CN113402372A - Cryptotanshinone derivative, preparation method thereof and application of cryptotanshinone derivative in reducing blood fat and resisting obesity - Google Patents

Abstract

The invention discloses a cryptotanshinone derivative, a preparation method thereof and application of the cryptotanshinone derivative in reducing blood fat and resisting obesity. The cryptotanshinone derivative has the structural formula

Description

Cryptotanshinone derivative, preparation method thereof and application of cryptotanshinone derivative in reducing blood fat and resisting obesity

Technical Field

The invention relates to a novel cryptotanshinone derivative and a preparation method and application thereof.

Background

The traditional Chinese medicine salvia miltiorrhiza has obvious effects of promoting blood circulation to remove blood stasis and dredging heart envelope, and is applied to clinic in various forms, such as injection, dripping pills, tablets and the like. The main active ingredients are water-soluble salvianolic acid and fat-soluble tanshinone compounds. Among tanshinone compounds, tanshinone IIA has been developed as a drug for treating angina pectoris, coronary heart disease and myocardial infarction. Myocardial fibrosis is generally a common pathological consequence caused by coronary atherosclerosis and is characterized by an excessive synthesis and pathological accumulation of extracellular matrix proteins in the myocardial tissue, resulting in myocardial stiffness and in diastolic heart failure.

Obesity refers to a physical state resulting from a certain accumulation of fat in the body. The criteria for obesity are usually measured by the Body Mass Index (BMI), i.e., the weight divided by the height squared (kg/m)²) When the BMI is more than 30kg/m²It is known as obesity. In recent years, obesity has prevailed globally. The world health organization states that over 19 million adults are overweight in 2016, 6.5 million people are obese, and that at least 280 million deaths per year can be attributed to overweight or obesity and exhibit a tendency to be under-aged. Obesity has become one of the most important public health problems in the 21 st century.

Research has shown that obesity is associated with the occurrence of many diseases, and can be roughly divided into two categories: one is the effects due to increased body fat mass (including sleep apnea, degenerative arthritis); the second is the influence caused by the increase of fat cells in the body (comprising cardiovascular diseases, type II diabetes, non-alcoholic fatty liver, cancer and the like). In 2013, the American medical society formally recognized obesity as a disease. However, the development and pathological process of obesity are complicated and caused by the interaction of multiple factors, including environmental factors (exercise and diet), genetic factors, endocrine imbalance or stress, epigenetic factors, insulin resistance, etc., which increases the difficulty of developing new drugs. At present, only five weight-reducing drugs orlistat, Lorcaserin hydrochloride, phentermine/topiramate compound preparations, and sugar-reducing drugs of bupropion naltrexone sustained-release tablets and liraglutide exist in the market. However, these antiobesity agents have significant side effects and safety and tolerance are yet to be confirmed. Therefore, the development of a novel anti-obesity drug which can effectively inhibit the differentiation of fat cells and reduce the lipid level in the fat cells and has low toxicity has important research significance and application value. At present, most of researches are focused on molecular design and chemical synthesis, and few researchers develop weight-reducing medicines through a natural product, namely a huge treasury.

No research shows that the existing cryptotanshinone derivative has the application of reducing blood fat and resisting obesity.

Disclosure of Invention

The invention aims to provide a novel cryptotanshinone derivative, a preparation method thereof and application of the cryptotanshinone derivative in reducing fat and resisting obesity.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided:

a cryptotanshinone derivative and its acceptable medicinal salt, the structural formula of the cryptotanshinone derivative is shown as formula I:

in a second aspect of the present invention, there is provided:

the process for preparing a cryptotanshinone derivative according to the first aspect of the present invention comprises the steps of:

s1) dissolving cryptotanshinone in organic solvent, adding oxalyl chloride, stirring and reacting completely;

s2) separating and purifying the reactant to obtain the compound shown in the formula I.

In some examples, the organic solvent is methylene dioxide.

In some examples, oxalyl chloride is added in excess relative to cryptotanshinone.

In some examples, the reaction temperature is 20 to 30 ℃.

In some examples, the separation and purification method is forward silica gel column chromatography, and the silica gel with 300-400 meshes is used as a stationary phase; 5% dichloromethane-petroleum ether mixed solvent is used as a mobile phase, and the compound of the formula I can be obtained by separation and purification.

In a third aspect of the present invention, there is provided:

a lipid-lowering or anti-obesity composition, wherein the active ingredient of the composition comprises at least one of cryptotanshinone derivatives according to the first aspect of the present invention and pharmaceutically acceptable salts thereof.

In some examples, the composition is an oral formulation.

In a fourth aspect of the present invention, there is provided:

the cryptotanshinone derivative and the acceptable medicinal salt thereof in the first aspect of the invention are applied to the preparation of the composition for reducing blood fat or resisting obesity.

The invention has the beneficial effects that:

the inventor prepares a novel cryptotanshinone derivative (a compound shown in a formula I) for the first time, and researches show that the cryptotanshinone derivative can effectively inhibit lipogenesis, has good lipid-lowering activity, and is expected to be developed into a novel lipid-lowering anti-obesity drug.

Drawings

FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of a compound of formula I;

FIG. 2 is a carbon spectrum and DEPT135 spectrum of a compound of formula I;

FIG. 3 shows the results of the effect of different concentrations of a compound of formula I on the rate of decrease of triglyceride TG levels;

FIG. 4 shows the results of oil red O staining after treatment with 1. mu.M of a compound of formula I;

FIG. 5 shows the results of oil red O staining after treatment with 5. mu.M of a compound of formula I;

FIG. 6 shows the results of oil red O staining after treatment with 10. mu.M of a compound of formula I.

Detailed Description

The invention obtains the subsidy of the project name 'research and development of innovative drugs and key technology for serious diseases related to inflammation' (number 2017BT01Y 093).

The technical scheme of the invention is further explained by combining experiments.

Preparation and isolation of cryptotanshinone derivatives (compounds of formula I):

dissolving cryptotanshinone in dichloromethane, adding oxalyl chloride with 2 times of equivalent weight, and stirring at room temperature for reaction for 24 hours to obtain the compound. Spin-drying the obtained reaction solution in a rotary evaporator, and then separating by using a forward silica gel column chromatography, wherein the silica gel with the size of 300-400 meshes is used as a stationary phase; 5 percent of dichloromethane-petroleum ether mixed solvent is used as a mobile phase to obtain the compound shown in the formula I

The structure confirmation data of the cryptotanshinone derivative (compound shown in the formula I) are as follows:

¹H NMR(CDCl3,400MHz)δ7.96(1H,d,J＝8.3Hz,H-9),7.88(1H,d,J＝8.3Hz,H-10),4.53(1H,dd,J＝10.6and 6.4Hz,H-16a),4.44(1H,dd,J＝10.6and 6.4Hz,H-16b),3.75(1H,dq,J＝14.2and 7.0Hz,H-15),3.23(2H,t,J＝6.34Hz,H-1),1.98(3H,s,H-17),1.85(2H,m,H-2),1.73(2H,m,H-3),1.39(3H,d,J＝7.3Hz,H-18),1.37(6H,s,H-19,20).¹³C NMR(CDCl3,400MHz)δ184.0(C,C-11),180.3(C,C-12),172.6(C,C-14),155.3(C,C-13),146.6(C,C-5),141.7(C,C-6),134.0(CH,C-10),132.8(C,C-8),130.0(C,C-7),125.9(CH,C-9),67.7(CH₂,C-16),39.0(CH₂,C-1),37.3(CH,C-15),36.1(C,C-4),32.1(CH₃×2,C-18,19),31.1(CH₂,C-3),20.7(CH₃,C-17),20.3(CH₂,C-2),15.3(CH₃,C-18).

HRMS(ESI)m/z:[M+H]⁺Calcd for C₂₀H₂₃ClO₃ 3147.1359；Found 347.1348.

effect of Compounds of formula I on the triglyceride content in adipocytes

The compound lipid-lowering test uses a mouse preadipocyte 3T3-L1 cell differentiation model, adopts oil red O staining and combines with triglyceride quantitative detection to evaluate the influence of the anti-obesity compound provided by the invention on the content of triglyceride in fat cells. 3T3-L1 preadipocytes, 5.0 x 10, in logarithmic growth phase⁴Cells/well, evenly inoculating to a 48-well plate, statically culturing in a cell culture box, and replacing the culture solution every two days. When the cells are fused at a growth rate close to 80%, the culture medium is replaced, the cells are cultured for 2 days until the cells are completely fused (Day 0), the DMEM complete culture medium containing the differentiation inducing liquid I (the DMEM culture medium containing 10% FBS and 1% double antibody) is replaced, and the temperature is 5% at 37%CO₂The cells were cultured for 3 days (Day 3). After 3 days, the culture was continued for 3 days by replacing the DMEM complete medium containing the differentiation-inducing liquid II (Day 6). For the drug intervention group, the compound solution of formula I was diluted to a certain concentration with DMEM complete culture containing differentiation-inducing solution as diluent, and added together with Day 0 and Day 3. The blank control group and the differentiation control group are added with DMSO solutions with equal volumes respectively. At Day 6, photographs were taken of oil red O staining and analysis of triglyceride content.

(1) Preparation of differentiation-inducing liquid

Differentiation-inducing liquid i: DMEM complete medium containing 500. mu.M 3-isobutyl-1-methyl-xanthine, 100ng/mL dexamethasone, 2. mu.g/mL insulin.

Differentiation-inducing liquid ii: DMEM complete medium containing 2. mu.M insulin containing the elicitor.

(2) Oil red O dyeing

When the cells are induced to differentiate to Day 6, the cells are rinsed 1 time by precooled PBS, and 4% frozen paraformaldehyde fixing solution is fixed for 60min at room temperature. Dyeing is carried out for 30min at room temperature by using 0.3% oil red O dyeing working solution. Deionized water rinsing 2-3 times at room temperature and photographing by an inverted microscope (40 times). And respectively adding 300uL of isopropanol solution into each hole, gently shaking a shaking table to extract the oil red O dye at room temperature for 30min, and respectively transferring 100 mu M dye solution to perform absorbance detection at 510 nm.

(3) Analysis of triglyceride content

After cell differentiation is finished, precooling PBS for rinsing for 2 times, removing PBS completely, adding a deionized solution containing 0.2% Triton X-100, standing for 1h at room temperature, collecting cell suspension, carrying out ultrasonic disruption for 10min, fully cracking cells, centrifuging, collecting supernatant, and determining the content of triglyceride according to the specification of the triglyceride detection kit.

(4) Results of the experiment

The experimental results (fig. 3-6) show that with the increase of the dosage of the compound of formula i, the red region stained by oil red O in the cells is significantly reduced, which indicates that the compound of formula i can effectively inhibit lipogenesis and has a certain dosage dependence.

The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.

Claims (9)

1. A cryptotanshinone derivative and its acceptable medicinal salt, the structural formula of the cryptotanshinone derivative is shown as formula I:

2. the process for preparing a cryptotanshinone derivative according to claim 1, comprising the steps of:

s1) dissolving cryptotanshinone in organic solvent, adding oxalyl chloride, stirring and reacting completely;

s2) separating and purifying the reactant to obtain the compound shown in the formula I.

3. The method of claim 2, wherein: the organic solvent is methane dioxide.

4. The method of claim 2, wherein: oxalyl chloride is added in excess relative to cryptotanshinone.

5. The method of claim 2, wherein: the reaction temperature is 20-30 ℃.

6. The method of claim 2, wherein: the separation and purification method is forward silica gel column chromatographic separation, and silica gel with 300-400 meshes is used as a stationary phase; 5% dichloromethane-petroleum ether mixed solvent is used as a mobile phase, and the compound of the formula I can be obtained by separation and purification.

7. A lipid-lowering or anti-obesity composition characterized by: an active ingredient in the composition comprises at least one of the cryptotanshinone derivative of claim 1 and its pharmaceutically acceptable salt.

8. The composition of claim 7, wherein: the composition is an oral preparation.

9. Use of a compound for the preparation of a lipid-lowering or anti-obesity composition, characterized in that: the compound is the cryptotanshinone derivative according to claim 1 and pharmaceutically acceptable salts thereof.

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