CN107892673B

CN107892673B - Ligustrazine derivative and preparation method and application thereof

Info

Publication number: CN107892673B
Application number: CN201711473452.1A
Authority: CN
Inventors: 齐菲; 夏俊霞; 张在军; 王亮
Original assignee: Shenzhen Xia Xi Wan Medicine Science And Technology Co Ltd
Current assignee: Shenzhen Xia Xi Wan Medicine Science And Technology Co Ltd
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2020-01-03
Anticipated expiration: 2037-12-29
Also published as: CN107892673A

Abstract

The invention provides a ligustrazine derivative and a preparation method and application thereof, the ligustrazine derivative can treat hypoxia injury induced by IAA, can be used for treating apoplexy and a plurality of neurodegenerative diseases, and has the functions of promoting the proliferation of neuronal cells and promoting the growth of cell synapses.

Description

Ligustrazine derivative and preparation method and application thereof

Technical Field

The invention relates to a ligustrazine derivative and a preparation method thereof.

Background

Ligustrazine (TMP) is an active ingredient extracted from plants such as Ligusticum chuanxiong (Ligusticum chuanxiong) of Umbelliferae, Curcuma wenyujin (Curcuma aromatica) of Zingiberaceae, and Jatropha podagrica (Jatropha podagrica) of Euphorbiaceae, and can be detected in fermented products such as vinegar, Chinese liquor and natto fermented by Bacillus subtilis. It has several biological activities, such as resisting tumor, resisting inflammation, resisting apoptosis and dilating blood vessel, etc. so that it is widely used in clinical treatment of cardiac and cerebral vascular diseases. However, the prior art lacks of researches on ligustrazine derivatives.

Disclosure of Invention

The invention aims to provide a ligustrazine derivative and a preparation method thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: a ligustrazine derivative has a structural formula shown in formula (I),

the invention provides a preparation method of the ligustrazine derivative, which is characterized in that the intermediate product is synthesized by mixing ligustrazine and dibenzoyl peroxide as follows:

then reacting with sulfur and sodium sulfide to obtain a target compound, and specifically comprising the following steps:

(1) mixing ligustrazine and dibenzoyl peroxide, adding solvent carbon tetrachloride, stirring at 30 ℃, adding NBS, and then refluxing and stirring; after the TCL detection reaction is finished, filtering while hot, adding saturated sodium bicarbonate water solution, extracting the filtrate for 2 times by using dichloromethane, and carrying out column chromatography to obtain a compound 2;

(2) dissolving sulfur and sodium sulfide in water, reacting for half an hour at 50 ℃, and then cooling to room temperature; adding DDAB, dropwise adding a chloroform solution containing the compound 2 prepared in the step (1), reacting at room temperature, after TCL detection reaction, adding water and dichloromethane for extraction, evaporating under reduced pressure to remove a solvent, and performing column chromatography to obtain a compound 3, namely the ligustrazine derivative;

compound mass spectrometry data are as follows:

1H NMR(400MHz,DMSO-d6,δppm):4.02(s,4H)；2.65(s,6H)；2.52(m,12H)；MS(ESI):335.7(C16H23N4S2,[M+H]+)。

preferably, the dosage form of the ligustrazine derivative is tablets, capsules or granules.

The invention provides a pharmaceutical composition, which comprises the ligustrazine derivative and a pharmaceutically acceptable carrier.

The invention provides application of the ligustrazine derivative in preparing a medicament for treating IAA-induced hypoxia injury.

The invention provides the application of the ligustrazine derivative in preparing the medicine for promoting the proliferation of neuronal cells.

The invention provides application of the ligustrazine derivative in preparing a medicine for promoting cell synapse growth.

The invention provides application of the ligustrazine derivative in preparing a medicament for treating neurodegenerative diseases.

Preferably, the neurodegenerative disease is selected from the group consisting of alzheimer's disease, parkinson's disease, huntington's disease, senile dementia, creutzfeldt-jakob disease.

The invention has the beneficial effects that: the invention provides a ligustrazine derivative which can treat hypoxia injury induced by IAA, has the function of promoting the proliferation of neuronal cells and the function of promoting the growth of cell synapses, and can be used for treating apoplexy and various neurodegenerative diseases.

Drawings

FIG. 1 is a graph of the effect of a ligustrazine derivative on the synapse of a cell;

FIG. 2 is an in vitro cytotoxicity test of ligustrazine derivatives;

wherein TMP is tetramethylpyridine.

Detailed Description

The preparation process of the ligustrazine derivative comprises the following steps:

the synthesis process of the compound I is as follows:

weighing 10mmol of ligustrazine and 0.5mmol of dibenzoyl peroxide (BPO), adding 10ml of solvent carbon tetrachloride into a single-neck flask, and stirring at 30 ℃. NBS (1.77g, 10mmol) was added portionwise, followed by stirring at reflux. After the TCL detection reaction was completed, the reaction mixture was filtered while hot, 10ml of a saturated aqueous solution of sodium bicarbonate was added, the filtrate was extracted with 10ml of dichloromethane for 2 times, and column chromatography was performed to obtain compound 2(1.98 g, yield 92%).

Sulfur (5mmol) and sodium sulfide (10mmol) were dissolved in water (2.5ml), reacted at 50 ℃ for half an hour, and then cooled to room temperature. DDAB (4 mol%) was added, and a chloroform solution (2.5ml) of Compound 2(10mmol) was added dropwise. Reacting at room temperature, and detecting the reaction completion by TCL. Water (5 ml) and dichloromethane (10 ml) were added and extracted 2 times, the solvent was distilled off under reduced pressure, and column chromatography was performed to obtain the objective product 3(3.17 g, 95% yield).

Will N₂a-APP cells according to 1X 10⁴The samples were inoculated in 6-well plates, 24h later, the medium was changed to 1% FBS, and the medium containing the test compound was added to final concentrations of 0.1, 1, 10. mu.M, and a blank solvent control and a positive compound TMP control were set. After 48h, the growth of synapses was observed and recorded under a microscope, and 10 fields were randomly selected for statistics.

As can be seen from FIG. 1, Compound 3 has the effect of promoting the growth of synapses in cells.

Western Blot detection of the influence of the ligustrazine derivative on APP, APP alpha, APP beta and BACE1 protein expression and tau phosphorylation

1. Drug treatment

N2a-APP cells are inoculated into a 6cm culture dish according to the number of 1 × 105/mL cells, after 24 hours, compounds with final concentration of 0.1, 1 and 10 μ M are added, after 24 hours of action, the culture medium is discarded and washed with cold PBS for 2 times, 300 μ L of cell lysate is added, lysis is carried out for 10min, and centrifugation is carried out for 10min at 10000rpm after collection. Taking the supernatant for BCA protein quantification.

2. Electrophoresis and transfer of membranes

Proteins were diluted with water and 5 Xloading buffer to an equal concentration of samples, e.g., each diluted to 2. mu.g/. mu.L, depending on the initial concentration of protein. Heating at 100 deg.C in metal bath for 5min to completely denature protein.

The electrophoresis conditions were set to constant voltage 90V for 20min, and constant voltage 140V continued until the blue band reached the bottom of the gel. The PVDF is used for membrane conversion, the constant current is generally set to be 100mA, and the membrane conversion time is 120min, if the molecular weight of the protein is more than 100kD, the current can be adjusted to be 120-160 mA.

3. Antibody immune recognition and imaging

Blocking the NC membrane in 5% skimmed milk powder for 40min, and incubating the corresponding primary antibody [ APP (Abcam, ab 32136); APP α (IBL,11088), APP β (IBL, 10321); pS396(Abcam, ab 109390); pS262(Thermo Fisher, 44750G); pS202(Abcam, ab 108387); BACE1(Abcam, ab 2077); GAPDH (Abcam, ab37168) ], overnight at 4 ℃. TBST washing 3 times, each time for 5-10 min. Secondary antibody incubation was continued for 40-60min, followed by 3 TBST washes. Development was carried out using ECL luminescence solution (Millipore).

Experiments show that the ligustrazine Dimer (DTMP) promotes the generation of APP alpha and reduces the phosphorylation level of Tau-T202.

In vitro cytotoxicity test of ligustrazine derivative

Taking N in logarithmic growth phase₂a neuron cells, inoculating the cells in a 96-well culture plate, adjusting the cell concentration to 3X 10⁴Per/mL, 100. mu.L of cell suspension was added per well. Setting blank group, control group andgroups of samples of different concentrations (equal ratio set concentrations of 0.1, 0.3, 1, 3, 10, 30, 100, 300, 1000. mu.M, respectively) were provided with 4-6 parallel wells per group. Placing at 37 ℃ and 5% (V/V) CO₂After culturing for 48h, adding 10 mu LCCK-8 to each well, continuously culturing for 4h, detecting the absorbance (OD) value of each well at the wavelength of 450nm by using a microplate reader, and calculating the growth inhibition rate and the half inhibition concentration IC according to the formula₅₀：

Percent growth inhibition [% ] - (1-average OD value in experimental group/average OD value in control group) × 100%

IC₅₀The calculation uses a nonlinear regression calculation formula in Graph Pad and a 4-parameter calculation method of variable slope to obtain IC₅₀The value is obtained.

The calculation model is Y ═ Bottom + (Top-Bottom)/(1+10^ ((LogEC)₅₀-X) × HillSlope)), where Y is the inhibition at a particular concentration, X is the current log concentration, and Bottom and Top are the minimum and maximum inhibition.

As shown in fig. 2, the cytotoxicity exhibited by the ligustrazine dimer at higher concentrations.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of ligustrazine derivatives is characterized by comprising the following specific steps: (1) mixing ligustrazine and dibenzoyl peroxide, adding solvent carbon tetrachloride, stirring at 30 ℃, adding NBS, and then refluxing and stirring; after the TCL detection reaction is finished, filtering while hot, adding saturated sodium bicarbonate water solution, extracting the filtrate for 2 times by using dichloromethane, and carrying out column chromatography to obtain a compound 2; (2) dissolving sulfur and sodium sulfide in water, reacting for half an hour at 50 ℃, and then cooling to room temperature; adding DDAB, dropwise adding a chloroform solution containing the compound 2 prepared in the step (1), reacting at room temperature, after TCL detection reaction is finished, adding water and dichloromethane for extraction, evaporating under reduced pressure to remove a solvent, and performing column chromatography to obtain a target compound;