A piperazine-naphthylcarboxamide compound, its preparation method and application

Abstract

This invention discloses a piperazine-naphthylcarboxamide compound, its preparation method, and its applications, belonging to the field of pharmaceutical synthesis. The structural formula of the piperazine-naphthylcarboxamide compound is shown in general formula I: wherein R1 is selected from NH2. Further chemical modification of the biologically active piperazine-naphthylcarboxamide core group yields many compounds with even higher biological activity, expanding the wide application of this type of compound in biomedicine and its prospects for drug formulation development. This type of compound 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 low doses (nanomolar), and can effectively inhibit STAT3 protein phosphorylation, indicating that this type of compound has the potential to be developed into an anti-tumor drug.

Description

Technical Field

[0001] This invention belongs to the field of drug synthesis, specifically relating to a piperazine naphthamide compound, its preparation method, and its application. Background Technology

[0002] While significant progress has been made in clinical cancer treatment, the heterogeneity and continuous evolution of tumors, coupled with low response rates and the tendency for drug resistance to develop, have resulted in a severe shortage of effective treatments. Conventional cytotoxic chemotherapy drugs such as paclitaxel, cisplatin, carboplatin, and capecitabine offer advantages in terms of broad-spectrum anticancer activity and efficacy, but they also suffer from significant side effects and are prone to early drug resistance. Therefore, actively exploring and developing new anticancer drugs is of great clinical significance.

[0003] This application synthesizes a class of piperazine-naphthylcarboxamide compounds with novel structural formulas. Through biological analysis, it was found that these compounds, at extremely low doses, 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 effectively inhibit STAT3 protein phosphorylation. Therefore, further development of these compounds will have significant implications for applications in tumor therapy. Summary of the Invention

[0004] The purpose of this invention is to provide a piperazine naphthamide compound, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A piperazine-naphthylcarboxamide compound, with the structural formula shown in general formula I:

[0007]

[0008] Among them, R1 is selected from NH2,

[0009] That is, the above-mentioned piperazine naphthamide compounds are specifically compounds with the following structures:

[0010]

[0011] The above-mentioned piperazine naphthoamide compounds form biologically acceptable salts with at least one of acetic acid, dihydrofolate, 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.

[0012] The synthetic route for preparing the above-mentioned piperazine naphthoamide compounds is shown below:

[0013]

[0014] The specific synthesis steps are as follows:

[0015] (1) Compound 1, compound 2, HBTU and DIEA were dissolved in DMF and stirred at room temperature until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation, slurryed with ethyl acetate, filtered, and the solid was collected to obtain compound 3.

[0016] (2) Compound 3, compound 4 and potassium carbonate were dissolved in DMF. After the reaction was completed by stirring at 70-90°C, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation, slurryed with ethyl acetate, filtered, and the solid was collected to obtain compound 5.

[0017] (3) Dissolve compound R1H and compound 5 in ethanol, stir at 80-90℃ until the reaction is complete, then evaporate directly to dryness and pass through column chromatography to obtain the target compound.

[0018] 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 and R1H is 1:(1~1.5).

[0019] The above-mentioned piperazine naphthamide compounds and their biologically acceptable salts are used in the preparation of STAT3 protein inhibitors.

[0020] The above-mentioned piperazine naphthamide compounds and their biologically acceptable salts are used in the preparation of antitumor drugs.

[0021] Preferably, the antitumor drug refers to a drug for treating breast cancer, liver cancer, pancreatic cancer, stomach cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, diffuse large B-cell lymphoma, etc.

[0022] Specifically, this invention synthesizes a class of piperazine-naphthylcarboxamide compounds with novel structures, namely RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD2024JH012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019. The inhibitory effects of these compounds on the proliferation of various cancer cells were detected by the CCK-8 assay; they also effectively inhibited STAT3 protein phosphorylation.

[0023] The results show that compounds RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD2024JH012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 of the present invention can effectively inhibit the proliferation of breast cancer, liver cancer, pancreatic cancer, and gastric cancer cells, and can effectively inhibit STAT3 protein phosphorylation.

[0024] In summary, this invention provides a novel piperazine naphthamide compound and its derivatives for use in tumor treatment and their potential molecular mechanisms. Attached Figure Description

[0025] Figure 1 The results show the effects of different concentrations of RD2024JH003 on protein expression in MDA-MB-468 cells;

[0026] Figure 2 The results are the analysis of the interaction between RD2024JH003 and STAT3 protein molecules and key amino acids. Detailed Implementation

[0027] To make the technical objectives, technical solutions, and beneficial effects of the present invention clearer, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0028] In the method for synthesizing compound I of this invention, the various raw materials used in the reaction can be prepared by those skilled in the art based on existing knowledge, or can be obtained by methods known in the literature, or can be commercially available. The intermediates, raw materials, reagents, reaction conditions, etc., used in the above reaction scheme can be appropriately modified based on the existing knowledge of those skilled in the art.

[0029] In this invention, unless otherwise stated, (i) the temperature is expressed in degrees Celsius (°C), and the operation is carried out at room temperature; more specifically, room temperature refers to 20-30°C; (ii) the organic solvent is dried using a common drying method, and the solvent is evaporated using a rotary evaporator under reduced pressure, with a bath temperature not exceeding 50°C; the developing solvent and eluent are both in volume ratio; (iii) the reaction process is monitored by thin-layer chromatography (TLC); and (iv) the final product has satisfactory proton nuclear magnetic resonance (1H-NMR).

[0030] Example 1: The synthesis of all compounds follows the route below.

[0031] The specific synthesis method, taking compound RD2024JH003 as an example, has the following structural formula:

[0032]

[0033] The compound RD2024JH003 is named (4-(2-hydroxy-3-(isopropylamino)propoxy)naphthalen-1-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone.

[0034] Its synthetic route is as follows:

[0035]

[0036] Step 1. (4-hydroxynaphthalen-1-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 3)

[0037] Compound 1 (2.0 g, 10.63 mmol, 1.0 eq), compound 2 (2.92 g, 10.63 mmol, 1.0 eq), HBTU (4.85 g, 12.57 mmol, 1.2 eq), and DIEA (4.15 g, 31.38 mmol, 3.0 eq) were dissolved in 30 mL of DMF. The mixture was stirred at room temperature for 3 hours, and the reaction was monitored by TLC until 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 evaporated to dryness. The solution was purified by slurrying with 15 mL of ethyl acetate and filtered. The solid was collected and dried in an oven at 80 °C to give 4.20 g of white solid compound 3, with a yield of 88.9%.

[0038] 1 H NMR (CDCl3, 300MHz) δ: 9.02 (s, 1H), 8.25 (d, J = 8Hz, 1H), 7.78 (d, J = 8Hz, 1H), 7.53 (m, 2H), 7.22 (d, J = 8Hz, 1H), 7.18 (d, J =8Hz,2H),6.86(d,J=8Hz,2H),6.77(d,J=8Hz,1H),4.32(m,2H),4.20-4.13(m,3H),3.95-3.88(m,2H),3.04-2.96(m,5H)

[0039] Step 2. (4-(oxiran-2-ylmethoxy)naphthalen-1-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone (Compound 5)

[0040] Compound 3 (1.0 g, 2.25 mmol, 1.0 eq), compound 4 (0.23 g, 2.47 mmol, 1.1 eq), and potassium carbonate (373 mg, 2.70 mmol, 1.2 eq) were dissolved in 30 mL of DMF. The mixture was stirred at 80 °C for 12 hours. TLC monitoring showed that the starting materials had reacted completely and new spots had formed. 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 evaporated to dryness. The solution was purified by slurrying with 25 mL of ethyl acetate and filtered. The solid was collected and dried in an oven at 80 °C to give 730 mg of white solid compound 5, with a yield of 64.6%.

[0041] 1H NMR (CDCl3, 300MHz) δ: 8.26 (d, J = 8Hz, 1H), 7.81 (d, J = 8Hz, 1H), 7.57 (m, 2H), 7.25 (d, J = 8Hz, 1H), 7.23 (d, J = 8Hz, 2H), 6.89 (d, J =8Hz,2H),6.68(d,J=8Hz,1H),4.28(m,3H),4.21-4.13(m,3H),3.90-3.88(m,2H),3.48(s,2H),3.01-2.93(m,5H),2.52(m,2H)

[0042] Step 3.4-(2-hydroxy-3-(isopropylamino)propoxy)naphthalen-1-yl)(4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)methanone(RD2024JH003)

[0043] Compound 5 (300 mg, 0.6 mmol, 1.0 eq) and isopropylamine (42.5 mg, 0.72 mmol, 1.2 eq) were dissolved in 20 mL of anhydrous ethanol and reacted at 80 °C for 2 hours. TLC monitoring showed that the starting material had reacted completely and new spots had appeared. The reaction solution was evaporated to dryness, stirred with silica gel, and passed through a column (DCM / MeOH = 1 / 0 to 30 / 1 eluent). The eluent containing the target compound was collected, evaporated to dryness, and 120 mg of RD2024JH003, a white solid, was obtained, with a yield of 35.8%.

[0044] 1 H NMR (CDCl3, 300MHz) δ: 8.27 (d, J = 8Hz, 1H), 7.80 (d, J = 8Hz, 1H), 7.55 (m, 2H), 7.24 (d, J = 8Hz, 1H), 7.20 (d, J = 8Hz, 2H), 6.88 (d, J = 8Hz, 2H), 6.7 9(d,J=8Hz,1H),4.34(m,3H),4.20-4.10(m,2H),3.96-3.90(m,4H),3. 47(s,2H),3.06-2.99(m,5H),2.55(m,2H),2.26(brs,2H),1.24(m,6H).

[0045] The synthesis of RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD2024JH012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 can refer to the method in Example 1, except that the raw material isopropylamine is replaced with the corresponding amine in the last step.

[0046] Example 2, RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD202 Inhibitory effects of 4JH012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 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.

[0047] MDA-MB-468, HepG2, BxPC-3, SGC7901, H460, KYSE450, HeLa, MM.1S, and OCI-LY3 cells in logarithmic growth phase were collected, counted, and the cell suspension concentration was adjusted to 5 × 10⁻⁶. 4The concentration of cells / mL was increased to 100 μL per well in a 96-well cell culture plate. Using DMSO as a solvent control, the compounds RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, and RD2024JH009 described in this invention were added. Compounds H012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 were diluted with DMSO and added to the culture wells to achieve final concentrations of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10 μmol / L, respectively. After culturing for 72 h, 10 / 20 μL of CCK-8 solvent was added to each well, and the cells were incubated at 37°C for 3 h. The OD values ​​at an absorption wavelength of 450 nm were measured using a microplate reader, and the results were recorded. A cell growth curve was plotted with the compound dosage on the x-axis and the absorbance value on the y-axis. The statistical results of the half-maximal inhibitory concentration (IC50) of the compounds against tumor cells are shown in Table 1 below.

[0048] Table 1. CCK-8 detects RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD 2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD Inhibitory effects of RD2024JH012, RD2024JH013, RD2024JH014, RD2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 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.

[0049]

[0050]

[0051]

[0052]

[0053] The table shows: RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD2024JH012, RD2024JH013, RD2024JH014, RD2024JH Compounds 015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 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. In particular, their antitumor activity is stronger in breast cancer, multiple myeloma, and diffuse large B-cell lymphoma cells. This application uses RD2024JH003 as an example to conduct a preliminary study on the antitumor mechanism of these compounds.

[0054] Example 3: Inhibitory effect of RD2024JH003 on STAT3 phosphorylation and C-myc expression in MDA-MB-468 cells

[0055] 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 Single-cell suspensions of 2 mL / well were seeded into 6-well plates. The plates were incubated overnight at 37°C with 5% CO2. Different concentrations (0, 0.3, 1, 3, 10 μM) of RD2024JH003 and 0.3 μM RDf001 were added as positive controls. After culturing for another 24 h, cells were lysed using RIPA lysis buffer and proteins were collected.

[0056] II. Cell Collection and Lysis: a. Discard the upper culture medium and wash the cells twice with pre-cooled PBS. Add 100 μL of pre-cooled RIPA cell lysis buffer (protease inhibitor and PMSF are added to the lysis buffer at a ratio of 1:100 and mixed well in advance) to each well.

[0057] b. Lyse on ice for 3 min, scrape cells off with a cell scraper and collect in a 1.5 mL EP tube; lyse on ice for 30 min, vortexing every 6 min. c. Centrifuge at 12000g for 10 min at 4℃. d. Transfer the cell supernatant to a new EP tube. Divide the cell supernatant into two parts: take 5 μL and add it to a 1.5 mL EP tube for BCA protein content measurement, then add 45 μL of 1×PBS and mix well for later use; for the remaining cell supernatant quantification, take 80 μL of each part, add 20 μL of 5×SDS Loading Buffer, mix well, boil in boiling water for 10 min, centrifuge, and then load or store at -20℃.

[0058] e. Protein concentration determination steps: (1) Preparation of BCA working solution: Calculate the total amount of A and B mixed working solution required based on the number of standards and samples to be tested. Prepare the working solution according to the volume ratio of BCA reagent A to B of 50:1, and vortex to mix well for later use.

[0059] (2) Dilute protein standard with 1×PBS:

[0060]

[0061]

[0062] (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 pre-prepared BCA working solution to each well and mix thoroughly. Remember not to blow air to generate bubbles. Tightly close the 96-well plate and incubate at 37°C for 30 min.

[0063] (4) Remove the 96-well plate and allow it to return to room temperature for 3-5 minutes. Measure the absorbance of A562 using a microplate reader and save the obtained values ​​in an Excel spreadsheet. Plot a standard curve and calculate the protein content per 1 μL of each sample for protein loading.

[0064] III. SDS-PAGE: (1) Fix the adhesive plate and prepare 10% SDS-PAGE separation adhesive.

[0065] Prepare the separating gel according to the table below: 10 mL

[0066] Deionized water 4.0mL 30% (m / v) Acrylamide 3.3mL 1.5M Tris-HCl (pH 8.8) buffer 2.5mL 10% (m / v) SDS 0.1mL 10% (m / v) APS 0.1mL TEMED 4μL Total 10mL

[0067] (2) Add the mixed separating gel to two gel plates respectively, up to 1.0 cm from the top, fill the gel plates with anhydrous ethanol, and let stand for 30-45 minutes.

[0068] (3) After the gel is separated, pour out the remaining anhydrous ethanol and use filter paper to absorb the remaining anhydrous ethanol.

[0069] (4) Prepare 5 mL of 5% concentrated gel according to the table below.

[0070]

[0071]

[0072] (5) Slowly add the prepared concentrated glue into the glue plate to avoid air bubbles, insert the comb, and let it stand for 30 to 45 minutes.

[0073] (6) Take out the protein sample, heat it in a water bath at 100℃ for 5 minutes, and centrifuge it at 10000 rpm for 5 minutes.

[0074] (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the comb, and add the processed protein samples into the sample well in sequence, 50 μg of protein per well.

[0075] (8) Electrophoresis at 80V for 40 minutes.

[0076] (9) Change the voltage to 120V for electrophoresis for about 1.5 hours until bromophenol blue runs out of the colloid;

[0077] IV. Western blot: (1) After electrophoresis, rinse the SDS-PAGE gel once in TBST buffer and soak the protein gel in transfer buffer. (2) Moisten 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 tightly, and place it into the transfer apparatus. If there are air bubbles between each layer, gently roll them out with a glass tube. (3) Turn on the transfer apparatus and transfer at a constant current of 300mA for 80min. (4) Place the membrane in TBST buffer and rinse 3 times, 8min each time. (5) Block with 20mL of 5% BSA-TBST blocking solution at room temperature for 2h. (6) Add primary antibody and incubate overnight at 4℃ and 60rpm. (7) Wash the membrane three times with TBST at room temperature and 60rpm for 10min each time. (8) Add secondary antibody and incubate at room temperature for 1h. (9) Wash the membrane three times with TBST at room temperature and shake at 60 rpm for 10 min each time. (10) Take 1 mL each of chemiluminescent substrate solution A and solution B and develop the color at room temperature for 2 min. (11) Use filter paper to absorb the liquid on the membrane and expose it on the machine.

[0078] V. Reagent Preparation:

[0079] (1) 10% SDS: Weigh 1g of high-purity (electrophoresis grade) SDS into a 10mL centrifuge tube, add about 8mL of deionized water, heat to dissolve, and make up to 10mL. Store at room temperature.

[0080] (2) 10% Ammonium persulfate (AP): Weigh 1g of ammonium persulfate, add about 10mL of deionized water and stir to dissolve, then store at 4℃.

[0081] (3) 5× Electrophoresis buffer: Weigh 15.1g Tris, 94g Glycine and 5.0g SDS into a beaker, add 1L of double-distilled water to dissolve, store at room temperature, and dilute 5 times before use.

[0082] (4) Transfer buffer: Weigh 5.8g of Tris, 11.6g of glycine and 0.75g of SDS into a beaker, add 700mL of double-distilled water, dissolve and bring the volume up to 800mL, and finally add 200mL of methanol.

[0083] (5) 1.5 mol / L Tris-HCl, 100 mL: Dissolve 18.15 g of Tris base in 80 mL of water, adjust the pH to 8.8 with 4 N HCl, and make up to 100 mL.

[0084] (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.

[0085] (7) TBS buffer: Weigh 8.8g of NaCl into 800mL of distilled water, dissolve it, add 10mL of 1mol / L TrisHCl (pH 7.5), and bring the volume up to 1L. Store at room temperature.

[0086] (8) TBST buffer: Add 500 μL of 20% Tween20 to 1L of TBS buffer to make the final concentration of Tween20 0.1%. Prepare fresh before use.

[0087] (9) Blocking buffer, antibody dilution buffer: Add 5% skim milk powder or BSA to TBST buffer, prepare fresh before use.

[0088] like Figure 1 The results showed that treatment with RD2024JH003 at 10 μM, 3 μM, 1 μM, and 0.3 μM effectively downregulated the expression levels of p-STAT3 (Y705), p-STAT3 (S727), and the downstream target proteins of STAT3, C-MYC and CyclinD1.

[0089] The results in summary indicate that RD2024JH001, RD2024JH002, RD2024JH003, RD2024JH004, RD2024JH005, RD2024JH006, RD2024JH007, RD2024JH008, RD2024JH009, RD2024JH010, RD2024JH011, RD2024JH012, RD2024JH013, RD2024JH014, and RD Drugs 2024JH015, RD2024JH016, RD2024JH017, RD2024JH018, and RD2024JH019 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. They can also effectively downregulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727), and the downstream target proteins of STAT3, C-MYC and Cyclin D1. Therefore, these drugs have good anti-cancer effects and development potential.

[0090] Following the general approach to drug development (first conducting routine in vitro screening for antitumor drugs, and then conducting targeted research), the compounds of this invention can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be prepared as antitumor drugs by mixing with human-acceptable salts or with pharmaceutical carriers.

[0091] Example 4: Binding ability analysis of RD2024JH003 to STAT3 target protein

[0092] I. Documentation Preparation for Target Proteins and Ligand Molecules

[0093] The protein crystal structure was downloaded from the PDB database (http: / / www.rcsb.org / ), PDB number 6NJS. Water molecules were removed from the protein using UltraEdit software, redundant chains were deleted, and the original ligand molecules were extracted. The individual protein and ligand files were saved in .pdb format, resulting in the files 6njs.pdb and RD2024JH003.pdb.

[0094] II. Pretreatment of target proteins and ligand molecules.

[0095] Open AutoDockTools and perform operations such as water removal, hydrogen addition, charge calculation, and atom type addition on the 6njs.pdb file. Save the processed file as a .pdbqt file to obtain the "6njs.pdbqt" file.

[0096] The process involves adjusting the charge of the ligand molecule RD2024JH003.pdb file, determining the root of the ligand, and selecting the torsion bonds of the ligand. The processed file is then saved as a .pdbqt file, resulting in the file "RD2024JH003.pdbqt".

[0097] III. Create the docking information file.

[0098] Create a configuration file named 6njs.conf in the 6NJS folder. This file should contain the detailed parameters used for integration.

[0099] receptor=6njs.pdbqt

[0100] ligand=RD2024JH003.pdbqt

[0101] center_x = 13.24

[0102] center_y = 54.43

[0103] center_z = 0.27

[0104] size_x = 20.6

[0105] size_y = 31.1

[0106] size_z = 23.1

[0107] energy_range = 4

[0108] exhaustiveness = 12

[0109] num_modes = 9

[0110] IV. Perform the calculation.

[0111] Press Win+R to open the Run window, type "cmd" to open the command prompt window. The default folder is usually the C drive. Type "E:" and press Enter to change it to the E drive.

[0112] Next, type "CD E:\AutoDock\6NJS" and press Enter to enter the folder. Then type "vina--config6njs.conf" and press Enter to perform molecular docking calculations and obtain the file RD2024JH003_out.pdbqt.

[0113] V. Analyze the docking results.

[0114] Export the RD2024JH003_out.pdbqt file obtained in step four as the RD2024JH003_out.pdb file. This file is the output file of the docking calculation. Use other visualization software to view the docking results.

[0115] The obtained RD2024JH003_out.pdb file was imported into LigPlot software for intermolecular interaction and key amino acid analysis of RD2024JH003. Figure 2 The results showed that the SH2 domains of RD2024JH003 and STAT3 have strong affinity, specifically forming extensive hydrogen bond interactions with amino acids Arg609, Gln635, and Glu625.

[0116] Finally, it should be noted that the above embodiments are only for illustration and not for limiting the technical solutions of the present invention. Any equivalent substitutions, modifications or partial substitutions that do not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A piperazine-naphthoamide compound, characterized in that, The structural formula is shown in general formula I: Among them, R1 is selected from NH2, 2. The biologically acceptable salt formed by the piperazine naphthoamide compound of claim 1 with at least one of acetic acid, dihydrofolate, 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 piperazine-naphthoamide compound according to claim 1, characterized in that, The synthesis route is shown below: The specific synthesis steps are as follows: (1) Compound 1, compound 2, HBTU and DIEA were dissolved in DMF and stirred at room temperature until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation, slurryed with ethyl acetate, filtered, and the solid was collected to obtain compound 3. (2) Compound 3, compound 4 and potassium carbonate were dissolved in DMF. After the reaction was completed by stirring at 70-90°C, the reaction solution was diluted with ethyl acetate and washed with saturated brine. The organic phase was dried by rotary evaporation, slurryed with ethyl acetate, filtered, and the solid was collected to obtain compound 5. (3) Dissolve compound R1H and compound 5 in ethanol, stir at 80-90℃ until the reaction is complete, then evaporate directly to dryness and pass through column chromatography to obtain the target compound.

4. The method for preparing piperazine-naphthylcarboxamide compounds 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 and R1H is 1:(1~1.5).

5. The use of the piperazine naphthamide compounds of claim 1 or 2 and their biologically acceptable salts in the preparation of STAT3 protein inhibitors.

6. The use of the piperazine naphthamide compounds of claim 1 or 2 and their biologically acceptable salts in the preparation of antitumor drugs, characterized in that: The anti-tumor drugs mentioned refer to drugs used to treat breast cancer, liver cancer, pancreatic cancer, stomach cancer, lung cancer, esophageal cancer, cervical cancer, multiple myeloma, and diffuse large B-cell lymphoma.

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