A pyrazolo pyrazine compound and a preparation method and application thereof

Abstract

This invention belongs to the field of pharmaceutical chemistry, specifically relating to a pyrazolopyrazine compound, its preparation method, and its applications. The pyrazolopyrazine compound of this invention is a compound with the structure shown in formula (I) or a pharmaceutically acceptable salt thereof. The compound provided by this invention has a novel pyrazolopyrazine chemical skeleton. Activity evaluation results show that the compound of this invention exhibits good inhibitory activity against both SHP2 and HDAC6, demonstrating excellent dual-target inhibitory effects. Therefore, this invention can provide new candidate compounds and research foundation for SHP2-HDAC6 dual-target inhibitors or antitumor drugs, and at the same time has important scientific significance and application value for overcoming drug resistance to existing single-target inhibitors, improving the treatment effect of solid tumors, and promoting the clinical translation of dual-target antitumor drugs.

Description

Technical Field

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

[0002] Malignant tumors are one of the leading causes of death worldwide, with their incidence and mortality rates showing an increasing trend year by year, seriously threatening human health. Targeted therapy with small molecule drugs, with its advantages of high specificity, significant efficacy, and relatively controllable adverse reactions, has become a core development direction in the treatment of malignant tumors after surgery, chemotherapy, and radiotherapy.

[0003] Among them, protein tyrosine phosphatase 2 (SHP2) and histone deacetylase 6 (HDAC6) are both well-established important oncoproteins and immunomodulators, participating in the regulation of multiple key biological processes such as cancer cell proliferation, survival, invasion and metastasis, and immune escape. They are two core hot targets in the current field of anti-tumor drugs.

[0004] SHP2 is a non-receptor protein tyrosine phosphatase encoded by the proto-oncogene PTPN11, located upstream of the RAS-ERK signaling pathway. It can fully activate this pathway to control cancer cell growth and survival, and its abnormal activation is closely related to the development and progression of various malignant tumors, including non-small cell lung cancer, triple-negative breast cancer, and pancreatic ductal adenocarcinoma. In the development of SHP2 inhibitors, early catalytic site inhibitors suffered from poor subtype selectivity due to the highly conserved catalytic pocket of the phosphatase, and their highly polar pharmacophores resulted in extremely low cell membrane permeability and oral bioavailability, making them long considered difficult targets for drug development. Subsequent allosteric inhibitors, such as SHP099, exert their effects by binding to an allosteric site outside the catalytic pocket and stabilizing the self-inhibitory conformation of SHP2, thus solving these problems. Currently, 16 compounds, including JAB-3312, RMC-4630, and TNO-155, have entered clinical trials. However, multiple clinical studies have shown that SHP2 single-target inhibitors have not shown significant anti-tumor efficacy when used alone, and their clinical application currently mainly relies on combination with other drugs such as KRAS inhibitors and PD-1 antibodies.

[0005] HDAC6 is a unique member of the histone deacetylase family, mainly distributed in the cytoplasm. It participates in tumor progression by regulating the acetylation levels of non-histone substrates such as α-tubulin, Hsp90, and STAT3. Compared with broad-spectrum HDAC inhibitors, selective HDAC6 inhibitors avoid interference with class I nuclear HDACs, significantly reducing serious adverse reactions such as myelosuppression, cardiotoxicity, and hepatotoxicity. Currently, several highly active HDAC6 inhibitors, such as ACY-1215, ACY-241, and Tubastatin A, have entered clinical trials. However, single-target HDAC6 inhibitors require high blood concentrations to achieve effective antitumor effects, which can easily lead to off-target effects, further increasing the risk of adverse reactions.

[0006] Regarding combination therapy, studies have confirmed that the combined use of SHP2 inhibitors and HDAC inhibitors can produce a stronger anti-tumor effect by synergistically inhibiting the RAS-ERK signaling pathway. When the ERK pathway is inhibited, the sensitivity of tumor cells to HDAC inhibitors is significantly increased. However, combination therapy has problems such as large differences in pharmacokinetic characteristics, complex drug interactions, and cumbersome dosing regimens, leading to poor patient compliance. In addition, the incidence of adverse reactions with combination therapy is significantly higher than that with monotherapy, which limits its widespread clinical application.

[0007] In the research of dual-target inhibitors, various dual-target anti-tumor inhibitors, such as SHP2-CDK4, SHP2-NAMPT, and HDAC6-LSD1, have been successfully developed both domestically and internationally. Furthermore, Fang Hao's team reported the first SHP2-HDAC dual-target inhibitor; however, this compound is a broad-spectrum HDAC inhibitor, lacking specific selection for the HDAC6 subtype, and therefore cannot avoid the serious adverse reactions caused by broad-spectrum HDAC inhibitors. To date, there are no publicly reported SHP2-HDAC6 dual-target inhibitors, both domestically and internationally, indicating a significant technological gap in this field.

[0008] Therefore, how to develop a class of compounds with excellent dual-target inhibitory activity against SHP2 and HDAC6 to address the shortcomings of existing single-target inhibitors, such as limited efficacy, poor adherence to combination therapy, and insufficient selectivity and numerous adverse reactions of existing dual-target inhibitor subtypes, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0009] To address the shortcomings of the prior art, the present invention aims to provide a pyrazolopyrazine compound, its preparation method, and its applications. The pyrazolopyrazine compound provided by the present invention possesses a pyrazolopyrazine structural skeleton and exhibits good inhibitory activity against both SHP2 and HDAC6, providing a novel structural option for the development of SHP2-HDAC6 dual-target drugs.

[0010] The objective of this invention is achieved by the following technical solution: A pyrazoloid compound, which is a compound of formula (I) or a pharmaceutically acceptable salt thereof: ; In equation (I), R is -LZ; where L is C2~C 12 straight-chain alkylene groups, C2~C 12 One of the branched alkylene groups, m-phenylene, p-phenylene, p-tolyl, and p-ethylphenyl; Z is one of -C(=O)NH(C6H4)NH2·HCl, -C(=O)NHNH2, and -C(=O)NHOH.

[0011] To enhance the inhibitory activity of the compound against both SHP2 and HDAC6 proteins, the pyrazolopyrazine compound is further selected from compounds with the following structures: , , , , , , , , , , , , , , , , , , , , , , , , , .

[0012] The compounds of this invention can also be in the form of pharmaceutically acceptable salts, which can be rationally selected by those skilled in the art as needed. Further, the pharmaceutically acceptable salts are one or more selected from the following: hydrochloride, hydrobromide, sulfate, phosphate, borate, methanesulfonate, p-toluenesulfonate, naphthalenesulfonate, benzenesulfonate, citrate, lactate, pyruvate, tartrate, acetate, trifluoroacetate, maleate, succinate, amygdalinate, fumarate, salicylate, and phenylacetate.

[0013] A method for preparing the pyrazoloid compound as described above includes the following steps: ; Step (a): Under inert gas protection, 3-chloro-4-iodopyridine-2-amine and methyl mercaptopropionate are reacted in a solvent at 80-120°C for 4-12 h in the presence of palladium catalyst, phosphine ligand and organic base, and then purified to obtain intermediate A. Step (b): The intermediate A is reacted with sodium ethoxide in a solvent at 0-30°C for 10-24 hours to obtain intermediate B; Step (c): 6-chloro-1H-pyrazolo[3,4-B]pyrazine is reacted with halosuccinimide in a solvent at 60-100°C for 2-8 h to obtain intermediate C; Step (d): The intermediate C is reacted with tert-butyl (4-methylpiperidin-4-yl)carbamate in a solvent at 60-90°C for 2-8 h, and then purified to obtain intermediate D; Step (e): The intermediate D is reacted in a solvent under acidic conditions at 0-30°C for 2-8 hours to obtain intermediate E; Step (f): The intermediate E is reacted with the diacid monomethyl ester compound in a solvent at 0-30°C for 12-48 h in the presence of a condensing agent and an organic base to obtain intermediate F; Step (g): Under inert gas protection, intermediate F and intermediate B are reacted in a solvent at 60-120°C for 5-15 h in the presence of a copper catalyst, ligand and inorganic base, and then purified to obtain intermediate G. Step (h): The intermediate G is reacted in a solvent at 0-30°C for 4-12 hours in the presence of an inorganic base to obtain intermediate H; the intermediate H is then reacted with... N-Boc-1,2-phenylenediamine is reacted in a solvent at 40-80°C for 4-12 h in the presence of a condensing agent and an organic base to obtain intermediate I; intermediate I is then reacted in a solvent at 0-30°C for 10-24 h under acidic conditions to obtain the pyrazolopyrazine compound shown in formula (I). Alternatively, in step (h), the intermediate G is reacted with hydrazine hydrate in a solvent at 60-100°C for 6-15 h to obtain the pyrazolopyrazine compound shown in formula (I). Alternatively, in step (h), the intermediate G is reacted with hydroxylamine in a solvent at -5 to 30°C for 4 to 24 hours in the presence of a base to obtain the pyrazolopyrazine compound shown in formula (I).

[0014] Further, in step (a), the molar ratio of 3-chloro-4-iodopyridin-2-amine, methyl mercaptopropionate, palladium catalyst, phosphine ligand, and organic base is 1:1~2:0.01~0.05:0.05~0.15:1.5~3.0; in step (b), the molar ratio of intermediate A to sodium ethoxide is 1:1~2; in step (c), the molar ratio of 6-chloro-1H-pyrazolo[3,4-B]pyrazine to halosuccinimide is 1:1.5~2.5; in step (d), the molar ratio of intermediate C to (4-methylpiperidin-4-yl)carbamate tert-butyl ester is 1:1~2; in step (f), the molar ratio of intermediate E to diacid monomethyl ester compound is 1~2:1~2; in step (g), the molar ratio of intermediate F to intermediate B is 1:1~3.

[0015] Further, in step (f), the diacid monomethyl ester compound is selected from one of monomethyl glutarate, monomethyl adipic acid, monomethyl pimecrolate, monomethyl octanoate, monomethyl azelaic acid, monomethyl isophthalic acid, monomethyl terephthalic acid, monomethyl 4-(3-carboxypropyl)phenylacetic acid, and monomethyl 4-(2-carboxyethyl)phenylacetic acid; in step (g), the copper catalyst is cuprous iodide, and the ligand is 1,10-o-phenanthroline.

[0016] Furthermore, the solvent is selected from 1,4-dioxane, tetrahydrofuran, acetonitrile, etc. N,N -Dimethylformamide, N One or more of methylpyrrolidone, ethyl acetate, methanol, ethanol, and water.

[0017] The use of a pyrazoloid compound as described above in the preparation of a drug that targets and inhibits SHP2 and / or HDAC6.

[0018] The use of a pyrazoloid compound as described above in the preparation of a medicament for treating cancer, wherein the cancer is one of gastric cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, or cervical cancer.

[0019] A medicament for treating cancer, the medicament comprising at least the pyrazoloid compound as described above, and pharmaceutically acceptable excipients or carriers; the cancer being one of gastric cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, or cervical cancer.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: The compounds provided by this invention have a novel pyrazolopyrazine chemical skeleton. Activity evaluation results show that the compounds of this invention exhibit good inhibitory activity against both SHP2 and HDAC6. Several preferred compounds show good synergistic inhibitory effects against SHP2 and HDAC6, demonstrating significant target inhibition.

[0021] The method for preparing pyrazoloid compounds provided by this invention is mild and uses readily available raw materials, making it suitable for compound preparation and application.

[0022] Therefore, given that SHP2 and HDAC6 are important targets for tumor therapy, the compounds of this invention can be used to prepare inhibitors or antitumor drugs based on the dual targets of SHP2 and HDAC6. This provides new candidate compounds and a research basis for novel therapeutic drugs with a synergistic antitumor mechanism based on the dual targets of SHP2 and HDAC6. At the same time, it has important scientific significance and application value for overcoming the drug resistance of existing single-target inhibitors, improving the treatment effect of solid tumors, and promoting the clinical translation of dual-target antitumor drugs. Detailed Implementation

[0023] The present invention will be further described below with reference to specific embodiments. It should be noted that the following embodiments are merely illustrative of the present invention and not intended to limit it. Without conflict, the various embodiments or technical features described below can be arbitrarily combined to form new embodiments. Unless otherwise specified, the reagents or raw materials used in the following embodiments are commercially available.

[0024] In the following examples, the structures of the compounds were determined by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS). The NMR spectrometer used was a Bruker DPX-400 superconducting NMR spectrometer (Sweden), with tetramethylsilane (TMS) as an internal standard; the HRMS spectrometer was a Waters-Micromass Q-Tof mass spectrometer. The structures of the compounds involved in the following examples were determined by melting point determination, 1 H NMR, 13 Confirmed by modern spectroscopic methods such as C NMR and HRMS.

[0025] The pyrazoloid compounds involved in Examples 1-27 of the present invention have the general structural formula shown in formula (I): ; In equation (I), R is -LZ; where L is C2~C 12 straight-chain alkylene groups, C2~C 12 One of the branched alkylene groups, m-phenylene, p-phenylene, p-tolyl, and p-ethylphenyl; Z is one of -C(=O)NH(C6H4)NH2·HCl, -C(=O)NHNH2, and -C(=O)NHOH.

[0026] Furthermore, the pyrazoloid compounds involved in Examples 1-27 of this invention have the following general reaction routes: .

[0027] Example 1 A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1c: .

[0028] The preparation method of compound 1c includes the following steps: (a) 3-Chloro-4-iodopyridin-2-amine (8.0 g, 1 eq.), methyl 3-mercaptopropionate (4.18 mL, 1.2 eq.), N,N-diisopropylethylamine (DIPEA, 10.95 mL, 2 eq.), tris(dibenzylacetone)dipalladium (0.86 g, 0.03 eq.), and 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene) (1.64 g, 0.09 eq.) were added to a 250 mL three-necked flask, along with 120 mL of 1,4-dioxane as a solvent. The entire reaction was carried out under nitrogen protection at 100 °C for 6 h. The reaction was monitored by TLC until complete (evolving solvent was petroleum ether:ethyl acetate, i.e., PE:EA = 1:1). After cooling to room temperature, the mixture was concentrated and then purified by column chromatography at a PE:EA ratio of 4:1 to obtain intermediate A.

[0029] (b) Intermediate A (13.072 g, 1 eq.) was placed in a 250 mL round-bottom flask, dissolved in 150 mL of tetrahydrofuran, and then placed in an ice bath. Once the system cooled to 0 °C, sodium ethoxide (23.61 mL, 1.2 eq.) was added dropwise, and the mixture was stirred overnight at room temperature (10–24 h). The reaction was monitored by TLC (PE:EA = 1:2) to ensure complete reaction. The mixture was concentrated under reduced pressure, and dichloromethane was added to precipitate the product. The solid was collected by filtration and dried to obtain intermediate B.

[0030] (c) Add 6-chloro-1H-pyrazolo[3,4-B]pyrazine (5 g, 1 eq.) and N-iodosuccinimide (12.15 g, 1.67 eq.) to a 250 mL round-bottom flask, along with 50 mL of [unspecified ingredient]. N,N Using dimethylformamide (DMF) as a solvent, the reaction was heated at 80°C for 4 h, and the reaction was monitored by TLC (PE:EA = 1:1) to ensure complete reaction. After the reaction system cooled to room temperature, it was stirred in an ice bath for 10 min, and 290 mL of 2 M sodium bisulfite solution was added dropwise. The reaction was then carried out in an ice bath for 1 h. After the reaction, the solid was collected by filtration and dried to obtain intermediate C.

[0031] (d) Intermediate C (10.49 g, 1 eq.) and tert-butyl (4-methylpiperidin-4-yl)carbamate (8.32 g, 1.2 eq.) were added to a 250 mL round-bottom flask, along with 135 mL of N-methylpyrrolidone (NMP). The mixture was heated at 75 °C for 4 h, and the reaction was monitored by TLC (PE:EA = 1:1) to ensure complete reaction. After the system cooled to room temperature, water was added to precipitate the product. The solid was collected by filtration and dried to obtain intermediate D.

[0032] (e) Intermediate D (15 g, 1 eq.) was added to a 250 mL round-bottom flask, and 23 mL of ethyl acetate was added as a solvent. Then, 23 mL of ethyl acetate solution of hydrochloric acid (4 M) was added dropwise to the system, and the mixture was stirred at room temperature for 4 h. The reaction was monitored by TLC (PE:EA = 1:1) to ensure complete reaction. The solid was collected by filtration, dried, and intermediate E was obtained.

[0033] (f) The monomethyl diacid ester compound (specifically, monomethyl pimecrolate, 591.65 μL, 1 eq.) and DIPEA (2.33 mL, 4 eq.) were added to a 100 mL round-bottom flask, along with 15 mL of DMF as solvent. After stirring at room temperature for 0.5 h, the condensation reagent HATU (1.27 g, 1 eq.) was added, followed by stirring at room temperature for 1 h. Then, intermediate E (1.2 g, 1 eq.) was added, and the mixture was stirred at room temperature for 24 h. The reaction was monitored by TLC (PE:EA = 1:1) to ensure complete reaction. Water was added to the system to precipitate the product. The solid was collected by filtration, dried, and the intermediate F was obtained.

[0034] (g) Intermediate F (985 mg, 1 eq.), intermediate B (699.35 mg, 2 eq.), anhydrous potassium carbonate (793.98 mg, 3 eq.), cuprous iodide (83.88 mg, 0.23 eq.), and 1,10-o-phenanthroline (79.34 mg, 0.23 eq.) were placed in a 100 mL round-bottom flask. 50 mL of 1,4-dioxane was added as a solvent. The air in the system was replaced with nitrogen, and the reaction was kept under nitrogen protection. The mixture was heated at 100 °C for 7 h, and the reaction was monitored for completeness by TLC (PE:EA = 1:4). The reaction mixture was concentrated, crude silica gel was added, and the mixture was purified by column chromatography (PE:EA = 1:4) to obtain intermediate G.

[0035] (h) Intermediate G (240 mg, 1 eq.) and sodium hydroxide (168.15 mg, 10 eq.) were added to a 50 mL round-bottom flask, along with 16 mL of methanol and 3 mL of water as solvents. The mixture was stirred at room temperature for 6 h, and the reaction was monitored by TLC (using dichloromethane:methanol, i.e., DCM:MeOH = 10:1 as the developing solvent) to monitor the completeness of the reaction. The system was then evaporated to dryness, and 2 mL of water was added to the system. The pH was adjusted to 3 with hydrochloric acid, and the mixture was stirred at room temperature for 20 min. The solid was collected by vacuum filtration and dried to obtain intermediate H. Intermediate H (150 mg, 1 eq.) and DIPEA (196.07 μL, 4 eq.) were added to a 50 mL round-bottom flask, along with 5 mL of DMF as a solvent. The mixture was stirred at room temperature for 30 min, then HATU (107 mg, 1 eq.) was added, and the mixture was stirred at room temperature for 1 h. N -Boc-1,2-phenylenediamine (117.21 mg, 2 eq.) was heated at 60 °C for 8 h, and the reaction was monitored by TLC (using EA as the developing solvent) to ensure complete reaction. After the system cooled to room temperature, it was extracted three times with ethyl acetate and water, washed once with saturated brine, and the organic phase was evaporated to dryness. The sample was then stirred with coarse silica gel and purified by column chromatography (PE:EA = 1:1 ratio) to obtain the starting material. N -Boc-1,2-phenylenediamine, and then purified by passing the product through a P:E ratio of 1:4 to obtain intermediate I; Intermediate I (83 mg) was added to a 25 mL round-bottom flask, along with 2.5 mL of ethyl acetate as solvent and 1 mL of ethyl hydrochloride solution (4 M). The mixture was stirred overnight at room temperature, and the reaction was monitored by TLC (using EA as the developing solvent) to ensure complete reaction. The solid was collected by filtration, dried, and the product was given as compound 1c.

[0036] The characterization results of compound 1c are as follows: N 1-(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo [3,4-b]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 7 -(2-aminophenyl)heptanediamide. Brown solid, yield 65.3%, melting point 223.6-223.8 °C. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.04 (s, 1H), 10.43 (s, 1H), 8.49 (s, 1H), 8.39 (s, 1H), 7.69 (d, J =6.8 Hz, 1H), 7.58 (s, 1H), 7.55 – 7.46 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.26(t, J = 7.7 Hz, 1H), 6.05 (d, J = 6.9 Hz, 1H), 4.09 (d, J = 13.5 Hz, 2H), 3.36 (t, J = 12.2 Hz, 2H), 2.39 (t, J = 7.5 Hz, 2H), 2.24 (d, J = 13.2 Hz, 2H), 2.14 (t, J =7.4 Hz, 2H), 1.63 (d, J = 8.3 Hz, 2H), 1.57 – 1.44 (m, 4H), 1.32 (s, 2H), 1.29 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 172.42, 155.29, 154.27, 153.33, 150.22,145.34, 134.39, 133.05, 130.66, 129.29, 125.52, 124.74, 113.69, 110.38,109.21, 50.86, 41.05, 35.74, 34.76, 27.72, 26.11, 25.91, 25.80, 24.84, 18.61.HR-MS(ESI): Calcd. C 29 H 35 ClN 10 O2S, [M+H] + m / z: 623.2432, found: 623.2435.

[0037] Example 2

[0038] A pyrazoloid compound, with the structural formula shown below, is designated as compound 1a: .

[0039] The preparation method of compound 1a is basically the same as that in Example 1, except that monomethyl pimecrolate in step (f) is replaced with monomethyl glutarate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1a.

[0040] The characterization results of compound 1a are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo [3,4-b]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 5 -(2-aminophenyl)glutaramide. Yellow solid, yield 98.4%, melting point 223.6-223.8 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ14.05 (s, 1H), 10.35 (s, 1H), 8.50 (s, 1H), 8.27 (s, 1H), 7.70 – 7.62 (m,2H), 7.50 – 7.40 (m, 2H), 7.34 (t, J = 7.9 Hz, 1H), 7.27 (t, J= 7.7 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 4.10 (d, J = 13.4 Hz, 2H), 3.39 (t, J = 12.4 Hz, 2H), 2.41 (t, J = 7.5 Hz, 2H), 2.31 – 2.16 (m, 4H), 1.92 – 1.81 (m, 2H), 1.56 – 1.42(m, 2H), 1.32 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.16, 171.83, 155.28,153.38, 150.24, 145.35, 134.50, 133.21, 131.87, 130.76, 129.30, 125.81,125.32, 124.75, 110.37, 109.23, 59.82, 50.90, 41.07, 35.46, 35.25, 26.08,21.41, 14.13. HR-MS(ESI): Calcd. C 27 H 31 ClN 10 O2S, [M+H] + m / z: 595.2119, found: 595.2124.

[0041] Example 3

[0042] A pyrazoloid compound, with the structural formula shown below, is designated as compound 1b: .

[0043] The preparation method of compound 1b is basically the same as that in Example 1, except that monomethyl pimecrolate in step (f) is replaced with monomethyl adipic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1b.

[0044] The characterization results of compound 1b are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo [3,4- b]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 6 -(2-aminophenyl)adipamide. Pale yellow solid, yield 77.8%, melting point 214.7-216.5 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ14.03 (s, 1H), 10.24 (s, 1H), 8.49 (s, 1H), 8.11 (s, 1H), 7.65 (s, 1H), 7.59(s, 1H), 7.43 (s, 1H), 7.34 (s, 1H), 7.23 (s, 2H), 6.01 (s, 1H), 4.08 (s,2H), 3.37 (s, 2H), 2.41 (s, 2H), 2.23 (s, 2H), 2.17 (s, 2H), 1.59 (s, 4H),1.49 (s, 2H), 1.30 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.42, 171.97,154.66, 153.35, 150.58, 145.32, 133.81, 131.27, 129.46, 126.68, 124.74,110.23, 50.85, 35.92, 35.47, 25.13. HR-MS(ESI): Calcd. C 28 H 33 ClN 10 O2S, [M+H] + m / z: 609.2275, found: 609.2275.

[0045] Example 4

[0046] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1d: .

[0047] The preparation method of compound 1d is basically the same as that in Example 1, except that monomethyl pimelic acid in step (f) is replaced with monomethyl succinate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1d.

[0048] The characterization results of compound 1d are as follows:N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H -pyrazolo [3,4-b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 8 -(2-aminophenyl)octanediamide. Yellow solid, yield 71.1%, melting point 225.5-226.0 ℃. 1 H NMR (400 MHz, DMSO- d 6 )δ 14.05 (s, 1H), 10.32 (s, 1H), 8.50 (s, 1H), 8.25 (s, 1H), 7.67 (d, J = 6.7Hz, 1H), 7.53 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.42 (d, J = 7.9 Hz, 1H), 7.32(t, J = 7.6 Hz, 1H), 7.25 (t, J = 7.6 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 4.09 (d, J =13.6 Hz, 2H), 3.38 (d, J = 12.2 Hz, 2H), 2.39 (t, J = 7.6 Hz, 2H), 2.24 (d, J =13.4 Hz, 2H), 2.12 (t, J = 7.4 Hz, 2H), 1.60 (d, J = 9.8 Hz, 2H), 1.50 (d, J = 10.2Hz, 4H), 1.30 (s, 7H). 13 C NMR (101 MHz, DMSO- d 6) δ 172.60, 172.11, 155.12,153.39, 150.33, 145.33, 133.92, 131.77, 129.32, 125.40, 124.79, 110.31,110.08, 50.79, 29.69, 28.46, 26.63, 25.46, 24.87, 23.66. HR-MS(ESI): Calcd.C 30 H 37 ClN 10 O2S, [M+H] + m / z: 637.2588, found: 637.2594.

[0049] Example 5

[0050] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1e: .

[0051] The preparation method of compound 1e is basically the same as that in Example 1, except that monomethyl pimecrolate in step (f) is replaced with monomethyl azelaic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1e.

[0052] The characterization results of compound 1e are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 9 -(2-aminophenyl)nonanediamide. Yellow solid, yield 73.8%, melting point 219.3-219.8 °C. 1 H NMR (400 MHz, DMSO- d 6 )δ 14.03 (s, 1H), 10.36 (s, 1H), 8.50 (s, 1H), 8.31 (s, 1H), 7.68 (d, J = 6.9Hz, 1H), 7.55 – 7.48 (m, 2H), 7.45 (d, J = 7.9 Hz, 1H), 7.35 (t, J= 7.7 Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 4.09 (d, J = 13.7 Hz, 2H), 3.36(t, J = 12.2 Hz, 2H), 2.38 (t, J = 7.6 Hz, 2H), 2.24 (d, J = 13.4 Hz, 2H), 2.11 (t, J = 7.4 Hz, 2H), 1.60 (s, 2H), 1.49 (s, 4H), 1.29 (s, 9H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.56, 172.12, 155.00, 153.36, 150.36, 145.31, 134.78, 131.64,130.67, 129.36, 125.70, 124.75, 113.71, 110.30, 59.80, 50.77, 36.15, 35.73,28.57, 25.97, 25.49, 24.89. HR-MS(ESI): Calcd. C 31 H 39 ClN 10 O2S, [M+H] + m / z:651.2745, found: 651.2734.

[0053] Example 6

[0054] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1f: .

[0055] The preparation method of compound 1f is basically the same as that in Example 1, except that monomethyl pimecrolate in step (f) is replaced with monomethyl isophthalic acid monomethyl ester, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1f.

[0056] The characterization results of compound 1f are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H-pyrazolo[3,4- b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 3 -(2-aminophenyl)isophthalamide. Yellow solid, yield 76.7%, melting point 219.7-220.6 °C. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.07 (s, 1H), 10.88 (s, 1H), 8.75 (s, 1H), 8.53 (s, 1H), 8.31 (s, 1H),8.18 (d, J = 7.8 Hz, 1H), 8.13 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H), 7.69 – 7.60 (m,3H), 7.53 (d, J = 7.8 Hz, 1H), 7.43 (t, J = 7.7 Hz, 1H), 7.35 (t, J = 7.7 Hz, 1H), 6.04 (d, J = 6.8 Hz, 1H), 4.17 (d, J = 13.4 Hz, 2H), 3.53 (t, J = 12.0 Hz, 2H), 2.57 (d, J = 13.1 Hz, 2H), 1.64 (t, J = 11.5 Hz, 2H), 1.46 (s, 3H). 13 C NMR (101MHz, DMSO- d 6 ) δ 166.31, 165.37, 154.95, 153.46, 150.36, 145.35, 135.62,133.96, 133.52, 131.42, 129.36, 127.75, 126.25, 124.80, 124.70, 110.29,110.09, 52.08, 26.56. HR-MS(ESI): Calcd. C 30 H 29 ClN 10 O2S, [M+H] +m / z: 629.1962, found: 629.1963.

[0057] Example 7

[0058] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1g: .

[0059] The preparation method of compound 1g is basically the same as that in Example 1, except that monomethyl pimecrolate in step (f) is replaced with monomethyl terephthalate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1g.

[0060] The characterization results of 1g of compound are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 4 -(2-aminophenyl)terephthalamide. White solid, yield 38.4%, melting point 217.7-218.8 °C. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.04 (s, 1H), 10.59 (s, 1H), 8.53 (s, 1H), 8.18 (d, J = 8.1 Hz, 2H),8.07 (s, 1H), 8.02 (d, J = 6.5 Hz, 1H), 7.99 (d, J = 8.2 Hz, 2H), 7.66 (d, J = 6.7Hz, 1H), 7.55 (d, J = 5.0 Hz, 1H), 7.40 (d, J = 4.8 Hz, 1H), 7.35 – 7.28 (m, 2H), 6.01 (d, J = 6.7 Hz, 1H), 4.16 (d, J = 13.4 Hz, 2H), 3.49 (t, J= 12.0 Hz, 2H),2.47 (s, 2H), 1.65 (t, J = 11.4 Hz, 2H), 1.46 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 )δ 166.36, 165.11, 154.66, 153.49, 150.61, 145.33, 138.65, 135.65, 133.95,131.18, 129.46, 128.71, 128.42, 126.68, 124.85, 124.75, 110.27, 110.10,51.97, 34.81, 33.44, 26.39, 25.16. HR-MS(ESI): Calcd. C 30 H 29 ClN 10 O2S, [M+H] + m / z:629.1962, found: 629.1970.

[0061] Example 8

[0062] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1h: .

[0063] The preparation method of compound 1h is basically the same as that in Example 1, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(3-carboxypropyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1h.

[0064] The characterization results of compound 1h are: 4-(2-((1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)amino)-2-oxoethyl)- N -(2-aminophenyl)benza-mide. Brown solid, yield 72.2%, melting point 244.8-245.3 ℃. 1 H NMR (400 MHz, DMSO- d 6) δ 14.05 (s, 1H), 10.46 (s, 1H), 8.48 (s, 1H), 8.02(d, J = 8.2 Hz, 3H), 7.65 (d, J = 6.5 Hz, 1H), 7.53 (s, 1H), 7.45 (d, J = 7.8 Hz,3H), 7.41 – 7.28 (m, 2H), 6.03 (d, J = 6.8 Hz, 1H), 4.09 (d, J = 13.1 Hz, 2H),3.57 (s, 2H), 3.33 (d, J = 12.8 Hz, 2H), 2.23 (d, J = 13.4 Hz, 2H), 1.51 (t, J =11.7 Hz, 2H), 1.31 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 170.02, 165.85,154.72, 153.42, 150.61, 145.40, 141.40, 131.71, 131.21, 129.59, 128.17,124.86, 110.35, 51.25, 43.22. HR-MS(ESI): Calcd. C 31 H 31 ClN 10 O2S, [M+H] + m / z:643.2119, found: 643.2126.

[0065] Example 9

[0066] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 1i: .

[0067] The preparation method of compound 1i is basically the same as that in Example 1, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(2-carboxyethyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 1i.

[0068] The characterization results of compound 1i are: 4-(3-((1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ]pyrazin-6-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropyl)- N -(2-aminophenyl)benzamide. Brown solid, yield 71.6%, melting point 207.5-208.3℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 14.11 (s, 1H), 10.52 (s, 1H), 8.48 (s, 1H), 8.38 (s, 1H), 8.04 (d, J = 8.2 Hz, 2H), 7.70 – 7.62 (m, 2H), 7.57 – 7.50 (m,2H), 7.44 – 7.37 (m, 3H), 7.35 – 7.30 (m, 1H), 6.03 (d, J = 6.8 Hz, 1H), 4.06(d, J = 13.3 Hz, 2H), 3.21 (t, J = 12.0 Hz, 2H), 2.91 (t, J = 7.6 Hz, 2H), 2.22 (d, J = 13.5 Hz, 2H), 1.46 (t, J = 11.8 Hz (2H), 1.28 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 171.43, 165.56, 155.21, 153.36, 150.25, 145.34, 131.88, 131.20, 129.30,127.71, 127.27, 124.76, 110.36, 110.09, 50.88, 31.20, 26.68. HR-MS(ESI):Calcd. C 32 H 33 ClN 10 O2S, [M+H] +m / z: 657.2275, found: 657.2270.

[0069] Example 10

[0070] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2c: .

[0071] The preparation method of compound 2c is basically the same as that in Example 1, except that steps (a) to (g) are the same as in Example 1; step (h) is adjusted as follows: intermediate G (100 mg, 1 eq.) is added to a 50 mL round-bottom flask, 5 mL of ethanol is added as a solvent, and then hydrazine hydrate (1.11 mL, 100 eq.) is added. The system is heated at 80 °C for 9 h. The reaction is monitored by TLC until complete. After the system cools to room temperature, 1 mL of water is added to the round-bottom flask, the mixture is concentrated under reduced pressure, rotated to near dryness, water is added to precipitate the solid, the solid is collected by vacuum filtration, and dried to obtain compound 2c.

[0072] The characterization results of compound 2c are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b yrazi-n-6-yl)-4-methylpiperidin-4-yl)-7-hydrazineyl-7-oxoheptanamide. Light brown solid, yield 60%, melting point 219.5-220.2 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.79 (s, 1H), 8.90 (s, 1H), 8.45 (s, 1H), 7.56 (d, J = 5.4 Hz, 1H), 7.44 (s, 1H), 6.37 (s, 2H), 5.73 (d, J = 5.3 Hz, 1H), 4.08 (d, J = 14.0 Hz,3H), 3.30 (s, 2H), 2.21 (d, J = 13.4 Hz, 2H), 2.08 (t, J = 7.4 Hz, 2H), 1.99(t, J= 7.6 Hz, 2H), 1.54 – 1.43 (m, 6H), 1.29 (s, 3H), 1.22 (d, J = 7.6 Hz, 2H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.44, 171.60, 155.51, 153.23, 146.33,145.58, 145.09, 132.41, 131.64, 124.80, 109.70, 108.39, 50.77, 41.00, 36.07,34.78, 33.35, 28.33, 25.95, 25.28, 25.04. HR-MS(ESI): Calcd. C 23 H 31 ClN 10 O2S, [M+H] + m / z: 547.2119, found: 547.2110.

[0073] Example 11

[0074] A pyrazoloid compound, with the structural formula shown below, is designated as compound 2a: .

[0075] The preparation method of compound 2a is basically the same as that in Example 10, except that monomethyl pimecrolate in step (f) is replaced with monomethyl glutarate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2a.

[0076] The characterization results of compound 2a are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ] p-yrazin-6-yl)-4-methylpiperidin-4-yl)-5-hydrazineyl-5-oxopentanamide. Light brown solid, yield 70.6%, melting point 183.5-184.0 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.79 (s, 1H), 8.97 (s, 1H), 8.45 (s, 1H), 7.55 (d,J = 5.5 Hz, 1H),7.47 (s, 1H), 6.37 (s, 2H), 5.72 (d, J = 5.4 Hz, 1H), 4.33 (s, 1H), 4.08 (d, J =13.6 Hz, 2H), 2.21 (d, J = 13.5 Hz, 2H), 2.09 (t, J = 7.6 Hz, 2H), 2.01 (s, 2H),1.77 – 1.65 (m, 2H), 1.50 (t, J = 12.0 Hz, 2H), 1.29 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 172.06, 171.35, 155.52, 153.26, 146.35, 145.59, 145.10, 132.32,131.66, 124.81, 109.69, 108.41, 50.82, 41.01, 35.68, 34.81, 32.94, 25.96,21.79. HR-MS(ESI): Calcd. C 21 H 27 ClN 10 O2S, [M+H] + m / z: 519.1806, found: 519.1805.

[0077] Example 12

[0078] A pyrazoloid compound, with the structural formula shown below, is designated as compound 2b: .

[0079] The preparation method of compound 2b is basically the same as that in Example 10, except that monomethyl pimecrolate in step (f) is replaced with monomethyl adipic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2b.

[0080] The characterization results of compound 2b are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- bp-yrazin-6-yl)-4-methylpiperidin-4-yl)-6-hydrazineyl-6-oxohexanamide. White solid, 70% yield, melting point 164.7-165.3 °C. 1 H NMR (400 MHz, DMSO- d 6 ) δ13.78 (s, 1H), 8.91 (s, 1H), 8.45 (s, 1H), 7.56 (d, J = 5.4 Hz, 1H), 7.44 (s,1H), 6.36 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.13 (s, 1H), 4.08 (d, J = 13.4 Hz,2H), 3.37 (s, 2H), 2.21 (d, J = 13.5 Hz, 2H), 2.09 (d, J = 7.2 Hz, 2H), 2.00 (d, J = 6.7 Hz, 2H), 1.54 – 1.42 (m, 6H), 1.29 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ172.33, 171.55, 155.52, 153.24, 146.35, 145.11, 131.65, 124.81, 108.40,50.80, 36.03, 34.48, 33.35, 25.28, 24.96. HR-MS(ESI): Calcd. C 22 H 29 ClN 10 O2S, [M+H] + m / z: 533.1962, found: 533.1965.

[0081] Example 13

[0082] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2d: .

[0083] The preparation method of compound 2d is basically the same as that in Example 10, except that monomethyl pimelic acid in step (f) is replaced with monomethyl succinate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2d.

[0084] The characterization results of compound 2d are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b p-yrazin-6-yl)-4-methylpiperidin-4-yl)-8-hydrazineyl-8-oxooctanamide. White solid, yield 60%, melting point 141.0-141.7 °C. 1 H NMR (400 MHz, DMSO- d 6 ) δ13.79 (s, 1H), 8.90 (s, 1H), 8.45 (s, 1H), 7.56 (d, J = 5.4 Hz, 1H), 7.44 (s,1H), 6.36 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.19 (s, 1H), 4.08 (d, J = 13.5 Hz,2H), 3.30 (s, 2H), 2.21 (d, J = 13.4 Hz, 2H), 2.08 (t, J = 7.4 Hz, 2H), 1.98 (t, J = 7.4 Hz, 2H), 1.55 – 1.42 (m, 6H), 1.29 (s, 3H), 1.25 – 1.19 (m, 4H). 13 C NMR (101 MHz, DMSO-) d 6 ) δ 172.50, 171.66, 155.52, 153.25, 146.35, 145.61, 145.10,132.44, 131.65, 124.81, 109.71, 108.39, 50.77, 41.02, 36.18, 34.77, 33.44,28.48, 25.96, 25.45, 25.21. HR-MS(ESI): Calcd. C24 H 33 ClN 10 O2S, [M+H] + m / z:561.2275, found: 561.2278.

[0085] Example 14

[0086] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2e: .

[0087] The preparation method of compound 2e is basically the same as that in Example 10, except that monomethyl pimecrolate in step (f) is replaced with monomethyl azelaic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2e.

[0088] The characterization results of compound 2e are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ]-yrazin-6-yl)-4-methylpiperidin-4-yl)-9-hydrazineyl-9-oxononanamide. Brown solid, yield 68.2%, melting point 146.0-146.4 ℃. 1 H NMR (400 MHz, DMSO- d 6 )δ 13.75 (s, 1H), 8.90 (s, 1H), 8.45 (s, 1H), 7.55 (d, J = 5.4 Hz, 1H), 7.45 (s,1H), 6.36 (s, 2H), 5.72 (d, J = 5.5 Hz, 1H), 4.08 (d, J = 14.4 Hz, 3H), 3.30 (s,2H), 2.22 (d, J = 13.5 Hz, 2H), 2.08 (t, J = 7.1 Hz, 2H), 1.97 (t, J = 7.5 Hz, 2H), 1.55 – 1.41 (m, 6H), 1.29 (s, 3H), 1.23 (s, 6H). 13 C NMR (101 MHz, DMSO-d 6 ) δ172.53, 171.66, 155.51, 153.24, 146.39, 145.17, 144.68, 133.19, 131.52,124.86, 110.60, 108.38, 50.77, 36.19, 33.41, 28.57, 25.49, 25.22. HR-MS(ESI):Calcd. C 25 H 35 ClN 10 O2S, [M+H] + m / z: 575.2432, found: 575.2436.

[0089] Example 15

[0090] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2f: .

[0091] The preparation method of compound 2f is basically the same as that in Example 10, except that monomethyl pimecrolate in step (f) is replaced with monomethyl isophthalic acid monomethyl ester, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2f.

[0092] The characterization results of compound 2f are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ] p-yrazin-6-yl)-4-methylpiperidin-4-yl)-3-(hydrazinecarbonyl)benzamide. Brown solid, yield 72.7%, melting point 175.9-176.3 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ13.81 (s, 1H), 9.86 (s, 1H), 8.50 (s, 1H), 8.23 ​​(s, 1H), 7.99 – 7.89 (m, 3H),7.53 (t, J = 9.9 Hz, 2H), 6.37 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.63 (s, 1H), 4.16 (d, J= 13.6 Hz, 2H), 3.47 (t, J = 12.3 Hz, 2H), 2.45 (d, J = 13.1 Hz, 2H), 1.63 (t, J = 11.9 Hz, 2H), 1.43 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 166.71,165.52, 155.53, 153.35, 146.35, 145.60, 145.13, 136.10, 133.25, 130.13,129.40, 128.31, 126.05, 124.85, 109.72, 108.41, 51.85, 41.13, 34.74, 25.84.HR-MS(ESI): Calcd. C 24 H 25 ClN 10 O2S, [M+H] + m / z: 553.1649, found: 553.1659.

[0093] Example 16 A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2g: .

[0094] The preparation method of compound 2g is basically the same as that in Example 10, except that monomethyl pimecrolate in step (f) is replaced with monomethyl terephthalic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2g.

[0095] The characterization results of compound 2g are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ] p-yrazin-6-yl)-4-methylpiperidin-4-yl)-4-(hydrazinecarbonyl)benzamide. White solid, yield 88.2%, melting point 175.7-176.1 °C. 1 H NMR (400 MHz, DMSO- d 6) δ9.90 (s, 1H), 8.48 (s, 1H), 7.97 (s, 1H), 7.88 (s, 4H), 7.56 (d, J = 5.4 Hz,1H), 6.37 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.57 (s, 2H), 4.13 (d, J = 13.3 Hz, 2H), 3.45 (t, J = 12.1 Hz, 2H), 2.45 (d, J = 12.9 Hz, 2H), 1.70 – 1.57 (m, 2H), 1.43 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 166.51, 165.15, 155.52, 153.35,146.35, 145.81, 145.59, 145.11, 138.06, 135.27, 132.47, 127.53, 126.74,124.84, 109.72, 108.94, 108.41, 51.85, 41.12, 34.71, 25.78. HR-MS(ESI):Calcd. C 24 H 25 ClN 10 O2S, [M+H] + m / z: 553.1649, found: 553.1656.

[0096] Example 17

[0097] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2h: .

[0098] The preparation method of compound 2h is basically the same as that in Example 10, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(3-carboxypropyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2h.

[0099] The characterization results of compound 2h are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4-b ] p-yrazin-6-yl)-4-methylpiperidin-4-yl)-2-(4-(hydrazinecarbonyl)phenyl)acetamide. Light brown solid, yield 54.6%, melting point 156.6-157.5 ℃. 1 H NMR (400 MHz, DMSO- d 6 ) δ 13.78 (s, 1H), 9.70 (s, 1H), 8.45 (s, 1H), 7.82(s, 1H), 7.74 (d, J = 7.8 Hz, 2H), 7.56 (d, J = 5.4 Hz, 1H), 7.33 (d, J = 7.9 Hz,2H), 6.37 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.52 (s, 1H), 4.08 (d, J = 13.7 Hz,2H), 3.49 (s, 2H), 3.28 (s, 2H), 2.20 (d, J = 13.4 Hz, 2H), 1.52 (t, J = 11.9 Hz, 2H), 1.30 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ 169.88, 165.90, 155.49, 153.24,146.33, 145.10, 144.72, 140.08, 133.36, 131.41, 128.11, 126.04, 124.89,110.59, 108.41, 51.11, 26.50. HR-MS(ESI): Calcd. C 25 H 27 ClN 10 O2S, [M+H] + m / z:567.1806, found: 567.1808.

[0100] Example 18

[0101] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 2i: .

[0102] The preparation method of compound 2i is basically the same as that in Example 10, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(2-carboxyethyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 2i.

[0103] The characterization results of compound 2i are as follows: N -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1 H -pyrazolo[3,4- b ] p-yrazin-6-yl)-4-methylpiperidin-4-yl)-3-(4-(hydrazinecarbonyl)phenyl)propenamide. Brown solid, yield 60%, melting point 168.5-169.2 ℃. 1 HNMR (400 MHz, DMSO- d 6 ) δ 13.76 (s, 1H), 9.65 (s, 1H), 8.41 (s, 1H), 7.72 (d, J = 7.7 Hz, 2H), 7.56 (d, J = 5.4 Hz, 1H), 7.49 (s, 1H), 7.28 (d, J = 7.9 Hz, 2H), 6.37 (s, 2H), 5.74 (d, J = 5.3 Hz, 1H), 4.47 (s, 1H), 4.03 (d, J = 13.4 Hz, 2H), 3.18 (t, J = 12.2 Hz, 2H), 2.85 (t, J = 7.4 Hz, 2H), 2.44 (t, J = 7.6 Hz, 2H), 2.17(d, J = 13.4 Hz, 2H), 1.45 (d, J = 11.8 Hz (2H), 1.26 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 171.43, 165.83, 155.52, 153.21, 146.38, 145.61, 145.13, 144.68,132.39, 131.59, 130.93, 128.25, 127.53, 126.96, 124.84, 109.75, 108.41,50.89, 40.86, 37.22, 34.72, 31.09, 25.98. HR-MS(ESI): Calcd. C 26 H 29 ClN 10 O2S, [M+H] + m / z: 581.1962, found: 581.1952.

[0104] Example 19

[0105] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 3c: .

[0106] The preparation method of compound 3c is basically the same as that in Example 1, except that steps (a) to (g) are the same as in Example 1; step (h) is adjusted as follows: intermediate G (100 mg, 1 eq.) is placed in a 50 mL round-bottom flask, with 2 mL of methanol and 1 mL of dichloromethane as solvents, cooled to 0 °C in an ice bath, and 80.94 μL (5 eq.) of 50% hydroxylamine aqueous solution is added, and the mixture is kept in an ice bath for 10 min. A 50 mg / mL NaOH methanol solution is prepared. The NaOH methanol solution (187 μL, 1.25 eq.) is added dropwise to the ice bath system until the pH of the system is between 10 and 11, and the reaction is carried out at room temperature for 6 h. The system is then cooled to 0 °C, 80.94 μL (5 eq.) of 50% hydroxylamine aqueous solution is added, the mixture is kept in an ice bath for 10 min, and the NaOH methanol solution is added dropwise until the pH of the system is between 10 and 11, and the reaction is carried out at room temperature until the reaction is complete. The solvent was then concentrated and evaporated to dryness. 2 mL of water was added to the system, and the mixture was placed in an ice bath at 0 °C. Hydrochloric acid was added to adjust the pH to neutral, and a precipitate was formed. The solid was collected by vacuum filtration and dried to obtain compound 3c.

[0107] The characterization results of compound 3c are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 7-hydroxyheptanediamide. Pale yellow solid, yield 52.4%, melting point 177.2-178.1 °C. 1 H NMR (400MHz, DMSO-d6) δ 13.78 (s, 1H), 10.32 (s, 1H), 8.65 (s, 1H), 8.45 (s, 1H), 7.56 (d, J = 5.4 Hz, 1H), 7.44 (s, 1H), 6.36 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.08 (d, J = 13.4 Hz, 2H), 2.21 (d, J = 13.6 Hz, 2H), 2.08 (t, J = 7.5Hz, 2H), 1.92 (t, J = 7.4 Hz, 2H), 1.54 – 1.42 (m, 6H), 1.29 (s, 3H), 1.23(s, 2H). 13 C NMR (101 MHz, DMSO-d6) δ 172.42, 169.06, 155.51, 153.22, 146.35,145.59, 145.14, 132.41, 131.59, 124.82, 109.72, 108.39, 50.78, 41.01, 36.07,34.78, 32.18, 28.28, 25.94, 25.25, 24.94. HR-MS(ESI): Calcd. C 23 H 30 ClN9O3S, [M+H] + m / z: 548.1959, found: 548.1957.

[0108] Example 20

[0109] A pyrazoloid compound, with the structural formula shown below, is designated as compound 3a: .

[0110] The preparation method of compound 3a is basically the same as that in Example 19, except that monomethyl pimecrolate in step (f) is replaced with monomethyl glutarate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3a.

[0111] The characterization results of compound 3a are as follows: N 1-(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 5 -hydroxyglutaramide. Brown solid, yield 26.5%, melting point 175.5-176.3 °C. 1 H NMR (400 MHz, DMSO- d 6) δ 8.44 (s, 1H), 7.74 – 7.65 (m, 1H), 7.55 (d, J = 5.4 Hz, 1H), 7.50(s, 1H), 6.36 (s, 2H), 5.72 (d, J = 5.4 Hz, 1H), 4.08 (d, J = 13.6 Hz, 2H), 2.21(d, J = 13.5 Hz, 2H), 2.14 – 2.01 (m, 3H), 1.95 (t, J = 7.5 Hz, 1H), 1.74 – 1.67(m, 2H), 1.51 (d, J = 12.1 Hz, 2H), 1.29 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6 ) δ172.00, 168.83, 155.51, 153.24, 146.38, 145.60, 145.17, 132.39, 131.53,124.84, 109.73, 108.39, 50.82, 41.01, 35.59, 34.78, 31.81, 25.97, 21.71. HR-MS(ESI): Calcd. C 21 H 26 ClN9O3S, [M+H] + m / z: 520.1646, found: 520.1641.

[0112] Example 21

[0113] A pyrazoloid compound, with the structural formula shown below, is designated as compound 3b: .

[0114] The preparation method of compound 3b is basically the same as that in Example 19, except that monomethyl pimelic acid in step (f) is replaced with monomethyl adipic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3b.

[0115] The characterization results of compound 3b are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 6 -hydroxyadipamide. Pale yellow solid, yield 43.9%, melting point 187.2-188.2 °C. 1 H NMR (400 MHz, DMSO-d6) δ 10.39 (s, 1H), 8.69 (s, 1H), 8.45 (s, 1H), 7.53 (d, J = 24.7 Hz,2H), 6.36 (s, 2H), 5.73 (s, 1H), 4.08 (s, 2H), 2.22 (d, J = 13.7 Hz, 2H), 2.10 (s, 2H), 1.95 (s, 2H), 1.47 (s, 6H), 1.30 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 172.30, 169.02, 155.47, 153.27, 146.33, 145.15, 144.70, 133.32, 131.56,124.89, 110.54, 108.37, 50.83, 40.96, 36.05, 32.20, 26.57. HR-MS(ESI): Calcd.C 22 H 28 ClN9O3S, [M+H] + m / z: 534.1803, found: 534.1805.

[0116] Example 22

[0117] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 3d: .

[0118] The preparation method of compound 3d is basically the same as that in Example 19, except that monomethyl pimelic acid in step (f) is replaced with monomethyl succinate, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3d.

[0119] The characterization results of compound 3d are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 8 -hydroxyoctanediamide. Pale yellow solid, yield 49.9%, melting point 182.3-184.0 ℃. 1 H NMR (400MHz, DMSO-d6) δ 8.44 (s, 1H), 7.55 (d, J = 5.4 Hz, 1H), 7.44 (s, 1H), 6.35(s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.07 (d, J = 13.3 Hz, 2H), 2.21 (d, J =13.6 Hz, 2H), 2.08 (t, J = 7.5 Hz, 2H), 1.92 (t, J = 7.5 Hz, 2H), 1.54 – 1.42(m, 6H), 1.29 (s, 3H), 1.23 (s, 4H). 13 C NMR (101 MHz, DMSO-d6) δ 172.51,169.11, 155.51, 153.23, 146.40, 145.20, 131.50, 124.87, 108.39, 50.77, 36.19,32.27, 28.44, 25.45, 25.11. HR-MS(ESI): Calcd. C 24 H 32 ClN9O3S, [M+H] + m / z:562.2116, found: 562.2124.

[0120] Example 23

[0121] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 3e: .

[0122] The preparation method of compound 3e is basically the same as that in Example 19, except that monomethyl pimecrolate in step (f) is replaced with monomethyl azelaic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3e.

[0123] The characterization results of compound 3e are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)-N 9 -hydroxynonanediamide. Pale yellow solid, yield 34.6%, melting point 184.3-185.1 °C. 1 H NMR (400MHz, DMSO-d6) δ 8.44 (s, 1H), 7.55 (d, J = 5.3 Hz, 1H), 7.44 (s, 1H), 6.35(s, 2H), 5.72 (d, J = 5.3 Hz, 1H), 4.07 (d, J = 13.4 Hz, 2H), 2.21 (d, J =13.6 Hz, 2H), 2.08 (s, 2H), 1.91 (s, 2H), 1.47 (s, 6H), 1.29 (s, 3H), 1.23(s, 6H). 13 C NMR (101 MHz, DMSO-d6) δ 172.51, 169.07, 155.51, 153.22, 146.46,145.58, 145.31, 132.33, 131.42, 124.94, 109.71, 108.36, 50.77, 41.05, 36.19,34.77, 32.23, 29.07, 28.58, 28.53, 25.97, 25.49, 25.14. HR-MS(ESI): Calcd.C 25 H 34 ClN9O3S, [M+H] + m / z: 576.2272, found: 576.2282.

[0124] Example 24

[0125] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 3f: .

[0126] The preparation method of compound 3f is basically the same as that in Example 19, except that monomethyl pimecrolate in step (f) is replaced with monomethyl isophthalic acid monomethyl ester, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3f.

[0127] The characterization results of compound 3f are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 3 -hydroxyisophthalamide. Yellow solid, yield 66.9%, melting point 223.7-224.3 °C. 1 H NMR (400 MHz, DMSO-d6) δ 13.88 (s, 1H), 8.48 (d, J = 2.5 Hz, 1H), 8.29 (d, J = 19.9 Hz, 1H), 8.07 (s, 1H), 8.02 – 7.76 (m, 3H), 7.55 (d, J = 5.4 Hz, 1H), 7.52 – 7.33(m, 1H), 6.36 (s, 1H), 5.73 (d, J = 5.4 Hz, 1H), 4.14 (s, 2H), 3.46 (s, 2H),2.46 (s, 2H), 1.62 (t, J = 11.8 Hz, 2H), 1.43 (s, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 169.86, 167.64, 166.63, 163.61, 155.52, 153.35, 146.42, 145.64, 145.15,135.87, 135.42, 132.65, 131.50, 130.22, 128.48, 124.86, 109.77, 108.40,51.94, 51.65, 41.20, 34.75, 25.87. HR-MS(ESI): Calcd. C 24 H 24 ClN9O3S, [M+H] + m / z:554.1490, found: 554.1499.

[0128] Example 25

[0129] A pyrazoloid compound, with the structural formula shown below, is designated as compound 3g: .

[0130] The preparation method of compound 3g is basically the same as that in Example 19, except that monomethyl pimecrolate in step (f) is replaced with monomethyl terephthalic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3g.

[0131] The characterization results of compound 3g are as follows: N 1 -(1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b] -pyrazin-6-yl)-4-methylpiperidin-4-yl)- N 4 -hydroxyterephthalamide. Pale yellow solid, yield 43.7%, melting point 204.2-205.1 °C. 1 H NMR (400MHz, DMSO-d6) δ 8.48 (s, 1H), 7.95 (s, 1H), 7.92 (d, J = 5.6 Hz, 1H), 7.85 (d, J = 14.6 Hz, 3H), 7.77 (d, J = 7.4 Hz, 1H), 7.56 (d, J = 5.4 Hz, 1H), 6.36 (s, 2H), 5.73 (d, J = 5.4 Hz, 1H), 4.13 (d, J = 13.3 Hz, 2H), 2.44 (s, 2H), 1.62 (s, 2H), 1.43 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 167.17, 166.54, 155.52, 153.34,146.39, 145.60, 145.17, 132.45, 131.53, 128.79, 127.54, 126.72, 124.86,109.72, 108.40, 51.85, 51.70, 41.16, 34.74, 25.81. HR-MS(ESI): Calcd.C 24 H 24 ClN9O3S, [M+H] + m / z: 554.1490, found: 554.1494.

[0132] Example 26

[0133] A pyrazoloid compound, with the structural formula shown below, is denoted as compound 3h: .

[0134] The preparation method of compound 3h is basically the same as that in Example 19, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(3-carboxypropyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3h.

[0135] The characterization results of compound 3h are as follows: 4-(2-((1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4-methylpiperidin-4-yl)amino)-2-oxoethyl)-N-hydroxybenzamide. Yellow solid, yield 54.9%, melting point 193.5-194.4 ℃. 1H NMR(400 MHz, DMSO-d6) δ 8.44 (s, 1H), 7.81 (d, J = 19.7 Hz, 2H), 7.68 (d, J =7.9 Hz, 2H), 7.56 (d, J = 5.3 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.31 – 7.25(m, 1H), 6.31 (s, 2H), 5.75 (d, J = 5.4 Hz, 1H), 4.07 (d, J = 13.3 Hz, 2H), 3.49 (d, J = 7.6 Hz, 4H), 2.21 (d, J = 13.5 Hz, 2H), 1.52 (s, 2H), 1.30 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 169.88, 164.12, 155.51, 153.21, 146.37,145.60, 145.13, 140.14, 132.41, 131.59, 130.90, 129.20, 128.95, 128.38,126.83, 124.85, 109.77, 108.43, 51.13, 51.09, 42.99, 41.00, 34.73, 25.86. HR-MS(ESI): Calcd. C 25 H 26 ClN9O3S, [M+H] + m / z: 568.1646, found: 568.1645.

[0136] Example 27 A pyrazoloid compound, with the structural formula shown below, is designated as compound 3i: .

[0137] The preparation method of compound 3i is basically the same as that in Example 19, except that monomethyl pimelic acid in step (f) is replaced with monomethyl 4-(2-carboxyethyl)phenylacetic acid, while the other process parameters in steps (a) to (h) remain unchanged, thereby preparing compound 3i.

[0138] The characterization results of compound 3i are as follows: 4-(3-((1-(3-((2-amino-3-chloropyridin-4-yl)thio)-1H-pyrazolo[3,4-b]pyrazin-6-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropyl)-N-hydroxybenzamide. Yellow solid, yield 51%, melting point 193.1-194.0 ℃. 1 H NMR(400 MHz, DMSO-d6) δ 8.43 (s, 1H), 7.85 (s, 1H), 7.66 (d, J = 7.6 Hz, 2H), 7.56 (s, 1H), 7.48 (s, 1H), 7.28 (s, 1H), 7.22 (s, 1H), 6.31 (s, 2H), 5.75(s, 1H), 4.03 (d, J = 13.3 Hz, 2H), 2.85 (s, 2H), 2.44 (s, 2H), 2.18 (d, J =13.2 Hz, 2H), 1.49 (s, 2H), 1.27 (s, 3H). 13 C NMR (101 MHz, DMSO-d6) δ 171.41,155.49, 153.19, 146.35, 145.58, 145.13, 132.37, 131.56, 130.44, 129.25,128.25, 127.61, 126.81, 124.82, 109.74, 108.40, 50.91, 40.94, 37.26, 34.74,31.06, 25.90. HR-MS(ESI): Calcd. C 26 H 28 ClN9O3S, [M+H] + m / z: 582.1803, found: 582.1805.

[0139] Experimental examples, SHP2 and HDAC6 inhibitory activity tests This experimental example examines the inhibitory activity of pyrazoloids prepared in Examples 1-27 against SHP2 and HDAC6 to evaluate their potential application in dual-target drugs.

[0140] The SHP2 activity assay was performed as follows: Compounds 1a-1i, 2a-2i, and 3a-3i prepared in the above examples were weighed and prepared into 10 mM stock solutions using dimethyl sulfoxide (DMSO), which were then stored at 4 °C for later use. During the experiment, the stock solutions were diluted to the required concentration with DMSO and reaction buffer to obtain the test samples. The reaction buffer was 60 mmol / L HEPES buffer, pH 7.2, containing 75 mmol / L NaCl, 75 mmol / L KCl, 1 mmol / L EDTA, 0.05% Tween-20, and 5 mmol / L dithiothreitol (DTT). The test samples were co-incubated with SHP2 protein and peptide p-IRS1 at room temperature, followed by the addition of the fluorescent substrate DiFMUP reaction. The fluorescence signal at wavelengths of 355 nm and 460 nm was monitored using a microplate reader, and the inhibition rate against SHP2 was calculated. Simultaneously, the fitted IC50 was calculated using GraphPad software. 50 value.

[0141] The HDAC6 activity assay was performed as follows: Compounds 1a-1i, 2a-2i, and 3a-3i prepared in the above examples were weighed and prepared into 10 mM stock solutions using dimethyl sulfoxide (DMSO), which were stored at 0 °C for later use. During the experiment, the stock solutions were diluted to the required concentration with DMSO to obtain the test samples. Recombinant HDAC6 protein, fluorescent reagent, BSA, and incubation buffer were then added to the test samples, and the mixture was incubated at 37 °C for 40 min. Trypsin was then added, and the mixture was incubated on a shaker at room temperature for 15 min. Fluorescence values ​​at wavelengths of 360 nm and 460 nm were monitored using a microplate reader. The inhibition rate was calculated by measuring the fluorescence intensity of the compounds at different concentrations. The IC50 values ​​of the compounds were obtained by calculation and fitting using GraphPad Prism 6.0 software. 50 .

[0142] The inhibition rate (%) was calculated as follows: (fluorescence intensity of the 100% group - fluorescence intensity of the compound group) / (fluorescence intensity of the 100% group - fluorescence intensity of the blank group) × 100%. The inhibitory activities of different pyrazolidine compounds against SHP2 and HDAC6 are shown in Table 1. In Table 1, "--" indicates that this test was not performed. SAHA is a positive control compound for HDAC6 target inhibitors; SHP099 is a positive control compound for SHP2 target inhibitors.

[0143] Table 1. Inhibitory activities of different pyrazolidine compounds on SHP2 and HDAC6

[0144] As shown in Table 1, the enzymatic activity evaluation results indicate that the pyrazolopyrazine compounds provided in this invention exhibit potent inhibitory activity against both SHP2 and HDAC6. Compounds 3c, 3e, and 3h are particularly preferred, achieving nanomolar-level dual inhibition of both targets. Among them, compound 3c shows the half-maximal inhibitory concentration (IC50) for both SHP2 and HDAC6. 50 The IC50 values ​​for compounds 3e and 34.36 nM and 16.17 nM, respectively; 50 The values ​​were 19.19 nM and 39.27 nM, respectively; the IC50 of the compound at 3 h was... 50 The values ​​are 50.29 nM and 9.27 nM, respectively.

[0145] In summary, the compounds provided by this invention possess a novel pyrazolopyrazine backbone and exhibit excellent dual-protein inhibitory activity. Their inhibitory efficacy on some indicators has reached or surpassed that of some previously reported clinical SHP2 single-target inhibitors or HDAC6 single-target inhibitors. Therefore, the compounds of this invention can provide novel chemical entities for the development of next-generation anti-tumor drugs and the exploration of combined SHP2 and HDAC6 inhibitory therapy strategies. They also lay a solid experimental foundation for the structural optimization and mechanistic study of SHP2-HDAC6 dual-target inhibitors, possessing significant scientific value and broad clinical application prospects.

[0146] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A pyrazolopyrazine compound, characterized in that, Compounds of the structure shown in formula (I) or pharmaceutically acceptable salts thereof: ； In equation (I), R is -LZ; where L is C2~C 12 straight-chain alkylene groups, C2~C 12 One of the branched alkylene groups, m-phenylene, p-phenylene, p-tolyl, and p-ethylphenyl; Z is one of -C (=O)NH(C6H4)NH2·HCl, -C (=O) NHNH2, and -C (=O) NHOH.

2. The pyrazoloid compound according to claim 1, characterized in that, The pyrazolidine compounds are selected from compounds with the following structures: 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 、 。 3. The pyrazoloid compound according to claim 1, characterized in that, The pharmaceutically acceptable salts are one or more of the following: hydrochloride, hydrobromide, sulfate, phosphate, borate, methanesulfonate, p-toluenesulfonate, naphthalenesulfonate, benzenesulfonate, citrate, lactate, pyruvate, tartrate, acetate, trifluoroacetate, maleate, succinate, amygdalinate, fumarate, salicylate, and phenylacetate.

4. A method for preparing a pyrazoloid compound as described in claim 1 or 2, characterized in that, Includes the following steps: ； Step (a): Under inert gas protection, 3-chloro-4-iodopyridine-2-amine and methyl mercaptopropionate are reacted in a solvent at 80-120°C for 4-12 h in the presence of palladium catalyst, phosphine ligand and organic base, and then purified to obtain intermediate A. Step (b): The intermediate A is reacted with sodium ethoxide in a solvent at 0-30°C for 10-24 hours to obtain intermediate B; Step (c): 6-chloro-1H-pyrazolo[3,4-B]pyrazine is reacted with halosuccinimide in a solvent at 60-100°C for 2-8 h to obtain intermediate C; Step (d): The intermediate C is reacted with tert-butyl (4-methylpiperidin-4-yl)carbamate in a solvent at 60-90°C for 2-8 h, and then purified to obtain intermediate D; Step (e): The intermediate D is reacted in a solvent under acidic conditions at 0-30°C for 2-8 hours to obtain intermediate E; Step (f): The intermediate E is reacted with the diacid monomethyl ester compound in a solvent at 0-30°C for 12-48 h in the presence of a condensing agent and an organic base to obtain intermediate F; Step (g): Under inert gas protection, intermediate F and intermediate B are reacted in a solvent at 60-120°C for 5-15 h in the presence of a copper catalyst, ligand and inorganic base, and then purified to obtain intermediate G. Step (h): The intermediate G is reacted in a solvent at 0-30°C for 4-12 hours in the presence of an inorganic base to obtain intermediate H; the intermediate H is then reacted with... N -Boc-1,2-phenylenediamine is reacted in a solvent at 40-80°C for 4-12 h in the presence of a condensing agent and an organic base to obtain intermediate I; intermediate I is then reacted in a solvent at 0-30°C for 10-24 h under acidic conditions to obtain the pyrazolopyrazine compound shown in formula (I). Alternatively, in step (h), the intermediate G is reacted with hydrazine hydrate in a solvent at 60-100°C for 6-15 h to obtain the pyrazolopyrazine compound shown in formula (I). Alternatively, in step (h), the intermediate G is reacted with hydroxylamine in a solvent at -5 to 30°C for 4 to 24 hours in the presence of a base to obtain the pyrazolopyrazine compound shown in formula (I).

5. The method for preparing pyrazoloid compounds according to claim 4, characterized in that, In step (a), the molar ratio of 3-chloro-4-iodopyridin-2-amine, methyl mercaptopropionate, palladium catalyst, phosphine ligand, and organic base is 1:1~2:0.01~0.05:0.05~0.15:1.5~3.0; in step (b), the molar ratio of intermediate A to sodium ethoxide is 1:1~2; in step (c), the molar ratio of 6-chloro-1H-pyrazolo[3,4-B]pyrazine to halosuccinimide is 1:1.5~2.5; in step (d), the molar ratio of intermediate C to (4-methylpiperidin-4-yl)carbamate tert-butyl ester is 1:1~2; in step (f), the molar ratio of intermediate E to diacid monomethyl ester compound is 1~2:1~2; in step (g), the molar ratio of intermediate F to intermediate B is 1:1~3.

6. The method for preparing pyrazoloid compounds according to claim 4, characterized in that, In step (f), the diacid monomethyl ester compound is selected from one of monomethyl glutarate, monomethyl adipic acid, monomethyl pimecrolate, monomethyl octanoate, monomethyl azelaic acid, monomethyl isophthalic acid, monomethyl terephthalic acid, monomethyl 4-(3-carboxypropyl)phenylacetic acid, and monomethyl 4-(2-carboxyethyl)phenylacetic acid; in step (g), the copper catalyst is cuprous iodide, and the ligand is 1,10-o-phenanthroline.

7. The method for preparing pyrazoloid compounds according to claim 4, characterized in that, The solvent is selected from 1,4-dioxane, tetrahydrofuran, acetonitrile, etc. N,N -Dimethylformamide, N One or more of methylpyrrolidone, ethyl acetate, methanol, ethanol, and water.

8. The use of a pyrazoloid compound as described in claim 1 or 2 in the preparation of a medicament that targets and inhibits SHP2 and / or HDAC6.

9. The use of a pyrazolopyrazine compound as described in claim 1 or 2 in the preparation of a medicament for treating cancer, characterized in that, The cancer mentioned is one of the following: stomach cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, or cervical cancer.

10. A drug for treating cancer, characterized in that, The drug comprises at least the pyrazoloid compound as described in claim 1 or 2, and a pharmaceutically acceptable excipient or carrier; the cancer is one of gastric cancer, pancreatic cancer, breast cancer, lung cancer, prostate cancer, colorectal cancer, ovarian cancer, liver cancer, or cervical cancer.

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