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

Abstract

The application provides a 1-methyl-6-pyrazinyl indole-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-pyrazinyl indole-2-carboxamide derivative is shown in general formula I: wherein R1 is selected from the group containing the 1-methyl-6-pyrazinyl indole-2-carboxamide parent nucleus group with biological activity, further chemical modification of the 1-methyl-6-pyrazinyl indole-2-carboxamide parent nucleus group generates a plurality of compounds with higher biological activity, and the application expands the wide application of the compounds in biological medicine and the development prospect 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, diffuse large B-cell lymphoma and the like at a low dose (nanomole), can effectively inhibit STAT3 protein phosphorylation, and indicate that the compounds have the prospect 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-pyrazinylindole-2-carboxamide derivative, a preparation method and application thereof. BACKGROUND

[0002] Cancer has become an important cause of premature death and shortened life expectancy of the global population. In 2020, there were about 19.3 million cases of cancer diagnosed worldwide, and nearly 10 million people died of cancer. Experts predict that by 2035, the number of cancer patients worldwide will increase by half. Breast cancer has replaced lung cancer as the world's largest cancer. There are as many as 2.26 million new cases of breast cancer worldwide. In 2020, about 0.9057 million people worldwide were diagnosed with liver cancer, and about 0.8302 million people died. It is estimated that by 2040, the number of people diagnosed with and dying from primary liver cancer may increase by more than 55%. In addition, the incidence of gastric cancer and pancreatic cancer is also high, and the prognosis is still poor. At present, although there has been great progress in the treatment of cancer in clinical practice, due to the heterogeneity and continuous evolution of tumors, the response rate of most drugs is low, and drug resistance is easy to occur, resulting in a lack of effective treatment drugs. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin and capecitabine have the advantages of wide anti-cancer spectrum and good efficacy, but have large toxic side effects and are prone to early drug resistance. Therefore, it is of great clinical significance to actively explore and develop new anticancer drugs.

[0003] The present application synthesizes a class of 1-methyl-6-pyrazinylindole-2-carboxamide derivatives with a completely new structural formula. 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-pyrazinylindole-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-pyrazinylindole-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 1-methyl-6-pyrazinylindole-2-carboxamide derivative is specifically a compound having the following structure:

[0010]

[0011]

[0012] The 1-methyl-6-pyrazinylindole-2-carboxamide derivative described above forms a biologically acceptable salt with 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.

[0013] The method for preparing the 1-methyl-6-pyrazinylindole-2-carboxamide derivative described above has the following synthetic route:

[0014]

[0015] The specific synthesis steps are as follows:

[0016] (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 evaporated, slurried with ethyl acetate, filtered, and the solid is dried to obtain compound 3;

[0017] (2) Compound 3, compound 4, Pd(dppf)Cl2, and AcOK are dissolved in DMF, and after the reaction is completed at 80-100°C, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, and column chromatography is performed to obtain compound 5;

[0018] (3) Compound 5, compound 6, Pd(dppf)Cl2, and Na2CO3 are dissolved in DMSO and H2O, and after the reaction is completed at 80-90°C, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, and column chromatography is performed to obtain compound 7;

[0019] (4) Compound 7, compound 8, 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 evaporated, and slurried with ethyl acetate, filtered, and the solid is 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, Pd(dppf)Cl2 and AcOK is 1:2:(0.02-0.07):3; in step (3), the molar ratio of compound 5, compound 6, Pd(dppf)Cl2 and Na2CO3 is 1:(1-1.5):(0.02-0.07):(1-1.5); in step (4), the molar ratio of compound 7, compound 8, Pd(PPh3)2Cl2, CuI and triethylamine is 1:(1-1.5):(0.01-0.02):(0.01-0.03):5.

[0021] Use of the above-mentioned 1-methyl-6-pyrazinylindole-2-carboxamide compound and its biologically acceptable salt in the preparation of STAT3 protein inhibitor.

[0022] Use of the above-mentioned 1-methyl-6-pyrazinylindole-2-carboxamide derivative and its biologically acceptable salt in the preparation of antitumor drug.

[0023] Preferably, the antitumor drug refers to the drug for treating breast cancer, liver cancer, pancreatic cancer, gastric cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma and the like.

[0024] Specifically, the present application synthesizes a class of 1-methyl-6-pyrazinylindole-2-carboxamide derivatives RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 and the like with a completely new structure. The CCK-8 method is used to detect the proliferation inhibition effect of the compounds on various cancer cells; and the compounds can effectively inhibit STAT3 protein phosphorylation.

[0025] The results show that the compounds RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 of the application can effectively inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer and gastric cancer cells and effectively inhibit the phosphorylation of STAT3 protein.

[0026] In summary, the application provides a new 1-methyl-6-pyrazinylindole-2-carboxamide derivative and its use and potential molecular mechanism in tumor treatment. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 6 is the result of the effect of different concentrations of RDG-2001 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 more clear, the technical solutions of the application are further described below in combination with the drawings and specific examples.

[0029] In the method for synthesizing the compound of formula I in the application, various raw materials used in the reaction can be prepared by those skilled in the art according to existing knowledge, or can be prepared by a method known from the literature, or can be commercially available. The intermediates, raw 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 application, unless otherwise specified, (i) the temperature is expressed in degrees Celsius (℃), and the operation is carried out at room temperature; more specifically, the room temperature refers to 20-30℃; (ii) the organic solvent is dried by a commonly used drying method, and the evaporation of the solvent uses a rotary evaporator for evaporation under reduced pressure, and the bath temperature is not higher than 50℃; the developing agent and the eluent are both volume ratios; (iii) the reaction process is tracked by thin layer chromatography (TLC); (iv) the final product has a satisfactory proton nuclear magnetic resonance (1H-NMR).

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

[0032] The specific synthesis method is exemplified by the compound RDG-2001, and the structural formula is as follows:

[0033]

[0034] The name of compound RDG-2001 is (1-methyl-6-(5-(3-morpholinoprop-1-yn-1-yl)pyrazin-2-yl)-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone,

[0035] The synthetic route thereof is as follows:

[0036]

[0037] Step 1. (6-bromo-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, 10.21 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 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 and filtering with 15 mL ethyl acetate, and dried in an oven at 80°C to obtain 4.03 g of white solid compound 3 with a yield of 82.3%.

[0039] 1 H NMR (CDCI3, 300 MHz) δ: 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. (1-methyl-6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indol-2-yl)

[0041] (4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0042] Compound 3 (1.0 g, 1.96 mmol, 1.0 eq), compound 4 (0.995 g, 3.92 mmol, 2.0 eq), AcOK (0.58 g, 5.88 mmol, 3.0 eq) and Pd(dppf)Cl2(71 mg, 0.098 mmol, 0.05 eq) were dissolved in 30 mL DMF, the reaction was stirred at 100 °C for 3 hours, TLC monitoring reaction was completed. The reaction 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 column chromatography was performed with petroleum ether / ethyl acetate = 10 / 1 ~ 1 / 1 (volume ratio) to obtain 0.57 g of white solid compound 5 with a yield of 52.3%.

[0043] Step 3. (6-(5-bromopyrazin-2-yl)-1-methyl-1H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)

[0044] piperazin-1-yl)methanone (Compound 7)

[0045] Compound 5 (0.5 g, 0.90 mmol, 1.0 eq), compound 6 (0.235 g, 0.99 mmol, 1.1 eq), Na2CO3(0.142 g, 1.35 mmol, 1.5 eq) and Pd(dppf)Cl2(33 mg, 0.045 mmol, 0.05 eq) were dissolved in 20 mL DMSO and 5 mL water, the reaction was stirred at 80 °C for 3 hours, TLC monitoring reaction was completed. The reaction 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 column chromatography was performed with petroleum ether / ethyl acetate = 8 / 1 ~ 1 / 1 (volume ratio) to obtain 0.35 g of white solid compound 7 with a yield of 66.0%.

[0046] 1H NMR (CDC13, 300 MHz) δ: 8.604-8.601 (s, 1H), 8.494-8.491 (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).

[0047] Step 4. (1 -methyl-6-(5-(3-morpholinoprop-1 -yn-1 -yl)pyrazin-2-yl)-1 H-indol-2-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1 -yl)methanone (RDG-2001 )

[0048] Compound 7 (0.30 g, 0.50 mmol, 1.0 eq), compound 8 (0.11 g, 0.76 mmol, 1.5 eq), Pd(PPh3)Cl2(3.58 mg, 0.05 mmol, 0.01 eq), CuI (7.46 mg, 0.15 mmol, 0.03 eq) and triethylamine (850 mg, 2.50 mmol, 5.0 eq) were dissolved in 30 mL DMF, protected by nitrogen, stirred at 80 °C for 12 hours, TLC monitoring showed that the raw material reacted completely, and a new spot was generated. The reaction liquid was diluted with 200 mL ethyl acetate, washed with saturated brine three times (200 mL*3), the organic phase was dried and rotary evaporated, purified by 25 mL ethyl acetate and filtered, the solid was collected and dried in the oven at 80 °C to obtain 290 mg of yellow solid compound RDG-2001, the yield was 83.1 %.

[0049] 1H NMR (CDC13, 400 MHz) δ: 9.05 (d, J = 4.0 Hz, 1H), 8.74 (d, J = 4.0 Hz, 1H), 8.12 (s, 1H), 7.85-7.67 (m, 2H), 7.32-7.27 (m, 2H), 6.91 (J = 8.0 Hz, 2H), 6.61 (s, 1H), 4.38-4.32 (q, J = 16 Hz, J = 16 Hz, 2H), 3.92 (s, 2H), 3.80-3.78 (m, 6H), 3.62 (s, 2H), 3.52-3.49 (m, 2H), 2.71 (t, J = 4 Hz, 3H), 2.49 (m, 4H), 1.60 (m, 4H)

[0050] The synthesis of RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 can refer to the method of Example 1, only need to replace the morpholinyl in compound 8 with the corresponding substituted alkynyl compound in the last step.

[0051] Example 2, RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 on the proliferation inhibition effect 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

[0052] 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 x 10 4The cells were cultured in 96-well cell culture plates 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-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 described in the present application were diluted with DMSO and added to the culture wells, so that the final concentration of the compounds in the system was 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 incubation was carried out 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 the cell growth curve. The statistical results of the half-inhibitory rate (IC50 value) of the compounds on tumor cells are shown in Table 1 below:

[0053] Table 1. CCK-8 detection of the proliferation inhibition effect of RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 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

[0054]

[0055]

[0056]

[0057]

[0058] The table shows: RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 have good proliferation inhibition effect 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, diffuse large B-cell lymphoma cell tumor inhibition activity is stronger, the present application takes RDG-2001 as an example to preliminarily study the antitumor mechanism of this kind of compound.

[0059] Example 3: Inhibition of STAT3 phosphorylation and C-MYC, CyclinD1 expression in MDA-MB-468 cells by RDG-2001

[0060] I. Cell culture and drug administration: a. Take the logarithmic growth period of MDA-MB-468 cells, adjust the density to 2x10 5 6-well plates. b. Incubate in a 37℃ incubator overnight, add different concentrations (final concentration is 0, 10, 30, 100, 300nM) of RDG-2001, DMSO as negative control, 30nM RDf001

Chinese full 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) methanone

[0061] II. Cell collection and lysis: a. Discard the supernatant, 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, scrape the cells with a cell scraper and collect them in a 1.5 mL EP tube; lysis on ice for 30 min, vortex every 6 min. c. 4°C, 12000 g centrifugation for 10 min. d. Transfer the cell supernatant to a new EP tube. 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, then centrifuge and load or store in a -20°C refrigerator.

[0062] 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, vortex to mix well, and reserve for later use.

[0063] (2) 1 x PBS dilution of protein standard:

[0064]

[0065]

[0066] (3) Take 25 μL of the protein standard solution and the sample supernatant diluted with PBS (10-fold dilution) and add them to a new 96-well plate. Then add 200 μL of the prepared BCA working solution 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.

[0067] (4) Take out the 96-well plate and restore it to room temperature for 3-5 min, measure the A562 absorbance value on the enzyme marker, and save the obtained value 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.

[0068] Prepare the separation gel according to the following table: 10 mL

[0069] 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

[0070] (2) Add the mixed separation gel to two gel plates, add to a position 1.0 cm from the top, fill the gel plate with anhydrous ethanol, and stand for 30-45 min.

[0071] (3) After the gelation, pour out the remaining absolute ethanol and use filter paper to absorb the remaining absolute ethanol.

[0072] (4) Prepare 5% concentrated gel 5 mL according to the following table

[0073] 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

[0074] (5) Slowly add the prepared concentrated gel into the gel plate to avoid air bubbles, insert the comb, and stand for 30-45 min.

[0075] (6) Take out the protein sample, heat in a 100°C water bath for 5 min, and centrifuge at 10000 rpm for 5 min.

[0076] (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.

[0077] (8) Electrophorese at 80 V for 40 min.

[0078] (9) Change the voltage to 120 V and electrophorese for about 1.5 h until the bromophenol blue runs out of the gel.

[0079] Four, Western-blot: (1) Place the electrophoresed SDS-PAGE gel in 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 them out. (3) Open the membrane transfer instrument, transfer at 300 mA constant current for 80 min. (4) Place the membrane in 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, incubate overnight at 4°C and 60 rpm. (7) Wash the membrane with TBST three times at room temperature and 60 rpm for 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 for 10 min each time. (10) Take 1 mL of chemiluminescent 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.

[0080] Five, reagent preparation:

[0081] (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.

[0082] (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.

[0083] (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.

[0084] (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.

[0085] (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.

[0086] (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.

[0087] (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.

[0088] (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.

[0089] (9) Blocking solution, antibody dilution: Add 5% skim milk or BSA to TBST buffer. Prepare fresh each time.

[0090] The test results are shown in detail in Figure 1 As shown in the results of Figure 1 , treatment with 10 nM, 30 nM, 100 nM, and 300 nM of RDG-2001 can effectively down-regulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727), and the downstream target protein C-MYC, CyclinD1 of STAT3.

[0091] In summary, RDG-2001, RDG-2002, RDG-2003, RDG-2004, RDG-2005, RDG-2006, RDG-2007, RDG-2008, RDG-2009, RDG-2010, RDG-2011, RDG-2012, RDG-2013, RDG-2014, RDG-2015, RDG-2016, RDG-2017, RDG-2018 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 proteins C-MYC, CyclinD1. Therefore, such drugs have good anti-cancer effect and development potential.

[0092] According to the general approach of drug development (first, conventional anti-tumor in vitro screening is carried out, and then targeted research is carried out), 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.

[0093] Finally, it should be noted that the above examples are only for illustration and do not 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-pyrazinylindole-2-carboxamide derivative, having a structure as shown in general formula I: ###0001### wherein R1is selected from R1is selected from 2. A biologically acceptable salt of the 1-methyl-6-pyrazinylindole-2-carboxamide derivative of claim 1 with 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-pyrazinylindole-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 stirring at room temperature until the reaction is complete, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, slurried with ethyl acetate, filtered, and the solid is dried to obtain compound 3; (2) Compound 3, compound 4, Pd(dppf)Cl2 and AcOK are dissolved in DMF, and after stirring at 80-100°C until the reaction is complete, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, and column chromatography is performed to obtain compound 5; (3) Compound 5, compound 6, Pd(dppf)Cl2 and Na2CO3 are dissolved in DMSO and H2O, and after stirring at 80-90°C until the reaction is complete, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, and column chromatography is performed to obtain compound 7; (4) Compound 7, compound 8, Pd(PPh3)2Cl2, CuI and triethylamine are dissolved in DMF, and after stirring at 70-90°C until the reaction is complete, the reaction solution is diluted with ethyl acetate, washed with saturated brine, the organic phase is rotary evaporated, and slurried with ethyl acetate, filtered, and the solid is dried to obtain the compound shown in formula I.

4. The process for the preparation of 1-methyl-6-pyrazinylindole-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, Pd(dppf)Cl2 and AcOK is 1:2:(0.02-0.07):3; in step (3), the molar ratio of compound 5, compound 6, Pd(dppf)Cl2 and Na2CO3 is 1:(1-1.5):(0.02-0.07):(1-1.5); in step (4), the molar ratio of compound 7, compound 8, 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-pyrazinylindole-2-carboxamide derivative of claim 1 or the biologically acceptable salt of the 1-methyl-6-pyrazinylindole-2-carboxamide derivative of claim 2 in the preparation of a STAT3 protein inhibitor.

6. Use of the 1-methyl-6-pyrazinylindole-2-carboxamide derivative of claim 1 or the biologically acceptable salt of the 1-methyl-6-pyrazinylindole-2-carboxamide derivative of claim 2 for the production of an antitumor agent, 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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