A steroidal compound and its use in the treatment of neurodegenerative diseases

Abstract

The present application relates to a kind of steroid compound and its application in treating neurodegenerative diseases, the steroid compound has the structure shown in formula I: The steroid compound of structure I is isolated from the whole grass of Solanum nigrum by yeast fermentation for the first time, it shows better neurotrophic factor (NGF) activity and acetylcholinesterase inhibitory activity, is expected to be developed as the drug or functional food of neurotrophic and neuroprotective function, for treating neurodegenerative diseases (such as senile dementia).

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a steroid compound and its application in the treatment of neurodegenerative diseases. Background Technology

[0002] Black nightshade is a commonly used traditional Chinese medicine for clearing heat and detoxifying. The Compendium of Materia Medica records: "Black nightshade is bitter, cold, slightly glycoside-containing, slippery, non-toxic, and found everywhere." Modern research shows that black nightshade possesses various pharmacological activities, including anti-tumor, anti-inflammatory, antibacterial, antiviral, antipyretic, and analgesic effects. This invention, through yeast fermentation, isolates a novel steroidal compound from black nightshade for the first time. This compound exhibits good nerve growth factor (NGF)-like activity and acetylcholinesterase inhibitory activity, and holds promise for development as a drug or functional food with neurotrophic and neuroprotective functions for the treatment of neurodegenerative diseases (such as Alzheimer's disease). Summary of the Invention

[0003] This invention provides a steroid compound, its stereoisomer, its tautomer, or a pharmaceutically acceptable salt thereof, characterized in that the steroid compound has the structure shown in Formula I:

[0004]

[0005] Another embodiment of the present invention provides a method for preparing a steroid compound with the structure of Formula I above, characterized by comprising the following steps:

[0006] (1) After the dried black nightshade whole herb is crushed and sterilized, it is mixed with dry yeast and placed in a sterile fermentation tank. Sterile water is added, the mixture is stirred evenly, sealed, and fermented at 35-37℃ for 24-48 hours to obtain the fermented product.

[0007] (2) After sterilizing the fermentation product obtained in step (1), filter it, collect the filtrate, extract it 1-3 times with an equal volume of dichloromethane, and then combine the dichloromethane phases and concentrate it to obtain an extract.

[0008] (3) The extract obtained in step (2) was separated by normal phase silica gel column chromatography, Sephadex LH-20 gel column chromatography and high performance liquid chromatography to obtain the steroidal compound with formula I.

[0009] In step (1), the amount of dry yeast used is 2.0%-2.5% of the weight of the whole black nightshade herb, and the amount of sterile water used is 3.0-5.0 times the weight of the whole black nightshade herb; in step (3), the stationary phase in the normal phase silica gel column chromatography is 200-300 mesh silica gel, the mobile phase is 10%-15% ethyl acetate / petroleum ether mixed solvent, and the elution volume is 4-5 column volumes; the eluent used in the Sephadex LH-20 gel column chromatography is CH2Cl2:MeOH = 1:1, and the elution volume is 3 column volumes; the chromatographic column used in the high performance liquid chromatography is a semi-preparative C 18 The chromatographic column was Kromasil, 7 μm, 10 × 250 mm, and the flow rate was 2 mL / min. The mobile phase was an 80% methanol / water mixture. All percentages of the mixed solvent mentioned above are volume percentages.

[0010] Another embodiment of the present invention provides a pharmaceutical composition for the prevention and / or treatment of neurodegenerative diseases, characterized in that the pharmaceutical composition uses a steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof of the structure of Formula I as the active ingredient.

[0011] Another embodiment of the present invention provides a pharmaceutical composition for the prevention and / or treatment of neurodegenerative diseases, characterized in that the pharmaceutical composition comprises a steroidal compound, stereoisomer, tautomer, or a pharmaceutically acceptable salt thereof of the structure of Formula I as the active ingredient; the pharmaceutical composition further comprises other ingredients having NGF-like activity and / or other acetylcholinesterase inhibitors. It further comprises pharmaceutical excipients (e.g., pharmaceutically acceptable carriers, excipients, diluents).

[0012] Another embodiment of the present invention provides the use of steroidal compounds, stereoisomers, tautomers or pharmaceutically acceptable salts of the above-described Formula I structure in the preparation of medicaments for the prevention and / or treatment of neurodegenerative diseases.

[0013] Another embodiment of the present invention provides the use of steroidal compounds, stereoisomers, tautomers or pharmaceutically acceptable salts of the above-described Formula I structure in the preparation of medicaments that promote nerve growth and / or provide neuroprotection.

[0014] Another embodiment of the present invention provides the use of steroidal compounds, stereoisomers, tautomers or pharmaceutically acceptable salts thereof of the above-described Formula I structure in the preparation of NGF-like active pharmaceutical ingredients.

[0015] Another embodiment of the present invention provides the use of steroidal compounds, stereoisomers, tautomers or pharmaceutically acceptable salts thereof of the structure of Formula I in the preparation of acetylcholinesterase inhibitors.

[0016] In this invention, the term "pharmaceutically acceptable salt" refers to the addition salt of a nontoxic inorganic or organic acid and / or base, see "Salt selection for basic drugs", Int. J. Pharm. (1986), 33, 201–217.

[0017] The dry yeast used in this invention is preferably a highly active dry yeast (e.g., Angel Yeast highly active dry yeast). The neurodegenerative disease used in this invention is preferably Alzheimer's disease.

[0018] The advantages of this invention are: for the first time, a steroidal compound of formula I was isolated from the whole plant of Solanum nigrum using yeast fermentation. This compound exhibits good nerve growth factor (NGF) activity and acetylcholinesterase inhibitory activity, and is expected to be developed as a drug with neurotrophic and neuroprotective functions for the treatment of neurodegenerative diseases (such as Alzheimer's disease). Attached Figure Description

[0019] Figure 1 This is a graph showing the neurite differentiation rate of PC12 cells 48 hours after the addition of the test compound. C represents the negative control 0.5% DMSO, NGF represents the positive control 40 ng / mL NGF, I-100 represents 100 μg / mL of compound I, and I-200 represents 200 μg / mL of compound I.

[0020] Figure 2 It is a compound of formula I 1 ¹H NMR (600MHz, CDCl₃) image.

[0021] Figure 3 It is a compound of formula I 1 Magnified H NMR (600MHz, CDCl3) image.

[0022] Figure 4 It is a compound of formula I 1 Magnified H NMR (600MHz, CDCl3) image.

[0023] Figure 5 It is a compound of formula I 13 C NMR (150MHz, CDCl3) plot.

[0024] Figure 6 It is a compound of formula I 13 Magnified C NMR (150MHz, CDCl3) image.

[0025] Figure 7 It is a compound of formula I 13 Magnified C NMR (150MHz, CDCl3) image. Detailed Implementation

[0026] To facilitate a further understanding of the present invention, the following embodiments are provided for more detailed description. However, these embodiments are only for a better understanding of the invention and are not intended to limit the scope or implementation principles of the invention. The implementation of the present invention is not limited to the following. The dry yeast used in the embodiments of the present invention is commercially available Angel high-activity dry yeast.

[0027] Example 1

[0028] (1) Take dried black nightshade whole herb (1.0kg), crush and sterilize it, mix it with dry yeast (20g, Angel high-activity dry yeast) and place it in a sterile fermentation tank, add sterile water (3.0kg), stir evenly, seal, and ferment at 35℃ for 48h to obtain fermented product.

[0029] (2) After sterilizing the fermentation product obtained in step (1), filter it, collect the filtrate, extract it twice with an equal volume of dichloromethane, combine the dichloromethane phases and concentrate it to obtain an extract.

[0030] (3) The extract obtained in step (2) is first separated by normal-phase silica gel column chromatography. The stationary phase is 200-300 mesh silica gel, and the mobile phase is a 15% ethyl acetate / petroleum ether mixed solvent. After elution for 4 column volumes, the eluent is concentrated and then separated by Sephadex LH-20 gel column chromatography. The eluent is CH2Cl2:MeOH = 1:1. After elution for 3 column volumes, the eluent is concentrated and then separated by high-performance liquid chromatography. The stationary phase is semi-preparative C 18 A Kromasil column (7 μm, 10 × 250 mm) was used at a flow rate of 2 mL / min with an 80% methanol / water mixture as the mobile phase; all solvent percentages are volume percentages. The resulting steroid compound of formula I (16.8 mg, colorless crystals) was obtained. Structural confirmation data:

[0031]

[0032] Formula I: HRESIMS m / z 425.3021[M+H] + (calcd forC 28 H 41 O3, 425.3050). 1 H NMR (600MHz, CDCl3)δ H5.74 (1H, s, H-4), 5.72 (1H, s, H-7), 5.25 (1H, dd, J = 18.0, 6.4 Hz, H-22), 5.14 (1H, dd, J = 18.0, 6.2 Hz, H-23), 2.57 - 2.50 (2H, m, H-2a, H-9), 2.48 - 2.42 (1H, m, H-25), 2.22 - 2.16 (1H, m, H-1a), 2.15 - 2.08 (2H, m, H-12a, H-14), 2.06 - 1.95 (2H, m, H-1b, H-20), 1.87 - 1.76 (3H, m, H-15, H-24), 1.75 - 1.66 (1H, m, H-16a), 1.58 - 1.50 (2H, m, H-11), 1.48 - 1.42 (2H, m, H-2b, H-12b), 1.40 - 1.32 (2H, m, H-16b, H-17), 1.23 (3H, s, H-19), 1.02 (3H, d, J = 6.6 Hz, H-21), 0.91 (3H, d, J = 6.5 Hz, H-28), 0.83 (3H, d, J = 6.5 Hz, H-27), 0.82 (3H, d, J = 6.5 Hz, H-26), 0.63 (3H, s, H-18); 13 13C NMR (150 MHz, CDCl3) δ C 198.4 (C, C-3), 173.8 (C, C-5), 162.4 (C, C-6), 159.4 (C, C-8), 134.7 (CH, C-22), 132.8 (CH, C-23), 114.6 (CH, C-4), 113.3 (CH, C-7), 58.1 (CH, C-14), 56.3 (CH, C-17), 47.2 (CH, C-9), 47.0 (C, C-13), 42.8 (CH, C-24), 40.3 (C, C-10), 40.2 (CH, C-20), 39.1 (CH2, C-12), 33.9 (CH2, C-1), 33.1 (CH2, C-2), 33.0 (CH, C-25), 27.7 (CH2, C-16), 25.4 (CH2, C-15), 22.6 (CH2, C-11), 21.0 (CH3, C-21), 20.0 (CH3, C-19), 20.0 (CH3, C-26), 19.6 (CH3, C-27), 17.6 (CH3, C-28), 12.4 (CH3, C-18).

[0033] Example 2

[0034] (1) Take the whole plant of Solanum nigrum (1.0kg), crush and sterilize it, mix it with dry yeast (25g, Angel high-activity dry yeast) and place it in a sterile fermentation tank, add sterile water (5.0kg), stir evenly, seal, and ferment at 37℃ for 24h to obtain fermented product.

[0035] (2) After sterilizing the fermentation product obtained in step (1), filter it, collect the filtrate, extract it three times with an equal volume of dichloromethane, and then combine the dichloromethane to concentrate it into an extract.

[0036] (3) The extract obtained in step (2) is first separated by normal-phase silica gel column chromatography. The stationary phase is 200-300 mesh silica gel, and the mobile phase is 10% ethyl acetate / petroleum ether mixed solvent. After elution for 5 column volumes, the eluent is concentrated and then separated by Sephadex LH-20 gel column chromatography. The eluent is CH2Cl2:MeOH = 1:1. After elution for 3 column volumes, the eluent is concentrated and then separated by high-performance liquid chromatography. The stationary phase is semi-preparative C 18 A Kromasil column (7 μm, 10 × 250 mm) was used at a flow rate of 2 mL / min with an 80% methanol / water mixture as the mobile phase; all percentages of the mixed solvent are volume percentages. The resulting steroid compound of formula I (13.2 mg, colorless crystals) was obtained. The structural confirmation data were consistent with those of Example 1.

[0037] Example 3: Assay of NGF-like activity of the steroid compound of formula I in PC12 cells, an in vitro screening model of Alzheimer's disease.

[0038] Preparation of CM medium: Add 25 mL of fetal bovine serum, 50 mL of horse serum and 1% of penicillin and streptomycin to 425 mL of DMEM high glucose medium (containing sodium pyruvate), gently shake to mix, and store at 4°C for later use.

[0039] Preparation of PBS: Dissolve 4.9g NaCl, 0.1g KCl, 0.575g Na2HPO4 and 0.01g KH2PO4 in 500mL miliQ ultrapure water, mix well and sterilize, then refrigerate at 4℃ until use.

[0040] PC12 cell resuscitation: First, preheat CM medium in a 37°C water bath. Quickly remove PC12 cells frozen in liquid nitrogen and thaw them in a 37°C water bath until ice crystals appear, approximately 1-2 minutes. Then, rapidly transfer the cell suspension from the cryopreservation tube completely to a 15mL centrifuge tube containing 4mL of CM medium. Mix thoroughly by pipetting and centrifuge (800 rpm, 3 minutes). Remove the supernatant, add another 5mL of CM medium, and repeat the process once. Leave the cell pellet, add 1mL of CM medium to the centrifuge tube, mix thoroughly by pipetting, and count cells using a hemocytometer. Count the cells at a concentration of 2×10⁶ cells. 5 One cell was seeded in a 100 mm cell culture dish containing 10 mL of CM medium and placed in an incubator at 37°C with 5% CO2. The CM medium was replaced after 24 h and 48 h, and the cells were subcultured after 72 h.

[0041] PC12 cell passage procedure: First, preheat the required CM medium and PBS solution in a 37°C water bath for 30 min. Remove the culture dish from the CO2 incubator, remove the CM medium with a pipette, and then wash the culture dish twice with 10 mL of PBS solution to remove any remaining medium. Add another 10 mL of PBS and incubate for 10 min. After resuspending the cells by pipetting, transfer the suspension to a 15 mL centrifuge tube and centrifuge (800 rpm, 3 min). Add 1 mL of CM medium, mix well by pipetting, and count the cells using a hemocytometer. In a 24-well plate, add 1 mL of CM medium and 2 × 10⁶ cells to each well. 4 Cells were collected and bioactivity was tested after 24 hours.

[0042] Bioactivity assay: First, the sample to be tested (Formula I) was prepared into solutions of different concentrations (100 μg / mL, 200 μg / mL) using DMSO. During the test, 0.5% DMSO was used as a negative control, and 40 ng / mL NGF was used as a positive control. Specifically, 1 mL of DMEM solution containing 0.5% DMSO and the sample was used to replace the original culture medium in each well of a 24-well plate, and the plate was placed in a CO2 incubator. Every 24 hours, for six consecutive days, the morphological changes of the cells were observed under an inverted microscope, and the neurite differentiation rate (magnification: ×200) was recorded. The neurite differentiation rate (%) was equal to the ratio of the number of cells with a neurite length greater than twice the cell body length to the total number of surviving cells in a field of view, multiplied by 100%. For counting, approximately 100 cells were required per field of view, and three random locations were selected for counting. The results were statistically analyzed and plotted. Figure 1The results showed that the compound of formula I exhibited extremely strong NGF-like activity at concentrations of 100 μg / mL (I-100) and 200 μg / mL (I-200), especially at a concentration of 200 μg / mL, where its neurite differentiation rate reached 76.8%, which was superior to the activity of 40 ng / mL NGF (70.5%). Therefore, the compound of formula I of this invention showed strong NGF-like activity in the in vitro screening model of Alzheimer's disease, PC12 cells, and holds promise for development into a drug for the treatment of Alzheimer's disease and other neurodegenerative diseases.

[0043] Example 4: Assay for the acetylcholinesterase inhibitory activity of Formula I steroidal compounds (Ellman assay)

[0044] The compound of formula I was dissolved in DMSO to prepare a 10 mM solution, which was then diluted to a 1 mM solution. The steroid compound of formula I was prepared to a 1 mmol / mL solution, which was then diluted to a 50 μmol / mL test solution. Acetylcholinesterase was diluted with 0.1 M phosphate buffer (PB, pH 8.0) to a 0.1 U / mL solution for later use. ATCI and DTNB were prepared into 6.25 mM working solutions using the above-mentioned PB solution.

[0045] The enzyme reaction was performed in 96-well plates. The negative control group was treated with 110 μL of PB, 10 μL of 10% DMSO, and 40 μL of acetylcholinesterase working solution. The positive control group was treated with 110 μL of PB, 10 μL of tacrine (6.66 μM), and 40 μL of acetylcholinesterase working solution. The drug group was treated with 110 μL of PB, 10 μL of a type I steroid compound (50 μM), and 40 μL of acetylcholinesterase working solution. The reactions were incubated at 30 °C for 20 min. Background values ​​were read three times at 412 nm using a microplate reader. Then, 40 μL of a 1:1 mixture of ATCI and DTNB was added to begin the assay, and the absorbance at 412 nm was measured every 30 seconds over 1 hour. Samples with absorbance values ​​approximately 1 from the negative control group were selected, and the acetylcholinesterase inhibition rate (I%) was calculated using the following formula.

[0046] I%=[(A0-A1)-(A-A1)] / (A0-A1)×100%

[0047] Where A0 is the absorbance value of the negative control group, A1 is the background value, and A is the absorbance value of the drug group.

[0048] When the inhibition rate is greater than 50%, the IC is calculated. 50 The specific method involves preparing a series of gradient concentrations of the test compound (Formula I, positive control), detecting them according to the above method, calculating the inhibition rate I, and plotting the IC50. 50 value.

[0049] The inhibitory activity of the steroid compound of formula I of this invention against acetylcholinesterase is IC 50 The concentration was 8.64 ± 0.14 μM; the IC50 of the positive control drug tacrine was 8.64 ± 0.14 μM. 50 It is 0.28±0.03μM.

Claims

1. Use of a steroidal compound of Formula I or a pharmaceutically acceptable salt thereof in the preparation of an acetylcholinesterase inhibitor, the structure of which is shown below: 。 2. The use according to claim 1, characterized in that... The acetylcholinesterase inhibitors are used for the prevention and / or treatment of neurodegenerative diseases.

3. The use according to claim 2, characterized in that... The neurodegenerative disease mentioned is selected from Alzheimer's disease.

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