阅读说明：本技术 一种柚皮苷衍生物、制备方法及其在制备治疗心血管类疾病的药物中的应用 (Naringin derivative, preparation method and application thereof in preparation of medicine for treating cardiovascular diseases ) 是由 李峰 李文保 于 2021-03-15 设计创作，主要内容包括：本发明提供一种柚皮苷衍生物,其具有通式(I)所示结构,本发明还提供了所述柚皮苷衍生物的制备方法和在制备心血管类疾病的药物中的应用,通过药理学实验证明,上述柚皮苷衍生物具有显著减肥、降血脂的生物学活性。柚皮苷衍生物具有一定的减肥、减脂的趋势,并且均具有良好的安全性,对于开发新型的减肥、降血脂等心血管类疾病的治疗药物具有重要意义。(The invention provides a naringin derivative which has a structure shown in a general formula (I), and also provides a preparation method of the naringin derivative and application of the naringin derivative in preparation of medicaments for treating cardiovascular diseases. The naringin derivative has certain tendency of losing weight and reducing fat, has good safety, and has important significance for developing novel medicaments for treating cardiovascular diseases such as losing weight, reducing blood fat and the like.)

1. A naringin derivative is characterized in that: it has a structure shown in a general formula (I):

；

in the formula: r is selected fromOrWherein n = 1-5.

2. The naringin derivative of claim 1, wherein: the naringin derivatives are specifically P-1 and P-3, and the structural formulas are as follows:

。

3. a process for preparing the naringin derivative of claim 2, wherein: the preparation method of the P-1 comprises the following steps:

dissolving naringin in acetone, performing condensation reaction with salicylic acid protected by benzyl under the action of a condensing agent to obtain naringin derivative P-2, and removing the benzyl protection to obtain P-1;

the structural formula of the P-2 is as follows:

；

the preparation method of the P-3 comprises the following steps: naringin is dissolved in N, N-dimethyl formamide, and then is subjected to etherification reaction with bromoethanol under the action of alkali to obtain a naringin derivative P-3.

4. The method of claim 3, wherein: the condensing agent is one or more of dicyclohexylcarbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and diisopropylcarbodiimide; the alkali is one or more of potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, triethylamine, diisopropylethylamine or 4-dimethylaminopyridine.

5. The method of claim 4, wherein: the condensing agent is one or two of dicyclohexylcarbodiimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; the alkali is one or more of potassium carbonate, potassium hydroxide and triethylamine.

6. The method of claim 3, wherein: the preparation method of the P-1 comprises the following steps: reacting naringin, condensing agent and 2- (benzyloxy) benzoic acid at room temperature for 24h, filtering to obtain mother liquor, adding palladium-carbon, hydrogenating, reacting at room temperature for 24h, filtering, concentrating the mother liquor, evaporating to dryness, and performing column chromatography to obtain P-1.

7. The method of claim 6, wherein: the molar ratio of the naringin to the condensing agent is 1: 1.2; the molar ratio of the naringin to the 2- (benzyloxy) benzoic acid is 1:1, and the mass ratio of the naringin to the palladium-carbon is 10: 1; the palladium carbon is 10wt% of palladium carbon.

8. The method of claim 3, wherein: dissolving naringin in a solvent, adding alkali, stirring at room temperature for reaction for 2h, adding bromoethanol, reacting at 80 ℃ overnight, cooling, extracting, and purifying by column chromatography to obtain P-3; the molar ratio of the naringin to the alkali is 1: 2.1; the molar ratio of the naringin to the bromoethanol is 1:1.

9. The use of the naringin derivative of claim 1 in the manufacture of a medicament for the treatment of cardiovascular disease.

10. Use according to claim 9, characterized in that: the medicine is used for losing weight and reducing blood fat.

Technical Field

The invention belongs to the technical field of chemistry and medicine, relates to a medicinal compound, a preparation method and application thereof, and particularly relates to a naringin derivative, a preparation method thereof and application thereof in preparing a medicament for treating cardiovascular diseases.

Background

Naringin, also known as naringin and isohesperidin, is derived from immature or near mature dry pericarp and pulp of Citrus plant of Rutaceae, and is a natural flavonoid compound. In recent years, research on clinical application of naringin finds that the naringin plays a positive role in treating anti-inflammatory pain easing, weight losing, resisting cancers and preventing cardiovascular diseases.

Obesity is a disorder of fat metabolism accompanied by inflammatory reactions, and is a major risk factor for inducing many chronic diseases, such as diabetes, cardiovascular diseases, and cancer. Many drugs for treating obesity are classified into appetite suppression, energy consumption increase, intestinal digestion and absorption inhibition, and plants and other products under study, such as obesity gene products, according to their action mechanisms. The total of 6 medicines are on the market abroad for treating obesity, and in addition, sibutramine and rimonabant are on the market and are withdrawn from the market, because the two medicines cause serious adverse reactions: sibutramine can cause an increase in cardiovascular disease, while rimonabant can lead to risks of depression and suicide.

Cardiovascular and cerebrovascular diseases such as hypertension, cerebral infarction and atherosclerosis pose serious threats to human health. Naringin can reduce blood viscosity, reduce thrombosis, protect cardiovascular system and cerebrovascular system, improve cardiac function, and regulate cardiovascular system.

Although naringin has various pharmacological activities, the clinical application of naringin is limited by the defects of low in-vivo bioavailability and the like, so that the naringin is chemically modified and reformed on the basis of the structure of the naringin to enhance the druggability, and the naringin has important significance for developing novel medicaments for treating cardiovascular diseases such as weight loss, blood fat reduction and the like.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a naringin derivative and a preparation method thereof; in addition, the invention also provides application of the naringin derivative in preparing medicines for treating cardiovascular diseases, such as losing weight, reducing blood fat and the like.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides a naringin derivative which has a structure shown in a general formula (I):

；

in the formula: r is selected fromOr(ii) a Wherein n = 1-5.

Specifically, n is 1, 2, 3, 4 or 5.

Further, the naringin derivatives are P-1 and P-3, and the structural formulas are as follows:

the invention also provides a preparation method of the naringin derivative, and the preparation method of the P-1 comprises the following steps:

performing condensation reaction on naringin and salicylic acid protected by benzyl under the action of a condensing agent to obtain a naringin derivative P-2, and removing the benzyl protection to obtain P-1;

；

the preparation method of the P-3 comprises the following steps: the naringin and bromoethanol are subjected to etherification reaction under the action of alkali to prepare a naringin derivative P-3.

Further, the condensing agent is one or more of Dicyclohexylcarbodiimide (DCC), 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and Diisopropylcarbodiimide (DIC); the alkali is one or more of potassium carbonate, sodium carbonate, cesium carbonate, potassium hydroxide, sodium hydroxide, triethylamine, diisopropylethylamine and 4-dimethylaminopyridine.

Further, the condensing agent is one or two of Dicyclohexylcarbodiimide (DCC) and 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC); the alkali is one or more of potassium carbonate, potassium hydroxide and triethylamine.

The preparation method of the P-1 comprises the following steps: reacting naringin with condensing agent and 2- (benzyloxy) benzoic acid at room temperature for 22-26h, filtering to obtain mother liquor, adding palladium carbon, hydrogenating, reacting at room temperature for 22-26h, filtering, concentrating the mother liquor, evaporating to dryness, and performing column chromatography to obtain P-1.

The molar ratio of the naringin to the condensing agent is 1: 1.1-1.3; the molar ratio of the naringin to the 2- (benzyloxy) benzoic acid is 1:0.9-1.1, and the mass ratio of the naringin to the palladium carbon is 9-11: 1; the palladium carbon is 9-11wt% of palladium carbon.

Dissolving naringin in a solvent, adding alkali, stirring at room temperature for reaction for 1.8-2.2h, adding bromoethanol, reacting at 78-82 ℃ overnight, cooling, extracting, and purifying by column chromatography to obtain P-3; the molar ratio of the naringin to the alkali is 1: 2-2.2; the molar ratio of the naringin to the bromoethanol is 1: 0.9-1.1.

The invention also provides application of the naringin derivative in preparing a medicament for treating cardiovascular diseases.

Furthermore, the medicine is used for losing weight and reducing blood fat.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention provides a naringin derivative, a preparation method thereof and application thereof in preparing a medicament for treating cardiovascular diseases. The naringin derivatives have certain tendency of losing weight and reducing fat, have good safety and have further development value.

Drawings

Figure 1 shows the effect of test samples on body weight of rats in the high fat model, wherein p <0.01vs. blank, # p <0.05, # p <0.01vs. model.

Figure 2 is a graph of the effect of test samples on food intake in high fat model rats, wherein # p <0.05 vs.

Figure 3 is a graph of the effect of test samples on body fat in rats in the high fat model, where p <0.01vs. blank, # p <0.05, vs. model.

Fig. 4 is a graph of the effect of test samples on blood lipids in high lipid model rats, wherein p <0.01vs. blank, # p <0.05, # p <0.01vs. model;

wherein 4a is the effect of the test sample on high fat model rat Triglycerides (TG);

4b is the effect of the test sample on high fat model rat cholesterol (TCHO);

4C is the effect of the test sample on low density lipoprotein-cholesterol (LDL-C) in a high fat model rat;

4d is the effect of the test sample on high density lipoprotein cholesterol (HDL-C) in a high fat model rat.

FIG. 5 is a graph showing the effect of test samples on aortic root lipid accumulation in a high lipid model rat;

wherein 5a is a picture of aortic root lipid accumulation of a blank group of rats;

5b is a picture of aortic root lipid accumulation of a rat in a model group;

5c is the effect of the test sample on the lipid accumulation at the root of the aorta of a rat with a high fat model in the orlistat group;

5d is the influence of the simvastatin group tested samples on the lipid accumulation of the aortic root of a rat in a high-fat model;

5e is the influence of the test samples of the P-1 group on the lipid accumulation of the aortic root of a rat in a high-fat model;

5f is the effect of the test samples of group P-3 on lipid accumulation in the aortic root of rats in the high lipid model.

Detailed Description

The present invention will be further described with reference to the following examples.

Example 1: the synthetic route of the naringin derivative P-1 is as follows:

；

adding naringin (5.80 g, 10 mmol) into acetone (50 mL), sequentially adding Dicyclohexylcarbodiimide (DCC) (2.47 g, 12 mmol) and 2- (benzyloxy) benzoic acid (2.28 g, 10 mmol), reacting at room temperature for 24 hours, filtering to remove white solid, directly adding (0.58 g) 10% palladium-carbon into mother liquor without purification, hydrogenating, reacting at 25 ℃ for 24 hours, filtering, concentrating and evaporating the mother liquor, and performing column chromatography to obtain P-1 with the yield of 55%.

¹H NMR (500 MHz, DMSO-d ₆) δ = 12.64 (s, 1H), 10.85 (s, 1H), 8.45 (d, 1H), 8.10 (dd, 1H), 7.61-7.53 (m, 1H), 7.35 (d, 2H), 7.29 (d, 2H), 7.22 (t, 1H), 6.37 (d, 1H), 6.20 (d, 1H), 5.51 (d, 1H), 5.31 (d, 1H), 5.10 (d, 1H), 4.77-4.75 (m, 2H), 4.65 (t, 1H), 4.51 (d, 1H), 4.29 (t, 1H), 4.20 (d, 1H), 3.70-3.67 (m, 1H), 3.61 (s, 1H), 3.56-3.50 (m, 2H), 3.49-3.45 (m, 2H), 3.43-3.38 (m, 3H), 3.32-3.26 (m, 3H), 1.13 (s, 3H) ppm. ESI-MS：（m/z，%）= 701 [M+H]⁺。

Example 2: the synthetic route of the naringin derivative P-3 is as follows:

dissolving naringin (5.80 g, 10 mmol) in N, N-dimethylformamide (30 mL), adding potassium carbonate (28.4 g, 21 mmol), stirring at room temperature for reaction for 2h, adding bromoethanol (13.1 g, 10 mmol), reacting at 80 deg.C overnight, pouring into cold water, extracting with dichloromethane, and purifying by column chromatography to obtain P-3 with yield of 65%.

¹H NMR (500 MHz, DMSO-d ₆) δ = 7.30 (d, 2H), 6.89 (d, 2H), 6.37 (d, 1H), 6.20 (d, 1H), 5.51 (d, 1H), 5.31 (d, 1H), 5.10 (d, 1H), 4.78- 4.75 (m, 2H), 4.65 (t, 1H), 4.51 (d, 1H), 4.33-4.29 (m, 3H), 4.20 (d, 1H), 3.70 – 3.67 (m, 3H), 3.61 (s, 1H), 3.58-3.50 (m, 2H), 3.46-3.41 (m, 2H), 3.45-3.38 (m, 3H), 3.33-3.26 (m, 3H), 1.12 (s, 3H) ppm. ESI-MS：（m/z，%）= 625 [M+H]⁺。

Example 3: in vitro lipid-lowering activity test for measuring naringin derivatives

1. Experimental materials:

DMEM medium, American FBS serum, glutamine, penicillin, streptomycin, 96-well plate, human liver cancer cell HepG2, oil red O, isopropanol, DMSO and the like.

2. The experimental method comprises the following steps:

1) 12000 cells of HepG2 cells in the logarithmic growth phase are inoculated in a 96-well plate at 100 mu l/well;

2) after 12h, the fusion degree reaches 70-80%, and then the culture medium is replaced by serum-free DMEM medium, 80 mu l of the culture medium is used for starving for 12 h. After 12h, the blank was added to 20. mu.l serum-free medium and the other groups were added to 10. mu.l/well of inducer OA (final concentration 80. mu.M). On the basis, the model group is supplemented with 10 mul of serum-free culture, the administration group is added with 10 mul of the compound to be tested, the final concentration is 10 mul, and the culture box is incubated for 24 h;

3) after 24h incubation, removing the culture medium, washing with PBS (room temperature) buffer solution for 1 time, adding 80 μ l of 4% paraformaldehyde fixing solution into each well, fixing at room temperature for 0.5h, washing with PBS for 1 time, rinsing with 60% isopropanol for 10min, adding 60 μ l of 0.3% oil red O (Sigma O0625) dye solution into each well, dyeing at room temperature for 1h, and washing with PBS buffer solution for 3 times;

4) dissolved in DMSO, 100. mu.l/well, and OD measured at 358nm with microplate reader.

3. The results are shown in Table 1:

as can be seen from Table 1, the OD values in the P-1 and P-3 groups were the highest, indicating that the results of in vitro lipid lowering were better.

Example 4: test for determining in vivo lipid-lowering activity of naringin derivatives

1. Experimental materials:

test compounds: the concentration of P-1 and P-3 is 100mg/kg

Experimental animals: male SD rats 72, 100 ± 5g in weight, purchased from denppone experimental animal breeding ltd, license number: SCXK ru 20140007.

2. Experimental methods

All rats were fed basal diet for 7 days, fasted for 12h, and then randomly divided into 8 groups by weight: blank group, model group, positive drug orlistat group (40 mg/kg), positive drug simvastatin group (5 mg/kg), P-1 group (100 mg/kg), P-3 group (100 mg/kg), 9 rats in each group, wherein the blank group is continuously fed with basal feed, the other groups are fed with high fat feed, blood is collected to detect blood lipid level after feeding for 4 weeks, and the success of molding is determined by combining the weight gain level.

After 4 weeks of high fat feeding, each group was given a corresponding sample for gavage for 1 time/day for 4 consecutive weeks, during which time body weight and food intake were recorded. Grouping and processing is shown in table 2 below:

the treatment refers to the intragastric administration according to the specification.

After 4 weeks of administration, fasting for 12h, 10% chloral hydrate anesthesia of rats in each group, blood was taken from abdominal aorta, serum was centrifuged at 2500rpm for 15min, Triglyceride (TG), cholesterol (TCHO), low density lipoprotein cholesterol (LDL-C) and high density lipoprotein cholesterol (HDL-C) contents were measured, the abdominal cavity was opened to take out epididymis and perirenal fat, and wet weight was weighed and recorded on an electronic balance.

Taking the heart of each group of rats, freezing and slicing the cross section of the aortic root, fixing the heart in 10% neutral formalin for 15min, washing the heart with distilled water for 2min, staining the heart with 60% isopropanol water solution for 2s, staining the heart with oil red O working solution for 15min, staining the heart with 60% isopropanol water solution for 2s, washing the heart with distilled water for 2min, staining the heart with Mayer's hematoxylin for 8min, washing the heart with water for 1min, differentiating the heart for 2s, washing the heart with water for 1min, returning blue for 1s, washing the heart with water for 1min, sealing the heart with glycerol, and.

3. Test results and analysis

(1) Effect of test samples on weight and food intake of high-fat model rats

The initial body weights of the rats in all groups are balanced, the difference is not significant, and the body weight of the rats is obviously increased compared with that of a blank group after the rats are fed with high-fat feed. After the sample is treated for 4 weeks, the rats in each group are in good condition, the positive drug orlistat and the samples P-1 and P-3 can obviously reduce the body weight of the rats, the samples P-1 and P-3 can obviously reduce the food intake of the rats (P <0.05), and the body weight and the food intake of the other groups have no significant difference. The samples P-1 and P-3 were presumed to have weight-loss activity, and the mechanism thereof might be related to the inhibition of intake (Table 3, FIGS. 1 and 2).

Note: model p <0.01vs. blank, # p <0.05, # p <0.01vs. model

(2) Influence of test sample on body fat of rat with high fat model

The fat coefficient is the percentage of fat pad around kidney and testis and the weight of the rat, and reflects the fat amount in the rat and the obesity degree of the rat. As can be seen from Table 4, the fat weight and fat coefficient of the model group were significantly increased (P <0.01) compared to the blank group, indicating that the feeding of high-fat diet can aggravate the accumulation of fat in the body of rats; compared with the model group, the orlistat and the samples P-1 and P-3 have obvious reduction and obvious difference (P <0.05) in fat weight and fat coefficient, which indicates that the samples P-1 and P-3 can reduce the fat accumulation in the body of the rat.

Note: p <0.01vs. blank, # p <0.05, vs. model.

(3) Influence of test sample on blood lipid of high-fat model rat

After 4 weeks of administration, TCHO and LDL-C levels were significantly increased (P <0.01) and HDL-C levels were significantly decreased (P <0.01) in the model group compared to the blank group, indicating that the hypercholesterolemic and hyperlipidemic models of the rat fed with the high-fat diet were successfully replicated.

Simvastatin, a positive drug, significantly reduced TCHO and LDL-C levels in rats (P <0.05), and P-3 reduced LDL-C and increased HDL-C levels, which were significantly different from the model group (P <0.05) (Table 5, FIG. 4).

Note: model p <0.01vs. blank, # p <0.05, # p <0.01vs. model

(4) Effect of test samples on lipid accumulation in aortic root of rat in high-lipid model

The aortic root is dyed with oil red O, so that the vessel wall of a blank group has no lipid accumulation, the lipid accumulation phenomenon in the blood vessel of a model group is obvious, after the drug treatment is carried out for 4 weeks, the rats of the orlistat and simvastatin groups have no lipid accumulation, each sample group has the lipid accumulation phenomenon of different degrees, the dyeing result of the oil red O is basically consistent with the blood lipid result of each group of the rats (figure 5), and the result shows that the samples have a certain lipid-lowering tendency.

(5) And (3) safety evaluation: in vivo experiments show that all the rats do not die, which indicates that all the tested compounds have good safety.

The experiment shows that in the experimental model, the compound P-3 has the effects of reducing weight, inhibiting food intake, reducing body fat accumulation and regulating apolipoprotein level at the administration concentration of 100 mg/kg; the compound P-1 has the effects of reducing weight, inhibiting food intake and reducing body fat accumulation; all tested compounds show certain tendency of losing weight and reducing fat on different indexes, have good safety and have further development value.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions.