Use of an amide compound for the manufacture of a medicament for the treatment of Alzheimer's disease

Abstract

The application discloses application of an amide compound in preparation of a drug for treating Alzheimer's disease, belongs to the technical field of medicines, and the amide compound is N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-formamide. The application adopts the application of the amide compound in preparation of the drug for treating Alzheimer's disease, and N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-formamide realizes precise intervention on diseases through regulation of cholinergic system functional defects and inhibition of oxidative stress neuron damage, improves the treatment effect through a synergistic effect, and provides a new idea for treatment of Alzheimer's disease.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the application of an amide compound in the preparation of drugs for treating Alzheimer's disease. Background Technology

[0002] Alzheimer's disease (AD), a neurodegenerative disease that seriously threatens the cognitive function of the elderly, faces key challenges in its treatment due to its complex pathological mechanisms and the limited efficacy of single-target drugs. On the one hand, cholinergic system dysfunction is one of the core pathological features of AD. Acetylcholine (ACh), an important neurotransmitter in the brain, depends on the dynamic balance between acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The abnormal changes in AD, such as the gradual decline in AChE activity and the compensatory increase in BChE, lead to an imbalance in ACh catabolism, which in turn exacerbates cognitive decline. Traditional single-target cholinesterase inhibitors, because they only target the activity of a single enzyme, cannot adapt to this dynamic change, making it difficult to achieve comprehensive regulation of the cholinergic system and prone to side effects. On the other hand, oxidative stress-induced neuronal damage is an important driving factor in the progression of AD. Excessive reactive oxygen species (such as H2O2) can cause oxidative modification of biomolecules such as proteins, DNA, and lipids in brain tissue, inducing mitochondrial dysfunction and ultimately leading to neuronal apoptosis, further aggravating the disease process.

[0003] Therefore, developing a novel therapeutic drug that can simultaneously target the two core pathological aspects of cholinergic system dysfunction and oxidative stress damage, and achieve precise intervention in Alzheimer's disease through synergistic effects, has become a key direction for breaking through existing treatment bottlenecks, improving efficacy, and reducing side effects. Summary of the Invention

[0004] The purpose of this invention is to provide an application of amide compounds in the preparation of drugs for treating Alzheimer's disease. N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide achieves precise multi-target intervention of the disease by regulating cholinergic system dysfunction and inhibiting oxidative stress neuronal damage, and enhances the therapeutic effect through synergistic effects, providing a new approach for the treatment of Alzheimer's disease.

[0005] To achieve the above objectives, this invention provides the application of an amide compound in the preparation of a drug for treating Alzheimer's disease. The amide compound is N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide, PubChem CID 39965569, and its structural formula is:

[0006] .

[0007] Preferably, Alzheimer's disease is a neurodegenerative disease caused by cholinergic system dysfunction and oxidative stress-induced neuronal damage.

[0008] Furthermore, cholinergic system dysfunction manifests as abnormal dynamic changes in the activities of acetylcholinesterase and butyrylcholinesterase.

[0009] Preferably, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide treats Alzheimer's disease by modulating cholinergic system dysfunction and inhibiting oxidative stress-induced neuronal damage.

[0010] Furthermore, the way to regulate the dysfunction of the cholinergic system is by inhibiting the dual activity of acetylcholinesterase and butyrylcholinesterase.

[0011] The present invention also provides a drug for treating Alzheimer's disease, comprising N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide and a drug carrier, wherein the drug carrier may be any known to those skilled in the art.

[0012] Therefore, the application of the above-mentioned amide compound in the preparation of drugs for treating Alzheimer's disease has the following beneficial effects:

[0013] (1) In this invention, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide achieves precise multi-target intervention of the disease by regulating the functional defects of the cholinergic system and inhibiting oxidative stress neuronal damage. It enhances the therapeutic effect through synergistic effect and provides a new idea for the treatment of Alzheimer's disease.

[0014] (2) In this invention, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide achieves precise regulation of the cholinergic system by dually inhibiting the activities of AChE and BChE. The amide compound exhibits concentration-dependent enhancement in the inhibition of AChE and BChE activities, which can adapt to the abnormal dynamic changes of the two enzymes during the course of the disease, reduce the excessive degradation of ACh from the source, maintain neurotransmitter balance, and thus improve cognitive function decline. This overcomes the shortcomings of traditional single-target inhibitors, which cannot comprehensively regulate the cholinergic system and are prone to side effects.

[0015] (3) In this invention, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide exerts a neuroprotective effect by inhibiting oxidative stress-induced neuronal apoptosis. This amide compound, through dose-dependent intervention, can significantly improve the survival rate of damaged cells and effectively inhibit cell apoptosis. By reducing the damage of oxidative stress to neurons, it stabilizes cell structure and function, thereby delaying the loss of neurons in the disease process.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 This invention relates to the effect of amide compounds on the survival rate of H2O2-damaged SK-N-SH cells;

[0018] Figure 2 This is a fluorescence staining microscope image of SK-N-SH cells damaged by H2O2 after intervention with the amide compounds of this invention;

[0019] in, Figure 2 In the image, 'a' is a fluorescence staining micrograph of SK-N-SH cells from the control group. Figure 2 In the image, b is a fluorescence staining micrograph of SK-N-SH cells from the model group. Figure 2 c in the image is a fluorescence staining micrograph of SK-N-SH cells in the amide compound group;

[0020] Figure 3 This invention relates to the effect of amide compounds on the apoptosis rate of H2O2-damaged SK-N-SH cells;

[0021] Figure 4 This invention relates to the concentration-dependent inhibitory effect of amide compounds on AChE and BChE. Detailed Implementation

[0022] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0023] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0024] In this invention, unless otherwise specified, the test materials and instruments are all conventional test materials in the field and can be purchased through commercial channels.

[0025] Example 1

[0026] Neuroblastoma cells (SK-N-SH cells) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 U / mL streptomycin. The SK-N-SH cells were incubated in a 37°C incubator containing 5% CO2. When the cells reached 80% confluence, they were passaged and seeded into culture plates.

[0027] Cell viability was determined using the MTT assay (microenzyme reaction colorimetric method with tetramethylazoazole salts).

[0028] After passage, the dispersed cells were divided at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL into 96-well plates, with a volume of 200 μL per well. After 8 hours, the medium was replaced with serum-free medium and allowed to stand overnight. Subsequently, the cells were divided into a control group, a model group, and an amide compound group, with the amide compound group further divided into low-dose, medium-dose, and high-dose groups. Each group was configured with 5 replicates, and the process was repeated 3 times.

[0029] Control group: 20 μL of 5 mg / mL MTT solution was added to each well and incubated at 37°C in an incubator containing 5% CO2 (volume concentration) for 4 hours. The culture medium in the 96-well plate was removed by flipping the plate, ensuring that all residual culture medium was completely aspirated from the bottom of the plate. Then, 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well, and the plate was shaken at 37°C for 15 minutes to promote complete dissolution of crystals. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0030] Model group: H2O2 (final concentration 150 μM) was added to each well. After 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well, and the plate was incubated at 37°C in an incubator containing 5% CO2 (volume concentration) for 4 hours. The culture medium in the 96-well plate was removed by flipping the plate, ensuring that the residual culture medium at the bottom of the plate was completely aspirated. Then, 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well, and the plate was shaken at 37°C for 15 minutes to promote complete dissolution of crystals. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0031] Low-dose group: Cells were treated with N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide at a final concentration of 0.1 μM for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C in an incubator containing 5% CO2 (volume concentration) for another 4 hours. The culture medium in the 96-well plate was removed by flipping the plate, ensuring that all residual culture medium was thoroughly aspirated from the bottom of the plate. Then, 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well, and the plate was shaken at 37°C for 15 minutes to promote complete dissolution of crystals. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0032] Medium-dose group: Cells were treated with N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide at a final concentration of 1 μM for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C in an incubator containing 5% CO2 (volume concentration) for another 4 hours. The culture medium in the 96-well plate was removed by flipping the plate, ensuring that all residual culture medium was thoroughly aspirated from the bottom of the plate. Then, 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well, and the plate was shaken at 37°C for 15 minutes to promote complete dissolution of crystals. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0033] High-dose group: Cells were treated with N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide at a final concentration of 10 μM for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C in an incubator containing 5% CO2 (volume concentration) for another 4 hours. The culture medium in the 96-well plate was removed by flipping the plate, ensuring that all residual culture medium was thoroughly aspirated from the bottom of the plate. Then, 150 μL of dimethyl sulfoxide (DMSO) solution was added to each well, and the plate was shaken at 37°C for 15 minutes to promote complete dissolution of crystals. Finally, the absorbance (OD value) of each well was measured at 570 nm using a microplate reader.

[0034] Cell viability was calculated based on the absorbance values ​​of each group, and the results are as follows: Figure 1 As shown. From Figure 1 As can be seen, compared with the control group, the cell survival rate of the model group was significantly reduced to 50% after treatment with 150 μM H2O2 for 24 hours (this result is statistically significant). p<0.001); Four hours before H2O2 treatment, cells were treated with different doses of N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide, and a dose-dependent increase in cell survival was observed. Compared with the model group, the cell survival rates of the low-dose, medium-dose, and high-dose groups were increased to 61%, 73%, and 80%, respectively (this result was statistically significant, in descending order of importance). p <0.05, p <0.01, p <0.001).

[0035] Cell apoptosis rate was detected using the Hoechest 33342 fluorescence staining method.

[0036] After passage, the dispersed cells were divided at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of 1 / mL into 96-well plates, with a volume of 200 μL per well. After 8 hours, the medium was replaced with serum-free medium and allowed to stand overnight. Subsequently, the cells were divided into a control group, a model group, and an amide compound group, with the amide compound group further divided into low-dose, medium-dose, and high-dose groups. Each group was configured with 5 replicates, and the process was repeated 3 times.

[0037] Control group: Hoechest 33342 was added to each well to achieve a final intracellular concentration of 10 μg / mL, and the cells were incubated at 37°C for 30 minutes. Then, a 4% paraformaldehyde solution was mixed with the culture medium at a volume ratio of 1:3 and added to the wells to fix the cells for 10 minutes. The cells were washed twice with phosphate-buffered saline (PBS), followed by the addition of a small amount of PBS.

[0038] Model group: 150 μM H₂O₂ was added to each well. After 24 hours, Hoechst 33342 was added to each well to achieve a final intracellular concentration of 10 μg / mL. The cells were incubated at 37°C for 30 minutes. Then, a 4% (v / v) paraformaldehyde solution was mixed with the culture medium at a 1:3 (v / v) ratio and added to the wells to fix the cells for 10 minutes. The cells were washed twice with phosphate-buffered saline (PBS), followed by the addition of a small amount of PBS.

[0039] Low-dose amide compound group: Cells were treated with 0.1 μM N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, Hoechest 33342 was added to each well to achieve a final intracellular concentration of 10 μg / mL, and the cells were incubated at 37°C for 30 minutes. Then, 4% paraformaldehyde solution was mixed with culture medium at a volume ratio of 1:3 and added to the wells to fix the cells for 10 minutes. The cells were washed twice with phosphate-buffered saline (PBS), followed by the addition of a small amount of PBS.

[0040] In the amide compound dosage group: Cells were treated with N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide at a final concentration of 1 μM for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, Hoechest 33342 was added to each well to achieve a final intracellular concentration of 10 μg / mL, and the cells were incubated at 37°C for 30 minutes. Then, a 4% (v / v) paraformaldehyde solution was mixed with the culture medium at a volume ratio of 1:3 and added to the wells to fix the cells for 10 minutes. The cells were washed twice with phosphate-buffered saline (PBS), followed by the addition of a small amount of PBS.

[0041] High-dose amide compound group: Cells were treated with 10 μM N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide for 4 hours, followed by the addition of 150 μM H2O2. After 24 hours, Hoechest 33342 was added to each well to achieve a final intracellular concentration of 10 μg / mL, and the cells were incubated at 37°C for 30 minutes. Then, 4% paraformaldehyde solution was mixed with culture medium at a volume ratio of 1:3 and added to the wells to fix the cells for 10 minutes. The cells were washed twice with phosphate-buffered saline (PBS), followed by the addition of a small amount of PBS.

[0042] SK-N-SH cells from the control group, model group, and amide compound group were observed under a fluorescence microscope with a maximum excitation wavelength of 361 nm and a maximum emission wavelength of 460 nm. Normal cells exhibited uniform blue fluorescence, while apoptotic cells showed condensed crescent-shaped, lobed, or fragmented fluorescence in their nuclei. The number of positive cells was counted in three randomly selected fields of view, and the average value was calculated to determine the apoptosis rate.

[0043] Fluorescence microscopy images of SK-N-SH cells in the control group, model group, and amide compound group are shown below. Figure 2As shown, the calculated apoptosis rates for the control group, model group, and amide compound group are as follows: Figure 3 As shown. From Figure 2 and Figure 3 As can be seen from the fluorescence microscope, after ultraviolet light excitation, the control group cells showed uniform blue fluorescence, and the apoptosis rate was only 4%; while in the model group, after 24 hours of H2O2 treatment, some cells showed typical apoptotic characteristics, with cell nuclei condensed into crescent shapes or fragmented into pieces, fluorescence intensity significantly increased, and the apoptosis rate surged to 28%, which was highly significant compared with the control group. p <0.001). Further intervention with 0.1 μM, 1 μM, and 10 μM N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide resulted in apoptosis rates of 19%, 11%, and 9%, respectively, which were statistically significant compared to the model group. p <0.05, p <0.001, p <0.001). This indicates that the inhibitory effect of N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide on apoptosis is significantly dose-dependent, and its anti-apoptotic effect gradually increases with the increase of the intervention dose.

[0044] Example 2

[0045] The inhibitory effect of N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide on the activities of AChE and BChE was determined by the Ellman colorimetric method.

[0046] The experiment was conducted in a 96-well plate, with three replicates in each well, and experimental, blank, and control groups were set up.

[0047] Experimental group: First, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide was prepared into a stock solution with DMSO, and then serially diluted with PBS buffer to obtain amide compound solutions with concentrations of 3nM, 10nM, 30nM, 100nM, 300nM, 1000nM, 3000nM, and 10000nM. Add 30 μL of PBS buffer, 10 μL of amide compound solution, and 20 μL of AChE / BChE enzyme solution extracted from rat brain to the wells sequentially (for example, add 30 μL of PBS buffer, 10 μL of 3 nM amide compound solution, and 20 μL of AChE / BChE enzyme solution to the first well; add 30 μL of PBS buffer, 10 μL of 10 nM amide compound solution, and 20 μL of AChE / BChE enzyme solution to the second well; and so on). Pre-incubate at 37°C for 10 minutes to allow the amide compound to fully bind with the enzyme. Then add 60 μL of acetylthiocholine substrate to initiate the enzymatic reaction. After reacting for 15 minutes, add 80 μL of LTNB chromogenic reagent to terminate the reaction and develop color.

[0048] Blank group: Add 60 μL of PBS buffer to the wells, followed by 60 μL of acetylthiocholine substrate. After 15 minutes, add 80 μL of DTNB chromogenic reagent and develop the color.

[0049] Control group: 40 μL of PBS buffer and 20 μL of AChE / BChE enzyme solution extracted from rat brain were added to the wells in sequence and pre-incubated at 37°C for 10 minutes. Then, 60 μL of acetylthiocholine substrate was added to start the enzymatic reaction. After 15 minutes of reaction, 80 μL of DTNB chromogenic reagent was added to terminate the reaction and develop color.

[0050] The absorbance value A of each well was measured at a wavelength of 412 nm using an ELISA reader, according to the formula:

[0051] Inhibition rate = [1-(A 实验 -A 空白 ) / (A 对照 -A 空白 )]×100%;

[0052] The inhibition rate of enzyme activity by different concentrations of N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide was calculated. A curve was plotted with the logarithm of the N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide concentration (C) on the x-axis and the inhibition rate on the y-axis. The results are shown below. Figure 4As shown, nonlinear regression analysis was performed using software such as GraphPad Prism to fit the dose-response curve, and then the concentration (IC50 value) of the amide compound when the inhibition rate was 50% was calculated.

[0053] To ensure the reliability of the experimental results, the volume concentration of DMSO was controlled to not exceed 1% throughout the entire experimental process, and all experiments were independently repeated 3 times.

[0054] from Figure 4 As can be seen, the inhibitory activity of N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide against AChE and BChE showed a concentration-dependent increase. Using GraphPad Prism software, a nonlinear regression analysis was performed on the data based on a Log (Inhibitor) vs. Response model. The results showed that the half-maximal inhibitory concentration (IC50) of this amide compound against AChE was 35.2 nM (95% confidence interval: 29.6–41.8 nM), the Hill coefficient was 0.84, and the coefficient of determination R0 of the fitted curve was [value missing]. 2 The efficacy was 0.98; the half-maximal inhibitory concentration (IC50) for BChE was 376.8 nM (95% confidence interval: 316.6–448.6 nM), the Hill coefficient was 0.77, and the R-value was [missing value]. 2 The value was also 0.98. The above results show a high goodness of fit and statistically reliable experimental results. Therefore, N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide exhibits highly efficient inhibitory activity against both AChE and BChE, and its inhibitory activity against AChE is more significant than that against BChE.

[0055] Therefore, this invention utilizes the above-mentioned amide compound in the preparation of drugs for treating Alzheimer's disease. N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide achieves precise multi-target intervention of the disease by regulating cholinergic system dysfunction and inhibiting oxidative stress neuronal damage, and improves the therapeutic effect through synergistic effect, providing a new approach for the treatment of Alzheimer's disease.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The use of an amide compound in the preparation of a drug for treating Alzheimer's disease, characterized in that: The amide compound is N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide, with the following structural formula: 。 2. The application of the amide compound according to claim 1 in the preparation of a drug for treating Alzheimer's disease, characterized in that: Alzheimer's disease is a neurodegenerative disease caused by cholinergic system dysfunction and neuronal damage due to oxidative stress.

3. The application of the amide compound according to claim 2 in the preparation of a drug for treating Alzheimer's disease, characterized in that: Cholinergic system dysfunction manifests as abnormal dynamic changes in the activity of acetylcholinesterase and butyrylcholinesterase.

4. The use of an amide compound according to claim 1 in the preparation of a drug for treating Alzheimer's disease, characterized in that: N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide treats Alzheimer's disease by modulating cholinergic system dysfunction and inhibiting oxidative stress-induced neuronal damage.

5. The use of an amide compound according to claim 4 in the preparation of a drug for treating Alzheimer's disease, characterized in that: The way to regulate cholinergic system dysfunction is by inhibiting the dual activity of acetylcholinesterase and butyrylcholinesterase.

6. A drug for treating Alzheimer's disease, characterized in that: Including N-[(1S)-1-(furan-2-yl)ethyl]-6-nitro-2,3-dihydro-1,4-benzodioxin-7-carboxamide and drug carriers.

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