-2-one)oxy)propylamine hydrobromide;

[0022]

[0023] N,N,N-Trimethyl-3-((-2H-chromen-2-one)oxy)propylamine hydroiodide;

[0024]

[0025] N,N,N-Trimethyl-4-((-2H-chromen-2-one)oxy)butylamine hydroiodide;

[0026]

[0027] N,N,N-Trimethyl-3-((8-(isopentenyl)-(2H-chromen-2-one)oxy)propylamine hydrobromide;

[0028]

[0029] 6-Methoxy-N,N,N-trimethyl-2-(2H-chromen-2-one)oxy)ethylamine hydrobromide.

[0030] In a second aspect, the present invention also provides the use of the above-mentioned coumarin derivatives in the preparation of drugs for preventing and treating central nervous system inflammation or neurodegenerative diseases. Wherein, the central nervous system inflammation includes meningitis, encephalitis and myelitis. The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

[0031] In a third aspect, the present invention also provides a pharmaceutical composition comprising an effective amount of the above-mentioned coumarin derivatives and a pharmaceutically acceptable carrier.

[0032] The pharmaceutically acceptable carrier of the present invention refers to diluents, preservatives, fillers, flow regulators, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, aromatics, antibacterial agents, antifungal agents, lubricants and dispersants generally accepted in the art, which specifically depends on the nature of the administration method and the dosage form.

[0033] The pharmaceutical compositions of the present invention can be administered in oral dosage forms such as tablets, capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions. They can be administered intravenously (bolus or infusion), intraperitoneally, subcutaneously, or intramuscularly, all of which are well known to those skilled in the art of pharmacy.

[0034] Compared with the prior art, the present invention has the following excellent effects:

[0035] The present invention provides a coumarin derivative having a structure shown in general formula (I) by structural modification and transformation of coumarin and choline model compounds. The solubility of the coumarin derivative with this structure in water is significantly improved, thus solving the problem of low solubility of existing coumarin compounds.

[0036] The present invention constructs a screening model for abnormal activation of BV2 and PC12 cells activated by LPS in vitro, detects the gene expression levels of inflammatory factors TNF-α, IL-6, IL-1β and IL-10 by qRT-PCR, and detects the inflammation level in the cell supernatant by ELISA. The results show that the coumarin derivatives of the present invention exhibit strong anti-inflammatory activity in inhibiting inflammatory factors and promoting the secretion of anti-inflammatory factors, and can be used as drugs for preventing and treating central nervous system inflammation or neurodegenerative diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the ESI-MS (m / Z) of N,N,N-trimethyl-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethanamine hydrobromide;

[0038] Figure 2 N,N,N-trimethyl-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethylamine hydrobromide 1 H-NMR;

[0039] Figure 3 N,N,N-trimethyl-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethylamine hydrobromide 13 C-NMR;

[0040] Figure 4 is the ESI-MS (m / Z) of N,N,N-trimethyl-2-(2H-chromen-2-one)oxy)ethylamine hydrobromide;

[0041] Figure 5 is the ESI-MS (m / Z) of N,N,N-trimethyl-3-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)propylamine hydrobromide;

[0042] Figure 6 is the ESI-MS (m / Z) of N,N,N-trimethyl-3-((-2H-chromen-2-one)oxy)propylamine hydrobromide;

[0043] Figure 7 The effects of different compounds on the proliferation activity of BV2 and PC12 cells;

[0044] Figure 8 ELISA was used to detect the secretion of inflammatory factors in the supernatant of PC12 cells;

[0045] Fig. 9 ELISA was used to detect the secretion of inflammatory factors in the supernatant of BV2 cells;

[0046] Fig.10 qRT-PCR was used to detect the expression levels of inflammatory genes in PC12 cells;

[0047] Fig.11 qRT-PCR was used to detect the expression level of inflammatory genes in BV2 cells. DETAILED DESCRIPTION

[0048] The present invention is further described below by means of specific implementation methods. The following examples are specific implementation methods of the present invention, but the implementation methods of the present invention are not limited to the following examples.

[0049] Embodiment 1:

[0050] Synthesis route:

[0051]

[0052] Synthesis steps:

[0053] Synthesis of 7-(2-(dimethylamino)ethoxy)quinolin-2(1H)-one

[0054] 7-Hydroxy-1H-quinolin-2-one (0.8 g, 5 mmol), 2-chloro-N,N-dimethylethylamine hydrochloride (0.7 g, 5 mmol), 1.7 g anhydrous potassium carbonate and 20 ml acetone were added into a flask and refluxed for 5 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was washed with acetone. The filtrates were combined and the acetone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 0.7 g of white solid with a yield of 61.2%.

[0055] ESI-MS (m / Z): C 13 H 16 N2O2,232.12,[M + H] + =233.0;

[0056] 1 H-NMR (400MHz, CD3OD), δ: 7.77-7.79 (d, J=8.8Hz, 1H), 7.47-7.49 (d, J=8.8Hz, 1H), 6.77-6.83 (m, 2H ),6.33-6.36(d,J=8.8Hz,1H),4.16-4.13(t,J=5.6Hz,2H),2.89-2.92(t,J=5.6Hz,2H),2.41(s,6H);

[0057] 13 C-NMR (100MHz, DMSO-d6), δ: 164.27, 160.87, 141.23, 140.06, 129.26, 117.86, 114.56, 112.14, 98.49, 64.77, 57.18, 44.02.

[0058] N,N,N-Trimethyl-2-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)ethylamine hydrobromide (Compound 4)

[0059] 7-(2-(Dimethylamino)ethyl)quinolin-2(1H)-one (0.2 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 10 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was monitored by TLC. After the reaction was completed, the filter cake was filtered and recrystallized from methanol-ethyl acetate to obtain 0.25 g of an off-white solid.

[0060] The results are as follows Figure 1-3 :

[0061] ESI-MS (m / Z): C 14 H 19 N2O2 + ,247.14,[M] + =247.2;

[0062] 1 H-NMR (400MHz, CD3OD), δ: 7.78-7.80 (d, J=8.8Hz, 1H), 7.51-7.53 (d, J=8.8Hz, 1H), 6.83-6.87 (m, 2H ),6.35-6.37(d,J=8.8Hz,1H),4.50-4.51(t,J=5.6Hz,2H),3.83-3.85(t,J=5.6Hz,2H),3.22(s,9H);

[0063] 13 C-NMR (100MHz, DMSO-d6), δ: 164.16, 159.69, 141.14, 139.99, 129.49, 117.73, 115.00, 111.84, 98.92, 64.98, 62.06, 53.38, 53.35, 53.31.

[0064] Embodiment 2:

[0065] Synthesis route:

[0066]

[0067] Synthesis steps:

[0068] Synthesis of 7-(2-(dimethylamino)ethoxy)-2H-chromen-2-one

[0069] 7-Hydroxycoumarin (0.8 g, 5 mmol), 2-chloro-N,N-dimethylethylamine hydrochloride (0.7 g, 5 mmol), 1.8 g anhydrous potassium carbonate and 30 ml acetone were added into a flask and refluxed for 6 h. The degree of reaction was detected by TLC. After the reaction was completed, the filter was filtered and the filter cake was washed with acetone. The filtrates were combined and the acetone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 0.8 g of a white solid.

[0070] ESI-MS (m / Z): C 13 H 15 NO3,233.11,[M+H] + =234.1.

[0071] 1 H-NMR (400MHz, CD3OD), δ: 7.79-7.81 (d, J=8.0Hz, 1H), 7.45-7.47 (d, J=8.0Hz, 1H), 6.79-6.81 (m, 2H ),6.25-6.27(d,J=8.0Hz,1H),4.15-4.12(t,J=5.6Hz,2H),2.91-2.94(t,J=5.6Hz,2H),2.45(s,6H);

[0072] 13 C-NMR (100MHz, CD3OD), δ: 165.13, 160.56, 140.53, 140.01, 129.35, 119.98, 114.87112.06, 98.41, 64.57, 57.61, 44.22.

[0073] N,N,N-Trimethyl-2-(2H-chromen-2-one)oxy)ethylamine hydrobromide (Compound 2)

[0074] 7-(2-(Dimethylamino)ethoxy)-2H-chromen-2-one (0.2 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 20 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was recrystallized from methanol-ethyl acetate to obtain 0.24 g of an off-white solid.

[0075] like Figure 4 As shown, ESI-MS (m / z): C 14 H 18 NO3 + ,248.13;[M] + =248.3.

[0076] 1H-NMR (400MHz, CD3OD), δ: 7.76-7.78 (d, J=8.0Hz, 1H), 7.52-7.54 (d, J=8.0Hz, 1H), 6.84-6.89 (m, 2H ),6.34-6.36(d,J=8.0Hz,1H),4.49-4.51(t,J=5.6Hz,2H),3.85-3.88(t,J=5.6Hz,2H),3.24(s,9H);

[0077] 13 C-NMR (100MHz, CD3OD), δ: 165.21, 160.01, 141.25, 140.03, 129.51, 117.79, 115.03, 111.79, 98.89, 64.99, 62.12, 53.41, 53.39, 53.37.

[0078] Embodiment 3:

[0079] Synthesis route:

[0080]

[0081] Synthesis steps:

[0082] Synthesis of 7-(2-(dimethylamino)propoxy)quinolin-2(1H)-one

[0083] 7-Hydroxy-1H-quinolin-2-one (0.8 g, 5 mmol), 3-chloro-N,N-dimethylpropylamine hydrochloride (0.8 g, 5 mmol), 1.7 g anhydrous potassium carbonate and 25 ml butanone were added into a flask and refluxed for 6 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was washed with butanone. The filtrates were combined and the butanone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 0.9 g of a white solid.

[0084] ESI-MS (m / Z): C 14 H 18 N2O2 + ,246.14;[M] + =247.2.

[0085] 1H-NMR (400MHz, DMSO-d6), δ: 7.78-7.80 (d, J=8.8Hz, 1H), 7.46-7.48 (d, J=8.8Hz, 1H), 6.76-6.79 (m, 2 H),6.26-6.28(d,J=8.8Hz,1H),4.05-4.17m,2H),2.49(s,6H),2.25-2.29(m,2H),1.76-1.79(m,2H);

[0086] 13 C-NMR (100MHz, DMSO-d6) δ: 165.39, 160.89, 140.89, 140.11, 129.38, 119.99, 114.91112.11, 99.42, 74.57, 57.69, 44.29, 27.15.

[0087] N,N,N-Trimethyl-3-((2-oxo-1,2-dihydroquinolin-7-yl)oxy)propylamine hydrobromide (Compound 3)

[0088] 7-(3-(Dimethylamino)propoxy)-2H-chromen-2-one (0.25 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 20 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was detected by TLC. After the reaction was completed, the filter cake was filtered off and recrystallized from methanol-ethyl acetate to obtain 0.15 g of an off-white solid.

[0089] like Figure 5 As shown, ESI-MS (m / Z): C 15 H 21 N2O2 + ,261.16,[M] + =261.1.

[0090] 1 H-NMR (400MHz, CD3OD), δ: 7.78-7.80 (d, J=8.0Hz, 1H), 7.53-7.55 (d, J=8.0Hz, 1H), 6.86-6.88 (m, 1H), 6.81-6 .83(m,1H),6.31-6.33(d,J=8.0Hz,1H),4.11-4.15(m,2H),3.26(s,9H),2.31-2.36(m,2H),1.93-1.96(m,2H);

[0091] 13C-NMR (100MHz, CD3OD) δ: 165.39, 160.18, 141.31, 139.93, 129.59, 117.81, 115.15, 111.49, 98.99, 74.89, 57.49, 53.51, 53.49, 53.47, 27.16.

[0092] Embodiment 4:

[0093] Synthesis route:

[0094]

[0095] Synthesis steps:

[0096] Synthesis of 7-(3-(dimethylamino)propoxy)-2H-chromen-2-one

[0097] 7-Hydroxycoumarin (0.8 g, 5 mmol), 3-chloro-N,N-dimethylpropylamine hydrochloride (0.8 g, 5 mmol), 1.9 g anhydrous potassium carbonate and 30 ml acetone were added into a flask and refluxed for 6 h. The degree of reaction was detected by TLC. After the reaction was completed, the filter was filtered and the filter cake was washed with acetone. The filtrates were combined and the acetone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 0.7 g of a white solid.

[0098] ESI-MS (m / Z): C 14 H 17 NO3,247.12,[M+H] + =248.5.

[0099] 1 H-NMR (400MHz, DMSO-d6), δ: 7.83-7.85 (d, J=8.8Hz, 1H), 7.56-7.58 (d, J=8.8Hz, 1H), 6.79-6.83 (m, 2 H),6.46-6.48(d,J=8.0Hz,1H),4.09-4.19m,2H),2.51(s,6H),2.27-2.31(m,2H),1.77-1.81(m,2H);

[0100] 13 C-NMR (100MHz, DMSO-d6) δ: 166.19, 160.79, 140.79, 140.23, 129.33, 119.90, 114.92112.18, 99.53, 74.86, 57.35, 44.56, 27.63.

[0101] N,N,N-Trimethyl-3-((-2H-chromen-2-one)oxy)propylamine hydrobromide (Compound 1)

[0102] 7-(3-(Dimethylamino)propoxy)-2H-chromen-2-one (0.25 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 20 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was detected by TLC. After the reaction was completed, the solvent was recovered under reduced pressure and recrystallized from methanol-ethyl acetate to obtain 0.19 g of an off-white solid.

[0103] like Figure 6 As shown, ESI-MS (m / Z): C 15 H 20 NO3 + ,262.14,[M] + =262.0.

[0104] 1 H-NMR (400MHz, CD3OD), δ: 7.84-7.86 (d, J=8.6Hz, 1H), 7.74-7.76 (d, J=8.0Hz, 1H), 6.98-7.00 (m, 1H), 6.91-6 .93(m,1H),6.51-6.53(d,J=8.0Hz,1H),4.13-4.15(m,2H),3.27(s,9H),2.35-2.38(m,2H),1.95-1.98(m,2H);

[0105] 13 C-NMR (100MHz, CD3OD) δ: 164.99, 160.83, 141.42, 139.97, 129.53, 117.91, 115.25, 111.53, 98.98, 74.93, 57.49, 53.51, 53.49, 53.47, 27.23.

[0106] Embodiment 5:

[0107] Synthesis route:

[0108]

[0109] Synthesis steps:

[0110] Synthesis of 7-(3-(dimethylamino)propoxy)-8-(isopentenyl)-2H-chromen-2-one

[0111] 7-Hydroxy-8-(isopentenyl)-coumarin (1.15 g, 5 mmol), 3-chloro-N,N-dimethylpropylamine hydrochloride (0.8 g, 5 mmol), 2.1 g of anhydrous potassium carbonate and 30 ml of acetone were added into a flask and refluxed for 6 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was washed with acetone. The filtrates were combined and the acetone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 1.1 g of a white solid.

[0112] ESI-MS (m / Z): C 19 H 25 NO3, 315.18, [M+H]+ = 316.1;

[0113] 1 H-NMR(400MHz, DMSO-d6),7.80(d,J=8.0Hz,1H),7.44(dd,J=8.0,4.0Hz,1H),6.68(d,J=2.0Hz,1H),6.19(dd,J=17.6,1 0.9Hz,1H),5.28(d,J=17.7Hz,1H),4.06(m,2H),3.54,(m,2H),2.35(m,2H),2.27(s,6H),1.89(m,2H),1.56(s,6H)ppm;

[0114] 13 C-NMR(100MHz,CD3OD)δ:161.66,160.30,152.99,144.01,132.85,126.40,121 .28,118.12,115.3,113.14,107.53,74.86,57.35,44.56,27.63,22.10,18.12.

[0115] N,N,N-Trimethyl-3-((8-(isopentenyl)-(2H-chromen-2-one)oxy)propylamine hydrobromide (Compound 5)

[0116] 7-(3-(Dimethylamino)propoxy)-2H-chromen-2-one (0.3 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 20 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was detected by TLC. After the reaction was completed, the solvent was recovered under reduced pressure and recrystallized from methanol-ethyl acetate to obtain 0.3 g of an off-white solid.

[0117] ESI-MS (m / Z): C 20 H 28 NO3+, 330.21, [M] + =330.1;

[0118] 1 H-NMR(400MHz, CD3OD),7.80-7.82(d,J=8.0Hz,1H),7.44-7.46(dd,J=8.0,4.0Hz,1H),6.68-6.70(d,J=2.0Hz,1H),6.19-6.23(dd ,J=17.6,10.9Hz,1H),5.26(d,J=17.7Hz,1H),4.08(m,2H),3.52,(m,2H),2.37(m,2H),3.27(s,9H),1.89(m,2H),1.59(s,6H)ppm;

[0119] 13 C-NMR(100MHz,CD3OD)δ:162.36,161.30,151.89,144.11,132.65,126.41,121.56,118 .42,115.32,113.15,107.78,74.89,57.35,53.23,53.21,53.19,27.66,22.13,18.19.

[0120] Embodiment 6:

[0121] Synthesis route:

[0122]

[0123] Synthesis steps:

[0124] Synthesis of 6-methoxy-7-(2-(dimethylamino)ethoxy)-2H-chromen-2-one

[0125] 6-Methoxy-7-hydroxy-coumarin (0.96 g, 5 mmol), 2-chloro-N,N-dimethylethylamine hydrochloride (0.7 g, 5 mmol), 3 g of anhydrous potassium carbonate and 30 ml of acetone were added into a flask and refluxed for 6 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was washed with acetone. The filtrates were combined and the acetone was recovered under reduced pressure. The target product was separated by column chromatography to obtain 0.8 g of a white solid.

[0126] ESI-MS (m / Z): C 14 H 17 NO4,263.11,[M+H] + =264.1.

[0127] 1H-NMR (400MHz, DMSO), δ: 7.81-7.83 (d, J = 8.0 Hz, 1H), 7.46 (s, 1H), 6.32 (s, 1H), 6.24-6.27 (d, J =8.0Hz,1H),4.15-4.17(t,J=5.6Hz,2H),3.81(s,3H),2.93-2.96(t,J=5.6Hz,2H),2.45(s,6H);

[0128] 13 C-NMR (100MHz, DMSO), δ: 165.13, 158.56, 140.36, 140.12, 139.89, 129.42, 114.85112.42, 98.99, 64.89, 57.42, 56.13, 44.45.

[0129] 6-Methoxy-N,N,N-trimethyl-2-(2H-chromen-2-one)oxy)ethylamine hydrobromide (Compound 6)

[0130] 6-methoxy-7-(2-(dimethylamino)ethoxy)-2H-chromen-2-one (0.23 g, 1 mmol), methyl bromide (0.25 g, 3 mmol), and 20 ml of a 10:1 mixed solvent of dichloromethane and methanol were added to a round-bottom flask and stirred at room temperature for 24 h. The degree of reaction was detected by TLC. After the reaction was completed, the mixture was filtered and the filter cake was recrystallized from methanol-ethyl acetate to obtain 0.24 g of an off-white solid.

[0131] ESI-MS (m / z): C 15 H 20 NO4 + ,278.13;[M] + =278.3.

[0132] 1 H-NMR (400MHz, D2O), δ: 7.76-7.78 (d, J=8.0Hz, 1H), 7.52-7.54 (d, J=8.0Hz, 1H), 6.84-6.89 (m, 2H) ,6.34-6.36(d,J=8.0Hz,1H),4.49-4.51(t,J=5.6Hz,2H),3.85-3.88(t,J=5.6Hz,2H),3.24(s,9H);

[0133] 13C-NMR (100MHz, D2O), δ: 164.93, 159.32, 140.23, 140.01, 127.53, 116.94, 111.32, 99.89, 65.29, 61.12, 58.6, 53.40, 53.38, 53.36.

[0134] Example 7: Solubility Test

[0135] 200 mg of the compound prepared in Example 1-6 was added to a 5 mL glass vial, and then 5 mL of solvent (water, methanol, ethyl acetate) was added, and the vials were sealed. Two samples were prepared for each sample, and the solubility was determined at 20°C. Each sample vial was placed in a constant temperature magnetic stirrer, kept at a constant temperature of 20°C, and stirred for 24 hours. Then, a 2 mL syringe was used to take the upper layer of liquid, and after filtering through a filter membrane, 1 mL of the filtrate was taken by a pipette into a 25 mL volumetric flask and diluted to volume. Chromatographic conditions and system suitability test: Octadecylsilane bonded silica gel was used as a filler, and methanol-0.5% phosphoric acid solution (48:52) was used as the mobile phase; the detection wavelength was 323 nm; the results are shown in Table 1:

[0136] Table 1 Solubility test results of compounds 1-6 in water, methanol and ethyl acetate

[0137] Serial number Water (mg / ml) Methanol (mg / ml) Ethyl acetate (mg / ml) Compound 1 12.98 16.07 Obviously insoluble, not tested Compound 2 15.07 20.12 Obviously insoluble, not tested Compound 3 14.8 19.19 Obviously insoluble, not tested Compound 4 11.36 18.37 Obviously insoluble, not tested Compound 5 9.12 22.18 Obviously insoluble, not tested Compound 6 12.02 20.98 Obviously insoluble, not tested

[0138] From the results in Table 1, it can be seen that the solubility of the coumarin derivatives 1-6 prepared by the present invention in water is 9-15 mg / mL, which is significantly higher than that of existing coumarin derivatives, thus broadening their application in the fields of biomedicine and medicine.

[0139] Example 7: Biological Activity Test

[0140] 1. Experimental Materials

[0141] High-glucose DMEM (Gibco), fetal bovine serum (Anhui Kangyuan Biotechnology Co., Ltd.), penicillin-streptomycin (Shandong Sikejie Biotechnology Co., Ltd.), trypsin (Biyuntian), CCK8 (Shandong Sikejie Biotechnology Co., Ltd., enhanced).

[0142] 2. Cell Culture

[0143] PC12 and BV2 cells were cultured in high-glucose DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37°C and 5% CO2. The medium was changed every 2 days, and when the cell density reached 70-80%, the cells were digested with 0.25% trypsin and passaged at a ratio of 1:3 to maintain the optimal growth state of the cells.

[0144] 3. Preparation of concentration gradients of different compounds

[0145] Compound 1: 3.14 mg of compound 1 (molecular weight 341 g / mol) was weighed and dissolved in 50 μL of dimethyl sulfoxide (DMSO), and then 950 μL of DMEM medium was added to obtain a 1 mM stock solution of compound 1. Compound 1 solutions were gradiently diluted with DMEM to 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0146] Compound 2: 3.27 mg of compound 2 (molecular weight 327 g / mol) was weighed and dissolved in 50 μL of DMSO, and then 950 μL of DMEM medium was added to obtain a 1 mM stock solution of compound 2. Compound 2 solutions were graded diluted with DMEM to 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0147] Compound 3: 3.4 mg of compound 3 (molecular weight 340 g / mol) was weighed and dissolved in 50 μL of DMSO, followed by addition of 950 μL of DMEM medium to obtain a 1 mM stock solution of compound 3. Compound 3 solutions were gradiently diluted with DMEM to 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0148] Compound 4: 3.26 mg of compound 4 (molecular weight 326 g / mol) was weighed and dissolved in 50 μL of DMSO, and 950 μL of DMEM medium was added to obtain a 1 mM stock solution of compound 4. Compound 4 solutions were graded diluted with DMEM to 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM.

[0149] 4. Cytotoxicity

[0150] In this study, the CCK8 method was used to evaluate the effects of compound 1, compound 2, compound 3, compound 4, almotriptan, butylphthalide and 7-methoxycoumarin on the proliferation activity of BV2 and PC12 cells (almotriptan, butylphthalide and 7-methoxycoumarin were used as positive controls). 4 / mL, add 100μL of cell suspension to each well of 96-well plate, and incubate overnight in a 37°C, 5% CO2 incubator to allow cells to adhere to the wall. Discard the culture medium, and add the above compounds with different concentration gradients (6.25μM, 12.5μM, 25μM, 50μM, 100μM, 200μM) except for the normal group, and treat in the incubator for 24 hours. By measuring the absorbance value at a wavelength of 450nm, calculate the cell survival rate at each concentration to screen out the compound concentration that has no significant toxicity to the two cells.

[0151] 5.ELISA to detect the secretion of inflammatory factors

[0152] In order to evaluate the effects of compound 1, compound 2, compound 3, compound 4, almotriptan, butylphthalide, and 7-methoxycoumarin on the levels of inflammatory factors secreted by BV2 and PC12 cells under LPS stimulation, the concentration of inflammatory factors in the cell culture supernatant after treatment was detected by enzyme-linked immunosorbent assay (ELISA). In the experiment, the BV2 and PC12 cell suspensions were adjusted to 10^5 / mL, and 2mL of cell suspension was added to each well of a 6-well plate, and incubated overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. The culture medium was discarded and replaced with a culture medium containing different compounds for 24 hours. After 24 hours, after stimulating with 10μg / mL of LPS for 4h, the cell culture supernatant was collected and centrifuged to obtain the supernatant for ELISA detection. A commercial ELISA kit was used according to the manufacturer's instructions, and the concentration of inflammatory factors in the supernatant was quantitatively analyzed by the standard curve to evaluate the regulatory effect of different concentrations of compounds on the secretion of cellular inflammatory factors.

[0153] 6. qRT-PCR detection of the expression level of inflammation-related genes

[0154] In order to evaluate the effects of compound 1, compound 2, compound 3, compound 4, almotriptan, and 7-methoxy-butylphthalide on the gene expression of inflammatory factors in BV2 and PC12 cells stimulated by LPS, qRT-PCR technology was used to detect the expression level of inflammatory factors in cells after treatment. In the experiment, the suspension of BV2 and PC12 cells was adjusted to 105 / mL, and 2mL of cell suspension was added to each well of the 6-well plate, and incubated overnight in a 37°C, 5% CO2 incubator to allow the cells to adhere to the wall. The culture medium was discarded, and except for the normal group, the remaining groups were replaced with culture medium containing different compounds for 24 hours. After 24 hours, after stimulating with 10μg / mL of LPS for 4h, the cells were collected and total RNA was extracted and reverse transcribed into cDNA. Subsequently, real-time fluorescence quantitative PCR amplification was performed by specific primers (primer sequences are shown in Table 2), and the relative expression of the target gene relative to the reference gene was calculated by the ΔΔCt method, so as to analyze the regulatory effect of different compounds on the cellular inflammatory response.

[0155] Table 2 PCR primer sequences

[0156]

[0157]

[0158] Note: R stands for rat, M stands for mouse

[0159] 7. Experimental Results

[0160] The results of CCK8 experiments are as follows Figure 7 As shown, at a concentration of 200 μM, compound 1, compound 2, compound 3, almotriptan, butylphthalide and 7-methoxycoumarin all showed significant inhibitory effects on the proliferation of BV2 cells; at a concentration of 100 μM, compound 2 and almotriptan still had an inhibitory effect on the proliferation of BV2 cells. At a lower concentration (50 μM), all compounds did not show a significant inhibitory effect on the proliferation of BV2 cells. For PC12 cells, at a concentration of 200 μM, compound 3, almotriptan and 7-methoxycoumarin significantly inhibited cell proliferation, while at a concentration of 100 μM, only 7-methoxycoumarin showed an inhibitory effect. At a concentration of 50 μM, each compound had no significant inhibitory effect on the proliferation of PC12 cells. Based on the above results, 50 μM was selected as the dosing concentration for subsequent experiments to ensure that the concentration of the compound in BV2 and PC12 cells would not cause significant cytotoxicity, thereby more accurately evaluating its anti-inflammatory activity.

[0161] The effects of compound 1, compound 2, compound 3, compound 4, almotriptan, butylphthalide and 7-methoxycoumarin on the secretion of inflammatory factors in BV2 and PC12 cells stimulated by LPS were evaluated by ELISA. Figure 8-9It was shown that compound 1, compound 2, compound 3, compound 4, almotriptan, butylphthalide and 7-methoxycoumarin can inhibit the secretion of inflammatory factors induced by LPS stimulation. At the same time, different compounds showed significant differences in inhibiting the secretion of TNF-α, IL-6, IL-1β and promoting the secretion of IL-10. Among them, compound 1, 7-methoxycoumarin and butylphthalide showed strong anti-inflammatory activity in inhibiting inflammatory factors and promoting the secretion of anti-inflammatory factors. The differences in the secretion levels of these inflammatory factors may be closely related to the structural characteristics of the compounds. The structure of compound 4 is substituted on the benzopyran structure compared with other compounds, resulting in its weaker activity in inhibiting inflammatory factors (such as TNF-α, IL-6 and IL-1β). In contrast, compound 1 and 7-methoxycoumarin have similar structural characteristics and no additional nitrogen heterocyclic substitutions on the benzopyran ring, which may make them show stronger effects in inhibiting multiple inflammatory factors. In particular, compound 1 showed the highest promoting effect in the secretion of IL-10, which may be attributed to its simpler structure, which makes it more active in targeting specific inflammatory pathways.

[0162] To further evaluate the effects of compound 1, compound 2, compound 3, compound 4, 7-methoxycoumarin, almotriptan, and butylphthalide on the gene expression of inflammatory factors in BV2 and PC12 cells stimulated by LPS, we used qRT-PCR technology to detect the gene expression levels of inflammatory factors in the treated cells. Figure 10-11 The results showed that the regulatory effects of each compound on the gene expression of inflammatory factors were consistent with the results of protein level detection by ELISA. Specifically, compounds 1, 7-methoxycoumarin and butylphthalide showed significant regulatory effects in inhibiting the gene expression of TNF-α, IL-6, IL-1β and promoting the gene expression of anti-inflammatory factor IL-10, further verifying the potential of these compounds in anti-inflammatory activity. The relationship between the structure of the compound and its anti-inflammatory effect was further confirmed through the consistency analysis of qRT-PCR and ELISA results, providing a more comprehensive experimental basis for the development of anti-inflammatory drugs.

Claims

1. Use of a coumarin derivative in the preparation of a drug for preventing and treating central neuritis or neurodegenerative diseases, wherein the coumarin derivative is selected from: , , or .

2. The use according to claim 1, characterized in that: The central nervous system inflammation includes meningitis, encephalitis and myelitis.

3. The use according to claim 1, characterized in that: The neurodegenerative diseases include Alzheimer's disease and Parkinson's disease.

4. The use according to claim 1, wherein the drug comprises an effective amount of the coumarin derivative according to claim 1 and a pharmaceutically acceptable carrier.

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