Azacyclonol Compounds, Their Preparation Methods and Applications in the Preparation of Drugs for Neurodegenerative Diseases

Abstract

The present invention discloses azone compounds, a preparation method thereof, and an application thereof in the preparation of neuroprotective drugs. The azone compounds are isolated from the fermentation product of a strain of Penicillium sclerotiorum ( Penicillium sclerotiorum ) UJNMF0503, with the preservation number of CGMCC No. 40172, have neuroprotective activity, and have good application prospects in the development of drugs with neuroprotective effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of marine biomedicine, and particularly relates to a kind of azone compounds, a preparation method thereof, and an application thereof in drugs for treating neurodegenerative diseases. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease and the most common movement disorder disease. The prevalence rate of people over 65 years old in China is nearly 2%. The main pathological features of PD are the selective degeneration of dopaminergic neurons in the substantia nigra compacta, the reduction of striatal dopamine level, and the formation of Lewy bodies in the cytoplasm, ultimately leading to clinical symptoms such as resting tremor, muscle rigidity, bradykinesia, and postural instability. It has been found that many factors such as mitochondrial dysfunction, oxidative stress, glutamate toxicity, misfolding of proteins, and abnormal deposition of iron are closely related to the onset and progression of PD. However, until now, the etiology, pathogenesis, and pathways of PD have not been fully understood.

[0003] 6-OHDA (6-hydroxydopamine lesion) is a hydroxylated derivative of catecholamine, and its structure is similar to that of catecholamine. It is a neurotoxin that can effectively cause the degeneration of dopamine neurons, and can highly selectively cause the acute degeneration of sympathetic adrenergic nerve endings. It is a classical model for the study of the pathogenesis of Parkinson's disease and drug development. Summary of the Invention

[0004] The present invention provides an azone compound having protective activity against 6-OHDA-lesioned PC12 cells, and its structural formula is shown as formula (I):

[0005]

[0006] Formula (I).

[0007] Another object of the present invention is to provide a preparation method of the above compound.

[0008] To achieve the above object, the present invention adopts the following technical solutions.

[0009] A kind of Penicillium sclerotiorum Penicillium sclerotiorum UJNMF 0503, which is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No. 40172.

[0010] A kind of Penicillium sclerotiorum Penicillium sclerotiorum The application of UJNMF 0503 and its fermentation product in the production of drugs for treating neurodegenerative diseases.

[0011] The said Penicillium sclerotiorum Penicillium sclerotiorumAfter being cultured with UJNMF 0503, fermentation products can be produced. The fermentation products contain substances with protective activity against 6-OHDA-damaged PC12 cells, and their fermentation broth can be used to produce drugs for treating neurodegenerative diseases or as additives for treating neurodegenerative diseases.

[0012] Penicillium sclerotiorum Penicillium sclerotiorum The azone-like compounds produced by UJNMF 0503 have the structure shown in formula (I):

[0013]

[0014] Formula (I).

[0015] The preparation method of the above azone-like compounds includes the following steps:

[0016] (1) Fermentation of Penicillium sclerotiorum Penicillium sclerotiorum UJNMF 0503;

[0017] (2) Obtaining a fermentation extract by extracting with ethyl acetate from the fermentation product obtained in step (1);

[0018] (3) Separating the extract in step (2) by methods such as normal-phase silica gel column, flash medium-pressure chromatography column, high-performance liquid chromatography, etc.

[0019] The present invention provides the use of the extract in step (2), or the compound shown in formula (I) in the preparation of drugs for treating neurodegenerative diseases or drug intermediates.

[0020] The present invention provides a drug for treating neurodegenerative diseases containing the extract in step (2), or the compound shown in formula (I). The anti-neurodegenerative disease drug also includes pharmaceutically acceptable excipients. The drug for treating neurodegenerative diseases may also include other active ingredients to enhance the neuroprotective effect.

[0021] The present invention has the following advantages:

[0022] The azone-like compounds of the present invention can be obtained by Penicillium sclerotiorum fermentation extraction and separation of UJNMF 0503, have neuroprotective activity, and have application potential in the preparation of anti-neurodegenerative disease drugs.

[0023] Biological preservation information

[0024] Penicillium sclerotiorum ( Penicillium sclerotiorum)UJNMF 0503 was deposited on May 13, 2022 at the China General Microbiological Culture Collection Center (CGMCC), with the deposit address being Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, and the deposit number: CGMCC No. 40172. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Main 1 H- 1 H COSY and HMBC correlation information of the compound shown in formula (I);

[0026] Figure 2 Main NOESY correlation information of the compound shown in formula (I);

[0027] Figure 3 ECD spectrum of the compound shown in formula (I);

[0028] Figure 4 For Penicillium sclerotiorum Effect of the fermentation extract of UJNMF 0503 and the compound shown in formula (I) on the survival rate of PC12 cells damaged by 6-OHDA. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be further described below in conjunction with examples and drawings, but the present invention is not limited by the following examples. The experimental methods in the examples, unless otherwise specified, are all conventional techniques in the art, and the experimental reagents are all commercially purchased.

[0030] Example 1 Preparation of Fermentation Extract

[0031] Preparation method of seed medium: 200 mL of potato leachate, 20 g of glucose, 30 g of sea salt, and made up to 1 L with water. The medium was filled into 20 500 mL Erlenmeyer flasks, about 150 mL per flask, and autoclaved at 121 °C for 25 minutes, and reserved for use.

[0032] Preparation method of PDB fermentation medium: 200 mL of potato leachate, 20 g of glucose, 30 g of sea salt, and made up to 1 L with water. Placed in 1 L Erlenmeyer flasks, 350 mL of liquid medium per flask, a total of 150 flasks. Autoclaved at 121 °C for 25 minutes, and reserved for use.

[0033] Pick an appropriate amount of fungus with a sterile bamboo stick Penicillium sclerotiorumThe strain UJNMF 0503 was inoculated into the seed medium and cultured in a shaker at 28 °C (160 rpm) for 3 days to obtain a seed solution. Then, 10 mL of the seed solution was inoculated into a 1 L Erlenmeyer flask containing PDB medium using a pipette. After static culture at 28 °C for 30 days, the fermentation product was collected.

[0034] The fermentation product was filtered through gauze to be divided into thalli and broth. The thalli were soaked in 95% ethanol, and after the ethanol in the leaching solution was recovered, the remaining aqueous phase was extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract A. The broth was extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to obtain ethyl acetate extract B. A and B were combined to obtain the total extract.

[0035] Example 2 Preparation of azacyclonol compounds

[0036] 70 L of liquid medium was obtained according to the method of Example 1. The fermentation product was filtered through gauze to be divided into thalli and broth. The thalli were soaked in 95% ethanol, and after the ethanol in the leaching solution was recovered, the remaining aqueous phase was extracted with ethyl acetate, and then concentrated under reduced pressure to obtain ethyl acetate extract A. The broth was extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to obtain ethyl acetate extract B. A and B were combined to obtain 200 g of the total extract. The ethyl acetate extract was subjected to column chromatography using macroporous resin D101 as the adsorbent material, and ethanol / water was used as the eluent, and elution was carried out at volume ratios of 30%, 50%, 75%, and 90%. The elution solvent was recovered to obtain 4 fractions (Fr.1-Fr.4).

[0037] Fr.2 was further subjected to normal-phase silica gel (200-300 mesh) column chromatography using dichloromethane-methanol as the eluent. According to the thin-layer chromatography situation, each fraction was combined, and the elution solvent was recovered to obtain 8 fractions (Fr.2-1-Fr.2-8). Fr.2.4 was eluted through a medium-pressure RP-C18 column chromatography with a gradient (methanol / water 25:75-100:0) to obtain 6 fractions (Fr.2.4.1-Fr.2.4.6). Fr.2.4.3 was passed through Sephadex LH-20 (methanol) to obtain 2 fractions (Fr.2.4.3.1-Fr.2.4.3.2). Fr.2.4.3.2 was passed through Rp C-18 semi-preparative high-performance liquid chromatography (methanol / water, v / v 55:45, 3 mL / min) at t R = 17.8 min to isolate a pure compound (2.0 mg). The compound was a pale yellow gum, soluble in chloroform, methanol, DMSO, insoluble in water, and the specific rotation value was [α] 27 D +173.0 (c 0.15, MeOH).

[0038] The isolated compound was subjected to high-resolution mass spectrometry (HR-ESIMS),1 1H NMR, 13 13C NMR, 2D 1 1H- 1 1H-1H COSY, HSQC, and HMBC analyses were performed to determine the planar structure. The relative and absolute configurations of the compound were then determined by combining the NOESY signals and ECD spectrum. The 1 1H and 13 13C NMR data of the compound are shown in Table 1. The main 1 1H- 1 1H COSY and HMBC correlation information is shown in Figure 1 , and the main NOESY signals are shown in Figure 2 , and the ECD spectrum is shown in Figure 3 .

[0039] Table 1 1H and 1 13C NMR data of the compound in CDCl3 13

[0040]

[0041] Compound structure identification:

[0042] Yellow gum. High-resolution electrospray ionization mass spectrometry (HRESIMS) gave a quasi-molecular ion peak at m / z 385.1417 ([M + H]+ + , calcd 385.1412), suggesting a molecular formula of C 19 19H 25 ClO6 with an unsaturation degree of 7. NMR data analysis revealed that the compound was similar to the reported compound peniaphilone A, but with a different C-3 side chain. 1 1H- 1 HCOSY spectrum showed correlations of H-13 with H-12, H2-14, and H3-17, H2-14 with H-15, HMBC spectrum showed the correlation of H2-15 with C-11, H3-16 with C-10 / C-11 / C-12. Combining the NMR data of C11-C17, it was speculated that the compound contained a dimethyltetrahydrofuran fragment attached to C-11. Further, based on the HMBC correlations of H-16 with C-10, H-9 with C-3 / C-4 / C-11 and the 1 1H- 1 1H COSY correlation of H-9 with H-10, the side chain structure at C-3 was deduced as shown in the figure.

[0043] In the NOESY spectrum, the correlation between H-8a and H3-18 indicated that H-8a and H3-18 were on the same side of the plane and in the axial position. Further, through J8,8a (3.1 Hz) It is speculated that H-8 and H-8a are on the same side of the plane; J 12,13 (10.3 Hz) It shows that H-12 and H-13 are in the trans-axial positions of the tetrahydropyran ring. The NOESY correlation between H-12 and H3-16 indicates that they are on the same side of the plane. Additionally, by comparing the ECD spectra of this compound with that of the known compound peniaphilone A, it is speculated that the configuration of this compound is as shown in formula (I). This is the first report on the structure of the tetrahydropyran ring at the C-3 position in azanone compounds.

[0044] The structure of the compound is as follows:

[0045]

[0046] Formula (I).

[0047] Example 3 Neuroprotective activity test of the compound shown in formula (I)

[0048] The neuroprotective activity of the compound shown in formula (I) was determined using PC12 cells (rat adrenal pheochromocytoma). 6-OHDA was used as a neurotoxin to induce nerve cell damage and stimulate cell death. The MTT method was used in the experiment to determine the cell viability and detect whether the compound has neuroprotective activity under the action of 6-OHDA. The cells were seeded into 96-well plates at a density of 1.5×10 5 / mL (100 μL per well) and incubated at 37 °C and 5% CO2 for 24 hours. A blank control group (DMSO), a control group (6-OHDA), and an experimental group (6-OHDA + 50 μM fermentation extract or compound) were set up. After pre-treating the cells for 6 hours respectively, an appropriate amount of 6-OHDA was added to stimulate the cells for 13 hours. Finally, MTT was added and incubated in the incubator in the dark for 4 hours, and the absorbance (OD value) at 490 nm was read to calculate the survival rate of PC12 cells.

[0049] The experimental results showed that at a test concentration of 50 μM, Penicillium sclerotiorum after treatment with the fermentation extract of UJNMF 0503 and the compound shown in formula (I), the survival rate of PC12 cells was increased compared with the control group, indicating that Penicillium sclerotiorum the fermentation extract of UJNMF 0503 and the compound shown in formula (I) can reduce 6-OHDA-induced cell damage, have a certain neuroprotective effect, and have the potential for the development of drugs against neurodegenerative diseases.

Claims

1. A kind of azone compound, whose structure is shown in formula (I): Formula (I).

2. A method for preparing a compound as claimed in claim 1, characterized in that, Comprising the following steps: (1) Ferment Penicillium sclerotiorum ( Penicillium sclerotiorum ) UJNMF 0503 to obtain a fermentation product: Use a sterile bamboo stick to pick an appropriate amount of Penicillium sclerotiorum UJNMF 0503 strain and inoculate it into the seed medium. Incubate it on a shaker at 28 °C for 3 days to obtain a seed liquid. Then, use a pipette to inoculate 10 mL of the seed liquid into a 1-L Erlenmeyer flask containing PDB medium. After static incubation at 28 °C for 30 days, collect the fermentation product; The preparation method of the fermentation product is as follows: Preparation method of the seed medium: 200 mL of potato extract, 20 g of glucose, 30 g of sea salt, make up the volume to 1 L with water, and sterilize it at 121 °C under high-pressure steam for 25 minutes; The preparation method of the PDB medium is: 200 mL of potato extract, 20 g of glucose, 30 g of sea salt, make up the volume to 1 L with water, and sterilize it at 121 °C under high-pressure steam for 25 minutes; (2) Extract the fermentation product obtained in step (1) with ethyl acetate to obtain a fermentation extract: The fermentation product is filtered through gauze into thalli and broth. The thalli are soaked in 95% ethanol, and the remaining aqueous phase is obtained after recovering ethanol from the leaching solution. Then, it is extracted with ethyl acetate, and concentrated under reduced pressure to obtain ethyl acetate extract A. The broth is extracted three times with an equal volume of ethyl acetate, and concentrated under reduced pressure to obtain ethyl acetate extract B. A and B are combined to obtain the fermentation extract; (3) Subject the fermentation extract obtained in step (2) to column chromatography using macroporous resin D101 adsorption material, and use ethanol / water as the eluent to perform elution at volume ratios of 30%, 50%, 75%, and 90%. Recover the elution solvent to obtain 4 fractions Fr.1 - Fr.4; Fr.2 is further subjected to normal-phase silica gel column chromatography using dichloromethane - methanol as the eluent. According to the thin-layer chromatography situation, each fraction is combined, and the elution solvent is recovered to obtain 8 fractions Fr.2-1 - Fr.2-8; Fr.2.4 is subjected to gradient elution with methanol / water 25:75 - 100:0 through medium-pressure RP-C18 column chromatography to obtain 6 fractions Fr.2.4.1 - Fr.2.4.6; Fr.2.4.3 is passed through a Sephadex LH-20 column using methanol as the eluent to obtain 2 fractions Fr.2.4.3.1 - Fr.2.4.3.2; Fr. 2.4.3.2 was obtained by Rp C-18 semi-preparative high performance liquid chromatography using methanol / water (55:45, v / v) as the mobile phase at a flow rate of 3 mL / min and eluting at t R = 17.8 min; The Penicillium sclerotiorum ( Penicillium sclerotiorum ) UJNMF 0503 is deposited in the China General Microbiological Culture Collection Center with the deposit number of CGMCC No. 40172.

3. Use of a kind of azone compound as described in claim 1 and the fermentation extract as described in step (2) of claim 2 in the preparation of a drug for treating neurodegenerative diseases.

4. A drug for treating neurodegenerative diseases, characterized in that, Containing an effective amount of the azone compound as described in claim 1 as the active ingredient; or, Containing an effective amount of the fermentation extract of Penicillium sclerotiorum ( Penicillium sclerotiorum ) UJNMF 0503 as the active ingredient described in step (2) of claim 2.

5. The drug according to claim 4, characterized in that, It further includes pharmaceutically acceptable excipients.

6. The application according to claim 3, characterized in that The neurodegenerative disease is Parkinson's disease.

7. The medicament according to claim 4 or 5, characterized in that, The neurodegenerative disease is Parkinson's disease.

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