Furanone derivative with neuroprotective activity as well as preparation method and application of furanone derivative
By extracting and purifying compound 1 and compound 2 from the culture medium after Penicillium paxilli fermentation, the problem of lack of effective neuroprotective drugs in the prior art was solved. The compound significantly improved cell survival and inhibited cell apoptosis, providing targeted Al New treatment options for neurological diseases such as Zheimer's disease.
Patent Information
- Application Number
- CN202311852849.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-29
- Publication Date
- 2025-01-10
AI Technical Summary
There are no fundamental solutions to existing neurological diseases and injuries treatments, especially for diseases such as Alzheimer's disease and stroke, with effective neuroprotective drugs lacking.
A derivative of furanone with neuroprotective activity was developed, and the culture medium after Penicillium paxilli fermentation was extracted with ethyl acetate, columnar chromatography and high-performance liquid phase to prepare compound 1 and 2 for the preparation of treatment. Neuroprotective drugs, especially for OGD/R-induced pheochromocytoma cell damage and Alzheimer's disease.
Compounds 1 and 2 significantly improve the survival rate of PC12 cells induced by OGD/R, inhibit apoptosis, have good neuroprotective activity, and provide new therapeutic options, especially in Alzheimer's disease and other nervous systems. Disease has application prospects.
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Figure CN119285587A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of medical technology, and specifically relates to a furanone derivative with neuroprotective activity, a preparation method and use thereof. Background Art
[0002] In drug discovery and development, natural products are an important source of drugs or drug lead compounds. Almost 60% of clinically approved drugs are directly or indirectly derived from natural products. Among them, natural product drugs produced by microorganisms have extremely high biological activity and component diversity, and are one of the hot areas for the development of new drugs. Through fermentation, extraction and purification and other technical means, drug compounds naturally produced by microorganisms can be obtained and then used in drug development.
[0003] At present, a large number of antibiotics such as penicillin are derived from microbial natural products. In addition, many other drugs such as cyclosporin A, echinocandin B and lovastatin are also derived from microbial natural products. The biodiversity and chemical diversity of microbial natural products have greatly enriched the sources of drugs and created more opportunities and possibilities.
[0004] As the trend of population aging intensifies, the prevalence of neurological diseases and injuries continues to increase. For example, the prevalence of Alzheimer's disease is on the rise worldwide, and stroke is also considered the second leading cause of death in the world. At present, there is still no fundamental solution to the treatment of neurological diseases and injuries. By discovering and developing new active compounds, drug targets and treatment strategies, innovation in neuroprotective drugs can be promoted and new treatment options can be provided. Therefore, the research and development of neuroprotective drugs is urgent to meet the growing demand for treatment, and the market prospects are very promising. Summary of the invention
[0005] The technical problem to be solved by the present invention is to provide a furanone derivative with neuroprotective activity, a preparation method and use thereof, which can be used to treat nerve damage, protect damaged cells and improve the therapeutic effect.
[0006] The embodiment of the present invention provides a furanone derivative, wherein the furanone derivative is compound 1 or compound 2, and the structural formula of compound 1 is The structural formula of compound 2 is .
[0007] As one embodiment, the esters of the furanone derivatives are also included.
[0008] The embodiment of the present invention further provides a medicine, comprising the furanone derivatives and pharmaceutically acceptable excipients.
[0009] Preferably, the dosage form of the drug is tablets, capsules, granules, pills, injections or gels.
[0010] The present invention also provides a method for preparing the furanone derivatives having neuroprotective activity, wherein the Penicillium paxilli The culture medium after fermentation is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; the crude extract is subjected to column chromatography and eluted with methanol water to obtain a crude extract A; the crude extract A is separated by silica gel column chromatography, using petroleum ether-ethyl acetate as an eluent, and gradient eluting the sample layer of the silica gel; during the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 1:0, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1, and 10 components Fr.A1-A10 are obtained after the fraction is monitored by TLC spot plate; The fraction Fr.A2 was passed through a small silica gel and gradient eluted using a petroleum ether-dichloromethane-ethyl acetate system. During the gradient elution, the volume ratios of petroleum ether, dichloromethane, and ethyl acetate were 5:1:0, 1:1:0, 0:1:0, 0:10:1, 0:10:2, 0:2:1, 0:1:1, and 0:0:1, to obtain 7 fractions Fr.A2.1-Fr.A2.7; Fraction Fr.A2.5 was purified by HPLC to obtain compound 2; Fraction Fr.A3 was purified by high performance liquid chromatography to obtain compound 1.
[0011] As one embodiment, the Penicillium Penicillium paxilli The method for preparing the fermented culture medium is as follows: Penicillium paxilli Inoculate into SDA medium, then inoculate into rice medium, and after cultivation, obtain Penicillium mycoides Penicillium paxilli The culture medium after fermentation; The rice culture medium is obtained by mixing rice and water and then sterilizing by high pressure.
[0012] As one embodiment, during column chromatography, elution is performed with methanol aqueous solution having a volume concentration of 80% and 100% in sequence.
[0013] As one embodiment, the Penicillium Penicillium paxilli It can be purchased from China General Microbiological Culture Collection Center, with the collection number CGMCC 3.11477.
[0014] The present invention provides a use of the furanone derivatives, wherein the furanone derivatives are used to prepare a neuroprotective drug. Preferably, the furanone derivatives are used to prepare a protective drug against OGD / R-induced pheochromocytoma cell damage. Preferably, the furanone derivatives are used to prepare a drug for treating or preventing Alzheimer's disease.
[0015] Compared with the prior art, the present invention has the following advantages and beneficial effects: PC12 cells are a cell line derived from rat pheochromocytoma and are commonly used in neurobiology and cell biology research. PC12 cells originally originated from the peripheral nervous system and have properties similar to those of nerve cells.
[0016] Compounds 1 and 2 of the present invention are furanone compounds. Common furanone compounds include methoxybenzyl furanone (MBF), vanillone (VK), etc., which are usually used for antibacterial and anti-inflammatory effects. The inventors found that the furanone derivatives of the present invention have good neuroprotective activity, which is not possessed by common furanone derivatives. At the same time, the inventors also found that compounds 1 and 2 of the present invention have significantly higher activity in neuroprotection than compound 3, which is very similar to it in structure, and it is of great significance for further research on neuroprotection.
[0017] The invention can significantly improve the survival rate of PC12 cells induced by OGD / R, inhibit the apoptosis of PC12 cells induced by OGD / R, and has a good application prospect in neuroprotective drugs. BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 is the HR-ESI-MS spectrum of compound 1.
[0019] Figure 2 For compound 1 1 H NMR (600 MHz, CD3OD) spectroscopy.
[0020] Figure 3 For compound 1 13 C NMR and DEPT (150 MHz, CD3OD) spectra.
[0021] Figure 4 is the HR-ESI-MS spectrum of compound 2.
[0022] Figure 5 For compound 2 1 H NMR (500 MHz, CDCl3) spectroscopy.
[0023] Figure 6 For compound 2 13 C NMR and DEPT (125 MHz, CDCl3) spectra.
[0024] Figure 7 The cytotoxic effects of compounds 1–3 on PC12 cells.
[0025] Figure 8Effects of compounds 1–3 on the cell viability of OGD / R-induced PC12 cells (concentration range: 0.01–10 μ M).
[0026] Fig. 9 Effects of compounds (10 μM) on antioxidant defense indicators of PC12 cells. (A) Effects of compounds on LDH release. (B) Effects of compounds on SOD activity. (C) Effects of compounds on MDA levels. (D) Effects of compounds on GSH levels. Different letters above the bar graphs indicate significant differences (after ANOVA test, p <0.05). The results are expressed as mean ± standard deviation (n=3). * p <0.01 compared with the control group; # p <0.05, ## p <0.01 compared with the model group.
[0027] Fig.10 The results of TUNEL staining analysis of the protective effect of compounds 1-3 on OGD / R-induced PC12 cell apoptosis (200× magnification, Fig.10 The scale bar in the middle is 50 μm. Blue: PC12 cell nuclei stained with DAPI. Green: TUNEL staining of cell nuclei showing DNA fragmentation. DETAILED DESCRIPTION
[0028] Example 1 The experimental instruments and reagents used in Example 1 are shown in Table 1.
[0029] Table 1 Experimental instruments and reagents
[0030] The preparation method of the compound comprises the following steps: SDA medium: glucose (20g / L), peptone (10g / L), yeast powder (1g / L), agar (15 g / L). Weigh the above reagents accurately, add 1L of tap water, stir evenly, pour into 4 500 mL conical flasks (about 250 mL per bottle), cover with a breathable membrane and tie with a rubber band, and finally put it in an autoclave for sterilization at 121°C for 30 min. After sterilization, the culture medium is cooled to about 50°C, poured into a sterile culture dish (about half the height of the culture dish) on a clean bench, and left to cool to room temperature for use.
[0031] Preparation of rice culture medium: Take 500 mL conical flasks, put 80 g of rice in each flask and add 100 mL of tap water, mix well, cover with a breathable membrane and tie with a rubber band, put into an autoclave and sterilize at 121°C for 30 min. After sterilization, cool to room temperature for use (a total of 7.0 kg of rice is used for this strain, which is evenly distributed into 70 500 mL conical flasks).
[0032] Penicillium mycoides Penicillium paxilli The mycelium (purchased from China General Microbiological Culture Collection Administration Center, with the collection number CGMCC 3.11477) was inoculated on SDA medium and cultured at 25°C for 8 days. After it grew well, a small steel shovel was used to cut an appropriate amount and inoculated into SDA liquid culture medium under sterile conditions (the SDA liquid culture medium in a 500 mL conical flask should not exceed 300 mL). The inoculated liquid culture was incubated at room temperature and 120 r / min for 5 days. Then it was aseptically inoculated into the sterilized rice culture medium, with about 5 mL of bacterial liquid per bottle, and cultured at room temperature for 30 days. Observe once every 2 days to prevent contamination.
[0033] Penicillium mycoides Penicillium paxilli After the fermentation of the strain was completed, the fermented culture medium was collected, extracted 8 times with 10 L ethyl acetate, and concentrated under reduced pressure to obtain 75.0 g of crude extract. The total extract was chromatographed on a macroporous resin D101 column and eluted with 80% and 100% methanol aqueous solutions, respectively, to obtain two crude extracts, Fr. A and Fr. B. Fr. A (55.0 g) was separated by silica gel column chromatography, and the dry sample was applied to silica gel (200-300 mesh). Petroleum ether-ethyl acetate was used as the eluent, and the sample layer of silica gel was gradient eluted (mobile phase volume ratio v / v, 1:0, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1). After the flow was monitored by TLC spot plate, 10 components Fr. A1~A10 were obtained. Fraction Fr. A2 (0.6 g) was further passed through small silica gel and gradient eluted with petroleum ether-dichloromethane-ethyl acetate system (v / v / v, 5:1:0, 1:1:0, 0:1:0, 0:10:1, 0:10:2, 0:2:1, 0:1:1, 0:0:1) to obtain 7 fractions Fr. A2.1 to Fr. A2.7. Fraction Fr. A2.5 was purified by semi-preparative HPLC (0-17 min, acetonitrile / water, 28%, 3 mL / min, 210 nm) to obtain compound 2 (15.0 mg, t R= 15.7 min); fraction Fr. A3 was purified by semi-preparative HPLC (0-23 min, methanol / water, 40%, 3 mL / min, 210 nm) to obtain two oily compounds 1 (10.0 mg, t R = 17.1 min), 3 (22.0 mg, t R = 21.3 min).
[0034] The structural formula of compound 1-3 is as follows: .
[0035] Compound 1: penicifuranone A
[0036] Transparent oily compound, easily soluble in methanol and chloroform, HPLC-UV (CH3CN-H2O) λ max : 287 nm, IR(KBr) υ max : 3413, 2929, 1694, 1614, 1214, 1134, 959 cm −1 , HRESIMS suggests that its molecular formula is C 10 H 16 O4( m / z : 201.1135 [M+H] + , calcd. for C 10 H 17 O4, 201.1121), the degree of unsaturation is 3.
[0037] Compound 1 1 H NMR spectrum (600 MHz, CD3OD) shows 15 proton signals. In addition to one oxygen-linked methine proton δ H 3.76 (1H, m, H-7), the other 14 protons showed splitting signals, including: 1 methoxy proton δ H 3.33 / 3.34 (3H, s, 7-OCH3); 3 methyl protons: δ H 1.22 / 1.23 (3H, d, J = 3.4 Hz, H-8), δ H 1.65 / 1.66 (3H, s, H-9), δH 1.41 / 1.42 (3H, s, H-10); 1 methylene proton: δ H 2.59 / 2.60 (1H, dd, J = 14.0, 8.7 Hz, H a -6), δ H 2.81 / 2.83 (1H, dd, J = 14.0, 6.7 Hz, H b -6).
[0038] Compound 1 13 C NMR spectrum (150 MHz, CD3OD) shows 10 carbon signals in total. Five of them show splitting signals. Combining DEPT and HSQC spectra, it can be concluded that the compound contains 1 carbonyl carbon ( δ C 205.2 / 205.2), 1 group of olefinic carbon ( δ C 110.0 / 109.9, 186.7 / 186.6), 1 hemiketal carbon ( δ C 104.2), 3 methyl carbons ( δ C 19.7 / 19.8,5.7, 22.2), 1 methoxy carbon ( δ C 56.8), 1 oxygen-linked methylene carbon ( δ C 75.7), 1 methylene carbon ( δ C 36.9 / 36.9).
[0039] Since the experimental ECD spectrum (200–400 nm) of compound 1 did not show obvious Cotton effect and the specific rotation was approximately equal to 0, it was shown that compound 1 was 2 S *,7 R * and 2 S *,7 S * Racemate.
[0040] In summary, the planar structure of compound 1 has been confirmed to be a new furanone derivative, named penicifuranone A.
[0041] Table 2 Proton spectrum (600 MHz) and carbon spectrum (150 MHz) data of compound 1 (CD3OD)
[0042] Compound 2: 2-hydroxy-2,4-dimethyl-5-propylfuran-3 (2H)-one
[0043] Transparent oil, easily soluble in chloroform, its molecular formula is C9H 14 O3( m / z : 171.10162 [M+H] + , calcd. forC9H 15 O3, 171.1016), and its NMR data were comprehensively analyzed (Table 3), and compound 2 was identified as 2-hydroxy-2,4-dimethyl-5-propylfuran-3 (2H)-one, which is a new natural product.
[0044] Compound 3: 2,3-dihydro-2-hydroxy-2,4-dimethyl-5- trans -propenylfuran-3-one
[0045] Yellow oil, easily soluble in chloroform, its molecular formula is C9H 12 O3( m / z : 169.08598 [M+H] + , calcd. forC9H 13 O3, 169.0859), and its NMR data were comprehensively analyzed (Table 3), and compound 3 was identified as 2,3-dihydro-2-hydroxy-2,4-dimethyl-5- trans -propenylfuran-3-one.
[0046] Table 3 H and C NMR data of compounds 2 and 3
[0047] Note: The NMR data test conditions of compound 2 are 1 H NMR 500 MHz, 13 C NMR 125 MHz (CDCl3), the NMR data test conditions of compound 3 are 1 H NMR 600 MHz, 13 C NMR 150 MHz (CD3OD).
[0048] Example 2 Study on the protective effect of compounds 1-3 on OGD / R-induced pheochromocytoma cell (PC12 cell) damage Experimental instruments and reagents Cell lines PC12 cells (purchased from Procell Life Science Technology Co., Ltd.) Experimental instruments Table 4 Experimental instruments and manufacturers
[0049] Experimental reagents and consumables Table 5 Reagents, consumables and manufacturers
[0050] Experimental methods 1. Cell culture and OGD / R model PC12 cells were collected at 1×10 4 Cells were added to a 96-well plate at a density of 10 cells / well in a medium containing 10% fetal bovine serum (FBS) and 1% double antibody (penicillin-streptomycin), and then cultured at 37°C and 5% CO2 for 24 hours. After the culture was completed, the cells were transferred to an anaerobic environment containing 5% CO2 and 95% N2 and cultured at 37°C for 6 hours, and then the cell culture medium was replaced with serum-free glucose-free RPMI-1640. After 6 hours of OGD treatment, the cells were switched to a normoxic incubator with serum-free high-glucose RPMI-1640 for 24 hours.
[0051] 2. Cell viability test The cell viability assay was performed using the CCK-8 method. PC12 cells were incubated for 6 h under hypoxia. After the test compound was serially diluted from 10 μM to 0.01 μM in serum-free medium, different concentrations of the test compound were added to each well and incubated under normal conditions for 24 h. After that, the medium was replaced with 10 μL of CCK8 solution (Bioss, China) per well. The cells were then incubated at 37°C for 2 h. The optical density (OD) at 450 nm was measured using a microwell reader (Perkinelmer, Enspire). Cell viability was expressed as a percentage of the control group.
[0052] 3. Determination of LDH, MDA, SOD and GSH contents PC12 cells were cultured at 1×10 5Cells were seeded into 6-well culture plates at a density of 100 cells / well. After different treatments, cells were collected and the activities of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were detected according to the instructions of the commercial kits of Nanjing Jiancheng Bioengineering Institute (Nanjing, China).
[0053] 4. Cell apoptosis detection The experiment used TUNEL staining to detect the apoptosis of PC12 cells under different treatments. 4 The density of cells / well was inoculated into 24-well culture plates. After OGD / R treatment, immunofluorescence TUNEL kit (Beyotime, China) was used for detection according to the manufacturer's instructions. The cell nucleus was clearly visible and stained with DAPI (Solarbio, China). Images were acquired by confocal laser scanning microscopy (Zesis, Germany).
[0054] Data analysis The data in this study are presented as the mean ± SD of at least three independent experiments. One-way ANOVA followed by LSD test was performed using Graphpad Prism software. p Values less than 0.01 were considered statistically significant. Significant difference: * p <0.01 vs. control group, # p <0.05, ## p <0.01 vs. model group.
[0055] Experimental Results Neuroprotective activity results analysis: The effects of compounds 1-3 on the survival rate of PC12 cells induced by OGD / R were evaluated by CCK-8 assay. Figure 7 ) showed that no toxic effects of these compounds were observed on PC12 cells at the tested concentrations.
[0056] Compounds 1 and 2 significantly increased the survival rate of PC12 cells under OGD / R conditions at all tested concentrations. The EC50 values of each compound were calculated by graphpad. The EC50 values of compounds 1, 2, and 3 were 1.16μM, 0.56μM, and >50μM, respectively. The EC50 values of compounds 1 and 2 were much smaller than the EC50 value of compound 3.
[0057] Figure 8 The results showed that compared with the normal group, OGD / R modeling led to a significant decrease in PC12 cell viability ( p<0.01); Compounds 1 and 2 can significantly improve OGD / R damage in PC12 cells at a concentration of 10μM-0.1μM. The higher the concentration, the stronger the damage improvement effect. Compound 3 has a significant improvement effect on OGD / R damage in PC12 cells at a concentration of 1μM, and the cell survival rate is 86.87% ( p <0.01), at other concentrations, compound 3 had no improvement effect on OGD / R injury in PC12 cells.
[0058] Fig. 9 (A) OGD / R-induced PC12 cells showed increased LDH release, while treatment with the four compounds at 10 µM concentration significantly reduced the amount of LDH release.
[0059] Furthermore, we examined the effects of compounds 1–3 on the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) in OGD / R-induced PC12 cells to evaluate their efficacy in alleviating OGD / R-induced oxidative stress in PC12 cells. Fig. 9 (B)-9(D) showed that OGD / R induction significantly reduced SOD and GSH activities, accompanied by an increase in MDA levels, compared with the control group. However, after compound treatment, the activities of SOD and GSH increased significantly, while the MDA level decreased significantly, which was significantly different from the model group. It is worth noting that compound 3 treatment did not show significant differences in SOD, MDA, and GSH levels compared with OGD / R-induced cells.
[0060] Apoptosis assays were performed using all test compounds at 10 μM concentration. Fig.10 ) showed that after OGD / R treatment, the number of TUNEL-positive cells (evidenced by green nuclear staining) was significantly increased compared with normal PC12 cells. Interestingly, compounds 1 and 2 (10 μM) had a significant effect in reducing the number of TUNEL-positive cells compared with the model, indicating that they have the potential to inhibit OGD / R-induced apoptosis in PC12 cells.
[0061] It can be seen from the figure that the compounds 1 and 2 described in the present invention have significantly better protective effects on OGD / R-induced PC12 cell damage than compound 3.
[0062] A person skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of protection of the present application is limited to these examples. In line with the concept of the present application, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of different aspects of one or more embodiments of the present application as described above, which are not provided in detail for the sake of simplicity.
[0063] One or more embodiments of the present application are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the present application. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of one or more embodiments of the present application should be included in the protection scope of the present application.
Claims
1. A furanone derivative having neuroprotective activity, characterized in that: The furanone derivative is compound 1 or compound 2, and the structural formula of compound 1 is The structural formula of compound 2 is .
2. The furanone derivative according to claim 1, characterized in that: Also included are esters of the furanone derivatives.
3. A drug, characterized in that: The invention comprises the furanone derivative as claimed in claim 1 or 2 and a pharmaceutically acceptable auxiliary material.
4. The drug according to claim 3, characterized in that: The dosage form of the medicine is tablet, capsule, granule, pill, injection or gel.
5. A method for preparing the furanone derivatives according to claim 1, characterized in that: Penicillium mycoides Penicillium paxilli The culture medium after fermentation is extracted with ethyl acetate and concentrated under reduced pressure to obtain a crude extract; the crude extract is subjected to column chromatography and eluted with methanol water to obtain a crude extract A; the crude extract A is separated by silica gel column chromatography, using petroleum ether-ethyl acetate as an eluent, and gradient eluting the sample layer of silica gel; during the gradient elution, the volume ratio of petroleum ether to ethyl acetate is 1:0, 20:1, 10:1, 5:1, 2:1, 1:1, 0:1; and after monitoring the fraction by TLC spot plate, 10 components Fr.A1-A10 are obtained; The fraction Fr.A2 was passed through a small silica gel and gradient eluted using a petroleum ether-dichloromethane-ethyl acetate system. During the gradient elution, the volume ratios of petroleum ether, dichloromethane, and ethyl acetate were 5:1:0, 1:1:0, 0:1:0, 0:10:1, 0:10:2, 0:2:1, 0:1:1, and 0:0:1, to obtain 7 fractions Fr.A2.1-Fr.A2.7; Fraction Fr.A2.5 was purified by HPLC to obtain compound 2; Fraction Fr.A3 was purified by high performance liquid chromatography to obtain compound 1.
6. The method for preparing the furanone derivatives according to claim 5, characterized in that: Penicillium Penicillium paxilli The method for preparing the fermented culture medium is as follows: Penicillium paxilli Inoculate into SDA medium, then inoculate into rice medium, and after cultivation, obtain Penicillium mycoides Penicillium paxilli The culture medium after fermentation; The rice culture medium is obtained by mixing rice and water and then sterilizing by high pressure.
7. The method for preparing the furanone derivatives according to claim 5, characterized in that: During column chromatography, elution was performed with 80% and 100% methanol aqueous solutions by volume.
8. A use of the furanone derivatives as claimed in claim 1 or 2, characterized in that: The furanone derivatives are used for preparing medicines for treating nerve damage.
9. The use of the furanone derivatives according to claim 8, characterized in that: The furanone derivatives are used for preparing protective drugs for resisting OGD / R-induced pheochromocytoma cell damage.
10. The use of the furanone derivatives according to claim 8, characterized in that: The furanone derivatives are used for preparing medicines for treating or preventing Alzheimer's disease.