Dnmt1-hdac dual-targeting inhibitors, preparation method and application thereof

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By designing and synthesizing a dual-target inhibitor of DNMT1 and HDAC, the drug interaction limitation problem of combining DNMT and HDAC inhibitors in the treatment of cancer in existing technologies has been solved. Selective inhibition of DNMT1 and HDAC has been achieved, significantly inhibiting tumor growth and demonstrating significant anti-tumor activity and research value.

CN119306700BActive Publication Date: 2026-06-02SHANDONG UNIV +1

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Patent Information

Authority / Receiving Office: CN · China
Patent Type: Patents(China)
Current Assignee / Owner: SHANDONG UNIV
Filing Date: 2024-08-28
Publication Date: 2026-06-02

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Abstract

The present application relates to a kind of DNMT1-HDAC dual-target inhibitor as shown in formula (I) and its preparation method and application, the present application compound not only shows good DNMT1 and HDAC enzyme inhibitory activity, and also has significant antitumor activity to solid tumor and blood tumor.This kind of compound is as the first reported DNMT1 and HDACs-based dual-target inhibitor, has great prospect in cancer treatment.

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Description

Technical Field

[0001] This invention relates to a DNMT1-HDAC dual-target inhibitor, its preparation method, and its application, belonging to the field of pharmaceutical technology. Background Technology

[0002] Cancer is a disease characterized by genetic abnormalities, often exhibiting resistance to treatment and a high mortality rate. However, epigenetics is increasingly recognized as a contributing factor to cancer behavior, which cannot be explained solely by genetic alterations. Typically, the heterogeneity of tumor cells in terms of growth potential, invasiveness, malignant transformation capacity, and resistance to treatment interventions can be attributed to interference with epigenetic regulation. Compared to normal cells, cancer cells induce extensive and profound changes in the epigenome, including DNA methylation, histone acetylation, and chromatin remodeling. Among these, abnormal histone tail lysine acetylation and hypermethylation of CpG islands in tumor suppressor gene promoter regions are driving factors and biomarkers of tumor progression and poor prognosis.

[0003] DNA methylation is regulated by DNA methyltransferases (DNMTs). The human DNMT family comprises five members, which can be further divided into two groups: canonical members (DNMT1, DNMT3A, and DNMT3B), which can add s-adenosyl-L-methionine (SAM)-derived methyl groups to the C-5 position of CpG islands of cytosine, and atypical members (DNMT2 and DNMT3L), which lack catalytic activity. Unlike DNMT3A and DNMT3B, which establish methylation patterns de novo, DNMT1 is responsible for maintaining DNA methylation and transferring the methylation pattern from the pattern strand to the daughter strand. Furthermore, compared to other DNMTs, DNMT1 levels are dominant in both normal tissues and metastatic cancer tissues. Currently, DNMT inhibitors are divided into covalent nucleoside inhibitors and non-nucleoside reversible inhibitors. The former are represented by azacitidine (AZA), decitabine (DAC), and sibraline, while the latter are represented by RG108 and SGI-1027.

[0004] Histone deacetylases (HDACs) remove acetyl groups from lysine residues in histone tails or non-histone substrates. Eighteen HDACs have been identified in humans and classified into four classes based on their catalytic mechanism and intracellular location: zinc-dependent class I (HDAC1, 2, 3, 8), class IIa (HDAC4, 5, 7, 9), class IIb (HDAC6, 10), class IV (HDAC11), and NAD+. +Class III (Sirtuin 1-7) HDAC inhibitors. To date, HDAC inhibitors are commonly used to treat hematologic malignancies, and some inhibitors have been approved by the FDA, including vorinostat (SAHA), belistat (PDX101), pabistat (LBH589), chidamide (CS055), and romidesin (FK 228).

[0005] DNMT inhibitors and HDAC inhibitors are used in combination to treat various cancers, such as acute myeloid leukemia (AML), multiple myeloma (MM), diffuse large B-cell lymphoma (DLBCL), T-cell lymphoma (TCL), and myelodysplastic syndromes. For example, the clinical efficacy observed in AML patients treated with the combination of azacitidine and psambisarine is superior to that of psambisarine monotherapy. While the combination of DNMT and HDAC inhibitors has certain advantages in the treatment of AML and in tumor immunotherapy, drug-drug interactions limit the realization of synergistic effects, such as complex dosing regimens and negative impacts on pharmacokinetics. In contrast, designing dual-target DNMT / HDAC inhibitors can circumvent these potential drawbacks. However, currently reported dual-target DNMT / HDAC inhibitors have poor inhibitory effects on either DNMT or HDAC and lack subtype selectivity for DNMT1, resulting in no significant therapeutic advantage compared to monotherapy.

[0006] Therefore, developing an effective dual inhibitor of DNMT1 / HDAC is of great clinical significance. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a DNMT1-HDAC dual-target inhibitor, its preparation method, and its application.

[0008] The technical solution of the present invention is as follows:

[0009] A DNMT1-HDAC dual-target inhibitor, wherein the inhibitor has the structural formula shown in Formula (I) or a pharmaceutically acceptable salt of Formula (I):

[0010]

[0011] Wherein, X is an aromatic heterocycle or one of the following linking groups:

[0012]

[0013] A can be one of the following structures:

[0014]

[0015] R1 is a hydroxyl group or a 2-aminophenyl group;

[0016] R2 is one of the following structures

[0017]

[0018] R3 is a hydrogen atom with one of the following structures:

[0019]

[0020] The DNMT1-HDAC dual-target inhibitor is selected from one of the following compounds:

[0021] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -Hydroxybutyramide (11a);

[0022] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutaramide (11b);

[0023] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -Hydroxyhexadiamide (11c);

[0024] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 7 -Hydroxyheptanamide (11d);

[0025] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 8 -Hydroxyoctadiamide (11e);

[0026] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 9 -Hydroxynonadiamide (11f);

[0027] N 1-(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -Hydroxy-p-toluamide (11g);

[0028] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-4-((4-(hydroxycarbamoyl)benzyl)oxy)benzamide (11h);

[0029] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11i);

[0030] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)glutaramide (11j);

[0031] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -(4-(hydroxycarbamoyl)phenyl)hexamethylenediamide (11k);

[0032] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11l);

[0033] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)glutaramide (11m);

[0034] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N4 -(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)butadiamide (11n);

[0035] 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-hydroxybenzamide (11o);

[0036] (E)-3-4-(((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (11p);

[0037] 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(hydroxycarbamoyl)phenyl)carbamoyl)phenyl)piperidine 4-ammonium salt (11q);

[0038] (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)carbamoyl)benzyl)piperidine-4-ammonium salt (11r);

[0039] (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)oxo)benzyl)piperidine-4-ammonium salt (11s);

[0040] 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((7-(hydroxyamino)-7-oxohepyl)oxo)benzyl)piperidine-4-ammonium salt (11t);

[0041] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-N-hydroxypyrimidine-5-carboxamide (11u);

[0042] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (11v);

[0043] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15a)

[0044] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15b);

[0045] (E)-3-(4-((4-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (19a);

[0046] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (19b);

[0047] (E)-3-(4-((((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide (23a);

[0048] (R,E)-3-(4-((((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide((R)-23a);

[0049] (S,E)-3-(4-((((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide((S)-23a);

[0050] 2-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)-N-hydroxypyrimidine-5-carboxamide (23b);

[0051] (E)-3-(4-((4-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (27);

[0052] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(2-aminophenyl)pentanediamide (30a);

[0053] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(2-aminophenyl)terephthalamide (30b);

[0054] 4-(2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-2-oxoethyl)-N-(2-aminophenyl)benzamide (30c);

[0055] 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-(2-aminophenyl)benzamide (30d);

[0056] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-(2-aminophenyl)pyrimidine-5-carboxamide (30e);

[0057] N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 4 -Hydroxybutyramide (40a);

[0058] N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 5 -Hydroxyglutaramide (40b);

[0059] N 1-(3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)propyl)-N 4 -Hydroxybutyramide (40c);

[0060] 2-((2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (40d);

[0061] N 1 -(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutamate (44a);

[0062] (E)-(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutaramide (44b);

[0063] (E)-3-(4-(((1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (44c);

[0064] 2-(4-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperazin-1-yl)N-hydroxypyrimidine-5-carboxamide (48).

[0065] The DNMT1-HDAC dual-target inhibitor is selected from one of the following structures:

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] A second objective of this invention is to provide a method for preparing a DNMT1-HDAC dual-target inhibitor, the method comprising any of the following methods:

[0073] The preparation method of compound 11a-11V is as follows:

[0074]

[0075] Reagents and conditions: (a) p-Toluenesulfonyl chloride, N,N-diisopropylethylamine, 4-dimethylaminopyridine, acetonitrile, 80°C, 6 h; (b) n-Propanal, N-methylmorpholine, anhydrous ethanol, room temperature, 30 h; (c) N,N-diisopropylethylamine, N,N-dimethylformamide, 85°C, 5 h; (d) Isoamyl nitrite, copper chloride, acetonitrile, 80°C, 2.5 h; (e) Aminopiperidine derivative, carbonic acid Potassium, dichloromethane, 4 hours; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (g) 1) 9a-n, isobutyl chloroformate, N-methylmorpholine, dichloromethane / tetrahydrofuran (1:1), room temperature, 6 hours; 2) 9o-t, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 3) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (h) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours;

[0076] The preparation methods of compounds 15a, 15b, 19a, and 19b are as follows:

[0077]

[0078] Reagents and conditions: (a) aminopiperidine derivative, potassium carbonate, dichloromethane, 4 h; (b) 4 M hydrochloric acid / ethyl acetate, room temperature, 12 h; (c) 9 u, potassium carbonate, acetonitrile, 80 °C, 6 h; (d) 4 M hydrochloric acid / ethyl acetate, room temperature, 12 h; (e) piperazine-1-tert-butylcarboxylate, potassium carbonate, dichloromethane, 4 h; (f) 4 M hydrochloric acid / ethyl acetate, room temperature, 12 h; (g) 1) 9 p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; 2) 9 u, potassium carbonate, acetonitrile, 80 °C, 6 h; (h) 4 M hydrochloric acid / ethyl acetate, room temperature, 12 h;

[0079] The preparation methods of compounds 23a, (R)-23a, (S)-23a, 23b, 27, and 30a-e are as follows:

[0080]

[0081] Reagents and conditions: (a) Piperidine-4-aminomethyl tert-butylcarbamate, potassium carbonate, dichloromethane, 4 h; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (c) 1) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; 2) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (e) tert-butyl(2-(methylamino)ethyl)carbamate, potassium carbonate, dichloromethane, 4 h; (f) 4M hydrochloric acid / acetic acid Ethyl ester, room temperature, 12 hours; (g) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; (h) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (i) 1) 28a-c, isobutyl chloroformate, N-methylmorpholine, dichloromethane / tetrahydrofuran (1:1), room temperature, 6 hours; 2) 28d, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 3) 28e, potassium carbonate, acetonitrile, 80°C, 6 hours; (j) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours;

[0082] The preparation method of compound 40a-d is as follows:

[0083]

[0084] Reagents and conditions: (a) Methanesulfonyl chloride, triethylamine, dichloromethane, room temperature, 4 h; (b) diisobutylaluminum hydride, tetrahydrofuran, -20 °C, 6 h; (c) p-Toluenesulfonyl chloride, triethylamine, DMAP, acetonitrile, 80 °C, 4 h; (d) 4, Triethylamine, DMF, 85 °C, 5 h; (e) Isoamyl nitrite, copper chloride, acetonitrile, 80 °C, 2.5 h; (f) 2-Methylamino derivative, potassium carbonate, dichloromethane, 4 h; (g) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (h) 1) 9a or 9b, isobutyl chloroformate, N-methylmorpholine, dichloromethane / tetrahydrofuran (1:1), room temperature, 6 h; 2) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (i) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h;

[0085] The preparation methods of compounds 44a-c and 48 are as follows:

[0086]

[0087] Reagents and conditions: (a) tert-butyl 4-carbamate, potassium carbonate, dichloromethane, 4 h; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (c) 1) 9b, isobutyl chloroformate, N-methylmorpholine, dichloromethane / tetrahydrofuran (1:1), room temperature, 6 h; 2) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; 3) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (e) piperazine-1-tert-butylcarboxylate, potassium carbonate, dichloromethane, 4 h; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (g) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (h) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h.

[0088] A pharmaceutical composition comprising a therapeutically effective amount of the said dual-target inhibitors of DNMT1 and HDAC and pharmaceutically acceptable excipients.

[0089] The application of the DNMT1-HDAC dual-target inhibitor in the preparation of drugs for treating target-related tumor diseases.

[0090] The tumors mentioned are 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, or myeloma.

[0091] Technical features and advantages of the present invention:

[0092] 1. The compounds of the present invention not only exhibit good inhibitory activity against DNMT1 and HDAC enzymes, and can inhibit the activity of DNMT1 and HDAC in cells, but also have strong in vitro and in vivo antitumor activity, and can significantly delay tumor growth.

[0093] 2. The compounds of this invention, as the first reported dual-target antitumor drugs based on DNMT1 and HDAC, have further development and research value. Attached Figure Description

[0094] Figure 1 The graph shows the in vivo antitumor activity of compound (R)-23a and the combination therapy (GSK5032 and SAHA) against human acute monocytic leukemia cells MV4-11. In the graph, A: changes in body weight of tumor-bearing mice; B: tumor volume growth curve of tumor-bearing mice.

[0095] Figure 2 The graph shows the in vivo antitumor activity of compound (R)-23a and the combination therapy (GSK5032 and SAHA) against MC38 colon cancer cells in mice. In the graph, A: changes in body weight of tumor-bearing mice; B: tumor volume growth curve of tumor-bearing mice. Detailed Implementation

[0096] The features of the present invention are further described below through examples, but the present invention is not limited to the following examples.

[0097] The reagents, raw materials, or intermediates used in the examples are all commercially available or can be prepared according to literature methods. Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer.

[0098] Example 1: Preparation of Intermediate 6

[0099] 2-Amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate (2)

[0100] 2-Hydroxy-2-phenylacetamide (10 g, 66.15 mmol), N,N-diisopropylethylamine (DMAP, 25.65 g, 198.46 mmol), and 4-dimethylaminopyridine (1.21 g, 9.92 mmol) were added to acetonitrile (100 mL) with 4-methylbenzenesulfonyl chloride (18.92 g, 99.23 mmol). The mixture was stirred at room temperature for 6 h, and then the solvent was removed by rotary evaporation. The residue was washed with 100 mL of water, and the mixture was extracted with ethyl acetate (30 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compound 2. The synthesis methods of compounds (R)-2, (S)-2, and 34 were the same as those described above.

[0101] White solid, yield 77.2%. 1 H NMR (400MHz, DMSO-d6) δ7.77(s,2H),7.48(d,J=7.9Hz,1H),7.43(d,J=8.1Hz,4H),7.34(s,4H),5.68(s,1H),2.40(s,3H).

[0102] (R)-2-amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate ((R)-2)

[0103] White solid, yield 69.1%. 1 H NMR (400MHz, DMSO) δ7.79–7.73(m,3H),7.45–7.38(m,3H),7.38–7.28(m,5H),5.68(s,1H),2.40(s,3H).

[0104] (S)-2-amino-2-oxo-1-phenylethyl-4-methylbenzenesulfonate ((S)-2)

[0105] White solid, yield 71.5%. 1H NMR (400MHz, DMSO) δ7.80–7.72(m,3H),7.47–7.38(m,3H),7.37–7.28(m,5H),5.68(s,1H),2.40(s,3H).

[0106] 4-Methylmorpholine-4-ammonium salt-6-amino-3,5-dicyano-4-ethylpyridine-2-thioester (4)

[0107] Propanal (870 mg, 14.98 mmol) was added to a solution of 2-cyanoacetamide (3 g, 29.96 mmol) and N-methyl porphyrin (3.03 g, 29.96 mmol) in EtOH (40 mL). The mixture was stirred at room temperature for 30 hours. The reaction mixture was filtered, the filter cake was collected and thoroughly washed with ethanol, and dried overnight to obtain compound 4.

[0108] Brown solid, yield 30.4%. 1 H NMR (400MHz, DMSO-d6) δ7.58(s,2H),3.67(s,2H),2.72(s,2H),2.62(q,J=7.5Hz,2H),2.51(s,3H),1.19(t,J=7.6Hz,3H).

[0109] 2-((6-amino-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide (5)

[0110] Compound 4 (661 mg, 1.96 mmol) and N,N-diisopropylethylamine (760 mg, 5.88 mmol) were dissolved in DMF (5 mL), and then compound 2 (598 mg, 1.96 mmol) was added. The mixture was heated to 85 °C and reacted for 5 hours, followed by rotary evaporation to remove the solvent. The residue was washed with 50 mL of water, and the mixture was extracted with ethyl acetate (15 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compound 5. The synthesis methods of compounds (R)-5, (S)-5, and 35 were the same as those described above. The product was a pale yellow solid with a yield of 68.1%. 1 H NMR (400MHz, DMSO-d6) δ7.90(s,2H),7.73(s,1H),7.59(d,J=7.3Hz,2H),7.41–7.27(m,4H),5.56(s,1H),2.69(q,J=7.5Hz,2H),1.18(t,J=7.6Hz,3H).

[0111] (R)-2-((6-amino-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide((R)-5)

[0112] Yellow solid, yield 40.7%. 1 H NMR (400MHz, DMSO) δ7.90(s,2H),7.73(s,1H),7.63–7.55(m,2H),7.37–7.28(m,4H),5.57(s,1H),2.68(t,J=7.6Hz,2H),1.18(t,J=7.6Hz,3H).

[0113] (S)-2-((6-amino-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide((S)-5)

[0114] Yellow solid, yield 45.1%. 1 H NMR (400MHz, DMSO) δ7.90(s,2H),7.73(s,1H),7.62–7.55(m,2H),7.41–7.26(m,4H),5.56(s,1H),2.68(t,J=7.6Hz,2H),1.18(t,J=7.6Hz,3H).

[0115] 2-((6-chloro-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide (6)

[0116] Compound 5a (402 mg, 1.19 mmol) and copper chloride (797 mg, 5.93 mmol) were dissolved in acetonitrile (20 mL), and then amyl nitrite (278 mg, 2.37 mmol) was added. The mixture was heated to 80 °C and reacted for 2.5 hours, followed by rotary evaporation to remove the solvent. The residue was washed with 100 mL of water, and the mixture was extracted with ethyl acetate (30 mL × 3). The separated organic phase was dried over anhydrous sodium sulfate and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compound 6. The synthesis of compounds (R)-6, (S)-6, and 36 was performed using the same procedure. The product was a pale yellow solid with a yield of 31.3%. 1 H NMR (400MHz, Chloroform-d) δ7.61–7.47(m,3H),7.40(d,J=6.6Hz,4H),5.63(s,1H),2.99(q,J=7.5Hz,2H),1.36(t,J=7.7Hz,3H).

[0117] (R)-2-((6-chloro-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide((R)-6)

[0118] Pale yellow solid, yield 31.7%. 1H NMR (400MHz, DMSO) δ7.99(s,1H),7.61–7.51(m,2H),7.43(s,1H),7.38(dd,J=8.2,6.3 Hz,2H),7.37–7.28(m,1H),5.69(s,1H),2.86(q,J=7.6Hz,2H),1.23(t,J=7.7Hz,3H).

[0119] (S)-2-((6-chloro-3,5-dicyano-4-ethylpyridin-2-yl)thio)-2-phenylacetamide((S)-6)

[0120] Pale yellow solid, yield 30.1%. 1 H NMR (400MHz, DMSO) δ7.99 (s, 1H), 7.61–7.54 (m, 2H), 7.45–7.28 (m, 4H), 5.69 (s, 1H), 2.86 (q, J = 7.6Hz, 2H), 1.23 (t, J = 7.7Hz, 3H).

[0121] Example 2: Preparation of intermediate 7a

[0122] Compound 6 (200 mg, 0.56 mmol) and potassium carbonate (232 mg, 1.68 mmol) were added to a CH2Cl2 solution (10 mL) containing tert-butyl piperidine-4-carbamate (224 mg, 1.12 mmol). The mixture was stirred at room temperature for 4 hours and then washed with 100 mL of water. The organic phase was then separated, dried over anhydrous sodium sulfate, and concentrated to give the crude product. The crude product was purified by silica gel column chromatography to give 7a. The synthesis of compounds 7b, 12a, 12b, 16, 20, (R)-20, (S)-20, 24, 37a-c, 41, and 45 was performed using the same procedure.

[0123] tert-butyl(1-6((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridinyl)piperidinyl)carbamate (7a)

[0124] White solid, yield 68.5%. 1 H NMR (400MHz, DMSO) δ7.91(s,1H),7.52(d,J=7.4Hz,2H),7.41–7.33(m,4H),6.94(d,J=7.7Hz,1H),5.52(s,1H),4.4 5(d,J=13.3Hz,2H),3.61(s,2H),2.76(q,J=7.5Hz,2H),1.88(d,J=12.9Hz,2H),1.41(s,9H),1.21(t,J=7.5Hz,3H).

[0125] tert-Butyl(1-6((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridinyl)piperidinyl(methyl)carbamate (7b)

[0126] White solid, yield 69.5%. 1 H NMR (400MHz, DMSO) δ7.91(s,1H),7.52(d,J=7.4Hz,2H),7.42–7.34(m,4H),6.94(d,J=7.7Hz,1H),5.52(s,1H),4.4 5(d,J=13.3Hz,2H),3.61(s,2H),2.76(q,J=7.5Hz,2H),1.88(d,J=12.9Hz,2H),1.41(s,9H),1.21(t,J=7.5Hz,3H).

[0127] Example 3: Preparation of intermediate 8a

[0128] 1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidine-4-ammonium salt (8a)

[0129] Compound 7a (292 mg, 0.56 mmol) was added to saturated ethyl hydrochloride (10 mL) and stirred at room temperature for 12 hours. After filtration, the filter cake was purified by recrystallization from ethyl acetate (20 mL) to give 8a. The synthetic methods for compounds 8b, 13a, 13b, 17, 21, (R)-21, (S)-21, 25, 38a-c, 42, and 46 conform to this procedure. The product is a white solid with a yield of 85.8%. 1 H NMR (400MHz, DMSO) δ8.22(s,3H),8.01(s,1H),7.54(d,J=7.4Hz,2H),7.41–7.32(m,4H),5.58(s,1H),4.59(d,J=13. 7Hz,2H),3.32–3.20(m,2H),2.77(q,J=7.6Hz,2H),2.09(d,J=12.6Hz,2H),1.65–1.57(m,2H),1.22(t,J=7.6Hz,3H).

[0130] 1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-methylpiperidine-4-ammonium salt (8b)

[0131] White solid, yield 90.4%. 1H NMR (400MHz, DMSO) δ9.03(s,2H),8.01(s,1H),7.53(d,J=7.4Hz,2H),7.39(dd,J=14.7,7.3Hz,4H),5.57(s,1H),4.63(d,J=13.6Hz,2H),3 .20(t,J=12.2Hz,3H),2.77(q,J=7.6Hz,2H),2.57(t,J=5.3Hz,3H),2.17(d,J=12.6Hz,2H),1.60(t,J=12.7Hz,2H),1.21(t,J=7.6Hz,3H).

[0132] Example 4: Preparation of intermediate 9a-f

[0133] O-(tetrahydro-2H-pyran-2-yl)hydroxylamine (1 g, 8.54 mmol), the corresponding cyclic anhydride (8.54 mmol), and triethylamine (2.6 g, 25.61 mmol) were added to 15 mL of CH2Cl2 and stirred at room temperature for 3 hours. The organic phase was then washed with saturated NaCl solution (30 mL), dried over anhydrous Na2SO4, and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain compounds 9a-f.

[0134] 4-O-4-(((tetrahydro-2H-pyran-2-yl)oxo)amino)butyric acid (9a)

[0135] White solid, yield 53%. 1 H NMR (400MHz, DMSO) δ4.79 (d, J=3.3Hz, 1H), 3.95–3.87 (m, 1H), 3.50 (dd, J=10.2, 5.7Hz, 1H),2.37(t,J=7.0Hz,2H),2.20(t,J=7.1Hz,2H),1.72–1.57(m,3H),1.57–1.44(m,3H).

[0136] 5-O-5-(((tetrahydro-2H-pyran-2-yl)oxo)amino)valeric acid (9b)

[0137] White solid, yield 45.7%. 1 H NMR(400MHz,DMSO)δ4.81(d,J=3.7Hz,1H),3.95–3.89(m,1H),3.50(dd,J=10.1,5.5Hz,1H ),2.19(t,J=7.4Hz,2H),2.02(t,J=7.4Hz,2H),1.74–1.60(m,5H),1.52(d,J=11.6Hz,3H).

[0138] 6-Oxo-6-(((tetrahydro-2H-pyran-2-yl)oxo)amino)hexanoic acid (9c)

[0139] White solid, yield 48.3%. 1 H NMR (400MHz, DMSO) δ10.92(s,1H),4.81(d,J=3.2Hz,1H),3.96–3.88(m,1H),3.50(dd,J=10 .3,5.3Hz,1H),2.22–2.15(m,2H),2.03–1.95(m,2H),1.71–1.58(m,3H),1.56–1.43(m,7H).

[0140] 7-Oxo-7-(((tetrahydro-2H-pyran-2-yl)oxo)amino)heptanoic acid (9d)

[0141] A colorless, oily liquid with a yield of 48.1%. 1 H NMR (400MHz, DMSO) δ12.01(s,1H),10.90(s,1H),4.79(d,J=3.3Hz,1H),3.95–3.87(m,1H),3.50(dd,J=10.1,5. 5Hz,1H),2.18(t,J=7.4Hz,2H),1.97(t,J=7.1Hz,2H),1.71–1.59(m,3H),1.53–1.39(m,7H),1.24–1.17(m,2H).

[0142] 8-Oxo-8-(((tetrahydro-2H-pyran-2-yl)oxo)amino)octanoic acid (9e)

[0143] White solid, yield 46%. 1 H NMR (400MHz, DMSO) δ10.90 (s, 1H), 4.80 (d, J = 3.2Hz, 1H), 3.96–3.88 (m, 1H), 3.53–3.45 (m, 1H), 2.28 (t,J=7.4Hz,2H),1.96(t,J=7.3Hz,2H),1.68–1.60(m,3H),1.54–1.39(m,7H),1.24(h,J=3.9Hz,4H).

[0144] 9-Oxo-9-(((tetrahydro-2H-pyran-2-yl)oxo)amino)decanoic acid (9f)

[0145] White solid, yield 64.3%. 1H NMR (400MHz, DMSO) δ11.97(s,1H),10.90(s,1H),4.80(d,J=3.3Hz,1H),3.95–3.89(m,1H),3.49(s,1H),2 .19(t,J=7.4Hz,2H),1.96(d,J=7.4Hz,2H),1.69–1.60(m,3H),1.52–1.45(m,7H),1.24(d,J=4.0Hz,6H).

[0146] Example 5: Preparation of intermediate 9g-9u

[0147] 4-((tetrahydro-2H-pyran-2-yl)carbamoyl)benzoic acid (9g)

[0148] The corresponding carboxylic acid (5.31 mmol), EDCI (1.23 g, 6.4 mmol), DMAP (843 mg, 6.9 mmol), and pyranolamine (622 mg, 5.31 mmol) were added to 15 mL of CH2Cl2 and reacted at room temperature for 8 hours. After the reaction was complete, the reaction solution was washed successively with 1 M hydrochloric acid aqueous solution (100 mL) and saturated sodium chloride aqueous solution (100 mL). The organic phase was then dried over anhydrous Na2SO4 and concentrated. Purification by silica gel column chromatography yielded 9 g of the product. The synthesis of compound 9h-u followed the same procedure. The product was a colorless oily liquid with a yield of 70.8%. 1 H NMR (400MHz, DMSO) δ11.81(s,1H),8.02(d,J=8.1Hz,2H),7.87(d,J=8.2Hz,2H),5.03(d, J=3.1Hz,1H),4.06(t,J=9.5Hz,1H),3.56–3.51(m,1H),1.73(s,3H),1.59–1.51(m,3H).

[0149] 4-((4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)benzyl)oxo)benzoic acid (9h)

[0150] White solid, yield 90.2%. 1H NMR (400MHz, DMSO) δ12.67(s,1H),11.67(s,1H),7.93–7.87(m,2H),7.79(d,J=8.0Hz,2H),7.55(d,J=8.1Hz,2H),7.14– 7.07(m,2H),5.26(s,2H),5.00(d,J=3.0Hz,1H),4.11–4.01(m,1H),3.57–3.48(m,1H),1.72(s,3H),1.57–1.51(m,3H).

[0151] 4-O-4-((4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)amino)butyric acid (9i)

[0152] White solid, yield 44.5%. 1 H NMR (400MHz, DMSO) δ11.52(s,1H),10.25(s,1H),7.87–7.53(m,4H),4.98(d,J=3.1Hz,1H),4.08–4.02(m ,1H),3.51(d,J=11.4Hz,1H),2.58(t,J=6.9Hz,2H),2.52(s,2H),1.82–1.63(m,3H),1.57–1.49(m,3H).

[0153] 5-Oxo-5-((4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)amino)valeric acid (9j)

[0154] White solid, yield 27.9%. 1 H NMR (400MHz, DMSO) δ11.54(s,1H),10.17(s,1H),7.95–7.53(m,4H),4.98(d,J=3.1Hz,1H),4.09–4.01(m,1H),3.52(dd ,J=9.8,5.4Hz,1H),2.38(t,J=7.4Hz,2H),2.28–2.22(m,2H),1.85–1.78(m,2H),1.74–1.67(m,3H),1.59–1.51(m,3H).

[0155] 6-Oxo-6-((4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)amino)hexanoic acid (9k)

[0156] Pale yellow solid, yield 54%. 1H NMR (400MHz, DMSO) δ11.50(s,1H),10.15(s,1H),7.87–7.57(m,4H),4.98(d,J=3.2Hz,1H),4.12–4.00( m,1H),3.57–3.44(m,1H),2.34(t,J=7.1Hz,2H),2.22(t,J=7.3Hz,2H),1.71(s,3H),1.60–1.48(m,7H).

[0157] 4-O-4-((4-(((tetrahydro-2H-pyran-2-yl)carbamoyl)benzyl)amino)butyric acid (9l)

[0158] White solid, yield 54.2%. 1 H NMR (400MHz, DMSO) δ11.52(s,1H),10.22(s,1H),7.72(d,J=8.7Hz,2H),7.66(d,J=8.8Hz,2H),4.97(d,J=3.2Hz,1H),4.06 (s,1H),3.70(t,J=6.5Hz,2H),3.51(d,J=11.4Hz,1H),2.53(s,2H),2.07–2.01(m,2H),1.71(s,3H),1.54(d,J=5.7Hz,3H).

[0159] 5-O-5-((4-(((tetrahydro-2H-pyran-2-yl)carbamoyl)benzyl)amino)valeric acid (9m)

[0160] White solid, yield 48.1%. 1 H NMR (400MHz, DMSO) δ11.63(s,1H),8.45(t,J=6.0Hz,1H),7.72(d,J=8.1Hz,2H),7.32(d,J=8.0Hz,2H),4.99(d,J=3.1Hz,1 H),4.30(d,J=5.9Hz,2H),4.07(d,J=8.6Hz,1H),3.59(s,1H),2.24–2.17(m,4H),1.77–1.69(m,5H),1.54(d,J=5.7Hz,3H).

[0161] 4-Oxo-4-((4-(3-Oxo-3-(((tetrahydro-2H-pyran-2-yl)oxo)amino)propyl-1-en-1-yl)phenyl)amino)butyric acid (9n)

[0162] White solid, yield 38.0%. 1H NMR (400MHz, DMSO) δ12.15(s,1H),11.18(s,1H),10.15(s,1H),7.63(d,J=8.3Hz,2H),7.51(d,J=8.3Hz,2H),7.42(d,J=15.6Hz,1H),6.39(d,J =15.9Hz,1H),4.90(s,1H),3.96(s,1H),3.54(dd,J=11.1,4.8Hz,1H),2.58(t,J=6.6Hz,2H),2.53(d,J=5.8Hz,2H),1.69(s,3H),1.54(s,3H).

[0163] 4-Formyl-N-((tetrahydro-2H-pyran-2-yl)oxo)benzamide (9o)

[0164] White solid, yield 57.2%. 1 H NMR (400MHz, DMSO) δ11.88(s,1H),10.09(s,1H),8.04–7.94(m,4H),5.04(d,J=2.8Hz,1H ),4.10–4.03(m,1H),3.54(dd,J=11.3,4.6Hz,1H),1.79–1.71(m,3H),1.61–1.53(m,3H).

[0165] (E)-3-(4-formylphenyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)acrylamide (9p)

[0166] White solid, yield 39.1%. 1 H NMR (400MHz, DMSO) δ11.37(s,1H),10.02(s,1H),7.95(d,J=8.0Hz,2H),7.81(d,J=8.0Hz,2H),7.58(d,J=15.9Hz ,1H),6.67(d,J=15.9Hz,1H),4.93(s,1H),3.97(s,1H),3.55(dd,J=11.1,5.5Hz,1H),1.70(s,3H),1.55(s,3H).

[0167] 4-Formyl-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)benzyl)benzamide (9q)

[0168] White solid, yield 31.6%. 1H NMR (400MHz, DMSO) δ11.61(s,1H),10.09(s,1H),9.33(t,J=5.9Hz,1H),8.08(d,J=8.2Hz,2H),8.02(d,J=8.2Hz,1H),7 .73(d,J=8.2Hz,2H),7.42(d,J=8.0Hz,2H),4.98(s,1H),4.55(d,J=6.0Hz,2H),4.06(s,1H),1.72(s,2H),1.55(s,2H).

[0169] 4-Formyl-N-(4-(3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxo)amino)propyl-1-en-1-yl)benzyl)benzamide (9r)

[0170] White solid, yield 47.7%. 1 H NMR (400MHz, DMSO) δ11.24(s,1H),10.09(s,1H),9.24(t,J=6.0Hz,1H),8.09(d,J=8.2Hz ,1H),8.02(d,J=8.1Hz,1H),7.95(d,J=8.3Hz,1H),7.74(d,J=8.3Hz,1H),7.55(d,J=7.7H z,2H),7.48(d,J=15.5Hz,1H),7.37(dd,J=8.0,5.1Hz,2H),6.48(d,J=15.8Hz,1H),4.91 (s,1H),4.52(t,J=6.8Hz,2H),3.96(s,1H),3.55(s,1H),1.73–1.66(m,3H),1.54(s,3H).

[0171] (E)-3-(4-((4-formylphenoxy)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)acrylamide(9s)

[0172] White solid, yield 52.8%. 1 H NMR (400MHz, DMSO) δ11.27(s,1H),9.87(s,1H),7.92–7.84(m,2H),7.62(d,J=7.9Hz,2H),7.55–7.46(m,3H),7.26–7.18(m,2H), 6.53(d,J=15.8Hz,1H),5.27(s,2H),4.92(s,1H),3.98(d,J=9.7Hz,1H),3.54(d,J=11.5Hz,1H),1.75–1.67(m,3H),1.54(s,3H).

[0173] 7-(4-Formylphenoxy)-N-((tetrahydro-2H-pyran-2-yl)oxo)heptanamide (9t)

[0174] White solid, yield 62.9%. 1 H NMR (400MHz, DMSO) δ10.92(s,1H),9.86(s,1H),7.91–7.81(m,2H),7.15–7.07(m,2H),4.81(d,J=3.2Hz,1H),4.07(t,J=6.4Hz,2H),3.95–3.88(m,1 H),3.52–3.45(m,1H),2.01–1.97(m,2H),1.73(p,J=6.7Hz,3H),1.68–1.6 0(m,3H),1.51(q,J=7.6Hz,6H),1.43–1.37(m,2H),1.31(q,J=4.5Hz,2H).

[0175] 2-Chloro-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (9u)

[0176] White solid, yield 29.4%. 1 H NMR (400MHz, DMSO) δ11.46 (s, 1H), 8.66 (s, 2H), 4.95 (d, J = 3.3Hz, 1H), 4.08–3.97 (m, 1H), 1.75–1.65 (m, 3H), 1.57–1.50 (m, 3H).

[0177] Example 6: Preparation of intermediates 10a-10n

[0178] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -((tetrahydro-2H-pyran-2-yl)oxo)succinamide (10a)

[0179] Add 9a (190 mg, 0.875 mmol) and NMM (110 mg, 1.09 mmol) to THF (10 mL), then add isobutyl chlorocarbonate (120 mg, 0.875 mmol) and stir at 0 °C for 0.5 h. Add the resulting filtrate dropwise to CH₂Cl₂ (10 mL) containing 8a (200 mg, 0.437 mmol) and N-methylmorpholine (44 mg, 0.437 mmol) at 0 °C. Stir the mixture at room temperature for 6 h, then remove the solvent by rotary evaporation. Wash the residue with 100 mL of water and extract with ethyl acetate (30 mL × 3). Wash the organic phase successively with 1 M hydrochloric acid aqueous solution (100 mL), saturated sodium bicarbonate aqueous solution (100 mL), and saturated sodium chloride aqueous solution (100 mL). Dry the organic phase with anhydrous Na₂SO₄ and concentrate. 10a was obtained by silica gel column chromatography purification. The synthesis of compounds 10b-n, 29a-c, 39a-c, and 43a followed the same procedure. The product was a white solid in 44.1% yield. 1 H NMR (400MHz, DMSO) δ10.97 (s, 1H), 7.91 (d, J = 8.4Hz, 2H), 7.55–7.48 (m, 2H), 7.44–7 .30(m,4H),5.52(s,1H),4.79(s,1H),4.42(s,2H),3.91(s,2H),3.49(d,J=11.2Hz,3 H),2.76(q,J=7.4Hz,2H),2.32(t,J=7.7Hz,2H),2.24(d,J=7.2Hz,2H),1.88(d,J=1 2.7Hz,2H),1.64(s,3H),1.50(s,3H),1.43(t,J=10.3Hz,2H),1.20(t,J=7.6Hz,3H).

[0180] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -((tetrahydro-2H-pyran-2-yl)oxo)glutaramide (10b)

[0181] White solid, yield 50.7%. 1H NMR (400MHz, DMSO) δ10.98 (s, 1H), 8.00 (s, 1H), 7.90 (d, J = 7.6Hz, 1H), 7.56–7.51 (m, 2H), 7. 41–7.30(m,4H),5.56(s,1H),4.82(s,1H),4.48–4.37(m,2H),3.91(d,J=11.5Hz,2H),3.55– 3.43(m,3H),2.76(q,J=7.6Hz,2H),2.08(t,J=7.5Hz,2H),2.00(t,J=7.5Hz,2H),1.89(d,J= 13.5Hz,2H),1.78–1.74(m,2H),1.69–1.61(m,3H),1.54–1.40(m,5H),1.20(t,J=7.5Hz,3H).

[0182] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -((tetrahydro-2H-pyran-2-yl)oxo)hexadiamide(10c)

[0183] White solid, yield 60.4%. 1 1H NMR (400MHz, DMSO) δ10.91(s,1H),7.92(s,1H),7.83(d,J=7.6Hz,1H),7.57–7.49(m,2H ),7.40–7.31(m,4H),5.53(s,1H),4.80(s,1H),4.43(s,2H),3.91(s,2H),3.49(d,J=11. 4Hz,1H),3.09(d,J=7.2Hz,2H),2.76(q,J=7.6Hz,2H),2.07(s,2H),1.99(d,J=6.5Hz,2H ),1.89(d,J=12.7Hz,2H),1.63(s,3H),1.53–1.40(m,7H),1.21(dd,J=10.9,7.4Hz,5H).

[0184] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 7 -((tetrahydro-2H-pyran-2-yl)oxo)heptanediamide (10d)

[0185] White solid, yield 66.2%. 1H NMR (400MHz, DMSO) δ10.91(s,1H),7.94(s,1H),7.83(d,J=7.6Hz,1H),7.55–7.49(m,2H),7. 42–7.30(m,4H),5.53(s,1H),4.79(t,J=3.0Hz,1H),4.49–4.37(m,2H),3.97–3.84(m,2H),3 .49(d,J=11.4Hz,1H),3.37(s,2H),2.75(q,J=7.5Hz,2H),2.05(t,J=7.4Hz,2H),1.97(d,J= 7.3Hz,2H),1.88(d,J=12.7Hz,2H),1.68–1.58(m,3H),1.53–1.39(m,9H),1.24–1.17(m,5H).

[0186] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 8 -((tetrahydro-2H-pyran-2-yl)oxo)octadiamide (10e)

[0187] White solid, yield 31.1%. 1 H NMR(400MHz,DMSO)δ10.92(s,1H),7.94(s,1H),7.90–7.76(m,1H),7.55–7 .49(m,2H),7.48–7.17(m,4H),5.53(s,1H),4.84(s,1H),4.57–4.41(m,2H) ,4.00–3.87(m,2H),3.55–3.51(m,3H),2.76(d,J=7.8Hz,2H),2.34–2.26(m ,4H),2.05(t,J=7.4Hz,2H),1.86(s,2H),1.53–1.40(m,10H),1.25(m,7H).

[0188] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 9 -((tetrahydro-2H-pyran-2-yl)oxo)octadiamide (10f)

[0189] White solid, yield 83%. 1H NMR (400MHz, DMSO) δ10.91(s,1H),7.94(s,1H),7.83(d,J=7.6Hz,1H),7.60–7.49(m,2H) ,7.46–7.29(m,4H),5.54(s,1H),4.79(s,1H),4.44(dd,J=13.1,8.9Hz,2H),3.96–3.86(m ,2H),3.49(d,J=11.0Hz,1H),2.76(q,J=7.6Hz,2H),2.06(t,J=7.4Hz,2H),1.97(d,J=7.4 Hz,2H),1.88(d,J=12.6Hz,2H),1.69–1.58(m,3H),1.56–1.38(m,9H),1.22–1.17(m,9H).

[0190] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -((tetrahydro-2H-pyran-2-yl)oxo)terephthalamide (10g)

[0191] White solid, yield 48.2%. 1 H NMR (400MHz, DMSO) δ11.79(s,1H),8.51(d,J=7.8Hz,1H),7.95(d,J=7.8Hz,2H),7.85(d ,J=8.2Hz,2H),7.52(d,J=7.5Hz,2H),7.44–7.30(m,4H),5.55(s,1H),5.02(d,J=3.3Hz, 1H),4.58(t,J=14.1Hz,2H),4.27–4.13(m,1H),4.08(d,J=7.7Hz,1H),3.54(q,J=6.6Hz, 3H),2.77(q,J=7.6Hz,2H),2.05–1.97(m,2H),1.77–1.53(m,8H),1.19(t,J=7.5Hz,3H).

[0192] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-4-((4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)oxo)benzamide (10h)

[0193] White solid, yield 41.4%. 1H NMR (400MHz, DMSO) δ11.67 (s, 1H), 8.21 (d, J = 7.8Hz, 1H), 7.94 (s, 1H), 7.82 (dd ,J=21.7,8.3Hz,4H),7.53(t,J=7.8Hz,4H),7.47–7.25(m,4H),7.09(d,J=8.6Hz ,2H),5.54(s,1H),5.25(s,2H),5.00(s,1H),4.57(t,J=13.7Hz,2H),4.17(s,2H ),2.77(t,J=7.6Hz,2H),1.99(s,2H),1.69–1.49(m,8H),1.21(t,J=7.5Hz,3H).

[0194] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)butyramide(10i)

[0195] White solid, yield 38.6%. 1 H NMR (400MHz, DMSO) δ11.56(s,1H),10.36(s,1H),8.04(s,1H),7.71(q,J=8.7Hz,4H),7.53(d,J =7.4Hz,2H),7.40–7.31(m,5H),5.57(s,1H),4.98(s,1H),4.43(d,J=13.0Hz,2H),3.91(s,2H) ,3.51(d,J=11.8Hz,2H),2.75(d,J=7.7Hz,2H),2.61(t,J=6.9Hz,2H),2.43(t,J=7.2Hz,2H),1 .89(d,J=12.5Hz,2H),1.71(s,2H),1.54(s,3H),1.45(t,J=12.6Hz,3H),1.22(t,J=7.6Hz,3H).

[0196] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)glutaramide(10j)

[0197] White solid, yield 39.2%. 1H NMR (400MHz, DMSO) δ11.54(s,1H),10.32(s,1H),8.01(s,2H),7.71(q,J=8.7Hz,4H),7.52(d,J= 7.4Hz,2H),7.42–7.30(m,5H),5.57(s,1H),4.98(s,1H),4.41(d,J=13.0Hz,2H),4.05(d,J=7.6 Hz,1H),3.91(s,1H),3.51(d,J=11.7Hz,2H),2.75(q,J=7.7Hz,2H),2.61(t,J=6.9Hz,2H),2.41 (t,J=7.0Hz,2H),1.87(d,J=12.6Hz,2H),1.71(s,2H),1.66–1.30(m,8H),1.20(t,J=7.6Hz,3H).

[0198] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)phenyl)hexadiamide(10k)

[0199] White solid, yield 41.4%. 1 H NMR (400MHz, DMSO) δ11.60(s,1H),10.52(s,1H),8.13(s,1H),8.06(d,J=7.6Hz,1H),7.73(s,4 H),7.58–7.52(m,2H),7.40–7.29(m,4H),5.61(s,1H),4.98(s,1H),4.42(d,J=13.3Hz,2H),4.1 0–4.01(m,1H),3.91(s,1H),3.51(d,J=11.2Hz,3H),2.75(q,J=7.6Hz,2H),2.40(t,J=6.8Hz,2 H),2.18–2.10(m,2H),1.87(d,J=12.8Hz,2H),1.71(s,3H),1.55(s,9H),1.20(t,J=7.6Hz,3H).

[0200] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)benzyl)succinamide (10l)

[0201] White solid, yield 42.5%. 1 H NMR (400MHz, DMSO) δ11.60(s,1H),8.46(t,J=6.0Hz,1H),8.00(s,1H),7.94(d,J=7.5Hz,1H),7.72(d, J=7.9Hz,2H),7.52(d,J=7.5Hz,2H),7.40–7.31(m,6H),5.56(s,1H),4.98(s,1H),4.40(d,J=10.8Hz, 2H),4.30(d,J=6.0Hz,2H),4.05(d,J=6.2Hz,1H),3.90(s,1H),3.54–3.43(m,2H),2.75(q,J=7.6Hz,2 H),2.43–2.33(m,4H),1.87(d,J=12.5Hz,2H),1.71(s,2H),1.62–1.35(m,6H),1.21(d,J=7.6Hz,3H).

[0202] N 1 -(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)benzyl)glutaramide(10m)

[0203] White solid, yield 41.3%. 1 H NMR (400MHz, DMSO) δ11.58(s,1H),8.42(t,J=6.0Hz,1H),7.98(s,1H),7.92(d,J=7.5Hz,1H),7.70(d, J=7.9Hz,2H),7.48(d,J=7.5Hz,2H),7.42–7.32(m,6H),5.57(s,1H),4.96(s,1H),4.40(d,J=10.8Hz, 2H),4.38(d,J=6.0Hz,2H),4.08(d,J=6.2Hz,1H),3.92(s,1H),3.52–3.41(m,2H),2.76(q,J=7.6Hz,2 H),2.41–2.31(m,4H),1.88(d,J=12.5Hz,2H),1.72(s,2H),1.64–1.31(m,8H),1.21(t,J=7.6Hz,3H).

[0204] (E)-N 1-(1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(3-oxo-3-(((tetrahydro-2H-pyran-2-yl)oxo)amino)propyl-1-en-1-yl)phenyl)butadiamide(10n)

[0205] White solid, yield 35.9%. 1 H NMR (400MHz, DMSO) δ11.22(s,1H),10.22(s,1H),7.99(d,J=8.2Hz,2H),7.65(d,J=8.3Hz,2H) ,7.59–7.45(m,4H),7.45–7.24(m,5H),6.40(d,J=15.8Hz,1H),5.55(s,1H),4.90(s,1H),4.42 (s,2H),3.93(s,2H),3.53(d,J=11.8Hz,2H),2.76(q,J=7.0Hz,2H),2.59(t,J=7.2Hz,2H),2. 43(t,J=7.2Hz,2H),1.93–1.85(m,2H),1.69(s,3H),1.58–1.39(m,5H),1.21(t,J=7.6Hz,3H).

[0206] Example 7: Preparation of intermediate 10o-10t

[0207] 4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)benzamide(10o)

[0208] 8a (350 mg, 0.766 mmol) and 9o (287 mg, 1.15 mmol) were added to a mixed solvent of MeOH / CH2Cl2 (1:1, 10 mL). After reacting at room temperature for 9 hours, sodium triacetoxyborohydride (649 mg, 3.06 mmol) was added, and the mixture was stirred at room temperature for another 3 hours. After removing the solvent by rotary evaporation, the residue was washed with 50 mL of water, and the mixture was extracted with ethyl acetate (30 mL × 3). The separated organic phase was washed successively with 1 M hydrochloric acid aqueous solution (100 mL), saturated sodium bicarbonate aqueous solution (100 mL), and saturated sodium chloride aqueous solution (100 mL). The organic phase was then dried over anhydrous Na2SO4 and concentrated. Purification by silica gel column chromatography yielded 10o. The synthesis of compounds 10p-t, 18a, 22, (R)-22, (S)-22, 26, 29d, and 43b followed the same procedure. White solid, yield 41.2%. 1 H NMR (400MHz, DMSO) δ11.87(s,1H),9.58(d,J=6.5Hz,2H),8.04(s,1H),8.00(d,J=8.0Hz,4H),7.95(d,J= 8.0Hz,2H),7.51(d,J=7.5Hz,2H),7.36(m,4H),5.61(s,1H),5.03(d,J=2.8Hz,1H),4.65(d,J=13.4Hz,2 H),4.26(t,J=6.0Hz,2H),4.08(m,1H),3.53–3.42(m,2H),3.52–3.39(m,1H),3.22(d,J=13.0Hz,2H),2. 77(q,J=7.6Hz,2H),2.29(d,J=12.5Hz,2H),1.85–1.69(m,5H),1.58–1.53(m,3H),1.21(t,J=7.7Hz,3H).

[0209] (E)-3-(4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)acrylamide(10p)

[0210] White solid, yield 39.6%. 1H NMR (400MHz, DMSO) δ11.23 (s, 1H), 7.93 (d, J = 2.1Hz, 1H), 7.56–7.49 (m, 4H), 7.47–7.39 ( m,3H),7.39–7.28(m,4H),6.48(d,J=15.7Hz,1H),5.53(s,1H),4.91(s,1H),4.39(d,J=13 .1Hz,2H),3.96(s,1H),3.54(dd,J=10.0,5.5Hz,2H),2.75(q,J=7.3Hz,3H),1.94(d,J=1 2.5Hz,2H),1.70(s,3H),1.54(d,J=4.8Hz,3H),1.44–1.32(m,2H),1.20(t,J=7.6Hz,3H).

[0211] 4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)benzyl)benzamide(10q)

[0212] White solid, yield 38.2%. 1 H NMR (400MHz, DMSO) δ11.20(s,1H),9.21(t,J=6.1Hz,1H),7.99(s,1H),7.98(d,J=8.0Hz,2H),7.80–7.70( m,4H),7.54–7.52(m,2H),7.41–7.27(m,6H),5.61(s,1H),4.88(s,1H),4.66(d,J=13.4Hz,2H),4.53(d,J =5.9Hz,2H),4.28(t,J=6.1Hz,2H),3.91(s,1H),3.36(m,1H),3.20(t,J=12.8Hz,2H),2.77(q,J=7.5Hz,2 H),2.30(d,J=12.4Hz,2H),1.77(dt,J=15.2,7.2Hz,2H),1.65(s,3H),1.54(s,3H),1.21(t,J=7.8Hz,3H)

[0213] (E)-4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)carbamoyl)propyl-1-en-1-yl)benzyl)benzamide(10r)

[0214] White solid, yield 35.1%. 1 H NMR (400MHz, DMSO) δ11.25(s,1H),9.07(t,J=6.1Hz,1H),7.95(s,1H),7.89(d,J=8.0Hz ,2H),7.62–7.43(m,7H),7.43–7.27(m,6H),6.48(d,J=15.9Hz,1H),5.54(s,1H),4.91( s,1H),4.47(dd,J=27.3,9.5Hz,4H),3.92(d,J=29.0Hz,4H),2.75(q,J=7.5Hz,2H),2.0 3–1.95(m,2H),1.70(s,3H),1.53(d,J=5.0Hz,3H),1.42(s,2H),1.20(t,J=7.6Hz,3H).

[0215] (E)-3-(4-((4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenoxy)methyl)phenyl)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)acrylamide(10s)

[0216] White solid, yield 33.8%. 1 H NMR (400MHz, DMSO) δ11.29(s,1H),7.98(s,1H),7.60(d,J=7.9Hz,2H),7.51(dd,J=15.5,7.7Hz,5H) ,7.39(dd,J=8.2,6.2Hz,4H),7.37–7.30(m,2H),7.03(d,J=8.1Hz,2H),6.53(d,J=15.9Hz,1H),5.56 (s,1H),5.15(s,2H),4.92(s,1H),4.51(d,J=13.1Hz,2H),3.93(d,J=24.3Hz,2H),3.57–3.51(m,1H ),3.23(s,2H),2.76(d,J=7.6Hz,2H),2.08(s,2H),1.70(s,2H),1.54(s,5H),1.21(t,J=7.6Hz,3H).

[0217] 7-(4-(((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenoxy)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)benzamide(10t)

[0218] White solid, yield 32.1%. 1 H NMR(400MHz,DMSO)δ10.90(s,1H),7.93(s,1H),7.52(d,J=7.4Hz,2H),7.45– 7.22(m,6H),6.92(s,2H),5.54(s,1H),4.80(s,1H),4.47(s,2H),3.95(t,J= 6.5Hz,2H),3.48(s,2H),2.76(q,J=7.5Hz,2H),1.99(t,J=7.3Hz,2H),1.74– 1.59(m,5H),1.55–1.34(m,8H),1.31(d,J=7.4Hz,2H),1.21(t,J=7.6Hz,3H).

[0219] Example 8: Preparation of intermediate 10u

[0220] 8a (356 mg, 0.78 mmol), 9u (242 mg, 0.94 mmol), and K₂CO₃ (162 mg, 1.17 mmol) were added to acetonitrile (10 mL), and the reaction was carried out at 80 °C for 6 hours. The reaction solution was diluted with 50 mL of water and extracted with ethyl acetate (20 mL × 3). The separated organic phase was dried over anhydrous Na₂SO₄ and concentrated to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 10u. The synthesis of compounds 10v, 14a, 14b, 18b, 22b, 29e, 39d, 43c, and 47 were performed using the same procedure.

[0221] 2-((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (10u)

[0222] White solid, yield 29.6%. 1 H NMR (400MHz, DMSO) δ11.51(s,1H),8.66(s,2H),7.97–7.89(m,2H),7.51(d,J=7.5Hz,2H),7.44–7.30(m,4H),5.53(s,1H),4.95(s,1H), 4.55(s,2H),4.18(s,1H),4.03(s,1H),2.76(q,J=7.3Hz,2H),2.03(d,J=13.3Hz,2H),1.71(s,3H),1.55(s,5H),1.21(t,J=7.6Hz,3H).

[0223] 2-((1-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-(4-(((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (10u)

[0224] White solid, yield 31.1%. 1 H NMR (400MHz, DMSO) δ11.54(s,1H),8.71(s,2H),7.92(s,1H),7.52(d,J=7.3H z,2H),7.44–7.31(m,4H),5.55(s,1H),4.98(d,J=16.4Hz,2H),4.74(d,J=13. 5Hz,2H),4.04(s,1H),3.51(s,1H),3.27(d,J=12.0Hz,2H),3.02(s,3H),2.77 (q,J=7.3Hz,3H),1.75(d,J=27.5Hz,7H),1.55(s,3H),1.22(t,J=7.5Hz,3H).

[0225] Example 9: Preparation of the target compound

[0226] Compound 10a (234 mg, 0.378 mmol) obtained in Example 6 was dissolved in saturated ethyl hydrochloride (10 mL), stirred at room temperature for 12 h, filtered, and the filter cake was purified by recrystallization with EA or MeOH (20 mL) to obtain 11a. The synthesis methods of compounds 11b-v, 15a, 15b, 19a, 19b, 23a, (R)-23a, (S)-23a, 23b, 27, 30a-e, 40a-d, 44a-c and 48 were the same as those steps.

[0227] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 11a-Hydroxybutyramide

[0228] White solid, yield 44.1%, mp 170-171℃. 1H NMR (400MHz, DMSO) δ10.30(s,1H),8.02–7.93(m,2H),7.53(d,J=7.4Hz,2H),7.42–7.30(m,4H),5.56(s,1H),4.48–4.37(m,2H),3.90(s,1H),3.4 1–3.30(m,2H),2.76(q,J=7.5Hz,2H),2.33(t,J=7.4Hz,2H),2.20(t,J= 7.4Hz,2H),1.92–1.83(m,2H),1.48–1.39(m,2H),1.18(t,J=7.5Hz,3H). 13 C NMR (101MHz, DMSO) δ174.32,174.06,170.81,170.04,169.99,164.87,164.78,164.40,158.46,136.66,129.12,128.67,128.63 ,116.43,115.15,93.78,87.59,53.64,47.47,46.93,45.68,32.04,30.55,29.62,29.24,27.58,14.01.HRMS-ESI(m / z)calc.for C 26 H 28 N7O4S[M–H] - 534.1929,found 534.1932.HPLC t R = 6.19 min (99.89% purity).

[0229] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 11b-hydroxyglutaramide

[0230] White solid, yield 55.8%, mp 171-173℃. 1H NMR (400MHz, DMSO) δ10.31(s,1H),8.01(s,1H),7.91(d,J=7.6Hz,1H),7.57–7.50 (m,2H),7.43–7.30(m,4H),5.56(s,1H),4.45–4.41(m,2H),3.91(d,J=8.6Hz,1H), 3.40–3.30(m,2H),2.76(q,J=7.5Hz,2H),2.08(t,J=7.4Hz,2H),2.01–1.94(m,2H ),1.91–1.85(m,2H),1.76–1.85(m,2H),1.51–1.40(m,2H),1.21(t,J=7.5Hz,3H). 13 C NMR (101MHz, DMSO) δ174.66,171.46,170.00,164.79,164.40,158.45,136.69,129.11,128.66,128.61,116.43,115.15,93.7 7,87.56,53.63,46.96,45.79,45.59,34.97,33.48,33.22,32.06,32.00,27.58,21.19,14.01,8.90.HRMS-ESI(m / z)calc.for C 27 H 30 N7O4S[MH] + 548.2085,found 548.2088.HPLC t R = 6.65 min (97.33% purity).

[0231] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 11c-hydroxyhexamethylenediamide

[0232] White solid, yield 34.5%, mp 173-175℃. 1H NMR (400MHz, DMSO) δ8.05(s,1H),7.94(d,J=7.6Hz,1H),7.54(d,J=7.4Hz,2H),7.43–7.30(m,4H),5.58(s,1H),4.43(d,J=14.0Hz,2H),3.91(s,1H) ,3.23–3.15(m,2H),2.76(q,J=7.6Hz,2H),2.07(d,J=7.6Hz,2H),1.98(d, J=9.2Hz,2H),1.88(d,J=12.7Hz,2H),1.48(s,6H),1.18(t,J=7.5Hz,3H). 13 C NMR (101MHz, DMSO) δ171.98,170.04,169.60,164.81,164.37,158.46,136.83,129.05,128.69,128.56,116.44,115.17,93 .77,87.54,53.59,47.04,46.95,45.54,35.87,32.71,32.06,28.84,27.57,25.70,25.52,14.00.HRMS-ESI(m / z)calc.for C 28 H 32 N7O4S[MH] + 562.2242,found 562.2246.HPLC t R = 6.49 min (98.83% purity).

[0233] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 7 11d-Hydroxyheptanamide

[0234] White solid, yield 59.6%, mp 173-175℃. 1H NMR (400MHz, DMSO) δ10.47(s,1H),8.70(s,1H),8.14(s,1H),7.97(d,J=7.5Hz,1H) ,7.55(d,J=7.2Hz,2H),7.41–7.30(m,4H),5.62(s,1H),4.44(d,J=14.3,2H),3.90 (s,1H),3.33(d,J=13.4Hz,2H),2.75(q,J=7.5Hz,2H),2.07(t,J=7.3Hz,2H),1.95 (t,J=7.3Hz,2H),1.87(d,J=12.6Hz,2H),1.60–1.36(m,6H),1.21(q,J=7.1Hz,5H). 13 C NMR (101MHz, DMSO) δ171.94,170.05,169.57,164.81,164.38,158.45,136.78,129.07,128.69,128.58,116.44,115.16,93 .74,87.52,53.59,47.02,46.94,45.54,35.78,32.62,32.06,28.68,27.57,25.53,25.39,14.01.HRMS-ESI(m / z)calc.for C 29 H 35 N7O4S[M+Na] + 600.2375,found 600.2379.HPLC t R =12.41min (98.19% purity).

[0235] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 8 11e-Hydroxyoctadiamide

[0236] Pale yellow solid, yield 35.2%, mp 177-179℃. 1H NMR (400MHz, DMSO) δ10.46 (s, 1H), 8.70 (s, 1H), 8.15 (s, 1H), 7.97 (d, J = 7.5Hz, 1H), 7 .55(d,J=7.3Hz,2H),7.35(dq,J=13.2,7.4Hz,4H),5.62(s,1H),4.51–4.38(m,2H),3 .90(s,1H),3.34(s,2H),2.75(q,J=7.5Hz,2H),2.06(q,J=6.9Hz,2H),1.94(t,J=7.2 Hz,2H),1.87(d,J=12.6Hz,2H),1.471.61–1.35(m,6H),1.23(dd,J=12.8,8.4Hz,7H). 13 C NMR (101MHz, DMSO) δ171.98,170.04,169.60,164.81,164.37,158.46,136.83,129.05,128.69,128.56,116.44,115.17,93 .77,87.54,53.59,47.04,46.95,45.54,35.87,32.71,32.06,28.84,27.57,25.70,25.52,14.00.HRMS-ESI(m / z)calc.for C 30 H 37 N7O4S[M+H] + 592.2706,found 592.2708.HPLC t R = 6.89 min (99.98% purity).

[0237] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 9 -Hydroxynonadiamide (11f)

[0238] White solid, yield 48.2%, mp 177-179℃. 1H NMR (400MHz, DMSO) δ10.37(s,1H),7.98(s,1H),7.86(d,J=7.6Hz,1H),7.56–7.49(m,2H),7.43–7.29(m,4H),5.55(s,1H),4.47–4.42(m,2H) ),3.34(t,J=12.2Hz,2H),2.76(q,J=7.6Hz,2H),2.06(t,J=7.4Hz,2H),1.98–1.83(m,4H),1.55–1.35(m,6H),1.21(dd,J=14.7,7.1Hz,9H). 13 C NMR (101MHz, DMSO) δ171.97,169.99,169.57,164.78,164.40,158.48,136.67,129.12,128.67,116.43,115.14,93.79,87 .60,53.64,47.00,45.56,35.91,32.70,32.04,29.00,28.97,28.93,27.57,25.74,25.56,14.01.HRMS-ESI(m / z)calc.for C 31 H 39 N7O4S[M+Na] + 628.2682, found 628.2681. HPLC t R = 11.64 min (99.9% purity).

[0239] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -Hydroxy-p-toluamide (11g)

[0240] White solid, yield 67.6%, mp 214-216℃. 1H NMR (400MHz, DMSO) δ11.37(s,1H),8.50(d,J=7.7Hz,1H),7.98(s,1H),7.93(d,J =8.3Hz,2H),7.84(d,J=8.2Hz,2H),7.56–7.49(m,2H),7.44–7.29(m,4H),5.56( s,1H),4.65–4.52(m,2H),4.29–4.17(m,1H),3.34(q,J=4.3Hz,2H),2.77(q,J=7 .3Hz,2H),1.98(d,J=6.2Hz,2H),1.67(q,J=12.3Hz,2H),1.21(t,J=7.5Hz,3H)). 13 C NMR (101MHz, DMSO) δ169.98,165.47,164.87,164.44,163.90,158.51,137.20,136.62,135.48,129.15,128.68,128.65,127. 86,127.29,116.45,115.14,93.83,87.64,53.68,47.33,47.23,46.73,31.96,31.86,27.60,14.03.HRMS-ESI(m / z)calc.for C 30 H 29 N7O4S[M+Na] + 606.1905,found 606.1908.HPLC t R = 6.73 min (97.35% purity).

[0241] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-4-((4-(hydroxycarbamoyl)benzyl)oxy)benzamide (11h)

[0242] White solid, yield 53.0%, mp 202-204℃. 1H NMR (400MHz, DMSO) δ11.34(s,1H),8.33(d,J=7.6Hz,1H),8.19(s,1H),7.89(d,J=8.5Hz,2H) ,7.80(d,J=7.9Hz,2H),7.54(dd,J=16.1,7.7Hz,4H),7.43–7.30(m,4H),7.07(d,J=8.5Hz,2H ),5.65(s,1H),5.24(s,2H),4.64–4.52(m,2H),4.25–4.13(m,1H),3.30(d,J=13.0Hz,2H),2. 76(q,J=7.5Hz,2H),1.98–1.92(m,2H),1.66(dd,J=26.5,12.7Hz,2H),1.21(t,J=7.6Hz,3H). 13 C NMR(101MHz,DMSO)δ170.05,165.53,164.89,164.39,160.84,158.50,140 .34,136.80,132.73,129.71,129.06,128.70,128.57,127.87,127.57,12 7.49,116.46,115.18,114.72,93.78,87.59,69.20,60.24,53.62,47.49, 47.32,46.58,32.04,27.59,21.25,14.56,14.03.HRMS-ESI(m / z)calc.for C 37 H 34 N7O5S[MH] - 688.2348,found 688.2348.HPLC t R = 4.86 min (99.92% purity).

[0243] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11i)

[0244] White solid, yield 28.2%, mp 225-227℃. 1H NMR(400MHz,DMSO)δ11.12(s,1H),10.33(s,1H),8.93(s,1H),8.08–7.97(m,2H),7 .68(q,J=8.7Hz,3H),7.53(d,J=7.4Hz,2H),7.43–7.27(m,4H),5.57(s,1H),4.43( d,J=13.1Hz,2H),3.91(s,1H),2.75(q,J=7.5Hz,2H),2.61(t,J=7.1Hz,2H),2.43( t,J=7.2Hz,2H),1.88(d,J=12.7Hz,2H),1.54–1.37(m,2H),1.20(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ170.03,164.76,164.36,163.09,158.45,138.23,136.73,135.03,130.31,129.10,128.68,12 8.60,127.95,119.84,116.46,115.16,93.68,87.58,53.64,48.02,30.29,27.56,14.00.HRMS-ESI(m / z)calc.forC 33 H 33 N8O5S[MH] + 653.2300, found 653.2302.HPLC t R = 8.39 min (99.06% purity).

[0245] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)glutaramide (11j)

[0246] White solid, yield 49.3%, mp 214-216℃. 1H NMR (400MHz, DMSO) δ10.25 (s, 1H), 8.02 (s, 1H), 7.94 (d, J = 7.6Hz, 1H), 7.70 (t, J = 6. 1Hz,4H),7.53(d,J=7.5Hz,2H),7.41–7.29(m,4H),5.56(s,1H),4.43(t,J=10.4Hz, 2H),3.92(d,J=10.4Hz,1H),3.40–3.28(m,2H),2.75(q,J=7.5Hz,2H),2.37(t,J=7. 4Hz,2H),2.15(t,J=7.4Hz,2H),1.92–1.79(m,4H),1.51–1.37(m,2H),1.20(s,3H). 13 C NMR (101MHz, DMSO) δ171.79,171.50,169.99,164.79,164.39,158.46,142.35,136.68,129.11,128.67,128.10,127.38,118 .72,116.43,115.15,93.77,87.57,53.63,46.97,45.60,36.20,35.12,32.08,27.58,21.64,14.01.HRMS-ESI(m / z)calc.for C 34 H 36 N8O5S[M+H] + 669.2612,found 669.2615.HPLC t R = 10.52 min (97.52% purity).

[0247] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -(4-(hydroxycarbamoyl)phenyl)hexamethylenediamide (11k)

[0248] White solid, yield 33.1%, mp 211-213℃. 1H NMR (400MHz, DMSO) δ11.10(s,1H),10.19(s,1H),7.99–7.95(m,1H),7.90(d,J=7.6Hz,1H),7.7 4–7.64(m,4H),7.52(d,J=7.4Hz,2H),7.40–7.31(m,4H),5.54(s,1H),4.44(t,J=10.4Hz,2H),3 .97–3.89(m,1H),3.32(d,J=12.1Hz,2H),2.75(q,J=7.5Hz,2H),2.35(t,J=6.8Hz,2H),2.11(t ,J=6.8Hz,2H),1.97–1.81(m,2H),1.62–1.53(m,4H),1.47–1.37(m,2H),1.20(t,J=7.6Hz,3H). 13 CNMR(101MHz,DMSO)δ172.01,171.79,169.98,164.78,164.40,158.46,142.33,136.65,129.12,128.66,128.63,128.11,127.40,118 .72,116.43,115.14,93.79,87.59,53.66,46.97,45.60,36.73,35.75,32.08,27.58,25.48,25.18,14.01.HRMS-ESI(m / z)calc.forC 35 H 37 N8O5S[MH] - 681.2613, found 681.2617. HPLC t R = 6.4 min (97.15% purity).

[0249] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11l)

[0250] White solid, yield 28.7%, mp 207-209℃. 1H NMR(400MHz,DMSO)δ11.18(s,1H),8.41(t,J=6.0Hz,1H),7.97–7.87(m,2H),7.7 0(d,J=7.9Hz,2H),7.51(d,J=7.4Hz,2H),7.42–7.29(m,6H),5.53(s,1H),4.47–4 .35(m,2H),4.30(d,J=5.9Hz,2H),3.95–3.87(m,1H),2.76(q,J=7.5Hz,2H),2.4 6–2.31(m,4H),1.88(d,J=12.7Hz,2H),1.51–1.36(m,2H),1.21(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ171.97,171.14,169.98,164.78,164.56,164.41,158.49,143.45,136.62,131.65,129.15,128.66,127.43,127 .33,116.43,115.14,93.81,87.63,53.67,46.93,45.64,42.22,32.07,31.98,31.24,31.15,27.58,14.01.HRMS-ESI(m / z)calc.for C 34 H 35 N8O5S[MH] - 667.2466,found 667.2467.HPLC t R = 11.36 min (99.30% purity).

[0251] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)glutaramide (11m)

[0252] White solid, yield 43.2%, mp 193-195℃. 1H NMR (400MHz, DMSO) δ11.19(s,1H),8.40(t,J=6.0Hz,1H),7.96(s,1H),7.88(d,J=7.6Hz,1H),7.70( d,J=8.1Hz,2H),7.52(d,J=7.1Hz,2H),7.42–7.26(m,6H),5.54(s,1H),4.48–4.37(m,2H),4.29(d,J =5.9Hz,2H),3.96–3.89(m,1H),3.37–3.31(m,2H),2.76(q,J=7.7Hz,2H),2.17(t,J=7.5Hz,2H),2. 10(t,J=7.5Hz,2H),1.94–1.83(m,2H),1.80–1.72(m,2H),1.48–1.40(m,2H),1.20(t,J=7.6Hz,3H). 13 CNMR(101MHz,DMSO)δ172.34,171.56,169.99,164.79,164.54,164.40,158.47,143.47,136.65,131.68,129.85,129.13,128.66,127.47,12 7.37,116.44,115.14,93.78,87.59,53.65,46.94,45.59,42.20,35.36,35.19,32.09,32.02,27.58,22.05,14.02.HRMS-ESI(m / z)calc.for C 35 H 37 N8O5S[MH] - 681.2613,found 681.2618.HPLC t R = 6.21 min (99.91% purity).

[0253] (E)-N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)butyramide(11n)

[0254] White solid, yield 30.8%, mp 212-214℃. 1H NMR(400MHz,DMSO)δ10.71(s,1H),10.14(s,1H),8.01–7.90(m,2H),7.64(d,J=8.3Hz,2H),7 .51(t,J=8.9Hz,4H),7.36(dt,J=22.5,7.6Hz,5H),6.36(d,J=15.8Hz,1H),5.54(s,1H),4.47 –4.36(m,2H),3.95–3.88(m,1H),3.34(d,J=12.0Hz,2H),2.76(q,J=7.5Hz,2H),2.60(t,J=7. 1Hz,2H),2.43(t,J=7.1Hz,2H),1.94–1.82(m,2H),1.54–1.39(m,2H),1.21(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ171.18,171.03,170.00,164.77,164.40,163.52,158.46,140.94,138.48,136.62,129.82,129.14,128.66,119 .41,117.65,116.43,115.14,93.79,87.60,53.66,46.92,45.71,32.13,32.08,32.01,30.71,27.58,14.02.HRMS-ESI(m / z)calc.for C 35 H 35 N8O5S[MH] + 679.2457,found 679.2462.HPLC t R = 6.44 min (97.25% purity).

[0255] 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-hydroxybenzamide(11o)

[0256] White solid, yield 26%, mp 201-203℃. 1H NMR (400MHz, DMSO) δ10.76(s,1H),9.03(s,1H),7.95(s,1H),7.53(dd,J=7.8,5.8Hz,4H),7.45(dd,J=11.7,4.0Hz,3H),7.41–7.28(m,4H),6.46(d,J =15.8Hz,1H),5.55(s,1H),4.43(d,J=13.3Hz,2H),3.86(s,2H),3.27(s,2 H), 2.86 (s, 1H), 2.75 (q, J = 7.6Hz, 2H), 1.44 (s, 2H), 1.20 (t, J = 7.6Hz, 3H). 13 CNMR(101MHz,DMSO)δ170.05,164.94,164.40,164.05,158.62,136.67,135.58,133.57,130.72,130.55,129.94,129.15,128.70,128.6 4,127.56,116.32,115.00,94.43,88.17,54.16,53.64,47.05,46.39,46.27,45.90,28.41,27.59,13.98,8.93.HRMS-ESI(m / z)calc.for C 30 H 32 N7O3S[M+H] + 570.2287,found 570.2297.HPLC t R = 9.58 min (97.78% purity).

[0257] (E)-3-4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide(11p)

[0258] White solid, yield 29.3%, mp 199-201℃. 1H NMR (400MHz, CDCl3) δ9.63 (s, 2H), 8.11 (d, J = 3.6Hz, 1H), 7.75–7.66 (m, 4H), 7.59 (d, J = 7. 2Hz,2H),7.53(d,J=15.8Hz,1H),7.47–7.36(m,4H),6.59(dd,J=15.9,1.7Hz,1H),5.65(s, 1H),4.70(d,J=13.4Hz,2H),4.28(t,J=6.0Hz,2H),3.47(d,J=6.0Hz,1H),3.24(t,J=12.9H z,2H),2.81(q,J=7.6Hz,2H),2.39–2.30(m,2H),1.88–1.73(m,2H),1.26(t,J=7.7Hz,3H). 13 C NMR (101MHz, DMSO) δ170.05,164.92,164.41,162.92,158.59,137.97,136.60,135.86,133.65,131.13,129.18,128.70,128.13,120.50 ,116.34,115.01,94.36,88.13,60.24,54.01,53.62,47.10,46.36,46.25,28.45,27.60,21.25,14.56,14.01.HRMS-ESI(m / z)calc.for C 32 H 33 N7O3S[M+H] + 596.2438,found 596.2440.HPLC t R = 8.11 min (98.52% purity).

[0259] 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(hydroxycarbamoyl)benzyl)carbamoyl)benzyl)piperidine 4-ammonium salt (11q)

[0260] White solid, yield 28%, mp 197-199℃. 1H NMR (400MHz, DMSO) δ11.20(s,1H),9.66(s,2H),9.24(t,J=6.0Hz,1H),8.06(s,1H),7.98(d,J=8 .0Hz,2H),7.73(dd,J=10.7,8.1Hz,4H),7.58–7.52(m,2H),7.44–7.31(m,6H),5.61(s,1H),4.6 6(d,J=13.4Hz,2H),4.53(d,J=5.9Hz,2H),4.28(t,J=6.1Hz,2H),3.45(m,1H),3.20(t,J=12.8H z,2H),2.77(q,J=7.5Hz,2H),2.30(d,J=12.4Hz,2H),1.85–1.75(m,2H),1.21(t,J=7.8Hz,3H). 13 CNMR(101MHz,DMSO)δ170.05,166.17,164.92,164.51,164.41,158.59,143 .32,136.62,135.76,134.92,131.78,130.53,129.89,129.18,128.70,128 .01,127.68,127.55,127.42,116.34,115.02,94.36,88.14,54.09,53.62, 46.99,46.37,46.26,42.89,28.46,27.61,14.02.HRMS-ESI(m / z)calc.for C 38 H 39 N8O4S[M+H] + 703.2816,found 703.2821.HPLC t R = 4.04 min (96.86% purity).

[0261] (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)carbamoyl)benzyl)piperidine-4-ammonium salt(11r)

[0262] White solid, yield 68.4%, mp 207-208℃. 1H NMR (400MHz, DMSO) δ10.86(s,1H),9.77(s,2H),9.26(t,J=5.9Hz,1H),8.13(s,1H),7.99(d,J=7.9Hz, 2H),7.77(d,J=8.0Hz,2H),7.54(dd,J=13.3,7.6Hz,4H),7.44–7.24(m,7H),6.48(d,J=15.8Hz,1H),5 .63(s,1H),4.66(d,J=13.2Hz,2H),4.51(d,J=5.9Hz,2H),4.28(t,J=6.0Hz,2H),3.25–3.16(m,2H),3 .11–3.16(m,1H),2.77(q,J=7.7Hz,2H),2.36–2.27(m,2H),1.83–1.70(m,2H),1.21(t,J=7.6Hz,3H). 13 CNMR(101MHz,DMSO)δ170.07,166.15,164.92,164.40,163.20,158.57,141. 58,138.45,136.65,135.74,134.96,133.90,130.54,129.16,128.70,128.66 ,128.25,127.98,127.96,119.19,116.34,115.03,94.34,88.11,54.07,53.6 0,46.97,46.38,46.26,42.92,28.43,27.61,14.01.HRMS-ESI(m / z)calc.for C 40 H 41 N8O4S[M+H] + 729.2975, found 729.2979. HPLC t R = 4.02 min (97.99% purity).

[0263] (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)oxo)benzyl)piperidine-4-ammonium salt(11s)

[0264] White solid, yield 58.4%, mp 187-189℃. 1H NMR (400MHz, DMSO) δ10.83(s,1H),9.35(s,2H),8.05(s,1H),7.58(d,J=7.9Hz,2H),7.54(d,J=7 .7Hz,4H),7.50–7.46(m,2H),7.46–7.30(m,5H),7.08(d,J=8.5Hz,2H),6.49(d,J=15.8Hz,1H), 5.60(s,1H),5.18(s,2H),4.65(d,J=13.3Hz,2H),4.14(t,J=5.9Hz,2H),3.39(s,2H),3.19(t,J =13.0Hz,2H),2.77(q,J=7.6Hz,2H),2.25(s,2H),1.72(t,J=13.7Hz,2H),1.21(t,J=7.6Hz,3H). 13 CNMR(101MHz,DMSO)δ170.04,164.92,164.41,159.07,158.59,138.69,138.26,136.60,134.91,132.14,129.18,128.69,128.55,128.06,124 .67,119.80,116.33,115.44,115.00,94.37,88.13,69.27,53.77,53.63,46.98,46.35,46.26,28.46,27.60,14.01.HRMS-ESI(m / z)calc.for C 40 H 41 N8O4S[M+Na] + 724.2682,found 724.2676.HPLC t R = 6.45 min (97.29% purity).

[0265] 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((7-(hydroxyamino)-7-oxoheptyl)oxo)benzyl)piperidine-4-ammonium salt(11t)

[0266] White solid, yield 45.2%, mp 191-193℃. 1H NMR (400MHz, DMSO) δ10.38(s,1H),9.36–9.31(m,2H),8.05(s,1H),7.53(t,J=8.4Hz,4H),7.46–7.33(m ,4H),7.00(d,J=8.2Hz,2H),5.60(s,1H),4.65(d,J=13.3Hz,2H),4.14(t,J=6.0Hz,2H),3.98(t,J=6.5H z,2H),3.20(t,J=12.7Hz,2H),2.77(q,J=7.5Hz,2H),2.27(d,J=10.7Hz,2H),1.95(t,J=7.3Hz,2H),1. 78–1.66(m,5H),1.54–1.48(m,2H),1.43–1.38(m,2H),1.34–1.27(m,2H),1.20(dt,J=14.5,7.3Hz,5H). 13 C NMR (101MHz, DMSO) δ170.07,169.58,164.92,164.41,159.57,158.58,136.60,132.12,129.17,128.69,124.16,116.33,115.02,114.98,9 4.34,88.10,67.94,53.71,53.62,47.00,46.37,46.26,32.66,28.95,28.78,28.45,27.60,25.69,25.52,14.01.HRMS-ESI(m / z)calc.for C 36 H 43 N7O4S[M+H] + 670.3170, found 670.3177. HPLC t R = 7.85 min (98.24% purity)

[0267] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-N-hydroxypyrimidine-5-carboxamide (11u)

[0268] White solid, yield 38.3%, mp 185-187℃. 1H NMR (400MHz, DMSO) δ8.66(s,2H),7.95(s,1H),7.90(d,J=7.8Hz,1H),7.52(d,J=7.4Hz,2H),7.41–7.31(m,4H),5.55(s,1H),4.58–4.49(m,2H) ,4.18(t,J=9.2Hz,1H),3.37(dd,J=11.2,6.5Hz,2H),2.77(q,J=7.6Hz,2H),2.03(d,J=12.8Hz,2H),1.65–1.55(m,2H),1.22(t,J=7.6Hz,3H). 13 C NMR(101MHz,DMSO)δ170.04,164.80,164.48,162.35,158.42,136.51,129.17,128.68,116.45,1 15.39,115.15,93.77,87.55,53.63,47.76,46.99,31.77,27.58,14.00.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+Na] + 580.1811,found 580.1810.HPLC t R = 5.74 min (97.25% purity).

[0269] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (11v)

[0270] White solid, yield 36.1%, mp 185-187℃. 1 H NMR (400MHz, DMSO) δ11.20(s,1H),9.03(s,1H),8.73(s,2H),8.07(s,1H),7.55(d,J=7.4Hz,2H),7.39–7.30(m,4H),5.61(s, 1H), 5.00 (s, 1H), 4.76 (d, J = 13.2Hz, 2H), 3.01 (s, 3H), 2.77 (q, J = 8.0Hz, 2H), 1.81 (d, J = 22.1Hz, 4H), 1.22 (t, J = 7.6Hz, 3H). 13C NMR (101MHz, DMSO) δ170.02,164.80,164.40,161.99,158.37,157.48,136.73,129.11,128.66,116.51,1 15.17,114.92,93.84,87.63,53.64,52.34,47.62,29.49,29.06,27.59,14.01.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+Na] + 597.1965,found 594.1967.HPLC t R = 4.52 min (95.29% purity)

[0271] tert-Butyl-4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidine-1-carbamate (12a)

[0272] White solid, yield 38.3%. 1 H NMR(400MHz,DMSO)δ7.89(s,1H),7.53–7.46(m,2H),7.44–7.28(m,4H),5.48(s,1H),4.69(s,1H),4.05 (s,2H),3.13(s,3H),2.78(q,J=7.7Hz,2H),1.67(d,J=9.4Hz,4H),1.41(s,9H),1.22(t,J=7.6Hz,3H).

[0273] tert-Butyl-4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)methyl)(methyl)amino)piperidine-1-carbamate (12b)

[0274] White solid, yield 42.8%. 1 H NMR(400MHz,DMSO)δ7.89(s,1H),7.53–7.46(m,2H),7.44–7.28(m,4H),5.48(s,1H),4.69(s,1H),4.05 (s,2H),3.13(s,3H),2.78(q,J=7.7Hz,2H),1.67(d,J=9.4Hz,4H),1.41(s,9H),1.22(t,J=7.6Hz,3H).

[0275] 4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidine-1-ammonium salt (13a)

[0276] White solid, yield 56.2%. 1 H NMR (400MHz, DMSO) δ8.82(d,J=37.0Hz,2H),8.08(s,1H),7.53(d,J=7.5Hz,2H),7.46–7.34(m,4H),5.53(s,1H),4.89(s,1H),3. 17(s,3H),3.15–3.05(m,2H),2.80(t,J=7.6Hz,2H),2.09(dd,J=19.2,8.3Hz,2H),1.80(t,J=14.8Hz,2H),1.23(t,J=7.6Hz,3H).

[0277] 4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)methyl)(methyl)amino)piperidine-1-ammonium salt (13b)

[0278] White solid, yield 41.7%. 1 H NMR (400MHz, DMSO) δ8.74(s,2H),8.04(s,1H),7.54(d,J=7.1Hz,2H),7.45–7.34(m,4H),5.48(s,1H),3.89(dd,J=14.0,8.3Hz,1H),3.63–3 .56(m,1H),3.27(d,J=12.3Hz,2H),2.83–2.72(m,3H),2.03(d,J=9.4Hz,1H),1.75–1.66(m,2H),1.56–1.42(m,2H),1.22(t,J=7.7Hz,3H).

[0279] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidin-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (14a)

[0280] White solid, yield 37.2%. 1H NMR (400MHz, DMSO) δ11.51(s,1H),8.70(s,2H),7.90(s,1H),7.51(d,J=7. 0Hz,2H),7.43–7.32(m,4H),5.52(s,1H),4.96(s,1H),4.87(d,J=12.6Hz,3 H),4.04(s,1H),3.53(d,J=11.4Hz,1H),3.12(s,4H),2.80(t,J=7.6Hz,2H) ,1.80–1.74(m,3H),1.73–1.69(m,2H),1.55(s,3H),1.23(t,J=7.6Hz,3H).

[0281] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)methyl)(methyl)amino)piperidin-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (14b)

[0282] White solid, yield 45.3%. 1 H NMR (400MHz, DMSO) δ11.51 (s, 1H), 8.69 (s, 2H), 7.92 (s, 1H), 7.46 (d, J = 7.5Hz, 2H), 7.41–7. 32(m,4H),7.21(dd,J=8.4,6.3Hz,1H),5.52(d,J=4.4Hz,1H),4.96(d,J=3.4Hz,1H),4.76(t, J=15.8Hz,2H),4.03(s,1H),3.92(dd,J=14.1,7.8Hz,1H),3.55(dd,J=14.3,6.9Hz,2H),2.9 5–2.83(m,2H),2.77(d,J=7.7Hz,2H),2.10(s,1H),1.75–1.49(m,8H),1.21(t,J=7.6Hz,3H).

[0283] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15a)

[0284] White solid, yield 37.2%, mp 172-173℃. 1H NMR(400MHz,DMSO)δ11.28(s,1H),9.06(s,1H),8.73(s,2H),8.24(s,1H),7.60–7.50(m,2H),7.45–7.30(m,4H),5 .66(s,1H),4.87(d,J=25.6Hz,3H),3.10(s,5H),2.77(d,J=7.6Hz,2H),1.72(d,J=34.2Hz,4H),1.27–1.18(m,3H). 13 C NMR (101MHz, DMSO) δ170.13,164.41,162.37,161.71,158.61,157.72,136.58,129.09,128.65,128.56,116.75,115 .30,114.85,93.61,87.03,56.28,53.48,43.05,42.96,33.60,29.00,28.75,27.62,14.00.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+Na] + 594.2007, found 594.2002. HPLC t R = 4.67 min (96.98% purity).

[0285] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15b)

[0286] White solid, yield 32.0%, mp 173-175℃. 1 H NMR (400MHz, DMSO) δ8.71 (s, 2H), 8.15 (s, 1H), 7.50 (d, J = 7.5Hz, 2H), 7.36 (s, 1H), 7.28(t,J=7.5Hz,2H),7.18(t,J=7.3Hz,1H),5.61(s,1H),4.74(t,J=16.8Hz,2H), 3.93(dd,J=14.2,7.8Hz,1H),3.54(dd,J=17.9,8.3Hz,2H),3.38(s,2H),2.87(q,J =13.4Hz,3H),2.76(q,J=7.4Hz,2H),2.08(s,1H),1.68–1.56(m,2H),1.20(m,5H). 13C NMR (101MHz, DMSO) δ170.13,164.50,164.34,161.67,157.91,157.66,144.73,136.87,134.34,129.01,128.52,128.39,116 .90,115.33,114.62,93.22,86.12,56.76,53.48,43.79,41.14,35.49,29.15,29.01,27.63,13.97.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+Na] + 608.2160,found 608.2155.HPLC t R = 4.55 min (95.28% purity).

[0287] tert-Butyl-4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazine-1-carbamate (16)

[0288] White solid, yield 81.2%. 1 H NMR(400MHz,DMSO)δ7.90(s,1H),7.54–7.49(m,2H),7.45–7.29(m,5H),5.53(s,1H),3.89(t ,J=5.3Hz,4H),3.25–3.18(m,4H),2.77(q,J=7.6Hz,3H),1.39(s,9H),1.21(t,J=7.5Hz,3H).

[0289] 4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazine-1-ammonium salt (17)

[0290] White solid, yield 90.2%. 1 H NMR (400MHz, DMSO) δ11.23(s,1H),7.87(s,1H),7.57(d,J=7.7Hz,2H),7.54–7.46(m,3H),7.43–7.27(m,6H),6.50(d,J=15.9Hz,1H),5.51(s, 1H),4.92(s,1H),3.97(s,1H),3.90(s,4H),3.57(s,2H),3.53(s,1H), 2.76(q,J=7.6Hz,2H),1.70(s,3H),1.55(s,3H),1.20(t,J=7.6Hz,3H).

[0291] (E)-3-(4-((4-(6-((2-amino-2-oxo-1-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)acrylamide(18a)

[0292] White solid, yield 48.8%. 1 H NMR (400MHz, DMSO) δ11.23(s,1H),7.87(s,1H),7.57(d,J=7.7Hz,2H),7.54–7.46(m,3H),7.43–7.27(m,6H),6.50(d,J=15.9Hz,1H),5.51(s, 1H),4.92(s,1H),3.97(s,1H),3.90(s,4H),3.57(s,2H),3.53(s,1H), 2.76(q,J=7.6Hz,2H),1.70(s,3H),1.55(s,3H),1.20(t,J=7.6Hz,3H).

[0293] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (18b)

[0294] White solid, yield 50.2%. 1 H NMR (400MHz, DMSO) δ11.63(s,1H),8.77(s,2H),7.98(s,1H),7.54(d,J=7.5Hz,2H),7.40(dd,J=15.2,7.7Hz,4H),5.59 (s,1H),4.98(s,1H),4.03(s,6H),4.00(s,4H),2.79(q,J=7.6Hz,2H),1.72(s,3H),1.55(s,3H),1.23(t,J=7.6Hz,3H).

[0295] (E)-3-(4-((4-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (19a)

[0296] White solid, yield 30.3%, mp 187-189℃. 1H NMR (400MHz, DMSO) δ10.81(s,1H),9.04(s,1H),7.93(s,1H),7.55(d,J=7.7Hz,2H),7.50(d,J=7.3Hz,2H),7.46(d,J=15.5Hz,1H) ,7.40–7.29(m,7H),6.48(d,J=15.8Hz,1H),5.54(s,1H),3.90(s,4H),3.56(s,2H),2.75(q,J=7.4Hz,2H),1.20(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ169.96,164.79,164.44,158.51,136.55,129.97,129.13,128.64,127.91,119.3 4,116.37,115.07,94.07,87.85,61.74,53.63,52.86,47.93,27.55,13.98.HRMS-ESI(m / z)calc.for C 31 H 31 N7O3S[M+Na] + 604.2058,found 604.2059.HPLC t R = 5.16 min (95.27% purity).

[0297] 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (19b)

[0298] White solid, yield 31.2%, mp 211-213℃. 1 H NMR (400MHz, DMSO) δ11.16(s,1H),8.75(s,2H),7.97(s,1H),7.54(d,J=7.1Hz,2H),7.45–7.34(m,4H),5.58( s,1H),4.04(dd,J=7.3,3.6Hz,4H),3.98(dd,J=7.3,3.6Hz,4H),2.79(q,J=7.5Hz,2H),1.23(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ170.02,164.91,164.39,162.19,161.68,160.15,158.62,157.62,136.42,129.21,128.7 3,128.70,116.37,115.52,115.06,94.14,87.93,53.71,47.31,43.54,27.57,14.01.HRMS-ESI(m / z)calc.for C 26 H 25 N9O3S[M+Na] + 566.1659, found 566.1655. HPLC t R = 4.51 min (96.89% purity).

[0299] tert-butyl-4-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)-1-carbamate (20)

[0300] White solid, yield 77.8%. 1 H NMR (400MHz, DMSO) δ7.92(s,1H),7.55–7.48(m,2H),7.41–7.30(m,4H),6.93(t,J=5.9Hz,1H),5.53(s,1H),4.55(d,J=13.3Hz,2 H),3.11(t,J=12.3Hz,2H),2.86(t,J=6.1Hz,2H),2.75(q,J=7.5Hz,2H),1.76(d,J=12.8Hz,3H),1.39(s,9H),1.25–1.16(m,5H).

[0301] tert-butyl-(R)-((1-(6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)-1-carbamate((R)-20)

[0302] White solid, yield 63.5%. 1H NMR (400MHz, DMSO) δ7.92(s,1H),7.52(dt,J=6.0,1.4Hz,2H),7.42–7.28(m,4H),6.94(q,J=5.7Hz,1H),5.54(s,1H),4.55(d,J=13.3Hz ,2H),3.10(d,J=10.5Hz,2H),2.85(q,J=6.2Hz,2H),2.75(q,J=7.5Hz,2H),1.80–1.68(m,3H),1.38(d,J=5.7Hz,9H),1.29–1.15(m,5H).

[0303] tert-butyl-(S)-((1-(6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)-1-carbamate((S)-20)

[0304] White solid, yield 66.2%. 1 H NMR (400MHz, DMSO) δ7.92(s,1H),7.55–7.46(m,2H),7.42–7.28(m,4H),6.93(t,J=5.9Hz,1H),5.54(s,1H),4.55(d,J=13.2Hz,2H) ,3.11(t,J=12.6Hz,2H),2.85(q,J=6.3Hz,2H),2.75(q,J=7.6Hz,2H),1.76–1.70(m,2H),1.38(d,J=5.6Hz,9H),1.29–1.15(m,5H).

[0305] (1-(6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methylammonium salt (21)

[0306] White solid, yield 88.2%. 1 H NMR (400MHz, DMSO) δ7.94(s,1H),7.81(s,3H),7.52(d,J=7.5Hz,2H),7.43–7.31(m,4H),5.54(s,1H),4.58(d,J=1 3.2Hz,2H),3.17(d,J=5.0Hz,2H),3.10(s,2H),2.76(d,J=8.2Hz,2H),1.88(t,J=13.2Hz,3H),1.35–1.17(m,5H).

[0307] (R)-(1-(6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methylammonium salt((R)-21)

[0308] White solid, yield 67.6%. 1 H NMR(400MHz,DMSO)δ8.08(s,4H),7.54(d,J=7.5Hz,2H),7.43–7.29(m,4H),5.57(s,1H),4.57(d,J=13.4Hz,2H), 3.14(q,J=13.7Hz,2H),2.76(q,J=6.0Hz,4H),1.88(d,J=12.5Hz,3H),1.43–1.28(m,2H),1.20(t,J=7.6Hz,3H).

[0309] (S)-(1-(6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methylammonium salt((S)-21)

[0310] White solid, yield 58.5%. 1 H NMR (400MHz, DMSO) δ8.11(s,4H),7.57–7.52(m,2H),7.41–7.32(m,4H),5.58(s,1H),4.57(d,J=13.5Hz,2H ),3.14(q,J=13.7Hz,2H),2.79–2.73(m,4H),1.91–1.86(m,3H),1.40–1.30(m,2H),1.20(t,J=7.6Hz,3H).

[0311] (E)-3-(4-((((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-(tetrahydro-2H-pyran-2-yl)oxo)acrylamide(22a)

[0312] White solid, yield 53.2%. 1H NMR (400MHz, DMSO) δ11.22 (s, 1H), 7.92 (s, 1H), 7.51 (d, J = 8.3Hz, 4H), 7.45–7. 34(m,5H),6.48(d,J=15.6Hz,1H),5.52(s,1H),4.91(s,1H),4.57(d,J=13.5Hz, 2H),3.96(s,1H),3.76(s,1H),3.53(d,J=11.4Hz,1H),3.17–3.03(m,2H),2.72 (d,J=7.6Hz,2H),1.85(s,2H),1.69(s,3H),1.54(s,3H),1.20(t,J=7.5Hz,3H).

[0313] (E)-3-(4-((((1-(6-((((R)-2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-(tetrahydro-2H-pyran-2-yl)oxo)acrylamide((R)-22a)

[0314] White solid, yield 33.0%. 1 H NMR (400MHz, DMSO) δ11.22(s,1H),7.92(s,1H),7.58–7.48(m,5H),7.48–7.28(m ,8H),6.50(d,J=15.8Hz,1H),5.53(s,1H),4.91(s,1H),4.56(d,J=13.3Hz,2H), 3.96(s,1H),3.81(s,2H),3.61–3.47(m,2H),3.17–3.04(m,2H),2.75(q,J=7.5H z,2H),1.87(d,J=12.2Hz,2H),1.70(s,3H),1.54(s,3H),1.19(t,J=7.7Hz,3H).

[0315] (E)-3-(4-((((1-(6-((((S)-2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-(tetrahydro-2H-pyran-2-yl)oxo)acrylamide((S)-22a)

[0316] White solid, yield 35.2%. 1H NMR (400MHz, DMSO) δ11.26(s,1H),7.95(s,1H),7.57(d,J=7.9Hz,2H),7.52(d,J=7.3H z,3H),7.47(d,J=7.3Hz,2H),7.41–7.32(m,4H),6.51(d,J=15.8Hz,1H),5.55(s,1H), 4.92(s,1H),4.46(d,J=13.1Hz,2H),3.90(s,4H),3.05(s,2H),2.76(q,J=7.5Hz,2H), 2.02(s,2H),1.72(d,J=10.7Hz,3H),1.55(s,3H),1.47(s,2H),1.20(t,J=7.4Hz,3H).

[0317] 2-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (22b)

[0318] White solid, yield 48.3%. 1 H NMR (400MHz, DMSO) δ11.49(s,1H),8.63(d,J=11.4Hz,2H),7.98(t,J=6.2Hz,1H ),7.92(s,1H),7.51(d,J=7.6Hz,2H),7.41–7.29(m,4H),5.53(s,1H),4.95(s,1 H),4.57(d,J=13.4Hz,2H),4.03(s,1H),3.28(s,2H),2.75(q,J=7.4Hz,3H),1. 95(s,1H),1.83(d,J=12.8Hz,2H),1.71(s,3H),1.54(s,3H),1.35–1.22(m,5H).

[0319] (E)-1-(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N-(4-(3-(hydroxyamino)3-oxopropane-1-en-1-yl)benzyl)methylammonium salt (23a)

[0320] White solid, yield 33.2%, mp 203-205℃. 1H NMR(400MHz,DMSO)δ10.85(s,1H),9.36(s,2H),8.02(s,1H),7.63(s,4H),7.57–7.45( m,3H),7.43–7.28(m,4H),6.53(d,J=15.8Hz,1H),5.56(s,1H),4.56(d,J=13.3Hz,2H) ,4.17(t,J=5.6Hz,2H),3.14(q,J=13.0Hz,2H),2.84(d,J=6.6Hz,2H),2.75(q,J=7.6H z,2H),2.15(s,1H),1.92(d,J=12.8Hz,2H),1.37–1.24(m,2H),1.20(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ170.03,164.81,164.34,162.93,158.51,137.98,136.69,135.86,133.39,131.31,129.11,128.68,128.61,128.05 ,120.48,116.43,115.14,93.77,87.73,53.64,51.57,50.46,49.06,47.86,47.72,32.84,30.00,27.56,14.01.HRMS-ESI(m / z)calc.for C 33 H 35 N7O3S[M+H] + 610.2570,found 610.2552.HPLC t R = 5.76 min (97.46% purity).

[0321] (R,E)-1-(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N-(4-(3-(hydroxyamino)3-oxopropan-1-en-1-yl)benzyl)methylammonium salt((R)-23a)

[0322] White solid, yield 50.2%, mp 201-202℃. 1H NMR(400MHz,DMSO)δ10.78(s,1H),9.04(s,1H),7.94(s,1H),7.58(d,J=7.8Hz,2H), 7.53(d,J=1.9Hz,2H),7.51–7.42(m,3H),7.42–7.28(m,4H),6.48(d,J=15.8Hz,1H), 5.54(s,1H),4.56(d,J=13.2Hz,2H),3.99(s,2H),3.14(d,J=8.8Hz,2H),2.75(q,J= 7.6Hz,2H),2.67(s,2H),1.98(s,1H),1.89(d,J=13.2Hz,2H),1.21(q,J=7.1Hz,5H). 13 C NMR(101MHz,DMSO)δ170.03,164.83,164.30,162.96,158.50,137.93,136.77 ,135.84,133.40,131.36,131.31,129.07,128.73,128.68,128.56,127.99,1 20.53,116.42,115.14,93.77,87.73,56.49,53.64,51.48,50.41,47.90,47. 73,45.78,32.81,30.05,27.55,19.03,13.99,8.88.HRMS-ESI(m / z)calc.forC 33 H 35 N7O3S[M+H] + 610.2570,found 610.2572.HPLC t R = 5.78 min (98.02% purity).

[0323] (S,E)-1-(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N-(4-(3-(hydroxyamino)3-oxopropan-1-en-1-yl)benzyl)methylammonium salt((S)-23a)

[0324] White solid, yield 47.5%, mp 202-204℃. 1H NMR(400MHz,DMSO)δ10.86(s,1H),9.29(s,2H),8.00(s,1H),7.63(s,3H),7.53(dd, J=7.0,1.9Hz,2H),7.50(s,1H),7.41–7.30(m,4H),6.53(d,J=15.8Hz,1H),5.56(s, 1H),4.56(d,J=13.3Hz,2H),4.17(t,J=5.6Hz,2H),3.21–3.07(m,3H),2.85(d,J=6. 0Hz, 2H), 2.75 (q, J = 7.6Hz, 2H), 2.14 (s, 1H), 1.96–1.88 (m, 2H), 1.31–1.17 (m, 5H). 13 C NMR(101MHz,DMSO)δ170.03,164.83,164.31,162.95,158.50,137.95,136 .75,135.83,133.40,131.35,131.30,129.08,128.75,128.68,128.57,12 8.00,120.50,116.43,115.15,93.76,87.73,56.49,53.63,51.49,50.41, 47.89,47.73,32.81,30.06,30.03,27.55,14.00.HRMS-ESI(m / z)calc.for C 33 H 35 N7O3S[2M+H] + 1219.5122,found 1219.5129.HPLC t R = 5.82 min (99.24% purity).

[0325] 2-(((1-(6-(((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)-N-hydroxypyrimidine-5-carboxamide (23b)

[0326] White solid, yield 35.1%, mp 178-180℃. 1H NMR (400MHz, DMSO) δ8.65(s,2H),8.07(s,1H),7.96(s,1H),7.52(dd,J=7.1,1.9Hz,2H),7.40–7.28(m,4H),5.55(s,1H),4.58(d,J =13.3Hz,2H),3.29(d,J=5.7Hz,2H),3.12(d,J=12.6Hz,2H),2.80–2.72(m,2H),2.00–1.92(m,1H),1.89–1.80(m,2H),1.21(m,5H). 13 C NMR (101MHz, DMSO) δ170.03,164.69,164.37,162.85,158.42,157.61,136.73,129.09,128.67,128.58,116.48,115 .20,93.58,87.50,53.61,49.06,48.10,46.36,45.90,35.53,30.25,30.12,27.56,14.00.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+H] + 572.2155,found 572.2150.HPLC t R = 11.25 min (99.40% purity).

[0327] tert-Butyl(2-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)ethyl)carbamate (24)

[0328] White solid, yield 85.2%. 1 H NMR (400MHz, DMSO) δ7.87 (s, 1H), 7.57–7.48 (m, 2H), 7.45–7.29 (m, 4H), 6.96 (t, J = 6.1Hz, 1H), 5.59 (s, 1H), 3. 87–3.74(m,2H),3.40(s,3H),3.21(t,J=5.7Hz,2H),2.76(q,J=7.5Hz,2H),1.34(s,9H),1.23(d,J=7.5Hz,3H).

[0329] 2-((6-((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)ethyl-1-ammonium salt (25)

[0330] White solid, yield 91.2%. 1 H NMR(400MHz,DMSO)δ8.27(s,4H),7.65–7.57(m,2H),7.47–7.31(m,4H),4.09(s,2H) ), 3.43 (d, J = 1.1Hz, 3H), 3.09 (s, 2H), 2.78 (q, J = 7.5Hz, 2H), 1.21 (d, J = 7.5Hz, 3H).

[0331] (E)-3-(4-(((2-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)ethyl)amino)methyl)phenyl)-N-(tetrahydro-2H-pyran-2-yl)oxo)acrylamide(26)

[0332] White solid, yield 51.5%. 1 H NMR (400MHz, DMSO) δ11.23(s,1H),7.96(s,1H),7.50(d,J=7.0Hz,4H),7.44(s,1H),7.42–7.27(m,6H),6.47(d,J=15.9Hz,1H),5.52(s,1H),4.91(s ,1H),3.99–3.91(m,2H),3.85(s,1H),3.72(s,2H),3.53(d,J=11.3Hz,2H ),2.74(q,J=7.4Hz,2H),1.70(s,3H),1.54(s,3H),1.18(d,J=7.1Hz,3H).

[0333] (E)-2-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)-N-(4-(3-(hydroxyamino)-3-oxopropane-1-en-1-yl)benzyl)ethyl-1-ammonium salt (27)

[0334] White solid, yield 32.7%, mp 197-199℃. 1H NMR (400MHz, DMSO) δ10.84(s,1H),9.51(s,2H),9.09(s,1H),8.32(s,1H),7.71–7.58(m,6H),7.55–7.52(m,1H),7.48(d,J=15.8Hz,1H),7 .41–7.32(m,3H),6.52(d,J=15.9Hz,1H),5.80(s,1H),4.26–4.11(m,4H),3.28–3.20(m,2H),2.79(q,J=7.6Hz,2H),1.21(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ170.55,164.48,163.01,158.18,138.04,136.44,135.92,133.44,131.14,129.17,128.79,128.7 1,128.12,120.48,116.82,115.15,93.97,87.12,52.94,50.58,48.06,44.46,27.62,13.96.HRMS-ESI(m / z)calc.for C 28 H 29 N9O3S[M+H] + 570.2282,found 570.2284.HPLC t R = 5.18 min (96.49% purity).

[0335] Example 10: Preparation of target compounds 30a-e

[0336] 5-((2-((tert-butoxycarbamoyl)amino)phenyl)amino)-5-oxovaleric acid (28a)

[0337] The corresponding carboxylic acid (2.66 mmol), EDCI (615 mg, 3.2 mmol), DMAP (422 mg, 3.45 mmol), and tert-butyl-2-aminophenylcarboxylate (555 mg, 2.66 mmol) were added to 15 mL of CH2Cl2 and reacted at room temperature for 8 hours. After the reaction was complete, the reaction solution was washed successively with 1 M hydrochloric acid aqueous solution (100 mL) and saturated sodium chloride aqueous solution (100 mL). The organic phase was then dried over anhydrous Na2SO4 and concentrated. Purification by silica gel column chromatography yielded 28a. The synthesis of compounds 28d-e followed the same procedure. The products were white solids in 25.8% yield. 1H NMR (400MHz, DMSO) δ12.10(s,1H),9.46(s,1H),8.32(s,1H),7.54(d,J=8.0Hz,1H),7.41(d,J=7.8Hz,1 H),7.19–7.05(m,2H),2.40(t,J=7.3Hz,2H),2.29(t,J=7.4Hz,2H),1.83(p,J=7.3Hz,2H),1.46(s,9H).

[0338] 4-((2-((tert-butoxycarbamoyl)amino)phenyl)carbamoyl)benzoic acid (28b)

[0339] White solid, yield 78.1%. 1 H NMR (400MHz, DMSO) δ13.31 (s, 1H), 9.98 (s, 1H), 8.74 (s, 1H), 8.11–8.05 (m, 4H), 7.56 (t, J = 9.4Hz, 2H), 7.21–7.14 (m, 2H).

[0340] 2-(4-((2-((tert-butoxycarbamoyl)amino)phenyl)carbamoyl)phenyl)acetic acid(28c)

[0341] White solid, yield 96.4%. 1 H NMR (400MHz, DMSO) δ9.83 (s, 1H), 8.69 (s, 1H), 7.90 (d, J = 8.2Hz, 2H), 7.57–7 .51(m,2H),7.46–7.40(m,2H),7.22–7.15(m,2H),3.69(s,2H),1.45(s,9H).

[0342] tert-butyl (2-(4-formylbenzamido)phenyl)carbamate (28d)

[0343] Yellow solid, yield 43.9%. 1 H NMR (400MHz, DMSO) δ10.13(s,1H),10.01(s,1H),8.74(s,1H),8.15(d,J=8.0Hz ,2H),8.07(d,J=8.3Hz,2H),7.58–7.50(m,2H),7.25–7.11(m,2H),1.44(s,9H).

[0344] tert-Butyl(2-(2-chloropyrimidin-5-carbamate)phenyl)carbamate (28e)

[0345] Yellow solid, yield 72.7%. 1H NMR (400MHz, DMSO) δ10.13(s,1H),10.01(s,1H),8.74(s,1H),8.15(d,J=8.0Hz ,2H),8.07(d,J=8.3Hz,2H),7.59–7.51(m,2H),7.26–7.15(m,2H),1.44(s,9H).

[0346] tert-Butyl(2-(5-((1-(6-(((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-5-oxopentanamino)phenyl)carbamate (29a)

[0347] White solid, yield 84.3%. 1 H NMR(400MHz,DMSO)δ9.43(s,1H),8.38(s,1H),7.96–7.87(m,2H),7.58–7 .49(m,3H),7.47–7.29(m,5H),7.19–7.08(m,2H),5.53(s,1H),4.52–4.3 9(m,2H),3.94(s,1H),2.76(q,J=7.5Hz,2H),2.37(t,J=7.4Hz,2H),2.16 (t,J=7.5Hz,2H),1.94–1.80(m,4H),1.46(s,9H),1.22(t,J=6.4Hz,3H).

[0348] tert-Butyl(2-(4-((1-(6-(((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)carbamoyl)benzamido)phenyl)carbamate (29b)

[0349] White solid, yield 82.2%. 1 H NMR (400MHz, DMSO) δ10.01(s,1H),8.76(s,1H),8.58(d,J=7.6Hz,1H),8.05(q,J= 8.6Hz,5H),7.57–7.52(m,4H),7.39(t,J=7.3Hz,2H),7.34(dd,J=4.9,2.3Hz,2H), 7.24–7.14(m,2H),5.58(s,1H),4.60(t,J=13.5Hz,2H),4.23(s,1H),2.78(q,J=7. 6Hz,2H),2.03(s,2H),1.68(t,J=11.9Hz,2H),1.45(s,9H),1.23(t,J=7.6Hz,3H).

[0350] tert-Butyl(2-(4-(2-((1-(6-(((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-2-oxoethyl)benzamido)phenyl)carbamate (29c)

[0351] White solid, yield 75.5%. 1 H NMR (400MHz, DMSO) δ9.86 (s, 1H), 8.71 (s, 1H), 8.27 (d, J = 7.5Hz, 1H), 7.99–7.95 (m, 1H), 7 .91(d,J=8.0Hz,2H),7.58–7.49(m,4H),7.44(d,J=8.0Hz,2H),7.41–7.29(m,4H),7.25–7. 15(m,2H),5.55(s,1H),4.49–4.39(m,2H),3.95–3.89(m,1H),3.53(s,2H),3.38(d,J=11.2 Hz,2H),2.76(q,J=7.5Hz,2H),1.91(d,J=12.8Hz,2H),1.44(s,9H),1.21(t,J=7.5Hz,3H).

[0352] tert-Butyl(2-(4-(((1-(6-(((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)benzamido)phenyl)carbamate (29d)

[0353] White solid, yield 50.2%. 1 H NMR (400MHz, DMSO) δ9.83 (s, 1H), 8.71 (s, 1H), 7.93 (d, J = 6.4Hz, 3H), 7.61–7.51 (m, 6H), 7.41–7.30 (m, 4H), 7.23–7.15 (m, 2H), 5.54 (s ,1H),4.43(s,2H),3.88(s,2H),3.30(d,J=11.4Hz,2H),2.75(q,J=7.5Hz,3H),2.02–1.90(m,2H),1.45(s,11H),1.20(t,J=7.6Hz,3H).

[0354] tert-Butyl(2-(2-(((1-(6-(((2-amino-2-oxo-phenethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)pyrimidine-formamido)phenyl)carbamate (29e)

[0355] White solid, yield 46.0%. 1 H NMR (400MHz, DMSO) δ9.71 (s, 1H), 8.92 (s, 2H), 8.57 (s, 1H), 7.93 (s, 1H), 7.61 (d, J = 7. 8Hz,1H),7.54(d,J=7.1Hz,2H),7.48(dd,J=7.9,1.6Hz,1H),7.44–7.30(m,4H),7.21– 7.15(m,2H),5.57(s,1H),5.09–4.99(m,1H),4.77(d,J=13.3Hz,2H),3.30(s,2H),3.0 6(s,3H),2.78(q,J=7.5Hz,2H),1.93–1.76(m,4H),1.45(s,9H),1.23(t,J=7.6Hz,3H).

[0356] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(2-Aminophenyl)glutaramide (30a)

[0357] White solid, yield 74.2%, mp 174-176℃. 1 H NMR (400MHz, DMSO) δ9.09 (s, 1H), 7.89 (dd, J = 12.0, 4.8Hz, 2H), 7.58–7.43 (m, 3H), 7.41–7.2 8(m,4H),7.24–7.05(m,2H),6.95–6.85(m,1H),6.72(d,J=7.9,1H),6.54(t,J=7.5,1H),5.5 3(s,1H),4.46–4.41(m,2H),3.94(s,1H),3.37(d,J=12.6Hz,2H),2.77(q,J=7.3Hz,2H),2.3 4(t,J=7.4Hz,2H),2.30–2.17(m,2H),2.05–1.80(m,4H),1.45(s,2H),1.21(t,J=7.5Hz,3H). 13C NMR (101MHz, DMSO) δ171.62,171.51,171.27,169.98,164.79,164.41,158.5 0,155.11,142.37,136.64,129.13,128.66,126.18,125.82,123.98,121.63 ,116.60,116.42,116.32,115.11,93.85,87.63,53.69,46.96,45.62,35.62 ,35.32,32.07,28.47,27.57,24.13,22.03,13.99.HRMS-ESI(m / z)calc.for C 33 H 36 N8O3S[M+Na] + 647.2529,found 647.2529.HPLC t R = 10.24 min (99.93% purity).

[0358] N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(2-Aminophenyl)terephthalamide (30b)

[0359] White solid, yield 76.5%, mp 247-249℃. 1 H NMR (400MHz, DMSO) δ10.64(s,1H),8.66(d,J=7.6Hz,1H),8.32–7.99(m,6H),7.67–7.25(m,12H),5.63(s,1H),4.65–4.54(m,2H) ,4.26–4.18(m,1H),3.34(d,J=12.1Hz,2H),2.77(q,J=7.6Hz,2H),2.06–1.98(m,2H),1.80–1.60(m,2H),1.22(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ170.05,165.45,165.38,164.91,164.40,158.53,137.68,136.79,136.41,129.08,128.71,128.58,128.52,127. 82,127.76,126.95,123.70,116.46,115.17,93.82,87.64,53.63,47.43,47.27,46.83,31.92,27.60,14.04.HRMS-ESI(m / z)calc.for C 36 H 34 N8O3S[M+Na] + 681.2372, found 681.2374. HPLC t R = 11.46 min (99.31% purity).

[0360] 4-(2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-2-oxoethyl)-N-(2-aminophenyl)benzamide (30c)

[0361] White solid, yield 79.2%, mp 245-247℃. 1 H NMR (400MHz, DMSO) δ10.41(d,J=11.4Hz,1H),8.34(t,J=7.0Hz,1H),8.04(td,J=9.7,4.1H z,3H),7.53(d,J=7.4Hz,3H),7.44(d,J=8.0Hz,2H),7.40–7.27(m,7H),5.57(d,J=2.2Hz,1 H),4.45(t,J=10.5Hz,2H),3.92(d,J=9.9Hz,1H),3.37(dd,J=13.6,10.5Hz,2H),2.76(q, J=7.6Hz,2H),1.91(d,J=12.4Hz,2H),1.48(t,J=14.4Hz,2H),1.24–1.18(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ170.00,169.46,166.02,164.80,164.40,158.48,141.21,136.70,132.32,129.39,129.11,128.67,128.48,127 .61,127.02,116.45,115.14,93.82,87.61,53.64,46.89,45.89,42.72,31.96,27.59,21.25,14.57,14.02.HRMS-ESI(m / z)calc.for C 36 H 34 N8O3S[M–H] - 671.2558,found 671.2559.HPLC t R = 5.18 min (98.52% purity).

[0362] 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-(2-aminophenyl)benzamide (30d)

[0363] White solid, yield 70.2%, mp 221-223℃. 1 H NMR (400MHz, DMSO) δ10.21(s,1H),9.82(s,2H),8.13(d,J=8.5Hz,3H),7.81(d,J=7 .9Hz,2H),7.56(d,J=7.4Hz,2H),7.47–7.27(m,5H),7.14(s,2H),6.98(s,1H),5.63 (s,1H),4.67(d,J=13.1Hz,2H),4.32(d,J=6.1Hz,2H),3.22(d,J=13.8Hz,2H),2.77 (q,J=7.6Hz,2H),2.33(d,J=11.7Hz,2H),1.88–1.75(m,2H),1.22(t,J=7.5Hz,3H). 13C NMR (101MHz, DMSO) δ170.06,165.43,164.93,164.40,158.60,136.66,136.10,134.89,130.46,129.17,128.71,128.64,127.54, 127.08,116.34,115.03,94.36,88.14,55.42,54.09,53.61,46.97,46.40,46.28,28.43,27.61,14.02.HRMS-ESI(m / z)calc.for C 36 H 36 N8O2S[M+Na] + 645.2755,found 645.2756.HPLC t R = 7.35 min (97.74% purity).

[0364] 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-(2-aminophenyl)pyrimidine-5-carboxamide (30e)

[0365] White solid, yield 70.2%, mp 217-219℃. 1 H NMR(400MHz,DMSO)δ9.52(s,1H),8.93(s,2H),7.95(s,1H),7.57–7.50(m,2H ),7.40–7.31(m,4H),7.14(d,J=7.8Hz,1H),7.01–6.93(m,1H),6.77(d,J=7.9 Hz,1H),6.59(t,J=7.5Hz,1H),5.57(s,1H),4.95(s,2H),4.76(d,J=13.3Hz,2 H),3.05(s,3H),2.83–2.75(m,2H),1.91–1.77(m,4H),1.22(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ170.01,164.79,164.43,163.23,162.03,158.42,143.80,136.60,129.16,128.64,127.38,127.06,123.26,116. 65,116.57,116.51,116.37,115.16,93.89,87.70,53.71,52.45,47.63,29.58,29.09,29.04,27.60,14.01.HRMS-ESI(m / z)calc.for C 34 H 34 N 10 O2S[MH] - 645.2489,found 645.2494.HPLC t R = 5.21 min (96.94% purity).

[0366] Example 11: Preparation of target compounds 40a-d, 44a-c, and 48

[0367] Methyl 4-(N-methylmethanesulfonamide)benzoate (32)

[0368] Methanesulfonyl chloride (1.04 g, 9.08 mmol) was slowly added to 10 mL of CH₂Cl₂ containing 31 (1 g, 6.05 mmol) at 0 °C, and the reaction was carried out at room temperature for 1.5 h. The reaction solution was diluted with 50 mL of CH₂Cl₂, and the organic phase was washed successively with 1 M hydrochloric acid aqueous solution (100 mL), saturated sodium bicarbonate aqueous solution (100 mL), and saturated sodium chloride aqueous solution (100 mL). The organic phase was then dried over anhydrous Na₂SO₄ and concentrated. 32 was purified by silica gel column chromatography to obtain a pale yellow solid in 83.5% yield. 1 H NMR (400MHz, DMSO) δ8.02–7.94(m,2H),7.59–7.51(m,2H),3.86(s,3H),3.30(s,3H),3.01(s,3H).

[0369] N-(4-(hydroxymethyl)phenyl)-N-methylmethanesulfonamide (33)

[0370] DIBAL (8.22 mmol) was added to a 32 g (1 g, 4.11 mmol) THF solution, and the mixture was stirred at -20 °C for 6 hours. After the reaction was completed, the reaction was quenched with 30 mL of 10% NaOH solution. The mixture was then extracted with ethyl acetate (10 mL × 3), and the organic phase was concentrated to give the crude product. The crude product was purified by column chromatography to give a 33 g gray solid, with a yield of 68.9%. 1 H NMR (400MHz, DMSO) δ7.40–7.30(m,4H),5.22(t,J=5.7Hz,1H),4.49(d,J=5.7Hz,2H),3.22(s,3H),2.92(s,3H).

[0371] 4-(N-methylmethylsulfonamido)benzyl-4-methylbenzenesulfonate (34)

[0372] Pale yellow solid, yield 45.8%. MS-ESI (m / z) calc. for C 16 H 19 N1O5S2[M+H] + 370.1.

[0373] N-(4-(((6-amino-3,5-dicyano-4-ethylpiperidin-2-yl)thio)methyl)phenyl)-N-methylmethanesulfonamide (35)

[0374] Brownish-yellow solid, yield 71.6%. 1 H NMR (400MHz, DMSO) δ7.55–7.50(m,2H),7.35–7.30(m,2H),4.46(s,2H),3.21(s,3H),2.93(s,3H),2.69(q,J=7.8Hz,2H),1.18(t,J=7.6Hz,3H).

[0375] N-(4-(((6-chloro-3,5-dicyano-4-ethylpiperidin-2-yl)thio)methyl)phenyl)-N-methylmethanesulfonamide (36)

[0376] Brownish-yellow solid, yield 31.3%. 1 H NMR (400MHz, DMSO) δ7.51(d,J=7.2Hz,2H),7.42–7.33(m,2H),4.58(s,2H),3.21(s,3H),2.94(s,3H),2.88(q,J=8.2Hz,2H),1.25(t,J=8.6Hz,3H).

[0377] tert-Butyl(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl)carbamate (37a)

[0378] Yellow solid, yield 79.7%. 1 H NMR (400MHz, DMSO) δ7.44(d,J=8.4Hz,2H),7.41–7.34(m,2H),6.94(t,J=6.0Hz,1H),4.52(s,2H),3.83(t,J=6.1H z,2H),3.39(s,3H),3.20(d,J=8.2Hz,5H),2.92(s,3H),2.77(q,J=7.6Hz,2H),1.31(s,9H),1.21(t,J=7.6Hz,3H).

[0379] tert-Butyl(3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)propyl)carbamate (37b)

[0380] Yellow solid, yield 77.2%. 1 H NMR (400MHz, DMSO) δ7.45(d,J=8.2Hz,2H),7.37(dd,J=8.3,1.5Hz,2H),6.85(d,J=5.6Hz,1H),4.52(s,2H),3.73(t,J=7.4Hz ,2H),3.21(s,3H),2.94(dd,J=8.3,3.5Hz,5H),2.77(q,J=7.6Hz,2H),1.79–1.71(m,2H),1.33(s,9H),1.21(t,J=7.6Hz,3H).

[0381] tert-Butyl(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl)(methyl)carbamate (37c)

[0382] Yellow solid, yield 74.0%. 1 H NMR (400MHz, DMSO) δ7.43(d,J=8.3Hz,1H),7.37(d,J=8.3Hz,1H),4.53(s,1H),3.91(d,J=25. 3Hz,1H),3.37(d,J=7.7Hz,3H),2.91(s,2H),1.38(s,2H),1.31(s,2H),1.22(d,J=6.8Hz,5H).

[0383] 2-((3,5-Dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl-1-ammonium salt (38a)

[0384] Pale yellow solid, yield 90.1%. 1 H NMR (400MHz, DMSO) δ8.01(s,3H),7.46(d,J=8.3Hz,2H),7.38(d,J=8.5Hz,2H),4.54(s,2H),3.99(t,J=6.4Hz,2 H), 3.45 (s, 3H), 3.22 (s, 3H), 3.10 (d, J = 7.1Hz, 2H), 2.94 (s, 3H), 2.80 (q, J = 7.6Hz, 3H), 1.23 (t, J = 7.5Hz, 3H).

[0385] 3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)propyl-1-ammonium salt (38b)

[0386] Pale yellow solid, yield 84.1%. 1 H NMR (400MHz, DMSO) δ7.95 (s, 3H), 7.46 (d, J = 8.4Hz, 2H), 7.39 (d, J = 8.3Hz, 2H), 4.54 (s, 2H), 3.99 (t, J = 6.4Hz, 2 H),3.45(s,3H),3.23(s,3H),3.11(t,J=6.5Hz,2H),2.94(s,3H),2.80(q,J=7.6Hz,2H),1.23(t,J=7.6Hz,3H).

[0387] 2-((3,5-Dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)propyl-1-ammonium salt (38c)

[0388] Pale yellow solid, yield 85.0%. 1 H NMR (400MHz, DMSO) δ8.82(s,2H),7.50–7.43(m,2H),7.43–7.35(m,2H),4.56(s,2H),4.04(t,J=6.3Hz,2H),3.46(s,4H ),3.22(s,3H),3.17(q,J=6.8Hz,3H),2.94(s,3H),2.80(q,J=7.6Hz,2H),2.54(d,J=5.3Hz,3H),1.23(t,J=7.5Hz,3H).

[0389] N 1 -2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 4 -((tetrahydro-2H-pyran-2-yl)oxo)succinamide (39a)

[0390] Pale yellow solid, yield 31.8%. 1 H NMR (400MHz, DMSO) δ10.89(s,1H),7.94(t,J=5.9Hz,1H),7.45(d,J=8.5Hz,2H),7.37(d,J=8. 4Hz,2H),4.79(s,1H),4.51(s,2H),3.89(d,J=11.1Hz,1H),3.82(t,J=6.2Hz,2H),3.48(d,J= 11.6Hz,1H),3.38(s,3H),3.29(d,J=6.1Hz,1H),3.21(s,3H),2.92(s,3H),2.78(q,J=7.5Hz, 2H), 1.98 (dt, J = 20.0, 7.5Hz, 4H), 1.65 (p, J = 7.8Hz, 5H), 1.50 (s, 3H), 1.22 (t, J = 7.6Hz, 3H).

[0391] N 1 -2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 5 -((tetrahydro-2H-pyran-2-yl)oxo)glutaramide (39b)

[0392] Pale yellow solid, yield 44.2%. 1 H NMR (400MHz, DMSO) δ10.86 (s, 1H), 7.96 (t, J = 6.1Hz, 1H), 7.44 (d, J = 8.4Hz, 2H) ,7.37(d,J=8.3Hz,2H),4.77(s,1H),4.49(s,2H),3.81–3.74(m,2H),3.40(s,3H ),3.29(d,J=6.1Hz,3H),3.20(s,3H),2.91(s,3H),2.76(q,J=7.5Hz,2H),,1.98 (dt,J=20.0,7.5Hz,4H),1.71–1.64(m,5H),1.50(s,3H),1.22(t,J=7.6Hz,3H).

[0393] N1 -2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)propyl)-N 4 -((tetrahydro-2H-pyran-2-yl)oxo)succinamide (39c)

[0394] Pale yellow solid, yield 39.5%. 1 H NMR (400MHz, DMSO) δ10.95(s,1H),7.89(s,1H),7.48–7.41(m,2H),7.41–7.34(m,2H),4. 78(s,1H),4.52(s,2H),3.90(s,1H),3.73(s,2H),3.48(s,2H),3.22(s,3H),3.05(d,J=6 .1Hz,2H),2.93(d,J=1.9Hz,3H),2.77(d,J=8.0Hz,2H),2.42(d,J=4.7Hz,2H),2.26(s,2 H),2.21(d,J=6.8Hz,2H),1.75(s,2H),1.62(s,3H),1.49(s,3H),1.21(t,J=7.7Hz,3H).

[0395] 2-((2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)(methyl)amino)ethyl)(methyl)amino)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (39d)

[0396] Pale yellow solid, yield 39.2%. 1 H NMR(400MHz,DMSO)δ11.49(s,1H),8.62(s,2H),7.44–7.32(m,4H),5.75(s, 1H),4.93(s,1H),4.47(s,2H),4.08(s,2H),4.02(s,1H),3.91(t,J=5.8Hz,2 H),3.52(d,J=11.4Hz,1H),3.35(s,3H),3.21(s,3H),3.08(s,3H),2.92(s,3 H),2.72(d,J=7.6Hz,10H),1.71(s,3H),1.54(s,3H),1.17(t,J=7.6Hz,3H).

[0397] N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N4 -Hydroxybutyramide (40a)

[0398] Pale yellow solid, yield 21.2%, mp 152-154℃. 1 H NMR (400MHz, DMSO) δ10.39(s,1H),8.68(d,J=1.6Hz,1H),8.04(t,J=5.9Hz,1H), 7.45(d,J=8.2Hz,2H),7.38(d,J=8.3Hz,2H),4.52(s,2H),3.81(t,J=6.2Hz,2H) ,3.39(s,3H),3.30(d,J=6.1Hz,3H),3.22(s,3H),2.93(s,3H),2.78(q,J=7.5Hz ,2H),2.25(dd,J=8.8,6.6Hz,2H),2.15(t,J=7.4Hz,2H),1.22(t,J=7.5Hz,3H). 13 C NMR (101MHz, DMSO) δ171.93,168.79,164.72,164.54,157.63,141.30,135.82,129.88,126.83,116.99,115 .36,93.51,86.00,51.69,38.18,36.93,35.47,33.45,31.06,28.17,27.62,14.00.HRMS-ESI(m / z)calc.for C 34 H 34 N 10 O2S[M+Na] + 596.1726,found 596.1728.HPLC t R = 3.98 min (96.78% purity).

[0399] N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 5 -Hydroxyglutaramide (40b)

[0400] Pale yellow solid, yield 30%, mp 117-119℃. 1H NMR (400MHz, DMSO) δ10.37(s,1H),8.68(s,1H),7.90(t,J=5.6Hz,1H),7.44(d,J=8.2 Hz,2H),7.37(d,J=8.1Hz,2H),4.52(s,2H),4.03(td,J=8.6,5.8Hz,1H),3.73(t,J=7 .5Hz,2H),3.22(s,3H),3.05(q,J=6.4Hz,3H),2.93(s,3H),2.77(q,J=7.4Hz,2H),2. 30–2.24(m,2H),2.16(t,J=7.4Hz,2H),1.76(t,J=7.5Hz,2H),1.22(t,J=7.9Hz,3H). 13 C NMR (101MHz, DMSO) δ171.56,168.89,164.80,164.59,157.44,141.32,135.81,129.87,126.82,116.93,115.38, 93.41,85.92,50.28,38.18,36.50,35.49,33.43,31.04,28.25,27.64,14.56,13.99.HRMS-ESI(m / z)calc.forC 26 H 33 N7O5S2[M+Na] + 610.1836, found 586.1831.HPLC t R = 4.09 min (95.64% purity).

[0401] N 1 -(3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)propyl)-N 4 -Hydroxybutyramide (40c)

[0402] Pale yellow solid, yield 39.7%, mp 110-112℃. 1H NMR (400MHz, DMSO) δ10.41(s,1H),8.70(s,1H),8.04(t,J=5.9Hz,1H),7.45(d,J= 8.2Hz,2H),7.38(d,J=8.3Hz,2H),4.52(s,2H),3.82(t,J=6.2Hz,2H),3.39(s,3H) ,3.31(q,J=6.1Hz,3H),3.22(s,3H),2.93(s,3H),2.78(q,J=7.5Hz,2H),2.01(t, J=7.6Hz,2H),1.92(t,J=7.4Hz,2H),1.66(p,J=7.5Hz,2H),1.22(t,J=7.5Hz,3H). 13 C NMR (101MHz, DMSO) δ172.49,169.17,164.75,164.53,157.66,141.31,135.80,129.90,126.81,116.97,115.34, 93.52,86.00,51.71,38.19,36.85,35.53,35.19,33.50,32.22,27.61,21.81,13.98.HRMS-ESI(m / z)calc.forC 26 H 33 N7O5S2[MH] - 586.1887, found 586.1883. HPLC t R = 7.85 min (98.24% purity).

[0403] 2-((2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (40d)

[0404] Orange-yellow solid, yield 27%, mp 155-157℃. 1 H NMR (400MHz, DMSO) δ11.05(s,1H),9.00(s,1H),8.61(s,2H),7.42(d,J=8.2Hz,2H),7.36(d,J=8.2Hz,2H),4.48(s,2H),4.12–4.0 5(m,2H),3.91(t,J=5.9Hz,2H),3.35(s,3H),3.32(s,3H),3.07(s,3H),2.93(s,3H),2.72(q,J=7.7Hz,2H),1.17(t,J=7.6Hz,3H). 13C NMR (101MHz, DMSO) δ172.49,169.17,164.75,164.53,157.66,141.31,135.80,129.90,126.81,116.97,115.34 ,93.52,86.00,51.71,38.19,36.85,35.53,35.19,33.50,32.22,27.61,21.81,13.98.HRMS-ESI(m / z)calc.for C 27 H 31 N9O4S2[MH] - 608.1845,found 608.1640.HPLC t R = 9.90 min (98.58% purity).

[0405] tert-Butyl(1-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidin-4-yl)carbamate (41)

[0406] White solid, yield 73.5%. 1 H NMR (400MHz, DMSO) δ7.44(d,J=8.3Hz,2H),7.37(d,J=8.3Hz,2H),6.90(d,J=7.8Hz,1H),4.50(s,2H),4.39(d,J=13.5Hz,2H),3.60(s,1H),3.2 7(s,1H),3.21(s,3H),2.92(s,3H),2.76(q,J=7.9Hz,2H),1.85(d,J=12.7Hz,2H),1.44(d,J=12.6Hz,2H),1.39(s,9H),1.21(t,J=7.6Hz,3H).

[0407] 1-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidine-4-ammonium salt (42)

[0408] White solid, yield 80.8%. 1H NMR (400MHz, DMSO) δ8.12(s,3H),7.45(d,J=8.4Hz,2H),7.38(d,J=8.4Hz,2H),4.53(d,J=6.8Hz,4H),3.24(d,J=1 7.7Hz,5H),2.94(s,3H),2.78(q,J=7.6Hz,2H),2.06(d,J=12.6Hz,2H),1.67–1.54(m,2H),1.22(t,J=7.6Hz,3H).

[0409] N 1 -(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -((tetrahydro-2H-pyran-2-yl)oxo)glutaramide (43a)

[0410] White solid, yield 37%. 1 H NMR (400MHz, DMSO) δ10.91(s,1H),7.81(d,J=7.6Hz,1H),7.44(d,J=8.3Hz,2H),7.37(d,J=8.4Hz,2H),4.8 0(s,1H),4.50(s,2H),4.37(d,J=13.4Hz,2H),3.91(d,J=9.6Hz,2H),3.49(d,J=11.6Hz,1H),3.37(d,J=12 .4Hz,2H),3.21(s,3H),3.17(d,J=5.2Hz,1H),2.93(s,3H),2.77(q,J=7.6Hz,2H),2.06(t,J=7.5Hz,2H),1 .99(t,J=7.4Hz,2H),1.87(d,J=12.8Hz,2H),1.77–1.60(m,5H),1.54–1.37(m,5H),1.22(t,J=7.6Hz,3H).

[0411] (E)-3-(4-(((1-(3,5-dicyano-4-ethyl-6-((N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-((tetrahydro-2H-pyran-2-yl)oxo)acrylamide(43b)

[0412] White solid, yield 45%. 1H NMR (400MHz, DMSO) δ11.21(s,1H),7.52(t,J=7.6Hz,3H),7.44(d,J=8.6Hz,2H),7.41(d,J =7.9Hz,2H),7.37(d,J=8.4Hz,2H),6.48(d,J=15.8Hz,1H),4.91(s,1H),4.51(s,2H),4.36 (d,J=13.3Hz,2H),3.97(s,1H),3.77(s,2H),3.21(s,3H),2.92(s,3H),2.77(d,J=7.5Hz, 2H),1.95(s,2H),1.70(s,3H),1.55(s,3H),1.37(d,J=10.5Hz,2H),1.22(t,J=7.7Hz,3H).

[0413] 2-((1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidin-4-yl)amino)-N-((tetrahydro-2H-pyran-2-yl)oxo)pyrimidine-5-carboxamide(43c)

[0414] White solid, yield 40.9%. 1 H NMR (400MHz, DMSO) δ11.51(s,1H),8.64(s,2H),7.91(d,J=7.9Hz,1H),7.45(d,J=8.5Hz,2H),7.41–7.33(m,2H),4.95(s,1H),4.48(d,J=22.6Hz,4H ),4.18(s,1H),3.20(s,3H),2.92(s,3H),2.78(q,J=7.6Hz,2H),1.99(d,J =12.3Hz,2H),1.71(s,3H),1.56(d,J=12.7Hz,5H),1.22(t,J=7.6Hz,3H).

[0415] N 1 -(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutaramide (44a)

[0416] White solid, yield 23.5%, mp 149-151℃. 1H NMR (400MHz, DMSO) δ10.41(s,1H),8.73–8.66(m,1H),7.91(d,J=7.7Hz,1H),7.44(d,J=8.2Hz,2H ),7.37(d,J=8.3Hz,2H),4.50(s,2H),4.38(d,J=13.4Hz,2H),3.91(s,1H),3.21(s,3H),3.17(d,J =5.2Hz,1H),2.93(s,3H),2.77(q,J=7.6Hz,2H),2.06(t,J=7.5Hz,2H),1.96(dt,J=14.7,7.4Hz, 3H), 1.86 (d, J = 12.7Hz, 2H), 1.70 (p, J = 7.7Hz, 2H), 1.44 (q, J = 11.4Hz, 2H), 1.21 (t, J = 7.6Hz, 3H). 13 C NMR (101MHz, DMSO) δ171.42,169.22,165.05,164.49,158.59,141.25,136.09,129.79,126.83,116.44,115.18,94.38,87. 83,60.23,46.93,45.50,38.18,35.46,35.23,33.53,33.24,32.18,31.92,31.72,27.60,21.92,21.03,14.59,14.03.HPLC t R = 9.90 min (98.58% purity).

[0417] (E)-(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutaramide (44b)

[0418] White solid, yield 27.2%, mp 164-166℃. 1H NMR (400MHz, DMSO) δ10.81 (s, 1H), 9.07 (s, 1H), 7.55 (d, J = 7.8Hz, 2H), 7.50–7. 41(m,5H),7.38(d,J=8.1Hz,2H),6.48(d,J=15.7Hz,1H),4.51(s,2H),4.43(d,J =13.2Hz,2H),3.90(s,1H),3.29(d,J=11.9Hz,3H),3.21(s,3H),2.92(s,3H),2. 77(q,J=7.5Hz,2H),2.04(d,J=12.1Hz,2H),1.49(s,2H),1.21(t,J=7.6Hz,3H). 13 C NMR (101MHz, DMSO) δ165.04,164.52,163.17,158.44,141.25,138.45,136.14,129.81,127.91,126.83,1 16.47,115.19,94.33,87.74,53.23,46.47,38.20,35.48,33.49,27.60,14.02.HRMS-ESI(m / z)calc.for C 33 H 37 N7O4S2[M+H] + 660.2368,found 660.2371.HPLC t R = 5.47 min (96.01% purity).

[0419] (E)-3-(4-(((1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (44c)

[0420] White solid, yield 29%, mp 137-139℃. 1H NMR (400MHz, DMSO) δ11.07(s,1H),9.00(s,1H),8.63(s,2H),7.82(d,J=7.8Hz ,1H),7.45(d,J=8.1Hz,2H),7.37(d,J=8.1Hz,2H),4.51(s,2H),4.46(s,2H), 4.17(s,1H),3.44(d,J=7.1Hz,2H),3.20(s,3H),2.92(s,3H),2.78(q,J=7.7H z, 2H), 1.99 (d, J = 12.3Hz, 2H), 1.57 (d, J = 11.7Hz, 2H), 1.22 (t, J = 7.5Hz, 3H). 13 C NMR (101MHz, DMSO) δ165.07,164.55,162.54,159.83,158.50,141.22,136.12,129.77,126.85,116.48,115.20,94 .37,87.76,64.00,55.39,47.58,46.98,38.17,35.42,33.51,31.71,27.61,14.69,14.03.HRMS-ESI(m / z)calc.for C 28 H 31 N9O4S2[MH] - 620.1840,found 620.1838.HPLC t R = 2.27 min (99.37% purity).

[0421] tert-Butyl 4-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridinyl)piperazine-1-carbamate (45)

[0422] White solid, yield 81.1%. 1 H NMR (400MHz, DMSO) δ7.46(d,J=8.2Hz,2H),7.38(d,J=8.2Hz,2H),4.53(s,2H),3.86(t,J=5.2Hz,4H ),3.46(s,3H),3.22(s,3H),2.93(s,3H),2.77(q,J=7.5Hz,2H),1.42(s,9H),1.22(t,J=7.5Hz,3H).

[0423] 4-(3,5-Dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperazine-1-ammonium salt (46)

[0424] White solid, yield 86.1%. 1 H NMR(400MHz,DMSO)δ9.42(s,2H),7.46(d,J=8.0Hz,2H),7.38(d,J=8.2Hz,2H),4.54(s,2H) ,4.05(q,J=6.5Hz,4H),3.23(s,7H),2.94(s,3H),2.84–2.75(m,2H),1.24(d,J=7.2Hz,3H).

[0425] 2-(4-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)benzyl)thio)pyridin-2-yl)piperazin-1-yl)-N-((tetrahydro-2-H-pyran-2-yl)oxo)pyrimidine-5-carboxamide (47)

[0426] White solid, yield 49.2%. 1 H NMR (400MHz, DMSO) δ12.05(s,1H),11.56(s,1H),9.05(s,2H),7.47(d,J=8 .3Hz,2H),7.38(d,J=8.4Hz,2H),4.96(s,1H),4.55(s,2H),4.02–3.99(m,4 H),3.96(dd,J=7.1,3.5Hz,4H),3.55(s,2H),3.21(s,3H),2.92(s,3H),2. 80(q,J=7.8Hz,2H),1.74–1.71(m,3H),1.55(s,3H),1.23(t,J=7.6Hz,3H).

[0427] 2-(4-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)benzyl)thio)pyridin-2-yl)piperazin-1-yl)N-hydroxypyrimidine-5-carboxamide (48)

[0428] White solid, yield 33%, mp 170-171℃. 1 H NMR (400MHz, DMSO) δ8.73(s,2H),7.47(d,J=8.4Hz,2H),7.38(d,J=8.5Hz,2H),4.55(s,2H),4.00(dd,J=7.2,3 .5Hz,4H),3.95(dd,J=7.1,3.6Hz,4H),3.21(s,3H),2.92(s,3H),2.80(q,J=7.6Hz,2H),1.23(t,J=7.6Hz,3H). 13CNMR(101MHz,DMSO)δ165.14,164.47,161.72,158.70,157.58,141.27,136.09,129.79,126.81,116.41, 115.57,115.12,94.78,88.00,47.19,43.34,38.19,35.54,33.62,27.58,14.01.HRMS-ESI(m / z)calc.for C 27 H 29 N9O4S2[MH] - 606.1688, found 606.1683.HPLCt R = 9.16 min (99.65% purity).

[0429] Experimental Example 1: Inhibitory Activity of Target Compound on HDACs

[0430] 1. Materials:

[0431] 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

[0432] 2. Method:

[0433] Buffer preparation:

[0434] The formula is 15mM Tris-HCl (pH=8.0), 250μM EDTA, 250mM NaCl, and 10% glycerol.

[0435] Preparation of Trypsin solution:

[0436] The formula is 10 mg / mL pancreatin, buffer, 2 μL BH589.

[0437] Preparation of substrate solution:

[0438] The substrate was dissolved in DMSO to prepare a 30 mM stock solution, which was then diluted to 300 μM with buffer.

[0439] The DMSO content is approximately 1%.

[0440] Diluting the enzyme solution:

[0441] Dilute the enzyme solution with a buffer at a ratio of 1:80.

[0442] Preparation of compound solutions:

[0443] Dilute the compounds (test compound and positive control SAHA) to a final concentration using a buffer.

[0444] Preparation and determination of 100% and blank:

[0445] 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.

[0446] Determination of the inhibitory activity of compounds against HDACs:

[0447] 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)

[0448] Table 1. HDAC-inhibiting activity of the target compounds

[0449]

[0450] 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

[0451] b Untested

[0452] Experimental Example 2: Inhibitory Activity of Target Compound on DNMT1

[0453] 1. Materials:

[0454] MT1 hypo-SFRP1-Luc cells; 96-well all-black microplates; RPMI 1640 medium; 10% fetal bovine serum (Hyclone, USA); PBS buffer; Luciferase assay reagent, positive control drugs SAHA and GSK5032.

[0455] 2. Method:

[0456] Inhibition of DNMT1 restored the expression of the hypermethylated SFRP1 gene. Since SFRP1 inserts into the luciferase gene, its protein expression level could be determined by detecting fluorescence intensity. MT1hypo-SFRP1-Luc cells (5400 cells per well) were seeded in black-well plates and cultured for 16 hours in a constant-temperature incubator (37°C, 5% CO2). Then, different concentrations of compound dilutions prepared with the corresponding cell culture media were added, and the plates were incubated for another 72 hours. Finally, luciferase assay reagent was added to each well, and the corresponding fluorescence intensity was detected using a microplate reader. Inhibitory activity was expressed as fold change (fluorescence intensity of the compound group / fluorescence intensity of the blank control group) (Table 2).

[0457] Table 2. DNMT1 inhibitory activity of the target compound

[0458]

[0459] a A. Fold change less than 100; B. Fold change between 100 and 200; C. Fold change between 200 and 300; D. Fold change greater than 300.

[0460] Experiment Example 3: Antiproliferative Activity Experiment of Target Compound

[0461] 1. Materials:

[0462] MV4-11 (human acute myeloid leukemia cells), KG-1 (human acute myeloid leukemia cells), THP-1 (human monocytic leukemia cells), Jurkat (human T-lymphocytic leukemia cells), K562 (human chronic myeloid leukemia cells), MC38 (mouse colon cancer cells), 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 drugs SAHA and GSK5032, 96-well plates.

[0463] 2. Method:

[0464] 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).

[0465] Table 3. Antiproliferative activity of the compounds

[0466]

[0467] a A, IC 50 Greater than 20 μM; B, IC 50 Between 4 μM and 20 μM; C, IC 50 Between 0.4 μM and 4 μM; D, IC 50 Below 0.4μM

[0468] Experimental Example 4: Therapeutic Effect of Compounds on Human Acute Myeloid Leukemia

[0469] 1. Materials:

[0470] Cell line: Human acute myeloid leukemia cells (MV4-11).

[0471] Laboratory animals: Six-week-old female BALB / c nude mice, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0472] 2. Method:

[0473] 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 28 days. The treatment group received 30 mg / kg / day, the combination therapy group received 30 mg / kg / day + 30 mg / kg / day, and 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 volume in the treatment group / average tumor volume in the control group) × 100%.

[0474] 3. Experimental Results:

[0475] Test results are available Figure 1 The target compound (R)-23a showed the best tumor inhibition rate (TGI) of 98%, which was significantly better than the combination therapy group of the positive control drug GSK5032 and SAHA.

[0476] Experimental Example 5: Therapeutic Effect of Compounds on Colon Cancer in Mice

[0477] 1. Materials:

[0478] Cell line: Mouse colon cancer cells (MC38).

[0479] Laboratory animals: Six-week-old female BALB / c nude mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0480] 2. Method:

[0481] Approximately 1.2 × 10 6 One MC38 cell was resuspended in 100 μL of serum-free high-glucose medium 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 14 consecutive days. The treatment group received 30 mg / kg / day, the combination therapy group received 30 mg / kg / day + 30 mg / kg / day, and 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 volume in the treatment group / average tumor volume in the control group) × 100%.

[0482] 3. Experimental Results:

[0483] Test results are available Figure 2 The target compound (R)-23a showed a tumor inhibition rate (TGI) of 82% in a mouse colon cancer model, which was superior to the positive control drug GSK5032 and its combination therapy with SAHA.

Claims

1. A dual-target inhibitor of DNMT1-HDAC, characterized in that, The inhibitor is selected from one of the following compounds: N 1 - (1 -(6-((2-amino-2-oxo- 1 -phenylethyl)thio)-3,5-dicyano-4- ethylpyridin-2-yl)piperidin-4-yl)-N 4 - hydroxybutanediamide (11a); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutamate (11b); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -Hydroxyhexadiamide (11c); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 7 -Hydroxyheptanamide (11d); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 8 -Hydroxyoctadiamide (11e); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 9 -Hydroxynonadiamide (11f); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -Hydroxy-p-toluidine (11g); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-4-((4-(hydroxycarbamoyl)benzyl)oxy)benzamide (11h); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11i); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)pentanediamide (11j); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 6 -(4-(hydroxycarbamoyl)phenyl)hexamethylenediamide (11k); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(hydroxycarbamoyl)phenyl)butyramide (11l); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(4-(hydroxycarbamoyl)phenyl)glutaramide (11m); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)phenyl)butadiamide (11n); 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-hydroxybenzamide (11o); (E)-3-(4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (11p); 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(hydroxycarbamoyl)phenyl)carbamoyl)phenyl)piperidine 4-ammonium salt (11q); (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)carbamoyl)benzyl)piperidine-4-ammonium salt (11r); (E)-1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((4-(3-(hydroxyamino)-3-oxopropyl-1-en-1-yl)benzyl)oxo)benzyl)piperidine-4-ammonium salt (11s); 1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)-N-(4-((7-(hydroxyamino)-7-oxohepyl)oxo)benzyl)piperidine-4-ammonium salt (11t); 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-N-hydroxypyrimidine-5-carboxamide (11u); 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (11v); 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15a) 2-(4-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)(methyl)amino)piperidin-1-yl)-N-hydroxypyrimidine-5-carboxamide (15b); (E)-3-(4-((4-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)methyl)phenyl)-N-hydroxyacrylamide (19a); 2-(4-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (19b); (E)-3-(4-((((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide (23a); (R,E)-3-(4-((((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide((R)-23a); (S,E)-3-(4-((((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)methyl)phenyl)-N-hydroxyacrylamide((S)-23a); 2-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)methyl)amino)-N-hydroxypyrimidine-5-carboxamide (23b); (E)-2-((6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)(methyl)amino)-N-(4-(3-(hydroxyamino)-3-oxopropane-1-en-1-yl)benzyl)ethyl-1-ammonium salt (27); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 5 -(2-aminophenyl)pentanediamide (30a); N 1 -(1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)-N 4 -(2-aminophenyl)terephthalamide (30b); 4-(2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)-2-oxoethyl)-N-(2-aminophenyl)benzamide (30c); 4-(((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)amino)methyl)-N-(2-aminophenyl)benzamide (30d); 2-((1-(6-((2-amino-2-oxo-1-phenylethyl)thio)-3,5-dicyano-4-ethylpyridin-2-yl)piperidin-4-yl)(methyl)amino)-N-(2-aminophenyl)pyrimidine-5-carboxamide (30e); N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 4 -Hydroxybutyramide (40a); N 1 -(2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)-N 5 -Hydroxyglutamate (40b); N 1 -(3-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)propyl)-N 4 -Hydroxybutyramide (40c); 2-((2-((3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)(methyl)amino)ethyl)(methyl)amino)-N-hydroxypyrimidine-5-carboxamide (40d); N 1 -(1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)-N 5 -Hydroxyglutamate (44a); (E)-3-(4-(((1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethanesulfonyl)benzyl)thio)pyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (44b) (E)-3-(4-(((1-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperidin-4-yl)amino)methyl)phenyl)-N-hydroxyacrylamide (44c); 2-(4-(3,5-dicyano-4-ethyl-6-((4-(N-methylmethylsulfonylamino)phenyl)thio)pyridin-2-yl)piperazin-1-yl)-N-hydroxypyrimidine-5-carboxamide (48).

2. The method for preparing the DNMT1-HDAC dual-target inhibitor according to claim 1, wherein the preparation method comprises any of the following methods: The preparation method of compound 11a-11V is as follows: The preparation methods of compounds 15a, 15b, 19a, and 19b are as follows: The preparation methods of compounds 23a, (R)-23a, (S)-23a, 23b, 27, and 30a-e are as follows: The preparation method of compound 40a-d is as follows: The preparation methods of compounds 44a-c and 48 are as follows: 。 3. The preparation method according to claim 2, characterized in that, In the preparation method of compound 11a-11V, The reagents and conditions are as follows: (a) p-Toluenesulfonyl chloride, N,N-diisopropylethylamine, 4-dimethylaminopyridine, acetonitrile, 80°C, 6 hours; (b) n-Propanal, N-methylmorpholine, anhydrous ethanol, room temperature, 30 hours; (c) N,N-diisopropylethylamine, N,N-dimethylformamide, 85°C, 5 hours; (d) Isoamyl nitrite, copper chloride, acetonitrile, 80°C, 2.5 hours; (e) Aminopiperidine derivative, potassium carbonate, dichloromethane, 4 hours; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (g) 1) 9a-n, (e) Isobutyl chloroformate, N-methylmorpholine, 1:1 dichloromethane / tetrahydrofuran, room temperature, 6 hours; (f) 9°C-t, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; (h) 9°C, potassium carbonate, acetonitrile, 80°C, 6 hours; (e) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; The structure of the aminopiperidine derivative in step (e) is as follows: , R=H or Me.

4. The preparation method according to claim 2, characterized in that, In the preparation methods of compounds 15a, 15b, 19a, and 19b, The reagents and conditions are as follows: (a) aminopiperidine derivative, potassium carbonate, dichloromethane, 4 hours; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (c) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (e) piperazine-1-tert-butylcarboxylate, potassium carbonate, dichloromethane, 4 hours; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; (g) 1) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 2) 9u, potassium carbonate, acetonitrile, 80°C, 6 hours; (h) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours; The structure of the aminopiperidine derivative in step (a) is... n = 0 or 1.

5. The preparation method according to claim 2, characterized in that, In the preparation methods of compounds 23a, (R)-23a, (S)-23a, 23b, 27, and 30a-e, The reagents and conditions are as follows: (a) piperidine-4-aminomethyl tert-butylcarbamate, potassium carbonate, dichloromethane, 4 h; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (c) 1) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; 2) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (e) tert-butyl(2-(methylamino)ethyl)carbamate, potassium carbonate, dichloromethane, 4 h; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (g) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; (h) 4M Hydrochloric acid / ethyl acetate, room temperature, 12 hours; (i) 1) 28a-c, isobutyl chloroformate, N-methylmorpholine, 1:1 dichloromethane / tetrahydrofuran, room temperature, 6 hours; 2) 28d, sodium triacetoxyborohydride, acetic acid, room temperature, 12 hours; 3) 28e, potassium carbonate, acetonitrile, 80°C, 6 hours; (j) 4M hydrochloric acid / ethyl acetate, room temperature, 12 hours.

6. The preparation method according to claim 2, characterized in that, In the preparation method of compound 40a-d, The reagents and conditions are as follows: (a) methanesulfonyl chloride, triethylamine, dichloromethane, room temperature, 4 h; (b) diisobutylaluminum hydride, tetrahydrofuran, -20 °C, 6 h; (c) p-toluenesulfonyl chloride, triethylamine, DMAP, acetonitrile, 80 °C, 4 h; (d) 4, triethylamine, DMF, 85 °C, 5 h; (e) isoamyl nitrite, copper chloride, acetonitrile, 80 °C, 2.5 h; (f) 2-methylamino derivative, potassium carbonate, dichloromethane, 4 h; (g) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (h) 1) 9a or 9b, isobutyl chloroformate, N-methylmorpholine, 1:1 dichloromethane / tetrahydrofuran, room temperature, 6 h; 2) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (i) 4M Hydrochloric acid / ethyl acetate, room temperature, 12 hours; the structure of the 2-methylamino derivative in step (f) is as follows: R=H or CH3, n=2 or 3.

7. The preparation method according to claim 2, characterized in that, In the preparation methods of compounds 44a-c and 48, The reagents and conditions are as follows: (a) tert-butyl 4-carbamate, potassium carbonate, dichloromethane, 4 h; (b) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (c) 1) 9b, isobutyl chloroformate, N-methylmorpholine, 1:1 dichloromethane / tetrahydrofuran, room temperature, 6 h; 2) 9p, sodium triacetoxyborohydride, acetic acid, room temperature, 12 h; 3) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (d) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (e) piperazine-1-tert-butylcarboxylate, potassium carbonate, dichloromethane, 4 h; (f) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h; (g) 9u, potassium carbonate, acetonitrile, 80 °C, 6 h; (h) 4M hydrochloric acid / ethyl acetate, room temperature, 12 h.

8. A pharmaceutical composition comprising a therapeutically effective amount of the DNMT1-HDAC dual-target inhibitor of claim 1 and a pharmaceutically acceptable excipient.

9. The use of the DNMT1-HDAC dual-target inhibitor according to claim 1 in the preparation of a medicament for treating target-related tumor diseases; The tumor is lung cancer, colon cancer, breast cancer, lymphoma, leukemia, or myeloma.