Ploretin derivative and application of phloretin derivative in preparation of medicine for treating diabetes mellitus

Abstract

The invention discloses a phloretin derivative and application thereof in preparation of a medicine for treating diabetes, relates to the technical field of medicines, and in particular relates to a compound as shown in a formula (1) and alpha-glucosidase inhibitory activity of the compound. Pharmacological experiment results show that the compound shown in the formula (1) has extremely high alpha-glucosidase inhibitory activity, so that the generation of glucose is inhibited, and the pharmacological action of resisting diabetes mellitus is exerted through the action mechanism.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a phloretin derivative and its medical use. Background Art

[0002] Type 2 diabetes mellitus (T2DM) is a serious chronic metabolic disease with long-term hyperglycemia, which is characterized by insulin resistance and insufficient insulin secretion by pancreatic β-cells leading to hyperglycemia. According to the survey of the World Health Organization, the number of global diabetes patients exceeds 420 million, and it is expected that this figure will exceed 642 million by 2040. Long-term uncontrolled hyperglycemia can lead to a variety of serious complications, including eye, kidney, heart and vascular diseases. Postprandial blood glucose control is an important means to delay the occurrence of complications. The change of blood glucose level comes from the decomposition of carbohydrates catalyzed by enzymes such as α-glucosidase and α-amylase. α-Glucosidase is an important membrane-bound enzyme located on the surface membrane of the small intestine brush border and can hydrolyze 1,4-α-glucosidic bonds to generate α-glucose. Therefore, α-glucosidase inhibitors can delay the intake of carbohydrates, thereby keeping postprandial blood glucose within the normal range, and thus are widely used in the treatment of T2DM patients. Currently, some α-glucosidase inhibitors (acarbose, voglibose and miglitol) are used to treat type 2 diabetes, but these drugs have a wide range of side effects including stomachache, diarrhea, flatulence, allergic reactions, etc.

[0003] Natural products usually have a wide range of biological activities due to their unique chemical structures. Among them, chalcone compounds are widely present in many plants, and studies have shown that chalcone compounds (including phloretin) have preventive and therapeutic effects on type 2 diabetes. Its main pharmacological mechanism of action is related to its inhibitory effects on α-glucosidase and α-amylase. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention aims to propose a new phloretin derivative with more efficient inhibitory effects on α-glucosidase and α-amylase, and solves the problem of low inhibition efficiency by modifying known compounds.

[0005] The first aspect of the present invention is to provide a compound, and the compound is a phloretin derivative shown in formula (1):

[0006]

[0007] Further, in the compound, R is a saturated hydrocarbon group or a halogen atom with 1 to 10 carbon atoms;

[0008] Further, R is a halogen atom, and more specifically, R is F.

[0009] The second aspect of the present invention is to provide a method for preparing the compound described in the first aspect. The method is synthesized according to the following route:

[0010]

[0011] Furthermore, the specific steps of the method are as follows:

[0012] 1) Using phloretin and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate as raw materials, reacting at room temperature for 6 - 10 h under the catalysis of triethylamine and acetonitrile to obtain a monosubstituted phloretin derivative;

[0013] 2) Subsequently, through silica gel column chromatography, separating and purifying with ethyl acetate / petroleum ether as the developing agent.

[0014] Furthermore, the molar ratio of phloretin to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1 - 1.5; preferably 1:1.3;

[0015] Furthermore, after the reaction in step 1), the product is obtained by rotary evaporation and drying;

[0016] Furthermore, in step 2), the ratio of ethyl acetate / petroleum ether is 1:3 - 7 (v / v); preferably 1:5 (v / v).

[0017] The third aspect of the present invention is to provide a pharmaceutical composition for treating diabetes. The pharmaceutical composition contains the compound described in the first aspect and a pharmaceutically acceptable carrier. It also contains adjuvants. The adjuvants are at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant, or a lubricant. The dosage form of the pharmaceutical composition is tablets, pills, capsules, oral liquids, or injections.

[0018] Fourthly, the present invention also provides the use of the compound described in the first aspect in the preparation of an α-glucosidase inhibitor.

[0019] Furthermore, the use is for non-therapeutic purposes.

[0020] Fifthly, the present invention also provides the application of the compound described in the first aspect in the preparation of a drug for treating diabetes.

[0021] Sixthly, the present invention also provides the application of the method described in the second aspect in the preparation of a drug for treating diabetes.

[0022] Compared with the prior art, the present invention has the following advantages

[0023] The present invention obtains compound (1) by modifying phloretin-like substances, which can significantly inhibit α-glucosidase activity, thereby inhibiting the conversion of starch into glucose and having extremely high anti-diabetic potential. Description of the Drawings

[0024] Figure 1 Determination of the IC50 value of the compound against α-glucosidase.

[0025] Figure 2 Fluorescence quenching of the compound against α-glucosidase.

[0026] Figure 3 Inhibition of starch digestion by the compound. Note: * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001, **** represents P < 0.0001.

[0027] Figure 4 Determination of blood glucose and oral glucose tolerance by the compound. Detailed Description of the Invention

[0028] The following further elaborates on the concept and technical effects of the present invention in combination with specific embodiments to fully understand the purpose, features, and effects of the present invention. The methods are conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. It should be noted that, without conflict, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0029] Example 1: Preparation of Phloretin Derivative

[0030] Synthesize the compound shown in Formula I according to the following process:

[0031]

[0032] The specific preparation process is as follows: Add 27.4 mg (0.1 mmol) of phloretin and 42 mg (0.13 mmol) of 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate to a 10 ml round-bottom flask. Pipette 1 ml of acetonitrile into the flask, and then add 22 μl (0.16 mmol) of triethylamine. Stir the reaction at room temperature for 8 h. After the reaction is completed, evaporate the reaction solution to dryness and separate and purify it by silica gel column chromatography using ethyl acetate / petroleum ether (1:5, v / v) as the eluent.

[0033] The physical and chemical data of the obtained compound (1) and 1 1H-NMR, 13 13C-NMR, 19 19F NMR data are as follows:

[0034] Compound (1) 1 H NMR (500 MHz, CDCl3) δ 10.25 (s, 1H), 7.27 (d, J = 8.9 Hz, 1H), 7.22–7.16 (m, 1H), 6.34 (s, 1H), 3.39 (t, J = 7.5 Hz, 1H), 2.99 (t, J = 7.5 Hz, 1H), 1.99 (d, J = 33.1 Hz, 1H); 13 C NMR (126 MHz, CDCl3) δ 205.05, 163.03, 153.85, 148.46, 142.06, 130.44, 120.86, 109.64, 100.80, 45.93, 29.26; 19 F NMR (471 MHz, CDCl3) δ 39.64, 37.35; Molecular formula: C 15 H 12 F 2 O 9 S 2 ; Molecular weight: 438.3688.

[0035] Example 2: Determination of the IC50 value of compound (1) against α-glucosidase

[0036] After weighing compound (1) and phloretin separately, they were dissolved in DMSO to prepare a stock solution of 20 mmol / L and serially diluted to 10 mmol / L, 5 mmol / L, 2.5 mmol / L, 1.25 mmol / L, 0.625 mmol / L, 0.3125 mmol / L, and 7.8125 mmol / L. Different concentrations of compound (10 μL), 0.5 U / ml enzyme solution (40 μL), and 0.1 mM phosphate buffer at pH 6.8 (100 μL) were pre-incubated in a 96-well plate at 37 °C for 10 min. Then, 50 μL of substrate (pNαGP, 0.6 mM) was added to each well and incubated at 37 °C for 20 min. The absorbance was measured at 405 nm to detect the change in enzyme activity. At the same time, acarbose was set as a positive control.

[0037] The results are as Figure 1 shown, and it can be seen from Figure 1 that the inhibitory activity of the derivatized compound (1) against α-glucosidase is higher than that of the original compound phloretin (190.48 μM), and at the same time, it is much higher than the efficacy of the medicinal acarbose (562.22 μM).

[0038] Table 1

[0039]

[0040] Example 3: Fluorescence Quenching Experiment of Compound (1) on α-Glucosidase

[0041] α-Glucosidase (1.0 mL, 2 U / mL) was titrated with inhibitor solutions at different concentrations (1.0 mL, 0 - 1000 mM). After equilibration for 5 min, fluorescence measurements were performed. The fluorescence intensity of the reaction solution was measured at different temperatures (305.15, 310.15, and 315.15 K) using a fluorescence spectrometer (F-7100 type, Tokyo, Japan). The excitation and emission slit widths were set to 5.0 nm, the excitation wavelength was 280 nm, and the emission wavelength range was 290 nm - 500 nm.

[0042] The results are as Figure 2 shown. The fluorescence quenching of α-glucosidase can be used to characterize the affinity between the compound and the enzyme. α-Glucoside has a strongest endogenous fluorescence around 340 nm caused by Trp and Tyr residues. Therefore, the binding degree can be determined by measuring the effect of the compound on the emission spectra of the two enzymes. As can be seen from Figure 2 it, as the concentration of the compound increases, the fluorescence intensity of the enzyme decreases significantly, indicating that all three compounds can quench the intrinsic fluorescence of the enzyme and bind to it. However, by comparing the effects of the compounds on the enzyme fluorescence at the same concentration, it can be seen that compound (1) has a greater effect on the fluorescence quenching value of α-glucosidase, which also indicates that compound (1) has a stronger affinity and interaction force with α-glucosidase, thus having a stronger inhibitory effect on the enzyme.

[0043] Example 4: In Vitro Starch Digestion Inhibition Experiment

[0044] The effect of the inhibitor on the in vitro starch digestibility was determined using a slightly modified Englyst method. Corn starch (300 mg) and guar gum (25 mg) were added to a 50 mL centrifuge tube and dissolved in 7.5 mL of distilled water. After boiling in a boiling water bath for 10 min and cooling to room temperature, sodium acetate buffer (2.5 mL, 0.4 M, pH 5.2, containing 0.18% (w / v) CaCl2) was added. After equilibrating the sample tubes at 37 °C for 15 min, a mixture of fresh porcine pancreatic protease extract, amyloglucosidase, and inhibitor (5.5 mL) was added to hydrolyze the starch. At the same time, the group without inhibitor and the acarbose group were used as blank and positive controls. At 20 min, 60 min, 120 min, and 240 min, 250 μL of the starch hydrolysate was taken from the centrifuge tube and added to 10.0 mL of 66% (v / v) ethanol. Glucose production was measured using a d-glucose assay kit (GOPOD).

[0045] The results are as Figure 3As shown, the results indicate that compound (1) can significantly inhibit α-glucosidase activity. At 20 min, 60 min, 120 min, and 240 min, compound (1) reduced the glucose production rate by 17.74%, 24.98%, 11.97%, and 43.24% respectively compared to acarbose. At 60 min, 120 min, and 240 min, compound (1) reduced the glucose production rate by 40.88%, 27.19%, and 35.77% respectively compared to phloretin. This shows that compound (1) is more capable of inhibiting the conversion of starch into glucose and has extremely high antidiabetic potential.

[0046] Table 2

[0047]

[0048] Example 5 Oral Glucose Tolerance Test

[0049] C57 / BL mice at 5 - 6 weeks of age were used as experimental subjects. Before the formal experiment, they were acclimated to the experimental environment for one week. The animal experiment was conducted in a specific pathogen-free (SPF) environment. The Experimental Animal Management and Welfare Ethics Committee of Beijing Huayuan Times Technology Co., Ltd. approved this study (Ethical Review Batch Number: HYSD2023 - 04). The C57 / BL mice were randomly divided into a normal group, an acarbose group, a phloretin group, and a compound (1) group, with 8 mice in each group. Before the oral glucose tolerance test, all mice were fasted for 12 h, and they had free access to water during this period. Phloretin, compound (1), and acarbose were dissolved in physiological saline containing 10% DMSO. The gavage dose was set at 20 mg / kg. The mice in the normal group were gavaged with an equal volume of physiological saline. 5 min after administration, the blood glucose levels of each group of mice were measured at 0, 30, 60, 90, and 120 minutes after gavage with a starch solution at a dose of 2 mg / kg. Subsequently, an OGTT curve was plotted and the area under the curve (AUC) was calculated.

[0050] After the oral glucose tolerance test ( Figure 4 ), the results showed that acarbose and compound intervention could effectively inhibit the increase in postprandial blood glucose. During the entire digestion process, by calculating the area under the curve of the 2-hour postprandial blood glucose curve ( Figure 4 ), it can be concluded that compared to phloretin, compound (1) can reduce the blood glucose fluctuation by 23.9% in postprandial blood glucose. Compared to acarbose, compound (1) can reduce the blood glucose fluctuation by 11.9% in postprandial blood glucose. The ability of compound (1) to control the blood glucose level at 2 hours after a meal is significantly better than that of the parent compound phloretin, and compound (1) is more conducive to controlling the increase in postprandial blood glucose during the entire digestion process.

[0051] Table 3

[0052]

[0053] The embodiments described above are only a part of the embodiments of the present invention, rather than all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative efforts shall fall within the scope of protection of the present invention.

Claims

1. A compound, wherein the compound is a phloretin derivative represented by formula (1): It is characterized in that The R is F.

2. A method for preparing the compound according to claim 1, characterized in that: The method described is synthesized according to the following route:

3. The method according to claim 2, characterized in that The specific steps of the method are: 1) Using phloretin and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate as raw materials, reacting at room temperature for 6 to 10 hours under the catalysis of triethylamine and acetonitrile to obtain a monosubstituted phloretin derivative; 2) The product was then separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether as a developing solvent.

4. The method according to claim 3, characterized in that The molar ratio of phloretin to 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate is 1:1 to 1.

5.

5. A pharmaceutical composition for treating diabetes, characterized in that: The pharmaceutical composition comprises the compound according to claim 1 and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that Also contains adjuvants.

7. The pharmaceutical composition according to claim 6, characterized in that The auxiliary agent is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant or a lubricant.

8. The pharmaceutical composition according to claim 6 or 7, characterized in that The dosage form of the pharmaceutical composition is tablet, pill, capsule, oral solution or injection.

9. Use of the compound according to claim 1 or the method according to any one of claims 2 to 4 in the preparation of an α-glucosidase inhibitor, characterized in that: The use described is for non-therapeutic purposes in vitro.

10. Use of the compound according to claim 1 or the method according to any one of claims 2 to 4 in the preparation of a drug for treating diabetes.

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