Preparation method and application of indole diterpenoid compounds and their derivatives derived from endophytic fungi of Schisandra chinensis

Abstract

The present application discloses a preparation method and application of an indole diterpenoid compound and its derivatives derived from an endophytic fungus of Schisandra chinensis, which have any of the following structures: The above substances all have 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, HMGCR) degradation activity, which can not only significantly reduce the HMGCR protein level, but also inhibit the compensatory increase of HMGCR caused by statins.

Description

Technical Field

[0001] The present application relates to the technical field of organic synthesis of natural products, and in particular to a method for preparing an indole diterpenoid compound derived from an endophytic fungus of Schisandra chinensis and its derivatives, and their application in preventing and / or treating hypercholesterolemia, anti-atherosclerosis and anti-nonalcoholic fatty hepatitis. Background Art

[0002] Cardiovascular disease is considered the number one cause of death threatening human health. Its occurrence and development are closely related to the level of cholesterol in human serum, especially low-density lipoprotein cholesterol. As the key rate-limiting enzyme in the process of cholesterol synthesis in the body, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is located in the endoplasmic reticulum of cells and is responsible for catalyzing the production of mevalonic acid, a precursor of cholesterol synthesis. This enzyme has been widely concerned as an important target for the development of lipid-lowering drugs. For example, statins reduce cholesterol levels in the human body by inhibiting HMGCR protein, and they are still commonly used clinical drugs to play a lipid-lowering role.

[0003] However, excessive use of statins can cause a compensatory increase in HMGCR protein, which not only weakens the cholesterol-lowering effect of statins themselves, but also brings a series of side effects such as muscle damage and drug-induced diabetes. Therefore, by finding small molecule compounds to promote HMGCR protein degradation to reduce protein levels, it is possible to solve the problem of drug resistance that occurs during the use of statins. At the same time, the combination of HMGCR protein degraders and statins is expected to become a new treatment for lowering blood lipids.

[0004] Natural products play an important role in the discovery of new drug lead compounds. As one of the important sources of natural products, the secondary metabolites produced by endophytic fungi in plants are not only rich and diverse in structural types, some of which have novel, unique and complex skeletons, but also a large number of compounds exhibit significant biological activity. For example, indole diterpenoids derived from endophytic fungi have always attracted widespread attention due to their novel and complex structures and excellent anti-cancer, antibacterial, antiviral and hypoglycemic activities.

[0005] In addition, endophytic fungi only need to be obtained from a small number of living plants. They have the characteristics of short growth cycle, repeatability and easy large-scale cultivation, which can bring new opportunities for the development and utilization of certain key protected plants with extremely small wild populations.

[0006] However, there are no reports on the use of endophytic fungal secondary metabolites to degrade HMGCR protein.

[0007] The information disclosed in the background technology section is only intended to enhance the understanding of the overall background of the application, and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to ordinary technicians in the field. Summary of the invention

[0008] In response to the above technical problems, the present application provides a method for preparing and applying indole diterpenoid compounds and their derivatives derived from endophytic fungi of Schisandra chinensis, explores the secondary metabolites of endophytic fungi in Schisandra chinensis, a key protected wild plant, and solves the problems of chemical structure and sample quantity of the compounds. It also provides new structural small molecules for finding HMGCR protein degradation agents, and provides a new idea for the sustainable development and utilization of plant resources.

[0009] The present invention provides a plant endophytic fungus Penicillium sp.DG23, which is isolated from Schisandra chinensis macrocarpa, wherein the Schisandra chinensis macrocarpa was collected by the inventor from Hekou Yao Autonomous County, Honghe Hani and Yi Autonomous Prefecture, Yunnan Province in May 2020.

[0010] The present application provides an indole diterpenoid compound derived from an endophytic fungus of Schisandra chinensis and its derivatives having any of the following structures:

[0011]

[0012] After experimental verification, the above series of substances can significantly promote the degradation of HMGCR protein and assist statins in lowering cholesterol levels. Compound schipenindolene A (abbreviated as Spid A) contains a novel bipyrrole ring, and compound schipenindolene G (abbreviated as Spid G) is a new type of paxilline-type indole diterpenoid compound substituted with 2,18-oxidative cleavage-20-position isopentenyl. They and the known compounds 20-isopentenylpaxilline (abbreviated as Ippe) and paspaline (abbreviated as Ppe) are all isolated from the fermentation of the endophytic fungus Penicillium sp.DG23 of Schisandra macrocarpa. Compound schipenindolene A can be obtained from its precursor by simple organic synthesis, while compounds schipenindolenes J–L (abbreviated as Spid J, Spid K, Spid L) are all obtained from their precursor 20-isopentenylpaxilline by chemical transformation.

[0013] Another aspect of the present application also provides an application of the indole diterpenoid compounds and their derivatives derived from the endophytic fungi of Schisandra chinensis as mentioned above, which is the application of the indole diterpenoid compounds and their derivatives derived from the endophytic fungi of Schisandra chinensis in the preparation of drugs for inhibiting cellular cholesterol synthesis.

[0014] Preferably, the compound schipenindolene A is used in the preparation of a preparation for promoting HMGCR protein degradation and reducing endogenous cholesterol levels;

[0015] Preferably, the compound schipenindolene A is used in combination with a statin in the preparation of a medicament for preventing and / or treating hypercholesterolemia, atherosclerosis, and / or non-alcoholic steatohepatitis;

[0016] Preferably, the statin is selected from at least one of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin.

[0017] Preferably, the indole diterpenoid compounds and derivatives thereof derived from endophytic fungi of Schisandra chinensis are used in the preparation of drugs for preventing and / or treating hypercholesterolemia, atherosclerosis, and / or non-alcoholic steatohepatitis.

[0018] The above series of drugs can be added to related preparations having the above functions, or can be added to related preparations simultaneously with statins to exert the above effects.

[0019] Another aspect of the present application provides a pharmaceutical composition comprising an effective amount of the above-mentioned compound schipenindolene A.

[0020] Another aspect of the present application also provides a method for preparing indole diterpenoid compounds and their derivatives derived from the above-mentioned endophytic fungus of Schisandra chinensis, comprising: isolating indole diterpenoid compounds from the fermentation product of strain Penicillium sp.DG23; or synthesizing indole diterpenoid compounds and their derivatives using substances isolated from the fermentation product of strain Penicillium sp.DG23 as raw materials; strain Penicillium sp.DG23 is an endophytic fungus of Schisandra chinensis; the preservation number of strain Penicillium sp.DG23 is: CGMCC No.40906.

[0021] Preferably, the preparation of the fermentation product comprises the following steps: inoculating the strain into a seed culture medium after expansion culture, and culturing at 28°C and 170 rpm for 7 days to obtain a seed fermentation liquid of the strain; inoculating the seed fermentation liquid into a rice culture medium, and culturing at a constant temperature of 28°C for 30 days to obtain a fermentation product;

[0022] Preferably, the seed culture medium is prepared by adding 22.4 g potato flour and 1.6 g glucose per liter of culture medium, with the remainder being water; and the rice culture medium is prepared by mixing 8 g rice with 9 mL water.

[0023] Preferably, the separation comprises the following steps:

[0024] (1) extracting the fermentation product of Penicillium sp. DG23 with methanol, concentrating it, extracting the concentrate with ethyl acetate, recovering the solvent under reduced pressure to obtain an extract, separating the extract by silica gel column chromatography, detecting it by thin layer chromatography, and collecting it in sections to obtain six parts, namely, A, B, C, D, E and F;

[0025] (2) recrystallizing the collected portion B to obtain compound 20-isopentenylpaxilline;

[0026] (3) separating the collected portion B by ODS medium-pressure column chromatography, and collecting the eight portions B1, B2, B3, B4, B5, B6, B7 and B8;

[0027] (4) after the collected fractions B5 and B6 are combined, they are separated by silica gel column chromatography, and detected by thin layer chromatography, nine fractions B5(6)-1, B5(6)-2, B5(6)-3, B5(6)-4, B5(6)-5, B5(6)-6, B5(6)-7, B5(6)-8 and B5(6)-9 are collected in sections;

[0028] (5) purifying the collected B5(6)-9 by semi-preparative HPLC to obtain compound schipenindolene A;

[0029] (6) The collected B7 fractions were separated by ODS medium pressure column chromatography, and detected by thin layer chromatography, and a total of 23 fractions B7-(1-23) were obtained;

[0030] (7) purifying the collected fraction B7-17 by semi-preparative HPLC to obtain compound schipenindolene G;

[0031] (8) Purifying the collected fraction B7-20 by semi-preparative HPLC to obtain compound paspaline;

[0032] Preferably, the eluent used for silica gel column chromatography in step (1) is a gradient elution of chloroform / acetone from a volume ratio of 1:0 to 0:1;

[0033] Preferably, the solvent used in the recrystallization method of step (2) is acetone; the eluent of the ODS medium-pressure column chromatography in step (3) is a gradient elution of methanol / water from a volume ratio of 30% to 100%; the eluent of the silica gel column chromatography in step (4) is a gradient elution of petroleum ether / acetone from a volume ratio of 50:1 to 0:1; the eluent of the ODS medium-pressure column chromatography in step (6) is a gradient elution of methanol / water from a volume ratio of 70% to 100%;

[0034] Preferably, the semi-preparative purification in step (5) is performed using a 5 μm, 10.0×250 mm COSMOSIL Cholester Packed column, 72% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and the compound schipenindolene A is separated at a retention time of 18.8 min;

[0035] The semi-preparative purification in step (7) was performed using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 58% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and the compound schipenindolene G was isolated at a retention time of 34.8 min;

[0036] The semi-preparative purification in step (8) was performed using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 84% CH 3 CN / H 2 O isocratic elution, a flow rate of 3 mL / min, and the compound paspaline was isolated at a retention time of 28.6 min.

[0037] Preferably, the step a) or b) is to use the substance isolated from the fermentation product of strain Penicillium sp.DG23 as the raw material to synthesize the indole diterpenoid compounds and their derivatives:

[0038] a) Using 7-hydroxypaxilline-13-ene separated from the fermentation product of strain Penicillium sp.DG23 as a raw material, a Maillard reaction was performed to obtain a compound schipenindolene A; the structural formula of 7-hydroxypaxilline-13-ene is:

[0039]

[0040] b) using the compound 20-isopentenylpaxilline isolated from the fermentation product of strain Penicillium sp. DG23 as a raw material for oxidation reaction to obtain compound schipenindolene J, compound schipenindolene K, and compound schipenindolene L; the structural formula of 20-isopentenylpaxilline is:

[0041]

[0042] Preferably, the reaction conditions in step a) are to add ammonium acetate and glacial acetic acid to the reaction system and react at 95° C. until the reaction solution changes from yellow to dark red, and the reaction is completed;

[0043] Preferably, in step a), 7-hydroxypaxilline-13-ene is dissolved in methanol and molecular sieves are added before the reaction;

[0044] Preferably, after the reaction in step a), the obtained substance is quenched with a saturated Na2CO3 solution, extracted with EtOAc, washed with saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and semi-preparatively purified to obtain a white solid compound schipenindolene A;

[0045] Preferably, the purification conditions are to use a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 70% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and a retention time of 10.1 min;

[0046] Preferably, the preparation of the compound schipenindolene J in step b) comprises the following steps: dissolving the compound 20-isopentenylpaxilline in dichloromethane, adding m-chloroperbenzoic acid, reacting at room temperature until the reaction is complete as detected by TLC, and then quenching with a Na2SO3 solution, extracting the obtained substance with EtOAc, washing with saturated brine, drying with anhydrous Na2SO4, filtering, concentrating, and semi-preparative purification to obtain a white solid compound schipenindolene J; the purification conditions are using a 5μm, 9.4×250mm Zorbax SB-C18 chromatographic column, 50% CH3CN / H2O isocratic elution, a flow rate of 3mL / min, and a retention time of 30.0min;

[0047] Preferably, the preparation of compounds schipenindolenes K and L in step b) comprises the following steps: after dissolving compound 20-isopentenylpaxilline in acetone, adding saturated sodium bicarbonate solution at 0°C and reacting at 0°C for 0.5h, adding potassium monopersulfate complex salt dissolved in 0.7mL water and continuing to react until the reaction is complete as detected by TLC, diluting with water, extracting with EtOAc, washing with saturated brine, drying with anhydrous Na2SO4, filtering, concentrating and semi-preparative purification to obtain white solid compounds schipenindolenes K and L; the purification conditions are using a 5μm, 9.4×250mm Zorbax SB-C18 chromatographic column, 70% CH3CN / H2O isocratic elution, a flow rate of 3mL / min, and retention times of 5.3min and 6.7min, respectively.

[0048] The beneficial effects of this application include:

[0049] 1) The present application provides a method for preparing indole diterpenoid compounds and derivatives thereof derived from endophytic fungi of Schisandra chinensis, wherein compounds schipenindolene A, schipenindolene G, 20-isopentenylpaxilline and paspaline are all isolated from endophytic fungi of Schisandra chinensis for the first time, and their absolute and relative configurations are determined by nuclear magnetic resonance, mass spectrometry, circular dichroism (CD) and quantum chemical calculation methods. The above compounds can be artificially synthesized by the method provided by the present application. On the one hand, they can be obtained by repeated fermentation of microorganisms, and on the other hand, the compound schipenindolene A can be obtained by Maillard reaction using compound 7-hydroxypaxilline-13-ene as a raw material. The derivatives schipenindolenes J–L can be obtained from the precursor 20-isopentenylpaxilline by oxidation reaction.

[0050] 2) The application of indole diterpenoid compounds and their derivatives derived from endophytic fungi of Schisandra chinensis provided in the present application. The indole diterpenoid compounds and their derivatives provided in the present application can significantly promote the degradation of HMGCR protein. Among them, the compound schipenindolene A can be used in combination with statins to lower cholesterol levels and can be well used in the preparation of cholesterol-lowering drugs. The preparation method is simple to operate and has good repeatability, has application prospects, and also provides a feasible strategy for the development and application of key protected wild plants.

[0051] 3) The application of indole diterpenoid compounds and their derivatives derived from endophytic fungi of Schisandra chinensis provided in this application. The indole diterpenoid compounds and their derivatives provided in this application all have 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase, HMGCR) degradation activity, which can not only significantly reduce the level of HMGCR protein, but also inhibit the compensatory increase of HMGCR caused by statins. Schipenindolene A can significantly reduce the level of endogenous cholesterol, and the level of endogenous cholesterol is further reduced after combined use with statins. The compounds schipenindolene A and schipenindolenes J-L in the present invention are synthesized for the first time, and their preparation methods are simple and reproducible, providing alternative compounds for the development of new cholesterol-lowering drugs.

[0052] The strain deposit information of the fungus Penicillium sp.DG23 described in the present invention: The strain deposit information of the fungus Penicillium sp.DG23: Name of the deposit unit: China General Microbiological Culture Collection Management Center; Address of the deposit unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; Deposit date: October 30, 2023; Deposit number: CGMCC No.40906; Strain classification name: Penicillium sp. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The compound schipenindolene A provided in this application 13 C NMR spectrum;

[0054] Figure 2 A local enlarged view of the compound schipenindolene A provided in the present application 13 C NMR spectrum;

[0055] Figure 3 The compound schipenindolene A provided in this application 1 H NMR spectrum;

[0056] Figure 4 The HSQC spectrum of the compound schipenindolene A provided in this application;

[0057] Figure 5 The HMBC spectrum of the compound schipenindolene A provided in this application;

[0058] Figure 6 The compound schipenindolene A provided in this application 1H- 1 H COSY spectrum;

[0059] Figure 7 ROESY spectrum of schipenindolene A, a compound provided in this application;

[0060] Figure 8 The compound schipenindolene A provided in this application 1 H- 1 Schematic diagram of H COSY (indicated by bold keys), HMBC (indicated by arrows) and ROESY (indicated by double-headed arrows);

[0061] Fig. 9 The experimental and calculated ECD spectra of the compound schipenindolene A provided in this application;

[0062] Fig.10 HR-ESIMS spectrum of schipenindolene A, a compound provided in this application;

[0063] Fig.11 The ECD spectrum of the compound schipenindolene A provided in this application;

[0064] Fig.12 The UV spectrum of the compound schipenindolene A provided in this application;

[0065] Fig.13 The compound schipenindolene G provided in this application 13 C NMR spectrum;

[0066] Fig.14 A local enlarged view of the compound schipenindolene G provided in this application 13 C NMR spectrum;

[0067] Fig.15 The compound schipenindolene G provided in this application 1 H NMR spectrum;

[0068] Fig.16 The HSQC spectrum of the compound schipenindolene G provided in this application;

[0069] Fig.17 The HMBC spectrum of the compound schipenindolene G provided in this application;

[0070] Fig.18The compound schipenindolene G provided in this application 1 H- 1 H COSY spectrum;

[0071] Fig.19 ROESY spectrum of schipenindolene G, a compound provided in this application;

[0072] Fig. 20 The compound schipenindolene G provided in this application 1 H- 1 Schematic diagram of H COSY (indicated by bold keys), HMBC (indicated by arrows) and ROESY (indicated by double-headed arrows);

[0073] Fig.21 The experimental and fitted ECD spectra of the compound schipenindolene G provided in this application;

[0074] Fig. 22 HR-ESIMS spectrum of schipenindolene G, a compound provided in this application;

[0075] Fig.23 The ECD spectrum of the compound schipenindolene G provided in this application;

[0076] Fig.24 The UV spectrum of the compound schipenindolene G provided in this application;

[0077] Fig.25 The indole diterpenoid compound provided in this application has the activity result of degrading HMGCR-GFP.

[0078] Fig.26 The compound schipenindolene A (Spid A) provided in the present application degrades HMGCR protein and inhibits the activity of cellular cholesterol synthesis. DETAILED DESCRIPTION

[0079] The present application is further described in detail below in conjunction with the accompanying drawings and embodiments, but the present application is not limited in any way. Any changes or improvements made based on the teachings of the present application fall within the scope of protection of the present application.

[0080] Example

[0081] Unless otherwise specified, the materials and instruments used in the following examples were obtained from commercial channels; the detection methods used were all existing methods unless otherwise specified.

[0082] Example 1 Obtaining the Fermentation Product of Penicillium sp. DG23

[0083] (1) Preparation of seed fermentation broth: Penicillium sp. DG23 was inoculated on a plate culture medium using the four-point method and then cultured in a constant temperature incubator at 28°C for 7 days. The colony on the culture medium was then cut into pieces of approximately 0.5 × 0.5 × 0.5 cm. 3 The small pieces were placed in a 500 mL conical flask filled with 200 mL seed culture medium, and cultured in a constant temperature shaking incubator at 28°C and 170 rpm for 7 days to obtain the seed fermentation liquid of the strain, wherein the seed culture medium contained 22.4 g potato flour, 1.6 g glucose, and the remainder water per liter.

[0084] (2) Preparation of the fermentation product of Penicillium sp. DG23: The seed fermentation liquid obtained above was added to a rice culture medium placed in a flask, with an inoculation amount of about 20 mL, and then placed in a constant temperature incubator at 28° C. and cultured for 30 days to obtain a rice fermentation product of the strain. The rice culture medium was prepared by mixing 80 g of rice with 90 mL of water per bottle.

[0085] Example 2 Isolation of schipenindolene A, schipenindolene G, 20-isopentenylpaxilline and paspaline from fermentation products of strains

[0086] (1) The fermented product of the strain is extracted with methanol, concentrated and then extracted with ethyl acetate, and the solvent is recovered under reduced pressure to obtain an extract. The extract is dissolved with a small amount of methanol and mixed with an appropriate amount of silica gel, and then separated by silica gel column chromatography. The eluent is chloroform / acetone with a volume ratio of 1:0 to 0:1. According to thin layer chromatography detection, six parts A, B, C, D, E and F are collected in sections.

[0087] (2) The collected portion B was dissolved in a small amount of methanol and then mixed with an appropriate amount of octadecylsilane bonded silica gel (ODS), and then separated by ODS medium-pressure column chromatography. The eluent was methanol / water with a gradient elution ratio from 30% to 100% by volume. The eight portions B1, B2, B3, B4, B5, B6, B7 and B8 were collected in segments.

[0088] (3) The collected part B is made into a saturated acetone solution, and then cooled and recrystallized to directly obtain the compound

[0089] 20-isopentenylpaxilline;

[0090] (4) The collected B5 and B6 fractions were dissolved in a small amount of methanol and mixed with an appropriate amount of silica gel. The fractions were separated by silica gel column chromatography. The eluent was petroleum ether / acetone with a gradient elution volume ratio of 50:1 to 0:1. According to the thin layer chromatography detection, the nine fractions B5(6)-1, B5(6)-2, B5(6)-3, B5(6)-4, B5(6)-5, B5(6)-6, B5(6)-7, B5(6)-8 and B5(6)-9 were collected in sections.

[0091] (5) The collected B5-9 was purified by semi-preparative HPLC using a 5 μm, 10.0×250 mm COSMOSIL Cholester Packed column, 72% CH 3 CN / H 2 O isocratic elution, a flow rate of 3 mL / min, and a retention time of 18.8 min to obtain the compound schipenindolene A.

[0092] (6) The collected B7 fraction was dissolved in a small amount of methanol and then mixed with an appropriate amount of ODS, and then separated by ODS medium-pressure column chromatography. The eluent was methanol / water with a gradient elution ratio of 70% to 100%. According to the thin layer chromatography detection, B7-(1–23) was collected in sections to obtain a total of 23 fractions;

[0093] (7) The B7-17 fraction collected in the step was purified by semi-preparative HPLC using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 58% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and a retention time of 34.8 min to obtain the compound schipenindolene G;

[0094] (8) The B7-20 fraction collected in the step was purified by semi-preparative HPLC under the conditions of 5 μm, 9.4×250 mm Zorbax SB-C18 column, 84% CH 3 CN / H 2 O isocratic elution, flow rate 3 mL / min, and the compound paspaline was obtained at a retention time of 28.6 min.

[0095] Example 3 Structural Identification of Compound Schipenindolene A

[0096] Perform the following tests according to the existing common operations, and refer to the results Figures 1 to 12 .

[0097] The compound schipenindolene A prepared in Examples 1 and 2 is a white solid. According to the quasi-molecular ion peak m / z 413.2240 [MH] in the HR-ESIMS spectrum, -, the calculated value is 413.2235, and the molecular formula of the compound is determined to be C 27 H 30 N2O2, unsaturation is 14. Combined 1 H and 13 CNMR and HSQC spectra suggested that the compound contained 1 keto carbonyl, 14 olefinic carbons (6 of which were protonated), 3 quaternary carbons (1 of which was oxidized), 1 methine, 4 methylene and 4 methyl groups. 1 H NMR showed that the compound contained one imino group (δ H 7.81) and four special aromatic proton signals (δ H 7.48,δ H 7.11,δ H 7.11 and δ H 7.34), indicating that the compound contains a 2,3-disubstituted indole moiety, which is 1 H- 1 The relevant signals of H COSY and HMBC spectra were confirmed. Further analysis of the NMR signals of the compound suggested that it may have similar structural fragments to the isolated compound 7-hydroxypaxilline-13-ene. For example, the double bond between C-12 and C-13 in the structure can be obtained by using the corresponding 1 H- 1 The H COSY and HMBC spectra were confirmed. However, the NMR spectrum of schipenindolene A showed another unique imine signal (δ H 9.12) and four obvious aromatic carbon signals (1 methine), indicating that this compound has an additional trisubstituted pyrrole ring compared with the known compound 7-hydroxypaxilline-13-ene. The HMBC spectrum confirmed the existence of this special structural fragment: H2-5 is related to C-7, H2-6 is related to C-11, H-10 is related to C-9, C-7 and C-12, and H-13 is related to C-11 ( Figure 8 ). In summary, through the detailed analysis of the one-dimensional and two-dimensional NMR spectra, the NMR data of the compound schipenindolene A can be assigned, the results are shown in Table 1, and its planar structure can be determined.

[0098] As for the relative configuration of the compound schipenindolene A, it is first assumed that H-15 in the structure is in β configuration. At the same time, the ROESY spectrum of the compound shows that H-15 and H3-25, H3-25 and H-5β, and H3-24 and H-5α have related signals. It is inferred that H-15 and H3-25 are in β configuration and H3-24 is in α configuration, such as Fig. 9Then, by comparing the experimental and calculated ECD spectra of the compound, its absolute configuration was finally determined to be 3S, 4S, and 15S, as shown in Fig. 9 So far, the chemical structure of the compound schipenindoleneA has been completely determined, as shown in the figure below.

[0099]

[0100] Table 1. Compound schipenindolene A 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (Chloroform-d)

[0101]

[0102] Example 4 Synthesis of compound schipenindolene A

[0103] The compound schipenindolene A was directly obtained by using the compound 7-hydroxypaxilline-13-ene (A) separated in Example 2 as a starting material through a key Maillard reaction, as follows:

[0104]

[0105] Synthesis steps: Compound A (3.0 mg) was dissolved in 0.5 mL of methanol and an appropriate amount of molecular sieves was added. Then, 0.006 g of ammonium acetate and 0.03 mL of glacial acetic acid were added. The mixture was reacted at 95° C. for 24 h. When the reaction solution turned from yellow to dark red, TLC detected that the reaction was complete. The mixture was quenched with saturated Na2CO3 solution (1.5 mL), extracted with EtOAc (3×1 mL), washed with saturated brine (1 mL), dried over anhydrous Na2SO4, filtered, concentrated, and semi-preparatively purified using a 5 μm, 9.4×250 mm ZorbaxSB-C18 column, 70% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and a retention time of 10.1 min to obtain a white solid compound schipenindolene A.

[0106] Characterization data of schipenindolene A: R f =0.48(silica,CHCl3 / Me2CO 4:1);[α] 2 D 4.0 :-196.36(MeOH,c 0.11); HRESIMS at m / z 437.2196([M+Na]+ ,calcd.for 437.2199). 1 H NMR (800MHz, Chloroform-d) δ = 9.16 (s, 1H), 7.82 (s, 1H), 7.48 (d, J = 7.8, 1H), 7.34 (d, 7.5, 1H ),7.11(overlapped,2H),7.05(s,1H),6.01(t,J=3.5,1H),3.09(m,1H),2.90(overlapped,1 H),2.89(overlapped,1H),2.85(dd,J=16.3,5.5,1H),2.49(dd,J=13.0,10.6,1H),2.38(m,1 H),2.27(m,1H),1.88(dd,J=12.6,4.8,1H),1.62(overlapped,6H),1.19(s,3H),1.01(s,3H); 13 C NMR(200MHz,Chloroform-d)δ=193.5,149.1,140.4,134.6,134.2,126.4,125.1,122.4,120.9,119.9,11 8.8,118.6,118.5,113.5,111.6,74.8,48.7,45.8,40.7,32.0,29.7,29.6,27.5,27.4,21.2,20.2,14.7.

[0107] Example 5 Structural Identification of Compound Schipenindolene G

[0108] The following tests were performed according to the existing common operation, and the results were obtained. Figures 13-24 The body is as follows:

[0109] The compound schipenindolene G prepared in Examples 1 and 2 is a white solid. Its molecular formula is as follows: - , 548.3023, calculated value is 548.3018) determined as C 33 H 43 NO6, the degree of unsaturation is 13. Through further analysis 1 H and 13CNMR data suggest that the compound may be a derivative of 20-isopentenylpaxilline, but the active NH signal and an olefinic carbon signal are missing in the DMSO-d6 spectrum, and are replaced by a carbonyl and a methoxy signal. Therefore, it is speculated that the molecule is a new type of 2,18-oxidative cleavage-20-isopentenyl-substituted paxilline-type indole diterpenoid compound, and its 2-position amide carbonyl is further methylated to form a unique eight-membered imine ring. This structure is also confirmed by the correlation of H3-2-OCH3 with C-2, H-16 with C-18, H2-17 with C-3 and C-19, and H3-25 with C-2 and C-16 in the HMBC spectrum ( Fig.17 ), and the assigned NMR data are shown in Table 2. In addition, the relative configuration and absolute configuration of the compound were determined by ROESY spectrum and calculated ECD method, respectively ( Fig. 20 and Fig.21 ).

[0110]

[0111] Table 2. Compounds of schipenindolene G 1 H NMR (800 MHz) and 13 C NMR (200 MHz) data (DMSO-d6)

[0112]

[0113]

[0114] Example 6 Synthesis of schipenindolenes JL and its identification

[0115] Synthesis of compounds schipenindolenes J–L: The isolated compound 20-isopentenylpaxilline was used as a starting material and further oxidized to obtain compounds schipenindolenes J–L. The preparation process is as follows:

[0116]

[0117] The synthesis steps are as follows:

[0118] (1) Preparation of compound schipenindolene J: Compound 20-isopentenylpaxilline (110.0 mg) was dissolved in 5 mL of dichloromethane, and then 0.065 g of m-chloroperbenzoic acid (m-CBPA) was added. The mixture was allowed to react at room temperature for 0.5 h. After TLC detection showed that the reaction was complete, Na2SO3 solution was used for quenching (Na2SO3 was 72.0 mg), and the mixture was extracted with EtOAc (3×3 mL). The mixture was washed with saturated brine (3 mL), dried over anhydrous Na2SO4, filtered, concentrated, and semi-preparatively purified using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 50% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and a retention time of 30.0 min to obtain the white solid compound schipenindolene J.

[0119] (2) Preparation of compounds schipenindolenes K and L: Compound 20-isopentenylpaxilline (28.0 mg) was dissolved in 1.5 mL of acetone, and then 0.93 mL of saturated sodium bicarbonate solution was added at 0°C. The mixture was reacted at 0°C for 0.5 h, followed by the addition of 0.068 g of potassium monopersulfate complex salt (oxone) dissolved in 0.7 mL of water. After TLC detection showed that the reaction was complete, the mixture was diluted with water, extracted with EtOAc (3×1.5 mL), washed with saturated brine (2 mL), dried over anhydrous Na2SO4, filtered, concentrated, and semi-preparatively purified using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 70% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and white solid compounds schipenindolenes K and L were isolated at retention times of 5.3 min and 6.7 min, respectively.

[0120] The following identification was performed according to the existing common operation. The structural identification data of schipenindolene J: Whitesolid; R f =0.31(silica,Chloroform / Me2CO 2:1); 19.68(MeOH,c 0.10); HRESIMS atm / z 550.2812([MH] - ,calcd.for 550.2810). 1 H NMR (500 MHz, DMSO-d6):δ H8.93(s,1H,H-1),7.19(t,J=7.8Hz,1H,H-23),6.91(dd,J=7.8,0.8Hz,1H,H-22),6.79(dd,J=7.8,0.8Hz,1H,H-24),5.76(d,J=1.9Hz,1H,H-12),5.13(m,1H,H-32),5.04(s,1H,H-14-OH),4.66(dd,J=10.2,8.1Hz,1H,H-8),4.29(s,1H,H-28-OH),3.62(d,J=1.9Hz,1H,H-10),3.32(overlapped,1H,H-31),3.04(dd,J=13.2,3.9Hz,1H,H-18α),2.58(m,1H,H-17),2.51(d,J=13.2Hz,1H,H-18β),2.33(td,J=13.3,3.9Hz,1H,H-6α),2.07(m,1H,H-7a),1.77(m,1H,H-15β),1.70(d,J=0.8Hz,3H,H-34),1.67(s,3H,H-35),1.64(overlapped,1H,H-6β),1.63(s,3H,H-26),1.51(overlapped,1H,7b),1.51(overlapped,1H,H-15α),1.50(overlapped,1H,H-16),1.17(s,3H,H-30),1.13(s,3H,H-29),0.65(s,3H,H-27). 13 C NMR(125MHz,DMSO-d6):δ C204.9(C,C-19),197.3(C,C-11),167.5(C,C-13),151.0(C,C-2),137.5(C,C-25),136.6(C,C-21),135.5(C,C-20),132.6(C,C-33),12 9.0(CH,C-23),125.3(CH,C-22),122.9(CH,C-32),120.7(CH,C-24),119.9(CH,C-12),88.9(C,C-4),83.3(CH,C-10),75.5(C,C-14),72 .4(CH,C-8),70.8(C,C-28),48.5(CH2,C-18),45.5(C,C-5),41.4(CH,C-17),32.4(CH2,C-31),28.9(CH2,C-15),27.7(CH2,C-7),25.9( CH3,C-29),25.8(CH3,C-30),25.4(CH3,C-34),25.1(CH2,C-6),24.1(CH2,C-16),17.9(CH3,C-35),17.8(CH3,C-27),14.6(CH3,C-26).

[0121] The following identification was performed according to the existing common operation. The structural identification data of schipenindolene K: Whitesolid; R f =0.25(silica,PE / Me2CO 2:1); -259.87(MeOH,c 0.15); HRESIMS at m / z580.2920([M+COOH] - ,calcd.for 580.2916). 1 H NMR (500 MHz, DMSO-d6):δ H7.24(t,J=7.4Hz,1H,H-22),7.20(d,J=7.4Hz,1H,H-23),6.98(d,J=7.4Hz,1H,H-21),5.80(s,1H,H-11),5.56(s,1H,H-1),5.02(s,1H,H-13-OH),4.78(t,J=9.0Hz,1H,H-32),4.34(s,1H,H-27-OH),3.66(s,1H,H-9),3.47(overlapped,1H,H-16),2.99(t,J=6.0Hz,1H,H-31),2.93(dd,J=14.6,6.0Hz,1H,30a),2.84(dd,J=14.6,6.0Hz,1H,H-30b),2.62(m,1H,H-5α),2.18(m,1H,H-6α),2.06(dd,J=11.8,3.3Hz,1H,H-17β),1.98(m,1H,H-14a),1.79(m,1H,H-15a),1.73(overlapped,1H,H-6β),1.70(overlapped,1H,H-14b),1.61(overlapped,1H,H-15b),1.59(overlapped,1H,H-5β),1.52(t,J=12.0Hz,1H,H-17α),1.35(s,3H,H-34),1.30(s,3H,H-25),1.26(s,3H,H-33),1.20(s,3H,H-29),1.16(s,3H,H-28),1.02(s,3H,H-26). 13 C NMR(125MHz,DMSO-d6):δ C201.3(C,C-2),197.6(C,C-10),168.4(C,C-12),157.7(C,C-24),139.1(C,C-19),134.8(C,C-20),129.2(CH,C-22),125.8(CH,C-21), 119.0(C,C-11),118.3(CH,C-23),87.8(C,C-18),83.5(CH,C-9),76.0(C,C-13),72.5(CH,C-7),71.0(C,C-27),63.0(CH,C-31),58.4( C,C-32),49.6(C,C-3),45.0(CH,C-16),43.3(C,C-4),35.6(CH2,C-17),32.9(CH2,C-14),30.9(CH2,C-30),28.2(CH2,C-6),26.0(CH3 ,C-28),25.81(CH3,C-29),25.78(CH2,C-5),24.6(CH3,C-33),20.7(CH2,C-15),19.9(CH3,C-26),19.2(CH3,C-25),18.9(CH3,C-34).

[0122] The following identification was performed according to the existing common operation. The structural identification data of schipenindolene L: White solid; R f =0.25(silica,PE / Me2CO 2:1); -66.33(MeOH,c 0.12); HRESIMS at m / z598.3028([M+COOH] - ,calcd.for 598.3022). 1 H NMR (600 MHz, DMSO-d6):δ H6.89(t,J=7.7Hz,1H,H-22),6.35(s,1H,H-1),6.32(d,J=7.7Hz,1H,H-21),6.26(d,J=7.7Hz,1H,H-23),5.76(d,J=1.8Hz,1H,H-11),5.11(s,1H,H-18-OH),4.74(s,1H,H-13-OH),4.71(dd,J=10.0,8.3Hz,1H,H-7),4.61(s,1H,H-2-OH),4.33(s,1H,H-27-OH),3.64(d,J=1.8Hz,1H,H-9),2.97(overlapped,1H,H-31),2.97(overlapped,1H,H-30a),2.70(dd,J=16.1,8.7Hz,1H,H-30b),2.51(m,1H,H-16),2.44(m,1H,H-5α),2.07(overlapped,1H,H-6α),2.08(overlapped,1H,H-17β),1.76(overlapped,1H,H-14a),1.73(overlapped,1H,H-17α),1.71(overlapped,1H,H-6β),1.56(overlapped,1H,H-5β),1.55(overlapped,1H,H-14b),1.55(overlapped,2H,H-15),1.34(s,3H,H-34),1.26(s,3H,H-33),1.19(s,3H,H-29),1.16(s,3H,H-28),1.13(s,3H,H-26),0.90(s,3H,H-25). 13 C NMR(150MHz,DMSO-d6):δ C197.8(C,C-10),170.2(C,C-12),150.6(C,C-24),134.4(C,C-20),129.7(C,C-19),129.1(CH,C-22),118.9(CH,C-11),116.0(CH,C-21 ),105.0(CH,C-23),101.7(C,C-2),85.3(C,C-18),83.5(CH,C-9),77.3(C,C-13),72.5(CH,C-7),71.0(C,C-27),63.2(CH,C-31),58.2 (C,C-32),54.4(C,C-3),43.9(C,C-4),43.0(CH2,C-17),37.8(CH,C-16),30.4(CH2,C-14),30.3(CH2,C-30),28.1(CH2,C-6),26.3(CH 2,C-5),26.0(CH3,C-28),25.7(CH3,C-29),24.7(CH3,C-33),21.84(CH3,C-26),21.78(CH2,C-15),18.9(CH3,C-34),13.6(CH3,C-25).

[0123] Example 7 Activity test of indole diterpenoids in degrading HMGCR-GFP

[0124] This example provides a test for the HMGCR-GFP degradation activity of the indole diterpenoid compound. This method uses the CHO-7 / HMGCR-GFP cell line to determine the HMGCR degradation activity of the compound prepared in the example of the present application.

[0125] The CHO-7 / HMGCR-GFP cell line is a stable transfection expressing HMGCR-GFP fusion protein in Chinese hamster ovary epithelial cells CHO-7. The specific method is as follows:

[0126] (1) Cell inoculation. CHO-7 / HMGCR-GFP cells in good growth condition were inoculated in 24-well plates. The culture medium was DMEM:F12 (1:1) (containing 0.5% FBS, 5% defatted serum, 100 units / mL Penicillin-Streptomycin). The cell density was 1.2×10 per well. 4 The cells were cultured in a 37°C, 5% CO2 incubator for 28 hours.

[0127] (2) Cell administration. The culture medium was removed by aspiration, and the cells were washed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Then, delipidated serum DMEM / F12 culture medium (containing 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonic acid, 100 units / mL Penicillin-Streptomycin) containing different concentrations of the compounds prepared in the examples of the present application was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours.

[0128] (3) Quantitative detection of HMGCR-GFP fluorescence intensity. After 16 hours, the culture medium was removed and the plates were rinsed once with PBS. Then, 150 μL of Reporter Lysis Buffer (Promega, E397A) was added to each well. The plates were then placed in a -80°C freezer and thawed at room temperature after 1 hour. After thawing, the plates were placed on a micro-oscillator for 10 minutes. Then, 100 μL of the lysate was drawn and added to a 96-well black ELISA plate (PerkinElmer, 6005329). The fluorescence intensity of HMGCR-GFP was measured using a BioTek Cytation reader (BioTek Instruments, Inc, USA) with an excitation light of 485 nm and an emission light of 535 nm. The compounds prepared according to the examples of the present application were used in a concentration gradient of 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, 10 μM, and 20 μM, and the DMSO group was standardized as 100%, and the half-effective inhibitory concentration DC was calculated using GraphPad Prism 8 software. 50 The above method was used to determine the activity of HMGCR degradation. Fig.25 (c) as shown.

[0129] Example 8 Activity test of indole diterpenoids in degrading HMGCR-GFP

[0130] The activity of the compound prepared in the examples of the present application in degrading HMGCR was determined using the CHO-7 / HMGCR-GFP cell line. The CHO-7 / HMGCR-GFP cell line is a Chinese hamster ovary epithelial cell CHO-7 that stably transfects and expresses the HMGCR-GFP fusion protein. The specific method is as follows:

[0131] (1) Cell seeding. CHO-7 / HMGCR-GFP cells in good growth condition were seeded in a 12-well plate containing a collagen-coated round cover slip. The culture medium was DMEM:F12 (1:1) (containing 5% FBS, 100 units / mL Penicillin-Streptomycin). The cell density was 1.2×10 per well. 4 The cells were cultured in a 37°C, 5% CO2 incubator for 28 hours.

[0132] (2) Cell administration. The culture medium was removed by aspiration, and the cells were washed twice with PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4). Then, delipidated serum DMEM / F12 culture medium (containing 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonic acid, 100 units / mL Penicillin-Streptomycin) containing different concentrations of the compounds prepared in the examples of the present application was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours.

[0133] (3) HMGCR-GFP fluorescence photography. The specific method is as follows: aspirate the culture medium, wash once with PBS buffer, fix with 4% paraformaldehyde (PFA) for 30 minutes, and then wash twice with PBS buffer. Then transfer the round coverslip with cells to the well plate cover, add 100 μL of 10% FBS-containing PBS diluted ethanol-dissolved Filipin (50 μg / mL) staining solution to each coverslip, and incubate at room temperature in the dark for 2 hours. After staining, rinse three times with PBS, then fix the coverslip on the slide with mounting medium (Mowiol 4-88) and mark it. After drying in the dark, observe and photograph the intracellular HMGCR-GFP fluorescence under the microscope of ImageXpress Pico automated imaging system. The DMSO group was standardized as 100%, and the HMGCR-GFP fluorescence intensity was quantified using Image J (NIH) software. The HMGCR-GFP degradation bar graph was drawn with GraphPad Prism 8 software. The activity of HMGCR degradation was determined by the above method, and the degradation effect was as follows: Fig.25 As shown in (a and b).

[0134] Example 9 Activity test of compound schipenindolene A (Spid A) in degrading HMGCR protein in Huh-7 cells

[0135] The specific method is as follows:

[0136] (1) Cell inoculation and administration. The specific method is as follows: Huh-7 cells in good growth state are inoculated into a 60 mm cell culture dish, the culture medium is DMEM (containing 10% FBS, 100 units / mL Penicillin-Streptomycin), and the cell density is 4.5×10 per dish. 5 The cells were cultured in a 37°C, 5% CO2 incubator. After 28 hours, the culture medium was removed and the cells were washed twice with PBS buffer. Then, a serum-free DMEM medium (containing 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonic acid, and 100 units / mL Penicillin-Streptomycin) containing different concentrations of the compounds prepared in the examples of the present application was added and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours.

[0137] (2) Extraction of whole cell protein. The specific method is as follows: aspirate the original culture medium, rinse once with PBS, then scrape the cells in the culture dish with a cell scraper and transfer them to a 15 ml centrifuge tube, centrifuge at 4°C, 1000g for 3 minutes. Then discard the supernatant, resuspend the cells in each tube with 1 mL of pre-cooled PBS and transfer them to a new 1.5 mL centrifuge tube, centrifuge at 4°C, 1000g for 3 minutes. Aspirate the supernatant, add 120 μL of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1.5% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) to each tube, and then shear the cells 30 times with a No. 7 syringe. After shearing, centrifuge at 4°C, 15000g for 10 min. After centrifugation, add 90 μL membrane protein solubilizing buffer (62.5 mM Tris-HCl, pH 6.8, 15% SDS, 8 M urea) and 60 μL 4×SDS Loading Buffer (150 mM Tris-HCl, pH 6.8, 12% SDS, 30% glycerol, 0.02% bromophenol blue, 6% β-mercaptoethanol) and incubate at 37°C for 30 min.

[0138] (3) Western Blot was used to detect the degradation of HMGCR protein. The specific method is as follows:

[0139] 1) Sample loading: 20 μL of each protein sample was loaded into the wells of 8% SDS-PAGE gel.

[0140] 2) Electrophoresis: Set the voltage to 85V for 40 minutes to allow the protein to enter the stacking gel, then adjust the voltage to 120V for 1 hour to allow the protein to enter the separation gel for separation.

[0141] 3) Transfer: After electrophoresis, take out the gel and place it into the transfer device in the order of filter paper, nitrocellulose membrane, gel, and filter paper. Be careful to remove bubbles and transfer the membrane at a constant voltage of 110 volts for 1.5 hours.

[0142] 4) Blocking: After the transfer, the nitrocellulose membrane with protein was immersed in TBST buffer (20 mM Tris-HCl (pH 7.4)), rinsed once with (150 mM NaCl and 0.05% Tween-20), and then immersed in TBST buffer containing 5% skimmed milk powder and incubated on a shaker at room temperature for 30 minutes.

[0143] 5) Primary antibody incubation: After blocking, wash the membrane three times with TBST buffer. Add 1:4000 diluted HMGCR antibody (IgG proteintech, rabbit polyclonal antibody that recognizes Huh-7 cell HMGCR) and incubate overnight at 4°C on a shaker.

[0144] 6) Secondary antibody incubation: After the primary antibody incubation is completed, the antibody is recovered and the membrane is washed 5 times with TBST, 5 minutes each time. Then, a 1:5000 diluted goat anti-rabbit HRP-conjugated secondary antibody is added and incubated at room temperature for 3 hours. Wash 5 times with TBST, 5 minutes each time.

[0145] 7) Protein development: Cover the nitrocellulose membrane with ECL luminescent substrate, seal it with plastic wrap, and take pictures with ChemiDocXRS gel imaging analyzer (Bio-Rad).

[0146] 8) Quantification of grayscale value of HMGCR protein: The obtained grayscale images were quantitatively analyzed using Image J (NIH) software, with the non-drug group set as 100%, and GraphPad Prism 8 software was used to draw a histogram of HMGCR protein degradation.

[0147] 9) The result is as follows Fig.26 As shown in (a and b).

[0148] Example 10 Detection of the effect of compound schipenindolene A (Spid A) on cholesterol accumulation level in HepG2 cells

[0149] The specific method is as follows:

[0150] (1) Cell inoculation and drug administration. The specific method is as follows: HepG2 cells in good growth state are inoculated into a 12-well plate containing a collagen-coated round cover glass. The culture medium is DMEM (containing 10% FBS, 100 units / mL Penicillin-Streptomycin). The cell density is 2.5×10 per well. 4 The cells were cultured in a 37°C, 5% CO2 incubator. After 28 hours, the culture medium was removed and 1 ml of serum-free DMEM culture medium (containing 1% hydroxypropyl-β-cyclodextrin, 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonate, 100 units / mL Penicillin-Streptomycin) was added to each well. After 2 hours, the cells were washed twice with PBS buffer, and then the serum-free DMEM culture medium (containing 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonate, 100 units / mL Penicillin-Streptomycin) containing different concentrations of the compounds prepared in the examples of the present application was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours.

[0151] (2) Cell fixation and sealing. The specific method is as follows: aspirate the culture medium, wash once with PBS buffer, fix with 4% paraformaldehyde (PFA) for 30 minutes, wash twice with PBS buffer, then transfer the round coverslip with cells to the well plate cover, add 100μL of PBS containing 10% FBS diluted ethanol dissolved Filipin (50μg / mL) staining solution to each coverslip, and incubate at room temperature in the dark for 2 hours. Rinse with PBS buffer for 5 times, then add 100μL of PBS diluted SYBR Green (5μg / mL) staining solution to each coverslip, and incubate at room temperature in the dark for 10 minutes. After staining, rinse with PBS for 3 times, then fix the coverslip on the slide with sealing medium (Mowiol4-88) and mark it. After drying in the dark, observe the accumulation of cholesterol in the cells under a Nikon ECLIPSE Ti microscope. The results are as follows. Fig.26 (c and d).

[0152] Example 11 Determination of the effect of compound schipenindolene A (Spid A) on the content of total cholesterol and free cholesterol in Huh-7 cells

[0153] The specific method is as follows:

[0154] (1) Cell inoculation and administration. The specific method is as follows: Huh-7 cells in good growth state are inoculated into a 60 mm cell culture dish. The culture medium is DMEM (containing 10% FBS, 100 units / mL Penicillin-Streptomycin). The cell density is 6.5×10 per dish. 5 The cells were cultured in a 37°C, 5% CO2 incubator. After 28 hours, the culture medium was removed and 1 mL of serum-free DMEM culture medium (containing 1% hydroxypropyl-β-cyclodextrin (HPCD), 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonate, and 100 units / mL Penicillin-Streptomycin) was added to each well. After 2 hours, the cells were washed twice with PBS buffer, and then the serum-free DMEM culture medium (containing 5% delipidated serum, 2 μM lovastatin, 20 μM mevalonate, and 100 units / mL Penicillin-Streptomycin) containing different concentrations of the compounds prepared in the examples of the present application was added, and the cells were cultured in a 37°C, 5% CO2 incubator for 16 hours.

[0155] (2) Extraction of intracellular cholesterol. The specific method is as follows: aspirate the original culture medium, rinse once with PBS, then scrape the cells in the culture dish with a cell scraper and transfer them to a 15 mL centrifuge tube, centrifuge at 4°C, 1000g for 3 minutes. Then discard the supernatant, resuspend the cells in each tube with 1 mL of pre-cooled PBS and transfer them to a new 1.5 mL centrifuge tube, centrifuge at 4°C, 1000g for 3 minutes. Aspirate the supernatant, add 300 μL of RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1.5% NP-40, 0.1% SDS, and 0.5% sodium deoxycholate) to each tube, and then shear the cells 30 times with a No. 7 syringe. Pipette 2 μL of lysis buffer into a new 1.5 mL centrifuge tube for determination of total cell protein content. Add 1 mL of chloroform:methanol solution (2:1) to the remaining lysis buffer and rotate at 20 rpm on a shaker at room temperature for 2 hours. After the rotation is completed, centrifuge at 15000g for 10 minutes at room temperature to separate the organic phase and the aqueous phase, remove the upper aqueous phase, and then pipette 500 μL of the lower organic phase into a 1.5 mL centrifuge tube, place it on a nitrogen blower, and blow dry the organic phase with nitrogen. After blowing dry, add 80 μL of anhydrous ethanol to dissolve the contents.

[0156] (3) Determination of total cholesterol and free cholesterol content in cells. The specific method is as follows: take 10 μL of anhydrous ethanol containing extracted intracellular cholesterol and a graded diluted cholesterol standard, add 90 μL of working solution (300 μM Amplex Red, 2U / mL cholesterol oxidase) prepared in reaction buffer to free cholesterol, and add 90 μL of working solution (300 μM Amplex Red, 2U / mL horseradish peroxidase, 2U / mL cholesterol oxidase) prepared in reaction buffer to total cholesterol, react at 37°C in the dark for half an hour, mix well and add 90 μL to each well of a 96-well plate. Use a BioTek Cytation reader (BioTekInstruments, Inc, USA) to detect the fluorescence intensity of the generated Resorufin under the conditions of an excitation wavelength of 560 nm and an emission wavelength of 590 nm. Calculate the cholesterol content in the sample based on the cholesterol standard, and then divide it by the protein content to obtain the relative quantification of the cholesterol level in the cell, reflecting the activity of the compound in inhibiting cellular cholesterol synthesis. The results are as follows: Fig.26 (e and f).

[0157] Example 12: Schipenindolene A-containing preparation

[0158] 1. Take the compound schipenindolene A, add excipients in a weight ratio of 1:1, and granulate and tablet.

[0159] 2. Take the compound schipenindolene A, add excipients in a weight ratio of 1:2 to the excipients, and granulate and tablet.

[0160] 3. Take the compound schipenindolene A and prepare capsules according to conventional capsule preparation methods.

[0161] 4. Take the compound schipenindolene A and make it into tablets according to the following method:

[0162] The tablets include: compound schipenindolene A 100 mg, starch 0.25 g, corn syrup 0.15 g, magnesium stearate 0.005 g;

[0163] 5. Capsules: Take 100 mg of schipenindolene A, 0.7 g of starch, and 0.007 g of magnesium stearate. Preparation method: Mix the compound with the auxiliary agent, sieve, mix evenly in a suitable container, and fill the resulting mixture into hard gelatin capsules.

[0164] 6. The nasal spray includes: schipenindolene A 80 mg, sodium oxide 8 mg, EDTA 1 mg, uranyl phosphate buffer (pH 6.5) 1 mg, polysorbate 60 10 mg, and double distilled water 2 mL.

[0165] Preparation: Add one ingredient at a time to an appropriate volume of redistilled water under stirring until completely dissolved, then add another ingredient. After adding water to 2 mL, filter the solution on a sterile filter, fill into bottles and divide according to appropriate dosages.

[0166] 7. Dropping pills: Take 1g of compound schipenindolene A and 9g of polyethylene glycol 6000. Preparation method: Weigh compound schipenindolene A according to the above prescription, add appropriate amount of anhydrous ethanol, dissolve it with slight heat, add it to the prescribed amount of polyethylene glycol melt (60℃ water bath insulation), stir and mix evenly until the ethanol evaporates, keep it in a 60℃ water bath for 30 minutes, wait for all bubbles to be removed, transfer the above mixed melt with all bubbles removed into a liquid storage cylinder, control the dripping speed, drip it drop by drop into the condensate under the condition of 80-85℃ insulation, wait for the condensate to be completely condensed, pour off the condensate, collect the dropping pills, drain and remove the condensate on the pills with filter paper, place it in a silica gel dryer or dry it naturally.

[0167] Embodiment 13

[0168] The difference from Example 12 is that the compound used is schipenindolene G.

[0169] Embodiment 14

[0170] The difference from Example 12 is that the compound used is schipenindolene J.

[0171] Embodiment 15

[0172] The difference from Example 12 is that the compound used is schipenindolene k.

[0173] Example 16

[0174] The difference from Example 12 is that the compound used is schipenindolene L.

[0175] Embodiment 17

[0176] The difference from Example 12 is that atorvastatin is added to the prepared tablets at a mass ratio of schipenindolene A to atorvastatin of 1:1.

[0177] Although the present application has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. An indole diterpenoid compound derived from endophytic fungi of Schisandra chinensis and its derivatives, characterized in that: Has any of the following structures:

2. A use of the indole diterpenoid compounds and their derivatives derived from the endophytic fungi of Schisandra chinensis as claimed in claim 1, characterized in that: The invention relates to the application of indole diterpenoid compounds and their derivatives derived from endophytic fungi of Schisandra macrocarpa in the preparation of drugs for inhibiting cellular cholesterol synthesis.

3. The application according to claim 2, characterized in that: Application of the compound schipenindolene A in the preparation of a preparation for promoting HMGCR protein degradation and reducing endogenous cholesterol levels.

4. The application according to claim 2, characterized in that: Use of the compound schipenindolene A in combination with statins in the preparation of a drug for preventing and / or treating hypercholesterolemia, atherosclerosis, and / or non-alcoholic fatty hepatitis.

5. The use according to claim 4, characterized in that: The statin is selected from at least one of lovastatin, simvastatin, pravastatin, fluvastatin, atorvastatin and rosuvastatin.

6. The use according to claim 2, characterized in that: Application of indole diterpenoid compounds and derivatives thereof derived from endophytic fungi of Schisandra chinensis in the preparation of drugs for preventing and / or treating hypercholesterolemia, atherosclerosis, and / or non-alcoholic fatty hepatitis.

7. A pharmaceutical composition, characterized in that The method comprises an effective amount of the compound schipenindolene A as claimed in claim 1.

8. A method for preparing indole diterpenoid compounds and derivatives thereof derived from endophytic fungi of Schisandra chinensis as claimed in claim 1, characterized in that: include: An indole diterpenoid compound is isolated from the fermentation product of strain Penicillium sp.DG23; or an indole diterpenoid compound and its derivatives are synthesized using the substance isolated from the fermentation product of strain Penicillium sp.DG23 as a raw material; the deposit number of strain Penicillium sp.DG23 is: CGMCC No.40906; The strain Penicillium sp.DG23 is an endophytic fungus of Schisandra macrocarpa; The preparation of the fermentation product comprises the following steps: the strain is expanded and cultured, inoculated into a seed culture medium, and cultured at 28°C and 170 rpm for 7 days to obtain a seed fermentation liquid of the strain; the seed fermentation liquid is inoculated into a rice culture medium, and cultured at a constant temperature of 28°C for 30 days to obtain a fermentation product; The seed culture medium is prepared by adding 22.4 g potato flour and 1.6 g glucose per liter of culture medium, and the balance is water; the rice culture medium is prepared by mixing 8 g rice and 9 mL water; The separation includes the following steps: (1) extracting the fermentation product of Penicillium sp.DG23 with methanol, concentrating it, extracting the concentrate with ethyl acetate, recovering the solvent under reduced pressure to obtain an extract, separating the extract by silica gel column chromatography, and detecting it by thin layer chromatography, collecting six parts A, B, C, D, E and F in sections, wherein part A is the compound 7-hydroxypaxilline-13-ene; (2) recrystallizing the collected portion B to obtain compound 20-isopentenylpaxilline; (3) separating the collected portion B by ODS medium-pressure column chromatography, and collecting the eight portions B1, B2, B3, B4, B5, B6, B7 and B8; (4) after the collected fractions B5 and B6 are combined, they are separated by silica gel column chromatography, and detected by thin layer chromatography, nine fractions B5(6)-1, B5(6)-2, B5(6)-3, B5(6)-4, B5(6)-5, B5(6)-6, B5(6)-7, B5(6)-8 and B5(6)-9 are collected in sections; (5) Purifying the collected B5(6)-9 by semi-preparative HPLC to obtain compound schipenindolene A; (6) The collected B7 fractions were separated by ODS medium pressure column chromatography, and detected by thin layer chromatography, and a total of 23 fractions B7-(1-23) were obtained; (7) purifying the collected fraction B7-17 by semi-preparative HPLC to obtain compound schipenindolene G; (8) The collected fraction B7-20 was purified by semi-preparative HPLC to obtain the compound paspaline.

9. The preparation method according to claim 8, characterized in that: In step (1), the eluent used for silica gel column chromatography is chloroform / acetone with a gradient elution from a volume ratio of 1:0 to 0:

1.

10. The preparation method according to claim 8, characterized in that: The solvent used in the recrystallization method of step (2) is acetone; the eluent for the ODS medium-pressure column chromatography in step (3) is methanol / water with a gradient elution from a volume ratio of 30% to 100%; the eluent for the silica gel column chromatography in step (4) is petroleum ether / acetone with a gradient elution from a volume ratio of 50:1 to 0:1; the eluent for the ODS medium-pressure column chromatography in step (6) is methanol / water with a gradient elution from a volume ratio of 70% to 100%.

11. The preparation method according to claim 8, characterized in that: The semi-preparative purification in step (5) is performed using a 5 μm, 10.0×250 mm COSMOSIL Cholester Packed column, 72% CH 3 CN / H 2 O isocratic elution, a flow rate of 3 mL / min, and the compound schipenindolene A is isolated at a retention time of 18.8 min; The semi-preparative purification in step (7) was performed using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 58% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and the compound schipenindolene G was isolated at a retention time of 34.8 min; The semi-preparative purification in step (8) was performed using a 5 μm, 9.4×250 mm Zorbax SB-C18 column, 84% CH 3 CN / H 2 O isocratic elution, a flow rate of 3 mL / min, and the compound paspaline was isolated at a retention time of 28.6 min.

12. The preparation method according to claim 8, characterized in that: The indole diterpenoid compounds and their derivatives are synthesized by using the substances separated from the fermentation product of strain Penicillium sp. DG23 as raw materials as step a) or b): a) Using 7-hydroxypaxilline-13-ene separated from the fermentation product of strain Penicillium sp.DG23 as a raw material, a Maillard reaction was performed to obtain a compound schipenindolene A; the structural formula of 7-hydroxypaxilline-13-ene is: b) using the compound 20-isopentenylpaxilline isolated from the fermentation product of strain Penicillium sp. DG23 as a raw material for oxidation reaction to obtain compound schipenindolene J, compound schipenindolene K, and compound schipenindolene L; the structural formula of 20-isopentenylpaxilline is: The reaction conditions in step a) are as follows: adding ammonium acetate and glacial acetic acid to the reaction system and reacting at 95° C. until the reaction solution changes from yellow to dark red, and the reaction is completed; a) in step a, 7-hydroxypaxilline-13-ene is dissolved in methanol and molecular sieves are added to carry out reaction; a) after the reaction in step a, the obtained substance is quenched with saturated Na2CO3 solution, extracted with EtOAc, washed with saturated brine, dried over anhydrous Na2SO4, filtered, concentrated, and semi-preparatively purified to obtain a white solid compound schipenindolene A; The purification conditions were a 5 μm, 9.4 × 250 mm Zorbax SB-C18 column, 70% CH3CN / H2O isocratic elution, a flow rate of 3 mL / min, and a retention time of 10.1 min; b) The step of preparing the compound schipenindolene J comprises the following steps: dissolving the compound 20-isopentenylpaxilline in dichloromethane, adding m-chloroperbenzoic acid, reacting at room temperature until the reaction is complete after TLC detection, and then quenching with a Na2SO3 solution, extracting the obtained substance with EtOAc, washing with saturated brine, drying with anhydrous Na2SO4, filtering, concentrating, and semi-preparative purification to obtain a white solid compound schipenindolene J; the purification conditions are using a 5μm, 9.4×250mm ZorbaxSB-C18 chromatographic column, 50% CH3CN / H2O isocratic elution, a flow rate of 3mL / min, and a retention time of 30.0min; b) The step of preparing compounds schipenindolenes K and L comprises the following steps: after dissolving compound 20-isopentenylpaxilline in acetone, adding saturated sodium bicarbonate solution at 0°C and reacting at 0°C for 0.5h, adding potassium monopersulfate complex salt dissolved in 0.7mL water and continuing to react until the reaction is complete as detected by TLC, diluting with water, extracting with EtOAc, washing with saturated brine, drying with anhydrous Na2SO4, filtering, concentrating and semi-preparative purification to obtain white solid compounds schipenindolenes K and L; the purification conditions are using a 5μm, 9.4×250mm Zorbax SB-C18 chromatographic column, 70% CH3CN / H2O isocratic elution, a flow rate of 3mL / min, and retention times of 5.3min and 6.7min, respectively.

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