Chrysin and lipoic acid splicing compound as well as preparation method and application thereof

Abstract

The invention relates to a chrysin and lipoic acid splicing compound as well as a preparation method and application thereof. The chrysin and lipoic acid splicing compound is named as CL-1. The CL-1 shows an obvious protective effect in nerve cell oxidative damage and inflammatory damage models, and the reduction amplitude of TNF-alpha and IL-6 levels is obviously higher than that of monomer chrysin and lipoic acid, so that the anti-inflammatory and anti-nerve damage effects of the CL-1 are superior to those of monomer drugs, and the CL-1 has the potential of treating neurodegenerative diseases such as Alzheimer's disease. Meanwhile, the provided preparation method is designed according to the structural characteristics of chrysin and lipoic acid, and CL-1 can be efficiently synthesized on a large scale.

Description

Technical Field

[0001] The present invention relates to the technical field of heterocyclic compounds, and particularly to a chrysin and lipoic acid conjugate, a preparation method thereof, and an application thereof. Background Art

[0002] Alzheimer's disease (AD), commonly known as "senile dementia", is a neurodegenerative disease with extremely insidious onset and progressive development. At different stages of AD, patients will successively present a series of symptoms such as continuous decline in memory, severe impairment of language expression and comprehension functions, disorders in visual-spatial perception and cognition, abnormal brain function, and significant changes in personality and behavior. The self-care ability of AD patients will gradually decline with the progression of the disease, which will undoubtedly bring a heavy social and economic burden to the global health system. Nowadays, AD has ranked fourth among the causes of death in the elderly, second only to cardiovascular diseases, tumors, and strokes, seriously threatening the life and health and quality of life of the elderly.

[0003] AD is a chronic disease involving a variety of complex pathophysiological changes. Currently, the main hypotheses include abnormal deposition of amyloid proteins, hyperphosphorylation of tau proteins, neuroinflammation, abnormal cholinergic nerves, abnormal gene inheritance, and mitochondrial dysfunction oxidative stress, etc. These hypotheses have explained the pathogenesis of AD from different perspectives, but due to the complexity of the disease itself, no unified conclusion has been formed yet.

[0004] In terms of clinical treatment, representative drugs for AD include donepezil, galantamine, memantine, etc. However, these existing drugs generally have problems such as high prices, unsatisfactory treatment effects, and more side effects. This makes doctors often face the dilemma of limited treatment means and poor treatment effects during the actual clinical treatment process, and it is also difficult for patients to obtain an ideal treatment experience and rehabilitation effect. Therefore, there is an urgent need to develop new, efficient, safe, and affordable treatment drugs clinically to meet the treatment needs of the majority of patients and bring new hope for conquering this disease. Summary of the Invention

[0005] The purpose of the present invention is to disclose a chrysin and lipoic acid conjugate, a preparation method thereof, and an application thereof, so as to solve one or more technical problems existing in the prior art, and provide at least one beneficial choice or create conditions.

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

[0007] The second aspect of the present invention is to provide a preparation method of the CL-1.

[0008] The third aspect of the present invention lies in providing the application directions of the said CL-1.

[0009] The compound CL-1 described in the first aspect of the present invention is formed by splicing chrysin and lipoic acid, and its structural formula is shown as formula (Ⅰ):

[0010] (Ⅰ).

[0011] Chrysin (5,7-dihydroxyflavone), as a polyphenolic compound, has a wide range of pharmacological activities, including anti-inflammatory, antioxidant, and neuroprotective effects. However, chrysin has drawbacks such as poor water solubility and low bioavailability, which limit its further application.

[0012] Lipoic acid (LA), also known as α-lipoic acid (ALA), is a dithiol compound with both lipophilic and hydrophilic properties. It can be converted into dihydrolipoic acid in the body, and both have multiple key functions such as antioxidant, metabolic regulation, and anti-inflammatory effects.

[0013] The said CL-1 is a conjugate of chrysin and lipoic acid, which can maintain the pharmacophores of both. Experimental results have shown that the said CL-1 is non-toxic at a specific concentration, can protect nerve cells from oxidative damage, and can also have an anti-inflammatory effect on the inflammatory response of nerve cells.

[0014] The preparation method described in the second aspect of the present invention is to dissolve chrysin and lipoic acid in a solvent, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP) to carry out a reaction. After the reaction is completed, the organic phase is separated and retained, and the said CL-1 is obtained after purification. EDCI activates the carboxylic acid of lipoic acid; DMAP acts as a nucleophilic catalyst to accelerate the esterification of the phenolic hydroxyl group at the C-7 position of chrysin and the carboxyl group of lipoic acid.

[0015]

[0016] In a further application embodiment, the molar ratio of the said chrysin to the said lipoic acid is (0.9~1):1.

[0017] In a further application embodiment, the molar ratio of the said lipoic acid to the said 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 1:(1.1~1.5).

[0018] In a further application embodiment, the molar ratio of the said chrysin to the said 4-dimethylaminopyridine is 1:(0.1~0.5).

[0019] In a further application embodiment, the said solvent is dichloromethane.

[0020] In a further application embodiment, the duration of the reaction is not less than 24 hours.

[0021] In a further application embodiment, the purification is to dry and concentrate the organic phase and then perform silica gel column chromatography treatment.

[0022] The application described in the third aspect of the present invention is to use the CL-1 for preparing a drug.

[0023] In a further application embodiment, the prepared drug has the efficacy of protecting nerve cells.

[0024] In a further application embodiment, the prepared drug has the efficacy of treating neurodegenerative diseases.

[0025] By using the H2O2-induced human neuroblastoma SH-SY5Y cell injury model and the LPS-induced microglial BV2 cell injury model, the effects of the CL-1 on cell viability and inflammatory factors were observed, providing a theoretical basis for its application in the treatment of related neurological diseases such as Alzheimer's disease. The experimental results showed that the CL-1 had significant neuroprotective activity and an obvious dose-effect relationship, suggesting its application prospect in the preparation of drugs for treating neurodegenerative diseases.

[0026] The beneficial effects of the present invention are as follows: (1) The CL-1 described in the present invention showed significant protective effects in both the nerve cell oxidative injury and inflammatory injury models. The reduction amplitude of the TNF-α and IL-6 levels was significantly higher than that of monomeric chrysin and lipoic acid, indicating that the anti-inflammatory and anti-neural injury effects of the CL-1 were superior to those of monomeric drugs and it had the potential to treat neurodegenerative diseases such as Alzheimer's disease.

[0027] (2) The preparation method provided by the present invention was designed according to the structural characteristics of chrysin and lipoic acid and could efficiently and massively synthesize the CL-1. Description of the Drawings

[0028] Figure 1 is the 1H NMR spectrum of the CL-1 described in Example 1; Figure 2 is the 13C NMR spectrum of the CL-1 described in Example 1; Figure 3 is the bar graph showing the effect of the CL-1 described in Example 2 on the viability of SH-SY5Y cells; Figure 4 is the bar graph showing the effect of different concentrations of H2O2 on the survival rate of the cell injury model in Example 2; Figure 5 is the bar graph showing the effect of the CL-1, chrysin and lipoic acid on the viability of SH-SY5Y cells in Example 2; Figure 6 It is a bar graph showing the effect of CL-1 described in Example 3 on the viability of BV2 cells; Figure 7 It is a bar graph showing the effect of CL-1, chrysin and lipoic acid described in Example 3 on the concentration of inflammatory factors in BV2 cells. Detailed implementation method

[0029] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0030] Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0031] Example 1: Preparation and analysis of the spliced compound.

[0032] Dissolve 2.5 g of chrysin and 2.2 g of lipoic acid in 1 L of dichloromethane. Under the condition of water bath, add 0.6 g of DMAP and 2.9 g of EDCI to the reaction system. After reacting for 24 hours, add 200 mL of water to the reaction system to quench the reaction, and then transfer it to a separatory funnel for liquid separation. The remaining organic phase is dried over anhydrous sodium sulfate, concentrated, and then subjected to silica gel column chromatography to obtain 3.8 g of the said CL-1, with a yield of 86%. The hydrogen spectrum of the product is as Figure 1 shown, and the data are: 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). The carbon spectrum is as Figure 2 shown, and the data are: 1313C 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.

[0033] Example 2: Study on the protective effect of CL-1 against oxidative damage of nerve cells.

[0034] Since the effect of the chrysin and lipoic acid conjugate on Alzheimer's disease has not been reported, a cell injury model of H2O2-induced human neuroblastoma SH-SY5Y cells was used to observe the effect of CL-1 on cell viability.

[0035] (1) Cell culture and drug preparation The SH-SY5Y cells were evenly seeded in a 75 cm 2 adherent cell culture flask, and cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody in an incubator at 37 °C and 5% CO2. When the cells reached 80% of the bottom area of the flask, cell passage was carried out, and cells in the logarithmic growth phase were selected for the experiment. The CL-1, chrysin and lipoic acid were prepared into a stock solution of 200 mmol / L with DMSO and then diluted to the experimental concentration with the medium.

[0036] (2) Detection of the effect of CL-1 on the activity of SH-SY5Y cells by MTT method After digesting the cells in the logarithmic growth phase, according to 1×10 4Cells were seeded at a density of [number of cells] per well in a 96-well culture plate. The first column was without cells and only contained culture medium as the blank group. The second column contained cells in a normal physiological state as the control group. The other columns contained cells in a normal physiological state as the dosing groups, with 6 replicate wells in each group. When the cell density reached about 80%, the dosing groups were given different concentrations of the CL-1 (1, 25, 50, 100, 150, 200 μmol / L), the control group was given a medium containing the solvent. After 24 hours of intervention culture, 10 μL of MTT (5 mg / mL) solution was added to each well. After incubating at 37°C for 4 hours, the liquid in the well plate was discarded, and 150 μL of DMSO was added to each well and incubated at 37°C for 15 minutes. The OD value of each well at a wavelength of 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated.

[0037] Cell survival rate = [(OD value of the dosing group - OD value of the blank group) / (OD value of the control group - OD value of the blank group)] * 100%.

[0038] The results were as Figure 3 shown. When the concentration of the CL-1 ≥ 150 μmol / L, the cell growth was significantly inhibited. However, in the concentration range of 1 - 100 μmol / L, the CL-1 did not inhibit the proliferation of cells, that is, no cytotoxicity was produced. Therefore, two concentrations of 50 μmol / L and 100 μmol / L were selected for subsequent experiments.

[0039] (3) Establishment of an H2O2-induced oxidative damage model in SH-SY5Y cells Cells in the logarithmic growth phase were seeded in a 96-well plate at a concentration of 1×10 4 / well. When the cell confluence reached about 80%, the cells were treated with 0, 50, 100, 200, 300, 500 μmol / L of H2O2. After 24 hours, the absorbance value was measured by the MTT method.

[0040] The MTT results were as Figure 4 shown. As the concentration of H2O2 increased, the cell activity decreased significantly and showed a concentration-dependent trend. When the concentration of H2O2 was 300 μmol / L, the cell survival rate was 58.9%. Therefore, a 300 μmol / L H2O2 was selected to induce SH-SY5 cells to establish an oxidative stress model.

[0041] (4) Protective effect of the CL-1 on the H2O2-induced cell damage model The experiment was divided into two different concentrations of 50 μmol / L and 100 μmol / L. Each concentration included a control group, a model group, a conjugate group, a chrysin group, and a lipoic acid group. In addition, a blank group without cells and only containing cell culture medium was set up. The cells were seeded at a density of 1×10 4The concentration of cells in the wells was plated in a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours. When the cell confluence reached about 80%, the CL-1 was added to the conjugate group, and the corresponding drugs were added to the chrysin group and the lipoic acid group for pretreatment for 2 hours. Subsequently, the model group, the conjugate group, the chrysin group, and the lipoic acid group were further cultured with H2O2 at a final concentration of 300 μmol / L for 24 hours, and the absorbance OD value was measured by the MTT method and the survival rate was calculated.

[0042] The MTT results are as Figure 5 shown. After preparing a nerve injury model by inducing SH-SY5Y cells with H2O2, compared with the control group, cell proliferation in the model group was inhibited, and cell viability was significantly reduced, and there was a statistical difference ( P <0.001). Compared with the model group, treatment with different concentrations of the CL-1 could significantly increase the survival rate of cells, showing a concentration-dependent manner, and the improvement amplitude of the conjugate was greater than that of the chrysin group and the lipoic acid group. It shows that the CL-1 has a certain protective effect on the damage of SH-SY5Y cells induced by H2O2, and the anti-nerve injury effect is better than that of chrysin and lipoic acid.

[0043] Data are expressed as mean ± standard deviation. ANOVA one-way analysis of variance and LSD pairwise comparison were used among multiple groups, and GraphPad prism statistical software was used for graphing analysis. P < 0.05 was considered to be statistically significant.

[0044] Example 3: Effect of CL-1 on LPS-induced neuroinflammatory response.

[0045] Neuroinflammation plays a key role in the occurrence and development of various neurological diseases. Microglia are central nervous immune cells and will be activated by stimuli such as pathogens and injuries, and are commonly activated by lipopolysaccharide (LPS). BV2 cells are a commonly used mouse microglial cell line, and inflammation will be induced after being stimulated by LPS, releasing inflammatory factors such as TNF-α and IL-1β to damage nerve cells. This study observed the effect of the CL-1 on LPS-induced inflammatory damage of BV2 cells.

[0046] (1) Cell culture BV2 cells were seeded in DMEM medium containing 10% fetal bovine serum and 1% double antibody and cultured in an incubator at 37°C and 5% CO2. When the cells grew to the logarithmic growth phase, they were used for subsequent experiments.

[0047] (2) Detection of cell viability in each group by MTT method After digesting the cells in the logarithmic phase, according to 5×10 3Cells were seeded at a density of

[0048] per well in a 96-well plate and incubated overnight in an incubator, followed by treatment with different concentrations (5, 10, 25, 50 μmol / L) of the CL-1. The control group was given a medium containing the vehicle, and each group had 5 replicate wells. After 24 hours of treatment, 10 μL of MTT (5 mg / mL) solution was added to each well. After incubation at 37°C for 4 hours, the liquid in the well plate was discarded, 150 μL of DMSO was added to each well, and the mixture was incubated at 37°C for 15 minutes. The OD value of each well at a wavelength of 490 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell survival rate was calculated. Figure 6 The cytotoxicity of the CL-1 against BV2 cells was detected by MTT assay. The results are shown in

[0049] (3) Detection of the contents of inflammatory factors TNF-α and IL-1β by ELISA The experimental groups included a control group, a model group, a conjugate group, a chrysin group, and a lipoic acid group. Cells in the logarithmic growth phase were digested and seeded at a density of 5×10 3 per well in a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 hours. The conjugate group was treated with 50 μmol / L of the CL-1, and the chrysin group and the lipoic acid group were pretreated with 50 μmol / L of the corresponding drugs for 2 hours, respectively. Subsequently, the model group, the conjugate group, the chrysin group, and the lipoic acid group were treated with LPS (1 μg / mL) for 24 hours. The culture supernatants of the cells in each group were collected and the contents of TNF-α and IL-1β were detected according to the instructions of the ELISA kit.

[0050] The results are shown in Figure 7 Compared with the control group, the levels of TNF-α and IL-6 in the cell supernatants of the model group were significantly increased, indicating that LPS promoted the release of inflammatory factors from BV2 cells, leading to inflammatory damage of nerve cells. Compared with the model group, the CL-1 group, the chrysin group, and the lipoic acid group all reduced the levels of inflammatory factors to varying degrees, but the CL-1 had a significantly higher reduction in TNF-α and IL-6 than chrysin and lipoic acid, indicating that its anti-inflammatory and anti-neural effects were superior to chrysin and lipoic acid.

[0051] The data were expressed as mean ± standard deviation. One-way analysis of variance (ANOVA) and LSD pairwise comparison were used for multiple groups, and GraphPad prism statistical software was used for graph analysis. A P value < 0.05 was considered statistically significant.

[0052] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, in any regard, the embodiments should be regarded as exemplary and non-restrictive. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention.

Claims

1. A chrysin and lipoic acid conjugate, characterized in that The splicing product has the structure shown in formula (I). （Ⅰ）。 2. The preparation method of the chrysin and lipoic acid conjugate according to claim 1, characterized in that, Chrysin and lipoic acid are dissolved in a solvent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4-dimethylaminopyridine are added for reaction. After the reaction is completed, the organic phase is separated and retained, and obtained after purification.

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

1.

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

6. According to the preparation method described in claim 2, characterized in that, The solvent is dichloromethane.

7. The preparation method according to claim 2, characterized in that, The duration of the reaction is not less than 24 hours.

8. The preparation method according to claim 2, wherein The purification is to dry and concentrate the organic phase and then perform silica gel column chromatography treatment.

9. Use of the chrysin and lipoic acid splicing product according to claim 1 in the preparation of a drug for protecting nerve cells.

10. Use of the chrysin and lipoic acid splicing product according to claim 1 in the preparation of a drug for treating neurodegenerative diseases.

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