Application of a 8-pyrrolidone-substituted flavanol natural product

Abstract

The present invention belongs to the field of medical technology, and specifically relates to the application of a natural product of 8-pyrrolidone-substituted flavanols. Experiments have confirmed that the compounds pouzoflavone A (Formula I) and pouzoflavone B (Formula II) of the present invention can significantly inhibit the activity of ACLY enzyme in vitro to reduce the biosynthesis of cholesterol. The compound is further expected to be developed as a drug for preventing or treating hypercholesterolemia. #imgabs0#

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to the application of a natural product of 8-pyrrolidone-substituted flavanols. Background Art

[0002] Hypercholesterolemia is a type of hyperlipidemia. In modern life, persistently high low-density lipoprotein cholesterol has become an important risk factor for inducing and promoting the development of atherosclerotic diseases, and is the main cause of the increase in the incidence and mortality of coronary heart disease. At present, the first-choice drugs for the treatment of hypercholesterolemia are statins and fibrates. Statins mainly competitively inhibit the rate-limiting enzyme HMG-CoA reductase in endogenous cholesterol synthesis, block the intracellular hydroxymevalonate metabolic pathway, reduce intracellular cholesterol synthesis, and increase serum cholesterol clearance. However, statins have potential side effects, mainly myopathy (rhabdomyolysis) and impaired liver function. Fibrates can activate peroxisome proliferator-activated receptors (PPARα), induce lipoprotein esterase expression, promote triglyceride hydrolysis in triglyceride-rich lipoprotein particles, and lead to a decrease in plasma very low-density lipoprotein, but in clinical practice, there are also side effects of rhabdomyolysis and abnormal liver function. The side effects of these drugs greatly limit the clinical application of lipid-lowering drugs. Although drugs for lowering LDL-C are constantly being updated, other non-statin drugs have poor effects on lowering LDL-C, resulting in patients at risk of ASCVD failing to achieve the goal of lowering LDL-C. Therefore, the present invention urgently needs to discover new lipid-lowering lead compounds with new regulatory mechanisms.

[0003] As a central metabolic enzyme, ACLY plays a connecting role in the process of glucose catabolism and lipid metabolism. It catalyzes the production of acetyl-CoA and oxaloacetate, and hydrolyzes ATP into ADP and phosphate. Its product, acetyl-CoA, is an indispensable substance in the biosynthesis of fatty acids and cholesterol. A large number of studies have shown that ACLY can be used as a promising therapeutic target for lowering cholesterol and protecting against atherosclerosis. In 2020, the U.S. Food and Drug Administration (FDA) approved the use of bempedoic acid in patients with heterozygous familial hypercholesterolemia and atherosclerotic cardiovascular disease. This is the first FDA-approved ACLY inhibitor, and the clinical trial results of the drug provide strong evidence for the drugability of this target.

[0004] Flavanol components are widely distributed in the plant kingdom, but there are relatively few structures with pyrrolidone groups substituted at the C-8 position, and there are relatively few studies on the biological activity of this type of structure. In the literature related to the structure of the compound in the present invention (Chem. Pharm. Bull. 2009, 57: 397-400), the compound exists in the form of a mixture of diastereomers (ratio of 2: 1), and no pure monomer compound is isolated separately, and its inhibitory effect on advanced glycation end products (AGE) is investigated. In the present invention, by separating the diastereomers and characterizing their structures, it is reported for the first time that it regulates cholesterol biosynthesis in hepatocytes by inhibiting the activity of ACLY enzyme to reduce the cholesterol level of hepatocytes, reflecting the biological activity of reducing cholesterol. Summary of the invention

[0005] The purpose of the present invention is to provide an application of an 8-pyrrolidone-substituted flavanol natural product, and to report for the first time that it regulates cholesterol biosynthesis in hepatocytes by inhibiting the activity of ACLY enzyme to reduce the cholesterol level in hepatocytes, thereby reflecting the biological activity of lowering cholesterol.

[0006] The specific technical solutions of the present invention are as follows:

[0007] A 8-pyrrolidone-substituted flavanol natural product pouzoflavone A, wherein the structural formula of the flavanol natural product pouzoflavone A is as shown in Formula I:

[0008]

[0009] A 8-pyrrolidone-substituted flavanol natural product pouzoflavone B, wherein the structural formula of the flavanol natural product pouzoflavone B is as shown in Formula II:

[0010]

[0011] The 8-pyrrolidone-substituted flavanol natural product pouzoflavone A (Formula I) of the present invention can inhibit the activity of ACLY enzyme at the molecular level. The experimental results show that the compound has a strong inhibitory effect on the activity of ACLY enzyme at the in vitro molecular level. Its IC 50 The value was 7.74±2.84μM.

[0012] The 8-pyrrolidone-substituted flavanol natural product pouzoflavone B (Formula II) of the present invention can inhibit the activity of ACLY enzyme at the molecular level. The experimental results show that the compound has a strong inhibitory effect on the activity of ACLY enzyme at the in vitro molecular level. Its IC50 The value was 5.61±0.22μM.

[0013] The 8-pyrrolidone-substituted flavanol natural product pouzoflavone A (Formula I) of the present invention inhibits the biosynthesis of cholesterol at the cellular level. In a 25 μg / mL ox-LDL-induced HepG2 cell hyperlipidemia model, the compound can significantly reduce the cholesterol level of hepatocytes at a concentration of 10 to 40 μM.

[0014] The 8-pyrrolidone-substituted flavanol natural product pouzoflavone B (Formula II) of the present invention inhibits the biosynthesis of cholesterol at the cellular level. In a 25 μg / mL ox-LDL-induced HepG2 cell hyperlipidemia model, the compound can significantly reduce the cholesterol level of hepatocytes at a concentration of 10 to 40 μM.

[0015] Another object of the present invention is to provide a use of an 8-pyrrolidone-substituted flavanol natural product pouzoflavone A or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating hypercholesterolemia.

[0016] Another object of the present invention is to provide a use of an 8-pyrrolidone-substituted flavanol natural product pouzoflavone B or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating hypercholesterolemia.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The present invention reports for the first time that it regulates cholesterol biosynthesis in liver cells by inhibiting the activity of ACLY enzyme to reduce the cholesterol level of liver cells, thus embodying the biological activity of reducing cholesterol. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the compound pouzoflavone A (Formula I) of the present invention;

[0020] Figure 2 The carbon-NMR spectrum of the compound pouzoflavone A (Formula I) of the present invention;

[0021] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the compound pouzoflavone B (Formula II) of the present invention;

[0022] Figure 4 The carbon NMR spectrum of the compound pouzoflavone B (Formula II) of the present invention;

[0023] Figure 5 Schematic diagram of the compound pouzoflavone A (Formula I) of the present invention significantly reducing the cholesterol level of HepG2 hepatocytes induced by ox-LDL at the in vitro cellular level (10 μM to 40 μM), and 1 μM atorvastatin was used as a positive control. The data are expressed as the percentage of the NC group and the mean ± standard error (SD) of the mean. **p<0.01 and ***p<0.001 compared with the model group, n=3-4 for all groups;

[0024] Figure 6 Schematic diagram of the compound pouzoflavone B (Formula II) of the present invention significantly reducing the cholesterol level of HepG2 hepatocytes induced by ox-LDL at the in vitro cellular level (10 μM to 40 μM), and 1 μM atorvastatin was used as a positive control. The data are expressed as the percentage of the NC group and the mean ± standard error (SD) of the mean. **p<0.01 and ***p<0.001 compared with the model group, n=3-4 for all groups. DETAILED DESCRIPTION

[0025] In order to facilitate the understanding of the technical solution of the present invention, a detailed description is given below in conjunction with the accompanying drawings and specific embodiments.

[0026] The terms used in the present invention, unless otherwise specified, generally have the meanings that are commonly understood by those of ordinary skill in the art.

[0027] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

[0028] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0030] As described in the following examples, one skilled in the art can prepare the compounds of the present invention.

[0031] Example 1

[0032] Preparation of the compound pouzoflavone A (Formula I) of the present invention:

[0033] The fresh root of Pouzolziazeylanica (L.) Benn. was chopped and ultrasonically extracted with methanol (10L×3 times) at room temperature for 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the alcohol extract of Pouzolziazeylanica. The alcohol extract was separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution, 10:1-0:1) to obtain 6 sub-fractions (Fr.1-6), followed by dichloromethane / methanol gradient elution (10:1-5:1) to obtain 5 sub-fractions (Fr.7-11). The Fr.10 component (1563.7 mg) was separated by semi-preparative HPLC (20% acetonitrile isocratic elution) to obtain the compound pouzoflavoneA (Formula I, 3.36 mg).

[0034] The physicochemical data of the compound pouzoflavone A (Formula I) are as follows: yellow amorphous powder, easily soluble in organic solvents such as methanol and ethanol. Its positive ion detection mode HRTOFMS shows a quasi-molecular ion peak of m / z 374.1218 [M+H] + (calcdfor C 19 H 20 O7N), the spectral data of the compound are shown in Table 1, and the NMR spectrum is shown in Appendix Figure 1 and attached Figure 2 .

[0035] Example 2

[0036] Preparation of the compound pouzoflavone B (Formula II) of the present invention:

[0037] The fresh root of Pouzolziazeylanica (L.) Benn. was chopped and ultrasonically extracted with methanol (10L×3 times) at room temperature for 1 hour each time. The extracts were combined and concentrated under reduced pressure to obtain the alcohol extract of Pouzolziazeylanica. The alcohol extract was separated by silica gel column chromatography (petroleum ether / ethyl acetate gradient elution, 10:1-0:1) to obtain 6 sub-fractions (Fr.1-6), followed by dichloromethane / methanol gradient elution (10:1-5:1) to obtain 5 sub-fractions (Fr.7-11). The Fr.10 component (1563.7 mg) was separated by semi-preparative HPLC (20% acetonitrile isocratic elution) to obtain compound pouzoflavone B (Formula II, 4.57 mg).

[0038] The physicochemical data of compound pouzoflavone B (Formula II) are as follows: yellow amorphous powder, easily soluble in organic solvents such as methanol and ethanol. The spectral data of the compound are shown in Table 1, and the NMR spectrum is shown in the attached Figure 3 and attached Figure 4 .

[0039] Table 1 H Spectrum (400 MHz) and C Spectrum (100 MHz) NMR Signals of Compounds Pouzoflavone A (Formula I) and Pouzoflavone B (Formula II) (Measured in Deuterated Methanol)

[0040]

[0041]

[0042] Example 3

[0043] The compound pouzoflavone A (Formula I) of the present invention inhibits ACLY enzyme at the in vitro molecular level.

[0044] Test method: ADP-Glo ​​kinase method.

[0045] Reaction system: Set up full enzyme wells (1μL 10% DMSO + 2μL ACLY + 2μL substrate) and Blank wells (1μL 10% DMSO + 2μL Assay Buffer + 2μL substrate) and experimental groups as follows: ① 1μL compound + 2μL enzyme + 2μL substrate at 37℃ for 30min; ② Add 2.5μL ADP-GLO E2 and react at 37℃ for 70min; ③ Add 5μL E3 and react at room temperature in the dark for 10min; ④ ACLY program detection. Results After analysis, the IC of the compound 50 The value was 7.74±2.84 μM, indicating that the compound exhibited a strong inhibitory effect on ACLY enzyme activity at the in vitro molecular level.

[0046] Example 4

[0047] The compound pouzoflavone B (Formula II) of the present invention inhibits ACLY enzyme at the in vitro molecular level.

[0048] Test method: ADP-Glo ​​kinase method.

[0049] Reaction system: Set up full enzyme wells (1μL 10% DMSO + 2μL ACLY + 2μL substrate) and Blank wells (1μL 10% DMSO + 2μL Assay Buffer + 2μL substrate) and experimental groups as follows: ① 1μL compound + 2μL enzyme + 2μL substrate at 37℃ for 30min; ② Add 2.5μL ADP-GLO E2 and react at 37℃ for 70min; ③ Add 5μL E3 and react at room temperature in the dark for 10min; ④ ACLY program detection. Results After analysis, the IC of the compound 50 The value was 5.61±0.22 μM, indicating that the compound exhibited a strong inhibitory effect on ACLY enzyme activity at the in vitro molecular level.

[0050] Example 5

[0051] The compound pouzoflavone A (Formula I) of the present invention significantly reduces the cholesterol level of HepG2 liver cells induced by ox-LDL at the in vitro cell level.

[0052] Cell culture and administration: HepG2 cells were cultured at approximately 3×10 5 100 cells / well were inoculated in a 12-well plate, 1 mL of cell suspension was added to each well, and cultured at 37°C, 5% CO2 for 16 hours; after the culture medium in the 12-well plate was removed, DMEM culture medium without FBS was added again, and double antibodies were added, and each group was added in the following manner: blank group (NC), model group (the final concentration of ox-LDL induction was 25 μg / mL), compound group (the final concentration of ox-LDL induction was 25 μg / mL, and the final concentration of compound I in the present invention was 10, 20 and 40 μM). After the addition was completed, gently shake it and culture it at 37°C, 5% CO2 for 48 hours.

[0053] TC detection of high cholesterol HepG2 cells: After collecting the samples, use the extraction method to extract them. When TC needs to be detected in the cells, the total protein content of the cells is detected using the BSA method.

[0054] Protein content detection: The sample was diluted 6 times, treated with G250, and the 595nm (OD) value was measured using an enzyme marker.

[0055] TC detection: Add 100 μL of anhydrous ethanol to the evaporated sample, take 30 μL of sample, and measure the 600-700nm (OD) value with an enzyme marker after processing with the kit.

[0056] The results are as follows Figure 5 As shown, in the 25 μg / mL ox-LDL-induced HepG2 cell hyperlipidemia model, the compound pouzoflavoneA (Formula I) can significantly reduce the cholesterol level of hepatocytes at a concentration of 10 to 40 μM.

[0057] Example 6

[0058] The compound pouzoflavone B (Formula II) of the present invention significantly reduces the cholesterol level of HepG2 liver cells induced by ox-LDL at the in vitro cell level.

[0059] Cell culture and administration: HepG2 cells were cultured at approximately 3×10 5100 cells / well were inoculated in a 12-well plate, 1 mL of cell suspension was added to each well, and cultured at 37°C, 5% CO2 for 16 hours; after the culture medium in the 12-well plate was removed, DMEM culture medium without FBS was added again, and double antibodies were added, and each group was added in the following manner: blank group (NC), model group (the final concentration of ox-LDL induction was 25 μg / mL), compound group (the final concentration of ox-LDL induction was 25 μg / mL, and the final concentration of compound II in the present invention was 10, 20 and 40 μM). After the addition was completed, gently shake it and culture it at 37°C, 5% CO2 for 48 hours.

[0060] TC detection of high cholesterol HepG2 cells: After collecting the samples, use the extraction method to extract them. When TC needs to be detected in the cells, the total protein content of the cells is detected using the BSA method.

[0061] Protein content detection: The sample was diluted 6 times, treated with G250, and the 595nm (OD) value was measured using an enzyme marker.

[0062] TC detection: Add 100 μL of anhydrous ethanol to the evaporated sample, take 30 μL of sample, and measure the 600-700nm (OD) value with an enzyme marker after processing with the kit.

[0063] The results are as follows Figure 6 As shown, in the 25 μg / mL ox-LDL-induced HepG2 cell hyperlipidemia model, the compound pouzoflavone B (Formula II) can significantly reduce the cholesterol level of hepatocytes at a concentration of 10 to 40 μM.

[0064] In summary, the above examples illustrate that the 8-pyrrolidone-substituted flavanol natural products pouzoflavone A (Formula I) and pouzoflavone B (Formula II) in the present invention can regulate the cholesterol biosynthesis of hepatocytes by inhibiting ACLY enzyme, so as to reduce the cholesterol level of hepatocytes and demonstrate the biological activity of lowering cholesterol.

[0065] The above are only preferred embodiments of the present invention. The protection scope of the present invention shall be based on the scope defined by the claims. Several improvements and modifications made by those skilled in the art without departing from the spirit and scope of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. Use of a 8-pyrrolidone-substituted flavanol natural product pouzoflavone A or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for treating hypercholesterolemia, characterized in that: The structural formula of the flavanol natural product pouzoflavone A is shown in Formula I: 。 2. A use of a 8-pyrrolidone-substituted flavanol natural product pouzoflavone B or a pharmaceutically acceptable salt thereof as the sole active ingredient in the preparation of a drug for treating hypercholesterolemia, wherein the structural formula of the flavanol natural product pouzoflavone B is as shown in Formula II: 。

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