Anti-AD compounds and preparation thereof

Abstract

The invention discloses a novel derivative as well as a preparation method and medical application thereof. The derivative can be prepared through a one-pot reaction. Experimental data show that the derivative provided by the invention can protect nerve cell injury caused by A beta, and has a prospect of being developed into a medicine for treating AD (Alzheimer's disease). The derivative and the preparation method and medical application thereof are not disclosed in the prior art.

Description

BACKGROUND

[0001] Alzheimer's disease (AD) is a progressive neurodegenerative disease, clinically characterized by memory impairment, aphasia, agnosia and behavioral changes, and is a serious threat to the health of the elderly. AD has a high incidence, a long course and great harm. With the intensification of global aging, AD has become one of the important diseases in the world, and has become a major public health and scientific problem affecting China's economic and social development. Currently, the commonly used AD treatment drugs in clinical practice are acetylcholinesterase inhibitors (donepezil, rivastigmine, huperzine A and galantamine, etc.) and NMDA receptor antagonist memantine, but these drugs can only improve the symptoms and cannot delay or prevent the development of the disease or cure AD. The pathogenesis of AD is complex, and there are many influencing factors. The currently recognized pathological feature is the neurotoxicity of senile plaques formed by excessive deposition of β-amyloid (Aβ) in the brain, which leads to the death and loss of cholinergic neurons in the central nervous system. Therefore, inhibiting Aβ deposition or reducing the damage of Aβ to neuron cells is one of the main directions of AD drug development.

[0002] Silybin mainly exists in the fruits of Silybum marianum and has certain neuroprotective effect. However, its anti-AD activity is weak and the effect is poor, which directly affects its application in the development of anti-AD drugs. The currently reported derivatives are not substituted in the E ring. It is generally believed that the substitution of the E ring has a great and unpredictable impact on the properties of the parent compound. Some documents have introduced various groups, but only some halogen or aromatic ring substituents have good effects. SUMMARY

[0003] The present application aims to utilize the particularity of the silybin skeleton to chemically modify its structure and unexpectedly obtain a compound with greatly enhanced anti-AD activity. Specifically, the first purpose of the present application is to provide a silybin derivative, and the second purpose is to provide the medical use of the derivative.

[0004] The above purpose of the present application is achieved by the following technical solutions: The silybin derivative or its pharmaceutically acceptable salt as shown in the following chemical structure 1: .

[0005] The preparation route and steps of the above compound 1 are as follows:

[0006] The molar amounts of each substrate used in the preparation can be the same, the temperature can be room temperature, and the solvent used can be DCM and HFIP in a volume ratio of 1:1. The steps can be as follows: first, ethyl glyoxylate and thiomorpholine are stirred at room temperature, then silymarin is added and the reaction continues to obtain the target compound shown in structural formula 1. As an example, the ethyl glyoxylate and thiomorpholine are first dissolved in DCM.

[0007] The present invention also discloses the pharmaceutical use of the above-mentioned compound 1 in the preparation of anti-AD drugs. The present invention unexpectedly discovered that the modified compound 1 can greatly protect against nerve cell damage caused by Aβ and has the prospect of being developed into a drug for the treatment of AD. Detailed Implementation

[0008] Example 1: Preparation and structural characterization of compound 1

[0009] Preparation of Compound 1: Ethyl glyoxylate (102 mg, 1 mmol) and thiomorpholine (103 mg, 1 mmol) were dissolved in 2 mL of dichloromethane (DCM) and stirred at room temperature for 3 hours. Then, silymarin (482 mg, 1 mmol) and 2 mL of hexafluoroisopropanol (HFIP) were added, and the mixture was stirred overnight at room temperature. After the reaction was complete, the solvent was removed under reduced pressure. The crude product was then dissolved in acetic acid and washed twice with saturated brine. The organic phase was dried, concentrated, and purified by column chromatography (petroleum ether / ethyl acetate = 1:1–1:2) to obtain Compound 1 (200 mg, yield 30%) as a white solid. Spectral data: 1 HNMR (500 MHz, Chloroform- d ) δ11.76 (m, 1H), 7.18 (m, 1H), 7.06 (m, 2H), 6.95 (m, 3H), 6.00 (m, 1H), 4.97(m, 2H), 4.67 (d, J = 4.5 Hz, 1H), 4.52 (m, 1H), 4.16 – 4.23 (m, 2H), 4.05(m, 1H), 3.93 (s, 3H), 3.81 (m, 1H), 3.56 (1, 1H), 2.89 (m, 4H), 2.76 (m,4H), 1.26 (t, J = 7.2 Hz, 3H). 13 CNMR (125 MHz, Chloroform- d) δ 195.73, 169.49,169.46, 168.30, 168.26, 162.87, 161.51, 161.50, 161.48, 147.00, 146.98,146.38, 144.13, 144.10, 143.86, 129.35, 129.31, 127.80, 121.07, 120.94,120.77, 117.26, 117.19, 116.52, 114.78, 109.68, 109.63, 99.87, 99.70, 97.10,96.96, 82.97, 82.82, 72.30, 72.21, 72.15, 72.06, 65.19, 65.15, 61.79, 61.56,56.04, 52.67, 27.53, 14.10, 14.08. ESI-MS (m / z): 670.18[M+1] + . Example 2: Anti-AD activity test of compound 1 1. All experimental materials were either publicly available or conventionally prepared, including: Aβ 25-35 Purchased from Beijing Bio-Sens Biotechnology Co., Ltd.; mouse hippocampal neuronal cells HT22 purchased from Shanghai Cell Bank, Chinese Academy of Sciences; Aβ 1-42 The ELISA kit was purchased from Elabscience (China) Co., Ltd.; the APPswe overexpression lentivirus was purchased from Shanghai Jikai Gene Technology Co., Ltd.

[0010] Aβ 25-35 Preparation of oligomers: 1 mg of Aβ 25-35 Dissolve in 1 mL of sterile ddH2O to prepare 1 mg / mL Aβ. 25-35 The solution was aged in a 37°C incubator for 7 days.

[0011] 2. Experimental Methods 1) Cell Culture Remove mouse neuroblastoma cells N2a that were frozen in liquid nitrogen, quickly place them in a 37°C water bath and shake until the cells thaw. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add a small amount of culture medium and gently pipette to mix. The culture medium is DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics. Transfer the cells to a culture dish and culture in a cell culture incubator at 37°C and 5% CO2.

[0012] 2) Compound 1 on Aβ 25-35 Evaluation of the protective effect against induced neuronal damage N2a cells in the logarithmic growth phase were used to prepare a solution with a concentration of 1.0 × 10⁻⁶. 5 Cell suspension of cells / mL was seeded into 96-well plates at 100 μL per well. After culturing for 24 h, the cells were treated according to the following groups, with 5 replicates per group: Control group: Replace with fresh culture medium and continue culturing for 3 hours; Aβ 25-35 Group (model group): Replace with fresh culture medium and continue culturing for 3 hours; Donepezil hydrochloride group (positive control group): The culture medium was replaced with fresh medium containing 20 μM donepezil hydrochloride and cultured for another 3 hours; Silymarin group: Replace with fresh medium containing 20 μM silymarin and continue culturing for 3 hours; Group 1 of compounds: Replace with fresh culture medium containing 10, 20, and 40 μM of compound 1 and continue culturing for 3 hours.

[0013] After continuing incubation for another 3 hours according to the above grouping, Aβ 25-35 Group 1, donepezil hydrochloride group, silymarin group, and compound 1 group were supplemented with condensed Aβ at a final concentration of 15 μM. 25-35 The control group was given an equal volume of solvent and cultured for 24 hours. Then, each group was given 20 μL of MTT solution (concentration of 5 mg / mL) and cultured at 37°C for another 4 hours.

[0014] After culturing for another 4 hours, the supernatant in the wells was aspirated, and 150 μL of DMSO was added to each well. The mixture was shaken for 10 minutes to fully dissolve the crystals. The absorbance (OD value) of each well was measured at a wavelength of 490 nm using an enzyme-linked immunosorbent assay (ELISA) reader. The average OD value of the six replicates was used to calculate the cell viability. The control group was recorded as 100%.

[0015] 3) Compound 1 affects intracellular Aβ in APPswe-stabilized HT22 cells. 1-42 Effects of secretion Log-phase HT22 cells were seeded into 96-well plates at a density of 4000-5000 cells per well and cultured for 12 hours. After cell adhesion, an appropriate amount of virus was added for cell infection based on the MOI value and viral titer of lentivirus infection of HT22 cells. Simultaneously, polybrene (50 µg / mL) was added to improve transfection efficiency. The cell culture plates were incubated for another 12 hours. The virus mixture in the infected wells was then removed and replaced with fresh culture medium. After 48 hours of culture in the fresh medium, the medium was replaced with drug-containing medium containing 2 µg / mL puromycin. Successfully infected cells were screened for resistance for 5-7 passages. Cells were then seeded into 6-well plates. After cell adhesion, the cells were treated with the following groups: control group, APPswe stable HT22 transfection group (model group), 20 μM donepezil hydrochloride group (positive control group), 20 μM silymarin group, and compound 1 group (concentrations of 10, 20, and 40 μM, respectively). Cells were collected 24 hours after drug administration, and total protein was extracted and Aβ was detected using an ELISA kit. 1-42 Calculate the relative content of the concentration.

[0016] 3. Data processing and analysis Cell viability was calculated using SPSS 19.0 software. One-way ANOVA was used for comparisons among multiple groups, and the q-test was used for pairwise comparisons between groups. P <0.05 indicates a statistically significant difference.

[0017] 4. Experimental Results The cell viability of each group is shown in Table 1 (Note: compared with the control group). # P <0.05, ## P <0.01, ### P <0.001; compared with the model group* P <0.05,** P <0.01, *** P <0.001).

[0018] Table 1. Effect of compound 1 on Aβ 25-35 Effects of induced HT22 cell damage

[0019] We also tested it without Aβ 25-35 The effects of compound 1 on cell viability under intervention are shown in Table 2: Table 2. Effect of compound 1 on N2a cell viability

[0020] Aβ cells in each group1-42 The relative secretion levels are shown in Table 3 (Note: compared with the control group). # P <0.05, ## P <0.01, ### P <0.001; compared with the model group* P <0.05,** P <0.01, *** P <0.001).

[0021] Table 3. Effects of compound 1 on Aβ in APPswe-stabilized HT22 cells 1-42 The effect of secretion

Claims

1. Use of compound 1, as shown in the following chemical structural formula, or a pharmaceutically acceptable salt thereof, in the preparation of anti-Alzheimer's disease drugs: 。 2. A method for preparing compound 1, comprising the following reaction: 。 3. The method as described in claim 2, characterized in that, The molar amounts of each substrate used in the preparation were the same.

4. The method as described in claim 2, characterized in that, The reaction temperature is room temperature.

5. The method as described in claim 2, characterized in that, The reaction solvents are dichloromethane and hexafluoroisopropanol.

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