Simple coumarin compounds, methods of making and using the same

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to a simple coumarin obtained from *Pseudoflavinus*, its preparation method, and its applications. The compound is shown in Formula 1 and its pharmaceutically acceptable salts and isomers; R 1 It is hydrogen, hydroxyl, or C1-C8 alkoxy; R 2 The group is composed of hydrogen, hydroxyl, C1-C8 alkoxy, aldehyde, fragment A, fragment B, or a 6-membered ring containing at least one heteroatom formed with an adjacent hydroxyl group, and the heterocycle is substituted by at least one substituent, which may be the same or different and selected from hydroxyl and C1-C3 alkyl groups; R 3 The compounds are hydrogen, hydroxyl, C2-C8 alkenyl, or fragment C. The structures of the simple coumarin compounds, their preparation methods, and their applications in the preparation of drugs for the prevention and treatment of neurodegenerative diseases are described.

Description

Simple coumarin compounds, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a simple coumarin obtained from false wampee, its preparation method, and its application. Background Technology

[0002] False wampee (Clausena excavate Burm.f.) is a plant belonging to the genus Clausena in the family Rutaceae. There are about 30 species of plants in the genus Clausena worldwide, distributed in Asia, Africa and Oceania. In my country, there are about 10 species and 2 varieties, one of which is introduced and cultivated. It is mainly distributed in Yunnan, Guangdong and Guangxi provinces.

[0003] *Phallus arvensis* is a shrub about 1-2 meters tall, commonly found in sparse forests or hillside thickets from plains to altitudes of up to 1,000 meters. Its roots are traditionally used to treat snake bites, possessing detoxifying properties; its fruit can be eaten fresh and has cooling, anti-inflammatory, and spleen-strengthening effects; its leaves are commonly used to regulate qi, relieve pain, dispel wind, eliminate dampness, and treat malaria. The various types of coumarins and carbazole alkaloids present in *Phallus arvensis* are the key material basis for its therapeutic effects. Modern pharmacological studies have found that they possess antitumor, anti-inflammatory, antibacterial, antiviral, hypoglycemic, and antioxidant activities. Summary of the Invention

[0004] The purpose of this invention is to provide a simple coumarin obtained from false wampee, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A simple coumarin compound, the compound as shown in Formula 1, and its pharmaceutically acceptable salts and isomers;

[0007]

[0008] R1 is hydrogen, hydroxyl, or C1-C8 alkoxy; R2 is hydrogen, hydroxyl, C1-C8 alkoxy, aldehyde, fragment A, fragment B, or a 6-membered ring containing at least one heteroatom formed with an adjacent hydroxyl group, and the heterocycle is substituted by at least one substituent, which may be the same or different and selected from hydroxyl, C1-C3 alkyl; R3 is hydrogen, hydroxyl, C2-C8 alkenyl, or fragment C.

[0009] Preferably, the compound is a compound of Formula 1 and its pharmaceutically acceptable salts or isomers; wherein, R1 is hydrogen, hydroxyl, or methoxy; R2 is hydrogen, hydroxyl, methoxy, or aldehyde, fragment A or fragment B, or a 6-membered ring containing two heteroatoms formed with an adjacent hydroxyl group, and the heterocycle is substituted by at least one substituent, which may be the same or different and selected from hydroxyl and C1-C3 alkyl groups; R3 is hydrogen, hydroxyl, isopentenyl, or fragment C.

[0010] More preferably, the compound is the following compounds and their pharmaceutically acceptable salts or isomers;

[0011]

[0012] One of the simple coumarin compounds described above is obtained by extracting the compound from *Pseudoflavinus* using an organic solvent and then purifying it to obtain the compound described in Formula 1.

[0013] To elaborate further,

[0014] (1) The root of the false wampee was repeatedly extracted with organic solvents, and the extracts were combined and recovered to obtain the crude extract;

[0015] (2) The crude extract obtained in step (1) was separated by silica gel column chromatography and gradient elution was performed using a mixed solvent as the eluent to obtain the eluent.

[0016] (3) The eluent obtained in step (2) above is subjected to ODS column chromatography and eluted by a mobile phase gradient.

[0017] (4) The eluent obtained in step (3) above was further separated by HPLC to obtain simple coumarin 1, 2, 3.

[0018] In step (1), the organic solvent is one or more of methanol, ethanol, chloroform or dichloromethane; wherein the material-to-liquid ratio is 1:5 to 1:30 g / mL.

[0019] Furthermore, in the preparation method of the new simple coumarin compounds 1-3, the extraction method in step (1) is reflux extraction or ultrasonic extraction 2-5 times. The volume concentration of methanol used is 60%-100%, preferably 80%-90%. The volume concentration of ethanol used is 60%-100%, preferably 80%-95%. The material-to-liquid ratio is 1:5-1:30 g / mL, preferably 1:10-1:15.

[0020] In step (2), the mixed solvents are: a petroleum ether-ethyl acetate mixture with a volume ratio of 100:5 to 1:1; a petroleum ether-acetone mixture with a volume ratio of 100:5 to 1:1; a dichloromethane-ethyl acetate mixture with a volume ratio of 100:1 to 5:1; a dichloromethane-acetone mixture with a volume ratio of 100:1 to 5:1; a chloroform-ethyl acetate mixture with a volume ratio of 100:1 to 5:1; and a chloroform-acetone mixture with a volume ratio of 100:1 to 5:1.

[0021] The elution solvent used in step (3) is a methanol-water mixture with a volume ratio of 50:50 to 100:0 or an acetonitrile-water mixture with a volume ratio of 30:70 to 90:10.

[0022] In the preparation method of the new simple coumarin compounds 1-3, the volume ratio of the methanol-water mixed solvent in step (3) is preferably 60:40 to 90:10; the volume ratio of the acetonitrile-water mixed solvent is preferably 40:60 to 80:20.

[0023] In step (4), a methanol-water mixed solvent with a volume ratio of 60:40 to 90:10 or an acetonitrile-water mixed solvent with a volume ratio of 40:60 to 80:20 is used.

[0024] In the preparation method of the new simple coumarin compounds 1-3, the volume ratio of the methanol-water mixed solvent in step (4) is preferably 70:30 to 90:10; the volume ratio of the acetonitrile-water mixed solvent is preferably 60:40 to 80:20.

[0025] A pharmaceutical composition comprising an active ingredient and pharmaceutically acceptable adjuvants and a carrier; wherein the active ingredient comprises a simple coumarin compound of Formula 1 and its pharmaceutically acceptable salts and isomers.

[0026] One application is the use of the simple coumarin compound of Formula 1 and its pharmaceutically acceptable salts, isomers, or pharmaceutical compositions in the preparation of medicaments for the prevention and treatment of neurodegenerative diseases.

[0027] Advantages of this invention:

[0028] This invention used an in vitro BV-2 cell model to test anti-inflammatory activity and evaluated the neuroinflammatory inhibitory activity of the prepared simple coumarin compounds 1-3. The results showed that these novel simple coumarin compounds 1 and 2 possess significant anti-neuroinflammatory activity and can be used to develop chemopreventive or therapeutic agents for neuroinflammation.

[0029] This invention provides for the first time a method for preparing and identifying four new simple coumarins using the root of *Pseudolarix amabilis* as raw material, and systematically evaluates their anti-neuroinflammatory activity, elucidating their application in the development of chemopreventive and therapeutic drugs related to neuroinflammation. Detailed Implementation

[0030] The following embodiments will further illustrate the present invention, but are not intended to limit the invention.

[0031] The silicone column model used in the following format example is 10cm*200cm.

[0032] Example 1

[0033] (1) 1500g of false yellow peel root was extracted three times by heating and refluxing with 95% ethanol (the amount used was 30L), and the crude extract of the extract was recovered under reduced pressure.

[0034] (2) The crude extract of 95% ethanol obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with chloroform-acetone mixed solvent at ratios of 100:1, 100:3, 100:5, 10:1, and 5:1 to obtain eluents of different polarities under different gradients.

[0035] (3) The eluents of the chloroform-acetone mixed solvent 100:3 to 10:1 in step (2) were separated by silica gel column chromatography and eluted sequentially with petroleum ether-ethyl acetate mixed solvent 100:5, 10:1, 7:1, 5:1, 2:1 and 1:1, and the eluents of each gradient were collected.

[0036] (4) The petroleum ether-ethyl acetate 10:1 to 2:1 fraction obtained in step (3) above is subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water at 50:50, 65:35, 80:20, and 90:10, and the eluents of each gradient are collected.

[0037] (5) The methanol-water fraction obtained in step (4) above (65:35 to 80:20) was separated by HPLC-UV chromatography, with detection at 210 nm, a flow rate of 3 mL / min, and a mobile phase of methanol:water = 72:28, to obtain compound 1 (t). R =30min)(yield 0.0000075%), compound 2 (t R =26 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =13.4min) and compound 3b(t R =14.9 min)(Total yield 0.000012%).

[0038] The structures of compounds 1, 2, 3a and 3b were identified based on their physicochemical properties and spectroscopic data.

[0039] The structural identification data of compound 1 are as follows:

[0040] A white amorphous powder (methanol) exhibits blue fluorescence under a 365nm ultraviolet light. -9.3 (c 0.50, CH3OH). HR-ESI-MS gives a quasi-molecular ion peak at m / z 333.1335 [M+H]. + (calcd.333.1338for C 18 H 21 O6), the molecular formula of the compound was determined to be C 18 H 20 O6 has an unsaturation degree of 9.

[0041] 1 In 1H NMR (600MHz, CDCl3), the low-field region yields a characteristic hydrogen signal at position 4 of coumarin H-3: δ H 6.19 (1H, d, J = 9.6 Hz, H⁻³), 7.98 (1H, d, J = 9.6 Hz, H⁻⁴); Group 1 1,1-dimethylallyl substituted hydrogen signal: δ H 6.25(1H,dd,J=17.4,10.6Hz,H-2”),4.90(1H,d,J=17.4Hz,H-3”a),4.88(1H,d,J=10.6Hz,H-3”b),1.66(3H,s,C H 3-4”), 1.66(3H,s,C H 3-5”); 2 active hydrogen signals: δ H 5.71(1H,br s,5-O H ), 3.16(1H,d,J=7.9Hz,3'-O H ); 1 hydroxymethyl hydrogen signal: δ H 5.17 (1H, d, J = 7.9 Hz, H-3'). The high-field region provides two methyl hydrogen signals: δ H 1.41(3H,s,C H 3-4'), 1.34(3H,s,C H 3-5').

[0042] 13 The 150 MHz C NMR (CDCl3) yielded 18 carbon signals, including one set of coumarin parent nucleus carbon signals: δ C161.3(C-2), 111.7(C-3), 138.6(C-4), 103.3(C-4a), 139.8(C-5), 124.0(C-6), 144.3(C-7), 115.2(C-8), 148.1(C-8a); Group 1 1,1-dimethylallyl substituted carbon signal δ C 41.5(C-1”),149.6(C-2”),108.6(C-3”),29.8( C H3-4”), 29.6( C H3-5”); the rest are aliphatic carbon signals, including two oxygen-bound carbon signals δ. C 75.7 (C-2'), 93.4 (C-3')

[0043] and 2 methyl carbon signals δ C 21.8 ( C H3-4'), 22.8( C H3-5').

[0044] In HMBC, δ H 5.71(5-O H ) and δ C Long-range correlations of 139.8 (C-5), 124.0 (C-6), and 103.3 (C-4a) confirmed that the hydroxyl group is attached to the C-5 position of the coumarin core; δ H 1.66(C H 3-4”), 1.66(C H 3-5”) and δ C 115.2 (C-8) exhibits long-range correlation, suggesting that the 1,1-dimethylallyl group is linked to the C-8 position; δ H 1.41(C H 3-4') and δ C 75.7 (C-2'), 93.4 (C-3'), 22.8 ( C H3-5') exhibits long-range correlation, δ H 1.34(C H 3-5') and δ C 75.7 (C-2'), 93.4 (C-3'), 21.8 ( C H3-4') exhibits long-range correlation, δ H 3.16(3'-O H ) and δ C 75.7 (C-2'), 93.4 (C-3') long-range correlation, δ H 5.17(H-3') and δ C 75.7 (C-2') long-range correlation suggests that the structure contains an oxygen-substituted isobutanol segment; δ H5.17(H-3') and δ C A long-range correlation exists between 124.0 (C-6), suggesting that the isobutanol structural fragment is connected to the coumarin core at C-6 and C-7 positions via a dioxane. In summary, the planar structure of compound 1 was determined.

[0045] The structure contains one chiral carbon atom. By comparing the measured and calculated ECD data, the absolute configuration of the chiral carbon in compound 1 was determined to be 3'S. A Sci-finder search revealed compound 1 to be a novel compound not previously reported in the literature. It was named clauexcatin A, and its NMR data were assigned (Table 1).

[0046] The structural identification data of compound 2 are as follows:

[0047] A white amorphous powder (methanol) exhibits blue fluorescence under a 365 nm UV lamp. HR-ESI-MS yields a quasi-molecular ion peak at m / z 289.1079 [M+H]. + (calcd.289.1076for C 16 H 17 O5), the molecular formula of the compound was determined to be C 16 H 16 O5, its degree of unsaturation is calculated to be 9.

[0048] 1 In 1H NMR (600MHz, DMSO-d6), the low-field region yields characteristic hydrogen signals at positions 3 and 4 of coumarin H-3: δ H 6.32 (1H,d,J=9.7Hz,H⁻³), 8.06 (1H,d,J=9.7Hz,H⁻⁴); 1 active hydrogen signal: δ H 12.98 (1H,br s,7-O) H ); 1 aldehyde hydrogen signal: δ H 10.19(1H,s,6-C H O); Group 1 1,1-Dimethylallyl substituted hydrogen signal: δ H 6.25(1H,dd,J=17.4,10.6Hz,H-2'),4.89(1H,d,J=17.4Hz,H-3'a),4.86(1H,d,J=10.6Hz,H-3'b),1.61(6H,s,C H (3-4', 5'). The high-field region also provided a methoxy hydrogen signal: δ H 4.02(3H,s,5-OC H 3).

[0049] 13In C NMR (150 MHz, DMSO-d6), 16 carbon signals were observed, including one group of coumarin parent nucleus carbon signals: δ C 158.7 (C-2), 112.0 (C-3), 139.0 (C-4), 106.5 (C-4a), 161.2 (C-5), 111.2 (C-6), 164.1 (C-7), 116.9 (C-8), 158.6 (C-8a); 1 aldehyde carbon signal: δ C 195.0(6- C HO); Group 1 1,1-dimethylallyl substituted carbon signal: δ C 40.7(C-1'),148.5(C-2'),109.0(C-3'),28.9×2( C H3-4',5'); 1 methoxy carbon signal: δ C 66.5(5-O C H3).

[0050] In HMBC, δ H 8.06 (H-4), 4.02 (5-OC) H 3) with δ C 161.2 (C-5) long-range correlation suggests that the C-5 position of the coumarin core is substituted with a methoxy group; δ H 1.61(C H 3-4',5') and δ C There is a long-range correlation between 116.9 (C-8), which, combined with the NOESY spectrum, δ H 1.61(C H 3-4',5') and δ H 12.98(7-O H There is a correlation, δ H 10.19(6-C H O) and δ respectively H 4.02(5-OC H 3), δ H 12.98(7-O H The presence of a correlation indicates that the aldehyde group is attached at C-6, the hydroxyl group at C-7, and the 1,1-dimethylallyl group at C-8. Based on these findings, the structure of compound 2 was determined. A Sci-finder search revealed compound 2 to be a novel compound not previously reported in the literature. It was named clauexcatin B, and its NMR data were assigned (Table 1).

[0051] Table 1. NMR data assignments for compounds 1 and 2.

[0052]

[0053] The structural identification data of compounds 3 (3a and 3b) are as follows:

[0054] It is a colorless oil (methanol) that fluoresces blue under a 365 nm UV lamp. HR-ESI-MS yields a quasi-molecular ion peak at m / z 259.0977 [MH]. - (calcd.259.0965for C 15 H 15 O4), the molecular formula of the compound was determined to be C 15 H 16 O4 has an unsaturation degree of 8.

[0055] 1 In 1H NMR (600MHz, DMSO-d6), two sets of characteristic hydrogen signals at position 4 of coumarin H-3 were observed in the low-field region: δ H 6.16(1H,d,J=9.5Hz,H-3),7.90(1H,d,J=9.5Hz,H-4) and δ H 6.15 (1H, d, J = 9.5 Hz, H-3'), 7.92 (1H, d, J = 9.5 Hz, H-4'); Two isolated aromatic proton signals: δ H 7.77(1H,s,H-5),6.71(1H,s,H-8) and δ H 7.63 (1H, s, H-5'), 6.71 (1H, s, H-8'); 1 pair of trans-olefin hydrogen signals: δ H 6.70 (1H, d, J = 16.2 Hz, H-9), 6.25 (1H, d, J = 16.2 Hz, H-10); 1 pair of cis-olefin hydrogen signals: δ H 6.43 (1H, d, J = 12.8 Hz, H-9'), 5.57 (1H, d, J = 12.8 Hz, H-10'). Two sets of methoxy hydrogen signals δ were also provided in the high-field region. H 3.09(3H,s,11-OC H 3) with δ H 2.96(3H,s,11'-OC H 3) and 2 groups of methyl hydrogen signals δ H 1.29(6H,s,C H 3-12,13) ​​and δ H 1.22(6H,s,C H 3-12', 13').

[0056] 13 Thirty carbon signals were observed in the 150 MHz C NMR (DMSO-d6) spectrum, including two groups of coumarin parent nucleus carbon signals: δ C160.4(C-2),111.0(C-3),144.6(C-4),110.9(C-4a),126.0(C-5),122.3(C-6),158.4(C-7),102.2(C-8),154.5(C-8a) and δ C 160.4 (C-2'), 111.0 (C-3'), 144.8 (C-4'), 110.2 (C-4a'), 130.2 (C-5'), 122.2 (C-6'), 158.7 (C-7'), 101.5 (C-8'), 154.5 (C-8a'); 2 groups of olefin carbon signals δ C 121.8 (C-9), 135.3 (C-10) and δ C 124.5 (C-9'), 136.7 (C-10'); the remaining carbon signals are: 2 groups of quaternary carbon signals δ C 74.7(C-11) and δ C 75.2 (C-11'); 2 groups of methoxy carbon signals δ C 49.7(11-O C H3) and δ C 49.5(11'-O C H3); 2 sets of methyl carbon signals δ C 25.8×2( C H3-12,13) ​​and δ C 26.9×2( C H3-12',13').

[0057] In HMBC, δ H 3.09(11-OC H 3) with δ C There is a long-range correlation between 74.7 (C-11), δ H 1.29(C H 3-12,13) ​​and δ C There is a long-range correlation between 135.3(C-10) and 74.7(C-11), δ H 6.70(H-9) and δ C There is a long-range correlation between 74.7 (C-11), δ H 6.25(H-10) and δ C 121.8(C-9), 74.7(C-11), 25.8( C The presence of H3-12,13) ​​indicates the existence of a 3-methoxyisopentenyl structural fragment in the structure. δ H 6.70(H-9) and δ C A correlation exists between 126.0 (C-5) and 158.4 (C-7), δ H7.77(H-5) and δ C A long-range correlation was found at 122.3 (C-6), confirming that the 3-methoxyisoprene group is attached to the C-6 position of the coumarin core. In summary, the structure of compound 3a was determined. In the HMBC spectrum, δ... H 2.96(11'-OC H 3) with δ C There is a long-range correlation between 75.2(C-11'), δ H 1.22(C H 3-12',13') and δ C There is a long-range correlation between 136.7(C-10') and 75.2(C-11'), δ H 6.43(H-9') and δ C There is a long-range correlation between 75.2(C-11'), δ H 5.57(H-10') and δ C 75.2(C-11'), 26.9( C The presence of a correlation (H3-12',13') confirms the existence of a 3-methoxyisopentenyl structural segment in the structure. δ H 5.57(H-10') and δ C A correlation exists at 122.2(C-6'), δ H 6.43(H-9') and δ C A correlation exists at 130.2(C-5'), δ H 7.63(H-5') and δ C A long-range correlation was found at 124.5 (C-9'), confirming that the 3-methoxyisoprenyl structural fragment is attached to the C-6' position of the coumarin core. In summary, the structure of compound 3b was determined. HPLC and NMR data revealed that compound 3 is a mixture that undergoes rapid conversion at room temperature, with an equilibrium ratio of approximately 5:3. A Sci-finder search identified compounds 3a and 3b as novel compounds not previously reported in the literature; they were named trans-clauexcatin C and cis-clauexcatin C, respectively, and their NMR data were assigned (Table 2).

[0058] Table 2. NMR data assignments for compounds 3a and 3b

[0059]

[0060] Example 2

[0061] (1) 1000g of false yellow peel root was extracted 4 times by heating and refluxing with 90% ethanol (the amount used was 15L), and the crude extract of the extract was recovered under reduced pressure.

[0062] (2) The crude extract of 90% ethanol obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with dichloromethane-acetone mixed solvent at ratios of 100:1, 100:3, 100:5, 10:1, and 5:1 to obtain eluents of different polarities under different gradients.

[0063] (3) The eluents of the dichloromethane-acetone mixed solvent 100:3 to 100:5 in step (2) were separated by silica gel column chromatography and eluted sequentially with petroleum ether-acetone mixed solvent 100:8, 10:1, 8:1, 5:1, 3:1, 2:1, 1:1, and the eluents of each gradient were collected.

[0064] (4) The petroleum ether-acetone 10:1 to 2:1 fraction obtained in step (3) above is subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water at 50:50, 60:40, 70:30, 80:20, and 90:10, and the eluents of each gradient are collected.

[0065] (5) The methanol-water 70:30 fraction obtained in step (4) above was separated by HPLC-UV chromatography at a flow rate of 3 mL / min and a mobile phase of methanol:water = 70:30 to obtain compound 1 (t). R =37min)(yield 0.0000075%), compound 2 (t R =39 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =14.7min) and compound 3b(t R =16.3min)(Total yield 0.000012%).

[0066] The structural identification methods for compounds 1-3 are described in Example 1.

[0067] Example 3

[0068] (1) 2000g of false yellow peel root was extracted 4 times with dichloromethane by heating and ultrasonication (the amount used was 30L), and the crude extract of the extract was recovered under reduced pressure.

[0069] (2) The crude dichloromethane extract obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with petroleum ether-acetone mixed solvents of 100:5, 100:8, 10:1, 5:1, 2:1 and 1:1 to obtain eluents of different polarities under different gradients.

[0070] (3) The petroleum ether-ethyl acetate 10:1 to 2:1 fraction obtained in step (2) was subjected to ODS chromatography and eluted with gradients of acetonitrile-water mixed solvents of 40:60, 60:40, 70:30, 80:20 and 90:10, and the eluents of each gradient were collected.

[0071] (4) The acetonitrile-water 60:40 fraction obtained in step (3) above was separated by HPLC-UV chromatography at a flow rate of 2.5 mL / min and a mobile phase of acetonitrile:water = 65:35 to obtain compound 1 (t R =21.1 min)(yield 0.0000075%), compound 2 (t R =24.8 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =13.4min) and compound 3b(t R =15.3min)(Total yield 0.000012%).

[0072] The structural identification methods for compounds 1-3 are described in Example 1.

[0073] Example 4

[0074] (1) 500g of false yellow peel root was extracted three times by heating and refluxing with chloroform (the amount used was 10L), and the crude extract of the extract was recovered under reduced pressure.

[0075] (2) The crude chloroform extract obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with dichloromethane-ethyl acetate mixed solvent at ratios of 100:1, 100:3, 100:5, 100:7, 10:1, 7:1, and 5:1 to obtain eluents of different polarities under different gradients.

[0076] (3) The fractions of dichloromethane-ethyl acetate mixed solvent 100:1 to 100:5 in step (2) were subjected to ODS chromatography and eluted with a gradient of methanol-water mixed solvents of 50:50, 60:40, 80:20, and 90:10, and the eluents of each gradient were collected.

[0077] (4) The methanol-water 80:20 fraction obtained in step (3) above was separated by HPLC-UV chromatography at a flow rate of 3.5 mL / min and a mobile phase of methanol:water = 70:30 to obtain compound 1 (t). R =31.5 min)(yield 0.0000075%), compound 2 (t R =32.7 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =13.6min) and compound 3b(t R =15.0 min)(Total yield 0.000012%).

[0078] The structural identification methods for compounds 1-3 are described in Example 1.

[0079] Example 5

[0080] (1) 2500g of false yellow peel root was extracted 4 times by heating and refluxing with 90% methanol (the amount used was 25L), and the crude extract of the extract was recovered under reduced pressure.

[0081] (2) The 90% methanol crude extract obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with chloroform-ethyl acetate mixed solvents of 100:1, 100:3, 100:5, 100:7, 10:1, and 5:1 to obtain eluents of different polarities under different gradients.

[0082] (3) The eluents of the chloroform-ethyl acetate mixed solvent 100:1 to 100:5 in step (2) were separated by silica gel column chromatography and eluted sequentially with petroleum ether-acetone mixed solvent 100:5, 100:7, 10:1, 8:1, 5:1, 2:1 and 1:1, and the eluents of each gradient were collected.

[0083] (4) The petroleum ether-acetone 10:1 to 2:1 fraction obtained in step (3) above is subjected to ODS chromatography and eluted with a gradient of mixed solvents of methanol-water at 50:50, 60:40, 70:30, 80:20 and 90:10, and the eluents of each gradient are collected.

[0084] (5) The methanol-water fraction obtained in step (4) above (70:30 to 80:20) was separated by HPLC RID-10A at a flow rate of 4 mL / min and a mobile phase of acetonitrile:water = 55:45 to obtain compound 1 (t R =28.0 min)(yield 0.0000075%), compound 2 (t R =31.3 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =16.6min) and compound 3b(t R =19.0 min)(Total yield was 0.000012%).

[0085] The structural identification methods for compounds 1-3 are described in Example 1.

[0086] Example 6

[0087] (1) 1000g of false yellow peel root was extracted three times by heating and refluxing with 95% ethanol (the amount used was 10L), and the crude extract of the extract was recovered under reduced pressure.

[0088] (2) The crude extract of 95% ethanol obtained in step (1) above was subjected to silica gel column chromatography and gradient elution with petroleum ether-ethyl acetate mixed solvents of 10:1, 8:1, 5:1, 3:1, 2:1 and 1:1 to obtain eluents of different polarities under different gradients.

[0089] (3) The petroleum ether: ethyl acetate obtained in step (2) above is mixed in a ratio of 10:1 to 2:1 and subjected to ODS chromatography. The mixture is eluted with a gradient of mixed solvents of acetonitrile-water at ratios of 40:60, 50:50, 60:40, 70:30, and 80:20. The eluents of each gradient are collected.

[0090] (4) The acetonitrile-water fraction obtained in step (3) above (40:60 to 60:40) was mixed and separated by HPLC-UV chromatography at a flow rate of 3.5 mL / min and a mobile phase of methanol:water = 60:40 to obtain compound 1 (t). R =37.0 min)(yield 0.0000075%), compound 2 (t R =44.2 min)(yield 0.000005%), and a pair of cis-trans isomers 3a(t R =18.5min) and compound 3b(t R =20.6 min)(Total yield 0.000012%).

[0091] The structural identification methods for compounds 1-3 are described in Example 1.

[0092] Example 7 Test on the anti-neuroinflammatory activity of the new simple coumarins 1-3 prepared above (1) Experimental principle:

[0093] Microglia are a major component of the brain's innate immune system, mainly distributed in the gray matter of the cerebellum, cerebrum and spinal cord, accounting for about 10% to 15% of all glial cells, and play an important role in maintaining the homeostasis of the central nervous system and protecting against pathogens [1]. Under normal physiological conditions, microglia are in a resting state, maintaining the stability of the brain environment to protect neurons. Under endogenous or exogenous pathological damage, such as stimulation by factors such as infection, injury, misfolded proteins, etc., they are rapidly activated into an activated state and release pro-inflammatory factors such as nitric oxide (NO), interleukin-6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor α (TNF-α). Over-activated microglia will lead to the continuous occurrence of neuroinflammatory response, eventually causing neuronal damage or even neuronal death, and persistent neuroinflammation is closely related to the occurrence and development of various central nervous system diseases. This invention constructs an in vitro LPS-activated BV-2 microglia abnormal activation screening model, and uses the activation of microglia to release NO as an indicator to evaluate the anti-inflammatory activity of new compounds 1-3.

[0094] (2) Experimental methods:

[0095] ① Culture of mouse microglia BV-2

[0096] All glassware and metal instruments (culture flasks, pipettes, solution bottles, etc.) used in cell culture and model establishment were autoclaved at 121°C for 30 minutes to thoroughly remove LPS contamination. A cell culture medium containing 10% fetal bovine serum was prepared using DMEM as a base. Microglia were cultured at approximately 2.0 × 10⁶ cells / day. 5 Cells / mL were passaged in 5% CO2, 37°C culture flasks. By day 3, adherent cells occupied approximately 70%-80% of the bottom area of ​​the culture flask. The adherent cells were then digested with trypsin and passaged to another culture flask. BV-2 cells that had been cryopreserved and thawed at -80°C were used as the first generation, and BV2 cells from the 3rd to 8th generations were selected for experiments.

[0097] ② Drug preparation method

[0098] All test compounds were in solid form and dissolved in DMSO. A stock solution with a concentration of 100 mM was prepared and stored at -20°C. Before use, the solutions were diluted with DMDM ​​medium to 100 μM, 30 μM, 10 μM, and 1 μM, respectively. The final DMSO concentration was <1‰.

[0099] ③ Griess method for detecting the inhibitory effect of compounds on LPS-activated microglia

[0100] BV2 microglia in the logarithmic growth phase were harvested and their density adjusted to 2.0 × 10⁶ cells / year using fresh DMDM ​​medium containing 10% fetal bovine serum. 5 Cells were seeded at 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator. After 24 h of adherent culture, the medium was replaced with serum-free fresh medium, and the cells were treated with the drug. Each compound was administered at doses of 100 μM, 30 μM, 10 μM, and 1 μM in combination with LPS. A blank control was also included. The final LPS concentration in each treatment group was 100 ng / mL. After 24 h of cell culture following drug administration, the supernatant was collected, and NO2 in the supernatant was detected using the Griess colorimetric method. - content.

[0101] ④ The effect of the compound on the survival rate of microglia was detected by MTT assay.

[0102] BV2 microglia cultured in the logarithmic growth phase were used to adjust the cell density to 2.0 × 10⁶ cells / year using fresh DMDM ​​medium containing 10% fetal bovine serum. 5Cells were seeded at 100 μL / well in 96-well plates and cultured at 37°C in a 5% CO2 incubator. After 24 h of adherent culture, the medium was replaced with fresh medium, and drug treatment was performed simultaneously. Each compound was administered at doses of 100 μM, 30 μM, 10 μM, and 1 μM in combination with LPS. A blank control was also included. The final LPS concentration in each treatment group was 100 ng / mL. After drug treatment, cells were cultured for another 24 h, then MTT solution (10 μL / well) was added to the cell culture medium. Cells were co-incubated with 0.25 mg / mL MTT at 37°C for 3 h. The culture medium was then removed, and 150 μL of DMSO solution was added. The optical density (OD) value was measured. Data processing was performed using the corresponding software of the microplate reader. The average OD value of three wells for each sample was calculated, and the cell viability (CV%) was calculated using the average value according to the following formula.

[0103] Cell viability % = (Average OD value of sample group / Average OD value of blank control group) × 100%

[0104] ⑤ Statistical methods

[0105] All data were analyzed using SPSS statistical software. Results are expressed as mean ± standard error. To assess overall differences, one-way ANOVA was used to analyze homogeneity of variance between groups, combined with Dunnett's test for inter-group comparisons. The Levene test was used to test homogeneity of variance in a multi-sample study. When p > 0.05, the variances were homogeneous; Dunnett's two-tailed t-test was used to assess differences in means among groups. When p < 0.05, the variances were unequal; Dunnett's T3 test was used to assess differences in means among groups.

[0106] ⑥IC 50 Calculation method

[0107] IC was calculated by fitting parameters such as dose and inhibition rate using nonlinear regression. 50 .

[0108] (3) Experimental results: see Table 3

[0109] Table 3. Experimental results on the inhibitory effect of compounds 1-3 on microglia activation.

[0110]

[0111] Significance: *P<0.05, **P<0.01, ***P<0.001 compared with the LPS-induced group.

[0112] The results showed that the new compounds 1 (10 μM, 30 μM, 100 μM), 2 (30 μM, 100 μM), and 3 (10 μM, 30 μM, 100 μM) prepared in Examples 1-6 could significantly inhibit the release of NO from LPS-induced overactivated BV2 microglia.

Claims

1. A coumarin compound, characterized in that: The compounds are the following compounds and their pharmaceutically acceptable salts; 。 2. A method for preparing the coumarin compound according to claim 1, characterized in that: (1) 1500 g of *Pyrrosia lingua* root was extracted three times by heating and reflux with 95% ethanol, with a volume of 30 L. The crude extract was recovered under reduced pressure. (2) The crude extract obtained in step (1) was subjected to silica gel column chromatography and eluted with a gradient of chloroform-acetone mixed solvents at ratios of 100:1, 100:3, 100:5, 10:1, and 5:1 to obtain eluents of different polar fractions under different gradients. (3) The eluents of chloroform-acetone mixed solvents at ratios of 100:3 to 10:1 in step (2) were separated by silica gel column chromatography and eluted sequentially with a petroleum ether-ethyl acetate mixed solvent at ratios of 100:

5. Elution at ratios of 10:1, 7:1, 5:1, 2:1, and 1:1, and collection of each gradient eluent; (4) The petroleum ether-ethyl acetate 10:1~2:1 fraction obtained in step (3) above is subjected to ODS chromatography and eluted with a gradient of 50:50, 65:35, 80:20, and 90:10 methanol-water mixed solvents, and collection of each gradient eluent; (5) The methanol-water 65:35~80:20 fraction obtained in step (4) above is separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and a mobile phase of methanol:water = 72:28, to obtain compound 1, compound 2, a pair of cis-trans isomers compound 3a and compound 3b; the t of compound 1 R = 30 min, yield 0.0000075%; t of compound 2 R = 26 min, yield 0.000005%; t of compound 3a R = 13.4 min, t of compound 3b R = 14.9 min, total yield 0.000012%; or, (1) 1000 g of false yellow peel root was extracted 4 times by heating and refluxing with 90% ethanol, the amount of which was 15 L, and the crude extract of the extract was recovered under reduced pressure; (2) the crude extract of 90% ethanol obtained in step (1) above was subjected to silica gel column chromatography, and gradient elution was performed with dichloromethane-acetone mixed solvent 100:1, 100:3, 100:5, 10:1, 5:1 to obtain eluents of different polarity fractions under different gradients; (3) the eluents of dichloromethane-acetone mixed solvent 100:3~100:5 in step (2) were separated by silica gel column chromatography, and successively separated with petroleum ether-acetone mixed solvent 100:8 Elution at ratios of 10:1, 8:1, 5:1, 3:1, 2:1, and 1:1, and collection of each gradient eluent; (4) The petroleum ether-acetone 10:1~2:1 fraction obtained in step (3) above is subjected to ODS chromatography and eluted with a gradient of 50:50, 60:40, 70:30, 80:20, and 90:10 methanol-water mixed solvents, and collection of each gradient eluent; (5) The methanol-water 70:30 fraction obtained in step (4) above is separated by HPLC-UV chromatography at a flow rate of 3 mL / min and a mobile phase of methanol:water = 70:30, to obtain compound 1, compound 2, a pair of cis-trans isomers compound 3a and compound 3b; the t of compound 1 R = 37 min, yield 0.0000075%; t of compound 2 R = 39 min, yield 0.000005%; t of compound 3a R = 14.7 min, t of compound 3b R = 16.3 min, total yield 0.000012%.

3. A pharmaceutical composition, characterized in that, The composition comprises an active ingredient and pharmaceutically acceptable adjuvants and a carrier; wherein the active ingredient is a coumarin compound as described in claim 1 and a pharmaceutically acceptable salt thereof.

4. An application of the compound according to claim 1, characterized in that: The use of the coumarin compounds of claim 1 and their pharmaceutically acceptable salts in the preparation of medicaments for the prevention and treatment of neurodegenerative diseases.

5. The use of the pharmaceutical composition according to claim 3, characterized in that: The use of the pharmaceutical composition of claim 3 in the preparation of a medicament for the prevention and treatment of neurodegenerative diseases.

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