A-ring opened nortriterpenoid compounds in Ganoderma lucidum and its pharmaceutical composition and application

Abstract

This invention provides four A-ring-opening triterpenoid compounds, their pharmaceutical compositions, preparation methods, and applications. It belongs to the field of pharmaceutical technology. Experimental results show that the novel four A-ring-opening triterpenoid compounds ganolearins A‒D of this invention activate the AMPK-ULK1 signaling pathway, inducing autophagy to clear the pathological microtubule-associated protein tau, revealing their potential value in the treatment of Alzheimer's disease (AD).

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a compound ganolearins AD (1-4), a pharmaceutical composition thereof, a preparation method thereof, and an application thereof in pharmaceutical manufacturing. Background Art

[0002] Ganoderma has been used to treat various diseases. At the same time, it is also an important source of some novel terpenoid compounds. Therefore, it is favored by modern pharmacologists, chemists and botanists. Due to its efficacy of "long-term consumption makes the body light and immortal, and prolongs life", many pharmacologists pay attention to the role of Ganoderma in anti-aging and treatment of other elderly diseases such as Alzheimer's disease (AD), and explore its key active substance basis. Ganoderma contains rich chemical components, among which Ganoderma triterpenoid compounds are considered to be one of its main active ingredients. Studies have shown that Ganoderma triterpenoids can reverse cognitive impairment, alleviate LPS-induced neuroinflammation and neuronal apoptosis, and can also clear Aβ amyloid protein and pathological Tau protein by activating autophagy. This shows that Ganoderma triterpenoids have significant advantages in the treatment of neurodegenerative diseases.

[0003] AD, as a common neurodegenerative disease, seriously threatens people's health and social life. Intraneuronal neurofibrillary tangles (NFTs) formed by massive aggregated amyloid β (Aβ) and phosphorylated Tau (MAPT / Tau) proteins are two main pathological features of AD. Although the cause of AD is not fully understood, eliminating Aβ (amyloid) and / or MAPT / tau protein aggregates is one of the most promising strategies to improve the symptoms of the disease. Studies have shown that autophagy is a key process for clearing faulty proteins, and cognitive impairment in AD patients can be alleviated by activating autophagy. Therefore, inducing autophagy may become one of the feasible therapeutic approaches for treating AD.

[0004] The applicant's previous research found that the Ganoderma triterpenes ganoapplin A and ganoapplin B can significantly activate the autophagy system to clear pathological Tau proteins, suggesting that these two compounds can play a role in treating AD by activating autophagy. Based on this, continuing to focus on the role of Ganoderma triterpenes in activating autophagy to treat AD, the present application discovered four novel triterpenes from Ganoderma lucidum that were rearranged after the opening of the A ring. Ganolearins AD (1-4) can induce autophagy to clear pathological Tau proteins by activating the AMPK-ULK1 signaling pathway, indicating that these four compounds have the potential to treat AD. The structures of these four compounds are reported for the first time and have not been reported in the literature. Therefore, there have been no reports on the compound ganolearins AD (1-4), its preparation method, and its application in pharmaceutical manufacturing, nor on pharmaceutical compositions with it as an active ingredient. Summary of the invention

[0005] The purpose of the present invention is to provide compounds ganolearins AD (1-4) and their preparation methods and applications in pharmaceuticals. Compounds 1-4 have potential anti-Alzheimer's disease (AD) effects. Also provided are pharmaceutical compositions containing the compounds as active ingredients, their preparation methods and applications in pharmaceuticals.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] The present invention first discloses the structures of four compounds, the names of the compounds are ganolearins AD (1-4), and the compounds are ganoderma triterpenoid compounds with the following structural formula:

[0008]

[0009] The compound is obtained by extraction from Ganoderma lucidum or by artificial synthesis.

[0010] The present invention also provides:

[0011] 1) A pharmaceutical composition of four novel compounds, comprising ganolearins AD (1-4) or pharmaceutically acceptable derivatives and salts thereof.

[0012] 2) The pharmaceutical composition is a pharmaceutical preparation, which is composed of ganolearins AD (1-4) or its pharmaceutically acceptable derivatives and salts thereof as active ingredients and a pharmaceutically acceptable carrier.

[0013] Pharmaceutically acceptable carriers include: solid preparations including tablets, capsules, pills, granules, etc.; semi-solid preparations including ointments, suppositories, etc.; liquid preparations including solutions, injections, sprays, etc.; ophthalmic preparations including eye drops, ophthalmic gels, etc.

[0014] 3) Provided are four methods for preparing triterpene compounds or pharmaceutical compositions, the methods comprising:

[0015] Take the Ganoderma lucidum, crush it, and reflux extract it with 20 times the volume of 95% ethanol. The extract is decompressed to recover the ethanol to obtain an extract. The extract is suspended in water and extracted with an equal volume of ethyl acetate for 4 times. After the ethyl acetate layer is concentrated, it is separated by normal phase silica gel and eluted with dichloromethane / methanol to obtain five fractions of 100:0, 80:1, 50:1, 20:1, 10:1 and 5:1. A→F; the Fr.C part was treated with a normal phase silica gel column, with dichloromethane / acetone as the eluent, and after merging, 5 parts Fr.C1→Fr.C5 were obtained; Fr.C3 was treated with a reverse phase silica gel column, eluted with methanol / water 35%-100%, and 8 parts Fr.C3-1→Fr.C3-8 were obtained after merging. The Fr.C3-1 part was separated with normal phase silica gel to obtain 5 parts Fr.C3-1a→Fr.C3-1d, of which Fr.C3-1d was purified by semi-preparative HPLC to obtain compound 4 (acetonitrile / water=53:48, v / v). In addition, Fr.C3-2 was treated with gel LH-20, and eluted with methanol to obtain 6 parts: Fr.C3-2a-2f. Fr.3C-2d was also purified by semi-preparative HPLC to obtain compound 1 (acetonitrile / water=40:60, v / v).

[0016] The Fr.C4 fraction was first treated with a normal phase silica gel column, and the eluent was dichloromethane / methanol (80:1, 50:1, 20:1, 10:1 and 5:1) to obtain 5 fractions Fr.C4-1→Fr.C4-5. Among them, Fr.C4-2 was treated with gel and eluted with methanol to obtain 5 fractions (Fr.C4-2a→Fr.C4-2e). Fr.C4-2c was purified by semi-preparative HPLC to obtain compound 2 (acetonitrile / water = 55:45, v / v). Similarly, Fr.C4-2e was prepared by semi-preparative HPLC to obtain compound 3, and the elution conditions were acetonitrile / water = 42:58, v / v. The flow rate in the above semi-preparative process was 3 mL / min.

[0017] The preparation of the pharmaceutical composition also includes taking one or any combination of compounds 1-4 and then adding a pharmaceutically acceptable carrier; or further preparing the compound into a pharmaceutically acceptable derivative and a salt thereof and then adding a pharmaceutically acceptable carrier.

[0018] 4) Provided is the use of the new compound described in 1) in the preparation of a drug for treating AD.

[0019] The fifth aspect of the present invention provides the second aspect

[0020] 5) Provided is the use of the pharmaceutical composition of the new compound described in 2) in the preparation of a drug for treating AD.

[0021] 6) The compound ganolearins AD (1-4) provided by the present invention is extracted and separated from Ganoderma lucidum or prepared by artificial synthesis, and has a good effect in treating AD.

[0022] 7) Provided is the use of the compound ganolearins AD (1-4) or the pharmaceutical composition described in 1) in the preparation of a drug for treating AD-related diseases.

[0023] When used as medicine, the compound of the present invention can be used directly or in the form of a pharmaceutical composition. The pharmaceutical composition contains 0.1-99%, preferably 0.5-90% of the compound of the present invention, and the rest is a pharmaceutically acceptable, non-toxic and inert pharmaceutical carrier to humans and animals.

[0024] The pharmaceutical carrier is one or more solid, semi-solid and liquid diluents, fillers and pharmaceutical product adjuvants. The pharmaceutical composition of the present invention is used in the form of a dosage per unit body weight. The drug of the present invention can be administered in three forms: injection (intravenous injection, intramuscular injection), oral administration and dripping pills.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] 1. The present invention provides four new compounds ganolearins AD (1-4), which fill the gap in the prior art.

[0027] 2. The present invention provides a method for preparing the compound ganolearins AD (1-4), which has readily available raw materials, is easy to operate, has a high yield, and is suitable for industrial production.

[0028] 3. The present invention provides a pharmaceutical composition with the compound ganolearins AD (1-4) as an active ingredient, providing a candidate drug for the treatment of AD.

[0029] 4. The compounds ganolearins AD (1-4) of the present invention can induce autophagy by activating the AMPK-ULK1 signaling pathway, clearing pathological tau protein, thereby exerting an anti-AD effect.

[0030] 5. The compound ganolearins AD (1-4) can be used as a drug to treat AD and other related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the structure of the compound ganolearins AD (1-4) of the present invention;

[0032] Figure 2 The crystal structures of the compounds ganolearin B (2) and ganolearin D (4) of the present invention;

[0033] Figure 3 The compounds of the present invention, ganolearins AD (1-4), promote the autophagic flux of SH-SY5Y mCherry-GFP-LC3 cells;

[0034] Figure 4 The compounds ganolearins AD (1-4) of the present invention promote autophagy and inhibit the expression of pathological molecule Tau P301S in SH-SY5Y MAPT cells;

[0035] Figure 5 The inhibitory effect of the compound ganolearins AD (1-4) of the present invention on mTOR;

[0036] Figure 6 The compounds of the present invention, ganolearins AD (1-4), activate the AMPK-ULK1 signaling pathway;

[0037] Figure 7 The present invention is a molecular docking of the compounds ganolearins AD (1-4) of the present invention and AMPK. DETAILED DESCRIPTION

[0038] The following is a further description of the essential content of the present invention with reference to the accompanying drawings, but the present invention is not limited thereto. Simple improvements made to the present invention according to the essence of the present invention all belong to the scope of the present invention.

[0039] Embodiment 1:

[0040] Isolation and purification of compound ganolearins AD (1-4).

[0041] Take the Ganoderma lucidum, crush it, and reflux extract it with 20 times the volume of 95% ethanol. The extract is decompressed to recover the ethanol to obtain an extract. The extract is suspended in water and extracted with an equal volume of ethyl acetate for 4 times. After the ethyl acetate layer is concentrated, it is separated by normal phase silica gel and eluted with dichloromethane / methanol to obtain five fractions of 100:0, 80:1, 50:1, 20:1, 10:1 and 5:1. A→F; the Fr.C part was treated with a normal phase silica gel column, with dichloromethane / acetone as the eluent, and after merging, 5 parts Fr.C1→Fr.C5 were obtained; Fr.C3 was treated with a reverse phase silica gel column, eluted with methanol / water 35%-100%, and 8 parts Fr.C3-1→Fr.C3-8 were obtained after merging. The Fr.C3-1 part was separated with normal phase silica gel to obtain 5 parts Fr.C3-1a→Fr.C3-1d, of which Fr.C3-1d was purified by semi-preparative HPLC to obtain compound 4 (acetonitrile / water=53:48, v / v). In addition, Fr.C3-2 was treated with gel LH-20, and eluted with methanol to obtain 6 parts: Fr.C3-2a-2f. Fr.3C-2d was also purified by semi-preparative HPLC to obtain compound 1 (acetonitrile / water=40:60, v / v).

[0042] The Fr.C4 part was first treated with a normal phase silica gel column, and the eluent was dichloromethane / methanol (80:1, 50:1, 20:1, 10:1 and 5:1) to obtain 5 parts Fr.C4-1→Fr.C4-5. Among them, Fr.C4-2 was treated with gel and eluted with methanol to obtain 5 fractions (Fr.C4-2a→Fr.C4-2e). Fr.C4-2c was purified by semi-preparative HPLC to obtain compound 2 (acetonitrile / water = 55:45, v / v). Similarly, Fr.C4-2e was prepared by semi-preparative HPLC to obtain compound 3, and the elution conditions were acetonitrile / water = 42:58, v / v. The flow rate in the above semi-preparative process was 3mL / min. The structure of the compound was identified as follows:

[0043]

[0044] Compound ganolearin A(1): white powder (MeOH); [α] 24 D 22.75(c 0.16,MeOH); UV(MeOH); λ max (logε): 201 (3.72), 234 (3.88) and 297 (2.87); 1 H NMR and 13 C NMR: data see Table 1; HRMS (ESI-TOF) m / z: 401.2322 [M+H] + (calcd for C24 H 32 O5,401.2323).

[0045] Compound ganolearin B (2): colorless needles (MeOH); [α] 20 D +51.13(c 0.13,MeOH); UV(MeOH)λ max (logε):277(2.09),195(2.03); 1 H NMR and 13 C NMR data are shown in Table 1; HRMS (ESI-TOF) m / z: 469.2564 [M+Na] + (calcd for C 26 H 38 O6,469.2561).

[0046] Compound ganolearin C(3): white powder (MeOH); [α] 24 D -53.39(c 0.23,MeOH); UV(MeOH); λ max (logε):202(3.68) and 273(3.80); 1 H NMR and 13 C NMR data are shown in Table 1; HRMS (ESI-TOF) m / z: 457.2602 [MH] - (calcd for C 27 H 38 O6,457.2596).

[0047] Compound ganolearin D(4): colorless crystals (MeOH); [α] 24 D 12.67(c 0.18,MeOH); UV(MeOH); λ max (logε): 202(3.25) and 256(3.33); 1 H NMR and 13 C NMR data are shown in Table 1; HRMS (ESI-TOF) m / z: 471.2143 [MH] - (calcd for C 25 H 36 O6,471.2143).

[0048] Table 1. Compounds 1-4 1 H and 13 C NMR data

[0049]

[0050] a :CDCl3,600 / 150MHz

[0051] Embodiment 2:

[0052] Any one or a combination of the compounds ganolearins AD (1-4) is added with an injection solvent according to a conventional method, and then finely filtered, sealed and sterilized to prepare an injection solution.

[0053] Embodiment 3:

[0054] Any one of the compounds ganolearins AD (1-4) or a combination thereof is used as a pharmaceutical active ingredient, and several excipients are used as auxiliary ingredients for preparing combined pharmaceutical tablets, and tablet samples containing 1-100 mg of the pharmaceutical ingredient per tablet are prepared according to a certain ratio.

[0055] Embodiment 4:

[0056] Any one or a combination of the compounds ganolearins AD (1-4) can be prepared into capsules by conventional methods with various pharmaceutical excipients:

[0057] The invention relates to the preparation of a pharmaceutical combination capsule preparation containing any one of the compounds ganolearins AD (1-4) or a combination thereof as an active ingredient. The invention relates to a capsule preparation containing 1-100 mg of the compound ingredient in each capsule by using any one of the compounds ganolearins AD (1-4) as the active ingredient and several excipients as auxiliary ingredients for preparing the combination capsule preparation.

[0058] Embodiment 5:

[0059] Take 1 part of any one of the compounds ganolearins AD (1-4) or a combination thereof and 10 parts of non-dairy creamer, mix them well, and prepare a solid beverage according to a conventional method.

[0060] Embodiment 6:

[0061] Activity evaluation of compounds ganolearins AD (1-4).

[0062] Cell culture and treatment.

[0063] SH-SY5Y mCherry-GFP-LC3 and SH-SY5Y MAPT cells were provided and cultured by Kunming Institute of Zoology, Chinese Academy of Sciences. The cells were cultured in Dulbecco's modified medium (Gbico, 11140050) supplemented with 10% fetal bovine serum (FBS), 1×MEM non-essential amino acid solution, 100U / ml penicillin and 100mg / ml streptomycin in a humidified incubator at 37°C, 5% CO2 and 95% humidity. The cells were seeded in preheated growth medium in 6-well plates. Chemical agents were applied directly to the culture medium for treatment, and cells were harvested 24 hours after treatment for the next analysis.

[0064] Construction of SH-SY5Y cells stably expressing the mutant MAPT (p.P301S) gene (SH-SY5YMAPT cells).

[0065] The CDS region of the MAPT gene tagged with mCherry was cloned into the PLVX vector of the lti-xTet-On Advanced Inducible Expression System (Clontech). The mutant MAPT-p.P301S was introduced into the PLVX-MAPT vector by site-directed mutagenesis PCR. SH-SY5Y cells were maintained in DMEM (Gibco, USA, 11875) supplemented with 10% fetal bovine serum (Gibco, USA, 11875), 1×MEM non-essential amino acid solution (Gibco, 11140050), 100 U / ml penicillin and 100 mg / ml streptomycin (Gibco, USA, 11965), and cultured in a humidified atmosphere incubator at 37°C with 5% CO2. HEK293T cells were cultured in DMEM containing 10% heat-inactivated fetal bovine serum. SH-SY5Y cells stably expressing mutant MAPT (P301S) were constructed using the lentix Tet-On Advanced Inducibleexpression System (Clontech) software. In summary, the response lentiviral system consisted of the mutant PLVX-MAPT P301S construct, the packaging plasmid psPAX2 (Addgene, England, 12260), and the envelope plasmid PMD2.G (Addgene, England, 12259). The regulatory lentiviral system consisted of the PLVX-Tet-On-Advanced vector, psPAX2, and PMD2.G. Lentiviral supernatants were prepared from HEK293T cells and infected SH-SY5Y cells at a ratio of 4:1 with the response lentivirus and the regulatory virus. Cells were selected in medium containing 500 μg / mL puromycin.

[0066] The cell viability was determined using the CCK-8 method.

[0067] For cell viability assay, CCK-8 kit was used as described by the manufacturer (Beyotime, C0038). Briefly, SH-SY5Y mCherry-GFP-LC3 cells were plated at 4×10 3 Cells were seeded in 96-well culture plates. After 24 hours of treatment with compounds 1-4, CCK-8 reagent was added to each well. Cell viability was assessed by measuring the optical density at 450 nm.

[0068] Flow cytometric analysis.

[0069] SH-SY5Y mCherry-GFP-LC3 cells are cells stably expressing a triple fusion protein (red fluorescent protein (mCherry), green fluorescent protein (GFP), and the autophagosome marker LC3, which directly reflects the intensity of autophagic flux, to detect the biological activity of compounds 1-4. In the absence of autophagy, these cells show yellow fluorescence, which is due to the co-expression of red mCherry and green GFP. When the autophagic process proceeds smoothly, the autophagosome fuses with the lysosome to form the autophagosome, and the acidic lysosomal environment quenches the fluorescence of the acid-sensitive GFP, while mCherry is not affected, and the autophagosome exhibits red fluorescence. Therefore, the red fluorescence in the cell can indicate the formation of autolysosomes. The higher the red fluorescence and the lower the green fluorescence, the better the flux smoothness of the autophagosome. SH-SY5Y mCherry-GFP-LC3 cells were cultured in DMEM supplemented with 10% fetal bovine serum (Gibco-BRL, 10099-141) at 37°C, 5% carbon dioxide and 95% humidity in an incubator. SH-SY5YmCherry-GFP-LC3 cells were cultured in 12-well plates for 24 h, and 5 μM and 20 μM compounds were added directly to the culture medium. After 24 hours of treatment, the cells were harvested and fixed with 4% paraformaldehyde. The autophagic flux of fixed cells was then detected by flow cytometry. Data were analyzed using FlowJo software (FlowJo, LLC).

[0070] Western blot analysis.

[0071] SH-SY5Y and SH-SY5Y MAPT cells were lysed with RIPA lysis buffer (Beyotime, P0013). Protein concentration was determined using a BCA protein assay kit (Beyotime, P0012). 20 μg of protein was separated using 12% or 8% (vol / vol) SDS-polyacrylamide gel, electrophoretically transferred to a polyvinylidene difluoride membrane (Bio-Rad, L1620177 Rev D), and soaked in 5% (vol / vol) skim milk for 2 h at room temperature. The membrane was incubated with primary antibodies (ACTB [Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., TA-09]); GAPDH, glyceraldehyde-3-phosphate dehydrogenase [Proteintech, 60004-1-Ig]; LC3 [Proteintech, 14600-1-AP]; phospho-AMPKβ1(ser108)(E8N3N); AMPKα(D63G4)[Cell Signaling Technology, 5832]; phospho-AMPKα(thr172)(D4D6D)[Cell[Cell Signaling Technology, 20081]; AMPKβ1 / 2(57C12); mTOR(7C10); phospho-mTOR(ser2448)(D9C2)[Cell Signaling Technology, 20081]; Technology, 5536]; phospho-mTOR (ser2481); phospho-ULK1 (ser757) (D7O6U) [Cell Signaling Technology, 2014]; phospho-ULK1 (ser317) [Cell Signaling Technology, 37762]; ULK1 (D8H5) [Cell Signaling Technology, 8054]; phospho-ULK1 (ser555) [Cell Signaling Technology, 5869]; phospho-ULK1 (ser777) [Sigma-Aldrich, ABC213]; SQSTM1, sequestosome 1 [Elabscience, EAP3350] and Tau (D1M9X) [Cell Signaling Technology, 46687S]) at 4°C overnight.The membranes were incubated with TBST (tris-buffered saline [Cell Signaling Technology, 9997], Tween 20 [0.1%]; Sigma, P1379]) for 5 min each time and reacted with peroxidase-conjugated anti-mouse (474-1806) or anti-rabbit (474-1516) IgG (1:5000; KPL) at room temperature. The epitopes were visualized using the ECL Western blot detection kit (Millipore, WBKLS0500). ImageJ software (National Institutes of Health, Bethesda, Maryland, USA) was used for density determination. GAPDH or ACTB was used as a control to quantify the density of the target protein.

[0072] Confocal imaging analysis.

[0073] SH-SY5Y mCherry-GFP-LC3 cells were placed in glass-bottomed cell dishes (NEST, 801001) and cultured overnight in DMEM supplemented with 10% FBS, 1×MEM non-essential amino acid solution (Gibco, 11140050), 100U / ml penicillin and 100mg / ml streptomycin. To evaluate the tandem fluorescent LC3, cells were washed three times with PBS and fixed with 4% paraformaldehyde after 24h of treatment with rapamycin, compound 1, 2, 3 or 4. Fixed intact cells were captured using a FluoView 1000 confocal microscope (Olympus, America).

[0074] Flow cytometry analysis showed that compounds 1-4 significantly increased autophagic flux ( Figure 3 ). In addition, CCK-8 experiments showed that they were not toxic to SH-SY5Y mCherry-GFP-LC3 cells ( Figure 3 ).

[0075] In SH-SY5Y MAPT cells treated with 1-4, the levels of lipidated (pe-conjugated) forms of MAP1LC3 / LC3 (microtubule-associated protein 1 light chain 3; LC3-II) / LC3-I and SQSTM1 (sequestosome 1) proteins decreased in a dose-dependent manner ( Figure 4 Meanwhile, compounds 1-4 reduced the level of Tau P301S in SH-SY5Y MAPT cells ( Figure 4). In addition, BAFA1 (Bafilomycin A1), a vacuolar (V)-type ATPase inhibitor, blocked the fusion of autophagosomes with lysosomes and the accumulation of LC3-II, further demonstrating the role of 1-4-induced autophagy in MAPT / Tau clearance in SH-SY5YMAPT cells. Treatment with BAFA1 (20 nM) alone increased LC3-II / LC3-I but had no significant effect on Tau P301S. BAFA1 reversed the decrease in Tau P301S levels induced by compounds 1-4 ( Figure 4 ). These results suggest that compounds 1-4 reduce TauP301S by activating autophagy.

[0076] In addition, the present invention also studied the mechanism by which compounds 1-4 regulate autophagy. First, the present invention examined whether these compounds would affect the activity of mTOR. Rapamycin (an inhibitor of mTOR) was used as a positive control. The results showed that the activity of mTOR was not affected by compounds 1-4, as there was no significant change in the protein levels of mTOR, pmTOR2448, and pmTOR2481 in SH-SY5Y MAPT cells ( Figure 5 ).

[0077] Since AMPK activation is one of the key pathways for autophagy induction, recent studies have reported that mTOR inactivation is not always required for autophagy, and AMPK can also directly phosphorylate and activate Ser317, Ser555 and Ser777 of ULK1, thereby initiating autophagy. In the present invention, compounds 1-4 significantly increased the protein levels of pAMPKα, pAMPKβ1, pULKser777, pULKser317, and pULKser555 in SH-SH5Y MAPT cells, while after treatment with compounds 1-4, the protein levels of AMPKα, AMPKβ1, AMPKβ2, ULK1, and pULKser757 did not change significantly ( Figure 6 ).

[0078] Molecular docking revealed the interaction mode between compounds 1-4 and AMPK, and their binding energy range was between -7.7 and -8.2 kcal / mol. Compounds 1-4 bind to the key domain of AMPK containing ARG263 ( Figure 7 ), thereby affecting the phosphorylation of AMPK. These results suggest that compounds 1-4 induce autophagy by activating the AMPK-ULK1 pathway rather than the mTOR-dependent signaling pathway.

Claims

1. A compound represented by the following structural formula, 2. The method for preparing the compound ganolearins AD (1-4) according to claim 1, characterized in that: The method comprises the following steps: taking the Ganoderma lucidum, crushing it, refluxing and extracting it with 20 times the volume of 95% ethanol, recovering the ethanol from the extract under reduced pressure to obtain an extract, suspending the extract in water, extracting it with an equal volume of ethyl acetate for 4 times, concentrating the ethyl acetate layer, separating it with normal phase silica gel, eluting it with dichloromethane / methanol, and obtaining five fractions of 100:0, 80:1, 50:1, 20:1, 10:1 and 5:1 A→F; the Fr.C part was treated with a normal phase silica gel column with dichloromethane / acetone as the eluent, and after merging, 5 parts Fr.C1→Fr.C5 were obtained; Fr.C3 was then treated with a reverse phase silica gel column and eluted with methanol / water 35%-100%, and 8 parts Fr.C3-1→Fr.C3-8 were obtained after merging. The Fr.C3-1 part was separated with normal phase silica gel to obtain 5 parts Fr.C3-1a→Fr.C3-1d, of which Fr.C3-1d was purified by semi-preparative HPLC to obtain compound 4. In addition, Fr.C3-2 was treated with gel LH-20 and eluted with methanol to obtain 6 parts: Fr.C3-2a-2f; Fr.C3-2d was also purified with Compound 1 was purified by semi-preparative HPLC; the Fr.C4 part was first treated with a normal phase silica gel column, and the eluent was dichloromethane / methanol in a volume ratio of 80:1, 50:1, 20:1, 10:1 and 5:1 to obtain 5 parts Fr.C4-1→Fr.C4-5, among which Fr.C4-2 was treated with gel and eluted with methanol to obtain 5 fractions Fr.C4-2a→Fr.C4-2e; Fr.C4-2c was purified by semi-preparative HPLC to obtain compound 2; similarly, Fr.C4-2e was prepared by semi-preparative HPLC to obtain compound 3, and the elution conditions were acetonitrile / water in a volume ratio of 42:58; the flow rate in the above semi-preparative process was 3 mL / min.

3. Use of the compound ganolearins AD (1-4) according to claim 1 in the preparation of a drug for treating Alzheimer's disease.

4. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the compound ganolearins AD (1-4) as claimed in claim 1 or a pharmaceutically acceptable carrier thereof.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition is a pharmaceutical preparation, which is composed of ganolearins AD (1-4) and its salts as active ingredients and a pharmaceutically acceptable carrier.

6. Use of the pharmaceutical composition according to claim 4 or 5 in the preparation of a drug for treating Alzheimer's disease.

7. The method for preparing the pharmaceutical composition according to claim 4 or 5, characterized in that: The method comprises the following steps: taking the Ganoderma lucidum, crushing it, refluxing and extracting it with 20 times the volume of 95% ethanol, recovering the ethanol from the extract under reduced pressure to obtain an extract, suspending the extract in water, extracting it with an equal volume of ethyl acetate for 4 times, concentrating the ethyl acetate layer, separating it with normal phase silica gel, eluting it with dichloromethane / methanol, and obtaining five fractions of 100:0, 80:1, 50:1, 20:1, 10:1 and 5:1 A→F; the Fr.C part was treated with a normal phase silica gel column and dichloromethane / acetone as the eluent, and after merging, 5 parts Fr.C1→Fr.C5 were obtained; Fr.C3 was then treated with a reverse phase silica gel column and eluted with methanol / water 35%-100%, and 8 parts Fr.C3-1→Fr.C3-8 were obtained after merging. The Fr.C3-1 part was separated with normal phase silica gel to obtain 5 parts Fr.C3-1a→Fr.C3-1d, of which Fr.C3-1d was purified by semi-preparative HPLC to obtain compound 4. In addition, Fr.C3-2 was treated with gel LH-20 and eluted with methanol to obtain 6 parts: Fr.C3-2a-2f; Fr.3C-2d was also purified by semi-preparative HPLC to obtain compound Compound 1; the Fr.C4 part was first treated with a normal phase silica gel column, and the eluent was dichloromethane / methanol in a volume ratio of 80:1, 50:1, 20:1, 10:1 and 5:1 to obtain 5 parts Fr.C4-1→Fr.C4-5, among which Fr.C4-2 was treated with gel and eluted with methanol to obtain 5 fractions Fr.C4-2a→Fr.C4-2e; Fr.C4-2c was purified by semi-preparative HPLC to obtain compound 2; similarly, Fr.C4-2e was prepared by semi-preparative HPLC to obtain compound 3, and the elution conditions were acetonitrile / water in a volume ratio of 42:58; the flow rate in the above semi-preparative process was 3 mL / min; then compound 1-4 or a salt thereof was taken and a pharmaceutically acceptable carrier was added.

8. Use of the compound ganolearins AD (1-4) according to claim 1 or the pharmaceutical composition according to claim 4 or 5 in the preparation of a drug for treating Alzheimer's disease-related diseases.

Images