A phloretin derivative and its use in preparing medicine for treating diabetes

Abstract

The present invention provides a phloretin derivative and its use in preparing a medicament for treating diabetes, relating to the field of medical technology, and particularly to a compound represented by formula (1) and its α-glucosidase inhibitory activity. Pharmacological experimental results show that the compound represented by formula (1) has extremely high α-glucosidase inhibitory activity, thereby inhibiting the production of glucose, and further exerting an anti-diabetic pharmacological effect through this mechanism of action.

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 severe chronic metabolic disease characterized by long-term hyperglycemia, characterized by insulin resistance and insufficient insulin secretion from pancreatic beta cells, leading to hyperglycemia. According to the World Health Organization, there are over 420 million people with diabetes worldwide, and this number is expected to 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 measure to delay the onset of these complications. Changes in blood glucose levels result from the breakdown 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 brush border of the small intestine that hydrolyzes 1,4-α-glucosidic bonds to produce α-glucose. Therefore, α-glucosidase inhibitors can delay carbohydrate intake, thereby maintaining postprandial blood glucose within a normal range, and are therefore widely used in the treatment of patients with T2DM. Currently, some α-glucosidase inhibitors (acarbose, voglucose, and miglitol) are used to treat type 2 diabetes, but these drugs have a wide range of side effects including stomach pain, diarrhea, flatulence, allergic reactions, etc.

[0003] Natural products, due to their unique chemical structures, often possess a wide range of biological activities. Chalcone compounds, among them, are widely found in numerous plants. Studies have shown that chalcones (including phloretin) have preventive and therapeutic effects on type 2 diabetes. Their primary pharmacological mechanism of action is related to their inhibitory effects on α-glucosidase and α-amylase. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention aims to propose a new phloretin derivative with a more efficient inhibitory effect on α-glucosidase and α-amylase, and solves the problem of low inhibition efficiency by modifying the known compounds.

[0005] The first aspect of the present invention is to provide a compound, which is a phloretin derivative represented by formula (1):

[0006]

[0007] Furthermore, in the compound, R is a C1-C10 saturated hydrocarbon group or a halogen;

[0008] Furthermore, the R is halogen, and further, the 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, wherein the method is synthesized according to the following route:

[0010]

[0011] Furthermore, the method comprises the following specific steps:

[0012] 1) Using phloretin and 1-(fluorosulfonyl)-2,3-dimethyl-1H-imidazol-3-ium trifluoromethanesulfonate as raw materials, the reaction was catalyzed by triethylamine and acetonitrile at room temperature for 6 to 10 hours to obtain a monosubstituted phloretin derivative;

[0013] 2) The product was then separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether as a developing solvent.

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

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

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

[0017] A third aspect of the present invention provides a pharmaceutical composition for treating diabetes, comprising the compound described in the first aspect and a pharmaceutically acceptable carrier. The composition further comprises an adjuvant. The adjuvant is 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 pharmaceutical composition is in the form of a tablet, a pill, a capsule, an oral solution, or an injection.

[0018] In a fourth aspect, the present invention further provides 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] In a fifth aspect, the present invention further provides use of the compound described in the first aspect in the preparation of a drug for treating diabetes.

[0021] In a sixth aspect, the present invention also provides the use of the method described in the second aspect in preparing a drug for treating diabetes.

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

[0023] The present invention transforms phloretin to obtain compound (1), which can significantly increase α-glucosidase activity, thereby inhibiting the conversion of starch into glucose and having extremely high anti-diabetic potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The IC50 values ​​of the compounds against α-glucosidase were determined.

[0025] Figure 2 The compounds quench the fluorescence of 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 Effects of compounds on blood glucose and oral glucose tolerance tests. DETAILED DESCRIPTION

[0028] The following is a further description of the concept of the present invention and the technical effects produced in conjunction with specific embodiments, so as to fully understand the purpose, features and effects of the present invention. The methods described are all conventional methods unless otherwise specified. The materials described 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 undue limitations of the present invention. It should be noted that, unless there is a conflict, the embodiments of the present invention and the features in the embodiments can be combined with each other.

[0029] Example 1: Preparation of phloretin derivatives

[0030] The compound shown in formula I was synthesized according to the following process:

[0031]

[0032] The specific preparation process is as follows: 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 were added to a 10 ml round-bottom flask. 1 ml of acetonitrile was pipetted into the flask, followed by 22 μl (0.16 mmol) of triethylamine. The mixture was stirred at room temperature for 8 hours. After the reaction, the reaction solution was dried and separated and purified by silica gel column chromatography using ethyl acetate / petroleum ether (1:5, v / v) as the developing solvent.

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

[0034] Compound (1) 1 H NMR (500MHz, CDCl3) δ10.25 (s, 1H), 7.27 (d, J = 8.9Hz, 1H), 7.22–7.16 (m, 1H), 6. 34(s,1H),3.39(t,J=7.5Hz,1H),2.99(t,J=7.5Hz,1H),1.99(d,J=33.1Hz,1H); 13 C NMR (126MHz, CDCl3) δ205.05,163.03,153.85,148.46,142.06,130.44,120.86,109.64,100.80,45.93,29.26; 19 F NMR (471MHz,CDCl3)δ39.64,37.35;Molecular formula: C 15 H 12 F2O9S2; molecular weight: 438.3688.

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

[0036] Compound (1) and phloretin were weighed separately and dissolved in DMSO to prepare a 20 mmol / L stock solution. The stock solution was then 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, pH 6.8 phosphate buffer (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 microwell and incubated at 37°C for 20 min. The absorbance at 405 nm was measured to detect changes in enzyme activity. Acarbose was also used as a positive control.

[0037] The results are as follows Figure 1 As shown by Figure 1 It can be seen that the inhibitory activity of the derived compound (1) on α-glucosidase is higher than that of the original compound phloretin (190.48 μM), and is also 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 of varying concentrations (1.0 mL, 0–1000 mM). Fluorescence was measured after 5 min of equilibration. 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, 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 290–500 nm.

[0042] The results are as follows Figure 2 As shown in Figure 1, the fluorescence quenching of α-glucosidase can be used to characterize the affinity of a compound to the enzyme. α-glucosidase has a strongest intrinsic fluorescence at around 340 nm caused by Trp and Tyr residues. Therefore, the degree of binding can be determined by measuring the effect of the compound on the emission spectra of the two enzymes. Figure 2 As can be seen from the figure, the fluorescence intensity of the enzyme decreases significantly with increasing compound concentrations, 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 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 with α-glucosidase, thereby exerting a stronger inhibitory effect on the enzyme.

[0043] Example 4 In vitro starch digestion inhibition experiment

[0044] The effects of inhibitors on in vitro starch digestibility were 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. The mixture was boiled in a boiling water bath for 10 min, cooled to room temperature, and sodium acetate buffer (2.5 mL, 0.4 M, pH 5.2, containing 0.18% (w / v) CaCl2) was added. After equilibration at 37°C for 15 min, fresh porcine trypsin extract, amyloglucosidase, and an inhibitor cocktail (5.5 mL) were added to hydrolyze the starch. Simultaneously, groups without inhibitors and acarbose served as blank and positive controls. At 20, 60, 120, and 240 min, 250 μL of the starch hydrolyzate was collected from the centrifuge tube and 10.0 mL of 66% (v / v) ethanol was added. Glucose production was measured using a d-glucose assay kit (GOPOD).

[0045] The results are as follows Figure 3As shown, the results show 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 with 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 with phloretin. This shows that compound (1) can better inhibit the conversion of starch into glucose and has extremely high anti-diabetic potential.

[0046] Table 2

[0047]

[0048] Example 5 Oral Glucose Tolerance Test

[0049] 5- to 6-week-old C57 / BL mice were used as experimental subjects. They were acclimated to the experimental environment for one week before the formal experiments. Animal experiments were conducted in a specific pathogen-free (SPF) environment. This study was approved by the Laboratory Animal Care and Welfare Ethics Committee of Beijing Huayuan Times Technology Co., Ltd. (Ethics Review Approval Number: HYSD2023-04). C57 / BL mice were randomly divided into a normal group, an acarbose group, a phloretin group, and a compound (I) group, with 8 mice in each group. Before the oral glucose tolerance test, all mice fasted for 12 hours, with free access to water during this period. Phloretin, compound (I), and acarbose were dissolved in saline containing 10% DMSO. The oral dose was set at 20 mg / kg. The normal group mice were gavaged with an equal volume of saline. Five minutes after drug administration, blood glucose levels were measured in each group at 0, 30, 60, 90, and 120 minutes after gavage with a 2 mg / kg starch solution. OGTT curves were then plotted, and the area under the curve (AUC) was calculated.

[0050] Oral glucose tolerance test ( Figure 4 ), the results showed that acarbose and compound intervention can effectively inhibit the increase of blood sugar after meal. During the whole digestion process, by calculating the area under the blood sugar curve 2 hours after meal ( Figure 4 ), it can be concluded that compared to phloretin, compound (I) can reduce blood sugar fluctuations after a meal by 23.9%. Compared to acarbose, compound (I) can reduce blood sugar fluctuations after a meal by 11.9%. Compound (I) has significantly better control of blood sugar levels 2 hours after a meal than the parent compound phloretin, and compound (I) is more conducive to controlling the rise in blood sugar after a meal throughout the digestive process.

[0051] Table 3

[0052]

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

Claims

1. A pharmaceutical composition for treating diabetes, characterized in that: The pharmaceutical composition comprises a compound and a pharmaceutically acceptable carrier, wherein the compound is a phloretin derivative represented by formula (1): The R is F; Also contains adjuvants.

2. The pharmaceutical composition according to claim 1, wherein 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.

3. The pharmaceutical composition according to claim 1 or 2, wherein The dosage form of the pharmaceutical composition is tablet, pill, capsule, oral solution or injection.

4. Use of a compound in the preparation of an α-glucosidase inhibitor, characterized in that: The compound is a phloretin derivative represented by formula (1): The R is F; The use is for non-therapeutic purposes in vitro.

5. Use of a compound in the preparation of a drug for treating diabetes, characterized in that: The compound is a phloretin derivative represented by formula (1): The R is F.

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