A class of compounds with antidiabetic activity, their preparation methods and applications

Abstract

The present invention relates to compounds having antidiabetic activity, their preparation methods and uses. The compounds are analogues of diprotin A, have strong antidiabetic activity, belong to DPP-IV inhibitors, can improve blood insulin levels, and open up a new way for the treatment of diabetes. Applying the compounds to the preparation of foods, health products and drugs for preventing and / or treating diabetes has high social value and great significance.

Description

[0001] This patent application is a divisional application of a Chinese invention patent application with the application number 2021116040348 and the patent name "A Class of Compounds with Antidiabetic Activity and Their Applications" (filing date: December 24, 2021). Its content is incorporated herein by reference in its entirety. Technical Field

[0002] The present invention relates to the technical field of compounds with antidiabetic activity, and specifically relates to a class of compounds with antidiabetic activity, their preparation methods and applications. Background Art

[0003] Type II diabetes (T2DM) is a common disease of metabolic disorders of blood glucose, cholesterol, protein, water, and electrolyte levels, as well as low cell function, impaired insulin secretion, and insulin resistance. Patients with poor blood glucose control are prone to various diabetic complications, including renal failure, ketoacidosis, and diabetic non-ketotic hyperosmolar syndrome. Blood glucose regulation is a highly complex process controlled by multiple enzymes, hormones, and nerves. It has been found that among many enzymes, there is a close relationship between blood glucose levels and the activity of dipeptidyl peptidase IV (DPP–IV).

[0004] DPP–IV is a well-known drug target for the treatment of type II diabetes because it degrades glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) with high selectivity in vivo. At the same time, DPP–IV inhibitors can enhance the activity of exogenous GLP-1 and GIP, thereby improving blood insulin levels. Some synthetic antidiabetic molecules, such as sitagliptin, linagliptin, and gemigliptin, have been approved for the treatment of type II diabetes by DPP–IV inhibition. Currently, a large number of type II diabetes patients are generated globally every year. The disease has a long course and requires strong control, bringing pain and trouble to more and more patients. Therefore, finding more compounds with antidiabetic activity is of great significance for the treatment of diabetes. Summary of the Invention

[0005] The purpose of the present invention is to provide a class of compounds with antidiabetic activity, their preparation methods and applications in view of the problem that diabetic patients are troubled by the disease. The compounds disclosed in the present invention have strong antidiabetic activity, opening up a new way for the prevention and treatment of diabetes.

[0006] In order to achieve the above purpose, the technical solution adopted by the present invention is:

[0007] A class of compounds with antidiabetic activity, and the compounds are analogs of diprotin A.

[0008] The analogs of diprotin A disclosed by the present invention have strong anti-diabetic activity, belong to DPP-IV inhibitors, can improve blood insulin levels, and provide new options for the treatment of diabetes.

[0009] Furthermore, the compound is selected from at least one of the following structural formulas;

[0010]

[0011]

[0012] Furthermore, the compound is selected from at least one of the following structural formulas;

[0013]

[0014]

[0015] Furthermore, the compound is selected from at least one of the following structural formulas;

[0016]

[0017] Furthermore, the compound is isolated from Gynura divaricata (L.) DC. but not limited to it. Preferably, the compound is isolated from Gynura divaricata (L.) DC.

[0018] Another object of the present invention is to provide the application of the above compound.

[0019] Such as the application of the above compound with anti-diabetic activity in food or health products.

[0020] Such as the application of the above compound with anti-diabetic activity in the preparation of a drug, and the drug is a drug for preventing and / or treating diabetes.

[0021] Another object of the present invention is to provide a class of drugs containing the above compound.

[0022] A class of drugs, the drugs contain the above compound with anti-diabetic activity.

[0023] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are:

[0024] 1. The present invention discovers anti-diabetic active ingredients from Gynura divaricata (L.) DC. which can be used as both medicine and food, characterizes the chemical structures of 25 diprotin A analogs, and verifies their anti-diabetic activities; the compounds to be protected by the present invention belong to DPP-IV inhibitors, IC 50The value is 0.40 mg / mL; molecular docking studies also confirmed the interaction between the diprotin A analog and DPP-IV; in addition, cell experiments and animal experiments also demonstrated that the compounds disclosed in the present invention can improve blood insulin levels and have good hypoglycemic effects, opening up a new way for the treatment of diabetes.

[0025] 2. The present invention also discloses the application of the compound with antidiabetic activity, which has high social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the molecular network of the diprotin A analog.

[0027] Figure 2 are the secondary mass spectra and possible fragmentation pathways of diprotin A (a), formula (2) (b), and formula (3) (c).

[0028] Figure 3 are the DPP-IV inhibition rates of different concentrations of the diprotin A analog.

[0029] Figure 4 is the molecular docking score chart of 25 diprotin A analogs.

[0030] Figure 5 is the schematic diagram of the molecular docking interaction between DPP-IV and three compounds, namely diprotin A, formula (2), and formula (3).

[0031] Figure 6 is the effect of the diprotin A analog in Example 2 on the GLP-1 level in NCI-H716 cells. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0032] The present invention will be described in detail below with reference to the accompanying drawings.

[0033] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0034] In the following examples, LC-MS grade acetonitrile and LC-MS grade formic acid were purchased from Fisher Scientific (Massachusetts, USA). Ethanol was purchased from Titan Scientific (Shanghai, China). Methanol was purchased from Jinshan Chemical Reagents (Chengdu, China). Disodium hydrogen phosphate anhydrous was purchased from Keshijia (Chengdu, China). Hydrochloric acid was purchased from Xilong Chemical (Chengdu, China). A Milli-Q water purification system (Billerica, MA, USA) was used to produce ultrapure water. Gynura divaricata was collected from Luxian County (Sichuan, China) and identified by its morphological characteristics.

[0035] Example 1

[0036] Natural products play a crucial role in modern life sciences and new drug development. Discovering active compounds from natural products has attracted the attention of scientists, but it remains a huge challenge due to the extremely complex nature of natural products.

[0037] Rapid Analysis of Antidiabetic Components in Gynura divaricata

[0038] Step 1: Take the Gynura divaricata sample and prepare a test solution using a methanol-aqueous solution as the solvent.

[0039] Soak 1 kg of Gynura divaricata in 20 L of methanol–water (50:50, v / v -1 ) containing 0.1% hydrochloric acid for two weeks. Filter, concentrate, and freeze-dry the extract (GD-E) of Gynura divaricata for further processing.

[0040] Step 2: Dissolve the test solution prepared in Step 1 and analyze it by ultra-high performance liquid chromatography–mass spectrometry to obtain the mass spectrometry information of the crude extract.

[0041] Analyze GD-E by ultra-high performance liquid chromatography. This process uses an Inertsil C 18 chromatographic column (100×2.1 mm, 3 μm), the column oven temperature is set at 40 °C, an aqueous solution of 0.1% formic acid (A) and acetonitrile (B) are used as the mobile phase, and gradient elution is carried out at a flow rate of 0.3 mL / min. The gradient elution program is as follows: 5% B for 0–2 min, 5–70% B for 2–18 min, 50–100% B for 18–20 min, and 100% B for 20–25 min.

[0042] The eluent of ultra-high performance liquid chromatography was introduced into a mass spectrometer, and the X500R Q-TOF mass spectrometer was used to collect the first-order and second-order mass spectrometry data of the compounds. The electrospray ionization (ESI) parameters were as follows: temperature: 500 °C; ion source gas 1 and 2: 50 psi; curtain gas: 35 psi; CAD gas: 7 psi. The specific IDA settings were: collision energy 40 V, maximum candidate ions 10; intensity threshold: 400 cps; full scan mass range: 100 - 1500 Da; ion spray voltage: 5500 V.

[0043] Step 3: Import the mass spectrometry data obtained in Step 2 into the GNPS platform, use the GNPS database to characterize the chemical components in the test solution by searching the library, and then confirm with the compounds reported in the literature to determine the potential active ingredients.

[0044] SCIEX OS 1.4 software was used to collect and output the original data files. MSconver software was used to convert the original data files into the mzXML format and establish a molecular network (MN). The MN operation parameters were as follows: precursor ion mass deviation ±0.02 Da; product ion mass deviation ±0.02 Da; minimum paired cosine value: 0.7; minimum cluster size: 2; minimum matching fragment ions: 6.

[0045] GNPS formed a visual network diagram based on the similarity degree of the compound second-order mass spectrometry data, where one node represented one compound, and the colors of different nodes represented different sources or attributes. Nodes with similar structures (analogues) were clustered in a cluster. There was a cosine value between two nodes; the higher the cosine (0 - 1), the more similar the structures. Using this method, diprotin A was successfully identified. Diprotin A is a known DPP-IV inhibitor, with an IC 50 of 1.6 - 5.8 μg / mL, and is usually used as a positive control in DPP-IV inhibition tests; it can also prevent the degradation of GLP-1, thereby exerting a hypoglycemic effect.

[0046] Step 4: Enrich the potential active ingredient analogues from the Gynura divaricata extract by strong cation exchange SPE method.

[0047] First, dissolve the Gynura divaricata extract in ethanol-water (20:80, v / v -1 ) containing 0.1% hydrochloric acid. Use a strong cation exchange solid-phase extraction (SPE) column for separation. First, activate the column with methanol, and then continue to activate the column with methanol-water (20:80, v / v 2 HPO 4 at 250 mmol / L Na -1 ). Before loading the sample, use methanol-water (20:80, v / v -1)Wash the chromatographic column to remove residual Na 2 HPO 4 . After sample loading, wash the column with methanol - water (50:50, v -1 ) to remove neutral compounds. Use methanol - water (20:80, v / v 2 HPO 4 containing 150 mmol / L Na -1 ) for elution. Desalt using C 18 (10 μm, Acchrom, China) as the packing material, and collect the methanol eluate.

[0048] Step 5: Take the methanol eluate, analyze it by ultra - performance liquid chromatography, then introduce the eluate into a mass spectrometer to obtain the mass spectrometry data of the test sample solution; then upload the mass spectrometry data into the GNPS platform to construct a molecular network and characterize the potential active ingredients.

[0049] As Figure 1 shown, solid circles represent compounds identified by library search, which were identified as diprotin A because the cosine value was set to >0.7, indicating that other nodes are analogs of diprotin A. As Figure 2 (a) shows, diprotin A generates ions m / z 229.1566, 86.0968, 72.0812, and 70.0815, and fragment assignments are made. Among them, m / z 72.0812 and 70.0815 are characteristic ions of the N - containing 5 - membered ring, and these are used for auxiliary analysis of analogs. When the molecule fragments, isoleucine and valine are removed, generating M–113 Da and M - 99 Da ions respectively. In addition, diprotin A analogs show a neutral loss of 159 Da due to the elimination of isoleucine and formic acid composition. As Figure 2 (b), the parent ion generated by formula (1) is m / z 328.2238 (–2.2 ppm, C 16 H 29 N 3 O 4 ), generating daughter ions similar to those of diprotin A, including m / z 229.1561, 86.0968, 72.0812, and 70.0815. The fragment ion at m / z 229.1561 corresponds to [M + H–99] + . In addition, the molecular weight of formula (1) is 13 Da smaller than that of diprotin A. Therefore, by comparing MS and MS / MS data, formula (1) was identified as diprotin C. In the positive mode, as Figure 2 (c) shows, formula (2) has a peak at m / z 243.1709 (2.4 ppm, C 12 H22 N 2 O 3 ) generates [M+H] + ions, and by cleavage, generates m / z 144.1028 ([M+H–99] + ) ions and fragment ions (m / z 70, 72) identical to those of formula (1). Therefore, the structure of formula (2) was successfully characterized. Based on the above fragmentation pathways, 25 diprotin A analogs were preliminarily characterized, as shown in Table 1.

[0050] Table 1 Structures and molecular information of 25 diprotin A analogs

[0051]

[0052]

[0053]

[0054]

[0055] Step 6: Determine the DPP–IV inhibitory activity of the potential bioactive analogs to obtain IC 50 ; and perform molecular docking of these analogs with DPP–IV to obtain several molecular docking binding energy values to verify the antidiabetic activities of several potential bioactive analogs.

[0056] Molecular docking technology, as a computer-based method, can be used to predict drug-enzyme interactions. The interactions between diprotin A analogs and DPP–IV (PDB crystal structure: 1WCY) were studied using the molecular docking method. A higher score (binding energy value) indicates a more reasonable and stable interaction between the ligand and the protein. The preparation steps for all receptor proteins are as follows: remove water molecules, add hydrogen atoms to the protein, and apply the CHARMM force field. All compounds used for docking were prepared using the "Prepare Ligand" module in DS3.1. The molecular docking scoring statistical data are listed in Figure 4 . All diprotin A analogs interacted with DPP–IV to varying degrees, indicating that they are potential inhibitors of DPP–IV. DPP–IV appears as a dimer and forms two openings, providing channels leading to the cavity, which is the exact binding site for the 25 analogs. The scores of three analogs were higher than that of diprotin A. As Figure 5 shown in, in Table 1, formula (6)( Figure 5b) Stably docked inside the cavity of DPP–IV and interacting with several amino acid residues, the docking value is 40.5567 kcal / mol. Equation (6) forms five conventional hydrogen bonds with the amino acid residues Ser209, Glu 205, Arg 125, Ser 630, and Tyr 547, three carbon-hydrogen bonds with the amino acid residues Glu 206, Glu205, and Tyr 547, and two hydrophobic interactions with the amino acid residues His 126 and His 740. Similarly, Equation (3)( Figure 5 a) and Equation (23)( Figure 5 c) also form hydrogen bonds and hydrophobic interactions with the amino acids in DPP–IV. Other compounds also interact with DPP–IV through hydrogen bonds and hydrophobic interactions.

[0057] The inhibitory activity of DPP–IV was determined using a DPP–IV inhibitor screening kit (Merck, Germany). The total volume of the reaction mixture was 100 μL. First, the sample was dissolved using the buffer provided in the DPP–IV kit. Then, 25 μL of the sample and 1 μL of DPP–IV enzyme were added to 49 μL of DPP–IV detection buffer in a 96-well black plate. The mixture was homogenized and incubated at 37 °C for 10 min. Subsequently, 25 μL of the enzyme reaction mixture (2 μL of DPP–IV substrate added to 23 μL of buffer) was added to each well and mixed. Finally, it was detected using a microplate reader (JIYUANBIO-TECH, China).

[0058] The inhibitory activity of DPP–IV was calculated as follows:

[0059]

[0060]

[0061] Where:

[0062] FLU1 is the fluorescence intensity at T1; FLU2 is the fluorescence intensity at T2; SlopeSM is the slope of the sample inhibition group; SlopeEC is the slope of the enzyme control group. The ΔFLU value of the irreversible DPP–IV inhibitor is 0, and the relative inhibition rate is 100%.

[0063] The known DPP–IV enzyme inhibitor (sitagliptin) was used as a positive control to determine the inhibitory effect of the diprotin A analog on DPP–IV, and the IC 50 value was calculated. As Figure 3 shown, the IC 50was 0.40 mg / mL and showed a dose-dependence (n = 3). By directed synthesis, several compounds with higher molecular docking scores (3, 6, 10, 12, and 16) were obtained, and the IC 50 of each compound against DPP–IV was tested, and the results are shown in Table 2.

[0064] Table 2 IC 50 (μg / mL) of six representative diprotin A analogs against DPP–IV n = 5

[0065] Group Sitagliptin 3 6 10 12 16 23 <![CDATA[IC 50 > 0.18±0.07 150±5.05 120±6.32 310±6.12 301±7.36 450±8.08 160±7.07

[0066] Example 2

[0067] The analogs of diprotin A obtained in step 5 of Example 1 and six monomeric compounds obtained by directed synthesis were subjected to cell experiments.

[0068] NCI-H716 cells were cultured in 12-well plates at a density of 1×10 6 cells / mL. After 48 h, the supernatant was aspirated, 1 mL of buffer and the test drug were added. The positive drug was alogliptin (2 μg / mL), and the test drug was a mixture of 25 compounds at concentrations of 100, 200, and 300 μg / mL. After incubation at 37 °C for 2 h, the supernatant was aspirated, and the GLP–1 content was detected using an ELISA kit. The test results of the diprotin A analogs are as Figure 6 shown. The analogs of diprotin A could increase the GLP-1 concentration in a concentration-dependent manner. When the concentration was 200 μg / mL, the GLP-1 concentration was 7.33 ± 0.44 pmol / L, and when the concentration was 300 μg / mL, the GLP-1 concentration was 7.87 ± 0.25 pmol / L, both showing significant differences (P < 0.05). Alogliptin increased the GLP-1 concentration from 6.73 ± 0.14 pmol / L to 7.45 ± 0.12 pmol / L, and it was significant (P < 0.05). The test results of the monomeric compounds at a concentration of 50 μg / mL on the GLP–1 concentration are shown in Table 3.

[0069] Table 3 Effects of diprotin A analogs and six representative monomeric compounds on GLP–1 concentration (pmol / L) n = 5

[0070] Table 3

[0071] Group Control Group Positive Group 3 6 GLP–1 6.8±0.23 7.35±0.54 7.53±0.36** 7.51±0.11** Group 10 12 16 23 GLP–1 7.02±0.33* 7.23±0.02* 6.89±0.36 7.56±0.12**

[0072] Note: Compared with the control group, *P < 0.05, **P < 0.01

[0073] Example 3

[0074] The analogs of diprotin A obtained by separating in step 5 in Example 1, namely the mixture of 25 compounds numbered 2 - 26 in Table 1 and 6 monomeric compounds obtained by directed synthesis, were subjected to animal experiments.

[0075] Male rats of the same body weight and age were used. After feeding the rats with a high - fat diet for 4 weeks, they were fasted but allowed to drink water for 6 h, and then intraperitoneally injected with 30 mg / kg STZ (0.5 mL / 100 g) at one time. They continued to be fed with a high - fat diet for 4 weeks. Blood glucose was measured, and the successfully modeled group was selected for grouped administration, and the diet was changed to a normal diet. 72 diabetic rats with successful modeling (blood glucose value 15 - 20 mmol / L) were randomly divided into 9 groups: alogliptin (3 mg / kg) group, model group, and drug groups. The drug groups were divided into the diprotin A analog group (20 mg / kg) and 6 monomeric compound groups (5 mg / kg). Another 8 normal male rats of the same batch were used as the normal group. After continuous intravenous injection for 7 days in the first 7 groups, the normal group was given an equal volume of normal saline; after fasting for 5 h, blood glucose was measured, and then 2 g / kg glucose was injected respectively. Blood was taken from the rat tail tip at 30 min, 60 min, 90 min, and 120 min after injection, and blood glucose was measured using a blood glucose meter (Johnson & Johnson OneTouch Ultra). The results are shown in Table 4.

[0076] Table 4 Glucose tolerance of diprotin A analogs and 6 monomeric compounds

[0077]

[0078] Note: Compared with the model, *P < 0.05, **P < 0.01

[0079] Table 5 Fasting blood glucose (mmol / L) of diprotin A analogs and 6 monomeric compounds n = 8

[0080] Group Normal Group Positive Group Model Group Diprotin A Analogue 3 Blood Glucose 4.61±0.80 14.32±1.33 17.80±1.25 16.19±0.32* 14.45±1.51** Group 6 10 12 16 23 Blood Glucose 15.02±1.44** 16.88±0.21* 15.96±1.33** 15.39±0.16** 14.13±2.01**

[0081] Note: Compared with the model group, *P < 0.05, **P < 0.01

[0082] As shown in Table 4, the blood glucose values at each point in the glucose tolerance test of the model group rats were higher than those of the normal group rats (P < 0.05). As shown in Table 5, after administration, the diprotin A analogs and 6 monomeric compounds significantly reduced the blood glucose level (P < 0.05).

[0083] The present invention uses the above method to discover compounds with anti-diabetic activity from natural products of Gynura divaricata, and efficiently and selectively isolates 25 analogues of diprotin A, and characterizes their anti-diabetic activity; the compounds to be protected by the present invention belong to DPP-IV inhibitors, with an IC 50 value of 0.40 mg / mL; molecular docking studies also confirm the interaction between diprotin A analogues and DPP-IV. Diprotin A analogues promote the release of GLP-1 from NCI-H716 cells, showing a significant effect. Animal experiments find that the compounds disclosed by the present invention have an obvious effect on lowering blood sugar, can improve blood insulin levels, and open up a new way for the treatment of diabetes.

[0084] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A class of compounds with antidiabetic activity, characterized in that, the compound is an analogue of diprotin A; the structural formula of the compound is:

2. Use of the compound with antidiabetic activity according to claim 1 in the preparation of a drug, wherein the drug is a drug for preventing and / or treating diabetes.

3. Use of the compound with antidiabetic activity according to claim 1 in the preparation of a health product for assisting in reducing blood sugar.

4. A class of drugs, characterized in that, the drug contains an effective amount of the compound with antidiabetic activity according to claim 1.

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