Preparation method and application of dual-mechanism ERK inhibitor

Abstract

The invention belongs to the technical field of design and synthesis of drugs, and particularly relates to a preparation method and application of a dual-mechanism ERK inhibitor. The invention develops and synthesizes a series of compounds (EPII-1-15), and discovers a part of compounds with excellent inhibition effects from the series of compounds. Wherein the compound EPI-15 has inhibitory activity on a human colon cancer cell COLO-205, a human melanoma cell A375, a human skin cancer cell A2058 and a human colon cancer cell HT-29. Besides, the invention also explores a synthesis path of the series of compounds and intermediates thereof, realizes efficient synthesis of the series of compounds and intermediates thereof, and provides a synthesis thought and method with reference value for subsequent drug research and development.

Description

[0001] This application is a divisional application. The application number of the original application is 202410683186.9, and the original application date is May 29, 2024. The entire contents of the original application are incorporated into this application by reference. Technical Field

[0002] The invention belongs to the technical field of drug design and synthesis, and specifically relates to a preparation method and application of a dual-mechanism ERK inhibitor. Background Art

[0003] The mitogen-activated protein kinase (MAPKs) signal transduction pathway is an important signal transduction pathway that transmits cell surface signals to the cell nucleus. This pathway affects the transcription and regulation of genes in animal cells, thereby causing biological reactions such as cell proliferation, differentiation, transformation and apoptosis. [1] At present, five MAPKs signaling pathways have been discovered in mammalian cells, including extracellular signal-regulated kinases (ERK, Extracellular regulated protein kinases), c-Jun N-terminal kinase (JNK / SAPK, c-Jun N-terminal kinase / stress-activated protein kinase), p38 kinase isozymes (p38A, p38B, p38C and p38D), ERK3 / ERK4 and ERK5. [2] The RAS-RAF-MEK-ERK signal transduction pathway (ERK pathway) is a protein kinase cascade pathway responsible for amplifying and regulating mitotic signals, which can be activated by a variety of mitogens or other extracellular sources (such as growth factors, insulin, osmotic pressure and cytokines). [3,4] .

[0004] The RAS-RAF-MEK-ERK cascade signal transduction pathway is activated in a variety of tumor cells and has become a hot topic in cancer treatment. However, upstream kinase inhibitors can develop severe drug resistance during clinical use, leading to treatment failure. Inhibitors targeting the downstream key node ERK kinase can, on the one hand, reverse the acquired drug resistance caused by inhibiting upstream targets. On the other hand, compared with other upstream kinase inhibitors in the MAPK pathway, ERK inhibitors are less likely to develop drug resistance. Therefore, designing ERK inhibitors with good selectivity and high activity has greater clinical potential and prospects.

[0005] Extracellular signal-regulated kinase 1 / 2 (ERK1 / 2) is a serine / threonine kinase that can be activated by phosphorylation by MEK1 and MEK2. So far, two ERK isoforms have been identified in the RAS-RAF-MEK-ERK pathway: ERK1 (360 amino acids) and ERK2 (379 amino acids), and their structures and sequences have a high degree of similarity (85%). [5] , and are ubiquitously expressed in tissues and model systems, but the expression level of ERK2 is higher than that of ERK1.

[0006] MEK1 / 2 activates ERK1 / 2 by dual phosphorylation of the Tyr187 and Thr185 residues in the ERK1 / 2 active domain. Its dual phosphorylation triggers two different functional modes. Activated ERK1 / 2 can catalyze the phosphorylation of many cytoplasmic downstream substrates (including RSK, which is often used as a biomarker for ERK1 / 2 activity and inhibition); secondly, phosphorylation of ERK1 / 2 can lead to its own translocation to the nucleus, where it phosphorylates and activates nuclear substrates (such as transcription factors) [6] .

[0007] ERK2 kinase has multiple conformations in solvent, and the distribution of its conformational states can be regulated by the structural types of different inhibitors. When bound to a catalytic ERK inhibitor, its Tyr36 located in front of the P-Loop ring can form a Pi-Pi conjugation with Tyr64, closing the second pocket and placing it in a state such as Figure 1 (a) is the "out" state; when bound to the dual-mechanism ERK inhibitor, Tyr36 and Tyr64 below the P-Loop are in an "in" state, forming Figure 1 (b) The second pocket shown [7] The difference between dual-mechanism and single-mechanism ERK inhibitors in regulating ERK1 / 2 is as follows Figure 2 shown.

[0008] At the same time, a large number of biological activity studies have shown that dual-mechanism ERK inhibitors that can induce ERK kinase to form a second pocket can not only block the catalytic activity of ERK, but also block the phosphorylation of ERK kinase by MEK, thereby preventing pERK (phosphorylated ERK) from transferring to the nucleus; while catalytic ERK inhibitors can only block the catalytic activity of ERK1 / 2, but cannot prevent themselves from being phosphorylated by MEK. Therefore, the design and development of dual-mechanism ERK inhibitors may have better inhibitory efficacy than ATP-competitive ERK inhibitors.

[0009] References:

[0010] [1](a)Johnson GL,Lapadat R.Mitgen-activated protein kinase pathwaysmediated by ERK,JNK and p38 protein kinases.Science.2002,298:1911-1912;(b)NixP,Hisamoto N,et al.Axon regeneration requires coordinate activation of p38and JNK MAPK pathways.PANS.2011,108(26):10738-10743.

[0011] [2](a)Javelaud D,Mauviel A.Crosstalk mechanisms between the mitogenactivated protein kinase pathways and Smad signaling downstream of TGF-beta:implications for carcinogenesis.Oncogene.2005,24(37):5742-5750;(b)Akasaka E,Takekoshi S,Horikoshi Y,et al.Protein Oxidative damage and Heme Oxygenase inSunlight-exposed Human Skin:Roles of MAPK Responses to Oxidative Stress.TokaiJournal of Experimental&Clinical Medicine.2010,35(4):152-164.

[0012] [3]Roskoski R,Jr.ERK1 / 2MAP kinases:structure,function,andregulation.Pharmacol Res.2012;66(2):105-143.

[0013] [4]Raman M,Chen W,Cobb MH.Differential regulation and properties ofMAPKs.Oncogene.2007;26(22):3100-3112.

[0014] [5] Boulton TG, Cobb MH. Identification of multiple extracellular signal-regulated kinases (ERKs) with antipeptide antibodies. Cell Regul. 1991, 2 (5): 357-371.

[0015] [6] Rodríguez, J.; Crespo, P. Working Without Kinase Activity:

[0016] Phosphotransfer-Independent Functions of Extracellular Signal-Regulated Kinases.Sci.Signaling.2011,4(196),re3.

[0017] [7]Heightman TD,Berdini V,et al..Fragment-Based Discovery of aPotent,Orally Bioavailable Inhibitor That Modulates the Phosphorylation andCatalytic Activity of ERK1 / 2.J.Med.Chem.2018,61:4978-4992. Summary of the invention

[0019] In order to develop a more efficient dual-mechanism ERK inhibitor, the present invention developed and synthesized a series of compounds (EPII-1 to 15), and found some compounds with excellent inhibitory effects.

[0020] The first aspect of the present invention is to provide a targeted inhibitor, specifically a dual-mechanism ERK inhibitor, wherein the dual-mechanism ERK inhibitor is a compound having a structure shown in Formula EPII-2:

[0021]

[0022] In addition, the dual-mechanism ERK inhibitor is preferably a compound with the structure shown in Formula EPII-15:

[0023]

[0024] Furthermore, the dual-mechanism ERK inhibitor may also be a pharmaceutically acceptable salt formed by a compound having a structure represented by Formula EPII-2 or Formula EPII-15.

[0025] The second aspect of the present invention is to provide the use of the dual-mechanism ERK inhibitor. The dual-mechanism ERK inhibitor can be used to prepare drugs for treating cancer.

[0026] Furthermore, in the drug for treating cancer, the dual mechanism ERK inhibitor can be used as the only active ingredient.

[0027] Furthermore, in the drug for treating cancer, the dual-mechanism ERK inhibitor can also be used as an active ingredient of the drug together with other ingredients having ERK inhibitory effects.

[0028] The third aspect of the present invention is to provide a drug for treating cancer, wherein the drug contains an active ingredient, and the active ingredient is the above-mentioned dual-mechanism ERK inhibitor.

[0029] Furthermore, the drug is prepared from active ingredients and pharmaceutically acceptable excipients. The pharmaceutically acceptable excipients may have certain physiological activity, but the addition of the ingredient will not change the dominant role of the dual-mechanism ERK inhibitor in the treatment of the disease, but only play an auxiliary role, which helps to utilize the activity of the dual-mechanism ERK inhibitor.

[0030] Furthermore, pharmaceutically acceptable excipients include any one or more combinations of carriers, binders, wetting agents, disintegrants, excipients, fillers, emulsifiers, cosolvents, solubilizers, osmotic pressure regulators, surfactants, antioxidants, antibacterial agents, colorants, pH adjusters, buffers, and coating materials.

[0031] Furthermore, the cancer may be colon cancer, melanoma, or skin cancer.

[0032] Beneficial Effects

[0033] The present invention designs a series of compounds (EPII-1 to 15), explores and realizes the efficient synthesis of the series of compounds and their intermediates, and provides synthetic ideas and methods with reference value for subsequent drug research and development.

[0034] The present invention has found some compounds with excellent ERK inhibitory activity in the designed series of compounds.

[0035] Among the series of compounds designed by the present invention, compound EPII-15 has inhibitory activity against human colon cancer cells COLO-205, human melanoma cells A375, human skin cancer cells A2058 and human colon cancer cells HT-29, among which the proliferation inhibition activity against cells COLO-205 is 1.083 μM, and it has good solubility, so it has good development prospects as a potential anticancer drug.

[0036] Among the series of compounds designed by the present invention, compound EPII-2 also has a certain proliferation inhibitory effect on human melanoma cells A375, human skin cancer cells A2058 and human colon cancer cells COLO-205, and also has good solubility, but its activity on human colon cancer cells HT-29 is relatively weak. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 (a) Schematic diagram showing ERK2 kinase in the "out" state; Figure 1 (b) is a schematic diagram showing ERK2 kinase in the "in" state.

[0038] Figure 2 Shown are the differences in the regulation of ERK1 / 2 by dual- and single-mechanism ERK inhibitors. DETAILED DESCRIPTION

[0039] The present invention is further illustrated by specific examples below. These examples are exemplary and are intended to illustrate the problem and explain the present invention, but are not intended to be limiting.

[0040] Reagents and instruments

[0041] RY-1 melting point instrument from Tianjin Analytical Instrument Factory; Nicolet Impact 410 infrared spectrometer, KBr pellet; 1 H-NMR was performed using BRUKER AM-300 and AM-500 nuclear magnetic resonance instruments with TMS as the internal standard; HP 1100 mass spectrometer; Agilent 1100 series LC / MSD Trap SL; a Waters Micros Q-TOF high-resolution analytical instrument; a full-wavelength microplate reader (BioTek); FSH-2A adjustable high-speed homogenizer (Dasheng Experimental Instrument Co., Ltd.); TG1650-WS desktop high-speed centrifuge (Shanghai Luxiang Centrifuge Co., Ltd.); Beckman ultra-speed low-temperature centrifuge (L-80XP) (Beckman Coulter Co., Ltd., USA); SHIMADZU-HPLC instrument (processing software LabSolutions LC / GC 5.71, Shimadzu); and C18-WR (4.6×150 mm, 5 μm) chromatographic column.

[0042] The silica gel used for column chromatography was a 200-300 mesh silica gel column layer (Anhui Liangchen Silica Gel Desiccant Factory); TLC silica gel plates were purchased from Yantai Institute of Chemical Industry, model HSGF 254; anhydrous THF was prepared by reflux treatment with Na; other commonly used reagents were commercially available analytical grade reagents; potassium dihydrogen phosphate (KH2PO4) (Nanjing Chemical Reagent Co., Ltd.); disodium hydrogen phosphate (Na2HPO4·12H2O) (Nanjing Chemical Reagent Co., Ltd.); potassium chloride (KCl) (Nanjing Chemical Reagent Factory No. 1); Tris-HCl buffer at pH 7.4; Sprague-Dawley rat liver homogenate (homemade, 1.00 mg / mL, 4.50 mg / ml, stored at -80°C); chromatographically pure acetonitrile (TEDIA, USA); 0.1 M potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer at pH = 7.4 (containing 3.00 mmol / L magnesium chloride and 1.00 mmol / L EDTA); Kits: TC (96t), TG (96t), LDL-C (96t), purchased from Nanjing Jiancheng Bioengineering Institute.

[0043] Synthesis of compounds

[0044] The synthesis of drugs and intermediates is carried out with reference to the general reaction formula. The amounts of reagents involved in the general reaction formula are schematic and can be scaled proportionally or adjusted to a certain extent.

[0045] The synthesis of key intermediates a30 and a39 refers to reaction formula 1:

[0046]

[0047] The reaction conditions in general formula 1 are:

[0048] (a) Hydrogen chloride gas, anhydrous ethanol;

[0049] (b)tert-butyl 1-methylhydrazine-1-carboxylate(a23), pyridine;

[0050] (c) trifluoroacetic acid, dichloromethane, 25°C, 1 h;

[0051] (d) formic acid;

[0052] (e) Pd(dppf)2Cl2, potassium carbonate, ethylene glycol dimethyl ether / water;

[0053] (f) trifluoroacetic acid, dichloromethane, 25 °C, 1 h;

[0054] (g) diethylamine, cuprous iodide, potassium carbonate, 125°C;

[0055] (h) Triethylamine, dichloromethane, 0°C.

[0056] The synthesis of key intermediates a35 and a38 refers to general formula 2:

[0057]

[0058] The reaction conditions in general formula 2 are:

[0059] (a)(i) tert-butyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (a27), Pd(dppf)2Cl2, potassium carbonate, ethylene glycol dimethyl ether / water or (ii) tert-butyl piperazine-1-carboxylate (a36), Pd(OAc)2, BINAP, cesium carbonate, toluene;

[0060] (b) Trifluoroacetic acid, dichloromethane, 25°C, 1 h.

[0061] The synthesis of key intermediate a62 refers to general formula 3:

[0062]

[0063] The reaction conditions in general formula 3 are:

[0064] (a) LDA, anhydrous tetrahydrofuran, -78 °C;

[0065] (b) trifluoroacetic acid, dichloromethane;

[0066] (c) methanol;

[0067] (d) (Boc)2O, triethylamine, dichloromethane;

[0068] (e) Lithium hydroxide solution (2.0 M), tetrahydrofuran / methanol. The general synthesis formula of the key intermediate a72 is as follows:

[0069]

[0070] The reaction conditions in general formula 4 are:

[0071] (a) (Boc)2O, triethylamine, methanol, room temperature;

[0072] (b) Pinacol borate, Pd(dppf)2Cl2, potassium acetate, dimethyl sulfoxide, 80℃, N2;

[0073] (c) Pd(PPh3)4, sodium bicarbonate, 1,4-dioxane / water, 120 °C, N2;

[0074] (d) trifluoroacetic acid, dichloromethane;

[0075] (e) Chloroacetyl chloride, triethylamine, dichloromethane, 0°C.

[0076] The synthesis of target compounds EPII-1 to 8 and EPII-10 to 13 refers to general formula 5:

[0077]

[0078] In general formula 5:

[0079]

[0080]

[0081] The reaction conditions in general formula 5 are:

[0082] (a) Pd(PPh3)4, potassium phosphate, tetrahydrofuran / water, 90°C, 10h;

[0083] (b) NBS, AIBN, benzene, 80 °C, 4 h;

[0084] (c) triethylamine, acetonitrile, 85°C, 8h;

[0085] (d) (i) LiOH.H2O, tetrahydrofuran / water or (ii) NaOH, methanol / water, 30°C;

[0086] (e) a29 / a32 / a35 / a38 / 2-(piperazin-1-yl)ethan-1-ol(a40a) / 2-(2-(piperazin-1-yl)ethoxy)ethan-1-ol(a40b) / 2-(2-aminoethoxy)ethan-1-ol(a41), EDCI, 1-hydroxybenzotriazole, N,N-diisopropylethylamine, dichloromethane, room temperature;

[0087] (f) (i) sec-butyl alcohol, 125°C, 12h or (ii) dimethyl sulfoxide, DIEA, 140°C, 12h.

[0088] The synthesis of target compounds EPII-9 and EPII-10 refers to general formula 6:

[0089]

[0090] The reaction conditions in general formula 6 are:

[0091] (a) EDCI, HOBt, N,N-diisopropylethylamine, dichloromethane, room temperature;

[0092] (b) NaN3, N,N-dimethylformamide, rt; (c) 2-(prop-2-yn-1-yloxy)ethan-1-ol (a45a) or 2-methylbut-3-yn-2-ol (a45b), CuI, acetonitrile / water;

[0093] (d) sec-Butanol, 125°C, 12h.

[0094] The synthesis of target compounds EPII-14 and EPII-15 refers to general formula 7:

[0095]

[0096] The reaction conditions in general formula 7 are:

[0097] (a) i-PrI, K2CO3, N,N-dimethylformamide;

[0098] (b) B2Pin2, Pd(dppf)2Cl2, KOAc, dimethyl sulfoxide;

[0099] (c) Pd(PPh3)4, Na2CO3, toluene / ethanol / water, 100°C;

[0100] (d) Pd / C, H2, isopropanol / toluene;

[0101] (e) HATU, N,N-diisopropylethylamine, N,N-dimethylformamide;

[0102] (f) trifluoroacetic acid, dichloromethane;

[0103] (g) a39 (for the synthesis of EPⅡ-14) or a72 (for the synthesis of EPⅡ-15), N,N-diisopropylethylamine, N,N-dimethylformamide, 50°C.

[0104] For example, the synthetic process of EPII-15 is taken as an example:

[0105] a49 (1.0 g, 5.75 mmol), potassium carbonate (2.4 g, 17.25 mmol) and a50 (1.5 g, 8.63 mmol) were dissolved in DMF (5 mL), and the reaction was stirred at room temperature for 7 h. After the reaction of the raw material a49 was completed, it was detected by TLC (V petroleum ether: V ethyl acetate = 3:1), and then diluted with water (15 mL) and ethyl acetate (20 mL), and allowed to stand for separation. The organic layer was washed with water (5 mL*3), dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain the residue. Column chromatography (V petroleum ether: V ethyl acetate = 20:1-5:1) (10 mL) gave 287 mg of an off-white solid with a yield of 23.1% and mp 104.8-106.0°C.

[0106] 1 H-NMR (300MHz, CDCl3) δ (ppm): 7.46 (d, J = 2.7Hz, 1H, ArH), 7.36 (dd, J = 9.6, 2.7Hz, 1H, ArH), 6.54 (d, J = 9.6Hz, 1H, ArH), 5.31-5.22 (m, 1H, CH ),1.40(d,J=6.8Hz,6H,CH(CH3)2).

[0107] a51 (200 mg, 0.93 mmol), pinacol borate (284 mg, 1.12 mmol), Pd(dppf)Cl2 (68 mg, 0.09 mmol) and potassium acetate (274 mg, 2.79 mmol) were dissolved in DMSO (2 mL) and reacted at 100 °C under nitrogen for 3 h. After the reaction of raw material a51 was completed as detected by TLC (V petroleum ether: V ethyl acetate = 3:1), the mixture was cooled to room temperature and directly used in the next step without treatment.

[0108] a52 (245 mg, 0.93 mmol), a48 (450 mg, 0.93 mmol), Pd(pph3)4 (108 mg, 0.09 mmol) and sodium carbonate (493 mg, 4.65 mmol) were dissolved in a mixed solution of VH2O:Vtoluene:Vethanol=1:1:1 (2 ml), and reacted at 100°C for 4 h under nitrogen protection. After the reaction of raw material a52 was completed, it was cooled to room temperature, diluted with water (10 mL) and ethyl acetate (10 mL), filtered through diatomaceous earth, and the filtrate was allowed to stand for stratification. The organic layer was washed with water (4 mL*3), and the residue was dried under reduced pressure to obtain a residue, which was separated by column chromatography (Vpetroleum ether:Vethyl acetate=10:1~3:1) to obtain 280 mg of a yellow solid with a yield of 55.7% and mp147.8~149.5°C.

[0109] 1H-NMR (300MHz, DMSO-d6) δ (ppm): 8.78 (d, J=2.3Hz, 1H, ArH), 8.25 (d, J=2.6Hz, 1H, ArH), 7.98 (dd, J=9.4, 2.2Hz, 1H, ArH), 7 .80(dd,J=9.4,2.5Hz,1H,ArH),7.44-7.33(m,9H,ArH),7.22-7.19(m,6H,ArH),6.58(t,J=9.7Hz,2HArH),5.14-5.09(m,1H, CH ),1.39(d,J=6.7Hz,6H,CH (CH3)2 ).

[0110] a54 (1.89 g, 3.49 mmol) and 50% wet Pd / C (0.19 g, 10%) were dissolved in a mixed solution of V toluene:V isopropanol = 1:2 (5 mL), and hydrogen was passed through. The reaction was carried out at room temperature for 13 h. After the reaction of the raw material a54 was completed, it was detected by TLC (V dichloromethane:V methanol = 20:1), and the mixture was filtered through diatomaceous earth. The filtrate was dried under reduced pressure to obtain a residue, which was separated by column chromatography (V dichloromethane:V methanol = 100:1-80:1) to obtain 1.20 g of an off-white solid with a yield of 67.3% and mp 86.1-87.8°C.

[0111] 1 H-NMR (300MHz, DMSO-d6) δ (ppm): 7.98 (d, J=2.4Hz, 1H, ArH), 7.76 (dd, J=9.4, 2.4Hz, 1H, ArH), 7.39-7.33 (m, 9H, ArH),7.25-7.21(m,6H,ArH),6.99-6.97(m,1H,ArH),6.59-6.50(m,2H),6.19(d,J=9.0Hz,1H),5.18-5.12(m,1H, CH ),1.42(d,J=6.7Hz,6H,CH (CH3)2 ).

[0112] a63 (1.04 g, 2.03 mmol), a62 (531 mg, 2.03 mmol), HATU (772 mg, 2.03 mmol) and DIEA (289 mg, 2.23 mmol) were dissolved in DMF (3 mL) and stirred at room temperature for 12 h. After the reaction of the raw material a63 was completed by TLC (petroleum ether: ethyl acetate = 1:1), water (9 mL) was added for dilution, and ethyl acetate (5 mL*3) was used for extraction. The organic layers were combined and the solvent was dried under reduced pressure to obtain a residue, which was purified by column chromatography (V petroleum ether: V ethyl acetate = 10:1~1:1) to obtain 1.12 g of a light yellow solid, with a yield of 73.2% and mp of 194.1~195.8°C.

[0113] 1 H NMR (300MHz, CDCl3) δ (ppm) 8.96 (d, J = 4.0Hz, 1H, ArH), 8.46 (d, J = 5.6Hz, 1H, ArH), 7.98 (d, J = 1.9Hz, 1H, ArH), 7.89 (d, J = 9.3Hz, 1H, ArH ),7.38-7.31(m,9H,ArH),7.26-7.23(m,6H,ArH),6.90(d,J=9.4Hz,1H),6.71(d,J=9.4Hz,1H),6.39(d,J=9.1Hz,1H),5.43-5.34(m,1H, CH (CH3)2),4.02-3.96(m,1H,N( CH 2)2),3.72-3.58(m,3H,N( CH 2)2),2.71-2.68(m,1H,C CH 2),2.16(s,3H, SCH3 ),2.12-2.08(m,1H C CH 2),1.51(s,9H,C( CH3)3 ),1.47(d,J=6.8Hz,6H,CH(CH3)2).

[0114] a64 (100 mg, 7.26 mmol) was dissolved in dichloromethane (1.5 mL), and trifluoroacetic acid (0.5 mL) and water (0.25 mL) were added. The reaction was stirred at room temperature for 12 h. After the reaction was completed as detected by TLC (V petroleum ether: V ethyl acetate = 1:1), the temperature was lowered to 0°C, saturated sodium carbonate (1 mL) was added to quench, the solvent was evaporated under reduced pressure, water (1 mL) was added to the residue, and extraction was performed with ethyl acetate (3 mL*6). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure, and directly used for the next step without further treatment.

[0115] a65 (200 mg, 0.49 mmol), a72 (137 mg, 0.49 mmol) and DIEA (380 mg, 2.94 mmol) were dissolved in DMF (3 mL), and the temperature was raised to 50°C for 12 h. TLC (V dichloromethane: V methanol = 20:1) detected that the reaction was complete, and the reaction solution was cooled to room temperature, diluted with water (6 mL), extracted with ethyl acetate (4 mL*3), and the organic layers were combined, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure, and separated by column chromatography (V eluent: V dichloromethane: methanol = 100:1-20:1) to obtain 132 mg of off-white solid, yield: 41.2%, mp 140.0-141.5°C.

[0116] 1 H-NMR (300MHz, CDCl3) δ (ppm): 9.04 (s, 1H, NH), 8.31 (s, 1H, CO NH ),8.06-7.94(m,6H,ArH),7.55-7.45(m,3H,ArH),7.34(s,1H,ArH),6.77(d,J=9.3Hz,1H,ArH),5.41(hept,J=7.4Hz,1H, CH (CH3)2),3.97(s,3H,N CH3 ),3.69-3.62(m,2H,N CH2 CO), 3.50-3.47(m,1H,NH CH 2),3.38-3.32(m,1H,NH CH 2),2.99-2.71(m,6H,N( CH2)2 ,NHCH2 CH2 ),2.68-2.64(m,1H, CH 2CH2N),2.11(s,3H),2.05-2.03(m,1H, CH 2CH2N),1.51-1.49(m,6H,CH(CH3)2)

[0117] 13C-NMR (75MHz, CDCl3) δ (ppm): 171.13, 170.33, 162.54, 161.78, 144.33, 141.88, 140.13, 139.21, 138.28, 131.90, 131.49, 129.18, 129.09 ,126.46,121.28,120.71,120.46,113.64,110.89,110.81,63.35,58 .36,57.68,53.56,46.90,40.02,36.65,36.28,35.33,22.07,13.90.

[0118] HRMS(ESI):m / z[M+H] + .Calcd for C 34 H 40 N9O3S:654.2969; Found:654.2977.

[0119] IR(cm -1 ):3270.40,2922.83,1660.16,1592.97,1533.53,1501.75,1447.29,1427.55,1401.05,1259.10,957.26,833.77,751.45.

[0120] The synthesis of key intermediate 30 refers to general formula 8:

[0121]

[0122] For example, the synthesis process of intermediate compound a30 is taken as an example:

[0123] 4-Bromobenzonitrile (a21) (5 g, 27.8 mmol) was suspended in anhydrous ethanol (30 mL). Hydrogen chloride gas was vigorously introduced at 0-5°C for 10 min, and then slowly introduced for 5 h. After the reaction was completed, TLC (V dichloromethane: V methanol = 30:1) was used to detect the completion of the reaction. Part of the solvent was evaporated under reduced pressure, and the mixture was stirred at room temperature for 30 min. A large amount of solid precipitated, and 1.45 g of white solid product a22 was obtained by suction filtration. The yield was 19.7%, and the mp was 188.3-190.1°C.

[0124] 1 H-NMR (300MHz, CDCl3) δ (ppm): 12.72 (s, 1H, HCl), 12.00 (s, 1H, CNH), 8.28 (d, J = 8.6Hz, 2H, ArH), 7.72 (d, J = 8.6Hz, 2H, ArH), 4.94 (q, J = 7.0Hz, 2H, OCH2 CH3), 1.62(t, J=7.0Hz, 2H, OCH2 CH3) .

[0125] Dissolve a22 (5 g, 18.9 mmol) and 1-tert-butyloxycarbonyl-1-methylhydrazine (a23) (3.3 g, 22.7 mmol) in pyridine (10 mL) and react at room temperature for 18 h. After the reaction is completed, TLC (V petroleum ether: V ethyl acetate = 1:1) is used to detect the completion of the reaction. The solvent is dried under reduced pressure, and ether (10 mL) is added to the residue for 2 h. Filter by suction to obtain 4.97 g of white solid a24, with a yield of 80.1% and mp of 174.1-175.7°C.

[0126] 1 H-NMR(300MHz,DMSO-d6+D2O)δ(ppm):7.85-7.81(m,2H,ArH),7.66-7.62(m,2H,ArH),3.07(s,3H,NC H3 ),1.38(s,9H,C(C H3)3 ).

[0127] Dissolve a24 (4 g, 12.19 mmol) in dichloromethane (20 mL), add trifluoroacetic acid (5 mL), and stir at room temperature for 3 h. After the reaction is completed, detect by TLC (V dichloromethane: V methanol = 10:1). The solvent is dried under reduced pressure, and the residue is adjusted to pH 8-9 with saturated sodium bicarbonate solution, stirred for 30 min, solid precipitated, and 2.56 g of white solid a25 is obtained by suction filtration, yield: 92.1%, mp 109.8-111.9 °C.

[0128] 1 H-NMR (300MHz, DMSO-d6) δ (ppm): 7.77 (d, J=8.5Hz, 2H, ArH), 7.66 (d, J=8.5Hz, 2H, ArH), 2.63 (s, 3H, NHC H3 ).

[0129] Add a25 (600 mg, 2.63 mmol) to formic acid (5 mL) and heat under reflux for 3 h. After the reaction is completed, TLC (V dichloromethane: V methanol = 20:1) is used to detect the completion of the reaction. Cool to room temperature, adjust the pH to 7-8 with saturated sodium bicarbonate solution, extract with ethyl acetate (5 mL*3), combine the organic layers, dry over anhydrous sodium sulfate, filter, and decompress and spin-dry the solvent to obtain 310 mg of white solid a26, yield: 49.5%, mp>260°C.

[0130] 1H-NMR (300MHz, CDCl3) δ (ppm): 8.07 (s, 1H, triazole-H), 7.98 (d, J = 8.6Hz, 2H, ArH), 7.59 (d, J = 8.5Hz, 2H, ArH), 3.98 (s, 3H, NC H3 ).

[0131] Add a26 (200 mg, 0.84 mmol), a27 (288 mg, 0.93 mmol), Pd(dppf)2Cl2 (31 mg, 0.042 mmol) and potassium carbonate (348 mg, 2.52 mmol) to V ethylene glycol dimethyl ether: V water = 5:1 (10 ml), evacuate with N2 protection, heat to 80 ° C and react for 18 hours. After the reaction is completed, TLC (V dichloromethane: V methanol = 30:1) is used to detect the completion of the reaction. Cool to room temperature, filter with diatomaceous earth, decompress and spin-dry the solvent, and separate the residue by column chromatography (eluent: dichloromethane: methanol = 200:1-50:1) to obtain 220 mg of white solid a28, yield: 77.0%, mp 117.9-119.8 ° C.

[0132] 1 H-NMR (300MHz, CDCl3) δ (ppm): 8.09-8.07 (m, 3H, triazole-H, ArH), 7.59 (d, J = 7.9Hz, 2H, ArH), 6.19-6.15 (m, 1H, tetrahydropyridine-H), 4.12-4.11 (m, 2H, N CH2 CHC),4.01(s,3H,NC H3 ),3.67(t,J=5.6Hz,2H,N CH2 CH2C), 2.62-2.59(m,2H,NCH2 CH2 C),1.52(s,9H,C(C H3)3 ).

[0133] Dissolve a28 (2.38 g, 7.0 mmol) in dichloromethane (16 mL), add trifluoroacetic acid (4 mL), and stir at room temperature for 3 h. After the reaction is completed, detect by TLC (V dichloromethane: V methanol = 10:1). The solvent is dried under reduced pressure, and the residue is adjusted to pH 8-9 with saturated sodium bicarbonate solution, stirred for 30 min, solid precipitated, and 1.35 g of white solid a29 is obtained by suction filtration, with a yield of 80.3%, and mp 203.8-204.3 °C.

[0134] 1H-NMR (300MHz, CDCl3) δ (ppm): 8.08 (s, 1H, triazole-H), 8.06 (d, J = 8.5Hz, 2H, ArH), 7.49 (d, J = 8.5Hz, 2H, ArH), 6.27-6.24 (m, 1H, tetrahydropyridine-H), 3.99 (s, 3H, NC H3 ),3.60-3.57(m,2H,N CH2 CH2C),3.16(t,J=5.7Hz,2H,N CH2 CH2C), 2.55-2.49(m,2H,NCH2 CH2 C).

[0135] A29 (500 mg, 2.08 mmol) was dissolved in dichloromethane (5 mL), and the temperature was lowered to 0°C, and triethylamine (421 mg, 4.16 mmol) and chloroacetyl chloride (353 mg, 3.12 mmol) were added. After stirring at 0°C for 1 h, TLC (V dichloromethane: V methanol = 20:1) detected that the reaction of the raw material a29 was complete, and water (2 mL) was added to quench the reaction, and dichloromethane was extracted (3 mL*3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain the residue, which was separated by column chromatography (V dichloromethane: V methanol = 200:1-50:1) to obtain 320 mg of light yellow solid a30, with a yield of 48.6%, and mp 206.1-208°C.

[0136] 1 H-NMR (300MHz, CDCl3) δ (ppm): 8.19 (s, 1H), 8.12 (d, J = 7.9Hz, 2H, ArH), 7.50 (d, J = 7.8Hz, 2H, ArH), 6.22-6.16 (m, 1H, tetrahydropyridine-H), 4.32-4.19 (m, 4H, Cl CH2 CON CH2 ),4.04(s,3H,N CH3 ),3.91-3.80(m,2H,CON CH2 ),2.75-2.67(m,2H,CH2 CH2 ).

[0137] For example, the synthesis process of intermediate compound a39 is taken as an example:

[0138]

[0139] A26 (200 mg, 0.84 mmol), potassium carbonate (234 mg, 1.68 mmol), cuprous iodide (8 mg, 0.042 mmol) and diethylamine (5 mL) were added to a sealed tube and heated to 123°C for 18 h. After the reaction was completed, the solvent was dried under reduced pressure and the residue was separated by column chromatography (eluent: V dichloromethane: V methanol = 200: 1-30: 1) to obtain 70 mg of a light yellow solid a32, with a yield of 38.4% and mp 86.8-88.5°C.

[0140] 1 H-NMR(300MHz,DMSO-d6)δ(ppm):8.35(s,1H,triazole-H),7.70(d,J=8.6Hz,2H,ArH),6.61(d,J=8.6Hz,2H,ArH),5.91(t,1H,J=5.1Hz,1H,NH),3.84(s,3H,NC H3 ),3.16-3.10(m,2H,NH CH2 CH2), 2.80(t, J=6.1Hz, 2H, NH2 CH2 CH2).

[0141] a32 (1.07 g, 4.94 mmol) was dissolved in dichloromethane (10 mL), the temperature was lowered to 0°C, triethylamine (1.0 g, 9.88 mmol) and chloroacetyl chloride (837 mg, 7.41 mmol) were added, and the mixture was stirred at 0°C for 1 h. After the reaction of the raw material a32 was completed, TLC (V dichloromethane: V methanol = 20:1) was used to detect that the reaction was complete, and water (5 mL) was added to quench the mixture. The mixture was extracted with dichloromethane (5 mL*3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain the residue, which was separated by column chromatography (V dichloromethane: V methanol = 200:1-50:1) to obtain 575 mg of yellow solid a39. The yield was 39.6%, and the mp was 128.0-130.0°C.

[0142] 1 H-NMR (300MHz, CDCl3) δ (ppm): 8.00 (s, 1H, ArH), 7.92 (d, J = 8.8Hz, 2H, ArH), 6.67 (d, J = 8.7Hz, 2H, ArH), 4.07 (s, 2H, Cl CH2 CO),3.93(s,3H,N CH3 ),3.60(q,J=5.9Hz,2H,NH CH2 ),3.39(t,J=5.8Hz,2H,CH2 CH2 NH).

[0143] The synthesis of key intermediate a35 refers to general formula 10:

[0144]

[0145] For example, the synthesis process of intermediate compound a35 is taken as an example:

[0146] Add a33 (2.56 g, 12.81 mmol), a27 (3.74 g, 12.10 mmol), Pd(dppf)2Cl2 (407 mg, 0.56 mmol) and potassium carbonate (4.56 g, 33.0 mmol) to V ethylene glycol dimethyl ether: V water = 5:1 (30 ml), evacuate with N2 protection, heat to 80 ° C and react for 18 hours. After the reaction is completed, TLC (V petroleum ether: V ethyl acetate = 8:1) is used to detect the completion of the reaction. Cool to room temperature, filter with diatomaceous earth, decompress and spin-dry the solvent, and separate the residue by column chromatography (eluent: V petroleum ether: V ethyl acetate = 50:1-40:1) to obtain 2.53 g of white solid a34, yield: 69.2%, mp 90.0-92.0 ° C.

[0147] Dissolve a34 (1.0 g, 3.31 mmol) in dichloromethane (3 mL), add trifluoroacetic acid (3 mL), and stir at room temperature for 1 h. After the reaction is completed, detect by TLC (V dichloromethane: V methanol = 30:1). The solvent is dried under reduced pressure, and the residue is adjusted to pH 8-9 with saturated sodium bicarbonate solution, stirred for 30 min, solid precipitated, and 0.667 g of white solid a35 is obtained by suction filtration, yield: 99.6%, mp 122.0-124.0°C.

[0148] 1 H-NMR (300MHz, CDCl3) δ (ppm): 7.28 (d, J=8.6Hz, 2H, ArH), 6.70 (d, J=8.7Hz, 2H, ArH), 5.99-5.97 (m, 1H, tetrahydropyridine-H), 3.55-3.54 (m, 2H, N CH2 CHC),3.13(t,J=5.8Hz,2H,N CH2 CH2C),2.95(s,6H,N( CH3)2 ),2.49-2.45(m,2H,NCH2 CH2 C).

[0149] For example, the synthesis process of intermediate compound a38 is taken as an example:

[0150]

[0151] Add a33 (2.20 g, 11.00 mmol), a36 (2.26 g, 12.13 mmol), Pd(OAc)2 (264 mg, 1.18 mmol), BINAP (517 mg, 0.83 mmol) and cesium carbonate (5.38 g, 16.51 mmol) to toluene (30 mL), evacuate with N2 protection, heat to 100 ° C and react for 20 h. After the reaction is completed, TLC (V petroleum ether: V ethyl acetate = 8:1) is used to detect the completion of the reaction. Cool to room temperature, filter with diatomaceous earth, decompress and spin-dry the solvent, and separate the residue by column chromatography (eluent: V petroleum ether: V ethyl acetate = 20:1-10:1) to obtain 957 mg of light yellow solid a37, yield: 28.7%, mp 80.0-82.0 ° C.

[0152] 1 H-NMR(300MHz,DMSO-d6)δ(ppm):6.84(d,J=8.6Hz,2H,ArH),6.68(d,J=8.7Hz,2H,ArH),3.45-3.41(m,4H,N( CH2)2 ),2.91-2.87(m,4H,N( CH2)2 ),2.50(s,6H,N( CH3)2 ),1.41(s,9H,C(C H3)3 ).

[0153] Dissolve a37 (300 mg, 0.98 mmol) in dichloromethane (3 mL), add trifluoroacetic acid (2 mL), and stir at room temperature for 1 h. After the reaction is completed, detect by TLC (V dichloromethane: V methanol = 20:1). The solvent is dried under reduced pressure, and the residue is adjusted to pH 8-9 with saturated sodium bicarbonate solution, stirred for 30 min, solid precipitated, and 196 mg of yellow solid a38 is obtained by suction filtration, yield: 97.2%, mp 98.5-100.0 °C.

[0154] 1 H-NMR(300MHz,DMSO-d6)δ(ppm):6.81(d,J=8.7Hz,1H,ArH),6.67(d,J=9.0Hz,1H,ArH),2.85-2.83(m,8H,piperazine-H),2.77(s,6H,N( CH3)2 ).

[0155] The synthesis of key intermediate a62 refers to reaction formula 12:

[0156]

[0157] Exemplarily, the synthetic operation process of key intermediate a62 is:

[0158] a56 (1.0 g, 4.20 mmol) was dissolved in anhydrous tetrahydrofuran (3 mL), cooled to -78°C, and LDA (2.31 mL, 4.62 mmol) was added. After stirring for 2 h at the same temperature, dimethyl disulfide (0.98 mL, 10.50 mmol) was added. After stirring for 10 min at -78°C, the mixture was moved to room temperature and stirred for 18 h. After the reaction of the raw material a56 was completed, saturated ammonium chloride solution (3 mL) was added to quench the reaction. After stirring for 5 min, the mixture was extracted with ethyl acetate (4 mL*3). The organic layers were combined and the solvent was evaporated under reduced pressure to obtain the residue. The residue was separated by column chromatography (V petroleum ether: V ethyl acetate = 10:1 to 3:1) to obtain 387 mg of colorless oil a58. The yield was 33.5%.

[0159] 1 H-NMR (300MHz, CDCl3) δ (ppm): 3.92-3.72 (m, 4H, O CH3 ,N CH 2),3.69-3.42(m,3H,N( CH2)2 ),2.51-2.42(m,1H,C CH 2),2.18(s,3H,S CH3 ),2.15-2.07(m,1H,,C CH 2),1.50(s,9H,C( CH3)3 ).

[0160] Dissolve a58 (2.0 g, 7.26 mmol) in dichloromethane (5 mL), add trifluoroacetic acid (5 mL), and stir at room temperature for 2 h. After the reaction is completed, detect by TLC (V petroleum ether: V ethyl acetate = 5:1), dry the solvent under reduced pressure, add water (6 mL) to the residue, adjust the pH to 8-9 with solid sodium bicarbonate, stir for 30 min, extract with ethyl acetate (5 mL*6), combine the organic layers, add anhydrous sodium sulfate to dry, filter, dry under reduced pressure, and obtain a59 as a colorless oil, which is directly used for the next step without treatment.

[0161] Dissolve a59 (1.27 g, 7.26 mmol) in methanol (6 mL), add L-Tartaric acid (a53) (1.1 g, 7.26 mmol), heat to 75°C and reflux for 1 h, then cool and stand for crystallization, filter to obtain 1.03 g of white solid a60, total yield of two steps: 33.4%, mp 200.1~201.5°C.

[0162] 1 H NMR(300MHz,D2O)δ3.75-3.71(m,4H, CH 2, CH3 ),3.56-3.39(m,3H, CH2 , CH 2),2.58-2.46(m,1H, CH 2),2.25-2.16(m,1H, CH 2),2.07(s,3H, CH3 ).

[0163] a60 (1.39 g, 4.26 mmol) was dissolved in dichloromethane (5 mL), and the temperature was lowered to 0°C. Triethylamine (1.1 g, 10.65 mmol) and (Boc)2O (1.2 g, 5.54 mmol) were added. The mixture was stirred at room temperature for 14 h. After completion of the reaction, the mixture was detected by TLC (V petroleum ether: V ethyl acetate = 5:1), diluted with water (4 mL), extracted with dichloromethane (4 mL*6), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain 1.10 g of colorless oil a61. The yield was 93.9%.

[0164] 1 H-NMR (300MHz, CDCl3) δ (ppm): 3.90-3.84 (m, 1H, N CH 2),3.81(s,3H,O CH3 ),3.65-3.47(m,3H,N( CH2)2 ),2.53-2.43(m,1H,C CH 2),2.17(s,3H,S CH3 ),2.15-2.07(m,1H,,C CH 2),1.50(s,9H,C( CH3)3 ).

[0165] a61 (2.0 g, 7.26 mmol) was dissolved in a mixed solvent (5 mL) of V tetrahydrofuran:V methanol = 1:1. LiOH.H2O (1 M aqueous solution, 21.78 mmol) was added and the reaction was stirred at room temperature for 14 h. After the reaction was completed, the solvent was evaporated under reduced pressure. The pH was adjusted to 3 with 1N HCl. After stirring for 30 minutes, the mixture was extracted with ethyl acetate (5 mL*6). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain 1.52 g of colorless oil a62. The yield was 80.1%.

[0166] 1 H-NMR(300MHz,CDCl3)δ(ppm):3.95-3.91(m,1H,N( CH 2)2),3.68-3.52(m,3H,N( CH2)2),2.56-2.46(m,1H,C CH 2),2.24(s,3H, SCH3 ),2.17-2.08(m,1H,C CH 2),1.51(s,9H,C( CH3)3 ).

[0167] The synthesis of key intermediate a72 refers to reaction formula 13:

[0168]

[0169] A66 (2.10 g, 10.50 mmol) was dissolved in dichloromethane (5 mL), and the temperature was lowered to 0°C. Triethylamine (4.30 g, 42.60 mmol) and (Boc)2O (4.62 g, 21.00 mmol) were added. The mixture was stirred at room temperature for 14 h. After the reaction was completed, the solvent was dried under reduced pressure to obtain the residue, which was purified by column chromatography (V petroleum ether: V ethyl acetate = 100:1-20:1) to obtain 2.50 g of white solid a67, with a yield of 79.2%, and mp 60.0-61.8°C.

[0170] 1 H NMR (300MHz, CDCl3-d) δ7.46(d,J=8.3Hz,2H,ArH),7.11(d,J=8.3Hz,2H,ArH),4.57(s,1H,NH),3.39(q,J=6.7Hz,2H,Ph CH2 ),2.79(t,J=7.0Hz,2H,CH2 CH2 ),1.47(s,9H,C (CH3)3 ).

[0171] a67 (200 mg, 0.67 mmol), bipyralidin (257 mg, 1.01 mmol), Pd(dppf)2Cl2 (49 mg, 0.07 mmol) and potassium acetate (197 mg, 2.01 mmol) were dissolved in DMSO (2 mL), and the mixture was reacted at 80°C for 16 h under nitrogen protection. After the reaction of the raw material a67 was completed, it was cooled to room temperature, diluted with water (10 mL), extracted with ethyl acetate (15 mL*1), dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a residue, which was purified by column chromatography (V petroleum ether: V ethyl acetate = 200:1-80:1) to obtain 210 mg of white solid a68, with a yield of 90.3%, and mp 82.1-84.0°C.

[0172] 1H-NMR (300MHz, CDCl3) δ (ppm): 7.80 (d, J=7.9Hz, 2H, ArH)., 7.25 (d, J=7.6Hz, 2H, ArH), 4.56 (s, 1H, NH), 3.43-3.39 (m, 2H, CH2 NH), 2.86(t, J=7.0Hz, 2H, Ph CH2 ),1.48(s,9H,C (CH3)3 ),1.39(s,12H,B2Pin2-H).

[0173] a68 (1.60 g, 4.61 mmol), a69 (747 mg, 4.61 mmol), Pd(pph3)4 (533 mg, 0.46 mmol) and sodium bicarbonate (775 mg, 9.22 mmol) were dissolved in a mixed solvent (6 mL) of V1,4-dioxane:Vwater=3:1, and the mixture was sealed at 120°C for 13 h under nitrogen protection. After the reaction of the raw material a68 was completed, it was cooled to room temperature, diluted with water (5 mL), extracted with ethyl acetate (6 mL*3), the organic layers were combined, dried over anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure to obtain a residue, which was purified by column chromatography (Vpetroleum ether:Vethyl acetate=10:1~1:1) to obtain 1.2 g of a yellow solid with a yield of 86.2% and mp150.1~152.3°C.

[0174] 1 H-NMR (300MHz, CDCl3) δ (ppm): 8.08-7.95 (m, 3H, ArH), 7.25 (d, J = 8.0Hz, 2H, ArH), 4.78 (s, 1H, NH), 3.92 (s, 3H, N CH3 ),3.44-3.35(m,2H, CH2 NH), 2.82(t, J=7.0Hz, 2H, Ph CH2 ),1.43(s,9H,C (CH3)3 ).

[0175] Dissolve a70 (2.0 g, 6.61 mmol) in dichloromethane (5 mL), add trifluoroacetic acid (5 mL), and stir at room temperature for 2 h. After the reaction is complete, detect by TLC (V petroleum ether: V ethyl acetate = 1:1), and dry the solvent under reduced pressure to obtain a yellowish solid a71 trifluoroacetate, which is directly used in the next step without further treatment.

[0176] a71 (516 g, 1.63 mmol) was dissolved in dichloromethane (3 mL), cooled to 0°C, triethylamine (330 g, 3.26 mmol) and chloroacetyl chloride (277 mg, 2.45 mmol) were added, and the mixture was stirred at 0°C for 1 h. After the reaction of the raw material a71 was completed, TLC (V dichloromethane: V methanol = 20:1) detected that the reaction was complete, water (4 mL) was added to quench the mixture, and dichloromethane was extracted (3 mL*3). The organic layers were combined, dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain the residue, which was separated by column chromatography (V dichloromethane: V methanol = 200:1-100:1) to obtain 170 mg of yellow solid a72, with a yield of 37.4%, and mp 130.0-132.0°C.

[0177] 1 H-NMR (300MHz, CDCl3) δ (ppm): 8.10 (s, 1H, ArH), 8.08 (d, J = 8.2Hz, 2H, ArH), 7.34-7.31 (d, J = 2.0Hz, 2H, ArH), 6.68 (s, 1H, NH), 4.07 (s, 2H, CH2 CO),4.01(s,3H,N CH3 ),3.64(q,J=6.7Hz,2H, CH2 NH), 2.93(t, J=7.0Hz, 2H, Ph CH2 ).

[0178] Exemplarily, the synthetic operation process of the target compound EPII-6 is:

[0179]

[0180] a14a (12.9 g, 86.9 mmol), a13 (20.0 g, 72.4 mmol), tetrakistriphenylphosphine palladium (3.2 g, 3.6 mmol) and potassium phosphate (46.1 g, 217.2 mmol) were dissolved in 1,4-dioxane / water (400 / 40 mL), and heated to 90°C under nitrogen protection for 10 h. After the reaction of the raw materials was complete as detected by TLC (V petroleum ether: V ethyl acetate = 3:1), ethyl acetate (40 mL) was added for separation, and the solvent was dried to obtain the residue, which was separated by column chromatography (V petroleum ether: V ethyl acetate = 50:1-20:1) to obtain 13.2 g of white solid a15a, with a yield of 69.4% and mp 140.1-141.9°C.

[0181] (300MHz, CDCl3) δ (ppm): 8.65 (d, J = 5.3 Hz, 1H), 8.61-8.60 (m, 1H), 8.16 (dd, J = 9.8, 1.8Hz,1H),7.68(d,J=5.3Hz,1H),7.41(d,J=8.1Hz,1H),3.95(s,3H),2.68(s,3H).

[0182] Dissolve the raw material a15a (10 g, 38.1 mmol) and NBS (10.2 g, 57.11 mmol) in benzene (150 mL), add azobisisobutyl cyanide (1.3 g, 7.6 mmol), and heat to 80°C for 6 h. TLC (V petroleum ether: V ethyl acetate = 1:1) was used to detect that the raw material had reacted completely. Cool to room temperature, concentrate to obtain a red oil, and directly use it in the next step without treatment.

[0183] A16a (13.0 g, 38.1 mmol), glycine methyl ester hydrochloride (a17, 4.8 g, 38.1 mmol) and triethylamine (11.6 g, 114.21 mmol) were dissolved in acetonitrile 4 (10 mL) and heated under reflux for 6 h. After the reaction was completed, the solvent was dried to obtain the residue, which was separated by column chromatography (V petroleum ether: V ethyl acetate = 5:1 to 1:1) to obtain 5.6 g of light yellow solid a18a, with a yield of 46.3% and mp of 182.2 to 183.9 °C.

[0184] (300MHz, CDCl3) δ (ppm): 8.71 (d, J = 5.3Hz, 1H), 8.51-8, 50 (m, 1H), 8.46 (dd, J = 9.7, 1.7Hz ,1H),7.76(d,J=5.3Hz,1H),7.64(d,J=8.0Hz,1H),4.64(s,2H),4.46(s,2H),3.80(s,3H).

[0185] a18a (2.0 g, 6.29 mmol) was dissolved in 1,4-dioxane (10 mL), and LiOH.H2O (0.66 g, 15.73 mmol) aqueous solution (6 mL) was added under ice bath, and the mixture was moved to room temperature for reaction for 2 h. After the reaction was completed, the 1,4-dioxane was partially dried under reduced pressure, and the residue was adjusted to pH 2-3 with 6N HCl to precipitate solids. The mixture was stirred for 30 min, and solids precipitated. 1.56 g of golden solid a19a was obtained by suction filtration, with a yield of 81.7%, and mp>250°C.

[0186] (300MHz, DMSO-d6)δ(ppm):8.84(d,J=5.3Hz,1H),8.48-8.43(m,2H),8.30(d,J=5.3Hz,1H),7.82(d,J=8.0Hz,1H),4.62(s,2H),4.32(s,2H).

[0187] a19a (216 mg, 1.02 mmol), a32 (300 mg, 0.99 mmol), EDCI (229 mg, 1.19 mmol), HOBt (161 mg, 1.19 mmol), DIEA (155 mg, 1.19 mmol) were dissolved in dichloromethane (7 mL) and stirred at room temperature for 24 h. After the reaction was completed, dichloromethane (6 mL) was added and washed with water (5 mL * 3), the organic layer was dried, filtered, and the filtrate was evaporated under reduced pressure to give 270 mg of white solid a42b, yield: 54.3%, mp 221.9~223.7℃.

[0188] (300MHz, CDCl3) δ (ppm): 8.69 (d, J = 5.1Hz, 1H), 8.41-8.38 (m, 2H), 7.97 (s, 1H), 7.78 (d, J = 8.5Hz, 2H), 7.71 (d, J = 5.4Hz, 1H) ,7.56(d,J=8.2Hz,1H),6.57(d,J=8.9Hz,2H),4.54(s,2H),4.27(s,2H),3.94(s,3H),3.59-3.53(m,2H),3.40-3.35(m,2H).

[0189] a42b (100 mg, 0.20 mmol), 4-aminotetrahydropyran (a53b, 40.5 mg, 0.40 mmol) and DIEA (53 mg, 0.40 mmol) were dissolved in sec-butyl alcohol (3 mL), sealed and heated to 125°C for 12 h. TLC (V dichloromethane: V methanol = 30:1) detected that the reaction was complete, the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and column chromatography (eluent: dichloromethane: methanol = 200:1-40:1) was used for separation to obtain 70 mg of white solid EPII-6, yield: 61.7%, mp218.5-220.2°C.

[0190] (300MHz, CDCl3) δ (ppm): 8.44 (s, 1H), 8.38 (d, J = 5.3Hz, 1H), 8.26 (dd, J = 7.5, 1.7Hz, 1H ),7.99(s,1H),7.86(d,J=8.5Hz,2H),7.52(d,J=8.2Hz,1H),7.04(d,J=5.0Hz,1H),6.62 (d,J=8.5Hz,2H),4.48(s,1H),4.28(s,2H),4.21-4.15(m,1H),4.06-4.02(m,2H),3.94 (s,3H),3.65-3.53(m,4H),3.36(t,J=6.2Hz,2H),2.15-2.09(m,2H),1.66-1.60(m,2H).

[0191] (75MHz, DMSO-d6)δ(ppm):168.70,168.35,163.20,162.11,162.04,149.70,145.42,144.93,137.35,132.83,130.49 ,127.37,124.37,121.45,118.97,114.94,112.21,106.16,66.58,51.22,47.10,45.43,42.29,36.14,32.83,27.27.

[0192] Exemplarily, the synthetic operation process of the target compound EPII-2 is:

[0193]

[0194] a14b (1g, 5.45mmol), a13 (1.26g, 4.54mmol), Pd(PPh3)4 (263mg, 0.227mmol) and potassium phosphate (2.9g, 13.62mmol) were dissolved in 1,4-dioxane / water (15 / 5mL), and heated to 85℃ for 10h under nitrogen protection. After the reaction of raw material a13 was completed by TLC (V petroleum ether: V ethyl acetate = 3:1), it was cooled to room temperature, diluted with a small amount of water, extracted with ethyl acetate (15mL*3), the organic layers were combined, the solvent residue was dried under reduced pressure, and purified by column chromatography (V petroleum ether: V ethyl acetate = 80:1) to obtain 1.29g of white solid, yield: 95.6%, mp 129.0~130.0℃.

[0195] 1H-NMR (400MHz, CDCl3) δ (ppm): 8.65 (s, 1H, ArH), 8.61-8.60 (d, J = 2.0Hz, 1H, ArH), 7.96 (dd ,J=10.0,2.0Hz,1H,ArH),7.41(d,J=8.0Hz,1H,ArH),3.93(s,3H,OCH3),2.69(s,3H,CH3).

[0196] Dissolve a15b (0.6 g, 2.02 mmol) and NBS (539.13 mg, 3.03 mmol) in benzene (150 mL), add azobisisobutyl cyanide (66.34 mg, 0.404 mmol), and heat to 80°C for 10 h. TLC (V petroleum ether: V ethyl acetate = 5:1) was used to detect that the reaction of the raw material a15b was complete. Cool to room temperature and concentrate to obtain a red oil, which was directly used in the next step without treatment.

[0197] a16b (759.6 mg, 2.02 mmol), a17 (253.5 mg, 2.02 mmol) and Et3N (612.9 mg, 6.06 mmol) were dissolved in acetonitrile (4 mL) and heated under reflux for 6 h. After the reaction of the raw material a16b was complete, the temperature was lowered to stop the reaction, and the solvent was dried under reduced pressure to obtain a residue, which was separated by column chromatography (V petroleum ether: V ethyl acetate = 5:1 to 1:1) to obtain 555 mg of a light yellow solid, with a yield of 78.0% and mp 113.5 to 115.0°C.

[0198] 1 H NMR (300MHz, CDCl3-d) δ (ppm): 8.69 (s, 1H, ArH), 8.44 (s, 1H, ArH), 8.11 (d, J = 8.1Hz, 1H, ArH), 7.63 (d, J = 8.1Hz, 1H, ArH), 4.63 (s, 2H, N CH2 ),4.46(s,2H, CH2 N),3.78(s,3H,O CH3 )

[0199] Dissolve a18b (200 mg, 0.57 mmol) in 1,4-dioxane (3 mL), add LiOH.H2O (60 mg, 1.43 mmol) aqueous solution (1.8 mL) in an ice bath, stir at this temperature for 10 min, and then move to room temperature for reaction for 3 h. After the reaction of raw material a18b is complete, TLC (V dichloromethane: V methanol = 20:1) is used to detect that part of 1,4-dioxane is dried under reduced pressure, and the residue is adjusted to pH = 2-3 with 6N HCl, stirred for 30 min, solid precipitates, and is filtered and dried to obtain 78 mg of white solid, yield: 40.5%, mp 209.5-210.5℃.

[0200] 1 H NMR (300MHz, DMSO-d6) δ (ppm): 12.98 (s, 1H, COOH), 9.03 (s, 1H, ArH), 8.14 (s, 1H, ArH), 8.07 (d, J = 7.6Hz, 1H), 7.83 (d, J = 8.3Hz, 1H), 4.64 (s, 2H, N CH2 ),4.33(s,2H, CH2 N).

[0201] a19b (500 mg, 1.5 mmol), a29 (360 mg, 1.5 mmol), EDCI (350 mg, 1.8 mmol), HOBt (250 mg, 1.8 mmol), DIEA (240 mg, 1.8 mmol) were dissolved in dichloromethane (5 mL), stirred at room temperature for 24 h, and after the reaction of the raw material a19b was complete, dichloromethane (5 mL) was added, and the mixture was washed with water (5 mL) for 1 h. * 3), the organic layer was dried under reduced pressure, and the residue was separated by column chromatography (V dichloromethane: V methanol = 100: 1-50: 1) to obtain 214 mg of a white solid, with a yield of 25.5%, mp 243.0-244.5°C.

[0202] 1 H NMR(400MHz, CDCl3-d)δ(ppm):8.70-8.67(m,1H,ArH),8.45-8.44(m,1H,ArH),8.08 -8.06(m,4H,ArH),7.63-7.62(m,1H,ArH),7.46-7.44(m,2H,ArH),6.18-6.13(m,1H, CH C),4.70(s,2H,N CH2 ),4.58-4.53(m,2H,N CH2 ),4.28(s,2H,N CH2),3.98(s,3H,N CH3 ),3.86-3.82(m,2H,N CH2 ),2.68-2.61(m,2H,NCH2 CH2 ).

[0203] a42f (40 mg, 0.07 mmol), 4-aminotetrahydropyran (28.83 mg, 0.29 mmol) and DIEA (18.46 mg, 0.143 mmol) were dissolved in sec-butyl alcohol (2 mL), sealed and heated to 125°C for 12 h. TLC (V dichloromethane: V methanol = 20:1) detected that the reaction of the raw material a42f was complete, the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and column chromatography (V dichloromethane: V methanol = 200:1-30:1) was used for separation to obtain 26 mg of a white solid, yield: 58.6%, mp 254.5-255.5°C.

[0204] 1 H-NMR(300MHz,DMSO-d6)δ(ppm):8.51(s,1H,ArH),8.44(s,1H,ArH),8.04-7.96(m,4H,ArH),7.75(d ,J=8.4Hz,1H,ArH),7.57-7.54(m,2H,ArH),6.29(s,1H,tetrahydropyridine-H),4.59-4.54(m,4H, CH2 N CH2 ),4.29-4.15(m,2H,N CH2 CH), 3.92-3.85(m,6H,N CH3 , CH NH,N CH2 CH2),3.76-3.73(m,2H,CH2O CH2 ),3.41-3.37(m,2H,CH2O CH2 ),2.66-2.54(m,2H,NCH2 CH2 ),1.87-1.83(m,2H, CH2 CHCH2), 1.55-1.51(m,2H,CH2CH CH2 ).

[0205] 13C-NMR(75MHz,Acetic-d4)δ(ppm):169.74,167.74,160.38,145.52,144.50,141.34,135.50,134.44,132.86,131.57,128.40,126.5 3,125.14,124.67,122.99,120.76,115.97,115.73,104.31,99.06,66.06,51.68,47.26,35.99,31.92,26.88,19.64,19.38,19.12.

[0206] HRMS(ESI):m / z[M+H] + .Calcd for C 33 H 34 ClN8O3:625.2437; Found:625.2431.

[0207] IR(cm -1 ):3433.30,1686.10,1664.13,1570.18,1437.63,1402.78,1201.06,1083.91,757.78.

[0208] ERK2 enzyme inhibition activity assay

[0209] Reagents: ERK2 enzyme was purchased from Signalchem, USA; ATP detection kit was purchased from Promega, USA; ATP was purchased from Roche; the assay buffer solution was prepared in-house in the laboratory.

[0210] Instruments: CA-13901-1 clean bench is a product of Shanghai Purification Equipment Co., Ltd.; BT 124S 1 / 100000 balance is a product of Sartorius; HF safe 1500 continuous sampler is a product of eppendorf; multifunctional microplate reader is a product of Molecular Devices.

[0211] All compounds were dissolved in DMSO to prepare 10mM stock solution, and then the compounds were added to the screening system. The detection concentration range of the compounds was 0.05nM-1μM, and they were diluted in a 3-fold gradient, with two replicates for each concentration. The experimental results were converted into activity percentages, and the dose-effect curve was drawn. The inhibition IC was calculated using GRAPHPAD PEISM 5 nonlinear regression. 50 value.

[0212] The enzyme reaction system is composed of 20mM Hepes (pH 7.5), 10mM MgCl2, 1mM EGTA, 0.02% Brij35, 0.02mg / ml BSA, 0.1mM Na3VO4, 2mM DTT, 10μM ATP, kinase, kinase substrate; add different concentrations of screening compounds at the same time to form a 50μl reaction system, react at room temperature for 2h, and then use the luciferase method to detect the ADP content in the system. After reacting for another 5min, the chemiluminescent signal is detected on the MD-SpectraMax M5 multifunctional microplate reader. The intensity of the chemiluminescent signal value is proportional to the inhibition of enzyme activity. The detected chemiluminescent signal value is substituted into the formula:

[0213] Enzyme activity percentage (%) = {(Lu drug-Lu background) / (Lu enzyme-Lu background)} x 100% and then processed with GraphpadPrism 5 software to calculate the IC of the compound. 50 value.

[0214] The structures of compounds EPII-1 to EPII-5 and their inhibitory activities against EKR2:

[0215]

[0216]

[0217] The preliminary results of the ERK2 kinase inhibitory activity test showed that the R2 substituent was a chlorine atom or a hydrogen atom, which had little effect on the ERK2 inhibitory activity; when R1 was replaced by a methoxy group from a tetrahydropyran-4-amino group or a 1-methylpyrazole-4-amino group, the ERK2 inhibitory activity was basically lost. Although compounds EPII-1 to EPII-4 have good ERK2 inhibitory activity, the solubility of such compounds is very poor due to the large size of the compound molecules and the strong rigidity of the structure.

[0218] Furthermore, using compounds EPII-1 and EPII-2 as lead compounds, the right fragments were modified by ring opening or introduction of tertiary amine structures to design and synthesize compounds EPII-6 to EPII-13. The structures of compounds EPII-6 to EPII-13 and their inhibitory activities against EKR2:

[0219]

[0220]

[0221] The preliminary results of ERK2 inhibitory activity test showed that when R 2 When R is a fatty side chain (EPII-7 and EPII-8), the inhibitory activity against ERK2 is almost completely lost;2 When N,N-dimethylamino (EPII-12) replaced 1-methyl-1,2,4-triazol-3-yl, the activity decreased significantly; compounds EPII-9 and EPII-10 had too short aromatic side chains of their R2 substituents, which greatly reduced their inhibitory activity against ERK2. 50 =20 nM) had the best activity.

[0222] In order to improve the physicochemical properties, the present invention further designed and synthesized compound EPII-14 and compound, whose structures and inhibitory activities against EKR2 are as follows:

[0223]

[0224] The results showed that compound EPII-14 (IC 50 =19 nM) and compound EPII-15 (IC 50 =56nM) had better ERK2 inhibitory activity and good solubility.

[0225] Human tumor cell proliferation inhibition experiment

[0226] Reagents: Imatinib (Sigma, St. Louis, MO, USA); fetal bovine serum (FBS) (GIBCO, Invitrogen Corporation, NY, USA), Leibovitz's L-15 medium, RPMI-1640, McCoy's 5a (GIBCO, Invitrogen Corporation, NY, USA), penicillin (Sigma, St. Louis, MO, USA), streptomycin (Sigma, St. Louis, MO, USA), and MTT (Shengxing Biotechnology Co., Ltd.).

[0227] Instruments: FORMA 700 ultra-low temperature refrigerator, Thermo Company; YC-300L medicine storage cabinet, Zhongke Meiling Cryogenic Technology Co., Ltd.; Direct-Q with pump ultrapure water instrument, Millopore Company; SW-CJ-2FD ultra-clean workbench: Suzhou Purification Equipment Co., Ltd.; Berthold LB941 microplate multifunctional microplate reader, Berthold Company.

[0228] A375, Colo-205, A2058, and HT-29 were purchased from Shanghai Cell Bank.

[0229] Concentration selection of II series compounds: 0.016μM, 0.049μM, 0.148μM, 0.444μM, 1.333μM, 4.00μM, 12μM, 60μM.

[0230] Cell Culture

[0231] The cells were subcultured for 10-15 generations in a medium containing penicillin (final concentration of 100U / ml), streptomycin (final concentration of 100μg / ml), and 10% FBS (II series compounds: A2058, A375: DMEM, Colo-205: RPMI-1640, HT-29: McCoy's 5A). When the cells were 90% confluent, the old medium was discarded, and the cells were washed twice with 2ml PBS. After discarding PBS, 2ml of 0.25% (w / v) Trypsin-0.53mM EDTA mixed digestion solution was added and observed under a microscope for about 30s. When the cells became round, 2ml of complete medium was quickly added to terminate digestion. The cells were gently blown and collected. Centrifuged at 800rpm, 4℃ for 5min, the supernatant was discarded, the cells were resuspended with complete medium, cultured in bottles, and the medium was changed every other day.

[0232] MTT assay

[0233] Cells in logarithmic growth phase were collected at 1×10 5 cells / well were inoculated in a 96-well plate and cultured at 37°C and 5% CO2 until the cells were 90% confluent, and then incubated in serum-free medium for 2 hours to synchronize the cells. Subsequently, the supernatant was discarded, and the culture medium containing each compound (II series compounds: 0.016μM, 0.049μM, 0.148μM, 0.444μM, 1.333Μm, 4.00μM, 12μM, 60μM) was added and incubated (II series compounds were incubated for 96 hours). 4 hours before the end of incubation, 20μl of MTT solution (5mg / ml) was added to each well. After the incubation, the supernatant of each well was discarded, 150μL of DMSO was added to each well, and the cells were shaken on a cell shaker for 10 minutes. After the crystals were fully dissolved, the OD was measured using an enzyme marker. 570 .

[0234] Proliferation inhibition activity percentage (%) = (OD value of drug administration - OD value of background) / (OD value of control - OD value of background) x 100%

[0235] The drug concentration was used as the horizontal axis, and the percentage of proliferation inhibition activity corresponding to each concentration was used as the vertical axis. Graphpd Prism5 was used for nonlinear regression to calculate the inhibitory IC of each compound. 50 value.

[0236] Compounds EPII-1, EPII-2, EPII-6, EPII-14 and EPII-15 have antiproliferative effects on four ERK pathway activated cell lines:

[0237]

[0238] Compounds EPII-1, EPII-2, EPII-6, EPII-14 and EPII-15 with good ERK2 inhibitory activity were selected to determine their anti-proliferation abilities against tumor cell lines containing RAS-RAF-MEK-ERK mutations, human colon cancer cells Colo-205, human melanoma cells A375, human skin cancer cells A2058 and human colon cancer cells HT-29. The experimental results showed that EPII-1 only had a certain inhibitory activity on the proliferation of A375 cells; EPII-2 had a certain proliferation inhibitory effect on A375, A2058 and COLO-205, but its activity on HT-29 was weaker; EPIII-6 had a certain proliferation inhibitory effect on A375 and COLO-205, a weaker inhibitory effect on A2058, and basically lost its proliferation inhibitory activity on HT-29; EPII-14 had poor proliferation inhibitory activity on the four tumor cells; EPII-15 had a certain inhibitory activity on the four tumor cells, among which the proliferation inhibitory activity on COLO-205 cells was 1.083μM.

[0239] The above embodiments are exemplary, and their purpose is to illustrate the technical concept and features of the present invention so that people familiar with the technology in this field can understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a dual-mechanism ERK inhibitor, characterized in that: The dual-mechanism ERK inhibitor is a compound of formula EPII-2: The compound of formula EPII-2 is prepared according to the following reaction path:

2. The method for preparing the dual-mechanism ERK inhibitor according to claim 1, characterized in that: The compound of formula EPII-2 is prepared according to the following reaction steps: 1 part by mass of compound a14b, 1.26 parts by mass of compound a13, 0.263 parts by mass of Pd(PPh3)4 and 2.9 parts by mass of potassium phosphate were dissolved in a mixed solvent of 1,4-dioxane and water, wherein the volume ratio of 1,4-dioxane to water was 15:5, and heated to 85°C under protective gas conditions for reaction. After the reaction was complete, the mixture was cooled to room temperature, diluted with water, extracted with ethyl acetate to obtain an organic phase, and the residue obtained after the solvent was dried under reduced pressure was purified by column chromatography to obtain a white solid compound a15b; 0.6 parts by mass of a15b and 0.539 parts by mass of NBS were dissolved in benzene, 0.066 parts by mass of azobisisobutyl cyanide were added, and the mixture was heated to 80°C for reaction. After the reaction was complete, the mixture was cooled to room temperature and concentrated to obtain a red oily substance containing compound a16b. The red oily substance was directly used in the next step without further treatment. A certain amount of a red oily substance containing 0.759 parts by mass of a16b, 0.253 parts by mass of compound a17 and 0.612 parts by mass of Et3N were dissolved in acetonitrile, heated under reflux for reaction, and after the reaction was complete, the temperature was lowered to stop the reaction, and the solvent was evaporated under reduced pressure to obtain a residue, which was separated by column chromatography to obtain a light yellow solid compound a18b; 0.2 parts by mass of a18b was dissolved in 1,4-dioxane, and 0.06 parts by mass of LiOH was added under ice bath. . The mixture was stirred for 10 min in an aqueous solution of H2O, and then moved to room temperature for reaction. After the reaction was complete, part of 1,4-dioxane was dried under reduced pressure, and the residue was adjusted to pH 2-3 with HCl, and stirred. The precipitated solid was filtered and dried to obtain a white solid compound a19b. 0.5 parts by mass of a19b, 0.36 parts by mass of compound a29, 0.35 parts by mass of EDCI, 0.25 parts by mass of HOBt, and 0.24 parts by mass of DIEA were dissolved in dichloromethane, and the mixture was stirred at room temperature for reaction. After the reaction was completed, dichloromethane was added, and the mixture was washed with water. The organic layer was then dried under reduced pressure, and the residue was separated by column chromatography to obtain a white solid compound a42f. 0.04 mass parts of a42f, 0.028 mass parts of 4-aminotetrahydropyran and 0.018 mass parts of DIEA were dissolved in sec-butanol, sealed and heated to 125°C for reaction. After the reaction was complete, the reaction solution was cooled to room temperature, the solvent was evaporated under reduced pressure, and separated by column chromatography to obtain a white solid, i.e., a compound with the structure of formula EPII-2.

3. The method for preparing the dual-mechanism ERK inhibitor according to claim 1, characterized in that: The compound a29 used in the reaction path is prepared according to the following reaction path:

4. A method for preparing a dual-mechanism ERK inhibitor, characterized in that: The dual-mechanism ERK inhibitor is a compound of formula EPII-15: The compound of formula EPII-15 is prepared according to the following reaction path:

5. The method for preparing the dual-mechanism ERK inhibitor according to claim 4, characterized in that: The reaction conditions in the reaction path for preparing the compound of formula EPII-15 are as follows: a: i-PrI, K2CO3, N,N-dimethylformamide; b: B2Pin2, Pd(dppf)2Cl2, KOAc, dimethyl sulfoxide; c: Pd(PPh3)4, Na2CO3, toluene / ethanol / water, 100°C; d: Pd / C, H2, isopropanol / toluene; e: HATU, N,N-diisopropylethylamine, N,N-dimethylformamide; f: trifluoroacetic acid, dichloromethane; g: N,N-diisopropylethylamine, N,N-dimethylformamide, 50℃.

6. The method for preparing the dual-mechanism ERK inhibitor according to claim 4, characterized in that: The compound of formula EPII-15 is prepared according to the following reaction steps: 1.0 parts by mass of compound a49, 2.4 parts by mass of potassium carbonate and 1.5 parts by mass of compound a50 were dissolved in DMF, and the mixture was stirred at room temperature for reaction. After the reaction was complete, water and ethyl acetate were added for dilution, and the mixture was allowed to stand for separation. The organic layer was washed with water, dried over anhydrous sodium sulfate, filtered, and dried under reduced pressure to obtain a residue, which was then subjected to column chromatography to obtain solid compound a51; 0.2 parts by mass of a51, 0.284 parts by mass of biboric acid pinacol ester, 0.068 parts by mass of Pd(dppf)Cl2 and 0.274 parts by mass of potassium acetate were dissolved in DMSO, and reacted at 100°C under protective gas conditions. After the reaction was complete, the mixture was cooled to room temperature to obtain a mixture containing compound a52, which was directly used for the next step without treatment; A certain amount of the above mixture containing 0.245 parts by mass of a52, 0.45 parts by mass of compound a48, 0.108 parts by mass of Pd(pph3)4 and 0.493 parts by mass of sodium carbonate were dissolved in a mixed solution of H2O:toluene:ethanol, with a volume ratio of H2O:toluene:ethanol being 1:1:1, and reacted at 100°C under protective gas conditions. After the reaction was complete, the mixture was cooled to room temperature, diluted with water and ethyl acetate, filtered through diatomaceous earth, and the filtrate was allowed to stand for stratification. The organic layer was washed with water and dried under reduced pressure to obtain a residue, which was separated by column chromatography to obtain a yellow solid compound a54; 1.89 parts by mass of a54 and 0.19 parts by mass of 50% wet Pd / C were dissolved in a mixed solution of toluene and isopropanol; the mass content of Pd in ​​Pd / C was 10%, and the volume ratio of toluene to isopropanol in the mixed solution was 1:2; hydrogen was then introduced, and the reaction was carried out at room temperature. After the reaction was complete, diatomaceous earth was used for filtration, and the filtrate was decompressed and dried to obtain a residue, which was separated by column chromatography to obtain an off-white solid compound a63; 1.04 parts by mass of a63, 0.531 parts by mass of a62, 0.772 parts by mass of HATU and 0.289 parts by mass of DIEA were dissolved in DMF, stirred at room temperature for reaction, diluted with water after the reaction was complete, extracted with ethyl acetate, collected the organic layer, and dried the solvent under reduced pressure to obtain the residue, which was purified by column chromatography to obtain a light yellow solid compound a64; 0.1 parts by mass of a64 was dissolved in dichloromethane, trifluoroacetic acid and water were added, and the mixture was stirred at room temperature for reaction. After the reaction was completed, the mixture was cooled to 0°C and saturated sodium carbonate was added for quenching. The solvent was evaporated under reduced pressure, and water was added to the residue. The organic layer was extracted with ethyl acetate and collected. Anhydrous sodium sulfate was added for drying, and the mixture was filtered and evaporated under reduced pressure to obtain a mixture containing compound a65, which was directly used for the next step without further treatment. A certain amount of the above mixture containing 0.2 mass parts of a65, 0.137 mass parts of compound a72 and 0.38 mass parts of DIEA were dissolved in DMF and heated to 50°C for reaction. After the reaction was complete, the reaction solution was cooled to room temperature, diluted with water, extracted with ethyl acetate and the organic layer was collected, dried with anhydrous sodium sulfate, filtered, and the solvent was evaporated under reduced pressure. After separation by column chromatography, 132 mg of solid was obtained, with a yield of 41.2%, mp140.0~141.5°C.

7. The method for preparing the dual-mechanism ERK inhibitor according to claim 4, characterized in that: The compound a72 used in the reaction path was prepared according to the following reaction path:

8. The method for preparing a dual-mechanism ERK inhibitor according to claim 4, characterized in that: The compound a62 used in the reaction path is prepared according to the following reaction path:

9. A use of a dual mechanism ERK inhibitor, characterized in that: The dual-mechanism ERK inhibitor is selected from compounds of EPII-2 structure or EPII-15 structure: The dual-mechanism ERK inhibitor is used in preparing medicine for treating colon cancer, melanoma or skin cancer.

10. The use of the dual mechanism ERK inhibitor according to claim 9, characterized in that: The dual-mechanism ERK inhibitor is prepared into a pharmaceutically acceptable salt and loaded into a pharmaceutically acceptable carrier to manufacture a drug for treating colon cancer, melanoma or skin cancer.

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