Compound with anti-diabetic activity and application thereof

Abstract

The invention relates to a compound with anti-diabetic activity and application thereof, the compound is an analogue of diprotin A, the compound has strong anti-diabetic activity, belongs to a DPP-IV inhibitor, can improve blood insulin level, and opens up a new way for treatment of diabetes; the compound is applied to preparation of food, health care products and medicines for preventing or / and treating diabetes, and has high social value and important significance.

Description

[0001] This patent application is a divisional application of a Chinese invention patent application with the application number 202310280725.X and the patent title "A Class of Compounds with Anti-Diabetic Activity, Their Preparation Methods and Applications" (application date: December 24, 2021). Its content is incorporated herein by reference in its entirety.

[0002] Among them, the original parent case of the invention patent with the application number 202310280725.X is a Chinese invention patent application with the application number 2021116040348 and the patent title "A Class of Compounds with Anti-Diabetic Activity and Their Applications" (application date: December 24, 2021). Technical Field

[0003] The present invention relates to the technical field of compounds with anti-diabetic activity, and specifically relates to a class of compounds with anti-diabetic activity and their applications. Background Art

[0004] 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).

[0005] 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 anti-diabetic 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 anti-diabetic activity is of great significance for the treatment of diabetes. Summary of the Invention

[0006] The purpose of the present invention is to provide a class of compounds with anti-diabetic activity and their applications in view of the problem that diabetic patients are troubled by the disease. The compounds disclosed in the present invention have strong anti-diabetic activity, opening up a new way for the prevention and treatment of diabetes.

[0007] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A class of compounds with anti-diabetic activity, and the compounds are analogs of diprotin A.

[0009] 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 a new option for the treatment of diabetes.

[0010] Furthermore, the compounds are selected from at least one of the following structural formulas;

[0011]

[0012]

[0013] Furthermore, the compounds are selected from at least one of the following structural formulas;

[0014]

[0015]

[0016] Furthermore, the compounds are selected from at least one of the following structural formulas;

[0017]

[0018] Furthermore, the compounds are isolated from Gynura divaricata (L.) DC. but not limited to Gynura divaricata (L.) DC. Preferably, the compounds are isolated from Gynura divaricata (L.) DC.

[0019] Another object of the present invention is to provide the application of the above compounds.

[0020] The application of the compounds with anti-diabetic activity as described above in foods or health products.

[0021] The application of the compounds with anti-diabetic activity as described above in the preparation of drugs, and the drugs are drugs for preventing and / or treating diabetes.

[0022] Another object of the present invention is to provide a class of drugs containing the above compounds.

[0023] A class of drugs, and the drugs contain the compounds with anti-diabetic activity as described above.

[0024] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0025] 1. The present invention has discovered anti - diabetic active ingredients from Gynura divaricata which is both a medicine and a food, characterized the chemical structures of 25 diprotin A analogues, and verified their anti - diabetic activities; 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 have also confirmed the interaction between diprotin A analogues and DPP–IV; in addition, cell experiments and animal experiments have also proved that the compounds disclosed by the present invention can improve blood insulin levels and have good hypoglycemic effects, opening up a new way for the treatment of diabetes.

[0026] 2. The present invention also discloses the application of compounds with anti - diabetic activities, which has high social value. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the molecular network of diprotin A analogues.

[0028] Figure 2 are the secondary mass spectra and possible cleavage pathways of diprotin A (a), formula (2) (b) and formula (3) (c).

[0029] Figure 3 is the DPP–IV inhibition rate of diprotin A analogues at different concentrations.

[0030] Figure 4 is the molecular docking score chart of 25 diprotin A analogues.

[0031] Figure 5 is the schematic diagram of the molecular docking interaction between DPP–IV and three compounds of diprotin A, formula (2) and formula (3).

[0032] Figure 6 is the effect of the diprotin A analogue in Example 2 on the GLP - 1 level in NCI - H716 cells. DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in detail below with reference to the accompanying drawings.

[0034] 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.

[0035] 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). Anhydrous disodium hydrogen phosphate was purchased from Keshitest (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.

[0036] Example 1

[0037] 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 due to the extremely complex nature of natural products, this remains a huge challenge.

[0038] Rapid analysis of anti-diabetic components in Gynura divaricata

[0039] Step 1: Take a sample of Gynura divaricata and prepare a test solution using a methanol-aqueous solution as the solvent.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Introduce the eluent of ultra-high performance liquid chromatography into a mass spectrometer, and use an X500R Q-TOF mass spectrometer to collect the first-order and second-order mass spectrometry data of compounds. The electrospray ionization (ESI) parameters are as follows: temperature: 500 °C; ion source gases 1 and 2: 50 psi; curtain gas: 35 psi; CAD gas: 7 psi. The specific IDA settings are: collision energy 40 V, maximum number of candidate ions 10; intensity threshold: 400 cps; full scan mass range: 100 - 1500 Da; ion spray voltage: 5500 V.

[0044] 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 database searching, and then confirm with the compounds reported in the literature to determine the potential active ingredients.

[0045] Use SCIEX OS 1.4 software to collect and output the original data files. Use MSconver software to convert the original data files into the mzXML format and establish a molecular network (MN). The MN operation parameters are 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 number of matching fragment ions: 6.

[0046] GNPS forms a visual network diagram based on the similarity of the second-order mass spectrometry data of compounds. One node represents one compound, and the colors of different nodes represent different sources or attributes. Nodes with similar structures (analogues) are clustered in one cluster. There is 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.

[0047] Step 4: Enrich the potential active ingredient analogues from the Gynura divaricata extract by strong cation exchange SPE method.

[0048] 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 ) of 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.

[0049] Step 5: Take the methanol eluate, analyze it by ultra-high 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.

[0050] As Figure 1 shown, solid circles represent compounds identified by library search and 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 produces ions m / z 229.1566, 86.0968, 72.0812, and 70.0815, and fragmentation assignments are made. Among them, m / z 72.0812 and 70.0815 are characteristic ions of the N5-membered ring containing N, and these are used to assist in the 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 the MS and MS / MS data, formula (1) was identified as diprotin C. In the positive mode, as Figure 2 (c) shows, formula (2) is at m / z 243.1709 (2.4 ppm, C 12 H22 N 2 O 3 [M+H] ions are generated at the position of + and m / z 144.1028 ([M+H–99] + ) ions and the same fragment ions (m / z 70, 72) as those in formula (1) are generated by cleavage. Therefore, the structure of formula (2) was successfully characterized. Based on the above fragmentation pathways, 25 diprotin A analogues were preliminarily characterized, as shown in Table 1.

[0051] Table 1 Structures and molecular information of 25 diprotin A analogues

[0052]

[0053]

[0054]

[0055]

[0056] Step 6. The potential active ingredient analogues were assayed for DPP–IV inhibitory activity to obtain IC 50 ; and these analogues were docked with DPP–IV to obtain a number of molecular docking binding energy values to verify the antidiabetic activities of several potential active ingredient analogues.

[0057] Molecular docking technology, as a computer-based method, can be used to predict drug-enzyme interactions. The interactions between diprotin A analogues and DPP–IV (PDB crystal structure: 1WCY) were studied by 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: removing water molecules, adding hydrogen atoms to the protein, and applying 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 analogues 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 analogues. The scores of three analogues were higher than that of diprotin A. As Figure 5 shown in Table 1, formula (6) ( Figure 5b) Stably docked in the cavity of DPP–IV and interacted with several amino acid residues, with a docking value of 40.5567 kcal / mol. Equation (6) formed five conventional hydrogen bonds with amino acid residues Ser209, Glu 205, Arg 125, Ser 630, and Tyr 547, three carbon-hydrogen bonds with amino acid residues Glu 206, Glu205, and Tyr 547, and two hydrophobic interactions with amino acid residues His 126 and His 740. Similarly, Equation (3)( Figure 5 a) and Equation (23)( Figure 5 c) also formed hydrogen bonds and hydrophobic interactions with amino acids in DPP–IV. Other compounds also interacted with DPP–IV through hydrogen bonds and hydrophobic interactions.

[0058] 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 with 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. After mixing well, it was 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).

[0059] The inhibitory activity of DPP–IV was calculated as follows:

[0060]

[0061] Where:

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

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

[0064] Table 2 IC of 6 representative diprotin A analogs against DPP–IV 50 (μg / mL) 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 analog of diprotin A obtained in step 5 of Example 1 and the 6 monomeric compounds synthesized 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 concentration was a mixture of 25 compounds at 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 can increase the GLP-1 concentration in a concentration-dependent manner. When the concentration is 200 μg / mL, the GLP-1 concentration is 7.33 ± 0.44 pmol / L, and when the concentration is 300 μg / mL, the GLP-1 concentration is 7.87 ± 0.25 pmol / L, both with 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 6 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 analogues of diprotin A obtained by separating in Step 5 in Example 1, i.e., 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 a high - fat diet for 4 weeks, they were fasted for 6 h without water deprivation and then intraperitoneally injected with 30 mg / kg STZ (0.5 mL / 100 g) once. They continued to be fed 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. Seventy - two 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 further divided into the Diprotin A analogue group (20 mg / kg) and the 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 analogues 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 analogues and 6 monomeric compounds n = 8

[0080] Group Normal group Positive group Model group Diprotin A analog 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 rats in the model group were higher than those of the normal group rats (P < 0.05). As shown in Table 5, after administration, the Diprotin A analogues and 6 monomeric compounds significantly reduced the blood glucose level (P < 0.05).

[0083] Using the above - mentioned method, the present invention discovered compounds with anti - diabetic activity from the natural products of Gynura divaricata, and efficiently and selectively separated 25 analogues of diprotin A, and characterized their anti - diabetic activity; the compounds to be protected by the present invention belong to DPP - IV inhibitors, IC50 The value is 0.40 mg / mL; molecular docking studies also confirmed the interaction between the diprotin A analog and DPP-IV. The diprotin A analog promotes the release of GLP-1 from NCI-H716 cells, showing a significant effect. Animal experiments found that the compounds disclosed in 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 compound is selected from at least one of the following structural formulas:

2. Use of the compound with antidiabetic activity according to claim 1 in the preparation of a drug, which 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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