ASGR1 small-molecule inhibitor for preventing and treating hypercholesteremia as well as preparation method and application of ASGR1 small-molecule inhibitor

Abstract

The invention relates to an ASGR1 (sialoglycoprotein receptor 1) small-molecule inhibitor for preventing and treating hypercholesteremia as well as a preparation method and application of the ASGR1 small-molecule inhibitor. ASGR1 is a main subtype of a sialoglycoprotein receptor, is specifically expressed in the liver, mediates endoswallowing of blood asialoglycoprotein and transports the blood asialoglycoprotein to lysosome degradation, and inhibition or function deletion of ASGR1 is related to low cholesterol and reduction of CVD (Chemical Vapor Deposition) risk. The compound (I) provided by the invention is an ASGR1 inhibitor, can be used for preventing or / and treating cardiovascular diseases related to hypercholesterolemia and the like, can be used for preparing medicines for reducing the degree of fatty liver caused by high-fat diet and relieving liver injury, can be used for preparing medicines for reducing subcutaneous fat and visceral fat accumulation, and can be used for preparing medicines for preventing or / and treating cardiovascular diseases related to hypercholesterolemia and the like. The compound can be used for preparing medicines for reducing TC and LDL-C contents in plasma.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of medicines, and particularly relates to an ASGR1 small molecule inhibitor for preventing and treating hypercholesterolemia and a preparation method and application thereof. BACKGROUND

[0002] Cardiovascular disease (CVD) is a common chronic disease. According to the statistics of the World Health Organization, about 17 million people die of CVD every year, more than any other cause of death, so it is particularly important and urgent to develop drugs for treating CVD. Atherosclerosis is an important cause of CVD, and its occurrence and development is a long-term and very complex pathological process, and its pathogenesis mainly includes abnormal deposition of cholesterol in the arterial blood vessels, generation of inflammatory reactions and activation of the immune system, etc. A series of research evidences show that hypercholesterolemia is an extremely important risk factor for atherosclerotic diseases, and many studies show that cholesterol-lowering treatment can effectively reduce the mortality rate of cardiovascular diseases.

[0003] Based on the causes of atherosclerosis, a variety of treatment approaches are derived, such as through exercise, reducing the bad habits of smoking, drinking and high-fat and high-sugar diet, and using related drugs such as antihypertensive drugs, anti-inflammatory drugs or statin lipid-lowering drugs, etc. However, there are still many pain points in the drug treatment of atherosclerosis, such as unsatisfactory effect, side effects, etc. Statin drugs are currently the first-line drugs for preventing and treating atherosclerosis in clinical practice. In addition to the lipid-lowering function, it also has multiple effects on atherosclerotic plaques, it can stabilize the plaques and inhibit the oxidation of low-density lipoprotein (LDL) and the release of local NO. However, studies have shown that although statin drugs can reduce the mortality rate of cardiovascular events, the reduction is not obvious, and long-term and large-scale use of statin drugs can cause transaminase and creatine kinase to rise, causing liver and kidney function damage and rhabdomyolysis, and also causing adverse reactions such as new-onset diabetes. By finding drugs with new mechanisms of action for statin drugs, alternative drugs, combination drugs or alternating drugs can be used to alleviate the adverse reactions caused by long-term and single drug use.

[0004] In recent years, natural medicines such as flavonoids and active alkaloids have shown great potential and market prospects, and have attracted more and more attention from researchers. Black pepper, as a natural seasoning, has a long history of consumption all over the world; Piper longum is used as a traditional Mongolian medicine to treat hyperlipidemia and coronary heart disease. Piperine is the main component in black pepper and piper longum, and has immunomodulatory, antioxidant, anti-lipid metabolism disorder, anti-inflammatory, uric acid regulation and other pharmacological activities. Among them, the lipid-lowering effect of piperine has been confirmed, and it is a compound with great potential for drug development. Piperine has great potential as a lipid-lowering drug, but piperine has the problems of high effective dose and reproductive toxicity at high dose, therefore, based on these pain points, it is necessary to screen or synthesize derivatives based on piperine as a prototype, in order to obtain a lead drug with higher lipid-lowering activity and higher safety. SUMMARY

[0005] In view of the deficiencies of the prior art, the present application aims to provide a novel piperine derivative, which can be used as an ASGR1 small molecule inhibitor for preventing and treating hypercholesterolemia, and the inhibitor can efficiently and specifically inhibit the activity of ASGR1, thereby effectively reducing the cholesterol level. Meanwhile, the present application also provides a preparation method of the small molecule inhibitor and its application in preventing and treating hypercholesterolemia and the like, so as to meet the clinical demand for safe and effective drugs for treating hypercholesterolemia.

[0006] The technical scheme of the present application mainly includes the following contents: A compound, the structural formula of the compound is shown as the following formula I:

[0007] I.

[0008] The synthesis method of the compound comprises the following steps: (1) potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium are sequentially added to a toluene mixed solution of 3,5-dimethoxyiodobenzene and N,N-dibutyl-1,3-pentadienylamide; and the temperature of the reaction system is increased to 100 DEG C; (2) saturated sodium bicarbonate aqueous solution is added to quench the reaction, and the water phase is extracted with ethyl acetate; (3) the organic phases are combined and washed with saturated sodium chloride aqueous solution, and dried with anhydrous sodium sulfate; (4) after filtration, the filtrate is concentrated, separated and purified by column chromatography, and the eluent is a mixture of ethyl acetate and petroleum ether, to obtain a light yellow oily liquid product, which is the compound.

[0009] Preferably, in step (1), the reaction time is 6 hours.

[0010] Preferably, the volume ratio of the ethyl acetate and petroleum ether is 1:4.

[0011] Preferably, the molar ratio of 3,5-dimethoxyiodobenzene, N,N-dibutyl-1,3-pentadienylamide, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium is 2.68:2.68:5.34:0.13.

[0012] A medicine containing the compound and / or pharmaceutically acceptable salt of the compound, and pharmaceutically acceptable adjuvant. The medicine can be prepared into various dosage forms, such as oral dosage forms (such as tablets, capsules) or injections, according to actual needs.

[0013] The compound is used for preparing an ASGR1 protein inhibitor.

[0014] Further, the application of the compound further includes at least one of the following (1) to (4); (1) the application in preparing a medicine for preventing and treating hypercholesterolemia; (2) the application in promoting cell cholesterol efflux; (3) the application in increasing the expression level of cholesterol efflux transporters ABCG5, ABCA1, ABCG8 and / or SR-B1 protein; (4) the application in preparing a preparation for promoting animal fat metabolism; (5) the application in preparing a preparation for improving small intestinal villus damage caused by high-fat diet.

[0015] Further, the cell includes Huh-7 cells and / or HEK-293T cells.

[0016] Further, in the application (1) and (2), the concentration of the compound is 1-10 μM.

[0017] Further, the fat includes subcutaneous fat, visceral fat, liver fat, blood fat and / or fecal fat.

[0018] The beneficial effects of the present application are: The ASGR1 small molecule inhibitor of the present application can be specifically combined with ASGR1, effectively inhibit the function of ASGR1, and play a significant role in promoting cholesterol efflux of cells expressing ASGR1, thereby significantly reducing the cholesterol level and achieving the purpose of preventing and treating hypercholesterolemia.

[0019] The ASGR1 small molecule inhibitor of the present application has good safety. Studies have shown that PD with a concentration of 1-30 μM has no obvious toxic effect on the growth of HEK-293T cells.

[0020] The ASGR1 small molecule inhibitor of the present application has a low effective dose, and a piperine derivative of only 0.3 μM can produce a significant effect on the cholesterol efflux of Huh-7 cells, and when used for treating hypercholesterolemia in lipid-loaded mice, the dosage can be as low as 1-10 mg / kg.

[0021] The preparation method of the present application has mild reaction conditions, is easy to control, does not require expensive instruments and equipment and complex processes, and has a relatively low production cost, which is conducive to large-scale production and clinical popularization and application. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 Synthesis path and structure of piperine derivative PD.

[0023] Figure 2 NMR-carbon spectrum of piperine derivative PD.

[0024] Figures 3-5 NMR-hydrogen spectrum of piperine derivative PD.

[0025] Figure 6 Effect of piperine derivative PD on cholesterol efflux of Huh-7 cells. (A) Effect of PD on the viability of Huh-7 cells was determined by resazurin conversion method (n=3); (B) Cholesterol efflux activity of PD (0.1, 0.3, 1, 3, 10 μM) in Huh-7 cells was determined by Bodipy-Cholesterol fluorescence labeling method (n=4). All data are expressed as mean ± standard deviation, * p <0.05,** p <0.01,*** p <0.001, n.s. indicates no statistical significance (one-way ANOVA).

[0026] Figure 7 Verification of ASGR1 overexpression type HEK-293T cell model and effect of piperine derivative PD on cholesterol efflux thereof. (A) Effect of PD on the viability of HEK-293T cells was determined by resazurin conversion method (n=3); (B) Verification of ASGR1 overexpression type HEK-293T cell model (n=3); (C) Cholesterol efflux activity of PD (1, 3, 10 μM) in ASGR1 overexpression type HEK-293T cells was determined by Bodipy-Cholesterol fluorescence labeling method (n=5). All data are expressed as mean ± standard deviation, * p <0.05,*** p <0.001,**** p<0.0001, n.s. means no statistical significance. Data were analyzed by one-way ANOVA for comparison between groups, except that data in group (B) were analyzed by Student's t test.

[0027] Figure 8 : Effects of piperine derivative PD on ASGR1 protein and its pathway-related proteins in ASGR1 overexpressed HEK-293T cells.

[0028] Figure 9 : Effects of piperine derivative PD on body weight, food intake and organ index of mice. (A) Changes in body weight of mice; (B) Changes in average food intake of mice after administration; (C) Table of organ index. Data in (A / B) were expressed as mean value, and significant difference was found between the high-fat model group (two-way ANOVA), *p < 0.05; data in (C) were expressed as mean value ± standard deviation (n = 6-8), and significant difference was found between groups (one-way ANOVA).

[0029] Figure 10 : Effects of piperine derivative PD on subcutaneous and visceral fat of mice. (A) Representative Micro-CT abdominal cross-sectional images of mice, in which white color represents bone or food in gastrointestinal tract, dark gray color represents fat, and light gray color represents viscera; (B) Representative Micro-CT modeling images of mice, in which yellow color represents subcutaneous fat, and green color represents visceral fat; (C) Relative volume of subcutaneous fat (mm 3 / g); (D) Relative volume of visceral fat (mm 3 / g). Data were expressed as mean value ± standard deviation (n = 3), and significant difference was found between the high-fat model group. p <0.05,** p <0.01,*** p <0.001,**** p <0.0001, n.s. means no statistical significance (one-way ANOVA).

[0030] Figure 11 : Verification of high-fat diet mouse model. (A) Serum TC level (mM); (B) Serum LDL-C level (mM). Data were expressed as mean value ± standard deviation (n = 8), p <0.01,**** p <0.0001, n.s. means no statistical significance (Student's t test).

[0031] Figure 12Effect of piperine derivative PD on serum lipid levels in mice. (A) Serum TC level (mM); (B) Serum LDL-C level (mM); (C) Serum TBA level (mM); (D) Serum TG level (mM); (E) Serum HDL-C level (mM). Data were expressed as mean ± SD (n = 6-8), and significant difference compared with the high-fat model group. p <0.05, p <0.01, p <0.001, p <0.0001, n.s. means no statistical significance (one-way ANOVA).

[0032] Figure 13 Effect of piperine derivative PD on fecal lipid levels in mice. (A) Fecal TC level (mmol / g prot); (B) Fecal TG level (mmol / g prot). Data were expressed as mean ± SD (n = 6-7), and significant difference compared with the high-fat model group. p <0.01, p <0.001, p <0.0001, n.s. means no statistical significance (one-way ANOVA).

[0033] Figure 14 Effect of piperine derivative PD on the morphology of the jejunum villi in mice. The figure is a representative image of H&E staining of mouse jejunum sections (magnification of 100 and 200 times; scale bar, 100 µm).

[0034] Figure 15 Effect of piperine derivative PD on liver lipid in mice. (A) Representative liver morphology of mice in each group; (B) Representative H&E staining of liver sections of mice in each group (original magnification of 200 times; scale bar, 100 µm).

[0035] Figure 16 Effect of piperine derivative PD on liver lipid in mice. (A) Liver TC level (mmol / g tissue); (B) Liver TBA level (mmol / g tissue); (C) Liver TG level (mmol / g tissue). Data were expressed as mean ± SD (n = 6-8), and significant difference compared with the high-fat model group. p <0.01, p <0.001, p <0.0001, n.s. means no statistical significance (one-way ANOVA).

[0036] Figure 17 : Effects of piperine derivative PD on the expression levels of ASGR1 and its pathway-related proteins. (A) ASGR1 protein expression level; (B) LXRa protein expression level; (C) ABCA1 protein expression level; (D) ABCG5 protein expression level; (E) ABCG8 protein expression level; (F) SR-B1 protein expression level. Data are expressed as mean ± standard deviation (n = 6), and significant differences were observed compared with the high-fat model group. p <0.05, p <0.01, p <0.001, n.s. represents no statistical significance (one-way ANOVA).

[0037] Figure 18 : Piperine derivative PD inhibits the mRNA expression level of mouse liver ASGR1. Data are expressed as mean ± standard deviation (n = 6), and significant differences were observed compared with the high-fat model group. n.s. represents no statistical significance (one-way ANOVA).

[0038] Figure 19 : Interaction affinity of piperine derivative PD with ASGR1 protein and molecular docking. DETAILED DESCRIPTION

[0039] In order to better understand the technical content of the present application, the present application will be further described below in combination with specific examples and drawings.

[0040] The Pd(dppf)Cl2 is [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium. The iodide is 3,5-dimethoxyiodobenzene, the amide is N,N-dibutyl-1,3-pentadienylamide, the EtOAc is ethyl acetate, and the petroleum ether is petroleum ether.

[0041] Example 1 Synthesis of piperine derivative The synthesis route of the piperine derivative is shown in Figure 1 The synthesis steps are as follows: (1) At 22 °C, K2CO3 (738 mg, 5.34 mmol) and Pd(dppf)Cl2 (98 mg, 0.13 mmol) were sequentially added to a mixed solution of the iodide (707 mg, 2.68 mmol) and the amide (560 mg, 2.68 mmol) in toluene (20 mL). The temperature of the reaction system was raised to 100 °C, and stirring was performed for 6 hours.

[0042] (2) The reaction was quenched by adding saturated NaHC03 aqueous solution (20 mL), and the aqueous phase was extracted with EtOAc (3 x 50 mL); (3) The organic phase was combined and washed with saturated NaCl aqueous solution (2 x 50 mL) and dried over anhydrous Na2S04.

[0043] (4) The filtrate after filtration was concentrated and purified by column chromatography with eluent of EtOAc: petroleum ether = 1:4 to obtain the product (730 mg, 2.11 mmol, 79%) as a yellowish oil.

[0044] The obtained product was determined to be piperine derivative PD, and its structural formula is as follows:

[0045] The compound has 1 H NMR, 13 C NMR, and HRMS data are as follows: 1 H NMR (400 MHz, CDCl3): δ = 0.80-1.03 (m, 6H), 1.21-1.44 (m, 4H), 1.45-1.72 (m, 4H), 3.18-3.49 (m, 4H), 3.79 (s, 6H), 6.39 (d, J = 14.5Hz, 1H), 6.39 (t, J = 2.2Hz, 1H), 6.59 (d, J = 2.2Hz, 2H), 6.75 (d, J = 15.5Hz, 2H), 6.89 (dd, J1 = 15.5Hz, J2 = 10.1Hz, 1H), 7.43 (dd, J1 = 14.5Hz, J2 = 10.0Hz, 1H). 13 C NMR (400 MHz, CDCl3): δ = 13.9 13.9 20.1 20.3 30.1 32.0 46.6 47.955.3 55.4 101.0 104.9 104.9 121.4 127.5 138.4 138.6 142.0 160.9 161 166.0 HRMS: [M+H] + = 346.2380 Example 2 Effect of piperine derivative on Huh-7 cells To determine the safe concentration of the piperine derivative in the experiment, we used the resazurin transformation method to determine the effect of the piperine derivative on the viability of Huh-7 cells. The results showed that the piperine derivative PD at concentrations of 1-10 μM had no significant toxic effect on the growth of Huh-7 cells. Figure 6 We then examined the effect of piperine derivatives at safe concentrations on cholesterol efflux activity in Huh-7 cells, and the results showed that only 0.3 μM of piperine derivatives could produce a significant cholesterol efflux-promoting effect (A). Figure 6 B), its EC 50 The concentration was 0.3165 μM, indicating a much higher activity than the precursor compound piperine (EC). 50 (Approximately 37 μM).

[0046] Example 3 Effects of piperine derivatives on ASGR1-overexpressing HEK-293T cells Based on previous research results, we hypothesize that the cholesterol efflux-promoting ability of piperine derivatives in PD is achieved by inhibiting ASGR1. Studies have shown that ASGR1 is only expressed in the liver. To avoid interference from endogenous ASGR1 expression, we constructed an ASGR1-overexpressing HEK-293T cell model to investigate the relevant mechanisms of PD's cholesterol efflux-promoting effect.

[0047] First, we determined the safe dose of PD for HEK-293T cell growth using a rezin transformation assay. The study showed that PD at concentrations of 1-30 μM had no significant toxic effect on HEK-293T cell growth. Figure 7 A). We then overexpressed ASGR1 in HEK-293T cells via transfection and verified the success of the modeling using Western blotting. The results showed that the ASGR1 protein expression level was significantly increased in transfected cells, and the overexpression model was successfully established. Figure 7 Consistent with the Huh-7 cell experiments, we examined the effect of PD at safe concentrations on cholesterol efflux in an ASGR1 overexpression model. Compared to the control group, cholesterol efflux was reduced in ASGR1-overexpressing HEK-293T cells. 1–10 μM PD reversed the decrease in cholesterol efflux induced by ASGR1 overexpression, and only 1 μM PD significantly increased cholesterol efflux in overexpressing cells. Figure 7 C).

[0048] To investigate the mechanism by which PD promotes cholesterol efflux, we used 10 μM PD to study its effects on ASGR1 levels and downstream target protein levels in wild-type and ASGR1-overexpressing HEK-293T cells. The ASGR1 protein level results were consistent with the model validation experiments; wild-type HEK-293T cells showed almost no ASGR1 expression, indicating that the study of this pathway is not affected by endogenous ASGR1 expression. Figure 8 A). 10 μM PD can inhibit the ASGR1 protein level in HEK-293T cells overexpressing and upregulate its LXRα protein expression level. Figure 8 (A, B), and can increase the protein expression of cholesterol efflux proteins ABCG5 and ABCA1 through LXRα ( Figure 8 (C, D).

[0049] Example 4: Effects of piperine derivative PD on subcutaneous and visceral fat levels in mice on a high-fat diet 4.1 Weight gain and average food intake in mice on a high-fat diet Mice were divided into a normal diet group (ND) and a high-fat diet group (HFD) and fed the corresponding diets. The model was established after 18 weeks, and the modeling results were assessed. After confirming successful establishment of the hyperlipidemia mouse model, mice in the high-fat diet group were randomly divided into a high-fat model group (HFD), a positive control group (Atorvastatin, 10 mg / kg), and a PD administration group, including low-dose (1 mg / kg), medium-dose (3 mg / kg), and high-dose (10 mg / kg) groups, and administered the corresponding drugs via gavage. The control group received the same volume of CMC-Na solution, continuing for 18 weeks. Mouse weight and food intake were recorded 1-2 times per week, with recordings consistently between 3-4 PM. Measurements taken at the same time period reduce errors, and since mice are nocturnal, most mice have not yet eaten at 4 PM, resulting in minimal weight fluctuations and more accurate measurements. For the same reason, feed changes and gavage administration were also performed during this period, as increased food intake and activity at night, along with vigorous metabolism, facilitate drug absorption and efficacy.

[0050] From the start of feeding the mice, we obtained changes in mouse body weight and food intake by detecting and recording mouse body weight and feed weight. Mice fed a normal diet reached a plateau in body weight at around 16 weeks, while mice fed a high-fat diet showed stable weight gain. From the successful establishment of the high-fat diet mouse model to the end of gavage administration, there were significant differences in body weight changes between the ND group and the HFD group, while there were no significant differences in body weight changes between the atorvastatin and PD groups and the HFD group. Figure 9 (A). Meanwhile, there were no significant differences in food intake among the groups of mice, indicating that PD does not affect normal food intake in mice, suggesting that its lipid-lowering effect is achieved by influencing lipid metabolism processes rather than reducing total lipid intake. Figure 9B).

[0051] Organ indices can reflect the functional status of organs to some extent and are often used to evaluate the side effects and toxicity of long-term drug administration on the organs of test animals. They are statistically expressed as the percentage of each internal organ's weight (kg) to the animal's body weight (kg). The results showed no significant differences in organ indices for the heart, liver, spleen, lungs, and kidneys among the different groups of mice after PD administration. Figure 9 (C). This indicates that long-term administration of PD has no significant toxic side effects and will not affect any organs. Combined with changes in body weight and food intake, this also shows that the PD dosage is within a safe range.

[0052] 4.2 Piperine derivatives (PD) significantly reduced subcutaneous and visceral fat levels in mice fed a high-fat diet. Based on changes in body weight, we used Micro-CT to scan the viscera and subcutaneous fat of mice. By taking a cross-section of the abdomen at the penultimate lumbar vertebra, we observed a significant increase in visceral fat in the HFD group compared to the ND group. Administration of atorvastatin and medium- to high-dose PD alleviated the accumulation of visceral fat induced by a high-fat diet. Figure 10 A).

[0053] The scan data were imported into Analyze 12.0 & AVW 12.0, and a 3D model was built to quantify fat volume. After modeling and rendering, corresponding to the abdominal cross-sectional image, the visceral fat volume of the HFD group mice was significantly increased compared to the ND group, and the volume decreased accordingly after drug administration. Figure 10 Modeling and quantitative analysis showed that visceral fat in mice on a high-fat diet increased by 0.88 times compared to a normal diet. Compared with the HFD group, subcutaneous and visceral fat in mice in the atorvastatin group and the medium and high dose PD groups decreased significantly, with subcutaneous fat decreasing by 35.71%, 37.20%, and 43.69%, respectively, and visceral fat decreasing by 43.54%, 33.95%, and 41.58%, respectively. Figure 10 (C, D).

[0054] Example 5: Effects of piperine derivative PD on serum lipid metabolism in mice on a high-fat diet 5.1 Validation of the high-fat diet mouse model We used a high-fat diet mouse model to verify the lipid-lowering activity of PD. Mice were fed a high-fat diet for 18 weeks to establish the model. Before testing, mice were fasted for 12 hours, and blood was collected by tail clipping. Serum lipid levels were measured using an in vitro assay kit (enzyme-linked immunosorbent assay). Plasma TC and LDL-C levels in the HFD group were significantly higher than those in the ND group (…). Figure 11 The levels of α and β increased by 0.16 and 0.39 times, respectively. These results indicate that the mouse model of hyperlipidemia was successfully established.

[0055] 5.2 Piperine derivative PD reduces blood lipid levels in high-fat diet mice After modeling success, intragastric administration for 18 weeks, mice were fasted for 12 h before testing, enucleation blood was detected by in vitro assay kit (immunoenzyme linked method) to detect serum lipid content. The plasma TC, LDL-C and TBA levels of ND group mice were lower than those of high-fat control group by 49.28%, 67.85% and 39.65% Figure 12 (A-C). After treatment with PD, plasma TC and LDL-C were significantly decreased compared with the high-fat control group. Among them, low-dose PD could make LDL-C decrease by 43.94%. Medium-dose PD could make TC and LDL-C decrease by 25.10% and 49.05%, respectively. High-dose PD had the same effect as the same dose of positive drug atorvastatin, which could make plasma TC and LDL-C decrease by 28.42% and 58.51%, respectively Figure 12 The lipid-lowering effect of piperine has been widely confirmed, and 25 mg / kg of piperine can reduce the plasma TC, TG and LDL-C of hyperlipidemic mice. Piperine derivatives at doses of 3 and 10 mg / kg can produce lipid-lowering effects and have the same effect as atorvastatin at the same dose, suggesting that they have better lipid-lowering potential and have the potential to be developed into clinical drugs. In the mice of the administration group, as the concentration of PD increased, the number of mice with high HDL-C levels also increased. There was no significant difference, suggesting that PD may have certain potential in improving HDL-C Figure 12 , E).

[0056] Example 6 Effect of piperine derivative PD on fecal lipid metabolism and small intestinal villus damage in high-fat diet mice 6.1 Piperine derivative PD promotes fecal lipid excretion in high-fat diet mice Cholesterol and its metabolites are excreted into the small intestine with bile and excreted out of the body with feces, which is the last step of cholesterol metabolism and is very important for maintaining lipid homeostasis in the body. After the administration ended, we used an in vitro assay kit (immunoenzyme linked method) to biochemically analyze the fresh feces of mice and found that the TC and TG levels of HFD group mice were 1.30 and 1.78 times higher than those of ND group, respectively, while atorvastatin and high-dose PD could make the fecal cholesterol excretion of high-fat diet mice increase by 38.6% and 34.93%, respectively Figure 13 (A, B). This shows that PD can produce long-term stable lipid-lowering effect by promoting fecal lipid excretion.

[0057] 6.2 Piperine derivative PD improves small intestinal villus damage caused by high-fat diet Based on the positive effect of PD on cholesterol excretion, we studied the jejunum of mice after PD treatment. Some studies have shown that hypercholesterolemia can increase the level of TNF-α inflammatory factor, thus causing the body to be in a state of chronic inflammation and leading to impaired intestinal barrier function. We proved this by H&E staining of the jejunum. The small intestinal villi of the jejunum of mice in the normal diet group were arranged in an orderly, complete and relatively slender structure; the small intestinal villi of the jejunum of mice in the high-fat control group were shortened and thickened, with necrosis at the end and becoming loose and hypertrophic; and the small intestinal villi damage caused by high-fat diet was improved after treatment with PD and atorvastatin. The small intestinal villi of the jejunum of mice in the PD high-dose group returned to a slender shape, the damage at the end of the villi was repaired, and the arrangement between the villi became tight again Figure 14 ).

[0058] Example 7 Effect of piperine derivative PD on lipid accumulation and lipid metabolism in the liver of high-fat diet mice 7.1 Piperine derivative PD improves liver steatosis caused by high-fat diet Liver steatosis is closely related to hyperlipidemia, and excessive accumulation of fat in the liver causes hyperlipidemia, and the increase in TC and LDL-C in hyperlipidemia also causes liver steatosis. We analyzed the liver in multiple dimensions. Morphological analysis showed that the liver of mice in the ND group was reddish brown, and the liver of mice in the HFD group was yellowish brown and larger in volume, indicating that high-fat diet caused lipid accumulation and pathological changes in the liver of mice Figure 15 , A). This phenomenon was alleviated after treatment with the positive drug atorvastatin and PD, and the yellowish brown of the liver subsided and the volume decreased. Observation of the H&E staining section of the liver showed that, similarly to the morphology, the liver cells of mice in the ND group were arranged tightly, while the liver cells of mice in the HFD group were arranged in disorder, with a large number of fat droplets deposited and a large area of ballooning degeneration phenomenon, and the fat droplets were significantly reduced and the cells were arranged tightly after treatment with the positive drug or high-dose PD Figure 15 , B).

[0059] 7.2 Piperine derivative PD reduces liver lipid levels in high-fat diet mice The lipid content in the liver of mice was determined by biochemical method. After 18 weeks of intragastric treatment, compared with the ND control group, the TC and TBA levels in the liver of mice fed with HFD increased by 1.69 times and 0.75 times Figure 16; A, B), which is consistent with other similar literatures. PD showed excellent ability to reduce liver TBA, and the medium dose of PD could significantly reduce the TBA of HFD mice by 36.67%. At the same time, the high dose of PD could significantly reduce the TC and TBA levels of HFD mice, which were 23.08% and 45.23% lower than those of the high-fat control group, respectively, and the efficacy was comparable to that of the positive drug atorvastatin. The TG of the HFD group was significantly increased by 0.62 times compared with the ND group, but there was no significant difference in TG among the TG groups of the HFD mice Figure 16 , C). The above results of liver lipid profile are consistent with the results of liver morphology and H&E staining.

[0060] Example 8 Effect of Piperine Derivative PD on Protein Expression Level of ASGR1-LXRa-RCT in Liver Based on the current research and our previous cell experiments, we hypothesized that the lipid-lowering mechanism of PD is to inhibit liver ASGR1 and then regulate the downstream pathway to promote cholesterol efflux.

[0061] First, we detected the effect of PD on the protein expression level of ASGR1 in liver. Consistent with the cell experiment, PD could inhibit the protein expression level of ASGR1. Compared with the HFD group, only the medium dose of PD could significantly reduce the protein level of ASGR1 by 19.61%, and the high dose of PD could significantly reduce it by 31.87% ( Figure 17 , A). ASGR1 with low protein level can inhibit the ubiquitination of LXRa protein by regulating the AMPK / mTORC1 signaling pathway, increase the protein level of LXRa, and then increase the expression of RCT-related proteins directly regulated by it. Therefore, we detected the protein levels of LXRa and its downstream RCT targets ABCA1, SR-B1, ABCG5 / ABCG8, and the results showed that the medium and high doses of PD could significantly up-regulate the protein levels of the above targets ( Figure 17 , B-F). This indicates that PD can down-regulate the protein level of liver ASGR1 and up-regulate the expression of cholesterol efflux-related proteins in this pathway in vivo.

[0062] Example 9 Effect of Piperine Derivative PD on mRNA Expression Level of ASGR1 in Liver In order to study whether the effect of PD on down-regulating ASGR1 protein is through regulating the gene level of the target, we detected the mRNA expression level of ASGR1 by PD. The results showed that there was no significant difference between each treatment group compared with the HFD group ( Figure 18 ).

[0063] Example 10 Interaction Affinity and Molecular Docking of Piperine Derivative PD and ASGR1 Protein The affinity between PD and ASGR1 proteins was determined using Biacore and the protein-coupled assay was performed using a CM5 chip-based method. The results showed that the KD value of the positive control GalNac with ASGR1 was 7.82e-6 (…). Figure 19 The dissociation constant KD between PD and ASGR1 protein is 3.39e-6 (A), while the dissociation constant KD between PD and ASGR1 protein is 3.39e-6 (A). Figure 19 The positive control (B) showed a stronger affinity of PD for ASGR1 than the negative control (B). The PYMOL molecular graphics system was used to simulate intermolecular binding. The results showed that the optimal binding conformation had a docking score of -4.15, with the binding site located at the edge of the ASGR1 protein, linked to TRP243 and ARG239 of the ASGR1 protein via two hydrogen bonds. Figure 19 The above results indicate that PD and ASGR1 protein have a moderate binding affinity, and PD can act directly on ASGR1 protein as a small molecule drug.

[0064] The above description is only a part of the embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A compound, characterized in that, The structural formula of the compound is shown as formula I below: I。 2. The method of synthesizing the compound of claim 1, characterized in that, The method comprises the following steps: (1) sequentially adding potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium to a toluene mixed solution of 3,5-di-methoxy-iodobenzene and N,N-dibutyl-1,3-pentadienylamide; increasing the temperature of the reaction system to 100 DEG C and stirring for 6 hours; (2) adding saturated aqueous sodium bicarbonate solution to quench the reaction and extracting the aqueous phase with ethyl acetate; (3) washing the combined organic phase with saturated aqueous sodium chloride solution and drying with anhydrous sodium sulfate; (4) concentrating the filtered filtrate and purifying by column chromatography, using a mixture of ethyl acetate and petroleum ether as the eluent to obtain the product in the form of a light yellow oily liquid.

3. The method of synthesis of claim 2, wherein, The volume ratio of the ethyl acetate and petroleum ether is 1:

4.

4. The method of synthesis of claim 2, wherein, The molar ratio of 3,5-di-methoxy-iodobenzene, N,N-dibutyl-1,3-pentadienylamide, potassium carbonate and [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium is 2.68:2.68:5.34:0.

13.

5. A medicament, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The medicament contains the compound of claim 1 and / or a pharmaceutically acceptable salt of the compound, and further contains a pharmaceutically acceptable excipient.

6. Use of the compound of claim 1 or the medicament of claim 5 in the preparation of an ASGR1 protein inhibitor.

7. Use of a compound according to claim 1 or a medicament according to claim 5, characterized in that, The use comprises at least one of the following (1) to (5); (1) use in the preparation of a medicament for preventing and treating hypercholesterolemia; (2) use in promoting the efflux of cellular cholesterol; (3) use in regulating the expression level of cholesterol efflux transporters ABCG5, ABCA1, ABCG8 and / or SR-B1 protein; (4) use in the preparation of a preparation for promoting the metabolism of animal fat; (5) use in the preparation of a preparation for improving small intestinal villus damage caused by a high-fat diet.

8. Use according to claim 7, characterized in that, The cells include Huh-7 cells and / or HEK-293T cells.

9. Use according to claim 7, characterized in that, In (1) and (2), the concentration of the compound used is 1-10 μM.

10. Use according to claim 7, characterized in that, The fat includes subcutaneous fat, visceral fat, liver fat, blood fat and / or fecal fat.

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