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

Abstract

The application provides a 1-methyl-6-oxylindole-2-carboxamide derivative, a preparation method and application thereof, and belongs to the field of drug synthesis. The structural formula of the 1-methyl-6-oxylindole-2-carboxamide derivative is shown in general formula I: wherein R1 is selected from G=C or N; and W=C or N. The 1-methyl-6-oxylindole-2-carboxamide parent nucleus group with biological activity contained in the 1-methyl-6-oxylindole-2-carboxamide derivative is further chemically modified to generate a plurality of compounds with higher biological activity, thereby expanding the wide application of the compounds in biological medicine and the development prospects of drug preparations. The compounds 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 at a low dose (nanomole), and can effectively inhibit the phosphorylation of STAT3 protein, which indicates that the compounds have the prospects of being developed into anti-tumor drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of drug synthesis, and particularly relates to a 1-methyl-6-oxylindole-2-carboxamide derivative, a preparation method and application thereof. BACKGROUND

[0002] Cancer has become an important cause of premature death and life shortening of global population. At present, although great progress has been made in the treatment of cancer in clinic, due to the heterogeneity and continuous evolution of tumors, the response rate of most drugs is low, and drug resistance is prone to occur, so that effective treatment drugs are still in short supply. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin and capecitabine have the advantages of wide anti-cancer spectrum and good efficacy, but have great toxic side effects and are prone to early drug resistance. Therefore, it is of important clinical significance to actively explore and develop new anticancer drugs.

[0003] The present application synthesizes a class of 1-methyl-6-oxylindole-2-carboxamide derivatives with a novel structure. Through some biological technology analysis, it is found that the compounds can significantly inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells at very low doses, and can effectively inhibit STAT3 protein phosphorylation. Therefore, further development of such compounds will have important significance in the application of tumor treatment. SUMMARY

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

[0005] In order to achieve the above purpose, the present application adopts the following technical solutions:

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

[0007]

[0008] wherein R1 is selected from

[0009] G=C or N;

[0010] W=C or N.

[0011] That is, the above-mentioned 1-methyl-6-oxylindole-2-carboxamide derivative is specifically a compound with the following structure:

[0012]

[0013] A biologically acceptable salt of 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 with the 1-methyl-6-oxoindole-2-carboxamide derivative described above.

[0014] A preparation method of the 1-methyl-6-oxoindole-2-carboxamide derivative described above, and a synthetic route is as follows:

[0015]

[0016] Specific synthesis steps are as follows:

[0017] (1) Compound 1, compound 2, HBTU and DIEA are dissolved in DMF, and after the reaction is completed at room temperature, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered, and dried to obtain compound 3;

[0018] (2) Compound 3, compound 4 and potassium carbonate are dissolved in DMF, and after the reaction is completed at 70-90°C, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered, and dried to obtain compound 5;

[0019] (3) Compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine are dissolved in DMF, and after the reaction is completed at 70-90°C, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered, and dried to obtain the compound shown in formula I.

[0020] Further, 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 and potassium carbonate is 1:(1-1.5):(1-1.5); in step (3), the molar ratio of compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.03):(0.01-0.09):5.

[0021] The 1-methyl-6-oxoindole-2-carboxamide compound and the biologically acceptable salt thereof described above are used for preparing a STAT3 protein inhibitor.

[0022] The 1-methyl-6-oxoindole-2-carboxamide derivative and the biologically acceptable salt thereof described above are used for preparing an antitumor drug.

[0023] Preferably, the antitumor 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.

[0024] Specifically, the application synthesizes a class of 1-methyl-6-oxylindole-2-carboxamide derivatives RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018, etc. with a completely new structure. The proliferation inhibition effect of the compounds on various cancer cells is detected by the CCK-8 method; and the phosphorylation of STAT3 protein can be effectively inhibited.

[0025] Results show that the compounds RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 of the application 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.

[0026] In summary, the application provides a new 1-methyl-6-oxylindole-2-carboxamide derivative and its derivatives for tumor treatment and potential molecular mechanisms. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is the result of the effect of different concentrations of RDG-1001 on the protein expression of MDA-MB-468 cells. DETAILED DESCRIPTION

[0028] In order to make the technical objects, technical solutions and beneficial effects of the application clearer, the technical solutions of the application will be further described below in combination with the drawings and specific embodiments.

[0029] In the process for synthesizing the compound of formula I according to the present application, the various starting materials used in the reaction can be prepared by those skilled in the art according to the existing knowledge, or can be prepared by the methods known in the art, or can be purchased commercially. The intermediates, starting materials, reagents, reaction conditions used in the above reaction scheme can be appropriately changed according to the existing knowledge of those skilled in the art.

[0030] In the present application, unless otherwise specified, (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20-30 °C; (ii) the organic solvent is dried by the commonly used drying method, and the evaporation of the solvent is carried out by rotary evaporation under reduced pressure, and the bath temperature is not higher than 50 °C; the developing agent and the eluent are both by volume; (iii) the reaction process is tracked by thin layer chromatography (TLC); (iv) the final product has a satisfactory proton nuclear magnetic resonance (H-NMR). 1

[0031] Example 1: Synthesis of all compounds according to the following route

[0032] The specific synthesis method is exemplified by compound RDG-1001, the structural formula of which is as follows:

[0033]

[0034] The name of compound RDG-1001 is (1-methyl-6-((5-(3-morpholinoprop-1-yn-1-yl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone,

[0035] The synthesis route thereof is as follows:

[0036]

[0037] Step 1. (6-hydroxy-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (compound 3)

[0038] ​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 DMF, the reaction was stirred at room temperature for 3 hours, and the reaction was monitored by TLC. The reaction solution was diluted with 200 mL ethyl acetate, washed with saturated brine three times (200 mL*3), the organic phase was dried and rotary evaporated, and the solid was collected by purifying with 15 mL ethyl acetate and filtering. After drying in an oven at 80 °C, 4.20 g of white solid compound 3 was obtained with a yield of 88.9%.

[0039] 1 H NMR (CDC13, 300 MHz) δ: 9.02 (s, 1H), 8.25 (d, J = 8 Hz, 1H), 7.78 (d, J = 8 Hz, 1H), 7.53 (m, 2H), 7.22 (d, J = 8 Hz, 1H), 7.18 (d, J = 8 Hz, 2H), 6.86 (d, J = 8 Hz, 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).

[0040] Step 2. (6-((5-bromopyrazin-2-yl)oxy)-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2- trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0041] Compound 3 (5.0 g, 11.17 mmol, 1.0 eq), compound 4 (2.69 g, 11.17 mmol, 1.0 eq) and potassium carbonate (1.85 g, 13.41 mmol, 1.2 eq) were dissolved in 50 mL DMF, and the reaction was stirred at 80 °C for 2 hours. The reaction was monitored by TLC. The reaction solution was diluted with 300 mL ethyl acetate, washed with saturated brine three times (200 mL*3), the organic phase was dried and rotary evaporated, and the solid was collected by purifying with 18 mL ethyl acetate, filtering and drying the filter cake to obtain 5.20 g of yellow solid compound 5 with a yield of 71.4%.

[0042] 1H NMR (DMSO-d6, 300 MHz) δ: 8.79 (d, J = 1.2 Hz, 1H), 8.67 (d, J = 1.2 Hz, 1H), 7.69 (d, J = 8.6 Hz, 1H), 7.51 (d, J = 1.9 Hz, 1H), 7.32 (s, 1H), 7.30 (s, 1H), 7.05 (s, 1H), 7.04 (s, 1H), 7.02 (dd, J = 8.5, 2.1 Hz, 1H), 7.01 (s, 1H), 4.76 (d, J = 8.9 Hz, 2H), 3.74 (s, 3H), 3.67 (s, 4H), 3.51 (s, 2H), 2.44 (s, 4H).

[0043] Step 3. (1-methyl-6-((5-(3-morpholinoprop-1-yn-1-yl)pyrazin-2-yl)oxy)-1H-indol-2- yl)(4-(4-(trifluoroethoxy)benzyl)piperazin-1-yl)methanone (RDG-1001)

[0044] (2,2,2-

[0045] trifluoroethoxy)benzyl)piperazin-1-yl)methanone (RDG-1001)

[0046] Compound 5 (200 mg, 0.33 mmol, 1.0 eq), compound 6 (45.56 mg, 0.36 mmol, 1.1 eq), Pd(PPh3)2Cl2(4.56 mg, 0.006 mmol, 0.02 eq), Cul (3.15 mg, 0.016 mmol, 0.05 eq) and TEA (167 mg, 1.65 mmol, 5.0 eq) were dissolved in 10 mL DMF, stirred at 80 °C for 48 hours, TLC monitored the reaction was completed. The reaction liquid was diluted with 300 mL ethyl acetate, washed with saturated brine three times (200 mL*3), the organic phase was dried and rotary evaporated, the crude product was purified by column (volume ratio, dichloromethane / methanol = 70 / 1~20 / 1) to obtain 182 mg of yellow solid compound RDG-1001, yield 85.0%.

[0047] 1H NMR (CDC13, 400 MHz) δ: 8.37 (d, J = 8.0 Hz, 1H), 8.18 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 8.0 Hz, 1H), 7.28 (d, J = 8.0 Hz, 3H), 7.16 (s, 1H), 6.93-6.91 (m, 2H), 6.60 (s, 1H), 4.38-4.32 (q, J = 16 Hz, J = 16 Hz, 2H), 3.80-3.75 (m, 8H), 3.56-3.49 (m, 4H), 2.65 (t, J = 4 Hz, 3H), 2.49 (m, 3H), 1.60 (m, 5H).

[0048] The synthesis of RDG-1001, RDG-1002, RDG-1003, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 can refer to the method of Example 1, only need to change the morpholinyl of compound 6 to the corresponding substituted alkynyl compound in the last step.

[0049] The synthesis of RDG-1004, RDG-1005 can refer to the method of Example 1, only need to change the compound 4 to the corresponding heterocyclic dibromide in the second step, and change the morpholinyl of compound 6 to the corresponding substituted alkynyl compound in the last step.

[0050] Example 2, RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 on breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cell proliferation inhibition effect

[0051] Collect MDA-MB-468, HepG2, BxPC-3, SGC7901, H460, KYSE450, Hela, MM.1S, OCI-LY3 cells in logarithmic growth phase respectively, count, adjust the concentration of cell suspension to 5 × 10 4The cells were cultured in a 96-well cell culture plate at a density of 1 x 104 / mL, and the volume of each well was 100 μL. DMSO was used as a solvent control, and the compounds RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 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 72 h of continuous culture, 10 / 20 μL of CCK-8 solvent was added to each well, and the mixture was incubated at 37°C for 3 h. The OD value at an absorption wavelength of 450 nm was measured by an enzyme marker, and the results were recorded. The dose of the compound was used as the horizontal coordinate, and the absorbance value was used as the vertical coordinate to draw a cell growth curve. The statistical results of the half-inhibitory rate (IC50 value) of the compounds on tumor cells are shown in Table 1.

[0052] Table 1. CCK-8 detection of the proliferation inhibition effect of RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 on breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma, and the like

[0053]

[0054]

[0055]

[0056] The table shows that RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, and RDG-1018 all exhibit good 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. Their antitumor activity is particularly strong in breast cancer, multiple myeloma, and diffuse large B-cell lymphoma cells. This application uses RDG-1001 as an example to conduct a preliminary study on the antitumor mechanism of this type of compound.

[0057] Example 3: Inhibitory effect of RDG-1001 on STAT3 phosphorylation and Cyclin D1 expression in MDA-MB-468 cells

[0058] I. Cell Culture and Drug Administration: a. Take MDA-MB-468 cells in logarithmic growth phase and adjust the density to 2×10⁻⁶. 5 indivual

[0059] a) Seed 2 mL of single-cell suspension per well into 6-well plates. b) Incubate overnight at 37°C. Add different concentrations (to final concentrations of 0, 10, 30, 60, and 100 nM) of RDG-1001. DMSO serves as a negative control, and 30 nM RDG-1001 [full Chinese name: 1-methyl-6-((5-((4-(trifluoromethyl)phenyl)ethynyl)pyrazin-2-yl)oxy)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methyl ketone] as a positive control. c) Continue culturing for 24 h, then lyse the cells with RIPA lysis buffer and collect proteins.

[0060] II. Cell collection and lysis: a. Discard the supernatant and wash the cells twice with pre-cooled PBS. Add 100 μL of pre-cooled RIPA cell lysis buffer (protease inhibitors and PMSF are added to the lysis buffer at a ratio of 1:100 in advance) to each well. b. Lysis on ice for 3 min, and scrape the cells with a cell scraper and collect them in a 1.5 mL EP tube; lysis on ice for 30 min, and vortex every 6 min. c. Centrifuge at 4°C, 12000 g for 10 min. d. Transfer the cell supernatant to a new EP tube. d. Divide the cell supernatant into two parts: take 5 μL and add to a 1.5 mL EP tube for BCA protein content determination, and add 45 μL of 1 x PBS and mix well for later use; take 80 μL of the remaining cell supernatant, add 20 μL of 5 x SDS loading buffer, mix well, and boil in boiling water for 10 min, and then centrifuge and load or store in a -20°C refrigerator.

[0061] e. Protein concentration determination steps: (1) BCA working solution preparation: according to the number of standard samples and samples to be determined, calculate the total amount of A and B mixed working solution required. Prepare the working solution at a ratio of BCA reagent A to B of 50:1, and vortex to mix well for later use.

[0062] (2) 1 x PBS dilution of protein standard:

[0063]

[0064]

[0065] (3) Take 25 μL of the protein standard solution and the sample supernatant diluted with PBS (10-fold dilution) and add to a new 96-well plate. Then add 200 μL of the BCA working solution prepared in advance and mix well. Remember not to blow to generate bubbles, tightly cover the 96-well plate, and react in a 37°C incubator for 30 min.

[0066] (4) Take out the 96-well plate and restore it to room temperature for 3-5 min, and measure the absorbance value of A562 on the enzyme marker instrument, and save the obtained values in an Excel table. Make 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 10% SDS-PAGE separation gel.

[0067] Prepare the separation gel according to the following table: 10 mL

[0068] Deionized water 4.0 mL 30% (m / v) Acrylamide 3.3 mL 1.5 M Tris-HCl (PH 8.8) buffer 2.5 mL 10% (m / v) SDS 0.1 mL 10% (m / v) APS 0.1 mL TEMED 4 μL Total 10 mL

[0069] (2) Put the mixed separating gel into two gel plates, add to 1.0 cm from the top, fill the gel plate with anhydrous ethanol, and stand for 30-45 min.

[0070] (3) After the separating gel is set, pour out the remaining anhydrous ethanol, and use filter paper to absorb the remaining anhydrous ethanol.

[0071] (4) Prepare 5% concentrated gel 5 mL according to the following table

[0072] Deionized water 2.77 mL 30% (m / v) Acrylamide 830 μL 0.5 M Tris-HCl (PH 6.8) buffer 1.26 mL 10% (m / v) SDS 50 μL 10% (m / v) APS 50 μL TEMED 5 μL Total 5 mL

[0073] (5) Slowly add the prepared concentrated gel into the gel plate to avoid air bubbles, insert the comb, and stand for 30-45 min.

[0074] (6) Take out the protein sample, heat in a 100°C water bath for 5 min, and centrifuge at 10,000 rpm for 5 min.

[0075] (7) Fix the gel plate in the electrophoresis tank, add SDS-PAGE electrophoresis buffer, pull out the comb, and add the treated protein sample into the sample tank in order, 50 μg of protein per well.

[0076] (8) Electrophorese at 80 V for 40 min.

[0077] (9) Change the voltage to 120 V and electrophorese for about 1.5 h until the bromophenol blue runs out of the gel.

[0078] Four, Western-blot: (1) Put the electrophoresed SDS-PAGE gel into TBST buffer for once, and soak the protein gel in the transfer buffer. (2) Soak a layer of cotton pad in the membrane transfer buffer, clamp it on the membrane transfer instrument with tweezers, and place them in order according to the blackboard, cotton pad, filter paper, protein gel, PVDF membrane, filter paper, cotton pad, and whiteboard, clamp tightly, and place in the membrane transfer instrument. If there are air bubbles between each layer, use a glass tube to gently roll to remove the air bubbles. (3) Open the membrane transfer instrument, transfer at 300 mA constant current for 80 min. (4) Put the membrane into TBST buffer, and rinse for 3 times, 8 min each time. (5) Use 5% BSA-TBST blocking solution 20 mL, and block at room temperature for 2 h. (6) Add the primary antibody, and incubate overnight at 4°C and 60 rpm. (7) Wash the membrane with TBST three times at room temperature and 60 rpm, 10 min each time. (8) Add the secondary antibody, and incubate at room temperature for 1 h. (9) Wash the membrane with TBST three times at room temperature and 60 rpm, 10 min each time. (10) Take 1 mL of chemiluminescence substrate solution A and solution B, and develop color at room temperature for 2 min. (11) Use filter paper to absorb the liquid on the membrane, and expose the film.

[0079] Five, reagent preparation:

[0080] (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.

[0081] (2) 10% Ammonium persulfate (AP): Weigh 1 g of ammonium persulfate and add about 10 mL of deionized water. Stir to dissolve and store at 4°C.

[0082] (3) 5x electrophoresis buffer: Weigh Tris 15.1 g, glycine 94 g, and SDS 5.0 g into a beaker, add 1 L of double distilled water to dissolve, and store at room temperature. Dilute 5 times when used.

[0083] (4) Transfer buffer: Weigh Tris 5.8 g, glycine 11.6 g, and SDS 0.75 g into a beaker, add 700 mL of double distilled water, dissolve, and dilute to 800 mL. Finally, add 200 mL of methanol.

[0084] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g of Tris base in 80 mL of water, adjust to pH 8.8 with 4N HCl, and dilute to 100 mL.

[0085] (6) 0.5 mol / L Tris-HCl, 1000 mL: Weigh 60.5 g of Tris base, add water to 850 mL, and add concentrated hydrochloric acid while stirring until completely dissolved. Adjust the pH to 6.8 and add water to 1 L.

[0086] (7) TBS buffer: Weigh NaCl 8.8 g into 800 mL of distilled water, dissolve, add 10 mL of 1 mol / L Tris-HCl (pH 7.5), dilute to 1 L, and store at room temperature.

[0087] (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 fresh each time.

[0088] (9) Blocking solution, antibody dilution: Add 5% skim milk or BSA to TBST buffer. Prepare fresh each time.

[0089] The detection results are shown in detail in Figure 1 As shown in the results of Figure 1 , treatment with 30 nM, 60 nM, and 100 nM of RDG-1001 can effectively down-regulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727), and CyclinD1, a downstream target protein of STAT3.

[0090] In summary, RDG-1001, RDG-1002, RDG-1003, RDG-1004, RDG-1005, RDG-1006, RDG-1007, RDG-1008, RDG-1009, RDG-1010, RDG-1011, RDG-1012, RDG-1013, RDG-1014, RDG-1015, RDG-1016, RDG-1017, RDG-1018 can significantly inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and other cells, and can effectively down-regulate the expression levels of p-STAT3(Y705), p-STAT3(S727) and STAT3 downstream target protein CyclinD1. Therefore, such drugs have good anticancer effect and development potential.

[0091] According to the general approach of drug development (first, conventional anti-tumor in vitro screening, then targeted research), the compounds of the present application can be applied to cancer treatment drugs related to abnormal cell proliferation, and anti-tumor drugs can be prepared by mixing with human body acceptable salt or with pharmaceutical carriers.

[0092] Finally, it should be noted that the above examples are only for illustration and not to limit the technical solutions of the present application, any equivalent replacement and modification or partial replacement of the present application without departing from the spirit and scope of the present application should be covered within the scope of protection of the claims of the present application.

Claims

1. A 1-methyl-6-oxoindole-2-carboxamide derivative, characterized in that, The structural formula is shown in general formula I: G = C or N; W = C or N.

2. The biologically acceptable salt of the 1-methyl-6-oxylindole-2-carboxamide derivative of 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. A process for the preparation of the 1-methyl-6-oxoindole-2-carboxamide derivatives according to claim 1, characterized in that, The synthetic route is shown as follows: The specific synthesis steps are as follows: (1) Compound 1, compound 2, HBTU and DIEA are dissolved in DMF, and after the reaction is completed at room temperature, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered and dried to obtain compound 3; (2) Compound 3, compound 4 and potassium carbonate are dissolved in DMF, and after the reaction is completed at 70-90℃, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered and dried to obtain compound 5; (3) Compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine are dissolved in DMF, and after the reaction is completed at 70-90℃, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary dried, and then the solid is washed with ethyl acetate, filtered and dried to obtain the compound shown in formula I.

4. The process for the preparation of 1-methyl-6-oxoindole-2-carboxamide derivatives 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 and potassium carbonate is 1:(1-1.5):(1-1.5); in step (3), the molar ratio of compound 5, compound 6, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.03):(0.01-0.09):

5.

5. The use of the 1-methyl-6-oxylindole-2-carboxamide derivative of claim 1 or the biologically acceptable salt of the 1-methyl-6-oxylindole-2-carboxamide derivative of claim 2 in the preparation of a STAT3 protein inhibitor.

6. Use of the 1-methyl-6-oxoindole-2-carboxamide derivative of claim 1 or the biologically acceptable salt of the 1-methyl-6-oxoindole-2-carboxamide derivative of claim 2 for the production of an antitumor medicament, characterized in that: The antitumor 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.

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