Compounds having anti-diabetic activity and use thereof

Abstract

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

Description

[0001] The present application is a divisional application of the Chinese invention patent application with the application number 202310280725.X and the patent name "A kind of compound with anti-diabetic activity and its preparation method and application" (the application date is December 24, 2021), the content of which is incorporated herein by reference in its entirety.

[0002] The original parent application of the invention patent with the application number 202310280725.X is the Chinese invention patent application with the application number 2021116040348 and the patent name "A kind of compound with anti-diabetic activity and its application" (the application date is December 24, 2021). TECHNICAL FIELD

[0003] The present application relates to the technical field of compounds with anti-diabetic activity, and specifically relates to a kind of compound with anti-diabetic activity and its application. BACKGROUND

[0004] Type II diabetes (T2DM) is a common disease of metabolic disorders of blood glucose, cholesterol, protein, water, and electrolyte levels, as well as cell dysfunction, 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. Studies have found that among the numerous enzymes, blood glucose levels are closely related to the activity of dipeptidyl peptidase IV (DPP-IV).

[0005] DPP-IV is a well-known drug target for treating type II diabetes because it degrades glucagon-like peptide (GLP-1) and glucose-dependent insulinotropic peptide (GIP) with high selectivity in the body. At the same time, DPP-IV inhibitors can enhance the activity of external 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 through DPP-IV inhibition. Currently, there are a large number of type II diabetes patients worldwide each year, and the disease course is long, requiring strong control, which causes pain and distress to an increasing number of patients. Therefore, finding more compounds with anti-diabetic activity is of great significance for the treatment of diabetes. SUMMARY

[0006] The present application aims to provide a kind of compound with anti-diabetic activity and its application to address the problem of patients with diabetes suffering from pain. The compound disclosed in the present application has strong anti-diabetic activity, opening up a new way for the prevention and treatment of diabetes.

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

[0008] A class of compounds with antidiabetic activity, said compounds being analogs of diprotin A.

[0009] The diprotin A analogs disclosed in this invention have strong anti-diabetic activity, belong to the DPP-IV inhibitors, and can improve blood insulin levels, providing a new option for the treatment of diabetes.

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

[0011]

[0012]

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

[0014]

[0015]

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

[0017]

[0018] Furthermore, the compound is isolated from, but not limited to, Panax notoginseng. Preferably, the compound is isolated from Panax notoginseng.

[0019] Another object of the present invention is to provide applications of the above-mentioned compounds.

[0020] The use of the compounds with antidiabetic activity described above in the preparation of a drug, wherein the drug is a drug for the prevention and / or treatment of diabetes.

[0021] Another object of the present invention is to provide a class of drugs comprising the above-mentioned compounds.

[0022] A class of drugs comprising the compounds described above that have antidiabetic activity.

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

[0024] 1. This invention discovered antidiabetic active ingredients in Panax notoginseng (a medicinal and edible herb), characterized the chemical structures of 25 diprotinin A analogs, and verified their antidiabetic activity; the compounds to be protected by this invention belong to DPP-IV inhibitors, IC50...50 The value was 0.40 mg / mL; molecular docking studies also confirmed the interaction between diprotin A analogues and DPP-IV; in addition, cell experiments and animal experiments have also demonstrated that the compounds disclosed in this invention can improve blood insulin levels and have a good hypoglycemic effect, opening up new avenues for the treatment of diabetes.

[0025] 2. This invention also discloses the application of compounds with anti-diabetic activity, which has high social value. Attached Figure Description

[0026] Figure 1 It is a molecular network of diprotin A analogues.

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

[0028] Figure 3 The DPP-IV inhibition rate of different concentrations of diprotin A analogues is shown.

[0029] Figure 4 This is a molecular docking score diagram of 25 diprotin A analogues.

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

[0031] Figure 6 This describes the effect of diprotin A analogues from Example 2 on GLP-1 levels in NCI-H716 cells. Detailed Implementation

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

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the 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 merely illustrative and not intended to limit the 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 Testing (Chengdu, China). Anhydrous disodium hydrogen phosphate was purchased from Keshi (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. *Panax notoginseng* was collected from Luxian County, Sichuan Province, China, and its species was identified based on morphological characteristics.

[0035] Example 1

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

[0037] Rapid analysis of anti-diabetic components in gynura medica less

[0038] Step 1: Take a sample of Panax notoginseng and prepare a test solution using methanol-water solution as the solvent.

[0039] Soak 1 kg of white-backed Panax notoginseng in 20 L of methanol-water solution containing 0.1% hydrochloric acid (50:50, vv). -1 The extract of Panax notoginseng (GD-E) was filtered, concentrated, and freeze-dried for further processing over two weeks.

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

[0041] GD-E was analyzed using ultra-high performance liquid chromatography (UHPLC). The procedure employed Inertsil C... 18 A chromatographic column (100 × 2.1 mm, 3 μm) was used, and the column oven temperature was set to 40 °C. A 0.1% formic acid aqueous solution (A) and acetonitrile (B) were used as the mobile phases, and gradient elution was performed at a flow rate of 0.3 mL / min. The gradient elution program was as follows: 0–2 min 5% B, 2–18 min 5–70% B, 18–20 min 50–100% B, and 20–25 min 100% B.

[0042] The eluent from ultra-high performance liquid chromatography (UHPLC) was introduced into a mass spectrometer, and primary and secondary mass spectrometry data of the compounds were acquired using an X500R Q-TOF mass spectrometer. Electrospray ionization (ESI) parameters were as follows: temperature: 500℃; ion source gases 1 and 2: 50 psi; curtain gas: 35 psi; CAD gas: 7 psi. IDA settings were: collision energy 40 V, maximum candidate ion 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 database, and then confirm them with compounds reported in the literature to determine the potential active ingredients.

[0044] Raw data files were acquired and output using SCIEX OS 1.4 software. MSconver software was used to convert the raw data files to mzXML format and construct a molecular network (MN). MN operating parameters were as follows: precursor ion mass deviation ±0.02 Da; daughter ion mass deviation ±0.02 Da; minimum pairing cosine: 0.7; minimum cluster size: 2; minimum matched fragment ion: 6.

[0045] GNPS uses a network diagram to visualize the similarity of secondary mass spectrometry data of compounds. Each node represents a compound, and different colors indicate different sources or properties. Nodes with similar structures (analogs) cluster together. A cosine value exists 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 IC50 value of [missing information]. 50 The concentration is 1.6–5.8 μg / mL, and it is usually used as a positive control in the DPP–IV inhibition assay; it can also prevent the degradation of GLP-1, thereby exerting a hypoglycemic effect.

[0046] Step 4: Enrich potential active ingredient analogs from Panax notoginseng extract using strong cation exchange (SPE) method.

[0047] First, dissolve the white-backed Panax notoginseng extract in an ethanol-water mixture containing 0.1% hydrochloric acid (20:80, vv). -1 In this study, a strong cation exchange solid-phase extraction (SPE) column was used for separation. The column was first activated with methanol, followed by separation with 250 mmol / L Na₂HPO₄ in a methanol-water (20:80, vv) solution. -1 Continue to activate the column. Before loading the sample, use methanol-water (20:80, v / v) -1The column was washed to remove residual Na₂HPO₄. After sample loading, the column was rinsed with methanol-water (50:50, v / v). -1 The column was washed to remove neutral compounds. A methanol-water solution (20:80, v / v) containing 150 mmol / L Na₂HPO₄ was used. -1 Elution with C 18 (10μm, Acchrom (China) was used as the packing material for desalting, and the methanol eluent was collected.

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

[0049] like Figure 1 As shown, solid circles represent compounds identified through library search, specifically diprotin A. Since the cosine value is set >0.7, it indicates that other nodes are analogues of diprotin A. Figure 2 As shown in (a), diprotin A generates ions with m / z values ​​of 229.1566, 86.0968, 72.0812, and 70.0815, and fragmentation is performed. Among these, m / z values ​​of 72.0812 and 70.0815 are characteristic ions containing an N5-membered ring, and these are used to assist in the analysis of analogs. During molecular fragmentation, isoleucine and valine are removed, generating M–113 Da and M–99 Da ions, respectively. Furthermore, diprotin A analogs exhibit a neutral loss of 159 Da due to the elimination of isoleucine and formic acid components. Figure 2 In (b), the precursor ion generated by equation (1) is m / z 328.2238 (–2.2ppm, C 16 H 29 N3O4 produces fragment ions similar to diprotin A fragments, 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]. + Furthermore, the molecular weight of formula (1) is 13 Da smaller than that of diprotin A. Therefore, formula (1) was identified as diprotin C by comparing MS and MS / MS data. In positive mode, as Figure 2 In (c), equation (2) is obtained at m / z 243.1709 (2.4ppm, C 12 H 22 [M+H] is generated at N2O3. + Ions, produced via cleavage, have an m / z of 144.1028 ([M+H–99)).+ The structure of formula (2) was successfully characterized by the presence of the same fragment ions (m / z 70, 72) as those in formula (1). Based on the above fragmentation pathway, 25 diprotin A analogues were preliminarily characterized, as shown in Table 1.

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

[0051]

[0052]

[0053]

[0054]

[0055] Step 6: Perform DPP-IV inhibitory activity assay on the potential active ingredient analog to obtain the IC50. 50 These analogues were then molecularly docked with DPP-IV to obtain several molecular docking binding energy values, in order to verify the antidiabetic activity of several potential active ingredient analogues.

[0056] Molecular docking, a computer-based method, can be used to predict drug-enzyme interactions. This study employed molecular docking to investigate the interaction between diprotin A analogues and DPP-IV (PDB crystal structure: 1WCY). Higher binding energy scores indicate a more rational and stable interaction between the ligand and protein. The preparation steps for all receptor proteins were as follows: removal of water molecules, addition of hydrogen atoms to the protein, and application of a CHARMM force field. All compounds used for docking were prepared using the "Prepare Ligands" module in DS3.1. Molecular docking score statistics are listed below. Figure 4 All diprotinin A analogues interacted with DPP-IV to varying degrees, suggesting they are potential inhibitors of DPP-IV. DPP-IV occurs as a dimer and forms two openings, providing a channel to the cavity, which is the exact binding site for the 25 analogues. Three analogues scored higher than diprotinin A. Figure 5 As shown in Table 1, equation (6) ( Figure 5b) Stably docks within the cavity of DPP–IV and interacts with several amino acid residues, with a docking value of 40.5567 kcal / mol. Formula (6) forms five conventional hydrogen bonds with amino acid residues Ser209, Glu205, Arg125, Ser630, and Tyr547, three C-H bonds with amino acid residues Glu206, Glu205, and Tyr547, and two hydrophobic interactions with amino acid residues His126 and His740. Similarly, Formula (3) Figure 5 a) and formula (23) Figure 5 c) It also forms hydrogen bonds and hydrophobic interactions with 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 with the DPP-IV kit. Then, 25 μL of 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 thoroughly mixed and incubated at 37°C for 10 min. Subsequently, 25 μL of the enzyme reaction mixture (2 μL of DPP-IV substrate in 23 μL of buffer) was added to each well and mixed. Finally, the sample was detected using a microplate reader (JIYUANBIO-TECH, China).

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

[0059]

[0060] in:

[0061] FLU1 represents the fluorescence intensity at T1; FLU2 represents the fluorescence intensity at T2; SlopeSM represents the slope of the sample inhibition group; SlopeEC represents 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%.

[0062] Using a known DPP-IV enzyme inhibitor (sitagliptin) as a positive control, the inhibitory effect of diprotin A analogues on DPP-IV was determined, and the IC50 was calculated. 50 Value. For example... Figure 3 As shown, the IC of diprotin A analogues 50 The concentration was 0.40 mg / mL, and the effect was dose-dependent (n=3). Several compounds (3, 6, 10, 12, and 16) with high molecular docking scores were synthesized by directed synthesis, and the IC50 of each compound inhibiting DPP–IV was tested. 50The results are shown in Table 2.

[0063] Table 2. IC50 of 6 representative diprotin A analogues, DPP-IV 50 (μg / mL) n = 5

[0064] Group Sitagliptin 3 6 10 12 16 23 IC 50 ]]> 0.18±0.07 150±5.05 120±6.32 310±6.12 301±7.36 450±8.08 160±7.07

[0065] Example 2

[0066] Cell experiments were conducted using the diprotin A analogue obtained in step 5 of Example 1 and the six directionally synthesized monomeric compounds.

[0067] NCI-H716 cells were used at a rate of 1×10 6 GLP-1 was cultured at a density of 1 / mL in 12-well plates. After 48 hours, the supernatant was aspirated, and 1 mL of buffer and the test drug were added. The positive drug was alogliptin (2 μg / mL). A mixture of 25 compounds with test drug concentrations of 100, 200, and 300 μg / mL was added. The mixture was incubated at 37°C for 2 hours. The supernatant was then aspirated, and the GLP-1 content was detected using an ELISA kit. The results of the diprotinin A analogue test are as follows: Figure 6 As shown in Table 3, analogues of Diprotin A significantly increased GLP-1 concentration in a concentration-dependent manner. At a concentration of 200 μg / mL, the GLP-1 concentration was 7.33 ± 0.44 pmol / L, and at 300 μg / mL, it was 7.87 ± 0.25 pmol / L (P < 0.05). Alogliptin significantly increased the GLP-1 concentration from 6.73 ± 0.14 pmol / L to 7.45 ± 0.12 pmol / L (P < 0.05). The results of GLP-1 concentration tests for monomeric compounds at a concentration of 50 μg / mL are shown in Table 3.

[0068] Table 3. Effects of diprotin A analogues and six representative monomeric compounds on GLP-1 concentration (pmol / L) n=5 Table 3

[0069] 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**

[0070] Note: Compared with the control group, *P<0.05, **P<0.01

[0071] Example 3

[0072] Animal experiments were conducted on the 25 compounds of diprotin A analogues obtained in step 5 of Example 1, namely the compounds numbered 2-26 in Table 1, and the 6 directed-synthesized monomeric compounds.

[0073] Male rats of similar weight and age were fed a high-fat diet for 4 weeks, followed by a 6-hour fast (with unlimited water) and a single intraperitoneal injection of 30 mg / kg STZ (0.5 mL / 100 g). The high-fat diet was continued for another 4 weeks, and blood glucose levels were measured. The successfully modeled group was selected for further treatment, and then switched to a normal diet. Seventy-two diabetic rats with successfully established models (blood glucose levels 15–20 mmol / L) were randomly divided into nine groups: alogliptin (3 mg / kg), a model group, and a drug group. The drug group consisted of a Diprotin A analog group (20 mg / kg) and six monomeric compound groups (5 mg / kg). Eight normal male rats from the same batch were used as the normal control group. After 7 consecutive days of intravenous administration to the first seven groups, the normal control group received an equal volume of saline. Blood glucose was measured after a 5-hour fast, followed by an injection of 2 g / kg glucose. Blood samples were collected from the tail tip of the rats at 30, 60, 90, and 120 minutes after injection, and blood glucose was measured using a Johnson & Johnson OneHand Microplate glucometer. The results are shown in Table 4.

[0074] Table 4. Glucose tolerance of Diprotin A analogues and 6 monomeric compounds

[0075]

[0076] Note: Compared with the model, *P<0.05, **P<0.01

[0077] Table 5. Effects of Diprotin A analogues and 6 monomeric compounds on fasting blood glucose (mmol / L) n=8

[0078] 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**

[0079] Note: Compared with the model group, *P<0.05, **P<0.01

[0080] As shown in Table 4, the blood glucose levels at all points during the glucose tolerance test in the model group rats were higher than those in the normal group rats (P<0.05). As shown in Table 5, after administration, Diprotin A analogues and six monomeric compounds significantly reduced blood glucose levels (P<0.05).

[0081] This invention utilizes the above-described method to discover compounds with antidiabetic activity from the natural product of Panax notoginseng, and efficiently and selectively isolates 25 diprotinin A analogs, characterizing their antidiabetic activity. The compounds to be protected in this invention belong to the DPP-IV inhibitor category, IC50. 50The value was 0.40 mg / mL; molecular docking studies also confirmed the interaction between diprotin A analogues and DPP-IV. Diprotin A analogues promoted the release of GLP-1 from NCI-H716 cells, which had a significant effect. Animal experiments found that the compounds disclosed in this invention have a significant effect on lowering blood sugar and can improve blood insulin levels, opening up new avenues for the treatment of diabetes.

[0082] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within 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: Equation (9); Equation (11); Equation (22).

2. The use of the compound with antidiabetic activity as described in claim 1 in the preparation of a medicament, wherein the medicament is a medicament for the prevention and / or treatment of diabetes.

3. A class of drugs, characterized in that, The drug comprises an effective amount of the compound with antidiabetic activity as described in claim 1.

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