Compound K802 and application thereof in preparation of diabetes medicines
Compound (1) was screened using virtual screening technology, which solved the problem of large side effects of existing α-glucosidase inhibitors, achieved efficient inhibition of α-glucosidase activity, significantly reduced postprandial blood glucose, and had a better therapeutic effect on type 2 diabetes.
Patent Information
- Application Number
- CN202510978667.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-11-25
AI Technical Summary
Existing alpha-glucosidase inhibitors such as acarbose, vogose, and miglitol have a wide range of side effects and have failed to effectively inhibit alpha-glucosidase activity, resulting in poor postprandial blood glucose control.
Compound (1) was screened from a large compound library using virtual screening technology and its activity was verified. It was found that it could significantly inhibit α-glucosidase activity, including high affinity binding to α-glucosidase and strong inhibitory effect.
Compound (1) showed significant α-glucosidase inhibitory effects in in vitro and in vivo experiments, reduced postprandial blood glucose levels, reduced side effects, and had greater anti-diabetic potential.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of medicine, and particularly relates to a compound as shown in formula (1) and application thereof in preparation of a drug for treating diabetes. BACKGROUND
[0002] Type 2 diabetes (T2DM) is a serious chronic metabolic disease with long-term hyperglycemia, which is characterized by insulin resistance and insufficient insulin secretion from pancreatic b cells, leading to hyperglycemia. 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 in glucose level in the blood comes from the decomposition of carbohydrates catalyzed by enzymes such as alpha-glucosidase and alpha-amylase. Alpha-glucosidase is an important membrane-bound enzyme located on the surface membrane of the small intestinal brush border, which can hydrolyze 1,4-alpha-glucoside bonds to generate alpha-glucose. Therefore, alpha-glucosidase inhibitors can delay the intake of carbohydrates, so that the postprandial blood glucose remains within the normal range, and thus are widely used in the treatment of T2DM patients. At present, some alpha-glucosidase inhibitors (acarbose, voglibose and miglitol) are used for the treatment of type 2 diabetes, but these drugs have a wide range of side effects including stomach pain, diarrhea, flatulence, allergic reactions, etc.
[0003] Virtual screening is currently the main means of new drug research and development, which is mainly used for studying large compound libraries, and can quickly screen small molecules with high potential for interaction with target proteins for later biochemical evaluation in vitro. Therefore, using virtual screening technology can quickly screen high-potential alpha-glucosidase inhibitors from large compound libraries. At the same time, combined with experimental verification, new compounds that can interact with target proteins can be greatly improved. SUMMARY
[0004] In view of the deficiencies of the prior art, the present application aims to provide a new compound with higher inhibitory effect on alpha-glucosidase, so as to solve the deficiencies of current clinical drugs.
[0005] The first aspect of the present application provides a compound, which is a compound as shown in formula (1):
[0006]
[0007] The compound is named 4-hydroxy-5-(3-(4-methoxyphenyl)-5-methyl-1-phenyl-1H-pyrazol-4-yl)phthalonitrile.
[0008] The second aspect of the present application provides a pharmaceutical composition for treating diabetes, which comprises the compound of the first aspect and a pharmaceutically acceptable carrier.
[0009] The third aspect, the present application also provides the use of the compound of the first aspect in the preparation of an α-glucosidase inhibitor.
[0010] Further, the use is a non-therapeutic use.
[0011] The fourth aspect, the present application also provides the use of the aforementioned compound or pharmaceutical composition in the preparation of a drug for treating diabetes.
[0012] Compared with the prior art, the present application has the following advantages,
[0013] The present application can significantly inhibit the activity of α-glucosidase by screening the compound (1) from a large compound library, thereby inhibiting the conversion of starch into glucose, and has a very high anti-diabetic potential. BRIEF DESCRIPTION OF DRAWINGS
[0014] Figure 1 Verification of virtual screening results.
[0015] Figure 2 Determination of IC50 value of the compound on α-glucosidase.
[0016] Figure 3 Fluorescence quenching of the compound on α-glucosidase.
[0017] Figure 4 Molecular docking of the compound with α-glucosidase
[0018] Figure 5 Inhibition of starch digestion by the compound. DETAILED DESCRIPTION
[0019] The concept and technical effects of the present application will be further described below in combination with specific examples, so as to fully understand the purposes, features and effects of the present application. The methods are all conventional methods unless otherwise specified. The materials can be obtained from public commercial channels unless otherwise specified. The illustrative examples of the present application and their descriptions are used to explain the present application and do not constitute an improper limitation on the present application. It should be noted that the examples in the present application and the features in the examples can be combined with each other without conflict.
[0020] Example 1: Compound screening
[0021] Autodock Vina was used to perform virtual screening on 100K+ compounds in a representative diverse library from ChemDiv database. In addition, 2QMJ and 1CXW were selected from protein database as crystal structures for docking of a-glucosidase and a-amylase. The compounds were docked with a-glucosidase and a-amylase respectively to obtain the scoring results, and the compounds with the docking score of a-glucosidase <-9.0 and the docking score of a-amylase >-5.0 were selected, synthesized by ChemDiv Company, and verified for activity. The synthesis method of compound (1) is as follows:
[0022]
[0023] Subsequently, the inhibitory effect of the screened compounds on the two digestive enzymes was determined at a concentration of 250 μM, and the results are shown in Table 1 and Table 2. Figure 1 Among the five screened compounds, compound (1) showed obvious a-glucosidase specific inhibitor effect, and the inhibitory effect on a-glucosidase reached 91.1% at a concentration of 250 μM.
[0024] Table 1: Compound structure and a-glucosidase and a-amylase inhibition rate
[0025]
[0026]
[0027] Example 2: Determination of IC50 value of compound (1) on a-glucosidase
[0028] The in vitro a-glucosidase activity was determined by spectrophotometry. A phosphate buffer solution with pH 6.8 was used to prepare 0.5 U / mL a-glucosidase and 0.6 mM p-nitrophenyl-α-glucopyranoside (pNαGP). All test chemicals, including the standard drug acarbose, were dissolved in DMSO to form a 10 mM stock solution. Gradient dilution was performed with phosphate buffer solution to obtain sample solutions with different concentrations. First, different concentrations of compound (10 μL), enzyme solution (40 μL), and potassium phosphate buffer solution (100 μL) were pre-incubated at 37°C for 10 min in a 96-well plate. Then, 50 μL of substrate (pNαGP, 0.6 mM) was added to each microwell, and the absorbance was measured at 405 nm after incubation at 37°C for 20 min to detect the change in enzyme activity. Acarbose and DMSO were used as standard inhibitors and control inhibitors, respectively.
[0029] The formula for calculating the enzyme inhibitory activity of the tested compound is as follows:
[0030]
[0031] The IC50values of the test compounds were calculated by non-linear fitting (logit method).
[0032] The results are shown in Table 1. Figure 2 As can be seen from Table 1, the inhibitory activity of compound (1) (74.67 μM) on α-glucosidase is much higher than that of the medicinal acarbose (562.22 μM). Figure 2
[0033] Example 3: Fluorescence quenching experiment of compound (1) on α-glucosidase
[0034] The α-glucosidase (1.0 mL, 2 U / mL) was titrated with different concentrations (1.0 mL, 0-1000 mM) of inhibitor solution, and the fluorescence measurement was carried out after 5 min of equilibration. The fluorescence intensity of the reaction solution was measured at different temperatures (305.15, 310.15 and 315.15 K) by a fluorescence spectrometer (F-7100, Tokyo, Japan). The excitation and emission slit widths were set at 5.0 nm, the excitation wavelength was 280 nm, and the emission wavelength range was 290-500 nm.
[0035] The results are shown in Table 2. Figure 3 As can be seen from Table 2, the fluorescence quenching of α-glucosidase can be used to characterize the affinity of the compound to the enzyme. The α-glucosidase has a strongest intrinsic fluorescence at about 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 spectrum of the two enzymes. As can be seen from Table 2, the fluorescence intensity of the enzyme is significantly reduced with the increase of the concentration of the compound, indicating that the compound can quench the intrinsic fluorescence of the enzyme and bind to it. However, by comparing the effect of the compound on the fluorescence of the enzyme at the same concentration, it can be seen that compound (1) has a greater effect on the fluorescence quenching of α-glucosidase, which also indicates that the affinity and interaction force of compound (1) to α-glucosidase are stronger, thereby having a stronger inhibitory effect on the enzyme. Since Ksv can reflect the interaction (binding) of the inhibitor with α-glucosidase, the higher the Ksv value, the higher the binding performance of the inhibitor. As can be seen from Table 2, the Ksv value of compound (1) is much higher than that of acarbose at different temperatures, further indicating that compound (1) has a stronger affinity to α-glucosidase. Figure 3 Table 2 Affinity determination of the compound to α-glucosidase at different temperatures
[0036]
[0037] Example 4 Molecular docking of the compound to α-glucosidase
[0038]
[0039] AutoDock Vina was used for molecular docking in this experiment. The first step was to prepare the protein crystal structure. 2QMJ was selected as the crystal structure for a-glucosidase docking from the protein database, and the protein crystal structure was processed by removing water molecules and adding hydrogen atoms. The second step was to prepare small molecules. The three-dimensional structure of the inhibitor was drawn using ChemBiodraw Ultra 14.0 (Waltham Perkin Elmer Instruments Co., MA, USA) and the energy of the small molecule was minimized. The third step was to generate a docking box for the center and use AutoDock Vina for docking. Ten poses were generated during the docking process, and the pose with the highest Glide score was selected to study the interaction between the inhibitor and a-glucosidase.
[0040] The results, as shown in Figure 4 The binding of the clinical drug acarbose in the active site mainly involves multiple hydrogen bonds, especially with the acarbose ring. The interaction mainly occupies the -1 and +1 binding sites, and the same phenomenon is also found in the docking of the screened compounds. Compound (1) mainly forms hydrogen bonds or π-π interactions with TYR605, GLC603 and TRP406 of the a-glucosidase active pocket, as well as multiple amino acid residues to form hydrophobic interactions, thus forming a strong binding effect.
[0041] Example 4 In vitro starch digestion inhibition experiment
[0042] The effect of the inhibitor on the in vitro starch digestion rate 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 distilled water. Boil in a boiling water bath for 10 min, cool to room temperature, and add sodium acetate buffer (2.5 mL, 0.4 M, pH 5.2, containing 0.18% (w / v) CaCl2). After equilibrating the sample tube at 37°C for 15 min, add a fresh porcine trypsin extract, starch glycosidase and inhibitor mixture (5.5 mL) to hydrolyze the starch. At the same time, the group without adding the 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 starch hydrolysate was taken from the centrifuge tube and added to 10.0 mL of 66% (v / v) ethanol. The glucose production was determined using a d-glucose assay kit (GOPOD).
[0043] The results are shown in Table 3, which show that compound (1) can significantly inhibit α-glucosidase activity. At 20 min, 60 min, 120 min and 240 min, compound (1) reduced glucose production by 7.83%, 4.10%, 9.00% and 3.73% respectively, as compared to acarbose. This shows that compound (1) can more effectively inhibit the conversion of starch to glucose, and has a high anti-diabetic potential
[0044] Table 3 GOPOD assay for determination of glucose production
[0045]
[0046]
[0047] Example 5 In vivo starch digestion inhibition experiment
[0048] Intervention experiment on normal mice Figure 5 Pre-administration of compound (1) and acarbose can effectively reduce the rapid rise in blood glucose level within 30 minutes before starch suspension consumption (P < 0.01, Figure 5 A), and some interesting additional findings are also revealed. First, we observed that compound (1) has a control postprandial blood glucose effect comparable to acarbose, and has an effect of alleviating the side effects of acarbose. This shows that compound (1) is a very potential alternative treatment option for those who have difficulty in starch digestion and postprandial blood glucose control. AUC represents the overall blood glucose response over a period of time, and the lower the AUC curve value, the smaller the postprandial blood glucose fluctuation and the lower the harm to diabetic patients. When comparing the area under the curve (AUC) of the control group and the compound (1) or acarbose treatment group, the results show that the difference is statistically significant (P < 0.001). At the same time, the area under the curve of the AUC curve of the compound (1) group is significantly close to that of the acarbose group, and the blood glucose fluctuation reaches the treatment effect of the acarbose group. In this case, it clearly shows that both compound (1) and acarbose can more effectively reduce postprandial blood glucose levels as compared to no treatment.
[0049] The above-described embodiments are only some of the embodiments of the present application, but not all the embodiments, and other embodiments obtained by those skilled in the art based on the embodiments of the present application without creative labor are within the scope of protection of the present application.
Claims
1. A compound, the compound being represented by formula (1): ###0001### (1) 2. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the compound of claim 1 and a pharmaceutically acceptable carrier.
3. The pharmaceutical composition of claim 2, wherein It further comprises an adjuvant.
4. The pharmaceutical composition of claim 3, wherein The adjuvant is at least one of a sustained release agent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant or a lubricant.
5. The pharmaceutical composition according to claim 3 or 4, wherein The dosage form of the pharmaceutical composition is a tablet, a pill, a capsule, an oral solution or an injection.
6. Use of the compound of claim 1 or the pharmaceutical composition of any one of claims 2 to 5 in the preparation of an α-glucosidase inhibitor.
7. Use of the compound of claim 1 or the pharmaceutical composition of any one of claims 2 to 5 in the preparation of a medicament for treating diabetes.