Pharmaceutical composition for preventing and / or treating neurodegenerative diseases

Abstract

The invention belongs to the field of medicines, and particularly relates to a pharmaceutical composition for preventing and / or treating neurodegenerative diseases, the pharmaceutical composition takes a steroid compound with a structure as shown in a formula I, a stereoisomer, a tautomer or pharmaceutically acceptable salt thereof as an active ingredient; the structure of the formula I is shown in the specification.

Description

[0001] According to the non-unitary examination opinion with document number 2025071001508890 issued by the State Intellectual Property Office, the applicant files the present divisional application for Chinese Patent Application No. 202211646818.1, the original application date of which is December 21, 2022, and the original application title of which is a steroidal compound and its application in treating neurodegenerative diseases. TECHNICAL FIELD

[0002] The present application belongs to the field of medicine, and specifically relates to a pharmaceutical composition for preventing and / or treating neurodegenerative diseases. BACKGROUND

[0003] Solanum nigrum is a commonly used heat-clearing and detoxifying traditional Chinese medicine. The Compendium of Materia Medica records that Solanum nigrum is bitter, cold, slightly sweet, slippery, and non-toxic, and is everywhere. Modern research shows that Solanum nigrum has various pharmacological activities such as anti-tumor, anti-inflammatory, antibacterial, anti-viral, and antipyretic analgesic. A new steroidal compound is isolated from Solanum nigrum for the first time by yeast fermentation, which shows good nerve growth factor (NGF) activity and acetylcholinesterase inhibition activity, and is expected to be developed as a drug or functional food for neurotrophic and neuroprotective functions, and used for treating neurodegenerative diseases (such as Alzheimer's disease). SUMMARY

[0004] The present application provides a steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof, characterized in that the steroidal compound has the structure shown in formula I:

[0005]

[0006] Another embodiment of the present application provides a preparation method of the steroidal compound with the above structure of formula I, characterized in that it comprises the following steps:

[0007] (1) After the dried Solanum nigrum whole plant is crushed and sterilized, it is mixed with dry yeast and placed in a sterile fermentation tank, sterile water is added, and after stirring uniformly, it is sealed and fermented at 35-37℃ for 24-48h to obtain a fermentation product;

[0008] (2) The fermentation product obtained in step (1) is sterilized, filtered, and the filtrate is collected, extracted with an equal volume of dichloromethane for 1-3 times, and then the dichloromethane phase is concentrated to obtain an extract;

[0009] (3) The extract obtained in step (2) is sequentially subjected to normal phase silica gel column chromatography, Sephadex LH-20 gel column chromatography, and high performance liquid chromatography to obtain the steroidal compound with the structure of formula I.

[0010] The amount of dry yeast in step (1) is 2.0%-2.5% of the mass of the whole plant of Solanum nigrum L., and the amount of sterile water is 3.0-5.0 times the mass of the whole plant of Solanum nigrum L.; in step (3), the stationary phase in the normal-phase silica gel column chromatography separation is 200-300 mesh silica gel, the mobile phase is a 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 separation is CH2Cl2:MeOH=1:1, and the elution volume is 3 column volumes; and the high-performance liquid chromatography separation uses a semi-preparative C 18 The chromatographic column is Kromasil, 7 μm, 10×250 mm, the flow rate is 2 mL / min, and 80% methanol / water mixed solvent is used as the mobile phase; the percentages in the above mixed solvents are all volume percentages.

[0011] Another embodiment of the present application provides a pharmaceutical composition for preventing and / or treating neurodegenerative diseases, characterized in that the pharmaceutical composition comprises the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I as an effective ingredient.

[0012] Another embodiment of the present application provides a pharmaceutical composition for preventing and / or treating neurodegenerative diseases, characterized in that the pharmaceutical composition comprises the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I as an effective ingredient; the pharmaceutical composition further comprises other ingredients having pseudo-NGF activity and / or other acetylcholinesterase inhibitors. Further, the pharmaceutical composition further comprises pharmaceutical adjuvants (such as pharmaceutically acceptable carriers, excipients, diluents).

[0013] Another embodiment of the present application provides the use of the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I in the preparation of a medicament for preventing and / or treating neurodegenerative diseases.

[0014] Another embodiment of the present application provides the use of the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I in the preparation of a medicament or functional food for promoting nerve growth and / or neuroprotection.

[0015] Another embodiment of the present application provides the use of the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I in the preparation of a pseudo-NGF active medicament.

[0016] Another embodiment of the present application provides the use of the steroidal compound, stereoisomer, tautomer or pharmaceutically acceptable salt thereof having the structure of formula I in the preparation of an acetylcholinesterase inhibitor.

[0017] 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.

[0018] 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.

[0019] 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 or functional food with neurotrophic and neuroprotective functions for the treatment of neurodegenerative diseases (such as Alzheimer's disease). Attached Figure Description

[0020] 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.

[0021] Figure 2 It is a compound of formula I 1 ¹H NMR (600MHz, CDCl₃) image.

[0022] Figure 3 It is a compound of formula I 1 Magnified H NMR (600MHz, CDCl3) image.

[0023] Figure 4 It is a compound of formula I 1 Magnified H NMR (600MHz, CDCl3) image.

[0024] Figure 5 It is a compound of formula I 13 C NMR (150MHz, CDCl3) plot.

[0025] Figure 6 It is a compound of formula I 13 Magnified C NMR (150MHz, CDCl3) image.

[0026] Figure 7 It is a compound of formula I 13 Magnified C NMR (150MHz, CDCl3) image. Detailed Implementation

[0027] In order to facilitate further understanding of the present application, the following examples are provided to make a more detailed description of the present application. However, these examples are only for better understanding of the present application and not intended to limit the scope or principles of the present application, and the embodiments of the present application are not limited to the following. The dry yeast used in the examples of the present application is commercially purchased Angel high-activity dry yeast.

[0028] Example 1

[0029] (1) After the dried Solanum nigrum L. whole plant (1.0 kg) was ground and sterilized, it was mixed with dry yeast (20 g, Angel high-activity dry yeast) and placed in a sterile fermentation tank. Sterile water (3.0 kg) was added, and after stirring uniformly, it was sealed and fermented at 35°C for 48 h to obtain a fermentation product;

[0030] (2) The fermentation product obtained in step (1) was sterilized, filtered, and the filtrate was extracted twice with an equal volume of dichloromethane, and then the dichloromethane phase was concentrated to obtain an extract;

[0031] (3) The extract obtained in step (2) was first subjected to normal phase silica gel column chromatography separation, the stationary phase was 200-300 mesh silica gel, and the mobile phase was a mixture of 15% ethyl acetate / petroleum ether, eluted for 4 column volumes. The eluate was concentrated and then subjected to Sephadex LH-20 gel column chromatography separation, the eluent was CH2Cl2:MeOH = 1:1, eluted for 3 column volumes. The eluate was concentrated and then subjected to high performance liquid chromatography separation, the stationary phase was semi-preparative C 18 chromatography column (Kromasil, 7 μm, 10 x 250 mm), the flow rate was 2 mL / min, and 80% methanol / water mixture was used as the mobile phase; the above-mentioned percentage is volume percentage, to obtain the steroidal compound of formula I (16.8 mg, colorless crystal). The structure confirmation data:

[0032]

[0033] Formula I: +2.3 (c, 0.15, MeOH), HRESIMS m / z 425.3021 [M+H] + (calcd for C 28 H 41 N3O3, 425.3050). 1 H NMR (600 MHz, 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-la), 2.15-2.08 (2H, m, H-12a, H-14), 2.06-1.95 (2H, m, H-lb, 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-l l), 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 C NMR (150 MHz, CDC13) 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-l), 33.1 (CH2, C-2), 33.0 (CH, C-25), 27.7 (CH2, C-16), 25.4 (CH2, C-15), 22.6 (CH2, C-l l), 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).

[0034] Example 2

[0035] (1) After the whole plant of Solanum nigrum L. (1.0 kg) was crushed and sterilized, it was mixed with dry yeast (25 g, Angel high-activity dry yeast) and placed in a sterile fermentation tank. Sterile water (5.0 kg) was added, and the mixture was stirred uniformly, sealed, and fermented at 37 °C for 24 h to obtain a fermentation product;

[0036] (2) The fermentation product obtained in step (1) was sterilized, filtered, and the filtrate was extracted with an equal volume of dichloromethane three times. The dichloromethane was combined and concentrated to obtain an extract;

[0037] (3) The extract obtained in step (2) was first subjected to normal-phase silica gel column chromatography separation, with 200-300 mesh silica gel as the stationary phase and 10% ethyl acetate / petroleum ether mixed solvent as the mobile phase. Five column volumes were eluted, and the eluate was concentrated and then subjected to Sephadex LH-20 gel column chromatography separation with CH2Cl2:MeOH = 1:1 as the eluent. Three column volumes were eluted, and the eluate was concentrated and then subjected to high-performance liquid chromatography separation with semi-preparation C 18 chromatography column (Kromasil, 7 μm, 10 x 250 mm) at a flow rate of 2 mL / min, with 80% methanol / water mixed solvent as the mobile phase; the above-mentioned percentage is the volume percentage, to obtain the steroidal compound of formula I (13.2 mg, colorless crystal). The structure confirmation data are consistent with those of Example 1.

[0038] Example 3 In vitro screening model PC12 cell test of the steroidal compound of formula I of the present application for NGF-like activity in senile dementia

[0039] Preparation of CM medium: 25 mL of fetal bovine serum, 50 mL of horse serum, and 1% of double-antibiotic (10000 U / mL of penicillin and 10000 μg / mL of streptomycin) were added to 425 mL of DMEM high-sugar medium (containing sodium pyruvate), and the mixture was shaken uniformly at 4 °C in a refrigerator for standby use.

[0040] Preparation of PBS: 4.9 g of NaCl, 0.1 g of KCl, 0.575 g of Na2HPO4, and 0.01 g of KH2PO4 were dissolved in 500 mL of miliQ ultrapure water, mixed uniformly, sterilized, and stored in a refrigerator at 4 °C for standby use.

[0041] 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.

[0042] 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.

[0043] 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 treating Alzheimer's disease and other neurodegenerative diseases, or as a functional food for neurotrophic purposes.

[0044] Example 4: Assay for the acetylcholinesterase inhibitory activity of Formula I steroidal compounds (Ellman assay)

[0045] 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.

[0046] 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.

[0047] I%=[(A0-A1)-(A-A1)] / (A0-A1)×100%

[0048] 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.

[0049] 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.

[0050] 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. 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 Formula I as the active ingredient. The structure of Formula I is as follows:

2. The pharmaceutical composition according to claim 1, characterized in that... The pharmaceutical composition also includes other components with NGF-like activity and / or other acetylcholinesterase inhibitors.

3. The pharmaceutical composition according to any one of claims 1-2, characterized in that... This also includes pharmaceutical excipients (such as pharmaceutically acceptable carriers, excipients, and diluents).

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