A pyrazine compound, a preparation method and application thereof

Abstract

The application belongs to the technical field of pharmaceutical chemistry, and particularly relates to a pyrazine compound and a preparation method and application thereof. The compound provided by the application is screened in a virtual screening and pharmacophore manner, has a novel structure, has good inhibitory activity on MNK1, and the above compound makes up for the problems of insufficient ideal activity and insufficient diversity of chemical structure of existing MNK1 inhibitors, and the like.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically relating to a pyrazine compound, its preparation method, and its application. Background Technology

[0002] Mitogen-activated protein kinase-interacting kinase (MNK1) plays a crucial role in cell signaling and carcinogenesis. Through its unique DFD motif and self-inhibitory conformation, MNK1 plays a vital role in regulating eIF4E phosphorylation and influencing mRNA translation, thereby participating in various cellular events, including cell proliferation, survival, and metastasis. Given the overexpression of MNK1 in various tumors and its significant association with poor prognosis, the development of specific MNK1 inhibitors has become an important direction in anti-tumor drug development.

[0003] Despite increased understanding of the structure and function of MNK1, research progress has been minimal. There remains a pressing need to develop new compounds that specifically inhibit MNK1 kinase activity, particularly interfering with MNK1's role in cancer. Although potential MNK1 inhibitors have been investigated for two decades, none have yet reached the market. Currently, only three are undergoing clinical trials for the treatment of solid tumors and leukemia (BAY1143269, eFT508, and ETC-206). Clearly, there is a need to develop additional potent and selective inhibitors of MNK1 to meet clinical and research needs. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a pyrazine compound, its preparation method and application, so as to solve the problems of insufficient development of existing MNK1 inhibitors, such as unsatisfactory activity and insufficient diversity of chemical structures.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is: a pyrazine compound, the structure of which is shown in Formula I; Formula I Wherein, R1 and R2 are Cl, substituted or unsubstituted benzene rings, substituted or unsubstituted pyridine, and R1 and R2 are not both Cl; The substituted benzene ring and the substituted pyridine are substituted by one or two substituents independently selected from the following: In this context, * indicates a connection site.

[0006] Specifically, the pyrazine compounds are: 1-(4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea (compound 1); N-(2,4-Difluorophenyl)-3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide (compound 2); 4-(4-(6-(3-(trifluoromethyl)phenyl)pyrazin-2-yl)benzyl)morpholine (compound 3); 1-(5-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)pyridin-2-yl)-3-(p-tolyl)urea (compound 4); 1-(3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea (compound 5); N-(2,4-difluorophenyl)-4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide (compound 6); Ethyl 3-(6-chloropyrazin-2-yl)benzoate (compound 7); 3-(6-(4-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)ethyl benzoate (compound 8); Ethyl 3-(6-(4-(trifluoromethyl)phenyl)pyrazin-2-yl)benzoate (compound 9); 3-(6-(3-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)ethyl benzoate (compound 10).

[0007] In addition, the present invention provides a pharmaceutical composition comprising the above-mentioned pyrazine compound or its pharmaceutically acceptable salt, isomer, hydrate, and at least one pharmaceutically acceptable carrier.

[0008] Furthermore, the present invention also provides a method for preparing the above-mentioned pyrazine compounds, which includes the following steps:

[0009] Based on the above technical solutions, the present invention may have the following further specific options or optimizations.

[0010] Specifically, in steps c and d, the solvent is water or ethanol, the catalyst is an inorganic base and tetra(triphenylphosphine)palladium, and the reaction is carried out at 80-100℃ for 10-20 hours under nitrogen protection.

[0011] Preferably, the inorganic base is cesium carbonate, potassium carbonate, sodium carbonate, or sodium hydroxide. In step c, the ratio of the feed material 2,6-dichloropyrazine to dioxoborane compounds is 1:(1-3) by molar amount.

[0012] It should be noted that the above synthesis method has been carefully optimized in terms of reaction conditions, type and amount of alkali, and feed ratio to improve reaction yield and product purity.

[0013] In addition, the present invention also provides the use of the above-mentioned pyrazine compounds, specifically for use in the preparation of MNK1 inhibitors, drugs that inhibit MNK1-related signaling pathways, and anticancer drugs.

[0014] Specifically, the compounds provided by this invention exhibit significant inhibitory activity against a variety of tumor cells, including but not limited to: highly metastatic human liver cancer cells (MHCC97-H cells), human lung cancer cell line (A549 cells), human breast cancer cells (MCF7), human leukemia cells (K562), cervical cancer cells (HeLa), ovarian cancer cells (SKOV3), gastric adenocarcinoma cells (AGS), and prostate cells (PC-3). Therefore, this invention provides the use of the above-mentioned compounds, their pharmaceutically acceptable salts, their hydrates, or the pharmaceutical compositions of this invention in the preparation of drugs for treating liver cancer, lung cancer, breast cancer, leukemia, cervical cancer, ovarian cancer, gastric adenocarcinoma, and prostate cancer.

[0015] It should be noted that the compounds provided in this invention were obtained through virtual screening and pharmacophore-based methods. By deeply analyzing the structure and function of MNK1, its key role in tumor cell biology was clarified, leading to systematic and rational inhibitor design and synthesis research. Based on a thorough understanding of the MNK1 structure, especially its unique DFD motif and self-inhibitory conformation, a pharmacophore model with clear pharmacodynamic characteristics was constructed. Using Discovery Studio software for calculation and optimization, novel compounds with pyrazine as the core parent ring and specific R1 and R2 side chains were designed. Through virtual docking screening, compounds with excellent scores and potential activity were selected for subsequent synthesis. These structural characteristics are the result of mutual verification between the inventors' virtual docking design and compound synthesis, resulting in compounds with very high activity, good selectivity, better biotolerance, and promising drug development prospects. Attached Figure Description

[0016] Figure 1 Compound 01 1 HNMR spectrum.

[0017] Figure 2 Compound 01 13 CNMR spectrum.

[0018] Figure 3 Compound 02 1 H NMR spectrum.

[0019] Figure 4 Compound 02 13 C10 NMR spectrum.

[0020] Figure 5 HRMS plot of compound 02.

[0021] Figure 6 Compound 03 1 H NMR spectrum.

[0022] Figure 7 Compound 03 13 C10 NMR spectrum.

[0023] Figure 8 HRMS plot of compound 03.

[0024] Figure 9 Compound 04 1 H NMR spectrum.

[0025] Figure 10 Compound 04 13 C10 NMR spectrum.

[0026] Figure 11 HRMS plot of compound 04.

[0027] Figure 12 Compound 05 1 H NMR spectrum.

[0028] Figure 13 HRMS plot of compound 05.

[0029] Figure 14 Compound 06 1 H NMR spectrum.

[0030] Figure 15 HRMS plot of compound 06.

[0031] Figure 16 Compound 07 1 H NMR spectrum.

[0032] Figure 17 HRMS plot of compound 07.

[0033] Figure 18 Compound 08 1 H NMR spectrum.

[0034] Figure 19 Compound 08 13 C10 NMR spectrum.

[0035] Figure 20 HRMS plot of compound 08.

[0036] Figure 21 Compound 09 1 H NMR spectrum.

[0037] Figure 22 Compound 09 13 C10 NMR spectrum.

[0038] Figure 23 HRMS plot of compound 09.

[0039] Figure 24 Compound 10 1 H NMR spectrum.

[0040] Figure 25 Compound 10 13 C10 NMR spectrum.

[0041] Figure 26 Virtual docking diagram of compound 01.

[0042] Figure 27 Virtual docking diagram of compound 02.

[0043] Figure 28 Virtual docking diagram of compound 08. Detailed Implementation

[0044] To better understand the present invention, the following description, in conjunction with the accompanying drawings and specific embodiments, further clarifies the content of the present invention, but the content of the present invention is not limited to the following embodiments.

[0045] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0046] Example 1: This invention provides a method for synthesizing pyrazine compounds. Taking the synthesis of 1-(3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea (compound 5) as an example, the specific operation steps are as follows: Reaction c: In a 100 mL single-necked flask, 0.50 g (3.38 mmol) of 2,6-dichloropyrazine, 1.40 g (10.14 mmol) of K₂CO₃, and 1.79 g (5.07 mmol) of 1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxoborheptacyclopentan-2-yl)phenyl)-3-(p-tolyl)urea were dissolved in dioxane (15... 1 mL of water was added to the solution. Then 0.19 g (0.17 mmol) of tetra(triphenylphosphine)palladium was added, and the mixture was reacted overnight at 85 °C under nitrogen atmosphere with five venting and five purging cycles. The reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was evaporated to dryness, washed with 30 mL of water, extracted with 30 mL × 3 dichloromethane, dried over anhydrous sodium sulfate, and purified by column chromatography (V dichloromethane / V methanol = 30:1) to give 0.32 g of 1-(3-(6-chloropyrazin-2-yl)phenyl)-3-(p-tolyl)urea (white solid), with a yield of 27.7%. ¹H NMR (400 MHz, Chloroform-d) δ 8.95 (s, 1H), 8.61 (s, 1H), 8.03 – 7.96 (m, 2H), 7.51 (d, J = 8.2 Hz, 2H). 3.78 – 3.72 (m,4H), 3.60 (s, 2H), 2.50 (t, J = 4.6 Hz, 4H). Reaction d: In a 100 mL single-necked flask, 0.30 g (0.89 mmol) of 1-(3-(6-chloropyrazin-2-yl)phenyl)-3-(p-tolyl)urea, 0.37 g (2.66 mmol) of K₂CO₃, and 0.32 g (1.06 mmol) of 4-(4-morpholinomethyl)phenylboronic acid pinacol ester were dissolved in 15 mL of dioxane solution, and 1 mL of water was added. Then, 0.05 g (0.04 mmol) of tetra(triphenylphosphine)palladium was added, and the reaction was carried out overnight at 85 °C under nitrogen atmosphere with five venting and five evacuation cycles. The progress of the reaction was monitored by TLC. After the reaction was complete, the reaction solution was evaporated to dryness, washed with 30 mL of water, extracted with 30 mL of dichloromethane, dried over anhydrous sodium sulfate, and purified by column chromatography (V dichloromethane / V methanol = 10:1) to obtain 0.32 g of 1-(3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea, with a yield of 76.4%.

[0047] Example 2: Compound 01 (1-(4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea) The synthesis method was consistent with that of compound 05 in Example 1. The parent ring starting material for compound 01 was 2,6-dichloropyrazine, with side chains of 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)benzyl)morpholine and 1-(3-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)phenyl)-3-(p-tolyl)urea. The product was a white powder. Overall yield: 14.7%. 1H NMR (400 MHz, DMSO-d6) δ 9.13 (d, J = 14.4 Hz, 2H), 8.92 (s, 1H), 8.64 (s, 1H), 8.25 – 8.13 (m, 4H), 7.69 – 7.61 (m, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.41–7.34 (m, 2H), 7.11 (d, J = 8.2 Hz, 2H), 3.60 (t, J = 4.6 Hz, 4H), 3.55 (d, J = 4.4 Hz, 2H), 2.40 (t, J = 4.7 Hz, 4H), 2.26 (s, 3H).13C NMR (101MHz, DMSO-d6)δ152.87, 150.65, 150.60, 142.23, 140.39, 139.91, 139.67, 137.39,135.34, 131.35, 129.97, 129.68, 129.49, 127.98, 127.18, 118.90, 118.59,66.69, 62.53, 53.68, 20.83. Example 3: Compound 02 (N-(2,4-difluorophenyl)-3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide) The preparation method for compound 05 in Example 1 was consistent with that for compound 02. The parent ring starting material for compound 02 was 2,6-dichloropyrazine, and the side chains were 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)morpholine and N-(2,4-difluorophenyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzamide. The product was a white powder with an overall yield of 15.2%. 1H NMR (400 MHz, Chloroform-d) δ 9.03 (d, J = 2.2 Hz, 2H), 8.44 (td, J = 9.3,8.8, 5.7 Hz, 1H), 8.32 (d, J = 8.1 Hz, 2H), 8.10 (dd, J = 27.5, 8.2 13C NMR (101 MHz, DMSO-d6)δ165.63, 150.97, 150.20, 141.13, 140.82, 140.64, 136.74, 135.07, 134.98,130.66, 130.03, 129.86, 129.79, 129.11, 129.08, 129.01, 128.98, 127.32,126.54, 111.88, 111.85, 111.66, 111.63, 105.18, 104.92, 104.67, 66.68, 62.51,53.68. HRMS(ESI):m / z[M+H]+ calcd for C22H24N4O2: 487.1867. found: 487.1952.

[0048] Example 4: Compound 03 (4-(4-(6-(3-(trifluoromethyl)phenyl)pyrazin-2-yl)benzyl)morpholine) The preparation method was consistent with that of compound 05 in Example 1. The parent ring starting material for the synthesis of compound 03 was 2,6-dichloropyrazine, and the side chains were 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)morpholine and 4,4,5,5-tetramethyl-2-(3-(trifluoromethyl)phenyl)-1,3,2-dioxaborane. The product was a white powder with an overall yield of 20.2%. 1H NMR (400MHz, Chloroform-d) δ 9.03 (d, J = 11.4 Hz, 2H), 8.29 (d, J = 8.1 Hz, 2H), 8.14 (d, J = 8.0 Hz, 2H), 7.82 (d, J = 8.1 Hz, 2H), 7.49 (t, J = 7.1 Hz, 2H),3.86–3.61 (m, 6H), 2.58 (s, 4H).13C NMR (101 MHz, DMSO-d6)δ151.02, 149.31,141.69, 141.07, 140.33, 134.96, 132.00, 131.91, 130.08, 129.29, 129.17,128.13, 127.32, 126.40, 126.36, 73.98, 66.64, 62.45, 53.64, 25.42. HRMS(ESI):m / z[M+H]+ calcd for C22H24N4O2: 400.1558. found: 400.1642.

[0049] Example 5: Compound 04 (1-(5-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)pyridin-2-yl)-3-(p-tolyl)urea) The preparation method for compound 05 in Example 1 was consistent with that used in the synthesis of compound 04. The parent ring starting material was 2,6-dichloropyrazine, and the side chains were 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)benzyl)morpholine and 1-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborin-2-yl)pyridin-2-yl)-3-(p-tolyl)urea. The product was a white powder, with an overall yield of 18.1%. 1H NMR (400 MHz, Chloroform-d) δ 9.57 (s, 1H), 9.06 (d, J = 2.3 Hz, 1H), 8.96 (d, J = 16.0 Hz, 2H), 8.44 (dd, J = 8.7, 2.4 Hz, 1H), 8.12 (d, J =7.9 Hz, 2H), 7.55 (t, J = 7.6 Hz, 4H), 7.21 (d, J = 8.0 Hz, 2H), 7.15 (d, J =8.7 Hz, 1H), 3.77 (t, J = 4.6 Hz, 4H), 3.63 (s, 2H), 2.53 (t, J = 4.6 Hz,4H), 2.38 (s, 3H).13C NMR (101 MHz, Chloroform-d)δ153.88, 145.00, 140.11,138.81, 136.94, 135.66, 135.16, 133.30, 129.85, 129.54, 127.01, 120.48,112.34, 75.06, 67.03, 63.09, 53.68, 24.88, 20.91. HRMS(ESI):m / z[M+H]+ calcdfor C22H24N4O2: 481.2274. found: 481.2355.

[0050] Example 6: Compound 06 (N-(2,4-difluorophenyl)-4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide) The preparation method for compound 05 in Example 1 was consistent with that used in the synthesis of compound 06. The parent ring starting material was 2,6-dichloropyrazine, and the side chains were 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzyl)morpholine and N-(2,4-difluorophenyl)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzamide. The overall yield was 16.7%. 1H NMR (400MHz, Chloroform-d) δ 9.03 (d, J = 2.2 Hz, 2H), 8.44 (td, J = 9.3, 8.8, 5.7Hz, 1H), 8.32 (d, J = 8.1 Hz, 2H), 8.10 (dd, J = 27.5, 8.2 Hz, 5H), 7.72 –7.44 (m, 3H), 6.97 (tdd, J = 11.2, 7.5, 3.1 Hz, 2H), 3.77 (t, J = 4.6 Hz, 4H), 3.63 (s, 2H), 2.53 (t, J = 4.6 Hz, 4H). HRMS(ESI):m / z[M+H]+ calcd forC22H24N4O2: 487.1867. found: 487.1952.

[0051] Example 7: Compound 07 (ethyl 3-(6-chloropyrazin-2-yl)benzoate) The preparation method of compound 05 in Example 1 was consistent with that of compound 07. The parent ring raw material for the synthesis of compound 07 was 2,6-dichloropyrazine, and the side chain was ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzoate. The product was a white powder with an overall yield of 34.7%. 1H NMR (400 MHz, Chloroform-d) δ 8.99 (s, 1H), 8.66 (dt, J = 3.7,1.8 Hz, 1H), 8.56 (s, 1H), 8.25 (dq, J = 7.8, 1.8 Hz, 1H), 8.17 (dq, J = 7.8,1.6 Hz, 1H), 7.60 (td, J = 7.8, 1.7 Hz, 1H), 4.44 (q, J = 7.1 Hz, 2H), 1.43(t, J = 7.1 Hz, 3H).HRMS(ESI):m / z[M+H]+ calcd for C22H24N4O2: 263.0509. found: 263.0594.

[0052] Example 8: Compound 08 (ethyl 3-(6-(4-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)benzoate) The preparation method for compound 05 in Example 1 was consistent with that for compound 08. The parent ring starting material for the synthesis of compound 08 was 2,6-dichloropyrazine, and the side chains were ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)benzoate and N-(2,4-difluorophenyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)benzamide. The product was a white powder with an overall yield of 19.2%. 1HNMR (400 MHz, Chloroform-d) δ 9.11 (d, J = 7.8 Hz, 2H), 8.83 (q, J = 2.0 Hz, 1H), 8.47 (dt, J = 8.6, 4.2 Hz, 1H), 8.44–8.38 (m, 1H), 8.38–8.32 (m, 2H), 8.23 ​​(dq, J = 7.8, 1.7 Hz, 1H), 8.14–8.07 (m, 2H), 7.58 (td, J = 7.3, 1.5 Hz, 2H), 7.02–6.92 (m, 2H), 4.49 (q, J = 7.1 Hz, 2H), 1.48 (t, J = 7.1 Hz, 2H).13C NMR (101 MHz, Chloroform-d)δ166.21, 164.89, 150.93, 150.29, 140.79,140.53, 139.80, 136.54, 135.38, 132.15, 132.05, 132.01, 131.99, 131.90,131.45, 131.35, 131.09, 131.05, 129.29, 129.24, 128.60, 128.48, 128.10,128.06, 127.90, 127.48, 123.31, 123.23, 111.55, 111.51, 111.33, 111.29,103.98, 103.75, 103.48, 61.37, 52.43, 14.39. HRMS(ESI):m / z[M+H]+ calcd forC22H24N4O2: 460.1394. found: 460.1475.

[0053] Example 9: Compound 09 (ethyl 3-(6-(4-(trifluoromethyl)phenyl)pyrazin-2-yl)benzoate) The preparation method of compound 05 in Example 1 was consistent with that used in the synthesis of compound 09. The parent ring starting material was 2,6-dichloropyrazine, and the side chains were ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborane-2-yl)benzoate and 4,4,5,5-tetramethyl-2-(3-(trifluoromethyl)phenyl)-1,3,2-dioxaborane. The product was a white powder with an overall yield of 20.5%. 1H NMR (400MHz, Chloroform-d) δ 9.09 (d, J = 20.2 Hz, 2H), 8.81 (t, J = 1.8 Hz, 1H), 8.56 – 8.05 (m, 4H), 7.93 – 7.56 (m, 3H), 4.48 (q, J = 7.1 Hz, 2H), 1.47 (t,J = 7.1 Hz, 3H).13C NMR (101 MHz, Chloroform-d)δ166.19, 150.97, 150.23,140.87, 140.49, 139.64, 136.49, 131.46, 131.34, 131.07, 129.25, found: 373.1173.

[0054] Example 10: Compound 10 (ethyl 3-(6-(3-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)benzoate) The preparation method was consistent with that of compound 05 in Example 1. The parent ring starting material for the synthesis of compound 10 was 2,6-dichloropyrazine, and the side chains were ethyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)benzoate and N-(2,4-difluorophenyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboran-2-yl)benzamide. The product was a white powder with an overall yield of 13.7%. 1H NMR (400 MHz, Chloroform-d) δ 9.09 (s, 2H), 8.74 (dt, J = 33.6,1.8 Hz, 2H), 8.53 – 8.31 (m, 3H), 8.26 – 8.09 (m, 2H), 8.01 (dt, J = 7.9, 1.4Hz, 1H), 7.68 (dt, J = 21.9, 7.8 Hz, 3H), 7.05–6.82 (m, 2H), 4.46 (q, J = 7.1Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H).13C NMR (101 MHz, DMSO-d6)δ165.92, 165.62, 150.37, 150.02, 141.59, 141.40, 136.84, 136.56, 135.06, 132.52, 132.00, 131.90, 131.31, 130.99, 130.66, 130.11, 129.91, 129.87, 129.29, 129.17, 129.05, 129.02, 128.92, 127.88, 126.66, 111.88, 111.84, 111.66, 111.62, 105.17, 104.93, 104.67, 61.52, 14.58. Example 11: Enzyme inhibitory activity study Enzyme activity was assessed using the ADP-Glo ​​kinase assay kit. MNK1 was obtained from Carna Biosciences, and the ADP-Glo ​​kinase assay kit (Promega) was used. The reaction buffer consisted of 15 mM HEPES (pH 7.4), 20 mM NaCl, 1 mM EGTA, 10 mM MgCl2, 0.1 mg / mL BGG, and 0.02% Tween-20. The terminal concentrations of MNK1 enzyme, substrate peptide (TATKSGSTTKNR), and ATP were 10 nM, 100 μM, and 300 μM, respectively. Compounds were screened by serial dilutions in the presence of 1% DMSO.

[0055] MNK1 enzyme and the compound were added to 384-well plates (Corning, New York, NY, USA) and incubated at room temperature for 10 minutes. Then, substrate peptides and ATP were added, and the plates were pre-incubated at room temperature for 50 minutes. ADP-Glo ​​reagent was then added, the reaction was cooled, and the plates were incubated for 40 minutes. Finally, kinase detection reagent was added, and the plates were incubated for 40 minutes to generate a luminescent signal. The luminescent signal was measured using a Spectramax 190 microplate reader (Molecular Devices, Valley, CA, USA), and the inhibition rate was calculated.

[0056] Table 1. Enzyme inhibition rates of compounds at concentrations of 10 nM and 100 nM. As shown in the table above, the inhibition rates of different compounds at 10 nM and 100 nM concentrations revealed significant differences in activity. The positive control drug Tomivosertib (eFT-508) exhibited inhibition rates of 81% and 95% at 10 nM and 100 nM concentrations, respectively, demonstrating its highly efficient inhibitory effect on MNK1 enzyme and providing a reliable reference benchmark for the experiment. Compound 01 showed the most outstanding performance, achieving an inhibition rate of 79.70% at 10 nM and further increasing to 96.28% at 100 nM, exceeding the activity of the positive control drug Tomivosertib, indicating its strong MNK1 inhibitory potential. Compounds 10, 05, and 06 also exhibited good inhibitory activity.

[0057] Example 12: Cytotoxicity studies To determine the inhibitory effect of compound 01 on K562 cells (IC50), 50 The MTT assay was used to perform the experiment. First, human leukemia cells (K562), human lung cancer cell line (A549 cells), and human breast cancer cells (MCF7) were cultured to the logarithmic growth phase, and the cell concentration was adjusted to 5 × 10³ cells / well. 100 μL of the solution was seeded into each well of a 96-well plate. Then, different concentrations of the compound working solution were added, with three replicates. An equal volume of DMSO was added to the control group. The plates were incubated at 37°C with 5% CO2 for 24 hours. After incubation, 20 μL of MTT solution (5 mg / mL) was added to each well, and incubation continued for 3-4 hours. Then, the culture medium was aspirated, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader. Cell viability was calculated, and dose-response curves were plotted. The IC50 was calculated using nonlinear regression fitting. 50Values. The experiment used RPMI 1640 medium (Gibco), MTT reagent (Sigma-Aldrich, catalog number M2128), DMSO (Sigma-Aldrich), and other reagents, as well as equipment such as a CO2 incubator (Thermo Fisher) and a microplate reader (Thermo Fisher). Strict aseptic technique was maintained throughout the experiment, and the results were repeated three times to ensure reliability.

[0058] Table 2. Results of in vitro inhibitory effects of the compounds on K562, A549, and MCF7 cells. In K562, A549, and MCF7 cell inhibition assays, compounds 01, 10, 05, and 06 were preferred for their IC50 values. 50 The values ​​showed that these compounds all exhibited good inhibitory activity. In experiments targeting A549 and MCF7 cells, compounds 01, 10, and 05 all demonstrated superior inhibitory activity compared to the control, Tomivosertib.

[0059] Example 13: Virtual docking of compounds Virtual docking studies of compound 1-(4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea (hereinafter referred to as compound 01) with protein MNK1 were conducted using Discovery Studio software (see the instruction manual appendix). Figure 26 The docking results showed that compound 01 successfully inserted into the hydrophobic protein cavity composed of amino acids PHE124, LEU55, LEU127, GLY130, ALA134, and GLU174. This cavity provided a favorable binding site for compound 01, thereby affecting the activity of MNK1. Through docking model analysis, we found that compound 01 formed a stable hydrogen bond with amino acid SER131, which may play a key role in the binding affinity and specificity of compound 01.

[0060] The hydrogen bond formed between compound 01 and SER131 may help stabilize the orientation of compound 01 at the binding site, thereby enhancing its interaction with MNK1. Furthermore, the cavity into which compound 01 is inserted consists of various amino acids, including hydrophobic amino acids (such as PHE124, LEU55, LEU127, GLY130, ALA134) and charged amino acids (such as GLU174), suggesting that compound 01 may bind to MNK1 through multiple interactions (such as hydrophobic interactions, hydrogen bonds, and possible electrostatic interactions). Subsequently, virtual docking diagrams of compounds 02 and 08 also confirm this structure-activity relationship (see appendix to the specification). Figure 27 ,28 ).

[0061] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pyrazine compound, characterized in that, The structure of the compound is shown in Formula I; Formula I Wherein, R1 and R2 are Cl, substituted or unsubstituted benzene rings, substituted or unsubstituted pyridine, and R1 and R2 are not both Cl; The substituted benzene ring and the substituted pyridine are substituted by one or two substituents independently selected from the following: In this context, * indicates a connection site.

2. The pyrazine compound according to claim 1, characterized in that: The pyrazine compounds are specifically: 1-(4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea; N-(2,4-Difluorophenyl)-3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide; 4-(4-(6-(3-(trifluoromethyl)phenyl)pyrazin-2-yl)benzyl)morpholine; 1-(5-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)pyridin-2-yl)-3-(p-tolyl)urea; 1-(3-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)phenyl)-3-(p-tolyl)urea; N-(2,4-Difluorophenyl)-4-(6-(4-(morpholinomethyl)phenyl)pyrazin-2-yl)benzamide; Ethyl 3-(6-chloropyrazin-2-yl)benzoate; 3-(6-(4-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)ethyl benzoate; Ethyl 3-(6-(4-(trifluoromethyl)phenyl)pyrazin-2-yl)benzoate; Ethyl 3-(6-(3-(2,4-difluorophenyl)carbamoyl)phenyl)pyrazin-2-yl)benzoate.

3. A pharmaceutical composition, characterized in that: It comprises the pyrazine compound of claim 1 or 2 or its pharmaceutically acceptable salt, isomer, hydrate, and at least one pharmaceutically acceptable carrier.

4. A method for preparing a pyrazine compound as described in claim 1 or 2, characterized in that, Includes the following steps: 。 5. The method for preparing pyrazine compounds according to claim 4, characterized in that: In steps c and d, the solvent is water or ethanol, the catalyst is an inorganic base and tetra(triphenylphosphine)palladium, and the reaction is carried out at 80-100℃ for 10-20 hours under nitrogen protection.

6. The method for preparing pyrazine compounds according to claim 5, characterized in that: The inorganic base is cesium carbonate, potassium carbonate, sodium carbonate, or sodium hydroxide. In step c, the ratio of the feed material 2,6-dichloropyrazine to dioxoborane compounds is 1:(1-3) by molar amount.

7. Use of the pyrazine compounds as described in claim 1 or 2 in the preparation of MNK1 inhibitors, drugs that inhibit MNK1-related signaling pathways, and anticancer drugs.

8. Use of the pyrazine compounds as described in claim 1 or 2 in the preparation of drugs for treating liver cancer, lung cancer, breast cancer, leukemia, cervical cancer, ovarian cancer, gastric adenocarcinoma, and prostate cancer.

Images