A chimeric compound of chrysin and thioctic acid, and a preparation method and application thereof

Abstract

This invention relates to a juglansin and lipoic acid conjugate, its preparation method, and its applications. The juglansin and lipoic acid conjugate is named CL-1. CL-1 exhibits significant protective effects in models of oxidative and inflammatory damage in nerve cells, with a significantly greater reduction in TNF-α and IL-6 levels than the monomeric juglansin and lipoic acid, indicating that CL-1 has superior anti-inflammatory and anti-neural damage effects compared to the monomeric drugs, and possesses the potential to treat neurodegenerative diseases such as Alzheimer's disease. The provided preparation method is designed specifically for the structural characteristics of juglansin and lipoic acid, enabling efficient and large-scale synthesis of CL-1.

Description

Technical Field

[0001] This invention relates to the field of heterocyclic compound technology, and in particular to a succinic acid and lipoic acid composite, its preparation method and application. Background Technology

[0002] Alzheimer's disease (AD), commonly known as "senile dementia," is a neurodegenerative disease with an extremely insidious onset and progressive development. At different stages of AD, patients experience a series of symptoms, including persistent memory loss, severe impairment of language expression and comprehension, visual-spatial perception and cognition disturbances, abnormal brain function, and significant changes in personality and behavior. The self-care ability of AD patients gradually declines as the disease progresses, undoubtedly placing a heavy social and economic burden on global health systems. Currently, AD is the fourth leading cause of death among the elderly, after cardiovascular disease, cancer, and stroke, seriously threatening their health and quality of life.

[0003] Alzheimer's disease (AD) is a chronic disease involving multiple complex pathophysiological changes. Current main hypotheses include abnormal amyloid deposition, tau protein hyperphosphorylation, neuroinflammation, cholinergic neuronal abnormalities, genetic abnormalities, and mitochondrial dysfunction and oxidative stress. These hypotheses explain the pathogenesis of AD from different perspectives, but due to the complexity of the disease itself, a unified conclusion has not yet been reached.

[0004] In clinical treatment, representative drugs for Alzheimer's disease (AD) include donepezil, galantamine, and memantine. However, these existing drugs generally suffer from high prices, unsatisfactory treatment effects, and numerous side effects. This often leaves doctors facing a dilemma of limited treatment options and poor treatment outcomes in actual clinical practice, and patients also struggle to achieve ideal treatment experiences and recovery results. Therefore, there is an urgent clinical need to develop new, highly effective, safe, and affordable treatment drugs to meet the treatment needs of a wide range of patients and bring new hope for conquering this disease. Summary of the Invention

[0005] The purpose of this invention is to disclose a compound of apigenin and lipoic acid, its preparation method and application, in order to solve one or more technical problems existing in the prior art and provide at least one beneficial option or create conditions.

[0006] The first aspect of the present invention is to provide a compound named CL-1.

[0007] A second aspect of the present invention is to provide a method for preparing the CL-1.

[0008] A third aspect of the present invention is to provide the application directions of the CL-1.

[0009] The compound CL-1 described in the first aspect of this invention is composed of succinin and lipoic acid, and its structural formula is shown in formula (I):

[0010]

[0011] (I).

[0012] Fibroin (5,7-dihydroxyflavonoid), as a polyphenolic compound, possesses a wide range of pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, fibroin suffers from poor water solubility and low bioavailability, which limits its further application.

[0013] Lipoic acid (LA), also known as α-lipoic acid (ALA), is a disulfide compound that is both fat-soluble and water-soluble. It can be converted into dihydrolipoic acid in the body. Both have a variety of key functions such as anti-oxidation, metabolism regulation and anti-inflammation.

[0014] The CL-1 is a compound of salicylic acid and lipoic acid, which retains the pharmacophores of both. Experiments have shown that CL-1 is non-toxic at specific concentrations, can protect nerve cells from oxidative damage, and also has an anti-inflammatory effect on the inflammatory response of nerve cells.

[0015] The preparation method described in the second aspect of this invention involves dissolving salicumin and lipoic acid in a solvent, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to the solvent, reacting the solutions, separating and retaining the organic phases, and purifying the solution to obtain CL-1. EDCI activates the carboxylic acid group of lipoic acid; DMAP acts as a nucleophilic catalyst, accelerating the esterification of the phenolic hydroxyl group at the C-7 position of salicumin with the carboxyl group of lipoic acid.

[0016]

[0017] In a further application embodiment, the molar ratio of the salicylic acid to the thioctic acid is (0.9~1):1.

[0018] In a further application embodiment, the molar ratio of the lipoic acid and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1.1~1.5).

[0019] In a further application embodiment, the molar ratio of the salicylic acid to the 4-dimethylaminopyridine is 1:(0.1~0.5).

[0020] In a further application embodiment, the solvent is dichloromethane.

[0021] In a further implementation, the reaction duration is no less than 24 hours.

[0022] In a further application embodiment, the purification involves drying and concentrating the organic phase, followed by silica gel column chromatography.

[0023] The third aspect of the invention describes the use of the CL-1 in the preparation of a drug.

[0024] In a further application implementation, the prepared drug has the effect of protecting nerve cells.

[0025] In a further application implementation, the prepared drug has the effect of treating neurodegenerative diseases.

[0026] Using H2O2-induced SH-SY5Y neurocytoma cell injury models and LPS-induced BV2 microglia cell injury models, the effects of CL-1 on cell viability and inflammatory factors were observed, providing a theoretical basis for its application in the treatment of Alzheimer's disease and other related neurological diseases. Experimental results showed that CL-1 possesses significant neuroprotective activity and a clear dose-response relationship, suggesting its potential application in the preparation of drugs for treating neurodegenerative diseases.

[0027] The beneficial effects of this invention are:

[0028] (1) The CL-1 described in this invention showed significant protective effects in both oxidative and inflammatory models of nerve cells. The reduction in TNF-α and IL-6 levels was significantly greater than that of monomeric succinate and lipoic acid, indicating that the anti-inflammatory and anti-neural damage effects of CL-1 are superior to those of monomeric drugs and have the potential to treat neurodegenerative diseases such as Alzheimer's disease.

[0029] (2) The preparation method provided by the present invention is designed based on the structural characteristics of salicylic acid and thioctic acid, and can efficiently and on a large scale synthesize the CL-1. Attached Figure Description

[0030] Figure 1 It is the proton spectrum of CL-1 described in Example 1;

[0031] Figure 2 It is the carbon spectrum of CL-1 described in Example 1;

[0032] Figure 3 This is a bar chart showing the effect of CL-1 on the viability of SH-SY5Y cells as described in Example 2;

[0033] Figure 4 This is a bar chart showing the effect of different concentrations of H2O2 on the survival rate of the cell damage model in Example 2;

[0034] Figure 5 This is a bar chart showing the effects of CL-1, succinic acid, and lipoic acid on the viability of SH-SY5Y cells as described in Example 2;

[0035] Figure 6 This is a bar chart showing the effect of CL-1 on BV2 cell viability as described in Example 3;

[0036] Figure 7 This is a bar chart showing the effects of CL-1, apigenin, and lipoic acid on the concentration of inflammatory factors in BV2 cells, as described in Example 3. Detailed Implementation

[0037] The following embodiments further illustrate the content of the present invention, but should not be construed as limiting the present invention. Any modifications and substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the present invention are within the scope of the present invention.

[0038] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0039] Example 1: Preparation and analysis of spliced ​​compounds.

[0040] 2.5 g of salicylic acid and 2.2 g of lipoic acid were dissolved in 1 L of dichloromethane. Under water bath conditions, 0.6 g of DMAP and 2.9 g of EDCI were added to the reaction system. After reacting for 24 hours, the reaction was quenched with 200 mL of water, and then the mixture was separated in a separatory funnel. The retained organic phase was dried over anhydrous sodium sulfate, concentrated, and then subjected to silica gel column chromatography to obtain 3.8 g of the CL-1, with a yield of 86%. The 1H NMR spectrum of the product is shown below. Figure 1 As shown, the data is: 1 H NMR (500 MHz, CDCl3) δ 12.73 (s,1H), 7.94–7.86 (m, 2H), 7.60–7.50 (m, 3H), 6.86 (d, J = 2.1 Hz, 1H), 6.74 (s,1H), 6.57 (d, J = 2.0 Hz, 1H), 3.63–3.60 (m, 1H), 3.23–3.19 (m, 1H), 3.16–3.11(m, 1H), 2.62 (t, J = 7.4 Hz, 2H), 2.54–2.46 (m, 1H), 1.99–1.91 (m, 1H), 1.87–1.72 (m, 4H), 1.60–1.50 (m, 2H). Carbon spectra are as follows: Figure 2 As shown, the data is: 13 C NMR (125 MHz, CDCl3) δ 183.04, 171.14, 164.87, 162.06, 156.93, 156.15, 132.35, 131.12, 129.35, 126.58, 109.07, 106.29, 105.61, 101.20, 56.44, 40.43, 38.71, 34.76, 34.33, 28.84, 24.68. Mass spectrometry data are as follows: HRMS (ESI): m / z calcd for C 23 H 22 NaO5S2 + [M+Na] + : 465.0801,found: 465.0800.

[0041] Example 2: Study on the protective effect of CL-1 against oxidative damage to nerve cells.

[0042] Since the effects of succinic acid and lipoic acid conjugates on Alzheimer's disease have not been reported, the effect of CL-1 on cell viability was observed using a H2O2-induced human neurocytoma SH-SY5Y cell damage model.

[0043] (1) Cell culture and drug preparation

[0044] Spread SH-SY5Y cells evenly on a 75cm surface. 2 Adherent cell culture flasks were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibiotics at 37°C in a 5% CO2 incubator. Cells were passaged when they reached 80% confluence with the bottom of the flask, and cells in the logarithmic growth phase were selected for experiments. The CL-1, succinate, and lipoic acid were prepared as a 200 mmol / L stock solution using DMSO, which was then diluted with culture medium to the experimental concentration.

[0045] (2) The effect of CL-1 on the viability of SH-SY5Y cells was detected by MTT assay.

[0046] After digesting cells in the logarithmic growth phase, they were processed at a rate of 1×10⁻⁶. 4Cells were seeded per well in a 96-well plate. The first column contained no cells but culture medium as a blank control. The second column contained cells under normal physiological conditions as a control group. The other columns contained cells under normal physiological conditions as treatment groups, with 6 replicates per group. When the cell density reached approximately 80%, the treatment groups were given different concentrations of CL-1 (1, 25, 50, 100, 150, 200 μmol / L), while the control groups were given culture medium containing solvent. After 24 hours of intervention culture, 10 μL of MTT (5 mg / mL) solution was added to each well, and the plates were incubated at 37°C for 4 hours. The liquid in the plates was then discarded, and 150 μL of DMSO was added to each well, followed by incubation at 37°C for 15 minutes. The OD value of each well at 490 nm was measured using a microplate reader, and cell viability was calculated.

[0047] Cell viability = [(OD value of drug-treated group - OD value of blank group) / (OD value of control group - OD value of blank group)] * 100%.

[0048] The results are as follows Figure 3 As shown, when the concentration of CL-1 is ≥150 μmol / L, cell growth is significantly inhibited. However, in the concentration range of 1~100 μmol / L, CL-1 does not inhibit cell proliferation, i.e., it does not produce cytotoxicity. Therefore, 50 μmol / L and 100 μmol / L were selected for subsequent experiments.

[0049] (3) Establishment of H2O2-induced oxidative damage model of SH-SY5Y cells

[0050] Take cells in the logarithmic growth phase at a ratio of 1×10 4 The concentration of H2O2 was seeded into 96-well plates. When the cell coverage reached about 80%, the cells were treated with H2O2 at concentrations of 0, 50, 100, 200, 300, and 500 μmol / L. The absorbance was measured by the MTT assay after 24 hours.

[0051] MTT results are as follows Figure 4 As shown, cell viability decreased significantly with increasing H2O2 concentration, exhibiting a concentration-dependent trend. The cell survival rate was 58.9% when the H2O2 concentration was 300 μmol / L; therefore, 300 μmol / L H2O2 was chosen to induce oxidative stress in SH-SY5 cells to establish an oxidative stress model.

[0052] (4) The protective effect of CL-1 on the H2O2-induced cell damage model

[0053] The experiment was conducted at two different concentrations: 50 μmol / L and 100 μmol / L. Each concentration included a control group, a model group, a splice group, a guar gum group, and a lipoic acid group. A blank group was also included, containing only cell culture medium and no cells. Cells were cultured at a concentration of 1×10⁶... 4 The concentration of each cell was seeded into 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. When the cell coverage reached approximately 80%, the splice group was treated with CL-1, while the guar gum group and lipoic acid group were pretreated with their respective drugs for 2 hours. Subsequently, the model group, splice group, guar gum group, and lipoic acid group were incubated with H2O2 at a final concentration of 300 μmol / L for 24 hours. The absorbance (OD) value was measured using the MTT assay, and the viability was calculated.

[0054] MTT results are as follows Figure 5 As shown, after inducing SH-SY5Y cells with H2O2 to establish a neural injury model, compared with the control group, cell proliferation in the model group was inhibited, cell viability was significantly reduced, and the difference was statistically significant. P <0.001). Compared with the model group, treatment with different concentrations of CL-1 significantly increased cell survival rate in a concentration-dependent manner, and the increase in the number of splice cells was greater than that in the apigenin group and the lipoic acid group. This indicates that CL-1 has a certain protective effect against H2O2-induced SH-SY5Y cell damage, and its anti-neural damage effect is better than that of apigenin and lipoic acid.

[0055] Data are expressed as mean ± standard deviation. One-way ANOVA and LSD pairwise comparisons were performed between multiple groups. GraphPad Prism statistical software was used for plotting and analysis. A p-value < 0.05 was considered statistically significant.

[0056] Example 3: Effect of CL-1 on LPS-induced neuronal inflammatory response.

[0057] Neuroinflammation plays a crucial role in the development and progression of various neurological diseases. Microglia are central nervous system immune cells that are activated by pathogens, injury, and other stimuli, often through lipopolysaccharide (LPS). BV2 cells are a commonly used mouse microglia cell line; stimulation with LPS triggers an inflammatory response, releasing inflammatory factors such as TNF-α and IL-1β that damage nerve cells. This study observes the effect of CL-1 on LPS-induced inflammatory damage in BV2 cells.

[0058] (1) Cell culture

[0059] BV2 cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody and cultured at 37°C in a 5% CO2 incubator. Cells were used in subsequent experiments after reaching the logarithmic growth phase.

[0060] (2) MTT assay was used to detect cell viability in each group.

[0061] After digesting the cells in the logarithmic growth phase, they were processed at a ratio of 5 × 10⁻⁶. 3 Cells were seeded at 1000 cells / well in 96-well plates and cultured overnight. Then, different concentrations (5, 10, 25, 50 μmol / L) of CL-1 were administered. The control group received culture medium containing the solvent. Each group had 5 replicates. After 24 hours of treatment, 10 μL of MTT (5 mg / mL) solution was added to each well, and the plates were incubated at 37°C for 4 hours. The liquid in the plates was discarded, and 150 μL of DMSO was added to each well. The plates were incubated at 37°C for 15 minutes. The OD value of each well was measured at 490 nm using a microplate reader, and cell viability was calculated.

[0062] The cytotoxicity of CL-1 against BV2 cells was detected by MTT assay, and the results are as follows: Figure 6 As shown, the CL-1 does not cause damage to BV2 cells and has no toxic effects in the range of 0~50 μmol / L.

[0063] (3) ELISA method for detecting the levels of inflammatory factors TNF-α and IL-1β

[0064] The experimental groups included a control group, a model group, a splice group, a succinic acid group, and an alpha-lipoic acid group. After digestion, logarithmic-phase cells were collected and processed at a ratio of 5 × 10⁻⁶ cells / day. 3 Cells were seeded per well in 96-well plates and incubated at 37°C with 5% CO2 for 24 hours. The splice group was pretreated with 50 μmol / L CL-1, while the guar gum group and lipoic acid group were pretreated with 50 μmol / L of their respective drugs for 2 hours. Subsequently, the model group, splice group, guar gum group, and lipoic acid group were treated with LPS (1 μg / mL) for 24 hours. The cell culture supernatant from each group was collected, and the levels of TNF-α and IL-1β were measured according to the ELISA kit instructions.

[0065] The results are as follows Figure 7 As shown, compared with the control group, the levels of TNF-α and IL-6 in the cell supernatant of the model group were significantly increased, indicating that LPS promotes the release of inflammatory factors in BV2 cells, leading to inflammatory damage to nerve cells. Compared with the model group, the CL-1 group, apigenin group, and lipoic acid group all reduced the levels of inflammatory factors to varying degrees. However, the reduction of TNF-α and IL-6 by CL-1 was significantly greater than that by apigenin and lipoic acid, indicating that its anti-inflammatory and anti-neurogenic effects were superior to those of apigenin and lipoic acid.

[0066] Data are expressed as mean ± standard deviation. One-way ANOVA and LSD pairwise comparisons were performed between multiple groups. GraphPad Prism statistical software was used for plotting and analysis. A p-value < 0.05 was considered statistically significant.

[0067] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A compound of salinomycin and lipoic acid, characterized in that, The splice has a structure as shown in formula (I). （Ⅰ）。 2. The method for preparing the salicylic acid and thioctic acid conjugate according to claim 1, characterized in that, The succinic acid and lipoic acid were dissolved in a solvent, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine were added to react. After the reaction was completed, the organic phase was separated and retained, and the product was purified.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the salicylic acid to the thioctic acid is (0.9~1):

1.

4. The preparation method according to claim 2, characterized in that, The molar ratio of the lipoic acid and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1.1~1.5).

5. The preparation method according to claim 2, characterized in that, The molar ratio of the salicylic acid to the 4-dimethylaminopyridine is 1:(0.1~0.5).

6. The preparation method according to claim 2, characterized in that, The solvent is dichloromethane.

7. The preparation method according to claim 2, characterized in that, The reaction lasts for no less than 24 hours.

8. The preparation method according to claim 2, characterized in that, The purification process involves drying and concentrating the organic phase, followed by silica gel column chromatography.

9. The use of the salicylic acid and thioctic acid compound of claim 1 in the preparation of a drug for protecting nerve cells.

10. The use of the salicylic acid and thioctic acid conjugate of claim 1 in the preparation of a medicament for treating neurodegenerative diseases.

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