Flavaglines compounds as well as preparation method and application thereof

Abstract

The invention relates to a Flavaglines compound as well as a preparation method and application thereof, in particular to a Flavaglines compound extracted from Aglaia indica and a preparation method and application thereof, and belongs to the technical field of medicines. The invention provides a method for preparing and identifying 15 new flavaglines derivatives by taking branches and leaves, especially dry branches and leaves, of Aglaia Bilutea L. as raw materials for the first time, systematically evaluates the activity of the flavaglines in the aspect of neuroprotection, and provides a new method for preparing and identifying the 15 new flavaglines derivatives in the aspect of neuroprotection, so that the new flavaglines derivatives can be used for preparing and identifying the new flavaglines derivatives in the aspect of neuroprotection, and the new flavaglines can be used for preparing and identifying the new flavaglines in the aspect of neuroprotection in the aspect of neuroprotection in the aspect of neuroprotection. The invention illustrates the application of the compound in the aspect of developing and treating the neurodegenerative diseases.

Description

Technical Field

[0001] The present invention relates to a class of Flavaglines compounds, their preparation methods and applications, and particularly to Flavaglines compounds extracted from Aglaia perviridis Hiern, their preparation methods and applications, belonging to the field of pharmaceutical technology. Background Art

[0002] Aglaia perviridis Hiern is a plant of the genus Aglaia Lour. in the family Meliaceae. It is produced in southern and southeastern Yunnan of China and grows in valley rainforests, monsoon rainforests and evergreen broad-leaved forests. In addition, it is also distributed in India and Sikkim.

[0003] Currently, various types of compounds such as alkaloids (flavaglines, bisamides), terpenoids (triterpenes, sesquiterpenes), steroids, flavonoids, etc. have been extracted and isolated from Aglaia perviridis Hiern. These compounds have novel and diverse structural skeletons and extensive and significant biological activities. Among them, flavaglines are unique chemical components in plants of the genus Aglaia and are one of the chemotaxonomic markers of plants in this genus. Based on the biosynthetic pathway, flavaglines compounds can be divided into three skeleton types: cyclopenta[b]benzofuran, cyclopenta[bc]benzopyran, and benzo[b]oxepine. Flavaglines have various biological activities such as anti-inflammatory, anti-tumor and antiviral, which have attracted extensive attention from pharmacologists and medicinal chemists. Flavaglines have various biological activities such as anti-inflammatory, anti-tumor and antiviral, which have attracted extensive attention from pharmacologists and medicinal chemists. Summary of the Invention

[0004] The present invention provides a method for extracting Flavaglines compounds from Aglaia perviridis Hiern, and the application of such compounds in drugs for preventing or treating neurodegenerative diseases.

[0005] A class of Flavaglines compounds with the following chemical structural formula and their pharmaceutically acceptable salts,

[0006]

[0007] A preparation method of a class of Flavaglines compounds, comprising the following steps:

[0008] S1. The branches and leaves of Aglaiaperviridis Hiern are extracted with methanol or ethanol solution, and the extract is recovered to obtain a crude extract. The obtained crude extract is dissolved in water and then extracted with an organic solvent to obtain extracts with different polarities. The obtained extracts are separated by silica gel column chromatography and eluted with a mixed solvent gradient. The obtained fractions are separated by polyamide column chromatography and eluted with a methanol-water or ethanol-water mixed solvent as the mobile phase gradient.

[0009] S2. The fractions obtained in step S1 are separated by ODS column chromatography and eluted with a methanol-water or acetonitrile-water mixed solvent as the mobile phase gradient.

[0010] S3. The methanol-water or acetonitrile-water eluates obtained in step S2 are further separated by HPLC and eluted with a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as the mobile phase gradient to obtain racemic mixture compounds 1, 2, 4, 5, 6, 7, 8 and compound 3. Among them, the obtained racemic mixture compounds 1, 2, 4, 5, 6, 7, 8 of flavaglines are subjected to chiral resolution by HPLC to obtain compounds 1a, 1b, 2a, 2b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b.

[0011] The branches and leaves of Aglaiaperviridis Hiern used in the present invention can be dry branches and leaves or fresh branches and leaves. Dry branches and leaves are preferred. Dry branches and leaves have higher extraction efficiency than fresh branches and leaves, are easy to transport and store, and the content of active ingredients is relatively stable.

[0012] Preferably, in step S1, the extraction method with methanol or ethanol solution is heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction 2 - 5 times. The volume concentration of the ethanol solution is 70% - 95%, the volume concentration of the methanol solution is 60% - 90%, and the material-liquid ratio is 1:8 - 1:20 g / mL.

[0013] Preferably, in step S1, the organic solvent extraction is as follows: according to the volume ratio of the aqueous phase to the organic phase of 1:1 - 1:5, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate, and n-butanol are used to extract 3 - 5 times in turn, and the above organic solvents are recovered under reduced pressure.

[0014] Preferably, the mixed solvent used in the silica gel column chromatography is one of the gradient elutions of a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol, and a mixed solvent of dichloromethane and methanol.

[0015] Furthermore, the volume ratio of petroleum ether to ethyl acetate is 10:1 to 1:1, the volume ratio of petroleum ether to acetone is 8:1 to 1:1, the volume ratio of dichloromethane to acetone is 100:1 to 1:1, the volume ratio of the mixed solvent of chloroform and acetone is 50:1 to 1:1, the volume ratio of dichloromethane to methanol is 20:1 to 2:1, and the volume ratio of chloroform to methanol is 10:1 to 2:1.

[0016] Preferably, in the step S1, the volume ratio of methanol to water is 1:9 to 1:9, and the volume ratio of ethanol to water is 1:9 to 1:9.

[0017] Preferably, in the steps S2 and S3, the volume ratio of methanol to water is 2:8 to 8:2, and the volume ratio of acetonitrile to water is 1:9 to 9:1.

[0018] Preferably, in the step S3, the chiral chromatography column separation solvent is a mixed solvent of n-hexane and absolute ethanol, and its volume ratio is 40:60 to 80:20.

[0019] Another object of the present invention is to provide a pharmaceutical composition comprising the above-mentioned Flavaglines compounds.

[0020] A pharmaceutical composition comprising a Flavaglines compound having the following chemical structural formula, its pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

[0021]

[0022] Another object of the present invention is to provide the use of the above-mentioned Flavaglines compounds, their pharmaceutically acceptable salts or pharmaceutical compositions in the preparation of drugs for preventing or treating neurodegenerative diseases.

[0023] The beneficial effects of the present invention are as follows: The present invention for the first time provides a method for preparing and identifying 15 new flavaglines derivatives using the branches and leaves of Aglaia perviridis, especially the dry branches and leaves, and systematically evaluates their activities in neuroprotection, and clarifies their application in the development and treatment of drugs for neurodegenerative diseases. The present invention evaluated the anti-neuroinflammation of the prepared flavaglines compounds 1 to 8 using an LPS-induced overactivation model of BV-2 microglia. The results showed that especially the new compounds 2, 3, 5 and 6 could inhibit the release of NO from LPS-induced overactivated BV-2 microglia, showing significant anti-neuroinflammatory activity. Therefore, the new flavaglines compounds prepared in the present invention can be applied in the development of drugs for treating neurodegenerative diseases. Detailed embodiments

[0024] The following non-limiting examples can enable those of ordinary skill in the art to more comprehensively understand the present invention, but do not limit the present invention in any way.

[0025] In the test methods described in the following examples, unless otherwise specified, they are all conventional methods; the reagents and materials, unless otherwise specified, can all be obtained from commercial channels.

[0026] A method for preparing a class of Flavaglines compounds, comprising the following steps:

[0027] (1) The dried branches and leaves of Aglaiaperviridis Hiern are extracted by heating under reflux with 70% - 95% ethanol or 60% - 90% methanol, or by heating and ultrasonic extraction, and the extract is recovered to obtain a crude extract;

[0028] (2) After the crude extract obtained in step (1) is dissolved in water, it is extracted 3 - 5 times in turn with petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate, and n-butanol according to the volume ratio of the aqueous phase to the organic phase of 1:1 - 1:5 to obtain extracts with different polarities;

[0029] (3) The extracts obtained in the above step (2) are separated by silica gel column chromatography, and gradient elution is carried out with a mixed solvent of petroleum ether and ethyl acetate at 100:10 - 1:1, or a mixed solvent of petroleum ether and acetone at 10:1 - 1:1, or a mixed solvent of petroleum ether and acetone at 8:1 - 1:1, or a mixed solvent of dichloromethane and acetone at 100:1 - 1:1, or a mixed solvent of chloroform and acetone at 50:1 - 1:1, or a mixed solvent of chloroform and methanol at 10:1 - 2:1, or a mixed solvent of dichloromethane and methanol at 20:1 - 1:1;

[0030] (4) The fractions obtained in the above step (3) are separated by polyamide column chromatography, and gradient elution is carried out with a mixed solvent of methanol - water at 1:9 - 1:9 or ethanol - water at 1:9 - 1:9 as the mobile phase;

[0031] (5) The fractions obtained in the above step (4) are separated by ODS column chromatography, and gradient elution is carried out with a mixed solvent of methanol - water at 2:8 - 8:2 or acetonitrile - water at 1:9 - 9:1 as the mobile phase;

[0032] (6) The methanol and water, acetonitrile and water eluates obtained in the above step (5) are further separated by HPLC, and gradient elution is carried out with a mixed solvent of methanol and water at 2:8 - 8:2 or a mixed solvent of acetonitrile and water at 3:7 - 7:3 as the mobile phase to obtain racemic mixture compounds 1, 2, 4, 5, 6, 7, 8 and compound 3;

[0033] (7) The compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b obtained from the racemic mixture of flavaglines compounds 1, 2, 4, 5, 6, 7, 8 in step (6) by HPLC chiral resolution, with the solvent being a mixed solvent of n-hexane and absolute ethanol, and the volume ratio of the mixed solvent being 40:60 to 80:20.

[0034] For the preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the extraction method in step (1) is heating under reflux with methanol extraction, heating under reflux with ethanol extraction, or heating and ultrasonic extraction for 2 to 5 times, and the solvent used is ethanol at 70% to 95% or methanol at 60% to 90%, preferably ethanol at 75% to 85% or methanol at 65% to 85%. The solid-liquid ratio is 1:8 to 1:20 g / mL, preferably 1:10 to 1:15.

[0035] For the preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, for the organic solvent extraction method in step (2), the crude extract is dissolved in water, and according to the volume ratio of the aqueous phase to the organic phase of 1:1 to 1:5, preferably 1:1 to 1:3, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate, and n-butanol are used to extract 3 to 5 times in sequence, preferably 5 times, and the above organic solvents are recovered under reduced pressure.

[0036] For the preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the volume ratio of the mixed solvent of petroleum ether and ethyl acetate, or petroleum ether and acetone in step (3) is 10:1 to 1:1, preferably 8:1 to 1:1; the volume ratio of the mixed solvent of dichloromethane and acetone, or chloroform and acetone, or dichloromethane and methanol, or chloroform and methanol is 100:1 to 1:1, preferably 10:1 to 2:1.

[0037] For the preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the volume ratio of the mixed solvent of methanol and water in step (4) is 1:9 to 9:1, preferably 3:7 to 9:1, and the volume ratio of the mixed solvent of ethanol and water is 1:9 to 1:9, preferably 3:7 to 9:1.

[0038] The preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the volume ratio of the mixed solvent of methanol and water in step (5) is 2:8 to 8:2, preferably 3:7 to 7:3, and the volume ratio of the mixed solvent of acetonitrile and water is 1:9 to 9:1, preferably 2:8 to 8:2.

[0039] The preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the volume ratio of the mixed solvent of methanol and water in step (6) and step (7) is 2:8 to 8:2, preferably 4:6 to 7:3, and the volume ratio of the mixed solvent of acetonitrile and water is 3:7 to 7:3, preferably 4:6 to 7:3.

[0040] The preparation method of the novel flavaglines compounds 1a, 1b, 2a, 2b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b provided by the present invention, the chiral column chromatography separation solvent in step (6) and step (7) is a mixed solvent of n-hexane and absolute ethanol, and its volume ratio is 40:60 to 80:20, preferably 45:55 to 70:30.

[0041] Example 1

[0042] (1) 1000 g of dried branches and leaves of Aglaia perviridis were extracted by heating under reflux with 80% ethanol for 3 times (dosage: 8 L), and the extract was recovered under reduced pressure to obtain a crude extract;

[0043] (2) The 80% ethanol crude extract obtained in the above step (1) was dissolved in water and successively extracted with chloroform, ethyl acetate, and n-butanol. Each organic phase was extracted 3 times, and the volume ratio of the aqueous phase to the organic phase was 1:1 each time to obtain extracts of different polar parts;

[0044] (3) The ethyl acetate extract in step (2) was separated by silica gel column chromatography and eluted successively with mixed solvents of petroleum ether and ethyl acetate at 10:1, 8:1, 5:1, 4:1, 2:1, and 1:1;

[0045] (4) The fractions of petroleum ether:ethyl acetate (8:1 to 2:1) obtained in the above step (3) were subjected to polyamide column chromatography and eluted with gradient of mixed solvents of methanol-water at 1:9, 3:7, 5:5, 7:3, and 9:1;

[0046] (5) The fractions of methanol:water (5:5 to 7:3) obtained in the above step (4) were subjected to ODS column chromatography and eluted with gradient of mixed solvents of methanol-water at 2:8, 4:6, 6:4, and 8:2;

[0047] (6) The methanol: water (6:4 - 8:2) fraction obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was methanol: water = 70:30, to obtain the racemic mixture compound 1 (t R = 16.4 min) (yield 0.00021‰), compound 2 (t R = 20.5 min) (yield 0.00018‰), compound 3 (t R = 25.4 min) (yield 0.00009‰), compound 7 (t R = 27.1 min) (yield 0.00005‰), and compound 8 (t R = 35.4 min) (yield 0.00004‰). The racemic mixture of compound 1 was further separated by HPLC chiral column chromatography, and eluted with n-hexane: absolute ethanol (60:40) as the mobile phase to obtain new 1a (21.4 min), 1b (29.3 min) (yields each 0.0001‰); the racemic mixture of compound 2 was further separated by HPLC chiral column chromatography, and eluted with n-hexane: absolute ethanol (50:50) as the mobile phase to obtain new 2a (5.3 min), 2b (8.2 min) (yields each 0.00009‰); the racemic mixture of compound 7 was further separated by HPLC chiral column chromatography, and eluted with n-hexane: absolute ethanol (50:50) as the mobile phase to obtain new 7a (7.2 min), 7b (9.4 min) (yields each 0.000026‰); the racemic mixture of compound 8 was further separated by HPLC chiral column chromatography, and eluted with n-hexane: absolute ethanol (70:30) as the mobile phase to obtain new 8a (17.6 min), 8b (19.4 min) (yields each 0.00002‰).

[0048] (7) The methanol: water (2:8 - 4:6) fraction obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was methanol: water = 45:55, to obtain compound 4 (t R = 41.9 min) (yield 0.00013‰), compound 5 (t R = 46.1 min) (yield 0.00022‰), and compound 6 (t R= 49.8 min) (yield 0.00013‰). The racemic mixture of compound 4 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (70:30) as the mobile phase to obtain new 4a (3.5 min), 4b (8.5 min) (yields 0.00006‰ each); the racemic mixture of compound 5 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (60:40) as the mobile phase to obtain new 5a (21.5 min), 5b (29.4 min) (yields 0.00011‰ each); the racemic mixture of compound 6 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (70:30) as the mobile phase to obtain new 6a (4.8 min), 6b (6.5 min) (yields 0.00006‰ each).

[0049] The structures of compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b were identified based on their physicochemical properties and spectral data.

[0050] Since compounds 1a and 1b are a pair of enantiomers obtained by chiral column resolution and their hydrogen-carbon data are completely consistent, their structural analyses were performed simultaneously. The structural identification data of compounds 1a and 1b are as follows:

[0051] White oil (methanol), 1a: (c 0.50, methanol), 1b: (c 0.50, methanol). HR-ESI-MS gave the quasi-molecular ion peak m / z 671.2361 [M+Na] + (calcd. 671.2369 for C 38 H 36 N2O8Na), indicating that its molecular formula is C 38 H 36 N2O8. 1 In 1H NMR (600 MHz, CDCl3), 4 sets of benzene ring hydrogen signals were observed in the low field region, namely 2 sets of monosubstituted benzene ring hydrogen signals: δ H 7.44 (2H, m, H-2”,6”), 7.24 (1H, m, H-4”), 7.21 (2H, m, H-3”,5”), and δ H 7.75 (2H, m, H-20,24), 7.49 (1H, m, H-22), 7.43 (2H, m, H-21,23); 1 set of 1,4-disubstituted benzene ring hydrogen signals: δ H6.65 (2H, d, J = 9.0 Hz, H-3', 5'), 6.44 (2H, d, J = 9.0 Hz, H-2', 6'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H 6.28 (1H, d, J = 1.8 Hz, H-9), 6.00 (1H, d, J = 1.8 Hz, H-7); One set of mutually coupled methine hydrogen signals can be observed in the high field region: δ H 3.96 (1H, d, J = 4.8 Hz, H-3), 3.79 (1H, d, J = 4.8 Hz, H-2); Three methoxy hydrogen signals: δ H 3.85 (3H, s, 8-OC H 3), 3.84 (3H, s, 6-OC H 3), 3.73 (3H, s, 4'-OC H 3); One set of 1,4-butanediamide hydrogen signals: δ H 3.61 (2H, m, H2-13), 3.50 (1H, m, H-16a), 3.41 (1H, m, H-16b), 1.68 (2H, m, H2-14), 1.60 (2H, m, H2-15).

[0052] 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including: Two sets of monosubstituted benzene ring carbon signals: δ C 134.8 (C-1”), 128.8 (C-4”), 128.7×2 (C-3”, 5”), 125.3×2 (C-2”, 6”), and δ C 134.7 (C-19), 131.5 (C-22), 128.7×2 (C-21, 23), 127.1×2 (C-20, 24); One set of 1,2,3,5-tetrasubstituted benzene ring carbon signals: δ C 173.7 (C-9a), 170.0 (C-8), 159.7 (C-6), 104.1 (C-5a), 93.8 (C-7), 89.0 (C-9); One set of 1,4-disubstituted benzene ring carbon signals: δ C 159.1 (C-4'), 129.3 (C-1'), 128.7×2 (C-2', 6'), 114.4×2 (C-3', 5'); One set of 1,4-butanediamide carbon signals: δ C 177.4 (C-11), 167.7 (C-18), 39.6 (C-16), 38.7 (C-13), 26.4 (C-15), 25.4 (C-14); Three methoxy carbon signals: δ C 56.3 (8-OC H3), 56.2 (6 - O C H3), 55.4 (4'-O C H3); In addition, there are also 2 sp 2 hybridized quaternary carbon signals: δ C 193.3 (C - 5), 173.9 (C - 10); 1 sp 3 hybridized quaternary carbon signal: δ C 90.4 (C - 2); and 2 methylene carbon signals: δ C 56.9 (C - 3), 48.6 (C - 4).

[0053] The hydrogen - carbon data were assigned using the HSQC spectrum (Table 1). In the HMBC spectrum, δ H 7.75 (H - 20, 24) and 3.50 (H - 16a), 3.41 (H - 16b) are respectively in long - range correlation with δ C 167.7 (C - 18), δ H 3.61 (H2 - 13) is in long - range correlation with δ C 177.4 (C - 11), 173.9 (C - 10), confirming the connecting groups at both ends of the 1,4 - butanediamide fragment; δ H 6.44 (H - 2', 6') is in long - range correlation with δ C 48.6 (C - 2), confirming the connecting position of the 1,4 - substituted benzene ring; δ H 6.00 (H - 7) is in long - range correlation with δ C 193.5 (C - 5), 170.0 (C - 8), 159.7 (C - 6), 104.1 (C - 5a), 89.0 (C - 9), δ H 6.27 (H - 9) is in long - range correlation with δ C 173.7 (C - 9a), 170.0 (C - 8), 104.1 (C - 5a), 93.8 (C - 7), δ H 7.44 (H - 2”, 6”) is in long - range correlation with δ C 90.4 (C - 4), δ H 3.85 (8 - OC H 3) is in long - range correlation with δ C 170.0 (C - 8), δ H 3.84 (6 - OC H 3) is in long - range correlation with δ C 159.7 (C - 6), confirming the connecting groups of the 1,2,3,5 - tetrasubstituted benzene ring. Finally, combined with the d in the ACD / Structure Elucidator software A (13 C), d N ( 13 C), and d I ( 13 C) scoring value, and δ H 3.96 (H-3) and δ C 193.5 (C-5), 173.9 (C-10), 90.4 (C-4), δ H 3.79 (H-2) and δ C The HMBC correlation signals between 177.4 (C-11), 90.4 (C-4) and 3.79 (H-2) determined the planar structures of compounds 1a and 1b. In addition, based on the NOESY correlations between δ H 3.96 (H-3) and δ H 6.44 (H-2', 6'), δ H 3.79 (H-2) and δ H 7.44 (H-2”, 6”) and combined with the computational NMR and statistical analysis methods, the relative configurations of compounds 1a and 1b were confirmed to be 2R*, 3R*, 4S*. Through the analysis of the optical rotation and ECD spectra, it was determined that compounds 1a and 1b were a pair of enantiomers, and chiral resolution was successfully carried out on them. The absolute configurations of 1a and 1b were determined by comparing the measured and calculated ECD spectra. Among them, compound 1a had negative Cotton effects at 208, 232 and 290 nm, and compound 1b had positive Cotton effects at 208, 232 and 296 nm. After searching in Scifinder, they were new compounds not reported in the literature, and they were named (-)-flavaperviridis A and (+)-flavaperviridis A respectively.

[0054] Table 1 NMR data assignment of compounds 1a and 1b

[0055]

[0056] Since compounds 2a and 2b were a pair of enantiomers obtained by chiral column resolution and their hydrogen-carbon data were exactly the same, the structural analysis of both of them was carried out simultaneously. The structural identification data of compound 2 are as follows:

[0057] White oil (methanol), 2a: (c 0.50, methanol), 2b: (c 0.50, methanol). HR-ESI-MS gave the quasi-molecular ion peak m / z 623.2764 [M + H] + (calcd. 623.2757 for C 37 H 39N2O7), its molecular formula can be speculated as C 37 H 38 N2O7. 1 In 1H NMR (600 MHz, CDCl3), four sets of benzene ring hydrogen signals were observed in the low field region, including two sets of monosubstituted benzene ring hydrogen signals: δ H 7.77 (2H, m, H-20, 24), 7.51 (1H, m, H-22), 7.46 (2H, m, H-21, 23), and δ H 7.05 (3H, m, H-3”, 4”, 5”), 6.98 (2H, m, H-2”, 6”); one set of 1,4-disubstituted benzene ring hydrogen signals: δ H 7.44 (2H, d, J = 8.4 Hz, H-2', 6'), 6.87 (1H, d, J = 8.4 Hz, H-3', 5'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H 6.38 (1H, d, J = 2.4 Hz, H-7), 6.08 (1H, d, J = 2.4 Hz, H-5); in the high field region, a set of ABX coupling system hydrogen signals could be observed: δ H 4.52 (1H, dd, J = 12.0, 3.6 Hz, H-3), 2.78 (1H, dd, J = 15.0, 12.0 Hz, H-2a), 2.72 (1H, dd, J = 15.0, 3.6 Hz, H-2b); three methoxy hydrogen signals: δ H 3.92 (3H, s, 8-OC H 3), 3.79 (3H, s, 4'-OC H 3), 3.78 (3H, s, 6-OC H 3); one set of 1,4-butanediamide hydrogen signals: δ H 3.27 (2H, m, H2-16), 3.17 (1H, m, H-13a), 2.95 (1H, m, H-13b), 1.24 (4H, m, H2-14, 15). 13 13C NMR (150 MHz, CDCl3) gave a total of 37 carbon signals, including four sets of benzene ring carbon signals, namely two sets of monosubstituted benzene ring carbon signals, one set of 1,2,3,5-tetrasubstituted benzene ring carbon signals, and one set of monosubstituted benzene ring carbon signals; one set of 1,4-butanediamide carbon signals; three methoxy carbon signals, one ester carbonyl carbon signal: δ C 177.4 (C-3a), one methine carbon signal: δ C 50.6 (C-3), one methylene carbon signal: δ C 39.0 (C-2), and one quaternary carbon signal: δ C61.4 (C-8b). The hydrogen-carbon data were assigned using the HSQC spectrum (Table 2). In the HMBC spectrum, δ H 7.77 (H-20, 24) and 3.27 (H2-16) showed long-range correlations with δ C 167.7 (C-18) respectively. δ H 3.17 (H-13a), 2.95 (H-13b) showed long-range correlations with δ C 172.0 (C-11), confirming the connection position of a monosubstituted benzene; δ H 6.98 (H-2”, 6”) showed long-range correlations with δ C 50.6 (C-3), confirming the connection position of another monosubstituted benzene ring; δ H 7.44 (H-2', 6') showed long-range correlations with δ C 61.4 (C-8b), confirming the connection position of a 1,4-disubstituted benzene ring; δ H 6.38 (H-7) showed long-range correlations with δ C 162.3 (C-6), 157.4 (C-8), 106.8 (C-8a), 89.6 (C-5). δ H 6.08 (H-5) showed long-range correlations with δ C 162.3 (C-6), 155.2 (C-4a), 106.8 (C-8a), 94.9 (C-7). δ H 3.92 (8-OC H 3) showed long-range correlations with δ C 157.4 (C-8). δ H 3.78 (6-OC H 3) showed long-range correlations with δ C 162.3 (C-6), confirming the connecting groups of a 1,2,3,5-tetrasubstituted benzene ring. Using the ACD / Structure Elucidator software, based on the HMBC correlation signals of δ H 4.52 (H-3) with δ C 177.4 (C-3a), 172.0 (C-11), 106.8 (C-8a), 61.4 (C-8b), δ H 2.78 (H-2a), 2.72 (H-2b) with δ C 172.0 (C-11), 61.4 (C-8b) and d A ( 13 C), d N ( 13 C), and d I ( 13C) Scoring values were used to determine the planar structures of compounds 2a and 2b. Subsequently, the relative configurations of compounds 2a and 2b were confirmed as 3aS*, 8bS* by NMR calculation combined with statistical analysis methods. By analyzing the optical rotation and ECD spectra, it was determined that compounds 2a and 2b were a pair of enantiomers, and chiral resolution was successfully performed on them. The absolute configurations of 2a and 2b were determined by comparing the measured and calculated ECD spectra. Among them, compound 2a had a positive Cotton effect at 244 nm, and compound 2b had a negative Cotton effect at 245 nm. After searching in Scifinder, they were new compounds not reported in the literature, and they were named (-)-flavaperviridis B and (+)-flavaperviridis B respectively.

[0058] Table 2 NMR data attribution of compounds 2a and 2b

[0059]

[0060] The structure identification data of compound 3 are as follows:

[0061] White oil (methanol), (c 0.50, CH3OH). HR-ESI-MS gave the molecular ion peak m / z 592.2174 [M + CH3OH - H2O + H] + (calcd. 592.2183 for C 32 H 34 NO 10 ), m / z 614.2000 [M + CH3OH - H2O + Na] + (calcd. 614.2002 for C 32 H 33 NO 10 Na), and its molecular formula was speculated to be C 31 H 31 NO 10 , and the degree of unsaturation was 17. 1 In 1H NMR (600 MHz, CDCl3), three sets of benzene ring hydrogen signals were observed in the low field region, including one set of singly substituted benzene ring hydrogen signals: δ H 7.16 (1H, m, H-4”), 7.12 (2H, m, H-3”, 5”), 7.03 (2H, m, H-2”, 6”); one set of 1,4-disubstituted benzene ring hydrogen signals: δ H 7.21 (2H, d, J = 9.0 Hz, H-3', 5'), 6.75 (2H, d, J = 9.0 Hz, H-2', 6'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H6.28 (1H, d, J = 2.4 Hz, H-9), 6.26 (1H, d, J = 2.4 Hz, H-7); A set of coupled methine hydrogen signals can be observed in the high-field region: δ H 4.28 (1H, d, J = 4.8 Hz, H-4), 3.95 (1H, d, J = 4.8 Hz, H-3); Three methoxy hydrogen signals: δ H 3.78 (3H, s, 8 - OC H 3), 3.74 (3H, s, 4'-OC H 3), 3.65 (3H, s, 16 - OC H 3); Three sets of methylene hydrogen signals: δ H 3.84 (1H, m, H-13a), 3.72 (1H, m, H-13b), 2.02 (2H, m, H2-15), 1.51 (2H, m, H2-14). 13 13C NMR (150 MHz, CDCl3) gives a total of 31 carbon signals, including three sets of benzene ring carbon signals, namely one set of monosubstituted benzene ring carbon signals: δ C 134.3 (C-1”), 130.1×2 (C-2”, 6”), 128.5×2 (C-3”, 5”), 128.1 (C-4”); One set of 1,2,3,5-tetrasubstituted benzene ring carbon signals: δ C 160.8×2 (C-6, 8), 155.5 (C-9a), 114.9 (C-5a), 103.4 (C-9), 97.4 (C-7); One set of 1,4-disubstituted benzene ring carbon signals: δ C 159.3 (C-4'), 131.2 (C-1'), 127.2×2 (C-2', 6'), 113.5×2 (C-3', 5'); Three methoxy carbon signals: δ C 56.6 (8 - O C H3), 55.4 (4'-O C H3), 51.8 (16 - O C H3); One set of methyl γ-aminobutyrate carbon signals: δ C 173.3 (C-16), 167.7 (C-11), 40.4 (C-13), 31.2 (C-15), 22.5 (C-14); One ketone carbonyl carbon signal: δ C 190.0 (C-5); One carboxylic carbonyl carbon signal: δ C 170.3 (C-10), Two methine carbon signals: δ C 65.3 (C-4), 48.6 (C-3), and one sp 3 hybridized quaternary carbon signal: δ C84.9 (C-2). The hydrogen-carbon data were assigned using the HSQC spectrum (Table 3). In the HMBC spectrum, δ H 3.84 (H-13a) and 3.72 (H-13b) showed long-range correlations with δ C 31.2 (C-15) and 22.5 (C-14) respectively. δ H 3.65 (16-OC H 3), 2.02 (H2-15), and 1.51 (H2-14) showed long-range correlations with δ C 173.3 (C-16) respectively, confirming the structural fragment of methyl 4-aminobutyrate; 7.03 (H-2”,6”) showed a long-range correlation with δ C 48.6 (C-3), confirming the connection position of the monosubstituted benzene ring; δ H 6.26 (H-7) showed long-range correlations with δ C 160.8×2 (C-6,8), 114.9 (C-5a), and 103.4 (C-9). δ H 6.28 (H-9) showed long-range correlations with δ C 155.5 (C-9a), 114.9 (C-5a), and 97.4 (C-7). δ H 7.21 (H-2',6') showed a long-range correlation with δ C 84.9 (C-2), confirming the connecting group of the 1,2,3,5-tetrasubstituted benzene ring; δ H 3.78 (8-OC H 3) showed a long-range correlation with δ C 160.8 (C-8). δ H 3.74 (4'-OC H 3) showed a long-range correlation with δ C 159.3 (C-4'). Combining with the NOESY correlations between δ H 3.78 (8-OC H 3) and δ H 6.28 (H-9), 6.26 (H-7), it was confirmed that the remaining two methoxy groups were substituted at the C-8 and C-4' positions. Using the ACD / Structure Elucidator software, based on the HMBC correlation signals of δ H 3.95 (H-3) with δ C 190.0 (C-5), δ H 4.28 (H-4) with δ C 190.0 (C-5), 167.7 (C-11), 84.9 (C-2), the planar structure of compound 3 was finally confirmed.

[0062] In the NOESY spectrum, δ H 7.21 (H-2', 6'), 4.28 (H-4) showed correlation signals with δ H 3.96 (H-3), indicating that the benzene ring at C-2 was substituted and H-3 and H-4 were on the same side of the heptacyclic ring plane. Thus, the relative configuration of the compound was confirmed as 2R*, 3S*, 4S*. The absolute configuration was confirmed as 2R, 3S, 4S by ECD calculation. After searching in Scifinder, it was found to be a new compound not reported in the literature and was named flavaperviridis C.

[0063] Table 3 NMR data assignment of Compound 3

[0064]

[0065] Since Compounds 4a and 4b were a pair of enantiomers obtained by chiral column separation and their hydrogen and carbon data were exactly the same, the structural analysis of both was carried out simultaneously. The structural identification data of Compound 4 are as follows:

[0066] White oil (methanol), 4a: (c 0.50, methanol), 4b: (c 0.50, methanol). HR-ESI-MS gave the quasi-molecular ion peak m / z 673.2528 [M+Na] + (calcd. 673.2526 for C 38 H 38 N2O8Na), suggesting that its molecular formula was C 38 H 38 N2O8. 1 In 1H NMR (600 MHz, CD3OD), four sets of benzene ring hydrogen signals were observed in the low field region, including two sets of singly substituted benzene ring hydrogen signals: δ H 7.77 (2H, m, H-20, 24), 7.51 (1H, m, H-22), 7.44 (2H, m, H-21, 23), and δ H 7.03 (3H, m, H-3”, 4”, 5”), 6.90 (2H, m, H-2”, 6”); one set of 1,4-disubstituted benzene ring hydrogen signals: δ H 6.98 (2H, d, J = 9.0 Hz, H-2', 6'), 6.68 (1H, d, J = 9.0 Hz, H-3', 5'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H 6.40 (1H, d, J = 1.8 Hz, H-5), 6.16 (1H, d, J = 1.8 Hz, H-7); an isolated methine hydrogen signal was observed in the high field region: δ H4.22 (1H, s, H-3); signals of three methoxy hydrogens: δ H 3.84 (3H, s, 6-OC H 3), 3.75 (3H, s, 8-OC H 3), 3.69 (3H, s, 4'-OC H 3); signals of one set of 1,4-butanediamide hydrogens: δ H 3.31 (2H, m, H2-16), 3.19 (1H, m, H-13a), 3.15 (1H, m, H-13b), 1.52 (4H, m, H2-14, 15). 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including one set of carbon signals of the cyclopenta[b]benzofuran nucleus: δ C 206.8 (C-1), 101.1×2 (C-2, 3a), 90.0 (C-8b), 52.1 (C-3); four sets of benzene ring carbon signals, one set of 1,4-butanediamide carbon signals; and three methoxy carbon signals. The hydrogen-carbon data were assigned using the HSQC spectrum (Table 4). In the HMBC spectrum, δ H 6.98 (H-2', 6') was correlated with δ C 101.2 (C-3a), δ H 6.90 (H-2”, 6”) was correlated with δ C 52.1 (C-3), δ H 4.22 (H-3) was correlated with δ C 168.2 (C-11), 101.2 (C-2, 3a), confirming that the 1,4-disubstituted benzene ring, monosubstituted benzene ring and bisamide side chain are connected to C-3a, C-3 and C-2 positions respectively; the carbon chemical shifts δ C 206.8 (C-1), 101.1 (C-2) and 168.2 (C-11) and 1 In the 1H NMR (600 MHz, CDCl3) spectrum, keto-enol tautomerism was exhibited, indicating the presence of an α,β-unsaturated enol fragment in the compound. In summary, the planar structures of compounds 4a and 4b were confirmed. According to the biosynthetic pathway, 8b-OH and 3a-aryl are on the same side; δ H6.94 (H-2',6') and 6.97 (H-2'',6'') are at a higher field, indicating that C-3a and the benzene ring at C-3 are on the same side. Thus, the relative configurations of compounds 4a and 4b were determined. By analyzing the optical rotation and ECD spectra, it was determined that compounds 4a and 4b are a pair of racemates, and chiral resolution was successfully carried out on them. The absolute configurations of 4a and 4b were determined by comparing the measured and calculated ECD spectra. Among them, compound 4a has a positive Cotton effect at 308 nm and negative Cotton effects at 246 and 263 nm, while 4b has positive Cotton effects at 226 and 261 nm and a negative Cotton effect at 309 nm. After searching in Scifinder, they are new compounds not reported in the literature, and they were named (-)-flavaperviridis D and (+)-flavaperviridis D respectively.

[0067] Table 4 NMR data assignment of compounds 4a and 4b

[0068]

[0069] Since compounds 5a and 5b are a pair of enantiomers obtained by chiral column resolution and their hydrogen and carbon data are exactly the same, their structural analysis was carried out simultaneously. The structural identification data of compounds 5a and 5b are as follows:

[0070] White oil (methanol), 5a: (c 0.50, methanol), 5b: (c 0.50, methanol). HR-ESI-MS gave the quasi-molecular ion peak m / z 649.2553 [M+H] + (calcd. 649.2550 for C 38 H 37 N2O8), and its molecular formula was speculated to be C 38 H 36 N2O8. 1 In 1H NMR (600 MHz, CDCl3), four sets of benzene ring hydrogen signals were observed in the low field region, namely two sets of monosubstituted benzene ring hydrogen signals: δ H 7.75 (2H, m, H-20,24), 7.48 (1H, m, H-22), 7.40 (2H, m, H-21,23), and δ H 7.32 (1H, m, H-4''), 7.23 (4H, m, H-2'',3'',5'',6''); one set of 1,4-disubstituted benzene ring hydrogen signal: δ H7.27 (2H, d, J = 9.0 Hz, H-2', 6'), 6.89 (1H, d, J = 9.0 Hz, H-3', 5'); and one set of hydrogen signals of 1,2,3,5-tetrasubstituted benzene ring: δ H 6.17 (1H, d, J = 1.8 Hz, H-5), 6.08 (1H, d, J = 1.8 Hz, H-7); Three methoxy hydrogen signals can be observed in the high field region: δ H 3.82 (3H, s, 6-OC H 3), 3.79 (3H, s, 4'-OC H 3), 3.78 (3H, s, 8-OC H 3); One set of 1,4-butanediamide hydrogen signals: δ H 3.48 (2H, m, H2-16), 3.42 (1H, m, H-13a), 3.37 (1H, m, H-13b), 1.67 (4H, m, H2-14, 15). 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including one set of carbon signals of the cyclopenta[b]benzofuran nucleus: δ C 200.7 (C-1), 171.9 (C-3), 131.8 (C-2), 98.6 (C-3a), 86.5 (C-8b). The chemical shift values of C-1, C-2 and C-3 indicate the presence of an α,β-unsaturated ketone fragment; Four sets of benzene ring carbon signals, one set of 1,4-butanediamide carbon signals; and three methoxy carbon signals. The hydrogen-carbon data were assigned using the HSQC spectrum (Table 5). In the HMBC spectrum, δ H 7.27 (H-2', 6') is correlated with δ C 98.6 (C-3a), δ H 7.23 (H-2”, 6”) is correlated with δ CThere is a correlation with 171.9 (C-3), and it is confirmed that the 1,4-substituted benzene ring, monosubstituted benzene ring, and bisamide side chain are connected to C-3a, C-3, and C-2 positions respectively. According to the biosynthetic pathway, 8b-OH and 3a-aryl are on the same side. Thus, the relative configurations of compounds 5a and 5b are determined. Through the analysis of optical rotation and ECD spectra, it is determined that compounds 5a and 5b are a pair of enantiomers, and their chiral resolution is successfully carried out. The absolute configurations of 5a and 5b are determined by comparing the measured and calculated ECD spectra respectively. Among them, compound 5a has a negative Cotton effect at 269 nm and a positive Cotton effect at 312 nm, while 5b has a positive Cotton effect at 269 nm and a negative Cotton effect at 311 nm. After searching in Scifinder, they are new compounds not reported in the literature, and they are named (-)-flavaperviridis E and (+)-flavaperviridis E respectively.

[0071] Since compounds 6a and 6b are a pair of enantiomers obtained by chiral column separation and their hydrogen-carbon data are completely the same, their structural analysis is carried out simultaneously. The structural identification data of compounds 6a and 6b are as follows:

[0072] White oil (methanol), 6a: (c 0.50, methanol), 6b: (c 0.50, methanol). HR-ESI-MS gives the quasi-molecular ion peak m / z 649.2547 [M+H] + (calcd. 649.2550 for C 38 H 37 N2O8), and it can be speculated that its molecular formula is C 38 H 36 N2O8. 1 In 1H NMR (600 MHz, CDCl3), 4 sets of benzene ring hydrogen signals are observed in the low field region, including 2 sets of monosubstituted benzene ring hydrogen signals: δ H 7.74 (2H, m, H-20, 24), 7.50 (1H, m, H-22), 7.42 (2H, m, H-21, 23), and δ H 7.57 (2H, m, H-2”, 6”), 7.37 (3H, m, H-3”, 4”, 5”); 1 set of 1,4-substituted benzene ring hydrogen signals: δ H 7.37 (2H, d, J = 9.0 Hz, H-2', 6'), 6.92 (1H, d, J = 9.0 Hz, H-3', 5'); and 1 set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H6.26 (1H, d, J = 1.8 Hz, H-5), 6.09 (1H, d, J = 1.8 Hz, H-7); Three methoxy hydrogen signals can be observed in the high-field region: δ H 3.80 (6H, s, 6,8-OC H 3), 3.76 (3H, s, 4'-OC H 3); One set of 1,4-butanediamide hydrogen signals: δ H 3.37 (1H, m, H-13a), 3.30 (2H, m, H2-16), 3.20 (1H, m, H-13b), 1.45 (1H, m, H-14a), 1.40 (1H, m, H-14b), 1.30 (2H, m, H2-15). 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including one set of cyclopenta[b]benzofuran nucleus carbon signals: δ C 199.7 (C-1), 152.3 (C-3), 143.6 (C-2), 97.2 (C-3a), 86.0 (C-8b). The chemical shift values of C-1, C-2 and C-3 suggest the presence of an α,β-unsaturated ketone fragment; four sets of benzene ring carbon signals, one set of 1,4-butanediamide carbon signals; and three methoxy carbon signals. The hydrogen-carbon data were assigned using the HSQC spectrum (Table 5). Compared with the 1 H and 13 13C NMR data of compounds 5a and 5b, the NMR data of the two are very similar, only showing obvious differences in the chemical shifts of H-2” / H-6”, C-2, and C-3. It is speculated that compounds 6a and 6b are positional isomers of compounds 5a and 5b at the C-2 and C-3 positions. In the HMBC spectrum, δ H 7.37 (H-2',6') is correlated with δ C 97.2 (C-3a), δ H 7.57 (H-2”,6”) is correlated with δ CRelated to 143.6 (C-2), a 1,4-substituted benzene ring, a monosubstituted benzene ring, and a bisamide side chain were confirmed to be connected to C-3a, C-2, and C-3 positions respectively. According to the biosynthetic pathway, 8b-OH is on the same side as 3a-aryl. Thus, the relative configurations of compounds 6a and 6b were determined. By optical rotation and ECD spectral analysis, it was determined that compounds 6a and 6b are a pair of enantiomers, and their chiral resolution was successfully carried out. The absolute configurations of 6a and 6b were determined by comparing the measured and calculated ECD spectra respectively. Among them, compound 6a has negative Cotton effects at 217 and 274 nm and a positive Cotton effect at 246 nm, while 6b has positive Cotton effects at 220 and 274 nm and a negative Cotton effect at 242 nm. After searching in Scifinder, they are new compounds not reported in the literature, and they were named (-)-flavaperviridis F and (+)-flavaperviridis F respectively.

[0073] Table 5 NMR data attribution of compounds 5a, 5b, 6a, and 6b

[0074]

[0075]

[0076] Since compounds 7a and 7b are a pair of enantiomers obtained by chiral column resolution and their hydrogen and carbon data are exactly the same, their structural analysis was carried out simultaneously. The structural identification data of compounds 7a and 7b are as follows:

[0077] White oil (methanol), 7a: (c 0.50, methanol), 7b: (c 0.50, methanol). HR-ESI-MS gave a quasi-molecular ion peak at m / z 651.2708 [M+H] + (calcd. 651.2706 for C 38 H 39 N2O8), and its molecular formula was speculated to be C 38 H 38 N2O8. 1 1H NMR (600 MHz, CDCl3) gave a set of characteristic hydrogen signals of flavaglines compounds: δ H 4.11 (1H, d, J = 4.2 Hz, H-3), 3.56 (1H, d, J = 4.2 Hz, H-4); in addition, 4 sets of benzene ring hydrogen signals were observed in the low field region, which were 2 sets of monosubstituted benzene ring hydrogen signals: δ H7.80 (2H, m, H-20, 24), 7.46 (1H, m, H-22), 7.37 (2H, m, H-21, 23), and δ H 7.11 (2H, m, H-2”, 6”), 7.08 (3H, m, H-3”, 4”, 5”); one set of 1,4-substituted benzene ring hydrogen signals: δ H 6.97 (2H, d, J = 9.0 Hz, H-2', 6'), 6.63 (1H, d, J = 9.0 Hz, H-3', 5'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H 6.37 (1H, d, J = 1.8 Hz, H-9), 6.21 (1H, d, J = 1.8 Hz, H-7); three methoxy hydrogen signals can be observed in the high field region: δ H 3.87 (3H, s, 6-OC H 3), 3.85 (3H, s, 8-OC H 3), 3.73 (3H, s, 4'-OC H 3); one set of 1,4-butanediamide hydrogen signals: δ H 3.51 (1H, m, H-16a), 3.37 (2H, m, H-13a, 16b), 2.36 (1H, m, H-13b), 1.75 (1H, m, H-15a), 1.66 (3H, m, H-14a, 14b, 15a). 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including one set of cyclopenta[bc]benzopyran nucleus carbon signals: δ C 90.2 (C-10), 87.7 (C-2), 85.4 (C-5), 59.5 (C-4), 50.9 (C-3), four sets of benzene ring carbon signals, one set of 1,4-butanediamide carbon signals; and three methoxy carbon signals. The hydrogen-carbon data were assigned using the HSQC spectrum (Table 6). The chemical shift of C-10 and the isolated methine hydrogen signal that disappeared in the hydrogen spectrum indicated that C-10 was a quaternary carbon signal. Compared with the 1 H and 13 13C NMR data of the reported compound cyclofoveoglin, the NMR data of the two were very similar, only showing obvious differences in the coupling constant 3 J H-3 / H-4 , and the hydrogen chemical shifts H-9 / H-2' / H-6', indicating that compounds 7a and 7b were C-3 epimers of the compound cyclofoveoglin. In the HMBC spectrum, δ H 3.58 (10-O H ) was correlated with δ C 87.7 (C-2), δH 5.51 (5-O H ) is correlated with δ C 90.2 (C-10), 85.4 (C-5), δ H 3.56 (H-4) is correlated with δ C 90.2 (C-10), δ H 4.11 (H-3) is correlated with δ C 87.7 (C-2), confirming the cyclopenta[bc]benzopyran nucleus; δ H 6.97 (H-2',6') is correlated with δ C 87.7 (C-2), δ H 7.11 (H-2”,6”) is correlated with δ C 50.9 (C-3), δ H 4.11 (H-3) is correlated with δ C 174.1 (C-11), confirming the 1,4-substituted benzene ring, with the monosubstituted benzene ring and the bisamide side chain connected to C-2, C-3, and C-4 positions respectively; δ H 3.37 (H-13a) is correlated with δ C 90.2 (C-10), indicating that C-10 is connected to N-12, forming a new ring system. According to the coupling constant, the relative configuration of C-3 and C-4 is 3S*, 4S*; due to the rigidity of the polycyclic fusion, the relative configurations of C-2, C-4, C-5, and C-10 are homodromous. Therefore, the relative configurations of compounds 7a and 7b are determined to be 2R*, 3S*, 4S*, 5R*, 10R*.

[0078] Through the analysis of the optical rotation and ECD spectra, it was determined that compounds 7a and 7b are a pair of enantiomers, and their chiral resolution was successfully carried out. The absolute configurations of 7a and 7b were determined by comparing the measured and calculated ECD spectra. Among them, compound 7a has a negative Cotton effect at 217 nm and a positive Cotton effect at 232 nm, and 7b has a positive Cotton effect at 217 nm and a negative Cotton effect at 231 nm. After searching in Scifinder, they are new compounds not reported in the literature, and they are named (2S,3R,4R,5S,10S)-flavaperviridis G and (2R,3S,4S,5R,10R)-flavaperviridis G respectively.

[0079] Table 6 NMR data assignment of compounds 7a and 7b

[0080]

[0081] Since compounds 8a and 8b are a pair of enantiomers obtained by chiral column separation and their hydrogen-carbon data are exactly the same, the structural analysis of both was carried out simultaneously. The structural identification data of compounds 8a and 8b are as follows:

[0082] White oil (methanol), 8a: (c 0.50, methanol), 8b: (c 0.50, methanol). HR-ESI-MS gave the quasi-molecular ion peak m / z 651.2708 [M+H] + (calcd. 651.2706 for C 38 H 39 N2O8), suggesting the molecular formula is C 38 H 36 N2O8. 1 In 1H NMR (600 MHz, CDCl3), four sets of benzene ring hydrogen signals were observed in the low field region, including two sets of monosubstituted benzene ring hydrogen signals: δ H 7.74 (2H, m, H-3'',7''), 7.30 (1H, m, H-5''), 7.21 (2H, m, H-4'',6''), and δ H 7.03 (1H, m, H-7'), 6.88 (2H, m, H-6',8'), 6.79 (2H, m, H-5',9'); one set of 1,4-disubstituted benzene ring hydrogen signals: δ H 7.59 (2H, d, J = 8.4 Hz, H-3',5'), 6.88 (2H, d, J = 8.4 Hz, H-2',6'); and one set of 1,2,3,5-tetrasubstituted benzene ring hydrogen signals: δ H 6.41 (1H, d, J = 1.8 Hz, H-8), 6.17 (1H, d, J = 1.8 Hz, H-6); in the high field region, a set of ABX coupling system hydrogen signals was observed: δ H 5.63 (1H, d, J = 11.4 Hz, H-3'''), 3.23 (1H, dd, J = 18.0, 11.4 Hz, H-2'''a), 2.65 (1H, d, J = 18.0 Hz, H-2'''b); three methoxy hydrogen signals: δ H 3.91 (3H, s, 4'-OCH H 3), 3.85 (3H, s, 7-OCH H 3), 3.74 (3H, s, 5-OCH H 3); one set of 1,4-butanediamide hydrogen signals: δ H3.68 (1H, m, H-5”a), 3.60 (2H, m, H-2”a, 5”b), 3.51 (1H, m, H-2”b), 2.06 (1H, m, H-4”a), 1.93 (1H, m, H-3”a), 1.83 (2H, m, H-3”b, H-4”b). 13 13C NMR (150 MHz, CDCl3) gave a total of 38 carbon signals, including a set of characteristic flavonol C-ring carbon signals: δ C 175.3 (C-4), 156.5 (C-2), 137.2 (C-3); four sets of benzene ring carbon signals, namely two sets of monosubstituted benzene ring carbon signals, one set of 1,2,3,5-tetrasubstituted benzene ring carbon signals, and one set of 1,4-disubstituted benzene ring carbon signals; one set of 1,4-butanediamide carbon signals; three methoxy carbon signals, one oxygenated methylene carbon signal and one methylene carbon signal. By comparing the NMR data, 18 carbon signals were basically consistent with those of the compound 5,7,4'-trimethoxyflavonol

[28] , and the other 20 carbon signals were very similar to the NMR data of the compound pyramidatine. According to the hydrogen chemical shift δ H 5.63 (H-3”'), 3.23 (H-2”'a), 2.65 (H-2”'b) and the carbon chemical shift δ C 78.5 (C-3”'), 43.4 (C-2”'), it was speculated to be the double bond reduction product of pyramidatine. Therefore, compounds 8a and 8b are the adducts of 5,7,4'-trimethoxyflavonol and tetrahydropyramidatine. The hydrogen-carbon data were assigned using the HSQC spectrum (Table 8). In the HMBC spectrum, δ H 5.63 (H-3”') was correlated with δ C 170.5 (C-1”'), 137.8 (C-3), and δ H 6.79 (H-5”', 9”') was correlated with δ C 78.5 (C-3”'), indicating that the two units are connected through C-3-O-C-3”'. Thus, the planar structures of compounds 8a and 8b were confirmed.

[0083] Through the analysis of optical rotation and ECD spectra, it was determined that compounds 8a and 8b are a pair of racemates, and chiral resolution was successfully carried out on them. The absolute configurations of 8a and 8b were determined by comparing the measured and calculated ECD spectra. Among them, compound 8a has a negative Cotton effect at 235 and 265 nm and a positive Cotton effect at 254 nm, while 8b has a positive Cotton effect at 225 and 268 nm and a negative Cotton effect at 251 nm. After searching in Scifinder, they are new compounds not reported in the literature, and they were named (-)-flavaperviridis H and (+)-flavaperviridis H respectively.

[0084] Table 7 NMR data attribution of compounds 8a and 8b

[0085]

[0086]

[0087] Example 2

[0088] (1) 500 g of dried branches and leaves of Aglaia perviridis were extracted by heating under reflux with 95% ethanol twice (dosage: 5 L), and the extract was recovered under reduced pressure to obtain a crude extract.

[0089] (2) The 95% ethanol crude extract obtained in the above step (1) was dissolved in water and successively extracted with petroleum ether, ethyl acetate, and n-butanol. Each organic phase was extracted 5 times, and the volume ratio of the aqueous phase to the organic phase was 1:5 each time, to obtain extracts of different polar parts.

[0090] (3) The ethyl acetate extract in step (2) was separated by silica gel column chromatography and eluted successively with a mixed solvent of petroleum ether and acetone at ratios of 8:1, 5:1, 4:1, 2:1, and 1:1.

[0091] (4) The fractions of petroleum ether:acetone (8:1 - 2:1) obtained in the above step (3) were subjected to polyamide column chromatography and eluted with a gradient of mixed solvents of methanol - water at ratios of 3:7, 5:5, 7:3, and 9:1.

[0092] (5) The fractions of methanol:water (7:3 - 9:1) obtained in the above step (4) were subjected to ODS column chromatography and eluted with a gradient of mixed solvents of methanol - water at ratios of 3:7, 5:5, 7:3, and 9:1.

[0093] (6) The fractions of methanol:water (7:3 - 9:1) obtained in the above step (5) were separated and prepared by HPLC - RID chromatography at a flow rate of 3 mL / min, and the mobile phase was acetonitrile:water = 63:37, to obtain compound 2 (t R = 18.4 min) (yield 0.00022‰), compound 3 (tR = 22.3 min) (yield 0.00012‰), compound 7 (t R = 24.2 min) (yield 0.00005‰), and compound 8 (t R = 29.7 min) (yield 0.00005‰). The racemic mixture of compound 2 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (52:48) as the mobile phase to obtain new 2a (6.4 min), 2b (9.4 min) (yields 0.00011‰ each); the racemic mixture of compound 7 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (53:47) as the mobile phase to obtain new 7a (10.2 min), 7b (15.1 min) (yields 0.00002‰ each); the racemic mixture of compound 8 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (62:38) as the mobile phase to obtain new 8a (7.1 min), 8b (8.3 min) (yields 0.00002‰ each).

[0094] (7) The methanol: water (3:7 - 5:5) fraction obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was acetonitrile: water = 40:60, to obtain compound 1 (t R = 63.7 min) (yield 0.00023‰), compound 4 (t R = 31.8 min) (yield 0.00014‰), compound 5 (t R = 37.4 min) (yield 0.00024‰), compound 6 (t R = 42.9 min) (yield 0.00015‰). The racemic mixture of compound 1 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (62:38) as the mobile phase to obtain new 1a (26.2 min), 1b (31.4 min) (yields 0.00011‰ each); the racemic mixture of compound 4 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (72:28) as the mobile phase to obtain new 4a (5.7 min), 4b (12.9 min) (yields 0.00007‰ each); the racemic mixture of compound 5 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (58:42) as the mobile phase to obtain new 5a (17.2 min), 5b (22.7 min) (yields 0.00012‰ each); the racemic mixture of compound 6 was further separated by HPLC chiral column chromatography, eluted with n-hexane: absolute ethanol (72:28) as the mobile phase to obtain new 6a (7.9 min), 6b (9.1 min) (yields 0.00007‰ each).

[0095] The structural identification methods of flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b are shown in Example 1.

[0096] Example 3

[0097] (1) 1000 g of dried branches and leaves of Aglaia perviridis were extracted by heating under reflux with 70% ethanol three times (dosage: 15 L), and the extract was recovered under reduced pressure to obtain a crude extract;

[0098] (2) The 70% ethanol extract obtained in the above step (1) was extracted with organic solvents, successively extracted with cyclohexane, dichloromethane, ethyl acetate, and n-butanol, each organic phase was extracted 3 times, and the volume ratio of the aqueous phase to the organic phase was 1:2 each time, to obtain extracts of different polar parts;

[0099] (3) The ethyl acetate extract obtained in the above step (2) was separated by silica gel column chromatography, and eluted successively with a mixed solvent of dichloromethane and acetone at 100:1, 50:1, 20:1, 10:1, 8:1, 5:1, 4:1, 2:1, 1:1;

[0100] (4) The dichloromethane:acetone (10:1 - 2:1) fraction obtained in the above step (3) was subjected to polyamide column chromatography, and gradient elution was carried out with a mixed solvent of methanol - water at 3:7, 5:5, 7:3, 9:1;

[0101] (5) The methanol:water (5:5 - 7:3) fraction obtained in the above step (4) was subjected to ODS chromatography, and gradient elution was carried out with a mixed solvent of acetonitrile - water at 2:8, 4:6, 6:4, 8:2;

[0102] (6) The acetonitrile:water (6:4 - 8:2) fraction obtained in the above step (5) was separated and prepared by HPLC - UV chromatography, detected at 210 nm, the flow rate was 3 mL / min, and the mobile phase was methanol:water = 73:27, to obtain compound 3 (t R = 21.9 min) (yield 0.00008‰), compound 7 (t R = 23.5 min) (yield 0.00004‰), and compound 8 (t R= 28.4 min) (yield 0.00004 ‰). The racemic mixture of compound 7 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (55:45) as the mobile phase to obtain new 7a (13.9 min), 7b (20.1 min) (yields 0.00002 ‰ each); the racemic mixture of compound 8 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (65:35) as the mobile phase to obtain new 8a (9.3 min), 8b (10.2 min) (yields 0.00002 ‰ each).

[0103] (7) The fraction of acetonitrile: water (2:8 - 4:6) obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was methanol: water = 48:52, to obtain compound 1 (t R = 62.2 min) (yield 0.0002 ‰), compound 2 (t R = 75.2 min) (yield 0.00016 ‰), compound 4 (t R = 32.0 min) (yield 0.00014 ‰), compound 5 (t R = 37.4 min) (yield 0.00023 ‰), and compound 6 (t R = 41.3 min) (yield 0.00012 ‰). The racemic mixture of compound 1 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (55:45) as the mobile phase to obtain new 1a (13.2 min), 1b (16.4 min) (yields 0.0001 ‰ each); the racemic mixture of compound 2 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (55:45) as the mobile phase to obtain new 2a (9.1 min), 2b (15.3 min) (yields 0.00008 ‰ each); the racemic mixture of compound 4 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (75:25) as the mobile phase to obtain new 4a (7.2 min), 4b (15.2 min) (yields 0.00007 ‰ each); the racemic mixture of compound 5 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (55:45) as the mobile phase to obtain new 5a (11.0 min), 5b (14.3 min) (yields 0.00011 ‰ each); the racemic mixture of compound 6 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (75:25) as the mobile phase to obtain new 6a (9.3 min), 6b (12.8 min) (yields 0.00006 ‰ each).

[0104] The structural identification methods of flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b can be found in Example 1.

[0105] Example 4

[0106] (1) 1500 g of dried Aglossa rotundifolia (Bl.) O. Ktze. branches and leaves were extracted by heating under reflux with 60% methanol 4 times (dosage: 18 L), and the extract was recovered under reduced pressure to obtain a crude extract.

[0107] (2) The methanol extract obtained in the above step (1) was extracted with organic solvents. It was successively extracted with cyclohexane, ethyl acetate, and n-butanol at a volume ratio of water phase to organic phase of 1:4, and extracted 4 times respectively to obtain extracts of different polar parts.

[0108] (3) The ethyl acetate extract obtained in the above step (2) was separated by silica gel column chromatography and eluted successively with a mixed solvent of chloroform and acetone at 50:1, 20:1, 10:1, 8:1, 5:1, 4:1, 2:1, 1:1.

[0109] (4) The chloroform:acetone (10:1 - 2:1) fraction obtained in the above step (3) was subjected to polyamide column chromatography and eluted with a gradient of a mixed solvent of ethanol - water at 1:9, 3:7, 5:5, 7:3, 9:1.

[0110] (5) The ethanol:water (5:5 - 7:3) fraction obtained in the above step (4) was subjected to ODS chromatography and eluted with a gradient of a mixed solvent of acetonitrile - water at 1:9, 3:7, 5:5, 7:3, 9:1.

[0111] (6) The acetonitrile:water (7:3 - 9:1) fraction obtained in the above step (5) was separated and prepared by HPLC - UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was methanol:water = 68:32, to obtain the racemic mixture compounds 1 (t R = 19.3 min) (yield 0.00019‰), compound 2 (t R = 23.8 min) (yield 0.00014‰), compound 3 (t R = 27.6 min) (yield 0.00007‰), compound 7 (t R = 30.9 min) (yield 0.00004‰), and compound 8 (t R= 39.4 min) (yield: 0.00003‰). The racemic mixture of compound 1 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (57:43) as the mobile phase to obtain new 1a (16.9 min) and 1b (22.4 min) (yields: 0.00009‰ each); the racemic mixture of compound 2 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (60:40) as the mobile phase to obtain new 2a (19.7 min) and 2b (30.9 min) (yields: 0.00007‰ each); the racemic mixture of compound 7 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (45:55) as the mobile phase to obtain new 7a (3.4 min) and 7b (4.9 min) (yields: 0.00002‰ each); the racemic mixture of compound 8 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (60:40) as the mobile phase to obtain new 8a (4.6 min) and 8b (5.7 min) (yields: 0.00001‰ each).

[0112] (7) The fraction of acetonitrile: water (3:7 - 5:5) obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was methanol: water = 50:50, to obtain compound 4 (t R = 19.6 min) (yield: 0.0001‰), compound 5 (t R = 21.7 min) (yield: 0.00017‰), and compound 6 (t R = 24.3 min) (yield: 0.0001‰). The racemic mixture of compound 4 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (80:20) as the mobile phase to obtain new 4a (13.7 min) and 4b (32.6 min) (yields: 0.00005‰ each); the racemic mixture of compound 5 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (57:43) as the mobile phase to obtain new 5a (15.3 min) and 5b (20.5 min) (yields: 0.00008‰ each); the racemic mixture of compound 6 was further separated by HPLC chiral column chromatography and eluted with n-hexane: absolute ethanol (80:20) as the mobile phase to obtain new 6a (19.4 min) and 6b (25.7 min) (yields: 0.00005‰ each).

[0113] The structural identification methods of flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b are shown in Example 1.

[0114] Example 5

[0115] (1) 2000 g of dried branches and leaves of Garcinia renigera are extracted by heating and ultrasonic treatment with 80% methanol 5 times (dosage: 30 L), and the extract is recovered under reduced pressure to obtain a crude extract;

[0116] (2) The methanol extract obtained in the above step (1) is extracted with organic solvents. Dichloromethane, ethyl acetate, and n-butanol are used to extract with a volume ratio of aqueous phase to organic phase of 1:1, and each is extracted 5 times to obtain extracts of different polar parts;

[0117] (3) The chloroform extract obtained in the above step (2) is separated by silica gel column chromatography and eluted successively with a mixed solvent of dichloromethane and methanol at 20:1, 10:1, 8:1, 5:1, 4:1, and 2:1;

[0118] (4) The fraction of dichloromethane:methanol (10:1 - 3:1) obtained in the above step (3) is subjected to polyamide column chromatography and eluted with a gradient of a mixed solvent of ethanol-water at 3:7, 5:5, 7:3, and 9:1;

[0119] (5) The fraction of methanol:water (5:5 - 7:3) obtained in the above step (4) is subjected to ODS chromatography and eluted with a gradient of a mixed solvent of acetonitrile-water at 2:8, 4:6, 6:4, and 8:2;

[0120] (6) The fraction of acetonitrile:water (6:4 - 8:2) obtained in the above step (5) is separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile:water = 60:40, to obtain the racemic mixture compound 1 (t R = 17.2 min) (yield 0.00023‰), 2 (t R = 22.4 min) (yield 0.0002‰), compound 3 (t R = 26.8 min) (yield 0.00012‰), 7 (t R = 28.5 min) (yield 0.00006‰), and 8 (t R= 37.9 min) (yield 0.00004‰). The racemic mixture of Compound 1 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (50:50) as the mobile phase to obtain new 1a (6.1 min), 1b (8.2 min) (yields 0.00011‰ each); the racemic mixture of Compound 2 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (58:42) as the mobile phase to obtain new 2a (16.1 min), 2b (24.3 min) (yields 0.0001‰ each); the racemic mixture of Compound 7 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (48:52) as the mobile phase to obtain new 7a (6.1 min), 7b (7.6 min) (yields 0.00003‰ each); the racemic mixture of Compound 8 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (67:33) as the mobile phase to obtain new 8a (13.4 min), 8b (17.2 min) (yields 0.00002‰ each).

[0121] (7) The fraction of acetonitrile: water (2:8 - 4:6) obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was acetonitrile: water = 42:58, to obtain Compound 4 (t R = 26.8 min) (yield 0.00015‰), 5 (t R = 30.4 min) (yield 0.00024‰) and 6 (t R = 33.7 min) (yield 0.00014‰). The racemic mixture of Compound 4 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (77:23) as the mobile phase to obtain new 4a (10.3 min), 4b (19.4 min) (yields 0.00007‰ each); the racemic mixture of Compound 5 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (53:47) as the mobile phase to obtain new 5a (8.4 min), 5b (11.3 min) (yields 0.00012‰ each); the racemic mixture of Compound 6 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (77:23) as the mobile phase to obtain new 6a (16.2 min), 6b (21.4 min) (yields 0.00007‰ each).

[0122] The structural identification methods of flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b are shown in Example 1.

[0123] Example 6

[0124] (1) 2500 g of dried branches and leaves of Horsfieldia kingii are extracted 3 times by heating and ultrasonic extraction with 90% methanol (dosage: 50 L), and the extract is recovered under reduced pressure to obtain a crude extract;

[0125] (2) The methanol extract obtained in the above step (1) is extracted with organic solvents. It is successively extracted with petroleum ether, chloroform, ethyl acetate, and n-butanol at a volume ratio of water phase to organic phase of 1:3, and extracted 3 times respectively to obtain extracts of different polar parts;

[0126] (3) The ethyl acetate extract obtained in the above step (2) is separated by silica gel column chromatography and eluted successively with a mixed solvent of chloroform and methanol at 10:1, 8:1, 5:1, 4:1, 2:1;

[0127] (4) The chloroform:methanol (10:1 - 3:1) fraction obtained in the above step (3) is subjected to polyamide column chromatography and eluted with a gradient of mixed solvents of ethanol - water at 3:7, 5:5, 7:3, 9:1;

[0128] (5) The ethanol:water (5:5 - 7:3) fraction obtained in the above step (4) is subjected to ODS chromatography and eluted with a gradient of mixed solvents of acetonitrile - water at 1:9, 3:7, 5:5, 7:3, 9:1;

[0129] (6) The acetonitrile:water (7:3 - 9:1) fraction obtained in the above step (5) is separated and prepared by HPLC - UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase is acetonitrile:water = 65:35, to obtain the racemic mixture compounds 1 (t R = 9.1 min) (yield 0.00025‰), 2 (t R = 13.7 min) (yield 0.00022‰), compound 3 (t R = 14.9 min) (yield 0.00014‰), 7 (t R = 15.2 min) (yield 0.00007‰), and 8 (t R= 22.6 min) (yield 0.00004‰). The racemic mixture of Compound 1 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (58:42) as the mobile phase to obtain new 1a (17.8 min), 1b (24.7 min) (yields 0.00012‰ each); the racemic mixture of Compound 2 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (57:43) as the mobile phase to obtain new 2a (13.8 min), 2b (21.4 min) (yields 0.00011‰ each); the racemic mixture of Compound 7 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (58:42) as the mobile phase to obtain new 7a (19.3 min), 7b (27.9 min) (yields 0.00003‰ each); the racemic mixture of Compound 8 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (72:28) as the mobile phase to obtain new 8a (21.5 min), 8b (26.3 min) (yields 0.00002‰ each).

[0130] (7) The fraction of acetonitrile: water (3:7 - 5:5) obtained in the above step (5) was separated and prepared by HPLC-UV chromatography, detected at 210 nm, with a flow rate of 3 mL / min, and the mobile phase was acetonitrile: water = 45:55, to obtain Compound 4 (t R = 20.3 min) (yield 0.00017‰), 5 (t R = 22.4 min) (yield 0.00026‰) and 6 (t R = 24.8 min) (yield 0.00016‰). The racemic mixture of Compound 4 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (75:25) as the mobile phase to obtain new 4a (7.2 min), 4b (15.2 min) (yields 0.00008‰ each); the racemic mixture of Compound 5 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (50:50) as the mobile phase to obtain new 5a (5.1 min), 5b (7.2 min) (yields 0.00013‰ each); the racemic mixture of Compound 6 was further separated by HPLC chiral column chromatography, eluting with n-hexane: absolute ethanol (70:30) as the mobile phase to obtain new 6a (4.8 min), 6b (6.5 min) (yields 0.00008‰ each).

[0131] The structural identification methods of flavaglines compounds 1a, 1b, 2a, 2b, 3, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b are shown in Example 1.

[0132] Example 7. Anti-neuroinflammatory activity test of the newly prepared flavaglines compounds 1-8 in Examples 1-6. (1) Experimental principle: The chronic inflammatory response mediated by the over-activation of microglia is an important link in the occurrence and development of neurodegenerative diseases. Therefore, inhibiting the over-activated microglia may become a new target for drug discovery. LPS can activate microglia to release NO, TNF-α, IL-6, IL-1β, etc. In this experiment, an in vitro screening model of abnormally activated BV-2 microglia induced by LPS was established, and the anti-neuroinflammatory activity of the newly prepared flavaglines compounds 1-8 was evaluated with the NO release amount as an index.

[0133] (2) Experimental methods:

[0134] ① Culture of mouse microglial cell line BV-2

[0135] 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 min to completely remove the contaminated LPS. A cell culture medium containing 10% fetal bovine serum was prepared based on DMEM medium. Microglia were passaged at a concentration of about 2.0×10 5 cells / mL in a 5% CO2, 37 °C culture flask. By the third day, the adherent cells accounted for about 70-80% of the bottom area of the culture flask. The adherent cells were digested with trypsin and passaged to another culture flask. The BV-2 cells after cryopreservation and resuscitation in an ultra-low temperature freezer at -80 °C were used as the first generation, and the 3rd-8th generation BV-2 cells were selected for the experiment.

[0136] ② Method for preparing the drug

[0137] The compounds to be tested were all dissolved in DMSO. A stock solution (100 mM) was prepared and stored at -20 °C. When in use, it was diluted with DMEM culture medium to 0.001 μM, 0.01 μM, 0.1 μM, and 1 μM in sequence. The final concentration of DMSO was <1‰.

[0138] ③ Detection of the inhibitory effect of the compound on LPS-activated microglia by the Griess method

[0139] Take BV-2 microglia in the logarithmic growth phase and adjust the cell density to 2.0×10 with fresh DMEM culture medium containing 10% fetal bovine serum 5cells / mL, inoculated into 96-well plates, 100 μL / well, and cultured in an incubator at 37 °C with 5% CO₂. After the cells adhered and were cultured for 24 h, they were changed to fresh serum-free culture medium, and drug treatment was carried out simultaneously. The test concentrations of the compound were 0.001 μM, 0.01 μM, 0.1 μM, 1 μM, which were co-administered with LPS. At the same time, a blank control was set. The final concentration of LPS in each drug administration group was 100 ng / mL. After the cells were treated with the drug and cultured for another 24 h, the supernatant was collected, and the NO content in the supernatant was detected by the Griess colorimetric method. 2- Content.

[0140] ④ Detection of the effect of the compound on the viability of microglial cells by the MTT method

[0141] BV-2 microglial cells cultured in the logarithmic growth phase were taken, and the cell density was adjusted to 2.0×10 5 cells / mL, inoculated into 96-well plates, 100 μL / well, and cultured in an incubator at 37 °C with 5% CO₂. After the cells adhered and were cultured for 24 h, they were changed to fresh culture medium, and drug treatment was carried out simultaneously. The compound doses of 100 μM, 30 μM, 10 μM, 1 μM were co-administered with LPS. At the same time, a blank control was set. The final concentration of LPS in each drug administration group was 100 ng / mL. After the cells were treated with the drug and cultured for 24 h, then MTT solution was added to the cell solution, 10 μL / well, and the cells and 0.25 mg / mL MTT were incubated together at 37 °C for 3 h. The culture medium was aspirated, and then 150 μL of DMSO solution was added to measure its optical density OD value. Data processing was carried out using the corresponding software of the microplate reader, and the average value of the OD values of 3 wells of each sample was calculated. The cell viability (cell viability, CV%) was calculated using the average value according to the following formula.

[0142] Cell viability % = [Average value of OD values of the sample group / Average value of OD values of the blank control group] × 100%

[0143] ⑤ Statistical method

[0144] All data were analyzed using the SPSS (27.0) statistical software package. The results were expressed as mean ± standard error. To evaluate the overall difference, the homogeneity of variance of the group means was analyzed by the One-Way ANOVA analysis method, and the group means were compared by combining the Dunnett’s test analysis method. The Levene test was used for the homogeneity test of variances of multiple samples. When p > 0.05, the variances were homogeneous, and the Dunnett’s two-sided T test was used to analyze the differences in the means of multiple groups. When p < 0.05, the variances were heterogeneous, and the Dunnett T3 test was used to analyze the differences in the means of multiple groups.

[0145] ⑥IC 50 Calculation method

[0146] Use non - linear regression fitting to calculate IC with parameters such as each dose and inhibition rate 50 (see Table 10).

[0147] (3) Experimental results:

[0148] The experimental results are shown in Table 8, Table 9 and Table 10.

[0149] Table 8 Effects of Flavaglines compounds 1 - 8 on the viability of BV - 2 microglial cells

[0150]

[0151]

[0152] Note: *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS - induced group; ### P < 0.001 compared with the control group. Mino: positive drug. The concentrations of Mino are 1, 10, 30, 100 μM

[0153] Table 9 Effects of Flavaglines compounds 1 - 8 on the release of NO from LPS - activated BV - 2 microglial cells

[0154]

[0155] Note: *P < 0.05, **P < 0.01, ***P < 0.001 compared with the LPS - induced group; ### P < 0.001 compared with the control group. Mino: Minocycline, positive drug.

[0156] Table 10 Effects of Flavaglines compounds 1 - 8 on the release of NO from LPS - activated BV - 2 microglial cells

[0157]

[0158] Note: Mino: Minocycline, positive drug.

[0159] It can be seen from the results that the new Flavaglines compounds 2 (10 μM), 3 (1 μM, 10 μM, 30 μM), 5 (30 μM), 6 (1 μM, 10 μM, 30 μM, 100 μM) prepared in Embodiments 1 - 6 can significantly inhibit the release of NO from LPS - induced over - activated BV - 2 microglial cells.

Claims

1. A class of Flavaglines compounds with the following chemical structural formula and their pharmaceutically acceptable salts, characterized in that 2. The preparation method of the Flavaglines compound according to claim 1, characterized in that, The following steps are included: S1, The branches and leaves of Aglaiaperviridis Hiern are extracted with methanol or ethanol solution, and the extract is recovered to obtain a crude extract; the obtained crude extract is dissolved in water and extracted with an organic solvent to obtain extracts with different polarities; the obtained extracts are separated by silica gel column chromatography, gradient eluted with a mixed solvent, and the obtained fractions are separated by polyamide column chromatography, gradient eluted with a mixed solvent of methanol-water or ethanol-water as the mobile phase; S2, The fractions obtained in step S1 are separated by ODS column chromatography, gradient eluted with a mixed solvent of methanol-water or acetonitrile-water as the mobile phase; S3, The methanol-water or acetonitrile-water eluate obtained in step S2 is further separated by HPLC, gradient eluted with a mixed solvent of methanol and water or a mixed solvent of acetonitrile and water as the mobile phase to obtain racemic mixture compounds 1, 2, 4, 5, 6, 7, 8 and compound 3. Among them, the obtained flavaglines racemic mixture compounds 1, 2, 4, 5, 6, 7, 8 are subjected to chiral resolution by HPLC to obtain compounds 1a, 1b, 2a, 2b, 4a, 4b, 5a, 5b, 6a, 6b, 7a, 7b, 8a, 8b.

3. The method according to claim 2, wherein: In step S1, the extraction method with the methanol or ethanol solution is heating reflux ethanol extraction, heating reflux methanol extraction or heating ultrasonic extraction 2 - 5 times, the volume concentration of the ethanol solution is 70% - 95%, the volume concentration of the methanol solution is 60% - 90%, and the solid-liquid ratio is 1:8 - 1:20 g / mL.

4. The method according to claim 2, wherein In step S1, the organic solvent extraction is as follows: according to the volume ratio of the aqueous phase to the organic phase of 1:1 - 1:5, petroleum ether or cyclohexane, dichloromethane or chloroform, ethyl acetate, and n-butanol are used to extract 3 - 5 times in turn, and the above organic solvents are recovered under reduced pressure.

5. The method according to claim 2, characterized in that, The mixed solvent used in the silica gel column chromatography is one of gradient elution of a mixed solvent of petroleum ether and ethyl acetate, a mixed solvent of petroleum ether and acetone, a mixed solvent of chloroform and acetone, a mixed solvent of dichloromethane and acetone, a mixed solvent of chloroform and methanol, and a mixed solvent of dichloromethane and methanol.

6. The method according to claim 5, wherein The volume ratio of the petroleum ether to the ethyl acetate is 10:1 - 1:1, the volume ratio of the petroleum ether to the acetone is 8:1 - 1:1, the volume ratio of the dichloromethane to the acetone is 100:1 - 1:1, the volume ratio of the mixed solvent of chloroform and acetone is 50:1 - 1:1, the volume ratio of the dichloromethane to the methanol is 20:1 - 2:1, and the volume ratio of the chloroform to the methanol is 10:1 - 2:

1.

7. The method according to claim 2, wherein In step S1, the volume ratio of the methanol to the water is 1:9 - 1:9, and the volume ratio of the ethanol to the water is 1:9 - 1:9; in steps S2 and S3, the volume ratio of the methanol to the water is 2:8 - 8:2, and the volume ratio of the acetonitrile to the water is 1:9 - 9:

1.

8. The method according to claim 2, wherein In step S3, the chiral column resolution solvent is a mixed solvent of n-hexane and absolute ethanol, and its volume ratio is 40:60 - 80:

20.

9. A pharmaceutical composition, characterized in that, Comprising the flavaglines compound described in claim 1, its pharmaceutically acceptable salts and pharmaceutically acceptable carriers.

10. Use of the compound described in claim 1, its pharmaceutically acceptable salts or the pharmaceutical composition described in claim 9 in the preparation of a medicament for preventing or treating neurodegenerative diseases.

Images