A compound containing an imidazo[1,2-a]pyrazine fragment, preparation method and application

Abstract

This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a compound containing an imidazo[1,2-a]pyrazine fragment, its preparation method, and its applications. The compound containing the imidazo[1,2-a]pyrazine fragment of this invention exhibits significant inhibitory activity against SYK, and also significantly inhibits the proliferation of tumor cells, particularly breast cancer cells, ovarian cancer cells, and liver cancer cells. Furthermore, it can induce apoptosis in ovarian cancer cells, thus providing a novel option for SYK inhibitors used in the treatment of solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical synthesis technology, specifically relating to a compound containing an imidazo[1,2-a]pyrazine fragment, its preparation method, and its application. Background Technology

[0002] Protein kinases regulate various signal transduction processes by adding phosphate groups to proteins, making them important cellular targets in the development of novel chemical drugs for cancer treatment. Abnormal kinase activity is closely related to various cancers, and small molecule inhibitors of protein kinases have been widely used in cancer therapy. Spleen tyrosine kinase (SYK) plays a crucial survival role in various receptor-mediated signal transductions and is a core regulator of many inflammatory cell and immune signaling pathways. Studies have shown that SYK is considered a potential therapeutic target for immune-related diseases such as rheumatoid arthritis, asthma / rhinitis, and malignant tumors. Recent studies have also revealed that abnormal SYK activation is associated with various malignant tumors. Research also suggests that SYK may be a potential oncogenic driver in lung cancer. Inhibition of SYK can significantly reduce toxin-induced liver fibrosis, related hepatocellular damage, and intrahepatic inflammation, and inhibit the occurrence of liver cancer. In recent years, several SYK inhibitors have entered clinical trials. Fostamatinib was once considered one of the most promising small molecule SYK inhibitors for the treatment of rheumatoid arthritis, but it failed due to limited efficacy in phase II studies and numerous adverse reactions in phase III studies. PRT062607 is currently in Phase II clinical trials, but no further data has been reported. Entospletinib (GS9973) has shown clinical activity as a monotherapy in Phase II clinical trials for patients with relapsed chronic lymphocytic leukemia (CLL) and is being developed in combination with the PI3K δ inhibitor Idelalisib. TAK-659 is an inhibitor that simultaneously inhibits SYK and FLT3 and is currently undergoing clinical trials for advanced solid tumors and lymphomas. The development of selective SYK inhibitors has always been challenging. Most SYK inhibitors are primarily used to treat inflammation-related diseases such as asthma, arthritis, and allergic diseases, but there are few selective SYK inhibitors that inhibit SYK for the treatment of solid tumors, requiring further development and research. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a compound containing an imidazo[1,2-a]pyrazine fragment, its preparation method, and its applications.

[0004] The first aspect of the present invention provides a compound containing an imidazo[1,2-a]pyrazine fragment, the structural formula of which is shown in formula (1): Formula (1); R1 is any substituent among A01~A03; A01, A02, A03, A04, A05, A06, A07, A08, A09, A10, A11, A12, A13; The compound is used to inhibit SYK.

[0005] A second aspect of the present invention provides a method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment, comprising the following steps: when R1 is A01, the preparation method of the SYK inhibitor is as follows: Starting with p-fluoronitrobenzene, intermediate MA01 was prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediate MA01 and intermediate n2 were mixed in a molar ratio of 1:1 to 1.5, coupled via Suzuki coupling, and deprotected to obtain a compound containing the A01 substituent. The structural formula of M1 is as follows: .

[0006] In another preferred embodiment, when R1 is A02~A04, the compound is prepared as follows: Starting from p-fluoronitrobenzene, 2-fluoro-5-nitropyridine, and 2-fluoro-5-nitrotoluene, respectively, intermediates MA02, MA03, and MA04 were prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA02, MA03, and MA04 were mixed with intermediate n2 in a molar ratio of 1:1 to 1.5, and then Suzuki coupling and deprotection were performed to obtain compounds containing substituents A02, A03, and A04, respectively.

[0007] In another preferred embodiment, when R1 is A05~A08, the compound is prepared as follows: Starting from p-fluoronitrobenzene (M3), 2-fluoro-5-nitropyridine (M4), 3,4-difluoronitrobenzene (M5), and 2-fluoro-5-nitrotoluene (M6), intermediates MA05 to MA08 were prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA05, MA06, MA07, MA08, and intermediate n2 were mixed in a molar ratio of 1:1 to 1.5 and then coupled with Suzuki coupling and deprotection to obtain compounds containing substituents A05, A06, A07, and A08, respectively.

[0008] In another preferred embodiment, when R1 is A09~A11, the compound is prepared as follows: Starting from p-fluoronitrobenzene, 3,4-difluoronitrobenzene, and 2-fluoro-5-nitrotoluene, intermediates MA09, MA10, and MA11 were obtained by sequentially undergoing aromatic nucleophilic substitution, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was obtained by Boc protection and Miyaura borate esterification. Intermediates MA09, MA10, MA11, and n2 were mixed in a molar ratio of 1:1 to 1.5 and then subjected to Suzuki coupling and deprotection to obtain compounds containing substituents A09, A10, and A11, respectively.

[0009] In another preferred embodiment, when R1 is A12 and A13, the compound is prepared as follows: Starting from p-fluoronitrobenzene and 2-fluoro-5-nitrotoluene, intermediates MA12 and MA13 were prepared by sequentially undergoing aromatic nucleophilic substitution, hydrolysis, reductive amination, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA12 and MA13 were mixed with intermediate n2 in a molar ratio of 1:1 to 1.5, respectively, and then coupled by Suzuki coupling and deprotection to obtain compounds containing substituents A12 and A13, respectively.

[0010] A third aspect of this invention provides the use of the compound containing the imidazo[1,2-a]pyrazine fragment in the preparation of a SYK inhibitor. Preferably, the SYK inhibitor is obtained by diluting a compound containing the imidazo[1,2-a]pyrazine fragment. The dilution is performed using dimethyl sulfoxide.

[0011] The fourth aspect of the present invention provides the use of the compound containing the imidazo[1,2-a]pyrazine fragment in the preparation of anticancer drugs.

[0012] In another preferred embodiment, the anticancer drug is any one of the drugs for treating ovarian cancer, breast cancer, and liver cancer.

[0013] In another preferred embodiment, the content of the compound containing the imidazo[1,2-a]pyrazine fragment in the drug is 1 μM to 10 μM. Specifically, the compound containing the imidazo[1,2-a]pyrazine fragment is obtained by dilution with dimethyl sulfoxide reagent.

[0014] Compared with the prior art, the present invention has the following beneficial effects: The compound containing the imidazo[1,2-a]pyrazine fragment provided by this invention exhibits excellent inhibitory activity against splenic tyrosine kinase (SYK). When R1 is A13, the half-maximal inhibitory concentration (IC50) of the compound is as low as 10.90 nM, significantly superior to the positive control compound Lanraplenib (IC50). 50 =13.19 nM), indicating that this class of compounds is a highly effective SYK inhibitor. Regarding antitumor activity, the compounds of this invention exhibit significant inhibitory effects on various solid tumor cell lines. Proliferation inhibition experiments against human hepatocellular carcinoma HepG2, human breast cancer cells MDA-MB-231, and human ovarian cancer cells SK-OV-3 showed that, with R1 set to A13, the half-maximal inhibitory concentrations (IC50) of the compounds against the three tumor cell lines were 4.49 μM, 64.48 μM, and 10.39 μM, respectively, while the corresponding values ​​for the positive control Lanraplenib were 44.13 μM, greater than 100 μM, and 55.71 μM, respectively. Particularly in HepG2 and SK-OV-3 cells, the inhibitory activity of the compounds was nearly 10-fold and more than 5-fold higher than the positive control, respectively, demonstrating excellent antitumor activity and good tumor selectivity. The compounds containing the imidazo[1,2-a]pyrazine fragment provided by this invention not only exhibit potent inhibitory activity against SYK kinase but also demonstrate excellent anti-proliferative activity against various solid tumor cells. Their mechanism of action involves inducing tumor cell apoptosis and arresting cell cycle progression. Furthermore, this series of compounds exhibits good metabolic stability and in vivo anti-tumor effects, providing ideal lead compounds and drug candidates for the development of novel SYK inhibitors for the treatment of solid tumors such as liver cancer, breast cancer, and ovarian cancer, and possessing significant clinical application potential and development value. Attached Figure Description

[0015] Figure 1 Figure A shows the results of the apoptosis experiment; Figure B shows the results of the control group; Figure C shows the results of the compound A13 at a concentration of 2.5 μM; Figure D shows the results of the compound A13 at a concentration of 5 μM; Figure E shows the results of the compound A13 at a concentration of 10 μM; Figure F shows the results of the Lanraplenib at a concentration of 2.5 μM; and Figure F shows the bar charts corresponding to A through E.

[0016] Figure 2 Figure A shows the results of the cell cycle experiment; Figure B shows the results of the control group; Figure C shows the results of the compound A13 at a concentration of 2.5 μM; Figure D shows the results of the compound A13 at a concentration of 5 μM; Figure E shows the results of the compound A13 at a concentration of 10 μM; Figure E shows the results of the Lanraplenib at a concentration of 2.5 μM; and Figure F shows the bar charts corresponding to A through E.

[0017] Figure 3 This is a diagram showing the results of a clone formation experiment.

[0018] Figure 4 The results are from experiments using the SK-OV-3 tumor-bearing mouse model; A is a physical image, and B is a line graph.

[0019] Figure 5 This is a schematic diagram of the structure of Lanraplenib. Detailed Implementation

[0020] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0021] The most commonly used SYK inhibitor is Lanraplenib, whose structure is as follows: Figure 5 As shown, this invention improves the existing structure of Lanraplenib by replacing its Part A substituent, thereby obtaining a new compound with higher inhibitory activity, better antitumor activity, and good tumor selectivity. The following describes a compound containing an imidazo[1,2-a]pyrazine fragment, its preparation method, and its applications.

[0022] 1. Preparation of compounds containing imidazo[1,2-a]pyrazine fragments The structural formula of the compound containing the imidazo[1,2-a]pyrazine fragment is (1); Formula (1); R1 is any substituent among A01~A03; A01, A02, A03, A04, A05, A06, A07, A08, A09, A10, A11, A12, A13. For the compounds corresponding to the different substituents mentioned above, they shall be named according to the corresponding substituents. For example, a compound containing the substituent A01 shall be denoted as compound A01.

[0023] The specific synthetic route for the target compound A01 is as follows: Starting with p-fluoronitrobenzene (M3), intermediate MA01 was obtained through sequential aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was obtained through Boc protection and Miyaura borate esterification. Intermediate MA01 and intermediate n2 were then coupled via Suzuki coupling and deprotection to obtain the target compound A01. The reaction equations are as follows:

[0024] .

[0025] The letters above the arrows in the above reaction process indicate the reaction conditions, and the same applies below. Here, a represents N,N-dimethylformamide (DMF) and K₂CO₃, 80℃; b represents trifluoroacetic acid (TFA), dichloromethane (DCM), 25℃ (room temperature, rt); c represents triethyl orthoformate, formyl hydrazine, and acetic acid. Acid), methanol (MeOH), reflux; d is H2, Pd / C, MeOH, 40℃; e is (Boc)2O, 4-dimethylaminopyridine (DMAP), tetrahydrofuran (THF), rt; f is B2(Pin)2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-dioxane, reflux; g is N,N-diisopropylethylamine (DIPEA), isopropanol (IPA), 85℃; h is PdCl2(dppf)·CH2Cl2, K3PO4, 1,4-Dioxane / H2O, reflux; i is TFA, DCM, rt.

[0026] The specific synthesis method of A01 is as follows: The preparation process of the intermediate (1-(4-nitrophenyl)azacyclobutane-3-yl)carbamate tert-butyl ester (ma2) is as follows: At room temperature (25℃), 10.0 g of the product was added sequentially to p-fluoronitrobenzene (M3, 0.07 mol), and 18.3 g of the product was added to 3- N- tert-Butoxycarbonylaminocyclobutylamine (ma1, 0.11 mol) and 29.4 g of potassium carbonate (0.21 mol) were dissolved in 50 mL of DMF, and the mixture was heated to 80 °C and reacted for 4 h. After the reaction was complete, the reaction solution was cooled to room temperature (25 °C) and slowly poured into 500 mL of ice water. The mixture was stirred for 0.5 h, filtered, and the filter cake was washed with water and dried to obtain 19.74 g of yellow solid, which was ma2, with a yield of 95.0%. LC-MS (ESI) m / z (%): 294.1 [M+H] + .

[0027] The preparation of intermediate 1-(4-nitrophenyl)azacyclobutane-3-amine (ma3) was as follows: Under ice bath conditions, 10 g of compound ma2 (0.034 mol) was dissolved in 30 mL of dichloromethane, and 5 mL of trifluoroacetic acid was slowly added dropwise. After the addition was complete, the mixture was stirred for 10 min, and then reacted at 25 °C for 4 h. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to 7-8. The mixture was extracted with dichloromethane (30 mL × 2), and the organic phases were combined. The solutions were washed successively with water (30 mL × 2) and saturated brine (30 mL × 2). The organic layer was evaporated to dryness to obtain 5.27 g of a yellow solid, which was ma3, with a yield of 80.1%. LC-MS (ESI) m / z (%): 194.1 [M+H] + .

[0028] Intermediate 4-(1-(4-nitrophenyl)azacyclobutane-3-yl)-4 H The preparation process of -1,2,4-triazole (1a) is as follows: At 0℃, 3g of compound ma3 (0.015mol), 3.45g of triethyl orthoformate (0.023mol), 1.4g of formyl hydrazide (0.023mol), and 3mL of acetic acid were dissolved in 20mL of acetonitrile. The mixture was kept at 0℃ for 10 minutes, and then the temperature was increased to 80℃ for 8 hours. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to the reaction solution to adjust the pH to 7-8. The mixture was extracted with dichloromethane (20mL×2), and the organic phase was collected. The organic layer was washed successively with water (20mL×2) and saturated brine (20mL×2). The organic layer was evaporated to dryness to obtain 2.89g of yellow solid, i.e., intermediate 1a, with a yield of 76.1%. LC-MS (ESI) m / z (%): 246.1 [M+H] + .

[0029] The preparation of intermediate 4-(3-(4H-1,2,4-triazol-4-yl)azacyclobutan-1-yl)aniline (1b) was as follows: Under hydrogen atmosphere, 2.0 g of intermediate 1a (8.1 mmol) was dissolved in 10 mL of methanol, and 0.1 g of palladium on carbon (10% m / m) was added. The reaction was carried out at 40 °C for 4 h under hydrogen atmosphere. After the reaction was complete, the mixture was filtered, the filter cake was washed with a small amount of dichloromethane, the filtrate was recovered, and the organic layer was evaporated to dryness to obtain 1.40 g of a light yellow solid, which was 1b, with a yield of 80.1%. LC-MS (ESI) m / z (%): 216.1 [M+H] + .

[0030] intermediate N -(4-(3-(4 H -1,2,4-triazol-4-yl)azacyclobutane-1-yl)phenyl)-6-bromoimidazole[1,2- a The specific preparation process of pyrazine-8-amine (MA01) is as follows: At room temperature (25°C), 1.0 g of compound 1b (4.7 mmol), 1.5 g of DIPEA (11.6 mmol), and 3.21 g of compound M2 (11.6 mmol) were added to 10 mL of isopropanol solution, and the temperature was raised to 85°C for 8 h. After the reaction was complete, the mixture was cooled to room temperature (25°C), and the reaction solution was poured into 100 mL of ice water. The mixture was stirred at 25°C for 0.5 h, filtered, and the filter cake was washed with a small amount of water. The filter cake was collected, dried, and 1.63 g of a light yellow solid was obtained, which was MA01, with a yield of 86.2%. LC-MS (ESI) m / z (%): 411.1 [M+H] + .

[0031] The preparation process of intermediate (6-bromopyrazin-2-yl) tert-butyl carbamate (n1) is as follows: At room temperature (25°C), 1.0 g of compound M1 (5.8 mmol) and 0.07 g of DMAP (0.58 mmol) were dissolved in 5 mL of tetrahydrofuran. 2.77 g of ditert-butyl dicarbonate (12.8 mmol) was slowly added dropwise with stirring. After the addition was complete, the reaction was carried out at room temperature (25°C) for 4 h. After the reaction was complete, 50 mL of water was added, and the mixture was stirred at room temperature (25°C) for 0.5 h. The mixture was then filtered, and the filter cake was washed with water. The filter cake was placed in a mixed solvent of 10 mL of methanol / 1 M NaOH aqueous solution (1:1), and stirred at room temperature (25°C) for 1 h. 20 mL of water was added, and the mixture was stirred for 10 min. The mixture was then filtered, the filter cake was washed with water, collected, and dried to obtain 1.21 g of white solid, which was n1, with a yield of 76.9%. LC-MS (ESI) m / z (%): 274.0 [M+H] + .

[0032] The preparation of intermediate (6-(4,4,5,5-tetramethyl-1,3,2-dioxoboronyl-2-yl)pyrazin-2-yl)tert-butyl carbamate (n2) was carried out as follows: At room temperature (25°C), 2.0 g of compound n1 (7.3 mmol), 2.05 g of pinacol diborate (8.1 mmol), 0.6 g of PdCl2 (dppf)·DCM (0.73 mmol), and 2.5 g of potassium acetate (25.5 mmol) were added to 10 mL of dry 1,4-dioxane. The reaction was carried out at 90°C for 3 h under nitrogen protection. After the reaction was completed, the mixture was cooled to room temperature (25°C), filtered through a diatomaceous earth filter, the filter cake was washed with ethyl acetate, the filtrate was concentrated, 30 mL of n-heptane was added and stirred, and the mixture was filtered to obtain 1.25 g of a grayish-yellow solid, which was n2 (yield 53.2%). LC-MS (ESI) m / z (%): 322.2 [M+H] + .

[0033] Intermediate (6-(8-((4-(3-(4H-pyrazol-4-yl)azacyclobutane-1-yl)phenyl)amino)imidazo[1,2- a The specific preparation process of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A01) is as follows: At room temperature of 25℃, 0.25g of compound MA01 (0.61mmol), 0.29g of n2 (0.9mmol), 0.05g of PdCl2(dppf)·DCM (0.06mmol), and 0.13g of K3PO4 (1.83mmol) were dissolved in 5mL of 1,4-dioxane / water (10:1) mixed solvent and reacted at 95℃ for 8h under nitrogen protection. After the reaction was complete, the reaction solution was cooled to room temperature (25°C), filtered through a diatomaceous earth filter, and the filter cake was washed with 1,4-dioxane. The filtrate was washed successively with water (10 mL × 2) and saturated brine (10 mL × 2). The organic phase was collected, dried by rotary evaporation, and 0.24 g of a pale yellow solid, namely M'A01, was obtained, with a yield of 75.2%. LC-MS (ESI) m / z (%): 526.2 [M + H] + .

[0034] target compound N -(4-(3-(4 H -1,2,4-triazol-4-yl)azacyclobutane-1-yl)phenyl)-6-(6-aminopyrazin-2-yl)imidazo[1,2- aThe preparation of pyrazine-8-amine (A01) was carried out as follows: 0.2 g of compound M'A01 (0.38 mmol) was dissolved in 5 mL of dichloromethane at room temperature (25 °C). The reaction solution was placed in an ice bath and stirred for 5 min. 0.5 mL of trifluoroacetic acid was slowly added dropwise. After the addition was complete, the mixture was stirred for 10 min and then moved to room temperature (25 °C) for 4 h. The reaction was monitored by TLC until complete. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8. The mixture was extracted with dichloromethane (5 mL × 2), and the organic phase was collected. The organic phase was washed successively with water (10 mL × 2) and saturated brine (10 mL × 2). The organic phase was evaporated to dryness, and the crude product was purified by column chromatography to obtain 0.11 g of a white solid, which was A01, with a yield of 70.1%, mp: 172.5-173.6 °C; LC-MS (ESI) m / z (%): 426.2 [M+H]. + .

[0035] The synthetic route for target compounds A02~A04 is as follows: Starting with p-fluoronitrobenzene (M3), 2-fluoro-5-nitropyridine (M4), and 2-fluoro-5-nitrotoluene (M6), intermediates MA02~MA04 were obtained through sequential aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was obtained through Boc protection and Miyaura borate esterification. Intermediates MA02~MA04 and intermediate n2 were then coupled via Suzuki coupling and deprotection to obtain target compounds A02~A04. The overall reaction equation is as follows:

[0036] .

[0037] Where a is DMF, K2CO3, 80℃; b is TFA, DCM, rt; c is Triethyl orthoformate, Formylhydrazine, Acetic Acid, MeOH, reflux; d is H2, Pd / C, MeOH, 40℃; e is (Boc)2O, DMAP, THF, rt; f is B2(Pin)2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-Dioxane, reflux; g is DIPEA, IPA, 85℃; h is PdCl2(dppf)·CH2Cl2, K3PO4, 1,4-Dioxane / H2O, reflux; i is TFA, DCM, rt.

[0038] The specific synthesis methods for A02~A04 are as follows: The preparation of the intermediate (1-(4-nitrophenyl)piperidin-4-yl)carbamate tert-butyl ester (ma5) is as follows: At room temperature (25°C), 10.0 g of p-fluoronitrobenzene (M3, 0.07 mol), 21.3 g of 4-tert-butyloxycarbonylaminopiperidine (ma4, 0.11 mol), and 29.4 g of potassium carbonate (0.21 mol) were dissolved in 50 mL of DMF, and the mixture was heated to 80°C and reacted for 4 h. After the reaction was completed, the reaction solution was cooled to room temperature (25°C) and slowly poured into 500 mL of ice water. The mixture was stirred at room temperature (25°C) for 0.5 h, filtered, the filter cake was washed with water, and dried to obtain 21.17 g of yellow solid, which is ma5, with a yield of 93.0%. LC-MS (ESI) m / z (%): 322.1 [M+H] + .

[0039] The preparation of the intermediate (1-(5-nitropyridin-2-yl)piperidin-4-yl)tert-butyl carbamate (MA6) was as follows: Following the preparation method of MA5, using M4 and MA4 as raw materials, 20.69 g of yellow solid MA6 was obtained, with a yield of 91.2%. LC-MS (ESI) m / z (%): 323.1 [M+H] + .

[0040] The preparation of the intermediate (1-(2-methyl-4-nitrophenyl)piperidin-4-yl)tert-butyl carbamate (MA7) was as follows: Following the preparation method of MA5, using M6 and MA4 as raw materials, 19.52 g of yellow solid MA7 was obtained, with a yield of 90.3%. LC-MS (ESI) m / z (%): 336.1 [M+H] + .

[0041] The preparation of the intermediate (1-(2-fluoro-4-nitrophenyl)piperidin-4-yl)tert-butyl carbamate (MA8) was as follows: Following the preparation method of MA5, using M5 and MA4 as raw materials, 19.00 g of yellow solid MA8 was obtained, with a yield of 89.1%. LC-MS (ESI) m / z (%): 340.1 [M+H] + .

[0042] Preparation of intermediate 1-(4-nitrophenyl)piperidin-4-amine (MA9): Following the preparation method of MA3, using MA5 as a raw material, yellow solid MA9 was prepared in a yield of 78.7%. LC-MS (ESI) m / z (%): 222.1 [M+H] + .

[0043] The preparation of intermediate 1-(5-nitropyridin-2-yl)piperidin-4-amine (ma10) was as follows: Following the preparation method of ma3, using ma6 as a raw material, a yellow solid ma10 was obtained with a yield of 74.5%. LC-MS (ESI) m / z (%): 223.1 [M+H] + .

[0044] The intermediate 1-(2-methyl-4-nitrophenyl)piperidin-4-amine (MA11) was prepared as follows: Following the preparation method of MA3, using MA7 as a raw material, a yellow solid MA11 was obtained with a yield of 68.9%. LC-MS (ESI) m / z (%): 236.1 [M+H] + .

[0045] Intermediate 1-(4-nitrophenyl)-4-(4 H The preparation of -1,2,4-triazol-4-yl)piperidine (2a) is as follows: Following the preparation method of 1a, using ma9 as raw material, a yellow solid was obtained with a yield of 75.0%. LC-MS (ESI) m / z (%): 274.1 [M+H] + .

[0046] Intermediate 2-(4-(4) H The preparation of -1,2,4-triazol-4-yl)piperidin-1-yl)-5-nitropyridine (3a) is as follows: Following the preparation method of 1a, using ma10 as raw material, a yellow solid 3a was obtained with a yield of 75.6%. LC-MS (ESI) m / z (%): 275.1 [M+H] + .

[0047] Intermediate 1-(2-methyl-4-nitrophenyl)-4-(4 H The preparation of -1,2,4-triazol-4-yl)piperidine (4a) is as follows: Following the preparation method of 1a, using ma11 as the raw material, a yellow solid 4a was obtained with a yield of 73.2%. LC-MS (ESI) m / z (%): 288.1 [M+H] + .

[0048] Preparation of intermediate 4-(4-(4H-1,2,4-triazol-4-yl)piperidin-1-yl)aniline (2b): Following the preparation method of 1b, using 2a as the starting material, a yellow solid 2b was prepared in a yield of 79.0%. LC-MS (ESI) m / z (%): 244.1 [M+H] + .

[0049] The preparation of intermediate 4-(4-(4H-1,2,4-triazol-4-yl)piperidin-1-yl)aniline (3b) is as follows: Following the preparation method of 1b, using 3a as the raw material, a yellow solid 3b was obtained with a yield of 66.8%. LC-MS (ESI) m / z (%): 245.1 [M+H] + .

[0050] The preparation of intermediate 4-(4-(4H-1,2,4-triazol-4-yl)piperidin-1-yl)aniline (4b) is as follows: Following the preparation method of 1b, using 4a as the raw material, a yellow solid 4b was obtained with a yield of 69.1%. LC-MS (ESI) m / z (%): 258.1 [M+H] + .

[0051] intermediate N -(4-(4-(4 H -1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)-6-bromoimidazole[1,2- a The preparation of pyrazine-8-amine (MA02) is as follows: Following the preparation method of MA01, using 2b as raw material, a yellow solid MA02 was obtained with a yield of 85.0%. LC-MS (ESI) m / z (%): 439.1 [M+H] + .

[0052] intermediate N -(6-(4-(4 H -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)-6-bromoimidazole[1,2- a The preparation of pyrazine-8-amine (MA03) is as follows: Following the preparation method of MA01, using 3b as raw material, a yellow solid MA03 was obtained with a yield of 80.3%. LC-MS (ESI) m / z (%): 440.1 [M+H] + .

[0053] intermediate N -(4-(4-(4 H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)-6-bromoimidazole[1,2- a The preparation of pyrazine-8-amine (MA04) is as follows: Following the preparation method of MA01, using 4b as raw material, a yellow solid MA04 was obtained with a yield of 82.1%. LC-MS (ESI) m / z (%): 453.1 [M+H] + .

[0054] Intermediate 6-(8-((4-(4) H-1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A02) is as follows: Following the preparation method of M'A01, using MA02 and n2 as raw materials, a yellow solid M'A02 was obtained with a yield of 70.3%. LC-MS (ESI) m / z (%): 554.2 [M+H] + .

[0055] Intermediate (6-(8-((6-(4-(4) H -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)amino)imidazo[1,2-] a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A03) is as follows: Following the preparation method of M'A01, using MA03 and n2 as raw materials, a yellow solid M'A03 was obtained with a yield of 71.2%. LC-MS (ESI) m / z (%): 555.2 [M+H] + .

[0056] Intermediate (6-(8-((4-(4) H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A04) is as follows: Following the preparation method of M'A01, using MA04 and n2 as raw materials, a yellow solid M'A04 was obtained with a yield of 78.3%. LC-MS (ESI) m / z (%): 568.2 [M+H] + .

[0057] target compound N -(4-(4-(4 H -1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)-6-(6-aminopyrazin-2-yl)imidazo[1,2- a The preparation of pyrazine-8-amine (AO2) is as follows: Following the preparation method of AO1, using M'AO2 as raw material, a white solid AO2 was obtained with a yield of 72.3%, mp: 180.6-181.8℃; LC-MS (ESI) m / z (%): 454.2 [M+H] + .

[0058] target compound N -(6-(4-(4 H-1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)-6-(6-aminopyrazin-2-yl)imidazo[1,2- a The preparation of pyrazine-8-amine (AO3) is as follows: Following the preparation method of AO1, using M'AO3 as raw material, a white solid AO3 was obtained with a yield of 70.5%, mp: 170.2-171.3°C; LC-MS (ESI) m / z (%): 455.2 [M+H] + .

[0059] target compound N -(4-(4-(4 H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)-6-(6-aminopyrazin-2-yl)imidazo[1,2- a The preparation of pyrazine-8-amine (A04) was as follows: Following the preparation method of A01, using M'A04 as the raw material, a white solid was obtained with a yield of 65.3%, mp: 160.1-161.3°C; LC-MS (ESI) m / z (%): 468.2 [M+H] + .

[0060] The synthetic route for target compounds A05-A08 is as follows: Starting with p-fluoronitrobenzene (M3), 2-fluoro-5-nitropyridine (M4), 3,4-difluoronitrobenzene (M5), and 2-fluoro-5-nitrotoluene (M6), intermediates MA05-MA08 were obtained through sequential aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was obtained through Boc protection and Miyaura borate esterification. Intermediates MA05-MA08 and intermediate n2 were then coupled via Suzuki coupling and deprotection to obtain target compounds A05-A08. The overall reaction equation is as follows:

[0061] .

[0062] Wherein, a is DMF, K2CO3, 80℃; b is TFA, DCM, rt; c is acetylhydrazide, 1,1-dimethoxy-N,N-dimethylethylamine, Acetic Acid, acetonitrile (ACN), reflux; d is H2, Pd / C, MeOH, 40℃; e is (Boc)2O, DMAP, THF, rt; f is B2(Pin)2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-Dioxane, reflux; g is 4-dimethylaminopyridine (DIPEA), IPA, 85℃; h is PdCl2(dppf)·CH2Cl2, K3PO4, 1,4-Dioxane / H2O, reflux; i is TFA, DCM, rt.

[0063] The specific synthesis methods for A05~A08 are as follows: Intermediate 4-(3,5-dimethyl-4 H The preparation of 1,2,4-triazol-4-yl)-1-(4-nitrophenyl)piperidine (5a) is as follows: At room temperature (25°C), 1.5 g of acetylhydrazine (0.021 mol) and 2.7 g of 1,1-dimethoxy- N,N 0.02 mol of dimethylethylamine was dissolved in 10 mL of acetonitrile. The mixture was heated to 50 °C and reacted for 30 min. 3.0 g of 1-(4-nitrophenyl)piperidin-4-amine (MA9, 0.014 mol) was added, followed by 3 mL of acetic acid. The reaction temperature was increased to 80 °C and the reaction was carried out for 8 h. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8. The mixture was extracted with dichloromethane (20 mL × 2), and the organic phase was collected. The organic phase was washed successively with water (15 mL × 2) and saturated brine (15 mL × 2). The organic phase was evaporated to dryness to give 2.86 g of a yellow solid, which was 70.1% of 5a. LC-MS (ESI) m / z (%): 302.1 [M+H] + .

[0064] Intermediate 2-(4-(3,5-dimethyl-4-) H The preparation of -1,2,4-triazol-4-yl)piperidin-1-yl)-5-nitropyridine (6a) is as follows: Following the preparation method of 5a, using ma10 as the raw material, a yellow solid 6a was obtained with a yield of 65.3%. LC-MS (ESI) m / z (%): 303.1 [M+H] + .

[0065] Intermediate 4-(3,5-dimethyl-4 HThe preparation of 1,2,4-triazol-4-yl)-1-(2-fluoro-4-nitrophenyl)piperidine (7a) is as follows: Following the preparation method of 5a, using MA11 as the raw material, a yellow solid 7a was obtained with a yield of 64.3%. LC-MS (ESI) m / z (%): 320.1 [M+H] + .

[0066] Intermediate 4-(3,5-dimethyl-4 H The preparation of 1,2,4-triazol-4-yl)-1-(2-methyl-4-nitrophenyl)piperidine (8a) is as follows: Following the preparation method of 5a, using MA12 as the raw material, a yellow solid 8a was obtained with a yield of 68.9%. LC-MS (ESI) m / z (%): 316.1 [M+H] + .

[0067] Intermediate 4-(4-(3,5-dimethyl-4-) H The preparation of 1,2,4-triazol-4-yl)piperidin-1-yl)aniline (5b) is as follows: Following the preparation method of 1b, using 5a as the raw material, a yellow solid 5b was obtained with a yield of 71.2%. LC-MS (ESI) m / z (%): 272.1 [M+H] + .

[0068] Intermediate 6-(4-(3,5-dimethyl-4) H The preparation of -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-amine (6b) is as follows: Following the preparation method of 1b, using 6a as the raw material, a yellow solid was obtained with a yield of 65.7%. LC-MS (ESI) m / z (%): 273.1 [M+H] + .

[0069] Intermediate 4-(4-(3,5-dimethyl-4-) H The preparation of 1,2,4-triazol-4-yl)piperidin-1-yl)-3-fluoroaniline (7b) is as follows: Following the preparation method of 1b, using 7a as the raw material, a yellow solid 7b was obtained with a yield of 68.6%. LC-MS (ESI) m / z (%): 290.1 ​​[M+H] + .

[0070] Intermediate 4-(4-(3,5-dimethyl-4-) H The preparation of 1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylaniline (8b) is as follows: Following the preparation method of 1b, using 8a as the raw material, a yellow solid 8b was obtained with a yield of 61.5%. LC-MS (ESI) m / z (%): 286.1 [M+H] + .

[0071] intermediate 6-bromo- N -(4-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (MA05) is as follows: Following the preparation method of MA01, using 5b as raw material, a yellow solid MA05 was obtained with a yield of 65.2%. LC-MS (ESI) m / z (%): 467.1 [M+H] + .

[0072] intermediate 6-bromo- N -(6-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)imidazo[1,2- a The preparation of pyrazine-8-amine (MA06) is as follows: Following the preparation method of MA01, using 6b as raw material, a yellow solid MA06 was obtained with a yield of 66.7%. LC-MS (ESI) m / z (%): 468.1 [M+H] + .

[0073] intermediate 6-bromo- N -(4-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-fluorophenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (MA07) is as follows: Following the preparation method of MA01, using 7b as raw material, a yellow solid MA07 was obtained with a yield of 66.1%. LC-MS (ESI) m / z (%): 485.1 [M+H] + .

[0074] intermediate 6-bromo- N -(4-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (MA08) was as follows: Following the preparation method of MA01, using 8b as the raw material, a yellow solid MA08 was obtained with a yield of 73.4%. LC-MS (ESI) m / z (%): 481.1 [M+H] + .

[0075] Intermediate 6-(8-((4-(4-(3,5-dimethyl-4) H-1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A05) is as follows: Following the preparation method of M'A01, using MA05 and n1 as raw materials, a yellow solid M'A05 was obtained with a yield of 60.8%. LC-MS (ESI) m / z (%): 582.2 [M+H] + .

[0076] Intermediate 6-(8-((6-(4-(3,5-dimethyl-4) H -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)amino)imidazo[1,2-] a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A06) is as follows: Following the preparation method of M'A01, using MA06 and n1 as raw materials, a yellow solid M'A06 was obtained with a yield of 68.9%. LC-MS (ESI) m / z (%): 583.2 [M+H] + .

[0077] Intermediate 6-(8-((4-(4-(3,5-dimethyl-4) H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-fluorophenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A07) is as follows: Following the preparation method of M'A01, using MA07 and n1 as raw materials, a yellow solid M'A07 was obtained with a yield of 60.8%. LC-MS (ESI) m / z (%): 600.2 [M+H] + .

[0078] Intermediate 6-(8-((4-(4-(3,5-dimethyl-4) H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A08) is as follows: Following the preparation method of M'A01, using MA08 and n1 as raw materials, a yellow solid M'A08 was obtained with a yield of 70.1%. LC-MS (ESI) m / z (%): 596.2 [M+H] + .

[0079] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(3,5-dimethyl-4- H-1,2,4-triazol-4-yl)piperidin-1-yl)phenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (A05) is as follows: Following the preparation method of A01, using M'A05 as raw material, a white solid A05 was obtained with a yield of 72.3%, mp: 187.2-188.6°C; LC-MS (ESI) m / z (%): 482.2 [M+H] + .

[0080] The target compound is 6-(6-aminopyrazin-2-yl)- N -(6-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)pyridin-3-yl)imidazo[1,2- a The preparation of pyrazine-8-amine (A06) is as follows: Following the preparation method of A01, using M'A06 as raw material, a white solid A06 was obtained with a yield of 70.6%, mp: 177.5-178.6°C; LC-MS (ESI) m / z (%): 483.2 [M+H] + .

[0081] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-fluorophenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (A07) is as follows: Following the preparation method of A01, using M'A07 as raw material, a white solid A07 was obtained with a yield of 75.8%, mp: 161.1-162.3°C; LC-MS (ESI) m / z (%): 500.2 [M+H] + .

[0082] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(3,5-dimethyl-4- H -1,2,4-triazol-4-yl)piperidin-1-yl)-3-methylphenyl)imidazo[1,2- a The preparation of pyrazine-8-amine (A08) was as follows: Following the preparation method of A01, using M'A08 as raw material, a white solid A08 was obtained with a yield of 76.2%, mp: 167.5-168.8°C; LC-MS (ESI) m / z (%): 496.2 [M+H] + .

[0083] The synthetic route for target compounds A09-A11 is as follows: Starting with p-fluoronitrobenzene (M3), 3,4-difluoronitrobenzene (M5), and 2-fluoro-5-nitrotoluene (M6), intermediates MA09-MA11 were obtained through sequential aromatic nucleophilic substitution, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was obtained through Boc protection and Miyaura borosilicate esterification. Intermediates MA09-MA11 and n2 were then coupled via Suzuki coupling and deprotection to obtain target compounds A09-A11. The overall reaction equations are as follows:

[0084] .

[0085] Where a is DMF, K2CO3, 80℃; b is H2, Pd / C, MeOH, 40℃; c is IPA, DIPEA, 85℃; d is (Boc)2O, DMAP, THF, rt; e is B2(Pin)2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-Dioxane, reflux; f is PdCl2(dppf)·CH2Cl2, K3PO4, 1,4-Dioxane / H2O, reflux; g is TFA, DCM, rt.

[0086] The specific synthesis methods for A09~A11 are as follows: The preparation of intermediate 1-(methanesulfonyl)-4-(4-nitrophenyl)piperazine (9a) was as follows: At room temperature (25°C), 1.5 g of p-fluoronitrobenzene (M3, 0.01 mol) and 2.62 g of 1-methanesulfonylpiperazine (0.016 mol) were dissolved sequentially in 10 mL of DMF, and 4.4 g of potassium carbonate (0.03 mol) was added. The mixture was heated to 80°C and reacted for 4 h. After the reaction was complete, the reaction solution was cooled to room temperature (25°C) and slowly poured into 100 mL of ice water. The mixture was stirred at 25°C for 0.5 h, filtered, and the filter cake was washed with water and dried to obtain 2.79 g of a yellow solid, i.e., intermediate 9a, with a yield of 92.0%. LC-MS (ESI) m / z (%): 286.0 [M+H] + .

[0087] The preparation of intermediate 1-(2-fluoro-4-nitrophenyl)-4-(methanesulfonyl)piperazine (10a) was as follows: Following the preparation method of 9a, using M5 as raw material, a yellow solid 10a was obtained with a yield of 69.8%. LC-MS (ESI) m / z (%): 304.1 [M+H] + .

[0088] The preparation of intermediate 1-(2-methyl-4-nitrophenyl)-4-(methanesulfonyl)piperazine (11a) was as follows: Following the preparation method of 9a, using M6 as raw material, a yellow solid 11a was obtained with a yield of 70.8%. LC-MS (ESI) m / z (%): 300.1 [M+H] + .

[0089] The preparation of intermediate 4-(4-(methanesulfonyl)piperazin-1-yl)aniline (9b) is as follows: Following the preparation method of 1b, using 9a as the raw material, a yellow solid 9b was obtained with a yield of 69.0%. LC-MS (ESI) m / z (%): 256.1 [M+H] + .

[0090] The preparation of intermediate 3-fluoro-4-(4-(methanesulfonyl)piperazin-1-yl)aniline (10b) is as follows: Following the preparation method of 1b, using 10a as the starting material, a yellow solid 10b was obtained with a yield of 71.5%. LC-MS (ESI) m / z (%): 274.1 [M+H] + .

[0091] The preparation of intermediate 3-methyl-4-(4-(methanesulfonyl)piperazin-1-yl)aniline (11b) is as follows: Following the preparation method of 1b, using 11a as the raw material, a yellow solid 11b was obtained with a yield of 59.0%. LC-MS (ESI) m / z (%): 270.1 [M+H] + .

[0092] intermediate 6-bromo- N -(4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (MA09) was as follows: Following the preparation method of MA01, using 9b as the raw material, a yellow solid MA09 was obtained with a yield of 65.2%. LC-MS (ESI) m / z (%): 451.1 [M+H] + .

[0093] intermediate 6-bromo- N -(3-fluoro-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (MA10) is as follows: Following the preparation method of MA01, using 10b as raw material, a yellow solid MA10 was obtained with a yield of 69.0%. LC-MS (ESI) m / z (%): 469.1 [M+H] + .

[0094] intermediate 6-bromo- N-(3-methyl-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (MA11) is as follows: Following the preparation method of MA01, using 11b as raw material, a yellow solid MA11 was obtained with a yield of 77.8%. LC-MS (ESI) m / z (%): 465.1 [M+H] + .

[0095] Intermediate (6-(8-((4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)amino)imidazo[1,2-] a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A09) is as follows: Following the preparation method of M'A01, using MA09 as raw material, a yellow solid M'A09 was obtained with a yield of 73.2%. LC-MS (ESI) m / z (%): 566.2 [M+H] + .

[0096] Intermediate (6-(8-((3-fluoro-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A10) is as follows: Following the preparation method of M'A01, using MA10 as raw material, a yellow solid M'A10 was obtained with a yield of 78.0%. LC-MS (ESI) m / z (%): 584.2 [M+H] + .

[0097] Intermediate (6-(8-((3-methyl-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)amino)imidazo[1,2-] a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A11) is as follows: Following the preparation method of M'A01, using MA11 as the raw material, a yellow solid M'A11 was obtained with a yield of 76.5%. LC-MS (ESI) m / z (%): 580.2 [M+H] + .

[0098] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (A09) is as follows: Following the preparation method of A01, using M'A09 as raw material, a white solid A09 was obtained with a yield of 70.2%, mp: 196.5-197.8°C; LC-MS (ESI) m / z (%): 466.1 [M+H]+ .

[0099] The target compound is 6-(6-aminopyrazin-2-yl)- N -(3-fluoro-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (A10) is as follows: Following the preparation method of A01, using M'A10 as the raw material, a white solid A10 was obtained with a yield of 75.4%, mp: 176.2-177.6°C; LC-MS (ESI) m / z (%): 484.1 [M+H] + .

[0100] The target compound is 6-(6-aminopyrazin-2-yl)- N -(3-methyl-4-(4-(methanesulfonyl)piperazin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (A11) is as follows: Following the preparation method of A01, using M'A11 as raw material, a white solid A11 was obtained with a yield of 80.2%, mp: 182.7-183.9°C; LC-MS (ESI) m / z (%): 480.1 [M+H] + .

[0101] The synthetic routes for target compounds A12 and A13 are shown in Scheme 3-5: Starting with p-fluoronitrobenzene (M3) and 2-fluoro-5-nitrotoluene (M6), intermediates MA12 and MA13 were obtained sequentially via aromatic nucleophilic substitution, hydrolysis, reductive amination, nitro reduction, and aromatic nucleophilic substitution. Starting with M1, intermediate n2 was obtained via Boc protection and Miyaura borate esterification. Intermediates MA12 and MA13 were then coupled with intermediate n2 via Suzuki coupling, followed by deprotection to obtain target compounds A12 and A13. The overall reaction equations are as follows:

[0102] .

[0103] Where a is DMF, K2CO3, 80℃; b is acetone, H2O, p-toluenesulfonic acid, 70℃; c is NaBH(OAc)3, Acetic Acid, DCM, rt; d is H2, Pd / C, MeOH, 40℃; e is (Boc)2O, DMAP, THF, rt; f is B2(Pin)2, PdCl2(dppf)·CH2Cl2, KOAc, 1,4-Dioxane, reflux; g is N,N-diisopropylethylamine (DIPEA), IPA, 85℃; h is PdCl2(dppf)·CH2Cl2, K3PO4, 1,4-Dioxane / H2O, reflux; i is TFA, DCM, rt.

[0104] The specific synthesis methods for A12~A13 are as follows: The preparation of intermediate 8-(4-nitrophenyl)-1,4-dioxa-8-azaspiro[4.5]decane (ma16) was as follows: At room temperature (25°C), 5.0 g of p-fluoronitrobenzene (M3, 0.035 mol), 7.6 g of 4-piperidinone ethylene glycol (ma14, 0.053 mol), and 14.68 g of potassium carbonate (0.106 mol) were dissolved in 20 mL of DMF, and the mixture was heated to 80°C and reacted for 4 h. After the reaction was complete, the reaction solution was cooled to room temperature (25°C) and slowly poured into 200 mL of ice water. The mixture was stirred at 25°C for 0.5 h, filtered, and the filter cake was washed with water and dried to obtain 8.42 g of yellow solid ma16, with a yield of 90.0%. LC-MS (ESI) m / z (%): 265.1 [M+H] + .

[0105] The preparation of intermediate 8-(2-methyl-4-nitrophenyl)-1,4-dioxa-8-azaspiro[4.5]decane (ma17) was as follows: Following the preparation method of ma16, using M6 as raw material, yellow solid ma17 was obtained with a yield of 88.8%. LC-MS (ESI) m / z (%): 279.1 [M+H] + .

[0106] The preparation of intermediate 1-(4-nitrophenyl)piperidin-4-one (ma18) was as follows: At room temperature (25℃), 5.0 g of intermediate ma16 (0.035 mol) and 0.64 g of p-toluenesulfonic acid (0.007 mol) were dissolved in an acetone:water solution with a volume ratio of 4:1 (25 mL). The mixture was heated to 70℃ and reacted for 4 h. The reaction was monitored by TLC until complete. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8. The mixture was extracted with dichloromethane (10 mL × 2), and the organic phase was collected. The organic phase was washed successively with water (15 mL × 2) and saturated brine (15 mL × 2). The organic phase was evaporated to dryness to obtain 3.5 g of yellow solid ma18, with a yield of 70.1%. LC-MS (ESI) m / z (%): 221.2 [M+H]. + .

[0107] The intermediate 1-(2-methyl-4-nitrophenyl)piperidin-4-one (MA19) was prepared as follows: Following the preparation method of MA18, using MA17 as a raw material, a yellow solid MA19 was obtained with a yield of 75.8%. LC-MS (ESI) m / z (%): 235.1 [M+H] + .

[0108] The preparation of intermediate 4-(3-isopropoxy-1-yl)-1-(4-nitrophenyl)piperidine (12a) was as follows: At room temperature (25°C), 3.0 g of intermediate MA18 (0.014 mol) and 2.47 g of 3-isopropoxyazacyclobutane (0.017 mol) were dissolved in dichloromethane. 1.5 mL of glacial acetic acid was added dropwise, and the reaction was carried out at 25°C for 1 h. Then, 5.78 g of sodium triacetoxyborohydride (0.028 mol) was slowly added in portions, and the reaction was carried out at 25°C for 4 h. After the reaction was complete, saturated sodium bicarbonate aqueous solution was added to adjust the pH to 7-8. The mixture was extracted with dichloromethane, and the organic phase was collected. The organic phase was washed successively with water (15 mL × 2) and saturated brine (15 mL × 2). The organic phase was evaporated to dryness to obtain 3.26 g of brown solid 12a, with a yield of 75.1%. LC-MS (ESI) m / z (%): 320.2 [M+H]. + .

[0109] The preparation of intermediate 4-(3-isopropoxy-1-yl)-1-(2-methyl-4-nitrophenyl)piperidine (13a) was as follows: Following the preparation method of 12a, using MA19 as a raw material, a brown solid 13a was obtained with a yield of 66.8%. LC-MS (ESI) m / z (%): 334.2 [M+H] + .

[0110] The preparation of intermediate 4-(4-(3-isopropoxyacetidin-1-yl)piperidin-1-yl)aniline (12b) is as follows: Following the preparation method of 1b, using 12a as the raw material, a yellow solid 12b was obtained with a yield of 75.6%. LC-MS (ESI) m / z (%): 290.2 [M+H] + .

[0111] The preparation of intermediate 4-(4-(3-isopropoxyacetidin-1-yl)piperidin-1-yl)-3-methylaniline (13b) was as follows: Following the preparation method of 1b, using 13a as the starting material, a yellow solid 13b was obtained with a yield of 70.1%. LC-MS (ESI) m / z (%): 304.2 [M+H] + .

[0112] intermediate 6-bromo- N -(4-(4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (MA12) is as follows: Following the preparation method of MA01, using 12b as raw material, a yellow solid MA12 was obtained with a yield of 66.1%. LC-MS (ESI) m / z (%): 485.1 [M+H] + .

[0113] intermediate 6-bromo- N -(4-(4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)-3-methylphenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (MA13) is as follows: Following the preparation method of MA01, using 13b as the raw material, a yellow solid MA13 was obtained with a yield of 67.2%. LC-MS (ESI) m / z (%): 499.1 [M+H] + .

[0114] Intermediate (6-(8-((4-(4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)phenyl)amino)imidazo[1,2- a The preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A12) is as follows: Following the preparation method of M'A01, using MA12 and n1 as raw materials, a yellow solid M'A12 was obtained with a yield of 68.4%. LC-MS (ESI) m / z (%): 600.2 [M+H] + .

[0115] Intermediate (6-(8-((4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)-3-methylphenyl)amino)imidazo[1,2-] aThe preparation of pyrazin-6-yl)pyrazin-2-yl)tert-butyl carbamate (M'A13) is as follows: Following the preparation method of M'A01, using MA13 and n1 as raw materials, a yellow solid M'A13 was obtained with a yield of 70.1%. LC-MS (ESI) m / z (%): 614.2 [M+H] + .

[0116] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)phenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (A12) is as follows: Following the preparation method of A01, using M'A12 as raw material, a white solid A12 was obtained with a yield of 70.2%, mp: 185.3-186.5°C; LC-MS (ESI) m / z (%): 500.2 [M+H] + .

[0117] The target compound is 6-(6-aminopyrazin-2-yl)- N -(4-(4-(3-isopropoxypyrazin-1-yl)piperidin-1-yl)-3-methylphenyl)imidazo[1,2-] a The preparation of pyrazine-8-amine (A13) was as follows: Following the preparation method of A01, using M'A13 as raw material, a white solid A13 was obtained with a yield of 77.7%, mp: 166.2-167.5°C; LC-MS (ESI) m / z (%): 514.3 [M+H] + The NMR information of the compound of formula (1) prepared above is shown in Table 1.

[0118] Table 1 NMR information of compounds in formula (1) 1. Regarding the results of SYK inhibition (IC) 50 ) The HTRF KinEASE TK kit was used as the kinase assay, with Lanraplenib (purchased from TargetMol Chemicals Inc.) as the positive control. Assays were performed on 384-well plates, each well containing Hepes buffer, Brij35, EGTA, MgCl2, DTT, DMSO, ATP, SYK, and the appropriately diluted analyte compound, using a 5-fold dilution, with 500 nM as the maximum concentration.

[0119] Prepare 2×ATP / substrate and 2× kinase solutions using kinase reaction buffer. Transfer 100 nm of the compound dilution to a 384 assay plate using an Echo 655; centrifuge, add 5 μL of 2× kinase solution to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 10 min. Add 5 μL of 2× substrate and ATP solution to the 384 assay plate, centrifuge at 1000 rpm for 1 min, and incubate at 25°C for 60 min. Prepare 2× XL665 and antibody assay reagents with assay buffer. Add 10 μL of kinase assay reagent to the assay plate and incubate at 25°C. Centrifuge at 1000 rpm for 1 min and incubate at 25°C for 1 h. Read the fluorescence signals at 620 nm (Cryptate) and 665 nm (XL665) as BMG. Calculate IC50 by fitting the inhibition value and the logarithm of the compound concentration to a nonlinear regression (dose-response-variable slope) using GraphPad 7.0. 50 The results are shown in Table 3.

[0120] Table 3 SYK Inhibition Results As can be seen from the results in Table 3, the compounds of the present invention have significant inhibitory activity against SYK, with Example A13 showing better inhibitory activity than Lanraplenib.

[0121] 2. Results of tumor cell experiments CCK-8 assay for breast cancer cell viability: Ovarian cancer cells SK-OV-3, breast cancer cells MDA-MB-231, and liver cancer cells HepG2 were seeded at 3000 cells per well in 96-well plates, with 3 replicates per group, and incubated for 24 h. After cell attachment, different concentrations of compounds were added, as shown in Table 1. After culturing for 24 h, 10 µL of CCK-8 reagent was added to each well, and incubation continued for 2-4 h. The OD value of each well at 450 nm was then measured using a microplate reader, and the results are shown in Table 4. Cell viability was calculated using Formula 1. Formula 1: Cell viability (%) = [OD (drug added) - OD (blank)] / [OD (control) - OD (blank)] × 100. OD (dosedated): Absorbance of wells containing cells, CCK-8 solution, A01-A13, and Lanraplenib; OD (blank): Absorbance of wells containing culture medium and CCK-8 solution but without cells; OD (control): Absorbance of wells containing cells and CCK-8 solution but without A01-A13 and Lanraplenib.

[0122] Table 4 Tumor cell inhibition results Table 5 shows the IC50 values ​​for three types of tumor cells. 50 value The results above show that the compounds of the present invention have significant inhibitory effects on three types of tumor cells, and the inhibitory activity of Example A13 is significantly better than that of Lanraplenib.

[0123] 3. Apoptosis experiment SK-OV-3 cells were stored at 4 × 10⁻⁶ cells per well. 5 Cells were seeded in six-well plates and incubated for 24 h until cell attachment. Then, 2 mL of 2.5 μM, 5 μM, and 10 μM A13 (drug-treated group), 10 μM Lanraplenib (positive control), and culture medium (control group) were added. After 24 h of incubation, cells were harvested. Cells were resuspended in 1× binding buffer, centrifuged at 1000 rpm for 5 min, and then resuspended in 1× binding buffer to prepare a single-cell suspension. 100 μL of suspension was transferred to EP tubes. 5 μL of FITC-Annexin V was added to each EP tube, and the cells were incubated at 25°C in the dark for 30 min. 5 μL of Propidium Iodide was added, and the cells were incubated at 25°C in the dark for 5 min. Cells were then resuspended in 300 μL of 1× binding buffer, and apoptosis was detected by flow cytometry. Results are as follows: Figure 1 As shown. From Figure 1 It can be seen that compound A13 induces apoptosis in a dose-dependent manner. Compared with the blank control and Lanraplenib, A13 showed significantly higher activity in inducing apoptosis with increasing concentration.

[0124] 4. Cell cycle experiment SK-OV-3 cells were stored at 4 × 10⁻⁶ cells per well. 5 Cells were seeded in six-well plates and incubated for 24 h until cell attachment. Then, 2 mL of 2.5 μM, 5 μM, and 10 μM A13 (drug-treated group), 10 µM Lanraplenib (positive control), and culture medium (control group) were added, respectively. After 24 h of culture, cells were harvested. Cells were washed once with PBS, resuspended in PBS, and pre-chilled anhydrous ethanol (approximately 75% concentration) was added dropwise while vortexing. The cells were then fixed overnight at -20°C. The fixed cells were centrifuged at 1500 rpm for 10 min at 4°C, washed once with pre-chilled PBS, and RNase was added to a concentration of 10 μg / mL. The cells were incubated at 37°C for 30 min. PI was then added to a final concentration of 50 μg / mL, and the cells were incubated at 4°C in the dark for 30 min. Cell cycle was then analyzed by flow cytometry. Results are as follows: Figure 2 As shown, from Figure 2It can be seen that compound A13 arrests SKOV-3 cells in the S phase, inhibits cell cycle progression, and this effect is dose-dependent.

[0125] 5. Cloning experiment SK-OV-3 cells were seeded at 1000 cells per well in six-well plates and incubated for 24 h until cell attachment. Then, 2 mL of 2.5 μM, 5 μM, and 10 μM A13 (drug-treated group), 10 μM Lanraplenib (positive control), and culture medium (control group) were added, respectively. After 14 days of treatment, the culture medium was discarded. Cells were fixed with 4% paraformaldehyde at 4°C for 30 min. The cells were washed twice with distilled water for 2 min each time. They were then stained with crystal violet for 10 min. After washing with distilled water, photographs were taken, and the cells were counted. Results are shown below. Figure 3 As shown, from Figure 3 It can be seen that compound A13 inhibits the clonogenic ability of SKOV-3 cells in a dose-dependent manner.

[0126] 6. In vitro liver microsomal stability test The human liver microsomes used in the experiment were obtained from Biovit, and the NADPH was obtained from Oriental Yeast. Human liver microsomes were pre-warmed for 5 min in a 37°C water bath with a constant temperature shaker to thaw and set aside. NADPH was weighed and dissolved in an appropriate amount of magnesium chloride solution to prepare a 2 mM solution. A mixed solution of the incubation system (excluding β-NADPH) was prepared and dispensed in 180 µL / tubes (50 µL / tube for the negative control group and 120 µL / tube for the positive control group). For the 0 min sample: 30 µL of the liver microsome and substrate mixture was taken from both the experimental group and the positive control group as the 0 min sample. 300 µL of internal standard working precipitant was added, followed by 30 µL of NADPH solution. For other samples: 150 µL of NADPH solution was added to initiate the reaction (50 µL of magnesium chloride solution was added to the negative control group). After incubation at 37°C for 5, 15, 30, and 60 min (60 min for the negative control group at 37°C), 60 µL of the mixture was removed and 300 µL of internal standard precipitant was added. For the positive control group: 90 µL of NADPH solution was added. The reaction was initiated with NADPH solution and incubated in a 37°C water bath for 5-15 minutes. 60 µL of the incubator was then added to 300 µL of a precipitant containing the internal standard. For the Neat sample: 297 µL of water was pipetted, 1500 µL of the internal standard working solution was added, and 3 µL of the test compound working solution was added. All samples were vortexed and centrifuged. 150 µL of the supernatant was added to 150 µL of water, vortexed, and the compound content was analyzed by LC-MS / MS. The half-life (T0) was calculated using first-order kinetics. 1 / 2 The results are shown in Table 6.

[0127] Table 6. Metabolic stability of compound A13 in human and rat liver microsomes. Note: CL int microsome represents the intrinsic clearance rate, and remaining represents the amount remaining.

[0128] 7. SK-OV-3 tumor-bearing mouse model experiment Six-week-old female BALB / c-nu mice (Tai'an Hongfeier Biotechnology Co., Ltd.; SCXK (Lu) 2024-0006) were housed in an SPF-grade environment with a temperature maintained at 22-25℃ and humidity at approximately 50%. Drinking water, feed, and experimental supplies were all sterilized. Logarithmically growing SK-OV-3 cells were collected, washed with PBS, digested with trypsin to prepare a single-cell suspension, centrifuged, washed twice with PBS, resuspended in PBS to prepare a single-cell suspension, and inoculated into the axillae of mice at a density of 3 × 10⁻⁶ cells / mL. 7 One cell per mouse. Until the mouse tumor reaches 100 mm in size. 3 Mice that successfully received the drug were randomly divided into a control group, a low-dose A13 group, a high-dose A13 group, and a positive control group. Mice in the control group were administered 0.2 mL of PBS by gavage; mice in the low-dose A13 group were administered 30 mg / kg A13 by gavage; mice in the high-dose A13 group were administered 60 mg / kg A13 by gavage; and mice in the positive control group were administered 60 mg / kg Lanraplenib by gavage. All four groups received the drug once daily for a total of 14 times. Tumor volume was measured using a microclamp, and mouse weight was recorded every three days. Results are as follows: Figure 4 As shown in the figure, A13 can significantly inhibit tumor growth, and its effect is better than that of Lanraplenib.

[0129] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A compound containing an imidazo[1,2-a]pyrazine fragment, characterized in that, The structural formula of the compound is shown in formula (1); Equation (1); R1 can be any one of the substituents from A01 to A13; A01, A02, A03, A04, A05, A06, A07, A08, A09, A10, A11, A12, A13； The compound is used to inhibit SYK.

2. A method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment as described in claim 1, characterized in that, Includes the following steps: When R1 is A01, the compound is prepared as follows: Starting from p-fluoronitrobenzene, intermediate MA01 was prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediate MA01 and intermediate n2 were coupled by Suzuki coupling and deprotection to obtain a compound containing the A01 substituent. The structural formula of M1 is as follows: 。 3. The method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment according to claim 2, characterized in that, When R1 is A02~A04, the preparation method of the compound is as follows: Starting from p-fluoronitrobenzene, 2-fluoro-5-nitropyridine, and 2-fluoro-5-nitrotoluene, intermediates MA02, MA03, and MA04 were prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA02, MA03, and MA04 were coupled with intermediate n2 via Suzuki coupling and deprotection to obtain compounds containing substituents A02, A03, and A04, respectively.

4. The method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment according to claim 2, characterized in that, When R1 is A05~A08, the preparation method of the compound is as follows: Starting from p-fluoronitrobenzene, 2-fluoro-5-nitropyridine, 3,4-difluoronitrobenzene, and 2-fluoro-5-nitrotoluene, intermediates MA05, MA06, MA07, and MA08 were prepared by sequentially undergoing aromatic nucleophilic substitution, deprotection, cyclization, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borosilicate esterification. Intermediates MA05, MA06, MA07, and MA08 were coupled with intermediate n2 via Suzuki coupling and deprotection to obtain compounds containing substituents A05, A06, A07, and A08, respectively.

5. The method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment according to claim 2, characterized in that, When R1 is A09~A11, the preparation method of the compound is as follows: Starting from p-fluoronitrobenzene, 3,4-difluoronitrobenzene, and 2-fluoro-5-nitrotoluene, respectively, intermediates MA09, MA10, and MA11 were prepared by sequentially undergoing aromatic nucleophilic substitution, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA09, MA10, and MA11 were coupled with intermediate n2 via Suzuki coupling and deprotection to obtain compounds containing substituents A09, A10, and A11, respectively.

6. The method for preparing the compound containing the imidazo[1,2-a]pyrazine fragment according to claim 2, characterized in that, When R1 is A12 and A13, the compound is prepared as follows: Starting from p-fluoronitrobenzene and 2-fluoro-5-nitrotoluene, respectively, intermediates MA12 and MA13 were prepared by sequentially undergoing aromatic nucleophilic substitution, hydrolysis, reductive amination, nitro reduction, and aromatic nucleophilic substitution. Starting from M1, intermediate n2 was prepared by Boc protection and Miyaura borate esterification. Intermediates MA12 and MA13 were coupled with intermediate n2 via Suzuki coupling and deprotection to obtain compounds containing substituents A12 and A13, respectively.

7. The use of a compound containing an imidazo[1,2-a]pyrazine fragment as described in claim 1 in the preparation of a SYK inhibitor.

8. The use of a compound containing an imidazo[1,2-a]pyrazine fragment as described in claim 1 in the preparation of an anticancer drug.

9. The application according to claim 8, characterized in that, The anticancer drug is any one of the drugs for treating ovarian cancer, breast cancer, or liver cancer.

10. The application according to claim 8, characterized in that, The drug contains compounds with an imidazo[1,2-a]pyrazine fragment in an amount of 1 μM to 10 μM.

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