SHP2 / HDAC dual-target inhibitors, preparation method and application thereof

Abstract

The application discloses a SHP2 / HDAC dual-target inhibitor with the following structure, a preparation method and application thereof. The compound of the application not only shows good SHP2 and HDAC enzyme inhibition activity, but also has good anti-proliferation activity on solid tumors and blood tumors, and can obviously inhibit tumor growth. The application further provides application of the SHP2 / HDAC dual-target inhibitor in preparation of a drug for treating a tumor disease related to SHP2 / HDAC dual-target and RAS signal pathway overactivation.

Description

Technical Field

[0001] This invention relates to an SHP2 / HDAC dual-target inhibitor, its preparation method, pharmaceutical composition, and pharmaceutical uses, belonging to the field of pharmaceutical technology. Background Technology

[0002] The non-receptor protein tyrosine phosphatase SHP2, encoded by the PTPN11 gene, is an important oncogenic factor. SHP2 maintains a self-inhibitory conformation through the interaction between its N-terminal SH2 domain and PTP domain, restricting substrate entry into its catalytic site. When a bisphosphonotyrosine peptide (e.g., IRS-1) binds to the SH2 domain, the interaction between the SH2 and PTP domains is disrupted, exposing the PTP catalytic domain and increasing the dephosphorylase activity of SHP2. SHP2 plays a role in many cancer-related signaling pathways, such as RAS-RAF-ERK, PI3K-AKT, JAK-STAT, NF-κB, and mTOR. Activating mutations in SHP2 occur in various cancer types, including juvenile myelomonocytic leukemia, B-cell acute lymphoblastic leukemia, acute myeloid leukemia, breast cancer, lung cancer, liver cancer, colon cancer, neuroblastoma, and melanoma. Therefore, SHP2 is a promising target for cancer therapy. To date, several SHP2 inhibitors are undergoing clinical trials.

[0003] Histone deacetylases (HDACs) are important epigenetic regulators, playing a crucial role in chromosomal structural modification and gene expression regulation by modulating the deacetylation of various substrates, including histones and non-histone proteins. Currently, 18 subtypes of HDACs have been identified in four major classes, with class I HDACs being most closely associated with cancer development and progression. HDACs are overexpressed in various types of cancer and have become important targets for anticancer therapy in recent years. To date, five HDAC inhibitors have been approved for clinical treatment of hematologic malignancies, such as cutaneous T-cell lymphoma (CTCL), peripheral T-cell lymphoma, and multiple myeloma.

[0004] The RAS-ERK pathway is a major signaling cascade in cancer. As an oncogene, highly active SHP2 can fully activate the RAS-ERK pathway, promoting cancer cell survival and excessive proliferation. Studies have shown that the combination of SHP2 inhibitors and ERK signaling pathway inhibitors not only enhances anti-tumor effects but also effectively overcomes resistance to ERK signaling pathway inhibitors. On the other hand, studies have found that the combination of HDAC inhibitors and RAS inhibitors can also synergistically inhibit ERK signaling pathway activation, exerting a stronger anti-tumor effect. This suggests that the combination of SHP2 inhibitors and HDAC inhibitors may have stronger anti-tumor activity compared to single-target inhibitors. We used MV4-11 cells to study the combination of SHP2 inhibitor (SHP099) and HDAC inhibitor (SAHA). The results showed that the combination therapy had better anti-proliferative activity than single therapy (SHP099, IC50). 50 =1.75μM; SAHA,IC 50 =0.50μM; SHP099+SAHA,IC 50 =0.24μM). Based on the above research, this invention designs and synthesizes a series of SHP2 / HDAC dual-target inhibitors in order to obtain stronger anti-tumor molecules, and conducts in-depth research on the application of this type of dual-target inhibitor. Summary of the Invention

[0005] The purpose of this invention is to provide a dual-target inhibitor of SHP2 / HDAC; another purpose of this invention is to provide a method for preparing and using a dual-target inhibitor of SHP2 / HDAC.

[0006] Technical solution:

[0007] I. SHP2 / HDAC Dual-Target Inhibitor

[0008] This invention provides an SHP2 / HDAC dual-target inhibitor or a pharmaceutically acceptable salt having the structure shown in Formula I:

[0009]

[0010] In general formula I, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, and bromine;

[0011] A represents furan, thiophene, pyrrole, thiazole, imidazole, oxazole, thiazole, isoxazole, isothiazole, pyridine, pyrazine, pyrimidine, pyridazine, pyran, piperazine, indole, quinoline, pteridine, acridine, benzimidazole, isoquinoline, pyrazinopyrazole, and pyrazinopyrazole.

[0012] R3 is a hydrogen atom or a carbonyl group;

[0013] X is one of a saturated straight-chain hydrocarbon group, aryl group, or one of the following linking groups with 0 to 10 carbon atoms:

[0014]

[0015] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0016] R4 is a hydroxyl group or a 2-aminophenyl group;

[0017] R5 is a hydrogen atom or has the following structure:

[0018]

[0019] R6 is a hydrogen atom or a carbonyl group;

[0020] Y is one of a saturated straight-chain hydrocarbon group, phenyl group, or one of the following linking groups with 0 to 10 carbon atoms:

[0021]

[0022] n=0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

[0023] R7 is a hydroxyl group or a 2-aminophenyl group. The compounds of general formula I described above are preferably one of the following:

[0024] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -Hydroxymalonamide (8a)

[0025] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 4 -Hydroxysuccinamide (8b)

[0026] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 5 8-Hydroxyglutaramide (8c)

[0027] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 6 -Hydroxyhexanediamine (8d)

[0028] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 78-Hydroxyheptanamide (8e)

[0029] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 8 -Hydroxyoctadiamide (8f)

[0030] 5-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-N-hydroxypentanamide (8g)

[0031] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -Hydroxyisophthalamide (8h)

[0032] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 4 -Hydroxyterephthalamide (8i)

[0033] N 1 -(2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-2-oxoethyl)-N 4 -Hydroxyterephthalamide (8j)

[0034] (E)-N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(4-(3-(hydroxyamino))-3-oxoprop-1-en-1-yl)benzyl)malonamide (8k)

[0035] (E)-N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(3-(3-(hydroxyamino))-3-oxoprop-1-en-1-yl)benzyl)malonamide (8l)

[0036] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(4-(hydroxycarbamoyl)benzyl)malonamide (8m)

[0037] (E)-3-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide(8n)

[0038] 3-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-hydroxybenzamide (8o)

[0039] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-hydroxybenzamide (8p)

[0040] (E)-3-(4-(2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)phenyl)-N-hydroxyacrylamide (8q)

[0041] (E)-3-(4-((2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)methyl)phenyl)-N-hydroxyacrylamide(8r)

[0042] (E)-4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)benzamide(8s)

[0043] (E)-4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)benzyl)benzamide(8t)

[0044] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 2-Aminophenyl)malonamide (8u)

[0045] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(2-aminophenyl)benzamide (8v)

[0046] The compound of general formula I of the present invention can be prepared into a pharmaceutically acceptable salt by known methods, wherein the salt is a salt prepared by mixing the compound of formula I with an acid or a base;

[0047] Suitable acid addition salts are formed from acids that form non-toxic salts. Representative acid addition salts include, but are not limited to, acetates, adipicates, alginates, citrates, aspartate, benzoates, benzenesulfonates, bisulfates, bicarbonates, butyrates, camphorates, camphorsulfonates, carbonates, citrates, digluconate, glycerol phosphates, hemisulfates, heptarates, hexanoates, formates, fumarates, gluconates, glucurons, glutamates, hydrochlorides, hydrobromide, hydroiodates, and isethionate. Lactate, maleate, malate, malonate, methanesulfonate, nicotate, 2-naphthalenesulfonate, nicotinate, nitrate, orotate, oxalate, palmitate, pyrate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, sucrose, stearate, succinate, sulfate, tartrate, thiocyanate, phosphate, hydrogen phosphate, dihydrogen phosphate, p-toluenesulfonate, trifluoroacetate, and undecanoate. II. Preparation methods of SHP2 / HDAC dual-target inhibitors.

[0048] The preparation method of SHP2 / HDAC dual-target inhibitors involves the following steps: Compound 1 and Compound 2 undergo a Suzuki coupling reaction to obtain intermediate 3, which is then protected by a Boc protecting group to obtain intermediate 4; 4 undergoes a nucleophilic substitution reaction with tert-butyl (4-methylpiperidin-4-yl)carbamate to obtain intermediate 5, followed by removal of the Boc protecting group to obtain intermediate 6; 6 is condensed with monomethyl esters of different carbon lengths or carboxylic acid intermediates containing aryl groups under HATU conditions and then ammonolyzed in a methanol solution of hydroxylamine to obtain target compounds 8a-8f, 8h-8m; 6 undergoes a nucleophilic substitution reaction with different bromine-containing intermediates and then ammonolyzed in a methanol solution of hydroxylamine to obtain target compounds 8g, 8n-8t; 6 is condensed with o-phenylenediamine under HATU conditions to obtain target compounds 8u and 8v.

[0049] The reaction route is as follows:

[0050]

[0051] Reagents and reaction conditions: (a) DPPF palladium dichloride, K3PO4, 1,4-dioxane / water, 90℃, 12h; (b) di-tert-butyl carbonate anhydride, DMAP, dichloromethane, room temperature, 8h; (c) (4-methylpiperidin-4-yl) tert-butyl carbamate, DIPEA, DMF, 85℃, 8h; (d) 4M HCl / EtOAc, room temperature, 8h; (e) HATU, DIPEA, DMF, room temperature, 8h; (f) hydroxylamine in methanol solution, room temperature, 6h.

[0052] The second reaction route is as follows:

[0053]

[0054] Reagents and reaction conditions: (a) HATU, DIPEA, DMF, room temperature, 8 hours; (b) K2CO3, DMF, room temperature or 80°C, 8 hours; (c) methanol solution of hydroxylamine, room temperature, 6 hours;

[0055] The third reaction route is as follows:

[0056]

[0057] Reagents and reaction conditions: (a) HATU, DIPEA, DMF, room temperature, 8 hours; (b) 4M sodium hydroxide / water, 50℃, 1 hour.

[0058] III. Application of SHP2 / HDAC Dual-Target Inhibitors

[0059] A pharmaceutical composition comprising a therapeutically effective amount of one or more of the aforementioned SHP2 / HDAC dual-target inhibitors, and pharmaceutical excipients or carriers.

[0060] The use of the SHP2 / HDAC dual-target inhibitor in the preparation of SHP2 inhibitors.

[0061] The use of the SHP2 / HDAC dual-target inhibitor in the preparation of histone deacetylase inhibitors.

[0062] The use of the SHP2 / HDAC dual-target inhibitor in the preparation of drugs for tumor diseases related to the overactivation of the SHP2 / HDAC dual-target and RAS signaling pathways.

[0063] The tumors mentioned include lung cancer, liver cancer, non-small cell lung cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, lymphoma, esophageal cancer, gastrointestinal cancer, nasopharyngeal carcinoma, leukemia, glioma, prostate cancer, myeloma, and KRAS-mutated tumors.

[0064] Beneficial effects

[0065] The compounds of this invention not only exhibit excellent SHP2 and HDAC enzyme inhibitory activity, inhibiting SHP2 and HDAC activity and downregulating ERK phosphorylation levels intracellularly, but also possess broad-spectrum in vitro antitumor activity, significantly delaying tumor growth. They can be applied to tumors with abnormal ERK signaling pathways mediated by SHP2 and HDAC. As the first reported dual-target antitumor drugs based on SHP2 and HDAC, these compounds have significant potential for further development and research. Attached Figure Description

[0066] Figure 1 The graph shows the in vivo antitumor activity of compounds 8r and 8t against human acute monocytic leukemia cells MV4-11. In the graph, A represents the tumor volume of tumor-bearing mice; B represents the change in body weight of tumor-bearing mice; C represents the tumor mass of tumor-bearing mice; and D represents the tumor volume growth curve of tumor-bearing mice. Detailed Implementation

[0067] All reagents, raw materials, or intermediates used in this invention are commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. The invention is further illustrated below with reference to examples, but is not limited thereto.

[0068] Example 1: Preparation of Intermediate 6

[0069] 6-Chloro-3-(2,3-dichlorophenyl)pyrazine-2-amine (3)

[0070] 2,3-Dichlorophenylboronic acid (1 g, 5.24 mmol), 2-amino-3-bromo-6-chloropyrazine (1.092 g, 5.24 mmol), PdCl2(dppf) (383 mg, 0.524 mmol), and K3PO4 (3.333 g, 51.72 mmol) were dissolved in 20 mL of 1,4-dioxane / water solution and stirred at 90 °C for 12 hours under nitrogen protection. After cooling to room temperature, the reaction mixture was filtered through diatomaceous earth, and 20 mL of water was added. The mixture was then extracted three times with EtOAc, and the organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (PE / EA = 100:1-20:1) to give 1.2 g of a yellow solid, yield: 90%. 1 HNMR (400MHz, Chloroform-d) δ8.02(s,1H),7.60(dd,J=7.8,1.7Hz,1H),7.37(t,J=7.7Hz,1H),7.32(dd,J=7.6,1.7Hz,1H),4.66(s,2H).

[0071] tert-Butyl (tert-Butoxycarbonyl) (6-Chloro-3-(2,3-Dichlorophenyl)pyrazin-2-yl)carbamate (4)

[0072] Compound 3 (100 mg, 0.364 mmol), (Boc)₂O (199 mg, 0.91 mmol), and DMAP (5 mg, 0.036 mmol) were dissolved in DCM (5 mL) and stirred at room temperature for 8 hours. The reaction mixture was washed with 1 M HCl and brine, dried over anhydrous magnesium sulfate, and the crude product was purified by column chromatography (PE / EA = 100:1–30:1) to give 107 mg of a yellow solid, yield: 62%. 1 H NMR (400MHz, Chloroform-d) δ8.67(s,1H),7.56(dd,J=7.9,1.6Hz,1H),7.34(dd,J=7.7,1.6Hz,1H),7.28(d,J=7.8Hz,1H),1.37(s,18H).

[0073] tert-Butyl (tert-Butoxycarbonyl) (6-(4-((tert-Butoxycarbonyl)amino)-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-yl)carbamate (5)

[0074] Compound 4 (193 mg, 0.406 mmol), tert-butyl (4-methylpiperidin-4-yl)carbamate (105 mg, 0.487 mmol), and DIPEA (0.1 mL, 0.61 mmol) were dissolved in DMF (2 mL) and stirred at 85 °C for 8 hours. The reaction mixture was diluted with EtOAc and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give a crude oil. The crude product was purified by column chromatography (PE / EA = 100:1-5:1) to give 288 mg of a yellow oil, yield: 90%. 1 H NMR (400MHz, DMSO-d6) δ8.42(s,1H),7.67(dd,J=8.0,1.3Hz,1H),7.42(t,J=7.9Hz,1H),7.29(dd,J=7.7,1.3Hz,1H),6.67 (s,1H),3.98–3.85(m,2H),3.33(d,J=10.5Hz,2H),2.15(d,J=12.5Hz,2H),1.53–1.44(m,2H),1.40(s,9H),1.28(s,18H).

[0075] 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazine-2-amine hydrochloride (6)

[0076] A solution of compound 5 (288 mg, 0.44 mmol) in 4 M HCl / EtOAc (20 mL) was stirred at room temperature for 8 hours. The solution was filtered to give a yellow solid (40 mg), yield: 21%. 1 H NMR (400MHz, DMSO-d6) δ7.61(d,J=7.9Hz,1H),7.48(s,1H),7.38(t,J=7.8Hz,1H),7.30(d,J=7 .1Hz,1H),5.58(s,2H),3.72–3.60(m,2H),3.51–3.45(m,2H),1.48–1.35(m,4H),1.09(s,3H). 13 C NMR (101MHz, DMSO) δ153.90,153.22,133.48,132.73,132.59,132.07,131.83,129.15,118.29,110.50,56.45,52.36,34.55,22.85,19.03.HRMS (ESI + m / z, calcd for C 16 H 19 Cl2N5([M+H) + )352.1090,found:352.1053.

[0077] Example 2: Preparation of intermediates 7a-7f, 7h-7m

[0078] 3-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropionic acid methyl ester (7a)

[0079] 6 (158 mg, 0.406 mmol), 3-methoxy-3-oxopropionic acid (0.05 mL, 0.447 mmol), HATU (155 mg, 0.406 mmol), and DIPEA (0.28 mL, 1.627 mmol) were dissolved in DMF (5 mL) and stirred at room temperature for 8 hours. The reaction mixture was then diluted with EtOAc and washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to give a crude product. The crude product was purified by column chromatography (DCM / MeOH = 100:1–50:1) to give 106 mg of a white solid, yield: 60%. 1H NMR(400MHz,Chloroform-d)δ7.62(s,1H),7.50(dd,J=7.6,1.9Hz,1H),7.32(dd,J=7.6,1.9Hz,1H),7.30(d,J=7.7Hz,1H),7.17(s,1 H),4.23(s,2H),3.97–3.94(m,2H),3.76(s,3H),3.31(s,2H),3.28–3.21(m,2H),2.25–2.21(m,2H),1.74–1.67(m,2H),1.48(s,3H).

[0080] 4-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-4-oxobutyrate methyl ester (7b)

[0081] 7b was synthesized in the same manner as 7a, with a yield of 70%. 1 H NMR(400MHz,Chloroform-d)δ7.61(s,1H),7.50(dd,J=7.6,1.9Hz,1H),7.33(dd,J=7 .6,1.9Hz,1H),7.29(d,J=7.7Hz,1H),5.55(s,1H),4.23(s,2H),3.88(dt,J=13.4,4.4 Hz,2H),3.68(s,3H),3.28(ddd,J=13.5,10.6,3.0Hz,2H),2.67(t,J=6.6Hz,2H),2.4 6(t,J=6.6Hz,2H),2.18(d,J=13.8Hz,2H),1.70(td,J=10.3,5.3Hz,2H),1.45(s,3H).

[0082] Methyl 5-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-5-oxovalerate (7c)

[0083] 7c was synthesized in the same manner as 7a, with a yield of 60%. 1H NMR(400MHz,Chloroform-d)δ7.61(s,1H),7.50(dd,J=7.6,1.9Hz,1H),7.33(dd,J=7.6, 1.9Hz,1H),7.29(d,J=7.6Hz,1H),5.41(s,1H),4.25(s,2H),3.86(dt,J=13.3,4.4Hz,2H) ,3.67(s,3H),3.30(ddd,J=13.3,10.4,3.0Hz,2H),2.40(t,J=7.1Hz,2H),2.21(dt,J=19 .4,10.5Hz,4H),1.97(q,J=7.2Hz,2H),1.71(ddd,J=14.0,10.3,4.1Hz,2H),1.46(s,3H).

[0084] Methyl 6-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-6-oxohexanoate (7d)

[0085] 7d was synthesized in the same manner as 7a, with a yield of 69%. 1 H NMR (400MHz, Chloroform-d) δ7.61 (s, 1H), 7.50 (dd, J = 7.6, 2.0Hz, 1H), 7.34 –7.31(m,1H),7.31–7.27(m,1H),5.39(s,1H),4.26(s,2H),3.86(dt,J=13.8, 4.7Hz,2H),3.67(s,3H),3.30(ddd,J=13.5,10.3,3.2Hz,2H),2.38–2.33(m,2 H),2.23–2.15(m,4H),1.75–1.70(m,2H),1.67(t,J=3.6Hz,4H),1.46(s,3H).

[0086] 7-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-7-oxoheptanoate (7e)

[0087] 7e was synthesized in the same manner as 7a, with a yield of 43%. 1H NMR(400MHz,Chloroform-d)δ7.61(s,1H),7.50(dd,J=7.6,1.9Hz,1H),7.33(dd,J =7.6,1.9Hz,1H),7.29(d,J=7.6Hz,1H),5.20(s,1H),4.23(s,2H),3.85(dt,J=13. 4,4.4Hz,2H),3.66(s,3H),3.36–3.24(m,2H),2.32(t,J=7.4Hz,2H),2.17(q,J=7. 3Hz, 4H), 1.78–1.70 (m, 2H), 1.69–1.63 (m, 4H), 1.46 (s, 3H), 1.38 (q, J = 8.1Hz, 2H).

[0088] 8-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-8-oxooctanoic acid methyl ester (7f)

[0089] 7f was synthesized in the same manner as 7a, with a yield of 56%. 1 H NMR (400MHz, Chloroform-d) δ7.61 (s, 1H), 7.50 (dd, J = 7.6, 1.9 Hz, 1H), 7.33 (dd, J = 7. 6,1.9Hz,1H),7.30(d,J=7.6Hz,1H),5.20(s,1H),4.23(s,2H),3.86(dt,J=13.3,4.3Hz ,2H),3.66(s,3H),3.34–3.25(m,2H),2.31(t,J=7.5Hz,2H),2.17(q,J=9.8,7.8Hz,4H ),1.72(td,J=10.1,5.2Hz,2H),1.63(s,4H),1.46(s,3H),1.35(dt,J=7.2,3.8Hz,4H).

[0090] 3-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)carbamoyl)methyl benzoate (7h)

[0091] 7h was synthesized in the same manner as 7a, with a yield of 74%. 1H NMR(400MHz,Chloroform-d)δ8.34(s,1H),8.16(d,J=7.8Hz,1H),8.01(d,J=7.8Hz,1H),7.63(s ,1H),7.54(t,J=7.8Hz,1H),7.50(dd,J=7.7,1.8Hz,1H),7.33(dd,J=7.7,1.8Hz,1H),7.29(d,J= 7.7Hz,1H),5.98(s,1H),4.25(s,2H),3.95(s,3H),3.91(dt,J=13.4,4.5Hz,2H),3.44–3.38(ddd ,J=13.9,10.1,4.0Hz,2H),2.36–2.33(m,2H),1.86(ddd,J=13.9,10.1,4.0Hz,2H),1.60(s,3H).

[0092] 4-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)carbamoyl)methyl benzoate (7i)

[0093] 7i was synthesized in the same manner as 7a, with a yield of 74%. 1 H NMR(400MHz,Chloroform-d)δ8.10(d,J=8.4Hz,2H),7.80(d,J=8.4Hz,2H),7.63( s,1H),7.50(dd,J=7.6,2.0Hz,1H),7.33(dd,J=7.6,2.0Hz,1H),7.30(d,J=7.6Hz ,1H),5.94(s,1H),4.25(s,2H),3.95(s,3H),3.91(dd,J=11.3,6.7Hz,2H),3.43– 3.37(m,2H),2.40–2.30(m,3H),1.86(ddd,J=14.1,10.1,4.2Hz,2H),1.60(s,3H).

[0094] 4-((2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-2-oxoethyl)carbamoyl)methyl benzoate (7j)

[0095] 7j was synthesized in the same manner as 7a, with a yield of 56%. 1H NMR(400MHz,Chloroform-d)δ8.09(d,J=8.2Hz,2H),7.88(d,J=8.3Hz,2H),7.66(t,J=5.0Hz,1H) ,7.57(s,1H),7.49(dd,J=7.6,2.0Hz,1H),7.32(dd,J=7.7,2.0Hz,1H),7.27(t,J=7.6Hz,1H),6.8 5(s,1H),4.28(s,2H),4.18(d,J=4.9Hz,2H),3.94(s,3H),3.92–3.82(m,2H),3.27(ddd,J=13.6, 10.6, 3.0Hz, 2H), 2.23 (dt, J=13.6, 3.6Hz, 2H), 1.69 (ddd, J=14.2, 10.6, 4.2Hz, 2H), 1.48 (s, 3H).

[0096] Methyl(E)-3-(4-((3-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropionamido)methyl)phenyl)acrylate (7k)

[0097] 7k was synthesized in the same manner as 7a, with a yield of 50%. 1 H NMR(400MHz,Chloroform-d)δ7.65(d,J=16.1Hz,1H),7.60(s,1H),7.51–7.46(m,3H),7 .36–7.29(m,2H),7.26(s,2H),7.02(d,J=6.5Hz,1H),6.96(s,1H),6.41(d,J=16.0Hz,1H ),4.47(d,J=5.9Hz,2H),4.26(s,2H),3.93–3.87(m,2H),3.79(s,3H),3.27–3.21(m,2H) ,3.20(s,2H),2.20(d,J=13.6Hz,2H),1.69(ddd,J=14.2,10.6,4.0Hz,2H),1.45(s,3H).

[0098] Methyl(E)-3-(3-((3-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropionamido)methyl)phenyl)acrylate (7l)

[0099] 7l was synthesized in the same manner as 7a, with a yield of 58%. 1H NMR(400MHz,Chloroform-d)δ7.66(d,J=16.0Hz,1H),7.59(s,1H),7.50(dd,J=7.7,1.9Hz,1H) ,7.41(d,J=9.1Hz,2H),7.35–7.27(m,4H),7.04(t,J=5.9Hz,1H),6.93(s,1H),6.43(d,J=16.0 Hz,1H),4.46(d,J=5.9Hz,2H),4.25(s,2H),3.89(dt,J=13.6,4.5Hz,2H),3.80(s,3H),3.30–3 .20(m,2H),3.20(s,2H),2.20–2.16(m,2H),1.68(ddd,J=14.2,10.5,4.2Hz,2H),1.45(s,3H).

[0100] 4-((3-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-3-oxopropamido)methyl)methyl)benzoate (7m)

[0101] 7m was synthesized in the same manner as 7a, with a yield of 41%. 1 H NMR(400MHz,Chloroform-d)δ7.99(d,J=8.2Hz,2H),7.59(s,1H),7.49(dd,J=7.7,1.8Hz,1H),7.34–7.28(m,5H),6.99(s,1H),4.50(d,J=5.9 Hz,2H),4.28(s,2H),3.90(s,3H),3.87(t,J=4.3Hz,2H),3.28–3.22(m ,2H),3.21(s,2H),2.20–2.16(m,2H),1.73–1.66(m,2H),1.44(s,3H).

[0102] Example 3: Preparation of intermediates 7g, 7n-7t, 7v

[0103] Methyl 5-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)valerate (7g)

[0104] Compound 6 (89 mg, 0.23 mmol), methyl 5-bromopentanoate (54 mg, 0.27 mmol), and K₂CO₃ (95 mg, 0.69 mmol) were dissolved in 3 mL of DMF and stirred at 80 °C for 8 hours. After the reaction was complete, the reaction mixture was poured into water and extracted with EtOAc. The combined EtOAc was washed with saturated brine and dried over anhydrous magnesium sulfate. The crude product was purified by column chromatography (DCM / MeOH = 100:1–30:1) by vacuum distillation to give 50 mg of white solid, yield: 47%. 1 H NMR (400MHz, DMSO-d6) δ7.62(dd,J=8.0,1.3Hz,1H),7.48(s,1H),7.39(t,J=7.8Hz,1H),7.31(dd,J=8.0,1.3Hz,1H),5.59(s,2H),3.58(s,3H) ,3.55(s,3H),3.39(s,2H),2.55–2.52(m,2H),2.32(t,J=7.4Hz,2H),1 .60(dt,J=15.5,7.5Hz,4H),1.42(dq,J=14.1,6.3Hz,4H),1.10(s,3H).

[0105] Methyl(E)-3-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)phenyl)acrylate (7n)

[0106] Compound 6 (136 mg, 0.35 mmol), methyl (E)-3-(4-(bromomethyl)phenyl)acrylate (99 mg, 0.388 mmol), and K₂CO₃ (146 mg, 1.05 mmol) were dissolved in 3 mL of LMF and stirred at room temperature. The reaction mixture was poured into water and extracted with EtOAc. The combined EtOAc was washed with saturated brine and dried over anhydrous magnesium sulfate. The crude product was purified by column chromatography (DCM / MeOH = 100:1–50:1) by vacuum distillation to give 117 mg of a yellow solid, yield: 63%. 1HNMR (400MHz, Chloroform-d) δ7.69 (d, J = 16.0 Hz, 1H), 7.62 (s, 1H), 7.49 (d, J = 8. 2Hz,3H),7.41(d,J=8.1Hz,2H),7.33(dd,J=7.6,1.7Hz,1H),7.29(d,J=7.7Hz,1H) ,6.42(d,J=16.0Hz,1H),4.20(s,2H),3.80(s,3H),3.77(s,2H),3.73(dd,J=11.5, 6.5Hz,2H),3.64–3.56(m,2H),1.76–1.69(m,2H),1.67–1.61(m,2H),1.25(s,3H).

[0107] 3-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)benzoate (7o)

[0108] 7o was synthesized in the same manner as 7n, with a yield of 71%. 1 H NMR(400MHz,Chloroform-d)δ8.03(s,1H),7.92(d,J=7.8Hz,1H),7.62(s,1H),7.6 0(d,J=7.7Hz,1H),7.49(dd,J=7.8,1.4Hz,1H),7.40(t,J=7.7Hz,1H),7.33(dd,J=7 .8,1.4Hz,1H),7.29(d,J=7.7Hz,1H),4.20(s,2H),3.92(s,3H),3.80(s,2H),3.76– 3.70(m,2H),3.65–3.58(m,2H),1.76–1.70(m,2H),1.68–1.62(m,2H),1.26(s,3H).

[0109] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)benzoate (7p)

[0110] 7p was synthesized in the same manner as 7n, with a yield of 42%. 1H NMR(400MHz,Chloroform-d)δ7.99(d,J=8.1Hz,2H),7.62(s,1H),7.49(dd,J=7.9,1.6Hz,1H),7.46(d,J=8.1Hz,2H),7.33(dd,J=7.6,1.5H z,1H),7.29(d,J=7.7Hz,1H),4.20(s,2H),3.91(s,3H),3.81(s,2H),3.78–3.69(m,2H),3.63–3.57(m,2H),1.76–1.61(m,4H),1.26(s,3H).

[0111] Methyl(E)-3-(4-(2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)phenyl)acrylate (7q)

[0112] 7q was synthesized in the same manner as 7g, with a yield of 62%. 1 H NMR (400MHz, DMSO-d6) δ10.07(s,1H),7.72–7.66(s,4H),7.65–7.56(m,2H),7.50(s,1H),7.39(t,J=7.8Hz,1H),7.31(dd,J=7.6,1.2Hz,1H),6. 54(d,J=16.0Hz,1H),5.60(s,2H),3.71(s,3H),3.62–3.55(m,4H),3.32 (s,2H),2.36(s,1H),1.66–1.57(m,2H),1.53–1.47(m,2H),1.10(s,3H).

[0113] Methyl(E)-3-(4-((2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)methyl)phenyl)acrylate (7r)

[0114] 7r was synthesized in the same manner as 7g, with a yield of 53%. 1H NMR(400MHz,DMSO-d6)δ8.40(t,J=6.1Hz,1H),7.67–7.63(m,3H),7.62–7.61 (m,1H),7.48(s,1H),7.38(t,J=7.8Hz,1H),7.32–7.30(m,3H),6.61(d,J=16. 1Hz,1H),5.59(s,2H),4.35(d,J=6.1Hz,2H),3.71(s,3H),3.61–3.51(m,4H), 3.16(s,2H),2.21(s,1H),1.59–1.56(m,2H),1.52–1.39(m,2H),1.05(s,3H).

[0115] Methyl(E)-3-(4-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)benzamido)phenyl)acrylate (7s)

[0116] 7s was synthesized in the same manner as 7n, with a yield of 75%. 1 H NMR (400MHz, DMSO-d6) δ10.37(s,1H),7.92(d,J=8.1Hz,2H),7.87(d,J=8.6Hz,2H),7.72( d,J=8.6Hz,2H),7.66–7.60(m,2H),7.56(d,J=8.1Hz,2H),7.50(s,1H),7.39(t,J=7.8Hz, 1H),7.31(d,J=6.6Hz,1H),6.57(d,J=16.0Hz,1H),5.60(s,2H),3.72(s,3H),3.67–3.64( m,2H),3.56–3.51(m,2H),1.99(s,1H),1.70–1.66(m,2H),1.52–1.47(m,2H),1.17(s,3H).

[0117] Methyl(E)-3-(4-((4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)benzoylamino)methyl)phenyl)acrylate (7t)

[0118] 7t was synthesized in the same manner as 7n, with a yield of 54%. 1H NMR(400MHz, DMSO-d6)δ9.02(t,J=5.9Hz,1H),7.84(d,J=8.1Hz,2H),7.72–7.65(m, 3H),7.62(d,J=6.4Hz,1H),7.54–7.46(m,3H),7.44–7.27(m,4H),6.61(d,J=16.0Hz, 1H),5.59(s,2H),4.49(d,J=5.7Hz,2H),3.74(s,2H),3.72(s,3H),3.67–3.63(m,2H) ,3.56–3.48(m,2H),1.90(s,1H),1.68–1.65(m,2H),1.51–1.46(m,2H),1.16(s,3H).

[0119] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)benzoic acid (7v)

[0120] 6 (233 mg, 0.6 mmol), methyl 4-(bromomethyl)benzoate (151 mg, 0.66 mmol), and K₂CO₃ (249 mg, 1.8 mmol) were dissolved in 3 mL of DMF at room temperature for 8 hours. The reaction mixture was then poured into water and extracted with EtOAc. The combined EtOAc was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated to give a solid. This solid was then dissolved in 10 mL of a mixed solvent (4 M NaOH / EtOH = 1:1) and stirred at 50 °C for 1 hour to remove the organic solvent. The residual aqueous solution was acidified to pH 5-6 with 1 M HCl, producing a yellow precipitate. Filtering yielded 100 mg of a yellow solid, which was used directly in the next reaction without purification; yield: 34%. 1 H NMR (400MHz, DMSO-d6) δ9.35(s,1H),7.99(d,J=8.0Hz,2H),7.75(d,J=7.5Hz,2H),7.63(d,J=8.0Hz,1H),7.57(s,1H),7.40(t,J=7 .8Hz,1H),7.30(d,J=7.6Hz,1H),5.70(s,2H),4.31–4.28(m,2H),4.24(s,2H),3.13–2.99(m,2H),2.07–1.95(s,4H),1.58(s,3H).

[0121] Example 4: Preparation of target compound 8a-8v

[0122] N 1-(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -Hydroxymalonamide (8a)

[0123] KOH (11.2 g, 200 mmol) and NH₂OH-HCl (9.2 g, 132.4 mmol) were dissolved in 28 mL and 48 mL of anhydrous MeOH, respectively, to obtain solutions A and B. Solution A was then added dropwise to solution B at 0°C and stirred for 30 minutes. The precipitate (KCl) was then filtered off, yielding an NH₂OK solution. Compound 7a (106 mg, 0.23 mmol) was dissolved in NH₂OK solution (5 mL) and stirred at room temperature for 6 hours. After the reaction was complete, the solvent was evaporated under vacuum. The residue was acidified to pH 7 with 1 M HCl, the precipitate was filtered off and dried to obtain the crude product. The crude product was purified by column chromatography (DCM / MeOH = 100:1-20:1) to give 60 mg of a yellow solid, yield: 57%, mp 158-160°C. 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.88(s,1H),7.77(s,1H),7.61(d,J=8.0Hz,1H),7.50(s,1H),7.39(t,J=7.8Hz,1H),7.30(d ,J=7.5Hz,1H),5.60(s,2H),3.88–3.85(m,2H),3.21–3.16(m,2H),2.91(s,2H),2.15–2.11(m,2H),1.50–1.45(m,2H),1.32(s,3H). 13 C NMR(101MHz,DMSO)δ166.82,153.61,151.11,139.87,132.49,132.00,131.10, 130.17,128.69,125.25,117.12,51.62,41.93,40.53,35.16,26.32.HRMS(ESI － m / z, calcd for C 19 H 22 Cl2N6O3([M-H) － )451.1058,found:451.1062.

[0124] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 4 -Hydroxysuccinamide (8b)

[0125] 8b was synthesized in the same manner as 8a, with a yield of 38%. mp 238-240℃. 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),7.87(s,1H),7.61(d,J=7.5Hz,1H),7.49(s,1H),7.41–7.34(m,1H),7.30(d,J=7.6Hz,1 H),5.59(s,2H),3.85–3.82(m,2H),3.29–3.08(m,2H),2.37–2.32(m,2H),2.30–1.88(m,4H),1.57–1.38(m,2H),1.29(s,3H). 13 C NMR(101MHz,DMSO)δ171.70,169.00,153.60,151.10,139.87,132.49,132.00,131 .09,130.16,128.69,125.16,117.10,51.28,35.23,31.99,28.48,26.38.HRMS(ESI － m / z, calcd for C 20 H 24 Cl2N6O3([M-H) － )465.1214,found:465.1210.

[0126] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 5 8-Hydroxyglutaramide (8c)

[0127] 8c was synthesized in the same manner as 8a, with a yield of 45%. mp 214-216℃. 1 H NMR(400MHz,DMSO-d6)δ10.37(s,1H),8.70(s,1H),7.62(dd,J=8.0,1.6Hz,1 H),7.50(s,1H),7.45–7.35(m,2H),7.30(dd,J=7.7,1.6Hz,1H),5.63(s,2H) ,3.88–3.82(m,2H),3.25–3.11(m,2H),2.19–2.06(m,4H),1.95(t,J=7.5Hz, 2H), 1.70 (p, J=7.5Hz, 2H), 1.45 (ddd, J=14.1, 10.5, 4.1Hz, 2H), 1.30 (s, 3H). 13C NMR(101MHz,DMSO)δ172.35,169.16,153.56,151.10,139.87,132.50,132.00,131.09 ,130.15,128.68,125.18,117.08,51.28,36.06,35.31,32.24,26.36,22.24.HRMS(ESI － m / z, calcd for C 21 H 26 Cl2N6O3([M-H) － )479.1371,found:479.1361.

[0128] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 6 -Hydroxyhexanediamine (8d)

[0129] 8d was synthesized in the same manner as 8a, with a yield of 40%. mp 144-146℃. 1 H NMR (400MHz, DMSO-d6) δ10.33(s,1H),8.67(s,1H),7.62(dd,J=7.9,1.6Hz,1H),7.51(s,1H),7.43–7.36(m,2H),7.31(dd,J=7.7,1.6Hz,1H),5.62 (s,2H),3.85(dt,J=13.7,4.5Hz,2H),3.18(ddd,J=13.5,10.5,3.0Hz,2H ),2.19–2.07(m,4H),1.97–1.94(m,2H),1.52–1.43(m,6H),1.31(s,3H). 13 C NMR (101MHz, DMSO) δ172.64,169.48,153.53,151.10,139.87,132.51,132.00,131.09,13 0.15,128.68,125.17,117.08,51.27,36.46,35.25,32.66,26.34,25.70,25.34.HRMS(ESI － m / z, calcd for C 22 H 28 Cl2N6O3([M-H) － )493.1527,found:493.1510.

[0130] N 1-(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 7 8-Hydroxyheptanamide (8e)

[0131] 8e was synthesized in the same manner as 8a, with a yield of 43%. mp 124-126℃. 1 H NMR (400MHz, DMSO-d6) δ10.22(s,1H),8.69(s,1H),7.61(d,J=7.8Hz,1H),7.50(s,1H),7.39(t,J=7.8Hz,1H),7.34(s,1H),7.30(d,J=7.0Hz,1H),5 .58(s,2H),3.85–3.82(m,2H),3.18(t,J=10.8Hz,2H),2.19–2.04(m,4H), 1.93(t,J=7.2Hz,2H),1.56–1.41(m,6H),1.30(s,3H),1.26–1.20(m,2H). 13 CNMR(101MHz,DMSO)δ172.74,169.42,153.54,151.10,139.88,132.49,132.00,131.09,130.1 5,128.69,125.17,117.07,51.24,36.53,35.25,32.61,28.76,26.34,25.71,25.41.HRMS(ESI － m / z, calcd for C 23 H 30 Cl2N6O3([M-H) － )507.1683,found:507.1682.

[0132] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 8 -Hydroxyoctadiamide (8f)

[0133] 8f was synthesized in the same manner as 8a, with a yield of 34%. mp 146-148℃. 1H NMR(400MHz,DMSO-d6)δ10.25(s,1H),8.63(s,1H),7.61(d,J=7.9Hz,1H),7.50 (s,1H),7.39(t,J=7.8Hz,1H),7.34(s,1H),7.30(d,J=6.8Hz,1H),5.58(s,2H), 3.85–3.82(m,2H),3.18(t,J=10.9Hz,2H),2.17–2.13(m,2H),2.08(t,J=7.3Hz ,2H),1.92(t,J=7.2Hz,2H),1.48–1.47(m,6H),1.30(s,3H),1.24–1.17(s,4H). 13 C NMR (101MHz, DMSO) δ172.81,169.49,153.55,151.10,139.87,132.49,131.99,131.09,130.1 6,128.69,125.18,117.07,51.23,36.64,35.24,32.71,28.90,26.37,25.91,25.57.HRMS(ESI － m / z, calcd for C 24 H 32 Cl2N6O3([M-H) － )521.1840,found:521.1825.

[0134] 5-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-N-hydroxypentanamide (8g)

[0135] 8g was synthesized in the same manner as 8a, yield: 53%. mp 156-158℃. 1 H NMR (400MHz, DMSO-d6) δ9.79(s,1H),7.61(d,J=7.9Hz,1H),7.47(s,1H),7.38(t,J=7.8Hz,1H),7.30(d,J=7.5Hz,1H),5.57(s,2H),3 .63–3.60(m,2H),3.48–3.43(m,2H),2.45(t,J=6.6Hz,2H),1.94(t,J=7.3Hz,2H),1.57–1.54(m,4H),1.43–1.34(m,4H),1.06(s,3H). 13C NMR(101MHz,DMSO)δ169.46,153.66,151.08,139.93,132.51,132.01,131.09,130.11,12 8.66,124.88,117.11,50.21,40.83,40.69,36.49,32.79,30.68,25.67,23.75.HRMS(ESI + )m / z,calcdfor C 21 H 28 Cl2N6O2([M+H) + )467.1723,found:467.1718.

[0136] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -Hydroxyisophthalamide (8h)

[0137] Synthesized in the same manner as 8a over 8h, yield: 45%. mp 210-212℃. 1 H NMR (400MHz, DMSO-d6) δ9.94(s,1H),8.23(s,1H),7.96–7.80(m,3H),7.61(dd,J=8.0,1.6Hz,1H),7.53(s,1H),7.45(t,J=7.6Hz,1H),7.39(t,J =7.8Hz,1H),7.31(dd,J=7.6,1.6Hz,1H),5.61(s,2H),3.93–3.87(m,2H ),3.31–3.26(m,2H),2.42–2.39(m,2H),1.64–1.53(m,2H),1.44(s,3H). 13 CNMR(101MHz,DMSO)δ167.22,163.62,153.64,151.12,139.88,136.36,132.50,132.00,131.10,130 .16,129.39,128.70,128.45,126.10,125.23,117.15,52.29,52.11,40.84,35.22,26.19.HRMS(ESI － m / z, calcd for C 24 H 24 Cl2N6O3([M-H) － )513.1214,found:513.1208.

[0138] N1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 4 -Hydroxyterephthalamide (8i)

[0139] 8i was synthesized in the same manner as 8a, with a yield of 55%. mp 190-192℃. 1 H NMR (400MHz, DMSO-d6) δ10.21(s,1H),7.90–7.81(m,5H),7.62(dd,J=8.0,1.6Hz,1H),7.53(s,1H),7.40(t,J=7.8Hz,1H),7.31 (dd,J=7.6,1.6Hz,1H),5.62(s,2H),3.93–3.87(m,2H),3.31–3.24(m,2H),2.41–2.37(m,2H),1.64–1.55(m,2H),1.44(s,3H). 13 C NMR(101MHz,DMSO)δ166.86,153.63,151.12,139.88,132.50,132.00,131.09,130.16, 128.70,127.88,126.94,125.25,117.14,52.33,52.18,40.82,35.20,26.17.HRMS(ESI － m / z, calcd for C 24 H 24 Cl2N6O3([M-H) － )513.1214,found:513.1203.

[0140] N 1 -(2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)-2-oxoethyl)-N 4 -Hydroxyterephthalamide (8j)

[0141] 8j was synthesized in the same manner as 8a, with a yield of 51%. mp 184-186℃. 1H NMR(400MHz,DMSO-d6)δ9.47(s,1H),8.80(s,1H),8.66(s,1H),7.98–7.90(m,2H),7.87–7.79(m,2H),7.67–7.54(m,2H),7.51(s,1H),7.40(t,J =7.8Hz,1H),7.32(dd,J=7.6,1.7Hz,1H),5.62(s,2H),3.98–3.80(m,4H ),3.27–3.16(m,2H),2.19–2.16(m,2H),1.54–1.44(m,2H),1.34(s,3H). 13 C NMR (101MHz, DMSO) δ169.00,166.93,166.34,153.60,151.11,139.87,132.50,132.00,131.10,130.16, 129.43,128.70,127.76,127.19,127.01,125.23,117.13,51.57,51.37,43.53,35.25,26.44.HRMS(ESI － m / z, calcd for C 26 H 27 Cl2N7O4([M-H) － )570.1429,found:570.1409.

[0142] (E)-N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(4-(3-(hydroxyamino))-3-oxoprop-1-en-1-yl)benzyl)malonamide (8k)

[0143] 8k was synthesized in the same manner as 8a, with a yield of 60%. mp 176-178℃. 1H NMR (400MHz, DMSO-d6) δ10.07(s,1H),8.49(s,1H),7.70(s,1H),7.61(d,J=7.9Hz ,1H),7.51(s,1H),7.47(d,J=7.4Hz,2H),7.39(t,J=7.8Hz,1H),7.37–7.17(m,4H) ,6.45(d,J=15.9Hz,1H),5.60(s,2H),4.30(d,J=5.6Hz,2H),3.89–3.85(m,2H),3 .25–3.17(m,2H),3.14(s,2H),2.16–2.13(m,2H),1.51–1.46(m,2H),1.33(s,3H). 13 C NMR (101MHz, DMSO) δ167.78,167.11,153.61,151.11,141.01,139.86,134.12,132.49,132.00,131.10,130 .17,128.70,128.17,127.78,125.25,119.40,117.11,51.63,44.75,42.45,40.54,35.17,26.34.HRMS(ESI － )m / z,calcdfor C 29 H 31 Cl2N7O4([M-H) － )610.1742,found:610.1738.

[0144] (E)-N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(3-(3-(hydroxyamino))-3-oxoprop-1-en-1-yl)benzyl)malonamide (8l)

[0145] 8l was synthesized in the same manner as 8a, with a yield of 64%. mp 190-192℃. 1H NMR(400MHz,DMSO-d6)δ9.81(s,1H),8.50(s,1H),7.69(s,1H),7.61(d,J=7 .9Hz,1H),7.50(s,1H),7.46–7.34(m,3H),7.34–7.11(m,4H),6.48(d,J=15. 5Hz,1H),5.59(s,2H),4.30(d,J=5.2Hz,2H),3.88–3.85(m,2H),3.20–3.18( m,2H),3.14(s,2H),2.15–2.12(m,2H),1.48(t,J=10.2Hz,2H),1.32(s,3H). 13 C NMR (101MHz, DMSO) δ167.80,167.09,153.60,151.11,140.40,139.87,132.49,132.00,131.10,130.16,129 .33,128.69,128.42,126.54,126.31,125.22,117.11,51.62,44.79,42.54,40.53,35.18,26.36.HRMS(ESI － m / z, calcd for C 29 H 31 Cl2N7O4([M-H) － )610.1742,found:610.1735.

[0146] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 3 -(4-(hydroxycarbamoyl)benzyl)malonamide (8m)

[0147] 8m was synthesized in the same manner as 8a, with a yield of 46%. mp 180-182℃. 1H NMR (400MHz, DMSO-d6) δ10.75(s,1H),8.59(s,1H),7.77(d,J=7.1Hz,1H),7.7 1(d,J=8.0Hz,2H),7.63–7.59(m,1H),7.50(s,1H),7.39(t,J=7.8Hz,1H),7.34 –7.27(m,3H),5.60(s,2H),4.32(d,J=5.7Hz,2H),3.89–3.85(m,2H),3.23–3.2 0(m,2H),3.16(s,2H),2.17–2.13(m,2H),1.48(t,J=10.1Hz,2H),1.33(s,3H). 13 C NMR(101MHz,DMSO)δ167.83,167.14,164.13,153.61,151.11,139.86,132.49,132.00,131.10, 130.17,128.70,127.38,127.17,125.23,117.12,51.65,44.81,42.38,35.16,26.35.HRMS(ESI － m / z, calcd for C 27 H 29 Cl2N7O4([M-H) － )584.1585,found:584.1580.

[0148] (E)-3-(4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide(8n)

[0149] 8n was synthesized in the same manner as 8a, with a yield of 42%. mp 166-168℃. 1 H NMR (400MHz, DMSO-d6) δ10.71(s,1H),9.00(s,1H),7.60(dd,J=7.6,1.3Hz,1H),7.56–7.46(m,3H),7.45–7.35(m,4H),7.31(dd,J=7.6,1.3Hz,1 H),6.43(d,J=15.8Hz,1H),5.56(s,2H),3.70(s,2H),3.67–3.64(m,2H) ,3.54–3.50(m,2H),1.68–1.65(m,2H),1.51–1.45(m,2H),1.16(s,3H). 13C NMR (101MHz, DMSO) δ163.18,153.66,151.10,144.14,139.93,133.59,132.49,132.00,131.11,130 .12,129.06,128.68,127.67,124.93,118.88,117.09,50.93,45.32,40.68,36.39,25.75.HRMS(ESI － m / z, calcd for C 26 H 28 Cl2N6O2([M-H) － )525.1578,found:525.1571.

[0150] 3-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-hydroxybenzamide (8o)

[0151] 8o was synthesized in the same manner as 8a, with a yield of 42%. mp 152-154℃. 1 H NMR (400MHz, DMSO-d6) δ11.16(s,1H),9.00(s,1H),7.79(s,1H),7.61(dd,J= 7.6,1.4Hz,1H),7.58(s,1H),7.53(d,J=7.6Hz,1H),7.49(s,1H),7.41–7.35 (m,2H),7.31(dd,J=7.6,1.4Hz,1H),5.57(s,2H),3.72(s,2H),3.66–3.63(m ,2H),3.60–3.49(m,2H),1.69–1.66(m,2H),1.52–1.47(m,2H),1.18(s,3H). 13 C NMR (101MHz, DMSO) δ164.76,153.65,151.10,142.61,139.93,133.09,132.49,132.01,131.39,131. 11,130.12,128.68,128.49,127.23,125.37,124.94,117.08,51.05,45.42,36.36,25.63.HRMS(ESI － m / z, calcd for C 24 H 26 Cl2N6O2([M-H) － )499.1422,found:499.1417.

[0152] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-hydroxybenzamide (8p)

[0153] 8p was synthesized in the same manner as 8a, with a yield of 48%. mp 156-158℃. 1 H NMR(400MHz,DMSO-d6)δ11.13(s,1H),8.97(s,1H),7.89–7.69(m,2H),7.61(d d,J=8.0,1.6Hz,1H),7.49(s,1H),7.46(d,J=8.1Hz,2H),7.39(t,J=7.8Hz,1H ),7.31(dd,J=7.7,1.6Hz,1H),5.56(s,2H),3.73(d,J=6.0Hz,2H),3.67–3.62 (m,2H),3.55–3.49(m,2H),1.68–1.65(m,2H),1.52–1.45(m,2H),1.16(s,3H). 13 C NMR (101MHz, DMSO) δ164.58,153.65,151.10,145.80,139.93,132.50,132.01,131.40,131.11,130.12, 129.55,128.68,128.41,128.18,127.11,124.95,117.09,51.05,45.24,40.68,36.35,25.64.HRMS(ESI － m / z, calcd for C 24 H 26 Cl2N6O2([M-H) － )499.1422,found:499.1417.

[0154] (E)-3-(4-(2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)phenyl)-N-hydroxyacrylamide (8q)

[0155] 8q was synthesized in the same manner as 8a, with a yield of 50%. mp 176-178℃. 1H NMR (400MHz, DMSO-d6) δ10.08(s,1H),7.69(d,J=8.6Hz,2H),7.62(d,J=8.4Hz,3H),7.51–7.47(m,2H),7.39(t,J=7.8Hz,1H),7. 31(d,J=7.4Hz,1H),6.44(d,J=16.0Hz,1H),5.60(s,2H),3.59–3.57(s,4H),1.68–1.58(m,2H),1.54–1.47(m,2H),1.10(s,3H). 13 C NMR (101MHz, DMSO) δ171.59,168.98,153.64,153.57,151.09,139.93,132.50,132.01,131.10,130.12 ,128.67,128.57,125.03,124.96,119.70,117.09,50.88,50.57,46.03,43.39,36.31,25.38.HRMS(ESI + m / z, calcd for C 27 H 29 Cl2N7O3([M+H) + )570.1781,found:570.1691.

[0156] (E)-3-(4-((2-((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)acetamido)methyl)phenyl)-N-hydroxyacrylamide(8r)

[0157] 8r was synthesized in the same manner as 8a, with a yield of 62%. mp 164-166℃. 1 H NMR (400MHz, DMSO-d6) δ10.09(s,1H),8.37(t,J=6.0Hz,1H),7.61(d,J=7.9H z,1H),7.47(d,J=10.9Hz,2H),7.38(t,J=7.8Hz,1H),7.34–7.09(m,4H),6.4 3(d,J=16.0Hz,1H),5.59(s,2H),4.32(d,J=5.4Hz,2H),3.61–3.51(m,4H),3 .15(s,2H),2.20(s,1H),1.58–1.55(m,2H),1.49–1.38(m,2H),1.05(s,3H). 13C NMR (101MHz, DMSO) δ172.62,162.68,153.63,151.09,139.92,137.34,132.50,132.00,131.10,130 .13,128.68,128.15,127.70,124.95,119.54,117.10,50.69,45.31,42.18,36.32,25.39.HRMS(ESI + m / z, calcd for C 28 H 31 Cl2N7O3([M+H) + )584.1938,found:584.1914.

[0158] (E)-4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)benzamide(8s)

[0159] 8s was synthesized in the same manner as 8a, with a yield of 58%. mp 178-180℃. 1 H NMR (400MHz, DMSO-d6) δ10.32(s,1H),7.92(d,J=7.9Hz,2H),7.84(d,J=8.3Hz,2H),7 .61(d,J=7.9Hz,1H),7.55(d,J=7.7Hz,4H),7.50(s,1H),7.43–7.35(m,2H),7.31(d, J=7.5Hz,2H),6.40(d,J=15.6Hz,1H),5.59(s,2H),3.77(s,2H),3.68–3.64(m,2H),3 .53(t,J=9.7Hz,2H),1.99(s,1H),1.70–1.66(m,2H),1.52–1.47(m,2H),1.17(s,3H). 13 C NMR (101MHz, DMSO) δ166.02,163.32,153.68,151.10,146.95,140.84,139.94,133.25,132.52,132.02,131.11,130.58, 130.13,128.67,128.43,128.35,128.02,124.95,120.80,118.11,117.12,50.88,45.34,40.72,36.50,25.88.HRMS(ESI +m / z, calcd for C 33 H 33 Cl2N7O3([M+H) + )646.2094,found:646.2042.

[0160] (E)-4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)benzyl)benzamide(8t)

[0161] 8t was synthesized in the same manner as 8a, with a yield of 64%. mp 166-168℃. 1 H NMR (400MHz, DMSO-d6) δ10.72 (s, 1H), 9.08–8.94 (m, 1H), 7.85 (d, J = 8.0Hz, 2H), 7. 61(d,J=7.9Hz,1H),7.54–7.43(m,5H),7.43–7.36(m,2H),7.34(d,J=8.0Hz,2H),7 .30(d,J=7.6Hz,1H),6.44(d,J=16.0Hz,1H),5.59(s,2H),4.49(d,J=5.5Hz,2H),3 .76(s,2H),3.67–3.51(m,4H),1.69–1.66(m,1H),1.52–1.50(m,2H),1.18(s,3H). 13 C NMR (101MHz, DMSO) δ166.66,163.21,153.64,151.11,141.76,139.93,138.43,133.89,132.96,132.53,132.02,131.10,130 .13,128.68,128.45,128.22,127.94,127.58,124.98,119.14,117.12,51.31,45.23,42.89,40.71,36.29,25.41.HRMS(ESI + m / z, calcd for C 34 H 35 Cl2N7O3([M+H) + )660.2251,found:660.2198.

[0162] N 1 -(1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)-N 32-Aminophenyl)malonamide (8u)

[0163] 8u was synthesized in the same manner as 7a, with a yield of 40%. mp 228-230℃. 1 H NMR(400MHz,DMSO-d6)δ9.24(s,1H),7.75(s,1H),7.67–7.58(m,1H),7.51(s,1H),7.3 9(t,J=7.8Hz,1H),7.35–7.26(m,1H),7.10(d,J=7.7Hz,1H),6.91(t,J=7.1Hz,1H),6. 70(d,J=7.5Hz,1H),6.51(t,J=7.6Hz,1H),5.60(s,2H),4.93(s,2H),3.89–3.86(m,2H ),3.28(s,2H),3.24–3.19(m,2H),2.17–2.14(m,2H),1.53–1.48(m,2H),1.35(s,3H). 13 C NMR (101MHz, DMSO) δ167.42,166.70,153.61,151.11,143.06,139.89,132.51,132.02,131.09,130.16,128 .68,126.77,126.37,125.29,123.29,117.17,116.39,115.92,51.76,45.12,40.59,35.22,26.32.HRMS(ESI － m / z, calcd for C 25 H 27 Cl2N7O2([M-H) － )526.1531,found:526.1526.

[0164] 4-(((1-(6-amino-5-(2,3-dichlorophenyl)pyrazin-2-yl)-4-methylpiperidin-4-yl)amino)methyl)-N-(2-aminophenyl)benzamide (8v)

[0165] 8V was synthesized in the same manner as 7A, with a yield of 31%. mp 166-168℃. 1H NMR(400MHz,DMSO-d6)δ9.61(s,1H),7.94(d,J=7.9Hz,2H),7.61(dd,J=7.9,1.4Hz,1H) ,7.58–7.45(m,3H),7.39(t,J=7.8Hz,1H),7.31(dd,J=7.6,1.4Hz,1H),7.17(d,J=7.6H z,1H),7.01–6.94(m,1H),6.78(d,J=7.9Hz,1H),6.60(t,J=7.5Hz,1H),5.58(s,2H),4. 87(s,2H),3.80(s,2H),3.67–3.54(m,3H),1.71–1.68(m,2H),1.52(s,2H),1.20(s,3H). 13 C NMR (101MHz, DMSO) δ165.70,153.64,151.11,143.55,139.93,133.33,132.53,132.02,131.10,130.14,128.68,128 .40,128.08,127.11,126.89,125.02,123.97,117.15,116.82,116.69,55.36,45.24,40.74,36.28,25.40.HRMS(ESI － m / z, calcd for C 30 H 31 Cl2N7O([M-H)) － )574.1894,found:574.1891.

[0166] Example 5: The inhibitory activity of the target compound against HDACs in this invention

[0167] 1. Materials:

[0168] HDACs enzymes (HeLa cell nuclear extract); Boc-Lys(Ac)-AMC substrate; trypsin; EDTA; Trichostatin A (TSA, 0.3 mM, soluble in dimethyl sulfoxide); 96-well black microplate; glycerol; ultrapure water

[0169] 2. Method:

[0170] Buffer preparation:

[0171] The formula is 15mM Tris-HCl (pH=8.0), 250μM EDTA, 250mM NaCl, and 10% glycerol.

[0172] Preparation of Trypsin solution:

[0173] The formulation is 10 mg / mL pancreatin, buffer, 2 μM TSA.

[0174] Preparation of substrate solution:

[0175] The substrate was dissolved in DMSO to prepare a 30 mM stock solution, which was then diluted to 300 μM with buffer.

[0176] The DMSO content is approximately 1%.

[0177] Diluting the enzyme solution:

[0178] Dilute the enzyme solution with a buffer at a ratio of 1:80.

[0179] Preparation of compound solutions:

[0180] Dilute the compounds (test compound and positive control SAHA) to a final concentration using a buffer.

[0181] Preparation and determination of 100% and blank:

[0182] Mix 50 μL HDAC buffer with 10 μL enzyme solution, add 40 μL substrate after 5 minutes, and react at 37 °C for 0.5 hours. Then add 100 μL Trypsin solution to terminate the reaction. After reacting at 37 °C for 20 minutes, measure the fluorescence intensity at (390 nm / 460 nm) to obtain 100% absorption. Add 40 μL substrate to 60 μL buffer, react at 37 °C for 0.5 hours, add 100 μL Trypsin solution, and react at 37 °C for 20 minutes. Measure the fluorescence intensity at (390 nm / 460 nm) to obtain blank absorption.

[0183] Determination of the inhibitory activity of compounds against HDACs:

[0184] 50 μL of drug-containing buffer was mixed with 10 μL of enzyme solution and incubated for 5 minutes. Then, 40 μL of substrate was added and the reaction was carried out at 37°C for 0.5 hours. The reaction was then terminated by adding 100 μL of Trypsin solution and incubated at 37°C for 20 minutes. Fluorescence intensity was measured at (390 nm / 460 nm), and the inhibition rate and IC50 were calculated using GraphPad Prism software and formulas. 50 value.

[0185] (Table 1)

[0186] Table 1. HDAC-inhibiting activity of the target compounds

[0187]

[0188] a IC 50 The value is expressed as the mean of two independent measurements ± standard deviation.

[0189] Example 6: Inhibitory activity of the target compound against SHP2 in this invention

[0190] 1. Materials:

[0191] SHP2 WT Full-length protein; DiFMUP; black ELISA plate; pIRS-1 peptide; HEPES; NaCl; KCl; EDTA;

[0192] Tween20; DTT.

[0193] 2. Method:

[0194] Buffer preparation:

[0195] The formula consists of 60 mM HEPES (pH 7.5), 75 mM NaCl, 75 mM KCl, 1 mM EDTA, and 0.05% Tween 20.

[0196] 5mM DTT.

[0197] Compound inhibits SHP2 WT Activity determination:

[0198] Add 0.5 nM SHP2 to the black orifice plate. WT 1 μM of dityrosine phosphorylated peptide and different concentrations of the compound were incubated at room temperature for 30 minutes. Then, 10 μM DiFMUP was added and incubated for another 30 minutes (buffer: 60 mM HEPES pH 7.2, 75 mM NaCl, 75 mM KCl, 1 mM EDTA, 0.05% Tween-20, and 5 mM DTT). Finally, 5 μL of 50 μM bpV (Phen) (Sigma, #SML0889) solution was added to quench the reaction. The fluorescence intensity at 450 nm (λex = 350) was read using a multi-function reader, and the IC50 value was calculated using GraphPad Prism software. (Table 2)

[0199] Table 2. SHP2 inhibitory activity of the target compounds

[0200]

[0201] a IC 50 The value is expressed as the mean of two independent measurements ± standard deviation.

[0202] Example 7: Antiproliferative activity experiment of the target compound in this invention

[0203] 1. Materials:

[0204] Four cell lines were used: BxPC-3 (human pancreatic cancer cells), SW 1990 (human pancreatic cancer cells), AsPC-1 (human pancreatic cancer cells), MV4-11 (human acute monocytic leukemia cells), and KYSE520 (human esophageal squamous cell carcinoma cells). The cell lines were treated with 10% fetal bovine serum (Hyclone, USA) at a concentration of 2.5 g / L. -1 Trypsin (Gibco, USA), CCK-8, modified RPMI 1640 medium, DMEM medium (Hyclone, USA), positive control drug HDAC inhibitor SAHA, positive control drug SHP2 inhibitor (SHP099), 96-well plate.

[0205] 2. Method:

[0206] Cells were cultured using standard methods, and cells in logarithmic growth phase were collected for experiments. Logarithmically growing solid tumor cells were diluted to 4 × 10⁶ cells / mL using RPMI 1640 or DMEM medium containing 10% fetal bovine serum. 3 pcs·mL -1 (Hematologic malignancy cells diluted to 1×10) 4 pcs·mL -1 Afterwards, the cells were seeded into 96-well plates (100 μL per well), with no cells added as blank wells. The plates were then incubated at 37°C (5% CO2) for 8 hours. The target compound solution and positive control solution prepared with the culture medium were added, with the blank wells designated as 100% wells. After incubation at 37°C (5% CO2) for 72 hours, 20 μL of CCK-8 was added. Three hours later, the absorbance of each well was measured at 450 nm using a microplate reader, and the inhibition rate and IC50 were calculated. 50 Values. (Table 3)

[0207] Table 3. Antiproliferative activity of selected compounds against tumor cells

[0208]

[0209] a IC 50 The value is expressed as the mean of two independent measurements ± standard deviation.

[0210] Example 8: Study on the therapeutic effect of the compound of the present invention on human acute monocytic leukemia cells

[0211] 1. Materials:

[0212] Cell line: Human acute monocytic leukemia cells (MV4-11).

[0213] Experimental animals: Six-week-old female BALB / c nude mice.

[0214] 2. Method:

[0215] Approximately 2×10 7 One MV4-11 cell was resuspended in 50 μL of serum-free high-glucose medium and 50 μL of matrix gel, and subcutaneously implanted into the right axillary region of BALB / c nude mice. When the tumor volume reached approximately 100 mm², the cells were cultured. 3 Oral administration was initiated and continued for 20 consecutive days. The treatment group received 40 mg / kg / day, while the control group received an equal volume of saline. Tumor size and mouse weight were measured daily during the treatment period. Tumor volume was calculated using the formula: V(mm²) 3 = Length (mm) × Width 2 (mm) / 2; The formula for calculating the tumor growth inhibition value (TGI) is: TGI = (1 - average tumor weight in the treatment group / average tumor weight in the control group) × 100%.

[0216] 3. Experimental Results:

[0217] The results are as follows Figure 1 As shown, the tumor inhibition rates of the target compounds 8r and 8t were 44% and 64%, respectively, and their tumor inhibition effects were significantly better than those of the positive control drug. Among them, 8t had the best effect and was also better than the combination of positive control drugs.

Claims

1. A dual-target inhibitor of SHP2 / HDAC, characterized in that, Compounds having the general formula I structure or their pharmaceutically acceptable salts: In general formula I, R1 and R2 are each independently selected from hydrogen, fluorine, chlorine, and bromine; A represents pyrimidine or pyrazine; R3 is a hydrogen atom or a carbonyl group; X is one of the following linking groups: n＝0，1，2，3，4，5，6，7，8，9，10； R4 is a hydroxyl group or a 2-aminophenyl group; R5 is H.

2. The SHP2 / HDAC dual-target inhibitor as described in claim 1, characterized in that, One of the following: 8a: 8b: 8c: 8d: 8e: 8f: 8g: 8h: 8i: 8j: 8k： 8l： 8m: 8n: 8o: 8p: 8q： 8r： 8s： 8t: 8u: 8v： 3. A method for preparing an SHP2 / HDAC dual-target inhibitor as described in claim 2, comprising the following steps: Compounds 1 and 2 undergo a Suzuki coupling reaction to give intermediate 3, which is then protected by a Boc protecting group to give intermediate 4. Intermediate 4 undergoes a nucleophilic substitution reaction with tert-butyl (4-methylpiperidin-4-yl)carbamate to give intermediate 5, followed by removal of the Boc protecting group to give intermediate 6. Intermediate 6 is condensed with monomethyl esters of different carbon lengths or aryl carboxylic acid intermediates under HATU conditions and then ammonolyzed in a methanol solution of hydroxylamine to give target compounds 8a-8f, 8h-8m. Intermediate 6 undergoes a nucleophilic substitution reaction with different bromine-containing intermediates and then ammonolyzed in a methanol solution of hydroxylamine to give target compounds 8g, 8n-8t. Intermediate 6 is condensed with o-phenylenediamine under HATU conditions to give target compounds 8u and 8v. Synthesis Route 1: Reagents and reaction conditions: (a) DPPF palladium dichloride, K3PO4, 1,4-dioxane / water, 90℃, 12h; (b) di-tert-butyl carbonate anhydride, DMAP, dichloromethane, room temperature, 8h; (c) (4-methylpiperidin-4-yl) tert-butyl carbamate, DIPEA, DMF, 85℃, 8h; (d) 4M HCl / EtOAc, room temperature, 8h; (e) HATU, DIPEA, DMF, room temperature, 8h; (f) hydroxylamine in methanol solution, room temperature, 6h. Synthesis Route 2: Reagents and reaction conditions: (a) HATU, DIPEA, DMF, room temperature, 8 hours; (b) K2CO3, DMF, room temperature or 80°C, 8 hours; (c) methanol solution of hydroxylamine, room temperature, 6 hours; Synthesis Route 3: Reagents and reaction conditions: (a) HATU, DIPEA, DMF, room temperature, 8 hours; (b) 4M sodium hydroxide / water, 50℃, 1 hour.

4. A pharmaceutical composition comprising a therapeutically effective amount of one or more SHP2 / HDAC dual-target inhibitors as described in claim 1 or 2, or a pharmaceutically acceptable salt thereof, and pharmaceutical excipients or carriers.

5. Use of the SHP2 / HDAC dual-target inhibitor according to claim 1 or 2 in the preparation of SHP2 inhibitors.

6. Use of the SHP2 / HDAC dual-target inhibitor according to claim 1 or 2 in the preparation of histone deacetylase inhibitors.

7. Use of the SHP2 / HDAC dual-target inhibitor of claim 1 or 2 in the preparation of a medicament for treating tumor diseases related to the above-mentioned targets and the overactivation of the RAS signaling pathway.

8. The use according to claim 7, wherein the tumor is lung cancer, liver cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, lymphoma, esophageal cancer, gastrointestinal cancer, nasopharyngeal carcinoma, leukemia, glioma, prostate cancer, or myeloma.

9. The use according to claim 8, wherein the tumor is non-small cell lung cancer or a KRAS-mutated tumor.

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