Deep-sea indole alkaloid and polyketide hybrids and their preparation and application in inhibiting cell ferroptosis and preventing and treating ferroptosis-related diseases

Abstract

The present invention belongs to the field of biomedicine technology, and discloses a class of indole alkaloid and polyketide hybrids derived from deep-sea fungi, a preparation method thereof, and an application in inhibiting cell ferroptosis and preventing and treating ferroptosis-related diseases. The indole alkaloid and polyketide hybrids of the present invention and their derivatives or pharmaceutically acceptable salts thereof are new hybrid skeleton compounds composed of prenylated indole alkaloids and C9 polyketones, which are a class of alkaloid secondary metabolites with novel structures and are very rare in nature. The present invention has discovered for the first time a new hybrid skeleton compound composed of prenylated indole alkaloids and C9 polyketones, and confirmed that it can downregulate HMOX1 protein and inhibit lipid peroxidation through targeted action, thereby inhibiting cell ferroptosis, and can be used to prepare targeted drugs that inhibit cell ferroptosis, which is of great significance for the treatment of ferroptosis-related diseases and the development of new drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a class of indole alkaloids and polyketide hybrids derived from deep-sea fungi, a preparation method thereof, and applications in inhibiting cell ferroptosis and preventing and treating ferroptosis-related diseases. Background Art

[0002] Ferroptosis is an iron-dependent, novel form of programmed cell death, distinct from apoptosis, necrosis, and autophagy. The primary mechanism of ferroptosis is lipid peroxidation of highly expressed unsaturated fatty acids on the cell membrane, catalyzed by ferrous iron or esteroxygenases, leading to cell death. Reduced activity of GPX4, a core enzyme in the antioxidant system (glutathione system), is a key factor in lipid peroxidation and ferroptosis. Ferroptosis is closely associated with numerous pathophysiological processes, such as aging, neurodegenerative diseases, stroke, and ischemia-reperfusion injury. With increasing research, key genes regulating ferroptosis have been discovered. Studies have shown that NRF2 (nuclear factor E2-related factor 2) can influence intracellular glutathione synthesis by upregulating SLC7A11 (cystine / glutamate antiporter) and GCLM (glutamate cysteine ​​ligase), forming the NRF2 / SLC7A11 / GCLM axis, thereby inhibiting lipid peroxidation and ferroptosis. In addition, HMOX1 (heme oxygenase 1) is also a key regulator in the ferroptosis pathway. Overexpression of HMOX1 can lead to increased intracellular iron concentrations, thereby inducing ferroptosis.

[0003] Existing studies have shown that ferroptosis is closely related to a variety of diseases, including acute kidney injury, neurodegenerative diseases, autoimmune diseases, and inflammation. Some ferroptosis inhibitors, such as deferiprone, are undergoing clinical trials for the treatment of amyotrophic lateral sclerosis (ALS). However, overall, research on ferroptosis and its inhibitors is still in its early stages, and small molecule ferroptosis inhibitors with selectivity, efficacy, and safety await further discovery and development. Summary of the Invention

[0004] In order to overcome the above-mentioned shortcomings and deficiencies of the prior art, the primary object of the present invention is to provide a class of indole alkaloid-polyketide hybrids and derivatives thereof or pharmaceutically acceptable salts thereof.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned indole alkaloid-polyketide hybrid.

[0006] The indole alkaloid and polyketide hybrid of the invention is separated from the fermentation product of Aspergillus ochraceus and has the advantages of being environmentally friendly, simple steps, high product purity and the like.

[0007] Another object of the present invention is to provide the use of the above-mentioned indole alkaloid-polyketide hybrid or a pharmaceutically acceptable salt thereof, particularly in inhibiting cell ferroptosis.

[0008] The indole alkaloid and polyketide hybrid of the present invention or a pharmaceutically acceptable salt thereof has cell ferroptosis inhibitory activity and can be used for the preparation and development of drugs for preventing and treating ferroptosis-related diseases.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] In a first aspect of the present invention, a class of indole alkaloid-polyketide hybrids and derivatives thereof or pharmaceutically acceptable salts thereof are provided, having a structure shown in Formula I:

[0011]

[0012] The second aspect of the present invention provides a method for preparing the above-mentioned indole alkaloid-polyketide hybrid and its derivatives, specifically, isolating them from Aspergillus ochraceus fermentation products.

[0013] Furthermore, the Aspergillus ochraceus has a deposit number of MCCC 3A00521 and is deposited in the Marine Culture Collection of China (MCCC).

[0014] The Aspergillus ochra fermentation broth is specifically prepared by fermenting Aspergillus ochra in a fermentation medium to obtain a fermented product.

[0015] The fermentation can be carried out at room temperature and for a period of 42-52 days.

[0016] The fermentation medium comprises: per liter of the medium, 20.0 g of mannitol, 3.0 g of yeast extract, 0.5 g of KH2PO4, 10.0 g of monosodium glutamate, 0.3 g of MgSO4·7H2O, 20.0 g of maltose, 1.0 g of corn steep liquor, and a pH of 7.5.

[0017] Furthermore, Aspergillus ochraceus can be cultured on a PDA plate at 28° C. for 3-4 days to obtain mycelium; the mycelium is inoculated into a culture medium containing PDB to obtain seed liquid after culture; and the seed liquid is inoculated into a liquid fermentation medium for fermentation.

[0018] Furthermore, the separation method comprises the following steps:

[0019] The fermentation product of Aspergillus ochraceus is extracted with ethyl acetate, the resulting extract is dissolved in methanol, extracted with petroleum ether, and the petroleum ether extract is discarded to obtain a defatted extract; the defatted extract is separated by normal phase silica gel column chromatography and gradient eluted with a dichloromethane-methanol system to obtain three fractions (Fr.1-Fr.3); fraction Fr.2 is sequentially purified by ODS column chromatography (methanol-water system), Sephadex LH-20 dextran gel chromatography column (methanol elution), and then by semi-preparative HPLC (acetonitrile-water, 40-60%) to obtain the indole alkaloid-polyketide hybrid and its derivatives of the present invention.

[0020] The invention provides a method for separating and obtaining indole alkaloids and polyketide hybrids and derivatives thereof from Aspergillus ochraceus fermentation products, which is of great significance for the development of marine drugs.

[0021] The third aspect of the present invention provides one of the uses of the above-mentioned indole alkaloid-polyketide hybrid and its derivatives or pharmaceutically acceptable salts in the preparation of the following products: (1) cell ferroptosis inhibitors; (2) drugs for preventing and treating ferroptosis-related diseases.

[0022] Furthermore, the cell ferroptosis refers to cell ferroptosis induced by RSL3, FINO2 or Erastin.

[0023] Furthermore, the ferroptosis-related diseases include neurodegenerative diseases, inflammatory bowel disease, atherosclerosis, acute pancreatitis, acute / drug-induced liver injury, acute kidney injury, viral infection, etc.

[0024] The indole alkaloid-polyketide hybrids and their derivatives or pharmaceutically acceptable salts thereof of the present invention can downregulate HMOX1 protein and inhibit lipid peroxidation through targeted effects, thereby inhibiting cell ferroptosis. They can be used to prepare targeted drugs that inhibit cell ferroptosis, which is of great significance for the treatment of ferroptosis-related diseases and the development of new drugs.

[0025] Furthermore, the drugs are the same or different and respectively include therapeutically effective amounts of indole alkaloid and polyketide hybrids and derivatives thereof or pharmaceutically acceptable salts thereof.

[0026] Furthermore, the drugs, whether the same or different, can be made into various pharmaceutical dosage forms using conventional methods. These dosage forms include: tablets, sugar-coated tablets, film-coated tablets, enteric-coated tablets, capsules, hard capsules, soft capsules, oral liquids, lozenges, granules, electuary preparations, pills, pills, suspensions, alcohol preparations, tinctures, drops and other oral dosage forms, as well as injections and other non-oral dosage forms, such as injections.

[0027] Furthermore, the drugs, whether the same or different, may also contain one or more pharmaceutically acceptable carriers or excipients.

[0028] Furthermore, the carrier or auxiliary material may include a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, a disintegrant, a preservative, and the like.

[0029] In a fourth aspect, the present invention provides a pharmaceutical composition for preventing and treating ferroptosis-related diseases, comprising the indole alkaloid-polyketide hybrid and its derivatives or pharmaceutically acceptable salts thereof.

[0030] The fifth aspect of the present invention provides a ferroptosis inhibitor comprising the indole alkaloid and polyketide hybrid and its derivatives or pharmaceutically acceptable salts thereof.

[0031] Compared with the prior art, the present invention has the following advantages:

[0032] The present invention provides a new skeleton compound StephaochratidinA (SCA, formula I), which is isolated from the fermentation product of deep-sea Aspergillus ochraceus. SCA is a new hybrid skeleton compound composed of isopentenylated indole alkaloids and C9 polyketones. It is a type of alkaloid secondary metabolite with a novel structure and is very rare in nature. The present invention discovered for the first time a new hybrid skeleton compound composed of isopentenylated indole alkaloids and C9 polyketones, which is of great significance for the discovery and research of new drugs for the prevention and treatment of ferroptosis-related diseases. The method of the present invention for isolating compound formula I from the fermentation broth has the advantages of environmental protection, simple steps, and high product purity. The present invention uses CellTiter-Lumi TM Luminescence, real-time quantitative PCR (QPCR), and Western blot analysis demonstrated that the indole alkaloid-polyketide hybrids of the present invention inhibited ferroptosis mediated by various ferroptosis-inducing agents in various cell lines by inhibiting lipid peroxidation and downregulating HMOX1 expression. Therefore, the novel backbone compound provided by the present invention has promising application prospects in the preparation of drugs for the prevention and treatment of ferroptosis-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0034] Figure 1It is the single crystal diffraction structure diagram of the compound of formula I of the present invention.

[0035] Figure 2 This is the effect of the compound of formula I of the present invention on RSL3-induced ferroptosis in A375 cells.

[0036] Figure 3 The present invention shows the effect of the compound of formula I on RSL3-induced ferroptosis in 786-O cells.

[0037] Figure 4 This is the effect of the compound of formula I of the present invention on RSL3-induced ferroptosis in H1299 cells.

[0038] Figure 5 The present invention shows the effect of the compound of formula I on erastin-induced ferroptosis in A375 cells.

[0039] Figure 6 This is the effect of the compound of formula I of the present invention on FINO2-induced ferroptosis in A375 cells.

[0040] Figure 7 This is the dose-dependent effect of the compound of formula I of the present invention on RSL3-induced ferroptosis in A375 cells.

[0041] Figure 8 The inhibitory effect of the compound of formula I of the present invention on lipid peroxidation induced by RSL3.

[0042] Figure 9 This is the effect of the compound of formula I of the present invention on the expression level of HMOX1 protein induced by RSL3.

[0043] Figure 10 This is the effect of the compound of formula I of the present invention on the expression level of the HMOX1 gene induced by RSL3.

[0044] Among them, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0045] The present invention will be further described in detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the materials involved in the following examples can be obtained from commercial channels. The methods described are conventional methods unless otherwise specified.

[0046] Example 1: Preparation and structural characterization of indole alkaloid-polyketide hybrids:

[0047] (1) Aspergillus ochraceus (deposited in the China Marine Microbial Culture Collection Center, with the deposit number MCCC 3A00521) was cultured on a PDA plate at 28°C for 3-4 days. Fresh mycelia were then inoculated into a culture medium containing 400 mL of PDB. After 24 h, 10 mL of the seed solution was inoculated into 1 L conical flasks (100 flasks). Each 1 L of liquid fermentation medium contained 20.0 g of mannitol, 3.0 g of yeast extract, 0.5 g of KH2PO4, 10.0 g of MSG, 0.3 g of MgSO4·7H2O, 20.0 g of maltose, and 1.0 g of corn steep liquor, with a pH of 7.5.

[0048] (2) The fermented product obtained in step (1) was extracted with ethyl acetate, the obtained extract was dissolved in methanol and extracted with petroleum ether to remove oil, and the petroleum ether extract was discarded to obtain 38 g of defatted extract;

[0049] (3) The crude extract obtained in step (2) was separated by normal phase silica gel column chromatography, and gradient elution was performed with a petroleum ether-ethyl acetate system (100:0, 98:2, 95:5, 90:10) to obtain three crude fractions (Fr.1 to Fr.3);

[0050] (4) The crude fraction Fr.2 (6.1 g) obtained in step (3) was purified by ODS column chromatography (methanol-water system, 5-100%), Sephadex LH-20 polyacrylamide gel chromatography (methanol elution), and then by semi-preparative HPLC (acetonitrile-water, 40-60%) to obtain the compound of formula I.

[0051] The compound of formula I is a colorless crystal. Its molecular formula is determined to be C based on its main ion peak in high-resolution mass spectrometry. 35 H 39 N3O7. 1 H NMR (400 MHz), 13 C NMR data (100 MHz) (Table 1, deuterated dimethyl sulfoxide) and DEPT and HMBC spectra showed 35 carbon signals. The planar structure of the compound was determined by detailed two-dimensional data. Finally, X-ray single crystal diffraction technology ( Figure 1 ) determined the absolute configuration of the compound of formula I and named it stephaochratidin A (SCA).

[0052] Table 1 Compound 1 1 H and 13 C NMR data

[0053]

[0054] The cell lines involved in the following examples are all common cell models in ferroptosis-related research and are widely used in the development of ferroptosis inhibitors. As can be seen from the examples, the compound of formula I of the present invention can effectively protect cells from ferroptosis and has low cytotoxicity (for example, in Example 5, 40 μM of the compound of formula I can still protect cells from ferroptosis after treating cells for 24 hours).

[0055] Example 2: Effect of the compound of formula I on RSL3-induced ferroptosis in A375 cells

[0056] (1) Human malignant melanoma cells (A375 cells) were routinely digested, resuspended in culture medium, and pipetted into a single-cell suspension. 8,000 cells were then seeded into a 96-well plate at a volume of 100 μL per well.

[0057] (2) Cultured in a 37°C, 5% CO2 incubator for 12 h, and then treated with 40 μM of the compound of formula I and 10 μM of ferrostatin-1 (Fer-1, positive control) for 1 h, respectively. DMSO was used as a solvent control.

[0058] (3) RSL3 (0.3 μM) was then added to stimulate the cells for 4 h;

[0059] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0060] The results are as follows Figure 2 As shown, the compound of formula I has significant inhibitory activity on RSL3-induced ferroptosis in A375 cells, with a cell survival rate of 80.4%.

[0061] Example 3: Effect of the compound of formula I on RSL3-induced ferroptosis in 786-O cells

[0062] (1) Human renal clear cell carcinoma cells (786-O cells) were routinely digested, resuspended in culture medium, and pipetted into a single-cell suspension. 6000 cells were then seeded into a 96-well plate at a volume of 100 μL per well.

[0063] (2) Cultured in a 37°C, 5% CO2 incubator for 12 h, and then treated with 40 μM of the compound of formula I and 10 μM of ferrostatin-1 (Fer-1, positive control) for 1 h, respectively. DMSO was used as a solvent control.

[0064] (3) RSL3 (0.3 μM) was then added to stimulate the cells for 6 h;

[0065] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0066] The results are as follows Figure 3 As shown, the compound of formula I has significant inhibitory activity on RSL3-induced ferroptosis in 786-O cells, with a cell survival rate of 97.2%.

[0067] Example 4: Effect of the compound of formula I on RSL3-induced ferroptosis in H1299 cells

[0068] (1) Human lung cancer cells (H1299 cells) were routinely digested, resuspended in culture medium, and pipetted into a single-cell suspension. 8,000 cells were then seeded into a 96-well plate at a volume of 100 μL per well.

[0069] (2) Cultured in a 37°C, 5% CO2 incubator for 12 h, and then treated with 40 μM of the compound of formula I and 10 μM of ferrostatin-1 (Fer-1, positive control) for 1 h, respectively. DMSO was used as a solvent control.

[0070] (3) RSL3 (0.3 μM) was then added to stimulate the cells for 14 h;

[0071] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0072] The results are as follows Figure 4 As shown, the compound of formula I has significant inhibitory activity on RSL3-induced ferroptosis in 786-O cells, with a cell survival rate of 87.8%.

[0073] Example 5: Effect of the compound of formula I on erastin-induced ferroptosis in A375 cells

[0074] (1) After conventional digestion, A375 cells were resuspended in culture medium and pipetted into a single-cell suspension. 8000 cells were then seeded into a 96-well plate with a volume of 100 μL per well.

[0075] (2) Cultured in a 37°C, 5% CO2 incubator for 12 h, and then treated with 40 μM of the compound of formula I and 10 μM of ferrostatin-1 (Fer-1, positive control) for 1 h, respectively. DMSO was used as a solvent control.

[0076] (3) Erastin (20 μM) was then added to stimulate the cells for 24 h;

[0077] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0078] The results are as follows Figure 5As shown, the compound of formula I has significant inhibitory activity on erastin-induced ferroptosis in A375 cells, with a cell survival rate of 65.6%.

[0079] Example 6: Effect of the compound of formula I on FINO2-induced ferroptosis in A375 cells

[0080] (1) After conventional digestion, A375 cells were resuspended in culture medium and pipetted into a single-cell suspension. 8,000 cells were then seeded into a 96-well plate with a volume of 100 μL per well.

[0081] (2) Cultured in a 37°C, 5% CO2 incubator for 12 h, and then treated with 40 μM of the compound of formula I and 10 μM of ferrostatin-1 (Fer-1, positive control) for 1 h, respectively. DMSO was used as a solvent control.

[0082] (3) FINO2 (20 μM) was then added to stimulate the cells for 18 h;

[0083] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0084] The results are as follows Figure 6 As shown, the compound of formula I has significant inhibitory activity on FINO2-induced ferroptosis in A375 cells, with a cell survival rate of 64.0%.

[0085] Example 7: Dose-dependent effect of the compound of formula I on RSL3-induced ferroptosis in A375 cells

[0086] (1) After routine digestion, 786-O cells were resuspended in culture medium and pipetted into a single-cell suspension. 8,000 cells were then seeded into a 96-well plate with a volume of 100 μL per well.

[0087] (2) Cultured in an incubator at 37°C, 5% CO2 for 12 h, and then treated with different concentrations of compound I for 1 h, with DMSO as a solvent control;

[0088] (3) Then, RSL3 (0.3 μM) was added to stimulate the cells for 12 h;

[0089] (4) At the corresponding time, the ATP content of the cells was detected (G7570, Promega).

[0090] The results are as follows Figure 7 As shown in Figure 2, the compound of formula I has a significant dose-dependent effect on RSL3-induced ferroptosis in A375 cells. 50 The value was 15.4 μM.

[0091] Example 8: Inhibitory effect of the compound of formula I on RSL3-induced lipid peroxidation

[0092] (1) A735 cells were grown at 4.5×10 5 Cells were seeded into 6-well plates with a volume of 100 μL per well;

[0093] (2) Cultured in an incubator at 37°C, 5% CO2 for 12 h, and then treated with the compound of formula I (40 μM) and Fer-1 (10 μM, positive control) for 1 h;

[0094] (3) RSL3 (0.3 μM) was then added to stimulate the cells for 6 h;

[0095] (4) Treat cells with BODIPY 581 / 591C11 dye 30 min before the end of incubation;

[0096] (5) Digest the cells with trypsin and resuspend them in PBS;

[0097] (6) The lipid peroxidation rate was analyzed using flow cytometry and calculated using FlowJo software.

[0098] The results are as follows Figure 8 As shown, the compound of formula I significantly inhibited RSL3-induced lipid peroxidation, with a lipid peroxidation rate of 12.2%, indicating that the compound of formula I inhibited cell ferroptosis by inhibiting lipid peroxidation.

[0099] Example 9: Effect of the compound of formula I on the expression level of HMOX1 protein induced by RSL3

[0100] (1) After conventional digestion, A375 cells were resuspended in culture medium and pipetted into single cell suspension. Then, 1.5×10 5 Cells were seeded into 12-well plates with a volume of 1 mL per well.

[0101] (2) Cultured in an incubator at 37°C, 5% CO2 for 12 h, and then pretreated with the compound of formula I (40 μM) for 1 h, with DMSO as a control;

[0102] (3) Then, RSL3 (0.3 μM) was added to stimulate the treated cells for 2 h, 4 h, 6 h, and 8 h respectively;

[0103] (4) Collect cells, lyse, and analyze by western blot.

[0104] The results are as follows Figure 9 As shown, the compound of formula I significantly downregulated the expression of HMOX1, thereby inhibiting ferroptosis.

[0105] Example 10: Effect of the compound of formula I on the expression level of HMOX1 gene induced by RSL3

[0106] (1) After conventional digestion, A375 cells were resuspended in culture medium and pipetted into single cell suspension. Then, 1×10 5 Cells were seeded into 12-well plates with a volume of 1 mL per well.

[0107] (2) Cultured in an incubator at 37°C, 5% CO2 for 12 h, then pretreated with the compound of formula I (40 μM) for 1 h, with DMSO as a control;

[0108] (3) Then, RSL3 (0.3 μM) was added to stimulate the cells for 6 h;

[0109] (4) The cells were harvested and the expression levels of the indicated genes were analyzed by RT-qPCR.

[0110] The results are as follows Figure 10 As shown, the compound of formula I significantly down-regulated the expression level of HMOX1 gene, further indicating that the compound of formula I inhibits ferroptosis by down-regulating HMOX1.

[0111] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A deep-sea indole alkaloid and polyketide hybrid or a pharmaceutically acceptable salt thereof, characterized in that It has the structure shown in formula I:

2. A method for preparing the indole alkaloid-polyketide hybrid according to claim 1, characterized in that Specifically, it is isolated from the fermentation product of Aspergillus ochraceus; the Aspergillus ochraceus has a preservation number of MCCC3A00521 and is preserved in the China Marine Microbial Culture Collection Center.

3. Use of the indole alkaloid-polyketide hybrid or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a cell ferroptosis inhibitor.

4. Use of the indole alkaloid-polyketide hybrid or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a medicament for preventing and treating ferroptosis-related diseases.

5. The use according to claim 3, characterized in that: The cell ferroptosis refers to cell ferroptosis induced by RSL3, FINO2 or Erastin.

6. The use according to claim 4, characterized in that: The ferroptosis-related diseases include at least one of neurodegenerative diseases, inflammatory bowel disease, atherosclerosis, acute pancreatitis, acute / drug-induced liver injury, acute kidney injury, and viral infection.

7. The use according to claim 4 or 6, characterized in that: The drug comprises a therapeutically effective amount of an indole alkaloid and polyketide hybrid or a pharmaceutically acceptable salt thereof.

8. The use according to claim 4 or 6, characterized in that: The medicine further contains one or more pharmaceutically acceptable carriers or excipients.

9. A pharmaceutical composition for preventing and treating ferroptosis-related diseases, characterized in that The invention comprises the indole alkaloid and polyketide hybrid according to claim 1 or a pharmaceutically acceptable salt thereof.

10. A ferroptosis inhibitor, characterized in that The invention comprises the indole alkaloid and polyketide hybrid according to claim 1 or a pharmaceutically acceptable salt thereof.

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