A 1-methyl-6-alkynyl indole-2-carboxamide derivative, its preparation method and application

Abstract

The present invention discloses a 1-methyl-6-alkynyl indole-2-carboxamide derivative, a preparation method, and an application thereof, belonging to the field of drug synthesis. The structural formula of the 1-methyl-6-alkynyl indole-2-carboxamide derivative is shown in General Formula I: #imgabs0# wherein R1 is selected from #imgabs1##imgabs2##imgabs3#. The biologically active 1-methyl-6-alkynyl indole-2-carboxamide nucleus group contained therein is further chemically modified to produce a variety of compounds with higher biological activity, thereby expanding the wide application of such compounds in biomedicine and the development prospects of pharmaceutical preparations. Such 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 at low doses (nanomolar), and can effectively activate AMPK protein phosphorylation, indicating that such compounds have the prospect of being developed into anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of drug synthesis, and in particular relates to a 1-methyl-6-alkynyl indole-2-carboxamide derivative, a preparation method and application thereof. Background Art

[0002] Cancer is currently one of the leading causes of death worldwide and has become a major disease that seriously threatens human health and hinders socioeconomic development. Despite extensive and intensive research in targeted anti-tumor drug therapies, many types of cancer still have a poor prognosis and are incurable. Therefore, the development of new anti-tumor drugs is imperative. AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and plays an important role in diabetes, cancer, and vascular disease. Targeted AMPK therapies have been widely used to treat a variety of metabolic disorders, including diabetes. Because AMPK has been shown to have tumor suppressor effects in certain conditions, several small molecules that trigger AMPK activation have been tested in preclinical cancer models. For example, the clinically validated diabetes drug canagliflozin activates AMPK and inhibits cell proliferation, survival, and tumorigenicity in prostate and lung cancer cells. The small molecule AMPK activator MT63-78 inhibits adipogenesis, the mTORC1 pathway, and the tumorigenic potential of prostate cancer cells.

[0003] This application synthesizes a novel class of 1-methyl-6-alkynyl indole-2-carboxamide derivatives. Biological analysis reveals that these compounds, at extremely low doses, can significantly inhibit the proliferation of cells in 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 activate AMPK protein phosphorylation. Therefore, further development of these compounds will be of great significance for their application in tumor treatment. Summary of the Invention

[0004] The purpose of the present invention is to provide a 1-methyl-6-alkynyl indole-2-carboxamide derivative, a preparation method and application thereof.

[0005] Based on the above objectives, the present invention adopts the following technical solutions:

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

[0007]

[0008] Wherein, R1 is selected from

[0009] That is, the above-mentioned 1-methyl-6-alkynyl indole-2-carboxamide derivative is specifically a compound with the following structure:

[0010]

[0011]

[0012]

[0013] A biologically acceptable salt formed by the above-mentioned 1-methyl-6-alkynyl indole-2-carboxamide derivative 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, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediaminetetraacetic acid.

[0014] The preparation method of the above-mentioned 1-methyl-6-alkynyl indole-2-carboxamide derivative and the synthetic route are as follows:

[0015]

[0016] The specific synthesis steps are as follows:

[0017] (1) Compound 1, Compound 2, HBTU, and DIEA were dissolved in DMF and stirred at room temperature. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 3;

[0018] (2) Compound 3, compound 4, Pd(PPh3)2Cl2, CuI and triethylamine were dissolved in DMF and stirred at 70-90°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 and slurried with ethyl acetate, filtered, and the solid was dried to obtain the target compound.

[0019] Furthermore, in step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:(1-1.5):3; in step (2), the molar ratio of compound 3, compound 4, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.02):(0.01-0.03):5.

[0020] Use of the above-mentioned 1-methyl-6-alkynyl indole-2-carboxamide compounds and biologically acceptable salts thereof in the preparation of AMPK protein activators.

[0021] Application of the above-mentioned 1-methyl-6-alkynyl indole-2-carboxamide derivatives and biologically acceptable salts thereof in the preparation of anti-tumor drugs.

[0022] Preferably, 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, diffuse large B-cell lymphoma, etc.

[0023] Specifically, the present invention synthesizes a class of 1-methyl-6-alkynyl indole-2-carboxamide derivatives RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, RD24M027, etc. with a completely new structure. The CCK-8 method was used to detect the proliferation inhibitory effect of this type of compound on various cancer cells; and it can effectively activate AMPK protein phosphorylation.

[0024] The results showed that the compounds of the present invention RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, R D24M016, RD24M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 can effectively inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, and gastric cancer cells, and can effectively activate AMPK protein phosphorylation.

[0025] In summary, the present invention provides a new 1-methyl-6-alkynyl indole-2-carboxamide derivative and the use and potential molecular mechanism of its derivatives in tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the result of the effect of different concentrations of RD24M002 on protein expression in MDA-MB-468 cells in Example 3;

[0027] Figure 2The curves of the changes in tumor volume over the administration time of the AMPK activators ASP4132 and RD24M002 in the MDA-MB-468 subcutaneous xenograft tumor model in Example 4 are as follows;

[0028] Figure 3 1 is a curve showing changes in mouse body weight over administration time in the MDA-MB-468 subcutaneous xenograft tumor model in Example 4 when AMPK activators ASP4132 and RD24M002 are administered. DETAILED DESCRIPTION

[0029] 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.

[0030] 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.

[0031] 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).

[0032] Example 1: The synthesis of all compounds is as follows

[0033] The specific synthesis method takes compound RD24M002 as an example, and the structural formula is as follows:

[0034]

[0035] The name of compound RD24M002 is (1-methyl-6-(pyridin-3-ylethynyl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone.

[0036] Its synthetic route is as follows:

[0037]

[0038] Step 1. (6-bromo-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 3)

[0039] Compound 1 (2.0 g, 10.21 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 room temperature for 3 hours. The reaction was monitored by TLC for completion. The reaction solution was diluted with 200 mL of ethyl acetate and washed three times with saturated brine (200 mL*3). The organic phase was dried and spin-dried, then purified by slurrying with 15 mL of ethyl acetate and filtered. The solid was collected and dried in an 80°C oven to obtain 4.03 g of compound 3 as a white solid, with a yield of 82.3%.

[0040] 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=8H z,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).

[0041] Step 2.(1-methyl-6-(pyridin-3-ylethynyl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone(RD24M002)

[0042] Compound 3 (1.0 g, 1.96 mmol, 1.0 eq), compound 4a (0.32 g, 2.16 mmol, 1.1 eq), Pd(PPh3)2Cl2 (13.8 mg, 0.02 mmol, 0.01 eq), CuI (7.46 mg, 0.04 mmol, 0.02 eq), and triethylamine (991 mg, 9.80 mmol, 5.0 eq) were dissolved in 30 mL of DMF under nitrogen atmosphere. The mixture was stirred at 80°C for 12 hours. TLC monitoring showed complete reaction of the starting materials, with the formation of new spots. 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 and spin-dried, then purified by slurrying with 25 mL of ethyl acetate and filtered. The solid was collected and dried in an oven at 80°C to obtain 950 mg of compound RD24M002 as a yellow solid (81.2% yield).

[0043] 1 H NMR (CDCl3, 400MHz) δ: 7.86-7.81 (m, 4H), 7.66 (d, J = 8.4Hz, 1H), 7.48 (d, J = 2.0Hz, 1H), 7.29 (d, J = 8.8Hz, 2H), 7.04-6. 89(m,3H),6.69(d,J=0.4Hz,1H),4.74(q,J=8.8Hz,2H),3.73(s,3H),3.66(broad,4H),3.48(s,2H),2.42(broad,4H).

[0044] The synthesis of RD24M001, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026 and RD24M027 can refer to the method of Example 1, except that in the last step, 3-ethynylpyridine (compound 4a) is replaced by an alkynyl compound having the corresponding substituent.

[0045] Example 2, RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 inhibit 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.

[0046] 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 RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, 4M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 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 using a microplate reader. The results were recorded and 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:

[0047] Table 1. CCK-8 detects RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD2 4M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M01 6. RD24M017, RD24M018, RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 inhibit 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

[0048]

[0049]

[0050]

[0051]

[0052]

[0053] The table shows: RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, RD24M018, RD24M019, RD24M020, 24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 all 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. The tumor inhibitory activity is stronger. This application uses RD24M002 as an example to conduct a preliminary study on the anti-tumor mechanism of this type of compound.

[0054] Example 3: Effects of RD24M002 on AMPK Phosphorylation and Downstream Target Protein Activation in MDA-MB-468 Cells I. Cell Culture and Drug Addition: a. MDA-MB-468 cells in the logarithmic growth phase were adjusted to a single-cell suspension density of 2×105 cells / mL and seeded into 6-well plates with 2 mL of cell suspension per well. b. Incubate overnight in a 37°C incubator and add RD24M002 at different concentrations (final concentrations of 0, 3, 10, 30, and 100 nM). DMSO served as a negative control. c. After a further 24 hours of culture, cells were lysed with RIPA buffer and protein was collected.

[0055] II. Cell Harvest and Lysis: a. Discard the supernatant and wash the cells twice with pre-chilled PBS. Add 100 μL of pre-chilled RIPA cell lysis buffer (add protease inhibitors and PMSF to the lysis buffer at a 1:100 ratio and mix thoroughly) to each well. b. Lyse on ice for 3 minutes. Scrape the cells with a cell scraper and collect them into a 1.5 mL EP tube. Keep lysing on ice for 30 minutes, vortexing every 6 minutes. c. Centrifuge at 12,000 g for 10 minutes at 4°C. d. Transfer the cell supernatant to a fresh EP tube. d. Divide the cell supernatant into two parts: add 5 μL to a 1.5 mL EP tube for BCA protein analysis, then add 45 μL of 1× PBS and mix thoroughly for later use. For the remaining cell supernatant, take 80 μL of each aliquot, add 20 μL of 5× SDS Loading Buffer, mix thoroughly, boil in boiling water for 10 minutes, centrifuge, and load onto the sample or store in a -20°C refrigerator.

[0056] e. Protein concentration determination steps: (1) Preparation of BCA working solution: Based on the number of standards and samples to be tested, calculate the total amount of BCA reagent A and B working solution required. Prepare the working solution with a volume ratio of BCA reagent A to B of 50:1, vortex and mix thoroughly.

[0057] (2) 1× PBS diluted protein standard:

[0058]

[0059] (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.

[0060] (4) Remove the 96-well plate and return it to room temperature for 3-5 minutes. Measure the absorbance of A562 on a microplate reader and save the obtained values ​​in an Excel spreadsheet. Create a standard curve and calculate the protein content of 1 μL of each sample for protein loading. III. SDS-PAGE: (1) Fix the gel plate and prepare a 10% SDS-PAGE separation gel.

[0061] Prepare separation gel according to the following table: 10 mL

[0062]

[0063]

[0064] (2) Add the mixed separation gel to two gel plates, adding it to a position 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let it stand for 30 to 45 minutes.

[0065] (3) After separation and gelation, pour out the remaining anhydrous ethanol and absorb the remaining anhydrous ethanol with filter paper.

[0066] (4) Prepare 5 mL of 5% concentrated gel according to the table below

[0067] Deionized water 2.77mL 30% (m / v) Acrylamide 830μL 0.5M Tris-HCl (pH 6.8) buffer 1.26mL 10% (m / v) SDS 50 μL 10% (m / v) APS 50 μL TEMED 5μL Total 5mL

[0068] (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.

[0069] (6) Take out the protein sample, heat it in a water bath at 100°C for 5 min, and centrifuge it at 10,000 rpm for 5 min.

[0070] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, pull out the comb, and add the processed protein samples into the sample tank in order, with 50 μg of protein per well.

[0071] (8) Electrophoresis at 80V for 40min.

[0072] (9) Change the voltage to 120 V and run the electrophoresis for about 1.5 hours until the bromophenol blue runs out of the colloid;

[0073] 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.

[0074] 5. Reagent preparation:

[0075] (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, dilute to 10 mL, and store at room temperature.

[0076] (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.

[0077] (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.

[0078] (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.

[0079] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g of Tris base in 80 mL of water and adjust the pH to 8.8 with 4 N HCl. The volume is then adjusted to 100 mL.

[0080] (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.

[0081] (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.

[0082] (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.

[0083] (9) Blocking solution, antibody diluent: Add 5% skim milk powder or BSA to TBST buffer and prepare it before use.

[0084] The above test results are detailed in Figure 1 ,like Figure 1 The results showed that treatment with 10nM, 30nM, and 100nM of RD24M002 could effectively activate AMPK phosphorylation and the phosphorylation of AMPK's downstream target protein ACC.

[0085] In summary, the results show that RD24M001, RD24M002, RD24M003, RD24M004, RD24M005, RD24M006, RD24M007, RD24M008, RD24M009, RD24M010, RD24M011, RD24M012, RD24M013, RD24M014, RD24M015, RD24M016, RD24M017, RD24M018 RD24M019, RD24M020, RD24M021, RD24M022, RD24M023, RD24M024, RD24M025, RD24M026, and RD24M027 can significantly inhibit 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, and can effectively activate AMPK phosphorylation and AMPK's downstream target protein ACC phosphorylation. Therefore, this class of drugs has good anti-cancer effects and development potential.

[0086] 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 by mixing with human-acceptable salts or with pharmaceutical carriers.

[0087] Example 4: In vivo pharmacodynamic study of RD24M002 on triple-negative breast cancer MDA-MB-468 subcutaneous xenograft tumor model

[0088] The in vivo antitumor effect of compound RD24M002 on triple-negative breast cancer cells MDA-MB-468 was evaluated by constructing a nude mouse xenograft tumor model of MDA-MB-468 cells and administering compound RD24M002 for treatment.

[0089] Establishment of MDA-MB-468 cell transplant tumor model in nude mice:

[0090] (1) MDA-MB-468 cells were cultured according to conventional cell culture methods. The culture method is as follows:

[0091] Observe under a microscope and subculture when the cell coverage in the culture dish reaches 80%-90%. In a biosafety cabinet, discard the original culture medium, add 2mL of trypsin, and digest at 37°C for 2 minutes. Once the cells have rounded, add an equal volume of complete culture medium to terminate digestion. Use a pipette to detach the cells by pipetting. Then, transfer the cell suspension to a 15mL centrifuge tube and centrifuge at 900 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the cells in 2mL of culture medium, pipette to mix thoroughly, and divide evenly between two 100mm cell culture dishes for continued culture.

[0092] (2) MDA-MB-468 cell collection

[0093] ① Aspirate the old culture medium in the culture dish and wash it once with PBS.

[0094] ② Add 2 mL of trypsin-EDTA digestion solution containing 0.25% trypsin solution and 0.02% EDTA (0.53 mM) to the culture dish.

[0095] ③ Place the culture flask in a 37°C incubator for digestion. After 2 minutes, place the culture flask under an inverted microscope for observation. If the cytoplasm shrinks or the intercellular space increases, the digestion should be stopped immediately.

[0096] ④Add complete culture medium to terminate digestion.

[0097] ⑤ Use a pipette tip to absorb the culture medium in the dish and gently blow the cells at the bottom of the dish repeatedly to separate them from the bottom of the dish to form a cell suspension.

[0098] ⑥After counting with a counting plate, adjust the cell suspension to 5×10 7 The collected MDA-MB-468 cells were placed on ice for later use.

[0099] (3) MDA-MB-468 tumor cell inoculation

[0100] Six-week-old, 18-20g BALB / c-nude female mice were inoculated with MDA-MB-468 tumor cells. The skin at the inoculation site was disinfected with alcohol pads. After the cell suspension was mixed thoroughly, a syringe was drawn up with a desired amount of the suspension and 100μL / mouse was inoculated into the axillary fat pad. Once the tumor had reached approximately 13-15 mm in diameter, the mouse was sacrificed. The body surface was soaked in 75% alcohol. The tumor tissue was then dissected with sterile scissors and forceps. The tumor tissue was then immersed in an appropriate amount of saline, ensuring the surface was moistened. The tumor capsule was removed and the tumor tissue was cut open. The necrotic central portion of the tumor was removed, retaining the healthy, fish-like tumor tissue. The tumor was then cut into 1 mm3 pieces and inserted into the inoculation needle using forceps. The needle was then inserted subcutaneously into the nude mouse's axilla. Once the needle reached the desired location, the solid needle at the center of the inoculation needle was used to push the tumor tissue piece out. The inoculated animal was returned to its original cage for maintenance, maintaining a clean environment to prevent infection.

[0101] Tumors were passaged using the above method. By the third generation, all nude mice had subcutaneous transplanted tumors, and the size of the tumors was similar. The tumor growth and tumor formation rate of each subcutaneous transplanted tumor mouse were recorded.

[0102] (4) Treatment of tumor-bearing mice

[0103] Ten days after inoculation, mice were divided into groups when their tumors reached approximately 100 cubic millimeters. The mice were evenly divided into three groups of five mice each, based on tumor size. The mice were dosed according to the groupings listed below, with free access to food and water. The experiment was terminated when the tumors in the control group reached approximately 1000 cubic millimeters. The axillary tumors were removed and weighed. The mice were dosed according to the following groupings.

[0104] RD24M002 suspension Negative control (blank suspension) ASP4132 suspension Drug concentration 1mg / mL / 1mg / mL Dosage 5mg / kg 5mg / kg 5mg / kg Number of mice 5 5 5 Route of administration Oral administration Oral administration Oral administration

[0105] The suspensions in the above table are prepared as follows:

[0106] The formulation process of AMPK activator RD24M002 suspension and blank suspension is as follows:

[0107] Components Blank preparation dosage Dosage for 1mg / mL preparation API 0mg 3mg PEG-400 3mL 3mL

[0108] Process: Take each component of the prescription into a 5mL EP tube and sonicate until clear.

[0109] The preparation formula and process of ASP4132 suspension are as follows:

[0110]

[0111] Process: Take each component of the prescription into a 5mL EP tube and ultrasonicate until clear. Preparation of 6% HP-β-CD: Dissolve 6g HP-β-CD in 100mL deionized water.

[0112] Note: When preparing the preparation, the raw material drug is converted according to the purity of each raw material drug. Since the raw material drugs of the above compounds are limited, in order to reduce the loss of the preparation preparation test, the preparation for animal model is fully prepared according to the above prescription ratio during preparation preparation, and the preparation volume is divided into EP tubes according to 3 days of use, sealed with sealing film, and stored at -80℃ for reconstitution when needed.

[0113] (5) Weight changes: Every day, the weight changes of mice were recorded and growth curves were drawn. The results are shown in the figure below. Figure 2 As shown in the table above, the drug concentration, dosage, number of test animals and administration route of the RD24M002 suspension group, the blank suspension group, and the positive control group ASP4132 suspension group.

[0114] (6) Tumor growth inhibition rate

[0115] The test indicator is to examine whether tumor growth can be inhibited, delayed, or cured. During the trial, the tumor diameter is measured with a vernier caliper twice a week. The formula for calculating tumor volume is: V = 0.5a × b 2 , a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of the mice was monitored at the same time. At the end of the experiment, the tumors were removed, weighed, and photographed.

[0116] The anti-tumor efficacy of the compound was evaluated by relative tumor proliferation rate T / C (%), relative tumor volume (Relative tumor volume, RTV) and tumor growth inhibition rate IR (%), which reflected the tumor growth inhibition situation. The calculation formula is as follows: T / C (%) = (average tumor volume of the test group after administration / average tumor volume of the test group before administration) / (average tumor volume of the control group / average tumor volume of the control group when grouping) × 100%. T and C represent the relative tumor volumes of the treatment group and the control group, respectively. If T / C% ≤ 40%, and P < 0.05 after statistical processing, the drug is considered to be effective; RTV = Vt / V0, where V0 is the tumor volume measured before group administration (i.e., d0), and Vt is the tumor volume measured each time after the start of administration; the curve of tumor volume change with administration time is shown in Figure 2. Figure 3 shown.

[0117] (7) Statistical analysis

[0118] Summary statistics were presented using the mean ± standard error of the mean (SEM). Statistical analysis was performed using Graphpad Prism 8.0 or SPSS 20.0, with p < 0.05 considered significant. Pairwise comparisons between groups were performed using one-way analysis of variance, with homogeneity of variance tested using the LSD test and heterogeneity of variance tested using the Games-Howell test.

[0119] (8) Result analysis

[0120] like Figure 2 and Figure 3 As shown, the body weights of mice in the blank control (SUS-Control) and RD24M002 groups remained stable throughout the dosing period, with no mice experiencing weight loss exceeding 15%. Furthermore, no significant abnormalities were observed in the mice's activity or food intake. These results demonstrate that RD24M002 suspension (RD24M002 SUS) exhibits good in vivo safety at an oral dose of 5 mg / kg. Based on the results of measuring the tumor volume of each group of mice at the end of the experiment, the T / C of the 5 mg / kg dose group of RD24M002 suspension was 34.66% at 28 days of administration, and P was less than 0.05. The T / C of the 5 mg / kg dose group of the positive control ASP4132 suspension (ASP4132SUS) was 43.40% at 28 days of administration, and P was less than 0.05. However, the T / C did not reach below 40%, which did not meet the efficacy standard, and the efficacy was weaker than RD24M002. This shows that RD24M002 achieved an effective therapeutic level in the MDA-MB-468 mouse subcutaneous transplant tumor model at an oral dose of 5 mg / kg (T / C <40% and statistical difference indicates effectiveness). This indicates that RD24M002 suspension has good in vivo safety at an oral dose of 5 mg / kg, and has a significant therapeutic effect in the MDA-MB-468 mouse subcutaneous transplant tumor model, and its efficacy is better than that of the positive control ASP4132.

[0121] 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-alkynyl indole-2-carboxamide derivative, characterized in that: The structural formula is shown in general formula I: Wherein, R1 is selected from 2. A biologically acceptable salt of the 1-methyl-6-alkynyl indole-2-formyl derivative according to claim 1 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, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid and ethylenediaminetetraacetic acid.

3. The method for preparing the 1-methyl-6-alkynyl indole-2-carboxamide derivative according to claim 1, characterized in that: 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 room temperature. After the reaction was complete, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was spin-dried and slurried with ethyl acetate, filtered, and the solid was dried to obtain Compound 3; (2) Compound 3, compound 4, Pd(PPh3)2Cl2, CuI and triethylamine were dissolved in DMF and stirred at 70-90°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 and slurried with ethyl acetate, filtered, and the solid was dried to obtain the target compound.

4. The method for preparing the 1-methyl-6-alkynyl indole-2-carboxamide derivative according to claim 3, characterized in that: In step (1), the molar ratio of compound 1, compound 2, HBTU and DIEA is 1:1:(1-1.5):3; in step (2), the molar ratio of compound 3, compound 4, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.02):(0.01-0.03):

5.

5. Use of the 1-methyl-6-alkynyl indole-2-carboxamide derivative and a biologically acceptable salt thereof according to claim 1 or 2 in the preparation of an AMPK protein activator.

6. Use of the 1-methyl-6-alkynyl indole-2-carboxamide derivative and a biologically acceptable salt thereof according to claim 1 or 2 in the preparation of an anti-tumor drug, characterized in that: The anti-tumor drugs are drugs for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma and diffuse large B-cell lymphoma.

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