5alpha, 8alpha-peroxysterol compound, preparation method thereof and application of 5alpha, 8alpha-peroxysterol compound in preparation of drugs for inhibiting neuroinflammation

Abstract

The invention provides a 5alpha, 8alpha-peroxysterol compound, or a stereoisomer, a tautomer, a geometric isomer, a deuterated compound and a pharmaceutically acceptable salt thereof, the structural formula of the 5alpha, 8alpha-peroxysterol compound is as follows: # imgabs0 #, the # imgabs1 # represents a single bond or a double bond, and the # imgabs2 # represents a single bond or a double bond; r1 is selected from # imgabs2 # R1a, R2a and R3a are respectively selected from hydrogen and C1-6 alkyl groups; and R2 is selected from a hydroxyl group and a C1-6 alkoxy group. The invention also provides an application of the 5alpha, 8alpha-peroxysterol compound in preparation of an anti-inflammatory drug, especially an anti-neuroinflammation drug.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceutical technology, and in particular to a 5α,8α-epoxysterol compound, a preparation method thereof, and an application thereof in the preparation of a drug for inhibiting neuroinflammation. Background Art

[0002] Inflammatory response is a defensive response of the body to various stimuli (such as infection, injury, etc.). Generally speaking, a moderate inflammatory response is beneficial to the body, which can effectively eliminate pathogens, repair tissue damage and maintain health. However, when the inflammatory response gets out of control or lasts for too long, it may cause a series of adverse consequences, including toxic shock, blood pressure drop, electrolyte disorder, local swelling, heat, pain, fever, etc., and may even cause serious damage to the body.

[0003] At present, there are already many anti-inflammatory drugs on the market, such as non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, etc. However, these drugs generally have problems such as large side effects, inconsistent curative effects, and inability to provide effective treatment for all patients, and it is difficult to meet the special needs of the treatment of nervous system diseases. Therefore, there is an urgent need to develop drugs with smaller side effects, strong specificity, and good anti-inflammatory effects, especially drugs that can target neuroinflammation.

[0004] In the nervous system, chronic inflammation is considered to be one of the core pathological mechanisms of many neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, stroke, etc.). Excessive neuroinflammation will not only accelerate neuron damage, but may also exacerbate the progression of the disease.

[0005] Therefore, developing anti-inflammatory drugs for the nervous system, especially anti-inflammatory drugs with neuroprotective effects, has important clinical significance. Summary of the Invention

[0006] The present invention aims to solve at least one of the technical problems existing in the prior art to some extent. Thus, in the first aspect of the present invention, the present invention provides a 5α,8α-epoxysterol compound, or a stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt thereof. The structural formula of the 5α,8α-epoxysterol compound is shown as follows:

[0007] Wherein, represents a single bond or a double bond; R1 is selected from R 1a , R 2a , R 3a are independently selected from hydrogen, C1-6 alkyl; R2 is selected from hydroxyl, C1-6 alkoxy.

[0008] Preferably, the structural formula of the 5α,8α-epoxysterol compound is as shown in Formula I-1 or Formula I-2:

[0009] Preferably, R1 is selected from representing a single bond or a double bond.

[0010] Preferably, R1 is selected from

[0011]

[0012] Preferably, the 5α,8α-epoxysterol compound is selected from the following structures:

[0013]

[0014] In the second aspect of the present invention, the present invention provides a method for preparing the 5α,8α-epoxysterol compound described in the first aspect of the present invention, comprising the following steps:

[0015] Step 1): Cut the Sarcophyton glaucum into pieces, soak it with acetone, extract, concentrate under reduced pressure to obtain a crude acetone extract, extract with diethyl ether, and concentrate under reduced pressure to obtain a diethyl ether extract;

[0016] Step 2): Subject the diethyl ether extract to silica gel column chromatography, gel column chromatography separation, and high performance liquid chromatography separation and purification to obtain the 5α,8α-epoxysterol compound.

[0017] Preferably, the silica gel column chromatography is eluted with a mixed solvent of petroleum ether and ethyl acetate, and the gel column chromatography is eluted with a mixed solvent of petroleum ether, dichloromethane and methanol.

[0018] In the third aspect of the present invention, the present invention provides a pharmaceutical preparation comprising the above-mentioned 5α,8α-epoxysterol compound or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt.

[0019] In the fourth aspect of the present invention, the present invention provides an application of the above-mentioned 5α,8α-epoxysterol compound or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt in the preparation of anti-inflammatory drugs.

[0020] Preferably, the anti-inflammatory drug is an anti-neuroinflammatory drug.

[0021] The beneficial effects of the present invention are as follows:

[0022] 1. The present invention provides a 5α,8α-epoxysterol compound, which is a new compound and has anti-inflammatory activity.

[0023] 2. The present invention also provides the use of the 5α,8α-epoxysterol compounds in the preparation of anti-inflammatory drugs. The research and development of such drugs will provide new treatment options for neuroinflammation. At the same time, this will also enhance China's competitiveness in the global pharmaceutical market and promote the innovative development of the domestic pharmaceutical industry. Detailed implementation manners

[0024] The present invention will be further described below in conjunction with specific embodiments. However, the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. For those conditions not specified in the following embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The methods used, unless otherwise specified, are all conventional methods well known in the art. The consumables and reagents used, unless otherwise specified, are all commercially available. Unless otherwise stated, the professional and scientific terms used herein have the same meaning as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content can also be applied to the present invention.

[0025] Some of the reagents and equipment used in the present invention are shown in Table 1 below:

[0026] Table 1

[0027]

[0028]

[0029] Example 1 Preparation method of 5α,8α-epoxysterol compounds [[ID=2L]]

[0030] (1) Thaw the Sarcophyton sp. collected from the waters of Weizhou Island, Hainan Province, China in 2009, directly cut it into pieces, soak it in acetone (1L×4) and extract it by ultrasonic wave 4 times (each ultrasonic time is 30 min, and the water temperature is 25-30 °C). Combine the extracts, concentrate them under reduced pressure to obtain an acetone crude extract, and extract the acetone crude extract with anhydrous ether until the supernatant is colorless. Concentrate it under reduced pressure to obtain an anhydrous ether extract.

[0031] (2) Subject the anhydrous ether extract to silica gel column chromatography (200-300 mesh) treatment, and elute it with organic solvents petroleum ether and ethyl acetate in a (0-100%) gradient to collect the eluate to obtain 7 fractions (A-G); Subject fraction D to Sephadex LH-20 gel column chromatography and elute it with petroleum ether:dichloromethane:methanol volume ratio of 2:1:1 to obtain fractions DA and DB;

[0032] (3) Subject fraction DB to silica gel column chromatography with petroleum ether and ethyl acetate (20:1→1:1) to obtain 7 sub-fractions, namely DB1 to BD7;

[0033] (4) The sub-component DB7 was separated and purified by reverse-phase high-performance liquid chromatography to obtain Compounds 1-5. The chromatographic conditions were as follows: the chromatographic column was an Agilent Eclipse XDB-C18 column (5 mm, 9.4×250 mm); the mobile phase was acetonitrile: water = 90:10, isocratic elution, and the flow rate was 3 mL / min; the detection wavelength was 210 nm. The retention time of Compound 1 was 18.0 min, the retention time of Compound 2 was 15.5 min, the retention time of Compound 3 was 22.0 min, the retention time of Compound 4 was 17.5 min, and the retention time of Compound 5 was 16.2 min.

[0034] (5) Through spectral analysis, the chemical structural formulas of 5α,8α-epidioxyergosta-9(11)-dehydropodocasterol F (Compound 1), 5α,8α-epidioxyergosta-6,9,25-trien-3β-ol (Compound 2), 5α,8α-epidioxyergosta-6,25-dien-3β-ol (Compound 3), 5α,8α-epidioxy-22-methylergosta-6,22,25-trien-3β-ol (Compound 4), and asinisterol (Compound 5) were determined as follows:

[0035]

[0036] Example 2 Anti-inflammatory activity study of 5α,8α-epidioxyergosta-9(11)-dehydropodocasterol F (Compound 1), 5α,8α-epidioxyergosta-6,9,25-trien-3β-ol (Compound 2), 5α,8α-epidioxyergosta-6,25-dien-3β-ol (Compound 3), 5α,8α-epidioxy-22-methylergosta-6,22,25-trien-3β-ol (Compound 4), and asinisterol (Compound 5):

[0037] I. Model construction

[0038] Microglial BV2 cells are widely distributed in the central nervous system and are the main cells for the nervous system to exert immune functions. Under physiological conditions, they play a monitoring role and maintain a stable internal environment. BV2 cells are sensitive to external stimuli. Once there is a slight pathological change in the central nervous system, microglial cells can be activated. Activated BV2 cells, on the one hand, play a phagocytic role, phagocytosing cell debris and secreting growth factors to promote nerve repair. On the other hand, they can also secrete inflammatory factors, glutamate, NO, etc., aggravating the inflammatory response and causing secondary damage. After stimulating BV2 cells with 100 ng / mL lipopolysaccharide (LPS), a large amount of inflammatory factors, including NO, are released. The Griess reagent method is used to detect the content of NO in the supernatant to preliminarily judge the level of the inflammatory response of BV2 cells.

[0039] In this experiment, the anti-inflammatory activity of 5α,8α-epoxysterols was tested using an LPS-induced pro-inflammatory differentiation model of BV2 cells.

[0040] II. Experimental Methods

[0041] 1. Inflammatory model of lipopolysaccharide (LPS)-induced nitric oxide (NO) production in BV2 cells

[0042] (1) After digestion with 0.25% trypsin, BV2 cells were suspended in high-glucose DMEM medium containing 10% fetal bovine serum.

[0043] (2) BV2 cells were seeded in 96-well culture plates at a density of 2×10 5 cells / ml, with an inoculation volume of 100 μl / well, and cultured in a 37°C incubator with 5% CO2.

[0044] (3) After 24 hours of culture, the culture medium of each group was replaced with fresh high-glucose DMEM medium containing 10% fetal bovine serum.

[0045] (4) In the drug treatment groups, 10 μl of the test compound at the corresponding concentration was added to each well. The normal control group and the LPS model group were added with high-glucose DMEM medium containing 10% fetal bovine serum (10 μl / well).

[0046] (5) After 2 hours of incubation, 10 μl of 0.001 mg / ml LPS was added to each well in the drug treatment groups and the LPS model group, with a final concentration of 100 ng / ml. The normal control group was added with high-glucose DMEM medium containing 10% fetal bovine serum (10 μl / well).

[0047] (6) After continued culture for 24 hours, 50 μl of the culture medium supernatant from each well was mixed with 50 μl of Greiss reagent and reacted at room temperature for 15 min. The OD value of each group was measured at a wavelength of 540 nm. NO inhibition rate (%) = [1 - (OD 药物组 - OD 正常对照组 ) / (OD LPS组 - OD 正常对照组 )] × 100%.

[0048] 2. Effect of the compound on cell viability

[0049] (1) After digestion with 0.25% trypsin, BV2 cells were suspended in high-glucose DMEM medium containing 10% fetal bovine serum.

[0050] (2) At a density of 1.25×10 4BV2 cells were seeded in 96-well culture plates at a density of cells / ml, with an inoculation volume of 100 μl / well, and cultured in a 37 °C constant temperature incubator containing 5% CO2.

[0051] (3) After 24 hours of BV2 cell culture, the culture medium of each group was replaced with fresh DMEM high-glucose medium containing 10% fetal bovine serum.

[0052] (4) In the dosing groups, the corresponding concentration of the test compound (10 μl / well) was added, and the normal control group was added with DMEM high-glucose medium containing 10% fetal bovine serum (10 μl / well).

[0053] (5) After continued culture for 24 hours, 5 mg / ml MTT (10 μl / well) was added for live cell staining.

[0054] (6) After incubation for 3 hours, the culture medium was discarded, 100% DMSO (100 μl / well) was added, and it was shaken on a shaker to dissolve it completely.

[0055] (7) The OD values of each group were measured at a wavelength of 490 nm. Cell survival rate (%) = OD 药物组 / OD 正常对照组 × 100%.

[0056] III. Experimental Results

[0057] As shown in Table 2, 5α,8α-epoxysterol compounds showed certain in vitro anti-inflammatory activities. The positive control was indomethacin (40 μM).

[0058] Table 2 Inhibitory effect of 5α,8α-epoxysterol compounds (20 μM) on NO production in LPS-induced BV2 cells

[0059]

[0060]

[0061] Table 3 shows the cytotoxicity results of Compounds 1-5 on BV2 cells.

[0062] Table 3 Cytotoxic effect of 5α,8α-epoxysterol compounds (20 μM) on BV2 cells

[0063]

[0064] It can be seen from the above data results that 5α,8α-epoxysterol compounds, especially Compounds 1, 2, 3, 4, and 5, have certain anti-inflammatory activities, especially neuroprotective anti-inflammatory activities; and when BV2 cells were treated with Compounds 1, 2, 3, 4, and 5 for 24 hours, the cell survival rate was greater than 85%.

[0065] In summary, 5α,8α-epoxysterol compounds have the potential to be developed into new anti-inflammatory drugs and are worthy of further in-depth research to develop this compound into an anti-inflammatory drug or a lead compound.

[0066] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention, and all of them should be included in the protection scope of the present invention.

Claims

1. A 5α,8α-epoxysterol compound, or a stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the 5α,8α-epoxysterol compound is as follows: Wherein, represents a single bond or a double bond; R1 is selected from R 1a , R 2a , R 3a are each independently selected from hydrogen, C1-6 alkyl; R2 is selected from hydroxy, C1-6 alkoxy.

2. The 5α,8α-peroxysterol compound according to claim 1, or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt, characterized in that, The structural formula of the 5α,8α-epoxysterol compound is as shown in Formula I-1 or Formula I-2:

3. The 5α,8α-peroxysterol compound according to claim 1, or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt, characterized in that, R1 is selected from representing a single bond or a double bond.

4. The 5α,8α-peroxysterol compound according to claim 1, or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt, characterized in that, R1 is selected from 5. The 5α,8α-peroxysterol compound according to claim 1, or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt, characterized in that, The 5α,8α-epoxysterol compound is selected from the following structures:

6. A method for preparing a 5α,8α-epoxysterol compound according to any one of claims 1-5, characterized in that, Comprising the following steps: Step 1): Cut the Sarcophyton glaucum into pieces, soak it with acetone, extract, concentrate under reduced pressure to obtain a crude acetone extract, extract with ether, and concentrate under reduced pressure to obtain an ether extract; Step 2): Subject the ether extract to silica gel column chromatography, gel column chromatography separation, and high performance liquid chromatography separation and purification to obtain the 5α,8α-epoxysterol compound.

7. The method for preparing a 5α,8α-peroxysterol compound according to claim 6, characterized in that, The silica gel column chromatography is eluted with a mixed solvent of petroleum ether and ethyl acetate, and the gel column chromatography is eluted with a mixed solvent of petroleum ether, dichloromethane and methanol.

8. A pharmaceutical preparation comprising the 5α,8α-epoxysterol compound or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt according to any one of claims 1-5.

9. Use of the 5α,8α-epoxysterol compound or its stereoisomer, tautomer, geometric isomer, deuterated compound, pharmaceutically acceptable salt according to any one of claims 1-5 in the preparation of an anti-inflammatory drug.

10. The application according to claim 9, characterized in that, The anti-inflammatory drug is an anti-neuroinflammatory drug.

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