Pyrrolopyridone alkaloid compounds, and preparation method and application thereof

Abstract

The application discloses pyrrolopyridine alkaloid compounds, a preparation method and application thereof, and belongs to the technical field of medicines. Pyrrolopyridine alkaloid compounds with an activity of resisting acute liver injury induced by paracetamol are extracted from fermentation products of a marine fungus Aspergillus aculeatinus WHUF0198. The compounds improve the cell survival rate of liver cells of mice treated by paracetamol, and reduce the glutamic-pyruvic transaminase level in cell supernatant. The pyrrolopyridine alkaloid compounds can be used for preparing medicines for treating acute liver injury. The application has a high potential economic benefit in view of the current situation that there are few types of medicines for treating acute liver injury caused by paracetamol.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to a pyrrolopyridone alkaloid compound isolated from a fermentation product of a pseudo-tubercular aspergillus fungus, a preparation method thereof, and application of the pyrrolopyridone alkaloid compound in preparation of a medicine for treating acute liver injury. BACKGROUND

[0002] Acute liver injury (ALI) is characterized by symptoms such as liver cell damage, necrosis, apoptosis and liver dysfunction occurring in a short period of time. ALI can be triggered by various factors, including drugs, viral infection, liver surgery and chemical toxins. Drug-induced liver injury (DILI) is the main cause of ALI, accounting for more than 50% of cases, and DILI refers to liver injury caused by various drugs, their metabolites or excipients, etc. Drugs that often cause DILI are acetaminophen (APAP), amiodarone, steroid drugs, antimetabolites, valproic acid, nicotinic acid, statins, etc. Among them, the combination of APAP and isoniazid and rifampicin is the most common cause of DILI, and the main pathogenesis is the decompensation of the antioxidant stress system and the accumulation of ROS, which further damages the function of liver cell mitochondria.

[0003] ALI caused by APAP occurs suddenly, often accompanied by liver failure, hepatic encephalopathy and other serious complications, leading to severe oxidative stress and persistent inflammatory response, and may lead to death if not treated in time. Therefore, it is of great significance to develop new drugs with multiple targets and multiple action pathways to treat ALI caused by APAP. N-acetyl-L-cysteine (NAC) is the most commonly used drug in clinical settings to treat APAP poisoning, but its use is complicated due to a narrow dosing window and potential serious side effects. Therefore, it is of great clinical significance to find a less side-effect and more effective drug to treat APAP-induced liver injury.

[0004] Pyrrolopyridone alkaloids are a class of alkaloid compounds with a 2-pyridone pyran skeleton. Natural and artificially synthesized compounds of this class have a wide range of biological activities, such as anti-tumor, antibacterial, antifungal and anti-inflammatory activities. Pyrrolopyridone compounds with unsaturated branched-chain substitutions on the pyran ring have only 14 kinds reported in the literature, and they have Ca 2+Inhibitory effect, NO release inhibitory effect. Due to its unique biological activity, this class of compounds has attracted great attention from synthetic and medicinal chemists. However, the research on pyrrolopyridone alkaloids with unsaturated side chains is quite limited at present, and there is no report on the ALI improving activity of such compounds. Therefore, it is of great research value to explore pyrrolopyridone derivatives with ALI improving activity from natural sources. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide pyrrolopyridone alkaloids with anti-APAP-induced acute liver injury activity extracted from the fermentation product of marine fungus Aspergillus aculeatinus WHUF0198 and its preparation method and pharmaceutical application. The pyrrolopyridone alkaloids 1-7, 9, 10, 12-15 provided by the present application can improve the cell survival rate of AML-12 cells (mouse liver cells) treated by APAP and reduce the alanine aminotransferase (ALT) level in the cell supernatant, and have the potential to develop into drugs for treating acute liver injury induced by APAP. The present application provides a lead compound for developing drugs for treating DILI.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] The present application extracts 17 pyrrolopyridone alkaloid compounds from the fermentation product by culturing marine fungus Aspergillus aculeatinus WHUF0198 alone and co-culturing with mangrove-derived fungus Penicillium sp. DM27 fungus.

[0008] The pyrrolopyridone alkaloid compounds provided by the present application are compounds 1-17 shown in the following structural formula.

[0009] The structural formula of compounds 1, 2, 3 and 4 is as follows:

[0010] The group combination of compounds 1, 2, 3 and 4 is shown in the following table respectively:

[0011] No. [R1] [R2] Compound 1 CH3 CH3 Compound 2 CH2OH CH3 Compound 3 COOH CH3 Compound 4 CH3 COOH

[0012] The structural formula of compounds 5, 6, 7, 8, 9, 10 and 11 is as follows:

[0013] The group combination of compounds 5, 6, 7, 8, 9, 10 and 11 is shown in the following table respectively:

[0014]

[0015]

[0016] Structural formula of compound 12, 13, 14, 15:

[0017] The group combinations of compound 12, 13, 14, 15 are shown in the following table respectively:

[0018] No. [R1] [R2] Compound 12 CH2OH CH3 <!-- 2 -->]]> Compound 13 COOH CH3 Compound 14 CH3 COOH Compound 15 CH2OCHO CH3

[0019] Structural formula of compound 16:

[0020] Structural formula of compound 17:

[0021] The preparation method of the pyrrolopyridone alkaloid compounds comprises the following steps: inoculating Aspergillus aculeatinus WHUF0198 strain alone or simultaneously with Penicillium sp. DM27 into a liquid culture medium to carry out single fermentation or co-culture fermentation respectively, and separating the pyrrolopyridone alkaloid compounds from the fermentation product.

[0022] Further, the preparation method of the pyrrolopyridone alkaloid compounds comprises the following steps: inoculating Aspergillus aculeatinus WHUF0198 strain alone or simultaneously with Penicillium sp. DM27 into a liquid culture medium to carry out single fermentation or co-culture fermentation, separating the fermentation broth and mycelium, stirring the mycelium, ultrasonic extracting with acetone, recovering the acetone, and then combining the fermentation broth and the mycelium after concentrating under reduced pressure. The combined liquid is extracted with ethyl acetate, concentrated under reduced pressure to obtain a crude extract; or the combined liquid is added to a macroporous resin for adsorption, and the macroporous resin adsorbing the metabolites is subjected to column chromatography to obtain components containing different methanol contents by gradient elution with methanol / water, and the pyrrolopyridone alkaloid compounds are determined by detecting the characteristic absorption peak of the pyrrolopyridone alkaloid by HPLC-DAD and analyzing the existence of nitrogen atoms by LC-MS. These components are combined and concentrated under reduced pressure to obtain a crude extract. Then, the crude extract is separated by silica gel column chromatography to obtain smaller components, and then the components containing the characteristic peak of the pyrrolopyridone alkaloid compound are purified by semi-preparative liquid chromatography to obtain the pyrrolopyridone alkaloid compounds.

[0023] The pyrrolopyridone alkaloid compound has an effect of resisting acute liver injury, and based on this, the application further provides application of the pyrrolopyridone alkaloid compound in preparation of a medicine for treating acute liver injury, wherein the pyrrolopyridone alkaloid compound comprises one or more of the compounds 1-17.

[0024] The application further provides a medicine for treating acute liver injury, which comprises the pyrrolopyridone alkaloid compound and can further comprise a pharmaceutically acceptable adjuvant; and the pyrrolopyridone alkaloid compound comprises one or more of the compounds 1-17.

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

[0026] Firstly, the culture conditions of the single culture of the strain Aspergillus aculeatinus WHUF0198 and the culture conditions of the co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp.DM27 are easy to control and have good reproducibility, and a sufficient amount of the pyrrolopyridone alkaloid compound can be obtained through fermentation, so as to solve the problems of few natural source acquisition channels and complicated artificial synthesis steps of the compound.

[0027] Secondly, the preparation method of the alkaloid compound comprises the following steps: ultrasonic extraction of mycelium by using acetone, macroporous resin HP20 adsorption extraction of fermentation liquor, macroporous resin column gradient solvent elution, silica gel column chromatography, Sephadex LH-20 gel column chromatography and semi-preparative high performance liquid chromatography separation and purification, etc. The required separation materials and equipment are simple and common, and the purification process is efficient and feasible.

[0028] Thirdly, the pyrrolopyridone alkaloid compound prepared by the application has a significant repairing effect on mouse liver cell damage caused by paracetamol, and can be used for preparing a medicine for treating acute liver injury caused by paracetamol, and the potential economic benefits are rich in view of the current situation of few types of medicines for treating acute liver injury caused by paracetamol. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 Normal mouse liver cells AML-12, APAP-induced liver injury AML-12 cells, cell survival rates of APAP-induced liver injury cells treated by different concentrations of compounds 1-7 and 9-16.

[0030] Figure 2Figure 9 is the alanine aminotransferase (ALT) level of normal mouse hepatocyte AML-12, APAP-induced liver injury AML-12 cells, and supernatant of APAP-induced liver injury cells treated with compounds 1-7 and 9-16. (c = 5 mM, n = 3-6). Data are expressed as mean SD. All compounds significantly reduced the ALT level *P < 0.05, **P < 0.01, ***P < 0.001.

[0031] Figure 3 Figure 10 is a Venn diagram of acute liver injury (ALI) disease-related targets and compound 12-related targets.

[0032] Figure 4 Figure 11 is a GO analysis of compound 12 targets.

[0033] Figure 5 Figure 12 is a KEGG analysis of compound 12 targets.

[0034] Figure 6 Figure 13 is a network diagram of ALI-related binding sites of compound 12. Color depth represents the degree of association.

[0035] Figure 7 Figure 14 is the binding energy of compound 12 and core target proteins.

[0036] Figure 8 Figure 15 is the molecular docking of compound 12 and ALI-related target proteins. (A) AKT, (B) HSP90AA1, (C) MTOR, (D) BCL2, (E) PIK3CA, (F) STAT3, (G) SRC, (H) TNF-a, (I) GRB2, (J) EGFR. DETAILED DESCRIPTION

[0037] The following examples are intended to further illustrate the present application and are not intended to limit the present application. Unless otherwise indicated, the techniques utilized in the examples are routine techniques well known in the art.

[0038] Example 1: Preparation of Pyrrolopyridone Alkaloid Compounds

[0039] A. Strain Fermentation

[0040] Strain Aspergillus aculeatinus WHUF0198 fermentation alone: Aspergillus aculeatinus WHUF0198 (the strain has been disclosed in the following articles: Wu J, Zhang H, He LM, et al, A New Fusicoccane-Type Norditerpene and a New Indone from the Marine-Derived Fungus Aspergillus aculeatinus WHUF0198 [J]. Chemistry & Biodiversity. 2021, 18(10): e2100562; Wu J, Wang F, He LM, et al, Aculeaquamide A, Cytotoxic Paraherquamide from the Marine Fungus Aspergillus Aculeatinus WHUF0198 [J]. Natural Product Research. 2022, 36(17), 4382-4387; Wu J, Shui H, Zhang M, et al. Aculeaxanthones A-E, New Xanthones from the Marine-Derived Fungus Aspergillus Aculeatinus WHUF0198 [J]. Frontiers in Microbiology. 2023, 14.) was inoculated in a culture dish containing PDA solid medium, and incubated at 28°C in a biochemical incubator for 7 days. Then the solid medium was divided into 1 cm x 1 cm blocks, inoculated into a 1L conical flask containing 300 mL of PDB liquid medium for fermentation, the fermentation temperature was 28°C, the fermentation time was 28 days, and the total fermentation volume was 100 flasks. PDA solid medium formula: potato glucose water 26g (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), agar 20g, water 1000mL, pH 6.5-7; PDB liquid medium formula: potato glucose water 26g (Qingdao High-tech Industrial Park Haibo Biotechnology Co., Ltd.), sea salt 15g, water 1000mL, pH 6.5-7.

[0041] Co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27: Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27 (this strain has been disclosed in the following article: He LM, Zhao ZF, Zhang MK, et al, Peniciisoquinoline A: A new tetrahydroisoquinoline from mangrove-derived Penicillium sp. DM27 fungus [J]. Journal of Holistic Integrative Pharmacy. 2022, 3(3): 222-227; Dong Zhidong, Isolation and screening of two special habitat fungi and research on metabolic products of Aspergillus foetidus 094102 [D]. Wuhan University, 2017.) were inoculated in Petri dishes containing PDA solid medium, and incubated at 28°C in a biochemical incubator for 7 days. Then the solid medium was divided into 1 cm x 1 cm blocks, and inoculated into 1 L conical flasks containing 300 mL PDB liquid medium at a ratio of Aspergillus aculeatinus WHUF0198: Penicillium sp. DM27 = 1:3 for fermentation. The fermentation temperature was 28°C, the fermentation time was 28 days, and the total fermentation volume was 100 flasks. The medium formula was the same as that of separate culture.

[0042] B. Fermentation product extraction

[0043] After the fermentation time was reached, the fermentation broth and mycelium in the conical flask were separated by filter cloth. The mycelium was cut into small pieces and completely immersed in acetone. It was placed in an ultrasonic cleaner and ultrasonicated for 30 minutes. After ultrasonication, the mycelium was stirred and ultrasonicated again. This process was repeated 3 times. The acetone extract and mycelium were separated by filtration. The mycelium was again immersed in acetone and the above ultrasonic extraction steps were repeated. The acetone extract and mycelium were obtained by filtration again. The acetone extracts obtained by ultrasonic extraction twice were combined, concentrated by evaporation under reduced pressure, acetone was recovered, and the concentrated liquid and fermentation broth were combined. The above steps are applicable to the extraction of fermentation products of Aspergillus aculeatinus WHUF0198 and co-culture of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27.

[0044] The fermentation combined liquid of Aspergillus aculeatinus WHUF0198 single fermentation was extracted with equal amount of ethyl acetate (EA) for three times, and then the EA layer was evaporated under reduced pressure to obtain dry crude extract 18.0 g.

[0045] For the fermentation combined liquid of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27 co-culture, 2L HP20 macroporous resin was added to the above combined liquid, and secondary metabolite adsorption extraction was carried out by stirring for 8 hours. After stirring, the macroporous resin adsorbed with the secondary metabolites of the strain was obtained by filtration. The macroporous resin was loaded into a glass chromatography column, and then eluted with 30%, 50%, 70%, 90%, and 100% methanol aqueous solution in turn, with a volume of 5L for each proportion. After elution, the 70%, 90%, and 100% eluents were combined and evaporated under reduced pressure to obtain dry crude extract 10.1 g.

[0046] C. Isolation and preparation of pyridopyranone alkaloid compounds.

[0047] For the crude extract of Aspergillus aculeatinus WHUF0198. The dry extract was subjected to column chromatography (CC) using 200-300 mesh normal silica gel as packing material and eluted with dichloromethane / methanol (DCM / MeOH) (volume ratio gradient of 80:1, 40:1, 20:1, 10:1, 5:1 and 1:1) using 250 mL receiving flask to collect the eluted samples. Thin layer chromatography (TLC) analysis was performed on each sample using silica gel plate, a straight line was drawn on the silica gel plate at a distance of 1 cm from the top using a pencil, and each sample was spotted on the line at different positions from left to right using a capillary glass tube (0.3 mm) and the sample numbers were marked on the plate using a pencil after the samples were dried. The appropriate developing solvent was prepared and the developing solvent system was DCM:MeOH (0.1% formic acid) = 100:1 to 15:1, and the components in each sample were well separated on the plate after development. The silica gel plate with samples was placed in the developing jar with developing solvent, and the side with samples faced down. The plate was removed when the developing solvent reached 1 cm from the top. The plate was placed in the UV detection box and the developed samples were observed under 254 nm and 365 nm UV light. The samples with almost identical developed spots were combined, and the combined samples were dried. Six major fractions (Fr. 1-6) were obtained. The TLC analysis described below was performed according to the above procedure. The effect of each fraction on cell survival rate was evaluated using mouse hepatocytes induced by APAP at a concentration of 100 μg / mL, and the results showed that the cell death rate of Fr. 5 and Fr. 6 was significantly reduced compared with the non-drug group. These two fractions were further separated. Fr. 5 (2.2 g) was separated by silica gel column chromatography using DCM / EA as eluent with a gradient of 20:1, 15:1, 10:1, 5:1, 0:1; and EA / MeOH, 10:1, 5:1, 2:1. Twenty-six sub-fractions (Fr. 5.a-Fr. 5.z) were obtained by TLC analysis and combined. At a concentration of 100 μg / mL, the cell survival rate of APAP-induced mouse hepatocytes was tested, and Fr. 5.k (256 mg) showed a significantly reduced cell death rate compared with the non-drug group. Then, semi-preparative high performance liquid chromatography (HPLC) was used to prepare the fraction, and the Sepax GP-C-18 (5 μm, 10 x 250 mm) column was used in the preparation process. The mobile phase conditions were as follows: 60-100% (V:V) methanol / water eluted at a flow rate of 3 mL / min for 30 min, and compounds 1 (3 mg, retention time 15.2 min) and 5 (3 mg, retention time 22.3 min) were separated.The fraction 6 (3.7 g) was separated by silica gel column chromatography using a gradient of eluent EtOAc / MeOH (20:1, 15:1, 10:1, 5:1, 0:1, V / V) and each eluted fraction was combined by TLC analysis to give 20 sub-fractions (Fr.6.a-Fr.6.t). Each fraction was tested for its effect on APAP-induced cell viability in mouse hepatocytes and Fr.6.c and Fr.6.f showed a significant decrease in cell death compared to the non- administered group. Fr.6.c (408 mg) was separated by gel column chromatography using Sephadex LH-20 (this packing material was used for all gel column chromatography) and eluted with DCM / MeOH (1:1, V / V) and each eluted fraction was combined by TLC analysis to give 10 sub-fractions (Fr.6.c.1-10). Fr.6.c.5 (26 mg) was prepared by semi-preparative HPLC using a mobile phase of 70-80% (V:V) methanol / water at a flow rate of 3 mL / min over 30 min and compound 6 (2 mg, retention time 24.3 min) was isolated. Under the same chromatographic conditions, semi-preparative HPLC was used to isolate compound 8 (8 mg, retention time 18.6 min), 9 (2 mg, retention time 20.3 min) and 10 (6 mg, retention time 1.6.7 min) from Fr.6.c.6 (35 mg). Under the same chromatographic conditions, compound 17 (1 mg, retention time 15.8 min) was prepared from Fr.6.c.10 (19 mg). Fr.6.f (270 mg) was prepared using a mobile phase of 70-100% (V:V) methanol / water at a flow rate of 3 mL / min over 30 min to give 16 (4 mg, retention time 17.6 min).

[0048] For the crude extract of Aspergillus aculeatinus WHUF0198 and Penicillium sp. DM27 co-culture (10.1 g). The crude extract was separated by column chromatography (CC) using 200-300 mesh normal silica gel as packing material eluted with dichloromethane / methanol (DCM / MeOH) (volume ratio gradient 100:1, 40:1, 20:1, 10:1 and 5:1) using 250 mL receiving flask for eluted samples. Each sample was analyzed by silica gel thin layer chromatography (TLC) analysis and combined to get five sub-fractions (Fr. B.1 - Fr. B.5). Fr. B.2 (468 mg) was separated by gel column chromatography eluted with DCM / MeOH (1:1, V / V) to get three sub-fractions (Fr. B.2.1 - Fr. B.2.3). Fr. B.2.2 (50 mg) was subjected to semi-preparative HPLC with mobile phase condition 70-80% (V:V, 0.1% formic acid) methanol / water at a flow rate of 3 mL / min for 40 min to get compound 12 (11 mg, retention time 12.9 min). Fr. B.2.3 (70 mg) was subjected to semi-preparative HPLC with mobile phase condition 40-70% (V:V, 0.1% formic acid) acetonitrile / water at a flow rate of 3 mL / min for 40 min to get compound 13 (2 mg, retention time 16.8 min), compound 14 (2 mg, retention time 15.5 min), compound 15 (2 mg, retention time 1.23 min) at different retention times. Fr. B.4 (1.03 g) was separated into seven sub-fractions (Fr. B.4.1 - Fr. B.4.4) using gel column chromatography and TLC analysis. Fr. B.4.3 (331 mg) was separated into five sub-fractions (Fr. B.4.3.1 - Fr. B.4.3.5) using silica gel column chromatography eluted with DCM / MeOH (500:1, 100:1, 80:1, 50:1, 10:1, 5:1) and combined by TLC analysis. Compound 11 (3 mg, retention time 33.6 min) was obtained from Fr. B.4.3.1 (21 mg) by semi-preparative HPLC with mobile phase condition 70-80% (V:V, 0.1% formic acid) methanol / water at a flow rate of 3 mL / min for 40 min. Compounds 6 (2 mg, retention time 34.3 min), 9 (2 mg, retention time 30.7 min) and 10 (4 mg, retention time 26.5 min) were obtained from Fr. B.4.3.5 (21 mg) by semi-preparative HPLC with mobile phase condition 65% (V:V) methanol / water at a flow rate of 3 mL / min for 40 min.Fr. B.4.4 (96 mg) was separated by silica gel column chromatography eluting with a gradient of DCM / MeOH, 40:1, 20:1, 10:1, and the eluted fractions were combined based on TLC analysis to give three subfractions (Fr. B.4.4.1 - Fr. B.4.4.3). Using semi-preparative HPLC with a mobile phase condition of 60-100% (V:V, 0.1% formic acid) methanol / water at a flow rate of 3 mL / min for 20 min, compound 3 (2 mg, retention time 13.1 min) and compound 4 (2 mg, retention time 13.8 min) were obtained from Fr. B.4.4.2 (26 mg). Compound 2 (2 mg, retention time 24.6 min) was purified from Fr. b.4.4.3 (38 mg) by semi-preparative HPLC with a mobile phase condition of 30-50% (V:V, 0.1% formic acid) acetonitrile / water at a flow rate of 3 mL / min for 40 min. Fr. B.5 (324 mg) was separated by gel column chromatography and combined based on TLC analysis to give six subfractions (Fr. B.5.1 - Fr. B.5.6). Using semi-preparative HPLC with a mobile phase condition of 40-80% (V:V, 0.1% formic acid) acetonitrile / water at a flow rate of 3 mL / min for 30 min, compound 7 (3.5 mg, retention time 22.1 min) was isolated from Fr. B.5.4 (50.3 mg).

[0049] C. Structure identification

[0050] The structures of each compound were analyzed by NMR, mass spectrometry, etc. The results are shown below.

[0051] Compound 1: yellow powder; [a] 2 D 0 -36 (c 0.02, MeOH); 1 H and 13 C NMR data are shown in Table 1; HR-ESI-MS (m / z): 328.1932 [M-H] - (calcd for C 20 H 26 NO3, 328.2277); ECD (c 21 mM, MeOH), λ max (Δε) 296 (-0.40), 275 (+3.01), 243 (-2.79), 228 (+1.33), 213 (-37.14) nm.

[0052] Compound 2: white powder; [a] 2 D 0 -54 (c 0.04, MeOH); 1H and 13 C NMR data are listed in Table 1; HR-ESI-MS (m / z): 344.1857 [M-H] - (calcd for C 20 H 26 NO4, 344.1862); ECD (c 2.9 mM, MeOH), λ max (Δε) 295 (-3.14), 275 (+2.98), 245 (+0.16), 226 (+1.84), 212 (-21.62) nm.

[0053] Compound 3: white powder; [a] 2 D 0 -57 (c 0.04, MeOH); 1 H and 13 C NMR data are listed in Table 1; HR-ESI-MS (m / z): 344.1857 [M-H] - (calcd for C 20 H 24 NO5, 358.1655); ECD (c 2.8 mM, MeOH), λ max (Δε) 298 (-0.41), 268 (+4.70), 243 (-2.35), 228 (+2.17), 213 (-50.68) nm.

[0054] Compound 4: white powder; [a] 2 D 0 -12 (c 0.13, MeOH); 1 H and 13 C NMR data are listed in Table 2; HR-ESI-MS (m / z): 358.1652 [M-H] - (calcd for C 20 H 24 NO5, 358.1655); ECD (c 2.2 mM, MeOH), λ max (Δε) 294 (-2.35), 271 (+2.31), 245 (-2.22), 230 (+1.59), 215 (-35.95) nm.

[0055] Compound 5: white powder; [a] 2 D 0 -19.5 (c 0.07, MeOH); 1 H and 13C NMR data see 2; HR-ESI-MS (m / z): 366.2038 [M+Na] + (calcd for C 21 H 29 NO3Na, 366.2045); ECD (c 17 mM, MeOH), λ max (Δε) 275 (+0.72), 245 (-4.09), 228 (+3.50), 215 (-30.28) nm.

[0056] Compound 6: white powder; [a] 2 D 0 -9 (c 0.01, MeOH); 1 H and 13 C NMR data see Table 2; HR-ESI-MS (m / z): 382.1981 [M+Na] + (calcd for C 21 H 29 NO4Na, 382.1994); ECD (c 16 mM, MeOH), λ max (Δε) 293 (-1.13), 264 (+0.20), 246 (-1.06), 229 (+1.49), 214 (-16.80) nm.

[0057] Compound 7: yellow powder; [a] 2 D 0 -31.5 (c 0.10, MeOH); 1 H and 13 CNMR data see Table 3; HR-ESI-MS (m / z): 398.1944 [M+Na] + (calcd for C 21 H 29 NO5Na, 398.1943); ECD (c 3.6 mM, MeOH), λ max (Δε) 300 (-1.12), 276 (+5.44), 242 (-1.53), 229 (+0.78), 215 (-30.75) nm.

[0058] Compound 8: white powder; [a] 2 D 0 -12 (c 0.03, MeOH); 1 H and 13C NMR data are listed in Table 3; HR-ESI-MS (m / z): 396.1778 [M+Na] + (calcd for C 21 H 27 NO5Na, 396.1787); ECD (c 10 mM, MeOH), l max (Δε) 267 (+5.14), 242 (-1.53), 229 (+0.78), 215 (-30.71) nm.

[0059] Compound 9: white powder; [a] 2 D 0 -27 (c 0.13, MeOH); 1 H and 13 C NMR data are listed in Table 3, 4; HR-ESI-MS (m / z): 396.1778 [M+Na] + (calcd for C 21 H 27 NO5Na, 396.1787); ECD (c 16 mM, MeOH), l max (Δε) 293 (-4.57), 270 (-2.05), 246 (-2.93), 229 (+3.08), 213 (-26.68) nm.

[0060] Compound 10: white powder; [a] 2 D 0 -12 (c 0.13, MeOH); 1 H and 13 C NMR data are listed in Table 4; HR-ESI-MS (m / z): 396.1772 [M+Na] + (calcd for C 21 H 27 NO5Na, 396.1787); ECD (c 20 mM, MeOH), l max (Δε) 292 (-1.11), 273 (+0.86), 244 (-0.91), 229 (+1.48), 215 (-15.7) nm.

[0061] Compound 11: white powder; [a] 2 D 0 -4 (c 0.04, MeOH); 1 H and 13C NMR data are listed in Table 4; HR-ESI-MS (m / z): 388.2103 [M+H] + (calcd for C 22 H 30 NO5, 388.2124); ECD (c 2.6 mM, MeOH), λ max (Δε) 295 (-1.09), 274 (+2.86), 244 (-1.56), 229 (+0.17), 215 (-22.91) nm.

[0062] Compound 12: yellow oil; [a] 2 D 0 -18 (c 0.07, MeOH); 1 H and 13 C NMR data are listed in Table 5; HR-ESI-MS (m / z): 376.2118 [M+H] + (calcd for C 21 H 30 NO5, 376.2124); ECD (c 2.7 mM, MeOH), λ max (Δε) 295 (-1.13), 265 (+2.73), 245 (-2.58), 230 (-0.51), 215 (-17.66) nm.

[0063] Compound 13: yellow oil; [a] 2 D 0 -26 (c 0.03, MeOH); 1 H and 13 C NMR data are listed in Table 5; HR-ESI-MS (m / z): 412.1745 [M+Na] + (calcd for C 21 H 27 NO6Na, 412.1736); ECD (c 2.6 mM, MeOH), λ max (Δε) 295 (-2.10), 266 (+1.69), 246 (-2.88), 228 (+0.85), 215 (-18.21) nm.

[0064] Compound 14: yellow oil; [a] 2 D 0 -38 (c 0.03, MeOH); 1 H and 13C NMR data are listed in Table 5; HR-ESI-MS (m / z): 390.1919 [M+H] + (calcd for C 21 H 28 NO6, 390.1917); ECD (c 2.6 mM, MeOH), l max (De) 294 (-2.43), 264 (+3.74), 245 (-2.30), 229 (+1.97), 213 (-36.86) nm.

[0065] Compound 15: white powder; [a] 2 D 0 -29 (c 0.03, MeOH); 1 H and 13 C NMR data are listed in Table 5; HR-ESI-MS (m / z): 404.2067 [M+H] + (calcd for C 22 H 30 NO6, 404.2073); ECD (c 2.5 mM, MeOH), l max (De) 296 (-1.52), 264 (+3.23), 245 (-2.60), 230 (-0.44), 215 (-18.14) nm.

[0066] Compound 16: white powder; [a] 2 D 0 -16 (c 0.07, MeOH); 1 H and 13 C NMR data are listed in Table 6; HR-ESI-MS (m / z): 444.2083 [M+Na] + (calcd for C 26 H 29 NO4Na, 444.2151); ECD (c 20 mM, MeOH), l max (De) 281 (-0.70), 265 (-0.48), 247 (-0.83), 227 (+0.05), 215 (-0.03) nm.

[0067] Compound 17: white powder; [a] 2 D 0 -31.5 (c 0.07, MeOH); 1 H and 13C NMR data are reported in Table 6; HR-ESI-MS (m / z): 378.1876 [M+Na] + (calcd for C 22 H 27 NO3Na, 378.1883); ECD (c 25 mM, MeOH), λ max (Δε) 260 (-2.04), 248 (-1.45), 221 (-3.09), 211 (-6.96) nm.

[0068] Table 1. H and C NMR data of compounds 1, 2, 3 1 H and 13 C NMR data

[0069]

[0070] Table 2. H and C NMR data of compounds 4, 5, 6 1 H and 13 C NMR data

[0071]

[0072] Table 3. H and C NMR data of compounds 7, 8, 9 1 H and 13 C NMR data

[0073]

[0074]

[0075] Table 4. H and C NMR data of compounds 9, 10, 11 1 H and 13 C NMR data

[0076]

[0077] Table 5. H and C NMR data of compounds 12, 13, 14, 15 1 H and 13 C NMR data

[0078]

[0079]

[0080] Table 6. H and C NMR data of compounds 16, 17 1 H and 13 C NMR data

[0081]

[0082]

[0083] Example 2: Application of pyridopyranone alkaloid compounds to the protective activity of APAP-induced acute liver injury

[0084] 1. Mouse liver parenchymal cell AML-12 cell culture

[0085] AML-12 cells were cultured in 10 cm cell culture dishes with complete culture medium in a 37°C, 5% CO2 incubator, and the cells were changed every other day. The specific steps are as follows: take out the AML-12 cell culture dish from the incubator, and discard the old culture medium in the culture dish; add 2 mL of PBS and discard; add 7 mL of complete culture medium; and return to the incubator for culture.

[0086] 2. MTT method for screening the activity of each component in the separation process of the fermentation crude extract

[0087] a. Culture and experimental grouping of AML-12 cells: the cell culture experiment steps are the same as above; the experiment is divided into blank control group, APAP model group, and fermentation crude extract administration group according to the difference in cell treatment.

[0088] b. Preparation of APAP drug: accurately weigh 0.4548 g of APAP, add 30 mL of PBS and dissolve thoroughly, filter with a 0.22 μm sterile filter to obtain a 100 mM concentration of APAP stock solution for use.

[0089] c. APAP treatment of AML-12 cells for 12 h to simulate in vivo ALI environment: dilute the 100 mM APAP solution 10 times with complete culture medium to prepare a 10 mM APAP working solution; after separation by silica gel column chromatography, accurately weigh 2 mg of each component, add 10 μL of DMSO to dissolve thoroughly, and prepare a 200 mg / mL drug stock solution, which is diluted with the APAP working solution to prepare a 100 mg / mL working solution for use.

[0090] d. MTT method for detecting AML-12 cell damage: AML-12 cells were seeded in a 96-well plate, and 100 μL of complete culture medium, 10 mM APAP working solution, and 10 mM APAP working solution containing extract were added, respectively, at 100 μL / well, and incubated at 5% CO2, 37°C for 24 h. The experimental groups were control group, model group, and extract administration group (n=5). After 24 h, the drug-containing medium was aspirated, and 100 μL of complete culture medium and 20 μL of MTT solution (5 mg / mL, i.e. 0.5% MTT) were added to each well, and incubated for another 4 h. The culture was terminated after 4 h, and the culture medium in the wells was carefully aspirated. 150 μL of dimethyl sulfoxide was added to each well, and shaken on a shaker for 10 min to dissolve the crystals thoroughly. The absorbance of each well at 490 nm was detected by a microplate reader.

[0091] 3. MTT method for detecting the effect of compounds 1-7 and compounds 9-16 on the survival rate of APAP-induced AML-12 cells

[0092] Precisely weigh 1 mg of each compound, dissolve it in DMSO according to the molecular weight, and add 10 mM APAP working solution to prepare a working solution with a compound concentration gradient of 50, 25, 10, and 5 μM for standby. MTT method for detecting AML-12 cell damage: AML-12 cells were inoculated in a 96-well plate, and 100 μL / well of complete medium, 10 mM APAP working solution, and 10 mM APAP working solution containing different concentrations of each compound were added, respectively. The cells were cultured at 5% CO2 and 37°C for 24 h, and the experimental groups were divided into a control group, a model group, and a compound administration group (n = 5). After 24 h, the drug-containing medium was aspirated, and 100 μL of complete medium and 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) were added to each well. The cells were cultured for another 4 h, after which the culture medium was carefully aspirated from the wells. Then, 150 μL of dimethyl sulfoxide was added to each well, and the wells were shaken on a shaker for 10 min to fully dissolve the crystals. The absorbance of each well at 490 nm was detected using an enzyme label instrument.

[0093] 4. Detection of alanine aminotransferase (ALT) in the supernatant of AML-12 cells induced by compounds 1-7, 9-10, and 12-15

[0094] Precisely weigh 1 mg of each compound, dissolve it in DMSO according to the molecular weight, and add 10 mM APAP working solution to prepare a working solution with a compound concentration gradient of 50, 25, 10, and 5 μM for standby. MTT method for detecting AML-12 cell damage: AML-12 cells were inoculated in a 96-well plate, and 100 μL / well of complete medium, 10 mM APAP working solution, and 10 mM APAP working solution containing different concentrations of each compound were added, respectively. The cells were cultured at 5% CO2 and 37°C for 24 h, and the experimental groups were divided into a control group, a model group, and a compound administration group (n = 5). After 24 h, the drug-containing medium was aspirated, and 100 μL of complete medium and 20 μL of MTT solution (5 mg / mL, i.e., 0.5% MTT) were added to each well. The cells were cultured for another 4 h, after which the culture medium was carefully aspirated from the wells. Then, 150 μL of dimethyl sulfoxide was added to each well, and the wells were shaken on a shaker for 10 min to fully dissolve the crystals. The absorbance of each well at 490 nm was detected using an enzyme label instrument.

[0095] 5. KEGG and GO analysis

[0096] The mechanism of compound 12 for improving acute liver injury (ALI) was further studied by network pharmacology and structure virtual screening method, and 105 target points were identified by SwissTargetPrediction database. Subsequently, search of OMIM and Genecards online databases resulted in 8547 target points related to ALI. The intersection of these data visualized in Venny plot revealed 95 possible targets of compound 12 for improving ALI ( Figure 3 ). GO biological function and KEGG pathway enrichment analysis of these common targets were analyzed using the clusterProfiler package in R (v.4.3.2). GO analysis results showed that most of the identified target proteins were related to various protein kinases, such as 3-kinase / protein kinase B, protein kinase complex, serine / threonine-protein kinase, protein serine kinase, protein tyrosine kinase and heptad repeat kinase ( Figure 4 ). In addition, KEGG results showed that most of these targets were enriched in PI3K-Akt signaling pathway, Rap1 signaling pathway, FOXO signaling pathway and EGFR signaling pathway ( Figure 5 ). At the same time, protein-protein interaction (PPI) network was constructed using STRING database and visualized using Cytoscape software, and 10 core target points (PIK3CA, MTOR, GRB2, BCL2, HSP90AA1, STAT3, EGFR, SRC, TNF and AKT1, Figure 6 ) were determined. Molecular docking was performed to explore the interaction between compound 12 and selected targets, and the binding energy of compound 12 with each target protein and the molecular docking are shown in Figure 7 and Figure 8 , and the lowest docking energy (-8.35 kcal / mol) was observed with the target EGFR protein. Analysis revealed the interaction between compound 12 and EGFR, indicating that compound 12 improves acute liver injury through the potential mechanism of regulating downstream cascade reactions such as ras / raf / MEK / MAPK, PI3K / Akt and STAT pathways.

[0097] 6. Experimental results

[0098] The cell survival rate results of normal mouse hepatocytes AML-12, APAP-induced liver injury AML-12 cells, and APAP-induced liver injury cells treated with different concentrations (5, 10, 25, 50 μM) of compounds 1-7 and 9-16 are shown in Figure 1The changes in cell survival rate at 5 μM and 10 μM dosing concentrations were expressed by cell protection rate, and Table 7 was calculated. The cell survival rate experiment showed that compounds 1-7, 9-10 and 12-15 significantly improved the cell survival rate of APAP-induced liver injury cells. Alanine aminotransferase (ALT) is an enzyme located in hepatocytes, which is released into the blood during liver injury and is a specific marker of acute lung injury. In order to determine the cell protection activity of bioactive compounds, the ALT content in the cell supernatant under the administration of compounds 1-7, 9-10 and 12-15 was further tested, and the results are shown in Figure 2 Table 8. The reduction rate of ALT level in APAP-induced AML-12 cells by compounds 1-7, 9-10 and 12-15

[0099] Table 7. Cell protection rate of compounds 1-7 and compounds 9-16 on APAP-induced liver injury cells

[0100]

[0101] Table 8. The reduction rate of ALT level in APAP-induced AML-12 cells by compounds 1-7, 9-10 and 12-15

[0102]

[0103]

[0104] The above examples are only used to help illustrate the present application, and the embodiments of the present application are not limited by the above examples. Any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and shall be included in the protection scope of the present application.

Claims

1. A pyrrolopyridone alkaloid compound, characterized in that: Any one of compounds 1-10, 12-15 shown in the following structural formula: Structural formulas of compounds 1, 2, 3, 4: ; The group combinations of compounds 1, 2, 3, and 4 are shown in the following tables, respectively: Structural formulas of compounds 5, 6, 7, 8, 9, 10: ; The group combinations of compounds 5, 6, 7, 8, 9, and 10 are shown in the following tables, respectively: Structural formulas of compounds 12, 13, 14, 15: ; The group combinations of compounds 12, 13, 14, and 15 are shown in the following tables, respectively:

2. The method for preparing the pyranopyridone alkaloid compound according to claim 1, characterized in that: Comprising the following steps: The Aspergillus aculeatinus WHUF0198 strain is inoculated alone or simultaneously with Penicillium sp. DM27 into a liquid culture medium for separate fermentation or co-cultivation fermentation, respectively, and the pyrrolopyridone alkaloid compounds are isolated from the fermentation products.

3. The method for preparing pyranopyridone alkaloids according to claim 2, characterized in that: Comprising the following steps: Will Aspergillus aculeatinus WHUF0198 strain, inoculated alone or with... Penicillium sp. DM27 was simultaneously inoculated into liquid culture medium for individual or co-fermentation. The fermentation broth and mycelium were separated. The mycelium was crushed and extracted with acetone by ultrasonication. The acetone was recovered, concentrated under reduced pressure, and then combined with the fermentation broth. The combined liquid is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; or the combined liquid is added to a macroporous resin for adsorption, and the macroporous resin is subjected to column chromatography using a methanol / water gradient elution to obtain components containing different methanol contents, and the components containing pyridopyranone alkaloids are determined. These components are combined and concentrated under reduced pressure to obtain a crude extract; The crude extract is separated into smaller components by silica gel column chromatography, followed by gel column chromatography separation. The parts containing pyridopyranone alkaloid compounds are subjected to semi-preparative liquid phase purification to obtain pyridopyranone alkaloid compounds.

4. The pyridopyranone alkaloid compound of claim 1 for use in the preparation of a medicament for treating acute liver injury.

5. Use according to claim 4, characterized in that: The pyridopyranone alkaloid compound is one or more of compounds 1-10, 12-15 in claim 1.

6. Use according to claim 4, characterized in that: The acute liver injury is drug-induced liver injury.

7. Use according to claim 4, characterized in that: The acute liver injury is acetaminophen-induced acute liver injury.

8. A medicament for treating acute liver injury, characterized by: The pyridopyranone alkaloid compound is one or more of compounds 1-10, 12-15 in claim 1.

9. The medicament according to claim 8, characterized in that: The acute liver injury is drug-induced liver injury.

10. The medicament according to claim 8 or 9, characterized in that: The acute liver injury is acetaminophen-induced acute liver injury. The pyridopyranone alkaloid compound is one or more of compounds 1-10, 12-15 in claim 1. The acute liver injury is drug-induced liver injury. The acute liver injury is acetaminophen-induced acute liver injury. The pyridopyranone alkaloid compound is one or more of compounds 1-10, 12-15 in claim 1. The acute liver injury is drug-induced liver injury. The acute liver injury is acetaminophen-induced acute liver injury.

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