Aryl alkynyl compounds, methods of making and using the same

Abstract

The application discloses aryl alkyne compounds, a preparation method and application thereof, and a structure formula is shown in general formula I: wherein, A ring is selected from B ring is selected from The experimental results of immunoblotting show that the compounds can specifically inhibit the phosphorylation of STAT3 and the expression of target proteins CyclinD1 and BCL-XL and the like. The compounds can significantly inhibit the proliferation of various tumor cells such as gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma and multiple myeloma. Therefore, the compounds have the prospects of being developed into JAKs / STATs signal pathway inhibitors, targeted anti-tumor drugs and drugs for treating STATs signal related immunological diseases.

Description

Technical Field

[0001] This invention belongs to the field of tumor targeted therapy, specifically relating to a class of arylkyne compounds, their preparation methods, and applications. Background Technology

[0002] Signal transducers and activators of transcriptions (STATs) are a family of seven transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5, STAT6, and STAT7) that regulate the expression of proteins encoding key proteins throughout the proteome, thereby participating in the regulation of cellular metabolism, intercellular communication, immune responses, and the cell cycle. STAT activation is typically initiated by ligand binding to cell surface receptors, followed by kinase-dependent phosphorylation of tyrosine (pY) residues. This leads to STAT-STAT dimerization mediated by the Src2 homology (SH2) domain, which rapidly translocates to the nucleus, binds to DNA elements, and initiates transcription of downstream genes. Recent studies have shown that STATs not only transmit signals to control transcription but also regulate mitochondrial anabolic and catabolistic metabolism and can influence nuclear functional division and genome integrity. Previous research has reported that gain-of-function mutations (i.e., somatic or desired variations) in STAT proteins are fundamental to tumor cell carcinogenesis. Driver mutations in the STAT3 pathway exist in human lung cancer, breast cancer, prostate cancer, colorectal cancer, liver cancer, and hematological malignancies, leading to abnormally enhanced STAT3 activity, which is closely related to malignant tumor progression and poor survival prognosis. Targeting STAT3 is considered a promising cancer treatment strategy. Currently, only three JAKs inhibitors are approved internationally for the treatment of immune diseases, and the application of JAK / STATs signaling inhibitors in cancer is still in the research and development stage.

[0003] To develop STAT3-targeted antitumor drugs, we recently synthesized a class of arylkyne compounds with novel structures. Through biological analysis, we found that these compounds significantly inhibited STAT3 signaling phosphorylation and activation, downregulated the expression of key proteins related to cell cycle and apoptosis, and suppressed cell proliferation in various tumor cell lines, including gastric cancer, pancreatic cancer, and breast cancer, demonstrating extremely strong antitumor activity.

[0004] This invention aims to reveal the antitumor effects and potential pharmacological mechanisms of a class of arylalkynyl compounds and their derivatives, as well as the potential applications of such compounds in the clinical treatment of gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma. Summary of the Invention

[0005] The purpose of this invention is to provide a class of aryl alkynes, their preparation methods, and applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A class of arylalkynyl compounds, with the structural formula shown in general formula I:

[0008] Among them, ring A is selected from

[0009] Ring B is selected from

[0010] The aforementioned aryl alkynes are specifically compounds with the following structures:

[0011]

[0012]

[0013] Biologically acceptable salts formed by the above-mentioned aryl alkynes 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.

[0014] The synthetic route for preparing the above-mentioned arylaceyne compounds is as follows:

[0015]

[0016] The synthesis steps are as follows:

[0017] (1) Compound 1, compound 2, DIEA and HBTU were dissolved in DMF. After the reaction was completed by stirring at room temperature, the reaction solution was poured into water, and a precipitate was formed. The solid was filtered, collected and dried to obtain compound 3.

[0018] (2) Compound 3, compound 4, triethylamine, Pd(PPh3)2Cl2 and CuI were dissolved in DMF and stirred at 70-90℃ until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with brine. The organic phase was dried, stirred and passed through a column to obtain compound 5.

[0019] (3) Dissolve compound 5 in methanol, add ethyl acetate solution of hydrogen chloride, stir at room temperature until the reaction is complete, and directly evaporate the reaction solution to obtain compound 6.

[0020] (4) Dissolve compound 6 and potassium carbonate in DMF, add compound 7, stir at 40-60℃ until the reaction is complete, dilute the reaction solution with ethyl acetate, wash with saline, dry the organic phase, stir and pass the sample through a column to obtain the compound shown in formula I.

[0021] Preferably, in step (1), the molar ratio of compound 1, compound 2, DIEA and HBTU is 1:1:2:(1.0~1.5); in step (2), the molar ratio of compound 3, compound 4, triethylamine and Pd(PPh3)2Cl2 and CuI is 1:2:3:0.02:0.01; in step (3), the molar ratio of compound 5 and hydrogen chloride is 1:10; in step (4), the molar ratio of compound 6, potassium carbonate and compound 7 is 1:(2~4):(1.0~1.5).

[0022] The use of the above-mentioned aryl yne compounds and their biologically acceptable salts in the preparation of antitumor drugs, wherein the antitumor drugs are drugs for treating tumors associated with STAT3 signaling.

[0023] Preferably, the drug for treating tumors related to STAT3 signaling refers to a drug for treating gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma.

[0024] Another object of the present invention is to provide a class of small molecule compounds with targeted antitumor activity.

[0025] The tumor can specifically be a tumor with high STAT3 expression or constitutive activation, including but not limited to gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma.

[0026] Specifically, this invention synthesizes a class of arylalynyl compounds RDn001, RDn002, and RDn with novel structures.

[0027] Compounds 003, RDn004, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014, and RDn015 were used. The inhibitory effect of these compounds on tumor cell proliferation was detected by the MTT assay, and their inhibitory effect on STAT3 signaling was demonstrated by Western blotting.

[0028] The results show that the compounds RDn001, RDn002, RDn003, RDn004, RDn005, RDn006, and RDn of the present invention...

[0029] RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014 and RDn015 can effectively inhibit the proliferation of gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma and multiple myeloma cells.

[0030] In summary, this invention provides a novel arylaceyne compound and its derivatives for use in tumor treatment and their potential molecular mechanisms. Attached Figure Description

[0031] Figure 1 These are Western blot results of RDn001 (0, 10, 30 and 100 nM) after 24 h of treatment in different tumor cell lines. Detailed Implementation

[0032] 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.

[0033] 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.

[0034] In this invention, unless otherwise stated, wherein: (i) 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 in volume ratio; (iii) the reaction process is monitored by thin-layer chromatography (TLC); and (iv) the final product exhibits satisfactory proton nuclear magnetic resonance (NMR) values. 1 H-NMR).

[0035] Example 1: Synthesis of compound RDn004

[0036]

[0037] The compound RDn004 is named (4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)(6-((4-(trifluoromethyl)phenyl)ethynyl)pyridin-3-yl)methanone.

[0038] Its synthetic route is as follows:

[0039]

[0040] Step 1. tert-butyl 4-(6-bromonicotinoyl)piperazine-1-carboxylate (intermediate 3)

[0041] Starting material 1D (1 g, 4.95 mmol, 1.0 eq), starting material 2 (0.92 g, 4.95 mmol, 1.0 eq), DIEA (1.28 g, 9.90 mmol, 2.0 eq), and HBTU (2.26 g, 5.94 mmol, 1.2 eq) were dissolved in 20 mL of LDM and stirred at room temperature for 2 hours. TLC (PE / EA = 2 / 1, Rf / product = 0.25) showed that the starting materials had reacted completely and new spots were formed. The reaction solution was poured into 100 mL of water, and a precipitate formed. The precipitate was filtered, collected, and dried to give intermediate 3D (1.60 g, 87.4%), a white solid.

[0042] Step 2.tert-butyl 4-(6-((4-(trifluoromethyl)phenyl)ethynyl)nicotinoyl)piperazine-1-carboxylate (Intermediate 5)

[0043] Intermediate 3D (1 g, 2.70 mmol, 1.0 eq), starting material 4D (0.92 g, 5.40 mmol, 2.0 eq), triethylamine (0.82 g, 8.10 mmol, 3.0 eq), Pd(PPh3)2Cl2 (38 mg, 0.054 mmol, 0.02 eq) and CuI (20 mg, 0.108 mmol, 0.01 eq) were dissolved in 20 mL of LMF and stirred at 80 °C. The reaction was carried out for 12 hours; TLC (DCM / MeOH = 20 / 1, Rf / product = 0.35) showed that the starting material had reacted completely and new spots were generated; the reaction solution was diluted with 100 mL of ethyl acetate and washed three times with saturated saline (100 mL * 3). The organic phase was dried, stirred and passed through a column, and eluted with (DCM / MeOH = 100 / 1 ~ 20 / 1) to obtain intermediate 5D (620 mg, 50.2%), a grayish-white solid.

[0044] Step 3.1-(4-(2-(4-(((4-aminonaphthalen-1-yl)oxy)methyl)phenoxy)ethyl)piperazin-1-yl)-2-phenylethan-1-one (Intermediate 6)

[0045] Intermediate 5D (620 mg, 1.35 mmol, 1.0 eq) was dissolved in 20 mL of methanol, and ethyl hydrogen chloride solution (4 mol / L, 3.37 mL, 13.5 mmol, 10.0 eq) was added. The mixture was stirred at room temperature for 2 hours. TLC (DCM / MeOH = 20 / 1, Rf / product = 0.01) showed that the starting material had reacted completely and a new spot was formed. The reaction solution was directly evaporated to dryness to obtain intermediate 6D (484 mg, 99%), a white solid.

[0046] Step 4. (4-(4-(2,2,2-trifluoroethoxy)benzyl)piperazin-1-yl)(6-((4-(trifluoromethyl)phenyl)ethynyl)pyridin-3-yl)methanone(RDn004)

[0047] Intermediate 6D (400 mg, 1.11 mmol, 1.0 eq) and potassium carbonate (461 mg, 3.34 mmol, 3.0 eq) were dissolved in 20 mL of LDM, and then starting material 7 (300 mg, 1.34 mmol, 1.2 eq) was added. The mixture was stirred at 50 °C for 12 hours. TLC (DCM / MeOH = 20 / 1, Rf / product = 0.2) showed that the starting material had reacted completely and a new spot was formed. The reaction solution was diluted with 100 mL of ethyl acetate and washed three times (100 mL * 3) with saturated saline solution. The organic phase was dried, stirred, and passed through a column. The solution was eluted with (DCM / MeOH = 100 / 1 ~ 20 / 1) to obtain RDn004 (420 mg, 68.9%), a grayish-white solid.

[0048] 1 H NMR (CDCl3, 400MHz) δ: 8.16 (d, J = 8.7Hz, 2H), 7.69 (d, J = 8.6Hz, 1H), 7.32 (d, J = 8.0Hz, 2H), 7.24 (s, 1H), 7.20 (d, J = 8.8Hz, 2H), 6. 99(dd,J=8.5,1.8Hz,1H),6.94(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0049] The synthesis methods for RDn001, RDn002, RDn003, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014, and RDn015 are as described in Example 1.

[0050] RDn001, white solid, yield 75.3%. 1 H NMR (CDCl3, 400MHz) δ: 8.86 (d, J = 8.7Hz, 2H), 8.53 (d, J = 8.6Hz, 1H), 7.30 (d, J = 8.0Hz, 2H), 7.26 (s, 1H), 7.14 (d, J = 8.8Hz, 2H), 6. 80(dd,J=8.5,1.8Hz,1H),6.90(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0051] RDn002, a light yellow solid, with a yield of 65.1%. 1 H NMR(CDCl3,400MHz)δ:8.15(d,J=8.7Hz,2H),7.68(d,J=8.6Hz,1H),7.30(d,J=8.0Hz,2H),7.28-7.25(m,4H),7.24(s,1H),7 .20(d,J=8.8Hz,2H),6.92(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0052] RDn003, a white solid, with a yield of 55.7%. 1 H NMR (CDCl3, 400MHz) δ: 8.88 (d, J = 8.7Hz, 2H), 8.52 (d, J = 8.6Hz, 1H), 7.34 (d, J = 8.0Hz, 2H), 7.24 (s, 1H), 7.12 (d, J = 8.8Hz, 2H), 6. 82(dd,J=8.5,1.8Hz,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0053] RDn005, brown solid, yield 32.7%. 1¹H NMR (CDCl₃, 400MHz) δ: 8.18 (d, J = 8.7Hz, 2H), 7.68 (d, J = 8.6Hz, 1H), 7.30 (d, J = 8.0Hz, 2H), 7.26 (s, 1H), 7.10 (s, 1H), 6.96 (s, 1H), 6.90 (d, J = 8.4Hz, 2H), 4.37 (q, J = 8.1Hz, 2H), 3.87–3.77 (m, 7H), 3.56–3.54 (m, 2H), 2.53–2.50 (m, 4H). RDnO₀₆, white solid, yield 68.9%. 1 H NMR(CDCl3,400MHz)δ:8.88(d,J=8.7Hz,2H),8.52(d,J=8.6Hz,1H),7.34-7.24(m,3H),7.12(d,J=8.8Hz,2H),6.82(dd,J =8.5,1.8Hz,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0054] RDn007, a grayish-white solid, with a yield of 70.2%. 1 H NMR (CDCl3, 400MHz) δ: 8.86 (d, J = 8.7Hz, 2H), 8.53 (d, J = 8.6Hz, 1H), 7.30 (d, J = 8.0Hz, 2H), 7.26 (s, 1H), 7.14 (d, J = 8.8Hz, 2H), 6. 80(dd,J=8.5,1.8Hz,1H),6.90(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0055] RDn008, a grayish-white solid, with a yield of 55.6%. 1 H NMR(CDCl3,400MHz)δ:8.15(d,J=8.7Hz,2H),7.68(d,J=8.6Hz,1H),7.30(d,J=8.0Hz,2H),7.28-7.25(m,3H),7.24(s,1H),7 .20(d,J=8.8Hz,2H),6.92(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0056] RDn009, a grayish-white solid, with a yield of 45.8%. 1 H NMR(CDCl3,400MHz)δ:8.17(d,J=8.7Hz,2H),7.65(d,J=8.6Hz,1H),7.35(d,J=8.0Hz,2H),7.30-7.25(m,3H),7.24(s,1H),7 .20(d,J=8.8Hz,2H),6.92(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0057] RDn010, a white solid, with a yield of 45.8%. 1 H NMR(CDCl3,400MHz)δ:8.88(d,J=8.7Hz,2H),8.52(d,J=8.6Hz,1H),7.34(d,J=8.0Hz,2H),7.24(s,1H),7.12(d,J=8.8Hz,2H),6 .82(s,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),4.19(s,3H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0058] RDn011, white solid, yield 72.8%. 1 H NMR(CDCl3,400MHz)δ:8.88(d,J=8.7Hz,2H),8.52(d,J=8.6Hz,1H),7.34(d,J=8.0Hz,2H),7.24(s,1H),7.12(d,J=8.8Hz,2H),6 .82(s,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.85(s,6H),2.53-2.50(m,4H).

[0059] RDn012, a white solid, with a yield of 72.8%. 1H NMR (CDCl3, 400MHz) δ: 8.16 (d, J = 8.7Hz, 2H), 7.69 (d, J = 8.6Hz, 1H), 7.32 (d, J = 8.0Hz, 2H), 7.24 (s, 1H), 7.20 (d, J = 8.8Hz, 2H), 6. 99(dd,J=8.5,1.8Hz,1H),6.94(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0060] RDn013, white solid, yield 58.3%. 1 H NMR(CDCl3,400MHz)δ:8.88(d,J=8.7Hz,2H),8.52(d,J=8.6Hz,1H),7.34(d,J=8.0Hz,2H),7.24(s,1H),7.12(d,J=8.8Hz,2H),6 .82(s,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),4.19(s,3H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0061] RDn014, a light yellow solid, with a yield of 67.9%. 1 H NMR (CDCl3, 400MHz) δ: 8.88 (d, J = 8.7Hz, 2H), 8.52 (d, J = 8.6Hz, 1H), 7.34 (d, J = 8.0Hz, 2H), 7.24 (s, 1H), 7.12 (d, J = 8.8Hz, 2H), 6. 82(dd,J=8.5,1.8Hz,1H),6.66(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0062] RDn015, a light yellow solid, with a yield of 67.9%. 1H NMR(CDCl3,400MHz)δ:8.15(d,J=8.7Hz,2H),7.68(d,J=8.6Hz,1H),7.30(d,J=8.0Hz,2H),7.28-7.25(m,3H),7.24(s,1H),7.20(d ,J=8.8Hz,2H),6.92(d,J=8.4Hz,2H),4.37(q,J=8.1Hz,2H),4.18(s,3H),3.87-3.77(m,7H),3.56-3.54(m,2H),2.53-2.50(m,4H).

[0063] Example 2: Inhibitory effects of RDn001, RDn002, RDn003, RDn004, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014, and RDn015 on the proliferation of gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma cells.

[0064] Tumor cells in the logarithmic growth phase were collected separately, and the cell suspension concentration was adjusted to 5 × 10⁻⁶. 4 Cells / mL were added to 96-well cell culture plates, with a volume of 100 μL per well. Using DMSO as a solvent control, the novel aryl alkynes RDn001, RDn002, RDn003, RDn004, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014 and RDn015 described in this invention were diluted with DMSO and added to the culture wells to achieve final concentrations of 0.1, 0.3, 1, 3, 10, 30, 100 and 300 (μmol / L), respectively. After culturing for another 48 hours, 10 μL of MTT solvent (5 mg / mL) was added to each well, and the mixture was incubated at 37°C for 4 hours. The culture supernatant was discarded, and 150 μL of LDMSO was added to each well. The mixture was shaken for 10 minutes to decolorize, and the OD value at an absorption wavelength of 490 nm was measured using a microplate reader. The results were recorded, and 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 RDn001, RDn002, RDn003, RDn004, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014, and RDn015 against tumor cells are shown in Table 1.

[0065] Table 1: IC50 values ​​(nM) of RDn series compounds in different tumor cell lines

[0066]

[0067]

[0068] The results in Table 1 show that RDn001, RDn002, RDn003, RDn004, RDn005, RDn006, RDn007, RDn008, RDn009, RDn010, RDn011, RDn012, RDn013, RDn014 and RDn015 all have good inhibitory effects on the proliferation of tumor cells such as gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma and multiple myeloma. In particular, RDn001 has the strongest antitumor activity in lymphoma OCI-LY3.

[0069] Example 3: RDn001 can downregulate STAT3 phosphorylation and target protein expression in OCI-LY3 cells.

[0070] I. Cell Culture and Drug Administration: OCI-LY3 cells in logarithmic growth phase were harvested and adjusted to a density of 4 × 10⁻⁶ cells / year. 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, and then different concentrations (0, 10, 30, 100 nM) of RDn001 were added. After culturing for another 24 h, the cells were lysed using RIPA lysis buffer and proteins were collected.

[0071] II. Cell Collection and Lysis: a. Discard the supernatant and wash cells twice with pre-chilled PBS. Add 100 μL of pre-chilled RIPA cell lysis buffer (protease inhibitor and PMSF are added to the lysis buffer at a 1:100 ratio and mixed beforehand) to each well. b. Lyse on ice for 3 min, scrape cells off with a cell scraper, and collect them 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, 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℃.

[0072] 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 test samples. 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.

[0073] (2) Dilute protein standards with 1×PBS: (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 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. (4) Remove the 96-well plate and allow it to return to room temperature for 3-5 min. Measure the absorbance value of A562 on a microplate reader and save the obtained value in an Excel spreadsheet. Plot a standard curve and calculate the protein content of 1 μL of each sample for protein loading.

[0075] III. SDS-PAGE: (1) Fix the adhesive plate and prepare 10% SDS-PAGE separating adhesive.

[0076] Prepare the separating gel according to the table below: 10 mL

[0077] 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

[0078] (2) Add the mixed separating gel to two gel plates, filling them to 1.0 cm from the top. Fill the plates with anhydrous ethanol and let them stand for 30–45 minutes. (3) After the separating gel has solidified, pour off the remaining anhydrous ethanol and blot it clean with filter paper. (4) Prepare 5 mL of 5% stacking gel according to the table below.

[0079] 30% (m / v) Acrylamide 830μL 0.5M Tris-HCl (pH 6.8) buffer 1.26mL 10% (m / v) SDS 50μL 10% (m / v) APS 50μL TEMED 5μL Total 5mL

[0080] (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.

[0081] (6) Remove the protein sample, heat in a 100℃ water bath for 5 min, and centrifuge at 10000 rpm for 5 min. (7) Fix the gel plate into the electrophoresis tank, add SDS-PAGE electrophoresis buffer, remove the comb, and add the processed protein sample into the sample well in sequence, 50 μg of protein per well. (8) Electrophoresis at 80V for 40 min. (9) Change the voltage to 120V and electrophoresis for about 1.5 h until bromophenol blue runs off the colloid;

[0082] 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 for 10 min each time at room temperature and shake at 60 rpm. (10) Take 1 mL each of chemiluminescent substrate solution A and solution B, and develop the color at room temperature for 2 min. (11) Blot the liquid off the membrane with filter paper, expose the membrane on the microscope, and see the results for details. Figure 1 V. Reagent Preparation:

[0083] (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.

[0084] (2) 10% Ammonium persulfate (AP): Weigh 1g of ammonium persulfate, add about 10mL of deionized water and stir to dissolve, then store at 4℃.

[0085] (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.

[0086] (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.

[0087] (5) 1.5 mol / L Tris-HCl, 100 mL: dissolve 18.15 gtris in 80 mL of water, adjust to 8.8 with 4N HCl, and make up to 100 mL.

[0088] (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.

[0089] (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.

[0090] (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.

[0091] (9) Blocking buffer, antibody dilution buffer: Add 5% skim milk powder or BSA to TBST buffer, prepare fresh before use.

[0092] Figure 1 The results showed that treatment with RDn001 at 10 nM, 30 nM and 100 nM could effectively downregulate the expression levels of p-STAT3 (Y705), p-STAT3 (S727) and target proteins Cyclin D1 and BCL-XL.

[0093] The results in summary demonstrate that arylaceyne compounds, represented by RDn001, can significantly inhibit the growth of gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma cells, and also exhibit significant inhibitory effects on STAT3 and its related proteins, showing promising anticancer activity. Following the general drug development pathway (first conducting routine in vitro antitumor screening, followed by targeted research), the compounds of this invention can be applied to cancer treatments related to abnormal cell proliferation, and can be prepared as antitumor drugs by salt formation in human-acceptable environments or by mixing with pharmaceutical carriers.

[0094] 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 and modifications or partial substitutions made to the present invention 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 class of arylkyne compounds, characterized in that, The structural formula is shown in general formula I: ， I Among them, ring A is selected from , , , , , , , , , , , , , , , or ; Ring B is selected from , , , , , or .

2. The arylaceyne compound according to claim 1, characterized in that, Specifically, compounds with the following structures: ， 。 3. A biologically acceptable salt formed by the arylalkynyl compound of claim 1 or 2 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.

4. The method for preparing the arylaceyne compound according to claim 1 or 2, characterized in that, The synthesis route is as follows: ， The synthesis steps are as follows: (1) Compound 1, compound 2, DIEA and HBTU were dissolved in DMF and stirred at room temperature until the reaction was complete. The reaction solution was then poured into water, and a precipitate was formed. The solid was filtered, collected and dried to obtain compound 3. (2) Compound 3, compound 4, triethylamine, Pd(PPh3)2Cl2 and CuI were dissolved in DMF and stirred at 70-90℃ until the reaction was complete. The reaction solution was diluted with ethyl acetate and washed with brine. The organic phase was dried, stirred and passed through a column to obtain compound 5. (3) Dissolve compound 5 in methanol, add ethyl acetate solution of hydrogen chloride, stir at room temperature until the reaction is complete, and directly evaporate the reaction solution to obtain compound 6; (4) Dissolve compound 6 and potassium carbonate in DMF, add compound 7, stir at 40~60℃ until the reaction is complete, dilute the reaction solution with ethyl acetate, wash with saline, dry the organic phase, stir and pass the sample through a column to obtain the compound shown in formula I.

5. The method for preparing arylaceyne compounds according to claim 4, characterized in that, In step (1), the molar ratio of compound 1, compound 2, DIEA and HBTU is 1:1:2:(1.0~1.5); in step (2), the molar ratio of compound 3, compound 4, triethylamine and Pd(PPh3)2Cl2 and CuI is 1:2:3:0.02:0.01; in step (3), the molar ratio of compound 5 and hydrogen chloride is 1:10; in step (4), the molar ratio of compound 6, potassium carbonate and compound 7 is 1:(2~4):(1.0~1.5).

6. The use of the arylkyne compounds according to any one of claims 1 to 3 and their biologically acceptable salts in the preparation of antitumor drugs, characterized in that, The antitumor drug is a drug for treating diseases related to abnormal STAT3 cell signaling, specifically drugs for treating gastric cancer, pancreatic cancer, breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, and multiple myeloma.

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