FLT3-HDAC dual-target inhibitor, its preparation method and application

Abstract

This invention relates to FLT3-HDAC dual-target inhibitors, their preparation methods, and applications. The FLT3-HDAC dual-target inhibitors have the structure shown in Formula I. The compounds of this invention not only exhibit good FLT3 and HDAC enzyme inhibitory activity, but also demonstrate certain anti-proliferative activity against solid tumors and hematological malignancies in vitro, especially showing a strong anti-tumor effect against acute myeloid leukemia. These compounds, as dual-target anti-tumor drugs based on FLT3 and HDACs, have further research value.

Description

Technical Field

[0001] This invention relates to a dual-target inhibitor of FLT3-HDAC, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Acute myeloid leukemia (AML) is caused by abnormal proliferation, clonal differentiation, and differentiation of cells in the hematopoietic system, characterized by genetic heterogeneity. Despite significant improvements in AML treatment strategies, from intensive chemotherapy (cytarabine + anthracyclines) to targeted small molecule drugs, the prospects for AML treatment remain bleak due to drug resistance and disease relapse. FMS-like tyrosine kinase 3 (FLT3) mutations are closely associated with poor clinical outcomes and treatment resistance, particularly in relapsed / refractory AML (R / R AML) patients. Therefore, the combination of FLT3 inhibitors and other anti-AML drugs is a common strategy to address drug resistance, improve efficacy, and enhance overall survival (OS) in AML patients, such as dual and triple therapies. The former is represented by giretinib with 7+3 induction chemotherapy (NCT02236013), giretinib with azacitidine (NCT02752035), and giretinib with venetumab (NCT03625505); the latter includes the combination of giretinib, azacitidine, and venetumab (NCT05520567), and the combination of quezatidine, decitabine, and venetumab (NCT03661307). In recent years, related studies have reported that synergistic inhibition of FLT3 and histone deacetylase (HDAC) can enhance the therapeutic response to FLT3-mutant AML cells and reduce drug resistance. These results suggest that dual inhibition of FLT3 and HDAC holds promise as an effective strategy for treating FLT3-driven AML.

[0003] FLT3 is a receptor tyrosine kinase (RTK) that plays a crucial role in regulating multiple signal transduction cascades, including the Janus kinase (JAK) / STAT5, phosphatidylinositol 3-kinase (PI3K) / protein kinase B (AKT), and rat sarcoma (RAS) / mitogen-activated protein kinase (MAPK) pathways. AML patients carrying FLT3 mutations are more likely to relapse compared to those carrying wild-type FLT3 (FLT3-WT). Approximately 25% of adult AML patients carry FLT3 internal tandem repeat (FLT3-ITD) mutations, and 10% carry FLT3 tyrosine kinase domain (FLT3-TKD) mutations. Notably, treatment for FLT3-mutant relapsed / relapsed AML typically involves the use of FLT3 inhibitors such as midoshuin, quezzartinib, and gipritinib alone or in combination with other inhibitors. On the other hand, HDACs possess the ability to scavenge lysine acetyl groups from histone or non-histone substrates, and are subdivided into four branches based on intracellular localization and functional mechanisms: class I (HDAC1, 2, 3, 8), class IIa (HDAC4, 5, 7, 9), class IIb (HDAC6, 10), class III (Sirtuin1-7), and class IV (HDAC11). The catalytic activity of class I, IIa, and IV HDAC members is zinc ion dependent, while the catalytic activity of class III HDAC members is NAD+ dependent. + To date, five HDAC inhibitors have been approved by the FDA for clinical use in hematologic malignancies, including chidamide (CS055), romidesin (FK 228), vorita (SAHA), belistat (PDX101), and pabistata (LBH-589).

[0004] While the combination of FLT3 and HDAC inhibitors offers certain advantages in treating AML, potential adverse drug-drug interactions limit the realization of synergistic effects, such as complex dosing regimens and negative impacts on pharmacokinetics. In contrast, dual-target FLT3 / HDAC inhibitors can circumvent these potential drawbacks. However, currently reported dual-target FLT3 / HDAC inhibitors do not offer significant therapeutic advantages compared to single FLT3 inhibitors.

[0005] Therefore, developing a highly effective dual-target inhibitor of FLT3-HDAC is of significant clinical importance. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a dual-target inhibitor of FLT3-HDAC, its preparation method, and its applications. This invention is achieved through the following technical solution:

[0007] FLT3-HDAC dual-target inhibitor, or its pharmaceutically acceptable salt, or its stereoisomer, characterized in that the FLT3-HDAC dual-target inhibitor has the structure shown in Formula I:

[0008]

[0009] In Formula I, R1 is a hydroxyl group or a 2-aminophenyl group.

[0010] X is an aromatic heterocyclic ring or one of the following linking groups:

[0011]

[0012] A can be one of the following structures:

[0013]

[0014] R2 is a hydrogen atom or one of the following structures.

[0015]

[0016] According to a preferred embodiment of the present invention, in the FLT3-HDAC dual-target inhibitor formula I,

[0017] R1 is a hydroxyl group or a 2-aminophenyl group;

[0018] X is one of the following linking groups:

[0019]

[0020] A is piperazinyl or piperidin-4-ylpiperazinyl;

[0021] R2 is tetrahydropyranoamine.

[0022] FLT3-HDAC dual-target inhibitors are selected from one of the following compounds:

[0023] 6-Ethyl-3-((4-(4-(4-(5-(hydroxyamino)-5-oxopentanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11a);

[0024] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxohexanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11b);

[0025] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxoheptanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11c);

[0026] 6-Ethyl-3-((4-(4-(4-(8-(hydroxyamino)-8-oxooctanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11d);

[0027] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxohexyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11e);

[0028] 6-Ethyl-3-((4-(4-(4-(7-(hydroxyamino)-7-oxohepyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11f);

[0029] 6-Ethyl-3-((4-(4-(4-(8-(hydroxyamino)-8-oxooctyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11g);

[0030] 6-Ethyl-3-((4-(4-(4-(4-(hydroxycarbamoyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11h);

[0031] (E)-6-ethyl-3-((4-(4-(4-(4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11i);

[0032] (E)-6-ethyl-3-((4-(4-(4-((4-((4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)carbamoyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11j);

[0033] 2-(4-(1-(4-((3-carbamoyl-5-ethyl-6((tetrahydro-2H-pyran-4-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)N-hydroxypyrimidine-5-carboxamide (14);

[0034] 3-((4-(4-(4-(((2-aminophenyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-6-ethyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (17a);

[0035] N-(2-aminophenyl)-2-(4-(1-(4-((3-carbamoyl-5-ethyl-6((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperidin-1-yl)pyrimidin-5-carboxamide (17b);

[0036] 6-Ethyl-3-((4-(4-(7-(hydroxyamino)-7-oxoheptanoyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25a);

[0037] 6-Ethyl-3-((4-(4-(8-(hydroxyamino)-8-oxooctanoyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25b);

[0038] 6-Ethyl-3-((4-(4-(3-(hydroxyamino)-3-oxopropyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25c);

[0039] 6-Ethyl-3-((4-(4-(4-(hydroxyamino)-4-oxobutyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25d);

[0040] 6-Ethyl-3-((4-(4-(5-(hydroxyamino)-5-oxopentyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25e);

[0041] 6-Ethyl-3-((4-(4-(6-(hydroxyamino)-6-oxohexyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25f);

[0042] 6-Ethyl-3-((4-(4-(7-(hydroxyamino)-7-oxoheptyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25g);

[0043] 6-Ethyl-3-((4-(4-(8-(hydroxyamino)-8-oxooctyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25h);

[0044] (E)-6-ethyl-3-((4-(4-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25i);

[0045] (E)-6-ethyl-3-((4-(4-(4-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)amino)-2-oxobutyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25j);

[0046] (E)-6-ethyl-3-((4-(4-(2-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25k);

[0047] (E)-6-ethyl-3-((4-(4-(3-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)propyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25l);

[0048] (E)-6-ethyl-3-((4-(4-(4-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)butyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25m);

[0049] 2-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25n);

[0050] 2-((3-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)propyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (25o);

[0051] 2-(4-((4-((3-carbamoyl-5-ethyl-6-(((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25p).

[0052] 2-(4-((4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25q)

[0053] A second objective of this invention is to provide a method for preparing a dual-target inhibitor of FLT3-HDAC, comprising the following steps:

[0054] Compound 1 reacts with 4-aminotetrahydropyran to give compound 2. Simultaneously, compounds 3 and 18 react with 1-fluoro-2-methoxy-4-nitrobenzene to give compounds 4 and 19. Compounds 4 and 19 are further reduced to compounds 5 and 20. Compound 2 reacts with either compound 5 or 20 via a Buchwald-Hartwig coupling reaction to give compounds 6 and 21. Compounds 6 and 21 are oxidized and deprotected by the Boc protecting group to give compounds 8 and 23. Compounds 8 and 23 continue to react with 9a-u, 12, 15a, and 15b to generate 10a-j, 13, 16a, 16b, and 24a-q. The ester groups of compounds 10a-j and 24a-q can be converted to hydroxamic acid groups in an alkaline hydroxylamine solution to give target compounds 11a-j and 25a-q. Compounds 13, 16a, and 16b are deprotected in an acidic environment to give target compounds 14, 17a, and 17b.

[0055] A method for preparing an FLT3-HDAC dual-target inhibitor, wherein the preparation method comprises any of the following methods:

[0056] The preparation methods of compounds 11a-11j are as follows:

[0057]

[0058] Reagents and conditions: (a) 4-aminotetrahydropyran, triethylamine, acetonitrile, 80°C, 6 hours; (b) 1-fluoro-2-methoxy-4-nitrobenzene, potassium carbonate, N,N-dimethylformamide, 100°C, 8 hours; (c) 10% Pd / C, tetrahydrofuran, room temperature, 24 hours; (d) palladium acetate, 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene, cesium carbonate, 1,4-dioxane, 100°C, 12 hours; (e) 30% hydrogen peroxide, potassium carbonate, dimethyl sulfoxide, room temperature, 12 hours; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (g) 1) 9a-d, EDCI, DMAP, room temperature, 8 hours; 2) 9e-j, potassium carbonate, acetonitrile, 80°C, 6 hours; (h) NH₂OH, KOH, anhydrous methanol, room temperature, 4 hours;

[0059] The preparation methods for compounds 14, 17a, and 17b are as follows:

[0060]

[0061] Reagents and conditions: (a) 12, triethylamine, acetonitrile, 80°C, 8 hours; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (c) 15a and 15b, triethylamine, acetonitrile, 80°C, 8 hours; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours.

[0062] The preparation method of compound 25a-q is as follows:

[0063]

[0064] Reagents and conditions: (a) 1-fluoro-2-methoxy-4-nitrobenzene, potassium carbonate, N,N-dimethylformamide, 100°C, 8 h; (b) 10% Pd / C, tetrahydrofuran, room temperature, 24 h; (c) 2, palladium acetate, 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene, cesium carbonate, 1,4-dioxane, 100°C, 12 h; (d) 30% hydrogen peroxide, potassium carbonate, dimethyl sulfoxide, room temperature, 12 h; (e) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (f) 1) 9c and 9d, EDCI, DMAP, room temperature, 8 h; 2) 9e-u, potassium carbonate, acetonitrile, 80°C, 6 h; (g) NH2OH, KOH, anhydrous methanol, room temperature, 4 h.

[0065] A pharmaceutical composition comprising a therapeutically effective amount of the aforementioned FLT3-HDAC dual-target inhibitor and pharmaceutically acceptable excipients.

[0066] The application of the FLT3-HDAC dual-target inhibitor in the preparation of drugs for treating target-related tumor diseases.

[0067] The tumors mentioned include lung cancer, liver cancer, non-small cell lung cancer, colon cancer, skin cancer, pancreatic cancer, ovarian cancer, breast cancer, bladder cancer, lymphoma, esophageal cancer, gastrointestinal cancer, nasopharyngeal carcinoma, leukemia, and myeloma.

[0068] Beneficial effects

[0069] 1. The compounds of the present invention not only exhibit good FLT3 and HDAC enzyme inhibitory activities, but also inhibit FLT3 and HDAC activities and downregulate ERK phosphorylation levels in cells, and have strong in vitro antitumor activity.

[0070] 2. Based on the potent FLT3 inhibitor gipritinib, this invention evaluated the effects of gipritinib, SAHA, and their combination on the viability of AML cells carrying FLT3 mutations. A more effective FLT3 / HDAC dual-target inhibitor was successfully developed. This is the first reported dual-target anti-tumor drug based on DNMT1 and HDAC, and its tumor-suppressing effect is significantly better than the combination of gipritinib and SAHA. Attached Figure Description

[0071] Figure 1 To assess the in vivo antitumor activity of MOLM-13 xenografted into BALB / c nude mice;

[0072] Where A represents the change in tumor volume in mice during the treatment period, B represents the tumor volume in mice after the treatment period, and C represents the tumor weight in mice after the treatment period. Detailed Implementation

[0073] 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.

[0074] Example 1: Preparation of Intermediate 8

[0075] 3-Bromo-6-ethyl-5-((tetrahydro-2H-pyran-4-group)amino)pyrazine-2-carboxamide (2)

[0076] 4-Aminotetrahydropyran (1.64 g, 16.23 mmol) was added to a 50 mL acetonitrile solution of compound 1 (4 g, 16.23 mmol) and triethylamine (4.92 g, 48.69 mmol), and the reaction was carried out at 80 °C for 6 h. The solvent was then removed by rotary evaporation under reduced pressure, and the residue was washed with 100 mL of water. The aqueous phase was then extracted with ethyl acetate (30 mL × 3). The separated organic phase was dried over anhydrous Na₂SO₄ and concentrated to give the crude product. The crude product was purified by silica gel column chromatography to give compound 2 in 89.4% yield. 1 H NMR (400MHz, DMSO) δ7.67 (d, J=7.7Hz, 1H), 4.18–4.05 (m, 1H), 3.94–3.86 (m, 2H), 3.46–3.37 (m,2H),2.64(q,J=7.3Hz,2H),1.82–1.74(m,2H),1.69–1.58(m,2H),1.17(t,J=7.3Hz,3H).

[0077] tert-Butyl-4-(1-(2-methoxy-4-nitrophenyl)piperidin-4-yl)piperazine-1-carboxylate (4)

[0078] 1-Fluoro-2-methoxy-4-nitrobenzene (2.1 g, 10.23 mmol) was added to a DMF solution (25 mL) of compound 3 (2.76 g, 10.23 mmol) and potassium carbonate (2.83 g, 20.45 mmol), and the reaction was carried out at 100 °C for 8 hours. The reaction solution was then diluted with 250 mL of water, followed by extraction of the aqueous phase with ethyl acetate (80 mL × 3). The separated organic phase was dried over anhydrous Na₂SO₄ and concentrated to give the crude product. The crude product was purified by silica gel column chromatography to give compound 4 in 92.8% yield. The synthesis of compound 19 followed the same procedure. 1 H NMR (400MHz, DMSO) δ7.96(s,1H),7.82(dd,J=8.9,2.6Hz,1H),7.68(d,J=2.6Hz,1H),7.01(d,J=8.9Hz,1H),3.91(s,3H),3.71(d, J=12.0Hz,2H),3.30(d,J=5.4Hz,2H),2.74(s,4H),2.46(t,J=5.0Hz,5H),1.83(d,J=12.1Hz,2H),1.60–1.52(m,2H),1.40(s,9H).

[0079] tert-Butyl-4-(1-(4-amino-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate (5)

[0080] 10% Pd / C was added to a THF solution of compound 4, and the reaction was carried out at room temperature for 24 hours under a hydrogen atmosphere. The reaction solution was then filtered with diatomaceous earth, and the filtrate was collected, concentrated, and the crude product was obtained. The crude product was purified by silica gel column chromatography to obtain compound 5 in 91.7% yield. The synthesis of compound 20 was carried out using the same procedure. 1 H NMR (400MHz, DMSO) δ7.95(s,1H),6.60(d,J=8.4Hz,1H),6.21(d,J=2.3Hz,1H),6.05(dd,J=8.3,2.3Hz,1H),4.67(s,2H), 3.67(s,3H),3.29(s,2H),3.14(d,J=11.0Hz,2H),2.46–2.41(m,5H),1.77–1.69(m,2H),1.56–1.47(m,2H),1.39(s,9H).

[0081] tert-Butyl-4-(1-(4-((3-cyano-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate (6)

[0082] Compound 2 was added to an anhydrous 1,4-dioxane solution (50 mL) of compound 5, palladium acetate, (9,9-dimethyl-9H-oxanthracene-4,5-diyl)bis(diphenylphosphine), and cesium carbonate. The reaction mixture was heated to 100 °C for 12 hours under nitrogen protection. The reaction solution was then diluted with 200 mL of water, followed by extraction of the aqueous phase with ethyl acetate (80 mL × 3). The separated organic phase was dried over anhydrous Na₂SO₄ and concentrated to give the crude product. The crude product was purified by silica gel column chromatography to give compound 6 in 64.3% yield. The synthesis of compound 21 followed the same procedure. 1 H NMR (400MHz, DMSO) δ8.60(s,1H),7.15(dd,J=8.5,,2.3Hz,1H),3.75(s,2H),3.24(s,2H),2.60–2.52(m,7H),2.45(d, J=5.4Hz,3H),2.33(s,1H),1.76(d,J=12.0Hz,4H),1.57(dd,J=16.0,7.9Hz,4H),1.40(s,9H),1.12(t,J=7.4Hz,3H).

[0083] tert-Butyl-4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazine-1-carboxylate (7)

[0084] In a solution of compound 6 (1 g, 1.61 mmol) and potassium carbonate (555 mg, 4.03 mmol) in dimethyl sulfoxide, 30% hydrogen peroxide (1 g, 3.21 mmol) was slowly added dropwise. After the addition was complete, the reaction was carried out at room temperature for 12 hours. The mixture was then diluted with 150 mL of water and the aqueous phase was extracted with ethyl acetate (40 mL × 3). The organic phase was dried over anhydrous magnesium sulfate and concentrated to give the crude product, which was then purified by silica gel column chromatography to give compound 7. The synthesis of compound 21 was carried out using the same procedure. A bright yellow solid was obtained in 52.4% yield. 1 HNMR (400MHz, DMSO) δ11.02 (s, 1H), 7.53 (s, 1H), 7.30–7.20 (m, 2H), 7.02 (d, J = 2. 3Hz,1H),6.80(dd,J=15.9,8.1Hz,2H),4.10(s,1H),3.93(d,J=11.1Hz,2H),3.81 (s,3H),3.39(s,2H),2.62–2.54(m,6H),2.46(d,J=5.5Hz,4H),2.33(s,1H),1.82 (dd,J=24.7,12.3Hz,4H),1.65–1.57(m,4H),1.40(s,9H),1.19(t,J=7.3Hz,3H).

[0085] 4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-ammonium salt(8)

[0086] Compound 7 (498 mg, 0.78 mmol) was added to a 4 M HCl / EtOAc solution, and the reaction was stirred at room temperature for 12 hours. The reaction mixture was then filtered, and the filter cake was recrystallized from ethyl acetate to give compound 8. Compound 21 was synthesized using the same procedure. It was a yellow solid in 88.9% yield. 1H NMR(400MHz,DMSO)δ11.46(s,1H),10.16(s,2H),9.83(s,2H),7.62(s,2H),7.50– 7.33(m,1H),7.24(d,J=1.9Hz,1H),6.98(s,1H),4.13(s,1H),4.00(s,3H),3.94(d d,J=12.2,8.3Hz,2H),3.68(s,4H),3.55(s,4H),3.38(dd,J=12.7,10.6Hz,2H),2 .62(q,J=7.3Hz,2H),1.92–1.82(m,2H),1.75–1.65(m,2H),1.21(t,J=7.3Hz,3H).

[0087] Example 2: Preparation of target compounds 11a-j and 25a-i

[0088] Methyl 5-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-5-pentanoate (10a)

[0089] Compound 9a (49 mg, 0.33 mmol) was added to a 15 mL solution of compound 8 (171 mg, 0.28 mmol), EDCI (69 mg, 0.36 mmol), and DMAP (103 mg, 0.84 mmol) in dichloromethane. After addition, the reaction mixture was allowed to react at room temperature for 8 hours. Afterward, the reaction solution was washed sequentially with saturated sodium bicarbonate and saturated sodium chloride solutions to separate the organic phase. The organic phase was dried over anhydrous magnesium sulfate and concentrated to give the crude product, which was then purified by silica gel column chromatography to give compound 10a. The synthesis of compounds 10b-d, 24a, and 24b followed the same procedure. The product was a yellow solid with a yield of 91.4%. 1 H NMR (400MHz, DMSO) δ11.02 (s, 1H), 7.54 (d, J = 3.3Hz, 1H), 7.30–7.21 (m, 2H), 7.0 2(d,J=2.4Hz,1H),6.81(t,J=8.7Hz,2H),4.12(s,1H),3.94(s,2H),3.81(s,3H), 3.59(s,3H),3.44–3.39(m,6H),2.58(d,J=7.4Hz,2H),2.45(d,J=5.3Hz,2H),2. 34(t,J=7.5Hz,4H),1.85–1.76(m,4H),1.71–1.51(m,6H),1.19(t,J=7.4Hz,3H).

[0090] Methyl 6-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-6-hexanoate (10b)

[0091] Yellow solid, yield 84.2%. 1 H NMR (400MHz, DMSO) δ11.02 (s, 1H), 7.53 (d, J = 3.2Hz, 1H), 7.33–7.20 (m, 2H), 7.02 (d,J=2.3Hz,1H),6.85–6.76(m,2H),4.11(s,1H),3.92(d,J=11.3Hz,2H),3.81(s ,3H),3.58(s,3H),3.47–3.37(m,6H),2.59(t,J=7.2Hz,2H),2.47–2.42(m,2H),2 .32(t,J=7.2Hz,4H),1.86–1.75(m,4H),1.69–1.47(m,8H),1.19(t,J=7.3Hz,3H).

[0092] 7-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-6-heptanoic acid ethyl ester (10c)

[0093] Yellow solid, yield 73.5%. 1 H NMR (400MHz, DMSO) δ11.02(s,1H),7.53(s,1H),7.30–7.21(m,2H),7.02(d,J=2.3Hz,1H),6.85–6.76(m,2H),4.04(q,J=7.1Hz,4H),3.92(d,J=10.9 Hz,2H),3.81(s,3H),3.42(s,6H),2.57(t,J=7.4Hz,2H),2.46(s,2H),2.3 7–2.28(m,4H),1.85–1.73(m,4H),1.69–1.43(m,10H),1.25–1.16(m,5H).

[0094] 8-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-6-octanoic acid methyl ester (10d)

[0095] Yellow solid, yield 71.2%.1 H NMR (400MHz, DMSO) δ11.02(s,1H),7.53(s,1H),7.30–7.22(m,2H),7.02(d,J=2.3Hz, 1H),6.81(t,J=8.8Hz,2H),4.11(d,J=7.4Hz,1H),3.92(d,J=11.1Hz,2H),3.81(s,3H) ,3.58(s,3H),3.42(s,6H),2.61–2.56(m,2H),2.29(dd,J=8.5,6.2Hz,4H),1.87–1.76 (m,4H),1.66–1.55(m,4H),1.50(q,J=8.1Hz,6H),1.26(s,2H),1.19(t,J=7.3Hz,3H).

[0096] 6-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl hexanoate (10e)

[0097] Compound 9e (194 mg, 0.93 mmol) was added to an acetonitrile solution of compound 8 (281 mg, 0.46 mmol) and triethylamine (85 mg, 0.84 mmol). After addition, the reaction was carried out at 80 °C for 8 hours. The solvent was removed by evaporation under reduced pressure to obtain the crude product, which was purified by silica gel column chromatography to obtain compound 10e. The synthesis of compounds 10f-j, 13, 16a, 16b, 24c-i, 27a-c, 30a-f, 36a-c, 41, and 43 was carried out using the same procedure.

[0098] Yellow solid, yield 74.4%. 1 H NMR (400MHz, DMSO) δ11.02(s,1H),7.53(s,1H),7.30–7.21(m,2H),7.02(d,J=2.3Hz,1H) ,6.81(t,J=8.4Hz,2H),4.11(s,1H),3.92(d,J=10.6Hz,2H),3.81(s,3H),3.58(s,3H),3 .39(s,2H),2.61–2.57(m,2H),2.37–2.27(m,6H),1.85(d,J=14.1Hz,4H),1.63(d,J=8.3 Hz,2H),1.52(q,J=7.7Hz,4H),1.41(s,2H),1.25(d,J=8.4Hz,2H),1.19(t,J=7.4Hz,3H).

[0099] 7-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl heptanoate (10f)

[0100] Yellow solid, yield 83%. 1 H NMR (400MHz, DMSO) δ11.02(s,1H),7.53(s,1H),7.30–7.20(m,2H),7.02(d,J=2.3Hz ,1H),6.80(dd,J=13.1,8.1Hz,2H),4.11(d,J=7.5Hz,1H),3.92(d,J=11.0Hz,2H),3 .81(s,3H),3.58(s,3H),3.37(d,J=9.8Hz,2H),2.58(q,J=7.4Hz,8H),2.38–2.22(m ,6H),1.85(d,J=13.5Hz,4H),1.70–1.46(m,8H),1.27(s,4H),1.19(t,J=7.3Hz,3H).

[0101] 8-(4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperidin-1-yl)methyl octanoate (10g)

[0102] Yellow solid, yield 67.7%. 1 H NMR (400MHz, DMSO) δ11.03 (s, 1H), 7.54 (d, J = 3.1Hz, 1H), 7.31–7.21 (m, 2H), 7.03 (d,J=2.3Hz,1H),6.82(t,J=8.3Hz,2H),3.93(d,J=11.1Hz,2H),3.81(s,3H),3.58 (s,3H),3.39(s,2H),2.59(s,2H),2.29(dd,J=9.4,5.4Hz,6H),1.85(d,J=12.7Hz, 4H),1.69–1.60(m,4H),1.53(d,J=7.2Hz,4H),1.27(s,6H),1.19(t,J=7.3Hz,3H).

[0103] 4-((4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl benzoate (10h)

[0104] Yellow solid, yield 73.1%. 1 H NMR (400MHz, DMSO) δ7.95(d,J=7.9Hz,2H),7.56–7.45(m,3H),7.32–7.21(m,2H),6.82(dd,J=13.0,8.1Hz,2H),4.11(q,J=5.3Hz,2H),3.9 2(d,J=11.2Hz,2H),3.85(s,2H),3.81(s,2H),3.38(s,2H),2.59(q,J=7.3Hz,2H),1.85(d,J=13.1Hz,4H),1.67–1.59(m,4H),1.20(s,3H).

[0105] (E)-3-(4-((4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl)phenyl)methyl acrylate (10i)

[0106] Yellow solid, yield 75.8%. 1 H NMR(400MHz,DMSO)δ11.04(s,1H),7.74–7.62(m,3H),7.54(s,1H),7.38(s,2H),7 .31–7.20(m,2H),7.03(d,J=2.3Hz,1H),6.81(dd,J=12.4,8.1Hz,2H),6.64(d,J=1 5.9Hz,1H),4.12(s,1H),3.93(s,2H),3.81(s,3H),3.73(s,3H),3.60(s,2H),3.3 8(s,2H),2.59(t,J=7.4Hz,4H),1.85(d,J=13.3Hz,4H),1.65(s,4H),1.20(s,3H).

[0107] (E)-3-(4-((4-((4-(1-(4-((3-carbamoyl-5-ethyl-6-(((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl)benzamido)methyl)phenyl)methyl)methacrylate(10j)

[0108] Yellow solid, yield 50.9%. 1H NMR (400MHz, DMSO) δ11.04(s,1H),9.09(s,1H),7.88(d,J=7.8Hz,2H),7.73–7.61(m,4H),7.55(s,1H), 7.43(s,2H),7.36(d,J=8.0Hz,2H),7.31–7.22(m,2H),7.03(d,J=2.2Hz,1H),6.82(t,J=8.1Hz,2H),6.6 2(d,J=16.1Hz,1H),4.50(d,J=5.9Hz,2H),4.12(q,J=5.2Hz,2H),3.92(d,J=9.0Hz,2H),3.81(s,3H),3 .72(s,3H),3.39(s,2H),2.58(d,J=7.4Hz,4H),1.85(d,J=12.7Hz,4H),1.73–1.55(m,4H),1.18(s,3H).

[0109] 6-Ethyl-3-((4-(4-(4-(5-(hydroxyamino)-5-oxopentanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11a)

[0110] Potassium hydroxide (3.3 g, 50 mmol) was added to an anhydrous methanol solution containing hydroxylamine hydrochloride (2.35 g, 33.5 mmol). After the addition was complete, the reaction was carried out at 0 °C for 0.5 h. The reaction solution was then filtered, and the filtrate was collected. The filtrate was added dropwise to an anhydrous methanol solution containing compound 10a (166 mg, 0.25 mmol). After the addition was complete, the reaction was carried out at room temperature for 4 h. After the reaction was complete, the solvent was removed by evaporation under reduced pressure. The resulting residue was dissolved in 5 mL of water and the pH was adjusted to approximately 7 with 1 M hydrochloric acid. A solid precipitated, which was filtered, and the filter cake was collected. The latter was recrystallized from anhydrous ethanol to give compound 11a. The synthesis of compounds 11b-j, 25a-i, 28a-c, 31a-f, 37a-c, and 42 was carried out using the same procedure. Yellow solid, yield 63.8%, mp 190-192 °C. 1H NMR (400MHz, DMSO) δ11.03(s,1H),10.37(s,1H),8.69(s,1H),7.54(d,J=3.3Hz,1H),7.33– 7.19(m,2H),7.02(d,J=2.3Hz,1H),6.81(t,J=7.3Hz,2H),4.17–4.05(m,1H),3.95–3.86(m ,2H),3.81(s,3H),3.46–3.38(m,4H),2.58(d,J=7.3Hz,2H),2.47–2.43(m,2H),2.33–2.26 (m,3H),1.99(t,J=7.3Hz,2H),1.88–1.76(m,4H),1.74–1.51(m,6H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.45,170.12,169.38,152.74,152.21,150.61,136.90,135.81,131.97,118.73,112.12,105.25,66.91 ,61.58,56.16,50.98,49.53,49.03,48.04,45.75,41.86,32.64,32.06,28.64,24.67,21.38,11.42.HRMS-ESI(m / z)calc.for C 33 H 49 N9O6[M+H] + 668.3833,found 668.3829.HPLC t R = 6.80 min (98.66% purity).

[0111] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxohexanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11b)

[0112] Yellow solid, yield 53.3%, mp 196-198℃. 1H NMR(400MHz,DMSO)δ11.03(s,1H),10.36(s,1H),8.70(s,1H),7.62–7.47(m,1H),7.35–7.21 (m,2H),7.02(d,J=2.3Hz,1H),6.81(t,J=7.9Hz,2H),4.18–4.06(m,1H),3.97–3.88(m,2H), 3.81(s,3H),3.42(q,J=8.5Hz,4H),2.58(q,J=7.3Hz,2H),2.47–2.42(m,2H),2.29(q,J=7.3 Hz,3H),1.95(t,J=6.8Hz,2H),1.90–1.74(m,4H),1.70–1.41(m,8H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.76,170.12,169.45,152.75,152.21,150.61,136.91,135.81,131.96,118.74,112.14,112.08,105.28,66.91,61 .58,56.17,50.98,49.58,49.07,48.04,45.84,41.87,32.65,32.59,32.40,28.66,25.36,24.86,24.66,11.42.HRMS-ESI(m / z)calc.forC 34 H 51 N9O6[M+H] + 682.3989,found 682.3985.HPLC t R = 8.76 min (97.28% purity).

[0113] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxoheptanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11c)

[0114] Yellow solid, yield 33.3%, mp 185-187℃. 1H NMR (400MHz, DMSO) δ11.03(s,1H),10.34(s,1H),8.68(s,1H),7.54(d,J=3.2Hz,1H),7.32–7 .20(m,2H),7.02(d,J=2.3Hz,1H),6.81(t,J=8.2Hz,2H),4.16–4.06(m,1H),3.92(d,J=10.9H z,2H),3.81(s,3H),3.47–3.38(m,4H),2.61–2.56(m,2H),2.46(d,J=5.6Hz,2H),2.29(q,J= 11.0Hz,3H),1.93(t,J=7.3Hz,2H),1.89–1.75(m,4H),1.69–1.42(m,9H),1.33–1.15(m,5H). 13 C NMR (101MHz, DMSO) δ170.82,170.12,169.52,152.74,152.21,150.61,136.90,135.81,131.95,118.72,112.12,112.06,105.25,66.91,61.57 ,56.16,50.98,49.57,49.06,48.04,45.83,41.85,32.68,32.65,32.60,28.85,28.65,25.48,25.02,24.67,11.42.HRMS-ESI(m / z)calc.forC 35 H 53 N9O6[M+H] + 696.4147, found 696.4142. HPLC t R = 8.14 min (97.26% purity).

[0115] 6-Ethyl-3-((4-(4-(4-(8-(hydroxyamino)-8-oxooctanoyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11d)

[0116] Yellow solid, yield 38.2%, mp 190-192℃. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.32(s,1H),8.64(s,1H),7.52(d,J=3.2Hz,1H),7.29–7.18(m,2H),7.03 (d,J=2.4Hz,1H),6.80(dd,J=17.8,8.1Hz,2H),4.17–4.06(m,1H),3.96–3.88(m,2H),3.81(s,3H),3.39(s,4 H),3.36(d,J=2.1Hz,2H),2.58(q,J=7.3Hz,2H),2.45(d,J=11.7Hz,2H),2.35–2.24(m,3H),1.93(t,J=7.4Hz ,2H),1.88–1.76(m,4H),1.66–1.58(m,9H),1.53–1.39(m,4H),1.25(d,J=6.8Hz,4H),1.19(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO) δ170.82,170.12,169.52,152.74,152.21,150.61,136.90,135.81,131.95,118.72,112.12,112.06,105.25,66.91,61.5 7,56.16,50.98,49.57,49.06,48.04,45.83,41.85,32.68,32.65,32.60,28.85,28.65,25.48,25.02,24.67,11.42.HRMS-ESI(m / z)calc.for C 36 H 55 N9O6[M+H] + 710.4298,found 710.4293.HPLC t R = 6.28 min (95.77% purity).

[0117] 6-Ethyl-3-((4-(4-(4-(6-(hydroxyamino)-6-oxohexyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11e)

[0118] Pale red solid, yield 22%, mp 198-200℃. 1H NMR(400MHz,DMSO)δ11.03(s,1H),10.35(s,1H),7.53(s,1H),7.34–7.15(m,2H),7.04(s,1H), 6.80(t,J=8.4Hz,2H),4.12(d,J=10.0Hz,1H),3.93(d,J=11.0Hz,2H),3.81(s,3H),3.40–3.33 (m,2H),2.59(q,J=7.4Hz,2H),2.46(d,J=10.0Hz,4H),2.34(s,2H),2.22(q,J=9.2Hz,4H),1.9 8–1.77(m,6H),1.69–1.60(m,2H),1.54–1.46(m,4H),1.38(q,J=7.5Hz,2H),1.25–1.16(m,5H). 13 CNMR(101MHz,DMSO)δ170.12,169.53,152.74,152.21,150.62,136.97,135.78,131.93,118.69,112.13,112.10,105.33,66.91,61.55,58.38,5 8.29,56.18,53.78,50.97,49.12,48.04,46.13,32.73,32.66,28.84,27.04,26.53,25.58,24.67,11.83,11.41,8.30.HRMS-ESI(m / z)calc.for C 34 H 53 N9O5[M+H] + 668.4198, found 668.4193. HPLC t R = 10.63 min (96.46% purity).

[0119] 6-Ethyl-3-((4-(4-(4-(7-(hydroxyamino)-7-oxoheptyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11f)

[0120] Pale yellow solid, yield 30%, mp 183-185℃. 1H NMR (400MHz, DMSO) δ11.04(s,1H),10.42(s,1H),7.54(d,J=3.3Hz,1H),7.33–7.21(m,2H) ,7.03(d,J=2.3Hz,1H),6.83(dd,J=8.1,5.4Hz,2H),4.15–4.06(m,1H),3.92(dd,J=10.3, 4.1Hz,2H),3.82(s,3H),3.38(d,J=11.3Hz,2H),2.86(s,8H),2.64–2.56(m,4H),1.98–1. 82(m,6H),1.70–1.61(m,4H),1.57–1.45(m,4H),1.29–1.23(m,4H),1.19(t,J=6.6Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.53,152.72,152.21,150.59,136.55,135.95,132.02,118.78,112.10,111.99,105.13,66.91,61.65,60 .23,57.04,56.13,51.75,50.60,48.04,34.08,32.63,28.77,28.18,26.74,25.45,24.69,21.24,14.56,11.43.HRMS-ESI(m / z)calc.for C 35 H 55 N9O5[M+H] + 682.4356,found 682.4351.HPLC t R = 8.62 min (99.25% purity).

[0121] 6-Ethyl-3-((4-(4-(4-(8-(hydroxyamino)-8-oxooctyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11g)

[0122] Pale yellow solid, yield 31.7%, mp 198-200℃. 1H NMR (400MHz, DMSO) δ11.03(s,1H),10.34(s,1H),8.75(s,1H),7.53(s,1H),7.34–7.16(m,2H),7.04(s ,1H),6.80(t,J=8.9Hz,2H),4.19–4.08(m,1H),3.93(d,J=11.0Hz,2H),3.81(s,3H),3.38(d,J=11.5H z,2H),2.59(q,J=7.4Hz,2H),2.46(d,J=11.3Hz,4H),2.33(s,2H),2.28–2.13(m,4H),1.94(t,J=7.3H z,2H),1.90–1.74(m,4H),1.64(d,J=14.2Hz,2H),1.58–1.44(m,4H),1.38(s,2H),1.28–1.16(m,9H). 13 C NMR (101MHz, DMSO) δ170.12,169.57,152.74,152.21,150.62,136.97,135. 78,131.91,118.68,112.14,112.09,105.32,66.91,61.55,60.22,58.48,5 6.18,53.80,50.98,49.15,48.04,32.74,32.66,29.17,29.05,28.85,27.3 6,26.79,25.57,24.67,21.23,14.56,11.41,8.36.HRMS-ESI(m / z)calc.for C 36 H 57 N9O5[M+H] + 696.4147,found 696.4142.HPLC t R = 6.94 min (99.58% purity).

[0123] 6-Ethyl-3-((4-(4-(4-(4-(hydroxycarbamoyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11h)

[0124] Pale yellow solid, yield 35.3%, mp 218-220℃. 1H NMR (400MHz, DMSO) δ11.18(s,1H),11.02(s,1H),8.99(s,1H),7.72(d,J=7.7Hz,2H),7.52(s,1H) ),7.38(d,J=7.7Hz,2H),7.26(d,J=8.6Hz,1H),7.23–7.18(m,1H),7.03(d,J=2.3Hz,1H),6.80(d d,J=16.2,8.1Hz,2H),4.12(s,1H),3.92(d,J=11.0Hz,2H),3.81(s,3H),3.53(s,2H),3.37(d,J= 11.5Hz,2H),2.59(q,J=7.4Hz,2H),2.00–1.74(m,4H),1.74–1.49(m,4H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.12,152.77,152.23,150.62,135.96,132.04,129.19,127.34,118.83,112.17,112.11 ,105.30,66.90,56.19,50.65,48.86,48.05,45.79,32.65,29.48,24.68,11.43,8.98.HRMS-ESI(m / z)calc.for C 36 H 49 N9O5[M+H] + 668.3881,found 668.3875.HPLC t R = 7.48 min (95.18% purity).

[0125] (E)-6-ethyl-3-((4-(4-(4-(4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11i)

[0126] Yellow solid, yield 30%, mp 220-222℃. 1H NMR (400MHz, DMSO) δ11.01(s,1H),10.76(s,1H),9.03(s,1H),7.51(d,J=7.7Hz,3H),7.33(d,J =7.8Hz,2H),7.28–7.18(m,2H),7.03(s,1H),6.79(dd,J=17.5,8.0Hz,2H),6.46(d,J=15.7Hz, 1H),4.11(d,J=9.3Hz,1H),3.92(d,J=10.0Hz,2H),3.80(s,3H),3.47(s,2H),2.63–2.52(m,6H ),2.39(s,2H),2.29(s,1H),1.83(t,J=14.0Hz,4H),1.71–1.51(m,4H),1.19(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO)δ170.13,163.23,152.72,152.21,150.61,140.15,138.54 ,136.82,135.84,134.06,131.96,129.84,127.82,119.13,118.71,112.11,1 12.07,105.25,66.91,62.09,61.65,60.23,56.15,53.11,50.85,48.98,48.0 4,32.64,28.57,24.68,21.22,14.55,11.62,11.40.HRMS-ESI(m / z)calc.for C 38 H 51 N9O5[M+H] - 714.4089, found 714.4093.HPLCt R = 7.63 min (97.72% purity).

[0127] (E)-6-ethyl-3-((4-(4-(4-((4-((3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)carbamoyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (11j)

[0128] Yellow solid, yield 38.5%, mp 205-206℃. 1H NMR (400MHz, DMSO) δ11.01(s,1H),9.03(t,J=6.1Hz,1H),7.86(d,J=7.9Hz,2H),7.55–7.44(m,3H),7.37(dd,J= 19.3,8.1Hz,4H),7.29–7.18(m,2H),7.02(d,J=2.3Hz,1H),6.79(dd,J=17.0,8.0Hz,2H),6.43(d,J=15.8Hz,1H) ,4.49(d,J=5.9Hz,2H),4.12(s,1H),3.92(d,J=10.8Hz,2H),3.80(s,3H),3.50(s,2H),3.40(d,J=7.7Hz,2H),2. 58(q,J=7.4Hz,4H),2.39(s,4H),2.27(s,1H),1.83(t,J=14.2Hz,4H),1.69–1.50(m,4H),1.19(t,J=7.3Hz,3H). 13 C NMR(101MHz,DMSO)δ170.13,166.59,163.19,152.73,152.21,150.62,142.31,141.74, 138.45,136.93,135.79,133.86,133.39,131.95,129.11,128.19,127.94,127.68,119. 14,118.69,112.12,112.06,105.25,66.91,62.17,61.53,58.25,56.15,53.55,50.96, 49.05,48.04,45.98,42.86,32.65,28.77,24.69,11.42,8.23.HRMS-ESI(m / z)calc.for C 46 H 58 N 10 O5[M+H] + 847.4546,found 847.4542.HPLC t R = 9.62 min (99.97% purity).

[0129] Example 3: Preparation of target substances 14, 17a, and 17b

[0130] 2-(4-(1-(4-((3-carbamoyl-5-ethyl-6((tetrahydro-2H-pyran-4-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (13)

[0131] Yellow solid, yield 33.3%, mp 167-169℃. 1 H NMR(400MHz,DMSO)δ11.55(s,1H),11.03(s,1H),9.38(s,2H),8.69(s,1H),7.54 (s,1H),7.26(d,J=17.4Hz,2H),7.03(s,1H),6.87–6.77(m,2H),4.96(s,1H),4.1 2(s,2H),3.93(d,J=10.4Hz,2H),3.82(s,3H),3.53(d,J=11.8Hz,2H),2.59(d,J= 7.3Hz, 4H), 1.86 (d, J = 12.2Hz, 4H), 1.72 (s, 4H), 1.65–1.51 (m, 6H), 1.24 (s, 3H).

[0132] 2-(4-(1-(4-((3-carbamoyl-5-ethyl-6((tetrahydro-2H-pyran-4-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)N-hydroxypyrimidine-5-carboxamide (14)

[0133] Compound 13 was added to 10 mL of 4 M hydrochloric acid / ethyl acetate and reacted at room temperature for 12 hours. The reaction mixture was filtered, and the filter cake was recrystallized from ethanol to give compound 14. Compounds 17a, 17b, and 44 were synthesized using the same procedure. The product was a brown solid, 28.1% yield, mp 216-218 °C. 1 H NMR (400MHz, DMSO) δ11.93(s,1H),11.32(d,J=48.7Hz,2H),8.78(s,2H),7.59(d,J=29.4Hz ,2H),7.36(s,1H),7.20(s,1H),6.94(d,J=7.5Hz,1H),4.84(d,J=13.7Hz,2H),4.16–4.10( m,1H),3.94(d,J=14.9Hz,5H),3.61(d,J=12.9Hz,8H),3.42–3.35(m,2H),2.62(q,J=7.4Hz ,2H),2.36(s,4H),1.87(dd,J=12.2,4.0Hz,2H),1.69–1.60(m,2H),1.20(t,J=7.3Hz,3H). 13C NMR(101MHz,DMSO)δ170.13,166.59,163.19,152.73,152.21,150.62,142.31,141.74, 138.45,136.93,135.79,133.86,133.39,131.95,129.11,128.19,127.94,127.68,119. 14,118.69,112.12,112.06,105.25,66.91,62.17,61.53,58.25,56.15,53.55,50.96, 49.05,48.04,45.98,42.86,32.65,28.77,24.69,11.42,8.23.HRMS-ESI(m / z)calc.for C 33 H 45 N 11 O5[MH] - 674.3503, found 674.3498. HPLC t R = 10.16 min (99.32% purity).

[0134] tert-Butyl(2-(4-((4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)methyl)benzamido)phenyl)carbamate (16a)

[0135] Yellow solid, yield 70.7%. 1 H NMR (400MHz, DMSO) δ11.05(s,1H),9.87(s,1H),9.65(s,2H),8.70(s,1H),7.96(d,J=7.9Hz,2H),7.52 (dd,J=14.3,7.4Hz,4H),7.29(d,J=8.5Hz,1H),7.27–7.13(m,4H),7.04(s,1H),6.82(dd,J=14.5,8.0 Hz,2H),4.10(t,J=5.3Hz,1H),3.92(d,J=11.3Hz,2H),3.82(s,3H),3.68(s,2H),3.50(s,2H),3.39(s ,4H),2.62–2.53(m,6H),2.14(s,2H),1.85(d,J=12.8Hz,4H),1.64(s,4H),1.45(s,9H),1.23(s,3H).

[0136] tert-Butyl(2-(4-((4-(1-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperidin-1-yl)methyl)pyrimidin-5-carbamoyl)phenyl)carbamate (16b)

[0137] Yellow solid, yield 77.2%. 1 H NMR (400MHz, DMSO) δ11.04(s,1H),9.67(s,1H),8.94(s,2H),8.62(s,1H),7.61(d,J=8.1 Hz,1H),7.54(s,1H),7.50–7.46(m,1H),7.31–7.17(m,4H),7.12(t,J=7.7Hz,2H),7.03( s,1H),6.83(dd,J=14.8,8.2Hz,2H),4.13–4.10(m,1H),3.92(d,J=9.2Hz,2H),3.82(s,3 H),2.59(d,J=7.4Hz,4H),1.85(d,J=12.7Hz,4H),1.63(s,4H),1.45(s,9H),1.23(s,3H).

[0138] 3-((4-(4-(4-(((2-aminophenyl)benzyl)piperazin-1-yl)piperidin-1-yl)-3-methoxyphenyl)amino)-6-ethyl-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (17a)

[0139] Yellow solid, yield 28.3%, mp 174-176℃. 1 H NMR (400MHz, DMSO) δ11.42(s,1H),10.69(s,1H),8.23(d,J=8.0Hz,2H),7.87(d,J=7.9Hz,2H),7.68 –7.54(m,3H),7.51(d,J=7.8Hz,1H),7.45–7.33(m,3H),7.32–7.13(m,2H),6.96(d,J=7.5Hz,1H),4 .55(s,2H),4.12(s,1H),3.95(d,J=20.4Hz,5H),3.73(s,8H),3.43–3.33(m,2H),2.62(q,J=7.4Hz, 2H), 2.34 (d, J=8.5Hz, 4H), 1.86 (dd, J=12.4, 4.1Hz, 2H), 1.73–1.61 (m, 2H), 1.19 (d, J=7.3Hz, 3H). 13C NMR(101MHz,DMSO)δ170.12,165.62,152.75,152.21,150.61,143.61,135 .90,133.86,131.98,129.15,128.23,127.15,126.91,123.86,118.78,116 .72,116.61,112.14,112.06,105.24,66.91,61.77,56.16,50.76,48.96, 48.04,45.98,32.65,28.43,26.82,24.68,11.42.HRMS-ESI(m / z)calc.for C 42 H 54 N 10 O4[M+H] + 763.4349, found 763.4345. HPLC t R = 5.67 min (97.79% purity).

[0140] N-(2-aminophenyl)-2-(4-(1-(4-((3-carbamoyl-5-ethyl-6((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperidin-4-yl)piperidin-1-yl)pyrimidine-5-carboxamide (17b)

[0141] Yellow solid, yield 27%, mp 238-240℃. 1 H NMR (400MHz, DMSO) δ11.02 (d, J = 14.3Hz, 1H), 9.55 (s, 1H), 8.92 (s, 2H), 7.54 (s, 1H) ),7.35–7.18(m,2H),7.17–7.09(m,1H),7.08–6.90(m,2H),6.90–6.68(m,3H),6.58 (t,J=8.1Hz,1H),4.96(s,2H),4.11(s,1H),3.95–3.75(m,7H),3.35(q,J=11.2Hz, 8H),2.68–2.55(m,4H),1.85(d,J=13.4Hz,4H),1.62(s,4H),1.18(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ170.12,163.11,161.77,158.62,152.76,152.22,150.61,143.78,131.99,127.36,127.02,123.27,118.80,116.53,11 6.37,112.15,112.07,105.25,66.91,56.17,50.81,48.89,48.05,45.99,32.65,28.35,24.68,11.42,11.31,9.01.HRMS-ESI(m / z)calc.for C 39 H 50 N 12 O4[M+H] + 763.4349, found 763.4345. HPLC t R = 10.97 min (99.93% purity).

[0142] tert-Butyl-4-(2-methoxy-4-nitrobenzene)piperazine-1-carboxylic acid ester (19)

[0143] Yellow solid, yield 87.3%. 1 H NMR (400MHz, DMSO) δ7.84 (dd, J=8.8, 2.6Hz, 1H), 7.70 (d, J=2.6Hz, 1H), 7.03 (d, J= 8.9Hz,1H),3.91(s,3H),3.46(t,J=5.0Hz,4H),3.15(d,J=5.4Hz,4H),1.42(s,9H).

[0144] tert-Butyl-4-(4-amino-2-methoxyphenyl)piperazine-1-carboxylic acid ester (20)

[0145] Pale yellow solid, yield 88.1%. 1 H NMR (400MHz, DMSO) δ6.61(d,J=8.3Hz,1H),6.23(d,J=2.3Hz,1H),6.06(dd,J=8.3,2.4 Hz,1H),4.76(s,2H),3.68(s,3H),3.42–3.37(m,5H),2.76–2.70(m,5H),1.41(s,9H).

[0146] tert-Butyl-4-(4-((3-cyano-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazine-1-carboxylate (21)

[0147] Yellow solid, yield 41.5%. 1 H NMR(400MHz,DMSO)δ8.67(s,1H),7.20(dd,J=8.6,2.3Hz,1H),7.14–7.05(m,2 H),6.80(d,J=8.6Hz,1H),3.94(s,1H),3.92–3.84(m,2H),3.76(s,3H),3.45( s,4H),3.25(t,J=11.9,2H),2.94–2.78(m,4H),2.56(q,J=7.3Hz,2H),1.76(d d,J=12.2,4.1Hz,2H),1.70–1.61(m,2H),1.42(s,9H),1.13(t,J=7.3Hz,3H).

[0148] tert-Butyl-4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazine-1-carboxylate (22)

[0149] Yellow solid, yield 57.5%. 1 H NMR (400MHz, DMSO) δ11.06(s,1H),7.54(d,J=3.5Hz,1H),7.32(dd,J=8.6,2.3Hz,1H),7. 24(d,J=3.2Hz,1H),7.02(d,J=2.3Hz,1H),6.82(dd,J=14.7,8.0Hz,2H),4.12(d,J=8.0H z,1H),3.92(d,J=9.8Hz,2H),3.82(s,3H),3.45(s,4H),3.40(s,2H),2.94–2.81(m,4H), 2.58(d,J=7.4Hz,2H),1.85(d,J=12.6Hz,2H),1.71–1.54(m,2H),1.19(t,J=7.3Hz,3H).

[0150] 4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazine-1-ammonium salt (23)

[0151] Bright yellow solid, yield 85.5%. 1H NMR(400MHz,DMSO)δ11.18(s,1H),9.42(s,2H),7.14–7.05(m,2H),6.87(s,1H),4.13(s,1H),3.96–3.90(m,2H), 3.87(s,3H),3.49–3.32(m,5H),2.63–2.57(m,2H),1.90–1.81(m,2H),1.72–1.60(m,2H),1.20(t,J=7.4Hz,3H).

[0152] Methyl 7-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)-7-heptanoate (24a)

[0153] Yellow solid, yield 78.1%. 1 H NMR (400MHz, DMSO) δ11.99(s,1H),11.06(s,1H),7.55(s,1H),7.32(dd,J=8.5,2.7Hz,1H),7.25(s,1H),7 .03(d,J=2.7Hz,1H),6.86–6.78(m,2H),4.15–4.09(m,1H),3.92(d,J=11.6Hz,2H),3.83(d,J=2.1Hz,3H), 3.58(s,3H),2.87(d,J=22.9Hz,4H),2.62–2.55(m,2H),2.37–2.25(m,6H),2.19(td,J=7.4,2.2Hz,2H),1 .86(d,J=12.7Hz,2H),1.70–1.60(m,2H),1.50(p,J=6.9Hz,7H),1.32–1.25(m,6H),1.20(t,J=7.4Hz,3H).

[0154] 8-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)-8-octanoic acid methyl ester (24b)

[0155] Yellow solid, yield 75%. 1H NMR (400MHz, DMSO) δ11.99(s,1H),11.06(s,1H),7.55(s,1H),7.32(dd,J=8.5,2.7Hz,1H),7.25(s,1H),7 .03(d,J=2.7Hz,1H),6.86–6.78(m,2H),4.15–4.09(m,1H),3.92(d,J=11.6Hz,2H),3.83(d,J=2.1Hz,3H), 3.58(s,3H),2.87(d,J=22.9Hz,4H),2.62–2.55(m,2H),2.37–2.25(m,6H),2.19(td,J=7.4,2.2Hz,2H),1 .86(d,J=12.7Hz,2H),1.70–1.60(m,2H),1.50(p,J=6.9Hz,7H),1.32–1.25(m,6H),1.20(t,J=7.4Hz,3H).

[0156] 3-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)ethyl propionate (24c)

[0157] Yellow solid, yield 81.3%. 1 H NMR (400MHz, DMSO) δ11.00 (s, 1H), 7.54 (d, J = 3.2Hz, 1H), 7.27 (dd, J = 8.5, 2.3Hz, 1H),7.22(d,J=3.3Hz,1H),7.03(d,J=2.3Hz,1H),6.81(dd,J=8.1,5.9Hz,2H),4.0 7(q,J=7.1Hz,4H),3.92(d,J=11.3Hz,2H),3.80(s,3H),2.90(s,4H),2.60(dd,J=1 3.3, 6.3Hz, 4H), 1.85 (d, J = 12.3Hz, 2H), 1.68–1.58 (m, 2H), 1.19 (t, J = 7.0Hz, 6H).

[0158] 4-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl butyrate (24d)

[0159] Yellow solid, yield 83.6%. 1H NMR(400MHz,DMSO)δ11.00(s,1H),7.54(d,J=3.2Hz,1H),7.27(dd,J=8.5,2.3Hz,1H), 7.22(d,J=3.3Hz,1H),7.03(d,J=2.3Hz,1H),6.81(dd,J=8.1,5.9Hz,2H),4.07(q,J=7 .1Hz,4H),3.92(d,J=11.3Hz,2H),3.80(s,3H),2.90(s,4H),2.60(dd,J=13.3,6.3Hz, 4H), 1.85 (d, J=12.3Hz, 2H), 1.63 (qd, J=12.1, 4.5Hz, 2H), 1.19 (dd, J=7.6, 6.4Hz, 6H).

[0160] Methyl 5-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)valerate (24e)

[0161] Yellow solid, yield 82.2%. 1 H NMR(400MHz,DMSO)δ11.00(s,1H),7.54(d,J=3.2Hz,1H),7.27(dd,J=8.5,2.3Hz,1H), 7.22(d,J=3.3Hz,1H),7.03(d,J=2.3Hz,1H),6.81(dd,J=8.1,5.9Hz,2H),4.07(q,J=7 .1Hz,4H),3.92(d,J=11.3Hz,2H),3.80(s,3H),2.90(s,4H),2.60(dd,J=13.3,6.3Hz, 4H), 1.85 (d, J=12.3Hz, 2H), 1.63 (qd, J=12.1, 4.5Hz, 2H), 1.19 (dd, J=7.6, 6.4Hz, 6H).

[0162] 6-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl hexanoate (24f)

[0163] Yellow solid, yield 77%. 1H NMR (400MHz, DMSO) δ11.08 (s, 1H), 7.56 (s, 1H), 7.33 (d, J = 8.7Hz, 1H), 7.26 (s, 1H) ),7.07(s,1H),6.89–6.79(m,2H),4.12(s,1H),3.93(d,J=11.4Hz,2H),3.83(s,3 H),3.59(s,3H),3.13–3.06(m,4H),2.59(q,J=7.4Hz,4H),2.32(t,J=7.3Hz,2H), 1.85(d,J=12.8Hz,2H),1.73–1.60(m,4H),1.55(s,2H),1.31(s,2H),1.18(s,3H).

[0164] 7-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl heptanoate (24g)

[0165] Yellow solid, yield 79.3%. 1 H NMR (400MHz, DMSO) δ11.01(s,1H),7.52(s,1H),7.30–7.18(m,2H),7.04(d,J=2.3Hz,1H),6.82(d ,J=8.6Hz,1H),6.77(d,J=7.4Hz,1H),3.92(d,J=10.8Hz,2H),3.80(s,3H),3.58(s,3H),3.39(s, 2H),2.92(s,2H),2.58(q,J=7.4Hz,4H),2.30(t,J=7.3Hz,2H),1.85(d,J=12.4Hz,2H),1.63(dd, J=11.8,4.2Hz,2H),1.52(d,J=7.6Hz,2H),1.44(s,2H),1.35–1.25(m,4H),1.19(t,J=7.3Hz,3H).

[0166] 8-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl octanoate (24h)

[0167] Yellow solid, yield 71.4%. 1H NMR (400MHz, DMSO) δ11.03(s,1H),7.54(d,J=3.4Hz,1H),7.31–7.21(m,2H),7.04(d,J=2.3Hz ,1H),6.81(t,J=8.4Hz,2H),4.17–4.06(m,1H),3.96–3.88(m,2H),3.81(s,3H),3.58(s,3H), 3.38(dd,J=12.0,2.1Hz,2H),2.92(s,4H),2.58(q,J=7.4Hz,2H),2.31(d,J=7.3Hz,2H),1.90 –1.81(m,2H),1.67–1.57(m,2H),1.56–1.43(m,4H),1.31–1.25(m,6H),1.19(t,J=7.4Hz,3H).

[0168] (E)-3-(4-((4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)phenyl)methyl acrylate (24i)

[0169] Yellow solid, yield 80.6%. 1 H NMR (400MHz, DMSO) δ11.01(s,1H),7.72–7.62(m,3H),7.52(s,1H),7.39(d,J=7.8Hz,2H),7.31 –7.24(m,1H),7.20(s,1H),7.03(d,J=2.3Hz,1H),6.83(d,J=8.6Hz,1H),6.77(d,J=7.6Hz,1H) ,6.62(d,J=16.0Hz,1H),3.92(d,J=10.3Hz,2H),3.80(s,3H),3.73(s,3H),3.56(s,2H),2.93( s,4H),2.58(q,J=7.4Hz,4H),1.85(d,J=12.6Hz,2H),1.70–1.57(m,2H),1.18(t,J=7.6Hz,3H).

[0170] 6-Ethyl-3-((4-(4-(7-(hydroxyamino)-7-oxoheptanoyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25a)

[0171] Pale yellow solid, yield 31.3%, mp 184-185℃. 1H NMR (400MHz, DMSO) δ11.04(s,1H),10.35(s,1H),8.64(s,1H),7.53(s,1H),7.30(d,J=8.1Hz,1H),7.22(s,1 H),7.04(d,J=2.3Hz,1H),6.81(dd,J=12.8,8.0Hz,2H),4.12(s,1H),3.92(d,J=10.9Hz,2H),3.83(s,3H),3 .57(s,4H),3.40(s,2H),2.90(s,4H),2.62–2.56(m,2H),2.32(t,J=7.4Hz,2H),1.96(q,J=8.7Hz,2H),1.85 (d,J=12.7Hz,2H),1.64(d,J=12.5Hz,2H),1.55–1.46(m,4H),1.25(d,J=13.1Hz,2H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ171.01,170.11,169.39,152.82,152.21,150.57,136.34,135.99,132.10,118.96,112.15,112.00,10 5.12,66.90,56.18,51.47,51.02,48.06,45.77,41.74,32.64,28.88,25.51,25.04,24.69,11.41.HRMS-ESI(m / z)calc.for C 30 H 44 N8O6[M+H] + 613.3771,found 613.3775.HPLC t R = 7.19 min (98.56% purity).

[0172] 6-Ethyl-3-((4-(4-(8-(hydroxyamino)-8-oxooctanoyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25b)

[0173] Pale red solid, yield 40.7%, mp 164-166℃. 1H NMR (400MHz, DMSO) δ11.06(s,1H),10.34(s,1H),8.67(d,J=1.6Hz,1H),7.54(d,J=3.1Hz,1H),7.35–7.23(m,2H), 7.03(d,J=2.3Hz,1H),6.82(t,J=7.9Hz,2H),4.12(d,J=8.2Hz,1H),3.92(d,J=10.4Hz,2H),3.83(s,3H),3.57(s,4 H),3.38(d,J=12.0Hz,2H),2.90(s,2H),2.84(s,2H),2.58(q,J=7.3Hz,2H),2.32(t,J=7.4Hz,2H),1.93(t,J=7.4H z,2H),1.85(d,J=12.3Hz,2H),1.70–1.57(m,2H),1.49(d,J=6.9Hz,4H),1.32–1.23(m,4H),1.19(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO) δ171.07,170.12,169.61,152.86,152.22,150.59,136.35,136.04,132.09,118.99,112.21,112.09,105.25,66.9 0,56.21,51.49,51.04,48.07,45.81,41.77,32.73,32.70,32.66,28.99,28.92,25.51,25.21,24.67,11.40.HRMS-ESI(m / z)calc.for C 31 H 46 N8O6[MH] - 625.3438,found 625.3434.HPLC t R = 7.59 min (95.52% purity).

[0174] 6-Ethyl-3-((4-(4-(3-(hydroxyamino)-3-oxopropyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25c)

[0175] Yellow solid, yield 35.1%, mp 167-168℃. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.44(s,1H),8.75(s,1H),7.53(d,J=3.3Hz,1H),7 .31–7.18(m,2H),7.05(d,J=2.3Hz,1H),6.81(dd,J=15.8,8.0Hz,2H),4.12(h,J=6.4H z,1H),3.93(dd,J=11.5,3.6Hz,2H),3.82(s,3H),3.40(s,2H),2.94(s,4H),2.59(q,J =7.3Hz,4H),2.20(s,2H),1.90–1.81(m,2H),1.68–1.59(m,2H),1.20(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,152.70,152.21,150.61,135.95,132.03,118.53,112.20,112.1 4,105.38,66.90,56.17,53.16,50.61,48.04,32.64,24.67,11.42.HRMS-ESI(m / z)calc.for C 26 H 38 N8O5[M+H] + 543.3049,found 543.3054.HPLC t R = 10.97 min (99.93% purity).

[0176] 6-Ethyl-3-((4-(4-(4-(hydroxyamino)-4-oxobutyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25d)

[0177] Yellow solid, yield 30.9%, mp 169-171℃. 1H NMR (400MHz, DMSO) δ11.05(s,1H),10.45(s,1H),8.73(s,1H),7.54(s,1H),7.30(d,J=8. 6Hz,1H),7.23(s,1H),7.05(s,1H),6.82(dd,J=14.0,8.0Hz,2H),4.16–4.07(m,1H),3.9 2(d,J=11.2Hz,2H),3.82(s,3H),3.38(d,J=11.9Hz,2H),3.02(s,4H),2.59(q,J=7.3Hz, 2H), 2.03(t,J=7.2Hz,2H),1.92–1.72(m,4H),1.69–1.57(m,2H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.20,152.68,152.21,150.58,136.19,132.09,118.61,112.13,112.08,1 05.25,66.90,57.03,56.17,52.90,49.86,48.06,32.63,30.39,24.68,11.42.HRMS-ESI(m / z)calc.forC 27 H 40 N8O5[M+H] + 557.3162, found 557.3157.HPLC t R = 9.18 min (99.13% purity).

[0178] 6-Ethyl-3-((4-(4-(5-(hydroxyamino)-5-oxopentyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25e)

[0179] Yellow solid, yield 34%, mp 167-169℃. 1H NMR (400MHz, DMSO) δ11.00(s,1H),10.32(s,1H),8.63(s,1H),7.51(d,J=3.3Hz,1H),7.25(dd,J=8.6,2.3Hz,1H),7.18(d ,J=3.2Hz,1H),7.05(d,J=2.3Hz,1H),6.82(d,J=8.6Hz,1H),6.75(d,J=7.5Hz,1H),4.18–4.06(m,1H),3.97–3.86(m,2H), 3.81(s,3H),3.41–3.32(m,2H),2.92(s,4H),2.58(q,J=7.4Hz,2H),2.48(s,2H),2.30(t,J=7.1Hz,2H),1.97(t,J=7.2Hz, 2H), 1.85 (dd, J=12.9, 4.2Hz, 2H), 1.68–1.58 (m, 2H), 1.51 (q, J=7.4Hz, 2H), 1.43 (q, J=7.6Hz, 2H), 1.19 (t, J=7.4Hz, 3H). 13 C NMR (101MHz, DMSO) δ170.11,169.20,152.68,152.21,150.58,136.19,132.09,118.61,112.13,112.08, 105.25,66.90,57.03,56.17,52.90,49.86,48.06,32.63,30.39,24.68,11.42.HRMS-ESI(m / z)calc.for C 28 H 42 N8O5[M+H] + 571.3317, found 571.3312. HPLC t R = 7.12 min (95.58% purity).

[0180] 6-Ethyl-3-((4-(4-(6-(hydroxyamino)-6-oxohexyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25f)

[0181] Yellow solid, yield 37.2%, mp 169-171℃. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.35(s,1H),8.69(s,1H),7.54(d,J=3.2Hz,1H),7.29–7.22(m,2H),7 .03(d,J=2.4Hz,1H),6.81(t,J=7.4Hz,2H),4.12(d,J=9.5Hz,1H),3.92(d,J=11.4Hz,2H),3.80(s,3H),2. 91(s,4H),2.59(t,J=7.3Hz,2H),2.28(t,J=7.3Hz,2H),1.95(t,J=7.3Hz,2H),1.85(d,J=12.6Hz,2H),1.6 8–1.59(m,2H),1.51(q,J=7.4Hz,2H),1.44(q,J=7.0Hz,2H),1.26(q,J=7.8Hz,2H),1.19(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO) δ170.12,169.50,152.70,152.21,150.61,136.61,135.81,131.98,118.44,112.22,112.13,105.42 ,66.90,58.39,56.17,53.62,50.95,48.04,32.72,32.65,27.03,26.51,25.58,24.67,11.42.HRMS-ESI(m / z)calc.for C 29 H 44 N8O5[M+H] + 585.3472, found 585.3467. HPLC t R = 8.60 min (99.22% purity).

[0182] 6-Ethyl-3-((4-(4-(7-(hydroxyamino)-7-oxoheptyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25g)

[0183] Yellow solid, yield 31.9%, mp 199-201℃. 1H NMR (400MHz, DMSO) δ11.06(s,1H),10.36(s,1H),8.67(s,1H),7.54(d,J=3.2Hz,1H),7.31(d,J=8.6Hz,1H),7.2 3(d,J=3.3Hz,1H),7.05(d,J=2.3Hz,1H),6.83(dd,J=17.4,8.0Hz,2H),4.11(p,J=5.3Hz,1H),3.93(dd,J=11.4, 3.7Hz,2H),3.82(s,3H),3.38(d,J=10.6Hz,2H),3.02(s,4H),2.59(q,J=7.3Hz,2H),1.95(t,J=7.3Hz,2H),1.86 (dd,J=12.9,4.1Hz,2H),1.71–1.54(m,4H),1.50(t,J=7.1Hz,2H),1.28(d,J=6.9Hz,4H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.53,152.68,152.21,150.57,136.43,132.14,118.72,112.19,112. 01,105.19,66.89,56.18,48.06,32.63,28.72,26.65,25.42,24.69,11.42.HRMS-ESI(m / z)calc.for C 30 H 46 N8O5[M+H] + 599.3626, found 599.3621. HPLC t R = 8.60 min (99.22% purity).

[0184] 6-Ethyl-3-((4-(4-(8-(hydroxyamino)-8-oxooctyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25h)

[0185] Yellow solid, yield 39.6%, mp 205-207℃. 1H NMR (400MHz, DMSO) δ11.03(s,1H),10.34(s,1H),8.67(s,1H),7.54(d,J=3.1Hz,1H),7.31–7.21(m,2H) ,7.03(d,J=2.3Hz,1H),6.81(t,J=7.7Hz,2H),4.18–4.07(m,1H),3.92(d,J=11.4Hz,2H),3.81(s,3H),3 .39(s,2H),2.93(s,4H),2.61–2.57(m,2H),2.34(s,2H),1.94(t,J=7.3Hz,2H),1.89–1.82(m,2H),1.6 9–1.60(m,2H),1.54–1.44(m,4H),1.25(d,J=12.5Hz,6H),1.19(t,J=7.3Hz,3H),1.13(t,J=7.3Hz,2H). 13 C NMR (101MHz, DMSO) δ170.10,169.56,152.68,152.21,150.56,132.14,118.73,112.19,111.99,105.16, 66.89,56.18,49.06,48.06,32.70,32.64,28.87,26.77,25.49,24.69,11.42.HRMS-ESI(m / z)calc.for C 31 H 48 N8O5[M+H] + 613.3767, found 613.3762. HPLC t R = 7.66 min (99.55% purity).

[0186] (E)-6-ethyl-3-((4-(4-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25i)

[0187] Yellow solid, yield 38.1%, mp 200-201℃. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.79(s,1H),9.04(s,1H),7.53(d,J=6.3Hz,3H),7.45(d,J=15.7Hz,1 H),7.37(d,J=7.7Hz,2H),7.31–7.20(m,2H),7.03(d,J=2.2Hz,1H),6.81(dd,J=13.2,8.0Hz,2H),6.47(d, J=15.8Hz,1H),4.19–4.05(m,1H),3.92(dd,J=11.7,3.6Hz,2H),3.80(s,3H),3.54(s,2H),3.39(s,2H),2 .94(s,4H),2.59(q,J=7.2Hz,2H),1.85(dd,J=12.6,4.1Hz,2H),1.68–1.60(m,2H),1.19(t,J=7.2Hz,3H). 13 C NMR (101MHz, DMSO) δ170.12,163.29,152.73,152.21,150.61,138.61,136.50,135.90,134.07,132.01,129.93,127.84,119 .15,118.55,112.17,112.12,105.34,66.90,62.24,56.16,53.37,50.93,48.04,32.64,24.67,11.42.HRMS-ESI(m / z)calc.C 33 H 42 N8O5[M+H] + 631.3312,found 631.3306.HPLC t R = 6.43 min (95.75% purity).

[0188] (E)-6-ethyl-3-((4-(4-(4-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)amino)-2-oxobutyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25j)

[0189] Pale yellow solid, yield 37.9%. 1H NMR(400MHz,DMSO)δ11.05(s,1H),10.84(s,1H),9.05(s,1H),8.47(s,1H),7.63–7.39(m,4H), 7.36–7.17(m,4H),7.05(s,1H),6.83(d,J=8.6Hz,2H),6.49(d,J=15.8Hz,1H),4.29(d,J=5.8Hz ,2H),4.12(s,1H),3.98–3.88(m,2H),3.82(s,3H),3.03(s,4H),2.76(s,3H),2.61–2.56(m,2H ), 2.23 (t, J = 7.4Hz, 2H), 1.85 (d, J = 15.5Hz, 4H), 1.65 (d, J = 12.4Hz, 2H), 1.19 (t, J = 7.3Hz, 3H). 13 C NMR (101MHz, DMSO) δ172.28,170.12,163.23,152.69,152.22,150.59,141.66,138.45,136.13,133.86,132.09,128.19,127.92,119 .21,118.58,112.14,105.31,66.90,57.21,56.19,52.97,49.96,48.06,42.30,33.42,32.63,24.69,11.42.HRMS-ESI(m / z)calc.for C 37 H 49 N9O6[MH] - 713.4716,found 713.4712.HPLC t R = 2.78 min (96.57% purity).

[0190] (E)-6-ethyl-3-((4-(4-(2-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25k)

[0191] Pale yellow solid, yield 34.5%. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.67(s,1H),8.98(s,1H),7.52(dd,J=12.1,5.7Hz,3H),7.41(d,J=15.7Hz,1H ),7.30–7.20(m,2H),7.05(d,J=2.3Hz,1H),7.00(d,J=8.3Hz,2H),6.81(dd,J=14.3,8.0Hz,2H),6.32(d,J=15.8H z,1H),4.16(s,2H),4.11(t,J=8.4Hz,1H),3.92(d,J=11.5Hz,2H),3.81(s,3H),3.39(s,2H),2.95(s,4H),2.79(s ,2H),2.67(s,2H),2.58(d,J=7.4Hz,2H),1.85(dd,J=12.8,4.1Hz,2H),1.70–1.57(m,2H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.12,163.66,159.97,152.71,152.21,150.62,138.49,136.43,135.93,132.01,129.52,127.93,118.52,117. 04,115.40,112.23,112.14,105.42,66.90,65.96,56.98,56.18,53.82,50.74,48.04,32.65,24.67,11.42.HRMS-ESI(m / z)calc.for C 34 H 44 N8O6[M+H] + 661.3416,found 661.3411.HPLC t R = 11.59 min (99.81% purity).

[0192] 2-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25l)

[0193] Pale yellow solid, yield 33.1%. 1H NMR (400MHz, DMSO) δ11.46(s,1H),8.79(s,2H),7.63(s,2H),7.22(s,1H),6.97(d,J=7.4Hz,1H),4.13(s,1H),4.05–3.88(m ,7H),3.60(s,4H),2.62(q,J=7.3Hz,2H),1.86(dd,J=12.9,3.5Hz,2H),1.67(qd,J=12.1,4.5Hz,3H),1.20(t,J=7.4Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,163.63,160.13,152.69,152.21,150.58,138.48,136.27,132.10,129.52,127.83,118.67,11 6.99,115.34,112.17,112.08,105.26,67.73,66.90,56.18,48.05,32.64,26.72,24.68,11.42.HRMS-ESI(m / z)calc.forC 36 H 48 N8O6[M+H] + 689.3725, found 689.3722. HPLC t R = 6.40 min (96.92% purity).

[0194] 6-Ethyl-3-((4-(4-(2-((4-(hydroxyamino)-4-oxobutyl)(methyl)amino)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25m)

[0195] Pale yellow solid, yield 31.7%. 1H NMR (400MHz, DMSO) δ11.02(s,1H),10.35(s,1H),8.65(s,1H),7.53(s,1H),7.29–7.20(m,2H),7. 04(d,J=2.3Hz,1H),6.80(t,J=8.7Hz,2H),4.11(s,1H),3.91(t,J=6.8Hz,2H),3.81(s,3H),3.39 (s,2H),2.91(s,4H),2.59(t,J=7.3Hz,2H),2.43(s,4H),2.28(t,J=7.2Hz,2H),2.15(s,2H),1.9 7(q,J=8.6Hz,2H),1.90–1.81(m,2H),1.63(q,J=6.8Hz,4H),1.23(s,2H),1.19(t,J=7.3Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.35,152.71,152.21,150.60,136.40,135.93,132.01,118.49,112.19,112.15,105.39,66 .90,60.23,56.19,53.62,50.70,48.04,45.96,32.65,32.37,24.68,23.13,21.24,14.56,11.43.HRMS-ESI(m / z)calc.for C 31 H 49 N9O5[M+H] + 614.3756,found 614.3751.HPLC t R = 6.88 min (97.52% purity).

[0196] 6-Ethyl-3-((4-(4-(2-((4-(hydroxycarbamoyl)benzyl)(methyl)amino)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25n)

[0197] Pale yellow solid, yield 35.2%. 1H NMR (400MHz, DMSO) δ11.22(s,1H),11.05(s,1H),7.73(d,J=7.8Hz,2H),7.56(d,J=3.3Hz,1H),7. 42(d,J=7.6Hz,2H),7.29(d,J=9.3Hz,2H),7.04(d,J=2.4Hz,1H),6.87–6.77(m,2H),4.18–4.08(m ,1H),3.97–3.89(m,2H),3.81(s,3H),3.63(s,2H),3.05(q,J=7.5Hz,4H),2.96(s,2H),2.58(q,J =7.4Hz,5H),2.20(s,2H),1.89–1.81(m,2H),1.63(qd,J=12.4,4.5Hz,2H),1.20(d,J=3.7Hz,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.00,152.69,152.21,150.60,136.32,135.96,132.02,118.51,112.13,105.34 ,66.90,56.17,53.33,50.63,48.05,45.90,32.64,30.09,24.68,14.56,11.43,9.21.HRMS-ESI(m / z)calc.for C 34 H 47 N9O5[M+H] + 662.3451,found 662.3746.HPLC t R = 8.41 min (97.95% purity).

[0198] (E)-6-ethyl-3-((4-(4-(2-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)(methyl)amino)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25o)

[0199] Pale yellow solid, yield 37.2%. 1H NMR (400MHz, DMSO) δ11.04(s,1H),10.76(s,1H),7.56–7.51(m,2H),7.45(d,J=15.8Hz,1H),7.38(s,1 H),7.26(dd,J=14.9,6.0Hz,2H),7.05(d,J=2.4Hz,1H),6.82(t,J=8.6Hz,2H),6.46(d,J=15.8Hz,1H) ,4.11(s,1H),3.96–3.89(m,2H),3.81(s,3H),3.51(s,2H),3.06(d,J=7.4Hz,4H),2.96(s,2H),2.58( q,J=7.3Hz,5H),2.22(s,2H),1.85(dd,J=13.1,4.1Hz,2H),1.63(qd,J=12.1,4.5Hz,2H),1.19(s,3H). 13 C NMR (101MHz, DMSO) δ170.11,169.00,152.69,152.21,150.60,136.32,135.96,132.02,118.51,112.13,105.34 ,66.90,56.17,53.33,50.63,48.05,45.90,32.64,30.09,24.68,14.56,11.43,9.21.HRMS-ESI(m / z)calc.for C 36 H 49 N9O5[M+H] + 688.3898, found 688.3895. HPLC t R = 9.40 min (97.47% purity).

[0200] 2-((3-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)propyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (25p)

[0201] Pale yellow solid, yield 37.2%. 1H NMR (400MHz, DMSO) δ11.06(s,1H),9.00(s,1H),8.68(s,2H),7.54(d,J=3.4Hz,1H),7.30(d,J=8.4H z,1H),7.24(d,J=3.1Hz,1H),7.05(d,J=2.3Hz,1H),6.83(dd,J=11.5,8.1Hz,2H),4.13(s,1H),3.92 (dd,J=11.2,3.6Hz,2H),3.82(s,3H),3.69(d,J=7.5Hz,2H),3.42–3.36(m,2H),3.16(s,3H),2.96(s ,2H),2.59(q,J=7.4Hz,4H),1.88–1.82(m,2H),1.64(qd,J=12.2,4.3Hz,2H),1.19(t,J=7.4Hz,3H). 13 CNMR(101MHz,DMSO)δ170.11,169.00,152.69,152.21,150.60,136.32,135.96,132.02,118.51,112.13,105.3 4,66.90,56.17,53.33,50.63,48.05,45.90,32.64,30.09,24.68,14.56,11.43,9.21.HRMS-ESI(m / z)calc.for C 32 H 45 N 11 O5[M+H] + 664.3651, found 664.3646.HPLC t R = 8.77 min (96.88% purity).

[0202] 2-(4-((4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25q)

[0203] Pale yellow solid, yield 35.3%. 1H NMR (400MHz, DMSO) δ11.05(d,J=10.9Hz,2H),8.99(s,1H),8.66(s,2H),7.54(d,J=3.3Hz,1H),7.30(d,J=8.5 Hz,1H),7.26–7.21(m,1H),7.04(d,J=2.4Hz,1H),6.82(dd,J=13.4,8.0Hz,2H),4.71(d,J=12.8Hz,2H),4.16– 4.06(m,1H),3.96–3.89(m,2H),3.82(s,3H),3.42–3.35(m,2H),2.94(d,J=12.0Hz,6H),2.59(q,J=7.4Hz,2H) ,2.19(s,2H),1.86(dd,J=12.9,3.9Hz,4H),1.64(qd,J=12.1,4.3Hz,2H),1.19(t,J=7.3Hz,3H),1.06(s,2H). 13 C NMR (101MHz, DMSO) δ170.11,169.00,152.69,152.21,150.60,136.32,135.96,132.02,118.51,112.13,105.34 ,66.90,56.17,53.33,50.63,48.05,45.90,32.64,30.09,24.68,14.56,11.43,9.21.HRMS-ESI(m / z)calc.forC 34 H 47 N 11 O5[M+H] + 690.3802,found 690.3798.HPLC t R = 9.87 min (95.99% purity).

[0204] Experimental Example 1: Inhibitory Activity of Target Compound on HDACs

[0205] 1. Materials:

[0206] HDACs enzymes (HeLa cell nuclear extract); Boc-Lys(Ac)-AMC substrate; trypsin; EDTA; LBH-589 (10mM, soluble in dimethyl sulfoxide); 96-well black microplate; glycerol; ultrapure water

[0207] 2. Method:

[0208] Buffer preparation:

[0209] The formula is 15mM Tris-HCl (pH=8.0), 250μM EDTA, 250mM NaCl, and 10% glycerol.

[0210] Preparation of Trypsin solution:

[0211] The formula is 10 mg / mL pancreatin, buffer, 2 μL BH589.

[0212] Preparation of substrate solution:

[0213] The substrate was dissolved in DMSO to prepare a 30 mM stock solution, which was then diluted to 300 μM with buffer.

[0214] The DMSO content is approximately 1%.

[0215] Diluting the enzyme solution:

[0216] Dilute the enzyme solution with a buffer at a ratio of 1:80.

[0217] Preparation of compound solutions:

[0218] Dilute the compounds (test compound and positive control SAHA) to a final concentration using a buffer.

[0219] Preparation and determination of 100% and blank:

[0220] Mix 50 μL 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.

[0221] Determination of the inhibitory activity of compounds against HDACs:

[0222] 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 Values. (Table 1) Table 1. HDAC inhibitory activity of the target compounds.

[0223]

[0224]

[0225] a A, IC 50 Greater than 5 μM; B, IC 50 Between 1 μM and 5 μM; C, IC 50 Between 0.2 μM and 1 μM; D, IC 50 Below 0.2μM

[0226] As shown in Table 1, compounds 11j, 25b, 25h-k, and 25o-q exhibited strong inhibitory activity against the HDAC mixed enzyme, comparable to the positive control drug SAHA. The remaining compounds also showed good inhibitory activity against the HDAC mixed enzyme.

[0227] Experimental Example 2: Inhibitory Activity of Target Compound on FLT3

[0228] 1. Materials:

[0229] FLT3 enzyme; 96-well black microplate; 40 mM Tris buffer (pH 7.4); MgCl2; bovine serum albumin (BSA); dithiothreitol (DTT); adenosine triphosphate (ATP); FLT3 ligand; positive control drug giretinib.

[0230] 2. Method:

[0231] The target compound at a specified concentration (5 μL) was added to a 50 μL reaction mixture containing 40 mM Tris (pH = 7.4), 10 mM MgCl2, 0.1 mg / ml BSA, 1 mM DTT, 10 μM ATP, FLT3 enzyme, and enzyme substrate, and incubated at 30 °C for 40 min. The assay was then performed according to the protocol of the kinase-glo Plus fluorescent kinase assay kit. Kinase inhibitory activity was directly proportional to the amount of ATP remaining. Fluorescence intensity was detected using a microplate reader, and inhibitory activity was calculated using GraphPad Prism software (Table 2).

[0232] Table 2. FLT3 inhibitory activity of the target compounds

[0233]

[0234]

[0235] a A, IC 50 Greater than 50 nM; B, IC 50Between 5 nM and 50 nM; C, IC 50 Between 2 nM and 5 nM; D, IC 50 Below 2nM

[0236] As shown in Table 2, compounds 25e-h and 25k-q exhibited the strongest inhibitory effects on FLT3 enzyme activity, comparable to the positive control drug giretinib. Furthermore, compounds 11c, 25a-d, and 25j also showed strong inhibitory activity against FLT3, with IC50 values ​​of [missing value]. 50 The value is between 2 and 5 nM.

[0237] Experiment Example 3: Antiproliferative Activity Experiment of Target Compound

[0238] 1. Materials:

[0239] Six cell lines: MV4-11, MOLM-13, KG-1, THP-1, Jurkat, and K562, 10% fetal bovine serum (Hyclone, USA), 2.5 g / L. -1 Trypsin (Gibco, USA), CCK-8, modified RPMI 1640 medium, DMEM medium (Hyclone, USA), positive control SAHA, GSK3685032, 96-well plate.

[0240] 2. Method:

[0241] 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).

[0242] Table 3. Antiproliferative activity of selected compounds

[0243]

[0244]

[0245] aA, IC 50 Greater than 20 μM; B, IC 50 Between 2 μM and 20 μM; C, IC 50 Between 0.2 μM and 2 μM; D, IC 50 Below 0.2μM

[0246] As shown in Table 3, compounds 25g, 25h, 25j, 25k, and 25o-q exhibited good inhibitory activity against both FLT3 and HDAC mixed enzymes. These compounds effectively inhibited the proliferation of FLT3-ITD-mutant acute myeloid leukemia cells, such as MV4-11 and MOLM-13 cells, with effects comparable to giretinib and stronger than SAHA. Furthermore, compounds 25h, 25k, and 25o-q also showed strong anti-proliferative activity against other types of acute leukemia (KG-1, THP-1, and Jurakat) and chronic myeloid leukemia cell lines, with effects superior to giretinib and comparable to SAHA. Therefore, these compounds have further research value.

[0247] Experiment Example 4: Study on the therapeutic effect of compounds on human acute myeloid leukemia cells

[0248] 1. Materials:

[0249] Cell line: Human acute myeloid leukemia cells (MOLM-13).

[0250] Laboratory animals: Six-week-old female BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0251] 2. Method:

[0252] Approximately 2×10 7 MOLM-13 cells were resuspended in 50 μL of serum-free 1640 medium and 50 μL of Matrigel, 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 9 consecutive days. Treatment groups received 10 mg / kg / day (25 p) and 10 mg / kg / day + 30 mg / kg / day (gefitinib + SAHA), while the control group received an equal volume of saline. Tumor size and mouse weight were measured daily during treatment. 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%.

[0253] 3. Experimental Results:

[0254] The results are as follows Figure 1As shown, the tumor inhibition rate of the target compound 25p was best at 64%, which was superior to the combination therapy group of the positive control drugs giretinib and SAHA.

Claims

1. A dual-target inhibitor of FLT3-HDAC or a pharmaceutically acceptable salt thereof, wherein the FLT3-HDAC dual-target inhibitor is selected from one of the following compounds: (E)-6-ethyl-3-((4-(4-(4-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)amino)-2-oxobutyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25j); (E)-6-ethyl-3-((4-(4-(2-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)ethyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25k); (E)-6-ethyl-3-((4-(4-(3-(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenoxy)propyl)piperazin-1-yl)-3-methoxyphenyl)amino)-5-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-carboxamide (25l); 2-((3-(4-(4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)propyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (25o); 2-(4-((4-((3-carbamoyl-5-ethyl-6-((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25p); 2-(4-((4-((3-carbamoyl-5-ethyl-6-(((tetrahydro-2H-pyran-4-yl)amino)pyrazin-2-yl)amino)-2-methoxyphenyl)piperazin-1-yl)methyl)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (25q).

2. The preparation method of the FLT3-HDAC dual-target inhibitor according to claim 1 includes the following steps: The preparation methods of compounds 25j-l and 25o-q are as follows: 。 3. The preparation method according to claim 2, characterized in that, During the preparation of 25j-l and 25o-q, Reagents and conditions: (a) 1-fluoro-2-methoxy-4-nitrobenzene, potassium carbonate, N,N-dimethylformamide, 100°C, 8 hours; (b) 10% Pd / C, tetrahydrofuran, room temperature, 24 hours; (c) 2, palladium acetate, 4,5-bis(diphenylphosphine-9,9-dimethyloxanthracene, cesium carbonate, 1,4-dioxane, 100°C, 12 hours; (d) 30% hydrogen peroxide, potassium carbonate, dimethyl sulfoxide, room temperature, 12 hours; (e) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (f) 9n-p, 9s-u, potassium carbonate, acetonitrile, 80°C, 6 hours; (g) NH2OH, KOH, anhydrous methanol, room temperature, 4 hours.

4. A pharmaceutical composition comprising a therapeutically effective amount of the FLT3-HDAC dual-target inhibitor of claim 1 and a pharmaceutically acceptable excipient.

5. The use of the FLT3-HDAC dual-target inhibitor of claim 1 in the preparation of a medicament for treating target-related tumor diseases, wherein the tumor is leukemia.

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