AMPK agonists and uses thereof

Abstract

The invention discloses an AMPK agonist and application thereof, and belongs to the technical field of medicinal chemistry, the AMPK agonist simultaneously has anthrone and benzimidazole structures and can be synthesized by taking anthrone compounds, NaH, epichlorohydrin and benzimidazole as raw materials, the synthesis steps are simple and efficient, the AMPK agonist is combined with a beta-regulatory subunit of AMPK to directly activate an AMPK signal channel, and the AMPK agonist can be used for preparing a medicine for treating AMPK. The compound can be used for regulating cell metabolism balance and preventing or treating AMPK-mediated diseases by activating AMPK, particularly can be used for inducing tumor cell apoptosis and inhibiting tumor growth, and has a wide application prospect in the field of tumor treatment.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to an AMPK agonist and its uses. Background Technology

[0002] AMPK (AMP-Activated Protein Kinase) is a key kinase regulating energy metabolism and inflammatory responses in the body. As an energy sensor, it participates in various cell signaling pathways and regulates the metabolism of glucose, fatty acids, and proteins. AMPK proteins exist as a heterotrimeric complex, including an α-catalytic subunit, a β-regulatory subunit, and a γ-regulatory subunit. AMPK is also an important target for the treatment of various diseases. The mechanism of action of AMPK-targeted drugs is to regulate biological processes such as energy metabolism and cell growth by targeting AMPK. AMPK-targeted drugs are mainly divided into two categories: AMPK agonists and AMPK inhibitors.

[0003] Common AMPK agonists include acalide-1 (AICAR) and metformin. AICAR is currently the most widely used direct AMPK activator; however, AICAR has difficulty crossing the cell membrane and requires transport protein mediation, resulting in poor permeability, low selectivity, and low bioavailability. Although metformin is a classic AMPK agonist, its direct molecular target is presenilin enhancer 2 (PEN2), which is an intermediate protein in AMPK activation. Therefore, metformin primarily activates AMPK indirectly, lacking a direct targeting mechanism.

[0004] In the prior art, researchers have also developed various AMPK agonists for the prevention or treatment of AMPK-mediated diseases. For example, Chinese patent document CN110372638A discloses a piperazine AMPK agonist, which exhibits significant agonistic activity against AMPK at both in vitro biochemical and cellular levels, potentially activating the AMPK signaling pathway by activating adiponectin receptors. Chinese patent document CN109512803A discloses 10-gingerol, a natural AMPK agonist that can spatially bind to AMPK (forming five hydrogen bonds with amino acid residues ARG-83, ASP-88, ASN-48, ILE-46, and LYS-29 of adenosine monophosphate-activated protein kinase), enhancing AMPK activity. Chinese patent document CN113121379A discloses that benzyloxyarylamide and piperidine urea compounds with specific structures have potent AMPK agonist activity, and that they do not cause myocardial hypertrophy when used as AMPK agonists.

[0005] AMPK activation is a viable approach to cancer treatment, and the development of small-molecule AMPK agonists with high activity and low toxicity is of great clinical significance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a novel AMPK agonist. This AMPK agonist directly activates the AMPK signaling pathway by binding to the β-regulatory subunit of AMPK, thereby regulating cellular metabolic balance. It can also induce tumor cell apoptosis and inhibit tumor growth by activating AMPK, and has broad application prospects in the field of AMPK-mediated disease prevention / treatment.

[0007] The specific technical solution adopted is as follows:

[0008] The use of the compound shown in formula (I), or its hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester or prodrug, in the preparation of a medicament, wherein the medicament is used as an AMPK agonist to activate the AMPK signaling pathway;

[0009]

[0010] The present invention also provides the use of the compound of formula (I), or its hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester or prodrug, in the preparation of a medicament for the prevention and / or treatment of AMPK-mediated diseases.

[0011] Through extensive screening and identification, the inventors discovered that small molecule compounds with the aforementioned specific structures can directly activate the AMPK signaling pathway and regulate cellular metabolic homeostasis by binding to the β-regulatory subunit of AMPK, exhibiting significant agonistic activity towards AMPK. These compounds were renamed AK2817 (the compound shown in formula (I)). In tumor cells, the AMPK signaling pathway can inhibit tumor progression through pathways such as generating reactive oxygen species (ROS), inhibiting the mTOR (mammalian target of rapamycin) signaling pathway, and regulating autophagy.

[0012] AMPK-mediated diseases include various tumors such as colon cancer, liver cancer, lung cancer, and melanoma, as well as diabetes.

[0013] Experimental results have shown that the AMPK agonist AK2817 exhibits remarkable anti-tumor effects, demonstrating a certain inhibitory effect on various cancers, including colon cancer, liver cancer, and lung cancer.

[0014] Furthermore, the preparation method of the compound shown in formula (I) includes the following steps:

[0015] (1) In an organic solvent system, anthrone compounds react with NaH, and then epichlorohydrin is added to continue the reaction. After the reaction is completed, an intermediate is obtained through post-processing.

[0016] (2) Under alkaline conditions, the intermediate obtained in step (1) is reacted with benzimidazole. After the reaction is completed, the compound shown in formula (I) is obtained through post-treatment.

[0017] The structural formulas of anthrone compounds are shown below:

[0018]

[0019] Preferably, in step (1), the anthrone compound reacts with NaH at a temperature of -20 to 4°C for 5 to 15 minutes. After the resulting reaction solution is heated to 60 to 80°C, epichlorohydrin is added and the reaction continues for 4 to 6 hours.

[0020] Preferably, in step (2), the reaction temperature between the intermediate and benzimidazole is 70-80°C and the reaction time is 5-8h.

[0021] The present invention also provides a pharmaceutical composition comprising a compound of formula (I), or a hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or prodrug thereof.

[0022] Furthermore, the pharmaceutical composition may also include pharmaceutically acceptable excipients or carriers.

[0023] Furthermore, the pharmaceutical composition is formulated into a pharmaceutically acceptable dosage form, including but not limited to tablets, granules, capsules, powders, or injections.

[0024] The present invention also provides the use of the pharmaceutical composition in the preparation of drugs that regulate the AMPK signaling pathway, AMPK-mediated disease prevention products, or AMPK-mediated disease treatment products.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) This invention screened and identified an AMPK direct agonist (AMPK agonist AK2817), whose AMPK agonistic effect is significantly superior to existing agonists. AK2817 demonstrated highly efficient AMPK agonistic ability in various in vitro models, with effects significantly superior to commonly used AMPK agonists A769662 and Phenformin. Experiments showed that AK2817 can significantly increase p-AMPK (Thr172) levels at concentrations as low as 20 μM, far exceeding A769662 and Phenformin, exhibiting stronger agonistic activity and higher efficacy. Furthermore, AK2817 showed activation of downstream p-ACC of AMPK at a dosage of 5 μM, and the activation effect was superior to that of A769662 at 20 μM. This indicates that AK2817 has a better dose-response relationship, reduces the risk of potential toxic side effects, and has better prospects for drug development.

[0027] (2) The AMPK agonist AK2817 provided by the present invention is suitable for the prevention and treatment of multiple types of tumors. It is a pan-cancer AMPK agonist that has shown significant AMPK agonist activity and excellent anti-tumor effect in both in vivo and in vitro experiments. Its combined use with immunotherapy, targeted therapy and other methods can help to further improve the effect of cancer treatment. Attached Figure Description

[0028] Figure 1 The results show the molecular docking of the AMPK agonist AK2817 with the AMPK complex under different models (Model 1 and Model 2).

[0029] Figure 2 Western blot was used to detect the activation of the AMPK pathway in MEF cells 24 hours after treatment with different concentrations of AK2817 (left panel) and different AMPK agonists (right panel).

[0030] Figure 3 Western blot analysis was performed to detect AMPK activation in HCT116 cells 24 hours after treatment with different concentrations of AK2817 (left panel) and different AMPK agonists (right panel).

[0031] Figure 4 Apoptosis of HCT116 cells was detected by flow cytometry.

[0032] Figure 5 Western blot was used to detect AMPK pathway activation in HepG2 cells after treatment with 5 μM, 10 μM, and 20 μM AMPK agonists AK2817, MK8722, and A769662 for 1 hour.

[0033] Figure 6 In the diagram, A represents the activation of the AMPK pathway in A549 cells after treatment with different concentrations of AK2817 for 48 hours using Western blot; B represents the cell cycle changes in A549 cells after AK2817 treatment using flow cytometry PI staining; and C represents the effect of AK2817 on A549 cell apoptosis using flow cytometry Annexin V / PI staining.

[0034] Figure 7 The left image shows the subcutaneous tumor growth curves of A549 in the control group and different experimental groups, and the right image shows the tumor weight statistics on day 21.

[0035] In the figure, the statistical analysis results ns indicate no statistically significant difference, * indicates p value < 0.05, *** indicates p value < 0.001, and **** indicates p value < 0.0001. Detailed Implementation

[0036] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0037] The operating methods described in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art.

[0038] Specifically, the AMPK agonist AK2817 was synthesized using the following method:

[0039]

[0040] The anthrone compound shown in the above formula (3 g, 13.4 mmol, 1.0 equivalent) was dissolved in dry N-methylpyrrolidone (NMP) (13.5 mL), and NaH (0.54 g, 13.4 mmol, 1.0 equivalent, 60% by mass) was added. The mixture was stirred at 0 °C for 10 minutes, and then the reaction solution was heated to 70 °C. Epichlorohydrin (1.23 g, 13.4 mmol, 1.0 equivalent) was then added, and the reaction was continued at 70 °C for 5 hours with stirring. The mixture was then quenched with ice water and filtered. The filter cake was dried and purified by silica gel column chromatography with dichloromethane as the eluent to give an intermediate (1 g, yield 26%) as a yellow solid.

[0041]

[0042] The intermediate obtained in the above steps (0.5 g, 1.78 mmol) was dissolved in anhydrous ethanol (120 mL), and benzimidazole (0.232 g, 1.96 mmol, 1.1 equivalents) and potassium carbonate (0.346 g, 2.49 mmol, 1.4 equivalents) were added. The mixture was stirred at 80 °C for 6 hours, cooled to room temperature, diluted with ethyl acetate (300 mL), and washed with water (500 mL). The organic phase was then washed with saturated sodium chloride solution (100 mL) and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated under reduced pressure, and the residue was purified by C18 column chromatography with 0.1% trifluoroacetic acid (TFA) solution as eluent to give AMPK agonist AK2817 (0.16 g, yield 22.8%) as a yellow solid.

[0043] The 1H NMR spectrum of the AMPK agonist AK2817 is as follows: 1 H NMR (400MHz, DMSO-d6) δ9.17(s,1H),8.23–8.20(m,3H),7.97–7.91(m,3H),7.81(d,J=7.2Hz,1H),7.67(d ,J=2.8Hz,1H),7.50–7.48(m,3H),5.75(s,1H),4.73–4.70(m,1H),4.57–4.51(m,1H),4.34–4.28(m,3H).

[0044] Example 1

[0045] The three-dimensional structure of the AMPK complex was obtained from the PDB protein database. The structure of the AMPK agonist AK2817 ligand was plotted using ChemDraw. Molecular docking of the ligand with the AMPK complex was performed using AutoDock Vina software. The docking results were visualized and analyzed based on parameters such as docking score. The results are as follows: Figure 1 As shown, this indicates that the AMPK agonist AK2817 binds to the β-regulatory subunit of AMPK, which is close to the binding site of the existing AMPK agonist MK-8722.

[0046] Example 2

[0047] MEF cells (mouse embryonic fibroblasts) were treated with different concentrations of the AMPK agonist AK2817 (0, 5, 10, and 20 μM) for 24 hours. Simultaneously, a control group (DMSO) and four AMPK agonist treatment groups (AK2817, MK8722, A769662, and Phenformin, each at 20 μM) were also established, with treatment lasting 24 hours. After treatment, total protein was extracted from the cells, subjected to SDS-PAGE electrophoresis, and transferred to a membrane. Western blot was used to detect the expression levels of p-ACC, ACC, p-AMPK, AMPK, and β-actin to assess the activation of the AMPK pathway.

[0048] The results are as follows Figure 2 As shown, the above results indicate that the AMPK agonist AK2817 can activate AMPK in mouse fibroblasts (MEF) and has the strongest effect among commonly used AMPK agonists, similar to the AMPK agonist MK-8722.

[0049] Example 3

[0050] HCT116 colorectal cancer cells were treated with the AMPK agonist AK2817 (0, 20, and 30 μM) for 24 hours. Simultaneously, a control group (DMSO) and four AMPK agonist treatment groups (AK2817, MK8722, A769662, and Phenformin, 30 μM each) were established, with treatment time of 24 hours for each group. After treatment, total cellular protein was extracted and analyzed by SDS-PAGE electrophoresis and Western blot to detect the expression of p-AMPK, AMPK, and β-actin, and to evaluate the activation effect of the AMPK pathway.

[0051] The results are as follows Figure 3 As shown above, the results indicate that the AMPK agonist AK2817 can activate AMPK in HCT116 colorectal cancer cells, and is stronger than phenformin at the same concentration (30 μM), and similar to MK-8722.

[0052] Example 4

[0053] Flow cytometry analysis was performed using Annexin V-FITC / PI double staining. HCT116 colorectal cancer cells were seeded in 6-well plates and treated with 20 μM AMPK agonist AK2817 for 24 hours after cell adhesion. After treatment, adherent and suspension cells were collected, stained with the Annexin V-FITC / PI apoptosis detection kit, and then analyzed by flow cytometry.

[0054] The results are as follows Figure 4As shown above, the results indicate that 20 μM of the AMPK agonist AK2817 can induce apoptosis in HCT116 colorectal cancer cells.

[0055] Example 5

[0056] HepG2 liver cancer cells were treated with three different AMPK agonists, AK2817, MK8722, and A769662 (0, 5, 10, and 20 μM), for 1 hour. After treatment, total protein was extracted from the cells and analyzed by SDS-PAGE electrophoresis and Western blot to detect the expression of p-AMPK, AMPK, and β-actin.

[0057] The results are as follows Figure 5 As shown above, the results indicate that the AMPK agonist AK2817 can activate AMPK and its downstream signal ACC in HepG2 liver cancer cells, and its activation intensity is stronger than that of the AMPK agonist A769662 and similar to that of the AMPK agonist MK8722.

[0058] Example 6

[0059] A549 lung cancer cells were seeded in 12-well plates and treated with different concentrations of AK2817 for 48 hours after adhesion. Total protein was extracted after treatment, and the expression levels of p-AMPK and AMPK were detected by Western blot to assess AMPK pathway activation. Simultaneously, cells were collected, and apoptosis was analyzed by flow cytometry using Annexin V-FITC / PI double staining. Cell cycle distribution was detected using PI single staining to evaluate the effect of AK2817 on cell proliferation and cell cycle progression.

[0060] The results are as follows Figure 6 As shown in AC, the above results indicate that the AMPK agonist AK2817 can significantly activate AMPK in lung cancer cells A549, induce apoptosis, and inhibit the cell cycle.

[0061] Example 7

[0062] Each experimental mouse was subcutaneously injected with 1×10⁻⁶ mmol / L on its right back. 6 A tumor model was constructed using A549 lung cancer cells, and the tumor volume was increased to approximately 50 mm. 3Mice were randomly divided into four groups and injected intraperitoneally with 100 μL of control solvent (PEG300:Tween20:physiological saline = 4.5:0.5:5), 10 mg / kg AK2817, 30 mg / kg AK2817, and 60 mg / kg AK2817, respectively. Treatment doses were administered once daily for 5 days, followed by a 2-day break, for a total of 21 days. Tumor size was recorded after 21 days, and tumors were removed and weighed after 20 days.

[0063] The results are as follows Figure 7 As shown above, the results indicate that the AMPK agonist AK2817 can still exert an anti-tumor effect in mice and can significantly inhibit the progression of the nude mouse A549 subcutaneous tumor model.

[0064] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The use of a compound of formula (I), or its hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or prodrug, in the preparation of a medicament, characterized in that, The drug is used as an AMPK agonist to activate the AMPK signaling pathway; 2. The use of the compound of formula (I), or its hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or prodrug, in the preparation of a medicament, characterized in that, The aforementioned drug is used for the prevention and / or treatment of AMPK-mediated diseases; 3. The use according to claim 2, characterized in that, AMPK-mediated diseases include tumors or diabetes, including colorectal cancer, liver cancer, lung cancer, or melanoma.

4. The use according to claim 1 or 2, characterized in that, The preparation method of the compound shown in formula (I) includes the following steps: (1) In an organic solvent system, anthrone compounds react with NaH, and then epichlorohydrin is added to continue the reaction. After the reaction is completed, an intermediate is obtained through post-processing. (2) Under alkaline conditions, the intermediate obtained in step (1) is reacted with benzimidazole. After the reaction is completed, the compound shown in formula (I) is obtained through post-treatment. The structural formulas of anthrone compounds are shown below:

5. The use according to claim 4, characterized in that, In step (1), the anthrone compounds react with NaH at a temperature of -20 to 4°C for 5 to 15 minutes. The resulting reaction solution is heated to 60 to 80°C and epichlorohydrin is added to continue the reaction for 4 to 6 hours.

6. The use according to claim 4, characterized in that, In step (2), the intermediate and benzimidazole react at a temperature of 70-80°C for 5-8 hours.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a compound of formula (I), or a hydrate, solvate, pharmaceutically acceptable salt, pharmaceutically acceptable ester, or prodrug thereof; 8. The pharmaceutical composition according to claim 7, characterized in that, It also includes pharmaceutically acceptable excipients or carriers; and formulates the pharmaceutical composition into a pharmaceutically acceptable dosage form, including tablets, granules, capsules, powders, or injections.

9. Use of the pharmaceutical composition according to claim 7 in the preparation of a medicament that regulates the AMPK signaling pathway, an AMPK-mediated disease prevention product, or an AMPK-mediated disease treatment product.

10. The use according to claim 9, characterized in that, AMPK-mediated diseases include tumors or diabetes, including colorectal cancer, liver cancer, lung cancer, or melanoma.

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