A compound, its preparation and use

Abstract

The application discloses a novel derivative, a preparation method and medical application thereof, and the derivative can be prepared by a one-pot reaction. Experimental data show that the derivative provided by the application can protect nerve cell damage caused by A beta, and has a prospect of development into a drug for treating AD. The derivative, the preparation method and the medical application thereof are not disclosed in the prior art.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and relates to a new compound, in particular to a silybin derivative and a preparation method and medical use thereof. BACKGROUND

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease, and is characterized by memory impairment, aphasia, agnosia and behavior change and other dementia. AD has a high incidence, a long course and great harm, and has become one of the important diseases in the world and a major public health and scientific problem affecting the economic and social development of China with the global aging. The currently used AD treatment drugs in the clinic mainly include acetylcholinesterase inhibitors (donepezil, rivastigmine, huperzine A and galantamine) and NMDA receptor antagonist memantine, but these drugs can only improve the related symptoms and cannot delay and prevent the development of the disease or achieve the effect of curing 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 brain amyloid beta (Aβ), thereby causing 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.

[0003] 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, and it is generally considered that the substitution of the E ring has a great and unpredictable effect on the properties of the parent compound. Some documents introduce various groups, but only some halogen or aromatic ring substituents have good effects. SUMMARY

[0004] The present application aims to utilize the particularity of the silybin skeleton to chemically modify the 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.

[0005] The above purpose of the present application is achieved by the following technical scheme:

[0006] A silybin derivative as shown in the following chemical structural formula 1 or a pharmaceutically acceptable salt thereof

[0007]

[0008] The preparation route and steps of the above compound 1 are as follows:

[0009]

[0010] The molar amount of each substrate used in the preparation can be the same, the temperature can be room temperature, the solvent used can be DCM and HFIP, the volume ratio of the two can be 1:1, and the step can be stirring ethyl glyoxylate and thiomorpholine at room temperature first, then adding the asiaticoside to continue the reaction to obtain the target compound shown in structural formula 1. As an example, the ethyl glyoxylate and thiomorpholine are first dissolved in DCM.

[0011] The application also discloses the medical use of the compound 1 for preparing an anti-AD drug. It is unexpectedly found that the compound 1 thus modified can greatly protect the nerve cell damage caused by A beta, and has the prospect of developing into a therapeutic drug for AD. DETAILED DESCRIPTION

[0012] Example 1: Preparation and structural characterization of compound 1

[0013]

[0014] Preparation of compound 1: ethyl glyoxylate (102 mg, 1 mmol) and thiomorpholine (103 mg, 1 mmol) were dissolved in 2 mL of dichloromethane (DCM), stirred at room temperature for 3 hours, then asiaticoside (482 mg, 1 mmol) and 2 mL of hexafluoroisopropanol (HFIP) were added, and stirring was continued at room temperature overnight. After the reaction was completed, the solvent was removed under reduced pressure, then the crude product was dissolved in ethyl acetate, washed twice with saturated brine. The organic phase was dried and concentrated, then purified by column chromatography (petroleum ether / ethyl acetate = 1:1-1:2) to obtain compound 1 (200 mg, yield 30%), white solid. Spectral data: 1 HNMR (500 MHz, Chloroform-d) δ 11.76 (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). 13CNMR (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] + .

[0015] Example 2: Anti-AD activity test of compound 1

[0016] 1. All experimental materials are obtained by public access or prepared conventionally, wherein:

[0017] Aβ 25-35 HT22 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences; Aβ 1-42 ELISA Kit was purchased from Elabscience (China) Co., Ltd.; APPswe overexpression lentivirus was purchased from Shanghai Jikai Gene Technology Co., Ltd.

[0018] Aβ 25-35 Preparation of oligomers: 1 mg of Aβ 25-35 was dissolved in 1 mL of sterile ddH2O to prepare a 1 mg / mL Aβ 25-35 solution, which was aged in a 37°C incubator for 7 days.

[0019] 2. Experimental methods

[0020] 1) Cell culture

[0021] The mouse neuroblastoma cells N2a stored in liquid nitrogen were taken out and quickly placed in a 37°C water bath for oscillation until the cells were thawed, then centrifuged at 1000 rpm for 5 min, the supernatant was discarded, a small amount of culture medium containing 10% fetal bovine serum and 1% double antibody was added and gently blown and sucked to uniform, the cells were transferred to a culture dish and cultured in a 37°C, 5% CO2 cell incubator.

[0022] 2) Anti-AD activity test of compound 125-35 Evaluation of protective effect of induced nerve cell injury

[0023] N2a cells in logarithmic growth phase were prepared into a cell suspension with a concentration of 1.0 x 10 5 After 24 h of culture, the cells were treated according to the following grouping, with 5 replicate wells in each group:

[0024] Control group: the medium was replaced with fresh medium and the cells were cultured for another 3 h;

[0025] Aβ 25-35 Model group: the medium was replaced with fresh medium and the cells were cultured for another 3 h;

[0026] Donepezil hydrochloride group (positive drug group): the medium was replaced with fresh medium containing 20 μM donepezil hydrochloride and the cells were cultured for another 3 h;

[0027] Silybin group: the medium was replaced with fresh medium containing 20 μM silybin and the cells were cultured for another 3 h;

[0028] Compound 1 group: the medium was replaced with fresh medium containing 10, 20 or 40 μM compound 1 and the cells were cultured for another 3 h.

[0029] After 3 h of culture according to the above grouping, the Aβ 25-35 in the model group, donepezil hydrochloride group, silybin group and compound 1 group was added to a final concentration of 15 μM, and the same volume of solvent was added to the control group, and the cells were cultured for another 24 h. 25-35 After 24 h of culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were cultured at 37°C for another 4 h.

[0030] After 4 h of culture, the supernatant in each well was removed, 150 μL of DMSO was added to each well, and the cells were shaken for 10 min to fully dissolve the crystals. The optical absorbance (OD value) of each well was determined on an enzyme-linked immunoassay instrument at a wavelength of 490 nm. The average OD value of 6 replicate wells was calculated, and the cell survival rate was calculated, with the control group being taken as 100%.

[0031] 3) Effect of compound 1 on Aβ 1-42 secretion in APPswe stably transfected HT22 cells

[0032] Logarithmic growth phase of HT22 cells were selected and inoculated into 96-well plates at 4000-5000 cells per well, and adherent culture was performed for 12 h. After the cells adhered, the virus was added in an appropriate amount according to the MOI value and virus titer of lentivirus infection of HT22 cells to infect the cells, and a final concentration of 50 μg / mL of transfection reagent polybrene was added to improve the transfection efficiency. The cell culture plate was placed in the incubator for continuous culture for 12 h, and then the virus mixture in the virus infected well was removed and replaced with fresh culture solution for continuous culture. After 48 h of culture in the new culture solution, the culture medium containing 2 μg / mL puromycin was replaced, and the successfully infected cells were subjected to resistance screening for 5-7 generations. Then the cells were inoculated into a 6-well plate, and after the cells adhered, drug treatment was performed, and the groups were as follows: control group, APPswe stable HT22 group (model group), 20 μM donepezil hydrochloride group (positive drug group), 20 μM gynostemma pentaphyllum group, and compound 1 group (concentrations were 10, 20, and 40 μM, respectively). The cells were collected 24 h after drug addition, and the total protein was extracted for detection of Aβ 1-42 concentration by an ELISA kit.

[0033] 3. Data processing and analysis

[0034] The cell survival rates of each group were calculated by SPSS19.0 software, and one-way ANOVA test was used for comparison among multiple groups, and q test was used for comparison between groups. P<0.05 was considered statistically significant.

[0035] 4. Experimental results

[0036] The cell survival rates of each group are 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).

[0037] Table 1. Effect of compound 1 on Aβ 25-35 induced HT22 cell damage

[0038]

[0039] We also tested the effect of compound 1 on cell viability without the intervention of Aβ 25-35 , and the results are shown in Table 2:

[0040] Table 2. Effect of compound 1 on N2a cell viability

[0041]

[0042] Aβ 1-42The relative secretion amount is 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).

[0043] Table 3. Effect of compound 1 on Aβ secretion of APPswe stable-transfected HT22 cells 1-42

[0044]

[0045] ​

Claims

1. Compound 1, or a pharmaceutically acceptable salt thereof, as shown in the following chemical structural formula: 。

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