A 1-methyl-6-chromenone indole-2-carboxamide compound, its preparation method and application

Abstract

The present invention discloses a 1-methyl-6-chromenone-indole-2-carboxamide compound, its preparation method, and application. The biologically active 1-methyl-6-chromenone-indole-2-carboxamide nucleus contained in the compound can be further chemically modified to produce a variety of compounds with higher biological activity, expanding the wide application of such compounds in biomedicine and the development prospects of pharmaceutical preparations. At low nanomolar doses, these compounds can significantly inhibit the proliferation of cells such as breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma, and can effectively inhibit STAT3 protein phosphorylation, indicating that these compounds have the potential to be developed as anti-tumor drugs. Furthermore, these compounds can also activate AMPK phosphorylation, indicating that these compounds also have great potential for treating metabolic diseases such as obesity, non-alcoholic fatty liver disease, and diabetes.

Description

Technical Field

[0001] The present invention belongs to the field of clinical treatment of indications such as tumor targeted therapy and metabolic disease-related diabetes and obesity, and specifically relates to a 1-methyl-6-chromenone indole-2-carboxamide compound, a preparation method and application thereof. Background Art

[0002] Cancer has become a major cause of premature death and shortened lifespan worldwide. In 2020, approximately 19.3 million cases of cancer were diagnosed worldwide, resulting in nearly 10 million deaths from cancer. Experts predict that by 2035, the number of cancer patients worldwide will increase by half. Breast cancer has overtaken lung cancer as the world's leading cancer, with 2.26 million new cases globally. In 2020, approximately 905,700 people were diagnosed with liver cancer worldwide, resulting in approximately 830,200 deaths. It is projected that by 2040, the number of diagnoses and deaths from primary liver cancer could increase by over 55%. Furthermore, gastric and pancreatic cancers also have high incidence rates and remain poor prognoses. While significant advances have been made in clinical cancer treatment, the heterogeneity and continuous evolution of tumors result in low response rates to most drugs and the development of drug resistance, resulting in a significant shortage of effective treatments. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin, and capecitabine offer advantages such as broad anticancer spectrum and good efficacy, but they also present significant side effects and are prone to early drug resistance. Therefore, actively exploring and developing new anticancer drugs is of great clinical significance.

[0003] AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and plays an important role in diabetes, cancer, and vascular diseases. Targeting AMPK has been widely used in the treatment of various metabolic disorders, including diabetes.

[0004] This application synthesizes a class of 1-methyl-6-chromenoindole-2-carboxamide compounds with a novel structural formula. Through biological analysis, it was found that these compounds, at extremely low doses, can significantly inhibit the proliferation of cells such as breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma. They can also effectively inhibit STAT3 protein phosphorylation and activate AMPK protein phosphorylation. Therefore, further development of these compounds will be of great significance in the treatment of tumors and metabolic diseases. Summary of the Invention

[0005] The purpose of the present invention is to provide a 1-methyl-6-chromenone indole-2-carboxamide compound, a preparation method and application thereof.

[0006] A 1-methyl-6-chromenoindole-2-carboxamide compound, the structural formula of which is shown in general formula I:

[0007]

[0008] Wherein, R1 and R2 are selected from H, -OH, -NH2, -NO2, -OCH3, F, Cl, Br,

[0009] The above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compound is specifically a compound with the following structure:

[0010]

[0011]

[0012]

[0013] A biologically acceptable salt formed by the above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compound and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

[0014] The preparation method of the above-mentioned 1-methyl-6-chromenone indole-2-carboxamide compounds, the synthetic route is as follows:

[0015] The specific synthesis steps are as follows:

[0016] (1) Compound 1, Compound 2, HBTU, and DIEA were dissolved in DMF and stirred at 20-30°C. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried, slurried with ethyl acetate, and filtered to obtain Compound 3;

[0017] (2) Compound 3, compound 4, Pd(dppf)Cl2 and potassium acetate were dissolved in DMF and stirred at 90-120°C. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation and subjected to column chromatography to obtain compound 5.

[0018] (3) Compound 5, compound 6, Pd(dppf)Cl2 and sodium carbonate were dissolved in DMSO and stirred at 90-120°C until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation and purified by column chromatography to obtain the compound of formula I.

[0019] Furthermore, in step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:

[0020] (1-1.5):3; in step (2), the molar ratio of compound 3, compound 4, potassium acetate and Pd(dppf)Cl2 is 1:2:3:0.05; in step (3), the molar ratio of compound 5, compound 6, sodium carbonate and Pd(dppf)Cl2 is 1:(1-1.5):

[0021] (1~1.5):0.05.

[0022] Use of the above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compounds or biologically acceptable salts thereof in the preparation of STAT3 protein phosphorylation inhibitors.

[0023] The use of the above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compounds or their biologically acceptable salts in the preparation of anti-tumor drugs, wherein the anti-tumor drugs refer to drugs for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma, etc.

[0024] Use of the above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compounds or biologically acceptable salts thereof in the preparation of drugs for activating AMPK.

[0025] The use of the above-mentioned 1-methyl-6-chromenoindole-2-carboxamide compounds or biologically acceptable salts thereof in the preparation of metabolic drugs. The drugs are drugs for treating metabolic diseases such as obesity, non-alcoholic fatty liver disease, and diabetes.

[0026] Specifically, the present invention synthesizes a class of 1-methyl-6-chromenone indole-2-carboxamide compounds RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD24V1013, RD24V1014, RD24V1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019, RD24V1020, RD24V1021, RD24 V1022, RD24V1023, RD24V1024, RD24V2001, RD24V2002, RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD 24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2 017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, RD24V2023, etc. The CCK-8 method was used to detect the proliferation inhibitory effect of this type of compound on various cancer cells; it can effectively inhibit STAT3 protein phosphorylation and activate AMPK phosphorylation.

[0027] The results showed that the compounds of the present invention RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD24V1013, RD24V1014, RD24V1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019, RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V1025, RD24V1026 2002, RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, and RD24V2023 can effectively inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, and gastric cancer cells, and can effectively inhibit the phosphorylation of STAT3 protein and activate AMPK phosphorylation.

[0028] In summary, the present invention provides a new 1-methyl-6-chromenoindole-2-carboxamide compound and its derivatives for the treatment of tumors and metabolic diseases and their potential molecular mechanisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The result is that RD24V1001 can effectively inhibit STAT3 protein phosphorylation;

[0030] Figure 2 This is the result of RD24V1001 activating AMPK protein phosphorylation. DETAILED DESCRIPTION

[0031] In order to make the technical purpose, technical solution and beneficial effects of the present invention more clear, the technical solution of the present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0032] In the method for synthesizing the compound of formula I of the present invention, the various raw materials used in the reaction can be prepared by those skilled in the art based on prior knowledge, or can be prepared by methods known in the literature, or can be purchased commercially. The intermediates, raw materials, reagents, reaction conditions, etc. used in the above reaction schemes can be appropriately modified based on the prior knowledge of those skilled in the art.

[0033] In the present invention, unless otherwise specified: (i) temperatures are expressed in degrees Celsius (°C), and operations are carried out at room temperature; more specifically, room temperature refers to 20-30°C; (ii) organic solvents are dried using conventional drying methods, and the solvent is evaporated using a rotary evaporator under reduced pressure with a bath temperature not higher than 50°C; the developing solvent and the eluent are both in volume ratios; (iii) the reaction process is tracked by thin layer chromatography (TLC); and (iv) the final product has satisfactory proton nuclear magnetic resonance (1H-NMR).

[0034] Example 1: The synthesis of all compounds is as follows

[0035] The specific synthesis method takes compound RD24V1001 as an example, and the structural formula is as follows:

[0036]

[0037] Its synthetic route is as follows:

[0038]

[0039] Step 1. (4-hydroxynaphthalen-1-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 3)

[0040] Compound 1 (2.0 g, 10.63 mmol, 1.0 eq), compound 2 (2.92 g, 10.63 mmol, 1.0 eq), HBTU (4.85 g, 12.57 mmol, 1.2 eq), and DIEA (4.15 g, 31.38 mmol, 3.0 eq) were dissolved in 30 mL of DMF and stirred at 25°C for 3 hours. Completion of the reaction was monitored by TLC. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL x 3). The organic phase was dried, slurried with 15 mL of ethyl acetate, and filtered to obtain 4.20 g of compound 3 as a white solid, with a yield of 88.9%.

[0041] 1H NMR(CDCl3,300MHz)δ:8.27(d,J=8Hz,1H),7.80(d,J=8Hz,1H),7.55(m,2H),7.24(d,J=8Hz,1H ),7.20(d,J=8Hz,2H),6.88(d,J=8Hz,2H),6.79(d,J=8Hz,1H),4.34(m,3H),4.20-4.10(m,2H)

[0042] Step 2. (1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0043] Compound 3 (5 g, 9.80 mmol, 1.0 eq), compound 4 (4.98 g, 19.59 mmol, 2.0 eq), potassium acetate (2.88 g, 29.39 mmol, 3.0 eq), and Pd(dppf)Cl2 (350 mg, 0.05 eq) were dissolved in 50 mL of DMF and reacted at 100°C for 12 hours. TLC monitoring showed that the reaction of the raw materials was complete and new spots were generated. The reaction solution was diluted with 500 mL of ethyl acetate and washed three times with saturated brine (500 mL*3). The organic phase was dried and spin-dried, then passed through a column (DCM / MeOH = 1 / 0 to 30 / 1). The eluent containing compound 5 was collected and spin-dried to obtain 5 g of white solid compound 5, with a yield of 91.56%.

[0044] 1 H NMR(CDCl3,300MHz)δ:8.25(d,J=8Hz,1H),7.78(d,J=8Hz,1H),7.53(m,2H),7.22(d,J=8Hz,1H),7.18(d,J=8Hz,2H), 6.86(d,J=8Hz,1H),4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),2.85(s,3H),3.04-2.96(m,5H),1.2(m,12H).

[0045] Step 3.7-(1-methyl-2-(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazine-1-carbonyl)-1H-indol-6-yl)-4H-chromen-4-one(RD24V1001)

[0046] Compound 5 (1.20 g, 2.15 mmol, 1.0 eq), compound 6-1 (532.91 mg, 2.37 mmol, 1.1 eq), sodium carbonate (342.25 mg, 1.5 eq), and Pd(dppf)Cl2 (75 mg, 0.05 eq) were dissolved in 20 mL of DMSO and reacted at 100°C for 10 hours. TLC monitoring showed complete reaction of the starting materials with the formation of new spots. The reaction solution was spin-dried, mixed with silica gel, and passed through a column (eluted with DCM / MeOH = 1 / 0 to 30 / 1). The eluent containing the product was collected and spin-dried to yield RD24V1001, 120 mg, as a light yellow solid, 58.1%.

[0047] 1 H NMR(CDCl3,300MHz)δ:8.25(d,J=8Hz,1H),7.87(d,J=8Hz,1H),7.78(d,J=8Hz,1H),7.53(m,2H),7.33((d,J=8Hz,2H),7.22(d,J=8Hz,2H),7. 18(d,J=8Hz,2H),6.86(d,J=8Hz,1H),6.35(d,J=8Hz,1H)4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),2.85(s,3H),3.04-2.96(m,5H).

[0048] Part of the intermediate 6 in this application was purchased from commercial channels, and part was obtained through the following synthetic route:

[0049]

[0050] The specific synthesis process is as follows:

[0051] a. Compound 7, compound 8 and LDA were dissolved in tetrahydrofuran and stirred at -78 ° C. The reaction was completed, and the reaction solution was quenched with ammonium chloride solution, washed with saturated brine, dried, and subjected to column chromatography to obtain compound 9. The molar ratio of compound 7, compound 8 and LDA was 1:1:2.

[0052] b. Compound 9 and Tf2O were dissolved in dichloromethane and stirred at 0°C until the reaction was complete. The reaction solution was rotary dried and column chromatography was performed to give compound 6a. The molar ratio of compound 9 and Tf2O was 1:1.5.

[0053] c. Compound 11, oxalyl chloride and pyridine were dissolved in DMF and stirred at room temperature until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried and purified by column chromatography to give compound 12. The molar ratio of compound 11, oxalyl chloride and pyridine was 1:2:0.5.

[0054] d. Compound 12 was dissolved in DMF and stirred at 100°C until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried and purified by column chromatography to obtain compound 6b.

[0055] Example 2, RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD24V1013, RD24V1014, RD24V1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019, RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V2001, RD24V2002 2. RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, and RD24V2023 have an inhibitory effect on the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma cells.

[0056] MDA-MB-468, HepG2, BxPC-3, SGC7901, H460, KYSE450, Hela, MM.1S, and OCI-LY3 cells in the logarithmic growth phase were collected and counted, and the cell suspension concentration was adjusted to 5×10 4 / mL, added to a 96-well cell culture plate, with a volume of 100 μL per well. Using DMSO as a solvent control, the compounds RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, and RD24V1012 described in the present invention were added to a 96-well cell culture plate, with a volume of 100 μL per well. , RD24V1013, RD24V1014, RD24V1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019 , RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V2001, RD24V2002, RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, R D24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, and RD24V2023 were diluted with DMSO and added to the culture wells so that the final concentrations of the compounds in the system were 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 (μmol / L), respectively. After continuing to culture for 72 hours, 10 / 20 μL of CCK-8 solvent was added to each well and incubated at 37°C for 3 hours. The OD value at an absorption wavelength of 450nm was measured and the results were recorded. The cell growth curve was drawn with the dose of the compound as the horizontal axis and the absorbance value as the vertical axis. The statistical results of the half-maximal inhibition rate (IC50 value) of the compound on tumor cells are shown in Table 1 below:

[0057] Table 1. CCK-8 detects RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1 006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD2 4V1013, RD24V1014, RD24V1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019 , RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V2001, RD24V2 RD24V2002, RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, and RD24V2023 have inhibitory effects on the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma cells.

[0058]

[0059]

[0060]

[0061]

[0062] The table shows: RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD24V1013, RD24V1014, RD24V 1015, RD24V1016, RD24V1017, RD24V1018, RD24V1019, RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V2001, RD24V2002, RD24V2003, RD24V2004, RD24V2005, RD24V 2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V 2021, RD24V2022, and RD24V2023 have good proliferation inhibitory effects on breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells, especially in breast cancer, multiple myeloma, and diffuse large B-cell lymphoma cells, where the tumor inhibitory activity is stronger. This application uses RD24V1001 as an example to conduct a preliminary study on the anti-tumor mechanism of this type of compound.

[0063] Example 3: Effect of RD24V1001 on STAT3 and AMPK phosphorylation in MDA-MB-468 cells

[0064] 1. Cell culture and drug addition: a. Take MDA-MB-468 cells in the logarithmic growth phase and adjust the density to 2×10 5 A single-cell suspension of 100 cells / mL was seeded into 6-well plates at 2 mL per well. The cells were incubated overnight at 37°C in a 5% CO2 incubator. RD24V1001 was added at various concentrations (0, 3, 10, 30, and 100 nM, respectively). DMSO was used as a negative control. c. After an additional 24 hours of culture, the cells were lysed with RIPA buffer and the protein was collected.

[0065] II. Cell Harvest and Lysis: a. Discard the supernatant medium and wash the cells twice with pre-chilled PBS. Add 100 μL of pre-chilled RIPA cell lysis buffer (protease inhibitors and PMSF are pre-mixed at a 1:100 ratio) to each well.

[0066] b. Lyse on ice for 3 minutes, scrape the cells with a cell scraper, and collect them in a 1.5 mL EP tube; place on ice for 30 minutes and vortex every 6 minutes.

[0067] c. Centrifuge at 12,000 g for 10 min at 4°C. d. Transfer the cell supernatant to a new EP tube.

[0068] d. Divide the cell supernatant into two parts: take 5 μL and add it to a 1.5 mL EP tube for BCA protein content measurement, then add 45 μL of 1× PBS and mix thoroughly for later use; take 80 μL of the remaining cell supernatant, add 20 μL of 5× SDS Loading Buffer, mix thoroughly, and boil in boiling water for 10 minutes. After centrifugation, load the sample or store in a -20°C refrigerator.

[0069] e. Protein concentration determination steps:

[0070] (1) Preparation of BCA working solution: Calculate the total amount of mixed working solution A and B required based on the number of standards and samples to be tested. Prepare the working solution at a volume ratio of 50:1 for BCA reagent A and B, vortex and mix thoroughly, and set aside.

[0071] (2) 1× PBS diluted protein standard:

[0072]

[0073]

[0074] (3) Add 25 μL each of the protein standard solution and the sample supernatant diluted with PBS (10-fold dilution) to a new 96-well plate. Then, add 200 μL of the previously prepared BCA working solution to each well and mix thoroughly. Be careful not to create bubbles by pipetting. Cover the 96-well plate tightly and incubate in a 37°C incubator for 30 min.

[0075] (4) Remove the 96-well plate and return it to room temperature for 3–5 min. Measure the absorbance of A562 on a microplate reader and save the values ​​in an Excel spreadsheet. Create a standard curve and calculate the protein content of 1 μL of each sample for protein loading.

[0076] 3. SDS-PAGE: (1) Fix the gel plate and prepare 10% SDS-PAGE separation gel.

[0077] Prepare separation gel according to the following table: 10 mL

[0078] Deionized water 4.0mL 30% (m / v) Acrylamide 3.3mL 1.5M Tris-HCl (pH 8.8) buffer 2.5mL 10% (m / v) SDS 0.1mL 10% (m / v) APS 0.1mL TEMED 4 μL Total 10mL

[0079] (2) Add the mixed separation gel to two gel plates, add it to 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let it stand for 30 to 45 minutes. (3) After the separation gel is solidified, pour out the remaining anhydrous ethanol and absorb the remaining anhydrous ethanol with filter paper. (4) Prepare 5 mL of 5% concentrated gel according to the table below

[0080]

[0081]

[0082] (5) Slowly add the prepared concentrated glue to the glue plate to avoid bubbles, insert a comb, and let it stand for 30 to 45 minutes.

[0083] (6) Remove the protein sample, heat in a 100°C water bath for 5 minutes, and centrifuge at 10,000 rpm for 5 minutes. (7) Fix the gel plate to the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the comb, and add the processed protein sample to the sample tank in order, 50 μg of protein per well. (8) Electrophoresis at 80V for 40 minutes. (9) Change the voltage to 120V and electrophoresis for about 1.5 hours until the bromophenol blue runs out of the colloid;

[0084] IV. Western-blot: (1) Rinse the SDS-PAGE gel after electrophoresis in TBST buffer once, and soak the protein gel in transfer buffer. (2) Soak a cotton pad in membrane transfer buffer, clamp it onto the transfer apparatus with tweezers, and place it in the order of blackboard, cotton pad, filter paper, protein gel, PVDF membrane, filter paper, cotton pad and whiteboard, clamp it, and place it on the transfer apparatus. If there are bubbles between each layer, use a glass tube to gently roll them out. (3) Turn on the transfer apparatus and transfer at a constant current of 300mA for 80 minutes. (4) Place the membrane in TBST buffer and rinse 3 times, 8 minutes each time. (5) Block with 20mL of 5% BSA-TBST blocking solution at room temperature for 2 hours. (6) Add primary antibody and incubate at 4℃ 60rpm overnight. (7) Wash the membrane three times with TBST at room temperature on a shaker at 60rpm, 10 minutes each time. (8) Add secondary antibody and incubate at room temperature for 1 hour. (9) Wash the membrane three times with TBST at room temperature, shaking at 60 rpm, for 10 minutes each time. (10) Take 1 mL each of chemiluminescent substrate solution A and solution B and develop the color at room temperature for 2 minutes. (11) Blot the liquid on the membrane with filter paper and expose it to light.

[0085] 5. Reagent preparation:

[0086] (1) 10% SDS: Weigh 1 g of high-purity (electrophoresis grade) SDS into a 10 mL centrifuge tube, add about 8 mL of deionized water, heat to dissolve, and dilute to 10 mL. Store at room temperature.

[0087] (2) 10% ammonium persulfate (AP): Weigh 1 g of ammonium persulfate, add approximately 10 mL of deionized water, stir to dissolve, and store at 4°C.

[0088] (3) 5× electrophoresis buffer: Weigh 15.1 g of Tris, 94 g of Glycine, and 5.0 g of SDS into a beaker, add 1 L of double-distilled water to dissolve, store at room temperature, and dilute 5-fold before use.

[0089] (4) Transfer buffer: Weigh 5.8 g of Tris, 11.6 g of glycine, and 0.75 g of SDS into a beaker, add 700 mL of double-distilled water, dissolve and adjust the volume to 800 mL, and finally add 200 mL of methanol.

[0090] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g of Tris in 80 mL of water and adjust the pH to 8.8 with 4 N HCl. The volume is then adjusted to 100 mL.

[0091] (6) 0.5 mol / L Tris-HCl, 1000 mL: Weigh 60.5 g of Tris base, add water to 850 mL, add concentrated hydrochloric acid and stir until completely dissolved, then adjust the pH to 6.8 and add water to 1 L.

[0092] (7) TBS buffer: Weigh 8.8 g of NaCl in 800 mL of distilled water, dissolve it, add 10 mL of 1 mol / L TrisHCl (pH 7.5), adjust the volume to 1 L, and store at room temperature.

[0093] (8) TBST buffer: Add 500 μL of 20% Tween 20 to 1 L of TBS buffer to make the final concentration of Tween 20 0.1%. Prepare it before use.

[0094] (9) Blocking solution, antibody diluent: Add 5% skim milk powder or BSA to TBST buffer and prepare it before use.

[0095] like Figure 1 The results showed that treatment with 30nM and 100nM of RD24V1001 could effectively downregulate the expression levels of p-STAT3(Y705), p-STAT3(S727) and STAT3's downstream target proteins C-MYC and CyclinD1.

[0096] like Figure 2 The results showed that treatment with 10nM and 30nM of RD24V1001 could effectively activate the phosphorylation of AMPK and the phosphorylation of AMPK's downstream target protein ACC.

[0097] In summary, the results show that RD24V1001, RD24V1002, RD24V1003, RD24V1004, RD24V1005, RD24V1006, RD24V1007, RD24V1008, RD24V1009, RD24V1010, RD24V1011, RD24V1012, RD24V1013, RD24V1014, RD24V1015, RD24V1016, R D24V1017, RD24V1018, RD24V1019, RD24V1020, RD24V1021, RD24V1022, RD24V1023, RD24V1024, RD24V 2001, RD24V2002, RD24V2003, RD24V2004, RD24V2005, RD24V2006, RD24V2007, RD24V2008, RD24V2009, RD24V2010, RD24V2011, RD24V2012, RD24V2013, RD24V2014, RD24V2015, RD24V2016, RD24V2017, RD24V2018, RD24V2019, RD24V2020, RD24V2021, RD24V2022, and RD24V2023 can significantly inhibit breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, and cervical cancer. It can inhibit the proliferation of cells such as cancer, multiple myeloma, diffuse large B-cell lymphoma, and can effectively downregulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727) and STAT3's downstream target proteins C-MYC and CyclinD1. Therefore, this type of drug has good anti-cancer effects and development potential; in addition, this type of compound can also activate AMPK phosphorylation, so this type of compound also has great application potential in metabolic diseases (such as weight loss and non-alcoholic fatty liver disease).

[0098] According to the general approach of drug development (conventional anti-tumor in vitro screening first, followed by targeted research), the compounds of the present invention can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be prepared as anti-tumor drugs and metabolic disease drugs by mixing with human-acceptable salts or with pharmaceutical carriers.

[0099] Finally, it should be noted that the above embodiments are only used to illustrate and not to limit the technical solutions of the present invention. Any equivalent substitutions of the present invention and any modifications or partial substitutions that do not depart from the spirit and scope of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A 1-methyl-6-chromenoindole-2-carboxamide compound, characterized in that: The structural formula is shown in general formula I: Wherein, R1 and R2 are selected from H, -OH, -NH2, -NO2, -OCH3, F, Cl, Br, 2. The 1-methyl-6-chromenoindole-2-carboxamide compound according to claim 1, characterized in that Specifically, the compound has the following structure:

3. A biologically acceptable salt formed by the 1-methyl-6-chromenoindole-2-carboxamide compound according to claim 1 or 2 and at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, diphosphoric acid, hydrobromic acid, nitric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, succinic acid, ascorbic acid, boric acid, lactic acid, p-toluenesulfonic acid, salicylic acid and ethylenediaminetetraacetic acid.

4. The method for preparing the 1-methyl-6-chromenone indole-2-carboxamide compound according to claim 1, wherein: The synthetic route is as follows: The specific synthesis steps are as follows: (1) Compound 1, Compound 2, HBTU, and DIEA were dissolved in DMF and stirred at 20-30°C. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried, slurried with ethyl acetate, and filtered to obtain Compound 3; (2) Compound 3, compound 4, Pd(dppf)Cl2 and potassium acetate were dissolved in DMF and stirred at 90-120°C. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation and subjected to column chromatography to obtain compound 5. (3) Compound 5, compound 6, Pd(dppf)Cl2 and sodium carbonate were dissolved in DMSO and stirred at 90-120°C until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation and purified by column chromatography to obtain the compound of formula I.

5. The method for preparing 1-methyl-6-chromenoindole-2-carboxamide compounds according to claim 4, characterized in that: In step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:(1-1.5):3; step (2) In the step (3), the molar ratio of compound 3, compound 4, potassium acetate and Pd(dppf)Cl2 is 1:2:3:0.05; in the step (3), the molar ratio of compound 5, compound 6, sodium carbonate and Pd(dppf)Cl2 is 1:(1-1.5):(1-1.5):0.

05.

6. Use of the 1-methyl-6-chromenoindole-2-carboxamide compound or a biologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a STAT3 protein phosphorylation inhibitor.

7. Use of the 1-methyl-6-chromenoindole-2-carboxamide compound or a biologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of an anti-tumor drug, characterized in that: The anti-tumor drug refers to a drug for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma.

8. Use of the 1-methyl-6-chromenoindole-2-carboxamide compound or a biologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of a drug for activating AMPK.

9. Use of the 1-methyl-6-chromenoindole-2-carboxamide compound or a biologically acceptable salt thereof according to any one of claims 1 to 3 in the preparation of metabolic drugs, characterized in that: The medicine is a medicine for treating diabetes and obesity.

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