Yunaconitine derivative and application thereof in preparation of hypoglycemic and antioxidant drugs

Abstract

The invention discloses a yunaconitine derivative and application of the yunaconitine derivative in preparation of hypoglycemic and antioxidant drugs, the yunaconitine derivative has a chemical structure as shown in a formula 1a or 1b, and the invention further provides application of the yunaconitine derivative in preparation of PTP1B inhibitors. The dosage form of the medicine is selected from at least one of a tablet, a capsule, a granule, a dripping pill, a suspension, syrup, an enteric-coated preparation, an emulsion suspension and an injection. The yunaconitine derivative disclosed by the invention is high in bioavailability and clinical use value, provides a candidate lead compound for research and development of novel preparations of hypoglycemic and antioxidant drugs, and has potential application value in the fields of hypoglycemic and antioxidant targeted drugs, probe design and fluorescent biomarkers.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to dianhydrolycorine derivatives and their application in the preparation of hypoglycemic and antioxidant drugs. Background Art

[0002] Diabetes is a common metabolic disease characterized by hyperglycemia, which can be caused by genetic or environmental factors and is mainly divided into type I diabetes and type II diabetes. The occurrence of long-term hyperglycemia in diabetic patients will further cause serious damage to multiple organs of the human body, such as the kidneys, liver, cardiovascular system, nervous system and other organs. On the one hand, free radicals are a class of highly reactive molecules that can damage cell membranes, proteins and DNA, leading to oxidative stress. In the case of diabetes, hyperglycemia will induce the generation of free radicals, and free radicals cause cell damage and trigger molecular mechanisms through various pathways, leading to the occurrence and development of diabetic complications. On the other hand, protein tyrosine phosphatase 1B (PTP1B) is a member of the PTP family and plays an important role in catalyzing protein tyrosine dephosphorylation and regulating various cell signaling pathways. Studies have found that PTP1B can negatively regulate the insulin and leptin signaling pathways, and PTP1B gene knockout mice show increased β-cell proliferation levels, increased insulin secretion, enhanced insulin sensitivity, improved blood glucose levels and obesity resistance. A large number of studies have shown that oxidative stress is closely related to insulin secretion, and cell damage caused by oxidative stress will ultimately lead to diabetic complications. Therefore, it is necessary to design and study dual-target inhibitors that can both lower blood sugar and have antioxidant effects.

[0003] Natural products are an important source of drug lead compounds and functional organic molecules. Screening drug lead compounds from natural products has always been an effective way for new drug research and development. Due to their wide range of biological activities and low toxicity and side effects, natural products have always been an important source for drug research and development. Therefore, the active ingredients in traditional Chinese medicine have become a current research hotspot. Among them, dianhydrolycorine is a C19 diterpenoid alkaloid that is almost ubiquitous in Yunnan Province. It is a natural product extracted from plants of the genus Aconitum and has a wide range of biological activities, including anti-inflammatory, analgesic, antipyretic and immunomodulatory properties. However, compared with aconitine (LD 50 = 0.12 - 0.27 mg / kg), mesaconitine (LD 50 = 0.1 - 0.13 mg / kg) and hypaconitine (LD 50 = 0.47 mg / kg), the median lethal dose of dianhydrolycorine injected intravenously into mice at 0.05 mg / kg is higher. In addition, the bioavailability of dianhydrolycorine is only 27.4%, and the high toxicity and low utilization rate of dianhydrolycorine limit its availability.

[0004] By modifying the nitrogen atom, C-8, and C-3 positions of yunaconitine, different substituents were attached at different positions, and the inhibitory effect of the compounds on PTP1B was measured using the enzyme-linked immunosorbent assay - double antibody sandwich method. The antioxidant activity of the drugs was detected using 1,1-diphenyl-2-picrylhydrazyl, providing a good starting point for the development of novel hypoglycemic and antioxidant drugs. Summary of the Invention

[0005] In view of this, the main object of the present invention is to provide a yunaconitine derivative with obvious hypoglycemic and antioxidant activities.

[0006] Another object of the present invention is to provide the use of the above-mentioned yunaconitine derivative in the preparation of hypoglycemic and antioxidant drugs.

[0007] The above objects of the present invention are achieved by the following technical solutions:

[0008] The present invention provides a yunaconitine derivative having a chemical structure shown in Formula 1a or 1b:

[0009]

[0010] In Formulas 1a and 1b, R 1 , R 2 , R 3 are as shown in the following table respectively:

[0011]

[0012]

[0013] The present invention also provides a preparation method of the yunaconitine derivative, and its synthetic route is shown in Formula 2:

[0014]

[0015] In Formula 2, for the yunaconitine derivatives with different numbers, R is selected from the substituents shown as follows:

[0016]

[0017] The present invention also provides the use of the yunaconitine derivative or its pharmaceutically acceptable salt in the preparation of hypoglycemic and antioxidant drugs. Preferably, the drug uses the yunaconitine derivative or its pharmaceutically acceptable solvate as the active ingredient, and the weight percentage content is 5% - 100%.

[0018] Preferably, the drug includes a pharmaceutically acceptable carrier or diluent.

[0019] "Pharmaceutically acceptable carrier" refers to the inactive ingredients in a drug, including but not limited to calcium carbonate, calcium phosphate, various sugars such as lactose, mannitol, etc., starch, cyclodextrin, magnesium stearate, cellulose, magnesium carbonate, acrylic polymer or methacrylic polymer, gel, water, polyethylene glycol, propylene glycol, ethylene glycol, castor oil or hydrogenated castor oil or polyethoxylated hydrogenated castor oil, sesame oil, corn oil, peanut oil.

[0020] "Pharmaceutically acceptable diluent" includes but not limited to starch (such as corn starch, wheat starch, potato starch, etc.), lactose, dextrin, sucrose, pregelatinized starch, microcrystalline cellulose, inorganic salts (such as calcium hydrogen phosphate, calcium sulfate, calcium carbonate, etc.) and mannitol.

[0021] Preferably, the dosage form of the drug is selected from at least one of tablets, capsules, granules, dripping pills, suspensions, syrups, enteric preparations, emulsion suspensions and injections.

[0022] The present invention also provides a drug for lowering blood sugar and antioxidation, the drug comprising a yunaconitine derivative or a pharmaceutically acceptable solvate thereof, the structural formula of the yunaconitine derivative being as described above, having an obvious inhibitory effect on PTP1B and having good antioxidant activity, and being a novel anti-sugar and antioxidant drug.

[0023] The present invention also provides the use of a yunaconitine derivative or a pharmaceutically acceptable salt thereof in the preparation of a preparation with hypoglycemic and antioxidant activities.

[0024] The present invention also provides the use of the yunaconitine derivative or a pharmaceutically acceptable salt thereof in the preparation of a PTP1B inhibitor.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The present invention confirms through in vitro experiments that the yunaconitine derivative has an obvious inhibitory effect on PTP1B and has good antioxidant activity, providing a candidate lead compound for the research and development of hypoglycemic and antioxidant drugs.

[0027] (2) The yunaconitine derivative of the present invention not only has the potential to be developed into a new generation of hypoglycemic and antioxidant drugs, but also has potential application value in the fields of hypoglycemic and antioxidant targeted drugs, probe design and fluorescence biological labeling. Description of the Drawings

[0028] Figure 1 It is the result of detecting the free radical scavenging effect of derivatives 2 and 25 at different action concentrations in Example 5.

[0029] Figure 2 It is the chemical structure of the yunaconitine derivative as shown in formula 1a or 1b.

[0030] Figure 3 It is a synthetic route of aconitine derivatives with chemical structures shown in Formula 1a or 1b. Detailed implementation manners

[0031] In order to more fully understand and demonstrate the technical solutions, objectives, and advantages of the present invention, the technical effects produced by the present invention will be further described in detail and completely below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of them. It should be noted that for those of ordinary skill in the art, other embodiments obtained without departing from the concept of the present invention all fall within the protection scope of the present invention.

[0032] Unless otherwise specified in the following embodiments, the reagents and materials used are all commercially available.

[0033] The following embodiments provide an aconitine derivative with a chemical structure shown in Formula 1a or 1b ( Figure 2 ):

[0034]

[0035] In Formulas 1a and 1b, R 1 , R 2 , R 3 are respectively shown in the following table:

[0036]

[0037]

[0038] In the following embodiments, there is no special requirement for the preparation method of the aconitine derivative, and it can be prepared using methods well-known to those skilled in the art. The preferred synthetic route is shown in Formula 2 ( Figure 3 ):

[0039]

[0040] In Formula 2, for aconitine derivatives with different numbers, R is selected from the substituents shown below:

[0041]

[0042] Example 1

[0043] (1) Preparation of Compound 1: Dissolve aconitine (30 mg, 0.045 mmol) and N-bromosuccinimide (0.135 mmol) in ethanol and react at room temperature for 12 h.

[0044] (2) After the reaction was completed, the pH value was adjusted to pH = 10 with concentrated ammonia water, then poured into water, and extracted with dichloromethane (3 × 30 mL). The organic layer was dried over anhydrous Na 2 SO 4 , filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain Compound 1 (1: yield 73.1%).

[0045] Example 2

[0046] (1) Preparation of Compounds 2 - 3: At room temperature, yunaconitine (30 mg, 0.045 mmol) was dissolved in tetrahydrofuran, and methanol / ethanol was added under acidic (p-toluenesulfonic acid) conditions. The resulting mixture was reacted at room temperature for 48 h.

[0047] (2) The reaction mixture was poured into water and extracted with ethyl acetate (3 × 30 mL). Then, the organic layer was dried over anhydrous Na 2 SO 4 , filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain Compounds 2 - 3 (2: yield 62.5%; 3: yield 43.8%).

[0048] Example 3

[0049] (1) Preparation of Compound 4: Yunaconitine (30 mg, 0.045 mmol) was dissolved in dichloromethane, and Dess-Martin periodinane (22.91 mg, 0.054 mmol) was added, and the reaction was carried out at room temperature for 5 h.

[0050] (2) The reaction mixture was diluted with cold water and extracted with ethyl acetate (3 × 30 mL). Then, the organic layer was dried over anhydrous Na 2 SO 4 , filtered, and the filtrate was evaporated under reduced pressure. Finally, it was purified by silica gel column chromatography to obtain Compound 4 (4: yield 67.1%).

[0051] Example 4

[0052] (1) Preparation of Compound 5-25: Dissolve yunaconitine (30 mg, 0.045 mmol) in dichloromethane, and add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (77.62 mg, 0.305 mmol), 4-dimethylaminopyridine, and the corresponding acid or acid anhydride (butyric anhydride: 8.64 mg, 0.0546 mmol; valeric anhydride: 10.16 mg, 0.0546 mmol; caproic anhydride: 11.7 mg, 0.0546 mmol; methoxyacetic acid: 16.5 mg, 0.183 mmol; ethoxyacetic acid: 19.1 mg, 0.183 mmol; furoic acid: 20.5 mg, 0.183 mmol; 5-methyl-2-furoic acid: 23.07 mg, 0.183 mmol; 3-bromofuran-2-carboxylic acid: 34.9 mg, 0.183 mmol; 5-nitro-2-furoic acid: 28.7 mg, 0.183 mmol; thiophene-2-carboxylic acid: 23.5 mg, 0.183 mmol; 5-methyl-2-thiophenecarboxylic acid: 26.0 mg, 0.183 mmol; 2-chlorothiophene-5-carboxylic acid: 29.8 mg, 0.183 mmol; 2-pyridinecarboxylic acid: 22.5 mg, 0.183 mmol; benzoic acid: 22.3 mg, 0.183 mmol; 4-(trifluoromethyl)benzoic acid: 34.8 mg, 0.183 mmol; 4-chlorobenzoic acid: 28.7 mg, 0.183 mmol; 2-iodoxybenzoic acid: 51.2 mg, 0.183 mmol; trans-cinnamic acid: 27.1 mg, 0.183 mmol; succinic anhydride: 18.3 mg, 0.183 mmol; glutaric anhydride: 20.9 mg, 0.183 mmol; adipic anhydride: 23.4 mg, 0.183 mmol), and react at room temperature for 10 h.

[0053] (2) After the reaction is completed, pour the reaction mixture into water and extract with ethyl acetate (3×30 mL). Then, dry the organic layer over anhydrous Na 2 SO 4 4, filter, and evaporate the filtrate under reduced pressure. Finally, purify by silica gel column chromatography to obtain Compound 5-25 (5: yield 54.2%; 6: yield 60.3%; 7: yield 49.1%; 8: yield 72.5%; 9: yield 69.1%; 10: yield 82.3%; 11: yield 79.3%; 12: yield 67.8%; 13: yield 42.5%; 14: yield 74.3%; 15: yield 78.3%; 16: yield 57.9%; 17: yield 74.3%; 18: yield 64.3%; 19: yield 83.2%; 20: yield 53.2%; 21: yield 82.1%; 22: yield 53.2%; 23: yield 57.3%; 24: yield 45.2%; 25: yield 32.6%).

[0054] Example 5

[0055] Inhibitory effect of yunaconitine derivatives on PTP1B:

[0056] In this example, the ELISA method was used to test the inhibitory activity of yunaconitine derivatives on PTP1B. Oleanolic acid was used as a positive drug for PTP1B inhibitory activity. The concentration of DMSO (10%) in the test wells had no significant effect on PTP1B inhibitory activity. The specific steps are as follows:

[0057] 1. Prepare yunaconitine derivative samples: Dissolve yunaconitine derivatives in 100 μL of DMSO, and then add 900 μL of distilled water to prepare a sample stock solution with a concentration of 1000 μM.

[0058] 2. Sample dilution: Dilute the sample stock solution with a sample diluent to a concentration of 10 μM and store it at 4 °C for later use.

[0059] 3. Prepare the reaction solution: Divide it into a blank group, a control group, and an experimental group. The specific grouping is as follows:

[0060] (1) Experimental group: 25 μL of the sample solution to be tested, 25 μL of PTP1B solution;

[0061] (2) Control group: 25 μL of buffer solution, 25 μL of PTP1B solution;

[0062] (3) Blank group: 50 μL of buffer solution.

[0063] Incubate 30 μL of the substrate solution at 37 °C for 10 minutes to prepare the reaction solution.

[0064] 4. Prepare the enzyme-linked immunosorbent assay (ELISA) plate: Carefully add 25 μL of the reaction solution to a 96-well ELISA plate containing the PTP1B solid-phase antibody and incubate it at a constant temperature of 37 °C for 30 minutes.

[0065] 5. Empty the ELISA plate: After incubation, empty the liquid in the ELISA plate and gently pat it dry.

[0066] 6. Washing: Wash the ELISA plate with the diluted washing solution, repeat the washing 5 times to ensure thorough washing, and pat the ELISA plate dry.

[0067] 7. Enzyme reaction: Add 25 μL of the HRP enzyme-labeled reagent to each well to initiate the enzyme reaction and incubate it at a constant temperature of 37 °C for 30 minutes.

[0068] 8. Empty the ELISA plate again: After incubation, empty the incubation liquid in the well plate and wash it with the diluted washing solution, repeat the washing 5 times.

[0069] 9. Color reaction: Add 25 μL of chromogenic reagent A and 25 μL of chromogenic reagent B to each well to complete the color reaction.

[0070] 10. Termination of reaction: Terminate the reaction by adding 25 μL of termination solution to each well.

[0071] 11. Absorbance measurement: Use a microplate reader to record the absorbance value of each well within 15 minutes. The measurement wavelength is 450 nm. Read the absorbance value (OD), and perform statistical analysis on the data using GraphPad Prism 6, and calculate the inhibition rate and IC 50 .

[0072] The results are shown in Table 1 and Table 2, indicating that yunaconitine derivatives have an obvious inhibitory effect on PTP1B. Among them, Compound 2 and Compound 25 have a relatively strong inhibitory effect on PTP1B.

[0073] Table 1

[0074]

[0075]

[0076]

[0077] Table 2

[0078] Compound <![CDATA[IC 50 a (μM)]]> 2 7.724±0.072 25 8.981±0.039 Oleanolic acid 6.682±0.008

[0079] Example 5

[0080] Antioxidant activity determination

[0081] 1. Prepare a reaction mixture by mixing 100 μL of 0.05 μM ethanol DPPH solution with 100 μL of compounds dissolved in ethanol at different concentrations (0 - 200 μM).

[0082] 2. After incubating the mixture in the dark at room temperature for 30 minutes, measure the absorbance of the mixture at 517 nm. Calculate the EC50 value, which represents the effective concentration of the sample that inhibits 50% of the DPPH free radicals, and is expressed in μM.

[0083] The results are as Figure 1 shown. Compared with the raw material Yunaconitine, Compound 25 has the best antioxidant effect. The free radical scavenging rate can reach 90% at 50 μM, and the free radical scavenging rate increases with the increase of the concentration of Compound 25, indicating that Compound 25 may be a potential antioxidant.

[0084] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A dianconitine derivative having a chemical structure as shown in Formula 1a or 1b: In formula 1a and 1b, R1, R2, and R3 are respectively as shown in the following table.

2. Use of the dianconitine derivative or its pharmaceutically acceptable salt according to claim 1 in the preparation of hypoglycemic and antioxidant drugs.

3. The use according to claim 2, characterized in that: The drug uses the dianconitine derivative or a pharmaceutically acceptable solvate thereof as claimed in claim 1 as an active ingredient, with a weight percentage of 5% to 100%.

4. The use according to claim 2, characterized in that: The medicament includes a pharmaceutically acceptable carrier or diluent.

5. The use according to claim 2, characterized in that: The dosage form of the drug is selected from at least one of tablets, capsules, granules, pellets, suspensions, syrups, enteric-coated preparations, emulsion suspensions and injections.

6. A hypoglycemic and anti-oxidant drug, characterized in that: The drug comprises the dianconitine derivative according to claim 1 or a pharmaceutically acceptable solvate thereof.

7. Use of the dianconitine derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a hypoglycemic and antioxidant preparation.

8. Use of the dianconitine derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a PTP1B inhibitor.

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